A.M. Holohean

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.

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.

Activation of 5-HT1C/2 receptors depresses polysynaptic reflexes and excitatory amino acid-induced motoneuron responses in frog spinal cord

Sucrose gap recordings from the ventral roots of isolated, hemisected frog spinal cords were used to evaluate the effects of high concentrations of serotonin (5-HT) and alpha-methyl-5-HT (alpha-Me-5-HT) on the changes in motoneuron potential produced by dorsal root stimulation and by excitatory amino acids and agonists. Bath application of 5-HT in concentrations of 10 microM or greater produced a concentration-dependent motoneuron depolarization. Polysynaptic ventral root potentials evoked by dorsal root stimuli were reduced in both amplitude and area by 5-HT or alpha-Me-5-HT (both 100 microM). This may result from a reduction of the postsynaptic sensitivity of motoneurons to excitatory amino acid transmitters because 5-HT significantly depressed motoneuron depolarizations produced by addition of L-glutamate and L-aspartate to the superfusate. Similarly, 5-HT reduced depolarizations produced by the excitatory amino acid agonists N-methyl-D-aspartate (NMDA), quisqualate, alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA), and kainate. alpha-Me-5-HT reduced NMDA depolarizations. Tetrodotoxin (TTX) did not affect the ability of 5-HT to attenuate NMDA or kainate depolarizations, but did eliminate the 5-HT-induced attenuation of quisqualate and AMPA depolarizations. The glycine receptor site associated with the NMDA receptor did not appear to be affected by 5-HT because saturation of the site by excess glycine did not alter the 5-HT-induced depression of NMDA responses. The 5-HT1C/2 antagonist ketanserin and the 5-HT1A/2 antagonist spiperone significantly attenuated the 5-HT-induced depression of NMDA-depolarizations.(ABSTRACT TRUNCATED AT 250 WORDS)

Modulation of frog spinal cord interneuronal activity by activation of 5-HT3 receptors

Motoneuron membrane potentials were recorded from the ventral roots of isolated, hemisected frog spinal cords using sucrose gap techniques. The effects of the selective 5-HT3 agonist 2-methyl-serotonin (2-Me-5HT) on the changes in motoneuron membrane potential produced by dorsal root stimulation and by superfusion of excitatory amino acid agonists were evaluated. Application of 2-Me-5HT (100 microM) did not alter motoneuron membrane potential, but did substantially reduce (approximately 20%) the polysynaptic ventral root potentials evoked by dorsal root stimulation. 2-Me-5HT did not change motoneuron depolarizations generated by addition to the Ringers solution of the excitatory amino acid agonists AMPA (10-30 microM), kainate (30 microM), or t-ACPD (100 microM), but NMDA-induced motoneuron depolarizations (100 microM) were significantly and reversibly reduced (approximately 20%) by exposure to 2-Me-5HT (100 microM). 2-Me-5HT-evoked decreases of NMDA depolarizations were blocked by the 5-HT3 antagonists ICS 205 930 (50-100 microM) and D-tubocurarine (3-10 microM), but not by MDL 72222 (20-100 microM), the 5-HT2 receptor antagonist ketanserin (10 microM), or the 5-HT1A/5-HT2A antagonist spiperone (10 microM). Two lines of evidence support the hypothesis that the effects of 2-Me-5HT are generated by an indirect mechanism involving interneurons: (1) TTX (0.781 microM) eliminated the effect of 2-Me-5HT on NMDA-induced motoneuron depolarizations, and (2) 2-Me-5HT reduced spontaneous ventral root potentials that result from interneuronal discharges. We attempted to establish the identity of a putative transmitter released by interneurons responsible for the effects on NMDA-depolarizations produced by 2-Me-5HT, but the AMPA receptor antagonist, CNQX (10 microM), the GABAA receptor antagonist, bicuculline (50 microM), the GABAB receptor antagonist, saclofen (100 microM), the opioid antagonist, naloxone (100 microM), and the adenosine antagonists, CPT (20-100 microM) and CSC (10-100 microM) did not alter 2-Me-5HT-induced reductions of NMDA-depolarizations. In sum, the site of interaction between 2-Me-5HT and NMDA appears to be at interneuronal locus, but the mechanism remains unclear.

Serotonin and GABA-induced depolarizations of frog primary afferent fibers

The interaction of gamma-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 microM) to the Ringers 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 microM 5-HT were mimicked by the 5-HT1A agonists 8-OH-DPAT (8-hydroxy-2-(n-dipropylamino)tetralin) and ipsapirone. The effects of ipsapirone were reversed by the 5-HT1A antagonist spiperone. The decreases of GABA-depolarizations produced by high doses of 5-HT were duplicated by application of alpha-methyl-5-HT, a 5-HT1C/2 agonist and reversed by superfusion of the cord with manserin, a 5-HT1C/2 antagonist. The presumptive 5-HT1A receptor-mediated effects of 1.0 microM 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 Ringers solution. In contrast, the putative 5-HT1C/2 receptor actions of 100 microM 5-HT and alpha-methyl-5-HT were substantially reduced by TTX and are presumably caused by activation of receptors located on interneurons. GABAB 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.(ABSTRACT TRUNCATED AT 250 WORDS)

After-hyperpolarizations produced in frog motoneurons by excitatory amino acid analogues.

After-hyperpolarizations (AHPs) produced in frog motoneurons by applications of the excitatory amino acid analogues quisqualate (QUIS), N-methyl-D-aspartate (NMDA), and kainate (KA) were studied in the isolated hemisected frog spinal cord using sucrose gap techniques. AHPs were present following 98% of QUIS-induced depolarizations, but were seen in only 35% and 15% of NMDA- and KA-evoked responses respectively. AHPs produced by QUIS are produced both by direct effects of QUIS on motoneuron membranes and by indirect effects mediated through a synaptic process involving interneurons. Thus, application of Mg2+, Mn2+, or tetrodotoxin (TTX) in concentrations sufficient to block synaptic transmission and interneuronal firing, reduced, but did not abolish the AHPs produced by QUIS. In contrast, NMDA- and KA-AHPs appear to be entirely mediated by indirect means as block of synaptic transmission and interneuronal firing eliminated AHPs produced by these substances. Exposure of the cord to Mn2+ after addition of TTX did not affect the size of QUIS-AHPs. In the presence of TTX, QUIS-AHPs were reduced or completely blocked by addition of dinitrophenol (DNP) and sodium cyanide, by dihydro-ouabain, by removal of K+ from the superfusate, by cooling, and by replacement of 50% of the external Na+ with Li+. The results suggest that the QUIS-AHPs are largely the result of the direct effect of the excitatory amino acid agonist on motoneuron membranes and is caused by activation of an electrogenic Na+ pump. AHPs following depolarizations evoked by NMDA and KA are presumably the result of indirect actions of these latter analogues on interneurons.