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Differences in sensitivity to neuroleptic blockade: Medial forebrain bundle versus frontal cortex self-stimulation
91
Behavioural Brain Research, 36 (1990) 91-96 Elsevier BBR00989
Differences in sensitivity to neuroleptic blockade: medial forebrain bundle versus frontal cortex self-stimulation Dale Corbett Division of Basic Medical Sciences and Department of Psychology, Memorial University of Newfoundland, St. John's, N.F. (Canada) (Received 17 April 1989) (Accepted 25 April 1989)
Key words: Self-stimulation; Prefrontal cortex; Medial forebrain bundle; Dopamine
The effects of systemic injections of the dopamine receptor antagonist, c/s-flupenthixol were tested on intracranial self-stimulation at electrode sites in the medial forebrain bundle and the medial prefrontal cortex. Changes in the reward effectiveness of the brain stimulation were assessed using a curve-shift paradigm. Low to moderate doses of cis-flupenthixol (0.05, 0.1 and 0.15 mg/kg) consistently produced larger upward shifts in the rate-frequency function for medial forebrain bundle than for medial prefrontal self-stimulation. At the highest doses of c/s-flupenthixol (0.15 and 0.2 mg/kg), some of the medial forebrain bundle rats failed to respond, whereas all medial prefrontal rats responded at these doses. These results demonstrate that medial forebrain bundle self-stimulation is much more dependent on dopamine systems than is prefrontal cortex self-stimulation.
INTRODUCTION
It is now well established that dopamine neurons play an important role in the mediation of intracranial self-stimulation (ICSS) and psychomotor stimulant r e w a r d 2-4,7A5,21,3°,31. With regard to the relative importance of the different dopamine pathways, it would seem that it is the mesolimbic dopamine pathway that underlies the above reward effects. Lesions or intracerebral receptor blockade of this pathway attenuates ICS S and stimulant self-administration 3,15,18,2°,21,23. However, there is some question as to the importance of the mesolimbic dopamine system at brain stimulation reward sites distal to the medial forebraln bundle (MFB) since lesions of the dopamine pathways have little effect on medial
(MFC) 6,20,25 or substantia nigra ICS S 12. Instead, corticofugal projections seem to be of more importance at these two ICS S sites 6"24. In addition, whereas intracerebral inactivation of the dopamine projections to the nucleus accumbens produce a significant impairment of MFB ICSS ~s'27,2s, they do not markedly affect MFC ICSS 23. Nonetheless, it remains possible that other terminal projections of the mesolimbic dopamine system (e.g. diagonal band nuclei, amygdala) are critical for M F C ICS S. To test this possibility animals with either MFB or MFC ICSS electrodes were given systemic injections of the dopamine antagonist cis-flupenthixol. It was reasoned that if MFC ICSS is less dependent on a dopaminergic substrate than MFB ICSS, this should be reflected as a reduced senprefrontal
Correspondence: D. Corbett, Division of Basic Medical Sciences, Faculty of Medicine, Memorial University of Newfoundland, St. John's, N.F. Canada, A1B 3V6. 0166-4328/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
92 sitivity of MFC ICSS to dopaminergic receptor blockade. MATERIALS AND METHODS
Surgery Eighteen male Sprague-Dawley rats weighing 300-350 g at the time of surgery were implanted with 250 #m stainless steel, monopolar electrodes (Plastic Products Inc.) under Somnotol anesthesia (65mg/kg) in combination with 2~o Xylocalne. Eleven rats had electrodes aimed at the MFB (ant. - 2 . 3 mm, lat. + 2 . 0 m m and depth - 8 . 8 mm from skull surface) while the remaining 7 animals had electrodes aimed at the M F C (ant. + 4.2 mm, lat. + 0.6 mm and depth - 3.5 mm from skull surface). Stereotaxic coordinates were derived from bregma with the skull level. All rats were housed individually in plastic cages and maintained on a 12: 12h reverse day-night cycle. Food and water were freely available. Following a minimum of 3 days of postoperative recovery, the rats were trained to leverpress for brain stimulation.
Behavioral testing The rats were trained to press a lever in order to deliver 0.5-s trains of 0.1-ms monophasic, cathodal pulses delivered at a rate of 100 Hz. Currents were adjusted individually to ensure vigorous responding. Two groups of MFB animals were used in this experiment, a high current group (405 + 83.9#A) and a low current (225.7 + 61 #A) group. Testing was conducted in Plexiglas and stainless steel chambers (30 cm × 24 cm × 24 cm) equipped with a retractable lever mounted on a side wall. Once the animals responded consistently, rate-frequency testing began. This phase of testing consisted of lowering the frequency from a high level (e.g. 100 Hz) every 60 s in 0.1 log unit steps until the animal ceased responding. At the end of each frequency presentation the lever retracted for 5 s, the frequency was lowered to the next level, the lever extended and one free priming stlmulataon was delivered to the animal. Delivery of the stimulation parameters and recording of lever-presses was performed by an Apple IIe ,
.
