A comparative study of the immunohistochemical localization of a presumptive proctolin-like peptide, thyrotropin-releasing hormone and 5-hydroxytryptamine in the rat central nervous system

A proctolin (PROC)-like peptide was studied immunohistochemically in the hypothalamus, lower brainstem and spinal cord of the rat using an antiserum against PROC conjugated to thyroglobulin. Neuronal cell bodies containing PROC-like immunoreactivity (PROC-LI) were observed in the dorsomedial, paraventricular and supraoptic nuclei of the hypothalamus and in the nucleus raphe magnus, nucleus raphe pallidus, nucleus raphe obscurus and nucleus interfascicularis nervi hypoglossi in the medulla oblongata. Fibers containing PROC-LI were seen in the median eminence and in other hypothalamic nuclei, and in the lower brainstem in cranial motor nuclei including the dorsal motor nucleus of the vagus nerve, the motor trigeminal nucleus, the facial nucleus and nucleus ambiguous, and in lower numbers in the nucleus of the solitary tract and locus coeruleus. Fibers containing PROC-LI were also located in the spinal cord, in the intermediolateral cell column at thoracic levels and in the ventral horns at all levels of the spinal cord. After transection of the spinal cord, all PROC-immunoreactive fibers below the lesion disappeared. Following injection of Fast blue into the thoracic spinal cord, retrogradely labeled cells in the nuclei raphe pallidus, obscurus and magnus and nucleus interfasciculari nervi hypoglossi were seen to contain PROC-LI. PROC-LI had a similar distribution as thyrotropin-releasing hormone (TRH)-LI in the above-mentioned areas and coexistence of TRH-LI and PROC-LI was shown in cell bodies in the hypothalamus and medulla oblongata. PROC-LI could also be shown to coexist with 5-hydroxytryptamine (5-HT)-LI in neuronal cell bodies in the lower brainstem. The results demonstrate the occurrence of a PROC-like peptide in the mammalian nervous system, and these neurons seem to be at least largely identical to previously described TRH systems. A possible involvement of the PROC-like peptide in spinal motor control is discussed in relation to the well-established role of PROC in control of motor behavior in insects and invertebrates.     

Some anatomical observations on the projections from the hypothalamus to brainstem and spinal cord: an HRP and autoradiographic tracing study in the cat

The hypothalamus is closely involved in a wide variety of behavioral, autonomic, visceral, and endocrine functions. To find out which descending pathways are involved in these functions, we investigated them by horseradish peroxidase (HRP) and autoradiographic tracing techniques. HRP injections at various levels of the spinal cord resulted in a nearly uniform distribution of HRP-labeled neurons in most areas of the hypothalamus except for the anterior part. After HRP injections in the raphe magnus (NRM) and adjoining tegmentum the distribution of labeled neurons was again uniform, but many were found in the anterior hypothalamus as well. Injections of 3H-leucine in the hypothalamus demonstrated that: The anterior hypothalamic area sent many fibers through the medial forebrain bundle (MFB) to terminate in the ventral tegmental area of Tsai (VTA), the rostral raphe nuclei, the nucleus Edinger-Westphal, the dorsal part of the substantia nigra, the periaqueductal gray (PAG), and the interpeduncular nuclei. Further caudally a lateral fiber stream (mainly derived from the lateral parts of the anterior hypothalamic area) distributed fibers to the parabrachial nuclei, nucleus subcoeruleus, locus coeruleus, the micturition-coordinating region, the caudal brainstem lateral tegmentum, and the solitary and dorsal vagal nucleus. Furthermore, a medial fiber stream (mainly derived from the medial parts of the anterior hypothalamic area) distributed fibers to the superior central and dorsal raphe nucleus and to the NRM, nucleus raphe pallidus (NRP), and adjoining tegmentum. The medial and posterior hypothalamic area including the paraventricular hypothalamic nucleus (PVN) sent fibers to approximately the same mesencephalic structures as the anterior hypothalamic area. Further caudally two different fiber bundles were observed. A medial stream distributed labeled fibers to the NRM, rostral NRP, the upper thoracic intermediolateral cell group, and spinal lamina X. A second and well-defined fiber stream, probably derived from the PVN, distributed many fibers to specific parts of the lateral tegmental field, to the solitary and dorsal vagal nuclei, and, in the spinal cord, to lamina I and X, to the thoracolumbar and sacral intermediolateral cell column, and to the nucleus of Onuf. The lateral hypothalamic area sent many labeled fibers to the lateral part of the brainstem and many terminated in the caudal brainstem lateral tegmentum, including the parabrachial nuclei, locus coeruleus, nucleus subcoeruleus, and the solitary and dorsal vagal nuclei.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Thyrotropin-releasing hormone-immunoreactive projections to the dorsal motor nucleus and the nucleus of the solitary tract of the rat.

