Activation of neurones in the prefrontal cortex by brain-stimulation reward in the rat

Brain Research, 60 (1973) 351-368 © Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

ACTIVATION OF NEURONES IN THE PREFRONTAL BRAIN-STIMULATION REWARD IN THE RAT

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E D M U N D T. ROLLS AND STEVEN J. COOPER*

University of Oxford, Department of Experimental Psychology, Oxford (Great Britain) (Accepted March 13th, 1973)

SUMMARY

To investigate reward pathways in the brain, recordings were made from neurones while electrical stimulation was applied to brain-stimulation reward sites in the rat. Single units in most areas of the neocortex were not activated by the stimulation. In the sulcal prefrontal cortex (which forms the dorsal bank of the rhinal sulcus) and in the medial prefrontal cortex (which forms the medial pregenual wall of the hemisphere) single units were activated by the stimulation. This indication that prefrontal neurones are involved in brain-stimulation reward received support from the observation that the neurones were activated in self-stimulation of many different sites - - the lateral hypothalamus, the midbrain tegmentum ventrolateral to the central grey, the nucleus accumbens, and the medial prefrontal cortex. Many of the prefrontal units were directly (probably antidromically)excited, with short latencies (1-10 msec), by stimulus pulses applied to self-stimulation sites other than the nucleus accumbens. Many prefrontal units were trans-synaptically activated with longer latencies (2-30 msec) by stimulus pulses applied to the self-stimulation sites. Further evidence that the prefrontal neurones are involved in brain-stimulation reward is that units in the prefrontal cortex were in general not activated from non-reward sites in the midbrain tegmentum. Also neurones in many other areas of the neocortex did not appear to be activated by the brain-stimulation reward.

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. Brain-stimulation reward of some sites is closely related to eating and drinking behaviour, and to emotional * Present address: Queen's University of Belfast, Department of Psychology, Belfast, N. Ireland.

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behaviour 14. It is therefore of interest to determine the neural basis of brain-stimulation reward 14. One approach is to determine the effects of brain-stimulation reward on neurones in different areas of the brain. In most areas in which recordings have been made neurones are not activated by rewarding lateral hypothalamic stimulation in any specific way 14. But two main areas have been found in which neurones are directly or trans-synaptically activated by the stimulation. One area is the midbrain 9, and through neurones here arousal is produced10,11. In the second activated area, the amygdala t2, the neuronal activation is probably closely related to eating, drinking and rewardS,12,1a, 16 produced by the stimulation. We now describe experiments which show that neurones in the prefrontal cortex are activated in self-stimulation of a number of different self-stimulation sites. The experiments indicate that the activation of prefrontal neurones is closely related to reward produced by electrical stimulation of the brain. The prefrontal cortex in the rat, defined as the projection field of the mediodorsal nucleus (MD) of the thalamus, has been identified by Leonard 6 using the FinkHeimer silver technique for tracing degenerating fibres. The sulcal prefrontal cortex forms the dorsal bank of the rhinal sulcus. It is reciprocally connected with the medial division of MD and in this respect is similar to the caudal orbitofrontal cortex of the rhesus monkey. The medial prefrontal cortex forms the medial wall of the hemisphere anterior and dorsal to the genu of the corpus caUosum. It is reciprocally related with the lateral division of MD. The dorsal sector of this medial cortex (the 'shoulder' region) may be comparable to the caudal prefrontal cortex (Brodmann's area 8, the frontal eye field) of the monkey, while the remainder of the medial cortex may be homologous with the major part of the monkey's frontal convexity cortex (Leonard 6 p. 338). There has been little previous electrophysiological work on the role of the prefrontal cortex in brain-stimulation reward. During single unit recordings made in many different brain areas, Ito and Olds 4 found 4 units which were probably in the medial prefrontal cortex and were activated in posterior hypothalamic self-stimulation. No recordings appear to have been made in the sulcal prefrontal cortex.

