Nerve growth factor receptor immunoreactive profiles in the normal, aged human basal forebrain: colocalization with cholinergic neurons

A monoclonal antibody raised against the receptor for nerve growth factor (NGF) has been used to map the distribution of NGF receptor-containing profiles within the human basal forebrain of four male and three female elderly patients without neurologic or psychiatric illness. Immunohistochemically processed tissue reveals a continuum of NGF receptor-positive neurons located within the medial septum, vertical and horizontal limb nuclei of the diagonal band, and nucleus basalis. NGF receptor-containing neurons are also found within the bed nucleus of the stria terminalis, the anterior commissure, the internal capsule, and the internal and external medullary laminae of the globus pallidus. Virtually all (greater than 95%) NGF receptor-containing neurons colocalize with the specific cholinergic marker choline acetyltransferase (ChAT) or the nonspecific marker acetylcholinesterase (AChE). Conversely, a few cholinergic perikarya are found which are not NGF receptor positive (and vice versa). These findings demonstrate that human basal forebrain neurons on which NGF receptor immunoreactivity is detected are primarily cholinergic and analogous to the nonhuman primate Ch1-Ch4 subgroups of Mesulam et al. (J. Comp. Neurol., 214:170-197, '83). NGF receptor-containing fiber tracts are observed emanating from the medial septum and vertical limb nucleus of the diagonal band coursing medially within the fornix. Another fascicle originating mainly from the nucleus basalis and travelling within the external capsule enroute to the cortex is observed innervating all cortical layers. Comparison of NGF receptor- and ChAT-containing neurons reveals cholinergic perikarya within the striatal complex, whereas virtually no NGF receptor-containing neurons are found in these structures. An occasional displaced NGF receptor-containing neurons is seen in the ventrolateral portion of the putamen and the white matter underlying the nucleus accumbens. These data are discussed in terms of the relationship of NGF receptor- and ChAT-containing neurons within the basal forebrain and in terms of the possible functional significance of NGF in normal and diseased brain.     

Galanin-like immunoreactivity within the primate basal forebrain: differential staining patterns between humans and monkeys.

Galanin-like immunoreactivity (GAL-ir) was examined within the basal forebrain and adjacent regions of eight young adult New World monkeys (Cebus apella), one aged Old World monkey (Macaca mulatta), and eight humans without clinical or pathological evidence of neurological disease. All monkeys demonstrated similar patterns of immunoreactive profiles characterized by a continuum of GAL-ir magnocellular neurons located within the medial septum, diagonal band nuclei, and nucleus basalis. Colocalization experiments revealed that most (greater than 90%) of GAL-ir basal forebrain neurons also expressed the receptor for nerve growth factor (NGFR), an excellent marker for primate cholinergic basal forebrain neurons. A few smaller parvicellular GAL-ir neurons were also observed within the monkey basal forebrain. In contrast, identical cytochemical experiments revealed that virtually none of the magnocellular neurons within the basal forebrain of humans were GAL-ir. Rather, a network of GAL-containing fibers and terminal-like profiles were observed encompassing the magnocellular cholinergic neurons in humans. This immunohistochemical species difference does not appear to be mediated by procedural or technical factors since human brains contained numerous GAL-ir perikarya and fibers within adjacent regions including the bed nucleus of the stria terminalis and medial hypothalamus. These data demonstrate that there is a prominent phylogenetic transformation in primates with respect to the processing of GAL-mediated information. This species difference potentially relates to the severe basal forebrain degeneration reported in human dementias and illustrates the possible need for a reevaluation of the use of monkeys as an animal model of human basal forebrain-related cognitive dysfunction.     

