Intracellular recording in avian brain of a nicotinic response that is insensitive to K-bungarotoxin.

We examined nicotinic acetylcholine receptors in the avian brain using a combination of autoradiographic and intracellular electrophysiological techniques. We found that the lateral spiriform nucleus (SPL) in the mesencephalon has a very high density of 3H-nicotine binding sites but no detectable 125I-K-bungarotoxin (125I-K-BuTx) or 125I-alpha-bungarotoxin (125I-alpha-BuTx) bindings sites. Intracellular recordings in brain slices revealed that SPL neurons depolarize in response to nicotine and carbachol (in the presence of atropine). These depolarizations were blocked by the classic nicotinic antagonists d-tubocurarine and dihydro-beta-erythroidine. As predicted for nicotinic receptors with a high affinity for nicotine, neither K-BuTx nor alpha-BuTx blocked these nicotinic responses. Thus, although the existence of high-affinity 3H-nicotine binding sites has been known for some time, we now report the in situ detection of a functional nicotinic receptor that has a high affinity for nicotine and is K-BuTx-insensitive.

Nicotinic receptor-mediated biphasic effect on neuronal excitability in chick lateral spiriform neurons.

Local neuronal circuits integrate synaptic information with different excitatory or inhibitory time windows. Here we report that activation of nicotinic acetylcholine receptors (nAChRs) leads to biphasic effects on excitability in chick lateral spiriform (SPL) neurons during whole cell recordings in brain slices. Carbachol (100 microM in the presence of 1 microM atropine) produced an initial short-term increase in the firing rates of SPL neurons (125+/-14% of control) that was mediated by postsynaptic nAChRs. However, after 3 min exposure to nicotinic agonists, the firing rate measured during an 800 ms depolarizing pulse declined to 19+/-7% (100 microM carbachol) or 26+/-8% (10 microM nicotine) of the control rate and remained decreased for 10-20 min after washout of the agonists. Similarly, after 60 s of electrically-stimulated release of endogenous acetylcholine (ACh) from cholinergic afferent fibers, there was a marked reduction (45+/-5% of control) in firing rates in SPL neurons. All of these effects were blocked by the nAChR antagonist dihydro-beta-erythroidine (30 microM). The inhibitory effect was not observed in Ca(2+)-free buffer. The nAChR-mediated inhibition depended on active G-proteins in SPL neurons and was prevented by the GABA(B) receptor antagonist phaclofen (200 microM), while the GABA(B) receptor agonist baclofen (10 microM) decreased firing rate in SPL neurons to 13+/-1% of control. The inhibitory response thus appears to be due to a nAChR-mediated enhancement of presynaptic GABA release, which then activates postsynaptic GABA(B) receptors. In conclusion, activation of nAChRs in the SPL initiates a limited time window for an excitatory period, after which a prolonged inhibitory effect turns off this window. The prolonged inhibitory effect may serve to protect SPL neurons from excessive excitation.

Fast excitatory nicotinic transmission in the chick lateral spiriform nucleus.

The lateral spiriform nucleus (SpL) in the chick mesencephalon contains functional nicotinic receptors and receives a cholinergic fiber projection. We now use double-label immunohistochemistry to demonstrate that choline acetyltransferase-immunopositive fibers in the SpL and in the cholinergic fiber tract lateral to the nucleus are associated with fibers expressing the alpha5 and/or alpha3 nicotinic receptor subunits as determined by mAb35 immunoreactivity. This morphological evidence suggests that there might be synapses between the cholinergic fibers and the dendrites of SpL neurons. Whole-cell recordings from SpL neurons in current-clamp mode revealed EPSPs evoked by stimulation of the cholinergic fiber tract lateral to the SpL. These EPSPs increased in amplitude in the presence of bicuculline. Further addition of the nicotinic antagonist dihydro-beta-erythroidine (DHbetaE) to the buffer significantly attenuated them. Almost all of the remaining EPSP was blocked by 6,7-dinitroquinoxaline-2,3-dione. In the presence of an antagonist cocktail that isolated the nicotinic responses, a fast, monosynaptic nicotinic EPSP or EPSC was evoked. In some neurons, the nicotinic EPSP resulted in the generation of an action potential. The nicotinic nature of the evoked response was confirmed by blockade of the EPSPs or EPSCs with nicotinic antagonists, including DHbetaE, D-tubocurare, and mecamylamine. The nicotinic response was insensitive to low concentrations (10-100 nM) of methyllycaconitine, indicating that typical alpha7-containing receptors were not involved. The results demonstrate that endogenously released acetylcholine generates EPSPs that can elicit action potentials by acting at postsynaptic nicotinic receptors on SpL neurons.

