Ethological study of the effects of tetrahydroaminoacridine (THA) on social recognition in rats

Psychopharmacology (1994) 114:644-650 Psychopharmacology © Springer-Verlag 1994

Ethological study of the effects of Tetrahydroaminoacridine (THA) on social recognition in rats Giiles Gheusi, Rose-Marie Bluthe, Glyn Goodall, Robert Dantzer

Neurobiologie Int6grative, INSERM U. 394, Domaine de Carreire-Rue Camille Saint Sa6ns, F-33077 Bordeaux Cedex, France

Received: 16 April 1993 / Final version: 28 September 1993

Abstract. Two major difficulties confront ethophar- macological investigations on cognitive abilities such as social recognition in drug-treated animals involved in free social interactions. The first concerns the choice of the most relevant behaviours, those reflecting the cognitive abilities attributed to the animals and assessing the specificity of the drug activity, and those reflecting non-specific drug effects. The second refers to the experimenter's awareness that in contrast to physical objects, social stimuli respond to drug-treated subjects and that their own level of responsiveness may influence the changes of drug-treated subjects' social interest. In addition, their contribution may vary according to the different treatments the drug-treated subjects receive. In examining the effects of tetrahydroaminoacridine (THA) at doses of 0.3, 1 and 3 mg/kg on the ability of adult male rats to recognize previously encountered conspecifics, we attempted to take into consideration such difficulties. A detailed behavioural profile of drug-treated rats was reported to separate specific from non-specific effects of THA. In addition, rats were assigned an index of responsibility for contact which takes into account the interactive dimension of each dyad and allows relevant comparisons between different treatments. The doses of THA which were found to decrease the duration of exploration of a familiar juvenile were also found to decrease the number of contacts initiated by the drug-treated subjects. THA induced a relative increase in body care by comparison to saline treatment. However, it had no effect of locomotor activity and rearing of the subjects. These findings enable dissociation of the effects of THA on cognitive versus non-cognitive processes.

Key words: Social recognition - Cognition - Social interaction - Ethological method - Tetrahydroaminoacridine - Rat

Despite controversy concerning its precise actions, THA (9-amino-l,2,3,4-tetrahydroacridine) has been demon- strated to improve memory both in animals (Bartus et al.

Correspondence to." G. Gheusi

1983; Flood et al. 1985; Fitten et al. 1987, 1988; Hodges et al. 1990) and humans (Summers et at. 1981, 1986; Kaye et al. 1982; Weintraub and Standish 1988). Different mech- anisms of action of THA through cholinergic systems have been proposed. THA was first reported to be a cholinesterase inhibitor by Shaw and Bentley (1953). This was subsequently confirmed both in vitro and in vivo (for a review see Freeman and Dawson 1991; and Hodges et al. 1990 for controversial results). Other work suggested that THA not only acts as a cholinesterase inhibitor but interacts with M2 muscarinic and nicotinic receptors (Perry et al. 1988; Nordberg et al. 1989) which are important for the regulation of the release of Ach in the cholinergic terminal. Furthermore, THA has K+chan - nel blocking properties (Osterrieder 1987; Druckarch et al. 1987; Stevens and Cotman 1987), thus increasing changes in biogenic amines (dopamine, norepinephrine) and their metabolites (Tachiki et al. 1988). Besides the encouraging results showing the efficacy of THA in Alzheimer's disease patients, undesirable side effects such as nausea and liver damage have also been reported (Kumar 1988).

Because of its memory enhancing ability, THA has been chosen in the present study to assess the role of specific and non-specific factors in drug-induced changes in sodal recognition. Social recognition assesses the ability of adult male rats to recognize juvenite conspecifics. All else being equal, rats spend more time investigating a novel conspecific than a familiar one. Thus, re-exposure to the same juvenile after a short interval leads to a dra- matic reduction of investigatory behaviour by adult male rats. By contrast, when a different conspecific is presented following the same interval, the duration of investigation is comparable to the one displayed during the first exposure (Thor and Holloway 1982).

