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Development of neural circuitry relies on precise matching between correct synaptic partners and appropriate synaptic strength tuning. Adaptive developmental adjustments may emerge from activity and calcium-dependent mechanisms. Calcium/calmodulin-dependent protein kinase II (CaMKII) has been associated with developmental synaptic plasticity, but its varied roles in different synapses and developmental stages make mechanistic generalizations difficult. In contrast, we focused on synaptic development roles of CaMKII in a defined sensory-motor circuit. Thus, different forms of CaMKII were expressed with UAS-Gal4 in distinct components of the giant fiber system, the escape circuit of Drosophila, consisting of photoreceptors, interneurons, motoneurons, and muscles. The results demonstrate that the constitutively active CaMKII-T287D impairs development of cholinergic synapses in giant fiber dendrites and thoracic motoneurons, preventing light-induced escape behavior. The locus of the defects is postsynaptic as demonstrated by selective expression of transgenes in distinct components of the circuit. Furthermore, defects among these cholinergic synapses varied in severity, while the glutamatergic neuromuscular junctions appeared unaffected, demonstrating differential effects of CaMKII misregulation on distinct synapses of the same circuit. Limiting transgene expression to adult circuits had no effects, supporting the role of misregulated kinase activity in the development of the system rather than in acutely mediating escape responses. Overexpression of wild-type transgenes did not affect circuit development and function, suggesting but not proving that the CaMKII-T287D effects are not due to ectopic expression. Therefore, regulated CaMKII autophosphorylation appears essential in central synapse development, and particular cholinergic synapses are affected differentially, although they operate via the same nicotinic receptor.

In contrast to its well-established actions as an organizer of synaptic differentiation at the neuromuscular junction, the proteoglycan agrin is still in search of a function in the nervous system. Here, we report an entirely unanticipated role for agrin in the dual modulation of electrical and chemical intercellular communication that occurs during the critical period of synapse formation. When applied at the developing splanchnic nerve-chromaffin cell cholinergic synapse in rat adrenal acute slices, agrin rapidly modified cell-to-cell communication mechanisms. Specifically, it led to decreased gap junction-mediated electrical coupling that preceded an increase in nicotinic synaptic transmission. This developmental switch from predominantly electrical to chemical communication was fully operational within one hour and depended on the activation of Src family-related tyrosine kinases. Hence, agrin may play a pivotal role in synaptogenesis in promoting a rapid switch between electrical coupling and synaptic neurotransmission.

The aim of this study was to examine the architecture of the uveal nerves in the sclera and suprachoroid of human eyes. Eyes from 17 adult human donors were investigated. The uveal nerves in different regions (retrobulbar, intrascleral, suprachoroidal, pars plana) were prepared and studied by light and electron microscopy. In addition, immunohistochemistry was performed for various neuronal markers. The long uveal nerves showed a characteristic suprachoroidal location with no branches supplying the choroid. It was found that typically they are composed of myelinated (75%) and non-myelinated (25%) nerve fibres. They mainly contain aminergic and sensory nerve fibres. A separate set of cholinergic non-myelinated nerve fibre bundles runs parallel with these long uveal nerves. The short uveal nerves supply the suprachoroidal nerve plexus with approximately 13% of their nerve fibres. The nerves and the branches supplying the choroid appear as mixed nerves containing sympathetic, parasympathetic and sensory axons. This study therefore provides new information about the quantity, type and distribution of myelinated and non-myelinated nerve fibres in the posterior uvea of the human eye., (Copyright 2004 Anatomical Society of Great Britain and Ireland)

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.

In the present study, we examined the ability of estrogen to enhance cholinergic neurite arborization in vitro and identified the signal transduction cascade associated with this effect. Basal forebrain primordia collected from rat pups on postnatal day 1 were cultured for 2 weeks and then treated with 5 nm 17beta-estradiol for 24 hr. Cholinergic neurons were identified immunocytochemically with an antibody against the vesicular acetylcholine transporter and digitally photographed. Morphological analysis indicated that female cultures respond to estrogen treatment with an increase in total neurite length per neuron (4.5-fold over untreated controls) and in total branch segment number per neuron (2.3-fold over controls). In contrast, there was no change in total neurite length per neuron in male cultures, and we also observed a decrease in total branch segment number per neuron (0.5-fold below controls). Detailed histograms indicated that estrogen increases primary and secondary branch length and number and also increases terminal neuritic branches to the seventh order in female cultures. In a second set of experiments, we investigated the signal transduction cascade involved in this response, and found that an upstream extracellular signal-regulated kinase (ERK) inhibitor blocked the ability of estrogen to enhance outgrowth in female cultures. Our study provides strong evidence in support of the fact that the ERK pathway is required for estrogen-induced structural plasticity in the cholinergic system of female rats. Understanding the intracellular processes that underlie the response of cholinergic neurons to estrogen provides a necessary step in elucidating how cholinergic neurons can be particularly susceptible to degeneration in postmenopausal women.

