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Effects of chlordiazepoxide, diazepam and chlorpromazine on conditioned emotional behaviour and conditioned neuronal activity in limbic, hypothalamic and geniculate regions
Nrwopharmacology,
1975, 14. 413425.
Pergamon
Press. Printed
in Gt. Briram
EFFECTS OF C~LOR~IAZEPOXIDE, DIAZEPAM AND CHLORPROMAZINE ON CONDITIONED EMOTIONAL BEHAVIOUR AND CONDITIONED NEURONAL ACTIVITY IN LIMBIC, HYPOTHALAMIC AND GENICULATE REGIONS* M. UlvlEMoTOtand M. E. OLDS Division of Biology, California Institute of Technology, Pasadena, California 91109 (Accepted 24 August
1974)
Summary-The effects of chlorpromazine, chlordiazepoxide and diazepam on conditioned emotional behaviour and neuronal activity correlated with it, were studied in the rat. Conditioned behaviour was produced by brain aversive stimulation in the central grey region. In the freely moving animal, neuronal activity was studied in the amygdala, the hippocampus, the hypothalamus, and the geniculate nuclei. Effects of the drugs were compared on the background rate of firing before the presentation of the conditioned stimulus in each trial, and on the change in the background rate correlated with the presentation of the conditioned stimulus after the subject was conditioned. Rehavioural responses for a food reward that had been suppressed during convict-conditioning were reinstated in approximately SOY0of the subjects after chlordiazepoxide (5 and 10 mg/kg) and in 20% after diazepam (25 and 5 mg/kg). Responding was not reinstated after chlorpromazine (1 and 2 mg/kg). The background rate of firing was reduced by chlordiazepoxide and chlorpromazine, but not by diazepam. The change in unit activity correlated with the presentation of the conditioned stimulus was reduced or abolished by chlordiazepoxide and diazepam, but not by chlorpromazine except in the hippocampus at the 2 mg/kg dose. The neuronal activity in the amygdala during the background period and during the conditioned stimulus-presentation was influenced by chlordiazepoxide to a greater extent than by the other two compounds. The significance of these results is discussed in terms of a possible relationship between the effects of the drugs on behaviour and on the rate of cellular discharge.
An interesting effect of the benzodiazepines is the disinhibition they produce on previously-suppres~d responses in an operant task, where reward and punishment are interpolated. Chlorpromazine, on the other hand, has no effect on punished responses but reduces the number of rewarded responses (GELLER, KOLAK and SEIFERS, 1962; MARGULES and STEIN,1967,196s ; DAVIDSON and COOK,1969 ; WEDEKIND, 1969 ; HEISE, LAUGHLIN and KELLER, 1970; SCHALLEK, SCHLOSSERand RANDALL, 1972). WEDEKIND (1969) attributed the release of suppressed behaviour by the minor tranquilizers, to a general breakdown of inhibition. MARGULES and STEIN(1968) showed that the nature of the punishing stimulus is unimportant; in their tests, although the stimuli employed to cause punishment varied from brain aversive stimulation to the adulteration of milk by the addition of quinine, suppressed behaviour was nonetheless released by oxazepam, a minor tranquilizer, but not by chlorpromazine, a neuroleptic, The value of these findings would be enhanced if the behavioural effects of these drugs were correlated with neurophysiological action. UMEMOTO, MURAI, KODAMA and KIDO (1970) have described patterns of neuronal activity characteristic of conflict behaviour such as that described by BRADY (1956, 1960). In the present tests, we used methods developed by OLDS (1973) for recording neuronal activity in order to study the effects of the benzodiazepines simultaneously on conflict behaviour and on the patterns of neuronal discharge associated with it. * Research supported in part by USPHS Grant MH-16978. T Present address: Shionogi Research Laboratory. Osaka. Japan. 413
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Neuronal activity was studied in four regions. The first two were the amygdala and the hippocampus. They were selected because they are generally presumed to be sites of action of the benzodiazepines (SCHALLEKand KUEHN, 1960; MORILLO,1962) and of chlorpromazine (PRESTON,1956; BROCKE,HORNYKIWIECZand SIGG, 1969). The third region studied was the hypothalamus. There is accumulating evidence for a direct action by these compounds in this region (GUERRERO-FIGUEROA, GALLANT, GUERRERO-FIGUEROA and GALLANT, 1973; OLDS, 1959; OLDS, 1966; DE WIED, 1967). Finally, neuronal activity in the geniculate nuclei was studied to determine if these compounds influence the neuronal excitability in regions not directly implicated in the regulation of emotional behaviour.
METHODS Animals
Male albino rats of the Holtzman strain with initial body weight of 25&300 g were used. They were housed in individual cages with food and water available ad libitum until 3-4 days after surgery, when the animals were placed on a 23-hr feeding schedule. Most of the food consumed during the duration of the tests was obtained during behavioural training in a conflict test. Surgery
Thirty-one rats were used. Each was implanted with 7 nichrome wire electrodes for recording (62.5 pm). These were aimed at sites in the amygdala, the hippocampus, the hypothalamus, and the geniculate nuclei. Each animal was also implanted with one set of twisted stainless steel wires (250 pm) in the central grey region for the production of aversive stimulation. An uninsulated stainless steel wire was implanted in the rostra1 regions of the brain at the level of the olfactory bulb to serve as an indifferent electrode for monopolar recording of unit activity. The stereotaxic coordinates were selected from the K~NIG and KLIPPELAtlas (1963). Surgery was carried out under pentobarbital sodium anaesthesia (50 mg/kg, i.p.). The details of the surgical methods have recently been described elsewhere (OLDS, 1973). The sites of stimulation and recording were verified at the end of the experiments on histological material processed by the frozen method and stained with either cresyl violet or Weil stain. The results are based on data obtained from animals in which the recording probes were lodged in the amygdala, hippocampus, hypothalamus and geniculate region. All stimulating probes were in the central grey region. Extracellular recording and stimulation techniques
The methods described by OLDS (1973) were used. The animal was able to move freely in the test chamber and to depress a lever to obtain food, while at the same time unit activity derived from three probes was recorded. The signal from the preamplifier was fed into a double-window discriminator by means of a conventional amplifier. The amplitude and the fall-time of the action potential was used to select the signal from the baseline activity. The output from the double-window discriminator was a 1-msec binary pulse that was counted and recorded on a pen-writing oscillograph. The animal’s responses and other event-markers were also recorded in the same manner. The extracellular activity from three microprobes was recorded simultaneously with the bar-pressing behaviour of the animal. Electrical brain stimulation was applied to the central grey region of the midbrain in a region bordering the reticular formation (Fig. 1 F). The stimulator was a Grass S-4 apparatus that delivered unidirectional pulses of 3-msec duration in trains of 60 Hz. The intensity of the stimulation was determined for each animal individually as that level just sufficient to produce a withdrawal reaction. All animals in which such a reaction was not observed during preliminary tests for the aversive properties of midbrain brain stimulation, were not used in the drug tests.
Neuronal activity during contlict behaviour
Behavioural
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training
After recovery from surgery (3-5 days) the subjects were screened for unit activity. When this activity was of a ratio of 2:l or higher in relation to the baseline “noise” on at least three of the seven probes implanted for recording in’ each animal, and furthermore was not correlated with the movements of the animal, then behavioural training was begun. The subjects learned to press a lever on a fixed-interval schedule (10 set) of food reinforcement. Usually this training was done overnight. When lever-pressing became stabilized, a light stimulus (conditioned stimulus) was introduced without brain stimulation. The light stimulus was presented every 90 set and was of lo-set duration. Time was allowed for any neuronal and ~havioural responses to the stimulus to extinguish (habituation; usually between 48-72 trials), and then a 0.2~set electrical train in the central grey region of the brain (aversive stimulation) was introduced at the end of each conditioned stimulus presentation. The intensity of the brain stimulus was adjusted to the minimum level necessary for the appearance of a withdrawal reaction. When the brain stimuli failed to produce this effect on behaviour in a subject, the subject was eliminated from the experiment. In the other subjects, training proceeded until the lever-pressing response for the food reward was suppressed, and until a change in the rate of firing in relation to the background rate appeared during the conditioned stimulus presentation. Data analysis
The score for the background activity was the number of units discriminated in the loset interval before each light stimulus presentation in blocks of six trials before the administration of the drugs and at various times after. The effects of the drugs were expressed as the percentages of the scores before injection (see panels B in Figs. 68). The score for the neuronal response (conditioned stimulus-correlated activity) was the ratio between the number of units in the lo-see interval of the conditioned stimulus presentation and the number of units in the IO-set interval immediately preceding the conditioned stimulus presentation, for blocks of six trials. The number of units in the conditioned stimulus-segments was subtracted from the number of units in the segments preceding the conditioned stimulus, and the result was divided by the number in the segments preceding the conditioned stimulus. The inflection ratio shifted in the positive direction when there was an increase in the rate of discharge the conditioned stimulus presentation, and it shifted toward - 1.0 when the rate of firing decreased during the conditioned stimulus presentation. To compute group scores, the negative inflection ratios were converted to absolute values. Ratios were computed for the period before the injection of the drugs and at various times thereafter. Effects were determined by comparing the inflection ratios at various times after the injection of the drugs with the inflection ratios before injection. The statistical significance of the changes was evaluated with the Student t test for paired comparison. Drugs
Chlordiazepoxide was tested at 5 and 10 mg/kg, diazepam at doses of 2.5 and 5 mg/kg, and chlorpromazine at doses of 1 and 2 mg/kg. The doses are given as the salts. Each drug was dissolved in 0.9% saline; the volume injected for chlordiazepoxide and chlorpromazine was Iess than O-5ml. For diazepam, the volume was less than 1 ml and the drug was in suspension. Control injections of physiological saline were made in a separate group of animals. No significant effects were observed on behaviour or neuronal activity in the control group.
