Research report
Effects of gonadal hormones and persistent pain on non-spatial working memory in male and female rats

https://doi.org/10.1016/S0166-4328(01)00195-4Get rights and content

Abstract

There are indications of a modulatory role carried out by gonadal hormones and pain in cognitive functions. We have examined this issue in male and female rats by assessing the impact of gonadectomy and persistent pain on the object recognition test. Intact and gonadectomized male and female rats were exposed to an open field (15 min) in which three objects were placed (Trial 1); the same test was repeated 2 h later (Trial 2), after the replacement of a “familiar” object with a novel one. Three days later (Day 2), the same procedure was repeated (Trial 3 and 4 with 2 h in between) but half of the animals were exposed to formalin-injection immediately before Trial 3. The latency, frequency and duration of approaching the three objects were recorded in each trial and compared by sex, gonadectomy and formalin treatment. The results showed that gonadectomized males and females had lower levels of approach to all objects and less locomotor/exploratory activity than intact animals in all experimental trials; their behaviour was not affected by repetition of the test or by pain. On Day 1, intact males showed a higher level of approach to the novel object than females. In intact males, the 2 h delay between the first and second trial failed to induce any significant modification of exploration of the novel object with respect to the familiar one, while in intact females the novel object was approached much less than the familiar one. Similarly on Day 2, the novel object was approached for a longer time by intact males than by all the other groups. In conclusion, our data show that physiological levels of circulating gonadal hormones significantly affected the performance of male but not female rats when exposed to the object recognition test.

Introduction

In humans as well as in experimental animals, the main gonadal hormones are androgen and estrogen. The effects of gonadal hormones on the central nervous system (CNS) have been defined as ‘organizational’ and ‘activational’ [35]. The former, due to the perinatal action of gonadal hormones, are permanent and different in the two sexes; these effects are evident not only in the primary and secondary sex characteristics, but also in the CNS where many structures are anatomically and functionally different between males and females [29], [36]. The activational effects are instead transient and depend on the gonadal hormone levels. Study of the various sex differences induced by gonadal hormones includes the analysis of cognitive aspects such as learning and memory processes [7], [27]. Women perform better than men on verbal and memory tasks, whereas men excel in spatial tasks [9], [10]. Rodents, commonly used to study cognitive functions, also show sex differences: males tend to perform better than females in a variety of spatial [28] and memory tasks [42], while females show a better performance in a visual memory test [15]. Studies of maze performance indicate that estradiol exerts an activational effect on spatial memory in females [25]; these actions have been attributed to the estrogen-induced effects on dendritic spine density and synapses numbers [18], [46], [47], neurotrophic mechanisms [13], [17], [44] and NMDA receptor protein levels [14] in the hippocampus, a brain area repeatedly shown to be involved in spatial performance, arousal, learning and memory. In addition to these functions, the hippocampal formation also participates in pain processes [3], [22]. Indeed hippocampal activity is changed by persistent pain and these effects are different in males and females [2], [5].

The purpose of the present study, conducted with a 2×2 test paradigm, was to determine whether gonadal hormones affect working memory, i.e. the type of memory whose impairment is first noted in Alzheimer's Disease in humans and which is impaired in aging rats [39]. In our case, working memory requires the rat to realize that an object in the experimental apparatus has been replaced by a novel one.

The Object Recognition Test (ORT) measures a form of non-spatial working memory, based on spontaneous exploratory activity, which may be considered episodic memory [11]. An important issue rarely considered in this type of experiment is the testing of animals under strictly physiological conditions. In particular, the testing of rodents during the light phase obliges the animals to perform cognitive tasks during their sleeping period. Restriction of food is also commonly used and the animals are rarely familiar with the testing apparatus.

We hypothesized that the ORT might induce different behavioural responses in male and female rats and that the lack of circulating gonadal hormones, as observed after gonadectomy, may change these responses. In addition, since painful conditions are known to change levels of attention and the ability to memorize [21], we tested ORT with male and female rats experiencing a tonic, long-lasting painful stimulus. In the first part of the experiment, intact and gonadectomized female and male rats were first placed in an open field with three objects and then were re-exposed to the same open field with a different combination of objects. In the second part of the experiment, the effect of a persistent nociceptive stimulation (formalin test) [3] applied just before the beginning of the test was evaluated.

Section snippets

Subjects

Sixty-four male and female Wistar rats, aged 3 months at the onset of behavioural testing (Harlan, Correzzana, Italy) were housed four per cage, same sex, to allow social interactions. They were given unlimited access to laboratory chow and water, and were maintained on a 12:12 light:dark cycle (light off at 7.00 h).

Gonadectomy: The rats were anaesthetised with pentobarbital (40 mg/kg, i.p.) and bilaterally gonadectomized (GDX) or subjected to a sham surgical procedure (Intact) as previously

Experimental Day 1: effects of sex and gonadectomy on object recognition (Trial 1)

Once inside the open field apparatus, the rats moved around in the cage approaching the three objects (sniffing, marking, licking) or simply walking around them. The latency of approach (Table 1) did not differ between the sexes; the significance of the factor Gonadectomy for OBJ 1 (P<0.006) was due to the lower latency of approach to this object in GDX animals than in intact ones. There were no differences for the other objects. The frequency of approach (Table 1) also did not differ between

Discussion

The main results of the present experiment are the sex- and GDX-induced differences in the behaviours related to the object recognition test apparatus and the modulatory effect on object recognition exerted by formalin treatment.

Acknowledgements

This work was supported by University of Siena and MURST (ex-40% funds).

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