Cholinergic neurons in the basal forebrain of aged female mice

Aging is associated with at least down-regulation of several cellular functions and diminished responsiveness to internal and external signals, and possibly with direct cell death. Consequently, pharmacological manipulations may be less effective in aged than in young organisms. In the present study, we investigated whether the basal forebrain cholinergic neurons and the estrogen receptor alpha (ERalpha) which they contain respond to changes in estrogen availability in aged female mice. The mice were sham-operated, ovariectomized, or ovariectomized and treated with 17beta-estradiol at the age of 18 months. Three months later, the mice were perfused and brain sections were double immunostained for choline acetyltransferase (ChAT) and ERalpha. Cell counting with a stereological method revealed that changes in the estrogen level have no effect on the total number of ChAT-immunoreactive (ir) neurons in the basal forebrain. However, the percentage of ChAT-ir neurons containing ERalpha-ir was higher in the ovariectomized mice than in the sham-operated or estrogen-treated mice. This was specific for the medial septum and vertical diagonal band of Broca. The findings indicate that even at old age the ERalphas in cholinergic cells are able to respond to changes in estrogen levels, though in a region-specific manner. This is naturally important for studies aiming to develop therapies for the elderly.     

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

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

Increased neuronal metabolic activity and estrogen receptors in the vertical limb of the diagonal band of Broca in Alzheimer's disease: relation to sex and aging

Changes in the interaction between sex hormones and the cholinergic system are presumed to play a role in cognitive decline in aging and Alzheimer's disease (AD). The hippocampus is one of the most strongly affected brain structures in AD and the vertical limb of the diagonal band of Broca (VDB) is its major source of innervation. In the present study we found, surprisingly, for the first time that the neuronal metabolic activity as measured by the size of the Golgi apparatus in the VDB gradually increases after the age of 50 years in controls and that this process starts earlier and is more pronounced in Alzheimer's disease patients. Neuronal metabolic activity in the VDB was significantly higher in AD than in control patients younger than 70 years of age and was higher in control patients over 70 years than in control patients younger than 70 years of age. The activation of VDB neurons during aging was accompanied by an increased nuclear estrogen receptor (ER) beta staining, which was stronger in patients over 70 years of age than in younger subjects (in both controls and AD patients). Interestingly, as in the nucleus basalis of Meynert, nuclear ERalpha expression was markedly enhanced in AD patients compared to controls independent of age. In addition, evidence was found for the influence of APOE genotype on ERalpha and ERbeta staining in the human VDB in aging and in AD. APOE genotype was positively correlated (epsilon 2 < epsilon 3 < epsilon 4) with the percentage of cytoplasm ERalpha-positive VDB neurons in elderly control male and female subjects and with both nuclear and cytoplasm ERbeta-positive neurons in control women. In conclusion, the VDB is compensatory activated and shows more nuclear ER expression in aging and AD in a sex- and APOE genotype-dependent way. So neither global degeneration or a strongly decreased neuronal metabolism nor a lack of sex hormone receptors in the VDB seems to contribute to the decline in cognition in aging or AD in which the hippocampus plays such a crucial role.     

Lesion‐induced transneuronal plasticity of the cholinergic innervation in the adult rat entorhinal cortex

