Subset of neurons characterized by the presence of NADPH-diaphorase in human substantia innominata

The substantia innominata encompasses an area of the basal forebrain that is ventral to the lenticular nucleus and anterior commissure, medial to the claustrum and external capsule, and lateral to the hypothalamus. The nucleus basalis of Meynert consists primarily of large acetylcholinesterase (AchE)-positive neurons embedded within the substantia innominata. Damage to these neurons may be important in the pathogenesis of cortical dysfunction in Alzheimer's disease. In order to characterize other neuronal elements in the substantia innominata and their relationship to the nucleus basalis, we chose to study a biochemically distinct neuronal subset containing the enzyme nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d). The substantia innominata was blocked from six normal brains obtained postmortem and fixed in neutral-buffered formalin at 4 degrees C for 48 hours. Free-floating 50-micron sections from several levels were stained for NADPH-d or AchE activities. Selected sections were double stained for NADPH-d and AchE. NADPH-d activity was present in a network of pleomorphic neurons that extended through all levels of the substantia innominata and into the striatum and amygdala. NADPH-d neurons were particularly numerous at the level of the anterior commisure and were closely associated with the cholinergic neurons of the nucleus basalis. They were not seen in the ventral pallidum, or the vertical limb of the diagonal band of Broca or in the islands of Calleja. The cell bodies of NADPH-d neurons were quite varied in shape, ranging from ovoid to fusiform, and about half the cells were bipolar. Where neuronal density was high, their dendrites formed an interlacing pattern. NADPH-d-positive fibres were seen coursing through the external capsule, hypothalamus, and amygdala. This novel set of neurons in the substantia innominata may be part of a more extensive network that interacts with the magnocellular basal forebrain system at the level of the nucleus basalis. Whether other neurotransmitters are present within these neurons and whether NADPH-d neurons are involved in Alzheimer's disease remain to be elucidated.     

Organization of cerebral cortical afferent systems in the rat. II. Magnocellular basal nucleus

The organization of the magnocellular basal nucleus (MBN) projection to cerebral cortex in the rat has been studied by using cytoarchitectonic, immunohistochemical, and retrograde and anterograde transport methods. The distribution of retrogradely labeled basal forebrain neurons after cortical injections of wheat germ agglutinin-horseradish peroxidase conjugate was essentially identical to that of neurons staining immunohistochemically for choline acetyltransferase. These large (20-30 micrometers perikaryon diameter) multipolar neurons were found scattered through a number of basal forebrain cell groups: medial septal nucleus, nucleus of the diagonal band of Broca, magnocellular preoptic nucleus, substantia innominata, and globus pallidus. This peculiar distribution mimics the locations of pathways by which descending cortical fibers enter the diencephalon. Each cortical area was innervated by a characteristic subset of MBN neurons, always located in close association with descending cortical fibers. In many instances anterogradely labeled descending cortical fibers appeared to ramify into diffuse terminal fields among MBN neurons which were retrogradely labeled by the same cortical injection. Double label experiments using retrograde transport of fluorescent dyes confirmed that MBN neurons innervate restricted cortical fields. Anterograde autoradiographic transport studies after injections of 3H-amino acids into MBN revealed that MBN axons reach cerebral cortex primarily via two pathways: (1) The medial pathway, arising from the medial septal nucleus, nucleus of the diagonal band, and medial substantia innominata and globus pallidus MBN neurons, curves dorsally rostral to the diagonal band nucleus, up to the genu of the corpus callosum. Most of the fibers either directly enter medial frontal cortex or turn back over the genu of the corpus callosum into the superficial medial cingulate bundle. Many of these fibers enter anterior cigulate or retrosplenial cortex, but some can be traced back to the splenium of the corpus callosum, where a few enter visual cortex but most turn ventrally and sweep into the hippocampal formation. Here they are joined by other fibers which, at the genu of the corpus callosum, remain ventrally located and run caudally through the dorsal fornix into the hippocampus. (2) The lateral pathway arises in part from medial septal, diagonal band, and magnocellular preoptic neurons whose axons sweep laterally through the substantia innominata to innervate primarily piriform, perirhinal, and endorhinal cortex. Some of these fibers may also enter the hippocampal formation from the entorhinal cortex via the ventral subiculum.(ABSTRACT TRUNCATED AT 400 WORDS)     

