Isolated Frog Spinal Cord Research Articles

Effects of Glycine and GABA on Monosynaptic EPSP in Frog Motoneurons

We report here our studies of the effects of the inhibitory transmitters glycine and GABA on monosynaptic EPSP recorded in response to microstimulation of presynaptic fibers (PF PSP) close to the bodies of motoneurons in the isolated frog spinal cord. Monosynaptic PF PSP had two components, mediated by AMPA/KA and NMDA receptors. Blockade of NMDA receptors was used to analyze the characteristics of the AMPA/KA and NMDA components of PF PSP. The amplitude of the NMDA component was 20% of PF PSP amplitude. The areas under the curves of the AMPA/KA and NMDA components were 78% and 22% respectively of the area of the PF PSP. Glycine and GABA had little effect on PF PSP, but significantly decreased the decay time constant (τdecay), by an average of 33.4 ± 4.0% (n = 18) on application of glycine and by 40.2 ± 3.6% (n = 18) on application of GABA. Blockade of NMDA receptors with DL-2-amino-5-phosophonovaleric acid (AP5) decreased the effects of glycine and GABA on the time constant. Effects were reversible and the amplitude and time characteristics of responses recovered in normal solution. Assessment of the inhibitory influences of glycine and GABA on each of the components of PF PSP showed that suppression of the NMDA component produced a greater proportionate effect than suppression of the AMPA/KA component. These results show that the inhibitory effects of glycine and GABA on the monosynaptic EPSP of motoneurons are mediated mainly by the NMDA component, with a small influence from the AMPA/KA component.

Presynaptic serotonergic modulation of spontaneous and miniature synaptic activity in frog lumbar motoneurons

In this study we investigated the effect of 5-HT on the spontaneous and miniature synaptic activity in lumbar motoneurons from isolated frog spinal cord using intracellular recording. 5-HT increased the frequency of the spontaneous and miniature postsynaptic potentials (mPSPs). The effect of 5-HT on different subpopulations of mPSPs was multidirectional: it increased the frequency of glutamatergic excitatoty mPSP by 18 % and decreased the frequency of glycinergic inhibitory mPSP by 28 %, but had no effect on the frequency of GABAergic inhibitory mPSP. The amplitude and kinetic parameters of any subpopulation of mPSPs did not change. The data obtained show that 5-HT regulates the probability of glutamate and glycine release from presynaptic terminals ending at the frog spinal motoneurons. 5-HT shifts the ba- lance between synaptic excitation and inhibition in the spinal neural network toward excitation. Thus, 5-HT participates in control of motor output and provides its facilitation.

Inhibitory Regulation of Responses of Glutamate Receptors in Frog Motoneurons

Experiments on isolated frog spinal cords (Rana ridibunda) with intracellular recording of potentials from lumbar motoneurons were performed to study interactions between the effects of exogenous excitatory (glutamate and its agonists NMDA, AMPA, kainate) and inhibitory (GABA, glycine) amino acids. Application of glycine and GABA induced reductions in responses to application of glutamate to a mean of 45.8 ± 2.9% (n = 12) and 67.8 ± 3.9% (n = 16), responses to AMPA to 13.9 ± 4.3% (n = 9) and 32.1 ± 8.3% (n = 12), responses to KA to 36.8 ± 8.2% (n = 7) and 48.0 ± 11.8% (n = 6), responses to NMDA to 7.7 ± 3.5% (n = 9) and 18.1 ± 3.8% (n = 14) of control baseline levels respectively. Similar patterns were seen on sequential application of excitatory amino acids (EAA) after glycine and GABA and on application of a mixture of GABA and glycine with an EAA. The specific glycine receptor antagonist strychnine blocked the effect of suppression of EAA responses on the background of glycine but not GABA administration, while the GABAA receptor blocker gabazine decreased the effect of suppression of EAA responses induced by application of GABA. These results point to a difference in the inhibitory influences on excitatory reactions mediated by different types of glutamate receptors and particularly the predominance of suppression of excitatory reactions mediated by NMDA receptors and the relatively weak suppression of responses mediated by kainate receptors. The inhibitory effect of glycine was about twice that of GABA (at equal concentrations).

