Cutaneous mechanosensory neurons detect mechanical stimuli that generate touch and pain sensation. Although opioids are generally associated only with the control of pain, here we report that the opioid system in fact broadly regulates cutaneous mechanosensation, including touch. This function is predominantly subserved by the delta opioid receptor (DOR), which is expressed by myelinated mechanoreceptors that form Meissner corpuscles, Merkel cell-neurite complexes, and circumferential hair follicle endings. These afferents also include a small population of CGRP-expressing myelinated nociceptors that we now identify as the somatosensory neurons that coexpress mu and delta opioid receptors. We further demonstrate that DOR activation at the central terminals of myelinated mechanoreceptors depresses synaptic input to the spinal dorsal horn, via the inhibition of voltage-gated calcium channels. Collectively our results uncover a molecular mechanism by which opioids modulate cutaneous mechanosensation and provide a rationale for targeting DOR to alleviate injury-induced mechanical hypersensitivity.

ABSTRACTThe neuropeptide galanin is implicated in regulation of affective behavior, including modulation of 5‐HT signaling. Here, we investigated, by use of microdialysis in freely moving rats, the effects of intracerebral (i.c.) and intracerebroventricular (i.c.v.) infusions of galanin on basal extracellular 5‐HT levels in medial prefrontal cortex (mPFC), CA1 area of ventral hippocampus (vHPC), central amygdaloid nucleus (CeA), ventromedial hypothalamic nucleus ventrolateral part (VMHvl), and ventromedial caudate putamen (CPu). These results were compared with a parallel immunohistochemical analysis of the distribution of galanin, 5‐HT, and noradrenaline (NA) nerve terminals, and with data on galanin receptors. Galanin i.c.v. significantly decreased the 5‐HT levels in mPFC to 79% and in vHPC to 72%. Local infusions of galanin caused a long‐lasting decrease in 5‐HT levels in vHPC to 88%, and a moderate decrease in CeA, whereas the 5‐HT levels in mPFC significantly increased to 121%. These effects of i.c. galanin correlated well with the density of 5‐HT and galanin nerve terminals and galanin receptors autoradiography in mPFC, vHPC, and CeA. No effects of i.c. or i.c.v. galanin on 5‐HT levels were observed in CPu or VMHvl, in agreement with the low numbers of galanin‐positive terminals and low/moderate galanin receptor density. Galanin was often found to coexist in NA, but could never be detected in 5‐HT terminals. Together the results show a neuroanatomical correlation between the effects of galanin infusions on 5‐HT release and distribution of galanin and its receptors, and that i.c.v. and i.c. administration can give opposite effects on 5‐HT release. Synapse 68:179–193, 2014. © 2014 Wiley Periodicals, Inc.

Neuromedin U (NMU) plays an important role in a number of physiological processes, but the relative contribution of its two known receptors, NMUR1 and NMUR2, is still poorly understood. Here we report the existence of a SNP T1022→A (Val341→Glu) in the third exon of the rat Nmur1 gene that leads to an inactive receptor. This SNP is present within the coding region of the highly conserved NPXXY motif found within all class A type G protein-coupled receptors and translates to an NMUR1 receptor that is not expressed on the cell surface. Genetic analysis of the Nmur1 gene in a population of Sprague-Dawley rats revealed that this strain is highly heterogeneous for the inactivating polymorphism. The loss of functional NMUR1 receptors in Sprague-Dawley rats homozygous for the inactive allele was confirmed by radioligand binding studies on native tissue expressing NMUR1. The physiological relevance of this functional genomics finding was examined in two nociceptive response models. The pronociceptive effects of NMU were abolished in rats lacking functional NMUR1 receptors. The existence of naturally occurring NMUR1-deficient rats provides a novel and powerful tool to investigate the physiological role of NMU and its receptors. Furthermore, it highlights the importance of verifying the NMUR1 single nucleotide polymorphism status for rats used in physiological, pharmacological or toxicological studies conducted with NMUR1 modulators.