Thyrotropin-releasing hormone-immunoreactive nerve terminals heavily innervate the dorsal motor nucleus and nucleus of the solitary tract, whereas cell bodies containing thyrotropin-releasing hormone residue most densely in the hypothalamus and raphe nuclei. By using double-labeling techniques accomplished by retrograde transport of Fluoro-Gold following microinjection into the dorsal motor nucleus/nucleus of the solitary tract combined with immunohistochemistry for thyrotropin-releasing hormone, it was demonstrated that thyrotropin-releasing hormone-immunoreactive neurons projecting to the dorsal motor nucleus/nucleus of the solitary tract reside in the nucleus raphe pallidus, nucleus raphe obscurus, and the parapyramidal region of the ventral medulla, but not in the paraventricular nucleus of the hypothalamus. The parapyramidal region includes an area along the ventral surface of the caudal medulla, lateral to the pyramidal tract and inferior olivary nucleus and ventromedial to the lateral reticular nucleus. Varying the position of the Fluoro-Gold injection site revealed a rostral to caudal topographic organization of these raphe and parapyramidal projections.     

Retrograde study of hypocretin-1 (orexin-A) projections to subdivisions of the dorsal raphe nucleus in the rat

A retrograde tracer, WGA-apo-HRP-gold (WG), was injected into each subdivision of the dorsal raphe (DR) nucleus, and subsequent orexin-A immunostaining was performed for the tuberal region of the hypothalamus in order to investigate orexin projections to the DR. Similar to previous studies, the majority of orexin-single-labeled neurons were observed at the dorsal half of the lateral hypothalamus (LH), the circle around the fornix, i.e., perifornical nucleus (PeF), and the area dorsal to the fornix. The present study reports that hypothalamic neurons exhibited differential projections to each subdivision of the DR. Following WG injections into rostral DR, WG-single-labeled cells were observed at the dorsal half of the LH as well as dorsomedial hypothalamic nucleus. The major input to the intermediate DR originates from the ventromedial portion of the LH, PeF, and the area dorsal to the PeF, whereas one to lateral wing DR derived from PeF as well as the ventrolateral portion of the LH. Following WG injections into caudal DR, WG-single-labeled cells were located at ventromedial LH and the ventrolateral portion of the posterior hypothalamus. Following WG injections into each DR subdivision, WG/orexin-double-labeled neurons were observed at LH, PeF, and the area dorsal to the PeF. Only a few double-labeled cells were observed in dorsomedial and posterior hypothalamic nuclei. Our observations suggest that various hypothalamic neurons differentially project to each subdivision of the DR, a portion of which is orexin-immunoreactive. These orexin-immunoreactive DR-projecting hypothalamic neurons might have wake-related influences over a variety of brain functions subject to DR efferent regulation, including affective behavior, autonomic control, nociception, cognition, and sensorimotor integration.     

