The potent mGlu receptor antagonist LY341495 identifies roles for both cloned and novel mGlu receptors in hippocampal synaptic plasticity

Understanding the roles of metabotropic glutamate (mGlu) receptors has been severely hampered by the lack of potent antagonists. LY341495 (2S-2-amino-2-(1S,2S-2-carboxycyclopropyl-1-yl)-3-(xanth-9-yl)propanoic acid) has been shown to block group II mGlu receptors in low nanomolar concentrations (Kingston, A.E., Ornstein, P.L., Wright, R.A., Johnson, B.G., Mayne, N.G., Burnett, J.P., Belagaje, R., Wu, S., Schoepp, D.D., 1998. LY341495 is a nanomolar potent and selective antagonist at group II metabotropic glutamate receptors. Neuropharmacology 37, 1–12) but can be used in higher concentrations to block all hippocampal mGlu receptors, identified so far by molecular cloning (mGlu_{1–5, 7,8}). Here we have further characterised the mGlu receptor antagonist activity of LY341495 and have used this compound to investigate roles of mGlu receptors in hippocampal long-term potentiation (LTP) and long-term depression (LTD). LY341495 competitively antagonised DHPG-stimulated PI hydrolysis in AV12-664 cells expressing either human mGlu₁ or mGlu₅ receptors with K_i-values of 7.0 and 7.6 μM, respectively. When tested against 10 μM -glutamate-stimulated Ca²⁺ mobilisation in rat mGlu₅ expressing CHO cells, it produced substantial or complete block at a concentration of 100 μM. In rat hippocampal slices, LY341495 eliminated 30 μM DHPG-stimulated PI hydrolysis and 100 μM (1S,3R)-ACPD-inhibition of forskolin-stimulated cAMP formation at concentrations of 100 and 0.03 μM, respectively. In area CA1, it antagonised DHPG-mediated potentiation of NMDA-induced depolarisations and DHPG-induced long-lasting depression of AMPA receptor-mediated synaptic transmission. LY341495 also blocked NMDA receptor-independent depotentiation and setting of a molecular switch involved in the induction of LTP; effects which have previously been shown to be blocked by the mGlu receptor antagonist (S)-MCPG. These effects may therefore be due to activation of cloned mGlu receptors. In contrast, LY341495 did not affect NMDA receptor-dependent homosynaptic LTD; an effect which may therefore be independent of cloned mGlu receptors. Finally, LY341495 failed to antagonise NMDA receptor-dependent LTP and, in area CA3, NMDA receptor-independent, mossy fibre LTP. Since in the same inputs these forms of LTP were blocked by (S)-MCPG, a novel type of mGlu receptor may be involved in their induction.     

Modulation of endocannabinoid-mediated long-lasting disinhibition of striatal output by cholinergic interneurons

The frequency and duration of glutamatergic inputs to the striatum are strong determinants of the net effect of retrograde endocannabinoid (eCB) signaling, and key factors in determining if long-term depression (LTD) has a net disinhibitory or inhibitory action in striatum. Low to moderate frequency stimulation in the dorsolateral striatum elevates eCB levels to an extent that primarily depresses transmitter release at inhibitory synapses, leading to a long-lasting disinhibition (DLL) of synaptic output. The aim of this study was to further characterize the basic features of endocannabinoid-mediated DLL of striatal output induced by moderate frequency stimulation (5 Hz, 60 s). DLL was inhibited in slices treated with the group 1 metabotropic glutamate receptor (mGluR) antagonists MPEP (40 μM) and CPCCOEt (40 μM), the dopamine D2 receptor antagonist sulpiride (5 μM), the L-type calcium channel blocker nifedipine (20 μM), the nicotinic receptor antagonist mecamylamine (10 μM), the muscarinic agonist oxotremorine sesquifumarate (10 μM), and strychnine (0.1 μM). Strychnine did not block DLL induced by WIN55,212-2 (250 nM), showing that glycine receptor-mediated modulation of eCB signaling occurs upstream from CB₁R activation. Scopolamine (10 μM) restored DLL in strychnine-treated slices, suggesting that inhibition of glycine receptors on cholinergic interneurons could modulate eCB signaling by enhancing muscarinic receptor activation and reducing the opening of L-type calcium channels in response to depolarization. These data suggests that similar activation points are required for stimulation-induced DLL as for LTD at excitatory striatal synapses, and that cholinergic interneurons are key modulators of stimulation-induced eCB signaling in the striatum.     

