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.     

Decreased hyperthermic effect of MK-801 in selectively bred hypercholinergic rats

The Flinders Sensitive Line (FSL) of rats has been selectively bred to have increased sensitivity to cholinergic agonists. However, these rats exhibit altered responsiveness to a number of noncholinergic agents, such as apomorphine, buspirone and ethanol. This study compared the FSL and control Flinders Resistant Line (FRL) rats in terms of their hyperthermic response to the phencyclidine (PCP) receptor agonist, MK-801 (0.2 mg/kg SC) and their MK-801 binding characteristics. We have found that FSL rats react with a delayed hyperthermia, having a significantly lower hyperthermia for the first 120 min of observation. Thereafter the response does not differ in FSL and FRL rats. Both groups had similar affinities and numbers of [3H]MK-801 binding sites in the hippocampus/cerebral cortex. Pretreatment with scopolamine (1 mg/kg SC) failed to affect MK-801-induced hyperthermia in either line of rats. These findings suggest that selective breeding of FSL rats attenuated the secondary mechanisms involved in the PCP receptor-mediated hyperthermic response. However, by itself cholinergic supersensitivity does not appear to be a major factor in the blunted responsiveness of FSL rats to MK-801.     

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.     

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.     

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.     

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.     

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 serotonergic 5-HT1A and 5-HT1B ligands on ventral pallidal neuronal activity

To clarify the role of the 5-HT system in limbic outputs, the present study compared the effects of the 5-HT1A agonist 8-OH-DPAT and the 5-HT1B agonist CP-94253 with the non-selective 5-HT agonist TFMPP on the firing rate of ventral pallidal (VP) neurons recorded in chloral hydrate-anesthetized rats. 8-OH-DPAT (0.25-256 microg/kg i.v.) dose-dependently enhanced (9/26 neurons) or suppressed (8/26) activity, and the 5-HT1A antagonist (+)WAY-100135 often attenuated these responses. TFMPP (0.011-1.453 mg/kg i.v.) dose-dependently reduced the firing rate of 7/8 VP neurons tested. In contrast, CP-94253 (0.013-12.8 mg/kg i.v.) had little or no effect. In sum, these data suggest that the 5-HT1A receptor appears to be particularly important in influencing limbic outputs mediated via the VP.     

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.     

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.     

Quantitative hippocampal structural changes following electroconvulsive seizure treatment in a rat model of depression

Objective: The pathophysiology of depression and the effects of antidepressant treatment are hypothesized to be related to hippocampal structural changes. This study aims to investigate the effect of electroconvulsive seizures on behavior and hippocampal structure in a rat model of depression. Methods: Flinders Sensitive Line (FSL) and Flinders Resistant Line (FRL) rats were treated daily for 10 days with either electroconvulsive seizures or sham treatment. The behavior was evaluated using the forced swim test. Design‐based stereological methods were used to quantify the hippocampal volume and the numbers of neurons and glial cells in specific hippocampal subregions. Results: The basal level of hippocampal volume and neuron number differed significantly between the two rat strains, and a trend toward the FSL strain having more glial cells was found. The structural differences found between the sham‐treated animals were counteracted by electroconvulsive seizure (ECS) treatment, which also normalized the behavior. ECS treatment increased the number of glial cells in hilus significantly in the FRL rats and with the same tendency for the FSL rats. Conclusion: Our results indicate that along with hippocampal neurogenesis, gliogenesis may also be involved in the pathophysiology of depression and in the effect of antidepressant treatment. The underlying mechanisms remain unknown, and further investigations are required to clarify whether the structural changes are necessary to induce a therapeutic effect of antidepressant treatment or if they rather represent an epiphenomenon. Synapse, 2012. © 2012 Wiley Periodicals, Inc.     

