Conserved Hypothalamic c-Fos Activation Pattern Induced by the mGlu5 Receptor Antagonist MPEP during Peri-pubertal Development in Mice

• Abstract
• Introduction
• Materials and Methods
• Animals
• Cell counting and statistical analysis
• Results
• Discussion
• References

Abstract

Introduction: 2-Methyl-6-(phenylethynyl)pyridine (MPEP) is a selective mGlu5 receptor (mGluR5) antagonist intensively studied as potential novel anxiolytic drug. In the adult, MPEP activates stress-related areas, including the paraventricular nucleus of the hypothalamus (PVNh). However, it is unknown if MPEP targets similar structures in the juvenile brain as well.

Methods: Here we examined by immunohistochemical methods the induction pattern of the neuronal activity marker c-Fos by MPEP at peri-pubertal stages (postnatal day P16, P24, P32 and P40) in C57BL6/N mice.

Results: Despite the previously reported sharply diminished hypothalamic mGluR5 expression during postnatal development, we found a highly conserved PVNh activation by MPEP together with c-Fos expression in the extended amygdala. Interestingly, MPEP also robustly activated the paraventricular nucleus of the thalamus (PVT) and suprachiasmatic nucleus (SCN), regions associated with the modulation of circadian rhythms.

Discussion: These results indicate a conserved activation pattern induced by MPEP in the young vs. adult brain especially in brain areas regulating stress and circadian rhythms and may be of importance regarding the effect of mGluR5 antagonists in the treatment of mood disorders during juvenile development.

Introduction

The selective antagonist of the metabotropic mGluR5 MPEP [1] is intensively studied as an alternative anxiolytic drug that lacks the side effects of benzodiazepines [2]. Its mechanism of action is only partly understood. Profiling of the expression of the immediate early gene c-Fos as a marker of neuronal activity [3] and pathology [4] [5] [6] represents a valid tool in the classification of psychoactive substances [7]. Drugs belonging to the same therapeutic group (anxiolytics, antidepressants or antipsychotics) induce c-Fos in very similar areas, respectively, even if they bind to different classes of receptors [7]. Accordingly, anxiolytic drugs induce c-Fos expression only in a few (stress-related) brain regions, especially in the PVNh [8]. Antidepressants activate the central amygdala (CeA) and the bed nucleus of stria terminalis (BNST) that together form the extended amygdala [9] [10]. Interestingly MPEP, which elicits both anxiolytic and antidepressant effects [11] [12], triggers specific c-Fos expression in all 3 of these regions [13].

Up-to-date c-Fos profiling of psychoactive agents is limited to adult stages. However, similar analyses during development would be important to understand the neural substrates of therapeutic action in the juvenile brain since peri-pubertal stages represent a period of vulnerability for developing affective disorders. mGluR5 shows important variations in its distribution during postnatal development [14] [15]. High mGluR5 expression occurs postnatally especially in the cortex, striatum, hippocampus, cerebellum and hypothalamus and decreases substantially towards very low/absent levels in the adult brain [14]. Considering this, it appears not surprising that MPEP activates only a few regions in the adult brain. However, since mGluR5 expression is significantly higher during development, it would be interesting to determine the MPEP-triggered c-Fos expression pattern in the young brain. To address this issue we compared the distribution of c-Fos expression after acute treatment with MPEP at several time points from P16 to P40.

Materials and Methods

Animals

Experiments were performed using C57BL6/N mice purchased from Charles River (Sulzfeld, Germany). Pups of each group were analyzed at different time points (P16, P24, P32, P40). These time points were chosen in a way that they comprise both pre- and post-pubertal stages, considering that the onset of puberty in mice is around P30 [16]. We therefore selected 2 representative time points before and 2 time points after P30. We followed the time points used by other authors for analysis of brain development during this period: P16-P23, P24-P31, P32-P39 [17]. Animals were supplied with food and water ad libitum as described earlier [18] . All efforts were made to minimise animal suffering and to reduce the number of animals used. All experiments were approved by the German Committee on Animal Care and Use and were carried out in accordance with the local Animal Welfare Act and the European Communities Council Directive 2010/63/EU.

