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Psychophysical method for mapping behavioral substrates using a moveable electrode
Brain Research Bulletin, Vol. 8, pp. 693-701, 1982. Printed in the U.S.A.
Psychophysical Method for Mapping Behavioral Substrates Using a Moveable Electrode’ E. MILIARESSIS,
Ecole de Psychologie,
P. P. ROMPRfi
AND A. DURLVAGE
35 McDougal Lane, Universitt d’ottawa, Received
21 December
Ottawa, KIN 6N5 Canada
1981
MILIARESSIS, E., P. P. ROMPRI? AND A. DURIVAGE. Psychophysical method for mapping behavioral substrates using a moveable electrode. BRAIN RES. BULL. 8(6) 693-701, 1982.-With the use of moveable electrodes, 28 rat mesencephalic sites were examined for self-stimulation behavior (SS). The relative importance of each site in SS was established according to (1) a traditional method which consists of comparing sites based on the rates of responding (2) a psychophysical procedure based on the pulse frequency required at each site in order for the stimulation to elicit a criterion behavioral performance. It was shown that anatomical conclusions reached by the use of behavioral output procedures depend purely on the arbitrary choice of stimulation parameters. It was also demonstrated that the combination of moveable electrodes with psychophysical measurements results in enhanced mapping resolution and enables one to trace the boundaries of behaviorally relevant structures and pathways with significantly better confidence.
Moveable electrodes
Brain stimulation
Brain mapping
AN important goal in brain mapping research is the identification of behaviorally relevant neuronal nuclei and pathways. This task can be accomplished with variable accuracy depending on the availability of efficient techniques and procedures. For instance, rough localization of relevant structures in large brain areas can be performed by electrical stimulation using fixed electrodes and simple behavioral measurements. However, fine delineation of critical boundaries within a circumscribed brain region is not attainable by these techniques. First, there is a great deal of uncertainty about the location of stimulated sites when across-animal comparisons are made. Second, establishment of brain mapping based on behavioral responding following stimulation is subject to significant distortion due to the lack of linearity between stimulation strength and behavioral output (behavioral floor and ceiling properties). In the present work we will show that when the strength of the stimulation is held constant in all stimulated sites, the resulting anatomical conclusions will be dependent on the arbitrary choice of stimulation parameters. On the other hand, if current intensity is varied from site to site so that the maximum behavioral output is examined, a good deal of anatomical resolution will be lost due to lack of precision concerning current spread. While rough localization of behaviorally-relevant sites in large brain areas has been a fruitful1 task for the last fifty years, correlations with some presently well identified pathways (for instance monoaminergic bundles) would require application of stimulation techniques which can provide one with significantly better anatomical resolution.
Self-stimulation
Psychophysical measurements
In the present work, some of the limitations of traditional mapping techniques will be examined in the light of data obtained in self-stimulation behavior (SS) in the rat. In addition, alternative solutions consisting basically of the use of moveable electrodes in combination with psychophysical measurements will be proposed.
METHOD
Subjects
and Surgery
Under general anesthesia with Nembutal (40 mg/kg), three male, adult Sprague-Dawley rats were implanted with a small moveable electrode (wire dia. 0.25 mm) described elsewhere [9]. With the skull held horizontal between bregma and lambda, stereotaxic coordinates were as follows: Rat 699,0.6 mm rostral to lambda, 0.0 mm lateral to the midline, 7.0 mm below the skull surface; Rat 711, 1.5 mm, 0.0, 7.0; Rat 713, 0.2 mm, 0.0, 7.7.
Stimulation
Parameters
Electrical stimulation of the brain consisted of trains (400 msec in duration) of cathodal pulses of constant intensity (400 PA) and duration (0.1 msec) and variable frequency. The stimulation was delivered by a constant-current generator [ 101 triggered by depression of a lever located in a transparent SS box. Self-administration of the stimulation was subject to an FI, 1 set schedule.
‘The authors are supported by NSERC (E. M.), FCAC (P. P. R.) and OGS (A. D.).
Copyright @ 1982 ANKHO
International Inc .-0361-9230/82/060693-09$03.00/O
MILIARESSIS,
694
Nil
ROMPRE AND DURIVAGE
Low
Medium
High
RAT 699
Stimulation
effectiveness
according
B
C
I :
!
: : +
: : i
61
13
7
,‘4
$6 Number
of pulses/train
FIG. 1. Self-stimulation responding as a function of the number of pulses per stimulation train for 7 brain sites in rat 699. Each site is identified by a number which refers to the position of the electrode shown in Fig. 7. The intersections of lines A, B and C with each curve show the amount of responding that would have been observed by three hypothetical investigators, each one using a different, single fixed frequency (16, 22 and 30 pulses/train respectively). Line D illustrates the use of a constant-behavior procedure. The right pane1 of the figure shows the stimulation effectiveness per brain site, as inferred by investigators A, B, C and D.
Procedure Following two weeks of post-operative recovery, rats were introduced into the SS box. Using a starting pulse frequency of 50 Hz, the investigator attempted to shape the animal for SS by triggering the stimulation each time the animal approached the lever. If the attempt was not successfil, the frequency was gradually increased up to 100 Hz. If this maximum frequency failed to elicit SS, the testing procedure for this brain site was considered complete. The electrode was then lowered down by 160 Frn and the animal tested again 24 hr later using the same procedure. Once SS was observed in a firs& site, the electrode was kept in the same position for one week. During this period the animal was allowed to self-stimulate for two consecutive hours per day, at a rate of approximately 75% of his maximum performance. This relatively long training period applied to the first SS location was intended to insure complete learning of the task before testing subsequent sites. Following completion of the training period, pulse frequency/SS response functions (F/R functions) were determined according to the following procedure: The rate of SS responses for each frequency was examined in 3 min trials separated by 2 min of rest. The pulse frequency was varied in ascending, then in descending order. Its total range was large enough to cover the animal’s complete responding capability, from zero to the maximum performance. Immediately before each 3 min
test, the animal was primed with a few stimulations and the testing period was initiated as soon as the animal made the first SS response. After a maximum of 20 primes, the testing period began even if the rat failed to resume SS. The mean of the ascending and descending series was used to draw the F/R function for the brain site under investigation. At the completion of the test, the electrode was lowered by 160 pm and the new site was examined 24 hr later according to the same procedure. At the end of the experiments, the animals were sacrificed by an overdose of Nembutal and their brains removed and stored for three days in 10% Fotmalin. Following this period, the brains were sliced (28 &rn thick) in a cryostat. The wet brain sections containing the track of the electrodes were examined under microscope immediately following the slicing process. RESULTSAND DISCUSSION
The left panel of Fig. 1 shows SS data from rat 699. The number of reinforcements per test period was plotted as a function of the number of pulses per stimulation train. Each curve was obtained from a different dorso-ventral position of the electrode. The lines labelled A, B and C are intended to show that divergent anatomical conclusions might havebeen reached by three investigators using different stimulation parameters. Investigators A, B and C use basically the same experimental paradigm: the effectiveness of the stimulation
695
BRAIN MAPPING
effectiveness
Stimulation
according to
Ai\ A
B
C
D
3 3 4 ._ In 5 .cZ 6 m 7
Number
of pulses / train
FIG. 2. Self-stimulation responding as a function of the number of pulses per stimulation train for 10 brain sites in rat 711. Further details in Fig. 1.
