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The effects of procaine injections into MFB-LHA during self-stimulation of septum and barpress for food… Madryga, Frederick John 1970

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THE EFFECTS OF PROCAINE INJECTIONS INTO MFB-LE. DURING SELF-STIMULATION OF SEPTUM AND BARPRESS FOR FOOD IN THE RAT ty Frederick John Madryga B.A., University of British Columbia A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF ARTS in the Department of Psychology We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA September, 1S70 In presenting th i s thes is in pa r t i a l fu l f i lment o f the requirements for an advanced degree at the Univers i ty of B r i t i s h Co 1umbia, I agree that the L ibrary shal l make it f ree ly ava i lab le for reference and study. I fur ther agree that permission for extensive copying of th i s thes is for scho la r l y purposes may be granted by the Head of my Department or by his representat ives . It is understood that copying or pub l i ca t ion of th is thes is fo r f inanc ia l gain sha l l not be allowed without my wr i t ten permission. Department of P s y c h o l o g y The Univers i ty of B r i t i s h Columbia Vancouver 8, Canada •Sfipt.ftTnhftr 1970 ABSTRACT The possibility that regions of the medial forebrain bundle-lateral hypothalamic area (MFB-LHA) are concerned with mediating the rewarding effect of electrical stimulation of the brain was examined by making unilateral injections of 2% procaine hydrochloride (2 ul) into this region during bar pressing for elec t r i c a l stimulation in the septum or preoptic area. When the injection was made into the same side of the brain as the stim-ulating electrode, there was a strong suppression of self-stim-ulating rates as compared to the effect of isotonic saline injections, and the effect occurred with injections anywhere along the entire length of the MFH-LHA. The finding that the suppression was significantly less severe when the injections were made into the MFB-LHA on the side of the brain opposite to the electrode suggests that the suppression was not due to general disruptive effects. As a further control for nonspecific disturbances, similar injections were made in animals bar-pressing for food reward; the results showed that these injections suppressed bar-pressing for food but only when the injections were made into those regions of the MFB-LHA which have been shown to be involved in feeding behavior. These results suggest that the entire MFB-LHA is a part of a system mediating the rewarding effect of ele c t r i c a l stimulation of the septum and preoptic area. i i TABLE OF CONTENTS Page Table of Contents . . . . . . . . . . . . . . . . . . . . . i i Lis t of Figures . . . . . . . . . . . . . . . . . . . . . . iv Acknowledgment ., . . . . . . v Introduction . . . . . . . . . . o . . . . . . . 1 Method 4 Subjects, Surgery, Histology, Apparatus Training and Testing Procedure Scoring Procedure and Statistical Analysis Results Effect of Procaine. Injections on Self-Stimulation Location and Strength of Procaine Effects Variation in Electrode Location and the Effect of Procaine The Effect of Variation in- Damage at the Injection Site Pellet 2" ( . • • • a * o a o e o e * o c * o * * ' l « " * i ' * Effect of Procaine Injections on Lever Press for Food The Effects of Deprivation, Reinforcement Schedule and Results of Histology Discussion . . . . . . . . , . . . . . . . . . . . . . . . . . 28 Appendix Table I; Electrode Location, Intensity of Stimulation, Baseline Rate of Responding and Magnitude of Effects of Procaine Injections in Animals Pressing Lever for Electrical Stimulation . . . . . . . . . . . 34 Table II: Means and Standard Seviations for Procaine„ Saline and Extinction Treatments over four 5 Minute Time Periods during Responding for Electrical Stimulation of the Brain . . . . . . 35 i i i TABLE OF CONTENTS (continued) Table III: Summary of Analysis of Variance of Scores for Procaine, Saline and Extinction Conditions during Responding for Electrical Stimulation „ . . 36 Table IV: Simple Main Effects Analysis of Variance of Scores for Procaine, Saline and Extinction Treatments Ipsilateral and Contralateral to the Stimulating Electrode . . . 37 Table V: Simple Main Effects Analysis of Variance of Scores for Injection Sites Ipsilateral and Contralateral to the Stimulating Electrode during 5 Minute Time Periods . 38 Table VI: Simple Main Effects Analysis of Variance of Scores for Procaine, Saline and Extinction Treatments over 5 Minute Time Periods during Responding for Electrical Stimulation . . 39 Table VII: Location of Electrode, Baseline Rate of Responding and Magnitude of Effects of Procaine Injections during Responding for Food Reward 40 Table VIII: Means and Standard Deviations for Procaine, Saline and Extinction Treatments over four 5 Minute. Tim-Periods during Responding for Food Reward 41 Table IX: Summary of Analysis of Variance of Scores for Procaine, Saline and Extinction Conditions during Responding for Food Reward . . . 42 Table X: Simple Main Effects Analysis of Variance of Scores for Procaine, Saline and Extinction Treatments over 5 Minute Time Periods during Responding for Food Reward „ . . 43 i v LIST OF FIGURES Page Figure 1. The effect of stopping the stimulating current (extinction) s injecting procaine, or injecting isotonic saline into the MFB-LHA Ipsilateral or contralateral to the stimulating electrode on the rate of bar-pressing for electrical stimulation . H Figure 2. The distribution of points along the. MFB-LHA where the effect of procaine injections on ele c t r i c a l stimulation was examined 15 Figure 3. Individual records from two animals showing the effect of the various treatment conditions on the bar press rate for electrical stimulation along with a histological section through the injection site 17 Figure 4. The effect of stopping food reinforcement (extinction), injecting procaine, or injecting isotonic saline into the MFB-IiiA on the rate of bar-pressing for food re-inforcement ' 22 Figure 5. The distribution of points along the MFB-LHA where the effect of procaine injections on rate of bar-pressing for food reinforcement was examined 25 V ACKNOWLEDGMENT The author would like to particularly acknowledge the advice, encouragement