Retrograde Axonal Tracing Research Articles

Neuronal connections of orbital cortex in rats: topography of cortical and thalamic afferents.

The cortical and thalamic afferent connections of rat orbital cortex were investigated using fluorescent retrograde axonal tracers. Each of the four orbital areas has a distinct pattern of connections. Corticocortical connections involving the ventral and ventrolateral orbital areas are more extensive than those of the medial and lateral orbital areas. The medial orbital area has cortical connections with the cingulate, medial agranular (Fr2) and posterior parietal (PPC) cortices. The ventral orbital area has connections with the cingulate area, area Fr2, secondary somatic sensory area Par2, PPC, and visual areas Oc2M and Oc2L. The ventrolateral orbital area (VLO) receives cortical input from insular cortex, area Fr2, somatic sensory areas Par1 and Par2, PPC and Oc2L. The lateral orbital area has cortical connections limited to the agranular and granular insular areas, and Par2. Thalamic afferents to the four orbital fields are also topographically organized, and are focused in the submedial and mediodorsal nuclei. The ventrolateral orbital area receives input from the entirety of the submedial nucleus, whereas the other orbital areas receive input from its periphery only. Each orbital area is connected with a particular segment of the mediodorsal nucleus. The medial orbital area receives its principal thalamic afferents from the parataenial nucleus, the dorsocentral portion of the mediodorsal nucleus, and the ventromedial portion of the submedial nucleus. The ventral orbital area receives input from the lateral segment of the mediodorsal nucleus, the rostromedial portion of the submedial nucleus, and the central lateral nucleus. Thalamic afferents to the ventrolateral orbital area arise from the entirety of the submedial nucleus and from the lateral segment of the mediodorsal nucleus. The lateral orbital area receives thalamic afferents from the central segment of the mediodorsal nucleus, the ventral portion of the submedial nucleus, and the ventromedial nucleus. The paraventricular, ventromedial, rhomboid and reuniens nuclei also provide additional input to the four orbital areas. The connections of the ventrolateral orbital area are interpreted in the context of its role in directed attention and allocentric spatial localization. The present findings provide anatomical support for the view that areas Fr2, PPC and VLO comprise a cortical network mediating such functions.

Sympathetic vasoconstrictor neurons projecting from the guinea-pig superior cervical ganglion to cutaneous or skeletal muscle vascular beds can be distinguished by soma size

We have used a combination of retrograde axonal tracing and intracellular dye injections to determine the soma size of sympathetic vasoconstrictor neurons projecting from the superior cervical ganglion to the cutaneous vascular bed of the eartips, or to the vascular beds of the masseter muscle, of guinea-pigs. Neurons projecting to vasculature of the masseter muscle had a cross-sectional area of 956 ± 295 μm 2 (mean ± SD; n = 45 cells) and were significantly larger than neurons projecting to the vasculature of the eartip skin (mean cross-sectional area ± SD, 604 ± 251 μm 2; n = 39 cells). These results are consistent with physiological observations showing that muscle vasoconstrictor neurons have faster conduction velocities than cutaneous vasoconstrictor neurons. Furthermore, they suggest that muscle vasoconstrictor neurons may innervate a larger volume of vasculature compared with cutaneous vasoconstrictor neurons.

Identification of uterine-related sympathetic neurons in the rat inferior mesenteric ganglion: neurotransmitter content and afferent input

