Estrogen receptor immunoreactivity within subregions of the rat forebrain: neuronal distribution and association with perikarya containing choline acetyltransferase

Administration of the neuroactive steroid hormone estrogen has been shown to effect cholinergic basal forebrain neuronal function. Antibodies directed against the estrogen receptor alpha (ERalpha) revealed dark (type 1) and light (type 2) nuclear positive neurons within the islands of Calleja, endopiriform nucleus, lateral septum, subfields of the cholinergic basal forebrain, bed nucleus of the stria terminalis, striohypothalamic region, medial preoptic region, periventricular, ventromedial, arcuate and tuberal mammillary nuclei of the hypothalamus, reuniens and anterior medial thalamic nuclei, amygdaloid complex, piriform cortex and subfornical organ. In contrast, only a few scattered ERalpha labeled neurons were found in cortex and hippocampus. ERalpha stained cell bodies were not seen in the striatum. Counts of ERalpha labeled neurons in intact female rats revealed significantly more type 2 neurons within the basal forebrain subfields. Quantitation of ERalpha immunoreactive neurons revealed a significant decrease in the relative number of type 1 neurons within the medial septum (MS), horizontal limb of the diagonal band (HDB) and substantia innominata/nucleus basalis (SI/NB) following ovariectomy. Quantitation following choline acetyltransferease (ChAT) immunohistochemistry revealed a significant decrease in the number of ChAT positive neurons within the MS, HDB and SI/NB, but not VDB following ovariectomy. Following ovx, the percentage of double labeled cholinergic basal forebrain neurons also declined significantly within the MS, VDB, HDB and SI/NB. These observations suggest that estrogen effects a subpopulation of cholinergic basal forebrain neurons and may provide insight into the biologic actions of this steroid in Alzheimer's disease.     

Efferents from medial basal forebrain and hypothalamus in the rat. I. An autoradiographic study of the medial preoptic area.

Efferent projections from the medial and periventricular preoptic area, bed nucleus of the stria terminalis and nuclei of the diagonal band were traced using tritiated amino acid autoradiography in albino rats. Medial and periventricular preoptic area efferents were not restricted to short-axon projections. Ascending projections from the medial preoptic area (mPOA) were traced through the diagonal band into the septum. Descending mPOA axons coursed in the medial parts of the medial forebrain bundle. Projections to most hypothalamic nuclei, including the arcuate nucleus and median eminence, were observed. In the midbrain, mPOA efferents were distributed in the central grey, raphe nuclei, ventral tegmental area and reticular formation. Projections from the mPOA were also observed to the amygdala through the stria terminalis, to the lateral habenula through the stria medullaris, and to the periventricular thalamus. Axons of the most medial and periventricular preoptic area (pvPOA) neurons had a distribution similar to more lateral mPOA neurons but their longest-axoned projections were weaker. The pvPOA did not send axons through the stria medullaris but did project more heavily than the more lateral mPOA to the arcuate nucleus and median eminence. Projections from the bed nucleus of the stria terminalis (nST) were in most respects similar to those from the medial preoptic area, with the major addition of a projection to the accessory olfactory bulb. The nuclei of the diagonal band of Broca (nDBB) gave a different pattern of projections than mPOA or nST, projecting, for instance, to the medial septum and hippocampus. Descending nDBB efferents ran in the ventral portion of the medial forebrain bundle. Among hypothalamic cell groups, only the medial mammillary nuclei received nDBB projections. nDBB efferents also distributed in the medial and lateral habenular nuclei and the mediodorsal thalamic nucleus.     

Cholinergic neurons of the laterodorsal tegmental nucleus: efferent and afferent connections

