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The objective of the present investigation is to study the involvement of nitric oxide (NO) in 3-nitropropionic acid (3-NPA)-induced depression of spinal reflexes. Experiments were conducted on preparations of hemisected spinal cord isolated from 4 to 8 day old rats. Stimulation of a dorsal root evoked reflex potentials (monosynaptic, MSR; polysynaptic, PSR) in the corresponding segmental ventral root. Superfusion of 3-NPA (3.4 mM) depressed the spinal reflexes in a time-dependent manner and the reflexes were abolished after 35 min. The time required to produce 50% depression of the reflexes (T-50) was 17.8+/-5.3 min for MSR and 17.5+/-2.1 min for PSR. L-NAME (Nomega-nitro-L-arginine methyl ester; 100 microM), a nitric oxide synthase inhibitor, antagonized the 3-NPA (3.4 mM)-induced depression of reflexes and increased the T-50 values (34 and 30 min for MSR and PSR, respectively) significantly (P<0.05). In addition, hemoglobin (Hb, 100 microM), a NO scavenger, blocked the 3-NPA-induced depression of reflexes significantly (P<0.05). T-50 values in Hb pretreated cords were 57 and 45 min for MSR and PSR, respectively which were greater than the cords pretreated with L-NAME. The nitrite (NO(2)(-)) content of the 3-NPA exposed cords was 84 microM/g of tissue which was significantly greater than the control (13 microM/g; P<0.05). Pretreatment of cords with L-NAME or Hb antagonized the 3-NPA-induced increase in NO(2)(-). The results indicate that NO produced by 3-NPA is involved in the 3-NPA-induced depression of spinal reflexes.

Effect of 3-nitropropionic acid (3-NPA) and ischemia (glucose- and O(2)-free solution) on synaptic transmission in hemisected spinal cord from 4 to 8 day old rats was examined in vitro. Stimulation of a dorsal root (L3-5 segments) evoked monosynaptic (MSR) and polysynaptic reflex (PSR) potentials in the segmental ventral root. Superfusion of 3-NPA (0.17-3.4 mM) depressed the reflexes in a concentration- and time-dependent manner. At 3.4 mM of 3-NPA, the reflexes were abolished by 35 min. Time required to produce 50% depression (T-50) was around 170, 80, 40 and 17 min for MSR and 110, 70, 25 and 16 min for PSR at 0.17, 0.51, 1.7 and 3.4mM of 3-NPA, respectively. Ischemia also produced a time-dependent depression of reflexes and abolished them by 35 min and the T-50 values were around 18 min. Presence of creatine phosphate (10mM) in the superfusing medium did not alter the time course of 3-NPA-induced depression of reflexes but prolonged the ischemia-induced depression. dl-2-amino-5-phosphonovaleric acid (NMDA receptor antagonist; 10 microM) failed to block the 3-NPA (3.4 mM)-induced depression of reflexes, but blocked the ischemia-induced depression. The results indicate that 3-NPA-induced depression of spinal reflexes does not involve NMDA receptors and is different from ischemia-induced depression.

The purpose of the present study was to investigate the 5-HT(2C) receptor-mediated effects on the spinal monosynaptic mass reflex activities and also its functional interactions with 5-HT(1A) receptors in anesthetized, acutely spinalized mammalian adult spinal cord in vivo. Intravenous administration of (+/-)-2,5-dimethoxy-4-iodoamphetamine hydrochloride (DOI) (0.1 mg/kg), an agonist of 5-HT(2A/2C) receptors, significantly increased the excitability of spinal motoneurons as reflected by an increase in the spinal monosynaptic mass reflex amplitude to 150-200% of the control. 5-HT(2A/2C) receptor-induced motoneuron excitability was slow, persistent and long-lasting for more than 2h that was significantly inhibited by 5-HT(2C) receptor specific antagonist SB 242084 administered 10 min prior to DOI. Simultaneous administration of DOI (0.1 mg/kg, i.v.) along with (+/-)-8-hydroxy dipropylaminotetraline hydrobromide (8-OH-DPAT) (0.1 mg/kg, i.v.) completely inhibited DOI-induced spinal monosynaptic mass reflex facilitation. In another separate study, administration of 8-OH-DPAT (0.1 mg/kg, i.v.) at the maximum response of DOI also inhibited the motoneuron's excitability; however, the inhibition lasted only for a period of 40-60 min after administration of 8-OH-DPAT, after which the spinal monosynaptic mass reflex amplitude reached its maximum level. These findings suggest that the 5-HT(2C) receptor is primarily involved in the mediation of the long-lasting excitability of spinal motoneurons and possibly interacts with its functional counterpart, 5-HT(1A) receptors in the mammalian adult spinal cord.

The adenosine A(1) receptor is involved in spinal cord antinociception. As its role at supraspinal sites is not well known, we studied the systemic effects of its agonist N-cyclopentyl-adenosine (CPA) in single motor units from adult-spinalized, intact and sham-spinalized rats. CPA was not effective after spinalization, but it was very effective in intact animals (ID50: 92+/-1.3 microg/kg, noxious pinch) and over 10-fold more potent in sham-spinalized animals (ID50 of 8.3+/-1 microg/kg). Wind-up was also inhibited by CPA. We also studied the effect of CPA in the immature spinal cord preparation, where CPA dose-dependently inhibited responses to low (IC50s: 9+/-0.7 and 7.7+/-1.3 nM) and high intensity stimulation (IC50s: 4.9+/-0.5 and 12.1+/-2 nM). We conclude that the integrity of the spinal cord is crucial for the antinociceptive activity of systemic CPA in adult rats but not in immature rats, not yet influenced by a completely developed supraspinal control.