Drug treatment and immunohistochemistry

Mice (n=6 for each treatment) were injected with either vehicle (0.9% NaCl, 5 ml/kg) or MPEP (30 mg/kg, i.p.) (Abcam, Germany). We did not notice any obvious behavioral abnormality using this dose of MPEP at peri-pubertal stages. Of note was that this dose of MPEP was previously used without inducing deleterious side effects even in younger mice (P7) treated chronically for 3–5 days [19]. In a previous c-Fos brain mapping study done at adult stages, we found that higher doses of MPEP than used in behavioral studies are needed for robust c-Fos induction [13]. After 2 h, animals were anesthetized with pentobarbital and transcardially perfused with 4% paraformaldehyde. Brains were removed and postfixed for 24 h, as described previously [20] [21]. 50 µm free-floating coronal sections were washed in 0.1 M phosphate buffer saline containing 0.3% Triton X-100 (PBST), pH 7.4, immersed in 0.3% hydrogen peroxidase for 20 min, and washed again before being incubated with the primary anti-c-Fos (polyclonal rabbit; Calbiochem, La Jolla, CA, diluted 1:10 000) antibody in PBST for 24 h at 4°C. After washes with PBST, sections were incubated with secondary antibody (biotinylated anti-rabbit IgG antibody, Vector laboratories), diluted 1:400 in PBST with 4% normal goat serum for 2 h at room temperature (RT) and the reaction was visualized using nickel-3,3'-diaminobenzidine (DAB), as described previously [22].

Cell counting and statistical analysis

Cell counting was performed blind to the treatment using a light microscope (LEICA TCS-NT). Quantification of c-Fos immunoreactive cells was performed at × 40 magnification in the regions where we found high MPEP-triggered c-Fos expression: PVNh, BNST, CeA, lateral septum (LS), paraventricular nucleus of the thalamus (PVT) and suprachiasmatic nucleus (SCN), as described previously [13]. The statistical analysis was performed using the statistical program PASW 23 for Windows. In all experiments, the mean number of c-Fos-expressing cells (±SEM) was estimated in 6 animals for each treatment group, and differences between groups were determined by one-way ANOVA followed by a Bonferroni post-test comparison of means, with p<0.05 considered statistically significant.

Results

As in adult animals, we found a highly specific MPEP-induced c-Fos expression pattern restricted to only few brain regions. In contrast, only very few scattered c-Fos-positive cells were found in control animals injected with saline (data not shown). At all time points investigated, high c-Fos expression was detected in the PVNh after MPEP treatment ([Fig. 1a–d]). The quantification of c-Fos-expressing cells revealed a comparable expression level at all ages investigated, i. e., from P16 to P40 ([Fig. 1e]). In contrast to the situation in the adult, we found rather low levels of MPEP-induced c-Fos expression in the extended amygdala, but increasing expression levels during development from P16 to P40 as quantified both in the CeA ([Fig. 1f]) and BNST ([Fig. 1g]). In addition, we also detected a significant c-Fos expression in the LS ([Fig. 1h]). Interestingly, we found that in the CeA, BNST and LS c-Fos expression increased from P16 to P24 followed by a significant decrease at P32 ([Fig. 1f–h]). Besides the PVNh, other brain areas with high accumulations of MPEP-induced c-Fos expression were the PVT ([Fig. 2a–c]) and the SCN ([Fig. 2d]). Compared to all these regions, only low to absent c-Fos expression was visible in neocortical and hippocampal areas (data not shown).

Discussion

Here we show that the mGluR5 antagonist MPEP elicits a highly specific neuronal activation pattern during peri-pubertal development. To our knowledge, the present study represents the first systematic investigation of the neuronal activation pattern elicited by a substance with anxiolytic properties at this developmental stage.

We found a robust and conserved c-Fos induction in the PVNh. This is important because even in the adult the PVNh represents the main brain area activated by anxiolytics, both by MPEP [13] and benzodiazepines [8]. The PVNh plays a crucial role in the regulation of the HPA axis by secretion of the corticotropin-releasing hormone (CRH) [23]. MPEP increases corticosterone concentrations by a yet unknown mechanism [24]. It was proposed that mGluR5 antagonists disinhibit CRH-releasing neurons in the PVNh acting on local GABAergic interneurons [25]. However, the MPEP-induced activation of the PVNh instead appears to be the result of local network effects, since mGluR5, although expressed in several other hypothalamic nuclei, was not found in the PVNh [26]. The high c-Fos induction in the PVNh both in the young and adult brain is also intriguing considering the sixfold decrease in hypothalamic mGluR5 expression from postnatal to adult ages [15]. Future studies may clarify the mechanisms underlying the developmentally conserved PVNh activation; one possibility is that putative differences in the excitatory/inhibitory drive of the PVNh during development may compensate the effect of the concomitant mGluR5 downregulation.

