GABAergic basal forebrain neurons project to the neocortex: the localization of glutamic acid decarboxylase and choline acetyltransferase in feline corticopetal neurons

Our objective was to determine whether GABAergic and cholinergic basal forebrain neurons project to the neocortex. The retrograde connectivity marker wheat germ agglutinin lectin-bound horseradish peroxidase was injected into the neocortex of adult cats. Histo- and immunohistochemical methods were combined to label sequentially connectivity and transmitter markers (glutamic acid decarboxylase; choline acetyltransferase) in forebrain neurons. The labels of each marker were identified by correlative light and electron microscopy. Two principal types of doubly labeled neurons were demonstrated. The connectivity marker was colocalized with glutamic acid decarboxylase or choline acetyltransferase. The neurons were located in the basal forebrain. Their ultrastructural, cellular, and regional organization supported 2 conclusions. (1) GABAergic basal forebrain neurons project to the neocortex. This is important new morphological evidence for the origin of inhibitory neocortical afferents from a subcortical brain site. (2) The GABAergic and cholinergic basal forebrain neurons projecting to the neocortex exhibit remarkable structural similarities. The transmitter diversity of these intertwined neocortical afferents may be significant for the pathology and treatment of human neurological disorders such as Alzheimer's disease.     

AMPA-induced Lesions of the Basal Forebrain Differentially Affect Cholinergic and Non-cholinergic Neurons: Lesion Assessment Using Quantitative In Situ Hybridization Histochemistry

The direct and transynaptic effects of lesions of the basal forebrain induced by α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and ibotenic acid were investigated using quantitative in situ hybridization histochemistry. Probes complementary to the sequences of choline acetyltransferase mRNA, glutamate decarboxylase mRNA and preproenkephalin mRNA were used to assess direct lesion effects within the basal forebrain and probes for postsynaptic M-1 and M-3 muscarinic receptors were used to assess long-term changes in neocortical muscarinic receptor mRNA expression following cholinergic deafferentation. AMPA-induced basal forebrain lesions destroyed significantly more neurons that expressed choline acetyltransferase mRNA than ibotenic acid-induced lesions (90 versus 60%), but significantly fewer neurons which expressed either glutamate decarboxylase or preproenkephalin mRNA (61 versus 83% reduction in glutamate decarboxylase mRNA and 56 versus 79% reduction in preproenkephalin mRNA). AMPA-induced lesions did, however, destroy a significant proportion of the neurons which expressed glutamate decarboxylase and preproenkephalin mRNA (-60%). The neurons spared following AMPA-induced lesions were typically situated dorsolaterally within the dorsal pallidum, although neurons expressing glutamate decarboxylase or preproenkephalin mRNA were frequently observed within the areas of greatest cholinergic neuronal loss, i.e. the region of the nucleus basalis magnocellularis. These findings suggest that there is a population of non-cholinergic pallidal neurons which are insensitive to AMPA but not to ibotenic acid, reflecting a possibly heterogeneous distribution of NMDA and non-NMDA subtypes of glutamate receptors within the rat basal forebrain. AMPA-induced lesions of the basal forebrain were, however, without significant effect on the levels of expression of M-1 and M-3 muscarinic receptor mRNAs in the cerebral neocortex.     

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

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

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.     

Calbindin-D28k Immunoreactivity within the Cholinergic and GABAergic Projection Neurons of the Basal Forebrain

The purpose of this study was to determine whether the calcium binding protein calbindin-D_28k was present within the cortically projecting basal forebrain neurons of various rodent species not previously examined. Double-label immunocytochemistry was performed using antibodies against calbindin-D_28k and choline acetyltransferase (ChAT) to detect the presence of the calcium binding protein within the cholinergic basal forebrain neurons of various species (i.e., humans, rats, mice, gerbils, guinea pigs). Antibodies against calbindin-D_28k, ChAT, and glutamic acid decarboxylase (GAD) were also used in combination with a cortically injected retrograde tracer (Fluoro-Gold) to determine whether calbindin-D_28k immunoreactive (IR) neurons within the basal forebrain projected to the frontoparietal cortex. The nucleus basalis of rats was examined for the presence of calbindin-D_28k IR within the GABAergic basal forebrain neurons. All species examined had cholinergic, GABAergic, and calbindinergic neurons within the basal forebrain; however, only the cholinergic neurons within the human nucleus basalis of Meynert were also immunoreactive for calbindin-D_28k. Although all rodent species had both cholinergic and GABAergic basal forebrain neurons that contained the Fluoro-Gold dye, none of the calbindin-D_28k IR neurons, detected using monoclonal and polyclonal antibodies, were found to contain the retrograde tracer. These results indicate that the cortically projecting cholinergic and GABAergic basal forebrain neurons within these rodent species do not contain calbindin-D_28k. Therefore, age- and disease-related loss of nucleus basalis projection neurons may not be mediated by alterations in calbindin-D_28k. Thus, rodents may not serve as an accurate model for either human aging or Alzheimer's disease as calbindin-D_28k is not found within the cortically projecting neurons of rodent species.     

