Genetic recombination in disgust-associated bitter taste-responsive neurons of the central nucleus of amygdala in male mice.

A bitter substance induces specific orofacial and somatic behavioral reactions such as gapes in mice as well as monkeys and humans. These reactions have been proposed to represent affective disgust, and therefore, understanding the neuronal basis of the reactions would pave the way to understand affective disgust. It is crucial to identify and access the specific neuronal ensembles that are activated by bitter substances, such as quinine, the intake of which induces disgust reactions. However, the method to access the quinine-activated neurons has not been fully established yet. Here, we show evidence that a targeted recombination in active populations (TRAP) method, induces genetic recombination in the quinine-activated neurons in the central nucleus of the amygdala (CeA). CeA is one of the well-known emotional centers of the brain. We found that the intraoral quinine infusion, that resulted in disgust reactions, increased both cFos-positive cells and Arc-positive cells in the CeA. By using Arc-CreER;Ai3 TRAP mice, we induced genetic recombination in the quinine-activated neurons and labelled them with fluorescent protein. We confirmed that the quinine-TRAPed fluorescently-labelled cells preferentially coexpressed Arc after quinine infusion. Our results suggest that the TRAP method can be used to access specific functional neurons in the CeA.

Genetic Access to Gustatory Disgust-Associated Neurons in the Interstitial Nucleus of the Posterior Limb of the Anterior Commissure in Male Mice.

Orofacial and somatic disgust reactions are observed in rats following intraoral infusion of not only bitter quinine (innate disgust) but also sweet saccharin previously paired with illness (learned disgust). It remains unclear, however, whether these innate and learned disgust reactions share a common neural basis and which brain regions, if any, host it. In addition, there is no established method to genetically access neurons whose firing is associated with disgust (disgust-associated neurons). Here, we examined the expression of cFos and Arc, two markers of neuronal activity, in the interstitial nucleus of the posterior limb of the anterior commissure (IPAC) of male mice that showed innate disgust and mice that showed learned disgust. Furthermore, we used a targeted recombination in active populations (TRAP) method to genetically label the disgust-associated neurons in the IPAC with YFP. We found a significant increase of both cFos-positive neurons and Arc-positive neurons in the IPAC of mice that showed innate disgust and mice that showed learned disgust. In addition, TRAP following quinine infusion (Quinine-TRAP) resulted in significantly more YFP-positive neurons in the IPAC, compared to TRAP following water infusion. A significant number of the YFP-positive neurons following Quinine-TRAP were co-labeled with Arc following the second quinine infusion, confirming that Quinine-TRAP preferentially labeled quinine-activated neurons in the IPAC. Our results suggest that the IPAC activity is associated with both innate and learned disgust and that disgust-associated neurons in the IPAC are genetically accessible by TRAP.

The COUP-TFII/Neuropilin-2 is a molecular switch steering diencephalon-derived GABAergic neurons in the developing mouse brain.

The preoptic area (POa) of the rostral diencephalon supplies the neocortex and the amygdala with GABAergic neurons in the developing mouse brain. However, the molecular mechanisms that determine the pathway and destinations of POa-derived neurons have not yet been identified. Here we show that Chicken ovalbumin upstream promoter transcription factor II (COUP-TFII)-induced expression of Neuropilin-2 (Nrp2) and its down-regulation control the destination of POa-derived GABAergic neurons. Initially, a majority of the POa-derived migrating neurons express COUP-TFII and form a caudal migratory stream toward the caudal subpallium. When a subpopulation of cells steers toward the neocortex, they exhibit decreased expression of COUP-TFII and Nrp2. The present findings show that suppression of COUP-TFII/Nrp2 changed the destination of the cells into the neocortex, whereas overexpression of COUP-TFII/Nrp2 caused cells to end up in the medial part of the amygdala. Taken together, these results reveal that COUP-TFII/Nrp2 is a molecular switch determining the pathway and destination of migrating GABAergic neurons born in the POa.

