Actions of phencyclidine and its thienylpyrrolidine analogue on synaptic transmission and axonal conduction in the central nervous system of the cockroach Periplaneta americana

The effects of phencyclidine (PCP) and its thienylpyrrolidine analogue (TCPY) were tested on conduction processes in the isolated axon of giant interneurone 2 (GI 2) of the cockroach Periplaneta americana and on binding of [³H]PCP and [¹²⁵I]α-bungarotoxin to membranes from Periplaneta brain and nerve cord. Their actions on synaptic transmission between cercal sensory neurones and GI 2, where acetylcholine is the likely neurotransmitter, were also examined. PCP suppressed both sodium and potassium currents in the axonal membrane at 5.0 × 10^?4 M. Block was reversible on rebathing the axon in normal saline. TCPY exerted similar effects on the axon, though at slightly higher concentrations. Excitatory postsynaptic potentials (EPSPs) recorded from GI 2 in response to electrical stimulation of cercal nerve XI were progressively blocked by 5.0 × 10^?4 M PCP following a brief initial enhancement (?10%) of EPSP amplitude. The depolarizing response of GI 2 to ionophoretically applied acetylcholine was also blocked at this concentration, indicating a postsynaptic action of PCP at the acetylcholine receptor-ion channel of GI 2. TCPY also blocked synaptic transmission at synapses between cercal afferents and GI 2, but, in contrast to the actions of PCP, EPSP block was accompanied by depolarization. PCP and TCPY inhibited [³H]PCP binding to nerve cord and brain membranes with multiple affinities, suggesting multiple molecular targets. They also modified aspects of the kinetics of [¹²⁵I]α-bungarotoxin binding to the nicotinic acetylcholine receptor in these membranes and enhanced conversion of the receptor to the high affinity desensitized state. At higher concentrations they also inhibited [¹²⁵I]α-bungarotoxin binding. PCP was more potent than TCPY in inhibiting [³H]PCP binding but less potent on [¹²⁵I]α-bungarotoxin binding. Thus PCP and TCPY, which are structurally very similar, interact with several molecular targets in insect neuronal membranes, including sodium and potassium channels and acetylcholine receptors.     

Differential accessibility to two insect neurones doe not account for differences in sensitivity to α-bungarotoxin

The nicotinic acetylcholine receptor probe α-bungarotoxin (1.0 × 10^?7 M) blocks the depolarising response to ionophoretic application of acetylcholine onto the cell body membrane of the fast coxal depressor motoneurone (D_f) of desheathed cockroach (Periplaneta americana) metathoracic ganglia, but at the same concentration is completely ineffective in blocking the depolarising action of acetylcholine on dorsal unpaired median (DUM) neurones in the same ganglion. The possibility that this is due to differences in accessibility of the toxin to the neurones has been tested by a combination of ionophoretic injection of horseradish peroxidase into single neurones with a study of the distribution of the exogenous tracer lanthanum, which is of similar effective size to α-bungarotoxin. The peripherally located cell body membranes and the fine axonal processes of D_f and DUM neurones of desheathed metathoracic ganglia are equally accessible to lanthanum. Differential accessibility to the two cell types does not account therefore for the differences in sensitivity to α-bungarotoxin.     

Actions of synthetic piperidine derivatives on an insect acetylcholine receptor/ion channel complex

The actions of synthetic piperidine derivatives on the response to ionophoretically-applied acetylcholine (ACh) have been tested on the cell body membrane of the fast coxal depressor motoneurone (F_f) of the cockroach Periplaneta americana. The cis form and the cis (80%):trans (20%) mixture of 2-methyl-6-undecyl piperidine were the most effective (the half-maximal blocking action of the mixed isomers was estimated to be 6.3 × 10^?5 M). Less potent was the cis (50%):trans (50%) mixture of 2-methyl-6-tridecyl piperidine. However, pure cis 2-methyl-6-tridecyl piperidine was even less effective than the mixed isomers, indicating that, in the case of the tridecyl derivative, the trans form was largely responsible for the block of the ACh response.Cis 2-Methyl-6-undecyl piperidine failed to inhibit the binding of N-[propionyl-³H] propionylated α-bungarotoxin to metathoracic ganglion homogenates at concentrations up to 1.0 × 10^?4 M. Also, block of ACh-induced current by 2-methyl-6-undecyl piperidine (cis 80%:trans 20%) was largely independent of membrane potential in the range ?120 mV to ?60 mV, indicating an interaction with the closed ACh receptor/ion channel complex at a site which, in the case of the cis isomer, is separate from the binding site for α-bungarotoxin.     

