Cerebellar Nuclei Receiving Orofacial Proprioceptive Signals through the Mossy Fiber Pathway from the Supratrigeminal Nucleus in Rats.

Proprioception from muscle spindles is necessary for motor function executed by the cerebellum. In particular, cerebellar nuclear neurons that receive proprioceptive signals and send projections to the lower brainstem or spinal cord play key roles in motor control. However, little is known about which cerebellar nuclear regions receive orofacial proprioception. Here, we investigated projections to the cerebellar nuclei from the supratrigeminal nucleus (Su5), which conveys the orofacial proprioception arising from jaw-closing muscle spindles (JCMSs). Injections of an anterograde tracer into the Su5 resulted in a large number of labeled axon terminals bilaterally in the dorsolateral hump (IntDL) of the cerebellar interposed nucleus (Int) and the dorsolateral protuberance (MedDL) of the cerebellar medial nucleus. In addition, a moderate number of axon terminals were ipsilaterally labeled in the vestibular group Y nucleus (group Y). We electrophysiologically detected JCMS proprioceptive signals in the IntDL and MedDL. Retrograde tracing analysis confirmed bilateral projections from the Su5 to the IntDL and MedDL. Furthermore, anterograde tracer injections into the external cuneate nucleus (ECu), which receives other proprioceptive input from forelimb/neck muscles, resulted in only a limited number of ipsilaterally labeled terminals, mainly in the dorsomedial crest of the Int and the group Y. Taken together, the Su5 and ECu axons almost separately terminated in the cerebellar nuclei (except for partial overlap in the group Y). These data suggest that orofacial proprioception is differently processed in the cerebellar circuits in comparison to other body-part proprioception, thus contributing to the executive function of orofacial motor control.

Ipsi- and contralateral corticocortical projection-dependent subcircuits in layer 2 of the rat frontal cortex.

In the neocortex, both layer 2/3 and layer 5 contain corticocortical pyramidal cells projecting to other cortices. We previously found that among L5 pyramidal cells of the secondary motor cortex (M2), not only intratelencephalic projection cells but also pyramidal tract cells innervate ipsilateral cortices and that the two subtypes are different in corticocortical projection diversity and axonal laminar distributions. Layer 2/3 houses intratelencephalically projecting pyramidal cells that also innervate multiple ipsilateral and contralateral cortices. However, it remained unclear whether layer 2/3 pyramidal cells can be divided into projection subtypes each with distinct innervation to specific targets. In the present study we show that layer 2 pyramidal cells are organized into subcircuits on the basis of corticocortical projection targets. Layer 2 corticocortical cells of the same projection subtype were monosynaptically connected. Between the contralaterally and ipsilaterally projecting corticocortical cells, the monosynaptic connection was more common from the former to the latter. We also found that ipsilaterally and contralaterally projecting corticocortical cell subtypes differed in their morphological and physiological characteristics. Our results suggest that layer 2 transfers separate outputs from M2 to individual cortices and that its subcircuits are hierarchically organized to form the discrete corticocortical outputs.NEW & NOTEWORTHY Pyramidal cell subtypes and their dependent subcircuits are well characterized in cortical layer 5, but much less is understood for layer 2/3. We demonstrate that in layer 2 of the rat secondary motor cortex, ipsilaterally and contralaterally projecting corticocortical cells are largely segregated. These layer 2 cell subtypes differ in dendrite morphological and intrinsic electrophysiological properties, and form subtype-dependent connections. Our results suggest that layer 2 pyramidal cells form distinct subcircuits to provide discrete corticocortical outputs.

Cell type-specific inhibitory inputs to dendritic and somatic compartments of parvalbumin-expressing neocortical interneuron.

