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.

The Cerebellar Cortex Receives Orofacial Proprioceptive Signals from the Supratrigeminal Nucleus via the Mossy Fiber Pathway in Rats.

Proprioceptive sensory information from muscle spindles is essential for the regulation of motor functions. However, little is known about the motor control regions in the cerebellar cortex that receive proprioceptive signals from muscle spindles distributed throughout the body, including the orofacial muscles. Therefore, in this study, we investigated the pattern of projections in the rat cerebellar cortex derived from the supratrigeminal nucleus (Su5), which conveys orofacial proprioceptive information from jaw-closing muscle spindles (JCMSs). Injections of an anterograde tracer into the Su5 revealed that many bilateral axon terminals (rosettes) were distributed in the granular layer of the cerebellar cortex (including the simple lobule B, crus II and flocculus) in a various sized, multiple patchy pattern. We could also detect JCMS proprioceptive signals in these cerebellar cortical regions, revealing for the first time that they receive muscle proprioceptive inputs in rats. Retrograde tracer injections confirmed that the Su5 directly sends outputs to the cerebellar cortical areas. Furthermore, we injected an anterograde tracer into the external cuneate nucleus (ECu), which receives proprioceptive signals from the forelimb and neck muscle spindles, to distinguish between the Su5- and ECu-derived projections in the cerebellar cortex. The labeled terminals from the ECu were distributed predominantly in the vermis of the cerebellar cortex. Almost no overlap was seen in the terminal distributions of the Su5 and ECu projections. Our findings demonstrate that the rat cerebellar cortex receives orofacial proprioceptive input that is processed differently from the proprioceptive signals from the other regions of the body.

Fluorochromized tyramide-glucose oxidase as a multiplex fluorescent tyramide signal amplification system for histochemical analysis.

Tyramide signal amplification (TSA) is a highly sensitive method for histochemical analysis. Previously, we reported a TSA system, biotinyl tyramine-glucose oxidase (BT-GO), for bright-filed imaging. Here, we develop fluorochromized tyramide-glucose oxidase (FT-GO) as a multiplex fluorescent TSA system. FT-GO involves peroxidase-catalyzed deposition of fluorochromized tyramide (FT) with hydrogen peroxide produced by enzymatic reaction between glucose and glucose oxidase. We showed that FT-GO enhanced immunofluorescence signals while maintaining low background signals. Compared with indirect immunofluorescence detections, FT-GO demonstrated a more widespread distribution of monoaminergic projection systems in mouse and marmoset brains. For multiplex labeling with FT-GO, we quenched antibody-conjugated peroxidase using sodium azide. We applied FT-GO to multiplex fluorescent in situ hybridization, and succeeded in labeling neocortical interneuron subtypes by coupling with immunofluorescence. FT-GO immunofluorescence further increased the detectability of an adeno-associated virus tracer. Given its simplicity and a staining with a high signal-to-noise ratio, FT-GO would provide a versatile platform for histochemical analysis.

Protocol for multi-scale light microscopy/electron microscopy neuronal imaging in mouse brain tissue.

An imaging technique across multiple spatial scales is required for extracting structural information on neurons with processes of meter scale length and specialized nanoscale structures. Here, we present a protocol combining multi-scale light microscopy (LM) with electron microscopy (EM) in mouse brain tissue. We describe tissue slice preparation and LM/EM dual labeling with EGFP-APEX2 fusion protein. We then detail ScaleSF tissue clearing and successive LM/EM imaging. Our protocol allows for deciphering structural information across multiple spatial scales on neurons. For complete details on the use and execution of this protocol, please refer to Furuta et al. (2022).

A Tissue Clearing Method for Neuronal Imaging from Mesoscopic to Microscopic Scales.

