Hemisected Frog Spinal Cord Research Articles

Role of metabotropic glutamate receptors in the depression of GABA-mediated depolarization of frog primary afferent terminals

Sucrose gap recordings from the dorsal roots of isolated, hemisected frog spinal cords were used to determine the effects of metabotropic l-glutamate receptor activation on primary afferent terminals by (±)-1-amino- trans-1,3-cyclopentane-dicarboxylic acid ( t-ACPD). Dorsal root potentials evoked by ventral root volleys were significantly reduced by t-ACPD (30 μM), as were GABA- and muscimol-induced afferent terminal depolarizations. The effects of t-ACPD on GABA-depolarizations depended upon activation of group I metabotropic glutamate receptors, i.e. the effects were blocked by the group I/II antagonist ( RS)- α-methyl-4-carboxyphenylglycine, but not by the group II antagonist α-methyl-(2 S,3 S,4 S)- α-(carboxycyclopropyl)-glycine or the group III antagonist α-methyl-( S)-2-amino-4-phosphonobutyrate and were mimicked by the group I agonist 3,5-dihydroxyphenylglycine but were not mimicked by the group III agonist ( S)-2-amino-4-phosphonobutyrate. Increasing the intracellular concentration of 3′,5′-cyclic adenosine monophosphate with 8-bromo-cAMP, forskolin, and 3-isobutyl-1-methylxanthine significantly reduced GABA depolarizations, but the protein kinase inhibitors Rp-adenosine 3,5-cyclic monophosphothioate triethylamine and N-[2-( p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide did not alter t-ACPD's depression of GABA depolarizations. The actions of t-ACPD on GABA depolarizations were neither mimicked nor blocked by phorbol-12-myristate 13-acetate, thapsigargin, staurosporine, or arachidonic acid, presumptive indications that the effects of t-ACPD did not involve phosphoinositide hydrolysis, the release of Ca 2+ from intracellular stores, or the formation of arachidonate. t-ACPD's effects on GABA depolarizations were blocked by 20 mM Mg 2+, the broad spectrum l-glutamate antagonist kynurenate, and the selective N-methyl- d-aspartate antagonist D(−)-2-amino-5-phosphonovaleric acid, but not by the non- N-methyl- d-aspartate antagonist 6-cyano-7-nitroquinoxaline-2,3-dione. Low concentrations of N-methyl- d-aspartate (10 μM) mimicked the effect of t-ACPD on GABA responses. These results suggest that t-ACPD's depression of GABA depolarizations involves an indirect, three-stage mechanism that includes activation of Group I metabotropic glutamate receptors on interneurons and/or on afferent terminals, the release of l-glutamate from the latter structures, and the activation of N-methyl- d-aspartate receptors on primary afferent terminals. The depression of GABA depolarizations caused by the release of l-glutamate from afferent terminal and/or interneurons leads to a block of presynaptic inhibition (produced in the frog spinal cord by GABA) resulting in a positive feed-forward amplification of reflex transmission.

Intrinsic NMDA-induced oscillations in motoneurons of an adult vertebrate spinal cord are masked by inhibition.

Low-frequency membrane potential oscillations were induced in motoneurons (MNs) of isolated hemisected frog spinal cords during N-methyl-D-aspartate (NMDA) application. Oscillations required the presence of physiological Mg2+ and preincubation with strychnine, whereas incubation with bicuculline or phaclofen was not effective. Oscillations were evident in intracellular recordings from single MNs and simultaneous extracellular recordings from lumbar ventral roots. In Mg(2+)-free solution, MNs exhibited irregular transient membrane potential depolarizations that were blocked by D,L-2-amino-5-phosphonopentanoic acid (APV) but not by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). Generation and maintenance of membrane potential oscillations required specific NMDA receptor activation. Oscillations were antagonized by APV but not by CNQX. Strychnine preincubation was required for NMDA to induce oscillations, but was not critical in maintaining them, because oscillations persisted after removal of strychnine. Therefore oscillations are suggested to be an inherent property of the spinal neuronal circuitry. Tetrodotoxin (TTX) blocked spike activity and had a bimodal effect on membrane potential oscillations. Oscillations initially were blocked by TTX, but reappeared spontaneously after 10-40 min. This suggests that maintenance of oscillations, once evoked, does not involve MN firing. Na+ entry through TTX-insensitive Na+ channels and/or NMDA receptor channels, trans-membrane Ca2+ flux, Ca2+ release from intracellular stores, and Ca2+ activated K+ channels were critical in controlling the amplitude and frequency of membrane potential oscillations. It is hypothesized that these unmasked intrinsic oscillations in adult frog spinal cord MNs may represent a premetamorphic spinal oscillator involved in tadpole swimming that becomes suppressed during metamorphosis as strychnine-sensitive inhibition becomes more pronounced.