The anesthetic urethane blocks excitatory amino acid responses but not GABA responses in isolated frog spinal cords

The anesthetic urethane is commonly used in physiological experiments. We tested urethane's actions on GABA receptors on the primary afferents in the spinal cord, which are one of the few areas in the adult central nervous system (CNS) that are depolarized by GABA, and on ligand-gated excitatory amino acid (EAA) receptors located on motoneurons. Both receptor types are critically important during anesthetic immobilization. We used the isolated hemisected spinal cord of the frog in a sucrose gap chamber to record glutamate-, N-methyl-D-aspartate (NMDA)-, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-, kainate-, and gamma-aminobutyric acid (GABA)-induced depolarizations of the dorsal root (DR) and ventral root (VR). DR potentials (DRPs) and VR potentials (VRPs) evoked by single supramaximal afferent stimuli were also studied. Urethane (10-80 mM) was applied for 10-30 min. Urethane depressed EAA responses on the motoneurons in a dose-dependent manner. At a clinical anesthetic concentration (10 mM), EAA-induced depolarizations were reduced by 8.1 ± 2.2 % (n = 7, P = 0.025), but increasing the concentration to 40 mM revealed a larger, 24.7 ± 3 % (n = 53, P = 0.0001) depressing effect of urethane on all EAA responses in the motoneurons. However, GABA and K(+) responses recorded in the DR were not altered by the presence of 10 or 40 mM urethane. Evoked DRPs and VRPs were reduced by urethane and spontaneous DR and VR potentials were suppressed by 10 or blocked by 40 mM urethane. Urethane appears to be selective for EAA-, sparing GABA responses at a clinical anesthetic concentration. Only a 10 % reduction of EAA activity seems to be necessary to induce anesthesia.

Three types of inhibitory miniature potentials in frog spinal cord motoneurons: Possible GABA and glycine cotransmission

Miniature inhibitory postsynaptic potentials (mIPSP) of motoneurons in isolated frog spinal cord were recorded in conditions of blockade of the conduction of nerve spikes and ionotropic glutamate receptors (TTX, 1 microM, CNQX, 25 microM, D-AP5, 50 microM). Three types of mIPSP were identified: those with fast and slow time characteristics and mIPSP with two-component decays. Two-component mIPSP accounted for 8.7% of all selected responses, fast mIPSP for 64.5%, and slow mIPSP for 26.8%. Blockade of GABA(A) receptors with bicuculline (20 microM) led to decreases in the numbers of slow and two-component mIPSP and an increase in the number of mIPSP with fast kinetics. Strychnine (1 microM), a blocker of glycine receptors, led to a reduction in the number of fast receptors and an increase in the number of slow potentials. These data suggest that frog spinal cord motoneurons have three types of inhibitory mIPSP, mediated by GABA, glycine, and simultaneous release of these two transmitters from the same presynaptic terminals.

Recruitment of motor neuronal persistent inward currents shapes withdrawal reflexes in the frog

The details of behaviour are determined by the interplay of synaptic connectivity within neuronal circuitry and the intrinsic membrane properties of individual neurones. One particularly dramatic intrinsic property displayed by neurones in many regions of the nervous system is membrane potential bistability, in which transient excitation of a neurone results in a persistent depolarization outlasting the initial excitation. Here we characterize the contribution of such intrinsic bistability, also referred to as plateau properties and mediated by persistent inward currents (PICs), in spinal motor neurones to the production of withdrawal behaviours in the frog. We performed experiments on the isolated frog spinal cord with attached hindlimb. This preparation allowed the simultaneous monitoring of muscle activations during motor behaviour and intracellular neuronal recordings. We found that PICs, following their potentiation by serotonin (5-HT), are recruited and contribute to the production of withdrawal behaviours. These properties conferred a voltage-dependent prolongation to the duration of motor neuronal activity. Consistent with this potentiation of motor neuronal PICs, 5-HT also increased the duration of evoked muscle activations. This behavioural potentiation, as well as the expression of PICs in individual neurones, was reduced following antagonism of L-type Ca(2+) channels. These results demonstrate that PICs in motor neurones can be recruited during the production of behaviour and play a role in specifying the temporal details of motor output.

Extracellular diffusion parameters in spinal cord and filum terminale of the frog