The present experiments were designed to determine the effect that lesions of the basal forebrain cholinergic system exert on cholinergic interneurons within the entorhinal cortex (EC) in the rat. Unilateral infusion of 192 IgG-saporin into the nucleus of the horizontal diagonal band of Broca (HDB) decreased the number of ipsilateral choline acetyltransferase immunoreactive (ChAT-ir) neurons by 54%. Two–four weeks after the lesion, the ipsilateral EC exhibited a moderate but significant loss of ChAT-ir fibres and interneurons. Adjacent sections revealed a parallel loss of vasoactive intestinal polypeptide (VIP) immunoreactivity. Cell counts in the cingulate cortex were unaffected, suggesting that this effect was indeed specific to the main target area for HDB neurons. Ibotenic acid lesions also induced a significant 36% decrease in the number of cholinergic neurons in the ipsilateral HDB, and disappearance of ChAT terminals in the EC, whereas the number of ChAT-ir neurons in the EC was unchanged. Since ibotenic acid affects all cells and not only cholinergic ones, our results suggest that the specific degeneration of cholinergic neurons in the HDB after 192 IgG-saporin treatment could be inducing transsynaptic effects on their targets. Injections of 192 IgG-saporin directly into the EC also lesioned the cholinergic projection from the HDB, but had no effect on the intrinsic population. Eight weeks after immunolesion, the number of interneurons immunoreactive for ChAT and VIP in the EC had returned to normal values, and persisted for as long as 6 months after the lesion. By contrast, ChAT-ir neurons in the HDB were permanently lost. Our results suggest that the transient down-regulation of the cholinergic phenotype in entorhinal cortex interneurons could be a manifestation of activity-dependent plasticity, and that the loss of cholinergic innervation from the basal forebrain could be responsible for these effects through an imbalance of inputs. We hypothesize that the recovery of the phenotypic expression of entorhinal interneurons could be due to a recovery in their innervation, perhaps from sprouting axons in the same fields, belonging to surviving cholinergic neurons in the basal forebrain.     

Degeneration of beta-amyloid-associated cholinergic structures in transgenic APP SW mice

Cholinergic dysfunction is a consistent feature of Alzheimer's disease, and the interrelationship between beta-amyloid deposits, inflammation and early cholinergic cell loss is still not fully understood. To characterize the mechanisms by which beta-amyloid and pro-inflammatory cytokines may exert specific degenerating actions on cholinergic cells ultrastructural investigations by electron microscopy were performed in brain sections from transgenic Tg2576 mice that express the Swedish double mutation of the human amyloid precursor protein and progressively develop beta-amyloid plaques during aging. Both light and electron microscopical investigations of the cerebral cortex of 19-month-old transgenic mice revealed a number of pathological tissue responses in close proximity of beta-amyloid plaques, such as activated microglia, astroglial proliferation, increased number of fibrous astrocytes, brain edema, degeneration of nerve cells, dendrites and axon terminals. Ultrastructural detection of choline acetyl transferase (ChAT)-immunostaining in cerebral cortical sections of transgenic mice clearly demonstrated degeneration of ChAT-immunoreactive fibres in the environment of beta-amyloid plaques and activated glial cells suggesting a role of beta-amyloid and/or inflammation in specific degeneration of cholinergic synaptic structures.     

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.     

Estrogen receptor alpha and vasopressin in the paraventricular nucleus of the hypothalamus in Peromyscus

The purpose of this study was to determine the presence of estrogen receptor alpha (ERalpha) and the relationship between neurons that express ERalpha and produce vasopressin (AVP) in the paraventricular nucleus of the hypothalamus (PVN) in new world mice of the genus Peromyscus. Brains were collected from male and female Peromyscus californicus, Peromyscus leucopus, Peromyscus maniculatus, and Peromyscus polionotus, and double labeled for the expression of ERalpha and AVP immunoreactivity (IR). The number of cells expressing ERalpha-IR and AVP-IR was determined in the medial and posterior region of the PVN. The results indicate that Peromyscus is the first taxonomic group reported to have ERalpha widely distributed in the PVN, occurring in both medial and posterior regions of the PVN. While estrogen can regulate the production of AVP, AVP and ERalpha were rarely colocalized. There was, however, a significant inverse relationship between the number of cells that expressed ERalpha-IR and the number expressing AVP-IR. There were no sex differences in the expression of ERalpha-IR or AVP-IR.     