at each brain site is inferred by the behavioral performance obtained by the use of constant intensity (400 PA) and a single, fixed pulse frequency (16, 22 and 30 pulses/train respectively). In other words, the complete F/R functions were not examined as in this figure. The behavioral performance that would have been obtained by these investigators is shown by the intersection of lines A, B and C with each curve. Thus, investigator A, using a fixed number of pulses of 16 would have concluded that only site 5 supports SS behavior. On the other hand, investigator B would have been unable to detect any difference between sites 1 to 3 and sites 4 to 7. At the other extreme, investigator C, using a higher pulse frequency would have concluded that the stimulation has the same effectiveness at all seven sites. Line D illustrates a different procedure as used by a fourth investigator. In this case, the overall F/R curves were first obtained for each individual site (curves shown in Fig. 1). The stimulation effectiveness (E) at each brain site was then inferred by the pulse period required to elicit a constant amount of responding, arbitrarily set at 70 reinforcements per test period (line D). The right panel of the figure illustrates the comparative anatomical picture of this experiment as obtained by the four hypothetical investigators. For A, B and C, the stimulation effectiveness at each site is labelled high, medium, low or nil, as usually found in the literature. In the case of D, the density corresponding at each site was made proportional to the pulse period required to elicit the criterion performance. Figure 2 demonstrates essentially the same phenomenon in a different rat. The right panel of the figure shows the expected marked discrepencies due to the lack of dis-
criminability associated with the procedure used by the first three investigators. Figure 3 shows data from a third animal. Since the inconsistencies due to direct output measurements can be easily replicated, the data of rat 713 will instead be used to illustrate problems associated with the constant-behavior procedure. First, there is a progressive depression of the behavioral ceiling throughout sites 6 to 11. Observation of the animal during testing suggests that this phenomenon was due to contamination of SS by simultaneous stimulus-bound motoric activity. Second, concomitant to the ceiling depression, there seems to be an abnormal reduction in curve slopes. This means that in each curve, the slope reduction is higher than what should be expected by calculation of Weber’s fraction (see discussion). The consequence of this phenomenon is that calculations of the relative stimulation effectiveness between sites will be dependent on the arbitrary choice of the criterion performance. The severity of this artifact in the case of rat 713 is documented in Fig. 4. The largest variation occurs at site 11 where E shifts from 0.37 to 0.22 when the criterion performance varies from zero to 70 reinforcements. In ordei to circumvent this difficulty, calculation of E values was also attempted by the use of the following criteria: (a) Edmond and Gallistel’s constant proportion (for instance 50% of the maximum performance or MSO) and (b) theoretical zero behavior. In both cases, the required number of pulses was calculated by the use of the linear regressions of each FIR curve. For instance, the required number of pulses corresponding to the theoretical zero behavior (or eo) was obtained by the intersection of each re-
MILIARESSIS,
696
ROMPRk
AND DURIVAGE
2
RAT
713
/
MY----’
Number
of
pulses
/train
.
FIG. 3. Self-stimulation responding as a function of the number of pulses per stimulation train for 11 brain sites in rat 713. Note the progressive ceiling depression through sites 6 to 11.
RAT
713
Bre in
.
sites
.
m .
7
6
9 10 11
r
0
6
,
70
10 Criterion
(Reinforcements
/ 3 min.)
FIG. 4. Relative stimulation effectiveness (E) at brain sites 6 to 11 as a function of the behavioral criterion. For a given site X, E was calculated using the ratio, Pulse period required at site X/Pulse period required at sites 1 to 5 (pooled together).
BRAIN
697
MAPPING
8
l 0
l 0
l 0 0 0
FIG. 5. Relative stimulation effectiveness at sites 6 to 11of rat 713 as inferred by the use of M50 and Go procedures. For explanation see
text.
gression line with the abscissa. The rationale of 80 is ilhtstrated in Fig. 6 and will be developed in the discussion. Figure 5 shows the relative stimulation effectiveness in sites 6 to 11 (compared to sites 1 to 5 pooled together) as obtained by these two procedures. It can be observed that these methods generate similar results with however slightly lower estimates for the MS0 procedure. We suspect that this difference was due to a small artificial ceiling depression, mainly exerted on the reference sites,_due to the constraint imposed by the use of the FI-1 sec. schedule. This means that the sites that are subject to this effect will show higher than normal stimulation effectiveness, for the artifactual ceiling depression will contribute to provide lower pulse numbers for the MS0 criterion. In the case of rat 713, the above schema also predicts that the consequent E reductions will roughly increase throughout sites 6 to 11. Figures 7,8 and 9 show the successive electrode positions for each rat as well as the required pulse periods obtained by the aforementioned two procedures. It can be observed that both techniques seem to be sensitive to smalI changes in stimulation effectiveness. Especially in the case of rats 711 and 713 where the brain tested areas were large enough, the U-shaped progressive changes in the required pulse period provide a fairly detailed picture of the relative importance of each site in SS behavior. GENERAL
DISCUSSION
The purpose of the present work was to examine some of
CO FIG. 6. Simulation of two f~que~cy/res~nse curves in selfstimulation, obtained in hypothetical sites A and B. The solid lines represent the regression of the linear portion of each curve. Like the empirical data of rat 713, (see results), site B shows a ceiling depression concomittant to a reduction in the intercept (C,) on the axis of behavior. The broken line shows the theoretical position of the regression line of B in the absence of motoric cont~ination. Theta zero is the intercept of the regression line of B on the axis of pulses. Note that this intercept is common to the theoretical and empirical regression lines. The theoretical (broken) line was obtained by pivoting the regression of B on 80 point so that C, joins Co.
the limitations associated with the use of different brain mapping procedures and to propose alternative solutions. Some relevant questions are discussed below. Are Behavioral Output Measurements
of Any Use?
