Retrograde study of projections from the tuberomammillary nucleus to the dorsal raphe and the locus coeruleus in the rat

In the first series of experiments, a retrograde tracer, WGA-apo-HRP-gold (WG), was injected into the dorsal raphe (DR) or the locus coeruleus (LC) and adenosine deaminase immunostaining was subsequently performed for the tuberomammillary nucleus (TMN) in order to investigate projections from the TMN to the two brainstem monoaminergic nuclei. Following rostral DR injections, the majority of retrogradely labeled cells were located in the dorsomedial and ventrolateral subdivisions of the TMN. At middle DR levels, midline injections resulted in labeling mainly in the ventrolateral subdivision, whereas lateral wing injections produced labeling mostly in ventral and caudal TMN subdivisions. When injections were made in the caudal DR, only a few cells were observed along the rostro-caudal extent of the TMN. On the other hand, following rostral LC injections, labeled neurons were observed mainly in ventrolateral and ventral subdivisions of TMN. For principal LC injections, labeled cells were observed mostly in ventrolateral, ventral, and caudal TMN subdivisions, whereas for injections at caudal pole of LC, only a few cells were located along the rostro-caudal extent of the TMN. In the second series of experiments, an iontophoretic injection of fluorogold (FG) into the DR was paired with a pressure injection of WG into the LC to investigate the collateral distribution of TMN axonal fibers to DR and LC. Double-labeled cells were observed in ventrolateral, ventral, and caudal TMN subdivisions. The present study indicated that there exists a robust projection from the TMN to the DR or the LC and that some TMN neurons have axon collaterals projecting to both DR and LC. The TMN neurons with such axon collaterals might provide simultaneous, possibly more efficient, way of controlling the brainstem monoaminergic nuclei, thus influencing various sleep and arousal states of the animal.     

Neuronal connections between the cerebellar nuclei and hypothalamus in Macaca fascicularis: cerebello-visceral circuits.

The purpose of this study was to identify the basic pattern of interconnections between the cerebellar nuclei and hypothalamus in Macaca fascicularis. The distribution of retrogradely labeled cells and anterogradely filled cerebellofugal axons in the hypothalamus of M. fascicularis was investigated after pressure injections of a horseradish peroxidase mixture (HRP + WGA-HRP) in the cerebellar nuclei. Following injections in the lateral, anterior, and posterior interposed cerebellar nuclei retrogradely labeled cells were present in the following areas (greatest to least concentration): lateral and dorsal hypothalamic areas, dorsomedial nucleus, griseum periventriculare hypothalami, supramammillary and tuberomammillary nuclei, posterior hypothalamic area, ventromedial nucleus and periventricular hypothalamus, around the medial mammillary nucleus, lateral mammillary nucleus, and infundibular nucleus. Cell labeling was bilateral with an ipsilateral preponderance. In these same experiments anterogradely labeled cerebellar efferent fibers terminated in the contralateral posterior, dorsal and lateral hypothalamic areas, and the dorsomedial nucleus. In these regions retrogradely labeled hypothalamic cells were occasionally found in areas that also contained anterogradely filled cerebellar axons. This suggests a partial reciprocity in this system. In addition, sparse numbers of labeled cerebellar fibers recross in the hypothalamus to distribute to homologous areas ipsilateral to the injection site. Subsequent to an injection in the medial cerebellar nucleus (NM), cell labeling was present in more rostral hypothalamic levels including the lateral and dorsal hypothalamic areas, the dorsomedial nucleus, around or in fascicles of the column of the fornix, and in the periventricular hypothalamic area. Although no fastigiohypothalamic fibers were seen in this study, on the basis of information available from the literature it is likely that such a connection exists in primates. In summary, hypothalamic projections to NM originated mainly from rostral to midhypothalamic levels, whereas those projections to the lateral three cerebellar nuclei came from mid and more caudal levels. The existence of direct hypothalamic projections to cerebellar nuclei in M. fascicularis and of cerebellofugal projection to some hypothalamic centers indicates that circuitry is present through which the cerebellum may influence visceral functions. Furthermore, the fact that projections to NM versus the other cerebellar nuclei originate from somewhat different regions of the hypothalamus would suggest that the visceral functions modulated by each pathway is not the same.     