In contrast to adult stages, we found relatively low MPEP-triggered c-Fos induction in other areas involved in the regulation of anxiety and stress like the extended amygdala [27]. The reason for the different activation of the PVNh vs. CeA/BNST by MPEP is unknown. Of relevance in this context could be the phenotypic difference between CRH-releasing neurons in the PVNh and BNST, with the first being glutamatergic and the latter GABAergic [28]. Additionally, we found MPEP-triggered c-Fos expression in the LS, which also plays an important modulatory role in the stress response and stress-induced persistent anxiety [29] [30]. Interestingly, the c-Fos expression in the extended amygdala and LS showed significant developmental variations with a sharp decrease (U-shape) around puberty initiation (P30). The reason for these differences is not known.

Finally, we found strong and conserved MPEP-induced activation in the PVT and SCN in addition to the PVNh. The PVT is strongly interconnected with the amygdala and the BNST and participates in stress and mood regulation [31]. On the other hand, the PVT relays circadian timing information from the SCN, also receiving inputs from the retina [32]. The specific effect of MPEP on these nuclei appears interesting, considering that mGluR5 regulates circadian activity rhythms [33] [34]. The relevance of the present findings for effects on circadian rhythms specifically during puberty needs to be unraveled by future investigations.

In conclusion, our results indicate a conserved activation pattern induced by MPEP in the juvenile brain, especially in brain areas regulating stress and circadian rhythms. These data may be of importance regarding the effect of mGluR5 antagonists in the treatment of mood disorders during juvenile development. To our knowledge, the anxiolytic and/or antidepressant effect of MPEP was not yet investigated at these stages. Of note, age-dependent differential effects of mGluR5 on anxiety vs. mood were shown previously: mice lacking mGluR5 display a sharp increase in anxiety, but no depression-like phenotype during aging [35]. Therefore, future studies should evaluate possible age-specific differences in the anxiolytic/antidepressive effect of MPEP in animal models and humans.

Conflict of Interest

The authors declare no conflict of interest.

Acknowledgements

This work was supported by an Olympia-Morata grant of the University of Heidelberg to I.I., and grants from the Deutsche Forschungsgemeinschaft (GA427/11-1) and Collaborative Research Center (Sonderforschungsbereich) 636 of the University of Heidelberg to P.G.