Differential effects of advancing age on neurotransmitter cell loss in the substantia nigra and striatum of C57BL/6N mice

The present study was carried out to examine the extrapyramidal motor system of C57BL/6N mice for age-related cell loss in cholinergic neurons of the striatum (ST) and dopaminergic (DA) neurons of the substantia nigra (SN). Immunocytochemistry using antibodies against tyrosine hydroxylase (TH) or choline acetyltransferase (CAT) were used to identify DA or cholinergic neurons of the SN and ST in 6 age groups of young (3 months), middle (6, 10, 20 months) and old (25, 30 months) aged mice. We found that while there was a small decline (11%) in the total number of DA neurons of the SN with age, this decrease did not reach statistical significance. In contrast, the total number of cholinergic neurons of the ST significantly decreased between 25 and 30 months of age with the largest cell loss (38%) found in the rostral ST. In addition, the loss of cholinergic neurons in 30-month-old mice was paralleled by a decline in the mean cross-sectional area of the cell soma and nucleus of remaining cholinergic neurons. These data suggest that advancing age has a differential effect on neurotransmitter neurons of the SN and ST and supports the notion that cell loss is not an inevitable characteristic of senescence but is brain region- and cell type-specific. In addition, these data are consistent with the hypothesis that the proliferation of striatal dendrites described previously in aged C57BL/6N mice may result, in part, from a compensatory growth of these processes secondary to age-related cell loss of striatal neurons.     

Neuronal and glial beta-secretase (BACE) protein expression in transgenic Tg2576 mice with amyloid plaque pathology

We measured tissue distribution and expression pattern of the beta-site amyloid precursor protein (APP)-cleaving enzyme (BACE) in the brains of transgenic Tg2576 mice that show amyloid pathology. BACE protein was expressed at high levels in brain; at lower levels in heart and liver; and at very low levels in pancreas, kidney, and thymus and was almost absent in spleen and lung when assayed by Western blot analysis. We observed strictly neuronal expression of BACE protein in the brains of nontransgenic control mice, with the most robust immunocytochemical labeling present in the cerebral cortex, hippocampal formation, thalamus, and cholinergic basal forebrain nuclei. BACE protein levels did not differ significantly between control and transgenic mice or as a result of aging. However, in the aged, 17-month-old Tg2576 mice there was robust amyloid plaque formation, and BACE protein was also present in reactive astrocytes present near amyloid plaques, as shown by double immunofluorescent labeling and confocal laser scanning microscopy. The lack of astrocytic BACE immunoreactivity in young transgenic Tg2576 mice suggests that it is not the APP overexpression but rather the amyloid plaque formation that stimulates astrocytic BACE expression in Tg2576 mice. Our data also suggest that the neuronal overexpression of APP does not induce the overexpression of its metabolizing enzyme in neurons. Alternatively, the age-dependent accumulation of amyloid plaques in the Tg2576 mice does not require increased neuronal expression of BACE. Our data support the hypothesis that neurons are the primary source of beta-amyloid peptides in brain and that astrocytic beta-amyloid generation may contribute to amyloid plaque formation at later stages or under conditions when astrocytes are activated.     

Distribution of choline acetyltransferase (ChAT) immunoreactivity in the central nervous system of a chondrostean, the siberian sturgeon (Acipenser baeri)

All studies to date of cholinergic systems of bony fishes have been done in teleosts. To gain further insight into the evolution of the cholinergic systems of bony fishes, we have studied the brain of a chondrostean fish, the Siberian sturgeon (Acipenser baeri, Brandt), by using an antibody against choline acetyltransferase (ChAT). This study showed the presence of ChAT-immunoreactive (ChAT-ir) neurons in the preoptic region (parvocellular and magnocellular preoptic nuclei and suprachiasmatic nucleus), the periventricular and tuberal hypothalamus, the saccus vasculosus, the dorsal thalamus, and the habenula. The mesencephalic tegmentum contained ChAT-ir cells in the torus semicircularis and torus lateralis. The isthmus contained several cholinergic populations: the nucleus isthmi, the lateral nucleus of the valvula, the secondary visceral nucleus, and the dorsal tegmental nucleus. The motor neurons of the cranial nerves and the spinal motor column were strongly immunoreactive. The medial (sensory) trigeminal nucleus also contained a ChAT-ir neuronal population. The distribution of ChAT-ir neurons in the sturgeon brain showed some notable differences with that observed in teleosts, such as the absence of cholinergic cells in the telencephalon and the optic tectum. Several brain regions were richly innervated by ChAT-ir fibers, particularly the telencephalon, optic tectum, thalamus, posterior tubercle, and interpeduncular nucleus. The hypothalamo-hypophyseal tract, the tract of the saccus vasculosus, the fasciculus retroflexus, and an isthmo-mesencephalo-thalamic tract were the most conspicuous cholinergic bundles. Comparative analysis of these results suggests that teleosts have conserved most traits of the cholinergic system of the sturgeon, having acquired new cholinergic populations during evolution.     