and financial support of Dr. D.J. Albert throughout a l l phases of this research. Thanks are also due to Dr. Roderick Wong who read and c r i t i c a l l y evaluated the writing of the thesis as a member of the Thesis Committee and my wife Patricia Madryga for her emotional support and help in preparing the manuscript. The medial forebrain bundle-lateral hypothalamic area (MFB-LHA) i s generally regarded as an important part of the system that mediates the rewarding effect of e l e c t r i c a l stimulation of the brain. This conclusion i s supported by several lines of indirect evidence. The di s t r i b u t i o n of MFB-LHA connections strongly p a r a l l e l s the d i s t r i b u t i o n of l o c i exhibiting the self-stimulation phenomenon (Guillery, 1957°, Nauta, 1958; Nauta, 1960; Olds, 1956: Olds and Olds, 1963; Olds, Travis and Schwing, 1960). Self-stimulation rates i n the MFB-LHA are high, occur at low i n t e n s i t i e s , and are generally resistant to sat i a t i o n (Olds, 1958a, Olds, 1958b) . Further, many experiments using a variety of techniques have implicated the MFB-LHA i n such behaviors as eating, drinking, sex, aggression and r e a c t i v i t y to e l e c t r i c shock (Grossman, 1967;, Harvey and L i n t s , 1965), Although the above results strongly suggest an important role for. the MFB-LHA i n motivational processes, direct attempts to demonstrate that t h i s region is- essential for the self-stimulation effect have, produced contradictory r e s u l t s . The strongest evidence against the MFB-LHA as being c r i t i c a l for the preservation of self-stimulation has been, presented'by Valenstein and Campbell (1966). They • report ' that', extensive lesions; i n the MFB-LRA do.not stop self-stimulation from septal or olfactory s i t e s when the animals are allowed s u f f i c i e n t time for postoperative recovery. In support cf this conclusion, Kant (1969) has recently reported that chronic lesions i n the anterior areas of the MFB-LHA do not decrease septal self-stimulation. Reports have-. also ; appeared that.lesions i n the MFB-LHA do not decrease l a t e r a l hypothalamic self-stimulation (Lorens, 1966; Umemoto, 1968). These negative findings 2 contrast with other substantial investigations showing that the rewarding character of self-stimulation i s quite sensitive to disturbances i n th i s region. The most important of these experiments indicate that lesions i n the MFB-LHA quite consistently and severely attenuate or abolish hypothalamic self-stimulation (Olds and Olds, 1964; Olds and Olds, 1969). These results are p a r t i a l l y supported by those of Boyd and Gardner (1967) which, while f a i l i n g to implicate, s p e c i f i c a l l y the MFB-LHA, agree that some lesions i n this general region can sub-s t a n t i a l l y decrease self-stimulation rates. The contradictory results generated by lesion studies evaluating the role of the MFB-LHA i n self-stimulation have been generally attributed to differences i n the size and location of the lesions used and to differences i n the degree of postoperative recovery allowed (Boyd and Gardner, 1967; Olds and Olds, 1969; Valenstein, 1966; Umemoto, 1968). In view of these d i f f i c u l t i e s with the. lesion method, i t was decided to' re-examine the role of the MFB-LHA i n self-stimulation of the septal area using microinjections of procaine hydrochloride into the MFB-LHA. to block synaptic transmission. The use of chemical injections has the advantage that while the disturbance of MFB-LHA a c t i v i t y i s almost immediate, i t i s of short duration. This allows for a within subjects experimental design, repeated testing, and minimizes the p o s s i b i l i t y for reorganization of the neural substrate mediating the behavior. Un i l a t e r a l injections were used since the rewarding character of the u n i l a t e r a l stimulation appears to be mediated primarily by the ac-t i v i t y on one side of the brain (Boyd and Gardner, 1967; Olds and Olds, 1969) . The use of u n i l a t e r a l injections also has the advantage that 3 injections made on the side of the brain contralateral to the electrode provide a potential means of evaluating the general dis-ruptive effects of the procaine injections. In a further attempt to evaluate the disruptive effects arising from unilateral injections of procaine, an additional experiment examined the effects of such injections on bar-pressing for food. 4 METHOD A total of 42 male hooded rats (Quebec Breeding Farms) weighing from 210 to 310 grams were used in the self-stimulation experiment and 20 additional rats weighing from 230 to 310 grams were used to evaluate the effect of procaine on lever press performance reinforced with food. Data is reported from 19 animals in the ele c t r i c a l stim-ulation experiment and from 10 animals in the food experiment. The animals that were discarded from the experiments dislodged their electrode assembly (n=4), did not self-stimulate (n=12), became i l l and were sacrificed (n=8), developed seizures (n=3), or persistently circled toward the injected side of the brain during the procaine treatment (n=6). Surgery was performed using Sodium Pentobarbital anesthesia (50 mg/lOOg). Bipolar stainless steel stimulating electrodes (0.01 in dia.) insulated except at the tip were implanted in various parts of the septum and preoptic area. Stainless steel cannulas (23 ga) for chemical injections were also implanted bilaterally at various locations along the MFB-LHA. A stainless steel wire with a cap was placed in each injection cannula to prevent passage of foreign material and plugging. When an animal had completed the testing cordLtions i t was sacrificed with an overdose of ether. The brain was removed and placed in 10% formalin. Several days later the brain was placed in 20% ethanol for 24 hours, sectioned at 40 microns and stained with thionin. The apparatus for self-stimulation consisted of a lever box measuring 11 X 12 X 25 in . high idLth a grid floor, aluminum walls on 3 sides and a plexiglass door. Leads made of light hearing aid wire descended to the animal through a hole in the ceiling. E l e c t r i c a l 5 stimulation was provided by stimulators delivering 60 hz sine wave current. Duration of the stimulation, for each lever press