The distribution and morphological characteristics of serotonergic cells in the brain of monotremes

The distribution and cellular morphology of serotonergic neurons in the brain of two species of monotremes are described. Three clusters of serotonergic neurons were found: a hypothalamic cluster, a cluster in the rostral brainstem and a cluster in the caudal brainstem. Those in the hypothalamus consisted of two groups, the periventricular hypothalamic organ and the infundibular recess, that were intimately associated with the ependymal wall of the third ventricle. Within the rostral brainstem cluster, three distinct divisions were found: the dorsal raphe nucleus (with four subdivisions), the median raphe nucleus and the cells of the supralemniscal region. The dorsal raphe was within and adjacent to the periaqueductal gray matter, the median raphe was associated with the midline ventral to the dorsal raphe, and the cells of the supralemniscal region were in the tegmentum lateral to the median raphe and ventral to the dorsal raphe. The caudal cluster consisted of three divisions: the raphe obscurus nucleus, the raphe pallidus nucleus and the raphe magnus nucleus. The raphe obscurus nucleus was associated with the dorsal midline at the caudal-most part of the medulla oblongata. The raphe pallidus nucleus was found at the ventral midline of the medulla around the inferior olive. Raphe magnus was associated with the midline of the medulla and was found rostral to both the raphe obscurus and raphe pallidus. The results of our study are compared in an evolutionary context with those reported for other mammals and reptiles.     

Organization of neural inputs to the suprachiasmatic nucleus in the rat

The circadian timing of the suprachiasmatic nucleus (SCN) is modulated by its neural inputs. In the present study, we examine the organization of the neural inputs to the rat SCN using both retrograde and anterograde tracing methods. After Fluoro-Gold injections into the SCN, retrogradely labeled neurons are present in a number of brain areas, including the infralimbic cortex, the lateral septum, the medial preoptic area, the subfornical organ, the paraventricular thalamus, the subparaventricular zone, the ventromedial hypothalamic nucleus, the posterior hypothalamic area, the intergeniculate leaflet, the olivary pretectal nucleus, the ventral subiculum, and the median raphe nuclei. In the anterograde tracing experiments, we observe three patterns of afferent termination within the SCN that correspond to the photic/raphe, limbic/hypothalamic, and thalamic inputs. The median raphe projection to the SCN terminates densely within the ventral subdivision and sparsely within the dorsal subdivision. Similarly, areas that receive photic input, such as the retina, the intergeniculate leaflet, and the pretectal area, densely innervate the ventral SCN but provide only minor innervation of the dorsal SCN. A complementary pattern of axonal labeling, with labeled fibers concentrated in the dorsal SCN, is observed after anterograde tracer injections into the hypothalamus and into limbic areas, such as the ventral subiculum and infralimbic cortex. A third, less common pattern of labeling, exemplified by the paraventricular thalamic afferents, consists of diffuse axonal labeling throughout the SCN. Our results show that the SCN afferent connections are topographically organized. These hodological differences may reflect a functional heterogeneity within the SCN. J. Comp. Neurol. 389:508–534, 1997. © 1997 Wiley-Liss, Inc.     

CNS cell groups projecting to sympathetic outflow of tail artery: neural circuits involved in heat loss in the rat

In the rat, ≈20% of total body heat-loss occurs by sympathetically mediated increases in blood flow through an elaborate system of arteriovenous anastomoses in the skin of its tail. In this study, the CNS cell groups that regulate this sympathetic outflow were identified by the viral transneuronal labeling method. Pseudorabies virus was injected into the wall of the ventral tail artery in rats that had their cauda equina transected to eliminate the somatic innervation of the tail. After 4–7 days survival, the pattern of CNS transneuronal labeling was studied. Sympathetic preganglionic neurons in the T11–L2 (mainly L1) levels of the intermediolateral cell column (IML) were labeled by 4 days. After 5 days, sympathetic pre-motor neurons (i.e., supraspinal neurons that project to the IML) were identified near the ventral medullary surface; some of these contained serotonin immunoreactivity. Additional groups of the sympathetic premotor areas were labeled by 6 days post-injection, including the rostral ventrolateral medulla (C1 adrenergic neurons), rostral ventromedial medulla, caudal raphe nuclei (serotonin neurons in the raphe pallidus and magnus nuclei), A5 noradrenergic cell group, lateral hypothalamic area and paraventricular hypothalamic area (oxytocin-immunoreactive neurons). Seven days after the PRV injections, additional cell groups in the telencephalon (viz., bed nucleus of the stria terminalis, medial and lateral preoptic areas and medial preoptic nucleus), diencephalon (viz., subincertal nucleus, zona incerta as well as dorsal, dorsomedial, parafascicular, posterior and ventromedial hypothalamic nuclei) and midbrain (viz., periaqueductal gray matter, precommissural nucleus, Edinger–Westphal nucleus and ventral tegmental area) were labeled. The discussion is focused on the CNS cell groups involved in the control of body temperature and fever.     

