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Contrasting effects of hypothalamic and nucleus accumbens septi self-stimulation on brain stem single unit activity and cortical arousal
BRAIN RESEARCH
275
C O N T R A S T I N G EFFECTS OF H Y P O T H A L A M I C A N D N U C L E U S A C C U M B E N S SEPTI S E L F - S T I M U L A T I O N ON B R A I N STEM SINGLE UNIT ACTIVITY AND CORTICAL AROUSAL
EDMUND T. ROLLS
University of Oxford, Department of Experimental Psychology, South Parks Road, Oxford (Great Britain) (Accepted February l lth, 1971)
INTRODUCTION
Electrical stimulation of some brain sites is rewarding, in that animals learn to press a bar or to run in a runway to obtain the stimulation. Recent electrophysiological experiments have provided evidence that two separate neural systems are activated in hypothalamic self-stimulation3,1°. Neurones in the limbic system form part of one system 1°. In a separate system, brain stem neurones are directly excited by the hypothalamic stimulation. At least some of these neurones are antidromically excited by the stimulation. There is evidence that these neurones drive other neurones in the brain stem and finally activate brain stem and thalamic neurones which have firing rates which are closely correlated with arousal. Through this latter system, the hypothalamic stimulation produces arousal 1°A1. Self-stimulation of the hypothalamic sites used in these electrophysiological experiments contrasts with self-stimulation of sites in the rhinencephalon, for example, the amygdala and hippocampus. Stimulation of brain regions near the medial forebrain bundle (MFB) between the levels of the pre-optic area and the midbrain, including hypothalamic sites, is associated with hyperactivity, high rates of self-stimulation, lack of satiation, and sometimes with stimulus-bound motivational behaviour. In contrast, stimulation of some rhinencephalic sites is associated with hypoactivity, lower rates of self-stimulation, some satiation seen in a decline in self-stimulation rate over time, and sometimes with motivational behaviour which occurs after stimulation 7,s. Stimulation of the nucleus accumbens septi region is associated with hypoactivity and low rates of self-stimulation (personal observation), and it can be considered as an example of a rhinencephalic self-stimulation site. It has been shown that stimulation of diencephalic ~e.g., posterior hypothalamic) self-stimulation sites influences the activity of many brain stem units, but that stimulation in the region of the nucleus accumbens septi influences very few brain stem units 13. To further analyse the role in self-stimulation of the brain stem arousal system traced in earlier experiments~0,11, the effects of stimulation of nucleus accumbens Brain Research, 31 (1971) 275-285
276
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septi and hypothalamic self-stimulation sites on this brain stem arousal ~ystem arc compared in this paper. These two regions were chosen for comparison becau>c the3, are examples of the rhinencephalic and MFB self-stimulation sites, through which contrasting types of behaviour are elicited. METHODS
Albino male rats weighing 250-350 g were implanted with monopolar electrodes aimed at self-stimulation sites, tested for self-stimulation, and about one week later used in an acute electrophysiological experiment performed under urethane anaesthesia. As the methods used in these experiments have been described u. only a brief account is given here.
Preparation of anhnalsfor the acute electrophysiological experiments Under Equi-Thesin (Jensen-Salsbery Labs. Inc.) anaesthesia (2 ml/kg, intraperitoneally) the dorsal surface of the cranium was exposed and a rectangle of bone stretching from lambda to 3 mm anterior to bregma, and from the midline to the lateral ridge, was removed unilaterally. This large exposure of the dura was to allow the insertion ofmicroelectrodes in the later acute experiments. Two monopolar stimulating electrodes made of No. 00 stainless steel insect pins insulated to within 0.5 m m of the tip were lowered to self-stimulation sites and were held in position by an electrode holder cemented to 3 screws placed in the skull on the contratateral side. The level-head co-ordinates of the anterior electrode, aimed at the nucleus accumbens, septi, were 1.6 m m anterior to bregma, lateral 0.5-1.0 mm, and 5,0 mm beneath the surface of the dura; and of the posterior electrode, aimed at the medial forebrain bundle between the levels of the lateral and posterior hypothalamus, 3.4 mm posterior to bregma, lateral 1.5 ram, and 7.8 m m beneath the surface of the dura. Further details of the electrode sites are given under Results. After two days the rats were tested for self-stimulation. They were placed in an aluminium cage 10 in. × 8 in. × 10 in. which contained a bar I in. ~i 0.5 in. A 0.3 sec train of 0.1 msec constant current cathodal pulses, at a frequency of 100 c/sec, was applied to the electrode under test through flexible wires when the rat pressed the lever. Current return was by one of the screws placed in the skull. MFB electrodes classed as positive for self-stimulation supported self-stimulation rates of more than 30 responses/min, and positive nucleus accumbens septi sites produced rates of more than l0 responses/rain. Only animals with one or both electrodes positive for s e l f stimulation were used in the electrophysiological experiments. Control experiments on MFB animals which did not self-stimulate are described elsewhere n. For the acute electrophysiological experiments, the animals were anaesthetized with an intraperitoneal injection of 20~o urethane solution (l.2 g/kg) and placed in a stereotaxic instrument. Body temperature was maintained by wrapping the animal in cellulose wadding. Stainless steel screws were placed in the skull over frontal and occipital cortex to record the electroencephalogram (EEG). Single unit activity was
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recorded extracellularly with tungsten 4 or glass capillary microelectrodes. The latter were filled with 10~,, sodium chloride, and had a DC resistance of 0.5-4.0 M ~ . A differential pre-amplifier (AI0I, Isleworth Electronics, Waddesdon, England) was fed from two separate field effect transistor (FET) amplifiers. One amplifier was connected to the microelectrode, and the other to a silver wire resting on the exposed surface of the dura which recorded stimulus artefact. By adjusting the relative gains of these two amplifiers, it was possible to reject most of the stimulus artefact from the single unit channel. Further clarification of short latency single unit responses was obtained by setting the high pass filter at 1 kc/sec. The output of the lsleworth preamplifier was connected to a Tektronix 502A oscilloscope, from which single unit activity could be directly photographed or stored on magnetic tape. The oscilloscope also provided standard pulses which corresponded to each action potential and which were connected to a ratemeter. The EEG, standard pulses, and unit firing rate were recorded on an Ultraviolet polygraph (type 3006, S.E. Labs., Feltham, England).