Age-related shrinkage of cortically projecting cholinergic neurons: A selective effect

The number and size of basal forebrain neurons that provide the cholinergic innervation for the cerebral cortex, amygdala, and hippocampus were studied in young and aged mice. The results showed that these neurons became substantially smaller with increasing age. This effect was relatively selective, since the immediately adjacent cholinergic neurons in the striatum did not show a change of similar magnitude. The shrinkage of these basal forebrain neurons may account for the decline of cholinergic innervation that occurs with age. In the material that we examined, aging did not influence the number of cholinergic neurons in the basal forebrain, only their size. It seems, therefore, that the age-related changes in cholinergic function (and their putative behavioral consequences) are not associated with a substantial component of irreversible cell death.     

Stereologic analysis of estrogen receptor alpha (ER alpha) expression in rat hypothalamus and its regulation by aging and estrogen

The estrogen receptor alpha (ERalpha) in the hypothalamus plays important roles in the regulation of reproductive development, physiology, and behavior. However, the expression of the ERalpha may change during aging or in response to varying estrogen levels. The present study measured changes in the numbers of ERalpha-expressing cells in specific hypothalamic and preoptic nuclei of ovariectomized female Sprague-Dawley rats at three ages (young [3-4 months], middle-aged [10-12 months], or old [24-26 months]) and with or without estrogen replacement. Numbers of ERalpha-immunoreactive neurons were quantified in four regions relevant to reproductive function: the anteroventral periventricular nucleus (AVPV), medial preoptic nucleus (MPN), arcuate nucleus (ARH), and ventromedial nucleus (VMN), using an unbiased stereologic approach. In the AVPV and VMN, significant age-related increases in the numbers of ERalpha-expressing cells from the middle-aged to the old group were detected, and no differences were observed in the MPN and ARH, indicating that ERalpha neuron number is maintained or even elevated during aging. No significant effects of estrogen on ERalpha cell number were detected in any of the four regions studied. Therefore, ERalpha cell number in the rat hypothalamus and preoptic area changes with aging in a region-specific manner.     

Impairment of cholinergic neurotransmission in adult and aged transgenic Tg2576 mouse brain expressing the Swedish mutation of human beta-amyloid precursor protein

To address the question of whether beta-amyloid peptides also affect cholinergic neurotransmission in vivo, brain tissue from transgenic Tg2576 mice with Alzheimer plaque pathology at ages ranging from 7 to 24 months were examined by immuno- and histochemical staining for choline acetyltransferase (ChAT) and acetycholinesterase (AChE), by assaying cholinergic enzyme activities and high-affinity choline uptake as well muscarinic and nicotinic cholinergic receptor binding levels by quantitative autoradiography. Cortical and hippocampal activities of AChE and ChAT were not different between transgenic mice and non-transgenic littermates regardless of the postnatal ages examined. However, high-affinity choline uptake was reduced in the hippocampus of 21-month-old transgenic mice. In brains of 8-month-old transgenic mice which do not yet demonstrate cortical beta-amyloids, reduced binding levels of cortical and hippocampal M1-muscarinic cholinergic receptors were observed, which were still reduced in 17-month-old transgenic mouse brains with high plaque load as compared to non-transgenic littermates. M2-muscarinic cholinergic receptor binding was hardly affected in brains from 8-month-old transgenic mice, but in 17-month-old transgenic mice reduced cortical and hippocampal binding levels were observed as compared to non-transgenic controls. Decreased cortical nicotinic cholinergic receptor binding was detected in 17-month-old transgenic mice. The development of changes in cholinergic synaptic markers in transgenic Tg2576 mouse brain before the onset of progressive plaque deposition provides in vivo evidence of a modulatory role of soluble beta-amyloid on cholinergic neurotransmission and may be referred to the deficits in learning and memory also observed in these mice before significant plaque load.     

