Synaptic potentials in motoneurons during fictive swimming in spinal Xenopus embryos

Embryos spinalized at the 3rd to 6th postotic myotome and immobilized in 10(-4) M tubocurarine can respond to a brief skin stimulus with motor root activity suitable for swimming. Embryos spinalized at the more caudal levels give shorter episodes of fictive swimming. We have previously described the synaptic inputs to motoneurons during fictive swimming in intact embryos (23). In the present paper we look to see if similar synaptic inputs are present in spinal embryos and are therefore spinal in origin. All motoneuron firing during fictive swimming is associated with a tonic depolarization that falls away slowly once firing stops, is increased by hyperpolarizing current, and is reduced by depolarizing current. A slow depolarizing potential evoked by lower levels of skin stimulation has similar properties and rate of fall. In 1-2 mM PDA, an excitatory amino acid antagonist, only a small remnant of the depolarization remains, and motoneuron firing stops. The NMDA antagonist 50 microM APV reduces the depolarization less but also blocks firing. Motoneurons fire one spike per swimming cycle, in phase with nearby motor root discharge. Spikes are preceded by a depolarizing prepotential. This increases with hyperpolarizing current, which can block the spike to reveal an underlying depolarizing potential. In phase with motor root discharge on the opposite side of the body, motoneurons receive a midcycle inhibitory postsynaptic potential, which increases with depolarizing current, decreases with hyperpolarizing current, and is blocked by 10(-6) M strychnine. Strychnine, 5 X 10(-7) M, leads first to broadening of motor root bursts then to loss of the alternating swimming pattern of activity, which is replaced by synchronous bursts on both sides of the body. We conclude that the synaptic inputs to motoneurons during fictive swimming in spinal embryos are very similar in properties and pharmacology to those in intact embryos. These inputs, including the tonic depolarization always associated with motoneuron firing during swimming, must be at least partly spinal in origin.     

Serotonin and L-norepinephrine as mediators of altered excitability in neonatal rat motoneurons studied in vitro.

The actions of serotonin on the membrane properties of motoneurons and on the synaptic responses evoked by stimulating the segmental dorsal root have been investigated using intracellular recording in a neonatal rat hemisected spinal cord preparation in vitro. Superfusion with serotonin produced concentration-dependent depolarizations (EC50 32.1 microM) with an apparent increase in input resistance and increase in motoneuron excitability. During serotonin depolarizations an increase in membrane noise was seen. At higher serotonin concentrations repetitive firing was induced. Sensitivity to serotonin was enhanced by blockade of neuronal uptake with citalopram, when the EC50 was 1.4 microM. The depolarization was mimicked by alpha-methyl-5-hydroxytryptamine (EC50 11.7 microM). Serotonin depolarizations were blocked by ketanserin (0.1 and 1 microM), ritanserin (1 microM), spiperone (0.1 and 1 microM) and LY 53857 (1 microM). A norepinephrine-induced depolarization of motoneurons, which was mimicked by L-phenylephrine and antagonized by prazosin, is probably mediated by an alpha 1-adrenoceptor. An inhibitory action of serotonin was also apparent. The frequency and amplitude of spontaneous postsynaptic potentials and the response following dorsal root stimulation were markedly reduced. This action was mimicked by 5-carboxamidotryptamine and 8-hydroxy-2-(n-dipropylamino)tetralin, but was not antagonized by ketanserin (1 microM), ritanserin (1 microM), methiothepin (1 microM), metergoline (1 microM), spiperone (1-10 microM) or 21-009 (1-10 microM). It is proposed that the depolarization and increase in excitability of spinal motoneurons is mediated by a serotonin (5-HT2) receptor subtype.     

Slow excitatory post-synaptic potentials in myenteric AH neurons of the guinea-pig ileum are reduced by the 5-hydroxytryptamine7 receptor antagonist SB 269970