Nerve growth factor receptor mRNA distribution in human brain: normal levels in basal forebrain in Alzheimer's disease

Nerve growth factor (NGF) receptor mRNA was found to be widely distributed throughout the human central nervous system, with the highest levels in the basal forebrain; this suggests that NGF may function as a retrograde trophic messenger for basal forebrain magnocellular cholinergic nerve cells. The degeneration of the latter constitutes one of the main features of Alzheimer's disease and it may be responsible for some of the cognitive impairment that characterizes the disease. No evidence was obtained for an insufficient synthesis of NGF receptor mRNA in the basal forebrain in Alzheimer's disease, where NGF receptor-like immunoreactivity was confined to neuronal cell bodies. NGF could thus be therapeutically beneficial. It could be expected to induce basal forebrain cholinergic cells to hypertrophy, synthesize more choline acetyltransferase and extend neurites.     

Effects of lesion of the cholinergic basal forebrain nuclei on the activity of glutamatergic and GABAergic systems in the rat frontal cortex and hippocampus

Summary The effects of cholinergic basal forebrain lesions on the activity of the glutamatergic and GABAergic systems were investigated in the rat frontal cortex and hippocampus. Bilateral quisqualic acid injections in the nucleus basalis magnocellularis (NBM) at the origin of the main cholinergic innervation to the neocortex induced a cholinergic deficit in the cerebral cortex 15 days later, as shown by the marked selective decrease in cortical choline acetyltransferase (CAT) activity observed. Concurrent alterations in the kinetic parameters of high affinity glutamate uptake consisting mainly of a decrease in the V max were observed in the cerebral cortex. These changes presumably reflect a decreased glutamatergic transmission and provide support for the hypothesis that cortical glutamatergic neurons may undergo the influence of cholinergic projections from the NBM. Surprisingly, similar alterations in the glutamate uptake process were found to occur at hippocampal level in the absence of any significant change in the hippocampal cholinergic activity. These data indicate that the NBM may contribute to regulating hippocampal glutamatergic function without interfering with the hippocampal cholinergic innervation that mainly originates in the medial septal area-diagonal band (MSA-DB) complex. No change in parameters of GABAergic activity, namely the glutamic acid decarboxylase (GAD) activity and high affinity GABA uptake, were observed in any of the structures examined. In a second series of experiments involving bilateral intraventricular injections of AF 64 A, marked survival time-dependent decreases in CAT and high affinity choline uptake activities but no significant change in the high affinity glutamate uptake rate were observed in the hippocampus. No significant change in either parameters of cholinergic activity or in the glutamate uptake was concurrently observed in the cerebral cortex. The GABAergic activity was again unaffected whatever the survival time and the structure considered. Taken as a whole, these data suggest that basal forebrain projections originating in the NBM may play a major role in regulating glutamatergic but not GABAergic function in both the cerebral cortex and the hippocampus; whereas the glutamatergic and GABAergic activities in these two structures may not be primarily under the influence of the cholinergic projections from the MSA-DB complex.Abbreviations NBM nucleus basalis magnocellularis - MSA-DB medial septal area-diagonal band - CAT choline acetyltransferase - GAD glutamic acid decarboxylase - GABA gamma-aminobutyric acid Laboratoire associé à l'Université Aix-Marseille II     

Nerve growth factor does not influence the expression of β amyloid precursor protein mRNA in rat brain: in vivo and in vitro studies

We investigated the effect of NGF on amyloid precursor protein (APP) mRNA levels in the rat septal/nucleus basalis system. Total APP mRNA and APP 695 mRNA were determined in basal forebrain primary cell cultures exposed acutely and chronically to NGF (150–300 ng/ml) and, in vivo, in the septal area and striatum of rat pups after multiple intracerebroventricular injections of NGF. The trophic factor was able to affect cholinergic neurons in both paradigms, as evidenced by the significant increase of choline acetyltransferase (ChAT) activity induced by NGF in cell cultures (+80%) and in the striatum (+240%) of rat pups. In spite of this effect, no significant change of APP mRNA expression was observed in neuronal cultures and brain tissues. These data indicate that the neurotrophic effect of NGF on forebrain cholinergic neurons is not always associated with an alteration of APP expression.     