A detailed protocol is provided here to visualize neuronal structures from mesoscopic to microscopic levels in brain tissues. Neuronal structures ranging from neural circuits to subcellular neuronal structures are visualized in mouse brain slices optically cleared with ScaleSF. This clearing method is a modified version of ScaleS and is a hydrophilic tissue clearing method for tissue slices that achieves potent clearing capability as well as a high-level of preservation of fluorescence signals and structural integrity. A customizable three dimensional (3D)-printed imaging chamber is designed for reliable mounting of cleared brain tissues. Mouse brains injected with an adeno-associated virus vector carrying enhanced green fluorescent protein gene were fixed with 4% paraformaldehyde and cut into slices of 1-mm thickness with a vibrating tissue slicer. The brain slices were cleared by following the clearing protocol, which include sequential incubations in three solutions, namely, ScaleS0 solution, phosphate buffer saline (-), and ScaleS4 solution, for a total of 10.5-14.5 h. The cleared brain slices were mounted on the imaging chamber and embedded in 1.5% agarose gel dissolved in ScaleS4D25(0) solution. The 3D image acquisition of the slices was carried out using a confocal laser scanning microscope equipped with a multi-immersion objective lens of a long working distance. Beginning with mesoscopic neuronal imaging, we succeeded in visualizing fine subcellular neuronal structures, such as dendritic spines and axonal boutons, in the optically cleared brain slices. This protocol would facilitate understanding of neuronal structures from circuit to subcellular component scales.

Multi-scale light microscopy/electron microscopy neuronal imaging from brain to synapse with a tissue clearing method, ScaleSF.

The mammalian brain is organized over sizes that span several orders of magnitude, from synapses to the entire brain. Thus, a technique to visualize neural circuits across multiple spatial scales (multi-scale neuronal imaging) is vital for deciphering brain-wide connectivity. Here, we developed this technique by coupling successive light microscopy/electron microscopy (LM/EM) imaging with a glutaraldehyde-resistant tissue clearing method, ScaleSF. Our multi-scale neuronal imaging incorporates (1) brain-wide macroscopic observation, (2) mesoscopic circuit mapping, (3) microscopic subcellular imaging, and (4) EM imaging of nanoscopic structures, allowing seamless integration of structural information from the brain to synapses. We applied this technique to three neural circuits of two different species, mouse striatofugal, mouse callosal, and marmoset corticostriatal projection systems, and succeeded in simultaneous interrogation of their circuit structure and synaptic connectivity in a targeted way. Our multi-scale neuronal imaging will significantly advance the understanding of brain-wide connectivity by expanding the scales of objects.

Efferent and afferent connections of supratrigeminal neurons conveying orofacial muscle proprioception in rats.

The supratrigeminal nucleus (Su5) is a key structure for controlling jaw movements; it receives proprioceptive sensation from jaw-closing muscle spindles (JCMSs) and sends projections to the trigeminal motor nucleus (Mo5). However, the central projections and regulation of JCMS proprioceptive sensation are not yet fully understood. Therefore, we aimed to reveal the efferent and afferent connections of the Su5 using neuronal tract tracings. Anterograde tracer injections into the Su5 revealed that the Su5 sends contralateral projections (or bilateral projections with a contralateral predominance) to the Su5, basilar pontine nuclei, pontine reticular nucleus, deep mesencephalic nucleus, superior colliculus, caudo-ventromedial edge of the ventral posteromedial thalamic nucleus, parafascicular thalamic nucleus, zona incerta, and lateral hypothalamus, and ipsilateral projections (or bilateral projections with an ipsilateral predominance) to the intertrigeminal region, trigeminal oral subnucleus, dorsal medullary reticular formation, and hypoglossal nucleus as well as the Mo5. Retrograde tracer injections into the Su5 demonstrated that the Su5 receives bilateral projections with a contralateral predominance (or contralateral projections) from the primary and secondary somatosensory cortices, granular insular cortex, and Su5, and ipsilateral projections (or bilateral projections with an ipsilateral predominance) from the dorsal peduncular cortex, bed nuclei of stria terminalis, central amygdaloid nucleus, lateral hypothalamus, parasubthalamic nucleus, trigeminal mesencephalic nucleus, parabrachial nucleus, juxtatrigeminal region, trigeminal oral and caudal subnuclei, and dorsal medullary reticular formation. These findings suggest that the Su5, which receives JCMS proprioception, has efferent and afferent connections with multiple brain regions that are involved in emotional and autonomic functions as well as orofacial motor functions.

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.

Widespread corticopetal projections from the oval paracentral nucleus of the intralaminar thalamic nuclei conveying orofacial proprioception in rats.

