Retrograde Transport Studies Research Articles

Structural Aspects of the Involvement of the Brainstem Nuclei in the Processing of Functionally Diverse Information Streams Passing through the Basal Ganglia

The structural basis for information processing in the morphofunctional system of the basal ganglia is considered on the basis of analysis of retrograde axonal transport studies of the projections between functionally diverse parts of the brainstem nuclei and functionally diverse segments of basal ganglia structures. The organization of projections between these structures in the dog brain revealed topical elements pointing to the possibility of the segregated transmission of information between the limbic and motor segments of the brainstem nuclei and the corresponding areas of the basal ganglia. However, these projection systems are dominated by convergent transmission of functionally diverse information, providing evidence that information of different modalities may be integrated not solely in the main, but in all the nuclei of the morphofunctional basal ganglia system. Labeled sparsely branched long-axon “reticular” neurons in the brainstem nuclei projecting to the basal ganglia also provide evidence of the integrative function of the nuclei of interest. The possibility that these data could be used to create new models helping us to understand the functioning of the basal ganglia in health and disease is discussed.

A monoclonal natural human IgM protects axons in the absence of remyelination.

BackgroundWhereas demyelination underlies early neurological symptoms in multiple sclerosis (MS), axonal damage is considered critical for permanent chronic deficits. Intracerebral infection of susceptible mouse strains with Theiler’s murine encephalomyelitis virus (TMEV) results in chronic induced demyelinating disease (TMEV-IDD) with progressive axonal loss and neurologic dysfunction similar to progressive forms of MS. We previously reported that treatment of chronic TMEV-IDD mice with a neurite outgrowth-promoting natural human antibody, HIgM12, improved brainstem NAA concentrations and preserved functional motor activity. In order to translate this antibody toward clinical trial, we generated a fully human recombinant form of HIgM12, rHIgM12, determined the optimal in vivo dose for functional improvement in TMEV-IDD, and evaluated the functional preservation of descending spinal cord axons by retrograde labeling.FindingsSJL/J mice at 45 to 90 days post infection (dpi) were studied. A single intraperitoneal dose of 0.25 mg/kg of rHIgM12 per mouse is sufficient to preserve motor function in TMEV-IDD. The optimal dose was 10 mg/kg. rHIgM12 treatment protected the functional transport in spinal cord axons and led to 40 % more Fluoro-Gold-labeled brainstem neurons in retrograde transport studies. This suggests that axons are not only present but also functionally competent. rHIgM12-treated mice also contained more mid-thoracic (T6) spinal cord axons than controls.ConclusionsThis study confirms that a fully human recombinant neurite outgrowth-promoting monoclonal IgM is therapeutic in a model of progressive MS using multiple reparative readouts. The minimum effective dose is similar to that of a remyelination-promoting monoclonal human IgM discovered by our group that is presently in clinical trials for MS.

Analysis of the Conduction of Information and Its Integration in the Projection Systems Connecting the Basal Ganglia and the Rostromedial Tegmental Nucleus of the Dog Brain

Analysis of retrograde axonal transport studies of the projections between individual parts of the rostral and caudal sectors of the rostromedial tegmental nucleus (RMTg) and functionally diverse segments of striatal and pallidal structures was used to study the structural basis of information processing in the morphofunctional system of the basal ganglia. Topical elements were seen in the organization of the projections between these structures in the dog brain, suggesting the possibility of segregated conduction of information between the limbic segments of the RMTg and the limbic areas of the striopallidum. However, most striopallidal structures showed convergence of projection fibers running from neurons in functionally diverse parts of the RMTg, which may be evidence that functionally diverse information is integrated within these structures. Labeled sparsely branching, long-axon, “reticular” neurons of the RMTg projecting to striopallidal structures also provided evidence of the integrative function of the nucleus of interest. We evaluate data on the possible pathways conducting specific motor and limbic information and its integration in the structures of this system and the possibility of using them to create new models promoting our understanding of the functioning of the basal ganglia in health and disease.

