Location of a ligand recognition site of FMRFamide-gated Na(+) channels.

The second FMRFamide-gated Na(+) channel (HtFaNaC), from Helisoma trivolvis, has been cloned. HtFaNaC has some different pharmacological properties to HaFaNaC, from Helix aspersa, which has enabled a rational approach to be made to start to identify the FMRFamide recognition site. Several chimeras were made by switching sections between the channels. The differences in sensitivity to FMRFamide, and amiloride, were assessed after expression in Xenopus oocytes. The data suggest that a recognition site for FMRFamide, and the potentiating action of amiloride, resides in a sequence of about 120 amino acids in the extracellular loop proximal to the first transmembrane segment.

Histochemical localisation of FMRFamide-gated Na+ channels in Helisoma trivolvis and Helix aspersa neurones.

FMRFamide-gated Na+ channels of molluscan neurones belong to the ENa/Deg family of channels which have diverse functions. FMRFamide (Phe-Met-Arg-Phe-NH2) Na+ channels were detected electrophysiologically in specified neurones of Helix (Helix aspersa) and Helisoma (Helisoma trivolvis), and clones (FaNaCs) subsequently identified. We have now made a study to determine the distribution of mRNA for the clones HaFaNaC (Helix) and HtFaNaC (Helisoma) in the nervous systems of these species using standard in situ hybridization techniques. Immunohistochemical experiments were also made using an HtFaNaC antibody to detect the channel protein in Helisoma neurones. Many neurones in the central ganglia, including those which exhibit the FMRFamide Na+ current, stained for FaNaC-mRNA, suggesting a much wider distribution of the channel than was indicated by the earlier work. An immunoreactive response to the channel antibody was also observed in some Helisoma neurones, such as the giant dopamine neurone of the left pedal ganglion, also shown to possess HtFaNaC-mRNA and to exhibit the FMRFamide Na+ current. Taken together, these experiments suggest that the clones HaFaNaC and HtFaNaC are major, if not the only, subunits of the FMRFamide-gated Na+ channel detected electrophysiologically in the identified neurones of these species. However, fewer neurones in Helisoma reacted with the HtFaNaC-antibody than those which exhibited message for the channel. This discrepancy may be due to a difference in sensitivity of the two techniques, or because not all of the channel mRNA is normally expressed as a membrane protein.

Cloning and expression of a FMRFamide-gated Na(+) channel from Helisoma trivolvis and comparison with the native neuronal channel.

We have cloned a cDNA encoding a Phe-Met-Arg-Phe-NH(2) (FMRFamide)-gated Na(+) channel from nervous tissue of the pond snail Helisoma trivolvis (HtFaNaC) and expressed the channel in Xenopus oocytes. The deduced amino acid sequence of the protein expressed by HtFaNaC is 65 % identical to that of the FMRFamide-gated channel cloned from Helix aspersa (HaFaNaC). HtFaNaC expressed in oocytes was less sensitive to FMRFamide (EC(50) = 70 microM) than HaFaNaC (EC(50) = 2 microM). The two had a similar selectivity for Na+. The amplitude of the FMRFamide response of HtFaNaC was increased by reducing the extracellular concentration of divalent cations. The conductance of the two channels was similar, but the mean open time of unitary events was shorter for expressed HtFaNaC compared to expressed HaFaNaC. Each channel was susceptible to peptide block by high agonist concentrations. In marked contrast to HaFaNaC and other amiloride-sensitive Na(+) channels, amiloride, and the related drugs benzamil and 5-(N-ethyl-N-isopropyl)-amiloride (EIPA), enhanced the FMRFamide response in oocytes expressing HtFaNaC cRNA. The potentiating effects of EIPA and benzamil were greater than those of amiloride. Unitary current analysis showed that with such drugs, there was channel blockade as well as an increased probability of channel opening. The similar permeability of the oocyte-expressed HtFaNaC and the Helisoma neuronal channel, and the susceptibility of both to agonist blockade and blockade by divalent cations, suggest that the channels are the same. However, neuronal channels were less susceptible to enhancement by amiloride analogues and in some patches were more sensitive to FMRFamide than expressed HtFaNaC.