Chronic fluoxetine treatment has a larger effect on the density of a serotonin transporter in the Flinders Sensitive Line (FSL) rat model of depression than in normal rats

The 5‐hydroxytryptamine system is thought to play a crucial role in the pathophysiology of depression and represents the target for selective 5‐HT reuptake inhibitors (SSRIs). Flinders Sensitive Line (FSL) and Flinders Resistant Line (FRL) rats were bred from Sprague‐Dawley (SPD) rats to produce strains with increased (FSL) or decreased (FRL) sensitivity to the cholinesterase inhibitor. The FSL rats have been identified as a good model of depression. Many studies in normal rats showed that chronic treatments with SSRIs reduce the densities of SERT. The objective of the present investigation was to assess the influence of chronic fluoxetine treatment on SERT density (Bmax; fmol/mg) in the FSL rat model of depression, relative to that in the FRL rats and SPD rats. FSL, FRL and SPD rats were randomly assigned into groups receiving the vehicle or 10 mg/kg of fluoxetine i.p. for 14 days. Binding was assessed by incubating the brain sections in a buffer containing 20 pM of [¹²⁵I]‐RTI‐55 [[¹²⁵I] (?) ‐2β‐carbomethoxy‐3β‐(4‐iodophenyl)tropane and 200 nM of GBR12935 [1‐(2‐(diphenylmethoxy)ethyl)‐4‐(3‐phenylpropyl)piperazine]. The fluoxetine treatment reduced B_max in all three rat strains when the saline and respective fluoxetine groups were compared (e.g., the FSL‐SAL relative to FSL‐FLX groups). Chronic fluoxetine treatment reduces the densities of SERT in the FSL rats to a larger extent than in the normal SPD control rats. Synapse 64:231–240, 2010. © 2009 Wiley‐Liss, Inc.     

Altered neuroendocrine response and gastric dysmotility in the Flinders Sensitive Line rat

In the search for animal models that can replicate some features of functional dyspepsia (FD) patients, we turned our interest to the Flinders Sensitive Line (FSL) rat. Gastric motility disturbances prevalent in FD patients as well as urine corticosterone and plasma prolactin were measured following buspirone challenge. Flinders Resistant Line (FRL) rat was used as control. The results show that the FSL rats have a disturbed gastric motility, reflected as both an increased gastric accommodation rate and gastric volume during gastric distension as well as a delayed gastric emptying, the latter possibly as a consequence of the former. Lipid administration resulted in a significant increase in maximal gastric volume only in the FRL rats. Both the corticosterone response to buspirone and the 24-h urinary output of corticosterone were normal in FSL rats. Similar to FD patients, the FSL rat showed supersensitivity to buspirone in the increase in prolactin release. Although FSL rats show some features similar to a subset of FD patients, the increased gastric accommodation contrasts to the reduced accommodation often seen in FD patients. Further studies are warranted to determine the relevance of this rat strain as a model for FD.     

Mapping the differences in the brain concentration of brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) in an animal model of depression

Antidepressant drugs as well as electroconvulsive stimuli can significantly influence brain concentrations of neurotrophic factors. However, it is not known whether the baseline brain concentrations of neurotrophic factors are altered in human subjects suffering from affective disorders or whether there are sex differences in concentrations of neurotrophins in human brain. In order to elucidate some of these questions, we measured by ELISA brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) in an animal model of depression, the Flinders Sensitive Line (FSL) rats and their controls, the Flinders Resistant Line (FRL). Altered BDNF and NGF concentrations were found in frontal cortex, occipital cortex, and hypothalamus of depressed FSL compared to FRL control rats. Furthermore, different levels of these neurotrophins were also found in the male and female brain. Cumulatively these observations suggest that BDNF and NGF may play a role in depression and, hypothetically, different brain regional concentrations of BDNF and NGF in male and female animals may be relevant to gender differences in vulnerability to depression.     

