Morphologic characteristics of physiologically defined neurons in the cat trigeminal nucleus principalis

Although the principalis nucleus (Vp) contains trigeminothalamic and internuclear tract cells, the functional and morphologic differences between the two kinds of neurons have remained unsettled. The present study was aimed to address these problems by using the intracellular horseradish peroxidase injection technique in the cat. Of 20 neurons stained, 7 and 13 were located in the dorsomedial subnucleus (Vpd) and ventrolateral subnucleus (Vpv) of Vp, respectively. The Vpd neurons received input from the intraoral structures only but the Vpv neurons from the intraoral or facial structures. Nineteen neurons could be divided as class I and class II, based on the branching pattern of their stem axons. Class I (eight neurons) had an ascending stem axon without branching. Class II was divided into two subclasses (IIa and IIb). Class IIa (eight neurons) had an ascending stem axon from which branches were given off. Their branches formed a local-circuit restricted to the lower brainstem. Class IIb (three neurons) had a stem axon that formed the local-circuit only. The dendritic morphology was indistinguishable between different classes of neurons and between the subdivisions. Although the dendritic arborization pattern was governed by the location of the somata, it was suggested to be also important elements for determining primary afferent arborizations. In the brainstem nuclei, the jaw-closing motor nucleus received the highest density of projections from class II neurons with the receptive field involving the periodontal ligaments. The present study provides new findings that Vp neurons could be divided into three distinct populations and suggests that each population exerts a distinct function with respect to sensory discrimination, sensorimotor reflexes, or both. J. Comp. Neurol. 401:308–328, 1998. © 1998 Wiley-Liss, Inc.     

Tumor necrosis factor-alpha inhibits physiologically identified dorsal motor nucleus neurons in vivo

Our previous studies have shown that tumor necrosis factor-alpha (TNF-alpha) activates solitary nucleus neurons involved in vago-vagal reflex control of gastric motility. Here, we describe the dual role of TNF-alpha as also modulating neurons in the dorsal motor nucleus of the vagus (DMN) that respond to gastric distention. A large majority of physiologically identified DMN neurons are rapidly and completely inhibited by exposure to TNF-alpha, suggesting that TNF-alpha may induce gastric stasis by functioning as a hormone that modulates both portions of this reflex pathway during illness.     

Morphological and functional properties of trigeminal nucleus oralis neurons projecting to the trigeminal motor nucleus of the cat

Horseradish peroxidase (HRP) was injected into the somata located in the rostrodorsomedial part (Vo.r) of the trigeminal nucleus oralis; an axonal projection to the trigeminal motor nucleus (Vmo) was demonstrated in two Vo.r neurons. The two neurons differed in their morphological and functional properties. The first Vo.r neuron responded to stimulation of low-threshold mechanoreceptors and its stem axon gave off massive axon collaterals that issued terminal branches to the dorsolateral subdivision of Vmo, Vo.r, and the medial and lateral parts of the lower brainstem reticular formation. The second Vo.r neuron was activated by stimulation of the tooth pulp or lingual nerve at twice longer latency than that of the first neuron. This stem axon was divided into two main ascending and one descending branches, and one of the main ascending branches was further bifurcated into two branches. The main non-bifurcated ascending branch gave off 4 collaterals, two of which sent terminal branches into the dorsolateral subdivision of Vmo and others into the Vo.r and juxta-trigeminal regions. The somato-dendroarchitectonic differences were also described in the two Vo.r neurons stained.     

Glycine-immunoreactive synaptic terminals in the nucleus tractus solitarii of the cat: ultrastructure and relationship to GABA-immunoreactive terminals.

