Acute stress differentially affects corticotropin-releasing hormone mRNA expression in the central amygdala of the "depressed" flinders sensitive line and the control flinders resistant line rats

Preclinical and clinical evidence suggests that neuropeptides play a role in the pathophysiology of mood disorders. In the present study, we investigated the involvement of the peptides corticotropin-releasing hormone (CRH), neuropeptide Y (NPY) and nociceptin/orphanin FQ (N/OFQ) and of their receptors in the regulation of emotional behaviours. In situ hybridization experiments were performed in order to evaluate the mRNA expression levels of these neuropeptidergic systems in limbic and limbic-related brain regions of the Flinders Sensitive Line (FSL) rats, a putative genetic animal model of depression. The FSL and their controls, the Flinders Resistant Line (FRL) rats, were subjected to one hour acute restraint and the effects of the stress exposure, including possible strain specific changes on these neuropeptidergic systems, were studied. In basal conditions, no significant differences between FSL and FRL rats in the CRH mRNA expression were found, however an upregulation of the CRH mRNA hybridization signal was detected in the central amygdala of the stressed FRL, compared to the non stressed FRL rats, but not in the FSL, suggesting a hypoactive mechanism of response to stressful stimuli in the "depressed" FSL rats. Baseline levels of NPY and N/OFQ mRNA were lower in the FSL rats compared to the FRL in the dentate gyrus of hippocampus and in the medial amygdala, respectively. However, the exposure to stress induced a significant upregulation of the N/OFQ mRNA levels in the paraventricular thalamic nucleus, while in the same nucleus the N/OFQ receptor mRNA expression was higher in the FSL rats. In conclusion, selective alterations of the NPY and N/OFQ mRNA in limbic and limbic-related regions of the FSL rats, a putative animal model of depression, provide further support for the involvement of these neuropeptides in depressive disorders. Moreover, the lack of CRH activation following stress in the "depressed" FSL rats suggests a form of allostatic load, that could alter their interpretation of environmental stimuli and influence their behavioural response to stressful situations.     

The opposite effect of a 5-HT(1B) receptor agonist on 5-HT synthesis, as well as its resistant counterpart, in an animal model of depression

Flinders Sensitive Line (FSL) rat is as an animal model of depression with altered parameters of the serotonergic (5-HT) system function (5-HT synthesis rates, tissue concentrations, release, receptor density and affinity), as well as an altered sensitivity of these parameters to different 5-HT based antidepressants. The effects of acute and chronic treatments with the 5-HT(1B) agonist, CP-94253 on 5-HT synthesis, in the FSL rats and the Flinders Resistant Line (FRL) controls were measured using α-[(14)C]methyl-L-tryptophan (α-MTrp) autoradiography. CP-94253 (5mg/kg), or an adequate volume of saline, was injected i.p. as a single dose in the acute experiment or delivered via the subcutaneously implanted osmotic minipump (5 mg/kg/day for 14 days) in the chronic experiment. The acute treatment with CP-94253 significantly decreased the 5-HT synthesis in both the FRL and FSL rats, with a more widespread effect in the FRL rats. Chronic treatment with CP-94253 significantly decreased 5-HT synthesis in the FRL rats, while 5-HT synthesis in the FSL rats was significantly increased throughout the brain. In both the acute and chronic experiment, the FRL rats had higher brain 5-HT synthesis rates, relative to the FSL rats. The shift in the direction of the treatment effect from acute to chronic, using the 5-HT(1B) agonist, CP-94253, on 5-HT synthesis in the FSL model of depression, with an opposite effect on the control FRL rats, suggests the differential adaptation of the 5-HT system in the FSL and FRL rats to chronic stimulation of 5-HT(1B) receptors.     