Migratory pathways of GABAergic interneurons when they enter the neocortex.

Inhibitory gamma-aminobutyric-acid-containing interneurons play important roles in the functions of the neocortex. During rodent development, most neocortical interneurons are generated in the subpallium and migrate tangentially toward the neocortex. They migrate through multiple pathways to enter the neocortex. Failure of interneuron migration through these pathways during development leads to an abnormal distribution and abnormal functions of interneurons in the postnatal brain. Because of recent discoveries regarding the novel origins and migratory pathways of neocortical interneurons, in this article we review the literature on the migratory pathways of interneurons when they enter the neocortex.

Leucine-rich glioma inactivated 1 (Lgi1), an epilepsy-related secreted protein, has a nuclear localization signal and localizes to both the cytoplasm and the nucleus of the caudal ganglionic eminence neurons.

Leucine-rich glioma inactivated 1 (Lgi1) is a secreted synaptic protein that organizes a transsynaptic protein complex throughout the brain. Mutations in the Lgi1 gene have been found in patients with autosomal dominant lateral temporal lobe epilepsy (ADLTE). Although a large number of studies have focused on the expression and function of Lgi1 in the postnatal brain, information regarding its functions and distribution during development remains sparse. Here we report that Lgi1 mRNA is preferentially expressed in the caudal ganglionic eminence (CGE) of the early embryonic telencephalon, and LGI1 protein is unexpectedly localized in the nucleus of dissociated CGE neurons. Using bioinformatics analysis, we found that LGI1 contains a putative nuclear localization signal (NLS) in its leucine-rich repeat C-terminal domain. Furthermore, we show that the transient expression of Lgi1 in CGE neurons resulted in nuclear translocation of the LGI1 protein, and a mutation in the NLS led to the retention of LGI1 in the cytoplasm. We also confirmed that the NLS sequence of LGI1 had the ability to mediate the nuclear localization by using the NLS-containing fusion protein. Interestingly, when Lgi1 was expressed in neurons obtained from the medial ganglionic eminence or cerebral cortex, almost no nuclear localization of LGI1 was observed. These results raise the possibility of a novel role of Lgi1 within embryonic neurons through nuclear translocation and may provide insight into its potential effects on the development of the central nervous system and ADLTE pathogenesis.

GABAergic interneuron migration and the evolution of the neocortex.

A neocortex is present in all mammals but is not present in other classes of vertebrates, and the neocortex is extremely elaborate in humans. Changes in excitatory projection neurons and their progenitors within the developing dorsal pallium in the most recent common ancestor of mammals are thought to have been involved in the evolution of the neocortex. Our recent findings suggest that changes in the migratory ability of inhibitory interneurons derived from outside the neocortex may also have been involved in the evolution of the neocortex. In this article we review the literature on the migratory profile of inhibitory interneurons in several different species and the literature on comparisons between the intrinsic migratory ability of interneurons derived from different species. Finally, we propose a hypothesis about the mammalian-specific evolution of the migratory ability of interneurons and its potential contribution to the establishment of a functional neocortex.

GABAergic precursor transplantation into the prefrontal cortex prevents phencyclidine-induced cognitive deficits.