Subcellular relationships between cholinergic terminals and estrogen receptor-alpha in the dorsal hippocampus

Cholinergic septohippocampal neurons are affected by circulating estrogens. Previously, we found that extranuclear estrogen receptor-alpha (ERalpha) immunoreactivity in presynaptic profiles had an overlapping distribution with cholinergic afferents in the rat hippocampal formation. To determine the subcellular relationships between cholinergic presynaptic profiles and ERalpha, hippocampal sections were dually immunolabeled for vesicular acetylcholine transporter (VAChT) and ERalpha and examined by electron microscopy. Within the hippocampal formation, immunoreactivities for VAChT and ERalpha both were presynaptic, although their subcellular targeting was distinct. VAChT immunoreactivity was found exclusively within presynaptic profiles and was associated with small synaptic vesicles, which usually filled axon terminals. VAChT-labeled presynaptic profiles were most concentrated in stratum oriens of the hippocampal CA1 region and dentate inner molecular layer and hilus. In contrast, ERalpha immunoreactivity was found in clusters affiliated either with select vesicles or with the plasmalemma within preterminal axons and axon terminals. ERalpha-immunoreactive (IR) presynaptic profiles were more evenly distributed between hippocampal lamina than VAChT-IR profiles. Quantitative ultrastructural analysis revealed that VAChT-IR presynaptic profiles contained ERalpha immunoreactivity (ranging from 3% to 17%, depending on the lamina). Additionally, VAChT-IR presynaptic profiles apposed ERalpha-IR dendritic spines, presynaptic profiles, and glial profiles; many of the latter two types of profiles abutted unlabeled dendritic spines that received asymmetric (excitatory-type) synapses from unlabeled terminals. The presence of ERalpha immunoreactivity in cholinergic terminals suggests that estrogen could rapidly and directly affect the local release and/or uptake of acetylcholine. The affiliation of cholinergic terminals with excitatory terminals near ERalpha-labeled dendritic spines or glial profiles suggests that alterations in acetylcholine release could indirectly affect estrogen-modulated structural plasticity.     

Distribution of choline acetyltransferase immunoreactivity in the brain of an elasmobranch, the lesser spotted dogfish (Scyliorhinus canicula)

