Extra- and intracellular HRP analysis of the organization of extraocular motoneurons and internuclear neurons in the guinea pig and rabbit

The distribution of extraocular motoneurons and abducens and oculomotor internuclear neurons was determined in guinea pigs by injecting horseradish peroxidase (HRP) into individual extraocular muscles, the abducens nucleus, the oculomotor nucleus, and the cerebellum. Motoneurons in the oculomotor nucleus innervated the ipsilateral inferior rectus, inferior oblique, medial rectus, and the contralateral superior rectus and levator palpebrae muscles. Most motoneurons of the trochlear nucleus projected to the contralateral superior oblique muscle although a small number innervated the ipsilateral superior oblique. The abducens and accessory abducens nuclei innervated the ipsilateral rectus and retractor bulbi muscles, respectively. The somata of abducens internuclear neurons formed a cap around the lateral and ventral aspects of the abducens nucleus. The axons of these internuclear neurons terminated in the medial rectus subdivision of the contralateral oculomotor nucleus. At least two classes of guinea pig oculomotor internuclear interneurons exist. One group, located primarily ventral to the oculomotor nucleus, innervated the abducens nucleus and surrounding regions. The second group, lying mainly in the dorsal midline area of the oculomotor nucleus, projected to the cerebellum. Intracellular staining with HRP demonstrated similar soma-dendritic organization for oculomotor and trochlear motoneurons of both guinea pigs and rabbits. Dendrites of oculomotor motoneurons radiated symmetrically from the soma to cover approximately one-third of the entire nucleus, and each motoneuron sent at least one dendrite into the central gray overlying the oculomotor nucleus. In both species, a small percentage of oculomotor motoneurons possessed axon collaterals that terminated both within and outside of the nucleus. The dendrites of trochlear motoneurons extended into the medial longitudinal fasciculus and the reticular formation lateral to the nucleus. Our data on the topography of motoneurons and internuclear neurons in the guinea pig and soma-dendritic organization of motoneurons in the guinea pig and rabbit show that these species share common organizational and morphological features. In addition, comparison of these data with those from other mammals reveals that dendritic complexity (number of dendrites per motoneuron) of extraocular motoneurons exhibits a systematic increase with animal size.     

Abducens internuclear and ascending tract of deiters inputs to medial rectus motoneurons in the cat oculomotor nucleus: synaptic organization

Abducens internuclear and ascending tract of Deiters (ATD) inputs to medial rectus motoneurons in the oculomotor nucleus are important for conjugate horizontal movements. In the present study, the organization of these separate populations of neurons and their synaptic connections with medial rectus motoneurons in the cat oculomotor nucleus have been examined by light and electron microscopy by using retrograde and anterograde axonal tracers. Consistent with the patterns of retrograde horseradish peroxidase labeling, the abducens internuclear projection is predominantly, if not exclusively, contralateral, whereas the ATD projection is exclusively ipsilateral, as demonstrated by anterograde autoradiographic and biocytin labeling. Both populations of synaptic endings contain spheroidal synaptic vesicles and establish synaptic contacts with modest postsynaptic densifications. In addition, ATD synaptic endings frequently are associated with subjunctional dense bodies and subsurface cisternae. The two populations of excitatory inputs differ, however, in their soma-dendritic distribution. The majority of abducens internuclear synaptic endings contact distal dendrites, whereas the majority of ATD synaptic endings contact proximal dendrites or somata. Abducens internuclear synaptic endings furthermore have a higher density of mitochondria than ATD synaptic endings. The more proximal location of ATD synaptic endings is consistent with the faster rise time and earlier reversal to polarizing currents of ATD excitatory postsynaptic potentials in comparison to those evoked by the abducens internuclear pathway as determined electrophysiologically. Given the differences in the physiologic signals conveyed by the abducens internuclear (eye velocity and eye position) and ATD (head velocity) pathways, the findings in this study suggest that the soma-dendritic stratification of the two inputs to medial rectus motoneurons may provide a means for the separate control of visuomotor and vestibular functions, respectively.     

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.     