Ionophoretic application of acetylcholine on to the dendrites of an identified giant interneurone (GI 1) in the cockroach Periplaneta americana

Acetylcholine was ionophoretically applied within the neuropile of the sixth abdominal ganglion of the cockroach Periplaneta americana and depolarizing responses were recorded from an identified giant interneurone (GI 1) using the oil-gap, single-fibre technique. Estimated peak concentrations in the range 1.0 × 10^?11M ? 1.0 × 10^?8 M acetylcholine were present at the receptors, when this putative neurotransmitter was ionophoretically-applied, at threshold doses, to regions of the neuropile containing dendritic branches of GI 1. Using a stepping micro-drive unit for positioning the ionophoretic micropipette, it was shown that profiles of acetylcholine sensitivity correlated with the distribution of dendritic branches of GI 1 revealed by cobalt staining of the cell under test. It is concluded that acetylcholine receptors are widely distributed throughout the dendritic branches of GI 1 in the sixth abdominal ganglion. The results demonstrate the possibility of investigating the effects of locally-applied putative transmitter molecules and pharmacological agents on synaptic membranes of an identified neurone in the central nervous system of an insect.     

GABA and taurine sensitivity of cockroach giant interneurone synapses: Indirect evidence for the existence of a GABA uptake mechanism

The effects of exogenous GABA and taurine were studied on the cercal afferent-giant interneurone synapses (G.I. 2) located in the neuropile of the sixth abdominal ganglion of the cockroach, Periplaneta americana L. The decrease in excitatory synaptic potentials and the increase in postsynaptic membrane conductance due to GABA were enhanced by lowering the temperature of the saline, by using Na⁺ pump inhibitors, Na⁺ free salines or by agents blocking GABA uptake. The action of temperature was studied for taurine. Implications of these results for the identification of a metabolically dependent GABA uptake mechanism into glial cells are discussed.     

The effects of amantadine on liposomally reconstituted nicotinic acetylcholine receptor

The effects of amantadine on liposomally reconstituted nicotinic acetylcholine receptor function were studied. At 1 × 10^?4M, the drug blocked 85% of the carbamylcholine-induced cation influx into liposomes, but left 90% of the αbungarotoxin binding intact. In addition, amantadine was shown to be a non-competitive inhibitor of membrane bound acetylcholinesterase. These experiments are relevant to the mechanism of action of amantadine at the motor end plate, where it produces electrophysiological changes compatible with an inhibition of cholinergic agonist mediated ion flux.     

Effect of acetylcholine on membrane potential of the horizontal cells of the goldfish retina and the electroretinogram of the frog

The action of acetylcholine on the horizontal cells of the goldfish retina and the electro-retinogram of the frog was studied. Acetylcholine in concentrations of 1·10⁻⁹–1·10⁻³ M depolarized these cells. The maximal level of depolarization never reached zero level of the membrane potential and was about equal to the membrane potential in darkness. In a concentration of 1·10⁻²–5·10⁻² M acetylcholine suppressed the b- and d-waves of the frog electro-retinogram, and as a result the stable P_III component was isolated from the ERG. A mediator role is ascribed to acetylcholine in the synapses of the outer plexiform layer.     

