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Gene expression programs and concomitant chromatin regulation change dramatically during the maturation of postmitotic neurons. Subnuclear positioning of gene loci is relevant to transcriptional regulation. However, little is known about subnuclear genome positioning in neuronal maturation. Using cultured murine hippocampal neurons, we found genomic locus 14qD2 to be enriched with genes that are upregulated during neuronal maturation. Reportedly, the locus is homologous to human 8p21.3, which has been extensively studied in neuropsychiatry and neurodegenerative diseases. Mapping of the 14qD2 locus in the nucleus revealed that it was relocated from the nuclear periphery to the interior. Moreover, we found a concomitant decrease in lamin B1 expression. Overexpression of lamin B1 in neurons using a lentiviral vector prevented the relocation of the 14qD2 locus and repressed the transcription of the Egr3 gene on this locus. Taken together, our results suggest that reduced lamin B1 expression during the maturation of neurons is important for appropriate subnuclear positioning of the genome and transcriptional programs., (Copyright © 2021 Elsevier B.V. and Japan Neuroscience Society. All rights reserved.)

During embryonic development, GABAergic interneurons, a main inhibitory component in the cerebral cortex, migrate tangentially from the ganglionic eminence (GE) to cerebral cortex. After reaching the cerebral cortex, they start to extend their neurites for constructing local neuronal circuits around the neonatal stage. Aberrations in migration or neurite outgrowth are implicated in neurological and psychiatric disorders such as epilepsy, schizophrenia and autism. Previous studies revealed that in the early phase of cortical development the neural population migrates tangentially from the GE in the telencephalon and several genes have been characterized as regulators of migration and specification of GABAergic interneurons. However, much less is known about the molecular mechanisms of GABAergic interneurons-specific maturation at later stages of development. Here, we performed genome-wide screening to identify genes related to the later stage by flow cytometry based-microarray (FACS-array) and identified 247 genes expressed in cortical GABAergic interneurons. Among them, Dgkg, a member of diacylglycerol kinase family, was further analyzed. Correlational analysis revealed that Dgkg is dominantly expressed in somatostatin (SST)-expressing GABAergic interneurons. The functional study of Dgkg using GE neurons indicated alteration in neurite outgrowth of GABAergic neurons. This study shows a new functional role for Dgkg in GABAergic interneurons as well as the identification of other candidate genes for their maturation., (Copyright © 2017 Elsevier Ireland Ltd and Japan Neuroscience Society. All rights reserved.)

Neurons in the prepositus hypoglossi nucleus (PHN), which is involved in controlling horizontal gaze, show distinct firing patterns in response to depolarizing current pulses. Although the firing patterns are commonly used to classify neuron types, whether the classified PHN neurons show differences in voltage response properties when stimulated with various types of current inputs remains unclear. In this study, we investigated the response properties of PHN neurons to various current stimuli using whole-cell recordings in rat brainstem slices. In response to pulse currents, neurons that exhibited oscillatory firing (OSC type) showed greater gain than other types, and neurons with a low firing rate (LFR type) showed strong overshooting firing responses to ramp currents. In response to triangular ramp currents, the late-spiking type and the LFR type showed a marked hysteretic frequency-current relationship. In response to sinusoidal currents, the gain was larger in the OSC type than in the other types, although the gain and phase of all types of neurons were similarly modulated by an increase in the input frequency. These findings suggest that distinct neuron types show distinct response properties, depending on the type of stimulus. These neuron types may represent the functionally different populations in the PHN., (Copyright © 2017 Elsevier Ireland Ltd and Japan Neuroscience Society. All rights reserved.)

Multicellular neuronal activities should be investigated to reveal the dynamics of the neuronal circuit. Optical recording from neuronal populations is suitable for recording multicellular activities. We fabricated the prototype of the micro-imaging probe in combination with a gradient index lens and image fiber. This probe has a smaller diameter than traditional probes. We found an optimal optical configuration for maximizing the efficiency of the imaging probe. Using this optical configuration with the prototype of the imaging probe, the fluorescence images were captured from neurons expressing green fluorescent protein in a cerebellar block preparation, and the calcium-dependent images were sampled in a mouse brain slice preparation. Our optical system would facilitate the in vivo imaging studies with less invasive manners using thinner optic fiber than previously made., (Copyright © 2012 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.)

