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Activity of neurons in the anteromedial cortex during rewarding brain stimulation, saccharin consumption and orienting behavior
Behavioural Brain Research, 8 (1983) 69-84
69
Elsevier Biomedical Press
ACTIVITY OF N E U R O N S IN T H E A N T E R O M E D I A L C O R T E X D U R I N G R E W A R D I N G BRAIN S T I M U L A T I O N , S A C C H A R I N C O N S U M P T I O N A N D O R I E N T I N G BEHAVIOR
J O H N P. KANKI, TERRY L. M A R T I N and HARRY M. S I N N A M O N *
Laboratory of Neuropsychology, Wesleyan University, Middletown, CT06457 (U.S.A.) (Received February 22nd, 1982) (Revised version received September 28th, 1982) (Accepted November 26th, 1982)
Key words: anteromedial cortex - reward - brain stimulation - saccharin consumption - orienting behavior - rat
SUMMARY
Single neuronal activity in the deep layers of the prelimbic and dorsal anterior cingulate regions of the anteromedial cortex (AMC) was recorded through chronically implanted fine wires (25 #m diameter) during reward-related behavior in five rats. Tests were conducted in a transparent plastic cylindrical chamber (28 cm in diameter) in which the water-deprived rats initiated and terminated trains of 50 Hz rewarding electrical stimulation to the ventral tegmental area or the medial forebrain bundle and approached and consumed a 0.01~o saccharin solution. The rats were also presented with non-rewarding visual, olfactory and tactile stimuli which did not elicit approach but did elicit orienting behavior, i.e., head fixation in the direction of the stimulus, tracking head movements or tactile contact with the snout accompanied by sniffing. Of 60 neurons tested during intracranial self-stimulation (ICS S), 43 showed consistent activity changes within 1 sec of onset. Decreases in activity were most common (32 of 43) but increases that appeared to be synaptically mediated (n --- 9) or antidromically mediated (n -- 2) were also observed. Neurons tested with two ICSS sites were likely to show the same response (28 of 30). Of 52 neurons recorded when the rat drank saccharin, 24 changed in activity with 16 showing decreases. Of 45 neurons tested during orienting behavior to the non-rewarding stimuli, 15 increased and 7
* To whom correspondence and requests for reprints should be addressed. 0166-4328/83/0000-0000/$03.00 © 1983 Elsevier Science Publishers
70 decreased in activity. Comparisons were made of activity during ICSS to that during drinking and orienting behavior. Neurons that decreased during drinking were likely to also decrease during ICS S. Neurons that decreased during orienting were likely to decrease also during ICS S and neurons that increased during ICS S were likely to increase during orienting. These results indicate that decreases in activity of A M C neurons may be significant in the registration of rewarding states and that orienting behavior appears to be an important correlate to increased activity in these neurons.
1N T R O D U C T I O N
The anteromedial cortex (AMC) of the rat consists of regions which are primary targets of the dorsomedial nucleus of the thalamus [2, 11, 12]. According to the cytoarchitectonic boundaries delineated by Krettek and Price [11 ], the AMC includes the anterior cingulate cortex, both the dorsal (ACd) and ventral (ACv) parts, the prelimbic area (PL) and the medial precentral region (PRCM). These regions together with the dorsal perirhinal cortex which also receives projections from the dorsomedial nucleus [22, 24] have been proposed to be the homolog of the primate prefrontal cortex [ 12]. S everal lines of evidence indicate that the AMC has a particularly important role in motivation. It is one of the few cortical regions that support ICSS [ 15, 19]. Neurons in the region respond antidromically and synaptically at short latency to pulses of ICS S current [7, 18, 21 ]. Longer duration responses, both increases and decreases in activity, with relatively long and variable latencies have also been described in anesthetized rats [21]. The AMC receives projections from the dopaminergic system that has cell bodies in the ventral tegmental area of the midbrain and the substantia nigra [13]. These regions also support ICSS [3, 4], although it is not clear that the dopaminergic fibers are essential [20]. Lesions of the AMC produce a variety of effects which are complex but at least indicate possible dysfunctions in reward processes. For example, Glass et al. [5] showed that rats with AMC lesions failed to show the normal facilitatory effects that prior feeding in a goal box has on learning to approach that goal box in a runway situation. Gurowitz et al. [6] showed that the running speed of rats with AMC lesions was less sensitive than that of controls to a reduction in the magnitude of reward. When reward is omitted on alternate trials in a runway, rats with AMC lesions fail to show the normal pattern of fast and slow running [ 1]. In a barpressing situation on a fixed interval schedule, they respond more than controls to omitted reward [ 14]. This experiment addressed several questions. First, it was important to determine the type of activity changes associated with reward in the freely behaving rat. Ito and Olds' [7] early chronic study reported short latency
71 responses but not the long term responses described in acute studies [21]. Another important question was the relationship between the response to ICSS and the activity patterns during more conventional rewards. To this end, neural activity during consumption of sweetened water was compared to that during ICSS. As a first step in characterizing the sensory properties of neurons responsive to ICSS, a range of stimuli was presented to the animal. These stimuli were not motivationally potent but did generally elicit orienting responses and provided a second class of correlates for comparison to the ICSS correlates. METHODS
Subjects and surgery The rats (n = 5, Fischer-Dark Agouti) were caged individually with free access to food but not to water. They were anesthetized and mounted in a stereotaxic device. Stainless steel screws were inserted in the skull and an uninsulated wire terminating in an Amphenol pin was attached to serve as a ground connection. The recording electrode bundle was mounted in a drive assembly (see below) which was secured to the skull with dental cement. The bundle passed through a l-ram hole drilled in the skull over the AMC and penetrated the brain to a depth of approximately 1 ram. For brain stimulation, two bipolar stimulation electrodes were aimed for the medial forebrain bundle (MFB) and the ventral tegmental area of the midbrain (VTA) ipsilateral to the recording bundle or to the VTA bilaterally. The electrodes were formed by twisting two 125-pro stainless steel wires, insulated with Teflon except at the cross-section of the tips. Amphenol pins connected to the electrodes were inserted into an Amphenol connector strip along with the ground pin. The entire assembly composed of the recording drive and the connector strip was secured to the skull with dental cement.
Apparatus Recordings were made with the rat in a transparent Plexiglas cylinder which was 28 cm in diameter and 36 cm high. In the wall of the chamber were three 1.5 cm diameter holes which were spaced 90 ° apart and 2 cm above the floor through which the stimulation onset rod was inserted. On the inner wall of the chamber opposite the middle hole, a 15 x 16 cm copper plate was located 1.5 cm from the floor. Contact with the plate by the rat caused offset of the brain stimulation. A cable of Microdot wires carrying recording and stimulation lines connected the rat to a commutator. Constant-current stimulation was provided by 0.3 msec square wave pulses at 50 Hz for a train duration determined by the rat. The current levels effective for maintaining consistent ICSS varied between sites from 70 to 600 #A. The rat initiated the ICSS train by completing a low current circuit between ground and a 2-mm rod which protruded 2 cm into the chamber through any one of three
72 chamber holes. A 0.5-sec delay was imposed between the initial contact with the rod and the onset of stimulation; the delay allowed the temporal separation of a neural activity change associated with the operant response from one associated with the onset of ICS S. The rat terminated the ICS S train by contacting the metal plate on the wall of the chamber. The contact was detected by a low current circuit and again a 0.5-sec delay was imposed between the operant response and the actual offset of the train.
