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 GABA_A and GABA_B 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.

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.

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 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.

Motogens

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].

Chemorepellants

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].

Chemoattractants

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.

Neurotransmitters

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 GABA_A and GABA_B, 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 GABA_A 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.