Corticotropin-releasing factor-containing afferents to the inferior colliculus of the rat brain

Using a modified cobalt-glucose oxidase-diaminobenzidine (Co-GOD) method in a combination of horseradish peroxidase (HRP) retrograde tracing and immunohistochemistry, a widespread localization of corticotropin releasing factor-like immunoreactive (CRFI) structures in the rat inferior colliculus (IC), and a CRFI-containing pathway from the subthalamus and the hypothalamus to the IC have been observed. By means of the modified Co-GOD method, CRFI cells were detected in almost all the subdivisions of the IC, including the dorsomedial part of the central nucleus, the ventrolateral part of the central nucleus, the pericentral nucleus and the external nucleus. Neural processes with CRFI were observed in all of the above areas. Following HRP injection into the IC, double-labeled cells which contained a homogeneous brown immunoreaction product of CRF and a granular black reaction product of retrogradely transported HRP were identified in the lateral hypothalamic area (LH), zona incerta (ZI) and perifornical hypothalamic area (PeF). These double-labeled cells provide direct evidence for CRFI projections from the LH, ZI and PeF to the IC. Thus, the present study supports the view that CRF may act as a neurotransmitter or neuromodulator in the brain.     

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.     

Efferent projections of the nucleus accumbens in the rat with special reference to subdivision of the nucleus: biotinylated dextran amine study

The nucleus accumbens (Acb) of the rat has been divided immunohistochemically into shell and core, and further, it was subdivided into several portions in relation to functional significance. In this report, the efferent projection of each subdivision of the Acb was examined using biotinylated dextran amine as an anterograde tracer. In rostral Acb, the dorsomedial shell mainly projected to the dorsomedial ventral pallidum (VP), lateral hypothalamus (LH) and substantia nigra pars compacta (SNc), while the ventromedial shell projected to the ventromedial VP, lateral preoptic area, LH and ventral tegmental area (VTA). The dorsal core of rostral Acb projected to the caudate putamen, dorsolateral VP, globus pallidus (GP), LH, and substantia nigra pars reticulata (SNr). In the middle to caudal Acb, the dorsomedial shell mainly projected to the dorsomedial VP, LH and VTA, the ventromedial shell projected to the ventromedial VP, substantia innominata, VTA, SNc and retrorubral area, and the ventrolateral shell projected to the ventrolateral VP and SNc. Furthermore, the ventromedial shell projected to the parabrachial nucleus (PB). The dorsomedial core projected to the dorsal VP, LH, SNc and SNr, and the ventral and lateral core sent axons to the dorsolateral VP, GP and SNc. From the point of view of projection patterns, shell and core are distinct throughout the rostro-caudal extent of the Acb. The ventrolateral shell at the caudal Acb was clearly differentiated. A direct projection from the ventromedial shell of the Acb to PB was also recognised.     

Glutamatergic afferent projections to the dorsal raphe nucleus of the rat

Based on WGA-apo-HRP-gold (WG) retrograde tracing, the present study revealed that different subdivisions of the dorsal raphe (DR) such as dorsomedial, ventromedial, lateral wing, and caudal regions receive unique, topographically organized afferent inputs, that are more restricted than previously reported. Phaseolus vulgaris leucoagglutinin anterograde tracing studies confirmed that the medial prefrontal cortex provides the major afferent input to each subdivision of the DR. Double-labeling studies combining WG tracing and glutamate immunostaining indicated that the medial prefrontal cortex, various hypothalamic nuclei including perifornical, lateral, and arcuate nuclei, and several medullary regions such as lateral and medial parabrachial nuclei, and laterodorsal tegmental nucleus provide the major glutamatergic input to each subregion of the DR. It should be noted that the degree of glutamatergic input from these afferent sites was specific for each DR subdivision. The present findings indicated that dorsomedial, ventromedial, lateral wing, and caudal subdivisions of the DR receive excitatory inputs from both cortical and subcortical sites which might be involved in regulation or modulation of a broad range of systems, including sensory and motor functions, arousal and sleep-wake cycle, biorhythmic, cognitive, and affective behaviors.     