Recording technique Extracellular activity was differentially recorded across selected pairs of the four wires in the electrode bundle. The electrode bundle was composed of four 25-#m diameter, Teflon-coated, stainless steel wires which had been twisted together. The bundle was fixed into a longitudinal slot of a brass guide screw (size 6/32). The end of the bundle extended 10 mm beyond the bottom and was cut transversely with scissors to expose the recording surface. The guide screw and attached electrode bundle was inserted into a custom made drive assembly which allowed downward movement of the bundle without rotation. The drive consisted of a threaded Delrin spur gear, a Delrin sleeve which fit over the hub of the spur gear, and a metal key sized to fit snugly into the slot of the guide screw. One revolution of the spur gear provided 780 #m of downward travel by the electrode bundle. A short four-wire cable connected pins connected to the bundle to a miniature, differential, DC-coupled, 10-gain, preamplifier (F. Haer Co.) mounted near the base of the cable. A manual switch allowed for alternation between pairs of electrodes for differential recording. The output of the preamplifier was sent to a band pass filter which passed frequencies between 200 Hz and 10 kHz. The filtered electrical activity was led to an oscilloscope, an audio amplifier, and a voltage and fall time discriminator to produce standard 5-V pulses when spikes of critical amplitude and duration occurred. To avoid the inclusion of stimulation artifact or evoked field responses in the counts of neuronal spikes, the output of the window discriminator was suppressed for a selectable period (1-4 msec) after the onset of each stimulation pulse. This circuitry and the use of differential recording effectively eliminated the difficulties associated with stimulus artifact. Because the electrodes were very similar in impedance, the differential recording also effectively reduced the size of muscle potentials to insignificant levels. Discrimination was monitored continually by one experimenter throughout the course of the testing procedure to verify the reliability of discrimination. All recordings were from single neurons as judged by the consistency of spike height and waveform and the presence of an apparent refractory period. No tests were made of spikes that showed variable amplitudes, extremely fast baseline rates, or notches on the waveform that are characteristic of injured cells.
73 The output of the discriminator was directed to a ratemeter that reset once every second and was recorded on a polygraph along with written notes on the rat's behavior and stimulus conditions. Procedure Following 5 days of recovery from surgery, the rats were trained to selfstimulate and to drink 0.01 ~o saccharin solution when the ICSS bar or drinking tube were presented at either of the three chamber holes. Generally, two or three sessions were required to achieve reliable ICSS performance in which the rat would approach the onset bar with a short latency and terminate the stimulation train with an apparently purposeful behavior pattern which typically involved rearing and contacting the wall plate with the forepaws. Rats that did not have at least one electrode which supported such consistent ICSS performance were not tested further. Neurons with spontaneous or evoked activity were sought by manually advancing the recording electrode bundle in steps of approximately 65/~m and then monitoring the activity at each of two pairs of electrodes during selfstimulation. When the activity of a single neuron was discriminated, the testing sequence began with the determination of baseline activity rate during immobility. The rat was allowed to initiate at least four ICSS trains at a minimum of two chamber holes. Following the ICSS tests various stimuli were presented to the rat in no systematic order. Tactile stimulation was provided by probing the rat's dorsal and lateral body surface, face and vibrissae with a pencil. Olfactory stimulation was provided by presenting cotton swabs soaked in conspecific urine, rum or lemon flavoring which were waved in front of the rat's nostrils at a distance of about 2 cm. Visual stimulation was provided by moving a white wand with black spots (visual grid) in and out of the rat's field of vision. Auditory stimulation was provided by clicking a microswitch at a distance of about 0.5 m from the animal. This stimulus typically produced a startle response that was resistant to habituation. A positive motivational stimulus was provided by presenting a tube containing a 0.01 ~o saccharin solution at two or more of the chamber holes. The tube was moved to a different hole after 5-10 sec of drinking so that approach could be repeatedly elicited. Except the click, the stimuli all elicited orienting behavior on some presentations. Orienting behavior was defined as the pattern of head fixation and tracking that was usually accompanied by tactile contact by the snout and by sniffing of the object. Neural activity was also monitored during behaviors that were not evoked by the test stimuli. Examples of such behaviors were grooming, non-directed locomotion, and sniffing at chamber holes. Baseline activity determinations and the presentation of some stimuli were repeated as needed to confirm the correlations. The test procedure required between 30 and 60 min for each neuron and employed three experimenters, one to administer the test stimuli, one to monitor
74 the reliability of the discrimination, and one to record behaviors on the ratemeter record.
Histology At least 24 h after the final recording session the rat was anesthetized and perfused through the heart with 0.9~o saline followed by 10~o formalin. After fixation of the head with electrodes in situ for several days, the brain was removed and 32 #m transverse sections were cut with a cryostat microtome. The sections were stained with cresyl violet and viewed with a Bausch and Lomb projector. The track made by the single recording bundle in each rat was clearly marked through its entire length by gliosis. The path of the bundle was projected onto drawings adapted from the atlas of K0nig and Klippel [9]. The locations of recorded neurons were determined by reference to the deepest portion of the track and to the records made during movements of the electrode bundle. RESULTS
Location of neurons The locations of 69 single neurons that were isolated for a period long enough to characterize their correlates are shown in Fig. 1. All such neurons were found to lie in the deeper layers of cortex; electrode penetrations that passed through the more superficial layers of cortex were not successful in isolating the activity of single cells. In the figure, the numbers of cells tested within the AMC regions delineated by Krettek and Price [11] are shown at the various anterior-posterior levels. The regional distribution was: prelimbic region (PL), n = 40; dorsal anterior cingulate area (ACd), n -- 22; infralimbic region (IL),
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Fig. l. Distribution of single neurons recorded in various regions of cortex. Arrows indicate the approximate regional boundaries defined by Krettek and Price [11]. Brain drawings adapted from the K6nig and Klippel [9] atlas of the rat brain. Abbreviations: ACd, dorsal anterior cingulate area; IL, infralimbic area; MO, medial orbital area; PL, prelimbic area; PRCM, medial precentral area.
75
n = 3; and medial orbital area (MO), n = 4. Of these regions, the PL and ACd receive projections from the dorsomedial nucleus of the thalamus and only these 62 neurons are considered in these results. I C S S correlates
Rewarding brain stimulation in the medial forebrain bundle (MFB), the substantia nigra and the ventral tegmental area (VTA) of the midbrain (see Fig. 2) exerted widespread and potent effects on AMC neural activity. As shown in Table I, of the 60 cells tested, 43 showed clear activity changes within 1 sec of TABLE I
Activity patterns of neurons in the dorsal anterior cingulate and prelimbic regions of the anteromedial cortex during ICSS drinking and orienting An increase in the rate of neural activity is indicated by + ; a decrease is indicated by - . The two classes of orienting correlates are mutually exclusive.
