How many synapses are there in the different target regions of a typical cortical pyramidal cell?

How many synapses are there in the different target regions of a typical cortical pyramidal cell?

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I found the following pictures of axon trees:


source (axons are red)

but didn't find a concise answer to the following question:

How many (in relative terms) branches terminate and how many axon terminals (synapses) are there in the different target regions of a typical cortical pyramidal cell, i.e.

  • same cortical layer

  • other cortical layers

  • same cortical column

  • other cortical columns

  • more distant cortical regions (e.g. in the same gyrus)

  • subcortical regions

Pyramidal neurons: dendritic structure and synaptic integration

Pyramidal neurons have basal and apical dendrites, including an apical tuft. This preserved core structure suggests that they have conserved core functions, whereas structural variation in other areas suggests additional functional specialization.

A number of new methods for studying pyramidal-cell activation and circuitry are available. These include in vivo patch-clamp recording, optical activation and transgenic methods for activating, inactivating or labelling neurons and their connections.

Synaptic inputs from distinct sources occur onto separate dendritic domains. Defining the degree to which synapses that carry different kinds of information are segregated onto different dendritic domains remains an important challenge.

Most excitatory synapses onto pyramidal neurons occur on dendritic spines, but the structure of the synapses they receive differs between dendritic domains.

Dendritic integration of synaptic input depends on the dendritic domain that is targeted. Synapses distant from the soma tend to produce less synaptic depolarization, but this might be countered by increasing the conductance of distal synapses or by activating voltage-gated channels in dendrites. Synapses on small-diameter dendrites cause larger local voltage changes, which reduce the effectiveness of synaptic scaling but increase the activation of voltage-gated conductances.

Inhibitory synapses specifically target the axon, soma or different dendritic domains. Integration of inhibitory inputs also differs across cellular domains.

The intrinsic firing properties of pyramidal neurons vary considerably. Along with variation in dendritic structure and channel distributions, such variability suggests that different pyramidal neurons might carry out specialized functions.

Pyramidal-neuron dendrites contain voltage-gated channels that can influence synaptic integration. These channels can also support backpropagating action potentials and dendritically initiated spikes. Dendritic excitability is a general property of all pyramidal neurons studied so far, but the details differ between different types of pyramidal neurons. Although there is some evidence for dendritic excitability in vivo, much more work is needed in this area.

Activation of a small fraction of the tens of thousands of excitatory synapses on a pyramidal neuron can probably evoke dendritic spikes, but these events do not always propagate to the soma and the axon. The coupling of dendritic spikes to axonal action-potential firing probably depends on the pattern of synaptic activation. This results in forms of coincidence detection that are determined by dendritic structure and excitability.

Backpropagating action potentials and dendritic spikes are important signals for the induction of synaptic plasticity. Even single dendritic spikes can result in significant long-term potentiation or long-term depression.

Neurotransmitters can modulate pyramidal-neuron function. At least some forms of modulation affect various dendritic domains and their synaptic inputs in different ways.

Domain-specific properties in excitatory and inhibitory synaptic inputs, voltage-gated channels, dendritic excitability and neuromodulation all point to a multi-compartment model of pyramidal-neuron function. Elaborating simple models of pyramidal-neuron function based on these dendritic-domain-specific properties is a central challenge for the study of cortical function.



The mammalian central nervous system (CNS) contains many millions of neurons, each of which characteristically receives thousands of individual inputs from other neurons and in turn provides thousands of outputs to other neurons. The sites of input and output are the synapses, which can be structurally defined with electron microscopy. Individual synapses vary enormously in their biochemical properties and physiological effects. The transmitter substances emitted by neurons at synapses, the receptors activated by the neurotransmitters, and the ensuing physiological responses (excitatory, inhibitory facilitating, depressing strong, weak ionotropic, metabotropic) are all tremendously variable, and susceptible to modification by neural activity or during development.

A major question about neural synapses is as follows: Are all of the structurally defined synapses visible in electron micrographs physiologically potent? Since the early 1970's, more and more evidence has accumulated that suggests that they are not. In particular, two early observations pointed to the occurrence of ineffective or incomplete (and thus, potentially silent) synapses. First, ultrastructural studies of crustacean motor neurons showed many synapses that appeared to lack docked (releasable) synaptic vesicles, and there were many more synapses than quanta released during neural activity (Jahromi and Atwood 1974). Secondly, and more dramatically, in the mammalian CNS, circumstances were found in which receptive fields could be suddenly expanded, implying the existence of newly awakened (previously silent) synapses (Wall 1977). Such observations indicated a strong possibility that many of the individual synapses formed between pairs of neurons are effectively silent, and do not produce a physiological effect when the presynaptic neuron is activated. It could even be that a majority of synapses in many neural pathways are physiologically silent, but such synapses might be recruited to physiological effectiveness with specific patterns of neural activity or through actions of neuromodulators and hormones. If so, this type of recruitment could be a major mechanism in pathway consolidation, learning, and memory. One can imagine that in many parts of the nervous system, there is a large reserve of silent synapses, which can be jolted into an active state by the right combination of stimuli, and can then enhance transmission either transiently or permanently in the neural circuits to which they contribute. According to this view, the nervous system is normally operating well below capacity, but has the potential for great enhancement and reconfiguration of local circuits. Silent synapses provide one mechanism (perhaps a major one) for enhanced performance of the nervous system.

The potential importance of silent synapses is a stimulus to review evidence supporting their existence and their possible functional significance. Evidence for silent synapses, particularly in the mammalian CNS, has been reviewed in several recent articles (Malenka and Nicoll 1997, 1999 Malenka 1998 Malinow 1998). We wish to extend the scope of the discussion to include other well-studied examples that offer relevant evidence and additional opportunities. New examples and evidence are appearing at an increasing rate.

First, we examine some basic features of synapses that might be involved in creation of silent synapses then, we examine a selection of currently available evidence and finally, we review briefly possible mechanisms and functional significance.


Synapses have both structural and functional attributes that are interrelated. Electron microscopy permits resolution of structural specialization at points of contact between nerve cells and their postsynaptic targets. The structural specialization is thought to reflect molecular features that impart the capability for fast release of neurotransmitter (fast exocytosis) from the presynaptic neuron during an action potential. Most commonly, the structural definition of a synapse (Korn 1998) is taken to be the individual contact, comprising electron-dense pre- and postsynaptic membranes aligned rigidly with uniform (20–50 nm) separation (the synaptic cleft), and including variable specialized structures in both pre- and postsynaptic compartments. As illustrated in Figure 1, A and C, synaptic terminals or their varicosities often provide many individual synapses to a postsynaptic target cell, but in many cases, only a single synapse is present. In the literature, other uses of the term synapse appear these have been reviewed recently by Korn (1998). The totality of synapses (which may be many) between one neuron and another, or between a neuron and a non-neural target, is commonly termed a synaptic connection (for review, see Faber et al. 1991).

Structural features of synapses related to function. (A) Synapses of Drosophila neuromuscular junction, showing several synapses (S) on one terminal. Characteristic structural features include the specialized electron-dense synaptic membranes, presynaptic dense bodies (T-shaped in Drosophila) located at active zones on the synapses (arrows), and specialized subsynaptic reticulum (postsynaptic side of the synapses). Synaptic vesicles are more densely clustered at dense bodies than elsewhere, and not all synapses possess dense bodies note that in this section, only three of the six synapses show this structure. Scale bar, 1 μm. (B) Diagram of some of the essential molecular components of the presynaptic vesicular docking and fusion-promoting apparatus. The core complex (vesicular VAMP/synaptobrevin, and presynaptic SNAP-25 and syntaxin) is thought to be essential for fusion of vesicular and presynaptic membranes. Regulatory proteins, including synaptotagmin (a putative Ca 2+ receptor) and cysteine string proteins that appear to affect Ca 2+ sensitivity of release processes, modify the rate and calcium sensitivity of release. (Other known regulatory proteins are not shown in this diagram). Interaction of syntaxin with part of the presynaptic calcium channel (synprint site) is thought to enhance the coordination between calcium entry and vesicular fusion during fast exocytosis. (C) Diagram of two types of mammalian synapse: (1) small synapse on a dendritic spine, with one active zone (2) large caliciform somatic synaptic connection (calyx of Held), with many individual synapses and active zones. This structure has features in common with the arthropod neuromuscular junction (A). The individual spine synaptic connection that frequently comprises a single synapse is typical of the majority of synapses in the mammalian CNS. (DF) Details of the synaptic active zone in freeze-fracture replicas (crayfish neuromuscular junction, inhibitory nerve terminal after Govind et al. 1995). In D andE, small and large active zones, respectively, exhibit collections of prominent intramembranous particles (putative calcium and calcium-activated K channels) in p-face views. Images of vesicular release (arrows), captured during chemical fixation, appear around the periphery of an active zone in an e-face view (F). Scale bars, 0.2 μm.

Associated with the individual synapse's presynaptic membrane, dense projections often occur. Although their composition is not fully known, they appear to be focal points for accumulations of synaptic vesicles and are thus likely involved in directed docking and tethering of these vesicles, which are preferentially released at the margins of the presynaptic specializations in arthropod neuromuscular junctions (Fig.1F). The presynaptic subregion involved in preferential release of neurotransmitter from synaptic vesicles is generally termed the active zone, from studies on the neuromuscular junction (del Castillo and Katz 1956 Couteaux 1970).

In many synapses of the CNS, particularly those on dendritic spines, the entire presynaptic face of the synapse may constitute an active zone, whereas in other cases, several active zones occur at one synapse (Jahromi and Atwood 1974 Cooper et al. 1995). Synaptic vesicles are preferentially released at the margins of an active zone, as shown in Figure 1F, or within it, in the case of many central synapses. Calcium channels are highly concentrated in the active zone (Fig. 1, D and E). Closely associated with calcium channels in this region are specialized molecules on the vesicles and on the presynaptic membrane, which form a multimolecular complex (core complex Fig. 1B). The core complex, regulatory proteins, and calcium channel are thought to be mutually linked, and the entire ensemble is sometimes referred to as a secretosome (Bennett 1996). Regulatory molecules and calcium receptors are also present, generating complex molecular interactions (Wu et al. 1999). Only some of the relevant molecules are illustrated in Figure1B. When this complex is activated by Ca 2+ , fast exocytosis ensues. Regulatory systems—calcium buffers, calcium sequestration, and extrusion mechanisms—profoundly affect synaptic strength. Postsynaptically, the membrane is specialized for localization of ligand-binding receptors, probably anchored to cytoskeletal components (Van Rossum and Hanisch 1999). Arrays of receptors are often seen in regular alignments in freeze-fracture replicas (Franzini-Armstrong 1976).

The structural components are linked to functional aspects of synaptic transmission. Synaptic vesicles contain and release transmitter substances in quantal events voltage-activated calcium channels of the presynaptic active zone trigger impulse-evoked release when opened by nerve impulses postsynaptic receptors bind released transmitter molecules and initiate a postsynaptic response, usually either movement of ions into the postsynaptic cell through an ion channel (ionotropic response), or alteration of a second-messenger pathway (metabotropic response). Modification of any of the synaptic components alters the physiological performance of the synapse, rendering it more or less effective. Deficiencies or incomplete assembly of synaptic components can render the synapse silent (incapable of producing a functional response). Because deficiencies or lack of fine tuning may often occur at the molecular level, structural entities observed as synapses in electron micrographs may in fact be functionally silent, although ultrastructural clues to this situation may exist, as discussed below. Without additional critical evidence, one cannot accept counts of synapses in electron micrographs as being equivalent to the functional capabilities of the sampled network.


We envision several scenarios that could give rise to silent synapses (Fig. 2). A functional synapse would have all its normal structural components, and also possess an appropriate complement of the essential presynaptic molecules and postsynaptic receptors. Deficiencies in any of these components may affect the final operation of the synapse, rendering it physiologically silent. Thus, absence of, or interference with, any of several molecules of the presynaptic docking and fusion mechanisms (Fig. 2B) blocks synaptic transmission, as illustrated experimentally by studies ofDrosophila mutants (Littleton et al. 1993 Umbach et al. 1994) and injection of interfering peptides into nerve terminals (Bommert et al. 1993). Lack of functional receptors could also defeat successful neurotransmission (Fig. 2D). If the synapse is incompletely assembled, as could occur during development or senescence, it would not be able to perform physiologically (Fig. 2E), but could emerge into functionality either spontaneously or with a prodding stimulus.

Possible types of silent synapse. (A) Complete synapse, possessing all of the necessary pre- and postsynaptic components. (B) Synapse silent due to presynaptic molecular deficiency. (C) Conditionally silent mammalian synapse (NMDA receptors only). (D) Synapse silent due to postsynaptic deficiency (receptors nonfunctional). (E) Synapse in transition (postsynaptic components being assembled).

A special case that occurs often in the mammalian CNS is that of synapses lacking functional AMPA-type glutamate receptors, but possessing NMDA-type glutamate receptors (Fig. 2C). Such synapses may be very prevalent early in development, and decrease in occurrence thereafter, as discussed below. Although these synapses are often referred to as silent synapses, we think they should be termed conditionally silent synapses, because they can express a physiological response when the membrane is depolarized, but not when it is hyperpolarized close to the resting potential (Fig.3).

Conditionally silent synapses. Two experiments from mammalian CNS preparations to illustrate physiological participation of NMDA-only synapses as the membrane potential becomes depolarized. (A) EPSCs of synapses between neurons of CA3 and CA1 (rat hippocampus) appear at +55 mV, but not at the resting membrane potential, and are blocked by APV (from Malinow 1998). (B) EPSCs observed when the membrane potential was changed from −70 to +50 mV (synapses between thalamic afferents and layer IV neurons in barrel-field cortex of rat). EPSCs were blocked by APV, indicating that NMDA receptors caused them (from Isaac et al. 1997).

We conclude that two major criteria must be satisfied before one can claim the occurrence of silent synapses in a given neural pathway. First, synapses must be structurally present second, they must be shown not to produce a physiological response under the conditions in which the system normally operates. Because conjoint physiological and structural observations at the level of individual synapses are difficult to obtain experimentally, it is not surprising that many claims of silent synapses are inferential rather than rigorously proven. Often, the claims are based on statistical analyses of physiological data that leave open more than one interpretation. Nevertheless, the accumulating evidence, incomplete or circumstantial as it may be, is compelling.


Of the studies on mammalian neurons in which the possibility of silent synapses has been raised, the majority have examined developmental processes, or tissue-cultured neurons that may develop differently from neurons in the intact CNS studies of silent synapses in the mature nervous system are fewer in number. The general issue arises concerning the extent to which manifestations of silent synapses are attributable to a developmental sequence. Properties of long- and short-term plasticity differ in mature and immature synapses, and thus the age and developmental state of observed synapses is a significant variable.

Silent synapses have been implicated in brain plasticity of both young and mature animals. One could therefore ask whether silent synapses are more numerous in young, developing neurons or in mature ones? The dominant concept of the silent synapse in immature neurons of mammals (supported by many published studies) has been the synapse with NMDA-type glutamate receptors only, and lacking AMPA-type glutamate receptors (Fig. 2C). These receptors, due to their characteristic voltage and magnesium dependence, produce no postsynaptic current at the resting membrane potential even though release of the neurotransmitter (glutamate) occurs normally (Malenka and Nicoll 1997). There is convincing evidence for the occurrence of such silent synapses in the developing nervous system (Durand et al. 1996 Wu et al. 1996Isaac et al. 1997 Rumpel et al. 1998 and many other recent studies). As maturation progresses, these silent synapses become rare and are presumably replaced progressively by active ones (possessing AMPA receptors). The transformation is thought to involve recruitment of AMPA-type glutamate receptors into the postsynaptic membrane (Shi et al. 1999). Activation of the NMDA receptors by glutamate with consequent inflow of Ca 2+ is believed to be the major stimulus for recruitment of AMPA-type receptors.

There is only limited evidence for the presence of silent synapses in the mature mammalian brain. Immunogold labeling experiments suggest that ∼17% of synapses in the CA1 area of the hippocampus of mature rats and 28% in immature ones are devoid of a significant number of AMPA receptors (Nusser et al. 1998). It is not known whether the same synapses that lack AMPA receptors possess NMDA receptors, but if they do, they could be silent, under certain conditions. A few synapses with only NMDA receptor subunits have been reported (He et al. 1998). However, the NMDA-only silent synapses can only be considered conditionally silent, because on depolarization from the resting membrane potential, the synapses likely become instantly activated (Fig. 3). In the intact nervous system, such depolarization could occur through activation of adjacent excitatory AMPA synapses and, particularly in very young neurons, by means of depolarizing, GABA-dependent synapses (Ben Ari et al. 1997). Neurons in the intact brain normally have continuously fluctuating membrane potentials, and thus, NMDA-only synapses would be capable of producing a response much of the time. Moreover, even at rest, very young neurons are thought to possess relatively depolarized membrane potentials of around −60 mV, which could unmask the NMDA-dependent depolarization even in the absence of additional synaptic input (Verheugen et al. 1999).

In summary, silent synapses possessing NMDA and not AMPA receptors, which appear to be much more frequent in the developing nervous system, are not likely to be truly silent. More exactly, they are conditionally silent. Use of the term silent synapse for conditionally silent synapses can lead inadvertently to a rather confusing description in which, for example, the observed bursting activity in a developing cortical circuit has been attributed to the activity of silent synapses (Golshani and Jones 1999). A more useful concept of the truly silent synapse is a more restricted one. For example, the classical Hebbian synapse, which is ineffective until a period of combined pre- and postsynaptic activity makes it active (Hebb 1949), would fit the stricter definition. According to this concept, the NMDA receptor can be a suitable trigger for the associative strengthening of such synapses by the right combination of pre- and postsynaptic mechanisms.


