Brain Research, 155 (1978) 249-261 © Elsevier/North-HollandBiomedicalPress
249
THE LOCUS COERULEUS NORADRENERGIC SYSTEM - - EVIDENCE AGAINST A ROLE IN ATTENTION, HABITUATION, ANXIETY AND MOTOR ACTIVITY
T. J. CROW, J. F. W. DEAK1N, S. E. FILE, A. LONGDEN and S. WENDLANDT Division of Psychiatry, Clinical Research Centre, Northwick Park Hospital, Harrow, HA1 3UJ (Great Britain)
(Accepted February 16th, 1978)
SUMMARY Lesions of the locus coeruleus system were induced by combined stereotaxic injections of 6-OH-dopamine to the ascending fibres and just lateral to the locus coeruleus itself, to deplete the noradrenaline content of both the cerebral and cerebellar cortices. A group of rats with cortical noradrenaline concentrations of less than 30 ng/g were compared with a group with lesser destruction of the system (mean noradrenaline concentration 71 + 44 ng/g) and with controls (mean noradrenaline 347 ± 58 ng/g). Lesioned rats showed normal motor activity and exploration (assessed with a holeboard) and showed normal habituation of these behaviours. The lesioned rats gave no evidence of increased susceptibility to distracting auditory stimuli whilst licking for water, and the groups did not differ in their rate of habituation to these stimuli, or in dishabituation. In a social interaction test, lesioned animals showed a decrease in social contacts in an unfamiliar situation (interpreted as a response to anxiety) of similar magnitude to that seen in the control group. In this test, lesioned animals engaged in more 'aggressive' behaviour (boxing and wrestling) than did the controls. These findings are incompatible with hypotheses that the locus coeruleus system is an integral part of the physiological mechanisms which control gross motor behaviour, attention, habituation or anxiety. Together with previous findings with the benzodiazepines, the results with the social interaction test make it unlikely that the benzodiazepines exert their anxiolytic effects by inhibiting the locus coeruleus system.
INTRODUCTION The noradrenaline-containing cell bodies of the nucleus locus coeruleus in the mid-pontine region of the brain stem give rise to terminal networks innervating
250 telencephalic cortical structures (including the cerebral and hippocampal cortices) as well as the cerebellar cortex 35. However, the functions of this widely distributed system have remained obscure, and experimental findings on the effects of stimulation and ablation are in conflict. Several workers have suggested that the locus coeruleus system may function as part of a reward mechanism. Electrical self-stimulation can be obtained with electrodes either within or in close proximity to the locus coeruleus 9,z9,31, and this behaviour is associated with increased turnover of noradrenaline in the ipsilateral cerebral cortex ~. On the other hand, it has not been possible to demonstrate that selfstimulation with electrodes in the region of the locus coeruleus is abolished by 6-OHdopamine-induced lesions of the fibres ascending from the nucleus to innervate the forebrain 4. Similar problems arise in interpreting the effects of lesions of the system. Deficits in the acquisition of a running response for a food reward have been reported following bilateral electrolytic lesions of the locus coeruleus by some workersZ,3L However, such lesions appear not to impair performance in other learning situationsl,3L Moreover, when the cerebral cortical and hippocampal content of noradrenaline is depleted to a similar or greater extent by 6-OH-dopamine injected into the fibres (the 'dorsal bundle') ascending from the nucleus impairments of runway performance are not seen 25,a0. In some experiments 26, by contrast, a failure of extinction of a previously acquired response was observed within a closely spaced series of non-reward trials, both in a runway and other operant situations. Some technical problems may be relevant to these apparently discrepant findings. For example it seems possible, as suggested by Vogt aT, that monoamine systems possess a high degree of redundancy, and that the effects of ablations will become apparent only when the extent of destruction of the systems approaches 100 ~ . There is some evidence that this may be the case for ablations of ascending dopamine systems 11,z6. In assessing this possibility the sensitivity of techniques for estimating the destruction of the system (i.e. the limits of detection for noradrenaline in tissue samples) is of critical importance. A second problem concerns the extent to which collateral systems (e.g. the innervation of the cerebellar cortex) may be able to compensate for destruction of the major ascending pathway to the cerebral cortex and hippocampus. Here we report the effects of a recent and relatively extensive method 1° of lesioning the system on general motor behaviour, exploration and responsiveness to novel physical and social environments. The findings reveal one rather subtle effect of such lesions which has escaped previous detection; some negative findings are relevant to current concepts of locus coeruleus system function. METHODS