Electrophysiological methods The electrophysiological experiments were designed to compare tke effects of rewarding MFB and nucleus accumbens septi stimulation on brain stem sing!e units and on arousal. Midbrain, pontine or thalamic single unit activity was recorded with a microelectrode lowered through a slit made in the exposed dura. Cathodal stimulus pulses of 0.1 msec duration were applied to the self-stimulation electrodes at the lowest current for which the animals had previously shown a good rate of self-stimulation. Thus units activated by the electrical stimulation in the acute experiment were probably also activated in self-stimulation. Whenever a spontaneously active single unit was encountered, the effects of stimulation applied to a self-stimulation electrode were determined, and compared where possible with stimulation applied to the other electrode. As some activated units were not spontaneously active, stimulus pulses were applied frequently while the microelectrode was lowered through the brain. Effects of nucleus accumbens septi stimulation on brain stem single unit activity were analysed in detail in 8 rats, and of MFB stimulation in 18 rats. Thus the total numbers of units affected by nucleus accumbens septi and MFB stimulation are not directly comparable. For this reason only major differences between the two sites are presented here. Six rats had positive self-stimulation electrodes in both the nucleus accumbens septi and the MFB. Single units activated by the electrical stimulation were classed as directly, synaptically, or indirectly activated 11 Directly excited units passed under both the recording and the stimulating electrodes. The action potentials of these units followed single stimulus pulses with short, fixed latencies (usually less than 5 msec). Excitation was either in the ortho- or antidromic direction. Dromicity was determined where possible by the collision technique 11. When action potentials were from a directly excited unit, the latency and absolute refractory period of the unit were measured. The absolute refractory period is a useful parameter to characterize a population of neurones, and was measured using
Brain Research, 31 (1971) 275-285
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b
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s Fig. 1. a, Absence of collision in a synapticatlydriven unit. Action potentials occurring before a twicethreshold stimulus pulse (at 's') do not collide with and prevent the appearance of action potentials elicited by the pulse. This unit has a relatively short latency of approximately 2 msec. Each horizontal line on the left and right portions of (a) represents a single sweep of the oscilloscope beam. b, The characteristic variable latency of a synaptically driven unit is illustrated in these superimposed traces of the action potential following a twice-threshold stimulus pulse at 's'. Negative down for a and b, pulse pairs of more than twice threshold strength 11. Units activated by single stimulus pulses with a longer (usually in the range 2-15 msec) variable latency were classed as synaptically driven. These units are probably excited by the stimulus pulses through one or several synapses. The long variable latency of a synaptically driven unit is illustrated in Fig. lb. The stimulus artefact of twice threshold stimulus pulses is seen at 's'. Approximately l0 superimposed sweeps show that the single unit action potentials follow the stimulus pulses with a variable latency which has a mean value of about 7 msec. The unit shown in Fig. la has a relatively short latency of about 2 msec. Close inspection shows that the latency is variable (each horizontal line is a single sweep). The demonstration that spontaneously occurring action potentials which im mediately precede the stimulus pulse do not collide with the electrically elicited action potential is additional evidence that this type of unit is synaptically driven. indirectly driven units were not activated by single stimulus pulses, but only by trains of stimulus pulses. For example, the firing rate of an indirectly driven unit might show a post-stimulation increase or decrease when stimulation consisted of a 100 c/sec train of 0.1 msec pulses with a duration of 100 msec, given at the current for which the animal had previously self-stimulated. The latencies for the start of an effect on indirectly driven units were in the range of 20-300 msec. When an activated unit had been classed as directly, synaptically, or indirectly driven, the firing rate of the unit and the EEG were monitored, and the effects of intracranial stimulation and arousing stimuli on the unit were measured. The arousing stimuli used were a pinch to the hind leg, and inhalation of amyl nitrite. The latter procedure produces a drop in blood pressure, and acts as an arousing agent 5. As rewarding MFB stimulation produces cortical desynchronization, and the firing rate of the brain stem indirectly driven units is correlated with this arousal al, the effect of rewarding stimulation of the nucleus accumbens septi region on arousal was measured in all animals with nucleus accumbens electrodes. Recording sites were marked with tungsten microelectrodes by passing an anodal current of 30-60 #A for 60 sec through the electrode. At the termination of an experiBrain Research, 31 (1971) 275-285
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Fig. 2. Absolute refractory periods of brain stem units directly excited through self-stimulation eIectrodes in the region of the medial forebrain bundle (MFB, left) and nucleus accumbens septi (right). The ordinate of the right-hand histogram is twice the scale of the left-hand histogram. ment, the brain was fixed by an intracardiac perfusion of saline followed by 10% neutral formalin. After several days in 10% formalin the brain was removed and 50 # m frozen sections were cut, mounted, and stained with the Nissl procedure. The sites of the stimulating and recording electrodes were determined. Tracks made by capillary microelectrodes could be seen, and the site of a unit was estimated from the distance from the dorsal surface and base of the brain. RESULTS
Directly excited units Very few units in the midbrain and pontine tegmentum were directly excited by rewarding nucleus accumbens septi stimulation. This is in contrast to rewarding MFB stimulation, which directly excites a large population of brain stem neurones 11. Fig. 2 shows that in the 8 animals with nucleus accumbens septi electrodes, only 6 directly excited brain stem units were found for refractory period measurements. The values of the absolute refractory periods of this small number of units are not closely grouped, and it is not possible to characterize these units on the basis of refractory period. For comparison Fig. 2 shows that the brain stem units directly excited by rewarding MFB stimulation form a large population characterized by absolute refractory periods in the range 0.78-0.88 msec. The majority of the latter units were recorded in 18 animals. The very small number of brain stem units directly excited by nucleus accumbens septi stimulation is further emphasized in Fig. 3, in which the latencies of action potentials of brain stem units directly excited by MFB and nucleus accumbens septi Brain Research, 31 (1971) 275-285
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Fig. 3. Latencies of brain stem units directly excited (bar histograms) or synaptically driven (filled circles) through self-stimulation electrodes in the region of the MFB (left) at the level of the lateral hypothalamus and the nucleus accumbens septi (right). Fifteen of the 22 units synaptically driven by nucleus accumbens stimulation fright) were found in one animal - - thus very few units were synaptically driven in the remaining 7 animals.
