Y. Shinoda

Cerebellar input to corticothalamic neurons in layers V and VI in the motor cortex

To investigate whether corticothalamic (CT) neurons in the motor cortex (Mx) receive cerebellar input via the ventroanterior-ventrolateral nucleus of the thalamus (VA-VL), we recorded intracellular potentials from neurons in the Mx of anesthetized cats and examined effects of stimulation of the VA-VL and the brachium conjunctivum on them. After this electrophysiological identification, horseradish peroxide (HRP) was injected iontophoretically into the recorded neurons for morphological analysis. We identified 34 neurons as CT neurons by their antidromic response to stimulation of the VA-VL, of which 13 were layer VI CT neurons and 21 were layer V CT neurons. A majority of the CT neurons of both layers VI and V received monosynaptic excitatory postsynaptic potentials (EPSPs) from the VA-VL and di- or polysynaptic EPSPs from the cerebellum. The laminar distribution and morphological characteristics of single CT neurons receiving cerebellar input were analyzed on 19 HRP-labeled CT neurons. Eight layer V and six layer VI CT neurons were reconstructed from serial sections. All the reconstructed layer VI CT neurons were modified pyramidal neurons whose apical dendrites ended in layer III or V, and all the stained layer V CT neurons were typical pyramidal neurons, although the laminar and tangential distribution of recurrent collaterals of these neurons varied from neuron to neuron.

Innervation of multiple neck motor nuclei by single reticulospinal tract axons receiving tectal input in the upper cervical spinal cord

Axons of reticulospinal neurons (RSNs) activated monosynaptically by stimulation of the contralateral superior colliculus (SC) were stained with intraaxonal injection of horseradish peroxidase in the cat upper cervical spinal cord. Stem axons of single RSNs gave rise to multiple axon collaterals to laminae IX, VIII and VII over a few cervical segments. Single RSNs made contacts with retrogradely labeled neck motoneurons of different neck muscles. Therefore, RSNs were regarded as mediating output of the SC to functionally different groups of neck muscles simultaneously. The result gave evidence of neural implementation of a functional synergy for a neck movement at a single neuron level.

Morphology of single axons of tectospinal neurons in the upper cervical spinal cord

Morphology of single axons of tectospinal (TS) neurons was investigated by intraaxonal injection of horseradish peroxidase (HRP) at the upper cervical spinal cord of the cat. TS axons were electrophysiologically identified by their direct responses to stimulation of the contralateral superior colliculus (SC). None of these axons responded to thoracic stimulation at Th2. Three‐dimensional reconstructions of the axonal trajectories were made from 20 well‐stained TS axons at C1‐C3. Cell bodies of these axons were located in the intermediate or deep layers of the caudal two‐thirds of the SC. Usually, TS axons had multiple axon collaterals, and up to seven collaterals were given off per stem axon [2.7 ± 1.6 (mean ± S.D.); n = 20]. Collaterals had simple structures and ramified a few times mainly in the transverse plane. The number of terminals for each collateral was small. These collaterals terminated in the lateral parts of laminae V–IX, mainly in laminae VI, VII, and VIII. There were usually gaps free from terminal arborizations between adjacent collaterals, because the rostrocaudal spread of each collateral (mean = 700 μm) was narrower than the intercollateral interval (mean = 2,500 μm). Seven of the 19 TS axons had terminals in the lateral parts of laminae V–VIII, with little projection to lamina IX, and the other 12 axons had terminals in lamina IX besides the projection to the lateral parts of laminae V–VIII. Axon terminals in lamina IX did not appear to make contacts with the somata or proximal dendrites of retrogradely labeled motoneurons, but contacts were found with the somata of counterstained interneurons in the lateral parts of laminae V–VIII. Three spinal interneurons (two in lamina VIII and one in lamina V at C1) that received monosynaptic excitation from the SC were stained, and their axonal trajectories were reconstructed. They had multiple axon collaterals at C1‐C2 and mainly projected to laminae VIII and IX, with smaller projections to lamina VII. Many axon terminals of the interneurons were found in multiple neck motor nuclei, where some of them made contacts with retrogradely labeled motoneurons. The present finding provides evidence that the direct TS projection to the spinal cord may influence activities of multiple neck muscles, mainly via spinal interneurons, and may play an important role in control of head movement in parallel with the tectoreticulospinal system.

