Elevated Expression of HSP72 in the Prefrontal Cortex and Hippocampus of Rats Subjected to Chronic Mild Stress and Treated with Imipramine.

The HSP70 and HSP90 family members belong to molecular chaperones that exhibit protective functions during the cellular response to stressful agents. We investigated whether the exposure of rats to chronic mild stress (CMS), a validated model of depression, affects the expression of HSP70 and HSP90 in the prefrontal cortex (PFC), hippocampus (HIP) and thalamus (Thal). Male Wistar rats were exposed to CMS for 3 or 8 weeks. The antidepressant imipramine (IMI, 10 mg/kg, i.p., daily) was introduced in the last five weeks of the long-term CMS procedure. Depressive-like behavior was verified by the sucrose consumption test. The expression of mRNA and protein was quantified by real-time PCR and Western blot, respectively. In the 8-week CMS model, stress alone elevated HSP72 and HSP90B mRNA expression in the HIP. HSP72 mRNA was increased in the PFC and HIP of rats not responding to IMI treatment vs. IMI responders. The CMS exposure increased HSP72 protein expression in the cytosolic fraction of the PFC and HIP, and this effect was diminished by IMI treatment. Our results suggest that elevated levels of HSP72 may serve as an important indicator of neuronal stress reactions accompanying depression pathology and could be a potential target for antidepressant strategy.

Spontaneous head twitches in aged rats: behavioral and molecular study.

RATIONALE: We have discovered that rats at the age of 18 months begin to twitch their heads spontaneously (spontaneous head twitching, SHT). To date, no one has described this phenomenon. OBJECTIVES: The purpose of this study was to characterize SHT pharmacologically and to assess some possible mechanisms underlying SHT. METHODS: Wistar male rats were used in the study. Animals at the age of 18 months were qualified as HSHT (SHT ≥ 7/10 min observations) or LSHT (SHT < 7/10 min observations). Quantitative real-time PCR with TaqMan low-density array (TLDA) approach was adopted to assess the mRNA expression of selected genes in rat's hippocampus. RESULTS: HSHT rats did not differ from LSHT rats in terms of survival time, general health and behavior, water intake, and spontaneous locomotor activity. 2,5-dimethoxy-4-iodoamphetamine (DOI) at a dose of 2.5 mg/kg increased the SHT in HSHT and LSHT rats, while ketanserin dose-dependently abolished the SHT in the HSHT rats. The SHT was reduced or abolished by olanzapine, clozapine, risperidone, and pimavanserin. All these drugs have strong 5-HT2A receptor-inhibiting properties. Haloperidol and amisulpride, as antipsychotic drugs with a mostly dopaminergic mechanism of action, did not influence SHT. Similarly, escitalopram did not affect SHT. An in-depth gene expression analysis did not reveal significant differences between the HSHT and the LSHT rats. CONCLUSIONS: SHT appears in some aging rats (about 50%) and is permanent over time and specific to individuals. The 5-HT2A receptor strongly controls SHT. HSHT animals can be a useful animal model for studying 5-HT2A receptor ligands.

Psychosocial Crowding Stress-Induced Changes in Synaptic Transmission and Glutamate Receptor Expression in the Rat Frontal Cortex.

This study demonstrates how exposure to psychosocial crowding stress (CS) for 3, 7, and 14 days affects glutamate synapse functioning and signal transduction in the frontal cortex (FC) of rats. CS effects on synaptic activity were evaluated in FC slices of the primary motor cortex (M1) by measuring field potential (FP) amplitude, paired-pulse ratio (PPR), and long-term potentiation (LTP). Protein expression of GluA1, GluN2B mGluR1a/5, VGLUT1, and VGLUT2 was assessed in FC by western blot. The body's response to CS was evaluated by measuring body weight and the plasma level of plasma corticosterone (CORT), adrenocorticotropic hormone (ACTH), and interleukin 1 beta (IL1B). CS 3 14d increased FP and attenuated LTP in M1, while PPR was augmented in CS 14d. The expression of GluA1, GluN2B, and mGluR1a/5 was up-regulated in CS 3d and downregulated in CS 14d. VGLUTs expression tended to increase in CS 7d. The failure to blunt the effects of chronic CS on FP and LTP in M1 suggests the impairment of habituation mechanisms by psychosocial stressors. PPR augmented by chronic CS with increased VGLUTs level in the CS 7d indicates that prolonged CS exposure changed presynaptic signaling within the FC. The CS bidirectional profile of changes in glutamate receptors' expression seems to be a common mechanism evoked by stress in the FC.