GABA-immunoreactive internuclear neurons in the ocular motor system of lampreys

The presence of internuclear neurons in the abducens and oculomotor nuclei of lampreys [González, M.J., Pombal, M.A., Rodicio, M.C. and Anadón, R., Internuclear neurons of the ocular motor system of the larval sea lamprey, J. Comp. Neurol. 401 (1998) 1-15] indicates that coordination of eye movements by internuclear neurons appeared early during the evolution of vertebrates. In order to investigate the possible involvement of inhibitory neurotransmitters in internuclear circuits, the distribution of gamma-aminobutyric acid (GABA) in the extraocular motor nuclei of the lamprey was studied using immunocytochemical techniques. Small GABA-immunoreactive (GABAir) neurons were observed in the three ocular motor nuclei. Numerous GABAir neurons were observed in the group of internuclear neurons of the dorsal rectus oculomotor subnucleus. A second group of GABAir neurons was observed among and below the trochlear motoneurons. Two further groups of GABAir interneurons, periventricular and lateral, were located in the abducens nucleus among the cells of the caudal rectus and the ventral rectus motor subnuclei, respectively. In addition to the presence of GABAir neurons, in all the ocular motor nuclei the motoneurons were contacted by numerous GABAir boutons. Taken together, these results suggest that GABA is involved as a neurotransmitter in internuclear pathways of the ocular motor system of lampreys.     

Hippocampal and midline thalamic fibers and terminals in relation to the choline acetyltransferase-immunoreactive neurons in nucleus accumbens of the rat: a light and electron microscopic study

The synaptic interactions between terminals of allocorticostriatal and thalamostriatal fibers and the cholinergic neurons in the nucleus accumbens were investigated using degeneration and dual labelling immunocytochemistry in Wistar rats. The presumptive cholinergic neurons were labelled with antibodies directed against choline acetyltransferase and the afferent fibers were labelled anterogradely with Phaseolus vulgaris-leucoagglutinin. Fibers from the subiculum of the hippocampal formation and from the midline and intralaminar thalamus project densely into the medial nucleus accumbens where they overlap a relatively dense population of choline acetyltransferase-immunoreactive neurons. Varicosities containing Phaseolus vulgaris-leucoagglutinin juxtapose the immunoreactive neurons. To study the possibility that the cholinergic neurons could be the synaptic targets of these incoming fibers, the subiculum, the fornix, and the midline/intralaminar thalamus were lesioned in separate animals and brain sections were immunoprocessed for choline acetyltransferase and studied with the electron microscope. In addition, dual-labelling electron microscopic immunocytochemistry was employed. In total, 164 synaptic terminals from the subiculum/hippocampus and 130 from the midline/intralaminar thalamus were examined; all formed asymmetrical synaptic specializations. No hippocampal endings were seen to contact the somata or primary dendrites of the choline acetyltransferase-immunoreactive neurons; however, three were found in synaptic contact with distal, immunolabelled dendritic shafts. Most hippocampal terminals established contacts with unlabelled spines. Fifteen percent of the thalamic endings were found to synapse on the somata and the primary and distal dendrites of the choline acetyltransferase-immunoreactive neurons. The remaining thalamic terminals established synaptic junctions with small unlabelled dendrites or spines. These findings have important implications not only for our understanding of the synaptic organization of the hippocampal and thalamic projections to the nucleus accumbens, but also for the contribution of the cholinergic neurons to the circuitry of this nucleus.     

Motoneurons of twitch and nontwitch extraocular muscle fibers in the abducens, trochlear, and oculomotor nuclei of monkeys