J
computer system 5. This procedure yields a sigmoid-shaped curve from which one can derive the frequency that produces half-maximal responding. This value has been termed the locus of rise (LOR) and has been shown to be sensitive to manipulations that alter the reward value of the stimulation (e.g. current changes, amphetamine) but only minimally affected by treatments (e.g. non-specific lesions, partial paralysis) that disturb performance capability 8'~°'16"29. Test sessions were preceded by a 2-min warm-up period. When each animals' LOR varied less than 0.1 log unit over 3 consecutive days, the animals were considered stable. The MFB animals achieved this stability criterion after 19.1 _+ 6.34 (S.D.) days. The frontal group took an average of 29.0 + 8.78 (S.D.) days to meet the same criterion. The LOR values from these 3 days were used to derive the mean baseline LOR for each animal. The group baseline LOR values were used to assess all drug effects (see below). The animals were then pretreated with one of several doses of cis-flupenthixol (0.05, 0.1, 0.15 and 0.2mg/kg). Cis-flupenthixol was dissolved in physiological saline and was injected intraperitoneally 4 h prior to the test session. Control injections consisted of equal volumes of saline or the inactive trans-flupenthixot isomer. A minimum of 2 days separated each drug injection. Animals were not injected with drug if the LOR values on non-drug days varied by more than 0.1 log unit from the original mean baseline LOR value. Instead, the animals were tested until the LOR values returned to baseline levels. The data were analyzed by an Apple IIe computer. A least squares procedure was used to find the best fit for each rate-frequency function. On some test sessions the LOR was undefined (U). This occurred when the response rates were either zero or uniformly low across atl frequencies. Such conditions were typically encountered with the highest doses of cis-flupenthixol. Modified 99~o confidence intervals were constructed by muItiplying the group mean baseline LORs by their corresponding standard deviations 28. Any shift in LOR beyond these limits was considered significant.
93
Histology
TABLE II
At the conclusion of testing the animals were given an overdose of Somnotol and perfused with physiological saline followed by 10~o formalin. The brains were removed and stored in the same fixative prior to being sectioned at 40 #m in a cryostat. The cut sections were stained with Cresyl violet and then examined to verify electrode placements. All electrodes (not shown) were found to be localized to the MFB (n = 11) or to the medial wall of the prefrontal cortex ~ (n = 7).
Mean (+ S.D.) MFB (High and low current groups) and MFC response asymptotes following treatment with cis-flupenthixol (mg/kg) Numbers in parentheses refer to % of the baseline asymptotic level. The number of animals in each condition is as in Table 1.
Baseline Saline 0.05
RESULTS
The effects of cis-flupenthixol on MFB and prefrontal ICSS are shown in Tables 1 and 2 and in Fig. 1. It can be seen that the 3 lowest doses of the drug had greater effects on MFB than prefrontal ICSS. At each of these doses the increase in the mean LOR (i.e. reward reduction) was approximately twice as great at the MFB electrode sites (Table I). This was true for both the low and the high current MFB groups. In addition, a number of the MFB rats (mostly in the low current group) ceased responding in the warm-up period and did not complete their
0.1 0,15 0,2
MFB (high)
MFB (low)
48.8 + 7.8 (100%) 50.0 + 10.8 (102.5%) 46.6 + 13.8 (95.5%) 39.6 _+ 14.1 (81.2%) 37.7 + 8.9 (77.3%) 27.7 + 6.5 (56.8%)
58.2 + 11.1 (100%) 69.3 + 23.4 (119.1%) 62.8 + 38.2 (107.9%) 60.5 _+ 31.2 (103.9%) 61.3 + 26.6 (105,3%) 52.5 + 13.2 (90.2%)
49.9 + 16.0 (100%) 50.4 _+ 16.0 (101.0%) 46.0 + 19.2 (92.2%) 36.8 + 8.5 (73.8%) 29.0 + 8.5 (58.0%) -
CM 109 MFE
60 50 .=_ :B
g
g TABLE I
MFC
f ~
40 30
IO
_-/; 25
Mean (+_S.D.) MFB (High and low current groups) and MFC LOR (log units) values following treatment with cis-flupenthixol (mg/kg)
MFC
MFB (high)
MFB (low)
1.47 (7) 1.48 (7) 1.50 (7) 1.53 (7) 1.56 (7) 1.75 (7)
1.53 (4) 1.53 (4) 1.59 (4) 1.66 (4) 1.73 (4) 1.72 (3)
1.75 (7) 1.76 (7) 1.81 (7) 1.85 (4) 1.90 (2) (0)
.o5
20
0
Numbers in parentheses indicate the number of animals completing the test condition. *P < 0.01.
,olne
32
40
L//
50
63
,
7'9
I00
Frequency (HZ)
OC f36 MFC 50
40 30
Baseline Saline 0.05 0.1 0.15 0.2
_+ 0.07 _+ 0.08 _+ 0.1 + 0.11 + 0.12 + 0.09*
+ 0.03 + 0.1 _+ 0.05 _+ 0.08* + 0.06* + 0.06*
+ 0.02 20 •/5
+ 0.07
IO
+ 0.09*
O
+ 0.06* + 0.07*
_
16
T
20
25
32
t 4 0
.
.
.
50
63
79
Frequency (HZ)
Fig. 1. Representative rate-frequency curves illustrating the effects of e/s-flupenthixol (mg/kg) on MFB (low current group) and M F C ICSS. Note that the MFB rat ceases responding at frequencies that the M F C rat continues to press for.
