Quantitative analysis of the dendrites of cat phrenic motoneurons stained intracellularly with horseradish peroxidase

All the dendrites (N = 37) generated by four phrenic motoneurons were analyzed following intracellular injection of horseradish peroxidase. The dendritic arbors produced from each of these stem dendrites were studied in detail. The mean number of stem dendrites produced by a phrenic motoneuron was 9.7, their mean diameter was 6.0 micron, and their mean combined diameter was 58.3 micron. The length at which a phrenic motoneuronal dendrite terminated was 1,236 micron, with several end terminals extending more than 2 mm from the cell body. The mean value for the combined lengths of all segments originating from a single stem dendrite was 5.3 mm. A full spectrum of dendritic branching patterns was observed from simple (five unbranched) to complex, the latter producing up to ninth-order branches. Most terminal and nonterminal dendritic segments tapered, producing a mean diameter reduction of 34%, or approximately 9% per 100-micron length. All phrenic motoneurons exhibited a steady decrease in the combined dendritic parameter (sigma d3/2) with distance from the soma as a result of tapering and end-branch termination. The mean surface area and volume of a phrenic motoneuronal dendrite were 35.3 X 10(3) micron 2 and 25.9 X 10(3) micron 3, respectively. The dendrites constituted greater than 97% of the total phrenic motoneuronal surface area, with 75% of this area lying outside of a 300-micron radius from the cell body. The diameter of a stem dendrite was positively correlated with its combined dendritic length, number of terminal branches, dendritic surface area, and volume. Despite this strong correlation, the value of total dendritic surface area calculated using the power equation derived from the dendritic surface area versus stem dendritic diameter plot was not a consistent estimator of the total dendritic surface area directly measured for these four phrenic motoneurons. It is suggested that this inconsistency may be the result of a heterogeneity in the phrenic motoneuronal population and/or in the dendrites projecting to the different terminal fields.     

Morphology of masticatory motoneurons stained intracellularly with horseradish peroxidase

Masticatory motoneurons were identified electrophysiologically and stained with horseradish peroxidase (HRP). The masseter motoneurons could be divided into 3 groups on the basis of their dendritic morphology. In contrast, the digastric or mylohyoid motoneurons showed a similar dendritic configuration. These neurons had much developed dendritic trees in the dorsomedial than ventrolateral direction. The first group of the masseter motoneurons had their dendritic trees which extended radially in all directions with a slight preference to project rostrally. These somata were located in the center of the subdivision containing the masseter motoneurons. In the second group, their dendritic arbores had a polarity extending hemispherically. These neuronal somata were located in the medial, ventral, and lateral regions of the subdivision. For the masseter motoneurons in the two groups and jaw-opening motoneurons, the dendritic swellings were frequently observed in the distal branches. The third group had their dendritic trees which were much simpler in configurations with less tapering or branching than those of other neurons examined. Furthermore, a wide variety of dendritic spines and appendages, and no dendritic swellings, observed in the third group were distinct from other neurons stained. The dendritic trees of the jaw-closing and -opening motoneurons were confined to the individual subdivisions. There were no instances in which axon collaterals were observed for well-stained 16 axons.     

Distribution of dendrites from biventer cervicis and complexus motoneurons stained intracellularly with horseradish peroxidase in the adult cat