ICSS Drink Orient, general Orient, restricted
Cells tested
Correlated activity
Total
60 52 45 45
11 8 5 10
43 24 8 14
32 16 3 4
TABLE II
Abbreviations used in ratemeter figures Acts D G I LO
Objects and events
OR
Drink Groom Immobile without active sniffing Locomote in chamber, no apparent goal Orient head to object
RR S
Rear Sniff
I 2 3 AUD
Hole 1 Hole 2 Hole 3 Auditory stimulation
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stimulation onset. The more common changes were decreases in activity which appeared in 32 of the 43 cases. A dot display of spike activity showing a consistent decrease at the onset of ICSS in the ipsilateral MFB and the ipsilateral VTA is shown in Fig. 3. Note that consistent increases in activity closely followed the offset of MFB stimulation but appeared earlier with VTA stimulation. Similar 'rebound' effects were found in 11 of the 32 neurons that showed decreases during ICS S trains. The ratemeter record of a neuron showing decreased activity during ICSS in the contralateral substantia nigra is shown in Fig. 4. Note that the increases in activity occurring after stimulation offset appeared with a latency of
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Fig. 3. The upper two panels show dot displays of activity of neuron 3-13 in the prelimbic region showing changes during eight trials of ICSS in the ipsilateral MFB and again in the ipsilateral VTA. Below each dot display is an event marker. The upward triangle indicates the point at which the ICSS onset manipulandum was contacted by the rat. This operant response was followed 0.5 sec later by the onset of the stimulation train, indicated by the upward deflection of the event marker. Each dot display continues to the right resuming at a point 600 msec before the animal's offset response which consisted of contacting a metal plate on the chamber wall, indicated by a downward triangle. Current flow terminated 0.5 sec after the animal's offset response, indicated by the downward deflection on the event marker. Lower tracings are traces of spontaneous activity at different sweep speeds.
a b o u t 5 sec a n d a p p a r e n t l y were n o t correlated with a n y particular behavior. It is also n o t a b l e that similar patterns were o b s e r v e d during a n d after drinking saccharin. I n c r e a s e d activity during the first 1 sec o f I C S S w a s f o u n d in 11 o f 60 A M C n e u r o n s . T h e s e p a t t e r n s were & t h r e e types. First a n d m o s t c o m m o n were diffuse increases that s h o w e d n o consistent latency. F o r example, the neural activity r e p r e s e n t e d in Fig. 5 increased to ipsilateral V T A stimulation (lines 1-3). Less c o n s i s t e n t increases were f o u n d with contralateral substantia nigral I C S S (line 4) a n d n o c o n s i s t e n t c h a n g e w a s a s s o c i a t e d with drinking (line 3). A s e c o n d type o f
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Fig. 4. Continuous ratemeter record of activity of neuron 31-3 in the ACd showing a tonic decrease in activity during ICSS and during drinking of a saccharin solution. On line 2 the heavy event marker labeled S S 1 indicates that ICS S was initiated at hole 1 and continued for the duration corresponding to the length of the marker. On lines 3 and 4, ICSS was initiated at hole 2. On lines 4-6, a tube containing a saccharin solution (SAC 2) was presented at hole 2 and the rat drank (D) for duration of the presentation. The activity of the neuron also decreased during orienting behavior directed to the majority of test stimuli which included the tactile probe, the visual grid, and a cotton swab soaked in rum flavoring. Baseline, 0.61 spikes/sec; calibrations, 3 spikes/sec, 10 sec.
increase during ICSS was found with two neurons which responded at consistent latencies of 11 and 19 msec to ICSS pulses in the ipsilateral VTA and contralateral substantia nigra. Since the responses were not reliable at 50 Hz, they appeared to be synaptically mediated. The activity of both of these neurons increased during orienting behavior but did not change during drinking. The third type of increased response in two neurons appeared to be antidromically mediated as they reliably followed pulse frequencies of 333 Hz and had latencies of 6.5 and 7.8 msec from ICSS pulses in the ipsilateral VTA. Both neurons with antidromic responses increased in activity during drinking but showed no consistent change during orienting behavior. Of the 30 neurons tested with two ICS S sites, the vast majority (n = 28) showed similar patterns during the first 1 sec of stimulation in the two sites. Increases in activity were seen in 6 neurons, decreases in 18 neurons and no change in 4 neurons. The only exceptions were the two neurons which showed
79 21-7
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Fig. 5. Continuous ratemeter record of activity of neuron 21-7 in the PL showing an increased rate of discharge during ICSS in the ipsilateral VTA initiated at either hole 2 (lines 1 and 2) or at hole 1 (line 3). ICSS in the contralateral S N R produced a less consistent increase in activity (line 4). The pattern of activity during saccharin drinking was too variable to classify as an increase ( S A C 1, line 3 ).
antidromic responses to ipsilateral VTA stimulation but no response to contralateral ICSS in the substantia nigra.
Drinking correlates As shown in Table I, the activity of 24 of 52 AMC neurons changed during drinking of a saccharin solution. The majority of these changes were decreases which accounted for 16 of the 24 cases with correlated activity. The ratemeter record shown in Fig. 4 (lines 4-6) illustrates a typical case.
Orienting correlates Orienting behavior proved to be an unexpected but significant correlate of the activity in about half of the A M C neurons tested (see Table I). As used here, orienting behavior refers to the pattern of head fixation and tracking which is usually accompanied by tactile contact by the snout and by sniffing of the object. There appeared to be two classes of orienting correlates, general and restricted. In those classed as general orienting (8 of 45 neurons) neural activity changed during the majority of orienting responses to the majority of stimuli (at least two). In those classed as restricted orienting (14 of 45 neurons) activity changed during the majority of orienting behaviors to certain stimuli but showed no reliable change
80
during orienting to the majority of other test stimuli. As seen in Table I, increases in activity (n = 15) were more common than decreases (n = 7) during orienting. An example of neural activity with a particularly clear general orienting correlate is shown in Fig. 6. This neuron showed little or no activity during immobility, ICSS or drinking but showed marked increases to visual and auditory stimuli which produced orienting and investigatory behaviors. Note in line 1 of Fig. 6 that repeated presentation of the visual grid (VG) was associated with a progressively smaller increase in activity. This decline was associated with a parallel decline in the magnitude of the orienting response, i.e., habituation.
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Fig. 6. Continuous ratemeter record of activity of neuron 21-3 in the ACd showing an increased rate of discharge during orienting behavior directed to the majority of test stimuli. On line 1, the visual grid (VG) was presented to the rat on five trials during which orienting behavior progressively declined. On line 4 the visual grid was presented again and orienting behavior accompanied by increased neural activity returned. A novel auditory stimulus (AUD) provided by the rattling of keys elicited orienting which also was associated with increased neural activity (lines 4 and 5). Activity was generally absent during ICSS in the ipsilateral VTA (SS1, line 2) and during drinking of saccharin (SAC 1, line 3). This cell also increased in activity during orienting to the tactile probe, the saccharin tube and the experimenter's hand. There was no evidence of any lateral specificity. Baseline, 0 spikes/sec; calibrations, 30 spikes/sec, 10 sec.
81 T A B L E III
Comparisons of neural activiO,patterns o f A M C neurons during ICSS with patterns during drinking and orienting behavior S o m e n e u r o n s (n = 30) were tested with two I C S S sites a n d each test was considered an i n d e p e n d e n t d a t u m . T h e orienting category includes both the general and restricted orienting correlate classifications.
Pattern during ICSS +
-
Pattern during 1CSS 0
+
-
0
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+
5
3
2
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11
8
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-
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23
1
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-
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18
15
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0
2
22
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Relations between ICSS, drinking and orienting correlates The direction of the change in neural activity during drinking predicted to a moderate degree a neuron's response to the onset of ICS S. Taking those neurons that showed some change, during both drinking and ICS S, a Z Z-test showed that the patterns were not independent (Z 2 = 7.23, df = 7, P < 0.01). Specifically, neurons that decreased in activity during drinking were highly likely (23 out of 26cases) to also decrease in activity during ICSS. However, neurons that decreased in activity to ICSS were about as likely to be unchanged during drinking as they were to decrease. An overall comparison of changes during the two conditions is shown on the left side of Table III. The direction of the change in neural activity during orienting was also a moderately effective predictor of the direction of change during ICSS. Again taking those neurons showing some change in both conditions, the activity patterns were found not to be independent (Z z = 7.04, df = 7, P < 0.01). The clearest trends were that activity that decreased during orienting also decreased during ICSS and activity that increased during ICSS also increased during drinking. The right side of Table III shows the comparisons.
