Cholecystokinin-, galanin-, and corticotropin-releasing factor-like immunoreactive projections from the nucleus of the solitary tract to the parabrachial nucleus in the rat.

The parabrachial nucleus (PB) is the main relay for ascending visceral afferent information from the nucleus of the solitary tract (NTS) to the forebrain. We examined the chemical organization of solitary-parabrachial afferents by using combined retrograde transport of fluorescent tracers and immunohistochemistry for galanin (GAL), cholecystokinin (CCK), and corticotropin-releasing factor (CRF). Each peptide demonstrated a unique pattern of immunoreactive staining. GAL-like immunoreactive (-ir) fibers were most prominent in the "waist" area, the inner portion of external lateral PB, and the central and dorsal lateral PB subnuclei. Additional GAL-ir innervation was seen in the medial and external medial PB subnuclei. GAL-ir perikarya were observed mainly rostrally in the dorsal lateral, superior lateral, and extreme lateral PB. CCK-ir fibers and terminals were most prominent in the outer portion of the external lateral PB; some weaker labeling was also present in the central lateral PB. CCK-ir cell bodies were almost exclusively confined to the superior lateral PB and the "waist" area, although a few cells were seen in the K?lliker-Fuse nucleus. The distribution of CRF-ir terminal fibers in general resembled that of GAL, but showed considerably less terminal labeling in the lateral parts of the dorsal and central lateral PB, and the external medial and K?lliker-Fuse subnuclei. The CRF-ir cells were most numerous in the dorsal lateral PB and the outer portion of the external lateral PB; rostrally, scattered CRF-ir neurons were seen mainly in the central lateral PB. After injecting the fluorescent tracer Fast Blue into the PB, the distribution of double-labeled neurons in the NTS was mapped. GAL-ir cells were mainly located in the medial NTS subnucleus; 34% of GAL-ir cells were double-labeled ipsilaterally and 7% contralaterally. Conversely, 17% of the retrogradely labeled cells ipsilaterally and 16% contralaterally were GAL-ir. CCK-ir neurons were most numerous in the dorsomedial subnucleus of the NTS and the outer rim of the area postrema. Of the CCK-ir cells, 68% in the ipsilateral and 10% in the contralateral NTS were double-labeled, whereas 15% and 10%, respectively, of retrogradely labeled cells were CCK-ir. In the area postrema, 36% of the CCK-ir cells and 9% of the Fast Blue cells were double-labeled. CRF-ir neurons were more widely distributed in the medial, dorsomedial, and ventrolateral NTS subnuclei, but double-labeled cells were mainly seen in the medial NTS.(ABSTRACT TRUNCATED AT 400 WORDS)     

Distribution and hypothalamic projection of tyrosine-hydroxylase containing neurons of the nucleus of the solitary tract in the pigeon.

The avian nucleus of the solitary tract has an extensive subnuclear organization. Several subnuclear cell groups can be distinguished on the basis of cytoarchitectonic criteria. In general, the subnuclei of the medial division of the nucleus of the solitary tract receive gastrointestinal afferents, whereas the subnuclei of the lateral division of the nucleus of the solitary tract receive cardiopulmonary afferents. Forebrain afferents to the nucleus of the solitary tract are segregated to medial and lateral subnuclei, which are located at the periphery of the nucleus. These peripheral subnuclei of the nucleus of the solitary tract are also the source of ascending axonal projections to the forebrain. In this study, the tyrosine hydroxylase (initial enzyme for catecholamine synthesis) content of the anteromedial hypothalamic projecting neurons of the nucleus of the solitary tract is determined by use of a combined retrograde fluorescent dye-immunofluorescence method. Fast Blue implanted into the anteromedial hypothalamus (in the region of the nucleus periventricularis magnocellularis) resulted in the retrograde labeling of neurons in the caudal two-thirds of the nucleus of the solitary tract. At levels rostral to the obex, dye-labeled cells were mostly observed in the dorsally located subnuclei medialis superficialis pars posterior and lateralis dorsalis pars posterior and in the ventrally located subnucleus medialis ventralis pars posterior. More centrally located subnuclei contained few labeled cells, if any. For example, subnucleus medialis intermedius pars posterior only had a few retrogradely labeled cells, whereas the centrally located subnucleus medialis dorsalis pars posterior was almost devoid of labeled cells. At levels caudal to the obex, many retrogradely labeled neurons of the nucleus of the solitary tract were observed. Neurons immunoreactively labeled for tyrosine hydroxylase were mostly found within subnuclei, which contain anteromedial hypothalamic projection neurons. In subnuclei medialis superficialis pars posterior and lateralis dorsalis pars posterior, 87% of the retrogradely dye-labeled cells were also immunoreactively labeled, whereas in the caudal nucleus of the solitary tract (at levels caudal to the obex), 68% of the retrogradely labeled cells were immunoreactively labeled. Not all tyrosine hydroxylase containing cells had projections to the implantation site in the anteromedial hypothalamus since only 40% of the immunoreactive cells in the caudal nucleus of the solitary tract and 59% of the immunoreactive cells in the subnucleus medialis superficialis pars posterior were retrogradely labeled with Fast Blue.(ABSTRACT TRUNCATED AT 400 WORDS)     