The three-dimensional distribution of dendrites from the dorsal neck muscles biventer cervicis (BC) and complexus (CM) was examined in the adult cat using intracellular staining techniques. Motoneurons were electrophysiologically identified, stained with injection of horseradish peroxidase, and reconstructurcted from serial histological sections. The dendritic distributions of all motoneurons examined followed an orderly pattern. Many dendrites extended rostrally and caudally to form a complex parallel collection of dendrites in the ventromedial nucleus. Other dendrites projected dorsolaterally into the spinal accessory nucleus and lateral parts of lamina VII and VIII. Dorsomedial dendrites followed a path parallel to the medial border of the ventral horn and frequently terminated near the central canal. A few scattered dendrites were usually found directly dorsal to the soma in lamina VIII. This pattern of dendritic distribution differed distinctly from the dendritic distribution of motoneurons in other spinal regions. However, in all spinal regions, including the upper cervical spinal cord where BC and CM motoneurons were found, the pattern of dendritic distribution from different motoneurons was similar if their somata were located in the same region. For 15 motoneurons with well-stained dendrites, the mean rostral-caudal extent of the dendritic tree was 2,860 μm. The mean total dendritic length of three of these motoneurons measured 73,100 μm, almost four times larger than hindlimb motoneurons involved in planter reflexes. Despite the large size of the dendritic trees of BC and CM motoneurons, the surface areas of BC and CM cell bodies were smaller than most large hindlimb motoneurons. These quantitative differences in motoneuron dimensions may in turn be reflected by differences in the electrotonic properties of motoneurons in different motoneuron nuclei.     

Morphology of motoneurons in different subdivisions of the rat facial nucleus stained intracellularly with horseradish peroxidase

Horseradish peroxidase was injected into single facial motoneurons of the rat. Neurons were identified by antidromic stimulation of either the buccal or the marginal mandibular or the posterior auricular nerve branches. Motoneuronal cell bodies supplying the buccal branch were located in the lateral subdivision of the facial nucleus, those supplying the marginal mandibular branch were in the intermediate subdivision, and those supplying the posterior auricular branch were in the medial subdivision. Eleven motoneurons were reconstructed with a computer-assisted technique. Their soma diameters averaged 20 microns; the average number of primary dendrites was 7.9 and the combined lengths of the dendritic trees averaged 17,650 microns. There was no distinction between the three motoneuron groups in terms of these and other quantitative data. However, on the basis of reconstructed dendritic tree orientation (i.e., dendritic distribution), major differences were observed between motoneurons of the three groups. Dendrites from all groups extended beyond the boundaries of the facial nucleus into the reticular formation. The border between the intermediate and the lateral subdivision was crossed by some dendrites but the overlap was small. In contrast, no dendrite of a motoneuron in the medial subdivision entered the intermediate subdivision and vice versa. The dendritic extent was totally restricted by the borders between these two subdivisions. Outside the Nissl-defined nuclear border, however, dendrites from cells in adjacent subdivisions overlapped. It is concluded that the medial subdivision of the facial nucleus can be distinguished from the intermediate and lateral subdivisions not only by its sharp Nissl-defined border but also by the discrete organization of its dendritic field.     

Morphological analysis of cat masseteric motoneurons after intracellular staining with horseradish peroxidase

Intracellular injection of horseradish peroxidase (HRP) into 58 masseteric motoneurons identified by antidromic activation was performed in cats under pentobarbital anesthesia. Monosynaptic EPSPs were evoked by masseteric nerve stimuli in 52 cells, and were absent in the remaining six cells. The antidromic nature of the evoked spikes was confirmed by IS-SD separation observed at high frequency (50 Hz) stimulation. Motoneurons with monosynaptic excitation from masseter afferents showed IPSPs following stimulation of lingual and inferior alveolar nerves. Motoneurons which did not show monosynaptic excitation from masseter afferents showed no IPSPs from the above nerves. There were no differences in cell size or the number of stem dendrites between motoneurons with and without monosynaptic EPSPs. No recurrent collaterals were observed in any motor axons. Motoneurons with monosynaptic EPSPs were located at all rostrocaudal levels throughout the trigeminal motor nucleus, whereas motoneurons without such EPSPs were encountered only at the middle level. Dendrites of motoneurons with monosynaptic EPSPs did not extend into the medial portion of the nucleus where motoneurons innervating the anterior belly of the digastric muscle were located. In contrast, motoneurons without monosynaptic EPSPs had dendrite branches extending well into the medial part. The results show that there are two subpopulations of masseteric motoneurons that differ in peripheral inputs as well as dendritic morphology.     