Morphology of axon collaterals of single climbing fibers in the deep cerebellar nuclei of the rat

Projection of inferior olive (IO) neurons to the deep cerebellar nuclei (CN) was investigated in the rat by reconstructing single axons that were labeled with biotinylated dextran amine injected into the IO. All reconstructed terminal arborizations in the CN (n = 18) arose as collaterals from climbing fibers (CFs). One to six nuclear collaterals were given off from each of six CFs that were reconstructed along the nearly entire pathway backward from cortical terminal arborizations to the IO. Nuclear collaterals were much thinner (0.2-0.3 micron in diameter) than stem axons projecting to Purkinje cells (0.7-1.4 microns). The number of swellings per a single nuclear collateral ranged from 24 to 118 (n = 18). Terminal arborizations of nuclear collateral originating from a single CF spread for some hundreds of micrometers and occupied a localized portion within the CN.

Origin of Auditory Brainstem Responses in Cats: Whole Brainstem Mapping, and a Lesion and HRP Study of the Inferior Colliculus

In order to identify the origin of five waves of auditory brainstem responses (ABRs) in cats, we designed three experiments. In particular, we focused on the inferior colliculus (IC). Experiment 1: Evoked potential maps of the frontal sections of the brainstem from the level of the cochlear nuclei to the level of the ICs were constructed from the results of whole brainstem field-potential analysis comparing ABR peaks. Experiment 2: Both ICs were aspirated and the brainstem and midbrian were transected along the midline. Experiment 3: HRP was injected to the central nucleus of the IC to cause phase reversal of field potentials, and localization of brainstem auditory neurons projecting to the IC was studied. The results of these experiments suggest that each wave of the ABR is elicited from multiple sources of auditory brainstem nuclei and tracts only.

Serial electron microscopic reconstruction of axon terminals on physiologically identified thalamocortical neurons in the cat ventral lateral nucleus

The distribution of different types of terminals on different portions of single thalamocortical neurons (TCNs) was quantitatively investigated in the cat ventral lateral nucleus (VL) by the application of computer‐assisted three‐dimensional reconstruction from serial ultrathin sections. Single neurons in the VL were intracellularly penetrated with a glass micropipette filled with horseradish peroxidase (HRP), and were electrophysiologically identified as TCNs by their antidromic responses to stimulation of the motor cortex. These TCNs received monosynaptic excitation from the contralateral cerebellum. After electrophysiological identification, they were injected with HRP iontophoretically. The spatial distribution of terminals of different types on two identified TCNs was analyzed on serial ultrathin sections, some of which were stained by a postembedding immunogold technique by using a γ‐aminobutyric acid (GABA) antibody. Terminals that synapsed on the injected cells were categorized as LR terminals (GABA‐negative large axon terminals containing round vesicles), SR terminals (GABA‐negative small axon terminals containing round vesicles), P terminals (GABA‐positive axon terminals of various sizes containing pleomorphic vesicles), or PSDs (presynaptic dendrites). The order of dendritic branches of labeled TCNs was determined by computer‐assisted reconstruction from serial sections. LR terminals made contacts mainly with proximal dendrites of TCNs. SR terminals made contacts predominantly with distal dendrites, and were never found on somata or primary dendrites. P terminals were observed on somata and on every portion of the dendritic trees. Synapses formed by PSDs were concentrated on the proximal dendrites and sometimes formed synaptic triads with LR terminals. Only a few terminals were found on somata, all of which were P type. Therefore, terminals belonging to different classes were not uniformly distributed on the somata and dendrites of single TCNs. These results suggest that terminals originating from different sources may preferentially contact specific regions of TCNs in the VL, and their topographical locations reflect the electrophysiological response properties of the TCNs. J. Comp. Neurol. 388:613–631, 1997.