Animals and lesioning procedure 30 male hooded rats (Rattus norvegicus) weighing 200 ± 20 g at the time of operation were anaesthetized with tribromoethanol and either (i) had 4 stereotaxically-
251 placed intracerebral injections of 6-OH-dopamine (20 animals) or (ii) underwent the same procedure, including drilling of burr holes, but without the cannula being inserted into the brain (10 animals). In group (i), 6-OH-dopamine (8 #g in 2 #1 made up with ascorbic acid, 1 mg per ml) was injected at a site just lateral to the locus coeruleus (coordinates P --1.7, L 1.3, V +2.4 relative to the interaural plane, with tooth bar elevated 1 mm) and into the course of ascending fibres of the 'dorsal bundle' (coordinates P + 1.7, L 0.9, V +4.4). Previous experiments 1° had demonstrated that such lesions deplete noradrenaline in both the cerebral and cerebellar cortices. Animals were housed singly with food and water available ad libitum, except where otherwise stated. At the start of behavioural testing the mean weight of the controls was 188 g and that of the lesioned rats was 184 g; at the end of testing the mean weight of the controls was 219 g and that of the lesioned rats was 220 g. Noradrenaline assay. After completion of behavioural testing animals were decapitated after being concussed by a blow on the back, the brains were removed and the cerebral cortices were dissected off and frozen at --70 °C. Noradrenaline content was estimated by the radiometric enzymatic assay of Coyle and Henry n, in which the O-methylated derivative is formed in the presence of catechol-O-methyl transferase using [3H]S-adenosyl methionine as the methyl donor, the tritiated derivative being isolated by selective solvent extraction. With this technique we found the limit of detection in these tissue samples to be 18 ng/g.
Apparatus (a) Distraction to auditory stimuli. The test chamber, 19 x 19 x 26.5 cm, was enclosed in an acoustically insulated box. A slit in the end wall gave access to a water spout and a drinkometer recorded each lick. Experimental events were automatically programmed and tones delivered via a loudspeaker placed in the lid of the chamber at the water spout end. (b) Exploration. Testing took place in a 4-hole holeboard m, a wooden box with walls 45 cm high and floor 55 x 55 cm, with 4 equally-spaced holes in the floor, 3.8 cm in diameter. Objects were placed 2 cm below these holes. (c) Social interaction anxiety test. The test box was 65 x 65 cm with 47 cm high walls. The level of illuminance was 23.5 scotopic lux for the low level and 338 scotopic lux for the high level. Procedure (a) Distraction and habituation to tone stimuli. Rats were deprived of water for 36 h and for the next 2 days received water only in the test chamber and, immediately following the test session, in sufficient quantity to maintain a steady body weight. On the first day each rat was placed in the test chamber and given free access to the water spout for 20 min. On the second day, each rat's 200th lick turned on a control period of 9 see during which the number of licks (A) was counted. The next 200th lick turned on a 9 sec tone (7 kHz, 85 dB) and the number of licks made while the tone was on (B) was counted. A distraction ratio of (A-B)/A was calculated; this has a value of zero if there is no distraction at all to the tone and A = B, and a value of one if
252 there is total distraction, i.e. B = 0 . Habituation criterion was taken as 3 successive tone presentations producing ratios _< 0.10. Control and tone periods alternated until criterion was reached; this occurred on the first day of tone presentation for all rats. Further details of this procedure are given in 15. On the next day the rats were returned to the test chamber and given one presentation of the habituated tone, in order to test for 24-h retention of habituation. If a rat was distracted by this presentation further presentations were given until criterion was again reached. A series of control and tone periods then alternated in the following manner. First, a novel tone was presented (13 kHz, 85 dB) and distraction to this change in tone frequency was noted. The original tone was then presented again and any dishabituation (i.e. return of distraction to this tone) recorded. If necessary, criterion was re-established to this tone and then a second novel stimulus (7 kHz, 75 dB) was presented, followed by the original