Although analgesic action of xenon has been reported, little is known about the effect of xenon at the spinal cord, which plays a crucial role in nociceptive transmission. We studied the effect of xenon on nociceptive reflex (the slow ventral root potential) and the monosynaptic reflex in neonatal rat spinal cord in vitro in comparison with nitrous oxide. Xenon (30%) and nitrous oxide (30%) were applied for 17 min through superfusing artificial cerebrospinal fluid. Xenon and nitrous oxide significantly reduced the amplitude of nociceptive reflex by approximately 70% and approximately 25%, respectively. Xenon and nitrous oxide also significantly reduced the amplitude of the monosynaptic reflex by approximately 35% and approximately 15%, respectively. These results indicate that xenon suppressed the synaptic transmission at the spinal cord, especially those of the slow ventral root potential, which reflect nociceptive transmission.

In the spinal cord, various subtypes of serotonin (5-hydroxytryptamine; 5-HT) receptors are involved in the modulation of motor output. Although the excitatory role of 5-HT(2) receptors is known, the receptor subtypes mediating the inhibitory effect of 5-HT on monosynaptic reflex transmission remain unclear. In this study, segmental spinal reflexes were recorded to examine the receptor subtypes underlying 5-HT-mediated inhibition of monosynaptic reflex transmission in spinalized rats. Under conditions of monoamine oxidase blockade with clorgyline, the 5-HT precursor L-5-hydroxytryptophan depressed the monosynaptic reflex. 3-Hydroxybenzylhydrazine dihydrochloride (NSD-1015), a centrally active decarboxylase inhibitor, abolished this inhibition, confirming that the depression of the monosynaptic reflex by L-5-hydroxytryptophan was due to 5-HT. In the presence of GR127935 or isamoltane, which show high affinity for 5-HT(1B) receptors, L-5-hydroxytryptophan did not suppress the monosynaptic reflex, whereas 5-HT(1A), 5-HT(1D), 5-HT(2) and 5-HT(7) receptor antagonists did not alter the inhibitory effect of L-5-hydroxytryptophan. These results suggest that serotonergic depression of monosynaptic reflex transmission is mediated by 5-HT(1B) receptors.

Classical in vitro and in vivo models and electrophysiological techniques were used to investigate the role of AMPA- and NMDA-type glutamate receptors in various components of spinal segmental reflex potentials. In the rat hemisected spinal cord preparation, the AMPA antagonists NBQX and GYKI 52466 abolished the monosynaptic reflex (MSR) potential but caused only partial inhibition of the motoneuronal population EPSP. NMDA antagonists had no noticeable effect on the MSR in normal medium, but markedly depressed the late part of EPSP. However, an NMDA receptor antagonist sensitive monosynaptic response was recorded in magnesium-free medium at complete blockade of the AMPA receptors. In spinalized rats, the AMPA antagonists completely blocked all components of the dorsal root stimulation evoked potential. MK-801 (2mg/kg, i.v.) reduced monosynaptic responses in a frequency dependent way, with no effect at 0.03 Hz and 22% inhibition at 0.25 Hz. The reduction of the di- and polysynaptic reflex components was about 30% and did not depend on stimulation frequency. Long-latency reflex discharge responses, especially when evoked by train stimulation, were more sensitive to MK-801 than the polysynaptic reflex. These results suggest that glutamate activates MSR pathways through AMPA receptors. However, under certain conditions, NMDA receptors can modulate this transmission through plastic changes in the underlying neuronal circuits. AMPA and NMDA receptors play comparable roles in the mediation of longer latency reflex components.

(R)-8-Hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) depressed the monosynaptic reflex. This effect was not antagonized by 5-HT(1A) receptor antagonists. We examined whether 5-HT(1D) and 5-HT(7) receptors are involved in (R)-8-OH-DPAT-induced inhibition of the monosynaptic reflex in spinalized rats. Pretreatment with methiothepin and mesulergine, but not clozapine, inhibited (R)-8-OH-DPAT-induced monosynaptic reflex depression. Pretreatment with 2a-(4-phenyl-1,2,3,6-tetrahydropyridal)butyl)-2a,3,4,5-tetrahydrobenzo[c,d]indol-2(1H)-one (DR4004) and (R)-1-[(3-hydroxyphenyl)sulfonyl]-2-[2-(4-methyl-1-piperidinyl)ethyl]pyrolidine (SB-269970), new selective 5-HT(7) receptors antagonists, and N-[methoxy-3-(4-methyl-l-piperazinyl)phenyl]-2'-methyl-4'-(5-methyl-1,2,4-oxadiazol-3-yl)[1,1-biphenyl]-4-carboxamide (GR127935), a selective 5-HT(1D) receptor antagonist, had no effect on (R)-8-OH-DPAT-induced depression. These results suggested that 5-HT(7) and 5-HT(1D) receptors are not involved in (R)-8-OH-DPAT-induced monosynaptic reflex depression.

