Motoneuron depolarizations, paroxysmal activity, and reflex changes induced by rapid cooling of toad spinal cord

Vol. 112A.Nos. 314, pp. 517-525, 1995 Copyright 0 1995Elsevier Science Inc. Printed in Great Britain. All rights reserved 0300-%29/95$9.50+ .OO

Camp. Biochem. Physiol.

Pergamon 0300-9629(95)02021-B

Motoneuron depolarizations, paroxysmal activity, and reflex changes induced by rapid cooling of toad spinal cord Nelson L. Dal&* John C. Hackman Department of Neurology, Spinal Cord Pharmacology U.S.A.

and Robert A. Davidoff

University of Miami School of Medicine and Neurophysiology and Laboratories, Veteran’s Affairs Medical Center, Miami, FL 33101,

Sucrose gap recordings from the ventral roots of isolated, hemisected South American toad spinal cords were used to investigate the effects on motoneurons of rapid cooling from 21-22°C to 643°C. Such cooling produced large ventral root depolarizations that slowly decayed to baseline. During the repolarization, a series of large, slow, paroxysmal, motoneuron depolarizations were seen. Upon rewarming, large, long-lasting hyperpolarizations (unaffected by dihydro-ouabain) were observed. Cold markedly increased the duration of reflexes evoked by dorsal root stimulation. Mg’+ or tetrodotoxin reduced cold-induced motoneuron depolarizations by about 50%. Kynurenate or 1-2-amino-7-phosphono-heptanoic acid reduced cold-induced depolarizations by approximately the same amount and eliminated paroxysmal activity suggesting that synaptic effects caused by the release of excitatory amino acid transmitters and the subsequent activation of NMDA receptors are necessary for the generation of about one half of the cold-induced motoneuron depolarization, and for the production of paroxysmal motoneuron activity. Nonsynaptic mechanisms and cold-induced alteration of the resting membrane potential appear partly responsible for the remainder of the cold-induced depolarizations. Key words: Epileptiform cooling; toad. Comp. Biochem.

activity;

Excitatory

Physiol. 112A, 517-525,

amino acids; Motoneurons;

Spinal cord; Rapid

199.5.

Introduction Under appropriate conditions, the spinal cord, isolated from supraspinal structures, is capable of displaying intense, self-sustained convulsive activity that is very similar to that observed in intact animals (Schwindt and Grill, 1984; Somjen et al., 1978). In particular, con-

to: Robert A. Davidoff, M.D. Deoattme& of Neurology (D4-5). University of Miami Sc’hool of Medicine, P.O. Box 16960. Miami. FL 33101. U.S.A. Tel. (305) 243-6228; Fax (305) 545-7166. *Present Address: Department of Basic Sciences, Universidad Centroccidental, Barquisimeto 3001, Venezuela. Received 2 March 1995; revised and accepted 23 June 1995. Correspondence

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vulsants such as penicillin, strychnine, and bicuculline that cause seizures when applied to rostra1 CNS structures evoke seizures in both cat and amphibian spinal cords (Ryan et al., 1984; Schwindt and Crill, 1984; Somjen et al., 1978). Spinal seizures can also be produced by rapidly cooling the spinal cord in certain southern amphibians that are not normally exposed to cold environments (Da16 and Larson, 1991; Oz6rio de Almeida, 1943; Oz6rio de Almeida et al., 1943). The present experiments were performed to provide data about the electrophysiological mechanism and pharmacology of such coldinduced spinal seizures. For this purpose, we used DC sucrose gap recording from the ven-

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tral roots of the isolated, super-fused, hemisected toad spinal cord to determine the changes in motoneuron potential and activity produced by spinal cord cooling. A preliminary account of some of these findings has been reported (Da16 er al., 1993).

