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In a separate session, the same rat was tested for spatial working memory by placing it in two identical chambers (60 Ă 20 10 cm) and recording each rat's location for 4 min and its response to the presence of a hidden platform (A). The response to a hidden platform was measured after 3 d. For spatial working memory, the following parameters were measured: total duration of the platform-induced response (3 d), latency from the platform position to first movement of the rat (10â20 s), and latency from the first
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The animals in experimental group (n = 5) were
Buy alprazolam online canada tested for a general spatial ability (n = 9) and for spatial working memory (n = 8) before and after treatment. In each experimental group, the animals were treated with following drugs Restoril 30mg 90 pills US$ 320.00 US$ 3.56 (20 mg/kg/day, ip) and received the following treatments (20 mg/kg/day, ip) 7 d apart: clomipramine 30 mg/kg/day, s.c.; quinpirole 2.5 mg/kg b.i.d., i.p.; sertraline 20â40 mg/kg/day, fluoxetine 10 mg/kg, p.o.; and
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At the end of experimental period, brain was snap-frozen in liquid nitrogen and stored at â80 °C until it was used for immunohistochemical detection of α 2-adrenoceptors. Brain specimens were processed to determine the levels of brain α 2-adrenoceptors by immunohistochemically staining hippocampal sections with anti-α 2-adrenoceptor antibody (1:200; Abcam), and the percentage of α 2-adrenoceptors was measured by immunohistochemical methods (see below).
For the analysis of expression c-fos and N-methyl-d-aspartate (NMDA) receptors in the hippocampus and amygdala, rat brain tissue samples were homogenized and frozen in liquid nitrogen. The homogenates were then transferred to a microtiter plate and incubated in a buffer containing sodium orthovanadate (50 mM; Sigma Aldrich) and a protease inhibitor cocktail (50 mM tris-HCl, pH 8.3; 20 EGTA, 0.25% Triton X-100; 10 mM sodium orthovanadate; 1 SDS; phenylmethylsulfonyl fluoride; mM Na 2 EDTA; 150 NaCl; 0.1% sodium deoxycholate) at 4 °C overnight. After the incubation, protein extracts were incubated with an anti-c-fos antibody (1:1,000, Abcam) or an anti-NMDA antibody (1:1,000; Abcam) in a final volume of 1 ml for h at room temperature, followed by washing in tris (pH 8.0) pH 8.3 buffer for 2 min with a total of three subsequent washes in TBS, pH 8.0, at room temperature. The samples were then incubated with anti-rabbit Alexa Fluor 647-conjugated goat secondary antibody (1:1000; Vector Laboratories) at room temperature for 1 h. The samples were then washed in TBS, pH 8.0, several times and counterstained with hematoxylin eosin (Sigma) for visualization. The immunohistochemical analysis was performed as previously described [14].
In a further investigation based on our previous studies [13], [14], we tested the influence of treatment on expression NMDA and cFos receptors in the cortex of rat brain by in situ hybridization. tissue restoril 30 mg for sleep pill samples were digested in 1 h of trypsin (Roche Diagnostics, Mannheim, Germany) with protease inhibitors (100% ÎČ-mercaptoethanol, 1 mM dithiothreitol, MgCl 2 ).