Involvement of septal and striatal dopamine D-2 receptors in yawning behavior in rats

A behavioral study was performed in an attempt to understand the neuronal mechanisms involved in yawning behavior in rats. Subcutaneous injections of low doses of apomorphine (0.05–0.25 mg/kg) or piribedil (0.2–1.0 mg/kg), which preferentially activate presynaptic dopamine autoreceptors at those doses, evoked yawning. Marked yawning responses were also elicited by both 3-PPP (5–20 mg/kg, SC) and TL-99 (1–2 mg/kg, SC). SK & F 38393, a dopamine D-1 receptor agonist, at doses ranging from 0.1 to 8.0 mg/kg (SC) induced neither yawning nor stereotypy. However, bromocriptine (0.5–32.0 mg/kg, SC), a dopamine D-2 receptor agonist, induced yawning for which the dose-response curves showed a bell-shaped form. After a higher dose of 32 mg/kg (SC) bromocriptine, some rats occasionally showed sniffing and sawdust chewing. Yawning responses induced by systemic injection of apomorphine, piribedil, 3-PPP or bromocriptine were wholly suppressed after treatment with sulpiride (10 mg/kg SC), a dopamine D-2 receptor antagonist. Bilateral injections of apomorphine (20 g/side×2), piribedil (100 g/side×2) or 3-PPP (50, 100 g/side×2) into the striatum or septum also elicited marked yawning. The results indicate that low doses of apomorphine, piribedil, 3-PPP, TL-99 or bromocriptine elicit yawning by stimulating dopamine D-2 receptors and striatal and septal dopaminergic systems may be related to the occurrence of yawning behavior.     

Role of microcircuit structure and input integration in hippocampal interneuron recruitment and plasticity

The proper operation of cortical neuronal networks depends on the temporally precise recruitment of GABAergic inhibitory interneurons. Inhibitory cells receive convergent excitatory inputs from afferent pathways, as well as local collaterals of principal cells, and provide feedforward or feedback inhibition within the circuitry. Accumulating evidence indicates that recruitment of GABAergic cells is highly diverse among interneuron types. Differences in the properties of input synapses, dendritic architecture and membrane properties, as well as the rich repertoire of plasticity mechanisms contribute to this diversity. Efficient and precise recruitment of interneurons is thought to depend on the coincident occurrence of rapid synaptic responses and their faithful propagation to the action potential initiation site. However, slow inputs can also play important roles by facilitating the activation of interneurons by rapid synaptic inputs and supporting associative synaptic plasticity. Here we review how the diversity in the synaptic and integrative properties as well as dendritic geometry of hippocampal inhibitory cells impact on their activation. We further discuss how the various modes of interneuron recruitment can support the versatile cell type- and input-specific computational functions which appear to be adapted to the structure and the function of the network they are embedded in.This article is part of a Special Issue entitled ‘Synaptic Plasticity & Interneurons’.     

Functional interaction of NMDA and group I metabotropic glutamate receptors in negatively reinforced learning in rats

