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Usually nicotinic receptors in the central nervous system only influence the strength of a signal between neurons. At a few critical connections, for instance some of those involved in the flight response, nicotinic receptors not only modulate the signal, they actually determine whether a signal is conveyed or not. We show at one of the few such connections accessible for study, up to three different nicotinic receptor subtypes mediate the signal. The subtypes appear to be clustered in separate locations. Depending on the number and combination of the subtypes present the signal can range from short to long duration and from low to high amplitude. This provides a critical connection with a built-in plasticity and may enable it to adapt to a changing environment.

Anatomical studies indicate that synaptic inputs from many cortical and subcortical structures converge on neurons of the ventral tegmental area (VTA). Although in vitro electrophysiological studies have examined synaptic inputs to dopamine (DA) and non-DA neurons in the VTA, they have largely relied upon local electrical stimulation to activate these synapses. This provides little information regarding the distinct properties of synapses originating from different brain areas. Using whole-cell recordings in parasagittal rat brain slices that preserved subcortical axons from the pedunculopontine nucleus (PPN) to the VTA, we compared these synapses with those activated by intra-VTA stimulation. PPN-evoked currents demonstrated longer latencies than intra-VTA-evoked currents, and both VTA and PPN responses were mediated by GABA(A) and AMPA receptors. However, unlike VTA-evoked currents, PPN currents were exclusively mediated by glutamate in 25-40% of the VTA neurons. Consistent with a cholinergic projection from the PPN to the VTA, nicotinic acetylcholine receptors (nAChR) were activated by endogenous acetylcholine released during PPN, but not VTA, stimulation. This was seen as a reduction of PPN-evoked, and not VTA-evoked, synaptic currents by the alpha7-nAChR antagonist methyllycaconitine (MLA) and the agonist nicotine. The beta2-nAChR subunit antagonist dihydro-beta-erythroidine had no effect on VTA- or PPN-evoked synaptic currents. The effects of MLA on PPN-evoked currents were unchanged by the GABA(A) receptor blocker picrotoxin, indicating that alpha7-nAChRs presynaptically modulated glutamate and not GABA release. These differences in physiological and pharmacological properties demonstrate that ascending PPN and presumed descending inputs to VTA utilize distinct mechanisms to differentially modulate neuronal activity and encode cortical and subcortical information.

Dopaminergic (DAergic) neuronal activity in the ventral tegmental area (VTA) is thought to contribute generally to pleasure, reward, and drug reinforcement and has been implicated in nicotine dependence. nAChRs expressed in the VTA exhibit diverse subunit compositions, but the functional and pharmacological properties are largely unknown. Here, using patch-clamp recordings in single DAergic neurons freshly dissociated from rat VTA, we clarified three functional subtypes of nAChRs (termed ID, IID and IIID receptors) based on whole-cell current kinetics and pharmacology. Kinetic analysis demonstrated that comparing to ID, IID receptor-mediated current had faster activation and decay constant and IIID receptor-mediated current had larger current density. Pharmacologically, ID receptor-mediated current was sensitive to the alpha4beta2-nAChR agonist RJR-2403 and antagonist dihydro-beta-erythroidine (DHbetaE); IID receptor-mediated current was sensitive to the selective alpha7-nAChR agonist choline and antagonist methyllycaconitine (MLA); while IIID receptor-mediated current was sensitive to the beta4-containing nAChR agonist cytisine and antagonist mecamylamine (MEC). The agonist concentration-response relationships demonstrated that IID receptor-mediated current exhibited the highest EC(50) value compared to ID and IIID receptors, suggesting a relatively low agonist affinity of type IID receptors. These results suggest that the type ID, IID and IIID nAChR-mediated currents are predominately mediated by activation of alpha4beta2-nAChR, alpha7-nAChR and a novel nAChR subtype(s), respectively. Collectively, these findings indicate that the VTA DAergic neurons express diversity and multiplicity of functional nAChR subtypes. Interestingly, each DAergic neuron predominantly expresses only one particularly functional nAChR subtype, which may have distinct but important roles in regulation of VTA DA neuronal function, DA transmission and nicotine dependence.