Studies of the time-course of this form of memory show that it lasts for about 40 min in intact male rats (Sekigushi et al. 1991) and more than 2 h in castrated males and intact females (Bluth6 and Dantzer 1990; Bluth6 et al. 1990). In adult male rats, social recognition is based on chemosensory cues processed by the vomeronasal organ (Bluth6 and Dantzer 1993) and is modulated by androgen-dependent vasopressinergic neurons origin- ating from the bed nucleus of the stria terminalis and

projec t ing to the la tera l sep tum (Dantzer et al. 1988; Bluth6 and Dan t ze r 1992).

A m o n g the different advan tages it provides , the test of social recogni t ion is based on the spon taneous invest iga- to ry behav iou r of the subject. I t does not require posi t ive or negat ive exper imenta l reinforcements , or learning of rules. In addi t ion , it has been shown to be sensitive to noo t rop i c and cho l inomimet ic drugs (P6rio et al. 1989). Final ly , it offers the o p p o r t u n i t y to s tudy a form of work ing m e m o r y in the still poo r ly explored d o m a i n of social cogni t ion.

Beside our interest in invest igat ing the effect of T H A on social recogni t ion in rats, we a t t empted to address two l imi ta t ions of the m e t h o d o l o g y used to assess recogni t ion- m e m o r y processes in this test. The first is general ly ad- dressed to an imal models of cogni t ion and concerns the difficulty of separa t ing cogni t ive from non-cogni t ive (such as arousal) processes (Dawson et al. 1992). This r e m a r k is no t ewor thy in regard to the side effects of T H A repor ted in the l i terature. The second l imi ta t ion is more specific to the test of social recogni t ion. Since, in con t ras t to a physical object, a social s t imulus r e sponds to its inves t iga tor which in tu rn feeds back to him, one mus t consider the social interest of the exper imenta l subject as mo t iva t ed bo th by its spon taneous inves t iga tory behav iou r and by the activit ies (especially social contac ts and separat ions) d i sp layed by the social st imulus. In this sense, the experi- men ta l subject 's social exp lo ra t ion mus t be bet ter viewed as a relative, ra ther than absolute , measure of its social interest t owards a social s t imulus accord ing to the level of in te rac t ion tha t character izes the encounter . F r o m a phar - maco log ica l po in t of view, this cons idera t ion should prove to be more powerful as it is poss ible tha t the behav ioura l changes vary accord ing to the dose of the d rug admin i s t r a t ed and consequent ly in turn modi fy the non- t r ea t ed par tner ' s behav iou r (Dixon et al. 1990; G a o and Cut ler 1992a, b; Mitchel l and Redfern 1992). Keep ing these cons idera t ions in mind, we inves t iga ted the effect of T H A on social recogni t ion in male rats with two ma jo r objectives: 1) assessing m e m o r y processes in exper imenta l subjects based on rel iable measure of social interest, 2) p rov id ing in format ion a b o u t specificity of drug act ivi ty by record ing convenient behav ioura l i tems be longing to the exper imenta l subject 's repertoire . In this study, T H A was injected to adu l t male rats immedia te ly after a first pres- en ta t ion of a juveni le conspecific and thus before their re-exposure to the same juvenile. Consequent ly , T H A should no t affect the acquis i t ion phase of the identif ica- t ion processes, but the conso l ida t ion and poss ib ly re- t r ieval processes of s tored in format ion in adults .

Material and methods

Animals

Male Wistar rats (Charles River, France) weighing 250-270 g were used. Upon their arrival in the laboratory they were housed in groups of five in plastic cages (44 x 28 x 18 cm), at 22 _+ 2 °C on a reverse dark-light cycle (lights on from 4:00 p.m. to 4.00 a.m.). They were allowed a minimum of 14 days for habituation to their new environment. Food and drinking water were available ad libitum.

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Rats were housed singly in plastic cages (40 x 20 x 18 cm) at least 3 days before the tests began. Laboratory-reared juvenile male Wistar rats (4-5 weeks old) housed in groups of ten in a separate room served as social stimuli.