Afferent connections of the substantia innominata/basal nucleus of Meynert in carnivores and primates

Afferent connections to the substantia innominata/nucleus basalis complex of monkeys and cats were traced by using the method of retrograde transport of horseradish peroxidase (HRP). Altogether ten injections of HRP were performed in four monkeys (Saimiri sciureus, Callithrix jacchus, Galago senegalensis) and in four cats, with either vertical or oblique needle approaches. The entire brains excluding the olfactory bulbs and the cerebellum were then screened for labeled neurons. In both monkey and cat brains, many retrogradely labeled neurons could be detected in the amygdala, hypothalamus, midline thalamus, zona incerta, and the fields of Forel. Further but weaker labeling occurred in the medial septal nucleus, diagonal band of Broca, olfactory tubercle, paraventricular, anterior, mediodorsal, and central lateral thalamic nuclei, lateral habenula, ventral tegmental area of Tsai, interpeduncular nucleus, parabrachial, raphe, dorsal tegmental nucleus and the locus caeruleus. Cortically, prefrontal, insular, entorhinal, prepiriform, and periamygdaloid areas of both species showed considerable labeling as well as the whole temporal lobe of the monkeys used. The perirhinal and basal temporal cortex of all cats showed moderate labeling. In both monkeys and cats, extremely scarce labeling occurred within the cingulate, retrosplenial, and subicular cortex. From an anatomical point of view, the manifold connections of the substantia innominata/basal nucleus of Meynert found in this study underscore the participation of these nuclear groups in motivational, emotional, and cognitive (e.g. mnemonic) functions. Considering the widespread cortical efferents of this complex, it is suggested that the substantia innominata/nucleus basalis of Meynert serves the transmission of information arising within the limbic system to the whole neocortex.     

Cholinergic enzymes in neocortex, hippocampus and basal forebrain of non-neurological and senile dementia of alzheimer-type patients

Choline acetyltransferase (ChAT) activity and acetylcholinesterase (AChE) staining were examined in different cortical regions, hippocampal formation and basal forebrain of non-neurological controls and of patients afflicted with senile dementia of Alzheimer type (SDAT). Both enzymes showed a clear topographical distribution in the various regions studied. In SDAT cases, ChAT activity was reduced by 0–60% in the neocortex and by up to 97% in the hippocampus depending on the area and layer examined. In the nucleus of the diagonal band of Broca (NDB) and the medial septal nucleus (MSN), the activity was decreased by 65% and 55%, respectively; no significant change was found in the lateral septal nucleus (LSN), nucleus basalis of Meynert (NBM), substantia innominata (SI) and globus pallidus (GP). Comparable changes were seen in AChE staining. The results indicate that degeneration or dysfunction of cholinergic neurons in the medial septal area and possibly neocortex is an important characteristic of SDAT.     

Direct catecholaminergic-cholinergic interactions in the basal forebrain. III. Adrenergic innervation of choline acetyltransferase-containing neurons in the rat