Serotonin (5-HT) is a key modulator of neuronal excitability in the central and peripheral nervous system. In the enteric nervous system, 5-HT causes a slow depolarization in the intrinsic sensory neurons, but the receptor responsible for this has not been correlated with known gene products. The aim of this study was to determine whether the newly characterized 5-HT7 receptor may participate in the 5-HT-mediated depolarization of, and synaptic transmission to, the intrinsic sensory neurons of the guinea-pig ileum. Intracellular electrophysiological recordings were made from intrinsic sensory neurons identified as myenteric AH neurons from guinea-pig ileum. 5-HT (5 microM) applied to the cell body evoked both a fast depolarization (5-HT3 mediated) and/or a slow depolarization (5-HT1P-like). The 5-HT1/5/7 receptor agonist 5-carboxamidotryptamine (5-CT) (5 microM) evoked only a slow depolarization. When the fast depolarization evoked by 5-HT was blocked with granisetron (1 microM, 5-HT3 receptor antagonist), only a slow depolarization remained; this was abolished by the 5-HT7 receptor antagonist SB 269970 (1 microM, control: 14+/-2 mV, granisetron+SB 269970: -1+/-2 mV). The slow depolarization evoked by 5-CT was also significantly reduced by SB 269970 (control: 14+/-1 mV, SB 269970: 5+/-2 mV) suggesting a 5-HT7 receptor was activated by exogenous application of 5-CT and 5-HT. Slow excitatory postsynaptic potentials evoked by stimulating descending neural pathways (containing serotonergic fibers) were reduced by SB 269970 (control: 8+/-3 mV, SB 269970: 3+/-1 mV). However, SB 269970 had no effect on slow excitatory postsynaptic potentials evoked by stimulation of circumferential (tachykinergic) pathways (control: 7+/-1 mV, SB 269970: 6+/-1 mV). These data are consistent with the presence on enteric AH neurons of functional 5-HT7 receptors that participate in slow synaptic transmission.     

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.     

Measurement and analysis of postsynaptic potentials using a novel voltage-deconvolution method

This study investigated cellular and synaptic mechanisms of cholinergic neuromodulation in the in vitro lamprey spinal cord. Most spinal neurons tested responded to local application of acetylcholine (ACh) with depolarization and decreased input resistance. The depolarization persisted in the presence of either tetrodotoxin or muscarinic antagonist scopolamine and was abolished with nicotinic antagonist mecamylamine, indicating a direct depolarization through nicotinic ACh receptors. Local application of muscarinic ACh agonists modulated synaptic strength in the spinal cord by decreasing the amplitude of unitary excitatory and inhibitory postsynaptic potentials. The postsynaptic response to direct application of glutamate was unchanged by muscarinic agonists, suggesting a presynaptic mechanism. Cholinergic feedback from motoneurons was assessed using stimulation of a ventral root in the quiescent spinal cord while recording intracellularly from spinal motoneurons or interneurons. Mainly depolarizing potentials were observed, a portion of which was insensitive to removal of extracellular Ca2+, indicating electrotonic coupling. Hyperpolarizing potentials were also observed and were attenuated by the glycinergic antagonist strychnine, whereas depolarizing responses were potentiated by strychnine. Mecamylamine also reduced hyperpolarizing responses. The pharmacology of these responses suggests a Renshaw-like feedback pathway in lamprey. Immunohistochemistry for choline acetyltransferase, performed in combination with retrograde filling of motoneurons, demonstrated a population of nonmotoneuron cholinergic cells in the lamprey spinal cord. Thus endogenous cholinergic modulation of the lamprey spinal locomotor network is likely produced by both motoneurons and cholinergic interneurons acting via combined postsynaptic and presynaptic actions.     

Mechanisms underlying excitatory effects of thyrotropin-releasing hormone on rat hypoglossal motoneurons in vitro.

