Anna Pittaluga

The Use of Knock-Out Mice Unravels Distinct Roles for mGlu2 and mGlu3 Metabotropic Glutamate Receptors in Mechanisms of Neurodegeneration/Neuroprotection

Dual metabotropic glutamate 2/3 (mGlu2/3) receptor agonists have been examined with success in the clinic with positive proof of efficacy in several tests of anxiety and schizophrenia. Moreover, a large body of evidence has accumulated that these drugs have significant neuroprotective potential. An important discussion in the field deals with dissecting effects on mGlu2 versus effects on mGlu3 receptors, which is relevant for the potential use of subtype-selective agonists or allosteric activators. We addressed this issue using mGlu2 and mGlu3 receptor knock-out mice. We used mixed cultures of cortical cells in which astrocytes and neurons were plated at different times and could therefore originate from different mice. Cultures were challenged with NMDA for the induction of excitotoxic neuronal death. The mGlu2/3 receptor agonist, (−)-2-oxa-4-aminocyclo[3.1.0]hexane-4,6-dicarboxylic acid (LY379268), was equally neuroprotective in cultures containing neurons from wild-type, mGlu2−/−, or mGlu3−/− mice. Neuroprotection was instead abolished when astrocytes lacked mGlu3 receptors, unless neuronal mGlu2 receptors were also absent. The latter condition partially restored the protective activity of LY379268. Cultures in which neurons originated from mGlu2−/− mice were also intrinsically resistant to NMDA toxicity. In in vivo experiments, systemic administration of LY379268 protected striatal neurons against NMDA toxicity in wild-type and mGlu2−/− mice but not in mGlu3−/− mice. In addition, LY379268 was protective against nigrostriatal degeneration induced by low doses of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine only in mice lacking mGlu2 receptors. We conclude that neuroprotection by mGlu2/3 receptor agonists requires the activation of astrocytic mGlu3 receptors, whereas, unexpectedly, activation of mGlu2 receptors might be harmful to neurons exposed to toxic insults.

L-acetylcarnitine causes rapid antidepressant effects through the epigenetic induction of mGlu2 receptors

Epigenetic mechanisms are involved in the pathophysiology of depressive disorders and are unique potential targets for therapeutic intervention. The acetylating agent L-acetylcarnitine (LAC), a well-tolerated drug, behaves as an antidepressant by the epigenetic regulation of type 2 metabotropic glutamate (mGlu2) receptors. It caused a rapid and long-lasting antidepressant effect in Flinders Sensitive Line rats and in mice exposed to chronic unpredictable stress, which, respectively, model genetic and environmentally induced depression. In both models, LAC increased levels of acetylated H3K27 bound to the Grm2 promoter and also increased acetylation of NF-ĸB-p65 subunit, thereby enhancing the transcription of Grm2 gene encoding for the mGlu2 receptor in hippocampus and prefrontal cortex. Importantly, LAC reduced the immobility time in the forced swim test and increased sucrose preference as early as 3 d of treatment, whereas 14 d of treatment were needed for the antidepressant effect of chlorimipramine. Moreover, there was no tolerance to the action of LAC, and the antidepressant effect was still seen 2 wk after drug withdrawal. Conversely, NF-ĸB inhibition prevented the increase in mGlu2 expression induced by LAC, whereas the use of a histone deacetylase inhibitor supported the epigenetic control of mGlu2 expression. Finally, LAC had no effect on mGlu2 knockout mice exposed to chronic unpredictable stress, and a single injection of the mGlu2/3 receptor antagonist LY341495 partially blocked LAC action. The rapid and long-lasting antidepressant action of LAC strongly suggests a unique approach to examine the epigenetic hypothesis of depressive disorders in humans, paving the way for more efficient antidepressants with faster onset of action.

RELEASE-ENHANCING GLYCINE-DEPENDENT PRESYNAPTIC NMDA RECEPTORS EXIST ON NORADRENERGIC TERMINALS OF HIPPOCAMPUS

The release of [3H]noradrenaline ([3H]NA) in superfused rat hippocampal synaptosomes was increased concentration dependently by N-methyl-D-aspartate (NMDA). Glycine (1 microM), ineffective on its own, doubled the efficacy of NMDA with no significant change in affinity. The effects of NMDA alone and those of NMDA + glycine were antagonized by D-2-amino-5-phosphonopentanoate (D-AP5), Mg2+, (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohept-5,10-imine hydrogen maleate (MK-801) or 7-chloro-kynurenate (7-Cl-KYNA) but not by strychnine. The data provide a direct demonstration of the existence of presynaptic NMDA receptors.