tone. Rats were then returned to ad libitum water. (b) Exploration and its habituation. Exploration was measured in a holeboard with objects placed under the holes, as this provides a measure of directed exploration which is largely independent of changes in motor activity 19. Each rat was placed s'ngly in the holeboard for a 10-min trial on 3 successive days; testing took place between 17.30 and 20.00 h. The number of head-dips made, the time spent head-dipping and an activity score were recorded for each rat. Head-dipping was counted and timed automatically by means of infra-red cells placed just below the holes. Infra-red cells in the walls of the holeboard provided a measure of motor activity, a count being scored each time a beam was interrupted. Animals could be observed on a television monitor in an adjacent room, and on the first day an observer scored the incidence of grooming and number of rears. (c) Social interaction anxiety test. A test of anxiety has been developed in which time spent by pairs of male rats in active social interaction is a measure of the level of anxiety 16. Normal rats show less social interaction if tested in an unfamiliar box than if tested in a familiar one, and less social interaction as the illuminance of the box is increased. For this test, rats are housed singly and are tested with a partner of closely similar weight. Rats were therefore allocated to pairs within the control and lesioned groups on the basis of weight, and were scored by an observer who did not know to which group they belonged. Rats were first tested for social interaction in a box with which they were unfamiliar, under a high level of illuminance ( H U condition). Each pair of rats were placed in the centre of the box and observed for 10 min on a television monitor in an adjacent room. Time spent in active social contact was measured and the following behaviours scored: sniffing, following, nipping, grooming, mounting, kicking, boxing, wrestling, jumping on, crawling under or over the partner. During the tests for social interaction a measure of exploration was also obtained by scoring the time the rats spent sniffing or manipulating objects that were hung on the walls of the test box. At the end of each trial rats were returned to single cages, and the floor and walls of the test box were wiped and dried. Rats were tested in a random order between 14.00 and 17.00 h.
253 TABLEI D~tract~ntoachangemst~ulusandthedish~#ua~nproduced~th~
~ange
I ( n = 10)
H ( n = 9)
111(n = 9)
6 0.14 4- 0.09 2 0.06 4- 0.02
6 0.27 -4- 0.10 2 0.05 ± 0.02
6 0.15 4- 0.04 3 0.09 4- 0.03
4 0.13 -4- 0.03 5 0.19 4- 0.08
5 0.23 -4- 0.10 5 0.17 -4- 0.08
4 0.11 -4- 0.03 4 0.15 -4- 0.07
Change in tone frequency
No. of rats showing distraction Mean distraction ratio No. of rats dishabituating Mean dishabituation ratio Change in tone intensity
No. of rats showing distraction Mean distraction ratio No. of rats dishabituating Mean dishabituation ratio
The following day each rat was placed singly in the test box, under low illumination, for 10 min. The next day, pairs of rats were again tested for social interaction under low illumination and thus in a familiar environment (LF condition). RESULTS Noradrenaline assay
The group of control rats (group I) were found to have cortical noradrenaline concentrations between 274 and 479 ng/g (mean 347 4- 58 ng/g). Lesioned rats had concentrations between 18 ng/g (the limit of detectability) and 152 ng/g. This group was divided into two - - group II - - 9 rats with a cortical noradrenaline concentration between 30 and 250 ng/g (mean 71 4- 44 ng/g; 20 ~o of control levels) and group III - 9 rats with cortical noradrenaline less than 30 ng/g (mean 21 4- 4 ng/g; 6 ~o of control levels taking the 4 rats with levels of < 18 ng/g to be 18 ng/g). The results of the behavioural experiments were analyzed using this subdivision of the experimental group by cortical noradrenaline concentration. Behavioural testing (a) Distraction and habituation to tone stimuli. Lesioned rats had a slightly higher baseline rate of licking than did the control rats. The controls made a mean of 44.3 -+0.7 licks in 9 see, group II rats made a mean of 46.7 -4- 1.8 licks and group III rats 47.1 i 0.9 licks, the latter differed significantly from the controls (t = 2.49, df = 17, P < 0.05). Control rats had a mean distraction ratio of 0.86 4- 0.08 to the first tone presentation. Lesioned rats showed less distraction with a mean of 0.62 4- 0.12 for group II, and 0.56 4- 0.11 for group III (t = 2.25, df = 17, P < 0.05). There were no differences in the number of trials (stimulus presentations) needed to habituate to this tone. Control rats took a mean of 7.5 4- 2.4 trials, group II 6.4 4- 1.9 and group III 6.4 4- 2.1 trials.
