Biomedical subjects

A Nistri

Publications and source records attributed to A Nistri.

At least 19 recordsLinked to original sources

PubMed2006

The effects induced by the sulphonylurea glibenclamide on the neonatal rat spinal cord indicate a novel mechanism to control neuronal excitability and inhibitory neurotransmission.

BACKGROUND AND PURPOSE: Using the neonatal rat spinal cord in vitro, we investigated the action of glibenclamide, a drug possessing dual pharmacological effects, namely block of K(ATP) channels and of the cystic fibrosis transmembrane conductance regulator (CFTR). EXPERIMENTAL APPROACH: Intra- and extracellular recordings were performed on motoneurons and interneurons. RT-PCR and western immunoblotting were used to determine gene and protein expression. KEY RESULTS: Glibenclamide (50 microM) facilitated mono- and polysynaptic reflexes, hyperpolarized motoneuron resting potential, increased action potential amplitude, decreased Renshaw cell-mediated recurrent inhibition, and increased network excitability by depressing GABA- and glycine-mediated transmission. The action of glibenclamide was mimicked by tolbutamide (500 microM) or the CFTR blocker diphenylamine-2,2-dicarboxylic acid (500 microM). The action of glibenclamide was independent from pharmacological inhibition of the Na(+)-K(+) pump with strophanthidin (4 microM) and was associated with a negative shift in the extrapolated reversal potential for CI(-) dependent synaptic inhibition. On interneurons, intracellularly-applied 8-bromo-cAMP elicited an inward current and resistance decrease; effects antagonized by the selective CFTR antagonist, CFTR(inh)-172 (5 microM). RT-PCR and western immunoblotting indicated strong expression of the CFTR in neonatal rat spinal cord. CONCLUSIONS AND IMPLICATIONS: These data suggest the CFTR expressed in motoneurons and interneurons of the neonatal spinal cord is involved in the control of Cl(-) homeostasis and neuronal excitability. CFTR appeared to contribute to the relatively depolarized equilibrium potential for synaptic inhibition, an important process to control hyperexcitability and seizure-predisposition in neonates.

Animals↗

PubMed2005

Fictive locomotor patterns generated by tetraethylammonium application to the neonatal rat spinal cord in vitro.

Intrinsic spinal networks generate a locomotor rhythm characterized by alternating electrical discharges from flexor and extensor motor pools. Because this process is preserved in the rat isolated spinal cord, this preparation in vitro may be a useful model to explore methods to reactivate locomotor networks damaged by spinal injury. The present electrophysiological investigation examined whether the broad spectrum potassium channel blocker tetraethylammonium could generate locomotor-like patterns. Low (50-500 microM) concentrations of tetraethylammonium induced irregular, synchronous discharges incompatible with locomotion. Higher concentrations (1-10 mM) evoked alternating discharges between flexor and extensor motor pools, plus large depolarization of motoneurons with spike broadening. The alternating discharges were superimposed on slow, shallow waves of synchronous depolarization. Rhythmic alternating patterns were suppressed by blockers of glutamate, GABA(A) and glycine receptors, disclosing a background of depolarizing bursts inhibited by antagonism of group I metabotropic glutamate receptors. Furthermore, tetraethylammonium also evoked irregular discharges on dorsal roots. Rhythmic alternating patterns elicited by tetraethylammonium on ventral roots were relatively stereotypic, had limited synergy with fictive locomotion induced by dorsal root stimuli, and were not accelerated by 4-aminopyridine. Horizontal section of the spinal cord preserved irregular ventral root discharges and dorsal root discharges, demonstrating that the action of tetraethylammonium on spinal networks was fundamentally different from that of 4-aminopyridine. These results show that a potassium channel blocker such as tetraethylammonium could activate fictive locomotion in the rat isolated spinal cord, although the pattern quality lacked certain features like frequency modulation and strong synergy with other inputs to locomotor networks.

4-Aminopyridine↗

PubMed2005

Electrophysiological effects of 4-aminopyridine on fictive locomotor activity of the rat spinal cord in vitro.

