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.     

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.     

Nerve growth factor response to excitotoxic lesion of the cholinergic basal forebrain is slightly impaired in aged rats

Nerve growth factor (NGF) promotes survival and function of basal forebrain cholinergic neurons. We studied NGF and choline acetyltransferase (ChAT) activity after partial quisqualic acid induced lesions of the basal forebrain in 3 and 27 months-old rats, in order to investigate whether NGF-related regeneration is disturbed in old age. 2 weeks post lesion, ChAT activity decreased by 25 to 32% in adult and old rats. 3 months post lesion, the ChAT deficit receded in adult rats, but remained unchanged in old rats. 2 weeks post lesion, NGF levels were reduced by 36 to 44%, but there was no significant difference between adult and old rats. 3 months post lesion, we found increased NGF levels by 44% in the posterior cortex of adult rats. These results indicate that the compensatory NGF increase in the posterior cortex after partial cholinergic lesion of the basal forebrain is slightly impaired in old age.     

Acidic fibroblast growth factor mRNA is expressed by basal forebrain and striatal cholinergic neurons

Evidence for the importance of the basal forebrain cholinergic system in the maintenance of cognitive function has stimulated efforts to identify trophic mechanisms that protect this cell population from atrophy and dysfunction associated with aging and disease. Acidic fibroblast growth factor (aFGF) has been reported to support cholinergic neuronal survival and has been localized in basal forebrain with the use of immunohistochemical techniques. Although these data indicate that aFGF is present in regions containing cholinergic cell bodies, the actual site of synthesis of this factor has yet to be determined. In the present study, in situ hybridization techniques were used to evaluate the distribution and possible colocalization of mRNAs for aFGF and the cholinergic neuron marker choline acetyltransferase (ChAT) in basal forebrain and striatum. In single-labeling preparations, aFGF mRNA-containing neurons were found to be codistributed with ChAT mRNA⁺ cells throughout all fields of basal forebrain, including the medial septum/diagonal band complex and striatum. By using a double-labeling (colormetric and isotopic) technique, high levels of colocalization (over 85%) of aFGF and ChAT mRNAs were observed in the medial septum, the diagonal bands of Broca, the magnocellular preoptic area, and the nucleus basalis of Meynert. The degree of colocalization was lower in the striatum, with 64% of the cholinergic cells in the caudate and 33% in the ventral striatum and olfactory tubercle labeled by the aFGF cRNA. These data demonstrate substantial regionally specific patterns of colocalization and support the hypothesis that, via an autocrine mechanism, aFGF provides local trophic support for cholinergic neurons in the basal forebrain and the striatum. © 1996 Wiley-Liss, Inc.     

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.     

Nerve growth factor induces galanin gene expression in the rat basal forebrain: Implications for the treatment of cholinergic dysfunction

Nerve growth factor (NGF) is a potential treatment for cholinergic dysfunction associated with Alzheimer's disease (AD). In rats, NGF activates gene expression of the acetylcholine synthetic enzyme choline acetyltransferase (ChAT) and prevents age- and lesion-induced degeneration of basal forebrain (BF) cholinergic neurons. Cholinergic neurons in the BF coexpress galanin (GAL), a neuropeptide that has been shown to impair performance on memory tasks possibly through the inhibition of cholinergic memory pathways. NGF up-regulates both ChAT and GAL gene expression in cultured pheochromocytoma cells; however, the effect of chronic in vivo NGF administration on GAL gene expression within the BF has not been studied. We used in situ hybridization and quantitative autoradiography to assess GAL and ChAT gene expression within the BF of adult male rats following chronic intracerebroventricular infusion of NGF or cytochrome c. We now report that, in addition to stimulating ChAT gene expression, NGF strongly up-regulated the GAL gene in the rat cholinergic BF. NGF had no effect on GAL gene expression in other noncholinergic forebrain regions. NGF induction of GAL gene expression in the BF was specific, because gene expression for another neuropeptide, neurotensin, present within noncholinergic BF neurons was unchanged. Our data provide the first evidence that in vivo NGF administration up-regulates GAL gene expression in the cholinergic BF. These results suggest that the concurrent induction of GAL in the BF could limit the ameliorating actions of NGF on cholinergic dysfunction. J. Comp. Neurol. 379;563–570, 1997. © 1997 Wiley-Liss, Inc.     