94 evident only at the 0.2 mg/kg dose. With the exception of one rat, the asymptotes of the high current MFB animals were not greatly affected (i.e. <10~o) by the highest dose of cisflupenthixol. At the 0.2 mg/kg dose of cis-flupenthixol, both the MFB and the MFC rats began to show signs of sedation, ptosis and assumed hunched postures.
IlO
9O
% 0 50
30
tO
DISCUSSION
0 105
I
I 15
2
Cis- flupenthixol m g / k g Fig. 2. Mean response levels during the 2-min warm-up period prior to each drug test session. The MFC (solid) and MFB (open) response levels are expressed as a% of the saline control data. The baseline maximal rates of response were 48.8 + 7.8 responses/rain for the MFC group and 49.9 + 16.0 responses/min for the low current MFB group. The high current MFB group (data not shown) had baseline levels of 58.2 _+ 11.1 responses/min.
rate-frequency trial, resulting in an undefined run in several of the drug conditions (Table I). In contrast, the MFC rats responded normally at all but the highest dose of cis-flupenthixol which shifted the rate-frequency curve to the right. All MFC rats responded at several frequencies of each drug trial unlike the MFB rats (Table 1 and Fig. 1). Response levels during the 2-min warm-up period prior to each drug session also revealed differences between the MFC and MFB animals (Fig. 2). The performance of the low current MFB rats declined from 95.3~o at the 0.05 dose of cis-flupenthixol to a low of 47 ~ at the 0.2 mg/kg dose. The performance of the high current MFB animals (data not shown) was 90.6~o at the 0.05 dose and 75.8 ~o at the 0.2 mg/kg dose. The MFC rats maintained near normal responding at all doses of c/s-flupenthixol: 96.8~o at the 0.05 dose and 82.4~ at the 0.2 mg/kg dose. Changes in the asymptotic levels of responding at each dose of c/s-flupenthixol are shown in Table II. A large reduction (i.e. > 4 0 ~ ) in the MFB asymptotes (low current group) occurred at the 0.15mg/kg dose of cis-flupenthixol, whereas similar reductions in the MFC asymptotes were
The most important finding in the present study was the marked difference in sensitivity between MFB and MFC ICSS to neuroleptic challenge. The only dose of cis-flupenthixol that greatly affected prefrontal ICSS was one (0.2 nag/kg) that produced clear signs of sedation. In contrast, low doses of this neuroleptic that had no significant effect on prefrontal ICSS produced significant reductions in MFB reward pulse effectiveness in both the low and high current groups. These differences between MFB and prefrontal sites are especially impressive in view of findings that ammals prefer MFB to prefrontal ICSS over a wide range of supra-threshold current intensities 13-z2. In other words, the relative reward value of MFB stimulation is greater than that of prefrontal stimulation. It follows that MFB tCSS should be more resistant to dopamine receptor blockade than prefrontal ICSS, if dopamine neurons represent some critical synaptic stage in the reward system. The baseline LOR value of the MFC group (1.47 log units) was lower than that of the low current MFB group (1.75 log units) and this fact might be interpreted as indicating that the MFC stimulation was more rewarding or effective than the MFB stimulation. If so, then it would not be surprising that the cL~-flupenthixol had greater effects on MFB ICSS. This interpretation seems unlikely for two reasons. First, the only way to truly equate reward value between different ICSS sites is with a preference paradigm. When such a procedure has been applied to MFC and MFB ICSS such as by Hand and Franklin ~3, it has not been possible to equate MFB and MFC reward in every animal; MFB
95 ICSS appears to be more 'rewarding' than MFC ICSS. Second, in this latter study the stimulation parameters that yielded no clear cut preference between MFB and MFC ICSS were very similar to the parameters used in the present study which yielded nearly a 0.3 log unit difference between MFC and MFB LORS. It may be that like rate of response, the LOR measure is not very useful to gauge differences in reward magnitude between different ICS S sites. However, in order to rule out the possibility that the MFC stimulation was more rewarding than the low current MFB stimulation, a group of MFB animals was tested at high currents that generated LOR values comparable to those of the MFC animals (1.53 versus 1.47 log units respectively). The effects of cis-flupenthixol were again greater in the high current MFB animals than in the MFC animals. Thus it would seem that the most parsimonious explanation of the present findings is that the dopamine systems do not play an important role in prefrontal ICS S, This conclusion is consistent with the results of lesion studies 6,2°,2s, studies involving the intracerebral inactivation of mesolimbic and mesocortical dopamine pathways 23 and from studies examining the sensitivity of prefrontal ICSS to dopamine agonist drugs 26. Overall the data seem to suggest that dopamine's role may be restricted to a ventral MFB-medial brainstem system of ICSS sites. Similarly, a strong case can be made for dopamine mediation of psychomotor stimulant reward although data are not as compelling for other drugs of abuse TM. In the last decade there has been a trend to refine the behavioral paradigms used in ICSS studies such that simple performance effects (e.g. sedation, illness, etc.) can be distinguished from true alterations of reward pulse effectiveness (see refs. 14, 16, 29 for recent reviews). The rate-frequency method used in the present study rates among the best of the presently available reward assessment techniques. However, there still remains some doubt about the ability of these methods to detect 'higher level' performance effects such as drug-induced changes in task difficulty that accompany dopamine dysfunction. It is