Calcitonin gene-related peptide immunoreactivity in the visceral sensory cortex, thalamus, and related pathways in the rat

It has been proposed that calcitonin gene-related peptide (CGRP) may serve as a major neuromodulator in visceral sensory pathways, but its exact role in the visceral sensory thalamus and cortex has not been determined. We therefore examined the distribution of CGRP-like immunoreactive (CGRPir) innervation of the insular cortex and the parvicellular division of the ventroposterior nucleus of the thalamus (VPpc) in the rat by using immunohistochemistry for CGRP combined with retrograde transport of the fluorescent dye fluoro-gold. Modest numbers of CGRPir fibers were distributed in the dysgranular and agranular insular cortex, but few were observed in the granular insular cortex. The density of CGRPir innervation increased caudally along the rhinal fissue and was considerably greater in the perirhinal cortex. When fluoro-gold was injected into the insular cortex numerous retrogradely labeled neurons were seen in the VPpc, but few of these were CGRPir. Retrogradely labeled CGRPir neurons were, however, seen in the ventral lateral and medial parabrachial (PB) subnuclei. Injection of fluoro-gold into the perirhinal cortex (which is just caudal to the insular cortex along the rhinal fissure) resulted in many retrogradely labeled CGRPir neurons in the posterior thalamic region, including the subparafascicular, the lateral subparafascicular, and the posterior intralaminar nuclei. The VPpc was heavily innervated by CGRPir fibers but contained few CGRPir cell bodies. Injection of fluoro-gold into the VPpc resulted in many retrogradely labeled CGRPir neurons in the external medial PB subnucleus bilaterally, but with a contralateral predominance. Smaller numbers of retrogradely labeled CGRPir neurons were also observed in the ventrolateral PB subnucleus, bilaterally with an ipsilateral predominance. These results suggest that CGRP may be a neuromodulator in the ascending visceral sensory pathways from the PB to the VPpc and the insular cortex, but not between the latter two structures.     

Neuropeptide organization of the hypothalamic projection to the parabrachial nucleus in the rat

The hypothalamus is a major source of afferents to the parabrachial nucleus (PB), but the neurotransmitters in this pathway are largely unknown. In this study, we examine the neuropeptide immunoreactivities of neurons in the hypothalamus that project to the PB by using the combined retrograde fluorescence-immunofluorescence method. After injections of the fluorescent tracer fast blue into the PB, retrogradely labeled neurons were observed in the paraventricular, dorsomedial, ventromedial, median preoptic, and anteroventral periventricular hypothalamic nuclei; in the dorsal, retrochiasmatic, and lateral hypothalamic areas; and in the medial and lateral preoptic areas. Our results show that at least five distinct neuropeptide-immunoreactive cell populations in the hypothalamus project to the PB. In the perifornical lateral hypothalamus, many neurotensin (NT)-, corticotropin-releasing factor-, dynorphin (DYN)-, angiotensin II (AII)-, and galanin-like immunoreactive (-ir) neurons were retrogradely labeled. A cluster of retrogradely labeled neurons in the juxtacapsular lateral hypothalamus stained with an antiserum against alpha-melanocyte stimulating hormone (alpha MSH). Over 50% of the retrogradely labeled cells in the arcuate nucleus were adrenocorticotropin (ACTH)-or alpha MSH-ir. Many alpha MSH- and ACTH-ir, and a few DYN-, NT- and AII-ir neurons in the retrochiasmatic area were retrogradely labeled. Only small numbers of double-labeled neurons were found in the paraventricular nucleus, and, of these, enkephalin-ir and dynorphin-ir neurons were the most common. Somatostatin-ir cells in the hypothalamus were rarely double-labeled. The chemical coding of these hypothalamic projections to the PB may provide important clues to the functional organization of these descending pathways.     