METHODS

The methods in general follow those already describedg,11,1L Rats were implanted with monopolar electrodes and later tested for self-stimulation on each electrode. Then an acute electrophysiological experiment was performed on each rat, recording the activity of single neurones extraceUularly with capillary microelectrodes while stimulation was applied to a self-stimulation site. If a single unit was activated by the stimulation, tests were performed to determine whether it was directly excited, and if so whether ortho- or antidromically, or whether it was trans-synaptically excited. To ensure that all the units classed as activated were activated during self-stimulation, only units activated at currents which, or lower than those which, supported selfstimulation were used in the acute electrophysiological experiments. Analysis of the

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effects of self-stimulation of a number of different brain areas on neuronal activity in the prefrontal cortex was possible with the methods used.

Preparation of animals for the acute electrophysiological experiments Under Equithesin (Jensen-Salsbury Labs, Inc.) anaesthesia, male rats were implanted with monopolar stainless steel electrodes for stimulation insulated (except for 0.5 mm at the tip) with Insl-X varnish. Several electrodes were implanted in each rat. One was aimed at the lateral hypothalamus (level-head coordinates, 3.0 mm behind bregma, 1.5 mm lateral to the sagittal sinus, and 7.6 mm beneath the dura, i.e., --3.0:1.5:7.6 mm down). Other electrodes were aimed at the medial prefrontal cortex (+2.5:0.5:2.5 mm down), the midbrain tegmentum ventrolateral to the central grey (--5.5:1.2:5.5 mm down) and sometimes at the nucleus accumbens ( + 1.6:1.0:4.6 mm down). A large exposure of the dura was not made at this stage, but enough skull was removed round each electrode shaft to ensure that the dura could be exposed later for the insertion of microelectrodes without touching the stimulating electrodes. The electrodes were held in an electrode assembly which was connected to the skull contralateral to and thus clear of the stimulating electrodes. Two days later the rats were tested for self-stimulation. Each rat was placed in a cage which contained a lever. A 0.3 sec train of 0.1 msec constant current cathodal pulses, at a frequency of 100 Hz, was applied to the electrode under test when the rat pressed the lever. Current return was by the screws in the skull which held the electrodes. Lateral hypothalamic and tegmental electrodes classed as positive for self-stimulation supported self-stimulation rates of more than 30 lever presses/rain, and positive nucleus accumbens, and medial prefrontal cortex sites produced rates of more than I0 lever presses/min. The current and the voltage of the stimulating current which just supported self-stimulation were found for each positive electrode. For the acute electrophysiological experiments the animals were anaesthetized with an intraperitoneal injection of 20% urethane solution (1.2 g/kg) and placed in a stereotaxic instrument. Rectal temperature was maintained at 37 °C by wrapping the animal in cellulose wadding and by using an homeothermic electric blanket. Single unit activity was recorded extracellularly with glass capillary microelectrodes. These were filled with pontamine sky blue dye (6BX, George T. Gurr Ltd., London) made up as a 2% solution in 0.5 M sodium acetate ~ and had a DC resistance of 0.5-4.0 M f L A differential pre-amplifier (A101, 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 which rested on the exposed surface of the dura and which recorded stimulus artifact. By adjusting the relative gains of these two amplifiers, it was possible to reject most of the stimulus artifact from the single unit channel. Further clarification of short latency single unit responses was obtained by setting the high pass filter to 1 kHz. The output of the Isleworth 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.

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Electrophysiological methods Single unit activity in the region of the prefrontal cortex 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 animal had previously shown a good rate of self-stimulation. This procedure was to ensure that units activated by the electrical stimulation in the acute experiments were also activated in self-stimulation. Whenever a spontaneously active single unit was encountered, the effects of stimulation applied to a selfstimulation electrode were determined and compared, where possible, with stimulation applied to other electrodes. As some activated units were not spontaneously active, stimulus pulses were applied frequently while the microelectrode was lowered through the brain. Single units activated by the electrical stimulation were classed as directly or synaptically activated 9, as described below. 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 could be either in the orthoor antidromic direction. Dromicity was determined where possible by the collision techniqueL When action potentials were from a directly excited unit, the latency and absolute refractory period of the unit were measured (e.g., Fig. 1a). To ensure that the absolute refractory period was measured, the stimulating current of the pulse pairs was increased until no further reduction in the refractory period was produced. Under the stimulating conditions used, this occurred at twice-threshold or lower currentsL Units activated by single stimulus pulses with a longer and variable latency (usually in the range 2-50 msec) were classed as synaptically driven (e.g., Fig. lb). These units are probably trans-synaptically excited by the stimulus pulses through one or several synapses. Collision can not be demonstrated in synaptically driven units 11. RESULTS

