Microglial diversity along the hippocampal longitudinal axis impacts synaptic plasticity in adult male mice under homeostatic conditions.

The hippocampus is a plastic brain area that shows functional segregation along its longitudinal axis, reflected by a higher level of long-term potentiation (LTP) in the CA1 region of the dorsal hippocampus (DH) compared to the ventral hippocampus (VH), but the mechanisms underlying this difference remain elusive. Numerous studies have highlighted the importance of microglia-neuronal communication in modulating synaptic transmission and hippocampal plasticity, although its role in physiological contexts is still largely unknown. We characterized in depth the features of microglia in the two hippocampal poles and investigated their contribution to CA1 plasticity under physiological conditions. We unveiled the influence of microglia in differentially modulating the amplitude of LTP in the DH and VH, showing that minocycline or PLX5622 treatment reduced LTP amplitude in the DH, while increasing it in the VH. This was recapitulated in Cx3cr1 knockout mice, indicating that microglia have a key role in setting the conditions for plasticity processes in a region-specific manner, and that the CX3CL1-CX3CR1 pathway is a key element in determining the basal level of CA1 LTP in the two regions. The observed LTP differences at the two poles were associated with transcriptional changes in the expression of genes encoding for Il-1, Tnf-α, Il-6, and Bdnf, essential players of neuronal plasticity. Furthermore, microglia in the CA1 SR region showed an increase in soma and a more extensive arborization, an increased prevalence of immature lysosomes accompanied by an elevation in mRNA expression of phagocytic markers Mertk and Cd68 and a surge in the expression of microglial outward K+ currents in the VH compared to DH, suggesting a distinct basal phenotypic state of microglia across the two hippocampal poles. Overall, we characterized the molecular, morphological, ultrastructural, and functional profile of microglia at the two poles, suggesting that modifications in hippocampal subregions related to different microglial statuses can contribute to dissect the phenotypical aspects of many diseases in which microglia are known to be involved.

Fluoxetine effects on molecular, cellular and behavioral endophenotypes of depression are driven by the living environment.

Selective serotonin reuptake inhibitors (SSRIs) represent the most common treatment for major depression. However, their efficacy is variable and incomplete. In order to elucidate the cause of such incomplete efficacy, we explored the hypothesis positing that SSRIs may not affect mood per se but, by enhancing neural plasticity, render the individual more susceptible to the influence of the environment. Consequently, SSRI administration in a favorable environment promotes a reduction of symptoms, whereas in a stressful environment leads to a worse prognosis. To test such hypothesis, we exposed C57BL/6 mice to chronic stress in order to induce a depression-like phenotype and, subsequently, to fluoxetine treatment (21 days), while being exposed to either an enriched or a stressful condition. We measured the most commonly investigated molecular, cellular and behavioral endophenotypes of depression and SSRI outcome, including depression-like behavior, neurogenesis, brain-derived neurotrophic factor levels, hypothalamic-pituitary-adrenal axis activity and long-term potentiation. Results showed that, in line with our hypothesis, the endophenotypes investigated were affected by the treatment according to the quality of the living environment. In particular, mice treated with fluoxetine in an enriched condition overall improved their depression-like phenotype compared with controls, whereas those treated in a stressful condition showed a distinct worsening. Our findings suggest that the effects of SSRI on the depression- like phenotype is not determined by the drug per se but is induced by the drug and driven by the environment. These findings may be helpful to explain variable effects of SSRI found in clinical practice and to device strategies aimed at enhancing their efficacy by means of controlling environmental conditions.

Lymnaea stagnalis as model for translational neuroscience research: From pond to bench.

The purpose of this review is to illustrate how a reductionistic, but sophisticated, approach based on the use of a simple model system such as the pond snail Lymnaea stagnalis (L. stagnalis), might be useful to address fundamental questions in learning and memory. L. stagnalis, as a model, provides an interesting platform to investigate the dialog between the synapse and the nucleus and vice versa during memory and learning. More importantly, the "molecular actors" of the memory dialogue are well-conserved both across phylogenetic groups and learning paradigms, involving single- or multi-trials, aversion or reward, operant or classical conditioning. At the same time, this model could help to study how, where and when the memory dialog is impaired in stressful conditions and during aging and neurodegeneration in humans and thus offers new insights and targets in order to develop innovative therapies and technology for the treatment of a range of neurological and neurodegenerative disorders.

Vortioxetine Prevents Lipopolysaccharide-Induced Memory Impairment Without Inhibiting the Initial Inflammatory Cascade.