RESULTS
Recordings of unit activity were derived from microelectrodes (62.5 pm) implanted in the amygdala, the hippocampus, the hypothalamus, and the geniculate nuclei. Representative examples of histological material showing the electrode-tracks and the site of stimulation (indicated by an arrow) in these regions are shown in Figure 1, A-E. Stimulation
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and M. E. OLDS
Fig. I. A-E: the sites where neuronal activity was recorded. F: the site where stimulation applied to produce punishment. Am: amygdala; H: hippocampus; CL: lateral geniculate; medial geniculate; Hyp: hypothalamus; CG: central grey region.
was GM:
was applied via macroelectrodes (250pm, twisted wires) implanted in the central grey region, bordering the reticular formation (Fig. 1 k‘). Figure 2A shows an example of unit activity recorded in the amygdala with the display of the outputs of a window discriminator above some of the action potentials to show which spikes are “counted”. In Figure 2B, an example of several superimposed traces of the output of a neurone, the two gates used to discriminate this output from that of nearby
Neuronal
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behaviour
Fig. 2. Example of unit activity. A: The dots on top of the action potentials indicate that activity from two neurones was studied as shown by the different amplitude of the potentials. B: The time base was expanded to show the use of the two-window discriminator used to select the signals that were counted.
After
condltiomg
Rat-1381
‘I
11
I
f hght
I
1t ,
,I(
-1
lo
Cunrewarded lever press reworded lever press bmn stimulotlon (artifact)
Fig. 3. Example of the effect of aversive brain stimulation on a lever-pressing response rewarded with food. Responding was abolished before and during the presentation of the conditioned stimulus (light) but was reinstated after the brain stimulus.
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M. UMEMOTO and M. E. OLDS Table 1. Effects of drugs on conditioned lever-press response for food reward No drug Number tested
Drug Chlordiazepoxide Chlordiazepoxide Diazepam Chlordiazepoxide Chlorpromazine Chlordiazepoxide
5 10 25 5.0 I 2
Partial
Complete
depressiont
&p1.%i0!P
II 1.7 IO IO 10 12
8 IO 8 8 8 8
After drug Disinhibitionf
3 2 2 2 2 2
Peak time ofeKect after injection (min)
5 5 3 2 0 0
Number which showed complete depression of lever-pressing during entire period. t Number which showed lever-pressing during inter-conditioned stimulus period (non-conditioned period). $ Number showed lever-pressing during conditioned stimulus period and/or inter-conditioned period.
>I0 f-20 5 5 5 5
*
stimulus stimulus
neurones and the markings signifying that these spikes are “counted”, are shown to illustrate the methods of recording extracellular activity in the freely moving subject. Figure 3 illustrates the effects of introducing aversive brain stimulation on lever-pressing behaviour maintained by food reward. Before the introduction of brain stimulation, leverpressing behaviour was distributed about evenly between the inter-trial period and the conditioned stimulus presentation interval. After the interpolation of aversive brain stimulation at the end of the conditioned stimulus presentation, lever-pressing behaviour was either completely suppressed, or partially suppressed, although the intensity of stimulation was at the threshold level of eliciting a withdrawal reaction. The group results obtained with the introduction of aversive brain stimulation on lever-pressing for food reward are shown in Table 1. The majority of animals stopped responding altogether after brain stimulation was introduced. In a few subjects, when suppression was partial, responding occurred for a brief interval after the conditioned stimulus presentation. It was difficult to apply an intensity of stimulation that was low enough to elicit a just noticeable withdrawal reaction but not so low as to suppress completely the lever-pressing response for food. Total or partial suppression usually occurred after 1-5 trials. Figure 4 illustrates the effects of introducing aversive brain stimulation on the rate of cellular discharge. The rate of firing was sometimes increased and sometimes decreased
Control (before Hypothalamus
6
Hippocampus
fi
conditioning)
Rat -1540
Amygdola Behoviour
~
Conditioning
r38th IO
set
Fig. 4. Examples of neuronal conditioned responses in the hypothalamus, the hippocampus, and the amygdala. In all three regions the conditioned neuronal response was an increase in the rate offiring during the presentation of the conditioned stimulus while at the same time lever-responding was suppressed.
419
Neuronal activity during conflict behaviour
Fig. 5. Example of the disinhibiting effect of chlordiazepoxide (10 mg/kg) on a lever-pressing response suppressed by aversive brain stimulation. Before the drug, the animal responded after the application of the aversive stimulus only; after the drug, the animal responded during the conditioned stimulus and the inter-trial periods.
during the presentation of the conditioned stimulus, but mainly the change in the rate of discharge was to a great extent correlated with the presentation of the conditioned stimulus. The neuronal responses to the conditioned stimulus required about 30-50 trials for development, in contrast to the much smaller number of trials required for the development of the conditioned behavioural responses. The correlation between the appearance of the neuronal responses and the time of presentation of the conditioned stimulus was only partial. The neuronal responses lasted somewhat longer than the conditioned stimulus and started somewhat later. In a few cases, there was no conditioned neural response, but instead an unconditioned response. In other instances, the conditioned neuronal response was followed by an unconditioned one. Eflects of the drugs on behaviour
Chlordiazepoxide, at the 5 and 10 mg/kg doses, antagonized the suppression of responding for food reward in five subjects. An example of this effect on behaviour is shown in Figure 5. During the pre-injection period, the animal pressed for the food reward only during a brief interval of time after the presentation of the conditioned stimulus. Twenty-eight minutes after the injection of 10 mg/kg chlordiazepoxide, responding occurred during the inter-trial period and often during the period of the conditioned stimulus presentation. This effect lasted for 515 min and then suppression of responding returned to pre-injection levels. Less than half of the animals tested were disinhibited after chlordiazepoxide, at either the 5 or the 10 mg/kg dose. There was no dose-related effect on behaviour (Table 1). Diazepam was less effective than chlordiazepoxide on conditioned emotional behaviour. At the 2.5 mg/kg dose, three subjects were disinhibited, but at the 5 mg/kg dose, only two subjects lever-pressed for food during the inter-trial and the conditioned stimulus presentation periods (Table 1). The data suggest an inverse relation between the number of subjects disinhibited and the dose of diazepam injected. There was no correlation between cases of partial suppression of lever pressing for food and the disinhibiting effects of chlordiazepoxide and diazepam. Table 2. Number of units used in drug experiments Drug
Dose (mg/kg)
Amygdala
Chlordiazepoxide Chlordiazepoxide Diazepam Chlordiazepoxide Chlorpromazine Chlordiazepoxide
5 10 2.5 5.0 1 2
6 7 7 7 6 7
Hippocampus 8 I 6 6 6 7
L. Hypothalamus 6 8 6 6 6 7
Geniculate 6 6 7 6 6 I
M. UMEMOTO
420
CDP
l
p;1
0 n
andM.E.
