Isolated Frog Spinal Cord Research Articles

The action of LSD on the isolated frog spinal cord: Electrophysiological and ultrastructural data

1. In the isolated hemisected frog spinal cord, LSD induced a substantial enhancement of spontaneous dorsal and ventral root activity and a general decrease of both orthodromic and antidromic root potentials. 2. 2. These electrophysiological modifications were reversible. The LSD homologue compound—Lisuride—was without effect. 3. 3. Electron microscopy of the LSD-treated spinal cord revealed synaptic “omega” figures and terminals almost depleted of vesicles. 4. 4. The results suggest that LSD acts at the synaptic level favouring the spontaneous release of transmitters from those involved in the pathways tested.

The role of calcium ion in the l-glutamate-induced depolarization in the frog spinal cord

1. The role of Ca 2+ in l-glutamate-induced depolarization was investigated in the isolated frog spinal cord. 2. The size of a depolarization induced by l-glutamate (3 mM) was inversely related to the extracellular Ca 2+ concentration, but was reduced in a Ca 2+-free medium containing EGTA (0.3 mM). 3. l-Glutamate caused a marked depolarization in both ventral and dorsal roots, even in a NaCl-deficient medium (Ca 2+, 2.0 mM). The size of the depolarization was attenuated by a prolonged or repeated application of l-glutamate. Ca 2+ can be replaced by Sr 2+ or Mg 2+. 4. Concanavalin A (1 μM) prevents the development of desensitization to l-glutamate. 5. Present results suggest that Ca 2+ plays the role of a charge carrier for l-glutamate-induced depolarization and of a regulator or modulator for l-glutamate-receptor sensitivity. The roles are exaggerated in NaCl-free medium.

Physiologic basis for neuroelectric blocking activity in multiple sclerosis.

The physiologic basis for the blocking effect of sera from patients with multiple sclerosis on isolated frog spinal cord is shown to be due to an inhibition of synaptic transmission. There are no detectable effects of blocking sera on conduction in either normal or demyelinated nerve.

The presynaptic effects of valproic acid in the isolated frog spinal cord

The effects of the anticonvulsant valproic acid ( n-dipropylacetate, DPA) on frog primary afferent fibers was examined with sucrose gap recordings from the dorsal roots. Addition of DPA to the superfusate consistently reduced the amplitude and duration of the dorsal root potential. In contrast, DPA augmented the depolarization of dorsal roots produced by GABA, β-alanine and taurine. It also decreased afferent fiber ‘desensitization’ to GABA. DPA depressed the ability of K + and the excitatory amino acids glutamate and aspartate to depolarize afferent fibers. In addition, the compounds decreased the amount of K + released by tetanic stimulation of the dorsal root. The K +-evoked release of tritiated GABA from cord slices was initially reduced by exposure to DPA, but was then unaffected after a longer application of the anticonvulsant. On the other hand, the high affinity uptake of tritiated GABA and glycine were almost totally blocked by the addition of DPA to the incubating medium. In sum, DPA has complex effects on neuronal membranes. Some of these effects may serve to explain the anticonvulsant actions of this drug.

Disruption of vertebrate monosynaptic habituation by ethyl alcohol

The effects of ethyl alcohol on neuronal plasticity were examined using a model systems preparation of monosynaptic habituation: the lateral column to motoneuron pathway of the isolated frog spinal cord. Two fundamental effects of alcohol on this system were demonstrated. First, alcohol always depressed the ventral root responses to lateral column stimulation when stimulus intensity was held constant across conditions. This was accompanied by an increase in the amount of habituation produced as training progressed. Second, when stimulus intensity was increased in the alcohol condition so that responses were equated with pre-alcohol response amplitudes, the amount of habituation was markedly decreased. Results are discussed both in terms of proposed mechanisms of habituation and of possible pharmacological interactions with this simple example of response plasticity.

