Nicotinic synapses formed between chick ciliary ganglion neurons in culture resemble those present on the neurons in vivo.

We studied nicotinic synapses between chick ciliary ganglion neurons in culture to learn more about factors influencing their formation and receptor subtype dependence. After 4--8 days in culture, nearly all neurons displayed spontaneous excitatory postsynaptic currents (sEPSCs), which occurred at about 1 Hz. Neurons treated with tetrodotoxin displayed miniature EPSCs (mEPSCs), but these occurred at low frequency (0.1 Hz), indicating that most sEPSCs are actually impulse driven. The sEPSCs could be classified by decay kinetics as fast, slow, or biexponential and, reminiscent of the situation in vivo, were mediated by two major nicotinic acetylcholine receptor (AChR) subtypes. Fast sEPSCs were blocked by alpha-bungarotoxin (alpha Bgt), indicating dependence on alpha Bgt-AChRs, most of which are alpha 7 subunit homopentamers. Slow sEPSCs were unaffected by alpha Bgt, and were blocked instead by the alpha 3/beta 2-selective alpha-conotoxin-MII (alpha CTx-MII), indicating dependence on alpha 3*-AChRs, which lack alpha 7 and contain alpha 3 subunits. Biexponential sEPSCs were mediated by both alpha Bgt- and alpha 3*-AChRs because they had fast and slow components qualitatively similar to those comprising simple events, and these were reduced by alpha Bgt and blocked by alpha CTx-MII, respectively. Fluorescence labeling experiments revealed both alpha Bgt- and alpha 3*-AChR clusters on neuron somata and neurites. Colabeling with antisynaptic vesicle protein antibody suggested that some alpha 3*-AChR clusters, and a few alpha Bgt-AChR clusters are associated with synaptic sites, as is the case in vivo. These findings demonstrate the utility of ciliary ganglion neuron cultures for studying the regulation of nicotinic synapses, and suggest that mixed AChR subtype synapses characteristic of the neurons in vivo can form in the absence of normal inputs or targets.

Expression and channel properties of alpha-bungarotoxin-sensitive acetylcholine receptors on chick ciliary and choroid neurons.

Cell-specific expression of nicotinic acetylcholine receptors (AChRs) was examined using ciliary and choroid neurons isolated from chick ciliary ganglia. At embryonic days 13 and 14 (E13,14) the neurons can be distinguished by size, with ciliary neuron soma diameters exceeding those of choroid neurons by about twofold. Both neuronal populations are known to express two major AChR types: alpha3*-AChRs recognized by mAb35, that contain alpha3, alpha5, beta4, and occasionally beta2 subunits, and alpha-bungarotoxin (alphaBgt)-AChRs recognized and blocked by alphaBgt, that contain alpha7 subunits. We found that maximal whole cell current densities (I/C(m)) mediated by alphaBgt-AChRs were threefold larger for choroid compared with ciliary neurons, while alpha3*-AChR current densities were similar in the two populations. Different densities of total cell-surface alphaBgt-AChRs could not explain the distinct alphaBgt-AChR response densities associated with ciliary and choroid neurons. Ciliary ganglion neurons display abundant [(125)I]-alphaBgt binding ( approximately 10(6) sites/neuron), but digital fluorescence measurements revealed equivalent site densities on both populations. AChR channel classes having single-channel conductances of approximately 30, 40, 60, and 80 pS were present in patches excised from both ciliary and choroid neurons. Treating the neurons with alphaBgt selectively abolished the 60- and 80-pS events, identifying them as arising from alphaBgt-AChRs. Kinetic measurements revealed brief open and long closed durations for alphaBgt-AChR channel currents, predicting a very low probability of being open (p(o)) when compared with 30- or 40-pS alpha3*-AChR channels. None of the channel parameters associated with the 60- and 80-pS alphaBgt-AChRs differed detectably, however, between choroid and ciliary neurons. Instead calculations based on the combined whole cell and single-channel results indicate that choroid neurons express approximately threefold larger numbers of functional alphaBgt-AChRs (N(F)) per unit area than do ciliary neurons. Comparison with total surface [(125)I]-alphaBgt-AChR sites (N(T)), reveals that N(F)/N(T) << 1 for both neuron populations, suggesting that "silent" alphaBgt-AChRs predominate. Choroid neurons may therefore express a higher density of functional alphaBgt-AChRs by recruiting a larger fraction of receptors from the silent pool than do ciliary neurons.

