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.

Baclofen exacerbates epileptic myoclonus in kindled rats.

Electrically kindled rats exhibit myoclonic jerks (1-25/min for 30-90 min) when injected with 200 mg/kg i.p. cysteamine. L-Baclofen, 10 mg/kg i.p. injected 45 min prior was not anticonvulsant but rather exacerbated the myoclonus. Furthermore, baclofen itself induced myoclonus in 29% of kindled rats at 10 mg/kg and in 100% of kindled rats at 15 mg/kg. These findings suggest that caution should be exercised before proposing the use of baclofen as an anticonvulsant or indeed using baclofen in patients who are predisposed to seizures.

Actions of baclofen on components of the Ca-current in rat and mouse DRG neurones in culture.

1. Ca currents in rat and mouse sensory dorsal root ganglion (DRG) neurones were inhibited by concentrations of (-)-baclofen as low as 1 micron. The proportion of neurones responding to baclofen was low (less than 20%), except in young cultures of neonate rat DRG neurones (3 days in culture), where 86% of the neurones were responsive. 2. Three types of unitary Ca currents were observed in the rat DRG neurones, corresponding to the T-, N- and L-type currents of chick DRG neurones. 3. Baclofen produced two types of response on whole-cell currents of DRG neurones from both species. The first was on an early inactivating component of the Ca current. This early current was partially inactivated at a holding potential of -40 mV. It was also reduced during the second of a pair of depolarizing command pulses. The results suggest that this action of baclofen is due to an action on an N-type component of the current. The second response to baclofen persisted throughout the command step and was not reduced during the second of a pair of command pulses, indicating that this effect is due to an action on the L-type current. 4. Unitary or ensemble Ca currents recorded in cell-attached patches, on neurones previously shown to respond to baclofen in whole-cell clamp mode, were generally not inhibited by baclofen application external to the patch electrode. This indicates that a readily diffusible internal second messenger substance is probably not involved in coupling the GABAB receptor to the ion channels.

A simple method for measuring a picogram of acetylcholine using the clam (Mya arenaria) heart.

1. A simple and reliable method is described for assaying 1 pg or less of acetylcholine (ACh) using a strip of clam (Mya arenaria) heart.2. Clam heart preparations generally are extremely selective for ACh. The preparation described is at least 10(5) times less sensitive to the following: acetyl coenzyme A, adrenaline, choline, dopamine, gamma-aminobutyric acid, glutamic acid, histamine, 5-hydroxytryptamine and noradrenaline.3. Because this preparation is one of the most sensitive known for detecting ACh, it should prove useful for assaying particularly low levels of this substance in biological extracts.

Glutamic acid mimicking of synaptic inhibition on the giant serotonin neurone of the snail.

Stimulation of the snail's tentacle nerves results in hyperpolarization and inhibition of the 'giant serotonin-containing neurone'located in the brain. Glutamic acid mimics this effect. Available data indicate that the reversal potential for the glutamic acid effect is very similar to that of the synaptic action being about 5 to 8 mV more negative than the resting potential in each case.

Modulation of GABAA receptor activity by alphaxalone.

The modulation of the gamma-aminobutyric acidA (GABAA) receptor by alphaxalone has been investigated by use of voltage-clamp recordings from enzymatically isolated bovine chromaffin cells maintained in cell culture. Alphaxalone (greater than 30 nM) reversibly and dose-dependently potentiated the amplitude of membrane currents elicited by locally applied GABA (100 microM). The potentiation was not associated with a change in the reversal potential of GABA-evoked currents and was not influenced by the benzodiazepine receptor antagonist, Ro15-1788 (300 nM). At relatively high concentrations (greater than 1 microM), alphaxalone directly elicited a membrane current. It is concluded that such currents result from GABAA receptor activation since they were reversibly suppressed by bicuculline (3 microM), dose-dependently enhanced by phenobarbitone (100-500 microM), and had a similar reversal potential (approximately 0 mV) to currents elicited by GABA. Additionally, on outside-out membrane patches, alphaxalone activated single channel currents with amplitudes and a reversal potential similar to those evoked by GABA. Alphaxalone (30 nM-1 microM) had no effect upon the amplitude of membrane currents elicited by locally applied acetylcholine (ACh) (100 microM). However, higher concentrations of alphaxalone (10-100 microM) reversibly suppressed ACh-evoked currents, the IC50 for blockade being 20 microM. The beta-hydroxy isomer of alphaxalone, betaxalone (100 nM-1 microM), did not potentiate GABA-induced currents, nor did higher concentrations of the steroid (10-100 microM) directly evoke a membrane current. However, over the latter concentration range, betaxalone suppressed the amplitude of currents elicited either by GABA or ACh. The relevance of the present results to the anaesthetic action of alphaxalone is discussed together with the broader implications of steroidal modulation of the GABAA receptor.