stimulation are compared (bar histograms). One brain stem unit excited by nucleus accumbens stimulation included in this figure is absent from Fig. 2 because its refractory period was not measured. Synaptically activated units Both nucleus accumbens septi and MFB rewarding stimulation synaptically activated units in the midbrain and pontine tegmentum. The effects of stimulation in the two regions are compared in Fig. 3, in which the latencies of units from pulses applied to the electrodes are shown by the filled circles. Thirty-eight units were synaptically driven from the MFB electrodes, and 22 from the nucleus accumbens septi electrodes. Sixteen of the 22 synaptically activated units were recorded in one of the 8 nucleus accumbens septi animals. Thus in 7 of the 8 animals only a very weak synaptic input to the brain stem was found. This is therefore the typical finding. A further difference between the effects of the MFB and nucleus accumbens septi rewarding stimulations is that MFB stimulation has a powerful short latency input to the brain stem with 21 of 38 units having latencies of 1-4 msec, whereas nucleus accumbens septi stimulation has a more temporally diffuse input, with latencies of synaptically driven units evenly distributed between 3 and 10 msec. In the one animal with a strong input from the nucleus accumbens septi to the brain stem, convergence of input from both MFB and nucleus accumbens electrodes onto 9 brain stem units was found. A typical example has been illustrated in Fig. 1. The unit in (a) was synaptically driven from an MFB electrode with a latency of 2 msec and the same unit shown in (b)
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was synaptically driven from a nucleus accumbens electrode with a mean latency of 7 msec.
Indirectly driven units: the effects of rewarding nucleus accumbens septi stimulation on arousal Although many units in the brain stem and thalamus were indirectly driven by rewarding MFB stimulation 11, these units were not driven by nucleus accumbens septi stimulation. Similarly, the arousal (cortical desynchronization seen under urethane) which is produced by MFB stimulation, and which is correlated with the firing rates of the indirectly driven units, was not produced by rewarding nucleus accumbens septi stimulation. An example of the firing rate of an indirectly driven arousal unit is shown in Fig. 4. At 10A, ten 0.3-sec trains of 0.1 msec pulses at 100 pulses/sec at the current for which the animal had previously self-stimulated were applied to the anterior (nucleus accumbens septi) electrode. Trains of this nature were used because each such train given for a bar press had been sufficient to maintain a good rate of self-stimulation in the behavioural test. The single unit does not fire (unit trace), only stimulus artefact pulses are counted in the unit rate trace, and almost no change in the EEG is seen. In contrast, 10, 20 or 1 similar trains of stimulation applied to the posterior (MFB) electrode at the current for which the animal had previously self-stimulated produced a prolonged post-stimulation increase in the firing rate of the unit, which
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Fig. 4. Arousal is not produced by stimulation applied to a nucleus accumbens septi self-stimulation site. One, l0 or 20 trains of stimulation applied to the posterior, MFB, self-stimulation electrode (1 P, lOP, 20P) increased the firing rate of this unit (see 'unit' and 'rate' traces) and produced cortical desynchronization. Ten trains applied to the anterior, nucleus accumbens septi, electrode (10A) did not produce a similar arousal effect. (Only stimulus artefact was counted in the rate trace.) The same stimulation did not inhibit the arousal effect (20P followed by 10A). Each 0.3 sec train of pulses at 100 c/sec was above the threshold for self-stimulation. This unit, which was indirectly driven by MFB stimulation, had general arousal properties in that its firing rate was correlated with cortical synchronization/desynchronization, and stimuli which altered arousal level (e.g., amyl nitrite) always produced a correlated change in the firing rate of the unit.
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l. ROLLS
was correlated with a change of the EEG to the desynchronized (aroused) state. In the fourth presentation of nucleus accumbens stimulation at 10A, the current of the pulses was doubled (note the larger stimulus artefact on the EEG), yet the unit firing rate was not affected, nor was any prolonged change in the EEG seen. Inhalation of amyl nitrite, which is an arousing stimulus 5, produced cortical desynchronization and increased the firing rate of the unit. It was not possible to separate the firing rates of these indirectly driven units from arousal. In a test to determine whether the nucleus accumbens stimulation produced cortical synchronization, arousal produced by MFB stimulation was followed by nucleus accumbens stimulation (Fig. 4, 20P followed by 10A). No indication of a synchronizing influence of the nucleus accumbens stimulation was found in this type of test in any animal. In Fig. 4, the arousal unit fires about 40 sec after the fourth (twice threshold) stimulation applied to nucleus accumbens. A similar effect, of apparent arousal starting considerably after the termination of stimulation, was found in several other animals. The apparent arousal in this animal was associated with large waves in the EEG, which were associated with movement of the animal. The effect is temporally similar to the hyperactivity which follows the hypoactivity produced by repeated stimulation of nucleus accumbens septi reward sites.