Nicotinic acetylcholine receptor agonists promote survival and reduce apoptosis of chick ciliary ganglion neurons.

The abundance, diversity, and ubiquitous expression of neuronal nicotinic acetylcholine receptors (AChRs) suggest that many are involved in functions other than synaptic transmission. We now report that a major AChR class promotes neuronal survival. The 10-day survival of ciliary ganglion neurons in basal culture medium (MEM) was approximately 35%, but increased to approximately 75% in MEM containing nicotine (MEM/Nic) or carbachol, an effect similar to that achieved by chronic depolarization with KCl. Pharmacological experiments revealed that agonist-enhanced survival requires activation of AChRs sensitive to alpha-bungarotoxin (alphaBgt). alphaBgt-AChRs partly support neuronal survival by limiting apoptosis since fewer apoptotic neurons were observed in MEM/Nic compared to MEM. Moreover, nicotinic survival support was not further enhanced by fibroblast growth factor, as seen for KCl, but increased to 100% by adding PACAP, a trophic neuropeptide present in the ganglion. These results indicate that alphaBgt-AChR activation regulates neuronal survival and suggest a mechanism involving reduced apoptosis and interaction with an endogenous neuropeptide growth factor.

Pituitary adenylate cyclase-activating polypeptide activates a phospholipase C-dependent signal pathway in chick ciliary ganglion neurons that selectively inhibits alpha7-containing nicotinic receptors.

Neuropeptide receptors couple via G-proteins to two principal signaling pathways that elevate cAMP through adenylate cyclase (AC) or mobilize intracellular Ca(2+) through phospholipase C (PLC)-stimulated inositol phosphate (IP) turnover and production of inositol 1,4,5-trisphosphate (IP(3)). We showed previously that high-affinity receptors for pituitary adenylate cyclase-activating polypeptide (PACAP) are present on chick ciliary ganglion neurons and that receptor occupation increases cAMP production, resulting in enhanced acetylcholine sensitivity. After we suppressed AC activity and cAMP production with 2'-5' dideoxyadenosine, however, PACAP no longer increased acetylcholine sensitivity but instead reduced it, suggesting that an AC-independent signal pathway activated by PACAP inhibits some nicotinic acetylcholine receptors (AChRs). We now use fast-perfusion, imaging, and biochemical methods to identify the AChRs modulated by PACAP and to characterize the signal pathway responsible for their inhibition. Without previous AC block, both the rapidly desensitizing, alpha-bungarotoxin (alphaBgt)-sensitive alpha7-AChRs and the slowly desensitizing, alphaBgt-insensitive alpha3*-AChRs on the neurons were potentiated by PACAP. After AC blockade, however, PACAP inhibited alpha7-AChRs but left alpha3*-AChRs unaffected. The selective inhibition of alpha7-AChRs appeared to use a PLC signaling pathway because it was not seen after lowering PLC activity or buffering intracellular Ca(2+) and was mimicked by dialyzing neurons with an IP(3) receptor agonist. PACAP also induced IP turnover and increased [Ca(2+)](i) assessed directly with Fluo-3AM imaging. Given our previous findings that PACAP receptors couple to AC, the present results demonstrate a remarkable ability of a single neuropeptide to activate two signaling pathways and in so doing selectively regulate two classes of downstream ion channel targets.

Chick ciliary ganglion neurons contain transcripts coding for acetylcholine receptor-associated protein at synapses (rapsyn).

A peripheral membrane protein of approximately 43 kDa (rapsyn) clusters muscle nicotinic acetylcholine receptors (AChRs), but molecules relevant to clustering neuronal AChRs have not been identified. Here, we have detected rapsyn transcripts in the chick nervous system, localized rapsyn mRNA in ciliary ganglion (CG) neurons, which are known to cluster AChRs, and identified three rapsyn cDNAs derived from the ganglion. Our initial Northern blots, performed using a mouse probe, revealed rapsyn-like transcripts in chick muscle and brain. To develop species-specific probes, we prepared a chick rapsyn cDNA construct, Ch43K.1, that encodes a protein having extensive homology to mouse rapsyn. Using primers designed to anneal near the 5' and 3' boundaries of Ch43K.1, three prominent cDNAs were amplified from chick muscle templates by reverse transcriptase based-PCR. Products of similar size were also amplified using cDNA prepared from neuronal tissues expected to contain clustered AChRs (CG and brain), whereas none were detected using templates from tissues not displaying clustered AChRs (sensory ganglia and liver). In situ hybridization confirmed that rapsyn mRNA is expressed both in chick muscle fibers and in CG neurons. Sequencing the three cDNAs amplified from CG templates revealed the largest to be Ch43K.1, whereas the smaller two may represent splice variants. These findings suggest that multiple rapsyn-like molecules are involved in clustering the distinct AChRs expressed by muscle fibers and neurons.