We examined the effects of (R)- and (S)-8-hydroxy-2-(di-n-propylamino)tetralin hydrobromide (8-OH-DPAT) on the monosynaptic spinal reflex in rats. In intact rats, (R)-8-OH-DPAT (10 microg/kg, i.v.) enhanced the amplitude of the monosynaptic reflex, whereas at 100 microg/kg, it reduced the amplitude. (S)-8-OH-DPAT enhanced the monosynaptic reflex dose-dependently. In spinalized rats, (R)-8-OH-DPAT produced dose-dependent inhibition, but the (S)-enantiomer did not affect the monosynaptic reflex. Pretreatment with spiroxatrine or 1-(2-methoxyphenyl)-4-[4-(2-phthalimido)butyl]-piperazine (NAN-190) inhibited (R)-8-OH-DPAT-induced monosynaptic reflex enhancement in intact rats, as did 5-hydroxytryptamine (5-HT) depletion. Ketanserin reduced the effect of (R)-8-OH-DPAT. These pretreatment regimens had no effect on the monosynaptic reflex depression produced by the (R)-enantiomer in intact and spinalized rats. Pretreatment with prazosin inhibited (S)-8-OH-DPAT-induced monosynaptic reflex enhancement in intact rats, as did noradrenaline and 5-HT depletion. These results suggest that supraspinal 5-HT1A receptors and the descending serotonergic system are involved in the stimulatory effect of (R)-8-OH-DPAT on the monosynaptic reflex, while both the descending serotonergic and noradrenergic systems, the latter acting via alpha1-adrenoceptors, are involved in the effect of the (S)-enantiomer on this reflex.

1. The effects of N-methyl-D-aspartate (NMDA) receptor glycine-binding site antagonists 7-chlorokynurenate (7-Clkyn) and (+/-)-3-amino-1-hydroxy-2-pyrrolidone (HA-966) on spinal reflexes in an isolated spinal cord that was maintained in Mg(2+)-free medium in vitro were examined. The actions of 7-Clkyn and HA-966 were compared with those of the channel-site antagonist (i.e., dizocilpine) and NMDA-binding site antagonists--that is, 3-[(+/-)-2-carboxypiperazin-4-yl]-propyl-1-phosphonate (CPP) and DL-2-amino-5-phosphonovalerate (APV). 2. 7-Clkyn and HA-966 produced a selective depression of the polysynaptic reflex (PSR) while negligibly affecting the activity of the monosynaptic reflex (MSR). The PSR was also differentially suppressed by dizocilpine, CPP and APV. The PSR inhibitory activity of the NMDA antagonists was in the following order: dizocilpine > CPP > APV = 7-Clkyn > HA-966. 3. The inhibitory effects of 7-Clkyn on PSR were markedly antagonized by the simultaneous application of D-serine, an agonist for the NMDA receptor glycine-binding sites. However, PSR inhibition by dizocilpine and CPP was unaffected. 4. Inhibition of the PSR by 7-Clkyn persisted in the presence of strychnine, which markedly increased the PSR activity by itself. 5. These findings suggest that the NMDA receptor glycine-binding sites play a role in generating the NMDA receptor-mediated PSR in the spinal cord in vitro.

Ethanol, usually studied in relation to intoxication, is also capable of producing general anesthesia. The most common standard of anesthetic potency is the concentration which produces immobility in response to a noxious stimulus. This concentration will be referred to as the anesthetic concentration. Immobilization is a spinal effect. Ethanol effects were studied in spinal cord from 2-7-day-old rats at concentrations which included the anesthetic concentration in both adult rats (97 mM) and 6-7-day-old rats (235 mM). At neonatal but not adult anesthetic concentrations, ethanol depressed monosynaptic reflex amplitude (mediated by glutamate AMPA receptors + compound action potential). At both neonatal and adult anesthetic concentrations ethanol reversibly depressed the population excitatory postsynaptic potential (pEPSP) (glutamate AMPA and NMDA receptors), the slow ventral root potential (NMDA + metabotropic receptors), and the dorsal root potential (GABA(A) receptors, via glutamate-excited interneurons). Effects were greater on NMDA receptor-mediated components than on AMPA-receptor-mediated components of the pEPSP and greater on NMDA than on metabotropic receptor-mediated components of the slow ventral root potential. The profile of ethanol effects on spinal cord resembles that of inhalation general anesthetics. The results show that both AMPA and NMDA receptor-mediated transmission are sensitive to ethanol and that enhancement of GABAergic neurotransmission is overridden by depression of excitation to the interneurons. They provide no obvious explanation for ethanol's lower general anesthetic potency in the neonate.

The effects of a recently synthesized benzoyl-piperidine drug that enhances currents mediated by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)-type glutamate receptors were tested on monosynaptic and polysynaptic responses in hippocampal slices of the rat. Stimulation of perforant path inputs to the dentate gyrus evoked extracellular responses in field CA1 that had latencies and laminar profiles indicating that they were relayed through the trisynaptic intrahippocampal circuit. Under control conditions, trisynaptic field excitatory postsynaptic potentials did not show larger paired-pulse facilitation than monosynaptic responses and failed to exhibit frequency facilitation. Low concentrations of picrotoxin greatly enhanced trisynaptic responses and, under these conditions, frequency facilitation was obtained. Benzoyl-piperidine-12 (250 microM) had a three-fold greater effect on the amplitude of trisynaptic responses than on monosynaptic field excitatory postsynaptic potentials, indicating that the drug's effect is amplified across the successive stages of a polysynaptic circuit. The AMPA receptor modulator did not change the frequency characteristics of monosynaptic potentials and had only a modest influence on those of the trisynaptic response. The effect of benzoyl-piperidine-12 on trisynaptic responses was significantly greater when GABAergic inhibition was partially blocked with picrotoxin; the GABA blocker did not alter the effects of benzoyl-piperidine-12 on monosynaptic responses. These results indicate that centrally active AMPA receptor modulators are likely to have a greater influence on brain operations involving long chains of connections than on those mediated by simple reflex-like circuits, and will vary markedly in their effects depending upon the excitability of local interneurons.