Imipramine administration induces changes in the phosphorylation of FAK and PYK2 and modulates signaling pathways related to their activity.

BACKGROUND: Antidepressants can modify neuronal functioning by affecting many levels of signal transduction pathways that are involved in neuroplasticity. We investigated whether the phosphorylation status of focal adhesion kinase (FAK/PTK2) and its homolog, PYK2/PTK2B, and their complex with the downstream effectors (Src kinase, p130Cas, and paxillin) are affected by administration of the antidepressant drug, imipramine. The treatment influence on the levels of ERK1/2 kinases and their phosphorylated forms (pERK1/2) or the Gαq, Gα11 and Gα12 proteins were also assessed. METHODS: Rats were injected with imipramine (10 mg/kg, twice daily) for 21 days. The levels of proteins investigated in their prefrontal cortices were measured by Western blotting. RESULTS: Imipramine induced contrasting changes in the phosphorylation of FAK and PYK2 at Tyr397 and Tyr402, respectively. The decreased FAK phosphorylation and increased PYK2 phosphorylation were reflected by changes in the levels of their complex with Src and p130Cas, which was observed predominantly after chronic imipramine treatment. Similarly only chronic imipramine decreased the Gαq expression while Gα11 and Gα12 proteins were untouched. Acute and chronic treatment with imipramine elevated ERK1 and ERK2 total protein levels, whereas only the pERK1 was significantly affected by the drug. CONCLUSION: The enhanced activation of PYK2 observed here could function as compensation for FAK inhibition. GENERAL SIGNIFICANCE: These data demonstrate that treatment with imipramine, which is a routine in counteracting depressive disorders, enhances the phosphorylation of PYK2, a non-receptor kinase instrumental in promoting synaptic plasticity. This effect documents as yet not considered target in the mechanism of imipramine action.

Brief maternal separation affects brain α1-adrenoceptors and apoptotic signaling in adult mice.

Exposure to adversity during early life is a risk factor for the development of different mood and psychiatric disorders, including depressive-like behaviors. Here, neonatal mice were temporarily but repeatedly (day 1 to day 13) separated from mothers and placed in a testing environment containing a layer of odorless clean bedding (CB). We assessed in adult animals the impact of this early experience on binding sites and mRNA expression of α1-adrenergic receptor subtypes, heat shock proteins (HSPs) and proapoptotic and antiapoptotic members of the Bcl-2 family proteins in different brain regions involved in processing of olfactory information and rewarding stimuli. We found that repeated exposure to CB experience produced anhedonic-like behavior in terms of reduced saccharin intake and α1-adrenoceptor downregulation in piriform and somatosensory cortices, hippocampus, amygdala and discrete thalamic nuclei. We also found a selective decrease of α1B-adrenoceptor binding sites in the cingulate cortex and hippocampus and an increase of hippocampal α1A and α1B receptor, but not of α1D-adrenoceptor, mRNA levels. Moreover, while a significant decrease of antiapoptotic heat shock proteins Hsp72 and Hsp90 was identified in the prefrontal cortex, a parallel increase of antiapoptotic members of Bcl-2 family proteins was found at the hippocampal level. Together, these data provide evidence that the early exposure to CB experience produced enduring downregulation of α1-adrenoceptors in the prefrontal-limbic forebrain/limbic midbrain network, which plays a key role in the processing of olfactory information and reaction to rewarding stimuli. Finally, these data show that CB experience can "prime" the hippocampal circuitry and promote the expression of antiapoptotic factors that can confer potential neuroprotection to subsequent adversity.

α1-Adrenergic receptor subtypes in the central nervous system: insights from genetically engineered mouse models.