The ascending projections of cholinergic neurons in the laterodorsal tegmental nucleus (TLD) were investigated in the rat by using Phaseolus vulgaris leucoagglutinin (PHA-L) and wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP) anterograde tracing techniques. Two ascending pathways were identified after iontophoretic injections of PHA-L into the TLD. A long projection system courses through the dorsomedial tegmentum, caudal diencephalon, medial forebrain bundle, and diagonal band. Different branches of this system innervate the midbrain (superior colliculus, interstitial magnocellular nucleus of the posterior commissure, and anterior pretectal nucleus), the diencephalon (lateral habenular nucleus, parafascicular, anteroventral, anterodorsal, mediodorsal, and intralaminar thalamic nuclei), and the telencephalon (lateral septum and medial prefrontal cortex). The second system is shorter and more diffuse and innervates the median raphe, interpeduncular, and lateral mammillary nuclei. Retrograde tracing with WGA-HRP, combined with choline acetyltransferase immunohistochemistry, revealed that most of the TLD projections to the tectum, pretectum, thalamus, lateral septum, and medial prefrontal cortex are cholinergic. Afferents to the TLD were studied by anterograde and retrograde tracing techniques. Injection of tracers into the TLD retrogradely labelled neurons bilaterally in the midbrain reticular formation, the periaqueductal gray, the medial preoptic nucleus, the anterior hypothalamic nucleus, and the perifornical and lateral hypothalamic areas. Retrogradely labelled cells were also located bilaterally in the premammillary nucleus, paraventricular hypothalamic nucleus, zona incerta, and lateral habenular nucleus. In the telencephalon, the nucleus of the diagonal band and the medial prefrontal cortex contained retrogradely labelled neurons ipsilateral to the TLD injection site. The projections of the medial prefrontal cortex, the bed nucleus of the stria terminalis, and the lateral habenular nucleus to the TLD were confirmed in anterograde tracing studies. These findings indicate that the TLD gives rise to several ascending cholinergic projections that innervate diverse regions of the forebrain. Afferents to the TLD arise in hypothalamic and limbic forebrain regions, some of which appear to have reciprocal connections with the TLD. The latter include the lateral habenular nucleus and medial prefrontal cortex.     

Cholinergic and GABAergic afferents to the olfactory bulb in the rat with special emphasis on the projection neurons in the nucleus of the horizontal limb of the diagonal band

We have examined the location of cholinergic and GABAergic neurons that project to the rat main olfactory bulb by combining choline acetyltransferase (ChAT) and glutamic acid decarboxylase (GAD) immunohistochemistry with retrograde fluorescent tracing. Since many of the projection neurons are located in subcortical basal forebrain structures, where the delineation of individual regions is difficult, particular care was taken to localize projection neurons with respect to such landmarks as the ventral pallidum (identified on the basis of GAD immunoreactivity), the diagonal band, and medial forebrain bundle. In addition, sections with fluorescent tracers or immunofluorescence were counterstained for Nissl substance in order to correlate tracer or immunopositive neurons with the cytoarchitecture of the basal forebrain. The majority of the cholinergic bulbopetal neurons are located in the medial half of the nucleus of the horizontal limb of the diagonal band (HDB), whereas only a few are located in its lateral half. A substantial number of cholinergic bulbopetal cells are also found in the sublenticular substantia innominata. A small number of cholinergic bulbopetal neurons, finally, are located in the ventrolateral portion of the nucleus of the vertical limb of the diagonal band. At the level of the crossing of the anterior commissure, approximately 17% of the bulbopetal neurons in the HDB are ChAT-positive. The noncholinergic bulbopetal cells are located mainly in the lateral half of the HDB. GAD-containing bulbopetal neurons are primarily located in the caudal part of the HDB, especially in its lateral part. About 30% of the bulbopetal projection neurons in the HDB are GAD-positive. A few GAD-positive bulbopetal cells, furthermore, are located in the ventral pallidum, anterior amygdaloid area, deep olfactory cortex, nucleus of the lateral olfactory tract, lateral hypothalamic area, and tuberomamillary nucleus. The topography of bulbopetal neurons was compared to other projection neurons in the HDB. After multiple injections of fluorescent tracer in the neocortex, retrogradely labeled neurons were concentrated in the most medial part of the HDB, while neurons projecting to the olfactory and entorhinal cortices were located in the ventral part of the HDB. These results show that the cells of the HDB can be divided into subpopulations based upon projection target as well as transmitter content. Furthermore, these subpopulations correspond, at least to a considerable extent, to areas that can be defined on cyto- and fibroarchitectural grounds.     