Phencyclidine (PCP) is a noncompetitive NMDA receptor antagonist, and it induces schizophreniform cognitive deficits in healthy humans and similar cognitive deficits in rodents. Although the PCP-induced cognitive deficits appear to be accompanied and possibly caused by dysfunction of GABAergic inhibitory interneurons in the prefrontal cortex (PFC), the potential benefit(s) of GABAergic interneuron manipulations on PCP-induced cognitive deficits remains unexplored. In this study we show that when embryonic medial ganglionic eminence (MGE) cells, many of which differentiate into cortical GABAergic interneurons in situ, were grafted into the medial PFC (mPFC) of neonatal mice, they differentiated into a specific class of GABAergic interneurons and became functionally integrated into the host neuronal circuitry in adults. Prior MGE cell transplantation into the mPFC significantly prevented the induction of cognitive and sensory-motor gating deficits by PCP. The preventive effects were not reproduced by either transplantation of cortical projection neuron precursors into the mPFC or transplantation of MGE cells into the occipital cortex. The preventive effects of MGE cell transplantation into the mPFC were accompanied by activation of callosal projection neurons in the mPFC. These findings suggest that increasing GABAergic interneuron precursors in the PFC may contribute to the development of a cell-based approach as a novel means of modulating the PFC neuronal circuitry and preventing schizophreniform cognitive deficits.

Changes in cortical interneuron migration contribute to the evolution of the neocortex.

The establishment of the mammalian neocortex is often explained phylogenetically by an evolutionary change in the pallial neuronal progenitors of excitatory projection neurons. It remains unclear, however, whether and how the evolutionary change in inhibitory interneurons, which originate outside the neocortex, has been involved in the establishment of the neocortex. In this study, we transplanted chicken, turtle, mouse, and marmoset medial ganglionic eminence (MGE) cells into the embryonic mouse MGE in utero and compared their migratory behaviors. We found that the MGE cells from all of the species were able to migrate through the mouse neocortical subventricular zone and that both the mouse and marmoset cells subsequently invaded the neocortical cortical plate (CP). However, regardless of their birthdates and interneuron subtypes, most of the chicken and turtle cells ignored the neocortical CP and passed beneath it, although they were able to invade the archicortex and paleocortex, suggesting that the proper responsiveness of MGE cells to guidance cues to enter the neocortical CP is unique to mammals. When chicken MGE cells were transplanted directly into the neocortical CP, they were able to survive and mature, suggesting that the neocortical CP itself is essentially permissive for postmigratory development of chicken MGE cells. These results suggest that an evolutionary change in the migratory ability of inhibitory interneurons, which originate outside the neocortex, was involved in the establishment of the neocortex by supplying inhibitory components to the network.

Cortical GABAergic interneurons transiently assume a sea urchin-like nonpolarized shape before axon initiation.

Mature neurons polarize by extending an axon and dendrites. In vitro studies of dissociated neurons have demonstrated that axons are initiated from a nonpolarized stage. Dissociated hippocampal neurons form four to five minor neurites shortly after plating but then one of them starts to elongate rapidly to become the future axon, whereas the rest constitutes the dendrites at later stages. However, neuroepithelial cells as well as migrating neurons in vivo are already polarized, raising the possibility that mature neurons inherit the polarities of immature neurons of neuroepithelial or migrating neurons. Here we show that the axon of interneurons in mouse cortical explant emerges from a morphologically nonpolarized shape. The morphological maturation of cortical interneurons labeled by electroporation at an embryonic stage was analyzed by time-lapse imaging during the perinatal stage. In contrast to earlier stages, most interneurons at this stage show sea urchin-like nonpolarized shapes with alternately extending and retracting short processes. Abruptly, one of these processes extends to give rise to an outstandingly long axon-like process. Given that the interneurons exhibit typical polarized shapes during embryonic development, the present results suggest that axon-dendrite polarity develops from a nonpolarized intermediate stage.

CXCR4 is required for proper regional and laminar distribution of cortical somatostatin-, calretinin-, and neuropeptide Y-expressing GABAergic interneurons.

