Chick Ciliary Ganglion Neurons Research Articles

Nicotinic and muscarinic acetylcholine responses in the embryo chick ciliary ganglion cells

Nicotinic and muscarinic acetylcholine (ACh) responses were investigated in acutely dissociated chick ciliary ganglion neurons using the nystatin perforated patch clamp technique. ACh-induced a rapid transient inward current in 100% of the neurons at a holding potential of −60 mV. This rapid inward current was mimicked by nicotine but not by muscarine. The reversal potential of the rapid inward current was + 10.5 mV and the current was inhibited by d-tubocurarine and hexamethonium in a dose-dependent manner. In 57.6% of neurons, a slow inward current was also induced by ACh at a holding potential of −20 mV. This slow inward current was mimicked by muscarine but not by nicotine. The slow inward current became smaller at a hyperpolarized potential but not reversed, being consistent with the fact that this current was elicited by the inhibition of M-current. p-Fluorohexa-hydrosiladifenidol (P-F-HHSiD) strongly inhibited the slow inward current, suggesting that the current was elicited by the activation of M 3 receptors.

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.

Somatostatin-induced inhibition of neuronal Ca2+ current modulated by cGMP-dependent protein kinase

Neurotransmitter release is frequently regulated by peptides that modulate neuronal calcium channels. Whole-cell recordings show that the ion permeability and voltage dependence of these channels are controlled by a membrane-associated pathway involving GTP-binding proteins. Here we use perforated-patch recordings to show that, in addition to this pathway, the peptide somatostatin inhibits the calcium current in chick ciliary ganglion neurons by a second soluble pathway involving a cyclic GMP-dependent protein kinase (cGMP-PK). This somatostatin inhibition of Ca2+ current did not desensitize and was not characterized by the slowing of Ca(2+)-current activation (kinetic slowing) observed in whole-cell recordings. When cGMP-PK was inhibited, somatostatin inhibition of Ca2+ current resembled that observed with whole-cell recordings. cGMP agonists mimic the effect of somatostatin only in perforated patch recordings. An endogenous cGMP-PK therefore forms part of the mechanism by which somatostatin induces a sustained inhibition of neuronal calcium channels.

Target tissues and innervation regulate the characteristics of K+ currents in chick ciliary ganglion neurons developing in situ

The expression of appropriate ensembles of ionic channels is necessary for the differentiation and normal function of vertebrate neurons. Cell-cell interactions may regulate the expression and properties of ionic channels in embryonic neurons. Previous studies have shown that the expression of A-type K+ channels (IA) and Ca2+-activated K+ channels (lK[Ca]) is abnormal in chick ciliary ganglion neurons developing in vitro in the absence of normal cell-cell interactions. Other voltage-activated currents develop normally under these conditions. The present studies were designed to establish the role of the target tissues and the preganglionic innervation in regulating the expression of these currents in embryonic chick ciliary ganglion neurons developing in situ. Surgical manipulations were used to remove the developing optic vesicle, which contains the target tissues, the mid-dorsal region of the midbrain primordium, which contains the preganglionic nucleus, or both, all prior to the formation of the ciliary ganglion. IA and IK[Ca] were then examined in acutely isolated neurons that developed in ovo in the presence (OV+) or absence (OV-) of the normal target tissues, in the presence (MB+) or absence (MB-) of preganglionic innervation, and in the absence of both preganglionic innervation and target tissues (OV-/MB-). The amplitude of IA was unaffected by the operations. However, the activation and inactivation kinetics of IA were two- to threefold faster in OV- or OV-/MB- cells compared to neurons isolated from control OV+ ganglia at embryonic days 11-14 (E11-E14). There were no changes in the voltage dependence of activation or steady-state inactivation, or in the time course of recovery from inactivation. By contrast, neurons isolated from MB- ganglia expressed an IA with amplitude, voltage dependence, and kinetics that were indistinguishable from those of control MB+ and OV+ ganglia. Therefore, interactions with target tissues in the eye play a role in determining the characteristics of IA in developing ciliary ganglion neurons, whereas preganglionic innervation does not. Furthermore, the amplitude of IK[Ca] was reduced by 90-100% in OV-, MB-, and OV-/MB- neurons isolated at E12-E14 as compared to MB+ and OV+ controls. Voltage-activated Ca2+ currents were present at normal amplitudes in all of these neurons. Thus, the expression of IK[Ca] in chick ciliary ganglion neurons is regulated by both target tissue interactions and preganglionic innervation. Therefore, cell-cell interactions are necessary for the expression of a normal ensemble of ionic channels in chick ciliary ganglion neurons developing in situ.