Rationale The role of glutamatergic system in learning and memory has been extensively studied, and especially N-methyl-d-aspartate (NMDA) receptors have been implicated in different learning and memory processes. Less is known, however, about group I metabotropic glutamate (mGlu) receptors in this field. Recent studies indicated that the coactivation of both NMDA and group I mGlu receptors is required for the induction of long-term potentiation (LTP) and learning. Objective The purpose of the study is to evaluate if there is a functional interaction between NMDA and group I mGlu receptors in two different models of aversive learning. Methods Effects of NMDA, mGlu1, and mGlu5 receptor antagonists on acquisition were tested after systemic coadministration of selected ineffective doses in passive avoidance (PA) and fear-potentiated startle (FPS). Results Interaction in aversive learning was investigated using selective antagonists: (3-ethyl-2-methyl-quinolin-6-yl)-(4-methoxy-cyclohexyl)-methanone methanesulfonate (EMQMCM) for mGlu1, [(2-methyl-1,3-thiazol-4-yl)ethynyl]pyridine (MTEP) for mGlu5, and (+)-5-methyl-10,11-dihydro-5H-dibenzocyclohepten-5,10-imine maleate [(+)MK-801] for NMDA receptors. In PA, the coapplication of MTEP at a dose of 5 mg/kg and (+)MK-801 at a dose of 0.1 mg/kg 30 min before training impaired the acquisition tested 24 h later. Similarly, EMQMCM (2.5 mg/kg) plus (+)MK-801 (0.1 mg/kg), given during the acquisition phase, blocked the acquisition of the PA response. In contrast, neither the combination of MTEP (1.25 mg/kg) nor EMQMCM (5 mg/kg) plus (+)MK-801 (0.05 mg/kg) was effective on the acquisition assessed in the FPS paradigm. Conclusion The findings suggest differences in the interaction of the NMDA and mGlu group I receptor types in aversive instrumental conditioning vs conditioning to a discrete light cue.     

Differential regulation of synaptic transmission by mGlu2 and mGlu3 at the perforant path inputs to the dentate gyrus and CA1 revealed in mGlu2 -/- mice

Group II metabotropic glutamate (mGlu) receptors can act as presynaptic autoinhibitory receptors at perforant path inputs to the hippocampus under conditions of high frequency synaptic activation. We have used mGlu2 -/- mice to examine the relative roles of mGlu2 and mGlu3 in the regulation of perforant path synaptic transmission mediated by both the selective group II receptor agonist, DCG-IV, and by synaptically released glutamate. Field excitatory postsynaptic potentials evoked by stimulation of either the perforant path inputs to the dentate gyrus mid-moleculare or the CA1 stratum lacunosum moleculare were inhibited by DCG-IV with IC(50) values and maximum percentage inhibition of: 169 nM (60%) and 41 nM (72%) in wild-type mice and 273 nM (19%) and 116 nM (49%) in mGlu2 -/- mice, respectively. Activation of presynaptic group II mGlu autoreceptors by synaptically released glutamate, as revealed by a LY341495-mediated increase in the relative amplitude of a test fEPSP evoked after a conditioning burst, was observed in both the dentate gyrus and the stratum lacunosum of wild-type, but not mGlu2 -/- mice. These observations demonstrate that activation of mGlu3 receptors can regulate synaptic transmission at perforant path synapses but suggest that mGlu2 is the major presynaptic group II autoreceptor activated by synaptically released glutamate.     

Effects of mGlu1 receptor blockade on anxiety-related behaviour in the rat lick suppression test

Rationale Group I metabotropic glutamate receptor antagonists, which block both the mGlu1 and mGlu5 receptors, have been shown to have anxiolytic effects in the lick suppression test in rats.Objective The anxiolytic potential of the selective mGlu1 antagonist 3,4-dihydro-2H-pyrano[2,3]-quinolin-7-yl)(cis-4-methoxycyclohexyl)methanone (JNJ16259685) was investigated and compared with the mGlu5 antagonist MPEP.Methods Anxiety-related behaviour was assessed in lick suppression and in the elevated zero maze in rats. Non-specific effects on pain threshold, water intake and locomotor activity were also measured.Results Acute administration of JNJ16259685 or MPEP increased the number of licks (lowest active dose 2.5 mg/kg IP for each compound). JNJ16259685 did not increase water intake or reduce acute pain threshold, suggesting that the anxiolytic-like properties are specific. However, acute administration decreased locomotor activity. The effects of chronic administration of JNJ16259685 over 14 days (5 mg/kg bid) on lick suppression were comparable to those seen after acute administration, arguing against development of behavioural tolerance or sensitisation. Yet, there was a tendency for an increase in locomotor activity after cessation of chronic treatment. Acute co-administration of both JNJ16259685 and MPEP had additive effects on the number of licks. No anxiolytic-like properties of JNJ16259685 were observed in the elevated zero maze.Conclusion Our data suggest that the anxiolytic-like effects induced by group I metabotropic glutamate receptor antagonists are mediated through both mGlu1 and mGlu5 receptors. Rather than producing a general anxiolytic-like effect, the effects seen following mGlu1 antagonism seem task-dependent, as prominent effects were seen in a conflict procedure, but not in a task based on spontaneous exploration.     

