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.

Interaction between cholinergic neurons and substance P or calcitonin gene-related peptide terminals of the rat sacral intermediolateral nucleus: double immunostaining at the light and electron microscopic levels.

The relationships both between cholinergic neurons and substance P (SP) and between cholinergic neurons and calcitonin gene-related peptide (CGRP) terminals were examined in the rat sacral intermediolateral nucleus at the light and electron microscopic levels by means of double-immunostaining methods. Cholinergic neurons were labeled by a monoclonal antibody to choline acetyltransferase (CAT) with the avidin-biotin technique and stained bluish-green by indolyl-beta-galactoside reaction products with beta-galactosidase as a marker. On the same sections, SP or CGRP fibers were labeled by polyclonal antisera to SP or CGRP after application of the peroxidase-antiperoxidase (PAP) method and stained brown by the p-dimethylaminoazobenzene (DAB) reaction. After embedding in Epon, light and electron microscopic sections were examined. At the light microscopic level, CGRP-like immunoreactive (CGRP-I) fibers and SP-like immunoreactive (SP-I) fibers were found to pass through the lateral edge of the dorsal horn and then into the dorsal region of the sacral intermediolateral nucleus. In addition, SP-I fibers also extend from the dorsolateral funiculus into the entire sacral intermediolateral region. At the electron microscopic level, many axosomatic and axodendritic synapses were found between CAT-I structures and SP-I terminals in the intermediolateral nucleus, whereas most of the CGRP-I terminals in this area made axodendritic synapses with CAT-I dendrites. These results indicate that cholinergic neurons in the sacral intermediolateral nucleus receive direct synaptic input from SP-I and CGRP-I terminals.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Parasympathetic nerves in penile erectile tissue of the rat contain choline acetyltransferase.

Choline acetyltransferase (ChAT), a biochemical marker of cholinergic neurons, was measured in the erectile tissue of intact rats and in rats in which postganglionic fibers from the pelvic plexus were interrupted. ChAT activity in the denervated erectile tissue fell by 56% compared to control tissues. Acetylcholinesterase positive (AChE+) nerves also fell by about 48%. Penile neurons distal to the lesion probably account for the residual ChAT activity and remaining AChE+ nerve fibers in erectile tissue. These results indicate that acetylcholine is an important neurotransmitter in the regulation of penile erection.

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.

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.

Evidence for the existence of a population of arcuate neurons costoring choline acetyltransferase and tyrosine hydroxylase immunoreactivities in the male rat.

By combined immunoperoxidase and immunofluorescence histochemistry we have analyzed the distribution of choline acetyltransferase (ChAT) and tyrosine hydroxylase (TH) immunoreactive (IR) perikarya within the same sections of the mediobasal hypothalamus of the male rat. Evidence was obtained for the existence of perikarya costoring TH and ChAT immunoreactivities in both the dorsomedial and ventrolateral part of the arcuate nucleus and in the adjacent periarcuate nucleus at all rostrocaudal levels. The results strongly implicate interactions between dopamine and acetylcholine as well as acetylcholine and growth hormone releasing factor in dorsomedially and ventrolaterally located TH/ChAT costoring tuberoinfundibular neurons, respectively.

Embryonic spinal cord neurons develop preferential cholinergic projections to sympathetic ganglia explanted from appropriate levels of the neuraxis.