Before answering this question it should be recalled that there are basically two approaches associated with the use of the observed behavioral performance as the dependent variable. First, the use of fixed stimulation strength and second, the use of variable strength obtained by variations in pulse frequency or pulse intensity. In the present work we showed that the use of fixed pulse intensity and frequency leads to a variety of mapping conclusions depending on the arbitrary choice of the stimulation parameters. Distortion of refractory period measurements due to the use of fixed stimulation parameters has also been demonstrated earlier by Yeomans
MILIARESSIS,
ROMPRI? AND DURIVAGE
4’ s6’
-’
0
50
30
10
Pulse
Period
msec
FIG. 7. Schematic representation of a brain section showing the successive dorso-ventral positions of the electrode in rat 699. The right panel of the figure shows the pulse periods required at each stimulated site as inferred by the use of M50 and GO procedures (see text). The effectiveness of the stimulation within the investigated area is also represented by a density spectrum at the middle of the figure. Abbreviations: cc: crus cerebri; Im: lemniscus medialis; mr: nucleus medianus raphe; PCS: pedunculus cerebellaris superior.
Pulse
Period
(msec)
FIG. 8. Schematic representation of a brain section showing the successive electrode positions in rat 711. At the right panel, the required pulse periods are shown for each site. Additional details in Fig. 7. Abbreviations: cc: crus cerebri; Im: lemniscus medialis; sgcl: substantia grisea centralis lateralis; rn: nucleus ruber; sn: substantia nigra.
699
BRAIN MAPPING
,.
Rat
2. 3. 4. 5. (I7' 8' 9. 10. 11 . 1
30 Pulse
Period
JO
(msec)
FIG. 9. Schematic representation of a brain section showing the successive positions of the electrode in rat 713. At the right panel, the required pulse periods and effectiveness spectrum are shown. Additional details in Fig. 7. Abbreviations: ac: aqueductus cerebri; cc: crus cerebri; Ic: nucleus linearis, pars caudalis; lm: lemniscus medialis; PCS: pedunculus cerebellaris superior.
[13]. On the other hand, the use of variable pulse intensity (relatively common in the literature) presents some advantages over the first approach. For instance, had the current intensity been increased by investigator A, it would have been possible to observe that all seven sites in rat 699 (Fig. 1) can support equally high rates of SS. This technique may therefore be valuable in preliminary studies in which the main purpose consists of a rough localization of behaviorally active sites in large areas of the brain. As a matter of fact, a potion of the present knowledge on this matter is due to this procedure. However, an important limitation of this technique is that tine anatomical delineation is rendered difficult when the current spread is varied from site to site. Thus, in brain mapping studies, current intensity should be held constant. In this case, the m~imum behavior could be examined by increasing the pulse frequency. Again, had investigator A increased the frequency, as did investigator D, it would have been possible to observe that all seven sites in rat 699 can support equally high rates of SS. However, does equal responding mean equal neuronal density? As shown in Fig. 1, all tested sites of rat 699 support equally high rates of SS, however, the number of pulses required to achieve this result differs from site to site. This observation suggests that the proper way to infer the relative stimulation effectiveness would have been to compare the pulse frequency required for the stimulation to elicit the same level of responding at all sites. How About Behavioral Threshold Procedures? Behavioral threshold and constant behavior measurements are both psychophysical methods. Usually, the behavioral threshold procedure consists of looking for the current intensity required to induce a just observable behavioral re-
action. Again, constant intensity and variable frequency should be preferred. The difference, when compared to the procedure used in the present work by investigator D is that the required behavior is set at its lowest value (behavioral threshold). However, at such low responding level, the behavioral performance is usually very variable. For this reason, though threshold measurements are better compared to behavioral output techniques, they may be less reliab!e when compared to the procedure used in the present work. Are There Any Limitations in the Use of Constant Behavior? A main limitation that we have encountered is that estimation of the relative stimulation effectiveness between sites may somewhat vary depending on the criterion performance from which the calculations are drawn. The extent of such a variation has been documented in rat 713 and illustrated in Fig. 4. The reason for this variation is that the curve slopes deviate from their theoretical position. Indeed, it should be recalled that in order for the estimates to be independent from the criterion behavior, the linear portion of all F/R curves must pass through the same origin (C) in the axis of behavioral performance. In other words, these curve portions should be parallel when plotted on a semilog scale. (For instance, when the behavior increases from 20 to 40 bar presses per test period, Weber’s fraction which is the ratio (N pulses for behavior 40/N pulses for behavior 20) - 1, should be the same in all brain sites). Examination of the linear regressions in rat 713 shows that C intercept decreases progressively through sites 6 to 11. The fact that this change is highly correlated with the behavioral ceiling depression in each site jr=.997, p
MILIARESSIS,
700
the electrode moves out of target structure. It should however be stressed that the variation found at site 11 of rat 713 is one of the worst cases we have encountered. Actually, this variation represents a deviation from an otherwise ideal estimate. In spite of this limitation, the constant-behavior technique remains the best when compared to other more frequently used procedures. However, this is not to say that this technique should be definitely adopted, for there are at least two alternative ways which we believe may provide one with better estimates. These are Edmond and Gallistel’s half-maximum procedure (MSO) and the theoretical zero procedure, both applied in the results section. Support for the MS0 originates in experimental work which showed that the number of pulses required to maintain a half-maximum performance is reasonably independent from a variety of constant, extraneous factors, (difficulty of the task, debilitating drug treatment, health condition etc.see [2]). These factors, like the motoric effect in the case of rat 713 have been shown to depress the behavioral asymptote. It should be remembered however that in our experiment the motoric contaminating effect was produced by the same pulses that elicited reinforcement. This means that the severity of this effect was dependent on SS performance. In view of this fact, the question of whether the MS0 procedure operates a sufficient level of correction will require empirical work. That the application of this procedure does operate some correction is undoubtful: Visual inspection of Fig. 3 reveals that this procedure generates estimates of relative stimulation effectiveness that are higher when compared to those obtained by the use of an arbitrary constant criterion (procedure D). Furthermore, the correction effect seems to be proportional to the severity of contamination. The rationale for using what we call the theoretical zero behavior procedure is schematized in Fig. 6. In this Qure, we simulate two F/R functions obtained from hypothetical brain sites A and B. Like the empirical data of rat 713, function B shows a ceiling depression concomitant to a reduction in C intercept. As we mentioned before, the severity of the contaminating effect depends on SS performance. This means that at virtually zero rate of responding the contaminating effect should be very low. In other words, the contaminating effect rotates curve B on the point of intersection of the regression line with the axis of pulses. If this assumption is correct, the dotted line represents the position that should theoretically be occupied by B in the absence of contamination. The corrected function will necessarily generate an estimate of relative stimulation effectiveness which will be independent of the criterion performance. There are three considerations that make the above approach interesting: First, in calculating the relative stimulation effectiveness, the regression intercepts of the empirical functions on the abscissa can be used directly without any adjustment. Second, the number of pulses thereby obtained is very close to what is normally required to elicit a threshold behavioral reaction. Third, the variability in the number of pulses required for zero behavior should be low, for these numbers are obtained from a set of points (the regression line). Can Neuronal Measurements?