I. Shiga toxin B-subunit system: retrograde transport, intracellular vectorization, and more

Many intracellular transport routes are still little explored. This is particularly true for retrograde transport between the plasma membrane and the endoplasmic reticulum. Shiga toxin B subunit has become a powerful tool to study this pathway, and recent advances on the molecular mechanisms of transport in the retrograde route and on its physiological function(s) are summarized. Furthermore, it is discussed how the study of retrograde transport of Shiga toxin B subunit allows one to design new methods for the intracellular delivery of therapeutic compounds.

Analysis of retrograde transport in motor neurons reveals common endocytic carriers for tetanus toxin and neurotrophin receptor p75NTR.

Axonal retrograde transport is essential for neuronal growth and survival. However, the nature and dynamics of the membrane compartments involved in this process are poorly characterized. To shed light on this pathway, we established an experimental system for the visualization and the quantitative study of retrograde transport in living motor neurons based on a fluorescent fragment of tetanus toxin (TeNT HC). Morphological and kinetic analysis of TeNT HC retrograde carriers reveals two major groups of organelles: round vesicles and fast tubular structures. TeNT HC carriers lack markers of the classical endocytic pathway and are not acidified during axonal transport. Importantly, TeNT HC and NGF share the same retrograde transport organelles, which are characterized by the presence of the neurotrophin receptor p75NTR. Our results provide the first direct visualization of retrograde transport in living motor neurons, and reveal a novel retrograde route that could be used both by physiological ligands (i.e., neurotrophins) and TeNT to enter the central nervous system.

Detection of retrogradely transported WGA-HRP in axotomized adult hamster facial motoneurons occurs after initiation of the axon reaction.

We have previously shown that facial nerve transection at the stylomastoid foramen activates ribosomal RNA transcription in injured facial motoneurons (FMN) of the adult hamster within 30 minutes postoperative. The signal for the initiation of the nerve cell body response to injury in vertebrates is currently unknown. It has been hypothesized that the signal for initiating the injury response is dependent on retrograde transport, where the signal itself is either the loss of a repressor substance from the periphery or the loss of retrogradely transported target-derived factors. To examine if a retrograde transport-mediated signal would be sufficient to produce the rapid ribosomal effects observed in hamster FMN following injury, adult hamsters were subjected to right facial nerve axotomies, with the neuronal tracer wheat germ agglutinin horseradish peroxidase (WGA-HRP; M.W. 80,000) applied at the proximal stump of the transected nerve. At time points ranging from 0.5 to 24 hours postoperative (hpo), the animals were killed and brainstem sections containing bilateral facial nuclei processed for WGA-HRP label using the TMB method. The earliest time point at which WGA-HRP was detected in the axotomized facial nucleus occurred at 3 hpo. To eliminate molecular weight as a confounding factor, an additional retrograde transport study was performed using the smaller tracer, Fluoro-Gold (M.W. 532.59). Fluoro-Gold was not detected until well after the 3 hpo time point. Thus, it appears that initiation of the axon reaction in hamster FMN is likely to be independent of the retrograde transport properties of the injured neuron.

The timecourse of neuronal connections of the rotundoectostriatal pathway in chicks (Gallus gallus) during embryogenesis: A retrograde transport study

The avian retinotectofugal pathway has been suggested to be homologous to the mammalian retinotectofugal pathway. The projection of the nucleus rotundus upon the ectostriatum is equivalent to that of the pulvinar nucleus upon the extrastriate cortex in mammals. In this system, the optic tectum relays retinal input to the nucleus rotundus, which then ascends to the ectostriatum of the telencephalon. Given the fact that the chick retinotectofugal system becomes mature early during development, the present study attempted to investigate the timecourse of neuronal connections of the embryonic rotundoectostriatal pathway. We used multiple injections of cholera toxin B subunit (CTb) in the ectostriatum of chick embryos to retrogradely trace projections to the nucleus rotundus. We found CTb-labeled neurons in the nucleus rotundus at embryonic day 7.5-8. By embryonic day 8-8.5, increased numbers of CTb-labeled neurons were seen in the nucleus rotundus. It was noted that the time of this initial connection between the nucleus rotundus and the ectostriatum is nearly synchronous with that of the retinotectal and tectorotundal pathways, respectively (Crossland et al., 1975; Thanos & Bonhoeffer, 1987; Wu et al., 2000). These findings, combined with the present study, suggest that the retinotectofugal system becomes established, at least at a structural level, by embryonic day E8.