Nerve growth factor receptor immunoreactivity in the nonhuman primate (Cebus apella): distribution, morphology, and colocalization with cholinergic enzymes

A monoclonal antibody raised against the receptor for nerve growth factor (NGF) was used to examine the distribution and morphology of NGF receptor-containing neurons within the central nervous system of Cebus apella monkeys. Most somata demonstrating positive immunoreactivity were localized within the Ch1-4 regions of the basal forebrain. Neurons in the Ch1 region displayed morphological features typical of cholinergic medial septal neurons. These perikarya were primarily vertically oriented (40-50 micron along the vertical axis) with both apical and basal neuritic processes. Magnocellular (40-50 micron) neurons within the Ch2 (vertical limb of the diagonal band), Ch3 (horizontal limb of the diagonal band) and Ch4 (nucleus basalis of Meynert) regions were multipolar and had rounded perikarya that often displayed an eccentric nucleus. Fibers presumably originating from the Ch1-2 regions were observed throughout the fimbria-fornix system and were found to terminate preferentially within the CA1 and CA3 regions of the hippocampal formation and within the dentate gyrus of the hippocampus. An intense fiber network was also observed in the olfactory tubercle and other rhinencephalic structures, presumably originating from the Ch3 region of the basal forebrain. Beaded processes emanating from the Ch4 region primarily coursed within the external capsule and terminated preferentially within layers I, II, and IV of the cerebral cortex. In a pattern similar to that of cortical acetylcholinesterase (AChE) staining, NGF receptor immunopositive fibers were oriented in a tangential plane within the molecular layer of the cortex and in both a radial and tangential fashion within the cortical granular cell layers. In addition to neural innervation, there was an extensive vascular apposition by NGF receptor-containing neurites on both large caliber vessels and microcapillaries. NGF receptor immunoreactivity was extensively, but not exclusively, colocalized with choline acetyltransferase (ChAT) and AChE in the basal forebrain. A small population of cholinergic neurons were observed that were not NGF receptor-immunoreactive. Conversely, a few NGF receptor-containing neurons that were noncholinergic were also observed in this brain region. NGF receptor-containing somata were also identified in the putamen. The number of immunoreactive neurons observed in this structure, however, would not appear to be sufficient to account for the homologous NGF receptor binding densities described in rodents.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Nerve growth factor receptor-containing cholinergic neurons of the basal forebrain project to the thalamic reticular nucleus in the rat

The origin of nerve growth factor receptor-immunoreactive (NGFr-ir) fibers innervating the thalamic reticular nucleus (Rt) was here investigated in the rat using retrograde tracers in combination with immunocytochemistry. Neurons retrogradely labeled from Rt were scattered ipsilaterally throughout the medial septal nucleus and the other cell groups of the basal forebrain, which contained NGFr-ir cells; 10–20% of these retrogradely labeled neurons were also NGFr-ir. Furthermore, a few retrogradely labeled NGFr-ir cells were detected in the basal forebrain on the contralateral side. Retrograde tracing combined with a double immunocytochemical procedure revealed that all the NGFr-ir neurons labeled from Rt also displayed immunoreactivity for choline acetyltransferase. The present results demontrate that the NGFr-ir neurons of the basal forebrain which project to Rt are cholinergic. The possible functional implications of these findings are discussed.     