Ketamine and phenobarbital do not reduce the evoked-potential enhancement induced by electroconvulsive shock seizures in the rat.

Electroconvulsive shock (ECS) seizures provide an animal analog of electroconvulsive therapy (ECT). Repeated ECS seizures cause a long-lasting, and perhaps permanent, enhancement of entorhinal-dentate evoked potentials (EPs) in the rat. Recently it has been reported that ketamine protects against ECS-induced EP enhancement. The present study was designed to replicate these findings and to extend them by incorporating a phenobarbital group (to control for ketamine's partial diminution of seizures) and an animal test of antidepressant activity (the Porsolt test). Unexpectedly, we found that neither ketamine nor phenobarbital protected against ECS-induced enhancement of EPs. Both, however, diminished the 'therapeutic' effects of ECS, as modeled by the Porsolt test. These data suggest that the use of ketamine would not eliminate the unwanted effects of ECT and that it might diminish ECT's therapeutic benefits.

Mossy fiber sprouting induced by repeated electroconvulsive shock seizures.

The elicitation of repeated focal seizures (kindling) induces mossy fiber sprouting in the hippocampus of the rat. The present study investigated whether repeated generalized seizures also induce mossy fiber sprouting. Human psychiatric patients receive repeated generalized seizures during electroconvulsive therapy (ECT). Male Long-Evans rats received a course of eight electroconvulsive shock (ECS) seizures administered on a 48-h schedule over a course of 2 1/2 weeks. Control subjects received matched handling, but no stimulation. Fourteen days after the last ECS trial, all subjects were sacrificed and their brains subjected to Timm staining. Cell counts and area measures were also taken in the hilus. Significant sprouting, but not significant cell loss, was seen in the fascia dentata of the subjects that had received ECS.

Block of the helix FMRFamide-gated Na+ channel by FMRFamide and its analogues.

1. In Helix neurones high doses of Phe-Met-Arg-Phe-NH2 (FMRFamide) often evoke biphasic inward whole-cell currents with brief application, and suppression of the current with prolonged application. With outside-out patches, a transient early suppression of the unitary current amplitude was seen following application of high doses of FMRFamide. 2. Continuous application of a concentration of FMRFamide from 30 microM to 1 mM resulted in a reduction in the amplitude of the unitary currents and an increase in open state noise. There was also an increase in the occurrence of smaller, 'subconductance' currents with the higher concentrations of FMRFamide. Similar effects were seen with FMRFamide on FaNaC expressed in oocytes. The FMRFamide analogues FLRFamide and WnLRFamide were more effective in evoking the lower conductance state. These effects of agonist at high concentrations were voltage dependent suggesting channel block. 3. A similar effect was seen when one of the related peptides FKRFamide, FM(D)RFamide, nLRFamide or N-AcFnLRFamide was co-applied with a low FMRFamide concentration. However, the non-amidated peptides FKRF, FLRF and nLRF and also WMDFamide did not have this effect. 4. The inhibition of unitary currents induced by amiloride was qualitatively different from that produced by FMRFamide analogues with no obvious occurrence of subconductance levels. FMRFamide-gated channels were also blocked by guanidinium, but only at very high concentrations. 5. The results strongly suggest a partial inhibition of current flow through the FMRFamide- gated channel by some part of the agonist or the related antagonist peptide molecules.

Amygdala-kindled and electroconvulsive seizures alter hippocampal expression of the m1 and m3 muscarinic cholinergic receptor genes.