Density
be Inferred
by Behavioral
Establishment of the neuronal density of behaviorally relevant neuronal elements in different brain regions is
ROMPRE AND DURIVAGE
ideally the goal pursued in brain mapping studies. The answer to the question is that the relative density of relevant neurons can be approximated using a constant behavior procedure involving variations in pulse frequency. For instance, using this technique, one might be able to state that site A comprises twice as many relevant neuronal elements compared to site B. The rationale underlying this statement is briefly as follows: Suppose C the total number of firings per stimulation train required at the synapse in order for the system to produce a predetermined behavioral output (behavioral criterion). C can be obtained by an infinite number of combinations between number of neurons in the stimulated field (neuronal density, D) and number of pulses (Np). The greater the density, the smaller the required number of pulses. In other words, the product of D by Np is the constant C. The relation DxNp=C can also be written D=Cx(l/Np) i.e., the neuronal density is linearily related to the reciprocal of the number of pulses (or pulse period). In conclusion, within the same brain system, the relative neuronal density between stimulated sites can be inferred by the pulse period required to elicit a criterion behavior (for instance MS0 or Go). It should be remembered however that this rationale holds in condition that the following assumptions are true: (a) The synapse acts as a pulse counter (see [3,4]); (b) Current spread is reasonably isotropic throughout the investigated brain area; (c) Within this area, neurons that have different activation thresholds and/or unequal contribution to the behavioral reaction are randomly distributed. Can Critical Boundaries
be Delineated
with Precision?
This of course is an important and timely question. The answer is that this task can be accomplished satisfactorily when moveable electrodes are used in combination with psychophysical measurements. For instance, the boundaries of the hypothalamic substrates of drinking and eating behaviors were determined by Wise by application of a behavioral threshold technique [ 1I]. A slightly different approach would be the application of a trade-off paradigm: As soon as the first SS reaction is observed, the pulse frequency should be progressively increased so that the minimum effective current intensity be determined. For instance, the possibility that 100 PA are sufficient to elicit some SS would mean that the boundary lies within the radius of diffusion of this intensity. With cathodal rectangular pulses of 0.1 msec. in duration, the above current would activate SS neurons lying approximately at 200 pm from the center of the electrode (George Fouriezos, personal communication). The same procedure will be required for determination of the lower boundary of the structure. The task could theoretically be accomplished at once at the time of application of 80 procedure if one could set the current at a constant very low level (for instance 100 PA instead of 400 PA used in the present work). However, setting the current so low implies that E estimates be drawn from application of a range of very short interpulse intervals. However, such estimates will certainly be distorted, since the successive pulses will interfere with the neuronal excitability cycles. What are the Advuntages, Electrodes?
Zf Any,
in Using Moveable
There are several advantages, some of them have already been discussed in previous studies [I, 5, 8, 9, 121. With the moveable electrode used in the present work, (for technical description see [9]) up to 75, 80 pm apart dorsoventral sites
BRAIN
MAPPING
can be tested in the same brain. Besides the obvious economic and practical benefits, the controled tiny changes in electrode position in combination with low current intensities allow a level of anatomical resolution which would never be attained by the use of fixed electrodes. Histological reliability is another important advantage. This means that the spatial relationship between stimulated sites is accurately represented. As we stressed before, this is poorly accomplished when fixed electrodes belonging to different animals are used. Since the locations of the successive stimulated sites are inferred from the last position, care must be taken that the tip of the electrode is well localized and that there is no size alterations of the brain following histological treatments. Thus, moveable electrodes allow faster as well as better mapping work. Though examination of the anatomical corre-
lates of self-stimulation falls outside the scope of this presentation, it should be stressed that the SS data obtained in the raphe region in only two animals in the present work, (rats 699 and 713) provide us with more reliable and complete information when compared to our previous studies using several animals with fixed electrodes [6,7]. Following rough behavioral brain mapping obtained by the use of fixed electrodes and simple behavioral measurements, fine delineation of critical boundaries and approximation of the relative neuronal density within a circumscribed region requires the use of special electrodes in combination with advanced behavioral estimates. In the present work we showed that accurate brain mapping can be accomplished satisfactorily when moveable electrodes are used in combination with psychophysical measurements.
REFERENCES 1. Corbett, D. and R. A. Wise. Intracranial self-stimulation in relation to the ascending dopaminergic systems of the midbrain. A moveable electrode mapping study. Brain Res. 1%5: l-15, 1980. 2. Edmonds, D. E. and C. R. Gallistel. parametric analysis of self-stimulation reward in the rat: III. Effect of performance variables on the reward summation functions. J. camp. physiol. Psychol. 87: 876883, 1974. 3. Gallistel, C. R. Spatial and temporal summation in the neural circuit subserving brain stimulation reward. In: Brain S&FZ&tion Reward, edited by A. Wauquier and E. T. Rolls. New York: American Elsevier publishing Company, 1976, pp. 97-99. 4. Gallistel, C. R., P. Shizgal and J. S. Yeomans. A portrait of the substrate for self-stimulation. Psychol. Rev. 88: 228-273, 1981. 5. Mathis, G. and P. Schmitt. Dispositif B demeure pour la descente progressive d’une electrode dans le cerveau du rat. Physiol. Behav. 12: 177-180, 1974. 6. Miliaressis, E., A. Bouchard and D. M. Jacobowitz. Strong positive reward in median raphe: Specific inhibition by parachlorophenylalanine. Brain Res. 98: 194-201, 1975.
7. Miliaressis, E. Serotonexgic basis of reward in median raphe of the rat. Pharmac. B&hem. Behav. 7: 177-180, 1977. 8. Miliaressis, E. and A. Gratton. A chronic, moveable nonrotating electrode for brain stimulation in the rat. Physiof. Behav. 26: 891-894, 1981. 9. Miliaressis, E. A miniature moveable electrode for brain stimulation in small animals. Bruin Res. Bull. 7: 715-718, 1981. 10. Mundl, W. J. A constant-current stimulator. Pkysiol. Behav. 24: 991-993, 1980. 11. Wise, R. A. Spread of the current from monopolar stim~ation of the lateral hypothalamus. Am. .I. Physiol. 223: 545-548,1972. 12. Wise, R. A. Moveable electrode for chronic brain stimulation in the rat. Physiol. Behav. 16: 105-106, 1976. 13. Yeomans, J. S. Quantitative measurement of neural poststimulation excitability with behavioral methods. Physiol. Rehav. 15: 593-602, 1975.