Nerve growth factor-like immunoreactive profiles in the primate basal forebrain and hippocampal formation

The distribution of nerve growth factor (NGF), the prototypic neurotrophin, within the basal forebrain and hippocampal formation of young adult monkeys and aged humans was characterized with and affinity purified polyclonal β-NGF antibody raised against mouse β-NGF. In the basal forebrain of both primates, a granular NGF-like immunoreactive (ir) reaction product was observed within neurons of the medial septum, nucleus of the diagonal band, and nucleus basalis of Meynert. NGF-like immunoreactivity exclusively colocalized within p75 NGF receptor (NGFR) containing basal forebrain neurons. The intensity of NGF immunolabeling varied between cell bodies. Many NGF-ir perikarya were highly immunoreactive. In other basal forebrain neurons, NGF-like immunoreactivity was either undetectable or minimally expressed. In the hippocampus of both species, NGF-like immunoreactivity was mainly localized within the hilus of the dentate gyrus and within CA3 and CA2 hippocampal subfields. A marked diminution in NGF-like staining was seen in CA1. Within the hippocampal formation, NGF-like immunoreactivity was heaviest within the neuropil of stratum radiatum, intermediate in stratum oriens, and lightest in stratum pyramidal. NGF-like immunoreactivity was not found within the granule or pyramidal cells of the dentate gyrus and hippocampal formation, respectively. These findings demonstratre the presence of an NGF-like antigen in association with monkey and human magnocellular basal forebrain neurons and within their hippocampal target sites. This lends support to the hypothesis that NGF is internalized from sources located within target regions of the primate cholinergic basal forebrain neurons and is retrogradely transported to these cell bodies where the NGF trophic effect likely occurs.     

Ultrastructural characterization of choline acetyltransferase-containing neurons in the basal forebrain of rat: evidence for a cholinergic innervation of intracerebral blood vessels

The ultrastructural morphology and vascular associations of cholinergic neurons in the horizontal limb of the nucleus of the diagonal band of Broca (nDBBhl) and amygdala of rat were determined by the immunocytochemical localization of choline acetyltransferase (ChAT), the acetylcholine biosynthetic enzyme. Within the nDBBhl peroxidase reaction product was distributed throughout the cytoplasm of selectively labeled neuronal perikarya and dendrites. Labeled perikarya were characterized by an oval cell body (7-10 microns X 17-26 microns in diameter) in which was located a large nucleus and often a prominent nucleolus. Dendrites were by far the most numerous immuno-labeled profiles in the nDBBhl. The labeled dendrites had a cross-sectional diameter of 0.4-4.6 microns and contained numerous mitochondria and microtubules. Approximately 10% of all immunolabeled dendrites received synaptic contacts from unlabeled presynaptic boutons. In contrast to the relatively large number of ChAT-labeled dendrites within the nDBBhl, ChAT-positive axons were less frequently observed and immunolabeled axon terminals were never detected. The labeled axons had an outside diameter of 0.4-1.4 micron and were myelinated. The absence or relative paucity of immunolabeled terminals in the nDBBhl indicates that most if not all of the cholinergic perikarya within this nucleus are efferent projection neurons. The nDBB is known to have widespread projections to many areas of the neocortex, hippocampus, and amygdala. In the present study we examined the amygdala and observed many ChAT-labeled axon boutons. The immunolabeled varicosities contained numerous agranular vesicles and although ChAT-positive terminals were in direct contact with unlabeled neuronal elements within the amygdala, few if any synaptic densities were detected in a single plane of section. With respect to the vasculature, immunolabeled perikarya and dendrites within the nDBBhl and axon terminals in the amygdala were often in direct apposition to blood vessels. In many instances the labeled profile was observed lying directly on the basal lamina of a capillary endothelial cell. In no instance, however, were membrane densities observed. The presence of cholinergic neuronal elements contacting the vessel wall provides morphologic evidence suggesting that the neurogenic control of cerebral vasculature is in part mediated via a cholinergic mechanism.     

