Diurnal inhibition of NMDA-EPSCs at rat hippocampal mossy fibre synapses through orexin-2 receptors.

Diurnal release of the orexin neuropeptides orexin-A (Ox-A, hypocretin-1) and orexin-B (Ox-B, hypocretin-2) stabilises arousal, regulates energy homeostasis and contributes to cognition and learning. However, whether cellular correlates of brain plasticity are regulated through orexins, and whether they do so in a time-of-day-dependent manner, has never been assessed. Immunohistochemically we found sparse but widespread innervation of hippocampal subfields through Ox-A- and Ox-B-containing fibres in young adult rats. The actions of Ox-A were studied on NMDA receptor (NMDAR)-mediated excitatory synaptic transmission in acute hippocampal slices prepared around the trough (Zeitgeber time (ZT) 4-8, corresponding to 4-8 h into the resting phase) and peak (ZT 23) of intracerebroventricular orexin levels. At ZT 4-8, exogenous Ox-A (100 nm in bath) inhibited NMDA receptor-mediated excitatory postsynaptic currents (NMDA-EPSCs) at mossy fibre (MF)-CA3 (to 55.6 ± 6.8% of control, P = 0.0003) and at Schaffer collateral-CA1 synapses (70.8 ± 6.3%, P = 0.013), whereas it remained ineffective at non-MF excitatory synapses in CA3. Ox-A actions were mediated postsynaptically and blocked by the orexin-2 receptor (OX2R) antagonist JNJ10397049 (1 µm), but not by orexin-1 receptor inhibition (SB334867, 1 µm) or by adrenergic and cholinergic antagonists. At ZT 23, inhibitory effects of exogenous Ox-A were absent (97.6 ± 2.9%, P = 0.42), but reinstated (87.2 ± 3.3%, P = 0.002) when endogenous orexin signalling was attenuated for 5 h through i.p. injections of almorexant (100 mg kg(-1)), a dual orexin receptor antagonist. In conclusion, endogenous orexins modulate hippocampal NMDAR function in a time-of-day-dependent manner, suggesting that they may influence cellular plasticity and consequent variations in memory performance across the sleep-wake cycle.

Sublinear integration underlies binocular processing in primary visual cortex.

Although we know much about the capacity of neurons to integrate synaptic inputs in vitro, less is known about synaptic integration in vivo. Here we address this issue by investigating the integration of inputs from the two eyes in mouse primary visual cortex. We find that binocular inputs to layer 2/3 pyramidal neurons are integrated sublinearly in an amplitude-dependent manner. Sublinear integration was greatest when binocular responses were largest, as occurs at the preferred orientation and binocular disparity, and highest contrast. Using voltage-clamp experiments and modeling, we show that sublinear integration occurs postsynaptically. The extent of sublinear integration cannot be accounted for solely by nonlinear integration of excitatory inputs, even when they are activated closely in space and time, but requires balanced recruitment of inhibition. Finally, we show that sublinear binocular integration acts as a divisive form of gain control, linearizing the output of binocular neurons and enhancing orientation selectivity.

Do mice habituate to "gentle handling?" A comparison of resting behavior, corticosterone levels and synaptic function in handled and undisturbed C57BL/6J mice.

STUDY OBJECTIVES: "Gentle handling" has become a method of choice for 4-6 h sleep deprivation in mice, with repeated brief handling applied before sleep deprivation to induce habituation. To verify whether mice do indeed habituate, we assess how 6 days of repeated brief handling impact on resting behavior, on stress, and on the subunit content of N-methyl-D-aspartate receptors (NMDARs) at hippocampal synapses, which is altered by sleep loss. We discuss whether repeated handling biases the outcome of subsequent sleep deprivation. DESIGN: Adult C57BL/6J mice, maintained on a 12 h-12 h light-dark cycle, were left undisturbed for 3 days, then handled during 3 min daily for 6 days in the middle of the light phase. Mice were continuously monitored for their resting time. Serum corticosterone levels and synaptic NMDAR subunit composition were quantified. RESULTS: Handling caused a ∼25% reduction of resting time throughout all handling days. After six, but not after one day of handling, mice had elevated serum corticosterone levels. Six-day handling augmented the presence of the NR2A subunit of NMDARs at hippocampal synapses. CONCLUSION: Repeated handling induces behavioral and neurochemical alterations that are absent in undisturbed animals. The persistently reduced resting time and the delayed increase in corticosterone levels indicate that mice do not habituate to handling over a 1-week period. Handling-induced modifications bias effects of gentle handling-induced sleep deprivation on sleep homeostasis, stress, glutamate receptor composition and signaling. A standardization of sleep deprivation procedures involving gentle handling will be important for unequivocally specifying how acute sleep loss affects brain function.

NR2A at CA1 synapses is obligatory for the susceptibility of hippocampal plasticity to sleep loss.