Galanin immunoreactivity within the primate basal forebrain: Evolutionary change between monkeys and apes

Galanin immunoreactivity (GAL-ir) is differentially expressed within the basal forebrain of monkeys and humans. Most monkey magnocellular basal forebrain neurons colocalize GAL-ir. In contrast, virtually no human magnocellular basal forebrain neurons express GAL-ir. Rather, an extrinsic galaninergic fiber plexus innervates these neurons in humans. The present study examined the expression of GAL-ir within the basal forebrain of apes to establish the phylogenetic level at which this transformation occurs. The staining patterns of GAL-ir within the basal forebrain of both lesser (gibbons) and great (chimpanzee and gorilla) apes were compared to that previously observed within monkeys and humans. All apes displayed a pattern of basal forebrain GAL-ir indistinguishable from humans. GAL-ir was not expressed within ape basal forebrain magnocellular neurons as seen in monkeys. Rather like humans, a dense collection of GAL-ir fibers was seen in close apposition to magnocellular perikarya. In addition, a few GAL-ir parvicellular neurons were scattered within the ape basal forebrain. These data indicate that the evolutionary change in the expression of GAL-ir within the primate basal forebrain occurs at the branch point of monkeys and apes. © 1993 Wiley-Liss, Inc.     

The localization of nerve growth factor-like immunoreactivity in the adult rat basal forebrain and hippocampal formation.

The role of nerve growth factor (NGF) as a target derived neurotrophic agent for specific cell populations in the peripheral nervous system has been well documented and much evidence suggests that NGF may serve a similar neurotrophic role in the CNS supporting the cholinergic neurons of the basal forebrain. Previous attempts to localize NGF by immunocytochemical methods, however, have not yielded evidence confirming the regional distribution expected based upon reported levels of extractable NGF. In the present study, affinity purified polyclonal antibodies to beta-NGF and a modified immunohistochemical protocol were used to demonstrate specific NGF-like immunoreactivity in the adult rat hippocampal formation and basal forebrain. In the hippocampal formation, NGF-like immunoreactivity was localized primarily within the hilus of the dentate gyrus and within stratum lucidum of the CA3 and CA2 hippocampal subfields. Staining appeared to be associated with cell processes and was similar to the reported distribution of mossy fibers suggesting that granule cells may either serve as a primary source of hippocampal NGF or that mossy fibers selectively accumulate NGF produced by other cell populations. In the basal forebrain, NGF-like immunoreactivity was localized within neuronal cell bodies of the medial septum, diagonal band, and nucleus basalis of Meynert and was further demonstrated to colocalize exclusively with LNGF-R positive neurons. These findings demonstrate the presence of an NGF-like antigen in association with cholinergic neurons of the basal forebrain and strongly support the hypothesis that NGF may serve as an endogenous trophic factor for this adult neuronal population.     

Postmortem changes in the neuroanatomical characteristics of the primate brain: hippocampal formation

Comparative studies of the structural organization of the brain are fundamental to our understanding of human brain function. However, whereas brains of experimental animals are fixed by perfusion of a fixative through the vasculature, human or ape brains are fixed by immersion after varying postmortem intervals. Although differential treatments might affect the fundamental characteristics of the tissue, this question has not been evaluated empirically in primate brains. Monkey brains were either perfused or acquired after varying postmortem intervals before immersion-fixation in 4% paraformaldehyde. We found that the fixation method affected the neuroanatomical characteristics of the monkey hippocampal formation. Soma size was smaller in Nissl-stained, immersion-fixed tissue, although overall brain volume was larger as compared to perfusion-fixed tissue. Nonphosphorylated high-molecular-weight neurofilament immunoreactivity was lower in CA3 pyramidal neurons, dentate mossy cells, and the entorhinal cortex, whereas it was higher in the mossy fiber pathway in immersion-fixed tissue. Serotonin-immunoreactive fibers were well stained in perfused tissue but were undetectable in immersion-fixed tissue. Although regional immunoreactivity patterns for calcium-binding proteins were not affected, intracellular staining degraded with increasing postmortem intervals. Somatostatin-immunoreactive clusters of large axonal varicosities, previously reported only in humans, were observed in immersion-fixed monkey tissue. In addition, calretinin-immunoreactive multipolar neurons, previously observed only in rodents, were found in the rostral dentate gyrus in both perfused and immersion-fixed brains. In conclusion, comparative studies of the brain must evaluate the effects of fixation on the staining pattern of each marker in every structure of interest before drawing conclusions about species differences.     

