Characterization and Stage-Dependent Lineage Analysis of Intermediate Progenitors of Cortical GABAergic Interneurons.

Intermediate progenitors of both excitatory and inhibitory neurons, which can replenish neurons in the adult brain, were recently identified. However, the generation of intermediate progenitors of GABAergic inhibitory neurons (IPGNs) has not been studied in detail. Here, we characterized the spatiotemporal distribution of IPGNs in mouse cerebral cortex. IPGNs generated neurons during both embryonic and postnatal stages, but the embryonic IPGNs were more proliferative. Our lineage tracing analyses showed that the embryonically proliferating IPGNs tended to localize to the superficial layers rather than the deep cortical layers at 3 weeks after birth. We also found that embryonic IPGNs derived from the medial and caudal ganglionic eminence (CGE) but more than half of the embryonic IPGNs were derived from the CGE and broadly distributed in the cerebral cortex. Taken together, our data indicate that the broadly located IPGNs during embryonic and postnatal stages exhibit a different proliferative property and layer distribution.

Two-Phase Lineage Specification of Telencephalon Progenitors Generated From Mouse Embryonic Stem Cells.

Proper brain development requires precisely controlled phases of stem cell proliferation, lineage specification, differentiation, and migration. Lineage specification depends partly on concentration gradients of chemical cues called morphogens. However, the rostral brain (telencephalon) expands prominently during embryonic development, dynamically altering local morphogen concentrations, and telencephalic subregional properties develop with a time lag. Here, we investigated how progenitor specification occurs under these spatiotemporally changing conditions using a three-dimensional in vitro differentiation model. We verified the critical contributions of three signaling factors for the lineage specification of subregional tissues in the telencephalon, ventralizing sonic hedgehog (Shh) and dorsalizing bone morphogenetic proteins (BMPs) and WNT proteins (WNTs). We observed that a short-lasting signal is sufficient to induce subregional progenitors and that the timing of signal exposure for efficient induction is specific to each lineage. Furthermore, early and late progenitors possess different Shh signal response capacities. This study reveals a novel developmental mechanism for telencephalon patterning that relies on the interplay of dose- and time-dependent signaling, including a time lag for specification and a temporal shift in cellular Shh sensitivity. This delayed fate choice through two-phase specification allows tissues with marked size expansion, such as the telencephalon, to compensate for the changing dynamics of morphogen signals.

Sequential pattern of sublayer formation in the paleocortex and neocortex.

The piriform cortex (paleocortex) is the olfactory cortex or the primary cortex for the sense of smell. It receives the olfactory input from the mitral and tufted cells of the olfactory bulb and is involved in the processing of information pertaining to odors. The piriform cortex and the adjoining neocortex have different cytoarchitectures; while the former has a three-layered structure, the latter has a six-layered structure. The regulatory mechanisms underlying the building of the six-layered neocortex are well established; in contrast, less is known about of the regulatory mechanisms responsible for structure formation of the piriform cortex. The differences as well as similarities in the regulatory mechanisms between the neocortex and the piriform cortex remain unclear. Here, the expression of neocortical layer-specific genes in the piriform cortex was examined. Two sublayers were found to be distinguished in layer II of the piriform cortex using Ctip2/Bcl11b and Brn1/Pou3f3. The sequential expression pattern of Ctip2 and Brn1 in the piriform cortex was similar to that detected in the neocortex, although the laminar arrangement in the piriform cortex exhibited an outside-in arrangement, unlike that observed in the neocortex.

Formation of dorsal-ventral axis of the pallium derived from mouse embryonic stem cells.

