Cholinergic activation of the lobster cardiac ganglion

The frequency of rhythmic burst activity of the isolated lobster cardiac ganglion is increased by exogenously applied acetylcholine and muscarinic agonists. Responses of individual motor neurons isolated from the ganglion by transection consist of a slow depolarization and repetitive bursting. The pharmacological profile of the receptors mediating this response is similar to that of vertebrate neuronal muscarinic receptors. Isolated ganglia incubated in the presence of [3H]-choline (18-19 h) exhibited radiolabelled acetylcholine accumulation. It is suggested that ganglionic excitation may be accomplished by extrinsic or intrinsic activation of muscarinic receptors on the motor neurons.     

Role of non-quantum acetylcholine secretion in neural control of the membrane potential in skeletal muscle fibers of rats

Experiments on muscle fibers of the rat diaphragm (in vitro denervation) showed that their three-hour incubation in the cultural medium results in an 8-mV drop in the resting membrane potential (RMP). Addition of 5·10^–8 M carbacholine to the cultural medium, mimicing the effect of non-quantum acetylcholine, delayed depolarization of the denervated muscle. The effect of carbacholine could not be eliminated byd-tubocurarine (5·10^–6 M), a postsynaptic acetylcholine receptor blocker, and by ouabain (1·10^–4 M), and inhibitor of Na⁺, K⁺-ATPase of the membrane. At the same time, the effect could be completely eliminated by Mg²⁺ ions (5·10^–3 M), which blocked Ca²⁺ channels of the membrane, by N-nitroarginine (1·10^–4 M), which inhibited the enzyme NO-synthase, and by hemoglobin (2·10^–5 M), which inactivated the extracellular NO molecules. It is concluded that the released non-quantum acetylcholine can contribute to neural control of RMP of cross-striated muscle fibers via the Ca²⁺-dependent activation of NO synthesis in the sarcoplasm. The NO molecules can play the role of a retrograde signal indicative of the normal functioning of the neuromuscular synapse. The impairment of this link caused by a denervation-induced cessation of the non-quantum secretion can serve as a signal triggering the early changes in the muscle membrane following nerve transection.Neirofiziologiya/Neurophysiology, Vol. 27, No. 1, pp. 67–71, January–February, 1995.     

Depolarizing effects of dopamine on the primary afferent fibers of a segment isolated from the spinal cord of newborn rats

Effects of dopamine on dorsal root potentials were investigated during experiments on a segment of spinal cord isolated from 12- to 18-day-old rats. Applying dopamine to the brain was found to produce a slow, reversible, dose-dependent depolarization at primary afferent fiber terminals. This dopamine-induced depolarization was retained during complete blockade of synaptic transmission brought about by exchanging calcium ions in the perfusing fluid by magnesium or manganese ions. Minimum dopamine concentration required to produce this effect was 1·10^–10–1·10^–9 M. Peak amplitude of depolarization equaled 1.5 mV. Duration of this reaction ranged from 5.5 to 36.7 min, depending on the duration and concentration of dopamine application. Depolarizing response to dopamine differed considerably from GABA-induced dorsal root depolarization in amplitude and rate of rise. Haloperidol, a dopamine antagonist, reduced dopamine-induced dorsal root depolarization. Findings indicate that dopamine acts directly on the membrane of primary afferent fiber terminals, shifting membrane potential toward depolarization. This raises the possibility that dopaminergic brainstem-spinal pathways may exert an effect on sensory information transmission in segmental reflex arcs already traveling to the spinal cord.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 19, No. 6, pp. 741–748, November–December, 1987.     