Eye muscle fibers can be divided into two categories: nontwitch, multiply innervated muscle fibers (MIFs), and twitch, singly innervated muscle fibers (SIFs). We investigated the location of motoneurons supplying SIFs and MIFs in the six extraocular muscles of monkeys. Injections of retrograde tracers into eye muscles were placed either centrally, within the central SIF endplate zone; in an intermediate zone, outside the SIF endplate zone, targeting MIF endplates along the length of muscle fiber; or distally, into the myotendinous junction containing palisade endings. Central injections labeled large motoneurons within the abducens, trochlear or oculomotor nucleus, and smaller motoneurons lying mainly around the periphery of the motor nuclei. Intermediate injections labeled some large motoneurons within the motor nuclei but also labeled many peripheral motoneurons. Distal injections labeled small and medium-large peripheral neurons strongly and almost exclusively. The peripheral neurons labeled from the lateral rectus muscle surround the medial half of the abducens nucleus: from superior oblique, they form a cap over the dorsal trochlear nucleus; from inferior oblique and superior rectus, they are scattered bilaterally around the midline, between the oculomotor nucleus; from both medial and inferior rectus, they lie mainly in the C-group, on the dorsomedial border of oculomotor nucleus. In the medial rectus distal injections, a "C-group extension" extended up to the Edinger-Westphal nucleus and labeled dendrites within the supraoculomotor area. We conclude that large motoneurons within the motor nuclei innervate twitch fibers, whereas smaller motoneurons around the periphery innervate nontwitch, MIF fibers. The peripheral subgroups also contain medium-large neurons which may be associated with the palisade endings of global MIFs. The role of MIFs in eye movements is unclear, but the concept of a final common pathway must now be reconsidered.     

The motor nuclei and sensory neurons of the IIIrd, IVth, and VIth cranial nerves in the monitor lizard, Varanus exanthematicus

The motor nuclei of the oculomotor, trochlear, and abducens nerves of the reptile Varanus exanthematicus and the neurons that subserve the sensory innervation of the extraocular muscles were identified and localized by retrograde and anterograde transport of horseradish peroxidase (HRP). The highly differentiated oculomotor nuclear complex, located dorsomedially in the tegmentum of the midbrain, consists of the accessory oculomotor nucleus and the dorsomedial, dorsolateral, intermediate, and ventral subnuclei. The accessory oculomotor nucleus projects ipsilaterally to the ciliary ganglion. The dorsomedial, dorsolateral, and intermediate subnuclei distribute their axons to the ipsilateral orbit, whereas the ventral subnucleus, which innervates the superior rectus muscle, has a bilateral, though predominantly contralateral projection. The trochlear nucleus, which rostrally overlaps the oculomotor nuclear complex, is for the greater part a comma-shaped cell group situated lateral, dorsal, and medial to the medial longitudinal fasciculus. Following HRP application to the trochlear nerve, almost all retrogradely labeled cells were found in the contralateral nucleus. The nuclear complex of the abducens nerve consists of the principal and accessory abducens nuclei, both of which project ipsilaterally. The principal abducens nucleus is located just beneath the fourth ventricle laterally adjacent to the medial longitudinal fasciculus and innervates the posterior rectus muscle. The accessory abducens nucleus has a ventrolateral position in the brainstem in close approximation to the ophthalmic fibers of the descending trigeminal tract. It innervates the retractor bulbi and bursalis muscles. The fibers arising in the accessory abducens muscles form a loop in or just beneath the principal abducens nucleus before they join the abducens nerve root. The afferent fibers conveying sensory information from the extraocular muscles course in the oculomotor nerve and have their perikarya in the ipsilateral trigeminal ganglion, almost exclusively in its ophthalmic portion.     

The serotoninergic fibers in the dorsal lateral geniculate nucleus of the cat: distribution and synaptic connections demonstrated with immunocytochemistry

The distribution, morphology, and synaptic contacts of serotoninergic fibers were studied with immunocytochemical methods in the lateral geniculate complex of the cat. The serotonin-immunoreactive fibers are diffusely distributed throughout the main laminae of the dorsal lateral geniculate nucleus (dLGN) and the perigeniculate nucleus (PGN) and reach a particular density in the ventral lateral geniculate nucleus (vLGN). The labeled fibers are in most cases very thin and sometimes varicose. There is no obvious order in their distribution pattern except that they sometimes partially encircle the unlabeled cell bodies of the dLGN. The synaptic connections of the serotoninergic fibers were investigated mainly in the A laminae of the dLGN. Few synaptic complexes were found, most of them with asymmetric morphology. The postsynaptic elements were small dendritic profiles. Perisomatic serotoninergic fibers were seen, but no convincing synaptic contacts were found between labeled fibers and cell somata. In the dLGN, serotoninergic profiles were almost exclusively confined to the extraglomerular neuropile. In the PGN serotoninergic fibers also contacted dendritic profiles and formed asymmetrical synapses, but as in the geniculate, synaptic specializations were very rare.     