The telencephalon is one of the most-elaborated tissues. A broad variety of cell types is produced by spatiotemporally regulated mechanisms and is involved, in different combinations, in subregional formation. The dorsal half of the telencephalon, the pallium or cerebral cortex, is subdivided along the dorsal-ventral (D-V) axis into the medial, dorsal, lateral, and ventral pallium (MP, DP, LP and VP, respectively). An in vitro differentiation system has been achieved using mouse embryonic stem cells, and major telencephalic neurons can be obtained in this way; however, in using the in vitro differentiation system, many telencephalic neuron subtypes remain undifferentiated, although some of them are related to neuronal diseases. In the current study, we found that inhibiting the TGFbeta signal was efficient for neural induction. A continuous arrangement of Emx1+/Pax6-, Emx1+/Pax6+, and Emx1-/Pax6+ cells was achieved in Foxg1+ neuroepithelia, corresponding approximately to cortical progenitors derived from MP, DP/LP, and VP, respectively. A small portion of Dbx1+ cells resided in the VP fraction. These findings suggested that the D-V axis of the pallium was recapitulated in the in vitro-derived pallium.

Motor learning requires myelination to reduce asynchrony and spontaneity in neural activity.

Myelination increases the conduction velocity in long-range axons and is prerequisite for many brain functions. Impaired myelin regulation or impairment of myelin itself is frequently associated with deficits in learning and cognition in neurological and psychiatric disorders. However, it has not been revealed what perturbation of neural activity induced by myelin impairment causes learning deficits. Here, we measured neural activity in the motor cortex during motor learning in transgenic mice with a subtle impairment of their myelin. This deficit in myelin impaired motor learning, and was accompanied by a decrease in the amplitude of movement-related activity and an increase in the frequency of spontaneous activity. Thalamocortical axons showed variability in axonal conduction with a large spread in the timing of postsynaptic cortical responses. Repetitive pairing of forelimb movements with optogenetic stimulation of thalamocortical axon terminals restored motor learning. Thus, myelin regulation helps to maintain the synchrony of cortical spike-time arrivals through long-range axons, facilitating the propagation of the information required for learning. Our results revealed the pathological neuronal circuit activity with impaired myelin and suggest the possibility that pairing of noninvasive brain stimulation with relevant behaviors may ameliorate cognitive and behavioral abnormalities in diseases with impaired myelination.

Matrin-3 is essential for fibroblast growth factor 2-dependent maintenance of neural stem cells.

To investigate the mechanisms underlying the maintenance of neural stem cells, we performed two-dimensional fluorescence-difference gel electrophoresis (2D-DIGE) targeting the nuclear phosphorylated proteins. Nuclear phosphorylated protein Matrin-3 was identified in neural stem cells (NSCs) after stimulation using fibroblast growth factor 2 (FGF2). Matrin-3 was expressed in the mouse embryonic subventricular and ventricular zones. Small interfering RNA (siRNA)-mediated knockdown of Matrin-3 caused neuronal differentiation of NSCs in vitro, and altered the cerebral layer structure of foetal brain in vivo. Transfection of Matrin-3 plasmids in which the serine 208 residue was point-mutated to alanine (Ser208Ala mutant Matrin3) and inhibition of Ataxia telangiectasia mutated kinase (ATM kinase), which phosphorylates Matrin-3 Ser208 residue, caused neuronal differentiation and decreased the proliferation of neurosphere-forming stem cells. Thus, our proteomic approach revealed that Matrin-3 phosphorylation was essential for FGF2-dependent maintenance of NSCs in vitro and in vivo.

Inhibitory neuron-specific Cre-dependent red fluorescent labeling using VGAT BAC-based transgenic mouse lines with identified transgene integration sites.