Pharmacology of the neuronal nicotinic acetylcholine receptor of cultured Kenyon cells of the honeybee, <Emphasis Type="Italic">Apis mellifera</Emphasis>

We investigated the pharmacology of the nicotinic acetylcholine receptor of honeybee Kenyon cells, a subset of olfactory interneurons, which are crucial for olfactory learning and memory. Whole-cell currents were recorded using patch-clamp techniques. Pressure application of agonists induced inward currents in cultured Kenyon cells at holding potentials of –110 mV. Acetylcholine or carbamylcholine were full agonists, nicotine, epibatidine and cytisine were only partial agonists. Coapplications of these partial agonists with acetylcholine reduced the current amplitude. The most efficient antagonists were dihydroxy--erythroidine (EC₅₀=0.5 pmol·l^–1) and methyllycaconitine (EC₅₀=24 pmol·l^–1). The open channel blocker mecamylamine, d-tubocurarine and hexamethonium were rather weak blockers of the honeybee nicotinic response. Bath applications of the muscarinic antagonist atropine inhibited nicotinic currents dependent on concentration (EC₅₀=24.3 mol·l^–1). Muscarine, pilocarpine or oxotremorine (1 mmol·l^–1) did not induce any measurable currents. The non-cholinergic drugs strychnine, bicuculline and picrotoxin partially and reversibly blocked the acetylcholine-induced currents. Our results indicate the expression of only one nicotinic acetylcholine receptor subtype in cultured Kenyon cells. Muscarinic as well as non-cholinergic antagonists also inhibit the receptor function, distinguishing the honeybee nicotinic receptor from the typical nicotinic receptor of vertebrates and from many described insects receptors.     

Depolarizing effects of dopamine on motoneurons from a segment of spinal cord isolated from newborn rats

The effects on dorsal root potentials of applying dopamine to the perfusing fluid were investigated in experiments on a segment isolated from the spinal cord of 13- to 18-day-old rats. Dopamine induced slow, dose-dependent depolarization in motoneurons in 28 trials out of 32, retained in the solution blocking synaptic transmission. Threshold concentration of dopamine in the normal perfusing fluid measured 1·10^–6 M and 1·10^–5 M in a calcium-free perfusate containing magnesium or manganese ions. Depolarization was accompanied by an increased rate of motor discharges recorded from the ventral root. Segmental reflex response produced by dorsal root stimulation was depressed following depolarization. Hyperpolarization in response to dopamine was observed in 4 out of 32 experiments. Dopamine-induced electrotonic dorsal root potentials were suppressed by prior haloperidol application to the brain.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 19, No. 6, pp. 735–741, November–December, 1987.     

Physiological properties of nicotinic acetylcholine receptors of sympathetic ganglionic neurons

The basic properties of nicotinic acetylcholine receptors of the neurons of a sympathetic ganglion responsible for the performance by these receptors of their main function — initiation of an electric current through the postsynaptic membrane — and determining the particular features of the acetylcholine receptors of these neurons by contrast with receptors of other objects, are described. Stoichiometric relations of the recognition center of the acetylcholine receptors with the transmitter, the relative strength of various agonists, and the method of action of -bungarotoxin on this center are indicated; the "life-time" and conductance of the ion channel are described. On the basis of "life-time" two groups of acetylcholine receptors are distinguished: synaptic (long-living) and extrasynaptic (short-living). Selective blockers of acetylcholine receptors of ganglionic neurons, namely bis-ammonium compounds, have two types of effect (competitive and channel-blocking), caused by the action of the blocker on two different regions of the receptor molecule, respectively. Since the channel-blocking action develops at lower concentrations than the competitive, and since it correlates closely with the ganglion-blocking effect, it is concluded that it is the first of these which determines the properties of selective blockers of acetylcholine receptors.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 16, No. 3, pp. 319–326, May–June, 1984.     

Oxotremorine-M potentiates NMDA receptors by muscarinic receptor dependent and independent mechanisms

