A study of calretinin in Hirschsprung pathology, particularly in total colonic aganglionosis.

INTRODUCTION: Calretinin, a calcium-binding protein, has been reported to be an important new marker in Hirschsprung's disease (HD). The aim is to study the diagnostic value of Calretinin in total colonic aganglionosis (TA), prematurity, and superficial biopsy when nerve hyperplasia may not be accessed by ACE activity. METHODS: Records of patients diagnosed with HD at our institution from 1985 to 2010 were studied and patients with TA identified. We examined tissue samples from those TA, partial colectomies for HD, biopsies for suspicion of HD, and rectal tissue from aborted fetuses. Immunohistochemical analysis of Calretinin was compared with ACE gold standard method in all cases. RESULTS: In the majority of the cases, the diagnosis was ascertained by ACE activity and Calretinin staining. However, in 9 cases, the diagnosis was possible with Calretinin staining but not with ACE: in 4 TA because of the absence of nerve hyperplasia, and in 5 cases because the biopsies were too superficial to examine the nerve hyperplasia. In addition, Calretinin was expressed in the gut as early as 22 gestational weeks. CONCLUSION: The use of Calretinin staining may be superior to ACE activity, particularly in the context of TA, superficial biopsies, and prematurity, allowing earlier diagnosis.

Immunohistochemical demonstration of cholinergic structures in central ganglia of the slug (Limax maximus, Limax valentianus).

Immunohistochemical techniques were used to study the distribution of cholinergic neurons containing choline acetyltransferase of the common type (cChAT), the synthetic enzyme of acetylcholine, in the central nervous system of the slug Limax maximus and Limax valentianus. Because the antiserum applied here was raised against a recombinant protein encoded by exons 7 and 8 of the rat gene for ChAT, three methods were used in order to validate antibody specificity for the Limax counterpart enzyme. Western blot combined with ChAT activity assay following native gel electrophoresis and immunoprecipitation analysis both indicated that immunoreactive Limax brain molecules were capable of synthesizing acetylcholine. Western blot after denatured gel electrophoresis of Limax brain extracts revealed a single band of about 67kDa. All findings obtained with these three methods clearly indicated that the antiserum effectively recognized Limax cChAT. 1400 neuronal cell bodies positive for cChAT, mainly small to medium-sized, were found in various brain regions in the buccal, cerebral, pleural, parietal, visceral and pedal ganglia. cChAT immunoreactive nerve fibers were distributed extensively in the neuropil, connectives and commissures of these central ganglia. The map of cChAT-positive cells provided here are valuable for understanding the cholinergic mechanism in the slug brain, as well as giving an important hint to clarifying the mechanisms of learning and memory in higher vertebrates including humans.

Lesion of cholinergic neurons in nucleus basalis enhances response to general anesthetics.

Acetylcholine in the brain has been associated with consciousness and general anesthesia effects. We tested the hypothesis that the integrity of the nucleus basalis magnocellularis (NBM) affects the response to general anesthetics. Cholinergic neurons in NBM were selectively lesioned by bilateral infusion of 192IgG-saporin in adult, male Long-Evans rats, and control rats were infused with saline. Depletion of choline-acetyltransferase (ChAT)-immunoreactive cells in the NBM and decrease in optical density of acetylcholinesterase (AChE) staining in the frontal and visual cortices confirmed a significant decrease in NBM cholinergic neurons in lesioned as compared to control rats. AChE staining in the hippocampus and ChAT-positive neurons in the medial septum-vertical limb of the diagonal band were not different between lesioned and control rats. When a general anesthetic was administered, lesioned compared to control rats showed significantly longer duration of loss of righting reflex (LORR) after propofol (5 or 10mg/kg i.v.), pentobarbital (20 or 40 mg/kg i.p.) but not halothane (2%). However, the behavioral excitation, as indicated by horizontal movements, induced by halothane was reduced in lesioned as compared to control rats. Reversible inactivation of NBM with GABA(A) receptor agonist muscimol increased slow waves in the neocortex during awake immobility, and prolonged the duration of LORR and loss of tail-pinch response after propofol, pentobarbital and halothane. In summary, lesion of NBM cholinergic neurons or inactivation of the NBM prolonged the LORR response to general anesthetic drugs.