Electrophysiological experiments were performed on 14 rats. Each rat had at least 1 and up to 4 electrodes positive for self-stimulation. Up to 150 units activated by stimulation applied to the different electrodes were analysed in each animal in 3-day experiments. Recordings were made from many hundreds of units in each rat in order to determine the brain areas in which units are activated in self-stimulation. In most areas of the brain from which recordings have been made, including most of the neocortex, units were not directly or trans-synaptically activated by the stimulation. Areas in which activated units are found have been described previously s,11, and the activation of neurones in the prefrontal cortex is described here. Motor effects (e.g., leg flexion) could usually be elicited from electrodes which did not support self-stimulation. In the acute experiments stimulation at the motor effect intensity was applied to the electrodes to determine whether the effects from the self-stimulation sites were specific to these sites. The results provide evidence that neurones in the prefrontal cortex are activated in self-stimulation of the lateral hypo-

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thalamus, midbrain tegmentum, and nucleus accumbens, and provide a description of this activation and its relation to brain-stimulation reward.

Unit activity recorded in the prefrontal cortex An example of a unit in the sulcal prefrontal cortex directly driven by stimulus pulses applied to a lateral hypothalamic self-stimulation site is shown in Fig. la. With two pulses separated by 10 msec (top trace) action potentials, which were all-or-none, can be seen following each pulse with a short, fixed latency of 2.0 msec. As the intrapair interval of the pulse pairs was reduced, the second action potential became intermittent at 1.5 msec, and was almost never present at 1.35 msec. At the intra-pair interval of 1.35 msec a second action potential can be seen twice in Fig. la, lowest trace. Thus the refractory period of the unit was between 1.35 and 1.5 msec. This was the absolute value, because increasing the stimulating current further did not reduce the refractory period. Because of the good following to pulse pairs (i.e., short absolute refractory period) and because of the short fixed latency of the action potentials the

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unit was classified as directly excited by the stimulus pulses. The excitation of this type o f unit is probably antidromic (e.g., refs. 9 and 12), but because the units often lacked spontaneous activity it was not possible to perform collision tests on more than a few o f the neurones. Where collision tests were performed, antidromic activation was confirmed. A unit recorded in the sulcal prefrontal cortex and classified as trans-synaptically driven by stimulation applied to a midbrain tegmentum self-stimulation site is shown in Fig. lb. The action potentials occur with a longer, more variable latency after the stimulus pulses than that characteristic o f directly excited units. If a unit showed spontaneous activity and collision could not be demonstrated, this aided its classification as a synaptically activated unit 11. If a unit followed the second pulse o f a pulse pair only at long intra-pair intervals, this also led it to be classified as a synaptically activated unit. F o r prefrontal units 2.0 msec was considered a long intra-pair interval, although units with apparent absolute refractory periods o f as little as 1.5 msec were sometimes classed as synaptically activated because o f their other characteristics. Using these criteria it was possible to classify most activated units as directly or synaptically excited, but particularly in the prefrontal cortex some units were recorded with short latencies o f less than 4 msec whose classification was difficult. In cases where classification was difficult units were classed as trans-synapticalty activated.