The centrally acting muscle relaxant cyclobenzaprine was thought to be an alpha 2-adrenoceptor agonist that reduced muscle tone by decreasing the activity of descending noradrenergic neurons. In the present study, we examined the effects of cyclobenzaprine on descending neurons by measuring the monosynaptic reflex in rats. Cyclobenzaprine reduced the monosynaptic reflex amplitude dose dependently and this effect was not inhibited by the alpha 2-adrenoceptor antagonists idazoxan and yohimbine. Cyclobenzaprine-induced monosynaptic reflex depression was not attenuated by noradrenergic neuronal lesions produced by 6-hydroxydopamine. However, cyclobenzaprine inhibited monosynaptic reflex facilitation induced by (+/-)-1-(4-iodo-2,5-dimethoxyphenyl)-2-aminopropane, a 5-HT2 receptor agonist, in spinalized rats markedly, and 5-HT depletion by DL-p-chlorophenylalanine inhibited the depressive effect of cyclobenzaprine on the monosynaptic reflex. These results suggest that cyclobenzaprine is a 5-HT2 receptor antagonist and that its muscle relaxant effect is due to inhibition of serotonergic, not noradrenergic, descending systems in the spinal cord.

The efficacy of thyrotropin-releasing hormone (TRH) and its analogs to potentiate the spinal monosynaptic reflex was studied in isolated cords. The analogs examined were L-pyro-2-aminoadipyl-histidyl-thizolidine-4-carboxyamide (MK-771); pyroglutamyl-histidyl-prolineamide (TRH); pyroglutamyl-L-histidyl-3,3'-dimethyl-prolineamide (RX77368); (3-methyl-His2)TRH(methyl-TRH); gamma-buturolactone-gamma-carbonyl-histidyl-prolineamide citrate (DN-1417); pyroglutamyl-histidyl-proline (TRH-free acid); and histidyl-proline-diketopiperazine (cyclo(His-Pro)). The TRH analogs potentiated the monosynaptic reflex in a dose-dependent manner and the maximal potentiation occurred at about 1 microM. TRH-free acid potentiated the monosynaptic reflex but the maximal potentiation occurred at 100 times the TRH concentration. Cyclo(His-Pro) was totally ineffective. The concentration required to potentiate the monosynaptic reflex by 50% of the maximal response (EC50) was taken as an index for comparing various analogs in relation to TRH. The EC50 values of the analogs did not differ significantly from each other. However, the ratio of the mean value of an analog to that of TRH was of the following order: MK-771 (N- and C-terminally altered) > or = TRH > or = DN-1417 (N-terminal) > or = methyl-TRH > or = RX77368 (C-terminal) >>> TRH-free acid. Cyclo(His-Pro) was ineffective.

We examined the effects of intrathecally administered neuropeptide Y on the spinal nociceptive flexor reflex in decerebrate, spinalized, unanesthetized rats with intact sciatic nerves, or 11-39 days after unilateral transection of the sciatic nerve. In rats with intact sciatic nerve, intrathecal neuropeptide Y at low doses (10 and 100 ng) caused a brief facilitation of the flexor reflex. At a dose of 300 ng, the effect of neuropeptide Y on the flexor reflex was biphasic, i.e. a brief facilitation followed by slight depression. At higher doses (1 and 10 micrograms), the effect of neuropeptide Y was mainly inhibitory, causing substantial and usually prolonged depression of the flexor reflex magnitude. The reflex depression caused by intrathecal neuropeptide Y was not reversed by the opioid antagonist naloxone or the alpha 2 adrenoceptor antagonist atipamezole. Intrathecal neuropeptide Y at doses up to 1 and 10 micrograms had no effect on reflex facilitation caused by conditioning stimulation of C-fibers, intrathecal substance P or neurokinin A. Topical application of neuropeptide Y (1 microgram/microliter) failed to influence the monosynaptic reflex in normal rats. Eleven to 16 days after peripheral axotomy, the initial excitation of the flexor reflex to intrathecal neuropeptide Y was significantly enhanced in axotomized compared with normal rats. However, the depressive effect of neuropeptide Y on the flexor reflex was unchanged. Neuropeptide Y did not influence the monosynaptic reflex in axotomized rats at this period. In experiments performed on rats in which the sciatic nerve had been transected 31-39 days previously, the facilitatory effect of neuropeptide Y on the flexor reflex remained enhanced compared with normal rats. Furthermore, the inhibitory effect of neuropeptide Y also increased as 100 ng intrathecal neuropeptide Y was able to produce reflex depression in a similar fashion as 300 ng neuropeptide Y normally and the reflex depression caused by 1 microgram neuropeptide Y was stronger and longer lasting than in normal rats. Intrathecal neuropeptide Y (100 ng-10 micrograms) in rats with intact sciatic nerves caused a moderate decrease in spinal cord dorsal surface blood flow as measured with a laser Doppler flowmeter. This effect of neuropeptide Y was unchanged in axotomized rats. The present results support previous observations that spinal application of neuropeptide Y in normal rats caused antinociception. As the depressive effect of neuropeptide Y is independent of spinal opioid and alpha 2-adrenergic systems, it may be mediated by its own receptors.(ABSTRACT TRUNCATED AT 400 WORDS)