General Procedure

The procedure used has been previously described by Dantzer et al. (1987). An unfamiliar juvenile was placed in the home cage of an adult for 5 min. This was followed by a second 5-rain exposure to the same or a different juvenile 30 or 120 min later. The behaviour of the adult male was examined under red light on a video monitor in an adjacent room. Between two successive presentations juveniles were kept individually in small plastic cages (23 x 13 x 12 cm), the floor of which was covered with fresh bedding. Ten adult male rats were used in this experiment. Tests were conducted during the dark period of the cycle. They began at 8.30 a.m. and ended at least 1 h before the onset of the light period.

Before the start of the experiment adult male rats were subjected to two sessions, one per day, of familiarisation to the test conditions. They were put in the observation room under the camera and a juvenile rat was introduced in their home cage for a 5-rain period.

Durations of social and non-social behaviours displayed by the adult male were recorded by typing preset keys on the keyboard of an IBM microcomputer. Social investigatory behaviours consisted of close following, head sniffing, body sniffing, and anogenital sniffing. Non-social behaviours (i.e., not-directed toward the juvenile) included locomotor activity, rearing, and self-grooming.

We also recorded the number of occasions when contact was established by the adult (UA) and by the juvenile (UB). In the same way, the number of occasions when separation was due to the adult (SA) and by the juvenile (SB) was recorded. This enabled us to calculate an index measuring the adult's responsibility for contact (Ia). This index was calculated as follows (Martin and Bateson 1986):

Ia = UA/(UA + UB) - SA/(SA + SB)

Such an index has several advantages. It ranges from - 1 to + 1. A value of - 1 is obtained when only the juvenile initiates

contact and never separates from the adult and when, conversely, the adult never initiates contact and only separates from the juvenile. In contrast, a value of indicates + 1 that the adult male is totally responsible for contact and never responsible for separation which is always displayed by the juvenile. The major interest of such an index lies in allowing comparisons between different situations. In our case this index should allow one to see if the responsibility of the adult male for contact declines as the juvenile is better recognised following different doses of THA.

Experimental protocols

Pre-drug recognition. Before drug treatment a juvenile recognition procedure was carried out to make sure that the adult rats (n = 10) showed normal recognition for an inter-exposure interval (IEI) of 30 rain and no recognition for a 120-min interval. Only total durations of social investigation were recorded in this experiment.

Effects of THA on interaction with familiar juvenile. An unfamiliar juvenile was placed in the home cage of each adult male for 5 rain. THA was administrated intraperitoneally immediately after the first presentation of the juvenile. The same juvenile was presented to the adult male 2 h later for another 5-min period.

Effects of THA on interaction with non-familiar juvenile. In order to assess the specificity of THA, a juvenile different from that used during the first exposure was presented 2 h after the adult males received active doses of THA intraperitoneally. Any significant reduction in social exploration of the second juvenile was thus considered as reflecting non-specific effects of the drug (Dantzer et al. 1987).

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Drug treatment

THA (Sigma, St. Louis, Mo., USA) was dissolved in 0.9% saline to the appropriate concentration just before testing. Injections were given intraperitoneally (IP) in a volume of t ml/kg. Three doses were tested 0.3, 1 and 3 mg/kg. Placebo treatment consisted of the same volume of saline. Each rat received the three doses of THA and the vehicle alone in different sessions in a random order. Different treatments were separated by 3 days.

Statistical analysis

Statistical analysis was performed using non-parametric tests. The Friedman test was used to assess the variance of behavioural measures over different treatments. Paired comparisons were carried out using a two-tailed Wilcoxon T test according to the Conover method (Conover 1980).

