J.C. Hackman

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-5H- 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 microM) and 12-hydroxyibogamine (1 mM) blocked (85-90% of control) the ability of NMDA (100 microM, 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.

Changes in membrane potential of frog motoneurons induced by activation of serotonin receptor subtypes

Application of serotonin to the isolated, hemisected frog spinal cord resulted in two distinctive changes in motoneuron membrane potential: hyperpolarizations were produced by low concentrations (0.01-1.0 microM) and depolarizations by higher concentrations (3.0-100 microM). The hyperpolarizations appeared to be caused by a direct action of the amine upon motoneurons since exposure of spinal cord tetrodotoxin or magnesium ions in concentrations which blocked interneuronal firing and synaptic transmission, respectively did not reduce these responses. In contrast, depolarizations were significantly reduced by tetrodotoxin or magnesium indicating a large indirect component. The use of agonists and antagonists known to discriminate among different subtypes of serotonin receptors indicated that the hyperpolarizations were produced by activation of 5-HT1A receptors and the depolarizations were generated by activation of 5-HT2 and/or 5-HT1C receptors. Accordingly, the selective 5-HT1A agonists 8-hydroxy-2-(n-dipropylamino)tetralin and ipsapirone directly hyperpolarized motoneurons. The changes in potential produced by low concentrations of serotonin and by these agonists were blocked by the 5-HT1A receptor antagonists spiperone and spiroxatrine. In contrast, application of high concentrations of alpha-methyl-5-hydroxytryptamine, a serotonin analog which activates 5-HT1C and 5-HT2 receptor subtypes, depolarized motoneurons. These depolarizations, and those produced by high concentrations of serotonin, were blocked by the 5-HT1C/5-HT2 antagonists ketanserin, methysergide and mianserin. These observations indicate that serotonin can alter the membrane potential of motoneurons directly and indirectly by activation of both 5-HT1 and 5-HT2 receptor subtypes. Activation of different receptor subtypes depends upon the concentration of the amine.

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

The metabotropic glutamate receptor (mGluR) agonist trans‐(±)‐1‐amino‐1,3‐cyclopentanedicarboxylic acid (trans‐ACPD) (10–100 μM) depolarized isolated frog spinal cord motoneurones, a process sensitive to kynurenate (1.0 mM) and tetrodotoxin (TTX) (0.783 μM). 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 α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐proprionate (AMPA)‐ or kainate‐induced depolarizations. NMDA potentiation was blocked by (RS)‐α‐methyl‐4‐carboxyphenylglycine (MCPG) (240 μM), but not by α‐methyl‐(2S,3S,4S)‐α‐(carboxycyclopropyl)‐glycine (MCCG) (290 μM) or by α‐methyl‐(S)‐2‐amino‐4‐phosphonobutyrate (L‐MAP4) (250 μM), and was mimicked by 3,5‐dihydroxyphenylglycine (DHPG) (30 μM), but not by L(+)‐2‐amino‐4‐phosphonobutyrate (L‐AP4) (100 μM). Therefore, trans‐ACPDs facilitatory effects appear to involve group I mGluRs. 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 μM) and N‐(2‐aminoethyl)‐5‐isoquinolinesulphonamide HCl (H9) (77 μM) did not significantly reduce enhanced NMDA responses. Protein kinase C activation with phorbol‐12‐myristate 13‐acetate (5.0 μM) had no effect. Intracellular Ca2+ depletion with thapsigargin (0.1 μM) (which inhibits Ca2+/ATPase), 1,2‐bis(O‐aminophenoxy)ethane‐N,N,N′,N′‐tetracetic acid acetyl methyl ester (BAPTA‐AM) (50 μM) (which buffers elevations of [Ca2+]i), and bathing spinal cords in nominally Ca2+‐free medium all reduced trans‐ACPDs effects. The calmodulin antagonists N‐(6‐aminohexyl)‐5‐chloro‐1‐naphthalenesulphonamide (W7) (100 μM) and chlorpromazine (100 μM) diminished the potentiation. 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.

GABA: Presynaptic Actions

Activation of afferent spinal inputs not only excites secondary neurons (interneurons) and motoneurons but also results in inhibition of the transmission of other sensory impulses.* Although the mechanism of this inhibition has been the focus of considerable controversy in the past two decades, there is agreement that much of the effect—designated “presynaptic inhibition”—is produced at the level of the presynaptic terminal of afferent fibers.

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. Mn2+, 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-alpha-aminoadipate, D-2-amino-5-phosphonovalerate, gamma-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.