Recently the K+ channel blocker 4-aminopyridine (4-AP) has been suggested to be useful to improve motor deficits due to spinal cord lesions. There is, however, little basic research support for this action of 4-AP. In this study we have used as a model the neonatal mammalian spinal cord in vitro that generates a rhythmic activity termed fictive locomotion (induced by bath-application of NMDA + 5-HT) with phasic electrical discharges alternating between flexor and extensor motor pools and between left and right motoneurons within the same segment. When 4-AP was added in the presence of sub-threshold concentrations of NMDA + 5-HT, there was facilitation of fictive locomotion which appeared with alternating patterns on all recorded ventral roots (VR). Furthermore, in the presence of 4-AP, weak dorsal root (DR) stimuli, previously insufficient to activate locomotor patterns, generated alternating discharges from various VRs. The present data show that 4-AP could strongly facilitate the locomotor program initiated by neurochemicals or electrical stimuli, indicating that the spinal locomotor network is a very sensitive target for the action of 4-AP.

4-Aminopyridine↗

PubMed2005

Pre- and postsynaptic modulation of glycinergic and gabaergic transmission by muscarinic receptors on rat hypoglossal motoneurons in vitro.

The motor output of hypoglossal motoneurons to tongue muscles takes place in concert with the respiratory rhythm and is determined by the balance between excitatory glutamatergic transmission and inhibitory transmission mediated by glycine or GABA. The relative contribution by these transmitters is a phasic phenomenon modulated by other transmitters. We examined how metabotropic muscarinic receptors, widely expressed in the brainstem where they excite cranial motor nuclei, might influence synaptic activity mediated by GABA or glycine. For this purpose, using thin slices of the neonatal rat brainstem, we recorded (under whole-cell patch clamp) glycinergic or GABAergic responses from visually identified hypoglossal motoneurons after pharmacological block of glutamatergic transmission. Muscarine inhibited spontaneous and electrically induced events mediated by GABA or glycine. The amplitude of glycinergic miniature inhibitory postsynaptic currents was slightly reduced by muscarine, while GABAergic miniature inhibitory postsynaptic currents were unaffected. Motoneuron currents induced by focally applied GABA and glycine were depressed by muscarine with stronger reduction in glycine-mediated responses. Histochemical observations indicated the presence of M1, M2 and M5 subtypes of muscarinic receptors in the neonatal hypoglossal nucleus. These results suggest that muscarine potently depressed inhibitory neurotransmission on brainstem motoneurons, and that this action was exerted via preterminal and extrasynaptic receptors. Since the large reduction in inhibitory neurotransmission may contribute to overall excitation of brainstem motoneurons by muscarinic receptors, these data might help to understand the central components of action of antimuscarinic agents in preanesthetic medication or against motion sickness.

Animals↗

PubMed2005

Characteristics of the electrical oscillations evoked by 4-aminopyridine on dorsal root fibers and their relation to fictive locomotor patterns in the rat spinal cord in vitro.

4-Aminopyridine (4-AP) is suggested to improve symptomatology of spinal injury patients because it may facilitate neuromuscular transmission, spinal impulse flow and the operation of the locomotor central pattern generator (CPG). Since 4-AP can also induce repetitive discharges from dorsal root afferents, this phenomenon might interfere with sensory signals necessary to modulate CPG activity. Using electrophysiological recording from dorsal and ventral roots of the rat isolated spinal cord, we investigated 4-AP-evoked discharges and their relation with fictive locomotor patterns. On dorsal roots 4-AP (5-10 microM) induced sustained synchronous oscillations (3.3+/-0.8 s period) smaller than electrically evoked synaptic potentials, persistent after sectioning off the ventral region and preserved in an isolated dorsal quadrant, indicating their dorsal horn origin. 4-AP oscillations were blocked by tetrodotoxin, or 6-cyano-7-nitroquinoxaline-2,3-dione and d-amino-phosphonovalerate, or strychnine and bicuculline, suggesting they were network mediated via glutamatergic, glycinergic and GABAergic transmission. Isolated ventral horn areas could not generated 4-AP oscillations, although their intrinsic disinhibited bursting was accelerated by 4-AP. Thus, ventral horn areas contained 4-AP sensitive sites, yet lacked the network for 4-AP induced oscillations. Activation of fictive locomotion by either application of N-methyl-D-aspartate and serotonin or stimulus trains to a single dorsal root reversibly suppressed dorsal root oscillations induced by 4-AP. This suppression was due to depression of dorsal network activity rather than simple block of root discharges. Since dorsal root oscillations evoked by 4-AP were turned off when the fictive locomotor program was initiated, these discharges are unlikely to interfere with proprioceptive signals during locomotor training in spinal patients.

