Tyrosine hydroxylase and dopamine transporter expression in residual dopaminergic neurons: potential contributors to spontaneous recovery from experimental Parkinsonism

1-Methyl-4-phenyl-1,2,3,6-tetrahyrdropyridine (MPTP)-exposed cats develop severe Parkinsonism that spontaneously resolves in 4-6 weeks. The present study examined the extent to which compensatory changes in tyrosine hydroxylase (TH) and dopamine transporter (DAT) gene and protein expression may underlie this behavioral recovery. In normal cats, TH and DAT protein levels were higher in the dorsal vs. ventral striatum. Expression of DAT and TH mRNA was higher in substantia nigra pars compacta (SNc) than in the ventral tegmental area (VTA). In symptomatic parkinsonian animals, DAT and TH protein levels were significantly decreased in all striatal areas studied. TH and DAT mRNA expression in residual SNc neurons were decreased a mean 32% and 38%, respectively. DAT gene expression in residual VTA neurons in symptomatic animals was decreased 30% whereas TH gene expression was unaffected. In spontaneously recovered cats, TH protein levels were significantly higher than the levels in symptomatic cats only in the ventral striatum, whereas no increase in DAT protein levels were observed in any striatal area. Residual neurons in most ventral mesencephalic regions of recovered cats had increased TH mRNA expression but not increased DAT gene expression, compared with symptomatic animals. Thus, increased TH protein and mRNA and suppression of DAT protein and mRNA expression in the striatum and ventral mesencephalon were associated with functional recovery from MPTP-induced parkinsonism.     

Alterations in dopamine uptake sites and D1 and D2 receptors in cats symptomatic for and recovered from experimental parkinsonism

The administration of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to adult cats severely disrupts the dopaminergic innervation of the striatum. Animals display a parkinson-like syndrome, consisting of akinesia, bradykinesia, postural instability, and rigidity, which spontaneously recovers by 4–6 weeks after the last administration of MPTP. In this study we used quantitative receptor autoradiography to examine changes in DA uptake sites and DA receptors in the basal ganglia of normal, and symptomatic and recovered MPTP-treated cats. Consistent with the destruction of the nigrostriatal DA pathway, there was a severe loss of DA uptake sites, labeled with [³H]-mazindol, in the caudate nucleus (64–82%), nucleus accumbens (44%), putamen (63%), and substantia nigra pars compacta (SNc, 53%) of symptomatic cats. Following behavioral recovery, there were no significant changes in DA uptake site density. Significant increases of [³H]-SCH 23390 binding to D1 DA receptors were observed in the dorsal caudate (>24%; P < 0.05) of symptomatic cats and in all regions of the caudate-putamen (>30%; P < 0.05) of recovered animals. [³H]-SCH 23390 binding in tree substantia nigra pars reticulata was half of that in the striatum and showed no changes in symptomatic or recovered animals. No alterations in the binding of [¹²⁵1]-epidepride to D2 receptors was observed in any region of the striatum in either, symptomatic or recovered animals. [¹²⁵1]-Epidepride binding in the SNc was decreased by >36% (P < 0.05) following MPTP treatment. These data show that cats made parkinsonian by MPTP exposure have a significant decrease in the number of DA reuptake sites throughout the striatum and that recovery of sensorimotor function in these animals is not correlated with an increase in the number of striatal reuptake sites. Behavioral recovery, however, does seem to be correlated with a general elevation of Dl receptors throughout the striatal complex. The present data also show that direct correlations between changes in DA receptor regulation after a large DA depleting lesion and behavioral deficits or recovery from those deficits are difficult and that the relationships between DA receptors/transporters and behavior require further study. © 1995 Wiley-Liss, Inc.     

Differences in release and clearance of extracellular dopamine in the striatum after spontaneous or GM1-ganglioside-stimulated recovery from experimental Parkinsonism