1. The hypoglossal motor nucleus contains binding sites for the neuropeptide thyrotropin-releasing hormone (TRH) and is innervated by TRH-containing fibers. Although excitatory effects of TRH on hypoglossal motoneurons (HMs) have been described, the ionic mechanisms by which TRH exerts such effects have not been fully elucidated. Therefore, we investigated the effects of TRH on HMs in transverse slices of rat brainstem with intracellular recording techniques. 2. TRH was applied by perfusion (0.1-10 microM) or by pressure ejection (1.0 microM), while HMs were recorded in current or voltage clamp. In all cells tested, TRH caused a depolarization and/or the development of an inward current. These effects were fully reversible, dose dependent, and showed only modest desensitization with long applications. In addition, although TRH increased synaptic activity in many cells, the depolarizing response to TRH was maintained in tetrodotoxin (0.5-1.0 microM)-containing or in a nominally Ca(2+)-free perfusate containing 2 mM Mn2+. Thus TRH acts directly on HMs to cause the depolarization. 3. Hyperpolarizing current (or voltage) steps superimposed on the TRH-induced depolarization (or inward current) revealed a decreased input conductance. Extrapolated instantaneous current-voltage relationships obtained before and at the peak of the response to TRH intersected (i.e., reversed) at -101 mV, negative to the expected K+ equilibrium potential (EK). When extracellular [K+] was raised from 3 to 12 mM, the reversal potential was shifted in the depolarizing direction and the magnitude of the TRH-induced depolarization was diminished. Moreover, the TRH response was enhanced in size from depolarized potentials (i.e., further from EK). Taken together, these results indicate that TRH depolarizes HMs, in part, by decreasing a resting K+ conductance. 4. Similar to TRH, bath-application of 2 mM Ba2+ caused a depolarization associated with decreased conductance, suggesting that Ba2+ also blocks a resting K+ conductance. The Ba(2+)-sensitive and TRH-sensitive resting K+ conductances are apparently identical; in the presence of Ba2+, the customary TRH-induced decrease in conductance was occluded. 5. It is noteworthy that the TRH-induced inward current (ITRH), although diminished, was not entirely blocked by Ba2+. This second Ba(2+)-insensitive component of ITRH was not associated with a measurable change in input conductance. It was especially evident during current-clamp recordings, when the diminutive TRH-induced current was still capable of causing a substantial depolarization. The ionic basis of the residual TRH-induced inward current remains to be determined. 6. We investigated the functional consequences of these mechanisms of action of TRH on spike firing behavior of HMs.(ABSTRACT TRUNCATED AT 400 WORDS)     

APV-sensitive dorsal root afferent transmission to neonate rat sympathetic preganglionic neurons in vitro

1. Intracellular recordings were made from antidromically identified sympathetic preganglionic neurons (SPNs) in transverse thoracolumbar spinal cord slices from neonate (12- to 22-day-old) rats. 2. Electrical stimulation of dorsal roots or dorsal root entry zone elicited in SPNs an excitatory postsynaptic potential (EPSP) or multiple EPSPs of varying latencies. The EPSP could be graded by varying the stimulus intensity and, on reaching the threshold, discharged an action potential. 3. The dorsal root-evoked EPSPs had a mean synaptic latency of 2.6 ms (range: 1.2-11 ms), suggesting a polysynaptic pathway. The EPSPs were characteristically slow in onset with a mean rise time and half-decay time of 8.3 and 23 ms, respectively. 4. At the resting membrane potential of -50 to -60 mV, the amplitude of EPSPs recorded in normal (1.3 mM Mg2+) Krebs solution was reduced by membrane hyperpolarization or depolarization. In Mg2(+)-free solution, EPSPs were potentiated and reached threshold for spike discharge. 5. The EPSPs were suppressed by the nonselective glutamate receptor antagonist kynurenic acid (0.1-0.5 mM) and by the N-methyl-D-aspartate (NMDA) receptor antagonists D-2-amino-5-phosphonovaleric acid (APV; 1-10 microM) and ketamine (5-10 microM), but not by the quisqualate (QA)/kainate (KA) receptor antagonist 6,7-dinitroquinoxaline-2,3-dione (DNQX, 1-10 microM). The latter depressed the EPSPs elicited by stimulation of lateral funiculus in the same SPNs. 6. NMDA applied by pressure elicited a depolarization in the SPNs. In normal Krebs solution the response was voltage dependent with the peak amplitude occurring around -60 mV; conditioning depolarization or hyperpolarization diminished the response.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential roles of NMDA and non-NMDA receptors in synaptic responses of neurons in nucleus tractus solitarii of the rat.

The relative role of N-methyl-D-aspartate (NMDA) and non-NMDA receptors in synaptic responses of neurons in caudal nucleus tractus solitarii (cNTS) was delineated by immunohistochemical and electrophysiologic experiments in rats. Double immunohistochemical staining in in vivo experiments revealed that approximately 80% of cNTS neurons that showed Fos-like immunoreactivity induced by baroreceptor activation were generally also immunoreactive to non-NMDA receptor subunits GluR1 or GluR2. On the other hand, only 20% of Fos-labeled cNTS neurons showed immunoreactivity to NMDA receptor subunits NMDAR1 or NMDAR2. Stimulation of the ipsilateral solitary tract at suprathreshold intensity in slice preparations induced Fos expression in the cNTS and evoked either a single action potential or a complex synaptic response consisting of an initial action potential followed by a secondary slow depolarization. In a majority (70%) of cNTS neurons that exhibited the complex synaptic response, both the initial and secondary components were eliminated reversibly by 6-cyano-7-nitroquinoxaline-2,3-dione (20 microM). This non-NMDA antagonist also inhibited the single action potential manifested by the other population of cNTS neurons. On the other hand, only the secondary slow depolarization was blocked by D(-)-2-amino-5-phosphonopentanoic acid (250 microM) or potentiated by NMDA (1.7 microM). Our results suggested that NMDA and non-NMDA receptors are involved differentially in the synaptic responses of cNTS neurons. Non-NMDA receptors may be distributed predominantly on a majority of the second-order cNTS neurons that may receive primary baroreceptor afferent inputs. On the other hand, NMDA receptors are located primarily on higher-order neurons, which may be connected reciprocally with the second-order cNTS neurons.     

