Physiology & Behavior, Vol. 21, pp. 165-168. Pergamon Press and Brain Research Publ., 1978. Printed in the U.S.A.
Behavioural Measurements of Excitability Changes in Reward Sites in the Dog: A Frequency Dependent Effect A. W A U Q U I E R 1 A N D B. S A D O W S K I 2
Department of Pharmacology, Janssen Pharmaceutica Research Laboratories, B-2340 Beerse, Belgium and 2Polish Academy of Sciences, Laboratory of Applied Physiology, 00-730 Warsaw, Poland (Received 6 March 1978) WAUQUIER, A AND B. SADOWSKI. Behavioural measurements of excitability changes in reward sites of the dog: A frequency dependent effect. PHYSIOL. BEHAV. 21(2) 165-168, 1978.--The excitability changes of a heterogenous stimulation field, in self-stimulating dogs, were measured by a pulse-pair technique. Using such a technique behaviourally, results in a mixed measure of axonal and synaptic events. Excitability estimates, at various C-T intervals, vary systematically depending on the frequency (and/or current) used in a single placement. Using a frequency-threshold method allows a quantitative comparison of neuronal populations.
Neuronal excitability
Hypothalamus
Preopticarea
THE CLASSICAL method of measuring the refractory period in homogenous axons involves the application of a stimulatory pulse pair, the f'trst pulse being a conditioning or C-pulse, the second a test or T-pulse. The interval between the C- and T-pulses is then systematically varied. At relatively large C-T intervals both the C- and the T-pulse will elicit a neuronal response. At shorter intervals the T-pulse will fail to elicit a second response, thus giving an estimate of the refractoriness of the neuron. Increasing the intensity of the T-pulse in the relative refractory period will cause the axons to respond, but not if the T-pulse falls in the earlier absolute refractory period. If one assumes a correspondence between underlying neuronal events and overt behaviour, then it is possible by measuring changes in the behaviour, to infer the characteristics of neuronal events. One such application has been the measurement of the absolute refractory period of neurons sustaining lever-pressing for brain-stimulation reward (intracranial self-stimulation, ICS), by varying the C-T pulse interval of brain-stimulation [3, 6, 7, 9, 11, 13, 16, 17, 21, 22]. It is obvious that experimental and interpretational difficulties will arise in the transition of a method from homogenous axons to heterogenous stimulation fields composed of cell bodies, axons, dendrites, synapses and extracellular fluid. One problem relates to intensity; changes in current density in the stimulation field may recruit different numbers and unknown varieties of neuronal elements (spatial summation). Since single pulse stimulation does not result in behaviour another difficulty arises with regard to the frequency used; the higher the frequency the more likely it becomes that postsynaptic neurons will fire (temporal summation) [2, 4, 8, 15, 18]. Thus, true refractoriness may be masked by postsynaptic events.
Septum
Self-stimulation
Nevertheless an absolute refractory period has been reported for self-stimulation in the rat, but also for midbrain pain-systems [1, 5, 12] and stimulus bound-feeding [4]. Controversy has arisen however as to whether ICS is mediated by neurones which have different refractory periods [13, 16, 20] and whether the motivating and reinforcing properties of ICS might be separated at the neuronal level as proposed by Deutsch [6]. Previous experiments [15,20] have shown that ICS in the dog can be obtained from essentially the same placements as in the rat; in addition, the dog has a larger and more easily discernable behavioral repertoire. Most previous studies on refractory period have been confined to the rat and Wetzel [21] has questioned the validity of these studies after finding that multiple factors controlled the response in cats. We have therefore attempted a more rigorous examination of refractory period in the dog paying particular attention to the possible influence of stimulus frequency changes. METHOD
Adult male Labrador dogs aged 1 year at the time of surgery and bred in our laboratories were housed in pairs, in large cages, in which water was constantly available; standard food pellets were delivered once per day. For electrode implantation dogs were premeditated with 10 ml Hypnorm (10 mg fluanisone (R 2028) and 0.315 mg fentanyl dihydrogen citrate and 0.2 mg fentanyl base (R 4263) per ml) SC followed by 5 ml Nembutal (pentobarbital sodium, 60 mg/ml) IV. They were then intubated and artificially ventilated from a N20 blender (mark 4, Bird). If required, the dogs received an additional 2 ml of Nembutal to ensure sufficient anesthesia during surgery. After surgery the dogs were placed in a recovery room and treated daily with Dicastrepton 1500 IM for at least 5 days.