4-Aminopyridine↗

PubMed2004

Low micromolar concentrations of 4-aminopyridine facilitate fictive locomotion expressed by the rat spinal cord in vitro.

Upregulating the operation of spinal locomotor networks is one mechanism to restore, at least partially, lesion-impaired locomotion. We investigated if the K+ channel blocker 4-aminopyridine (4-AP) could facilitate spinal locomotor networks in addition to its well-known effect on motor nerve conduction. Fictive locomotor patterns were recorded from ventral roots (VRs) of the isolated spinal cord of the neonatal rat. 4-AP (0.1-50 microM) produced synchronous VR oscillations which did not develop into fictive locomotion. These oscillations had network origin, required intact glutamatergic transmission and were probably amplified via electrotonic coupling because of their depression by the selective gap junction blocker carbenoxolone. 4-AP (5 microM) slightly increased input resistance of lumbar motoneurons without affecting their action or resting potentials. Dorsal root (DR) evoked synaptic responses were enhanced (217 +/- 65%) by 5 microM 4-AP without changes in axon conduction. 4-AP (5 microM) accelerated fictive locomotion induced by N-methyl-d-aspartate (NMDA) and serotonin (5-HT) without altering cycle amplitude and facilitated the onset of fictive locomotion in the presence of sub-threshold concentrations of NMDA and 5-HT. Furthermore, in the presence of 4-AP, weak DR stimuli, previously insufficient to activate locomotor patterns, generated alternating VR discharges. Thus, although 4-AP per se could not directly activate the locomotor network of the spinal cord, it could strongly facilitate the locomotor program initiated by neurochemicals or electrical stimuli. These data suggest that the reported improvement by 4-AP in locomotor activity of spinal-injury patients may include activation of locomotor networks when low concentrations of this drug are administered in coincidence with appropriate stimuli.

4-Aminopyridine↗

PubMed2002

Evaluation of GluR2 subunit involvement in AMPA receptor function of neonatal rat hypoglossal motoneurons.

AMPA receptors (AMPAr) mediate fast synaptic responses to glutamate and, when they lack the GluR2 subunit, are strongly Ca2+ permeable and may increase intracellular Ca2+ levels. Because hypoglossal motoneurons possess restricted ability to buffer internal Ca2+ and are vulnerable to Ca2+ excitotoxicity, we wondered if, in these cells, any significant Ca2+ influx could be generated via AMPAr activity. Using whole cell patch-clamp recording from neonatal rat hypoglossal motoneurons, we tested the AMPAr properties conferred by GluR2 subunits, namely Ca2+ permeability, current rectification and sensitivity to pentobarbital or to the subunit-specific channel blockers, IEM-1460 and IEM-1925. We recorded membrane currents generated by the agonist, kainate, and compared them with those obtained from hippocampal pyramidal neurons (expressing GluR2-containing AMPAr) and from striatal giant aspiny or hippocampal interneurons (with GluR2-lacking AMPAr). Ca2+ vs. Na+ permeability of motoneuron AMPAr was relatively low (0.25 +/- 0.05), although higher than that of pyramidal neurons. With intracellularly applied spermine, significant inward rectification was absent from motoneurons. These data indicated the prevalence of functional GluR2 subunits. However, the sensitivity of motoneuron AMPAr to pentobarbital did not differ from that of GluR2-lacking AMPAr on interneurons. Motoneurons possessed sensitivity to IEM-1460 (IC50 = 90 +/- 10 microm) approximately 10-fold lower than striatal interneurons, although 10-fold higher than hippocampal pyramidal cells. IEM-1925 also reduced the amplitude of excitatory synaptic currents in brainstem slice motoneurons. We hypothesize that hypoglossal motoneuron AMPAr (moderately Ca2+ permeable because they contain few GluR2 subunits) may contribute to intracellular Ca2+ rises especially if persistent AMPAr activation (or the pathological GluR2 down-regulation) occurs.

Adamantane↗

PubMed2001

Calibration of agonist concentrations applied by pressure pulses or via rapid solution exchanger.

