RESUMEN
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.
Asunto(s)
Corteza Cerebelosa , Fibras Musgosas del Hipocampo , Ratas , Animales , Ratas Wistar , Terminales PresinápticosRESUMEN
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.
RESUMEN
Whisking and sniffing are predominant aspects of exploratory behaviour in rodents. Yet the neural mechanisms that generate and coordinate these and other orofacial motor patterns remain largely uncharacterized. Here we use anatomical, behavioural, electrophysiological and pharmacological tools to show that whisking and sniffing are coordinated by respiratory centres in the ventral medulla. We delineate a distinct region in the ventral medulla that provides rhythmic input to the facial motor neurons that drive protraction of the vibrissae. Neuronal output from this region is reset at each inspiration by direct input from the pre-Bötzinger complex, such that high-frequency sniffing has a one-to-one relationship with whisking, whereas basal respiration is accompanied by intervening whisks that occur between breaths. We conjecture that the respiratory nuclei, which project to other premotor regions for oral and facial control, function as a master clock for behaviours that coordinate with breathing.
Asunto(s)
Movimientos de la Cabeza/fisiología , Respiración , Olfato/fisiología , Vibrisas/fisiología , Animales , Relojes Biológicos/fisiología , Cara/anatomía & histología , Cara/fisiología , Femenino , Ácido Kaínico/administración & dosificación , Ácido Kaínico/farmacología , Masculino , Bulbo Raquídeo/citología , Bulbo Raquídeo/fisiología , Músculo Esquelético/fisiología , Ratas , Ratas Long-Evans , Vibrisas/inervaciónRESUMEN
UNLABELLED: The architectonic subdivisions of the brain are believed to be functional modules, each processing parts of global functions. Previously, we showed that neurons in different regions operate in different firing regimes in monkeys. It is possible that firing regimes reflect differences in underlying information processing, and consequently the firing regimes in homologous regions across animal species might be similar. We analyzed neuronal spike trains recorded from behaving mice, rats, cats, and monkeys. The firing regularity differed systematically, with differences across regions in one species being greater than the differences in similar areas across species. Neuronal firing was consistently most regular in motor areas, nearly random in visual and prefrontal/medial prefrontal cortical areas, and bursting in the hippocampus in all animals examined. This suggests that firing regularity (or irregularity) plays a key role in neural computation in each functional subdivision, depending on the types of information being carried. SIGNIFICANCE STATEMENT: By analyzing neuronal spike trains recorded from mice, rats, cats, and monkeys, we found that different brain regions have intrinsically different firing regimes that are more similar in homologous areas across species than across areas in one species. Because different regions in the brain are specialized for different functions, the present finding suggests that the different activity regimes of neurons are important for supporting different functions, so that appropriate neuronal codes can be used for different modalities.
Asunto(s)
Potenciales de Acción/fisiología , Relojes Biológicos/fisiología , Encéfalo/fisiología , Modelos Neurológicos , Red Nerviosa/fisiología , Neuronas/fisiología , Animales , Gatos , Simulación por Computador , Femenino , Haplorrinos , Masculino , Ratones , Ratas , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Especificidad de la EspecieRESUMEN
BACKGROUND: Excitatory interneurons account for the majority of neurons in laminae I-III, but their functions are poorly understood. Several neurochemical markers are largely restricted to excitatory interneuron populations, but we have limited knowledge about the size of these populations or their overlap. The present study was designed to investigate this issue by quantifying the neuronal populations that express somatostatin (SST), neurokinin B (NKB), neurotensin, gastrin-releasing peptide (GRP) and the γ isoform of protein kinase C (PKCγ), and assessing the extent to which they overlapped. Since it has been reported that calretinin- and SST-expressing cells have different functions, we also looked for co-localisation of calretinin and SST. RESULTS: SST, preprotachykinin B (PPTB, the precursor of NKB), neurotensin, PKCγ or calretinin were detected with antibodies, while cells expressing GRP were identified in a mouse line (GRP-EGFP) in which enhanced green fluorescent protein (EGFP) was expressed under control of the GRP promoter. We found that SST-, neurotensin-, PPTB- and PKCγ-expressing cells accounted for 44%, 7%, 12% and 21% of the neurons in laminae I-II, and 16%, 8%, 4% and 14% of those in lamina III, respectively. GRP-EGFP cells made up 11% of the neuronal population in laminae I-II. The neurotensin, PPTB and GRP-EGFP populations showed very limited overlap, and we estimate that between them they account for ~40% of the excitatory interneurons in laminae I-II. SST which is expressed by ~60% of excitatory interneurons in this region, was found in each of these populations, as well as in cells that did not express any of the other peptides. Neurotensin and PPTB were often found in cells with PKCγ, and between them, constituted around 60% of the PKCγ cells. Surprisingly, we found extensive co-localisation of SST and calretinin. CONCLUSIONS: These results suggest that cells expressing neurotensin, NKB or GRP form largely non-overlapping sets that are likely to correspond to functional populations. In contrast, SST is widely expressed by excitatory interneurons that are likely to be functionally heterogeneous.