Time of origin of cholinergic neurons in the rat basal forebrain

The timing of the final mitotic division of basal forebrain cholinergic neurons was studied by injecting [3H]thymidine into timed pregnant rats and processing the brains of their progeny as young adults for immunohistochemistry with a monoclonal antibody to choline acetyltransferase (ChAT) followed by autoradiography. ChAT-positive neurons located caudally in the basal forebrain were found to become postmitotic mostly on embryonic (E) days 12 and 13, whereas the peak final mitosis of more rostrally located ChAT-positive neurons occurred increasingly later, with the most rostral ChAT-immunoreactive neurons leaving their final mitotic cycles on E15 and E16. In all basal forebrain regions, cholinergic neurogenesis was complete by E17. These results indicate that the cholinergic neurons in the basal forebrain become postmitotic in a caudal-to-rostral gradient over about 5 days. The continuity of the gradient suggests that these cholinergic neurons may derive from the same germinal source.     

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.     

Cultured basal forebrain cholinergic neurons in contact with cortical cells display synapses,enhanced morphological features,and decreased dependence on nerve growth factor

Prior studies examining the dependence of basal forebrain cholinergic neurons (BFCNs) on nerve growth factor (NGF) for survival have reached differing conclusions depending on the experimental paradigm employed, suggesting the importance of environmental and developmental variables. The present study examined the NGF dependence of BFCNs and modulatory effects of target (cortical) neurons under the controlled conditions of dissociated cell cultures. Initial experiments found BFCNs (identified by using choline acetyltransferase immunocytochemistry) in pure basal forebrain (BF) cultures to be dependent on NGF between the 2nd and 4th week in vitro. During that developmental period, NGF deprivation for 3 days, induced by application of anti-NGF antibody, resulted in degeneration of over 80% of BFCNs, whereas at earlier or later times, BFCNs were largely resistant to NGF deprivation. When BF neurons were plated together with cortical neurons (as dissociated co-cultures), the BFCNs grew neuritic processes (labeled with acetylcholinesterase histochemistry) that appeared to specifically target cortical neurons; electron microscopy revealed that synapses formed between these cells. BFCNs in co-cultures were more resistant to NGF deprivation, were larger, and had much more extensive neuritic growth than BFCNs in pure BF cultures. The resistance of BFCNs to NGF deprivation provided by cortical neurons could be largely reproduced by addition of other trophic factors (brain-derived neurotrophic factor, BDNF; neurotrophin 3, NT3; neurotrophin 4/5, NT4/5; or glial-derived neurotrophic factor, GDNF) during NGF deprivation in pure BF cultures. These results suggest that developing BFCNs undergo a critical period requiring trophic influences that may be provided by NGF or other trophic factors, as well as unknown factors derived from cortical neurons. © 1996 Wiley-Liss, Inc.     

Ontogeny of cholinergic neurons in the mouse forebrain

The development of cholinergic neurons in the mouse forebrain was studied by immunocytochemistry with a monoclonal antibody to choline acetyltransferase (ChAT), the rate-limiting enzyme for acetylcholine synthesis. Since this antibody stained dividing cells in ventricular germinal zones as well as differentiating neurons, likely routes of migration could be inferred on the basis of the location of immunoreactive (IR) cells at different gestational ages. Germinal zones for cholinergic cells were observed in all ventricular zones of the forebrain with the ventral zones generating the earliest cells by gestational day 13.5 (GD13.5). On GD14, ChAT IR cells were visible in the germinal zones of the eye, olfactory ventricle, anterior horn, and dorsolateral aspect of the lateral ventricle, lateral ganglionic eminence, ventro- and dorsolateral third ventricle, and in the pineal anlage (epiphysis). ChAT IR neurons continued to develop in these and additional germinal zones on GD15, including the medial, dorsal, and dorsomedial walls of the lateral ventricle, and the medial and dorsal ganglionic eminence. On GD16, ChAT IR neurons were located in the prelimbic, pyriform, and parietal cortices and the lamina terminalis, and a cluster of IR cells was observed in the ventricular zone of the caudatopallial angle. On GD17-18, neurons in the anterior olfactory nucleus, olfactory tubercle, horizontal and vertical nucleus of the diagonal band, and medial septal nucleus stained more darkly and were multipolar, whereas immature bipolar neurons appeared to continue their migration into the hippocampus and along major fiber tracts, such as the corpus callosum, external capsule, fornix and anterior commissure. This study provides a comprehensive view of the zones of origin, probable routes of migration, and final destination of cholinergic neurons in the mouse forebrain.     