Electron microscopic observations of axon collateral synaptic endings of cat oculomotor motoneurons stained by intracellular injection of horseradish peroxidase

Motoneurons in the cat oculomotor nucleus have been identified electrophysiologically and stained by intracellular injection of horseradish peroxidase. Axon collateral arborizations with preterminal and terminal boutons identified by light microscopy corresponded to synaptic endings observed by electron microscopy. Despite variations in size and shape, synaptic endings showed similar ultrastructural features and established asymmetrical predominantly axodendritic synaptic contacts usually characterized by the presence of subjunctional dense bodies underlying the postsynaptic membrane densification.     

Physiological response properties of cells labeled intracellularly with horseradish peroxidase in cat ventral cochlear nucleus

To determine the correspondence between anatomical and physiological cell types in the ventral cochlear nucleus of the cat, intracellular injections of horseradish peroxidase were made into cells whose extracellular and intracellular responses to sound had been studied. Three identified cells responded to a short tone burst at their characteristic frequencies with an onset pattern. This pattern is characterized by a strong response to the onset of the stimulus. One was an octopus cell. The second cell, located in the octopus-cell area, was a giant cell with a few somatic spines and thin tapering dendrites; the intracellular record revealed that even in the absence of sound it received continuous synaptic input, while tones at characteristic frequency produced a sustained depolarization. A third cell, which had an onset response at low intensities and a chopper response at high intensities, was a stellate cell located in the intermediate acoustic stria with dendrites oriented parallel to the fiber tract. This cell had an unusually broad dynamic range in response to changes in intensity. Two cells with transient chopper response patterns were stellate cells in the posteroventral cochlear nucleus with many branched and beaded dendrites. Three cells with more sustained chopper response patterns were stellate cells in the anteroventral cochlear nucleus with fewer, less-branched, smooth dendrites. Two cells with primarylike responses to tones were bushy cells with numerous short, thin, highly branched dendrites in the posterior division of the anteroventral cochlear nucleus. Intracellular responses to tones at the characteristic frequency consisted of large brief depolarizations, which were not sustained. Another cell, which responded to tones in a phase-locked fashion, was also located in the anteroventral cochlear nucleus. It was a small, stellate cell with relatively few, smooth dendrites. The labeled cells largely support previous attempts at physiological-morphological correlations: (1) bushy cells exhibit primarylike pattern; (2) stellate cells exhibit chopper patterns; and (3) octopus cells exhibit an onset pattern. It was also demonstrated that more than one cell type can exhibit a particular response pattern.     

Afferent fibers in the hypoglossal nerve: a horseradish peroxidase study in the cat

The existence of afferent fibers in the cat hypoglossal nerve was studied by transganglionic transport of horseradish peroxidase (HRP). Injections of wheat germ agglutinin-conjugated HRP (WGA-HRP) into the hypoglossal nerve resulted in some retrograde labeling of cell bodies within the superior ganglia of the ipsilateral glossopharyngeal and vagal nerves. A few labeled cell bodies were also present ipsilaterally within the inferior ganglion of the vagal nerve and the spinal ganglion of the C1 segment. Some of the labeled glossopharyngeal and vagal fibers reached the nucleus of the solitary tract by crossing the dorsal portion of the spinal trigeminal tract. Others distributed to the spinal trigeminal nucleus pars interpolaris and to the ventrolateral part of the medial cuneate nucleus by descending through the dorsal portion of the spinal trigeminal tract. In the spinal cord these descending fibers, intermingling with labeled dorsal root fibers, distributed to laminae I, IV-V and VII-VIII of the C1 and C2 segments. Additional HRP experiments revealed that the fibers in laminae VII-VIII originate mainly from dorsal root of the C1 segment.     