Localization of 3H-nicotine, 125I-kappa-bungarotoxin, and 125I-alpha-bungarotoxin binding to nicotinic sites in the chicken forebrain and midbrain.

We have previously localized cholinergic cell bodies and fibers within the midbrain of the chicken with choline acetyltransferase immunohistochemistry. In a continuing effort to characterize the central cholinergic system, the present study examines the distribution of various nicotinic acetylcholine receptors in the forebrain and midbrain of the chicken. The binding of 3H-nicotine, 125I-kappa-bungarotoxin, and 125I-alpha-bungarotoxin was localized by film autoradiography in adjacent sections of the adult chicken brain, allowing a comparison of the distribution of different classes of nicotinic binding sites within the brain. Although all three ligands were often co-localized, there were areas that bound 3H-nicotine but not the 125I-neurotoxins, or vice versa. Very high densities of all three ligands were found in the hyperstriatum ventrale; the nucleus geniculatus lateralis, pars ventralis; the griseum tectale; the nucleus dorsolateralis anterior thalami; the nucleus lentiformis mesencephali, pars lateralis and pars medialis; the periventricular organ; and the stratum griseum et fibrosum superficiale, layer f of the optic tectum. The nucleus spiriformis lateralis had the highest levels of 3H-nicotine binding in the chicken brain, but it did not bind either of the two snake neurotoxins. On the other hand, high levels of both 125I-alpha-bungarotoxin and 125I-kappa-bungarotoxin binding were found in the nucleus semilunaris and the nucleus ovoidalis, but these areas contained little or no 3H-nicotine binding. No unique 125I-kappa-bungarotoxin sites, unrecognized by 125I-alpha-bungarotoxin, were identified by the low resolution autoradiography performed in this study. In general, nicotinic receptors were found in areas that have been reported to contain cholinergic cell bodies or fibers. Comparison of our results with the expression of neuronal nicotinic receptor subunits, as determined by in situ hybridization, suggests that many of the high affinity 3H-nicotine sites are localized presynaptically, as, for example, in the retinorecipient nuclei and the nucleus interpeduncularis. The lack of 125I-kappa-bungarotoxin binding in the presence of alpha-bungarotoxin indicates that the chicken brain has only very low levels of a unique kappa-bungarotoxin site. This is in marked contrast to chicken, frog, and rat autonomic ganglia, where a unique kappa-neurotoxin-sensitive receptor has been identified and shown to mediate nicotinic neurotransmission.

Immunohistochemical localization of choline acetyltransferase in the chicken mesencephalon.

Choline acetyltransferase, a specific marker for cholinergic neurons, has been immunohistochemically localized in the mesencephalon and in the caudal diencephalon of the chicken. A complete series of transverse sections through the mesencephalon is presented. In the diencephalon, cholinergic fibers were found in the stria medullaris, the fasciculus retroflexus, and the ventral portion of the supraoptic decussation. The nucleus triangularis and the nucleus geniculatus lateralis, pars ventralis also contained cholinergic fibers. Small cholinergic cell bodies were found in the medial habenula. In the pretectum, cholinergic fibers innervated the nucleus lentiformis mesencephali and the tectal gray. The nucleus spiriformis lateralis also contained cholinergic fibers, while most of the cell bodies in the nucleus spiriformis medialis were cholinergic. In the mesencephalon, labelled fibers were found in the nucleus intercollicularis and in all layers of the optic tectum except the stratum opticum. The highest density of tectal cholinergic fibers was in the stratum griseum et fibrosum superficiale (SGFS), layer f. Radial cells located in SGFS, layer i were also cholinergic. In the isthmic nuclei, cholinergic fibers were found in the pars magnocellularis, while the pars parvicellularis and the nucleus semilunaris contained labelled cells. The oculomotor, Edinger-Westphal, trochlear, and trigeminal motor nuclei all had cholinergic cell bodies. Cholinergic axons were present in the oculomotor and trochlear nerves. In the tegmentum, cell bodies were labelled in the nucleus mesencephalicus profundus, pars ventralis, while the nucleus interpeduncularis had dense cholinergic innervation. Our localization of cholinergic cell bodies and fibers has been compared with earlier autoradiographic and anatomical studies to help define cholinergic systems in the avian brain. For example, the results indicate that the chicken may have a cholinergic habenulointerpeduncular system similar to that reported in the rat. Establishing the cholinergic systems within the avian midbrain is important for designing future neurophysiological and pharmacological studies of cholinergic transmission in this region.