• References

• 1 Gasparini F, Lingenhohl K, Stoehr N et al. 2-Methyl-6-(phenylethynyl)-pyridine (MPEP), a potent, selective and systemically active mGlu5 receptor antagonist. Neuropharmacology 1999; 38: 1493-1503
  CrossrefPubMedSearch in Google Scholar
• 2 Tatarczynska E, Klodzinska A, Chojnacka-Wojcik E et al. Potential anxiolytic- and antidepressant-like effects of MPEP, a potent, selective and systemically active mGlu5 receptor antagonist. Br J Pharmacol 2001; 132: 1423-1430
  CrossrefPubMedSearch in Google Scholar
• 3 Sagar SM, Sharp FR, Curran T. Expression of c-fos protein in brain: metabolic mapping at the cellular level. Science 1988; 240: 1328-1331
  CrossrefPubMedSearch in Google Scholar
• 4 Kiessling M, Gass P. Immediate early gene expression in experimental epilepsy. Brain Pathol 1993; 3: 381-393
  CrossrefPubMedSearch in Google Scholar
• 5 Kiessling M, Gass P. Stimulus-transcription coupling in focal cerebral ischemia. Brain Pathol 1994; 4: 77-83
  PubMedSearch in Google Scholar
• 6 Gass P, Herdegen T. Neuronal expression of AP-1 proteins in excitotoxic-neurodegenerative disorders and following nerve fiber lesions. Prog Neurobiol 1995; 47: 257-290
  CrossrefPubMedSearch in Google Scholar
• 7 Sumner BE, Cruise LA, Slattery DA et al. Testing the validity of c-fos expression profiling to aid the therapeutic classification of psychoactive drugs. Psychopharmacology (Berl) 2004; 171: 306-321
  CrossrefPubMedSearch in Google Scholar
• 8 Salminen O, Lahtinen S, Ahtee L. Expression of Fos protein in various rat brain areas following acute nicotine and diazepam. Pharmacol Biochem Behav 1996; 54: 241-248
  CrossrefPubMedSearch in Google Scholar
• 9 Beck CH. Acute treatment with antidepressant drugs selectively increases the expression of c-fos in the rat brain. J Psychiatry Neurosci 1995; 20: 25-32
  PubMedSearch in Google Scholar
• 10 Morelli M, Pinna A, Ruiu S et al. Induction of Fos-like-immunoreactivity in the central extended amygdala by antidepressant drugs. Synapse 1999; 31: 1-4
  CrossrefPubMedSearch in Google Scholar
• 11 Li X, Need AB, Baez M et al. Metabotropic glutamate 5 receptor antagonism is associated with antidepressant-like effects in mice. J Pharmacol Exp Ther 2006; 319: 254-259
  CrossrefPubMedSearch in Google Scholar
• 12 Belozertseva IV, Kos T, Popik P et al. Antidepressant-like effects of mGluR1 and mglu5 receptor antagonists in the rat forced swim and the mouse tail suspension tests. Eur Neuropsychopharmacol 2007; 17: 172-179
  CrossrefPubMedSearch in Google Scholar
• 13 Inta D, Filipovic D, Lima-Ojeda JM et al. The mGlu5 receptor antagonist MPEP activates specific stress-related brain regions and lacks neurotoxic effects of the NMDA receptor antagonist MK-801: significance for the use as anxiolytic/antidepressant drug. Neuropharmacology 2012; 62: 2034-2039
  CrossrefPubMedSearch in Google Scholar
• 14 Catania MV, Landwehrmeyer GB, Testa CM et al Metabotropic glutamate receptors are differentially regulated during development. Neuroscience 1994; 61: 481-495
  CrossrefPubMedSearch in Google Scholar
• 15 Casabona G, Knopfel T, Kuhn R et al. Expression and coupling to polyphosphoinositide hydrolysis of group I metabotropic glutamate receptors in early postnatal and adult rat brain. Eur J Neurosci 1997; 9: 12-17
  CrossrefPubMedSearch in Google Scholar
• 16 Clarkson J, Boon WC, Simpson ER et al. Postnatal development of an estradiol-kisspeptin positive feedback mechanism implicated in puberty onset. Endocrinology 2009; 150: 3214-3220
  CrossrefPubMedSearch in Google Scholar
• 17 Insanally MN, Albanna BF, Bao S. Pulsed noise experience disrupts complex sound representations. J Neurophysiol 2010; 103: 2611-2617
  CrossrefPubMedSearch in Google Scholar
• 18 Chourbaji S, Zacher C, Sanchis-Segura C et al. Social and structural housing conditions influence the development of a depressive-like phenotype in the learned helplessness paradigm in male mice. Behav Brain Res 2005; 164: 100-106