The central adrenergic neurons have been suggested to play a role in the regulation of arousal and in the neuronal control of the cardiovascular system. To provide morphological evidence that these functions could be mediated via the basal forebrain, we performed correlated light and electron microscopic double-immunolabeling experiments using antibodies against phenylethanolamine N-methyltransferase (PNMT) and choline acetyltransferase, the synthesizing enzymes for adrenaline and acetylcholine, respectively. Most adrenergic/cholinergic appositions were located in the horizontal limb of diagonal band of Broca, within the substantia innominata, and in a narrow band bordering the substantia innominata and the globus pallidus. Quantitative analysis indicated that cholinergic neurons of the substantia innominata receive significantly higher numbers of adrenergic appositions than cholinergic cells in the rest of the basal forebrain. In the majority of cases, the ultrastructural analysis revealed axodendritic asymmetric synapses. By comparing the number and distribution of dopamine beta-hydroxylase (DBH)/cholinergic appositions, described earlier, with those of PNMT/cholinergic interactions in the basal forebrain, it can be concluded that a significant proportion of putative DBH/cholinergic contacts may represent adrenergic input. Our results support the hypothesis that the adrenergic/cholinergic link in the basal forebrain may represent a critical component of a central network coordinating autonomic regulation with cortical activation.     

Cholinergic neurons expressing substance P receptor (NK(1)) in the basal forebrain of the rat: a double immunocytochemical study

Cholinergic neurons expressing substance P receptor (SPR, NK(1)) were examined in the rat brain using double immunofluorescence. The distribution of SPR-like immunoreactive (SPR-LI) neurons completely overlapped with that of choline acetyltransferase (ChAT)-LI neurons in the medial septal nucleus, the nucleus of diagonal band of Broca, the magnocellular preoptic nucleus, the substantia innominata of basal forebrain, the caudate-putamen, and the ventral pallidum of the basal ganglia. In the mesopontine tegmentum and the cranial motor nuclei of the brainstem, the distribution of SPR-LI and ChAT-LI neurons was partially overlapping. Neurons showing both SPR-like and ChAT-like immunoreactivities, however, were predominantly found above basal forebrain regions and 82-90% of these ChAT-LI neurons displayed SPR-like immunoreactivity, in addition to the confirmatory observation that 100% of the ChAT-LI neurons exhibit SPR-like immunoreactivity in the basal ganglia. In contrast, neurons double-labeled for SPR-like and ChAT-like immunoreactivities were hardly detected in aforementioned regions of the brainstem. The present study has provided morphological evidence for direct physiological modulation of cholinergic neurons by tachykinins through substance P receptor in the basal forebrain of the rat.     

Neurons in the basal forebrain complex of the rat: a Golgi study

Several types of neurons are coexistent in the basal forebrain nuclear complex of the rat. In the medial septum and vertical limb of the diagonal band 3 classes of neurons occur which are characterized by varicose dendrites. In the horizontal limb of the diagonal band neurons with smooth dendrites and those with varicose dendrites are intermingled. We found 3 classes of neurons in the nucleus preopticus magnocellularis. A giant type with smooth and varicose dendrites occurs in this nucleus, but also in the substantia innominata. In the substantia innominata-nucleus basalis complex 4 classes of neurons with varicose dendrites and 2 classes with spiny dendrites have been observed. Our findings suggest that the nucleus of the vertical limb of the diagonal band forms a unit with the nucleus septi medialis, but is separated from the nucleus of the horizontal limb of the diagonal band by different neuronal composition. The nucleus preopticus magnocellularis is a separate nuclear structure characterized by a content of neurons different from those in the horizontal limb of the diagonal band and in most components also from the substantia innominata-nucleus basalis complex. There is some evidence that the cholinergic neurons have to be searched among those with varicose dendrites.     

Midbrain,diencephalic and cortical relationships of the basal nucleus of meynert and associated structures in primates