Modulation of frog spinal cord interneuronal activity by activation of 5-HT 3 receptors

Motoneuron membrane potentials were recorded from the ventral roots of isolated, hemisected frog spinal cords using sucrose gap techniques. The effects of the selective 5-HT 3 agonist 2-methyl-serotonin (2-Me-5HT) on the changes in motoneuron membrane potential produced by dorsal root stimulation and by superfusion of excitatory amino acid agonists were evaluated. Application of 2-Me-5HT (100 μM) did not alter motoneuron membrane potential, but did substantially reduce ( ∼ 20%) the polysynaptic ventral root potentials evoked by dorsal root stimulation. 2-Me-5HT did not change motoneuron depolarizations generated by addition to the Ringer's solution of the excitatory amino acid agonists AMPA (10–30 μM), kainate (30 μM), or t-ACPD (100 μM), but NMDA-induced motoneuron depolarizations (100 μM) were significantly and reversibly reduced ( ≈ 20%) by exposure to 2-Me-5HT (100 μM). 2-Me-5HT-evoked decreases of NMDA depolarizations were blocked by the 5-HT 3 antagonists ICS 205 930 (50–100 μM) and d-tubocurarine (3–10 μM), but not by MDL 72222 (20–100 μM), the 5-HT 2 receptor antagonist ketanserin (10 μM), or the 5-HT 1A/5-HT 2A antagonist spiperone (10 μM). Two lines of evidence support the hypothesis that the effects of 2-Me-5HT are generated by an indirect mechanism involving interneurons: (1) TTX (0.781 μM) eliminated the effect of 2-Me-5HT on NMDA-induced motoneuron depolarizations, and (2) 2-Me-5HT reduced spontaneous ventral root potentials that result from interneuronal discharges. We attempted to establish the identity of a putative transmitter released by interneurons responsible for the effects on NMDA-depolarizations produced by 2-Me-5HT, but the AMPA receptor antagonist, CNQX (10 μM), the GABA A receptor antagonist, bicuculline (50 μM), the GABA B receptor antagonist, saclofen (100 μM), the opioid antagonist, naloxone (100 μM), and the adenosine antagonists, CPT (20–100 μM) and CSC (10–100 μM) did not alter 2-Me-5HT-induced reductions of NMDA-depolarizations. In sum, the site of interaction between 2-Me-5HT and NMDA appears to be at interneuronal locus, but the mechanism remains unclear.

Glutathione suppresses spontaneous activity in the frog spinal cord.

Spontaneous electrical activity in the isolated hemisected frog spinal cord increased in the presence of the SH reductant dithiothreitol (DTT) was reversibly suppressed by the oxidant 5-5'-dithio-bis-(2 nitrobenzoic acid) (DTNB) and was irreversibly suppressed by the sulphydryl modifying agents N-ethyl-maleimide (NEM) and monobromotrimethylammonio bimane. Glutathione (GSH), an important natural low molecular weight thiol, reversibly suppressed spontaneous activity. Cords pretreated with glutathione and successively exposed to NEM or bimane maintained their normal electrical activity. This indicates that GSH had interacted with the exposed sulphydryls and prevented their reaction with NEM or bimane. Incubation with bimane resulted in fluorescence-labelled neurones in the dorsal and ventral horns, whereas samples pretreated with NEM or with GSH were not labelled. Neurones appeared again fluorescent in cords preincubated with GSH and sequentially exposed to NEM or bimane. Both electrophysiological and histochemical methods indicate that exposed membrane sulphydryls are involved in the genesis and/or modulation of spontaneous electrical potentials.

An examination of frog myelinated axons using intracellular microelectrode recording: the role of voltage-dependent and leak conductances on the steady-state electrical properties.