  CrossrefPubMedSearch in Google Scholar
• 19 Cruz-Martín A, Crespo M, Portera-Cailliau C. Delayed stabilization of dendritic spines in fragile X mice. J Neurosci 2010; 30: 7793-7803
  CrossrefPubMedSearch in Google Scholar
• 20 Herdegen T, Blume A, Buschmann T et al. Expression of ATF-2, SRF and CREB in the adult rat brain following generalized seizures, nerve fiber lesions and ultraviolet irradiation. Neuroscience 1997; 81: 199-212
  CrossrefPubMedSearch in Google Scholar
• 21 Bisler S, Schleicher A, Gass P et al. Expression of c-Fos, ICER, Krox-24 and JunB in the whisker-to-barrel pathway of rats: time course of induction upon whisker stimulation by tactile exploration of an enriched environment. J Chem Neuroanat 2002; 23: 187-98
  CrossrefPubMedSearch in Google Scholar
• 22 Gass P, Prior P, Kiessling M. Correlation between seizure intensity and stress protein expression after limbic epilepsy in the rat brain. Neuroscience 1995; 65: 27-36
  CrossrefPubMedSearch in Google Scholar
• 23 Filipovic D, Zlatkovic J, Inta D et al. Chronic isolation stress predisposes the frontal cortex but not the hippocampus to the potentially detrimental release of cytochrome c from mitochondria and the activation of caspase-3. J Neurosci Res 2011; 89: 1461-14670
  CrossrefPubMedSearch in Google Scholar
• 24 Johnson MP, Kelly G, Chamberlain M. Changes in rat serum corticosterone after treatment with metabotropic glutamate receptor agonists or antagonists. J Neuroendocrinol 2001; 13: 670-677
  CrossrefPubMedSearch in Google Scholar
• 25 Radley JJ, Sawchenko PE. A common substrate for prefrontal and hippocampal inhibition of the neuroendocrine stress response. J Neurosci 2011; 31: 9683-9695
  CrossrefPubMedSearch in Google Scholar
• 26 Scaccianoce S, Matrisciano F, Del Bianco P et al. Endogenous activation of group-II metabotropic glutamate receptors inhibits the hypothalamic-pituitary-adrenocortical axis. Neuropharmacology 2003; 44: 555-561
  CrossrefPubMedSearch in Google Scholar
• 27 Davis M, Walker DL, Miles L et al. Phasic vs sustained fear in rats and humans: role of the extended amygdala in fear vs anxiety. Neuropsychopharmacology 2010; 35: 105-135
  CrossrefPubMedSearch in Google Scholar
• 28 Dabrowska J, Hazra R, Guo JD et al. Central CRF neurons are not created equal: phenotypic differences in CRF-containing neurons of the rat paraventricular hypothalamus and the bed nucleus of the stria terminalis. Front Neurosci 2013; 7: 156
  PubMedSearch in Google Scholar
• 29 Singewald GM, Rjabokon A, Singewald N et al. The modulatory role of the lateral septum on neuroendocrine and behavioral stress responses. Neuropsychopharmacology 2011; 36: 793-804
  CrossrefPubMedSearch in Google Scholar
• 30 Anthony TE, Dee N, Bernard A et al. Control of stress-induced persistent anxiety by an extra-amygdala septohypothalamic circuit. Cell 2014; 156: 522-536
  CrossrefPubMedSearch in Google Scholar
• 31 Hsu DT, Kirouac GJ, Zubieta JK et al. Contributions of the paraventricular thalamic nucleus in the regulation of stress, motivation, and mood. Front Behav Neurosci 2014; 8: 73
  PubMedSearch in Google Scholar
• 32 Moga MM, Weis RP, Moore RY. Efferent projections of the paraventricular thalamic nucleus in the rat. J Comp Neurol 1995; 359: 221-238
  CrossrefPubMedSearch in Google Scholar
• 33 Park D, Lee S, Jun K et al. Translation of clock rhythmicity into neural firing in suprachiasmatic nucleus requires mGluR-PLCbeta4 signaling. Nat Neurosci 2003; 6: 337-338
  CrossrefPubMedSearch in Google Scholar
• 34 Gannon RL, Millan MJ. Positive and negative modulation of circadian activity rhythms by mGluR5 and mGluR2/3 metabotropic glutamate receptors. Neuropharmacology 2011; 60: 209-215
  CrossrefPubMedSearch in Google Scholar
• 35 Inta D, Vogt MA, Luoni A et al. Significant increase in anxiety during aging in mGlu5 receptor knockout mice. Behav Brain Res 2013; 241C: 27-31
  PubMedSearch in Google Scholar