254 Locus
coeruleus
lesions
'E /, 0
~.
o
40
"o
._o. 30
o
30
~: zo
~o
2o
"5
®
"o cl
E
lO
c 0
~E
0
,
,
,
2
3
~
Test
O'
,
,
1
2
3
Ooy
200
160 E 0
120
~,
80
Gp..~n
._> ~ 40
i
f
i
1
2
3
Test
Doy
Fig. 1. Mean number of head-dips, mean time (S) spent head-dipping and mean activity score during a 10-min test in the holeboard on 3 consecutive days. • 0 , Group I (controls); O O, Group II (mean N A 71 ng/g); A . . . . A, Group III (mean N A 21 rig/g).
All lesioned rats and all but one of the control rats showed 24-h retention of habituation. The one control rat needed a single tone presentation in order to regain habituation criterion. Table I shows the number of rats in each group that were distracted by the changes in stimulus frequency and intensity and the mean distraction ratios to these changes. It also shows the number of rats showing dishabituation and the mean ratios for this effect. The percentage of rats in the control group that were distracted by the changes in stimulus frequency and intensity are similar to those found previously 14,1s, and the lesioned rats did not differ significantly from the controls. There were no significant differences between the groups in their mean distraction ratios to the stimulus changes. Nor were there any group differences in the number of rats showing dishabituation or in the mean dishabituation ratios. (b) Exploration and its habituation. Fig. 1 shows the mean number of head-dips made, mean time spent head-dipping, and activity score for each of the 3 groups over each of 3 test days. These data were subjected to split-plot analyses of variance, with
255
15 v t-
fi.
o. "c2 i
10 '~
a
II
-r-
"X~ Cont rol
c
III 5
E i=
g 0
123~ 2.Stainperiods I I J I $
Fig. 2. Within-session habituation on day 1. The ordinate shows the mean time (in see) spent headdipping in each 2-5 min period; the abscissa shows each successive2-5 min period. • . . . . 0 , group I (controls); © ©, group II: A A, group III. the lesion as the between-groups factor (at 3 levels: control, group II, group III) and days (at 3 levels: 1, 2, 3) being the within-groups factor. The lesioned rats did not make significantly fewer head-dips (F2,24 = 1.40, P > 0.05), nor did they spend significantly less time head-dipping than did the controls (Fz,24 1.25, P > 0.05). There was no significant difference between the activity scores for the 3 groups (F2,~4 = 2.03, P > 0.05). Thus there was no evidence that the lesioned animals were less exploratory or less active than the controls. All groups showed significant habituation over days of number of head-dips (F2,4s = 19.5, P < 0.001), of time spent head-dipping (F2,4s =- 12.8, P < 0.001) and of activity (F2,4s = 19.5, P < 0.001). There was no significant interaction between the lesion factor and habituation of exploration (Fs4,48 = 1.43, = 2.08 for number of head dips, and time spent head dipping, respectively), and thus no evidence of impaired habituation in lesioned rats. There were no differences in within-session habituation on days 2 and 3 between the 3 groups. However, on day 1, although groups I and III showed a decrease in the time spent head-dipping from the first to the fourth time period, a different pattern was shown by the group II rats. These animals showed an initial period of sensitization, followed by habituation (Fig. 2). However, even analyzing the data for day 1 alone, this difference failed to reach significance. On a split-plot analysis of variance the interaction between the lesion factor and the time periods was not significant (F6,72 = 0.73), nor was the overall level of head-dipping significantly different for the lesioned rats (F2,,~4 = 0.08). Thus, this slight difference in the pattern of head-dipping on the first day's test was not significant; and, because the pattern shown by group III was the same as that shown by the controls, it was not related to the extent of NA depletion. There were no significant differences between groups in incidence of grooming or amount of rearing in the holeboard on trial 1. The mean incidence of grooming was
256 Locus .-~
coeruleus
m
600
5~
5o0
,,,-
c
0
0
7.0
G p.__m_m
249
THE LOCUS COERULEUS NORADRENERGIC SYSTEM - - EVIDENCE AGAINST A ROLE IN ATTENTION, HABITUATION, ANXIETY AND MOTOR ACTIVITY
T. J. CROW, J. F. W. DEAK1N, S. E. FILE, A. LONGDEN and S. WENDLANDT Division of Psychiatry, Clinical Research Centre, Northwick Park Hospital, Harrow, HA1 3UJ (Great Britain)
(Accepted February 16th, 1978)