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.

The wide range of actions of the FMRFamide-related peptides and the biological importance of peptidergic messengers.

The importance of peptides as intercellular messengers is discussed. The view is put forward that peptides evolved early in evolution as chemical messengers and that they have come to exert a wide range of actions. Using as an example the FMRFamide (Phe-Met-Arg-Phe-NH2) related peptide family of molluscs, the wide range of peptide actions on membrane currents is discussed and considered in relation to co-localization of peptides with low molecular weight (or "classical") intercellular messengers.

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.

Biological activity and receptor binding properties of some analogues of pQDPFLRFamide.

To investigate the role of the N-terminal region of the heptapeptide FMRFamide-like peptide, pQDPFLRFamide, three analogues were synthesized. The analogues [pQNPFLRFamide, pQDAibFLRFamide (Aib = aminoisobutyric acid) and pQDGFLRFamide] contained modifications at amino acid residues 2 and 3, which we believed might be critical for maintaining the bioactive conformation of the heptapeptide. The analogues were tested for their ability to bind to receptors in membranes from Helix aspersa circumoesophageal ganglia and for their biological effects on the isolated Helix heart, the Helix tentacle retractor muscle, and extensor-tibiae neuromuscular preparation of the locust. Schistocerca gregaria. The substitution of Asn for Asp2 and that of Aib for Pro3 were conservative with respect to retention of heptapeptide-like biological activity, whereas the substitution of Gly for Pro3 significantly improved the binding affinity of the peptide for the FMRFamide receptors and conferred on the peptide some characteristic FMRFamide-like biological activity. Thus, pQDPFLRFamide bioactivity may depend on a bent conformation in solution.

Biological activity and receptor binding properties of some C-terminally modified analogues of FMRFamide.

The functional role of the C-terminal amide group (-CONH2) of the molluscan regulatory peptide FMRFamide has been examined in two sets of analogues based on FnLKFamide and FnLRFamide (nL = norleucine). In each series the amide group was replaced by -CONHCH3, -CON(CH3)2, -CONHNH2, -COOCH3, -CH2OH, and -COOH. The analogues were tested for their ability to bind to receptors in membranes from Helix aspersa circumoesophageal ganglia and for their biological effects on the isolated Helix heart. The results indicate i) that agonist activity, but not binding to the receptor, requires the presence of the amide carbonyl group; ii) the hydrogen atoms of the amide group are not essential either for binding or for agonist activity (the mono- and dimethylamides were more effective than the parent compounds on both counts); iii) the is more effective an agonist than is the amide in stimulating Helix heart.

Behavioral actions of vasoactive intestinal peptide (VIP).

The effect of vasoactive intestinal peptide (VIP) was studied on fear-motivated behaviours, exploration of a novel environment and on novelty and ACTH-induced grooming. VIP was administered via a plastic cannula into the lateral ventricle. Retention of a step-through passive avoidance task was inhibited by 10 and 30 ng VIP injected 1 hour before the retention test. Extinction of pole-jumping active avoidance behaviour was facilitated by 10 and 100 ng VIP. Mild effects were observed in an open field test on exploration and grooming activity. In conclusion, VIP produces inhibitory effects on fear-motivated behaviours.

Rapid visualization of NMDA receptors in the brain: characterization of (+)-3-[125I]-iodo-MK-801 binding to thin sections of rat brain.