GABA is the main inhibitory neurotransmitter that participates in the regulation of cholinergic outflow to the airways. We have tested the hypothesis that a monosynaptic GABAergic circuit modulates the output of airway-related vagal preganglionic neurons (AVPNs) in the rostral nucleus ambiguus by using a dual-labeling electron microscopic method combining immunocytochemistry for glutamic acid decarboxylase (GAD) with retrograde tracing from the trachea. We also determined the effects of blockade of GABAA receptors on airway smooth muscle tone. The results showed that retrogradely labeled AVPNs received a significant GAD-immunoreactive (GAD-IR) terminal input. Out of a pooled total of 3,161 synaptic contacts with retrogradely labeled somatic and dendritic profiles, 20.2% were GAD-IR. GAD-IR terminals formed significantly more axosomatic synapses than axodendritic synapses (P < 0.02). A dense population of GABAergic synaptic contacts on AVPNs provides a morphological basis for potent physiological effects of GABA on the excitability of AVPNs. GAD-IR terminals formed exclusively symmetric synaptic specializations. GAD-IR terminals were significantly larger (P < 0.05) in both length and width than unlabeled terminals synapsing on AVPNs. Therefore, the structural characteristics of certain nerve terminals may be closely correlated with their function. Pharmacological blockade of GABAA receptors within the rostral nucleus ambiguus increased activity of putative AVPNs and airway smooth muscle tone. We conclude that a tonically active monosynaptic GABAergic circuit utilizing symmetric synapses regulates the discharge of AVPNs.

Background: Cholera toxin (CT) acts on intestinal epithelial cells both directly and indirectly via activation of a secretory neural reflex. The reflex may release acetylcholine as one of its final neurotransmitters. This opens up the possibility of a third mechanism of action for CT, namely a synergistic interaction between two secretagogues acting on different second messenger systems within the epithelial cell., Aims: To establish evidence for cholinergic innervation to human ileal epithelial cells and to investigate whether CT potentiates the action of acetylcholine on human intestinal epithelial cells., Methods: Transverse sections of human ileum were examined for mucosal cholinergic nerves and M3 muscarinic receptors using antibodies raised to choline acetyltransferase and M3 receptors. Short circuit current (Isc) responses and ion flux movements were elicited from T84 epithelial cell monolayers set up in Ussing chambers., Results: Immunohistochemistry of native human ileal mucosa revealed the presence of both cholinergic nerves and muscarinic M3 receptors located to the basolateral domain of epithelial cells. Secretory responses of T84 cell monolayers to acetylcholine were greatly potentiated in the presence of CT. This effect, substituting forskolin for CT, was mirrored by increases in basolateral 86Rb and apical 125I efflux. Charybdotoxin plus apamin reduced both Isc and 86Rb efflux evoked by acetylcholine, in the presence of forskolin., Conclusions: Human ileal mucosa receives a direct cholinergic innervation to its epithelial cells. Secretory effects of acetylcholine on epithelial cells are augmented in the presence of CT. Such a synergistic response is dependent on optimum opening of basolateral potassium channels by acetylcholine and apical chloride channels by CT. The interaction may contribute to the mechanism of action of cholera toxin induced secretory diarrhoea.

Forebrain cholinergic neurons play important roles as striatal local circuit neurons and basal telencephalic projection neurons. The genetic mechanisms that control development of these neurons suggest that most of them are derived from the basal telencephalon where Lhx8, a LIM-homeobox gene, is expressed. Here we report that mice with a null mutation of Lhx8 are deficient in the development of forebrain cholinergic neurons. Lhx8 mutants lack the nucleus basalis, a major source of the cholinergic input to the cerebral cortex. In addition, the number of cholinergic neurons is reduced in several other areas of the subcortical forebrain in Lhx8 mutants, including the caudate-putamen, medial septal nucleus, nucleus of the diagonal band, and magnocellular preoptic nucleus. Although cholinergic neurons are not formed, initial steps in their specification appear to be preserved, as indicated by a presence of cells expressing a truncated Lhx8 mRNA and mRNA of the homeobox gene Gbx1. These results provide genetic evidence supporting an important role for Lhx8 in development of cholinergic neurons in the forebrain.

The basal forebrain cholinergic system strongly influences both cortical plasticity and learning. Directly relating these two roles has proven difficult. New results indicate that nucleus basalis lesions prevent motor cortex map plasticity and impair skill learning. These results strengthen the hypothesis that nucleus basalis gates neural plasticity necessary for instrumental learning.

Nerve growth factor (NGF) is known to promote both the survival of cholinergic neurons after injury and the regeneration of damaged cholinergic axons. Recent evidence has implicated NGF in the regulation of cholinergic axonal sprouting by intact neurons projecting to the hippocampus of rats, sustaining a lesion of the entorhinal cortex. We explored the possibility that NGF may regulate this lesion-induced cholinergic sprouting by injecting recombinant adeno-associated virus (rAAV) vector expressing NGF and green fluorescent protein (GFP) into the dentate gyrus of rats that were subsequently given unilateral entorhinal lesions. Sprague Dawley rats were unilaterally injected with (1) rAAV vector expressing NGF and GFP or (2) rAAV vector expressing GFP. Fourteen days after injection, the animals received lesions of the entorhinal area ipsilateral to the virus injection. Four days after lesion, GFP expression and the septodentate sprouting response in the dentate gyrus were assessed. Optical densitometric analyses revealed a significant increase in acetylcholinesterase label (a marker for cholinergic septodentate sprouting) in the ipsilateral outer molecular layer of the dentate gyrus in rats injected with rAAV vector expressing NGF. Thus, NGF-expressing rAAV vector enhanced the sprouting response of intact cholinergic neurons after unilateral entorhinal lesions in rats.