5-HT2 receptor-mediated reversal of the inhibition of hippocampal long-term potentiation by acute inescapable stress

The serotonergic system is known to modulate and mediate many of the central nervous system effects of stress. Here we investigated the ability of serotonergic agents to reverse the inhibition of the induction of hippocampal long-term potentiation (LTP) caused by prior exposure to inescapable stress. Elevated platform stress prevented the induction of LTP in the CA1 area of anaesthetized rats. An agent that increases extracellular 5-HT concentration, fenfluramine (5 mg/kg, i.p.) enabled the induction of LTP in previously stressed animals. Consistent with a role for enhanced activation of 5-HT(2) receptors, the facilitatory effect of fenfluramine was prevented by the 5-HT(2) receptor antagonist cinanserin (30 mg/kg). Agents that directly activate 5-HT(2) receptors, including the 5-HT(2B) receptor agonist BW 723C86 (30 mg/kg) and the 5-HT(2C) receptor agonist MK-212 (3 mg/kg), mimicked the restorative effect of fenfluramine. Fenfluramine also opposed inhibition of LTP caused by the NMDA-receptor antagonist D-AP5 (100 nmol, i.c.v.) which suggests that the facilitatory action of serotonergic agents is not restricted to stress-mediated inhibition of LTP. These findings support an important role for activation of 5-HT(2) receptors by systemically applied agents to enable recovery from the inhibition of LTP by stress.     

Enhanced group III mGluR-mediated inhibition of pain-related synaptic plasticity in the amygdala

Pain has a strong emotional component. A key player in emotionality, the amygdala is also involved in pain processing. Our previous studies showed synaptic plasticity in the central nucleus of the amygdala (CeA) in a model of arthritic pain. Here, we address the role of group III metabotropic glutamate receptors (mGluRs) in the regulation of synaptic transmission in CeA neurons. Whole-cell current- and voltage-clamp recordings were made from neurons in the latero-capsular part of the CeA in brain slices from control rats and arthritic rats (>6 h postinduction). The latero-capsular part of the CeA is the target of the spino-parabrachio-amygdaloid pain pathway and is now designated as the "nociceptive amygdala". Monosynaptic excitatory postsynaptic currents (EPSCs) were evoked by electrical stimulation of afferents from the pontine parabrachial (PB) area. LAP4 decreased the amplitude of EPSCs more potently in CeA neurons from arthritic rats (EC(50)=1.2 nM) than in control animals (EC(50)=11.5 nM). The inhibitory effect of LAP4 was reversed by a selective group III mGluR antagonist (UBP1112). During the application of LAP4, paired-pulse facilitation was increased, while no significant changes in slope conductance and action potential firing rate of CeA neurons were observed. These data suggest that presynaptic group III mGluRs are involved in the regulation of synaptic plasticity in the amygdala in an arthritis pain model.     

