Morphology of lumbar motoneurons innervating hindlimb muscles in the turtle Pseudemys scripta elegans: an intracellular horseradish peroxidase study

Motoneurons in the turtle lumbar spinal cord, electrophysiologically identified as innervating a muscle belonging to a functional group, were injected with horseradish peroxidase by electrophoresis. A total of 45 motoneurons were reconstructed from transverse sections. Eleven motoneurons were identified as innervating knee extensor muscles, eight as innervating hip retractor and knee flexor muscles, 14 as supplying ankle and/or toe extensors, and 12 as innervating ankle and/or toe flexor muscles. The cell bodies were elongated and spindle-shaped in the transverse plane. The mean equivalent soma diameter was calculated to be 33.4 micrometers. The mean axon conduction velocity was 15.7 m/second. Significant, though rather weak, positive correlations were found between soma diameter, axon diameter, and axon conduction velocity. The axons of the reconstructed motoneurons did not reveal a recurrent axon collateral. However, a few unidentified motoneurons did possess such collaterals. The dendritic trees were restricted to the ipsilateral side of the cord, but reached out in lateral, ventral, and ventromedial directions to the subpial surface. Easily recognizable and characteristic dendrites were found both in the dorsal dendritic tree and in the dorsomedial dendritic tree. Correlations were calculated between the soma diameter and (1) the number of first-order dendrites, (2) the mean diameter of the first-order dendrites, and (3) the combined diameter of the first-order dendrites. In each case no correlations or only weak correlations were found. Fair correlations were observed between the diameter of a first-order dendrite and the number of terminal dendritic branches (r = .61) and the combined dendritic length (r = .78). However, correlations between the combined diameter of all first-order dendrites per neuron and the total number of terminal dendritic branches and the total combined dendritic length of a neuron were extremely weak. The overall appearance of turtle spinal motoneurons is comparable to that observed in other "lower" vertebrates such as frog and lizard. However, similarities are also observed between certain morphometric parameters in turtle and cat lumbar motoneurons.     

Morphology of retinogeniculate terminals in the turtle, Pseudemys scripta elegans

The dorsal lateral geniculate complex in turtles receives a bilateral, topographic projection from the retina and projects to the telencephalon. This study examined the morphology of individual retinogeniculate terminals that were filled with horseradish peroxidase by injections in the optic tract or optic tectum. A large number of retinogeniculate terminals were successfully filled and detailed drawings were prepared of 87 terminals. Terminals were classified into three types based on the size and number of varicosities in the terminal, and (if a terminal formed a spatially restricted arbor) the volume of the arbor. Type I retinogeniculate terminals form spatially restricted, large-volume arbors with a low density of large varicosities. Type II retinogeniculate terminals form small volume arbors with a high density of small varicosities. Type III retinogeniculate terminals, in contrast to types I and II, do not form spatially restricted arbors. Rather, they consist of sparsely branched axons that parallel the optic tract and contain scattered en passant varicosities. Plots of the distribution of different terminal types throughout the geniculate complex show that all three terminal types occur throughout the rostrocaudal and mediolateral extents of the complex. However, each terminal type has a preferential distribution with type II terminals being concentrated in the outer half of the neuropile, type I terminals in the inner half of the neuropile, and type III terminals in the cell plate. All three types can arise from axons that continue caudally to terminate in the tectum. These findings raise the possibility that various classes of retinal ganglion cells differ in their mode of termination within the geniculate complex, but the precise relation between the three types of retinogeniculate terminals and the classes of ganglion cells remains to be determined.     

Neuropeptide Y-immunoreactive amacrine cells in the retina of the turtle Pseudemys scripta elegans

