ACPD-mediated slow-onset potentiation is associated with cell death in the rat CA1 region in vivo

Slow-onset potentiation of synaptic transmission in the hippocampus in vitro and in vivo is induced by application of the metabotropic glutamate receptor (mGluR) agonist, 1S,3R-amino cyclopentane 2,3-dicarboxylic acid (ACPD). This study investigated the cellular response in the CA1 region of freely moving rats to ACPD application. Drugs were applied via the lateral cerebral ventricle, and measurements were obtained from the CA1 region via permanently implanted electrodes. ACPD (20 nmol/5 microl) produced a dose-dependent slow-onset potentiation in the CA1 region which lasted over 4 h. Histological evaluation at either 4 h or 7 days following ACPD-injection indicated that slow-onset potentiation was associated with gradual but marked cell death in the CA1 region. Whereas 20 nmol ACPD produced significant CAI neurotoxicity, concentrations which did not induce potentiation had little or no neurotoxic effect. Both the general mGluR antagonist R,S-alpha-methyl-carboxyphenylglycine (1 micromol/5 microl), and the group 1 mGluR antagonist (S)-4-carboxyphenylglycine (4CPG, 100 nmol/5 microl) significantly inhibited ACPD-induced neuropathology. In addition, 4CPG inhibited the expression of ACPD-mediated slow-onset potentiation. These results confirm previous findings that in the CA1 region in vivo, slow-onset potentiation is mediated group 1 mGluRs, and indicate that slow-onset potentiation may involve pathological processes.     

Influence of metabotropic glutamate receptor agonists on the inhibitory effects of adenosine A1 receptor activation in the rat hippocampus

1. Glutamate and other amino acids are the main excitatory neurotransmitters in many brain regions, including the hippocampus, by activating ion channel-coupled glutamate receptors, as well as metabotropic receptors linked to G proteins and second messenger systems. Several conditions which promote the release of glutamate, like frequency stimulation and hypoxia, also lead to an increase in the extracellular levels of the important neuromodulator, adenosine. We studied whether the activation of different subgroups of metabotropic glutamate receptors (mGluR) could modify the known inhibitory effects of a selective adenosine A₁ receptor agonist on synaptic transmission in the hippocampus. The experiments were performed on hippocampal slices taken from young (12–14 days old) rats. Stimulation was delivered to the Schaffer collateral/commissural fibres, and evoked field excitatory postsynaptic potentials (fe.p.s.p.) recorded extracellularly from the stratum radiatum in the CA1 area.
2. The concentration-response curve for the inhibitory effects of the selective adenosine A₁ receptor agonist, N⁶-cyclopentyladenosine (CPA; 2–50 nM), on the fe.p.s.p. slope (EC₅₀=12.5 (9.2–17.3; 95% confidence intervals)) was displaced to the right by the group I mGluR selective agonist, (R,S)-3,5-dihydroxyphenylglycine (DPHG; 10 μM) (EC₅₀=27.2 (21.4–34.5) nM, n=4). The attenuation of the inhibitory effect of CPA (10 nM) on the fe.p.s.p. slope by DHPG (10 μM) was blocked in the presence of the mGluR antagonist (which blocks group I and II mGluR), (R,S)-α-methyl-4-carboxyphenylglycine (MCPG; 500 μM). DHPG (10 μM) itself had an inhibitory effect of 20.1±1.9% (n=4) on the fe.p.s.p. slope.
3. The concentration-response curves for the inhibitory effects of CPA (2–20 nM) on the fe.p.s.p. slope were not modified either in the presence of the group II mGluR selective agonist, (2S,3S,4S)-α-(carboxycyclopropyl)glycine (L-CCG-I; 1 μM), or in the presence of the non-selective mGluR agonist (which activates both group I and II mGluR), (1S,3R)-1-aminocyclopentyl-1,3-dicarboxylate (ACPD; 100 μM). L-CCG-I had no consistent effects and ACPD (100 μM) decreased by 19.4±1.8% (n=4) the fe.p.s.p. slope.
4. The concentration-response curve for the inhibitory effects of CPA (2–100 nM) on the fe.p.s.p. slope (EC₅₀=8.2 (6.9–9.6) nM) was displaced to the right by the group III mGluR selective agonist, L-2-amino-4-phosphonobutyrate (L-AP4; 25 μM) (EC₅₀=17.7 (13.1–21.9) nM, n=4). The attenuation of the inhibitory effect of CPA (10 nM) on the fe.p.s.p. slope by L-AP4 (25 μM) was blocked in the presence of the mGluR antagonist (selective for the group III mGluR), (R,S)-α-methyl-4-phosphonophenylglycine (MPPG; 200 μM).
5. Both the direct effect of DHPG on synaptic transmission and the attenuation of the inhibitory effect of CPA (10 nM) were prevented in the presence of the protein kinase C selective inhibitors, staurosporine (1 μM) or chelerythrine (5 μM), and thus attributed to activation of protein kinase C.
6. The attenuation by L-AP4 (25 μM) of the inhibitory effect of CPA (10 nM) on the fe.p.s.p. slope was also prevented by the protein kinase C selective inhibitors, staurosporine (1 μM) or chelerythrine (5 μM), and thus attributed to activation of protein kinase C. But this effect seemed to be distinct from the direct effect of L-AP4 (25 μM) on synaptic transmission, which was not modified by the protein kinase C selective inhibitors.
7. We conclude that agonists of metabotropic glutamate receptors (Groups I and III) are able to attenuate the inhibitory effects of adenosine A₁ receptor activation in the hippocampus. This interaction may have pathophysiological relevance in hypoxia, in which there is marked release of both excitatory amino acids and the important endogenous neuroprotective substance, adenosine.