Results

Juvenile recognition before initiation of drug treatment in adult rats. Time dependency of social recognition

Adult males exposed to the same juvenile for the second time 30 min after the first exposure spent less time investi- gat ing it (mean _+ SEM: 84.3 4- 11.25) than on the first exposure (mean _+ SEM:129 + 8.8) (T = 2.8, P < 0.01). In contrast , when exposed to the same juvenile for the second time 120 min after the first exposure they spent as much time (mean _+ SEM:123 + 11.9) as they did during the first exposure (mean __ SEM: 121 +_ 8.3) (T = 1.17, P > 0.05). These results confirm that the subjects used in this experiment are able to recognize a juvenile 30 min after, but not 120 min after their exposure to this part icular juvenile. Thus, 120 rain represents a sufficient interval to investi- gate the effects of a potential proactive substance such as T H A on social recognit ion of adult male rats.

Effects of THA on interaction with familiar juvenile

Social investigatory behaviour in adult rats. Mean durations of investigatory behaviour during each exposure phase of the test session are plotted for each T H A treatment in Fig. 1. Evolut ion of this behaviour over the 5-rain test is shown, as well as totals for the entire test. Compar i - sons of social investigation times between the first and the second exposure to the same juvenile showed that there was no significant reduct ion in the durat ion of investiga- t ion 120 min following saline t reatment or T H A treatment at a dose of 0.3 mg/kg (respectively, T = 0.46, P > 0.05 and T = 0.56, P > 0.05). There was, however, a significant decrease in the dura t ion of investigation times of the same juvenile following both 1 and 3 mg/kg T H A (respectively, T = 2.39, P < 0.05 and T = 2.60, P < 0.01).

With the highest dose of THA, comparisons of mean dura t ion per minute of social investigation during the first and the second exposure clearly showed that recognit ion in adult males occurred immediately after the introduc- t ion of the juvenile (first minute, T = 2.59, P < 0.01 and second minute, T = 3.47, P < 0.05).

The dura t ion of different specific social behaviours during each exposure phase is illustrated as a function of T H A treatment in Table 1. Anogenital sniffing decreased in dura t ion during the second exposure to a familiar juvenile at the doses of 1 and 3 mg/kg (respectively, T = 2.59, P < 0.01 and T = 2.7, P < 0.01). Dura t ions of head and b o d y sniffing/grooming showed no significant variat ion between the first and the second exposure following each t reatment (0.15 < T < 1.88, P > 0.05). The subjects spent significantly less time in close following on the second exposure to the same juvenile, whatever the treatment (saline, T = 2 . 1 9 , P < 0 . 0 5 ; T H A 0.3mg/kg, T = 1.78, P = 0.07; T H A I mg/kg, T = 2.60, P < 0.01; T H A 3 mg/kg, r = 2.80, P < 0.01).

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Fig. 1. Effect of tet rahydroaminoacridine (THA) on social recognition in adult male rats (n = 10). Circles represent the mean ( ± SEM) duration per minute of investigation of the juvenile by adult rats on the first exposure (unfilled circles) and the second exposure (filled circles) carried out 120 min later, for saline and different doses of THA. Bars represent the mean ( +_ SEM) total duration of investigation of the juvenile by adult rats on the two exposure phases of each test session, for saline and different doses of THA. (Difference between first and second exposure: *P < O.05;**P < 0.01)

Table 1. Effect of tetrahydroaminoacridine (THA) on specific social behaviours directed by adult rats toward the juvenile. Values represent the mean ( ___ SEM) total duration of social behaviours of ten animals on the two 5-min exposure phases of each test session, for saline and different doses of THA. The second exposure took place 120 min after the end of the first one. (Difference between first and second exposure: NS, not significant; *P < 0.05; **P < 0.01)

Behavioural Doses 1st exposure 2nd exposure P categories (mg/kg) (same juvenile)

0 4.72 + 0.89 2.29 ,+ 0.48 * Following 0.3 3.08 _+ 0.38 2.03 +_ 0.51 NS

1 2.16 _+ 0.33 0.76 +_ 0.23 * 3 4.58 _+ 0.74 1.05 _+ 0.45 **

0 18.12 + 2.24 25.46 + 3.78 NS Head and body 0.3 16.93 ± 3.01 21.52 ± 3.52 NS sniffing 1 18.27 __ 3.55 23.08 + 2.39 NS

