Presence Of Muscarine Research Articles

Rhythmogenic networks are potently modulated by activation of muscarinic acetylcholine receptors in the rodent spinal cord.

Electrical stimulation of the spinal cord is a potent means for activating mammalian stepping in the absence of the descending control from the brain. Previously, we have shown that stimulation of pain delivering (Aδ) sacrocaudal afferents (SCA) has a powerful capacity to activate the sacral and lumbar rhythmogenic networks in the neonatal rodent spinal cord. Relatively little is known about the neural pathways involved in activation of the locomotor networks by Aδ afferents, on their mechanism of action and on the possibility to modulate their activity. We have shown that elevation of the endogenous level of acetylcholine at the sacral cord by blocking cholinesterase could modulate the SCA-induced locomotor rhythm in a muscarinic receptor-dependent mechanism. Here, we review these and more recent findings and report that controlled stimulation of SCA in the presence of muscarine is a potent activator of the locomotor network. The possible mechanisms involved in the muscarinic modulation of the locomotor rhythm are discussed in terms of the differential projections of sacral relay neurons, activated by SCA stimulation, to the lumbar locomotor rhythm generators, and to their target motoneurons. Altogether, our studies show that manipulations of cholinergic networks offer a simple and powerful means to control the activity of locomotor networks in the absence of supraspinal control. Cover Image for this issue: https://doi.org/10.1111/jnc.15079.

Evolution of the Toxins Muscarine and Psilocybin in a Family of Mushroom-Forming Fungi

Mushroom-forming fungi produce a wide array of toxic alkaloids. However, evolutionary analyses aimed at exploring the evolution of muscarine, a toxin that stimulates the parasympathetic nervous system, and psilocybin, a hallucinogen, have never been performed. The known taxonomic distribution of muscarine within the Inocybaceae is limited, based only on assays of species from temperate regions of the northern hemisphere. Here, we present a review of muscarine and psilocybin assays performed on species of Inocybaceae during the last fifty years. To supplement these results, we used liquid chromatography–tandem mass spectrometry (LC–MS/MS) to determine whether muscarine was present in 30 new samples of Inocybaceae, the majority of which have not been previously assayed or that originated from either the tropics or temperate regions of the southern hemisphere. Our main objective is to test the hypothesis that the presence of muscarine is a shared ancestral feature of the Inocybaceae. In addition, we also test whether species of Inocyabceae that produce psilocybin are monophyletic. Our findings suggest otherwise. Muscarine has evolved independently on several occasions, together with several losses. We also detect at least two independent transitions of muscarine-free lineages to psilocybin-producing states. Although not ancestral for the family as a whole, muscarine is a shared derived trait for an inclusive clade containing three of the seven major lineages of Inocybaceae (the Inocybe, Nothocybe, and Pseudosperma clades), the common ancestor of which may have evolved ca. 60 million years ago. Thus, muscarine represents a conserved trait followed by several recent losses. Transitions to psilocybin from muscarine-producing ancestors occurred more recently between 10–20 million years ago after muscarine loss in two separate lineages. Statistical analyses firmly reject a single origin of muscarine-producing taxa.

Modeling the layer V cortical pyramidal neurons showing theta-rhythmic firing in the presence of muscarine

In this study, we model a type of layer V pyramidal neurons in the prefrontal cortex of rats. In the presence of muscarine, the pyramidal neurons exhibit a self-sustained rhythmic firing in the theta frequency range (4– 7 Hz ) in response to a brief depolarizing step current. Since muscarine is known to activate the IP 3 receptors of the intracellular Ca 2+ store, the sustained firing seems to occur due to the activation of a certain Ca 2+-dependent current, presumably the Ca 2+-activated nonselective cationic current, during the IP 3-induced Ca 2+ release. Here we present a two-compartmental model neuron that shows the observed patterns of neuronal responses.

Cellular and synaptic effect of substance P on neonatal phrenic motoneurons.

