Effects of guanidine on synaptic transmission in the spinal cord of the frog

Summary The effects of guanidine on motoneurons of the isolated frog spinal cord were studied by adding the drug to the solution bathing the cord during intracellular recording. Guanidine (5·10^–4 M) did not alter the membrane potential of motoneurons.The main effect was a marked increase of the amplitudes and frequencies of small spontaneously occurring inhibitory postsynaptic potentials. The hyperpolarizing component of postsynaptic potentials evoked by stimulation of dorsal roots was also enhanced by guanidine. Higher concentrations of guanidine (5·10^–3 M) resulted in a very large and irreversible increase of the small spontaneously occurring inhibitory potentials, which now appeared in a regular, rhythmic pattern.The effects of guanidine could easily be blocked by increasing the magnesium ions (15 mM) in the bath solution.These results indicate that guanidine facilitates the release of an inhibitory transmitter in afferent terminals of the frog spinal cord either by a direct action on these terminals or indirectly by an action on nerve endings impinging on inhibitory interneurons.This work was supported by the Deutsche Forschungsgemeinschaft     

Antagonism of excitatory amino acid-induced responses and of synaptic excitation in the isolated spinal cord of the frog.

1. A range of compounds has been tested for excitatory amino acid agonist or antagonist activity and for effects on synaptic activity on isolated hemisected spinal cords of frogs. 2. L-Monoamino dicarboxylic acids of chain length up to 8 carbon atoms (L-alpha-aminosuberate) were all agonists. 3. Within a series of D-monoamino dicarboxylic acids, and with diamino dicarboxylic acids (mainly unresolved mixtures of diasteroisomers), there was a progression from agonist activity, for compounds of chain length equal to or shorter than glutamate, to antagonist activity, for compounds of longer chain length equal to or shorter than glutamate, to antagonist activity, for compounds of longer chain length, D-alpha-Aminosuberate (D alpha SD) was the most potent antagonist. 4. The antagonist actions of these substances showed a Mg2+--like selectivity with respect to depolarizations produced by different excitants. N-methyl-D-aspartate (NMDA) was the most susceptible agonist and quisqualate and kainate the least susceptible. Responses to other excitatory amino acids, including L-glutamate and L-aspartate, showed intermediate sensitivity to the antagonists. 5. A parallelism was observed between the relative potencies of mono- and diamino dicarboxylic acids as NMDA antagonists and their relative potencies as depressants of synaptic responses. 6. The results support the concept of different types of excitatory amino acid receptors, with NMDA and its antagonists acting predominantly on one type. These NMDA receptors are probably transmitter receptors activated by an excitatory amino acid transmitter.     

Effects of flurazepam on amino acid-evoked responses recorded from the lobster muscle and the frog spinal cord.

The effects of flurazepam hydrochloride on GABA- or glutamate-evoked responses recorded from the lobster muscle fibre and the frog isolated spinal cord were studied using electrophysiological techniques. On lobster muscle flurazepam (up to 100 μM) reversibly antagonized responses to bath-applied or iontophoretically-applied glutamate without any effect on GABA responses. Higher concentrations of flurazepam (>200 μM) sometimes increased the resting membrane conductance, an effect different from that of GABA in being insensitive to picrotoxin (0.5 μM). In the tetrodotoxin (TTX)-treated frog spinal cord flurazepam (5 μM) reversibly antagonized both glutamate- and GABA-evoked dorsal root depolarizations although a smaller dose (2.5 μM) clearly potentiated the action of GABA. Spontaneous fluctuations of the dorsal root d.c. level in the presence of flurazepam were also observed. It is suggested that flurazepam antagonized amino acid responses by blocking receptor-activated Na⁺ channels on the postsynaptic membranes. However, the potentiation of GABA action on the frog spinal cord may have involved either release of endogenous GABA or “sensitization” of spinal receptors to GABA, an action easily obscured by the predominant antagonistic effect of flurazepam at higher concentrations.     

Effects of ketamine on synaptic transmission in cat spinal cord.