We employed by light and electron microscopy to examine the innervation of the posterior cricoarytenoid muscle of the adult rat. The laryngeal nerve was found to often bifurcate into two different bundles. One contained large myelinated (motor) nerve fibers, which were located along the frontal (ventral) muscle surface and entered the muscle at its middle portion to form neuromuscular contacts with individual muscle fibers. The other nerve bundle consisting of clustered ganglion cells (20-30 microm in diameter) and their associated nonmyelinated and small-sized myelinated nerve fibers were mainly found on the dorsal side of the muscle and often ran along the peripheral clefts or depressions of the muscle surface. The nerve bundle often extended side branches, which entered the muscle to be distributed among muscle fibers and near arterioles. Some ganglion cells are considered to enter the muscle, accompanied by branched nerves. Intramuscular ganglion cells and their associated nerve fibers examined by electron microscopy were similar in fine structure to perimuscular ganglion cells and their associated nerve fibers. Nerve fibers contained abundant clear synaptic vesicles which were cholinergic in nature, and often formed synapses with both neighboring axons and the cell body of the ganglion cells. These findings suggest that, in the rat posterior cricoarytenoid muscle, perimuscular and intramuscular ganglion cells exist and may be involved in innervating and contracting smooth muscle cells of the arterioles, thus regulating the blood flow or intravascular pressure.

The innervation of the uterus is remarkable in that it exhibits physiological changes in response to altered levels in the circulating levels of sex hormones. Previous studies by our group showed that chronic administration of estrogen to rats during the infantile/prepubertal period provoked, at 28 days of age, an almost complete loss of norepinephrine-labeled sympathetic nerves, similar to that observed in late pregnancy. It is not known, however, whether early exposure to estrogen affects uterine cholinergic nerves. Similarly, it is not known to what extent development and estrogen-induced responses in the uterine cholinergic innervation are affected by the absence of sympathetic nerves. To address this question, in this study we analyzed the effects of infantile/prepubertal chronic estrogen treatment, chronic chemical sympathectomy with guanethidine, and combined sympathectomy and chronic estrogen treatment on developing cholinergic nerves of the rat uterus. Cholinergic nerves were visualized using a combination of acetylcholinesterase histochemistry and the immunohistochemical demonstration of the vesicular acetylcholine transporter (VAChT). After chronic estrogen treatment, a well-developed plexus of cholinergic nerves was observed in the uterus. Quantitative studies showed that chronic exposure to estrogen induced contrasting responses in uterine cholinergic nerves, increasing the density of large and medium-sized nerve bundles and reducing the intercept density of fine fibers providing myometrial and perivascular innervation. Estrogen-induced changes in the uterine cholinergic innervation did not appear to result from the absence/impairment of sympathetic nerves, because sympathectomy did not mimic the effects produced by estrogen. Estrogen-induced responses in parasympathetic nerves are discussed, considering the direct effects of estrogen on neurons and on changes in neuron-target interactions.

Aging is known to markedly affect the number and structural characteristics of both pre- and post-synaptic sites in the cerebral cortex. There is evidence that lamina V pyramidal neurons, and their basilar dendrites in particular, are affected by age-related decline. Furthermore, layer V is the area where the greatest overall age- related losses in the total population of synaptic boutons and of cholinergic boutons are observed. Since both pyramidal neurons and cortical cholinergic input are characteristically compromised in aging, we investigated whether aging altered the pattern of cholinergic boutons in apposition to the soma, proximal and distal basal dendrites of intracellularly labeled lamina V large pyramidal neurons in the parietal cortex of young and aged rats. We observed a significant age-related decrease in the population of both total and cholinergic boutons apposed to proximal and distal dendrites of layer V large pyramidal neurons. However, the age-related decreases of cholinergic presynaptic boutons were higher than those in the total bouton population apposed to the pyramidal neurons. The average decrease in cholinergic boutons in aged rats was 3.7-fold more pronounced than the diminution in the overall number of presynaptic boutons. Our results add important new evidence in support of the concept that the age-related learning and memory deficits are attributable, at least partially, to a decline in the functional integrity of the forebrain cholinergic systems.

Immunohistochemical study of the cholinergic innervation of the parvalbumin- and calbindin-containing cells in the hippocampus was conducted on 30 rat brains of various postnatal ages: P0, P4, P7, P14, P21, P30, P60 and P180. Sections with double immunostaining for vesicular acetylcholine transporter (VAChT; the marker of cholinergic cells, fibres and terminals) and parvalbumin (PV) or calbindin (CB) were analysed using confocal laser-scanning microscope. Obtained data demonstrate that the pattern of cholinergic innervation of calbindin- and parvalbumin-immunoreactive hippocampal neurones shows some differences. During development as well as in the adult species cholinergic terminals preferentially innervate CB-containing neurones, while cholinergic terminals on PV-containing cells were observed rarely. Cholinergic endings on the CB-ir neurones are localised both on their somata and dendrites, whereas on PV-ir cells they form synaptic contact predominantly with processes. In spite of the unquestionable cholinergic influence particularly on CB-ir cells, the number of cholinergic endings suggests that this input seems not to be crucial for the activity of the studied cell populations.

Akinesia and gait disturbances are particularly incapacitating for patients with Parkinson's disease. The anatomical and physiological substrates for these disturbances are poorly understood. The pedunculopontine nucleus (PPN) is thought to be involved in the initiation and modulation of gait and other stereotyped movements, because electrical stimulation and the application of neuroactive substances in the PPN can elicit locomotor activity in experimental animals. Glutamatergic neurones of the PPNd (pars dissipatus) are thought to be important regulators of the basal ganglia and spinal cord. The other component of the PPN, the cholinergic pars compacta (PPNc), is a principal component in a feedback loop from the spinal cord and limbic system back into the basal ganglia and thalamus. Electrophysiological studies suggest that 'bursting' glutamatergic PPNd neurones are related to the initiation of programmed movements while non-bursting cholinergic PPNc neurones are related to the maintenance of steady-state locomotion. Furthermore, since patients with Parkinson's disease have significant loss of PPN neurones and experimental lesions in the PPN of normal monkeys result in akinesia, the degeneration of PPN neurones or their dysfunction may be important in the pathophysiology of locomotor and postural disturbances of parkinsonism. The goal of this review is (i) to highlight the anatomical connections and physiological attributes of the PPN, (ii) to discuss how the function of these connections may be altered in the parkinsonian state, and (iii) to speculate how present and potential future therapy directed to the PPN might improve akinesia and gait difficulties in parkinsonian patients.