Desensitization of neuronal nicotinic acetylcholine receptors (nAChRs) can develop so rapidly to preclude functional studies when agonists are applied under steady-state conditions. To minimize receptor desensitization, we recorded from rat cultured chromaffin cells currents evoked by nicotine applied by pressure (via a fine tipped pipette) or through a multibarreled rapid solution exchanger. By comparing equi-amplitude, non-fading responses on the same cells, we constructed log 'dose'-response curves with the amount of nicotine expressed as either pulse duration (ms) or initial concentration (microM) inside the perfusion tubes. The linear part of these plots (10-50 ms or 20-100 microM) allowed extrapolating the initial nicotine concentration for a certain pulse duration within these limits. Potential dilution of nicotine in the bath was calculated with diffusion equations for a continuous point source: the maximal concentration of nicotine attainable by pressure application from a 100 microM containing pipette was 92 microM. We also calculated how diluted the same nicotine solution became following rapid superfusion and found it to be 46%. These results indicate that the amount of agonist applied by pressure to a relatively close cell could be calibrated in terms of effective concentration at membrane level and, with the present experimental arrangement, underwent limited dilution in the extracellular microenvironment.

Air Pressure↗

PubMed2001

Negative cross talk between anionic GABAA and cationic P2X ionotropic receptors of rat dorsal root ganglion neurons.

Using whole-cell patch-clamp recording and intracellular Ca(2+) imaging of rat cultured DRG neurons, we studied the cross talk between GABA(A) and P2X receptors. A rapidly fading current was the main response to ATP, whereas GABA elicited slowly desensitizing inward currents. Coapplication of these agonists produced a total current much smaller than the linear summation of individual responses (68 +/- 5% with 10 microm ATP plus 100 microm GABA). Occlusion was observed regardless of ATP response type. Neurons without functional P2X receptors manifested no effect of ATP on GABA currents (and vice versa). Occlusion was also absent in the presence of the P2X blocker trinitrophenyl-ATP (TNP-ATP) or of the GABA blocker picrotoxin, indicating a lack of involvement by metabotropic ATP or GABA receptors. Less occlusion was obtained when ATP was applied 2 sec after GABA than when GABA was applied after ATP. Changing the polarity of GABA currents by using intracellular SO(4)2- instead of Cl(-) significantly reduced the occlusion of ATP currents by GABA, suggesting an important role for Cl(-) efflux in this phenomenon. Occlusion was enhanced whenever intracellular Ca(2+) ([Ca(2+)](i)) was not buffered, indicating the cross talk-facilitating role of this divalent cation. Ca(2+) imaging showed that ATP (but not GABA) increased [Ca(2+)](i) in voltage-clamped or intact neurons. Our data demonstrated a novel Cl(-) and Ca(2+)-dependent interaction between cationic P2X and anionic GABA(A) receptors of DRG neurons. Such negative cross talk might represent a model for a new mechanism to inhibit afferent excitation to the spinal cord as GABA and ATP are coreleased within the dorsal horn.

Adenosine Triphosphate↗

PubMed2001

Evidence for increased extracellular K(+) as an important mechanism for dorsal root induced alternating rhythmic activity in the neonatal rat spinal cord in vitro.

Intracellular recording from rat lumbar motoneurones showed that patterned electrical stimulation of one dorsal root induced alternating patterns typical of fictive locomotion, accompanied by a reduction in the antidromic spike afterhyperpolarization (AHP). Since the AHP depended linearly on extracellular K(+) ([K(+)](o)), from the AHP fall we calculated that [K(+)](o) rose to 7.9+/-0.4 mM, a value within the 7-10 mM range known to elicit fictive locomotion. To confirm the specificity of this approach we also tested concentrations of N-methyl-D-aspartate (2 microM) below threshold for fictive locomotion, and we observed that, despite strong excitation, the estimated [K(+)](o) was 6 mM, i.e. outside the range for fictive locomotion. We suggest that synaptic activity induced by a certain pattern of electrical stimulation of afferent fibres evoked persistent elevation in [K(+)](o) which triggered the locomotor network to generate rhythmic patterns.

Animals↗

PubMed2001

Alternating rhythmic activity induced by dorsal root stimulation in the neonatal rat spinal cord in vitro.