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Interneuronas/química , Neuropéptidos/metabolismo , Asta Dorsal de la Médula Espinal/metabolismo , Animales , Calbindina 2/metabolismo , Péptido Liberador de Gastrina/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Masculino , Ratones Endogámicos C57BL , Modelos Biológicos , Neurotensina/metabolismo , Proteína Quinasa C/metabolismo , Precursores de Proteínas/metabolismo , Somatostatina/metabolismo , Taquicininas/metabolismoRESUMEN
Not only inhibitory afferent-dominant zone (IZ) of the ventral anterior-ventral lateral thalamic complex (VA-VL) but also the ventral medial nucleus (VM) is known to receive strong inputs from the basal ganglia and send axons to motor areas. We previously reported differences in axonal arborization between IZ neurons and the other VA-VL neurons in rats by single-neuron tracing with viral vectors. In the present study, the axonal arborization of single VM neurons was visualized by the same method, and compared with that of IZ neurons. VM neurons formed fewer axon collaterals in the striatum, but sent axon fibers more widely and more preferentially (79% of fibers) to layer 1 of cortical areas than IZ neurons. Furthermore, the VM seemed to contain at least 2 types of neurons; a major population of VM neurons sent axon fibers principally to motor-associated areas as VA-VL neurons did, and the other population projected mainly to orbital or cingulate areas. Although both VM and IZ neurons receive strong basal ganglia inputs, these results suggest that VM neurons, at a single neuron level, innervate the apical dendrites of cortical pyramidal neurons more intensely and more widely than IZ neurons.
Asunto(s)
Corteza Cerebral/citología , Neuronas/citología , Núcleos Talámicos Ventrales/citología , Vías Aferentes/citología , Animales , Axones/ultraestructura , Dendritas/ultraestructura , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
The lateral posterior thalamic nucleus (LP) is one of the components of the extrageniculate pathway in the rat visual system, and is cytoarchitecturally divided into three subdivisions--lateral (LPl), rostromedial (LPrm), and caudomedial (LPcm) portions. To clarify the differences in the dendritic fields and axonal arborisations among the three subdivisions, we applied a single-neuron labeling technique with viral vectors to LP neurons. The proximal dendrites of LPl neurons were more numerous than those of LPrm and LPcm neurons, and LPrm neurons tended to have wider dendritic fields than LPl neurons. We then analysed the axonal arborisations of LP neurons by reconstructing the axon fibers in the cortex. The LPl, LPrm and LPcm were different from one another in terms of the projection targets--the main target cortical regions of LPl and LPrm neurons were the secondary and primary visual areas, whereas those of LPcm neurons were the postrhinal and temporal association areas. Furthermore, the principal target cortical layers of LPl neurons in the visual areas were middle layers, but that of LPrm neurons was layer 1. This indicates that LPl and LPrm neurons can be categorised into the core and matrix types of thalamic neurons, respectively, in the visual areas. In addition, LPl neurons formed multiple axonal clusters within the visual areas, whereas the fibers of LPrm neurons were widely and diffusely distributed. It is therefore presumed that these two types of neurons play different roles in visual information processing by dual thalamocortical innervation of the visual areas.