The rat uterus is innervated by sensory and autonomic nerves. Sensory and sympathetic fibers travel in the hypogastric nerves and are associated with the thoracolumbar spinal cord levels T 13-L 3. The inferior mesenteric ganglion (IMG) contains the somata of sympathetic postganglionic neurons and some of these may project axons to the uterus. Sensory and parasympathetic fibers travel in the pelvic nerve and are associated with the lumbosacral cord levels L 6-S 1 and pelvic ganglion (PG). We previously reported data concerning the neurochemical anatomy of the PG with regard to the uterine innervation; the present study was undertaken to characterize the neurochemical anatomy of the IMG with regard to its involvement in uterine innervation. A retrograde axonal tracer was used to verify projections of axons of IMG neurons to the uterus. Immunostaining of cryostat sections of the IMG revealed neurons immunoreactive for neuropeptide Y (NPY) and for tyrosine hydroxylase (TH). Immunostaining for the synaptic terminal protein synapsin I (SYN) revealed numerous fine terminals immediately surrounding the principal neurons and in the interneuronal spaces. Varicosities immunoreactive for calcitonin gene-related peptide (CGRP), vasoactive intestinal polypeptide (VIP), enkephalin (ENK), substance P (SP) and galanin (GAL) appeared to be associated with principal neurons. Additional varicosities stained for nicotinamide adenine dinucleotide phosphate (reduced)-diaphorase (NADPH-d) and nitric oxide synthase (NOS), thus indicating sites of neuronal nitric oxide synthesis. This study revealed that the IMG contains uterine-related neurons and that some of the retrogradely labeled uterine-related neurons contain NPY, TH or both NPY/TH. In addition, uterine-related neurons received abundant afferent inputs indicated by SYN-immunoreactive (-ir) terminals and some of these varicosities labeled for GAL, CGRP, VIP, ENK, or NADPH-d/NOS.

Motoneurons of the injured spinal cord of the adult dog can grow lengthy axons into an autologous peripheral nerve graft. A retrograde axonal tracing study.

To our knowledge, the capacity of injured spinal neurons to regenerate axons into peripheral nerve autografts has not yet been documented with axonal tracing methods in large adult mammals such as dogs. In the present study, one end of an autologous peripheral nerve graft (PNG), 10-15 cm long, was introduced dorsally into the lumbar (L4) spinal cord of six adult beagle dogs, thus producing a small focal lesion. The other end of the PNG was driven outside the spinal cord, then crushed and tied to nearby peripheral tissue with non-absorbable suture. Clinical examination of the operated animals was performed throughout the postoperative period. In five animals (out of six), the neurological deficit induced by the grafting procedure disappeared within five days. Four months after surgery, application of horse radish peroxidase (HRP) to the transected peripheral tip of the PNG led to the retrograde axonal labelling of about 30 lumbar neurons. The labelled cells, which had extended lengthy (up to 10 cm) axons into the PNGs, were mainly located in the vicinity of the intraspinal tip of the grafted nerve. Upon specific criteria, most of them were characterized as motoneurons. As the surgical procedure probably left the original stem axon of these neurons uninjured, it is suggested that axonal production from the labelled motoneurons might have arisen either from collateral axonal sprouts or even directly from the neuronal soma.

Age-related changes in the morphology of preganglionic neurons projecting to the rat hypogastric ganglion.

The aim of this study was to investigate age-related changes in preganglionic neurons of the lumbar and sacral spinal cord of the male rat that may underlie impaired control of the urogenital system in old age. Preganglionic sympathetic and parasympathetic neurons of 4- and 24-month-old rats were identified by retrograde axonal tracing with cholera toxin subunit-B followed by immunocytochemistry. Labelled preganglionic neurons were scanned on the confocal microscope. Measurements were made of soma area, number of primary dendrites, number of dendritic branch points and total dendritic length. There were significant decreases in the number of dendritic branch points and total dendritic length of sympathetic preganglionic neurons in the aged rats compared to the adult group. The soma area and number of primary dendrites were not significantly different. Some cells exhibited signs of degeneration, such as swelling of the soma and distension of the proximal part of primary dendrites. No significant differences were found in any of the parameters measured for the parasympathetic neurons. The changes in dendritic morphology of sympathetic preganglionic neurons may reflect altered central and peripheral control of pelvic viscera in old age.

Distribution and origins of nitric oxide-producing nerve fibers in the dog tongue: correlated NADPH-diaphorase histochemistry and immunohistochemistry for calcitonin gene-related peptide using light and electron microscopy.