To determine whether cholinergic spinal cord neurons can develop preferential projections in vitro within sympathetic ganglia (SGs) of appropriate levels of the neuraxis, organotypic explants of fetal mouse spinal cord (E13) from cervical, thoracic (upper and lower) and lumbar segments were co-cultured with either pairs of neonatal SGs: the rostral superior cervical ganglion (SCG) or a caudally located upper lumbar ganglion (LG). After 3.5-4 weeks of co-culture, levels of the enzyme, choline acetyltransferase (ChAT), were measured in individual spinal cord explants and SCGs or double LGs (to match the target mass of a single SCG). Interaction was assumed to occur primarily between an SCG or LG doublet and the adjacent ipsilateral half of the co-cultured cord segment. An index of cholinergic interaction was defined as the ganglion ChAT activity per unit ChAT activity in half co-cultured cord segment. The index of interaction with the SCG was highest with the T1/T2 (1.4) as compared with the T10/T11 (0.79), L1/L2 (0.38) and C2/C3 (0.11) segments. In contrast, the index of cholinergic interaction with double LGs was highest with the more caudally located T10/T11 (0.62) cord segment as compared with the rostral T1/T2 (0.33), cervical C2/C3 (0.2) and lumbar L1/L2 (0.17) segments. Ganglion compound action potentials evoked in LGs by stimulation of the ipsilateral portion of T10/T11 cord were blocked by the ganglionic antagonist, hexamethonium, as previously observed in co-cultures of SCGs with T1/T2 cord. These results indicate that pools of preganglionic neurons in thoracic cord segments can develop in vitro preferential cholinergic projections within SGs of appropriate position. Cervical and lumbar cord segments which contain a preponderance of somatic motoneurons over preganglionic neurons did not interact as effectively with either type of SG. The preferential cholinergic projections from rostral thoracic cord explants within co-cultured SCGs and from caudal thoracic cord explants within co-cultured SCGs and from caudal thoracic cord explants within LGs may reflect some degree of positional preference intrinsic to embryonic spinal cord neurons and/or their appropriate target SGs, consistent with the positional specificity expressed by preganglionic neurons and SGs in situ.

Afferent and efferent connections of the cholinoceptive medial pontine reticular formation (region of the ventral tegmental nucleus) in the cat.

Following minor concussive brain injury when there is an otherwise general suppression of CNS activity, the ventral tegmental nucleus of Gudden (VTN) demonstrates increased functional activity (32). Electrical or pharmacological activation of a cholinoceptive region in this same general area of the medial pontine tegmentum contributes to certain components of reversible traumatic unconsciousness, including postural atonia (31, 32, 45). Therefore, in an effort to examine the neuroanatomical basis of the behavioral suppression associated with a reversible traumatic unconsciousness, the afferent and efferent connections of the VTN and putative cholinoceptive medial pontine reticular formation (cmPRF) were studied in the cat using the retrograde horseradish peroxidase (HRP), HRP/choline acetyltransferase (ChAT) double-labeling immunohistochemistry, and anterograde HRP and autoradiographic techniques. Based upon retrograde HRP labeling, the principal afferents to the VTN region of the cmPRF originated from the medial and lateral mammillary nuclei, and lateral habenular nucleus, and to a lesser extent from the interpeduncular nucleus, lateral hypothalamus, dorsal tegmental nucleus, superior central nucleus, and contralateral nucleus reticularis pontis caudalis. Other afferents, which were thought to have been labeled through spread of HRP into the medial longitudinal fasciculus (MLF), adjacent paramedian pontine reticular formation, or uptake by transected fibers descending to the inferior olive, included the nucleus of Darkschewitsch, interstitial nucleus of Cajal, zona incerta, prerubral fields of Forel, deep superior colliculus, nucleus of the posterior commissure, nucleus cuneiformis, ventral periaqueductal gray, vestibular complex, perihypoglossal complex, and deep cerebellar nuclei. In HRP/ChAT double labeling studies, only a very small number of cholinergic VTN afferent neurons were found in the medial parabrachial region of the dorsolateral pontine tegmentum, although the pedunculopontine and laterodorsal tegmental nuclei contained numerous single-labeled ChAT-positive cells. Anterograde HRP and autoradiographic findings demonstrated that the VTN gave rise almost exclusively to ascending projections, which largely followed the course of the mammillary peduncle (16,21) and medial forebrain bundle, or the tegmentopeduncular tract (4). The majority of fibers ascended to terminate in the medial and lateral mammillary nuclei, interpeduncular complex (especially paramedian subnucleus), ventral tegmental area, lateral hypothalamus, and the medial septum in the basal forebrain. Labeling that joined the mammillothalamic tract to terminate in the anterior nuclear complex of the thalamus was thought to occur transneuronally. Some projections were also observed to nucleus reticularis pontis oralis and caudalis, superior central nucleus, and dorsal tegmental nucleus adjacent to the VTN...

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.

Aberrant phosphorylation of neurofilaments accompanies transmitter-related changes in rat septal neurons following transection of the fimbria-fornix.