     

Metabotropic glutamate receptor subtypes mediating slow inward tail current (IADP) induction and inhibition of synaptic transmission in olfactory cortical neurones

1. The pharmacological features of the pre- and postsynaptic metabotropic glutamate receptors (mGluRs) present in the guinea-pig olfactory cortex, were examined in brain slices in vitro by use of a conventional intracellular current clamp/voltage clamp recording technique.
2. Bath-application of trans-aminocyclopentane-1,3-dicarboxylic acid (trans-ACPD) (50 μM) produced a sustained membrane depolarization, increase in cell excitability and induction of a post-stimulus inward (afterdepolarizing) tail current (I_ADP) (measured under ‘hybrid'' voltage clamp) similar to those evoked by the muscarinic receptor agonist oxotremorine-M (OXO-M, 2 μM).
3. L-Glutamate (0.25–1 mM, in the presence of 20 μM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 100 μM DL-amino-5-phosphono valeric acid (DL-APV)) or the broad spectrum mGluR agonists 1S,3R-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD, 10 μM), 1S,3S-ACPD (50 μM), ibotenate (Ibo; 25 μM, in the presence of 100 μM DL-APV), the selective mGluR I agonists (S)-3,5-dihydroxyphenylglycine ((S)-3,5-DHPG, 10 μM), (S)-3-hydroxyphenylglycine ((S)-3HPG, 50 μM), or quisqualate (10 μM, in the presence of 20 μM CNQX), but not the mGluR II agonist 2S,1′S,2′S-2-(2′-carboxycyclopropyl)-glycine (L-CCGI, 1 μM) or mGluR III agonist L(+)-2-amino-4-phosphonobutyric acid (L-AP4, 1 mM), were all effective in producing membrane depolarization and inducing a post-stimulus I_ADP. Unexpectedly, the proposed mGluR II-selective agonist (2S,1′R,2′R,3′R)-2-(2′,3′-dicarboxycyclopropyl)-glycine (DCG-IV, 10 μM, in the presence of 100 μM DL-APV) was also active.
4. The excitatory effects induced by 10 μM 1S,3R-ACPD were reversibly antagonized by the mGluR I/II antagonist (+)-α-methyl-4-carboxyphenylglycine ((+)-MCPG, 0.5–1 mM), as well as the selective mGluR I antagonists (S)-4-carboxyphenylglycine ((S)-4CPG) and (S)-4-carboxy-3-hydroxyphenyl glycine ((S)-4C3HPG) (both at 1 mM), but not the nonselective mGluR antagonist L(+)-2-amino-3-phosphonopropionic acid (L-AP3, 1 mM) or the selective mGluR III antagonist (S)-α-methyl-L-AP4 (MAP4, 1 mM).
5. The excitatory postsynaptic potentials (e.p.s.ps), induced by single focal stimulation of cortical excitatory fibre tracts, were markedly reduced by 1S,3R-ACPD or L-AP4 (both at 10 μM), and by the selective mGluR II agonists (mGluR I antagonists) (S)-4CPG or (S)-4C3HPG (both at 1 mM) but not (S)-3,5-DHPG or (S)-3HPG (both at 100 μM).
6. The inhibitory effects of 1S-3R-ACPD, but not L-AP4, were reversibly blocked by (+)-MCPG (1 mM), whereas those produced by L-AP4, but not 1S,3R-ACPD, were blocked by the selective mGluR III antagonist MAP4 (1 mM).
7. It is concluded that a group I mGluR is most likely involved in mediating excitatory postsynaptic effects, whereas two distinct mGluRs (e.g. group II and III) might serve as presynaptic inhibitory autoreceptors in the guinea-pig olfactory cortex.