Spinal and trigeminal dorsal horn projections to the parabrachial nucleus in the rat

We studied afferents to the parabrachial nucleus (PB) from the spinal cord and the spinal trigeminal nucleus pars caudalis (SNVc) in the rat by using the anterograde and retrograde transport of wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP). Injections of WGA-HRP into medial PB retrogradely labeled neurons in the promontorium and in lamina I of the dorsal rostral SNVc, while injections into lateral PB and the K?lliker-Fuse nucleus retrogradely labeled neurons in these areas as well as in lamina I throughout the caudal SNVc and spinal dorsal horn. Injections of WGA-HRP into the caudal SNVc and dorsal horn of the spinal cord resulted in terminal labeling in the dorsal, central, and external lateral subnuclei of PB and the K?lliker-Fuse nucleus, all of which are known to receive cardiovascular and respiratory afferent information. Injections of WGA-HRP into the promontorium and dorsal rostral SNVc resulted in terminal labeling in the same PB subnuclei, as well as in the medial and the ventral lateral PB subnuclei, which are sites of relay for gustatory information ascending from the medulla to the forebrain. The spinal and trigeminal projection to PB may mediate the convergence of pain, chemosensory, and temperature sensibilities with gustatory and cardiorespiratory systems in PB.     

Parabrachial nucleus projections to midline and intralaminar thalamic nuclei of the rat

The projections from the parabrachial nucleus to the midline and intralaminar thalamic nuclei were examined in the rat. Stereotaxic injections of the retrograde tracer cholera toxin-beta (CTb) were made in each of the intralaminar nuclei of the dorsal thalamus (the lateral parafascicular, medial parafascicular, oval paracentral, central lateral, paracentral, and central medial nuclei), as well as the midline thalamic nuclei (the paraventricular, intermediodorsal, mediodorsal, paratenial, rhomboid, reuniens, parvicellular part of the ventral posterior, and caudal ventral medial nuclei). The retrograde cell body labeling pattern within the parabrachial subnuclei was then analyzed. The paracentral thalamic nucleus received an input only from the internal lateral parabrachial subnucleus. However, this subnucleus also projected to all the other intralaminar thalamic nuclei, except for the central lateral thalamic nucleus, which received no parabrachial afferent inputs. The external lateral parabrachial subnucleus projected to the lateral parafascicular, reuniens, central medial, parvicellular part of the ventral posterior, and caudal ventromedial thalamic nuclei. Following CTb injections in the paraventricular thalamic nucleus, retrogradely labeled cells were found in the central lateral, dorsal lateral, and external lateral parabrachial subnuclei. The medial and ventral lateral parabrachial subnuclei projected to the oval paracentral, parafascicular, and rhomboid thalamic nuclei. Finally, the waist area of the parabrachial nucleus was densely labeled after CTb injections in the parvicellular part of the ventral posterior thalamic nucleus. Nociceptive, visceral, and gustatory signals may reach specific cortical and other forebrain sites via this parabrachial-thalamic pathway.     

Expression and colocalization of NADPH-diaphorase and Fos in the subnuclei of the parabrachial nucleus in rats following visceral noxious stimulation

To investigate whether neural nitric oxide synthase (nNOS) in the parabrachial nucleus (PB) is involved in processing visceral noxious stimulation, we mapped the distribution of histochemical staining for nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d), a marker for nNOS, and immunohistochemical staining for Fos, a neuronal activity marker, in the subnuclei of the PB following 2% formalin injection into the stomach of rats. NADPH-d and noxious-stimuli induced Fos staining were also examined in tissue containing PB cells labeled by the retrograde transport of fluogold (FG) injected into the central nucleus of the amygdala (CeA). We found that the number of Fos immunoreactive (Fos-IR) neurons was significantly increased in the dorsal lateral (dl), external lateral (el) and K?lliker-Fuse (KF) subnuclei of the PB. We observed that intensely labeled (type 1) NADPH-d positive neurons were mainly located in the rostral part of the PB; they extended long processes adjacent Fos-IR neurons, but no Fos/type 1 NADPH-d double-labeled neurons were seen. In contrast, lightly labeled (type 2) NADPH-d positive neurons were principally localized in the dl of the PB, in which a few Fos/type 2 NADPH-d double-labeled neurons were detected. Additionally, a large number of FG/Fos double-labeled neurons were observed to be surrounded closely by the intensive NADPH-d staining in the el of the PB. These results suggest that neurons in the el of the PB that project to the CeA are activated by visceral noxious stimulation and could be indirectly influenced by nitric oxide in the PB.     

Organization of cortical, basal forebrain, and hypothalamic afferents to the parabrachial nucleus in the rat.