was C.25 sec. Stimulation was monitored in some subjects across a 10K resistor in series with the animal using a Tektronix 565 Oscilloscope. Responses during self-stimulation were recorded in both boxes using electro-mechanical counters and cumulative recorders. The same apparatus xvas used testing the effect of procaine injections on bar-pressing for food except that a food magazine and a food tray were added. Chemicals were injected through a stainless steel needle (30 ga) which protruded 0.5 to 1.0 mm past the end of the guide cannula. The needle was connected by a piece of PE 50 polyethylene tubing to a micro-syringe graduated in steps of 1 microliter (Nakajima, 1964). Training and Testing Procedure Five to seven days following surgery, each animal was placed in the box and shaped to press the lever for electrical stimulation. When an animal began to press the lever i t was given one 30 min session per day u n t i l stable pressing rates were achieved. An animal x-ras judged to be stable i f i t s performance did not show a continuous increase in rate over 4 daily sessions and i f i t s performance did not vary more than 20% from i t s mean rate over these 4 days. Animals that did not self-stimulate were given numerous t r i a l s and lengthy periods in the box at several intensities before being discarded. Following attainment of stabilized rates of responding the animals began a series of conditions which included injections of 0.9% saline i p s i l a t e r a l (SI) or contralateral (SC) to the electrode^ and 2.0% procaine (in 0.54% saline) i p s i l a t e r a l (PI) or contralateral (PC) to ,the electrode, and extinction with the injection needle on the side of the brain i p s i l a t e r a l (EI) or contralateral (EC) to the electrode; With the chemical injections, the volume injected was always 2 microliters over a 1 min period. The animals were allowed to press for at least 20 min following the injection. During the extinction condition the injection needle was placed into one side of the brain and following 8 to 10 min of responding the current was turned off . by disconnecting the leads from the stimulator. Following 15 min of responding with the current off, the leads were reconnected and the animal was allowed to continue pressing for an additional 8 to 10 min. Occasionally an animal was not responding when the leads were re-connected. In this case the animal was l e f t u n t i l i t started pressing the lever. In no case did an animal pause for longer than 5 min. Evaluation of an animal Is performance during this recovery period was done on the basis of i t s responding during the 5 minutes following i t s f i r s t reinforced press. With a l l conditions a single h hr t r i a l was given per day and a rest day, in which the animals were allowed _ hr of reinforced responding without treatment, intervened between treatment t r i a l s . In a number of cases several successive rest days were given to allow recovery from previous treatments or anesthesia used to effect repairs on the surgical preparation. The conditions were run in two predetermined orders; Order 1. PI, SC, EI, PC, SI, EC: Order 2. PI, EC, SI, PC, EI, SC. Such ordering 7 prevented treatments on the same side of the brain from occurring d i r e c t l y after each other and insured that at least 5 days would elapse between procaine injections. Each animal was assigned to begin with one of the s i x "conditions within one of the"orders and continued through the assigned sequence of conditions on subsequent treatment days. Following completion of the assigned order the animal continued through the other order again beginning with the condition i t had o r i g i n a l l y been assigned. For example, i f an animal was assigned to begin with a PI treatment i n order 1, the order of conditions i t would follow would be: PI, SC, EI,PC, SI, EC, PI, EC, SI, PC, EI, SC. Assignments to orders of treatment, actual i n j e c t i o n procedures, surgery, histology, apparatus and scoring procedures used during the experiment evaluating the effects of procaine on bar press for food differed l i t t l e from those, used during the e l e c t r i c a l stimulation experiment. Electrodes that did not protrude above the surgical preparation were implanted i n the septal area i n a l l but 2 of these animals. Leads were connected to the animals by means of a c l i p which snapped on to a st e e l ring placed on the back of the surgical area with dental a c r y l i c . Following recovery from surgery, the animals were placed on a feeding schedule i n which they received 13.5 to 14.0 g of wet mash each day. The animals remained on this schedule through-out the experiment and the amount of food an animal received during each t r i a l was subtracted from the amount of food usually put i n the cage. After 7 days of adaptation to the feeding schedule each animal was placed i n the lever box and the lever was programmed to. deliver 45 mg food pellets on a continuous schedule of reinforcement. The lever was pressed 5~times by the experimenter and the animal was l e f t in the box u n t i l 100 or more reinforced responses occurred. On the following days the animals were given one \ hr session per day pressing on an FR-4 schedule u n t i l stable rates were achieved. The criterion for stable performance in this experiment was the same as for electrical stimulation. The. procedure for making saline and procaine injections, and for extinction, was the same as that used in the self-stimulation experiments. Scoring Procedure and Stat i s t i c a l Analysis Performance of an animal under the various conditions was scored directly from cumulative records by measuring the vertical excursion of the cumulative recorder pen during 8 to 10 min baseline and four 5 min time periods during and after treatment. The excursion of the pen during the baseline period was converted to a score for 5 min. Since the vertical excursion of the recorder pen yields figures that are directly proportional to the slope of the record, i t was not necessary to convert the excursion values to values for rate of responding. Scores obtained during the four 5 min time periods were expressed as a percentage of the score obtained during the baseline period and these percentages for each treatment were