The Place of Erythroidines in the History of Neuromuscular Blockers.

Between 1938 and 1951 erythroidine derivatives were seriously considered as alternatives to curare for the provision of muscle relaxation. This has been overlooked in the published history of anaesthesia. The first publication on the paralysing effect of an extract of Erythrina americana was in 1877, but this was in a Mexican journal, which was not widely read. Sixty years later erythroidine was isolated, and in 1938 it was first used clinically to treat spastic dystonia, preceding the use of Intocostrin for this purpose. By 1943 dihydro-ß-erythroidine was prepared in crystalline form, which was equipotent with curarine and of acceptable duration; it was used in clinical anaesthesia in 1946. In the 1940s curare was presented in solutions with potency stated in units, determined by bioassay, which was a disadvantage compared with the straightforward mg of dihydro-ß-erythroidine. However, by the early 1950s, improvement in the pharmaceutical presentation of d-tubocurarine and new neuromuscular blockers, displaced the erythroidines.

Nicotinic α4 Receptor-Mediated Cholinergic Influences on Food Intake and Activity Patterns in Hypothalamic Circuits.

Nicotinic acetylcholine receptors (nAChRs) play an important role in regulating appetite and have been shown to do so by influencing neural activity in the hypothalamus. To shed light on the hypothalamic circuits governing acetylcholine's (ACh) regulation of appetite this study investigated the influence of hypothalamic nAChRs expressing the α4 subunit. We found that antagonizing the α4ß2 nAChR locally in the lateral hypothalamus with di-hydro-ß-erythroidine (DHßE), an α4 nAChR antagonist with moderate affinity, caused an increase in food intake following free access to food after a 12 hour fast, compared to saline-infused animals. Immunocytochemical analysis revealed that orexin/hypocretin (HO), oxytocin, and tyrosine hydroxylase (TH)-containing neurons in the A13 and A12 of the hypothalamus expressed the nAChR α4 subunit in varying amounts (34%, 42%, 50%, and 51%, respectively) whereas melanin concentrating hormone (MCH) neurons did not, suggesting that DHßE-mediated increases in food intake may be due to a direct activation of specific hypothalamic circuits. Systemic DHßE (2 mg/kg) administration similarly increased food intake following a 12 hour fast. In these animals a subpopulation of orexin/hypocretin neurons showed elevated activity compared to control animals and MCH neuronal activity was overall lower as measured by expression of the immediate early gene marker for neuronal activity cFos. However, oxytocin neurons in the paraventricular hypothalamus and TH-containing neurons in the A13 and A12 did not show differential activity patterns. These results indicate that various neurochemically distinct hypothalamic populations are under the influence of α4ß2 nAChRs and that cholinergic inputs to the lateral hypothalamus can affect satiety signals through activation of local α4ß2 nAChR-mediated transmission.

Erythroidine alkaloids: a novel class of phytoestrogens.

Erythrina poeppigiana is a medicinal plant which is widely used in Asia, Latin America, and Africa in traditional remedies for gynecological complications and maladies. In continuation of studies for the discovery of novel phytoestrogens, four erythroidine alkaloids, namely α-erythroidine, ß-erythroidine, and their oxo-derivatives 8-oxo-α-erythroidine and 8-oxo-ß-erythroidine, were isolated and structurally characterized from the methanolic extract of the stem bark of E. poeppigiana. Due to the high amounts of erythroidines in the extract and considering the widespread utilization of Erythrina preparations in traditional medicine, the exploration of their estrogenic properties was performed. The estrogenicity of the isolated erythroidines was assayed in various estrogen receptor-(ER)-dependent test systems, including receptor binding affinity, cell culture based ER-dependent reporter gene assays, and gene expression studies in cultured cells using reverse transcription polymerase chain reaction techniques. α-Erythroidine and ß-erythroidine showed binding affinity values for ERα of 0.015 ± 0.010% and 0.005 ± 0.010%, respectively, whereas only ß-erythroidine bound to ERß (0.006 ± 0.010%). In reporter gene assays, both erythroidines exhibited a significant dose-dependent estrogenic stimulation of ER-dependent reporter gene activity in osteosarcoma cells detectable already at 10 nM. Results were confirmed in the MVLN cells, a bioluminescent variant of MCF-7 breast cancer cells. Further, α-erythroidine and ß-erythroidine both induced the enhanced expression of the specific ERα-dependent genes trefoil factor-1 and serum/glucocorticoid regulated kinase 3 in MCF-7 cells, confirming estrogenicity. Additionally, using molecular docking simulations, a potential mode of binding on ERα, is proposed, supporting the experimental evidences. This is the first time that an estrogenic profile is reported for erythroidine alkaloids, potentially a new class of phytoestrogens.