Muscarinic acetylcholine M1 receptors play an important role in synaptic plasticity in the hippocampus and cortex. Potentiation of NMDA receptors as a consequence of muscarinic acetylcholine M1 receptor activation is a crucial event mediating the cholinergic modulation of synaptic plasticity, which is a cellular mechanism for learning and memory. In Alzheimer's disease, the cholinergic input to the hippocampus and cortex is severely degenerated, and agonists or positive allosteric modulators of M1 receptors are therefore thought to be of potential use to treat the deficits in cognitive functions in Alzheimer's disease. In this study we developed a simple system in which muscarinic modulation of NMDA receptors can be studied in vitro. Human M1 receptors and NR1/2B NMDA receptors were co-expressed in Xenopus oocytes and various muscarinic agonists were assessed for their modulatory effects on NMDA receptor-mediated responses. As expected, NMDA receptor-mediated responses were potentiated by oxotremorine-M, oxotremorine or xanomeline when the drugs were applied between subsequent NMDA responses, an effect which was fully blocked by the muscarinic receptor antagonist atropine. However, in oocytes expressing NR1/2B NMDA receptors but not muscarinic M1 receptors, oxotremorine-M co-applied with NMDA also resulted in a potentiation of NMDA currents and this effect was not blocked by atropine, demonstrating that oxotremorine-M is able to directly potentiate NMDA receptors. Oxotremorine, which is a close analogue of oxotremorine-M, and xanomeline, a chemically distinct muscarinic agonist, did not potentiate NMDA receptors by this direct mechanism. Comparing the chemical structures of the three different muscarinic agonists used in this study suggests that the tri-methyl ammonium moiety present in oxotremorine-M is important for the compound's interaction with NMDA receptors.     

Cholinergic transmission from mechanosensory afferents to an identified nonspiking interneuron in the crayfish Procambarus clarkii girard

Pharmacological properties of excitatory synaptic transmission from mechanosensory afferents to an identifiable nonspiking interneuron of crayfish were studied by drug perfusion experiments using acetylcholine (ACh) agonists and antagonists. Application of carbachol, a general agonist of ACh, caused sustained depolarization of the interneuron and a decrease in the peak amplitude of its excitatory synaptic response to sensory stimulation on the soma side. Similar depolarization was observed during application of carbachol under the low-Ca²⁺, high-Mg²⁺ condition. The peak amplitude was also reduced by application of nicotine and tetramethylammonium, both of which also caused sustained depolarization of the inter-neuron. By contrast, perfusion of muscarinic agonists, muscarine, oxotremorine and pilocarpine, reduced the peak amplitude without affecting the membrane potential of the interneuron. Perfusion of nicotonic antagonists of ACh, d-tubocurarine and hexamethonium, caused reduction of the peak amplitude without any change in the membrane potential. A muscarinic antagonist atropine was also effective in blocking the synaptic transmission but at higher concentration than d-tubocurarine. The results suggest that the ACh receptors on the nonspiking interneuron belong to a previously characterized class of crustacean cholinergic receptors resembling the nicotinic subtype of vertebrates.     

Has nonquantal acetylcholine secretion from motor nerve endings a role in neurotrophic control of resting membrane potential in rat muscle fibers?

The resting membrane potential of fibers of the rat diaphragm was measured by a microelectrode technique 3 h after division of the phrenic nerve and incubation in culture medium for 5 days after denervation. The membrane potential was recorded in synaptic regions of fibers close to (2–3 mm) and distant from (9–11 mm) the site of nerve division. The membrane potential of the synaptic region of the close fibers 3 h after denervation became smaller, whereas that of the synaptic region of distant fibers did not change relative to the control. Placing the muscle 3 h after denervation into medium with carbamylcholine (1·10^–8 M), cGMP (1·10^–4 M), or dibutyryl-cGMP (1·10^–6 M) led to hyperpolarization of the synaptic region of the close fibers but did not change the resting potential in the synaptic region of the distant fibers, and abolished differences between them. Five days after division of the nerve, incubation of the muscle in a solution with the above-mentioned substances did not affect the resting membrane potential. Nonquantal release of acetylcholine from motor nerve endings, assessed by the amplitude of hyperpolarization of the postsynaptic membrane, induced by application of curarine against the background of acetylcholine esterase inhibition, 3 h after denervation was identical in the synaptic region of the close and distant fibers and did not differ from the control. It is postulated that the postdenervation fall of membrane potential of rat muscle fibers is not due to disturbance of nonquantal secretion of acetylcholine from motor nerve endings.S. V. Kurashov Kazan' Medical Institute, Ministry of Health of the USSR. Translated from Neirofiziologiya, Vol. 17, No. 3, pp. 358–365, May–June, 1985.     