Psychophysical Method for Mapping Behavioral Substrates Using a Moveable Electrode’ E. MILIARESSIS,
Ecole de Psychologie,
P. P. ROMPRfi
AND A. DURLVAGE
35 McDougal Lane, Universitt d’ottawa, Received
21 December
Ottawa, KIN 6N5 Canada
1981
MILIARESSIS, E., P. P. ROMPRI? AND A. DURIVAGE. Psychophysical method for mapping behavioral substrates using a moveable electrode. BRAIN RES. BULL. 8(6) 693-701, 1982.-With the use of moveable electrodes, 28 rat mesencephalic sites were examined for self-stimulation behavior (SS). The relative importance of each site in SS was established according to (1) a traditional method which consists of comparing sites based on the rates of responding (2) a psychophysical procedure based on the pulse frequency required at each site in order for the stimulation to elicit a criterion behavioral performance. It was shown that anatomical conclusions reached by the use of behavioral output procedures depend purely on the arbitrary choice of stimulation parameters. It was also demonstrated that the combination of moveable electrodes with psychophysical measurements results in enhanced mapping resolution and enables one to trace the boundaries of behaviorally relevant structures and pathways with significantly better confidence.
Moveable electrodes
Brain stimulation
Brain mapping
AN important goal in brain mapping research is the identification of behaviorally relevant neuronal nuclei and pathways. This task can be accomplished with variable accuracy depending on the availability of efficient techniques and procedures. For instance, rough localization of relevant structures in large brain areas can be performed by electrical stimulation using fixed electrodes and simple behavioral measurements. However, fine delineation of critical boundaries within a circumscribed brain region is not attainable by these techniques. First, there is a great deal of uncertainty about the location of stimulated sites when across-animal comparisons are made. Second, establishment of brain mapping based on behavioral responding following stimulation is subject to significant distortion due to the lack of linearity between stimulation strength and behavioral output (behavioral floor and ceiling properties). In the present work we will show that when the strength of the stimulation is held constant in all stimulated sites, the resulting anatomical conclusions will be dependent on the arbitrary choice of stimulation parameters. On the other hand, if current intensity is varied from site to site so that the maximum behavioral output is examined, a good deal of anatomical resolution will be lost due to lack of precision concerning current spread. While rough localization of behaviorally-relevant sites in large brain areas has been a fruitful1 task for the last fifty years, correlations with some presently well identified pathways (for instance monoaminergic bundles) would require application of stimulation techniques which can provide one with significantly better anatomical resolution.
Self-stimulation
Psychophysical measurements
In the present work, some of the limitations of traditional mapping techniques will be examined in the light of data obtained in self-stimulation behavior (SS) in the rat. In addition, alternative solutions consisting basically of the use of moveable electrodes in combination with psychophysical measurements will be proposed.
METHOD
Subjects
and Surgery
Under general anesthesia with Nembutal (40 mg/kg), three male, adult Sprague-Dawley rats were implanted with a small moveable electrode (wire dia. 0.25 mm) described elsewhere [9]. With the skull held horizontal between bregma and lambda, stereotaxic coordinates were as follows: Rat 699,0.6 mm rostral to lambda, 0.0 mm lateral to the midline, 7.0 mm below the skull surface; Rat 711, 1.5 mm, 0.0, 7.0; Rat 713, 0.2 mm, 0.0, 7.7.
Stimulation
Parameters
Electrical stimulation of the brain consisted of trains (400 msec in duration) of cathodal pulses of constant intensity (400 PA) and duration (0.1 msec) and variable frequency. The stimulation was delivered by a constant-current generator [ 101 triggered by depression of a lever located in a transparent SS box. Self-administration of the stimulation was subject to an FI, 1 set schedule.
‘The authors are supported by NSERC (E. M.), FCAC (P. P. R.) and OGS (A. D.).
Copyright @ 1982 ANKHO
International Inc .-0361-9230/82/060693-09$03.00/O
MILIARESSIS,
694
Nil
ROMPRE AND DURIVAGE
Low
Medium
High
RAT 699
Stimulation
effectiveness
according
B
C
I :
!
: : +
: : i
61
13
7
,‘4
$6 Number
of pulses/train
FIG. 1. Self-stimulation responding as a function of the number of pulses per stimulation train for 7 brain sites in rat 699. Each site is identified by a number which refers to the position of the electrode shown in Fig. 7. The intersections of lines A, B and C with each curve show the amount of responding that would have been observed by three hypothetical investigators, each one using a different, single fixed frequency (16, 22 and 30 pulses/train respectively). Line D illustrates the use of a constant-behavior procedure. The right pane1 of the figure shows the stimulation effectiveness per brain site, as inferred by investigators A, B, C and D.
Procedure Following two weeks of post-operative recovery, rats were introduced into the SS box. Using a starting pulse frequency of 50 Hz, the investigator attempted to shape the animal for SS by triggering the stimulation each time the animal approached the lever. If the attempt was not successfil, the frequency was gradually increased up to 100 Hz. If this maximum frequency failed to elicit SS, the testing procedure for this brain site was considered complete. The electrode was then lowered down by 160 Frn and the animal tested again 24 hr later using the same procedure. Once SS was observed in a firs& site, the electrode was kept in the same position for one week. During this period the animal was allowed to self-stimulate for two consecutive hours per day, at a rate of approximately 75% of his maximum performance. This relatively long training period applied to the first SS location was intended to insure complete learning of the task before testing subsequent sites. Following completion of the training period, pulse frequency/SS response functions (F/R functions) were determined according to the following procedure: The rate of SS responses for each frequency was examined in 3 min trials separated by 2 min of rest. The pulse frequency was varied in ascending, then in descending order. Its total range was large enough to cover the animal’s complete responding capability, from zero to the maximum performance. Immediately before each 3 min
test, the animal was primed with a few stimulations and the testing period was initiated as soon as the animal made the first SS response. After a maximum of 20 primes, the testing period began even if the rat failed to resume SS. The mean of the ascending and descending series was used to draw the F/R function for the brain site under investigation. At the completion of the test, the electrode was lowered by 160 pm and the new site was examined 24 hr later according to the same procedure. At the end of the experiments, the animals were sacrificed by an overdose of Nembutal and their brains removed and stored for three days in 10% Fotmalin. Following this period, the brains were sliced (28 &rn thick) in a cryostat. The wet brain sections containing the track of the electrodes were examined under microscope immediately following the slicing process. RESULTSAND DISCUSSION
The left panel of Fig. 1 shows SS data from rat 699. The number of reinforcements per test period was plotted as a function of the number of pulses per stimulation train. Each curve was obtained from a different dorso-ventral position of the electrode. The lines labelled A, B and C are intended to show that divergent anatomical conclusions might havebeen reached by three investigators using different stimulation parameters. Investigators A, B and C use basically the same experimental paradigm: the effectiveness of the stimulation
695
BRAIN MAPPING
effectiveness
Stimulation
according to
Ai\ A
B
C
D
3 3 4 ._ In 5 .cZ 6 m 7
Number
of pulses / train
FIG. 2. Self-stimulation responding as a function of the number of pulses per stimulation train for 10 brain sites in rat 711. Further details in Fig. 1.