SUMMARY Lesions of the locus coeruleus system were induced by combined stereotaxic injections of 6-OH-dopamine to the ascending fibres and just lateral to the locus coeruleus itself, to deplete the noradrenaline content of both the cerebral and cerebellar cortices. A group of rats with cortical noradrenaline concentrations of less than 30 ng/g were compared with a group with lesser destruction of the system (mean noradrenaline concentration 71 + 44 ng/g) and with controls (mean noradrenaline 347 ± 58 ng/g). Lesioned rats showed normal motor activity and exploration (assessed with a holeboard) and showed normal habituation of these behaviours. The lesioned rats gave no evidence of increased susceptibility to distracting auditory stimuli whilst licking for water, and the groups did not differ in their rate of habituation to these stimuli, or in dishabituation. In a social interaction test, lesioned animals showed a decrease in social contacts in an unfamiliar situation (interpreted as a response to anxiety) of similar magnitude to that seen in the control group. In this test, lesioned animals engaged in more 'aggressive' behaviour (boxing and wrestling) than did the controls. These findings are incompatible with hypotheses that the locus coeruleus system is an integral part of the physiological mechanisms which control gross motor behaviour, attention, habituation or anxiety. Together with previous findings with the benzodiazepines, the results with the social interaction test make it unlikely that the benzodiazepines exert their anxiolytic effects by inhibiting the locus coeruleus system.
INTRODUCTION The noradrenaline-containing cell bodies of the nucleus locus coeruleus in the mid-pontine region of the brain stem give rise to terminal networks innervating
250 telencephalic cortical structures (including the cerebral and hippocampal cortices) as well as the cerebellar cortex 35. However, the functions of this widely distributed system have remained obscure, and experimental findings on the effects of stimulation and ablation are in conflict. Several workers have suggested that the locus coeruleus system may function as part of a reward mechanism. Electrical self-stimulation can be obtained with electrodes either within or in close proximity to the locus coeruleus 9,z9,31, and this behaviour is associated with increased turnover of noradrenaline in the ipsilateral cerebral cortex ~. On the other hand, it has not been possible to demonstrate that selfstimulation with electrodes in the region of the locus coeruleus is abolished by 6-OHdopamine-induced lesions of the fibres ascending from the nucleus to innervate the forebrain 4. Similar problems arise in interpreting the effects of lesions of the system. Deficits in the acquisition of a running response for a food reward have been reported following bilateral electrolytic lesions of the locus coeruleus by some workersZ,3L However, such lesions appear not to impair performance in other learning situationsl,3L Moreover, when the cerebral cortical and hippocampal content of noradrenaline is depleted to a similar or greater extent by 6-OH-dopamine injected into the fibres (the 'dorsal bundle') ascending from the nucleus impairments of runway performance are not seen 25,a0. In some experiments 26, by contrast, a failure of extinction of a previously acquired response was observed within a closely spaced series of non-reward trials, both in a runway and other operant situations. Some technical problems may be relevant to these apparently discrepant findings. For example it seems possible, as suggested by Vogt aT, that monoamine systems possess a high degree of redundancy, and that the effects of ablations will become apparent only when the extent of destruction of the systems approaches 100 ~ . There is some evidence that this may be the case for ablations of ascending dopamine systems 11,z6. In assessing this possibility the sensitivity of techniques for estimating the destruction of the system (i.e. the limits of detection for noradrenaline in tissue samples) is of critical importance. A second problem concerns the extent to which collateral systems (e.g. the innervation of the cerebellar cortex) may be able to compensate for destruction of the major ascending pathway to the cerebral cortex and hippocampus. Here we report the effects of a recent and relatively extensive method 1° of lesioning the system on general motor behaviour, exploration and responsiveness to novel physical and social environments. The findings reveal one rather subtle effect of such lesions which has escaped previous detection; some negative findings are relevant to current concepts of locus coeruleus system function. METHODS