In relation to the question of synaptic development, the life span of individual synapses is relevant. Observations on several neuronal systems suggest that synapses may form and turn over rapidly in some cases For example, molluscan and leech neurons can apparently form new synapses within minutes when brought together in culture (Haydon 1988), and synapses in the optic system of insects undergo very rapid (sometimes diurnal) changes in size and number (Meinertzhagen 1993). In contrast, mammalian neuromuscular junctions, once established, remain in place with minor morphological alterations for a major portion of the lifetime of the animal (Balice-Gordon and Lichtman 1990). Conditions in culture differ from those in the intact brain, and the time course of individual synapse formation or dissolution is not well understood in the latter case. Published accounts indicate that synaptic age is variable and system or context specific.

In the mammalian CNS, the lifetime of a synapse is not well established. If silent synapses exist and if they are activated by activity to form a memory trace in the brain, one may wonder how long such a trace (if dependent on individual synapses) can last? Whereas synaptic connections in mature CNS are generally stable and maintained by homeostatic activity-dependent mechanisms (Turrigiano 1999), they can be potentiated for several weeks in the intact brain (Bliss and Lomo 1973). However, it is not clear if the individual synapses comprising the total synaptic connection can last that long, so the question of the synaptic turnover arises.

In some known systems, synapses can be formed continually during the lifetime of the animal. An example from the mammalian CNS concerns the generation of new neurons in the dentate gyrus of the hippocampus (Fig.4). Neurons giving rise to the perforant pathway that enters the hippocampal formation are thought to continuously form new synaptic junctions, even in the adult brain (. This results from production of new neurons (adult neurogenesis) within the dentate gyrus, a target of the perforant pathway. Thus, existing terminals of the perforant pathway probably sprout and form new synaptic junctions on newborn neurons. The physiological properties of these new synapses are different from those on mature neurons (Wang et al. Wojtowicz 1998). It is not known how long such synapses last, but some turnover likely occurs as granule cells age and lose dendritic branches, synapses must atrophy as well (Fig. 4). In the rat, mature granule neurons with sparse dendritic arborization can be observed in animals that are 1–2 months old (J.M. Wojtowicz, unpubl.). These could be cells whose dendrites are being phased out. Such cells may eventually be replaced by newly produced ones.

Synaptic turnover in the mammalian dentate gyrus. New neurons (1) are generated in the granule cell layer (GCL) throughout life (A, B). The new neurons grow dendrites that enlarge as the neuronal cell body migrates away from the hilus (2,3). Synapses (○) formed by the medial perforant path (MPP) afferents on new neurons (*) are younger than those already established on neurons that matured earlier. As neurons mature, some dendrites retract, degenerate, or are remodeled (4), and synapses are lost from such dendrites (●). (MOL) Molecular layer (SGZ) subgranular zone, located between GCL and hilus. (C) The existing synapses in young and mature neurons possess high or low capacity for plasticity respectively.

Another well-studied case in which synaptic turnover occurs throughout life is the excitatory (glutamatergic) motor neuron of crustaceans, which has many morphological features in common with central neurons (Atwood and Wojtowicz 1986). In the lobster, early stages of innervation have been documented, and the same identified neuron can be sampled in animals of different age. The neuron continues to grow throughout the life of the animal (50 years or more), creating new branches to keep up with muscle growth (Atwood and Govind 1990). Synapses are continuously replaced, and new ones formed, as growth occurs (Fig. 5). The same neuron thus possesses synapses of different age. The most distal varicosities on a nerve branch have lower quantal content, but just as many ultrastructurally defined synapses, as the more proximal varicosities, suggesting a higher proportion of silent synapses on younger varicosities (Cooper et al. 1995).

Formation and disappearance of synapses in a crustacean neuron. Identified motor neurons of lobsters and other crustaceans are present throughout life, but change progressively as the individual ages and becomes larger. Synapses formed in early development of a leg muscle (a) are lost and replaced by neural sheath as the axon grows and develops new branches. In turn, new synapses are formed on the younger branches (b, c). Thus, at all times, synapses of various ages are present on the neuron, with the younger synapses at the growing ends of the axonal branches (afterAtwood and Govind 1990).

The occurrence of synapses of different age on a single neuron, or within a population of neurons, is likely to affect the prevalence of silent synapses. Developing neurons or neuronal branches probably have a higher incidence of silent synapses. Synaptic generation or senescence creates synapses in which one or more of the underlying causes of synaptic silence are expressed (Fig. 2). We shall explore this theme further in selected examples.

Proprioceptive Sensations

Proprioception refers to the sense of knowing how one’s body is positioned in three-dimensional space.

Learning Objectives

Describe how propioception is the sense of the position of parts of our body in a three dimensional space

Key Takeaways

Key Points

  • Proprioception is the sense of the position of parts of our body and force being generated during movement.
  • Proprioception relies on two, primary stretch receptors: Golgi tendon organs and muscle spindles.
  • Muscle spindles are sensory receptors within the belly of a muscle that primarily detect changes in the length of this muscle. They convey length information to the central nervous system via sensory neurons. This information can be processed by the brain to determine the position of body parts.
  • The Golgi organ (also called Golgi tendon organ, tendon organ, neurotendinous organ, or neurotendinous spindle) is a proprioceptive sensory receptor organ that is located at the insertion of skeletal muscle fibers into the tendons of skeletal muscle.

Key Terms

  • alpha motor neuron: Large, multipolar lower motor neurons of the brainstem and spinal cord that are directly responsible for initiating muscle contraction.
  • proprioreceptor: A sensory receptor that responds to position and movement and that receives internal bodily stimuli.
  • posterior (dorsal) column-medial lemniscus pathway: A sensory pathway of the central nervous system that conveys localized sensations of fine touch, vibration, two-point discrimination, and proprioception from the skin and joints.
  • Law of Righting: A reflex rather than a law, this refers to the sudden movement of the head to level the eyes with the horizon in the event of an accidental tilting or imbalance of the body.
  • Golgi tendon organ: A proprioceptive sensory receptor organ that is located at the insertion of skeletal muscle fibers into the tendons of skeletal muscle.
  • muscle spindle: Sensory receptors within the belly of a muscle that primarily detect changes in the length of this muscle.
  • proprioception: The sense of the position of parts of the body, relative to other neighboring parts of the body.

Proprioception is the sense of the relative position of neighboring parts of the body and the strength of effort being employed in movement. It is distinguished from exteroception, perception of the outside world, and interoception, perception of pain, hunger, and the movement of internal organs, etc.

The initiation of proprioception is the activation of a proprioreceptor in the periphery. The proprioceptive sense is believed to be composed of information from sensory neurons located in the inner ear (motion and orientation) and in the stretch receptors located in the muscles and the joint-supporting ligaments (stance).

Conscious proprioception is communicated by the posterior ( dorsal ) column–medial lemniscus pathway to the cerebrum. Unconscious proprioception is communicated primarily via the dorsal and ventral spinocerebellar tracts to the cerebellum.

An unconscious reaction is seen in the human proprioceptive reflex, or Law of Righting. In the event that the body tilts in any direction, the person will cock their head back to level the eyes against the horizon. This is seen even in infants as soon as they gain control of their neck muscles. This control comes from the cerebellum, the part of the brain that affects balance.

Muscle spindles are sensory receptors within the belly of a muscle that primarily detect changes in the length of a muscle. They convey length information to the central nervous system via sensory neurons. This information can be processed by the brain to determine the position of body parts. The responses of muscle spindles to changes in length also play an important role in regulating the contraction of muscles.

Muscle spindle: Mammalian muscle spindle showing typical position in a muscle (left), neuronal connections in spinal cord (middle), and expanded schematic (right). The spindle is a stretch receptor with its own motor supply consisting of several intrafusal muscle fibers. The sensory endings of a primary (group Ia) afferent and a secondary (group II) afferent coil around the non-contractile central portions of the intrafusal fibers.

The Golgi organ (also called Golgi tendon organ, tendon organ, neurotendinous organ or neurotendinous spindle) is a proprioceptive sensory receptor organ that is located at the insertion of skeletal muscle fibers onto the tendons of skeletal muscle. It provides the sensory component of the Golgi tendon reflex.

The Golgi organ should not be confused with the Golgi apparatus—an organelle in the eukaryotic cell —or the Golgi stain, which is a histologic stain for neuron cell bodies.

Golgi tendon organ: The Golgi tendon organ contributes to the Golgi tendon reflex and provides proprioceptive information about joint position.

The Golgi tendon reflex is a normal component of the reflex arc of the peripheral nervous system. In a Golgi tendon reflex, skeletal muscle contraction causes the agonist muscle to simultaneously lengthen and relax. This reflex is also called the inverse myotatic reflex, because it is the inverse of the stretch reflex.

Although muscle tension is increasing during the contraction, alpha motor neurons in the spinal cord that supply the muscle are inhibited. However, antagonistic muscles are activated.


Nectin-3 and nectin-1 are expressed in upper layers of visual cortex

Previous studies have shown a role for nectin-3 and its binding partner nectin-1 in the formation and maintenance of hippocampal synapses [11, 26, 27]. Mizoguchi et al. (2002) demonstrated using immunoelectron microscopy that nectin-1 and nectin-3 are distributed asymmetrically at hippocampal synapses, with nectin-1 located at the presynaptic side of mossy fiber terminals and nectin-3 located postsynaptically along the dendrites of pyramidal cells (Fig. 1a) [11]. In situ hybridization (ISH) revealed that nectin-3 and nectin-1 had specific expression patterns not only in hippocampus, where nectin-3 is enriched in CA3 and nectin-1 is enriched in the dentate gyrus, but also in cortex, where nectin-3 and nectin-1 are both enriched in upper layer cortical neurons (P16 Fig. 1b). In cortex, neurons in L4 send axons to L2/3, and L2/3 neurons are highly reciprocally connected within this layer [53, 54]. Our ISH data confirm data found at Allen Brain Atlas, showing upper layer specific expression of nectin-3 beginning at E18.5 and continuing through adulthood (Fig. 1c, d, Additional file 1: Figure S1) [39]. Nectin-1 first shows strong L2/3 expression at P4 and is upper layer enriched at P14 and P28 (Additional file 1: Figure S1). These expression patterns suggest that, similar to hippocampus, nectin-1 and nectin-3 may also interact during the development of cortical circuits.

Nectin-3 knockdown at E15.5 increases dendritic spine density at P21

To knock down nectin-3 in developing neurons in vivo, we first designed new plasmid vectors allowing the Cre-dependent expression of nectin-3 short-hairpin RNA (shRNA) or scrambled control shRNA (Fig. 2a). We used previously published 19 bp siRNA sequences [41] to design our nectin-3 shRNA oligos and cloned these hairpins into the Cre-dependent pSico vector (Addgene plasmid #11578) [47]. pSico Cre-dependent shRNA constructs to nectin-3 (or scramble shRNA) contained loxP flanked GFP-stop sequences to prevent the expression of shRNA in the absence of Cre (Fig. 2a). shRNA constructs were co-electroporated with a Cre-expression plasmid and a Cre-dependent FLEX tdTomato construct (Fig. 2a). To test for knockdown of nectin-3, we co-transfected a nectin-3 expression plasmid with our new nectin-3 shRNA constructs (with the floxed GFP-Stop sequence removed, see Methods) into HEK-293 cells. Western blot analysis indicated that our newly designed nectin-3 shRNA constructs effectively reduced nectin-3 protein expression when normalized to a-tubulin and compared to cells co-transfected with a scrambled shRNA construct (technical replicates, N = 2, Fig. 2b). The nectin-3 expression construct used in this experiment led to very high nectin-3 expression in HEK-293 cells, making knockdown difficult to observe by eye (Fig. 2b, top). However, quantitative analysis using Image Studio software indicated consistently lower nectin-3 expression in cells where nectin-3 specific shRNA had been expressed (Fig. 2b, bottom). This is consistent with previously published data showing nectin-3 was knocked down in cultured cortical neurons using the same siRNA sequences [41].

Nectin-3 knockdown at E15.5 increases dendritic spine density at P21. a Co-electroporated expression constructs. b Top: Western blot showing nectin-3 expression after co-transfection of HEK-293 cells with a nectin-3 expression plasmid and either nectin-3 or scramble shRNA constructs. Bottom: Nectin-3 expression/intensity was quantified and normalized to α-tubulin. Expression is shown relative to average nectin-3 expression from the scramble shRNA condition. c Electroporation/shRNA expression occurred at E15.5. Dendritic spine densities were analyzed at P14, P21, and P35. d Electroporated neurons at P21 in visual cortex (10x image, scale bar = 500 μm). Inset top: Cells expressing Cre-dependent tdTomato did not express floxed GFP-stop from pSico (40x image, scale bar = 50 μm). Inset bottom: Representative V1 neuron (40x image, scale bar = 50 μm), and select apical and basal dendrites. e Differences in dendritic spine densities between control and Nec3-shRNA neurons. Error bars denote standard errors of the mean. From left to right, asterisks denote significant differences for pooled apical and basal dendrites (solid line) between time points or between conditions at P21 (Additional file 5). f Representative apical dendrites from P21 Nec3-shRNA and control neurons (63x image, scale bar = 10 μm). g Nec3-shRNA and control dendritic spine densities at P14. Apical and basal dendritic spine densities are plotted. Error bars denote standard error of the mean. Each dot represents a single dendrite. One apical and one basal dendrite per cell were counted (Control: N = 15 cells, 30 dendrites, 5 animals Nec3-shRNA: N = 16 cells, 32 dendrites, 6 animals). h Nec3-shRNA and control dendritic spine densities at P21. Significance is denoted by ‘*’ (Control: N = 17 cells, 34 dendrites, 5 animals Nec3-shRNA: N = 14 cells, 28 dendrites, 5 animals). i Nec3-shRNA and control dendritic spine densities at P35 (Control: N = 11 cells, 22 dendrites, 3 animals Nec3-shRNA: N = 10 cells, 20 dendrites, 5 animals)

To determine the effects of nectin-3 knockdown in vivo, we used in utero electroporation to introduce either or both nectin-3 shRNA1 and shRNA2 or scrambled shRNA (Fig. 2a, b) to developing L2/3 cortical neurons at E15.5 (Fig. 2b, c). pSico shRNA constructs were co-electroporated with a Cre-dependent tdTomato construct (at a 4:3 ratio) and a low concentration of a pCag-Cre plasmid (Fig. 2a, see methods for concentrations). Several previous studies have co-electroporated multiple plasmid constructs into developing mouse neurons with a high degree of efficiency [55,56,57,58]. One study found that when a 3:1 ratio of GFP to RFP expressing plasmids was co-electroporated, > 85% of GFP expressing cells also expressed RFP [55]. Since we were most concerned with the presence of our shRNA constructs, we used a higher concentration of these plasmids than any other construct (see methods). Using this paradigm, we are confident that the vast majority of cells expressing both the Cre-dependent tdTomato construct and the Cre construct (necessary to observe tdTomato) also expressed the more highly concentrated pSico shRNA expression plasmid. We further confirmed that cells expressing Cre-dependent tdTomato did not also express GFP, indicating the successful Cre excision of the GFP-stop sequence from the pSico construct (Fig. 2d, top inset). Since we demonstrated shRNA knockdown of nectin-3 when GFP-stop is excised from our constructs (allowing shRNA expression) in HEK cells, we have a high degree of confidence that nectin-3 knockdown was successful in tdTomato-positive cells (Fig. 2b).

After electroporation, animals were sacrificed at specific developmental time points, and we identified tdTomato expressing neurons in L2/3 of primary visual cortex (V1) for dendritic spine density analysis. Nectin-3 knockdown (or overexpression, Figs. 4 and 5) did not disrupt the migration of neurons to upper cortical layers (Fig. 2d, detailed migration analysis in Additional file 2: Figure S2). We selected two different dendrite types for dendritic spine density analysis: 1) secondary proximal apical dendrites extending sideways from the apical stalk, and 2) secondary basal dendrites at least one branch away from the soma (Fig. 2d, bottom inset). We assayed dendritic spine densities at three developmental time points: eye opening (P14), one week after eye opening (P21), and at the close of the critical period for ocular dominance plasticity (P35 Fig. 2c). This allowed us to assess nectin-3 function during critical periods in the development of visual cortex for synapse formation (P14 – P21) [18], as well as synaptic refinement (P21 – P35) [1, 22, 59].

Knocking down nectin-3 at E15.5 significantly impacted dendritic spine densities during postnatal development. When apical and basal dendrites were considered together, neurons electroporated with either scrambled shRNA (control) or nectin-3 shRNA constructs showed an increase in spine densities between P14 and P21, consistent with previous reports of synaptogenesis following eye opening in visual cortex (Control P14 – P21: p = 2.465e-05, Nec3-shRNA P14 – P21: p = 1.271e-10, Fig. 2e) [18]. Nectin-3 knockdown amplified this change, yielding significantly higher spine densities than control at P21 (Control – Nec3-shRNA: p = 0.00016, Fig. 2e, f, h). When analyzed separately, apical, but not basal, dendrites from Nec3-shRNA neurons showed a significant increase in spine densities relative to control neurons at P21 (Apical Control – Nec3-shRNA: p = 9.633e-05, Basal Control – Nec3-shRNA: p = 0.07893). We similarly found that knocking down both nectin-3 and nectin-1 increased dendritic spine densities relative to control neurons at P21, and that, again, apical dendrites were most significantly impacted (Control – Nec3 + Nec1-shRNA: p = 0.0014, Apical Control – Nec3 + Nec1-shRNA: p = 0.0014, Basal Control – Nec3 + Nec1-shRNA: p = 0.07063, Additional file 3: Figure S3). These results indicate that developing apical dendrites may be more sensitive to reduced nectin-3 expression than basal dendrites. Differences in dendritic spine densities between double knockdown and control neurons at P21 were present despite seeing no difference in dendrite complexity, as analyzed by Sholl analysis (Additional file 3: Figure S3f, g). By P35, the dendritic spine densities of Nec3-shRNA neurons were no longer significantly different from control neurons (Fig. 2i). Collectively, these results are consistent with a model where the extended knockdown of nectin-3 leads to a transient overproduction of weak spines after eye opening, which are readily pruned over the critical period for ocular dominance plasticity (P21 – P35) due to reduced stability.