Direct and indirect actions of dopamine on the membrane potential in medium spiny neurons of the mouse neostriatum

Many studies have shown dopamine (DA) to have a modulatory effect on neuronal excitability, which cannot be simply classified as excitatory or inhibitory in the neostriatum. To clarify whether the responses to DA (10-30 microM) are excitatory or inhibitory in the mouse medium spiny neurons, we examined the effects of DA agonists on the synchronous potential trajectory from the resting potential to the subthreshold potential. The DA-induced potential changes, which were estimated at the subthreshold potential (approximately -60 mV), were summarized as the combination of three kinds of responses: an initial hyperpolarization lasting approximately 1 min and a slow depolarization and/or hyperpolarization lasting more than 20 min. A D(1)-like receptor agonist, R(+)-6-chloro-7,8-dihydroxy-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrobromide (SKF81297, 1 microM) mainly induced the initial hyperpolarization and slow depolarization. A D(2)-like receptor agonist, trans-(-)-4aR-4,4a,5,6,7,8,8a,9-octahydro-5-propyl-1H-pyrazolo[3,4-g]quinoline hydrochloride (quinpirole, 1 microM), mainly induced the initial hyperpolarization and slow hyperpolarization. D(1)-like receptor antagonist R(+)-7-chloro-8-hydroxy-3-methyl1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrochloride (SCH23390, 1 microM) depressed both the initial hyperpolarization and slow depolarization. D(2)-like receptor antagonist sulpiride (1 microM) depressed all the DA-induced responses except for the slow depolarization. TTX (0.5 microM) abolished all the DA-induced responses. Bicuculline (20 microM) and atropine (1 microM) abolished the DA-induced initial hyperpolarization and slow depolarization, respectively. Either DL-2-amino-5-phosphonopentanoic acid (AP5; 100 microM) or 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 20 microM) blocked both the initial hyperpolarization and slow depolarization. The application of exogenous glutamate (Glu) mimicked the initial hyperpolarization and slow depolarization. These results suggest that the initial hyperpolarization is mainly due to GABA release via the cooperative action of D(1)- and D(2)-like receptors and Glu receptors in GABAergic interneurons, whereas the slow depolarization is mediated by acetylcholine (ACh) release via the cooperative action of mainly D(1)-like receptors and Glu receptors in cholinergic interneurons. The potential oscillation was generated at the subthreshold level in a Ba(2+)-, AP5-, CNQX-, bicuculline-, and atropine-containing medium. The oscillation depressed after the addition of TTX, Co(2+), or DA. In DA agonists, quinpirole rather than SKF81297 had a more depressive effect on the potential oscillation. These results indicate that the slow hyperpolarization is due to the suppression of noninactivating Na(+)-Ca(2+) conductances via mainly D(2)-like receptors in the medium spiny neurons. In conclusion, the DA actions on the medium spiny neurons show a transient inhibition by the activation of D(1)- and D(2)-like receptors in mainly GABAergic interneurons and a tonic excitation and/or inhibition by the activation of mainly D(1)-like receptors in cholinergic interneurons and by the activation of mainly D(2)-like receptors in the medium spiny neurons, respectively.     

Involvement of GABA and glycine in recurrent inhibition of spinal motoneurons.