Experiments were carried out on the in vitro brainstem-spinal cord preparation of the newborn rat to analyse the effects of substance P (SP) on phrenic motoneuron (PMN) activity. In current-clamp mode, SP significantly depolarized PMNs, increased their input resistance, decreased the rheobase current and shifted the firing frequency-intensity relationships leftwards, but did not affect spike frequency adaptation or single spike configuration. The neurokinin receptor agonist NK1 had SP-mimetic effects, whereas the NK3 and NK2 receptor agonists were less effective and ineffective, respectively. In a tetrodotoxin-containing aCSF, only SP or the NK1 receptor agonist were still active. No depolarization was observed when the NK1 receptor agonist was applied in the presence of muscarine. In voltage-clamp mode, SP or the NK1 receptor agonist produced an inward current (ISP) which was not significantly reduced by extracellular application of tetraethylammonium, Co2+, 4-aminopyridine or Cs+. In aCSF containing tetrodotoxin, Co2+ and Cs+, ISP was blocked by muscarine. No PMN displayed any M-type potassium current but only a current showing no voltage sensitivity over the range -100 to 0 mV, reversing near the expected EK +, hence consistent with a leak current. SP application to the spinal cord only (using a partitioned chamber) significantly increased the phrenic activity. Pretreatment with the NMDA receptor antagonist DL-2-amino-5-phosphonovaleric acid (AP5) decreased the C4 discharge duration and blocked the effect of SP, thus exhibiting an NMDA potentiation by SP. In conclusion, SP modulates postsynaptically the response of phrenic motoneurons to the inspiratory drive through the reduction of a leak conductance and the potentiation of the NMDA component of the synaptic input.

ANP has no postsynaptic effect on autonomic regulation of cardiac pacemaker rate in the rat.

We studied whether atrial natriuretic peptide (ANP) influences sinoatrial node pacemaker activity or whether it modifies the response to activation of postsynaptic autonomic receptors. Male Sprague-Dawley rats were anesthetized with pentobarbital sodium (45 mg/kg). Their hearts were removed quickly and placed in physiological salt solution. The atria were isolated; the right intra-atrial chamber was exposed to allow intracellular recording from sinoatrial node pacemaker cells. The tissue was placed in a temperature-regulated recording chamber and superfused with warmed oxygenated physiological salt solution. With use of standard microelectrode recording techniques, action potentials were recorded from spontaneously depolarizing cells in the presence of muscarine (62.5-500 nM) or norepinephrine (0.1 and 1.0 microM). Muscarine reduced the frequency of action potentials dose dependently, whereas norepinephrine increased their frequency. The addition of ANP (0.1-100 nM) to the superfusion had no effect on the frequency of action potentials during the superfusion of physiological salt solution or in the presence of either muscarine or norepinephrine. We conclude that ANP does not act on cardiac pacemaker cells to modulate the effect of neurotransmitters.

Characterization of a Calcium‐dependent Current Generating a Slow Afterdepolarization of CA3 Pyramidal Cells in Rat Hippocampal Slice Cultures

A depolarization-induced, slowly decaying inward current was examined in slice-cultured CA3 pyramidal cells by voltage-clamp techniques and microfluorometric measurements of cytosolic free Ca2+ concentration ([Ca2+]i). Action potentials elicited by intracellular injection of short-lasting (50-100 ms) depolarizing current pulses were followed by a slowly decaying afterhyperpolarization (AHP). After switching to voltage-clamp mode, short-lasting (50-100 ms) depolarizing voltage jumps from -60 mV to between -30 and 0 mV induced a slowly decaying outward aftercurrent (IAHP) which was depressed by bath application of muscarine (0.5 microM). In the presence of muscarine, the same depolarizations induced a slowly decaying afterdepolarization (ADP) or inward aftercurrent (IADP) in voltage-clamp mode. This current was also induced in the presence of trans(+/-)-1-amino-1,3-cyclopentanedicarboxylic acid (t-ACPD, 5 microM), an agonist of metabotropic glutamate receptors, but not in the presence of noradrenalin (5 microM), while both of these agonists depressed IAHP. IADP was depressed by reducing the external Ca2+ concentration from 3.8 to 0.5 mM, by external Co2+ (1 mM) and by external Cd2+ (10-100 microM). Combined voltage-clamp recordings and microfluorometric measurements of [Ca2+]i using the Ca2+ indicator fura-2 revealed that the amplitude of IADP was correlated with the amplitude of depolarization-induced Ca2+ influx. IADP was absent at membrane potentials < -90 mV, and reached maximal amplitudes at approximately -55 mV. Raising the extracellular K+ concentration from 2.7 to 13.5 mM increased the amplitude of IADP and resulted in a positively directed shift of the apparent reversal potential of IADP. When the external Na+ concentration was reduced from 157 to 33 or 18 mM the current reversed at more negative potentials and was reduced to 40 and 21%, respectively, of control amplitude. Lowering the external CI- concentration from 159 to 20 mM did not affect IADP. We conclude that IADP most likely represents a Ca(2+)-activated cation current, rather than a Ca2+ tail current, or an electrogenic Ca2+ extrusion current.