Histological determination of the areas enriched in cholinergic terminals and M2 and M3 muscarinic receptors in the mouse central auditory system.

Cholinergic projections to auditory system are vital for coupling arousal with sound processing. Systematic search with in situ hybridization and immunohistochemistry indicated that the ventral nucleus of the medial geniculate body and the nucleus of the brachium of the inferior colliculus constituted cholinergic synaptic sites in the brainstem auditory system, containing a significant number of cholinergic axon terminals and m2 receptor-expressing cell bodies.

Hypoglossal premotor neurons of the intermediate medullary reticular region express cholinergic markers.

The inspiratory drive to hypoglossal (XII) motoneurons originates in the caudal medullary intermediate reticular (IRt) region. This drive is mainly glutamatergic, but little is known about the neurochemical features of IRt XII premotor neurons. Prompted by the evidence that XII motoneuronal activity is controlled by both muscarinic (M) and nicotinic cholinergic inputs and that the IRt region contains cells that express choline acetyltransferase (ChAT), a marker of cholinergic neurons, we investigated whether some IRt XII premotor neurons are cholinergic. In seven rats, we applied single-cell reverse transcription-polymerase chain reaction to acutely dissociated IRt neurons retrogradely labeled from the XII nucleus. We found that over half (21/37) of such neurons expressed mRNA for ChAT and one-third (13/37) also had M2 receptor mRNA. In contrast, among the IRt neurons not retrogradely labeled, only 4 of 29 expressed ChAT mRNA (P < 0.0008) and only 3 of 29 expressed M2 receptor mRNA (P < 0.04). The distributions of other cholinergic receptor mRNAs (M1, M3, M4, M5, and nicotinic alpha4-subunit) did not differ between IRt XII premotor neurons and unlabeled IRt neurons. In an additional three rats with retrograde tracers injected into the XII nucleus and ChAT immunohistochemistry, 5-11% of IRt XII premotor neurons located at, and caudal to, the area postrema were ChAT positive, and 27-48% of ChAT-positive caudal IRt neurons were retrogradely labeled from the XII nucleus. Thus the pre- and postsynaptic cholinergic effects previously described in XII motoneurons may originate, at least in part, in medullary IRt neurons.

RanBPM is expressed in synaptic layers of the mammalian retina and binds to metabotropic glutamate receptors.

In the central nervous system, synaptic signal transduction depends on the regulation of neurotransmitter receptors by interacting proteins. Here, we searched for proteins interacting with two metabotropic glutamate receptor type 8 isoforms (mGlu8a and mGlu8b) and identified RanBPM. RanBPM is expressed in several brain regions, including the retina. There, RanBPM is restricted to the inner plexiform layer where it co-localizes with the mGlu8b isoform and processes of cholinergic amacrine cells expressing mGlu2 receptors. RanBPM interacts with mGlu2 and other group II and group III receptors, except mGlu6. Our data suggest that RanBPM might be associated with mGlu receptors at synaptic sites.

Heterogeneidad morfológica de la porción posterior del estriado humano / Morphological heterogeneity of the human posterior striatum