Vortioxetine is a novel multimodal antidepressant that modulates a wide range of neurotransmitters throughout the brain. Preclinical and clinical studies have shown that vortioxetine exerts positive effects on different cognitive domains and neuroprotective effects. Considering the key role of microglial cells in brain plasticity and cognition, we aimed at investigating the effects of pretreatment with vortioxetine in modulating behavioral and molecular effects induced by an immune challenge: peripheral injection of lipopolysaccharide (LPS). To this purpose, C57BL/6J male mice were first exposed to a 28-day standard diet or vortioxetine-enriched diet, which was followed by an acute immune challenge with LPS. Sickness symptoms and depressive-like behaviors (anhedonia and memory impairment) were tested 6 and 24 h after exposure to LPS, respectively. Moreover, the expressions of markers of immune activation and M1/M2 markers of microglia polarization were measured in the dorsal and ventral parts of the hippocampus. The pretreatment with vortioxetine did not affect both LPS-induced sickness behavior and anhedonia but prevented the deficit in the recognition memory induced by the immune challenge. At the transcriptional level, chronic exposure to vortioxetine did not prevent LPS-induced upregulation of proinflammatory cytokines 6 h after the immune challenge but rather seemed to potentiate the immune response to the challenge also by affecting the levels of expression of markers of microglia M1 phenotype, like cluster of differentiation (CD)14 and CD86, in an area-dependent manner. However, at the same time point, LPS injection significantly increased the expression of the M2 polarization inducer, interleukin 4, only in the hippocampus of animals chronically exposed to vortioxetine. These results demonstrate that a chronic administration of vortioxetine specifically prevents LPS-induced memory impairment, without affecting acute sickness behavior and anhedonia, and suggest that hippocampal microglia may represent a cellular target of this novel antidepressant medication. Moreover, we provide a useful model to further explore the molecular mechanisms specifically underlying cognitive impairments following an immune challenge.

Different synaptic location of mianserin and imipramine binding sites.

The high-affinity binding sites for mianserin and imipramine appear to be locate in different neurons of rat brain. Studies in which lesions were produced with 5,7-dihydroxytryptamine and other studies in which the 5-hydroxytryptamine content was decreased with p-chlorophenylalanine indicate that some of the imipramine binding sites are on serotonin axon terminals and others are on nonserotonergic synapses. The sites that bind mianserin are on postsynaptic serotonin sites as well as on synapses of other neuronal systems.

Modulation of neuroplasticity-related targets following stress-induced acute escape deficit.

Understanding resilience is a major challenge to improve current pharmacological therapies aimed at complementing psychological-based approaches of stress-related disorders. In particular, resilience is a multi-factorial construct where the complex network of molecular events that drive the process still needs to be resolved. Here, we exploit the acute escape deficit model, an animal model based on exposure to acute unavoidable stress followed by an escape test, to define vulnerable and resilient phenotypes in rats. Hippocampus and prefrontal cortex (PFC), two of the brain areas most involved in the stress response, were analysed for gene expression at two different time points (3 and 24 h) after the escape test. Total Brain-Derived Neurotrophic Factor (BDNF) was highly responsive in the PFC at 24-h after the escape test, while expression of BDNF transcript IV increased in the hippocampus of resistant animals 3 h post-test. Expression of memory enhancers like Neuronal PAS Domain Protein 4 (Npas4) and Activity-regulated cytoskeleton-associated protein (Arc) decreased in a time- and region-dependent fashion in both behavioural phenotypes. Also, the memory inhibitor Protein Phosphatase 1 (Ppp1ca) was increased in the hippocampus of resilient rats at 3 h post-test. Given the importance of neurotrophic factors and synaptic plasticity-related genes for the development of appropriate coping strategies, our data contribute to an additional step forward in the comprehension of the psychobiology of stress and resiliency.

Serine/threonine kinases as molecular targets of antidepressants: implications for pharmacological treatment and pathophysiology of affective disorders.

It is currently a widely accepted opinion that adaptive, plastic changes in the molecular and cellular components of neuronal signaling systems correlate with the effects on mood and cognition observed after long-term treatment with antidepressant drugs. Protein phosphorylation represents a key step for most signaling systems, and it is involved in the regulation of virtually all cellular functions. Two serine/threonine kinases, Ca2+ /calmodulin-dependent protein kinase II and cyclic AMP-dependent protein kinase, have been shown to be activated in the brain following antidepressant treatment. The changes in kinase activity are mirrored by changes in the phosphorylation of selected protein substrates in subcellular compartments (presynaptic terminals and microtubules), which, in turn, may contribute to the modulation of synaptic transmission observed with antidepressants. The molecular consequences of protein kinase activation may account for some of the alterations in neural function induced by antidepressants, and may suggest novel possible strategies of pharmacological intervention.