The effects of nicotine and tobacco particulate matter on dopamine uptake in the rat brain.

Cigarette smoking is the leading cause of preventable death worldwide. Recently, tobacco extracts have been shown to have a different pharmacological profile to nicotine alone and there is increasing evidence of a role for non-nicotinic components of cigarette smoke in smoking addiction. Nicotine is known to affect the uptake of dopamine in the brain of laboratory animals, but studies in the literature are often contradictory and little is known of the effects on non-nicotinic tobacco components on dopamine uptake. This study has examined the acute and chronic effects of nicotine and a tobacco extract (TPM) on dopamine uptake by the dopamine and norepinephrine transporters (DAT and NET) ex vivo using rotating disk electrode voltammetry, and quantified DAT and NET protein and mRNA expression in key brain regions. Nicotine (0.35 mg/kg) significantly decreased DAT function in the nucleus accumbens (NAc) at 30 min with no change in protein expression. This effect was sensitive to mecamylamine and DHßE but not MLA, indicating that it is dependent on α4 subunit containing nicotinic receptors. Furthermore, TPM, but not nicotine, increased DAT function in the dorsal striatum at 1 h in a nicotinic receptor independent manner with no change in DAT protein expression. At 1 h DAT mRNA in the ventral tegmental area was decreased by both acute and chronic TPM treatments.

MK-801 disrupts and nicotine augments 40 Hz auditory steady state responses in the auditory cortex of the urethane-anesthetized rat.

Patients with schizophrenia show marked deficits in processing sensory inputs including a reduction in the generation and synchronization of 40 Hz gamma oscillations in response to steady-state auditory stimulation. Such deficits are not readily demonstrable at other input frequencies. Acute administration of NMDA antagonists to healthy human subjects or laboratory animals is known to reproduce many sensory and cognitive deficits seen in schizophrenia patients. In the following study, we tested the hypothesis that the NMDA antagonist MK-801 would selectively disrupt steady-state gamma entrainment in the auditory cortex of urethane-anesthetized rat. Moreover, we further hypothesized that nicotinic receptor activation would alleviate this disruption. Auditory steady state responses were recorded in response to auditory stimuli delivered over a range of frequencies (10-80 Hz) and averaged over 50 trials. Evoked power was computed under baseline condition and after vehicle or MK-801 (0.03 mg/kg, iv). MK-801 produced a significant attenuation in response to 40 Hz auditory stimuli while entrainment to other frequencies was not affected. Time-frequency analysis revealed deficits in both power and phase-locking to 40 Hz. Nicotine (0.1 mg/kg, iv) administered after MK-801 reversed the attenuation of the 40 Hz response. Administered alone, nicotine augmented 40 Hz steady state power and phase-locking. Nicotine's effects were blocked by simultaneous administration of the α4ß2 antagonist DHßE. Thus we report for the first time, a rodent model that mimics a core neurophysiological deficit seen in patients with schizophrenia and a pharmacological approach to alleviate it.

Convergence of nicotine-induced and auditory-evoked neural activity activates ERK in auditory cortex.