at each brain site is inferred by the behavioral performance obtained by the use of constant intensity (400 PA) and a single, fixed pulse frequency (16, 22 and 30 pulses/train respectively). In other words, the complete F/R functions were not examined as in this figure. The behavioral performance that would have been obtained by these investigators is shown by the intersection of lines A, B and C with each curve. Thus, investigator A, using a fixed number of pulses of 16 would have concluded that only site 5 supports SS behavior. On the other hand, investigator B would have been unable to detect any difference between sites 1 to 3 and sites 4 to 7. At the other extreme, investigator C, using a higher pulse frequency would have concluded that the stimulation has the same effectiveness at all seven sites. Line D illustrates a different procedure as used by a fourth investigator. In this case, the overall F/R curves were first obtained for each individual site (curves shown in Fig. 1). The stimulation effectiveness (E) at each brain site was then inferred by the pulse period required to elicit a constant amount of responding, arbitrarily set at 70 reinforcements per test period (line D). The right panel of the figure illustrates the comparative anatomical picture of this experiment as obtained by the four hypothetical investigators. For A, B and C, the stimulation effectiveness at each site is labelled high, medium, low or nil, as usually found in the literature. In the case of D, the density corresponding at each site was made proportional to the pulse period required to elicit the criterion performance. Figure 2 demonstrates essentially the same phenomenon in a different rat. The right panel of the figure shows the expected marked discrepencies due to the lack of dis-
criminability associated with the procedure used by the first three investigators. Figure 3 shows data from a third animal. Since the inconsistencies due to direct output measurements can be easily replicated, the data of rat 713 will instead be used to illustrate problems associated with the constant-behavior procedure. First, there is a progressive depression of the behavioral ceiling throughout sites 6 to 11. Observation of the animal during testing suggests that this phenomenon was due to contamination of SS by simultaneous stimulus-bound motoric activity. Second, concomitant to the ceiling depression, there seems to be an abnormal reduction in curve slopes. This means that in each curve, the slope reduction is higher than what should be expected by calculation of Weber’s fraction (see discussion). The consequence of this phenomenon is that calculations of the relative stimulation effectiveness between sites will be dependent on the arbitrary choice of the criterion performance. The severity of this artifact in the case of rat 713 is documented in Fig. 4. The largest variation occurs at site 11 where E shifts from 0.37 to 0.22 when the criterion performance varies from zero to 70 reinforcements. In ordei to circumvent this difficulty, calculation of E values was also attempted by the use of the following criteria: (a) Edmond and Gallistel’s constant proportion (for instance 50% of the maximum performance or MSO) and (b) theoretical zero behavior. In both cases, the required number of pulses was calculated by the use of the linear regressions of each FIR curve. For instance, the required number of pulses corresponding to the theoretical zero behavior (or eo) was obtained by the intersection of each re-
MILIARESSIS,
696
ROMPRk
AND DURIVAGE
2
RAT
713
/
MY----’
Number
of
pulses
/train
.
FIG. 3. Self-stimulation responding as a function of the number of pulses per stimulation train for 11 brain sites in rat 713. Note the progressive ceiling depression through sites 6 to 11.
RAT
713
Bre in
.
sites
.
m .
7
6
9 10 11
r
0
6
,
70
10 Criterion
(Reinforcements
/ 3 min.)
FIG. 4. Relative stimulation effectiveness (E) at brain sites 6 to 11 as a function of the behavioral criterion. For a given site X, E was calculated using the ratio, Pulse period required at site X/Pulse period required at sites 1 to 5 (pooled together).
BRAIN
697
MAPPING
8
l 0
l 0
l 0 0 0
FIG. 5. Relative stimulation effectiveness at sites 6 to 11of rat 713 as inferred by the use of M50 and Go procedures. For explanation see
text.
gression line with the abscissa. The rationale of 80 is ilhtstrated in Fig. 6 and will be developed in the discussion. Figure 5 shows the relative stimulation effectiveness in sites 6 to 11 (compared to sites 1 to 5 pooled together) as obtained by these two procedures. It can be observed that these methods generate similar results with however slightly lower estimates for the MS0 procedure. We suspect that this difference was due to a small artificial ceiling depression, mainly exerted on the reference sites,_due to the constraint imposed by the use of the FI-1 sec. schedule. This means that the sites that are subject to this effect will show higher than normal stimulation effectiveness, for the artifactual ceiling depression will contribute to provide lower pulse numbers for the MS0 criterion. In the case of rat 713, the above schema also predicts that the consequent E reductions will roughly increase throughout sites 6 to 11. Figures 7,8 and 9 show the successive electrode positions for each rat as well as the required pulse periods obtained by the aforementioned two procedures. It can be observed that both techniques seem to be sensitive to smalI changes in stimulation effectiveness. Especially in the case of rats 711 and 713 where the brain tested areas were large enough, the U-shaped progressive changes in the required pulse period provide a fairly detailed picture of the relative importance of each site in SS behavior. GENERAL
DISCUSSION
The purpose of the present work was to examine some of
CO FIG. 6. Simulation of two f~que~cy/res~nse curves in selfstimulation, obtained in hypothetical sites A and B. The solid lines represent the regression of the linear portion of each curve. Like the empirical data of rat 713, (see results), site B shows a ceiling depression concomittant to a reduction in the intercept (C,) on the axis of behavior. The broken line shows the theoretical position of the regression line of B in the absence of motoric cont~ination. Theta zero is the intercept of the regression line of B on the axis of pulses. Note that this intercept is common to the theoretical and empirical regression lines. The theoretical (broken) line was obtained by pivoting the regression of B on 80 point so that C, joins Co.
the limitations associated with the use of different brain mapping procedures and to propose alternative solutions. Some relevant questions are discussed below. Are Behavioral Output Measurements
of Any Use?