Expression of m1 and m3 muscarinic cholinergic receptors mRNAs was examined in rat hippocampus following either: (1) kindling to five Stage 5 amygdala-kindled seizures; or (2) eight electroconvulsive shock (ECS) seizures. Twenty-four hours after the last seizure of either type, there was a significant decrease in both m1 and m3 mRNAs in CA1, CA3 and the dentate gyrus subfields of the hippocampus. Twenty-eight days after the last seizure of either type, there was a significant increase in m1 mRNAs in CA1, CA3, and the dentate gyrus; for m3 mRNAs, there was a significant increase in CA3 28 days after the last ECS seizure, and in CA1 and CA3 28 days after the last kindled seizure. These results suggest that seizures alter the cholinergic system in the hippocampus, and that some of the alterations are very long-lasting.

Single-channel currents of a peptide-gated sodium channel expressed in Xenopus oocytes.

1. Single-channel recordings were made from outside-out membrane patches of Xenopus oocytes injected with the cDNA clone FaNaCh, which encodes a peptide-gated Na+ channel from Helix aspersa. 2. The natural peptides FMRFamide and FLRFamide only activated unitary currents in oocytes injected with FaNaCh; the EC50 values were 1.8 and 11.7 microM, respectively. 3. The slope conductance of the channel was 9.2 pS for both peptides. 4. With FMRFamide, the open probability (Po) of the channel was 0.06 at 0.3 microM and 0.76 at 30 microM, whereas for FLRFamide the open probability increased from 0.04 at 1.8 microM to 0.49 at 50 microM. The Hill coefficient was greater than 1 for both peptides. 5. High concentrations of each peptide evoked very fast flickering between open and closed states which led to decreased unitary current amplitude. 6. At low doses, brief single openings and bursts of longer openings occurred. With higher doses, the occurrence of the brief openings declined and the number of longer openings increased; the duration of the longer openings was shorter with FLRFamide than with FMRFamide. 7. For each peptide, frequency distribution histograms of open events were best fitted by the sum of two exponential components, suggesting the existence of two open states of the channel. Closed events were fitted by the sum of three components, suggesting the existence of three closed states. 8. The data were analysed according to a five-state model in which the brief openings correspond to a single liganded open form of the channel and the longer openings to a doubly liganded open form. According to this interpretation, the greater whole-cell response observed with FMRFamide than with FLRFamide results mostly from a slower closing rate constant for the longer (doubly liganded) channel openings.

The first peptide-gated ion channel.

Patch-clamp experiments on the C2 neurone of Helix aspersa have shown that the neuropeptide Phe-Met-Arg-Phe-NH2 (FMRFamide) directly gates a Na+ channel. The channel is amiloride-sensitive. Activation of this channel is responsible for the fast excitatory action of the peptide. Using primers based on amiloride-sensitive epithelial Na+ channels, a complete cDNA sequence (FaNaCh) was cloned and sequenced from a Helix library. The sequence is predicted to have just two membrane-spanning regions and a large extracellular loop. When expressed in Xenopus laevis oocytes, the channel responded to FMRFamide. Taken together, these data provide the first evidence for a peptide-gated ion channel. Comparison of the properties of the expressed FaNaCh with the native neuronal channel show small differences in the sensitivities to some drugs and in channel conductance. It is not yet clear whether the native channel is a homo-oligomer or comprises other subunits. The peptide FKRFamide is an effective antagonist of FMRFamide on the expressed and neuronal channels. Nucleotide sequences encoding similar channel proteins occur in neurones of species as dissimilar as man and Caenorhabditis elegans. Some channels are thought to be associated with mechano-sensation, at least one is a proton-gated channel and others may also be ligand-gated channels.

Long-term enhancement of entorhinal-dentate evoked potentials following 'modified' ECS in the rat.

Electroconvulsive therapy (ECT) is widely used as a treatment for drug-resistant depression. The animal analogue of ECT is electroconvulsive shock (ECS) seizures. We have recently shown that repeated ECS seizures cause a long-lasting, perhaps permanent, enhancement in entorhinal-dentate evoked potentials in the rat. Our study, however, involved 'unmodified' ECS, whereas in clinical practice ECT is now usually given in its 'modified' form (with near-threshold currents, a short-acting barbiturate, muscle relaxant and oxygen). We have therefore repeated our experiments using modified ECS. Entorhinal-dentate evoked potentials were measured in Long-Evans rats before and after: (1) eight modified ECS seizures; or (2) eight sham modified ECS trials. Despite the use of the modified procedure, a significant and long-lasting enhancement in population spike amplitude was seen in the ECS group. We conclude that the modified procedure does not protect rats against the long-lasting enhancement of evoked potentials. Similar changes may be occurring in the brains of patients subjected to modified ECT.