Lesions of the fimbria-fornix (FF) have been reported to cause retrograde changes in neurons of the medial septal nucleus (MSN). To analyze the nature and time course of these events, we investigated changes in cytoskeletal elements (phosphorylated and non-phosphorylated neurofilament (NF) proteins) and transmitter-related enzymes (choline acetyltransferase (ChAT) in MSN neurons following FF transection. During the first week postlesion, ChAT immunoreactivity and size of many perikarya were reduced. Irregular, swollen cholinergic fibers appeared first at postlesion day 2 in caudal septum and soon spread rostrally, reaching rostral septum by day 7. A few perikarya developed abnormal accumulations of phosphorylated NFs. At postlesion days 7-10, many neurons did not stain for ChAT. Phosphorylated NFs were present in many perikarya. At this time, cell loss was apparent in Nissl-stained material. Cholinergic cell loss continued through postlesion weeks 6-8 but at a much slower rate than during the first week. Phosphorylated NF accumulations in MSN perikarya persisted until postlesion week 6, disappearing thereafter. Double-immunostaining procedures showed that MSN neurons expressed both ChAT and phosphorylated NF immunoreactivity at postlesion day 3; however, at days 7 and 14, cells that accumulated phosphorylated NFs did not stain for ChAT. The results of this study indicate that FF transection leads to perikaryal shrinkage with loss of ChAT immunoreactivity, perikaryal phosphorylation of NFs, cholinergic fiber abnormalities, and cell loss. Recent evidence suggests that reduction of transmitter markers and aberrant phosphorylation of NFs may be involved in the pathogenesis of several neurodegenerative disorders, including Alzheimer's disease. Therefore, FF transection provides a useful animal model for further investigations of complex disorders of the central nervous system that involve degeneration of transmitter-specific pathways.

In vitro electrophysiology of neurons in the lateral dorsal tegmental nucleus.

The lateral dorsal tegmental nucleus (LDT) provides ascending cholinergic projections to forebrain structures such as prefrontal cortex, septum, habenula, and thalamus, but relatively little is known of the physiology of LDT neurons. Intracellular recordings from LDT neurons in guinea pig brain slices found that most neurons fired action potentials either tonically or in bursts. The voltage dependent characteristics of the neurons suggest that a prolonged afterhyperpolarization due to an outward potassium current and a low-threshold calcium conductance contributed to these two modes of firing. Intracellular injections of Lucifer Yellow and subsequent staining for NADPH-diaphorase activity permitted positive identification of cholinergic neurons.

Immunohistochemical localization of choline acetyltransferase in the chicken mesencephalon.

Choline acetyltransferase, a specific marker for cholinergic neurons, has been immunohistochemically localized in the mesencephalon and in the caudal diencephalon of the chicken. A complete series of transverse sections through the mesencephalon is presented. In the diencephalon, cholinergic fibers were found in the stria medullaris, the fasciculus retroflexus, and the ventral portion of the supraoptic decussation. The nucleus triangularis and the nucleus geniculatus lateralis, pars ventralis also contained cholinergic fibers. Small cholinergic cell bodies were found in the medial habenula. In the pretectum, cholinergic fibers innervated the nucleus lentiformis mesencephali and the tectal gray. The nucleus spiriformis lateralis also contained cholinergic fibers, while most of the cell bodies in the nucleus spiriformis medialis were cholinergic. In the mesencephalon, labelled fibers were found in the nucleus intercollicularis and in all layers of the optic tectum except the stratum opticum. The highest density of tectal cholinergic fibers was in the stratum griseum et fibrosum superficiale (SGFS), layer f. Radial cells located in SGFS, layer i were also cholinergic. In the isthmic nuclei, cholinergic fibers were found in the pars magnocellularis, while the pars parvicellularis and the nucleus semilunaris contained labelled cells. The oculomotor, Edinger-Westphal, trochlear, and trigeminal motor nuclei all had cholinergic cell bodies. Cholinergic axons were present in the oculomotor and trochlear nerves. In the tegmentum, cell bodies were labelled in the nucleus mesencephalicus profundus, pars ventralis, while the nucleus interpeduncularis had dense cholinergic innervation. Our localization of cholinergic cell bodies and fibers has been compared with earlier autoradiographic and anatomical studies to help define cholinergic systems in the avian brain. For example, the results indicate that the chicken may have a cholinergic habenulointerpeduncular system similar to that reported in the rat. Establishing the cholinergic systems within the avian midbrain is important for designing future neurophysiological and pharmacological studies of cholinergic transmission in this region.