THE BINDING INTERACTION BETWEEN α-LATROTOXIN FROM BLACK WIDOW SPIDER VENOM AND A DOG CEREBRAL CORTEX SYNAPTOSOMAL MEMBRANE PREPARATION

—The major toxin of black widow spider venom, α-latrotoxin, can be iodinated with ¹²⁵I with hardly any loss in biological activity. The radioactive toxin could bind specifically to a dog cerebral cortex synaptosomal membrane preparation but not to a dog liver plasma membrane preparation. The bound protein could be recovered from the neuronal membrane preparation in an unchanged form. Non-specific binding was only 6–10% of the total binding. The protein nature of the presumed receptor was indicated by the complete inhibition of the binding by either heating the membrane preparation at 70°C or treating the membrane with trypsin. Pre-incubation with 2%β-mercaptoethanol also completely inhibited the binding, while 70% inhibition was observed after pre-treatment with 10m M-EDTA or EGTA. From plots of the equilibrium binding data, it could be ascertained that the binding is non-cooperative, with an apparent equilibrium dissociation constant, K₁, of 1.0 nM. Kinetic data gave an apparent association rate constant of 8.2 × 10⁵ M^?1 s^?1. Dissociation followed a biphasic exponential with rate constants of 1.4 × 10^?3 and 5.2 × 10^?5s^?1 corresponding to half-lives of 8.2 min and 3.7 h. Possible schemes for the binding interaction were proposed. Based on the present results and on previous results which indicated that α-latrotoxin causes the release of all neurotransmitters and a depletion of the synaptic vesicle population in vertebrate synapses, a hypothetical mechanism of the action for the toxin was proposed, involving the binding of the toxin to a membrane protein receptor which interacts with filamentous proteins linking the synaptic vesicles to the axolemma.     

Presynaptic Control of the Synthesis and Release of Dopamine from Striatal Synaptosomes: A Comparison Between the Effects of 5-Hydroxytryptamine, Acetylcholine, and Glutamate

The effects of 5-HT and glutamate on dopamine synthesis and release by striatal synaptosomes were investigated and compared with the action of acetylcholine, which acts presynaptically on this system. 5-HT inhibited (28%) synthesis of [¹⁴C]dopamine from L-[U-¹⁴C]tyrosine, at 10-⁵M and above. This contrasts with the action of acetylcholine, which stimulated [¹⁴C]-dopamine synthesis by 24% at 10-⁴ M. Tissue levels of GABA were unaffected by either 5-HT or acetylcholine up to concentrations of 10-⁴ M. The inhibitory action of 5-HT (5 × 10^?5 M and 2 × 10^?4 M) on [¹⁹C]dopamine synthesis was completely abolished by methysergide (2 × 10^?6 M). Higher concentrations of methysergide (10^?4 M) or cyproheptadine (10^?5 M) inhibited [¹⁴C]dopamine synthesis by 28% and 25%, respectively, when added alone to synaptosomes. However, only methysergide prevented the further inhibition of synthesis caused by 5-HT. At concentrations of 2 × 10^?5 M and above, 5-HT stimulated [¹⁴C]dopamine release. This releasing action differed from that of acetylcholine, which occurred at lower concentrations (e.g., 10^?6 M). Methysergide (up to 10^?4 M) or cyproheptadine (2 × 10^?4 M) did not reduce the 5-HT (5 × 10^?5 M)-induced release of [¹⁴C]dopamine, but methysergide (10^?4 M) showed a potentiation (49%) of this increased release. The stimulatory effects of 5-HT (2 × 10^?5 M) and K⁺ (56 mM) on [¹⁴C]dopamine release were additive, indicating that two separate mechanisms were involved. However, when both agents were present the stimulatory effect of K⁺ (56 mM) on [¹⁴C]dopamine synthesis was not seen above the inhibitory effect of 5-HT. Glutamate (0.1-5 mM) did not affect [⁴C]dopamine release or its synthesis from L-[U-¹⁴C]tyrosine. It is concluded that 5-HT modulates the synthesis of dopamine in striatal nerve terminals through a presynaptic receptor mechanism, an action antagonised by methysergide. The releasing action of 5-HT apparently occurs through a separate mechanism which is also distinct from that involved in the response to K⁺ depolarisation.     

