A new model of depressive-like behaviour in rats and mice for preclinical drug discovery Morozova A.Y., Zubkov E.A., Chekhonin V.P. Department of Basic and Applied Neurobiology, V.P. Serbsky National Medical Research Center for Psychiatry and Narcology, Moscow, Russia

The aim of our study was to create the experimental model of depression in laboratory rodents, used chronic informational stress based on effect of multidirectional flow of information (20-45 kHz)

«Ultrasound model of depression» Animals: Wistar rats and Balb/c mice. Stress procedure: Experimental groups were exposed during 21 days to ultrasonic radiation by emitted frequencies: 20 - 45 kHz. After stress procedure behavioral tests were performed (Social interaction test, Forced swimming test (Porsolt test), Open Field test, Anhedonia Test). Than the expression of genes of 5-HT1A, 5-HT2A, SERT were analyzed in PFC, midbrain and hippocampus; BDNF and VEGF were analyzed in hippocampus by Quantative Real-time PCR Analysis. Immunohistochemical assay was conducted using the method of double immunofluorescence staining with monoclonal antibodies to VEGF and polyclonal antibodies to BDNF.

Modeling of depressive-like behaviour (experimental paradigm) Chronic unpredictable ultrasound radiation (21 days) Depressive-like behaviour Stress Situation of chronic informational uncertainty attained by exposure of ultrasonic signals of different frequencies 20 -25 kHz «negative» emotional state 25–45 kHz «neutral» emotional state For drug discovery For basic research in a fields of biochemistry and pathophysiology

Experiment design

Anhedonia test Comparison of sucrose preference (a), sucrose (b) and water consumption (c) and total liquid intake (d) between control, exposed to ultrasound (US) and treated with fluoxetine ultrasound exposed (US + fluox) groups of Wistar rats (column I) and Balb/c mice (column II). Preference index was calculated according to the formula: Vs / (Vs + Vw) ∗ 100%, where Vw is volume of consumed water, Vs is volume of consumed sweet water. Bars presented as M ± SEM, preference shown in percentages and other parameters in millilitres. Data compared using one-way ANOVA followed by post-hoc Fisher's LSD test. (a) US exposure significantly decreased sucrose preference index in both rats and mice ***(p b 0.001 vs Control), and fluoxetine treatment improved preference in rats *(p b 0.05) and mice **(p b 0.01) compared to respective US group. (b) Control rats and mice drank more 1% sucrose solution than their US groups ***(p b 0.001), but in treated with fluoxetine animals' sucrose intake raised only in mice US + fluox group *p b 0.05 compared to US group. (c) Control and US + fluox groups of rats drank less tap water than their US group (***p b 0.001 and **p b 0.01 respectively), while all mice groups did not have any significant differences by that parameter. (d) All groups of Wistar rats did not differ by total liquid intake, and only control group of Balb/c mice has shown an increase in this parameter compared to their US group **p b 0.01.

Forced swimming test (Porsolt test) Comparison of immobility time during the last 6 min of an 8 min swim session between control, exposed to ultrasound (US) and treated with fluoxetine ultrasound exposed (US + fluoxetine) groups of Wistar rats (A and C) and Balb/c mice (B). Bars presented as M ± SEM, immobility time shown in seconds. Data compared using one-way ANOVA followed by post-hoc Fisher's LSD test. “A” and “B” diagrams present rats and mice that were subjected to behavioural test on the next day after finishing of US exposure and treatments. “C” diagram shows separate group of Wistar rats that were returned to normal housing for 30 d after 21 d US exposure (also for control group that was housed in individual cages for 21 d).

Social interaction test Time of social contacts between the experimental animal and the juvenile male. This test compared control, exposed to ultrasound (US) and treated with fluoxetine ultrasound exposed (US + fluoxetine) groups of Wistar rats (A) and Balb/c mice (B). Bars presented as M ± SEM, time of social contacts shown in seconds. Data compared using one-way ANOVA followed by post-hoc Fisher's LSD test

«Open Field» Comparison of horizontal activity (travelled distance, A and B) and vertical activity (number of rearings, C and D) between control, exposed to ultrasound (US) and treated with fluoxetine ultrasound exposed (US + fluoxetine) groups of Wistar rats (A and C) and Balb/c mice (B and D). Bars presented as M ± SEM, Data compared using one-way ANOVA followed by post-hoc Fisher's LSD test.

Q PCR-RT Expression of the genes encoding 5HT1A, 5HT2A and SERT in prefrontal cortex (a), hippocampus (b) and midbrain (c) in control and exposed to ultrasound (US) Wistar rat's brain, measured by quantitative real-time PCR. GAPDH gene was selected as reference gene for the analysis. The relative expression level of target genes was calculated using the following formula 2−ΔΔCt ± 2±SD (ΔCt for each group). Data compared with Mann–Whitney test.

Expression of BDNF and VEGF genes and immunohistochemical staining with anti-BDNF and anti-VEGF antibodies in hippocampus. (A, B) Expression of the genes encoding BDNF and VEGF in control and exposed to ultrasound (US) Wistar rat's hippocampus, measured by quantitative real-time PCR. (C) Fluorescence intensity of BDNF and VEGF expressions in rat hippocampus (CA3). Immunohistochemical analysis of BDNF and VEGF in CA3 zone of rat hippocampus (D,F) Merged images. (E,G) Nuclei of cells stained with DAPI. (H) Immunofluorescence of VEGF in the US group, (J) immunofluorescence of VEGF in the control group, (I) immunofluorescence of BDNF in the US group, (K) immunofluorescence of BDNF in the control group. Laser scanning confocal microscopy. Scale bar, 20 mm

Conclusion: In the present study, it is shown that a depression-like state is induced in rats and mice by a stress-causing situation of chronic informational uncertainty attained by exposure of ultrasonic signals of different frequencies. Depression-like behavior manifests itself in reduced social activity in social interaction test and increased immobility in forced swimming test. The administrated Fluoxetine demonstrated it effectiveness. RT-qPCR and immunohistochemical assay showed dysregulation of serotonergic system and decreased one of the main neurotrophic factors – BDNF. The obtained data allow to conclude that this model meets the main requirements set to animal models of neuropsychiatric disease (face, predictive and construct validity) and can be used in studies of depression-like disorders and in pre-clinical development of new antidepressants.