Forebrain afferents to the cat posterior hypothalamus: a double labeling study

Using a double immunostaining technique with cholera toxin (CT) as a retrograde tracer, we examined the cells of origin and the histochemical nature of afferents to the cat posterior hypothalamus. After injection in the tuberomamillary nucleus, a number of CT-labeled cells were observed in: medial preoptic area, nuclei of the septum and the stria terminalis, amygdaloid complex, anterior hypothalamic, ventromedial hypothalamic and premamillary nuclei. CT injections in the lateral hypothalamic area gave an additional heavy labeling of neurons in: lateral preoptic area, nuclei of the diagonal band of Broca, substantia innominata, and nucleus accumbens. The posterior hypothalamus receives: 1) cholinergic inputs from the septum, the lateral preoptic area and the nuclei of the diagonal band of Broca; 2) dopaminergic afferents from A11, A13, and A14 groups; 3) histaminergic afferents from the posterior hypothalamus; and 4) peptidergic afferents such as methionin-enkephalin, galanin and neurotensin, substance P and corticotropin-releasing factor from the medial preoptic area, the nucleus of the stria terminalis and/or the posterior hypothalamic structures.     

Neural associations of the substantia innominata in the rat: afferent connections

The afferent connections of the substantia innominata (SI) in the rat were determined employing the anterograde axonal transport of Phaseolus vulgaris leucoagglutinin (PHA-L) and the retrograde transport of wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP), in combination with histochemical procedures to characterize the neuropil of the SI and identify cholinergic cells. Both neurochemical and connectional data establish that the SI is organized into a dorsal and a ventral division. Each of these divisions is strongly affiliated with a different region of the amygdala, and, together with its amygdalar affiliate, forms part of one of two largely distinct constellations of interconnected forebrain and brainstem cell groups. The dorsal SI receives selective innervation from the lateral part of the bed nucleus of the stria terminalis, the central and basolateral nuclei of the amygdala, the fundus of the striatum, distinctive perifornical and caudolateral zones of the lateral hypothalamus, and caudal brainstem structures including the dorsal raphe nucleus, parabrachial nucleus, and nucleus of the solitary tract. Projections preferentially directed to the ventral SI arise from the medial part of the bed nucleus of the stria terminalis, the rostral two-thirds of the medial nucleus of the amygdala, a large region of the rat amygdala that lies ventral to the central nucleus, the medial preoptic area, anterior hypothalamus, medialmost lateral hypothalamus, and the ventromedial hypothalamus. Both SI divisions appear to receive afferents from the dorsomedial and posterior hypothalamus, supramammillary region, ventral tegmental area, and the peripeduncular area of the midbrain. Projections to the SI whose selectivity was not determined originate from medial prefrontal, insular, perirhinal, and entorhinal cortex and from midline thalamic nuclei. Findings from both PHA-L and WGA-HRP experiments additionally indicate that cell groups preferentially innervating a single SI division maintain numerous projections to one another, thus forming a tightly linked assembly of structures. In the rat, cholinergic neurons that are scattered throughout the SI and in parts of the globus pallidus make up a cell population equivalent to the primate basal nucleus of Meynert (Mesulam et al.: Neuroscience 10:1185-1201, '83). PHA-L-filled axons, labelled from lectin deposits in the dorsal raphe nucleus, peripeduncular area, ventral tegmental area, or caudomedial hypothalamus were occasionally seen to approach individual cholinergic neurons int he SI, and to contact the surface of such cells with axonal varicosities (putative synaptic boutons.(ABSTRACT TRUNCATED AT 400 WORDS)     

Efferents of the medial preoptic area in the guinea pig: An autoradiographic study

Medial preoptic axons were traced into the diagonal band of Broca and septum, particularly lateral septum. Other labeled fibers could be followed dorsally from medial preoptic area injections adjacent to the stria medullaris, and in the periventricular fiber system and the stria terminalis and its bed nucleus. The anterior and medial amygdaloid nuclei were labeled by fibers via the stria terminalis and others arching over the optic tract and through the substantia innominata. The lateral habenula was labeled. Labeled periventricular fibers reached the periventricular nucleus of the thalamus. Descending efferents were traced principally below the fornix and in the adjacent lateral hypothalamus to label the anterior hypothalamus, the tuberal nuclei, and median eminence. Axons of the medial preoptic area joined the medial part of the medial forebrain bundle and distributed to the reticular formation and the central gray of the midbrain and pons. A small amount of contralateral connections were described.     