The immaturity of interactions between GABA-and benzodiazepine binding sites in the frog spinal cord

1. 1. In the isolated frog spinal cord, diazepam had little effect on dorsal root responses to both adjacent dorsal root stimulation and exogenous GABA. 2. 2. [ 3H]diazepam and [ 3H]muscimol bound specifically to the crude synaptic membrane of the spinal cord and both binding sites revealed interactions different from those reported in the case of the rat. 3. 3. [ 3H]muscimol binding to the freshly prepared membrane of the spinal cord showed two different K D values, and here, Triton X-100 treatment was without effect. 4. 4. The lack of the potentiation by diazepam of GABA-mediated synaptic effects in the frog spinal cord is probably due to an immaturity of the functional unit.

Antagonistic action of baclofen on substance P-induced depolarization in the isolated spinal cord of the frog

1. 1. The depolarization induced by substance P (2 × 10 −8 mol) on the ventral root of the isolated frog spinal cord was not affected by baclofen (10 −4 M) in the normal medium, whereas the depolarization in the tetrodotoxin-treated or Ca 2+-free, 1.8 mM Mg 2+ medium-perfused preparation was significantly reduced by baclofen. 2. 2. The depolarization induced by l-glutamate (2 × 10 −7 mol) was not affected by baclofen in normal and also in TTX- or Ca 2+-free, 1.8 mM Mg 2+-treated preparation. 3. 3. The results suggest that baclofen specifically antagonizes the depolarization induced by substance P at postsynaptic membrane.

On the postsynaptic action of glutamate in frog spinal motoneurons.

In the isolated frog spinal cord depolarization of motoneurons (MNs) induced by glutamate (GLUT) was not accompanied by measurable changes of neuronal input resistance when chemical synaptic transmission was blocked by Mn2+ or Mg2+. The GLUT depolarization was, however, paralleled by a considerable increase in K+ in the extracellular space. To clarify, whether the GLUT depolarization was exclusively due to a reduction of the transmembrane K+ gradient or whether ion conductances not detectable by measurements of neuronal input resistance were involved, membrane potential (MP) was plotted semilogarithmically versus extracellular K+ activity (aKe+). During experimental elevation of aKe+ the function delta MP/dec. delta aKe+ was found to agree fairly with the Nernst equation. The slope of this function was much steeper during GLUT superfusion, indicating an influx of positive ions. The elevation of aKe+ during the GLUT action can mimic postsynaptic effects by release of transmitter from presynaptic terminals synapsing with the recorded cell. In vivo preparations do not allow blockade of chemical synaptic transmission. Therefore, it is impossible to decide, whether the recorded cell is depolarized either postsynaptically by GLUT or by K+ release from surrounding GLUT sensitive cells. As an experimental proof of the postsynaptic GLUT action is not feasible in such preparations, the ubiquitous action of GLUT in the CNS may have been overestimated.

Electron probe X-ray microanalysis of intracellular sodium, potassium and chlorine contents in amphibian motoneurones

The intracellular electrolyte content was determined in motoneurones of the isolated frog spinal cord by X-ray microanalysis. The mean concentration of Na + was found to be 79 ± 4.7 and that of Cl − and K + 43 ± 5.0 and 88 ± 11.4 respectively (mmols/kg wet weight ± S. E.). The unexpectedly high intracellular Na + concentration obtained by the present technique suggests that it can be compartmentalized or bound within the cell. The high Cl − concentration may be due to compartmentalization and/or active chloride transport into the cell. Significant loss of intracellular K + from motoneurones (about 50–60 mM/kg wet weight) in chloride-free solution confirms the existence of a high intracellular Cl − concentration in the normal external environment, since K + efflux is probably coupled with Cl −. The loss of K + from motoneurones appears to be greater than from the surrounding tissue, suggesting that electrolyte contents in the neuropile and in the somata of the motoneurones are different.

Selective depression of excitatory amino acid induced depolarizations by magnesium ions in isolated spinal cord preparations.