Other correlates of AMC neural activity Few cells in the AMC had activity changes obviously correlated with simple motor or sensory aspects of behavior. Head movement correlates were found in 5 of 45 cells expressly tested for this correlate. Two cells showed an increase in activity during tactile stimulation which did not seem to be related to any motoric response. In one case activity changed during tactile stimulation of the contralateral hindlimb and in the other case to stimulation of the dorsal body surface.
~2 Twelve cells altered in activity during execution of the operant responses required for the initiation or termination of ICSS trains. Ten of these cells showed increases and the remaining two showed decreases e.g., Fig. 3. The majority of these neurons (9 of 12) had other correlates as well. In general, the changes associated with the operant responses were less obvious than the previously discussed correlates and required repetitive trials as in Fig. 3 to be detected. The activity of two cells had place correlates, i.e., activity increased whenever the animal was near the ICSS offset plate on the chamber wall. The increases did not appear to depend on the behavior of the animal but rather upon the animal's orientation to or presence at the plate. One of these cells showed other correlates, viz., decreases in activity during orienting to some test stimuli and increases during drinking. The activity of the other cell had no other detectable correlates. DISCUSSION
Rewarding brain stimulation exerted a variety of effects on the impulse pattern of AMC neurons in the behaving rat. The several patterns of response to ICS S included occasional short latency synaptic and antidromic discharges and more frequently responses, usually long term decreases, that had indeterminant latencies. Although the methods of this experiment were designed to allow a careful analysis of short latency events, the trend of the data was toward responses that were more diffuse and tonic. The prominence of such patterns is consistent with the results of a previous acute study [21] but appears to contrast with the findings of Ito and Olds [7] with awake rats and the findings of Rolls and Cooper [18] with anesthetized rats. In both of these studies stimulation was applied to the middle levels of the MFB which would be expected to activate the reciprocal monosynaptic paths connecting the AMC and the lateral hypothalamic area [8]. The majority of the stimulation sites in the present study were posterior to this region and this difference could account for the paucity of short latency responses in this study. The reason that the long term responses, particularly the decreases in activity, were not reported by Ito and Olds is not clear but their absence in the study by Rolls and Cooper [ 18] may have been due to the use of urethane anesthesia and single pulse stimulation. Neurons which showed either increases or decreases in activity during drinking were likely to show similar responses during ICSS. Because of their prevalence, the correspondence appeared to be stronger for mutual decreases, a finding which provides preliminary support for the possibility that pauses in the activity of certain AMC neurons might specifically correlate with the registration of rewarding events. However, several lines of evidence appear to indicate that increases rather than decreases in AMC activity are specific to reward. Direct stimulation of the AMC itself is rewarding [ 15, 19]. AMC neurons are synaptically
~3 and antidromically activated by ICSS in several midbrain and diencephalic sites [7, 18, 21]. Some AMC neurons in the present study as well as in a study by Niki and Watanabe [16] with monkeys increased during the presentation of a consummatory reward. At present both increases and decreases in AMC activity appear to be potentially important as motivationally specific patterns. It would seem that the eventual determination of the significance of these patterns requires a better understanding of the general behavioral role of the AMC. The behavioral role of the AMC, as reflected in the patterns of activity observed here, would seem to be at an integrative as opposed to an elemental level. There were few obVious signs of simple sensory or motor correlations nor were there indications of lateralized correlations that would be expected in cortical systems involved in such functions. The stimuli used were salient at least in terms of eliciting sensory responses in midbrain neurons in previous studies [ 10, 17]. Some correlations were seen between activity and discrete acts, particularly manipulation of the ICSS onset bar, but as a class they were weak and difficult to detect. Head movement correlates were easier to detect but neither were they particularly striking. In contrast, the molar behavior of orienting which is characterized by variable patterns of head fixation, tracking and investigation did relate in a clear way to changes in the activity of several neurons. Indicative of the strength and frequency of the orienting correlations is that they were so prominent in a study primarily designed to study the neural responses to motivational stimuli. In some cases the change in activity consistently occurred whenever the rat oriented and in other cases the change occurred during some orienting but not others. In either case, the best description of the correlate seemed to be in terms of the overall pattern of the behavior rather than its elements. Drinking correlates also seemed to reflect molar processes since tonic decreases in activity usually were seen against a low baseline rate. The increases in activity during drinking were also diffuse and apparently were not linked to the details of the consummatory pattern such as licking or chewing. In conclusion, the results of this experiment have provided basic information on the types of activity shown by AMC neurons to rewarding stimuli but at the same time they raise fundamental questions about the place of these activity patterns in the general function of the AMC. Particularly interesting is the relationship between reward-related activity and activity associated with orienting behaviors which appears to be an important correlate of increased activity in AMC neurons. ACKNOWLEDGEMENTS
This work was supported by a Wesleyan Project Grant to H.M.S.
84 REFERENCES 1 Barker, J. and Thomas, G.J., Effects of regional ablation of midline cortex on alternation learning by rats, Physiol. Behav., 1 (1966) 313-317. 2 Beckstead, R.M., Convergent thalamic and mesencephalic projections to the anterior medial cortex in the rat, J. comp. Neurol., 166 (1976) 403-416. 3 Corbett, D. and Wise, R.A., Intracranial self-stimulation in relation to the ascending dopaminergic systems of the midbrain: a moveable electrode mapping study, Brain Res., 185 (1980) 1-15. 4 Crow, T.J., A map of the rat mesencephalon for electrical self-stimulation, Brain Res., 36 (1972) 265-273. 5 Glass, D.H., Ison, J.R. and Thomas, G.R., Dysfunction in facilitation produced by prior classical conditioning in rats with lesions in midline cortex, J. comp. physiol. Psychol., 73 (1970) 385-391. 6 Gurowitz, E.M., Rosen, A.J. and Tessel, R.E., Incentive shift performance in cingulectomized rats, J. comp. physiol. Psychol., 70 (1970) 476-481. 7 Ito, M. and Olds, J., Unit activity during self-stimulation behavior, J. Neurophysiol., 34 (1971) 263-273. 8 Kita, H. and Oomura, Y., Reciprocal connections between the lateral hypothalamus and the frontal cortex in the rat: electrophysiological and anatomical observations, Brain Res., 213 (1981) 1-16. 9 K6nig, J. and Klippel, R., The Rat Brain: A Stereotaxic Atlas of the Forebrain and Lower Parts of the Brain Stem, Williams and Wilkins, Baltimore, MD, 1963. 10 Kootz, J.P. and Sinnamon, H.M., Neuronal activity in the midbrain reticular formation related to behavioral habituation and sensitization, Brain Res. Bull., 3 (1978) 533-539. 11 Krettek, J.E. and Price, J.L., The cortical projections of the mediodorsal nucleus and adjacent thalamic nuclei in the rat, J. comp. Neurol., 17 (1977) 157-192. 12 Leonard, C.M., The prefrontal cortex of the rat. I. Cortical projections of the mediodorsal nucleus. II. Efferent connections, Brain Res., 12 (1969) 321-343. 13 Lindvall, O., Dopamine pathways in the rat brain. In A.S. Horn, J. Korf, and B.H.K. Westernik (Eds.), The Neurology of Dopamine, Academic Press, New York, 1979. 14 McDonough, J.H., Jr. and Manning, F.J., The effects of lesions in amygdala or dorsomedial frontal cortex on reinforcement omission and noncontingent reinforcement in rats, Physiol. Ps)'chol.~ 7 (1979) 167-172. 15 Mora, F., The neurochemical substrates of prefrontal cortex self-stimulation: a review and an interpretation of some recent data, Life Sci., 22 (1978) 919-930. 16 Niki, H. and Watanabe, M., Prefrontal and cingulate unit activity during timing behavior in the monkey, Brain Res., 171 (1979) 213-224. 17 Pond, F.J., Sinnamon, H.M. and Adams, D.B., Single unit recording in the midbrain of rats during shock-elicited fighting behavior, Brain Res., 120 (1977) 469-484. 18 Rolls, E.T. and Cooper, S.J., Activation ofneurones in the prefrontal cortex by brain-stimulation reward in the rat, Brain Res., 60 (1973) 351-368. 19 Routtenberg, A., Forebrain pathways of reward in Rattus norvegicus, J. comp. physiol. Psychol., 75 (1971) 269-276. 20 Simon, H., Stinus, L., Tassin, J,P., Lavielle, L., Blanc, G., Thierry, A.-M., Glowinski, J. and LeMoal, M., Is the dopaminergic mesocorticolimbic system necessary for intracranial selfstimulation? Behav. Neural Biol., 27 (1979) 125-145. 21 Sinnamon, H.M., Cromarty, A.S. and Miller, C.A., Response of medial telencephalic neurons to stimulation in reinforcing sites in the medial forebrain bundle and ventral tegmental area, Physiol. Behav., 22 (1979) 555-562.