OLDS
5mg/kg IOmg/kg
Amygdalo
min Hypothalamus
IO
Geniculate
A
d
Fig. 6. Effects of chlordiazepoxide (CDP) on neuronal activity during the background period (B) and on neuronal activity during the conditioned stimulus (A). Chlordiazepoxide, 5 mg/kg: filled circle in A and hatched bar in B; chlordiazepoxide, 10 mg/kg: open circle in A and open bar in B. The results on background activity are shown as the percentage changes in the rate of unit firing in an interval of IO set before the presentation of the conditioned stimulus. The results on unit activity during the conditioned stimulus are expressed as ratios of the activity during the background period. * PS
0.05 compared
with controls.
Chlorpromazine had no disinhibiting effect on lever pressing for food suppressed by aversive brain stimulation. Table 2 shows the number of units tested with each drug in each region. In most cases the test was made on a single unit, but sometimes the electronic gates were adjusted to count the output of 2-3 neurones. In Table 2, such a test was nevertheless classified as a single experiment because the 2-3 units were outputs of a single discriminator. A unit was selected for study if its background rate of firing changed as a function of the conditioned stimulus presentation. The drug effects were analyzed separately for 10 set of the inter-trial period, or background activity, and for the conditioned stimulus presentation interval, or neuronal response. Effects of the drugs OIZthe rate ofjring activity)
durirlg the inter-trial
period
(background
neuronal
The effects of chlordiazepoxide on the background rate of discharge are shown in Figure 6, panels B. At each of the two doses tested (5 and 10 mg/kg) it reduced the rate of firing in the inter-trial period, in the lo-set interval that preceded the conditioned stimulus presentation. The earliest effects at the 5 mg/kg dose occurred in the geniculate nuclei; the earliest effects at the 10 mg/kg dose occurred in the amygdala and the hypothalamus. During the l&25 min interval after injection, the rate of discharge during the background period was significantly lower in the amygdala, the hippocampus, and the hypothalamus, at the 5 and 10 mg/kg doses. The time of peak effect of chlordiazepoxide was between 16 and 35 min after injection. This was the time when disinhibition of suppressed behaviour was most often observed. Approximately one hour after injection of chlordiazepoxide, at
Neuronal
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DZP
‘!Ti 0 fl
during
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behaviour
2.5 mg/hg 5 mg/hg Hlppocampus
Amygdalo
A
A=Condiiioned
0 10
IO 08
Activity
:08
Fig. 7. Effects of diazepam (DZP) on the background and the conditioned neuronal activity. Diazepam, 2.5 mg/kg: filled circle and hatched bar; diazepam, 5 mg/kg: open circle and open bar.
Hypothalom”s
I
T
A
T
Fig. 8. Effects of chlorpromazine (CPZ) on spontaneous and on conditioned neuronal activity. Chlorpromazine, 1mg/kg: filled circle and hatched bar; chlorpromazine. 2 mg/kg: open circle and open bar.
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both doses, the background rate was still depressed in the hypothalamus and the hippocampus. In the amygdala, the rate was depressed at the 10 mg/kg dose. Dose-response relationships were observed in the amygdala and the hypothalamus, but not in the hippocampus. In summary, chlordiazepoxide reduced the rate of discharge in the intertrial interval in three of the four regions investigated, but the effects were more pronounced and had an earlier onset at the 10 mg/kg dose in the amygdala and the hypothalamus. The largest reduction occurred in the hypothalamus at the 10 mg/kg dose. Neuronal activity in the amygdala, the hippocampus, and the hypothalamus, however, showed significant decrease at both doses in the 16-25 min period, which was the time of peak behavioural effect of the drug. The effects of diazepam (2.5 and 5 mg/kg) are shown in Figure 7, panels B. The background rate in the amygdala was reduced at the 2.5 mg/kg dose but not at the 5 mg,/kg dose. In the other three regions in this inter-trial interval, the rate of discharge was not significantly reduced. In the geniculate nuclei, at the 2.5 mg/kg dose, it was enhanced but not significantly so. The effects of chlorpromazine (1 and 2 mg/kg) on the background rate are shown in Figure 8B. The rate of discharge was reduced at each dose and in each region. The largest decreases and the earliest effects occurred in the hypothalamus. In this region the time of peak effect was 16-25 min, but the reduction was of almost similar magnitude 5665 min after injection. In the amygdala, the background rate was reduced at the 2 mg/kg dose throughout the test, except for the 25-35 min interval, when a reduction of similar magnitude also occurred at the 1 mg/kg dose. A dose-response relationship was less obvious in the amygdala than in the hypothalamus. In the 5665 min interval, the background rate was not significantly different from the pre-injection period. Thus, neuronal activity during the background period in the amygdala was less sensitive to the depressant action of chlorpromazine than was similar activity in the hypothalamus; a recovery of the rate of firing was observed in the amygdala, but not in the hypothalamus. In the hippocampus, the rate of unit discharge during background was depressed during the 1625 min interval only. There was no dose-related effect in this region. In the geniculate nuclei, the decrease was small and occurred later than in the amygdala, the hypothalamus, or the hippocampus at the 1 mg/kg dose. However, at the 2 mg/kg dose, the reduction was in the same range as in the other three regions. A dose-response effect was observed in this region. Recovery to the control rate did not occur in the 5665 min interval, as it did in the amygdala and hippocampus. In summary, chlorpromazine reduced the background rate of discharge in all four regions, but the reductions were largest in the hypothalamus, and lasted longest in the hypothalamus and in the geniculate nuclei. Drug efects on the change in the rate of discharge correlated with the conditioned stimulus presentation
The effects of chlordiazepoxide are shown in Figure 6A. The changes in rate that were conditioned stimulus-correlated were reduced or abolished after chlordiazepoxide at the two doses in the amygdala and in the hippocampus. Chlordiazepoxide had no effect in the geniculate region, and only transiently reduced changes in rate at the 10 mg/kg dose in the hypothalamus. The effect had an early onset in the amygdala and the hippocampus at the two doses, but lasted longer in the amygdala at the lower dose than in the hippocampus. A dose-response effect was observed mainly in the hippocampus. In the amygdala, the conditioned stimulus-correlated rate was almost totally abolished even at the low dose. At the low dose, the time of peak effect was in the 1625 min interval. At the high dose, the peak effect occurred earlier, ie., in the 615 min period after injection The effects of diazepam are shown in Figure 7A. The neuronal response was reduced after diazepam at both doses in all four regions. The peak effect occurred in the 16-25 min interval after injection. A clear dose-response relationship was observed in the amygdala and in the geniculate nuclei.
423
Neuronal activity during conflict behaviour
I.
to set
t
Fig. 9. Example of the effect of chlordi~e~xide (10 mgjkg) on the background and con~~oned neurona activity in the hypothalamus, hippocampus, and amygdala, and on behaviour. Levetpressing was not disinhibited after chlordiaze~xidc. The neuronal activity in the h~othalamus was reduced during the presentation of the conditioned stimulus (CS) and during the background period. In the hippocampus and the amygdala, the conditioned neuronal activity was reduced.
The earliest effects of the 2.5 mg/kg dose appeared in the geniculate nuclei, and of the S mg/kg dose in the amygdaIa and h~Fp~arnpus. Conditioned s~mulus-~or~lated pattern of neuronal activity in the hypothalamus compared with the other three regions was disrupted less, and any disruption occurred later, after diazepam. Figure 8A shows the effects of chlorpromazine at doses of I and 2 mg/kg, on the conditioned stimulus presentation pattern of firing. At the 1 mg/kg dose there was a transient, early d~sruptjon ofthis pattern in the amygdafa only; at the 2 mg/kg dose, ~hlo~rorn~~ne significantly modified the conditioned stimuius~orrelated change in the unit activity in the hippo~mpus throughout most of the test, and only transiently so in the geniculate region. Zn summary, chlorpromazine had relatively minor disruptive effects on the conditioned neuronal responses. Figure 9 illustrates the action of ~hlordi~epoxide administered at a dose of 10 mg/kg. The top four traces show the pattern of lever-pressing for food and of unit activity before the introduction of the aversive brain stimuli. The next four traces show the effect of introducing the aversive stimuli on these responses. The rate of discharge was higher during the presentation of the conditioned stimulus compared with the background rate in the interval that immediately preceded the conditioned stimulus. Lever-pressing for a food reward was totally suppressed. Thirty minutes after the adminis~ation of 10 mg/kg of chlordiazepoxide, the neuronal activity correlated with the conditioned stimulus presentation, was reduced or suppressed. The background neuronal activity was reduced in the hypothalamus, but not in the hippocampus or the amygdala. The unconditioned neuronal response in the h~otha~amus was reduced, but not to the same degree as was the conditioned response in this region,
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andM.E.