El estriado (núcleo caudado, NC, y putamen, Put) forma parte de los gangliosbasales, un conjunto de núcleos subcorticales cuya principal función es la planificacióny ejecución de los movimientos voluntarios. La información nerviosa procedentede la corteza cerebral alcanza el estriado formando tres canales distintos,denominados asociativo, sensorimotor y límbico. La parte posterior del estriado,que incluye el cuerpo, el giro y la cola del NC, además del último tercio del Putpostcomisural, ha recibido una escasa atención en cuanto a sus característicasquímicas y composición celular. El presente trabajo tiene como objetivo analizarla distribución de dos poblaciones de interneuronas (nitrérgicas y colinérgicas) en la parte posterior del estriado. Según nuestros resultados, ambas poblaciones presentanuna mayor densidad en la parte posterior del estriado que en la parteanterior, siendo más abundantes en el NC que en el Put. La región más densamentepoblada por las neuronas nitrérgicas es el giro del NC, mientras que las neuronascolinérgicas son especialmente abundantes en el cuerpo de dicho núcleo.Además, la organización de los dos grupos neuronales con respecto al compartimentoestriosomal es diferente en la parte posterior del estriado que en las regionesanteriores, y también varía según se trate de las neuronas nitrérgicas o de lascolinérgicas. En definitiva, nuestro estudio demuestra que la porción posterior delestriado puede llevar a cabo un procesamiento de la información tanto o máscomplejo que la parte anterior

The striatum (caudate nucleus, NC and putamen, Put) is a main part of thebasal ganglia, a group of subcortical nuclei whose main function is the planningand execution of voluntary movements. Nervous inputs from the cerebral cortexare divided into three different channels in the striatum, termed associative, sensorimotorand limbic. The posterior aspect of the striatum comprises the body,gyrus and tail of the NC, and the postcommissural Put, and has been very muchleft out of most of chemical and cellular studies. The present work is aimed atanalyzing the distribution of two populations of interneurons (nitrergic and cholinergic)in those striatal regions. According to our results, both populations aremore abundant in the posterior striatum than in its anterior aspects, with a higherdensity in the NC than in the Put. The gyrus of NC is the most populated regionby nitrergic cells, whereas cholinergic interneurons are especially abundant in thebody of NC. Furthermore, the organization of both interneuronal groups regardingthe striosomal compartment is different in the posterior striatum with respect toits anterior aspects, and this organization also varied when nitrergic or cholinergicinterneurons were analyzed. Overall, our study demonstrates that the posterioraspect of the striatum might carry out a more complex processing of the informationthan its anterior counterpart

Immunohistochemical and histochemical findings favoring the occurrence of autocrine/paracrine as well as nerve-related cholinergic effects in chronic painful patellar tendon tendinosis.

The pathogenesis of the pain in patellar tendon tendinosis ("jumper's knee") is unclear. We have recently presented new information about the sensory nervous system in the human patellar tendon, but there is very little information regarding the possible occurrence of a cholinergic system in this tendon. In the present study, specimens of pain-free normal tendons and chronically painful tendinosis tendons were examined by different immunohistochemical and histochemical methods. Antibodies against the M(2) receptor, choline acetyltransferase (ChAT), and vesicular acetylcholine transporter (VAChT) were applied, and staining for demonstration of activity of acetylcholinesterase (AChE) was also utilized. It was found that immunoreactions for the M(2) receptor could be detected intracellularly in both blood vessel cells and tenocytes, especially in tendinosis specimens. Furthermore, in the tendinosis specimens, some tenocytes were seen to exhibit immunoreaction for ChAT and VAChT. AChE reactions were seen in fine nerve fibers associated with small blood vessels in both the normal control tendons and the tendinosis tendons. The observations suggest that there is both a nerve related and a local cholinergic system in the human patellar tendon. As ChAT and VAChT immunoreactions were detected in tenocytes of tendinosis tendons, these cells might be a source of local acetylcholine (Ach) production. As both tenocytes and blood vessel cells were found to exhibit immunoreactions for the M(2) receptor, it is likely that both of these tissue cells may be influenced by ACh. Thus, in conclusion, there appears to be an upregulation of the cholinergic system, and an occurrence of autocrine/paracrine effects in this system, in the tendinosis patellar tendon.

A quantitative study of the brainstem cholinergic projections to the ventral part of the oral pontine reticular nucleus (REM sleep induction site) in the cat.