In a previous study (Herbert et al., J. Comp. Neurol. [1990];293:540-580), we demonstrated that the ascending afferent projections from the medulla to the parabrachial nucleus (PB) mark out functionally specific terminal domains within the PB. In this study, we examine the organization of the forebrain afferents to the PB. The PB was found to receive afferents from the infralimbic, the lateral prefrontal, and the insular cortical areas; the dorsomedial, the ventromedial, the median preoptic, and the paraventricular hypothalamic nuclei; the dorsal, the retrochiasmatic, and the lateral hypothalamic areas; the central nucleus of the amygdala; the substantia innominata; and the bed nucleus of the stria terminalis. In general, forebrain areas tend to innervate the same PB subnuclei from which they receive their input. Three major patterns of afferent termination were noted in the PB; these corresponded to the three primary sources of forebrain input to the PB: the cerebral cortex, the hypothalamus, and the basal forebrain. Hypothalamic afferents innervate predominantly rostral portions of the PB, particularly the central lateral and dorsal lateral subnuclei. The basal forebrain projection to the PB ends densely in the external lateral and waist subnuclei. Cortical afferents terminate most heavily in the caudal half of the PB, particularly in the ventral lateral and medial subnuclei. In addition, considerable topography organization was found within the individual projections. For example, tuberal lateral hypothalamic neurons project heavily to the central lateral subnucleus and lightly to the waist area; in contrast, caudal lateral hypothalamic neurons send a moderately heavy projection to both the central lateral and waist subnuclei. Our results show that the forebrain afferents of the PB are topographically organized. These topographical differences may provide a substrate for the diversity of visceral functions associated with the PB.     

A topographically organized gamma-aminobutyric acid projection from the ventral pallidum to the nucleus accumbens in the rat

Anatomical and electrophysiological studies have indicated that a reciprocal projection from the ventral pallidum back to the nucleus accumbens exists and has functional relevance. In this study, the topographical projection from the ventral pallidum to the nucleus accumbens was examined by using retrograde tracing with fluoro-gold iontophoresed in subcompartments of the nucleus accumbens in rats combined with either in situ hybridization for glutamic acid decarboxylase and preproenkephalin mRNA or substance P immunoreactivity. Deposits made into the medial nucleus accumbens preferentially labeled neurons in the medial ventral pallidum, while deposits into the dorsolateral nucleus accumbens, at or lateral to the anterior commissure, labeled primarily cells in the dorsal and lateral ventral pallidum. A mediolateral to rostrocaudal topography was also observed, with the medial deposits preferentially labeling cells in rostral ventral pallidum and the lateral deposits resulting in retrogradely labeled cells in the ventral pallidum below the crossing of the posterior anterior commissure (subcommissural) as well as below the globus pallidus (sublenticular). The majority of cells retrogradely labeled with fluoro-gold were double-labeled for glutamic acid decarboxylase mRNA. In contrast, very few retrogradely labeled neurons in the ventral pallidurn were double labeled for mRNA for preproenkephalin. These data demonstrate a topographically organized projection from the ventral pallidum, to the nucleus accumbens that is primarily γ-aminobutyric acid (GABA)-ergic and reciprocal to the GABAergic projection from the nucleus accumbens to the ventral pallidum. © 1994 Wiley-Liss, Inc.     

Local and commissural neuropeptide-containing projections of the nucleus of the solitary tract to the dorsal vagal complex in the pigeon

The neuropeptide content of neurons of the nucleus of the solitary tract (NTS), which have local and commissural projections to the dorsal motor nucleus of the vagus (DMNX) and to NTS, were demonstrated in the pigeon (Columba livia) by using a combined fluorescein-bead retrograde-transport-immunofluorescence technique. The specific peptides studied were bombesin, cholecystokinin, enkephalin, galanin, neuropeptide Y, neurotensin, and substance P. Perikarya immunoreactive for bombesin were located in the Medial tier subnuclei of NTS and the caudal NTS. Most galanin- and substance P-immunoreactive cells were found in subnucleus medialis ventralis. Cells immunoreactive for neuropeptide Y were found in the medial tier of NTS and in the lateral tier, especially in subnucleus lateralis dorsalis intermedius. The majority of enkephalin- and neurotensin-immunoreactive cells were found centrally in subnuclei medialis dorsalis and medialis intermedius. Cells immunoreactive for cholecystokinin were located in subnuclei laterolis dorsalis pars anterior, medialis superficialis, and the caudal NTS. Based on the presence of retrogradely labeled cells, numerous neurons of the medial tier of NTS, but extremely few lateral tier NTS neurons, had projections to the ipsilateral and contralateral DMNX and NTS. The number of retrogradely labeled NTS cells was always greater ipsilateral than contralaterally. The percentages of peptide-immunoreactive NTS cells that projected to the ipsilateral and contralateral DMNX were in the ranges of 29–61% and 10–48%, respectively. The percentages of peptide-immunoreactive NTS cells that projected to the contralateral NTS ranged from 13 to 60%. Peptide-immunoreactive NTS cells that have local and commissural projections to DMNX and NTS may act as interneurons in vagovagal reflex pathways and in the integration of visceral sensory and forebrain input to NTS and DMNX. © 1994 Wiley-Liss, Inc.     