The long-lasting descending inhibition of lumbar segmental reflexes in the neonatal rat spinal cord has been investigated in vitro by recording from lumbar ventral roots on stimulation of a single lumbar dorsal root. Descending inhibition was elicited by a single stimulus to the latero-ventral thoracic cord. A number of strategies were used to clarify the role of 5-hydroxytryptamine (5-HT) in inhibiting the monosynaptic reflex, the ipsilateral polysynaptic response and the contralateral fast response evoked on the opposite side of the lumbar cord. The 5-HT uptake inhibitor, citalopram (10 nM), potentiated both short-interval (0.5-2 s) inhibition and long-interval (5-100 s) inhibition of the monosynaptic reflex, and also inhibition of the polysynaptic response 10-100 s after the thoracic stimulus. Inhibition of the monosynaptic reflex was blocked by ketanserin (1 microM), spiperone (1 microM) and methiothepin (1 microM), but not by spiroxatrine (0.1 microM) or sulpiride (1 microM). Sumatriptan (20 nM) and methysergide (10 nM) enhanced inhibition of the monosynaptic reflex 0.2-1 s after the thoracic stimulus. It was concluded that 5-HT acting through 5-HT2A/2C receptors is the transmitter responsible for monosynaptic reflex inhibition, at intervals of 0.5-100 s, but a stronger stimulus to the thoracic cord may elicit a non-serotonergic component at intervals of 0.1-2 s. There was no unequivocal evidence that endogenous 5-HT activates 5-HT1 receptors to produce inhibition.(ABSTRACT TRUNCATED AT 250 WORDS)

Pituitary adenylate cyclase activating peptide (PACAP) is a novel vasoactive intestinal peptide-like peptide. It has a neuronal localization and occurs in two forms, PACAP-38 and the C-terminally truncated PACAP-27. In a recent report we described a dense accumulation of PACAP-27-immunoreactive nerve fibres in the superficial layer of the dorsal horn in the spinal cord and a few PACAP-27-immunoreactive nerve cell bodies in the spinal ganglia in the rat. Double-immunostaining showed that immunoreactive PACAP-27 occurred in a subpopulation of the calcitonin gene related peptide/substance P-containing cell bodies and fibres. In this study, PACAP-27 (0.47-0.63 pmol) given intrathecally produced a significant and long-lasting suppression of C-fibre-evoked flexion reflex produced by the electrical stimulation of the plantar nerve and recorded as a reflex discharge in the peroneal nerve. With a higher dose, 1.25 pmol, the magnitude of the response was not increased further but the effect became longer-lasting. PACAP-38 and vasoactive intestinal peptide were tested in doses up to 5 pmol and produced a significant and, in the case of PACAP-38, long-lasting suppression of the spinal flexion reflex. PACAP-27 seemed to be much more potent than PACAP-38 and vasoactive intestinal peptide. Intrathecal PACAP-27 was without effect on the monosynaptic stretch reflex (evoked by electrical stimulation of the gastrocnemius nerve and recorded as a reflex discharge in the sciatic nerve), suggesting that its effect on the flexion reflex is specific.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of substance P and the selective neurokinin-1 receptor antagonist (+/-)-CP-96,345 have been compared on in vitro spinal cord preparations from the rat and the gerbil. Substance P produced a concentration-dependent depolarization of motoneurons recorded from ventral roots of both species. The EC50 values (microM mean +/- S.E.M.) obtained in rat (0.95 + 1.0/-0.49) and gerbil (0.47 + 0.26/-0.17) preparations were comparable. The mean maximal depolarization (mV mean +/- S.E.M.) evoked in rat (2.07 + 0.26/-0.25) was approximately two-fold greater than that evoked in gerbil (1.21 + 0.15/-0.14) preparations. In the rat substance P had a biphasic effect (depression followed by potentiation) on the short latency probably monosynaptic reflex evoked by electrical stimulation of a dorsal root. In gerbil preparations substance P produced only potentiation of the monosynaptic reflex. The EC50 values (microM) mean +/- S.E.M.) for this potentiating action in rat (0.97 + 0.75/-0.43) and gerbil (0.46 + 3.6/-0.4) preparations were similar. This potentiation demonstrates a positive modulation of an endogenous excitatory probably glutamatergic transmission by substance P in the ventral horn of the spinal cord. The depressant phase observed in rat preparations may be related to the relative immaturity of myelination in rat ventral root fibres compared to the gerbil. The selective neurokinin-1 antagonist (+/-)-CP-96,345 was one hundred-fold less potent as an antagonist of substance P-induced depolarizations in the rat (pA2 4.69 +/- 0.18, n = 7) than in the gerbil (pA2 6.79 +/- 0.16, n = 5) spinal cord. This finding suggests that (+/-)-CP-96,345 may not act solely at the neurokinin-1 recognition site. In conclusion this study demonstrates that substance P modulates the monosynaptic reflex in the spinal cord presumably via activation of neurokinin-1 receptors.

Dopaminergic influence on spinal monosynaptic transmission was examined in rats. Monosynaptic mass reflex (MMR) was recorded from the ventral root L6 following supramaximal stimulation (0.2 Hz; 0.1 ms) to the ipsilateral dorsal root L6 in spinalized rat under pentobarbitone sodium (40 mg/kg, i.p.) anaesthesia. MMR was inhibited by intravenous administration of the dopaminergic agonist, apomorphine (50-200 ug/kg) in a dose-dependent manner. The attenuatory effect of apomorphine (200 ug/kg i.v.) on the reflex could be reversed by the dopaminergic antagonist haloperidol (0.5 mg/kg, i.v.). Under tetanic stimulation (200 Hz; 15s), the pretetanic relative inhibition induced by apomorphine (200 ug/kg, i.v.) was increased only for a short period immediately after the cessation of tetanic stimulation. The results indicate existence of presynaptic dopamine receptors on the afferent terminals converging on the motoneurone which may functionally modulate the spinal motor output.