The distribution and origins of nitric oxide (NO)-producing nerves in the dog tongue with reference to calcitonin gene-related peptide (CGRP)-containing sensory fibers were investigated using NADPH-diaphorase (NADPH-d) histochemistry and immunohistochemistry for CGRP and NO synthase combined with retrograde axonal tracing and denervation experiments. The ultrastructural relationships between NADPH-d-positive and CGRP-immunoreactive neuronal elements were also examined electron microscopically. NADPH-d-positive and CGRP-immunoreactive varicose fibers were found within the taste buds and surrounding the epithelia of the fungiform papillae, and they disappeared completely after severance of the lingual nerve. Following injection of fast blue into the subepithelial layer of the anterior two thirds of the tongue, retrogradely labeled neurons possessing NO synthase and/or CGRP immunoreactivities were mainly detected in the trigeminal ganglion. Some of the retrogradely labeled trigeminal cells showed the coexistence of NADPH-d reactivity and CGRP immunoreactivity, but in the geniculate ganglion neither NADPH-d reactivity nor NO synthase immunoreactivity was found instead of retrogradely labeled CGRP-immunoreactive neurons. The lingual artery and its branches, including the arteriovenous anastomoses, showed dense distributions of NADPH-d-positive fibers, most of which were unaffected by the denervation experiments. There were many small ganglia in the tongue, and virtually all ganglionic neurons were NADPH-d reactive. CGRP-immuno-reactive varicose fibers were also found around the vascular walls and within the intralingual ganglia. Ultrastructural analysis revealed a close distribution of NADPH-d-positive and CGRP-immunoreactive varicose fibers within the arterial walls, and synaptic contacts between CGRP-immunoreactive terminals and NADPH-d-positive intralingual ganglionic neurons. These results indicated that the taste buds of epithelia of fungiform papillae in the anterior two thirds of the dog tongue receive NADPH-d-positive and CGRP-immunoreactive sensory fibers from the trigeminal ganglion, and that perivascular NADPH-d-positive fibers mainly originate from intrinsic ganglia in the tongue. The ultrastructural findings suggest an intrinsic peripheral nerve-reflex mechanism in the regulation of the lingual vascular function by NO-producing postganglionic parasympathetic neurons and CGRP-containing sensory fibers.

Sensory and premotor connections of the orbital and medial prefrontal cortex of macaque monkeys

Sensory and premotor inputs to the orbital and medial prefrontal cortex (OMPFC) were studied with retrograde axonal tracers. Restricted areas of the lateral and posterior orbital cortex had specific connections with visual-, somatosensory-, olfactory-, gustatory-, and visceral-related structures. More medial areas received few direct sensory inputs. Within the lateral and posterior orbital cortex, area 12l received a substantial projection from visual areas in the inferior temporal cortex (TE). Area 12m received somatosensory input from face, digit, or forelimb regions in the opercular part of area 1-2, in area 7b, in the second somatosensory area (SII), and in the anterior infraparietal area (AIP). Areas 13m and 13l also received a projection from the opercular part of areas 1-2 and 3b. The posteromedial and lateral agranular insular areas (Iapm and Ial, respectively) received fibers from the ventral part of the parvicellular division of the ventroposterior medial nucleus of the thalamus (VPMpc) that may represent a visceral afferent system. The dorsal part of VPMpc projected to the adjacent gustatory cortex. These restricted inputs from several sensory modalities and the convergent corticocortical connections to orbital areas 13l and 13m suggest a network related to feeding. The OMPFC was also connected to premotor cortex in ventral area 6 (areas 6va and 6vb), in cingulate area 24c, and probably in the supplementary eye field. Area 6va projected to area 12m, whereas a region of area 6vb projected to area 13l. The region of the supplementary eye field projected to areas 12l, 12o, and 12r. Area Ial received fibers from area 24c. Lighter and more diffuse projections also reached wider areas of the OMPFC. For example, injections in several orbital areas labeled a few cells scattered through the anterior part of area TE and the superior temporal gyrus. There was also a projection to the intermediate agranular insular area (Iai) and to areas 13a and 12o from the apparently multimodal areas in the superior temporal sulcus and gyrus.