IgE-mediated 14C-serotonin release from rat mast cells modulated by morphine and endorphins

The formaldehyde method was used to examine the interaction of PGE₁ with morphine, β-endorphin and Met-enkephalin on rat mast cells by their effects on IgE-mediated ¹⁴C-serotonin release. PGE₁ (2×10^?8?2×10^?5 M) caused a dose-related inhibition of the mediator release 1 min after an antigen challenge, and morphine (3×10^?7?3×10^?5 M) reversed this PGE₁ effect dose-dependently and stereospecifically; naloxone (2×10^?4 M) antagonized this action of morphine. β-Endorphin (3×10^?7?10^?5 M) and Met-enkephalin (3×10^?6?10^?4 M) mimicked this morphine action dose-dependently and were antagonized by naloxone (2×10^?4 M). These results suggest that morphine and endorphins modulate immunological mediator release from rat mast cells through opioid receptors.     

Studies on the effects of insulin and acetylcholine on activation of glycogen synthase and on glycogenesis in hepatocytes

The addition of insulin (4.0 × 10^?11 M) or acetylcholine (10^?6 M) to isolated hepatocytes stimulated glycogen accumulation and this stimulation was more pronounced when the medium glucose was raised from 50 to 300 mg percent. Studies with [¹⁴C]-glucose showed a two-fold stimulation in glycogen synthesis by the addition of insulin (4.0 × 10^?11 M) or acetylcholine (10^?6 M). A sixteen percent increase in the activity of glycogen synthase was observed in cells incubated for 10 minutes with insulin (4.0 × 10^?11 M) or acetylcholine (10^?6 M), whereas at one hour incubation a 40 percent increase in activity was observed with the same concentration of insulin or acetylcholine. The effects of insulin and acetylcholine were not additive.     

Dendritic calcium accumulation regulates wind sensitivity via short‐term depression at cercal sensory‐to‐giant interneuron synapses in the cricket

An in vivo Ca²⁺ imaging technique was applied to examine the cellular mechanisms for attenuation of wind sensitivity in the identified primary sensory interneurons in the cricket cercal system. Simultaneous measurement of the cytosolic Ca²⁺ concentration ([Ca²⁺]_i) and membrane potential of a wind‐sensitive giant interneuron (GI) revealed that successive air puffs caused the Ca²⁺ accumulation in dendrites and diminished the wind‐evoked bursting response in the GI. After tetanic stimulation of the presynaptic cercal sensory nerves induced a larger Ca²⁺ accumulation in the GI, the wind‐evoked bursting response was reversibly decreased in its spike number. When hyperpolarizing current injection suppressed the [Ca²⁺]_i elevation during tetanic stimulation, the wind‐evoked EPSPs were not changed. Moreover, after suprathreshold tetanic stimulation to one side of the cercal nerve resulted in Ca²⁺ accumulation in the GI's dendrites, the slope of EPSP evoked by presynaptic stimulation of the other side of the cercal nerve was also attenuated for a few minutes after the [Ca²⁺]_i had returned to the prestimulation level. This short‐term depression at synapses between the cercal sensory neurons and the GI (cercal‐to‐giant synapses) was also induced by a depolarizing current injection, which increased the [Ca²⁺]_i, and buffering of the Ca²⁺ rise with a high concentration of a Ca²⁺ chelator blocked the induction of short‐term depression. These results indicate that the postsynaptic Ca²⁺ accumulation causes short‐term synaptic depression at the cercal‐to‐giant synapses. The dendritic excitability of the GI may contribute to postsynaptic regulation of the wind‐sensitivity via Ca²⁺‐dependent depression. © 2001 John Wiley & Sons, Inc. J Neurobiol 46: 301–313, 2001     

Studies on action mechanisms of a possible false cholinergic transmitter, (2-hydroxyethyl) methyldiethylammonium