In order to examine the effect of chronic neuroleptics on spinal dopaminergic system, rats were treated with haloperidol (0.5 mg/kg IP) for 21 days and the monosynaptic mass reflex (MMR) as well as dopamine (DA) metabolism were investigated. MMR, recorded from ventral root L6 following supramaximal stimulation to ipsilateral dorsal root L6 in spinalized rats, were found to be unaffected following chronic haloperidol treatment when compared to control. Apomorphine (0.1 mg/kg IV) caused 10-20% depression of MMR in control animals which was augmented to 40-50% in chronically haloperidol-treated animals suggesting an upregulation of DA receptors in the spinal cord. DA content of lumbar region of the spinal cord was unaffected whereas its major metabolite, homovanillic acid, was significantly reduced in chronic haloperidol-treated animals. This decreased utilization of DA may compensate the upregulation of DA receptors to maintain the physiological homeostasis of the spinal dopaminergic system.

Alpha 2-Adrenoceptors mediate analgesia in vivo. The present study explored the actions of the alpha 2-adrenoceptor agonists dexmedetomidine and clonidine on a nociceptive response in isolated neonatal rat spinal cord. Stimulation of a dorsal root generates a slow ventral root potential (slow VRP) at the corresponding ipsilateral ventral root. The slow VRP meets several criteria for a nociceptive response. Dexmedetomidine (10 nM) and clonidine (200 nM) depressed the slow VRP by approximately 80%. Dexmedetomidine's action was approximately linear over the concentration range 0.5-500 nM, whereas clonidine (20 nM-5 microM) exerted biphasic effects. The profile of agonist and antagonist effectiveness characterized the receptor(s) as alpha 2-adrenoceptors; the subtype could not be identified as either alpha 2A or alpha 2B. Naloxone pretreatment partially blocked dexmedetomidine's effect, suggesting a possible endogenous opiate involvement. Dexmedetomidine (0.5-2.0 nM) also depressed the VRP evoked by application of substance P to the cord, implicating postsynaptic as well as possible presynaptic actions. At high concentrations, dexmedetomidine (50-500 nM) depressed the monosynaptic reflex, probably through non-alpha 2-receptor(s). Results from the neonatal spinal cord correlate well with those from in vivo analgesia studies. They suggest an important direct spinal contribution to alpha 2-adrenoceptor-mediated analgesia.

1. In the lumbar spinal cord, 10(-6) M bicuculline reduces the inhibition of the dorsal root-ventral root (DR-VR) reflex produced by lateral stimulation of the spinal cord. The inhibition returns on washing in control artificial cerebrospinal fluid (ACSF). 2. Baclofen (10(-6) M) inhibits the monosynaptic DR-VR reflex which returns on washing in control ACSF. 3. This suggests an involvement of GABAA receptors in the lateral inhibition and GABAB receptors in the DR-VR reflex system. 4. The increased activity seen following bicuculline or tubocurarine probably resets the "set point" of neural activity to a lower level.

The effect of i.v. morphine on the spinal nociceptive flexor reflex was examined in decerebrate, unanesthetized rats in which the spinal cord was acutely or chronically transected. In acutely spinalized rats i.v. morphine dose dependently depressed the flexor reflex with an ED50 of 1.4 mg/kg. In rats in which the spinal cord was transected 1-3 days prior to the electrophysiological experiments, i.v. morphine had a similar depressive effect (ED50 = 1.1 mg/kg). However, 4-6 days after spinalization there was a 4-fold increase in morphine's depressive effect (ED50 = 0.35 mg/kg). The sensitivity to morphine was further increased 7-10 days post-spinalization with ED50 of 0.17 mg/kg. This supersensitivity was maintained up to 30-60 days. The reflex depression caused by morphine in both acutely and chronically spinalized rats could be reversed by naloxone. Morphine i.v. did not influence the monosynaptic reflex in either acutely or chronically spinalized rats. The present results support the clinical finding that patients with spinal lesions have an increased sensitivity to the antinociceptive and reflex depressive effects of systemic and intrathecal morphine.

The effects of GYKI 52466, a new 2,3-benzodiazepine with muscle relaxant and anticonvulsant properties, were investigated and compared to those of midazolam in electrophysiological experiments. The effects of the drugs on the reflex potentials evoked by afferent nerve stimulation and recorded from the spinal roots in unanesthetized spinal cats were studied. GYKI 52466 exerted a strong inhibitory effect on the monosynaptic as well as the polysynaptic ventral root reflexes, while the dorsal root responses decreased slightly. In contrast, midazolam markedly enhanced the dorsal root responses, did not modify the monosynaptic reflex and partially inhibited the polysynaptic reflex. The spontaneous firing of cerebellar Purkinje cells was depressed by midazolam, but not by GYKI 52466. These results suggest strongly that, contrary to the classical 1,4-benzodiazepines, potentiation of the GABA-A receptor-mediated inhibition does not play a significant role in the pharmacological actions of GYKI 52466.

beta-beta'-iminodipropionitrile (IDPN) neurotoxicity is morphologically characterized by the presence of giant axon swellings in the first proximal internodes of motor axons. The electrophysiological consequences of these proximal giant axon swellings on monosynaptic reflexes and recurrent inhibition were investigated along dorsal root-ventral root, medial gastrocnemius-ventral root and soleus-ventral root pathways of IDPN-intoxicated cats 35, 50 and 100 days following initial administration of the toxin (50 mg/kg/wk for 5 weeks). Monosynaptic reflex action potentials, normally relatively synchronous spike potentials, frequently appeared as doublet potentials which did not represent temporal fractionation of the spike potential. Latencies to the monosynaptic reflex action potentials were prolonged. Amplitudes of unconditioned monosynaptic reflex action potentials were significantly decreased at all time points, although there was a tendency for the amplitudes to recover with time. Post-tetanic potentiation was variously altered. Recurrent inhibition was reduced by 42-49% along monosynaptic reflex pathways. These results demonstrate that electrophysiological function in the spinal cord of IDPN-intoxicated cats is profoundly altered and the dysfunction partly results from the presence of the giant axon swellings.