Expression of Trk receptors in the oculomotor system of the adult cat

We examined the expression of the three Trk receptors for neurotrophins (TrkA, TrkB, and TrkC) in the extraocular motor nuclei of the adult cat by using antibodies directed against the full-Trk proteins in combination with horseradish peroxidase retrograde tracing. The three receptors were present in all neuronal populations investigated, including abducens motoneurons and internuclear neurons, medial rectus motoneurons of the oculomotor nucleus, and trochlear motoneurons. They were also present in the vestibular and prepositus hypoglossi nuclei. TrkA, TrkB, and TrkC immunopositive cells were found in similar percentages in the oculomotor and in the trochlear nuclei. In the abducens nucleus, however, a significantly higher percentage of cells expressed TrkB than the other two receptors, among both motoneurons (81.8%) and internuclear neurons (88.4%). The percentages obtained for the three Trk receptors in identified neuronal populations pointed to the colocalization of two or three receptors in a large number of cells. We used confocal microscopy to elucidate the subcellular location of Trk receptors. In this case, abducens motoneurons and internuclear neurons were identified with antibodies against choline acetyltransferase and calretinin, respectively. We found a different pattern of staining for each neurotrophin receptor, suggesting the possibility that each receptor and its cognate ligand may use a different route for cellular signaling. Therefore, the expression of Trk receptors in oculomotor, trochlear, and abducens motoneurons, as well as abducens internuclear neurons, suggests that their associated neurotrophins may exert an influence on the normal operation of the oculomotor circuitry. The presence of multiple Trk receptors on individual cells indicates that they likely act in concert with each other to regulate distinct functions.     

Interconnections between the primate cerebellum and midbrain near-response regions.

The goal of this study was to determine the pattern of the connections between the midbrain and cerebellum that may play a role in the modulation of the near-response in the macaque. Injection of the retrograde tracer wheat germ agglutinin conjugated horseradish peroxidase (WGA-HRP) into the physiologically identified midbrain near-response region, which includes the supraoculomotor area, labelled cells throughout the deep cerebellar nuclei. However, labelled cells were particularly concentrated in the ventrolateral corner of the contralateral posterior interposed nucleus and in the contralateral and, to a lesser extent, the ipsilateral fastigial nuclei. Subsequently, injections of WGA-HRP were used to define the midbrain terminations of the deep cerebellar nuclei. Fastigial nucleus injections labelled terminals in a band along the border between the oculomotor nucleus and the supraoculomotor area that included the Edinger-Westphal nucleus. Injections of the posterior interposed nucleus labelled terminals in the portion of the supraoculomotor area dorsal to the fastigial projection and did not involve the Edinger-Westphal nucleus. In both cases, the terminal label was primarily found contralaterally. In contrast, retrogradely labelled cells were primarily found ipsilaterally within the supraoculomotor area following cerebellar injections. Retrogradely labelled cells projecting to the deep nuclei were also found bilaterally in the anteromedian nucleus, along with sparse terminal label. Taken as a whole, these results demonstrate the presence of a highly specific pattern of labelling in the supraoculomotor area, which may indicate that the posterior interposed nucleus and the fastigial nucleus play different roles in the control of the near-response. Alternatively, these projections may subserve other functions, such as modulating the pupillary light reflex. The fact that the projection from the deep nuclei is primarily contralateral, while the supraoculomotor projection to the deep nuclei is primarily ipsilateral, suggests that this may not be a simple feedback system, but may instead be involved in balancing the gains in the two eyes. In sum, physiological experiments have indicated the presence of near-response neurons in the midbrain supraoculomotor area and have indicated that the cerebellum may play a role in modulating the components of the near-response, as well as activity in the intrinsic eye muscles. The present experiments suggest a pattern of connections that might subserve this cerebellar modulation.     