Limbic connections to the lateral preoptic area: a horseradish peroxidase study in the rat

Horseradish peroxidase, 13% Sigma Type VI, was administered iontophoretically to the lateral preoptic area (LPA) of male hooded rats. Animals were perfused intracardially on the following day and brains were removed and sliced in the coronal plane into 50 microns sections. Alternate sections were processed with DAB and BDH for the brown and blue reaction products and later examined by bright and dark field microscopy for the presence and location of retrogradely labeled neurons. Results indicate that there are a significant number of limbic efferent connections to the LPA. Afferents to the LPA originate in the prefrontal corex, nucleus accumbens, diagonal band and olfactory structures, lateral and medial septum, stria hypothalamic tract and stria terminalis, the magnocellular and medial preoptic nuclei, along the extent of the medial forebrain bundle in the LPA and LH, anterior and basolateral amygdala, ventromedial caudate-putamen, stria medullaris and lateral habenula, the stellatocellular-periventricular, ventromedial, arcuate and anterior hypothalamic nuclei, the perifornical area, zona incerta, ventral medial thalamic area, ventral tegmental area of Tsai, interpeduncular nucleus, reticular zone of the substantia nigra, mesencephalic periaqueductal gray and reticular formation, all aspects of the raphe nuclei and the locus coeruleus. Results are discussed in terms of known anatomical and neurophysiological data and the similar limbic inputs observed for lateral hypothalamic neurons which are found along the extent of the medial forebrain bundle.     

Efferent projections from the region of the medial zona incerta containing A13 dopaminergic neurons: a PHA-L anterograde tract-tracing study in the rat

Potential efferent projections of A₁₃ dopaminergic (DA) neurons were identified in the present study by examining the distribution of labelled fibers following iontophoretic injection of the anterogradely transported lectinPhaseolus vulgaris leucoagglutinin (PHA-L) into the medial zona incerta (MZI), the region of the diencephalon containing A₁₃ DA neuronal perikarya. One week after injection, PHA-L labelled fibers were found throughout the brain with the heaviest labelling occurring ipsilateral to the injection site in the anterior hypothalamic area, lateral hypothalamus, lateral preoptic area, horizontal diagonal band of Broca, and parvocellular region of the paraventricular nucleus. Moderate labelling was observed in the ipsilateral median preoptic nucleus, lateral septum, lateral aspect of the bed nucleus of the stria terminalis, and central nucleus of the amygdala. Moderate labelling was also found in the contralateral MZI and parvocellular region of the paraventricular nucleus. Light labelling was detected in the ipsilateral medial preoptic area, supraoptic nucleus, ventromedial nucleus, arcuate nucleus, vertical limb of the diagonal band of Broca, and in the contralateral lateral hypothalamus. Few immunopositive fibers were present in the dorsomedial nucleus of the hypothalamus or the magnocellular region of the paraventricular nucleus. These results reveal that neurons located in the MZI (possibly A₁₃ DA neurons) have ipsilateral efferent axonal projections to a variety of brain regions including the lateral hypothalamus, lateral preoptic area, and the limbic structures at the diencephalic-telencephalic juncture.     

Amygdaloid and pontine projections to the ventromedial nucleus of the hypothalamus

Amygdaloid and pontine projections to the feline ventromedial nucleus of the hypothalamus (HVM) were studied with retrograde transport of horseradish peroxidase (HRP) and anterograde transport of tritiated amino acids. Following injections of HRP into HVM, amygdaloid neurons were labeled in the ipsilateral cortical and medial nuclei and the ventral portion of the parvocellular part of the basal nucleus. In experiments in which HRP was injected into the tuberal hypothalamus following stria terminalis lesions, it was determined that amygdaloid neurons projecting to HVM by way of the stria terminalis were located in the cortical and medial nuclei while those projecting through another route, presumably the ventral amygdalofugal pathway, were found in the rostral part of the medial nucleus and the parvocellular basal nucleus. Following HRP injection into lateral hypothalamus at the level of HVM, labeled neurons were seen in the magnocellular basal nucleus. After preoptic injections, neurons containing the HRP reaction product were in cortical and medial nuclei and magnocellular and parvocellular parts of the basal nucleus. In addition to cells in the amygdala, rostral pontine neurons were labeled after HRP injections into HVM. The cells were located ipsilateral to the injection, mostly in the dorsal nucleus of the lateral lemniscus, lateral and dorsolateral to the brachium conjunctivum. The pontine cells labeled following HVM injections of HRP were different from those labeled following lateral hypothalamic and preoptic region injections. The pontine projection to HVM was confirmed using axoplasmic transport autoradiography. A mixture of tritiated leucine and tritiated proline was injected into the lateral pontine region labeled after HRP injections into HVM. Labeled axons ascending in the medial forebrain bundle terminated throughout the rostro-caudal extent of HVM.     

