Neurogenesis of basal forebrain cholinergic neurons in rat

The basal forebrain cholinergic system embodies a heterogeneous group of neurons distributed in the basal telencephalon that project topographically to the cortical mantle. We sought to examine the generation of these neurons to determine whether basal forebrain neurons have unique patterns of neurogenesis or, if, in contrast, they are born along general neurogenic gradients. The techniques of tritiated thymidine autoradiography and choline acetyltransferase (ChAT) immunocytochemistry were combined to determine the birthdays and neurogenic gradients of cholinergic cells in this region of rat brain. Cholinergic neurogenesis throughout the basal forebrain ranged from embryonic days 12 to 17 (E12-17). Neurogenesis in the nucleus basalis magnocellularis occurred over E12-16, with a peak day of generation on E13. The horizontal limb nucleus of the diagonal band which is located rostral to the nucleus basalis was generated over E12-17, with the majority of cells arising on E14-15. The rostral-most nuclei of the basal forebrain cholinergic system, the vertical limb of the diagonal band and the medial septum, were generated between E13-17, with peak days of neurogenesis on E15 and E15-16, respectively. These results were evaluated quantitatively and demonstrated that the basal forebrain cholinergic neurons were generated along the general caudal-to-rostral gradient previously described for all neurons in this brain region. The results of this study, in combination with those of similar investigations, emphasize that position-dependent epigenetic factors appear to be more potent determinants of the time of neuronal origin than factors which influence a cell's transmitter phenotype.     

Neurofibrillary degeneration of cholinergic and noncholinergic neurons of the basal forebrain in Alzheimer's disease

Two principal features of Alzheimer's disease (AD) are (1) the occurrence of neurofibrillary tangles (NFTs) and senile plaques, and (2) the loss of cortical cholinergic activity because of dysfunction of neurons in the basal forebrain cholinergic system. The relationship of these two abnormalities is an unresolved issue in the pathology of AD. We used polyclonal antibodies specific for paired helical filaments (PHFs), combined with acetylcholinesterase (AChE) histochemistry, to assess the cytoskeletal changes of cholinergic and noncholinergic neurons in the basal forebrain in AD. In both sporadic and familial AD, the nucleus basalis of Meynert (nbM) showed a marked decrease in AChE-positive (AChE+) perikarya and abundant immunoreactive NFTs. In double-labeling studies of the nbM, PHF reactivity was found both in surviving AChE+ neurons and in many AChE- NFTs that were not associated with microscopically recognizable cell structures. Some surviving AChE+ perikarya did not contain NFTs. Numerous NFTs and senile plaques were identified by PHF immunoreactivity in other basal forebrain areas, including subnuclei of the amygdala that showed low or absent AChE activity. We conclude that the dysfunction and death of cholinergic neurons in the nbM is associated with extensive NFT formation, including apparently residual NFTs in loci where nbM neurons once existed; and many noncholinergic neurons and neurites in the basal forebrain show NFT and senile plaque formation. The cytopathology of AD involves neurons of varying transmitter specificities, including cholinergic neurons in the basal forebrain.     

Morphological and electrophysiological characteristics of noncholinergic basal forebrain neurons

Cholinergic neurons in the basal forebrain are the focus of considerable interest because they are severely affected in Alzheimer's disease. However, both cholinergic and noncholinergic neurons are intermingled in this region. The goal of the present study was to characterize the morphology and in vivo electrophysiology of noncholinergic basal forebrain neurons. Neurons in the ventral pallidum and substantia innominata were recorded extracellularly, labeled juxtacellularly with biocytin and characterized for the presence of choline acetyltransferase immunoreactivity. Two types of ventral pallidal cells were observed. Type I ventral pallidal neurons had axons that rarely branched near the cell body and tended to have smaller somata and lower spontaneous firing rates than did type II ventral pallidal neurons, which displayed extensive local axonal arborizations. Subtypes of substantia innominata neurons could not be distinguished based on axonal morphology. These noncholineregic neurons exhibited local axon arborizations along a continuum that varied from no local collaterals to quite extensive arbors. Substantia innominata neurons had lower spontaneous firing rates, more variable interspike intervals, and different spontaneous firing patterns than did type II ventral pallidal neurons and could be antidromically activated from cortex or substantia nigra, indicating that they were projection neurons. Ventral pallidal neurons resemble, both morphologically and electrophysiologically, previously described neurons in the globus pallidus, whereas the substantia innominata neurons bore similarities to isodendritic neurons of the reticular formation. These results demonstrate the heterogeneous nature of noncholinergic neurons in the basal forebrain. J. Comp. Neurol. 394:186–204, 1998. © 1998 Wiley-Liss, Inc.     