Evidence for glycine as an inhibitory neurotransmitter of vestibular, reticular, and prepositus hypoglossi neurons that project to the cat abducens nucleus

The localization and distribution of brain-stem afferent neurons to the cat abducens nucleus has been examined by high-affinity uptake and retrograde transport of 3H-glycine. Injections of 3H-glycine selectively labeled (by autoradiography) only neurons located predominantly in the ipsilateral medial vestibular and contralateral prepositus hypoglossi nuclei, and in the contralateral dorsomedial reticular formation, the latter corresponding to the location of inhibitory burst neurons. The specificity of uptake and retrograde transport of 3H-glycine was indicated by the absence of labeling of the dorsomedial medullary reticular neurons ipsilateral and in close proximity to the injection site, where local uptake by diffusion could have occurred. The selectivity of uptake and transport was demonstrated by the absence of retrograde labeling following injections of 3H-GABA or 3H-leucine into the abducens nucleus. The immunohistochemical localization of glycine and GABA revealed a differential distribution of the 2 inhibitory neurotransmitter candidates in the extraocular motor nuclei. Glycine-immunoreactive staining of synaptic endings in the abducens nucleus was dense with a widespread soma-dendritic distribution but was sparse in the trochlear and oculomotor nuclei. By contrast, GABA-immunoreactive staining within the oculomotor and trochlear nuclei was associated with synaptic endings that were particularly prominent on the somata of motoneurons. GABA-immunoreactive staining in the abducens nucleus, however, was sparse. These differences between glycine- and GABA-immunoreactive staining in the extraocular motor nuclei were correlated with differences in the immunoreactivity of axons in the descending (glycine) and ascending (GABA) limbs of the medial longitudinal fasciculus. Glycine-immunoreactive neurons, furthermore, were observed in the same locations as neurons that were labeled autoradiographically by retrograde transport of 3H-glycine from the abducens nucleus. Electrophysiological recordings from abducens motoneurons and internuclear neurons revealed a marked reduction in the slow positivity of the orthodromic extracellular potential elicited by ipsilateral vestibular nerve stimulation following systemic administration of strychnine, an antagonist of glycine. Intracellular recordings demonstrated that the vestibular-evoked disynaptic inhibitory postsynaptic potentials in abducens neurons were effectively blocked by strychnine but were unaffected by picrotoxin, an antagonist of GABA.(ABSTRACT TRUNCATED AT 400 WORDS)     

Midbrain projections to the trigeminal, facial and hypoglossal nuclei in the opossum. A study using axonal transport techniques

It has been proposed (see Berntson and Micco for review) that circuits intrinsic to the midbrain play an important role in the elaboration and control of behaviors involving the motor nuclei of the trigeminal, facial and hypoglossal nerves (e.g. defense, threat, attack); but because of technical problems, it has been difficult to analyze their organization. Using the horseradish peroxidase technique we have localized those midbrain neurons which project to each of the above nuclei and by using the autoradiographic method we have plotted the intranuclear distribution of their axons. Using both techniques, we have seen that mesencephalic projections to oral-facial motor nuclei strongly favor the nucleus of the facial nerve. Cells ventral to the cerebral aqueduct, including the ventral periaqueductal gray, the interstitial nucleus of Cajal, the nucleus of Darkshchewitsch and the rostral oculomotor nucleus provide major midbrain-facial projections in the opossum. Their axons terminate densely and bilaterally within areas innervating auricular muscles and to a lesser extent, the platysma sheet. The projection to the caudal auricular area of the facial complex is particularly dense. Neurons within and dorsal to the red nucleus project to regions of the contralateral facial nucleus reported to supply buccolabial, zygomatic and cervical musculature. There is also a minor tectal projection to the facial nucleus. Direct projections to the hypoglossal nuclei also arise within the periaqueductal gray and interstitial nucleus, but if such regions influence the motor trigeminal nucleus, it is mainly by way of dendrites that extend outside the nucleus or by at least one synaptic delay. The mesencephalic nucleus of the trigeminal nerve, however, projects strongly to the motor trigeminal nucleus. These data are discussed in light of their possible functional significance.     