OLDS
DISCUSSION
The finding of these experiments was that the benzodiazepines, but not chlorpromazine, exerted a disinhibition action on the lever-pressing response for food in approximately half of the subjects and they simultaneously reduced the background rate of neuronal activity and also the conditioned stimulus-correlated rate of discharge. The results using the lever-pressing response are consistent with the findings reported previously that the benzodiazepines are effective in conflict-type behaviour, and that chlorpromazine is not (SCHALLEKet al., 1972). However, on the basis of these reports, a much larger proportion of the subjects had been expected to show disinhibition after the administration of the benzodiazepines. The rather moderate action of these compounds in the present tests must therefore be related to the use of brain stimulation in the central grey region to cause suppression of lever-pressing. MARGULESand STEIN (1967, 1968) showed that the nature of the punishing stimuli was unimportant. Our results differ from this finding. In general, there was little difference between an intensity which caused no behavioural effect and an intensity that caused total suppression of lever-pressing. It is possible that, had we achieved in most animals a suppression of lever-pressing limited to the conditioned stimulus period, the benzodiazepines would have acted more effectively as disinhibiting compounds. It was interesting that diazepam disinhibited three subjects at the 2.5 mg/kg dose, but only two at the 5 mg/kg dose. It is possible that a lower dose than the 2.5 mg/kg dose would have caused more subjects to be disinhibited. In relation to the main question of whether the drug-effects on the unit activity reveal something of their mode of action in a conflict test, the answer has to be cautious and more hypothetical than factual. This is so for several reasons. First was the observation of a loose correlation between the conditioned behavioural response to aversive brain stimulation and the conditioned neuronal response. The former appeared as soon as brain stimulation was introduced, the latter developed gradually over a large number of trials, its onset in each trial being later than the onset of the conditioned stimulus and often outlasting the conditioned stimulus. Second was the observation that diazepam, which disinhibited only 2-3 subjects, was the compound which antagonized most effectively the conditioned stimulus-induced change in the pattern of firing, and chlorpromazine, which was totally ineffective on lever-pressing behaviour, also antagonized the conditioned stimuluscorrelated neuronal activity in the hippocampus at the 2 mg/kg dose. Thus, on the basis of their effects on the conditioned stimulus-correlated neuronal activity, it would be impossible to separate the benzodiazepines, which sometimes cause disinhibition, from the non-disinhibiting phenothiazine. It is interesting to note, however, that the benzodiazepines were effectively antagonizing the conditioned neuronal activity in the amygdala, and that chlorpromazine had practically no such effect in this region. Another observation of possible significance was that conditioned neuronal activity in the hypothalamus and in the geniculate nuclei must have some complex relationship to the conditioned behavioural response because chlordiazepoxide, which disinhibited five subjects out of eleven at the 5 mg/kg dose, did not modify significantly the conditioned stimulus-correlated neuronal activity, while diazepam, at both doses, abolished the neuronal responses in the geniculate nuclei and reduced them in the hypothalamus. When the effects of the drugs on the background rate were analyzed, a clear pattern distinguishing the action of the benzodiazepines from that of chlorpromazine also failed to emerge. Chlordiazepoxide and chlorpromazine reduced the rate of unit discharge in the background period, but as was noted earlier, the first compound was effective on behaviour while the second was not. And diazepam, which had a very little effect on the background rate, had more effect on the lever-pressing behaviour than chlorpromazine, which did reduce the background rate. There were, however, two aspects of the drug effects that merit our consideration. The first was that the time of peak effect of the benzodiazepines on behaviour correlated well with their peak effect on the conditioned stimulus-correlated neuronal activity and on the activity during the background period. The second was that the two benzodiazepines
Neuronal activity during conflict behaviour
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reduced both the background rate of discharge and the conditioned neuronal responses, but only in the amygdala. It would seem, therefore, that their action there could be of significance to their action on lever-pressing. However, at present this is only a possibility because the relationship between the neuronal activity and the behaviour is complex and hence does not allow an easy interpretation of the drug effects on neuronal discharge. The findings point nevertheless to selective effects on conditioned and background neuronal activity at the time when the drugs were effective on behaviour, and therefore the data point in the direction that future work will have to take to explain their anti-anxiety effects. REFERENCES BRADY,J. V. (1956). A comparative approach to the evaluation of drug effects upon affective behaviour. Ann. N.y Acad. Sci. 64,632~643. BRADY.J. V. (1960). Emotional behaviour. In: Handbook of Physiology, Sec. I, Neurophysiology, Vol. 3 (FIELD, J., MACOUN,H. W. and HALL,V. E., Eds.), pp. 1529-1552. Am. Physiol. Sot., Baltimore. BRDCKE, F. T. V., HORNYKIWIECZ, 0. and SIGG, E. B. (1969). The Pharmacology of‘ Psychotherapeutic Drugs. Springer, New York. DAVIDSON, A. B. and COOK,L. (1969). Effects of combined treatment with trifluoperazone-HCI and amobarbital on punished behavior in rats. Psychopharrnucologia 15: 159-168. DE WIED, D. (1967). Chlorpromazine and endocrine function. Pharmuc. Rev. 19: 251-288. GELLER,I., KOLAK,J. T., JR. and SEIFERS,J. (1962). The effects of chlordiazepoxide and chlorpromazine on a punished discrimination. Psychopharmacologia 3: 374-385. GUERRERO-FIGUEROA, R., GALLANT,D. M., GUERRERO-FIGUEROA, C. and GALLANT,J. (1973). Electrophysiological analysis of the action of four benzodiazepine derivatives on the central nervous system. In: The Benzodiazepines (GARATTINI, S., MUSSINI,E. and RANDALL,L. O., Eds.), pp. 489-510. Raven Press, New York. HEISE,G. A., LAUGHLIN,N. and KELLER,C. (1970). A behavioral and pharmacological analysis of reinforcement withdrawal. Psychopharmacologia 16: 341-368. KGNIG,J. F. R. and KLIPPEL,R. A. (1963). The Rat Brain: A Stereotaxic Atlas of the Forebrain and Lower Parts qf‘the Brain Stem. Williams & Wilkins. Baltimore. MARGULES,D. L. and STEIN.L. (1967). Neuroleptics vs tranquilizers: evidence from animal behavior studies of mode and site of action. In: Neuropsychopharmacology (BRILL,H., COLE,J. O., DENIKER,P., HIPPINS,H. and BRADLEY, P. B., Eds.), Vol. 5, pp. 108-120. Elsevier, Amsterdam. MARGULES,D. L. and STEIN,L. (1968). Increase of “antianxiety” activity and tolerance of behavioral depression during chronic administration of oxazepam. Psychopharmacologia 13: 7480. MORILLO,A. (1962). Effects of benzodiazepines upon amygdala and hippocampus of cat. Int. J. Neuropharmac. 1: 353-359. OLIX, J. (1959). Studies of neuropharmacologicals by electrical and chemical manipulation of the brain in animals with chronically implanted electrodes. In: Neuro-psychopharmacology: Proc. 1st. Int. Congr. Neuropharmacol., Rome, 1958 (BRADLEY.P. B., DENIKER, P. and RADOUCO-THOMAS, C., Eds.), pp. 20-32. Elsevier, Amsterdam. OLDS,J. (1973). Multiple unit recordings from behaving rats. In: Eioelectric Recording Techniques, Part A. Cellular Processes and Brain Potentials (THOMPSON, R. F. and PATTERSON, M. E., Eds.), pp. 16s198. Academic Press, New York. OLDS, M. E. (1966). Facilitatory action of diazepam and chlordiazepoxide on hypothalamic reward behavior. J. camp. physiol. Psychol. 62: 136-140. PRESTON,J. B. (1956). Effects of chlorpromazine on the central nervous system of the cat: a possible neural basis for action. J. Pharmac. exp. Ther. 118: 10&l 15. SCHALLEK, W. and KUEHN,A. (1960). Effects of psychotropic drugs on limbic system of cat. Proc. SOC. exp. Biol. Med. 105: 115-l 17. SCHALLEK, W., S~HLOSSER, W. and RANDALL,L. 0. (1972). Recent developments in the pharmacology of the benzodiazepines. Adv. Pharmac. Chemother. 10: 119-182. U~MOTO, M., MURAI, Y., KODAMA,M. and KIDQ R. (1970). Neuronal discharge patterns in conditioned emotional response. Brain Res. 24: 347-351. WEDEKIND,P. W. (1969). Disinhibition effect of chlordiazepoxide. Psychonom. Sci. 15: 232-237.