Nectin-3 knockdown at

P19 increases dendritic spine density at P35

Early (E15.5) shRNA knockdown of nectin-3 yielded an overproduction of dendritic spines after eye opening, significantly increasing dendritic spine densities at P21 relative to control. On the other hand, dendritic spine densities between Nec3-shRNA and control neurons were not different at P35, indicating that the extra spines produced at P21 may have been removed over the critical period for ODP (P21 – P35). We next wanted to test whether the initial overproduction of spines at P21 after nectin-3 knockdown directly led to compensatory spine pruning in the following weeks. To accomplish this, we selectively knocked down nectin-3 near the start of the critical period for ODP and assessed dendritic spine densities at P35. For this experiment, we co-electroporated the same Cre-dependent Nec3-shRNA constructs (or control scramble shRNA) and a Cre-dependent tdTomato construct into transgenic CaMKII-Cre mice at E15.5 (Fig. 3a, c). In a previous study, CaMKII-Cre driven Cre/loxP recombination was first observed in cortex and hippocampus at P19 and expression was described as robust by P23 [42]. From this, we concluded that the knockdown of nectin-3 likely occurred in most cells around the start of the critical period for ocular dominance plasticity (P21). Both apical and basal dendritic spine densities were analyzed at P35, to allow time for full shRNA expression and knockdown of nectin-3 in L2/3 cortical neurons (Fig. 3b, c).

P19 increases dendritic spine density at P35. a A Cre-dependent shRNA construct to nectin-3 (or scramble shRNA construct) was co-electroporated with a Cre-dependent FLEX tdTomato plasmid (also expressing synaptophysin-EGFP) into developing CaMKII-Cre transgenic mice. b Both apical and basal dendrites on CaMKII-Cre/shRNA/tdTomato positive neurons were imaged at P35 (40x image, scale bar = 50 μm). c Mice were electroporated at E15.5 to target developing L2/3 neurons, but Cre recombination is not observed until

P19 in the CaMKII-Cre mouse line [42]. For this experiment, neurons develop normally until nectin-3 knockdown at

P19. Mice were sacrificed and neurons were imaged at P35. d Dendritic spine densities are significantly higher at P35 when nectin-3 is knocked down at

P19 using a CaMKII-Cre mouse. Each dot represents a single dendrite. One apical and one basal dendrite per cell were counted. Significance is denoted by ‘*’ (Additional file 5, CKII-control: N = 14 cells, 28 dendrites, 5 animals CKII-Nec3-shRNA: N = 16 cells, 32 dendrites, 6 animals). e Representative images of apical dendrites from control and Nec3-shRNA animals (63x image, scale bar = 10 μm)

Unlike early (E15.5) shRNA knockdown of nectin-3, late nectin-3 knockdown (

P19) significantly increased dendritic spine densities at P35 relative to control (p = 0.0107, Fig. 3d, e). While both apical and basal dendritic spine densities appeared to increase with nectin-3 knockdown, when considered separately, only apical dendrites showed a statistically significant increase relative to control (p = 0.0207, Fig. 3d, e, Additional file 5). The critical period for ODP is typically associated with experience dependent spine pruning in V1, though different dendrite types have been shown to undergo different degrees of pruning [19, 60]. While our results could indicate that nectin-3 knockdown disrupts pruning over the critical period for ODP, the increased spine densities observed could equally be the result of increased spinogenesis. It is also possible that, as with early (E15.5) knockdown, the increased spine densities observed with late knockdown of nectin-3 may be transient. We did not take measurements of spine densities after P35, but other studies have found that nectin-3 knockdown in adult hippocampus decreases dendritic spine densities [35, 36]. Further studies are necessary to determine whether the developmental knockdown of nectin-3 has long-term effects in L2/3 cortical neurons.

Nectin-3 overexpression at E15.5 decreases dendritic spine density

We next examined the effect of overexpressing nectin-3 in L2/3 cortical neurons over development. We generated a newly designed nectin-3 expression plasmid, which dramatically increased nectin-3 expression after transfection into HEK-293 cells (Fig. 4a, c). This nectin-3 overexpression construct was electroporated into developing L2/3 neurons at E15.5 (with a Cre-dependent tdTomato construct and a Cre-expression plasmid, Fig. 4a, b), and apical and basal dendritic spine densities were analyzed at P14, P21, and P35 (Fig. 4d). Dendritic spine densities on both control (scramble shRNA) and nectin-3 overexpression (Nec3-OE) neurons increased significantly between P14 and P21 (Control, P14 – P21: p = 2.47e-05 Nec3-OE, P14 – P21: p = 0.00017, Additional file 5). In addition, when all time points were considered together, Nec3-OE significantly decreased dendritic spine densities relative to control neurons (p = 0.0019, pooled apical and basal dendrites, Additional file 5, Fig. 4e). The greatest difference between Nec3-OE and control dendritic spine densities was observed at P35, and as with nectin-3 knockdown, spine densities were most significantly impacted on apical dendrites (pooled apical and basal, P35: p = 0.0492, apical dendrites alone, P35: p = 0.00136, Fig. 4i). Consistent with our shRNA experiments, these data further indicate that nectin-3 expression may restrict spinogenesis over development and/or facilitate pruning over the critical period for ODP (P21 – P35).

Nectin-3 overexpression beginning at E15.5 decreases dendritic spine density. a Co-electroporated expression constructs. b Electroporation/nectin-3 overexpression occurred at E15.5. Dendritic spine densities were analyzed at P14, P21, and P35. c Western blot analysis after transfection of HEK-293 cells with a nectin-3 overexpression vector. Greatly increased nectin-3 expression is observed relative to endogenous levels in HEK-293 cells. d Representative image of an electroporated neuron with select apical and basal dendrites circled (scale bar = 50 μm). e Nectin-3 overexpression produced an overall decrease in dendritic spine densities compared to control (scramble shRNA) neurons (pooled apical and basal dendrites, solid line). As with control neurons, spine densities significantly increased between P14 and P21, indicating spinogenesis was intact with nectin-3 overexpression. Significance is denoted by ‘*’, and p values are listed in Additional file 5. f Representative images of apical dendrites from control and Nec3-OE neurons at P35 (63x image, scale bar = 10 μm). g Nec3-OE and control dendritic spine densities at P14. Apical and basal dendritic spine densities are plotted. Error bars denote standard error of the mean. Each dot represents a single dendrite. One apical and one basal dendrite per cell were counted (Control: N = 15 cells, 30 dendrites, 5 animals Nec3-OE: N = 16 cells, 32 dendrites, 6 animals). h Nec3-OE and control dendritic spine densities were not significantly different at P21 (Control: N = 17 cells, 34 dendrites, 5 animals Nec3-OE: N = 14 cells, 28 dendrites, 5 animals). When Nec3-OE and control data were combined, apical dendritic spine densities were significantly lower than basal at P21. i Dendritic spine densities for Nec3-OE neurons trended lower than control neurons at P35 (pooled apical and basal dendrites). This result was significant when considering apical dendrites alone (Control: N = 11 cells, 22 dendrites, 3 animals Nec3-OE: N = 12 cells, 24 dendrites, 3 animals)

Our study indicates that the apical and basal dendrites we analyzed may experience different degrees of spinogenesis after eye opening. Individual t-tests were performed to determine whether apical and basal dendritic spine densities may differ within a single condition at a given age. Both control and Nec3-OE neurons demonstrated potential differences between apical and basal dendritic spine densities at P21 (Control, p = 0.0038 Nec3-OE, p = 0.029 Additional file 5, Metadata). When these datasets were combined in our statistical model (see methods), differences between apical and basal dendrites were highly significant (p = 4.14e-05, Additional file 5, Fig. 4g). This result suggests that the apical dendrites sampled in this study may not increase their spine densities after eye opening to the same degree as basal dendrites under the conditions of normal or above normal nectin-3 expression (Fig. 4e, h). It is important to note that this effect does not seem dependent on the overexpression of nectin-3, since no significant difference between Nec3-OE and control conditions was observed at P21 (Fig. 4g). Dendritic spine densities on apical dendrites continued to increase between P21 and P35 in the control condition alone. This indicates that, unlike other dendrite types [22, 61], proximal secondary apical dendrites (distinct from apical tufts) may be slower to increase dendritic spine densities after eye opening and may not undergo pruning over the critical period for ODP. Overexpression of nectin-3 most significantly decreased apical dendritic spine densities at P35, further indicating that the unique development of this dendrite type may be dependent on precise levels of nectin-3 expression. From this we conclude that dendrite type may also be an important consideration when evaluating developmental changes in dendritic spine densities following eye opening.

Overexpressing nectin-3 lacking the afadin binding domain decreases dendritic spine density

Nectin proteins consist of a three extracellular Ig domains, a transmembrane fragment, and multiple intracellular C-terminal domains with distinct and independent functions [11, 30, 62,63,64]. Most nectins, including nectin-3, have a conserved motif of four amino acids at their cytoplasmic tail that binds the PDZ domain of afadin [11, 27, 30, 40]. Nectin-3/afadin binding is required for the interaction of nectin-3 with the actin cytoskeleton and the organization of PAJs in cooperation with N-cadherin (Fig. 1a) [11, 26,27,28,29, 31, 65]. Nectins can also recruit and activate c-Src leading to the downstream activation of Cdc42 and Rac, which facilitate the formation of filopodia and lamellipodia, respectively [63, 66,67,68]. The nectin-1 C-terminus, but not the afadin binding site, was necessary for the activation of Cdc42 and Rac [66]. The extracellular domain of nectin-3 has also been shown to act independently of its C-terminus to bind nectin-1, leading to nectin-1 intracellular signaling and cadherin recruitment [63, 66, 69]. It is unclear which nectin-3 domain may regulate dendritic spine formation during critical periods of postnatal development.

We next tested whether the decreased spine densities observed with nectin-3 overexpression required an interaction between nectin-3 and afadin. For this experiment, we used in utero electroporation to overexpress a nectin-3 protein lacking the four C-terminal amino acids required to bind afadin (Nec3 Δafadin ) [11, 27, 30, 40]. Developing L2/3 neurons were electroporated with Nec3 Δafadin (plus Cre and Cre-dependent tdTomato plasmids, Fig. 5a) at E15.5, and dendritic spine densities were assayed at P14, P21, and P35 (Fig. 5b, c). Note that the Nec3 Δafadin protein should still bind nectin-1 and activate Cdc42 and Rac signaling [63, 66,67,68]. Similar to the Nec3-OE condition, Nec3 Δafadin produced an overall decrease in dendritic spine densities relative to control neurons (scramble shRNA) when all time points were considered together (p = 0.00199, Fig. 5d). Also similar to Nec3-OE, Nec3 Δafadin neurons had decreased (trending) dendric spine densities relative to control at P35 (pooled apical and basal dendrites, P35: p = 0.057), which was significant when apical dendrites were considered independently (apical dendrites alone, P35: p = 0.004). From this we conclude that nectin-3 utilizes an afadin-independent pathway to restrict spine formation during this developmental period.

Overexpression of Nec3 Δafadin at E15.5 restricts spine formation after eye opening. a Co-electroporated expression constructs. b Representative image of an electroporated neuron with select apical and basal dendrites circled (scale bar = 50 μm). c Electroporation/dominant negative inhibition of nectin-3/afadin binding (Nec3 Δafadin ) occurred at E15.5. Dendritic spine densities were measured at P14, P21, or P35. d Nec3 Δafadin produced an overall decrease in dendritic spine densities compared to control neurons when all time points were considered (pooled apical and basal dendrites, solid line). In addition, dendritic spine densities did not significantly increase between P14 and P21. Significance is denoted by ‘*’, and p values are listed in Additional file 5. e Representative images of basal dendrites at P21 from Nec3 Δafadin and control neurons (63x image, scale bar = 10 μm). f Representative images of apical dendrites at P35 from Nec3 Δafadin and control neurons (63x image, scale bar = 10 μm). g Nec3 Δafadin and control dendritic spine densities at P14. Apical and basal dendritic spine densities are plotted along with error bars denoting the standard errors of the mean (Control: N = 15 cells, 30 dendrites, 5 animals Nec3 Δafadin , N = 16 cells, 32 dendrites, 6 animals). h Nec3 Δafadin and control dendritic spine densities at P21. Overall dendritic spine densities trended lower at P21 in Nec3 Δafadin neurons compared to control with a p value barely above the Bonferroni corrected p value of 0.0055. When basal dendrites were considered alone, a significant decrease in dendritic spine densities was observed (Control: N = 17 cells, 34 dendrites, 5 animals Nec3 Δafadin : N = 12 cells, 24 dendrites, 3 animals). i Nec3 Δafadin and control dendritic spine densities at P35. When apical dendrites were considered alone Nec3 Δafadin spine densities were significantly lower than control (Control: N = 11 cells, 22 dendrites, 3 animals Nec3 Δafadin : N = 9 cells, 18 dendrites, 3 animals)

Unlike Nec3-OE, the greatest difference between Nec3 Δafadin and control neurons was observed one week following eye opening, at P21 (pooled apical and basal dendrites, p = 0.0056). Interestingly, this effect appeared to be driven by basal dendrites, which were significantly decreased relative to control at P21 (p = 0.003). Furthermore, Nec3 Δafadin was the only condition where we did not observe a significant developmental increase in dendritic spine densities between P14 and P21 (Fig. 5d, Nec3 Δafadin , P14 – P21: p = 0.1765). This result could suggest that, in the context of high nectin-3 expression, nectin-3-afadin binding may counteract the role of nectin-3 to suppress spine formation (see discussion). However, there are several possible explanations for why Nec3 Δafadin produced an enhanced phenotype at P21 relative to Nec3-OE, including differences in protein stability or expression levels between the two conditions. While further studies are necessary to fully understand the role of nectin-3-afadin binding in spinogenesis following eye opening, our results support an overall model whereby nectin-3 knockdown increases spine densities, and nectin-3 overexpression reduces spine densities, as described in Fig. 6 (representative images of dendrites and cells from all conditions and ages are shown in Additional file 4: Figure S4).

Hypothesized model summarizing results. After eye opening, L2/3 neurons experience an increase in dendritic spine densities. This period of spinogenesis may first require a weakening of synaptic adhesion at existing synapses. In this model, the over-stabilization of existing spines prevents new spine formation, while reduced synaptic adhesion facilitates new spine formation. It is further possible a similar but opposing mechanism facilitates pruning between P21 and P35 the strengthening of select spines by increased cell-adhesion facilitates the removal of neighboring weak spines. As a component of synaptic PAJs, nectin-3 may facilitate both the accumulation and removal of adhesion molecules at synaptic sites, perhaps through its association with afadin (created with


Glial cells, or neuroglia or simply glia, are the other type of cell found in nervous tissue. They are considered to be supporting cells, and many functions are directed at helping neurons complete their function for communication. The name glia comes from the Greek word that means “glue,” and was coined by the German pathologist Rudolph Virchow, who wrote in 1856: “This connective substance, which is in the brain, the spinal cord, and the special sense nerves, is a kind of glue (neuroglia) in which the nervous elements are planted.” Today, research into nervous tissue has shown that there are many deeper roles that these cells play. And research may find much more about them in the future.

There are six types of glial cells. Four of them are found in the CNS and two are found in the PNS. Table 2 outlines some common characteristics and functions.


Interneurons play a vital role in the wiring and circuitry of the developing nervous system of all organisms, both invertebrates and vertebrates alike. Generally speaking, an interneuron is a specialized type of neuron whose primary role is to form a connection between other types of neurons. They are neither motor neurons nor sensory neurons, and also differ from projection neurons in that projection neurons send their signals to more distant locations such as the brain or the spinal cord. Of great importance is that interneurons function to modulate neural circuitry and circuit activity [1–4]. A large majority of interneurons of the central nervous system are of the inhibitory type. In contrast to excitatory neurons, inhibitory cortical interneurons characteristically release the neurotransmitters gamma-aminobutyric acid (GABA) and glycine [5–7]. Cortical interneurons are so named for their localization in the cerebral cortex, which is defined as a sheet of outer neural tissue, that functions to cover the cerebrum and cerebellum structures in the brain.

This review will place an emphasis on the function and origin of GABAergic cortical interneurons of the developing nervous system. Within the overarching categorization of GABAergic interneurons there are also numerous interneuron subtypes that are largely categorized based on the surface markers they express. Three major cortical interneuron subtypes will be discussed: parvalbumin (PV)-expressing interneurons, a heterogeneous population of somatostatin (SST)-expressing interneurons, and a relatively recently identified population of the ionotropic serotonin receptor 5HT3a (5HT3aR)-expressing interneurons together, these three subtypes account for approximately 100 percent of the mouse neocortical GABAergic interneuronal population [8]. Although these interneurons home to their respective layers of the cerebral cortex, they are generated in various subpallial locations the primary origins of these interneuronal progenitors will be extensively discussed as well. The subsequent migratory routes that these interneuronal precursors take to the cerebral cortex are also covered here.