α1-Adrenergic receptors (α1-ARs) are important players in peripheral and central nervous system (CNS) regulation and function and in mediating various behavioral responses. The α1-AR family consists of three subtypes, α1A, α1B and α1D, which differ in their subcellular distribution, efficacy in evoking intracellular signals and transcriptional profiles. All three α1-AR subtypes are present at relatively high densities throughout the CNS, but the contributions of the individual subtypes to various central functions are currently unclear. Because of the lack of specific ligands, functionally characterizing the α1-ARs and discriminating between the three subtypes are difficult. To date, studies using genetically engineered mice have provided some information on subtype-related functions of the CNS α1-ARs. In this mini-review, we discuss several CNS processes where the α1-ARs role has been delineated with pharmacological tools and by studies using mutated mice strains that infer specific α1-AR subtype functions through evaluation of behavioral phenotypes.

Morphine-induced place preference affects mRNA expression of G protein α subunits in rat brain.

BACKGROUND: The conditioned place preference (CPP) test is an animal model serving to assess addictive potential of drugs in which environmental cues become associated with the subjective effects of drugs of abuse. Morphine, a known addictive drug, is an agonist of opioid receptors that couple to the G(i/o) family of guanine nucleotide-binding proteins (GP). We have recently found that chronic treatment with morphine affects mRNA levels of GPs that are not coupled to opioid receptors (OR). Therefore, in this study, we investigated the influence of morphine-induced CPP on mRNA expression of the Gα subunits, G(i/o), G(s), G(q/11), and G(12), in the rat prefrontal cortex (PFC) and nucleus accumbens (NAc) using standard PCR techniques. METHODS: CPP and NO-CPP experiments were conducted; Wistar rats were either subjected to the standard CPP procedure or were injected with morphine (or saline) in their home cage. All rats were decapitated 24 h after the last injection. RESULTS: We found that mRNA levels of Gα(q), Gα(11) and Gα(12) were increased after morphine in non-conditioned treatment in the PFC but remained unchanged in the NAc. In rats showing conditioned place preference to morphine, levels of Gα(i2) in the PFC and levels of Gα(oA) in the NAc were diminished by ≈58% and ≈30%, respectively (p < 0.05 vs. saline), but levels of Gα(s-l) in NAc were increased (≈60%, p = 0.05). CONCLUSION: Our data indicate that only G(i/o) and G(s) were specifically changed in animals after morphine-induced CPP, thus suggesting that the effect was related to learning environmental cues associated with morphine.

Effects of the noradrenergic neurotoxin DSP-4 on the expression of α1-adrenoceptor subtypes after antidepressant treatment.

We have previously reported that chronic imipramine and electroconvulsive treatments increase the α(1A)-adrenoceptor (but not the α(1B) subtype) mRNA level and the receptor density in the rat cerebral cortex. Furthermore, we have also shown that chronic treatment with citalopram does not affect the expression of either the α(1A)- or the α(1B)-adrenoceptor, indicating that the previously observed up-regulation of α(1A)-adrenoceptor may depend on the noradrenergic component of the pharmacological mechanism of action of these antidepressants. Here, we report that previous noradrenergic depletion with DSP-4 (50 mg/kg) (a neurotoxin selective for the noradrenergic nerve terminals) significantly attenuated the increase of α(1A)-adrenoceptor mRNA induced by a 14-day treatment with imipramine (IMI, 20 mg/kg, ip) and abolished the effect of electroconvulsive shock (ECS, 150 mA, 0.5 s) in the prefrontal cortex of the rat brain. The changes in the receptor protein expression (as reflected by its density) that were induced by IMI and ECS treatments were differently modulated by DSP-4 lesioning, and only the ECS-induced increase in α(1A)-adrenoceptor level was abolished. This study provides further evidence corroborating our initial hypothesis that the noradrenergic component of the action of antidepressant agents plays an essential role in the modulation of α(1A)-adrenoceptor in the rat cerebral cortex.

Effects of morphine and methadone treatment on mRNA expression of Gα(i) subunits in rat brains.

Methadone is clinically effective as substitution therapy in patients with opioid dependence. The diversity of methadone and morphine in their intracellular activity is postulated. We compared the effects of repeated daily treatment of Sprague-Dawley rats with morphine (10 mg/kg) and methadone (1 mg/kg) on the expression of the Gα(i1-i3) mRNAs in several rat brain areas using RT-qPCR. We found that both opioid receptor agonists decreased Gα(i3) mRNA in only the nucleus accumbens. Although there was no difference in the influence of morphine and methadone on Gα(i), our results indicate that among Gα(i) subunits, the Gα(i3) is specifically involved in the mechanism of action of both drugs.

