Nerve growth factor and phorbol esters increase the number of choline acetyltransferase-positive cells in two morphologically distinct classes of basal forebrain neurons in primary cultures.

Nerve growth factor (NGF) has been shown to be active in the CNS as a neurotrophic agent. Cholinergic neurons of the basal forebrain are one cell type in the CNS which have been identified as a target for NGF. When dissociated cell cultures from the basal forebrain were treated for 7 days with NGF (20 ng/100 microliters), the number of choline acetyltransferase (ChAT)-immunopositive cells was increased from 30 +/- 6 to 58 +/- 3. Cholinergic cells taken from the basal forebrain exhibit 3 different morphologies: stellate, pyramidal, and bipolar. The NGF treatment was found to increase the number of stellate cells from 7 +/- 2 to 23 +/- 2 and the number of pyramidal cells from 14 +/- 2 to 26 +/- 2, but had no effect on the number of bipolar cells. The activation of protein kinase C by phorbol 12-myristate, 13-acetate (TPA) also increased the number of ChAT-positive cells in a dose-dependent manner. A maximal increase was observed with 10 ng/ml of TPA which increased the number of positive cells from a basal level of 21 +/- 4 to 42 +/- 4. As was the case with NGF, only the stellate and pyramidal cells were affected by the phorbol ester treatment. In co-addition experiments, the cultures were treated with 10 ng/100 of NGF and 10 ng/ml of TPA, with the result that there was no further increase in the number of immunopositive cells over the NGF controls. These results suggest that the mechanisms by which NGF and TPA increase the number of ChAT-positive cells are interactive at some point. The effect of TPA at the higher doses of NGF was distinctly different. When cells were treated with 20 ng/100 microliters of NGF and 0.05-50 ng/ml of TPA, the NGF response was down-regulated to the level of the vehicle-treated controls.

Complete sequence of a cDNA encoding an active rat choline acetyltransferase: a tool to investigate the plasticity of cholinergic phenotype expression.

A cDNA clone encoding the complete sequence of an active rat choline acetyltransferase (ChoAcTase; acetyl-CoA:choline O-acetyltransferase, EC 2.3.1.6) has been isolated. Analysis of the deduced amino acid sequence reveals 85% and 31% identity with the porcine and Drosophila melanogaster enzymes, respectively. To further elucidate the molecular basis of neurotransmitter-related phenotypic plasticity, the expression of ChoAcTase mRNA was compared with that of tyrosine hydroxylase [TH; tyrosine 3-monooxygenase, L-tyrosine, tetrahydropteridine:oxygen oxidoreductase (3-hydroxylating), EC 1.14.16.2], in neurons from superior cervical ganglia grown in the following conditions: 1) normal medium, 2) high K+ medium, and 3) normal medium supplemented with 50% muscle-conditioned medium (CM). TH mRNA was expressed in all three media; its level rose in high K+ and decreased strikingly in the presence of CM. ChoAcTase mRNA could be visualized in CM, but fell to undetectable levels in normal and high K+ media. These results suggest that translational or post-translational mechanisms do not play a major role for the modulation of neurotransmitter-associated phenotype.

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.

Human reticular formation: cholinergic neurons of the pedunculopontine and laterodorsal tegmental nuclei and some cytochemical comparisons to forebrain cholinergic neurons.

Choline acetyltransferase immunohistochemistry showed that the human rostral brainstem contained cholinergic neurons in the oculomotor, trochlear, and parabigeminal nuclei as well as within the reticular formation. The cholinergic neurons of the reticular formation were the most numerous and formed two intersecting constellations. One of these, designated Ch5, reached its peak density within the compact pedunculopontine nucleus but also extended into the regions through which the superior cerebellar peduncle and central tegmental tract course. The second constellation, designated Ch6, was centered around the laterodorsal tegmental nucleus and spread into the central gray and medial longitudinal fasciculus. There was considerable transmitter-related heterogeneity within the regions containing Ch5 and Ch6. In particular, Ch6 neurons were intermingled with catecholaminergic neurons belonging to the locus coeruleus complex. The lack of confinement within specifiable cytoarchitectonic boundaries and the transmitter heterogeneity justified the transmitter-specific Ch5 and Ch6 nomenclature for these two groups of cholinergic neurons. The cholinergic neurons in the nucleus basalis (Ch4) and those of the Ch5-Ch6 complex were both characterized by perikaryal heteromorphism and isodendritic arborizations. In addition to choline acetyltransferase, the cell bodies in both complexes also had high levels of acetylcholinesterase activity and nonphosphorylated neurofilament protein. However, there were also marked differences in cytochemical signature. For example, the Ch5-Ch6 neurons had high levels of NADPHd activity, whereas Ch4 neurons did not. On the other hand, the Ch4 neurons had high levels of NGF receptor protein, whereas those of Ch5-Ch6 did not. On the basis of animal experiments, it can be assumed that the Ch5 and Ch6 neurons provide the major cholinergic innervation of the human thalamus and that they participate in the neural circuitry of the reticular activating, limbic, and perhaps also extrapyramidal systems.