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cortex, which forms the dorsal bank of the rhinal sulcus, units activated from lateral hypothalamic self-stimulation sites were recorded (see examples in Figs. 2 and 6). The track shown in Fig. 2 was the furthest posterior of the sulcal prefrontal tracks described in this study. The units in this track were activated with longer latencies than were usual from the lateral hypothalamus. The unit potentials recorded here and throughout the prefrontal cortex had the general form illustrated in Fig. 1 and were analysed as described above. The latencies of 85 units recorded in similar tracks in 8 animals are shown in Fig. 3a. The short latencies and the large number of the units recorded indicates a strong connection between lateral hypothalamic reward sites and the sulcal prefrontal cortex. The directly excited units probably reflect the antidromic activation of prefrontal neurones with axons passing caudally through the lateral hypothalamic site. The synaptically activated units may reflect an input through collateral fibres of the directly excited units, or they may reflect synaptic activation from afferent neurones with synapses in the prefrontal cortex but cell bodies in or caudal to the lateral hypothalamus. The absolute refractory periods of the directly excited units were long - - between 1.0 and 2.2 msec (see Fig. 3b). At least some of the units in these lateral tracks were in the sulcal prefrontal cortex, that is the cortex which receives a projection from the mediodorsal nucleus (MD) of the thalamus. It forms the dorsal bank of the rhinal sulcus (see also Fig. 6). Other units, although included in the analysis, were dorsal to the main region in which degeneration after MD lesions was found by Leonard6. Medial prefrontal cortex. An example of a track through the medial prefrontal cortex in a rat with a lateral hypothalamic electrode positive for self-stimulation is shown in Fig. 4. Thirty-two units activated by the stimulation at or below the self-

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stimulation intensity were recorded in the track, and their position along the track was fixed by a pontamine sky-blue mark at the deepest point where activated units were found (see Fig. 4), and by another mark (not shown in Fig. 4) at the most superficial activated unit recorded. In this track several activated units were often recorded at the same depth. The short latencies and the large number of the units indicate a strong connection between the lateral hypothalamic reward site and the medial prefrontal cortex. The latencies of 30 directly excited units recorded in 8 rats with lateral hypothalamic self-stimulation are shown in Fig. 5a. Many of the units have latencies between 2 and 4 msec, which is longer than the latencies of the units recorded in the sulcal prefrontal cortex. Sixty synaptically activated units recorded in 9 animals also had relatively short latencies (see Fig. 5a): most were between 3 and 10 msec. Although in the track shown in Fig. 4 more units than usual were recorded, the general similarity of the results found in the 9 rats emphasizes the powerful activation of the medial prefrontal cortex in lateral hypothalamic self-stimulation. Absolute refractory period measurements could be made in 23 of the directly excited units. The distribution of refractory periods is shown in Fig. 5b. The refractory periods are in general long, with 12 units having refractory periods greater than 1.5

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Activation of units from self-stimulation sites in the midbrain tegmentum Sulcal prefrontal cortex. Units in the sulcal prefrontal cortex were driven from the midbrain self-stimulation sites. A track in which directly and synaptically activated units were recorded is shown in Fig. 6, top left. Units activated from lateral hypothalamic and medial prefrontal cortex self-stimulation sites were also recorded in this track. It is characteristic of the prefrontal cortex that only rarely were individual units driven from more than one of the self-stimulation sites used, although units activated from different sites were in close proximity. The latencies of 51 units recorded in lateral tracks and activated from tegmental self-stimulation sites in 4 animals are shown in Fig. 7a. It is clear that a large number of units are directly activated with short latencies. No sulcal prefrontal cortex units were directly activated in 3 animals with tegmental motor effect electrodes. (In a further group of 5 animals with tegmental motor effect electrodes further posterior in the midbrain, no activated units were found in the sulcal prefrontal cortex of 4 animals but 18 activated units were found in one animal.) The absolute refractory periods of units directly excited from the self-stimulation sites in the midbrain tegmentum covered a wide range between 0.68 and 2.5 msec (see Fig. 7b). So that these results can be compared with earlier resultsg,11,12, bars have been drawn in Fig. 7b to include units with refractory periods of 0.68 up to 0.78 msec, and of 0.78 up to 0.90 msec. Medialprefrontal cortex. Activation of units in the medial prefrontal cortex was found from the tegmental self-stimulation sites. An example of a track in which directly and synaptically activated units were recorded is shown in Fig. 8. The latencies of 26 of these units recorded in 3 rats are shown in Fig. 9a. The activation of medial

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The absolute refractory periods of units directly excited from the self-stimulation sites in the midbrain tegmentum were long. Most were between 1.2 and 2.5 msec (see Fig. 9b).