1975, 14. 413425.
Pergamon
Press. Printed
in Gt. Briram
EFFECTS OF C~LOR~IAZEPOXIDE, DIAZEPAM AND CHLORPROMAZINE ON CONDITIONED EMOTIONAL BEHAVIOUR AND CONDITIONED NEURONAL ACTIVITY IN LIMBIC, HYPOTHALAMIC AND GENICULATE REGIONS* M. UlvlEMoTOtand M. E. OLDS Division of Biology, California Institute of Technology, Pasadena, California 91109 (Accepted 24 August
1974)
Summary-The effects of chlorpromazine, chlordiazepoxide and diazepam on conditioned emotional behaviour and neuronal activity correlated with it, were studied in the rat. Conditioned behaviour was produced by brain aversive stimulation in the central grey region. In the freely moving animal, neuronal activity was studied in the amygdala, the hippocampus, the hypothalamus, and the geniculate nuclei. Effects of the drugs were compared on the background rate of firing before the presentation of the conditioned stimulus in each trial, and on the change in the background rate correlated with the presentation of the conditioned stimulus after the subject was conditioned. Rehavioural responses for a food reward that had been suppressed during convict-conditioning were reinstated in approximately SOY0of the subjects after chlordiazepoxide (5 and 10 mg/kg) and in 20% after diazepam (25 and 5 mg/kg). Responding was not reinstated after chlorpromazine (1 and 2 mg/kg). The background rate of firing was reduced by chlordiazepoxide and chlorpromazine, but not by diazepam. The change in unit activity correlated with the presentation of the conditioned stimulus was reduced or abolished by chlordiazepoxide and diazepam, but not by chlorpromazine except in the hippocampus at the 2 mg/kg dose. The neuronal activity in the amygdala during the background period and during the conditioned stimulus-presentation was influenced by chlordiazepoxide to a greater extent than by the other two compounds. The significance of these results is discussed in terms of a possible relationship between the effects of the drugs on behaviour and on the rate of cellular discharge.
An interesting effect of the benzodiazepines is the disinhibition they produce on previously-suppres~d responses in an operant task, where reward and punishment are interpolated. Chlorpromazine, on the other hand, has no effect on punished responses but reduces the number of rewarded responses (GELLER, KOLAK and SEIFERS, 1962; MARGULES and STEIN,1967,196s ; DAVIDSON and COOK,1969 ; WEDEKIND, 1969 ; HEISE, LAUGHLIN and KELLER, 1970; SCHALLEK, SCHLOSSERand RANDALL, 1972). WEDEKIND (1969) attributed the release of suppressed behaviour by the minor tranquilizers, to a general breakdown of inhibition. MARGULES and STEIN(1968) showed that the nature of the punishing stimulus is unimportant; in their tests, although the stimuli employed to cause punishment varied from brain aversive stimulation to the adulteration of milk by the addition of quinine, suppressed behaviour was nonetheless released by oxazepam, a minor tranquilizer, but not by chlorpromazine, a neuroleptic, The value of these findings would be enhanced if the behavioural effects of these drugs were correlated with neurophysiological action. UMEMOTO, MURAI, KODAMA and KIDO (1970) have described patterns of neuronal activity characteristic of conflict behaviour such as that described by BRADY (1956, 1960). In the present tests, we used methods developed by OLDS (1973) for recording neuronal activity in order to study the effects of the benzodiazepines simultaneously on conflict behaviour and on the patterns of neuronal discharge associated with it. * Research supported in part by USPHS Grant MH-16978. T Present address: Shionogi Research Laboratory. Osaka. Japan. 413
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Neuronal activity was studied in four regions. The first two were the amygdala and the hippocampus. They were selected because they are generally presumed to be sites of action of the benzodiazepines (SCHALLEKand KUEHN, 1960; MORILLO,1962) and of chlorpromazine (PRESTON,1956; BROCKE,HORNYKIWIECZand SIGG, 1969). The third region studied was the hypothalamus. There is accumulating evidence for a direct action by these compounds in this region (GUERRERO-FIGUEROA, GALLANT, GUERRERO-FIGUEROA and GALLANT, 1973; OLDS, 1959; OLDS, 1966; DE WIED, 1967). Finally, neuronal activity in the geniculate nuclei was studied to determine if these compounds influence the neuronal excitability in regions not directly implicated in the regulation of emotional behaviour.
METHODS Animals
Male albino rats of the Holtzman strain with initial body weight of 25&300 g were used. They were housed in individual cages with food and water available ad libitum until 3-4 days after surgery, when the animals were placed on a 23-hr feeding schedule. Most of the food consumed during the duration of the tests was obtained during behavioural training in a conflict test. Surgery
Thirty-one rats were used. Each was implanted with 7 nichrome wire electrodes for recording (62.5 pm). These were aimed at sites in the amygdala, the hippocampus, the hypothalamus, and the geniculate nuclei. Each animal was also implanted with one set of twisted stainless steel wires (250 pm) in the central grey region for the production of aversive stimulation. An uninsulated stainless steel wire was implanted in the rostra1 regions of the brain at the level of the olfactory bulb to serve as an indifferent electrode for monopolar recording of unit activity. The stereotaxic coordinates were selected from the K~NIG and KLIPPELAtlas (1963). Surgery was carried out under pentobarbital sodium anaesthesia (50 mg/kg, i.p.). The details of the surgical methods have recently been described elsewhere (OLDS, 1973). The sites of stimulation and recording were verified at the end of the experiments on histological material processed by the frozen method and stained with either cresyl violet or Weil stain. The results are based on data obtained from animals in which the recording probes were lodged in the amygdala, hippocampus, hypothalamus and geniculate region. All stimulating probes were in the central grey region. Extracellular recording and stimulation techniques
The methods described by OLDS (1973) were used. The animal was able to move freely in the test chamber and to depress a lever to obtain food, while at the same time unit activity derived from three probes was recorded. The signal from the preamplifier was fed into a double-window discriminator by means of a conventional amplifier. The amplitude and the fall-time of the action potential was used to select the signal from the baseline activity. The output from the double-window discriminator was a 1-msec binary pulse that was counted and recorded on a pen-writing oscillograph. The animal’s responses and other event-markers were also recorded in the same manner. The extracellular activity from three microprobes was recorded simultaneously with the bar-pressing behaviour of the animal. Electrical brain stimulation was applied to the central grey region of the midbrain in a region bordering the reticular formation (Fig. 1 F). The stimulator was a Grass S-4 apparatus that delivered unidirectional pulses of 3-msec duration in trains of 60 Hz. The intensity of the stimulation was determined for each animal individually as that level just sufficient to produce a withdrawal reaction. All animals in which such a reaction was not observed during preliminary tests for the aversive properties of midbrain brain stimulation, were not used in the drug tests.
Neuronal activity during contlict behaviour
Behavioural
425
training
After recovery from surgery (3-5 days) the subjects were screened for unit activity. When this activity was of a ratio of 2:l or higher in relation to the baseline “noise” on at least three of the seven probes implanted for recording in’ each animal, and furthermore was not correlated with the movements of the animal, then behavioural training was begun. The subjects learned to press a lever on a fixed-interval schedule (10 set) of food reinforcement. Usually this training was done overnight. When lever-pressing became stabilized, a light stimulus (conditioned stimulus) was introduced without brain stimulation. The light stimulus was presented every 90 set and was of lo-set duration. Time was allowed for any neuronal and ~havioural responses to the stimulus to extinguish (habituation; usually between 48-72 trials), and then a 0.2~set electrical train in the central grey region of the brain (aversive stimulation) was introduced at the end of each conditioned stimulus presentation. The intensity of the brain stimulus was adjusted to the minimum level necessary for the appearance of a withdrawal reaction. When the brain stimuli failed to produce this effect on behaviour in a subject, the subject was eliminated from the experiment. In the other subjects, training proceeded until the lever-pressing response for the food reward was suppressed, and until a change in the rate of firing in relation to the background rate appeared during the conditioned stimulus presentation. Data analysis
The score for the background activity was the number of units discriminated in the loset interval before each light stimulus presentation in blocks of six trials before the administration of the drugs and at various times after. The effects of the drugs were expressed as the percentages of the scores before injection (see panels B in Figs. 68). The score for the neuronal response (conditioned stimulus-correlated activity) was the ratio between the number of units in the lo-see interval of the conditioned stimulus presentation and the number of units in the IO-set interval immediately preceding the conditioned stimulus presentation, for blocks of six trials. The number of units in the conditioned stimulus-segments was subtracted from the number of units in the segments preceding the conditioned stimulus, and the result was divided by the number in the segments preceding the conditioned stimulus. The inflection ratio shifted in the positive direction when there was an increase in the rate of discharge the conditioned stimulus presentation, and it shifted toward - 1.0 when the rate of firing decreased during the conditioned stimulus presentation. To compute group scores, the negative inflection ratios were converted to absolute values. Ratios were computed for the period before the injection of the drugs and at various times thereafter. Effects were determined by comparing the inflection ratios at various times after the injection of the drugs with the inflection ratios before injection. The statistical significance of the changes was evaluated with the Student t test for paired comparison. Drugs
Chlordiazepoxide was tested at 5 and 10 mg/kg, diazepam at doses of 2.5 and 5 mg/kg, and chlorpromazine at doses of 1 and 2 mg/kg. The doses are given as the salts. Each drug was dissolved in 0.9% saline; the volume injected for chlordiazepoxide and chlorpromazine was Iess than O-5ml. For diazepam, the volume was less than 1 ml and the drug was in suspension. Control injections of physiological saline were made in a separate group of animals. No significant effects were observed on behaviour or neuronal activity in the control group.