Stimulating and recording sites Examples of the locations of the MFB self-stimulation electrode tips used have been given elsewhere 12. They were close to the MFB at the level of the lateral or posterior hypothalamus. In stereotaxic co-ordinates 6 the anterior self-stimulation electrodes were close to A9650, lateral 0.5-1.0, and 0.0-0.5 mm below horizontal zero, in the region of the nuclei accumbens septi. Examples of the brain stem sites where units were directly and indirectly activated by MFB stimulation have been given elsewherO 1. The few brain stem units activated by nucleus accumbens stimulation were found in the more central parts of the same brain regions. In stereotaxic co-ordinates 6 the regions are A620--A2180, lateral 0.0-2.5 mm, and 1.0 mm above to 3.0 mm below horizontal zero, in the midbrain and pontine tegmentum. DISCUSSION
Because only units activated through self-stimulation electrodes at or below currents which produced self-stimulation were analysed in the acute experiments, it is likely that they were activated during self-stimulation. A large population of brain stem neurones was directly excited in MFB (hypothatamic) self-stimulation; but very few brain stem neurones were directly excited in self-stimulation of the nucleus accumbens septi region. Many brain stem neurones were synaptically excited in MFB self-stimulation except in one animal - - very few of its brain stem neurones were synaptically activated in self-stimulation of the nucleus accumbens septi region. Neo-
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cortical desynchronization (arousal) was produced in these acute experiments performed under urethane by trains of MFB stimulation, but not by trains of nucleus accumbens septi stimulation. The firing rates of units driven indirectly - - probably polysynaptically - - by MFB stimulation were correlated with the cortical desynchronization. The firing rates of these arousal units were very little affected by nucleus accumbens stimulation. These observations, that directly and synaptically activated brain stem units, and arousal produced by rewarding brain stimulation, are found in MFB but not in nucleus accumbens septi animals, are further evidence that arousal is produced by activation of a brain stem neural system in MFB self-stimulation 11. These observations also show that activation of this brain stem neural system is not necessary for self-stimulation. A priming effect of brain stimulation at a self-stimulation site can be measured by its effect on subsequent running speed for a fixed brain stimulation reward at the end of a runwayL The priming effect has many similarities with the arousal effect produced by MFB stimulation, and it has been suggested that the arousal effect partly mediates the priming effect11,12. The effects are similar in a number of ways: both are produced through MFB self-stimulation electrodes, are proactive effects of stimulation which continue after the end of stimulation, have a gradual decay to the prestimulation level, have greater magnitudes and durations when the number of stimulating trains is increased, and are produced by the direct excitation ofneurones with absolute refractory periods in the range of 0.78-1.1 msec. If activation of the brain stem neural system and the associated arousal do partly mediate thepriming effect, then stimulation of rewarding nucleus accumbens septi sites, which does not activate the brain stem arousal system, should not produce a similar priming effect. This has not yet been tested. Some of the differences between MFB and rhinencephalic self-stimulation s may be due to differences in the activation of the brain stem arousal system. As already stated, the nucleus accumbens septi region may be taken as an example of a rhinencephalic self-stimulation site. For example hyperactivity which occurs in MFB self-stimulation may be contrasted with the hypoactivity which occurs at least during stimulation of nucleus accumbens septi self-stimulation sites (personal observation). Using the refractory period of the directly excited brain stem neurones to manipulate the level of activation reaching the brain stem neural system, it was shown that activity in the brain stem arousal system greatly influenced the rate of self-stimulation 1'~. Thus activation of the brain stem arousal system may partly account for the much greater rates of self-stimulation at MFB than at rhinencephalic sites. This interpretation is supported by lesion evidence which indicates that the rate of MFB self-stimulation is reduced by lesions which probably interrupt the axons of the directly excited brain stem neutones 1,9. The brain stem arousal system may play a role in the lack of satiation characteristic of MFB self-stimulation, by maintaining the arousal of the animal. Finally, as stimulus-bound motivational behaviour is elicited from some MFB self-stimulation sites, but not from rhinencephalic self-stimulation sites, activation of the brain stem arousal system may be involved in stimulus-bound motivational behaviour. It has been shown that stimulation at posterior diencephalic (i.e., MFB and Brain Research, 31 (1971)275 285
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field H2 of Forel) self-stimulation sites influenced many more brain stem single units than stimulation at self-stimulation sites in the region of the nucleus accumbens septi 13. The experiments described here analyse the differences between rewarding MFB and nucleus accumbens septi stimulation on the brain stem arousal system traced inpreviousexperiments 1°,11. The main differences are in effects on brain stem directly excited units, and indirectly activated (arousal) units, with differences usuaJly appearing in effects on brain stem synaptically activated units. The present experiments also give an indication about the role of activation of the brain stem units. They provide further evidence that through these brain stem units cortical desynchronization (arousal) may be produced in MFB stimulation. Earlier experiments 11,~e indicate thal this arousal may partly mediate a priming effect in self-stimulation SUMMARY
Very few brain stem single units were directly or synaptically excited through self-stimulation electrodes in the region of the nucleus accumbens septi. This is in contrast to the large populations of brain stem single units directly and synapticalty excited through hypothalamic self-stimulation electrodes. The hypothalamic, but not the nucleus accumbens septi, stimulation produced arousal measured by cortical desynchronization and activation of single units with general arousal properties. These results were obtained in acute experiments performed under urethane anaesthesia on rats previously implanted and tested for self-stimulation. They support other evidence that arousal is produced in self-stimulation of the medial forebrain bundle region by activation of a brain stem neural system. This arousal continues after the end of stimulation and may play a role in a priming effect of brain stimulation reward. The experiments also show that activation of the brain stem arousal system is not necessary for self-stimulation, and thus indicate that activation of a different neural system may mediate the rewarding effect of brain stimulation reward. ACKNOWLEDGEMENT
The author is grateful to Mr. J. Broad for photographic assistance.
REFERENCES 1 BOYD, E. S., AND GARDNER, L. C., Effect of some brain lesions on intracranial self-stimulation in
the rat, Amer. J. Physiol., 213 (1967) 1044-1052. 2 GALLISTEL, C. R., The incentive of brain-stimulation reward, J. eomp. physiol. PsychoL, 69 (1969) 713-721. 3 GALLISTEL, C. R., ROLLS, E., ANO GREENE, D., Neuron function inferred from behavioral and electrophysiologieal estimates of refractory period, Seience, 166 (1969) 1028-1030. 4 HtJBEL, D. H., Tungsten microelectrode for recording from single units, Science, 125 (1957) 549-550. 5 KOMISARUK, B. R., McDONALD, P. G., WHITMOYER, D. 1., AND SAWYER, C. H., Et'i'ects of progesterone and sensory stimulation on EEG and neural activity in the rat, Exp, Neurol., 19 (1967) 494-507.
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6 KONIG, J. F. R., AND KLIPPEL, R. A., The Rat Brain, Williams and Wilkins, Baltimore, Md., 1963. 7 MILGRAM,N. W., Effects of hippocampal stimulation on feeding in the rat, Physiol. Behav., 4 (1969) 665-670. 80LDS, J., AND OLDS, M., Drives, rewards, and the brain. In New Directions in Psychology, 11"ol.1I, Holt, Rinehart and Winston, New York, 1965, pp. 327410. 90LDS, M. E., AND OLDS, J., Effects of lesions in medial forebrain bundle on self-stimulation behavior, Amer. J. Physiol., 217 (1969) 1253 1264. 10 RoLLs, E. T., Neural systems involved in intracranial self-stimulation, Brain Research, 24 (1970) 548. (Abstract.) I I ROLLS, E. T., Involvement of brainstem units in medial forebrain bundle self-stimulation, Physiol. Behav., 7 (1971) in press. 12 ROLLS, E. T., Absolute refractory period of neurons involved in MFB self-stimulation, Physiol. Behav., 7 (1971) in press. 13 ROUTTENBERG, A., AND HUANG, Y. H., Reticular formation and brainstem unitary activity: effects of posterior hypothalamic and septal-limbic stimulation at reward loci, Physiol. Behav., 3 (1968) 611 617.
