Katsuyasu Sakurai

Fabp7 maps to a quantitative trait locus for a schizophrenia endophenotype.

Deficits in prepulse inhibition (PPI) are a biological marker for schizophrenia. To unravel the mechanisms that control PPI, we performed quantitative trait loci (QTL) analysis on 1,010 F2 mice derived by crossing C57BL/6 (B6) animals that show high PPI with C3H/He (C3) animals that show low PPI. We detected six major loci for PPI, six for the acoustic startle response, and four for latency to response peak, some of which were sex-dependent. A promising candidate on the Chromosome 10-QTL was Fabp7 (fatty acid binding protein 7, brain), a gene with functional links to the N-methyl-D-aspartic acid (NMDA) receptor and expression in astrocytes. Fabp7-deficient mice showed decreased PPI and a shortened startle response latency, typical of the QTLs proposed effects. A quantitative complementation test supported Fabp7 as a potential PPI-QTL gene, particularly in male mice. Disruption of Fabp7 attenuated neurogenesis in vivo. Human FABP7 showed altered expression in schizophrenic brains and genetic association with schizophrenia, which were both evident in males when samples were divided by sex. These results suggest that FABP7 plays a novel and crucial role, linking the NMDA, neurodevelopmental, and glial theories of schizophrenia pathology and the PPI endophenotype, with larger or overt effects in males. We also discuss the results from the perspective of fetal programming.

The Neurogenesis-Controlling Factor, Pax6, Inhibits Proliferation and Promotes Maturation in Murine Astrocytes

Astrocytes serve various important functions in the CNS, but the molecular mechanisms of their generation and maturation are still enigmatic. Here, we show that Pax6, a key transcription factor that controls neurogenesis, also regulates proliferation, differentiation, and migration of astrocytes in the CNS. We first reveal that Pax6 is expressed in astrocytes during development as well as postnatally in the wild-type mouse. Astrocytes derived from Pax6 homozygous mutants (Sey/Sey) mice exhibited aberrant proliferation together with immature differentiation, both in vivo and in vitro, with higher migration potential in scratch-wound assays in vitro. Furthermore, a larger population of Sey/Sey astrocytes expresses neural stem cell markers such as nestin, Sox2, and prominin-1. These phenotypes of Pax6-deficient astrocytes putatively occur via higher Akt activity. Thus, the breakdown of Pax6 function induces the retention of neural stem-like characteristics and inhibits astrocyte maturation.

A Circuit for Motor Cortical Modulation of Auditory Cortical Activity

Normal hearing depends on the ability to distinguish self-generated sounds from other sounds, and this ability is thought to involve neural circuits that convey copies of motor command signals to various levels of the auditory system. Although such interactions at the cortical level are believed to facilitate auditory comprehension during movements and drive auditory hallucinations in pathological states, the synaptic organization and function of circuitry linking the motor and auditory cortices remain unclear. Here we describe experiments in the mouse that characterize circuitry well suited to transmit motor-related signals to the auditory cortex. Using retrograde viral tracing, we established that neurons in superficial and deep layers of the medial agranular motor cortex (M2) project directly to the auditory cortex and that the axons of some of these deep-layer cells also target brainstem motor regions. Using in vitro whole-cell physiology, optogenetics, and pharmacology, we determined that M2 axons make excitatory synapses in the auditory cortex but exert a primarily suppressive effect on auditory cortical neuron activity mediated in part by feedforward inhibition involving parvalbumin-positive interneurons. Using in vivo intracellular physiology, optogenetics, and sound playback, we also found that directly activating M2 axon terminals in the auditory cortex suppresses spontaneous and stimulus-evoked synaptic activity in auditory cortical neurons and that this effect depends on the relative timing of motor cortical activity and auditory stimulation. These experiments delineate the structural and functional properties of a corticocortical circuit that could enable movement-related suppression of auditory cortical activity.

Intersectional Cre Driver Lines Generated Using Split-Intein Mediated Split-Cre Reconstitution

Tissue and cell type highly specific Cre drivers are very rare due to the fact that most genes or promoters used to direct Cre expressions are generally expressed in more than one tissues and/or in multiple cell types. We developed a split-intein based split-Cre system for highly efficient Cre-reconstitution through protein splicing. This split-intein-split-Cre system can be used to intersect the expression patterns of two genes or promoters to restrict full-length Cre reconstitution in their overlapping domains. To test this system in vivo, we selected several conserved human enhancers to drive the expression of either Cre-N-intein-N, or intein-C-Cre-C transgene in different brain regions. In all paired CreN/CreC transgenic mice, Cre-dependent reporter was efficiently induced specifically in the intersectional expression domains of two enhancers. This split-intein based method is simpler to implement compared with other strategies for generating highly-restricted intersectional Cre drivers to study complex tissues such as the nervous system.