Asunto(s)
Corteza Cerebral/ultraestructura , Núcleos Talámicos Laterales/ultraestructura , Neuronas/ultraestructura , Vías Visuales/ultraestructura , Animales , Axones , Dendritas , Vectores Genéticos , Masculino , Técnicas de Trazados de Vías Neuroanatómicas , Ratas , Ratas Long-Evans , Ratas Wistar , Virus Sindbis/fisiologíaRESUMEN
This study focuses on the structure and function of the primary sensory neurons that innervate vibrissal follicles in the rat. Both the peripheral and central terminations, as well as their firing properties were identified using intracellular labelling and recording in trigeminal ganglia in vivo. Fifty-one labelled neurons terminating peripherally, as club-like, Merkel, lanceolate, reticular or spiny endings were identified by their morphology. All neurons responded robustly to air puff stimulation applied to the vibrissal skin. Neurons with club-like endings responded with the highest firing rates; their peripheral processes rarely branched between the cell body and their terminal tips. The central branches of these neurons displayed abundant collaterals terminating within all trigeminal nuclei. Analyses of three-dimensional reconstructions reveal a palisade arrangement of club-like endings bound to the ringwulst by collagen fibers. Our morphological findings suggest that neurons with club-like endings sense mechanical aspects related to the movement of the ringwulst and convey this information to all trigeminal nuclei in the brainstem.
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Mecanorreceptores/citología , Ganglio del Trigémino/citología , Vibrisas/fisiología , Animales , Fenómenos Electrofisiológicos , Imagenología Tridimensional , Espacio Intracelular/metabolismo , Masculino , Ratas , Ratas Wistar , Ganglio del Trigémino/fisiologíaRESUMEN
We previously demonstrated that the P2X7 receptor (P2X7R), a purinergic receptor, expressed by mouse cultured cortical astrocytes is constitutively activated without any exogenous stimulus, differing from the case of neurons. It is well known that astrocytic morphology differs between in vitro and in vivo situations, implying different functionalities. Brain acute slices are widely accepted as an in vitro experimental system that reflects in vivo cell conditions better than in vitro cell culture ones. We examined whether astrocytic P2X7Rs exhibited constitutive activation in mouse cortical slices. In acute cortical slices, P2X7R-immunoreactivity was detected in both glial fibrillary acidic protein-immunopositive astrocytes and microtubule-associated protein 2-immunopositive neurons. Astrocytic, but not neuronal, spontaneous uptake of propidium iodide, an indicator of P2X7R channel/pore activity, was inhibited by representative antagonists of P2X7R, but they had no effect on the uptake by astrocytes in membrane-permeabilized fixed slices. These findings indicate that astrocytes, but not neurons, in acute cortical slices exhibit constitutive activation of P2X7Rs under non-stimulated resting conditions as in the case of cell culture systems.
Asunto(s)
Astrocitos/metabolismo , Encéfalo/citología , Neuronas/metabolismo , Receptores Purinérgicos P2X7/metabolismo , Animales , Transporte Biológico , Células Cultivadas , Femenino , Regulación de la Expresión Génica/fisiología , Ratones , Propidio/farmacocinética , Receptores Purinérgicos P2X7/genética , Coloración y EtiquetadoRESUMEN
Large projection neurons in lamina III of the rat spinal cord that express the neurokinin 1 receptor are densely innervated by peptidergic primary afferent nociceptors and more sparsely by low-threshold myelinated afferents. However, we know little about their input from other glutamatergic neurons. Here we show that these cells receive numerous contacts from nonprimary boutons that express the vesicular glutamate transporter 2 (VGLUT2), and form asymmetrical synapses on their dendrites and cell bodies. These synapses are significantly smaller than those formed by peptidergic afferents, but provide a substantial proportion of the glutamatergic synapses that the cells receive (over a third of those in laminae I-II and half of those in deeper laminae). Surprisingly, although the dynorphin precursor preprodynorphin (PPD) was only present in 4-7% of VGLUT2 boutons in laminae I-IV, it was found in 58% of the VGLUT2 boutons that contacted these cells. This indicates a highly selective targeting of the lamina III projection cells by glutamatergic neurons that express PPD, and these are likely to correspond to local neurons (interneurons and possibly projection cells). Since many PPD-expressing dorsal horn neurons respond to noxious stimulation, this suggests that the lamina III projection cells receive powerful monosynaptic and polysynaptic nociceptive input. Excitatory interneurons in the dorsal horn have been shown to possess I(A) currents, which limit their excitability and can underlie a form of activity-dependent intrinsic plasticity. It is therefore likely that polysynaptic inputs to the lamina III projection neurons are recruited during the development of chronic pain states.