1. The depressant actions of Mg2+ and a range of other divalent ions on synaptic excitation and on responses produced by excitatory amino acids and other putative transmitters have been investigated in hemisected isolated spinal cords of frogs and neonatal rats. Some comparative studies were also made using the rat isolated superior cervical ganglion. 2. At concentrations above 10 microM, Mg2+ selectively antagonized N-methyl-D-aspartate (NMDA)-induced motoneurone depolarization as recorded from ventral roots of tetrodotoxin-blocked spinal cords. Depolarization evoked by quisqualate (unaffected by 20 mM-Mg2+) was resistant to the depressant action of these ions, while depolarizations evoked by other excitant amino acids were depressed to intermediate degrees. 3. Mn2+, Co2+ and Ni2+ had qualitatively similar actions to Mg2+; Mn2+ was somewhat less potent and Co2+ and Ni2+ more potent than Mg2+. The alkaline earth metal ions, Ca2+, Sr2+ and Ba2+, had very weak Mg2+-like actions. Ca2+ and Mg2+ acted additively in depressing amino acid-induced responses. 4. Mg2+ also depressed motoneurone responses evoked by noradrenaline, substance P and carbachol in the neonatal rat isolated spinal cord. However, none of these effects were as marked as the depression of NMDA-induced responses by Mg2+ in this preparation. Mg2+ did not depress motoneurone depolarization produced by 5-HT in the rat spinal cord or the depolarizing action of GABA on primary afferent terminals of the isolated frog spinal cord. 5. At concentrations producing marked depression of NMDA-induced responses, Mg2+ also depressed synaptic transmission in spinal cords in the absence of an effect on ganglionic transmission. At the same concentrations, Mn2+, Co2+ and Ni2+ depressed synaptic transmission in both preparations. 6. From the similarity in action between Mg2+ and the D-alpha-aminoadipate group of NMDA antagonists, it is suggested that the central depressant action of low concentrations of Mg2+ involves predominantly a postsynaptically mediated interference with the action of an excitatory amino acid transmitter.

The effects of increased extracellular potassium in the isolated spinal cord on the flexor reflex of the frog

The effects of superfusing the isolated frog spinal cord with various concentrations of K + on flexor reflexes were examined. The reflexes were evoked by pinching or by electrical stimulation. An increase in [K +] e from 2 to 6 mmol/l facilitates the flexor reflex. Higher [K +] e produces inhibition. It is suggested that the increase in [K +] e which may occur under physiological conditions facilitates the impulse transmission, and that larger increases in [K +] e which likely occur only under extreme conditions, result in inhibition.

Extracellular potassium accumulation and transmission in frog spinal cord

In the isolated frog spinal cord, intracellular recordings from motoneurons and neuroglia and extracellular recordings from dorsal and ventral roots were used to compare electrophysiological changes produced by tetanic stimulation of dorsal roots with those resulting from increasing extracellular potassium concentration, [K +] 0. Many of the after-effects of tetanic dorsal root stimulation could be mimicked by increasing [K +] 0. These include the following: depolarization of motoneurons and neuroglia, prolongation and depression of excitatory postsynaptic potentials, depression of dorsal root potentials, facilitation and depression of ventral root potentials, as well as increases in spontaneous synaptic activity and depression of antidromic spikes recorded from motoneurons. The levels of [K +] 0 necessary to produce effects comparable to those following maximal dorsal root stimulation are about twice those estimated from measurements with K-selective microelectrodes or glial depolarization, suggesting that both these methods underestimate the amount of potassium which accumulates in the narrow intercellular spaces between neurons and glia in the intermediate region of the spinal cord. It is concluded that, at least within the isolated frog spinal cord, K + accumulation is a significant factor modifying transmission following dorsal root tetanic stimulation and that its distribution is inhomogeneous.