1. Intracellular microelectrode current-clamp technique was used to study the steady-state membrane properties of single intact large primary afferent axons (conduction velocity > 10 m/s) attached to isolated hemisected frog spinal cord. 2. Hyperpolarizing electrotonic potentials (ETPs) had a slow complex multiphasic charging. This complex charging could be approximated by two time constants: one in the range of 70-210 ms, the other of < 20 ms. 3. Two regions of outward and inward rectification hyperpolarized to the resting membrane potential were observed, in addition to the previously characterized outward rectification active at potentials depolarized to resting membrane potential. The peak and steady-state input resistance of these axons in tetrodotoxin Ringer solution was on average 65.6 +/- 21.1 and 31.1 +/- 10.8 M omega, mean +/- SE, respectively. 4. Application of external tetraethylammonium (10-20 mM) significantly depolarized the axon and decreased the outward rectification just hyperpolarized to the resting membrane potential. This outward rectification could also be blocked by external barium ions (2-10 mM). 5. Activation of an inward or anomalous rectification in these axons was observed 300-600 ms after the start of a current pulse. In addition, a depolarizing afterpotential (DAP) (1-3 mV in amplitude, 500 ms-10 s in duration) was evident after a current pulse in which inward rectification had been activated. This DAP most likely reflected the slow inactivation of the inwardly rectifying conductance. 6. Inward rectification was blocked by external application of cesium ions (1-3 mM) but it was insensitive to external application of barium ions (2-10 mM). The blockade of the voltage attenuation was accompanied by a disappearance of the DAP and an increase in the charging time constant of the axon. This blockade resulted in a single linear voltage-current (V-I) relationship. Axons now had, on average, an input resistance of 114 +/- 19.1 M omega. 7. Reducing the concentration of external potassium ions increased both the peak and steady-state slope resistance. Reducing the external sodium concentration altered the ETPs and the V-I relationship little but it consistently reduced the magnitude and length of the DAP. These results are compatible with the hypothesis that anomalous rectification is a mixed ionic conductance dependent on potassium and sodium ions in the external media. 8. Overall, the V-I relationship of these intact axons had both linear and nonlinear regions reflecting the activity of numerous slowly activating and inactivating conductances. (ABSTRACT TRUNCATED AT 400 WORDS)

Activation of 5-HT 1C/2 receptors depresses polysynaptic reflexes and excitatory amino acid-induced motoneuron responses in frog spinal cord

Sucrose gap recordings from the ventral roots of isolated, hemisected frog spinal cords were used to evaluate the effects of high concentrations of serotonin (5-HT) and α-methyl-5-HT (α-Me-5-HT) on the changes in motoneuron potential produced by dorsal root stimulation and by excitatory amino acids and agonists. Bath application of 5-HT in concentrations of 10 μM or greater produced a concentration-dependent motoneuron depolarization. Polysynaptic ventral root potentials evoked by dorsal root stimuli were reduced in both amplitude and area by 5-HT or α-Me-5-HT (both 100 μM). This may result from a reduction of the postsynaptic sensitivity of motoneurons to excitatory amino acid transmitters because 5-HT significantly depressed motoneuron depolarizations produced by addition of l-glutamate and l-aspartate to the superfusate. Similarly, 5-HT reduced depolarizations produced by the excitatory amino acid agonists N-methyl- d-aspartate (NMDA), quisqualate, α-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA), and kainate. α-Me-5-HT reduced NMDA depolarizations. Tetrodotoxin (TTX) did not affect the ability of 5-HT to attenuate NMDA or kainate depolarizations, but did eliminate the 5-HT-induced attenuation of quisqualate and AMPA depolarizations. The glycine receptor site associated with the NMDA receptor did not appear to be affected by 5-HT because saturation of the site by excess glycine did not alter the 5-HT-induced depression of NMDA responses. The 5-HT 1C/2 antagonist ketanserin and the 5-HT 1A/2 antagonist spiperone significantly attenuated the 5-HT-induced depression of NMDA NMDA-depolarizations. The relatively selective 5-HT 1A antagonist spiroxatrine and the selective 5-HT 3 blocker MDL 72222 were without effect against the depressive effects of 5-HT. These observations support the notion that 5-HT putatively released from descending bulbospinal fibers can alter frog motoneuronal output. The data indicate that 5-HT 1C/2 receptors affect the afferent input to motoneurons and do so by attenuation of NMDA receptor-mediated synaptic processes.