Effects of ketamine on the evoked synaptic potentials and on the post-tetanic potentiation (PTP) of synaptic responses were studied in dorsal root-ventral root preparations of unanaesthetized spinal cats. Ketamine, in doses of 10–20 mg/kg, depressed the synaptic transmission, but enhanced the PTP responses in spinal cord. The enhancement of PTP responses after ketamine may be related to its seizure-inducing property.     

Effects of tiletamine on spinal cord synaptic transmission

The effect of tiletamine on synaptic transmission was studied in the dorsal root—ventral root preparation of unanesthetized spinal cats. Tiletamine, 10–20 mg/kg, reduced the evoked monosynaptic response of motoneurons with only slight depression on polysynaptic responses. It also augmented the post-tetanic potentiation (PTP) of monosynaptic responses, which may be related to its seizure-producing property. The effect of tiletamine developed within 5 min after i.v. administration and lasted for more than 1 hr.     

The action of acetylcholine antagonists on amino acid responses in the frog spinal cord in vitro.

1 The isolated hemisected frog spinal cord has been used to study the action of acetylcholine antagonists on amino acid responses by means of sucrose gap recording. 2 Primary afferents and motoneurones were shown to contain few, if any, cholinoceptors, since acetylcholine and carbachol responses were essentially abolished when synaptic transmission was blocked with magnesium ions or when action potentials were blocked by tetrodotoxin. 3 Curare antagonized the gamma-aminobutyric acid (GABA) and beta-alanine depolarizations of primary afferents and hyperpolarizing action of these amino acids on motoneurones. Nicotine also antagonized beta-alanine depolarizations and to a small extent GABA depolarizations of primary afferents. These actions are similar to but weaker than those obtained previously with picrotoxin. 4 Atropine selectively antagonized beta-alanine depolarizations of primary afferents and blocked beta-alanine and glycine hyperpolarizations of motoneurones. GABA responses were entirely resistant to the action of atropine. These actions are similar to but 50 times weaker than those obtained previously with strychnine. 5 Dihydro-beta-erythroidine, tetraethylammonium, and gallamine were entirely ineffective in antagonizing amino acid responses. Since these agents are known to block the dorsal root potential elicited by ventral root stimulation but have no effect on the amino acid responses of primary afferents, it is evident that a cholinergic step is involved in this pathway.     

Bicuculline and the frog spinal cord

1 The effects of bicuculline on dorsal and ventral root activity and upon the depressant effect of gamma-aminobutyric acid (GABA) and glycine on ventral root responses have been studied on the isolated spinal cord of the frog.2 In the absence of stimulation, the alkaloid induced a variety of activity of which the most notable was phasic simultaneous slow wave depolarization in the dorsal and ventral roots which could be reduced or suppressed by magnesium.3 With low concentrations of bicuculline, the adjacent dorsal root response evoked by a single stimulus was depressed maximally before an increase in the ventral root response could be discerned.4 The bicuculline-induced dorsal root activity (in the absence of stimulation) was still apparent at times when the evoked dorsal root response was reduced.5 Bicuculline did not differentiate between the depressant effects of GABA and glycine on the evoked ventral root responses.6 The excitant effects of bicuculline reported here did not appear to be attributable to specific antagonism of the postsynaptic depressant action of GABA.     

Cholinergic transmission in the frog spinal cord

The effect of acetylcholine and other drugs has been tested on the isolated and sagittally hemisected spinal cord of the frog (Rana temporaria). The release of acetylcholine from this preparation during stimulation of the spinal roots and whole hemicord has also been studied. After inactivation of the tissue cholinesterases acetylcholine was released spontaneously from the preparation at a steady rate of about 5.5 pmole/15 min/preparation. No increase in this release was obtained by stimulating the dorsal roots, but antidromic stimulation of the ventral roots always gave an increase in the rate of acetylcholine release up to 2.3 times the spontaneous level. Direct stimulation of the spinal cord did not alter the rate of acetylcholine release. Acetylcholine (1 mM) and eserine (10 mM) had weak excitatory actions and prolonged the reflex response of the preparation during dorsal root stimulation, but dihydro-β-erythroidine and atropine had no effect. The response evoked by antidromic ventral root stimulation was potentiated by eserine (10 μM) and depressed by acetylcholine (10 μM) and dihydro-β-erythroidine (1 μM) as in the toad preparation. The evidence supporting cholinergic transmission at some synapses in the spinal cord is discussed in relation to these results.     

