Isolated Frog Spinal Cord Research Articles

Properties of the depolarization induced by TRH in the isolated frog spinal cord

We observed the excitatory effects of thyrotropin-releasing hormone (TRH) on isolated, intraarterially perfused spinal cords of frogs. TRH (10 −7–10 −3 M) produced a transient depolarization and potentiated the spontaneous activities in both the ventral and dorsal roots. The TRH effects were related to both direct and indirect actions, and Na + deficiency attenuated the direct actions. Serotonin was the only neurotransmitter candidate with which TRH exhibited an interaction. Iproniazid potentiated the TRH effects, in normal Ringer's solution. These results indicate that the TRH depolarization due to direct actions is Na + dependent, and that indirect actions involve an interaction with a monoaminergic pathway.

Spinal seizures and excitatory amino acid-mediated synaptic transmission

In the isolated frog spinal cord penicillin or strychnine produced spinal seizures with spontaneous slow paroxysmal ventral root depolarizations (pVRDs) and superimposed motoneuron spikes. Mn 2+, tetrodotoxin, mephenesin and low [Na +] o suppressed pVRDs, an indication that paroxysmal activity requires intact excitatory synaptic transmission involving interneurons. Compounds reducing the release of amino acids ((—)baclofen) or interfering with the activation of N-methyl- d-aspartic acid (NMDA) receptors ( d,l-α-aminoadipate, d-2-amino-5-phosphonovalerate, γ- d-glutamylglycine) eliminated pVRDs. The results suggest that synaptic release of excitatory amino acids (e.g. l-glutamate, l-aspartate) and subsequent activation of specific receptors sensitive to the action of NMDA underlie spinal convulsions.

P-117) - Effects of enkephalin on the isolated frog spinal cord

P-117) - Effects of enkephalin on the isolated frog spinal cord

The actions of serotonin on frog primary afferent terminals and cell bodies

1. 1. The actions of serotonin (5-HT) were studied in the isolated frog spinal cord and dorsal root ganglion preparations. 2. 2. In the spinal cord. 5-HT increased the spontaneous activity recorded from dorsal roots, facilitated evoked spinal reflexes and produced fast and slow primary afferent depolarization (PAD). 3. 3. A direct action of 5-HT on primary afferent terminals is likely since 5-HT induced PAD remained in the presence of 1μM tetrodotoxin and 2 mM Mn 2+. 4. 4. The direct action of 5-HT on primary afferent terminals was blocked by methysergide and attenuated by concentrations of Mn 2+ in excess of that required to block transmitter release. 5. 5. Cell bodies of the dorsal root ganglion were also depolarized by 5-HT. A slow hyperpolarization occasionally followed the initial depolarization. The depolarizing action of 5-HT in the dorsal root ganglion was also attenuated by treatment with Mn 2+. 6. 6. It is concluded that 5-HT acts directly on frog primary afferents and that this influence may involve a calcium sensitive process. The dorsal root ganglion response to 5-HT appears to be a suitable model of the afferent terminal response.

Synaptic connexions between primary afferents and thoracic motoneurones in the frog.

Synaptic actions were evoked in thoracic motoneurones of the isolated frog spinal cord by 6th or 7th dorsal root volleys or impulses in single primary afferents. In a few cases the impaled motoneurones or the whole dorsal root were filled with horseradish peroxidase. Although HRP labelling has revealed that motoneurones have extensive dendritic arborizations extending into the dorsal horn, the zone in which thoracic dorsal root afferents terminate, there is no positive evidence for a direct link between them. Synaptic actions produced in thoracic motoneurones by dorsal root stimulation are mediated via interneurones and do not involve direct sensorimotor synapses. This conclusion is in agreement with results of simultaneous intracellular recording from motoneurones and individual dorsal root fibres afferent to them.