A protein of Mr 100,000 (MVP100) is highly enriched in the electromotor system of electric rays. Biochemical analysis indicates that MVP100 is contained in the cholinergic nerve terminals of Torpedo electric organ as part of a large cytosolic complex. On sucrose density gradient centrifugation MVP100 comigrates with synaptic vesicles or synaptosomes. It can be partially separated from synaptic vesicles by gel filtration or glycerol velocity gradient centrifugation. Within the complex MVP100 behaves like a hydrophobic protein and is protected against proteolytic attack. MVP100 can be immunodetected by an antibody against phosphotyrosine, and it becomes phosphorylated on incubation with [gamma-32P]ATP. By screening an electric ray electric lobe cDNA library the primary structure of MVP100 was analyzed. MVP100 is highly homologous to the major vault proteins of slime mold and rat and to the human lung resistance-related protein. Compared with non-neural tissues the expression of MVP100 is highest in brain and enriched in the electric lobe that contains the somata of the electromotor neurons. Immunoelectron microscopic analysis reveals a close association of MVP100 and synaptic vesicles in the nerve terminals of the electric organ.

A cholinergic locus has recently been identified consisting of a unique mammalian genomic arrangement containing the genes for choline acetyltransferase (ChAT) and a putative vesicular acetylcholine transporter (VAChT). Although transcripts for ChAT and VAChT protein have been localized in cholinergic neurons, little is known about the encoded VAChT protein. Here we describe production of highly specific rabbit polyclonal antibodies, generated using a VAChT C-terminus/glutathione-S-transferase fusion protein, and immunological characterization of the native VAChT protein. These antibodies specifically recognized full-length recombinant VAChT expressed in transfected HeLa cells by Western blotting, with the prominent immunoreactive band at 55 kDa. In rat brain homogenates, a single VAChT-immunoreactive band of approximately 70 kDa was predominant in known areas of cholinergic innervation, including striatum, cortex, hippocampus,and amygdala. Light microscopic immunocytochemistry revealed reaction product in cholinergic cell groups but not in noncholinergic areas. More significantly, immunoreactivity was also concentrated in axonal fibers in many regions known to receive prominent cholinergic innervation, such as cerebral cortex, hippocampus, amygdala, striatum, several thalamic nuclei, and brainstem regions. Electron microscopy using immunoperoxidase revealed that VAChT was localized in axon terminals, and using more precise immunogold techniques, to synaptic vesicles. In VAChT-positive perikarya, the immunogold particles were localized to the cytoplasmic face of the Golgi complex. These findings confirm that VAChT protein is expressed uniquely in cholinergic neurons, concentrated in synaptic vesicles, and at least for the C terminus, topologically oriented as predicted by models.

The distribution of the ciliary ganglion (CG) innervation to the pigeon choroid was determined immunohistochemically, using antisera against choline acetyltransferase (CHAT) and a neurofilament-related protein (the 3A10 antigen). Single-labeling revealed that the nerve fibers containing these two antigens were similarly distributed in the pigeon choroid, with the superior and temporal quadrants of the eye containing the most fibers. Both types of fibers surrounded and ramified on choroidal blood vessels. Additionally, CHAT+ varicosities were evident among vessels in the choroid and choriocapillaris. Double-label immunofluorescence revealed that CHAT and the 3A10 antigen were almost completely colocalized in choroidal nerve fibers, but absent from CHAT+ varicosities. Substance P-containing and calcitonin gene-related peptide-containing choroidal nerve fibers were poor in 3A10+ labeling. Transection of the postganglionic fibers of the CG reduced CHAT+ and 3A10+ nerve fibers in the choroid to 3-5% of normal abundance, with most of the residual fibers being located in the nasal and inferior quadrants. The present results suggest that the CG in pigeon preferentially influences choroidal blood flow in the superior and temporal parts of the eye, which are involved in high acuity and binocular vision.

1. The effects of A1 and A2a adenosine receptor agonists on the veratridine-evoked release of [3H]-acetylcholine ([3H]-ACh) from rat striatal synaptosomes was investigated by use of the A1-selective agonist, R-PIA and the 185 fold selective A2a agonist, CGS 21680. The effects of NECA, which is equipotent at both receptor subtypes, were also studied. 2. The evoked release of [3H]-ACh was significantly enhanced by the A2a agonist CGS 21680 but decreased by the A1 agonist, R-PIA. The effects of NECA were dependent on the concentration used, with high concentrations inhibiting and low concentrations enhancing the evoked release of [3H]-ACh. In the absence of any antagonists, the rank order of potency for these three drugs on increasing [3H]-ACh release was CGS 21680 > NECA > R-PIA. 3. The stimulatory effects of CGS 21680 and low NECA concentrations on evoked [3H]-ACh release were antagonized by the A2a receptor antagonists, CP66,713 (300 nM) and CGS 15943A (50 nM) whilst the inhibitory effects of R-PIA were reversed by the selective A1 antagonist, DPCPX (4 nM). In the presence of DPCPX, NECA greatly enhanced the evoked release of [3H]-ACh at all concentrations studied when, during such A1 receptor blockade, the rank order of potency was NECA >> CGS 21680 > R-PIA. 4. These results demonstrate that both A1 and A2a adenosine receptors modulate the veratridine-evoked release of [3H]-ACh from rat striatal synaptosomes.