Animals and lesioning procedure 30 male hooded rats (Rattus norvegicus) weighing 200 ± 20 g at the time of operation were anaesthetized with tribromoethanol and either (i) had 4 stereotaxically-
251 placed intracerebral injections of 6-OH-dopamine (20 animals) or (ii) underwent the same procedure, including drilling of burr holes, but without the cannula being inserted into the brain (10 animals). In group (i), 6-OH-dopamine (8 #g in 2 #1 made up with ascorbic acid, 1 mg per ml) was injected at a site just lateral to the locus coeruleus (coordinates P --1.7, L 1.3, V +2.4 relative to the interaural plane, with tooth bar elevated 1 mm) and into the course of ascending fibres of the 'dorsal bundle' (coordinates P + 1.7, L 0.9, V +4.4). Previous experiments 1° had demonstrated that such lesions deplete noradrenaline in both the cerebral and cerebellar cortices. Animals were housed singly with food and water available ad libitum, except where otherwise stated. At the start of behavioural testing the mean weight of the controls was 188 g and that of the lesioned rats was 184 g; at the end of testing the mean weight of the controls was 219 g and that of the lesioned rats was 220 g. Noradrenaline assay. After completion of behavioural testing animals were decapitated after being concussed by a blow on the back, the brains were removed and the cerebral cortices were dissected off and frozen at --70 °C. Noradrenaline content was estimated by the radiometric enzymatic assay of Coyle and Henry n, in which the O-methylated derivative is formed in the presence of catechol-O-methyl transferase using [3H]S-adenosyl methionine as the methyl donor, the tritiated derivative being isolated by selective solvent extraction. With this technique we found the limit of detection in these tissue samples to be 18 ng/g.
Apparatus (a) Distraction to auditory stimuli. The test chamber, 19 x 19 x 26.5 cm, was enclosed in an acoustically insulated box. A slit in the end wall gave access to a water spout and a drinkometer recorded each lick. Experimental events were automatically programmed and tones delivered via a loudspeaker placed in the lid of the chamber at the water spout end. (b) Exploration. Testing took place in a 4-hole holeboard m, a wooden box with walls 45 cm high and floor 55 x 55 cm, with 4 equally-spaced holes in the floor, 3.8 cm in diameter. Objects were placed 2 cm below these holes. (c) Social interaction anxiety test. The test box was 65 x 65 cm with 47 cm high walls. The level of illuminance was 23.5 scotopic lux for the low level and 338 scotopic lux for the high level. Procedure (a) Distraction and habituation to tone stimuli. Rats were deprived of water for 36 h and for the next 2 days received water only in the test chamber and, immediately following the test session, in sufficient quantity to maintain a steady body weight. On the first day each rat was placed in the test chamber and given free access to the water spout for 20 min. On the second day, each rat's 200th lick turned on a control period of 9 see during which the number of licks (A) was counted. The next 200th lick turned on a 9 sec tone (7 kHz, 85 dB) and the number of licks made while the tone was on (B) was counted. A distraction ratio of (A-B)/A was calculated; this has a value of zero if there is no distraction at all to the tone and A = B, and a value of one if
252 there is total distraction, i.e. B = 0 . Habituation criterion was taken as 3 successive tone presentations producing ratios _< 0.10. Control and tone periods alternated until criterion was reached; this occurred on the first day of tone presentation for all rats. Further details of this procedure are given in 15. On the next day the rats were returned to the test chamber and given one presentation of the habituated tone, in order to test for 24-h retention of habituation. If a rat was distracted by this presentation further presentations were given until criterion was again reached. A series of control and tone periods then alternated in the following manner. First, a novel tone was presented (13 kHz, 85 dB) and distraction to this change in tone frequency was noted. The original tone was then presented again and any dishabituation (i.e. return of distraction to this tone) recorded. If necessary, criterion was re-established to this tone and then a second novel stimulus (7 kHz, 75 dB) was presented, followed by the original tone. Rats were then returned to ad libitum water. (b) Exploration and its habituation. Exploration was measured in a holeboard with objects placed under the holes, as this provides