Mechanisms intrinsic to 5‐HT2B receptor‐induced potentiation of NMDA receptor responses in frog motoneurones

In the presence of NMDA receptor open‐channel blockers [Mg2+; (+)‐5‐methyl‐10,11‐dihydro‐5H‐dibenzo[a,d]cyclohepten‐5,10‐imine maleate (MK‐801); 1‐amino‐3,5‐dimethyladamantane (memantine)] and TTX, high concentrations (30–100 μM) of either 5‐hydroxytryptamine (5‐HT) or α‐methyl‐5‐hydroxytryptamine (α‐Me‐5‐HT) significantly potentiated NMDA‐induced depolarizations of frog spinal cord motoneurones. Potentiation was blocked by LY‐53,857 (10–30 μM), SB 206553 (10 μM), and SB 204741 (30 μM), but not by spiroxatrine (10 μM), WAY 100,635 (1–30 μM), ketanserin (10 μM), RS 102221 (10 μM), or RS 39604 (10–20 μM). Therefore, α‐Me‐5‐HTs facilitatory effects appear to involve 5‐HT2B receptors. These effects were G‐protein dependent as they were prevented by prior treatment with guanylyl‐5′‐imidodiphosphate (GMP‐PNP, 100 μM) and H‐Arg‐Pro‐Lys‐Pro‐Gln‐Gln‐D‐Trp‐Phe‐D‐Trp‐D‐Trp‐Met‐NH2 (GP antagonist 2A, 3–6 μM), but not by pertussis toxin (PTX, 3–6 ng ml−1, 48 h preincubation). This potentiation was not reduced by protein kinase C inhibition with staurosporine (2.0 μM), U73122 (10 μM) or N‐(2‐aminoethyl)‐5‐isoquinolinesulfonamide HCl (H9) (77 μM) or by intracellular Ca2+ depletion with thapsigargin (0.1 μM) (which inhibits Ca2+/ATPase). Exposure of the spinal cord to the L‐type Ca2+ channel blockers nifedipine (10 μM), KN‐62 (5 μM) or gallopamil (100 μM) eliminated α‐Me‐5‐HTs effects. The calmodulin antagonist N‐(6‐aminohexyl)‐5‐chloro‐1‐naphtalenesulfonamide (W7) (100 μM) diminished the potentiation. However, the calcium/calmodulin‐dependent protein kinase II (CaM Kinase II) blocker KN‐93 (10 μM) did not block the 5‐HT enhancement of the NMDA responses. In summary, activation of 5‐HT2B receptors by α‐Me‐5‐HT facilitates NMDA‐depolarizations of frog motoneurones via a G‐protein, a rise in [Ca2+]i from the entry of extracellular Ca2+ through L‐type Ca2+ channels, the binding of Ca2+ to calmodulin and a lessening of the Mg2+ ‐produced open‐channel block of the NMDA receptor.

Serotonin1A facilitation of frog motoneuron responses to afferent stimuli and toN-methyl-d-aspartate

The effects of serotonin and excitatory amino acids on motoneurons were examined by sucrose gap recordings from the ventral root of the isolated, hemisected frog spinal cord superfused with magnesium-free, carbonate-buffered Ringer solution. Low concentrations of serotonin (0.1 microM) and the serotonin1A agonist 8-hydroxy-2-(n-dipropylamino)tetralin (8-OH-DPAT; 0.01 microM) significantly increased the duration and amplitude of the polysynaptic components of ventral root potentials produced by dorsal root stimulation. The facilitations of the ventral root potentials were blocked by the serotonin1A antagonist spiroxatrine, but were unaffected by the serotonin2 antagonist ketanserin or the serotonin3 antagonist 1 alpha H,3 alpha,5 alpha H-tropan-3-yl-3,-dichlorobenzoate (MDL 72222). The actions of 0.1 microM serotonin on motoneuron depolarizations evoked by the putative excitatory amino acid transmitters L-glutamate and L-aspartate were quite variable, but in the presence of ketanserin (20 microM), small consistent increases in amino acid-induced motoneuron depolarizations were observed. 8-OH-DPAT significantly enhanced motoneuron depolarizations elicited by the selective excitatory amino acid agonist N-methyl-D-aspartate in both normal and tetrodotoxin-containing Ringer solution. Quisqualate-induced motoneuron depolarizations were also facilitated by 8-OH-DPAT in normal Ringer solution, but these increases were eliminated by addition of either tetrodotoxin or the N-methyl-D-aspartate antagonist D(-)-2-amino-5-phosphonovalerate to the Ringer superfusate. Kainate-depolarizations were not altered by low concentrations of serotonin or 8-OH-DPAT. Prior exposure of the cord to spiperone, but not ketanserin or MDL 72222 blocked the enhancement of N-methyl-D-aspartate-induced motoneuron depolarizations by 8-OH-DPAT.(ABSTRACT TRUNCATED AT 250 WORDS)

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

The effects of serotonin on the membrane potential of primary afferent terminals of isolated hemisected frog spinal cords was investigated by sucrose gap recordings from dorsal root. Serotonin produced two distinctive changes in primary afferent terminal membrane potential: modest (about 0.5 mV) hyperpolarizations in low concentrations (0.01-1.0 microM) and larger (about 1.0 mV) slow depolarizations in higher concentrations (3.0-100 microM). The hyperpolarizations appeared related to a direct activation of 5-HT1A receptors on afferent terminals. The depolarizations were attributed to both direct and indirect actions and appeared to be generated by activation of 5-HT2 and/or 5-HT1C receptors. The results suggest that 5-HT released from terminals in the frog dorsal horn could exert a modulatory action on the afferent input of the spinal cord, but different effects generated by activation of different 5-HT receptor subtypes are dependent upon the concentration of the amine.