Serotonin 1A facilitation of frog motoneuron responses to afferent stimuli and to N-methyl- d-aspartate

The effects of serotonin and excitatory amino acids on motoneurons were examined by sucrose gap recordings from the ventral root of the isolated, hemisected frog spinal cord superfused with magnesium-free, carbonate-buffered Ringer solution. Low concentrations of serotonin (0.1 μM) and the serotonin 1A agonist8-hydroxy-2-( n-dipropylamino)tetralin (8-OH-DPAT; 0.01 μM) significantly increased the duration and amplitude of the polysynaptic components of ventral root potentials produced by dorsal root stimulation. The facilitations of the ventral root potentials were blocked by the serotonin 1A antagonist spiroxatrine, but were unaffected by the serotonin 2 antagonist ketanserin or the serotonin 3 antagonist 1αH,3α,5αH-tropan-3-yl-3,-dichlorobenzoate (MDL 72222). The actions of 0.1 μM serotonin on motoneuron depolarizations evoked by the putative excitatory amino acid transmitters l-glutamate and l-aspartate were quite variable, but in the presence of ketanserin (20 μM), small consistent increases in amino acid-induced motoneuron depolarizations were observed. 8-OH-DPAT significantly enhanced motoneuron depolarizations elicited by the selective excitatory amino acid agonist N-methyl- d-aspartate in both normal and tetrodotoxin-containing Ringer solution. Quisqualate-induced motoneuron depolarizations were also facilitated by 8-OH-DPAT in normal Ringer solution, but these increases were eliminated by addition of either tetrodotoxin or the N-methyl- d-aspartate antagonist d(−)-2-amino-5-phosphonovalerate to the Ringer superfusate. Kainate-depolarizations were not altered by low concentrations of serotonin or 8-OH-DPAT. Prior exposure of the cord to spiperone, but not ketanserin or MDL 72222 blocked the enhancement of N-methyl- d-aspartate-induced motoneuron depolarizations by 8-OH-DPAT. In the presence of saturating concentrations of glycine, 8-OH-DPAT facilitated N-methyl- d-aspartate responses. Increases in extracellular potassium activity, measured with ion-selective microelectrodes inserted in the intermediate gray matter, evoked by repetitive stimulation of myelinated afferent fibers in the sciatic nerve and by application of N-methyl- d-aspartate were significantly enhanced by 8-OH-DPAT. These findings are compatible with the notion that serotonergic activation of serotonin 1A receptors plays a role in regulating the responsiveness of frog spinal motoneurons to excitatory afferent inputs and to N-methyl- d-aspartate-mediated synaptic transmission.

Dorsal root potentials in the isolated frog spinal cord: Amino acid neurotransmitters and magnesium ions

Sucrose gap techniques recorded dorsal root potentials evoked by supramaximal dorsal root stimulation in in vitro, hemisected frog spinal cords. In 0 mM Mg 2+ large (mean 13.0 mV), long lasting (mean 8.1 s) dorsal root potentials were recorded which consisted of two components: (1) an early component sensitive to picrotoxin, bicuculline, and low [Cl −] 0 and presumably produced by activation of GABA A receptors; and (2) a long-duration second component enhanced and lengthened by picrotoxin, bicuculline and low [Cl −] 0 and thought to result from increased intemeuron discharges resulting from depression of GABA-mediated pre- and postsynaptic inhibition. Both the early and late components were reduced by over 90% in amplitude and duration by 20 mM Mg 2+ or by kynurenate and bicuculline. The early component of the dorsal root potential may depend mainly upon activation of non-N-methyl- d-aspartate receptors, but the late component requires bothN-methyl- d-aspartate and non-N-methyl- d-aspartate receptors. Thus, theN-methyl- d-aspartate antagonist d-(−)-2-amino-5-phosphonovalerate caused only a modest reduction in the amplitude of the early dorsal root potential component while the nonN-methyl- d-aspartate antagonist 6-cyano-7-nitroquinoxaline-2,3-dione caused a much more substantial reduction. Exposure of the spinal cord to a “physiological” concentration of Mg 2+ (1.0 mM) greatly reduced the duration and somewhat reduced the amplitude of the dorsal root potential. The reduction of dorsal root potentials by 1.0 mM Mg 2+ appears to be caused by both pre- and postsynaptic factors. These include: (1a) reduction of evoked transmitter release; the reduction of the dorsal root potential by 1.0 mM Mg 2+ was partly reversed by doubling [Ca 2+] 0; (2a) block of theN-methyl- d-aspartate receptor ion channels; (3a) decrease of GABA-depolarization of primary afferent fiber terminals; GABA-depolarizations of terminals were depressed by 1.0 mM Mg 2+; and (4a) decreased release of K + by afferent volleys; exposure to 1.0 mM Mg 2+ reduced the increment in [K +] 0 produced by repetitive afferent stimulation. d-(−)-2-amino-5-phosphonovalerate had only minimal effects on dorsal root potentials in 1.0 mM Mg 2+ presumably because theN-methyl- d-aspartate receptor-ion channel complex is largely inactivated by the Mg 2+. In contrast, kynurenate and 6-cyano-7-nitroquinoxaline-2,3-dione substantially diminished dorsal root potential amplitude and duration.

