ABSTRACTS FOR KENNETH C. MARSHALL
MODULATION OF EXCITATORY SYNAPTIC TRANSMISSION IN LOCUS COERULEUS BY MULTIPLE PRESYNAPTIC METABOTROPIC GLUTAMATE RECEPTORS
G.R. Dubé and K.C. Marshall
Department of Physiology, Faculty of Medicine, University of Ottawa, Ottawa, Ontario, Canada K1H 8M5
Metabotropic glutamate receptors have been implicated in modulation of synaptic transmission in many different systems. This study reports the effects of selective activation of metabotropic glutamate receptors on synaptic transmission in intracellularly recorded locus coeruleus neurons in brain slice preparations. Perfusion of either L-2-amino-4-phosphonobutyric acid (L-AP4; 0.1-500 uM) or (±)-1-aminocyclopentane-trans-1,3,dicarboxylic acid (t-ACPD; 0.1-500 uM) caused a depression of excitatory postsynaptic potentials in a dose-dependent fashion to about 70% inhibition. Both agonists exerted their effects at relatively low concentrations with estimated EC50S of 2.6 uM and 11.5 uM for L-AP4 and t-ACPD, respectively. This inhibition was not observed with the potent group I metabotropic glutamate receptor agonist (RS)-3,5-dihydroxyphenylglycine (DHPG; 100 uM). Conversely, (R)-4-carboxy-3-hydroxyphenyl-glycine (4C-3H-PG), a group I antagonist/group II agonist, and 2R,4R-4-aminopyrrolidine-2,4-dicarboxylate (APDC), a novel and specific group II agonist, also caused an inhibition of excitatory postsynaptic potentials. Both t-ACPD and L-AP4 produced an increase in paired-pulse facilitation, and failed to change the locus coeruleus response to focally applied glutamate, indicating a presynaptic locus of action. The L-AP4 inhibition was antagonized by (S)-amino-2-methyl-4-phosphonobutanoic acid (MAP4; group III antagonist) but not by (RS)-alpha-methyl-4-carboxyphenylglycine [(RS)-MCPG; mixed antagonist], suggesting that this agonist acts through a type 4 metabotropic glutamate receptor. Conversely, t-ACPD was antagonized by MCPG and by ethyl glutamate (group II antagonist), but not by aminoindan dicarboxylic acid (AIDA; group I antagonist) or MAP4, suggesting that this agonist acts on a type 2 or 3 metabotropic glutamate receptor. Taken together, these results suggest that two pharmacologically distinct presynaptic metabotropic glutamate receptors function in an additive fashion to inhibit excitatory synaptic transmission in locus coeruleus neurons. These receptors may be involved in a feedback mechanism and as such may function as autoreceptors for excitatory amino acids.
ACTIONS OF EXCITATORY AMINO ACIDS ON MESENCEPHALIC TRIGEMINAL NEURONS
Kenneth A. Pelkey and Kenneth C. Marshall
Mesencephalic trigeminal neurons are primary sensory neurons of which the cell soma is located within the brain stem, and is associated with synaptic contacts. In previous studies it has been reported that these cells are resistant to kainic acid excitotoxicity, and have little or no responsiveness to exogenously applied glutamate or selective agonists. In an in vitro slice preparation with intracellular recording, we have found that these cells respond to pressure-applied glutamate, n-methyl-D-aspartic acid (NMDA), kainate (KA), and (R,S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA). The kainate and AMPA responses appear to be mediated by different receptors, at least in part, since they exhibit differing sensitivity to an AMPA receptor-selective antagonist. The agonists generally evoke larger responses than glutamate, and exhibit a long duration desensitization requiring about ten minutes for full recovery. Some cross-desensitization between the glutamate agonists is also observed. Mesencephalic trigeminal neurons exhibit high-frequency oscillatory activity during depolarizations that approach threshold potentials, and these could combine with transmitter-induced depolarizations to enhance excitability of these cells. Previous reports of non-sensitivity to glutamate and to kainate excitotoxicity are attributable to relatively small responses, and to the desensitization expressed by these neurons.