Morphology and physiology of abducens motoneurons and internuclear neurons intracellularly injected with horseradish peroxidase in alert squirrel monkeys

Axons of abducens motoneurons and internuclear neurons were penetrated with HRP-filled glass microelectrodes in alert squirrel monkeys. The firing rate of these axons and spontaneous eye movements were recorded and the axons were then injected with HRP for subsequent visualization of the recorded cells. Soma-dendritic and axon and axonal terminal morphology were studied for possible correlation with firing frequency. The physiology of squirrel monkey abducens neurons is qualitatively similar to their counterparts in the rhesus monkey and the cat, being primarily correlated with the position and velocity of the eyes. The locations of moto- and internuclear neurons are similar in the squirrel monkey and cat as are the axonal projections and terminals. However, squirrel monkey abducens cells are smaller than their feline counterparts and have dendrites that are confined to the cellular borders of the abducens nucleus. The size of the soma and proximal dendrites of moto- and internuclear neurons are poorly correlated with either their threshold for recruitment or their tonic eye position sensitivity. However, cells with smaller dendritic trees tended to have higher saccadic eye velocity sensitivity than those with larger trees. Three types of internuclear neurons were distinguishable upon the basis of their axon collaterals. All cells terminated within the medial rectus subdivision of the oculomotor nucleus. One class of cells did not give rise to collaterals before projecting to the oculomotor nucleus and the other classes gave rise to collaterals that terminated in the intermediate and/or caudal interstitial nuclei of the median longitudinal fasciculus. Within the IIIrd nucleus internuclear terminations were usually confined to a single subgroup of medial rectus motoneurons.     

Morphology of cat phrenic motoneurons as revealed by intracellular injection of horseradish peroxidase

The morphology of phrenic motoneurons (PMs) of adult cat was examined by utilizing the technique of intracellular injection of horseradish peroxidase. Twenty-one cells were reconstructed from serial sections in transverse, sagittal, and horizontal planes. The cell bodies were ellipsoid, with the major diameter oriented parallel to the longitudinal axis of the spinal cord. The dendrites of PMs are not distributed in a radially symmetric fashion, but rather project to four separate fields. The field containing the greatest number of dendrites extends rostrocaudally within the phrenic motor column. This collection of dendrites forms a rostrocaudal bundle in which the dendrites from neighboring PMs lie in close association with one another. The remaining dendrites project dorsolaterally, dorsomedially, and to a lesser extent, ventrally. The dorsolaterally directed dendrites form bundles upon entering the lateral funiculus with the dendrites from other PMs. Several of the dorsomedially directed dendrites cross to the contralateral spinal cord via the anterior commissure or central gray. A wide variety of dendritic spines and appendages was observed. There were no instances in which axon collaterals were observed for the 11 well-stained axons examined. The length of the initial segment of the axon was a function of the distance of the cell body from the ventral funiculus.     

Physilogy and morphology of substantia gelatinosa neurons intracellularly stained with horserdish peroxidase

Neurons in Rexed's layer II were physiologically characterized with natural and electrical stimuli applied to their cutaneous receptive field. The neurons were then intracellularly stained with horseradish peroxidase. Three general patterns of physiological responses were found Nociceptive specific neurons did not respond to gentle mechanical stimulation. Most responded exclusively to tissue-damaging stimuli. Some also responded to moderately heavy pressure, but these responded to noxious stimuli with an increased discharge frequency. Wide dynamic range neurons responded to both gentle mechanical stimulation and to tissue-damaging stimulation. Low-threshold mechanoreceptive neurons responded only to gentle mechanical stimulation. Some of the low-threshold mechanoreceptive neurons were innervated by primary afferents with unmyelinated axons. Excepting those low-threshold mechanoreceptive neurons with input form unmyelinated afferents, the patterns of primary afferent innervation of layer II neurons were similar to the patterns innervation that gave been found for neurons in layers I and IV-V. All nut 2 of the 22 neurons that we found were recognized as being of two general morphological types. Stalked cells had their perikarya situated along the superficial border of layer II. Most of their dendrites traveled ventrally while spreading out rostrocaudally. This gave their dendritic arbors a fan-like shape. Stalked cell axons arborized largely in layer I. Islet cell perikarya were found throughout layer II. Most of their dendrites traveled rostrocaudally. Their dendritic arbors were shaped like cylinders with their long axes parallel to the long axis of the spinal cord. Islet cell axons arborized in the immediate vicinity of their dendtritic territories, within layer II. Stalked cells and those islet cells whose dendritic arbors were largely contained within the superficial one-third of layer II (layer IIa) were either nociceptive specific or wide dynamic range neurons. The islet cells whose dendritic arbors were largely within the deeper two-thirds of layer II (layer IIb) were all low-threshold mechanoreceptive neurons. These observations suggest that layers IIa and IIb have different functional roles and that stalked cells and islet cells are separate and distinct components of the neural circuitry of the superficial dorsal horn.     