The ventral part of the cat oral pontine reticular nucleus (vRPO) is the site in which microinjections of small dose and volume of cholinergic agonists produce long-lasting rapid eye movement sleep with short latency. The present study determined the precise location and proportions of the cholinergic brainstem neuronal population that projects to the vRPO using a double-labeling method that combines the neuronal tracer horseradish peroxidase-wheat germ agglutinin with choline acetyltransferase immunocytochemistry in cats. Our results show that 88.9% of the double-labeled neurons in the brainstem were located, noticeably bilaterally, in the cholinergic structures of the pontine tegmentum. These neurons occupied not only the pedunculopontine and laterodorsal tegmental nuclei, which have been described to project to other pontine tegmentum structures, but also the locus ceruleus complex principally the locus ceruleus alpha and peri-alpha, and the parabrachial nuclei. Most double-labeled neurons were found in the pedunculopontine tegmental nucleus and locus ceruleus complex and, much less abundantly, in the laterodorsal tegmental nucleus and the parabrachial nuclei. The proportions of these neurons among all choline acetyltransferase positive neurons within each structure were highest in the locus ceruleus complex, followed in descending order by the pedunculopontine and laterodorsal tegmental nuclei and then, the parabrachial nuclei. The remaining 11.1% of double-labeled neurons were found bilaterally in other cholinergic brainstem structures: around the oculomotor, facial and masticatory nuclei, the caudal pontine tegmentum and the praepositus hypoglossi nucleus. The disperse origins of the cholinergic neurons projecting to the vRPO, in addition to the abundant noncholinergic afferents to this nucleus may indicate that cholinergic stimulation is not the only or even the most decisive event in the generation of REM sleep.

The acetylcholine innervation of cerebral cortex: new data on its normal development and its fate in the hAPP(SW,IND) mouse model of Alzheimer's disease.

To follow on prior studies of the cerebral cortex, we examined the acetylcholine innervation in the developing hippocampus of rat, by means of light and electron microscopic immunocytochemistry with a highly sensitive antibody against choline acetyltransferease. As in neocortex, the growth of this innervation mostly occurred within the first two weeks after birth. A preliminary ultrastructural survey indicated that a vast majority of these ChAT-immunostained axon varicosities were asynaptic during development as in the adult. In parallel, we quantified the cholinergic innervations of cerebral cortex and hippocampus in transgenic mice overexpressing human beta-amyloid peptide (hAPP(SW,IND)). A selective, widespread, plaque independent cholinergic denervation was thus demonstrated, first in hippocampus and then neocortex, in addition to a non-selective, plaque-dependent, local neurotoxic effect of aggregated beta-amyloid on ACh and 5-HT axons.

Neuronal nicotinic synapse assembly requires the adenomatous polyposis coli tumor suppressor protein.

Normal cognitive and autonomic functions require nicotinic synaptic signaling. Despite the physiological importance of these synapses, little is known about molecular mechanisms that direct their assembly during development. We show here that the tumor-suppressor protein adenomatous polyposis coli (APC) functions in localizing alpha3-nicotinic acetylcholine receptors (nAChRs) to neuronal postsynaptic sites. Our quantitative confocal microscopy studies indicate that APC is selectively enriched at cholinergic synapses; APC surface clusters are juxtaposed to synaptic vesicle clusters and colocalize with alpha3-nAChRs but not with the neighboring synaptic glycine receptors or perisynaptic alpha7-nAChRs on chick ciliary ganglion (CG) neurons. We identify PSD (postsynaptic density)-93, beta-catenin, and microtubule end binding protein EB1 as APC binding partners. PSD-93 and beta-catenin are also enriched at alpha3-nAChR postsynaptic sites. EB1 shows close proximity to and partial overlap with alpha3-nAChR and APC surface clusters. We tested the role of APC in neuronal nicotinic synapse assembly by using retroviral-mediated in vivo overexpression of an APC dominant-negative (APC-dn) peptide to block the interaction of endogenous APC with both EB1 and PSD-93 during synapse formation in CG neurons. The overexpressed APC-dn led to dramatic decreases in alpha3-nAChR surface levels and clusters. Effects were specific to alpha3-nAChR postsynaptic sites; synaptic glycine receptor and perisynaptic alpha7-nAChR clusters were not altered. In addition, APC-dn also reduced surface membrane-associated clusters of PSD-93 and EB1. The results show that APC plays a key role in organizing excitatory cholinergic postsynaptic specializations in CG neurons. We identify APC as the first nonreceptor protein to function in localizing nAChRs to neuronal synapses in vivo.

