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. 1999 Jan;126(1):333-41.
doi: 10.1038/sj.bjp.0702263.

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

A M Holohean  1 , J C Hackman, R A Davidoff
Affiliations

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

A M Holohean et al. Br J Pharmacol. 1999 Jan.

Abstract

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.

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Figures

Figure 1
Figure 1
trans-ACPD and 1S,3R-ACPD depolarize frog motoneurones. (a) Changes in frog motoneurone membrane potential (electrotonically-conducted along the IXth VR) produced by three concentrations (10, 30 and 100 μM, 30 s applications) of trans-ACPD. (b) Changes in frog motoneurone membrane potential produced by two concentrations (10 and 30 μM) of 1S,3R-ACPD (30 s applications). In these, and in all subsequent records, negativity is indicated by an upward pen deflection and signifies a depolarization of motoneurones whose axons exit from the cord in the IXth VR. The upward ‘spike-like' deflections of the baseline represent spontaneous ventral root potential (VRPs). Applications of trans-ACPD and 1S,3R-ACPD are indicated by bars below the baseline. Traces in a and b were obtained from two different spinal cords. Calibration bars apply to both a and b.
Figure 2
Figure 2
trans-ACPD-induced motoneurone depolarizations are blocked by kynurenate (KYN) and tetrodotoxin (TTX). (a) Application of trans-ACPD (30 μM, 30 s) before, during, and after addition of kynurenate (1.0 mM, 30 min) to Ringer's solution. (b) Depolarization produced by trans-ACPD (30 μM, 30 s) is diminished by TTX (0.783 μM). Subsequent addition of kynurenate (1.0 μM) had no further effect. Applications of trans-ACPD are indicated by bars below the baseline. Traces in a and b were obtained from two different spinal cords. Calibration bars apply to both a and b.
Figure 3
Figure 3
trans-ACPD potentiates polysynaptic reflexes in the frog spinal cord. Traces are ventral root potentials (VRPs) produced by single supramaximal shocks (arrows) applied to the afferent fibres in the sciatic nerve. Control VRP, VRP recorded during exposure to trans-ACPD (30 μM), and VRP obtained after return to normal Ringer's solution are shown. The experiment was carried out in Ringer's solution containing 1.0 mM Mg2+.
Figure 4
Figure 4
trans-ACPD facilitates NMDA-evoked motoneurone depolarizations in Ringer's solution containing Mg2+. (a) Applications of NMDA (100 μM, 10 s), AMPA (10 μM, 10 s), and kainate (KA) (30 μM, 10 s) in nominally Mg2+-free Ringer's solution before and after exposure to trans-ACPD (30 μM). (b) Same experiment carried out in another spinal cord in Ringer's solution containing 1.0 mM Mg2+. Both experiments were performed in Ringer's solution containing TTX (0.783 μM). Applications of iGluR agonists are indicated by arrows below the baseline. Calibration bars apply to both a and b.
Figure 5
Figure 5
trans-ACPD potentiates NMDA responses if the concentration of extracellular Mg2+ is sufficient to produce channel block. (a) trans-ACPD (t-ACPD) (30 μM) potentiates NMDA responses in a cord exposed to 100 μM Mg2+. Note the decreased amplitude of the NMDA response when the Ringer's contained 100 μM Mg2+. (b) trans-ACPD (30 μM) had no effect on NMDA-induced motoneurone depolarization in a second spinal cord exposed to 100 μM Mg2+; nor did 100 μM Mg2+ affect NMDA-responses. Both experiments were performed in Ringer's solution containing TTX (0.783 μM). Calibration bars apply to both a and b.
Figure 6
Figure 6
trans-ACPD-induced potentiation of NMDA responses is present in spinal cords exposed to NMDA receptor channel blockers. (a) Spinal cord exposed to MK-801 (6 μM). (b) Spinal cord exposed to memantine (MEM) (10 μM). Both experiments were performed in Ringer's solution containing TTX (0.783 μM). Applications of NMDA (100 μM, 5 s) are indicated by arrows below the baseline. Calibration bars apply to both a and b.
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