Differential expression of postsynaptic NMDA and AMPA receptor subunits in the hippocampus and prefrontal cortex of the flinders sensitive line rat model of depression

Glutamatergic abnormalities have recently been implicated in the pathophysiology of depression, and the ionotropic glutamate receptors in particular have been suggested as possible underlying molecular determinants. The Flinders Sensitive Line (FSL) rats constitute a validated model of depression with dysfunctional regulation of glutamate transmission relatively to their control strain Flinders Resistant Line (FRL). To gain insight into how signaling through glutamate receptors may be altered in the FSL rats, we investigated the expression and phosphorylation of AMPA and NMDA receptor subunits in an enriched postsynaptic fraction of the hippocampus and prefrontal cortex. Compared to the hippocampal postsynaptic fractions of FRL rats, FSL rats exhibited decreased and increased levels of the NMDA receptor subunits GluN2A and GluN2B, respectively, causing a lower ratio of GluN2A/GluN2B. The GluA2/GluA3 AMPA receptor subunit ratio was significantly decreased while the expression of the individual GluA1, GluA2, and GluA3 subunits were unaltered including phosphorylation levels of GluA1 at S831 and S845. There were no changes in the prefrontal cortex. These results support altered expression of postsynaptic glutamate receptors in the hippocampus of FSL rats, which may contribute to the depressive‐like phenotype of these rats.     

Effects of chronic 17beta-estradiol treatment on the serotonin 5-HT(1A) receptor mRNA and binding levels in the rat brain

Acute 17beta-estradiol treatment had been shown to downregulate the 5-HT(1A) receptor mRNA expression in limbic areas of the female rat brain. The aim of the present study was to determine the effects of chronic 17beta-estradiol treatment on 5-HT(1A) receptor mRNA expression and 5-HT(1A) receptor binding in ovariectomized female rats. Using in situ hybridization histochemistry, no alterations were found on the 5-HT(1A) receptor mRNA levels after the estradiol treatment (2 weeks). Radioligand autoradiographic studies using the selective 5-HT(1A) receptor antagonist [(3)H]WAY-100635 revealed reduced receptor binding in the amygdala, hippocampus, perirhinal cortex, and motor cortex after estradiol treatment, whereas no changes were observed in the piriform or retrosplenial cortex. Thus, the previous findings together with the present results indicate that estradiol-induced alterations in 5-HT(1A) receptor mRNA expression appears within hours, but diminishes with chronic treatment when significant changes on the receptor-protein level are apparent. The effects of estradiol treatment on the 5-HT(1A) receptor binding in the limbic areas suggest that estrogen can modulate functions such as learning, memory, cognition, emotional processing, and social behavior. Consequently, estradiol modulation of 5-HT(1A) receptor circuits might be a possible pathway for the estrogen influence in the expression of psychiatric and neurological disorders such as Alzheimer's disease, affective disorders, and schizophrenia.     

Acute citalopram has different effects on regional 5-HT synthesis in FSL, FRL, and SDP rats: an autoradiographic evaluation

In this study, we measured the effect of an acute treatment of citalopram on 5-hydroxytryptamine (5-HT) synthesis in a genetic rat model of depression, the Flinders Sensitive Line (FSL) rats, their counterparts, the Flinders Resistant Line (FRL) rats, and outbred Sprague-Dawley (SPD) rats, using the alpha-[(14)C]methyl-l-tryptophan (alpha-MTrp) autoradiographic method. A comparison of 5-HT synthesis in the FSL rats treated with citalopram (FSL-CTP) and those treated with saline (FSL-SAL) indicate that citalopram reduces global 5-HT synthesis in the FSL rats, as well as in all the brain areas investigated. The reduced synthesis was also observed in the dorsal raphe (DR) nucleus and the median raphe (MR) nucleus. The comparison of the synthesis between the citalopram-treated SPD rats (SPD-CTP) and the saline-treated SPD rats (SPD-SAL) revealed a global increase of 5-HT synthesis in the SPD-CTP group, as well as an increase in some terminal areas, but a reduction in the DR and the MR. In contrast to the reduction throughout the brain in the FSL rats, the FRL rats treated with citalopram (FRL-CTP), when compared to the saline group (FRL-SAL), showed a global increase of 5-HT synthesis, as well as in most of the terminal areas and in the DR and the MR. The reduction of 5-HT synthesis throughout the brain in the FSL rats is likely, in part, a result of reported supersensitivity of the 5-HT(1A) receptors. Comparing changes in the SPD, FRL, and FSL rats treated with citalopram to their respective controls (saline-treated rats), the FSL rats treated acutely with citalopram were the only rats that exhibited lower 5-HT synthesis rates in all of the limbic areas, the basal ganglia, and the neocortices. This may be related to the pathophysiological basis of depressive characteristics in FSL rats. The citalopram treatment produced unexpected results in the FRL rats: 5-HT synthesis was elevated not only in most of the terminal areas, but also in the cell body areas, the DR and MR. The increase of 5-HT synthesis throughout the brain in the FRL rats is likely, in part, a result of the reported subsensitivity of the 5-HT(1A) receptors, and possibly other sites through which 5-HT synthesis could be controlled (e.g., 5-HT(1B)). In addition differences in intracellular signaling could be at least in part responsible for these differences.     