Enhancement of sound-evoked responses in auditory cortex (ACx) following administration of systemic nicotine is known to depend on activation of extracellular-signaling regulated kinase (ERK), but the nature of this enhancement is not clear. Here, we show that systemic nicotine increases the density of cells immunolabeled for phosphorylated (activated) ERK (P-ERK) in mouse primary ACx (A1). Cortical injection of dihydro-ß-erythroidine reduced nicotine-induced P-ERK immunolabel, suggesting a role for nicotinic acetylcholine receptors located in A1 and containing α4 and ß2 subunits. P-ERK expressing cells were distributed mainly in layers 2/3 and more sparsely in lower layers, with many cells exhibiting immunolabel within pyramidal-shaped somata and proximal apical dendrites. About one-third of P-ERK positive cells also expressed calbindin. In the thalamus, P-ERK immunopositive cells were found in the nonlemniscal medial geniculate (MG) and adjacent nuclei, but were absent in the lemniscal MG. Pairing broad spectrum acoustic stimulation (white noise) with systemic nicotine increased P-ERK immunopositive cell density in ACx as well as the total amount of P-ERK protein, particularly the phosphorylated form of ERK2. However, narrow spectrum (tone) stimulation paired with nicotine increased P-ERK immunolabel preferentially at a site within A1 where the paired frequency was characteristic frequency (CF), relative to a second site with a spectrally distant CF (two octaves above or below the paired frequency). Together, these results suggest that ERK is activated optimally where nicotinic signaling and sound-evoked neural activity converge.

Synapse- and subtype-specific modulation of synaptic transmission by nicotinic acetylcholine receptors in the ventrobasal thalamus.

The rodent thalamic ventrobasal complex (VB) which is a subdivision of somatosensory thalamus receives two excitatory inputs through the medial lemniscal synapse, which is a sensory afferent synapse, and the corticothalamic synapse from layer VI of the somatosensory cortex. In addition, the VB also receives cholinergic inputs from the brain stem, and nicotinic acetylcholine receptors (nAChRs) are highly expressed in the VB. Little is known, however, how acetylcholine (ACh) modulates synaptic transmission at the medial lemniscal and corticothalamic synapses in the VB. Furthermore, it remains unclear which subtype of nAChRs contributes to VB synaptic transmission. We report here that the activation of nAChRs presynaptically depressed corticothalamic synaptic transmission, whereas it did not affect medial lemniscal synaptic transmission in juvenile mice. This presynaptic modulation was mediated by the activation of nAChRs that contained α4 and ß2 subunit, but not by α7 nAChRs. Moreover, galanthamine, an allosteric modulator of α4ß2α5 nAChR, enhanced the ACh-induced depression of corticothalamic excitatory postsynaptic currents (EPSCs), indicating that α4ß2α5 nAChRs at corticothalamic axon terminals specifically contribute to the depression of corticothalamic synaptic transmission.

Basal forebrain activation enhances cortical coding of natural scenes.

The nucleus basalis of the basal forebrain is an essential component of the neuromodulatory system controlling the behavioral state of an animal and it is thought to be important in regulating arousal and attention. However, the effect of nucleus basalis activation on sensory processing remains poorly understood. Using polytrode recording in rat visual cortex, we found that nucleus basalis stimulation caused prominent decorrelation between neurons and marked improvement in the reliability of neuronal responses to natural scenes. The decorrelation depended on local activation of cortical muscarinic acetylcholine receptors, whereas the increased reliability involved distributed neural circuits, as evidenced by nucleus basalis-induced changes in thalamic responses. Further analysis showed that the decorrelation and increased reliability improved cortical representation of natural stimuli in a complementary manner. Thus, the basal forebrain neuromodulatory circuit, which is known to be activated during aroused and attentive states, acts through both local and distributed mechanisms to improve sensory coding.

Hippocampal nAChRs mediate nicotine withdrawal-related learning deficits.

Nicotine modulation of learning may contribute to its abuse liability. The role of hippocampal nicotinic acetylcholine receptors (nAChRs) in the effects of acute, chronic and withdrawal from chronic nicotine on learning was assessed via intrahippocampal drug infusion in mice. Acute dorsal hippocampal nicotine infusion enhanced contextual fear conditioning. Conversely, chronic intrahippocampal infusion of a matched dose had no effect, and withdrawal from chronic infusion impaired learning. Thus, hippocampal functional adaptation, evidenced by learning deficits during abstinence, occurs with the transition from acute to chronic nicotine exposure. To investigate which hippocampal nAChRs mediate these adaptations, C57BL/6, beta2 nAChR subunit knockout (KO), and wildtype (WT) mice treated chronically with systemic nicotine received intrahippocampal dihydro-beta-erythroidine (a high affinity nAChR antagonist). Intrahippocampal dihydro-beta-erythroidine precipitated learning deficits in all but the KO mice. Therefore, the action of nicotine at hippocampal beta2 nAChRs mediates adaptations in hippocampal function that underlie withdrawal deficits in contextual fear conditioning.