Before answering this question it should be recalled that there are basically two approaches associated with the use of the observed behavioral performance as the dependent variable. First, the use of fixed stimulation strength and second, the use of variable strength obtained by variations in pulse frequency or pulse intensity. In the present work we showed that the use of fixed pulse intensity and frequency leads to a variety of mapping conclusions depending on the arbitrary choice of the stimulation parameters. Distortion of refractory period measurements due to the use of fixed stimulation parameters has also been demonstrated earlier by Yeomans
MILIARESSIS,
ROMPRI? AND DURIVAGE
4’ s6’
-’
0
50
30
10
Pulse
Period
msec
FIG. 7. Schematic representation of a brain section showing the successive dorso-ventral positions of the electrode in rat 699. The right panel of the figure shows the pulse periods required at each stimulated site as inferred by the use of M50 and GO procedures (see text). The effectiveness of the stimulation within the investigated area is also represented by a density spectrum at the middle of the figure. Abbreviations: cc: crus cerebri; Im: lemniscus medialis; mr: nucleus medianus raphe; PCS: pedunculus cerebellaris superior.
Pulse
Period
(msec)
FIG. 8. Schematic representation of a brain section showing the successive electrode positions in rat 711. At the right panel, the required pulse periods are shown for each site. Additional details in Fig. 7. Abbreviations: cc: crus cerebri; Im: lemniscus medialis; sgcl: substantia grisea centralis lateralis; rn: nucleus ruber; sn: substantia nigra.
699
BRAIN MAPPING
,.
Rat
2. 3. 4. 5. (I7' 8' 9. 10. 11 . 1
30 Pulse
Period
JO
(msec)
FIG. 9. Schematic representation of a brain section showing the successive positions of the electrode in rat 713. At the right panel, the required pulse periods and effectiveness spectrum are shown. Additional details in Fig. 7. Abbreviations: ac: aqueductus cerebri; cc: crus cerebri; Ic: nucleus linearis, pars caudalis; lm: lemniscus medialis; PCS: pedunculus cerebellaris superior.
[13]. On the other hand, the use of variable pulse intensity (relatively common in the literature) presents some advantages over the first approach. For instance, had the current intensity been increased by investigator A, it would have been possible to observe that all seven sites in rat 699 (Fig. 1) can support equally high rates of SS. This technique may therefore be valuable in preliminary studies in which the main purpose consists of a rough localization of behaviorally active sites in large areas of the brain. As a matter of fact, a potion of the present knowledge on this matter is due to this procedure. However, an important limitation of this technique is that tine anatomical delineation is rendered difficult when the current spread is varied from site to site. Thus, in brain mapping studies, current intensity should be held constant. In this case, the m~imum behavior could be examined by increasing the pulse frequency. Again, had investigator A increased the frequency, as did investigator D, it would have been possible to observe that all seven sites in rat 699 can support equally high rates of SS. However, does equal responding mean equal neuronal density? As shown in Fig. 1, all tested sites of rat 699 support equally high rates of SS, however, the number of pulses required to achieve this result differs from site to site. This observation suggests that the proper way to infer the relative stimulation effectiveness would have been to compare the pulse frequency required for the stimulation to elicit the same level of responding at all sites. How About Behavioral Threshold Procedures? Behavioral threshold and constant behavior measurements are both psychophysical methods. Usually, the behavioral threshold procedure consists of looking for the current intensity required to induce a just observable behavioral re-
action. Again, constant intensity and variable frequency should be preferred. The difference, when compared to the procedure used in the present work by investigator D is that the required behavior is set at its lowest value (behavioral threshold). However, at such low responding level, the behavioral performance is usually very variable. For this reason, though threshold measurements are better compared to behavioral output techniques, they may be less reliab!e when compared to the procedure used in the present work. Are There Any Limitations in the Use of Constant Behavior? A main limitation that we have encountered is that estimation of the relative stimulation effectiveness between sites may somewhat vary depending on the criterion performance from which the calculations are drawn. The extent of such a variation has been documented in rat 713 and illustrated in Fig. 4. The reason for this variation is that the curve slopes deviate from their theoretical position. Indeed, it should be recalled that in order for the estimates to be independent from the criterion behavior, the linear portion of all F/R curves must pass through the same origin (C) in the axis of behavioral performance. In other words, these curve portions should be parallel when plotted on a semilog scale. (For instance, when the behavior increases from 20 to 40 bar presses per test period, Weber’s fraction which is the ratio (N pulses for behavior 40/N pulses for behavior 20) - 1, should be the same in all brain sites). Examination of the linear regressions in rat 713 shows that C intercept decreases progressively through sites 6 to 11. The fact that this change is highly correlated with the behavioral ceiling depression in each site jr=.997, p
MILIARESSIS,
700
the electrode moves out of target structure. It should however be stressed that the variation found at site 11 of rat 713 is one of the worst cases we have encountered. Actually, this variation represents a deviation from an otherwise ideal estimate. In spite of this limitation, the constant-behavior technique remains the best when compared to other more frequently used procedures. However, this is not to say that this technique should be definitely adopted, for there are at least two alternative ways which we believe may provide one with better estimates. These are Edmond and Gallistel’s half-maximum procedure (MSO) and the theoretical zero procedure, both applied in the results section. Support for the MS0 originates in experimental work which showed that the number of pulses required to maintain a half-maximum performance is reasonably independent from a variety of constant, extraneous factors, (difficulty of the task, debilitating drug treatment, health condition etc.see [2]). These factors, like the motoric effect in the case of rat 713 have been shown to depress the behavioral asymptote. It should be remembered however that in our experiment the motoric contaminating effect was produced by the same pulses that elicited reinforcement. This means that the severity of this effect was dependent on SS performance. In view of this fact, the question of whether the MS0 procedure operates a sufficient level of correction will require empirical work. That the application of this procedure does operate some correction is undoubtful: Visual inspection of Fig. 3 reveals that this procedure generates estimates of relative stimulation effectiveness that are higher when compared to those obtained by the use of an arbitrary constant criterion (procedure D). Furthermore, the correction effect seems to be proportional to the severity of contamination. The rationale for using what we call the theoretical zero behavior procedure is schematized in Fig. 6. In this Qure, we simulate two F/R functions obtained from hypothetical brain sites A and B. Like the empirical data of rat 713, function B shows a ceiling depression concomitant to a reduction in C intercept. As we mentioned before, the severity of the contaminating effect depends on SS performance. This means that at virtually zero rate of responding the contaminating effect should be very low. In other words, the contaminating effect rotates curve B on the point of intersection of the regression line with the axis of pulses. If this assumption is correct, the dotted line represents the position that should theoretically be occupied by B in the absence of contamination. The corrected function will necessarily generate an estimate of relative stimulation effectiveness which will be independent of the criterion performance. There are three considerations that make the above approach interesting: First, in calculating the relative stimulation effectiveness, the regression intercepts of the empirical functions on the abscissa can be used directly without any adjustment. Second, the number of pulses thereby obtained is very close to what is normally required to elicit a threshold behavioral reaction. Third, the variability in the number of pulses required for zero behavior should be low, for these numbers are obtained from a set of points (the regression line). Can Neuronal Measurements?