The oval paracentral nucleus (OPC) was initially isolated from the paracentral nucleus (PC) within the intralaminar thalamic nuclei in rats. We have recently shown that the rat OPC receives proprioceptive inputs from jaw-closing muscle spindles (JCMSs). However, it remains unknown which cortical areas receive thalamic inputs from the OPC, and whether the cortical areas receiving the OPC inputs are distinct from those receiving inputs from the other intralaminar nuclei and sensory thalamic nuclei. To address this issue, we injected an anterograde tracer, biotinylated dextranamine (BDA), into the OPC, which was electrophysiologically identified by recording of proprioceptive inputs from the JCMSs. Many BDA-labeled axonal fibers and terminals from the OPC were ipsilaterally observed in the rostral and rostroventral regions of the primary somatosensory cortex (S1), the rostral region of the secondary somatosensory cortex (S2), and the most rostrocaudal levels of the granular insular cortex (GI). In contrast, a BDA injection into the caudal PC, which was located slightly rostral to the OPC, resulted in ipsilateral labeling of axonal fibers and terminals in the rostrolateral region of the medial agranular cortex and the rostromedial region of the lateral agranular cortex. Furthermore, injections of a retrograde tracer, Fluorogold, into these S1, S2, and GI regions, resulted in preferential labeling of neurons in the ipsilateral OPC among the intralaminar and sensory thalamic nuclei. These findings reveal that the rat OPC has widespread, but strong corticopetal projections, indicating that there exist divergent corticopetal pathways from the intralaminar thalamic nucleus, which process JCMS proprioceptive sensation.

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).

Proprioceptive thalamus receiving forelimb and neck muscle spindle inputs via the external cuneate nucleus in the rat.

Proprioceptive signals from body muscles have historically been considered to project to the rostrodorsal shell of the ventrobasal thalamic complex [the ventral posterolateral nucleus (VPL) and ventral posteromedial nucleus (VPM)]. However, we have recently found that proprioception from rat jaw-closing muscle spindles (JCMSs) is conveyed via the supratrigeminal nucleus to the caudo-ventromedial edge of the VPM, but not to the rostrodorsal shell of the VPM. Therefore, proprioception from other body muscles may also project to thalamic regions other than the rostrodorsal shell of the VPL. We thus examined the thalamic projection from the rat external cuneate nucleus (ECu), which receives proprioceptive inputs from forelimb and neck muscles. After injection of anterograde tracer into the ECu, axon terminals were contralaterally labeled in the ventromedial part (VPLvm) of the VPL, but not in the rostrodorsal shell of the VPL. After anterograde tracer injection into the cuneate nucleus (Cu), axon terminals were widely labeled in the contralateral VPL including the VPLvm. In the VPLvm, we electrophysiologically confirmed the proprioceptive inputs responsive to electrical stimulation of the ECu or median nerve and to the pressure of forelimb/neck muscles or wrist flexion. After retrograde tracer injection into the VPLvm, neurons were contralaterally labeled in the ECu and Cu. After retrograde tracer injection into the VPL where no such proprioceptive inputs were recorded, no ECu neurons were labeled. These findings indicate that proprioception from forelimb/neck muscle spindles and JCMSs is somatotopically transmitted to the ventromedial floor of the ventrobasal thalamic complex, but not to its rostrodorsal shell.

Ascending projection of jaw-closing muscle-proprioception to the intralaminar thalamic nuclei in rats.

An invasive intralaminar thalamic stimulation and a non-invasive application of oral splint are both effective in treating tic symptoms of patients with Tourette syndrome (TS). Therefore, these two treatments may exert some influence on the same brain region in TS patients. We thus hypothesized that the proprioceptive input arising from the muscle spindles of jaw-closing muscles (JCMSs), known to be increased by the application of oral splint, is transmitted to the intralaminar thalamic nuclei. To test this issue, we morphologically and electrophysiologically examined the thalamic projections of proprioceptive input from the JCMSs to the intralaminar thalamic nuclei of rats. We first injected an anterograde tracer, biotinylated dextranamine, into the electrophysiologically identified supratrigeminal nucleus, which is known to receive proprioceptive inputs from the JCMSs via the trigeminal mesencephalic neurons. A moderate number of biotinylated dextranamine-labeled axon terminals were bilaterally distributed in the oval paracentral nucleus (OPC) of the intralaminar thalamic nuclei. We also detected electrophysiological responses to the electrical stimulation of bilateral masseter nerves and to sustained jaw-opening in the OPC. After injection of retrograde tracer (cholera toxin B subunit or Fluorogold) into the OPC, neuronal cell bodies were retrogradely labeled in the rostrodorsal portion of the bilateral supratrigeminal nucleus. Here, we show that proprioceptive inputs from the JCMSs are conveyed to the OPC in the intralaminar nuclei via the supratrigeminal nucleus. This study can help to understand previously unrecognized pathways of proprioception ascending inputs from the brainstem to the thalamus, which may contribute to treatments of TS patients.