     

Dual Action of Metabotropic Glutamate Receptor Agonists on Neuronal Excitability and Synaptic Transmission in Spinal Ventral Horn Neurons In Vitro

A dual action of selective metabotropic glutamate receptor agonists on neuronal excitability and dorsal root-evoked excitatory (DR-EPSPs) and inhibitory (DR-IPSPs) neurotransmission is described for immature rat ventral horn neurons in vitro. Trans-1-Aminocyclopentane-1,3-dicarboxylate (trans-ACPD), its stereoisomer (1S,3R)-1-aminocyclopentane-1,3-dicarboxylate ((1S,3R)-ACPD) and (2S,3S,4S)-α-(carboxycyclopropyl)-glycine (MCPG)-sensitive depolarisation. An (1S,3R)-ACPD- or L-CCG-1-induced increase in intrinsic neuronal excitability was apparently independent of the depolarisation and was observed as (a) a fall in the threshold current required to elicit regenerative excitation and (b) an increased number of spikes to a fixed amplitude step depolarisation. The spike after-hyperpolarisation (AHP) duration and amplitude were reduced, suggesting an mGluR agonist action on potassium channels. Synaptic responses were depressed by the mGluR agonists. (1S,3R)-ACPD or L-CCG-1 reduced the mean ± S.E.M. peak amplitude of a subthreshold EPSP elicited by low-intensity stimuli likely to recruit only low-threshold sensory afferents. The peak amplitude of longer-latency EPSPs elicited by higher-intensity stimuli likely to recruit high-threshold afferents in addition was attenuated. (1S,3R)-ACPD- or L-CCG-1 reduced the peak amplitude of an IPSP evoked by dorsal root stimulation. These effects on synaptic transmission were likely to be due to the combined activation of postsynaptic and presynaptic metabotropic glutamate receptors. The implications of these data for the physiological role of spinal mGluRs is discussed. © 1997 Elsevier Science Ltd. All rights reserved.     

Comparison of the effects of low and high concentrations of group I metabotropic receptor agonists on field potentials in the hippocampal CA1 region in vitro

We compared the effects of low and high concentrations of the selective group I metabotropic glutamate receptor (mGluR) agonist (R,S)-3,5-dihydroxyphenylglycine (DHPG) and the nonselective mGluR agonist (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid ((1S,3R)-ACPD) on extracellularly recorded potentials which were evoked in the rat hippocampal CA1 region by stimulation of the Schaffer collateral/commissural pathway and on intracellularly recorded electrophysiological properties of CA1 neurons, in vitro. At low concentrations (2.5 and 5 microM) DHPG and (1S,3R)-ACPD increased while at high concentrations (20 and 50 microM) they decreased population spike amplitudes. Simultaneous recordings of population spikes in the CA1 cell layer and field excitatory postsynaptic potentials (fEPSPs) in stratum radiatum of the CA1 area revealed that the enhancement of the population spike amplitude is not associated with any change in the fEPSP slope, but the decrease in population spikes is accompanied with a decrease in the fEPSP slope, suggesting that at high concentrations both agents may attenuate excitatory synaptic transmission in CA1 cells. DHPG and (1S,3R)-ACPD had a number of direct excitatory effects on CA1 pyramidal cells like a concentration-dependent depolarization and an inhibition of the slow afterhyperpolarization, which in all probability underlay the increase in the amplitude of population spikes. At high concentrations, both mGluR agonists strongly depolarized CA1 cells indicating that depolarization block of cell discharges may underlay the reduction in the population spike amplitude. Furthermore, robust cell discharges induced by the strong depolarizations, activate several secondary processes which may significantly contribute to the action of high concentrations of DHPG and (1S,3R)-ACPD. Therefore, the effects of low and high concentrations of the studied mGluR agonists may involve different mechanisms, at low concentrations the effects can be directly related to the activation of postsynaptically localized group I mGluRs while at higher concentrations the contribution of indirect effects may predominate.     

Enhancement of NMDA responses by group I metabotropic glutamate receptor activation in striatal neurones

1. The interactions between N-methyl-D-aspartate (NMDA) and metabotropic glutamate receptors (mGluRs) were investigated in striatal slices, by utilizing intracellular recordings, both in current- and voltage-clamp mode.
2. Bath-application (50 μM) or focal application of NMDA induced a transient membrane depolarization, while in the voltage-clamp mode, NMDA (50 μM) caused a transient inward current. Following bath-application of the non-selective mGluR agonist 1S,3R-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD, 10 μM), NMDA responses were reversibly potentiated both in current (197±15% of control) and voltage-clamp experiments (200±18% of control).
3. Bath-application of the group I mGluR agonist (RS)-3,5-dihydroxyphenylglycine (3,5-DHPG, 10–300 μM) resulted in a dose-dependent potentiation of NMDA-induced membrane depolarization (up to 400±33% of control). This potentiation was either prevented by preincubation with (RS)-α-methyl-4-carboxyphenylglycine (RS-α-MCPG, 300 μM), or blocked when applied immediately after 3,5-DHPG wash-out.
4. Neither (2S,1′S,2′S)2-(2′-carboxycyclopropyl)glycine (L-CCG I, up to 100 μM) nor (2S,1′R,2′R,3′R)-2-(2,3-dicarboxycyclopropyl)-glycine (DCG-IV, 1 μM), agonists for group II mGluRs caused any change in NMDA responses. Likewise, L-serine-O-phosphate (L-SOP, 30 μM), agonist for group III mGluRs, did not affect the NMDA-induced depolarization.
5. The enhancement of the NMDA responses was mimicked by phorbol-12,13-diacetate (PDAc, 1 μM) which activates protein kinase C (PKC). The 3,5-DHPG-mediated potentiation of the NMDA-induced depolarization was prevented by preincubation with staurosporine (100 nM) or calphostin C (1 μM), antagonists of PKC.
6. Electrophysiological responses to α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor activation were not affected by agonists for the three-classes of mGluRs.
7. The present data suggest that group I mGluRs exert a positive modulatory action on NMDA responses, probably through activation of PKC. This functional interaction in the striatum appears of crucial importance in the understanding of physiological and pathological events, such as synaptic plasticity and neuronal death, respectively.