Possible excitatory amino acid afferents to nucleus raphe dorsalis of the rat investigated with retrograde wheat germ agglutinin and D-[3H]aspartate tracing

Evidence for excitatory amino acid afferents to nucleus raphe dorsalis (NRD) has been found with retrograde tracing techniques. For neuroanatomical definition of afferent sources to NRD, rats received stereotaxic injections of wheat germ agglutinin-horseradish peroxidase (WGA-HRP) or implantations of crystal WGA-HRP in glass micropipettes. Retrogradely transported WGA-HRP was visualized with the tetramethyl-benzidine method, and afferents to NRD were identified from 20 different brain regions. Large numbers of labeled cells appeared in the lateral hypothalamus, lateral habenular nucleus, ventral tegmental area, periaqueductal gray, parabrachial nuclei and nucleus raphe magnus. Important inputs were also noted from dorsomedial hypothalamus and the area surrounding the perihypoglossal nucleus. Smaller numbers of WGA-HRP labeled cells appeared in bed nucleus of stria terminalis, diagonal band of Broca, cuneiform nucleus, superior vestibular nucleus, pontine periventricular gray, and some hypothalamic and reticular areas. Another group of rats received microinjections of D-[3H]aspartate (D[3H]Asp) and autoradiography consistently revealed retrograde labeling of cell bodies in 4 of the regions indicated by the WGA-HRP experiments as afferents to NRD. The most prominent aggregation of D-[3H]Asp-labeled cells was found in the lateral habenular nucleus, indicating that this input operates with an excitatory amino acid as transmitter. Significant numbers of D-[3H]Asp-labeled cells were also found in substantia nigra, periaqueductal and pontine periventricular gray. After large D-[3H]Asp injections involving NRD as well as surrounding areas, labeled cells were observed in several additional areas. Some of these areas were considered as afferents to surrounding periaqueductal gray or dorsal tegmental nuclei, while others may represent NRD afferents with relatively lower affinity for D-[3H]Asp. Several afferents to NRD failed to label with D-[3H]Asp, including diagonal band of Broca, hypothalamic areas, ventral tegmental area, parabrachial nuclei, locus coeruleus and reticular areas.     

Subcortical projections to the centromedian and parafascicular thalamic nuclei in the cat

The primary objective of this study is to identify the totality of input to the centromedian and parafascicular (CM-Pf) thalamic nuclear complex. The subcortical projections upon the CM-Pf complex were studied in the cat with three different retrograde tracers. The tracers used were unconjugated horseradish peroxidase (HRP), horseradish peroxidase conjugated to wheat germ agglutinin (WGA-HRP), and rhodamine-labeled fluorescent latex microspheres (RFM). Numerous subcortical structures or substructures contained labeled neurons with all three tracing techniques. These labeled structures included the central nucleus of the amygdala; the entopeduncular nucleus; the globus pallidus; the reticular and ventral lateral geniculate nuclei of the thalamus; parts of the hypothalamus including the dorsal, lateral, and posterior hypothalamic areas and the ventromedial and parvicellular nuclei; the zona incerta and fields of Forel; parts of the substantia nigra including the pars reticularis and pars lateralis, and the retrorubral area; the pretectum; the intermediate and deep layers of the superior colliculus; the periaqueductal gray; the dorsal nucleus of the raphe; portions of the reticular formation, including the mesencephalic, pontis oralis, pontis caudalis, gigantocellularis, ventralis, and lateralis reticular nuclei; the nucleus cuneiformis; the marginal nucleus of the brachium conjunctivum; the locus coeruleus; portions of the trigeminal complex, including the principal sensory and spinal nuclei; portions of the vestibular complex, including the lateral division of the superior nucleus and the medial nucleus; deep cerebellar nuclei, including the medial and lateral cerebellar nuclei; and lamina VII of the cervical spinal cord. Moreover, the WGA-HRP and rhodamine methods (known to be more sensitive than the HRP method) revealed several afferent sources not shown by HRP: the anterior hypothalamic area, ventral tegmental area, lateral division of the superior vestibular nucleus, nucleus interpositus, and the nucleus praepositus hypoglossi. Also, the rhodamine method revealed labeled neurons in laminae V and VI of the cervical spinal cord.     