Dye and electrical coupling between frog motoneurons

Coupling between lumbar motoneurons in the isolated frog spinal cord was studied by using intracellular recordings and intracellular injections of Lucifer Yellow CH. Physiological studies revealed intra- and intersegmental, short latency electrical interactions between many motoneurons. Injections of Lucifer Yellow into motoneurons that were found to be coupled electrically revealed intra-and intersegmental dye-coupling.

Primary afferent depolarization in frog spinal cord is associated with an increase in membrane conductance.

The mechanism of primary afferent depolarization (PAD) was studied in the isolated frog spinal cord using intrafibre recording (microelectrodes filled with 0.6 M potassium sulfate) from large myelinated axons of dorsal roots. Standard current-clamp technique was used to obtain voltage-current (V-I) relationship. It was found that: (i) PAD is voltage dependent: its amplitude and rate of rise are increased with hyperpolarization; (ii) the slope of the linear part of the V-I curve obtained during PAD is decreased compared with the V-I curve at rest; (iii) the PAD equilibrium potential, estimated by extrapolation, ranged from -66 to -40 mV. These results suggest that PAD is associated with an increase in conductance of primary afferent terminals and thus seem to provide the first experimental evidence for the hypothesis that shunting of primary afferent membrane is the mechanism of presynaptic inhibition in the vertebrate nervous system.

Prolonged post-tetanic depolarization of frog dorsal root fibers

In the isolated frog spinal cord, tetanic stimulation of a dorsal root resulted in a sustained negative potential recorded from that root. This negative potential was followed by a large (5–12 mV) and slow (5–60 s) negative after-potential which persisted after the train of impulses and which was succeeded by a long-lasting positive potential. The negative after-potential was postulated to be largely a consequence of excess extracellular K + accumulated during the tetanus, because the negative after-potential and the extracellular [K +] measured with K +-sensitive microelectrodes were a function of the stimulating current, the stimulation frequency and the duration of the tetanus. Both measures were augmented by procedures which interfered with the clearance of K + from the extracellular space (addition of dinitrophenol or ouabain, partial substitution of Li + for Na +, elimination of K +from the superfusate and cooling). The negative after-potential and the liberation of much of the excess extracellular K + were reduced by increasing the superfusing divalent ion concentration with 4mM Ca 2+or 50–100 μM Mn 2+. Both measures were augmented in low Ca 2+-containing Ringer's solution. The results with low concentrations of Ca 2+, Na + and K + and with high concentrations of Ca 2+ indicate that the negative after-potential was not caused by a conductance increase for these cations. Reduction of extracellular [Cl −] did not change the negative after-potential making it unlikely that this anion is a factor in its production. The negative after-potential was not mediated by γ-aminobutyrate because its amplitude and duration were increased by the γ-aminobutyrate antagonist picrotoxin, and were reduced by pentobarbital which enhances the actions of γ-aminobutyrate. Application of the excitatory amino acid antagonists dl-α-aminoadipic acid and glutamic acid diethylester eliminated the negative after-potential and markedly reduced the amount of K + liberated by a tetanus. These results suggest that the K + release that produces the negative after-potential may result from the action of an excitatory amino acid such as l-glutamate or l-aspartate either synaptically released or released from nonsynaptic regions of afferent fibers.

Acute ethanol treatment modifies response properties and habituation of the DR-VR reflex in the isolated frog spinal cord.

Ethanol modification of habituation, a fundamental form of behavioral plasticity, was examined in the isolated frog spinal cord preparation. The polysynaptic dorsal root to ventral root (DR-VR) reflex response was assessed in normal Ringer's and at one of four ethanol concentrations. The reflex itself was facilitated at lower levels (0.025%) and depressed at higher levels (0.05-0.5%) of ethanol. Habituation, decrement of the polysynaptic ventral root response to repeated dorsal root stimulation, was reduced at all ethanol concentrations. Understanding the mechanisms of ethanol action involved in the disruption of simple forms of plasticity will help us to explain its actions on more complex forms of associational processes.

