Peptidergic neurons in the basal forebrain magnocellular complex of the rhesus monkey

The basal forebrain magnocellular complex of primates is defined by the presence of large, hyperchromic, usually cholinergic neurons in the nucleus basalis of Meynert and nucleus of the diagonal band of Broca. Because there is growing evidence for noncholinergic neuronal elements in the basal forebrain complex, five neuropeptides and the enzyme choline acetyltransferase were studied immunocytochemically in this region of rhesus monkeys. Galaninlike immunoreactivity coexists with choline-acetyl-transferase-like immunoreactivity in most large neurons and in some smaller neurons of the primate nucleus basalis and nucleus of the diagnonal band. Four other peptides show immunoreactivity in more limited regions of the basal forebrain complex, usually in separate smaller, noncholinergic neurons. Numerous small, somatostatinlike-immunoreactive neurons occupy primarily anterior and intermediate segments of the nucleus basalis, especially laterally and ventrally. Somewhat fewer, small neuropeptide Y-like-immunoreactive somata are found in the same regions. Neurons that show neurotensinlike immunoreactivity are slightly larger than cells that contain immunoreactivity for somatostatin or neuropeptide Y, but these neurons also occur mainly in anterior and intermediate parts of the nucleus basalis. Overall, the usually small, leucine-enkephalin-like-immunoreactive neurons are infrequent in the basal forebrain complex and are most abundant in the rostral intermediate nucleus basalis. Thus, neurons that appear to contain somatostatin, neuropeptide Y, neurotensin, or enkephalin mingle with cholinergic/galaninergic neurons only in some subdivisions of the nucleus basalis/nucleus of the diagonal band, and their distributions suggest that some of these small neurons could be associated with structures that overlap with cholinergic neurons of the labyrinthine basal forebrain magnocellular complex. We also have found light microscopic evidence for innervation of basal forebrain cholinergic neurons by boutons that contain galanin-, somatostatin-, neuropeptide Y-, neurotensin-, or enkephalinlike immunoreactivity. The origins and functions of these putative synapses remain to be determined.     

Noncollateral projections of basal forebrain neurons to frontal and parietal neocortex in primates

To test the hypothesis that axons of the basal forebrain cholinergic system collateralize to innervate widely separated areas of cortex, two distinct, retrogradely transported fluorescent dyes were injected into discrete neocortical regions of three macaques. In two monkeys, True Blue was injected into parietal cortex and Nuclear Yellow into frontal cortex; in a third monkey, placement of the dyes was reversed. Following these large (3-10 microliters total) injections, neurons single labeled with either Nuclear Yellow or True Blue were seen throughout most of the ipsilateral nucleus basalis of Meynert and nucleus of the diagonal band of Broca. Neurons projecting to either frontal or parietal cortex were most heavily concentrated in the anteromedial aspect of the basal forebrain. A small number of labeled neurons was also seen in the contralateral basal forebrain. Cells single labeled with either True Blue or Nuclear Yellow were frequently adjacent to one another, but in no case was a neuron labeled with both dyes. Thus, individual neurons of the basal forebrain complex do not appear to innervate both frontal and parietal lobes of monkeys. This finding is consistent with recent studies in rodents which suggest that basal forebrain neurons innervate relatively small, restricted cortical fields.     

Nerve growth factor mRNA-containing cells are distributed within regions of cholinergic neurons in the rat basal forebrain