Density
be Inferred
by Behavioral
Establishment of the neuronal density of behaviorally relevant neuronal elements in different brain regions is
ROMPRE AND DURIVAGE
ideally the goal pursued in brain mapping studies. The answer to the question is that the relative density of relevant neurons can be approximated using a constant behavior procedure involving variations in pulse frequency. For instance, using this technique, one might be able to state that site A comprises twice as many relevant neuronal elements compared to site B. The rationale underlying this statement is briefly as follows: Suppose C the total number of firings per stimulation train required at the synapse in order for the system to produce a predetermined behavioral output (behavioral criterion). C can be obtained by an infinite number of combinations between number of neurons in the stimulated field (neuronal density, D) and number of pulses (Np). The greater the density, the smaller the required number of pulses. In other words, the product of D by Np is the constant C. The relation DxNp=C can also be written D=Cx(l/Np) i.e., the neuronal density is linearily related to the reciprocal of the number of pulses (or pulse period). In conclusion, within the same brain system, the relative neuronal density between stimulated sites can be inferred by the pulse period required to elicit a criterion behavior (for instance MS0 or Go). It should be remembered however that this rationale holds in condition that the following assumptions are true: (a) The synapse acts as a pulse counter (see [3,4]); (b) Current spread is reasonably isotropic throughout the investigated brain area; (c) Within this area, neurons that have different activation thresholds and/or unequal contribution to the behavioral reaction are randomly distributed. Can Critical Boundaries
be Delineated
with Precision?
This of course is an important and timely question. The answer is that this task can be accomplished satisfactorily when moveable electrodes are used in combination with psychophysical measurements. For instance, the boundaries of the hypothalamic substrates of drinking and eating behaviors were determined by Wise by application of a behavioral threshold technique [ 1I]. A slightly different approach would be the application of a trade-off paradigm: As soon as the first SS reaction is observed, the pulse frequency should be progressively increased so that the minimum effective current intensity be determined. For instance, the possibility that 100 PA are sufficient to elicit some SS would mean that the boundary lies within the radius of diffusion of this intensity. With cathodal rectangular pulses of 0.1 msec. in duration, the above current would activate SS neurons lying approximately at 200 pm from the center of the electrode (George Fouriezos, personal communication). The same procedure will be required for determination of the lower boundary of the structure. The task could theoretically be accomplished at once at the time of application of 80 procedure if one could set the current at a constant very low level (for instance 100 PA instead of 400 PA used in the present work). However, setting the current so low implies that E estimates be drawn from application of a range of very short interpulse intervals. However, such estimates will certainly be distorted, since the successive pulses will interfere with the neuronal excitability cycles. What are the Advuntages, Electrodes?
Zf Any,
in Using Moveable
There are several advantages, some of them have already been discussed in previous studies [I, 5, 8, 9, 121. With the moveable electrode used in the present work, (for technical description see [9]) up to 75, 80 pm apart dorsoventral sites
BRAIN
MAPPING
can be tested in the same brain. Besides the obvious economic and practical benefits, the controled tiny changes in electrode position in combination with low current intensities allow a level of anatomical resolution which would never be attained by the use of fixed electrodes. Histological reliability is another important advantage. This means that the spatial relationship between stimulated sites is accurately represented. As we stressed before, this is poorly accomplished when fixed electrodes belonging to different animals are used. Since the locations of the successive stimulated sites are inferred from the last position, care must be taken that the tip of the electrode is well localized and that there is no size alterations of the brain following histological treatments. Thus, moveable electrodes allow faster as well as better mapping work. Though examination of the anatomical corre-
lates of self-stimulation falls outside the scope of this presentation, it should be stressed that the SS data obtained in the raphe region in only two animals in the present work, (rats 699 and 713) provide us with more reliable and complete information when compared to our previous studies using several animals with fixed electrodes [6,7]. Following rough behavioral brain mapping obtained by the use of fixed electrodes and simple behavioral measurements, fine delineation of critical boundaries and approximation of the relative neuronal density within a circumscribed region requires the use of special electrodes in combination with advanced behavioral estimates. In the present work we showed that accurate brain mapping can be accomplished satisfactorily when moveable electrodes are used in combination with psychophysical measurements.
REFERENCES 1. Corbett, D. and R. A. Wise. Intracranial self-stimulation in relation to the ascending dopaminergic systems of the midbrain. A moveable electrode mapping study. Brain Res. 1%5: l-15, 1980. 2. Edmonds, D. E. and C. R. Gallistel. parametric analysis of self-stimulation reward in the rat: III. Effect of performance variables on the reward summation functions. J. camp. physiol. Psychol. 87: 876883, 1974. 3. Gallistel, C. R. Spatial and temporal summation in the neural circuit subserving brain stimulation reward. In: Brain S&FZ&tion Reward, edited by A. Wauquier and E. T. Rolls. New York: American Elsevier publishing Company, 1976, pp. 97-99. 4. Gallistel, C. R., P. Shizgal and J. S. Yeomans. A portrait of the substrate for self-stimulation. Psychol. Rev. 88: 228-273, 1981. 5. Mathis, G. and P. Schmitt. Dispositif B demeure pour la descente progressive d’une electrode dans le cerveau du rat. Physiol. Behav. 12: 177-180, 1974. 6. Miliaressis, E., A. Bouchard and D. M. Jacobowitz. Strong positive reward in median raphe: Specific inhibition by parachlorophenylalanine. Brain Res. 98: 194-201, 1975.
7. Miliaressis, E. Serotonexgic basis of reward in median raphe of the rat. Pharmac. B&hem. Behav. 7: 177-180, 1977. 8. Miliaressis, E. and A. Gratton. A chronic, moveable nonrotating electrode for brain stimulation in the rat. Physiof. Behav. 26: 891-894, 1981. 9. Miliaressis, E. A miniature moveable electrode for brain stimulation in small animals. Bruin Res. Bull. 7: 715-718, 1981. 10. Mundl, W. J. A constant-current stimulator. Pkysiol. Behav. 24: 991-993, 1980. 11. Wise, R. A. Spread of the current from monopolar stim~ation of the lateral hypothalamus. Am. .I. Physiol. 223: 545-548,1972. 12. Wise, R. A. Moveable electrode for chronic brain stimulation in the rat. Physiol. Behav. 16: 105-106, 1976. 13. Yeomans, J. S. Quantitative measurement of neural poststimulation excitability with behavioral methods. Physiol. Rehav. 15: 593-602, 1975.