The Cellular and Mechanical Basis for Response Characteristics of Identified Primary Afferents in the Rat Vibrissal System.

Compared to our understanding of the response properties of receptors in the auditory and visual systems, we have only a limited understanding of the mechanoreceptor responses that underlie tactile sensation. Here, we exploit the stereotyped morphology of the rat vibrissal (whisker) array to investigate coding and transduction properties of identified primary tactile afferents. We performed in vivo intra-axonal recording and labeling experiments to quantify response characteristics of four different types of identified mechanoreceptors in the vibrissal follicle: ring-sinus Merkel; lanceolate; clublike; and rete-ridge collar Merkel. Of these types, only ring-sinus Merkel endings exhibited slowly adapting properties. A weak inverse relationship between response magnitude and onset response latency was found across all types. All afferents exhibited strong "angular tuning," i.e., their response magnitude and latency depended on the whisker's deflection angle. Although previous studies suggested that this tuning should be aligned with the angular location of the mechanoreceptor in the follicle, such alignment was observed only for Merkel afferents; angular tuning of the other afferent types showed no clear alignment with mechanoreceptor location. Biomechanical modeling suggested that this tuning difference might be explained by mechanoreceptors' differential sensitivity to the force directed along the whisker length. Electron microscopic investigations of Merkel endings and lanceolate endings at the level of the ring sinus revealed unique anatomical features that may promote these differential sensitivities. The present study systematically integrates biomechanical principles with the anatomical and morphological characterization of primary afferent endings to describe the physical and cellular processing that shapes the neural representation of touch.

Multiple Patterns of Axonal Collateralization of Single Layer III Neurons of the Rat Presubiculum.

The presubiculum plays a key role in processing and integrating spatial and head-directional information. Layer III neurons of the presubiculum provide strong projections to the superficial layers of the medial entorhinal cortex (MEC) in the rat. Our previous study revealed that the terminal distribution of efferents from layer III cells of the presubiculum was organized in a band-like fashion within the MEC, and the transverse axis of these zones ran parallel to the rhinal fissure. Identifying axonal branching patterns of layer III neurons of the presubiculum is important to further elucidate the functional roles of the presubiculum. In the present study, we visualized all axonal processes and terminal distributions of single presubicular layer III neurons in the rat, using in vivo injection of a viral vector expressing membrane-targeted palmitoylation site-attached green fluorescent protein (GFP). We found that layer III of the rat presubiculum comprised multiple types of neurons (n = 12) with characteristic patterns of axonal collateralization, including cortical projection neurons (n = 6) and several types of intrinsic connectional neurons (n = 6). Two of six cortical projection neurons provided two or three major axonal branches to the MEC and formed elaborate terminal arbors within the superficial layers of the MEC. The width and axis of the area of their terminal distribution resembled that of the band-like terminal field seen in our massive-scale observation. Two of the other four cortical projection neurons gave off axonal branches to the MEC and also to the subiculum, and each of the other two neurons sent axons to the subiculum or parasubiculum. Patterns of axonal arborization of six intrinsic connectional neurons were distinct from each other, with four neurons sending many axonal branches to both superficial and deep layers of the presubiculum and the other two neurons showing sparse axonal branches with terminations confined to layers III-V of the presubiculum. These data demonstrate that layer III of the rat presubiculum consists of multiple types of cortical projection neurons and interneurons, and also suggest that inputs from a single presubicular layer III neuron can directly affect a band-like zone of the MEC.

Intracellular labile zinc is a determinant of vulnerability of cultured astrocytes to oxidative stress.