The structure and connectivity of the basal nucleus of Meynert, the substantia innominata in which it lies, and certain related areas have been examined in New World and Old World Monkeys, using retrograde and anterograde axonal transport methods. Experiments using the retrograde, horseradish peroxidase method confirm the observations of Kievet and Kuypers ('75) that the basal nucleus and substantia innominata project directly, heavily and with a somewhat crude topography upon the neocortex. Experiments involving the anterograde, autoradiographic method show that the basal nucleus and substantia innominata form part of a complex pathway that links them together with the lateral hypothalamus, certain parts of the amygdala and the peripeduncular nucleus of the midbrain. The peripeduncular nucleus is often regarded as a part of the central auditory pathway; it gives rise to a fiber bundle of considerable size that ascends on the dorsal surface of the ipsilateral optic tract and terminates ultimately in the lateral hypothalamic area of both sides. As well as distributing fibers to the basal nucleus, substantia innominata and lateral hypothalamus, this pathway provides a heavy projection to a cytoarchitectonically distinct posterior part of the lateral nucleus of the amygdala, the medial and intercalated nuclei of the amygdala and a less dense projection to the bed nucleus of the stria terminalis. Certain parts of the hypothalamus and possibly the preoptic areas give rise to a complementary descending pathway that distributes fibers to the ipsilateral basal nucleus, substantia innominata and amygdala, and ends in the peripeduncular nuclei of both sides. Decussating fibers in both the ascending and descending pathways cross in the ventral supraoptic commissure. It is concluded that the basal nucleus should include most of the aggregated and unaggregated large cells that lie in the substantia innominata and which in places intrude upon the preoptic regions and the nucleus of the diagonal band of Broca. Together, these may form a complex that receives inputs from a variety of brainstem sources, and projects widely and diffusely upon all cortical structures of the telencephalon.     

Cholinergic neurons expressing substance P receptor (NK1) in the basal forebrain of the rat: a double immunocytochemical study

Cholinergic neurons expressing substance P receptor (SPR, NK₁) were examined in the rat brain using double immunofluorescence. The distribution of SPR-like immunoreactive (SPR-LI) neurons completely overlapped with that of choline acetyltransferase (ChAT)-LI neurons in the medial septal nucleus, the nucleus of diagonal band of Broca, the magnocellular preoptic nucleus, the substantia innominata of basal forebrain, the caudate-putamen, and the ventral pallidum of the basal ganglia. In the mesopontine tegmentum and the cranial motor nuclei of the brainstem, the distribution of SPR-LI and ChAT-LI neurons was partially overlapping. Neurons showing both SPR-like and ChAT-like immunoreactivities, however, were predominantly found above basal forebrain regions and 82–90% of these ChAT-LI neurons displayed SPR-like immunoreactivity, in addition to the confirmatory observation that 100% of the ChAT-LI neurons exhibit SPR-like immunoreactivity in the basal ganglia. In contrast, neurons double-labeled for SPR-like and ChAT-like immunoreactivities were hardly detected in aforementioned regions of the brainstem. The present study has provided morphological evidence for direct physiological modulation of cholinergic neurons by tachykinins through substance P receptor in the basal forebrain of the rat.     

Basal forebrain efferents reach the whole cerebral cortex of the cat

Efferent projections from the basal forebrain to the cat's cerebral cortex were traced with the retrograde horseradish peroxidase technique. Different areas of the cerebral cortex of 51 cats were injected with small amounts of horseradish peroxidase. The entire basal forebrain was screened for labeled neurons. Following all injections, retrogradely labeled neurons could be detected in either the medial septum, or the vertical and horizontal limb of the diagonal band of Broca, or the substantia innominata, or in several of these structures. All three basal forebrain structures project heavily to allocortical regions, but only weakly to neocortical regions. An exception is the medial prefrontal cortex which is densily innervated by the substantia innominata (i.e., comparably dense as allocortical regions are innervated by the substantia innominata). Large injections into he basal temporal cortex (including the perirhinal cortex) and into the insular cortex also led to a considerable number of labeled cells in the substantia innominata. The results indicate a widespread innervation of the cat's cerebral cortex by the basal forebrain. This diffuse projection to the cortex has recently been found also in monkeys and rats. Anatomical and functional implications of these projections in the cat are discussed and related to findings in other species.     