thus of interest to note that in the present study a number of the MFB animals ceased responding during the warm-up period preceding rate-frequency testing, while all MFC animals continued responding. In fact, the MFC animals showed near normal performance during the warm-up periods at all doses of cis-flupenthixol. In addition, large changes in MFC asymptotes were observed only at the highest dose of cis-flupenthixol. These observations suggest that the low dose neuroleptic-induced impairments in MFB ICSS cannot be attributed to some higher order performance impairment since the same doses did not significantly interfere with prefrontal ICSS. Thus it is clear that dopamine exerts a special influence on MFB ICSS. The precise nature of dopamine's role in MFB ICSS is still uncertain. Recently it has been reported that with the stimulation parameters most commonly used for ICSS there is little activation of dopamine terminal fields (e.g. nucleus accumbens) as assessed by 2-deoxyglucose metabolic studies 11. Further, it has been demonstrated that square wave stimulation, at short pulse widths (e.g. 0.1 ms), which are extensively used in ICSS research, produces little in the way of dopamine release as detected by in vivo voltammetry 17. Interestingly, sine-wave stimulation does result in significant dopamine release. These data raise the possibility that dopamine's role in brain stimulation reward may be of a permissive, modulatory nature ~1, a view already the subject of much research 9. These recent studies and the data reported here support the view of multiple reward systems ~9 rather than a single dopamine-gated reward system. ACKNOWLEDGEMENTS This research was supported by NSERC grant, A 1198, awarded to D.C. The author is grateful to Judy Furlong and Cynthia Mercer for technical assistance and to Dr. O. Svendsen of H. Lundbeck A/S, Copenhagen, Denmark for a generous supply of cis-flupenthixol. Carolyn Harley and Richard Neuman provided useful comments on an earlier draft of this paper.
96 REFERENCES 1 Beckstead, R.M., An autoradiographic examination of corticocortical and subcortical projections of the mediodorsal-projection (prefrontal) cortex in the rat, J. Comp. Neurol., 184 (1979) 43-62. 2 Bozarth, M.A., Neural basis of psychomotor stimulant and opiate reward: evidence suggesting involvement of a common dopaminergic system, Behav. Brain Res., 22 (1986) 107-116. 3 Bozarth, M.A. and Wise R.A., Intracranial self-administration of morphine into the ventral tegmental area, Life Sci., 28 (1981) 551-555. 4 Bozarth, M.A. and Wise, R.A., Heroin reward is dependent on a dopaminergic substrate, Life Sci., 29 (1981) 1881-1886. 5 Campbell, K.A., Evans, G. and Gallistel, C.R., A microcomputer-based method for physiologicallyinterpretable measurement of the rewarding efficacy of brain stimulation, Physiol. Behav,, 35 (1985) 395-403. 6 Corbett, D., Laferri6re, A. and Milner, P.M., Elimination of medial prefrontal cortex self-stimulation following transection of efferents to the sulcal cortex in the rat, Physiol. Behav., 29 (1982) 425-431. 7 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) i-15. 8 Edmonds, D.E. and Gallistel, C.R., Reward versus performance in self-stimulation: electrode-specific effects of alpha-methyl-p-tyrosine on reward in the rat, J. Comp. Physiol. PsychoL, 91 (1977) 962-974. 9 Fibiger, H.C., LePaine, F.G., Jakubovic, A. and Phillips, A.G., The role of dopamine in intracranial self-stimulation of the ventral tegmental area, J. Neurosci., 7 (1988) 3888-3896. 10 Gallistel, C.R. and Freyd, G., Quantitative determination of the effects of catecholaminergic agonists and antagonists on the rewarding efficacy of brain stimulation, Pharmacol. Biochem. Behav., 26 (1987) 731-741. 11 Gallistel, C.R., Gomita, Y., Yadin, E. and Campbell, K.A., Forebrain origins and terminations of the medial forebrain bundle metabolically activated by rewarding stimulation or by reward blocking doses of pimozide, J. Neurosci., 5 (1985) 1246-1261. 12 Gerfen, C.R., Staines, W.A., Clavier, R.M. and Fibiger, H.C., Neuroanatomical studies ofintracranial self-stimulation in the rat. In B.G. Hoebel and D. Novin (Eds.), The Neural Basis of Feeding and Reward, Haer Institute, Brunswick, ME, 1982, pp. 305-322. 13 Hand, T.H. and Franklin, K.B.J., The influence of amphetamine on preference for lateral hypothalamic versus prefrontal cortex or ventral tegmental area self-stimulation, Pharmacol. Biochem. Behav., 18 (1983)695-699. 14 Liebman, J.M., Discriminating between reward and performance: a critical review ofintracranial self-stimulation methodology, Neurosci. Biobehav. Rev., 7 (1983) 45-72. 15 Lyness, W.H., Friedle, N.M. and Moore, K.E., Destruction of dopaminergic nerve terminals in nucleus accumbens: effect on d-amphetamine self-administration, Pharmacol. Biochem. Behav., 11 (1979)553-556.