averaged over the results from two separate t r i a l s . In order to test the statis-t i c a l significance of the effects noted in the experiments a 3 factor (side of brain, treatment; time blocks) completely within subject analysis of variance was applied, to this data. Further analysis of significant F values was done using F-tests for simple main effects (Winer, 1962) and t-tests for related measures (Bruning and Kintz, 1968). The magnitude of the effect of the procaine injections at each site in both experiments was classified by expressing the excursion of the recorder pen during the f i r s t 10 min under procaine as a percentage of a score for saline derived in the same manner. Each classification was based on the average of performance on two separate t r i a l s . In the electrical stimulation experiment procaine was judged to have a strong effect i f performance was 0 - 40% that of saline; moderate 41 - 80%; weak 81% or greater. During lever press for food procaine was judged to have strong effects i f performance was 0 - 60% that of saline, and weak effects i f performance was 61% of saline or greater. 10 RESULTS Part 1: Effect of Procaine Injections on Self-Stimulation Data from animals were included in the s t a t i s t i c a l analysis of results only i f their cannulas were located in the MFB-LHA and i f injections or procaine at these sites did not cause obvious disturb-ances of behavior. The scores over the four 5 min time periods for these 14 animals appear in Fig. 1. This figure shows the effect of procaine injections i p s i l a t e r a l or contralateral to the injection Insert Figure 1 about here site in comparison with saline and extinction. The experiment was done with saline injections i p s i l a t e r a l or contralateral to the electrode and .during the extinction t r i a l s the injection needle was also placed into one side of the brain. However, since saline injections did not have differential effects in sites i p s i l a t e r a l and contralateral to the stimulating electrode and the same result was obtained with extinction, data from the two sides of the brain are combined for each of these treatments in Fig. 1. Procaine injected into the side of the brain with the electrode suppressed self-stimulation more than saline during each of the f i r s t two time periods (p <0.001). There was no significant difference bet-ween these treatments during either of the last two time periods (p >0.10). The injections of procaine contralateral to the electrode, which were intended to control for suppression of self-stimulation due to general disruption of behavior, did suppress self-stimulation more than did saline injections. However, this difference occurred primarily Figure 1. The effect of stopping the stimulating current (extinction), injecting procaine, or injecting isotonic saline into the MFB-LHA ipsil a t e r a l or contralateral to the stimulating electrode on the rate of bar-pressing for el e c t r i c a l stimulation. Because there was no laterality difference in the effect of extinction and saline injection, the ipsilateral and contralateral data for these two conditions is combined into a single curve. M E A N P E R C E N T O F B A S E L I N E 13 during the f i r s t 5 min (p <0.001). Thereafter responding during the procaine treatment on this side of the brain recovered rapidly and the differences were not significant during any of the f i n a l 3 time periods (p>0„05); Further the suppression of self-stimulation with procaine on the contralateral side was significantly less than with procaine on the ip s i l a t e r a l side during each of the f i r s t two time periods (p <0.05). There was no significant difference between the effects of these treatments during time periods 3 or 4 (p >0.10). As indicated by Fig. 1 extinction did not suppress self-stimulation more than procaine injections into the MFB-LHA on the same side of the brain as the stimulating electrode during the f i r s t time period (p >0.20) although extinction did suppress rates more than procaine during each of the next two 5 min intervals (p <0.01). The extinction treatment suppressed self-stimulation more during each of the three time periods than either procaine injections into the contra lateral side of the brain (p <0.05), or the saline treatment (p <0.001) Making ele c t r i c a l stimulation available to the animals again following extinction resulted in higher rates of self-stimulation than occurred, during the fourth time period under saline injections (p <0.01) or procaine injections i p s i l a t e r a l (p <0.05) or contralateral (p<0.05) to the stimulating electrode. Location and Strength of Procaine effects Figute 2 shows the location of the various injection sites. Each injection site i s coded (see Method for procedure) with respect 14 to whether procaine there suppressed pressing mildly (81 - 100% of the rate following saline injections), moderately (41 - 80%), or strongly (0 - 40%). This figure includes data for the 5 animals discarded from Insert Figure 2 about here thie previous s t a t i s t i c a l analysis because of assymetry of cannula implants or behavioral effects of procaine that appeared to be incompatible with pressing the lever. It i s apparent that strong and moderate effects of procaine upon self-stimulation occurred at sites located along the entire length of the MFB-LHA system. The figure also shews that the majority of sites (12 out of 15) yielding strong effects were located i p s i l a t e r a l to the stimulating electrode. Cannulas located in the very lateral part of the MFB-LHA appeared to give the strongest effect on self-stimulation at the level of or anterior to the ventromedial hypothalamus (VMH) but strong effects upon self-stimulation were observed throughout the MFB-LHA as placements were made more posterior. Figure 3 contains 2 photomicrographs of brain sections and selected cumulative recordings from animals that exhibited strong depression of rate under injections of procaine. Although the st a t i s t i c a l analysis indicated that extinction in general depressed self-stimulation more than procaine, the,recordings il l u s t r a t e that procaine injections could have an effect equal in magnitude to Insert Figure 3 about here » Figure 2. The dist r i b u t i o n