Group la primary afferent terminal (PAT) function was assessed in cats with acrylamide neuropathy. Spinal monosynaptic input-output relationships, critical input, the ability to maintain repetitive discharge at low frequencies, and apparent transmitter turnover parameters were determined in animals with subthreshold, moderate and severe degrees of acrylamide neuropathy (75, 150, and 300 mg/kg, respectively. The monosynaptic pathways of the soleus and medial gastrocnemius motoneuron pools were elicited by stimulation of their respective muscle nerves. The threshold for activation of the monosynaptic reflex (MSR) evoked by both SOL and MG nerves was increased 50% (critical input), regardless of dose regimen. Repetitive stimulation of the MSR at low frequencies (2-10 Hz) produces a slow decline in amplitude until it reaches a plateau. The height of the plateau attained in neuropathic cats was considerably lower than the controls. Determination of apparent transmitter turnover parameters in neuropathic cats showed that the constant fraction of neurotransmitter released from group la PAT remained the same while the rate of replenishment of transmitter available for release was attenuated in both SOL-VR and MG-VR pathways. The increase in the critical input and decrease in the rate of replenishment points up a functional defect in a group la PAT in cats with acrylamide neuropathy.

Dopamine (DA) is thought to have a neurotransmitter role in the spinal cord of the rat. Intrathecal administration of the DA receptor agonist apomorphine has previously been shown to reduce nocifensive responses. The present experiments investigated the site of action of apomorphine, and the mechanisms by which DA agonists apparently produce antinociception. Small doses of apomorphine (40-80 micrograms/kg) increased the tail flick latency (TFL) in lightly anaesthetised rats when given intrathecally and intravenously but not intracerebroventricularly. This effect is probably mediated via D2 receptors since the D2 agonist LY171555 had a similar effect whereas the D1 agonist SK&F 38393 was inactive. Furthermore the D2 antagonist sulpiride blocked the effects of apomorphine and LY171555. The spinal monosynaptic reflex was not modified by 150 micrograms/kg apomorphine suggesting that sensory rather than motor processes are being influenced. Pretreatment with the serotonin receptor antagonist methysergide prevented the apomorphine induced increase in TFL. It is concluded that exogenously applied DA agonist can result in antinociception in the spinal cord and that this effect may be dependent upon activity in the spinal 5-hydroxytryptaminergic and noradrenergic systems.

The effects of conditioning stimulation at various positions in the pons and the medulla on the lumbar spinal monosynaptic reflex (MSR) were examined in rats. The conditioning stimulation facilitated the MSR. Facilitation with long latency was completely blocked by phenoxybenzamine, chlorpromazine or haloperidol. Phenoxybenzamine and chlorpromazine inhibited the MSR. Haloperidol, on the other hand, augmented the MSR, and this effect became more potent after the brain stem had been transected at the upper part of the medulla oblongata. These results suggest that pontine noradreneric systems have a facilitatory influence on the MSR via alpha-adrenoceptors, and that alpha-blockers exert an inhibitory effect on the MSR by blocking these receptors.

Conditioning stimulation of the nucleus raphe in the rat medulla enhanced the monosynaptic reflex (MSR) and depressed the polysynaptic reflex (PSR) in separate time courses. The PSR decreasing effect was reduced in preparations pretreated with reserpine, PCPA, or 5,6-DHT. The MSR increasing effect was not altered by these three pretreatments. Intravenously administered LSD but not methysergide reduced the PSR decreasing effect with no influence on the MSR increasing effect. These two drugs had similar effects on the unconditioned responses; the MSR was decreased and the PSR was increased. Thus, the PSR decreasing effect seems to be mediated via a serotonergic pathway probably descending from the nucleus raphe.

1. The functional significance of subtypes of 5-hydroxytryptamine (5-HT) receptors was studied in the rat spinal reflex pathway. 2. Ketanserin had no effect on the mono- (MSR) or polysynaptic reflex (PSR) in spinal rats, but decreased the PSR in intact rats. 3. 8-Hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) and 5-methoxy-N,N-dimethyltryptamine (5-MeODMT) decreased the MSR and increased the PSR in spinal rats. 4. Ketanserin antagonized the effects of 5-MeODMT without antagonizing the effects of 8-OH-DPAT. 5. Cinanserin had similar effects to those of ketanserin. 6. These results suggest that both 5-HT1A and 5-HT2 receptors mediate MSR inhibition and PSR augmentation in the spinal reflexes of spinal rats, and that the 5-HT2 receptor has a supraspinal tonic excitatory influence on the PSR in intact rats.

1. The effects of phencyclidine (PCP) on spinal reflexes and the excitability of motoneuron somata were studied in rats. 2. PCP decreased both the monosynaptic reflex (MSR) and the polysynaptic reflex (PSR) in spinal rats as well as in intact rats. 3. The effects of PCP were not antagonized by monoaminergic and cholinergic antagonists. 4. A low dose of PCP antagonized the PSR augmentation but not the MSR augmentation caused by N-methyl-D-aspartate (NMDA). 5. In the excitability test, PCP decreased not only the monosynaptic response but also the excitability of motoneuron somata. 6. These results suggest that the inhibitory effects of PCP on spinal reflexes are not due to mediation of the monoaminergic and cholinergic systems, but partly due to its blockade of NMDA-type receptors and also to a hyperpolarizing action or a membrane-stabilizing action on the motoneuron soma.