The actions of the kappa 1 opioid agonist U-50,488 on presynaptic nerve terminals of the chick ciliary ganglion.

The actions of the kappa 1 opioid receptor agonist U-50,488 (trans-(+-)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]-benz ene - acetamide methane sulfonate) on the membrane properties of presynaptic calyciform nerve terminals of the chick ciliary ganglion were examined using intracellular recordings obtained from intact ganglion preparations maintained in vitro. U-50,488 produced a concentration-dependent (30-1000 microM) hyperpolarization with an apparent increase in input resistance. This hyperpolarization resulted from inhibition of the Na(+)-K+ inward rectifier, since it was blocked by 3 mM Cs+ and was not observed when terminals were depolarized beyond resting potential where inward rectification was voltage inactivated. A depolarizing effect on membrane potential with a further rise in input resistance was commonly observed at the highest perfused U-50,488 concentration (1 mM). The depolarizing event appears to result from a decrease in membrane potassium conductance, as the reversal potential for the response was estimated to be between -70 and -90 mV and the potassium channel blocker Ba2+ (1 mM) abolished the response. The kappa 1 opioid receptor agonist also blocked spontaneously occurring miniature hyperpolarizations in the terminals, which are considered to be due to a Ca(2+)-dependent K+ conductance. Most of the responses to U-50,488 were abolished in the presence of the kappa 1 receptor antagonist norbinaltorphimine. In conclusion, the excitability of presynaptic nerve terminals in the chick ciliary ganglion can be modulated by the inhibition of at least three separate ion conductances following activation of kappa 1 opioid receptor sites in the nerve terminal region.

Analysis of quantal content and quantal conductance in two populations of neurons in the avian ciliary ganglion.

The avian ciliary ganglion contains two populations of parasympathetic cells, termed the ciliary and choroid neurons. We have estimated the quantal contents of nicotinic excitatory postsynaptic potentials in both populations of neurons by several methods. The singly innervated ciliary neurons have quantal contents of 15-30. In contrast, the multiply innervated choroid cells have quantal contents of 4-7. Quantal conductance was also determined, using a parallel conductance model which takes into account the capacitance of the cell membrane. This analysis indicates that in both populations of neurons one quantum activates approximately 100 postsynaptic receptors. It is concluded that in autonomic ganglia singly innervated cells demonstrate a larger quantal content, consistent with a higher safety factor for neurotransmission, while quantal content in multiply innervated cells is generally much lower, allowing for considerable summation of presynaptic inputs. Further, in autonomic neurons many fewer postsynaptic receptors are activated by a single quantum than is the case at the neuromuscular junction.

Distinct muscarinic receptors enhance spontaneous GABA release and inhibit electrically evoked GABAergic synaptic transmission in the chick lateral spiriform nucleus.