GABAergic and other noncholinergic basal forebrain neurons,together with cholinergic neurons,project to the mesocortex and isocortex in the rat

The extrathalamic relay from the brainstem reticular formation to the cerebral cortex in the basal forebrain has been thought to be constituted predominantly, if not exclusively, by cholinergic neurons. In contrast, the septohippocampal projection has been shown to contain an important contingent of γ-aminobutyric acid (GABA)ergic neurons. In the present study, we investigated whether GABAergic neurons also contribute to the projection from the basal forebrain to neocortical regions, including the mesocortex (limbic) and the isocortex in the rat. For this purpose, retrograde transport of cholera toxin (CT) was examined from the medial prefrontal cortex for the mesocortex and from the parietal cortex for the isocortex and was combined with dual-immunohistochemical staining for either choline acetyltransferase (ChAT) or glutamic acid decarboxylase (GAD) in adjacent series of sections. Retrogradely labelled GAD⁺ neurons were codistributed with retrogradely labelled ChAT⁺ neurons through the basal forebrain from both the prefrontal and the parietal cortex, suggesting parallel, widespread cortical projections. The GAD⁺ cortically projecting cells were similar in size to the ChAT⁺ cells, thereby indicating that they comprise a contingent of the magnocellular basal cell complex. The proportions of retrogradely labelled neurons that were GAD⁺ (approximately one-third) were equal to or greater than those that were ChAT⁺ from both the prefrontal cortex and the parietal cortex. In addition, the total of GAD⁺ and ChAT⁺ neurons did not account for the total number of cortically projecting cells, indicating that another equivalent proportion of chemically unidentified noncholinergic neurons also contributes to the basalocortical projection. Accordingly, as in the allocortex, GABAergic, cholinergic, and other unidentified noncholinergic neurons may have the capacity to modulate activity in the mesocortex (limbic) and the isocortex through parallel, widespread projections. J. Comp. Neurol. 383:163-177, 1997. © 1997 Wiley-Liss, Inc.     

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.     

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.     

Few cholinergic neurons in the rat basal forebrain coexpress galanin messenger RNA

The concept that galanin (GAL) is cosecreted with acetylcholine (ACh) into the ventral hippocampus is a major component of the current model delineating GAL regulation of the cholinergic memory pathways in the rat. Although GAL-immunoreactivity coexists in 50–70% of cholinergic neurons in the basal forebrain (BF) of colchicine-treated rats, the actual coexistence of these neurotransmitters in the basal state may be lower, because colchicine treatment was recently shown to both induce GAL gene expression and inhibit choline acetyltransferase (ChAT) gene expression in this brain region. We have used single and double in situ hybridization histochemistry to examine the distribution and coexistence of GAL and ChAT mRNAs in the BF of male and female rats. Compared with other forebrain regions, few GAL mRNA-expressing neurons are present within the cholinergic fields of the BF. The greatest number of GAL mRNA-expressing cells in this region are located within the nucleus of the horizontal limb of the diagonal band; but, even in this region, they represent only a small percentage (<20%) of ChAT mRNA-expressing cells. Our results indicate that few cholinergic neurons in the rat BF coexpress GAL mRNA and suggest that, in the basal state, GAL is not widely cosecreted with ACh into hippocampal memory centers. J. Comp. Neurol. 391:248–258, 1998. © 1998 Wiley-Liss, Inc.     

Tamoxifen enhances choline acetyltransferase mRNA expression in rat basal forebrain cholinergic neurons

Novel estrogen-like molecules known as SERMs (selective estrogen receptor modulators) produce many of the beneficial estrogen-like actions without the detrimental side-effects. The SERM, tamoxifen, an estrogen-like molecule with both agonist and antagonist properties, is widely prescribed for the treatment of breast cancer. While the effects of tamoxifen are being evaluated in many peripheral tissues, its effects in the central nervous system (CNS) have been largely ignored. In the present study, we begin to evaluate the effects of tamoxifen in the rat basal forebrain, a region known to be highly responsive to estrogen. We compared the effects of short-term (24 h) tamoxifen treatment to that of estrogen on ChAT mRNA expression in cholinergic neurons. In addition, we examined the effect of tamoxifen in the presence and absence of estrogen. Our results indicate that tamoxifen enhances ChAT expression in a manner similar to that of estrogen in several basal forebrain regions. In contrast, tamoxifen exhibits antagonist properties with respect to estrogen-induction of progesterone receptor mRNA in the medial preoptic nucleus. These results indicate tamoxifen has estrogenic properties with respect to cholinergic neurons, suggesting a previously unidentified effect of this agent in the CNS.     