Inhibitory neurons are crucial for shaping and regulating the dynamics of the entire network, and disturbances in these neurons contribute to brain disorders. Despite the recent progress in genetic labeling techniques, the heterogeneity of inhibitory neurons requires the development of highly characterized tools that allow accurate, convenient, and versatile visualization of inhibitory neurons in the mouse brain. Here, we report a novel genetic technique to visualize the vast majority and/or sparse subsets of inhibitory neurons in the mouse brain without using techniques that require advanced skills. We developed several lines of Cre-dependent tdTomato reporter mice based on the vesicular GABA transporter (VGAT)-BAC, named VGAT-stop-tdTomato mice. The most useful line (line #54) was selected for further analysis based on two characteristics: the inhibitory neuron-specificity of tdTomato expression and the transgene integration site, which confers efficient breeding and fewer adverse effects resulting from transgene integration-related genomic disruption. Robust and inhibitory neuron-specific expression of tdTomato was observed in a wide range of developmental and cellular contexts. By breeding the VGAT-stop-tdTomato mouse (line #54) with a novel Cre driver mouse line, Galntl4-CreER, sparse labeling of inhibitory neurons was achieved following tamoxifen administration. Furthermore, another interesting line (line #58) was generated through the unexpected integration of the transgene into the X-chromosome and will be used to map X-chromosome inactivation of inhibitory neurons. Taken together, our studies provide new, well-characterized tools with which multiple aspects of inhibitory neurons can be studied in the mouse.

Autonomous and non-autonomous roles for ephrin-B in interneuron migration.

While several studies indicate the importance of ephrin-B/EphB bidirectional signaling in excitatory neurons, potential roles for these molecules in inhibitory neurons are largely unknown. We identify here an autonomous receptor-like role for ephrin-B reverse signaling in the tangential migration of interneurons into the neocortex using ephrin-B (EfnB1/B2/B3) conditional triple mutant (TMlz) mice and a forebrain inhibitory neuron specific Cre driver. Inhibitory neuron deletion of the three EfnB genes leads to reduced interneuron migration, abnormal cortical excitability, and lethal audiogenic seizures. Truncated and intracellular point mutations confirm the importance of ephrin-B reverse signaling in interneuron migration and cortical excitability. A non-autonomous ligand-like role was also identified for ephrin-B2 that is expressed in neocortical radial glial cells and required for proper tangential migration of GAD65-positive interneurons. Our studies thus define both receptor-like and ligand-like roles for the ephrin-B molecules in controlling the migration of interneurons as they populate the neocortex and help establish excitatory/inhibitory (E/I) homeostasis.

Targeted Interneuron Depletion in the Dorsal Striatum Produces Autism-like Behavioral Abnormalities in Male but Not Female Mice.

BACKGROUND: Interneuronal pathology is implicated in many neuropsychiatric disorders, including autism spectrum disorder (ASD) and Tourette syndrome (TS). Interneurons of the striatum, including the parvalbumin-expressing fast-spiking interneurons (FSIs) and the large cholinergic interneurons (CINs), are affected in patients with TS and in preclinical models of both ASD and TS. METHODS: To test the causal importance of these neuronal abnormalities, we recapitulated them in vivo in developmentally normal mice using a combination transgenic-viral strategy for targeted toxin-mediated ablation. RESULTS: We found that conjoint ~50% depletion of FSIs and CINs in the dorsal striatum of male mice produces spontaneous stereotypy and marked deficits in social interaction. Strikingly, these behavioral effects are not seen in female mice; because ASD and TS have a marked male predominance, this observation reinforces the potential relevance of the finding to human disease. Neither of these effects is seen when only one or the other interneuronal population is depleted; ablation of both is required. Depletion of FSIs, but not of CINs, also produces anxiety-like behavior, as has been described previously. Behavioral pathology in male mice after conjoint FSI and CIN depletion is accompanied by increases in activity-dependent signaling in the dorsal striatum; these alterations were not observed after disruption of only one interneuron type or in doubly depleted female mice. CONCLUSIONS: These data indicate that disruption of CIN and FSI interneurons in the dorsal striatum is sufficient to produce network and behavioral changes of potential relevance to ASD, in a sexually dimorphic manner.

Neural stem cells and neuro/gliogenesis in the central nervous system: understanding the structural and functional plasticity of the developing, mature, and diseased brain.