Substance P (neurokinin-1) receptor mRNA is selectively expressed in cholinergic neurons in the striatum and basal forebrain

In the striatum substance P (neurokinin-1) receptor, mRNA is selectively localized in large neurons that also express mRNA encoding choline acetyltransferase (ChAT) by in situ hybridization histochemistry. Substance P receptor mRNA is also localized in ChAT mRNA-containing neurons in the medial septum and basal forebrain cell groups. Thus, in the rat forebrain the substance P receptor appears to be expressed selectively by cholinergic neurons. Striatal neurons that contain substance P also utilize γ-aminobutyric acid (GABA) as a transmitter. These neurons make synaptic contact with striatal cholinergic neurons, which are shown here to express the substance P receptor, and with other GABAergic neurons in the striatum and substantia nigra, which express GABA receptors but not substance P receptors. This suggests that individual striatal neurons may differentially affect target neurons dependent on the receptors expressed by those target neurons.     

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.     

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.     

Transmitter cosynthesis by corticopetal basal forebrain neurons

The objective was to determine if corticopetal basal forebrain neurons could co-synthesize different transmitters. Histochemical labeling of a molecular marker of connectivity (wheat germ agglutinin lectin-bound horseradish peroxidase [HRP]; axonal uptake and retrograde transport from neocortex) and immunohistochemical labeling of molecular markers of transmitter synthesis (glutamic acid decarboxylase [GAD]: choline acetyltransferase [ChAT]) were combined in adult cats and examined by light microscopy. Adjoining partial profiles of the same neurons in the basal forebrain co-localized GAD + HRP and ChAT + HRP in adjacent faces of serial tissue sections. GAD + ChAT were also co-localized within individual profiles of neurons in the basal forebrain from single tissue sections. The results indicate that infrequent corticopetal neurons in the basal forebrain can produce both gamma-aminobutyric acid and acetylcholine.     

Developmental change in the nerve growth factor action from induction of choline acetyltransferase to promotion of cell survival in cultured basal forebrain cholinergic neurons from postnatal rats

Nerve growth factor (NGF), a well-characterized target-derived growth factor, has been postulated to promote neuronal differentiation and survival of the basal forebrain cholinergic neurons. In the present paper, we demonstrate that a developmental change in NGF action occurs in postnatal rat basal forebrain cholinergic neurons in culture. Firstly, NGF acts as maturation factor by increasing choline acetyltransferase (ChAT) activity and acts later as a survival factor. In dissociated cell cultures of septal neurons from early postnatal (P1-4) rats, ChAT activities were increased by the addition of NGF. That is, ChAT activities in P1 septal cells cultured for 7 days was increased 4-fold in the presence of NGF at a concentration of 100 ng/ml. However, the number of the acetylcholinesterase (AChE)-positive neurons was not significantly different between these groups. In contrast, septal neurons from P8 to P14 rats showed different responses to NGF. Although the P14 septal neurons in culture for 7 days without NGF lost about half of the ChAT activity during a 7-day cultivation, cells cultured with NGF retained the activity at the initial level. The number of AChE-positive neurons counted in cultures with NGF was much greater than the number without NGF. These results suggest that, during the early postnatal days, the action of NGF on the septal cholinergic neurons in culture changes from induction of ChAT activity to the promotion of cholinergic neuronal cell survival. During this developmental period in vivo, septal neurons are terminating their projections to the hippocampal formation. Similar NGF-regulated changes in cholinergic neurons were observed in cultured postnatal neurons from vertical limb of diagonal band. An analogy has been pointed out between the neuronal death of the basal forebrain cholinergic neurons and a similar neuronal death in senile dementia, especially Alzheimer's type. The work reported here might present a possibility that NGF could play a role in preventing the loss of the basal forebrain cholinergic neurons in this disease.     