Parvalbumin (PV)-producing fast-spiking neurons are well known to generate gamma oscillation by mutual chemical and electrical connections in the neocortex. Although it was clearly demonstrated that PV neurons form a dense gap junction network with each other not only at the proximal sites but also at the distal dendrites, comprehensive quantitative data on the chemical connections are still lacking. To elucidate the connectivity, we investigated inhibitory inputs to PV neurons in the somatosensory cortex, using the transgenic mice in which the dendrites and cell bodies of PV neurons were clearly visualized. We first examined GABAergic inputs to PV neurons by labeling postsynaptic and presynaptic sites with the immunoreactivities for gephyrin and vesicular GABA transporter. The density of GABAergic inputs was highest on the cell bodies, and almost linearly decreased to the distal dendrites. We then investigated inhibitory inputs from three distinct subgroups of GABAergic interneurons by visualizing the axon terminals immunopositive for PV, somatostatin (SOM), or vasoactive intestinal polypeptide (VIP). PV and SOM inputs were frequently located on the dendrites with the ratio of 2.5:1, but much less on the cell bodies. By contrast, VIP inputs clearly preferred the cell bodies to the dendrites. Consequently, the dendritic and somatic compartments of PV neurons received ∼60 and 62% of inhibitory inputs from PV and VIP neurons, respectively. This compartmental organization of inhibitory inputs suggests that PV neurons, together with gap junctions, constitute mutual connections at the dendrites, and that their activities are negatively controlled by the somatic inputs of VIP neurons.

Synaptic configuration and reconfiguration in the neocortex are spatiotemporally selective.

Brain computation relies on the neural networks. Neurons extend the neurites such as dendrites and axons, and the contacts of these neurites that form chemical synapses are the biological basis of signal transmissions in the central nervous system. Individual neuronal outputs can influence the other neurons within the range of the axonal spread, while the activities of single neurons can be affected by the afferents in their somatodendritic fields. The morphological profile, therefore, binds the functional role each neuron can play. In addition, synaptic connectivity among neurons displays preference based on the characteristics of presynaptic and postsynaptic neurons. Here, the author reviews the "spatial" and "temporal" connection selectivity in the neocortex. The histological description of the neocortical circuitry depends primarily on the classification of cell types, and the development of gene engineering techniques allows the cell type-specific visualization of dendrites and axons as well as somata. Using genetic labeling of particular cell populations combined with immunohistochemistry and imaging at a subcellular spatial resolution, we revealed the "spatial selectivity" of cortical wirings in which synapses are non-uniformly distributed on the subcellular somatodendritic domains in a presynaptic cell type-specific manner. In addition, cortical synaptic dynamics in learning exhibit presynaptic cell type-dependent "temporal selectivity": corticocortical synapses appear only transiently during the learning phase, while learning-induced new thalamocortical synapses persist, indicating that distinct circuits may supervise learning-specific ephemeral synapse and memory-specific immortal synapse formation. The selectivity of spatial configuration and temporal reconfiguration in the neural circuitry may govern diverse functions in the neocortex.

Parvalbumin-producing cortical interneurons receive inhibitory inputs on proximal portions and cortical excitatory inputs on distal dendrites.

To examine inputs to parvalbumin (PV)-producing interneurons, we generated transgenic mice expressing somatodendritic membrane-targeted green fluorescent protein specifically in the interneurons, and completely visualized their dendrites and somata. Using immunolabeling for vesicular glutamate transporter (VGluT)1, VGluT2, and vesicular GABA transporter, we found that VGluT1-positive terminals made contacts 4- and 3.1-fold more frequently with PV-producing interneurons than VGluT2-positive and GABAergic terminals, respectively, in the primary somatosensory cortex. Even in layer 4, where VGluT2-positive terminals were most densely distributed, VGluT1-positive inputs to PV-producing interneurons were 2.4-fold more frequent than VGluT2-positive inputs. Furthermore, although GABAergic inputs to PV-producing interneurons were as numerous as VGluT2-positive inputs in most cortical layers, GABAergic inputs clearly preferred the proximal dendrites and somata of the interneurons, indicating that the sites of GABAergic inputs were more optimized than those of VGluT2-positive inputs. Simulation analysis with a PV-producing interneuron model compatible with the present morphological data revealed a plausible reason for this observation, by showing that GABAergic and glutamatergic postsynaptic potentials evoked by inputs to distal dendrites were attenuated to 60 and 87%, respectively, of those evoked by somatic inputs. As VGluT1-positive and VGluT2-positive axon terminals were presumed to be cortical and thalamic glutamatergic inputs, respectively, cortical excitatory inputs to PV-producing interneurons outnumbered the thalamic excitatory and intrinsic inhibitory inputs more than two-fold in any cortical layer. Although thalamic inputs are known to evoke about two-fold larger unitary excitatory postsynaptic potentials than cortical ones, the present results suggest that cortical inputs control PV-producing interneurons at least as strongly as thalamic inputs.