Alz-50 immunoreactive neuropil differentiates hippocampal complex subfields in Alzheimer's disease

The topographic distribution of Alz-50 containing profiles was determined within the hippocampal formation and anterior parahippocampal gyrus by using a monoclonal antibody directed against the A68 protein in normal and Alzheimer's diseased (AD) brains. Although there was a paucity of immunoreactive neuropil in the normal hippocampal complex, there were a few Alz-50 positive neurons that occupied the hippocampal subfield, CA2. In most AD cases, Alz-50 immunoreactive neuropil was prominent in the outer two-thirds of the molecular layer of the dentate gyrus, although a few cases exhibited staining in the inner third of the molecular layer. CA2 was characterized by an increased density of neuropil staining within stratum pyramidale. The neuropil in subfield CA1 was stained densely with Alz-50 in strata oriens, pyramidale, and at the border between strata lacunosum-moleculare and radiatum. Alz-50 immunostained neurites occupied primarily the lateral two-thirds of the subiculum proper, whereas only sparse staining was seen in the adjacent presubiculum. Alz-50 neuropil and neuronal staining displayed three distinct laminar patterns along the mediolateral extent of the entorhinal cortex, whereas the perirhinal cortex exhibited a bilaminar pattern of immunoreactivity involving heavy staining in layers 1-3 as compared to layer 5. In general, the density of Alz-50 neurite staining in the neuropil appeared inversely proportional to the distribution of Alz-50 immunoreactivity within dendritic and somal compartments. Interestingly, the patterns of Alz-50 staining observed in the hippocampal complex in AD coincides with patterns of well-characterized afferent fiber pathways to these regions, thus further supporting the suggestion that hippocampal subfield specific pathology effectively disconnects medial temporal structures from adjacent neocortex in AD.     

Age-related decrease of nerve growth factor-like immunoreactivity in the basal forebrain of senescence-accelerated mice

The senescence-acceleratedmouse P10 (SAMP10) is a murine model of accelerated senescence characterized by the deterioration of learning and memory with advancing age. In the present study, we examined the distribution of nerve growth factor (NGF) immunohistochemically in SAMP10 mice and its control strain, SAMR1. In both strains, NGF-like immunoreactivity (NGF-IR) was observed in neurons throughout the entire forebrain and in glial cells in a particular location. In aged SAMP10 mice, each layer of the cerebral cortex retained its NGF-IR, although the thickness of the cortical mantle was markedly decreased in comparison with younger animals. There was an age-related decline in NGF-IR in the substantia innominata of SAMP10 mice at the age of 10 months, when compared to 2-month-old SAMP10. These results indicate age-related decrease of NGF in the basal forebrain in SAMP10 mice.     

Distribution of beta-nerve growth factor receptors in the human basal forebrain

The distribution of neurons expressing the receptor for beta-nerve growth factor has been examined immunohistochemically in serial coronal sections of basal forebrain from aged normal human subjects. Neurons expressing the receptor were observed in the nucleus of the diagonal band of Broca and in the anterior, the intermediate, and the posterior portions of the nucleus basalis of Meynert. Neurons could also be seen in the medial septal nucleus and embedded in myelinated fibre tracts such as those of the external capsule, cingulum, medullary laminae of the globus pallidus, ansa penduncularis, ansa lenticularis, and anterior commissure. In situ hybridization with a 35S cDNA probe to the human beta-nerve growth factor receptor confirms a neuronal location as the site of synthesis of beta-nerve growth factor receptors in the nucleus basalis of Meynert in a fifth brain. A high percentage of Nissl-stained hyperchromic magnocellular neurons expressed the receptor for beta-nerve growth factor, suggesting that most neurons in the human cholinergic magnocellular basal forebrain system express these receptors. Recent data suggest that beta-nerve growth factor functions as a neurotrophic factor in basal forebrain cholinergic neurons. In Alzheimer's disease there is known to be a reduction in cholinergic function and an apparent loss of neurons in the cholinergic nucleus basalis of Meynert. For this reason we have examined the distribution of receptors for beta-nerve growth factor in the normal human basal forebrain in order to form a basis for comparison to those with Alzheimer's disease.     