A loss in the necessary amount of sleep alters expression of genes and proteins implicated in brain plasticity, but key proteins that render neuronal circuits sensitive to sleep disturbance are unknown. We show that mild (4-6 h) sleep deprivation (SD) selectively augmented the number of NR2A subunits of NMDA receptors on postsynaptic densities of adult mouse CA1 synapses. The greater synaptic NR2A content facilitated induction of CA3-CA1 long-term depression in the theta frequency stimulation range and augmented the synaptic modification threshold. NR2A-knock-out mice maintained behavioral response to SD, including compensatory increase in post-deprivation resting time, but hippocampal synaptic plasticity was insensitive to sleep loss. After SD, the balance between synaptically activated and slowly recruited NMDA receptor pools during temporal summation was disrupted. Together, these results indicate that NR2A is obligatory for the consequences of sleep loss on hippocampal synaptic plasticity. These findings could advance pharmacological strategies aiming to sustain hippocampal function during sleep restriction.

Consequences of sleep deprivation on neurotransmitter receptor expression and function.

Several pieces of evidence suggest that sleep deprivation causes marked alterations in neurotransmitter receptor function in diverse neuronal cell types. To date, this has been studied mainly in wake- and sleep-promoting areas of the brain and in the hippocampus, which is implicated in learning and memory. This article reviews findings linking sleep deprivation to modifications in neurotransmitter receptor function, including changes in receptor subunit expression, ligand affinity and signal transduction mechanisms. We focus on studies using sleep deprivation procedures that control for side-effects such as stress. We classify the changes with respect to their functional consequences on the activity of wake-promoting and/or sleep-promoting systems. We suggest that elucidation of how sleep deprivation affects neurotransmitter receptor function will provide functional insight into the detrimental effects of sleep loss.

RANTES modulates the release of glutamate in human neocortex.

The effects of the recombinant chemokine human RANTES (hRANTES) on the release of glutamate from human neocortex glutamatergic nerve endings were investigated. hRANTES facilitated the spontaneous release of d [(3)H]D-aspartate ([(3)H]DASP-) by binding Pertussis toxin-sensitive G-protein-coupled receptors (GPCRs), whose activation caused Ca(2+) mobilization from inositol trisphosphate-sensitive stores and cytosolic tyrosine kinase-mediated phosphorylations. Facilitation of release switched to inhibition when the effects of hRANTES on the 12 mM K(+)-evoked [(3)H]D-ASP exocytosis were studied. Inhibition of exocytosis relied on activation of Pertussis toxin-sensitive GPCRs negatively coupled to adenylyl cyclase. Both hRANTES effects were prevented by met-RANTES, an antagonist at the chemokine receptors (CCRs) of the CCR1, CCR3, and CCR5 subtypes. Interestingly, human neocortex glutamatergic nerve endings seem to possess all three receptor subtypes. Blockade of CCR1 and CCR5 by antibodies against the extracellular domain of CCRs prevented both the hRANTES effect on [(3)H]D-ASP release, whereas blockade of CCR3 prevented inhibition, but not facilitation, of release. The effects of RANTES on the spontaneous and the evoked release of [(3)H]D-ASP were also observed in experiments with mouse cortical synaptosomes, which may therefore represent an appropriate animal model to study RANTES-induced effects on neurotransmission. It is concluded that glutamate transmission can be modulated in opposite directions by RANTES acting at distinct CCR receptor subtypes coupled to different transduction pathways, consistent with the multiple and sometimes contrasting effects of the chemokine.

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.

Experience-dependent changes in NMDA receptor composition at mature central synapses.

N-methyl-D-aspartate receptor (NMDAR) activation is obligatory for the induction of diverse forms of synaptic plasticity. The molecular composition and the function of NMDARs are themselves modified by synaptic activity, which, in turn, alters the ability of synapses to undergo subsequent plastic modification. This homeostatic control of synaptic plasticity is well-known for the experience-dependent development of sensory cortices. However, it is now becoming clear that NMDAR properties may not only be altered at juvenile, but also at mature synapses. Diverse types of behavioral manipulation, such as sensory experience, learning and sleep deprivation alter the NR2A/NR2B ratio of hippocampal or cortical NMDARs. As an additional facet to the dynamics of NMDAR function, NMDAR trafficking is regulated by G-protein-coupled neurotransmitter receptors implicated in learning and arousal, such as orexin and dopamine. These findings suggest that mature glutamatergic synapses may be modified by recent activity via alterations in synaptic NMDAR function. Rapid forms of NMDAR trafficking, perhaps controlled by the neurochemical environment featuring specific states of arousal and learning, may regulate plasticity and modulate cognitive abilities in adulthood.

Insufficient sleep reversibly alters bidirectional synaptic plasticity and NMDA receptor function.