Nicotine-induced intracellular calcium changes are not antagonized by α-bungarotoxin in adrenal medullary cells

The snake toxin α-bungarotoxin distinguishes between neuronal nicotinic receptor subtypes. In chick ciliary ganglion neurons, activation of α-bungarotoxin-sensitive nicotinic receptors has been proposed to produce elevations in intracellular calcium levels. In the present study we show that prolonged treatment with α-bungarotoxin did not affect the nicotine-evoked calcium response in suspended chromaffin cells. On the other hand, the classical nicotinic receptor blocker d-tubocurarine potently blocked nicotinic receptor mediated effects. The degree of inhibition of the nicotinic response observed with d-tubocurarine was not modified by prior treatment with α-bungarotoxin. These results suggest that nicotinic α-bungarotoxin receptors are not primarily involved in nicotine-mediated increases in intracellular calcium in bovine adrenal medullary cells.

Differential distribution of purine metabolizing enzymes between glia and neurons.

Previous studies showed that in cultured chick ciliary ganglion neurons and CNS glia, adenosine can be synthesized by hydrolysis of 5'-AMP and that the accumulation of the adenosine degradative products inosine and hypoxanthine was significantly greater in glial than in neuronal cultures. Furthermore, previous immunochemical and histochemical studies in brain showed that adenosine deaminase and nucleoside phosphorylase are localized in endothelial and glial cells but are absent in neurons; however, adenosine deaminase may be found in a few neurons in discrete brain regions. These results suggested that adenosine degradative pathways may be more active in glia. Thus, we have determined if there is a differential distribution of adenosine deaminase, nucleoside phosphorylase, and xanthine oxidase enzyme fluxes in glia, comparing primary cultures of central and ciliary ganglion neurons and glial cells from chick embryos. Hypoxanthine-guanine phosphoribosyltransferase and production of adenosine by S-adenosylhomocysteine hydrolase activity were also examined. Our results show that there is a distinct profile of purine metabolizing enzymes for glia and neurons in culture. Both cell types have an S-adenosylhomocysteine hydrolase, but it was more active in neurons than in glia. In contrast, in glia the enzymatic activities of xanthine oxidase (443 +/- 61 pmol/min/10(7) cells), nucleoside phosphorylase (187 +/- 8 pmol/min/10(7) cells), and adenosine deaminase (233 +/- 32 pmol/min/10(7) cells) were more active at least 100, 20, and five times, respectively, than in ciliary ganglion neurons and 100, 100, and nine times, respectively, than in central neurons.

Regulation of A-currents by cell-cell interactions and neurotrophic factors in developing chick parasympathetic neurones.

1. The developmental regulation of ion channel expression was studied in parasympathetic neurones isolated from the chick ciliary ganglion. Whole-cell patch clamp recordings were made from ciliary ganglion neurones that were removed from the embryo on the ninth embryonic day (E9) and maintained in dissociated cell culture for an additional 4 days. Previous studies have shown that the expression of a transient voltage-activated K+ current (IA) is regulated by unidentified environmental stimuli during these developmental stages. 2. The effect of interactions between neurones and target tissue on the expression of IA was tested by co-culturing ciliary ganglion neurones with chick striated muscle cells. Neurones from the nerve-muscle co-cultures expressed normal amplitudes of IA, but the neurones did not express normal levels of IA when they were plated onto lysed muscle fibres. 3. The effect of interactions between ganglionic neurones and non-neuronal ganglionic cells was tested by culturing ganglia as explants rather than as dissociated cells. Neurones isolated from the explant cultures did not express normal levels of IA. Similarly, when dissociated ganglionic neurones were co-cultured with fibroblasts isolated from embryonic chick skin, they did not express normal amplitudes of IA. 4. Chronic depolarization caused by growing ciliary ganglion neurones in the presence of elevated K+ concentrations did not allow for the normal expression of IA, although it did promote the survival of these neurones in vitro. 5. Addition of 40 ng ml-1 of recombinant human ciliary neurotrophic factor (CNTF) or basic fibroblast growth factor (bFGF) to the cell culture medium had no effect on IA expression in developing chick ciliary ganglion neurones. However, 40 ng ml-1 of acidic fibroblast growth factor (aFGF) stimulated the expression of IA. All trophic factors promoted the growth and survival of ciliary ganglion neurones in vitro. 6. Dissociated ciliary ganglion neurones were maintained in a culture medium containing an aqueous extract of the whole brain. Neurones developing under these conditions expressed normal levels of IA. The stimulatory activity of the brain extract was destroyed by heating. 7. The expression of IA in chick ciliary ganglion neurones developing in vitro can be regulated by soluble growth factors and by interactions with certain other cell types. Similar interactions may regulate the expression of IA in ciliary ganglion neurones developing in situ.