Postembedding immunogold labeling methods applied to ultrathin and semithin sections of cat dorsomedial medulla showed that neuronal perikarya, dendrites, myelinated and nonmyelinated axons, and axon terminals in the nucleus tractus solitarii contain glycine immunoreactivity. Light microscopic observations on semithin sections revealed that these immunoreactive structures were unevenly distributed throughout the entire nucleus. At the electron microscopic level, synaptic terminals with high levels of glycine-immunoreactivity, assumed to represent those releasing glycine as a neurotransmitter, were discriminated from terminals containing low, probably metabolic levels of glycine-immunoreactivity, by a quantitative analysis method. This compared the immunolabeling of randomly sampled terminals with a reference level of labeling derived from sampling the perikarya of dorsal vagal neurones. The vast majority of these "glycinergic" terminals contained pleomorphic vesicles, formed symmetrical synaptic active zones, and targeted dendrites. They appeared to be more numerous in areas of the nucleus tractus solitarii adjoining the tractus solitarius, but rather scarce caudally, medially, ventrally, and in the dorsal motor vagal nucleus. In a random analysis of the entire nucleus tractus solitarii, 26.2% of sampled terminals were found to qualify as glycine-immunoreactive. In contrast, boutons immunoreactive for gamma-aminobutyric acid (GABA) were more evenly distributed throughout the dorsal vagal complex and accounted for 33.7% of the synaptic terminals sampled. A comparison of serial ultrathin sections suggested three subpopulations of synaptic terminals: one containing high levels of both GABA- and glycine-immunoreactivities (21% of all terminals sampled), one containing only GABA-immunoreactivity (12.7%), and relatively few terminals (5.2%) that were immunoreactive for glycine alone. These results were confirmed by dual labeling of sections using gold particles of different sizes. This study reports the first analysis of the ultrastructure of glycinergic nerve terminals in the cat dorsal vagal complex, and the pattern of coexistence of glycine and GABA observed provides an anatomical explanation for our previously reported inhibitory effects of glycine and GABA on neurones with cardiovascular and respiratory functions in the nucleus tractus solitarii.     

Cerebellar and cerebral inputs to physiologically identified efferent cell groups in the red nucleus of the cat

Cells of the red nucleus of the cat have been classified into 4 projection sets, consisting of cells which could be fired antidromically from (1) only the contralateral spinal cord, (2) only the ipsilateral medulla, (3) both the contralateral spinal cord and the ipsilateral medulla, and (4) both the contralateral spinal cord and brachium conjunctivum. Each of these projection sets receives excitatory synaptic input from axons leaving the cerebellum in the superior cerebellar peduncle, crossing to the red nucleus of the opposite side, and sending axon collaterals — in at least some cases — to the thalamus. Most cells in each projection set are also excited following stimulation of the ipsilateral precruciate cerebral cortex. Rubral cells with medullary, cerebellar, and spinal-projecting axons are found dispersed throughout the rostrocaudal extent of the nucleus, although caudal cells are primarily rubrospinal and rubral cells sending axons to the ipsilateral medulla are predominantly located in the rostral portions of the red nucleus.     

Cerebral cortical projections to the reticular regions around the trigeminal motor nucleus in the cat

Cerebral cortical regions which send projection fibers to the reticular regions around the trigeminal motor nucleus were identified in the cat by the horseradish peroxidase (HRP) method. The reticular region around the trigeminal motor nucleus are known to contain many interneurons for masticatory motoneurons. After injections of HRP into the reticular regions around the trigeminal motor nucleus, HRP-labeled neuronal cell bodies in the cerebral cortex were found in layer V. They were distributed bilaterally in the orbitofrontal cortical regions, mainly in the rostral extension of the orbital gyrus close to the presylvian sulcus; more were located in the floor and lateral bank of the presylvian sulcus than in the crown of the orbital gyrus. After injections of HRP conjugated with wheat germ agglutinin (WGA-HRP) into these cortical regions, many labeled presumed axon terminals were distributed bilaterally in the reticular regions around the trigeminal motor nucleus; mainly in the region ventral to the trigeminal motor nucleus and in the intertrigeminal region between the main sensory trigeminal nucleus and the trigeminal motor nucleus. Terminal labeling in these regions was more prominent after WGA-HRP injection into the lateral bank of the presylvian sulcus than after WGA-HRP injection into the crown of the orbital gyrus. Thus, the present results indicate that the main part of the cortical region projecting directly to the reticular regions around the trigeminal motor nucleus in the cat is folded into the presylvian sulcus.     