Peptidergic hypothalamic afferents to the cat nucleus raphe pallidus as revealed by a double immunostaining technique using unconjugated cholera toxin as a retrograde tracer

Using a double immunostaining technique with unconjugated cholera toxin (CT) as a retrograde tracer, we have demonstrated in the cat that the nucleus raphe pallidus receives two major afferent projections from the hypothalamus: the preoptic periventricular nucleus; and the peri- and paraventricular zones of the posterior hypothalamic area. Some CT-labeled neurons in the preoptic periventricular nucleus showed Met-Enk-like immunoreactivity, while many CT-labeled neurons in the posterior hypothalamic area presented either corticotropin-releasing-factor-like or Met-Enk-like immunoreactivity.     

Retrograde double-labeling study of common afferent projections to the dorsal raphe and the nuclear core of the locus coeruleus in the rat

Common afferent projections to the dorsal raphe (DR) and locus coeruleus (LC) nuclei were analyzed in the rat by making paired injections of retrograde tracers, gold-conjugated and inactivated wheatgerm agglutinin-horseradish peroxidase (WGA-apo-HRP-gold) and Fluorogold (FG), into the DR and the nuclear core of the LC. Our results demonstrate that the largest number of double-labeled neurons was located at various preoptic regions including medial preoptic area, lateral preoptic nucleus, and ventrolateral preoptic nucleus. The majority of labeled cells were also observed at the lateral hypothalamus, where the number of labeled cells was comparable to that of neurons at the medial preoptic area or lateral preoptic nucleus. A few double-labeled cells were observed at various hypothalamic regions including anterior, medial tuberal, posterior, and arcuate nuclei, as well as mesencephalic areas including substantia nigra compacta and ventrolateral/lateral periaqueductal gray matter. Cells were also observed at prelimbic/infralimbic prefrontal cortices, diagonal band of Broca, bed nucleus of stria terminalis, and pontine/medullary regions including various raphe nuclei, Barrington's nucleus, gigantocellularis, paragigantocellularis, prepositus hypoglossi, subcoeruleus, and dorsomedial tegmental area. Although electrophysiological studies need to be performed, a large number of double-labeled neurons located at preoptic regions as well as lateral hypothalamus might have their major role in simultaneous control over these monoaminergic nuclei as a means of influencing various sleep and arousal states of the animal. Double-labeled cells at the other locations might be positioned to influence a variety of other functions such as analgesia, cognition, and stress responses.     

Possible excitatory amino acid afferents to nucleus raphe dorsalis of the rat investigated with retrograde wheat germ agglutinin and d-[H]aspartate tracing

Evidence for excitatory amino acid afferents to nucleus raphe dorsalis (NRD) has been found with retrograde tracing techniques. For neuroanatomical definition of afferent sources to NRD, rats received stereotaxic injections of wheat germ agglutinin-horseradish peroxidase (WGA-HRP) or implantations of crystal WGA-HRP in glass micropipettes. Retrogradely transported WGA-HRP was visualized with the tetramethyl-benizidine method, and afferents to NRD were identified from 20 different brain regions. Large numbers of labeled cells appeared in te lateral hypothalamus, lateral habenular nucleus, ventral tegmental area, periaqueductal gray, parabrachial nuclei and nucleus raphe magnus. Important inputs were also noted from dorsomedial hypothalamus and the area surounding the perihypoglosal nucleus. Smaller numbers of WGA-HRP labeled cells appeared in bed nucleus of stria terminals, diagonal band of Broca, cuneiform nucleus, superior vestibular nucleus, pontine periventricular gray, and some hypothalamic and reticular areas. Another group of rats received microinjections of d-[³H]aspartate (d[³H]Asp) and autoradiography consistently revealed retrogra labeling of cell bodies in 4 of the regions indicated by the WGA-HRP experiments as afferents to NRD. The most prominent aggregation of d-[³H]Asp-labeled cells was found in the lateral habenular nucleus, indicating that this input operates with an exitatory amino acids as transmitter. Significant numbers of d-[³H]Asp-labeled cells were also found in substantia nigra, periaqueductal and pontine periventricular gray. After large d-[³H]Asp injections involving NRD as well as surrounding areas, labeled cells were observed in several additional areas. Some of these areas were considered as afferents to sorrounding periaqueductal gray or dorsal tegmental nuclei, while others may represent NRD afferents ith relatively lower affinity for d-[³H]Asp. Several afferents to NRD failed to label with d-[³H]Asp, including diagonal band of Broca, hypothalamic areas, ventral tegmental area, parabrachial nuclei, locus coeruleus and reticular areas.     