Inhibition of muscarinic receptor-mediated Ca27#x002B; mobilization by phorbol 12-myristate 13-acetate in chick embryo cells

Summary A muscarinic cholinergic receptor is present on undifferentiated cells of the chick embryo. Stimulation of the muscarinic receptor with muscarinic agonists triggers intracellular Ca^2#x002B; mobilization. Here, we investigate the effect of phorbol 12-myristate 13-acetate (PMA) on the muscarinic receptor-mediated Ca^2#x002B; mobilization, which is monitored in cell suspensions of chick embryos of stage 24 by chlorotetracycline fluorescence. PMA inhibits the Ca^2#x002B; mobilization in a time-dependent and concentration-dependent manner without changing the ED₅₀ of acetylcholine. The concentration of PMA that gives halfmaximal inhibition is 3.1×10^–9 M PMA.     

The pharmacology of an insect ganglion: actions of carbamylcholine and acetylcholine.

1. Methods for presenting dose-response data for the ganglionic actions of cholinergic agonists (e.g. carbamylcholine) are compared, using the mannitol-gap technique for electrophysiological recording of synaptic events at the cercal nerve, giant fibre synapse of the sixth abdominal ganglion of the cockroach Periplaneta americana. 2. At concentrations around 10(-5)M, carbamylcholine has no effect on ganglionic polarization but potentiates the monosynaptic EPSP. At 10(-4)M and higher concentrations, ganglionic depolarization is accompanied by a reduction of EPSP. 3. Pretreatment with eserine (10(-6) M) considerably shifts the dose-response curve for acetylcholine so that synaptic transmission is consistently sensitive to 10(-6) M acetylcholine.     

High-Affinity Choline Transporter

The cholinergic neurons have long been a model for biochemical studies of neurotransmission. The components responsible for cholinergic neurotransmission, such as choline acetyltransferase, vesicular acetylcholine transporter, nicotinic and muscarinic acetylcholine receptors, and acetylcholine esterase, have long been defined as functional units and then identified as molecular entities. Another essential component in the cholinergic synapses is the one responsible for choline uptake from the synaptic cleft, which is thought to be the rate-limiting step in acetylcholine synthesis. A choline uptake system with a high affinity for choline has long been assumed to be present in cholinergic neurons. Very recently, the molecular entity for the high-affinity choline transporter was identified and is designated CHT1. CHT1 mediates Na⁺- and Cl^–-dependent choline uptake with high sensitivity to hemicholinium-3. CHT1 has been characterized both at the molecular and functional levels and was confirmed to be specifically expressed in cholinergic neurons.     

Ionic mechanisms underlying modulating effects of serotonin on acetylcholine-induced responses in Helix pomatia neurons

The ionic mechanisms underlying modulatory effects of serotonin on acetylcholine-response in identified and nonidentifiedHelix pomatia neurons were investigated using voltage-clamping techniques at the neuronal membrane. External application of 10^–5–10^–4 M serotonin to the membrane of neurons responding to application of acetylcholine depending on Na⁺ depolarization (D_Na response) reduced membrane conductivity during response to acetylcholine without changing reversal potential of acetylcholine-induced current. Acetylcholine (10^–6–10^–4 M) administration took place 1–3 min later. Neurons with response to acetylcholine application dependent on Cl⁺ depolarization (D_Cl response) or hyperpolarization (H_Cl response) behaved similarly. Analogous effects could be produced by external application of theophylline which, together with the latency and residual effect characteristic of serotonin action points to the participation of intracellular processes associated with the cellular cyclase system in the changes produced by serotonin in acetylcholineinduced response. Serotonin brought about a shift in reversal potential and an increase in the acetylcholine-induced current in those neurons where this response was associated with changed permeability at the membrane to certain types of ions. During two-stage acetylcholine-induced response of the D_Na-H_K type, serotonin inhibited the inward current stage. Mechanisms underlying modulatory serotonin action on acetylcholine-induced response in test neurons are discussed in the light of our findings.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 20, No. 1, pp. 57–64, January–February, 1988.     