Extracellular space (ECS) diffusion parameters were studied in isolated frog spinal cord grey matter and filum terminale (FT), that is predominantly composed of glial cells and axons. We compared the cell swelling induced by K(+) application, hypotonic stress and tetanic stimulation of afferent input. The ECS diffusion parameters, volume fraction alpha (alpha = ECS volume/total tissue volume), tortuosity lambda (lambda(2) = free/apparent diffusion coefficient in the tissue) and non-specific cellular uptake k', were determined by the real-time iontophoretic method using TMA(+)-selective microelectrodes. Stimulation-evoked changes in extracellular K(+) concentration ([K(+)](e)) were measured by K(+)-selective microelectrodes. Histological analysis revealed that in the central region of the FT, the cell density was lower than in SC, neurons and oligodendrocytes were scarce, GFAP-positive astrocytes were abundant, and they showed thicker and more densely stained processes than in spinal cord. In the FT, alpha was 58% higher and lambda significantly lower than in the spinal cord. In 50 mM K(+), alpha in spinal cord decreased from about 0.19 to 0.09, i.e., by 53%, whereas in FT from about 0.32 to 0.20, i.e., by only 38%; lambda increased significantly more in FT than in spinal cord. Hypotonic solution (175 mmol/kg(-1)) resulted in similar decreases in alpha, and there were no changes in lambda in either spinal cord or FT. Stimulation of VIII or IX dorsal root (DR) by 30 Hz evoked an increase in [K(+)](e) from 3 to 11-12 mM in spinal cord, but to only 4-5 mM in FT. In the spinal cord this stimulation led to a 30% decrease in alpha and a small increase in lambda whereas in the FT the decrease in alpha was only about 10% and no increase in lambda was found. We conclude that in spinal cord, a complex tissue with a higher density of cellular elements than the FT, 50 mM K(+), hypotonic stress as well as DR stimulation evoked a greater decrease in ECS volume than in FT. Nevertheless, the K(+)-induced increase in tortuosity was higher in FT, suggesting that a substantial part of the K(+)-evoked increase in lambda was due to astrocytic swelling.

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.

Properties of ibogaine and its principal metabolite (12-hydroxyibogamine) at the MK-801 binding site of the NMDA receptor complex

The putative anti-addiction alkaloid ibogaine and its principal metabolite 12-hydroxyibogamine appear to act at the (+)-5 methyl-10,11-dihydro-5 H-dibenzo[ a,d]cycloheten-5–10-imine maleate (MK-801) binding site in the N-methyl- d-aspartate (NMDA)-receptor cation channel. This conclusion is based on findings that both compounds competitively displaced specific [ 3H]MK-801 binding to membranes from postmortem human caudate and cerebellum and from frog spinal cord. Ibogaine was 4–6 fold more potent than its metabolite and both compounds were less potent (50–1000-fold) than MK-801 binding to the NMDA receptor. In addition, ibogaine (100 μM) and 12-hydroxyibogamine (1 mM) blocked (85–90% of control) the ability of NMDA (100μM, 5 s) to depolarize frog motoneurons in the isolated frog spinal cord. The prevention of NMDA-depolarizations in frog motoneurons showed use-dependency and was very similar to the block produced by MK-801. In view of the abilities of MK-801 to affect the responses to addictive substances in pre-clinical investigations, our results are compatible with the idea that the ability of ibogaine and 12-hydroxyibogamine to interrupt drug-seeking behavior may, in part, result from their actions at the MK-801 binding site.

Radiolabeling motoneuron proteins in the isolated frog spinal cord preparation

An in vitro method for radiolabeling protein in adult frog spinal motoneurons is described, with per cell incorporations which are 2–3 orders of magnitude higher than previously reported for mammalian brain neurons. In the procedure, isolated lumbar spinal cord preparations from Rana pipiens are labeled with 3H- l-leucine, motoneuron cell bodies are recovered and TCA-precipitated protein is analyzed by scintillation counting. The higher levels of labeling (> 90 cpm/cell body) allow one to quantify newly synthesized protein within individual or small groups of identified nerve cell bodies. Motoneuronal labeling correlates directly with cell body size, and other sources of variation in labeling and their control are identified and discussed.

Multiple mechanisms underlying nootropic drug-induced enhancement of neuronal responses mediated by activation of glutamate NMDA-type receptors

Nootropic drugs of various chemical structure (pyracetam, dimethylaminoethanol, ethymisol, and ambocarb) possessing antiamnesic activity were found to increase depolarization evoked by L-aspartate and the EPSP late components evoked by dorsal roots stimulation (DR-EPSP). Experiments were performed on motoneurons of the lumbar region of isolated frog spinal cord superfused with Mg2+-free saline. When ionic channels of NMDA-glutamate receptors were blocked with ketamine and Mg2+, the nootropic substances did not affect DR-EPSP and motoneuronal depolarization evoked by L-glutamate. Butamid, a protein kinase A inhibitor, blocked the ethymisol and pyracetam effects, whereas polymixin B, a protein kinase C inhibitor, blocked the ambocarb effect. Trifluoroperazine, a calmodulin inhibitor, blocked the effect of dimethylaminoethanol. Proline inhibited only the pyracetam effect. These results indicate that the NMDA-potentiating activity of the above nootropic drugs may be based on both intracellular phosphorylation of NMDA receptor-channel complexes, as mediated by protein kinases, and allosteric enhancement of affinities of these complexes to excitatory amino acids.