Orexin-A projections to the caudal medulla and orexin-induced c-Fos expression, food intake, and autonomic function

Orexin-expressing neurons in the hypothalamus project throughout the neuraxis and are involved in regulation of the sleep/wake cycle, food intake, and autonomic functions. Here we specifically analyze the anatomical organization of orexin projections to the dorsal vagal complex (DVC) and raphe pallidus and effects on ingestive behavior and autonomic functions of local orexin-A administration in nonanesthetized rats. Retrograde tracing experiments revealed that as many as 20% of hypothalamic orexin neurons project to the DVC, where they form straight varicose axon profiles, some of which are in close anatomical apposition with tyrosine hydroxylase (TH)-, glucagon-like peptide-1-, gamma-aminobutyric acid-, and nitric oxide synthase-immunoreactive neurons in a nonselective manner. Similar contacts were frequently observed with neurons of the nucleus of the solitary tract whose activation by gastrointestinal food stimuli was demonstrated by the expression of nuclear c-Fos immunoreactivity. Orexin-A administration to the fourth ventricle induced significant Fos-expression throughout the DVC compared with saline control injections, with about 20-25% of TH-ir neurons among the stimulated ones. Fourth ventricular orexin injections also significantly stimulated chow and water intake in nonfood-deprived rats. Direct bilateral injections of orexin into the DVC increased intake of palatable high-fat pellets. Orexin-ir fibers also innervated raphe pallidus. Fourth ventricular orexin-A (1 nmol) activated Fos expression in the raphe pallidus and C1/A1 catecholaminergic neurons in the ventral medulla and increased body temperature, heart rate, and locomotor activity. The results confirm that hypothalamomedullary orexin projections are involved in a variety of physiological functions, including ingestive behavior and sympathetic outflow.     

Afferent connections of the nucleus of the facial nerve in the cat detected using the technic of retrograde axonal transport of horseradish peroxidase

Neuronal populations in the brainstem and spinal cord as sources of fibre pathways to the facial nucleus were studied in adult cats by means of microionophoretic injections of horseradish peroxidase into restricted zones of the facial nucleus. Projection from nucleus nervi hypoglossi, nucleus praepositus hypoglossi, nucleus raphe pallidus, nucleus intercalatus, medial nucleus of the solitary tract, dorsal motor nucleus of the vagus, neurons of genu of the facial nerve, ipsilateral red nucleus and reticular formation of the midbrain to the facial nucleus are found. Projections from a number of other brain structures to the facial nucleus are confirmed. A topographical map of distribution of the brainstem and spinal cord afferents in the facial nucleus is proposed.     

Hypothalamic and ventral thalamic projections to the superior colliculus in the cat