The generation and differentiation of cholinergic neurons in rat caudate-putamen.

By combining [3H]thymidine autoradiography with choline acetyltransferase (ChAT) immunocytochemistry, we have determined the generation pattern of the large cholinergic neurons in the neostriatum. All of these neurons are produced between embryonic days 12 and 17 (E12-E17), with 75% of them being born between E13 and 15. Cholinergic neurons appeared to be among the earliest cells produced in the neostriatum when compared with previous generation studies of all neurons in the rat caudate-putamen. The caudal-to-rostral neurogenic gradient reported in previous investigations of all neurons was the only spatiotemporal gradient observed for cholinergic neurons. The generation peak for these cells was E13 caudally, and E15 rostrally. Additional immunocytochemical studies detected ChAT immunoreactivity within somata and primary dendrites of 1 day postnatal (1 dpn) rat neostriatum, and subsequently demonstrated a dramatic increase in the intensity of reaction product and the complexity of dendritic arborizations by 14 dpn. Large ChAT-positive neurons of the basal forebrain contained within the same specimens appeared to differentiate their cholinergic phenotype earlier than those in the neostriatum. However, recent generation studies of basal forebrain neurons combined with the present results have demonstrated that both cholinergic populations are produced simultaneously along the same neurogenic gradients. This then represents an example of cholinergic projection (basal forebrain system) and local circuit (neostriatum) neurons that share similar generation patterns but differ with respect to sequences of transmitter phenotype expression. Thus, for cholinergic forebrain neurons, a cell's position along the neurogenic gradient and its transmitter phenotype appear to be more closely associated with its birth date than its ultimate projection or rate of differentiation.

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.

Effect of septal ablation on choline acetyltransferase in the paraventricular nucleus.

To determine whether there is a cholinergic projection from the lateral septum to the paraventricular nucleus of the hypothalamus (PVH), a quantitative histochemical analysis of the effects of unilateral ablation (0.5 microliter of a 10 micrograms/ml solution of ibotenic acid) of the lateral septum (LSV) on the choline acetyltransferase (ChAT) activity of the PVH and surrounding perinuclear area was undertaken. Comparisons of the ChAT activity of the PVH region ipsilateral to the LSV lesion (lesioned side) with that of the PVH contralateral (nonlesioned side) to the ablated LSV showed a 20% reduction (p less than 0.05) of ChAT activity in 2 areas of the PVH on the lesioned side. When ChAT activity of LSV-ablated and intact control rats was compared, a 20% difference in activity (p less than 0.05) was noted in four areas on the nonlesioned side and a 35% difference (p less than 0.05) of ChAT activity was noted in five regions of the PVH on the lesioned side of LSV-ablated animals. Taken together these data suggest that the pathway from the LSV to the PVH contains a small, bilateral cholinergic component. However, the data also indicate that this is not the only cholinergic projection to the PVH.

Hydrodynamic molecular weight of solubilized cholinergic synaptic vesicle glycoprotein ATPase.

The Torpedo californica electric organ synaptic vesicle glycoprotein ATPase was solubilized with octaethyleneglycoldodecyl ether and stabilized with phosphatidylserine. The complex was analyzed by size exclusion chromatography and band sedimentation velocity ultracentrifugation in water/glycerol and deuterium oxide/glycerol density gradients. The complex was found to have a Stokes' radius of 79 +/- 0.7 A, a sedimentation velocity coefficient at 20 degrees C in water of 6.8 +/- 0.2S, a partial specific volume of 0.81 +/- 0.01 cm3/g, and a frictional coefficient of 1.6. The molecular weight of the solubilized complex was calculated to be 320,000 +/- 7,000 and that of the protein 210,000 +/- 9,000. The relationship of this latter value to the major transport ATPase types is discussed.