Regulation of production by primary cultures of rat forebrain astrocytes of a trophic factor important for the development of cholinergic neurons.

The possibility was examined for the production of a trophic factor by astrocytes important for the biochemical development of cholinergic neurons. The activity of choline acetyltransferase (ChAT) was used as a marker to study the maturation of cholinergic neurons, while the metabolic state of astrocytes was monitored in terms of glutamine synthetase activity. When the dissociated cells, derived from the septal-diagonal band region of embryonic rat brain, were cultured either on a preformed layer of highly enriched astrocytes or in the presence of astrocyte-conditioned medium, a marked increase was observed in the expression of ChAT activity. Similar effect was not noticed when cerebellar granule cells replaced the astrocytes. The production of cholinergic factor was decreased when the astroglial cells were induced to proliferate by epidermal growth factor, whereas its production was increased when quiescent astrocytes were treated with a low dose of cytosine arabinoside. The results would indicate that a trophic factor important for the development of cholinergic cells is produced by astrocytes, and that exogenously added agents influencing astroglial metabolism have a marked regulatory effect on the production of this trophic factor.

Brainstem projecting neurons in the rat basal forebrain: neurochemical, topographical, and physiological distinctions from cortically projecting cholinergic neurons.

Magnocellular regions of the basal forebrain contain cholinergic neurons that project to the cerebral cortex. Neurons in the same basal forebrain regions innervate the brainstem. The present study investigated whether these brainstem projecting neurons are cholinergic, project also to the cortex, and share similar physiological properties as cortically projecting neurons. Data with retrograde tracing from various regions of the pons, medulla, and cortex combined with choline acetyltransferase immunofluorescence indicated that: 1) brainstem projecting neurons are usually segregated from cortically projecting and/or cholinergic neurons in the basal forebrain, 2) virtually no brainstem projecting neurons in the basal forebrain are cholinergic, and 3) only rarely do basal forebrain neurons have axon collaterals that project to both cortex and brainstem. Extracellular recordings from basal forebrain neurons confirmed the paucity of axonal collateralization and the topographic segregation between cortically and brainstem projecting basal forebrain neurons, and, in addition, showed that brainstem projecting neurons have a slower mean conduction velocity than cortically projecting neurons. These observations suggest that basal forebrain neurons projecting to the brainstem (pons, medulla) and the cortex represent separate cell populations in terms of projections, neurotransmitter content, distribution, and physiological properties.

Localization of Drosophila neurons that contain choline acetyltransferase messenger RNA: an in situ hybridization study.

In situ hybridization with radiolabeled complementary RNA (cRNA) probes was used to determine the location of the messenger RNA (mRNA) encoding choline acetyltransferase (ChAT) in Drosophila nervous system. Areas in the cell-rich cortical regions of the cerebrum and optic lobes hybridized with substantial concentrations of the probe. This contrasted with the cell-sparse neuropil areas where no significant concentrations of probe were observed. Although most of the cortical regions were substantially labeled, there were regions within all of the areas where labeling was sparse or nonexistent. For example in the lamina, even though the monopolar cell layer appeared to be heavily labeled, there were some neuronal profiles that were not associated with the probe. Moreover, the epithelial glia that form an arch of cell profiles subjacent to the monopolar cells were not labeled, nor were amacrine neurons in the apex of the lamina near the external optic chiasma. The highest concentration of probe (approximately 140 grains/400 microns2) was observed in the laminar monopolar cell region and the cerebral cortical rind. The next most heavily labeled region (approximately 90 grains/400 microns2) occurred over cortical cells of the medulla-lobula. In the peripheral nervous system, label over the antennal sensory neurons amounted to about 75 grains/400 microns2, and the retinular cell layer of the compound eye exhibited about 60 grains/400 microns2. The control probe did not hybridize in significant quantities in either cellular or noncellular regions. This study presents evidence that large numbers of Drosophila cortical and primary sensory neurons contain the messenger RNA necessary for the production of ChAT, the acetylcholine-synthesizing enzyme. Further, our findings provide baseline information for use in ontogenetic studies of cholinergic neurons in Drosophila, and they also provide normative data for studying the effects of mutant alleles at the Cha or Ace loci upon the transcription of ChAT messenger RNA.