GABAergic neurons in the primate basal forebrain magnocellular complex

Hybridization histochemistry was used to detect messenger ribonucleic acid (mRNA) coding for glutamic acid decarboxylase, the synthesizing enzyme for gamma-aminobutyric acid (GABA), in neurons of the nucleus basalis of Meynert and nucleus of the diagonal band of Broca of one rhesus monkey and 4 baboons. GABAergic neurons were distributed among the unlabeled large, hyperchromic Nissl-stained neurons characteristic of this basal forebrain magnocellular complex, although they were infrequent within the dense islands of large cells. Most GABAergic cells were small to medium in size, but some were large and hyperchromic. These findings demonstrate a heterogeneous population of presumably inhibitory neurons in the basal forebrain magnocellular complex of primates.     

Nerve growth factor receptor and choline acetyltransferase colocalization in neurons within the rat forebrain: response to fimbria-fornix transection

Although it is well known that magnocellular cholinergic basal forebrain neurons are trophically responsive to nerve growth factor (NGF) and contain NGF receptors (NGFr), the exact distribution of forebrain NGFr-immunoreactive neurons and the degree to which cholinergic neurons are colocalized with them have remained in question. In this study we employed a very sensitive double-labelling method and examined in the same tissue section the distribution and cellular features of NGFr-positive and choline acetyltransferase (ChAT)-immunolabelled neurons within the rat basal forebrain. Throughout this region the majority of magnocellular basal forebrain neurons were immunoreactive for both NGFr and ChAT. However, a small percentage of neurons in the ventral portion of the vertical limb of the diagonal band of Broca were immunoreactive only for NGFr, whereas a larger population of magnocellular neurons in the substantia innominata exhibited only ChAT immunoreactivity. No NGFr-immunoreactive cells were found associated with ChAT-positive neurons in the striatum, neocortex, or hippocampus, and no single-labelled NGFr-immunoreactive neurons were found outside the basal forebrain area, except for a large number of positive-labelled cells along the ventricular walls of the third ventricle. In addition to its function in maintaining the normal integrity of the basal forebrain and cholinergic, peripheral sympathetic, and neural-crest-derived sensory neurons, NGF may also have a role in the growth of these neurons after damage to the nervous system. To examine this postulate the hippocampus was denervated of its septal input and examined 8 weeks later. Two populations of neurons were found to have undergone collateral sprouting--namely, the midline magnocellular cholinergic neurons of the dorsal hippocampus and the sympathetic noradrenergic neurons of the superior cervical ganglion. Both of these neuronal populations also stained strongly for NGFr. In contrast, the small intrinsic cholinergic neurons of the hippocampus exhibited neither sprouting response nor staining for NGFr. In view of these results, we suggest that the differing sprouting responses demonstrated by these three neuronal populations may be due to their responsiveness to NGF, as indicated by the presence or absence of NGF receptors.     

Nerve growth factor increases choline acetyltransferase activity in developing basal forebrain neurons

Nerve growth factor (NGF) is a neuronotrophic protein. Its effects on developing peripheral sensory and sympathetic neurons have been extensively characterized, but it is not clear whether NGF plays a role during the development of central nervous system neurons. To address this point, we examined the effect of NGF on the activity of neurotransmitter enzymes in several brain regions. Intracerebroventricular injections of highly purified mouse NGF had a marked effect on the activity of choline acetyltransferase (ChAT), a selective marker of cholinergic neurons. NGF elicited prominent increases in ChAT activity in the basal forebrain of neonatal rats, including the septum and a region which contains neurons of the nucleus basalis and substantia innominata. NGF also increased ChAT activity in the hippocampus and neocortex, terminal regions for the fibers of basal forebrain cholinergic neurons. In analogy with the response of developing peripheral neurons, the NGF effect was shown to be selective for basal forebrain cholinergic cells and to be dose-dependent. Furthermore, septal neurons closely resembled sympathetic neurons in the time course of their response to NGF. These observations suggest that endogenous NGF does play a role in the development of basal forebrain cholinergic neurons.     