Interleukin-6 is not expressed in activated microglia and in reactive astrocytes in response to lesion of rat basal forebrain cholinergic system as demonstrated by combined in situ hybridization and immunocytochemistry

Interleukin-6 may play an essential role in early inflammatory processes as response to degenerating cholinergic cells in the nucleus basalis of Meynert in patients suffering Alzheimer's disease. The cholinergic immunotoxin, 192IgG-saporin, was applied to produce selective and specific degenerations of basal forebrain cholinergic cells. To disclose the lesion-induced temporal cascade of the expression pattern of IL-6, and to reveal the cellular source for production and secretion of IL-6 in vivo after endogeneously induced basal forebrain cholinergic cell loss, both in situ hybridization and immunocytochemistry for IL-6 were performed. To identify the cell types expressing IL-6 mRNA, double labeling techniques were applied combining in situ hybridization technique with immunocytochemistry and lectin histochemistry for both micro- and astroglia and a number of neuronal markers including choline acetyltransferase, parvalbumin, and neurofilaments. In the intact brain, IL-6 is mainly localized in neurons, in particular in both cholinergic and GABAergic neurons of the basal forebrain. Although basal forebrain cholinergic lesion resulted in a dramatic increase in the number of micro- and astroglial cells at the lesion site, IL-6 expression could not be detected in any of the lesion-induced activated glial cell types. Moreover, cholinergic lesion led to a reduced number of IL-6-expressing cells in the basal forebrain, which is assumed to be due to the loss of cholinergic cells. The predominantly neuronal localization in rat brain suggests a role for IL-6 in activating micro- and astroglial cells in response to degenerating cholinergic neurons. J. Neurosci. Res. 51:223–236, 1998. © 1998 Wiley-Liss, Inc.     

Human nerve growth factor prevents degeneration of basal forebrain cholinergic neurons in primates

Basal forebrain cholinergic neurons respond to nerve growth factor (NGF), and it has been suggested that the administration of NGF might prevent their degereration in patients with Alzheimer's disease. One major prerequisite to be fulfilled before the consideration of clinical trials of NGF in patients with Alzheimer's disease is the demonstration that human NGF affects basal forebrain cholinergic neurons in primates. In the present study, we used a recombunant human nerve growth facotr (rhNGF), which we previousl showed to be active on rat basa forebrain cholinergic neurons, in nonhuman primates with a unilateral transection of the fornix (a well-established model for the induction of retrograde degenerative changes in septal cholinergic neurons). After the lesion, one group of animals received rhNGF and a second group received vehicle solution for 2 weeks. In animals receiving vehicle, the medical septal nucleus ipsilateral to the lesion showed reductions in numbers (55°) and size of cell bodies immunoreactive for NGF receptor and choline acetyltransferase. In Sissl stains, many cells showed frduced size and basophilia. The rhNGF completely prevented alternation in the number and size of NGF receptor—and choline acetyltransferase—immunnoreactive neurons in the medical septal nucles and reversed atrophy in a subpopulation of large, basophilic medical which we have previously used in the same primate lesion paradigm. The restoration of the phenotype of injured acetylcholine-dependent memory impariments that occur in aged nonhuman primates. In concert, results of the present investigation provide critical information for the futrue use of NGF in in patients with neurological disorders that affect NGF-responsive cells in the peripherl and central nervous system.     

GABAergic neurons in the primate basal forebrain magnocellular complex

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

Low voltage-activated calcium and fast tetrodotoxin-resistant sodium currents define subtypes of cholinergic and noncholinergic neurons in rat basal forebrain

Neurons of the basal forebrain (BF) possess unique combinations of voltage-gated membrane currents. Here, we describe subtypes of rat basal forebrain neurons based on patch-clamp analysis of low-voltage activated (LVA) calcium and tetrodotoxin-resistant (TTX-R) sodium currents combined with single-cell RT-PCR analysis. Neurons were identified by mRNA expression of choline acetyltransferase (ChAT+, cholinergic) and glutamate decarboxylase (GAD67, GABAergic). Four cell types were encountered: ChAT+, GAD+, ChAT+/GAD+ and ChAT-/GAD- cells. Both ChAT+ and ChAT+/GAD+ cells (71/75) displayed LVA currents and most (34/39) expressed mRNA for LVA Ca(2+) channel subunits. Ca(v)3.2 was detected in 31/34 cholinergic neurons and Ca(v)3.1 was expressed in 6/34 cells. Three cells expressed both subunits. No single neurons showed Ca(v)3.3 mRNA expression, although BF tissue expression was observed. In young rats (2-4 mo), ChAT+/GAD+ cells displayed larger LVA current densities compared to ChAT+ neurons, while these latter neurons displayed an age-related increase in current densities. Most (29/38) noncholinergic neurons (GAD+ and ChAT-/GAD-) possessed fast TTX-R sodium currents resembling those mediated by Na(+) channel subunit Na(v)1.5. This subunit was expressed predominately in noncholinergic neurons. No cholinergic cells (0/75) displayed fast TTX-R currents. The TTX-R currents were faster and larger in GAD+ neurons compared to ChAT-/GAD- neurons. The properties of ChAT+/GAD+ neurons resemble those of ChAT+ neurons, rather than of GAD+ neurons. These results suggest novel features of subtypes of cholinergic and noncholinergic neurons within the BF that may provide new insights for understanding normal BF function.     