Of particular interest as of late in this field of research is mapping the network of transcription factors that are responsible for specifying cortical interneurons and specific interneuronal subtype. This review is organized such that the transcription factors are organized and discussed based on the major interneuronal origins in the subpallium. The most arduous task that researchers face is understanding the mechanism by which each interneuron subtype is specified as well as the various genes and transcription factors that may be involved this is not helped by the fact that there are so many subtypes whose features often overlap with one another.

Of utmost importance with regard to cortical circuitry is the balance between excitatory inputs and inhibitory inputs that must be carefully maintained. Disrupting the balance of neural circuits may very well be a contributing factor toward the emergence of neuropsychiatric disorders, such as epilepsy, autism spectrum disorders, and intellectual disabilities, just to name a few [9]. This review focuses on one severe mental illness in particular, schizophrenia. Gaining an understanding of cortical interneurons within the “bigger picture” of neocortical circuitry may provide much needed information behind the etiologies of neurological disorders.

Role of GABAergic cortical interneurons

Given that the population of GABAergic interneurons in the brain is such a heterogeneous one, it is only logical that the many different classes of interneurons will have a myriad of roles to play in the adult nervous system. GABAergic neurons play an inhibitory role and synaptically release the neurotransmitter GABA in order to regulate the firing rate of target neurons. Neurotransmitter release typically acts through postsynaptic GABAA ionotropic receptors in order to trigger a neuronal signaling pathway.

This research field typically organizes interneuron role/function into three components: (1) afferent input, (2) intrinsic properties of the interneuron, and (3) targets of the interneuron. Generally speaking, interneurons receive input from various sources, including pyramidal cells as well as cells from other cortical and subcortical regions [10, 11]. With regard to output, cortical interneurons engage in feed-forward and feedback inhibition [12–14]. Regardless of the mode of output, the cortical interneuronal network is further complicated by the fact that a single cortical interneuron is capable of making multiple connections with its excitatory neuronal target(s) [15].

Cortical interneuron subtype

It is estimated that there are over 20 different subtypes of GABAergic interneurons in the cortex, and subtypes are also distinguished from one another based upon the calcium-binding proteins they express, which serve as markers (Table 1) [16–24]. Studies performed in both mouse and rat brain tissue have suggested that in particular, the calcium-binding protein known as parvalbumin, and the neuropeptide somatostatin, are two crucial markers in defining the most predominant interneuron subtypes within the cerebral cortex [16, 20, 22]. Importantly, the PV-expressing interneuron population is independent from the SST-expressing population, in that expression of these markers does not overlap [16, 20, 22, 25]. In addition to PV- and SST-positive GABAergic interneurons, which together comprise approximately 70% of the total GABAergic cortical interneuron population, another subgroup of interneurons that express 5HT3aR were found to comprise approximately 30% of all interneurons [25]. While these three interneuronal subpopulations account for nearly (if not all) 100% of all GABAergic cortical interneurons, it is also important to remember that each of these populations, especially the 5HT3aR-expressing population, is heterogeneous, and therefore expresses other proteins or neuropeptides that contribute to their characterization.

In the recent years there has been a push to create a consistent nomenclature for the varying interneuronal subtypes a 2005 conference in Petilla, Spain, was held to accomplish this task. A group of researchers known as the Petilla Interneuron Nomenclature Group (PING) convened to formulate a set of terminologies to describe the morphological, molecular, and physiological features of GABAergic cortical interneurons [16]. Morphologically speaking, cortical interneurons are described with regard to their soma, dendrites, axons, and the connections they make. Molecular features include transcription factors, neuropeptides, calcium-binding proteins, and receptors these interneurons express, among many others. Physiological characteristics include firing pattern, action potential measurements, passive or subthreshold parameters, and postsynaptic responses, to name a few [16]. The overarching goal of this conference and the resulting Petilla terminology is to create a uniform set of criteria by which interneurons can be described so as to reduce confusion between the findings by various research groups in this field. While results from the Petilla conference have shown that there are many criteria with which to define and distinguish classes of interneurons, this review will classify interneuron subtypes based on the markers they express, particularly PV, SST, or 5HT3aR, and elaborate upon each of these three groups.

Parvalbumin-expressing interneurons

The PV interneuron group represents approximately 40% of the GABAergic cortical interneuron population [8]. This population of interneurons possesses a fast-spiking pattern, and fire sustained high-frequency trains of brief action potentials [10, 16, 17, 22, 26]. Additionally, these interneurons possess the lowest input resistance and the fastest membrane time constant of all interneurons [10, 16, 17, 22, 23, 27, 28].

Two types of PV-interneurons make up the PV interneuron group: basket cells and chandelier cells [16, 22]. Far more is understood about basket cells, which are interneurons that make synapses at the soma and proximal dendrite of target neurons, and usually have multipolar morphology [16, 22]. Several studies have shown that fast-spiking basket neurons are the dominant inhibitory system in the neocortex, where they mediate the fast inhibition of target neurons, among many other functions [29–35]. As will be discussed in a later section, fast-spiking basket neurons likely play a large role in regulating the delicate balance between excitatory and inhibitory inputs in the cerebral cortex [36, 37].

Much less is known about the second subgroup of PV-expressing interneurons, the chandelier cells. Unlike basket neurons, chandelier cells target the axon initial segment of pyramidal neurons [16, 22]. Both basket cells and chandelier cells are fast-spiking, but they differ in electrophysiological properties, as reviewed by Woodruff et al[38]. Several relatively recent studies have suggested that in contrast to other interneurons, chandelier cells may be excitatory rather than inhibitory due to their depolarizing effects on membrane potential [38–40], although the functions of this subgroup have yet to be elucidated.

One research group has characterized a group of PV-expressing cells that is independent from chandelier and basket neurons in the mouse neocortex [41]. These interneurons were designated multipolar bursting cells, and differ from chandelier and basket cells in both electrophysiology and connectivity [41]. Multipolar bursting neurons possess synapses with pyramidal cells (or other multipolar bursting cells) that demonstrate a paired-pulse facilitation in contrast, chandelier and basket cells are usually strongly depressing [41]. While this group of interneurons holds promise as a third subgroup within the PV-expressing interneuron type, further investigation is warranted, as no other research groups have characterized these neurons.

Somatostatin-expressing interneurons

The SST-expressing interneuron group is the second-largest interneuron group in the mouse neocortex, representing roughly 30% of the total cortical interneuron population [8]. SST-positive interneurons are known as Martinotti cells, and possess ascending axons that arborize layer I and establish synapses onto the dendritic tufts of pyramidal neurons [22]. Martinotti cells are found throughout cortical layers II-VI, but are most abundant in layer V [14, 22, 42]. These interneurons function by exhibiting a regular adapting firing pattern but also may initially fire bursts of two or more spikes on slow depolarizing humps when depolarized from hyperpolarized potentials. In contrast to PV-positive interneurons, excitatory inputs onto Martinotti cells are strongly facilitating [43–47]. More details regarding the electrophysiology and firing patterns of SST-expressing Martinotti cells can be found in a review by Rudy et al[8].

Results from several studies have suggested that the SST interneuron population is a heterogeneous one. Ma et al generated a mouse line designated as X94, whereby GFP expression (encoded for by random insertion of the GFP gene) was controlled by the GAD67 promoter cells from this line express SST but differ from Martinotti cells in many aspects [48]. These cells were located in layers IV and V that, unlike, Martinotti cells, targeted cells from cortical layer IV [48]. X94 cells also possessed a lower input resistance relative to Martinotti cells, with spikes of a shorter duration and a stuttering firing pattern [48]. This evidence clearly suggests that there are other interneuronal subgroups within the SST subtype than just Martinotti cells. McGarry and colleagues have reported that another transgenic mouse line contains two distinct, SST-positive cells that primarily occupy layers II and III [49]. Like the X94 cells, these two populations of interneurons differ electrophysiologically than Martinotti cells, although additional research must be performed to truly determine their presence, as other research groups have not been able to duplicate these findings [26, 50, 51].

It is clear that there are likely additional subpopulations of SST-expressing cortical interneurons. This is bolstered by the observed differences in firing properties, expression of molecular markers, and connectivity of different neurons within this population [8, 49, 50, 52, 53]. Additional research in this field is warranted, as the SST-expressing population of GABAergic interneurons is such a large one.

5HT3aR Interneuron group

The third group of GABAergic cortical interneurons was initially characterized in a study by Lee et al, and is designated as the 5HT3aR interneuron group [25]. This study utilized both in situ hybridization and immunohistochemistry to demonstrate the existence of a population of GABAergic interneurons in the mouse cortex that express the 5HTa3 receptor, but neither PV nor SST this population accounts for approximately 30% of the GABAergic cortical interneuron population [25]. Due to its relatively recent discovery, this group has yet to be fully characterized, although it is evident that this population is a very heterogeneous one.

Within the 5HT3aR interneuron group are several subsets of interneurons that also express other protein or neuropeptide markers, one of them being vasoactive intestinal peptide (VIP) [22, 26, 54]. VIP-expressing interneurons are localized in cortical layers II and III, and while they express neither PV nor SST, Lee et al confirmed that this subset does indeed express the 5HTa3 receptor and accounts for approximately 40% of the 5HT3aR population [25]. VIP interneurons generally make synapses onto dendrites [25, 55, 56], and some have been observed to target other interneurons [57, 58]. Relative to all cortical interneurons, VIP interneurons possess a very high input resistance and are among the most excitable of interneurons [25, 55, 56].

There are several types of VIP cortical interneurons that differ in electrophysiological properties, but in general they possess a bipolar, bitufted and multipolar morphology [22, 26, 54, 56]. One VIP subtype in particular is commonly referred to as irregular-spiking neurons [17, 25, 55, 56, 59–61]. Irregular spiking interneurons possess a vertically oriented, descending axon that extends to deeper cortical layers, and have an irregular firing pattern that is characterized by action potentials occurring irregularly during depolarizations near threshold [17, 25, 55, 56, 59–61]. Additionally, irregular spiking cortical interneurons express the calcium-binding protein calretinin (CR) [56, 61], which is a marker that some SST-positive interneurons also possess (and interestingly, also a marker that neither VIP nor SST interneurons express) [25, 55, 56, 62].

There are several other subtypes of VIP-expressing 5HT3aR interneurons present in the cerebral cortex, which are nicely summarized by Rudy et al[8]. Briefly, among these are rapid-adapting [63], fast-adapting neurons [25, 56] and IS2 [61], as well as a minor population of VIP-positive basket cells with regular, bursting, or irregular-spiking firing patterns [22, 26, 64].

60% of cortical interneurons in the 5HT3aR-expressing group do not express VIP [25]. Of this VIP-negative 5HT3aR group, nearly 80% express the interneuronal marker reelin [56]. Neurogliaform cells are a type of cortical interneuron that belongs to this category: they are also known as spiderweb cells and express neuropeptide Y (NPY), with multiple dendrites radiating from a round soma [22, 65]. Neurogliaform cells are unique relative to other GABAergic cortical interneurons because they are capable of forming synaptic connections with each other as well as with other interneuronal types (as opposed to other interneurons that can only make synapses onto homologous neurons), thus solidifying their important role in regulating neural circuitry [66–68]. Furthermore, neurogliaform cells function by activating slow GABAA and GABAB receptors in order to provoke long lasting inhibitory postsynaptic potentials onto pyramidal neurons and other interneurons [65, 69]. Other subtypes exist within the 5HT3aR-positive, VIP-negative group as well see the 2010 review by Rudy et al[8]. While progress has certainly been made in distinguishing interneuron groups from one another, further research is definitely warranted.

Origin of GABAergic cortical interneurons

Cortical interneurons are born in the ganglionic eminences

Throughout embryogenesis, interneurons are primarily generated in a structure broadly termed the ganglionic eminence (GE) (Figure 1) [70]. The GE is a transitory brain structure located in the ventral area of the telencephalon, and is anatomically present during embryonic development. The GE becomes evident at approximately E11.5 in the developing murine system [71]. In total there are three ganglionic eminences: the medial ganglionic eminence (MGE), the caudal ganglionic eminence (CGE), and the lateral ganglionic eminence (LGE). The names of the different areas within the GE are based on their rostral-caudal location in the telencephalon. As embryonic development continues, the GEs grow and ultimately fuse, at which point they are no longer present in the mature brain. The MGE and CGE are the primary sources of cortical interneurons in the developing nervous system.

Origin of GABAergic Cortical Interneurons. Anatomy of the embryonic telencephalon at approximately embryonic day 13.5 (E13.5), showing the major origins of GABAergic cortical interneurons. A Sagittal (top) view of the telencephalon. The MGE is labeled in pink and represents Lhx6- Nkx2.1-positive areas. MGE-derived interneurons ultimately express either PV or SST in the cerebral cortex. The CGE is labeled in light blue and represents Lhx6-positive, Nkx2.1-negative areas. CGE-derived interneurons ultimately express 5HT3aR in the cortex. The gray-labeled area represents Dlx1/Mash1-expressing areas. The black dotted line represents the migratory route interneuron precursors take to the cortex. B Coronal view of the telencephalon. Interneuronal progenitors originating in the LGE are labeled in blue MGE-derived progenitors are labeled in green, and interneurons from the preoptic area are labeled in red. Abbreviations: CR, calretinin CGE, caudal ganglionic eminence IN, interneuron LGE, lateral ganglionic eminence MGE, medial ganglionic eminence OB, olfactory bulb PV, parvalbumin POA, preoptic area SST, somatostatin.

Several crucial studies during the 1980s demonstrated that contrary to the previous postulation that cortical interneurons originate in the underlying cortical ventricular zone, the majority of GABAergic interneurons are actually born in the ganglionic eminences. Van Eden and colleagues first showed, utilizing analysis of GABA-positive cells in the developing rodent cerebral cortex, that cells presumed to be GABAergic interneurons seemed to be migrating away from the subpallium [72]. This crucial observation spawned several other studies that subsequently discovered that indeed, interneurons must migrate from the subpallium to their final destinations in the cortex. To corroborate Van Eden et al’s findings, BrdU labeling of cells in the subpallium were found to accumulate in the cerebral cortex throughout the course of neurogenesis importantly, these BrdU labeled cells were GABA-positive, implicating these cells as cortical interneurons [73]. Additional studies utilizing fluorescent dye labeling of neurons and an in vitro migratory assay with DiI showed that these interneurons display the same migratory pattern from the ganglionic eminence to the mature cerebral cortex [74, 75]. Use of retroviruses to track neural cell lineage also contributed the early yet important study that excitatory and inhibitory neurons are actually generated independently from one another and do not share a common lineage [76, 77]. Rakic and Lombroso later confirmed that the telencephalon consists of two separate domains: one containing the GEs that serves as the source of inhibitory, GABAergic interneurons, and a second in which excitatory neurons are generated [78]. The formerly mentioned domain is also termed the subpallial compartment, whereas the latter is referred to as the pallial compartment. Later studies demonstrated that distinct cortical interneuron subtypes are derived from specific regions within the GEs, as will now be discussed.

Medial ganglionic eminence

The medial ganglionic eminence has long been regarded as a primary source of GABAergic cortical interneurons it is thought to be the site of origin of approximately

50-60% of the cortical interneuron population in mice [63, 79, 80]. With regard to interneuron subtype, the MGE gives rise to most of the PV- and SST-expressing interneuron population [80]. To break this down, studies have demonstrated that in vitro, approximately 65% of MGE-derived interneurons express parvalbumin, with the remainder of the MGE-derived interneuronal population (

35%) expressing somatostatin [81, 82]. In vivo experiments have confirmed these initial findings [63, 81, 83–85]. Of note, the SST-positive subset that is derived from the MGE is a heterogenous population with regard to morphology, electrophysiology, and expression of reelin, NPY, and/or CR [50, 56].

It remains to be elucidated how PV-expressing and SST-expressing GABAergic cortical interneurons are spatially segregated within the MGE itself, and to what extent this spatial segregation occurs. Flames et al have suggested that the MGE may actually consist of several progenitor domains, each of which may serve as an origin for unique classes of interneurons [84].

Caudal ganglionic eminence

The CGE itself is unique in that it is somewhat of a “hybrid” of the MGE and LGE: similar to the MGE, the ventral-most CGE expresses transcription factor Nk×2.1, and the dorsal region of the CGE expresses the transcription factor Gsh2, which is required for proper patterning of the LGE [86]. The caudal ganglionic eminence has been shown to be the second-greatest contributor of interneuron progenitors, producing approximately 30-40% of all cortical interneurons [87–89]. While this is a substantial percentage, the CGE proves to be a difficult place of origin to objectively study due to the challenge of consistently defining this region. Fate mapping CGE-derived interneurons and subsequently determining the exact proportion these interneurons make up within the whole cortical interneuron population has therefore proven to be quite problematic. Nery et al were the first to show, via in utero transplantation analyses, that a certain percentage of interneurons destined for the cortex indeed originates in the CGE [89]. Other studies have utilized both in vitro and in vivo experiments to confirm this initial discovery, and have further added that CGE-derived interneurons are bipolar or double-bouquet in morphology [63, 79]. These interneurons express CR (not SST) and/or VIP [63, 79]. Recently, Miyoshi and colleagues utilized genetic fate mapping approach to corroborate the finding that the CGE is indeed a source of cortical interneurons [56].