Quantitative ultrastructure of slowly adapting lingual afferent terminals in the principal and oral nuclei in the cat

Previous studies provide evidence that a structure/function correlation exists in the cytoarchitectonically different zones of the trigeminal sensory nuclei. To extend this relationship, we examined the ultrastructural features of trigeminal primary afferent neurons in the cat dorsal principal nucleus (Vpd) and the rostrodorsomedial oral nucleus (Vo.r) using intra-axonal labeling with horseradish peroxidase and morphometric analyses. All labeled boutons contained round synaptic vesicles. Eighty-two percent of the boutons in the Vo.r and 99% of the boutons in the Vpd were presynaptic to nonprimary dendrites. The remaining boutons in the Vo.r were presynaptic to somata (8%) or primary dendrites (10%). The average number of postsynaptic profiles per labeled bouton did not differ in the Vpd and Vo.r. Most labeled boutons in the two nuclei were postsynaptic to unlabeled axon terminals with pleomorphic vesicles (p-ending). The number of p-endings per labeled bouton was higher in the Vpd than Vo.r A morphometric analysis indicated that labeled bouton volume and apposed surface area were larger in the Vpd than Vo.r while active zone area and vesicle number did not differ. All these parameters were larger than those of p-endings in each nucleus. In both labeled boutons and p-endings, the parameters were positively correlated with bouton size. These results suggest that sensory information conveyed through trigeminal afferents is more strongly controlled at the level of the first synapse by presynaptic mechanisms in the Vpd than in the Vo.r, while the efficacy of transmission at primary afferent synapses does not differ.     

The morphology of physiologically identified GABAergic neurons in the somatic sensory part of the thalamic reticular nucleus in the cat

Neurons with somatic sensory receptive fields were examined electrophysiologically in the thalamic reticular nucleus of the cat. All cells had receptive fields much larger than those of neurons in the ventral posterior nucleus and were driven by less readily defined somesthetic stimuli. Response latencies to peripheral or medial lemniscal stimulation were, on average, longer than in the ventral posterior nucleus and suggested activation of the reticular nucleus cells by collaterals of thalamocortical relay cell axons arising in the ventral posterior nucleus. When injected intracellularly with horseradish peroxidase, reticular nucleus cells displayed thin axons with intrareticular collaterals and diffuse branches through much of the ventral posterior and posterior thalamic nuclei. Dendrites ended in processes resembling synaptic terminals. Electron microscopic immunocytochemistry of the same part of the reticular nucleus revealed processes immunoreactive for glutamic acid decarboxylase and identifiable as both collateral axon terminals and presynaptic dendrites of GABAergic reticular nucleus cells. These synaptically linked reticular nucleus cells and, in addition, immunoreactive somata and presynaptic dendrites received synapses from at least three varieties of nonimmunoreactive profiles.     

Tumor necrosis factor-α inhibits physiologically identified dorsal motor nucleus neurons in vivo

Our previous studies have shown that tumor necrosis factor-alpha (TNF-alpha) activates solitary nucleus neurons involved in vago-vagal reflex control of gastric motility. Here, we describe the dual role of TNF-alpha as also modulating neurons in the dorsal motor nucleus of the vagus (DMN) that respond to gastric distention. A large majority of physiologically identified DMN neurons are rapidly and completely inhibited by exposure to TNF-alpha, suggesting that TNF-alpha may induce gastric stasis by functioning as a hormone that modulates both portions of this reflex pathway during illness.     