Olanzapine improves deficient sensory inhibition in DBA/2 mice.

Most schizophrenia patients do not inhibit their P50 auditory evoked potential to the second of duplicate auditory stimuli, reflecting a failure to inhibit responses to irrelevant sensory input. Typical antipsychotic drugs do not improve this deficit while some atypical antipsychotics do. A previous study using an animal model, deficient P20-N40 (which corresponds to the human P50) inhibitory processing in DBA/2 mice found that sensory inhibition was improved by clozapine, the prototypical atypical antipsychotic, but not by haloperidol, a typical antipsychotic. The improvement after clozapine was mediated by alpha7 nicotinic receptors. The present study addresses whether another atypical antipsychotic, olanzapine, will also improve sensory inhibition deficits in the mouse model. In vivo electrophysiological recordings of the P20-N40 auditory evoked potential in anesthetized DBA/2 mice, which spontaneously exhibit a schizophrenia-like inhibitory processing deficit, were obtained after olanzapine alone (0.01, 0.033, 0.1, 0.33 mg/kg, IP) and the efficacious dose of olanzapine (0.033 mg/kg, IP) in combination with either the alpha7 nicotinic receptor antagonist alpha-bungarotoxin or the alpha4beta2 nicotinic receptor antagonist di-hydro-beta-erythroidine. All doses of olanzapine produced improved P20-N40 inhibitory processing in DBA/2 mice. The normalization observed after the 0.033 mg/kg dose of olanzapine was due to a selective decrease in response to the second auditory stimulus indicating an increase in inhibitory processing. This improvement was blocked by pre-administration of alpha-bungarotoxin but not di-hydro-beta-erythroidine. Like clozapine, olanzapine acts via alpha7 nicotinic receptors to elicit improved inhibitory processing of auditory stimuli.

Affective and somatic aspects of spontaneous and precipitated nicotine withdrawal in C57BL/6J and BALB/cByJ mice.

The aversive aspects of nicotine withdrawal are powerful motivational forces contributing to the tobacco smoking habit. We evaluated measures of affective and somatic aspects of nicotine withdrawal in C57BL/6J and BALB/cByJ mice. Nicotine withdrawal was induced by termination of chronic nicotine delivery through osmotic minipumps or precipitated with the nicotinic acetylcholine receptor (nAChR) antagonists mecamylamine or dihydro-beta-erythroidine (DHbetaE). A rate-independent discrete-trial intracranial self-stimulation threshold procedure was used to assess brain reward function. Anxiety-like behavior and sensorimotor gating were assessed in the light-dark box and prepulse inhibition (PPI) tests, respectively. Acoustic startle response and somatic signs of withdrawal were also evaluated. Spontaneous nicotine withdrawal after 14-day exposure to 10-40 mg/kg/day nicotine induced no alterations in anxiety-like behavior, startle reactivity, PPI, or somatic signs in either strain, and no changes in thresholds in C57BL/6J mice. Extended 28-day exposure to 40 mg/kg/day nicotine induced threshold elevations, increased somatic signs, and anxiety-like behavior 24 h post-nicotine in C57BL/6J mice; thresholds returned to baseline levels by day 4 in nicotine-exposed mice. Mecamylamine or DHbetaE administration induced threshold elevations in nicotine-exposed C57BL/6J mice compared with saline-exposed mice. In conclusion, administration of relatively high nicotine doses over prolonged periods of time induces both the affective and somatic aspects of spontaneous nicotine withdrawal in the mouse, while exposure to nicotine for shorter periods of time is sufficient for nAChR antagonist-precipitated nicotine withdrawal. The current study is one of the first to demonstrate reward deficits associated with both spontaneous and nAChR antagonist-precipitated nicotine withdrawal in C57BL/6J mice.

Spontaneous nicotine withdrawal potentiates the effects of stress in rats.