The effects of muscarinic agonists on GABAergic synaptic transmission were examined using whole-cell patch-clamp recording in chick brain slices containing the lateral spiriform nucleus. Bath application of muscarine (10 microM) both increased the frequency of spontaneous GABAergic postsynaptic currents and reduced the amplitude of evoked GABAergic polysynaptic postsynaptic currents elicited by focal afferent fiber electrical stimulation. Both of these muscarinic actions were reversible and dose-dependent. Two M(1) antagonists, telenzepine and pirenzipine, and to a lesser extent the M(2) antagonist methoctramine, protected against muscarine's inhibition of the evoked polysynaptic currents. Other M(2) antagonists (tripitramine and gallamine) as well as the M(3) antagonist 4-DAMP mustard (4-diphenylacetoxy-N-(2-chloroethyl)-piperidine hydrochloride) and an M(4) antagonist (tropicamide) provided little or no protection against muscarine in this assay. In contrast, 4-diphenylacetoxy-N-(2-chloroethyl)-piperidine hydrochloride, tropicamide and telenzepine, but not pirenzepine, methoctramine, tripitramine and gallamine, blocked muscarine's enhancement of spontaneous GABAergic currents. McN-A-343 [(4-hydroxy-2-butynyl)-1-trimethylammonium-m-chlorocarbanilate chloride] and CDD-0097 (5-propargyloxycarbonyl-1,4,5,6-tetrahydropyrimidine hydrochloride), two M(1) agonists, mimicked muscarine's inhibition of the evoked polysynaptic GABAergic currents but did not mimic muscarine's enhancement of spontaneous GABAergic currents. Both actions of muscarine persisted when slices were pretreated with pertussis toxin or N-ethylmaleimide, which inactivate G-proteins coupled to M(2) and M(4) receptors while leaving G-proteins coupled to M(1), M(3) and M(5) receptors intact. Muscarine had no significant effect on the amplitude of the direct postsynaptic current elicited by exogenous GABA in the presence of tetrodotoxin. The results demonstrate that distinct muscarinic receptors oppositely modulate GABAergic transmission in the lateral spiriform nucleus. The receptor mediating the inhibition of evoked GABAergic polysynaptic currents is pharmacologically similar to an M(1) receptor, while the enhancement of spontaneous GABAergic currents appears to be mediated by an M(3) receptor.

A novel choline-sensitive nicotinic receptor subtype that mediates enhanced GABA release in the chick ventral lateral geniculate nucleus.

Nicotinic acetylcholine receptors modulate the release of GABA, glutamate, acetylcholine and dopamine in the brain. Here we describe a novel choline-sensitive nicotinic acetylcholine receptor that mediates enhanced GABA release in the chick ventral lateral geniculate nucleus. Whole-cell recordings in slices demonstrated that choline (0.03-10 mM), generally considered an alpha7-selective agonist, and carbachol (3-300 microM), a non-selective cholinergic agonist, both increased the frequency of spontaneous GABAergic events in ventral lateral geniculate nucleus neurons. Tetrodotoxin (0.5 microM) partially reduced responses to carbachol, but eliminated responses to choline. During long-term (5 min) exposure to choline the GABA enhancement was maintained until choline was washed out. Choline (300 microM) enhanced the frequency of spontaneous GABAergic events by 4.28-fold in control artificial cerebrospinal fluid. This choline-mediated enhancement was significantly reduced by the following nicotinic acetylcholine receptor antagonists: 1 microM dihydro-beta-erythroidine (1.49-fold increase, P<0.001), 1 microM methyllycaconitine (1.53-fold, P<0.001) and 0.2 microM alpha-conotoxin ImI (1.84-fold, P<0.001). In contrast, no significant change was seen in the presence of 0.1 microM dihydro-beta-erythroidine, 0.1 microM methyllycaconitine, 0.1 microM alpha-bungarotoxin, 0.1 microM alpha-conotoxin MII, 0.1 microM kappa-bungarotoxin, or 1 microM alpha-conotoxin AuIB. These results indicate that choline, at concentrations as low as 100 microM, activates a nicotinic acetylcholine receptor that is distinct from the classical alpha7 nicotinic acetylcholine receptors previously known to be activated by choline.

Nicotinic receptors mediate increased GABA release in brain through a tetrodotoxin-insensitive mechanism during prolonged exposure to nicotine.