69
Elsevier Biomedical Press
ACTIVITY OF N E U R O N S IN T H E A N T E R O M E D I A L C O R T E X D U R I N G R E W A R D I N G BRAIN S T I M U L A T I O N , S A C C H A R I N C O N S U M P T I O N A N D O R I E N T I N G BEHAVIOR
J O H N P. KANKI, TERRY L. M A R T I N and HARRY M. S I N N A M O N *
Laboratory of Neuropsychology, Wesleyan University, Middletown, CT06457 (U.S.A.) (Received February 22nd, 1982) (Revised version received September 28th, 1982) (Accepted November 26th, 1982)
Key words: anteromedial cortex - reward - brain stimulation - saccharin consumption - orienting behavior - rat
SUMMARY
Single neuronal activity in the deep layers of the prelimbic and dorsal anterior cingulate regions of the anteromedial cortex (AMC) was recorded through chronically implanted fine wires (25 #m diameter) during reward-related behavior in five rats. Tests were conducted in a transparent plastic cylindrical chamber (28 cm in diameter) in which the water-deprived rats initiated and terminated trains of 50 Hz rewarding electrical stimulation to the ventral tegmental area or the medial forebrain bundle and approached and consumed a 0.01~o saccharin solution. The rats were also presented with non-rewarding visual, olfactory and tactile stimuli which did not elicit approach but did elicit orienting behavior, i.e., head fixation in the direction of the stimulus, tracking head movements or tactile contact with the snout accompanied by sniffing. Of 60 neurons tested during intracranial self-stimulation (ICS S), 43 showed consistent activity changes within 1 sec of onset. Decreases in activity were most common (32 of 43) but increases that appeared to be synaptically mediated (n --- 9) or antidromically mediated (n -- 2) were also observed. Neurons tested with two ICSS sites were likely to show the same response (28 of 30). Of 52 neurons recorded when the rat drank saccharin, 24 changed in activity with 16 showing decreases. Of 45 neurons tested during orienting behavior to the non-rewarding stimuli, 15 increased and 7
* To whom correspondence and requests for reprints should be addressed. 0166-4328/83/0000-0000/$03.00 © 1983 Elsevier Science Publishers
70 decreased in activity. Comparisons were made of activity during ICSS to that during drinking and orienting behavior. Neurons that decreased during drinking were likely to also decrease during ICS S. Neurons that decreased during orienting were likely to decrease also during ICS S and neurons that increased during ICS S were likely to increase during orienting. These results indicate that decreases in activity of A M C neurons may be significant in the registration of rewarding states and that orienting behavior appears to be an important correlate to increased activity in these neurons.
1N T R O D U C T I O N
The anteromedial cortex (AMC) of the rat consists of regions which are primary targets of the dorsomedial nucleus of the thalamus [2, 11, 12]. According to the cytoarchitectonic boundaries delineated by Krettek and Price [11 ], the AMC includes the anterior cingulate cortex, both the dorsal (ACd) and ventral (ACv) parts, the prelimbic area (PL) and the medial precentral region (PRCM). These regions together with the dorsal perirhinal cortex which also receives projections from the dorsomedial nucleus [22, 24] have been proposed to be the homolog of the primate prefrontal cortex [ 12]. S everal lines of evidence indicate that the AMC has a particularly important role in motivation. It is one of the few cortical regions that support ICSS [ 15, 19]. Neurons in the region respond antidromically and synaptically at short latency to pulses of ICS S current [7, 18, 21 ]. Longer duration responses, both increases and decreases in activity, with relatively long and variable latencies have also been described in anesthetized rats [21]. The AMC receives projections from the dopaminergic system that has cell bodies in the ventral tegmental area of the midbrain and the substantia nigra [13]. These regions also support ICSS [3, 4], although it is not clear that the dopaminergic fibers are essential [20]. Lesions of the AMC produce a variety of effects which are complex but at least indicate possible dysfunctions in reward processes. For example, Glass et al. [5] showed that rats with AMC lesions failed to show the normal facilitatory effects that prior feeding in a goal box has on learning to approach that goal box in a runway situation. Gurowitz et al. [6] showed that the running speed of rats with AMC lesions was less sensitive than that of controls to a reduction in the magnitude of reward. When reward is omitted on alternate trials in a runway, rats with AMC lesions fail to show the normal pattern of fast and slow running [ 1]. In a barpressing situation on a fixed interval schedule, they respond more than controls to omitted reward [ 14]. This experiment addressed several questions. First, it was important to determine the type of activity changes associated with reward in the freely behaving rat. Ito and Olds' [7] early chronic study reported short latency
71 responses but not the long term responses described in acute studies [21]. Another important question was the relationship between the response to ICSS and the activity patterns during more conventional rewards. To this end, neural activity during consumption of sweetened water was compared to that during ICSS. As a first step in characterizing the sensory properties of neurons responsive to ICSS, a range of stimuli was presented to the animal. These stimuli were not motivationally potent but did generally elicit orienting responses and provided a second class of correlates for comparison to the ICSS correlates. METHODS
Subjects and surgery The rats (n = 5, Fischer-Dark Agouti) were caged individually with free access to food but not to water. They were anesthetized and mounted in a stereotaxic device. Stainless steel screws were inserted in the skull and an uninsulated wire terminating in an Amphenol pin was attached to serve as a ground connection. The recording electrode bundle was mounted in a drive assembly (see below) which was secured to the skull with dental cement. The bundle passed through a l-ram hole drilled in the skull over the AMC and penetrated the brain to a depth of approximately 1 ram. For brain stimulation, two bipolar stimulation electrodes were aimed for the medial forebrain bundle (MFB) and the ventral tegmental area of the midbrain (VTA) ipsilateral to the recording bundle or to the VTA bilaterally. The electrodes were formed by twisting two 125-pro stainless steel wires, insulated with Teflon except at the cross-section of the tips. Amphenol pins connected to the electrodes were inserted into an Amphenol connector strip along with the ground pin. The entire assembly composed of the recording drive and the connector strip was secured to the skull with dental cement.