Studies on [3H] GABA and endogenous GABA release in rat cerebral cortex suggest the presence of autoreceptors of the GABAB type

The presence of autoreceptors for gamma-aminobutyric acid (GABA) in the CNS was reinvestigated using rat cortex synaptosomes prelabeled with [3H]GABA and exposed to GABA by superfusion in the presence of a new GABA uptake inhibitor, N-(4,4-diphenyl-3-butenyl)-nipecotic acid (SK&F 89976A). This compound itself did not increase the basal or the depolarization-evoked release of [3H]GABA. GABA reduced in a concentration-dependent way the release of [3H]GABA evoked by 15 mM K+. The effect was not antagonized by bicuculline, picrotoxin or by the new GABAA antagonist SR 95531. The GABAA agonist muscimol did not affect [3H]GABA release. This was reduced by (-)baclofen (but not by the (+) isomer) and the concentration-inhibition curve of (-)baclofen was superimposable on to that of GABA. Also the K+-evoked release of endogenous GABA was stereoselectively and concentration dependently inhibited by the (-) enantiomer of baclofen. It is concluded that the release of GABA from rat cortical nerve endings may be inhibited through the activation of autoreceptors which appear to belong to the GABAB type.

Antidepressant treatments and function of glutamate ionotropic receptors mediating amine release in hippocampus

Previous evidences showed that, besides noradrenaline (NA) and 5-hydroxytryptamine (5-HT), glutamate transmission is involved in the mechanism of action of antidepressants (ADs), although the relations between aminergic and glutamatergic systems are poorly understood. The aims of this investigation were to evaluate changes in the function of glutamate AMPA and NMDA receptors produced by acute and chronic administration of the two ADs reboxetine and fluoxetine, selective inhibitors of NA and 5-HT uptake, respectively. Rats were treated acutely (intraperitoneal injection) or chronically (osmotic minipump infusion) with reboxetine or fluoxetine. Isolated hippocampal nerve endings (synaptosomes) prepared following acute/chronic treatments were labelled with [(3)H]NA or [(3)H]5-HT and [(3)H]amine release was monitored during exposure in superfusion to NMDA/glycine, AMPA or K(+)-depolarization. Acute and chronic reboxetine reduced the release of [(3)H]NA evoked by NMDA/glycine or by AMPA. The NMDA/glycine-evoked release of [(3)H]NA was also down-regulated by chronic fluoxetine. Only acute, but not chronic, fluoxetine inhibited the AMPA-evoked release of [(3)H]5-HT. The release of [(3)H]NA and [(3)H]5-HT elicited by K(+)-depolarization was almost abolished by acute reboxetine or fluoxetine, respectively, but recovered during chronic ADs administration. ADs reduced NMDA receptor-mediated releasing effects in noradrenergic terminals after acute and chronic administration, although by different mechanisms. Chronic treatments markedly reduced the expression level of NR1 subunit in synaptic membranes. The noradrenergic and serotonergic release systems seem to be partly functionally interconnected and interact with glutamatergic transmission to down-regulate its function. The results obtained support the view that glutamate plays a major role in AD activity.

Age-related decrease of the NMDA receptor-mediated noradrenaline release in rat hippocampus and partial restoration by D-cycloserine

The release of [3H]noradrenaline ([3H]NA) evoked by N-methyl-D-aspartate (NMDA) from superfused rat hippocampus synaptosomes was monitored during aging. The maximal effects of NMDA decreased with age from 50% (1.5 months) to 10% enhancement (24 months). Quisqualic acid (100 microM) also enhanced [3H]NA release. Its effect decreased with age with a pattern partly different from that of NMDA. Glycine (1 microM) potentiated the [3H]NA releasing effect of 100 microM NMDA. Unexpectedly, the potentiation which amounted to 50% at 1.5 months, reached almost 200% and 300% in the 18- and 24-month-old rats, respectively, thus compensating in part for the age-related loss of the NMDA-induced effect. Concentration-response relationships for glycine at 3 vs. 24 months suggest that the glycine receptor is superresponsive in the aged brain. This may be due to more efficient glycine removal or/and to impaired release since uptake of the amino acid was increased by 350% in 24- vs. 3-month-old rats, while the K(+)-evoked tritium release from synaptosomes prelabeled with [3H]glycine was decreased. D-Cycloserine, although about 10 times less potent than glycine, strongly enhanced the NMDA-evoked [3H]NA release and may prove useful in cognitive deficits associated with aging and dementia.