The effects of nicotine on the spontaneous release of GABA from nerve terminals in the chick lateral spiriform nucleus were examined using whole cell patch-clamp recording in brain slices. Exposure to 1 microM nicotine produced an early immediate increase in the frequency of spontaneous postsynaptic GABAergic currents. This effect was blocked in the presence of 0.5 microM tetrodotoxin. However, a prolonged application of 0.1-1 microM nicotine (>3 min) caused a tetrodotoxin-insensitive increase in the frequency of spontaneous GABAergic currents. This late tetrodotoxin-insensitive effect was blocked by the nicotinic antagonists dihydro-beta-erythroidine (30 microM) and mecamylamine (10 microM), but not by methyllycaconitine (50-100 nM), indicating that activation of high affinity nicotine receptors was mainly responsible for this effect. This enhancement was inhibited by the high threshold Ca(2+) channel blocker Cd(2+) (100 microM), but not by dantrolene or ryanodine. The tetrodotoxin-insensitive enhancement of the frequency of GABA currents by nicotine was reduced by inhibition of cAMP-dependent protein kinase with HA1004 (30 microM), but not by inhibition of protein kinase C with staurosporine (1 microM), and was facilitated by forskolin (10 microM) or bromo-cAMP (50 microM). The results indicate that nicotine-enhanced GABA release can operate through both tetrodotoxin-sensitive and -insensitive mechanisms in a single brain region and that a second messenger cascade may be involved in the tetrodotoxin-insensitive enhancement by nicotine.

Muscarinic receptors mediate enhancement of spontaneous GABA release in the chick brain.

The functional role of muscarinic acetylcholine receptors in the lateral spiriform nucleus was studied in chick brain slices. Whole-cell patch-clamp recordings of neurons in the lateral spiriform nucleus revealed that carbachol enhanced GABAergic spontaneous inhibitory postsynaptic currents. The duration of the response to carbachol was significantly reduced after blockade of muscarinic receptors with atropine. In the presence of the nicotinic receptor antagonist dihydro-beta-erythroidine, carbachol produced a delayed but prolonged enhancement of spontaneous GABAergic inhibitory postsynaptic currents that was completely blocked by atropine. Muscarine also enhanced the frequency of spontaneous GABAergic inhibitory postsynaptic currents in a dose-dependent manner, but had no effect on inhibitory postsynaptic current amplitude. While 4-diphenylacetoxy-N-(2-chloroethyl)-piperidine hydrochloride, a M3 antagonist, completely blocked muscarine's effect, telenzepine, a M1 antagonist, and tropicamide, a M4 antagonist, only partially decreased the response to muscarine. Pirenzepine, a M1 antagonist, and methoctramine, a M2 antagonist, potentiated muscarine's enhancement of spontaneous GABAergic inhibitory postsynaptic currents. Muscarine's action was blocked by tetrodotoxin, cadmium chloride and omega-conotoxin GVIA, but was not affected by dihydro-beta-erythroidine, 6-cyano-7-nitroquinoxaline-2,3-dione, D(-)-2-amino-5-phosphonopentanoic acid, naloxone or fluphenazine. These results demonstrate that activation of both muscarinic and nicotinic acetylcholine receptors can enhance GABAergic inhibitory postsynaptic currents in the lateral spiriform nucleus. The muscarinic response has a slower onset but lasts longer than the nicotinic effect. The M3 receptor subtype is predominantly involved in enhancing spontaneous GABAergic inhibitory postsynaptic currents. These M3 receptors must be located some distance from GABA release sites, since activation of voltage-dependent sodium channels, and consequent activation of N-type voltage-dependent calcium channels, is required to trigger enhanced GABA release following activation of muscarinic receptors.

Lophotoxin: selective blockade of nicotinic transmission in autonomic ganglia by a coral neurotoxin.

Lophotoxin is a diterpene lactone isolated from gorgonian corals. The toxin has previously been shown to bind with high affinity to an acetylcholine recognition site located on skeletal muscle nicotinic receptors, producing an essentially irreversible blockade of neuromuscular transmission. Lophotoxin has also been shown to block nicotinic transmission in autonomic ganglia of the frog and in ileal strips of guinea pig and rabbit. The effects of lophotoxin have now been examined on neuronal nicotinic receptors in autonomic ganglia of the chick and rat. Low concentrations of lophotoxin (1 microM) produce a blockade of neuronal nicotinic transmission which is partially reversed by 3-5 h of washing out the toxin. The blockade produced by higher concentrations of lophotoxin (up to 32 microM) is not reversed during a similar washout period. Prior exposure to d-tubocurarine, a competitive nicotinic antagonist, can partially protect ganglia against exposure to lophotoxin. In contrast the local anesthetic QX-314, a noncompetitive nicotinic antagonist, does not protect ganglia against lophotoxin exposure. Lophotoxin binds to a site in ganglia identified by [125I]kappa-bungarotoxin which appears to be on the neuronal nicotinic receptor. Intracellular recordings reveal that lophotoxin has no effect on either muscarinic responses or on responses to gamma-aminobutyrate in autonomic ganglia. Passive and active membrane properties of the neurons are unaffected by lophotoxin except for the blockade of nicotinic responses. It is concluded that lophotoxin is a selective, high-affinity antagonist at the neuronal nicotinic receptor. The long-term nature of the blockade with lophotoxin suggests that the toxin will be of considerable value as a probe for characterizing the ganglionic nicotinic receptor.

