PSYCHOLOGICAL STRESS REDUCES CYCLIC 3′,5′‐ADENOSINE MONOPHOSPHATE LEVELS IN THE CEREBRAL CORTEX OF CONSCIOUS RATS, AS DETERMINED BY A NEW CRYOGENIC METHOD OF RAPID TISSUE FIXATION¹

Abstract

Abstract— Cryoplates were implanted on the surface of the cortex in 32 chronic rat preparations. These devices were used both to freeze and to extract small samples of tissue. Coolant was circulated through each device by small flexible polyethylene tubes. Two series of experiments were performed. In the first, the animals were unrestrained and showed no behavioral signs of stress during the freeze fixation. The temperature responses of the cryoplates were very rapid (−632°C/s), and samples more than 1 mm thick were frozen and extracted within a few hundred ms following the onset of cooling. Each sample was analyzed for 3′.5′‐adenosine monophosphate (cyclic AMP) and protein content. The results from the cryoplate group (25.6 ± 15.6pmol cyclic AMP/mg protein) were compared to those obtained from two other groups in which freeze fixation was produced by immersion in liquid nitrogen (13.6 ± 4.6pmol/mg protein) or decapitation into liquid nitrogen (18.6 + 7.6pmol/mg protein). In the second series of experiments, three types of stress (limb restraint, non‐adaptation to the experimental situation, and moderate cutaneous electric shock) were induced separately in order to determine the influence of each on cortical levels of cyclic AMP. Control animals were highly adapted to the experimental situation, freely moving and not shocked. The samples from each of the stressed groups showed a statistically significant (P ≤. 0.01) reduction in cyclic AMP in comparison with the level in the controls (control: 29.3 pmol/mg protein; restrained: 14.2pmol/mg protein; unadapted: 9.6pmol/mg protein; shocked: 7.1 pmol/mg protein). Thus, psychological and physical stress reduced cyclic AMP content in parietal cortex. Results from the second series of experiments suggest that the significantly higher mean and larger standard deviation of the cryoplate group in the first series are due to less psychological and physical stress being evoked by our method; different types of stress appear to account for the two different lower levels found in the immersion and decapitation groups. We believe that our method of cryogenic tissue fixation offers an improved approach to study of the neurochemical correlates of behavioral and neuroelectric events in the conscious animal.