It has been proposed that nerve growth factor (NGF) provides critical trophic support for the cholinergic neurons of the basal forebrain and that it becomes available to these neurons by retrograde transport from distant forebrain targets. However, neurochemical studies have detected low levels of NGF mRNA within basal forebrain areas of normal and experimental animals, thus suggesting that some NGF synthesis may actually occur within the region of the responsive cholinergic cells. In the present study with in situ hybridization and immunohistochemical techniques, the distribution of cells containing NGF mRNA within basal forebrain was compared with the distribution of cholinergic perikarya. The localization of NGF mRNA was examined by using a ³⁵S-labeled RNA probe complementary to rat preproNGF mRNA and emulsion autoradiography. Hybridization of the NGF cRNA labeled a large number of cells within the anterior olfactory nucleus and the piriform cortex as well as neurons in a continuous zone spanning the lateral aspects of both the horizontal limb of the diagonal band of Broca and the magnocellular preoptic nucleus. In the latter regions, large autoradiographic grain clusters labeled relatively large Nissl-pale nuclei; it did not appear that glial cells were autoradiographically labeled. Comparison of adjacent tissue sections processed for in situ hybridization to NGF mRNA and immunohistochemical localization of choline acetyltransferase (ChAT) demonstrated overlapping fields of cRNA-labeled neurons and ChAT-immunoreactive perikarya in both the horizontal limb of the diagonal band and magnocellular preoptic regions. However, no hybridization of the cRNA probe was observed in other principal cholinergic regions including the medial septum, the vertical limb of the diagonal band, or the nucleus basalis of Meynert. These results provide evidence for the synthesis of NGF mRNA by neurons within select fields of NGF-responsive cholinergic cells and suggest that the generally accepted view of “distant” target-derived neurotrophic support should be reconsidered and broadened.     

Apoptotic signals within the basal forebrain cholinergic neurons in Alzheimer's disease

A relatively early and substantial loss of basal forebrain cholinergic neurons is a constant feature of Alzheimer's disease (AD). However, the mechanisms that contribute to the selective vulnerability of these neurons are not fully delineated. In the present series of experiments, we determined the possible contribution of apoptotic processes and other pathologic cascades to the degeneration of the cholinergic neurons of the nucleus basalis of Meynert (NBM) in AD. In contrast to neurons in the frontal cortex which showed prominent DNA fragmentation as detected by the TUNEL method, no DNA fragmentation was observed within the NBM in any of the AD or normal brains. Similarly, immunoreactivity for the apoptotic signals Fas, Fas-ligand, Bax, Bcl-x, caspase-8, caspase-9 and caspase-3 was absent from the NBM of AD and control brains. In contrast, a substantial subpopulation of cholinergic neurons within the NBM in AD displayed prominent immunoreactivity for the apoptotic signal Fas-associated death domain (FADD) in the form of tangles. FADD immunoreactivity was also present in dystrophic neurites. FADD-positive tangle-like structures were localized in neurons which contained immunoreactivity for the cholinergic marker choline acetyltransferase (ChAT) and the low affinity neurotrophin receptor p75NTR. While many of the NBM cholinergic neurons in control brains contained immunoreactivity for the calcium binding protein calbindin-D28K (CB), the NBM neurons in AD displayed a substantial loss of CB immunoreactivity. Importantly, most of FADD-immunoreactive cholinergic neurons were devoid of CB immunoreactivity, and, conversely, most CB-positive cholinergic neurons had no FADD immunoreactivity. FADD immunoreactivity within the basal forebrain was colocalized with phosphorylated tau immunoreactive tangles and dystrophic neurites. In contrast, FADD immunoreactivity did not appear to be related to the primarily diffuse amyloid-beta deposits intermingled between cholinergic neurons in AD NBM. Finally, many CD68-positive microglia were observed surrounding the NBM cholinergic neurons in AD. In conclusion, the findings of the present study indicate that, while the FADD apoptotic signaling pathway may be triggered within the basal forebrain cholinergic neurons in AD, the apoptotic cascade is most likely aborted as no DNA fragmentation was detected and the executioner caspase-3 was not up-regulated within these neurons. The findings also suggest possible relationships between loss of CB, FADD expression and phosphorylation of tau within the basal forebrain cholinergic neurons in AD.     

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.     