Spontaneous dorsal root potentials arise from interneuronal activity in the isolated frog spinal cord

Spontaneous dorsal root potentials (sDRPs) were recorded from the dorsal roots of the isolated frog spinal cord using sucrose gap techniques. sDRPs were always negative (depolarizing) in sign and ranged in size from about 100 microV to 6.0 mV. The largest sDRPs were 25-40% of the amplitude of DRPs evoked by stimulation of adjacent dorsal roots. Hypoxia or accumulation of extracellular K+ ions did not appear responsible for the generation of this spontaneous activity since exposing the cord to unoxygenated Ringers solution decreased sDRPs and K+-sensitive microelectrodes indicated that only small changes in extracellular K+ (approximately 0.15 mM) were produced coincidently with the largest sDRPs. Chemically-mediated synaptic transmission was found to be necessary for the production of sDRPs because the addition of Mn2+ or Mg2+ ions or tetrodotoxin to the Ringers solution or reduction of its Na+ concentration blocked sDRPs, whereas application of 4-aminopyridine enhanced them. It did not seem that a direct action of GABA on afferent fiber terminals was responsible for the generation of spontaneous potentials since an increase in sDRPs was seen after: application of the GABA antagonists, bicuculline and picrotoxin; exposure to the glutamic acid decarboxylase inhibitor, semicarbazide (which significantly reduced the concentration of GABA in the cord); and lowering of the external Cl- concentration. Similarly taurine is probably not significant since the taurine antagonist, TAG, increased the amount of spontaneous activity. On the other hand, (--)-baclofen, which is thought to reduce excitatory amino acid release, D,L-alpha-aminoadipic acid, alpha, epsilon-diaminopimelic acid, and 2-amino-4-phosphonobutyric acid, which are believed to be selective postsynaptic excitatory amino acid antagonists, and [D-Pro2-D-Phe7-D-Trp9]-substance P, a postsynaptic blocker of the action of substance P, markedly and reversibly reduced sDRPs. Experiments were performed on isolated cords without supraspinal or afferent input; therefore sDRPs must be generated by intraspinal structures. It would seem that interneurons are responsible because addition of mephenesin or pentobarbital--compounds which inhibit polysynaptic reflex transmission involving interneurons--reduced the production of sDRPs. sDRPs may result from the action of excitatory transmitters such as L-glutamate, L-aspartate, or substance P released by interneuronal firing in the spinal cord. Moreover, because sDRPs were increased by application of yohimbine, corynanthine and propanolol and reduced by haloperidol, such interneurons may be under descending control of adrenergic and dopaminergic fibers.

Amino acid antagonists do not block the depolarizing effects of potassium ions on frog primary afferents

Abstract The effects of picrotoxin, bicuculline and strychnine on the dorsal root potential evoked by dorsal root stimulation and on the responses of primary afferents to elevated [K + ] and to γ-aminobutyric acid (and in some cases to β-alanine) were studied by sucrose gap recording from dorsal roots of the isolated hemisected frog spinal cord. Bicuculline and picrotoxin when applied in concentrations sufficient to block depolarizations of dorsal roots evoked by γ-aminobutyrate and the early phase (initial 150–200 ms) of the dorsal root potential did not alter the sensitivity of primary afferents to the depolarizing effects of K + . Strychnine produced inconsistent effects on the early component of the dorsal root potential, but in low or moderate concentrations ( −3 M) which antagonized β-alanine-induced depolarizations, did not affect K + -induced depolarizations. In high concentrations (10 −3 M), strychnine decreased the responses of dorsal roots to K + , β-alanine and γ-aminobutyrate. All the convulsants tested prolonged and augmented a late phase of the dorsal root potential. These observations indicate that primary afferent depolarization (as measured by the dorsal root potential) consists of two components which appear to be pharmacologically distinct. The results also indicate that the reduction of the early component of primary afferent depolarization by picrotoxin and bicuculline result from a blockage of the effects of γ-aminobutyrate on primary afferents rather than from a reduced sensitivity of these afferents to elevations in the concentration of extracellular K + . The ability of picrotoxin, bicuculline and strychnine to facilitate the late phase of the dorsal root potential, may be related to the ability of K + to contribute to the enhancement of the late component of primary afferent depolarization produced by convulsants.