The action of general anaesthetic agents on root responses of the frog isolated spinal cord

1. The action of volatile and barbiturate general anaesthetic agents on synaptic transmission in the frog isolated spinal cord has been studied by recording ventral root synaptic potentials and spike discharges evoked by volleys in a dorsal root and in the lateral column fibres.2. Some observations on the distribution of the lateral column fibres and the characteristics of the dorsal root potentials have been presented.3. Volatile agents depressed and eventually abolished all components of the ventral root responses. Failure of motoneurone discharge was the result of two factors, a decrease in the slope of the synaptic potential and an elevation of the critical depolarization required to trigger propagated impulses.4. Barbiturate compounds, in contrast, readily abolished polysynaptic components of the ventral root responses, but the short latency discharge produced by lateral column stimulation was potentiated, and was accompanied by a lowering of the firing threshold of motoneurones. The mechanism of this potentiation by barbiturate compounds is discussed.5. It is concluded that volatile agents act predominantly on the initial segment and subsynaptic elements of the motoneurone, whereas barbiturate compounds depress the presynaptic or postsynaptic components of interneuronal synapses.     

Mechanisms involved in the metabotropic glutamate receptor-enhancement of NMDA-mediated motoneurone responses in frog spinal cord

1. The metabotropic glutamate receptor (mGluR) agonist trans-(+/-)-1-amino-1,3-cyclopentanedicarboxylic acid (trans-ACPD) (10-100 microM) depolarized isolated frog spinal cord motoneurones, a process sensitive to kynurenate (1.0 mM) and tetrodotoxin (TTX) (0.783 microM). 2. In the presence of NMDA open channel blockers [Mg2+; (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate (MK801); 3,5-dimethyl-1-adamantanamine hydrochloride (memantine)] and TTX, trans-ACPD significantly potentiated NMDA-induced motoneurone depolarizations, but not alpha-amino-3-hydroxy-5-methylisoxazole-4-proprionate (AMPA)- or kainate-induced depolarizations. 3. NMDA potentiation was blocked by (RS)-alpha-methyl-4-carboxyphenylglycine (MCPG) (240 microM), but not by alpha-methyl-(2S,3S,4S)-alpha-(carboxycyclopropyl)-glycine (MCCG) (290 microM) or by alpha-methyl-(S)-2-amino-4-phosphonobutyrate (L-MAP4) (250 microM), and was mimicked by 3,5-dihydroxyphenylglycine (DHPG) (30 microM), but not by L(+)-2-amino-4-phosphonobutyrate (L-AP4) (100 microM). Therefore, trans-ACPD's facilitatory effects appear to involve group I mGluRs. 4. Potentiation was prevented by the G-protein decoupling agent pertussis toxin (3-6 ng ml(-1), 36 h preincubation). The protein kinase C inhibitors staurosporine (2.0 microM) and N-(2-aminoethyl)-5-isoquinolinesulphonamide HCI (H9) (77 microM) did not significantly reduce enhanced NMDA responses. Protein kinase C activation with phorbol-12-myristate 13-acetate (5.0 microM) had no effect. 5. Intracellular Ca2+ depletion with thapsigargin (0.1 microM) (which inhibits Ca2+/ATPase), 1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetracetic acid acetyl methyl ester (BAPTA-AM) (50 microM) (which buffers elevations of [Ca2+]i), and bathing spinal cords in nominally Ca2+-free medium all reduced trans-ACPD's effects. 6. The calmodulin antagonists N-(6-aminohexyl)-5-chloro-1-naphthalenesulphonamide (W7) (100 microM) and chlorpromazine (100 microM) diminished the potentiation. 7. In summary, group I mGluRs selectively facilitate NMDA-depolarization of frog motoneurones via a G-protein, a rise in [Ca2+]i from the presumed generation of phosphoinositides, binding of Ca2+ to calmodulin, and lessening of the Mg2+-produced channel block of the NMDA receptor.     