Apparatus Recordings were made with the rat in a transparent Plexiglas cylinder which was 28 cm in diameter and 36 cm high. In the wall of the chamber were three 1.5 cm diameter holes which were spaced 90 ° apart and 2 cm above the floor through which the stimulation onset rod was inserted. On the inner wall of the chamber opposite the middle hole, a 15 x 16 cm copper plate was located 1.5 cm from the floor. Contact with the plate by the rat caused offset of the brain stimulation. A cable of Microdot wires carrying recording and stimulation lines connected the rat to a commutator. Constant-current stimulation was provided by 0.3 msec square wave pulses at 50 Hz for a train duration determined by the rat. The current levels effective for maintaining consistent ICSS varied between sites from 70 to 600 #A. The rat initiated the ICSS train by completing a low current circuit between ground and a 2-mm rod which protruded 2 cm into the chamber through any one of three
72 chamber holes. A 0.5-sec delay was imposed between the initial contact with the rod and the onset of stimulation; the delay allowed the temporal separation of a neural activity change associated with the operant response from one associated with the onset of ICS S. The rat terminated the ICS S train by contacting the metal plate on the wall of the chamber. The contact was detected by a low current circuit and again a 0.5-sec delay was imposed between the operant response and the actual offset of the train.
Recording technique Extracellular activity was differentially recorded across selected pairs of the four wires in the electrode bundle. The electrode bundle was composed of four 25-#m diameter, Teflon-coated, stainless steel wires which had been twisted together. The bundle was fixed into a longitudinal slot of a brass guide screw (size 6/32). The end of the bundle extended 10 mm beyond the bottom and was cut transversely with scissors to expose the recording surface. The guide screw and attached electrode bundle was inserted into a custom made drive assembly which allowed downward movement of the bundle without rotation. The drive consisted of a threaded Delrin spur gear, a Delrin sleeve which fit over the hub of the spur gear, and a metal key sized to fit snugly into the slot of the guide screw. One revolution of the spur gear provided 780 #m of downward travel by the electrode bundle. A short four-wire cable connected pins connected to the bundle to a miniature, differential, DC-coupled, 10-gain, preamplifier (F. Haer Co.) mounted near the base of the cable. A manual switch allowed for alternation between pairs of electrodes for differential recording. The output of the preamplifier was sent to a band pass filter which passed frequencies between 200 Hz and 10 kHz. The filtered electrical activity was led to an oscilloscope, an audio amplifier, and a voltage and fall time discriminator to produce standard 5-V pulses when spikes of critical amplitude and duration occurred. To avoid the inclusion of stimulation artifact or evoked field responses in the counts of neuronal spikes, the output of the window discriminator was suppressed for a selectable period (1-4 msec) after the onset of each stimulation pulse. This circuitry and the use of differential recording effectively eliminated the difficulties associated with stimulus artifact. Because the electrodes were very similar in impedance, the differential recording also effectively reduced the size of muscle potentials to insignificant levels. Discrimination was monitored continually by one experimenter throughout the course of the testing procedure to verify the reliability of discrimination. All recordings were from single neurons as judged by the consistency of spike height and waveform and the presence of an apparent refractory period. No tests were made of spikes that showed variable amplitudes, extremely fast baseline rates, or notches on the waveform that are characteristic of injured cells.
73 The output of the discriminator was directed to a ratemeter that reset once every second and was recorded on a polygraph along with written notes on the rat's behavior and stimulus conditions. Procedure Following 5 days of recovery from surgery, the rats were trained to selfstimulate and to drink 0.01 ~o saccharin solution when the ICSS bar or drinking tube were presented at either of the three chamber holes. Generally, two or three sessions were required to achieve reliable ICSS performance in which the rat would approach the onset bar with a short latency and terminate the stimulation train with an apparently purposeful behavior pattern which typically involved rearing and contacting the wall plate with the forepaws. Rats that did not have at least one electrode which supported such consistent ICSS performance were not tested further. Neurons with spontaneous or evoked activity were sought by manually advancing the recording electrode bundle in steps of approximately 65/~m and then monitoring the activity at each of two pairs of electrodes during selfstimulation. When the activity of a single neuron was discriminated, the testing sequence began with the determination of baseline activity rate during immobility. The rat was allowed to initiate at least four ICSS trains at a minimum of two chamber holes. Following the ICSS tests various stimuli were presented to the rat in no systematic order. Tactile stimulation was provided by probing the rat's dorsal and lateral body surface, face and vibrissae with a pencil. Olfactory stimulation was provided by presenting cotton swabs soaked in conspecific urine, rum or lemon flavoring which were waved in front of the rat's nostrils at a distance of about 2 cm. Visual stimulation was provided by moving a white wand with black spots (visual grid) in and out of the rat's field of vision. Auditory stimulation was provided by clicking a microswitch at a distance of about 0.5 m from the animal. This stimulus typically produced a startle response that was resistant to habituation. A positive motivational stimulus was provided by presenting a tube containing a 0.01 ~o saccharin solution at two or more of the chamber holes. The tube was moved to a different hole after 5-10 sec of drinking so that approach could be repeatedly elicited. Except the click, the stimuli all elicited orienting behavior on some presentations. Orienting behavior was defined as the pattern of head fixation and tracking that was usually accompanied by tactile contact by the snout and by sniffing of the object. Neural activity was also monitored during behaviors that were not evoked by the test stimuli. Examples of such behaviors were grooming, non-directed locomotion, and sniffing at chamber holes. Baseline activity determinations and the presentation of some stimuli were repeated as needed to confirm the correlations. The test procedure required between 30 and 60 min for each neuron and employed three experimenters, one to administer the test stimuli, one to monitor
74 the reliability of the discrimination, and one to record behaviors on the ratemeter record.
Histology At least 24 h after the final recording session the rat was anesthetized and perfused through the heart with 0.9~o saline followed by 10~o formalin. After fixation of the head with electrodes in situ for several days, the brain was removed and 32 #m transverse sections were cut with a cryostat microtome. The sections were stained with cresyl violet and viewed with a Bausch and Lomb projector. The track made by the single recording bundle in each rat was clearly marked through its entire length by gliosis. The path of the bundle was projected onto drawings adapted from the atlas of K0nig and Klippel [9]. The locations of recorded neurons were determined by reference to the deepest portion of the track and to the records made during movements of the electrode bundle. RESULTS
Location of neurons The locations of 69 single neurons that were isolated for a period long enough to characterize their correlates are shown in Fig. 1. All such neurons were found to lie in the deeper layers of cortex; electrode penetrations that passed through the more superficial layers of cortex were not successful in isolating the activity of single cells. In the figure, the numbers of cells tested within the AMC regions delineated by Krettek and Price [11] are shown at the various anterior-posterior levels. The regional distribution was: prelimbic region (PL), n = 40; dorsal anterior cingulate area (ACd), n -- 22; infralimbic region (IL),
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Fig. l. Distribution of single neurons recorded in various regions of cortex. Arrows indicate the approximate regional boundaries defined by Krettek and Price [11]. Brain drawings adapted from the K6nig and Klippel [9] atlas of the rat brain. Abbreviations: ACd, dorsal anterior cingulate area; IL, infralimbic area; MO, medial orbital area; PL, prelimbic area; PRCM, medial precentral area.