Electrical stimuli applied to a single dorsal root (DR) of the neonatal rat spinal cord in vitro were used to test the possibility that the central pattern generator responsible for locomotion could be activated by synaptic inputs. Brief pulse trains evoked oscillatory patterns recorded from pairs of lumbar ventral roots. These patterns alternated rhythmically between the left and right sides and between predominantly flexor and extensor motoneuronal pools on the same side, thus displaying properties similar to the fictive locomotor pattern elicited by bath-applied excitatory transmitter agonists like NMDA and serotonin. Usually pulse trains rather than single pulses were necessary to induce these patterns, the period of which was independent of stimulation frequency (1-10 Hz) and only moderately dependent on stimulus intensity. Patterns reached a steady rhythm during the stimulus train, lasted for 50 +/- 20 s with gradual period lengthening and finally ceased. Since DR stimuli activated the central pattern generator for locomotion in the rat isolated spinal cord, it is suggested that sensory inputs from the periphery can reach the spinal locomotor network and trigger its operation.

Animals↗

PubMed2001

Characteristics of fast Na(+) current of hypoglossal motoneurons in a rat brainstem slice preparation.

Whole-cell patch clamp recordings were performed on hypoglossal motoneurons in a brainstem slice preparation from the neonatal rat brain to study the characteristics of the fast Na(+) current (I(Na)) which has not been hitherto investigated in these cells. To aid voltage clamping of I(Na), cells were bathed in low Na(+) solution, loaded intracellularly with Na(+) (to reverse the Na(+) gradient) or treated with a small dose (20 nM) of tetrodotoxin. In low extracellular Na(+) solution (Na(+) was replaced by choline or N-methyl-D-glucamine) I(Na) activated at membrane potentials positive to -45 mV and was half-maximally activated at -30 mV. Similar data were obtained when the Na(+) gradient was reversed or tetrodotoxin was applied. I(Na) rapidly activated (1--3.5 ms time constant) and inactivated (1.6 ms time constant at 0 mV) during membrane depolarization. Inactivation was strongly voltage-dependent (half inactivation at -44 mV) and developed mono-exponentially. Recovery from inactivation was bi-exponential with fast and slow time constants of 14 and 160 ms, respectively, at -58 mV. The rapid turning on of I(Na) was presumably responsible for the upstroke of the fast action potential generated by these cells while the slow phase of recovery from inactivation might modulate the ability to fire repetitively at high rate.

Action Potentials↗

PubMed2001

Depression of windup of spinal neurons in the neonatal rat spinal cord in vitro by an NK3 tachykinin receptor antagonist.

The effects of the NK3 tachykinin receptor antagonist SR 142801 on synaptic transmission and spike windup induced by trains of stimuli applied to a dorsal root were investigated with intra- and extracellular recording from the neonatal rat spinal cord in vitro. SR 142801 (10 microM) reduced the depolarization (recorded from lumbar ventral roots) induced by senktide (an NK3 agonist) more strongly than the one evoked by substance P methyl ester (SPMeO; an NK1 agonist). Nevertheless, after a long (>2 h) application time, SR 142801 largely depressed the response to SPMeO as well. When NK1 or NK3 receptors were blocked by >50% in the presence of SR 142801, there was also a significant reduction in the cumulative depolarization induced by repeated stimuli to a single dorsal root. This blocking action by SR 142801 was also observed in the presence of the N-methyl-D-aspartate (NMDA) receptor antagonist D-aminophosphonovalerate (APV) and the calcium channel blocker nifedipine. Intracellular data from lumbar motoneurons showed that the spike windup was the first and most sensitive target for the SR 142801 blocking effect. Increasing stimulus strength to dorsal root fibers could partly surmount such a block. SR 142801 per se had no direct action on fast synaptic transmission, membrane potential, or input resistance. These findings indicate that SR 142801 could lead to an early, large reduction in the windup of action potential discharge by motoneurons, suggesting its ability to suppress the reflex component of central sensitization evoked by repeated dorsal root stimuli.

Action Potentials↗

PubMed2001

Differential short-term changes in GABAergic or glycinergic synaptic efficacy on rat hypoglossal motoneurons.