The combined retrograde transport and unbiased stereological study of the claustrocortical connections in the rabbit

The quantitative analysis of the claustrocortical connections in the rabbit, labeled with the fluorescent retrograde tracer Fluoro-Gold (FG), was conducted by means of unbiased stereology. The FG was injected into selected regions of the motor, somatosensory, auditory and visual cortices and then a comparison of the various claustrocortical projections was carried out. This was achieved by comparing (1) the numerical densities of projecting neurones for each claustral projection zone and (2) the distribution of the labeled neurones throughout the rostro-caudal extent of the claustrum. No significant differences between the numerical densities of labeled neurones in the various projection zones are reported. The motor and primary somatosensory projections dominated in the anterior and central parts of the claustrum, whereas the secondary somatosensory, auditory and visual projections--in the posterior part. The difference in the distributions was significant (p < 0.001). Summarizing, the cortical projections in the claustrum, although varying topographically, do not reveal a quantitative differentiation. This may speak in favour of the integrative and modulating function of this structure in relationship to the neocortex.

Physiological evidence for caudal brainstem projections of jaw muscle spindle afferents.

Retrograde transport and intra-axonal labeling studies provide convincing evidence that jaw-muscle spindle afferents project to the caudal medulla by way of Probst's tract. However, functional properties of this caudal projection are not well understood. Extracellular recordings were made in cats at the level of the subnucleus interpolaris (Vi) to identify single units that showed consistent responses to ramp-and-hold stretches of the jaw. In this report, we present data from 20 central units with properties indicating that they received input from trigeminal muscle spindle afferents. All units were activated by gentle palpation of jaw muscles, and none had superficial receptive fields. Two groups of neurons could be defined based on their responses to passive jaw movements. One group (n=12) showed an obvious dynamic response (i.e., a higher level of activity at the onset of stretch than during the hold period). Activity was maintained during the hold phase, and the units stopped firing (unloaded) for a brief period upon jaw closure. The other group (n=8) lacked a dynamic response. Instead, they showed an increase in firing with onset of stretch that was maintained during the hold phase. Thirteen units, which were tested with more than three different jaw stretch speeds and/or amplitudes, were further characterized by analyzing dynamic index (DI) and mean firing rate (MFR) during each phase of the ramp-and-hold movement as well as interspike interval (ISI) variability. All but one unit with a dynamic response showed a speed-sensitivity. In all cases, the MFR was a more sensitive indicator of changes in jaw speed than DI. Neurons in the other group (5/5 tested) showed a high position-sensitivity, i.e., their firing rates varied as a function of amplitude of jaw opening. The percent change in ISI variability for all neurons ranged from 37-84%. The response characteristics of these central neurons were compared to known physiological properties of muscle spindle afferents. The results provided compelling evidence for jaw-muscle-spindle afferent projection onto these neurons. Reconstruction of recording sites showed that medial Vi, and the adjacent reticular formation, are likely recipients for the caudal projections from jaw-muscle-spindle afferents. We suggest that muscle spindle input to this region is well suited for influencing the coordination of motor behavior during feeding and for the integration and processing of kinesthetic information.