Antiserum directed against neuropeptide Y selectively labeled certain amacrine cells in the turtle retina. The cell types, sizes, dendritic stratification, regional distribution, and degrees of immunolabeling were examined. The results indicated that three morphologically distinct cell types were labeled: types A, B, and C. Computer rotation of digitized data from camera lucida drawings was used to study dendritic stratification. The type A somata were large (11.5 micron in diameter), well-stained, and located in the third tier of the inner nuclear layer. Type A somata gave rise to well-stained processes which arborized within the inner plexiform layer in strata 1 and 3 and at the border between strata 4 and 5. Processes in stratum 1 were sparse and delicate with small boutons. Processes in stratum 3 were numerous and often coarse, with many large and small boutons. At the border between strata 4 and 5 the processes were frequently numerous but slender, with many small boutons. Occasional immunolabeled processes were found in the ganglion cell layer. The somata of the type B cells were smaller (9.0 micron in diameter) and gave rise to single labeled processes which descended into the inner plexiform layer and divided quickly into two secondary processes. These secondary processes gave rise to lightly labeled dendritic fields which arborized primarily in strata 2 and 4. The type C cells were usually observed at the periphery of the retina and had large somata (12.0 micron in diameter) with simple, but very elongated, dendritic arborizations in strata 1, 4, and 5. Observations also showed that type A and B cells were often found in close proximity to each other and suggested that dendrites of these cells made contact with each other. The labeled neurons were distributed relatively evenly throughout the retina except for the visual streak where they were sparse. This study indicates that neuropeptide Y-like immunoreactivity is found in more than one anatomically distinct class of amacrine cells in the turtle retina.     

Primary afferent projections of the major splanchnic nerve to the spinal cord and gracile nucleus of the cat

Splanchnic afferent projections to the spinal cord and gracile nucleus were labeled following the application of HRP to the central cut end of the major splanchnic nerve. Labeled afferent fibers were detected in the ipsilateral dorsal column, in Lissauer's tract (LT), in laminae 1, 5, 7, and 10, and in the dorsal gray commissure at T1-T13 levels of the spinal cord. Afferent projections were not identified in laminae 2-4. Collaterals from LT projected ventrally along the lateral and medial margins of the dorsal horn (called lateral and medial pathways, respectively). Afferents in the lateral pathway formed small bundles, spaced rostrocaudally at intervals of 300-1,000 microns, which passed medially at the base of the dorsal horn into laminae 5, 7, and 10 and to the contralateral spinal cord. Some afferents in the lateral pathway projected to the intermediolateral nucleus where labeled sympathetic preganglionic neurons were located. Afferents in the medial pathway entered the lateral aspect of the dorsal column and projected as a group near the midline rostrally to the medulla. The dorsal column pathway terminated in the ventral gracile nucleus in four or five clusters, each occupying a region ranging in size from 0.01-0.1 mm3 and separated in the rostrocaudal axis by distances of 400-800 microns. These clusters were concentrated in the middle and caudal portions of the nucleus below the obex. A comparison of the present results with those from earlier experiments on the central projections of afferent fibers from the heart, kidney, and pelvic organs demonstrates a consistent pattern of visceral afferent termination in the thoracolumbar and sacral segments of the spinal cord. This is not unexpected, since visceral afferent pathways to different organs perform similar functions, such as the transmission of nociceptive information and the initiation of autonomic reflexes.     

Morphology of basal optic tract terminals in the turtle,Pseudemys scripta elegans

The morphologies of axon terminals of retinal ganglion cells projecting to the basal optic nucleus (BON) via the basal optic tract (BOT) were studied in the red-eared turtle. The BOT was visualized on the ventral surface of the brainstem in vitro, and either biotinylated dextran amine was injected extracellularly or neurobiotin was injected into physiologically identified axons during intracellular recordings. Up to 16 hours after tracer injection, the brains were fixed, sectioned parasagittally, and stained for biotin and Nissl substance. The diameters and depths of extracellularly filled axons were measured at three BON sites. Fourteen axons were reconstructed from serial sections with the aid of appropriate computer software. Analysis of extracellularly filled retinal axons revealed that about three times more axons were present just inside the rostral border of the BON compared with its caudal border. Thick (2–4 μm) axons were located within 100 μm from the ventral border, whereas thin (<2 μm) axons were found throughout the nucleus. Only the thinnest axons (<1 μm) extended caudally from the nucleus, indicating that some extracellularly labelled fibers passed through the BON. The intracellularly filled axons were more similar to the thick axons filled extracellularly and arborized entirely within the BON. All of the intracellularly filled axons had thick ventral trunks from which many thin branches extended dorsally and obliquely within the BON. The thin branches bifurcated repeatedly to form bead-like varicosities or boutons that often formed clusters within regions of 150 μm³ or less. These clusters may reflect areas of focused synaptic contact on BON cells with specific direction preferences. J. Comp. Neurol. 393:267–283, 1998. © 1998 Wiley-Liss, Inc.     

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).     