Organization of electrical coupling between frog lumbar motoneurons.

1. The organization of electrical coupling between lumbar motoneurons in the isolated frog spinal cord was investigated. Intracellular recordings were made from unidentified and identified thigh muscle motoneurons in segment 9. Excitatory postsynaptic potentials (EPSPs) elicited by stimulation of individual muscle nerves with dorsal roots cut (VR-EPSPs) were observed when the cord was perfused with either normal frog Ringer solution or calcium-free Ringer solution containing 2, 2.5, or 5 mM magnesium. 2. The VR-EPSPs recorded following stimulation of the homonymous muscle nerve were greater in amplitude and occurred with a shorter latency than the VR-EPSPs observed following stimulation of muscle nerves to different muscles. In addition, the latter VR-EPSPs had a shorter half-decay time. 3. Electrical connections made by motoneurons innervating synergistic muscles had a similar distribution, as indicated by the linear correlation observed between the amplitudes of VR-EPSPs in the same motoneuron elicited by stimulation of muscle nerves innervating synergistic muscles. No such linear correlation was observed between VR-EPSP amplitudes when muscle nerves innervating antagonistic muscles were stimulated. 4. Small-amplitude, spikelike potentials were often observed on top of the muscle nerve VR-EPSPs. In the case of VR-EPSPs elicited by stimulation of muscle nerves not containing the axon from the recorded motoneuron, the spikelike component accounted for a major part of the response. These spikelike potentials had a rapid time course (half-decay time, 2.5-5.9 ms) and in a few cases were observed to be all or none. These observations are consistent with dendritic action potentials in the recorded motoneuron as well as electrotonic coupling between a small number of neurons close to the recording site in the impaled motoneuron. 5. These results are interpreted to mean that a high degree of specificity exists in the organization of electrical coupling among frog lumbar motoneurons.

EFFECT OF TAURINE ON DEPOLARIZATIONS INDUCED BY L-GLUTAMATE AND OTHER EXCITATORY AMINO ACIDS IN THE ISOLATED SPINAL CORD OF THE FROG

The effect of taurine on depolarizing responses to L-glutamate (L-glu) was examined in isolated frog spinal cord using the sucrose gap method. Taurine (1 mM) reduced L-glu-induced depolarization in ventral and dorsal roots, even when calcium ion was deleted from or EGTA was added to the perfusate. GABA (1 mM) showed little or no effect on the L-glu response. A log dose-depolarization curve for L-glu was found to be shifted by taurine (1 mM) to the right in a non-parallel fashion. Strychnine blocked the taurine effect completely, while picrotoxin produced a partial reduction, and bicuculline exhibited no blockade at all. The depolarizations by 3-types of excitatory amino acids were also inhibited by taurine in the following order: N-methyl-D,L-aspartate >kainate>quisqualate. These results suggest a possibility that taurine but not GABA inhibits the depolarization mediated by excitatory amino acid receptors, and this may explain, in part, the inhibitory action of taurine in the central nervous system.

Effects of naloxone on spinal reflexes in the isolated frog spinal cord.

Naloxone (Nix) has generally been considered to be a specific antagonist of opiates. However, other effects of Nix have been reported recently. This study demonstrated the effects of Nix using the isolated intra-arterially perfused frog spinal cord and discussed characteristics of the opiate system in the spinal cord. Nix (more than 30 mM) potentiated spontaneous discharges recorded from the ventral root (VR). Resting potentials of VR and the dorsal root (DR) recorded by sucrose-gap method were not affected or slightly depolarized by Nix. Methionine-enkephlin (Enk), 30 mM, inhibited VR-DR potential (VR-DRP) significantly, whereas morphine only slightly inhibited VR-DRP even at 100 mM. It suggests that opiate receptors mediating this inhibitory effect on VR-DR neuronal network have higher affinity for Enk. Nix (more than 10 mM) dose-dependently inhibited VR-DRP, but partially antagonized the inhibitory effect of Enk. Nix had no effect on DR-DR network. Enk increased DR-DRP, although the effect was not significant. These observations suggest that the spinal opiate system may individually modulate two presynaptic inhibitory systems, i.e., DR-DR and VR-DR neuronal networks.