Transcription Factor Short Stature Homeobox 2 Is Required for Proper Development of Tropomyosin-Related Kinase B-Expressing Mechanosensory Neurons

Dorsal root ganglia (DRG) contain somatosensory neurons of diverse sensory modalities. Among these different types of sensory neurons, the molecular mechanisms that regulate the development and specification of touch neurons are the least well understood. We took a candidate approach and searched for transcription factors that are expressed in subsets of DRG neurons, and found that the transcription factor Shox2 (short stature homeobox 2) is expressed in subpopulations of TrkB (tropomyosin-related kinase B)- and Ret-expressing neurons at neonatal stages. Since TrkB is a known marker that is selectively expressed in touch sensory neurons, we decided to examine the function of Shox2 in specifying TrkB-positive DRG neurons. Conditional deletion of Shox2 in neural crest cells (which give rise to all DRG neurons) caused a 60 ∼ 65% reduction in the number of TrkB-expressing neurons. It also resulted in an increase in coexpression of TrkC in Ret-positive sensory neurons. Deletion of Shox2 in differentiating DRG neurons at later time points caused only a moderate reduction in TrkB expression. Overexpression of Shox2 in all neural crest cells resulted in a small increase in the number of TrkB-expressing neurons. Finally, Shox2 deletion also caused reduced touch sensory axonal innervation to layers III/IV of the spinal cord. Together, our findings identify Shox2 as an essential but not sufficient component of the transcription programs required in neural progenitor cells for the proper specification of subsets of TrkB-expressing touch/mechanosensory neurons.

Capturing and Manipulating Activated Neuronal Ensembles with CANE Delineates a Hypothalamic Social-Fear Circuit

We developed a technology (capturing activated neuronal ensembles [CANE]) to label, manipulate, and transsynaptically trace neural circuits that are transiently activated in behavioral contexts with high efficiency and temporal precision. CANE consists of a knockin mouse and engineered viruses designed to specifically infect activated neurons. Using CANE, we selectively labeled neurons that were activated by either fearful or aggressive social encounters in a hypothalamic subnucleus previously known as a locus for aggression, and discovered that social-fear and aggression neurons are intermixed but largely distinct. Optogenetic stimulation of CANE-captured social-fear neurons (SFNs) is sufficient to evoke fear-like behaviors in normal social contexts, whereas silencing SFNs resulted in reduced social avoidance. CANE-based mapping of axonal projections and presynaptic inputs to SFNs further revealed a highly distributed and recurrent neural network. CANE is a broadly applicable technology for dissecting causality and connectivity of spatially intermingled but functionally distinct ensembles.

Structure and chemical organization of the accessory olfactory bulb in the goat.

The structure and chemical composition of the accessory olfactory bulb (AOB) were examined in male and female goats. Sections were subjected to either Nissl staining, Klüver‐Barrera staining, lectin histochemistry, or immunohistochemistry for nitric oxide synthase (NOS), neuropeptide Y (NPY), tyrosine hydroxylase (TH), dopamine β‐hydroxylase (DBH), and glutamic acid decarboxylase (GAD). The goat AOB was divided into four layers: the vomeronasal nerve layer (VNL), glomerular layer (GL), mitral/tufted (M/T) cell layer (MTL), and granule cell layer (GRL). Quantitative and morphometric analyses indicated that a single AOB contained 5,000–8,000 putative M/T cells with no sex differences, whereas the AOB was slightly larger in males. Of the 21 lectins examined, 7 specifically bound to the VNL and GL, and 1 bound not only to the VNL, but also to the MTL and GRL. In either of these cases, no heterogeneity of lectin staining was observed in the rostrocaudal direction. NOS‐, TH‐, DBH‐, and GAD‐immunoreactivity (ir) were observed in the MTL and GRL, whereas NPY‐ir was present only in the GRL. In the GL, periglomerular cells with GAD‐ir were found in abundance, and a subset of periglomerular cells containing TH‐ir was also found. Double‐labeling immunohistochemistry revealed that virtually all periglomerular cells containing TH‐ir were colocalized with GAD‐ir. Anat Rec, 2007.