Neurons and glia in the central nervous system (CNS) originate from neural stem cells (NSCs). Knowledge of the mechanisms of neuro/gliogenesis from NSCs is fundamental to our understanding of how complex brain architecture and function develop. NSCs are present not only in the developing brain but also in the mature brain in adults. Adult neurogenesis likely provides remarkable plasticity to the mature brain. In addition, recent progress in basic research in mental disorders suggests an etiological link with impaired neuro/gliogenesis in particular brain regions. Here, we review the recent progress and discuss future directions in stem cell and neuro/gliogenesis biology by introducing several topics presented at a joint meeting of the Japanese Association of Anatomists and the Physiological Society of Japan in 2015. Collectively, these topics indicated that neuro/gliogenesis from NSCs is a common event occurring in many brain regions at various ages in animals. Given that significant structural and functional changes in cells and neural networks are accompanied by neuro/gliogenesis from NSCs and the integration of newly generated cells into the network, stem cell and neuro/gliogenesis biology provides a good platform from which to develop an integrated understanding of the structural and functional plasticity that underlies the development of the CNS, its remodeling in adulthood, and the recovery from diseases that affect it.

Altered proliferative ability of neuronal progenitors in PlexinA1 mutant mice.

Cortical interneurons are generated predominantly in the medial ganglionic eminence (MGE) and migrate through the ventral and dorsal telencephalon before taking their final positions within the developing cortical plate. Previously we demonstrated that interneurons from Robo1 knockout (Robo1(-/-)) mice contain reduced levels of neuropilin 1 (Nrp1) and PlexinA1 receptors, rendering them less responsive to the chemorepulsive actions of semaphorin ligands expressed in the striatum and affecting their course of migration (Hernandez-Miranda et al. [2011] J. Neurosci. 31:6174-6187). Earlier studies have highlighted the importance of Nrp1 and Nrp2 in interneuron migration, and here we assess the role of PlexinA1 in this process. We observed significantly fewer cells expressing the interneuron markers Gad67 and Lhx6 in the cortex of PlexinA1(-/-) mice compared with wild-type littermates at E14.5 and E18.5. Although the level of apoptosis was similar in the mutant and control forebrain, proliferation was significantly reduced in the former. Furthermore, progenitor cells in the MGE of PlexinA1(-/-) mice appeared to be poorly anchored to the ventricular surface and showed reduced adhesive properties, which may account for the observed reduction in proliferation. Together our data uncover a novel role for PlexinA1 in forebrain development.

Glutamate Decarboxylase 67 Deficiency in a Subset of GABAergic Neurons Induces Schizophrenia-Related Phenotypes.

Decreased expression of the GABA synthetic enzyme glutamate decarboxylase 67 (GAD67) in a subset of GABAergic neurons, including parvalbumin (PV)-expressing neurons, has been observed in postmortem brain studies of schizophrenics and in animal models of schizophrenia. However, it is unclear whether and how the perturbations of GAD67-mediated GABA synthesis and signaling contribute to the pathogenesis of schizophrenia. To address this issue, we generated the mice lacking GAD67 primarily in PV neurons and characterized them with focus on schizophrenia-related parameters. We found that heterozygous mutant mice exhibited schizophrenia-related behavioral abnormalities such as deficits in prepulse inhibition, MK-801 sensitivity, and social memory. Furthermore, we observed reduced inhibitory synaptic transmission, altered properties of NMDA receptor-mediated synaptic responses in pyramidal neurons, and increased spine density in hippocampal CA1 apical dendrites, suggesting a possible link between GAD67 deficiency and disturbed glutamatergic excitatory synaptic functions in schizophrenia. Thus, our results indicate that the mice heterozygous for GAD67 deficiency primarily in PV neurons share several neurochemical and behavioral abnormalities with schizophrenia, offering a novel tool for addressing the underlying pathophysiology of schizophrenia.

Specification of select hypothalamic circuits and innate behaviors by the embryonic patterning gene dbx1.