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with nucleus accumbens self-stimulation were classed as synaptically driven and had long latencies between 8 and 60 msec (see Fig. 10a). Medial prefrontal cortex. Units in the medial prefrontal cortex were transsynaptically activated by stimulation applied to nucleus accumbens self-stimulation sites. A track through the medial prefrontal cortex in an animal in which nucleus accumbens stimulation was rewarding and lateral hypothalamic stimulation elicited m o t o r effects is shown in Fig. 11. The latencies of the action potentials from nucleus accumbens stimulation are in the range 5-90 msec (see Fig. 10b). These latencies are much longer than those from the other self-stimulation sites.

Activation of units from self-stimulation sites in the medial prefrontal cortex Sulcal prefrontal cortex. A powerful activation of sulcal prefrontal cortex by stimulation applied to medial prefrontal cortex self-stimulation sites was found. An example of a track through the sulcal prefrontal cortex in which units were activated from the medial prefrontal cortex has been shown in Fig. 6. The latencies of 61 units activated from medial prefrontal self-stimulation sites are shown in Fig. 12a. Medial prefrontal cortex. As expected, activated units were recorded close to self-stimulation electrodes in the medial prefrontal cortex. An example of a track in which such units were recorded is shown in Fig. 8. The majority of these units were trans-synaptically activated and had latencies between 2 and 10 msec (see Fig. 12b). The very small number of directly excited units recorded under these conditions is of

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Fig. 12. a: the latencies of 61 units in the sulcal prefrontal cortex activated from self-stimulation sites in the medial prefrontal cortex. The units were recorded in 5 rats. Conventions as in Fig. 3. b: the latencies of 46 units in the medial prefrontal cortex activated from self-stimulation sites in the medial prefrontal cortex. The units were recorded in 5 rats. Conventions as in Fig. 3. interest, for it contrasts with the large number of directly excited units recorded in the same region during lateral hypothalamic or tegmental stimulation.

Convergence from different self-stimulation sites Only rarely were individual neurones in the prefrontal cortex activated from more than one of the self-stimulation sites. Only two neurones in the sulcal prefrontal cortex were activated from more than one site. One was synaptically activated from the lateral hypothalamus with a latency of 30 msec and synaptically activated from the nucleus accumbens with a latency of 40 msec. The other was directly excited from the lateral hypothalamus with a latency of 1 msec, and synaptically activated from the medial prefrontal cortex with a latency of 3.5 msec. In the medial prefrontal cortex only one neurone was activated from both lateral hypothalamic and tegmental sites. Three neurones were activated by both lateral hypothalamic and medial prefrontal cortex stimulation. Although convergence onto individual neurones was rarely seen, neurones activated from different self-stimulation sites were frequently close to each other in the sulcal and medial prefrontal cortex. This is clearly seen in Figs. 2, 6 and 8. These observations indicate that in the sulcal and medial prefrontal cortex neurones close to each other are activated in self-stimulation of the different brain sites, but that this activation did not in general reach the prefrontal cortex by the same neurones, and also that this activation did not spread trans-synaptically to activate the same neurones in the prefrontal cortex.

The sites of units in the prefrontal cortex activated in self-stimulation Suleal prefrontal cortex. Microelectrode tracks through the sulcal prefrontal cortex have been shown in Figs. 2 and 6. Localization of units using the pontamine sky blue method for marking was good, especially when more than one mark was