The afferent connections of the substantia innominata in the monkey, Macaca fascicularis

The afferent connections of the substantia innominata and the magnocellular nuclei within it (the nucleus of the horizontal limb of the diagonal band, NHDB, and the nucleus basalis of Meynert, NBM) have been studied with anterograde and retrograde axonal tracing techniques. Prominent inputs arise in the amygdaloid complex, restricted areas of the cerebral cortex, parts of the thalamus and hypothalamus, and nuclei of the lower brainstem. Autoradiographic tracing experiments indicate that the amygdaloid fibers are distributed throughout the NHDB and the NBM, and to a lesser extent to the ventral pallidum. Relatively few fibers innervate the more medially located nucleus of the vertical limb of the diagonal band (NVDB) and the medial septal nucleus. Visualization of the amygdalofugal fibers with the tracer PHA-L (Phaseolus vulgaris leuco-agglutinin) shows that they have varicosities resembling boutons en passant along their length in the substantia innominata. Retrograde tracing experiments using WGA-HRP indicate that the cells of origin of the projection from the amygdala are concentrated in the parvicellular basal nucleus, the caudal part of the magnocellular basal nucleus, the magnocellular accessory basal nucleus, and the central nucleus. Relatively few fibers to the substantia innominata arise in the rostrodorsal part of the magnocellular basal nucleus, or in the lateral or parvicellular accessory basal nuclei. Cortical cells projecting to the substantia innominata were retrogradely labeled in the orbitofrontal cortex (including areas 11-14 and 25), the rostral insula (especially the agranular area), the rostroventral temporal cortex (including areas 35, 36, and parts of TG and TE), and the piriform and entorhinal cortices. The projections from the orbital and rostral temporal cortex were confirmed with anterograde tracers. Projections to the substantia innominata were not found from the more lateral, dorsal or caudal parts of the cerebral cortex, although fibers from temporal area TA may pass through the dendritic field of the most caudal cells of the NBM. Diencephalic cells projecting to the substantia innominata are distributed diffusely throughout the preoptic area and hypothalamus, with higher concentration in the lateral preoptic area and in the pre-, supra-, and tubero-mammillary nuclei. Cells are also found in the midline thalamic nuclei and in the region between the peripeduncular and subparafascicular nuclei.(ABSTRACT TRUNCATED AT 400 WORDS)     

Descending projections of the basal forebrain in the rat demonstrated by the anterograde neural tracer Phaseolus vulgaris leucoagglutinin (PHA-L)

Descending pathways from the mediobasal forebrain were studied in the rat by injecting anterograde axonal tracer Phaseolus vulgaris leucoagglutinin into the substantia innominata and diagonal band of Broca. From both areas, positive fibers which varied in density were observed in the mediodorsal and ventral parts of the ventroposterior and ventromedial thalamic nuclei, the lateral habenula, the stria medullaris, the lateral hypothalamus and the ventral tegmental area. This descending complex appeared predominantly course through the medial forebrain bundle from which positive fibers ramified into the fasciculus thalamicus to distribute in the thalamic nuclei. A minor descending pathway through the stria medullaris was also noted which terminated in the lateral habenula and the mediodorsal thalamic nucleus. An obvious difference in terminal distribution in the medial habenula, mediodorsal thalamic nucleus and pons could be observed following substantia innominata or diagonal band injection.     

Loss of neurons in the nucleus basalis of Meynert in Alzheimer's disease,paralysis agitans and Korsakoff's disease

Summary The nucleus basalis of Meynert, the major source of cholinergic innervation of the cerebral cortex, was morphometrically investigated in 58 cases of neuropsychiatric disorders and compated to 14 controls. The results demonstrate a loss of neurons in the nucleus basalis of Meynert in Alzheimer's disease (70%), paralysis agitans (77%), and Korsakoff's disease (47%) but no marked reduction of neurons in postencephalitic parkinsonism, Huntington's disease, chronic alcoholism without dementia, schizophrenia and infantile brain damage. Neurons of the three subdivisions of the nucleus basalis of Meynert (the nucleus septi medialis, the nucleus of the diagonal band of Broca and the nucleus basalis Meynert neurons in the substantia innominata) may be affected in a different manner in different patients within a single group homogeneous with respect to the usual clinical and neuropathological diagnostic criteria. Cell loss in the basal forebrain is restricted to the large neurons of the nucleus basalis, the immediately adjacent neurons of the globus pallidus externus not being affected. The selective degeneration of these neurons provides the morphological correlate of the cortical cholinergic deficiency in these neuropathological conditions. The degeneration of this discrete cholinergic neuronal population in several disorders of higher cortical function is probably directly related to the progressive deterioration of memory and cognitive processes in affected patients.     