Findings from several studies suggest that 5HT3aR-expressing cortical interneurons are actually largely CGE-derived, as demonstrated by EGFP visualization in the 5HT3aR-BAC EGFP mouse [90–92]. Lee et al combined the Nkx2.1-BAC cre and the cre-dependent red R26R tdRFP reporter with 5HT3aR-BAC EGFP in order to exclude the MGE as a possible source of this population of interneurons [25]. Overlap of cells labeled in red and cells labeled in green was not observed, and since the Nk×2.1-BAC cre line labels MGE-derived cortical interneurons, the MGE can thus be discounted as a source of 5HT3aR interneurons [25]. Additionally, the Mash1-BAC CreER R26R tdRFP mouse line, which is CGE-specific, displayed significant and near complete overlap with 5HT3aR-expressing cells, confirming the CGE as the major source of 5HT3aR-positive cortical interneurons [25].

Embryonic preoptic area (POA)

A very recent study by Gelman et al has demonstrated that in addition to the ganglionic eminences, the embryonic preoptic area (POA) should also be considered a source of cortical GABAergic interneurons [93]. The POA is a region of the hypothalamus, and results from this study suggest that this area contributes approximately 10% of all GABAergic interneurons in the murine cerebral cortex.

Lateral ganglionic eminence

While it is largely agreed upon that the MGE and CGE serve as the primary source of cortical interneurons in the developing rodent nervous system, the possibility of the LGE as a third source has also been heavily debated. Results from several studies have allowed the conclusion that the LGE is at most a minor contributor of interneurons [85, 87, 94]. However, observations from a few studies do suggest otherwise: Sussel and colleagues reported that Nkx2.1 mutants, in which normal MGE tissue fails to form, show a 50% reduction in cortical interneuron numbers relative to wild-type [95]. If the MGE was the origin of the majority of cortical interneurons, only a 50% reduction implies that there are clearly other areas of the brain responsible for generating interneurons. More convincing evidence has shown that at E15.5, the LGE-like region in Nkx2.1 mutants demonstrates strong cellular migration to the developing cortex [87, 88]. BrdU labeling of neural progenitors also supports the notion of a cellular migratory route from the LGE to the cortex during embryogenesis, although only a portion of the BrdU labeled cells were also GABA-positive [87]. Additionally, Jimenez and colleagues discovered that when the MGE is removed in explants taken from rat embryos, cellular migration from the LGE to the cortex continued to be observed, suggesting that the migrating cells are not MGE cells merely passing through the LGE [96].

Specification of cortical interneurons

There has been a recent push in the study of GABAergic interneurons to understand and create a transcriptional network that regulates GABAergic interneuron development, migration to the cortex, and ultimately maturation to the appropriate adult phenotype. To understand the complexity of the transcriptional network and identify other candidate genes involved in the production and patterning of interneurons in the mammalian cortex, it is most logical to separate the various transcription factors based on the three primary origins of cortical interneurons: the medial ganglionic eminence, the caudal ganglionic eminence, and the embryonic preoptic area (Figure 2).

Specification of GABAergic Cortical Interneurons. With regard to the specification of MGE-derived interneuronal progenitors, several transcription factors play a role. Shh signaling activates Nk×2.1, which is the key transcription factor in specifying PV- and SST-positive interneurons from this region. Lh×6 and Lh×8 are transcription factors that lie downstream of Nk×2.1 they also aid in the specification of PV and SST interneurons (see text). Sox6 lies downstream of both Nkx2.1 and Lhx6/8. The Dlx homeobox family of genes play a key role in specification of CGE-derived cortical interneurons, although they also function to maintain the PV-expressing subset of MGE-derived interneurons (Dlx5 in particular). Arx is a homeobox transcription factor whose expression is directly affected by Dlx genes Arx seems to play a role in the migration of interneurons to the cortex. Gsx1 and Gsx2 are both required for the specification of cortical interneurons that originate in the CGE. Mash1 is a downstream transcription factor whose absence results in reduced cortical interneuron numbers it is required for proper function of the Notch ligand Delta1, which, in the Notch signaling pathway, serves to repress neuronal differentiation. The Dlx genes lie further downstream and play a crucial role in CGE-derived interneuron specification. The molecular mechanisms behind POA interneuron specification are unclear, although Nk×2.1 is expressed by interneurons derived from this area. Lh×6 is not expressed by these interneurons. Nk×5.1 was shown to affect the specification of NPY and Reelin interneurons.

Specification of MGE-derived GABAergic cortical interneurons

There is an extensively studied transcriptional network that plays a role in regulating proper development and specification of MGE-derived GABAergic cortical interneurons (Figure 2). While transcription factors such as the Dlx homeobox genes, Lh×6, and So×6 are crucial toward specification of the PV-positive and SST-positive subset of interneurons derived from the MGE, the major transcription factor that must be taken into account is Nk×2.1 [97–102], whose expression is localized and confined within the MGE [103, 104]. Nk×2.1 expression dictates the generation of both PV- and SST-expressing MGE progenitors, and maintenance of Nk×2.1 expression is activated by Sonic hedgehog (Shh) signaling [105]. More specifically, the level of Shh signaling within MGE interneuronal progenitor cells seems to be a determinant of what marker these neurons will express, either PV or SST: Xu et al have shown that high levels of Shh signaling preferentially give rise to SST-expressing interneurons, which in turn results in reduced production of PV-expressing interneurons [106]. Complete absence or conditional loss of Nk×2.1 results in reduction of PV- and SST-expressing interneurons, demonstrating its importance for MGE-derived interneuronal specification [99, 100, 102].

A second transcription factor whose role is crucial to MGE-derived interneuronal specification, and whose expression is also confined to the MGE, is Lh×6 [99, 100, 102]. Lh×6 was discovered to be a target of Nk×2.1 [107]. Mutational analyses with regard to Lh×6 have demonstrated its importance in determining the fate of both PV- and SST-positive interneurons: in the absence of Lhx6, MGE-derived neural progenitors are still able to migrate properly to the pallium, but most of these interneurons fail to express either PV or SST interestingly, an increase in NPY expression is observed [99, 100, 102]. Additionally, Lhx6-deficient interneurons are unable to properly integrate into their respective cortical layers this observation suggests that factors downstream of Lhx6 contribute to the process of cortical integration [102]. Despite these findings, the PV- and SST-expressing population of cortical interneurons is not completely eliminated in Lhx mutants, implying that PV-positive and SST-positive interneurons are not solely derived from the MGE [99, 100, 102]. Alternatively, transcription factors other than Nkx2.1 and Lh×6 may be important for the specification of MGE-derived, PV and SST interneurons this may partially compensate for the loss of Nk×2.1 and Lh×6. Additional studies should be performed to discern other origins of these subsets of cortical interneurons.

It is also important to take into account additional transcription factors that lie downstream of both Nk×2.1 and Lh×6 so as to obtain a better understanding of the specification of MGE-derived interneurons. Recent studies have done just that, and several transcription factors are suggested to act in conjunction with either Nk×2.1 or Lh×6. One such transcription factor is So×6. So×6 is an HMG-box-containing transcription factor that is expressed by MGE-derived GABAergic cortical interneurons mice lacking Sox6 do not possess PV-positive interneurons and have significant reductions of SST-positive interneurons [97, 98]. Interestingly, proper Lh×6 functioning is required for maintenance of So×6 expression in neurons that are actively migrating toward the cortex, but not in MGE-derived interneuronal progenitors [98, 108].

Another transcription factor that works in conjunction with Lh×6 is Lh×8, which lies downstream of Nk×2.1 and is co-expressed with Lhx6 in MGE-derived neuronal progenitors [95, 109]. While Lh×8-positive cells are not specified to become GABAergic cortical interneurons, mutational analyses of mice lacking both Lh×6 and Lh×8 provide some insight toward the Lh×8’s role in cortical interneuron specification. Lhx6/8 double mutants exhibit significantly reduced MGE-derived interneuron production and defective migration [108]. However, this phenotype is not observed in mice that are mutant for Lh×8 only [108].

The Dlx family of homeobox genes, specifically Dlx1, 2, 5, and 6, also play a role in the specification of MGE interneuronal progenitors, and are expressed in most subpallial neural progenitor cells [110, 111]. The Dlx genes will be discussed more extensively in the following section, seeing as they play a larger role in the specification of CGE-derived cortical interneurons. Dl×5 in particular is expressed in the PV-positive mature interneuronal subset [101]. Wang et al utilized transplantation experiments to demonstrate that loss of Dl×5 or both Dl×5 and 6 in mice manifests as a significant reduction in PV-expressing interneuron numbers, alteration in dendritic morphology, and epilepsy [101].

Specification of CGE-derived GABAergic cortical interneurons

As was previously mentioned, approximately 30% of GABAergic cortical interneurons originate from the CGE. While it was previously thought that the gene expression profile of the CGE was merely an extension of that of the LGE [80, 84, 110, 111], more recent work has suggested that the CGE possesses a unique transcriptional network of its own, making it a bona fide source of cortical interneurons, separate from any other source [111–113].

Of great importance in the specification of CGE-derived interneurons are the homeobox transcription factors known as Gs×1 and Gs×2, both of which are required for the specification of interneuronal progenitors in this region [106]. Xu et al have shown through conditional loss and gain of Gs×2 function that Gs×2 has a hand in the generation of CR-expressing bipolar cortical interneurons [106].

Investigation of genes downstream of Gs×1 and Gs×2 resulted in the discovery of Mash1, a basic-helix-loop-helix transcription factor also involved in the interneuron specification process. Mash1 mutants demonstrate significant neuronal loss early in development, as well as reduced cortical interneuron numbers [114]. Mash1 was found to be required to express the Notch ligand Delta1, whose function within the Notch signaling pathway serves to repress neuronal differentiation [114–116]. Thus, loss of Mash1 results in the premature differentiation of cells within the subventricular zone (SVZ) expressing the Dlx genes [114, 115].

As was mentioned in the previous section, the Dl× family of genes are key players in the specification of CGE-derived interneurons. The Dlx genes are located downstream of both Gsx2 and Mash1 [117–120]. With regard to interneuron specification, four Dl× genes – Dl×1, Dl×2, Dl×5, and Dl×6 – are expressed in the developing forebrain, and are central to the specification process [121]. Temporal expression of these four genes follows a Dl×2, Dl×1, Dl×5, and Dl×6 sequence [122, 123]. Gs×2 and Mash1 neural progenitor cells have been shown to simultaneously express Dl×1/2 [110, 115, 124]. Loss of Dl×1/2 function results in the failure of Dl×5/6 expression [110, 115, 124]. Importantly, these Dlx1/2 double mutants exhibit incorrect specification of the LGE/CGE, with inappropriate expression of ventral cortex markers [110, 111]. A detailed analysis of the transcriptional network within the Dlx1/2 double mutants has resulted in the investigation of almost 100 transcription factors that may play a role, both dependently and independently of the Dl× genes, in CGE-derived cortical interneuron specification [110, 111]. An outline of several candidate transcription factors is nicely reviewed by Gelman et al[125].

Specification of embryonic POA-derived GABAergic cortical interneurons

While the embryonic POA is similar to the MGE in that it expresses the transcription factor Nk×2.1, many POA cells do not express the transcription factor Lh×6 [84]. A study by Gelman et al has elegantly showed via fate mapping with a Cre line expressed from transcription factor Nk×5.1 that the POA serves as a source of GABAergic interneurons expressing NPY and/or reelin [126]. Interestingly, none of the other calcium-binding proteins, such as PV, SST, CR or VIP, were expressed in these POA-derived cortical interneurons [126]. It is possible that this population of NPY- and/or reelin-expressing cells may stem from both the CGE and the POA, but additional investigations should be carried out to confirm these speculations. Studies should also be performed to discover the transcription factors and the roles they play in specification of POA-derived cells.

Influence of non-autonomous cell factors on GABAergic interneuron identity

Non-cell autonomous factors and the role they possess in conferring GABAergic cortical interneuron identity must also be taken into account. Lodato et al have shown that certain excitatory neurons are able to control the distribution of GABAergic interneurons within the cortex [127]. Deletion of subcerebral projection neurons and subsequent replacement with callosal projection neurons results in the abnormal lamination of cortical interneurons, along with abnormal GABAergic inhibition [127]. Certain factors may also affect particular subsets of interneurons. Orthodenticle homeobox 2 (Otx2) is a homeoprotein whose expression is required to both open and close a critical period of neural plasticity in the visual cortex of PV-expressing interneurons in mice [128, 129]. It remains to be clarified whether other factors affect the specification of other interneuronal subsets in a non-cell-autonomous manner.

Differences in cortical interneuron origin and specification between rodents and primates

Studies in both primates and humans have determined that differences do exist between rodents and primates with regard to the origins and migratory routes of GABAergic interneurons. This will be briefly discussed, but for more detail refer to the review by Petanjek et al[130].

A few studies have shed light on the fact that the majority of primate GABAergic interneurons, in contrast to rodent cortical interneurons, may not actually originate solely in the GEs [131, 132]. In fact, cortical interneurons in primates have also been shown to have their origins in the proliferative zones of the dorsal telencephalon [131, 133–135]. Letinic and colleagues utilized retroviral labeling in organotypic slice cultures to demonstrate that there are two independent lineages of cortical interneurons in the fetal human forebrain [131]. However, whereas it was previously mentioned that the GE is the primary source of rodent interneurons, this study demonstrated that in a Dl×1/2-positive, Mash1-negative lineage of cortical interneurons in the fetal human forebrain, only 35% of the interneuron population in the proliferative ventricular zone and subventricular zone originates from the GE [131]. To support these findings, the same results were reported in the macaque monkey (Macaca rhesus, Macaca fascicularis) [134].

In addition to differing locations of interneuron origin between species, it is also important to realize that the transcription factors expressed by rodent interneurons and interneuronal precursors may not necessarily overlap with the transcriptional network that governs the development of human GABAergic interneurons. Research to better characterize human interneurons is underway. One such study investigated the expression of transcription factors Nk×2.1, Dlx1/2, Lh×6 and Mash1 in human fetal forebrains during the first half of gestation [136]. All transcription factors were expressed in both the GEs and the ventricular/subventricular zones, and expression was maintained up to 20 gestational weeks. Collectively the data suggest that cortical interneuron populations exist in multiple locations, both ventrally (as described in rodents) and dorsally in the VZ/SVZ [136]. The possibilities that the production of interneurons may follow a different time-course based on origin, or that different areas of production may contribute to the heterogeneity of the cortical interneuron population, should also be taken into account. Findings from a previous study utilizing cryosections and in vitro data also support the existence of multiple sources of cortical interneuron progenitors in the developing human brain [137]. The greater complexity characterizing progenitor populations most likely reflects the higher brain functioning characteristic of humans compared to other organisms.

Migration to the cerebral cortex

Once generated and specified in their respective origins within the ventral telencephalon, GABAergic cortical interneurons face the task of migrating to their ultimate destinations within the cerebral cortex [138]. It has been observed that GABAergic interneurons first begin a tangential route of migration at E12.5 in rodents, a time point that also happens to correspond with the early stages of neurogenesis [80, 139, 140]. At E12.5 in the mouse, an early population of interneurons reaches the cortex, migrating at the level of the preplate, while a second, larger proportion of interneurons migrates through the intermediate zone [139, 141, 142] (IZ). At a later time point of corticogenesis (E14-15), three migratory routes, called tangential migratory streams, are observed in the cortex: the marginal zone (MZ), subplate (SP), and the lower intermediate zone (IZ)/subventricular zone (SVZ) [87, 142, 143]. Migration is, for the most part, complete by birth, with exception of the RMS the migration route that neurons in the RMS take to reach the olfactory bulb will not be discussed in this review. Like the processes of interneuron generation and specification, migration from the subpallial origins to the cerebral cortex is a complicated undertaking, involving the activity of various motogens, chemotactic factors, transcription factors, as well as neurotransmitters [144, 145]. Each will now be briefly elaborated upon.


Motogens are secreted factors that influence newly specified interneurons in their ability to migrate [141]. One such example of a motogen is Hepatocyte Growth Factor (HGF), which was discovered to regulate the migratory abilities of subpallial-derived cortical interneurons. HGF has been found to be required for interneuron migration, and encourages interneurons to migrate away from their sites of origin [146]. mu-PAR nulls (mu-PAR is a required component of the HGF pathway) demonstrate significant deficits in interneuron migration from the GEs to the cortex [146, 147]. In their 2003 study Powell et al also presented the finding that HGF loss of function has an effect on some, but not all, subsets of interneurons [147].

In addition to HGF, the neurotrophin family of genes serve as motogens for migratory cortical interneurons. Various developmental studies have demonstrated that the cortex is positive for neurotrophin expression [148–151], and that the neurotrophin receptors TrkB and TrkC (tyrosine kinase receptors) are expressed by interneurons [152, 153]. One member of the neurotrophins is known as Brain-Derived Neurotrophic Factor (BDNF) [154]. Loss of proper BDNF signaling has been shown to result in a downregulation of cortical interneuron markers [155–157].

Lastly, the neurotrophin glial cell line-derived neurotrophic factor (GDNF), which is expressed in cortical interneuron migratory routes [158], acts as a motogen for GABAergic interneurons. A 2002 study demonstrated that members of the glial derived neurotrophic factor family bind to specific types of receptors known as GFRα1-4 [159]. Loss of GFRα1 in mice manifests in improper migration from the MGE and an inability to reach the cortex compared to wild type [159]. A more recent study proposed, using GFR α1 null mice, that GFRα1 signaling may serve to guide the development of a population of PV-expressing interneurons destined for the cortex [160].