GABAergic control of basal forebrain cholinergic neurons and memory

The involvement of the GABAergic innervation of basal forebrain neurons in the rats' conditional visual discrimination performance was examined. Performance in such a task is based on the subjects's ability to retrieve information about response rules, and previous experiments have demonstrated that basal forebrain lesions interfere with this ability. Following the acquisition of the task, chronic guide cannulae were stereotaxically implanted into the substantia innominata of both hemispheres, and the animals were retrained. Administration of the GABAA-agonist muscimol into the substantia innominata (0, 25, 50 ng/0.5 microliters/hemisphere) dose-dependently decreased the number of correct responses, increased the number of errors of omission, increased response latency, but did not affect side bias. Systemic co-administration of the cholinesterase inhibitor physostigmine (0, 0.1, 0.2 mg/kg; i.p.) exclusively interacted with the effects of muscimol on correct responding. Specifically, physostigmine dose-dependently intensified and attenuated the muscimol-induced reduction in correct responding. Although it cannot be excluded that alternative neuronal mechanisms were involved in the mediation of the effects of muscimol and their interaction with physostigmine, these findings support previous evidence indicating that the activity of basal forebrain cholinergic neurons is controlled by a GABAergic input, and that this neuronal link is involved in mnemonic processing.     

Fiber pathways of basal forebrain cholinergic neurons in monkeys

In rhesus monkeys, autoradiographic tracing methods, complemented by immunocytochemical and histochemical techniques, were used to delineate pathways by which cholinergic neurons of the nucleus basalis of Meynert (nbM) and nucleus of the diagonal band of Broca (ndbB) project to forebrain targets. Following injections of [3H]amino acids into these nuclei, 5 major fiber pathways were identified: axons of the nbM and ndbB project medially, principally within the cingulum bundle, to dorsomedial portions of the hemispheres; nbM and ndbB fibers exit laterally beneath the pallidum and striatum, enter the external and extreme capsules, and pass within the corona radiata to terminate in lateral and caudal regions of neocortex; axons coursing ventrally from the nbM project to portions of the temporal lobe, including the amygdala; some fibers pass through the fibrae pass orbitofrontales to the orbitofrontal cortex; and, finally axons of the nbM/ndbB project via the fimbria/rornix and a ventral pathway to the hippocampus. The presence of these 5 radiolabeled pathways arising from basal forebrain cholinergic neurons was confirmed by acetylcholinesterase histochemistry and choline acetyltransferase immunocytochemistry.     

Age-related variations in plasticity of rat basal forebrain cholinergic neurons after cortical lesions

The enzymatic activity of choline acetyltransferase (ChAT) in the nucleus basalis (NBM) of young rats (30 days old at the time of the operation) drops by 50% thirty days after cortical damage. This is followed by a spontaneous recovery of the enzymatic activity at 120 days after the lesion. In the present study, similar changes were observed in rats which were lesioned at maturity (4 months old). However, a different response was noted when surgery was performed on aged rats (2 years old at the time of operation). In these aged rats the drop in enzymatic activity in the NBM at 30 days post-lesion was as marked as in the young and mature animals, and no recovery was observed, even at 120 days. These results are discussed in the context of age-related neurodegenerative disease with cholinergic involvement.     

Galanin attenuates beta-amyloid (Abeta) toxicity in rat cholinergic basal forebrain neurons

In brains of Alzheimer's disease (AD) patients, expression of the neuropeptide galanin is significantly upregulated and galanin-immunoreactive fibers hypertrophy and hyperinnervate cholinergic neurons of the basal forebrain. However, the role of galanin in AD, whether it is detrimental or neuroprotective, remains controversial. In this study, using primary cultured neurons from the rat basal forebrain, we show that pretreatment with galanin protects cholinergic neurons against beta-amyloid-induced apoptotic cell death as judged by visual observation, MTT assay, Live/dead cell assay, TUNEL and cleaved caspase-3 staining. These effects are mimicked by the galanin receptor 2 (GALR2) agonist, AR-M1896. Western blot analysis revealed Abeta-induced decrease in phospho-PKC and phospho-Akt levels was reversed by galanin. Galanin also attenuated cleavage of caspases-3 and -9 following exposure to Abeta. These findings support a neuroprotective role for galanin and may have implications for development of compounds based on this peptide to treat AD.     