RESULTS
Recordings of unit activity were derived from microelectrodes (62.5 pm) implanted in the amygdala, the hippocampus, the hypothalamus, and the geniculate nuclei. Representative examples of histological material showing the electrode-tracks and the site of stimulation (indicated by an arrow) in these regions are shown in Figure 1, A-E. Stimulation
416
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Fig. I. A-E: the sites where neuronal activity was recorded. F: the site where stimulation applied to produce punishment. Am: amygdala; H: hippocampus; CL: lateral geniculate; medial geniculate; Hyp: hypothalamus; CG: central grey region.
was GM:
was applied via macroelectrodes (250pm, twisted wires) implanted in the central grey region, bordering the reticular formation (Fig. 1 k‘). Figure 2A shows an example of unit activity recorded in the amygdala with the display of the outputs of a window discriminator above some of the action potentials to show which spikes are “counted”. In Figure 2B, an example of several superimposed traces of the output of a neurone, the two gates used to discriminate this output from that of nearby
Neuronal
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behaviour
Fig. 2. Example of unit activity. A: The dots on top of the action potentials indicate that activity from two neurones was studied as shown by the different amplitude of the potentials. B: The time base was expanded to show the use of the two-window discriminator used to select the signals that were counted.
After
condltiomg
Rat-1381
‘I
11
I
f hght
I
1t ,
,I(
-1
lo
Cunrewarded lever press reworded lever press bmn stimulotlon (artifact)
Fig. 3. Example of the effect of aversive brain stimulation on a lever-pressing response rewarded with food. Responding was abolished before and during the presentation of the conditioned stimulus (light) but was reinstated after the brain stimulus.
418
M. UMEMOTO and M. E. OLDS Table 1. Effects of drugs on conditioned lever-press response for food reward No drug Number tested
Drug Chlordiazepoxide Chlordiazepoxide Diazepam Chlordiazepoxide Chlorpromazine Chlordiazepoxide
5 10 25 5.0 I 2
Partial
Complete
depressiont
&p1.%i0!P
II 1.7 IO IO 10 12
8 IO 8 8 8 8
After drug Disinhibitionf
3 2 2 2 2 2
Peak time ofeKect after injection (min)
5 5 3 2 0 0
Number which showed complete depression of lever-pressing during entire period. t Number which showed lever-pressing during inter-conditioned stimulus period (non-conditioned period). $ Number showed lever-pressing during conditioned stimulus period and/or inter-conditioned period.
>I0 f-20 5 5 5 5
*
stimulus stimulus
neurones and the markings signifying that these spikes are “counted”, are shown to illustrate the methods of recording extracellular activity in the freely moving subject. Figure 3 illustrates the effects of introducing aversive brain stimulation on lever-pressing behaviour maintained by food reward. Before the introduction of brain stimulation, leverpressing behaviour was distributed about evenly between the inter-trial period and the conditioned stimulus presentation interval. After the interpolation of aversive brain stimulation at the end of the conditioned stimulus presentation, lever-pressing behaviour was either completely suppressed, or partially suppressed, although the intensity of stimulation was at the threshold level of eliciting a withdrawal reaction. The group results obtained with the introduction of aversive brain stimulation on lever-pressing for food reward are shown in Table 1. The majority of animals stopped responding altogether after brain stimulation was introduced. In a few subjects, when suppression was partial, responding occurred for a brief interval after the conditioned stimulus presentation. It was difficult to apply an intensity of stimulation that was low enough to elicit a just noticeable withdrawal reaction but not so low as to suppress completely the lever-pressing response for food. Total or partial suppression usually occurred after 1-5 trials. Figure 4 illustrates the effects of introducing aversive brain stimulation on the rate of cellular discharge. The rate of firing was sometimes increased and sometimes decreased
Control (before Hypothalamus
6
Hippocampus
fi
conditioning)
Rat -1540
Amygdola Behoviour
~
Conditioning
r38th IO
set
Fig. 4. Examples of neuronal conditioned responses in the hypothalamus, the hippocampus, and the amygdala. In all three regions the conditioned neuronal response was an increase in the rate offiring during the presentation of the conditioned stimulus while at the same time lever-responding was suppressed.
419
Neuronal activity during conflict behaviour
Fig. 5. Example of the disinhibiting effect of chlordiazepoxide (10 mg/kg) on a lever-pressing response suppressed by aversive brain stimulation. Before the drug, the animal responded after the application of the aversive stimulus only; after the drug, the animal responded during the conditioned stimulus and the inter-trial periods.
during the presentation of the conditioned stimulus, but mainly the change in the rate of discharge was to a great extent correlated with the presentation of the conditioned stimulus. The neuronal responses to the conditioned stimulus required about 30-50 trials for development, in contrast to the much smaller number of trials required for the development of the conditioned behavioural responses. The correlation between the appearance of the neuronal responses and the time of presentation of the conditioned stimulus was only partial. The neuronal responses lasted somewhat longer than the conditioned stimulus and started somewhat later. In a few cases, there was no conditioned neural response, but instead an unconditioned response. In other instances, the conditioned neuronal response was followed by an unconditioned one. Eflects of the drugs on behaviour
Chlordiazepoxide, at the 5 and 10 mg/kg doses, antagonized the suppression of responding for food reward in five subjects. An example of this effect on behaviour is shown in Figure 5. During the pre-injection period, the animal pressed for the food reward only during a brief interval of time after the presentation of the conditioned stimulus. Twenty-eight minutes after the injection of 10 mg/kg chlordiazepoxide, responding occurred during the inter-trial period and often during the period of the conditioned stimulus presentation. This effect lasted for 515 min and then suppression of responding returned to pre-injection levels. Less than half of the animals tested were disinhibited after chlordiazepoxide, at either the 5 or the 10 mg/kg dose. There was no dose-related effect on behaviour (Table 1). Diazepam was less effective than chlordiazepoxide on conditioned emotional behaviour. At the 2.5 mg/kg dose, three subjects were disinhibited, but at the 5 mg/kg dose, only two subjects lever-pressed for food during the inter-trial and the conditioned stimulus presentation periods (Table 1). The data suggest an inverse relation between the number of subjects disinhibited and the dose of diazepam injected. There was no correlation between cases of partial suppression of lever pressing for food and the disinhibiting effects of chlordiazepoxide and diazepam. Table 2. Number of units used in drug experiments Drug
Dose (mg/kg)
Amygdala
Chlordiazepoxide Chlordiazepoxide Diazepam Chlordiazepoxide Chlorpromazine Chlordiazepoxide
5 10 2.5 5.0 1 2
6 7 7 7 6 7
Hippocampus 8 I 6 6 6 7
L. Hypothalamus 6 8 6 6 6 7
Geniculate 6 6 7 6 6 I
M. UMEMOTO
420
CDP
l
p;1
0 n
andM.E.
OLDS
5mg/kg IOmg/kg
Amygdalo
min Hypothalamus
IO
Geniculate
A
d
Fig. 6. Effects of chlordiazepoxide (CDP) on neuronal activity during the background period (B) and on neuronal activity during the conditioned stimulus (A). Chlordiazepoxide, 5 mg/kg: filled circle in A and hatched bar in B; chlordiazepoxide, 10 mg/kg: open circle in A and open bar in B. The results on background activity are shown as the percentage changes in the rate of unit firing in an interval of IO set before the presentation of the conditioned stimulus. The results on unit activity during the conditioned stimulus are expressed as ratios of the activity during the background period. * PS
0.05 compared
with controls.