Cholinergic and catecholaminergic afferents to the olfactory bulb in the hamster: a neuroanatomical, biochemical, and histochemical investigation

A series of neuroanatomical, biochemical, and histochemical studies have been conducted to determine the sources of cholinergic afferents to the main olfactory bulb (MOB) in the hamster. Following horseradish peroxidase (HRP) injections that are restricted to the MOB, retrograde neuronal labeling is observed bilaterally in the anterior olfactory nucleus, locus coeruleus, and raphe nuclei, and ipsilaterally in the ventral hippocampal rudiment, dorsal peduncular cortex, piriform cortex, nucleus of the lateral olfactory tract, anterior pole of the medial septal area and vertical limb of the diagonal band, nucleus of the horizontal limb of the diagonal band (HDB), and hypothalamus. Spread of HRP into the accessory olfactory bulb results in additional neuronal labeling ipsilaterally in the bed nucleus of the accessory olfactory tract, medial amygdaloid nucleus, and bed nucleus of the stria terminalis, and bilaterally in the posteromedial cortical amygdaloid nucleus. Retrograde tracing studies also have been conducted in cases with lesions in the basal forebrain or hypothalamus to assess the extent to which such lesions interrupt fibers of passage from other sources of centrifugal afferents, and the effects of such lesions on choline acetyltransferase (CAT) activity and catecholamine content in the MOB and on acetylcholinesterase (AChE) activity in the forebrain have been evaluated. Lesions in the basal forebrain reduce or eliminate CAT and AChE activity in the MOB in direct relationship to the extent of damage to the HDB. Norepinephrine (NE) content in the MOB also is reduced by basal forebrain lesions, but in relationship to damage of the medial forebrain bundle (MFB). The hypothalamic lesions have no effect on AChE activity in the forebrain or on CAT activity in the MOB, but they eliminate retrograde labeling in the locus coeruleus and raphe nuclei and reduce the NE content of the MOB to undetectable levels. The dopamine content of the MOB is not reduced by any of the lesions. Anterograde tracing studies have been conducted to compare the rostral projection patterns of the HDB with the distribution of AChE activity. Most of the rostrally directed axons travel in association with the MFB. A small component of axons travels in association with the lateral olfactory tract. Within the MOB, the axons terminate predominantly in the glomerular layer and in the vicinity of the internal plexiform layer. The projection and termination patterns of the HDB correspond well with the distribution of AChE activity. These various results indicate that the HDB is the major source of cholinergic afferents to the MOB.     

Afferent connections to the lateral hypothalamus: A horseradish peroxidase study in the rat

Horseradish peroxidase, 13% Sigma Type VI, was administered iontophoretically to the mid lateral hypothalamus (LH) of male hooded rats. Animals were perfused intracardially on the following day and brains were removed and sliced in the coronal or sagittal planes into 30–50 μm sections. Sections were processed with DAB and BDH for the brown and blue reaction products and later examined by bright and dark field microscopy for the presence and location of retrogradely labeled neurons. Results indicate that a significant number of afferent connections to the LH originate in the olfactory and accumbens nuclei, pyriform cortex, olfactory tracts, magnocellular and medial preoptic and anterior hypothalamic regions, stria terminalis, stria hypothalamic tract, diagonal tract of Broca, caudate-putamen and globus pallidus, internal capsule, lateral septal nuclei, lateral preoptic area and anterior medial forebrain bundle, the various amygdaloid nuclei, zona incerta, perifornical region, dorsal and ventral medial hypothalamic areas, supraoptic, paraventricular and periventricular nuclei, posterior hypothalamus and medial forebrain bundle, ventral thalamic nuclei, the fields of Forel, arcuate and mammillary nuclei, adjacent to the fasciculus retroflexus, in the ventral tegmental area of Tsai, interpeduncular nucleus, substantia nigra, mesencephalic reticular formation, periaqueductal gray, locus coeruleus and parabrachial region. Results are discussed in terms of previous anatomical and neurophysiological data, probable pathways, and the function of LH neurons.     