(2-Hydroxyethyl) methyldiethylammonium (DEC; Diethylcholine) was found to inhibit cholinergic fibers slowly, both in skeletal muscle (ED₅₀: 2.25 × 10^?5 M in chick biventer cervicis and 42 mg/kg in rat sciatic-gastrocnemius) and in smooth muscle preparations (ED₅₀: 7.7 × 10^?4 M in transmurally stimulated guinea-pig ileum) without having any effect on dose-response curves of acetylcholine to contract chick biventer cervicis, frog rectus abdominis and guinea-pig ileum. These results indicate that DEC acts at the prejunctional nerve fibers, but not at the postjunctional cholinergic receptor sites. DEC was acetylated efficiently both by choline acetyltransferase and by minced rat brain, suggesting that it can be acetylated to acetyl-DEC in the nerve ending. Acetyl-DEC was found to block acetylcholine actions competitively both in smooth and in skeletal muscle preparations (1 × 10^?3 ? 1 × 10^?2M) indicating that the acetylated product of DEC can serve as an antagonist at the cholinergic receptor site. It is therefore concluded that DEC is a false cholinergic transmitter.     

Activation and inhibition of the action potential Na+ ionophore of cultured rat muscle cells by neurotoxins.

Both myoblasts and myotubes in cultures of clonal rat muscle cells have action potential Na⁺ ionophore activity. The ionophore is activated by batrachotoxin (K_0.5 = 3 to 5 × 10^?7 M) and veratridine (K_0.5 = 4 to 6 × 10^?5 M) which compete for the same activation site. As in denervated rat muscle, the ionophore of cultured muscle is 100 fold more resistant to inhibition by tetrodotoxin (K_0.5 = 1.5 to 3 × 10^?6 M) and 20 fold more resistant to inhibition by saxitoxin (K_0.5 = 1.5 to 3 × 10^?7 M) than in nerve, innervated muscle, or cultured neuroblastoma cells.     

Selective depression of dopamine-induced depolarization by morphine and enkephalins in snail neurons

Morphine, met-enkephalin, and leu-enkephalin in a concentration of 1×10^?5 M depress rapidly and reversibly the amplitude of depolarization induced by dopamine application toHelix pomatia neurons; the effect is naloxone-dependent. The amplitudes of dopamine-induced hyperpolarization and also of the depolarization and hyperpolarization responses to acetylcholine application are unchanged under these circumstances. The hypothesis of blocking of chemosensitive sodium channels by enkephalins is discussed. It is suggested that this hypothesis is true for high concentrations of morphine and enkephalins (1×10^?4 to 1×10^?3 M). In lower concentrations (1×10^?5 M) morphine and enkephalins lead to modulation of the reponses to the action of neurotransmitters, evidently through their influence on the cyclic nucleotide system.     

Effect of chlordimeform on nerve-muscle system of the larva of the waxmoth, Galleria mellonella

The action of chlordimeform on the nerve-muscle preparation of the larvae of the waxmoth Galleria mellonella has been studied by means of microelectrodes. Exitatory junction potential evoked by nerve stimulation is reversibly suppressed by 2 × 10^?3 M chlordimeform, and spike-like component is abolished. The resting membrane potential of the muscle fibre and the action potential from the nerve terminal are not affected at 5 × 10^?3 M chlordimeform. The depolarizing membrane response caused by outward current and the effective membrane resistance are not appreciably affected. It appears that chlordimeform exerts its blocking action on the neuromuscular junction rather than the conductance mechanism of muscle fibre membrane.     

Membrane transport of L-triiodthyronine by human red cell ghosts.

L-T₃ transport has been investigated in human red cell ghosts. Determination of initial T₃ uptake revealed two separate saturable uptake systems, one with a K_m of 1.6 × 10^?8M, the other with a K_m of 3.3 × 10^?6M. Binding experiments resulted in two dissociation constants, 1.4 × 10^?7M.and 2.6 × 10^?6M. Uptake was dependent on the ghost volume, indicating an intravesicular location of T₃. The T₃ was concentrated 6 times by the ghosts. Ouabain reduced the uptake by the low K_m system, but was without effect on the high K_m system. Thus evidence is provided both of binding of T₃ to the ghost membrane and of its uphill transport across the membrane.     

A serotonin sensitive guanylate cyclase associated with specific neurotransmitter binding sites on isolated synaptic membranes from mature rat brain.