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C O N T R A S T I N G EFFECTS OF H Y P O T H A L A M I C A N D N U C L E U S A C C U M B E N S SEPTI S E L F - S T I M U L A T I O N ON B R A I N STEM SINGLE UNIT ACTIVITY AND CORTICAL AROUSAL
EDMUND T. ROLLS
University of Oxford, Department of Experimental Psychology, South Parks Road, Oxford (Great Britain) (Accepted February l lth, 1971)
INTRODUCTION
Electrical stimulation of some brain sites is rewarding, in that animals learn to press a bar or to run in a runway to obtain the stimulation. Recent electrophysiological experiments have provided evidence that two separate neural systems are activated in hypothalamic self-stimulation3,1°. Neurones in the limbic system form part of one system 1°. In a separate system, brain stem neurones are directly excited by the hypothalamic stimulation. At least some of these neurones are antidromically excited by the stimulation. There is evidence that these neurones drive other neurones in the brain stem and finally activate brain stem and thalamic neurones which have firing rates which are closely correlated with arousal. Through this latter system, the hypothalamic stimulation produces arousal 1°A1. Self-stimulation of the hypothalamic sites used in these electrophysiological experiments contrasts with self-stimulation of sites in the rhinencephalon, for example, the amygdala and hippocampus. Stimulation of brain regions near the medial forebrain bundle (MFB) between the levels of the pre-optic area and the midbrain, including hypothalamic sites, is associated with hyperactivity, high rates of self-stimulation, lack of satiation, and sometimes with stimulus-bound motivational behaviour. In contrast, stimulation of some rhinencephalic sites is associated with hypoactivity, lower rates of self-stimulation, some satiation seen in a decline in self-stimulation rate over time, and sometimes with motivational behaviour which occurs after stimulation 7,s. Stimulation of the nucleus accumbens septi region is associated with hypoactivity and low rates of self-stimulation (personal observation), and it can be considered as an example of a rhinencephalic self-stimulation site. It has been shown that stimulation of diencephalic ~e.g., posterior hypothalamic) self-stimulation sites influences the activity of many brain stem units, but that stimulation in the region of the nucleus accumbens septi influences very few brain stem units 13. To further analyse the role in self-stimulation of the brain stem arousal system traced in earlier experiments~0,11, the effects of stimulation of nucleus accumbens Brain Research, 31 (1971) 275-285
276
~ 3, r~()m~,~
septi and hypothalamic self-stimulation sites on this brain stem arousal ~ystem arc compared in this paper. These two regions were chosen for comparison becau>c the3, are examples of the rhinencephalic and MFB self-stimulation sites, through which contrasting types of behaviour are elicited. METHODS
Albino male rats weighing 250-350 g were implanted with monopolar electrodes aimed at self-stimulation sites, tested for self-stimulation, and about one week later used in an acute electrophysiological experiment performed under urethane anaesthesia. As the methods used in these experiments have been described u. only a brief account is given here.
Preparation of anhnalsfor the acute electrophysiological experiments Under Equi-Thesin (Jensen-Salsbery Labs. Inc.) anaesthesia (2 ml/kg, intraperitoneally) the dorsal surface of the cranium was exposed and a rectangle of bone stretching from lambda to 3 mm anterior to bregma, and from the midline to the lateral ridge, was removed unilaterally. This large exposure of the dura was to allow the insertion ofmicroelectrodes in the later acute experiments. Two monopolar stimulating electrodes made of No. 00 stainless steel insect pins insulated to within 0.5 m m of the tip were lowered to self-stimulation sites and were held in position by an electrode holder cemented to 3 screws placed in the skull on the contratateral side. The level-head co-ordinates of the anterior electrode, aimed at the nucleus accumbens, septi, were 1.6 m m anterior to bregma, lateral 0.5-1.0 mm, and 5,0 mm beneath the surface of the dura; and of the posterior electrode, aimed at the medial forebrain bundle between the levels of the lateral and posterior hypothalamus, 3.4 mm posterior to bregma, lateral 1.5 ram, and 7.8 m m beneath the surface of the dura. Further details of the electrode sites are given under Results. After two days the rats were tested for self-stimulation. They were placed in an aluminium cage 10 in. × 8 in. × 10 in. which contained a bar I in. ~i 0.5 in. A 0.3 sec train of 0.1 msec constant current cathodal pulses, at a frequency of 100 c/sec, was applied to the electrode under test through flexible wires when the rat pressed the lever. Current return was by one of the screws placed in the skull. MFB electrodes classed as positive for self-stimulation supported self-stimulation rates of more than 30 responses/min, and positive nucleus accumbens septi sites produced rates of more than l0 responses/rain. Only animals with one or both electrodes positive for s e l f stimulation were used in the electrophysiological experiments. Control experiments on MFB animals which did not self-stimulate are described elsewhere n. For the acute electrophysiological experiments, the animals were anaesthetized with an intraperitoneal injection of 20~o urethane solution (l.2 g/kg) and placed in a stereotaxic instrument. Body temperature was maintained by wrapping the animal in cellulose wadding. Stainless steel screws were placed in the skull over frontal and occipital cortex to record the electroencephalogram (EEG). Single unit activity was
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recorded extracellularly with tungsten 4 or glass capillary microelectrodes. The latter were filled with 10~,, sodium chloride, and had a DC resistance of 0.5-4.0 M ~ . A differential pre-amplifier (AI0I, Isleworth Electronics, Waddesdon, England) was fed from two separate field effect transistor (FET) amplifiers. One amplifier was connected to the microelectrode, and the other to a silver wire resting on the exposed surface of the dura which recorded stimulus artefact. By adjusting the relative gains of these two amplifiers, it was possible to reject most of the stimulus artefact from the single unit channel. Further clarification of short latency single unit responses was obtained by setting the high pass filter at 1 kc/sec. The output of the lsleworth preamplifier was connected to a Tektronix 502A oscilloscope, from which single unit activity could be directly photographed or stored on magnetic tape. The oscilloscope also provided standard pulses which corresponded to each action potential and which were connected to a ratemeter. The EEG, standard pulses, and unit firing rate were recorded on an Ultraviolet polygraph (type 3006, S.E. Labs., Feltham, England).