a measure of directed exploration which is largely independent of changes in motor activity 19. Each rat was placed s'ngly in the holeboard for a 10-min trial on 3 successive days; testing took place between 17.30 and 20.00 h. The number of head-dips made, the time spent head-dipping and an activity score were recorded for each rat. Head-dipping was counted and timed automatically by means of infra-red cells placed just below the holes. Infra-red cells in the walls of the holeboard provided a measure of motor activity, a count being scored each time a beam was interrupted. Animals could be observed on a television monitor in an adjacent room, and on the first day an observer scored the incidence of grooming and number of rears. (c) Social interaction anxiety test. A test of anxiety has been developed in which time spent by pairs of male rats in active social interaction is a measure of the level of anxiety 16. Normal rats show less social interaction if tested in an unfamiliar box than if tested in a familiar one, and less social interaction as the illuminance of the box is increased. For this test, rats are housed singly and are tested with a partner of closely similar weight. Rats were therefore allocated to pairs within the control and lesioned groups on the basis of weight, and were scored by an observer who did not know to which group they belonged. Rats were first tested for social interaction in a box with which they were unfamiliar, under a high level of illuminance ( H U condition). Each pair of rats were placed in the centre of the box and observed for 10 min on a television monitor in an adjacent room. Time spent in active social contact was measured and the following behaviours scored: sniffing, following, nipping, grooming, mounting, kicking, boxing, wrestling, jumping on, crawling under or over the partner. During the tests for social interaction a measure of exploration was also obtained by scoring the time the rats spent sniffing or manipulating objects that were hung on the walls of the test box. At the end of each trial rats were returned to single cages, and the floor and walls of the test box were wiped and dried. Rats were tested in a random order between 14.00 and 17.00 h.
253 TABLEI D~tract~ntoachangemst~ulusandthedish~#ua~nproduced~th~
~ange
I ( n = 10)
H ( n = 9)
111(n = 9)
6 0.14 4- 0.09 2 0.06 4- 0.02
6 0.27 -4- 0.10 2 0.05 ± 0.02
6 0.15 4- 0.04 3 0.09 4- 0.03
4 0.13 -4- 0.03 5 0.19 4- 0.08
5 0.23 -4- 0.10 5 0.17 -4- 0.08
4 0.11 -4- 0.03 4 0.15 -4- 0.07
Change in tone frequency
No. of rats showing distraction Mean distraction ratio No. of rats dishabituating Mean dishabituation ratio Change in tone intensity
No. of rats showing distraction Mean distraction ratio No. of rats dishabituating Mean dishabituation ratio
The following day each rat was placed singly in the test box, under low illumination, for 10 min. The next day, pairs of rats were again tested for social interaction under low illumination and thus in a familiar environment (LF condition). RESULTS Noradrenaline assay
The group of control rats (group I) were found to have cortical noradrenaline concentrations between 274 and 479 ng/g (mean 347 4- 58 ng/g). Lesioned rats had concentrations between 18 ng/g (the limit of detectability) and 152 ng/g. This group was divided into two - - group II - - 9 rats with a cortical noradrenaline concentration between 30 and 250 ng/g (mean 71 4- 44 ng/g; 20 ~o of control levels) and group III - 9 rats with cortical noradrenaline less than 30 ng/g (mean 21 4- 4 ng/g; 6 ~o of control levels taking the 4 rats with levels of < 18 ng/g to be 18 ng/g). The results of the behavioural experiments were analyzed using this subdivision of the experimental group by cortical noradrenaline concentration. Behavioural testing (a) Distraction and habituation to tone stimuli. Lesioned rats had a slightly higher baseline rate of licking than did the control rats. The controls made a mean of 44.3 -+0.7 licks in 9 see, group II rats made a mean of 46.7 -4- 1.8 licks and group III rats 47.1 i 0.9 licks, the latter differed significantly from the controls (t = 2.49, df = 17, P < 0.05). Control rats had a mean distraction ratio of 0.86 4- 0.08 to the first tone presentation. Lesioned rats showed less distraction with a mean of 0.62 4- 0.12 for group II, and 0.56 4- 0.11 for group III (t = 2.25, df = 17, P < 0.05). There were no differences in the number of trials (stimulus presentations) needed to habituate to this tone. Control rats took a mean of 7.5 4- 2.4 trials, group II 6.4 4- 1.9 and group III 6.4 4- 2.1 trials.