Galanin-immunoreactive synaptic terminals on basal forebrain cholinergic neurons in the rat

Previous studies have indicated that galanin is one of the most abundant peptides in the basal forebrain and that it has a significant modulatory influence on cholinergic transmission. The aim of the present study was to use a light electron microscopic correlation technique to determine whether galanin-immunoreactive terminals form synaptic contacts with basal forebrain cholinergic cells of the rat. Sections from fixed-perfused brains were stained at the light and electron microscopic levels for galanin and choline acetyltransferase immunoreactivity in the same section by using a dual-colour immunohistochemical method. The results showed that galanin-immunoreactive axonal terminals are unevenly distributed in the medial septal nucleus, the diagonal band, and the nucleus basalis. Galanin-positive synapses were most prominent on choline acetyltransferase-positive neurons in the lateral parts of the nucleus of the diagonal band and in the posterior half of the nucleus basalis, which is where there was the greatest overlap between the distribution of galanin-immunoreactive terminals and choline acetyltransferase-positive neurons. The origins of these galanin-positive terminals are not known, but the results confirm that the basal forebrain galaninergic system has a synaptic influence on basal forebrain cholinergic neurons in the rat. J. Comp. Neurol. 383:82–93, 1997. © 1997 Wiley-Liss, Inc.     

Expression of estrogen receptor-like immunoreactivity by different subgroups of basal forebrain cholinergic neurons in gonadectomized male and female rats

Recent studies have demonstrated that estrogen administration can produce significant increases in relative levels of choline acetyltransferase (ChAT) mRNA and protein in specific regions of the female, but not the male, rat basal forebrain. In the present study immunocytochemical techniques were used to identify and compare relative numbers of cholinergic neurons containing estrogen receptors within the medial septum, horizontal limb of the diagonal band of Broca, nucleus basalis magnocellularis, and striatum of gonadectomized male and female rats to determine whether there are differences in the percentage of cholinergic neurons expressing estrogen receptors which might contribute to the different regional- and sex-specific effects of estrogen which have been described. Counts of choline acetyltransferase-immunoreactive cells revealed significant regional differences in the average number of cholinergic neurons/section; however, no difference between males and females in the numbers of cholinergic neurons in each of the four regions analyzed was observed. Fifty to eighty percent of the cholinergic neurons detected in both males and females contained estrogen receptor-like immunoreactivity. A small but significant difference between males and females was detected with females having slightly more (10.5%) double-labeled cells than males overall. Individual comparisons revealed that significantly more (18–33%) double-labeled cells were detected in the horizontal limb of the diagonal band, but not in the medial septum, nucleus basalis, or striatum of females vs. males. There was also a small but significant regional difference in the percentage of double-labeled cells with the highest percentage (74.2%) detected in the striatum and the lowest percentage (63.4%) detected in the horizontal limb. None of these differences appear to account for the regional- and sex-specific effects of estrogen on cholinergic neurons which have been observed. We conclude that differences in the effects of estrogen on cholinergic neurons in males vs. females and in different Subregions of the female basal forebrain are not due to differences in the percentage of cholinergic neurons expressing estrogen receptors.     

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.     

Sex differences in androgen receptor immunoreactivity in basal forebrain nuclei of elderly and Alzheimer patients

The vertical limb of the diagonal band of Broca (VDB or Ch2) and the nucleus basalis of Meynert (NBM or Ch4) are major cholinergic nuclei of the human basal forebrain, a complex that is affected in Alzheimer's disease (AD). Sex hormones influence the function of these cholinergic neurons in animals and humans and we showed earlier that estrogen and androgen receptors (AR) are present in both the VDB and the NBM of young patients of 20-39 years of age. The aim of the present study was to investigate whether AR expression changes in relation to aging and AD. In both brain areas of male and female patients over the age of 56 nuclear staining had almost disappeared and cytoplasmic AR expression was decreased. This decrease was most pronounced in the VDB of men. In addition, the proportion of neurons showing cytoplasmic AR expression was higher in control aged women than in control aged men in both the VDB and the NBM. Surprisingly, cytoplasmic ARs were significantly decreased in the VDB and the NBM only in AD women and not in AD men. These observations suggest the possible involvement of androgens in the functional changes of the basal forebrain nuclei in aging and AD.     