of points along the MH'-LHA where the. effect cf procaine injections on e l e c t r i c a l stimulation was examined„ The effect of the injections i p s i l a t e r a l (top) and contralateral (bottom) to the stimulating, elec-trode are plotted separately. F i l l e d c i r c l e s represent site s where procaine suppressed bar-pressing by 60-100%; h a l f - f i l l e d c i r c l e s represent sit e s where bar-pressing was suppressed by 20-60%; and open c i r c l e s where the suppression was 0-20% of that folioxtfing saline i n j e c t i o n s . The hexagonals represent in j e c t i o n sites which were not-included i n the analysis of the data because they are assymetrical.. These symbols are coded i n the same way as the c i r c l e s . The brain sections are redrawn from DeGroot, (1959). I P S I L A T E R A L C O N T R A L A T E R A L •7r I—1 Figure 3, Individual records from two animals showing the effect of the various treatment conditions on the bar press rate for e l e c t r i c a l stimulation along with a h i s t o l o g i c a l section through the injection s i t e * In animal No.23 (top), the stimulating electrode was i n the nucleus' accumbens and the injection cannula was i n the posterior portion of' the MFB-LHA. In animal No.21 (bottom), the stimulating electrode was in the diagonal band of Broca and the i n j e c t i o n cannula was i n the anterior portion of the MFB-LHA. 19 extinction. Animal 23 in this figure displayed circling behavior when procaine was injected into the side of the brain i p s i l a t e r a l to the electrode. Circling in this animal was mild relative to the animals which were discarded for this reason and a subsequent injection of hypertonic NaCl (2 microliters; 3%) duplicated the turning behavior without depressing self-stimulation rate (see cumulative recordings Fig. 3). Such a result would seem to suggest that circling in this case did not indicate sufficient disruption to compete with s e l f -stimulation. It should be emphasized that both animal 21 and animal 23 were not chosen for this figure as representative examples of the effect of procaine in the various areas studied. Although the magnitude of the.reaction to procaine and some histological features in these two animals are consistent with data in several other animals, they do not reflect the wide range of response rates that occurred in the experiment or a tendency noted in some animals to decrease rate over t r i a l s . Variation in Electrode Location and the Effect of Procaine Histological examination revealed stimulating electrodes in the nucleus accumbens (n=6), diagonal band of Broca (n=5), lateral septum (n=2)s medial septum (n=l) and preoptic area (n=5). These electrode sites yielded an average rate of 21 bar presses per min (range=ll-55, SD=11) at intensities ranging from 35 to 110 micro-amperes rms. Procaine injections moderately or strongly depressed self-stimulation at some electrode sites in a l l these areas which 20 suggests that electrode placement was not the c r i t i c a l variable in determining the effect of the injections in this experiment. Similarly, procaine injections strongly depressed self-stimulation at both high and low bar press rates and at both high and low stimulation intensities. The Effect of Variation in Damage at the Injection Site Histological examination of injection sites revealed several factors that could conceivably cause depression of self-stimulation performance or create a lateralized effect of procaine such as occurred in the experiment. Some cannula sites showed evidence of passage of chemicals up the outer shell of the guide cannula (animal no. 21, Fig. 3) while others showed evidence of passage of chemicals toward the base of the brain (animal no. 23, Fig. 3). These patterns occurred equally as often on both sides of the brain and occurred at sites yielding different magnitudes of effect during procaine injections. Neither pattern was systematically related to the lateralized depression of self-stimulation caused by procaine. The area damaged by the injection was irregular in shape and stained a darker blue under thionin. The distance of the maximum lateral and dorsal extent of the affected area was measured by magnifying (14X) and projecting slides on a grid which was graduated in mm. The distance of this area in the anterior-posterior place was measured by counting the number of sections upon which the darker area occurred. This analysis resulted in average values of 0.4 mm in the lateral plane, 1.0 mm in the dorsal plane and 1.4 mm in the anterior-21 posterior plane. S t a t i s t i c a l analysis of the values for i p s i l a t e r a l and contralateral sites revealed no differences on these dimensions . ( a l l 't' values p >0.20). Comparisons of sites yielding strong, moderate and weak effects of procaine on self-stimulation rate showed no difference between these sites in anterior (F=1.26, df=2/25, p >0.10), lateral (F <1) or dorsal (F=2.06, df-2/25, p >0.10) extent of the discoloured area. Part 2: Effect of Procaine Injections on Lever Press for Food Evaluation of the effect of procaine injections into the MFB-LHA upon performance of a lever press task reinforced with food was done as an additional control experiment for the general disruptive effects of procaine injections. The procedure was the same as that for the self-stimulation experiment (see Method)„ Figure 4 shows performance during the procaine, saline, and extinction treatments for the 10 animals included in the analysis (see Method) of the experiment. Since s t a t i s t i c a l analysis indicated no significant differences in the effect of these treatments, dependent Insert Figure 4 about here on whether the treatment occurred on the same or opposite side as the electrode (F=2.46, df-2/18, p >0.10), data from each side of the brain arecombined in this figure. The only "significant effect in the experiment was a suppression of the lever press response by procaine injections relative to extinction (p <0.01) or saline (p <0.01) during ro N3 Figure 4. The effect of stopping food reinforcement (extinction), injecting procaine., or injecting isotonic saline into the MFB-LHA on the rate of bar-pressing for food reinforcement. The injections were unilateral in each case. 24 the f i r s t 5 min of the