M1 and M2 muscarinic receptors mediate excitation and inhibition of guinea‐pig intracardiac neurones in culture.

1. The effects of muscarine upon intracardiac neurones cultured from ganglia within the atria and interatrial septum of the newborn guinea-pig heart were studied using intracellular recording techniques. 2. Muscarine applied to the neuronal soma typically produced a biphasic change in membrane potential which consisted of a small hyperpolarization followed by a depolarization. In addition, muscarine (0.01-10 microM) inhibited the calcium-dependent, after-hyperpolarization (AHP) and greatly increased the number of action potentials that could be evoked by a given depolarizing current. 3. The hyperpolarization was associated with a decrease in input resistance and it reversed to become a depolarization at a potential of -86.5 mV. This response was antagonized by 4-diphenylacetoxy-N-methyl-piperidine (4-DAMP; 100 nM) and AF-DX 116 (500 nM), but was unaffected by pirenzepine (0.1-5 microM). 4. Two types of slow depolarization were observed in the presence of muscarine. The most common was associated with an increase in input resistance in the potential range -70 to -40 mV. Pirenzepine (100 nM) selectively antagonized this response, 4-DAMP (100 nM) similarly antagonized the response, but was non-selective. AF-DX 116 (0.5-5 microM) showed no antagonist effect. The less common depolarization (5% of cells) had a long latency and was associated with a decrease in input resistance. 5. Muscarine reduced the duration of the action potential and inhibited the AHP. Cadmium chloride (100 microM) mimicked these actions of muscarine. Application of muscarine immediately following a train of action potentials did not inhibit the AHP, suggesting that muscarine did not directly inhibit the calcium-activated potassium current (IK(Ca)). Muscarine-induced depression of the slow AHP was antagonized by 4-DAMP (100 nM) but was not antagonized by either pirenzepine (0.1-0.5 microM) or AF-DX 116 (0.5-5 microM). 6. It is concluded that the muscarine-induced depolarization of guinea-pig intracardiac neurones results from reduction of a potassium conductance similar to the M-conductance, through activation of M1 muscarinic receptors. The hyperpolarization results from an increase in potassium conductance, through activation of M2 muscarinic receptors.

Caffeine affects four different ionic currents in the bull‐frog sympathetic neurone.