Activation of α-adrenoceptors indirectly facilitates sodium pumping in frog motoneurons

The effects of clonidine on Na + pumping in motoneurons of the isolated frog spinal cord was investigated using sucrose gap recordings from ventral roots. Na + pump activity, induced in motoneurons either by tetanizing the dorsal root or by rapidly exposing the cord to normal medium following 30 min in K +-free Ringer's solution (K +-activated hyperpolarizations), was increased by application of clonidine (100 μM). These actions of clonidine were blocked by the preferential α 2-adrenergic antagonist yohimbine, but not by α 1-adrenergic antagonist prazosin or the β-blocker propranolol. Clonidine's effects on Na + pumping appeared to be indirect (presumably via interneurons) because its effects on K +-activated hyperpolarizations were reduced by tetrodotoxin (TTX) or high concentrations of Mg 2+. This indirect mechanism involved activation of non-NMDA excitatory amino acid receptors. Thus, in the presence of clonidine, CNQX, but not APH, limited the ability of clonidine to enhance K +-activated hyperpolarizations. The AMPA receptor may play a role in the process. K +-activated hyperpolarizations were augmented by the presence of AMPA; NMDA had no effect. The present results are consistent with the idea that activation of α 2-adrenoceptors produces the following: the release of excitatory amino acids from interneurons; the activation of non-NMDA receptors on motoneurons; increased Na + influx and loading and increased Na + pump activity.

Bulbospinal inhibition of PAD elicited by stimulation of afferent and motor axons in the isolated frog spinal cord and brainstem.

1. In the isolated spinal cord and brainstem of the frog, stimulation of the brainstem (BS) with trains of 3-4 pulses at 60-400 Hz produced dorsal root potentials (DRPs). The lowest threshold sites eliciting DRPs were located at the level of the obex up to about 2.5 mm rostrally, 0.5-1.2 mm laterally, between 0.5 and 1.6 mm depth. This region corresponds to the bulbar reticular formation (RF). 2. Stimulation of the RF with strengths below those required to produce DRPs, very effectively inhibited the DRPs produced by stimulation of a neighboring dorsal root (DR-DRPs) as well as the DRPs produced by antidromic stimulation of the central end of motor nerves (VR-DRPs). The inhibition was detectable 20 ms after the first pulse of the conditioning train, attained maximal values between 50 and 100 ms and lasted more than 250 ms. 3. Stimulation of the bulbar RF increased the negative response (N1 response) produced in the motor pool by antidromic activation of motoneurons. The time course of the facilitation of the N1 response resembled that of the reticularly-induced inhibition of the VR-DRPs and DR-DRPs. 4. The present series of observations supports the existence of reticulo-spinal pathways that are able to inhibit the depolarization elicited in afferent fibers by stimulation of other afferent fibers or by antidromic activation of motor axons. This inhibition appears to be exerted on the PAD mediating interneurons and is envisaged as playing an important role in motor control.

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.

Serotonin and GABA-induced depolarizations of frog primary afferent fibers

The interaction of γ-aminobutyric acid (GABA) and serotonin (5-HT) on primary afferent terminals of the isolated frog spinal cord was investigated by sucrose gap recordings from dorsal roots. Application of 5-HT (1.0–100 μM) to the Ringer's solution significantly reduced afferent terminal depolarizations elicited by concentrations of GABA ranging from 0.1 to 1.0 mM. The reductions of GABA-depolarizations which were produced by 1.0 μM 5-HT were mimicked by the 5-HT 1A agonists 8-OH-DPAT (8-hydroxy-2-( n-dipropylamino)tetralin) and ipsapirone. The effects of ipsapirone were reversed by the 5-HT 1A antagonists spiperone. The decreases of GABA-depolarizations produced by high doses of 5-HT were duplicated by application of α-methyl-5-HT 1 c/2 agonist and reversed by superfusion of the cord with mianserin, a 5-HT 1C/2 antagonist. The presumptive 5-HT 1A receptor-mediated effects of 1.0 μM 5-HT and 8-OH-DPAT appeared to result from a direct action on afferent terminals because the reduction of GABA responses was unchanged by addition of TTX to the Ringer's solution. In contrast, the putative 5-HT 1C/2 receptor actions of 100 μM 5-HT and α-methyl-5-HT were substantially reduced by TTX and are presumably caused by activation of receptors located on interneurons. GABA B receptors did not appear to be affected by addition of 5-HT at low or high concentrations because baclofen-induced afferent terminal hyperpolarizations remained unchanged during exposure to 5-HT. The effects of 5-HT on GABA-depolarizations were not caused by changes in GABA uptake or changes in the rate of desensitization of GABA receptors because: (1) afferent terminal depolarizations produced by the non-transportable GABA A receptor agonist muscimol were significantly reduced by 8-OH-DPAT and (2) the decrement in depolarization produced by repetitive exposure to GABA, which is caused by both ‘receptor’ desensitization and by neuronal transport of GABA, was unaffected by 5-HT. These observations are important in understanding the action of 5-HT on transmission of sensory information by afferent fibers.