Distribution of Acetylcholinesterase-Containing Neurons in the Basal Forebrain and Upper Brainstem of the Squirrel Monkey (Saimiri sciureus)

The topographical distribution of neurons containing acetylcholinesterase (AChE, EC 3.1.1.7) in the basal forebrain and upper brainstem of the squirrel monkey (Saimiri sciureus) was studied by means of Butcher's pharmacohistochemical technique which involves staining for AChE at various times after the systemic administration of the AChE inhibitor di-isopropylphosphorofluoridate (DFP). Only those neurons whose AChE staining was as intense as that of known cholinergic neurons present in the same material (e.g., neurons of cranial nerve nuclei) were examined and mapped. Three major collections of such strongly-stained AChE neurons were disclosed in squirrel monkey brain: one located in the striatum, the other lying along the ventralmost aspects of the basal forebrain, and a third one present within the midbrain-pontine tegmentum. The striatal AChE neurons vary in shape from fusiform with 2 thick processes to polygonal with 4-5 thinner processes. They are uniformly scattered throughout the caudate nucleus and putamen and represent only a small proportion of the total striatal cell population (4-6 cells/mm2). They most likely correspond to the aspiny type II cells described in Golgi material of monkey striatum. Similar neurons occur also in ventral striatal areas comprising nucleus accumbens septi and the deep polymorph layer of the olfactory tubercle. The second major AChE neuronal population is composed of the magnocellular neurons that form a somewhat continuous chain of neuronal aggregates extending rostrocaudally from the septal region to the caudal pole of the lentiform nucleus. It includes the neurons of the medial septal nucleus, the nucleus of the diagonal band of Broca and the nucleus basalis of Meynert, all displaying strikingly similar morphological and histochemical characteristics. The AChE neuronal population of nucleus basalis encroaches markedly upon the lateral hypothalamus laterally and the globus pallidus dorsally. The third important AChE cell collection occurs within the pedunculopontine nucleus area in upper brainstem. In that constellation, the AChE neurons are clustered in 2 continuous cell groups: one located dorsolaterally, the other lying ventromedially to the brachium conjunctivum. The thick processes of these neurons form impressive AChE neuronal networks that surround and pervade the brachium conjunctivum over long distances. This cell group, which is one of the most highly AChE reactive structures of the entire brain in the squirrel monkey, may provide a major cholinergic input to various basal ganglia structures, particularly the substantia nigra.     

Cells with varicose dendrites: a characteristic type of neurons in Golgi preparations of the rat cholinergic basal forebrain nuclei

Golgi preparations reveal that in the basal forebrain nuclei composed of medial septal nucleus, diagonal band, nucleus preopticus magnocellularis and substantia innominata including nucleus basalis magnocellularis of Meynert, the most frequently impregnated type of neuron is medium to large sized with long smooth or sparsely spined varicose dendrites. Some indications implying the cholinergic nature of those cells are discussed.     