The present report describes the organization of collicular afferents that arise within either the hypothalamus or the ventral thalamus. Following the placement of large injections of WGA-HRP into the superior colliculus of the cat, retrogradely labeled neurons are located within the reticular nucleus of the thalamus, the zona incerta, the fields of Forel, and throughout the hypothalamus. Although the dorsal hypothalamic area contains the largest number of labeled hypothalamic neurons, labeled cells are also found within the periventricular, paraventricular, dorsomedial, ventromedial, posterior, lateral, and anterior hypothalamic nuclei. A strikingly similar pattern of distribution of labeled neurons is also observed following placement of small injections of WGA-HRP that are restricted within the stratum griseum intermedium (SGI). In contrast, hypothalamic and ventral thalamic labeling is not seen after placement of injections within the stratum griseum superficiale. Following the placement of injections of tritiated anterograde tracers within the dorsal hypothalamic area, transported label is organized in two bands of clusters over the SGI. When injections of tritiated tracers are placed within the zona incerta, terminal label is also located over the SGI; however, the distribution of silver grains does not appear as clusters or distinct puffs. On the basis of the comparison of the cellular types that give rise to these projections and the differences in terminal distribution, we suggest that the hypothalamic and ventral thalamic projections to the superior colliculus are totally separate and unrelated pathways. The functional implications of the hypothalamotectal pathway are also discussed.     

Anatomical evidence for a strong ventral parabrachial projection to nucleus raphe magnus and adjacent tegmental field

Injections with [3H]leucine in the ventral parabrachial nuclei and nucleus K?lliker-Fuse of the cat revealed strong projections to the nucleus raphe magnus (NRM) and adjacent tegmentum, while similar injections in the adjacent nucleus subcoeruleus produced diffuse projections to large parts of the tegmentum, but not specifically to the NRM. Horseradish peroxidase (HRP) injections in the area of the NRM and adjacent tegmentum demonstrated many labeled neurons in the ventral parabrachial nuclei, nucleus K?lliker-Fuse and nucleus subcoeruleus. These results suggest that the inhibition of nociception induced by stimulation in the ventral parabrachial nuclei may be based on the projections of this area to NRM and adjacent tegmentum.     

Connections of the ventral telencephalon (subpallium) in the zebrafish (Danio rerio)

Connections of the medial precommissural subpallial ventral telencephalon, i.e., dorsal (Vd, interpreted as part of striatum) and ventral (Vv, interpreted as part of septum) nuclei of area ventralis telencephali, were studied in the zebrafish (Danio rerio) using two tracer substances (DiI or biocytin). The following major afferent nuclei to Vd/Vv were identified: medial and posterior pallial zones of dorsal telencephalic area, and the subpallial supracommissural and postcommissural nuclei of the ventral telencephalic area, the olfactory bulb, dorsal entopeduncular, anterior and posterior parvocellular preoptic and suprachiasmatic nuclei, anterior, dorsal and central posterior dorsal thalamic, as well as rostrolateral nuclei, periventricular nucleus of the posterior tuberculum, posterior tuberal nucleus, various tuberal hypothalamic nuclei, dorsal tegmental nucleus, superior reticular nucleus, locus coeruleus, and superior raphe nucleus. Efferent projections of the ventral telencephalon terminate in the supracommissural nucleus of area ventralis telencephali, the posterior zone of area dorsalis telencephali, habenula, periventricular pretectum, paracommissural nucleus, posterior dorsal thalamus, preoptic region, midline posterior tuberculum (especially the area dorsal to the posterior tuberal nucleus), tuberal (midline) hypothalamus and interpeduncular nucleus. Strong reciprocal interconnections likely exist between septum and preoptic region/midline hypothalamus and between striatum and dorsal thalamus (dopaminergic) posterior tuberculum. Regarding ascending activating/modulatory systems, the pallium shares with the subpallium inputs from the (noradrenergic) locus coeruleus, and the (serotoninergic) superior raphe, while the subpallium additionally receives such inputs from the (dopaminergic) posterior tuberculum, the (putative cholinergic) superior reticular nucleus, and the (putative histaminergic) caudal hypothamalic zone.     

Efferents and afferents of the ventral tegmental-A10 region studied after local injection of [H]leucine and horseradish peroxidase

The efferents and the afferents of the VMT-A10 region were studied by using anterograde ([³H]leucine) and retrograde (HRP) tracing techniques. In order to produce very small injections in various parts of the VMT-A10 region, a slow diffusion technique for [³H]leucine labelling and a microiontophoretic injection for horseradish peroxidase labelling were developed. According to the histochemical and biochemical data, the [³H]leucine anterograde results were separated into three main types of projections.