Effects of diltiazem and verapamil on responses to acetylcholine.

1. The calcium channel antagonists diltiazem and verapamil were found to alter the average lifetime of ion channels activated by acetylcholine (ACh). 2. Average channel lifetime was determined from the decay phase of miniature endplate currents at the neuromuscular junction of mouse hemidiaphragms and from direct recording of single channel currents activated by ACh from BC3H1 mouse tumour cells in culture. 3. Both diltiazem and verapamil reduced average channel lifetime in a dose-dependent manner. For each drug, concentrations as high as 20 microM-100 microM were required to decrease channel lifetime by 50%. 4. Single channel recording experiments also showed that both diltiazem and verapamil greatly decreased the frequency of opening events at concentrations as low as 2 microM to 5 microM. This finding is consistent with an enhancement of receptor desensitization.     

Effects of cyclobenzaprine on spinal synaptic transmission.

The effects of cyclobenzaprine on spinal synaptic transmission were studied in spinal unanesthetized cats. The drug, in cumulative doses up to 20 mg/kg, depressed monosynaptic reflexes evoked by stimulation at 0.2 Hz to a moderate degree. Polysynaptic reflexes were depressed to a similar extent. Monosynaptic reflexes evoked repeatedly at 5 and 10 Hz were depressed even after 5 mg/kg of cyclobenzaprine. Maximum potentiation of monosynaptic responses after tetanization was not affected. Neither direct postsynaptic nor recurrent inhibition was influenced by the drug. Presynaptic inhibition was slightly reduced. It is suggested that direct effects of cyclobenzaprine at the spinal level do not represent the primary site of action responsible for its antispastic activity.     

Quinoxalinediones selectively block quisqualate and kainate receptors and synaptic events in rat neocortex and hippocampus and frog spinal cord in vitro.

1. Two quinozalinediones, FG9041 and FG9065, which had previously been shown to displace binding to the quisqualate receptor, were tested on rat neocortex and frog spinal cord in vitro against depolarizations induced by quisqualate, kainate and N-methyl-D-aspartate (NMDA). In both preparations effects of quisqualate were reduced the most and those of NMDA the least. 2. The near unitary slopes of the Schild plots were consistent with a competitive type of interaction. pA2 values for FG9041 were estimated to be 6.6, 6.1 and 5.1 in frog cord and 5.9, 5.3 and and about 4 in the rat neocortex for quisqualate, kainate and NMDA antagonism, respectively. FG9065 gave equivalent pA2 values of 6.2, 5.6 and 4.5. 3. At concentrations, which were without effect on depolarizations induced by NMDA, FG9041 and FG9065 reduced or blocked synaptically-evoked field potentials in hippocampal and neocortical slices superfused with normal magnesium-containing medium. Since these synaptic components are also insensitive to NMDA antagonists, these results are consistent with their mediation by postsynaptic receptors of the quisqualate (or kainate) type. 4. By contrast, quinoxalinediones had only limited effects on spontaneous epileptiform activity seen in both neocortical and hippocampal preparations when superfused with magnesium-free medium. These burst discharges were, however, abolished by NMDA antagonists. 5. In the frog spinal cord the early component of the dorsal root to ventral root reflexes was selectively reduced by FG9041 whereas NMDA antagonists reduced the longer latency components. 6. Our results suggest that the quinoxalinediones are likely to be useful pharmacological probes for elucidating the role of non-NMDA receptors in the vertebrate central nervous system.     

Effects of proline and other neutral amino acids on ventral root potentials of the frog spinal cord in vitro.

