Research reportEffects of novelty and pain on behavior and hippocampal extracellular ACh levels in male and female rats
Introduction
The hippocampus plays an important role in processes such as arousal, learning and memory. Since physiological (aging) and pathological (neurodegenerative) alterations of the hippocampus can affect these functions, its circuits have been the object of several studies. Aversive stimuli have repeatedly been found to modulate the main hippocampal neuronal systems; for instance, electrophysiological data demonstrate that short-lasting nociceptive stimuli activate the septo-hippocampal cholinergic pathway, synchronize the hippocampal EEG and induce long-term depression of the hippocampal pyramidal neurons 16, 29, 30, 43. All these studies were carried out in males without consideration of the several anatomical and functional sex differences documented in rodents 5, 6, 23, 34, 39, 45. The important effects exerted by gonadal hormones on the hippocampal formation are indicated in rats by the peculiar features induced by estradiol in learning and memory processes 32, 37, 42, by the changes in c-Fos expression induced by testosterone in male rats exposed to a novel environment [28], by the estrogen-induced increase in choline acetyltransferase activity in ovariectomized females 19, 25, 33, by the fluctuations recorded in the multiunit activity of the hippocampus [26]and by the change in activity and seizure threshold observed in females during the estrous cycle 21, 44, 46.
Sex differences have repeatedly been described in behaviors shown by the animals in response to aversive stimuli 3, 7, 9. For instance, females are less anxious and more active than males when exposed to a novel environment [7]. In addition, while behavioral impairment in females is lower than in males after an acute stressful stimulus, repetition of the same stimulus shows females to be more affected than males [27]. These sex differences appear to be related to the levels of the hypothalamus–pituitary–adrenal-axis-related hormones [20]which differ between females and males both in basal conditions and after aversive stimuli 2, 31.
The aim of the present experiments was to detect sex differences in the septo-hippocampal cholinergic pathways in response to both novelty (Novelty+Object) and persistent pain (Formalin test). To this purpose we measured the extracellular ACh levels in the hippocampus (by the transversal microdialysis technique) and the animal's behavior during the same collecting periods. Freely-moving rats were subjected to two days of testing, on the first and the second day after the microdialysis probe implantation. On the first day the animals were placed in a new cage and after 30 min an object was added to the cage (Novelty/Object test). On the second day the Formalin test was carried out in the home cage, which limited the effects of `novelty'. In females the estrous cycle phase was determined at the end of each day of testing. These experiments have been presented as an abstract [14].
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Animals
Twelve male and 12 female Wistar rats, age matched, weighing 280–300 g and 240–260 g respectively, were used (Harlan-Nossan, Milan, Italy). The animals, housed in groups of 3 until surgery, were maintained on a 12-h light/dark schedule (lights off at 07.00 h), at 23°C and 53% humidity, with food and water available ad libitum, for at least one week before the beginning of the experiment. All experiments were carried out according to the International Association for the Study of Pain guidelines
Experimental Day 1
During the 1-h settling period and Baseline 1, females were clearly more active in their home cage than males, with a tendency to escape, to jump out of the cage and to bite the microdialysis tubes and minitubes. The vaginal smears carried out at the end of Day 1 showed 8 females in diestrus, 2 in estrus and 2 in proestrus.
Discussion
Utilization of the microdialysis technique in freely moving animals allowed us to study the ACh release in the dorsal hippocampus of male and female rats exposed to mild stressful stimuli (caused by changes in environment) and to an acute persistent painful stimulation (Formalin test). The results showed that: 1) on Day 1, females have higher levels of extracellular ACh release than males; this difference was no longer evident 48 h after surgery. 2) during the two days of testing, sex
Acknowledgements
This work was supported by a grant from MURST (funds 60% and 40%). I.C. wishes to thank `Fondi di Qualità' from Azienda Ospedaliera Senese.
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