Distribution of the vestibular neurons projecting to the oculomotor and trochlear nuclei in rabbits

Horseradish peroxidase and Fast Blue were injected into the oculomotor and trochlear nuclei of rabbits so as to study the distribution of vestibular neurons that project to these nuclei. After the oculomotor nucleus was injected, labelled neurons were found in the superior, medial, and descending vestibular nuclei as well as in cell group Y. In the superior nucleus, most of the neurons (510 +/- 46) were ipsilateral to the injection, although contralaterally labelled neurons were also observed (104 +/- 19) more peripherally. In cell group Y, 186 +/- 24 contralaterally labelled neurons were observed, whereas hardly any (8 +/- 3) were found on the ipsilateral side. The largest group of labelled neurons (811 +/- 65) constituted a neuronal band located contralaterally in the medial nucleus and rostral part of the descending nucleus. This band rostromedially continued with the caudal part of the group of internuclear neurons of the abducens nucleus. Only 190 +/- 31 neurons were labelled in the medial and descending nucleus ipsilateral to the injected oculomotor nucleus. After injection of the trochlear nucleus, labelled neurons were found in the ipsilateral superior nucleus and contralateral medial and descending nuclei: a few labelled cells were also observed in the ipsilateral medial and descending nuclei as well as in the contralateral cell group Y.     

Topography and synaptology of mamillary body projections to the mesencephalon and pons in the rat.

The anterograde and retrograde transport of horseradish peroxidase conjugated to wheat germ agglutinin (WGA-HRP) was used to study the anatomical organization of descending projections from the mamillary body (MB) to the mesencephalon and pons at light and electron microscopic levels. Injections of WGA-HRP into the medial mamillary nucleus resulted in dense anterograde and retrograde labeling in the ventral tegmental nucleus, while injections in the lateral mamillary nucleus resulted in dense anterograde labeling in the dorsal tegmental nucleus pars dorsalis and dense anterograde and retrograde labeling in the pars ventralis of the dorsal tegmental nucleus. Anterogradely labeled fibers in the mamillotegmental tract diverged from the principal mamillary tract in an extensive dorsocaudally oriented swath of axons which extended to the dorsal and ventral tegmental nuclei, and numerous axons turned sharply ventrally and rostrally to terminate topographically in the dorsomedial nucleus reticularis tegmenti pontis and rostromedial pontine nuclei. The anterograde labeling in these two precerebellar relay nuclei was distributed near the midline such that projections from the lateral mamillary nucleus terminated mainly dorsomedial to the terminal fields of projections from the medial mamillary nucleus. In the dorsal and ventral tegmental nuclei, labeled axon terminals contained round synaptic vesicles and formed asymmetric synaptic junctions primarily with small diameter dendrites and to a lesser extent with neuronal somata. A few labeled terminals contained pleomorphic vesicles and formed symmetric synaptic junctions with dendrites and neuronal somata. Labeled axon terminals were also frequently found in synaptic contact with retrogradely labeled dendrites and neuronal somata in the dorsal and ventral tegmental nuclei. These findings indicate that neurons in the dorsal and ventral tegmental nuclei are reciprocally connected with MB projection neurons. In the nucleus reticularis tegmenti pontis and medial pontine nuclei, labeled axon terminals contained round synaptic vesicles and formed asymmetric synaptic junctions primarily with small diameter dendrites. The present study demonstrates that projections from the medial and lateral nuclei of the MB are topographically organized in the mesencephalon and pons. The synaptic morphology of mamillotegmental projections suggests that they may have excitatory influences primarily on the distal dendrites of neurons in these brain regions.     

Dorsal mesencephalic projections to pons, medulla, and spinal cord in the cat: limbic and non-limbic components.