Subcellular distribution of L-type Ca2+ channels responsible for plateau potentials in motoneurons from the lumbar spinal cord of the turtle

L-type calcium channels mediate the persistent inward current underlying plateau potentials in spinal motoneurons. Electrophysiological analysis shows that plateau potentials are generated by a persistent inward current mediated by low threshold L-type calcium channels located in the dendrites. As motoneurons express L-type calcium channels of the CaV1.2 and CaV1.3 subtypes, we have investigated the subcellular distribution of these channels using antibody labelling. The plateau generating a persistent inward current is modulated by the activation of metabotropic receptors. For this reason, we also examined the relationship between CaV1.2 and CaV1.3 subunits in motoneurons and presynaptic terminals labelled with antibodies against synapsin 1a. Motoneurons in the spinal cord of the adult turtle were identified as large neurons, immunopositive for choline acetyltransferase, located in the ventral horn. In these neurons, CaV1.2 subunits were present in the cell bodies and axons. Patches of CaV1.3 subunits were seen in association with the cell membrane of the somata and both the proximal and distal dendrites. Double labelling with an antibody against synapsin 1a showed that CaV1.3 subunits, but not CaV1.2 subunits, were always located at synaptic sites. The distribution of CaV1.2 and CaV1.3 strongly suggests that the persistent inward current underlying plateau potentials in spinal motoneurons is mediated by CaV1.3 and not by CaV1.2. Our findings also show that CaV1.3 may be located in the somatic and dendritic membrane adjacent to particular presynaptic terminals.     

An electron microscopic study of terminals of rapidly adapting mechanoreceptive afferent fibers in the cat spinal cord

The intra-axonal horseradish peroxidase technique was used to examine the central terminals of 7 A beta primary afferent fibers from rapidly adapting (RA) mechanoreceptors in the glabrous skin of the cat's hindpaw. At the light microscopic level, labelled collaterals were seen to bear occasional boutonlike swellings, mostly (75-82%) of the en passant type. These swellings were distributed more or less uniformly from lamina III to a dorsal part of lamina VI in the dorsal horn, over a maximum longitudinal extent of about 4 mm. At the electron microscopic level, we observed that labelled boutons of RA afferent fibers were 1.0 to 3.3 micrometers in longest sectional dimension, and contained clear, round synaptic vesicles. They frequently formed asymmetric axospinous and axodendritic synapses and commonly appeared to receive contacts from unlabelled structures containing flattened or pleomorphic vesicles plus occasional large dense-cored vesicles. The examination of synaptic connectivity over the entire surface of individual boutons indicated that RA afferent boutons each made contacts with an average of one spine and one dendrite and, in addition, appeared to be postsynaptic to an average of two unlabelled vesicle-containing structures. This synaptic organization was, in general, more complex than that we had seen previously in Pacinian corpuscle (PC) and slowly adapting (SA) type I mechanoreceptive afferent fibers. Our findings indicate that RA, SA, and PC afferent terminals, while displaying some differential synaptic organizations, have many morphological and synaptological characteristics in common. These afferent terminals, in turn, seem to be generally distinguishable from the terminals of muscle spindle Ia afferents or unmyelinated primary afferents.     

Distribution of vesicular glutamate transporters in the brain of the turtle (Pseudemys scripta elegans)

The distribution of glutamatergic neurons has been extensively studied in mammalian and avian brains, but its distribution in a reptilian brain remains unknown. In the present study, the distribution of subpopulations of glutamatergic neurons in the turtle brain was examined by in situ hybridization using probes for vesicular glutamate transporter (VGLUT) 1–3. Strong VGLUT1 expression was observed in the telencephalic pallium; the mitral cells of the olfactory bulb, the medial, dorsomedial, dorsal, and lateral parts of the cerebral cortex, pallial thickening, and dorsal ventricular ridge; and also, in granule cells of the cerebellar cortex. Moderate to weak expression was found in the lateral and medial amygdaloid nuclei, the periventricular cellular layer of the optic tectum, and in some brainstem nuclei. VGLUT2 was weakly expressed in the telencephalon but was intensely expressed in the dorsal thalamic nuclei, magnocellular part of the isthmic nucleus, brainstem nuclei, and the rostral cervical segment of the spinal cord. The cerebellar cortex was devoid of VGLUT2 expression. The central amygdaloid nucleus did not express VGLUT1 or VGLUT2. VGLUT3 was localized in the parvocellular part of the isthmic nucleus, superior and inferior raphe nuclei, and cochlear nucleus. Our results indicate that the distribution of VGLUTs in the turtle brain is similar to that in the mammalian brain rather than that in the avian brain.     