test period. There was no significant difference in performance between the three treatments during the second and third 5 min time blocks of the test period. When the food reward became available after extinction,rate of responding rose to a higher level than rates under saline (p <0.01) or procaine (p <0.01) during the same time period. Figure 5 shows the injection sites located on sections taken from the atlas of DeGroot (1959) and coded for the strength of the procaine effect (see Method). Sites were classified as yielding either strong ((0 - 60%) of that following saline), or weak (61 - 100%) depression of performance. This dichotomy was chosen because the animals bar pressing for food tended to f a l l into two groups; in one of these the suppression by procaine was very brief ( 1 - 3 min) and in the other more comparable to that seen with self-stimulation (5 - 10 min) . It i s apparent from Fig. 5 that injection sites f e l l a l l along the path of MFB-LHA. The majority of sites injected showed some evidence of disruption due to procaine during the f i r s t 5 min time Insert Figure 5 about here period following the injection. Injection sites that gave strong effects were either at, or anterior to, the level of the ventromedial hypothalamus and were not anatomically differentiable from sites giving suppression of self-stimulation. In contrast, injection sites posterior to the level of the ventromedial hypothalamus gave relatively weak effects Figure 5. The distribution of points along the MFB-LHA where the effect of procaine injections on rate of bar-pressing for food reinforcement was examined. F i l l e d circles represent sites where procaine suppressed bar-pressing by 40-100%, open circles represent sites where bar-pressinp wr.s suppressed by 0-40?. The brain sections are redrawn from DeGroot (195S). 27 on lever press for food although these sites had strong effects on self-stimulation (Fig. 1); This distribution of areas giving strong suppression of bar pressing for food coincides closely with the distribution of brain regions in the hypothalamus concerned with feeding (Albert, Storlein, Wood, and Ehman, 1970; Booth, 1967). The Effects of Deprivation, Reinforcement Schedule and Results of Histology The use of relatively low deprivation (13.5 to 14.0 g of food per day) and an FR-4 schedule of reinforcement resulted in an average response rate of 21 responses/min (range=15-28, SD=5). Animals usually began showing periods 1 or 2 min in length in which they did not respond before the end of a % hr session. Body weight was maintained during the running of the experiment at approximately 90% of preoperative weight. Histological analysis of injection sites in these animals was done in the same manner as in the el e c t r i c a l stimulation experiment (see results of Study 1). This analysis resulted in values of 1.4 mm, 0.8 mm, and 0.7 mm for the average maximum extent of the lesion due to injections in a dorsal, anterior-posterior and lateral direction respectively. 28 DISCUSSION The results of this experiment show that a unilateral 2 micro-l i t e r injection of 2% procaine into the MFB-LHA can cause suppression of lever pressing for electrical stimulation in the septum or preoptic area. The suppression obtained with these injections occurred along the entire length of the MFB-LHA. The strongest suppression of bar pressing occurred when the injections were made into the MFB-LHA on the same side of the brain as the stimulating electrode. Unilateral injections were used in the experiment as a method of controlling for disruptive effects attributable to the injections of procaine. A stronger effect of procaine on the same side of the brain as the stimulating electrode suggests that general disruptive effects associated with the drug are not sufficient to account for the suppression of self-stimulation caused by injections on the ips i l a t e r a l side of the brain. Histological results indicated that the difference in suppression on the two sides of the brain did not occur as a result of variations in the location of the injection sites, differences in the size of the lesion caused by the injections or differences in pattern of spread of the injections. The fact that suppression of performance in the food rewarded task occurred in the regions of the MFB-LHA associated with feeding (Fig. 5) (Albert, Storlien, Wood, and Ehman, 1970; Booth, 1967) and not along i t s entire length is also consistent with the notion that general disrup-tive effects cannot entirely explain the effects of procaine on self-stimulation. Disruptive effects of procaine were also controlled for 29 by discarding data from animals which showed indications of disruption such as circlin g or motor dysfunction. These results lead to the tentative conclusion that the effect of injections of procaine on self-stimulation in this experiment is not explainable on the basis of general disturbances of behavior such as; motor dysfunction, dis-orientation, nausea, inability to perform the response, emotionality changes or forgetting. The finding of a suppression of self-stimulation somewhat independent of general disruption by injecting procaine into the MFB-LHA suggests that this structure is part of a system mediating the rewarding effect of electrical stimulation in the septum and pre-optic areas. There does not appear to be any particular section of the MFB-LHA that is uniquely important, since positive results were found along the entire length of the structure (Fig. 2). However, the possibility that the effects might also occur with injection sites around the MFB-LHA was not systematically investigated. The fact that extinction caused greater overall suppression of self-stimulation than* procaine indicates that blocking synaptic transmission in the MFB-LHA and stopping the ele c t r i c a l stimulation do not have identical effects. The greater effect of the extinction treatment i s presumably explain-able on the basis that procaine injections varied in the strength of their effects. The higher rates noted in the extinction condition during the fourth time period are probably due to the fact that scores for this time period were measured after the animals were responding again. It i s also possible that contrast effects (Panksepp and Trowill, 