Materials and Methods Experiments were performed on the spinal cords of adult toads (Bufo marinus) weighing 90 to 180 g (purchased from Lemberger, Oshkosh, WI, U.S.A.). These toads were captured in Santo Domingo in SeptemberOctober, kept in an amphibian facility at 21-22°C and fed with crickets until used. Toads were anesthetized with urethane (0.5 g/kg) (Pina-Crespo and Dal& 1992) After decapitation and laminectomy, the lumbar cord with attached IXth and Xth ventral and dorsal roots was quickly removed, and hemisected sagittally. A hemicord was mounted in a sucrose gap recording chamber (Barker et al., 1975) where the spinal cord and the intramedullary portion of the roots were continuously superfused at 10 ml/min with HCO;-buffered Ringer’s solution of the following composition: (in mM) NaCl, 114; CaCl,, 1.9; KCl, 2.0; NaHCO,, 10; glucose, 5.5. Mg2+ was omitted from the medium (except as noted in Results) to facilitate study of responses mediated by N-methyl-D-aspartate (NMDA) receptors (Ault et al., 1980). The medium was maintained at pH 7.4 by gassing it with 95% 02/5% CO, and kept at room temperature (21-22°C). After mounting in the chamber, spinal cords were kept in normal Ringer’s solution at room temperature for at least 1.0 hr before testing their reflex responses. Differential DC recordings of the electrotonically-conducted changes in the membrane potential of spinal motoneurons were obtained from the IXth ventral root (VR) placed across a 3-mm sucrose gap (Davidoff and Hackman, 1980). Calomel electrodes connected via agarRinger bridges were used to measure the difference in potential between the spinal cord bath and the end of the VR maintained in a pool of Ringer’s solution. The preparation was left ungrounded. The signals were amplified and recorded on a rectilinear pen recorder (model 2400 Gould Inc., Cleveland, OH, U.S.A., rise time <8 msec). The fibers in the IXth dorsal root (DR) were stimulated with supramaximal 1.O ms rectangular pulses delivered via bipolar silver/silver chloride electrodes applied to the sciatic nerve, which had been left in continuity with the DR. Electrotonically-controlled solenoid valves were used for rapid switching between

Ringer’s solution at room temperature, cold Ringer’s solution, and warm and cold Ringer’s solutions containing pharmacological agents. Drugs and amino acids were dissolved in Ringer’s solution shortly before use to minimize chemical degradation. The pH was adjusted when necessary. To cool the spinal cord, a bottle containing Ringer’s solution was connected to a two-meter long polyethylene coil submerged in crushed ice. The temperature could be altered by increasing the length of the coil or by adding salt to the crushed ice. The temperature inside the cord bath was continuously monitored using a 29gauge needle microprobe (model MT-29/3, Physitemp Instruments, Inc., Clifton, NJ, U.S.A.). Unless otherwise stated, spinal cords were cooled to between 6 and 8°C. The cooling took between 1 and 3 sec. Data are expressed as mean t SEM. The significance of differences was assessed using Student’s t-test for correlated means.

Results Figure 1A is a sucrose gap recording of the typical motoneuron membrane potential changes recorded from the VR of the toad spinal cord when the temperature of the Ringer’s solution used to superfuse the spinal cord was rapidly reduced from room temperature to 8°C. The change in temperature generated a long-lasting motoneuron depolarization. VR depolarizations as large as 21 mV (5.5 + 0.6 mV, N = 35) were recorded. Provided sufficient time was left for recovery (approximately one hr), repetitive cooling produced responses that varied less than 10% in a given cord. The cord could be cooled as many as 10 to 12 times without decrement. The amplitude of the cold-induced depolarization was usually larger than synaptically mediated ventral root potentials (VRPs) produced by supramaximal stimulation of the afferent fibers contained in the DR (DR-VRPs). A typical DR-VRP is illustrated in Figure 2A. Despite continued cooling, the membrane potential slowly returned to baseline levels. The repolarization phase lasted from 4 to 57 min (25.2 -+ 2.8 min, N = 35). During the descending phase of the coldinduced depolarization, considerable paroxysmal motoneuronal activity was recorded from the VRs (Figs. IA, lB, 3A,, 3B,, 5A). This activity consisted of a series (1 to 20) of slow, repetitive, depolarizations that were sometimes accompanied by motoneuron action potentials. The magnitude of the paroxysmal motoneuron potentials varied among different preparations, but in some spinal cords they