Cortical GABAergic interneurons are divided into various subtypes, with each subtype contributing to rich variety and fine details of inhibition. Despite the functional importance of each interneuron subtype, the molecular mechanisms that contribute to sorting them to their appropriate positions within the cortex remain unclear. Here, we show that the chemokine receptor CXCR4 regulates the regional and layer-specific distribution of interneuron subtypes. We removed Cxcr4 specifically in a subset of interneurons at a specific mouse embryonic developmental stage and analyzed the number of interneurons and their laminar distribution in 9 representative cortical regions comprehensively in adults. We found that the number of Cxcr4-deleted calretinin- and that of neuropeptide Y-expressing interneurons were reduced in most caudomedial and lateral cortical regions, respectively, and also in superficial layers. In addition, Cxcr4-deleted somatostatin-expressing interneurons showed a reduction in the number of superficial layers in certain cortical regions but of deep layers in others. These findings suggest that CXCR4 is required for proper regional and laminar distribution in a wider interneuron subpopulation than previously thought and may regulate the establishment of functional cortical circuitry in certain cortical regions and layers.

Random walk behavior of migrating cortical interneurons in the marginal zone: time-lapse analysis in flat-mount cortex.

Migrating neurons are thought to travel from their origin near the ventricle to distant territories along stereotypical pathways by detecting environmental cues in the extracellular milieu. Here, we report a novel mode of neuronal migration that challenges this view. We performed long-term, time-lapse imaging of medial ganglionic eminence (MGE)-derived cortical interneurons tangentially migrating in the marginal zone (MZ) in flat-mount cortices. We find that they exhibit a diverse range of behaviors in terms of the rate and direction of migration. Curiously, a predominant population of these neurons repeatedly changes its direction of migration in an unpredictable manner. Trajectories of migration vary from one neuron to another. The migration of individual cells lasts for long periods, sometimes up to 2 d. Theoretical analyses reveal that these behaviors can be modeled by a random walk. Furthermore, MZ cells migrate from the cortical subventricular zone to the cortical plate, transiently accumulating in the MZ. These results suggest that MGE-derived cortical interneurons, once arriving at the MZ, are released from regulation by guidance cues and initiate random walk movement, which potentially contributes to their dispersion throughout the cortex.

[Guidance mechanisms in neuronal and axonal migration].

During development, neurons migrate from their birthplace to their appropriate positions and extend their axons toward their proper targets. These events are crucial for the establishment of CNS cytoarchitectures and neural cuircuits essential for brain functions. Recent genetic and biochemical studies have led to the notion that neurons and axons appear to be guided by similar guidance mechanisms and molecules. They appear to be guided by at least four distinct mechanisms: contact attraction, chemoattraction, contact repulsion, and chemorepulsion. These mechanisms are mediated by evolutionarily conserved ligand-receptor system. Extracellualr guidance cues, including Netrins, Slits, Semaphorins and Ephrins, act as ligands, while membrane proteins which are expressed in migrating neurons or axons serve as receptors. A certain guidance cue can bind to several distinct receptors and vice versa, leading to elicit diverse responses in migrating neurons or axons with a relatively small number of guidance cues.

Multidirectional and multizonal tangential migration of GABAergic interneurons in the developing cerebral cortex.

Most GABAergic interneurons originate from the basal forebrain and migrate tangentially into the cortex. The migratory pathways and mode of interneuron migration within the developing cerebral cortex, however, previously was largely unknown. Time-lapse imaging and in vivo labelling with glutamate decarboxylase (GAD)67-green fluorescence protein (GFP) knock-in embryonic mice with expression of GFP in gamma-aminobutyric acid (GABA)ergic neurons indicated that multidirectional tangential (MDT) migration of interneurons takes place in both the marginal zone (MZ) and the ventricular zone (VZ) of the cortex. Quantitative analysis of migrating interneurons showed that rostrocaudally migrating neurons outnumber those migrating mediolaterally in both of these zones. In vivo labelling with a lipophilic dye showed that the MDT migration in the MZ occurs throughout the cortex over distances of up to 3 mm during a period of a few days. These results indicate that MZ cortical interneurons undergo a second phase of tangential migration in all directions and over long distances, after reaching the cortex by dorsomedial tangential migration. The MDT migration in the MZ may disperse and intermix interneurons within the cortex, resulting in a balanced distribution of interneuron subtypes.