Correspondence

• References

• 1 Gasparini F, Lingenhohl K, Stoehr N et al. 2-Methyl-6-(phenylethynyl)-pyridine (MPEP), a potent, selective and systemically active mGlu5 receptor antagonist. Neuropharmacology 1999; 38: 1493-1503
  CrossrefPubMedSearch in Google Scholar
• 2 Tatarczynska E, Klodzinska A, Chojnacka-Wojcik E et al. Potential anxiolytic- and antidepressant-like effects of MPEP, a potent, selective and systemically active mGlu5 receptor antagonist. Br J Pharmacol 2001; 132: 1423-1430
  CrossrefPubMedSearch in Google Scholar
• 3 Sagar SM, Sharp FR, Curran T. Expression of c-fos protein in brain: metabolic mapping at the cellular level. Science 1988; 240: 1328-1331
  CrossrefPubMedSearch in Google Scholar
• 4 Kiessling M, Gass P. Immediate early gene expression in experimental epilepsy. Brain Pathol 1993; 3: 381-393
  CrossrefPubMedSearch in Google Scholar
• 5 Kiessling M, Gass P. Stimulus-transcription coupling in focal cerebral ischemia. Brain Pathol 1994; 4: 77-83
  PubMedSearch in Google Scholar
• 6 Gass P, Herdegen T. Neuronal expression of AP-1 proteins in excitotoxic-neurodegenerative disorders and following nerve fiber lesions. Prog Neurobiol 1995; 47: 257-290
  CrossrefPubMedSearch in Google Scholar
• 7 Sumner BE, Cruise LA, Slattery DA et al. Testing the validity of c-fos expression profiling to aid the therapeutic classification of psychoactive drugs. Psychopharmacology (Berl) 2004; 171: 306-321
  CrossrefPubMedSearch in Google Scholar
• 8 Salminen O, Lahtinen S, Ahtee L. Expression of Fos protein in various rat brain areas following acute nicotine and diazepam. Pharmacol Biochem Behav 1996; 54: 241-248
  CrossrefPubMedSearch in Google Scholar
• 9 Beck CH. Acute treatment with antidepressant drugs selectively increases the expression of c-fos in the rat brain. J Psychiatry Neurosci 1995; 20: 25-32
  PubMedSearch in Google Scholar
• 10 Morelli M, Pinna A, Ruiu S et al. Induction of Fos-like-immunoreactivity in the central extended amygdala by antidepressant drugs. Synapse 1999; 31: 1-4
  CrossrefPubMedSearch in Google Scholar
• 11 Li X, Need AB, Baez M et al. Metabotropic glutamate 5 receptor antagonism is associated with antidepressant-like effects in mice. J Pharmacol Exp Ther 2006; 319: 254-259
  CrossrefPubMedSearch in Google Scholar
• 12 Belozertseva IV, Kos T, Popik P et al. Antidepressant-like effects of mGluR1 and mglu5 receptor antagonists in the rat forced swim and the mouse tail suspension tests. Eur Neuropsychopharmacol 2007; 17: 172-179
  CrossrefPubMedSearch in Google Scholar
• 13 Inta D, Filipovic D, Lima-Ojeda JM et al. The mGlu5 receptor antagonist MPEP activates specific stress-related brain regions and lacks neurotoxic effects of the NMDA receptor antagonist MK-801: significance for the use as anxiolytic/antidepressant drug. Neuropharmacology 2012; 62: 2034-2039
  CrossrefPubMedSearch in Google Scholar
• 14 Catania MV, Landwehrmeyer GB, Testa CM et al Metabotropic glutamate receptors are differentially regulated during development. Neuroscience 1994; 61: 481-495
  CrossrefPubMedSearch in Google Scholar
• 15 Casabona G, Knopfel T, Kuhn R et al. Expression and coupling to polyphosphoinositide hydrolysis of group I metabotropic glutamate receptors in early postnatal and adult rat brain. Eur J Neurosci 1997; 9: 12-17
  CrossrefPubMedSearch in Google Scholar
• 16 Clarkson J, Boon WC, Simpson ER et al. Postnatal development of an estradiol-kisspeptin positive feedback mechanism implicated in puberty onset. Endocrinology 2009; 150: 3214-3220
  CrossrefPubMedSearch in Google Scholar
• 17 Insanally MN, Albanna BF, Bao S. Pulsed noise experience disrupts complex sound representations. J Neurophysiol 2010; 103: 2611-2617
  CrossrefPubMedSearch in Google Scholar
• 18 Chourbaji S, Zacher C, Sanchis-Segura C et al. Social and structural housing conditions influence the development of a depressive-like phenotype in the learned helplessness paradigm in male mice. Behav Brain Res 2005; 164: 100-106