Reduced levels of acetylcholine receptor expression in chick ciliary ganglion neurons developing in the absence of innervation

Chick ciliary ganglion neurons receive innervation from a single source, the accessory oculomotor nucleus (AON), and nicotinic ACh receptors (AChRs) mediate chemical synaptic transmission through the ganglion. Previous experiments examining the developmental expression of AChRs in embryonic chick ciliary ganglion neurons in situ have shown that AChR levels increase substantially in the neurons at the time of innervation. Prior to synapse formation, few AChRs are detected in the neurons. In the present experiments, the role of presynaptic inputs in inducing an increase in AChRs was established by examining AChR levels in ciliary ganglion neurons that have been deprived of innervation by surgical ablation of the AON prior to synapse formation. AChR levels were dramatically reduced in neurons of input-deprived ganglia as compared to control innervated neurons at all developmental stages examined from embryonic day (ED) 5 to ED 12 as determined by indirect immunocytochemical labeling of frozen ganglion sections with the anti-AChR monoclonal antibody mAb 35, and light microscopy. In contrast, neuronal somata of input-deprived and control ganglia had equivalent levels of immunolabeling for three other components, a transmembrane glycoprotein of synaptic vesicles, SV2, and two microtubule-associated proteins, MAP 1B and MAP 2, from ED 5 up to ED 10. The results demonstrate that presynaptic inputs specifically increase the levels of AChR expression in developing neurons. In addition, changes in the levels of immunolabeling for AChRs, SV2, MAP 1B, and MAP 2 in neuronal somata after ED 10 demonstrate that other major developmental events also influence the levels of these components in neurons. Declines in the intensity of AChR, SV2, MAP 1B, and MAP 2 immunolabeling within a subset of neuronal somata in both operated and control ganglia at ED 10 and 12 coincide with the period of neuronal cell death. Increases in AChR labeling in the rest of the neuronal population of input-deprived ganglia at ED 12 suggest that, in addition to innervation, synapse formation with the peripheral target tissue influences AChR levels in developing neurons in situ.

Characteristics of multiple voltage‐activated K+ currents in acutely dissociated chick ciliary ganglion neurones.