The cholinergic basal forebrain in the ferret and its inputs to the auditory cortex

Cholinergic inputs to the auditory cortex can modulate sensory processing and regulate stimulus‐specific plasticity according to the behavioural state of the subject. In order to understand how acetylcholine achieves this, it is essential to elucidate the circuitry by which cholinergic inputs influence the cortex. In this study, we described the distribution of cholinergic neurons in the basal forebrain and their inputs to the auditory cortex of the ferret, a species used increasingly in studies of auditory learning and plasticity. Cholinergic neurons in the basal forebrain, visualized by choline acetyltransferase and p75 neurotrophin receptor immunocytochemistry, were distributed through the medial septum, diagonal band of Broca, and nucleus basalis magnocellularis. Epipial tracer deposits and injections of the immunotoxin ME20.4‐SAP (monoclonal antibody specific for the p75 neurotrophin receptor conjugated to saporin) in the auditory cortex showed that cholinergic inputs originate almost exclusively in the ipsilateral nucleus basalis. Moreover, tracer injections in the nucleus basalis revealed a pattern of labelled fibres and terminal fields that resembled acetylcholinesterase fibre staining in the auditory cortex, with the heaviest labelling in layers II/III and in the infragranular layers. Labelled fibres with small en‐passant varicosities and simple terminal swellings were observed throughout all auditory cortical regions. The widespread distribution of cholinergic inputs from the nucleus basalis to both primary and higher level areas of the auditory cortex suggests that acetylcholine is likely to be involved in modulating many aspects of auditory processing.     

Maternal choline supplementation differentially alters the basal forebrain cholinergic system of young‐adult Ts65Dn and disomic mice

Down syndrome (DS), trisomy 21, is a multifaceted condition marked by intellectual disability and early presentation of Alzheimer's disease (AD) neuropathological lesions including degeneration of the basal forebrain cholinergic neuron (BFCN) system. Although DS is diagnosable during gestation, there is no treatment option for expectant mothers or DS individuals. Using the Ts65Dn mouse model of DS that displays age‐related degeneration of the BFCN system, we investigated the effects of maternal choline supplementation on the BFCN system in adult Ts65Dn mice and disomic (2N) littermates at 4.3–7.5 months of age. Ts65Dn dams were maintained on a choline‐supplemented diet (5.1 g/kg choline chloride) or a control, unsupplemented diet with adequate amounts of choline (1 g/kg choline chloride) from conception until weaning of offspring; post weaning, offspring were fed the control diet. Mice were transcardially perfused with paraformaldehyde, and brains were sectioned and immunolabeled for choline acetyltransferase (ChAT) or p75‐neurotrophin receptor (p75^NTR). BFCN number and size, the area of the regions, and the intensity of hippocampal labeling were determined. Ts65Dn‐unsupplemented mice displayed region‐ and immunolabel‐dependent increased BFCN number, larger areas, smaller BFCNs, and overall increased hippocampal ChAT intensity compared with 2N unsupplemented mice. These effects were partially normalized by maternal choline supplementation. Taken together, the results suggest a developmental imbalance in the Ts65Dn BFCN system. Early maternal‐diet choline supplementation attenuates some of the genotype‐dependent alterations in the BFCN system, suggesting this naturally occurring nutrient as a treatment option for pregnant mothers with knowledge that their offspring is trisomy 21. J. Comp. Neurol. 522:1390–1410, 2014. © 2013 Wiley Periodicals, Inc.     

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.     

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.