Electrophysiological methods The electrophysiological experiments were designed to compare tke effects of rewarding MFB and nucleus accumbens septi stimulation on brain stem sing!e units and on arousal. Midbrain, pontine or thalamic single unit activity was recorded with a microelectrode lowered through a slit made in the exposed dura. Cathodal stimulus pulses of 0.1 msec duration were applied to the self-stimulation electrodes at the lowest current for which the animals had previously shown a good rate of self-stimulation. Thus units activated by the electrical stimulation in the acute experiment were probably also activated in self-stimulation. Whenever a spontaneously active single unit was encountered, the effects of stimulation applied to a self-stimulation electrode were determined, and compared where possible with stimulation applied to the other electrode. As some activated units were not spontaneously active, stimulus pulses were applied frequently while the microelectrode was lowered through the brain. Effects of nucleus accumbens septi stimulation on brain stem single unit activity were analysed in detail in 8 rats, and of MFB stimulation in 18 rats. Thus the total numbers of units affected by nucleus accumbens septi and MFB stimulation are not directly comparable. For this reason only major differences between the two sites are presented here. Six rats had positive self-stimulation electrodes in both the nucleus accumbens septi and the MFB. Single units activated by the electrical stimulation were classed as directly, synaptically, or indirectly activated 11 Directly excited units passed under both the recording and the stimulating electrodes. The action potentials of these units followed single stimulus pulses with short, fixed latencies (usually less than 5 msec). Excitation was either in the ortho- or antidromic direction. Dromicity was determined where possible by the collision technique 11. When action potentials were from a directly excited unit, the latency and absolute refractory period of the unit were measured. The absolute refractory period is a useful parameter to characterize a population of neurones, and was measured using
Brain Research, 31 (1971) 275-285
278
~: i~. ROLLS
b
S' s
S'
I IO0,uV lOrnsec
s Fig. 1. a, Absence of collision in a synapticatlydriven unit. Action potentials occurring before a twicethreshold stimulus pulse (at 's') do not collide with and prevent the appearance of action potentials elicited by the pulse. This unit has a relatively short latency of approximately 2 msec. Each horizontal line on the left and right portions of (a) represents a single sweep of the oscilloscope beam. b, The characteristic variable latency of a synaptically driven unit is illustrated in these superimposed traces of the action potential following a twice-threshold stimulus pulse at 's'. Negative down for a and b, pulse pairs of more than twice threshold strength 11. Units activated by single stimulus pulses with a longer (usually in the range 2-15 msec) variable latency were classed as synaptically driven. These units are probably excited by the stimulus pulses through one or several synapses. The long variable latency of a synaptically driven unit is illustrated in Fig. lb. The stimulus artefact of twice threshold stimulus pulses is seen at 's'. Approximately l0 superimposed sweeps show that the single unit action potentials follow the stimulus pulses with a variable latency which has a mean value of about 7 msec. The unit shown in Fig. la has a relatively short latency of about 2 msec. Close inspection shows that the latency is variable (each horizontal line is a single sweep). The demonstration that spontaneously occurring action potentials which im mediately precede the stimulus pulse do not collide with the electrically elicited action potential is additional evidence that this type of unit is synaptically driven. indirectly driven units were not activated by single stimulus pulses, but only by trains of stimulus pulses. For example, the firing rate of an indirectly driven unit might show a post-stimulation increase or decrease when stimulation consisted of a 100 c/sec train of 0.1 msec pulses with a duration of 100 msec, given at the current for which the animal had previously self-stimulated. The latencies for the start of an effect on indirectly driven units were in the range of 20-300 msec. When an activated unit had been classed as directly, synaptically, or indirectly driven, the firing rate of the unit and the EEG were monitored, and the effects of intracranial stimulation and arousing stimuli on the unit were measured. The arousing stimuli used were a pinch to the hind leg, and inhalation of amyl nitrite. The latter procedure produces a drop in blood pressure, and acts as an arousing agent 5. As rewarding MFB stimulation produces cortical desynchronization, and the firing rate of the brain stem indirectly driven units is correlated with this arousal al, the effect of rewarding stimulation of the nucleus accumbens septi region on arousal was measured in all animals with nucleus accumbens electrodes. Recording sites were marked with tungsten microelectrodes by passing an anodal current of 30-60 #A for 60 sec through the electrode. At the termination of an experiBrain Research, 31 (1971) 275-285
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SINGLE UNIT ACTIVITYAND REWARD MFB
N. accurnbens septi
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Fig. 2. Absolute refractory periods of brain stem units directly excited through self-stimulation eIectrodes in the region of the medial forebrain bundle (MFB, left) and nucleus accumbens septi (right). The ordinate of the right-hand histogram is twice the scale of the left-hand histogram. ment, the brain was fixed by an intracardiac perfusion of saline followed by 10% neutral formalin. After several days in 10% formalin the brain was removed and 50 # m frozen sections were cut, mounted, and stained with the Nissl procedure. The sites of the stimulating and recording electrodes were determined. Tracks made by capillary microelectrodes could be seen, and the site of a unit was estimated from the distance from the dorsal surface and base of the brain. RESULTS
Directly excited units Very few units in the midbrain and pontine tegmentum were directly excited by rewarding nucleus accumbens septi stimulation. This is in contrast to rewarding MFB stimulation, which directly excites a large population of brain stem neurones 11. Fig. 2 shows that in the 8 animals with nucleus accumbens septi electrodes, only 6 directly excited brain stem units were found for refractory period measurements. The values of the absolute refractory periods of this small number of units are not closely grouped, and it is not possible to characterize these units on the basis of refractory period. For comparison Fig. 2 shows that the brain stem units directly excited by rewarding MFB stimulation form a large population characterized by absolute refractory periods in the range 0.78-0.88 msec. The majority of the latter units were recorded in 18 animals. The very small number of brain stem units directly excited by nucleus accumbens septi stimulation is further emphasized in Fig. 3, in which the latencies of action potentials of brain stem units directly excited by MFB and nucleus accumbens septi Brain Research, 31 (1971) 275-285
280
t. ~.