     

Priming of group 2 metabotropic glutamate receptors facilitates induction of long-term depression in the dentate gyrus of freely moving rats

Activation of group 1 metabotropic glutamate receptors (mGluRs) has been shown to facilitate the induction of tetanically evoked long-term potentiation in vivo, whereas group 2 mGluR antagonists inhibit long-term depression (LTD) in the CA1 region. LTD has not been successfully demonstrated to date in the dentate gyrus of freely moving rats. In this study, it was found that 1-Hz low-frequency stimulation (LFS) when applied via a stimulating electrode chronically implanted in the perforant path, evoked short-term depression (STD) of field excitatory post-synaptic potentials and population spikes measured from the dentate gyrus granule cell layer. Application of the group 2 mGluR agonists (S)-4-carboxyphenylglycine (4C3HPG) or (2S,2′R,3′R)-2-(2′,3′-dicarboxycyclopropyl)glycine (DCG-IV) prior to LFS facilitated the expression of LTD. Application of the group 2 mGluR antagonist (2S)--ethylglutamic acid (EGLU) prior to agonist application prevented the facilitating effect of the agonists on LFS-induced depression. EGLU did not influence the expression of LFS-induced STD. Neither 4C3HPG, DCG-IV nor EGLU had an effect on basal synaptic transmission at the concentrations used. The present study demonstrates that priming of group 2 mGluRs leads to induction of persistent LTD in the dentate gyrus in vivo, consistent with a role for group 2 mGluRs in metaplasticity.     

Co-administration of (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid and arachidonic acid potentiates synaptic transmission in rat hippocampal slices

Perfusion of the 1S,3R isomer of trans-aminocyclopentane-1,3-dicarboxylic acid (t-ACPD, 50 μM), or arachidonic acid (10 μM), for 5 min produced only depression of the field excitatory postsynaptic potential recorded in the CA1 region of rat hippocampal slices from which the CA3 region had been removed. However, perfusion of t-ACPD and arachidonic acid in combination induced a rapid potentiation of the response which in 4/6 slices was maintained for at least 90 min.     

Short- and long-term depression at glutamatergic synapses on hippocampal interneurons by group I mGluR activation

Group I metabotropic glutamate receptors (mGluRs) are expressed by many interneurons of the hippocampus. Although they have been implicated in short- and long-term synaptic plasticity of glutamatergic transmission, their roles in modulating transmission to interneurons are incompletely understood. The selective group I mGluR agonist (S)-3,5-dihydroxyphenylglycine (DHPG) acutely depressed transmission at synapses in the feed-forward inhibitory pathway made by Schaffer collaterals on interneurons in the rat hippocampal CA1 sub-field. DHPG elicited a qualitatively similar depression at synapses made by pyramidal neuron axon collaterals on interneurons in the feedback circuit in stratum oriens. Selective blockers revealed a link from mGluR1 to reversible, and mGluR5 to long-lasting, depression. The acute DHPG-induced depression was consistently accompanied by an elevation in paired-pulse ratio, implying a presynaptic decrease in release probability. However, it was also attenuated by blocking G-protein and Ca²⁺ signalling within the postsynaptic neuron, arguing for a retrograde signalling cascade. The DHPG-evoked depression was unaffected by antagonists of CB1 and GABA_B receptors but was occluded when presynaptic P/Q-type Ca²⁺ channels were blocked. Finally, high-frequency stimulation delivered to an independent conditioning pathway evoked a heterosynaptic reversible depression, which was sensitive to group I mGluR antagonists. Group I mGluRs thus powerfully modulate synaptic excitation of hippocampal interneurons and mediate inter-synaptic cross-talk.This article is part of a Special Issue entitled ‘Synaptic Plasticity & Interneurons’.     