Anxiety is a common symptom of nicotine withdrawal in humans, and may predict an inability to abstain from cigarette smoking. It is not clear if self-reports of anxiety during abstinence reflect increased baseline anxiety and/or increased responses to exogenous stressors. We hypothesized that nicotine withdrawal selectively exacerbates reactivity to aversive stimuli in rodents. Here, we investigated the effect of withdrawal from chronic nicotine administration (3.16 mg/kg per day base, delivered via subcutaneous osmotic minipumps) in the light-enhanced startle (LES) test in Wistar rats. In this procedure, baseline startle responding in the dark is compared to startle responding when the chamber is brightly lit. Bright illumination is aversive for rats and potentiates the startle response. Hence, this procedure allows comparisons of withdrawal effects on startle reactivity between relatively neutral and stressful contexts. We found that spontaneous nicotine withdrawal (24 h post-pump removal) did not influence baseline startle responding, but produced a selective increase in LES. Precipitated nicotine withdrawal through injections of one of two nicotinic acetylcholine receptor (nAChR) antagonists, dihydro-beta-erythroidine hydrobromide (DHbetaE: 0, 1.5, 3, or 6 mg/kg) or mecamylamine (0, 1, 2, or 4 mg/kg), did not influence baseline startle responding or LES. These results suggest that spontaneous nicotine withdrawal selectively potentiates responses to anxiogenic stimuli, but does not by itself produce a strong anxiogenic effect. These findings support the hypothesis that nicotine withdrawal exacerbates stress responding, and indicate LES may be a useful model to examine withdrawal effects on anxiety.

Nicotinic control of axon excitability regulates thalamocortical transmission.

The thalamocortical pathway, a bundle of myelinated axons that arises from thalamic relay neurons, carries sensory information to the neocortex. Because axon excitation is an obligatory step in the relay of information from the thalamus to the cortex, it represents a potential point of control. We now show that, in adult mice, the activation of nicotinic acetylcholine receptors (nAChRs) in the initial portion of the auditory thalamocortical pathway modulates thalamocortical transmission of information by regulating axon excitability. Exogenous nicotine enhanced the probability and synchrony of evoked action potential discharges along thalamocortical axons in vitro, but had little effect on synaptic release mechanisms. In vivo, the blockade of nAChRs in the thalamocortical pathway reduced sound-evoked cortical responses, especially those evoked by sounds near the acoustic threshold. These data indicate that endogenous acetylcholine activates nAChRs in the thalamocortical pathway to lower the threshold for thalamocortical transmission and to increase the magnitude of sensory-evoked cortical responses. Our results show that a neurotransmitter can modulate sensory processing by regulating conduction along myelinated thalamocortical axons.

Increase in cochlear microphonic potential after toluene administration.

Human and animal studies have shown that toluene can cause hearing loss. In the rat, the outer hair cells are first disrupted by the ototoxicant. Because of their particular sensitivity to toluene, the cochlear microphonic potential (CMP) was used for monitoring the cochlea activity of anesthetized rats exposed to both noise (band noise centered at 4 kHz) and toluene. In the present experiment, the conditions were specifically designed to study the toluene effects on CMP and not those of its metabolites. To this end, 100-microL injections of a vehicle containing different concentrations of solvent were made into the carotid artery connected to the tested cochlea. Interestingly, an injection of 116.2-mM toluene dramatically increased in the CMP amplitude (approximately 4 dB) in response to an 85-dB SPL noise. Moreover, the rise in CMP magnitude was intensity dependent at this concentration suggesting that toluene could inhibit the auditory efferent system involved in the inner-ear or/and middle-ear acoustic reflexes. Because acetylcholine is the neurotransmitter mediated by the auditory efferent bundles, injections of antagonists of cholinergic receptors (AchRs) such as atropine, 4-diphenylacetoxy-N-methylpiperidine-methiodide (mAchR antagonist) and dihydro-beta-erythroidine (nAchR antagonist) were also tested in this investigation. They all provoked rises in CMP having amplitudes as large as those obtained with toluene. The results showed for the first time in an in vivo study that toluene mimics the effects of AchR antagonists. It is likely that toluene might modify the response of protective acoustic reflexes.

Activation of orexin neurons by acute nicotine.

The hypothalamus is a prominent central site of action of nicotine but the phenotype of nicotine-sensitive neurons in this region has not been fully described. Hypothalamic orexin neurons are important regulators of state-dependent behavior, arousal and feeding. Here, we treated rats with acute nicotine and quantitated Fos expression as a marker of neuronal activation. Nicotine increased the percentage of orexin neurons expressing Fos without a significant effect on non-orexin neurons. This effect was attenuated by the nicotinic antagonists mecamylamine and dihydro-beta-erythroidine, implicating alpha4beta2-containing nicotinic receptors. The orexin system is likely to play an important role in the coordination of physiological and behavioral responses to acute nicotine treatment.