1. Recurrent inhibitory postsynaptic potentials (IPSPs) were recorded intracellularly from chloride-loaded motoneurons in the isolated lumbar spinal cord of neonatal rats (day 5-day 12). This in vitro preparation exhibited an intact and functional recurrent inhibitory pathway that displayed characteristics previously described for this pathway in other species. 2. Although strychnine (1-5 microM) depressed the chloride-dependent recurrent synaptic potentials evoked by ventral root stimulation by 48.2 +/- 2.7% (mean +/- SE, n = 13), confirming that part of the recurrent IPSP is mediated by a glycinergic mechanism, in every case a residual strychnine-resistant synaptic potential was observed. 3. The gamma-aminobutyric acid (GABA) antagonist bicuculline, in low concentrations (2-10 microM), depressed the recurrent synaptic potentials in a dose-dependent manner by 27.0 +/- 4.3% (range 0-49%, n = 19). Application of bicuculline almost eliminated the strychnine-resistant component of the IPSP. However, in some motoneurons, a small synaptic potential remained after combined application of strychnine and bicuculline. 4. The selective antagonists of GABA uptake, (+/-)-nipecotic acid (1 mM) and guvacine (1 mM), increased the amplitude of recurrent synaptic potentials in 12 of 16 motoneurons by 37.2 +/- 7.2% (range 12.6-84.2%). 5. The excitatory amino acid antagonists kynurenic acid (1 mM), 6-cyano-7-nitroquinoxaline-2,3-dione [CNQX (10 microM)] and 6,7-dinitroquinoxaline-2,3-dione (10 microM) potentiated recurrent synaptic potentials in 5 of 7 motoneurons. However, CNQX (10-15 microM) in the presence of strychnine and bicuculline virtually abolished the synaptic potential remaining after application of the inhibitory amino acid antagonists. It is concluded that ventral root stimulation evokes a small excitatory amino acid-mediated synaptic potential in neonatal rat motoneurons. 6. An antidromic synaptic potential due to electrotonic coupling between motoneurons was unaffected by changes in membrane potential, chloride loading, or antagonists of glycine, GABA, excitatory amino acid, and acetylcholine receptors. 7. The results suggest that a major portion of the strychnine-resistant component of the IPSP is mediated by a GABAergic mechanism. It is concluded that both glycinergic and GABAergic mechanisms play a role in recurrent inhibition of motoneurons in the mammalian spinal cord. It is unknown whether these inhibitory amino acids are released by a single pool of Renshaw cells or by neurochemically distinct populations.     

Excitatory postsynaptic potentials evoked by ventral root stimulation in neonate rat motoneurons in vitro

1. Intracellular recordings were made from antidromically identified motoneurons in transverse (500 microns) lumbar spinal cord slices of neonatal (12-20 day) rats. 2. Electrical stimulation of ventral rootlets evoked, with or without an antidromic spike or initial segment potential, a depolarizing response (latency, 1-4.2 ms), a hyperpolarizing response (latency, 1.5-3.5 ms), or a combination of two preceding responses in 38, 6, and 8% of motoneurons investigated. 3. The hyperpolarizing response was reversibly eliminated by low Ca2+ (0.25 mM), d-tubocurarine (d-Tc; 10 microM) or strychnine (1 microM), suggesting that this response represents an inhibitory post-synaptic potential (IPSP) mediated by glycine or a related substance release from inhibitory interneurons subsequent to their activation by axon collaterals in a manner analogous to the Renshaw cell circuitry described for the cat motoneurons. 4. The depolarizing responses were excitatory postsynaptic potentials (EPSPs), because they could be graded by varying the stimulus intensity and were reversibly abolished in low Ca2+ solution. 5. Membrane hyperpolarization increased the amplitude of EPSPs, and the mean extrapolated reversal potential was -4 mV. 6. EPSPs were augmented, rather than diminished, by dihydro-beta-erythroidine (1 microM) or d-Tc, arguing against a role of recurrent motor axon collaterals in initiating the responses. 7. The conduction velocity of the fibers initiating the EPSPs ranged from 0.35 to 0.96 m/s, indicating that these fibers were unmyelinated. Furthermore, the EPSP exhibited a constant delay when the stimulus frequency was varied from 1 to 5 Hz, and the synaptic delay estimated by extrapolation was less than 1 ms, suggesting that it was a monosynaptic event. 8. After complete separation of the ventral and dorsal horns by a knife cut, stimulation of ventral rootlets could still evoke an EPSP in motoneurons. 9. Superfusion of the slices with the nonselective glutamate receptor antagonist kynurenic acid (0.2-1 mM) or the selective quisqualate/kainate receptor antagonist 6,7-dinitroquinoxaline-2,3-dione (DNQX) (0.5-1 microM) reversibly diminished the EPSPs. 10. EPSPs evoked by stimulation of dorsal and ventral rootlets exhibited different latency and waveform in the same motoneurons. 11. The results provide evidence that activation of ventral root afferents evoked an EPSP mediated by glutamate or a related substance in a population of motoneurons. Furthermore, the afferent pathway mediating the EPSP appears to be monosynaptic and confined to the ventral horn.     