Group I mGluRs modulate the pattern of non-synaptic epileptiform activity in the hippocampus

The hippocampus is well known for its susceptibility to epileptic seizures, in part because of its neuronal architecture that facilitates synchronization. Although synaptic networks are important for the genesis and spread of epileptiform activity, synchronization of neuronal activity can occur when action potential-dependent chemical synaptic transmission is absent. In particular, it is possible to induce epileptiform activity by perfusing hippocampal slices with a low-Ca(2+)/high-K(+) mediums. Using extracellular recording in area CA1 we have characterized the effects of metabotropic glutamate receptor (mGluR) activation on this non-synaptic bursting activity. Under control conditions, bursting occurred at intervals of 14-86 s with each burst comprising a long (up to 44 s) negative-going field potential of 2 to 13 mV superimposed upon which was sustained firing of population spikes. Activation of group I mGluRs by (S)-3,5-dihydroxyphenylglycine (DHPG) (25 microM) caused a dramatic increase in burst frequency (up to five-fold), which was accompanied by a decrease in the duration and amplitude of bursts. The selective mGluR(1) antagonist 2-methyl-4-carboxyphenylglycine (LY367385) and the selective mGluR(5) antagonist 2-methyl-6-(phenylethynyl)pyridine (MPEP) both restricted the increase in burst frequency induced by DHPG. However, only LY367385 inhibited the decrease in burst duration and amplitude. Combined application of both antagonists prevented all DHPG-induced changes in bursting activity. These data provide evidence for a role of both mGluR(1) and mGluR(5) subtypes in changing the frequency of non-synaptic bursting, with mGluR(1) alone causing alterations in burst duration and amplitude. These effects are likely to contribute to the group I mGluR-induced changes in synaptic epileptic activity that are already well documented.     

Brain ATP:l-methionine S-adenosyltransferase (MAT), S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH): regional distribution and age-related changes

The distribution of the activity of the enzyme methionine adenosyltransferase (ATP:l-methionine S-adenosyltransferase, EC 2.5.1.6, MAT) was investigated in human postmortem brains of individuals without a known history of neuropsychiatric disorders. The brain regions were the frontal, temporal, parietal and occipital cortices, nucleus caudatus, putamen, globus pallidus, thalamus and white matter. The activities in the nucleus caudatus and putamen were approximately 25% higher than the activities in the seven other brain regions, however, not on a statistically significant level. The apparent values of MAT K_m and V_max in the parietal cortex were 11.41 ± 3.51 μM methionine and 25.72 ± 3.90 nmol/mg protein/h, respectively. In the frontal cortex, a significant positive correlation between age and the activity of MAT was found (r = 0.997, P<0.01). Concerning MAT stability in the rat brain, there was a steady decrease in the activity with postmortem time in the brains kept for 0–72 h at room temperature (23°C), which reached the level of significance at 24 h. The activity did not change significantly when the brains were kept for 120 h at 4°C, or by freezing and thawing the tissue before analysis. In a parallel study in rats of different ages (2–22 months), a homogeneous distribution of SAM and SAH was observed in the cortex, striatum, midbrain, hypothalamus, brainstem and cerebellum. The lowest levels of SAM and the highest levels of SAH observed in the striatum gave the lowest SAM/SAH ratio. The SAH content of rat cerebral cortex was highest in the oldest group. The levels of S-adenosylmethionine (SAM) decreased by 38% and that of S-adenosylhomocysteine (SAH) increased by 200% when the brains were kept for 2 h at 23°C. In the brains kept for 24 h at 4°C, the levels of SAM decreased by 70% while those of SAH increased by 240%.     

A novel kainate receptor ligand [H]-(2S,4R)-4-methylglutamate: Pharmacological characterization in rabbit brain membranes

Since kainate evokes large non-desensitizing currents at α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, kainate is of limited use in discriminating between AMPA and kainate receptors. Following recent reports that (2S,4R)-4-methylglutamate is a kainate receptor-selective agonist, we have radiolabelled and subsequently characterized the binding of [³H]-(2S,4R)-4-methylglutamate to rabbit whole-brain membranes. [³H]-(2S,4R)-4-methylglutamate binding was rapid, reversible and labelled two sites (KD1 = 3.67 ± 0.50 nM/B_max1 = 0.54 ± 0.03 pmol/mg protein and K_D2 = 281.66 ± 12.33 nM/ B_max2 = 1.77 ± 0.09 pmol/mg protein). [³H]-(2S,4R)-4-methylglutamate binding was displaced by several non-NMDA receptor ligands: domoate > kainate -quisqualate -glutamate > 6-cyano-7-nitroquinoxaline-2, 3-dione (CNQX) (S)-AMPA = (S)-5-fluorowillardiine > NMDA. Neither the metabotropic glutamate receptor agonists (1S,3R)-ACPD or -AP4, together with the -glutamate uptake inhibitor -trans-2,4-PDC, influenced binding when tested at 100 μM. We conclude that [³H]-(2S,4R)-4-methylglutamate is a useful radioligand for labelling kainate receptors. It possesses high selectivity, and possesses a pharmacology similar to that for rat cloned low-affinity (Glu5 and 6) kainate receptor subunits.     