An in vitro study of the effects of serotonin on frog primary afferent terminals

The effects of serotonin on the membrane potential of primary afferent terminals of isolated hemisected frog spinal cords was investigated by sucrose gap recordings from dorsal root. Serotonin produced two distinctive changes in primary afferent terminal membrane potential: modest (about 0.5 mV) hyperpolarizations in low concentrations (0.01–1.0 μM) and larger (about 1.0 mV) slow depolarizations in higher concentrations (3.0–100 μM). The hyperpolarizations appeared related to a direct activation of 5-HT 1A receptors on afferent terminals. The depolarizations were attributed to both direct and indirect actions and appeared to be generated by activation of 5-HT 2 and/or 5-HT 1C receptors. The results suggest that 5-HT released from terminals in the frog dorsal horn could exert a modulatory action on the afferent input of the spinal cord, but different effects generated by activation of different 5-HT receptor subtypes are dependent upon the concentration of the amine.

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 μM) and depolarizations by higher concentrations (3.0–100 μM). 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-HT 1A receptors and the depolarizations were generated by activation of 5-HT 2 and/or 5-HT 1C receptors. Accordingly, the selective 5-HT 1A agonists8-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-HT 1A receptor antagonists spiperone and spiroxatrine. In contrast, application of high concentrations of alpha-methyl-5-hydroxytryptamine, a serotonin analog which activates 5-HT 1C and 5-HT 2 receptor subtypes, depolarized motoneurons. These depolarizations, and those produced by high concentrations of serotonin, were blocked by the 5-HT 1C/5-HT 2 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-HT 1 and 5-HT 2 receptor subtypes. Activation of different receptor subtypes depends upon the concentration of the amine.

Modulation of the NMDA receptor subtype function in hemisected baby rat spinal cord

The action of N-methyl-d-aspartate (NMDA) antagonistson motoneurons was studied in the isolated, hemisected frog spinal cord using sucrose gap techniques. NMDA-evoked motoneuron depolarizations were depressed by application of APV, APH, kynurenate, Mg2+ ions, ketamine, and MK-801. Upon returning to normal Ringer's solution after exposure to all antagonists (except MK-801), NMDA responses were significantly potentiated. Kainate- and quisqualate-induced depolarizations were unchanged. The facilitation appeared to result, at least in part, from a direct action on motoneuron membranes since it persisted in the presence of tetrodotoxin which eliminated interneuronal firing. However, indirect actions involving interneurons also contributed to the potentiation because NMDA-evoked changes in K+ release were increased following exposure to NMDA antagonists and return to normal medium. Reduction of temperature (7°C) which should reduce amino acid uptake did not affect results with APV. In addition, desensitization of NMDA responses was not altered by application of APV. The results indicate that NMDA antagonists have complex and long-lasting effects on the function of the NMDA receptor complex.

NMDA antagonists and potentiation of NMDA-induced motoneuron depolarizations in the isolated frog spinal cord

The action of N-methyl- d-aspartate (NMDA) antagonistson motoneurons was studied in the isolated, hemisected frog spinal cord using sucrose gap techniques. NMDA-evoked motoneuron depolarizations were depressed by application of APV, APH, kynurenate, Mg 2+ ions, ketamine, and MK-801. Upon returning to normal Ringer's solution after exposure to all antagonists (except MK-801), NMDA responses were significantly potentiated. Kainate- and quisqualate-induced depolarizations were unchanged. The facilitation appeared to result, at least in part, from a direct action on motoneuron membranes since it persisted in the presence of tetrodotoxin which eliminated interneuronal firing. However, indirect actions involving interneurons also contributed to the potentiation because NMDA-evoked changes in K + release were increased following exposure to NMDA antagonists and return to normal medium. Reduction of temperature (7°C) which should reduce amino acid uptake did not affect results with APV. In addition, desensitization of NMDA responses was not altered by application of APV. The results indicate that NMDA antagonists have complex and long-lasting effects on the function of the NMDA receptor complex.