Recently, we found that treatment of cultured mouse astrocytes of ddY-strain mice (ddY-astrocytes) with 400 µM H2O2 for 24 h increased the intracellular labile zinc level without cell toxicity. In contrast, 170 µM H2O2 for 12 h is reported to kill mouse astrocytes obtained from C57BL/6-strain mice (C57BL/6-astrocytes) with an increase in intracellular labile zinc. To clarify this discrepancy, we compared the intracellular zinc levels and cell toxicity in H2O2-treated ddY- and C57BL/6-astrocytes. Exposure of C57BL/6-astrocytes to 170 or 400 µM H2O2 for 12 h dose-dependently decreased the cell viability and administration of plasma membrane-permeable zinc chelator TPEN prevented the 170 µM H2O2-induced astrocyte death, while neither concentration of H2O2 killed ddY-astrocytes. The intracellular zinc level in H2O2-treated C57BL/6-astrocytes was higher than that in H2O2-treated ddY-astrocytes, and this increase was suppressed by TPEN. There was no apparent difference in the expression levels of zinc transporters ZIPs and ZnTs between the two types of astrocytes, while expression of zinc releasable channel TRPM7 was found on the plasma membrane in ddY-astrocytes, but not in C57BL/6-astrocytes, although the total cellular expression levels were almost the same. In addition, a TRPM7 blocker, 2-aminoethoxydiphenyl borate, increased the intracellular zinc level in H2O2-treated ddY-, but not C57BL/6-astrocytes. Collectively, it is suggested that vulnerability of astrocytes to oxidative stress depends on an increased level of intracellular labile zinc, and TRPM7 localized on the plasma membrane contributes, at least in part, to ameliorate the cell injury by decreasing the zinc level.

Expression of cholecystokinin by neurons in mouse spinal dorsal horn.

Excitatory interneurons account for the majority of dorsal horn neurons, and are required for perception of normal and pathological pain. We have identified largely non-overlapping populations in laminae I-III, based on expression of substance P, gastrin-releasing peptide, neurokinin B, and neurotensin. Cholecystokinin (CCK) is expressed by many dorsal horn neurons, particularly in the deeper laminae. Here, we have used immunocytochemistry and in situ hybridization to characterize the CCK cells. We show that they account for ~7% of excitatory neurons in laminae I-II, but between a third and a quarter of those in lamina III. They are largely separate from the neurokinin B, neurotensin, and gastrin-releasing peptide populations, but show limited overlap with the substance P cells. Laminae II-III neurons with protein kinase Cγ (PKCγ) have been implicated in mechanical allodynia following nerve injury, and we found that around 50% of CCK cells were PKCγ-immunoreactive. Neurotensin is also expressed by PKCγ cells, and among neurons with moderate to high levels of PKCγ, ~85% expressed CCK or neurotensin. A recent transcriptomic study identified mRNA for thyrotropin-releasing hormone in a specific subpopulation of CCK neurons, and we show that these account for half of the CCK/PKCγ cells. These findings indicate that the CCK cells are distinct from other excitatory interneuron populations that we have defined. They also show that PKCγ cells can be assigned to different classes based on neuropeptide expression, and it will be important to determine the differential contribution of these classes to neuropathic allodynia.

Expression of Calretinin Among Different Neurochemical Classes of Interneuron in the Superficial Dorsal Horn of the Mouse Spinal Cord.

Around 75% of neurons in laminae I-II of the mouse dorsal horn are excitatory interneurons, and these are required for normal pain perception. We have shown that four largely non-overlapping excitatory interneuron populations can be defined by expression of the neuropeptides neurotensin, neurokinin B (NKB), gastrin-releasing peptide (GRP) and substance P. In addition, we recently identified a population of excitatory interneurons in glabrous skin territory that express dynorphin. The calcium-binding protein calretinin is present in many excitatory neurons in this region, but we know little about its relation to these neuropeptide markers. Here we show that calretinin is differentially expressed, being present in the majority of substance P-, GRP- and NKB-expressing cells, but not in the neurotensin or dynorphin cells. Calretinin-positive cells have been implicated in detection of noxious mechanical stimuli, but are not required for tactile allodynia after neuropathic pain. Our findings are therefore consistent with the suggestion that neuropathic allodynia involves the neurotensin and/or dynorphin excitatory interneuron populations. Around a quarter of inhibitory interneurons in lamina I-II contain calretinin, and recent transcriptomic studies suggest that these co-express substance P. We confirm this, by showing that inhibitory Cre-expressing cells in a Tac1Cre knock-in mouse are calretinin-immunoreactive. Interestingly, there is evidence that these cells express low levels of peptidylglycine alpha-amidating monooxygenase, an enzyme required for maturation of neuropeptides. This may explain our previous finding that although the substance P precursor preprotachykinin A can be detected in some inhibitory interneurons, very few inhibitory axonal boutons are immunoreactive for substance P.