The vertebrate dorsal mesencephalon consists of the superior colliculus, the dorsal portion of the periaqueductal gray, and the mesencephalic trigeminal neurons in between. These structures, via their descending pathways, take part in various behavioral responses to environmental stimuli. This study was undertaken to compare the origins and trajectories of these pathways in the cat. Injections of horseradish peroxidase into the cervical spinal cord and upper medullary medial tegmentum retrogradely labeled cells mainly in the contralateral intermediate and deep superior colliculus, and in the ipsilateral dorsal and lateral periaqueductal gray and adjacent tegmentum. Only injections in the medullary lateral tegmental field labeled mesencephalic trigeminal neurons ipsilaterally. Autoradiographic tracing results, based on injections across the dorsal mesencephalon, revealed three efferent fiberstreams. A massive first fiberstream (limbic pathway), consisting of thin fibers, descended ipsilaterally from the dorsal and lateral periaqueductal gray and adjacent superior colliculus through the mesencephalic and pontine lateral tegmentum, terminating in these areas as well as in the ventral third of the caudal pontine and medullary medial tegmentum. A few fibers from the dorsal periaqueductal gray matter (PAG) were distributed bilaterally to the dorsal vagal, solitary, and retroambiguus nuclei. The second fiberstream (the predorsal bundle) descended contralaterally from the superior colliculus (SC) and consisted of both thick and thin labeled fibers. The thin fibers terminated bilaterally in the dorsomedial nucleus reticularis tegmenti pontis and the medial half of the caudal medial accessory inferior olive. The thick fibers targeted the contralateral dorsal two thirds of the caudal pontine and medullary medial tegmental fields, and the facial, abducens, lateral reticular, subtrigeminal, and prepositus hypoglossi nuclei. A few fibers recrossed the midline to terminate in the ipsilateral medial tegmentum. Caudal to the obex, fibers terminated laterally in the tegmentum and upper cervical intermediate zone. From the lateral SC, fibers terminated bilaterally in the lateral tegmental fields of the pons and medulla and lateral facial subnuclei. The third fiberstream (mesencephalic trigeminal or Probst tract) terminated in the supratrigeminal and motor trigeminal nuclei, and laterally in the tegmentum and upper cervical intermediate zone. In summary, neurons in the PAG and in the deep layers of the SC give rise to a massive ipsilateral descending pathway, in which a medial-to-lateral organization exists. A similar topographical pattern occurs in the crossed SC projections. The possibility that these completely different descending systems cooperate in producing specific defensive behaviors is discussed.     

Identification of abducens motoneurons, accessory abducens motoneurons, and abducens internuclear neurons in the chick by retrograde transport of horseradish peroxidase

The location of the motoneurons innervating the lateral rectus, pyramidalis, and quadratus muscles of the chick has been determined by application of horseradish peroxidase (HRP) to these muscles and their nerve branches, and internuclear neurons in the abducens nucleus have been identified by injection of HRP into the oculomotor nucleus. Quantitative results were obtained by means of a semiautomatic image analyzer. Lateral rectus motoneurons were observed only in the ipsilateral principal abducens nucleus, where they numbered 500-550, and quadratus and pyramidalis motoneurons only in the ipsilateral accessory abducens nucleus. The 325-375 internuclear neurons that appeared in the principal abducens nucleus contralateral to the oculomotor nucleus injected with HRP were practically confined to the rostral two thirds of the nucleus, where they tended to surround the lateral rectus motoneurons in dorsal or lateral positions, though a minority of interneurons also mingled with the motoneurons in the center or at the medial face of the nucleus. Most interneurons were small and elongated, but a minority of larger interneurons morphologically similar to the lateral rectus motoneurons were also distinguishable. The 100-110 quadratus motoneurons and the 45-55 pyramidalis motoneurons mingled in the accessory abducens nucleus were larger than the lateral rectus motoneurons and sent their axons into the ipsilateral abducens nerve.     

Distinct populations of neurons in the ventromedial periaqueductal gray project to the rostral ventral medulla and abducens nucleus.

Following microinjections of a colloidal gold complex into the nucleus paragigantocellularis (PGi) of the ventral medulla, and of latex microspheres into the nucleus abducens (Abd) of the same animal, retrogradely labeled neurons were identified in the region of the contralateral supraoculomotor nucleus (SOM) in the ventromedial periaqueductal gray (PAG). Neurons labeled from the Abd were found in the SOM in the ventromedial PAG throughout the midbrain, as well as scattered ventrally in the oculomotor nucleus. Neurons in the SOM area retrogradely labeled from the PGi were most numerous rostral to the dorsal raphe nucleus and extended throughout the level of the oculomotor nucleus. Direct comparison of the two labels revealed that the neurons that project to the Abd were located slightly more ventrally than PGi-projecting neurons. Almost no doubly labeled neurons were identified, although singly labeled neurons formed adjacent but separate populations. These results indicate that neurons in the SOM area projecting to the PGi are distinct from those projecting to the Abd, and that the PGi-projecting neurons are probably not pre-oculomotor neurons. Given their more dorsal location and the nature of their target neurons in the rostral ventrolateral medulla, PGi-projecting neurons may be related to visceromotor, as opposed to oculomotor functions.     

