Sex differences in benzodiazepine binding in the frontal cortex and amygdala of the rat 24 hours after restraint stress.

Marked sex differences have been reported in behavioural responses of rats 24 h after exposure to a brief period of restraint (RT) stress. In the present study, differences in benzodiazepine (BZ) binding between male and female rat litter-mates randomly allocated to control or RT groups were investigated 24 h after RT. Scatchard analysis, using [3H] flunitrazepam, was carried out on the the frontal cortex and amygdala. In the frontal cortex, females had a significantly lower affinity and a greater number of BZ receptors than males; males, but not females, showed increased affinity after RT. In the amygdala, there was a tendency towards a greater number of BZ receptors in females, with no effect of RT on receptor number or affinity. These results provide evidence of sex differences in BZ binding both under basal conditions and 24 h after RT, which could contribute to the behavioural sex differences already reported.

Formalin-induced changes in adrenocorticotropic hormone and corticosterone plasma levels and hippocampal choline acetyltransferase activity in male and female rats.

Formalin (10%) induces higher levels of licking and flexing in female than in male rats, as shown in the present study. In order to ascertain the neural and hormonal modifications that accompany these behavioural differences, we determined the activity of choline acetyltransferase in the hippocampus and the levels of adrenocorticotropic hormone and corticosterone in the plasma. Two concentrations of formalin were used (50 microliters; 0.1% or 10%). Formalin was injected subcutaneously in the dorsal part of the hindpaw, and the animal's behaviour was then recorded for 60 min in a familiar open-field apparatus. Hippocampal choline acetyltransferase activity did not differ between the two genders in controls, while a significant gender difference was present in both formalin-injected groups, with higher levels in females than in males. This was the result of a decrease in males but not in females. In contrast, adrenocorticotropic hormone was increased by both formalin concentrations in females; corticosterone was not affected by treatment in either gender. Results are discussed in the light of the morphological and functional differences between the two genders in the hippocampus and in the hypothalamo-pituitary-adrenal axis.

Differential effects of restraint and novelty on the social behaviour of female rats.

In a previous work, we have described an increase of agonistic behaviour 24 h after a single 30-min restraint (RT) in female rats. The present work was aimed at assessing whether this effect was actually due to physical immobilization or was a mere consequence of the exposure to a novel environment (the restraining box in the experimental room). Dioestrous females were either left undisturbed in their home cages (Control), restrained (RT), or subjected to the same experimental schedule as the restrained ones, but placed in a living cage instead of in the restraining device (Novelty). Twenty-four hours after treatment, the social behaviour of the experimental females was recorded during a 20-min encounter, in a neutral arena, with an unfamiliar conspecific female in the same oestrous cycle phase. Novelty did affect behaviour, but in a way completely different from RT: while RT increased the frequencies of agonism and other social behaviours, novelty caused a selective decrease of agonism. The effects of RT on the social behaviour of female rats appear therefore to be specific and independent of those caused by novelty.

Sex-related effects on behaviour and beta-endorphin of different intensities of formalin pain in rats.

The effects of two intensities of formalin pain on behaviour and beta-Endorphin (beta-EP) concentration in the brain and pituitary were studied in male and female rats. The animals were familiarized with the Hole-Board apparatus for 3 days, and then, after a subcutaneous injection of formalin (50 microliter, 0.1 or 10%) or Sham-injection (Control) in the hindpaw, they were tested in the Hole-Board for 60 min. Licking, Flexing and Paw-Jerk of the injected limb were recorded. beta-EP concentration was determined in the hypothalamus (HYP), the periaqueductal gray matter (PAG), the anterior pituitary (AP) and the neurointermediate lobe (NIL). Licking and Flexing durations were greater in females than males only with formalin 10%. Sex differences in beta-EP concentration between the Control groups were found in all tissues except the HYP; beta-EP levels were higher in females in the PAG and NIL, but greater in the AP in males. beta-EP concentration increased in males in the HYP and NIL with formalin 10%; in females, a decrease was found in the HYP with formalin 0.1%. The present results suggest that: (a) there are differences between males and females in the responses to formalin pain, and the nature (pattern and duration) of the sex differences varies according to the pain intensity; (b) there are differences in beta-EP concentration between the two sexes in control animals, and male and female rats also exhibit differences in the modifications of beta-EP in response to formalin-induced pain.

Behavioural effects of different intensities of formalin pain in rats.

The effects of two concentrations of formalin (0.1% and 10%) on Licking, Flexing and Paw-Jerk, and standard measures of activity, were studied in male rats during three experimental conditions: Box, Open-Field and Novel Object. Pain-evoked responses were present in all formalin-injected animals, with greater intensity in the group injected with formalin 10%. In this group Rearing and Olfactory Exploration were reduced with respect to the controls, locomotion was inhibited to the point that it was virtually absent during the last part of the test. In contrast, the lower formalin concentration not only failed to inhibit the locomotor/exploratory behaviours but also appeared to induce a general activation of behaviour, as suggested by the longer durations of Pendulum, the absence of Sleeping-like episodes and the higher number of Approaches to the object found in this group. The results of the present experiment underline the importance of a detailed analysis of behaviour in animal models of pain and support the view that the intensity of pain plays a crucial role in its behavioural effects.