Although the distribution of cholinergic cells is remarkably similar across the vertebrate species, no data are available on more primitive species, such as cartilaginous fishes. To extend the evolutionary analysis of the cholinergic systems, we studied the distribution of cholinergic neurons in the brain and rostral spinal cord of Scyliorhinus canicula by immunocytochemistry using an antibody against the enzyme choline acetyltransferase (ChAT). Western blot analysis of brain extracts of dogfish, sturgeon, trout, and rat showed that this antibody recognized similar bands in the four species. Putative cholinergic neurons were observed in most brain regions, including the telencephalon, diencephalon, cerebellum, and brainstem. In the retrobulbar region and superficial dorsal pallium of the telencephalon, numerous small pallial cells were ChAT-like immunoreactive. In addition, tufted cells of the olfactory bulb and some cells in the lateral pallium showed faint immunoreactivity. In the preoptic-hypothalamic region, ChAT-immunoreactive (ChAT-ir) cells were found in the preoptic nucleus, the vascular organ of the terminal lamina, and a small population in the caudal tuber. In the epithalamus, the pineal photoreceptors were intensely positive. Many cells of the habenula were faintly ChAT-ir, but the neuropil of the interpeduncular nucleus showed intense ChAT immunoreactivity. In the pretectal region, ChAT-ir cells were observed only in the superficial pretectal nucleus. In the brainstem, the somatomotor and branchiomotor nuclei, the octavolateral efferent nucleus, and a cell group just rostral to the Edinger-Westphal (EW) nucleus contained ChAT-ir neurons. In addition, the trigeminal mesencephalic nucleus, the nucleus G of the isthmus, some locus coeruleus cells, and some cell populations of the vestibular nuclei and of the electroreceptive nucleus of the octavolateral region exhibited ChAT immunoreactivity. In the reticular areas of the brainstem, the nucleus of the medial longitudinal fascicle, many reticular neurons of the rhombencephalon, and cells of the nucleus of the lateral funiculus were immunoreactive to this antibody. In the cerebellum, Golgi cells of the granule cell layer and some cells of the cerebellar nucleus were also ChAT-ir. In the rostral spinal cord, ChAT immunoreactivity was observed in cells of the motor column, the dorsal horn, the marginal nucleus (a putative stretch-receptor organ), and in interstitial cells of the ventral funiculus. These results demonstrate for the first time that cholinergic neurons are distributed widely in the central nervous system of elasmobranchs and that their cholinergic systems have evolved several characteristics that are unique to this group.     

Neuropathology of mice carrying mutant APP(swe) and/or PS1(M146L) transgenes: alterations in the p75(NTR) cholinergic basal forebrain septohippocampal pathway

Cholinergic basal forebrain (CBF) projection systems are defective in late Alzheimer's disease (AD). We examined the brains of 12-month-old singly and doubly transgenic mice overexpressing mutant amyloid precursor protein (APP(swe)) and/or presenilin-1 (PS1(M146L)) to investigate the effects of these AD-related genes on plaque and tangle pathology, astrocytic expression, and the CBF projection system. Two types of beta-amyloid (Abeta)-immunoreactive (ir) plaques were observed: type 1 were darkly stained oval and elongated deposits of Abeta, and type 2 were diffuse plaques containing amyloid fibrils. APP(swe) and PS1(M146L) mouse brains contained some type 1 plaques, while the doubly transgenic (APP(swe)/PS1(M146L)) mice displayed a greater abundance of types 1 and 2 plaques. Sections immunostained for the p75 NGF receptor (p75(NTR)) revealed circular patches scattered throughout the cortex and hippocampus of the APP(swe)/PS1(M146L) mice that contained Abeta, were innervated by p75(NTR)-ir neurites, but displayed virtually no immunopositive neurons. Tau pathology was not seen in any transgenic genotype, although a massive glial response occurred in the APP(swe)/PS1(M146L) mice associated with amyloid plaques. Stereology revealed a significant increase in p75(NTR)-ir medial septal neurons in the APP(swe) and PS1(M146L) singly transgenic mice compared to the APP(swe)/PS1(M146L) mice. No differences in size or optical density of p75(NTR)-ir neurons were observed in these three mutants. p75(NTR)-ir fibers in hippocampus and cortex were more pronounced in the APP(swe) and PS1(M146L) mice, while the APP(swe)/PS1(M146L) mice showed the least p75(NTR)-ir fiber staining. These findings suggest a neurotrophic role for mutant APP and PS1 upon cholinergic hippocampal projection neurons at 12 months of age.     

The caudal sublenticular region/anterior amygdaloid area is the only part of the rat forebrain and mesopontine tegmentum occupied by magnocellular cholinergic neurons that receives outputs from the central division of extended amygdala