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made in a track. The units activated from the self-stimulation sites were recorded mainly within the following region, using coordinates from K6nig and Klippel's atlas5: A 11050-A 10050: 2.0-3.5 mm lateral, and 1.0--4.5 mm above the interaural line. Thus the track illustrated in Fig. 2 is the most posterior one in which units were recorded in the sulcal prefrontal cortex in this study. Although many of the units in these tracks were in the dorsal bank of the rhinal sulcus, the sulcal prefrontal cortex as designated by Leonard ¢, other units just dorsal to this region were sometimes activated. Medialprefrontal cortex. Tracks through the medial prefrontal cortex have been shown in Figs. 4, 8 and 11. The activated units in these medial tracks were within the following region: A l1050-A 9410, 0.0-1.5 mm lateral, and 1.0-4.5 mm above the interaural line. On the dorsomedial convexity of the prefrontal cortex ('shoulder cortex') some activated units were recorded out to about 2.0 mm lateral. In general activated units appeared to be few in number in this 'shoulder cortex' region. This region has connections with the superior colliculus, and in this respect is similar to the frontal eye field of the monkey 6.

Self-stimulation sites Examples of lateral hypothalamic and nucleus accumbens self-stimulation sites reached with the coordinates used have been given elsewhere I°,15. Examples of the medial prefrontal cortex and midbrain tegmentum implant sites from which selfstimulation could be elicited are shown in Fig. 13. Most of the other tegmental sites were in the position ventrolateral to the central grey, rather than more ventral near the interpeduncular nucleus. DISCUSSION These results show that neurones in the sulcal and the medial prefrontal cortex are activated in self-stimulation of the lateral hypothalamus, the midbrain tegmentum, the nucleus accumbens and the medial prefrontal cortex. There is some evidence in the results that activation of these neurones in the prefrontal cortex is closely related to the self-stimulation. First, the main neocortical region in which neurones were found to be activated in self-stimulation was the prefrontal cortex. Activated neurones were not found in most other neocortical areas, except for the cingulate cortex 14. This finding is consistent with the known anatomy of neocortical-hypothalamic pathways 6,7. Second, in general the prefrontal neurones were not excited from sites in

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Fig. 13. Examples of self-stimulation sites in the medial prefrontal cortex (a and b) and the midbrain tegmentum (c). Outlines from K6nig and Klippel5, Figs. 9b, 12b and 49b.

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the midbrain tegmentum from which self-stimulation could not be elicited. Thus in only 1 of 8 animals with midbrain tegmental motor effect electrodes were activated units recorded in the sulcal prefrontal cortex. Third, the observation that neurones in the prefrontal cortex are activated in self-stimulation of many different brain sites also suggests that these neurones are involved in brain-stimulation reward. The electrophysiological experiments described here lead to the suggestion that neurones in the prefrontal cortex are involved in brain-stimulation reward. The suggestion has been further investigated in a number of ways. It has been shown that neurones in the prefrontal cortex are activated from far caudal self-stimulation sites, in the pontine tegmentum near the locus coeruleus (Cooper and Rolls, in preparation). If the prefrontal cortex is involved in brain-stimulation reward in the rat, then selfstimulation of it might be expected. Self-stimulation of' pregenual frontal cortex (i.e., medial prefrontal cortex) has been found (ref. 17 and present study), as also has selfstimulation of sulcal prefrontal cortex (ref. 19 and Rolls and Cooper, in prep.). These indications of the role of prefrontal cortex in brain-stimulation reward in the rat have been tested in experiments in which either the region of the sulcal or the medial prefrontal cortex has been anaesthetized by injections of procaine through previously implanted cannulae. Bilateral anaesthetization of the region of the sulcal prefrontal cortex (but not in our present experiments of the medial prefrontal cortex) stops selfstimulation of the lateral hypothalamus and the pontine tegmentum (Rolls and Cooper, in preparation). Anaesthetization of the neocortex dorsal to the sulcal prefrontal cortex, or the caudate nucleus, both of which are areas outside the activated region of prefrontal cortex described here, did not inhibit self-stimulation. Taken together, these experiments indicate that activity in the sulcal prefrontal cortex and brain-stimulation reward are closely related in the rat. Although the monkey prefrontal cortex shows many differences from that in the rat 6, it does appear to be related in some respects to brain-stimulation reward. For example, self-stimulation with electrodes in or near the orbito-frontal cortex can be obtained in the squirrel monkey, and units in the orbito-frontal cortex are activated in hypothalamic stimulation (personal observation). The results described here suggest that the prefrontal neural system activated in brain-stimulation reward has the following properties. Neurones with cell bodies in the sulcal and medial prefrontal cortex are antidromically excited in self-stimulation of the lateral hypothalamus and midbrain tegmentum. Although the collision technique 9,i2 could not be applied systematically in these experiments because the directly excited units often lacked spontaneous activity, antidromic activation of these units does seem likely because of the collision obtained with a small number of the units, and because of the nature of the action potentials. The units trans-synaptically activated by lateral hypothalamic and tegmental self-stimulation in this study may be activated by neurones afferent to the prefrontal cortex with axons passing through the self-stimulation sites; alternatively, the synaptically activated neurones may be driven by collateral axons of the directly excited neurones. In the case of nucleus accumbens stimulation, the activation appears to be through afferent neurones as directly excited neurones were not found in the prefrontal cortex.