Topography of the magnocellular basal forebrain system in human brain

In primates, the large neurons in the nucleus basalis of Meynert (nbM), nucleus of the diagonal band of Broca (dbB), and medial septum are part of a cholinergic system with direct projections to amygdala, hippocampus, and cortex. Recent evidence indicates that neurons of this system selectively degenerate in individuals with Alzheimer's disease (AD) and suggests that degeneration of these cells contributes to the loss of presynaptic cortical cholinergic markers which occurs in these patients. The present report describes the topographical distribution of these large intensely basophilic, basal forebrain neurons in human brain. Rostrally, neurons of this magnocellular system are present in the medial septum and the dorsal and ventral parts of the nucleus of the dbB. The largest number occur in the nbM, which is situated in the substantia innominata below the globus pallidus. Caudally, large nbM-type neurons are found along the ventral and lateral edges of the globus pallidus. Neurons of this type are also encountered in the white matter below the putamen and nucleus accumbens, at the edges of the anterio commissure, in the white matter laminae of the globus pallidus and within and at the medial edge of the genu of the interal capsule. Directions for dissection of this system in human brain are given in an Appendix.     

Topological analysis of the brainstem of the reedfish, Erpetoichthys calabaricus

The neuronal distribution of 7-aminobutyric acid (GABA) transaminase (GABA-T), the enzyne which metabolizes GABA, has been mapped in rat brain. The method involves staining for newly synthesized GABA-T by the previously established nitro blue tetrazolium technique in animals killed 8–48 hours after administration of gabaculine, an irreversible inhibitor of GABA-T. Neuronal staining is obscured by staining of other elements if initial suppression is inadequate or survival times postgabaculine are too long. With appropriate conditions, GABA-T-positive neuronal somata can be widely detected. The stained cells include neuronal groups previously reported to be GAB Aergic on the basis of glutamate decarboxylase (GAD)-col-chicine immunocytochemistry and other methods, i.e.: Purkinje, basket, Golgi, and stellate neurons of the cerebellum; basket and stellate neurons of the hippocampus; granule and periglomerular cells of the olfactory bulb; magnocellular neurons of the hypothalamus; and neurons of the striatum, pallidum, entopeduncular nucleus, cortex, medial septal area, diagonal band, substantia innominata, reticular nucleus of the thalamus, substantia nigra, and dorsal raphe. Other cells that stain intensely for GABA-T and may be GABAergic include neurons in the midlateral septal area, accumbens, the central medial and basal. nucler of the amyugdala, zona in certa, the brainstem reticular formation, central gray, interstitial nucleus of Cajal, and various thalamic nuclei including the periventricular, intralami-nar, rhomboid, and subparafascicular. Known non-GABA neuronal groups are negative for GABA-T staining under these conditions, reinforcing the hypothesis that GABA neurons are far more GABA-T intensive than other neurons.     

Heterogeneity and selectivity of the degeneration of cholinergic neurons in the basal forebrain of patients with Alzheimer's disease

Cholinergic neurons were studied by immunohistochemistry, with an antiserum against choline acetyltransferase (ChAT), in the basal forebrain (Ch1 to Ch4) of four patients with Alzheimer's disease (AD) and four control subjects. ChAT-positive cell bodies were mapped and counted in Ch1 (medial septal nucleus), Ch2 (vertical nucleus of the diagonal band), Ch3 (horizontal nucleus of the diagonal band) and Ch4 (nucleus basalis of Meynert). Compared to controls, the number of cholinergic neurons in AD patients was reduced by 50% on average. The interindividual variations in cholinergic cell loss were high, neuronal loss ranging from moderate (27%) to severe (63%). Despite the small number of brains studied, a significant correlation was found between the cholinergic cell loss and the degree of intellectual impairment. To determine the selectivity of cholinergic neuronal loss in the basal forebrain of AD patients, NPY-immunoreactive neurons were also investigated. The number of NPY-positive cell bodies was the same in controls and AD patients. The results (1) confirm cholinergic neuron degeneration in the basal forebrain in AD and the relative sparing of these neurons in some patients, (2) indicate that degneration of cholinergic neurons in the basal forebrain contributes to intellectual decline, and (3) show that, in AD, such cholinergic cell loss is selective, since NPY-positive neurons are preserved in the basal forebrain.     

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.