A new parcellation of the human thalamus on the basis of histochemical staining.

Serial sections of human thalami, cut in the 3 standard planes, were stained in alternating series for Nissl substance, myelin, cytochrome oxidase and acetylcholinesterase. Nissl and acetylcholinesterase-stained sections revealed a parcellation of the nuclei that could be correlated with that used in the macaque monkey thalamus. Human nuclei were accordingly re-named using the monkey nomenclature. Apart from differences of size, the nuclei of the human and monkey thalamus are remarkably similar. In the human ventral nuclear complex there is a very clear histochemical distinction between nuclei which, on the basis of comparison with the monkey, probably form the pallidal, cerebellar and lemniscal relays to premotor, motor and somatic sensory cortex, respectively. In the human somatic sensory relay nucleus there is a further clear cytoarchitectonic distinction between components that are probably equivalent to the relays for deep and cutaneous receptors in the equivalent monkey nucleus.

Afferents to the basal forebrain cholinergic cell area from pontomesencephalic--catecholamine, serotonin, and acetylcholine--neurons.

The afferent input to the basal forebrain cholinergic neurons from the pontomesencephalic tegmentum was examined by retrograde transport of wheatgerm agglutinin-horseradish peroxidase in combination with immunohistochemistry. Multiple tyrosine hydroxylase-, dopamine-beta-hydroxylase-, serotonin- and choline acetyltransferase-immunoreactive fibres were observed in the vicinity of the choline acetyltransferase-immunoreactive cell bodies within the globus pallidus, substantia innominata and magnocellular preoptic nucleus. Micro-injections of horseradish peroxidase-conjugated wheatgerm agglutinin into this area of cholinergic perikarya led to retrograde labelling of a large population of neurons within the pontomesencephalic tegmentum, which included cells in the ventral tegmental area, substantia nigra, retrorubral field, raphe nuclei, reticular formation, pedunculopontine tegmental nucleus, laterodorsal tegmental nucleus, parabrachial nuclei and locus coeruleus nucleus. Of the total population of retrogradely labelled neurons, a significant (approximately 25%) proportion were tyrosine hydroxylase-immunoreactive and found in the ventral tegmental area (A10), the substantia nigra (A9), the retrorubral field (A8), the raphe nuclei (dorsalis, linearis and interfascicularis) and the locus coeruleus nucleus (A6), Another important contingent (approximately 10%) was represented by serotonin neurons of the dorsal raphe nucleus (B7), the central superior nucleus (B8) and ventral tegmentum (B9). A small proportion (less than 1%) was represented by cholinergic neurons of the pedunculopontine (Ch5) and laterodorsal (Ch6) tegmental nuclei. These results demonstrate that pontomesencephalic monoamine neurons project in large numbers up to the basal forebrain cholinergic neurons and may represent a major component of the ventral tegmental pathway that forms the extra-thalamic relay from the brainstem through the basal forebrain to the cerebral cortex.

Immunohistochemical localization of cells containing nerve growth factor receptors in the different regions of the adult rat forebrain.

Conditions have been optimized for an immunohistochemical procedure for the localization of nerve growth factor receptor-containing cells in the brain. Using this immunohistochemical procedure, the normal morphology and distribution of the nerve growth factor receptor-containing neurons of the adult rat forebrain have been studied, and the findings compared with observations on the choline acetyltransferase-positive neurons present either in the immediately-adjacent sections or in the medially-divided half of the same section. Unlike in the peripheral nervous system, only neurons showed immunoreactivity to the nerve growth factor receptor in the brain. Both the nerve growth factor receptor- and choline acetyltransferase-immunoreactive cells appear to form a continuous anteroposterior band, which includes the olfactory tubercle, the medial septal nucleus, the vertical and horizontal limbs of the diagonal band and the basal nucleus. In each subdivision of the basal forebrain, the topographic organization, the localization, the intensity of the immunoreaction and the total cell number of nerve growth factor receptor- and of choline acetyltransferase-immunoreactive neurons were strikingly similar, indicating that nearly all nerve growth factor receptor-containing cells were cholinergic neurons. However, in the striatum, only about half the number of the choline acetyltransferase-positive cells showed immunopositive reactions to the nerve growth factor receptor, and, also, in the nerve growth factor receptor-containing neurons the intensity of the reaction product was much less than the choline acetyltransferase immunoreactivity. In the neurons of the basal forebrain nuclei, the choline acetyltransferase immunoreaction product was uniformly distributed on the cell bodies, while the nerve growth factor receptor immunoreaction product was present also as intensely stained granules on the cell somata and the dendrites. The mean diameter and the mean cross-sectional area of the nerve growth factor receptor-containing neurons were least in the medial septal nucleus and were greatest in the basal nucleus, and showed a gradation in cell size going from the medial septal nucleus through the nucleus of the diagonal band and extending more posteriorly to the basal nucleus.

