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Acetylcholine-activating pentameric nicotinic receptors (nAChRs) are an essential mode of neurotransmission in the enteric nervous system (ENS). In this study, we examined the functional development of specific nAChR subtypes in myenteric neurons usingWnt1-Cre;R26R-GCaMP3mice, where all enteric neurons and glia express the genetically encoded calcium indicator, GCaMP3. Transcripts encoding α3, α4, α7, β2, and β4 nAChR subunits were already expressed at low levels in the E11.5 gut and by E14.5 and, thereafter, α3 and β4 transcripts were the most abundant. The effect of specific nAChR subtype antagonists on evoked calcium activity in enteric neurons was investigated at different ages. Blockade of the α3β4 receptors reduced electrically and chemically evoked calcium responses at E12.5, E14.5, and P0. In addition to the α3β4 antagonist, antagonists to α3β2 and α4β2 also significantly reduced responses by P10–11 and in adult preparations. Therefore, there is an increase in the diversity of functional nAChRs during postnatal development. However, an α7 nAChR antagonist had no effect at any age. Furthermore, at E12.5 we found evidence for unconventional receptors that were responsive to the nAChR agonists 1-dimethyl-4-phenylpiperazinium and nicotine, but were insensitive to the general nicotinic blocker, hexamethonium. Migration, differentiation, and neuritogenesis assays did not reveal a role for nAChRs in these processes during embryonic development. In conclusion, there are significant changes in the contribution of different nAChR subunits to synaptic transmission during ENS development, even after birth. This is the first study to investigate the development of cholinergic transmission in the ENS.

Attention depends on cholinergic stimulation of nicotinic and muscarinic acetylcholine receptors in the medial prefrontal cortex. Pyramidal neurons in layer VI of this region express cholinergic receptors of both families and play an important role in attention through their feedback projections to the thalamus. Here, we investigate how nicotinic and muscarinic cholinergic receptors affect the excitability of these neurons using whole-cell recordings in acute brain slices of prefrontal cortex. Since attention deficits have been documented in both rodents and humans having genetic abnormalities in nicotinic receptors, we focus in particular on how the cholinergic excitation of layer VI neurons is altered by genetic deletion of either of two key nicotinic receptor subunits, the accessory α5 subunit or the ligand-binding β2 subunit. We find that the cholinergic excitation of layer VI neurons is dominated by nicotinic receptors in wild-type mice and that the reduction or loss of this nicotinic stimulation is accompanied by a surprising degree of plasticity in excitatory muscarinic receptors. These findings suggest that disrupting nicotinic receptors fundamentally alters the mechanisms and timing of excitation in prefrontal attentional circuitry.

In humans, tobacco withdrawal produces symptoms that contribute to the difficulty associated with smoking cessation. Nicotine withdrawal symptoms can also be observed in rodents. A major standing question is which nicotinic receptor subtypes and which areas of the brain are necessary for nicotine withdrawal to occur. Using knock-out mice, we previously showed that the beta4, but not the beta2 subunit of nicotinic acetylcholine receptors, is necessary for the somatic manifestations of nicotine withdrawal. Since the beta4 subunit is highly expressed in the medial habenula, we focused our studies on the medial habenula and its primary target, the interpeduncular nucleus. In particular, we studied nicotine withdrawal in mice lacking the alpha2 or the alpha5 nicotinic receptor subunits, which are highly expressed in the interpeduncular nucleus. We precipitated withdrawal by systemically injecting the nicotinic antagonist mecamylamine in mice chronically treated with nicotine. Both the alpha2 and the alpha5 null mutations abolished the somatic manifestations of nicotine withdrawal. In addition, in wild-type mice chronically treated with nicotine, mecamylamine precipitated withdrawal when microinjected into the habenula or the interpeduncular nucleus, but not into the cortex, ventral tegmental area or hippocampus. Our results demonstrate a major role for the habenulo-interpeduncular system and the nicotinic receptor subunits expressed therein, in nicotine withdrawal symptoms. Our data suggest that the efforts to develop new smoking cessation therapies should concentrate on these areas and receptor types.