Chlorpromazine had no disinhibiting effect on lever pressing for food suppressed by aversive brain stimulation. Table 2 shows the number of units tested with each drug in each region. In most cases the test was made on a single unit, but sometimes the electronic gates were adjusted to count the output of 2-3 neurones. In Table 2, such a test was nevertheless classified as a single experiment because the 2-3 units were outputs of a single discriminator. A unit was selected for study if its background rate of firing changed as a function of the conditioned stimulus presentation. The drug effects were analyzed separately for 10 set of the inter-trial period, or background activity, and for the conditioned stimulus presentation interval, or neuronal response. Effects of the drugs OIZthe rate ofjring activity)
durirlg the inter-trial
period
(background
neuronal
The effects of chlordiazepoxide on the background rate of discharge are shown in Figure 6, panels B. At each of the two doses tested (5 and 10 mg/kg) it reduced the rate of firing in the inter-trial period, in the lo-set interval that preceded the conditioned stimulus presentation. The earliest effects at the 5 mg/kg dose occurred in the geniculate nuclei; the earliest effects at the 10 mg/kg dose occurred in the amygdala and the hypothalamus. During the l&25 min interval after injection, the rate of discharge during the background period was significantly lower in the amygdala, the hippocampus, and the hypothalamus, at the 5 and 10 mg/kg doses. The time of peak effect of chlordiazepoxide was between 16 and 35 min after injection. This was the time when disinhibition of suppressed behaviour was most often observed. Approximately one hour after injection of chlordiazepoxide, at
Neuronal
activity
DZP
‘!Ti 0 fl
during
conflict
behaviour
2.5 mg/hg 5 mg/hg Hlppocampus
Amygdalo
A
A=Condiiioned
0 10
IO 08
Activity
:08
Fig. 7. Effects of diazepam (DZP) on the background and the conditioned neuronal activity. Diazepam, 2.5 mg/kg: filled circle and hatched bar; diazepam, 5 mg/kg: open circle and open bar.
Hypothalom”s
I
T
A
T
Fig. 8. Effects of chlorpromazine (CPZ) on spontaneous and on conditioned neuronal activity. Chlorpromazine, 1mg/kg: filled circle and hatched bar; chlorpromazine. 2 mg/kg: open circle and open bar.
421
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M. UMEMOTO and M. E. OLDS
both doses, the background rate was still depressed in the hypothalamus and the hippocampus. In the amygdala, the rate was depressed at the 10 mg/kg dose. Dose-response relationships were observed in the amygdala and the hypothalamus, but not in the hippocampus. In summary, chlordiazepoxide reduced the rate of discharge in the intertrial interval in three of the four regions investigated, but the effects were more pronounced and had an earlier onset at the 10 mg/kg dose in the amygdala and the hypothalamus. The largest reduction occurred in the hypothalamus at the 10 mg/kg dose. Neuronal activity in the amygdala, the hippocampus, and the hypothalamus, however, showed significant decrease at both doses in the 16-25 min period, which was the time of peak behavioural effect of the drug. The effects of diazepam (2.5 and 5 mg/kg) are shown in Figure 7, panels B. The background rate in the amygdala was reduced at the 2.5 mg/kg dose but not at the 5 mg,/kg dose. In the other three regions in this inter-trial interval, the rate of discharge was not significantly reduced. In the geniculate nuclei, at the 2.5 mg/kg dose, it was enhanced but not significantly so. The effects of chlorpromazine (1 and 2 mg/kg) on the background rate are shown in Figure 8B. The rate of discharge was reduced at each dose and in each region. The largest decreases and the earliest effects occurred in the hypothalamus. In this region the time of peak effect was 16-25 min, but the reduction was of almost similar magnitude 5665 min after injection. In the amygdala, the background rate was reduced at the 2 mg/kg dose throughout the test, except for the 25-35 min interval, when a reduction of similar magnitude also occurred at the 1 mg/kg dose. A dose-response relationship was less obvious in the amygdala than in the hypothalamus. In the 5665 min interval, the background rate was not significantly different from the pre-injection period. Thus, neuronal activity during the background period in the amygdala was less sensitive to the depressant action of chlorpromazine than was similar activity in the hypothalamus; a recovery of the rate of firing was observed in the amygdala, but not in the hypothalamus. In the hippocampus, the rate of unit discharge during background was depressed during the 1625 min interval only. There was no dose-related effect in this region. In the geniculate nuclei, the decrease was small and occurred later than in the amygdala, the hypothalamus, or the hippocampus at the 1 mg/kg dose. However, at the 2 mg/kg dose, the reduction was in the same range as in the other three regions. A dose-response effect was observed in this region. Recovery to the control rate did not occur in the 5665 min interval, as it did in the amygdala and hippocampus. In summary, chlorpromazine reduced the background rate of discharge in all four regions, but the reductions were largest in the hypothalamus, and lasted longest in the hypothalamus and in the geniculate nuclei. Drug efects on the change in the rate of discharge correlated with the conditioned stimulus presentation
The effects of chlordiazepoxide are shown in Figure 6A. The changes in rate that were conditioned stimulus-correlated were reduced or abolished after chlordiazepoxide at the two doses in the amygdala and in the hippocampus. Chlordiazepoxide had no effect in the geniculate region, and only transiently reduced changes in rate at the 10 mg/kg dose in the hypothalamus. The effect had an early onset in the amygdala and the hippocampus at the two doses, but lasted longer in the amygdala at the lower dose than in the hippocampus. A dose-response effect was observed mainly in the hippocampus. In the amygdala, the conditioned stimulus-correlated rate was almost totally abolished even at the low dose. At the low dose, the time of peak effect was in the 1625 min interval. At the high dose, the peak effect occurred earlier, ie., in the 615 min period after injection The effects of diazepam are shown in Figure 7A. The neuronal response was reduced after diazepam at both doses in all four regions. The peak effect occurred in the 16-25 min interval after injection. A clear dose-response relationship was observed in the amygdala and in the geniculate nuclei.
423
Neuronal activity during conflict behaviour
I.
to set
t
Fig. 9. Example of the effect of chlordi~e~xide (10 mgjkg) on the background and con~~oned neurona activity in the hypothalamus, hippocampus, and amygdala, and on behaviour. Levetpressing was not disinhibited after chlordiaze~xidc. The neuronal activity in the h~othalamus was reduced during the presentation of the conditioned stimulus (CS) and during the background period. In the hippocampus and the amygdala, the conditioned neuronal activity was reduced.
The earliest effects of the 2.5 mg/kg dose appeared in the geniculate nuclei, and of the S mg/kg dose in the amygdaIa and h~Fp~arnpus. Conditioned s~mulus-~or~lated pattern of neuronal activity in the hypothalamus compared with the other three regions was disrupted less, and any disruption occurred later, after diazepam. Figure 8A shows the effects of chlorpromazine at doses of I and 2 mg/kg, on the conditioned stimulus presentation pattern of firing. At the 1 mg/kg dose there was a transient, early d~sruptjon ofthis pattern in the amygdafa only; at the 2 mg/kg dose, ~hlo~rorn~~ne significantly modified the conditioned stimuius~orrelated change in the unit activity in the hippo~mpus throughout most of the test, and only transiently so in the geniculate region. Zn summary, chlorpromazine had relatively minor disruptive effects on the conditioned neuronal responses. Figure 9 illustrates the action of ~hlordi~epoxide administered at a dose of 10 mg/kg. The top four traces show the pattern of lever-pressing for food and of unit activity before the introduction of the aversive brain stimuli. The next four traces show the effect of introducing the aversive stimuli on these responses. The rate of discharge was higher during the presentation of the conditioned stimulus compared with the background rate in the interval that immediately preceded the conditioned stimulus. Lever-pressing for a food reward was totally suppressed. Thirty minutes after the adminis~ation of 10 mg/kg of chlordiazepoxide, the neuronal activity correlated with the conditioned stimulus presentation, was reduced or suppressed. The background neuronal activity was reduced in the hypothalamus, but not in the hippocampus or the amygdala. The unconditioned neuronal response in the h~otha~amus was reduced, but not to the same degree as was the conditioned response in this region,
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andM.E.