Further evidence in support of taurine as a mediator of synaptic transmission in the frog spinal cord

It has been reported that 6-aminomethyl-3-methyl-4H,1,2,4-benzothiadiazine-1,1-dioxide (AMBD, TAG) is a specific blocker of taurine and β-alanine responses in the central nervous system. We have re-examined the effect of AMBD on amino acid and synaptically evoked responses recorded from isolated hemisected frog spinal cords by means of the sucrose gap technique. When indirect responses were blocked by adding tetrodotoxin (0.2 μM) or manganese chloride (2 mM) to the normal Ringer solution, AMBD (0.01–0.5 mM) selectively antagonized taurine, β-alanine, hypotaurine and kojic amine evoked depolarizations of primary afferents at their intramedullary part (dorsal root terminals, DRT) and on dorsal root ganglia (DRG), without significantly affecting responses to glutamate (on DRT), glycine (on DRT) or GABA (on DRT and DRG). Depolarizing responses to taurine and β-alanine (1 mM) were depressed by up to 50% with 0.1 mM AMBD and often completely antagonized with 0.25 mM AMBD. In normal Ringer solution, AMBD selectively antagonized the dorsal root potential evoked by ventral root stimulation (VR-DRP, threshold at 0.02 mM AMBD, 90% block with 0.25 mM); other synaptic potentials increased in duration and/or amplitude, demonstrating a strong convulsant effect of AMBD. Thus, the depolarizing responses of taurine, β-lanine and hypotaurine on primary afferents are pharmacologically indistinguishable from the VR-DRP. These results are in agreement with the proposal that taurine or a taurine-like substance (possibly β-alanine or hypotaurine) is the mediator of VR-DRP in amphibian spinal cord.

Excitotoxicity, reflex responses, and evoked changes in extracellular potassium in the frog spinal cord

1. The effects of the excitatory amino acid agonists kainate (KA), quisqualate (QUIS), and N- methyl- d- aspartate (NMDA) were studied in vitro on the hemisected frog spinal cord. 2. Prolonged (1.0 hr) application of excitatory amino acid agonists (KA, 50 or 300 μM; QUIS, 30 μM; NMDA, 300 μM) significantly reduced the ventral root potentials (VRPs) and [K +] 0 evoked by a dorsal root tetanus (10 see, 25 Hz), by brief (10 sec) applications of the same agonists (KA, 30 μM; QUIS, 30 μM; NMDA, 300 μM), and by GABA (10 sec, 1.0 mM). 3. The effect was essentially irreversible and persisted despite 2–4 hr of washing. 4. Excitatory amino acid antagonists (APV, 30 μM and kynurenate, 2 mM) blocked the neurotoxic effects of the excitatory agonists NMDA and KA respectively, an observation which indicates the observed effects of the agonists require the activation of specific excitatory receptors. 5. TTX did not alter the neurotoxic effects of KA suggesting that interneuronal firing does not contribute to the observed changes. 6. Addition of high K + did not duplicate the effect of prolonged excitatory amino acid agonist exposure, an indication that elevation of K + does not cause the decreased responses. 7. Light microscopy did not provide any evidence of gross tissue damage. 8. The parallel reduction of postsynaptic responses and Δ[K +] 0 support the idea that elevation of extracellular [K +] by afferent stimuli results from interneuronal activity.