16 Miliaressis, E., Rompre, P.-P., Laviolette, P., Philippe, 1.. and Coulombe, D., The curve-shift paradigm in sell'stimulation, Physiol. Behav., 37 (1986) 85-91. 17 Millar, J., Stamford, J.A., Kruk, Z.L. and Wightman, R.M., Electrochemical, pharmacological, and electrophysiological evidence of rapid dopamine release and removal in the rat caudate nucleus following electrical stimulation of the medial forebrain bundle, Eur..L PharmacoL, 109 (1985) 341-348. 18 Mogenson, G.J., Takigawa, M., Robertson, A. and Wu, M., Self-stimulation of the nucleus accumbens and ventral tegmental area of Tsai attenuated by microinjections of spiroperidol into the nucleus accumbens, Brain Res., 171 (1979) 247-259. 19 Phillips, A.G., Brain reward circuitry: a case for separate systems, Brain Res. Bull., 12 (1984) 195-201. 20 Phillips, A.G. and Fibiger, H.C., The role of dopamine in maintaining intracranial self-stimulation in the ventral tegmentum, nucleus accumbens, and medial prefrontal cortex, Can. J. Psychol., 32 (1978) 58-66. 21 Roberts, D.C.S., Koob, G.F., Klonoff, P. and Fibiger, H.C., Extinction and recovery of cocaine self-administration following 6-hydroxydopamine lesions of the nucleus accumbens, Pharmacol. Bioehem. Behav., 12 (1980) 781-787. 22 Robertson, A., Laferri6re, A. and Franklin, K.B.J., Amphetamine and increases in current intensity modulate reward in the hypothalamus and substantia nigra but not in the prefrontal cortex, PhysioL Behav., 28 (1981) 869-872. 23 Robertson, A. and Mogenson, G.J., Evidence for a role for dopamine in self-stimulation of the nucleus accumbens of the rat, Can. J. Psychol., 32 (1978) 67-76. 24 Silverman, J.A. and Corbett, D., Prefrontal cortex lesions attenuate substantia nigra self-stimulation: a reward summation analysis, Behav. Brain Res., 32 (1989) 43-50. 25 Simon, H., Stinus, L., Tassin, J., Lavietle, S., Blanc, G., Thierry, A.M., Glowinski, J. and LeMoal, M., Is the dopaminergic mesocorticolimbic system necessary for intracranial self-stimulation? Behav. Neur. BioL, 27 (1979) 125-145. 26 Spence, S.J., Silverman, J.A. and Corbett, D,, Cortical and ventral tegmental systems exert opposing influences on self-stimulation from the prefrontal cortex, Behav. Brain Res., 17 (1985) 117-124. 27 Stellar, J.R. and Corbett, D., Effects of regional neuroleptic infusion suggest a role for nucleus accumbens in lateral hypothalamic self-stimulation reward, Brain Res., 477 (1989) 126-143. 28 Stellar, J.R., Kelley, A.E. and Corbett, D., Effects of peripheral and central dopamine blockade on lateral hypothalamic self-stimulation: evidence for both reward and motor deficits, Pharmacol. Biochem. Behav, 18 (1983) 433-442. 29 Stellar, J.R. and Stellar, E., The Neurobiology of Motivation and Reward, Springer, New York, 1985. 30 Wise, R.A., Action of drugs of abuse on brain reward systems, Pharmacol. Biochem. Behav., 13 Suppl. 1, (1980) 213-223. 31 Wise, R.A. and Bozarth, M.A., A psychomotor stimulant theory of addiction, Psychol. Rev., 94 (1987) 469-492.
Behavioural Brain Research, 36 (1990) 91-96 Elsevier BBR00989
Differences in sensitivity to neuroleptic blockade: medial forebrain bundle versus frontal cortex self-stimulation Dale Corbett Division of Basic Medical Sciences and Department of Psychology, Memorial University of Newfoundland, St. John's, N.F. (Canada) (Received 17 April 1989) (Accepted 25 April 1989)
Key words: Self-stimulation; Prefrontal cortex; Medial forebrain bundle; Dopamine
The effects of systemic injections of the dopamine receptor antagonist, c/s-flupenthixol were tested on intracranial self-stimulation at electrode sites in the medial forebrain bundle and the medial prefrontal cortex. Changes in the reward effectiveness of the brain stimulation were assessed using a curve-shift paradigm. Low to moderate doses of cis-flupenthixol (0.05, 0.1 and 0.15 mg/kg) consistently produced larger upward shifts in the rate-frequency function for medial forebrain bundle than for medial prefrontal self-stimulation. At the highest doses of c/s-flupenthixol (0.15 and 0.2 mg/kg), some of the medial forebrain bundle rats failed to respond, whereas all medial prefrontal rats responded at these doses. These results demonstrate that medial forebrain bundle self-stimulation is much more dependent on dopamine systems than is prefrontal cortex self-stimulation.