Toad spinal cord cooling

I 1mV B

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Fig. 1. Sucrose gap recordings from the ventral root illustrating some effects of rapid cooling of isolated toad spinal cords. In this and in all subsequent figures, upwards deflections represent motoneuron depolarization. The onset of cooling is indicated by a closed arrow. The onset of rewarming (return to Ringer’s solution at room temperature) is marked by an open arrow. A: Large, long-lasting cold-induced depolarization of toad motoneurons. The spinal cord was cooled from room temperature (21°C) to 8°C. Paroxysmal motoneuron depolarizations (some accompanied by motoneuron action potentials) are superimposed on the declining phase of the cold-induced depolarizations. B: Cold-induced depolarization (from 21” to 7°C) in another preparation. C: Small-amplitude, shorterlasting depolarization evoked in a third preparation by cooling to 10°C (C,). Only small amounts of spontaneous motoneuron firing are seen during cooling. A modest, shorter-lasting hyperpolarization is evoked by exposure of the spinal cord to Ringer’s solution at 21°C (CJ. D: The cord was rapidly rewarmed from 7°C to room temperature. Note the substantial, slow hyperpolarization generated during rewarming.

B

A 21°C

6°C

Fig. 2. Cold reduces the amplitude and increases the duration of evoked ventral root potentials (DR-VRPs). The fibers contained in the ninth DR were supramaximally stimulated by electrodes applied to the sciatic nerve, which had been left in continuity with the root. A: Control DR-VRP obtained with cord at room temperature. B: DR-VRP recorded 12 min after exposure to cold Ringer’s solution (6.O”C). Note the increased motoneuron firing during cooling.

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were 5.0 mV or more in size and as long as 200 set in duration. When the temperature of the spinal cord bath was only reduced to between 10 and 12”C, smaller amplitude, shorter duration motoneuron depolarizations were seen (Fig. IC,). These cold-induced depolarizations were not accompanied or followed by slow paroxysmal activity, but motoneuron action potentials were seen on the declining phase of the coldinduced depolarization. As seen in Fig. lD, when spinal cords that had been superfused with cold (6 to 8°C) Ringer’s solution were rewarmed to room temperature, long-lasting (17.9 & 2.4 min, N = 28), large (4.6 + 0.3 mV, N = 28) motoneuron hyperpolarizations were seen. Smaller, shorter-lasting hyperpolarizations were noted when the spinal cord temperature had been reduced to only 10 to 12°C and then rewarmed (Fig. 1CJ. Hyperpolarizations resulting from rewarming after exposure to low temperatures can result from increased activity of an electrogenic Na+ pump (Carpenter and Alving, 1968). Characteristically electrogenic Na+ pumps that involve Na+/K+ ATPase are blocked by ouabain. But, neither the amplitude (103 + 11% of control, N = 3) nor the duration (111 + 11%) of the hyperpolarizations produced by rewarming were significantly altered by exposure to dihydro-ouabain (10 PM) (not shown). Evoked VR potentials produced by supramaximal stimulation of afferent fibers (DRVRPs) consist of two components: a slow component produced by the electrotonic transmission of postsynaptic potentials in motoneurons and fast components that represent motoneuron action potentials conducted by VR axons. As seen in Fig. 2B, cooling of the toad spinal cord to between 6 and 8°C significantly prolonged the later DR-VRP components, which are polysynaptic in origin (Simpson, 1976) (231 + 36% of control, N = 7, P < 0.01). In addition, increased motoneuron firing was noted in the cooled toad spinal cords, despite the decreased amplitude of the slow postsynaptic potentials. To evaluate whether or not the motoneuron potential changes induced by cold were produced by a direct action on motoneurons or were generated by an indirect action caused by the release of endogenous neurotransmitters by interneurons or afferent fibers, we added either high concentrations of Mg2+ ions (10 mM) or tetrodotoxin (TTX; 0.625 PM) to the superfusate. When Ringer’s solution containing either Mg2+ or TTX was used to block synaptic transmission or interneuronal firing,