Nicotinic acetylcholine receptors in separate brain regions exhibit different affinities for methyllycaconitine.

The family of nicotinic acetylcholine receptors contains numerous subtypes. Since the subunit compositions of most native neuronal nicotinic receptors are unknown, an important method for distinguishing subtypes of functional neuronal receptors is based on pharmacological criteria, such as affinity for snake toxins. We have now examined the affinities of native chick nicotinic receptors for methyllycaconitine, a toxin purified from Delphinium. We find that methyllycaconitine is a potent antagonist at central nicotinic receptors located on Edinger-Westphal neurons, producing nearly complete functional blockade of nicotinic responses at 10 nM. In marked contrast, methyllycaconitine is 1000-fold less potent at blocking nicotinic responses in the lateral spiriform nucleus. Methyllycaconitine inhibits kappa-bungarotoxin-sensitive nicotinic receptors in ciliary ganglia at 0.5-1.0 microM. Radioligand binding studies also reveal heterogeneity in the affinity of the toxin for nicotinic receptors. Methyllycaconitine binds most avidly to [125I] alpha-bungarotoxin sites in brain (Ki = 5.4 nM), and is 200-fold less potent at muscle nicotinic receptors (IC50 = 1.1 microM). The least potent binding of the toxin is to [3H]nicotine sites in brain (Ki = 3.7 microM). Methyllycaconitine is thus a useful pharmacological tool for distinguishing certain subtypes of native nicotinic receptors. The relatively low affinity of the toxin for nicotinic receptors in the lateral spiriform nucleus is consistent with the known properties of these receptors, which include a high affinity for [3H]nicotine and a lack of sensitivity to alpha- and kappa-bungarotoxin. On the basis of high affinity for methyllycaconitine and insensitivity to alpha-bungarotoxin, the nicotinic receptors in the Edinger-Westphal nucleus are unlike any previously described nicotinic receptor subtype.

Nicotinic receptor activation facilitates GABAergic neurotransmission in the avian lateral spiriform nucleus.

Whole-cell patch clamp recordings were performed in embryonic chick brain slices to characterize responses to nicotinic receptor activation in the mesencephalic lateral spiriform nucleus. Using intracellular recording, we previously reported the presence of functional high-affinity nicotinic sites in this nucleus that are insensitive to blockade with kappa- and alpha-bungarotoxin. We now report that nicotinic agonists not only produce an inward current in these cells, but also elicit a massive increase in the frequency of spontaneous postsynaptic currents without changing the amplitude distribution or risetime and decay kinetics of these events. The nicotinic receptor antagonist, dihydro-beta-erythroidine, blocks both the postsynaptic inward current and the enhancement of spontaneous postsynaptic currents. The spontaneous currents reverse at or near the chloride ion equilibrium potential and are completely blocked by 10 microM bicuculline, indicating that these events are likely to be GABAergic inhibitory postsynaptic currents. The nicotinic agonist-induced enhancement in inhibitory postsynaptic current frequency is blocked by 1.0 microM tetrodotoxin, demonstrating that the effect is mediated through the activation of voltage-dependent sodium channels. Nicotinic receptors are widely distributed in the central nervous system and in some cases are thought to modulate the release of various neurotransmitters. Our results show that activation of nicotinic receptors facilitates inhibitory neurotransmission in the avian lateral spiriform nucleus by increasing the frequency of spontaneous GABAergic postsynaptic currents. These data support a role for nicotinic receptors in the regulation of GABA release from nerve terminals in this nucleus.

Different responses to opioids measured in terminals and somas of Edinger-Westphal neurons.