In our previous study on the prepositus hypoglossi nucleus (PHN), we found a neuronal subtype exhibiting a specific firing pattern in which the first interspike interval (ISI) was longer than that of the second, designated FIL (first interspike interval long) neurons. In the present study, we explored the ionic mechanisms underlying this firing pattern using whole-cell recordings of rat brainstem slice preparations. In addition to a longer first ISI, FIL neurons showed properties such as increased slow afterhyperpolarization (AHP) of the first spike relative to the second spike. The application of 4-aminopyridine (4-AP) shortened the longer first ISI and reduced the larger AHP of the first spike, but α-dendrotoxin affected neither the ISI nor the AHP. A voltage clamp study revealed that FIL neurons express transient outward currents with slow decay kinetics. When T-type Ca(2+) currents alone or T-type Ca(2+) plus persistent Na(+) currents were blocked, the FIL firing pattern changed to one with transient hyperpolarization and delayed spike generation characteristic of late-spiking neurons. These findings indicate that A-type K(+) currents showing slow decay, T-type Ca(2+) currents, and persistent Na(+) currents all contribute to the specific firing pattern of FIL neurons., (Copyright © 2012 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.)

Both down-regulation of glutamate decarboxylase 67 (GAD67) and maternal exposure to severe stress during pregnancy can increase the risk of schizophrenia and related psychotic disorders in the offspring. To investigate a gene-environment interaction, we performed the restraint-and-light stress to pregnant GAD67-GFP knock-in (GAD67(+/GFP)) and wild-type (GAD67(+/+)) mice three times a day for 45 min per session during gestational day (G) 15.0-17.5. The stress hormone (corticosterone) level of pregnant GAD67(+/GFP) mice (the overall GABA content is reduced because of the destruction of one allele of the endogenous GAD67 gene) was higher than that of GAD67(+/+), even without stress. The fetal body weights (GAD67(+/+)) in the GAD67(+/GFP) mothers were lower than those in the GAD67(+/+) mothers. GAD67(+/GFP) fetuses exhibited higher corticosterone (CORT) levels than GAD67(+/+) fetuses, even in non-stressed GAD67(+/+) mothers. Fetal body weight-decreases and CORT-increases by maternal stress (GAD67(+/+) mother) were significantly more in the GAD67(+/GFP) fetuses than the GAD67(+/+) fetuses. These results indicate that a GAD67 heterozygous deletion itself enhances vulnerability by many aspects, e.g., maternal stress, maternity, and being in utero. Thus, an abnormality in GAD67 could interact with environmental risk factors of psychiatric disorders, including schizophrenia., (Copyright © 2011. Published by Elsevier Ireland Ltd.)

GABA plays an important role in inhibitory neurotransmission. In the developing brain, GABA also acts as a paracrine chemical mediator. To evaluate the ambient GABA gradients in the brain, an enzyme-linked imaging system that consisted of GABase and NADP(+) was developed. In rat cerebellar slices, GABA release was observed in the layers containing GABAergic neurons. In telencephalic slices from embryonic GAD67-GFP knock-in mice, ambient GABA levels were high in the ganglionic eminence, where GABA cells are generated, but missing in homozygotes. This study indicates that this method will be useful to study the topography and dynamics of ambient GABA concentrations., (Copyright 2010 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.)

The mammalian central nervous system is populated with various types of neurons and glia. To investigate the functions and development of individual cells requires gene-expression analysis at the single-cell level. Here, we developed a microarray-based method for the gene-expression profiling of single cells and tested it for GABAergic neuron progenitors. Single GABAergic neuron progenitors were collected from the neocortex of GAD67-GFP knock-in mice by dissociation followed by the aspiration of GFP-positive cells. Complementary DNA from the single cells was amplified by a method in which Super SMART PCR and T7 RNA polymerase amplification were combined at a optimized condition. The cRNA was subjected to microarray hybridization and analysis, which yielded reliable and reproducible results.