3 17.50_+ 2.05 22.43 _+ 4.19 NS

0 94.99 _+ 6.16 93.08 ,+ 9.40 NS Anogenital 0.3 101.25 __ 7.97 90.73 ± 6.98 NS sniffing 1 105.17 ,+ 9.79 80.13 ,+ 9.65 *

3 99.66_+ 8.31 59.92_+ 7.97 **

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for establishing contact decreased as the dose of T H A increased and became negative at the doses of 1 and 3 mg/kg THA, indicat ing that in these cases the n u m b e r of occasions on which the adul t male broke contact was greater than the number of occasions on which it init iated contact. This decrease was due to two effects. Firstly, dur ing the second exposure to the same juveni le the number of occasions which the pair was uni ted by the adult male treated with a dose of 3 mg/kg tended to be smaller than when other doses were given and did no t significantly differ from the n u m b e r of contacts init iated by the juvenile (T = 1.01, P > 0.05). Secondly, dur ing the second exposure after the adult males received the highest doses of T H A (1 and 3 mg/kg), they expressed a significantly greater n u m b e r of separat ions than the juveni le (respectively, T = 2.50, P < 0.01 and T - - 2.14, P < 0.05). Fur - thermore, juveniles avoided contact with adult males to a lesser extent dur ing the second exposure than dur ing the first exposure, as shown by the reduct ion in the n u m b e r of separations they init iated (saline, T = 2.20, P < 0.05; T H A 0.3 mg/kg, T = 1.85, P = 0.06; T H A 1 mg/kg, T = 2.56, P = 0.01; T H A 3 mg/kg. T = 2.49, P < 0.05).

Changes in social interest of the adult and the juvenile males as a function of THA treatments

Changes in the index of responsibil i ty of the adult for establishing contact dur ing each exposure is shown as a funct ion of T H A t rea tment in Table 2. Compar i sons for the first exposure of the four different test sessions revealed no significant var ia t ion (Friedman, F = 0.49, df = 3/27, P = 0.69). Positive mean values in the first four exposures indicated that the pair came together predomi- nant ly as a result of the adult male's initiative. O n the second exposure the dose effect was significant (Friedman, F = 4.15, d f = 3/27, P = 0.01). The index of responsibil i ty

Non-social behaviours

The effects of drug t rea tment on the dura t ion of non- social behaviours in adult male rats are summarized in Table 3. Paired compar isons revealed that the dura t ion of locomot ion and rearing were affected neither by saline (respectively, T = 0.97, P > 0.05 and T = 0.76, P > 0.05) nor by T H A t rea tment (THA 0.3mg/kg, T = 0.56, P > 0.05 and T = 1.07, P > 0.05; T H A 1 mg/kg, T = 1.02, P > 0.05 and T = 1.68, P > 0.05; T H A 3 mg/kg, T = 0.66, P > 0.05 and T = 1.37, P > 0.05). A slight, bu t significant, decrease in time spent self-grooming was observed in

Table 2. Effect of tetrahydroaminoacridine (THA) on the number of occasions on which the pair is united or separated either by the adult mate or by the juvenile and on the index of responsibility for contact of the adult male. Values represent the mean ( ,+ SEM) number of contacts and separations initiated by the adults and by the juveniles (n = 10 in each case) and the mean ( + SEM) score of the index of responsibility for contact of the adult male (Ia) on the first and the second exposure 120 rain later, for saline and different doses of THA. Ia, index of responsibility of adult males for maintaining contact with the juvenile (see text). For contacts and separations, differences shown during each exposure phase of the test session are between adult male and juvenile (NS, not significant; *P < 0.05; **P < 0.01). For Ia, differences were assessed over the different treatments using the Friedman test: first exposure NS, not significant; 2nd exposure, **P < 0.0I)

1st exposure 2nd exposure (same juvenile)

Doses Adult Juvenile Adult (mg/kg)