Retinoic Acid Synthesizing Astrocytes within the Developing Spinal Cord: Correlative Microscopic Study

The Western Mosquitofish, Gambusia affinis affnis, a sexually dimorphic internal fertilizing species of the family Poeciliidae, shows distinct sexual dimorphism in the anal fin and its axial and appendicular support.The development of the regional vertebral differences (i.e. precaudal [vertebra bearing ribs] and caudal [vertebra bearing hemal spines]) in female and male G. a. affinis is generated through a development program of first posterionzing more anterior vertebra (formation of hemal spines more anteriorly) then antenonzing more posterior vertebra (loss of hemal spines and formation of parapophysis with pleural ribs) . This developmental program is reminiscent of effects obtained by disturbing developmental programs by exogenous treatment with retinoic acid.Recently we generated data suggesting that all-trans retinoic acid (tRA), a non-steroidal lipophilic molecule may play a role in the regulation and patterning of sexual dimorphic phenotype(s), an area considered the domain of steroid hormones.We wanted to identify the specific neural cell type(s) synthesizing endogenous tRA within the developing spinal cord which might coordinate sexually dimorphic patterning of spinal cord levels in G. a. affinis. To achieve this, we employed retrograde transport studies using 3,000 molecular weight (MW) dextran amine and biocytin combined with immunogold and/or lmmunohistochemistry to identify the specific neural type(s) which express tRA synthesizing enzymefs] (i.e. retinaldehyde dehydrogenase [RALDH]).

Study of shiga toxin B-fragment retrograde transport: Development of intracellular targeting tools with potential applications in cancer therapy

Study of shiga toxin B-fragment retrograde transport: Development of intracellular targeting tools with potential applications in cancer therapy

Synthesis of green fluorescent protein-ricin and monitoring of its intracellular trafficking.

We performed genetic engineering to fuse enhanced green fluorescent protein (EGFP) to the N terminus of RTA, expressed the fusion protein in Escherichia coli, purified and reassociated EGFP-RTA with plant RTB, and purified EGFP-ricin by size exclusion HPLC. The fusion heterodimer was able to bind galactosides, intoxicate cells, and show strong fluorescence. Mammalian cells incubated with EGFP-ricin showed strong cell surface fluorescence at 4 degrees C and, on incubation at 37 degrees C, distributed initially to endosomes and then to Golgi vesicles. Variable sensitivity of mammalian cells to ricin and ricin fusion proteins may be due in part to different patterns of intracellular routing. Cells were incubated with ricin or EGFP-ricin, and inhibition of protein synthesis was measured. Human hepatocellular carcinoma Hep3B cells were 10-fold more sensitive to ricin and 85-fold more sensitive to EGFP-ricin than human epidermoid carcinoma KB cells. Epifluorescence microscopy of cells incubated with EGFP-ricin showed greater localization of the fluorescence signal in the Golgi compartments in Hep3B cells than in KB cells. These data support a model requiring a Golgi-dependent step in cell intoxication by ricin. The work further identifies the usefulness of green fluorescent protein fusions in the study of retrograde transport of internalized peptides.

Trigeminal ganglion innervation of the cochlea—a retrograde transport study

Innervation patterns of sensory nerves from the trigeminal ganglion to the cochlear blood vessels were studied using retrograde transport of wheat germ agglutinin conjugated to horseradish peroxidase. Guinea-pigs ( n=7) were unilaterally implanted with an osmotic pump and a cannula for cochlear delivery of 2% or 20% wheat germ agglutinin–horseradish peroxidase (Group 1), 2% wheat germ agglutinin–horseradish peroxidase followed by 100 μmol capsaicin (Group 2), or vehicle alone. Histological sections of the trigeminal ganglia, the C1 and C2 dorsal ganglia, the superior and inferior ganglia of the glossopharyngeal nerve bilaterally, the midbrain and the brainstem were obtained after 48 h of infusion. In Group 1, a large number of labeled nerve cell bodies were observed in the anteromedial portion of the trigeminal ganglion and at the origin of the ophthalmic nerve. Some labeled cells were also found on the lateral side of the ophthalmic nerve, as well as on the medial side of the maxillary nerve root. Capsaicin pretreatment significantly reduced the density of labeled neurons in the trigeminal ganglion. A few labeled neurons were also found in the trigeminal brainstem nucleus complex and in certain auditory brainstem nuclei. No wheat germ agglutinin–horseradish peroxidase-positive cells were observed in the spinal C1 or C2 cervical ganglia or in the superior or inferior glossopharyngeal ganglia. In contrast, wheat germ agglutinin–horseradish peroxidase application to the middle ear resulted in labeled cells in the middle posterolateral portion of the trigeminal ganglia and in the superior ganglia of the glossopharyngeal nerve. These results provide the first direct evidence that the trigeminal ganglion sends projections to the cochlea.