Ethanol-induced Fos immunoreactivity in the extended amygdala and hypothalamus of the rat brain: focus on cholinergic interneurons of the nucleus accumbens.

BACKGROUND: The primary goal of this study was to investigate the effects of varying doses of ethanol on cellular activation, as measured by Fos immunoreactivity, in brain areas that have been implicated in the reinforcing and anxiolytic effects of substance abuse and dependence, namely, the extended amygdala and hypothalamus. Specific regions examined included the central nucleus of the amygdala, bed nucleus of the stria terminalis, substantia innominata, and nucleus accumbens of the extended amygdala, as well as the paraventricular nucleus of the hypothalamus. The cholinergic interneurons of the nucleus accumbens were of particular interest, because these cells have recently been reported to play a pivotal role in substance abuse. METHODS: Adult Sprague-Dawley rats underwent 10 days of handling and 5 days of habituation. Animals then received an injection of saline or 0.5, 1, or 2 g/kg of ethanol. Rats were perfused 2 hr after the injections, and brain sections were processed for single Fos or dual Fos/choline acetyltransferase immunolabeling procedures. The number of Fos-positive neurons was calculated from a 0.45-mm sample area from each of the brain regions examined. RESULTS: A dose of 2 g/kg of ethanol significantly increased the number of Fos-immunoreactive neurons in the central nucleus of the amygdala by 149%, in the shell nucleus accumbens by 80%, and in the paraventricular nucleus of the hypothalamus by 321%. Additionally, 1 g/kg of ethanol significantly increased the percentage of Fos-immunoreactive cholinergic neurons in the nucleus accumbens by 59%. CONCLUSIONS: The findings reported in this study reveal region-specific and dose-dependent changes in Fos immunoreactivity in the extended amygdala and hypothalamus and, more specifically, an increase in neuronal activation of cholinergic cells in the shell nucleus accumbens. These findings contribute to our current knowledge of the brain areas and cellular microcircuits involved in the underlying basis of substance abuse and dependence.

Effects of estrogen replacement therapy on cholinergic basal forebrain neurons and cortical cholinergic innervation in young and aged ovariectomized rhesus monkeys.

Estrogen modulates the function of cholinergic basal forebrain neurons in aged female rats. The present study tested the hypothesis that estrogen enhances the phenotype of cholinergic basal forebrain neurons and their cortical cholinergic innervation in young adult and aged ovariectomized rhesus monkeys. Sixteen monkeys (9 young and 7 aged) received two injections of estradiol cypionate or vehicle separated by 3 weeks. All monkeys were killed 1 day after the last injection. Quantitative immunofluorescence in the vertical limb of the diagonal band (VLDB) revealed enhanced optical density for choline acetyltransferase (ChAT) in both young and aged monkeys treated with estrogen. In contrast, optical density for low-affinity p75 neurotrophin receptor immunoreactivity in the VLDB did not change after estrogen treatment in either aged or young animals. Quantitative immunofluorescence for either ChAT or the low-affinity p75 neurotrophin receptor in the nucleus basalis Meynert failed to reveal differences between vehicle and estrogen treatment in either age group. Quantitative estimates of acetylcholinesterase (AChE) fiber density revealed that estrogen-treated aged monkeys but not their younger counterparts had decreased numbers of AChE-positive fibers in layer II of frontal, insular, and cingulate cortices. These data indicate that estrogen administered in a manner simulating natural hormonal cyclicity produces modest age-specific chemical phenotypic and regional changes in select neuronal subfields of the cholinergic basal forebrain and their cortical projection sites in nonhuman primates.