Juvenile

0 13.9 ,+ 1.11 4.1 ,+ 0.89 ** 0.3 12.3 __ 1.06 5.5 __ 0.97 **

Contacts 1 11.5 _+ 1.39 4.3 ± 0.54 ** 3 14.0 _+ 1.58 4.3 __+ 0.76 **

0 8.2 +_ 0.89 9.8 _+ 0.96 NS Separations 0.3 9.8 -I- 1.46 8.0 ± 1.07 NS

1 8.8 -1- 0.80 7.0 __ 0.87 NS 3 8.2 -I- t.45 10.1 +_ 1.61 NS 0 0.32 _+ 0.05

Ia 0.3 0.15 _+ 0.10 NS I 0.15 _+ 0.07 3 0.29 ± 0.12

tl.5 -+ 1.39 10.9 __ 1.45 12.2 ,+ 1.38 9.2 + 1.47

9.5 ,+ 1.27 10.2 ± 1.52 12.1 + 1.19 10.9 -+. 1.52

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4.2 _+ 0.73 ** 5.9 ___ 1.27 * 3.3 + 0.63 ** 6.4 + 0.65 NS

6.2 + 1.22 NS 6.6 ± 0.96 NS 3.4 ± 1.25 ** 4.7 ,., 0.92 *

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TaMe 3. Effects of tetrahydroaminoacridine (THA) on non-social activities shown by the adult males (n = 10). Values represent the mean ( ± SEM) total duration of non-social behaviours on the two 5-min exposure phases of each test session, for saline and different doses of THA. The second exposure took place 120 min after the end of the first one. (Difl?rence between first and second exposure: NS not significant; *P < 0.05)

Behavioural Doses 1st exposure 2nd exposure P categories (mg/kg) (same juvenile)

0 43.54 -t- 4.41 38.05 _+ 3.96 NS Locomotor 0.3 42.48 _+ 3.86 40.41 __ 3.02 NS activity 1 35.87 +_ 3.07 40.12 -F 2.88 NS

3 39.54 _+ 4.15 42.70 _+ 5.48 NS 0 96.33 _+ 9.87 92.07 _+ 14.26 NS

Rearing 0.3 106.55 _+ 10.87 101.32 _+ 9.69 NS 1 103.87 _+ 11.20 124.29 ± 12.37 NS 3 101.41 _+ 8 .30 112.22 ___ 12.19 NS 0 14.86 +__ 4.46 3.28 __ 1.12 *

Self 0.3 13.49 _+ 4.84 5.91 _+ 2.45 NS grooming 1 10.43 + 4.39 5.11 _+ 2.20 NS

3 14.68 + 4.64 25.55 _+ 8.15 NS

saline-treated subjects (T = 2.31, P < 0.05). N o significant variat ion appeared in terms of the durat ion of self-grooming between the first and second exposure to the same juvenile for all T H A doses (0.57 < T < 1.82, P > 0.05).

Effects of THA on interaction with unfamiliar juvenile

When adult rats were presented with a different juvenile 120 min after the initial presentat ion followed by treatment with an active dose of T H A (1 and 3 mg/kg) no significant variat ion in social investigation times could be detected (respectively, T = 0.66, P > 0.05 and T = 0.25, P > 0.05) (Fig. 2). Time-course of investigatory behaviour did no t differ between the two exposures to the different juveniles. Vehicle-treated subjects showed a greater dura- t ion of investigatory behaviour during the second exposure to a different juvenile ( T = 2.19, P < 0.05). When considering the index of responsibility for contact of the adult male, no significant variat ion appeared between the different exposures to different juveniles either before or after t reatment (first exposure, 0.1 < Ia < 0.28, Fr iedman, F = 1.23, df = 2/18, P > 0.05; second exposure to a different juvenile, 0.06 < Ia < 0.23, Fr iedman, F = 1.34, df= 2/18, P > 0.05). In all cases mean values of the index were positive, indicating a p redominan t contr ibut ion of the adults in establishing contact during the test sessions.