Estrogen receptors are expressed in a subset of tyrosine hydroxylase-positive neurons of the anterior preoptic region in the rainbow trout.

A double immunocytochemical procedure, with two different chromogens, was used to compare the respective distribution of estrogen receptor-immunoreactive cells and tyrosine hydroxylase-immunoreactive neurons on the same sections of the preoptic region of adult female rainbow trout (Oncorhynchus mykiss). Estrogen receptor-immunoreactive cells were observed in the anterior preoptic region surrounding the preoptic recess and its large lateral extensions. Tyrosine hydroxylase-immunoreactive cells were consistently detected in the ventral and ventrolateral walls of the preoptic recess, in an area that was named nucleus preopticus pars anteroventralis. Dopamine immunohistochemistry and Dil retrograde transport studies indicated that part of these catecholaminergic neurons are dopaminergic and could project to the pituitary. Double staining studies showed consistently that most estrogen receptor-positive cells located ventral to the large extensions of the preoptic recess are also tyrosine hydroxylase-positive, indicating that this region is a major target for estradiol feedback. The results are discussed in relation to the role of the nucleus preopticus pars anteroventralis in mediating the negative feedback actions of estradiol on the secretion of gonadotrophin (GTH2) secretion. A hypothesis is drawn in order to explain the synchronizing role of estradiol at the time of ovulation in rainbow trout.

Excitatory amino acid projections to the nucleus of the solitary tract in the rat: a retrograde transport study utilizing d-[ 3H]aspartate and [ 3H]GABA

Afferents to the nucleus tractus solitarius utilizing excitatory amino acid transmitters were described in rat brain by autoradiography following microinfusion and retrograde transport of d-[ 3H]aspartate. Parallel experiments with the injection of [ 3H]GABA were employed to establish the transmitter-selective nature of the retrograde labelling found with d-[ 3H]aspartate. Following infusion of d-[ 3H]aspartate, perikaryal labelling was heaviest in myencephalon, where at least 16 discrete nuclei were labelled. Heaviest labelling was localized bilaterally in the trigeminal nucleus with cells extending through its subdivisions and the entire rostrocaudal axis. Intense labelling was also obtained in the inferior olive, predominantly contralaterally, and non-perikaryal labelling noted. Vestibular, reticular and raphe nuclei contained heavily labelled perikarya. In cervical spinal cord, a moderate density of labelled cells was found in dorsal horn, adjoining the central canal (lamina X) and in the central cervical nucleus, along with appreciable labelling of processes and non-perikaryal labelling. The relative density of labelled perikarya in mesencephalic nuclei was much lower than found in myencephalon, although d-[ 3H]aspartate produced topographic and precise labelling of a small number of cells in the periaqueductal gray, medial parabrachial nucleus and Koelliker-Fuse nucleus. Only weak labelling was found in cortex and hypothalamus. Labelled cells were not consistently observed in other regions (stria terminalis, amygdala, fastigial nucleus, locus coeruleus and rostral ventrolateral medulla) known to innervate the nucleus tractus solitarius. Lower densities of labelled perikarya were found after the microinjection of [ 3H]GABA, and the only regions in which a small number of cells were labelled by both d-[ 3H]aspartate and [ 3H]GABA were trigeminal nucleus, reticular nuclei and raphe obscurus. An exception was the ventrolateral medulla, where [ 3H]GABA produced precise labelling in the nucleus ambiguus and facial nucleus consistent with previous evidence for a GABAergic pathway from this area to the nucleus tractus solitarius. Our findings confirm the selectivity of the retrograde transport of d-[ 3H]aspartate and [ 3H]GABA. Overall, the transport of d-[ 3H]aspartate revealed a complex topographic and convergent network of afferent pathways to the nucleus tractus solitarius likely to utilize an excitatory amino acid transmitter.