Neurotransmitter receptors on the axon terminals of a neuron can be located a considerable distance away from comparable receptors on the cell body or dendrites of the same neuron. We examined the effects of activating either nerve terminal receptors or those located on or near the somas of chick Edinger-Westphal neurons. Cell body responses were measured via intracellular recording in a brain slice preparation. To measure nerve terminal responses, intracellular recordings were obtained from the large, calyciform nerve endings in intact ciliary ganglia, which emanate from neurons of the lateral Edinger-Westphal nucleus. Cell bodies of Edinger-Westphal neurons responded to leucine-enkephalin with a dose-dependent hyperpolarization that was associated with a decrease in input resistance. In spontaneously active Edinger-Westphal somas, leucine-enkephalin caused marked inhibition of suprathreshold and subthreshold activity, indicating that, as with a number of other central neurons, the major effect of opioids was to reduce excitability. The response to opioids was sensitive to naloxone (1 microM) and was a direct effect, since it was not blocked by either 0.5 microM tetrodotoxin or 100 microM cadmium. More selective mu ([D-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin) and delta ([D-Ser2]-leucine-enkephalin-Thr and [D-Pen2,5]-enkephalin) opioid agonists produced effects similar to those of leucine-enkephalin. Opioids produced strikingly different effects in the nerve terminals of Edinger-Westphal neurons, where the major effect was a depolarization associated with a decrease in input resistance. The effects of opioids in the terminals were reduced in a low sodium buffer, indicating that they were dependent on the presence of extracellular sodium.(ABSTRACT TRUNCATED AT 250 WORDS)

alpha7-Containing nicotinic receptors are segregated to the somatodendritic membrane of the cholinergic neurons in the avian nucleus semilunaris.

Segregation of ion channels and neurotransmitter receptors is an important mechanism for determining the functionality of the nervous system. In the case of nicotinic acetylcholine receptors, electrophysiological and anatomical studies have demonstrated that these receptors can be located at the somatodendritic and the axon terminal portions of neurons. Functionally, somatodendritic nicotinic receptors mediate fast excitatory transmission and possibly regulate other cell functions, while presynaptic nicotinic receptors enhance the release of neurotransmitters from axon terminals. Neurons in the mesencephalic lateral spiriform nucleus of the chick do not appear to restrict the localization of nicotinic receptors to specific membrane compartments, since receptors containing alpha5 and/or beta2 subunits are found both on the cell bodies and on the axonal projections of these neurons [Torrao A. S. et al. (1996) Brain Res. 743, 154-161]. We report here that, in contrast to lateral spiriform neurons, neurons in the nucleus semilunaris do appear to compartmentalize nicotinic receptors. The cholinergic nucleus semilunaris neurons express a high density of alpha7-containing nicotinic receptors on their somas [Britto L. R. G. et al. (1992) J. comp. Neurol. 317, 325-340]. However, when we examined the projections of these neurons in the lateral spiriform nucleus, we found no evidence for expression of alpha7-containing receptors on the cholinergic fibers from nucleus semilunaris neurons. Furthermore, patch-clamp electrophysiological recording from lateral spiriform neurons indicated an absence of presynaptic alpha7-containing nicotinic receptors capable of modulating the release of acetylcholine. We conclude that neurons are capable of segregating alpha7-containing nicotinic receptors to specific areas of their plasma membrane. Such targeting of nicotinic receptors would play an important role in determining their functional role in neurons.

Characterization of a snake venom neurotoxin which blocks nicotinic transmission in the avian ciliary ganglion.

Bungarus multicinctus venom was fractionated by ion exchange chromatography and the various fractions were assayed for their ability to block synaptic transmission through the chick ciliary ganglion. alpha-Bungarotoxin purified from this venom failed to block transmission at 50 micrograms/ml. A second neurotoxin, which we designate Toxin F, blocked transmission at 1-3 micrograms/ml and also blocked ganglionic depolarizations induced by carbachol. Toxin F was clearly distinguishable from alpha-bungarotoxin on the basis of molecular weight (estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and isoelectric point. Binding assays revealed that 125I-labeled toxin F bound to two sites in the ciliary ganglion: one site that was shared by alpha-bungarotoxin and toxin F and another site that was recognized solely by toxin F. Carbachol and d-tubocurarine displaced only that [125I]toxin F bound to the shared site and had no effect on [125I]toxin F bound to the site recognized by toxin F alone. The results suggest that toxin F blocks synaptic transmission in the chick ciliary ganglion by a postsynaptic mechanism. Further study is required to determine whether this effect of toxin F is mediated through a direct interaction with ganglionic nicotinic receptors.