254 Locus
coeruleus
lesions
'E /, 0
~.
o
40
"o
._o. 30
o
30
~: zo
~o
2o
"5
®
"o cl
E
lO
c 0
~E
0
,
,
,
2
3
~
Test
O'
,
,
1
2
3
Ooy
200
160 E 0
120
~,
80
Gp..~n
._> ~ 40
i
f
i
1
2
3
Test
Doy
Fig. 1. Mean number of head-dips, mean time (S) spent head-dipping and mean activity score during a 10-min test in the holeboard on 3 consecutive days. • 0 , Group I (controls); O O, Group II (mean N A 71 ng/g); A . . . . A, Group III (mean N A 21 rig/g).
All lesioned rats and all but one of the control rats showed 24-h retention of habituation. The one control rat needed a single tone presentation in order to regain habituation criterion. Table I shows the number of rats in each group that were distracted by the changes in stimulus frequency and intensity and the mean distraction ratios to these changes. It also shows the number of rats showing dishabituation and the mean ratios for this effect. The percentage of rats in the control group that were distracted by the changes in stimulus frequency and intensity are similar to those found previously 14,1s, and the lesioned rats did not differ significantly from the controls. There were no significant differences between the groups in their mean distraction ratios to the stimulus changes. Nor were there any group differences in the number of rats showing dishabituation or in the mean dishabituation ratios. (b) Exploration and its habituation. Fig. 1 shows the mean number of head-dips made, mean time spent head-dipping, and activity score for each of the 3 groups over each of 3 test days. These data were subjected to split-plot analyses of variance, with
255
15 v t-
fi.
o. "c2 i
10 '~
a
II
-r-
"X~ Cont rol
c
III 5
E i=
g 0
123~ 2.Stainperiods I I J I $
Fig. 2. Within-session habituation on day 1. The ordinate shows the mean time (in see) spent headdipping in each 2-5 min period; the abscissa shows each successive2-5 min period. • . . . . 0 , group I (controls); © ©, group II: A A, group III. the lesion as the between-groups factor (at 3 levels: control, group II, group III) and days (at 3 levels: 1, 2, 3) being the within-groups factor. The lesioned rats did not make significantly fewer head-dips (F2,24 = 1.40, P > 0.05), nor did they spend significantly less time head-dipping than did the controls (Fz,24 1.25, P > 0.05). There was no significant difference between the activity scores for the 3 groups (F2,~4 = 2.03, P > 0.05). Thus there was no evidence that the lesioned animals were less exploratory or less active than the controls. All groups showed significant habituation over days of number of head-dips (F2,4s = 19.5, P < 0.001), of time spent head-dipping (F2,4s =- 12.8, P < 0.001) and of activity (F2,4s = 19.5, P < 0.001). There was no significant interaction between the lesion factor and habituation of exploration (Fs4,48 = 1.43, = 2.08 for number of head dips, and time spent head dipping, respectively), and thus no evidence of impaired habituation in lesioned rats. There were no differences in within-session habituation on days 2 and 3 between the 3 groups. However, on day 1, although groups I and III showed a decrease in the time spent head-dipping from the first to the fourth time period, a different pattern was shown by the group II rats. These animals showed an initial period of sensitization, followed by habituation (Fig. 2). However, even analyzing the data for day 1 alone, this difference failed to reach significance. On a split-plot analysis of variance the interaction between the lesion factor and the time periods was not significant (F6,72 = 0.73), nor was the overall level of head-dipping significantly different for the lesioned rats (F2,,~4 = 0.08). Thus, this slight difference in the pattern of head-dipping on the first day's test was not significant; and, because the pattern shown by group III was the same as that shown by the controls, it was not related to the extent of NA depletion. There were no significant differences between groups in incidence of grooming or amount of rearing in the holeboard on trial 1. The mean incidence of grooming was
256 Locus .-~
coeruleus
m
600
5~
5o0
,,,-
c
0
0
7.0
G p.__m_m