Glycine-immunoreactive terminals in the rat trigeminal motor nucleus: light- and electron-microscopic analysis of their relationships with motoneurones and with GABA-immunoreactive terminals

Post-embedding immunolabelling methods were applied to semi-thin and ultrathin resin sections to examine the relationships between glycine- and γ-aminobutyric acid (GABA)-immunoreactive terminals on trigeminal motoneurones, which were identified by the retrograde transport of horseradish peroxidase injected into the jaw-closer muscles. Serial sections were cut through boutons and alternate sections were incubated with antibodies to glycine and GABA. Light-microscopic analysis of semi-thin sections revealed a similar pattern of glycine and GABA-immunoreactive boutons along the motoneurone soma and proximal dendrites, and of immunoreactive cell bodies in the parvocellular reticular and peritrigeminal areas surrounding the motor nucleus. Immunoreactive synaptic terminals on motoneurones were identified on serial ultrathin sections at electron-microscopic level using a quantitative immunogold method. Three populations of immunolabelled boutons were recognized: boutons immunoreactive for glycine alone (32%), boutons immunoreactive for GABA alone (22%), and boutons showing co-existence of glycine and GABA immunoreactivities (46%). Terminals which were immunoreactive for glycine only contained a higher proportion of flattened synaptic vesicles than those which were immunoreactive for GABA only, which contained predominantly spherical vesicles. Terminals which exhibited both immunoreactivities contained a mixture of vesicle types. All three classes of terminal formed axo-dendritic and axo-somatic contacts onto retrogradely labelled motoneurones. A relatively high proportion (25%) of boutons that were immunoreactive for both transmitters formed synapses on somatic spines. However, only GABA-immunoreactive boutons formed the presynaptic elements at axo-axonic contacts: none of these were found to contain glycine immunoreactivity. These data provide ultrastructural evidence for the role of glycine and GABA as inhibitory neurotransmitters at synapses onto jaw-closer motoneurones, but suggest that presynaptic control of transmission at excitatory (glutamatergic) synapses on motoneurones involves GABAergic, but not glycinergic inhibition.     

Synaptic inputs to physiologically identified retinal X-cells in the cat.

The cat retina contains a number of different classes of ganglion cells, each of which has a unique set of receptive field properties. The mechanisms that underlie the functional differences among classes, however, are not well understood. All of the afferent input to retinal ganglion cells are from bipolar and amacrine cell terminals in the inner plexiform layer, suggesting that the physiological differences among cat retinal ganglion cells might be due to differences in the proportion of input that they receive from these cell types. In this study, we have combined in vivo intracellular recording and labeling with subsequent ultrastructural analysis to determine directly the patterns of synaptic input to physiologically identified X-cells in the cat retina. Our primary aim in these analyses was to determine whether retinal X-cells receive a characteristic pattern of bipolar and amacrine cell input, and further, whether the functional properties of this cell type can be related to identifiable patterns of synaptic input in the inner plexiform layer. We reconstructed the entire dendritic arbor of an OFF-center X-cell and greater than 75% of the dendritic tree of an ON-center X-cell and found that 1) both ON- and OFF-center X-cells are contacted with approximately the same frequency by bipolar and amacrine cell terminals, 2) each of these input types is distributed widely over their dendritic fields, and 3) there is no significant difference in the pattern of distribution of bipolar and amacrine cell synapses onto the dendrites of either cell type. Comparisons of the inputs to the ON- and the OFF-center cell, however, did reveal differences in the complexity of the synaptic arrangements found in association with the two neurons; a number of complex synaptic arrangements, including serial amacrine cell synapses, were found exclusively in association with the dendrites of the OFF-center X-cell. Most models of retinal X-cell receptive fields, because their visual responses share a number of features with those of bipolar cells, have attributed X-cell receptive field properties to their bipolar cell inputs. The data presented here, the first obtained from analyzing the inputs to the entire dendritic arbors of retinal X-cells, demonstrate that these retinal ganglion cells receive nearly one-half of their input from amacrine cells. These results clearly indicate that further data concerning the functional consequences of amacrine cell input are needed to understand more fully visual processing in the X-cell pathway.     