75
n = 3; and medial orbital area (MO), n = 4. Of these regions, the PL and ACd receive projections from the dorsomedial nucleus of the thalamus and only these 62 neurons are considered in these results. I C S S correlates
Rewarding brain stimulation in the medial forebrain bundle (MFB), the substantia nigra and the ventral tegmental area (VTA) of the midbrain (see Fig. 2) exerted widespread and potent effects on AMC neural activity. As shown in Table I, of the 60 cells tested, 43 showed clear activity changes within 1 sec of TABLE I
Activity patterns of neurons in the dorsal anterior cingulate and prelimbic regions of the anteromedial cortex during ICSS drinking and orienting An increase in the rate of neural activity is indicated by + ; a decrease is indicated by - . The two classes of orienting correlates are mutually exclusive.
ICSS Drink Orient, general Orient, restricted
Cells tested
Correlated activity
Total
60 52 45 45
11 8 5 10
43 24 8 14
32 16 3 4
TABLE II
Abbreviations used in ratemeter figures Acts D G I LO
Objects and events
OR
Drink Groom Immobile without active sniffing Locomote in chamber, no apparent goal Orient head to object
RR S
Rear Sniff
I 2 3 AUD
Hole 1 Hole 2 Hole 3 Auditory stimulation
SAC ( ) Saccharin tube presented hole ( ) SS ICSS initiated at hole ( ) ( ) (I) ipsilateral site (C) contralateral site VG Visual grid presented
at
76
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stimulation onset. The more common changes were decreases in activity which appeared in 32 of the 43 cases. A dot display of spike activity showing a consistent decrease at the onset of ICSS in the ipsilateral MFB and the ipsilateral VTA is shown in Fig. 3. Note that consistent increases in activity closely followed the offset of MFB stimulation but appeared earlier with VTA stimulation. Similar 'rebound' effects were found in 11 of the 32 neurons that showed decreases during ICS S trains. The ratemeter record of a neuron showing decreased activity during ICSS in the contralateral substantia nigra is shown in Fig. 4. Note that the increases in activity occurring after stimulation offset appeared with a latency of
77
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Fig. 3. The upper two panels show dot displays of activity of neuron 3-13 in the prelimbic region showing changes during eight trials of ICSS in the ipsilateral MFB and again in the ipsilateral VTA. Below each dot display is an event marker. The upward triangle indicates the point at which the ICSS onset manipulandum was contacted by the rat. This operant response was followed 0.5 sec later by the onset of the stimulation train, indicated by the upward deflection of the event marker. Each dot display continues to the right resuming at a point 600 msec before the animal's offset response which consisted of contacting a metal plate on the chamber wall, indicated by a downward triangle. Current flow terminated 0.5 sec after the animal's offset response, indicated by the downward deflection on the event marker. Lower tracings are traces of spontaneous activity at different sweep speeds.
a b o u t 5 sec a n d a p p a r e n t l y were n o t correlated with a n y particular behavior. It is also n o t a b l e that similar patterns were o b s e r v e d during a n d after drinking saccharin. I n c r e a s e d activity during the first 1 sec o f I C S S w a s f o u n d in 11 o f 60 A M C n e u r o n s . T h e s e p a t t e r n s were & t h r e e types. First a n d m o s t c o m m o n were diffuse increases that s h o w e d n o consistent latency. F o r example, the neural activity r e p r e s e n t e d in Fig. 5 increased to ipsilateral V T A stimulation (lines 1-3). Less c o n s i s t e n t increases were f o u n d with contralateral substantia nigral I C S S (line 4) a n d n o c o n s i s t e n t c h a n g e w a s a s s o c i a t e d with drinking (line 3). A s e c o n d type o f
78 31-3
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Fig. 4. Continuous ratemeter record of activity of neuron 31-3 in the ACd showing a tonic decrease in activity during ICSS and during drinking of a saccharin solution. On line 2 the heavy event marker labeled S S 1 indicates that ICS S was initiated at hole 1 and continued for the duration corresponding to the length of the marker. On lines 3 and 4, ICSS was initiated at hole 2. On lines 4-6, a tube containing a saccharin solution (SAC 2) was presented at hole 2 and the rat drank (D) for duration of the presentation. The activity of the neuron also decreased during orienting behavior directed to the majority of test stimuli which included the tactile probe, the visual grid, and a cotton swab soaked in rum flavoring. Baseline, 0.61 spikes/sec; calibrations, 3 spikes/sec, 10 sec.
increase during ICSS was found with two neurons which responded at consistent latencies of 11 and 19 msec to ICSS pulses in the ipsilateral VTA and contralateral substantia nigra. Since the responses were not reliable at 50 Hz, they appeared to be synaptically mediated. The activity of both of these neurons increased during orienting behavior but did not change during drinking. The third type of increased response in two neurons appeared to be antidromically mediated as they reliably followed pulse frequencies of 333 Hz and had latencies of 6.5 and 7.8 msec from ICSS pulses in the ipsilateral VTA. Both neurons with antidromic responses increased in activity during drinking but showed no consistent change during orienting behavior. Of the 30 neurons tested with two ICS S sites, the vast majority (n = 28) showed similar patterns during the first 1 sec of stimulation in the two sites. Increases in activity were seen in 6 neurons, decreases in 18 neurons and no change in 4 neurons. The only exceptions were the two neurons which showed
79 21-7
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Fig. 5. Continuous ratemeter record of activity of neuron 21-7 in the PL showing an increased rate of discharge during ICSS in the ipsilateral VTA initiated at either hole 2 (lines 1 and 2) or at hole 1 (line 3). ICSS in the contralateral S N R produced a less consistent increase in activity (line 4). The pattern of activity during saccharin drinking was too variable to classify as an increase ( S A C 1, line 3 ).
antidromic responses to ipsilateral VTA stimulation but no response to contralateral ICSS in the substantia nigra.
Drinking correlates As shown in Table I, the activity of 24 of 52 AMC neurons changed during drinking of a saccharin solution. The majority of these changes were decreases which accounted for 16 of the 24 cases with correlated activity. The ratemeter record shown in Fig. 4 (lines 4-6) illustrates a typical case.
Orienting correlates Orienting behavior proved to be an unexpected but significant correlate of the activity in about half of the A M C neurons tested (see Table I). As used here, orienting behavior refers to the pattern of head fixation and tracking which is usually accompanied by tactile contact by the snout and by sniffing of the object. There appeared to be two classes of orienting correlates, general and restricted. In those classed as general orienting (8 of 45 neurons) neural activity changed during the majority of orienting responses to the majority of stimuli (at least two). In those classed as restricted orienting (14 of 45 neurons) activity changed during the majority of orienting behaviors to certain stimuli but showed no reliable change
80
during orienting to the majority of other test stimuli. As seen in Table I, increases in activity (n = 15) were more common than decreases (n = 7) during orienting. An example of neural activity with a particularly clear general orienting correlate is shown in Fig. 6. This neuron showed little or no activity during immobility, ICSS or drinking but showed marked increases to visual and auditory stimuli which produced orienting and investigatory behaviors. Note in line 1 of Fig. 6 that repeated presentation of the visual grid (VG) was associated with a progressively smaller increase in activity. This decline was associated with a parallel decline in the magnitude of the orienting response, i.e., habituation.