Cryptic peptide derived from the rat neuropeptide FF precursor affects G-proteins linked to opioid receptors in the rat brain.

Recently, we reported the discovery of a novel amino acid sequence derived from the NPFF precursor NAWGPWSKEQLSPQA, which blocked the expression of conditioned place preference induced by morphine and reversed the antinociceptive activity of morphine (5mg/kg, s.c.) in the tail-immersion test in rats. Here, we name it as NPNA (Neuropeptide NA from its flanking amino acid residues). The synthetic peptide influenced the expression of mRNA coding for Galpha(i1), (i2), and (i3) subunits. The results provide further evidence that yet another bioactive sequence might be present within the NPFF precursor.

Effect of cocaine on responsiveness of alpha(1)-adrenergic receptors in rat cerebral cortex: modulation by GABA-mimetic drugs.

We investigated the effects of single doses of cocaine (10 mg/kg, ip) and the gamma-aminobutyric acid (GABA)-mimetics tiagabine (10 mg/kg, ip) and vigabatrin (150 mg/kg, ip) injected separately or concomitantly with cocaine, on the responsiveness of cerebral cortical alpha(1)-adrenergic receptors. The accumulation of noradrenaline-stimulated inositol phosphates was estimated in vitro at 2 and 24 h after the drug injection. Cocaine significantly enhanced alpha(1)-adrenergic receptor responsiveness to noradrenaline. Neither tiagabine nor vigabatrin influenced the accumulation of inositol phosphates. Finally, the cocaine-evoked augmentation of alpha(1)-adrenoceptor responsiveness was counteracted by tiagabine but not by vigabatrin. This effect may represent a characteristic feature of tiagabine, not necessarily shared by other GABA-mimetic drugs.

Does the presence of morphine counteract adaptive changes in expression of G-protein alpha subunits mRNA induced by chronic morphine treatment?

Opiate dependence develops due to changes in intracellular signaling caused by long-term exposure to morphine. Here we investigated changes in the mRNA expression of the main classes of G-protein alpha (Galpha) subunits in various brain regions in morphine-dependent rats. Rats received increasing doses of morphine, 10-50 mg/kg, b.i.d., for 14 days. G-protein alpha-subunit mRNA expression was determined shortly following the conclusion of chronic morphine administration (2 h after the final dose) and during withdrawal (48 h after the final dose). Significant changes in mRNA expression for Galpha subunits were observed in several brain areas during withdrawal, while the changes were much less evident or absent 2 h after the final drug injection. Changes in mRNA expression were particularly evident in the nucleus accumbens (increases in Galpha(12), Galpha(q), Galpha(11), and Galpha(o) during withdrawal, increase in Galpha(i) and decrease in Galpha(s) just following treatment). The direction of the changes, which were not all significant, for Galpha(12), Galpha(q), and Galpha(11) was generally consistent in the amygdala and prefrontal cortex; changes in G proteins coupled to the adenylyl cyclase cascade were less consistent. These results suggest that morphine dependence leads to alterations in intracellular signaling, which are reflected in changes in the expression of genes encoding various G proteins. The results may explain why signs of opiate dependence are not expressed during chronic administration of morphine, but only after cessation of the treatment.

Chronic treatment with citalopram does not affect the expression of alpha1-adrenergic receptor (alpha1-AR) subtypes.

We previously reported that chronic treatment with imipramine and electroconvulsive shock up-regulate the density and alpha1A-adrenergic receptor (alpha1A-AR) mRNA level in the rat prefrontal cortex, while the expression of the alpha1B subtype was unchanged. The present study examined whether repeatedly given citalopram, a selective serotonin reuptake inhibitor, induces any changes in the expression of alpha1A and alpha1B subtypes of alpha1-AR. The receptors density was assessed in the rat cerebral cortex by [3H]prazosin binding while the expression of alpha1A and alpha1B receptors' mRNA was measured in the rat prefrontal cortex by Northern blot analysis or competitive reverse transcription and polymerase chain reaction (RT-PCR), respectively. We did not find any changes in alpha1A- and alpha1B-AR density or mRNA expression in the investigated rat brain structures of citalopram-treated rats. Thus, it seems that up-regulation of alpha1A-AR subtype is characteristic only of those antidepressant agents in which a noradrenergic component is involved in their pharmacological mechanism of action.