The role of group I metabotropic glutamate receptors in acquisition and expression of contextual and auditory fear conditioning in rats - a comparison

Glutamatergic neurotransmission in the CNS plays a predominant role in learning and memory. While NMDA receptors have been extensively studied, less is known about the involvement of group I metabotropic glutamate receptors in this area. The purpose of the present study was to evaluate the contribution of mGluR1 and mGluR5 to both acquisition and expression of behaviours in contextual and auditory fear conditioning models. The effects of both receptor types were tested using selective antagonists: (3-ethyl-2-methyl-quinolin-6-yl)-(4-methoxy-cyclohexyl)-methanone methanesulfonate (EMQMCM) for mGluR1, and [(2-methyl-1,3-thiazol-4-yl)ethynyl]pyridine (MTEP) for mGluR5. Their effects on acquisition were compared to those of the NMDA receptor antagonist (+)MK-801, and the unselective muscarinic antagonist scopolamine, while diazepam and citalopram served as reference compounds in the expression experiments. EMQMCM (1.25 to 5 mg/kg) impaired acquisition of contextual fear conditioning (CFC), but not auditory fear conditioning (AFC). Similarly, administration of MTEP during the acquisition phase impaired learning in CFC at doses of 2.5 to 10 mg/kg, but was ineffective in AFC. When given before the retention test, both EMQMCM (1 and 3 mg/kg) and MTEP (3 mg/kg) impaired expression of CFC. In contrast, MTEP (2.5 and 5 mg/kg) blocked the expression of AFC, while EMQMCM was ineffective. In conclusion, group I mGlu receptors are shown to be involved in the acquisition of hippocampus-dependent CFC, but not hippocampus-independent AFC. Unlike mGluR5, mGluR1 does not seem be involved in expression of AFC.     

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.

     

mGlu1alpha receptors are co-expressed with CB1 receptors in a subset of interneurons in the CA1 region of organotypic hippocampal slice cultures and adult rat brain

Recent studies have demonstrated a functional interaction between group I metabotropic glutamate (mGlu) receptors and the cannabinoid system in the modulation of synaptic transmission. By using antisera directed against mGlu1alpha and CB1 cannabinoid receptors, we examined their distribution in the CA1 region of rat organotypic hippocampal slice cultures. Immunoreactive mGlu1alpha and CB1 elements were localized in non-principal cells, with a labeling distribution that was very similar to the pattern previously observed in the adult rat brain. Double-immunofluorescence staining and confocal microscopy showed that a subset of interneurons, mainly located in the stratum radiatum, was double-labeled for both mGlu1alpha and CB1 receptors. Co-localization of the two receptor subtypes was confirmed in hippocampal sections from adult rat brain. By using the "mirror technique" in adjacent sections, we observed that the double-labeled cells for mGlu1alpha and CB1 receptors were also immunopositive for the cholecystokinin peptide. Quantitative analysis revealed that in the stratum radiatum the majority (92%) of the CB1-positive cells and 19% of the mGlu1alpha-positive cells expressed both receptors. Triple immunofluorescence staining showed partial co-labeling of mGlu1alpha- and CB1-immunopositive cells with the vesicular glutamate transporter 3 or calbindin. Our results demonstrate that mGlu1alpha and CB1 receptors co-exist in a subpopulation of inhibitory neurons in the stratum radiatum of the hippocampus that is suggestive of the Schaffer collateral-associated interneurons. Hence, additional functional mechanisms underlying the cooperation between these two receptor subtypes may exist.     

Intermittent ethanol consumption depresses endocannabinoid-signaling in the dorsolateral striatum of rat

Recent research suggests that adaptations elicited by drugs of abuse share common features with traditional learning models, and that drugs of abuse cause long-term changes in behavior by altering synaptic function and plasticity. In this study, endocannabinoid (eCB) signaling in the dorsolateral striatum, a brain region vital for habit formation, was evaluated in acutely isolated brain slices from ethanol (EtOH)-consuming rats and control rats. EtOH-consuming rats had free access to a 20% EtOH solution for three 24 hour sessions a week during seven weeks and consumed an average of 3.4 g/kg per session. eCB-mediated long-lasting disinhibition (DLL) of population spike (PS) amplitude induced by moderate frequency stimulation was impaired in EtOH-consuming rats, and was not restored by the muscarinic receptor antagonist scopolamine (10 μM). The lack of DLL could be linked to a reduced GABA_A receptor tone, since bicuculline-mediated disinhibition of striatal output was significantly reduced in slices from EtOH-consuming rats. However, eCB signaling induced by high frequency stimulation (HFS) was also impaired in slices from EtOH-consuming rats and isolated control rats. Activation of presynaptic cannabinoid 1 receptors (CB1R) with WIN55,212-2 (250 nM, 1 μM) significantly modulated PS amplitude in slices from age-matched control rats while slices from EtOH-consuming rats remained unaffected, indicating that eCB signaling is inhibited at a level that is downstream from CB1R activation. Intermittent alcohol intake for seven weeks might thus be sufficient to modulate a presynaptic mechanism that needs to be synergized with CB1R activation for induction of long-term depression (LTD). In conclusion, alcohol consumption inhibits striatal eCB signaling in a way that could be of importance for understanding the neurological underpinnings of addictive behavior.This article is part of a Special Issue entitled ‘Synaptic Plasticity and Addiction’.     