  CrossrefPubMedSearch in Google Scholar
• 19 Cruz-Martín A, Crespo M, Portera-Cailliau C. Delayed stabilization of dendritic spines in fragile X mice. J Neurosci 2010; 30: 7793-7803
  CrossrefPubMedSearch in Google Scholar
• 20 Herdegen T, Blume A, Buschmann T et al. Expression of ATF-2, SRF and CREB in the adult rat brain following generalized seizures, nerve fiber lesions and ultraviolet irradiation. Neuroscience 1997; 81: 199-212
  CrossrefPubMedSearch in Google Scholar
• 21 Bisler S, Schleicher A, Gass P et al. Expression of c-Fos, ICER, Krox-24 and JunB in the whisker-to-barrel pathway of rats: time course of induction upon whisker stimulation by tactile exploration of an enriched environment. J Chem Neuroanat 2002; 23: 187-98
  CrossrefPubMedSearch in Google Scholar
• 22 Gass P, Prior P, Kiessling M. Correlation between seizure intensity and stress protein expression after limbic epilepsy in the rat brain. Neuroscience 1995; 65: 27-36
  CrossrefPubMedSearch in Google Scholar
• 23 Filipovic D, Zlatkovic J, Inta D et al. Chronic isolation stress predisposes the frontal cortex but not the hippocampus to the potentially detrimental release of cytochrome c from mitochondria and the activation of caspase-3. J Neurosci Res 2011; 89: 1461-14670
  CrossrefPubMedSearch in Google Scholar
• 24 Johnson MP, Kelly G, Chamberlain M. Changes in rat serum corticosterone after treatment with metabotropic glutamate receptor agonists or antagonists. J Neuroendocrinol 2001; 13: 670-677
  CrossrefPubMedSearch in Google Scholar
• 25 Radley JJ, Sawchenko PE. A common substrate for prefrontal and hippocampal inhibition of the neuroendocrine stress response. J Neurosci 2011; 31: 9683-9695
  CrossrefPubMedSearch in Google Scholar
• 26 Scaccianoce S, Matrisciano F, Del Bianco P et al. Endogenous activation of group-II metabotropic glutamate receptors inhibits the hypothalamic-pituitary-adrenocortical axis. Neuropharmacology 2003; 44: 555-561
  CrossrefPubMedSearch in Google Scholar
• 27 Davis M, Walker DL, Miles L et al. Phasic vs sustained fear in rats and humans: role of the extended amygdala in fear vs anxiety. Neuropsychopharmacology 2010; 35: 105-135
  CrossrefPubMedSearch in Google Scholar
• 28 Dabrowska J, Hazra R, Guo JD et al. Central CRF neurons are not created equal: phenotypic differences in CRF-containing neurons of the rat paraventricular hypothalamus and the bed nucleus of the stria terminalis. Front Neurosci 2013; 7: 156
  PubMedSearch in Google Scholar
• 29 Singewald GM, Rjabokon A, Singewald N et al. The modulatory role of the lateral septum on neuroendocrine and behavioral stress responses. Neuropsychopharmacology 2011; 36: 793-804
  CrossrefPubMedSearch in Google Scholar
• 30 Anthony TE, Dee N, Bernard A et al. Control of stress-induced persistent anxiety by an extra-amygdala septohypothalamic circuit. Cell 2014; 156: 522-536
  CrossrefPubMedSearch in Google Scholar
• 31 Hsu DT, Kirouac GJ, Zubieta JK et al. Contributions of the paraventricular thalamic nucleus in the regulation of stress, motivation, and mood. Front Behav Neurosci 2014; 8: 73
  PubMedSearch in Google Scholar
• 32 Moga MM, Weis RP, Moore RY. Efferent projections of the paraventricular thalamic nucleus in the rat. J Comp Neurol 1995; 359: 221-238
  CrossrefPubMedSearch in Google Scholar
• 33 Park D, Lee S, Jun K et al. Translation of clock rhythmicity into neural firing in suprachiasmatic nucleus requires mGluR-PLCbeta4 signaling. Nat Neurosci 2003; 6: 337-338
  CrossrefPubMedSearch in Google Scholar
• 34 Gannon RL, Millan MJ. Positive and negative modulation of circadian activity rhythms by mGluR5 and mGluR2/3 metabotropic glutamate receptors. Neuropharmacology 2011; 60: 209-215
  CrossrefPubMedSearch in Google Scholar
• 35 Inta D, Vogt MA, Luoni A et al. Significant increase in anxiety during aging in mGlu5 receptor knockout mice. Behav Brain Res 2013; 241C: 27-31
  PubMedSearch in Google Scholar