PURPOSE: This study was designed to assess differences in dopamine clearance rates and potassium chloride (KCl)-stimulated release in the striatum of cats that had either spontaneously recovered from 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP)-induced Parkinsonism or recovered after receiving GM1 ganglioside treatment. METHODS: A severe Parkinsonian motor disorder was produced in 17 adult cats by administration of MPTP for seven to ten days. Six MPTP-treated cats received daily GM1 administration (30 mg/kg, i.m.) for 6 weeks and eleven MPTP-treated cats were allowed to spontaneously recover over the same period of time. High-speed chronoamperometric electrochemical measurements were obtained from dorsal and ventral striatal regions in all animals. Dopamine clearance rates were obtained by measuring the clearance of pressure-ejected dopamine from the extracellular space and local potassium-induced release was studied by applying KCl to the tissue. RESULTS: Dopamine clearance rates recorded in all striatal areas in GM1-treated cats were significantly faster than dopamine clearance rates recorded in spontaneously recovered cats. In GM1-treated animals, electrochemical signals recorded in response to KCl stimulation were sig-nificantly greater in all striatal areas compared to spontaneously recovered animals. Reduction/oxidation (redox) ratios recorded in GM1- treated animals indicated dopamine to be the predominant electroactive species released in all striatal areas in response to KCl stimulation. Redox ratios recorded in the ventral striatum of spontaneously recovered cats also indicated dopamine to be the predominant electroactive spe-cies released in response to KCl stimulation. However, redox ratios recorded in the dorsal striatum of spontaneosuly recovered cats indicated serotonin to significantly contribute to the recorded signal. CONCLUSIONS: These results support previous observations that volume transmission may predominate dopaminergic signaling in the stria-tum of spontaneously recovered cats and suggest that a greater degree of synaptic transmission is possible in GM1-treated animals. While the functional significance of this partial restoration of dopaminergic synaptic transmission in the striatum remains to be determined, it may under-lie improved behavioral recovery observed following GM1 treatment.     

Synaptic connections between spinal motoneurons and dorsal root ganglion cells in the cat

Light- and electron-microscopical horseradish peroxidase (HRP) studies have been employed in conjunction with a degeneration study in order to clarify the origin and axonal passage of afferent synaptic terminals in cat dorsal root ganglia. After injection of HRP into ganglia (C3) without involvement of the ventral roots and spinal nerves, a few ipsilateral spinal ventral horn neurons (C3) were retrogradely labeled with HRP. The labeled neurons were localized in the dorsomedial and the ventromedial nuclei. Following ventral rhizotomy of C3, the afferent terminals in the ganglia (C3) anterogradely degenerated and contained accumulated and disintegrated neurofilaments, depleted, aggregated and enlarged synaptic vesicles. Subsequent to an HRP and wheat germ agglutinin (WGA)-HRP-mixture injection into the dorsal neck or suboccipital muscles, many spinal motoneurons (C3) were labeled retrogradely with an HRP mixture. On the other hand, the afferent synaptic terminals in ganglia contained the membrane-bound and electron-dense bodies which were anterogradely labeled with an HRP mixture in addition to the normal synaptic elements. The present findings strongly suggest that some spinal motoneurons send their axon collaterals to the dorsal root ganglia, in which the terminals of the axon collaterals directly synapse with the dorsal root ganglion cells.     

Innervation of the tooth pulp by the mesencephalic trigeminal nucleus in the cat: a retrograde horseradish peroxidase study

HRP was applied to the tooth pulp of 8 cats. Six were subjected to postoperative administration of the anti-inflammatory drug, prednisolone, whereas the remaining two were not. In all prednisolone-treated cats, labeled neurons were found in both the mesencephalic trigeminal nucleus and trigeminal ganglion, ipsilaterally. On the other hand, no labeled neurons were observed in the mesencephalic nucleus in cats receiving no steroid.     

Topography of locus ceruleus neurons projecting to the area dentata

The distribution of locus ceruleus neurons projecting to the dorsal and ventral area dentata was studied using the retrograde transport of horseradish peroxidase (HRP). Furthermore, the possibility that collaterals of single locus ceruleus neurons innervate both the dorsal and ventral area dentata was explored with a retrograde double-label technique using HRP and the fluorescent dye 4′,6-diamidino-2-phenylindole 2 HCl (DAPI). Fusiform and multipolar neurons of the pars dorsalis of the locus ceruleus (LC_d) were labeled after injection of HRP into the area dentata. The HRP-positive cells were found almost exclusively in the dorsal LC_d after dorsal area dentata injections whereas ventral injections labeled neurons throughout the LC_d. The highest density of LC_d neurons labeled from the ventral area dentata was observed in the dorsal half of the LC_d and thus overlapped with the distribution of neurons labeled from dorsal injections. Neurons containing both HRP and DAPI were observed in the dorsal LC_d suggesting that the topographic coincidence of neurons labeled from dorsal and ventral area dentata was in part a result of single neurons having terminals in both loci.     