Kappa-bungarotoxin: a probe for the neuronal nicotinic receptor in the avian ciliary ganglion.

The interaction of snake alpha-neurotoxins with neuronal membranes has been examined in the chick ciliary ganglion. Some, but not all, alpha-neurotoxins block nicotinic transmission in this ganglion. alpha-Bungarotoxin (ABgT), the major alpha-neurotoxin in the venom of Bungarus multicinctus, does not block transmission at high concentrations (1.2 microM) although it binds (Kd = 1 nM) to a pharmacologically nicotinic site in the ganglion. A toxin (kappa-bungarotoxin, KBgT) has been purified from the venom of Bungarus multicinctus. KBgT has a molecular weight of 6500 daltons and a pI of 9.1. KBgT is a potent inhibitor of nicotinic transmission in the ciliary ganglion, producing a reversible (overal several hours) blockade at 75 nM. Pre-exposure of ganglia to 1.2 microM ABgT does not prevent the effects of KBgT, indicating that the blockade occurs at a site distinct from that recognized by ABgT. Binding of [125I]KBgT to ciliary ganglia reveals two binding sites: one which has previously been characterized by [125I]ABgT and one which is not identified by [125I]ABgT. Both of these [125I]KBgT binding sites are blocked following pre-treatment of ganglia with the irreversible nicotinic affinity agent bromoacetylcholine. A two-site model is proposed to account for these observations. One site (the ABgT binding site) is seen by both ABgT and KBgT, and has as yet no physiological function associated with it. The second site is recognized only by the physiologically active KBgT, and may represent binding of the toxin to the physiologically detected nicotinic receptor.

Blockade of ganglionic transmission during synaptogenesis decreases alpha-bungarotoxin binding in the chick ciliary ganglion and iris.

The role of normal synaptic activity in the biochemical development of the nervous system has been examined in the chick embryo. Chlorisondamine, a ganglionic blocking drug, was administered in ovo during the period of synaptogenesis in the parasympathetic ciliary ganglion. Following treatment with chlorisondamine, nicotinic binding sites (as measured with [125I] alpha-bungarotoxin) were significantly reduced in both the ganglion and its end organ, the striated iris muscle. While the number of [125I] alpha-bungarotoxin binding sites eventually approached control levels in the iris, binding in the ciliary ganglion remained below normal values through hatching.

Properties of tachykinin receptors examined by intracellular recording from chick sympathetic ganglia.

The physiological and pharmacological properties of tachykinin receptors have been examined by intracellular recording from intact chick lumbar sympathetic ganglia in vitro. In these ganglia, both substance P and eledoisin are potent agonists, producing a slowly developing depolarization in ganglion neurons which is associated with an increase in input resistance and inhibition of the M-current. In contrast, physalaemin is a considerably less potent tachykinin in chick ganglia, and kassinin is without activity even at high doses. The rank order of potencies of tachykinin agonists is consistent from cell to cell, indicating that a single type of tachykinin receptor may be responsible for the observed responses. A putative tachykinin antagonist, (D-Arg1,D-Pro2,D-Trp7,9,Leu11)-substance P, exhibits no intrinsic activity in the ganglia but does block the responses to subsequently applied substance P and eledoisin. This tachykinin antagonist also inhibits a portion of the slow excitatory postsynaptic potential (EPSP) elicited in ganglion neurons by repetitive nerve stimulation. Since substance P has been identified within nerve fibers in the ganglia, it appears that this or another endogenous tachykinin mediates a portion of the slow EPSPs observed in the ganglia. Acetylcholine acting via muscarinic receptors is also capable of producing slow EPSPs in the ganglia, since perfusion with atropine can reduce the size of some slow EPSPs. It is concluded that chick sympathetic neurons contain both tachykinin and muscarinic receptors, and that these receptors are involved in slow synaptic responses in the ganglion neurons which increase the excitability of the cells.