Brown adipose tissue sympathetic nerve activity is potentiated by activation of 5-hydroxytryptamine (5-HT)1A/5-HT7 receptors in the rat spinal cord

In urethane-chloralose anesthetized, neuromuscularly blocked, ventilated rats, microinjection of NMDA (12 pmol) into the right fourth thoracic segment (T4) spinal intermediolateral nucleus (IML) immediately increased ipsilateral brown adipose tissue (BAT) sympathetic nerve activity (SNA; peak +492% of control), expired CO2 (+0.1%) heart rate (+48 beats min(-1)) and arterial pressure (+8 mmHg). The increase in BAT SNA evoked by T4 IML microinjection of NMDA was potentiated when it was administered immediately following a T4 IML microinjection of 5-hydroxytryptamine (5-HT, 100 pmol) or the 5-HT1A/5-HT7 receptor agonist, 8-OH-DPAT (600 pmol), (area under the curve: 184%, and 259% of the NMDA-only response, respectively). In contrast, T4 IML microinjection of the 5-HT2 receptor agonist, DOI (28 pmol) did not potentiate the NMDA-evoked increase in BAT SNA (101% of NMDA-only response). Microinjection into the T4 IML of the selective 5-HT1A antagonist, WAY-100635 (500 pmol), plus the 5-HT7 antagonist, SB-269970 (500 pmol), prevented the 5-HT-induced potentiation of the NMDA-evoked increase in BAT SNA. When administered separately, WAY-100635 (800 pmol) and SB-269970 (800 pmol) attenuated the 8-OH-DPAT-induced potentiation of the NMDA-evoked increase in BAT SNA through effects on the amplitude and duration of the response, respectively. The selective 5-HT2 receptor antagonist, ketanserin (100 pmol), did not attenuate the potentiations of the NMDA-evoked increase in BAT SNA induced by either 5-HT or 8-OH-DPAT. These results demonstrate that activation of 5-HT1A/5-HT7 receptors can act synergistically with NMDA receptor activation within the IML to markedly increase BAT SNA.     

P2Y1 receptors inhibit long-term depression in the prefrontal cortex

Long-term depression (LTD) is a form of synaptic plasticity that may contribute to information storage in the central nervous system. Here we report that LTD can be elicited in layer 5 pyramidal neurons of the rat prefrontal cortex by pairing low frequency stimulation with a modest postsynaptic depolarization. The induction of LTD required the activation of both metabotropic glutamate receptors of the mGlu1 subtype and voltage-sensitive Ca²⁺ channels (VSCCs) of the T/R, P/Q and N types, leading to the stimulation of intracellular inositol trisphosphate (IP3) receptors by IP3 and Ca²⁺. The subsequent release of Ca²⁺ from intracellular stores activated the protein phosphatase cascade involving calcineurin and protein phosphatase 1. The activation of purinergic P2Y₁ receptors blocked LTD. This effect was prevented by P2Y₁ receptor antagonists and was absent in mice lacking P2Y₁ but not P2Y₂ receptors. We also found that activation of P2Y₁ receptors inhibits Ca²⁺ transients via VSCCs in the apical dendrites and spines of pyramidal neurons. In addition, we show that the release of ATP under hypoxia is able to inhibit LTD by acting on postsynaptic P2Y₁ receptors. In conclusion, these data suggest that the reduction of Ca²⁺ influx via VSCCs caused by the activation of P2Y₁ receptors by ATP is the possible mechanism for the inhibition of LTD in prefrontal cortex.     