Responses of frog motoneurones in vitro to bath-applied amino acids were studied by means of extracellular recordings from ventral roots in the presence of tetrodotoxin. Proline, GABA or taurine produced Cl^?-dependent depolarizations or hyperpolarizations of the ventral roots; these effects were analysed in a quantitative manner and fitted by dose-response relationships. Proline was a hyperpolarizing agent more potent than GABA or taurine. The different polarity of the amino acid responses might have been due to changes in the Cl^? equilibrium potential. However, hyperpolarizations following high doses (>2 mM) of proline were probably mediated by a different mechanism (perhaps increased K⁺ permeability) as suggested by the different kinetics and ion-sensitivity of this phenomenon. Hyperpolarizations induced by proline were antagonized by strychnine but not by picrotoxin (the opposite was found in the case of GABA) while those due to taurine were reduced by either convulsant. Amino acid-evoked depolarizations were almost insensitive to convulsants. It is concluded that proline in low concentrations is a fairly potent amino acid and that it might have a physiological role in certain synaptic mechanisms at spinal level.     

Release of endogenous glutamate and aspartate from the frog spinal cord in vitro.

The release of the endogenous excitatory amino acids aspartate and glutamate from an in vitro slice preparation of the frog spinal cord was investigated by using high performance liquid chromatography (HPLC). Spinal slices were incubated at 4 degrees C to minimize amino acid uptake; samples of the bathing solution were collected at 10 min intervals for amino acid assay. In each experiment electrophysiological responses were recorded continuously from ventral roots after stimulation of an adjacent dorsal root, so that changes in the profile of amino acid release could be correlated with electrophysiological responses. At rest the release of glutamate and aspartate was 52.9 +/- 5.8 and 66.9 +/- 5.6 pmol/10 min, respectively, and was unaffected by low Ca2+ media or tetrodotoxin. After trains of high frequency stimulation applied to a dorsal root the release of glutamate and aspartate was significantly and reproducibly enhanced by 33 +/- 13 and 49 +/- 18%, respectively. The stimulus-dependent release was blocked by low Ca2+ media, tetrodotoxin or topical application of the neuropeptide thyrotropin releasing hormone. The present study provides direct evidence to support the role of glutamate and aspartate as neurotransmitters in the spinal cord.     

Antagonism by some antihistamines of the amino acid-evoked responses recorded from the lobster muscle fibre and the frog spinal cord.

1 The effects of some antihistamines on the lobster muscle fibre and the frog spinal cord were investigated using intracellular and extracellular recordings, respectively. 2. On lobster muscle, histamine H1-blockers reversibly antagonized responses to bath-applied glutamate, aspartate and quisqualate but not responses to gamma-aminobutyric acid (GABA). Iontophoretic glutamate potentials were also reduced. Histamine (up to 1 mM) had no effect on this preparation. 3 The H1-antagonists produced a small increase in muscle membrane conductance and a slight hyperpolarization. These effects were largely unchanged in a low C1- bathing solution. Procaine (1 mM) decreased membrane conductance and did not affect responses to GABA or glutamate. 4 The H2-antagonist burimamide blocked both glutamate and GABA-evoked responses on the lobster muscle without affecting resting potential or conductance. 5 In the frog cord, bath-applied histamine produced ventral root depolarizations and dorsal root hyperpolarizations (sometimes biphasic responses). These effects were reduced by tetrodotoxin (TTX) but not by antazoline (H1-blocker) or burimamide; the latter reversibly antagonized responses to both glutamate and GABA on TTX-treated cords while antazoline was ineffective. 6 It is suggested that antihistamines can act as non-specific amino acid antagonists by interacting at the level of the receptor-coupled ionophores.     

Alterations in spontaneous activity of the isolated frog spinal cord in Calcium-free environment.

Spontaneous electrical activity of the isolated frog spinal cord was examined in Ca2+-free environment. Spontaneous discharges from the ventral root altered in four distinguishable steps. The first step was an immediate increase in the rate of spontaneous discharges and the second was a gradual decrease. The third was the occurrence of rhythmical bursts, and the last, the appearance of continuous firing. The rhythmical bursts could be depressed by the addition of metabolic inhibitors (ouabain or dinitrophenol in a concentration of 5 x 10(-5) M) as well as of Ca2+-chelating agents (EDTA or EGTA in a concentration of 10(-3) M). Our results suggest that the occurrence of the rhythmical bursts requires a metabolic pumping process to redistribute Na+ and K+ across the membrane and a small amount of Ca2+ for transmitter secretion.