Nicotine is known to enhance learning and memory in hippocampus-dependent tasks such as contextual fear conditioning. The present study was designed to directly examine whether the hippocampus plays a role in mediating this enhancement and which nicotinic acetylcholine receptor (nAChR) subtypes localized to the hippocampus are critical for enhanced learning. Contextual fear conditioning consisted of two white noise conditioned stimuli presentations, each coterminating with a 2 s, 0.57 mA footshock separated by a 120 s intertrial interval. Nicotine (0.09, 0.18, and 0.35 microg per side) was bilaterally infused into the dorsal hippocampus before training and testing. Infusions of nicotine into the dorsal hippocampus produced a dose-dependent enhancement of contextual fear conditioning. To determine which nAChRs are critical to the enhancing effect of nicotine, the preferential alpha4beta2 nAChR antagonist, dihydro-beta-erythroidine (DHbetaE) (6.00 and 18.00 microg per side), or the preferential alpha7 nAChR antagonist, methyllycaconitine (MLA) (13.50 and 27.00 microg per side), was bilaterally infused into the dorsal hippocampus before systemic injections of nicotine (0.09 mg/kg). DHbetaE infusions dose-dependently blocked the enhancement of contextual fear conditioning by nicotine, whereas MLA infusions yielded an intermediate effect. In addition, neither DHbetaE nor MLA had an effect on contextual fear conditioning in the absence of systemic nicotine. The present results suggest a critical role for alpha4beta2 nAChRs in the dorsal hippocampus for mediating the enhancing effect of nicotine on contextual fear conditioning.

Increasing use of cannabis makes the search for medications to reduce cannabis abuse extremely important. Here, we show that homomeric α7nicotinic receptors are novel molecular entities that could be targeted in the development of new drugs for the treatment of cannabis dependence. In rats, systemic administration of the selective α7nicotinic acetylcholine receptor antagonist methyllycaconitine (MLA), but not the selective heteromeric non-α7nicotinic acetylcholine receptor antagonist dihydrobetaerythroidine, (1) antagonized the discriminative effects of δ-9-tetrahydrocannabinol (THC), the main active ingredient in cannabis, (2) reduced intravenous self-administration of the synthetic cannabinoid CB1 receptor agonist WIN55,212-2 [(R)-(+)-[2,3-dihydro-5-methyl-3[(4-morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxazinyl]-(1-naphthalenyl)methanone, mesylate salt], and (3) decreased THC-induced dopamine elevations in the shell of the nucleus accumbens. Altogether, our results indicate that blockade of α7nicotinic receptors reverses abuse-related behavioral and neurochemical effects of cannabinoids. Importantly, MLA reversed the effects of cannabinoids at doses that did not produce depressant or toxic effects, further pointing to α7nicotinic antagonists as potentially useful agents in the treatment of cannabis abuse in humans.

The mushroom bodies, bilaterally symmetric regions in the insect brain, play a critical role in olfactory associative learning. Genetic studies inDrosophilasuggest that plasticity underlying acquisition and storage of memory occurs at synapses on the dendrites of mushroom body Kenyon cells (Dubnau et al., 2001). Additional exploration of the mechanisms governing synaptic plasticity contributing to these aspects of olfactory associative learning requires identification of the receptors that mediate fast synaptic transmission in Kenyon cells. To this end, we developed a culture system that supports the formation of excitatory and inhibitory synaptic connections between neurons harvested from the central brain region of late-stageDrosophilapupae. Mushroom body Kenyon cells are identified as small-diameter, green fluorescent protein-positive (GFP+) neurons in cultures from OK107-GAL4;UAS-GFP pupae. In GFP+ Kenyon cells, fast EPSCs are mediated by α-bungarotoxin-sensitive nicotinic acetylcholine receptors (nAChRs). The miniature EPSCs have rapid rise and decay kinetics and a broad, positively skewed amplitude distribution. Fast IPSCs are mediated by picrotoxin-sensitive chloride conducting GABA receptors. The miniature IPSCs also have a rapid rate of rise and decay and a broad amplitude distribution. The vast majority of spontaneous synaptic currents in the cultured Kenyon cells are mediated byα-bungarotoxin-sensitive nAChRs or picrotoxin-sensitive GABA receptors. Therefore, these receptors are also likely to mediate synaptic transmission in Kenyon cellsin vivoand to contribute to plasticity during olfactory associative learning.