Localization of cholinergic innervation in guinea pig heart by immunohistochemistry for high-affinity choline transporters.

OBJECTIVE: Previous studies have used acetylcholinesterase (AChE) histochemistry to identify cholinergic nerves in the heart, but this enzyme is not a selective marker for cholinergic neurons. This study maps cholinergic innervation of guinea pig heart using a new antibody to the human high-affinity choline transporter (CHT), which is present only in cholinergic nerves. METHODS: Immunohistochemistry was used to localize CHTs in frozen and paraffin sections of heart and whole mount preparations of atrial ganglionated nerve plexus. AChE-positive nerve fibers were identified in sections from separate hearts for comparison. RESULTS: Control experiments established that the antibody to human CHT selectively labeled cholinergic neurons in the guinea pig. CHT-immunoreactive nerve fibers and AChE-positive nerves were very abundant in the sinus and AV nodes, bundle of His, and bundle branches. Both markers also delineated a distinct nerve tract in the posterior wall of the right atrium. AChE-positive nerve fibers were more abundant than CHT-immunoreactive nerves in working atrial and ventricular myocardium. CHT-immunoreactive nerves were rarely observed in left ventricular free wall. Both markers were associated with numerous parasympathetic ganglia that were distributed along the posterior atrial walls and within the interatrial septum, including the region of the AV node. CONCLUSIONS: Comparison of labeling patterns for CHT and AChE suggests that AChE histochemistry overestimates the density of cholinergic innervation in the heart. The distribution of CHT-immunoreactive nerve fibers and parasympathetic ganglia in the guinea pig heart suggests that heart rate, conduction velocity, and automaticity are precisely regulated by cholinergic innervation. In contrast, the paucity of CHT-immunoreactive nerve fibers in left ventricular myocardium implies that vagal efferent input has little or no direct influence on ventricular contractile function in the guinea pig.

Postnatal development of cholinergic afferents from the pedunculopontine tegmental nucleus to the lateralis medialis-suprageniculate nucleus of the feline thalamus.

The lateralis medialis-suprageniculate nuclear complex (LM-Sg) has been shown to receive cholinergic fibers from the pedunculopontine tegmental nucleus (PPT). The majority of terminals of these cholinergic fibers make simple synaptic contact with dendritic profiles, whereas some make contacts with the dendrites of projection neurons and GABAergic interneurons forming a glomerular synaptic complex. In the present study, we investigate the postnatal development of glomerular synaptic complexes in the LM-Sg in association with terminals of the PPT-thalamic projection fibers. We examined the postnatal development of cholinergic innervation as well as GABAergic interneuron innervation in the LM-Sg using antibodies against ChAT and GABA, respectively. Although choline acetyltransferase (ChAT)-positive neurons already exist in the PPT at birth (P0), ChAT-positive fibers in the LM-Sg were observed only after P7. These ChAT-positive fibers gradually increased in number, and almost reached the adult level by postnatal day 28 (P28). GABA-positive interneurons were scattered throughout the LM-Sg at P0, increased in size gradually and reached adult size by P14. Immature glomerulus-like synaptic arrangements appeared at P14. Definite glomeruli, in which ChAT-positive terminals are present, were observed at P28. These results emphasize that interneurons in the LM-Sg grow by P14, and then make neural circuits with cholinergic innervation within the glomerulus by 3-4 weeks.

Synaptic connections of cholinergic antennal lobe relay neurons innervating the lateral horn neuropile in the brain of Drosophila melanogaster.