Development of cholinergic pedunculopontine neurons in vitro: comparison with cholinergic septal cells and response to nerve growth factor, ciliary neuronotrophic factor, and retinoic acid

The well-documented role of nerve growth factor (NGF) in the function of cholinergic neurons in the mammalian basal forebrain can be regarded as a paradigm for the action of trophic substances on CNS neurons. Although several growth factors have been identified in recent years, the specificities and importance of such factors for the development of the nervous system are still unknown. In the present study it has been tested whether NGF affects the group of pedunculopontine cholinergic neurons. This population, which has been described in detail only recently, is located more caudally than but resembles, in some aspects, the basal forebrain cholinergic neurons. The cell bodies are located in the metencephalic pedunculopontine and dorsolateral tegmental nuclei. Similar to the forebrain cholinergic neurons, they are medium to large in size and ascend centrally with long axons. Projection areas are widespread throughout the mesencephalon and diencephalon. Dissociated pontine and septal cells of fetal rat brain (embryo ages E14 to E17) were grown in culture for 7 to 14 days in the presence or absence of NGF. Furthermore, a possible action of retinoic acid and ciliary neuronotrophic factor (CNTF) on cholinergic neurons of both the basal forebrain and the pontine area were tested. Differentiation of cultured cholinergic neurons was assessed by biochemical determination of choline acetyltransferase (ChAT) activity and by immunocytochemical staining for ChAT. NGF in concentrations of 1 to 1,000 ng/ml medium increased the number of immunostained cells and the staining intensity in ChAT immunocytochemistry and enhanced ChAT activity by at least 100% above control levels in septal cultures, thus confirming earlier results. In marked contrast, the same concentrations of NGF failed to influence ChAT activity or immunocytochemical staining in cultures of the pontine area. Retinoic acid (10(-8) M to 10(-5) M) and CNTF (0.2 and 2.0 ng/ml, corresponding to 1 and 10 trophic units, as defined in the ciliary ganglion cell assay) failed to enhance ChAT activity in either culture system and did not potentiate the NGF-mediated increase of ChAT activity in septal cultures. Our results, which indicate that pedunculopontine cholinergic neurons do not respond to NGF during development, are in line with those of NGF-receptor visualization studies that failed to demonstrate such receptors on cholinergic pontine cells in postnatal and adult rats. The findings further underline the specificity of NGF action in the central nervous system and, in particular, do not support the idea of transmitter-specific neurotrophic factors.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cholinergic projections from the basal forebrain to the basolateral amygdaloid complex: a combined retrograde fluorescent and immunohistochemical study

We have examined the location of cholinergic and non-cholinergic neurons that project to the rat basolateral amygdaloid nucleus by using choline acetyltransferase (ChAT) immunohistochemistry in combination with retrograde fluorescent tracing on the same tissue section. Since many tracer-and ChAT-positive neurons were identified in basal forebrain areas, including the ventral pallidum, we also stained many of the sections for glutamate decarboxylase, a suitable marker for the delineation of pallidal areas. Cholinergic neurons projecting to the basolateral amygdaloid nucleus were observed in a continuous territory stretching from the dorsal part of ventral pallidum, through sublenticular substantia innominata to ventral parts of globus pallidus and peripallidal areas. Non-cholinergic neurons projecting to the basolateral amygdaloid nucleus were found intermixed within the same structures and constitute approximately 25% of the amygdalopetal projection neurons in these ventral forebrain structures. Since amygdalopetal cholinergic neurons were demonstrated in areas generally recognized as giving rise to cholinergic projections to cerebral cortex, several retrograde double-labeling experiments with two different fluorescent tracers were performed for the purpose of detecting the possible existence of collateral projections. The results obtained showed that the cholinergic basal forebrain neurons in general project to only one forebrain region, and, furthermore, that the cholinergic system consists of partially overlapping subsets of neurons that project to various neocortical and allocortical areas and to the amygdaloid body.