R()LI,~
35 3O MFB
N. a c c u m b e n s
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5
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Fig. 3. Latencies of brain stem units directly excited (bar histograms) or synaptically driven (filled circles) through self-stimulation electrodes in the region of the MFB (left) at the level of the lateral hypothalamus and the nucleus accumbens septi (right). Fifteen of the 22 units synaptically driven by nucleus accumbens stimulation fright) were found in one animal - - thus very few units were synaptically driven in the remaining 7 animals.
stimulation are compared (bar histograms). One brain stem unit excited by nucleus accumbens stimulation included in this figure is absent from Fig. 2 because its refractory period was not measured. Synaptically activated units Both nucleus accumbens septi and MFB rewarding stimulation synaptically activated units in the midbrain and pontine tegmentum. The effects of stimulation in the two regions are compared in Fig. 3, in which the latencies of units from pulses applied to the electrodes are shown by the filled circles. Thirty-eight units were synaptically driven from the MFB electrodes, and 22 from the nucleus accumbens septi electrodes. Sixteen of the 22 synaptically activated units were recorded in one of the 8 nucleus accumbens septi animals. Thus in 7 of the 8 animals only a very weak synaptic input to the brain stem was found. This is therefore the typical finding. A further difference between the effects of the MFB and nucleus accumbens septi rewarding stimulations is that MFB stimulation has a powerful short latency input to the brain stem with 21 of 38 units having latencies of 1-4 msec, whereas nucleus accumbens septi stimulation has a more temporally diffuse input, with latencies of synaptically driven units evenly distributed between 3 and 10 msec. In the one animal with a strong input from the nucleus accumbens septi to the brain stem, convergence of input from both MFB and nucleus accumbens electrodes onto 9 brain stem units was found. A typical example has been illustrated in Fig. 1. The unit in (a) was synaptically driven from an MFB electrode with a latency of 2 msec and the same unit shown in (b)
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was synaptically driven from a nucleus accumbens electrode with a mean latency of 7 msec.
Indirectly driven units: the effects of rewarding nucleus accumbens septi stimulation on arousal Although many units in the brain stem and thalamus were indirectly driven by rewarding MFB stimulation 11, these units were not driven by nucleus accumbens septi stimulation. Similarly, the arousal (cortical desynchronization seen under urethane) which is produced by MFB stimulation, and which is correlated with the firing rates of the indirectly driven units, was not produced by rewarding nucleus accumbens septi stimulation. An example of the firing rate of an indirectly driven arousal unit is shown in Fig. 4. At 10A, ten 0.3-sec trains of 0.1 msec pulses at 100 pulses/sec at the current for which the animal had previously self-stimulated were applied to the anterior (nucleus accumbens septi) electrode. Trains of this nature were used because each such train given for a bar press had been sufficient to maintain a good rate of self-stimulation in the behavioural test. The single unit does not fire (unit trace), only stimulus artefact pulses are counted in the unit rate trace, and almost no change in the EEG is seen. In contrast, 10, 20 or 1 similar trains of stimulation applied to the posterior (MFB) electrode at the current for which the animal had previously self-stimulated produced a prolonged post-stimulation increase in the firing rate of the unit, which
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al
Unit
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I
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Fig. 4. Arousal is not produced by stimulation applied to a nucleus accumbens septi self-stimulation site. One, l0 or 20 trains of stimulation applied to the posterior, MFB, self-stimulation electrode (1 P, lOP, 20P) increased the firing rate of this unit (see 'unit' and 'rate' traces) and produced cortical desynchronization. Ten trains applied to the anterior, nucleus accumbens septi, electrode (10A) did not produce a similar arousal effect. (Only stimulus artefact was counted in the rate trace.) The same stimulation did not inhibit the arousal effect (20P followed by 10A). Each 0.3 sec train of pulses at 100 c/sec was above the threshold for self-stimulation. This unit, which was indirectly driven by MFB stimulation, had general arousal properties in that its firing rate was correlated with cortical synchronization/desynchronization, and stimuli which altered arousal level (e.g., amyl nitrite) always produced a correlated change in the firing rate of the unit.
Brain Research, 31 (1971) 275-285
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l. ROLLS
was correlated with a change of the EEG to the desynchronized (aroused) state. In the fourth presentation of nucleus accumbens stimulation at 10A, the current of the pulses was doubled (note the larger stimulus artefact on the EEG), yet the unit firing rate was not affected, nor was any prolonged change in the EEG seen. Inhalation of amyl nitrite, which is an arousing stimulus 5, produced cortical desynchronization and increased the firing rate of the unit. It was not possible to separate the firing rates of these indirectly driven units from arousal. In a test to determine whether the nucleus accumbens stimulation produced cortical synchronization, arousal produced by MFB stimulation was followed by nucleus accumbens stimulation (Fig. 4, 20P followed by 10A). No indication of a synchronizing influence of the nucleus accumbens stimulation was found in this type of test in any animal. In Fig. 4, the arousal unit fires about 40 sec after the fourth (twice threshold) stimulation applied to nucleus accumbens. A similar effect, of apparent arousal starting considerably after the termination of stimulation, was found in several other animals. The apparent arousal in this animal was associated with large waves in the EEG, which were associated with movement of the animal. The effect is temporally similar to the hyperactivity which follows the hypoactivity produced by repeated stimulation of nucleus accumbens septi reward sites.