Using whole cell patch-clamp recording from hypoglossal motoneurons of a neonatal rat brain slice preparation, we investigated short-term changes in synaptic transmission mediated by GABA or glycine. In 1.5 mM extracellular Ca(2+) [Ca(2+)](o), pharmacologically isolated GABAergic or glycinergic currents were elicited by electrical stimulation of the reticular formation. At low stimulation frequency, glycinergic currents were larger and faster than GABAergic ones. GABAergic currents were strongly facilitated by pulse trains at 5 or 10 Hz without apparent depression. This phenomenon persisted after pharmacological block of GABA(B) receptors. Glycinergic currents were comparatively much less enhanced than GABAergic currents. One possible mechanism to account for this difference is that GABAergic currents decayed so slowly that consecutive responses summated over an incrementing baseline. However, while synaptic summation appeared at > or =10-Hz stimulation, at 5 Hz strong facilitation developed with minimal summation of GABA-mediated currents. Glycinergic currents decayed so fast that summation was minimal. As [Ca(2+)](o) is known to shape short-term synaptic changes, we examined if varying [Ca(2+)](o) could differentially affect facilitation of GABA- or glycine-operated synapses. With 5 mM [Ca(2+)](o), the frequency of spontaneous GABAergic or glycinergic currents appeared much higher but GABAergic current facilitation was blocked (and replaced by depression), whereas glycinergic currents remained slightly facilitated. [Ca(2+)](o) manipulation thus brought about distinct processes responsible for facilitation of GABAergic or glycinergic transmission. Our data therefore demonstrate an unexpectedly robust, short-term increase in the efficiency of GABAergic synapses that can become at least as effective as glycinergic synapses.

Animals↗

PubMed2001

Neuronal bursting induced by NK3 receptor activation in the neonatal rat spinal cord in vitro.

Intracellular recording from lumbar motoneurons and extracellular recording from ventral roots of the neonatal rat isolated spinal cord were used to study the mechanisms responsible for the excitation mediated by NK3 tachykinin receptors. The selective NK3 agonists senktide or [MePhe7]neurokinin B induced a slow depolarization with superimposed oscillations (mean period +/- SD was 2.8 +/- 0.8 s) that, in the majority of cases, showed left-right alternation at segmental level and were synchronous between L2 and L5 of the same side. During agonist wash out (5-20 min) a delayed form of hyperexcitability emerged consisting of bursts lasting 8 +/- 2 s (average interburst interval 55 +/- 21 s) with superimposed oscillations usually with homosegmental alternation and heterosegmental synchronicity. Such bursting was accompanied by depression of GABAergic dorsal root potentials evoked by dorsal root stimulation and of the recurrent inhibitory postsynaptic potential recorded from motoneurons. Despite bursting, motoneuron membrane potential returned to baseline while input resistance was increased. Bursts were a network-dependent phenomenon triggered by previous NK3 receptor activation because bursting was suppressed by glutamate receptor antagonists and was insensitive to motoneuron membrane potential or subsequent application of an NK3 receptor antagonist. NK3 receptors operated synergistically with N-methyl-D-aspartate (NMDA) and 5-hydroxytryptamine (5-HT) to trigger fully alternating locomotor-like rhythms while NK3 receptor antagonism disrupted the same rhythm. In summary, in the neonatal rat spinal cord NK3 receptors could trigger rhythmic activity predominantly with alternation at segmental level but with synchronous coupling between ipsilateral motor pools. NK3 receptor activation could also facilitate fictive locomotor patterns induced by NMDA and 5-HT.

Animals↗

PubMed2000

Opposite changes in synaptic activity of organotypic rat spinal cord cultures after chronic block of AMPA/kainate or glycine and GABAA receptors.