A Descending Circuit Derived From the Superior Colliculus Modulates Vibrissal Movements.

The superior colliculus (SC) is an essential structure for the control of eye movements. In rodents, the SC is also considered to play an important role in whisking behavior, in which animals actively move their vibrissae (mechanosensors) to gather tactile information about the space around them during exploration. We investigated how the SC contributes to vibrissal movement control. We found that when the SC was unilaterally lesioned, the resting position of the vibrissae shifted backward on the side contralateral to the lesion. The unilateral SC lesion also induced an increase in the whisking amplitude on the contralateral side. To explore the anatomical basis for SC involvement in vibrissal movement control, we then quantitatively evaluated axonal projections from the SC to the brainstem using neuronal labeling with a virus vector. Neurons of the SC mainly sent axons to the contralateral side in the lower brainstem. We found that the facial nucleus received input directly from the SC, and that the descending projections from the SC also reached the intermediate reticular formation and pre-Bötzinger complex, which are both considered to contain neural oscillators generating rhythmic movements of the vibrissae. Together, these results indicate the existence of a neural circuit in which the SC modulates vibrissal movements mainly on the contralateral side, via direct connections to motoneurons, and via indirect connections including the central pattern generators.

Projection Patterns of Corticofugal Neurons Associated with Vibrissa Movement.

Rodents actively whisk their vibrissae, which, when they come in contact with surrounding objects, enables rodents to gather spatial information about the environment. Cortical motor command of whisking is crucial for the control of vibrissa movement. Using awake and head-fixed rats, we investigated the correlations between axonal projection patterns and firing properties in identified layer 5 neurons in the motor cortex, which are associated with vibrissa movement. We found that cortical neurons that sent axons to the brainstem fired preferentially during large-amplitude vibrissa movements and that corticocallosal neurons exhibited a high firing rate during small vibrissa movements or during a quiet state. The differences between these two corticofugal circuits may be related to the mechanisms of motor-associated information processing.

Cortical and Subcortical Projections from Granular Insular Cortex Receiving Orofacial Proprioception.

We have recently revealed that the proprioceptive signal from jaw-closing muscle spindles (JCMSs) is conveyed to the dorsal part of granular insular cortex rostroventrally adjacent to the rostralmost part of secondary somatosensory cortex (dGIrvs2) via the caudo-ventromedial edge (VPMcvm) of ventral posteromedial thalamic nucleus (VPM) in rats. However, it remains unclear to which cortical or subcortical structures the JCMS proprioceptive information is subsequently conveyed from the dGIrvs2. To test this issue, we injected an anterograde tracer, biotinylated dextranamine, into the electophysiologically identified dGIrvs2, and analyzed the resultant distribution profiles of labeled axon terminals in rats. Labeled terminals were distributed with an ipsilateral predominance. In the cerebral cortex, they were seen in the primary and secondary somatosensory cortices, lateral and medial agranular cortices and dorsolateral orbital cortex. In the basal ganglia, they were found in the caudate putamen, core part of accumbens nucleus, lateral globus pallidus, subthalamic nucleus, and substantia nigra pars compacta and pars reticulata. They were also observed in the central amygdaloid nucleus and extended amygdala (the interstitial nucleus of posterior limb of anterior commissure and the juxtacapsular part of lateral division of bed nucleus of stria terminalis). In the thalamus, they were seen in the reticular nucleus, ventromedial nucleus, core VPM, parvicellular part of ventral posterior nucleus, oval paracentral nucleus, medial and triangular parts of posterior nucleus, and zona incerta as well as the VPMcvm. These data suggest that the JCMS proprioceptive information through the dGIrvs2 is transmitted to the emotional 'limbic' regions as well as sensorimotor regions.