Whereas motogens affect the ability of interneurons to migrate, chemorepellants and chemoattractants serve to provide migratory cells with the information about which direction to migrate. Conceivably, chemorepellants serve as a type of guidance cue that will direct migrating interneurons away from a certain area. Wichterle and colleagues have shown through cell culture studies that in the telencephalon, the GEs exert a repulsive force on interneurons, allowing the postulation that chemorepellants are primarily expressed in the subpallium [161].

One such example of a chemorepellant is the family of ligands known as the semaphorins (Sema) [162]. The semaphorins are able to exert their repulsive forces upon interneurons because interneurons express neuropilins (Nrp1 and Nrp2) and plexin coreceptors, both of which recognize the semaphorins that are expressed within the LGE [162]. A 2003 study demonstrated that two semaphorins in particular, Sema 3A and 3 F, regulate the migratory capacity of GABAergic interneurons toward the cortex [163]. Additionally, Zimmer et al have shown that the chondroitin sulfate proteoglycan known as chondroitin-4-sulfate acts in concert with Sema 3A to repel migrating cortical interneurons within the LGE, further defining the boundary of the migratory route of interneurons [164].

Slit1 is a second chemorepellant whose expression is found in the ventricular zone and the subventricular zone of the GEs, as well as the embryonic POA [165–168]. Slit1 is able to exert its chemorepulsive effects due to the expression of its receptor, Roundabout (Robo1), on migrating cortical interneurons [165–168]. Analysis of the expression patterns of both Slit and Robo proteins are indicative that interneurons are repelled from their sites of origin in the GEs [169–172].

The last type of chemorepellant that will be discussed is Ephrin and its Ephrin receptor tyrosine kinases, which are repulsive cues for MGE-derived interneurons, as demonstrated both in vitro and in vivo[173, 174]. Ephrin A5 in particular is expressed in the ventricular zone of the GE, whereas its receptor, EphA4, is expressed by calbindin-expressing MGE-derived interneurons [173]. Interestingly, siRNA knockdown of the EphA4 receptor resulted in the inability of ephrin A3 to exert its repulsive effects upon interneurons within the MGE [174].


While inhibitory signals exhibited by chemorepellants are undoubtedly essential in defining the tangential migratory route cortical interneurons undertake, chemoatractive factors are just as important in aiding the migration process. Whereas chemorepellants are localized in the subpallium, chemoattractive molecules are present in the pallium. One such chemoattractant is the chemokine CXCL12, which carries out signaling through its receptors CXCR4 and CXCR7. Interneurons derived from the MGE are found to express both of these receptors [175]. The expression pattern of CXCL12 varies throughout development: its expression remains high in the marginal zone and subventricular zone until time point E14.5, but during later stages of corticogenesis its expression is significantly reduced in the subventricular zone (expression in the marginal zone remains unchanged) [176]. Li et al have described CXCL12’s function: it exerts its attractant force on MGE-derived interneurons, guiding them to the previously mentioned tangential migratory streams these cells remain there until intracortical migrations are received [177].

The previously mentioned gene NRG1, which is a susceptibility gene in schizophrenia, also serves as a chemoattractant in the interneuronal migration process. NRG1 was discovered to be essential for interneurons to leave the MGE, traverse through the LGE and into the cortical wall [178]. Flames et al more specifically demonstrated that different isoforms of NRG1 possess unique migratory roles [178]. When interneurons are exposed to an exogenous source of NRG1, neurons extend new neurites in the direction of the source [179], suggesting endogenous NRG1 present during development serves the same purpose. Reduction or loss of NRG1 in the forebrain prevents GABAergic interneurons from leaving the MGE [178]. Research has also been performed on the NRG receptor ErbB4, which is expressed on migratory interneurons [180]. In mature, conditional ErbB4 mutants, a reduced number of cortical interneurons is observed [178].

Transcription factors

While both motogenic and chemotactic factors have been recognized as classic influences on interneuron migration to the cortex, relatively recent studies have cited the roles of transcription factors in migration. One such transcription factor is the LIM homeodomain transcription factor Lh×6. MGE-derived interneurons that are actively migrating to the cerebral cortex express Lhx6 [143, 181]. Moreover, Lh×6 is also expressed in most PV- and SST-positive cortical interneurons in mice [83]. Utilization of Lhx6 knockdown using RNAi hindered the migration of cortical interneurons, lending strong support to the postulation that the Lh×6 gene somehow plays a role in migratory capability of interneurons [182]. Recent studies have also shown that Lh×6 may operate by promoting the expression of receptors such as ErbB4 (receptor for NRG1), CXCR4 and CXCR7 (receptors for chemokine CXCL12) [102].

While Nk×2.1 is known to be a key transcription factor for the specification of MGE-derived GABAergic interneurons [99, 107], it is also suggested to play a role in migration of these interneurons toward the cortex. Nobrega-Pereira and colleagues have shown that proper migration of MGE-derived interneurons to the cortex requires downregulation of Nk×2.1 expression [183]. Further confirming this observation is the finding that ectopic expression of Nk×2.1 in migrating MGE interneuron progenitors results in the inability of Sema3A and Sema3F to exert their repulsive effects [183]. Additional studies should be performed to further elucidate the mechanism by which Nk×2.1 acts in the migration process.

The Dlx family of homeobox genes is involved in migration of interneurons to the cortex [124, 184]. More specifically, Dl×1 and Dl×2 may repress the genes PAK3 and MAP2 in order to restrain neurite outgrowth [184]. PAK3 is a gene involved in regulating and maintaining cytoskeletal dynamics, and whose expression is low in migrating interneurons Dlx1/2 mutants show aberrant, increased PAK3 expression, with an inability to migrate to the cortex [184]. Reducing these aberrant levels of PAK3 restores the ability of these interneurons to migrate tangentially to the cortex [184]. Additionally, results from another study have shown that Dl×1 and Dl×2 may play a role in repression of Nrp2, the receptor for semaphorins [107], suggesting the Dlx genes have multiple modes of action to regulate migration.

Lastly,the homeobox transcription factor known as Arx is located downstream of the Dlx genes, and Dlx function has been shown to directly affect Arx expression: Dlx1/2 double mutants show a down-regulation of Arx expression [185]. In the absence of Arx, MGE-derived interneuron progenitors are unable to migrate toward the cortex, resulting in reduced numbers of interneurons in the cortex [186, 187]. Additionally, mice possessing conditional Arx mutations also have reduced cortical interneuron numbers and have epilepsy [188], demonstrating the importance of this transcription factor in interneuron specification within the MGE.


While their role in migration toward the cerebral cortex may seem unconventional, evidence is emerging that neurotransmitters do indeed aid GABAergic interneurons in their movement toward the cortex. The two major neurotransmitters that will be elaborated upon are GABA and dopamine. Migrating interneurons express the GABAA and GABAB, two main receptors for GABA [189–191]. Reducing GABA activity via neutralizing antibodies results in an accumulation of interneurons at the corticostriatal junction, and prevents their entry into the cortical wall [189]. A more recent study also showed that migrating interneurons have a higher affinity for GABA, relative to non-migrating interneurons that are localized in the MGE [191].

Like the GABA receptors, migrating cortical interneurons also express D1 and D2, which are the receptors for dopamine [192, 193]. D1 and D2 receptors, when activated, are known to have opposite functions from one another [192]. Generation of individual knockouts further confirms the inverse function of these receptors: D 1 nulls possess a decreased ability to migrate, allowing the conclusion that D1 normally functions to promote cortical interneuron migration. On the other hand, D 2 knockouts possess increased migratory capability, suggesting that its role is to inhibit migration of interneurons [192]. It is clear that additional studies must be performed to further elucidate the roles that neurotransmitters such as GABA and dopamine play in the migratory process, as the mechanisms by which they operate are still unknown.

Interestingly enough, while there are clearly many factors that influence the migratory route of GABAergic interneurons to the cortex, Sahara et al have demonstrated that the proportion of cortical interneurons, relative to excitatory neurons, stays the same from the beginning of neurogenesis until adulthood [194]. Data from this study suggests that approximately 1 in 5 interneurons migrating tangentially toward the cerebral cortex in mice are GAD67-positive, indicating they are inhibitory GABAergic interneurons [194].

It is important to note that once GABAergic interneurons successfully reach the cortex, a process referred to as “intracortical migration” must be carried out, whereby interneurons undergo different migratory routes within the cortex to reach their ultimate destinations. This process, as well as interneuron lamination, is beyond the scope of this review and will not be discussed. For a detailed discussion of these processes, refer to the review by Faux et al[195].

To date, the molecular mechanisms that regulate the termination of interneuron migration in the cortex are largely unknown. However, Bortone and Polleux have shown that the potassium-chloride cotransporter KCC2 plays an integral role in terminating interneuron migration [196]. When interneurons are actively migrating, ambient GABA and glutamate initially stimulate the motility through activation of GABAA and AMPA/NMDA receptors. However, once interneurons reach the cortex, upregulation of KCC2 results in a hyperpolarization of membrane potential, thereby serving as a stop signal that interneurons are able to sense [196]. Further investigations should be performed to further elucidate the mechanisms underlying termination of migration.

GABAergic dysfunction and neuropsychiatric disorders

As can be imagined, there is a delicate balance between excitatory and inhibitory inputs that must be carefully maintained with regard to cortical circuits. It is thus conceivable that a number of neuropsychiatric disorders may stem from an alteration or disruption in the balance of excitation vs. inhibition in the cortical circuitry. Additionally, a number of studies have shown that the lack of proper cortical interneuron specification may play a significant role in the development of neurological disorders. This may entail a deviation from either the course of interneuron development, or aberrant transcriptional regulation in the cortical interneuron specification process. Schizophrenia, a severe mental illness, will be utilized as an example of cortical circuitry gone awry. Understanding the effects of both GABAergic neurotransmission, alterations in inhibitory cortical circuits, and how they may be responsible for the clinical features observed in schizophrenia are paramount to this field of research.

Schizophrenia is a mental disorder that is characterized by a variety of symptoms, with cognitive deficits being recognized as both the core and enduring features of this illness [197]. Hallmark features of schizophrenia include auditory hallucinations, paranoid or bizarre delusions, disorganized speech and thinking, and social withdrawal. Additionally, working memory and attention are characteristically impaired in schizophrenic patients [197]. Onset of symptoms occurs in young adulthood. While environmental influences are widely suggested to play a contributory role in development of this illness, genetics and familial predisposition play a very significant role: a meta-analytic review on cognitive performance between relatives of schizophrenic patients and healthy control subjects demonstrated that the same cognitive deficits found in patients with schizophrenia are also found in non-affected relatives [198].

It was initially postulated that schizophrenic patients demonstrating GABAergic interneuron deficits had a significantly reduced number of interneurons relative to non-schizophrenic subjects [199]. While this may still hold true, this field of research is seeing a gradual shift toward the belief that GABAergic dysfunction in schizophrenia may be a result of disruption or imbalance of inhibitory neurocircuitry, rather than a sheer reduction in neuron number [9].

A common trend observed in independently performed studies has shown that only certain interneuronal subtypes seem to be affected in schizophrenia. Schizophrenic patients showed a marked reduction of GAD67 mRNA levels in PV-expressing interneurons of the prefrontal cortex, while GAD67 mRNA levels did not differ in CR-positive interneurons of schizophrenics and healthy controls [200, 201]. A number of studies have postulated that the population of PV-expressing GABAergic interneurons in people with schizophrenia may not be functioning at full capacity, and this may contribute to the cognitive deficits observed with this illness. An inability of PV-positive interneurons to function properly may result in disruption of inhibitory input onto pyramidal neurons, and impairment of synchronization in the gamma range [202, 203]. This theory is corroborated by the observation that schizophrenic patients, when asked to do working memory tasks, display abnormal gamma frequency oscillations in the prefrontal cortex relative to healthy, non-schizophrenic subjects [204–207].

With the knowledge that the parvalbumin-expressing subtype of GABAergic interneurons in particular is largely affected in schizophrenia, it is sensible to seek out factors that play a role in the development of the PV-positive subtype. One such protein is the receptor tyrosine protein kinase ErbB4, which is a receptor for the trophic factor Neuregulin 1 (NRG1). Research was initially focused on NRG1, which was first identified as a susceptibility gene for schizophrenia in an Icelandic population, and then confirmed as a susceptibility gene in an unrelated Scottish population [208]. NRG1 plays a variety of roles during neural development, including modulation of neuronal migration, synaptogenesis, gliogenesis, myelination, and neurotransmission [209]. In particular, NRG1 stimulates GABA release from interneurons, which inhibits pyramidal cells in the prefrontal cortex [210]. It can be imagined that disruption of NRG1 function can have greatly affect the balance between excitatory and inhibitory input. The possible mechanisms by which altered function of NRG1 and its receptor ErbB4 contribute to schizophrenia have been reviewed by Mei and Xiong [211].

With regard to schizophrenia, attention is shifted to NRG1’s receptor, ErbB4: this is largely due to the fact that ErbB4 is a receptor preferentially expressed by interneurons migrating tangentially from the ventral to the dorsal telencephalon [180] and by both embryonic and postnatal PV-expressing interneurons with chandelier and basket cell morphology [212, 213]. Importantly, in vitro and in vivo gain- and loss-of-function experiments prove that ErbB4 promotes the formation of axo-axonic inhibitory synapses over pyramidal neurons in a cell-autonomous manner [212]. More evidence suggests that ErbB4 exerts its effects on PV-positive interneurons: PV-ErbB4 -/- mice exhibit a schizophrenia-like phenotype much like that of NRG1-null and ErbB4-null mutants these mice are hyperactive and show impaired working memory [210]. Deletion of ErbB4 in PV-positive interneurons in these mice also results in fewer synapses being made onto pyramidal neurons [210], thus demonstrating the importance of this transmembrane receptor (as well as Neuregulin 1) in affecting the proper development of at least one subset of GABAergic interneurons.

An additional gene whose disruption predisposes to schizophrenia was first identified in 2000 in a large Scottish family, and named Disrupted-In-Schizophrenia 1 (DISC1) [214]. Utilization of DISC1 genetically engineered mice served as a model for mental illnesses such as schizophrenia, and analysis of these mice showed that dominant-negative DISC1 mice display behavioral abnormalities and a depression-like deficit [215]. Hikida et al also importantly show that DSC1 plays a role in the development of PV-expressing cortical interneurons: dominant-negative DISC1 mice possess pyramidal cells with reduced PV immunoreactivity in the cortex [215]. Another study using knockdown of DISC1 has generated the same results regarding decreased PV immunoreactivity [216]. DISC1 knockdown in prefrontal cortex pyramidal neurons during the pre- and perinatal stages results in abnormal postnatal mesocortical dopaminergic maturation, as well as behavioral abnormalities linked to disrupted cortical circuitry during adulthood [216]. A recent review by Porteus et al offers a detailed summary of the progress that several research groups have made in understanding the role DISC1 protein plays in neurosignaling and neurodevelopment since its initial discovery [217]. While the specific role that DISC1 plays in maintaining cortical interneuron development has yet to be elucidated, it is clear that it is needed to allow proper development of PV-expressing interneurons.

Dysbindin is another schizophrenia susceptibility gene, which plays a role in dopamine receptor trafficking [218]. Multipoint linkage analysis in an Irish population showing genetic variation in the 6p22.3 gene DTNBP1 (dystrobrevin-binding protein 1, the human ortholog of dysbindin) was found to be associated with schizophrenia, thereby spurring a number of investigations of this gene [219]. Findings from a subsequent study indicated that reduction in DTNBP1, frequently observed in schizophrenia, was linked to glutamatergic alterations in intrinsic hippocampal formation connections [220]. Another study corroborated the finding of dysbindin reduction: schizophrenic patients show decreased levels of dysbindin mRNA in multiple layers of the dorsolateral prefrontal cortex relative to healthy subjects [221]. Importantly, Dtnbp1-knockout mice possess cortical and striatal PV-expressing, fast-spiking interneurons with a significant reduction in excitability [218]. This therefore results in decreased inhibitory input to pyramidal neurons in layer V of the prefrontal cortex [218].

While it was initially believed that DISC1 and dysbindin both served as independent susceptibility genes to schizophrenia, a 2011 study investigated whether both DISC1 and dysbindin proteins converged onto a common pathway. Co-aggregation of the two proteins in postmortem brains of patients with mental disease, but not that of healthy patients, was observed, demonstrating that DISC1 and dysbindin do indeed directly interact with each other on a molecular level [222]. It is clear that there are many genetic components implicated in the development of schizophrenia, each of which serve to somehow tip the excitatory-inhibitory balance within the neural circuitry. It is also clear how important of a role GABAergic interneurons play in maintaining this balance. It is important, however, to remember that this is merely the tip of the iceberg – there are many other neuropsychiatric disorders such as epilepsy, autism spectrum disorders, and various intellectual disabilities, many of which are reviewed extensively in a review by the Marin research group [9], that possess some sort of alteration or disruption of balance within the developing nervous system.