Sensitivity of hippocampal interneurons and pyramidal cells to GABA and some of its agonists:in vitro experiments

Effects of GABA and its agonists baclofen and muscimol on the background spike activity of single hippocampal neurons were studied in rat brain slices using an intracellular recording technique. Interneurons localized in thestratum alveus-oriens and pyramidal neurons of thestratum pyramidale showed high sensitivity to GABA (mean ID₅₀=65 µM and 40 µM, ranges 10–140 µM and 3–200 µM), baclofen (ID₅₀=2.6 µM and 3.5 µM, ranges 0.6–20.0 µM and 0.4–30.0 µM), and muscimol (ID₅₀=0.85 µM and 0.21 µM, ranges 0.11–4.0 µM and 0.05–0.45 µM, respectively). Responses of hippocampal neurons to application of GABA or either of its agonists were predominantly inhibitory. A part of interneurons (30%) differed from pyramidal neurons in their irresponsivity or low sensitivity to baclofen applications. GABA- or muscimol-induced inhibition of spike activity in many pyramidal cells was preceded by a short-lasting excitation. Our findings indicate that a part of hippocampal interneurons are very poorly supplied with GABAb receptors. Inhibition of pyramidal cells evoked by activation of GABAa receptors probably develops against the background of accompanying depolarization, which in some cases can result in a provisional excitation of these neurons. The excitatory effects of GABA on the pyramidal cells are mediated by GABAa receptors.Neirofiziologiya/Neurophysiology, Vol. 27, No. 1, pp. 36–44, January–February, 1995.     

Naloxone-dependent depression by morphine of responses of snail neurons to serotonin

Morphine, added to the extracellular solution in a concentration of 1·10^–5 M, quickly and reversibly weakens the depolarizing and hyperpolarizing responses of neurons of the snailHelix lucorum evoked by 1·10^–6 M serotonin. The inhibitory effect of morphine is completely abolished by the addition of naloxone (1·10^–5 M), suggesting that opiate receptors are involved in the process. Interaction between morphine and serotonin is noncompetitive in type, as is shown by the character of the dose-effect curves recorded during the action of serotonin before and after morphine application.Institute of Psychiatry, Academy of Medical Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 13, No. 6, pp. 589–593, November–December, 1981.     

Pre- and post-synaptic modulation of neuromuscular transmission in smooth muscles by thiazole analogs of vitamin B1

The effects of structural analogs of vitamin B₁, thiazole derivatives with alkyloxycarbonylmethyl substituents at position 3, on neuromuscular transmission were studied in the smooth muscles of the guinea pig gastrointestinal tract. In the smooth muscles of the stomach, the studied compounds depressed excitatory cholinergic neuromuscular transmission. In the case of 3-hexyl-, 3-decyl-, and 3-dodecyloxycarbonylmethyl-4-methyl-5-(2-hydroxyethyl)-thiazole chlorides this effect was due to their presynaptic action, while in the case of 3-menthyloxycarbonylmethyl-4-methyl-5-(2-hydroxyethyl)thiazole chloride it was due to the block of muscarinic acetylcholine receptors in smooth muscle fibers. In the circular smooth muscles of the distal colon, 3-decyloxycarbonylmethyl-4-methyl-5-(2-hydroxyethyl)thiazole chloride blocked non-adrenergic inhibitory synaptic potentials (ISP) apparently through interaction with the ATP-sensitive acetylcholine receptors. In contrast, 3-hexyloxycarbonylmethyl-4-methyl-5-(2-hydroxyethyl)thlazole chloride enhanced postinhibitory excitation, without changing the ISP amplitude. Possible ways of pre- and post-synaptic modulations of neuromuscular transmission by thiazole derivatives are discussed. It has been suggested that the effects of these compounds are due to similarity of their structures to the structure of vitamin B₁.Neirofiziologiya/Neurophysiology, Vol. 27, No. 5/6, pp. 375–386, September–December, 1995.     