The hypothalamus integrates information required for the production of a variety of innate behaviors such as feeding, mating, aggression, and predator avoidance. Despite an extensive knowledge of hypothalamic function, how embryonic genetic programs specify circuits that regulate these behaviors remains unknown. Here, we find that in the hypothalamus the developmentally regulated homeodomain-containing transcription factor Dbx1 is required for the generation of specific subclasses of neurons within the lateral hypothalamic area/zona incerta (LH) and the arcuate (Arc) nucleus. Consistent with this specific developmental role, Dbx1 hypothalamic-specific conditional-knockout mice display attenuated responses to predator odor and feeding stressors but do not display deficits in other innate behaviors such as mating or conspecific aggression. Thus, activity of a single developmentally regulated gene, Dbx1, is a shared requirement for the specification of hypothalamic nuclei governing a subset of innate behaviors. VIDEO ABSTRACT.

Contribution of parvalbumin and somatostatin-expressing GABAergic neurons to slow oscillations and the balance in beta-gamma oscillations across cortical layers.

Cortical interneurons are classified into several subtypes that contribute to cortical oscillatory activity. Parvalbumin (PV)-expressing cells, a type of inhibitory interneuron, are involved in the gamma oscillations of local field potentials (LFPs). Under ketamine-xylazine anesthesia or sleep, mammalian cortical circuits exhibit slow oscillations in which the active-up state and silent-down state alternate at ~1 Hz. The up state is composed of various high-frequency oscillations, including gamma oscillations. However, it is unclear how PV cells and somatostatin (SOM) cells contribute to the slow oscillations and the high-frequency oscillations nested in the up state. To address these questions, we used mice lacking glutamate decarboxylase 67, primarily in PV cells (PV-GAD67 mice) or in SOM cells (SOM-GAD67 mice). We then compared LFPs between PV-GAD67 mice and SOM-GAD67 mice. PV cells target the proximal regions of pyramidal cells, whereas SOM cells are dendrite-preferring interneurons. We found that the up state was shortened in duration in the PV-GAD67 mice, but tended to be longer in SOM-GAD67 mice. Firing rate tended to increase in PV-GAD67 mice, but tended to decrease in SOM-GAD67 mice. We also found that delta oscillations tended to increase in SOM-GAD67 mice, but tended to decrease in PV-GAD67 mice. Current source density and wavelet analyses were performed to determine the depth profiles of various high-frequency oscillations. High gamma and ripple (60-200 Hz) power decreased in the neocortical upper layers specifically in PV-GAD67 mice, but not in SOM-GAD67. In addition, beta power (15-30 Hz) increased in the deep layers, specifically in PV-GAD67 mice. These results suggest that PV cells play important roles in persistence of the up state and in the balance between gamma and beta bands across cortical layers, whereas SOM and PV cells may make an asymmetric contribution to regulate up-state and delta oscillations.

Birthdate study of GABAergic neurons in the lumbar spinal cord of the glutamic acid decarboxylase 67-green fluorescent protein knock-in mouse.

Despite the abundance of studies on γ-aminobutyric acid (GABA) ergic neuron distribution in the mouse developing spinal cord, no investigation has been devoted so far to their birthdates. In order to determine the spinal neurogenesis of a specific phenotype, the GABAergic neurons in the spinal cord, we injected bromodeoxyuridine (BrdU) at different developmental stages of the glutamic acid decarboxylase (GAD)67-green fluorescent protein (GFP) knock-in mice. We thus used GFP to mark GABAergic neurons and labeled newly born cells with the S-phase marker BrdU at different embryonic stages. Distribution of GABAergic neurons labeled with BrdU was then studied in spinal cord sections of 60-day-old mice. Our birthdating studies revealed that GABAergic neurogenesis was present at embryonic day 10.5 (E10.5). Since then, the generation of GABAergic neurons significantly increased, and reached a peak at E11.5. Two waves for the co-localization of GABA and BrdU in the spinal cord were seen at E11.5 and E13.5 in the present study. The vast majority of GABAergic neurons were generated before E14.5. Thereafter, GABA-positive neuron generation decreased drastically. The present results showed that the birthdates of GABAergic neurons in each lamina were different. The peaks of GABAergic neurogenesis in lamina II were at E11.5 and E13.5, while in lamina I and III, they were at E13.5 and E12.5, respectively. The present results suggest that the birthdates of GABAergic neurons vary in different lamina and follow a specific temporal sequence. This will provide valuable information for future functional studies.