Delta and mu opioid receptors (DORs and MORs) are inhibitory G protein-coupled receptors that reportedly cooperatively regulate the transmission of pain messages by substance P and TRPV1-expressing pain fibers. Using a DOReGFP reporter mouse we now show that the DOR and MOR are, in fact, expressed by different subsets of primary afferents. The MOR is expressed in peptidergic pain fibers, the DOR in myelinated and nonpeptidergic afferents. Contrary to the prevailing view, we demonstrate that the DOR is trafficked to the cell surface under resting conditions, independently of substance P, and internalized following activation by DOR agonists. Finally, we show that the segregated DOR and MOR distribution is paralleled by a remarkably selective functional contribution of the two receptors to the control of mechanical and heat pain, respectively. These results demonstrate that behaviorally relevant pain modalities can be selectively regulated through the targeting of distinct subsets of primary afferent pain fibers.

We previously showed that prolonged morphine treatment and chronic inflammation both enhanced delta opioid receptor (deltaOR) cell surface density in lumbar spinal cord neurons. Here, we sought to determine whether administration of morphine to rats with chronic inflammation would further increase the bio-availability of deltaOR, and thereby the analgesic properties of the deltaOR agonist deltorphin, over that produced by inflammation alone. We found that chronic inflammation produced by injection of complete Freund's adjuvant (CFA) into the hind paw resulted in a bilateral increase in the binding and internalization of fluorescent deltorphin in neurons of the lumbar spinal cord as did prolonged morphine treatment [Morinville A, Cahill CM, Aibak H, Rymar VV, Pradhan A, Hoffert C, Mennicken F, Stroh T, Sadikot AF, O'Donnell D, Clarke PB, Collier B, Henry JL, Vincent JP, Beaudet A (2004a) Morphine-induced changes in delta opioid receptor trafficking are linked to somatosensory processing in the rat spinal cord. J Neurosci 24:5549-5559]. This effect was accompanied by an increase in the antinociceptive efficacy of intrathecal deltorphin as measured using the tail-flick test. Treatment of CFA-injected rats with morphine decreased the cell surface availability of deltaOR in neurons of the dorsal horn of the lumbar spinal cord as compared with treatment with CFA alone. Behaviorally, it significantly enhanced the antihyperalgesic effects of deltorphin (plantar test; % maximum possible antihyperalgesic effect (MPAHE)=113.5%+/-32.4% versus 26.1%+/-11.6% in rats injected with CFA alone) but strongly reduced the antinociceptive efficacy of the drug (tail-flick test; % maximum possible antinociceptive effect (MPE)=29.6%+/-3.6% versus 66.6%+/-6.3% in rats injected with CFA alone) suggesting that the latter, but not the former, is linked to the deltaOR trafficking events observed neuroanatomically. These results demonstrate that in chronic inflammation, the antihyperalgesic effects of deltaOR agonists may be enhanced by morphine pre-treatment. They also reveal a dichotomy between mechanisms underlying antihyperalgesic and antinociceptive effects of deltaOR agonists.

The expression of the galanin receptor-1 and -2 (Gal1 and Gal2) messenger ribonucleic acids (mRNAs) was studied in the lower spinal cord of rat by means of in situ hybridization, using ribonucleic acid probes (riboprobes). Naïve rats as well as rats with unilateral axotomy of the sciatic nerve or unilateral inflammation of the hindpaw were analyzed. In naïve rats, numerous Gal1 mRNA-positive (+) neurons were detected in lamina (L) I–III. In addition, several Gal1 mRNA+ neurons were seen in deeper layers, including the ventral horns, area X, and the lateral spinal nucleus. In contrast, few and comparatively weakly labeled Gal2 mRNA+ neurons were observed, mostly in the ventral horns and in area X, with fewer in the dorsal horn and in the sympathetic and parasympathetic intermediate lateral cell columns. Axotomy induced a strong increase in intensity and number of Gal2 mRNA+ motoneurons ipsilateral to the lesion. In contrast, nerve cut or hindpaw inflammation did not alter the expression of Gal1 or Gal2 in the dorsal horn. The present (and previous) results suggest that galanin, acting through Gal1 and Gal2 receptors, has a modulatory role on spinal excitability, not only via interneurons in superficial dorsal horn, but also on neurons in deep layers and area X, as well as on the sympathetic and parasympathetic outflow. Furthermore, the nerve injury-induced Gal2 upregulation in motor neurons suggests a role for galanin in survival/regeneration mechanisms.