Expression of Kv1.1 delayed rectifier potassium channels in Lec mutant Chinese hamster ovary cell lines reveals a role for sialidation in channel function.

Kv1.1 potassium (K+) channels contain significant amounts of negatively charged sialic acids. To examine the role of sialidation in K+ channel function, Chinese hamster ovary cell lines deficient in glycosylation (Lec mutants) were transfected with rat brain Kv1.1 cDNA. The K+ channel was functionally expressed in all cell lines, but the voltage dependence of activation (V1/2) was shifted to more positive voltages and the activation kinetics were slower in the mutant cell lines compared with control. A similar positive shift in V1/2 was recorded in control cells expressing Kv1.1 following treatment with sialidase or by raising extracellular Ca2+. In contrast, these treatments had little or no effect on the Lec mutants, which indicates that channel sialic acids appear to be the negative surface charges sensitive to Ca2+. The data suggest that sialic acid addition modifies Kv1.1 channel function, possibly by influencing the local electric field detected by its voltage sensor, but that these carbohydrates are not required for cell surface expression.

Expression of nicotinic acetylcholine receptors and subunit mRNA transcripts in cultures of neural crest cells.

The expression of neuronal phenotypic characteristics by neural crest-derived neurons is dependent upon both environmental cues and intrinsic properties. Neuronal nicotinic acetylcholine receptors (nAChRs) are expressed by most neural crest-derived peripheral neurons; nevertheless, the factors that influence and modulate the early expression of nAChRs on neuronal precursor cells are unknown. We used RT-PCR to analyze the temporal sequence of the appearance of transcripts encoding alpha 3, alpha 5, alpha 7, beta 2, and beta 4 nAChR subunits in cultures of quail neural crest cells. At 2 days in culture, mRNA encoding each of these nAChR subunits was detectable and, except for alpha 7, remained so through 12 days. The appearance of NAPA 73 immunoreactivity during neural crest cell migration suggested that some cells express neuronal characteristics early in development and that the expression of nAChR subunits may be cell autonomous for some neural crest-derived cells. Between 8 and 10 days in culture, a 2.0-kb transcript encoding alpha 3 subunit appears in addition to the expected 3.5-kb transcript that is present at 8 days. nAChR protein was detected immunocytochemically at 7 days and was observed in immunoblots by 9 days. Whole-cell patch clamp recordings demonstrated that functional nAChRs develop concurrently in a subset of neural crest-derived neurons. In addition, both neuronal and nonneuronal cells showed inward and delayed outward currents in response to step depolarization. The appearance of functional nAChRs on neural crest-derived cells, in vitro, occurs independently of innervation and of the components in growth medium. The data suggest that neural crest-derived cells express transcripts encoding nAChR subunits, possibly as an intrinsic property of some peripheral neurons, early in development.

Pituitary adenylate cyclase-activating polypeptide type I receptors mediate cyclic AMP-dependent enhancement of neuronal acetylcholine sensitivity.

Nicotinic acetylcholine (ACh) receptors (AChRs) on ciliary ganglion neurons are positively regulated by elevated cAMP levels. Vasoactive intestinal peptide (VIP) can act as a first messenger in the regulation, because application of 1 microM VIP rapidly increases both neuronal cAMP levels and ACh sensitivity. We now report that high affinity receptors for a close VIP relative, pituitary adenylate cyclase-activating polypeptide (PACAP), are present on ciliary ganglion neurons and mediate the cAMP-dependent modulation of AChRs. Consistent with the presence of PACAP type I receptors, binding studies revealed sites on the neurons having approximately 1000-fold higher affinity for the 38- and 27-amino acid forms of PACAP than for VIP, and cAMP radio-immunoassays demonstrated that PACAP38 and PACAP27 are approximately 600-fold more potent agonists for mobilizing neuronal cAMP than is VIP. In accord with their higher affinity and potency, PACAP38 and -27 (both at 10 nM) increased neuronal ACh sensitivity by approximately 50% within 10 min, whereas VIP at the same low concentration was ineffective. The increased ACh sensitivity induced by 10 nM PACAP38 or PACAP27 or 1 microM VIP depends on coincident increases in cAMP levels, because treatment of neurons with adenylate cyclase inhibitors blocked both effects. The findings demonstrate the presence of functional PACAP type I receptors on ciliary ganglion neurons that preferentially recognize PACAP38 and -27 over VIP and act via adenylate cyclase to initiate cAMP-dependent enhancement of AChR function. Finally, we detected PACAP38-like material in ciliary ganglia, suggesting a role for the peptide in modulating neuronal AChRs in vivo.