The alpha6 subunit of the nicotinic acetylcholine receptor (nAChR) is expressed at very high levels in dopaminergic (DA) neurons. However, because of the lack of pharmacological tools selective for alpha6-containing nAChRs, the role of this subunit in the etiology of nicotine addiction remains unknown. To provide new tools to investigate this issue, we generated an alpha6 nAChR knock-out mouse. Homozygous null mutants (alpha6-/-) did not exhibit any gross neurological or behavioral deficits. A careful anatomic and molecular examination of alpha6-/- mouse brains demonstrated the absence of developmental alterations in these animals, especially in the visual and dopaminergic pathways, where the alpha6 subunit is normally expressed at the highest levels. On the other hand, receptor autoradiography revealed a decrease in [3H]nicotine, [3H]epibatidine, and [3H]cytisine high-affinity binding in the terminal fields of retinal ganglion cells of alpha6-/- animals, whereas high-affinity [125I]alpha-conotoxinMII (alphaCtxMII) binding completely disappeared in the brain. Moreover, inhibition of [3H]epibatidine binding on striatal membranes, using unlabeled alphaCtxMII or cytisine, revealed the absence of alphaCtxMII-sensitive and cytisine-resistant [3H]epibatidine binding sites in alpha6-/- mice, although the total amount of binding was unchanged. Because alphaCtxMII, a toxin formerly thought to be specific for alpha3beta2-containing nAChRs, is known to partially inhibit nicotine-induced dopamine release, these results support the conclusion that alpha6 rather than alpha3 is the partner of beta2 in the nicotinic modulation of DA neurons. They further show that alpha6-/- mice might be useful tools to understand the mechanisms of nicotine addiction, although some developmental compensation might occur in these mice.

Perisynaptic Schwann cells (PSCs) play critical roles in regulating and stabilizing nerve terminals at the mammalian neuromuscular junction (NMJ). However, although these functions are likely regulated by the synaptic properties, the interactions of PSCs with the synaptic elements are not known. Therefore, our goal was to study the interactions between mammalian PSCsin situand the presynaptic terminals using changes in intracellular Ca2+as an indicator of cell activity. Motor nerve stimulation induced an increase in intracellular Ca2+in PSCs, and this increase ÿas greatly reduced when transmitter release was blocked. Furthermore, local application of acetylcholine induced Ca2+responses that were blocked by the muscarinic antagonist atropine and mimicked by the muscarinic agonist muscarine. The nicotinic antagonist α-bungarotoxin had no effect on Ca2+responses induced by acetylcholine. Local application of the cotransmitter ATP induced Ca2+responses that were unaffected by the P2 antagonist suramin, whereas local application of adenosine induced Ca2+responses that were greatly reduced by the A1 receptor antagonist 8-cyclopentyl-1,3-dimethylxanthine (CPT). However, the presence of the A1 antagonist in the perfusate did not block responses induced by ATP. Ca2+responses evoked by stimulation of the motor nerve were reduced in the presence of CPT, whereas atropine almost completely abolished them. Ca2+responses were further reduced when both antagonists were present simultaneously. Hence, PSCs at the mammalian NMJ respond to the release of neurotransmitter induced by stimulation of the motor nerve through the activation of muscarinic and adenosine A1 receptors.