Results from this study indicate that adult rat brain posesses guanylate cyclase activity sensitive to serotonin (5-HT) and localized in the synaptic plasma membrane. The enzyme appears to have multiple activation sites for 5-HT with specific activity maxima at the 5-HT concentrations of 5 × 10^?10M and 7 × 10^?8M respectively. The rates of guanosine-3′:5′-monophosphate (cyclic GMP) formation at these concentrations of 5-HT are, respectively, 170% and 307% above the endogenous or basal production rate of 2.7±0.3picomoles/minute/milligram of synaptosomal membrane protein. We have also been able to identify $\text{four}$ distinct types (Type #1, #2, #3, and #4) of high affinity, specific binding sites for 5-HT on isolated synaptosomal membranes from rat brain. Dissociation constants of 2.6 × 10^?10M, 2.5 × 10^?9M, 7.0 × 10^?9M, and 4.6 × 10^?8M, characterize the binding of 5-HT to our sites of Type #1 through Type #4 respectively. The specific, high affinity binding was saturated at 5-HT concentrations of 5 × 10^?10M for the Type #1 sites, 5 × 10^?9M for our Type #2 sites, 1 × 10^?8M for our Type #3 sites, and 7 × 10^?8M for our Type #4 sites. The 5-HT concentrations producing saturation of our specific binding sites of Type #1 and Type #4 are virtually identical to those that elicit the two maxima of 5-HT stimulated cyclic GMP production, indicating that a membrane-bound guanylase cyclase may be closely associated with certain 5-HT receptors and/or re-uptake sites.     

Spectrophotometric determination of reaction stoichiometry and equilibrium constants of metallochromic indicators: III. Antipyrylazo III complexing with Ca2+ and acetylcholine receptor protein

Stoichiometries, equilibrium constants and optical extinction coefficients of calcium-antipyrylazo III (An) complexing are determined with the analytical method described in article I of this series. Spcctrophotometric Ca titrations of An at the wavelengths 595 and 710 nm indicate overall dissociation equilibrium constants for the complexes CaAn, CaAn₂ and Ca₂An to be 4.5 × 10^?4 M, 1.1 × 10^?8 M² and 1.5× 10^?6 M², respectively, extrapolated to zero ionic strength. Ca titrations of solutions containing An plus acetylcholine receptor protein give clear evidence that An binds to the protein to a large extent in the presence of Ca²⁺; furthermore, addition of acetylcholine results in release of protein-bound Ca and An. This is the first reported indication that antipyrylazo III binds to biological material and questions the usefulness of this dye as a Ca indicator in biological systems.     

Acetylcholine potentiation of field stimulated rat vas deferens: A pre-synaptic muscarinic mechanism?

Acetylcholine (ACh) was found to markedly enhance the nerve stimulation induced twitch response of isolated, field-stimulated rat vas deferens (RVD). The ED₂₀₀ (concentration which enhances the twitch response to 200% of control) for this potentiation was 6 × 10^?6M with the maximum twitch response being increased by more than 3 fold (325 ± 30%). Carbachol (ED₂₀₀ = 8.5 × 10^?7M) showed identical results. With each drug the potentiation was competitively antagonized by atropine (10^?7?10^?5M). Physostigmine 10^?8?10^?6M) both enhanced the basal twitch response (215 ± 8% of control at 10^?5M) and the sensitivity of the RVD to ACh (ED₂₀₀ = 3.3 × 10^?7M) but not to carbachol. Atropine, on the other hand reduced the basal twitch response by 18 ± 3% at 10^?5M. Hemicholinium (10^?4M) also reduced the basal twitch responses by 23 ± 5%. ACh (10^?7M?10^?5M) did not modify the responses of unstimulated RVD to norepinephrine or KCl suggesting a pre-synaptic site of action. Taken together these results are compatible with the presence of a pre-junctional, excitatory muscarinic mechanism in the field stimulated RVD. That this cholinergic system may be of physiological significance is supported by the observations that atropine and hemicholinium depress while physostigmine enhances the twitch response in the absence of exogenous ACh.