Nav1.1 haploinsufficiency in excitatory neurons ameliorates seizure-associated sudden death in a mouse model of Dravet syndrome.

Dravet syndrome is a severe epileptic encephalopathy mainly caused by heterozygous mutations in the SCN1A gene encoding a voltage-gated sodium channel Nav1.1. We previously reported dense localization of Nav1.1 in parvalbumin (PV)-positive inhibitory interneurons in mice and abnormal firing of those neurons in Nav1.1-deficient mice. In the present study, we investigated the physiologic consequence of selective Nav1.1 deletion in mouse global inhibitory neurons, forebrain excitatory neurons or PV cells, using vesicular GABA transporter (VGAT)-Cre, empty spiracles homolog 1 (Emx1)-Cre or PV-Cre recombinase drivers. We show that selective Nav1.1 deletion using VGAT-Cre causes epileptic seizures and premature death that are unexpectedly more severe than those observed in constitutive Nav1.1-deficient mice. Nav1.1 deletion using Emx1-Cre does not cause any noticeable abnormalities in mice; however, the severe lethality observed with VGAT-Cre-driven Nav1.1 deletion is rescued by additional Nav1.1 deletion using Emx1-Cre. In addition to predominant expression in PV interneurons, we detected Nav1.1 in subpopulations of excitatory neurons, including entorhino-hippocampal projection neurons, a subpopulation of neocortical layer V excitatory neurons, and thalamo-cortical projection neurons. We further show that even minimal selective Nav1.1 deletion, using PV-Cre, is sufficient to cause spontaneous epileptic seizures and ataxia in mice. Overall, our results indicate that functional impairment of PV inhibitory neurons with Nav1.1 haploinsufficiency contributes to the epileptic pathology of Dravet syndrome, and show for the first time that Nav1.1 haploinsufficiency in excitatory neurons has an ameliorating effect on the pathology.

Voltage drives diverse endocannabinoid signals to mediate striatal microcircuit-specific plasticity.

The dorsolateral striatum and cannabinoid type 1 receptor (CB1) signaling mediate habitual action learning, which is thought to require a balance of activity in the direct and indirect striatal output pathways. However, very little is known about how the high CB1-expressing striatal inhibitory microcircuitry might contribute to long-term plasticity capable of sculpting direct and indirect pathway output. Using optogenetic and molecular interrogation of striatal GABAergic microcircuits, we examined voltage-dependent long-term depression of inhibitory synapses (iLTD) onto mouse and rat medium spiny projection neurons (MSNs). The observed iLTD involved recruitment of different endocannabinoid types and showed both presynaptic and postsynaptic selectivity for MSN subtypes, ultimately resulting in a powerful disinhibition of direct pathway MSNs. These results suggest a new role for voltage states in gating circuit-specific forms of synaptic plasticity and illuminate possible circuit dynamics underlying action control.

Limk2 mediates semaphorin signalling in cortical interneurons migrating through the subpallium.

En route to the neocortex, interneurons migrate around and avoid the developing striatum. This is due to the chemorepulsive cues of class 3 semaphorins (Sema3A and Sema3F) acting through neuropilin and plexin co-receptors expressed in interneurons. In a recent genetic screen aimed at identifying novel components that may play a role in interneuron migration, we identified LIM-kinase 2 (Limk2), a kinase previously shown to be involved in cell movement and in Sema7A-PlexinC1 signalling. Here we show that Limk2 is differentially expressed in interneurons, with a higher expression in the subpallium compared to cortex, suggesting it may play a role in their migration through the subpallium. Chemotactic assays, carried out with small interfering RNAs (siRNAs), revealed that Limk2-siRNA transfected interneurons are less responsive to Sema3A, but respond to Sema3F. Lack of responsiveness to Sema3A resulted in their aberrant invasion of the developing striatum, as demonstrated in brain slice preparations and in in utero electroporated mouse embryos with the same siRNAs. Our results reveal a previously unknown role for Limk2 in interneuron migration and Sema3A signalling.