VIP modulates neuronal nicotinic acetylcholine receptor function by a cyclic AMP-dependent mechanism.

Neuronal nicotinic ACh receptors (AChRs) mediate synaptic transmission throughout the nervous system, and are regulated by cellular processes and interactions that include second messenger signaling pathways. In the case of chick ciliary ganglion neurons, activation of the cAMP-dependent signaling pathway with cAMP analogs enhances ACh sensitivity in a manner consistent with an increase in the number of functional nicotinic receptors. We have now identified vasoactive intestinal peptide (VIP) as a neuromodulator or "first messenger" in the cAMP-mediated pathway that regulates neuronal AChRs. Using cAMP imaging and biochemical detection assays, we find that bath application of VIP elevates intracellular cAMP in freshly isolated ciliary ganglion neurons within minutes. The VIP treatment also enhances neuronal ACh sensitivity assessed with whole-cell recording. The enhanced ACh sensitivity produced by VIP appears with a short latency, similar to that associated with the increase in cAMP, and is not additive with the enhanced ACh sensitivity produced by bath application of a cAMP analog. In contrast, calcitonin gene-related peptide (CGRP), known to regulate muscle nicotinic AChRs via a cAMP-dependent pathway, has no detectable effect on levels of either cAMP or ACh sensitivity in the neurons. The results indicate that VIP enhances the ACh sensitivity of ciliary ganglion neurons via a cAMP-dependent signaling pathway, presumably by interaction with a specific receptor. Since VIP-like immunoreactivity is present in the presynaptic nerve terminals of avian ciliary ganglia, a VIP-like peptide could modulate AChRs in vivo.

Eye-extract factors promote the expression of acetylcholine sensitivity in chick dorsal root ganglion neurons.

We have examined the development of acetylcholine (ACh) responses in chick sensory neurons and their regulation by growth factors. Between Embryonic Days E13 and E18 in vivo, the proportion of freshly dissociated dorsal root ganglion (DRG) neurons that were appreciably sensitive to 500 microM ACh increased from approximately 40 to > 90% and the size of the ACh response per unit membrane area increased nearly 10-fold. Nerve growth factor (NGF) supports the survival, growth, and differentiation of central, sympathetic, and sensory neurons. NGF also promotes ACh sensitivity in PC12 cells and nodose ganglion neurons grown in cell culture. When E13 chick DRG neurons were maintained in culture with NGF for 4-6 days, however, only approximately 50% had appreciable ACh sensitivity, and large responses comparable to those observed at E18 in vivo were rare. Chick-eye extract contains a 21.5-kDa trophic factor termed growth-promoting activity (GPA) that supports survival of sympathetic and DRG neurons for 24 hr in culture and an activity of approximately 50 kDa that promotes increases in choline acetyltransferase activity, ACh sensitivity, and ACh receptors (AChRs) in ciliary ganglion neurons (Nishi and Berg, 1981; Halvorsen et al., 1991). In the present study, whole-eye extract supported full survival of E13 DRG neurons for up to 6 days in culture and promoted ACh sensitivity in > 90% of the neurons tested. GPA-containing eye-extract fractions and NGF individually supported full DRG neuron survival for up to 6 days in culture, but neither promoted ACh sensitivity. Adding eye-extract fractions containing material of approximately 50 kDa for 4-6 days to either GPA- or NGF-supplemented media led to appreciable ACh sensitivity in approximately 90% of the DRG neurons tested. The levels of ACh sensitivity observed in DRG neurons maintained either in whole-eye extract or in fractions containing 50-kDa material were close to those observed at an equivalent developmental age in vivo. The results indicate that components in the 50-kDa eye-extract fraction promote the ACh sensitivity of DRG neurons in culture and suggest that similar factors may influence the appearance of AChRs on the neurons in vivo.