Zinc modulates primary afferent fiber-evoked responses of ventral roots in neonatal rat spinal cord in vitro

Zinc ions (Zn(2+)) are known to modulate the functions of a variety of channels, receptors and transporters. We examined the effects of Zn(2+) on the reflex potentials evoked by electrical stimulation and responses to depolarizing agents in the isolated spinal cord of the neonatal rat in vitro. Zn(2+) at low concentrations (0.5-2 microM) inhibited, but at high concentrations (5 and 10 microM) augmented, a slow depolarizing component (slow ventral root potential). Zn(2+) had no effect on fast components (monosynaptic reflex potential; fast polysynaptic reflex potential). Unlike Zn(2+), strychnine (5 microM), a glycine receptor antagonist, and (S),9(R)-(-)-bicuculline methobromide (10 microM), a GABA(A) receptor antagonist, potentiated both fast polysynaptic reflex potential and slow ventral root potential. Zn(2+) (5 microM) did not affect depolarizing responses to glutamate and N-methyl-D-aspartate. Zn(2+) enhanced the substance P-evoked depolarization in the absence of tetrodotoxin (0.3 microM) but not in its presence. The dorsal root potential was inhibited by (S),9(R)-(-)-bicuculline methobromide (10 microM) but not by Zn(2+) (5 microM). The Zn(2+)-potentiated slow ventral root potential was inhibited by the N-methyl-D-aspartate receptor antagonists, ketamine (10 microM) and DL-2-amino-5-phosphaonovaleric acid (50 microM) but not by P2X receptor antagonists, pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (30 microM) and 2',3'-O-(2,4,6-trinitrophenyl)ATP (10 microM). Ketamine (10 microM) and DL-2-amino-5-phosphaonovaleric acid (50 microM) almost abolished spontaneous activities increased by Zn(2+). It is concluded that Zn(2+) potentiated slow ventral root potential induced by primary afferent stimulation, which was mediated by the activation of N-methyl-D-aspartate receptors but not by activation of P2X receptors or blockade of glycinergic and GABAergic inhibition. Zn(2+) does not seem to directly affect N-methyl-D-aspartate receptors. The release of glutamate from interneurons may play an important role in Zn(2+)-induced potentiation of slow ventral root potential in the spinal cord of the neonatal rat.     

Alpha-1 adrenoceptor agonists generate a "fast" NMDA receptor-independent motor rhythm in the neonatal rat spinal cord

Noradrenaline, a potent activator of rhythmogenic networks in adult mammals has not been reported to produce functional rhythmic patterns in isolated spinal cords of newborn rats. We now show that a "fast" (cycle time: 1-4 s) transient rhythm was induced in sacrococcygeal (SC) and rostral-lumbar spinal segments of the neonatal rat by bath-applied noradrenaline. The fast rhythm was blocked by 1 microM of the alpha1-adrenoceptor antagonist prazosin but not by 1-20 microM of the alpha2-adrenoceptor blocker yohimbine, it could be initiated and maintained by alpha1-adrenoceptor agonists, and it was accompanied by a slow nonlocomotor rhythm. Transection at the lumbosacral junction abolished the fast-thoracolumbar (TL) rhythm while the fast-SC and slow-TL rhythms were unaffected. The N-methyl-d-aspartate (NMDA) receptor antagonist 2-amino-5-phosphonopentanoic acid (AP5) abolished the slow- and did not interrupt the fast rhythm. Thus alpha1-adrenoceptor agonists induce an NMDA receptor-independent rhythm in the SC cord and modulate NMDA receptor-dependent rhythmicity in TL segments. Injection of current steps into S(2) and flexor-dominated L(2) motoneurons during the fast rhythm revealed a 20-30% decrease in input-resistance (R(N)), coinciding with contralateral bursting. The R(N) of extensor-dominated L(5) motoneurons did not vary with the fast rhythm. The rhythmic fluctuations of R(N) in L(2) motoneurons were abolished, but the alternating left-right pattern of the fast rhythm was unchanged in midsagittally split TL cords. We suggest that the locomotor generators were not activated during the fast rhythm, that crossed-inhibitory pathways activated by SC projections controlled the rhythmic decrease in R(N) in L(2) motoneurons, and that the alternating pattern of the split TL cord was maintained by excitatory SC projections.     

Effects of endothelin-1 on the membrane potential and slow waves in circular smooth muscle of rat gastric antrum.