Localization of parvalbumin,calretinin, and calbindin D-28k in identified extraocular motoneurons and internuclear neurons of the cat

Calcium-binding proteins have been shown to be excellent markers of specific neuronal populations. We aimed to characterize the expression of calcium-binding proteins in identified populations of the cat extraocular motor nuclei by means of immunohistochemistry against parvalbumin, calretinin, and calbindin D-28k. Abducens, medial rectus, and trochlear motoneurons were retrogradely labeled with horseradish peroxidase from their corresponding muscles. Oculomotor and abducens internuclear neurons were retrogradely labeled after horseradish peroxidase injection into either the abducens or the oculomotor nucleus, respectively. Parvalbumin staining produced the highest density of immunoreactive terminals in all extraocular motor nuclei and was distributed uniformly. Around 15–20% of the motoneurons were moderately stained with antibody against parvalbumin, but their axons were heavily stained, indicating an intracellular segregation of parvalbumin. Colchicine administration increased the number of parvalbumin-immunoreactive motoneurons to approximately 85%. Except for a few calbindin-immunoreactive trochlear motoneurons (1%), parvalbumin was the only marker of extraocular motoneurons. Oculomotor internuclear neurons identified from the abducens nucleus constituted a nonuniform population, because low percentages of the three types of immunostaining were observed, calbindin being the most abundant (28.5%). Other interneurons located within the boundaries of the oculomotor nucleus were mainly calbindin-immunoreactive. The medial longitudinal fascicle contained numerous parvalbumin- and calretinin-immunoreactive but few calbindin-immunoreactive axons. The majority of abducens internuclear neurons projecting to the oculomotor nucleus (80.7%) contained calretinin. Moreover, the distribution of calretinin-immunoreactive terminals in the oculomotor nucleus overlapped that of the medial rectus motoneurons and matched the anterogradely labeled terminal field of the abducens internuclear neurons. Parvalbumin immunostained 42% of the abducens internuclear neurons. Colocalization of parvalbumin and calretinin was demonstrated in adjacent semithin sections, although single-labeled neurons were also observed. Therefore, calretinin is proven to be a good marker of abducens internuclear neurons. From all of these data, it is concluded that parvalbumin, calretinin, and calbindin D-28k selectively delineate certain neuronal populations in the oculomotor system and constitute valuable tools for further analysis of oculomotor function under normal and experimental conditions. J. Comp. Neurol. 390:377–391, 1998. © 1998 Wiley-Liss, Inc.     

Development and organization of the ocular motor nuclei in the larval sea lamprey,Petromyzon marinus L.: An HRP study

Retrograde transport of horseradish peroxidase (HRP) after its application into the orbit was used to investigate the development of the different ocular motor nuclei in larvae of the sea lamprey (Petromyzon marinus) and to identify their regions of origin. In the smallest larvae studied (10–19 mm in length), the oculomotor and abducens neurons were ipsilateral to the site of HRP application, whilst trochlear neurons were contralateral. These motoneurons did not have dendritic processes. In larvae more than 19 mm in length, both ipsilateral and contralateral components were found in the oculomotor and trochlear nuclei; dendrites were present, and their length and branching increased with larval age. An adult-like pattern of topographic organization and dendritic arborization was reached in larvae of about 45–60 mm in length. In oculomotor neurons, medial dendrites appear first, then dorsolateral dendrites, and finally ventral dendries. Similarly, in trochlear neurons ventral and ventrolateral dendrites develop first, foll owed by dorsal dendrites that course either to the caudal optic tectum or to the terminal fields of the octaval and lateral line nerves in the cerebellar plate. Dorsal and ventral dendrites of the abducens neurons arise at the same time, but dorsal dendrites attain an adult-like morphology earlier. A few motoneurons showed ventricular attachments in larvae longer than 40 mm. The significance of these processes and their possible usefulness as a marker for the regions of origin of the ocular motor nuclei are discussed. Finally, the results presented here indicate that differentiation of the ocular motor nuclei in larval lampreys precedes and is independent of the maturation of the eye at transformation.