Modulation of glutamate receptors in response to the novel antipsychotic olanzapine in rats.

BACKGROUND: A disturbance in glutamate neurotransmission has been hypothesized in schizophrenia. Hence, the beneficial effects of pharmacological treatment may be related to adaptive changes taking place in this neurotransmitter system. METHODS: In this study, we investigated the modulation of ionotropic and metabotropic glutamate receptors in the rat brain following acute or chronic exposure to the novel antipsychotic olanzapine. RESULTS: In accordance with the clear distinction between classical and atypical drugs, olanzapine did not alter glutamate receptor expression in striatum. Chronic, not acute, exposure to olanzapine was capable of up-regulating hippocampal mRNA levels for GluR-B and GluR-C, two alpha-amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid (AMPA)-forming subunits. This effect could be relevant for the improvement of schizophrenic alterations, which are thought to depend on dysfunction of the glutamatergic transmission within the hippocampal formation. We also found that the expression of group II glutamate metabotropic receptors was up-regulated in the frontal cortex after chronic exposure to clozapine, and to a lesser extent olanzapine, but not with haloperidol. CONCLUSIONS: The adaptive mechanisms taking place in glutamatergic transmission might prove useful in ameliorating some of the dysfunction observed in the brain of schizophrenic patients.

cAMP binding proteins in the rat cerebral cortex after administration of selective 5-HT and NE reuptake blockers with antidepressant activity.

This study was undertaken to evaluate the cyclic adenosine monophosphate (cAMP) binding proteins in the cerebral cortex of rat after short- and long-term administration with antidepressants. Prolonged treatment with different antidepressants that inhibit serotonin or norepinephrine uptake such as fluoxetine and the (+) enantiomer of oxaprotiline, respectively, was able to induce an increase in the photoactivated incorporation of 8-N3-[32P]cAMP into a protein band with apparent molecular weight of 52,000 in both soluble and crude microtubule fraction. On the contrary, chronic treatment with the (-) enantiomer of oxaprotiline, which does not affect monoamine uptake, failed to produce this effect. Moreover, no changes were observed after acute or in vitro addition of antidepressants, suggesting that modification in the cAMP binding may be related to adaptive changes elicited by prolonged antidepressants treatment. In conclusion, our studies indicate that the cAMP binding protein associated with the crude microtubule fraction could be an intracellular target for the action of antidepressant drugs.

New insights into the biology of schizophrenia through the mechanism of action of clozapine.

Many studies have detected in the brain of schizophrenic patients various morphological and structural abnormalities in various regions and in particular in the cortical and limbic areas. These abnormalities might in part result from neurodevelopmental disturbances suggesting that schizophrenia might have organic causes. These abnormalities may be the primary event in schizophrenia and be responsible for altered dopaminergic, but not only dopaminergic, neurotransmission in these regions. If schizophrenia is in some way strictly related to brain morphological abnormalities it becomes hard to believe that a curative treatment will ever be possible. Considering this scenario, treatment of schizophrenia will be restricted to symptomatic and preventive therapy and therefore, more effective and better tolerated antipsychotics are necessary. The widely used classical antipsychotic drugs present some disadvantages. They do not improve all symptoms of schizophrenia, are not effective in all patients, produce a number of unpleasant and serious, and partly irreversible, motor side effects. The atypical antipsychotic clozapine constitutes a major advance in particular for patients not responding to conventional neuroleptics. To explain the unique therapeutic effect of clozapine many hypothesis have been proposed. Most of the explanations given so far assume that the D2 blockade is the basis for the antipsychotic activity of clozapine and that the difference in respect to other antipsychotics is due to the contribution of other receptor interactions. Considering the dopaminergic receptor, in particular the recently discovered D4 receptor subtype, it has been observed that even if several classical neuroleptics exhibit high affinity to the D4 receptor, clozapine is more selective for this subtype compared to D2 receptors. Moreover clozapine, differently from all other conventional neuroleptics, is a mixed but weak D1/D2 antagonist. This observation has prompted speculation that the synergism between D1 and D2 receptors might allow antipsychotic effects to be achieved below the threshold for unwanted motor side effects. Probably the D1 antagonistic activity exerted by clozapine at low doses enhances preferentially the extracellular concentration of dopamine in specific areas of the brain, such as the prefrontal cortex, where a dopaminergic hypoactivity has been suggested to be in part responsible for negative symptoms of schizophrenia. The clozapine enhancement of dopaminergic activity in this brain area might explain its efficacy against schizophrenia negative symptoms. However, it cannot be excluded that the affinities displayed by clozapine for other nondopaminergic receptors also contribute to its unique therapeutic profile. The various hypotheses mentioned in this review need to be further validated or disproved. The only way to do that is developing new drugs where the postulated mechanistic profile is specifically realized and to clinically test these compounds.

