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1 The metabotropic glutamate receptor (mGluR) agonist trans-(+)-1-amino-1,3-cyclopentanedicarboxylic acid (trans-ACPD) (10 ± 100 mM) depolarized isolated frog spinal cord motoneurones, a process sensitive to kynurenate (1.0 mM) and tetrodotoxin (TTX) (0.783 mM). 2 In the presence of NMDA open channel blockers [Mg 2+ ; (+)-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 signi®cantly potentiated NMDA-induced motoneurone depolarizations, but not a-amino-3-hydroxy-5-methylisoxazole-4-proprionate (AMPA)-or kainate-induced depolarizations. 3 NMDA potentiation was blocked by (RS)-a-methyl-4-carboxyphenylglycine (MCPG) (240 mM), but not by a-methyl-(2S,3S,4S)-a-(carboxycyclopropyl)-glycine (MCCG) (290 mM) or by a-methyl-(S)-2-amino-4-phosphonobutyrate (L-MAP4) (250 mM), and was mimicked by 3,5-dihydroxyphenylglycine (DHPG) (30 mM), but not by L(+)-2-amino-4-phosphonobutyrate (L-AP4) (100 mM). Therefore, trans-ACPD's facilitatory e ects appear to involve group I mGluRs. 4 Potentiation was prevented by the G-protein decoupling agent pertussis toxin (3 ± 6 ng ml 71 , 36 h preincubation). The protein kinase C inhibitors staurosporine (2.0 mM) and N-(2-aminoethyl)-5-isoquinolinesulphonamide HCl (H9) (77 mM) did not signi®cantly reduce enhanced NMDA responses. Protein kinase C activation with phorbol-12-myristate 13-acetate (5.0 mM) had no e ect. 5 Intracellular Ca 2+ depletion with thapsigargin (0.1 mM) (which inhibits Ca 2+ /ATPase), 1,2-bis(Oaminophenoxy)ethane-N,N,N',N'-tetracetic acid acetyl methyl ester (BAPTA-AM) (50 mM) (which bu ers elevations of [Ca 2+ ] i ), and bathing spinal cords in nominally Ca 2+ -free medium all reduced trans-ACPD's e ects. 6 The calmodulin antagonists N-(6-aminohexyl)-5-chloro-1-naphthalenesulphonamide (W7) (100 mM) and chlorpromazine (100 mM) diminished the potentiation. 7 In summary, group I mGluRs selectively facilitate NMDA-depolarization of frog motoneurones via a G-protein, a rise in [Ca 2+ ] i from the presumed generation of phosphoinositides, binding of Ca 2+ to calmodulin, and lessening of the Mg 2+ -produced channel block of the NMDA receptor.

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‐HT's 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‐HT's 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. British Journal of Pharmacology (2004) 143, 351–360. doi:

gamma-Aminobutyric acid (GABA)-activated channels in embryonic (5-8 wk old) human dorsal root ganglion (DRG) neurons in dissociated culture were characterized by whole cell and single-channel techniques. All DRG neurons when held at negative holding membrane potentials displayed inward current to micromolar concentrations of GABA applied by pressure pulses from closely positioned micropipettes. The current was directly proportional to the concentration of GABA (EC50, 111 microM; Hill coefficient, 1.7). DRG neurons also responded to micromolar concentrations of pentobarbital and alphaxalone but not to cis-4-aminocrotonic acid (CACA), glycine, or taurine. Baclofen (100 microM) affected neither the holding currents nor K+ conductance (when patch pipettes were filled with 130 mM KCl) caused by depolarizing pulses. Whole cell GABA-currents were blocked by bicuculline, picrotoxin, and t-butylbicyclophosphorothionate (TBPS; all at 100 microM). The reversal potential of whole cell GABA-currents was close to the theoretical Cl- equilibrium potential, shifting with changes in intracellular Cl- concentration in a manner expected for Cl--selective channels. The whole cell I-V curve for GABA-induced currents demonstrated slight outward rectification with nearly symmetrical outside and inside Cl- concentrations. Spectral analysis of GABA-induced membrane current fluctuations showed that the kinetic components were best fitted by a triple Lorentzian function. The apparent elementary conductance for GABA-activated Cl- channels determined from the power spectra was 22.6 pS. Single-channel recordings from cell-attached patches with pipettes containing 10 microM GABA indicated that GABA-activated channels have a main and a subconductance level with values of 30 and 19 pS, respectively. Mean open and closed times of the channel were characterized by two or three exponential decay functions, suggesting two or three open channel states and two closed states. Single channels showed a lack of rectification. The actions of GABA on cultured human embryonic DRG neurons are mediated through the activation of GABAA receptors with properties corresponding to those found in the CNS of human and other mammalian species but differing from those of cultured human adult DRG neurons.