Basal forebrain inputs to the interpeduncular nucleus in the rat studied with the Phaseolus vulgaris-leucoagglutinin tracing method

The connections between the basal forebrain and the interpeduncular nucleus (IP) were studied in the rat using the anterograde transport of Phaseolus vulgaris-leucoagglutinin (PHA-L). PHA-L was injected in the septum-diagonal band complex, the preoptic area, the substantia innominata, the globus pallidus, and the ventral pallidum. In a number of cases sections of the IP were double-immunostained for PHA-L and serotonin. Only following PHA-L injections in the medial septal nucleus and the nucleus of the vertical limb of the diagonal band, were substantial terminations found throughout all subnuclei of IP. Following injections in the medial and lateral preoptic area labeling was confined to the caudal part of IP. This finding suggests that the area in the basal forebrain that contributes to these projections is smaller than has been indicated by previous retrograde tracing studies. Labeled fibers reach the IP predominantly via the medial forebrain bundle. Only a very small number of fibers reaches the ventral mesencephalon, and possibly the IP, via the stria medullaris and the fasciculus retroflexus. The highest density of terminations was seen in the apical subnucleus. The apical subnucleus contains serotonin-immunoreactive neurons, in the direct vicinity of which varicosities on the labeled fibers were seen. This finding suggests innervation of the serotonergic neurons by fibers from the medial septum and preoptic area.     

Basal forebrain inputs to the interpeduncular nucleus in the rat studied with the Phaseolus vulgaris-leucoagglutinin tracing method

The connections between the basal forebrain and the interpeduncular nucleus (IP) were studied in the rat using the anterograde transport of Phaseolus vulgaris-leucoagglutinin (PHA-L). PHA-L was injected in the septum-diagonal band complex, the preoptic area, the substantia innominata, the globus pallidusand the ventral pallidum. In a number of cases sections of the IP were double-immunostained for PHA-L and serotonin. Only following PHA-L injections in the medial septal nucleus and the nucleus of the vertical limb of the diagonal band, were substantial terminations found throughout all subnuclei of IP. Following injections in the medial and lateral preoptic area labeling was confined to the caudal part of IP. This finding suggests that the area in the basal forebrain that contributes to these projections is smaller than has been indicated by previous retrograde tracing studies. Labeled fibers reach the IP predominantly via the medial forebrain bundle. Only a very small number of fibers reaches the ventral mesencephalonand possibly the IP, via the stria medullaris and the fasciculus retroflexus. The highest density of terminations was seen in the apical subnucleus. The apical subnucleus contains serotonin-immunoreactive neurons, in the direct vicinity of which varicosities on the labeled fibers were seen. This finding suggests innervation of the serotonergic neurons by fibers from the medial septum and preoptic area.     

Projections of the nucleus and tracts of the stria terminalis following lesions at the level of the anterior commissure.

The projections of the stria terminalis were traced with the Fink-Heimer stain following lesions at the level of the anterior commissure. The pre-commissural stria terminalis is amygdalofugal only, and projects to the nucleus of the anterior commissure, the medial preoptic area, the ventral portion of the capsule surrounding the ventromedial nucleus, and to the area closely adjacent to the periventricular nucleus by way of the medial corticohypothalamic tract. The postcommissural stria terminalis is both amygdalofugal and amygdalopetal. Its hypothalamic projection is to the lateral preoptic area and the bed nucleus of the stria terminalis, and to the lateral hypothalamus by way of the lateral preoptic area. The amygdaloid projection is mainly to the basolateral nucleus, with fewer terminations to the basomedial nucleus and the area surrounding the central nucleus. The projections of the bed nucleus of the stria terminalis are quite similar to the postcommissural stria, except for an additional projection to the magnocellular paraventricular and dorsal periventricular nuclei by way of the lateral filiform tract. The commissural stria terminalis projects contralaterally to cells within its fiber bundle and the posterior limb of the anterior commissure.     

Increased ipsilateral expression of Fos following lateral hypothalamic self-stimulation

Immunohistochemical labeling of Fos protein was used to visualize neurons activated by rewarding stimulation of the lateral hypothalamic level of the medial forebrain bundle (MFB). Following training and stabilization of performance, seven rats were allowed to self-stimulate for l h prior to anesthesia and perfusion. Brains were then processed for immunohistochemistry. Two control subjects were trained and tested in an identical manner except that the stimulator was disconnected during the final l h test. Among the structures showing a greater density of labeled neurons on the stimulated side of the brains of the experimental subjects were the septum, lateral preoptic area (LPO), medial preoptic area, bed nucleus of the stria terminalis, substantia innominata (SI), and the lateral hypothalamus (LH). Several of these structures, the LPO, SI, and LH, have been implicated in MFB self-stimulation by the results of psychophysical, electrophysiological, and lesion studies.     