Anxiety-Like Behavior of Prenatally Stressed Rats Is Associated with a Selective Reduction of Glutamate Release in the Ventral Hippocampus

Abnormalities of synaptic transmission and plasticity in the hippocampus represent an integral part of the altered programming triggered by early life stress. Prenatally restraint stressed (PRS) rats develop long-lasting biochemical and behavioral changes, which are the expression of an anxious/depressive-like phenotype. We report here that PRS rats showed a selective impairment of depolarization- or kainate-stimulated glutamate and [3H]d-aspartate release in the ventral hippocampus, a region encoding memories related to stress and emotions. GABA release was unaffected in PRS rats. As a consequence of reduced glutamate release, PRS rats were also highly resistant to kainate-induced seizures. Abnormalities of glutamate release were associated with large reductions in the levels of synaptic vesicle-related proteins, such as VAMP (synaptobrevin), syntaxin-1, synaptophysin, synapsin Ia/b and IIa, munc-18, and Rab3A in the ventral hippocampus of PRS rats. Anxiety-like behavior in male PRS (and control) rats was inversely related to the extent of depolarization-evoked glutamate release in the ventral hippocampus. A causal relationship between anxiety-like behavior and reduction in glutamate release was demonstrated using a mixture of the mGlu2/3 receptor antagonist, LY341495, and the GABAB receptor antagonist, CGP52432, which was shown to amplify depolarization-evoked [3H]d-aspartate release in the ventral hippocampus. Bilateral microinfusion of CGP52432 plus LY341495 in the ventral hippocampus abolished anxiety-like behavior in PRS rats. These findings indicate that an impairment of glutamate release in the ventral hippocampus is a key component of the neuroplastic program induced by PRS, and that strategies aimed at enhancing glutamate release in the ventral hippocampus correct the “anxious phenotype” caused by early life stress.

N‐methyl‐D‐aspartate autoreceptors respond to low and high agonist concentrations by facilitating, respectively, exocytosis and carrier‐mediated release of glutamate in rat hippocampus

Presynaptic NMDA autoreceptors regulating glutamate release have rarely been investigated. High‐micromolar N‐methyl‐D‐aspartate (NMDA) was reported to elicit glutamate release from hippocampal synaptosomes in a Ca2+‐independent manner by reversal of excitatory amino acid transporters. The aim of this work was to characterize excitatory amino acid release evoked by low‐micromolar NMDA from glutamatergic axon terminals. Purified rat hippocampal synaptosomes were prelabelled with [3H]D‐aspartate ([3H]D‐ASP) and exposed in superfusion to varying concentrations of NMDA in the presence of 1 μM glycine. The release of [3H]D‐ASP and also that of endogenous glutamate provoked by 10 μM NMDA were external Ca2+ dependent and sensitive to the NMDA channel blocker MK‐801 but insensitive to the glutamate transporter inhibitor DL‐TBOA, which, on the contrary, prevented the Ca2+‐independent release evoked by 100 μM NMDA. The NMDA (10 μM) response was blocked by 1 nM Zn2+ and 1 μM ifenprodil, compatible with the involvement of a NR1/NR2A/NR2B assembly, although the presence of two separate receptor populations, i.e., NR1/NR2A and NR1/NR2B, cannot be excluded. This response was strongly antagonized by submicromolar (0.01–1 μM) concentrations of kynurenic acid and was mimicked by quinolinic acid (1–100 μM) plus 1 μM glycine. Finally, the HIV‐1 protein gp120 potently mimicked the NMDA coagonists glycine and D‐serine, being significantly effective at 30 pM. In conclusion, glutamatergic nerve terminals possess NMDA autoreceptors mediating different types of release when activated by different agonist concentrations: low‐micromolar glutamate would potentiate glutamate exocytosis, whereas higher glutamate concentrations would also provoke carrier‐mediated release.

Somatostatin potentiates NMDA receptor function via activation of InsP3 receptors and PKC leading to removal of the Mg2+ block without depolarization