Effects of microwave irradiation and temperature on spontaneous ventral root discharges in prog spinal cord

Aperiodic firing independent of extraneous stimuli at rates varying between 3–8 and 65–100 Hz (spontaneous firing activity, or SFA) was recorded at ventral root filaments of isolated, sagitally hemisected frog spinal cord. Lowest activity level was observed at temperatures of 7–11°C and an increased rate at either higher or lower temperatures. Some consistent short-lasting changes in SFA were noted straight away during the course of thermal changes: heating and cooling the preparation increased and reduced discharge rate, respectively. Characteristic activity rate for a given temperature level would set in 1–3 min after this level had stabilized. Microwave radiation of the spinal cord (6.45 GHz; specific absorption rates: 0.1, 0.4, and 2.0 W/g; duration 5 min) brought about no significant alteration in SFA at a steady temperature level. Microwave heating of the preparation and raised temperature both produced the same effects in all trials. Results would indicate that a thermal mechanism underlies the microwave effects on SFA at the ventral roots of frog spinal cord.

Primary afferent activity, putative excitatory transmitters and extracellular potassium levels in frog spinal cord.

1. Changes in extracellular K+ activity were measured with ion-selective microelectrodes in the grey matter of the isolated hemisected frog spinal cord. The magnitude of the elevation of [K+]o (delta[K+]o) produced by repetitive stimulation (25 Hz, 10 s) of afferent fibres in the sciatic nerve was monotonically related to the strength of the electrical stimuli applied to the sciatic nerve. Repetitive stimulation of the largest diameter A alpha and A beta fibres, which were found histologically to comprise only 11% of the afferent axons in the dorsal root, elevated [K+]o to approximately 60% of the maximum level seen when all afferent fibres were stimulated. 2. Addition of Mg2+ (20 mM) to Ringer solution devoid of Mg2+ reduced delta[K+]o by over 85% suggesting that about 15% of delta[K+]o results from action potentials in presynaptic primary afferents. When 20 mM-Mg2+ was added to spinal cords bathed in Ringer solution containing a physiological (i.e. 1.0 mM) concentration of Mg2+, delta[K+]o was reduced by ca. 65-75% indicating that in spinal cords bathed in medium containing 'physiological' concentrations of Mg2+ about 25-35% of the K+ is released from primary afferent fibres. 3. Application of excitatory amino acids and agonists increased [K+]o with the following potency pattern: quisqualate greater than kainate greater than NMDA (N-methyl-D-aspartate) greater than glutamate greater than aspartate. 4. D(-)-2-Amino-5-phosphonovalerate (APV), an NMDA antagonist, reduced [K+]o by only about 50%, but kynurenate, an NMDA and non-NMDA antagonist, reduced [K+]o by approximately 85%; i.e. the same levels observed when synaptic transmission was blocked with 20 mM-Mg2+. These findings support the idea that synaptic release of excitatory amino acids such as L-glutamate and/or L-aspartate and subsequent activation of specific receptors by these putative transmitters are necessary for the postsynaptic component of delta[K+]o. 5. Addition of tachykinins elevated [K+]o but the effect appeared to require the participation of excitatory amino acids because it was blocked by APV and by kynurenate. 6. The finding that tetrodotoxin substantially reduced the ability of excitatory amino acid agonists and tachykinins to elevate [K+]o suggests that discharges in interneurones as a result of excitatory amino acid receptor activation are responsible for the postsynaptic component of delta[K+]o.

Effects of Peptides from Bovine Myelin Basic Protein on the Bioelectrical Activity of the Frog Spinal Cord

Both bovine and human myelin basic protein (MBP) have been shown to have electrophysiological activity. As MBP is susceptible to proteolytic degradation, our aim was to discover whether the resulting peptides retained this activity. Bovine MBP was completely cleaved by plasmin into at least nine peptides. The electrophysiological activities of this peptide mixture and of bovine MBP were directly compared on the hemisected frog spinal cord. The peptide mixture and intact bovine MBP had quantitatively and qualitatively similar effects (dose-dependent long-lasting depolarization, about 100 times more active than glutamate). Four peptides (molecular weights 14,000, 10,500, 8,000, 4,500) from thrombin or cathepsin D cleavage of bovine MBP also showed electrophysiological activity, positively correlated to their molecular weights. As MBP-like material occurs in increased concentrations in the cerebrospinal fluid during demyelinating diseases, peptides resulting from proteolytic degradation of MBP, e.g. in demyelinating foci of multiple sclerosis, might cause neuronal disturbances.