ACTIVITY-DEPENDENT ACTIVATION OF PRESYNAPTIC METABOTROPIC GLUTAMATE RECEPTORS IN LOCUS COERULEUS
G.R. Dubé and Kenneth C. Marshall
Synaptic activation of metabotropic glutamate receptors (mGluRs) in the locus coeruleus (LC) was investigated in adult rat brain slice preparations. Evoked excitatory postsynaptic potentials (EPSPs) resulting from stimulation of LC afferents were measured under current clamp from intracellularly recorded LC neurons. In this preparation, mGluR agonists (±)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (t-ACPD) and L(+)-2-amino-4-phosphonobutyric acid (L-AP4) activate distinct presynaptic mGluRs resulting in an inhibition of EPSPs. When two stimuli were applied to afferents at intervals greater than 200 ms, the amplitude of the second [test (T)] EPSP was identical in amplitude to the first [control(C)]. However, when a stimulation volley was delivered before T, the amplitude of the latter EPSP was consistently smaller than C. The activity-dependent depression (ADD) was dependent on the frequency and duration of the train and the interval between the train and T. ADD was potentiated in the presence of an excitatory amino acid (EAA) uptake inhibitor L-trans-pyrrolidine-2, 4-dicarboxylic acid (t-PDC, 100 uM) changing the T/C ratio from 0.84 ± 0.05 (mean ± SEM) in control, to 0.69 ± 0.04 in t-PDC (n=9). In the presence of t-PDC the depolarizing response of LC neurons to focally applied glutamate was also increased. Together, these results suggest that accumulation of EAA following synaptic stimulation may be responsible for ADD. To test whether ADD is a result of the activation of presynaptic mGluRs, the effect of selective mGluR antagonists on ADD was assessed. In the presence of t-PDC, bath applied (S)-amino-2-methyl-4-phosphonobutanoic acid (MAP4, 500 uM), a mGluR group III antagonist, significantly reversed the decrease in T/C ratio following a train stimulation [from 0.66 ± 0.04 to 0.81 ± 0.02 (mean ± SEM), n=5]. The T/C ratio in the presence of MAP4 was not different from that measured in the absence of a stimulation volley. Conversely, ethyl glutamic acid (EGLU, 500 uM), a mGluR group II antagonist, failed to alter the T/C ratio. Together, these results suggest that, in LC, group III presynaptic mGluR activation provides a feedback mechanism by which excitatory synaptic transmission can be negatively modulated during high frequency synaptic activity. Furthermore, this study provides functional differentiation between the presynaptic group II and III mGluR in LC and suggests that the group II mGluR may be involved in functions distinct from those of group III mGluRs.
TESTING OF METABOTROPIC GLUTAMATE INHIBITION OF EPSPs IN mGluR4 KNOCKOUT MICE
Gilles R. Dubé and Kenneth C. Marshall
It has been demonstrated that excitatory neurotransmission to locus coeruleus (LC) neurons of the rat mediated by ionotropic glutamate receptors can be modulated by metabotropic glutamate agonists acting through two different subtypes of metabotropic glutamate receptors (mGluR) (Dubé & Marshall, Neuroscience 80, 511-521, 1997). One of the mGluR involved in this modulation was activated by L-2-amino-4-phosphonobutyric acid (L-AP4), a selective group III mGluR agonist. On the basis of the L-AP4 concentration-response curve, it appeared that the effect was more likely to be mediated by the mGluR4 subtype, than the mGluR7.
Knockout mice with a deficient mGluR4 were provided by Dr. David R. Hampson (Univ. of Toronto), so that this hypothesis could be tested. Experiments similar to those previously performed on rats were carried out using horizontal brain stem slices from wild type and knockout mice, with intracellular recording, and electrical stimulation of the slice to evoked excitatory postsynaptic potentials (EPSPs). The electrical properties of LC neurons of the mice were similar to those observed earlier in rat tissue, except that the mean input resistance of the mice neurons was significantly greater than that of rats, and there was no apparent intrinsic oscillatory activity like that observed in rats. The depressions of EPSPs in LC neurons of wild-type mouse tissue by L-AP4 and by the group I and II agonist (±) -1-aminocyclopentane-trans-1,3, dicarboxylic acid (ACPD) were very similar to those observed in rat tissue, and were also indistinguishable from the effects in the tissue from knockout mice. We conclude that the depression of EPSPs in LC neurons by L-AP4 is not mediated by the mGluR4 subtype, though the possibility exists that the responsible receptor is different in rats and mice. It is likely, therefore that the L-AP4 effects are mediated by the mGluR7 or mGluR8, or an as yet unidentified mGluR subtype.
MESENCEPHALIC TRIGEMINAL NEURON RESPONSES TO GAMMA-AMINOBUTYRIC ACID
Abdallah Hayar a, Michael O. Poulter a,1, Kenneth Pelkey b, Paul Feltz a,2, Kenneth C. Marshall b,*
aLaboratoire de Physiologie Générale, Université Louis Pasteur, Strasbourg, France
bDepartment of Physiology, University of Ottawa, 451 Smyth Road, Ottawa, Ont., Canada, K1H 8M5
Mesencephalic trigeminal neurons are primary sensory neurons which have cell somata located within the brain stem. In spite of the presence of synaptic terminals on and around the cell somata, applications of a variety of neurotransmitter substances in earlier studies have failed to demonstrate responses. Using intracellular recording in a brain slice preparation, we have observed prominent depolarizations and decreases in input resistance in response to applications of gamma-aminobutyric acid (GABA) in most recorded mesencephalic trigeminal neurons. Those cells failing to respond were located deeply within the slice, and the low responsiveness was shown to be related to uptake of GABA in the slice. The responses were direct, since they remained during perfusion with a low calcium, high magnesium solution that blocks synaptic transmission. The responses were mimicked by the GABAA receptor agonist isoguvacine, and blocked by GABAA receptor antagonists. The GABAB receptor agonist baclofen evoked no changes in membrane potential or input resistance in neurons exhibiting depolarizations with GABA application. Tests of neuronal excitability during GABA applications indicated that the excitatory effects of the depolarization prevail over the depressant effects of the increase in membrane conductance. In situ hybridization histochemistry indicated that the GABAA receptors in Me5 cells are comprised of alpha2, beta2 and gamma2 subunits.