Spontaneous waves of excitation in the developing mammalian retina are mediated, to a large extent, by neurotransmission. However, it is unclear how the underlying neurotransmitter systems interact with each other to play specific roles in the formation of retinal waves at various developmental stages. In particular, it is puzzling why the waves maintain a similar propagation pattern even after underlying neurotransmitter systems have undergone drastic developmental changes. Using Ca(2+) imaging and patch clamp in a whole-mount preparation of the developing rabbit retina, we discovered two dramatic and coordinated transitions in the excitatory drive for retinal waves: one from a nicotinic to a muscarinic system, and the other from a fast cholinergic to a fast glutamatergic input. Retinal waves before the age of postnatal day 1 (P1) were blocked by nicotinic antagonists, but not by muscarinic or glutamatergic antagonists. After P3, however, the spontaneous wave, whose basic spatiotemporal pattern remained similar, was completely inhibited by muscarinic or glutamate antagonists, but not by nicotinic antagonists. We also found that the muscarinic drive, mediated primarily by M1 and M3 receptors, was particularly important for wave propagation, whereas the glutamatergic drive seemed more important for local excitation. Our results suggest (1) a novel mechanism by which a neurotransmitter system changes its functional role via a switch between two completely different classes of receptors for the same transmitter, (2) the cholinergic system plays a critical role in not only early but also late spontaneous waves, and (3) the continued participation of the cholinergic system may provide a network basis for the consistency in the overall propagation pattern of spontaneous retinal waves.

Exogenous application of acetylcholine elicits inward currents in hippocampal interneurons that are mediated via α-bungarotoxin-sensitive nicotinic acetylcholine receptors, but synaptic responses mediated via such receptors have never been reported in mammalian brain. In the present study, EPSCs were evoked in hippocampal interneurons in rat brain slices by electrical stimulation and were recorded by using whole-cell voltage-clamp techniques. Nicotinic EPSCs were isolated pharmacologically, using antagonists to block other known types of ligand-gated ion channels, and then were tested with either α-bungarotoxin or methyllycaconitine, which are selective antagonists for nicotinic acetylcholine receptors that contain the α7 receptor subunit. Each antagonist proved highly effective at reducing the remaining synaptic current. Evoked α7-mediated nicotinic EPSCs also were desensitized by superfusion with 1 μmnicotine, had extrapolated reversal potentials near 0 mV, and showed strong inward rectification at positive potentials. In several interneurons, methyllycaconitine-sensitive spontaneous EPSCs also were observed that exhibited a biphasic decay rate very similar to that of the α7-mediated evoked response. These studies provide the first demonstration of a functional cholinergic synapse in the mammalian brain, in which the primary postsynaptic receptors are α-bungarotoxin-sensitive nicotinic acetylcholine receptors.

Motoneurons undergo a phase of target-dependent cell death during development. In chick embryos, motoneuronal death is blocked by the application of the nicotinic antagonists d-tubocurare (dTC) or alpha- bungarotoxin (alpha BTX). Paralytic doses of these drugs also increase intramuscular nerve branch formation. To investigate the possibility that a neuronal rather than a muscle-type nicotinic ACh receptor (nAChR) might be responsible for the toxin-induced arrest of naturally occurring motoneuronal death, we compared the doses of dTC and alpha- BTX required for paralysis with those needed to protect motoneurons from cell death. We also measured the effects of dTC on the survival of retrogradely labeled motoneurons in culture, and of various doses of dTC on intramuscular nerve branch formation. Subparalytic doses of dTC caused small but significant increases in nerve branch number, while higher doses produced larger effects. In contrast, motoneuronal survival was already maximal at doses of dTC or alpha-BTX below those needed for a visible effect on limb movement. Moreover, dTC increased motoneuron survival in culture, in the absence of muscle cells and muscle-type nAChRs. Nicotinic blocking agents can therefore rescue motoneurons with minimal depression of neuromuscular transmission, suggesting that this effect may be mediated through neuronal, rather than muscle-type.