Modulation by albumin of neuronal cholinergic sensitivity.

Bovine serum albumin greatly enhanced the cholinergic response mediated by neuronal nicotinic acetylcholine receptors in chick ciliary ganglion neurons. The enhancement exceeded 5-fold in some experiments (mean +/- standard error, 3.26 +/- 0.43-fold) and was rapid, was dose dependent, and occurred without changes in the unitary conductance or the mean open time of the acetylcholine receptor channel. This lack of detectable change in permeation or kinetic properties suggests that bovine serum albumin might increase acetylcholine responses by increasing the number of functional receptors. The enhancement appears to be specific to the albumin molecule, because activity could not be removed by detergent extraction, gel filtration, or dialysis. Acetylcholine responses in these cells are known to be enhanced by a cAMP-dependent mechanism that converts existing acetylcholine receptors from a nonfunctional to a functional state. We found that the enhancement by bovine serum albumin occurred without an increase in cAMP and that pretreatment with membrane-permeable cAMP analogs prevented any additional enhancement of the cholinergic response by bovine serum albumin. These observations are consistent with a cAMP-dependent modulation of the enhancement produced by bovine serum albumin or a convergence of the two enhancement mechanisms onto a single pathway.

Changes in the number, function, and regulation of nicotinic acetylcholine receptors during neuronal development.

Acetylcholine receptor (AChR) development was examined in neurons freshly isolated from chick ciliary ganglia. Between Embryonic Day 8 (E8) and 16, both the ACh response per unit membrane and the density of surface AChRs increased, while the apparent affinity for ACh decreased. AChRs had single-channel conductances of 25 and 40 pS. The distribution of single-channel events shifted during development; at E8 events of both conductances were equally rare, while by E14 there were many events and most were 40 pS. The open durations of 25 and 40 pS events had two mean values. The open lifetimes of the 25 pS events did not change between E8 and E14, while the lifetimes of the 40 pS events increased, and by E14 most were long. The ACh response of the neurons also became sensitive to regulation by a cAMP-dependent mechanism at about E10. The observed changes may reflect developmental control over processing required for receptor regulation and differential expression of AChR subtypes.

Regulating the number and function of neuronal acetylcholine receptors.

Nicotinic acetylcholine receptors (AChRs) on vertebrate neurons represent a family of receptors distinct from the well-characterized AChR of skeletal muscle. New probes for neuronal AChRs are now being used to examine the regulation of receptor number and function. The results suggest that neuronal AChRs differ from muscle AChRs in regulation by presynaptic input and by at least one second messenger system, and that neuronal AChRs are additionally regulated by retrograde signals from the synaptic target tissue. The forms of regulation provide potential mechanisms by which cell-cell interactions could stabilize synaptic contacts on neurons and modulate synaptic function.

Cyclic AMP regulates the proportion of functional acetylcholine receptors on chicken ciliary ganglion neurons.

Previous studies have shown that the number of functional acetylcholine receptors (AcChoRs) on chicken ciliary ganglion neurons in culture is considerably smaller than the total number of AcChoRs detected on the neurons by labeled receptor probes. Here we use patch-clamp recording to show that a cAMP-dependent process enhances the AcCho response of the neurons by a mechanism likely to involve an increase in the number of functional AcChoRs. The increase occurs without requiring protein synthesis and without involving a detectable increase in the total number of AcChoRs on the cell surface measured with a labeled receptor probe. The results imply that the neurons have functional and nonfunctional pools of AcChoRs and that functional receptors can be recruited from intracellular receptors or from nonfunctional receptors on the cell surface by a cAMP-dependent process. A cAMP-dependent regulation of the number of functional neurotransmitter receptors would provide a reversible mechanism by which cell-cell interactions could modulate synaptic transmission in the nervous system.

The properties and regulation of functional acetylcholine receptors on chick ciliary ganglion neurons.