Multiple subtypes of nicotinic acetylcholine receptors (nAChRs) are expressed in the CNS. The amygdala complex, the limbic structure important for emotional memory formation, receives cholinergic innervation from the basal forebrain. Although cholinergic drugs have been shown to regulate passive avoidance performance via the amygdala, the neuronal subtypes and circuits involved in this regulation are unknown. In the present study, whole-cell patch-clamp electrophysiological techniques were used to identify and characterize the presence of functional somato-dendritic nAChRs within the basolateral complex of the amygdala. Pressure-application of acetylcholine (ACh; 2 mm) evoked inward current responses in a subset of neurons from both the lateral (49%) and basolateral nuclei (72%). All responses displayed rapid activation kinetics, and were blocked by the alpha7-selective antagonist methyllycaconitine. In addition, the alpha7-selective agonist choline induced inward current responses that were similar to ACh-evoked responses. Spiking patterns were consistent with pyramidal class I neurons (the major neuronal type in the basolateral complex); however, there was no correlation between firing frequency and the response to ACh. The local photolysis of caged carbachol demonstrated that the functional expression of nAChRs is located both on the soma and dendrites. This is the first report demonstrating the presence of functional nAChR-mediated current responses from rat amygdala slices, where they may be playing a significant role in fear and aversively motivated memory.

Neurotoxins such as aconitine (AC) bind to receptor site 2 on voltage-gated sodium channels and modify channel kinetics. Although AC modification typically induces hyperpolarizing shifts in sodium channel activation, the effects on channel inactivation seem to vary depending on the tissue origin of the channel. In the present study, the alpha subunits of human heart (hH1) and rat skeletal muscle (mu1) sodium channels were transiently expressed in human embryonic kidney (HEK293t) cells. Whole-cell currents were examined before and after AC modification of the channels to determine whether the toxin had isoform-specific effects on channel kinetics. The magnitudes of the hyperpolarizing shifts in steady-state current activation and inactivation were similar for AC-modified hH1 and mu1 channels, and AC modification did not alter the voltage dependence of macroscopic current decay of either channel subtype. There were two notable differences between hH1 and mu1 channels after AC modification. First, the steady-state availability of AC-modified mu1 channels decreased by 5-10% after very negative conditioning pulses. Second, AC-modified mu1 channels inactivated completely at all voltages, whereas AC-modified hH1 channels exhibited sustained inward currents at voltages near the threshold of current activation. Interestingly, AC-modified hH1 channels inactivated completely if the external solution did not contain sodium ions. The data demonstrate that AC modification affects the activation of hH1 and mu1 channels similarly but affects inactivation of the two channels distinctly. The results also imply that the reduced inactivation of AC-modified hH1 channels at least partially depends on the presence of extracellular sodium.

1. The whole-cell configuration of the patch-clamp technique was used to study the modulation of giant depolarizing potentials (GDPs) by nicotinic acetylcholine receptors (nAChRs) in CA3 hippocampal neurons in slices from postnatal day (P) 2-6 rats. 2. Bath application of nicotine increased GDP frequency in a concentration-dependent manner. For example, nicotine (0.5-1 microM) enhanced GDP frequency from 0.05 +/- 0.04 to 0.17 +/- 0.04 Hz. This effect was prevented by the broad-spectrum nicotinic receptor antagonist dihydro-beta-erythtroidine (DHbetaE, 50 microM) and partially antagonized by methyllycaconitine (MLA, 50 nM) a competitive antagonist of alpha7 nAChRs. GDP frequency was also enhanced by AR-17779 (100 microM), a selective agonist of alpha7 nAChRs. 3. The GABA(A) receptor antagonist bicuculline (10 microM) and the non-NMDA glutamate receptor antagonist DNQX (20 microM) blocked GDPs and prevented the effects of nicotine on GDPs. In the presence of DNQX, nicotine increased GABA-mediated synaptic noise, indicating that this drug may have a direct effect on GABAergic interneurons. 4. Bath application of edrophonium (20 microM), a cholinesterase inhibitor, in the presence of atropine (1 microM), increased GDP frequency, indicating that nAChRs can be activated by ACh released from the septo-hippocampal fibres. This effect was prevented by DHbetaE (50 microM). 5. In the majority of neurons tested, MLA (50 nM) and DHbetaE (50 microM) reduced the frequency of GDPs with different efficacy: a reduction of 98 +/- 11 and 61 +/- 29 % was observed with DHbetaE and MLA, respectively. In a subset of cells (40 % in the case of MLA and 17 % in the case of DHbetaE) these drugs induced a twofold increase in GDP frequency. 6. It is suggested that, during development, nAChRs modulate the release of GABA, assessed as GDPs, through distinct nAChRs. The rise of intracellular calcium via nAChRs would further strengthen GABA-mediated oscillatory activity. This can be crucial for consolidation of synaptic contacts and for the fine-tuning of the developing hippocampus.