Differences in dispersion of monkey optic nerve fibers according to their terminal layers in the lateral geniculate nucleus

Dispersion of retinogeniculate fibers in the monkey optic nerve was investigated in relation to their terminal layers in the lateral geniculate nucleus (LGN) using a retrograde axonal tracer (wheat germ agglutinin conjugated to horseradish peroxidase, WGA-HRP). Dispersion of fibers projecting to the parvocellular layer of the LGN (LGNp) is clearly less extensive compared with that of fibers projecting to the magnocellular layer of the LGN (LGNm) in the most distal part of the optic nerve. The occupation rate of retrogradely labeled fibers projecting from the periphery of the nasal retina to the LGNp increases only 1.2 times in the initial part of the nerve. In contrast, the occupation rate increases up to 1.6 times for labeled fibers projecting from a similar part of the nasal peripheral retina to the LGNm in the same part of the optic nerve. The morphological features of the labeled ganglion cells indicate that these fibers arise from Pβ and Pa ganglion cells, respectively.

Development of acetylcholinesterase-positive thalamic and basal forebrain afferents to embryonic rat neocortex.

By combining anterograde and retrograde axonal tracing with AChE histochemistry, we demonstrate the sources of AChE-positive afferents to embryonic neocortex, the pathways they use, their time of arrival into cortex, and their initial invasion of the cortical plate. Acetylcholinesterase (AChE) is expressed by two populations of cortical afferents: AChE is permanently present in basal forebrain fibers and has been reported to be transiently localized in axons of the principal sensory thalamic nuclei over the first few postnatal weeks beginning at the middle of the first week. We first detect AChE-positive afferents histochemically in neocortex on embryonic day seventeen (E17) and determine that they arise from the principal sensory thalamic nuclei. AChE histochemistry labels the entire length of developing thalamocortical axons, including their growth cones and branches. These AChE-positive afferents enter the neocortex by the internal capsule and take an intracortical pathway centered on the subplate layer. As soon as these axons are detected, some have already begun to extend AChE-positive collateral branches superficially toward the cortical plate. By E19, a few collaterals have entered the deep part of the cortical plate and by E21 have densely invaded all but its most superficial undifferentiated part. AChE-positive afferents from basal forebrain structures reach the neocortex by three routes: the external capsule, the internal capsule, and the cingulate bundle. Among basal forebrain components, only the substantia innominata and nucleus basalis of Meynert reach the cortex by the internal capsule. Afferents from these two sources reach neocortex on E18, but are a very minor component of the total population of AChE-positive afferents at this age. Afferents from other basal forebrain components do not reach neocortex until several days later. The spatial and temporal patterns of AChE expression in developing thalamocortical axons indicate that it is useful for delineating their innervation of the primary sensory areas of embryonic neocortex, and suggest that AChE may function in axon extension and cortical differentiation.

Efferent Innervation of the Rat Testis1

Previous assessments of the autonomic nerve supply of the male genital tract concluded that the testis received sympathetic input primarily from paraaortic ganglia, particularly the spermatic ganglion. We challenged this consensus by using retrograde axonal tracing to examine the source and distribution of efferent fibers reaching the testis of adult rats. We also used immunohistochemical methods to assess putative neurotransmitters in testicular neurons of the abdominal and pelvic ganglia. The results indicate the majority of retrogradely labeled cell profiles were localized within the major pelvic (38%) and pelvic accessory ganglia (37%), and only a few labeled cell profiles were present in the paraaortic and spermatic ganglia. Injection of FluoroGold and Fast Blue dyes into the respective right and left testis demonstrated that 17% of the neurons in pelvic ganglia were labeled when tracers were microinjected beneath the capsule of the contralateral testis. About 8% of the neurons were labeled both with FluoroGold and Fast Blue, suggesting that certain neurons can provide simultaneous input to the left and right testicles. Immunohistochemical results showed that tyrosine hydroxylase, a marker for noradrenergic fibers, was present in over 33% of the cell profiles labeled with either FluoroGold or Fast Blue. Some 27% of the fluorescent-labeled cell profiles were positive for neuropeptide Y, while 22% were immunoreactive for vasoactive intestinal polypeptide. No evidence for vasoactive intestinal polypeptide immunoreactivity was detected within the testis, but neuropeptide Y-immunoreactive fibers were present in the tunica albuginea and testicular vasculature. Catecholamine fluorescent fibers were distributed sparsely throughout the periphery of the testis in association with the capsule, vasculature, and interstitium.(ABSTRACT TRUNCATED AT 250 WORDS)

Parasympathetic preganglionic neurons in the spinal cord involved in uterine innervation are cholinergic and nitric oxide-containing.