Regulation of regional cerebral blood flow by cholinergic fibers originating in the basal forebrain

The intracranial neural vasodilative system of cholinergic fibers projecting from the basal forebrain to the cortex was discovered by Biesold, Inanami, Sato and Sato (Biesold, D., Inanami, O., Sato, A., Sato, Y., 1989. Stimulation of the nucleus basalis of Meynert increases cerebral cortical blood flow in rats. Neurosci. Lett. 98, 39-44) using laser Doppler flowmetry in anesthetized rats. This cholinergic vasodilative system, which operates by increasing extracellular ACh release, relies upon activation of both muscarinic and nicotinic cholinergic receptors in the parenchyma of the cortex. Further, the involvement of nitric oxide in this cholinergic vasodilation, indicates the necessity to this system of neurons, which contain nitric oxide synthase. The increase in cortical blood flow elicited by this cholinergic vasodilative system is independent of systemic blood pressure and is not coupled to cortical metabolic rates. This cholinergic vasodilative system may be activated by somatic afferent stimulation. Most of the data presented here were obtained in anesthetized animals.     

Lesions of cortical GABAergic interneurons and acetylcholine neurons in xeroderma pigmentosum group A

Xeroderma pigmentosum (XP) is a rare genetic disorder caused by inherited disturbances in the nucleotide excision repair system; patients with XP groups A (XP-A), B, D, and G were shown to have progressive neurological disturbances. Particularly, XP-A patients, which account for approximately half of Japanese XP patients, show severe neurological disorders, including mental retardation and epilepsy. Herein, we performed an immunohistochemical analysis of the number of GABAergic interneurons (GABAis), including calbindin-D28K, parvalbumin, and calretinin, in the cerebral cortex and acetylcholinergic neurons (AchNs) in the nucleus basalis of Meynert (NM) and in the pedunculopontine tegmental nucleus (PPN) in six autopsy cases of XP-A in order to investigate the relationships between mental dysfunction and GABAis and AchNs. The density and percentages of neurons that were immunoreactive for calbindin-D28K and parvalbumin were significantly reduced in the frontal and temporal cortices in XP-A cases, although the density of neurons that were immunoreactive for MAP2 did not differ from that in controls. Additionally, XP-A cases showed reduced AchNs in both the NM and the PPN. The observed reductions of cortical GABAis and AchNs may be involved in the mental disturbances, the higher occurrence of epilepsy, and/or the abnormalities in rapid eye movement sleep in patients with XP-A.     

The cholinergic system in the basal forebrain of the Atlantic white‐sided dolphin (Lagenorhynchus acutus)

The basal forebrain (BFB) cholinergic neurotransmitter system is important in a number of brain functions including attention, memory, and the sleep‐wake cycle. The size of this region has been linked to the increase in encephalization of the brain in a number of species. Cetaceans, particularly those belonging to the family Delphinidae, have a relatively large brain compared to its body size and it is expected that the cholinergic BFB in the dolphin would be a prominent feature. However, this has not yet been explored in detail. This study examines and maps the neuroanatomy and cholinergic chemoarchitecture of the BFB in the Atlantic white‐sided dolphin (Lagenorhynchus acutus). As in some other mammals, the BFB in this species is a prominent structure along the medioventral surface of the brain. The parcellation and distribution of cholinergic neural elements of the dolphin BFB was comparable to that observed in other mammals in that it has a medial septal nucleus, a nucleus of the vertical limb of the diagonal band of Broca, a nucleus of the horizontal limb of the diagonal band of Broca, and a nucleus basalis of Meynert. The observed BFB cholinergic system of this dolphin is consistent with evolutionarily conserved and important functions for survival.     