1. The well-developed cytoarchitecture of rat organotypic spinal cord culture makes it a suitable model to explore how persistent suppression of certain synaptic inputs might be compensated by increased synaptic efficacy (homeostatic plasticity). 2. Spontaneous or electrically evoked synaptic transmission of patch-clamped ventral horn interneurons was studied in control solution after blocking, for the second week in culture, AMPA/kainate receptors with CNQX or glycine and GABAA receptors with strychnine and bicuculline, or indiscriminately removing inputs with tetrodotoxin (TTX). 3. In untreated cells, spontaneous postsynaptic currents (PSCs) had fast (tau < 5 ms) or slow (tau > 10 ms) decay. A similar separation was observed when recording miniature currents (mPSCs). Slow decay PSCs were suppressed by strychnine plus bicuculline while fast decay events were eliminated by CNQX. 4. After chronic CNQX treatment the frequency of spontaneous, fast PSCs (of larger amplitude) or mPSCs was almost doubled with respect to control. These events were blocked by acutely applied CNQX, which unmasked slow PSCs. 5. After chronic TTX treatment neither the frequency nor the amplitude of spontaneous events was changed. 6. After chronic strychnine and bicuculline treatment the frequency and amplitude of all PSCs was decreased in most cells. mPSCs were also decreased in frequency. Spontaneous or electrically evoked currents acquired a larger component mediated by NMDA receptor activity. 7. The developing spinal network thus operated distinct homeostatic processes which led to strong enhancement in glutamatergic transmission after CNQX block or to broad downregulation of synaptic activity following chronic exposure to strychnine and bicuculline.

6-Cyano-7-nitroquinoxaline-2,3-dione↗

PubMed2000

Antagonism of nicotinic receptors of rat chromaffin cells by N,N, N-trimethyl-1-(4-trans-stilbenoxy)-2-propylammonium iodide: a patch clamp and ligand binding study.

The effect of the oxystilbene derivative F3 was tested on nAChRs of whole-cell patch-clamped rat chromaffin cells in vitro and of rat adrenal gland membranes using (125)I-epibatidine. F3 (30 nM) rapidly and reversibly blocked inward currents generated by pulse applications of nicotine, shifting the dose-response curve to the right in a parallel fashion without changing the maximum response. The action of F3 was voltage insensitive and not due to altered current reversal potential. The R isomer of F3 was more potent (IC(50) = 350+/-30 nM) than its S-enantiomer (IC(50) = 1.5+/-0.3 microM). Nicotine-evoked currents were insensitive to 10 microM alpha-bungarotoxin. Equi-amplitude currents evoked by nicotine or epibatidine were similarly antagonized by R-F3 in a reversible fashion. Epibatidine-evoked currents readily produced receptor desensitization. Adrenal membranes specifically bound (125)I-epibatidine with a single population of binding sites endowed with high affinity (K(D) = 159 pM) and B(max) of 6.5+/-1.3 fmol mg(-1) of protein. (125)I-epibatidine binding was specifically displaced by cytisine (K(i) = 68 nM) or ACh (K(i) = 348 nM). F3 specifically displaced (125)I-epibatidine binding although with lower affinity (K(i) = 29.6 microM) than in electrophysiological experiments. (125)I-epibatidine binding to rat adrenal tissue was insensitive to alpha-bungarotoxin which readily antagonized (125)I-epibatidine binding to bovine adrenal tissue. The present results suggest that F3 is a relatively potent and apparently competitive antagonist of nAChRs on rat chromaffin cells. Since previous studies have indicated that F3 targets different subtypes on chick neuronal tissue, it appears that nAChRs display interspecies differences to be considered for drug development studies.

Adrenal Glands↗

PubMed2000

Facilitation of cholinergic transmission by substance P methyl ester in the mouse hippocampal slice preparation.

Using sharp microelectrode recording from CA1 pyramidal neurons of the adult mouse hippocampal slice preparation, we studied the modulatory action of the selective neurokinin 1 (NK1) receptor agonist substance P methyl ester (SPME), a peptidase-resistant analogue of the peptide substance P (SP), on cholinergic responses. While SPME (0.1-1 microM) had only slight effects on membrane potential and input resistance of CA1 neurons, it largely and reversibly enhanced the membrane depolarization and oscillatory activity induced by the cholinergic agonist carbachol (CCh; 0.1-100 microM). This effect of SPME was prevented by the selective NK1 receptor antagonist SR 140333 (4 microM). In about half of the tested neurons the action of SPME was preserved in tetrodotoxin (TTX) solution, suggesting that it partly occurred at the level of pyramidal cells. Cholinergic slow excitatory postsynaptic potentials (sEPSPs) were reversibly enhanced by SPME which increased their amplitude and prolonged any associated bursting activity. This action was also blocked by SR 140333. The present results suggest that SPME largely enhances cholinergic activity in the mouse hippocampus, an effect which can help to explain, in this brain area, the recently reported facilitation of seizures by SP.

6-Cyano-7-nitroquinoxaline-2,3-dione↗