Role of mGluR 1 in synaptic plasticity impairment induced by maltol aluminium in rats

The main symptoms of Alzheimer's disease (AD) is the loss of learning and memory ability, of which biological basis is synaptic plasticity. Aluminium has been found to cause changes in synaptic plasticity, but its molecular mechanism was unclear. In this study, Sprague-Dawley rats were injected with aluminium maltol (Al(mal)₃) through the lateral ventricle to establish an AD-like model. Y-maze, electrophysiological measurements, Golgi staining, scanning electron microscopy, quantitative real-time polymerase chain reaction, and western blot techniques were used to investigate regulation of the metabolic glutamate receptor 1 (mGluR1) in synaptic plasticity impairment induced by Al(mal)₃. The results showed that Al(mal)₃ inhibited the induction and maintenance of long-term potentiation in the hippocampal CA1 region. During this process, the expression of mGluR1 was up-regulated and it inhibited the expression and phosphorylation of the N-methyl-D-aspartic acid receptors (NMDARs). This mainly affected NMDAR1 and NMDAR2B but did not affect protein kinase C expression.     

Effect of sildenafil on neuroinflammation and synaptic plasticity pathways in experimental autoimmune encephalomyelitis

Multiple sclerosis (MS) is a chronic immuno-inflammatory disease of the central nervous system characterized by demyelination and axonal damage. Cognitive changes are common in individuals with MS since inflammatory molecules secreted by microglia interfere with the physiological mechanisms of synaptic plasticity. According to previous data, inhibition of PDE5 promotes the accumulation of cGMP, which inhibits neuroinflammation and seems to improve synaptic plasticity and memory. The present study aimed to evaluate the effect of sildenafil on the signaling pathways of neuroinflammation and synaptic plasticity in experimental autoimmune encephalomyelitis (EAE). C57BL/6 mice were divided into three experimental groups (n = 10/group): (a) Control; (b) EAE; (c) EAE + sild (25 mg/kg/21 days). Sildenafil was able to delay the onset and attenuate the severity of the clinical symptoms of EAE. The drug also reduced the infiltration of CD4+ T lymphocytes and their respective IL-17 and TNF-α cytokines. Moreover, sildenafil reduced neuroinflammation in the hippocampus (assessed by the reduction of inflammatory markers IL-1β, pIKBα and pNFkB and reactive gliosis, as well as elevating the inhibitory cytokines TGF-β and IL-10). Moreover, sildenafil induced increased levels of NeuN, BDNF and pCREB, protein kinases (PKA, PKG, and pERK) and synaptophysin, and modulated the expression of the glutamate receptors AMPA and NMDA. The present findings demonstrated that sildenafil has therapeutic potential for cognitive deficit associated with multiple sclerosis.     

Pharmacological characterisation of 5-HT receptors positively coupled to adenylyl cyclase in the rat hippocampus