Identification and localization of the motor nuclei and sensory projections of the glossopharyngeal, vagus, and hypoglossal nerves of the cockatoo (Cacatua roseicapilla), Cacatuidae

Horseradish peroxidase (HRP) has been applied to the proximal severed ends of glossopharyngeal (N IX), vagus (NX), and hypoglossal (N XII) cockatoo in order to localize the motoneurons and sensory projections of these nerves which are involved in the control of the bird's feeding and phonatory behaviors. Application of HRP to N IX labeled four rhombencephalic nuclei: (1) a large-celled, retrofacial nucleus supplying M. geniohyoideus, the major tongue extensor; (2) a dorsal nucleus composed of medium-sized cells, projecting to most branches of N IX; (3) a ventrolateral nucleus supplying, amongst other structures, the floor of the pharynx and larynx; and (4) a ventral portion of the dorsal motor nucleus of the vagus. Neurons labeled by application of HRP to the cervical vagus comprise the classically defined dorsal motor nucleus and a ventrolateral medullary nucleus which is coextensive with that of the glossopharyngeus: together they probably constitute a nucleus ambiguus. Application of HRP to hypoglossal branches labeled a large nucleus intermedius (IM) and neurons ventral, ventrolateral, and caudal to it. The rostral third of IM supplies the lingual muscles, the caudal two-thirds the tracheosyringeal muscles. Many labeled neurons were found in the "jugular" ganglion following HRP treatment of each of the three nerves, especially N IX and N XII, which innervate the tongue. Central projections of these neurons are to nuclei of the descending trigeminus and to largely nonoverlapping portions of the principal trigeminal nucleus. It is hypothesized that these afferents provide sensory information necessary for the efficient processing and passage of food in the mouth.     

Striatal preprotachykinin gene expression reflects parkinsonian signs

Striatal preprotachykinin (PPT) gene expression was measured in MPTP-treated cats when symptomatic and during various stages of recovery from parkinsonism using in situ hybridization histochemistry. Animals expressing severe (1 week post-MPTP) or moderate (3 weeks post-MPTP) parkinsonian sensorimotor deficits had significantly reduced striatal PPT mRNA expression. In contrast, fully recovered animals (6 weeks post-MPTP) had striatal PPT mRNA levels that were not significantly different from normal. Thus, PPT gene expression in the striatum appears to reflect presence or absence of sensorimotor deficits in MPTP-treated cats.     

Upregulation of striatal D2 receptors in the MPTP-treated vervet monkey is reversed by grafts of fetal ventral mesencephalon: an autoradiographic study

Although neural transplantation holds promise as a treatment for Parkinson's disease, parkinsonian primates have generally exhibited inconsistent and incomplete recovery of motor functions following intrastriatal grafting of fetal ventral mesencephalon. One possible contributing factor to this variable response is lack of appropriate integration of donor neurons with host striatal circuitry with the result that there is insufficient dopamine release and postsynaptic dopamine receptor activation. This issue was examined by measuring the effect of transplanting fetal ventral mesencephalon to the striatum of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated (MPTP) monkeys on striatal D2 receptor binding. One year after receiving MPTP, D2 receptor binding was upregulated in the dorsal and ventral striatum of African green monkeys. Grafting of fetal ventral mesencephalon to the dorsal striatum of MPTP-treated monkeys 9 months before sacrifice, eliminated the D2 receptor upregulation in dorsal, but not ventral, region. Dopamine concentration in dorsal striatum of grafted MPTP-treated monkeys was significantly higher than in that region of MPTP-treated non-grafted monkeys. In addition, dopamine concentration was significantly higher in dorsal compared to ventral striatum of grafted MPTP-treated monkeys. These data, in addition to those from a previous autoradiographic study on dopamine uptake site density in these monkeys, strongly supports the hypothesis that ectopically placed ventral mesencephalon not only produces, but maintains the release of sufficient levels of dopamine to restore postsynaptic dopamine transmission in regions influenced by graft-derived dopamine.     