Regulation of ionotropic glutamate receptors in the rat brain in response to the atypical antipsychotic seroquel (quetiapine fumarate).

The interplay between dopamine and glutamate appears to be relevant in the etiopathology of schizophrenia. Although currently used antipsychotics do not interact with glutamatergic receptors, previous results have demonstrated that the expression profile of ionotropic glutamate receptors can be regulated by drugs such as haloperidol or clozapine. In the present investigation, the mRNA levels for NMDA and AMPA receptor subunits were measured after chronic treatment with the novel antipsychotic agent Seroquel (quetiapine fumarate, quetiapine) as compared to haloperidol and clozapine. Similarly to the prototype atypical clozapine, quetiapine reduced the mRNA expression for NR-1 and NR-2C, two NMDA forming subunits, in the nucleus accumbens. Furthermore, quetiapine, but not haloperidol or clozapine, increased the hippocampal expression for the AMPA subunits GluR-B and GluR-C. The differences between classical and atypical antipsychotics, as well as among the novel agents, might be relevant for specific aspects of their therapeutic activity and could provide valuable information for the role of glutamate in specific symptoms of schizophrenia.

Cyclic GMP inhibition of metabotropic glutamate receptor-induced phosphoinositide hydrolysis in mesencephalic neurons.

The effect of cGMP on metabotropic glutamate receptor-induced stimulation of phosphoinositide hydrolysis in mesencephalic neuronal cultures was evaluated by cell incubation with the stable analogue dibutyryl-cGMP (10 microM). A complete blockade of (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid- or quisqualate-induced inositol phosphate formation was observed. Ionotropic glutamate receptors in mesencephalic neurons activate cGMP formation and, through this intracellular messenger, they might control mGluR activity.

Effect of reboxetine treatment on brain cAMP- and calcium/calmodulin-dependent protein kinases.

Previous studies showed that the type II Ca(2+)/calmodulin- and cAMP-dependent protein kinases (CaMKII and PKA) are affected by long-term antidepressant treatment in presynaptic and somatodendritic compartments, respectively. This study describes the long-term effects of the selective noradrenaline reuptake inhibitor reboxetine on PKA and CaMKII, in both the microtubule and subsynaptosomal fractions of rat brain. Unlike other antidepressants, chronic reboxetine induced in the cerebrocortical soluble and microtubule fractions a decrease in the [(32)P]cAMP binding to the type II PKA regulatory subunit. No change in the cAMP-dependent endogenous phosphorylation of the protein substrate, microtubule-associated protein 2 was observed. In the hippocampal subsynaptosomal fractions (synaptic vesicles and synaptosomal membranes) reboxetine induced a robust increase in the activity but not in the expression of CaMKII. An increase in the calcium/calmodulin-dependent phosphorylation of presynaptic substrates was also detected. These findings showed that reboxetine modulates post-receptor signal transduction systems in rat brain.

Down-regulation of beta-adrenergic receptors following repeated injections of desmethylimipramine: permissive role of serotonergic axons.

The injection of desmethylimipramine (DMI) twice daily for 3 weeks reduced the density of beta-adrenergic receptor recognition sites located in crude synaptic membranes prepared from the cortex and hippocampus and attenuated the stimulation of the membrane-bound adenylate cyclase by isoproterenol. Both actions were abolished if prior to treatment with desmethylimipramine the serotonergic axons were destroyed by an intraventricular injection of 5,7-dihydroxytryptamine. These results show that the down-regulation of beta-adrenergic receptors elicited by repeated injections of desmethylimipramine occurs only if the serotonergic axons are intact.

On the mode of action of imipramine: relationship between serotonergic axon terminal function and down-regulation of beta-adrenergic receptors.