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Al

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J-ypv . B2

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Fig. 3. Magnesium ions and tetrodotoxin diminish cold-induced motoneuron depolarizations and block cold-induced paroxysmal motoneuron activity. A: Effects of Mg*+ ions. A,, Control depolarization induced by cooling to 7°C. Al, Mg2+ (10 mM) reduces the ability of cooling (6°C) to depolarize motoneurons. Note loss of superimposed, slow, paroxysmal motoneuron depolarizations and motoneuron firing. B: Actions of tetrodotoxin (TTX). B,, Control motoneuron depolarization produced by exposure to cold Ringer’s solution (6°C). B,, Repeat exposure to cold Ringer’s solution after addition of TTX (0.625 FM) to superfusate. Again, note the loss of superimposed, slow, paroxysmal motoneuron depolarizations and motoneuron firing. Traces in A and B recorded from two separate spinal cords.

creased the amplitude of cold-induced motoneuron depolarizations by 40 to 45% (Fig. 4A, Table 1). Kynurenate also shortened the duration of the motoneuron depolarizations by about 55%. Moreover, kynurenate effectively suppressed the paroxysmal motoneuron depolarizations produced by cooling. The same concentrations of kynurenate reversibly inhibited motoneuron responses to applied Lglutamate (1 .O mM, 10 set applications) by approximately 50% (N = 2) (Fig. 4B) d, I-2-Amino-7-phosphonohepatonoic acid (APH, 20-40 PM) diminished the amplitude

both the amplitude and duration of coldinduced motoneuron depolarizations were substantially reduced (Table 1) and the paroxysmal activity was eliminated (Fig. 3). In contrast , addition of ‘physiological’ concentrations of Mg2+ (1 .O mM) to the Ringer’s solution was less effective in reducing the amplitude and shortening cold-induced motoneuron depolarizations (not shown; Table 1) Davidoff et al., 1988). Mg2+ (1.0 mM) did, however, abolish the cold-enhanced motoneuron firing during the DR-VRP (not shown). Application of kynurenate (1 .O-2.0 mM) de-

Table 1. Effects of Mg *+, TTX. excitatory amino acid antagonists, and dihydro-ouabain on motoneuron depolarizations induced by ranid cooline Amplitude Mg2+ (10 mM) TTX (0.625 FM) Mg2+ (1.0 mM) Kynurenate (1 .O-2.0 mM) APH (20-40 uM)

56 50 77 57 64

t 6* * 5* ” 0.9’ Z!Z5** * 5**

Duration (3) (6) (3) (9) (4)

43 ? 2 55 ? 8 49 -t 16 44 * 12 51*11

(3) (3) (3) (5) (4)

Values expressed as percentage (mean ? SEM) of control VR depolarizations produced by rapid cooling. Each cord was used as its own control. Numbers in parentheses are numbers of preparations tested. *P < 0.05, **I’ < 0.01 (Student’s paired r-test for correlated means).