The ultrastructural morphology of the subthalamic-nigral axon terminals intracellularly labeled with horseradish peroxidase

The labeled axons of neurons intracellularly injected with horseradish peroxidase (HRP) in the rat subthalamic nucleus (STH) were studied with electron microscopy. The main axons and the afferent daughter branches were all myelinated. The morphology of the intrinsic axon terminals within STH was obscured by the dard HRP reaction products, but the labeled efferent STH terminals in the substantia nigra (SN) were revealed to contain small oval vesicles and formed asymmetrical synapses with dendrites of SN neurons.     

Nitrergic innervation of trigeminal and hypoglossal motoneurons in the cat

The present study was undertaken to determine the location of trigeminal and hypoglossal premotor neurons that express neuronal nitric oxide synthase (nNOS) in the cat. Cholera toxin subunit b (CTb) was injected into the trigeminal (mV) or the hypoglossal (mXII) motor nuclei in order to label the corresponding premotor neurons. CTb immunocytochemistry was combined with NADPH-d histochemistry or nNOS immunocytochemistry to identify premotor nitrergic (NADPH-d(+)/CTb(+) or nNOS(+)/ CTb(+) double-labeled) neurons. Premotor trigeminal as well as premotor hypoglossal neurons were located in the ventro-medial medullary reticular formation in a region corresponding to the nucleus magnocellularis (Mc) and the ventral aspect of the nucleus reticularis gigantocellularis (NRGc). Following the injection of CTb into the mV, this region was found to contain a total of 60 +/- 15 double-labeled neurons on the ipsilateral side and 33 +/- 14 on the contralateral side. CTb injections into the mXII resulted in 40 +/- 17 double-labeled neurons in this region on the ipsilateral side and 16 +/- 5 on the contralateral side. Thus, we conclude that premotor trigeminal and premotor hypoglossal nitrergic cells coexist in the same medullary region. They are colocalized with a larger population of nitrergic cells (7200 +/- 23). Premotor neurons in other locations did not express nNOS. The present data demonstrate that a population of neurons within the Mc and the NRGc are the source of the nitrergic innervation of trigeminal and hypoglossal motoneurons. Based on the characteristics of nitric oxide actions and its diffusibility, we postulate that these neurons may serve to synchronize the activity of mV and mXII motoneurons.     

Identification of the trochlear motoneurons by retrograde transport of horseradish peroxidase

A common technique for demonstrating projections in the brain is to electrically stimulate one part of the brain and record mass or field potentials from another part. We showed in the visual system of the cat, where connections between retina, lateral geniculate nucleus, and superior colliculus are very well known, that the recording of field potentials is not at all sufficient to demonstrate connections. The most prominent potential after electrical stimulation of the optic tract is the field potential created by the Y-ganglion cell fibers of the optic nerve. We recorded this potential in the optic nerve head of the eye, in the lateral geniculate nucleus, and in the superior colliculus. To our surprise, we also could record this potential 7 mm in front of the retina, with the electrode in the vitreous, and 5 mm above the lateral geniculate nucleus and the superior colliculus, where there are no direct inputs from the optic tract. These results show quite clearly that field potentials can “stray” much farther than the underlying anatomical structure projects.     