We have developed and characterized a method for the rapid autoradiographic determination of receptor sites for the non-competitive NMDA receptor antagonist, MK-801, using an iodinated form of the compound, (+)-3-[125I]-iodo-MK-801. The binding site was shown to exhibit those criteria necessary for its definition as a receptor site, i.e., the binding was saturable, of high affinity, easily reversible, and stereospecific. Saturation analysis of binding to thin brain sections revealed a Bmax of 108.1 +/- 10.5 fmol/mg protein and a Kd of 383 +/- 67 pM. The pharmacology of the interaction of the ligand with the binding site yielded good correlation between the potency of various substances to complete for the binding site and their ability to act as antagonists of NMDA. Autoradiographs of thin coronal brain sections using (+)-3-[125I]-iodo-MK-801 yielded high quality images in 24-48 h with a distribution of binding sites paralleling that reported for the tritiated form of the ligand, i.e., with high densities in the hippocampus, cerebral cortex and lateral septum. Other areas with significant binding included parts of the thalamus, the amygdala and the olfactory tubercules. Furthermore, due to its high specific activity, this ligand lends itself to the study of regions not rich in MK-801 binding sites, such as the diencephalon.

An inexpensive microcomputer system for analysis of single channel currents.

A description is given of a system for rapidly measuring the durations of open times and closed times of single channel currents obtained by patch clamp techniques. The apparatus required, a BBC microcomputer and a Unilab interface, is inexpensive and easy to use. An outline of the software is given and measurements of the accuracy of timings presented. Examples of analyses of single channel currents obtained from spinal neurones in cultures are also presented.

Some neurobiological applications of the BBC Model B microcomputer and Unilab 8-bit interface.

Four computer programs written for the BBC Model B microcomputer (coupled to a Unilab 8-bit interface) are discussed. These programs enable the system to be used as (1) a transient recorder, (2) a rapid signal averager, (3) a spike-train analyser and (4) an instrument for measuring the amplitude of single channel currents. Flow-charts illustrating the operation of each program are given along with a detailed discussion of how the programs may be used in the laboratory. The discussion is illustrated using recordings taken from experiments conducted on a range of neurobiological preparations.

Effects of intrastriatal hormones on the dorsal immobility response in the rat.

Previous research has shown that the dorsal immobility response (DIR) changes significantly in duration across the estrous cycle. In order to test whether gonadal steroid hormones act directly on the striatum to modulate this behavior, ovariectomized female Long-Evans hooded rats were given bilateral intrastriatal implants of 17 beta-estradiol, 2-hydroxyestradiol, progesterone, or cholesterol. These implants were made at one-week intervals, each animal receiving each treatment in a Latin square design. Four hours after each hormone implant, the animals were tested for the DIR. Only rats receiving the intrastriatal 17 beta-estradiol implant were significantly different from those receiving cholesterol, and showed greatly potentiated DIR's.

Prevention of cysteamine-induced myoclonus blocks the long-term inhibition of kindled seizures.

In kindled rats, the administration of cysteamine (CSH, 200 mg/kg, i.p.) 4 h prior to a kindled seizure leads to long-term (up to 10 days) inhibition of kindled seizures. CSH (200 mg/kg, i.p.) also induces myoclonic seizures in kindled rats. We suggest that the long-term inhibition of kindled seizures might be the result of the myoclonus, not the somatostatin depletion as previously suggested. Prior administration of the short-acting benzodiazepine midazolam (5 mg/kg, i.p.) eliminated the CSH-induced myoclonus and prevented the long-term inhibition of kindled seizures. These results suggest that the CSH-induced long-term inhibition of kindled seizures is the result of an interaction between the myoclonic seizure and a subsequent kindled seizure.

Induction and suppression of seizures by cysteamine in hippocampal kindled rats.

Cysteamine has a biphasic effect in kindled rats. At a dose of 200 mg/kg (i.p.), hippocampal kindled rats all have myoclonic seizures during the first hour after injection, while naive rats seldom do. Four hours after cysteamine, the animals exhibited stage 5 seizures when stimulated. However, kindled seizures could not subsequently be elicited for up to 10 days in these animals.

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.

Hippocampal kindling: corticosterone modulation of induced seizures.

The effect of adrenalectomy (ADX) and corticosterone replacement was studied on seizures induced by hippocampal kindling. A complex series of changes occurred in after-discharge (AD) and behavioural depression (BD) during the immediate hours after ADX, culminating at day 1 in markedly decreased AD and BD, which returned to normal over the next several days. These changes were normalized after replacement of the ADX group with low doses of corticosterone. It is concluded that the expression and maintenance of hippocampal kindled seizures is under short-term control of corticosterone.