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Toad spinal cord cooling Al

Cl

6 ‘C

??I

KYN + 6°C

C2

KYN + GLU

L

APH + 7’~

APH + NMDA

Fig. 4. Excitatory amino acid antagonists attenuate cold-induced motoneuron depolarizations, paroxysmal activity, and excitatory amino acid-induced depolarizations. A: Effects of kynurenate on coldinduced depolarizations. A,, Control motoneuron depolarization produced by exposure to cold Ringer’s solution (6°C). Al, Repeat exposure to cold Ringer’s solution 30 min after adding kynurenate (1.0 mM) to the superfusate. B: Effects of kynurenate on motoneuron depolarizations produced by short (10 set) applications of L-glutamate (GLU, 1.O mM) indicated by bars. B,, L-glutamate response in normal Ringer’s solution. BZ, Response after bathing cord in medium containing kynurenate (1.0 mM, 30 min). Experiment performed at room temperatures (21”). C: Effects of APH on cold-induced depolarizations. C,, Control exposure to cold Ringer’s solution (7°C). Cz, Similar cooling after addition of APH (20 WM. 20 min). D: Effects of APH on motoneuron potential changes evoked bv short (10 set) applications of NMDA (50 PM) indicated by bars. D,, Control NMDA response. D2, Response after exposure of spinal cord to APH (20 PM, 15 min). Experiment performed at room temperature (21”). Traces in A and B were obtained from experiments in two separate spinal cords. Traces in C and D were recorded during an experiment in the same cord.

of the motoneuron depolarizations induced by rapid cooling by about 45% and the duration by about 50%. APH also eliminated the coldinduced paroxysmal activity (Fig. 4C) and the cold-enhanced firing of motoneurons following DR stimulation (not shown). In addition, the same concentrations of APH almost eliminated motoneuron depolarizations induced by short applications of NMDA (50 PM, 10 set; N = 2) (Fig. 4D). Addition of dihydro-ouabain (DHO, 10 PM, 30 min) to the superfusate caused a slow depolarization of the motoneurons that ranged from 0.5 to 2.25 mV (N = 3) (Fig. 5B). These depolarizations did not resemble the large, rapid depolarizations obtained by cooling. If the cardiac glycoside was maintained in the superfusate, the motoneuron depolarizations generated by rapid cooling were reduced by approximately 20% (81 + 3% of control, N = 3, P < 0.05) (Fig. SC).

Discussion Cooling of central neural tissues is generally considered to have a depressant action, but the present results show that rapid cooling of the isolated tropical toad spinal cord produced large and prolonged VR depolarizations. These VR depolarizations represent motoneuron membrane potential changes electrotonitally conducted along VR axons. As such, they are a measure of the shifts of the membrane potential of the population of motoneurons included in a spinal segment. Motoneuron depolarizations following spinal cord cooling have previously been reported in the cat (Klee et al., 1974; Pierau et al., 1969). It is also known that evoked and spontaneous repetitive motoneuron firing occurs in the cat when the temperature is reduced (25°C or below) (Brooks et al., 1955; Grundfest, 1941). The finding that both TTX and Mg2+, added

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522 A

c

B

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DHO

OH0 + 7°C

I 1mV 200s

Fig. 5. Effects of dihydro-ouabain on cold-induced motoneuron depolarizations. A: Control depolarization produced by exposure to cold Ringer’s solution (7°C). B: Motoneuron depolarization caused by addition of dihydro-ouabain (DHO, 10 FM) to superfusate. Application of dihydro-ouabain indicated by bar. C: Cold-induced depolarization evoked after 30 min exposure to dihydroouabain.

to the medium in concentrations sufficient to interrupt chemically mediated synaptic transmission in isolated amphibian spinal cords, substantially reduced (by about 50%) but did not eliminate, cold-induced depolarizations suggests that approximately one half of the change in motoneuron membrane potential evoked by cold is generated by indirect effects exerted via interneurons and/or afferent terminals and the remaining one half by direct actions of cold on motoneuron membranes. In support of this idea are findings that in the isolated spinal cord-hind limb preparation, intrathecal injection of TTX (10 PM) did not eliminate the muscle contractions induced by cooling (Da16 and Larson, 1991). In the same preparation, after spinal superfusion with Mg*+ (lo-20 mM) cooling still induced some clonic movements of the isolated leg (Da16 and Martinez, 1987). The indirect effects of cold presumably involve the synaptic release of L-glutamate and/ or related amino acids from primary afferent terminals and internuncial neurons and the subsequent activation of specific excitatory amino acid receptors on motoneuron membranes. The presumption that the indirect effects of cold involve excitatory amino acids is based on: (1) the idea that much of the excitatory synaptic transmission in the amphibian spinal cord is mediated by excitatory amino acids; and (2) our finding that cold-induced depolarizations were substantially reduced by kynurenate, a nonselective, broad-spectrum antagonist of amino acid-induced excitations in the spinal cord (Elmslie and Yoshikami, 1985; Jahr and Yoshioka, 1986). Although excitatory amino acids activate both NMDA and nonNMDA receptors on spinal neurons, NMDA receptors seem to play the major role in the indirect generation of cold-induced de-