Ventral lateral geniculate nucleus afferents to the suprachiasmatic nucleus in the cat

The lateral geniculate complex innervates the hypothalamic suprachiasmatic nucleus (SCN). The location of neurons in the cat ventral lateral geniculate nucleus (vLGN) that give rise to the geniculohypothalamic tract has not been described. In this study, retrogradely labeled neurons were noted throughout the rostrocaudal extent of the medial vLGN following tracer injection into the SCN region. In addition, neuropeptide Y immunoreactive processes were also observed in the vLGN in this same medial zone and in the SCN. The data suggest that the medial zone of the cat vLGN may be homologous to the rodent intergeniculate leaflet (IGL).     

Distribution of bombesin-like immunoreactivity in the nucleus of the solitary tract and dorsal motor nucleus of the rat and human: Colocalization with tyrosine hydroxylase

Bombesin is a peptide neurotransmitter/neuromodulator with important autonomic and behavioral effects that are mediated, at least in part, by bombesin-containing neurons and nerve terminals in the nucleus of the solitary tract (NTS) and the dorsal motor nucleus of the vagus (DMV). The distribution of bombesin-like immunoreactive nerve terminals/fibers and cell bodies in relation to a viscerotopically relevant subnuclear map of this region was studied by using an immunoperoxidase technique. In the rat, bombesin fiber/terminal staining was heavy in an area that included the medial subnucleus of the NTS and the DMV over their full rostral-caudal extent. Distinctly void of staining were the gelatinous, central, and rostral commissural subnuclei and the periventricular area of the NTS, regions to which gastric, esophageal, cecal, and colonic primary afferents preferentially project. The caudal commissural and dorsal subnuclei had light bombesin fiber/terminal staining, as did the intermediate, interstitial, ventral, and ventrolateral subnuclei. With colchicine pretreatment, numerous cell bodies were stained in the medial and dorsal subnuclei, with fewer neurons in the caudal commissural, intermediate, interstitial, ventral, and ventrolateral subnuclei. Bombesin-like immunoreactive neurons were found in numerous other areas of the brain, including the ventrolateral medulla, the parabrachial nucleus, and the medial geniculate body. In the human NTS/DMV complex, the distribution of bombesin fiber/terminal staining was very similar to the rat. In addition, occasional bombesin-like immunoreactive neurons were labeled in a number of subnuclei, with clusters of neurons labeled in the dorsal and ventrolateral subnuclei. Double immunofluorescence studies in rat demonstrated that bombesin colocalizes with tyrosine hydroxylase in neurons in the dorsal subnucleus of the NTS. Bombesin does not colocalize with tyrosine hydroxylase in any other location in the brain. In conclusion, the distribution of bombesin in the NTS adheres to a viscerotopically relevant map. This is the anatomical substrate for the effects of bombesin on gastrointestinal function and satiety and its likely role in concluding a meal. The anatomic similarities between human and rat suggest that bombesin has similar functions in the visceral neuraxis of these two species. Bombesin coexists with catecholamines in neurons in the dorsal subnucleus, which likely mediate, in part, the cardiovascular effects of bombesin. © 1996 Wiley-Liss, Inc.     

Glycine-like immunoreactive input to sympathetic preganglionic neurons.