Clozapine improves deficient inhibitory auditory processing in DBA/2 mice, via a nicotinic cholinergic mechanism.

RATIONALE: Insufficient inhibitory processing of the P50 auditory evoked potential (AEP) is observed in most schizophrenia patients and is not improved by typical antipsychotic drugs, such as haloperidol. This inhibitory processing deficit is associated with a subnormal level of hippocampal alpha7 nicotinic receptors (nAChRs), and drugs that activate these receptors normalize the deficit. The atypical antipsychotic clozapine also normalizes this deficit in schizophrenia patients, but by an unknown mechanism. OBJECTIVE: Similar to schizophrenia patients, DBA/2 mice spontaneously exhibit a deficit in inhibitory processing of the P20-N40 AEP, which is a rodent analogue of the human P50 AEP. The present study determined whether clozapine improved this deficit in DBA/2 mice, and by what mechanism. METHOD: Using a conditioning-testing paradigm with paired auditory stimuli to assess inhibitory P20-N40 AEP processing in DBA/2 mice, the effects of clozapine (0.1, 1, 3.33, or 10 mg/kg, i.p.) and haloperidol (1 mg/kg, i.p.) were assessed. The effect of clozapine (1 mg/kg) was assessed alone and after pre-administration of either alpha-bungarotoxin, an alpha7 nAChR antagonist, or dihydro-beta-erythroidine, an alpha4beta2 nAChR antagonist. RESULTS: In a dose-dependent manner, clozapine improved the deficient inhibitory processing of the P20-N40 AEP normally exhibited by DBA/2 mice. Like alpha7 agonists, 1 mg/kg clozapine selectively increased the inhibition of the P20-N40 response to the second of paired auditory stimuli. The normalizing effect of 1 mg/kg clozapine was blocked by alpha-bungarotoxin, but not by dihydro-beta-erythroidine. Haloperidol did not improve DBA/2's deficient P20-N40 AEP processing. CONCLUSIONS: Clozapine improved the deficient inhibitory processing of the P20-N40 AEP in DBA/2 mice, apparently through stimulation of alpha7 nicotinic receptors. This effect was not shared by the typical antipsychotic haloperidol.

Nicotinic receptors in the rat prefrontal cortex: increase in glutamate release and facilitation of mediodorsal thalamo-cortical transmission.

The modulatory influence of nicotinic acetylcholine receptor (nAChRs) on thalamocortical transmission was characterized in the prelimbic area (PrL) of the rat prefrontal cortex. In the first experiment, rats received a unilateral excitotoxic lesion centred on the mediodorsal thalamic nucleus (MD), and were sacrificed 1 week later. The lesion resulted in a 40% reduction of 3H-nicotine autoradiographic labelling in the ipsilateral prefrontal cortex, particularly in areas that are innervated by the MD. Electrophysiological experiments were subsequently performed in non-lesioned anaesthetized animals, in order to study modulation of short- and long-latency responses of PrL neurons evoked by electrical stimulation of the MD. The short-latency responses result from activation of the MD-PrL pathway and are mediated via AMPA-type glutamatergic receptors, whereas the long-latency responses reflect activation of the recurrent collaterals of cortical pyramidal neurons, Iontophoretic application of nicotinic agonists (nicotine, DMPP) facilitated both types of response. Local application of the nAChR antagonists dihydro-beta-erythroidine, mecamylamine and methyllycaconitine, prevented both kinds of facilitation. Finally, intracerebral microdialysis experiments were performed in order to test for nicotinic modulation of extracellular glutamate concentrations in the PrL. Direct application of nicotine via the dialysis probe increased glutamate levels in a dose-dependent manner. This effect was blocked by local perfusion of dihydro-beta-erythroidine. These findings therefore provide anatomical and functional evidence for nAChR-mediated modulation of thalamocortical input to the prefrontal cortex. Such a mechanism may be relevant to the cognitive effects of nicotine and nicotinic antagonists.