polarizations because APH, an effective and selective competitive antagonist of spinal NMDA receptors, was almost as effective as kynurenate in reducing the motoneuron depolarization generated by exposure to cold medium (Evans et al., 1982). Our findings do not specify whether the augmented excitatory amino acid-mediated synaptic transmission caused by exposure to cold temperatures results from: (1) an increased release of excitatory amino acids, (2) a decreased uptake of excitatory amino acids, or (3) a conformational change in postsynaptic excitatory amino acid receptor structure stimulated by low temperatures. Multiple factors may be involved. For example, L-glutamate is released from toad spinal cord slices as a result of rapidly lowering the temperature of the medium (Da16 and Larson, 1991). In addition, low temperature has been shown to interfere with the active transport involved in the Lglutamate reuptake mechanism (Davidoff and Adair, 1975). The present work appears to show that a small, direct component of the cold-induced motoneuron depolarizations results from a decreased rate of active Na+ transport. Dihydroouabain, a blocker of electrogenic Na+ pumps that involve Na+/K+ ATPase, reduced coldinduced depolarizations by about one fifth. In some invertebrate and vertebrate neurons, the electrogenic sodium pump creates a net outward transmembrane current that directly contributes to the resting membrane potential (Carpenter and Alving, 1968; Gorman and Marmor, 1970a; Padjen and Smith, 1983; Jones, 1989). Previous data from our laboratory (Hackman et al., 1987) indicate that a portion (~8) of the observed decrease may be attributed to a decrease in the Na+ gradient. Thus, the electrogenic Na+ pump may directly contribute to the resting membrane potential in toad motoneurons and dihydroouabain did depolarize frog motoneurons. Because the rate of electrogenic Na+ pumping is a function of temperature, we assume that inhibition of electrogenic Na+ pumping by cold is responsible for a portion of the direct component of the toad motoneuron depolarization produced by rapid cooling (Thomas, 1972). The resting membrane potential of amphibian motoneurons is determined by the electrical potential generated by the diffusion of ions across the membrane, as calculated by the Goldman-Hodgkin-Katz voltage equation. According to the equation, the membrane potential is a function of the absolute temperature (“K) and a decrease in temperature results in membrane depolarization. A temperature de-