The population of GABAergic interneurons in the cerebral cortex is a clearly a diverse one, comprised of many different subtypes and functions. While each interneuronal subtype is characteristically unique with regard to function, electrophysiology, immunohistochemical profile, axonal targeting and firing pattern, the overlapping features between particular subtypes makes the method of categorizing each subset of GABAergic interneurons quite challenging. Most recent undertakings in the study of cortical interneurons have been concerned with constructing the transcriptional network of genes that are involved in the specification of these interneurons, in pre-migratory stages within the subpallium as well as during the migratory phase of interneuron precursors. Additional investigations must be performed in order to understand the specific transcription factors and signaling pathways involved in the specification of interneuronal fate. More specifically, studies must be undertaken to understand how the 5HT3aR-expressing interneuron lineage is specified before migration of these interneuronal progenitors to the cerebral cortex, as it is such a large proportion of the interneuron population. Additionally, not enough is known about the differential specification of PV- and SST-positive subgroups within the Nkx2.1-expressing lineage of interneuronal progenitors. The emergence of cutting edge genetic technologies should be utilized to target specific interneuronal subtypes and understand what key players will determine their fate and function. On a broader level, investigations must also be carried out to understand the differences in origin and specification of rodent vs. human GABAergic interneurons. Understanding the mechanisms behind human GABAergic interneuron specification may enable a large step forward in treating various neurological disorders linked to alterations/dysfunctions in interneuron populations. An increased understanding of the mechanism(s) by which GABAergic interneurons are able to integrate properly and seamlessly into the cortex after migration is complete is also warranted.


There are several advantages to using extracellular field recordings for the study of LTP. First, it is a relatively simple method that is suitable even for beginner electrophysiologists with little background or expertise. Second, it is a relatively noninvasive technique that does not disrupt the internal milieu of the neurons. This is in contrast to whole-cell recordings, where the experimenter runs the risk of washing out substances that are essential for LTP as the cell is dialyzed (Malinow and Tsien 1990 Isaac et al. 1996). Third, it is possible to generate stable recordings for a long period of time, up to several hours, which is technically more challenging with whole-cell recordings (Malinow and Tsien 1990 Watt et al. 2004 Sjöström and Häusser 2006). Finally, response variability is lowered with field potential measurements, which sample the activity of large numbers of neurons simultaneously. This averaging helps produce robust data sets rapidly. Figure 1 in Protocol: Long-Term Potentiation by Theta-Burst Stimulation Using Extracellular Field Potential Recordings in Acute Hippocampal Slices (Abrahamsson et al. 2016) provides a comparison between field excitatory postsynaptic potentials (fEPSPs) and EPSPs from whole-cell recordings.

Theta-burst stimulation is a highly influential LTP induction paradigm that is commonly used because it resembles physiological theta activity and because it is quite robust in brain regions as different as neocortex and hippocampus (Kirkwood et al. 1993). Another standard LTP induction paradigm uses uninterrupted high-frequency stimulation—tetanization—rather than theta-burst stimulation (e.g., the original LTP study by Bliss and Lømo [1973]).

Jun Ding

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Current Research and Scholarly Interests

The interplay between motor cortex, sensory cortex, thalamus and basal ganglia is essential for neural computations involved in generating voluntary movements. Our goal is to dissect the functional organization of motor circuits, particularly cortico-thalamo-basal ganglia networks, using electrophysiology, 2-photon microscopy, optogenetics, and genetic tools. The long-term scientific goal of the lab is to construct functional circuit diagrams and establish causal relationships between activity in specific groups of neurons, circuit function, animal motor behavior and motor learning, and thereby to decipher how the basal ganglia process information and guide motor behavior. We will achieve this by investigating the synaptic organization and function that involve the cortex, thalamus and basal ganglia at the molecular, cellular and circuit level. Currently, we are focusing on several questions:
How are excitatory and inhibitory inputs integrated in the striatum?
How do feed-forward and recurrent local inhibitions balance the excitation in the striatum?
How are functional maps modulated in motor behavior and motor learning?
Our goal is to bridge the gap between molecular or cellular events and the circuit mechanisms that underlie motor behavior. In addition, we aim to further help construct the details of psychomotor disorder ‘circuit diagrams,’ such as the pathophysiological changes in Parkinson’s disease.

2020-21 Courses

2017-18 Courses

Stanford Advisees

  • Doctoral Dissertation Reader (AC)
    Ehsan Dadgar-Kiani, Chung-ha Davis, Isabel Low, John Peters, Mark Plitt
  • Postdoctoral Faculty Sponsor
    Daniel Bloodgood, Fuu Jiun Hwang, Di Lu, Richard Roth, Mengjun Sheng, Yue Sun
  • Doctoral Dissertation Advisor (AC)
    Eddy Albarran, Konstantin Kaganovsky, Renzhi Yang
  • Doctoral Dissertation Co-Advisor (AC)
    Stephen Evans, David Wang

Graduate and Fellowship Programs

All Publications

  • Modulating the Electrical and Mechanical Microenvironment to Guide Neuronal Stem Cell Differentiation ADVANCED SCIENCE Oh, B., Wu, Y., Swaminathan, V., Lam, V., Ding, J., George, P. M. 2021


The application of induced pluripotent stem cells (iPSCs) in disease modeling and regenerative medicine can be limited by the prolonged times required for functional human neuronal differentiation and traditional 2D culture techniques. Here, a conductive graphene scaffold (CGS) to modulate mechanical and electrical signals to promote human iPSC-derived neurons is presented. The soft CGS with cortex-like stiffness (≈3 kPa) and electrical stimulation (±800 mV/100 Hz for 1 h) incurs a fivefold improvement in the rate (14d) of generating iPSC-derived neurons over some traditional protocols, with an increase in mature cellular markers and electrophysiological characteristics. Consistent with other culture conditions, it is found that the pro-neurogenic effects of mechanical and electrical stimuli rely on RhoA/ROCK signaling and de novo ciliary neurotrophic factor (CNTF) production respectively. Thus, the CGS system creates a combined physical and continuously modifiable, electrical niche to efficiently and quickly generate iPSC-derived neurons.


Behaviors are inextricably linked to internal state. We have identified a neural mechanism that links female sexual behavior with the estrus, the ovulatory phase of the estrous cycle. We find that progesterone-receptor (PR)-expressing neurons in the ventromedial hypothalamus (VMH) are active and required during this behavior. Activating these neurons, however, does not elicit sexual behavior in non-estrus females. We show that projections of PR+ VMH neurons to the anteroventral periventricular (AVPV) nucleus change across the 5-day mouse estrous cycle, with 3-fold more termini and functional connections during estrus. This cyclic increase in connectivity is found in adult females, but not males, and regulated by estrogen signaling in PR+ VMH neurons. We further show that these connections are essential for sexual behavior in receptive females. Thus, estrogen-regulated structural plasticity of behaviorally salient connections in the adult female brain links sexual behavior to the estrus phase of the estrous cycle.


Synaptic vesicle and active zone proteins are required for synaptogenesis. The molecular mechanisms for coordinated synthesis of these proteins are not understood. Using forward genetic screens, we identified the conserved THO nuclear export complex (THOC) as an important regulator of presynapse development in C.elegans dopaminergic neurons. In THOC mutants, synaptic messenger RNAs are retained in the nucleus, resulting in dramatic decrease of synaptic protein expression, near complete loss of synapses, and compromised dopamine function. CRE binding protein (CREB) interacts with THOC to mark synaptic transcripts for efficient nuclear export. Deletion of Thoc5, a THOC subunit, in mouse dopaminergic neurons causes severe defects in synapse maintenance and subsequent neuronal death in the substantia nigra compacta. These cellular defects lead to abrogated dopamine release, ataxia, and animal death. Together, our results argue that nuclear export mechanisms can select specific mRNAs and be a rate-limiting step for neuronal differentiation and survival.


Striatal spiny projection neurons (SPNs) receive convergent excitatory synaptic inputs from the cortex and thalamus. Activation of spatially clustered and temporally synchronized excitatory inputs at the distal dendrites could trigger plateau potentials in SPNs. Such supralinear synaptic integration is crucial for dendritic computation. However, how plateau potentials interact with subsequent excitatory and inhibitory synaptic inputs remains unknown. By combining computational simulation, two-photon imaging, optogenetics, and dual-color uncaging of glutamate and GABA, we demonstrate that plateau potentials can broaden the spatiotemporal window for integrating excitatory inputs and promote spiking. The temporal window of spiking can be delicately controlled by GABAergic inhibition in a cell-type-specific manner. This subtle inhibitory control of plateau potential depends on the location and kinetics of the GABAergic inputs and is achieved by the balance between relief and reestablishment of NMDA receptor Mg2+ block. These findings represent a mechanism for controlling spatiotemporal synaptic integration in SPNs.


Midbrain dopamine neurons are an essential component of the basal ganglia circuitry, playing key roles in the control of fine movement and reward. Recently, it has been demonstrated that γ-aminobutyric acid (GABA), the chief inhibitory neurotransmitter, is co-released by dopamine neurons. Here, we show that GABA co-release in dopamine neurons does not use the conventional GABA-synthesizing enzymes, glutamate decarboxylases GAD65 and GAD67. Our experiments reveal an evolutionarily conserved GABA synthesis pathway mediated by aldehyde dehydrogenase 1a1 (ALDH1a1). Moreover, GABA co-release is modulated by ethanol (EtOH) at concentrations seen in blood alcohol after binge drinking, and diminished ALDH1a1 leads to enhanced alcohol consumption and preference. These findings provide insights into the functional role of GABA co-release in midbrain dopamine neurons, which may be essential for reward-based behavior and addiction.


Dynamic adaptations in synaptic plasticity are critical for learning new motor skills and maintaining memory throughout life, which rapidly decline with Parkinson's disease (PD). Plasticity in the motor cortex is important for acquisition and maintenance of motor skills, but how the loss of dopamine in PD leads to disrupted structural and functional plasticity in the motor cortex is not well understood. Here we used mouse models of PD and two-photon imaging to show that dopamine depletion resulted in structural changes in the motor cortex. We further discovered that dopamine D1 and D2 receptor signaling selectively and distinctly regulated these aberrant changes in structural and functional plasticity. Our findings suggest that both D1 and D2 receptor signaling regulate motor cortex plasticity, and loss of dopamine results in atypical synaptic adaptations that may contribute to the impairment of motor performance and motor memory observed in PD.


Structured illumination microscopy (SIM) is a well established method for optical sectioning and superresolution. The core of structured illumination is using a periodic pattern to excite image signals. This work reports a method for estimating minor pattern distortions from the raw image data and correcting these distortions during SIM image processing. The method was tested with both simulated and experimental image data from two-photon Bessel light sheet SIM. The results proves the method is effective in challenging situations, where strong scattering background exists, SNR is low and the sample structure is sparse. Experimental results demonstrate restoring synaptic structures in deep brain tissue, despite the presence of strong light scattering and tissue-induced SIM pattern distortion. This article is protected by copyright. All rights reserved.


Multi-channel electrical recordings of neural activity in the brain is an increasingly powerful method revealing new aspects of neural communication, computation, and prosthetics. However, while planar silicon-based CMOS devices in conventional electronics scale rapidly, neural interface devices have not kept pace. Here, we present a new strategy to interface silicon-based chips with three-dimensional microwire arrays, providing the link between rapidly-developing electronics and high density neural interfaces. The system consists of a bundle of microwires mated to large-scale microelectrode arrays, such as camera chips. This system has excellent recording performance, demonstrated via single unit and local-field potential recordings in isolated retina and in the motor cortex or striatum of awake moving mice. The modular design enables a variety of microwire types and sizes to be integrated with different types of pixel arrays, connecting the rapid progress of commercial multiplexing, digitisation and data acquisition hardware together with a three-dimensional neural interface.


Action control is a key brain function determining the survival of animals in their environment. In mammals, neurons expressing dopamine D2 receptors (D2R) in the dorsal striatum (DS) and the nucleus accumbens (Acb) jointly but differentially contribute to the fine regulation of movement. However, their region-specific molecular features are presently unknown. By combining RNAseq of striatal D2R neurons and histological analyses, we identified hundreds of novel region-specific molecular markers, which may serve as tools to target selective subpopulations. As a proof of concept, we characterized the molecular identity of a subcircuit defined by WFS1 neurons and evaluated multiple behavioral tasks after its temporally-controlled deletion of D2R. Consequently, conditional D2R knockout mice displayed a significant reduction in digging behavior and an exacerbated hyperlocomotor response to amphetamine. Thus, targeted molecular analyses reveal an unforeseen heterogeneity in D2R-expressing striatal neuronal populations, underlying specific D2R's functional features in the control of specific motor behaviors.


How have complex brains evolved from simple circuits? Here we investigated brain region evolution at cell-type resolution in the cerebellar nuclei, the output structures of the cerebellum. Using single-nucleus RNA sequencing in mice, chickens, and humans, as well as STARmap spatial transcriptomic analysis and whole-central nervous system projection tracing, we identified a conserved cell-type set containing two region-specific excitatory neuron classes and three region-invariant inhibitory neuron classes. This set constitutes an archetypal cerebellar nucleus that was repeatedly duplicated to form new regions. The excitatory cell class that preferentially funnels information to lateral frontal cortices in mice becomes predominant in the massively expanded human lateral nucleus. Our data suggest a model of brain region evolution by duplication and divergence of entire cell-type sets.


Optical interrogation of voltage in deep brain locations with cellular resolution would be immensely useful for understanding how neuronal circuits process information. Here, we report ASAP3, a genetically encoded voltage indicator with 51% fluorescence modulation by physiological voltages, submillisecond activation kinetics, and full responsivity under two-photon excitation. We also introduce an ultrafast local volume excitation (ULoVE) method for kilohertz-rate two-photon sampling invivo with increased stability and sensitivity. Combining a soma-targeted ASAP3 variant and ULoVE, we show single-trial tracking ofspikes and subthreshold events for minutes in deep locations, with subcellular resolution and with repeated sampling over days. In the visual cortex, we use soma-targeted ASAP3 to illustrate cell-type-dependent subthreshold modulation by locomotion. Thus, ASAP3 and ULoVE enable high-speed optical recording of electrical activity in genetically defined neurons at deep locations during awake behavior.


Mounting evidence in animal models indicates potential for rejuvenation of cellular and cognitive functions in the aging brain. However, the ability to utilize this potential is predicated on identifying molecular targets that reverse the effects of aging in vulnerable regions of the brain, such as the hippocampus. The dynamic post-translational modification O-linked N-Acetylglucosamine (O-GlcNAc) has emerged as an attractive target for regulating aging-specific synaptic alterations as well as neurodegeneration. While speculation exists about the role of O-GlcNAc in neurodegenerative conditions, such as Alzheimer's disease, its role in physiological brain aging remains largely unexplored. Here, we report that countering age-related decreased O-GlcNAc transferase (OGT) expression and O-GlcNAcylation ameliorates cognitive impairments in aged mice. Mimicking an aged condition in young adults by abrogating OGT, using a temporally controlled neuron-specific conditional knockout mouse model, recapitulated cellular and cognitive features of brain aging. Conversely, overexpressing OGT in mature hippocampal neurons using a viral-mediated approach enhanced associative fear memory in young adult mice. Excitingly, in aged mice overexpressing neuronal OGT in the aged hippocampus rescued in part age-related impairments in spatial learning and memory as well as associative fear memory. Our data identify O-GlcNAcylaton as a key molecular mediator promoting cognitive rejuvenation.


Fast-spiking (FS) neurons can fire action potentials (APs) up to 1,000Hz and play key roles in vital functions such as sound location, motor coordination, and cognition. Here we report that the concerted actions of Kv3 voltage-gated K+ (Kv) and Na+ (Nav) channels are sufficient and necessary for inducing and maintaining FS. Voltage-clamp analysis revealed a robust correlation between the Kv3/Nav current ratio and FS. Expressing Kv3 channels alone could convert 30%-60% slow-spiking (SS) neurons to FS in culture. In contrast, co-expression of either Nav1.2 or Nav1.6 together with Kv3.1 or Kv3.3, but not alone or with Kv1.2, converted SS to FS with 100% efficiency. Furthermore, RNA-sequencing-based genome-wide analysis revealed that the Kv3/Nav ratio and Kv3 expression levels strongly correlated with the maximal AP frequencies. Therefore, FS is established by the properly balanced activities of Kv3 and Nav channels and could be further fine-tuned by channel biophysical features and localization patterns.


Loss of dopamine in Parkinson's disease is hypothesized to impede movement by inducing hypo- and hyperactivity in striatal spiny projection neurons (SPNs) of the direct (dSPNs) and indirect (iSPNs) pathways in the basal ganglia, respectively. The opposite imbalance might underlie hyperkinetic abnormalities, such as dyskinesia caused by treatment of Parkinson's disease with the dopamine precursor L-DOPA. Here we monitored thousands of SPNs in behaving mice, before and after dopamine depletion and during L-DOPA-induced dyskinesia. Normally, intermingled clusters of dSPNs and iSPNs coactivated before movement. Dopamine depletion unbalanced SPN activity rates and disrupted the movement-encoding iSPN clusters. Matching their clinical efficacy, L-DOPA or agonism of the D2 dopamine receptor reversed these abnormalities more effectively than agonism of the D1 dopamine receptor. The opposite pathophysiology arose in L-DOPA-induced dyskinesia, during which iSPNs showed hypoactivity and dSPNs showed unclustered hyperactivity. Therefore, both the spatiotemporal profiles and rates of SPN activity appear crucial to striatal function, and next-generation treatments for basal ganglia disorders should target both facets of striatal activity.


In Parkinson's disease (PD), dopamine depletion causes major changes in the brain, resulting in the typical cardinal motor features of the disease. PD neuropathology has been restricted to postmortem examinations, which are limited to only a single time of PD progression. Models of PD in which dopamine tone in the brain is chemically or physically disrupted are valuable tools in understanding the mechanisms of the disease. The basal ganglia have been well studied in the context of PD, and circuit changes in response to dopamine loss have been linked to the motor dysfunctions in PD. However, the etiology of the cognitive dysfunctions that are comorbid in PD patients has remained unclear until now. In this article, we review recent studies exploring how dopamine depletion affects the motor cortex at the synaptic level. In particular, we highlight our recent findings on abnormal spine dynamics in the motor cortex of PD mouse models through in vivo time-lapse imaging and motor skill behavior assays. In combination with previous studies, a role of the motor cortex in skill learning and the impairment of this ability with the loss of dopamine are becoming more apparent. Taken together, we conclude with a discussion on the potential role for the motor cortex in PD, with the possibility of targeting the motor cortex for future PD therapeutics. © 2017 International Parkinson and Movement Disorder Society.