1. The electrophysiological and pharmacological characteristics of the calcium current (ICa) in acutely dissociated magnocellular cholinergic basal forebrain neurones from 11- to 14-day-old post-natal rats were studied using the whole-cell patch-clamp technique. 2. All cells exhibited a small transient low-voltage-activated (LVA) current with half-activation and half-inactivation potentials of -40.2 and -49.3 mV and slope factors for activation and inactivation of 4.82 and 3.85 mV per e-fold change in membrane potential (Vm) respectively. Activation and inactivation rates for the LVA current were highly voltage dependent. For test potential changes from -50 to -20 mV, the time-to-peak of the current decreased from 39.1 to 6.4 ms, and the time constant of current decay decreased from 81.7 to 15.5 ms. 3. A high-voltage-activated (HVA) component of ICa could be elicited at threshold voltages between -46 and -30 mV from a holding potential (VH) of -80 mV. The HVA current peaked around 0 mV; a 10-fold increase in [Ca2+]o produced a 13 mV positive shift in the peak, whilst the amplitude of the current showed an approximately hyperbolic relationship to [Ca2+]o with half-saturation at 2.5 mM. The transient phase of the HVA current could be described by two exponential functions with time constants tau fast and tau slow of 16.2 and 301 ms. Steady-state inactivation of the transient and extrapolated true sustained (pedestal) components of HVA current were described by Boltzmann equations, with half-inactivation potentials (slope factors) of -47.3 mV, (9.04) and -29.2 mV (11.8) respectively. 4. omega-Conotoxin (omega-CgTX; 100 nM) irreversibly inhibited a kinetically distinct component of HVA current but had no effect upon the transient LVA current. The omega-CgTX-sensitive current could not be distinguished from the control HVA current by the voltage dependence of its activation or inactivation rates. 5. Low concentrations of amiloride (< or = 300 microM) or Ni2+ (< or = 5 microM) selectively inhibited the transient LVA current, with IC50 values of 97 and 5 microM respectively. Cd2+ (< or = 1 microM) selectively blocked a component of HVA current. At higher concentrations, Cd2+ and Ni2+ were non-selective and totally blocked all components of ICa. 6. The lanthanide ions Gd3+ and La3+ produced saturable incomplete block of the HVA current. Maximally effective concentrations of Gd3+ (100 microM) or La3+ (30 microM) inhibited 76.5 and 41.2% respectively of the sustained component of HVA current with corresponding IC50 values of 2.2 and 1.1 microM.(ABSTRACT TRUNCATED AT 400 WORDS)

In this study light and EM quantitative analysis were used to examine whether exogenous nerve growth factor (NGF) could affect terminal fields and synaptic connections in the adult rat brain in vivo. Adult rats received, immediately after unilateral decortication, 2.5S NGF (12 micrograms/day) or vehicle intracerebroventricularly for 7 days. Thirty days after the lesion cholinergic fiber length was quantified, using image analysis, in the remaining cortical area adjacent to the lesion site in each animal. Rats that had received vehicle showed a significantly reduced cortical choline acetyl-transferase-immunoreactive fiber network in the remaining cortex when compared with control animals. By contrast, the network in lesioned rats that had received 2.5S NGF was not different from control animals. Furthermore, the number of cortical choline acetyltransferase-immunoreactive varicosities, which decreased in vehicle-treated lesioned rats, significantly increased above control in lesioned rats that had received 2.5S NGF. At the ultrastructural level, 30 days after the lesion, animals that had received vehicle showed shrunken cholinergic boutons in cortical layer V and fewer synapses compared with control animals. Exogenous NGF, administered to lesioned rats, increased to supernormal levels both size of cholinergic boutons and number of synaptic contacts. These parameters were unaltered in unlesioned rats treated with NGF. This study demonstrates that exogenous NGF can cause significant compensatory changes in terminal fields and synaptic connections in the adult fully differentiated central nervous system.

Different conditions in the arrangement of the adrenal gland are observed in urodeles. The gland consists of islets scattered on the ventral surface of the kidneys, the amount, size and position of the islets varying consistently within different families and even within genera. The infraordinal variation also extends to the fine structure of the gland, as observed in 14 species belonging to 6 different families. The ultrastructural characteristics of chromaffin cells and their relationships with interrenal cells appear to be related to the phyletic position. In primitive urodeles (Sirenidae, Proteidae) the chromaffin cells are isolated or in small groups, mostly separated from interrenal cells and often in contact with renal cells. In neourodeles (Amphiumidae, Ambystomidae, Salamandridae, Plethodontidae) the chromaffin cells appear generally grouped and intermingled with steroidogenic cells. Some cytological characteristics of chromaffin cells, such as nerve supply and the shape and electron density of chromaffin granules exhibit a variability related to phyletic position.

Coronal vibratome sections of the rostral part of the medial septum (MS) and vertical limb of the diagonal band of Broca (VDB) nuclei were studied by an immunocytochemical technique using a monoclonal antibody against choline acetyltransferase (ChAT) and a double histochemical method for detection of acid phosphatase (AcPase) and nucleoside diphosphatase (NDPase) activity. The electron microscopic morphology of ChAT-immunoreactive and non-immunoreactive neurons was compared with similar neurons showing both AcPase and NDPase activity. ChAT-labeled and non-labeled neurons were well differentiated by the organization of the endomembrane system and especially by the structure of the rough endoplasmic reticulum (RER) and associated lamellar bodies. These results support the theory that the peculiar ultrastructure of the lamellar bodies in each neuron is related to the pattern of organization of the endomembrane system and its function. The significance of the lamellar bodies is discussed, and the data of the present work, together with findings described by other investigators. These data suggest that these bodies are predominant in efferent projection neurons in the basal forebrain nuclei.