Effects of changes in osmolality on spinal cord activity

The effects of change in the osmotic pressure of the bathing medium on central neural activity in the isolated, hemisected frog spinal cord were studied. The bathing medium was made hyperosmotic by either increasing all constituents (except NaHCO 3) proportionally or by adding a nonelectrolyte, mannitol; and hyposmotic by decreasing all constituents (except NaHCO 3) proportionally. Changes in osmotic pressure depressed the monosynaptic lateral column-evoked ventral root response (LC-VRR). Depression occurred with changes of only 25 mosmol/kg H 2O in bathing medium osmolality. This osmotic effect was fully expressed within the first 5 min of exposure. The osmotic effect was probably not due to a change in the excitability of the motoneurons as there was no change in the antidromic field potential recorded in the ventral horn following stimulation of the ventral root after exposure to the nonisosmotic bathing media that had depressed the LC-VRR. Exposure to nonisosmotic bathing media produced an increase in the excitability of the lateral column terminals as shown by an increase in the lateral column field potentials antidromically evoked by stimulation in the ventral horn. This increase mirrored the depression in the LC-VRR. The depression in the LC-VRR and the augmentation of the MN-LC are reversibly blocked by picrotoxin, a gamma-aminobutyric acid (GABA) antagonist. This suggests that the osmotic effect on isolated frog spinal cord may be due either to the release of endogenous GABA or perhaps to a change in extracellular K + concentration.

Hyperpolarization of frog primary afferent fibres caused by activation of a sodium pump.

1. In the isolated frog spinal cord repetitive stimulation of a lumbar dorsal root produced a sustained negative potential recorded from an adjacent inactive dorsal root by sucrose gap techniques. This negative potential was followed by a positive potential, an indication that the dorsal root terminals were hyperpolarized. Increasing the duration of the tetanus applied to the active root increased the amplitude and duration of the after-hyperpolarization which could be up to 6 mV and 3 min respectively. 2. The hyperpolarization presumably reflected an increased rate of active sodium pumping. Since it was reversibly reduced by metabolic inhibitors (dinitrophenol, NaCN) and cooling (Q10, 2 . 6) it was clearly dependent upon intact metabolic activity. In addition, a variety of procedures used to inhibit sodium pumps (including application of ouabain, elimination of potassium from the superfusate, and partial substitution of lithium for sodium ions) significantly and reversibly decreased the potential. 3. The hyperpolarization was not dependent upon intact chemical synaptic transmission since it could survive prolonged immersion of the cord in Ringer solution containing manganese or magnesium ions. 4. It is suggested that the hyperpolarization of inactive fibres resulted from a decreased extracellular potassium concentration in the dorsal horn produced as a result of a pumping mechanism which extruded sodium and transported potassium inwards by dorsal root fibres directly activated by the tetanus.

Spontaneous miniature potentials in primary afferent fibres

Intracellular recording from primary afferent fibres of the isolated frog spinal cord revealed the existence of spontaneous synaptic activity, related probably to the firing of presynaptic inhibitory fibres.

Facilitation of synaptic activity in the frog spinal cord produced by 4-aminopyridine

Reflex activity in the isolated frog spinal cord is increased after adding 4-aminopyridine (4-AP) to the bath. Depression of synaptic transmission by increased magnesium or EGTA is effectively antagonized by 4-AP. It is suggested that 4-AP facilitates synaptic transmission in the central nervous system by interacting with the calcium mechanism involved in transmitter release from presynaptic nerve terminals.

Some actions of catechol on synaptic transmission in the isolated spinal cord of the frog

The isolated frog spinal cord was used to investigate the synaptic effects of the convulsant agent catechol. Addition of the compound to the superfusate consistently enhanced orthodromic reflex activity recorded from ventral roots and augmented primary affarent depolarization. Concomitantly catechol altered the polarization changes produced in ventral and dorsal roots by putative neurotransmitter amino acids when these compounds were applied in Mg 2+-containing Ringer. Catechol reduced the hyperpolarizations induced in motoneurons by the neutral amino acids, GABA, β-alanine, taurine and glycine, but did not affect the depolarizations produced by the dicarboxylic amino acids, l-glutamate and l-aspartate. In contrast, catechol increased the dorsal root depolarizations elicited by both neutral and dicarboxylic amino acids and also the depolarizations produced by elevated potassium concentrations. Catechol did not bring about significant changes in the passive electrical properties of motoneurons or dorsal root fibers. In addition, it did not alter either the high affinity uptake or the depolarization-evoked release of tritiated GABA, glycine, l-glutamate and l-aspartate. It appears that the postsynaptic actions of catechol explain its ability to enhance spinal reflexes.

The effects of gallamine on field and dorsal root potentials produced by antidromic stimulation of motor fibres in the frog spinal cord.