Isolated Flinders Sensitive Line rats have decreased dopamine D2 receptor mRNA

Social isolation has profound effects on animal behavior and dopamine systems. We investigated the effect of social isolation on the dopamine receptor and neuropeptide mRNAs in the brain reward system in an animal model of depression, the Flinders Sensitive Line rats and Sprague-Dawley controls. We demonstrate that socially isolated but not group housed Flinders sensitive line rats had lower dopamine D2 receptor mRNA levels compared with Sprague-Dawley rats. Isolated and group housed Flinders Sensitive Line rats had higher levels of dopamine D1 receptor and substance P and enkephalin but not dynorphin mRNAs when compared with Sprague-Dawley rats. Our findings of decreased dopamine D2 receptor levels in socially isolated Flinders Sensitive Line rats suggest that low D2 receptor expression may play a role in pathophysiology of depression.     

Reduced metabotropic glutamate receptor 5 in the Flinders Sensitive Line of rats, an animal model of depression: an autoradiographic study

Depression is a brain disorder and there is still only a partial understanding of its underlying pathophysiology. Antidepressant medications with a fast onset have not yet been developed. In addition to the monoaminergic systems, the brain glutaminergic system has been implicated in the etiology of depression. Animal studies of depression have gained importance because they permit a more invasive manipulation of the subjects than human studies. In the present study, we measured the densities of the brain regional metabotropic glutaminergic receptor 5 (mGluR5) in the Flinders Sensitive Line (FSL) rat model of depression and two groups of control rats, the Flinders Resistant Line (FRL) and Sprague Dawley (SPD), the parent strain for both the FSL and FRL rats. The FSL rats showed lower densities of mGluR5 in many brain regions compared to either the SPD and/or FRL rats. In addition, the densities in the FRL rats were larger than in the SPD rats, suggesting possible problems in using FRL rats as controls. The presented data suggest that mGluR5 is lower in animal models of depression which could be related to the cognitive and emotional dysfunctions in the FSL rat model of depression and could be relevant to a better understanding of depression in humans.     

Effect of chronic estradiol,tamoxifen or raloxifene treatment on serotonin 5-HT1A receptor