The organization of glycine-like immunoreactive (GLY-LIR) processes was investigated within the sympathetic preganglionic neuropils of male Sprague-Dawley rats and pigeons (Columba livia). Sympathetic preganglionic neurons were retrogradely labeled with horseradish peroxidase following injections into the superior cervical ganglion in rats or into the avian homologue of the mammalian stellate ganglion (paravertebral ganglion 14) in pigeons. Glycine-like immunoreactivity was visualized using postembedding immunoperoxidase and immunogold labeling methods. The neuropils surrounding pigeon sympathetic preganglionic neurons in the principal preganglionic cell column (nucleus of Terni) and in the nucleus intercalatus contained numerous GLY-LIR puncta. Many of these processes appeared to be 'terminal-like' swellings which closely apposed retrogradely labeled preganglionic perikarya and proximal dendrites. GLY-LIR somal and dendritic processes were intermingled among retrogradely labeled preganglionic neurons in the nucleus of Terni. None of these GLY-LIR cells were retrogradely labeled. The neuropils surrounding sympathetic preganglionic neurons in the rat also contained numerous GLY-LIR puncta; there were, however, qualitative differences in the density of such profiles across the preganglionic subnuclei. Within the central autonomic and intercalated regions there were numerous GLY-LIR processes, many of which closely apposed retrogradely labeled sympathetic preganglionic somas and proximal dendrites. Within the principal preganglionic cell column, the nucleus intermediolateralis pars principalis (Ilp), there were very few GLY-LIR 'terminal-like' swellings closely apposed to cell bodies in regions of high somal packing density. In regions were this density diminished, GLY-LIR puncta closely apposed retrogradely labeled perikarya and proximal dendritic processes. GLY-LIR spinal neurons were never observed to be within Ilp proper but were present in areas immediately dorsal (lateral lamina V), medial and ventral (lateral lamina VII). GLY-LIR neurons were never retrogradely labeled. The ultrastructural features of GLY-LIR terminals within the sympathetic preganglionic neuropils of both vertebrates were nearly identical. GLY-LIR terminal boutons formed synaptic contacts with retrogradely labeled preganglionic somas as well as with large and medium-sized proximal dendrites. The majority of identified GLY-LIR terminals, however, contacted non-retrogradely labeled medium and small caliber dendrites within the preganglionic neuropils. Ninety-eight percent of GLY-LIR synapses formed symmetric specializations with the postsynaptic element. Ninety-six percent of the GLY-LIR terminal boutons contained some combination of pleomorphic vesicles. These light and electron microscopic observations support the hypothesis that glycine is localized in terminals presynaptic to sympathetic preganglionic perikarya and dendrites.(ABSTRACT TRUNCATED AT 400 WORDS)     

Afferent projections to the interpeduncular nucleus in the rat, as studied by retrograde and anterograde transport of wheat germ agglutinin conjugated to horseradish peroxidase

We examined the afferent projections to the subnuclei of the interpeduncular nucleus (IPN) in the rat by means of retrograde and anterograde transport of wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP). We observed locations of retrogradely labeled cells following injections of WGA-HRP into the IPN, and distributions of anterogradely labeled fibers and terminals within the IPN following injections into the areas that contain cells of origin of afferents. Results of the retrograde and anterograde experiments have clarified the detailed organization of the IPN afferents. A part of the nucleus incertus, located dorsomedial to the dorsal tegmental nucleus, projects to the contralateral half of the rostral subnucleus of the IPN; the pars caudalis of the dorsal tegmental nucleus projects sparsely to the rostral lateral, dorsal lateral, lateral, caudal, and apical subnuclei predominantly contralaterally; the laterodorsal tegmental nucleus, to most of the subnuclei predominantly contralaterally; the ventromedial central gray rostral to the dorsal tegmental nucleus and lateral to the dorsal raphe nucleus projects to the rostral lateral and dorsal lateral subnuclei predominantly contralaterally; the median raphe nucleus, substantially to all subnuclei; the medial habenular nucleus, in a topographic manner, to the rostral, central, and intermediate subnuclei, to the rostral lateral and lateral subnuclei predominantly ipsilaterally, and to the dorsal lateral subnucleus predominantly contralaterally; the supramammillary nucleus and areas around the origin of the mammillothalamic tract and near the third ventricle project sparsely to the ventral part of the rostral subnucleus and to the central, lateral, caudal and apical subnuclei; the nucleus of the diagonal band, sparsely to the rostral, central, dorsal lateral, caudal, and apical subnuclei. These differential projections of the afferents to the subnuclei of the IPN may reflect its complex functions within the limbic midbrain circuit.     

Basal forebrain cholinergic and noncholinergic projections to the thalamus and brainstem in cats and monkeys