1. The properties of voltage-activated K+ currents were examined using whole-cell recording techniques in acutely isolated chick ciliary ganglion neurones. 2. Application of depolarizing voltage pulses from a holding potential of -60 mV evoked sustained outward currents that inactivated with time constants of hundreds of milliseconds (IDR). Bath application of 10 mM tetraethylammonium (TEA) caused a 70-90% reduction of IDR. Application of depolarizing voltage steps from a holding potential of -120 mV revealed a second class of TEA-resistant outward currents. These currents activated quickly but inactivated completely within tens of milliseconds (IA). IA activated at more negative command potentials than IDR. However, IDR exhibited a steeper voltage dependence of activation than IA. 3. The midpoint of the steady-state inactivation curve of IA was between -95 and -110 mV. By contrast the midpoint of the steady-state inactivation curve of IDR was between -80 and -90 mV. It was not possible to produce a complete inactivation of IDR using prepulses of up to 2 s duration. 4. The time course of IA inactivation could only be fitted with double-exponential curves with time constants of 5-18 ms and 30-60 ms at membrane potentials positive to -30 mV. The inactivation of IA was slower at more positive membrane potentials because of a greater contribution of the long time constant. The individual time constants were not markedly voltage dependent. 5. Bath application of 5 mM 4-aminopyridine (4-AP) caused a 70-100% block of IA whereas 1 mM 4-AP was ineffective. Bath application of 560 nM alpha-dendrotoxin (DTX) produced a 50-70% reduction of IA, but application of 280 nM DTX had no effect on IA. 6. Application of 1 mM 4-AP produced a reversible 55-80% block of IDR measured at the end of a 500 ms depolarizing pulse. The 4-AP-sensitive components of IDR activated rapidly and exhibited a gradual inactivation with continued depolarization. The 4-AP-resistant components of IDR activated much more slowly and showed very little tendency to inactivate. Significant blockade of IDR was produced by 10 microM 4-AP. 7. The decay of IDR tail currents could only be fitted with double exponential curves with time constants of 3-6 and 40-60 ms, respectively. The fast and slow components of the tail currents behaved independently with respect to the duration of the depolarizing voltage step. 8. Application of 1 mM 4-AP eliminated the fast, but not the slow component of IDR tail currents.(ABSTRACT TRUNCATED AT 400 WORDS)

Fibroblast Growth Factors, Depolarization, and Substratum Interact in a Combinatorial Way to Promote Neuronal Survival

Neuronal survival in vivo may be determined by the combined effects of multiple agents rather than simply by the effect of an individual agent. This idea is supported by experimental evidence showing that neuronal survival can independently be influenced by target-derived factors as well as afferent inputs. To test this idea directly, cultured chick ciliary ganglion neurons were used to study the potentially interactive and combinatorial effects of trophic factors (acidic and basic fibroblast growth factor; FGF), depolarization (as would be expected from afferent activity), and substrate (laminin and collagen IV). Our results were consistent with the idea that combinatorial interactions between multiple agents may be critical in the regulation of cell survival. Exposure to either basic FGF (bFGF) or depolarization on a laminin substrate promoted neuron survival. However, bFGF did not promote survival and depolarization-mediated survival was significantly reduced when neurons were plated on collagen. The simultaneous addition of FGF and depolarization affected survival synergistically when plated on both laminin and collagen. Surprisingly, while survival by FGF or depolarization alone was greatly dependent on substrate, the simultaneous addition of FGF and depolarization appeared to greatly reduce this dependency on substrate. Taken together, these data demonstrate the potential importance of synergistic interactions between trophic factors and depolarizing stimuli.

Muscle membrane preparation restores sensitivity to acetylcholine in cultured chick ciliary ganglion neurons

Ciliary ganglion (CG) neurons grown in culture in the absence of muscle cells rapidly lose sensitivity to acetylcholine (ACh), while neurons grown in the presence of muscle or muscle cell membranes maintain sensitivity to ACh for extended periods of time. The present study examined whether exposure to muscle membrane preparation or stimulation of cAMP-dependent processes could restore sensitivity to ACh in cultured neurons which had lost responsiveness to ACh. CG neurons from 11- to 14-day-old chick embryos were grown on collagen substrate in the absence of muscle cells. Sensitivity to ACh was assessed by measuring peak current responses following application of ACh (IACh) to neurons under whole-cell voltage clamp. In control cultures IACh decreased from an average of 837 pA the day of plating to 145 pA following 4 days in culture. Stimulation of cAMP-dependent processes with forskolin and 3-isobutyl-1-methylxanthine (IBMX) or 8'Br-cAMP and IBMX had variable effects on IACh. These treatments increased peak IACh in some neurons maintained in culture for less than 48 h. Treatment with these agents decreased peak IACh in cultures which were more than 48 h old. Exposure of neurons, which had lost sensitivity to ACh in culture, to muscle membranes increased IACh 2- to 3-fold over 24 to 48 h. This membrane-induced restoration of sensitivity to ACh was blocked by exposure to the protein synthesis inhibitor cycloheximide. Stimulation of cAMP-dependent processes in neurons exposed to muscle membrane decreased IACh. In conclusion, these results indicate that some element associated with the membranes of muscle cells has the ability to restore ACh responsiveness to CG neurons which have become insensitive to ACh in culture.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of ciliary neuronotrophic factor on somatostatin expression in chick ciliary ganglion neurons