The innervation of the adrenal medulla has been investigated in normal Wistar rats from birth to old age and ultrastructural findings compared with biochemical markers of the cholinergic innervation of the adrenal gland and catecholamine storage. Morphological evidence of the immaturity of the innervation during the first postnatal week is provided and using quantitative morphometry the innervation of chromaffin cells is shown to reach a mean total of 5.4 synapses per chromaffin cell during the period 26 days to 12 weeks of age. The variation in contents of synaptic profiles is discussed in the light of recent work that demonstrates a major sensory as well as visceral efferent innervation of the gland. Adrenal medullary neurons usually occur in closely packed groups, intimately associated with Schwann cells. Axodendritic and axosomatic synapses on these neurons are described and the likely origin of axonal processes innervating the neurons discussed. In old age the density of innervation remains the same as in young adult animals even though the medulla shows evidence of hyperplasia and hypertrophy of individual chromaffin cells.

We studied the development of selective adrenergic and cholinergic neuroeffector transmission in primary cultures of isolated ventricular muscle cells. Explants of either thoracolumbar sympathetic ganglia or sacrococcygeal spinal cord were added to newborn rat ventricular cultures harvested prior to the onset of in vivo autonomic innervation. Neuronal growth, migration, and the formation of neuromuscular junctions were observed with light and scanning electron microscopy. Glyoxylic acid histofluorescence, reflecting catecholamine synthesis, was found in only the sympathetic neuromuscular cultures. Choline acetyltransferase activity was detected in both spinal cord and sympathetic neuromuscular cultures, but was significantly higher in the spinal cord neuromuscular cultures. The isolated ventricular muscle cells remained at a constant spontaneous contraction frequency, regardless of the type of culture preparation. Guanethidine sulfate application produced a positive chronotropic response, blocked by propranolol, in the sympathetic neuromuscular cultures, but not in the spinal cord neuromuscular cultures. Bethanechol sulfate produced a negative chronotropic response, blocked by atropine, in the spinal cord neuromuscular cultures, but not in the sympathetic neuromuscular cultures. Isolated ventricular muscle cells in the absence of neurons failed to respond to either agent. Direct microelectrode stimulation of adrenergic or cholinergic neurons likewise respectively produced either a positive or negative ventricular muscle cell chronotropic response. These studies are the first to establish the selective production of functional cholinergic and adrenergic innervation of isolated cardiac muscle cells in vitro.

An analysis of the innervation of arteriovenous anastomoses in the web of the foot of the Pekin Duck (Anas platyrhynchos) was compared with the innervation of the right atrium of the duck heart using histochemical, ultrastructural and morphometric techniques, before and after 6OHDA. The presence of intense catecholamine fluorescence and nerve terminals containing typical noradrenergic small dense-cored vesicles, together with the absence of fluorescence and degeneration of noradrenergic terminals after 6OHDA, indicated the presence of a dense adrenergic innervation at the periphery of the anastomoses. Ultrastructural and histochemical data gave support to the presence of a cholinergic innervation. There was evidence that arteriovenous anastomoses were innervated by non-adrenergic, non-cholinergic nerves, viz. after 6OHDA, the mean diameter, mean percentage and mean density of granular vesicles in axon profiles associated with anastomoses (107 . 25 nm, 22 . 34% and 12 . 73 vesicles micron-2, respectively) were significantly higher (P less than 0 . 001) than values in the atrium (87 . 13 nm, 9 . 92% and 5 . 51 vesicles micron-2, respectively) and axons associated with anastomoses contained large granular vesicles ranging up to 210 nm in diameter. This non-adrenergic non-cholinergic innervation may represent the non-adrenergic non-cholinergic vasodilatory nerves shown by pharmacological methods to be present in the foot of the Pekin Duck.

The mechanical and pharmacological characteristics of the cholinergic activation of the smooth muscle in the choroidal coat of the chick eye have been assessed in tissues isolated from birds 1 d posthatching using histological, electrophysiological, and immunological techniques. The choroidal coat is innervated by a dense network of cholinergic nerves that make en passant synapses with smooth muscle. Thirty-hertz stimulation of these nerves initiates red blood cell (RBC) movement in the vessels of the choroidal coat, and this activation is blocked by muscarinic ACh receptor (AChR) antagonists. Force-transducer recordings of nerve-induced contractions of this tissue have a slow onset and relaxation time course similar to those of smooth muscle contractions. Furthermore, since nearly half the cholinergic neurons innervating the choroid die within a defined period during development, the onset and pharmacology of this innervation were studied during embryogenesis. With a neural cytoskeletal-like immunostain, we demonstrated that choroid axons are present in peripheral tissue by stage (St) 29. Extracellular electrical recordings made after choroid nerve stimulation allowed us to distinguish axon from muscle responses. These procedures permitted us to examine the time course of the innervation of the smooth muscle. However, to visualize the postsynaptic smooth muscle response, it was necessary to treat the isolated preparation with tetraethylammonium chloride (TEA). Accordingly, TEA-enhanced electrical smooth muscle responses to single-nerve stimuli could be recorded only after St 39. Treatment of the nerve-muscle preparation with prostigmine allowed the recording of TEA-enhanced electrical activity as early as St 36 (1 d after the beginning of the normal choroid neuron death period). This synaptic activation was completely blocked by atropine or quinuclidinyl benzylate (QNB), and was not affected by alpha bungarotoxin (alpha BTX), indicating that, as in the posthatching tissue, neuromuscular transmission is mediated by muscarinic receptors. These results show that cholinergic muscarinic activation of the choroidal coat can occur as early as St 36, but that it is not as efficient as transmission later in embryogenesis.