Background Several clinical studies suggested that antipsychotic-based medications could ameliorate cognitive functions impaired in certain schizophrenic patients. Accordingly, we investigated the effects of various dopaminergic receptor antagonists – including atypical antipsychotics that are prescribed for the treatment of schizophrenia – in a model of toxicity using cultured hippocampal neurons, the hippocampus being a region of particular relevance to cognition. Results Hippocampal cell death induced by deprivation of growth medium constituents was strongly blocked by drugs including antipsychotics (10 -10 -10 -6 M) that display nM affinities for D 2 and/or D 4 receptors (clozapine, haloperidol, (±)-sulpiride, domperidone, clozapine, risperidone, chlorpromazine, (+)-butaclamol and L-741,742). These effects were shared by some caspases inhibitors and were not accompanied by inhibition of reactive oxygen species. In contrast, (-)-raclopride and remoxipride, two drugs that preferentially bind D 2 over D 4 receptors were ineffective, as well as the selective D 3 receptor antagonist U 99194. Interestingly, (-)-raclopride (10 -6 M) was able to block the neuroprotective effect of the atypical antipsychotic clozapine (10 -6 M). Conclusion Taken together, these data suggest that D2-like receptors, particularly the D 4 subtype, mediate the neuroprotective effects of antipsychotic drugs possibly through a ROS-independent, caspase-dependent mechanism.

The present study demonstrates that perikaryalδ-opioid receptors (δORs) in rat dorsal root ganglion (DRG) neurons bind and internalize opioid ligands circulating in the CSF. Using confocal and electron microscopy, we found that prolonged morphine treatment increased the cell surface density of these perikaryal δORs and, by way of consequence, receptor-mediated internalization of the fluorescent deltorphin (DLT) analog ω-Bodipy 576/589 deltorphin-I 5-aminopentylamide (Fluo-DLT) in all three types of DRG neurons (small, medium, and large). In contrast, chronic inflammatory pain induced by the injection of complete Freund's adjuvant (CFA) into one hindpaw selectively increased Fluo-DLT internalization in small and medium-sized DRG neurons ipsilateral to the inflammation. Based on our previous studies in the spinal cord of μ-opioid receptor (μOR) knock-out mice, it may be assumed that the enhanced membrane recruitment of δORs observed after sustained morphine is attributable to stimulation of μORs. However, the selectivity of the effect induced by inflammatory pain suggests that it involves a different mechanism, namely a modality-specific and pain-related activation of C and Aδ fibers. Indeed, stimulation by capsaicin of transient receptor potential vanilloid 1 receptors, which are selectively expressed by small diameter (< 600 μm 2 ) DRG neurons, increased Fluo-DLT internalization exclusively in this cell population. The present results, therefore, demonstrate that DRG neurons express perikaryal δORs accessible to CSF-circulating ligands and that the density and, hence, presumably also the responsiveness, of these receptors may be modulated by both pain-related stimuli and sustained exposure to μOR agonists.