Presynaptic supervision of cortical spine dynamics in motor learning.

In mammalian neocortex, learning triggers the formation and turnover of new postsynaptic spines on pyramidal cell dendrites. However, the biological principles of spine reorganization during learning remain elusive because the identity of their presynaptic neuronal partners is unknown. Here, we show that two presynaptic neural circuits supervise distinct programs of spine dynamics to execute learning. We imaged spine dynamics in motor cortex during learning and performed post hoc identification of their afferent presynaptic neurons. New spines that appeared during learning formed small transient contacts with corticocortical neurons that were eliminated on skill acquisition. In contrast, persistent spines with axons from thalamic neurons were formed and enlarged. These results suggest that pyramidal cell dendrites in motor cortex use a neural circuit division of labor during skill learning, with dynamic teaching contacts from top-down intracortical axons followed by synaptic memory formation driven by thalamic axons. Dual spine supervision may govern diverse skill learning in the neocortex.

Exclusive and common targets of neostriatofugal projections of rat striosome neurons: a single neuron-tracing study using a viral vector.

The rat neostriatum has a mosaic organization composed of striosome/patch compartments embedded in a more extensive matrix compartment, which are distinguished from each other by the input-output organization as well as by the expression of many molecular markers. The matrix compartment gives rise to the dual γ-aminobutyric acid (GABA)ergic striatofugal systems, i.e. direct and indirect pathway neurons, whereas the striosome compartment is considered to involve direct pathway neurons alone. Although the whole axonal arborization of matrix striatofugal neurons has been examined in vivo by intracellular staining, that of striosome neurons has never been studied at the single neuron level. In the present study, the axonal arborizations of single striosome projection neurons in rat neostriatum were visualized in their entirety using a viral vector expressing membrane-targeted green fluorescent protein, and compared with that of matrix projection neurons. We found that not only matrix but also striosome compartments contained direct and indirect pathway neurons. Furthermore, only striatonigral neurons in the striosome compartment projected directly to the substantia nigra pars compacta (SNc), although they sent a substantial number of axon collaterals to the globus pallidus, entopeduncular nucleus and/or substantia nigra pars reticulata. These results suggest that striosome neurons play a more important role in the formation of reward-related signals of SNc dopaminergic neurons than do matrix neurons. Together with data from previous studies in the reinforcement learning theory, our results suggest that these direct and indirect striosome-SNc pathways together with nigrostriatal dopaminergic neurons may help striosome neurons to acquire the state-value function.

Exclusive labeling of direct and indirect pathway neurons in the mouse neostriatum by an adeno-associated virus vector with Cre/lox system.

We developed an adeno-associated virus (AAV) vector-based technique to label mouse neostriatal neurons comprising direct and indirect pathways with different fluorescent proteins and analyze their axonal projections. The AAV vector expresses GFP or RFP in the presence or absence of Cre recombinase and should be useful for labeling two cell populations exclusively dependent on its expression. Here, we describe the AAV vector design, stereotaxic injection of the AAV vector, and a highly sensitive immunoperoxidase method for axon visualization. For complete details on the use and execution of this protocol, please refer to Okamoto et al. (2020).

Structural basis for noradrenergic regulation of neural circuits in the mouse olfactory bulb.

Olfactory input is processed in the glomerulus of the main olfactory bulb (OB) and relayed to higher centers in the brain by projection neurons. Conversely, centrifugal inputs from other brain regions project to the OB. We have previously analyzed centrifugal inputs into the OB from several brain regions using single-neuron labeling. In this study, we analyzed the centrifugal noradrenergic (NA) fibers derived from the locus coeruleus (LC), because their projection pathways and synaptic connections in the OB have not been clarified in detail. We analyzed the NA centrifugal projections by single-neuron labeling and immunoelectron microscopy. Individual NA neurons labeled by viral infection were three-dimensionally traced using Neurolucida software to visualize the projection pathway from the LC to the OB. Also, centrifugal NA fibers were visualized using an antibody for noradrenaline transporter (NET). NET immunoreactive (-ir) fibers contained many varicosities and synaptic vesicles. Furthermore, electron tomography demonstrated that NET-ir fibers formed asymmetrical synapses of varied morphology. Although these synapses were present at varicosities, the density of synapses was relatively low throughout the OB. The maximal density of synapses was found in the external plexiform layer; about 17% of all observed varicosities contained synapses. These results strongly suggest that NA-containing fibers in the OB release NA from both varicosities and synapses to influence the activities of OB neurons. The present study provides a morphological basis for olfactory modulation by centrifugal NA fibers derived from the LC.