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Dinorfinas/metabolismo , Red Nerviosa/citología , Vías Nerviosas/fisiología , Neuronas/fisiología , Médula Espinal/citología , Análisis de Varianza , Animales , Péptido Relacionado con Gen de Calcitonina/metabolismo , Masculino , Microscopía Electrónica de Transmisión , Neuronas/clasificación , Neuronas/citología , Terminales Presinápticos/metabolismo , Terminales Presinápticos/ultraestructura , Precursores de Proteínas/metabolismo , Ratas , Ratas Wistar , Receptores de Neuroquinina-1/metabolismo , Sinapsis/metabolismo , Sinapsis/ultraestructura , Proteína 2 de Transporte Vesicular de Glutamato/metabolismoRESUMEN
The rostral sector of the posterior thalamic nuclei (POm) is, together with the ventral posterior nuclei (VP), involved in somatosensory information processing in rodents. The POm receives inputs from the spinal cord and trigeminal nuclei and projects to the primary somatosensory (S1) cortex and other cortical areas. Although thalamocortical axons of single VP neurons are well known to innervate layer (L) 4 of the S1 cortex with distinct columnar organization, those of POm neurons have not been elucidated yet. In the present study, we investigated complete axonal and dendritic arborizations of single POm neurons in rats by visualizing the processes with Sindbis viruses expressing membrane-targeted fluorescent protein. When we divided the POm into anterior and posterior parts according to calbindin immunoreactivity, dendrites of posterior POm neurons were wider but less numerous than those of anterior neurons. More interestingly, axon fibers of anterior POm neurons were preferentially distributed in L5 of the S1 cortex, whereas those of posterior neurons were principally spread in L1 with wider and sparser arborization than those of anterior neurons. These results suggest that the POm is functionally segregated into anterior and posterior parts and that the 2 parts may play different roles in somatosensory information processing.
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Axones/ultraestructura , Corteza Cerebral/ultraestructura , Vías Nerviosas/ultraestructura , Núcleos Talámicos/ultraestructura , Animales , Vectores Genéticos/genética , Masculino , Ratas , Ratas Sprague-Dawley , Virus Sindbis/fisiología , TransfecciónRESUMEN
A characteristic feature of the somatosensory cortex in rodents is the presence of discrete cellular aggregates in layer 4 (barrels) that process input from the mystacial vibrissae. Just like thalamic cells that relay vibrissal information to the barrels, barrel cells display directional preference to whisker motion. The present study examined whether the projection of single thalamic cells into a barrel is consistent with the existence of an orderly map of direction preference. The direction preference of single thalamic cells was assessed, and axonal projections were visualized after juxtacellular labeling with biotinylated dextran. Results show that the terminal field of individual thalamic neurons in a barrel is markedly anisotropic and that the location of boutons with respect to the somatotopic map is either positively or negatively correlated with the angular tuning of the thalamic neuron. These results indicate that angular tuning is not represented across a systematic map with fixed anteroposterior/mediolateral coordinates in a barrel. The actual significance of the direction-dependent segregation of thalamocortical terminals in barrels may only come to light in the context of active sensing.