Recognition sites for [3H]imipramine and [3H]mianserin are located in different structures and regulate different neuronal functions. Recognition sites for [3H]imipramine are located on serotonergic terminals, are part of the supramolecular organization of the uptake mechanisms and can be down-regulated by prolonged administration of the drug. When the number of recognition sites for imipramine is down-regulated, uptake of 5-hydroxytryptamine (5HT) in rat brain hippocampal slices is increased. The presence of the binding sites for imipramine in 5HT terminals is essential to mediate the down-regulation of recognition sites for norepinephrine (NE) and NE-mediated stimulation of adenylate cyclase. Mianserin binds on a site that is modulated by 5HT, the number of its binding sites is not down-regulated by repeated treatment and, like imipramine, decreases the NE-dependent cyclase but not the number of beta-adrenergic receptor recognition sites. Repeated treatment with imipramine and mianserin down-regulated the number of 5HT2 recognition sites. Several lines of evidence indicate that binding site for mianserin is related but not identical to the 5HT2 receptor binding site.

Temporal sequence of changes in central noradrenergic system of rat after prolonged antidepressant treatment: receptor desensitization and neurotransmitter interactions.

It has been shown that different receptor components may be involved in the adaptive changes occurring in noradrenergic (NE) neurones after prolonged periods of exposure to antidepressant drugs. In this report the desensitization of NE-coupled adenylate cyclase (NE-AC). beta-adrenergic receptors and [3H]imipramine ([3H]-IMI) or [3H]desipramine ([3H]-DMI) binding sites have been temporally correlated with in vivo changes of NE utilization. Normetanephrine (NMN) was measured as indicator of NE synaptic events involved in antidepressant action. Concentrations of normetanephrine were increased after acute desipramine (DMI), viloxazine and mianserin administration. Following 3 days of treatment, the antidepressant-induced increase of normetanephrine became tolerant and NE neurones were resistant to the antidepressant effect until the 15th day of treatment. After two weeks, DMI elicited a significant decrease in the content of normetanephrine. A different pattern of changes has been found in the temporal modification of [3H]-IMI recognition sites, beta-adrenoceptors and NE-AC activity after chronic DMI treatment. Binding sites and receptors were down regulated after 10 days of treatment preceding the decrease in normetanephrine content. No down-regulation was observed in [3H]-DMI binding sites. Studies on the effects of antidepressants during brain maturation revealed that the mechanisms which cause desensitization of beta-receptors and [3H]-IMI binding sites appear in the early stages of postnatal life. Since [3H]-IMI and [3H]-DMI recognition sites have been shown to be located on serotonergic (5-HT) and noradrenergic neurones respectively, the interactions between NE and 5-HT neurones could represent possible mechanisms implicated in receptor desensitization. The experiments presented involving lesions of 5-HT neurones have clearly demonstrated that NE release in rat cerebral cortex is under a tonic serotonergic influence. Alterations in the chemico-physical properties of the synaptic membranes might be also taken in consideration for the mechanisms underlying receptor modulation. In fact, evidence is provided that in neural tissue phospholipid methylation can be affected. In conclusion, the temporal sequence of changes in cortical noradrenergic neurones, after chronic antidepressant treatment, has demonstrated that integrated mechanisms are operative for the function of the overall system.

Age-related variations in relative abundance of alternative spliced D2 receptor mRNAs in brain areas of two rat strains.

Age-related reduction in the steady-state levels of messenger RNA for D2(415) and D2(444), the alternatively spliced form of dopamine D2 receptors, was observed in different rat brain areas using the sensitive reverse transcription (RT)-polymerase chain reaction (PCR) technique. In both Sprague-Dawley and Wistar aged rats, the decrease was more pronounced in the D2(444) isoform mRNA thus leading to a changed ratio in striatum as well as in the hippocampus.

cAMP-dependent phosphorylation system after short and long-term administration of moclobemide.

Accumulating evidence suggested that signal transduction cascade including protein phosphorylation is implicated in the neurochemical action of antidepressant agents. Clinical data indicated that moclobemide, a short acting and reversible inhibitor of monoamino oxidase type. A, is an effective antidepressant medication. However, little is known about the intracellular effects of this compound. Thus, in the present study we assessed the binding of cAMP to cAMP-dependent protein kinase (PKA) in rat cerebral cortex following short and long-term administration of moclobemide. The results showed that 21 days of treatment with moclobemide significantly increased the specific [32P]-cAMP covalent binding into the soluble 52-54 kDa cAMP-receptor. This effect was not seen following 1, 5 and 12 days of treatment. These findings suggest that PKA could be implicated in the biochemical effects of moclobemide.