Conventional intracellular electrophysiological methods were used to study fast synaptic transmission in the myenteric plexus of guinea pig ileum in vitro. Fast excitatory postsynaptic potentials (fEPSPs) were evoked in 98 neurons following single stimuli applied to interganglionic connectives. The nicotinic antagonist hexamethonium (100 microM) reduced fEPSPs by 83% in 37 neurons; these fEPSPs were considered to be cholinergic. In 61 neurons, hexamethonium reduced fEPSPs by 33%; fEPSPs recorded in the presence of hexamethonium were considered to be noncholinergic. Similar data were obtained using the nicotinic antagonist mecamylamine (10 microM) to block fEPSPs. Hexamethonium or mecamylamine completely blocked depolarizations caused by acetylcholine (ACh) applied by ionophoresis. The P2 receptor antagonist suramin (1–300 microM) inhibited noncholinergic fEPSPs in 30 cells; the suramin IC50 was 4 microM. Suramin (100 microM) did not block depolarizations caused by ACh or 5-HT, but suramin blocked depolarizations caused by ATP. Hexamethonium did not block ATP-induced depolarizations. The estimated reversal potential for suramin-sensitive fEPSPs and ATP-induced depolarizations was -25 and -16 mV, respectively. ATP responses were reduced in low-sodium (26 mM) extracellular solution, suggesting that ATP activates a cation channel. These data indicate that in myenteric nerves ATP, in addition to ACh, contributes to fast synaptic transmission.

Acetylcholine and nicotine can modulate respiratory patterns by acting on nicotinic acetylcholine receptors (nAChRs) in the preBötzinger complex (preBötC). To further explore the molecular composition of these nAChRs, we studied a knock-in mouse strain with a leucine-to-alanine mutation in the M2 pore-lining region (L9′A) of the nAChR α4 subunit; this mutation renders α4-containing receptors hypersensitive to agonists. We recorded respiratory-related rhythmic motor activity from hypoglossal nerve (XIIn) and patch-clamped preBötC inspiratory neurons in anin vitromedullary slice preparation from neonatal mice. Nicotine affected respiratory rhythm at concentrations ∼100-fold lower in the homozygous L9′A knock-in mice compared with wild-type mice. Bath application of 5 nmnicotine increased the excitability of preBötC inspiratory neurons, increased respiratory frequency, and induced tonic/seizure-like activities in XIIn in L9′A mice, effects similar to those induced by 1 μmnicotine in wild-type mice. In L9′A mice, microinjection of low nanomolar concentrations of nicotine into the preBötC increased respiratory frequency, whereas injection into the ipsilateral hypoglossal (XII) nucleus induced tonic/seizure-like activity. The α4*-selective nAChR antagonist dihydro-β-erythroidine produced opposite effects and blocked the nicotinic responses. These data, showing that nAChRs in the preBötC and XII nucleus in L9'A mice are hypersensitive to nicotine and endogenous ACh, suggest that functional α4* nAChRs are present in the preBötC. They mediate cholinergic/nicotinic modulation of the excitability of preBötC inspiratory neurons and of respiratory rhythm. Furthermore, functional α4* nAChRs are present in XII nucleus and mediate cholinergic/nicotinic modulation of tonic activity in XIIn.

We present a functional characterization of a neuronal nicotinic receptor in the CNS using patch-clamp techniques and a preparation of acutely isolated neurons from the medial habenular nucleus of 10- to 20- d-old rats. The salient pharmacological and electrophysiological properties of this nicotinic response are (1) its association with a channel that is relatively nonselective for cations and has a unitary conductance of 26.2 (+)+/- 5pS at room temperature; (2) its insensitivity to alpha-bungarotoxin and to neuronal bungarotoxin; (3) its activation by the ganglionic nicotinic agonists nicotine, 1,1- dimethyl-4-phenylpiperazinium and cytisine and its blocking by several nicotinic antagonists, mecamylamine, hexamethonium, d-tubocurarine, and dihydro-beta-erythroidine. The combination of these properties has not been reported for any other known type of nicotinic receptor.

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.