Afferent pathways to points of self-stimulation in the medial prefrontal cortex of the rat as revealed by the horseradish peroxidase technique

Afferent projections to points of self-stimulation (SS) in the medial prefrontal cortex (MPC) of the rat were studied using the horseradish peroxidase (HRP) technique. Intracranial microinjections of HRP (30%) were delivered at the same stereotaxic points at which the electrodes eliciting SS were located. Retrogradely transported HRP labeled neurons in different thalamic, hypothalamic, mesencephalic and pontine areas. In the thalamus, labeled neurons were found in the dorsomedial, anteromedial, anteroventral, ventral, ventromedial, posteromedial, paratenial, parafascicular nuclei and n. reuniens. Labeled neurons in mesencephalic areas were found in the n. interpeduncularis, ventral tegmental area (AVT) and substantia nigra (SN). In the pons, labeled neurons were found in the locus coeruleus and in the periaqueductal gray. Other nuclei in which labeled neurons were also found were: lateral hypothalamus (LH), periventricular gray and zona incerta (ZI). Theoretically it is possible that all these afferent areas contribute to SS of MPC. This assumption is discussed and criticized in connection with previous literature on SS. It is suggested that only specific areas and their projections are good candidates for the neural mechanisms involved in the reward produced by electrical stimulation of the prefrontal cortex.     

'Non-specific' cholinesterase-containing neurons of the dorsal thalamus project to medial limbic cortex

Thalamocortical neurons that contain 'non-specific' cholinesterase (ChE) were studied with cholinesterase histochemistry and experimental axonal tracing techniques in adult rats. In addition to the presence of ChE that is ubiquitous in capillary endothelium, neurons that contain ChE are found in 3 distinct regions of the dorsal thalamus, the thalamic reuniens nucleus (Re), the anterior dorsal nucleus (AD) and a region that includes the lateral part of the central lateral nucleus (CL) and the ventral portion of the lateral dorsal nucleus (LD). ChE activity appears light in cerebral cortex in general but histochemical staining is slightly greater in neuropil of the cingulate gyrus. Anterograde transport techniques with autoradiography demonstrated that neurons in the LD-CL region project to anterior cingulate cortex and the dorsal retrosplenial area. Anterograde degeneration techniques demonstrated that AD projects primarily to ventral retrosplenial cortex. Injections of horseradish peroxidase (HRP) in the anterior cingulate cortex resulted in double labeled cells (cells containing both ChE and HRP reaction products) primarily in LD and CL. HRP injections into ventral retrosplenial cortex resulted in double labeled cells in AD and Re. HRP injections in the subiculum resulted in double labeled cells in Re. Lesions placed in the region of thalamocortical projections resulted in a loss of ChE in the ipsilateral cingulate gyrus, as measured both histochemically and enzymatically. The finding that neurons containing ChE project to medial limbic cortex suggests that the ChE may be involved in the function of the thalamocortical component of the limbic system.     

Thoracic expiratory motor neurons of the rat: localization and sites of origin of their premotor neurons

The expiratory motor neurons of the representative thoracic segment of the rat were examined as to their localization and sites of origin of their premotor neurons in the lower brainstem. Either T6 or T7 was selected as a representative target segment for horseradish peroxidase (HRP) injection. The intercostal motor neurons in the T6 or T7 were doubly labeled by HRP placed in the cut end of the internal intercostal nerve and True blue placed in the cut end of the external intercostal nerve. The thoracic expiratory motor neurons labeled by HRP were concentrated in the oblique zone running along the dorsal to ventrolateral direction in both the T6 and T7 segments. By contrast, the thoracic inspiratory motor neurons labeled by True blue were concentrated in the horizontal zone running along the bottom of the ventral horn in both the T6 and T7 segments. A small amount of HRP was iontophoretically injected through a double-barrel coaxial electrode to sites of the expiratory motor neurons which had been identified electrophysiologically. In 5 successful experiments, the HRP-labeled cells were bilaterally distributed in the para-ambiguus nucleus (59.0%), ventral subnucleus of the paramedian reticular nucleus (13.6%), and raphe nuclei (10.8%). In another experiment, it was found that the expiratory premotor neurons in the para-ambiguus nucleus were present in a narrow column over the entire length of the nucleus at a level between 1.0 mm rostral and 1.4 mm caudal to the obex.     