An in vivo fluorescent deltorphin (Fluo-DLT) internalization assay was used to assess the distribution and regulation of pharmacologically available delta opioid receptors (deltaORs) in the rat lumbar (L4-5) spinal cord. Under basal conditions, intrathecal injection of Fluo-DLT resulted in the labeling of numerous deltaOR-internalizing neurons throughout dorsal and ventral horns. The distribution and number of Fluo-DLT-labeled perikaryal profiles were consistent with that of deltaOR-expressing neurons, as revealed by in situ hybridization and immunohistochemistry, suggesting that a large proportion of these cells was responsive to intrathecally administered deltaOR agonists. Pretreatment of rats with morphine for 48 hr resulted in a selective increase in Fluo-DLT-labeled perikaryal profiles within the dorsal horn. These changes were not accompanied by corresponding augmentations in either deltaOR mRNA or (125)I-deltorphin-II binding levels, suggesting that they were attributable to higher densities of cell surface deltaOR available for internalization rather than to enhanced production of the receptor. Unilateral dorsal rhizotomy also resulted in increased Fluo-DLT internalization in the ipsilateral dorsal horn when compared with the side contralateral to the deafferentation or to non-deafferented controls, suggesting that deltaOR trafficking in dorsal horn neurons may be regulated by afferent inputs. Furthermore, morphine treatment no longer increased Fluo-DLT internalization on either side of the spinal cord after unilateral dorsal rhizotomy, indicating that microOR-induced changes in the cell surface availability of deltaOR depend on the integrity of primary afferent inputs. Together, these results suggest that regulation of deltaOR responsiveness through microOR activation in this region is linked to somatosensory information processing.

The neuropeptide neuromedin U (NMU) has been shown to have significant effects on cardiovascular, gastrointestinal and CNS functions. The peptide was first isolated from the porcine spinal cord and later shown to be present in spinal cords of other species. Little is known about the distribution of neuromedin U receptors (NMURs) in the spinal cord and the spinal action of the peptide. Here we report on the expression of NMURs and a potential role in nociception in the rat spinal cord using a combination of behavioral and electrophysiological studies.Receptor autoradiography showed that NMU-23 binding was restricted to the superficial layers of spinal cord, a region known to be involved in the control of nociception. In situ hybridization analysis indicated the mRNA of NMUR2 was located in the same region (laminae I and Ilo) as NMU-23 binding, while the mRNA for NMU receptor 1 was observed in a subpopulation of small diameter neurons of dorsal root ganglia. Intrathecal (i.t.) administration of neuromedin U-23 (0.4-4.0 nmol/10 mul) dose-dependently decreased both the mechanical threshold to von Frey hair stimulation and the withdrawal latency to a noxious thermal stimulus. Mechanical allodynia was observed between 10 and 120 min, peaking at 30 min and heat hyperalgesia was observed 10-30 min after i.t. administration of NMU-23. A similar mechanical allodynia was also observed following i.t. administration of NMU-8 (0.4-4 nmol/10 mul). A significant enhancement of the excitability of flexor reflex was induced by intrathecal administration of NMU-23 (4 nmol/10 mul). Evoked responses to touch and pinch stimuli were increased by 439+/-94% and 188+/-36% (P<0.01, n=6) respectively.The behavioral and electrophysiological data demonstrate, for the first time, a pro-nociceptive action of NMU. The restricted distribution of NMU receptors to a region of the spinal cord involved in nociception suggests that this peptide receptor system may play a role in nociception. (C) 2003 IBRO. Published by Elsevier Science Ltd. All rights reserved.

We examined the effect of galanin (10(-15) - 10(-7) M) on dispersed, mainly small-sized dorsal root ganglion (DRG) neurons in adult rats using whole-cell patch-clamp. Galanin and AR-M1896, a selective galanin type 2 receptor (GalR2) agonist, both significantly increased the number of action potentials in response to current pulses in 77% of the neurons, indicating an increase in excitability. Galanin also caused a rise in input resistance, decreased the holding current for -60 mV and depolarized the resting potential. In addition, Ca(2+) currents elicited by voltage steps were significantly increased by both galanin and AR-M1896 in nearly 70% of the cells. This enhancement was observed in 30% of the neurons in the presence of nimodipine or omega-conotoxin, but in each case approximately 60% less than without blocking either N- or L-type Ca(2+) channels, indicating modulation of both types of Ca(2+) channels. The percentage of small- and medium-sized neurons expressing GalR2 mRNA in DRGs in situ was similar to that showing increased excitability and Ca(2+) current after galanin application, i.e. approximately 70-80% of the neurons. The findings suggest that GalR2 has a role in controlling both the excitability, probably by inhibition of GIRK or leak K(+) channels, and Ca(2+) entry in a large population of presumably nociceptive neurons. The combination of the two effects, which possibly arise from separate biochemical pathways, would increase excitability and enhance intracellular Ca(2+) signalling which would enhance sensory transmission. These mechanisms involving GalR2 receptors may underlie the pronociceptive effects of galanin described in the literature.