Subregions of the periaqueductal gray topographically innervate the rostral ventral medulla in the rat.

Previous anatomical and physiological studies have revealed a substantial projection from the periaqueductal gray (PAG) to the nucleus paragigantocellularis (PGi). In addition, physiological studies have indicated that the PAG is composed of functionally distinct subregions. However, projections from PAG subregions to PGi have not been comprehensively examined. In the present study, we sought to examine possible topographic specificity for projections from subregions of the PAG to PGi. Pressure or iontophoretic injections of wheat germ agglutinin-conjugated horseradish peroxidase, or of Fluoro-Gold, placed into the PGi of the rat retrogradely labeled a substantial number of neurons in the PAG from the level of the Edinger-Westphal nucleus to the caudal midbrain. Retrogradely labeled neurons were preferentially aggregated in distinct subregions of the PAG. Rostrally, at the level of the oculomotor nucleus, labeled neurons were i) compactly aggregated in the ventromedial portion of the PAG corresponding closely to the supraoculomotor nucleus of the central gray, ii) in the lateral and ventrolateral PAG, and iii) in medial dorsal PAG. More caudally, retrogradely labeled neurons became less numerous in the dorsomedial PAG but were more widely scattered throughout the lateral and ventrolateral parts of the PAG. Only few retrogradely labeled neurons were found in the ventromedial part of the PAG at caudal levels. Injections of retrograde tracers restricted to subregions of the PGi suggested topography for afferents from the PAG. Injections into the lateral portion of the PGi yielded the greatest number of labeled neurons within the rostral ventromedial PAG. Medially placed injections yielded numerous retrogradely labeled neurons in the lateral and ventrolateral PAG. Injections placed in the rostral pole of the PGi (medial to the facial nucleus) produced the greatest number of retrogradely labeled neurons in the dorsal PAG. To examine the pathways taken by fibers projecting from PAG neurons to the medulla, and to further specify the topography for the terminations of these afferents in the PGi, the anterograde tracer Phaseolus vulgaris-leucoagglutinin was iontophoretically deposited into subregions of the PAG that contained retrogradely labeled neurons in the above experiments. These results revealed distinct fiber pathways to the rostral medulla that arise from the dorsal, lateral/ventrolateral, and ventromedial parts of the PAG. These injections also showed that there are differential but overlapping innervation patterns within the PGi. Consistent with the retrograde tracing results, injections into the rostral ventromedial PAG near the supraoculomotor nucleus yielded anterograde labeling immediately ventral to the nucleus ambiguus in the ventrolateral medulla, within the retrofacial portion of the PGi.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

The cortico-medial amygdala in the central nervous system organization of agonistic behavior

Previous studies suggested that the corticomedial amygdala is involved in agonistic behavior by affecting social learning processes. The present study shows that deficits in the avoidance of a dominant male rat conditioned by defeat were only observed after bilateral lesions restricted to the medial amygdala and not after destroying the cortical portion. These results are related to the specific afferent and efferent connections of the medial amygdala with other brain structures involved in the control of agonistic behavior. Within the corticomedial amygdala the medial amygdaloid nucleus was found to be a major recipient of afferents from the accessory olfactory bulb, but also a source of efferent projections to the ventrolateral aspects of the ventromedial hypothalamic nucleus and the ventral premammillary nucleus. A strong reciprocity exists in the connections between ventromedial hypothalamus and premammillary nuclei with the medial amygdala. The connections between the ventromedial hypothalamic and dorsal premammillary nucleus with the midbrain periaqueductal grey suggest an important role for the periaqueductal gray in the descending output in the anatomical substrate for agonistic behavior.     