Molecular pharmacology of homologues of ibotenic acid at cloned metabotropic glutamic acid receptors

We have studied the effects of the enantiomers of 2-amino-3-(3-hydroxyisoxazol-5-yl)propionic acid (homoibotenic acid, HIBO) and analogues substituted with a methyl, bromo or butyl group in the four position of the ring at cloned metabotropic glutamate (mGlu) receptors expressed in Chinese hamster ovary (CHO) cells. In contrast to the parent compound ibotenic acid, which is a potent group I and II agonist, the (S)-forms of homoibotenic acid and its analogues are selective and potent group I antagonists whereas the (R)-forms are inactive both as agonists and antagonists at group I, II, and III mGlu receptors. Interestingly, (S)-homoibotenic acid and the analogues display equal potency at both mGlu_1α and mGlu_5a with K_i values in the range of 97 to 490 μM, (S)-homoibotenic acid and (S)-2-amino-3-(4-butyl-3-hydroxyisoxazol-5-yl)propionic acid [(S)-4-butylhomoibotenic acid] displaying the lowest and highest potency, respectively. The homoibotenic acid analogues thereby differ from mGlu receptor antagonists derived from phenylglycine such as (S)-4-carboxyphenylglycine which only antagonizes mGlu_1α (K_i=18 μM) showing no effect at mGlu_5a (K_i >300 μM).     

mGluR1-mediated potentiation of NMDA receptors involves a rise in intracellular calcium and activation of protein kinase C

Potentiation of ionotropic glutamate receptor activity by metabotropic glutamate receptors (mGluRs) is thought to modulate activity at glutamatergic synapses in the hippocampus. However, the precise pathway by which this modulation occurs is not well understood. The present study tests the hypothesis that mGluR1-mediated potentiation of N-methyl-D-aspartate receptors (NMDARs) occurs via a phospholipase C (PLC)-initiated cascade. NMDAR functional activity was examined by whole-cell recording from Xenopus oocytes expressing recombinant NMDARs and mGluR1alpha. The mGluR1 agonist (1S,3R)-1-amino-cyclopentane-1,3-dicarboxylic acid (ACPD) significantly potentiated NMDA-elicited currents. mGluR1alpha-mediated potentiation of NMDA responses was eliminated by the PLC inhibitor U-73122. Buffering of intracellular Ca2+ by BAPTA-AM or depletion of intracellular Ca2+ by the Ca2+/ATPase inhibitor thapsigargin greatly reduced ACPD potentiation. ACPD potentiation was reduced by the specific protein kinase C (PKC) inhibitor Ro-32-0432 and eliminated by the broad spectrum kinase inhibitor staurosporine. ACPD produced no further potentiation after potentiation of NMDARs by the PKC-activating phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA). Thus, Group I mGluRs potentiate NMDA responses via activation of PLC; at least part of the potentiation is due to rise in intracellular Ca2+ and stimulation of PKC. Cytochalasin D, which disrupts the actin cytoskeleton, blocked ACPD-elicited chloride currents and ACPD-induced potentiation of NMDAR currents, consistent with a role for cytoskeletal protein(s) in the signaling pathway. As Group I mGluRs are localized to the perisynaptic region in juxtaposition to NMDARs at glutamatergic synapses, mGluR-mediated potentiation of NMDAR activity may play a role in synaptic transmission and plasticity including LTP.     

Postsynaptic α-adrenoceptors in the perfused canine saphenous vein in vitro

To study the relative localization of α₁- and α₂-adrenoceptors in relation to the intima and the adventitia of canine saphenous vein, a comparison was made of the potency of α₁- and α₂-adrenoceptors agonists applied by intraluminal and extraluminal route of perfused segments of that vessel. Noradrenaline was the most potent of the agonists used and was approximately as potent by intraluminal as by extraluminal route. Cocaine (12 μmol/1) caused supersensitivity to noradrenaline which was of about the same magnitude (threefold) irrespective of the route of administration of noradrenaline. The selective α₁-agonist phenylephrine was about 10 times less potent than noradrenaline and was also equieffective by both routes. The selective α₂-agonist UK-14,304, at concentrations lower than 0.3 μmol/l, caused very small responses and only in 3 out of 14 experiments. In all cases it caused responses at concentrations higher than 0.3 μmol/l. Cocaine did not change the sensitivity to either phenylephrine or UK-14,304. Thus, it is concluded that the results obtained with cocaine agreed with expectations for a homogeneously innervated tissue. Furthermore, α₁-adrenoceptors seem to predominate and to be evenly distributed throughout the media. The lack of responses to the low concentrations of UK-14,304—those selectively acting on α₂-adrenoceptors—was ascribed to the very low efficacy of this agonist on the distal part of the canine saphenous vein and to the tone created by the perfusion pressure which might be high enough to mask this small response.     

Pharmacology of postsynaptic metabotropic glutamate receptors in rat hippocampal CA1 pyramidal neurones.