Insufficient sleep impairs cognitive functions in humans and animals. However, whether long-term synaptic plasticity, a cellular substrate of learning and memory, is compromised by sleep loss per se remains unclear because of confounding factors related to sleep deprivation (SD) procedures in rodents. Using an ex vivo approach in C57BL/6J mice, we show that sleep loss rapidly and reversibly alters bidirectional synaptic plasticity in the CA1 area of the hippocampus. A brief (approximately 4 h) total SD, respecting the temporal parameters of sleep regulation and maintaining unaltered low corticosterone levels, shifted the modification threshold for long-term depression/long-term potentiation (LTP) along the stimulation frequency axis (1-100 Hz) toward the right. Reducing exposure to sensory stimuli by whisker trimming did not affect the SD-induced changes in synaptic plasticity. Recovery sleep reversed the effects induced by SD. When SD was combined with moderate stress, LTP induction was not only impaired but also occluded. Both electrophysiological analysis and immunoblotting of purified synaptosomes revealed that an alteration in the molecular composition of synaptically activated NMDA receptors toward a greater NR2A/NR2B ratio accompanied the effects of SD. This change was reversed after recovery sleep. By using an unparalleled, particularly mild form of SD, this study describes a novel approach toward dissociating the consequences of insufficient sleep on synaptic plasticity from nonspecific effects accompanying SD in rodents. We establish a framework for cellular models of cognitive impairment related to sleep loss, a major problem in modern society.

The human immunodeficiency virus-1 protein transactivator of transcription up-regulates N-methyl-D-aspartate receptor function by acting at metabotropic glutamate receptor 1 receptors coexisting on human and rat brain noradrenergic neurones.

We investigated the effects of the human immunodeficiency virus-1 transactivator of transcription (Tat) on the release of norepinephrine (NE) from human and rat brain synaptosomes. Tat could not evoke directly release of [3H]NE. In the presence of Tat (1 nM), N-methyl-D-aspartate (NMDA) concentrations unable to release (human synaptosomes) or slightly releasing (rat synaptosomes) [3H]NE became very effective. The NMDA/Tat-evoked release depends on NMDA receptors (NMDARs) since it was abolished by MK-801 (dizocilpine). Tat binding at NMDARs was excluded. The NMDA-induced release of [3H]NE in the presence of glycine was further potentiated by Tat. The release evoked by NMDA/glycine/Tat depends on metabotropic glutamate receptor 1 (mGluR1) activation, since it was halved by mGluR1 antagonists. Tat seems to act at the glutamate recognition site of mGluR1. Recently, Tat was shown to release [3H]acetylcholine from human cholinergic terminals; here, we demonstrate that this effect is also mediated by presynaptic mGluR1. The peptide sequence Tat41-60, but not Tat61-80, mimicked Tat. Phospholipase C, protein kinase C, and cytosolic tyrosine kinase are involved in the NMDA/glycine/Tat-evoked [3H]NE release. To conclude, Tat can represent a potent pathological agonist at mGlu1 receptors able to release acetylcholine from human cholinergic terminals and up-regulate NMDARs mediating NE release from human and rat noradrenergic terminals.

Trafficking of presynaptic AMPA receptors mediating neurotransmitter release: neuronal selectivity and relationships with sensitivity to cyclothiazide.

Postsynaptic glutamate AMPA receptors (AMPARs) can recycle between plasma membrane and intracellular pools. In contrast, trafficking of presynaptic AMPARs has not been investigated. AMPAR surface expression involves interactions between the GluR2 carboxy tail and various proteins including glutamate receptor-interacting protein (GRIP), AMPA receptor-binding protein (ABP), protein interacting with C kinase 1 (PICK1), N-ethyl-maleimide-sensitive fusion protein (NSF). Here, peptides known to selectively block the above interactions were entrapped into synaptosomes to study the effects on the AMPA-evoked release of [3H]noradrenaline ([3H]NA) and [3H]acetylcholine ([3H]ACh) from rat hippocampal and cortical synaptosomes, respectively. Internalization of pep2-SVKI to prevent GluR2-GRIP/ABP/PICK1 interactions potentiated the AMPA-evoked release of [3H]NA but left unmodified that of [3H]ACh. Similar potentiation was caused by pep2-AVKI, the blocker of GluR2-PICK1 interaction. Conversely, a decrease in the AMPA-evoked release of [3H]NA, but not of [3H]ACh, was caused by pep2m, a selective blocker of the GluR2-NSF interaction. In the presence of pep2-SVKI the presynaptic AMPARs on noradrenergic terminals lost sensitivity to cyclothiazide. AMPARs releasing [3H]ACh, but not those releasing [3H]NA, were sensitive to spermine, suggesting that they are GluR2-lacking AMPARs. To conclude: (i) release-regulating presynaptic AMPARs constitutively cycle in isolated nerve terminals; (ii) the process exhibits neuronal selectivity; (iii) AMPAR trafficking and desensitization may be interrelated.