An HRP study of the afferent connections to rat lateral hypothalamic region

Afferent connections to the lateral hypothalamic region in the rat were studied using horseradish peroxidase (HRP). HRP was injected iontophoretically by a parapharyngeal approach. After HRP injections into the lateral hypothalamic area, labeled cells were found mainly in the medial prefrontal and infralimbic cortices, lateral and dorsal septal nuclei, nucleus accumbens, bed nucleus of the stria terminalis, medial and lateral amygdaloid nuclei, lateral habenular nucleus, peripeduncular nucleus, ventral tegmental area, mesencephalic and pontine central gray, ventral nucleus of the lateral lemniscus, lateral parabrachial area, raphe nuclei and the nucleus locus coeruleus. Labeled cells following HRP injections into the lateral preoptic area were found mainly in the lateral and dorsal septal nuclei, nucleus accumbens, diagonal band, ventral part of the globus pallidus, bed nucleus of the stria terminalis, central amygdaloid nucleus, mesencephalic and pontine central gray, dorsal raphe nucleus, parabrachial area and the nucleus locus coeruleus. The intrahypothalamic connections were also discussed.     

Selective loss of subpopulations of ventral mesencephalic dopaminergic neurons in the monkey following exposure to MPTP

Tyrosine hydroxylase immunohistochemical examination of the mesencephalon of severely parkinsonian MPTP-treated macaque fascicularis monkeys revealed a marked loss of substantia nigra pars compacta (SNc) neurons in both medial and central portions of the nucleus with a relative sparing of neurons in the dorsal-most portions of the substantia nigra. These animals also sustained 20–65% loss of neurons in the substantia nigra pars lateralis area, ventral tegmental area (A-10), and the retrorubral area (A-8 cell group, and the parabrachialis pigmentosus region). These animals all had extreme striatal dopamine depletions. A monkey which received several small doses of MPTP and yet remained asypptomatic for a motor disorder (although it had demonstrable behavioral performance deficits) had only a loss only ventral SNc neurons, with no appreciable cells in associated ventral mesencephalic dopamine areas and no loss of striatal dopamine. These data suggest that the effects of MPTP are not as selective as originally thought and, more importantly, indicate that MPTP-induced parkinsonism in the primate may be more analogous to idiopathic Parkinson's disease, where cells other than SNc cells are affected. Furthermore, the present findings suggest that only certain mesencephalic dopamine neurons are susceptible to MPTP-induced damage. The unique characteristics of these neurons need to be elucidated.     

Distribution and size of thalamic neurons projecting to layer I of the auditory cortical fields of the cat compared to those projecting to layer IV

The distribution of thalamocortical neurons projecting to layer I of the cat auditory cortical fields was examined by the horseradish peroxidase (HRP) method. After HRP injection into layer I of the primary auditory cortex (AI), HRP-labeled neuronal cell bodies were distributed mainly in the medial, dorsal, and ventrolateral divisions of the medial geniculate nucleus (MGN) and suprageniculate nucleus (Sg), and additionally in the lateral and medial divisions of the posterior group of the thalamus (Pol and Pom), lateroposterior thalamic nucleus (Lp), and nucleus of the brachium of the inferior colliculus (BIN). After HRP injection into layer I of the second auditory cortex (AII), labeled neurons were seen mainly in the medial, dorsal, and ventrolateral divisions of the MGN and Sg and additionally in the Pom, Lp, and BIN. After HRP injection into layer I of the anterior auditory field (AAF), labeled neurons were located mainly in the medial and dorsal divisions of the MGN, Sg, Pol, and BIN, and additionally in the ventrolateral divisions of the MGN, Pom, and Lp. After HRP injection into layer I of the dorsal part of the posterior ectosylvian gyrus (Epd), labeled neurons were observed chiefly in the medial and dorsal divisions of the MGN, Sg, and Lp and additionally in the ventrolateral division of the MGN, Pom, and BIN. After HRP injection into layer I of the ventral part of the posterior ectosylvian gyrus (Epv), labeled neurons were distributed chiefly in the medial and dorsal divisions of the MGN and Pol and additionally in the ventrolateral division of the MGN, Sg, and BIN. Thus no labeled neurons were found in the ventral division of the MGN after HRP injection into layer I of all auditory cortical fields examined in the present study. The average soma diameters of neurons that were labeled after HRP injection into layer I were statistically smaller than those of neurons that were labeled after HRP injection into layer IV.     