(1) Projections to regions rich in DA terminals. These projections certainly correspond to the efferents of the dopaminergic A10 neurones. According to various injection sites, we have been able to identify mesolimbic projections originating from the VMT-A10, pars medialis and mesostriatal-mesolimbic projections originating from the VMT-A10, pars lateralis.

The mesolimbic projections include the prefrontal cortex, the medial part of the lateral septum, the interstitial nucleus of the stria terminalis, the accumbens nucleus and the olfactory tubercle. The mesostriatal-mesolimbic projections include the anteromedial part of the caudate nucleus, the cingular cortex, the entorhinal cortex, the amygdaloid complex, the accumbens nucleus, the olfactory tubercle and the piriform cortex to a lesser extent.

(2) Projections to regions suspected of containing DA terminals. These ascending and descending projections which could represent the dopaminergic efferents of the VMT-A10 neurones have been demonstrated. Ascending projections originating either from the VMT-A10 pars medialis or pars lateralis region were found in the claustrum, the nucleus of the tractus diagonalis, the olfactory nuclei, the lateral habenula, the medial hypothalamus and the median eminence. The projections observed in the medial hypothalamus included the periventricular region, the arcuate nucleus, the ventral part of the ventromedial nucleus and the dorsomedial nucleus. The labelling of the anteromedial part of the dorsal hippocampus appeared to originate from the VMT-A10, pars posterior. The projections to the medial hypothalamus, median eminence and hippocampus may have a great functional significance, but further proof of their dopaminergic nature is needed. Descending projections were found ipsilateally to the dorsal raphe and to the cerebellum, and bilaterally to the locus coeruleus. The projections to the cerebellum are distributed to the nuclei interpositus and dentatus and to the Purkinje cell layer and granular layer of the cortex. These results raise the problem of descending dopaminergic projections from the A10 neurones.

(3) Projections to regions not known to contain DA terminals. Anterior projections were found ipsilaterally to the supraoptic nucleus and bilaterally to the anterodorsal thalamic nucleus. Posterior projections were traced ipsilaterally to the limbic midbrain area, including the median raphe, the ventral and dorsal tegmental nucleus and the central gray.

The horseradish peroxidase experiment supplied some clues as to the posterior afferents of the VMT-A10 region. Some labelled cells were found ipsilaterally in the substantia nigra, the medain raphe and the ventral tegmental nucleus. Numerous cells were labelled ipsilaterally in the dorsal raphe nucleus, and nuclei interpositus and dentatus of the cerebellum, and contralaterally in the locus coeruleus. These structures are likely to play an important role in the modulation of the activity of VMT-A10 neurones.

The results of [³H]leucine and HRP experiments permitted us to demonstrate reciprocal connections between VMT-A10 region and anterior raphe nuclei, locus coeruleus and cerebellum.

Raphe serotonergic neurons projecting to the olfactory bulb contain galanin or somatostatin but not neurotensin

Retrograde axonal transport with [3H]5-HT has been developed as a specific tracing technique to identify serotonergic projections. This method, in combination with immunocytochemistry, offers considerable advantage of specificity and sensitivity to study pathways of multitransmitter-containing neurons. In this work, we studied the presence of galanin, somatostatin, and neurotensin in serotonergic neurons of dorsal and median raphe, which project to the olfactory bulbs. After [3H]5-HT injections into the rat olfactory bulbs, double galanin-immunoreactive and [3H]5-HT radiolabelled cells were located in the dorsal, lateral, and ventral region of dorsal raphe, but they were never seen in the median raphe. In the dorsal raphe, galanin-radiolabelled neurons represented 28% of the total number of radiolabelled cells. Double somatostatin-immunoreactive and radiolabelled neurons were located in the dorsal and median raphe. In the dorsal raphe, double somatostatin-radiolabelled neurons represented only 11% of the radiolabelled cells and they were mainly located ventral to the aqueduct. In the median raphe, 15% of radiolabelled cells were also immunopositive for somatostatin. In contrast, neurotensin-immunoreactive cells in the dorsal and median raphe were distributed among [3H]5-HT radiolabelled neurons but they were never radiolabelled. Our results show subpopulations of serotonergic raphe-olfactory bulb projection neurons containing either galanin or somatostatin, but not neurotensin.