Somatostatin-induced activation and up-regulation of N-methyl-D-aspartate receptor function: mediation through calmodulin-dependent protein kinase II, phospholipase C, protein kinase C, and tyrosine kinase in hippocampal noradrenergic nerve endings.

Somatostatin receptors and glutamate N-methyl-D-aspartate (NMDA) receptors coexist on hippocampal noradrenergic axon terminals. Activation of somatostatin receptors was previously found to positively influence the function of NMDA receptors regulating norepinephrine release. The somatostatin receptors involved were pharmacologically characterized as sst5 type in experiments in Mg2+-free solutions. Here, we first confirm the pharmacology of these receptors using selective sst5 ligands in Mg2+-containing solutions. Moreover, we show by Western blot that the sst5 protein exists on purified hippocampal synaptosomal membranes. We then investigated the pathways connecting the two receptors using as a functional response the release of norepinephrine from rat hippocampal synaptosomes in superfusion. The release of norepinephrine evoked by somatostatin-14 plus NMDA/glycine was partly prevented by the protein kinase C inhibitor GF109203X [dihydrochloride3-[1-[3-(dimethylamino)propyl]-1H-indol-3-yl]-4-(1H-indol-3-yl)-1H-pyrrole-2,5-dione] and by the nonreceptor tyrosine kinase (Src) inhibitors PP2 [3-(4-chlorophenyl)1-(1,1-dimethylethyl)-1H-pyrazolo[3,4-D]pyrimidin-4-amine] and lavendustin A; it was largely and almost totally abolished by the phospholipase C inhibitor U73122 [1-(6-[([17beta]-3-methoxyextra-1,3,5[10]-trien-17-yl)amino]hexyl)-1H-pyrrole-2,5-dione] and by the Ca2+/calmodulin-dependent protein kinase II (CaMKII) inhibitor KN93 [N-(2-[N-[4-chlorocinnamyl]-N-methyl-amino-methyl]phenyl)-N-(2-hydroxyethyl)-4-methoxy-benzene-sulfonamide-phosphate salt], respectively; and it was unaffected by the protein kinase A inhibitor H89 [N-(2-[p-bromocinnamylamino]ethyl)5-isoquinolinesulfonamide hydrochloride]. The norepinephrine release evoked by somatostatin-14/NMDA/glycine was inhibited when anti-phosphotyrosine antibodies had been entrapped into synaptosomes. Entrapping the recombinant activated tyrosine kinase pp60(c-Src) strongly potentiated the release of norepinephrine elicited by NMDA/glycine in Mg2+-free medium but failed to permit NMDA receptor activation in presence of external Mg2+ ions. The results suggest the involvement of CaMKII in the sst5 receptor-mediated activation of NMDA receptors in presence of Mg2+ and of the PLC/PKC/Src pathway in the up-regulation of the ongoing NMDA receptor activity.

External pH changes affect NMDA-evoked and spontaneous release of cholecystokinin, somatostatin and noradrenaline from rat cerebrocortical nerve endings.

It was previously reported that the K+-evoked release of somatostatin-like immunoreactivity (SRIF-LI) and of cholecystokinin-like immunoreactivity (CCK-LI) from superfused rat cerebrocortical synaptosomes can be enhanced by NMDA or D-serine alone. We here studied the effects of extraterminal pH changes on SRIF-LI and CCK-LI release. Lowering pH from 7.4 to 6.9 or 6.4 abolished the effects of NMDA or D-serine on the K+-evoked peptide release. Identical results were obtained when external pH was raised to 8 or 8.7. Sudden alkalinization of the superfusion medium, in absence of K+-depolarization, induced SRIF-LI or CCK-LI release which was insensitive to NMDA. Based on experiments in Ca2+-free medium and with voltage-sensitive Ca2+ channel (VSCC) blockers, the pH 8.7-induced release of SRIF-LI and CCK-LI was only in part (30-50%) dependent on external Ca2+ and Ca2+ channel activation. In contrast, the alkalinization-evoked release of [3H]noradrenaline was highly sensitive to external Ca2+ removal and to blockade of Ca2+ channels with omega-conotoxins. The pH 8.7-evoked SRIF-LI and CCK-LI was about halved in synaptosomes intoxicated with botulinum toxin C1. The results suggest that the pH-sensitive NMDA receptors mediating somatostatin and cholecystokinin release contain NR1 subunits lacking the exon-5 cassette. Alkalinization represents a novel releasing stimulus which elicits neuropeptide release in part by conventional exocytosis and largely by an external Ca2+-independent mechanism. Differently, the release of noradrenaline provoked by alkalinization occurs entirely by conventional exocytosis.