Overlapping Projections of Neighboring Direct and Indirect Pathway Neostriatal Neurons to Globus Pallidus External Segment.

Indirect pathway medium-sized spiny neurons (iMSNs) in the neostriatum are well known to project to the external segment of the globus pallidus (GPe). Although direct MSNs (dMSNs) also send axon collaterals to the GPe, it remains unclear how dMSNs and iMSNs converge within the GPe. Here, we selectively labeled neighboring dMSNs and iMSNs with green and red fluorescent proteins using an adeno-associated virus vector and examined axonal projections of dMSNs and iMSNs to the GPe in mice. Both dMSNs and iMSNs formed two axonal arborizations displaying topographical projections in the dorsoventral and mediolateral planes. iMSNs displayed a wider and denser axon distribution, which included that of dMSNs. Density peaks of dMSN and iMSN axons almost overlapped, revealing convergence of dMSN axons in the center of iMSN projection fields. These overlapping projections suggest that dMSNs and iMSNs may work cooperatively via interactions within the GPe.

Large Volume Electron Microscopy and Neural Microcircuit Analysis.

One recent technical innovation in neuroscience is microcircuit analysis using three-dimensional reconstructions of neural elements with a large volume Electron microscopy (EM) data set. Large-scale data sets are acquired with newly-developed electron microscope systems such as automated tape-collecting ultramicrotomy (ATUM) with scanning EM (SEM), serial block-face EM (SBEM) and focused ion beam-SEM (FIB-SEM). Currently, projects are also underway to develop computer applications for the registration and segmentation of the serially-captured electron micrographs that are suitable for analyzing large volume EM data sets thoroughly and efficiently. The analysis of large volume data sets can bring innovative research results. These recently available techniques promote our understanding of the functional architecture of the brain.

Preferential inputs from cholecystokinin-positive neurons to the somatic compartment of parvalbumin-expressing neurons in the mouse primary somatosensory cortex.

Parvalbumin-positive (PV+) neurons in the cerebral cortex, mostly corresponding to fast-spiking basket cells, have been implicated in higher-order brain functions and psychiatric disorders. We previously demonstrated that the somatic compartment of PV+ neurons received inhibitory inputs mainly from vasoactive intestinal polypeptide (VIP)+ neurons, whereas inhibitory inputs to the dendritic compartment were derived mostly from PV+ and somatostatin (SOM)+ neurons. However, a substantial number of the axosomatic inputs have remained unidentified. Here we show preferential innervation of the somatic compartment of PV+ neurons by cholecystokinin (CCK)+ neurons in the mouse primary somatosensory cortex. CCK+ neurons, a minor population of GABAergic neurons (3.2%), displayed no colocalization with PV or SOM immunoreactivity but partial overlap with VIP immunoreactivity (27.7%). Confocal laser scanning microscopy observation of CCK+ synaptic inputs to PV+ neurons revealed that CCK+ neurons preferred the somatic compartment to the dendritic compartment of PV+ neurons and provided approximately 33% of the axosomatic inhibitory inputs to PV+ neurons. Additionally, 20.9% and 12.1% of the axosomatic inputs were derived from CCK+/VIP+ and CCK+/VIP-negative (-) neurons, presumably double bouquet and large basket cells, respectively. Furthermore, the densities of the axosomatic inputs from CCK+ and/or VIP+ neurons to PV+ neurons were not significantly different among the cortical layers. The present findings suggest that, by preferentially innervating the cell bodies of PV+ neurons, both CCK+/VIP- basket and CCK+/VIP+ double bouquet cells might efficiently interfere with action potential generation of PV+ neurons, and that the two types of CCK+ neurons might have a large impact on cortical activity through PV+ neuron inhibition.

A carbon nanotube tape for serial-section electron microscopy of brain ultrastructure.