Impairment of semantic memory after basal forebrain and fornix lesion

Semantic memory impairment is classically associated with lesion of the anterior temporal lobe. We report the case of a patient with severe semantic knowledge impairment and anterograde amnesia after bilateral ischemic lesion of the fornix and of the basal forebrain following surgical clipping of an aneurysm of the anterior communicating artery. Fluorodeoxyglucose positron emission tomography (FDG-PET) showed a temporal hypometabolism. Severe semantic impairment is a rare complication after rupture of an anterior communicating artery aneurysm and may result from disconnection of the temporal lobe.     

Galanin mRNA in the nucleus basalis of Meynert complex of baboons and humans.

Galanin, a 29-amino acid peptide, has been shown by immunocytochemistry to occur in most large acetylcholinergic neurons of the complex that includes the nucleus basalis of Meynert and the nucleus of the diagonal band of Broca in nonhuman primates. In contrast, several studies have reported that most large neurons of the human nucleus basalis of Meynert complex appear to lack galanin immunoreactivity. We investigated this apparent species-difference by hybridization histochemistry for galanin messenger ribonucleic acid (mRNA) in humans and baboons. The results confirm previous immunocytochemical data; very few large neurons of the nucleus basalis of Meynert complex in humans contained detectable galanin messenger RNA, whereas most such cells in baboons were labeled by the oligodeoxynucleotide probe. The few labeled neurons in humans were primarily medial or ventral to the main body of the nucleus basalis of Meynert and corresponded in location to a minor population of relatively intensely labeled cells in baboons. These findings indicate that the indetectability of immunoreactive galanin in most cells of the nucleus basalis of Meynert complex in humans is due to a paucity or an absence of galanin messenger RNA and not to differences in posttranslational processing or transport of the peptide. Inasmuch as the probe labeled neurons in several other nuclei of both species, it is unlikely that differences in galanin messenger RNA sequences underlie the species-related disparity in hybridization in the nucleus basalis of Meynert complex. The indetectability of galanin messenger RNA in most cells of the human nucleus basalis of Meynert complex indicates that the expression of the galanin gene is regulated by as yet unidentified influences that differ in human and nonhuman primates. The varying phenotypes of galanin in primates suggest potentially important species-differences in the function of galanin in neurons of the nucleus basalis of Meynert complex.     

Fucoidan inhibits cellular and neurotoxic effects of beta-amyloid (A beta) in rat cholinergic basal forebrain neurons

The deposition of beta-amyloid protein (A beta), a 39-43 amino acid peptide, in the brain and a loss of cholinergic neurons in the basal forebrain are pathological hallmarks of Alzheimer's disease (AD). Seaweeds consumed in Asia contain Fucoidan, a sulfated polysaccharide. Fucoidan has been known to exhibit various biological actions, such as an anti-inflammatory and antioxidant action. In this study, using whole-cell patch clamp recordings we examined the effects of Fucoidan on A beta-induced whole-cell currents in acutely dissociated rat basal forebrain neurons. We further investigated whether Fucoidan is capable of blocking A beta neurotoxicity in primary neuronal cultures. In dissociated cells, bath application of A beta(25-35) (1 microM) caused a reduction of the whole-cell currents by 16%. Fucoidan, in a dose-dependent manner, blocks the A beta(25-35) reduction of whole-cell currents. Exposure of A beta(25-35) (20 microM) or A beta(1-42) (20 microM) to rat cholinergic basal forebrain cultures for 48 h resulted in 40-60% neuronal death, which was significantly decreased by pretreatment of cultures with Fucoidan (0.1-1.0 microM). Fucoidan also attenuated A beta-induced down-regulation of phosphorylated protein kinase C. A beta(1-42)-induced generation of reactive oxygen species was blocked by prior exposure of cultures to Fucoidan. Furthermore, A beta activation of caspases 9 and 3, which are signaling pathways implicated in apoptotic cell death, is blocked by pretreatment of cultures with Fucoidan. These results show that Fucoidan is able to block A beta-induced reduction in whole-cell currents in basal forebrain neurons and has neuroprotective effects against A beta-induced neurotoxicity in basal forebrain neuronal cultures.     