Numerous reports demonstrate the potency of estrogens to modulate brain function and their implications in schizophrenia and depression. The 5-HT(1A) receptor has been suggested to be implicated in depression and anxiety. Selective estrogen receptor modulators (SERMs), like tamoxifen and raloxifene, have estrogenic and/or antiestrogenic activity depending on the target tissue. Hence, SERMs have beneficial effects in skeleton and cardiovascular systems but act as antagonists in breast and uterus. The aim of the present study was thus to investigate in ovariectomized rats the effects of 17beta-estradiol, tamoxifen and raloxifene treatments on 5-HT(1A) receptor binding sites (agonist and antagonist) and mRNA levels in the hippocampal formation, prefrontal and cingulate cortex, as well as dorsal raphea nucleus which are known to express estrogen receptors (ER). Two weeks ovariectomy of female rats led to a 60% decrease of uterine weight, which was prevented by a 2-week 17beta-estradiol treatment; tamoxifen and raloxifene increased uterine weights by 35% and 15%, respectively, but significantly less than estradiol treatment. Specific binding to 5-HT(1A) receptors was determined by autoradiography of brain sections using the selective ligands: [3H]8-OH-DPAT and [3H]MPPF. Ovariectomy and hormone replacement therapy did not significantly affect 5-HT(1A) receptor agonist and antagonist specific binding sites as well as mRNA levels in all subregions of the hippocampus, prefrontal and cingulate cortex as well as dorsal raphea nucleus. Although the present treatments had functional effects as assessed with uterine weights, ovariectomy and estrogen-receptor directed drugs had no effect on hippocampal 5-HT(1A) receptors as compared to 5-HT(2A) receptors previously reported.     

Immobility-reducing effects of antidepressants in a genetic animal model of depression

Chronic treatment with the tricyclic antidepressants imipramine (15 mg/kg) and desmethylimipramine (5 mg/kg) significantly reduced the exaggerated immobility normally exhibited by the Flinders Sensitive Line (FSL) rats in the Forced Swim Test. The control group, Flinders Resistant Line (FRL) rats were only slightly affected. In contrast, chronic treatment with the anticholinesterase diisopropyl fluorophosphate at doses known to down regulate muscarinic receptors did not alter swim test immobility in either FSL or FRL rats. Our findings support the validity of the FSL rats as an animal model of depression and suggest that serotonergic and/or noradrenergic, but not cholinergic mechanisms, may underlie the exaggerated immobility of the FSL rats.     

Effects of maternal separation on neuropeptide Y and calcitonin gene-related peptide in "depressed" Flinders Sensitive Line rats: a study of gene-environment interactions

Interactions between genetic vulnerability to stress/depression and early life experience may play a crucial role in the pathogenesis of mood disorders. Here we explore this hypothesis by superimposing early life trauma in the form of maternal deprivation for 180 min per day from postnatal day 2 to 14 onto a genetic model of depression/susceptibility to depression, Flinders Sensitive Line (FSL) and their controls, Flinders Resistant Line (FRL) rats. We investigate effects on neuropeptide Y (NPY) and calcitonin gene-related peptide (CGRP) like immunoreactivity (LI) in 10 brain regions as these neuropeptides are affected by antidepressants and are altered in cerebrospinal fluid of depressed patients. NPY-LI was reduced while CGRP-LI was elevated in hippocampus and frontal cortex of "genetically depressed" FSL rats. The two peptides displayed a significant negative correlation in these regions that was strongest in the FSL strain. Maternal deprivation exacerbated the strain difference in hippocampal CGRP-LI, while it was without effect on NPY-LI. FSL rats had higher tissue concentration of both neuropeptides in periaqueductal grey and higher NPY-LI in caudate/putamen. Maternal deprivation selectively raised CGRP-LI in amygdala of the FRL control stain. Thus, in two brain regions implicated in the neurobiology of depression, hippocampus and frontal cortex, changes in CGRP-LI and NPY-LI were in opposite direction, and CGRP-LI appears to be more responsive to adverse experience. Our findings thus support the hypothesis that genetic disposition and developmental stress may contribute to the susceptibility to depression by exerting selective neuropeptide- and brain region-specific effects on adult neurobiology.     

Influence of estradiol, stress, and 5-HT2A agonist treatment on brain-derived neurotrophic factor expression in female rats.