Alzheimer's disease (AD) is a human neurological disorder characterized by an increasing loss of cognitive function and the presence of extracellular neuritic plaques composed of the beta-amyloid peptide (Abeta(1-42)). However, the link between these molecular correlates of AD and the loss of cognitive function has not been established. The pathology associated with AD includes the loss of basal forebrain cholinergic neurons, presynaptic terminals in the neocortex and hippocampus, and a decrease in the total amount of neuronal nicotinic acetylcholine receptors (nAChRs). This leads to the hypothesis that failure in the cholinergic system underlies the dementia seen in AD. Cognitive performance has been linked to nAChR function in the hippocampus, and the interneurons expressing nAChRs coordinate the activity of large numbers of principal cells and therefore have a powerful role in the regulation of hippocampal activity. We have found that Abeta(1-42) inhibits whole-cell and single-channel nicotinic currents from rat hippocampal interneurons by directly blocking the postsynaptic nAChR channels at concentrations as low as 100 nm. This inhibition appears specific for peptide sequence and neuronal nAChRs, and the magnitude of Abeta(1-42) inhibition is dependent on the nAChR channel subtype expressed. Thus, chronic inhibition of cholinergic signaling by Abeta(1-42) could contribute to the cognitive deficits associated with AD.

A nicotinic acetylcholine receptor (AChR) on bovine adrenal chromaffin cells in culture has previously been identified using the alpha- neurotoxin n-Bgt and the monoclonal antibody mAb 35. Here, we report that the cells have 2 classes of high-affinity binding sites for 3H- nicotine, one being associated with the AChR and the other being associated with the alpha-bungarotoxin binding component that is distinct from the AChR. Scatchard analysis of 3H-nicotine binding to the AChR site yields a KD of 20 +/- 3 nM and a Bmax of 104 +/- 12 fmol/mg protein. Nicotinic antagonists block 3H-nicotine binding to the AChR site with the same rank order of potency and affinity with which they block nicotine-induced catecholamine release from the cells. About 80% of the AChRs are on the cell surface, as judged by the distribution of both 3H-nicotine binding and 125I-mAb 35 binding to the receptor, and the ratio of nicotine/mAb 35 binding to the AChR on the cell surface is approximately 1:1. Chronic treatment of the cells with mAb 35 results in receptor modulation such that all of AChR-related nicotine binding is lost from the cell surface, and all of the functional response to nicotine is lost as well. The results confirm that 3H-nicotine binding is associated with AChRs on the cells. The 3H- nicotine binding observed to the AChR represents binding to a desensitized form of the receptor having increased affinity for agonists and unchanged affinity for antagonists. This conclusion derives from the following observations. The KiS for agonist competition of 3H-nicotine binding indicate agonist affinities several orders of magnitude greater than do the KDS measured for receptor activation. Exposing cultures to low concentrations of nicotine and substance P causes receptor densensitization, and the concentration dependence of the nicotine-induced desensitization displays an EC50 of 20 nM, in good agreement with the KD obtained from equilibrium binding studies with 3H-nicotine. In addition, the rate of 3H-nicotine binding is increased both by substance P, which enhances the rate of agonist- induced desensitization on adrenal chromaffin cells, and by preincubation with nicotine. The increased rate of association, together with the dissociation rate, yields a kinetically derived KD of 19 nM, again in good agreement with the KD obtained from equilibrium binding studies. These results demonstrate that the bovine adrenal chromaffin AChR is similar to AChRs from muscle and electric organ in undergoing an agonist-induced conversion to a desensitized state having increased affinity for agonists.