GABAergic basal forebrain neurons that express receptor for neurokinin B and send axons to the cerebral cortex

Neurons expressing neurokinin B (NK3) receptor in the basal forebrain region of rats were characterized histochemically by combining immunocytochemistry, in situ hybridization and retrograde labeling, and electrophysiologically by whole-cell clamp recording. NK3 receptor-immunoreactive neurons were found in the basal forebrain region including the substantia innominata, where axon terminals immunoreactive for preprotachykinin B, the precursor peptide of neurokinin B (NKB), were densely distributed. More than 90% of NK3 receptor-expressing neurons in the basal forebrain region showed signals for glutamate decarboxylase mRNA, indicating that almost all NK3 receptor-expressing neurons were gamma-aminobutyric acid (GABA)ergic neurons. On the other hand, only a few NK3 receptor-immunoreactive neurons showed immunoreactivity for choline acetyltransferase or parvalbumin in the substantia innominata, ventral pallidum, and globus pallidus, although the distribution of NK3 receptor-expressing neurons overlapped with those of cholinergic neurons and parvalbumin-positive neurons. After injection of wheat germ agglutinin into the cerebral cortex, NK3 receptor immunoreactivity was detected in about 25% of retrogradely labeled basal forebrain neurons, indicating that NK3 receptor-expressing neurons send projection fibers to the cerebral cortex. In the whole-cell clamp recording study, a selective NK3 receptor agonist evoked membrane depolarization or inward currents with decrease of input impedance in 10 of 100 cortically projecting neurons recorded in the basal forebrain region. Because NKB-producing striatal neurons send axons selectively to the basal forebrain region, the present results suggest that the release of NKB by those striatal neurons induces an inhibitory effect on cortical neurons via facilitation of GABAergic basal forebrain neurons expressing NK3 receptor.     

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.     

Direct, complex effects of estrogens on basal forebrain cholinergic neurons

Although controversial, estrogens remain one of the few agents purported to influence the incidence of Alzheimer's disease and one of their postulated mechanisms of action is their effects on basal forebrain cholinergic neurons. However, it is unclear whether the responses of cholinergic neurons to estrogens are direct or mediated via the retrograde influences of neurotrophins, known to be induced by estrogens in the hippocampus and neocortex. In the present study, we explore the issue of the primary site of action of estrogens by studying the regulation of expression of genes that characterize mature cholinergic neurons, i.e., choline acetyltransferase, trkA, and p75(NTR) in the medial septum and the nucleus basalis complex. In parallel, we study the hippocampal expression of NGF, BDNF, and NT-3, i.e., neurotrophins with known trophic roles on cholinergic neurons. Gene expression is studied by RT-PCR in ovariectomized female rats with and without estrogen supplementation within the physiological estradiol range and in rats with complete fimbria-fornix transactions treated with estrogen or vehicle. To clarify mechanisms of estrogen transduction in cholinergic neurons, we study the effects of estrogen treatment on fimbria-fornix-lesioned mice with genetic ablations of ER subtypes alpha and beta. The results of the present study suggest that, while estrogens do regulate BDNF expression in the hippocampus and neocortex, they also exert stimulatory non-trophic effects on basal forebrain cholinergic neurons, primarily on ChAT expression. Cholinergic neurons retain their ability to respond to estrogens after their complete separation from the hippocampus. The elimination of ERalpha alters significantly the phenotypic responsiveness of cholinergic neurons to estrogens, whereas elimination of ERbeta appears to have no effect. Our findings support the idea that estrogens directly enhance cholinergic neuron function and that ERalpha plays a significant role in transducing these regulatory effects.