The normal development of somatostatin (SOM) expression in neurons of the chick ciliary ganglion and the effects of ciliary neuronotrophic factor (CNTF) on SOM induction in cultured ciliary ganglion neurons, were studied by immunocytochemical techniques. SOM immunoreactivity was first detectable in some neurons of the ganglion at embryonic day (E)8 and between E14 to hatch, 44–46% of the neuronal population contained the peptide. It was inferred that essentially all choroid neurons, which constitute 50% of the neuronal population, contain SOM. Culture studies indicated that CNTF supported both the SOM positive choroid neurons and the SOM negative ciliary neurons. Although CNTF was necessary for the survival and maturation of cultured ciliary ganglion neurons, it did not influence either the induction or maintenance of SOM expression in these neurons. CNTF may instead act as a permissive factor, allowing the induction of SOM in neurons of the ciliary ganglion by other, more specific, factors.

Inhibition of the nicotinic acetylcholine response by serotonergic and muscarinic agents in chick ciliary ganglion neurones

Chick ciliary ganglion neurones were investigated by whole cell voltage clamp recordings. The ACh- or nicotine-induced inward current was partially inhibited by perfusing the neurones with 5-HT. This effect was rapid (⩽ 1 min), dose-dependent (50–1000 μM) and quickly reversible. The selective 5-HT 1A agonist 8-OH-DPAT (10 μM) reduced the nicotinic ACh response more potently, irrespective of the absence or presence of propranolol (1 μM), a known 5-HT 1A antagonist. Other serotonergic antagonists, like ICS 205–930 (1 μM), mianserin (10 μM) and methysergide (10 μM), also failed to antagonize the 5-HT-mediated decrease in the nicotinic response. Muscarine (50 μM) did not affect the nicotine-induced inward current but the muscarinic agonist oxotremorine (10 μM) also decreased the nicotine-induced inward current. Atropine, at small concentrations failed to block this effect but caused some reduction of the ACh response itself at larger (1–10 μM) concentrations. It is suggested that 5-HT may modulate synaptic transmission in ciliary ganglion neurones in vivo. The site of action of 5-HT, oxotremorine and atropine might be at or close to the ACh receptor complex, because of the fast onset and reversibility of the effects and lack of specificity for structurally different drugs.

Cyclic AMP and the nicotinic response of bovine adrenal chromaffin cells

The effects of cAMP analogs on the nicotinic responses of bovine adrenal chromaffin cells were examined by monitoring nicotine-induced whole cell currents. [ 3H]norepinephrine release, and membrane conductance changes. None of the three methods revealed an increased nicotinic response after treatment with cAMP analogs. The compounds did increase [ 3H]norepinephrine release from the cells but the effect was not exerted at the level of nicotinic receptors. Bovine adrenal chromaffin cells differ in this respect from chick ciliary ganglion neurons which do show increased nicotinic responses after treatment with cAMP analogs.

Differential expression of the TGF-beta isoforms in embryogenesis suggests specific roles in developing and adult tissues.

The TGF-beta's are multifunctional, pleiotropic molecules with major effects in control of cellular migration, cellular proliferation, and elaboration of extracellular matrix. Thus far, five distinct isoforms of TGF-beta have been described, each approximately 65-85% homologous and containing the characteristic 9 positionally conserved cysteine residues. Although the actions of the activated mature forms of the different isoforms on cells are qualitatively similar in most cases, there are a few examples of distinct activities. For example, TGF-beta's 1 and 3, but not TGF-beta 2, inhibit the growth of large vessel endothelial cells, and TGF-beta's 2 and 3, but not TGF-beta 1, inhibit the survival of cultured embryonic chick ciliary ganglionic neurons. In addition, selective targeting of the latent forms of the TGF-beta's is suggested by the observation that latent TGF-beta 2 is the prominent isoform found in body fluids such as amniotic fluid, breast milk, and the aqueous and vitreous humor of the eye; it is noteworthy in this regard that TGF-beta 2 is unique among various isoforms in that it lacks a RGD integrin-binding sequence in its precursor. The most dramatic differences in the TGF-beta isoforms are seen at the level of expression, where there is now a wealth of data demonstrating both spatially and temporally distinct expression of both the mRNAs and proteins in developing tissues, regenerating tissues, and in pathologic responses.(ABSTRACT TRUNCATED AT 250 WORDS)

Changes in the electrical properties of chick ciliary ganglion neurones during embryonic development.