1. Ionic mechanisms related to the caffeine-induced current (Icaffeine) were examined in the single isolated sympathetic neurones of the bull-frog. We used the 'concentration-jump' technique in combination with intracellular perfusion and a rapid external solution change, under single-electrode voltage-clamp conditions. 2. Icaffeine was pharmacologically separated into a tetraethylammonium (TEA)-sensitive transient outward current (ITO), a picrotoxin (PTX)-sensitive transient inward current (ITI) and a TEA- and PTX-insensitive sustained inward current (ISI). At low concentrations of caffeine, a sustained outward current (ISO) was observed instead of ISI. 3. All components of Icaffeine were abolished by intracellular perfusion of 30 mM-EGTA. Pre-treatment with A23187 or ryanodine or the simultaneous application of procaine either reduced or abolished all the components of Icaffeine in a dose-dependent manner. The concentration causing 50% inhibition (IC50) was 10(-8) M for A23187 and 2 mM for procaine. 4. The peak response of ITO increased abruptly at caffeine concentrations between 3 and 6 mM followed by saturation above 30 mM. A notch was observed on the rising phase of ITO. 5. The reversal potential (Ecaffeine) of ITO shifted 58 mV for a tenfold change of the extracellular K+ concentration. External application of TEA blocked ITO with an IC50 of 1 mM. ITO was relatively insensitive to apamin, 4-aminopyridine and muscarine. 6. In external solution containing 2 mM-Ca2+, ITO induced by 10 mM-caffeine recovered completely within 3 min from a previous exposure to caffeine. In the absence of extracellular Ca2+, there was little such recovery. A 5 min treatment in a Ca2+-free solution reduced ITO induced by the first application of caffeine by 5%. With a continuous application of 3 mM-caffeine, the amplitude of ITO induced by 10 mM-caffeine reduced in 1 min, and showed a partial recovery in 3 min. The amplitude of ITO increased by increasing the concentration of intracellular Cl-. 7. ITI was activated around the peak of ITO and was rapidly inactivated. ITI was evoked at caffeine concentrations of about 6-10 mM. When the intracellular Cl- concentration was changed, the amplitude of ITI behaved like a Cl- electrode. The Ecaffeine of ITI was close to the Cl- equilibrium potential (ECl). 8. ISI was a 'plateau' response and persisted for over 3 min. ISI was due to a decrease in K+ conductance. In the presence of muscarine (3 x 10(-5) M), ISI was occluded.(ABSTRACT TRUNCATED AT 400 WORDS)

Excitatory effect of noradrenaline on pacemaker cells in spinal cord primary cultures

Intracellular recordings were made from dissociated fetal mouse spinal cord neurons in primary culture. One particular type of neuron, with a large cell body (40–50 μm) and three to five thick neuntes, exhibited rhythmic electrical activity of two different types, consisting of either spontaneous burst discharges or tonic action potential firing. Both types of activity appeared to be triggered by an endogenous membrane potential oscillation. Micropressure application of noradrenaline (10 −5 M in the delivery pipette) onto the surface of such cells evoked, in a dose-dependent manner, an increase in the input resistance with a depolarization of the membrane potential. The response to NA was potential-dependent. The maximum change in input resistance was observed at membrane potential values between −60 mV and −45 mV and the response was suppressed at membrane potentials lower than −80 mV. No modification of the response was observed in the presence of 50 mM of tetraethylammonium. The extrapolated reversal potential, close to −90 mV, was modified by increasing extracellular K + concentration and unaltered by increasing the intracellular Cl − concentration. The decrease in K + conductance induced by noradrenaline was Ca 2+-dependent and reversibly suppressed by Ba 2+ (6 mM) and Cd 2+ (0.1 mM). This response to noradrenaline was suppressed in the presence of muscarine (10 μM) suggesting that noradrenaline decreases a K + conductance related to M current. The noradrenaline evoked increase in input resistance was mediated by activation of an α 1 receptor site. Prazosin, an α 1 antagonist and phentolamine, an α 1α 2 antagonist, reversibly suppressed the response in a competitive manner. Yohimbine, a competitive α 2 antagonist, also blocked the response, but in a noncompetitive manner. Clonidine, an α 2 agonist, isoprenaline, a β agonist and l-alprenolol, a β antagonist, had no effect.

Noradrenaline hyperpolarization and depolarization in cat vesical parasympathetic neurones.