The properties of acetylcholine receptor (AChR) channels on chick ciliary ganglion neurons in culture were examined using patch-clamp recording techniques. Acetylcholine (ACh) was applied by rapid microperfusion. Whole-cell current noise analysis revealed a single class of functional receptors on the neurons. Dose-response studies indicated a Kd of about 36 microM and a Hill coefficient of 1.5-1.7, predicting 2 ACh binding sites per receptor. Both fast and slow components of receptor desensitization were observed. Single-channel recordings from excised outside-out patches of soma membrane exposed to 2-5 microM ACh indicated a single-channel conductance of 40 pS, a reversal potential of -9 mV, a mean open duration of 1 msec, and an opening probability of 0.34. The kinetic behavior of the channels was provisionally described by a 3-closed, 1-open state model for receptor activation. In all of these properties, AChRs of ciliary ganglion neurons resemble those on skeletal muscle fibers. Growing the neurons in an elevated K+ concentration produced a 2-3-fold decrease in peak whole-cell currents induced by ACh under standard test conditions, without altering any of the single-channel properties described above. Neither changes in cholinesterase activity nor receptor distribution accounted for the decrease. Instead, calculations indicated that elevated K+ reduced the ACh response by decreasing the number of functional AChRs on the neurons. No K+-dependent decrease is observed, however, in the number of total receptors on the neurons detected either by a monoclonal antibody specific for the receptor or by an alpha-neurotoxin that binds to the receptor and blocks its function. Moreover, the number of receptors detected by the 2 probes is at least 10-fold greater than the calculated number of functional receptors. The findings suggest that only a small fraction of the AChRs on the neuronal surface is functional and that the cell can alter the ratio of functional and nonfunctional receptors in response to growth conditions.

Enkephalin and substance P modulate synaptic properties of chick ciliary ganglion neurons in cell culture.

Since enkephalin- and substance P-like immunoreactive materials have been identified in preganglionic terminals of the avian ciliary ganglion, we tested the effects of enkephalin and substance P directly on chick ciliary ganglion neurons in dissociated cell culture. Under these conditions the neurons form cholinergic synapses with each other that are spontaneously active. Both peptides modulate properties of membrane components associated with synaptic transmission between the neurons. Enkephalin causes a 60% reduction in the mean amplitude of the excitatory synaptic potentials, and the effect appears to be presynaptic in origin: enkephalin does not alter acetylcholine sensitivity on the neurons, but does inhibit Ca2+ influx as reflected by a 38% shortening of the Ca2+ component of the action potential. Both the reduction in synaptic potential amplitude and the shortening of the Ca2+ action potential produced by enkephalin are blocked by naloxone. Substance P, on the other hand, has no effect on Ca2+ action potentials but does reduce the time course of acetylcholine responses in the neurons by a mechanism consistent with enhanced receptor desensitization. Decay of the acetylcholine voltage response in the absence of substance P is described by a single exponential process with a time constant of 4-5 s. Coapplication of acetylcholine and substance P results in a second exponential decay process with a time constant of about 1 s that appears after a 200-400 ms lag period. Preincubation with substance P alone does not decrease the peak voltage response or shorten the lag, suggesting that either agonist or activated receptor is necessary for the substance P effect. These findings suggest modulatory roles for the peptides in ganglionic transmission.

Cholinergic modulation of an acetylcholine receptor-like antigen on the surface of chick ciliary ganglion neurons in cell culture.

Chick ciliary ganglion neurons have a membrane component that shares an antigenic determinant with the "main immunogenic region" of the alpha subunits in nicotinic ACh receptors from skeletal muscle and electric organ. Ultrastructural studies on antibody binding in the ganglion have shown that the cross-reacting antigen on the neuron surface is located predominantly in synaptic membrane. Biochemical studies have shown that the cross-reacting component has a number of other properties expected for the ganglionic nicotonic ACh receptor and that it is distinct from the alpha-bungarotoxin binding component in the tissue. Here we show that ciliary ganglion neurons grown in dissociated cell culture express a similar component that cross-reacts with monoclonal antibodies to ACh receptors, and that the number of antibody-binding sites on the neurons can be modulated by exposure to cholinergic agonists and a protein neurotoxin that reversibly inhibits ACh receptors on the neurons. In most, though not all, cases, levels of ACh sensitivity associated with the neurons are specifically comodulated in parallel with the changes in number of antibody binding sites. The results suggest that at least a portion of the cross-reacting sites on the surface of ciliary ganglion neurons is likely to represent nicotinic ACh receptors. The fact that in some instances levels of ACh sensitivity can be altered without changing the number of cross-reacting sites, however, leaves open the possibility that not all of the sites are associated with receptors or that the neurons can alter the proportion of receptors that is functional.

gamma-Aminobutyric acid receptors on chick ciliary ganglion neurons in vivo and in cell culture.