1969) and the injection of a volume into the brain during the saline 30 procaine treatments contributed to this difference. The results of. the present experiments can be viewed as providing a framework for considering some of the apparent contradictions in existing results on the effect of lesions in the MFB-LHA on self-stimulation. The fact that the present results indicate that the MFB-LHA i s involved in mediating the rewarding effect of electrical stimulation makes i t reasonable to expect that lesions in this area such as those used by Olds and Olds (1969) could suppress self-stimulation. At the same time, the fact that the system appears to include at least the whole MFB-LHA suggests that in many cases enough of the system might remain to allow postoperative recovery of self-stimulation (Valenstein and Campbell, 1966). These facts combined with problems such as defining the size, location and symmetry of the lesion and specifying the criterion for recovery of animals suggest that disparate results with lesions might be expected. An important limitation in the evidence presented here and in other studies i s the inab i l i t y to specify the nature of the disruption which suppresses self-stimulation. Elements of this problem have been alluded to before by several authors (Boyd and Gardner, 1967; Olds and Olds, 1969; Valenstein, 1964). This problem is becoming increasingly salient in view of recent findings that b i l a t e r a l lesions in the MFB-LHA besides influencing motivation can have rather general and complex effects. For example, lesions in this area can disrupt learning of avoidance behavior (Coscina. and Balagura, 1970), counting behavior in a Lashley 111 maze (Olds and Hogberg, 1964) or cause changes 31 in activity (Balagura, Wilcox and Coscina, 1969). With symmetrical bil a t e r a l lesions, the grossest disturbances of the brain mechanisms controlling behavior would be sufficiently apparent to lead to dis-carding many animals incapable of performing the response being studied. With unilateral or assymetrical lesions, however, severe disruption of brain function of one hemisphere might go unnoticed since the other side would control body functions. With unilateral chemical injections as in the present experiment, i t is also possible for complex disruptive effects to occur. With each of these methods there is no xjay of assessing whether disruption of self-stimulation is due to a disturbance of the motivational characteristics of the electrical stimulation. The approach to this problem that seems necessary i s one in which an attempt is made to manipulate the under-lying neural systems so as to demonstrate both inhibition and fac i l i t a t i o n of self-stimulation. 32 REFERENCES Albert, D.J., L.H. Storlien, D.J. Wood and G.K. Ehman. Further evidence for a complex system controlling feeding behavior. Physiol. Behav.,5; 732-739, 1970. Balagura, S., R.H. Wilcox, and D.V. Coscina. The effect of diencephalic lesions on food intake and motor activity. Physiol. Behav., 4: 629-633, 1969. Booth, D.A. Localization of the adrenergic feeding system in the rat diencephalon. Science, 157: 1574-1575, 1967. Boyd, E.S., and L.C. Gardner. Effect of some brain lesions on intracranial self-stimulation in the rat. Airier. J. Physiol., 213: 1044-1052, 1967. Bruning, J.L., and B.L. Kintz. Computational Handbook of Statistics. Glenview: Scott, Foresman and Co., 1968, pp. 12-15. Coscina, D.V., and S. Balagura. Avoidance and escape behavior of rats with aphagia produced by basal diencephalic lesions. Physiol. Behav., _5j 651-657, 1970. DeGroot, J. The rat forebrain in stereotaxic coordinates. Verh. k. red. Akad. Wet. 52: No. 4, 1959. Grossman, S.P. A textbook of physiological psychology. New York: John Wiley & Sons, 1967, pp. 338-563. Guillery, R.W. Degeneration in the hypothalamic connexions of the albino rat. J. Anat., 91: 91-115, 1957. Harvey, J.A., and C.E. Lints. Lesions in the medial forebrain bundle: delayed effects on sensitivity to electric shock. Science, 148: 250-252, 1965. Kant, K.J. Influences of amygdala and medial forebrain bundle on self-stimulation in the septum. Physiol. Behav., 4_: 777-784, 1969. Lorens, S.A. Effect of lesions in the central nervous system on lateral hypothalamic self-stimulation in the rat. J. comp. physiol. Psychol., 62: 256-262, 1966. Nakajima., S. Effects of chemical injection into the reticular formation of rats. J ; comp. physiol. Psychol., 58: 10-15, 1964. Nauta, W.J.H. Hippocampal projections and related neural pathways to the midbrain in the cat. Brain, 81: 319-340, 1958. 33 REFERENCES (continued) Nauta, W.J.H. Some, neural pathways related to the limbic system. In: E l e c t r i c a l Studies of the Unanesthetized Brain, edited by E.R. Ramey and D.S. O'Boherty. New York: Hoeber, I960, pp. 1-16. Olds. J.A. A preliminary mapping of e l e c t r i c a l reinforcing effects i n the rat brain. J. comp. physiol. Psychol., 49:281-285, 1956. Olds, J . Self-stimulation of the brain. Science, 127: 315-324, 1958a. Olds, J . Satiation effects i n self-stimulation of the brain. J . comp. physiol. Psychol., 51: 675-678, 1958b. Olds, J . , and M.E. Olds. The mechanisms of voluntary behavior. In: The Role of Pleasure i n Behavior, edited by R.G. Heath, New York: Hoeber, 1964, pp. 23-53. Olds, J . , R.P. Travis, and R.C. Schwing. Topographic organization of hypothalamic self-stimulation functions. J. comp. physiol., Psychol., 5J3: 23-32, 1960. Olds, M.E. and D. Hogberg, Subcortical lesions and maze retention i n the rat. Exp. Neurol., 10: 296-304, 1964. Olds, M;E. and J . Olds. Approach-avoidance analysis of rat diencephalon. J . Comp. Neurol. 120: 259-295, 1963. Olds, M.E., and J . Olds. Effects of lesions i n medial forebrain bundle on self-stimulation behavior, Amer.• J. Physiol., 217: 1253-1263, 1969. Umemoto, M. Self-stimulation of the- l a t e r a l hypothalamus after e l e c t r o l y t i c injury of the medial forebrain bundle i n the cat. Brain Res., 11: 325-335, 1963, Valenstein, E.S. Problems of measurement and interpretation with reinforcing brain stimulation. Psychol. Rev., 71: 415-437, 1964. Valenstein, E.S. The anatomical locus of reinforcement. In: Progress i n Physiological Psychology, edited by E, S t e l l a r and J . Sprague. New York: Academic Press, 1966, 1, pp.149-190. Valenstein, E.S., and J.F. Campbell. Medial forebrain bundle-l a t e r a l hypothalamic area and reinforcing brain stimulation. Am. J . Physiol., 210: 270-274, 1966. Winer, B.S. S t a t i s t i c a l principles i n experimental design. New York: McGraw-Hill, 1962, pp. 232-238. 