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Neuronas/citología , Terminales Presinápticos/fisiología , Corteza Somatosensorial/citología , Tálamo/citología , Vibrisas/inervación , Potenciales de Acción/fisiología , Animales , Anisotropía , Biotina/análogos & derivados , Biotina/metabolismo , Mapeo Encefálico , Dextranos/metabolismo , Estimulación Eléctrica/métodos , Masculino , Vías Nerviosas/fisiología , Ratas , Ratas WistarRESUMEN
Corticothalamic projection neurons in the cerebral cortex constitute an important component of the thalamocortical reciprocal circuit, an essential input/output organization for cortical information processing. However, the spatial organization of local excitatory connections to corticothalamic neurons is only partially understood. In the present study, we first developed an adenovirus vector expressing somatodendritic membrane-targeted green fluorescent protein. After injection of the adenovirus vector into the ventrobasal thalamic complex, a band of layer (L) 6 corticothalamic neurons in the rat barrel cortex were retrogradely labeled. In addition to their cell bodies, fine dendritic spines of corticothalamic neurons were well visualized without the labeling of their axon collaterals or thalamocortical axons. In cortical slices containing retrogradely labeled L6 corticothalamic neurons, we intracellularly stained single pyramidal/spiny neurons of L2-6. We examined the spatial distribution of contact sites between the local axon collaterals of each pyramidal neuron and the dendrites of corticothalamic neurons. We found that corticothalamic neurons received strong and focused connections from L4 neurons just above them, and that the most numerous nearby and distant sources of local excitatory connections to corticothalamic neurons were corticothalamic neurons themselves and L6 putative corticocortical neurons, respectively. These results suggest that L4 neurons may serve as an important source of local excitatory inputs in shaping the cortical modulation of thalamic activity.
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Neuronas/fisiología , Corteza Somatosensorial/fisiología , Tálamo/fisiología , Animales , Axones/fisiología , Masculino , Vías Nerviosas/citología , Vías Nerviosas/fisiología , Trazadores del Tracto Neuronal , Neuronas/citología , Ratas , Ratas Wistar , Corteza Somatosensorial/citología , Tálamo/citologíaRESUMEN
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.
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Dendritas/ultraestructura , Interneuronas/ultraestructura , Modelos Neurológicos , Animales , Corteza Cerebral/citología , Corteza Cerebral/metabolismo , Técnica del Anticuerpo Fluorescente , Inmunohistoquímica , Interneuronas/metabolismo , Ratones , Ratones Transgénicos , Microscopía Inmunoelectrónica , Técnicas de Cultivo de Órganos , Parvalbúminas/biosíntesis , Técnicas de Placa-ClampRESUMEN
To characterize connexin36 (Cx36)-expressing neurons of the adult rat somatosensory cortex, we examined fluorescence signals for Cx36 messenger RNA (mRNA) in 3 nonoverlapping subpopulations of γ-aminobutyric acid (GABA)ergic interneurons, which showed immunoreactivity for 1) parvalbumin (PV); 2) somatostatin (SOM); and 3) either calretinin (CR), vasoactive intestinal polypeptide (VIP), cholecystokinin (CCK), or choline acetyltransferase (ChAT). About 80% of PV-, 52% of SOM-, 37% of CR/VIP/CCK/ChAT-immunoreactive cells displayed Cx36 signals across all cortical layers, and inversely 64%, 25%, and 9% of Cx36-expressing neurons were positive for PV, SOM, or CR/VIP/CCK/ChAT, respectively. Notably, although almost all Cx36-expressing neurons in layer (L) 4, L5, and L6 were positive for one of these markers, a substantial proportion of those in L1 (91%) and L2/3 (10%) were negative for the markers tested, suggesting that other types of neurons might express Cx36. We further investigated the colocalization of Cx36 mRNA and α-actinin2 immunoreactivity, as a marker for late-spiking GABAergic neurons, by using mirror-image sections. Surprisingly, more than 77% of α-actinin2-positive cells displayed Cx36 signals in L1-L3, and about 49% and 13% of Cx36-expressing neurons were positive for α-actinin2 in L1 and L2/3, respectively. These findings suggest that all the subtypes of GABAergic interneurons might form gap junctions in the neocortex.
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Conexinas/biosíntesis , Neuronas GABAérgicas/metabolismo , Corteza Somatosensorial/metabolismo , Animales , Uniones Comunicantes/metabolismo , Inmunohistoquímica , Hibridación Fluorescente in Situ , Masculino , Ratas , Ratas Wistar , Proteína delta-6 de Union ComunicanteRESUMEN
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).
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Encéfalo , Neuronas , Animales , Ratones , Microscopía ElectrónicaRESUMEN
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.
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Encéfalo , Neuronas , Animales , Encéfalo/metabolismo , Imagenología Tridimensional/métodos , Ratones , Microscopía Confocal/métodosRESUMEN
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.
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Propiocepción , Animales , Corteza Insular , Núcleos Talámicos Intralaminares , Neuronas Motoras , Husos Musculares , Vías Nerviosas , Ratas , Ratas WistarRESUMEN
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.