Modulation of ligand-gated dopamine channels in Helix neurones.

Dopamine gates a fast excitatory response in Helix C2 neurones. Whole cell, and multiple unitary dopamine-gated currents showed variable decay rates and desensitization properties, suggesting the presence of more than one channel type. Manipulation of internal free [Ca2+] by various procedures (external zero Ca2+ or 1 mM Co2+, prolonged depolarization, A23187, or flufenamic acid), affected both the amplitude and decay time for the response, and also suggested the presence of separate fast and slowly decaying components. Responses were prolonged by intracellular fluoride a non specific phosphatase inhibitor, and attenuated and shortened by the protein kinase inhibitors H7 and staurosporine, and the calmodulin inhibitor W7. Phorbol ester potentiated and prolonged the response and this effect was reversibly antagonized by the specific protein kinase C inhibitor chelerythrine. Different dopamine-activated unitary currents were distinguished in outside-out patches by conductance (5, 8, 12 and 15pS), rate of recovery from desensitization, and pattern of openings. Discrimination of slow and fast components of the response was possible with apomorphine, ADTN, and caffeine. Paradoxically the dopamine antagonists chlorpromazine and spiperone, but not dopamine itself, stimulated sustained activity of 5pS unitary currents which did not desensitize in outside-out patches. Modulation of different channels underlying the fast dopamine response by protein kinase C, and possibly other mechanisms, provides a potent means of controlling excitatory dopaminergic synaptic transmission.

Actions of FMRFamide-related peptides on the gCa2+ of the C1 neuron in Helix aspersa.

The endogenous neuropeptides FMRFamide and FLRFamide (tetrapeptides) reversibly reduced a voltage-activated calcium current in the C1 neuron of Helix aspersa by an average of 20%. Two structurally related heptapeptides, pQDPFLRFamide and pQDPFLRIamide, both derived from another precursor protein in this species, did not reduce the current at all.

The peptide pQFYRFamide is encoded on the FMRFamide precursor of the snail Helix aspersa but does not activate the FMRFamide-gated sodium current.

The complete sequence of the FMRFamide precursor cDNA from Helix aspersa is reported here. Since the 5' end of this cDNA is identical to that of the precursor that encodes the heptapeptide analogs of FLRFamide, the two transcripts are probably derived by alternative RNA splicing. A novel pentapeptide, Glp-Phe-Tyr-Arg-Phe-NH2 (pQFYRFamide), predicted from the cDNA sequence, was purified from extracts of H. aspersa ganglia and identified by mass spectroscopy. Partial gene sequences for the FMRFamide precursors of two closely related pulmonate species-Cepaea nemoralis and Polydontes acutangula-were also determined and compared with the cDNA sequence of H. aspersa and a partial gene sequence previously determined from H. pomatia. Not only are the FMRFamide-related sequences and proteolytic processing sites conserved, but the linear arrangement of these landmarks is also retained. Synthetic pQFYRFamide has some effects on the isolated heart and on neuronal potassium currents in H. aspersa that are similar to those of FMRFamide, but it does not activate the neuronal FMRFamide-gated sodium channel.

Amygdala-kindled convulsions in suspended rats.

Amygdala-kindled Long-Evans rats were suspended in a harness--with all four feet off the ground--and their convulsions were triggered, videotaped, and scored. In the suspended subjects, it was found that kindled convulsions involve the hindlimbs as well as the forelimbs and that they involve tonus as well as clonus. These data contradict the commonly held view that kindled convulsions consist only of face and forelimb clonus. They suggest that kindled convulsions resemble the generalized, whole-body convulsions observed in the maximal electroshock model and other "maximal" seizure models.