Neuropeptide Y in brains of the Flinders Sensitive Line rat, a model of depression. Effects of electroconvulsive stimuli and d-amphetamine on peptide concentrations and locomotion

Neuropeptide Y (NPY), has been implicated in the pathophysiology of depression and the mechanisms of action of electroconvulsive treatment (ECT). In this series of experiments, we explored whether there are differences between Flinders Sensitive Line (FSL) rats, an animal model of depression, and controls, Flinders Resistant Line (FRL) in (1) baseline brain NPY-LI concentrations, (2) effects of ECS on locomotion and brain neuropeptides, (3) amphetamine effects on behavior, and (4) effects of ECS pretreatment on subsequent effects of amphetamine on behavior. Both strains were divided into two groups, receiving eight ECS or ShamECS. Twenty-four hours after the last session, animals were habituated in activity boxes for 45 min before given d-amphetamine (1.5 mg.kg(-1), subcutaneously) or vehicle. Locomotor activity was then recorded for an additional 45 min. Twenty-four hours later, rats were sacrificed by microwave irradiation, the brains dissected into frontal cortex, occipital cortex, hippocampus, hypothalamus and striatum, and the neuropeptides extracted and measured by radioimmunoassay. No differences between FSL and FRL rats in baseline locomotor activity were found. FSL compared to FRL animals showed a significantly larger locomotion increase following saline and a significantly smaller increase following amphetamine. ECS pretreatment significantly decreased the saline effects on locomotion in the FSL and the amphetamine effects in the FRL rats. 'Baseline' NPY-like immunoreactivity (LI) concentrations were lower in the hippocampus of the 'depressed' rats. ECS increased NPY-LI in frontal cortex, occipital cortex and hippocampus of both strains. The hippocampal NPY-LI increase was significantly larger in the FSL compared to FRL animals.     

Enhanced affective aggression in genetically bred hypercholinergic rats

Affective aggression was studied in pairs of Flinders Sensitive Line hypercholinergic rats (FSL) and Flinders Resistant Line (FRL) rats in shock-induced and apomorphine-induced fighting tests. FSL rats were significantly more aggressive in both tests. They had higher pain threshold, assessed by the jump-flinch method, than FRL rats. It is concluded that genetically developed cholinergic system supersensitivity has resulted in enhanced responsiveness to stimuli eliciting affective aggression.     

Early-life stress and antidepressants modulate peripheral biomarkers in a gene–environment rat model of depression

Background

Availability of peripheral biomarkers for depression could aid diagnosis and help to predict treatment response. The objective of this work was to analyse the peripheral biomarker response in a gene–environment interaction model of depression. Genetically selected Flinders Sensitive Line (FSL) rats were subjected to maternal separation (MS), since early-life trauma is an important antecedent of depression. An open-ended approach based on a proteomic analysis of serum was combined with the evaluation of depression-associated proteins.

Methods

Rats experienced MS and chronically received escitalopram (ESC) or nortryptiline (NOR). Serum proteins were compared by two-dimensional gel electrophoresis. Corticosterone, cytokines, BDNF and C-reactive protein (CRP) were measured by immunoassays.

Results

Comparing FSL with the control Flinders Resistant Line (FRL), Apo-AI and Apo-AIV, α1-macroglobulin, glutathione peroxidase and complement-C3 were significantly modulated. Significant increases were detected in leptin, interleukin (IL) 1α and BDNF. CRP levels were significantly reduced.The impact of early-life stress was assessed by comparing FSL + MS versus FSL. Apo-E, α1-macroglobulin, complement-C3, transferrin and hemopexin were significantly modulated.The effect of stress in antidepressant response was then evaluated. In the comparison FSL + ESC + MS versus FSL + ESC, albumin, α1-macroglobulin, glutathione peroxidase and complement-C3 were modulated and significant reductions were detected in IL4, IL6, IL10, CRP and BDNF. By comparing FSL + NOR + MS versus FSL + NOR proteins like Apo-AIV, pyruvate dehydrogenase, α1-macroglobulin, transferrin and complement-C3 showed different levels.

Conclusions

Lipid metabolism and immunity proteins were differently expressed in FSL in comparison with FRL. Exposure to MS induced changes in inflammation and transport proteins which became apparent in response to antidepressant treatments. Modulated proteins could suggest biomarker studies in humans.