Monosynaptic innervation of facial motoneurones by neurones of the parvicellular reticular formation.

In order to determine whether neurones in the parvicellular reticular formation are in direct synaptic contact with motoneurones innervating facial muscles, a combined retrograde and anterograde transport study was carried out in the rat. Animals received injections of the retrograde tracer cholera toxin B conjugated to horseradish peroxidase into facial muscles and of the anterograde tracer biocytin into the parvicellular reticular formation. The facial motor nucleus was then examined for anterograde and retrograde labelling in the light and electron microscopes. Retrogradely labelled neurones were found in the facial motor nucleus with a distribution that was dependent on the muscles injected. Terminals anterogradely labelled with biocytin from the parvicellular reticular formation was observed in the motor nucleus amongst the retrogradely labelled neurones. At the electron microscope, the retrogradely labelled cells were found to receive input from unlabelled terminals and from terminals that were anterogradely labelled from the injections of biocytin in the parvicellular reticular formation. The labelled terminals were 1-2 microns in diameter at the active zone and packed with spherical vesicles. They formed both symmetrical and asymmetrical synapses with their labelled or unlabelled targets. It is concluded that neurones in the parvicellular reticular formation form direct synaptic contact with motoneurones of facial muscles. This may represent a pathway by which the basal ganglia can directly influence orofacial movement, as the substantia nigra is known to project to that part of the reticular formation.

Chemical coding of neurons projecting to pelvic viscera in the male guinea pig: a study by retrograde transport and immunohistochemistry

Retrograde transport studies using Fast Blue dye demonstrated that the ductus deferens, seminal vesicle, prostate and rectum, but not the urinary bladder of the male guinea pig are at least in part innervated by the anterior major pelvic ganglion. In the ductus deferens, seminal vesicle and prostate innervation is derived from ipsilateral and contralateral ganglia. In addition to retrograde studies, dye-filled neurons were analysed immunohistochemically for neuronal markers and associations with specifically identified neuronal projections. Neurons of the ganglion projecting to the ductus deferens either contained tyrosine hydroxylase alone, tyrosine hydroxylase and neuropeptide Y, neuropeptide Y alone, neuropeptide Y and vasoactive intestinal peptide, or vasoactive intestinal peptide alone. These neurons were associated with three classes of neuronal projections, substance P-, leucine-enkephalin-, and methionine-enkephalin-immunoreactive. Neurons projecting to the seminal vesicles were similar to the neurons supplying the ductus deferens, except none of the seminal vesicle-specific neurons exhibited vasoactive intestinal peptide immunoreactivity. Neurons supplying the prostate were immunoreactive for either tyrosine hydroxylase or neuropeptide Y; these neurons were infrequently associated with the three classes of varicose neuronal projections. Neurons projecting to the rectum contained neuropeptide Y and were only associated with methionine-enkephalin immunoreactive neuronal projections in one animal.

Calcitonin gene-related peptide-like immunoreactive (CGRPI) elements in the circadian system of the mouse: an immunohistochemistry combined with retrograde transport study

This study was based on immunohistochemical detection of calcitonin gene-related peptide-like immunoreactive (CGRPI) neurons and fibers in the suprachiasmatic nucleus (SCN) and intergeniculate leaflet (IGL) of the mouse. CGRPI neurons and fibers were found within the ventrolateral part of the SCN, in the whole extent of IGL and sparsely distributed in ventral geniculate body. The presence of CGRPI structures in the SCN and IGL of the mouse further supports the hypothesis of differences in the content of neuroactive substances in the circadian clock between mammalian species. Fluorogold retrograde transport combined with CGRP immunofluorescence demonstrates that CGRPI neurons in the IGL constitute a part of IGL reciprocal connections.