In this issue of Neuron, Moehle et al. (2017) demonstrate that presynaptic muscarinic receptors counteract the effects of dopamine in an output nucleus of the basal ganglia. They provide intracellular, anatomical, and network-level mechanisms for this cholinergic-dopaminergic interplay.


Stress, a prevalent experience in modern society, is a major risk factor for many psychiatric disorders. Although sensorimotor abnormalities are often present in these disorders, little is known about how stress affects the sensory cortex. Combining behavioral analyses with in vivo synaptic imaging, we show that stressful experiences lead to progressive, clustered loss of dendritic spines along the apical dendrites of layer (L) 5 pyramidal neurons (PNs) in the mouse barrel cortex, and such spine loss closely associates with deteriorated performance in a whisker-dependent texture discrimination task. Furthermore, the activity of parvalbumin-expressing inhibitory interneurons (PV+ INs) decreases in the stressed mouse due to reduced excitability of these neurons. Importantly, both behavioral defects and structural changes of L5 PNs are prevented by selective pharmacogenetic activation of PV+INs in the barrel cortex during stress. Finally, stressed mice raised under environmental enrichment (EE) maintain normal activation of PV+ INs, normal texture discrimination, and L5 PN spine dynamics similar to unstressed EE mice. Our findings suggest that the PV+ inhibitory circuit is crucial for normal synaptic dynamics in the mouse barrel cortex and sensory function. Pharmacological, pharmacogenetic and environmental approaches to prevent stress-induced maladaptive behaviors and synaptic malfunctions converge on the regulation of PV+ IN activity, pointing to a potential therapeutic target for stress-related disorders.Molecular Psychiatry advance online publication, 1 August 2017 doi:10.1038/mp.2017.159.


Neural circuits involving midbrain dopaminergic (DA) neurons regulate reward and goal-directed behaviors. Although local GABAergic input is known to modulate DA circuits, the mechanism that controls excitatory/inhibitory synaptic balance in DA neurons remains unclear. Here, we show that DA neurons use autocrine transforming growth factor β (TGF-β) signaling to promote the growth of axons and dendrites. Surprisingly, removing TGF-β type II receptor in DA neurons also disrupts the balance in TGF-β1 expression in DA neurons and neighboring GABAergic neurons, which increases inhibitory input, reduces excitatory synaptic input, and alters phasic firing patterns in DA neurons. Mice lacking TGF-β signaling in DA neurons are hyperactive and exhibit inflexibility in relinquishing learned behaviors and re-establishing new stimulus-reward associations. These results support a role for TGF-β in regulating the delicate balance of excitatory/inhibitory synaptic input in local microcircuits involving DA and GABAergic neurons and its potential contributions to neuropsychiatric disorders.


Changes in basal ganglia plasticity at the corticostriatal and thalamostriatal levels are required for motor learning. Endocannabinoid-dependent long-term depression (eCB-LTD) is known to be a dominant form of synaptic plasticity expressed at these glutamatergic inputs however, whether eCB-LTD can be induced at all inputs on all striatal neurons is still debatable. Using region-specific Cre mouse lines combined with optogenetic techniques, we directly investigated and distinguished between corticostriatal and thalamostriatal projections. We found that eCB-LTD was successfully induced at corticostriatal synapses, independent of postsynaptic striatal spiny projection neuron (SPN) subtype. Conversely, eCB-LTD was only nominally present at thalamostriatal synapses. This dichotomy was attributable to the minimal expression of cannabinoid type 1 (CB1) receptors on thalamostriatal terminals. Furthermore, coactivation of dopamine receptors on SPNs during LTD induction re-established SPN-subtype-dependent eCB-LTD. Altogether, our findings lay the groundwork for understanding corticostriatal and thalamostriatal synaptic plasticity and for striatal eCB-LTD in motor learning.


Two-photon laser scanning microscopy (2PLSM) allows fluorescence imaging in thick biological samples where absorption and scattering typically degrade resolution and signal collection of one-photon imaging approaches. The spatial resolution of conventional 2PLSM is limited by diffraction, and the near-infrared wavelengths used for excitation in 2PLSM preclude the accurate imaging of many small subcellular compartments of neurons. Stimulated emission depletion (STED) microscopy is a superresolution imaging modality that overcomes the resolution limit imposed by diffraction and allows fluorescence imaging of nanoscale features. Here, we describe the design and operation of a superresolution two-photon microscope using pulsed excitation and STED lasers. We examine the depth dependence of STED imaging in acute tissue slices and find enhancement of 2P resolution ranging from approximately fivefold at 20 μm to approximately twofold at 90-μm deep. The depth dependence of resolution is found to be consistent with the depth dependence of depletion efficiency, suggesting resolution is limited by STED laser propagation through turbid tissue. Finally, we achieve live imaging of dendritic spines with 60-nm resolution and demonstrate that our technique allows accurate quantification of neuronal morphology up to 30-μm deep in living brain tissue.


The substantia nigra pars compacta and ventral tegmental area contain the two largest populations of dopamine-releasing neurons in the mammalian brain. These neurons extend elaborate projections in the striatum, a large subcortical structure implicated in motor planning and reward-based learning. Phasic activation of dopaminergic neurons in response to salient or reward-predicting stimuli is thought to modulate striatal output through the release of dopamine to promote and reinforce motor action. Here we show that activation of dopamine neurons in striatal slices rapidly inhibits action potential firing in both direct- and indirect-pathway striatal projection neurons through vesicular release of the inhibitory transmitter GABA (γ-aminobutyric acid). GABA is released directly from dopaminergic axons but in a manner that is independent of the vesicular GABA transporter VGAT. Instead, GABA release requires activity of the vesicular monoamine transporter VMAT2, which is the vesicular transporter for dopamine. Furthermore, VMAT2 expression in GABAergic neurons lacking VGAT is sufficient to sustain GABA release. Thus, these findings expand the repertoire of synaptic mechanisms used by dopamine neurons to influence basal ganglia circuits, show a new substrate whose transport is dependent on VMAT2 and demonstrate that GABA can function as a bona fide co-transmitter in monoaminergic neurons.


AgRP neuron activity drives feeding and weight gain whereas that of nearby POMC neurons does the opposite. However, the role of excitatory glutamatergic input in controlling these neurons is unknown. To address this question, we generated mice lacking NMDA receptors (NMDARs) on either AgRP or POMC neurons. Deletion of NMDARs from AgRP neurons markedly reduced weight, body fat and food intake whereas deletion from POMC neurons had no effect. Activation of AgRP neurons by fasting, as assessed by c-Fos, Agrp and Npy mRNA expression, AMPA receptor-mediated EPSCs, depolarization and firing rates, required NMDARs. Furthermore, AgRP but not POMC neurons have dendritic spines and increased glutamatergic input onto AgRP neurons caused by fasting was paralleled by an increase in spines, suggesting fasting induced synaptogenesis and spinogenesis. Thus glutamatergic synaptic transmission and its modulation by NMDARs play key roles in controlling AgRP neurons and determining the cellular and behavioral response to fasting.


The proper formation of synaptic connectivity in the mammalian brain is critical for complex behavior. In the striatum, balanced excitatory synaptic transmission from multiple sources onto two classes of principal neurons is required for coordinated and voluntary motor control. Here we show that the interaction between the secreted semaphorin 3E (Sema3E) and its receptor Plexin-D1 is a critical determinant of synaptic specificity in cortico-thalamo-striatal circuits in mice. We find that Sema3e (encoding Sema3E) is highly expressed in thalamostriatal projection neurons, whereas in the striatum Plxnd1 (encoding Plexin-D1) is selectively expressed in direct-pathway medium spiny neurons (MSNs). Despite physical intermingling of the MSNs, genetic ablation of Plxnd1 or Sema3e results in functional and anatomical rearrangement of thalamostriatal synapses specifically in direct-pathway MSNs without effects on corticostriatal synapses. Thus, our results demonstrate that Sema3E and Plexin-D1 specify the degree of glutamatergic connectivity between a specific source and target in the complex circuitry of the basal ganglia.


Striatal cholinergic interneurons are pivotal modulators of the striatal circuitry involved in action selection and decision making. Although nicotinic receptors are important transducers of acetylcholine release in the striatum, muscarinic receptors are more pervasive and have been more thoroughly studied. In this review, the effects of muscarinic receptor signaling on the principal cell types in the striatum and its canonical circuits will be discussed, highlighting new insights into their role in synaptic integration and plasticity. These studies, and those that have identified new circuit elements driven by activation of nicotinic receptors, make it clear that temporally patterned activity in cholinergic interneurons must play an important role in determining the effects on striatal circuitry. These effects could be critical to the response to salient environmental stimuli that serve to direct behavior.


Modulatory interneurons such as, the cholinergic interneuron, are always a perplexing subject to study. Far from clear-cut distinctions such as excitatory or inhibitory, modulating interneurons can have many, often contradictory effects. The striatum is one of the most densely expressing brain areas for cholinergic markers, and actylcholine (ACh) plays an important role in regulating synaptic transmission and cellular excitability. Every cell type in the striatum has receptors for ACh. Yet even for a given cell type, ACh affecting different receptors can have seemingly opposing roles. This review highlights relevant effects of ACh on medium spiny neurons (MSNs) of the striatum and suggests how its many effects may work in concert to modulate MSN firing properties.


Salient stimuli redirect attention and suppress ongoing motor activity. This attentional shift is thought to rely upon thalamic signals to the striatum to shift cortically driven action selection, but the network mechanisms underlying this interaction are unclear. Using a brain slice preparation that preserved cortico- and thalamostriatal connectivity, it was found that activation of thalamostriatal axons in a way that mimicked the response to salient stimuli induced a burst of spikes in striatal cholinergic interneurons that was followed by a pause lasting more than half a second. This patterned interneuron activity triggered a transient, presynaptic suppression of cortical input to both major classes of principal medium spiny neuron (MSN) that gave way to a prolonged enhancement of postsynaptic responsiveness in striatopallidal MSNs controlling motor suppression. This differential regulation of the corticostriatal circuitry provides a neural substrate for attentional shifts and cessation of ongoing motor activity with the appearance of salient environmental stimuli.


Two-photon laser scanning microscopy (2PLSM) has allowed unprecedented fluorescence imaging of neuronal structure and function within neural tissue. However, the resolution of this approach is poor compared to that of conventional confocal microscopy. Here, we demonstrate supraresolution 2PLSM within brain slices. Imaging beyond the diffraction limit is accomplished by using near-infrared (NIR) lasers for both pulsed two-photon excitation and continuous wave stimulated emission depletion (STED). Furthermore, we demonstrate that Alexa Fluor 594, a bright fluorophore commonly used for both live cell and fixed tissue fluorescence imaging, is suitable for STED 2PLSM. STED 2PLSM supraresolution microscopy achieves approximately 3-fold improvement in resolution in the radial direction over conventional 2PLSM, revealing greater detail in the structure of dendritic spines located approximately 100 microns below the surface of brain slices. Further improvements in resolution are theoretically achievable, suggesting that STED 2PLSM will permit nanoscale imaging of neuronal structures located in relatively intact brain tissue.


The two principal excitatory glutamatergic inputs to striatal medium spiny neurons (MSNs) arise from neurons in the cerebral cortex and thalamus. Although there have been many electrophysiological studies of MSN glutamatergic synapses, little is known about how corticostriatal and thalamostriatal synapses differ. Using mouse brain slices that allowed each type of synapse to be selectively activated, electrophysiological approaches were used to characterize their properties in identified striatopallidal and striatonigral MSNs. At corticostriatal synapses, a single afferent volley increased the glutamate released by a subsequent volley, leading to enhanced postsynaptic depolarization with repetitive stimulation. This was true for both striatonigral and striatopallidal MSNs. In contrast, at thalamostriatal synapses, a single afferent volley decreased glutamate released by a subsequent volley, leading to a depressed postsynaptic depolarization with repetitive stimulation. Again, this response pattern was the same in striatonigral and striatopallidal MSNs. These differences in release probability and short-term synaptic plasticity suggest that corticostriatal and thalamostriatal projection systems code information in temporally distinct ways, constraining how they regulate striatal circuitry.


Twenty years ago, striatal cholinergic neurons were central figures in models of basal ganglia function. But since then, they have receded in importance. Recent studies are likely to lead to their re-emergence in our thinking. Cholinergic interneurons have been implicated as key players in the induction of synaptic plasticity and motor learning, as well as in motor dysfunction. In Parkinson's disease and dystonia, diminished striatal dopaminergic signalling leads to increased release of acetylcholine by interneurons, distorting network function and inducing structural changes that undoubtedly contribute to the symptoms. By contrast, in Huntington's disease and progressive supranuclear palsy, there is a fall in striatal cholinergic markers. This review gives an overview of these recent experimental and clinical studies, placing them within the context of the pathogenesis of movement disorders.


Dopamine shapes a wide variety of psychomotor functions. This is mainly accomplished by modulating cortical and thalamic glutamatergic signals impinging upon principal medium spiny neurons (MSNs) of the striatum. Several lines of evidence suggest that dopamine D1 receptor signaling enhances dendritic excitability and glutamatergic signaling in striatonigral MSNs, whereas D2 receptor signaling exerts the opposite effect in striatopallidal MSNs. The functional antagonism between these two major striatal dopamine receptors extends to the regulation of synaptic plasticity. Recent studies, using transgenic mice in which cells express D1 and D2 receptors, have uncovered unappreciated differences between MSNs that shape glutamatergic signaling and the influence of DA on synaptic plasticity. These studies have also shown that long-term alterations in dopamine signaling produce profound and cell-type-specific reshaping of corticostriatal connectivity and function.


Parkinson disease is a neurodegenerative disorder whose symptoms are caused by the loss of dopaminergic neurons innervating the striatum. As striatal dopamine levels fall, striatal acetylcholine release rises, exacerbating motor symptoms. This adaptation is commonly attributed to the loss of interneuronal regulation by inhibitory D(2) dopamine receptors. Our results point to a completely different, new mechanism. After striatal dopamine depletion, D(2) dopamine receptor modulation of calcium (Ca(2+)) channels controlling vesicular acetylcholine release in interneurons was unchanged, but M(4) muscarinic autoreceptor coupling to these same channels was markedly attenuated. This adaptation was attributable to the upregulation of RGS4-an autoreceptor-associated, GTPase-accelerating protein. This specific signaling adaptation extended to a broader loss of autoreceptor control of interneuron spiking. These observations suggest that RGS4-dependent attenuation of interneuronal autoreceptor signaling is a major factor in the elevation of striatal acetylcholine release in Parkinson disease.


Long-term depression (LTD) of the synapse formed between cortical pyramidal neurons and striatal medium spiny neurons is central to many theories of motor plasticity and associative learning. The induction of LTD at this synapse is thought to depend upon D(2) dopamine receptors localized in the postsynaptic membrane. If this were true, LTD should be inducible in neurons from only one of the two projection systems of the striatum. Using transgenic mice in which neurons that contribute to these two systems are labeled, we show that this is not the case. Rather, in both cell types, the D(2) receptor dependence of LTD induction reflects the need to lower M(1) muscarinic receptor activity-a goal accomplished by D(2) receptors on cholinergic interneurons. In addition to reconciling discordant tracts of the striatal literature, these findings point to cholinergic interneurons as key mediators of dopamine-dependent striatal plasticity and learning.


Parkinson disease is a common neurodegenerative disorder that leads to difficulty in effectively translating thought into action. Although it is known that dopaminergic neurons that innervate the striatum die in Parkinson disease, it is not clear how this loss leads to symptoms. Recent work has implicated striatopallidal medium spiny neurons (MSNs) in this process, but how and precisely why these neurons change is not clear. Using multiphoton imaging, we show that dopamine depletion leads to a rapid and profound loss of spines and glutamatergic synapses on striatopallidal MSNs but not on neighboring striatonigral MSNs. This loss of connectivity is triggered by a new mechanism-dysregulation of intraspine Cav1.3 L-type Ca(2+) channels. The disconnection of striatopallidal neurons from motor command structures is likely to be a key step in the emergence of pathological activity that is responsible for symptoms in Parkinson disease.

Wiring the Brain

The trick, and the power of the technique, comes from the specificity with which you can direct that expression. This is based on the fact that different types of cells express different sets of genes. All genes have two main parts – one part is basically the recipe or code for a particular protein. The other part, which is encoded on a neighbouring piece of DNA, is the regulatory region – the instructions for when and where to make that protein and how much to make. Those two regions can be separated. You can cut out the DNA that makes up just the regulatory piece of one gene and hook it up to the protein-coding region for any other gene you like (in this case, a channelrhodopsin protein). Now you can take that fusion gene and introduce it to cells or transgenically introduce it to animals, like worms or flies or mice. Such animals will now express channelrhodopsin only in the cell types directed by the regulatory piece of DNA you chose. A variety of other molecular methods can also be used to achieve this goal, including resources based on binary systems like the Cre-LoxP recombinase system. (Fibre optics can then be used to target light to those cells in particular brain regions).

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Watch the video: PT631 Lecture 2 (January 2023).