The projections of basal forebrain neurons to the thalamus and the brainstem were investigated in cats and primates by using retrograde transport techniques and choline acetyltransferase (ChAT) immunohistochemistry. In a first series of experiments, the lectin wheat germ-agglutinin conjugated with horseradish peroxidase (WGA-HRP) was injected into all major sensory, motor, intralaminar, and reticular (RE) thalamic nuclei of cats and into the mediodorsal (MD) and pulvinar-lateroposterior thalamic nuclei of macaque monkeys. In cats numerous neurons of the vertical and horizontal limbs of the diagonal band nucleus and the substantia innominata (SI), including its rostromedial portion termed the ventral pallidum (VP), were retrogradely labeled after WGA-HRP injections in the rostral pole of the RE complex, the MD, and anteroventral/anteromedial (AV/AM) thalamic nuclei. Fewer retrogradely labeled cells were observed in the same areas after injections in the ventromedial (VM) thalamic nucleus, and none or very few after other thalamic injections. After RE, MD, and AV/AM injections, 7-20% of all retrogradely labeled cells in the basal forebrain were also ChAT positive, while none of the retrogradely labeled neurons following VM injections displayed ChAT immunoreactivity. The basal forebrain projection to the MD nucleus was shown to arise principally from VP in both cats and macaque monkeys. In a second series of experiments performed in cats, injections of WGA-HRP in the brainstem peribrachial (PB) area comprising the pedunculopontine nucleus led to retrograde labeling of a moderate number of neurons in the lateral part of the VP, SI, and preoptic area (POA), only a few of which displayed ChAT immunoreactivity. In addition, a large number of retrogradely labeled cells were observed in the bed nuclei of the anterior commissure and stria terminalis after PB injections. In a third series of experiments, the use of the retrograde double-labeling method with fluorescent tracers in squirrel monkeys allowed us to identify a significant number of basal forebrain neurons sending axon collaterals to both the RE thalamic nucleus and PB brainstem area, while no double-labeled neurons were disclosed after injections confined to the ventral anterior/ventral lateral (VA/VL) thalamic nuclei and PB area or following injections in the cerebral cortex and PB area. Our findings reveal the existence of cholinergic and noncholinergic basal forebrain projections to the thalamus and the brainstem in both cats and macaque monkeys. We suggest that these projections may play a crucial role in the control of thalamic functions in mammals.     

Neuronal projections to the medial preoptic area of the sheep,with special reference to monoaminergic afferents: Immunohistochemical and retrograde tract tracing studies

The preoptic area contains most of the luteinizing hormone releasing hormone immunoreactive neurons and numerous monoaminergic afferents whose cell origins are unknown in sheep. Using tract tracing methods with a specific retrograde fluorescent tracer, fluorogold, we examined the cells of origin of afferents to the medial preoptic area in sheep. Among the retrogradely labeled neurons, immunohistochemistry for tyrosine hydroxylase, dopamine-β-hydroxylase, phenylethanolamine N-methyltransferase, and serotonin was used to characterize catecholamine and serotonin fluorogold labeled neurons. Most of the afferents came from the ipsilateral side to the injection site. It was observed that the medial preoptic area received major inputs from the diagonal band of Broca, the lateral septum, the thalamic paraventricular nucleus, the lateral hypothalamus, the area dorsolateral to the third ventricle, the perimamillary area, the amygdala, and the ventral part of the hippocampus. Other numerous, scattered, retrogradely labeled neurons were observed in the ventral part of the preoptic area, the vascular organ of the lamina terminalis, the ventromedial part of the hypothalamus, the periventricular area, the area lateral to the interpeduncular nucleus, and the dorsal vagal complex. Noradrenergic afferents came from the complex of the locus coeruleus (A6/A7 groups) and from the ventro-lateral medulla (group A1). However, dopaminergic and adrenergic neuronal groups retrogradely labeled with fluorogold were not observed. Serotoninergic fluorogold labeled neurons belonged to the medial raphe nucleus (B8, B5) and to the serotoninergic group situated lateral to the interpeduncular nucleus (S4). In the light of these anatomical data we hypothesize that these afferents have a role in the regulation of several functions of the preoptic area, particularly those related to reproduction. Accordingly these afferents could be involved in the control of luteinizing hormone releasing hormone (LHRH) pulsatility or of preovulatory LHRH surge.     

Organization of cortical and subcortical projections to medial prefrontal cortex in the cat

We have analyzed the cortical and subcortical afferent connections of the medial prefrontal cortex (MPF) in the cat with the specific aim of characterizing subregional variations of afferent connectivity. Thirteen tracer deposits were placed at restricted loci within a cortical district extending from the proreal to the subgenual gyrus. The distribution throughout the forebrain of retrogradely labeled neurons was then analyzed. Within the thalamus, retrogradely labeled neurons were most numerous in the mediodorsal nucleus and in the ventral complex. The projection from each region exhibited continuous topography such that more medial thalamic neurons were labeled by tracer from more ventral and posterior cortical deposits. Marked retrograde labeling without any sign of topographic order occurred in a narrow medioventral sector of the lateroposterior nucleus. Several additional thalamic nuclei contained small numbers of labeled neurons. In a subset of nuclei closely affiliated with the limbic system (the parataenial, paraventricular, reuniens, and basal ventromedial nuclei), retrograde labeling occurred exclusively after deposits at extremely ventral and posterior cortical sites. Within the amygdala, retrogradely labeled neurons occupied the anterior basomedial nucleus, the posterior basolateral nucleus, and a narrow strip of the lateral nucleus immediately adjoining the basolateral nucleus. The number of labeled neurons was greater after more ventral deposits. Very ventral deposits resulted in extensive labeling of the cortical amygdala. Within the cerebral cortex, the distribution of labeled neurons depended on the location of the tracer deposit. Comparatively dorsal deposits produced prominent retrograde transport to the anterior and posterior cingulate areas, to the agranular insula, and to lateral prefrontal cortex. Comparatively ventral deposits gave rise to prominent labeling of the hippocampal subiculum, various parahippocampal areas, and prepiriform cortex. On the basis of afferent connections, it is possible to divide the cat's medial prefrontal cortex into an infralimbic component, MPFil, marked by strong afferents from prepiriform cortex and the cortical amygdala, and a dorsal component, MPFd, without afferents from these structures. Further, within MPFd, it is possible to define an axis, running from ventral and posterior to dorsal and anterior levels, along which limbic afferents gradually become weaker and projections from cortical association areas gradually become stronger.     