Morphology of physiologically identified thalamocortical relay neurons in the rat ventrobasal thalamus

The anatomical structure of physiologically identified neurons of the rat ventrobasal thalamus was studied in order to determine if there are morphologically distinct subsets of neurons that correlate with the somatosensory submodalities processed by these cells. Intracellular recordings were used to determine the modality and receptive field of a neuron, after which horseradish peroxidase was iontophoretically injected into the cell, allowing it to be histologically visualized. Computer-assisted measurements of the labeled cells were made to quantitatively analyze the dendritic structure. Cells were divided into physiological groups stimulated by whiskers, glabrous skin, furry skin, noxious stimulation, or joint rotation. Qualitatively, all cells appeared similar, with the same types of branching patterns. Dendritic spines and long, sinuous appendages were found on all distal dendrites. Quantitatively, no statistically significant differences in dendritic structure were found between functionally defined groups with the aid of a number of parameters, including a fitted dendritic ellipse. There was a weak correlation between somal cross-sectional area and receptive field size, suggesting larger cells processed larger receptive fields. In summary, the ventrobasal thalamus of the rat, in contrast to that of higher mammals, appears to contain only one major cell type and to have a very simple neuronal circuitry.     

Organization of corticogeniculate projections in the turtle, Pseudemys scripta

The efferent pathways from the visual cortex to the dorsal lateral geniculate complex of turtles have been studied by using the orthograde and retrograde transport of horseradish peroxidase (HRP). Injections of HRP in the lateral thalamus retrogradely label neurons throughout the visual cortex. The majority of labeled neurons have somata in layer 2 of the lateral part of dorsal cortex (D2); a minority have somata in layer 3. Labeled neurons in layer 2 tend to have vertically oriented, fusiform somata and dendrites that ascend into layer 1. Labeled neurons in layer 3 have fusiform somata and dendrites, both oriented horizontally. Injections of HRP in visual cortex orthogradely label corticofugal axons. Those projecting to the lateral geniculate complex course laterally from the visual cortex, pass through the striatum (occasionally bearing varicosities), and enter the diencephalon in the ventral peduncle of the lateral forebrain bundle. Individual axons leave the ventral peduncle and run dorsally in the transverse plane, entering the dorsal lateral geniculate complex from its ventral edge. They continue dorsally, principally in the cell plate of the geniculate complex, where they bear varicosities.     

The determination of dendrite morphology on lateral rectus motoneurons in cat

Previously developed morphometric analysis of motoneurons (Ulfhake and Kellerth, '81, J. Comp. Neurol. 202: 571-583) was applied to lateral rectus motoneurons (LRMs). Total dendrite size was approximated from a single stem dendrite measurement. Fifteen dendrites from nine LRMs of the principal abducens nucleus intracellularly stained with HRP were morphometrically analyzed. The diameters and lengths along the extent of the dendrite were measured to calculate the surface area, volume, and combined length of the process. Linear correlation of stem dendrite diameter to these size parameters produced r values of .80, .84, and .61, respectively. Although the regression lines could be used to estimate dendrite size from the stem dendrite diameter, two morphologically distinct types were found among the 83 dendrites of the nine cells. Six dendrites differed from the other 77. Therefore, these six and a representative sample of the more common dendrite (nine) were included in the measurements. The rare dendrites consistently branched at about 40 micron from the soma into a rostrally and a caudally directed secondary dendrite. The secondary dendrites branched less and reduced more in diameter by tapering. Also, these dendrites exhibited a higher than expected total dendrite size to stem diameter ratio compared to "regular" dendrites. Statistical correlations of the stem diameter to surface area or volume within each dendrite type showed clear increases in r values from those of all 15. Significant differences were found between the size parameters of the two types. These qualitative and quantitative differences should be considered in accurate motoneuron size determinations in the abducens nucleus.     

Identification of neuropeptides in pelvic and pudendal nerve afferent pathways to the sacral spinal cord of the cat