The purpose of this study was to elucidate parasympathetic preganglionic neurons in the spinal cord that project axons in pathways to the uterus and to reveal their neurotransmitter phenotype. "Uterine-related" neurons were identified by using a combination of retrograde axonal tracers: (1) Fluorogold injected into the ganglia of termination of preganglionic fibers, and (2) a transganglionic axonal tracer (pseudorabies virus) injected into the uterus. Immunohistochemistry was used to reveal virus-labeled neurons and their neurotransmitter marker. Double-labeled (Fluorogold+pseudorabies virus) "uterine" preganglionic neurons were identified in the sacral parasympathetic nucleus of the rat lumbosacral spinal cord. Subpopulations of neurons in the sacral parasympathetic nucleus were shown to be immunoreactive for choline acetyltransferase or nitric oxide synthase. Double-staining immunohistochemistry (for pseudorabies virus+neurotransmitter enzyme) revealed that some of the uterine-related preganglionic neurons were cholinergic and some nitric oxide synthase-containing. These results demonstrate a subpopulation of preganglionic parasympathetic neurons in the sacral parasympathetic nucleus that are involved in uterine innervation. In addition, both acetylcholine and nitric oxide could be used to modify activity in the postganglionic neurons, which directly innervate the uterus.

Isolation of mature olfactory neurones using retrograde labelling and flow cytometry

Currently we can observe an increasing interest for epithelial olfactory neurones. Several laboratories are involved in elucidation of the odorant-molecule recognition process and transduction cascade which brings information to the olfactory bulb. Others use this model, unique in mammals, to accumulate new knowledge on the neurogenesis phenomenon. Here we describe a simple and efficient method to purify mature olfactory neurone populations extracted from the nasal cavity. The approach relies on retrograde axonal tracing followed by flow cytometry sorting. For this purpose we inject a fluorescent dye (Fast Blue or diI C 18(3)) in the olfactory bulb of adult rats. Seven days later, we extract the nasal turbinates and separate the mucosa from the subjacent lamina propria. The tissue is enzymatically dissociated and the labelled cells are sorted with a flow cytometer. Purification of the mature olfactory neurones varies from 80 to 99%.

Neurochemical mechanisms underlying amygdaloid modulation of aggressive behavior in the cat

Studies designed to determine the respective roles of substance P, excitatory amino acids, and enkephalins in amygdaloid modulation of defensive rage behavior in the cat are presented. The basic design of these studies involved three stages. In stage I, cannula electrodes for stimulation and drug infusion were implanted into medial hypothalamic of midbrain periaqueductal gray (PAG) sites from which defensive rage behavior could be elicited. Then, a stimulating electrode was implanted into a site within the medial, basal, or central nuclear complex from which modulation of the defensive rage response could be obtained. Amygdaloid modulation of defensive rage was determined in the following manner: it employed the paradigm of dual stimulation in which comparisons were made of response latencies between alternate trials of dual (i.e., amygdala+medial hypothalamus [or PAG]) and single stimulation of the hypothalamus or PAG alone. Thus, stage I established the baseline level of modulation (i.e., facilitation or suppression of defensive rage) in the predrug stimulation period. In stage II, a selective or non-selective receptor antagonist for a given transmitter system was administered either peripherally or intracerebrally at the defensive rage site, after which time the same dual stimulation paradigm was then repeated over the ensuing 180 min postinjection period in order to determine the effects of drug delivery upon amydaloid modulation of defensive rage. Stage III of the study took place at the completion of the pharmacological testing phase. The retrograde axonal tracer, Fluoro-Gold, was microinjected into the defensive rage site within the medial hypothalamus or PAG, and following a 6-14 day survival period, animals were sacrificed and brains were processed for histological and immunocytochemical analyses for the neurotransmitters noted above. This procedure thus permitted identification of cells within the amygdala which were labeled retrogradely and which were also immunostained positively for substance P, excitatory amino acids, or enkephalin. For studies involving substance P, defensive rage was elicited from the medial hypothalamus and for studies examining the roles of excitatory amino acids and enkaphalin, defensive rage was elicited from the PAG. In the first study, facilitation of hypothalamically elicited defensive rage was obtained with dual stimulation of the medial nucleus of the amygdala. In separate experiments, the selective NK 1 non-peptide antagonist, CP 96,345, was administered both peripherally as well as intracerabrally into the hypothalamic defensive rage sites in doses of 0.5-4.0 mg/kg (i.p.) and 0.5-2.5 nmol (i.c.). Following drug delivery, the facilitatory effects of medial amygdaloid stimulation were blocked ina dose- and time-dependent manner in which the effects were noted as early as 5 min postinjection. The maximum drug dose (4.0 mg/kg) employed for peripheral administration resulted in a 42% reduction in the facilitatory effects of the medial amygdala (P<0.002). This drug, when microinjected directly into medial hypothalamic defensive rage sites at the maximum dose level of 2.5 nmol, resulted in an 84% reduction of the suppressive effects of amygdaloid stimulation (P<0.5) at 5 min postinjection. In the next study, an N-methyl-D-aspartate (NMDA) antagonist, DL-α-amino-7-phosphonoheptanoic acid (AP-7), was administered either peripherally (0.1-1.0 mg/kg) or intracerebrally (0.2 and 2.0 nmol) into PAG defensive rage sites