1. Whole-cell recording techniques were used to examine the expression of ionic currents in chick ciliary ganglion neurones dissociated acutely at various stages of embryonic development. Currents were also examined in dissociated cells that had been maintained in vitro for several days. 2. Voltage-activated, tetrodotoxin (TTX)-sensitive Na+ currents (INa) could be detected in all cells tested between stage 25 and stage 40 (embryonic days 4.5-14). INa increased in both amplitude and density throughout development, but no obvious changes in kinetics or sensitivity to TTX were observed. 3. High-threshold Ca2+ currents (ICa) were also detectable between stage 25 and stage 40. ICa increased in both amplitude and density throughout this time. No obvious changes in kinetics or voltage dependence were observed. 4. Delayed rectifier K+ currents (IDR) and A-currents (IA) could be detected in Ca(2+)-free salines, and distinguished on the basis of differences in kinetics, voltage dependence, and sensitivity to tetraethylammonium (TEA). IA was either absent, or present at very low densities at stages 26-30, but showed a sharp increase in density thereafter. In contrast, IDR was detectable as early as stage 25, and did not display a significant increase in density during development. 5. Ca(2+)-activated K+ currents (IK(Ca)) were either undetectable or present at very low density between stage 26 and stage 30 (embryonic days 5-9) but showed a large increase in amplitude and density thereafter. 6. Ionic currents were examined in age-matched cells dissociated acutely on embryonic day 13, or isolated on embryonic day 9 and maintained in vitro for an additional 4 days. Most of the cells maintained in culture for 4 days did not express detectable IK(Ca), and had significantly reduced IA compared to acutely isolated controls. The cultured cells expressed normal densities of IDR, ICa and INa. 7. All ionic currents increased in amplitude during normal embryonic development, and all but IDR increased in density. The largest change in density generally occurred between stages 30 and 40, during which time ciliary ganglion neurones form synapses with target tissues. 8. Isolation of ciliary neurones from the in ovo environment prevented the normal development of IA and IK(Ca), suggesting that the expression of these channels is controlled by one or more extrinsic environmental factors. In contrast, the normal expression of INa, ICa and IDR is not dependent upon extrinsic factors.

Screening of adrenal medullary neuropeptides for putative neurotrophic effects

Chromaffin granules, the secretory organelles of the neuron-like adrenal medullary chromaffin cells, have previously been shown to store and liberate neurotrophic activities that support in vitro survival of several neuron populations including those innervating the adrenal medulla. Molecules resembling fibroblast growth factor and ciliary neurotrophic factor have been identified among these activities. Since chromaffin granules store a variety of neuropeptides and many neuropeptides can have pleiotropic effects on neuronal growth and maintenance we have tested 24 different neuropeptides for their capacities to promote survival of embryonic chick ciliary, dorsal root and sympathetic ganglionic neurons. Peptides tested included several derivatives of proenkephalin (Leu- and met-enkephalin, fragments BAM 22, B, F and E), somatostatin, substance P, neuropeptide Y, neurotensin, VIP, bombesin, secretin, pancreastatin, dynorphin B, dynorphin 1–13, β-endorphin, alpha-, beta-, and gamma-MSH. Control cultures received saturating concentrations of ciliary neurotrophic or nerve growth factor (CNTF; NGF), or no trophic supplements. At 1 × 10−5 M leu- and met-enkephalin as well as somatostatin supported sympathetic neurons to the same extent as NGF. At the same concentrations, leuenkephalin, the proenkephalin fragments BAM 22 and E, and somatostatin maintained about half of the dorsal root ganglionic neurons supported by NGF, but were not effective on ciliary neurons. VIP promoted the survival of approximately 50% of the ciliary and embryonic day 10 dorsal root ganglionic neurons as compared to saturating amounts of CNTF, but required the presence of non-neuronal cells in the cultures to be effective. Neurotensin (1 × 10−5M) had a small effect on ciliary neurons. All other peptides had no effect on any neuron population tested. Our results suggest that neuropeptides that occur in the adrenal medulla may play a role for the maintenance of cholinergic, sensory, and sympathetic neurons innervating the adrenal medulla.