To assess the validity of rodent models for investigating the role of delta opioid receptors (DOR) in analgesia, the distribution of DOR binding and mRNA were compared between rodent and primate spinal cord and dorsal root ganglia (DRG), using receptor autoradiography and in situ hybridization, respectively. In mouse and rat spinal cord, [I-125]-deltorphin-labeled DOR binding sites were detected throughout the gray matter. In contrast, in primate and particularly in human spinal cord, DOR binding was mainly present in laminae I-II, with little to no binding in deeper layers. Accordingly, in rodent spinal cord, DOR mRNA was expressed by a large number of neurons distributed throughout the ventral and dorsal horns, whereas in the primate, DOR expression was significantly lower, as evidenced by a moderate number of labeled cells throughout the gray matter in monkey and by only few labeled cells in human, mainly in Clarke's column and lamina IX. Major species differences in DOR expression were also observed in primary afferent cells bodies. In rat DRG, intense DOR mRNA hybridization was primarily observed over large ganglion cells immunopositive for neurofilament 200. In contrast, in monkey and human DRG, DOR mRNA was primarily detected over small and medium-sized ganglion cells. These results demonstrate major differences in the expression and distribution of DOR in the spinal cord and DRG between mammalian species. Specifically, they point to a progressive specialization of DOR toward the regulation of primary somatosensory, namely nociceptive, inputs during phylogeny and suggest that the effects of DOR agonists in rodents may not be entirely predictive of their action in humans. (C) 2003 Wiley-Liss, Inc.

Recent molecular cloning studies have established the existence of a third rat galanin receptor subtype, GalR3, however its precise distribution in the mammalian central nervous system (CNS) is not well established. In the present study, we examined the regional and cellular distribution of GalR3 mRNA in the CNS of the rat by in situ hybridization. Our findings indicate that GALR3 mRNA expression in the rat brain is discrete and highly restricted, concentrated mainly in the preoptic/hypothalamic area. Within the hypothalamus, GalR3 expression was confined to the paraventricular, ventromedial and dorsomedial hypothalamic nuclei. In addition to these hypothalamic nuclei, GalR3 mRNA-expressing cells were observed in the medial septum/diagonal band of Broca complex, the bed nucleus of the stria terminalis, the medial amygdaloid nucleus, the periaqueductal gray, the lateral parabrachial nucleus, the dorsal raphe nucleus, the locus coeruleus, the medial medullary reticular formation and in one of the circumventricular organs, the subfornical organ. In the spinal cord, a faint but specific ISH signal was observed over the laminae I-II with a few moderately labeled cells distributed in laminae V and X. The neuroanatomical distribution of GalR3 suggests it might be involved in mediating documented effects of galanin on food intake, fluid homeostasis, cardiovascular function and nociception.

