Regional neurochemical characterization of the flinders sensitive line rat with regard to glutamate-nitric oxide and cGMP signalling pathways

Abstract

The serious nature of MDD has intensified the need to identify and elucidate new neurobiological targets for antidepressant drug action. Depression presents with evidence for degenerative pathology that relates to disturbances in excitatory glutamatergic pathways, particularly the N-methyl-D-aspartate (NMDA) receptormediated release of the pleiotropic molecule, nitric oxide (NO), and cyclic guanosine monophosphate (cGMP). The contribution of the glutamate-NO/cGMP pathway may realize great importance as a fundamental substrate underlying the pathophysiology of major depression. In the next generation of antidepressant drugs, the nitric oxide pathway could playa dynamic role in addressing urgent therapeutic needs. In this study, we have used a genetic model of depression, the Flinders Sensitive Line (FSL) rat, to investigate the surrogate markers of the NO/cGMP pathway. The aim was to determine whether the depressive-like behaviour of the hypercholinergic FSL rat is accompanied by altered activation of the NO/cGMP pathway. To this end, the extent to which the FSL and Flinders Resistant Line (FRL) rats differ neurochemically with regard to basal hippocampal and frontal cortical NOS-activity, as well as nitric oxide (NO) and cGMP accumulation, were determined. Additionally, select behavioural assessments were performed to confirm the anxiogenic phenotype of the FSL strain. For neurochemical determinations a sensitive fluorometric reversed phase highperformance liquid chromatographic (HPLC) assay was developed to analyze total nitrite and nitrate in brain tissue. Nitrate was enzymatically converted to nitrite before derivatization with 2,3-diaminonaphthalene (DAN). The stable and highly fluorescent product, 2,3-naphthotriazole (NAT), was quantified. Secondly, the quantity of the amino acid L-citrulline was measured by HPLC with electrochemical detection after o-phthalaldehyde (OPA) derivatization. L-citrulline formation was used as an index for nNOS activity. Finally, a direct, competitive enzyme immunoassay kit was used to determine the downstream activity of the NO-pathway in brain tissue. FSL rats were compared to FRL rats with respect to sensitivity to serotonin 5-HT1A . receptor-mediated hypothermia under our lab-conditions. The Open Field Test (OFT) behavioural assessment was performed to compare FSL with FRL groups under baseline conditions according to their level of inherent anxiety. The parameters used to measure anxiety were number of line crosses (locomotor activity), time spent in middle blocks and social interaction time between pairs of rats. As an additional behavioural assessment, the Forced Swim Test (FST) was performed to assess behavioural restraint measured as time of immobility. Basal cGMP levels in the frontal cortex were found to be significantly less in FSL than in FRL rats, whereas the levels in the hippocampus did not differ significantly. No other significant differences with respect to NO and nNOS activity were apparent in either of the brain areas. The hypothermia test confirmed a significantly greater decrease in temperature in the FSL rat than the FRL rat. The FST did not confirm any differences in immobility time between the two rat strains. In the OFT, FSL rat groups exhibited behaviour that indicated significantly more anxiety than FRL rats. Under basal conditions, FSL rats do not present with significant changes in markers of the NO cascade in the hippocampus and frontal cortex compared to FRL controls, including NOS activity as well as NO accumUlation. However, cGMP levels were found to be significantly lower in the frontal cortex of FSL rats versus FRL rats, although not in the hippocampus. Since the FSL rat is known to be hypercholinergic, these data support an interaction between the NO/cGMP pathway and the cholinergIc system in the frontal cortex but not hippocampus of FSL animals. The mechanisms and implications of such a mutual involvement need further clarification. Further, this anatomical differentiation may have important implications for understanding the role of NO in the depressive-like behaviour of the FSL rat and, indeed, may reveal more on the neurobiology and treatment of depression. Through the performed behavioural assessments, the FSL and FRL rats were successfully separated with respect to their anxiety phenotype as well as their heightened response to serotonergic challenge, thus confirming a contribution of both the serotonergic and cholinergic systems to the depressogenic nature of these animals. As concluding remark can be said that under normal basal conditions markers of the NO/cGMP signalling cascade are not altered in FSL vs FRL rats, although cGMP levels are reduced in the frontal cortex of FSL rats, supportive of an NO-independent mechanism of cGMP regulation, possibly involving ACh.