Reciprocal connections between subdivisions of the dorsal raphe and the nuclear core of the locus coeruleus in the rat

The interconnection between two brainstem monoaminergic nuclei, the dorsal raphe (DR) and the locus coeruleus (LC), was analyzed in the rat using retrograde tracing and immunocytochemistry. Gold-conjugated and inactivated wheatgerm agglutinin-horseradish peroxidase (WGA-apo-HRP-gold) was injected into subdivisions of the DR or rostro-caudal levels of the nuclear core of the LC, and labeled LC or DR neurons were identified by dopamine-beta-hydroxylase (DBH) or 5-hydroxytryptamine (5-HT) immunostaining, respectively. Within the LC-DR projection, the caudal principal LC projected to the caudal, ventromedial, and interfascicular DR. Mid-LC as well as caudal LC projected with an ipsilateral predominance to the lateral wing subdivision of the DR. A few rostral LC neurons projected to caudal, dorsomedial, and ventromedial DR. Within the DR-LC projection, the rostral LC received inputs mainly from the caudal, dorsomedial, and ventromedial DR. Mid-LC to caudal LC received projections from mid-DR to caudal DR, with the heaviest projection from the ipsilateral lateral wing as well as caudal DR. The DR-LC projection was substantially more robust than LC-DR and included both serotonergic and nonserotonergic components. Thus, the data demonstrate topographically ordered, reciprocal connectivity between DR and LC with particularly strong projections from DR to LC. Communication between these two brainstem monoaminergic nuclei may be critical for a variety of functions including sleep-wake regulation, vigilance, analgesia, cognition, and stress responses.     

Efferent Projections from the Ovarian Steroid Receptor-Containing Area of the Ventrolateral Hypothalamus in Female Guinea Pigs

The ventrolateral hypothalamus (VLH) in female guinea pigs includes a subset of neurons which contain estrogen and progestin receptors, and which are implicated in the regulation of female sexual behavior by steroid hormones. However, little is known about where these neurons project, and consequently which other brain areas are involved in sexual behavior in female guinea pigs. The anterograde tracer Phaseolus vulgaris -Leucoagglutinin was used to label efferents from the ovarian steroid receptor-containing part of the VLH. To identify the correct placement of the tracer specifically within the group of neurons containing estrogen receptors, medial hypothalamic sections were also immunostained for estrogen receptors. Forebrain areas receiving dense projections from the ventrolateral hypothalamus included the bed nucleus of the stria terminalis, medial preoptic area, anterior hypothalamic area, anterior ventromedial hypothalamus, and caudal ventrolateral hypothalamus. The midbrain central gray was also heavily labeled. Moderate innervation was observed in the forebrain in the basolateral amygdala, medial preoptic nucleus, lateroanterior hypothalamic nucleus, dorsal hypothalamic areas, posterior hypothalamus, zona incerta, and in the midbrain interspersed among the central and lateral tegmental tracts. The major efferent pathways from the VLH appeared to travel rostrally through the mediobasal hypothalamus and preoptic area, and caudally via the medial thalamic nuclei and periventricular fiber system. These findings are similar to those of previous studies tracing the efferents from the ventromedial nucleus in rats and from the lateral hypothalamus in guinea pigs. Many of these areas that receive input from the steroid receptor rich area within the VLH are likely to be involved in the regulation of female sexual behavior.     

Afferent connections to the amygdaloid complex of the rat and cat: II. Afferents from the hypothalamus and the basal telencephalon

The projections from the basal telencephalon and hypothalamus to each nucleus of the amygdaloid complex of the rat, and to the central amygdala of the cat, were investigated by the use of retrograde transport of horseradish peroxidase (HRP). The enzyme was injected stereotaxically by microiontophoresis, using three different approaches. The ventral pallidum (Heimer, '78) and ventral part of the globus pallidus were found to project to the lateral and basolateral nuclei of the amygdala. The substantia innominata projects diffusely to the entire amygdaloid complex, except to the lateral nucleus and the caudal part of the medial nucleus. The anterior amygdaloid area shows a similar projection field, the only difference being that this structure does not project to any parts of the medial nucleus. The dorsal subdivision of the nucleus of the lateral olfactory tract sends fibers to the ipsilateral as well as the contralateral basolateral nucleus, and possibly to the ipsilateral basomedial and cortical amygdala. The ventral subdivision of the nucleus of the lateral olfactory tract was massively labeled after an injection in the ipsilateral central nucleus, but this injection affected the commissural component of the stria terminalis. The nucleus of the horizontal limb of the diagonal band of Broca connects with the medial, central, and anterior cortical nuclei, whereas the bed nucleus of stria terminalis and medial preoptic area are related to the medial nucleus predominantly. The lateral preoptic area is only weakly labeled after intra-amygdaloid HRP injections. The hypothalamo-amygdaloid projections terminate preponderantly in the medial part of the amygdaloid complex. Thus, axons from neurons in the area dorsal and medial to the paraventricular nucleus of the hypothalamus distribute to the medial nucleus and intra-amygdaloid part of the bed nucleus of stria terminalis. Most of the amygdalopetal fibers from the ventromedial, ventral premammillary, and arcuate nuclei of the hypothalamus end in the medial nucleus, but some extend into the central nucleus. A few fibers from the ventromedial nucleus of the hypothalamus reach the basolateral nucleus. The lateral hypothalamic area projects heavily to the central nucleus, and more sparsely to the medial and basolateral nuclei. The dorsal hypothalamic area and supramammillary nucleus show restricted projections to the central and basolateral nuclei, respectively. There are only a modest number of crossed hypothalamo-amygdaloid fibers. Most of these originate in the ventromedial nucleus of the hypothalamus and terminate in the contralateral medial nucleus. The projections from the basal telencephalon and hypothalamus to the central nucleus of the amygdala of the cat are similar to the corresponding projections in the rat.     