Activation of melanocortin receptors accelerates the gonadotropin-releasing hormone pulse generator activity in goats

The present study aims to elucidate whether the central melanocortin receptors [melanocortin-3 and -4 receptors (MC3/4-R)] are involved in regulating GnRH pulse generator activity in female goats. The GnRH pulse generator activity was electrophysiologically assessed at the intervals of characteristic increases in multiple-unit activity (MUA volleys) in the mediobasal hypothalamus. In ovariectomized goats, all doses (0.02, 0.2 and 2 nmol) of MT II, an MC3/4-R agonist, injected into the lateral ventricle significantly shortened MUA volley intervals. The duration of the period during which MT II accelerated MUA volleys was positively correlated with the dose of MT II injected. The stimulatory effect of MT II on the GnRH pulse generator activity was attenuated in the presence of estrogen. Intracerebroventricular injection of SHU9119, an MC3/4-R antagonist, significantly prolonged MUA volley intervals at 1 nmol. MT II (0.2 nmol)-induced acceleration of MUA volleys was partially blocked by the antagonism of MC3/4-R with pre-administered SHU9119 (1 nmol). The present findings demonstrate that MC3/4-R are involved in maintaining GnRH pulse generator activity in goats.

A craniofacial-specific monosynaptic circuit enables heightened affective pain

Humans often rank craniofacial pain as more severe than body pain. Evidence suggests that a stimulus of the same intensity induces stronger pain in the face than in the body. However, the underlying neural circuitry for the differential processing of facial versus bodily pain remains unknown. Interestingly, the lateral parabrachial nucleus (PBL), a critical node in the affective pain circuit, is activated more strongly by noxious stimulation of the face than of the hindpaw. Using a novel activity-dependent technology called CANE developed in our laboratory, we identified and selectively labeled noxious-stimulus-activated PBL neurons and performed comprehensive anatomical input–output mapping. Surprisingly, we uncovered a hitherto uncharacterized monosynaptic connection between cranial sensory neurons and the PBL-nociceptive neurons. Optogenetic activation of this monosynaptic craniofacial-to-PBL projection induced robust escape and avoidance behaviors and stress calls, whereas optogenetic silencing specifically reduced facial nociception. The monosynaptic circuit revealed here provides a neural substrate for heightened craniofacial affective pain.The authors show that unlike body sensory neurons, craniofacial nociceptive neurons directly synapse with noxious-stimulus-activated lateral parabrachial neurons (PBL), which in turn project to multiple limbic centers processing emotions and affects. This monosynaptic pathway is both sufficient and necessary for craniofacial-pain-activated aversive behaviors.

Identification of midbrain neurons essential for vocal communication

Vocalizations are an essential medium for communication and courtship in numerous mammalian species ranging from mice to humans. In mammals, the midbrain PAG serves as an obligatory node in a vocalization-related network that spans the forebrain and brainstem1–3, as bilateral lesions of the PAG result in mutism2–5. Despite the PAG’s importance for vocal production, the identity, function, and connectivity of PAG neurons involved in vocalization has remained elusive, in part because the PAG is a functionally and anatomically heterogeneous structure that serves myriad roles including nociception, defensive behaviors, and autonomic regulation6–9. Here we used a viral genetic “tagging” method10,11 to identify a distinct subset of PAG neurons in the male mouse that are selectively activated during the production of ultrasonic vocalizations (USVs) elicited by female cues. Silencing these PAG-USV neurons rendered males mute without affecting their other courtship behaviors and also impaired their ability to attract female mice in a social choice assay. Activating these neurons using chemogenetic or optogenetic methods strongly elevated USV production, even in the absence of female cues. Notably, the timing of individual USVs was entrained to the expiratory phase of breathing but not to the pattern of optogenetic stimulation, suggesting that PAG-USV neural activity initiates and sets the duration of vocal bouts and recruits downstream premotor circuits that precisely pattern vocal output. Consistent with this idea, we found that PAG-USV neurons extend axons into pontine and medullary regions that are speculated to contain premotor central pattern generators important for vocalization3,12,13. These experiments establish the identity of the PAG neurons selectively required for USV production in mice, map their efferent connections, and demonstrate the communicative salience of male USVs in promoting female social affiliation.