The development of ACh- and GABA-activated currents in embryonic chick ciliary ganglion neurons in the absence of innervation in vivo

Chemical synaptic transmission in the chick ciliary ganglion is mediated by nicotinic ACh receptors. Ciliary ganglion neurons also express GABAA receptors, although there is no known source of GABAergic innervation of the ganglion, and the function of GABA receptors on these neurons is not known. We examined whether ACh and GABA receptors on embryonic chick ciliary ganglion neurons are regulated by presynaptic inputs. Whole-cell currents evoked by ACh or GABA in neurons soon after dissociation were taken to estimate the level of functional receptors in intact ganglia. We destroyed the accessory oculomotor nucleus (AON), the only source of synaptic input to the ganglion, on embryonic day (E) 4. We determined that 80% of the operations resulted in the virtual elimination of synaptic contacts in the ganglion, using P65 immunohistochemistry (a synaptic vesicle antigen) and direct ultrastructural examination. Previous experiments have shown that during normal development, ACh-activated currents increase over sevenfold between E6 and E18; GABA-activated currents increase only twofold, in proportion to cell size. We found that ACh-activated currents of uninnervated neurons at E14 and E18 were as large as control responses. Furthermore, ACh receptor-like molecules, visualized with monoclonal antibody 35 immunofluorescence, were concentrated in high density clusters on the surface of E18 neurons from AON-ablated embryos. GABA-activated currents were also not affected by AON destruction. We conclude that ACh and GABA receptors are not induced in embryonic chick ciliary ganglion neurons during development by contact with or soluble factors released from AON synaptic terminals.

An inward rectifier is present in presynaptic nerve terminals in the chick ciliary ganglion

Inwardly rectifying voltage-sensitive channels have been detected in the cell bodies and axons of a number of excitable cells. The question of whether similar channels exist at axon terminals has been a matter of speculation for some time. We now report the first direct evidence for the existence of inward rectifiers in vertebrate presynaptic nerve terminals. Following impalement with intracellular electrodes, the large calyciform nerve terminals innervating chick ciliary ganglion neurons exhibit pronounced inward rectification upon hyperpolarization that increases with increasing current strength. The response is blocked by 2 mM Cs +, but is insensitive to Ba 2+, tetraethylammonium and tetrodotoxin. The inward rectifier exhibits dependence on both Na + and K +, but is unaffected by altering extracellular Ca 2+. Ciliary neurons innervated by these nerve terminals display inward rectification with similar properties. We conclude that the inward rectifier present in these presynaptic nerve terminals resembles the H-current previously described in sensory ganglion neurons and the Q-current found in hippocampal pyramidal neurons. The presence of channels that are activated by hyperpolarization may serve to enhance the excitability of the calyciform nerve terminals, which are capable of relatively high frequencies (> 100 Hz) of discharge.

Ciliary neurotrophic factor enhances neuronal survival in embryonic rat hippocampal cultures

First described as a survival factor for chick ciliary ganglion neurons, ciliary neurotrophic factor (CNTF) has recently been shown to promote survival of chick embryo motor neurons. We now report neurotrophic effects of CNTF toward three populations of rat hippocampal neurons, the first demonstration of effects of CNTF upon rodent CNS neurons in culture. CNTF elicited an increase in the neurofilament content of hippocampal cultures prepared from embryonic day 18 (E18) rat brain. This was accompanied by increases of 2-, 28-, and 3-fold in the number of GABAergic, cholinergic, and calbindin-immunopositive cells, respectively. CNTF totally prevented the 67% loss of GABAergic neurons that occurred in control cultures over 8 d. CNTF also increased high-affinity GABA uptake and glutamic acid decarboxylase activity. Effects of CNTF were in all cases dose dependent, with maximal stimulation at approximately 100 pg/ml. When addition was delayed for 3 d, CNTF failed to elicit increases either in the number of cholinergic neurons or in GABA uptake.