Copyright © 1978 Brain Research Publications Inc.--0031-9384/78/0801-0165502.00/0
166
WAUQUIER AND SADOWSKI
The stainless steel electrodes consisted of a needle (0.5 mm dia.) with a wire inserted so as to protrude 1 mm beneath the point. All electrodes were insulated except for I/2 mm at the tip. The dogs were implanted with 3 bipolar electrodes in the anterior basal forebrain (lateral preoptic, nucleus accumbens, lateral septum) and 3 bipolar electrodes in the posterior forebrain (lateral and posterior hypothalamus, mammillary bodies). The coordinates were determined according the stereotaxic atlas of Dua Sharma et al. [9] and corrected for individual skull dimensions (Sadowski et al., in preparation). An uninsulatedjeweller's screw in the frontal bone served as an indifferent electrode for monopolar stimulation. Dogs were screened for self-stimulation 3--4 weeks after surgery, in a large cage equipped with a lever. The wires connecting the electrodes were attached to a stimulator (type ST stimulator, Janssen Scientific Instruments) via a large swivel device permitting the dog to move relatively freely. At least 2 out of 6-7 electrodes sustained self-stimulation. A total of eight electrodes in five dogs were selected. The stimulation parameters used during training consisted of a half second train of biphasic rectangular 1 msec pulses equally spaced and given at a frequency of 200 pulses per second (pps). The stimulation current varied between 100 and 500/~A, but was kept constant in each individual dog. After the initial training period of 2 weeks, the dogs were given further training in a 'Pavlov-stand' in which they were partially restrained but had access to a stainless steel 5 × 10 cm lever placed in front of them at a height of 20--25 cm from the floor. Lever-pressing delivered monophasic (negative going) pulses of constant current with C-pulses at an interval of 10 msec and T-pulses at 5 msec from the C-pulse (i.e. 200 pps frequency) through a monopolar electrode. A session consisted of fixed 3 min stimulation periods (trials) interspaced with intervals of 1 min or more. At the start of each trial the dogs were primed once at the given set of parameters used in the trial. If the dog continued leverpressing at a regular rate over the 3 min and persisted to press the lever during 5 to 10 sec after the stimulation was cut off, then the parameters used were considered to have reached a self-stimulation criterion. Three minute trials were selected since a too short period of lever-pressing might also reflect anticipation. Similary, the interval between trials had to be sufficiently long to minimize carry-over effects. RESULTS AND DISCUSSION
Frequency-Intensity The initial experiment sought to determine the optimal frequency-intensity combination for measuring refractory period. Monophasic negative going 0.2 msec pulses were given in a 0.5 see train at different frequencies (range 30-300 pps) and the current determined for which the dogs would fulfill criterion. In accordance with the results of Rolls [14] in the rat, increasing the frequency beyond 100-150 pps did not require a simultaneous increase in the intensity to fulfill the criterion. At high frequencies (e.g. 200 pps) the absolute refractory period could not be measured since further changes in C-T interval did not change the intensity n e e d e d to meet criterion.
0,2 r e s e t .
"T interval msec. rval : 30rnsec
20. 110 I00. 9080. 70605040. 302010Or--4l
is
,
,
,
,
,
,
,
,
I I .......
02 04 06 o8 10 1~ 1.4 16 C-T
interval
,
25
i
s0
/
is
in msec
FIG. 1. Number (mean _+ SE of 7 electrodes) of lever-pressing for brain-stimulation with pulse-pairs obtained during 3 min periods in dogs, as a function of intrapair intervals between pulses (C-T), expressed as a percentage of response obtained with a C-T interval of 15 msec (C-C interval was kept constant at 30 msec). measure the absolute refractory period. The standard frequency chosen was 67 pps, which means that from the beginning of one C-pulse to the beginning of the next there was an interval of 30 msec and between the end of the C- and the beginning of the T-pulse an interval of 15 msec (reference parameters) (Fig. 1). The pulse width chosen was 0.2 msec. These reference parameters gave effective ICS without resorting to excessive stimulatory currents (i.e. currents above 1 mA). Various C-T intervals between 0.2 and 2.5 msec and always including the values 0.2, 0.6 and 1.0 msec were then given in a random order, keeping the C-C interval constant at 30 msec, to determine whether one or two absolute refractory periods might be found. The same criterion of ICS was used as in the first experiment. One priming stimulus was given before each trial and the 3 min period commenced when the dog gave the first lever-press. The results of this experiment are shown in Fig. 1. With C-T intervals below 1 msec there was a low rate of responding, the lowest point being reached at C-T intervals of 0.61.0 msec depending on the electrode. Above I msec responding began to increase towards normal levels, which might be judged as the relative refractory period. The increase in responding at 1 msec and sometimes seen around C-T intervals of 2 msec might reflect a summation phenomena. The lowest C-T interval found (0.6 msec) should then be the refractory period, a finding which confirms Szabo's [17] results obtained in the rat.