Ligand-gated ion channels opened by 5-HT in molluscan neurones.

1. 5-Hydroxytryptamine (5-HT) activated a fast (70 ms to half maximum) and desensitizing inward current through non-selective channels conducting predominantly monovalent cations in neurons of Helix aspersa. 2. alpha-Methyl-5-HT was equipotent with 5-HT in activating this current, but the known selective agonists at vertebrate 5-HT3 receptors, 2-methyl-5-HT and arylbiguanides were ineffective (< 100 microM). 5-Methoxytryptamine which is inactive on vertebrate 5-HT3 receptors was a very weak agonist. 3. The responses were antagonized by the specific vertebrate 5-HT3 receptor blocker MDL-72222 (IC50 = 1 microM), but were only weakly affected by ondansetron (10 microM). The 5-HT2-type antagonist, ketanserin (< 5 microM) had no effect. The responses were also antagonized by the non-specific antagonists (+)-tubocurarine and strychnine. 4. Unitary currents through channels non-selective for monovalent cations, and with a conductance of 2pS, could be activated repeatedly by 5-HT or alpha-methyl-5-HT in outside-out patches from neurones exhibiting the fast 5-HT-activated current (I[5-HT]fast), even in the presence of 500 microM GDP-[beta S] in the recording pipette. This strongly supports direct-gating of these channels by 5-HT. The properties of these unitary currents resembled those of I[5-HT]fast. 5. The pharmacological properties of this molluscan 5-HT-operated, ligand-gated channel differed sufficiently from known vertebrate 5-HT3-type receptors to suggest that it represents a new class of 5-HT receptor.

Dopamine directly activates a ligand-gated channel in snail neurones.

Synaptically released dopamine is known to evoke fast as well as slow synaptic potentials in neurones of gastropod molluscs. Here evidence is presented that the fast excitatory response to dopamine is mediated by the direct activation of a ligand-gated channel: unitary currents were observed in outside-out patches of neurones exposed to dopamine, and the response persisted in the presence of intracellular guanosine 5'-o-(2-thiobiphosphate), GDP[beta-S], a condition known to block G-protein-coupled responses to dopamine and other agents. In whole-cell recordings, the fast response desensitized very rapidly; it was less desensitized in outside-out patches, suggesting dependence of desensitization on an intracellular factor. The response to dopamine was blocked by D-tubocurarine and strychnine (both probably by channel blockade), by apomorphine, chlorpromazine and relatively high doses of (+/-)-sulpiride and spiperone. The channel conducts predominantly monovalent cations. Unexpectedly, the fast response to dopamine was also observed in an identified dopaminergic neurone when maintained in isolation in culture. The receptors on the dopaminergic neurone were unevenly distributed, being more abundant on the axon-hillock, axon and neurite terminals.

Long-term changes in entorhinal-dentate evoked potentials induced by electroconvulsive shock seizures in rats.

Entorhinal-dentate evoked potentials were measured in rats before and after: (1) eight electroconvulsive shock (ECS) seizures, or (2) matched handling. In animals that received ECS, evoked potentials were significantly enhanced, as evidenced by a long-lasting increase in the amplitude of the population spike. This increase in population-spike amplitude lasted for at least 3 months after the last ECS trial. No evoked-potential changes were observed in the subjects that received matched handling. These data suggest that ECS seizures produce long-lasting, perhaps permanent, changes in the brain.

Properties of an identified dopamine-containing neurone in culture from the snail Helisoma.