Cells containing immunoreactive estrogen receptor-alpha in the human basal forebrain

The distribution of estrogen receptor protein-alpha (ER-alpha)-containing cells in the human hypothalamus and adjacent regions was studied using a monoclonal antibody (H222) raised against ER-alpha derived from MCF-7 human breast cancer cells. Reaction product was found in restricted populations of neurons and astrocyte-like cells. Neurons immunoreactive for ER-alpha were diffusely distributed within the basal forebrain and preoptic area, infundibular region, central hypothalamus, basal ganglia and amygdala. Immunoreactive astrocyte-like cells were noted within specific brain regions, including the lamina terminalis and subependymal peri-third-ventricular region. These data are consistent with the location of estrogen receptors in the basal forebrain of other species and the known effects of estrogens on the cellular functions of both neurons and supporting elements within the human hypothalamus and basal forebrain.     

The cortical relationships of certain basal ganglia and the cholinergic basal forebrain nuclei

Retrograde cellular degeneration has been found in the basal nucleus of Meynert in macaque monkeys after large lesions of the neocortex, and in the human brain after either hemidecortication or leucotomy. These observations may be relevant to the interpretation of the cellular degeneration in the basal nucleus in Alzheimer's disease.     

Neuronal responses related to reinforcement in the primate basal forebrain

In the present study neurones recorded in the substantia innominata, the diagonal band of Broca and a periventricular region of the basal forebrain responded differentially to stimuli signalling the availability of fruit juice or saline obtained by making lick responses in two different visual discrimination tasks. The activity of certain neurones reflected the rewarding nature of stimuli used to signal the availability of juice in the tasks, responding to the sight and delivery of both foods and syringes used to deliver juice in tests in which behavioural responses were irrelevant. The activity of other neurones reflected aversion, responding to task stimuli signalling availability of saline and to syringes used to deliver saline to the mouth. In another task an auditory cue that signalled the availability of juice elicited neuronal responses. These neurones also responded to a tone cue used to signal the onset of the trial, and during certain mouth and arm movements which the monkey used to obtain reinforcement. The responses of these differential neurones were similar in most respects in all 3 regions of the basal forebrain. Thus these neurones respond to a range of visual and auditory stimuli that monkeys have learned can be used to obtain reinforcement, but not on the basis of sensory properties such as shape or colour of the stimuli. We conclude that the reinforcement-related nature of the neuronal signal from the basal forebrain could be used to facilitate processing in cortical regions, optimising the functioning of sensory, motor and association cortices, thus increasing the probability of responding appropriately to learned environmental contingencies. We suggest that the properties of these neurones are due to afferent inputs from ventromedial regions of the prefrontal and temporal cortices and amygdala.     

Tyrosine-hydroxylase-containing neurons in the primate basal forebrain magnocellular complex.

Immunocytochemistry and in situ hybridization for tyrosine hydroxylase (TH) were used to study the distribution of putative catecholaminergic neurons in the basal forebrain magnocellular complex (BFMC) of monkeys and humans. Magnocellular TH-expressing neurons in the primate BFMC are distributed along a rostrocaudal gradient, with the largest proportion of these cells located in the medial septal nucleus and nucleus of the diagonal band of Broca; smaller TH-containing neurons generally follow the same distribution. These findings suggest that, within rostromedial segments of the BFMC, there is a distinct subpopulation of neurons that express catecholamine-synthesizing enzymes. Further research is necessary to establish whether these neurons utilize one or more catecholamines as neurotransmitters.     

Nerve growth factor receptor-mediated transport from cerebrospinal fluid to basal forebrain neurons

Recent data indicate that the neurons of the cholinergic basal forebrain (CBF) respond to nerve growth factor (NGF) with increased survival under experimental conditions and have NGF receptors which mediate the binding and retrograde transport of NGF from axon terminals to somata. Focal intraparenchymal injections of retrograde tracing agents into neuropil demonstrate that the distribution of axons from cholinergic nuclei to cortex and hippocampus is topographically restricted and largely ipsilateral. Monoclonal antibody 192, a well-characterized antibody which recognizes only the rat NGF receptor, was labelled with 125I and injected into a lateral ventricle of adult rats. Highly specific bilateral transport to numerous neurons of the CBF system was demonstrated by autoradiography. This result directly demonstrates that suitably targeted antibodies can be taken up by specific neuronal populations following intraventricular injection and implies that CBF neurons may be influenced by relatively high molecular weight substances injected into cerebrospinal fluid.     