34 TABLE I Electrode Location, Intensity of Stimulation, Baseline Rate of Responding and the Magnitude of the Effects of Procaine Injections into the MFB-LHA in Animals Pressing the Lever for Electrical Stimulation of the Brain. Intensity Rate Procaine^QQ Procaine^QQ Animal Electrode (Microamps.) (BP/min.) Saline Saline Site* (Ipsilateral) (Contralateral) 1 ACB 110 24 35.5 90.6 3 POA 50 55 65.1 92.2 4 DBB 60 43 21.0 67.1 5 LS 80 20 16.5 38.8 8 ACB 60 16 16.4 100.6 9 ACB 65 25 29.3 28.3 10 ACB 70 11 100.0 18.1 11 POA 65 20 40.0 89.1 12 ACB 60 15 90.7 102.4 14 POA 65 11 16.6 88.1 15 ACB 60 19 92.0 109.8 16 ACB 60 18 30.3 106.5 17 POA 80 19 39.9 55.1 18 DBB 40 18 73.2 68.4 19 MS 45 29 73.7 65.4 20 DBB 55 13 28.8 97.0 21 DBB 40 17 12.4 52.8 22 LS 60 17 57.2 84.6 23 DBB 40 26 8.3 48.4 24 LS 45 16 101.6 57.1 * A l l abbreviations from the atlas of DeGroot (1959) 35 TABLE II Means and Standard Deviations for Procaine, Saline and Extinction Treatments over four 5 Minute Time Periods during Responding for Electrical Stimulation of the Brain Condition Time Time Time Time Period Feriod Period Period 1 2 3 ' 4 Procaine Mean AO .4 38.0 57.3 66.6 Ipsilateral SD 20 .5 23.1 27.3 22.1 Procaine Mean 62 .0 63.5 68.7 72.1 Contralateral SD 20 .4 33.6 32.6 18.4 Saline Mean 80 .8 80.0 67.2 68.9 Ipsilateral SD 22 .6 15.6. 18.5 20.6 Saline Mean 95 .9 75.8 73.3 69.5 Contralateral SD 18 .0 16.7 19.A 19.5 Saline Mean 88 .3 77.9 70.2 69.2 (combined) SD 21 .5 • 16.0 18.9 19.7 Extinction Mean AO .3 15.8 12.9 84.0 (combined) SD 22 .A 9.9 7.6 20.6 36 TABLE III Summary of Analysis of Variance of Scores for Procaine, Saline and Extinction Conditions during Responding for Electrical Stimulation SOURCE MS df F P Total 335 Subject 13 Side* 4,033.12 1 5.10 < 0.05 Treatment 40,836.68 2 63.08 < 0.001 Time ** 12,552.93 3 23.09 < 0.001 Side x Treatment 1,836.63 2 3.61 •< 0.05 Side x Time 649.49 3 3,56 < 0.025 Treatment x Time 11,106.34 6 33.S5 < o.ooi Side x Treatment x Time 276.64 6 1.25 > 0.20. Side x Subject 790.18 13 Treatment x Subject 647.31 26 Time x Subject 525.25 39 Side x Treatment x Subject 507.74 26 Side x Time x Subject 181.97 39 Treatment x Time x Subject 329.99 78 Side x Treatment x Time x Subject 219.67 78 * Side of the brain in which treatment occurred ** Four 5 minute time periods 37 TABLE IV Simple Main Effects Analysis of Variance of Scores for Procaine, Saline and Extinction Treatments i p s i l a t e r a l and Contralateral to the Stimulating Electrode SOURCE MS df F P Simple Effects of Treatments for Ipsilateral 18,523.64 2 36.48 < 0.001 for Contralateral 24,637.25 2 48.52 < 0.001 Simple Effects of Side of the Brain for Procaine 8,097.10 1 15.95 < 0.001 for Saline 536.81 1 1.06 > 0.20 for Extinction 4.76 1 0.009 ns Error w 507.74 2.6 38 TABLE V Simple Main Effects Analysis of Variance of Scores for Injection Sites Ipsilateral and Contralateral to the Stimulating Electrode during 5 Minute Time Periods SOURCE MS df Simple effects of Time Periods for Ipsilateral 7,966.38 3 43.78 < 0.001 for Contralateral 5,236.05 3 28.77 < 0.001 Simple Effects of Side of the Brain for T 3,877.36 1 21.31 < 0.001 for T, 1,412.04 1 7.76 < 0.01 for T^ 691.44 1 3.80 > 0.10 for T, 0.76 1 0.004 ns 4 Error 181.97 39 w T - Time Period 39 TABLE VI Simple Main Effects Analysis of Variance of Scores for Procaine, Saline and Extinction Treatments over 5 Minute Time Periods during Responding for Electrical Stimulation SOURCE Time Periods for Procaine for Saline for Extinction Treatments for T for Tj for T^ for T. 4 Error w MS df F P 2,351.67 3 7.13 < 0.001 2,186.26 ,3 6.63 < 0.001 30,227.70 . .-3 91.60. < 0,001 17,743.22 .2 53.77 < 0.001 27,093.89 2 82,11 < 0.001 27,310.28 2 82.76 < 0.001 2,008.31 2 6.08 < 0.005 329.99 78 T - Time Period AO TABLE VII Location of Electrode, Baseline Rate of Responding and the Magnitude of the Effects of Procaine Injections into the MFB-LHA in Animals Pressing the Lever for Food Reward. R a t e Saline x^QQ Saline x^QQ Animal Electrode (Responses/ Procaine Procaine Site* minute) (Right)** (Left) 1- 6 - 25 37.3 94.A 2- 6 - 27 92.0 83.3 6-6 ACB 15 65.8 77.2 8-6 ACB 15 55.9 69.8 3- 7 ACB 2A 71.1 73.A A-7 DBB 22 86.1 75.8 6- 7 DBB 28 A7.3 15.7 7- 7 LS 18 61.3 A8.5 10-7 DBB 17 150.0 90.2 16-7 ACB 16 97.A 39.1 * A l l abbreviations from the atlas of DeGroot (1959) ** Ipsilateral in animals that had electrodes implanted. 41 TABLE VIII Means and Standard Deviations for Procaine, Saline anc Extinction Treatments over four 5 Minute Time -Periods during Responding for Food Reward Time Time Time Time Condition Period Period Period Period 1 2 3 4 Procaine Mean 43.3 67.3 69.1 62.3 SD 16.4 25.7 22.3 18.2 Saline Mean 86.8 75.5 63.1 57.3 SD 24.7 15.5 17.8 15.1 Extinction Mean 84.8 61.7 58.1 85.6 SD 33.1 28.5 36.7 18.9 42 TABLE IX Summary of Analysis of Variance of Scores for Procaine, Saline and Extinction Conditions during Responding for Food Reward SOURCE MS df F P Total 239 Subjects 9 Side* 988.08 1 0.89 ns Treatment 3,388.78 2 1.88 > 0.10 Time ** 683.55 3 1.34 > 0.20 Side x Treatment 1,351.49 2 .2.46 > 0.10 Side x Time 71.52 3 0.20 ns Treatment x Time 4,939.68 6 13.57 < 0 001 Side x Treatment x Time 119.7 3 6 0.44 ns Side x Subject 1,102.29 9 Treatment x Subject 1,802.02 18 Time x Subject 507.64 27 Side x Treatment x Subject 547.99 18 Side x Time x Subject 352.99 27 Treatment x Time x Subject 363.82 54 Side x Treatment x Time x Subject 269.59 54 * Side of the brain in which treatment occurred ** Four 5 minute time periods 43 TABLE X Simple Main Effects Analysis of Variance of Scores for Procaine, Saline and Extinction Treatments over 5 Minute Time Periods during Responding for Food Reward SOURCE MS df F P Simple Effects of Time Periods for Procaine 2,794.06 3 7.27 < 0,001 for Saline 3,467.75 3 9.03 < 0,001 for Extinction 4,301.08 3 11.20 < O.OOl Simple Effects of Treatments for T 12,078.25 2 31.46 < 0,001 for Ti 959.90 2 2.50 > 0,05 for T^ 599.21 2 1.56 > 0.20 for 4,570.44 2 11,90 < 0.001 Error 383.82 54 w T - Time Period 

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