Decreased horseradish peroxidase labeling in deafferented spinal motoneurons of the rat

It has been suggested^29,50 that the incorporation and retrograde transport of horseradish peroxidase (HRP) were linked to the level of neuronal activity. Therefore one could postulate that the motor impairment resulting from dorsal rhizotomy affects the HRP labeling of spinal motoneurons in the absence of morphological damage to the motor system. This hypothesis was tested in the adult rat by sectioning bilaterally the L₃-L₅ dorsal roots. 2–18 months after surgery, the L₄ radicular nerve was immersed in a solution of HRP. Labeled motoneurons were counted together with the motor axons of the L₄ ventral root and results were compared with values obtained in paired controls. Deafferentation resulted in a crippling deficit of lower movements with disuse atrophy of muscle fibers but had no effect on the fiber population of the sciatic nerve and the L₄ ventral root. Whereas in normal animals the L₄ HRP-labeled motoneurons represented 71.9–98.3% (average 85.4) of the motor axonal counts, in animals studied 4, 12 and 18 months after dorsal rhizotomy, the number of motoneurons containing HRP granules constituted only 20.1–55.7% (average 46.2) of the number of motor axons and many of the labeled cells were faintly stained. These findings, which may reflect either a decreased retrograde transport of HRP in deafferented motoneurons or an increased turnover of the enzyme in the cell body, call attention to the possibility that the degree of activity in neuronal pathways influences HRP labeling.     

Localization of masticatory motoneurons in the cat and rat by means of retrograde axonal transport of horseradish peroxidase.

Topographical localization of monotoneurons supplying the masticatory muscles was investigated in the cat and rat, utilizing retrograde axonal transport of horseradish peroxidase. Following injection of horseradish peroxidase in each masticatory muscle, motoneurons labelled with peroxidase were seen to be aggregated into a cluster within the motor nucleus of the trigeminal nerve. Such clusters of peroxidase-motoneurons innervating each masticatory muscle were demarcated more sharply in kittens than in adult animals. The pattern of the nuclear representation of the masticatory muscles was found to be essentially the same in the cat and rat; it could be summarized as follows: The motor nucleus of the trigeminal nerve could be divided cytoarchitectonically into the dorsolateral and ventromedial divisions; the former was seen in almost the whole rostrocaudal extent of the nucleus, while the latter was localized at the levels of caudal two thirds of the nucleus. In the dorsolateral division, the temporal muscle was represented dorsally and dorsomedially, the masseter muscle ventrolaterally, and the pterygoid muscles ventromedially. In the ventromedial division, the anterior digastric muscle was represented dorsomedially, and the mylohyoid muscle ventrolaterally. It was also confirmed that the motoneurons supplying the posterior digastric muscle were localized in the accessory facial nucleus.     

A horseradish peroxidase study of rat lingual motoneurons with axons passing through the cervical nerve

The peripheral course of axons of rat lingual motoneurons was studied by HRP injection into the hypoglossal nerve in combination with transecting of the hypoglossal and/or cervical nerve components of the hypoglossocervical plexus. Furthermore, soma sizes of labeled lingual motoneurons were compared in transverse section with those of labeled geniohyoid and thyrohyoid motoneurons, which are situated adjacent to the lingual motoneurons. We found that axons of the majority of lingual motoneurons lying in the main hypoglossal nucleus passed through the hypoglossal nerve throughout their course to the tongue. In a remaining small number of lingual motoneurons lying in a medial portion of the ventromedial subnucleus in the caudal fourth of the main hypoglossal nucleus, their axons passed through the first cervical nerve to the upper root of the ansa cervicalis to the hypoglossal nerve and then to its medial branch. The labeled lingual motoneurons with axons passing through the cervical nerve were intermingled with those whose axons passed through the hypoglossal nerve. The latter motoneurons, however, diminished in number while being traced caudally, and finally in the most caudal main hypoglossal nucleus the former motoneurons occupied a major part of this nucleus. The lingual motoneurons with axons passing through the cervical nerve were smaller in soma size than those with axons passing through the hypoglossal nerve. These two types of lingual motoneurons were both smaller in soma size than the geniohyoid and thyrohyoid motoneurons, and their soma shape was not as flat as that of the latter types of motoneurons.