The neuropathological features associated with Alzheimer's disease (AD) brain include the presence of extracellular neuritic plaques composed of amyloid beta protein (Abeta), intracellular neurofibrillary tangles containing phosphorylated tau protein and the loss of basal forebrain cholinergic neurons which innervate regions such as the hippocampus and the cortex. Studies of the pathological changes that characterize AD and several other lines of evidence indicate that Abeta accumulation in vivo may initiate phosphorylation of tau protein, which by disrupting neuronal network may trigger the process of neurodegeneration observed in AD brains. However, the underlying cause of degeneration of the basal forebrain cholinergic neurons and their association, if any, to Abeta peptides or phosphorylated tau remains mostly unknown. In the present study, using rat primary septal cultures, we have shown that aggregated Abeta peptides, in a time (18-96 h)- and concentration (0.7-60 muM)-dependent manner, induce toxicity and decrease choline acetyltransferase enzyme activity in cultured neurons. Using immunocytochemistry and immunoblotting, we have also demonstrated that Abeta treatment can significantly increase the phosphorylation of tau protein in septal cultures. At the cellular level, hyperphosphorylated tau is mostly apparent in the somatodendritic compartment of the neurons. Abeta peptide (10 muM), in addition to tau phosphorylation, also activates mitogen-activated protein kinase and glycogen synthase kinase-3beta, the two kinases which are known to be involved in the formation of hyperphosphorylated tau in the AD brain. Exposure to specific inhibitors of the mitogen-activated protein kinase (i.e. PD98059) or glycogen synthase kinase-3beta (i.e. LiCl) attenuated the hyperphosphorylation of the tau protein in cultured neurons.Given the evidence that tau phosphorylation can induce cell loss by disrupting neuronal cytoskeleton, it is likely that aggregated Abeta peptide triggers degeneration of septal neurons, including those expressing the cholinergic phenotype, by phosphorylation of the tau protein activated by mitogen-activated protein kinase and glycogen synthase kinase-3beta. These results, taken together, suggest that cultured septal cholinergic neurons are vulnerable to Abeta-mediated toxicity and tau phosphorylation may play an important role in Abeta-induced neurodegeneration. (C) 2002 IBRO. Published by Elsevier Science Ltd. All rights reserved.

As chemokines and their receptors are primarily expressed by glial cells in brain parenchyma, a model of glial cell proliferation may be useful to study the regulation of their expression in the brain. The well-established kainic acid seizure model was used in this study, focusing on the expression of the CCR5 chemokine receptor. Adult Sprague-Dawley rats were injected intraperitoneally with kainic acid (12 mg/kg), and in situ hybridization of CCR5 mRNA was performed at 12 h, 1, 3, or 7 days, posttreatment. Autoradiographic films and wet photographic emulsions demonstrated the very low expression of CCR5 mRNA in normal brain parenchyma, as well as in the microvasculature and ventricular/choroid plexus systems. After kainic acid treatment, brain CCR5 mRNA expression increased progressively from 12 h to 7 days, especially in the olfactory system, amygdaloid complex, thalamus, hippocampal formation, septum, and neocortex. This increase paralleled that of activated microglial cells as shown, using the microglial marker, OX-42. Moreover, CCR5 mRNA ISH combined with neuron-specific enolase immunocytochemistry showed that, in addition to its glial expression, CCR5 mRNA is expressed in neurons in the normal brain and, to a lesser extent, after kainate treatment due to neuronal losses. Finally, CCR5 protein is detected by immunocytochemistry in neurodegenerative areas in numerous glial cells, as well as in neurons, as clearly shown in the hippocampal formation. In summary, the chemokine receptor CCR5 is expressed by neuronal and non-neuronal cell types in the normal brain and is upregulated in both cell types after an insult.

Long-term exposure to nerve growth factor (NGF) is well established to have neurotrophic effects on basal forebrain cholinergic neurons, but its potential actions as a fast-acting neuromodulator are not as well understood. We report that NGF (0.1-100 ng/ml) rapidly (<60 min) and robustly enhanced constitutive acetylcholine (ACh) release (148-384% of control) from basal forebrain cultures without immediate persistent increases in choline acetyltransferase activity. More ACh was released in response to NGF when exposure was coupled with a higher depolarization level, suggesting activity dependence. In a long-term potentiation-like manner, brief NGF exposure (10 ng/ml; 60 min) induced robust and prolonged increases in ACh release, a capacity that was shared with the other neurotrophins. K252a (10-100 nm), BAPTA-AM (25 microm), and Cd(2+) (200 microm) prevented NGF enhancement of ACh release, suggesting the involvement of TrkA receptors, Ca(2+), and voltage-gated Ca(2+) channels, respectively. Forskolin (10 microm), a cAMP generator, enhanced constitutive ACh release but did not interact synergistically with NGF. Tetrodotoxin (1 microm) and cycloheximide (2 microm) did not prevent NGF-induced ACh release, indicative of action at the level of the cholinergic nerve terminal and that new protein synthesis is not required for this neurotransmitter-like effect, respectively. In contrast, after a 24 hr NGF treatment, distinct protein synthesis-dependent and independent effects on choline acetyltransferase activity and ACh release were observed. These results indicate that neuromodulator/neurotransmitter-like (protein synthesis-independent) and neurotrophic (translation-dependent) actions likely make distinct contributions to the enhancement of cholinergic activity by NGF.