Responses to noradrenaline (NA) applied by superfusion, ionophoresis or pressure pulse were analysed using conventional intracellular recording and voltage-clamp methods in cat vesical parasympathetic ganglia. NA (1 microM) hyperpolarized 60% of the neurones, depolarized 25%, and produced a biphasic potential, which comprised a membrane hyperpolarization followed by a membrane depolarization, in 10%. About 5% of the neurones did not respond to NA. The NA hyperpolarization was blocked by yohimbine (1 microM), an alpha 2-adrenoceptor antagonist, whereas the NA depolarization was blocked by prazosin (0.1-1 microM), an alpha 1-adrenoceptor antagonist. These data indicated that the NA hyperpolarization was mediated through alpha 2-adrenoceptors and the NA depolarization through alpha 1-adrenoceptors. The NA hyperpolarization was accompanied by an increase in conductance, while the NA depolarization was associated with a decrease in conductance measured under manual-clamp conditions. Similar conductance changes were observed under voltage clamp. NA hyperpolarizations became smaller as the membrane was hyperpolarized and reversed polarity beyond -100 mV. NA depolarizations also became smaller at hyperpolarized membrane potentials and reversed polarity around -90 mV. The NA responses were enhanced in low-K media and depressed in high-K Krebs solution. The NA hyperpolarization was blocked by the Ca antagonists, Cd, Mn and Co. Intracellular injection of EGTA caused a slowly developing, progressive block of the NA hyperpolarization. The NA depolarization was not affected by low Ca concentrations, Ca antagonists or intracellular injection of EGTA. In some neurones the NA depolarization was unmasked in solutions containing Ca antagonists and after intracellular EGTA injection. The NA hyperpolarization was depressed by intracellular injection and extracellular superfusion of Cs but not by TEA. Ba (10-100 microM) depressed the NA hyperpolarization by 30%. The NA depolarization persisted in the presence of muscarine (10 microM) and was not blocked by Cs or TEA but was depressed 70% by Ba (10 microM). These data are consistent with the hypotheses that alpha 2-adrenoceptor activation produces a membrane hyperpolarization that is mediated through a Ca-dependent K conductance, and that alpha 1-adrenoceptor activation produces a membrane depolarization through closure of a voltage-insensitive K channel.

Identification of delayed potassium and calcium currents in the rat sympathetic neurone under voltage clamp.

Post-ganglionic neurones of the isolated rat superior cervical ganglion were studied at 37 degrees C under two-electrode voltage-clamp conditions. Membrane depolarization beyond -40 mV from holding levels between -50 and -100 mV produced a delayed outward current which exhibited no inactivation within this voltage range. The current is carried primarily by K+ ions and its instantaneous I-V relation is linear. The total outward current could be separated into two distinct components on the basis of ion-substitution experiments. A voltage-dependent component of the delayed current, termed IK(V), is activated by membrane depolarization beyond -40 mV when Ca2+ fluxes are selectively blocked by Cd2+ or in Ca2+-free solution. IK(V) develops following first-order kinetics and rises to a peak with a voltage-dependent delay (239 ms at -30 mV and 23 ms at +10 mV). GK(V) attains a saturating value of the order of 17 mS/cm2 at about +20 mV and can be described in terms of a simple Boltzmann distribution for a single gating particle with a valency equal to +2.5. A second component of the delayed outward current, termed IK(Ca), depends on Ca2+ entry for its activation and was isolated as difference current before and after block of Ca2+ movements across the membrane. IK(Ca) is larger and faster than IK(V): it is strictly related to Ca2+ influx and also depends on membrane potential depolarization. A distinct Ca2+ current, ICa, was recorded from the neurone exposed to Na+-free or tetrodotoxin solution. ICa was activated by membrane depolarization beyond -30 mV and reached a maximum value near 0 mV. Its activation agrees with fourth-order kinetics and becomes faster with increasing depolarization. The Ca2+ current developed with a voltage-dependent time to peak of 2.9-1.8 ms and thereafter completely inactivated. The relationship between ICa and IK(Ca) is discussed. The Ca2+-k+ repolarizing system is expected to be mainly associated with action potentials arising from a depolarized neurone, whereas the IA current (Belluzzi, Sacchi & Wanke, 1985) dominates the repolarization mechanism at the normal membrane potential. The effect of muscarine was examined. Muscarine (10-50 microM) produced a fall in conductance with a voltage dependence similar to that exhibited by GK(Ca) and was ineffective when removing extracellular Ca2+ or adding Cd2+. A partial suppression of ICa by muscarine is demonstrated. It is suggested that the decrease of the outward current magnitude in the presence of muscarine may be accounted for qualitatively by the reduction in ICa.