Mutation causing autosomal dominant nocturnal frontal lobe epilepsy alters Ca2+ permeability, conductance, and gating of human alpha4beta2 nicotinic acetylcholine receptors.

A mutation (S247F) in the channel-lining domain (M2) of the alpha4 nicotinic acetylcholine receptor (AChR) subunit has previously been linked genetically to autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE). To better understand the functional significance of this mutation, we characterized the properties of mutant and wild-type human alpha4beta2 AChRs expressed in Xenopus oocytes. Both had similar expression levels and EC50 values for ACh and nicotine. Substantial use-dependent functional upregulation was found for mutant alpha4beta2 AChRs, but not for wild type. Mutant AChR responses showed faster desensitization, slower recovery from desensitization, less inward rectification, and virtually no Ca2+ permeability as compared with wild-type alpha4beta2 AChRs. Addition of the alpha5 subunit restored Ca2+ permeability to the mutant alpha4beta2alpha5 AChRs. At -80 mV, wild-type alpha4beta2 AChR single channel currents exhibited two conductances, each with two mean open times (gamma1 = 17 pS, tau1 = 3.7 msec, and tau2 = 23.4 msec; gamma2 = 28 pS, tau1 = 1.9 msec, and tau2 = 8.1 msec). In contrast, mutant AChRs exhibited only one conductance of 11 pS, with tau1 = 1.9 msec and tau2 = 4.1 msec. The net effect of the mutation is to reduce AChR function. This could result in the hyperexcitability characteristic of epilepsy if the mutant AChRs were part of an inhibitory circuit, e.g., presynaptically regulating the release of GABA. In the minority of AChRs containing the alpha5 subunit, the overall functionality of these AChRs could be maintained despite the mutation in the alpha4 subunit.

Chimaeric nicotinic-serotonergic receptor combines distinct ligand binding and channel specificities.

The neuronal nicotinic alpha 7 (nAChR) and 5-hydroxytryptamine (5HT3) receptors are ligand-gated ion channels with a homologous topological organization and have activation and desensitization reactions in common. Yet these homo-oligomeric receptors differ in the pharmacology of their binding sites for agonists and competitive antagonists, and in their sensitivity to Ca2+ ions. The alpha 7 channel is highly permeable to Ca2+ ions and external Ca2+ ions potentiate, in an allosteric manner, the permeability response to acetylcholine, as shown for other neuronal nAChRs. The 5HT3 channel, in contrast, is not permeable to Ca2+ ions, but blocked by them. To assign these properties to delimited domains of the primary structure, we constructed several recombinant chimaeric alpha 7-5HT3 receptors. We report here that one of the constructs expresses a functional receptor that contains the serotonergic channel still blocked by Ca2+ ions, but is activated by nicotinic ligands and potentiated by external Ca2+ ions.

Characterization of N-[3H]methylcarbamylcholine binding sites and effect of N-methylcarbamylcholine on acetylcholine release in rat brain.

The present experiments show that N-[3H]-methylcarbamylcholine ([3H]MCC) binds specifically and with high affinity to rat hippocampus, frontal cortex, and striatum. The highest maximal density of binding sites was apparent in frontal cortex and the lowest in hippocampus. [3H]MCC binding was potently inhibited by nicotinic, but not muscarinic, agonists and by the nicotinic antagonist dihydro-beta-erythroidine in all three brain regions studied. The effect of unlabeled MCC on acetylcholine (ACh) release from slices of rat brain was tested. The drug significantly enhanced spontaneous ACh release from slices of hippocampus and frontal cortex, but not from striatal slices. This effect of MCC to increase ACh release from rat hippocampus and frontal cortex was antagonized by the nicotinic antagonists dihydro-beta-erythroidine and d-tubocurarine, but not by alpha-bungarotoxin or by the muscarinic antagonist atropine. The MCC-induced increase in spontaneous ACh release from hippocampal and frontal cortical slices was not affected by tetrodotoxin. The results suggest that MCC might alter cholinergic transmission in rat brain by a direct activation of presynaptic nicotinic receptors on the cholinergic terminals. That this alteration of ACh release is apparent in hippocampus and frontal cortex, but not in striatum, suggests that there may be a regional specificity in the regulation of ACh by nicotinic receptors in rat brain.