Distribution and some projections of cholinergic neurons in the brain of the common marmoset, Callithrix jacchus.

The distribution of choline acetyltransferase-immunoreactive (ChAT-IR) neurons was studied in the brain of the common marmoset by using immunohistochemistry. ChAT-IR neurons were found in the medial septal nucleus, vertical and horizontal limb nuclei of the diagonal band, the nucleus basalis of Meynert, pedunculopontine nucleus and laterodorsal tegmental nucleus, and also in the striatum, habenula, and brainstem cranial nerve motor nuclei. The organization of ChAT-IR neurons in the basal forebrain, midbrain, and pons is consistent with the Ch1-Ch6 nomenclature introduced by Mesulam et al. ('83). The combination of the retrograde transport of HRP-WGA with ChAT immunohistochemistry revealed the distribution of neurons in the Ch4 cell group projecting to the dorsolateral prefrontal cortex. The activity of ChAT was highest in limbic cortical structures, such as the hippocampus, and lowest in association areas of the neocortex. Lesions at various loci in the basal forebrain resulted in differential patterns of ChAT loss in the cortex, which suggests some degree of topographical organization of Ch4 projections to the cortical mantle.

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.

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.

Basal forebrain neurons project to the cortical mantle of the European hedgehog (Erinaceus europaeus).

The basal forebrain projections to the cortical mantle of the hedgehog were traced using the WGA-HRP method. The nuclei of the vertical and horizontal limbs of the diagonal band of Broca, the nucleus basalis and the substantia innominata were found to project to the ipsilateral frontal, parietal and occipital cortices. These projections, apparently cholinergic, were not topographically organized.

Distribution of cholinergic neurons and fibers in the hypothalamus of the rat using choline acetyltransferase as a marker.

The distribution of choline-acetyltransferase-like immunoreactive structures in the rat hypothalamus and preoptic area was examined by using avidin-biotin immunocytochemistry. We found that the hypothalamus is richly innervated by the cholinergic neuron system. Sites containing cholinergic neurons of varying density were: medial and lateral preoptic areas, septohypothalamic nucleus, median preoptic area, lateral hypothalamus including the perifornical area, anterior hypothalamic nucleus, arcuate nucleus, dorsomedial hypothalamic nucleus, posterior hypothalamic nucleus, dorsal and ventral premammillary nuclei, neuropil mediodorsal to the anterior hypothalamic nucleus, neuropil ventral to the anterior hypothalamic nucleus and ventromedial hypothalamic nucleus, neuropil between lateral hypothalamus and ventromedial hypothalamus, and neuropil between dorsal premammillary nucleus and posterior hypothalamic nucleus. There were also many varicose and non-varicose fibers in the preoptic area and hypothalamus. Two kinds of varicose fibers, one with strong immunoreactivity and the other with weak immunoreactivity, were seen. Non-varicose fibers were also detected in the optic chiasma and habenulo-interpeduncular tract. These fibers were passing fibers.

Morphological characterization of cholinergic neurons in the horizontal limb of the diagonal band of Broca in the basal forebrain of the rat.

A monoclonal antibody against choline acetyltransferase (ChAT), the acetylcholine synthesizing enzyme, was used to identify cholinergic neurons in the nucleus of the horizontal limb of the diagonal band of Broca at the light and electron microscopic levels. ChAT-labelled somata were fusiform, triangular or round in shape and varied considerably in size. Depending on the type of the cell, one to four dendrites emerged from the soma, but an axon could rarely be seen. The nuclei of most cells were round or oval, showed invaginations and displayed prominent nucleoli. The karyoplasm of the larger fusiform and triangular neurons contained abundant organelles including parallel arrays of granular endoplasmic reticulum. The synaptic input to labelled perikarya and proximal dendrites was sparse. It consisted chiefly of asymmetrical synaptic contacts, sometimes with postjunctional densities, but a few symmetrical synapses were also noted. ChAT-positive axon terminals were not identified which suggests that axon collaterals are rare within the nucleus of the horizontal limb of the diagonal band of Broca.