In the chick ciliary ganglion, preganglionic terminals maintain cholinergic synapses on the choroid neurons and both cholinergic and electrical synapses on the ciliary neurons. The preganglionic terminals also contain enkephalin- and substance P-like immunoreactivity, suggesting that transmission through the ganglion is more complicated than is indicated by the known synaptic connections. We report here that embryonic chick ciliary ganglion neurons also have gamma-aminobutyric acid (GABA) receptors and that GABA applied to the ganglion can block transmission elicited by preganglionic stimulation. Studies on the neurons in cell culture indicate that the GABA response is mediated by GABAA receptors: GABA activates a Cl- conductance, and the response can be mimicked by muscimol and blocked by bicuculline or picrotoxin. The GABA receptors are regulated independently from acetylcholine (ACh) receptors on the neurons since the levels of ACh and GABA sensitivity are influenced differently by culture age and by chronic exposure to GABA or elevated K+ concentrations. Application of GABA to intact ciliary ganglia increases the membrane conductance of ganglionic neurons (as in culture), reduces to subthreshold the amplitude of excitatory postsynaptic potentials in the neurons elicited by preganglionic stimulation and completely blocks transmission through the ganglion. A native source of ligand for the receptors in vivo has yet to be identified.

Differential regulation of acetylcholine sensitivity and alpha-bungarotoxin-binding sites on ciliary ganglion neurons in cell culture.

Levels of acetylcholine (ACh) sensitivity and numbers of alpha-bungarotoxin (alpha-Bgt)-binding sites have been measured for chick ciliary ganglion neurons grown in cell culture under various conditions. The two properties were found not to change in parallel. Neurons maintained in culture medium supplemented with embryonic eye extract developed high levels of ACh sensitivity and low numbers of alpha-Bgt-binding sites, whereas neurons grown in medium containing elevated K+ concentrations displayed the reverse. Neurons from media containing both eye extract and elevated K+ concentrations had both low levels of sensitivity and low numbers of toxin sites. The growth conditions do not alter the basic binding properties of the ACh receptors and alpha-Bgt-binding sites. Both the ACh receptor dose-response characteristics and the pharmacological properties of the toxin-binding sites were similar for neurons grown in media containing eye extract or elevated K+ concentrations. The inhibitory effects of eye extract on development of alpha-Bgt-binding sites appeared to be specific: eye extract had previously been shown to stimulate neuronal growth and cholinergic development, and in the present study eye extract enhanced development of ACh sensitivity and had no effect on mechanisms responsible for binding and accumulation of tetanus toxin. Eye extract did not block alpha-Bgt binding in competition binding experiments and did not cause redistribution of toxin sites away from the neuronal soma. These results demonstrate that ACh sensitivity and alpha-Bgt-binding sites can be independently regulated on the neurons and suggest that the two membrane properties are associated with separate membrane components.

Conductance and dye permeability of a rectifying electrical synapse.

Electrical synapses provide a basis for efficient signal transmission in a wide variety of nervous systems. These synapses are composed of specialized cell-to-cell contacts known as nexuses or gap junctions which mediate the direct transfer of ions and small molecules between adjacent cell interiors by way of intercellular channels embedded in the junctional membrane. The crayfish giant motor synapse (GMS) was the first cell-to-cell junction clearly demonstrated to operate by an electrical mechanism. Current applied to the presynaptic lateral giant (LG) axon or to the neurite of the postsynaptic giant flexor motoneurone (MoG) spreads passively through the synapse into the adjacent neurone. Each GMS behaves like an electrical rectifier: its conductance is high when LG is positive with respect to MoG, and decreases dramatically when the sign of the trans-synaptic voltage is reversed. We have now examined GMS conductance and dye permeability at thoracic and abdominal levels of the crayfish nerve cord. At both levels, values of GMS chord conductance fit a simple Boltzmann model in which the conductance of individual synaptic channels is assumed to be voltage dependent. Moreover, thoracic synapses display higher limiting conductances than do those at an abdominal level, apparently as a result of their larger size. We also find that synapses at both locations are permeable to the fluorescent dye Lucifer yellow, even in conditions where electrical conductance is low. These results provide a framework for understanding the operation and functional limits of rectifying electrical synapses, and illustrate that dye permeability can be associated even with their relatively low conductance condition.