The actions of ACh in the medial habenular nucleus (MHb) were investigated using extra- and intracellular recording techniques in guinea pig thalamic slice maintained in vitro. Applications of ACh to MHb neurons resulted in rapid excitation followed by inhibition. Neither of these responses was abolished by blockade of synaptic transmission, indicating that they are consequences of ACh action directly on MHb cells. Local applications of the nicotinic agonists nicotine and cytisine caused long-lasting excitation, while applications of another nicotinic agonist, 1,1-dimethyl-4- phenylpiperazinium caused both the excitatory and inhibitory responses. Applications of the muscarinic agonists DL-muscarine and acetyl-beta- methylcholine did not consistently cause either the excitatory or inhibitory response. Adding the nicotinic antagonist hexamethonium to the bathing medium blocked both the excitatory and inhibitory ACh responses, while addition of the muscarinic antagonists atropine or scopolamine had no effect. These results indicate that the effects of ACh on MHb neurons are mediated by nicotinic receptors. Intracellular recordings revealed that ACh or nicotine cause an increase in membrane conductance associated with depolarizations that had an average reversal potential of -16 to -11 mV. These results indicate that the ACh-induced excitation is due to an increase in membrane cation conductance. The inhibitory response that follows ACh-induced depolarization and repetitive firing was associated with a hyperpolarization and an increase in membrane conductance. Similar postexcitatory inhibition could also be elicited by direct depolarization or by applications of glutamate, indicating that the hyperpolarizing response to ACh may be an endogenous postexcitatory potential that is not directly coupled to activation of nicotinic receptors. These results suggest that cholinergic transmission in the MHb may be largely of the nicotinic type. This nucleus may be of one of the major regions of the nervous system through which nicotine mediates its central effects.

Three radioligands have been commonly used to label putative nicotinic cholinoceptors in the mammalian central nervous system: the agonists [3H]nicotine and [3H]acetylcholine ([3H]ACh--in the presence of atropine to block muscarinic receptors), and the snake venom extract, [125I]-alpha-bungarotoxin([125I]BTX), which acts as a nicotinic antagonist at the neuromuscular junction. Binding studies employing brain homogenates indicate that the regional distributions of both [3H]nicotine and [3H]ACh differ from that of [125I]BTX. The possible relationship between brain sites bound by [3H]nicotine and [3H]ACh has not been examined directly. We have used the technique of autoradiography to produce detailed maps of [3H]nicotine, [3H]ACh, and [125I]BTX labeling; near-adjacent tissue sections were compared at many levels of the rat brain. The maps of high affinity agonist labeling are strikingly concordant, with highest densities in the interpeduncular nucleus, most thalamic nuclei, superior colliculus, medial habenula, presubiculum, cerebral cortex (layers I and III/IV), and the substantia nigra pars compacta/ventral tegmental area. The pattern of [125I]BTX binding is strikingly different, the only notable overlap with agonist binding being the cerebral cortex (layer I) and superior colliculus. [125I]BTX binding is also dense in the inferior colliculus, cerebral cortex (layer VI), hypothalamus, and hippocampus, but is virtually absent in thalamus. Various lines of evidence suggest that the high affinity agonist-binding sites in brain correspond to nicotinic cholinergic receptors similar to those found at autonomic ganglia; BTX- binding sites may also serve as receptors for nicotine and are possibly related to neuromuscular nicotinic cholinoceptors.

Nictonic cholinergic responses have been previously documented in mammalian retinal ganglion cells. In the present study, dissociated retinal cells were densely plated and grown in a specific batch of rat serum. When held at negative potentials during whole-cell recording with a patch electrode, the retinal ganglion cells located close to presumptive cholinergic amacrine cells in these cultures were found to respond to acetylcholine (ACh; 20–200 microM) with an apparent outward current in the presence of physiological salines on both sides of the membrane. Other nicotinic agonists (nicotine, carbachol) produced the same effect. Conductance measurements revealed that this apparent outward current was actually a decrease in a tonic inward current. Nicotinic antagonists such as d-turbocurarine (10 microM) and dihydro- beta-erythroidine (20 microM), when applied in dishes that had never been exposed to exogenous ACh, produced a similar decrease in a tonic inward current. The reversal potential of the tonic current suppressed by ACh was similar to the nicotinic current previously studied in these central neurons. Furthermore, purified acetylcholinesterase was capable of modulating the tonic inward current of the retinal ganglion cells. Lowering an excised patch of muscle into dense areas of the retinal cultures activated nicotinic channels in the muscle membrane, indicating the presence of endogenous ACh in the culture fluid. Divalent cations such as Co2+ blocked the tonic inward current of retinal ganglion cells in these cultures. Finally, direct biochemical measurements indicated that low levels of endogenous ACh (on the order of 0.5 microM near putative cholinergic amacrine cells) were present in the retinal cultures. Taken together, these results show that ACh was being spontaneously released into these cultures, resembling at least to some degree the tonic leakage of ACh found in the intact retina. This concentration of ACh was capable of tonically depolarizing the membrane potential of retinal ganglion cells exposed to this dosage. This culture system allows the study of trophic effects of ACh on a central mammalian neuron in a precisely controlled extracellular environment.