The connections of the septal region in the rat

The efferent, afferent and intrinsic connections of the septal region have been analyzed in the rat with the autoradiographic method. The lateral septal nucleus, which can be divided into dorsal, intermediate and ventral parts, receives its major input from the hippocampal formation and projects to the medial septal-diagonal band complex. The ventral part of the nucleus also sends fibers through the medial forebrain bundle to the medial preoptic and anterior hypothalamic areas, to the lateral hypothalamic area and the dorsomedial nucleus, to the mammillary body (including the supramammillary region), and to the ventral tegmental area. The medial septal nucleus/diagonal band complex projects back to the hippocampal formation by way of the dorsal fornix, fimbria, and possibly the cingulum. Both nuclei also project through the medial forebrain bundle to the medial and lateral preoptic areas, to the lateral hypothalamic area, and to the mammillary complex. The medial septal nucleus also sends fibers to the midbrain (the ventral tegmental area and raphe nuclei) and to the parataenial nucleus of the thalamus, while the nucleus of the diagonal band has an additional projection to the anterior limbic area. Ascending inputs to the medial septal nucleus/diagonal band complex arise in several hypothalamic nuclei and in the brainstem aminergic cell groups. The posterior septal nuclei (the septofimbrial and triangular nuclei) receive their major input from the hippocampal formation, and project in a topographically ordered manner upon the habenular nuclei and the interpeduncular nuclear complex. The bed nucleus of the stria terminalis receives its major input from the amygdala (Krettek and Price, '78); but other afferents arise from the ventral subiculum, the ventromedial nucleus, and the brainstem aminergic cell groups. The principal output of the bed nucleus is through the medial forebrain bundle to the substantia innominata, the nucleus accumbens, most parts of the hypothalamus and the preoptic area, the central tegmental fields of the midbrain, the ventral tegmental area, the dorsal and median nuclei of the raphe, and the locus coeruleus. The bed nucleus also projects to the anterior nuclei of the thalamus, the parataenial and paraventricular nuclei, and the medial habenular nucleus, and through the stria terminalis to the medial and central nuclei of the amygdala, and to the amygdalo-hippocampal transition area.     

Efferent connections of the substantia innominata in the rat

The efferent connections of the substantia innominata (SI) were investigated employing the anterograde axonal transport of Phaseolus vulgaris leucoagglutinin (PHA-L) and the retrograde transport of wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP). The projections of the SI largely reciprocate the afferent connections described by Grove (J. Comp. Neurol. 277:315-346, '88) and thus further distinguish a dorsal and a ventral division in the SI. Efferents from both the dorsal and ventral divisions of the SI descend as far caudal as the ventral tegmental area, substantia nigra, and peripeduncular area, but projections to pontine and medullary structures appear to originate mainly from the dorsal SI. Within the amygdala and hypothalamus, which receive widespread innervation from the SI, the dorsal SI projects preferentially to the lateral part of the bed nucleus of the stria terminalis; the lateral, basolateral, and central nuclei of the amygdala; the lateral preoptic area; paraventricular nucleus of the hypothalamus; and certain parts of the lateral hypothalamus, prominently including the perifornical and caudolateral zones described previously. The ventral SI projects more heavily to the medial part of the bed nucleus of the stria terminalis; the anterior amygdaloid area; a ventromedial amygdaloid region that includes but is not limited to the medial nucleus; the lateral and medial preoptic areas; and the anterior hypothalamus. Modest projections reach the lateral hypothalamus, with at least a slight preference for the medial part of the region, and the ventromedial and arcuate hypothalamic nuclei. Both SI divisions appear to innervate the dorsomedial and posterior hypothalamus and the supramammillary region. In the thalamus, the subparafascicular, gustatory, and midline nuclei receive a light innervation from the SI, which projects more densely to the medial part of the mediodorsal nucleus and the reticular nucleus. Cortical efferents from at least the midrostrocaudal part of the SI are distributed primarily in piriform, infralimbic, prelimbic, anterior cingulate, granular and agranular insular, perirhinal, and entorhinal cortices as well as in the main and accessory olfactory bulbs. The cells of origin for many projections arising from the SI were identified as cholinergic or noncholinergic by combining the retrograde transport of WGA-HRP with histochemical and immunohistochemical procedures to demonstrate acetylcholinesterase activity or choline acetyltransferase immunoreactivity. Most of the descending efferents of the SI appear to arise primarily or exclusively from noncholinergic cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Prefrontal cortical projections to the cholinergic neurons in the basal forebrain