Electrophysiological effects of endothelin-1 (ET-1) on circular smooth muscle of rat gastric antrum were investigated by using intracellular membrane potential recording techniques. ET-1 (10 nM) caused an initial hyperpolarization of the membrane which was followed by a sustained depolarization. ET-1 also increased the frequency but not the amplitude of slow waves. In the presence of the endothelin type A (ETA) receptor antagonist, BQ123 (1 microM), ET-1 (10 nM) depolarized the membrane and increased the frequency of slow waves, but without the initial hyperpolarization. The selective endothelin type B (ETB) receptor agonist, sarafotoxin S6c (10 nM), also depolarized the membrane and increased the frequency of slow waves. In the presence of the ETB receptor antagonist, BQ788 (1 microM), ET-1 (10 nM) hyperpolarized the membrane. However, in the presence of BQ788, ET-1 caused neither the depolarization nor the increase in the frequency of the slow waves. The ET-1-induced hyperpolarization was completely abolished by apamin (0.1 microM). In the presence of apamin, ET-1 depolarized the membrane and increased the frequency of slow waves. The ET-1-induced depolarization was significantly attenuated by 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS, 0.3 mM). The increase of the frequency by ET-1 was observed both in the presence and absence of DIDS. These results suggest that, ET-1 hyperpolarizes the membrane by the activation of Ca2+-activated K+ channels via ETA receptors, and depolarizes the membrane by the activation of Ca2+-activated Cl- channels via ETB receptors. ET-1 also appears to increase the frequency of slow waves via ETB receptors, however this mechanism would seem to be independent of membrane depolarization.     

Capsazepine, a novel capsaicin antagonist, selectively antagonises the effects of capsaicin in the mouse spinal cord in vitro.

The mouse hemisected spinal cord with attached dorsal roots and spinal ganglia in vitro preparation was used to investigate the effects of the capsaicin antagonist, capsazepine (2-[2-(4-chlorophenyl)ethylamino-thiocarbonyl]-7,8-dihydroxy-2,3,4 ,5- tetrahydro-1H-2-benzazepine). The spinal cord and the ganglia were separated by a perspex gap, allowing application of drugs separately to each compartment. Intracellular recordings were made from 37 cells in laminae II-VI of 12 to 20-day-old mice. Brief applications (30 s) of capsaicin (0.8 microM) excited dorsal horn neurones by activating small diameter primary afferent fibres. The response to capsaicin administered to the spinal cord or to the spinal ganglia was antagonised by the capsaicin antagonist, capsazepine (1.5 microM), administered to the same site. Excitatory postsynaptic potentials (EPSPs) evoked by electrical stimulation of the dorsal root were not affected by capsazepine. Capsazepine itself (5 microM) did not affect the membrane potential of the dorsal horn cells. Capsazepine did not depress the depolarization evoked by substance P. When capsazepine was applied to the spinal cord and capsaicin to the dorsal root ganglion the capsaicin effect was not antagonised. These data suggest that capsaicin-induced depolarization of spinal dorsal horn neurones was mediated via activation of a specific receptor on primary afferent neurones.     

Changes in membrane potential of frog motoneurons induced by activation of serotonin receptor subtypes

Application of serotonin to the isolated, hemisected frog spinal cord resulted in two distinctive changes in motoneuron membrane potential: hyperpolarizations were produced by low concentrations (0.01-1.0 microM) and depolarizations by higher concentrations (3.0-100 microM). The hyperpolarizations appeared to be caused by a direct action of the amine upon motoneurons since exposure of spinal cord tetrodotoxin or magnesium ions in concentrations which blocked interneuronal firing and synaptic transmission, respectively did not reduce these responses. In contrast, depolarizations were significantly reduced by tetrodotoxin or magnesium indicating a large indirect component. The use of agonists and antagonists known to discriminate among different subtypes of serotonin receptors indicated that the hyperpolarizations were produced by activation of 5-HT1A receptors and the depolarizations were generated by activation of 5-HT2 and/or 5-HT1C receptors. Accordingly, the selective 5-HT1A agonists 8-hydroxy-2-(n-dipropylamino)tetralin and ipsapirone directly hyperpolarized motoneurons. The changes in potential produced by low concentrations of serotonin and by these agonists were blocked by the 5-HT1A receptor antagonists spiperone and spiroxatrine. In contrast, application of high concentrations of alpha-methyl-5-hydroxytryptamine, a serotonin analog which activates 5-HT1C and 5-HT2 receptor subtypes, depolarized motoneurons. These depolarizations, and those produced by high concentrations of serotonin, were blocked by the 5-HT1C/5-HT2 antagonists ketanserin, methysergide and mianserin. These observations indicate that serotonin can alter the membrane potential of motoneurons directly and indirectly by activation of both 5-HT1 and 5-HT2 receptor subtypes. Activation of different receptor subtypes depends upon the concentration of the amine.     