Vascularization and innervation of the canine wrist joint synovial membrane.

The correlation between vascular distribution and synovial structures was investigated using corrosion cast of the synovial fold of the dog antebrachiocarpal joint (wrist joint) under scanning electron microscopy. Arterial branches arose from a main artery deep in the subsynovial layer, ascended toward the free margin of the fold, dividing into several branches, and finally formed a dense capillary network beneath the lining layer. Immunohistochemical study and a retrograde axonal tracing experiment revealed that almost all nerve fibers were associated with the arterial tree and that: (1) proximal parts of the arterial tree were innervated with neuropeptide Y (NPY)-containing noradrenergic sympathetic fibers, vasoactive intestinal peptide-containing non-catecholaminergic sympathetic fibers from the stellate ganglion and substance P (SP)- and calcitonin gene-related peptide (CGRP)-containing sensory fibers from the C7-T1 segments of the dorsal root ganglia; (2) more distal parts of the arterial branches were associated with NPY-immunoreactive sympathetic fibers and SP- and CGRP-immunoreactive sensory fibers; and (3) the most peripheral precapillary arterioles were accompanied by only SP- and CGRP-containing sensory fibers. These results indicate that synovial blood flow is regulated by at least three different nerve systems which possess regional differences.

Cutaneous distribution of sympathetic postganglionic fibers from stellate ganglion: A retrograde axonal tracing study using wheat germ agglutinin conjugated with horseradish peroxidase.

Sympathetic postganglionic cells of the stellate ganglion (SG) projecting to the skin in young dogs were labeled retrogradely with wheat germ agglutinin conjugated with horseradish peroxidase (WGA-HRP). After application of WGA-HRP into the dermis and/or subcutaneous tissue of the upper extremity, numerous labeled cells were observed in the SG. The sympathetic postganglionic fibers from the SG were distributed in the skin area between the third cervical vertebra and the level of the thirteenth rib. These fibers decreased in number toward the rostral and caudal skin area with a peak on the upper extremity. Sympathetic postganglionic cells in the SG projecting to these skin areas were found ipsilaterally to the injection side. No labeled cells were observed in the SG after injecting WGA-HRP into the epidermis and/or subcutaneous tissue of the face, the level of the second cervical vertebra, and the lower extremity.Sympathetic postganglionic neurons of the SG which project in the cervical and thoracic sympathetic trunks were labeled retrogradely with unconjugated horseradish peroxidase (HRP) applied to the cut end of the sympathetic trunks proximal to the SG. The application was made in the cervical sympathetic trunk (CST) caudal to the superior cervical ganglion (SCG). In the thoracic sympathetic trunk (TST), the application was made between the fourth and fifth thoracic sympathetic ganglia (T4 and T5), and between T10 and T11. Labeled cell bodies were observed in SG bilaterally.

Cutaneous distribution of sympathetic postganglionic fibers from stellate ganglion: A retrograde axonal tracing study using wheat germ agglutinin conjugated with horseradish peroxidase

Cutaneous distribution of sympathetic postganglionic fibers from stellate ganglion: A retrograde axonal tracing study using wheat germ agglutinin conjugated with horseradish peroxidase

Regulation of SC1/DM-GRASP during the migration of motor neurons in the chick embryo brain stem.