The nicotinic cholinergic antagonist, dihydro-beta-erythroidine, binds to two sites in rat cortical membranes with dissociation constants of 4 and 22 nM and respective apparent Bmax values of 52 and 164 fmol/mg of protein. Binding to the higher affinity site, defined by the use of 2 nM [3H]dihydro-beta-erythroidine, was saturable, reversible, and susceptible to protein denaturation. Binding was highest in the thalamus and lowest in the spinal cord and showed preferential enrichment in a synaptosomal subfraction of rat brain. Nicotine displaced [3H]dihydro-beta-erythroidine in a stereospecific manner, the (-)-isomer being approximately 6 times more potent than the (+)-isomer. The alkaloid nicotinic agonists, cytisine and lobeline, were potent inhibitors of binding, while acetylcholine in the presence of the cholinesterase inhibitor di-isopropylfluorophosphate was equipotent with (+)-nicotine. Binding was also inhibited by the muscarinic ligands, arecoline, atropine, and oxotremorine. The nicotinic antagonists mecamylamine, hexamethonium, and pempidine were essentially inactive in displacing [3H]dihydro-beta-erythroidine. These findings indicate that dihydro-beta-erythroidine binds to a nicotinic recognition site in rat brain which is neuromuscular, rather than ganglionic, in nature and that such binding is similar in several respects to that seen with nicotinic agonists. Whether such binding is to a nicotinic, as opposed to nicotinic cholinergic, recognition site or to a “common” nicotinic/muscarinic site is an issue that requires further study.

Although alpha-bungarotoxin (BGT), a common probe for nicotinic ACh receptors from vertebrate skeletal muscle, binds tightly to many autonomic ganglia, it fails to block nicotinic transmission in most of these ganglia. Recently, we have isolated a second toxin, toxin F, that blocks transmission in several autonomic ganglia, including the chick ciliary ganglion. 125I-Toxin F binds to 2 sites in the ciliary ganglion: one site that is also recognized by BGT and one site that is not. Since the presence of BGT fails to prevent the blocking effect of toxin F, the toxin F site not recognized by BGT most likely represents the neuronal nicotinic receptor. Accordingly, we have localized the binding of 125I-toxin F to both sites, using electron-microscopic autoradiography. After a 45 min incubation, 125I-toxin F binding sensitive to BGT was primarily localized extrasynaptically on the neuronal plasma membranes; however, by 4 hr, much of this site had been internalized. In contrast, the 125I-toxin F binding site not recognized by BGT was highly concentrated near synaptic membranes at both times. Pretreatment of ganglia with the classic nicotinic antagonists dihydro- beta-erythroidine (DHBE) and d-tubocurarine (DTC) significantly reduced 125I-toxin F binding to the toxin F-specific site. The average density of these sites on synaptic membranes was approximately 600 sites/micron 2. We conclude that toxin F binds to 2 pharmacologically distinct sites in the ciliary ganglion and that these sites are distributed differently over the plasma membrane of ciliary neurons. On the basis of the density of the toxin F-specific binding sites at synaptic membranes, we infer that the density of synaptic nicotinic receptors on these neurons is at least 20-fold lower than the density of nicotinic receptors at the vertebrate neuromuscular junction, as determined by BGT binding. These findings are consistent with those of electrophysiological studies, which also suggest low nicotinic receptor densities on ganglionic neurons.