Effect of taurine on depolarizations induced by L-glutamate and other excitatory amino acids in the isolated spinal cord of the frog.

The effect of taurine on depolarizing responses to L-glutamate (L-glu) was examined in isolated frog spinal cord using the sucrose gap method. Taurine (1 mM) reduced L-glu-induced depolarization in ventral and dorsal roots, even when calcium ion was deleted from or EGTA was added to the perfusate. GABA (1 mM) showed little or no effect on the L-glu response. A log dose-depolarization curve for L-glu was found to be shifted by taurine (1 mM) to the right in a non-parallel fashion. Strychnine blocked the taurine effect completely, while picrotoxin produced a partial reduction, and bicuculline exhibited no blockade at all. The depolarizations by 3-types of excitatory amino acids were also inhibited by taurine in the following order: N-methyl-D,L-aspartate greater than kainate greater than quisqualate. These results suggest a possibility that taurine but not GABA inhibits the depolarization mediated by excitatory amino acid receptors, and this may explain, in part, the inhibitory action of taurine in the central nervous system.

The effects of cyclic AMP on the membrane potentials in the isolated frog spinal cord

Membrane potential changes were recorded by the sucrose-gap method from the ventral as well as dorsal roots in the isolated perfused spinal cord of the frog. Cyclic AMP (l-5mM), dissolved in perfusion fluid, caused depolarization in both roots in preparations where spontaneous DC shifts were frequently observed. Adenosine phosphates (ATP,ADP,AMP ;3mM) exerted the same effects (sometimes a small hyperpolarization preceded the depolarization) in these preparations, although cyclic GMP and adenosine (3mM for each) hyperpolarized both roots. In preparations in which neurotransmission was blocked with tetrodotoxin (10-7), cyclic AMP failed to depolarize the ventral root, although ATP could still do so. Therefore, unlike the direct effect of ATP on the post-synap-tic membranes of the ventral horn cells, the excitatory effect of cyclic AMP may be trans-synaptic. In Ringer solution containing high concentration of Ca++, ATP and cyclic AMP (3mM for each) could depolarize the ventral root, although their amplitudes were greatly reduced. Therefore, their depolarizing actions may be partly ascribed to their Ca-chelating actions: but especially for cyclic AMP, the depolarizing action might be covered with decrement of spontaneous DC shifts caused by high concentration of Ca++.

Supersensitivity of the GABA receptor in the frog spinal cord, as induced by kainic acid

1. In the isolated frog spinal cord perfused with kainic acid (KA, 5 × 10 −4M) containing Ringer's solution, within 2 hr there were increases in the amplitude of the dorsal root depolarization, as induced by the GABA-agonists. 2. KA perfusion produced increases in the specific binding of [ 3H]muscimol to crude synaptic membranes and incubation with KA for 3 hr did not increase [ 3H]muscimol binding. 3. [ 3H]GABA was released from KA-treated spinal cord slices in the presence of high K +. 4. KA-induced supersensitivity of the dorsal root to GABA may relate to direct actions on primary afferent terminals and not to denervation of GABAergic neurons.

Changes of intracellular sodium and potassium ion concentrations in frog spinal motoneurons induced by repetitive synaptic stimulation

A post-tetanic membrane hyperpolarization following repetitive neuronal activity is a commonly observed phenomenon in the isolated frog spinal cord as well as in neurons of other nervous tissues. We have now used double-barrelled Na +- and K +-ion-sensitive microelectrodes to measure the intracellular Na +- and K +-concentrations and also the extracellular K +-concentration of lumbar spinal motoneurons during and after repetitive stimulation of a dorsal root. The results show that the posttetanic membrane hyperpolarization occurred at a time when the intracellular [Na +] reached its maximal value, intracellular [K +] had its lowest level and extracellular [K +] was still elevated. The hyperpolarization was blocked by ouabain and reduced by Li +. These data support the previous suggestion that an electrogenic Na +/K + pump mode may be the mechanism underlying the post-tetanic membrane hyperpolarization.