GABAergic and cholinergic neurons exhibit high-affinity nerve growth factor binding in rat basal forebrain

We have used dissociated, rat basal forebrain cultures to identify specific cell types that are potentially responsive to nerve growth factor (NGF). Expression of high-affinity NGF binding sites was examined. A subpopulation of cells containing choline acetyltransferase (CAT), the acetylcholine-synthesizing enzyme, exhibited high-affinity binding, employing combined immunocytochemistry and 125I-NGF radioautography. Unexpectedly, a gamma-aminobutyric acid (GABA)-containing cell group also expressed high-affinity binding. These cells that exhibit high-affinity binding appear to be neurons since they stain positively with the neuron marker, neuron-specific enolase, and negatively with the nonneuron marker, glial fibrillary acidic protein. Our observations suggest that NGF may regulate multiple brain systems and functions that have yet to be explored. Conversely, only subsets of cholinergic or GABA neurons expressed high-affinity binding, suggesting that these transmitter populations are composed of differentially responsive subpopulations.     

Specificity of 192 IgG-saporin for NGF receptor-positive cholinergic basal forebrain neurons in the rat

A monoclonal antibody to the rat nerve growth factor (NGF) receptor, 192 IgG, accumulates bilaterally and specifically in cholinergic basal forebrain (CBF) cells following intraventricular injection. An immunotoxin composed of 192 IgG linked to saporin (192 IgG-saporin) has been shown to destroy cholinergic neurons in the basal forebrain. We sought to determine if intraventricular 192 IgG-saporin affected choline acetyltransferase (ChAT) enzyme activity in the CBF terminal projection fields. ChAT assays from 192 IgG-saporin-treated animals showed significant time-dependent decreases in ChAT activity in the neocortex, olfactory bulb and hippocampus, compared to PBS- or OKT1-saporin-injected controls. ChAT and tyrosine hydroxylase activity in the striatum was always unchanged by 192 IgG-saporin. ChAT immunohistochemistry was confirmative of major cell loss in the CBF, while other cholinergic nuclei appeared unremarkable. The data provide further evidence of the selectivity of 192 IgG-saporin in abolishing cholinergic, NGF receptor-positive CNS neurons.     

pH-dependent actions of aluminum on voltage-activated sodium currents in snail neurons

The pH-dependent actions of aluminum(III) hydroxides (Al(III))on the voltage-activated sodium currents (VASCs) in the giant neurons of the pond snail Lymnaea stagnalis L. were studied by means of a conventional two-electrode voltage-clamp technique. The final concentration of Al(III) was 5-500 microM at pH 7.7, 6.9 or 6.0. A significant and concentration-dependent increase in the peak amplitude of the VASCs was recorded over the entire voltage range at pH 7.7 (EC50 = 100.7 +/- 33.7 microM, n = 9), without alteration of the gating properties. A concentration-dependent decrease in the peak amplitude (IC50 = 175.9 +/- 73.6 microM, n = 6) and concomitant increases in the time constants of activation and inactivation of the VASCs were recorded in slightly acidic media (pH 6.0), whereas there were no changes in the investigated parameters at pH 6.9. A significant increase in the V1/2 of the half-maximal current of the steady-state inactivation resulted on Al(III) application at pH 7.7, but not at pH 6.9 or 6.0. These results suggest that Al(III) can differentially up- and down-modulate the sodium current and related physiological functions to extents dependent on the pH-determined speciation of the Al(III) hydroxides present.     

NGF mRNA is expressed by GABAergic but not cholinergic neurons in rat basal forebrain

Nerve growth factor (NGF) supports the survival and biosynthetic activities of basal forebrain cholinergic neurons and is expressed by neurons within lateral aspects of this system including the horizontal limb of the diagonal bands and magnocellular preoptic areas. In the present study, colormetric and isotopic in situ hybridization techniques were combined to identify the neurotransmitter phenotype of the NGF-producing cells in these two areas. Adult rat forebrain tissue was processed for the colocalization of mRNA for NGF with mRNA for either choline acetyltransferase, a cholinergic cell marker, or glutamic acid decarboxylase, a GABAergic cell marker. In both regions, many neurons were single-labeled for choline acetyltransferase mRNA, but cells containing both choline acetyltransferase and NGF mRNA were not detected. In these fields, virtually all NGF mRNA-positive neurons contained glutamic acid decarboxylase mRNA. The double-labeled cells comprised a subpopulation of GABAergic neurons; numerous cells labeled with glutamic acid decarboxylase cRNA alone were codistributed with the double-labeled neurons. These data demonstrate that in basal forebrain GABAergic neurons are the principal source of locally produced NGF. © 1995 Wiley-Liss, Inc.     