The distribution of several neuropeptides, including vasoactive intestinal polypeptide (VIP), substance P, somatostatin, leucine enkephalin, methionine enkephalin, and cholecystokinin, in sacral afferent pathways of the cat was examined by immunohistochemical techniques. Certain peptides (substance P, somatostatin, and leucine enkephalin) could be demonstrated in normal dorsal root ganglion cells; however, topical administration or injections of colchicine solution into ganglia 36-56 hours prior to removal markedly increased the number of cells labeled and the intensity of staining. Other peptides (VIP, cholecystokinin, and methionine enkephalin) were only detected in significant numbers of cells following intraganglionic injections of colchicine. The distribution of peptides in dorsal root ganglion cells projecting to the pelvic nerve (visceral) and the pudendal nerve (somatic) was examined by retrograde dye labeling combined with immunohistochemistry. Fluorescent dyes were applied to the cut ends of the nerves 2 weeks prior to removal. A considerably higher percentage of pelvic nerve afferent neurons than pudendal nerve afferent neurons exhibited peptide immunoreactivity; e.g., VIP (42% vs. 10%), cholecystokinin (29% vs. 12%), substance P (24% vs. 21%), leucine enkephalin (30% vs. 24%), and methionine enkephalin (10% vs. 3%). Somatostatin was present in only a small percentage of either type of afferent neuron (0.3-2%). The total percentage of peptide-containing pelvic afferent neurons exceeded 100% (137%), suggesting that more than one peptide is present in some visceral afferent neurons. This has been confirmed in preliminary experiments. The peptide-containing cells were in general less than 40 micron in average diameter; however, a significant percentage of substance P and cholecystokinin neurons ranged from 40 to 60 micron in average diameter. VIP cells had the smallest average diameter (30 micron) whereas somatostatin cells had the largest average diameter (36 micron). Statistical analysis of cell sizes revealed that substance P cells projecting to the pelvic nerve were smaller than substance P cells sending axons into the pudendal nerve. On the other hand, VIP cells in the two afferent pathways were not significantly different in size. Sacral visceral and somatic afferent neurons contain a wide spectrum of neuropeptides, some of which (e.g., VIP and cholecystokinin) seem to be preferentially distributed in the visceral afferent systems.(ABSTRACT TRUNCATED AT 400 WORDS)     

Morphology and laminar organization of electrophysiologically identified neurons in the primary auditory cortex in the cat

The morphology of electrophysiologically identified neurons was examined in the primary auditory cortex (AI) of the cat. After stimulation of the medial geniculate nucleus (MG), second auditory cortex, posterior ectosylvian gyrus, contralateral AI, or corpus callosum, intracellular potentials were recorded from AI neurons, which were then injected intracellularly with horseradish peroxidase and recovered. Layer IV neurons, which receive MG fibers monosynaptically, are spiny and nonspiny stellate cells, small and medium-sized nonspiny tufted cells, and fusiform cells. They send their axons to layer III of the AI. Corticocortical AI neurons are medium-sized pyramidal cells in layer III. They receive axons from layer IV neurons of the AI and send their axons to layers I, II, IV, and V of the AI. Horizontal cells in layer I receive slow-conducting MG fibers monosynaptically, and send their axons to layer II of the AI. Stellate cells and small pyramidal cells in layer II receive afferent inputs polysynaptically from the MG. Layer II pyramidal cells receive afferent inputs from the MG via AI neurons in layers I and III, and send their axons to layers V and VI. The axons of layer II stellate cells were distributed within layer II. Pyramidal cells which send their axons to the MG are located in layers V and VI, distributing their axon collaterals to layers III-VI of the AI.     

The projection of the medial and posterior articular nerves of the cat's knee to the spinal cord

We studied the spinal projections of the medial and posterior articular nerves (MAN and PAN) of the knee joint in the cat with the aid of the transganglionic transport of horseradish peroxidase. The afferent fibers of the MAN entered the spinal cord via the lumbar dorsal roots L5 and L6 and those of the PAN entered via the dorsal roots L6 and L7. Within the dorsal root ganglia, most labeled neurons had small to medium diameters. A relatively higher number of medium-size cell bodies were labeled from the PAN than from the MAN. In the spinal cord labeled MAN afferent fibers and terminations were most dense in the L5 and L6 segments, and those of the PAN were most dense in L6 and L7, that is, in the respective segments of entry. Labeled afferent fibers from both nerves projected rostrally at least as far as L1 and caudally as far as S2. Labeled fibers were found in Lissauer's tract as well as in the dorsal column immediately adjacent to the dorsal horn. In the spinal gray matter, both nerves had two main projection fields, one in the cap of the dorsal horn in lamina I, the other in the deep dorsal horn in laminae V-VI and the dorsal part of lamina VII. Both nerves, but particularly the PAN, projected to the medial portion of Clarke's column. No projection was found to laminae II, III, and IV of the dorsal horn or to the ventral horn. Since these findings parallel observations on hindlimb muscle afferent fibers, the present data support the existence of a common pattern for the central distribution of deep somatic afferent fibers.