Ascending cholinergic projections and the central nucleus of the amygdala (CeA) have both been implicated in attentional and orienting mechanisms leading to adaptive behavioral responses. In view of this, the present study was carried out to identify relevant neuroanatomical relationships in the form of projections from the CeA and a related structure, the dorsolateral divison of the bed nucleus of the stria terminalis (dlBST), to parts of the basal forebrain and mesopontine tegmentum that contain magnocellular cholinergic neurons. The CeA and dlBST are components of the 'central division of extended amygdala'. Following injections of the anterogradely transported compounds, Phaseolus vulgaris-leucoagglutinin or biotinylated dextran amine, into the CeA or dlBST, sections were processed with immunohistochemical reagents to localize the anterograde tracer and choline acetyltransferase (ChAT). The trajectories of efferent projections from CeA and dlBST were qualitatively similar. Few ChAT-immunoreactive (ir) neurons were present within the extended amygdala or regions containing the dense terminations of its efferent projections, with the striking exception of the caudal sublenticular/anterior amygdaloid region. The ChAT-ir neurons there, however, were significantly smaller and weakly ChAT-ir as compared to those located outside of the dense extended amygdaloid terminations. In the mesopontine tegmentum, the robust downstream projection from the extended amygdala was centered medial to ChAT-ir neurons of the pedunculopontine tegmental nucleus. The differentiated character of the relationships between extended amygdala and forebrain and mesopontine districts containing ChAT-ir neurons that give rise to ascending projections may have significant implications for the control of cortical and diencephalic acetylcholine release and accompanying effects on attention, vigilance and locomotor activation.     

Estrogen treatment suppresses forebrain p75 neurotrophin receptor expression in aged,noncycling female rats

There is increasing evidence that estrogen has beneficial effects on cognition, both in humans and in rodents, and may delay Alzheimer's disease onset in postmenopausal women. Several rodent studies have utilised the ovariectomy model to show estrogen regulation of the p75 neurotrophin receptor, TrkA, and markers of acetylcholine synthesis in the cholinergic basal forebrain. We studied estrogenic effects in aged (16-17-month-old), noncycling rats. Estrogen treatment for 10 days drastically reduced p75(NTR) immunoreactivity in the rostral parts of the basal forebrain. The number of p75(NTR)-immunoreactive neurons was decreased, and those neurons remaining positive for p75(NTR) showed reduced p75(NTR) staining intensity. In vehicle-treated rats, almost all choline acetyltransferase-immunoreactive neurons were p75(NTR) positive (and vice versa), but, in estrogen treated rats, large numbers of choline acetyltransferase-immunoreactive cells were negative for p75(NTR). Similar levels of p75(NTR) down-regulation in the rostral basal forebrain were found when estrogen treatment was extended to 6 weeks. There was no reduction in the number of p75(NTR)-immunoreactive neurons in the caudal basal forebrain after 10 days of treatment. After 6 weeks of treatment, however, there was evidence of p75(NTR) down-regulation in the caudal basal forebrain. There was no evidence of hypertrophy or atrophy of cholinergic neurons even after 6 weeks of estrogen treatment. Considering the evidence for the role of p75(NTR) in regulating survival, growth and nerve growth factor responsiveness of cholinergic basal forebrain neurons, the results indicate an important aspect of estrogen's effects on the nervous system.     

Estrogen receptor-alpha mediates estradiol attenuation of ATP-induced Ca2+ signaling in mouse dorsal root ganglion neurons

A mechanism underlying gender-related differences in pain perception may be estrogen modulation of nociceptive signaling in the peripheral nervous system. In rat, dorsal root ganglion (DRG) neurons express estrogen receptors (ERs) and estrogen rapidly attenuates ATP-induced Ca2+ signaling. To determine which estrogen receptor mediates rapid actions of estrogen, we showed ERalpha and ERbeta expression in DRG neurons from wild-type (WT) female mice by RT-PCR. To study whether ERalpha or ERbeta mediates this response, we compared estradiol action mediating Ca2+ signaling in DRG neurons from WT, ERalpha knockout (ERalphaKO), and ERbetaKO mice in vitro. ATP, an algesic agent, induced [Ca2+]i transients in 48% of small DRG neurons from WT mice. 17beta-Estradiol (E2) inhibited ATP-induced intracellular Ca2+ concentration ([Ca2+]i) with an IC50 of 27 nM. The effect of E2 was rapid (5-min exposure) and stereo specific; 17alpha-estradiol had no effect. E2 action was blocked by the ER antagonist ICI 182,780 (1 microM) in WT mouse. Estradiol coupled to bovine serum albumin (E-6-BSA), which does not penetrate the plasma membrane, had the same effect as E2 did, suggesting that a membrane-associated ER mediated the response. In DRG neurons from ERbetaKO mice, E2 attenuated the ATP-induced [Ca2+]i flux as it did in WT mice, but in DRG neurons from ERalphaKO mice, E2 failed to inhibit the ATP-induced [Ca2+]i increase. These results show that mouse DRG neurons express ERs and the rapid attenuation of ATP-induced [Ca2+]i signaling is mediated by membrane-associated ERalpha.     