Uptake sites of horseradish peroxidase after injection into peritoneal structures: defining some pitfalls

Following injection of horseradish peroxidase (HRP) either into the jejunal wall or the peritoneal cavity, neurons in the dorsal motor nucleus of the vagus nerve, celiac, nodose and spinal ganglia, and ventral and lateral horns of the spinal cord from the mid-thoracic to lumbar segments were labeled. When HRP was injected into the wall of the exteriorized gut, neurons of the spinal cord were not labeled. Furthermore, there was a significant decrease in the number of labeled neurons in the dorsal motor nucleus and ganglia examined. These results indicate that HRP injected into the intestinal wall could leak into the peritoneal cavity and be taken up and transported to neuronal cell bodies by nerve fibers not terminating in the injection area. The leakage of HRP to nearby abdominal structures and its subsequent uptake by nerve fibers is attributed to the lack of a diffusion barrier across the surfaces of the intestinal wall and the abdominal structures. It is suggested that in applying the HRP techniques for the study of neuronal connections in the peripheral nervous system, it is essential that carefully planned control experiments be undertaken which can overcome the problem of mislabeling due to diffusion from the injection site.     

HRP study of the central components of the trigeminal nerve in the larval sea lamprey: organization and homology of the primary medullary and spinal nucleus of the trigeminus

The medullary and spinal connections of the trigeminal nerve of larval sea lampreys Petromyzon marinus were studied by anterograde and retrograde HRP transport after application into the orbit. Three components were found, all of them ipsilateral: 1) The motor nucleus was undivided in the larva, and its neurons possessed a rich dendritic tree. The single motor root was well separated from the sensory root. 2) The descending root was laterally located, and its fibers ran compactly to spinal levels. 3) Most medullary and many rostral spinal dorsal cells were labeled. Dorsal cells, which were mostly multipolar, had numerous mutual contacts. Some dorsal cell processes contacted the fourth ventricle. The name "primary medullary and spinal nucleus of the trigeminal nerve" (PMSV) is proposed for these dorsal cells. Medullary dorsal cells were not labeled by applying HRP at the level of spinal nerves, but application to the vagus nerve did label some. The possible relationship of this nucleus with the mesencephalic trigeminal nucleus of jawed vertebrates is discussed.     

Efferent projections from temperature sensitive recording loci within the marginal zone of the nucleus caudalis of the spinal trigeminal complex in the cat

The efferent projections from nucleus caudalis of the spinal trigeminal complex in cats were studied with retrograde and anterograde axonal transport techniques combined with localization of recording sites in the thalamus and marginal zone of nucleus caudalis to innocuous skin cooling. Results showed brainstem projections from nucleus caudalis to rostral levels of the spinal trigeminal complex, to the ventral division of the principal trigeminal nucleus, the parabrachial nucleus, cranial motor nuclei 7 and 12, solitary complex, contralateral dorsal inferior olivary nucleus, portions of the lateral reticular formation, upper cervical spinal dorsal horn and, lateral cervical nucleus. Projections to the thalamus included: a dorsomedial region of VPM (bilaterally) and to the main part of VPM and PO contralaterally. Neuronal activity was recorded in the dorsomedial region of VPM to cooling the ipsilateral tongue. HRP injections in this thalamic region retrogradely labeled marginal neurons in nucleus caudalis. These results show that marginal neurons of nucleus caudalis provide a trigeminal equivalent of spinothalamic projections to the ventroposterior nucleus in cats.     

Collateral branching in axonal projections to spinal cord from paramedian reticular nucleus neurons

Experiments were done in cats to identify neurons in the paramedian reticular nucleus (PRN) sending collateral axons to the region of the intermediolateral nucleus (IML) at different levels of the thoracic cord by using lectin-conjugated horseradish peroxidase (HRP) and double-labeling fluorochrome histochemistry to retrogradely label PRN neurons. Injections of Fast blue (FB) into the spinal cord at the T2 level centered in the region of the IML were coupled with injections of Nuclear yellow (NY) into the ipsilateral cord at either the T4 or T7 levels centered in the region of the IML. Neurons in the PRN retrogradely labeled after diffusion of HRP into the region of the IML at the T2 level were observed throughout the rostrocaudal extent of the ventral PRN. In addition, a few labeled neurons were noted in the ventral portion of the dorsal PRN. About 40% of the neurons in the PRN which were labeled with FB after an injection at the T2 level were also labeled with NY injected into the cord in further caudal segments. These data suggest that the PRN may exert its influence on the cardiovascular system partly through collateral axonal branches to widely separated populations of sympathetic preganglionic neurons in different spinal segmental levels.