Organization of ascending hypothalamic projections to the rostral forebrain with special reference to the innervation of cholinergic projection neurons

Axonal projections from hypothalamic nuclei to the basal forebrain, and their relation to cholinergic projection neurons in particular, were studied in the rat by using the anterograde tracer Phaseolus vulgaris-leucoagglutinin (PHA-L) in combination with choline acetyltransferase (ChAT) immunocytochemistry. Discrete iontophoretic PHA-L injections were delivered to different portions of the caudal lateral hypothalamus, as well as to various medial hypothalamic areas, including the ventromedial, dorsomedial, and paraventricular nuclei, and anterior hypothalamic and medial preoptic areas. The simultaneous detection of PHA-L-labeled fibers/terminals and ChAT-positive neurons was performed by using nickel-enhanced diaminobenzidine (DAB) and nonenhanced DAB as chromogens. Selected cases were investigated at the electron microscopic level. Ascending hypothalamic projections maintained an orderly lateromedial arrangement within the different components of the medial forebrain bundle, as well as with respect to their terminal projection fields (e.g., within the bed nucleus of the stria terminalis and lateral septal nucleus). The distribution pattern of hypothalamic inputs to cholinergic projection neurons corresponded to the topography of ascending hypothalamic axons. Axons originating from neurons in the far-lateral hypothalamus reached cholinergic neurons in a zone that extended from the dorsal part of the sublenticular substantia innominata (SI) caudolaterally, to the lateral portion of the bed nucleus of the stria terminalis rostromedially, encompassing a narrow band along the ventral part of the globus pallidus and medial portion of the internal capsule. Axons originating from cells in the medial portion of the lateral hypothalamus reached cholinergic cells primarily in more medial and ventral parts of the SI, and in the magnocellular preoptic nucleus and horizontal limb of the diagonal band nucleus (HDB). Axons from medial hypothalamic cells appeared to contact cholinergic neurons primarily in the medial part of the HDB, and in the medial septum/vertical limb of the diagonal band complex. Electron microscopic double-labeling experiments confirmed contacts between labeled terminals and cholinergic cells in the HDB and SI. Individual hypothalamic axons established synapses with both cholinergic and noncholinergic neuronal elements in the same regions. These findings have important implications for our understanding of the organization of afferents to the basal forebrain cholinergic projection system.     

CRFergic Innervation Of the Paraventricular Nucleus of the Rat Hypothalamus: A Tract-Tracing Study