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.     

Antagonism by (1,2,5,6-tetrahydropyridine-4-yl) methylphosphinic acid of synaptic transmission in the neonatal rat spinal cord in vitro: an electrophysiological study

The effect of the novel GABAc receptor antagonist (1,2,5,6-tetrahydropyridine-4-yl)methyl-phosphinic acid (TPMPA) on synaptic transmission and GABA-mediated responses was investigated with electrophysiological recordings from the in vitro spinal cord preparation of the neonatal rat. Bath-applied TPMPA (10 microM) had no effect on spinal reflexes evoked by dorsal root stimulation, on ventral root polarization level or amplitude of ventral root depolarizations induced by exogenously applied GABA (0.5 mM). TPMPA significantly attenuated the depressant action of GABA on spinal reflexes without changing responses induced by the GABA(A) receptor agonist isoguvacine (50 microM) or the GABA(B) receptor agonist baclofen (0.5-2 microM). Following block of GABA(A) receptors by bicuculline (20 microM) and of glycine receptors by strychnine (1 microM), regular bursting activity recorded from ventral roots developed spontaneously and persisted unchanged for many hours. This bursting pattern, which is generated at the level of the interneuronal network, was significantly slowed down by TPMPA, which also increased the duration of individual bursts and the number of intraburst oscillations. These results suggest that in the neonatal rat spinal cord some functional GABAc receptors exist: their role was clearly unmasked following pharmacological block of GABA(A) (and glycine) receptors. Under these conditions GABAc receptors appeared to contribute to the excitation of spinal interneurons supporting rhythmic bursting activity.     

Development and serotonergic modulation of NMDA bursting in rat trigeminal motoneurons

The development of N-methyl-D-aspartate (NMDA)-induced burst discharge in rat trigeminal motoneurons (TMNs) between postnatal days P1 and P10 was examined using whole cell patch-clamp recording methods in brain slices. Bath application of NMDA (50 microM) induced a Mg(2+)-dependent rhythmical bursting activity starting around P8. Prior to the onset of bursting, the membrane potential depolarized and the input resistance increased. Hyperpolarization of the membrane potential with extrinsic current demonstrated a narrow window of membrane potential where maintained rhythmical burst discharge was evident. In P1-P4 neurons, NMDA application produced membrane depolarization and a minimal change in input resistance, but no burst activity at any membrane potential. Voltage-clamp analysis indicated that the bursting activity was related to the presence or absence of a voltage-dependent Mg(2+) block and induction of a negative slope conductance (NSC) region in the I(NMDA)-V relationship. Regardless of age, reduction of extracellular Mg(2+) from 1 mM to 30 microM enhanced I(NMDA) at voltages negative to -60 mV. However, in 1 mM Mg(2+), P1-P4 neurons were devoid of a prominent NSC region compared with P8-P10 neurons, suggesting that the efficacy of depolarization in unblocking the NMDA receptors increased with age. NMDA bursting was not dependent on calcium influx through voltage-gated calcium channels (VGCC) but did require a minimal concentration of Ca(2+) in the bath. Intracellular bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid application suppressed burst discharge completely, suggesting that intracellular Ca(2+) directly, or via second-messenger systems, regulates NMDA receptor activity and bursting. Interestingly, NMDA bursting could be induced in P1-P4 neurons by simultaneous bath application of serotonin (5-HT, 10 microM), which by itself did not produce bursting, suggesting an "enabling" role for 5-HT. Voltage-clamp analysis demonstrated that the NMDA/5-HT bursting resulted from induction of an NSC in the I-V relationship of total membrane current. 5-HT by itself produced no such effect. The mechanisms for this effect were due to an enhancement of the NSC region of the I(NMDA)-V relationship and reduction of a presumed leak current by 5-HT. These data indicate that NMDA bursting in trigeminal motoneurons is developmentally regulated and subject to neuromessenger modulation. Control of the Mg(2+) sensitivity of the NMDA receptor and voltage-dependent block by neuromessengers could be an effective means to control the efficacy of glutamatergic synaptic drive to motoneurons during rhythmical oral-motor activity at early postnatal ages.