Mitochondria buffer non-toxic calcium loads and release calcium through the mitochondrial permeability transition pore and sodium/calcium exchanger in rat basal forebrain neurons

Mitochondria participate in intracellular Ca2+ buffering and signalling. They are also major mediators of cell death. Toxic Ca2+ accumulation in mitochondria is widely believed to initiate cell death in many cell types by opening the permeability transition pore (PTP). In non-neuronal cells, the PTP has been implicated as a Ca2+ release mechanism in physiological Ca2+ signalling. In neurons, Ca2+ release from mitochondria has been attributed primarily to mitochondrial Na+/Ca2+ exchange. Using fura-2 ratiometric microfluorimetry in acutely dissociated rat basal forebrain neurons, we show that mitochondria are able to buffer non-toxic Ca2+ loads arising from caffeine-sensitive internal stores or from extracellular influx through voltage gated channels. We also show that these non-toxic Ca2+ loads are reversibly released from mitochondria through the PTP and the Na+/Ca2+ exchanger. Evoked Ca2+ transients have characteristic peak and shoulder features mediated by mitochondrial buffering and release. Depolarizing mitochondria with carbonyl cyanide m-chlorophenylhydrazone (CCCP, 5 microM) causes release of mitochondrial Ca2+ and prevents Ca2+ uptake. In CCCP, the magnitudes of evoked Ca2+ transients are increased, and the peak and shoulder features are eliminated. The PTP antagonist, cyclosporin A, (CSA, 2 microM) and the Na+/Ca2+ exchange blocker, clonazepam, (CLO, 20 microM) reversibly inhibited both the shoulder features of evoked Ca2+ transients and Ca2+ transients associated with CCCP application. We suggest that central neuronal mitochondria buffer and release Ca2+ through the PTP and Na+/Ca2+ exchanger during physiological Ca2+ signalling. We also suggest that CLO blocks both the PTP and the mitochondrial Na+/Ca2+ exchanger.     

N-methylaspartate: an effective tool for lesioning basal forebrain cholinergic neurons of the rat

The ability of the excitotoxin, N-methyl-D,L-aspartic acid (NMA), to destroy basal forebrain cholinergic (BFC) neurons was evaluated. NMA (100 nmol) was directly injected into the peripallidum, a region containing a proportionately large number of cortically-projecting BFC neurons. Cholineacetyltransferase (ChAT) activity 10 days later was markedly and significantly reduced (up to 62%) in the cortex ipsilateral to the lesion. NMA induced a focal lesion affecting BFC neurons without damaging axons of passage or causing lesions distant from the site of injection. ChAT immunohistochemistry (IHC) was used to directly demonstrate loss of ChAT-positive neurons from the lesion site. This loss persisted at all survival times examined, from 2 days to 7.5 months post-injection.     

Effects of estrogen on basal forebrain cholinergic neurons and spatial learning

Estrogen receptors are expressed in several areas of the brain associated with cognition, including the basal forebrain cholinergic nuclei, and numerous reports have described improvements in memory in response to estrogen supplementation. The relationship between estrogen's effects on the basal cholinergic system and improvements in cognitive function, however, are obscure. We therefore undertook a study to determine the effects of estrogen on several parameters of the cholinergic system in ovariectomized rats and measured the concomitant effects on performance in the Barnes maze, a test of spatial memory. Six weeks of estradiol treatment caused an increase in choline acetyltransferase activity throughout the projection fields of the basal forebrain, including the hippocampal formation (14%), olfactory bulb (30%), and cerebral cortex (35%). Estrogen treatment also caused an increase in cell soma size of cholinergic neurons in the horizontal diagonal limb of the band of Broca and in the basal nucleus of Meynert. There was no change in the number of neurons positive for p75(NTR), nor in the level of p75(NTR) expression per neuron. Barnes maze performance was markedly improved after estradiol treatment, reinforcing the view that estrogen has beneficial cognitive effects, particularly on spatial memory. The beneficial cognitive effect was likely mediated in part by stimulation of the basal forebrain cholinergic system, especially in its neocortical projection, but was not associated with changes in the level of p75(NTR) expression.