INTRODUCTION
It is now well established that dopamine neurons play an important role in the mediation of intracranial self-stimulation (ICSS) and psychomotor stimulant r e w a r d 2-4,7A5,21,3°,31. With regard to the relative importance of the different dopamine pathways, it would seem that it is the mesolimbic dopamine pathway that underlies the above reward effects. Lesions or intracerebral receptor blockade of this pathway attenuates ICS S and stimulant self-administration 3,15,18,2°,21,23. However, there is some question as to the importance of the mesolimbic dopamine system at brain stimulation reward sites distal to the medial forebraln bundle (MFB) since lesions of the dopamine pathways have little effect on medial
(MFC) 6,20,25 or substantia nigra ICS S 12. Instead, corticofugal projections seem to be of more importance at these two ICS S sites 6"24. In addition, whereas intracerebral inactivation of the dopamine projections to the nucleus accumbens produce a significant impairment of MFB ICSS ~s'27,2s, they do not markedly affect MFC ICSS 23. Nonetheless, it remains possible that other terminal projections of the mesolimbic dopamine system (e.g. diagonal band nuclei, amygdala) are critical for M F C ICS S. To test this possibility animals with either MFB or MFC ICSS electrodes were given systemic injections of the dopamine antagonist cis-flupenthixol. It was reasoned that if MFC ICSS is less dependent on a dopaminergic substrate than MFB ICSS, this should be reflected as a reduced senprefrontal
Correspondence: D. Corbett, Division of Basic Medical Sciences, Faculty of Medicine, Memorial University of Newfoundland, St. John's, N.F. Canada, A1B 3V6. 0166-4328/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
92 sitivity of MFC ICSS to dopaminergic receptor blockade. MATERIALS AND METHODS
Surgery Eighteen male Sprague-Dawley rats weighing 300-350 g at the time of surgery were implanted with 250 #m stainless steel, monopolar electrodes (Plastic Products Inc.) under Somnotol anesthesia (65mg/kg) in combination with 2~o Xylocalne. Eleven rats had electrodes aimed at the MFB (ant. - 2 . 3 mm, lat. + 2 . 0 m m and depth - 8 . 8 mm from skull surface) while the remaining 7 animals had electrodes aimed at the M F C (ant. + 4.2 mm, lat. + 0.6 mm and depth - 3.5 mm from skull surface). Stereotaxic coordinates were derived from bregma with the skull level. All rats were housed individually in plastic cages and maintained on a 12: 12h reverse day-night cycle. Food and water were freely available. Following a minimum of 3 days of postoperative recovery, the rats were trained to leverpress for brain stimulation.
Behavioral testing The rats were trained to press a lever in order to deliver 0.5-s trains of 0.1-ms monophasic, cathodal pulses delivered at a rate of 100 Hz. Currents were adjusted individually to ensure vigorous responding. Two groups of MFB animals were used in this experiment, a high current group (405 + 83.9#A) and a low current (225.7 + 61 #A) group. Testing was conducted in Plexiglas and stainless steel chambers (30 cm × 24 cm × 24 cm) equipped with a retractable lever mounted on a side wall. Once the animals responded consistently, rate-frequency testing began. This phase of testing consisted of lowering the frequency from a high level (e.g. 100 Hz) every 60 s in 0.1 log unit steps until the animal ceased responding. At the end of each frequency presentation the lever retracted for 5 s, the frequency was lowered to the next level, the lever extended and one free priming stlmulataon was delivered to the animal. Delivery of the stimulation parameters and recording of lever-presses was performed by an Apple IIe ,
.
J
computer system 5. This procedure yields a sigmoid-shaped curve from which one can derive the frequency that produces half-maximal responding. This value has been termed the locus of rise (LOR) and has been shown to be sensitive to manipulations that alter the reward value of the stimulation (e.g. current changes, amphetamine) but only minimally affected by treatments (e.g. non-specific lesions, partial paralysis) that disturb performance capability 8'~°'16"29. Test sessions were preceded by a 2-min warm-up period. When each animals' LOR varied less than 0.1 log unit over 3 consecutive days, the animals were considered stable. The MFB animals achieved this stability criterion after 19.1 _+ 6.34 (S.D.) days. The frontal group took an average of 29.0 + 8.78 (S.D.) days to meet the same criterion. The LOR values from these 3 days were used to derive the mean baseline LOR for each animal. The group baseline LOR values were used to assess all drug effects (see below). The animals were then pretreated with one of several doses of cis-flupenthixol (0.05, 0.1, 0.15 and 0.2mg/kg). Cis-flupenthixol was dissolved in physiological saline and was injected intraperitoneally 4 h prior to the test session. Control injections consisted of equal volumes of saline or the inactive trans-flupenthixot isomer. A minimum of 2 days separated each drug injection. Animals were not injected with drug if the LOR values on non-drug days varied by more than 0.1 log unit from the original mean baseline LOR value. Instead, the animals were tested until the LOR values returned to baseline levels. The data were analyzed by an Apple IIe computer. A least squares procedure was used to find the best fit for each rate-frequency function. On some test sessions the LOR was undefined (U). This occurred when the response rates were either zero or uniformly low across atl frequencies. Such conditions were typically encountered with the highest doses of cis-flupenthixol. Modified 99~o confidence intervals were constructed by muItiplying the group mean baseline LORs by their corresponding standard deviations 28. Any shift in LOR beyond these limits was considered significant.
93
Histology
TABLE II
At the conclusion of testing the animals were given an overdose of Somnotol and perfused with physiological saline followed by 10~o formalin. The brains were removed and stored in the same fixative prior to being sectioned at 40 #m in a cryostat. The cut sections were stained with Cresyl violet and then examined to verify electrode placements. All electrodes (not shown) were found to be localized to the MFB (n = 11) or to the medial wall of the prefrontal cortex ~ (n = 7).