Several lines of evidence indicate that nerve growth factor is important for the development and maintenance of the basal forebrain cholinergic phenotype. In the present study, using rat primary embryonic basal forebrain cultures, we demonstrate the differential regulation of functional cholinergic markers by nerve growth factor treatment (24--96 h). Following a 96-h treatment, nerve growth factor (1--100 ng/mL) increased choline acetyltransferase activity (168--339% of control), acetylcholine content (141--185%), as well as constitutive (148--283%) and K(+)-stimulated (162--399%) acetylcholine release, but increased release was not accompanied by increased high-affinity choline uptake. Enhancement of ACh release was attenuated by vesamicol (1 microM), suggesting a vesicular source, and was abolished under choline-free conditions, emphasizing the importance of extracellular choline as the primary source for acetylcholine synthesized for release. A greater proportion of acetylcholine released from nerve growth factor-treated cultures than from nerve growth factor-naïve cultures was blocked by voltage-gated Ca(2+) channel antagonists, suggesting that nerve growth factor modified this parameter of neurotransmitter release. Cotreatment of NGF (20 ng/mL) with K252a (200 nM) abolished increases in ChAT activity and prevented enhancement of K(+)-stimulated ACh release beyond the level associated with K252a, suggesting the involvement of TrkA receptor signaling. Also, neurotrophin-3, neurotrophin-4 and brain-derived neurotrophic factor (all at 5--200 ng/mL) increased acetylcholine release, although they were not as potent as nerve growth factor and higher concentrations were required. High brain-derived neurotrophic factor concentrations (100 and 200 ng/mL) did, however, increase release to a level similar to nerve growth factor. In summary, long-term exposure (days) of basal forebrain cholinergic neurons to nerve growth factor, and in a less-potent fashion the other neurotrophins, enhanced the release of acetylcholine, which was dependent upon a vesicular pool and the availability of extracellular choline.

A detailed investigation of endogenous acetylcholine (ACh) release from primary embryonic septal cultures is described in this study. Applications of veratridine (25 microM) or increasing extracellular concentrations of K(+) (6-100 mM) induced robust increases of endogenous ACh release ( approximately 500-15,000 fmol/well/10 min). Release stimulated with K(+) (25 mM) was sustainable and did not differ significantly over 180 min. ACh release was dependent on extracellular choline and decreased proportionally to choline concentrations (0-10 microM). For example, after 30 min of stimulation with K(+) (25 mM), release in the absence of extracellular choline was approximately 25% of that associated with 10 microM choline. The vesicular transport blocker vesamicol (0-5 microM) almost completely prevented stimulated and basal ACh release at the highest concentration evaluated, which suggests a mostly vesicular mode of release in this model. The M(2)-like muscarinic receptor antagonist AF-DX 384 (0-10 microM) enhanced stimulated ACh release ( approximately 150% at the highest concentration evaluated), whereas the nonspecific muscarinic receptor agonist oxotremorine (0-10 microM) decreased stimulated release (approximately 60% at the highest concentration evaluated), suggesting that functional muscarinic autoreceptors exist in primary embryonic septal cultures. Novel findings concerning ACh release from primary embryonic septal cultures are reported herein, and the demonstration of ACh release gives further credit to the use of these cultures for studying cholinergic system functioning and in relation to physiology and pathology.