Distribution of four types of synapse on physiologically identified relay neurons in the ventral posterior thalamic nucleus of the cat

This study was aimed at providing quantitative data on the thalamic circuitry that underlies the central processing of somatosensory information. Four physiologically identified thalamocortical relay neurons in the ventral posterior lateral nucleus (VPL) of the cat thalamus were injected with horseradish peroxidase and subjected to quantitative electron microscopy after pre- or postembedding immunostaining for γ-aminobutyric acid to reveal synaptic terminals of thalamic inhibitory neurons. The four cells all had rapidly adapting responses to light mechanical stimuli applied to their receptive fields, which were situated on hairy or glabrous skin or related to a joint. Their dendritic architecture was typical of cells previously described as type I relay cells in VPL, and they lacked dendritic appendages. Terminals ending in synapses on the injected cells were categorized as RL (ascending afferent), F (inhibitory), PSD (presynaptic dendrite), and RS (mainly corticothalamic) types and were quantified in reconstructions of serial thin sections. RL and F terminals formed the majority of the synapses on proximal dendrites (approximately 50% each). The number of synapses formed by RL terminals declined on intermediate dendrites, but those formed by F terminals remained relatively high, declining to moderate levels (20–30%) on distal dendrites. RS terminals formed moderate numbers of the synapses on intermediate dendrites and the majority (< 60%) of the synapses on distal dendrites. Synapses formed by PSDs were concentrated on intermediate dendrites and were few in number (～6%). They formed synaptic triads with F terminals and rarely with RL terminals. On somata, only a few synapses were found, all made by F terminals. The total number of synapses per cell was calculated to be 5,584–8,797, with a density of 0.6–0.9 per micrometer of dendritic length. Of the total, RL terminals constituted approximately 15%, F terminals approximately 35%, PSD terminals approximately 5%, and RS terminals approximately 50%. These results provide the first quantitative assessment of the synaptic architecture of thalamic somatic sensory relay neurons and show the basic organizational pattern exhibited by representatives of the physiological type of relay neuron most commonly encountered in the VPL nucleus. © 1995 Wiley-Liss, Inc.     

Quantitative analysis and postsynaptic targets of GABA-immunoreactive boutons within the rat trigeminal motor nucleus

We have used the post-embedding immunogold labelling method using antibodies to gamma-aminobutyric acid (GABA) to obtain quantitative data on the distribution, frequency, postsynaptic targets and ultrastructural characteristics of GABA-immunoreactive (GABA-IR) boutons in the trigeminal motor nucleus of rats. We have also combined this method with horseradish peroxidase tracing to obtain specific evidence for termination of some GABA-IR boutons onto identified jaw-elevator motoneurones. Twenty-eight percent of all synapses in the motor nucleus involved GABA-IR boutons. Seventy-three percent of the GABA-IR boutons formed axo-dendritic synapses, 13% axo-somatic synapses and 14% axo-axonic synapses. Ninety-three percent of GABA-IR boutons formed symmetrical synapses. Overall, 58% of all boutons contained only flattened vesicles, while 26% contained round vesicles and 16% a mixture of vesicle types. Measurements of bouton cross sectional area, apposition length, and active zone length were obtained from serial reconstructions of 15 GABA-IR boutons and 30 unlabelled boutons. In each case mean values for GABA-IR boutons were significantly smaller than those for nonlabelled boutons.     

Morphology of physiologically identified rubrospinal axons in the spinal cord of the cat

Intra-axonal injection of HRP into physiologically identified rubrospinal neurons has shown that axon collaterals are given off at different cervical segments from stem axons. These collaterals spread in a delta-like fashion in laminae V–VII (occasionally in IX) and extend very widely in a rostrocaudal direction (1.0–5.1 mm).     

Noradrenaline-like terminals in the cat nucleus ventralis posterior of the thalamus

Noradrenaline-like immunoreactivity in the cat nucleus ventralis posterior of the thalamus was investigated using an indirect immunocytochemical technique. Specific antinoradrenaline antibodies, raised in rabbits, were used. It was first verified that these antibodies recognize noradrenaline cells bodies of the locus coeruleus and their ascending axons in the ascending noradrenergic tract. In the nucleus ventralis posterior itself, noradrenaline-like fibers were observed. They were either randomly distributed or grouped around nonlabeled cell bodies. These neurons were generally oblong and measured 60-80 microns. With electron microscopy, preliminary results showed immunoreactive fibers in close apposition to unlabeled cell bodies or dendrites. The precise nature of these profiles was sometimes difficult to ascertain, since experiments were done in presence of detergent. In some cases symmetric synapses might be observed between immunoreactive axon terminals and unlabeled dendrites. The specificity of the reaction is discussed in the light of several control experiments.     