Effects of formalin-induced pain on ACTH, beta-endorphin, corticosterone and interleukin-6 plasma levels in rats.

The behavioral and immunoendocrine effects of formalin-induced pain were studied in male rats following a subcutaneous injection of formalin (50 microliters; 0.1%, F01 groups, 10%, F10 groups) or sham injection (control groups). After treatment, animals were tested in a transparent open field for either 30 or 60 min and thereafter sacrificed by decapitation. Plasma was collected for adrenocorticotropic hormone (ACTH), corticosterone, beta-endorphin (beta-EP) and interleukin-6 (IL-6) determinations. Pain-evoked responses (licking, flexing, paw jerk), standard measures of activity (locomotion, rearing, olfactory exploration) and self-grooming were recorded. The higher formalin concentration induced stronger pain-evoked behavioral responses, paralleled by higher levels of ACTH, beta-EP and IL-6, but did not affect the other behavioral parameters. In contrast, the lower formalin concentration induced a marked increase in locomotion and rearing and a decrease in ACTH levels. In both formalin-injected groups, corticosterone did not differ from controls.

Effects of single restraint on the defensive behavior of male and female rats.

The effects of single aversive stimulation due to restraint (RT) on behavioral responses to unfamiliar conspecifics were studied in male and female rats. The Resident-intruder paradigm was adopted, RT animals and their controls playing the role of intruders. Introductory and agonistic behaviors of both intruders and residents were recorded. In males, RT increased both the number of subjects which showed freezing and freezing duration, and this was independent of the amount of aggression received by the residents. By contrast, no change was found in active defense. Increased passive defense was not paralleled by a complete inhibition of aggression. The latter was rare, but not absent, and occurred in RT males as often as in their controls. Females never showed freezing and, unlike males, resorted to a fully active defensive strategy. RT females were the preferential targets of residents' high-intensity aggression, but showed the same rate of defensive responding as control females. The crucial role played in studies of social behavior by testing conditions and mutual influences between the behavior of experimental subjects and residents are discussed.

Sex differences in the behavioural response to persistent pain in rats.

The behavioural response to formalin-induced persistent pain was examined in male and female rats both unfamiliar and familiar with the test apparatus. Rats were subcutaneously injected with 50 microliters of formalin (10%) in the hindpaw and placed in the test cage (60 min). Licking and Flexing duration and Paw-Jerk frequency were recorded. Licking and Flexing lasted longer in females than males, while Paw-Jerk occurred in both sexes with comparable frequencies. Flexing and Paw-Jerk were lower in animals unfamiliar with the test apparatus. Therefore, behavioural responses to pain appeared to be affected by sex and familiarization with the experimental setting in different and independent ways.

Social stress by repeated defeat: effects on social behaviour and emotionality.

The consequences of repeated defeat stress on social and non-social behaviours were assessed in male rats 24 h after the last defeat. Aggressive, defensive, introductory and affiliative items of both experimental animals and their opponents were recorded in a social behaviour test, while emotionality, exploration and general motor activity were scored in the Emergency, Hole-Board, and Elevated Plus-Maze tests. In addition to a dramatic loss of body weight, a selective inhibition of aggression was observed in the stressed experimental subjects, paralleled by decreased defence in their opponents. In the stressed animals, no change was found in other social and non-social behaviours; in particular, defence and emotionality were unaffected. This shows that, under our experimental conditions, the inhibition of aggression, which has often been reported to parallel an increase in defence after social and non-social aversive stimulation, was not dependent on a concomitant activation of a prevailing defensive motivational system, sustained by increased emotionality and fear. As the same result, namely a selective inhibition of aggression with no effect on defence, was obtained after exposure to a non-social stressor (restraint), the hypothesis is advanced that the threshold for stress-induced behavioural changes is lower for aggression than for any other behavioural and motivational system, including that leading to defence. The inhibition of aggression would therefore be a direct response to stress and not a by-product of the activation of a fear-based defensive system.

Decrease of hippocampal choline acetyltransferase activity induced by formalin pain.

The involvement of the hippocampal choline acetyltransferase (ChAT) activity in the response to tonic pain was investigated in rats injected with formalin, either 50 microliters 10% or 50 microliters 0.1%. Hippocampal ChAT activity was found to be reduced both 30 and 60 min after injection of the higher concentration of formalin but only 30 min after the lower one. Results indicate that the decrease in ChAT activity depends upon the presence of the nociceptive input rather than its magnitude. The hippocampal formation is involved in the specific behavioural response to pain, namely licking.