The single channel properties of neuronal nicotinic ACh receptors (nAChRs) were investigated in outside-out patches from CA1 stratum radiatum interneurons from thin slices of rat hippocampus. The application of ACh (10 microM to 1 mM) induced the opening of observable single channel currents with two distinct current levels, which corresponded to conductance levels of 38 +/- 3 and 62 +/- 2 pS. The 38 pS channel was observed in 10 % (n = 260) of patches, whereas the 62 pS channel was observed in 4 % of patches; these two channel types were most often observed independently. The alpha7-selective nAChR antagonist methyllycaconitine (MLA; 50 nM) reduced the open probability of the 38 pS channel by 73 %. In contrast, the 62 pS channel was unaffected by MLA, but instead was blocked by dihydro-beta-erythroidine (DHbetaE; 10 microM), a broad spectrum nAChR antagonist. These data suggest that rat hippocampal CA1 stratum radiatum interneurons in the slice possess at least two different types of functional nAChRs, an alpha7-containing subtype with a single channel conductance of 38 pS, and a non-alpha7 subtype with a single channel conductance of 62 pS.

Nicotinic acetylcholine receptors (nAChRs) that bind alpha-bungarotoxin (alpha Bgt) were studied on isolated rat superior cervical ganglion (SCG) neurons using whole-cell patch clamp recording techniques. Rapid application of ACh onto the soma of voltage clamped neurons evoked a slowly desensitizing current that was reversibly blocked by alpha Bgt (50 nM). The toxin-sensitive current constituted on average about half of the peak whole-cell response evoked by ACh. Nanomolar concentrations of methyllycaconitine blocked the alpha Bgt-sensitive component of the ACh-evoked current as did intracellular dialysis with an anti-alpha 7 monoclonal antibody. The results indicate that the slowly reversible toxin-sensitive response elicited by ACh arises from activation of an unusual class of alpha 7-containing receptor (alpha 7-nAChR) similar to that reported previously for rat intracardiac ganglion neurons. A second class of functional alpha 7-nAChR was identified on some SCG neurons by using rapid application of choline to elicit responses. In these cases a biphasic response was obtained, which included a rapidly desensitizing component that was blocked by alpha Bgt in a pseudo-irreversible manner. The pharmacology and kinetics of the responses resembled those previously attributed to alpha 7-nAChRs in a number of other neuronal cell types. Experiments measuring the dissociation rate of 125I-labelled alpha Bgt from SCG neurons revealed two classes of toxin-binding site. The times for toxin dissociation were consistent with those required to reverse blockade of the two kinds of alpha Bgt-sensitive response. These results indicate that rat SCG neurons express two types of functional alpha 7-nAChR, differing in pharmacology, desensitization and reversibility of alpha Bgt blockade.

1. Whole-cell clamp recordings of the compound synaptic current elicited by afferent stimulation of Schaffer collaterals showed that blockade of the NMDA, AMPA and GABAA receptor-mediated components by 6-nitro-7-sulphamoyl- benzo(f)quinoxaline-2,3-dione (NBQX), 3-((R)-2-carboxypiperazine-4-yl)propyl-1-phosphonate (R-CPP) and picrotoxin, respectively, left a small residual current in 39 out of 41 CA1 pyramidal neurones in organotypic cultures and 9 out of 16 CA1 cells in acutely prepared slices. 2. This current represented 2. 9 +/- 0.4 % of the compound evoked synaptic response in organoypic cultures and 1.4 +/- 0.5 % in slices. It was characterized by a slightly rectifying I-V curve and a reversal potential of 3.4 +/- 5. 1 mV. 3. This residual current was insensitive to blockers of GABAB, purinergic, muscarinic and 5-HT3 receptors, but it was essentially blocked by the nicotinic receptor antagonist d-tubocurarine (91 +/- 4 % blockade; 20 microM), and partly blocked by alpha-bungarotoxin (200 nM) and methyllycaconitine (10 nM), two antagonists with a higher selectivity for alpha7 subunit-containing nicotinic receptors (48 +/- 3 % and 55 +/- 11 % blockade, respectively). 4. The residual current was of synaptic origin, since it occurred after a small delay; its amplitude depended upon the stimulation intensity and it was calcium dependent and blocked by the sodium channel antagonist tetrodotoxin. 5. We conclude that afferent stimulation applied in the stratum radiatum evokes in some hippocampal neurones a small synaptic current mediated by activation of neuronal nicotinic receptors.