Some characteristics of baclofen-evoked responses of primary afferents in frog spinal cord

Baclofen has been shown to be a selective agonist for a subclass of GABA receptors (GABA B) in many regions of the vertebrate nervous system. On the intraspinal terminals of dorsal roots (DRT), it evokes a pure hyperpolarizing response. We have previously shown that the response of DRT to GABA and some of its analogs (e.g. kojic amine) in isolated frog spinal cord is dual in nature, consisting of a bicuculline-sensitive depolarizing component and a bicuculline-resistant hyperpolarizing component. Under the working hypothesis that the hyperpolarizing component of the GABA-evoked response is mediated by the activation of GABA B receptors, we have examined, using the sucrose gap technique, some characteristics of the response of DRT to baclofen. We have found that this response is stereospecific ( l-baclofen being about 100 times more potent than d-baclofen), dependent on [K] o (response amplitude inversely related to [K] o), blocked by barium (0.5 mM causing a reduction of the response amplitude to 37% of control), and is not significantly affected by 4-aminopyridine, nor by inorganic calcium channel blockers (manganese, cobalt, cadmium). Some proposed GABA B antagonists (δ-aminovaleric acid, δ-aminolaevulinic acid, phaclofen) are also rather ineffective at blocking it. These results are therefore consistent with the notion that the baclofen-evoked response of DRT is mediated by an increase in conductance to potassium ions.

Identification of two types of excitatory monosynaptic inputs in frog spinal motoneurones

Synaptic responses induced in motoneurones by stimulation of dorsal root (DR) and reticular formation (RF) were recorded intracellularly in the isolated frog spinal cord. Argiopine (spider neurotoxin, a selective blocker of glutamate receptors of the non-N-methyl-D-aspartate (non-NMDA) type) in concentrations from 3 x 10(-7) to 1 x 10(-5) M effectively inhibited di- and polysynaptic components of excitatory postsynaptic potentials (EPSPs) induced by DR and RF stimulation. The monosynaptic component of the RF response was inhibited by argiopine, whereas the monosynaptic component of DR response was actually enhanced. All types of EPSPs studied were blocked by kynurenate (1-2 x 10(-3) M). D,L-2-Amino-5-phosphonovaleric acid (1 x 10(-4) M) slightly diminished the amplitude only of polysynaptic DR responses. A special type of argiopine-resistant, non-NMDA glutamate receptor is assumed to be involved in the generation of an EPSP by the monosynaptic DR input.

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.

Excitatory transmitters, ventral root potentials and [K +] o in the isolated frog leg-spinal cord preparation

Motoneuron depolarizations and changes in [K +] o were studied in the isolated frog spinal cord in response to non-nociceptive (touch/pressure) and nociceptive (acetic acid, heat, pinch) stimulation of the skin of the leg. Kynurenate, APV ( d(−)-2-amino-5-phosphonovalerate), and 1.0 mM Mg 2+ blocked or substantially reduced responses produced by all types of stimuli. Spantide was minimally effective in reducing nociceptive responses and had insignificant effects on responses to innocuous stimuli. These observations are consistent with the notion that excitatory amino acids activating both N- methyl- d-aspartate (NMDA) and non-NMDA receptors participate in the reflex responses to cutaneous stimuli in the frog spinal cord.

Phencyclidine receptors and N-methyl-D-aspartate antagonsim: Electrophysiologic data correlates with known behaviours

Using cortical wedges and isolated frog spinal cords, the potency of a series of psychoactive phencyclidine (PCP) and sigma receptor ligands as antagonists of N-methyl-D-aspartate (NMDA) has been compared with their potency in neurochemical and behavioural studies. Phencyclidine receptor, but not sigma or kappa, ligands were selective antagonists of NMDA on both preparations. Combination studies suggested that dissociative anaesthetics and sigma benzomorphans act at the same site. The relative potencies of the drugs as NMDA antagonists correlated well with their potency in PCP receptor binding studies in vitro and in PCP discrimination studies in vivo.