N‐methyl‐D‐aspartate (NMDA) receptors exist on noradrenergic axon terminals and mediate enhancement of noradrenaline (NA) release. We here investigated modulation by somatostatin (SRIF, somatotropin release inhibiting factor) of the NMDA‐induced release of NA using superfused hippocampal synaptosomes. The NMDA response was increased by SRIF‐28 and SRIF‐14, but not SRIF‐28(1–14), whereas the release of [3H]‐NA elicited by α‐amino‐3‐hydroxy‐5‐methylisoxazide‐4‐propionic acid (AMPA) was unaffected. SRIF‐14 did not mimic glycine at the NMDA receptor but activated SRIF receptors sited on noradrenergic terminals. The SRIF‐14 effect was blocked by pertussis toxin but mimicked by mastoparan, a G‐protein activator. BIM‐23056, but not Cyanamid 154806, antagonized the SRIF‐14 effect. This effect was mimicked by L362855, a partial agonist at the sst5 subtype, but not by the new selective sst1–sst4 receptor agonists L797591, L779976, L796778 and L803087. Protein kinase C (PKC) inhibitors (H7, staurosporine, GF 209103X, cheleritrine and sphingosine) prevented the SRIF‐14 effect, while phorbol 12‐myristate 13‐acetate enhanced the NMDA response. SRIF‐14 permitted NMDA receptor activation in the presence of 1.2 mM Mg2+ ions, both in hippocampal synaptosomes and slices. Blockade of inositol‐1,4,5‐trisphosphate (InsP3) receptors with heparin abolished the effect of SRIF‐14. It is concluded that SRIF receptors, possibly of the sst5 subtype, can exert a permissive role on NMDA receptors colocalized on hippocampal noradrenergic terminals: activation of sst5 receptors is coupled to pertussis toxin‐sensitive G proteins enhancing phosphoinositide metabolism with activation of InsP3 receptors and PKC; NMDA receptor subunits might be phosphorylated with consequent removal of the Mg2+ block in absence of depolarization.

Differential desensitization of ionotropic non-NMDA receptors having distinct neuronal location and function

Abstract The release of tritium from rat hippocampal synaptosomes prelabeled with [3H]noradrenaline ([3H]NA) or [3H]5-hydroxytryptamine ([3H]5-HT) and from rat neocortex synaptosomes prelabeled with [3H]choline and the release of endogenous GABA and glutamate from rat neocortex synaptosomes were monitored during superfusion with media containing varying concentrations of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) or kainic acid. Concentration-dependent release potentiations were elicited by both excitatory amino acids (EAAs) in all the transmitter systems investigated. The releases evoked by 100 µM AMPA were, in all cases, almost totally dependent on external Ca2+ and sensitive to 6,7-dinitroquinoxaline-2,3-dione (DNQX), indicating involvement of non-NMDA receptors. When cyclothiazide, a drug able to prevent desensitization of AMPA-preferring receptors, was added to the superfusion medium (at 1 or 10 µM) concomitantly with 100 µM AMPA or kainate, the EAA-evoked release of [3H]NA was significantly enhanced. Concanavalin A, a lectin thought to prevent desensitization of kainate-preferring receptors, had no effect (up to 10 µM) on the release of [3H]NA evoked by AMPA or kainate. The effect of cyclothiazide was lost if, after an 8-min pretreatment, the drug was removed just before the AMPA stimulus. When added concomitantly with the EAAs, cyclothiazide potentiated the release of [3H]5-HT elicited by AMPA and, less so, that evoked by kainate. Concanavalin A was ineffective. Neither cyclothiazide (1 or 10 µM) nor concanavalin A (3 or 10 µM) could affect the release of [3H]ACh or endogenous GABA provoked by 100 µM AMPA or kainate, suggesting that the receptors involved do not desensitize. Exposure of neocortex synaptosomes to AMPA or kainate concomitantly with cyclothiazide caused endogenous glutamate release that did not differ from that evoked by the EAAs alone. In contrast, the effects of AMPA and kainate were potentiated by concanavalin A. The activity of the lectin (3 µM) persisted when it was applied for 8 min and then removed before the AMPA or kainate (100 µM) pulse. When hippocampal synaptosomes prelabeled with [3H]NA were subjected to three subsequent AMPA (100 µM) stimuli (S1, S2 and S3), the release of [3H]NA decreased dramatically from S1 to S3 (S3/S1 = 0.14 ± 0.04); a significant ‘protection’ of the AMPA effect was offered by 1 µM cyclothiazide (S3/S1 = 0.36 ± 0.06). This value did not differ from the S3/S1 ratio (0.38 ± 0.04) obtained in parallel experiments with 12 mM K+. The release evoked by high-K+ was insensitive to cyclothiazide. Finally, the effect of AMPA on the release of [3H]ACh did not respond to cyclothiazide also during three subsequent stimuli with 100 µM AMPA. To conclude: a) ionotropic non-NMDA receptors mediating enhancement of NA, 5-HT, ACh, GABA and glutamate release exist on the corresponding nerve terminals; b) the receptors present on noradrenergic and serotonergic neurons are AMPA-preferring receptors, whereas the glutamate autoreceptors resemble most the kainate-preferring subtype; the receptors mediating ACh and GABA release can not be subclassified at present; c) desensitization may not be a property of all non-NMDA ionotropic receptors. The receptors here characterized represent five models of native non-NMDA receptors suitable for pharmacological and molecular studies.