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The medial prefrontal region activated in self-stimulation corresponds well with that in which Leonard6 found degeneration after MD lesions. The lateral activated region is more extensive than that in which MD lesions produced degeneration. The present observation that medial and sulcal prefrontal cortex units were directly excited by electrodes in the lateral hypothalamus and in the midbrain tegmentum ventrolateral to the central grey is in agreement with Leonard's observation that fibre degeneration after lesions in the sulcal or medial prefrontal cortex can be traced past the hypothalamic to the tegmentum. The present results show that the axons activated in lateral hypothalamus self-stimulation are in general different to those activated in the tegmental self-stimulation. A single neurone was very rarely activated from these two sites. In contrast, single amygdaloid neurones are often activated by stimulation of both the lateral hypothalamus and the nucleus accumbenslL The absolute refractory periods of the prefrontal neurones activated in selfstimulation were in general long. Medial prefrontal units directly excited by the lateral hypothalamic stimulation had refractory periods in the range 0.9-2.2 msec, and all but 2 units had refractory periods of, or greater than, 1.1 msec (see Fig. 5b). All the sulcal prefrontal cortex units activated from the lateral hypothalamic sites had absolute refractory periods of, or greater than, 1.0 msec (see Fig. 3b). In contrast, midbrain units directly excited by the lateral hypothalamic stimulation have characteristic absolute refractory periods in the range 0.78-1.0 msec9. There is evidence that through these neurones arousal is producedg, 11. Amygdaloid neurones directly excited by lateral hypothalamic self=stimulation have characteristic absolute refractory periods in the range 0.58-0.68 mseclL These neurones may be involved in eating and drinking8,12,13,16. The finding that neurones in the prefrontal cortex excited by the lateral hypothalamic stimulation have refractory periods different from those characteristic of neurones activated in different brain areas is a further indication that absolute refractory period can be useful in separating populations of neurones. The refractory periods of medial prefrontal cortex units activated by the tegmental stimulation were longer than 1.0 msec (see Fig. 9b). The refractory periods of sulcal prefrontal neurones activated by the tegmental stimulation show a wide distribution between 0.68 and 2.5 msec (see Fig. 7b). The present conclusion developed from the electrophysiological findings, that the prefrontal cortex is involved in brain-stimulation reward, receives some support from work with neuroanatomical techniques. Using anterograde degeneration techniques Routtenberg17 followed degeneration from pregenual frontal (medial prefrontal) cortex self-stimulation sites in the rat through the cingulum, corpus callosum and internal capsule. It was suggested that this pathway might be involved in brain-stimulation reward. In the monkey degeneration from reward sites near the hypothalamus runs to the mediodorsal nucleus of the thalamus is, which is reciprocally related with the prefrontal cortex. The problem with this type of experiment is that when a lesion is made through a stimulation electrode in a reward site, there is no assurance that all the degeneration observed comes from neurones which were excited by the rewarding electrical stimulation. In the monkey particular behavioural deficits follow lesions of different regions