The effects of gallamine on the intraspinal field potentials and the dorsal root potentials produced by antidromic stimulation of motor fibres were studied in the isolated frog spinal cord preparation. After gallamine (10-(3) M), the duration of the negative field potential produced by antidromic activation of motoneurons (N1 response) was increased often without changing its amplitude. This resulted in an increased passive spread of the antidromic action potential towards the dorsal dendritic regions, where afferent fibres terminate. In the untreated spinal cord, stimulation of motor axons produced a late negative dorsal root potential (VR-DRP) which was depressed after gallamine administration. Abolition of the VR-DRP was frequently associated with the appearance of a short latency, conducted response, in the dorsal roots (EVR-DRP). The earliest component of the EVR-DRP had a latency ranging between 0.5 and 2.5 ms measured after the peak of the N1 response recorded at the motor nucleus. Such a brief latency of the EVR-DRP suggests that this response results from electrical interaction between motoneurons and afferent fibres. After gallamine, the primary afferent depolarization produced by orthodromic stimulation of sensory nerves facilitates the EVR-DRP without necessarily increasing the amplitude or duration of the N1 response. Also, gallamine appears to increase directly the excitability of the afferent fibre terminal arborizations. The nature of the electrical interaction between motoneuron dendrites and afferent fibre terminal arborizations is discussed in terms of two hypotheses: interaction by current flows and by electrical coupling.

Pentylenetetrazol and reflex activity of isolated frog spinal cord

The superfused in vitro frog spinal cord preparation was used to investigate the effects of pentylenetetrazol (PTZ) on the spinal cord. PTZ depressed monosynaptic, but augmented polysynaptic reflexes, and decreased primary afferent deplorization. Concurrently, in Ringer's solution containing sufficient magnesium or cobalt ions to block synaptic transmission, PTZ antagonized the hyperpolarizing effects on motoneurons and the depolarizing effects on primary afferent fibers of the inhibitory amino acids GABA, beta-alanine, taurine, and glycine. PTZ did not affect responses to the excitatory amino acids glutamic acid and aspartic acid. Furthermore, PTZ did not alter high affinity uptake by cord slices, K+ -evoked release of [3H]GABA from them, or the spinal concentration of GABA. These data suggest that PTZ may produce its excitatory effects by postsynaptic blockade of inhibitory processes mediated by GABA (and possibly by other amino acids).

Effect of a water soluble benzodiazepine on the responses of spinal neurones to acetylcholine and excitatory amino acid analogues

The effects of a new water soluble benzodiazepine RO-21-3981 on responses of spinal neurones in the pentobarbitone anaesthetised cat to acetylcholine, and the amino acid analogues N-methyl-D-aspartate and kainate were determined using the microiontophoretic technique. RO-21-3981 reduced the sensitivity of neurones to all three excitants. However, on spinal Renshaw cells iontophoretic RO-21-3981 reduced acetylcholine responses more than amino acid responses. Administered intravenously RO-21-3981 depressed dorsal root but not ventral root evoked responses of Renshaw cells. The possible mechanism responsible for the effects of RO-21-3981 are discussed. Within the mammalian central nervous system iontophoretically administered benzodiazepines have been studied mostly in respect of their interaction with the inhibitory amino acids GABA and glycine (Curtis, Game and Lodge, 1976; Curtis, Lodge, Johnston and Brand, 1976; Dray and Straughan, 1976; Steiner and Felix, 1976; Kozhechkin and Ostrovskaya, 1977). However, a recent study on the isolated frog spinal cord suggests that benzodiazepines may also interact with excitatory amino acids (Evans, Francis and Watkins, 1977). Hence, the present experiments describe the effect of a new water soluble benzodiazepine, RO-21-3981, administered iontophoretically on the excitatory responses of Renshaw cells and other spinal interneurones to acetylcholine and/or kainate and N-methyl-D-aspartate (NMDA). The latter two substances are potent analogues of L-glutamate and L-aspartate respectively. In addition, the effects of intravenously administered RO-21-3981 on synaptic responses of Renshaw cells evoked by ventral and dorsal root stimulation were investigated.