OLDS
DISCUSSION
The finding of these experiments was that the benzodiazepines, but not chlorpromazine, exerted a disinhibition action on the lever-pressing response for food in approximately half of the subjects and they simultaneously reduced the background rate of neuronal activity and also the conditioned stimulus-correlated rate of discharge. The results using the lever-pressing response are consistent with the findings reported previously that the benzodiazepines are effective in conflict-type behaviour, and that chlorpromazine is not (SCHALLEKet al., 1972). However, on the basis of these reports, a much larger proportion of the subjects had been expected to show disinhibition after the administration of the benzodiazepines. The rather moderate action of these compounds in the present tests must therefore be related to the use of brain stimulation in the central grey region to cause suppression of lever-pressing. MARGULESand STEIN (1967, 1968) showed that the nature of the punishing stimuli was unimportant. Our results differ from this finding. In general, there was little difference between an intensity which caused no behavioural effect and an intensity that caused total suppression of lever-pressing. It is possible that, had we achieved in most animals a suppression of lever-pressing limited to the conditioned stimulus period, the benzodiazepines would have acted more effectively as disinhibiting compounds. It was interesting that diazepam disinhibited three subjects at the 2.5 mg/kg dose, but only two at the 5 mg/kg dose. It is possible that a lower dose than the 2.5 mg/kg dose would have caused more subjects to be disinhibited. In relation to the main question of whether the drug-effects on the unit activity reveal something of their mode of action in a conflict test, the answer has to be cautious and more hypothetical than factual. This is so for several reasons. First was the observation of a loose correlation between the conditioned behavioural response to aversive brain stimulation and the conditioned neuronal response. The former appeared as soon as brain stimulation was introduced, the latter developed gradually over a large number of trials, its onset in each trial being later than the onset of the conditioned stimulus and often outlasting the conditioned stimulus. Second was the observation that diazepam, which disinhibited only 2-3 subjects, was the compound which antagonized most effectively the conditioned stimulus-induced change in the pattern of firing, and chlorpromazine, which was totally ineffective on lever-pressing behaviour, also antagonized the conditioned stimuluscorrelated neuronal activity in the hippocampus at the 2 mg/kg dose. Thus, on the basis of their effects on the conditioned stimulus-correlated neuronal activity, it would be impossible to separate the benzodiazepines, which sometimes cause disinhibition, from the non-disinhibiting phenothiazine. It is interesting to note, however, that the benzodiazepines were effectively antagonizing the conditioned neuronal activity in the amygdala, and that chlorpromazine had practically no such effect in this region. Another observation of possible significance was that conditioned neuronal activity in the hypothalamus and in the geniculate nuclei must have some complex relationship to the conditioned behavioural response because chlordiazepoxide, which disinhibited five subjects out of eleven at the 5 mg/kg dose, did not modify significantly the conditioned stimulus-correlated neuronal activity, while diazepam, at both doses, abolished the neuronal responses in the geniculate nuclei and reduced them in the hypothalamus. When the effects of the drugs on the background rate were analyzed, a clear pattern distinguishing the action of the benzodiazepines from that of chlorpromazine also failed to emerge. Chlordiazepoxide and chlorpromazine reduced the rate of unit discharge in the background period, but as was noted earlier, the first compound was effective on behaviour while the second was not. And diazepam, which had a very little effect on the background rate, had more effect on the lever-pressing behaviour than chlorpromazine, which did reduce the background rate. There were, however, two aspects of the drug effects that merit our consideration. The first was that the time of peak effect of the benzodiazepines on behaviour correlated well with their peak effect on the conditioned stimulus-correlated neuronal activity and on the activity during the background period. The second was that the two benzodiazepines
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reduced both the background rate of discharge and the conditioned neuronal responses, but only in the amygdala. It would seem, therefore, that their action there could be of significance to their action on lever-pressing. However, at present this is only a possibility because the relationship between the neuronal activity and the behaviour is complex and hence does not allow an easy interpretation of the drug effects on neuronal discharge. The findings point nevertheless to selective effects on conditioned and background neuronal activity at the time when the drugs were effective on behaviour, and therefore the data point in the direction that future work will have to take to explain their anti-anxiety effects. REFERENCES BRADY,J. V. (1956). A comparative approach to the evaluation of drug effects upon affective behaviour. Ann. N.y Acad. Sci. 64,632~643. BRADY.J. V. (1960). Emotional behaviour. In: Handbook of Physiology, Sec. I, Neurophysiology, Vol. 3 (FIELD, J., MACOUN,H. W. and HALL,V. E., Eds.), pp. 1529-1552. Am. Physiol. Sot., Baltimore. BRDCKE, F. T. V., HORNYKIWIECZ, 0. and SIGG, E. B. (1969). The Pharmacology of‘ Psychotherapeutic Drugs. Springer, New York. DAVIDSON, A. B. and COOK,L. (1969). Effects of combined treatment with trifluoperazone-HCI and amobarbital on punished behavior in rats. Psychopharrnucologia 15: 159-168. DE WIED, D. (1967). Chlorpromazine and endocrine function. Pharmuc. Rev. 19: 251-288. GELLER,I., KOLAK,J. T., JR. and SEIFERS,J. (1962). The effects of chlordiazepoxide and chlorpromazine on a punished discrimination. Psychopharmacologia 3: 374-385. GUERRERO-FIGUEROA, R., GALLANT,D. M., GUERRERO-FIGUEROA, C. and GALLANT,J. (1973). Electrophysiological analysis of the action of four benzodiazepine derivatives on the central nervous system. In: The Benzodiazepines (GARATTINI, S., MUSSINI,E. and RANDALL,L. O., Eds.), pp. 489-510. Raven Press, New York. HEISE,G. A., LAUGHLIN,N. and KELLER,C. (1970). A behavioral and pharmacological analysis of reinforcement withdrawal. Psychopharmacologia 16: 341-368. KGNIG,J. F. R. and KLIPPEL,R. A. (1963). The Rat Brain: A Stereotaxic Atlas of the Forebrain and Lower Parts qf‘the Brain Stem. Williams & Wilkins. Baltimore. MARGULES,D. L. and STEIN.L. (1967). Neuroleptics vs tranquilizers: evidence from animal behavior studies of mode and site of action. In: Neuropsychopharmacology (BRILL,H., COLE,J. O., DENIKER,P., HIPPINS,H. and BRADLEY, P. B., Eds.), Vol. 5, pp. 108-120. Elsevier, Amsterdam. MARGULES,D. L. and STEIN,L. (1968). Increase of “antianxiety” activity and tolerance of behavioral depression during chronic administration of oxazepam. Psychopharmacologia 13: 7480. MORILLO,A. (1962). Effects of benzodiazepines upon amygdala and hippocampus of cat. Int. J. Neuropharmac. 1: 353-359. OLIX, J. (1959). Studies of neuropharmacologicals by electrical and chemical manipulation of the brain in animals with chronically implanted electrodes. In: Neuro-psychopharmacology: Proc. 1st. Int. Congr. Neuropharmacol., Rome, 1958 (BRADLEY.P. B., DENIKER, P. and RADOUCO-THOMAS, C., Eds.), pp. 20-32. Elsevier, Amsterdam. OLDS,J. (1973). Multiple unit recordings from behaving rats. In: Eioelectric Recording Techniques, Part A. Cellular Processes and Brain Potentials (THOMPSON, R. F. and PATTERSON, M. E., Eds.), pp. 16s198. Academic Press, New York. OLDS, M. E. (1966). Facilitatory action of diazepam and chlordiazepoxide on hypothalamic reward behavior. J. camp. physiol. Psychol. 62: 136-140. PRESTON,J. B. (1956). Effects of chlorpromazine on the central nervous system of the cat: a possible neural basis for action. J. Pharmac. exp. Ther. 118: 10&l 15. SCHALLEK, W. and KUEHN,A. (1960). Effects of psychotropic drugs on limbic system of cat. Proc. SOC. exp. Biol. Med. 105: 115-l 17. SCHALLEK, W., S~HLOSSER, W. and RANDALL,L. 0. (1972). Recent developments in the pharmacology of the benzodiazepines. Adv. Pharmac. Chemother. 10: 119-182. U~MOTO, M., MURAI, Y., KODAMA,M. and KIDQ R. (1970). Neuronal discharge patterns in conditioned emotional response. Brain Res. 24: 347-351. WEDEKIND,P. W. (1969). Disinhibition effect of chlordiazepoxide. Psychonom. Sci. 15: 232-237.