Calretinin immunoreactive structures in the human hippocampal formation

The calcium-binding protein calretinin is present in an intrinsic GABAergic and an extrinsic non-GABAergic system in the rat and monkey hippocampal formation. Important species differences have been noted in hippocampal cell types immunostained for calretinin and the termination pattern of calretinin containing hypothalamic afferents in the hippocampus. In the present study, calretinin-containing neurons were visualized using immunocytochemistry in the human hippocampal formation of individuals which showed no significant neuropathological alterations. Calretinin-immunoreactivity was present exclusively in non-granule cells of the dentate gyrus and in non-pyramidal cells of Ammon's horn. Calretinin-positive neurons were found most frequently in the hilus of the fascia dentata and in strate radiatum and lacunosum-moleculare of CA1, whereas neurons in CA2 and CA3 were rarely immunostained. The majority of calretinin-immunoreactive neurons were small, bipolar or fusiform neurons. The dendritic trees of the calretinin-positive neurons were, for the most part, parallel to the dendrites of the principal cells. In the hilus, however, we observed cells with dendrites restricted to the hilar area. These dendrites were parallel to the granule cell layer. In the stratum lacunosum-moleculare, neurons with dendrites oriented parallel to the hippocampal fissure were frequently detected. In general, dendrites were smooth or sparsely spiny, displaying small conventional spines. The axons usually emerged from the proximal dendrite and could be followed over long distances. Axons were thin, had small varicosities and displayed only few collaterals which branched relatively far away from the cell body. Distinct bands of darkly stained calretinin-positive fibers occupied the innermost portion of the dentate molecular layer and the pyramidal cell layer of CA2. This distribution of calretinin-immunoreactive structures in the human hippocampus is similar to that observed in other primates but differs from that described in lower mammals, i.e., the rat. Our findings suggest that primates may share a common hippocampal calrtinin-containing system, presumably both the intrinsic GABAergic and the extrinsic hypothalamic non-GABAergic components. © 1995 Wiley-Liss, Inc.     

Nerve growth factor induces Fos-like immunoreactivity within identified cholinergic neurons in the adult rat basal forebrain

Immunocytochemical techniques were used to examine and compare the effects of intracerebroventricular administration of nerve growth factor (NGF) on Fos expression within identified cholinergic and non-cholinergic neurons located in different regions of the adult rat basal forebrain. Animals were killed 1, 3, 6, and 12 h after receiving NGF (0.5 or 5.0 μg) or vehicle into the left lateral ventricle and sections through the medial septum, diagonal band of Broca, nucleus basalis magnocellularis, and striatum were processed for the combined immunocytochemical detection of Fos and choline acetyltransferase (a marker for cholinergic neurons), or Fos and parvalbumin (a marker for gamma aminobutyric acid (GABA)-containing neurons). NGF produced a significant increase in the percentage of cholinergic neurons containing Fos-like immunoreactivity within all four regions examined. The largest increases were detected in the medial septum (47.8%) and the horizontal limb of the diagonal band of Broca (67.7%). In these areas, NGF-mediated induction of Fos-like immunoreactivity was detected as early as 3 h, peaked at 6 h, and was reduced by 12 h, postinfusion. Small but significant increases in the percentage of cholinergic neurons containing Fos-like immunoreactivity were also detected in the striatum (4.2%) and in the nucleus basalis magnocellularis (19.2%) 3–12 h following administration of the higher dose of NGF. No evidence for an NGF-mediated induction of Fos within parvalbumin-containing neurons was detected in any of the four regions at any of the time-points examined; however, evidence for an NGF-mediated induction of Fos within epithelial cells lining the lateral ventricle was observed. These data demonstrate that NGF induces Fos expression within cholinergic, and not parvalbumin-containing (GABAergic), neurons in the basal forebrain, and furthermore that intracerebroventricular administration of NGF influences the different subgroups of basal forebrain cholinergic neurons to different degrees. ©1977 Elsevier Science B.V. All rights reserved.