The pharmacological properties of 5-hydroxytryptamine (5-HT) receptors positively coupled to adenylyl cyclase in the rat hippocampus were investigated using selective agonists and antagonists. 5-HT (0.008–125 μM) stimulated cyclic AMP formation in homogenates of rat hippocampus in a concentration-dependent manner. The maximal increase in cyclic AMP formation occurred at 1 μM (141 ± 6%) and the half-maximal effect (EC₅₀) at 50 ± 22 nM. Cyclic AMP accumulation induced by 1 μM 5-HT was partly inhibited by the selective 5-HT_1A receptor antagonist WAY 100,635 (1 μM), the selective 5-HT₄ receptor antagonist SB 203,186 (1 μM), and the 5-HT_2A/C/ 5-HT₇ receptor antagonist mesulergine (25 μM). WAY 100,635, SB 203,186 and mesulergine inhibited the effect of 5-HT (1 μM) by 47%, 33% and 49%, respectively. The combination of WAY 100,635 (1 μM) with SB 203,186 (1 μM) or mesulergine (25 μM) resulted in stronger inhibition than with each antagonist alone, and the combination of all three antagonists produced almost total blockade (95%) of 5-HT-induced cyclic AMP accumulation. 5-Carboxamidotryptamine (5-CT; 0.008–125 μM), a 5-HT₁/5-HT₇ receptor agonist, and SDZ 216–454 (0.008– 125 μM), a selective 5-HT₄ receptor agonist, concentration-dependently stimulated cyclic AMP formation, but the maximal effect of each agonist was smaller than that of 5-HT alone. SDZ 216–454 (5 μM) and 5-CT (5 μM) in combination stimulated cyclic AMP formation in an additive manner. 8-OH-PIPAT and 8-OH-DPAT, two selective 5-HT_1A agonists, produced a small but significant increase in cyclic AMP formation at concentrations above 0.04 μM and 10 μM, respectively. These findings suggest that at least three 5-HT receptor subtypes, i.e. 5-HT_1A, 5-HT₇ and 5-HT₄ receptors, are involved in mediating 5-HT-induced cyclic AMP formation in rat hippocampus. Received: 28 October 1998 / Accepted: 16 March 1999     

Properties and distribution of muscarinic cholinergic receptors in rat striatal micropunched tissue homogenates

The properties of muscarinic receptors in rat striatal micropunched tissue homogenates were studied with a binding technique using [³H]quinuclidinyl benzilate (QNB) as ligand. Among the agonists, oxotremorine was the most potent inhinitor (ID₅₀ value, 2.20 × 10⁻⁵ M), followed by pilocarpine, acetylcholine, carbachol and bethanechol. No marked differences were found between the binding properties of QNB in the micropunched tissue homogenates and membrane fractions. Therefore, the detailed distribution of muscarinic receptors in the striatum was examined using the micropunched tissue homogenates. High levels of QNB binding in the striatum were found at dorso-lateral sites and low levels in the center regions of the striatum. The technique presented here for studying small regions of the striatum should be useful in further studies of the function of cholinergic neurons.     

Chronic antidepressant treatment increases the membrane expression of AMPA receptors in rat hippocampus

It has been proposed that potentiation of AMPA receptor (AMPAR) function may be useful in the treatment of depression. Here we studied the acute and chronic effect of the antidepressants desipramine and paroxetine, which differentially affect monoamine reuptake, on the expression of the AMPAR subunits GluR1 and GluR2/3, analyzed by Western blot, both in total and in membrane-enriched extracts from rat hippocampus. Acute antidepressant treatment did not produce any change in the expression of AMPAR subunits. In chronic treatments, rats were daily treated with the antidepressants (10 mg/kg/day) for 7, 14, or 21 days. In rats receiving either of the two antidepressant treatments for 21 consecutive days and killed 24 h after the last injection, an increase in GluR1 and GluR2/3 levels was found in the membrane fraction, with no significant change in the total extract, suggesting a trafficking of the AMPAR subunits from intracellular pools to synaptic sites in the hippocampus. This appeared to be a region-specific effect since no change in AMPAR subunit expression was found in the frontal cortex. Previously reported modifications in phosphorylating enzymes by chronic antidepressants could perhaps play a role in hippocampal membrane insertion of AMPAR subunits. When the survival time after the 21-day-treatment was longer — 72 instead of 24 h — the hippocampal membrane expression of GluR1, but not of GluR2/3 subunits, was still increased, as could be expected from the distinct mechanisms operating in synaptic delivery of GluR1 and GluR2/3 subunits. The antidepressant-induced increase in the number of GluR1- and GluR2/3-containing AMPARs at the synapses may indicate an enhanced AMPAR-mediated synaptic transmission which could help to counteract the alterations in neuronal connectivity which appear to underlie the pathophysiology of mood disorders.