Toad spinal cord cooling

crease from 21” to 6°C (from 294” to 279°K) would diminish the motoneuron membrane potential from -90 mV to approximately -85.4 mV (Barrett and Barrett, 1976). But, sucrose gap measurements from the VR record only a variable proportion of the changes in motoneuron membrane potential (Stampfli, 1954). We estimate that about one third of the actual changes in motoneuron membrane potential are reflected in VR recordings. We, therefore, believe that only a fraction of the large cold-induced depolarizations recorded from toad spinal cords are generated by changes predicted by the Nemst equation. On the other hand, passive ion permeabilities have been reported to vary with temperature in some neurons and it may well be that changes in ion permeabilities constitute a major factor in producing cold-induced motoneuron depolarizations (Carpenter, 1970; Gorman and Marmor, 1970b). Epileptiform activity can be generated in the isolated spinal cord of both mammals and amphibians by a variety of convulsant agents, such as strychnine, bicuculline, and penicillin (Ryan et al., 1984; Schwindt and Crill, 1984; Somjen et al., 1978). Spinal epileptiform activity generated in motoneurons by convulsants resembles the convulsive activity produced in supraspinal neurons in that, in both situations, recurrent spontaneous neuronal depolarizations that cause repetitive firing are seen (Schwindt and Grill, 1984; Somjen et al., 1978). Rapid cooling of the spinal cord of South American toads can also generate seizure activity (Ozorio de Almeida, 1943; Ozorio de Almeida et al., 1943). In particular, rapid cooling of the tropical toad spinal cord attached to an isolated hind limb by the sciatic nerve is reported to induce contractions in the gastrocnemius muscle that closely resemble epileptiform seizures (Da16 and Larson, 1991). The observations reported in this study show that the isolated tropical toad spinal cord generated sizeable, long-duration, paroxysmal motoneuron depolarizations when the temperature of the cord was lowered to between 6 and 8°C. These cold-induced paroxysmal depolarizations of motoneurons strongly resemble the discharges produced in the isolated frog spinal cord by convulsants (Davidoff, 1972a,b; Ryan et al., 1984). In addition, we have shown that the convulsive activity induced by cooling is abolished by an NMDA antagonist, as are the spinal seizures induced by convulsive agents (Ryan et al., 1984). The paroxysmal, cold-induced motoneuron depolarizations recorded from the VR are presumed to be the electrophysiological explanation for the epileptiform activity seen in the

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hind limbs of cooled South American toads. This idea is strengthened by previous observations that the tonic phase of the coldinduced hind limb epileptiform activity is inhibited by NMDA antagonists injected intrathecally and by superfusion with high concentrations of Mg2+ (Dalb and Larson, 1991; Da16 and Martinez, 1987). In the present experiments, the slower, polysynaptic components of evoked ventral root reflexes were highly affected by cold; in particular, the DR-VRP was markedly increased in duration. Amphibian spinal cords have the ability to preserve chemically mediated synaptic transmission even at low temperatures, and one could anticipate that cooling would increase DR-VRPs (Czeh and Dezso, 1982). For example, the evoked synaptic release of neurotransmitters is presumably increased by the well described capacity of cold to prolong action potentials (Klee et al., 1974). Second, prolonged action potentials would facilitate propagation of impulses across branch points in primary afferent fibers (Cruzblanca and Alvarez-Leefmans, 1989). Afferent fibers in amphibians divide repeatedly before they finally make synaptic contacts (Grantyn et al., 1984). Transmission in branched axons would also be facilitated by the increase in the space constant that occurs when the temperature is lowered (Del Castillo and Machne, 1953). Finally, as indicated above, the rate of uptake of L-glutamate (and other neurotransmitters) is diminished by low temperatures (Davidoff and Adair, 1975). It is difficult to compare the current results with previous investigations of reflex changes induced by spinal cord cooling in amphibians because there is no unity of opinion with regard to the effect of temperature on amphibian reflexes (Gergen and Gasteiger, 1967; Grinnell, 1966; Kolmodin and Skoglund, 1953; Tebecis and Phillis, 1968; Winterstein and Terzioglu, 1942). In large measure, we believe that the discrepancies are dependent upon different recording techniques, including variations in amplifier coupling. Sucrose gap DC recordings of reflexes from amphibian spinal cords-such as were used in the present studies -are considerably larger and longer than recordings of reflexes obtained in the conventional way with wire electrodes placed on two points on a VR. AC-coupled recording, conventionally used to record reflexes, can artifactually and substantially shorten the record by filtering out the slower components of the VRP; namely, those components that appear most sensitive to the effects of cooling (Hackman and Davidoff, 1991).

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Acknowledgments-This work was supported by USPHS Grant NS 17577 and Department of Veterans Affairs Medical Center Funds (MRIS 1769 and 3369). Travel to the U.S.A. for NLD was provided by Fundayacucho and CONICIT, Venezuela.

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