Chlorpromazine (CPZ) depressed mono-(MSR) and polysynaptic reflexes in anesthetized rats. The MSR remained unaltered after spinal transection (C1). After spinal transection, the inhibitory effect of CPZ on the MSR disappeared. After CPZ injection, spinal transection enhanced the MSR to pre-drug level. Reserpine pretreatment abolished the inhibitory effect of CPZ on the MSR. Phenoxybenzamine inhibited the MSR and, when pre-injected, markedly attenuated the effect of CPZ on the MSR. These results suggest that in anesthetized rats, CPZ blocks the alpha-adrenoceptor-mediated facilitatory component of the spontaneous tonic influence on the MSR.

Destruction of descending serotonergic nerve terminals containing thyrotropin-releasing hormone (TRH) was affected in rats by the intracisternal injection of 5,6-dihydroxytryptamine (5,6-DHT) two weeks before subsequent experiments. Although the level of TRH in the lumbar enlargement was significantly reduced in 5,6-DHT-treated rats, the effects of TRH on the monosynaptic reflex (MSR) and the polysynaptic reflex (PSR) in these rats were no different from those in control rats. MSR inhibition by 5-methoxy-N,N-dimethyltryptamine (5-MeODMT) was attenuated by 5,6-DHT treatment although there was no obvious difference in the effects of 5-MeODMT on the PSR between 5,6-DHT-treated and control rats. In 5,6-DHT-treated rats, L-5-hydroxytryptophan (5-HTP) markedly decreased the MSR and increased the PSR although the same doses of 5-HTP did not produce any effects on either the MSR or the PSR in control rats. In control rats, after administration of imipramine or clorgyline, 5-HTP produced effects similar to those observed in 5,6-DHT-treated rats. These results suggest that the supersensitivity to 5-HTP in 5,6-DHT-treated rats is due to a lack of 5-hydroxytryptamine (5-HT) uptake into 5-HT-containing nerve terminals rather than to a change in 5-HT receptors.

The effects of thyrotropin-releasing hormone (TRH), 5-methoxy-N,N-dimethyltryptamine (5-MeODMT) and L-5-hydroxytryptophan (5-HTP) were studied on the monosynaptic reflex (MSR) and the polysynaptic reflex (PSR) in acute and chronic spinal rats. Radioimmunoassay showed that while chronic spinal transection (for 2 weeks) caused the complete depletion of TRH in the ventral lumbar enlargement a certain level of TRH was maintained in the dorsal lumbar enlargement. This result suggested the existence of TRH-containing neurons in the dorsal horn other than the medullary raphe neurons descending to the spinal cord. The latency to the start of the MSR was shortened in chronic spinal rats and the amplitudes of the MSR and PSR were significantly greater than those in acute spinal rats. There was no obvious difference in the effects of TRH and 5-MeODMT on spinal reflexes of acute and of chronic spinal rats although marked supersensitivity to 5-HTP was observed in both the MSR and the PSR in chronic spinal rats. The supersensitivity to 5-HTP was considered to be due to a lack of 5-hydroxytryptamine (5-HT) uptake into 5-HT-containing nerve terminals rather than to a change in 5-HT receptors. It is suggested that TRH and 5-HT do not show any mutual requirement for each other in their effects on the spinal reflex since co-depletion of TRH and 5-HT did not change the effects of TRH and 5-MeODMT in chronic spinal rats. The coexistence of 5-HT and TRH in the descending spinal pathway is not considered to be significant for the control of spinal reflexes at the postsynaptic level.

The brain stem areas involved in the inhibitory effect of chlorpromazine (CPZ) on the monosynaptic reflex (MSR) in the lumbar spinal cord of rats were investigated following brain stem transection and focal thermo-lesioning. Transection of the medulla oblongata markedly reduced the effect of CPZ, while transection in the mid-brain or more anterior level did not alter the effect of CPZ. With regard to focal lesions at various sites of the brain stem, lesioning of the medial portion of the medulla oblongata most effectively attenuated the MSR inhibition by CPZ and also attenuated the MSR inhibition induced by phenoxybenzamine. Bilateral lesioning of the locus coeruleus did not attenuate the MSR inhibition by CPZ. These results suggest that the pathway involved in the inhibitory effect of CPZ on the MSR originates in the pons and passes through the medial portion of the medulla oblongata, and that the coerulo-spinal pathway is not the major pathway involved in the effect of CPZ.

The electrophysiology of organophosphorus induced delayed neurotoxicity was studied to determine the extent peripheral nerve changes affect monosynaptic reflex responses of the spinal cord. Cats were given a single dermal dose of 1500 mg/kg or two dermal doses of 500 mg/kg of tri-o-cresyl phosphate. Animals were observed for 60 days after which monosynaptic reflex (MSR) responses were recorded from L7 and S1 ventral roots after stimulation of the tibial or common peroneal nerves. Post-tetanic potentiated responses as well as ventral and dorsal root compound action potentials were also recorded. There was a significant decrease from control in the unconditioned MSR. The post-tetanic potentiated response was significantly decreased in the S1 ventral root, however, when expressed as a factor of potentiation of the unconditioned MSR, it was found to be increased, although not significantly. The dorsal and ventral root compound action potentials were also significantly decreased from control with their conduction velocities being no different from control The decrease in the unconditioned MSR as well as the dorsal and ventral root compound action potentials were attributed to peripheral nerve damage. The absence of any significant change in the post-tetanic potentiated response expressed as a factor of potentiation of the unconditioned MSR was attributed to a decrease in both the discharge zone as well as the subliminal fringe. The absence of any change in the conduction velocity indicates at least some of the large myelinated peripheral fibers were spared from significant damage.