Amygdala kindling induces nestin expression in the leptomeninges of the neocortex.

Nestin is an intermediate filament found in neurogenic progenitors and non-neuronal cells. Nestin-immunoreactivity (IR) in the brain often increases after brain damage. Here we show that amygdala kindling, which mimics the epileptic seizures, also induces nestin expression in the brain. Nestin-IR was greatly enhanced in the leptomeninges (pia and arachnoid maters) and neocortical parenchyma, but not much in the SVZ around the lateral ventricle, SGZ in the dentate gyrus, or the endothelial progenitor cells of blood vessels, fimbria, or choroid plexus after kindling. Electron microscopy revealed that nestin-IR in the leptomeninges was localized to granule cells, where it co-localized with GAD67-IR after electrical stimulation. The nestin-positive granule cells in the leptomeninges, especially around the emissary vein, were proliferative. However, nestin-IR in the neocortical parenchyma was expressed in NG2 glia and did not co-localize with GAD67-IR. Deletion of nestin-positive cells resulted in a high susceptibility to electrical stimulation. Consequently, almost all of the mice died or dropped out during kindling progression in 20 days, from naturally generated epileptic seizure or exhaustion. We speculate that the nestin-positive cells activated by amygdala kindling may involve in the protection of the brain from epilepsy.

Visualization of odor-induced neuronal activity by immediate early gene expression.

BACKGROUND: Sensitive detection of sensory-evoked neuronal activation is a key to mechanistic understanding of brain functions. Since immediate early genes (IEGs) are readily induced in the brain by environmental changes, tracing IEG expression provides a convenient tool to identify brain activity. In this study we used in situ hybridization to detect odor-evoked induction of ten IEGs in the mouse olfactory system. We then analyzed IEG induction in the cyclic nucleotide-gated channel subunit A2 (Cnga2)-null mice to visualize residual neuronal activity following odorant exposure since CNGA2 is a key component of the olfactory signal transduction pathway in the main olfactory system. RESULTS: We observed rapid induction of as many as ten IEGs in the mouse olfactory bulb (OB) after olfactory stimulation by a non-biological odorant amyl acetate. A robust increase in expression of several IEGs like c-fos and Egr1 was evident in the glomerular layer, the mitral/tufted cell layer and the granule cell layer. Additionally, the neuronal IEG Npas4 showed steep induction from a very low basal expression level predominantly in the granule cell layer. In Cnga2-null mice, which are usually anosmic and sexually unresponsive, glomerular activation was insignificant in response to either ambient odorants or female stimuli. However, a subtle induction of c-fos took place in the OB of a few Cnga2-mutants which exhibited sexual arousal. Interestingly, very strong glomerular activation was observed in the OB of Cnga2-null male mice after stimulation with either the neutral odor amyl acetate or the predator odor 2, 3, 5-trimethyl-3-thiazoline (TMT). CONCLUSIONS: This study shows for the first time that in vivo olfactory stimulation can robustly induce the neuronal IEG Npas4 in the mouse OB and confirms the odor-evoked induction of a number of IEGs. As shown in previous studies, our results indicate that a CNGA2-independent signaling pathway(s) may activate the olfactory circuit in Cnga2-null mice and that neuronal activation which correlates to behavioral difference in individual mice is detectable by in situ hybridization of IEGs. Thus, the in situ hybridization probe set we established for IEG tracing can be very useful to visualize neuronal activity at the cellular level.