The giant neurone in the left pedal ganglion of the snail Helisoma trivolvis is homologous with the giant dopaminergic neurone of Planorbis corneus, because the neurones have a very similar location and morphology, and react similarly with glyoxylic acid to produce an intense blue fluorescence, indicating the presence of dopamine. Each of these neurones is therefore referred to as a giant dopaminergic neurone, or GDN. Conditions for the extension of neurites and formation of chemical junctions in culture have been determined for the H. trivolvis GDN, and compared with other neurones from this species. The pattern of neurites that extended from the neurone was indistinguishable from that of another identified aminergic neurone, the large serotonergic neurone (LSN), but differed markedly from many other central neurones. However, the type of substrate also greatly affected the pattern of the neurites observed. Some of the electrical properties of the GDN in culture differed from those recorded in situ: peak spike amplitude was increased, spike half-width reduced and the firing pattern of the neurone was altered. However, the resting membrane potential was very similar. The GDN formed chemical and electrical junctions in culture. The chemical junctions formed were of the same type as those found in situ. They formed rapidly, within 18 h after plating, but were not stable and were lost within 48 h, to be replaced by a non-rectifying electrical junction. A chemical junction may form in either direction between the GDN and the LSN, but only rarely did such junctions allow transmission in both directions, as observed in situ. Experiments in which neurones were plated out at different times suggested that the direction of formation of the chemical junction was not dependent on the degree or state of neurite extension.

N-terminally extended FMRFamide-related peptides of Helix aspersa: processing of the precursor protein and distribution of the released peptides.

Sequencing of cDNA clones reveals a precursor protein that can be processed into 10 different hepta-FaRPs. Two of the peptides are previously undescribed and are N-terminally extended forms of-YMRFamide, making them the only methionine-containing peptides in the precursor. They are separated from the main cluster of hepta-FaRPs by a recognition site (RQKR) for the Golgi-resident proteolytic enzyme furin. Antisera raised against the synthetic peptide KQDPFLRFGK specifically stain the clusters of neurons in the parietal ganglia that have been shown to contain hepta-FaRP mRNA. These antisera recognize two major protein bands of 35 and 23 kDa on immunoblots. Evidence is presented to identify the larger band as the precursor protein and the smaller band as the fragment containing the main cluster of hepta-FaRPs produced after furin cleavage. A series of immunostaining bands of 22-13 kDa suggests sequential and non-preferential N- or C-terminal cleavage at the mainly monobasic (K and R) sites that link all of the peptide sequences throughout the 23-kDa fragment, to yield the preamidated hepta-FaRPs. Immunostaining of sections shows punctate staining in the perikarya of the parietal cluster neurons commensurate with label within the endoplasmic reticulum and Golgi apparatus. Staining is followed through the axons to many fibers in the nerve trunks and is picked up as fine processes within the skin. These observations indicate that the antiserum used here recognizes one or more of the processed hepta-FaRPs, a view confirmed by radioimmunoassay. The abundance of immunoreactive fibers within the skin suggests a major role for the peptides in this tissue.

The neuropeptide Phe-Met-Arg-Phe-NH2 (FMRFamide) directly gates two ion channels in an identified Helix neurone.

FMRFamide (i.e. Phe-Met-Arg-Phe-NH2) application to the C2 neurone of Helix caused a depolarizing response which consisted of a large, rapidly developing, and rapidly desensitizing inward current, underlain by a smaller, slower inward current which did not desensitize. Both currents were carried through sodium-selective channels which were insensitive to D-tubocurarine, and the to the fast sodium channel blockers tetrodotoxin (TTX) and lignocaine. Only the faster, desensitizing current could be blocked by amiloride. FMRFamide also activated two types of unitary inward currents with slightly differing amplitudes in outside-out patches taken from the C2 neurone, both through sodium-selective ion channels. Only the smaller unitary currents readily desensitized and were susceptible to block by amiloride, and they also activated more rapidly. Unitary currents of both types were recorded in outside-out patches in the absence of freely diffusible intracellular mediators, and were also activated when guanosine 5'-O-(2-thiodiphosphate) (GDP [beta-S]) was included in the recording pipette solution. This supports a tight receptor/channel coupling for both responses, with no involvement of GTP-binding proteins. Further, the very fast rate of activation of the smaller channels, which generally carry the major part of the FMRFamide-induced current, strongly indicates that these channels are ligand gated.