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of the prefrontal cortex1,2, 20. L e o n a r d 6 showed that the regions termed here prefrontal cortex in the rat had anatomical connections similar in some respects to the c o n n e c t i o n s of prefrontal cortex in the monkey. The present study is an a t t e m p t to clarify the connections a n d functions of this region in the rat, and taken with the other recent work described suggests that the prefrontal cortex may be i m p o r t a n t in brainstimulation reward. ACKNOWLEDGEMENTS This investigation was supported in part by Medical Research Council G r a n t No. 971/397/B. The authors t h a n k Miss S. Babrekar, Mr. J. Broad a n d Mr. I. Hughes for general, p h o t o g r a p h i c a n d histological assistance. S. J. C o o p e r was supported in part by the E u r o p e a n T r a i n i n g P r o g r a m in Brain a n d Behaviour Research. REFERENCES 1 BUTTER, C. M., Perseveration in extinction and in discrimination reversal tasks following selective frontal ablations in Macaca mulatta, Physiol. Behav., 4 (1969) 163-171. 2 BUTTERS, N., AND PANDYA, n . , Retention of delayed alternation: effect of selective lesions of sulcus principalis, Science, 165 (1969) 1271-1273. 3 HELLON, R. F., The marking of electrode tip positions in nervous tissue, J. Physiol. (Lond.), 214 (1971) 12P. 4 ITO, i . , AND OLDS, J., Unit activity during self-stimulation behavior, J. NeurophysioL, 34 (1971) 263-273. 5 KONIG, J. F. R., AND KLIPPEL, R. A., The Rat Brain, Williams and Wilkins, Baltimore, 1963. 6 LEONARD, C. M., The prefrontal cortex of the rat. I. Cortical projection of the mediodorsal nucleus. II. Efferent connections, Brain Research, 12 (1969) 321-343. 7 RAISMAN,G., Neural connexions of the hypothalamus, Brit. reed. Bull., 22 (1966) 197-201. 8 ROLLS,B. J., ANDROLLS,E. T., Effects of lesions in the basolateral amygdala on fluid intake in the rat, J. comp. physioL PsychoL, 83 (1973) 240-247. 9 ROLLS,E. T., Involvement of brainstem units in medial forebrain bundle self-stimulation, Physiol. Behav., 7 (1971) 297-310. 10 ROLLS,E. T., Absolute refractory period of neurons involved in MTFB self-stimulation, Physiol. Behav., 7 (1971) 311-315. 11 ROLLS,E. T., Contrasting effects of hypothalamic and nucleus accumbens septi self-stimulation on brain stem single unit activity and cortical arousal, Brain Research, 31 (1971) 275-285. 12 ROLLS,E. T., Activation of amygdaloid neurones in reward, eating and drinking elicited by electrical stimulation of the brain, Brain Research, 45 (1972) 365-381. 13 ROLLS,E. T., Refractory periods of neurons directly excited in stimulus-bound eating and drinking in the rat, J. comp. physioL Psychol., 82 (1973) 15-22. 14 ROLLS,E. T., The neural basis of brain-stimulationreward, Progr. Neurobiol., In press. 15 ROLLS, E. T., AND KELLY, P. a . , Neural basis of stimulus-bound locomotor activity in the rat, J. comp. physiol. PsychoL, 81 (1972) 173-182. 16 ROLLS,E. T., ANDROLLS,B. J., Altered food preferences after lesions in the baso-lateral region of the amygdala in the rat, J. comp. physiol. Psychol., 83 (1973) 248-259. 17 ROUTTENBERG,A., Forebrain pathways of reward in Rattus norvegicus, J. comp. physiol. Psychol., 75 (1971) 269-276. 18 ROUTTENBERG,A., GARDNER, E. L., AND HUANG, Y. H., Self-stimulation pathways in the monkey, Macaca mulatta, Exp. Neurol., 33 (1971) 213-224. 19 ROUTTENBERG, k., AND SLOAN, i . , Self-stimulation in the frontal cortex of Rattus norvegicus, Behav. Biol., 7 (1972) 567-572. 20 WARREN, J. M., AND AKERT, K. (Eds.), The Frontal Granular Cortex and Behavior, McGraw-Hill, New York, 1964.