The prefrontal cortex (PFC) projections to the basal forebrain cholinergic cell groups in the medial septum (MS), vertical and horizontal limbs of the diagonal band of Broca (VDB and HDB), and the magnocellular basal nucleus (MBN) in the rat were investigated by anterograde transport of Phaseolus vulgaris leuco-agglutinin (PHA-L) combined with acetylcholinesterase (AChE) histochemistry or choline acetyltransferase (ChAT) immunocytochemistry. The experiments revealed rich PHA-L-labeled projections to discrete parts of the basal forebrain cholinergic system (BFChS) essentially originating from all prefrontal areas investigated. The PFC afferents to the BFChS display a topographic organization, such that medial prefrontal areas project to the MS, VDB, and the medial part of the HDB, whereas the orbital and agranular insular areas predominantly innervate the HDB and MBN, respectively. Since the recurrent BFChS projection to the prefrontal cortex is arranged according to a similar topography, the relationship between the BFChS and the prefrontal cortex is characterized by reciprocal connections. Furthermore, tracer injections in the PFC resulted in anterograde labeling of numerous "en passant" and terminal boutons apposing perikarya and proximal dendrites of neurons in the basal forebrain, which were stained for the cholinergic marker enzymes. These results indicate that prefrontal cortical afferents make direct synaptic contacts upon the cholinergic neurons in the basal forebrain, although further analysis at the electron microscopic level will be needed to provide conclusive evidence.     

Cortical projection patterns of the medial septum-diagonal band complex

A detailed analysis of the cortical projections of the medial septum-diagonal band (MS/DB) complex was carried out by means of anterograde transport of Phaseolus vulgaris leucoagglutinin (PHA-L). The tracer was injected iontophoretically into cell groups of the medial septum (MS) and the vertical and horizontal limbs of the diagonal band of Broca (VDB and HDB), and sections were processed immunohistochemically for the intra-axonally transported PHA-L. The labeled efferents showed remarkable differences in regional distribution in the cortical mantle dependent on the position of the injection site in the MS/DB complex, revealing a topographic organization of the MS/DB-cortical projection. In brief, the lateral and intermediate aspects of the HDB, also referred to as the magnocellular preoptic area, predominantly project to the olfactory nuclei and the lateral entorhinal cortex. The medial part of the HDB and adjacent caudal (angular) part of the VDB are characterized by widespread, abundant projections to medial mesolimbic, occipital, and lateral entorhinal cortices, olfactory bulb, and dorsal aspects of the subicular and hippocampal areas. Projections from the rostromedial part of the VDB and from the MS are preponderantly aimed at the entire hippocampal and retrohippocampal regions and to a lesser degree at the medial mesolimbic cortex. Furthermore, the MS projections are subject to a clear mediolateral topographic arrangement, such that the lateral MS predominantly projects to the ventral/temporal aspects of the subicular complex and hippocampus and to the medial portion of the entorhinal cortex, whereas more medially located cells in the MS innervate more septal/dorsal parts of the hippocampal and subicular areas and more lateral parts of the entorhinal cortex. PHA-L filled axons have been observed to course through a number of pathways, i.e., the fimbria-fornix system, supracallosal stria, olfactory peduncle, and lateral piriform route (the latter two mainly by the HDB and caudal VDB). Generally, labeled projections were distributed throughout all cortical layers, although clear patterns of lamination were present in several target areas. The richly branching fibers were abundantly provided with both "boutons en passant" and terminal boutons. Both distribution and morphology of the labeled basal forebrain efferents in the prefrontal, cingulate, and occipital cortices closely resemble the distribution and morphology of the cholinergic innervation as revealed by immunohistochemical demonstration of choline acetyltransferase. In contrast, the labeled projections to the olfactory, hippocampal, subicular, and entorhinal areas showed a heterogeneous morphology. Here, the distribution of only the thin varicose projections resembled the distribution of cholinergic fibers.