Refractory Period Classically Determined Using an intensity approximately twice the threshold value and the corresponding frequency we attempted to
Frequency Dependend Refractory Periods If the absolute refractory period of axons is measured by
B E H A V I O U R A L L Y M E A S U R E D E X C I T A B I L I T Y C H A N G E S IN T H E DOG
F [pps) 67--i
C-C Irnsec) 30.
71-1
28
77q
26
S3-I
2&
91-4
22
100-
20
111-
18-q
125134-
16152
I
:00-]
°/o
.too
V/
-9o 8o~
.7o~ -so i;_
o
-30 ~ -2o .~ -lO ; -0
r--~#,'
15
,
,
,
167
,
,
II
,
~
I1.~
0.2 0.6 0.91.01.11.2 1.8 SP C-T interval in msec ( C - C = 3 0 m s e c ]
1 0°011
}
'01 30-.I
FIG. 2. Decrease of C-T interval or increase in frequency at different C-T intervals to reach criterion of self-stimulation. SP or single pulse represents stimulation with the T-pulse omitted, o--o: electrode in mammillary body; o--o: electrode in posterior hypothalamus.
the above method (for instance at a C-T interval of 0.6 msec) it is logical that only a doubling of frequency, i.e. halving the C-C interval, keeping the T-pulse in the absolute refractory period, would restore self-stimulation to criterion this assumes that the T-pulse does not induce neuronal activity in the absolute refractory period. This postulate was tested using a C-C interval of 30 msec and C-T intervals of 0.2, 0.6 or 1.0 msec and up to 2.5 msec. At these C-T intervals, the C-C interval was gradually reduced by steps of 1 msec until sell-stimulation returned to the reference criterion. As shown in Fig. 2 at any given C-T interval the C-C interval did not have to be halved in order to restore self-stimulation to the criterion. However, if the T-pulse is omitted ('single pulse condition'), the C-C interval must be shortened from 30 msec to 15 msec in order to restore ICS to criterion. This method not only provides another technique for measuring excitability changes but in addition proves that the T-pulse does have a function in recruiting neuronal activity. The slope of recovery when the C-T intervals are gradually increased may be a measure of the homogeneity of the stimulation field. A homogenous field would recover at a rapid rate, as most neurones would begin firing again once the absolute refractory period was past. On the other hand, a heterogenous field would recover slowly, as increasing C-T intervals would act differentially on the various neuronal elements (see also Yeomans [23]).
T-pulse Effectiveness With the parameters chosen in the last experiment (C-C interval 30 msec, 67 pps) the C-C interval had to be shortened to about 50% in order to reach the reference criterion. We then investigated whether the T-pulse effectiveness depends on the original C-C pulse intervals (or frequencies) used (Yeomans [23]). The intensity which fulfilled criterion of ICS was measured for different frequencies with the T-pulse occurring equidistant between the C-pulses (C-C intervals of 60, 50, 40, 30, 20, 10, 8 msec). At these parameters, shortening the C-T interval to 0.6 msec failed to produce responding, except in the high frequency range (/>150 pps). Increasing frequency
c f o,,r 0 /o °,'0 FIG. 3. Contribution of T-pulse: percentage increase in frequency (or shortening C-C interval) at different frequencies of pulse pair stimulations with a C-T interval of 0.6 msec; calculations according to Yeomans [23]. Electrode in lateral hypothalamus.
by shortening C-C interval again restored ICS. The amount by which the frequency had to be increased was dependent on the original C-C interval used. The contribution of the T-pulse was calculated for each C-C interval from the Yeomans' formula [23], i.e. relative effectiviness of the T-pulse is equal to (number of equidistant pulses in a single pulse condition/number of C-pulses in a C-T condition) -1, multiplied by 100 to express the values in percentages. The calculated contribution of the T-pulse are displayed in Fig. 3. At low frequencies, the T-pulses contributed about 30% to the C-pulses and this rose to 100% at 200 pulses per second. CONCLUSIONS The present experiments are evidence that using a pulsepair technique in behavioural measurements results in the measurement of a mixture of neuronal events. At least three hypotheses can be advanced to account for this. During the refractory period the T-pulse could initially have no depolarizing effect on the neurons but may cause the accumulation of electrical charge on the axons. Secondly the T-pulse may cause recruitment from dendrites [17,23]. Thirdly, C-T pulse summation may occur at the periphery of the stimulation field, where the C-pulse only produces subthreshold excitation. Local potential summation is dependent on the size of the stimulation field; our use of 0.2 msec pulses instead of 0.1 msec pulses would increase the excitation of the sub-liminal fringe. Additional experiments carried out by the second author have indeed shown that 'refractoriness' is better seen using 0.1 msec rather than 0.2 msec pulses which suggests that local potential summation overcomes refractoriness, although this may depend on the placement of the electrode.