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Fig. 6. Continuous ratemeter record of activity of neuron 21-3 in the ACd showing an increased rate of discharge during orienting behavior directed to the majority of test stimuli. On line 1, the visual grid (VG) was presented to the rat on five trials during which orienting behavior progressively declined. On line 4 the visual grid was presented again and orienting behavior accompanied by increased neural activity returned. A novel auditory stimulus (AUD) provided by the rattling of keys elicited orienting which also was associated with increased neural activity (lines 4 and 5). Activity was generally absent during ICSS in the ipsilateral VTA (SS1, line 2) and during drinking of saccharin (SAC 1, line 3). This cell also increased in activity during orienting to the tactile probe, the saccharin tube and the experimenter's hand. There was no evidence of any lateral specificity. Baseline, 0 spikes/sec; calibrations, 30 spikes/sec, 10 sec.
81 T A B L E III
Comparisons of neural activiO,patterns o f A M C neurons during ICSS with patterns during drinking and orienting behavior S o m e n e u r o n s (n = 30) were tested with two I C S S sites a n d each test was considered an i n d e p e n d e n t d a t u m . T h e orienting category includes both the general and restricted orienting correlate classifications.
Pattern during ICSS +
-
Pattern during 1CSS 0
+
-
0
Pattern
+
5
3
2
Pattern
+
11
8
5
During
-
2
23
1
During
-
0
10
0
Drinking
0
5
18
15
Orienting
0
2
22
8
Relations between ICSS, drinking and orienting correlates The direction of the change in neural activity during drinking predicted to a moderate degree a neuron's response to the onset of ICS S. Taking those neurons that showed some change, during both drinking and ICS S, a Z Z-test showed that the patterns were not independent (Z 2 = 7.23, df = 7, P < 0.01). Specifically, neurons that decreased in activity during drinking were highly likely (23 out of 26cases) to also decrease in activity during ICSS. However, neurons that decreased in activity to ICSS were about as likely to be unchanged during drinking as they were to decrease. An overall comparison of changes during the two conditions is shown on the left side of Table III. The direction of the change in neural activity during orienting was also a moderately effective predictor of the direction of change during ICSS. Again taking those neurons showing some change in both conditions, the activity patterns were found not to be independent (Z z = 7.04, df = 7, P < 0.01). The clearest trends were that activity that decreased during orienting also decreased during ICSS and activity that increased during ICSS also increased during drinking. The right side of Table III shows the comparisons.
Other correlates of AMC neural activity Few cells in the AMC had activity changes obviously correlated with simple motor or sensory aspects of behavior. Head movement correlates were found in 5 of 45 cells expressly tested for this correlate. Two cells showed an increase in activity during tactile stimulation which did not seem to be related to any motoric response. In one case activity changed during tactile stimulation of the contralateral hindlimb and in the other case to stimulation of the dorsal body surface.
~2 Twelve cells altered in activity during execution of the operant responses required for the initiation or termination of ICSS trains. Ten of these cells showed increases and the remaining two showed decreases e.g., Fig. 3. The majority of these neurons (9 of 12) had other correlates as well. In general, the changes associated with the operant responses were less obvious than the previously discussed correlates and required repetitive trials as in Fig. 3 to be detected. The activity of two cells had place correlates, i.e., activity increased whenever the animal was near the ICSS offset plate on the chamber wall. The increases did not appear to depend on the behavior of the animal but rather upon the animal's orientation to or presence at the plate. One of these cells showed other correlates, viz., decreases in activity during orienting to some test stimuli and increases during drinking. The activity of the other cell had no other detectable correlates. DISCUSSION
Rewarding brain stimulation exerted a variety of effects on the impulse pattern of AMC neurons in the behaving rat. The several patterns of response to ICS S included occasional short latency synaptic and antidromic discharges and more frequently responses, usually long term decreases, that had indeterminant latencies. Although the methods of this experiment were designed to allow a careful analysis of short latency events, the trend of the data was toward responses that were more diffuse and tonic. The prominence of such patterns is consistent with the results of a previous acute study [21] but appears to contrast with the findings of Ito and Olds [7] with awake rats and the findings of Rolls and Cooper [18] with anesthetized rats. In both of these studies stimulation was applied to the middle levels of the MFB which would be expected to activate the reciprocal monosynaptic paths connecting the AMC and the lateral hypothalamic area [8]. The majority of the stimulation sites in the present study were posterior to this region and this difference could account for the paucity of short latency responses in this study. The reason that the long term responses, particularly the decreases in activity, were not reported by Ito and Olds is not clear but their absence in the study by Rolls and Cooper [ 18] may have been due to the use of urethane anesthesia and single pulse stimulation. Neurons which showed either increases or decreases in activity during drinking were likely to show similar responses during ICSS. Because of their prevalence, the correspondence appeared to be stronger for mutual decreases, a finding which provides preliminary support for the possibility that pauses in the activity of certain AMC neurons might specifically correlate with the registration of rewarding events. However, several lines of evidence appear to indicate that increases rather than decreases in AMC activity are specific to reward. Direct stimulation of the AMC itself is rewarding [ 15, 19]. AMC neurons are synaptically
~3 and antidromically activated by ICSS in several midbrain and diencephalic sites [7, 18, 21]. Some AMC neurons in the present study as well as in a study by Niki and Watanabe [16] with monkeys increased during the presentation of a consummatory reward. At present both increases and decreases in AMC activity appear to be potentially important as motivationally specific patterns. It would seem that the eventual determination of the significance of these patterns requires a better understanding of the general behavioral role of the AMC. The behavioral role of the AMC, as reflected in the patterns of activity observed here, would seem to be at an integrative as opposed to an elemental level. There were few obVious signs of simple sensory or motor correlations nor were there indications of lateralized correlations that would be expected in cortical systems involved in such functions. The stimuli used were salient at least in terms of eliciting sensory responses in midbrain neurons in previous studies [ 10, 17]. Some correlations were seen between activity and discrete acts, particularly manipulation of the ICSS onset bar, but as a class they were weak and difficult to detect. Head movement correlates were easier to detect but neither were they particularly striking. In contrast, the molar behavior of orienting which is characterized by variable patterns of head fixation, tracking and investigation did relate in a clear way to changes in the activity of several neurons. Indicative of the strength and frequency of the orienting correlations is that they were so prominent in a study primarily designed to study the neural responses to motivational stimuli. In some cases the change in activity consistently occurred whenever the rat oriented and in other cases the change occurred during some orienting but not others. In either case, the best description of the correlate seemed to be in terms of the overall pattern of the behavior rather than its elements. Drinking correlates also seemed to reflect molar processes since tonic decreases in activity usually were seen against a low baseline rate. The increases in activity during drinking were also diffuse and apparently were not linked to the details of the consummatory pattern such as licking or chewing. In conclusion, the results of this experiment have provided basic information on the types of activity shown by AMC neurons to rewarding stimuli but at the same time they raise fundamental questions about the place of these activity patterns in the general function of the AMC. Particularly interesting is the relationship between reward-related activity and activity associated with orienting behaviors which appears to be an important correlate of increased activity in AMC neurons. ACKNOWLEDGEMENTS
This work was supported by a Wesleyan Project Grant to H.M.S.
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