Automated tape-collecting ultramicrotomy in conjunction with scanning electron microscopy (SEM) is a powerful approach for volume electron microscopy and three-dimensional neuronal circuit analysis. Current tapes are limited by section wrinkle formation, surface scratches and sample charging during imaging. Here we show that a plasma-hydrophilized carbon nanotube (CNT)-coated polyethylene terephthalate (PET) tape effectively resolves these issues and produces SEM images of comparable quality to those from transmission electron microscopy. CNT tape can withstand multiple rounds of imaging, offer low surface resistance across the entire tape length and generate no wrinkles during the collection of ultrathin sections. When combined with an enhanced en bloc staining protocol, CNT tape-processed brain sections reveal detailed synaptic ultrastructure. In addition, CNT tape is compatible with post-embedding immunostaining for light and electron microscopy. We conclude that CNT tape can enable high-resolution volume electron microscopy for brain ultrastructure analysis.

A Single Vector Platform for High-Level Gene Transduction of Central Neurons: Adeno-Associated Virus Vector Equipped with the Tet-Off System.

Visualization of neurons is indispensable for the investigation of neuronal circuits in the central nervous system. Virus vectors have been widely used for labeling particular subsets of neurons, and the adeno-associated virus (AAV) vector has gained popularity as a tool for gene transfer. Here, we developed a single AAV vector Tet-Off platform, AAV-SynTetOff, to improve the gene-transduction efficiency, specifically in neurons. The platform is composed of regulator and response elements in a single AAV genome. After infection of Neuro-2a cells with the AAV-SynTetOff vector, the transduction efficiency of green fluorescent protein (GFP) was increased by approximately 2- and 15-fold relative to the conventional AAV vector with the human cytomegalovirus (CMV) or human synapsin I (SYN) promoter, respectively. We then injected the AAV vectors into the mouse neostriatum. GFP expression in the neostriatal neurons infected with the AAV-SynTetOff vector was approximately 40-times higher than that with the CMV or SYN promoter. By adding a membrane-targeting signal to GFP, the axon fibers of neostriatal neurons were clearly visualized. In contrast, by attaching somatodendritic membrane-targeting signals to GFP, axon fiber labeling was mostly suppressed. Furthermore, we prepared the AAV-SynTetOff vector, which simultaneously expressed somatodendritic membrane-targeted GFP and membrane-targeted red fluorescent protein (RFP). After injection of the vector into the neostriatum, the cell bodies and dendrites of neostriatal neurons were labeled with both GFP and RFP, whereas the axons in the projection sites were labeled only with RFP. Finally, we applied this vector to vasoactive intestinal polypeptide-positive (VIP+) neocortical neurons, one of the subclasses of inhibitory neurons in the neocortex, in layer 2/3 of the mouse primary somatosensory cortex. The results revealed the differential distribution of the somatodendritic and axonal structures at the population level. The AAV-SynTetOff vector developed in the present study exhibits strong fluorescence labeling and has promising applications in neuronal imaging.

Structural basis for cholinergic regulation of neural circuits in the mouse olfactory bulb.

Odor information is regulated by olfactory inputs, bulbar interneurons, and centrifugal inputs in the olfactory bulb (OB). Cholinergic neurons projecting from the nucleus of the horizontal limb of the diagonal band of Broca and the magnocellular preoptic nucleus are one of the primary centrifugal inputs to the OB. In this study, we focused on cholinergic regulation of the OB and analyzed neural morphology with a particular emphasis on the projection pathways of cholinergic neurons. Single-cell imaging of a specific neuron within dense fibers is critical to evaluate the structure and function of the neural circuits. We labeled cholinergic neurons by infection with virus vector and then reconstructed them three-dimensionally. We also examined the ultramicrostructure of synapses by electron microscopy tomography. To further clarify the function of cholinergic neurons, we performed confocal laser scanning microscopy to investigate whether other neurotransmitters are present within cholinergic axons in the OB. Our results showed the first visualization of complete cholinergic neurons, including axons projecting to the OB, and also revealed frequent axonal branching within the OB where it innervated multiple glomeruli in different areas. Furthermore, electron tomography demonstrated that cholinergic axons formed asymmetrical synapses with a morphological variety of thicknesses of the postsynaptic density. Although we have not yet detected the presence of other neurotransmitters, the range of synaptic morphology suggests multiple modes of transmission. The present study elucidates the ways that cholinergic neurons could contribute to the elaborate mechanisms involved in olfactory processing in the OB. J. Comp. Neurol. 525:574-591, 2017. © 2016 Wiley Periodicals, Inc.