Presumed cholinergic projection neurons (PNs) in the brain of the fruit fly Drosophila melanogaster, immunoreactive to choline acetyltransferase (ChAT), convey olfactory information between the primary sensory antennal lobe neuropile and the mushroom body calyces, and finally terminate in the lateral horn (LH) neuropile. The texture and synaptic connections of ChAT PNs in the LH and, comparatively, in the smaller mushroom body calyces were investigated by immuno light and electron microscopy. The ChAT PN fibers of the massive inner antennocerebral tract (iACT) extend into all portions of the LH, distributing in a nonrandom fashion. Immunoreactive boutons accumulate in the lateral margins of the LH, whereas the more proximal LH exhibits less intense immunolabeling. Boutons with divergent presynaptic sites, unlabeled as well as ChAT-immunoreactive, appear to be the preponderant mode of synaptic input throughout the LH. Synapses of ChAT-labeled fibers appear predominantly as divergent synaptic boutons (diameters 1-3 microm), connected to unlabeled postsynaptic profiles, or alternatively as a minority of tiny postsynaptic spines (diameters 0.05-0.5 microm) among unlabeled profiles. Together these spines encircle unidentified presynaptic boutons of interneurons which occupy large areas of the LH. Thus, synaptic circuits in the LH differ profoundly from those of the PNs in the mushroom body calyx, where ChAT spines have not been encountered. Synaptic contacts between LH ChAT elements were not observed. The synaptic LH neuropile may serve as an output area for terminals of the ChAT PNs, their presynaptic boutons providing input to noncholinergic relay neurons. The significance of the postsynaptic neurites of the ChAT PNs is discussed; either local or other interneurons might connect the ChAT PNs within the LH, or PNs might receive inputs arising from outside the LH.

Cholinergic axons in the rat prostate and neurons in the pelvic ganglion.

Fluorogold or green fluorescent pseudorabies virus labeled postganglionic neurons in the pelvic ganglion that innervate the prostate gland. Small cholinergic neurons were demonstrated by immunohistochemistry (IHC) with antiserum against vesicular acetylcholine transferase (VAChT). Large, mainly adrenergic neurons, were surrounded by preganglionic cholinergic boutons. In the prostate, M3 type muscarinic receptors were found in the outer muscle layer surrounding the prostatic acini. The antiserum against VAChT marked the inner epithelial layer. Antisera against the vesicular monoamine transporters VMAT1 and VMAT2 demonstrated staining of the inner secretory layer and adrenergic fibers in the outer muscle layer, respectively, of the prostatic acini. These results provide new evidence for the presence of neural elements that have a cholinergic influence over the rat prostate gland.

Vesicular localization and activity-dependent trafficking of presynaptic choline transporters.

Presynaptic synthesis of acetylcholine (ACh) requires a steady supply of choline, acquired by a plasma membrane, hemicholinium-3-sensitive (HC-3) choline transporter (CHT). A significant fraction of synaptic choline is recovered from ACh hydrolyzed by acetylcholinesterase (AChE) after vesicular release. Although antecedent neuronal activity is known to dictate presynaptic CHT activity, the mechanisms supporting this regulation are unknown. We observe an exclusive localization of CHT to cholinergic neurons and demonstrate that the majority of CHTs reside on small vesicles within cholinergic presynaptic terminals in the rat and mouse brain. Furthermore, immunoisolation of presynaptic vesicles with multiple antibodies reveals that CHT-positive vesicles carry the vesicular acetylcholine transporter (VAChT) and synaptic vesicle markers such as synaptophysin and Rab3A and also contain acetylcholine. Depolarization of synaptosomes evokes a Ca2+-dependent botulinum neurotoxin C-sensitive increase in the Vmax for HC-3-sensitive choline uptake that is accompanied by an increase in the density of CHTs in the synaptic plasma membrane. Our study leads to the novel hypothesis that CHTs reside on a subpopulation of synaptic vesicles in cholinergic terminals that can transit to the plasma membrane in response to neuronal activity to couple levels of choline re-uptake to the rate of ACh release.