BACKGROUND: Estradiol affects neuronal plasticity, mood, and cognition. We examined the effects of the estrous cycle, acute and chronic estradiol treatments on BDNF mRNA expression in the hippocampus and cortex of female rats. The roles of 5-HT2A receptors and of stress on the BDNF mRNA regulation were also explored. METHODS: BDNF mRNA levels were measured using in situ hybridization at proestrus and estrus, and following acute and chronic estradiol treatment of acutely and chronically ovariectomized (OVX) female rats. Some rats were pretreated with 5-HT2A agonist and antagonist, and another group was subjected to two-hour immobilization stress. RESULTS: BDNF mRNA levels in the dentate gyrus and the medial prefrontal cortex were decreased during estrus, when estradiol levels are highest. Acute estradiol treatment decreased hippocampal BDNF mRNA in acutely OVX rats, but neither acute nor chronic estradiol had effect in chronically OVX rats. Estradiol pretreatment reduced the 5-HT2A receptor-mediated cortical upregulation in BDNF mRNA and did not effect the stress-induced down-regulation of BDNF mRNA in the dentate gyrus. CONCLUSIONS: The duration of the estradiol treatment and the duration of the ovarian hormone deprivation are important factors in the regulation of BDNF synthesis and possibly in the functional outcome of estrogen treatment.     

Electroconvulsive stimuli alter nerve growth factor but not brain-derived neurotrophic factor concentrations in brains of a rat model of depression

Nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) are proteins involved in neuronal survival and plasticity of dopaminergic, cholinergic and serotonergic neurons in the central nervous system (CNS). Moreover, it has been hypothesized that these molecules play a role in the pathophysiology as well as treatment of depression. Using an animal model of depression, the Flinders Sensitive Line (FSL) rats and their controls, the Flinders Resistant Line (FRL), we investigated the effects of electroconvulsive stimuli (ECS) on brain NGF and BDNF. ECS or SHAM ECS were administered eight times, with a 48-h interval between each treatment. NGF and BDNF were measured with enzyme-linked immunosorbent assay (ELISA). In the hippocampus ECS increased NGF concentration in FSL but not FRL rats. ECS decreased NGF concentration in the frontal cortex of FSL rats. In both FSL and FRL rats ECS increased NGF levels in the striatum. In contrast, ECS did not change BDNF concentration in hippocampus, frontal cortex and striatum of FSL and FRL rats. Our data support the notion that neurotrophin concentrations may be altered by ECS.     

Altered physiology of acid secretion in depression-prone Flinders rats results in exacerbated NSAID and stress-induced gastric damage

Background Flinders Sensitive Line (FSL) rats are characterized by hypersensitivity to cholinergic stimuli and have been extensively used for studying depressive disorders. A link between depression and peptic ulcers has long been established; however, there is a lack of data from animal models. Methods We studied the physiology of acid secretion in FSL and Flinders Resistant Line (FRL) rats in vivo and in vitro. We also examined the susceptibility of Flinders rats to water immersion restraint stress (WIRS) or NSAID‐induced gastric damage and explored the effect of an anticholinergic agent, atropine, in reversing this effect. Key Results Basal acid output was more than twofold greater in FSL compared with FRL rats in vivo, 213.5 and 92.8 μEq/3 h/100 g (P = 0.02), respectively. Carbachol was a more potent secretagog in vitro, and somatostatin was a less potent inhibitory agent, while paradoxically stimulating acid secretion over and above the carbachol response in gastric glands from FSL rats. The FSL rats were more susceptible to indomethacin and WIRS‐induced gastric mucosal damage compared with FRL rats. Atropine reduced acid output, which resulted in a reduction in indomethacin and stress‐induced gastric damage in FSL rats. Conclusions & Inferences Our study, for the first time, demonstrates that the altered vagally mediated physiology of acid secretion in depression‐prone FSL rats contributes to gastric hypersecretion and, consequently, results in exacerbated stress and NSAID‐induced gastric damage. Flinders rats may be a useful animal model for studying acid‐related and also gastrointestinal functional disorders in depression.     

