Neuochemical and USV measurements of the effects of morphine withdrawal in rats

Data was acquired to investigate the effects of morphine withdrawal in rats, both in the brain neurochemistry and ultrasonic vocalisation.

To this end, a following experiment was performed. 38 adult male Sprague–Dawley rats were used in the experiment, divided into four groups: Saline-Control (10), Morphine-Control (9), Saline-Withdrawal (10) and Morphine-Withdrawal (9). Morphine in dose 10.0 mg/kg (s.c.) was administered repeatedly to the morphine groups over a 14 day period; saline solution was administered repeatedly (1.0 ml/kg) to other groups in the same manner. Administration was performed in testing cages in a group of four animals. The testing room was situated in a remote part of the laboratory, and significantly different than home cage room, both in the lighting conditions and in the arrangement of spatial cues, which were constant throughout the whole experiment. All animals were kept for 30 min in the testing box after each saline or morphine injection.

On day 14, Morph-Control group and control Saline-Control group were exposed to the context of drug administration, and the ultrasonic vocalizations were recorded for 20 min. Withdrawal groups, 30 min after last injection, were left undisturbed (in their home cages), whilst they were subjected to 2-week withdrawal period. On day 28, the rats were re-exposed to the context of morphine/saline administration and ultrasonic vocalization was recorded. Each rat was immediately decapitated after USVs recording session, and its brain tissues were frozen. The USVs were recorded individually for each rat, in a dark room with dim red light.

USV recordings were processed using a proprietary RatRec Pro software; individual calls were extracted and counted. All identified episodes were of the "50 kHz appetitive" class, no "aversive 22 kHz" alarm calls were recorded.

For each rat, tissue samples of 6 brain structures were extracted: medial prefrontal cortex (mPFC), nucleus accumbens (NAcc), striatum (Cpu), hippocampus (Hipp), amygdala and ventral tegmental area (VTA).

In each sample, levels of 15 compounds were measured: norepinephrine (NA), 3-methoxy-4-hydroxyphenylglycol (MHPG), 3,4-dihydroxyphenylacetic acid (DOPAC), dopamine (DA), 5-hydroxyindoleacetic acid (5-HIAA), homovanillic acid (HVA), serotonin (5-HT), 3-methoxytyramine (3-MT), taurine, glutamine (Gln), glycine (Gly), aspartic acid (Asp), glutamic acid (Glu), alanine (Ala) and gamma-aminobutyric acid (GABA).