After-hyperpolarizations produced in frog motoneurons by excitatory amino acid analogues

After-hyperpolarizations (AHPs) produced in frog motoneurons by applications of the excitatory amino acid analogues quisqualate (QUIS), N-methyl- d-aspartate (NMDA), and kainate (KA) were studied in the isolated hemisected frog spinal cord using sucrose gap techniques. AHPs were present following 98% of QUIS-induced depolarizations, but were seen in only 35% and 15% of NMDA- and KA-evoked responses respectively. AHPs produced by QUIS are produced both by direct effects of QUIS on motoneuron membranes and by indirect effects mediated through a synaptic process involving interneurons. Thus, applications of Mg 2+, Mn 2+, or tetrodotoxin (TTX) in concentrations sufficient to block synaptic transmission and interneuronal firing, reduced, but did not abolish the AHPs produced by QUIS. In contrast, NMDA- and KA-AHPs appear to be entirely mediated by indirect means as block of synaptic transmission and interneuronal firing eliminated AHPs produced by these substances. Exposure of the cord to Mn 2+ after addition of TTX did not affect the size of QUIS-AHPs. In the presence of TTX, QUIS-AHPs were reduced or completely blocked by addition of dinitrophenol (DNP) and sodium cyanide, by dihydro-ouabain, by removal of K + from the superfusate, by cooling, and by replacement of 50% of the external Na + with Li +. The results suggest that the QUIS-AHPs are largely the result of the direct effect of the excitatory amino acid agonist on motoneuron membranes and is caused by activation of an electrogenic Na + pump. AHPs following depolarizations evoked by NMDA and KA are presumably the result of indirect actions of these latter analogues on interneurons.

Effects of high-pressure helium on gamma-[3H]aminobutyric acid release from the isolated frog spinal cord.

The application of high pressure in vivo causes a hyperexcitability syndrome involving tremors and convulsions. Drugs that potentiate GABA transmission protect animals against this syndrome. It is possible that changes in gamma-aminobutyric acid (GABA) transmission may underlie the hyperexcitability. We have therefore investigated the effects of pressure on the components of GABA transmission in vitro. After incubation with [3H]GABA, hemisected frog spinal cords were superfused inside a pressure chamber and perfusate fractions were collected every 10 min. Helium, at 50 or 100 atm, did not alter the spontaneous release of GABA, but if electrical stimulation had been applied previously, then pressure caused a prolonged increase in GABA release. Helium at 50 atm did not alter the evoked release during electrical stimulation, but at 100 atm this was increased. This increase was smaller in the absence of calcium. No corresponding changes in [14C]urea efflux were seen, suggesting that the effects were not due to nonselective membrane permeability changes. The results are consistent with the known effects of pressure on neuronal activity, such as repetitive firing, but they do not suggest a selective action on the GABA release process.

Potential changes of frog afferent terminals in response to dopamine

The actions of dopamine on the membrane potential of afferent fibers of the isolated hemisected frog spinal cord were studied by sucrose gap techniques. The most prominent effect seen after addition of dopamine to the superfusing Ringer's solution was a slow reversible hyperpolarization at concentrations as low as 0.01 μM; its amplitude and duration were dependent upon concentration and length of application. Biphasic responses with an initial dominant hyperpolarization and a much smaller, later depolarization were also noted and were particularly prominent when dopamine was applied at higher concentrations. Exposure of the cord to apomorphine, a non-selective agonist, to SKF 38393A, a D-1 selective agonist, or to LY-14186, a D-2 selctive agonist, hyperpolarized the dorsal root in a manner similar to that of dopamine, but only when the former compounds were applied at higher concentrations (100 μM or greater). Apomorphine also elicited a late depolarization. The non-selective dopamine antagonists, fluphenazine and haloperidol, reversibly reduced dopamine's actions. Similar effects were produced by the selective D-2 antagonists, sulpiride and metoclopramide, which had no effect on hyperpolarizations evoked by norepinephrine. Dopamine did not appear to activate adrenergic or serotonergic receptors, for its effects were not affected by yohimbine, corynanthine, propranolol, or methysergide. The effect of dopamine appeared to result from an action of the amine on both afferent fibers and interneurons. This inference was drawn because the potential changes produced by dopamine were substantially reduced, but never eliminated, by superfusion of the cord with solutions containing Mn 2+ ions, tetrodotoxin or mephenesin. Dopamine appears to have the ability to activate receptors in the spinal cord — an action which results in potential changes in afferents fibers. The data are compatible with the idea that dopamine modulates spinal sensory transmission.