The chicken retina has been used to examine the toxicity of a highly reactive chemical analog of choline, ethylcholine mustard aziridinium ion (ECMA). Following a single intravitreal injection, retinas were analyzed biochemically for CAT and AChE activities, and GABA, glycine, and dopamine levels. Retinas were also examined using histofluorescence for dopamine histochemistry, for AChE, and immunohistochemistry with antibodies to CAT, tyrosine hydroxylase, GABA, 5-HT, Leu-enkephalin, and somatostatin. A dose of 50 nmol ECMA caused a prolonged 70% depletion of CAT activity and a 40% depletion of AChE activity. The other biochemical parameters were unchanged. This result corresponds to the morphological finding that 2 populations of cholinergic cells were destroyed and that the AChE activity associated with their terminal arbors was lost. A third population of cholinergic cells, located towards the middle of the inner nuclear layer, was resistant to the toxic effects of ECMA. The other cell types, except for somatostatin-immunoreactive cells and photoreceptors, which showed transient effects, were unaffected. ECMA therefore appears to be a highly specific toxin for cholinergic cells in the retina.

A general model of the autonomic neuroeffector junction is proposed. In this model, emphasis is placed on the muscle effector bundle with electrotonic coupling between individual cells via gap junctions (or nexuses) and en passage release of transmitter from autonomic nerve varicosities. This release results in transmission to effector cells across junctional clefts ranging from about 20 nm in the vas deferens and iris to as much as 2000 nm in some large arteries. The ultrastructural identification of different autonomic nerve types is described. Current theories on the synthesis, storage, release, and inactivation of transmitter during cholinergic, adrenergic, and purinergic transmission are summarized. Some speculations are made about the possible involvement of purinergic nerves in the innervation of vessels and mast cells in the skin, and whether this involvement results in a functional link between ATP, histamine, bradykinin, and prostaglandin in cutaneous vasodilatation. Another possibility considered as the basis for this reflex is the release of substance P from sensory (pain) nerve collaterals in the skin.

Investigations of the nerves of the rabbit lung, by light and electron microscopy, showed a dense acetylcholinesterase-positive innervation of the bronchi through the bronchiolar level. Large nerve bundles were found to decrease in size as they progressed from extrachondral to subchondral connective tissue, forming complex networks of mostly terminal fibres in the muscle layer. In several instances single fibres penetrated the submucosal layer and approached the mucosa. Gangliocytes, which also reacted positively for cholinesterase, were visible in the vicinity of the large peribronchial bundles. Gangliocytes rarely were seen in association with the vasculature. Blood vessels received a much less dense cholinesterase-positive nerve supply than the bronchi. Single, non-terminal fibres were noted at the adventitiomedial junction of the pulmonary artery and vein. In addition, segments of nerve fibres (networks) were observed in the arterial and venous smooth muscle layers. Cholinesterase-positive innervation was even less extensive in the veins than in the arteries.

Selective autonomic denervations of the iris have been used to study the possible redistribution of adrenergic markers within adult nerve fiber systems and to reveal the cellular origin of a nonsympathetic fiber plexus induced to express such markers. The presence and distribution of fibers showing neuropeptide Y (NPY)- and tyrosine hydroxylase (TH)-like immunoreactivity was studied in the rat iris using stretch-prepared whole mounts. Normal irides contained a dense regular network of NPY-positive varicose fibers. Such fibers were regularly seen innervating blood vessels. The choroid membrane had a high number of fluorescent fibers. A similar, although slightly denser TH-positive fiber system was visualized in the iris. One or 2 days after surgical removal of the superior cervical ganglion, almost all NPY- and TH-positive fibers had disappeared, suggesting that most, if not all, NPY-positive fibers in the iris originate in the superior cervical ganglion. In irides from long-term sympathectomized animals, a high number of TH- and NPY-immunoreactive fibers had reappeared, while such irides were devoid of catecholamine-containing fibers, as evidenced by Falck-Hillarp histochemistry. The appearance of TH- and NPY-positive fibers in sympathetically denervated irides was clearly time dependent. The distribution of fluorescent fibers in irides from intact and sympathectomized animals showed obvious dissimilarities such as a lower fluorescence intensity and fewer varicose fibers in denervated irides. Furthermore, in irides from sympathectomized rats, TH- and NPY-positive fibers were not associated with blood vessels. Unilateral removal of the parasympathetic ciliary ganglion, which supplies the iris with cholinergic fibers, 3 days prior to sacrifice in animals bilaterally sympathectomized 1 month earlier, led to a drastic reduction in numbers of TH- and NPY-positive iris fibers on the ciliarectomized/sympathectomized side as compared to the sympathectomized-alone side. The present experiments thus suggest that adult cholinergic neurons in vivo are capable of expressing adrenergic characteristics under experimental conditions.