Projections from the nucleus tractus solitarii to the spinal cord.

Projections from the nucleus tractus solitarii (NTS) to the spinal cord were demonstrated in the male Sprague-Dawley rat. In retrograde transport studies, a horseradish peroxidase conjugate or a fluorescent dye, FluoroGold, were injected into midcervical or upper thoracic spinal segments. Most solitariospinal neurons were multipolar or bipolar and located between the obex and spinomedullary junction. Solitariospinal neurons were concentrated in proximity to the ventral border of the solitary tract and extended dorsally into the intermediate division and ventrolaterally into the intermediate reticular zone (IRt) of the lateral tegmental field. This subgroup predominantly projects to midcervical spinal segments. A subset of small neurons was retrogradely labeled from cervical or thoracic spinal segments in the medial commissural nucleus and contiguous with a periventricular group surrounding the central canal. In anterograde transport studies, iontophoretic deposits of Phaseolus vulgaris leucoagglutinin were centered stereotaxically on sites in NTS identified by retrograde transport data. The lectin was incorporated by neurons of the solitary complex and transported bilaterally by axons that emerged from the nucleus and entered the reticular formation. The solitario-reticular (transtegmental) pathway irradiated diagonally across the IRt and extended caudally into the cervical lateral funiculus and spinal gray. A small periventricular-spinal pathway also descended longitudinally to the neuraxis. Solitariospinal neurons project to superficial lamina of the dorsal horn, laminae VII and X and ventral horn. The projections are predominantly contralateral to phrenic and intercostal motor nuclei and ipsilateral to the intermediolateral cell column. The solitariospinal projection represents the shortest route in the central nervous system, other than the local intraspinal reflex, through which first order visceral afferents signal cardiorespiratory and alimentary motor nuclei.

Monosynaptic innervation of trigeminal motor neurones involved in mastication by neurones of the parvicellular reticular formation.

In order to determine whether neurones in the parvicellular reticular formation are in direct synaptic contact with motor neurones innervating masticatory muscles, a combined retrograde and anterograde transport study was carried out in the rat at both light and electron microscopic levels. The animals received injections of the retrograde tracers wheat germ agglutinin conjugated to horseradish peroxidase or cholera toxin B conjugated to horseradish peroxidase into the masticatory muscles and of the anterograde tracer biocytin into the ipsilateral parvicellular reticular formation. The trigeminal motor nucleus was then examined for both anterograde and retrograde labelling in the light and electron microscopes. Retrogradely labelled motor neurones were identified in the trigeminal motor nucleus. They were large and their locations within the nucleus depended on the muscle injected. In addition, terminals anterogradely labelled with the biocytin that was injected in the parvicellular reticular formation were identified throughout the motor nucleus. At the electron microscopic level, the retrogradely labelled cells were found to receive input both from distinct types of unlabelled terminals and from terminals that were anterogradely labelled from the parvicellular reticular formation. The labelled terminals comprised one of the four classes of afferent terminals, being 1-2 microns in diameter and densely packed with spherical vesicles. They formed mostly asymmetrical but also symmetrical synapses with the labelled perikarya and dendrites. Anterogradely labelled terminals were also observed to form both symmetrical and asymmetrical synaptic contacts with unlabelled structures in the motor nucleus. It is concluded that neurones in the parvicellular reticular formation form direct synaptic contact with motor neurones of masticatory muscles. This pathway may represent the anatomical substrate by which the reticular formation exerts at least part of its influence on mastication. Since the parvicellular reticular formation receives input from the substantia nigra pars reticulata, it is possible that this pathway represents a system whereby the basal ganglia directly influence orofacial movement.