It has been reported that corticotropin-releasing factor (CRF) may regulate its own biosynthesis in the paraventricular nucleus of the hypothalamus (PVH). Whether this CRF autoregulation is mediated by local circuitry or from extra-PVH CRF neuronal fibers terminating on CRF perikarya within the PVH is unknown. In the present study, we sought to determine the origin(s) of this CRF innervation using retrograde transport of wheat germ-conjugated-gold particles (WGA-apoHRP-Au) combined with immunohistochemistry for CRF. The rats also received colchicine (100 μg, icv) 5–7 days after tracer injection and were perfused 24 h later. Results of retrograde labeling with pressure injections of WGA-apoHRP-Au centered to PVH and subsequent immunohistochemical staining for CRF demonstrated numerous retrogradely labeled CRF neurons in the perifornical hypothalamic nucleus (PeF), the dorsolateral hypothalamic area (DA) (medial and lateral portions) and the dorsomedial nucleus of the hypothalamus (DMH). Smaller groups of CRF-ir neurons that were retrogradely labeled were found in the bed nuclei of the stria terminalis (BnST), the Barrington’s nucleus (Bar) and the dorsal raphé (DR). These CRFergic pathways to the PVH may represent an anatomical substrate underlying the function of the stress-integrative PVH neurons in the autonomic, behavioral and neuroendocrine regulation during the stress response, including CRF autoregulation.     

Efferents from medial basal forebrain and hypothalamus in the rat. II. An autoradiographic study of the anterior hypothalamus.

Using tritiated amino acid autoradiography, the efferent projections of the anterior hypothalamic area (AHA) were studied in albino rats. Axons from AHA neurons were not confined to local projections in the hypothalamus. Ascending AHA axons ran through the preoptic region, joined the diagonal band and distributed in the lateral septum. Descending AHA efferents within the hypothalamus coursed in a bundle ventromedial to the fornix. Projections were observed to the dorsomedial, ventromedial, arcuate and dorsal premammillary nuclei, and to the median eminence. Sweeping dorsomedially in the posterior hypothalamus, some AHA axons distributed in the central grey. AHA axons staying ventral projected to the supramammillary region, ventral tegmental area, raphe nuclei and midbrain reticular formation. Other AHA efferents distributed to the periventricular thalamus, to the medial amygdala via the stria terminalis or supraoptic commissure, and to the lateral habenula through the stria medullaris. For comparison with the AHA, efferent projections from the paraventricular nucleus (PVN) and from the ventromedial nucleus and adjacent basal hypothalamus (VMR) were studied. Projections from PVN neurons were not restricted to the median eminence and neurohypophysis. PVN efferents also distributed to many of the same regions as did those of the AHA but had somewhat different fiber trajectories and longer descending projections. VMR efferents were more widespread than those of the AHA, with projections extending into the lateral zona incerta and pontine reticular formation. Projections from the AHA were distinct from those of the medial preoptic area (mPOA). For example, while AHA axons descended in a bundle ventromedial to the fornix, mPOA axons ran in the medial forebrain bundle. Such anatomical differences may underlie experimentally demonstrated functional differences between the mPOA and AHA, for instance, in mediation of male and female sex behaviors.     

Projections from Ventrolateral Hypothalamic Neurons Containing Progestin Receptor- and Substance P-Immunoreactivity to Specific Forebrain and Midbrain Areas in Female Guinea Pigs

Many neurons within the ventrolateral hypothalamus in guinea pigs contain estrogen-induced progestin receptors as well as substance P. Retrograde tracing combined with immunocytochemistry was used to determine the specific projections of this subset of steroid- sensitive cells. Unilateral Fluoro-Gold injections into the dorsal midbrain, including the central gray, labeled a large proportion of the ventrolateral hypothalamic neurons immunoreactive for both progestin receptors and substance P (approximately 30%); substantially fewer of these neurons were labeled by unilateral Fluoro-Gold injections into the preoptic area (approximately 6%), medial amygdala (approximately 10%), or the bed nucleus of the stria terminalis (approximately 11 %). The projections of progestin receptor-immunoreactive neurons in the ventrolateral hypothalamus were similar to those of progestin receptor/substance P double-labeled neurons, while a slightly lower percentage of the ventrolateral hypothalamic, substance P-immunoreactive neurons tended to project to each of these areas. These pathways may prove to be components of the neural circuitry underlying a variety of functions influenced by gonadal steroid hormones and substance P, such as female sexual behavior, salt intake, nociception and aggression.     