Differential Inputs to the Perisomatic and Distal-Dendritic Compartments of VIP-Positive Neurons in Layer 2/3 of the Mouse Barrel Cortex.

The recurrent network composed of excitatory and inhibitory neurons is fundamental to neocortical function. Inhibitory neurons in the mammalian neocortex are molecularly diverse, and individual cell types play unique functional roles in the neocortical microcircuit. Recently, vasoactive intestinal polypeptide-positive (VIP+) neurons, comprising a subclass of inhibitory neurons, have attracted particular attention because they can disinhibit pyramidal cells through inhibition of other types of inhibitory neurons, such as parvalbumin- (PV+) and somatostatin-positive (SOM+) inhibitory neurons, promoting sensory information processing. Although VIP+ neurons have been reported to receive synaptic inputs from PV+ and SOM+ inhibitory neurons as well as from cortical and thalamic excitatory neurons, the somatodendritic localization of these synaptic inputs has yet to be elucidated at subcellular spatial resolution. In the present study, we visualized the somatodendritic membranes of layer (L) 2/3 VIP+ neurons by injecting a newly developed adeno-associated virus (AAV) vector into the barrel cortex of VIP-Cre knock-in mice, and we determined the extensive ramification of VIP+ neuron dendrites in the vertical orientation. After immunohistochemical labeling of presynaptic boutons and postsynaptic structures, confocal laser scanning microscopy revealed that the synaptic contacts were unevenly distributed throughout the perisomatic (<100 µm from the somata) and distal-dendritic compartments (≥100 µm) of VIP+ neurons. Both corticocortical and thalamocortical excitatory neurons preferentially targeted the distal-dendritic compartment of VIP+ neurons. On the other hand, SOM+ and PV+ inhibitory neurons preferentially targeted the distal-dendritic and perisomatic compartments of VIP+ neurons, respectively. Notably, VIP+ neurons had few reciprocal connections. These observations suggest different inhibitory effects of SOM+ and PV+ neuronal inputs on VIP+ neuron activity; inhibitory inputs from SOM+ neurons likely modulate excitatory inputs locally in dendrites, while PV+ neurons could efficiently interfere with action potential generation through innervation of the perisomatic domain of VIP+ neurons. The present study, which shows a precise configuration of site-specific inputs, provides a structural basis for the integration mechanism of synaptic inputs to VIP+ neurons.

Structural basis for serotonergic regulation of neural circuits in the mouse olfactory bulb.

Olfactory processing is well known to be regulated by centrifugal afferents from other brain regions, such as noradrenergic, acetylcholinergic, and serotonergic neurons. Serotonergic neurons widely innervate and regulate the functions of various brain regions. In the present study, we focused on serotonergic regulation of the olfactory bulb (OB), one of the most structurally and functionally well-defined brain regions. Visualization of a single neuron among abundant and dense fibers is essential to characterize and understand neuronal circuits. We accomplished this visualization by successfully labeling and reconstructing serotonin (5-hydroxytryptamine: 5-HT) neurons by infection with sindbis and adeno-associated virus into dorsal raphe nuclei (DRN) of mice. 5-HT synapses were analyzed by correlative confocal laser microscopy and serial-electron microscopy (EM) study. To further characterize 5-HT neuronal and network function, we analyzed whether glutamate was released from 5-HT synaptic terminals using immuno-EM. Our results are the first visualizations of complete 5-HT neurons and fibers projecting from DRN to the OB with bifurcations. We found that a single 5-HT axon can form synaptic contacts to both type 1 and 2 periglomerular cells within a single glomerulus. Through immunolabeling, we also identified vesicular glutamate transporter 3 in 5-HT neurons terminals, indicating possible glutamatergic transmission. Our present study strongly implicates the involvement of brain regions such as the DRN in regulation of the elaborate mechanisms of olfactory processing. We further provide a structure basis of the network for coordinating or linking olfactory encoding with other neural systems, with special attention to serotonergic regulation.

Sequence of Molecular Events during the Maturation of the Developing Mouse Prefrontal Cortex.