Four Convergent Patterns of Input from the Six Semicircular Canals to Motoneurons of Different Neck Muscles in the Upper Cervical Cord

This study was performed to investigate the pattern of input and the pathways from the six semicircular canals to motoneurons of various neck muscles in anesthetized cats. Intracellular postsynaptic potentials from neck motoneurons were recorded in response to electrical stimulation of the six ampullary nerves. The results showed that motoneurons of a particular neck muscle have a homogeneous convergent pattern of input from the six semicircular canals; there are four patterns of input from the six semicircular canals to motoneurons of various neck muscles; and the trisynaptic connection between the semicircular canal nerves and neck motoneurons was identified in addition to the disynaptic connection.

Projection pattern of single corticocortical fibers from the parietal cortex to the motor cortex.

Arborization of single corticocortical (CC) axons projecting from the parietal cortex to the motor cortex (Mx) was analysed using an intraaxonal staining technique in the cat. Stem axons arising from cell bodies in area 5 ramified repeatedly into numerous terminal branches in the Mx, forming 2-6 patches (0.2-0.8 mm in diameter) separated by a terminal-free gap. Axon terminals were distributed mainly in layers II and III and sparsely in layers V, VI and I. This feature is quite similar to that of thalamocortical axons and other corticocortical fibres. Thus the patchy organization may be a basic input structure for afferents of the Mx and play a role in generation of adequate motor output patterns in the Mx.

Three-dimensional analysis of cerebellar terminals and their postsynaptic components in the ventral lateral nucleus of the cat thalamus

Relationships among cerebellar terminals (CTs), dendrites of thalamocortical projection neurons (TCNs), and dendrites of local circuit neurons in the ventral lateral nucleus of the cat thalamus were analyzed quantitatively by observing several series of serial ultrathin sections and by using a computer‐assisted program for the three‐dimensional reconstruction from serial ultrathin sections. In pentobarbital‐anesthetized cats, CTs were labeled either by injections of wheat germ agglutinin conjugated to horseradish peroxidase (WGA‐HRP) into the cerebellar nuclei or by intra‐axonal injection of HRP after electrophysiological identification. By using two series of 133 and 73 serial sections, mutual relationships between 43 WGA‐HRP‐labeled CTs and their postsynaptic structures were analyzed based on their synaptic specializations and shapes of synaptic vesicles. Thirty‐nine of these CTs formed a synapse with one TCN dendrite, whereas only four CTs formed synapses with two TCN dendrites. These CTs also synapsed on dendrites containing pleomorphic synaptic vesicles (presynaptic dendrites). Single CTs synapsed on 0–6 presynaptic dendrites (2.2 ± 1.5, N = 43) through their whole extents, and about 40% of these presynaptic dendrites that were contacted by CTs established synaptic contacts with the same TCN dendrites on which the CTs synapsed. Thus, a CT, a presynaptic dendrite, and a TCN dendrite formed a triadic arrangement. Triadic arrangements were identified in approximately 60% of these 43 CTs. However, they rarely had a glomerulus‐like appearance, as described previously in the ventral lateral nucleus and other main thalamic relay nuclei. In another series of 83 and 43 serial sections along dendrites of TCNs, observations were focused on the triadic arrangement. Triadic arrangements were located evenly on the primary and secondary dendrites of TCNs. Computer‐assisted three‐dimensional reconstructions were made on one WGA‐HRP‐labeled CT and two intra‐axonally labeled CTs (a bouton en passant and a bouton terminal) with their surrounding neuronal elements, and complex spatial arrangement of neuronal processes became obvious. These results provide the quantitative assessment of synaptic arrangements among CTs, presynaptic dendrites, and TCN dendrites and reveal their spatial interrelations in the cat ventral lateral nucleus.