Endogenous opioid-immunoreactive neurons of the ventromedial hypothalamic nucleus concentrate estrogen in male and female rats

Estrogen stimulates expression of proenkephalin mRNA in neurons of the hypothalamic ventromedial nucleus, and evidence is accumulating that synaptic release of one of the peptide end products, met-enkephalin, influences events that regulate reproductive behavior. To address the question of whether estrogen acts directly on neurons that synthesize met-enkephalin or indirectly through a separate neuronal population, we combined estrogen autoradiography with endogenous opioid peptide (EOP) immunohistochemistry. In agreement with previous studies, the ventrolateral subdivision of the hypothalamic ventromedial nucleus was densely packed with EOP-immunoreactive cells. In males, 48% of the estrogen-concentrating cells of the ventrolateral subdivision of the hypothalamic ventromedial nucleus contained EOP, and, in females, 27% of the estrogen-concentrating cells contained EOP. These findings indicate that estrogen acts directly on neurons that express EOP and suggest a mechanism that underlies sexually differentiated reproductive behavior.     

The efferent connections of the ventromedial nucleus of the hypothalamus of the rat.

The efferent connections of the ventromedial nucleus of the hypothalamus (VMH) of the rat have been examined using the autoradiographic method. Following injections of small amounts (0.4-2.0 muCi) of tritium labeled amino acids, fibers from the VMH can be traced forward through the periventricular region, the medial hypothalamus and the medial forebrain bundle to the preoptic and thalamic periventricular nuclei, to the medial and lateral preoptic areas, to the bed nucleus of the stria terminalis and to the ventral part of the lateral septum. Some labeled axons continue through the bed nucleus of the stria terminalis into the stria itself, and hence to the amygdala, where they join other fibers which follow a ventral amygdalopetal route from the lateral hypothalamic area and ventral supraoptic commissure. These fibers terminate in the dorsal part of the medial amygdaloid nucleus and in the capsule of the central nucleus. A lesser number of rostrally directed fibers from the VMH crosses the midline in the ventral supraoptic commissure and contributes a sparse projection to the contralateral amygdala. Descending fibers from the VMH take three routes: (i) through the medial hypothalamus and medial forebrain bundle; (ii) through the periventricular region; and (iii) bilaterally through the ventral supraoptic commissure. These three pathways are interconnected by labeled fibers so that it is not possible to precisely identify their respective terminations. However, the periventricular fibers seem to project primarily to the posterior hypothalamic area and central gray, as far caudally as the anterior pole of the locus coeruleus, while the medial hypothalamic and medial forebrain bundle fibers apparently terminate mainly in the capsule of the mammillary complex, in the supramammillary nucleus and in the ventral tegmental area. The ventral supraoptic commissure fibers leave the hypothalamus closely applied to the medial edges of the two optic tracts. After giving off their contributions to the amygdala, they continue caudally until they cross the dorsal edge of the cerebral peduncle to enter the zona incerta. Some fibers probably terminate here, but others continue caudally to end in the dentral tegmental fields, and particularly in the peripeduncular nucleus. Within the hypothalamus, the VMH appears to project extensively to the surrounding nuclei. However, we have not been able to find evidence for a projection from the VMH to the median eminence. Isotope injections which differentially label the dorsomedial or the ventrolateral parts of the VMH have shown that most of the long connections (to the septum, amygdala, central tegmental fields and locus coeruleus) originate in the ventrolateral VMH, and there is also some evidence for a topographic organization within the projections of this subdivision of the nucleus.     

Efferents from the medial anterior hypothalamic area in the guinea pig

Medial anterior hypothalamic connections were studied with H³-proline and autoradiography. Most of the axons projected to other hypothalamic nuclei. The major pathways were found ventral medial to the fornix and in the periventricular tract. Substantial projections were apparent in the ventromedial and dorsomedial nuclei with less label in the arcuate nucleus. The dorsal premammillary nuclei were labeled bilaterally, particularly with more caudal injections of anterior hypothalamus. Efferents were evident in the posterior hypothalamus and continued into the central gray of the midbrain. Labeled fibers reached the ventral tegmental area and in the reticular formation were traced only through pons. Rostral projections were to the medial and lateral preoptic areas and ventral lateral septum. The bed nucleus of stria terminalis was labeled and a very few fibers reached the medial amygdaloid nucleus. The periventricular nucleus of thalamus was labeled.     

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.