Involvement of nitric oxide with the behavioural and molecular changes induced by exposure to an intense stressor: possible effect of cannabidiol

• Grant number: 22/00198-4
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• Start date: February 01, 2022
• End date: April 18, 2024
• Field of knowledge: Biological Sciences - Pharmacology - Neuropsychopharmacology
• Principal Investigator: Francisco Silveira Guimaraes
• Grantee: Mariana Gallio Fronza
• Host Institution: Faculdade de Medicina de Ribeirão Preto (FMRP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil
• Associated research grant: 17/24304-0 - New perspectives in the use of drugs that modify atypical neurotransmitters in the treatment of neuropsychiatric disorders, AP.TEM

Abstract

In the present project we will investigate whether cannabidiol (CBD) could favorably intervene in the long-term behavioral, molecular and inflammatory changes induced by exposure of rats to an intense stress situation called single prolonged stress (SPS). A complementary objective of the proposal is to investigate whether the nitrergic system is involved in these changes, and the impact of CBD on it. Wistar rats will undergo SPS, consisting of 2 hours of restraint, followed by 20 minutes of forced swimming. After 15 minutes of rest, they will be placed in a chamber with ether until loss of postural tone. Immediately after, they will be allocated in isolation. Twenty-four hours later, the animals will undergo the sucrose preference test (basal consumption will be evaluated before SPS) and, 7 days later, the forced swim test in order to verify stress coping strategies style. According to these results, the animals will be separated into stress responsive or resilient. The treatment with CBD will begin after the forced swim test. Daily injections of CBD (5 mg/kg, i.p., dose based on results of a previous study) will be given for seven days, with the last injection 24h before conditioning. All animals will undergo context aversive conditioning (MCC). After 24h, the expression and acquisition of the extinction response will be assessed for 15 min in the absence of shocks. To evaluate the consolidation of this extinction memory, after 24 h the animals will again be exposed to the box for 10 min. Generalization processes of fear responses in a context different from the conditioning context will also be analyzed for 5 min, 24h after conditioning.To determine synaptic changes related to the nitrergic and glutamatergic systems, the levels (mRNA and protein) of PSD-95, NMDA receptor subunits (GluN1 and GluN2), NF-kB p50/p65 (nuclear fraction) and nitrergic system-relataed molecules (nNOS, p-nNOS, iNOS, nNOS-AP) in post-traumatic stress disorder (PTSD)-related structures (prefrontal cortex, hippocampus, amygdala and striatum (negative control) will be measured. We will also assess S-nitrosylation, nitrite levels and GAPDH translocation to the nucleus, and activation of pro-apoptotic and inflammatory factors (by measuring p53, Caspase-1, Bax, IL-1², TNF-5ü and IL-6 levels). Finally, the involvement of nitric oxide in these changes will be investigated by genetic and pharmacological approaches.