1. Heterologous expression studies of the alpha5 subunit of the neuronal acetylcholine receptor (nAChR) gene family have demonstrated that it can participate in the function of ACh-gated channels if co-expressed with another alpha- and a beta-subunit. Previous studies also indicate prominent expression of alpha5 in both central and peripheral nervous systems. The participation of alpha5 in native nAChRs and its functional role in these channels is, however, unknown. 2. In this study, we present evidence that alpha5 has a role in at least two distinct subtypes of nAChR complexes expressed by embryonic chick sympathetic neurones. 3. alpha5 contributes not only to agonist but also to antagonist sensitivity of natively expressed nAChR channels. Functional deletion of the alpha5 subunit by antisense oligonucleotide treatment removes the nAChRs with relatively low affinity to ACh and cytisine. Deletion of alpha5 also eliminates channels that are blocked by the alpha7-specific antagonist methyllycaconitine (MLA) while increasing the percentage of current carried by nAChRs that are sensitive to alpha-bungarotoxin (alpha-BgTx). 4. Single channel analyses indicate that functional deletion of alpha5 results in the deletion of both the 'brief' and 'long' open duration, 50 pS subtypes of nAChR channels while increasing the expression of the 18 pS, alpha-BgTx-sensitive native nAChRs normally detected in sympathetic neurones at later developmental stages. 5. The biophysical and pharmacological profiles of native nAChRs revealed by this study and previous work are discussed in the context of a proposed model of the nAChR channels expressed by chick sympathetic neurones throughout development.

1. Many studies of the alpha7 subunit of the neuronal nicotinic acetylcholine receptor (nAChR) family have demonstrated that this alpha-bungarotoxin (alpha-BgTx)-binding neuronal receptor can participate in ACh-gated channels. Heterologous expression studies reveal that alpha7 subunits form homomeric channels of unusually high Ca2+ permeability. However, the physiological role of the alpha7 subunit in native neuronal nAChR channels is less clear. 2. We present evidence that the alpha7 subunit contributes to the function of at least three subtypes of native nAChR expressed by embryonic chick sympathetic neurones. These subtypes are functionally distinct from heterologously expressed homomeric alpha7 nAChRs as well as homomeric-like currents described in studies of hippocampal and parasympathetic neurones. 3. The proposed nAChRs differ from one another and from homomeric alpha7 nAChRs in their sensitivity to block by alpha7 subunit-specific antagonists: alpha-BgTx and methyllycaconitine (MLA). While MLA blocks 60 % of the macroscopic ACh response, alpha-BgTx inhibits a small component of the macroscopic current described by slow-on and slow-off kinetics. 4. Functional deletion of the alpha7 subunit by antisense oligonucleotide treatment eliminates the susceptibility of the nAChRs to block by both MLA and alpha-BgTx. 5. Single channel recordings combined with pharmacological and antisense-mediated 'deletion' techniques reveal that alpha-BgTx-sensitive alpha7-containing nAChRs have a small unitary conductance (18 pS), brief open time kinetics and relatively low open probability (Po). MLA-sensitive alpha7 nAChRs are characterized by a conductance of approximately 35 pS, intermediate burst duration, and a relatively high Po. 6. The third nAChR subtype deleted by alpha7 antisense treatment is characterized by a unitary conductance of 50 pS and prolonged opening duration. 7. We propose that these three populations of native alpha7-containing nAChRs are distinct heteromeric complexes that include other alpha and/or beta nAChR subunits.

1. The recently isolated compound methyllycaconitine (MLA) is a plant toxin which is a competitive inhibitor of nicotinic acetylcholine receptors (nAChRs). We found that homomeric alpha 7 receptors display a very high sensitivity to MLA with an IC50 in the picomolar range. 2. The competitive nature of the alpha 7 MLA blockade was reinforced by the observation that this compound has no action on wild-type serotoninergic receptors (5-HT3), whereas it is a powerful antagonist of chimaeric receptors alpha 7-5-HT3. 3. The time course of MLA inhibition of the wild-type (WT) alpha 7 follows a monotonic exponential decay whose time constant is proportional to the MLA concentration and could be described by a bimolecular mechanism with a forward rate constant (k+) of 2.7 x 10(7) S-1 M-1. In contrast, recovery from MLA inhibition displays an S-shaped time course that is incompatible with a simple bimolecular reaction. 4. Given the pentameric nature of the neuronal nicotinic receptors, a linear chain model, including five putative MLA binding sites corresponding to the homomeric nature of alpha 7, is proposed. 5. Both onset and recovery data obtained on the alpha 7 wild-type receptor are adequately described by this model assuming that a single MLA molecule is sufficient to block receptor function. 6. Analysis of MLA blockade and recovery of reconstituted heteromeric alpha 4 beta 2 receptors reveals, as expected, a time course compatible with only two binding sites for the toxin and, thus, further supports the validity of our model.