Mean (+ S.D.) MFB (High and low current groups) and MFC response asymptotes following treatment with cis-flupenthixol (mg/kg) Numbers in parentheses refer to % of the baseline asymptotic level. The number of animals in each condition is as in Table 1.
Baseline Saline 0.05
RESULTS
The effects of cis-flupenthixol on MFB and prefrontal ICSS are shown in Tables 1 and 2 and in Fig. 1. It can be seen that the 3 lowest doses of the drug had greater effects on MFB than prefrontal ICSS. At each of these doses the increase in the mean LOR (i.e. reward reduction) was approximately twice as great at the MFB electrode sites (Table I). This was true for both the low and the high current MFB groups. In addition, a number of the MFB rats (mostly in the low current group) ceased responding in the warm-up period and did not complete their
0.1 0,15 0,2
MFB (high)
MFB (low)
48.8 + 7.8 (100%) 50.0 + 10.8 (102.5%) 46.6 + 13.8 (95.5%) 39.6 _+ 14.1 (81.2%) 37.7 + 8.9 (77.3%) 27.7 + 6.5 (56.8%)
58.2 + 11.1 (100%) 69.3 + 23.4 (119.1%) 62.8 + 38.2 (107.9%) 60.5 _+ 31.2 (103.9%) 61.3 + 26.6 (105,3%) 52.5 + 13.2 (90.2%)
49.9 + 16.0 (100%) 50.4 _+ 16.0 (101.0%) 46.0 + 19.2 (92.2%) 36.8 + 8.5 (73.8%) 29.0 + 8.5 (58.0%) -
CM 109 MFE
60 50 .=_ :B
g
g TABLE I
MFC
f ~
40 30
IO
_-/; 25
Mean (+_S.D.) MFB (High and low current groups) and MFC LOR (log units) values following treatment with cis-flupenthixol (mg/kg)
MFC
MFB (high)
MFB (low)
1.47 (7) 1.48 (7) 1.50 (7) 1.53 (7) 1.56 (7) 1.75 (7)
1.53 (4) 1.53 (4) 1.59 (4) 1.66 (4) 1.73 (4) 1.72 (3)
1.75 (7) 1.76 (7) 1.81 (7) 1.85 (4) 1.90 (2) (0)
.o5
20
0
Numbers in parentheses indicate the number of animals completing the test condition. *P < 0.01.
,olne
32
40
L//
50
63
,
7'9
I00
Frequency (HZ)
OC f36 MFC 50
40 30
Baseline Saline 0.05 0.1 0.15 0.2
_+ 0.07 _+ 0.08 _+ 0.1 + 0.11 + 0.12 + 0.09*
+ 0.03 + 0.1 _+ 0.05 _+ 0.08* + 0.06* + 0.06*
+ 0.02 20 •/5
+ 0.07
IO
+ 0.09*
O
+ 0.06* + 0.07*
_
16
T
20
25
32
t 4 0
.
.
.
50
63
79
Frequency (HZ)
Fig. 1. Representative rate-frequency curves illustrating the effects of e/s-flupenthixol (mg/kg) on MFB (low current group) and M F C ICSS. Note that the MFB rat ceases responding at frequencies that the M F C rat continues to press for.
94 evident only at the 0.2 mg/kg dose. With the exception of one rat, the asymptotes of the high current MFB animals were not greatly affected (i.e. <10~o) by the highest dose of cisflupenthixol. At the 0.2 mg/kg dose of cis-flupenthixol, both the MFB and the MFC rats began to show signs of sedation, ptosis and assumed hunched postures.
IlO
9O
% 0 50
30
tO
DISCUSSION
0 105
I
I 15
2
Cis- flupenthixol m g / k g Fig. 2. Mean response levels during the 2-min warm-up period prior to each drug test session. The MFC (solid) and MFB (open) response levels are expressed as a% of the saline control data. The baseline maximal rates of response were 48.8 + 7.8 responses/rain for the MFC group and 49.9 + 16.0 responses/min for the low current MFB group. The high current MFB group (data not shown) had baseline levels of 58.2 _+ 11.1 responses/min.