The hindbrain of the chick embryo contains three classes of motor neurons: somatic, visceral, and branchial motor. During development, somata of neurons in the last two classes undergo a laterally directed migration within the neuroepithelium; somata translocate towards the nerve exit points, through which motor axons are beginning to extend into the periphery. All classes of motor neuron are immunopositive for the SC1/DM-GRASP cell surface glycoprotein. We have examined the relationship between patterns of motor neuron migration, axon outgrowth, and expression of the SC1/DM-GRASP mRNA and protein, using anterograde or retrograde axonal tracing, immunohistochemistry, and in situ hybridization. We find that as motor neurons migrate laterally, SC1/DM-GRASP is down-regulated, both on neuronal somata and axonal surfaces. Within individual motor nuclei, these lateral, more mature neurons are found to possess longer axons than the young, medial cells of the population. Labelling of sensory or motor axons growing into the second branchial arch also shows that motor axons reach the muscle plate first, and that SC1/DM-GRASP is expressed on the muscle at the time growth cones arrive.

Direct projections from the entopeduncular nucleus to the lower brainstem in the rat.

The present study reports the existence of projection fibers from the entopeduncular nucleus to the superior colliculus and lateral parts of the pontobulbar tegmental regions (so-called lateral tegmental field) in the rat, suggesting that the entopeduncular nucleus may control eye-head and orofacial movements via these projection fibers. The anterograde axonal tracing with Phaseolus vulgaris-leucoagglutinin has revealed that the entopedunculotectal fibers terminate, bilaterally, with an ipsilateral predominance, in the deep layers of the superior colliculus through its rostral one-third level and that the entopedunculotegmental fibers terminate, bilaterally, with an ipsilateral predominance, in the parabrachial area, reticular formation surrounding the trigeminal motor nucleus, and parvicellular, dorsal, and ventral reticular nuclei. The cells of origin of the entopedunculotectal and entopedunculotegmental projections have been identified by retrograde axonal tracing with Fluoro-Gold and cholera toxin B subunit. The entopedunculotectal or entopedunculotegmental fibers originate, respectively, from the dorsal or ventral part of the entopeduncular nucleus. Additionally, a series of fluorescent retrograde double-labeling experiments with Fast Blue and Diamidino Yellow have indicated that single entopeduncular nucleus neurons projecting to the superior colliculus or lateral tegmental field often send their axon collaterals to the lateral habenular nucleus. The entopedunculotectal fibers are assumed to control head movements, which may be provoked via the tectospinal fibers, and further to participate in eye movements as the nigrotectal fibers that have been known to arise from the substantia nigra pars reticulata to end in the deep layers of the superior colliculus primarily through its caudal two-thirds level. The entopedunculotegmental fibers are presumed to be involved in control of orofacial movements, because the sites of termination of the entopedunculotegmental fibers correspond well with the reported areas of distribution of premotor interneurons for the trigeminal motor, facial, and hypoglossal nuclei.

Cholinergic neurons in the ventral trapezoid nucleus project to the cochlear nuclei in the rat

A combination of retrograde axonal tract tracing and choline acetyltransferase immunocytochemistry was used to determine the cells of origin of the cholinergic innervation of the rat cochlear nucleus. The results showed that the cochlear nucleus receives a major cholinergic input from a group of small cells found in the ventral trapezoid nucleus. Experiments using an anterograde tracer confirmed the presence of a neuronal pathway from the ventral trapezoid nucleus to the cochlear nucleus and showed that this pathway travels via the trapezoid body.

A cholinergic propriospinal innervation of the rat spinal cord

Previous work has suggested the presence of a widespread, intrinsic cholinergic innervation of the spinal cord. A combination of retrograde axonal tract tracing and choline acetyltransferase immunocytochemistry was used to show that there is a propriospinal cholinergic innervation of the rat spinal cord that arises from short-range projections (spanning up to six spinal segments) of cholinergic neurons known as ‘central canal’ and ‘partition’ cells.