Specific social behaviours of adults presented with a different juvenile are summarized in Table 4. Time spent by adult males following the juveniles did not differ between the exposure phases of each test session (0.35 < T < 1.48, P > 0.05). N o significant decrease in dura t ion of head, b o d y or anogenital sniffing was observed in adults when exposed to a different juvenile. At most, a greater dura t ion of head, body and anogenital sniffing was detected in subjects treated with vehicle (head and b o d y sniffing, T = 2.09, P < 0.05; anogenital sniffing, T = 2.09, P < 0.05) with T H A 3 mg/kg (head and body sniffing, T = 2.70, P < 0.01). In addition, paired comparisons revealed that the dura t ion of non-social behaviours

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Fig. 2. Specificity of active doses (1 and 3 mg/kg) of tetrahydroaminoacridine (THA) on social recognition. Circles represent the mean ( _+ SEM) duration per minute of investigation of a juvenile by adult rats on the first exposure (unfilled circles) and of a different juvenile on the second exposure ~lled circles) carried out 120 min later, for saline and the two active doses of THA. Bars represent the mean ( ± SEM) total duration of investigation of the juveniles by adult rats on the two exposure phases of each test session, for saline and the two active doses of THA. (Difference between first and second exposure: *P < 0.05)

was no t affected by T H A treatment (0.15 < T < 0.56, P > 0.05). Al though a large inter-individual variability in time spent self-grooming could be observed, dura t ion of this behaviour tended to increase with the dose of THA.

Discussion

The present results show that T H A administered to adult male rats following the first presentat ion of a juvenile specifically decreases social interest towards the same juvenile when it is presented 2 h later. This effect is dose- dependent, the minimal effective dose being 1 mg/kg. These findings confirm those previously reported by Worms et al (1989). Paired compar isons of the time- course of the effects of T H A on durat ion of investigatory behaviour during the first and second exposure to the

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Table 4. Effect of active doses (1 and 3 mg/kg) of tetrahydroaminoacridine (THA) on social and non-social behaviours shown by the adult males (n = 10) presented with a different juvenile. Values represent the mean ( ± SEM) total duration of social and non-social behaviours on the first exposure to a juvenile and on the second exposure to a different juvenile carried out 120 rain later, for saline and the two active doses of THA. (Difference between first and second exposure: NS, not significant; *P < 0.05; **P < 0.01)

Behavioural Doses 1st exposure 2nd expdsure P categories (mg/kg) (different juvenile)

0 3.28 ± 0.53 2.64 _+ 0.52 NS Following 1 4.03 +_ 0.61 3.96 _+ 0.71 NS

3 3.04 + 0.55 2.58 + 0.43 NS Head and body 0 14.14 + 2.15 19.62 +_ 3.67 * sniffing 1 16.49 ± 2.70 18.53 ± 2.51 NS

3 14.2 ± 1.27 2t.02 +_ 1.66 ** Anogenital 0 91.43 _+ 10.44 t10.08 ± 13.04 * sniffing 1 100.3 ± 8.01 90.02 _+ 10.91 NS

3 112.22 _+ 7.91 107.34 ± 11.19 NS Locomotor 0 41.17 _+ 3.46 29.73 _+ 5.35 * activity 1 32.23 _+ 4.32 31.06 +_ 4.04 NS