rate-frequency trial, resulting in an undefined run in several of the drug conditions (Table I). In contrast, the MFC rats responded normally at all but the highest dose of cis-flupenthixol which shifted the rate-frequency curve to the right. All MFC rats responded at several frequencies of each drug trial unlike the MFB rats (Table 1 and Fig. 1). Response levels during the 2-min warm-up period prior to each drug session also revealed differences between the MFC and MFB animals (Fig. 2). The performance of the low current MFB rats declined from 95.3~o at the 0.05 dose of cis-flupenthixol to a low of 47 ~ at the 0.2 mg/kg dose. The performance of the high current MFB animals (data not shown) was 90.6~o at the 0.05 dose and 75.8 ~o at the 0.2 mg/kg dose. The MFC rats maintained near normal responding at all doses of c/s-flupenthixol: 96.8~o at the 0.05 dose and 82.4~ at the 0.2 mg/kg dose. Changes in the asymptotic levels of responding at each dose of c/s-flupenthixol are shown in Table II. A large reduction (i.e. > 4 0 ~ ) in the MFB asymptotes (low current group) occurred at the 0.15mg/kg dose of cis-flupenthixol, whereas similar reductions in the MFC asymptotes were
The most important finding in the present study was the marked difference in sensitivity between MFB and MFC ICSS to neuroleptic challenge. The only dose of cis-flupenthixol that greatly affected prefrontal ICSS was one (0.2 nag/kg) that produced clear signs of sedation. In contrast, low doses of this neuroleptic that had no significant effect on prefrontal ICSS produced significant reductions in MFB reward pulse effectiveness in both the low and high current groups. These differences between MFB and prefrontal sites are especially impressive in view of findings that ammals prefer MFB to prefrontal ICSS over a wide range of supra-threshold current intensities 13-z2. In other words, the relative reward value of MFB stimulation is greater than that of prefrontal stimulation. It follows that MFB tCSS should be more resistant to dopamine receptor blockade than prefrontal ICSS, if dopamine neurons represent some critical synaptic stage in the reward system. The baseline LOR value of the MFC group (1.47 log units) was lower than that of the low current MFB group (1.75 log units) and this fact might be interpreted as indicating that the MFC stimulation was more rewarding or effective than the MFB stimulation. If so, then it would not be surprising that the cL~-flupenthixol had greater effects on MFB ICSS. This interpretation seems unlikely for two reasons. First, the only way to truly equate reward value between different ICSS sites is with a preference paradigm. When such a procedure has been applied to MFC and MFB ICSS such as by Hand and Franklin ~3, it has not been possible to equate MFB and MFC reward in every animal; MFB
95 ICSS appears to be more 'rewarding' than MFC ICSS. Second, in this latter study the stimulation parameters that yielded no clear cut preference between MFB and MFC ICSS were very similar to the parameters used in the present study which yielded nearly a 0.3 log unit difference between MFC and MFB LORS. It may be that like rate of response, the LOR measure is not very useful to gauge differences in reward magnitude between different ICS S sites. However, in order to rule out the possibility that the MFC stimulation was more rewarding than the low current MFB stimulation, a group of MFB animals was tested at high currents that generated LOR values comparable to those of the MFC animals (1.53 versus 1.47 log units respectively). The effects of cis-flupenthixol were again greater in the high current MFB animals than in the MFC animals. Thus it would seem that the most parsimonious explanation of the present findings is that the dopamine systems do not play an important role in prefrontal ICS S, This conclusion is consistent with the results of lesion studies 6,2°,2s, studies involving the intracerebral inactivation of mesolimbic and mesocortical dopamine pathways 23 and from studies examining the sensitivity of prefrontal ICSS to dopamine agonist drugs 26. Overall the data seem to suggest that dopamine's role may be restricted to a ventral MFB-medial brainstem system of ICSS sites. Similarly, a strong case can be made for dopamine mediation of psychomotor stimulant reward although data are not as compelling for other drugs of abuse TM. In the last decade there has been a trend to refine the behavioral paradigms used in ICSS studies such that simple performance effects (e.g. sedation, illness, etc.) can be distinguished from true alterations of reward pulse effectiveness (see refs. 14, 16, 29 for recent reviews). The rate-frequency method used in the present study rates among the best of the presently available reward assessment techniques. However, there still remains some doubt about the ability of these methods to detect 'higher level' performance effects such as drug-induced changes in task difficulty that accompany dopamine dysfunction. It is
thus of interest to note that in the present study a number of the MFB animals ceased responding during the warm-up period preceding rate-frequency testing, while all MFC animals continued responding. In fact, the MFC animals showed near normal performance during the warm-up periods at all doses of cis-flupenthixol. In addition, large changes in MFC asymptotes were observed only at the highest dose of cis-flupenthixol. These observations suggest that the low dose neuroleptic-induced impairments in MFB ICSS cannot be attributed to some higher order performance impairment since the same doses did not significantly interfere with prefrontal ICSS. Thus it is clear that dopamine exerts a special influence on MFB ICSS. The precise nature of dopamine's role in MFB ICSS is still uncertain. Recently it has been reported that with the stimulation parameters most commonly used for ICSS there is little activation of dopamine terminal fields (e.g. nucleus accumbens) as assessed by 2-deoxyglucose metabolic studies 11. Further, it has been demonstrated that square wave stimulation, at short pulse widths (e.g. 0.1 ms), which are extensively used in ICSS research, produces little in the way of dopamine release as detected by in vivo voltammetry 17. Interestingly, sine-wave stimulation does result in significant dopamine release. These data raise the possibility that dopamine's role in brain stimulation reward may be of a permissive, modulatory nature ~1, a view already the subject of much research 9. These recent studies and the data reported here support the view of multiple reward systems ~9 rather than a single dopamine-gated reward system. ACKNOWLEDGEMENTS This research was supported by NSERC grant, A 1198, awarded to D.C. The author is grateful to Judy Furlong and Cynthia Mercer for technical assistance and to Dr. O. Svendsen of H. Lundbeck A/S, Copenhagen, Denmark for a generous supply of cis-flupenthixol. Carolyn Harley and Richard Neuman provided useful comments on an earlier draft of this paper.
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