Excitatory amino acids and potassium release in the frog spinal cord

Synaptic release of excitatory amino acids such as l-glutamate and/or l-aspartate and subsequent activation of specific receptors by these putative transmitters appears necessary for the release of K + by afferent stimulation in the isolated frog spinal cord. This conclusion is based on the findings that (−)baclofen, which is thought to reduce the presynaptic release of putative excitatory amino acid transmitters, and some amino dicarboxylic amino acids ( d,l-α-aminoadipic acid, 2-amino-4-phosphonobutyric acid, and d,l-α,ϵ-diaminopimelic acid), which are believed to interfere with the activation of receptors by these same excitatory amino acids, significantly attenuate the increment in extracellular K + evoked by tetanic dorsal root stimulation.

Combined morphological and electrophysiological description of connections between single primary afferent fibres and individual motoneurons in the frog spinal cord.

In experiments on the isolated frog spinal cord the relationship between the statistical properties of the unitary EPSP and the number of synaptic contacts was determined when the primary afferent fibre used in evoking the EPSP and the motoneuron in which it was recorded were both stained with HRP. The size of the chemical component of the EPSP corresponds to the number of presynaptic boutons. Less obvious numerical correlation exists between the number of contact zones and the number of binomial units.

An analysis of the epileptogenic potency of CO2+- its ability to induce acute convulsive activity in the isolated frog spinal cord.

The action of Co2+ on the isolated frog spinal cord was studied by extracellular application of the ion in the superfusing solution. A complete and reversible blockade of chemical synaptic transmission by Co2+ (3 mmol/l) could be achieved after a superfusion period of 20-30 min. During continued Co2+ application (greater than 60 min) the following effects upon the motoneuron membrane, dorsal root and ventral root fibres were observed. Motoneurons and ventral root fibers: 1. prolongation of initial segment action potential to a maximum of 30 ms, 2. blockade of the long afterhyperpolarization, 3. abolition of adaptation, 4. increased duration of fibre action potential in the ventral root, 5. backfiring after ventral root stimulation. Dorsal root fibres: 1. prolongation of the extraspinal fibre action potential, 2. marked prolongation of the action potential of the terminal region, 3. backfiring of multiple action potentials after dorsal root stimulation. Even in the presence of Co2+, when synaptic transmission was completely blocked, strong convulsive reactions of the isolated spinal cord were observed. Intracellular injection of Co2+ into motoneurons did not affect the action potential, but led to a shift of the EIPSP towards the membrane potential. The results indicate that the induction of convulsive reactions by Co2+ is mainly due to a prolongation of action potentials. The plateau-like deformation of the action potential of the initial segment membrane and presumably of the terminal region of nerve endings results in retrograde propagation of action potentials and in some cases induces oscillatory discharge of single neurons.

Lithium distribution across the membrane of motoneurons in the isolated frog spinal cord.

Lithium sensitive microelectrodes were used to investigate the transmembrane distribution of lithium ions (Li+) in motoneurons of the isolated frog spinal cord. After addition of 5 mmol.1(-1) LiCl to the bathing solution the extracellular diffusion of Li+ was measured. At a depth of 500 micrometers, about 60 min elapsed before the extracellular Li+ concentration approached that of the bathing solution. Intracellular measurements revealed that Li+ started to enter the cells soon after reaching the motoneuron pool and after up to 120 min superfusion, an intra - to extracellular concentration ratio of about 0.7 was obtained. The resting membrane potential and height of antidromically evoked action potentials were not altered by 5 mmol.1(-1) Li+.