Subthalamic nucleus of the monkey: connections and immunocytochemical features of afferents.

Retrograde and anterograde transport of wheat germ agglutinin-horseradish peroxidase (WGA-HRP) was studied in 7 squirrel monkeys with discrete injections of the subthalamic nucleus (STN). Injections labeled: (1) the lateral two-thirds of the nucleus (63% and 47%), (2) ventrolateral parts caudally (20%), (3) dorsomedial parts caudally (18%), (4) rostromedial parts (21%), (5) the medial third (38%) and (6) the lateral pole of the nucleus (9%). Afferents to the lateral two-thirds of the STN originated from two parallel cellular arrays in dorsal parts of the middle third of the lateral pallidal segment (LPS) and a single array in the rostral third of the LPS. Medial regions of the STN received input from cells in the rostral LPS. Small numbers of cells were retrogradely labeled in the centromedian-parafascicular (CM-PF) and the pedunculopontine (PPN) nuclei. No cells were labeled in the frontal cortex, the striatum, the substantia innominata (SI), the substantia nigra (SN) or the dorsal nucleus of the raphe. Virtually all pallidal neurons, including identified pallidosubthalamic neurons, were immunoreactive (IR) for gamma-aminobutyric acid (GABA). Pallidosubthalamic neurons were most numerous in regions of the LPS with the lowest density of leucine enkephalin-IR fibers. Substance P-IR fibers, found mainly in the medial pallidal segment, bore no relationship to pallidal afferents to the STN. Choline acetyltransferase-IR cells in the SI and the PPN were not retrogradely labeled with WGA-HRP granules. Anterograde transport in fibers and terminal fields surrounded retrogradely labeled cells in the LPS, suggesting a reciprocal relationship. The caudal third of the LPS and ventral region of the middle third of this nucleus, appeared to project few fibers to, or to receive few fibers from, the STN. A small number of STN efferents entered the medial border of the putamen, but no terminal fields were identified. STN projections to the pars reticulata of the SN appeared to represent about 10% of the projection to the LPS. No STN efferents were identified in the frontal cortex, the SI or the PPN. The hypothesis that STN afferents from the frontal cortex and CM-PF may represent collaterals of projections to other loci is discussed.     

Activation of the parabrachio-amygdaloid pathway by immune challenge or spinal nociceptive input: a quantitative study in the rat using Fos immunohistochemistry and retrograde tract tracing

Peripheral nociceptive stimulation results in activation of neurons in the pontine parabrachial nucleus (PB) of rats. Electrophysiological studies have suggested that noxiously activated PB neurons project to the amygdala, constituting a potential pathway for emotional aspects of pain. In the present study we examined this hypothesis by combining retrograde tract tracing with Fos immunohistochemistry. Cholera toxin subunit B was injected into the amygdala of rats. After a minimum of 48 hours the rats were given a subcutaneous injection of 100 microl of 5% formalin into one hindpaw and killed 60-90 minutes later. A dense aggregation of retrogradely labeled neurons was seen in the external lateral PB. Fos-expressing neurons were present preferentially in the central, dorsal, and superior lateral subnuclei as well as in the lateral crescent area, as described previously. There was little overlap between the retrogradely labeled and Fos-expressing populations and double-labeled neurons were rare. In contrast, systemic immune challenge by intravenous injection of bacterial wall lipopolysaccharide resulted in a Fos expression that overlapped the retrograde labeling in the external lateral PB, and many double-labeled neurons were seen. While these data provide direct functional anatomical evidence that nociceptive information from the hindlimb is relayed to the amygdala via the parabrachial nucleus, the number of parabrachio-amygdaloid neurons involved is small. Considering the widespread activation of parabrachio-amygdaloid neurons by a variety of visceral and humoral stimuli, the parabrachio-amygdaloid pathway thus appears to be more involved in the mediation of information related to viscerally and humorally elicited activity than in transmission of spinal nociceptive inputs.