Serotonergic axonal contacts on identified cat trigeminal motoneurons and their correlation with medullary raphe nucleus stimulation

The innervation of the trigeminal motor nucleus by serotonergic fibers with cell bodies in the raphe nuclei pallidus and obscurus suggests that activation of this pathway may alter the excitability of trigeminal motoneurons. Thus, we recorded intracellular responses from cat jaw-closing (JC) and jaw-opening (JO) α-motoneurons evoked by raphe stimulation and used a combination of intracellular staining of horseradish peroxidase (HRP) and immunohistochemistry at the light and electron microscopic levels to examine the distribution of contacts made by serotonin (5-HT)-immunoreactive boutons on the two motoneurons types. Electrical stimulation applied to the nucleus raphe pallidus-obscurus complex induced a monosynaptic excitatory postsynaptic potential (EPSP) in JC (masseter) α-motoneurons and an EPSP with an action potential in JO (mylohyoid) α-motoneurons. The EPSP rise-times (time to peak) and half widths were significantly longer in the JC than in the JO motoneurons. The EPSPs were suppressed by systemic administration of methysergide (2 mg/kg). Six JC and seven JO α-motoneurons were well stained with HRP. Contacts were seen between 5-HT-immunoreactive boutons and the motoneurons. The JC motoneurons received a significantly larger number of the contacts than did the JO motoneurons. The contacts were distributed widely in the proximal three-fourths of the dendritic tree of JC motoneurons but were distributed on more proximal dendrites in the JO motoneurons. At the electron microscopic level, synaptic contacts made by 5-HT-immunoreactive boutons on motoneurons were identified. The present study demonstrated that JC motoneurons receive stronger 5-HT innervation, and this correlates with the fact that raphe stimulation caused larger EPSPs among these neurons than among JO motoneurons. J. Comp. Neurol. 384:443–455, 1997. © 1997 Wiley-Liss, Inc.     

Anatomy of physiologically identified eye-movement-related pause neurons in the cat: pontomedullary region

Pause neurons (PNs) are inhibitory neurons close to the midline at the pontomedullary junction that fire tonically and then cease firing just prior to quick eye movements of visual or vestibular origin. Previous physiological evidence has shown that these neurons have a role of central importance in the generation of rapid eye movements in any direction and all major models of ocular motor control incorporate PNs as major elements. In this study in cats, we injected horseradish peroxidase intracellularly into somata or axons of physiologically identified PNs. After appropriate tissue preparation, cell body and axonal reconstructions were performed, with the aid of a camera lucida-equipped microscope. Fifty-three PNs were stained and reconstructed. These consisted of 17 cell bodies and dendrites and 36 axons. Seven of these included both cell bodies and axons. PN somas lay close to the midline in the nucleus raphe pontis and centralis superior, had extensive dendritic arborizations tending to arise from either pole of the elongated soma, and had axons which typically crossed the midline and bifurcated into long branches which extended rostrally and caudally, inferior to the medial longitudinal fasciculus. There were major terminal arborizations and boutons in areas just rostral and caudal to the abducens nucleus in areas where two types of premotor neurons, excitatory and inhibitory burst neurons, are concentrated. Many axosomatic contacts were noted. Other terminal arborizations and boutons were found close to the midline in a region rostral to abducens nucleus containing other neurons known to burst prior to quick eye movements, and in the nucleus reticularis gigantocellularis. Rostral stem axons could be traced to the level of the trochlear nucleus and inferior to the medial longitudinal fasciculus. The caudal stem axons could be traced parallel to the midline and inferior to the medial longitudinal fasciculus and as far caudally as the hypoglossal nucleus.