Electrophysiological investigation of the properties of acetylcholine receptors of frog sympathetic ganglionic neurons

Changes in membrane potential and conductance were studied in neurons of isolated sympathetic ganglia ofRana ridibunda during perfusion with cholinomimetics and cholinolytics. Activation of nicotinic (N) acetylcholine receptors by carbachol, suberyldicholine, and tetramethylammonium led to depolarization with an increase in conductance, whereas activation of muscarinic (M) acetylcholine receptors by perfusion with carbachol or 5-methylfurmethide, led to depolarization with a decrease or (less frequently) an increase in conductance. The M-cholinolytic atropine was shown to cause depolarization with an increase in conductance if perfusion with atropine was preceded by perfusion with carbachol.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 11, No. 5, pp. 475–482, September–October, 1979.     

Effects of aspartate,substance P,and serotonin on neuronal activity in the central gray substance:In vitro experiments

Effects of aspartate (2 · 10^–5 M), substance P (10^–7–10^–8 M), and serotonin (5-hydroxytryptamine, 5-HT; 5 · 10^–5 M) on the background activity of neurons in the central gray substance (CGS) were studied on slices of the rat midbrain. Aspartate and substance P (transmitters of nociceptive signals), and 5-HT (modulator of transmission of nociceptive influences) were found either to facilitate or to depress the activity of CGS neurons. The predominant effect of substance P or 5-HT applications to neurons of the dorsal CGS part was facilitation, and to neurons of the ventral CGS part, inhibition. The effects of aspartate application on studied CGS neurons were of varying nature, but inhibitory effects were found to prevail.The findings support our earlier hypothesis that assigned the studied neurons to spontaneously discharging inhibitory CGS interneurons, which control the activity of efferent CGS neurons. The role of tested substances in the regulation of CGS neuronal activity and the antinociceptive CGS effects is discussed.Neirofiziologiya/Neurophysiology, Vol. 25, No. 5, pp. 354–362, September–October, 1993.     

Identifying nicotinic and muscarinic cholinoreceptors at the soma of neuron RPa4 inHelix lucorum

Cholinoreceptors were identified at the somatic membrane of theHelix lucorum RPa4 neuron using intracellular recording techniques. Application of specific agonists of nicotinic (nicotine, cytisine) and muscarinic (muscarine, arecoline) cholinoreceptors to the soma produced neuronal depolarization. The depolarization produced by applying acetylcholine to the cell was of short duration and was often replaced by hyperpolarization. Both selective desensitization of receptors by nicotine and muscarine as well as receptor occupancy by cytisine and arecoline reduced acetylchloline-induced response. The nicotinic cholinoblocker d-tubocurarine substantially inhibited responses to nicotinic cholinomimetics, while atropine, a muscarinic cholinoblocker, depressed response to muscarinic cholinomimetics. Acetylcholine-induced response was inhibited by both cholinoblockers more or less equally. The presence at the RPa4 neuronal somatic membrane is postulated of standard nicotinic and muscarinic cholinoreceptors similar to those found in vertebrates.M. V. Lomonsov State University, Moscow. Translated from Neirofiziologiya, Vol. 20, No. 2, pp. 203–212, March–April, 1988.     

Comparative research on synaptic transmission in the sympathetic ganglia of rabbits and frogs during acetylcholinesterase inhibition

Organophosphorus inhibitor of acetylcholinesterase (AChE) armin (1 x 10(-6) M) induced a variety of pre- and postsynaptic effects resulting from the AChE inhibition and subsequent accumulation of acetylcholine (ACh) in the synaptic cleft. The intensity of postsynaptic effects (level of neuron depolarization, degree of action potential depression) was shown to be different in the ganglia of frog and rabbit. This could be explained by differences in the total amount of ACh released in response to nerve stimulation as well as at rest. Both muscarinic and nicotinic cholinoreceptors were involved in the process of sustained depolarization of the neurons in the rabbit superior cervical ganglion after AChE inhibition. In frog ganglion neurons the nicotinic receptors did not participate in depolarization evidently due to their fast desensitization. The activation of presynaptic muscarinic receptors resulted in decrease of ACh released by nerve stimulation seems to weaken depolarization and blockade of synaptic transmission in sympathetic ganglia treated by AChE inhibitors.