1. Activation of metabotropic glutamate receptors (mGluRs) in hippocampal CA1 pyramidal neurones leads to a depolarization, an increase in input resistance and a reduction in spike frequency adaptation (or accommodation). At least eight subtypes of mGluR have been identified which have been divided into three groups based on their biochemical, structural and pharmacological properties. It is unclear to which group the mGluRs which mediate these excitatory effects in hippocampal CA1 pyramidal neurones belong. We have attempted to address this question by using intracellular recording to test the effects of a range of mGluR agonists and antagonists, that exhibit different profiles of subtype specificity, on the excitability of CA1 pyramidal neurones in rat hippocampal slices. 2. (2S, 1'S,2'S)-2-(2'-carboxycyclopropyl)glycine (L-CCG1) caused a reduction in spike frequency adaptation and a depolarization (1-10 mV) associated with an increase in input resistance (10-30%) at concentrations (> or = 50 microM) that have been shown to activate mGluRs in groups I, II and III. Similar effects were observed with concentrations (50-100 microM) of (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid ((1S,3R)-ACPD) and (1S,3S)-ACPD that exhibit little or no activity at group III mGluRs but which activate groups I and II mGluRs. 3. Inhibition of the release of endogenous neurotransmitters through activation of GABAB receptors, by use of 200 microM (+/-)-baclofen, did not alter the effects of (1S,3R)-ACPD (50-100 microM), (1S,3S)-ACPD (100 microM) or L-CCG1 (100 microM). This suggests that mGluR agonists directly activate CA1 pyramidal neurones. 4. Like these broad spectrum mGluR agonists, the racemic mixture ((SR)-) or resolved (S)-isomer of the selective group I mGluR agonist 3,5-dihydroxyphenylglycine ((SR)-DHPG (50-100 microM) or (S)-DHPG (20-50 microM)) caused a reduction in spike frequency adaptation concomitant with postsynaptic depolarization and an increase in input resistance. In contrast, 2S,1'R,2'R,3'R-2-(2',3'-dicarboxycyclopropyl)glycine (DCG-IV; 100 microM) and (S)-2-amino-4-phosphonobutanoic acid (L-AP4; 100-500 microM), which selectively activate group II mGluRs and group III mGluRs, respectively, had no effect on the passive membrane properties or spike frequency adaptation of CA1 pyramidal neurones. 5. The mGluR antagonists (+)-alpha-methyl-4-carboxyphenylglycine ((+)-MCPG; 1000 microM) and (S)-4-carboxyphenylglycine ((S)-4CPG; 1000 microM), which block groups I and II mGluRs and group I mGluRs, respectively, had no effect on membrane potential, input resistance or spike frequency adaptation per se.(ABSTRACT TRUNCATED AT 400 WORDS)     

NMDA Receptors,mGluR5, and Endocannabinoids are Involved in a Cascade Leading to Hippocampal Long-Term Depression

In the CA1 region of the rat hippocampus, metabotropic glutamate receptor-5 (mGluR5) and cannabinoid-1 receptors (CB1Rs) are believed to participate in long-term synaptic depression (LTD). How mGluRs and CB1Rs interact to promote LTD remains uncertain. In this study, we examined LTD induced by CB1R agonists, mGluR5 agonists, and low-frequency electrical stimulation (LFS) of the Schaffer collateral pathway. Synthetic CB1R agonists induced robust LTD that was mimicked by the endocannabinoid (EC), noladin ether (NLDE), but not by anandamide. 2-Arachidonylglycerol (2AG) produced only a small degree of LTD. The selective mGluR5 agonist, namely (RS)-2-chloro-5-hydroxyphenylglycine (CHPG), also induced robust LTD, and CHPG and NLDE occluded each other''s effects. Similarly, CHPG and NLDE occluded LFS-induced LTD, and LTD resulting from all three treatments was blocked by a CB1R antagonist. CHPG-LTD and NLDE-LTD were insensitive to N-methyl--aspartate receptor (NMDAR) block, even though LFS-LTD requires NMDARs. LTD induced by LFS or CHPG, but not NLDE-LTD, was blocked by a selective mGluR5 antagonist. (RS)-3,5-dihydroxyphenylglycine (DHPG), a less selective group I mGluR agonist, also induced LTD, but its effects were not blocked by mGluR5 or CB1R antagonists. Furthermore, DHPG-LTD was additive with LFS-LTD and CHGP-LTD. These results suggest that NMDARs, mGluR5, and CB1Rs participate in a cascade that underlies LFS-LTD and that release of an EC and CB1R activation occur downstream of NMDARs and mGluR5. Furthermore, DHPG induces a form of LTD that differs mechanistically from LFS-induced depression.     