The serotonin-dopamine interaction is critical for fast-onset action of antidepressant treatment: in vivo studies in an animal model of depression

In the last decade, many new antidepressants have been developed that display a more rapid onset to clinical effects than classical antidepressants. However, the mechanism that enables some drugs to have a faster onset of action than others is poorly understood. The aim of the present study was to determine neural alterations that are specific to fast-acting antidepressant action using Flinders Sensitive Line (FSL) rats, an animal model of depression. Because of the central role of accumbal dopamine in the mediation of motivation and reward, our measurements were focused on dopaminergic neurotransmission in the nucleus accumbens (NAC). The authors found that 7-day treatment with nefazodone (a putative fast-onset antidepressant) but not with desipramine (a classical antidepressant) normalized immobility time in the swim test in FSL rats. Serotonin (5-HT)-induced dopamine release but not basal dopamine levels correlated with the improvement of depressive-like behavior. The authors conclude that the 5-HT-dopamine interaction is critical to the fast-onset action of antidepressant treatment.     

Social isolation increases number of newly proliferated cells in hippocampus in female flinders sensitive line rats

Genetic background influences the responsiveness to stress and plays a crucial role in the pathophysiology of depression. In an animal model of depression, Flinders Sensitive Line rats, and Sprague Dawley controls we analyzed if 7 weeks of social isolation of adult animals affect the number of newly proliferated cells in the dentate gyrus or mRNAs of Neuropeptide Y (NPY), the NPY-Y1 receptor, nociceptin, BDNF, and the serotonin 5HT1A and 5HT2A receptors, which are molecules involved in hippocampal plasticity. Since depressive illness more frequently affects women than men, and females seem to respond differently to stressful experiences than males, female rats were used in this study. Bromodeoxyuridine, which is a thymidin analogue that is incorporated into the DNA of newly formed cells, was administered during 9 days to even out the effects of hormonal fluctuations. Social isolation increased the number of newly proliferated Bromodeoxyuridine-immunoreactive cells in the Flinders Sensitive Line rats, whereas it had no impact on the number of cells in the Sprague Dawley strain. Group housed Sprague Dawley rats had a higher expression of BDNF, NPY, and the serotonin 5HT2A receptor mRNA than "depressed" Flinders Sensitive Line. Social isolation downregulated these molecules in Sprague Dawley but not in Flinders Sensitive Line rats thereby eliminating the differences between the two strains. We demonstrate strain and gender specific responses to stress induced regulation of factors important for hippocampal plasticity.     

Chronic inositol treatment reduces depression-like immobility of Flinders Sensitive Line rats in the forced swim test

Inositol, a precursor of the PIP cycle that was reported to have therapeutic effects in depressive patients and to be effective in two animal models of depression, was evaluated in the forced swim test using the genetic Flinders Sensitive Line (FSL) rats model of depression. Groups of rats were tested in a 2 x 2 design with Strain (FSL or Control) as one factor and Drug (Inositol or Placebo) as the second factor. Rats received chronic treatment (daily for 14 days) with inositol (1.2 g/kg) or placebo (1:2 glucose/mannitol solution). On day 14 rats were exposed to the forced swim test for 5 min and their behavior videotaped. Tapes were analyzed for three levels of activity: immobility, swimming, and vigorous struggle. Inositol countered the exaggerated immobility of FSL rats in the forced swim test, without affecting control animals. Data support our previous suggestion of inositol as a potential antidepressant.     

Pseudo-affective visceromotor responses and HPA axis activation following colorectal distension in rats with increased cholinergic sensitivity

We analysed visceromotor (VMR) and corticosterone responses to colorectal stimuli under control conditions and following acoustic stress in rats selectively bred for increased sensitivity to cholinergic agonists, the Flinders Sensitive Line (FSL) rats, compared with Flinders Resistant Line (FRL) rats. FSL rats demonstrated a significant VMR response at the smallest distension pressure, whereas no response was evident in FRL controls. FSL rats also demonstrated enhanced VMR responses at both larger distension levels compared with FRL rats. Colorectal distension (CRD) produced significant increases in serum corticosterone levels, which were comparable in FRL and FSL. Noise stress induced divergent corticosterone responses in FRL and FSL, but did not affect VMR to CRD in either group. These data suggest that FSL rats show altered VMR responses to CRD and disturbed hypothalamic-pituitary-adrenal axis responses to acute stress.