Recent progress in psychiatric research has accumulated many mouse models relevant to developmental neuropsychiatric disorders using numerous genetic and environmental manipulations. Since the prefrontal cortex (PFC) is essential for cognitive functions whose impairments are central symptoms associated with the disorders in humans, it has become crucial to clarify altered developmental processes of PFC circuits in these mice. To that end, we aimed to understand a sequence of molecular events during normal mouse PFC development. Expression profiles for representative genes covering diverse biological processes showed that while there were little changes in genes for neuroreceptors and synaptic molecules during postnatal period, there were dramatic increases in expression of myelin-related genes and parvalbumin gene, peaking at postnatal day (P) 21 and P35, respectively. The timing of the peaks is different from one observed in the striatum. Furthermore, evaluation of the circuitry maturation by measuring extracellular glutamate in PFC revealed that sensitivity to an NMDA antagonist became adult-like pattern at P56, suggesting that some of maturation processes continue till P56. The trajectory of molecular events in the PFC maturation described here should help us to characterize how the processes are affected in model mice, an important first step for translational research.

Convergence of Lemniscal and Local Excitatory Inputs on Large GABAergic Tectothalamic Neurons.

Large GABAergic (LG) neurons form a distinct cell type in the inferior colliculus (IC), identified by the presence of dense VGLUT2-containing axosomatic terminals. Although some of the axosomatic terminals originate from local and commissural IC neurons, it has been unclear whether LG neurons also receive axosomatic inputs from the lower auditory brainstem nuclei, i.e., cochlear nuclei (CN), superior olivary complex (SOC), and nuclei of the lateral lemniscus (NLL). In this study we injected recombinant viral tracers that force infected cells to express GFP in a Golgi-like manner into the lower auditory brainstem nuclei to determine whether these nuclei directly innervate LG cell somata. Labeled axons from CN, SOC, and NLL terminated as excitatory axosomatic endings, identified by colabeling of GFP and VGLUT2, on single LG neurons in the IC. Each excitatory axon made only a few axosomatic contacts on each LG neuron. Inputs to a single LG cell are unlikely to be from a single brainstem nucleus, since lesions of individual nuclei failed to eliminate most VGLUT2-positive terminals on the LG neurons. The estimated number of inputs on a single LG cell body was almost proportional to the surface area of the cell body. Double injections of different viruses into IC and a brainstem nucleus showed that LG neurons received inputs from both. These results demonstrated that both ascending and intrinsic sources converge on the LG somata to control inhibitory tectothalamic projections.

Preprodynorphin-expressing neurons constitute a large subgroup of somatostatin-expressing GABAergic interneurons in the mouse neocortex.

Dynorphins, leumorphin, and neoendorphins are preprodynorphin (PPD)-derived peptides and ligands for κ-opioid receptors. Using an antibody to PPD C-terminal, we investigated the chemical and molecular characteristics of PPD-expressing neurons in mouse neocortex. PPD-immunopositive neuronal somata were distributed most frequently in layer 5 and less frequently in layers 2-4 and 6 throughout neocortical regions. Combined labeling of immunofluorescence and fluorescent mRNA signals revealed that almost all PPD-immunopositive neurons expressed glutamic acid decarboxylase but not vesicular glutamate transporter, indicating their γ-aminobutyric acid (GABA)ergic characteristics, and that PPD-immunopositive neurons accounted for 15% of GABAergic interneurons in the primary somatosensory area. As GABAergic interneurons were divided into several groups by specific markers, we further examined the chemical characteristics of PPD-expressing neurons by the double immunofluorescence labeling method. More than 95% of PPD-immunopositive neurons were also somatostatin (SOM)-immunopositive in the primary somatosensory, primary motor, orbitofrontal, and primary visual areas, but only 24% were SOM-immunopositive in the medial prefrontal cortex. In the primary somatosensory area, PPD-immunopositive neurons constituted 50%, 79%, 55%, and 17% of SOM-immunopositive neurons in layers 2-3, 4, 5, and 6, respectively. Although SOM-expressing neurons contained calretinin-, neuropeptide Y-, nitric oxide synthase-, and reelin-expressing neurons as subgroups, only reelin immunoreactivity was detected in many PPD-immunopositive neurons. These results indicate that PPD-expressing neurons constitute a large subgroup of SOM-expressing cortical interneurons, and the PPD/SOM-expressing GABAergic neurons might serve not only as inhibitory elements in the local cortical circuit, but also as modulators for cortical neurons expressing κ-opioid and/or SOM receptors.