The role of NMDA receptors in regulating group II metabotropic glutamate receptor-mediated long-term depression in rat medial prefrontal cortex

Previous work has shown that brief application of group II metabotropic glutamate receptor (mGluR) agonist (2S,2′R,3′R)-2-(2′,3′-dicarboxycyclopropyl) glycine (DCG-IV) can induce long-term depression (LTD) of excitatory transmission on layer V pyramidal neurons of rat medial prefrontal cortex (mPFC). An unusual feature of this LTD is that it relies on activation of both group II mGluRs and N-methyl-d-aspartate receptors (NMDARs). However, it is not known whether other specific group II mGluR agonists also induce LTD and whether they depend on the conjoint activation of group II mGluRs and NMDARs. We show here that the ability of DCG-IV to induce LTD was mimicked by a more selective group II mGluR agonist, LY379268. The induction of LTD by a lower concentration of DCG-IV (0.2 μM) or LY379268 (0.03 μM) was blocked by the NMDAR antagonist APV or the interruption of synaptic stimulation during drug application. In contrast, application of a higher concentration of DCG-IV (1 μM) or LY379268 (0.1 μM) can induce LTD that was independent of synaptic NMDAR activation. These results suggest that although molecular cooperation between group II mGluRs and synaptic NMDARs may facilitate the induction of group II mGluR-mediated LTD at excitatory synapses onto mPFC layer V pyramidal neurons, enhancing group II mGluR activation may remove NMDAR involvement in this form of synaptic plasticity.     

DAMGO recognizes four residues in the third extracellular loop to discriminate between μ- and κ-opioid receptors

Previously, we reported that replacement of the region from the fifth transmembrane domain to the C-terminus of κ-opioid receptor with the corresponding region of μ-opioid receptor gives high affinity for [

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-Ala², N-MePhe⁴, Gly-ol⁵]enkephalin (DAMGO), a μ-opioid receptor-selective ligand, to the resultant chimeric receptor, suggesting that the difference in the amino acid sequence within this region is critical for the discrimination between μ- and κ-opioid receptors by DAMGO. In the present study, we constructed further six μ/κ-chimeric receptors and revealed that at least two separate regions around the third extracellular loop are critical for the discrimination between μ- and κ-opioid receptors by DAMGO. Furthermore, we constructed several mutant receptors by a site-directed mutagenesis technique and found that the difference between Glu²⁹⁷ of κ-opioid receptor and Lys³⁰³ of μ-opioid receptor in one region, and the difference between Ser³¹⁰, Tyr³¹² and Tyr³¹³ of κ-opioid receptor and Val³¹⁶, Trp³¹⁸ and His³¹⁹ of μ-opioid receptor in the other region, are critical for the discrimination between these receptors by DAMGO. The mutant receptor, κ (E297K+Y313H+Y312W+S310V), in which the Glu²⁹⁷, Ser³¹⁰, Tyr³¹² and Tyr³¹³ of κ-opioid receptor were changed to Lys, Val, Trp and His, respectively, bound to DAMGO with high affinity (K_d=8.7±1.2 nM) and efficiently mediated the inhibitory effect of DAMGO on intracellular cAMP accumulation. The present results showed that these four amino acid residues act as determinants for the discrimination between μ- and κ-opioid receptors by DAMGO.     

Group I metabotropic glutamate receptors reduce excitotoxic injury and may facilitate neurogenesis

Group I metabotropic glutamate receptor (mGluR) agonist DHPG reduced nerve cell death caused by their exposure to NMDA ("neuroprotective effect") and attenuated NMDA receptor-mediated currents [Blaabjerg, M., Baskys, A., Zimmer, J., Vawter, M. P., 2003b. Changes in hippocampal gene expression after neuroprotective activation of group I metabotropic glutamate receptors. Brain Research, Molecular Brain Research 117, 196-205.]. In the present study, we used organotypic hippocampal culture preparation to examine specific phospholipase C (PLC) inhibitor U73122 effects on DHPG-induced neuroprotection, changes in excitatory synaptic transmission associated with the neuroprotective DHPG treatment and a role of group I mGluR ligands in neurogenesis. Results show that short (10 min) DHPG treatment did not result in neuroprotection but significantly depressed field synaptic potentials (fEPSP) in the Schaffer collateral-CA1 pathway. The fEPSP depression was not affected by the PLC inhibitor U73122. In contrast, prolonged (2-h) treatment of cultures with DHPG induced a significant protective effect that was blocked by a PLC inhibitor U73122 but not by its inactive analog U73343. Voltage-clamp measurements of spontaneous miniature excitatory post-synaptic currents (EPSCs) recorded in CA1 neurons from cultures treated with DHPG (10 microM, 2 h) showed a significant reduction of the EPSC amplitude in DHPG-treated but not control (untreated) cultures. This reduction was completely abolished by U73122, suggesting a PLC involvement. Since activation of PLC is thought to be associated with cell proliferation, we investigated whether group I mGluR agonist DHPG or subtype antagonists LY367385 and MPEP have an effect on dentate granule cells expressing immature neuronal marker TOAD-64. DHPG (100 microM, 72 h) slightly but not significantly increased the number of TOAD-64 positive cells. The mGluR1 antagonists LY367385 (10 microM, 72 h) markedly decreased the number of TOAD-64 positive cells and mGluR5 antagonist MPEP (1 microM, 72 h) had no effect. These data suggest that (1) prolonged activation of group I mGluRs reduces nerve cell susceptibility to excitotoxic injury in a PLC-dependent manner; (2) this reduction is associated with a PLC-dependent depression of excitatory synaptic transmission; and (3) mGluR1 activation may facilitate neurogenesis.