Impairment of Basal Forebrain Cholinergic Neurons Associated with Aging and Long-Term Loss of Ovarian Function

Recent studies suggest that women are at greater risk for Alzheimer's disease than men and that estrogen replacement can help to reduce the risk and severity of Alzheimer's-related dementia in postmenopausal women. We have hypothesized that the increased risk for Alzheimer's-related dementia is due, in part, to the loss of ovarian function in postmenopausal women and to the effects that decreased levels of ovarian hormones have on basal forebrain cholinergic function. In the present study, the effects of aging and ovariectomy on cholinergic neurons in the rat basal forebrain were examined to determine (1) whether aging differentially affects cholinergic neurons in the basal forebrain of males vs females, and (2) whether long-term loss of ovarian function produces deficits in basal forebrain cholinergic function beyond those associated with aging and sex. In part I of the study, gonadally intact male and female rats were sacrificed at 13, 19, and 25 months of age and the effects of aging on cholinergic neurons in the medial septum (MS) and nucleus basalis magnocellularis (NBM) were compared. In part II of the study, female rats were ovariectomized at 13 months of age and then sacrificed 3 and 6 months later along with gonadally intact, age-matched controls. Adjacent sections through the MS and NBM were processed for either immunocytochemical detection of choline acetyltransferase (ChAT) and p75NTR-like immunoreactivity or forin situhybridization detection and quantification of ChAT and trkA mRNA. Results from part I revealed no significant effects of age on the relative size or density of cholinergic neurons in the MS and NBM of gonadally intact animals. Likewise, no significant effects on the relative numbers of cholinergic neurons expressing p75NTR protein were detected. However, a significant decrease in trkA mRNA was detected in the MS of gonadally intact females, but not males, between 13 and 25 months of age. No significant effects of aging on ChAT mRNA were detected. Results from part II revealed significant decreases in both ChAT and trkA mRNA in the MS and NBM of female rats sacrificed 6 months, but not 3 months, following ovariectomy relative to age-matched, gonadally intact controls. Short-term estrogen replacement initiated 6 months following ovariectomy and administered for 3 days prior to sacrifice partially restored ChAT mRNA levels in the MS and trkA mRNA levels in the NBM. These findings suggest that ovarian hormones play a role in maintaining normal levels of ChAT and trkA expression in the MS and NBM. The fact that ChAT mRNA was decreased in the MS and NBM at 6 months following ovariectomy suggests that long-term loss of ovarian function produces a decrease in the functional status of basal forebrain cholinergic neurons projecting to the hippocampus and cortex. In addition, we hypothesize that the decreases in trkA mRNA detected both in the MS as a function of aging, and in the MS and NBM in response to ovariectomy, reflect decreases in the production of high affinity nerve growth factor (NGF) receptors, and a decrease in the responsiveness of the cholinergic neurons to endogenous NGF. This, in turn, may increase the susceptibility of the cholinergic neurons to the effects of aging and disease and thereby contribute to basal forebrain cholinergic decline. We conclude that long-term loss of ovarian function combined with aging has a negative impact on basal forebrain cholinergic neurons. These effects may contribute to the risk and severity of cognitive decline associated with aging and Alzheimer's disease in postmenopausal women.