3 32.53 ± 6.50 28.4 + 3.55 NS 0 100.83 ± 15.42 112.84 ± 10.37 NS

Rearing 1 106.58 _+ 11.71 113.14 ± 5.66 NS 3 104.64 _+ 7.55 101.68 ± 12.78 NS

Self 0 11.76 _+ 8.19 5.79 ± 3.26 NS grooming 1 8.67 _+ 3.22 8.5 ± 3.63 NS

3 15.59 ± 5.65 15.29 ± 8.25 NS

same juvenile clearly show that the difference of attitude of adult males is immediate (i.e. within the first minute of the re-exposure phase of the test session). Close sniffing of the anogenital area of the social stimulus appeared to be the most salient index for assessing characteristics of the adult males' social investigation. This posture typically involves the head of the adult rat tilted to the side. It certainly facilitates the use of the vomeronasal organ (which represents the primary system by which intact male rats process and store socially relevant cues) for sensing olfac- tory cues fi'om the juvenile (Bluth6 and Dantzer 1993). In contrast, changes in close following by the adult males between the two exposure phases of the test session did not appear to provide a good measure of social assess- ment, since reduction of time spent following the juveniles appeared irrespective of the treatment administered. Since close following occurs in response to the displacement of the juveniles, reduction of time spent following by adult males could be easily understood on the basis of the reduction of the occurrence of separations by the former animals during the second exposure. This change in the juveniles' behavioural profile probably reflects habituation to the environmental situation between the two exposures.

The lack of a decrease in time spent investigating a different juvenile by adult rats treated 2 h earlier with effective doses of THA allow us to conclude that the THA-induced decrease in social investigation is not due to non-specific toxic effects. This aspect has been more precisely assessed in the present study by focusing on non- social activities displayed by adults given an effective dose of THA prior to re-exposure to the same juvenile. Indeed no significant change (impairment or increase) could be

detected in their locomotor activity and rearing, which are often regarded as reflecting arousal. These arguments are also supported by the lack of any significant change in non-social activities of adult males exposed to a different juvenile. The sole indication of possible non-specific effects of THA concerns the relative increase in body care, by comparison with saline treatment, observed during the second exposure either to the same or a different juvenile. It is difficult, however, to interpret self-grooming because of its heterogeneous causal factors. Depending on the context in which the subjects are placed, self-grooming has been classified in different categories such as increased or decreased arousal (Delius 1970; Gipsen and Isaacson 1981), thermoregulatory behaviours (Roberts et al. 1969; Tanaka et al. 1986) or social signalling ( Thiessen et al. 1983; Harr iman and Thiessen 1985). The great variability observed in body care duration in experimental subjects and the numerous ways in which these different functions may be intimately related (Thiessen 1988; Irvin et al. 1990; Dantzer and Bluth6 1992) render such a result difficult to interpret.

In addition to our use of an ethological approach, we attempted to assess the respective responsibility of each participant in establishing and maintaining contact during social encounters. This provides information concerning the supposed processes of recognition in adult males. An index such as the one we propose seems to be a rep- resentative and reliable tool insofar as it takes into account the level of interaction between the two partners and allows comparisons between different situations. The observed decrease of the index at the doses of 1 and 3 mg/kg THA indicates that adult male rats initiated a greater number of separations of the pair than contacts. This may be interpreted as a weaker interest by adults toward a previously investigated and familiar juvenile. This index therefore appears to be a reliable indicator for assessing recognition and memory in adult males. Fur- thermore, our results suggest that the decrease of social interest shown by adult males allowed the juveniles to approach and initiate contact more frequently. It should be stressed that despite the maintenance of contact en- sured by juveniles, adult males treated with effective doses of THA did not extend their investigatory behaviour toward the juveniles.

Finally, the present findings provide confirmatory support for previous studies from other laboratories util- izing the social recognition paradigm. At the present time, the test of social memory must be considered as a test of social recognition which cannot tell us whether juveniles are recognized precisely on the basis of their idiosyncrasic characteristics or more broadly on the basis of their fam- iliarity. Despite these limitations, this test is rapid to implement and is an appropriate method for studying drug action (impairing as well as facilitating effects) and the neurobiological bases of social cognition. Besides the primary concern of e thopharmacology in drug effects on behavioural patterns and social status (Miczek 1983), the test of social recognition extends the recent emergence of interest in how individuals know about, identify, classify and recognize each other. In conclusion, the present study broadly supports the enhancement of cognitive function of THA that has been reported with different

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neuropharmacologica l tests. This also suggests that research on cognitive processes should benefit from includ- ing models of social cognit ion.

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Ethological study of the effects of tetrahydroaminoacridine (THA) on social recognition in rats

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