Stimulating and recording sites Examples of the locations of the MFB self-stimulation electrode tips used have been given elsewhere 12. They were close to the MFB at the level of the lateral or posterior hypothalamus. In stereotaxic co-ordinates 6 the anterior self-stimulation electrodes were close to A9650, lateral 0.5-1.0, and 0.0-0.5 mm below horizontal zero, in the region of the nuclei accumbens septi. Examples of the brain stem sites where units were directly and indirectly activated by MFB stimulation have been given elsewherO 1. The few brain stem units activated by nucleus accumbens stimulation were found in the more central parts of the same brain regions. In stereotaxic co-ordinates 6 the regions are A620--A2180, lateral 0.0-2.5 mm, and 1.0 mm above to 3.0 mm below horizontal zero, in the midbrain and pontine tegmentum. DISCUSSION
Because only units activated through self-stimulation electrodes at or below currents which produced self-stimulation were analysed in the acute experiments, it is likely that they were activated during self-stimulation. A large population of brain stem neurones was directly excited in MFB (hypothatamic) self-stimulation; but very few brain stem neurones were directly excited in self-stimulation of the nucleus accumbens septi region. Many brain stem neurones were synaptically excited in MFB self-stimulation except in one animal - - very few of its brain stem neurones were synaptically activated in self-stimulation of the nucleus accumbens septi region. Neo-
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cortical desynchronization (arousal) was produced in these acute experiments performed under urethane by trains of MFB stimulation, but not by trains of nucleus accumbens septi stimulation. The firing rates of units driven indirectly - - probably polysynaptically - - by MFB stimulation were correlated with the cortical desynchronization. The firing rates of these arousal units were very little affected by nucleus accumbens stimulation. These observations, that directly and synaptically activated brain stem units, and arousal produced by rewarding brain stimulation, are found in MFB but not in nucleus accumbens septi animals, are further evidence that arousal is produced by activation of a brain stem neural system in MFB self-stimulation 11. These observations also show that activation of this brain stem neural system is not necessary for self-stimulation. A priming effect of brain stimulation at a self-stimulation site can be measured by its effect on subsequent running speed for a fixed brain stimulation reward at the end of a runwayL The priming effect has many similarities with the arousal effect produced by MFB stimulation, and it has been suggested that the arousal effect partly mediates the priming effect11,12. The effects are similar in a number of ways: both are produced through MFB self-stimulation electrodes, are proactive effects of stimulation which continue after the end of stimulation, have a gradual decay to the prestimulation level, have greater magnitudes and durations when the number of stimulating trains is increased, and are produced by the direct excitation ofneurones with absolute refractory periods in the range of 0.78-1.1 msec. If activation of the brain stem neural system and the associated arousal do partly mediate thepriming effect, then stimulation of rewarding nucleus accumbens septi sites, which does not activate the brain stem arousal system, should not produce a similar priming effect. This has not yet been tested. Some of the differences between MFB and rhinencephalic self-stimulation s may be due to differences in the activation of the brain stem arousal system. As already stated, the nucleus accumbens septi region may be taken as an example of a rhinencephalic self-stimulation site. For example hyperactivity which occurs in MFB self-stimulation may be contrasted with the hypoactivity which occurs at least during stimulation of nucleus accumbens septi self-stimulation sites (personal observation). Using the refractory period of the directly excited brain stem neurones to manipulate the level of activation reaching the brain stem neural system, it was shown that activity in the brain stem arousal system greatly influenced the rate of self-stimulation 1'~. Thus activation of the brain stem arousal system may partly account for the much greater rates of self-stimulation at MFB than at rhinencephalic sites. This interpretation is supported by lesion evidence which indicates that the rate of MFB self-stimulation is reduced by lesions which probably interrupt the axons of the directly excited brain stem neutones 1,9. The brain stem arousal system may play a role in the lack of satiation characteristic of MFB self-stimulation, by maintaining the arousal of the animal. Finally, as stimulus-bound motivational behaviour is elicited from some MFB self-stimulation sites, but not from rhinencephalic self-stimulation sites, activation of the brain stem arousal system may be involved in stimulus-bound motivational behaviour. It has been shown that stimulation at posterior diencephalic (i.e., MFB and Brain Research, 31 (1971)275 285
284
+. J. aot.~+
field H2 of Forel) self-stimulation sites influenced many more brain stem single units than stimulation at self-stimulation sites in the region of the nucleus accumbens septi 13. The experiments described here analyse the differences between rewarding MFB and nucleus accumbens septi stimulation on the brain stem arousal system traced inpreviousexperiments 1°,11. The main differences are in effects on brain stem directly excited units, and indirectly activated (arousal) units, with differences usuaJly appearing in effects on brain stem synaptically activated units. The present experiments also give an indication about the role of activation of the brain stem units. They provide further evidence that through these brain stem units cortical desynchronization (arousal) may be produced in MFB stimulation. Earlier experiments 11,~e indicate thal this arousal may partly mediate a priming effect in self-stimulation SUMMARY
Very few brain stem single units were directly or synaptically excited through self-stimulation electrodes in the region of the nucleus accumbens septi. This is in contrast to the large populations of brain stem single units directly and synapticalty excited through hypothalamic self-stimulation electrodes. The hypothalamic, but not the nucleus accumbens septi, stimulation produced arousal measured by cortical desynchronization and activation of single units with general arousal properties. These results were obtained in acute experiments performed under urethane anaesthesia on rats previously implanted and tested for self-stimulation. They support other evidence that arousal is produced in self-stimulation of the medial forebrain bundle region by activation of a brain stem neural system. This arousal continues after the end of stimulation and may play a role in a priming effect of brain stimulation reward. The experiments also show that activation of the brain stem arousal system is not necessary for self-stimulation, and thus indicate that activation of a different neural system may mediate the rewarding effect of brain stimulation reward. ACKNOWLEDGEMENT
The author is grateful to Mr. J. Broad for photographic assistance.
REFERENCES 1 BOYD, E. S., AND GARDNER, L. C., Effect of some brain lesions on intracranial self-stimulation in
the rat, Amer. J. Physiol., 213 (1967) 1044-1052. 2 GALLISTEL, C. R., The incentive of brain-stimulation reward, J. eomp. physiol. PsychoL, 69 (1969) 713-721. 3 GALLISTEL, C. R., ROLLS, E., ANO GREENE, D., Neuron function inferred from behavioral and electrophysiologieal estimates of refractory period, Seience, 166 (1969) 1028-1030. 4 HtJBEL, D. H., Tungsten microelectrode for recording from single units, Science, 125 (1957) 549-550. 5 KOMISARUK, B. R., McDONALD, P. G., WHITMOYER, D. 1., AND SAWYER, C. H., Et'i'ects of progesterone and sensory stimulation on EEG and neural activity in the rat, Exp, Neurol., 19 (1967) 494-507.
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6 KONIG, J. F. R., AND KLIPPEL, R. A., The Rat Brain, Williams and Wilkins, Baltimore, Md., 1963. 7 MILGRAM,N. W., Effects of hippocampal stimulation on feeding in the rat, Physiol. Behav., 4 (1969) 665-670. 80LDS, J., AND OLDS, M., Drives, rewards, and the brain. In New Directions in Psychology, 11"ol.1I, Holt, Rinehart and Winston, New York, 1965, pp. 327410. 90LDS, M. E., AND OLDS, J., Effects of lesions in medial forebrain bundle on self-stimulation behavior, Amer. J. Physiol., 217 (1969) 1253 1264. 10 RoLLs, E. T., Neural systems involved in intracranial self-stimulation, Brain Research, 24 (1970) 548. (Abstract.) I I ROLLS, E. T., Involvement of brainstem units in medial forebrain bundle self-stimulation, Physiol. Behav., 7 (1971) in press. 12 ROLLS, E. T., Absolute refractory period of neurons involved in MFB self-stimulation, Physiol. Behav., 7 (1971) in press. 13 ROUTTENBERG, A., AND HUANG, Y. H., Reticular formation and brainstem unitary activity: effects of posterior hypothalamic and septal-limbic stimulation at reward loci, Physiol. Behav., 3 (1968) 611 617.
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