Sympathetic neurons taken from rat superior cervical ganglia and grown in culture acquire cholinergic function under certain conditions. These cholinergic sympathetic neurons, however, retain a number of adrenergic properties, including the enzymes involved in the synthesis of norepinephrine (NE) and the storage of measurable amounts of NE. These neurons also retain a high affinity uptake system for NE; despite this, the majority of the synaptic vesicles remain clear even after incubation in catecholamines. The present study shows, however, that if these neurons are depolarized before incubation in catecholamine, the synaptic vesicles acquire dense cores indicative of amine storage. These manipulations are successful when cholinergic function is induced with either a medium that contains human placental serum and embryo extract or with heart-conditioned medium, and when the catecholamine is either NE or 5-hydroxydopamine. In some experiments, neurons are grown at low densities and shown to have cholinergic function by electrophysiological criteria. After incubation in NE, only 6% of the synaptic vesicles have dense cores. In contrast, similar neurons depolarized (80 mM K+) before incubation in catecholamine contain 82% dense-cored vesicles. These results are confirmed in network cultures where the percentage of dense-cored vesicles is increased 2.5 to 6.5 times by depolarizing the neurons before incubation with catecholamine. In both single neurons and in network cultures, the vesicle reloading is inhibited by reducing vesicle release during depolarization with an increased Mg++/Ca++ ratio or by blocking NE uptake either at the plasma membrane (desipramine) or at the vesicle membrane (reserpine). In addition, choline appears to play a competitive role because its presence during incubation in NE or after reloading results in decreased numbers of dense-cored vesicles. We conclude that the depolarization step preceding catecholamine incubation acts to empty the vesicles of acetylcholine, thus allowing them to reload with catecholamine. These data also suggest that the same vesicles may contain both neurotransmitters simultaneously.

The giant cholinergic neuron R2 of Aplysia was cultured in combination with identified neurons L11 and R15 and members of a group of left upper quadrant (LUQ) cells L2 to L6 from the abdominal ganglion. All of these neurons receive cholinergic input from other cells in vivo, but not from R2. In vitro, R2 reliably formed unidirectional chemical connections with these cells. Single action potentials in R2 produced a dual fast and slow inhibitory response in LUQ cells (L2 to L6), a dual fast inhibitory-slow excitatory response in L11, and a slow inhibitory response in R15. The connections formed on LUQ cells were characteristic of their cholinergic input, but the R2-L11 and the R2-R15 connections also had noncholinergic properties. Thus, unlike L10 which forms connections only with its normal targets in vitro, R2 forms strong chemical connections with other neurons which are not found in vivo. The properties of the R2 connections also suggest that it may release another neurotransmitter besides acetylcholine.

The cholinergic innervation of the human axillary sweat glands of hyperhidrotic patients was demonstrated by using the specific Karnovsky-Roots thiocholine method. The cholinergic innervation pattern was compared with the immunohistochemically demonstrated vasoactive intestinal polypeptide (VIP)-like activity at light and electron microscopic levels. The innervation patterns were identical in the light microscopic serial sections. In the electron microscope sections, VIP-like immunoreactivity was localized to the nerve terminals containing large, dense-cored vesicles 100-140 nm in size. No synapses were found, however positively stained nerve terminals were located immediately outside the basement membrane but close to the glandular secretory and myoepithelial cells, blood vessels, and occasionally the mast cells. Our results suggest the coexistence of the two neurotransmitters, acetylcholine and VIP, in the same nerves innervating both eccrine and apocrine sweat glands in human axillae.

Pure cholinergic nerve endings (synaptosomes) were isolated from the electric organ of Torpedo by a rapid procedure. These synaptosomes are approximately 3 micron in diameter. They contain an occasional mitochondrion, numerous synaptic vesicles, and sometimes an active zone is observed. No postynaptic membrane attachment is found. This nerve ending fraction is extremely pure as shown by morphological controls and biochemical data. It is rich in choline acetyltransferase (450 nmol/h per mg protein) and acetylcholine (ACh) (130 nmol/mg protein). The isolated endings retain their cytoplasmic components and they synthesize ACh and are stable in vitro for several hours, as shown by biochemical measurements and morphological analysis.

The neuromorphology and neuropharmacology of the human penis are only briefly described in literature. The present study was undertaken to define the adrenergic and cholinergic neuromorphology of the human corpus cavernosum (CC) and corpus spongiosum and to evaluate the in vitro response of the CC to pharmacologic stimulation. Human penile tissue was obtained from six transsexual patients undergoing penectomy. For morphologic study, the tissue was processed for (a) hematoxylin and eosin staining; (b) smooth muscle staining; (c) acetylcholinesterase localization; (d) glyoxylic acid histofluorescence; (e) electron microscopy; and (f) electron microscopy after glutaraldehyde dichromate fixation. In addition, strips of CC were placed in in vitro muscle chambers and tension changes recorded isometrically after stimulation with norepinephrine (NE) and acetylcholine. The CC contains abundant smooth muscle, numerous glyoxylic acidfluorescent (catecholaminergic) fibers and varicosities, and a scant distribution of acetylcholinesterase-positive fibers. Fewer of all these elements were present in the corpus spongiosum. No "polsters" were observed in the CC. Although glutaraldehyde-fixed controls exhibited no typical adrenergic vesicles (small, dense core, measuring 400-600 A in diameter), some small, strongly electron-dense vesicles were found in glutaraldehyde dichromate-fixed tissue and were thought to contain NE. A variety of other vesicles were also encountered. The addition of NE to the in vitro muscle chambers caused a dose-related contraction, which was blocked by pretreatment with phentolamine in all CC strips tested. Acetylcholine in high concentration produced minimal contraction in 2 of 24 strips. Our morphologic and pharmacologic data suggest that the sympathetic nervous system may affect erection by acting not only on the penile vasculature but also by direct action on the smooth muscle of the CC itself.