Utilizing slice preparations of GAD67-GFP knock-in mouse, in which GABAergic neurons are specifically labeled with GFP fluorescence, we studied electrophysiological characteristics of GABAergic neurons of IC by whole-cell patch clamp-recording combined with biocytin-intracellular-staining techniques. GABAergic neurons of IC fell into two distinct firing types; (1) tonic type neurons and (2) transient (phasic) type neurons. Tonic type neurons showed regularly repetitive discharge pattern in response to a long depolarizing current pulse (200 ms), and transient type neurons showed spike discharges just at the onset of current pulse. Most of neurons of both types showed depolarizing sag in response to hyperpolarizing current pulse, which were blocked by 0.1 mM ZD7288 (Ih blocker). All two types of tonic neurons showed an AHP, which was blocked by Cd2+ (0.1 mM) and high concentration of apamin (2 microM). One of tonic type neurons (BP) revealed a long delay in spike onset or a longer first spike interval when they were stimulated from hyperpolarized potentials. The remaining tonic neurons (RS) did not show this property. Tonic type neurons were distributed in all region of IC. Morphologically, they were not identical; heterogeneous in somatic diameter, dendritic field size and its orientation. One of transient type neurons (Th-) revealed an AHP after the spike. The other transient type neurons (Th+) showed a depolarization hump after the spike, which were blocked by 0.1-0.2 mM Ni2+. Th+ type neurons were found only in the dorsolateral region of IC, having small dendritic field. Th+ type neurons are likely to be a distinct, homogenous group of GABAergic neuron in IC.

The superior colliculus (SC) is a layered structure in the midbrain and is particularly rich in gamma-aminobutyric acid (GABA). The present investigation aimed to determine whether the development of GABAergic neurons in the SC is common to that of the neocortex in which they are produced in a distinct area called the ganglionic eminence and are transported by tangential migration. A green fluorescent protein (GFP) knock-in mouse was used in which a GFP gene was introduced into the gene for glutamic acid decarboxylase (GAD) 67 and all GABAergic neurons were fluorescent. At embryonic day (E) 11-14, GFP-positive cells increased strikingly. They were spindle-shaped with processes at both poles and oriented radially between the ventricular and pial surface, together with other GFP-negative cells. After the cutting of the embryonic SC, GFP-positive cells accumulated on one side of the injury as expected from their radial but not tangential migration. In the living slice preparations GFP-positive cells migrated radially during the observation. These results indicate that tangential migration of GABAergic neurons as observed in the neocortex is not applicable and that radial migration from the underlying ventricular zone is predominant in the SC. At E12-13, bundles of commissural GFP-positive fibers which appeared to originate outside the SC were distributed at the superficial layer. These superficial fibers were no longer observed at the later stages.

Stellate and basket cells in the molecular layer (ML) of the cerebellar cortex proliferate within the white matter (WH) during development. Developmental neuronal death has been documented on granule cells but has not been demonstrated on other GABAergic neurons. We investigated the migration and the cell death of stellate/basket cells further in glutamic acid decarboxylase 67/green fluorescent protein (GFP) knock-in mouse in which every GABAergic neuron was identified by its GFP fluorescence. Analyses were made in the first three postnatal weeks. In the WM, GFP-positive cells were abundant on postnatal day (P) 5-15 but scarce in P21. Stellate/basket cells increased in number in the ML until P15, corresponding to the growth of the ML. Administration of 5-bromo-2'deoxyuridine (BrdU) at P2-8 labeled many cells in the WM within 1h. After BrdU administration at P5, many BrdU-labeled GFP-positive cells were observed in the WM and the internal granular layer at P7, and in the ML at P9. These results support the proliferation of stellate/basket cells in the WM and their migration to the ML. Apoptosis of GABAergic interneurons was demonstrated in the ML and WM during the first two weeks. Their apoptotic loss will contribute to the adjustment of neuron number or elimination of any improper populations.