Cholinergic modulation of non-N-methyl-D-aspartic acid glutamatergic transmission in the chick ventral lateral geniculate nucleus.

Neurotransmission between glutamatergic terminals of retinal ganglion cells and principal neurons of the ventral lateral geniculate nucleus (LGNv) was examined with patch clamp recordings in chick brain slices during electrical stimulation of the optic tract. Since muscarinic and nicotinic receptors are present in high densities in LGNv, the present study examined possible roles of both receptors in modulating retinogeniculate transmission. During whole-cell recordings from LGNv neurons, acetylcholine (ACh, 100 microM) caused an initial increase in amplitudes of optic tract-evoked non-N-methyl-D-aspartic acid (NMDA) glutamatergic postsynaptic currents (PSCs). This increase was unchanged when 1 microM atropine was present, indicating that this initial enhancement of PSCs was due entirely to activation of nicotinic receptors. However, during washout of ACh the amplitudes of evoked PSCs became significantly decreased by 40.4+/-5.0% for several minutes before recovering to their original amplitudes, an effect blocked by 1 microM atropine. Exogenously applied muscarine (10 microM) markedly depressed optic tract-evoked PSCs, and this decrease in amplitude was blocked by atropine. In a second set of experiments, we examined effects of releasing endogenous ACh prior to optic tract stimulation. This was accomplished by stimulation of the lateral portion of LGNv via a separate conditioning electrode. Following a brief train of low intensity conditioning stimuli, non-NMDA glutamatergic PSCs evoked by optic tract stimulation were potentiated. However, at higher conditioning stimulus intensities the PSCs were markedly decreased compared with control, and this decrease was partially blocked by atropine (1 microM). Neither ACh nor muscarine altered amplitudes of PSCs elicited by exogenously applied glutamate. Muscarine significantly reduced the frequency but not the amplitudes of miniature PSCs, consistent with a presynaptic location for muscarinic receptors mediating these effects. Thus while activation of nicotinic receptors potentiates retinogeniculate transmission, activation of muscarinic receptors mediates depression of transmission, demonstrating a complex cholinergic modulation of sensory information in LGNv.

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.

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.

Postsynaptic nicotinic receptors on dopaminergic neurons in the substantia nigra pars compacta of the rat.

Previous studies have shown that application of nicotinic agonists in the substantia nigra pars compacta increases the firing rate of dopaminergic neurons. We have used intracellular recordings to show that the response of these neurons to nicotine is postsynaptic, since it persists in the presence of low-calcium buffer containing tetrodotoxin. Burst firing in the presence of nicotine was not observed. The presence of postsynaptic nicotinic receptors was confirmed by immunohistochemical localization of the alpha4 nicotinic receptor subunit on dendrites in the substantia nigra pars compacta. The majority of tyrosine hydroxylase-immunopositive neurons in the substantia nigra pars compacta were also immunopositive for the alpha4 subunit. Immunohistochemical localization of the alpha4 and beta2 subunits in adjacent brain sections produced similar patterns of staining. Electron micrographs clearly indicated the presence of alpha4 subunit at postsynaptic densities. The predominant role of nicotinic receptors in the central nervous system has been suggested to be the presynaptic modulation of neurotransmitter release [McGehee D. S. and Role L. W. (1995) A. Rev. Physiol. 57, 521-546]. Although several postsynaptic nicotinic responses have also been reported in the literature, it is unclear as to whether the postsynaptic nicotinic receptors mediating responses to exogenously applied agonists are involved in synaptic transmission. From our electrophysiological and immunohistochemical results, we conclude that alpha4-containing nicotinic receptors are found at synapses on dopaminergic neurons. These synapses are similar to the cholinergic synapses described at these neurons, suggesting that nicotinic receptors are important in modulating the excitability of dopaminergic neurons by direct synaptic transmission.

The membrane hyperpolarization of rat dorsolateral septal nucleus neurons is mediated by a novel nicotinic receptor.

The pharmacology, calcium dependence and G protein mediation of the membrane hyperpolarization of rat dorsolateral septal nucleus (DLSN) neurons in response to nicotinic agonists was examined to classify the nicotinic receptor mediating the response. Intracellular recording from DSLN neurons in a brain slice preparation was used to determine whether chlorisondamine, trimethaphan, cytisine or strychnine inhibited the membrane hyperpolarization in response to application of the nicotinic agonist 1,1-dimethyl-4-phenylpiperazinium (DMPP). Chlorisondamine was found to block the response only at a high concentration (500 microM) although strychnine (100 microM) was without effect. Cytisine was neither an effective agonist nor an antagonist (500 microM). Surprisingly, trimethaphan appeared to act as an agonist, rather than an antagonist, with a potency and efficacy similar to that reported for nicotine at this receptor. The response was dependent on intracellular calcium stores because it persisted in the absence of extracellular calcium but was blocked by intracellular injection of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). Injection of GTP gamma S into the neurons blocked the nicotinic response. Apamin, iberiotoxin and charybdotoxin reduced but did not block the response at concentrations that selectively block calcium-dependent potassium channels. These results indicate that the nicotinic response in DLSN neurons may be mediated by a metabotropic nicotinic receptor coupled to a calcium-dependent potassium channel through the activation of a G-protein and release of intracellular calcium stores. The unusual pharmacology of the nicotinic receptor on DLSN neurons indicates that it may be a novel receptor which has yet to be cloned.

The reducing agent dithiothreitol (DTT) does not abolish the inhibitory nicotinic response recorded from rat dorsolateral septal neurons.

Previous intracellular recordings have demonstrated that dorsolateral septal nucleus (DLSN) neurons express a novel nicotinic receptor which produces a direct membrane hyperpolarization when activated by nicotinic agonists. Activation of the classical excitatory nicotinic receptors has been shown to require a disulfide bond involving the cysteines at positions 192 and 193 of the alpha subunits of the receptor. Reduction of this cystine bond with dithiothreitol (DTT) abolishes agonist activation of excitatory nicotinic receptors. We have now examined whether DTT treatment of the inhibitory nicotinic receptor on DLSN neurons also abolishes the inhibitory nicotinic response. We find that the inhibitory response persists after treatment of the neurons with 1 mM DTT, even if the reduction is followed by alkylation of the receptor with bromoacetylcholine to prevent possible reformation of disulfide bonds. This result suggests that the agonist binding site on the inhibitory nicotinic receptor does not require an intact disulfide bond, similar to the bond on the alpha subunit of the excitatory nicotinic receptor, for agonist activation of the receptor. Some of these results have been previously reported in abstract form.

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.

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.

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.

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.