168 Since pulse-pair synaptic and axonal sizes and h e n c e the ICS. The technique
WAUQUIER AND SADOWSKI m e a s u r e m e n t s p r o d u c e a mixture of events, it is ditticult to evaluate axonal types o f neuronal systems operating in described in the present p a p e r allows a
quantitative c o m p a r i s o n of the excitability of neuronal populations, which may differ depending on the structure stimulated.
REFERENCES
1. Brauth, S. E. and E. C. Coons. Correlation of overt escape behaviour, multiunit thalamic activity, and midbrain lemniscai stimulation in rats. J. comp. physiol. Psychol. 89: 371-378, 1975. 2. Coons, E. E. and J. A. F. Cruce. Lateral hypothalamus: Food and current intensity in maintaining self-stimulation of hunger. Science 159: 1117-1119, 1968. 3. Coons, E. E., N. Schupf and L. G. Ungedeider. Uses of double-pulse stimulation behaviouraily to infer refractoriness, summation, convergence, and transmitter characteristics of hypothalamic reward systems. J. comp. physiol. Psychol. 90: 317-342, 1976. 4. Dennis, S. G. Adaptation of aversive brain-stimulation. It. Effects of current level and pulse frequency. Behav. Biol. 18: 515-530, 1976. 5. Dennis, S. G., J. S. Yeomans and J. A. Deutsch. Adaptation of aversive brain-stimulation. III. Excitability characteristics of behaviorally relevant neural substrates. Behav. Biol. 18: 531544, 1976. 6. Deutsch, J. A. Behavioural measurement of the neural refractory period and its application to intracranial self-stimulation. J. comp. physiol. Psychol. 58: 1-9, 1964. 7. Deutsch, J. A. The drive-reward theory of brain reward. In: Brain-Stimulation Reward, edited by A. Wauquier and E. T. Rolls. Amsterdam: North-Holland Publishing Company, 1976, pp. 593-600. 8. Deutsch, J. A. and R. Collins. Facilitation as a function of temporal spacing of stimuli in intracranial self-stimulation. Nature 208: 592-593, 1965. 9. Dua Sharma, S., K. N. Sharma and H. L. Jacobs. The canine brain in stereotaxic coordinates. Massachusetts: The M.I.T. Press, 1970. 10. Gallistel, C. R., E. T. Rolls and D. Greene. Neuron function inferred from behavioural and electrophysiological estimates of refractory period. Science 166: 1028--1030, 1969. 11. Halboth, P. H. and E. E. Coons. Behaviourai measurements of the neural poststimulation excitability cycle in the lateral hypothalamic eating system of the rat. J. comp. physiol. Psychol. 83: 429-433, 1973.
12. Hawkins, R. D. Behavioural measurements of neural refractory periods: A reappraisal. In: Brain-Stimulation Reward, edited by A. Wauquier and E. T. Rolls. Amsterdam: North-Holland Publishing Company, 1976, pp. 99-101. 13. Kestenbaum, R. S., J. A. Deutsch and E. E. Coons. Inference of refractory period, temporal summation, and adaptation from behaviour in chronic implants: midbrain pain systems. J. comp. physiol. Psychol. 83: 412-428, 1973. 14. Rolls, E. T. Absolute refractory period of neurons involved in MFB self-stimulation. Physiol. Behav. 7: 311-315, 1971. 15. Sadowski, B. Intracranial self-stimulation patterns in dogs. Physiol. Behav. 9: 189-193, 1972. 16. Smith, N. S. and E. G. Coons. Temporal summation and refractoriness in hypothalamic reward neurons as measured by selfstimulation behaviour. Science 169: 782-785, 1970. 17. Szab6, I., C. Lrnard and B. Kosaras. Drive decay theory of self-stimulation: refractory periods and axon diameters in hypothalamic reward loci. Physiol. Behav. 12: 329-343, 1974. 18. Szab6, I. and C. Lrnard. Two low threshold self-stimulation regions in the posterior hypothalamus. In: Brain-Stimulation Reward, edited by A. Wauquier and E. T. Rolls. Amsterdam: North-Holland Publishing Company, 1976, pp. 104-105. 19. Ungerleider, L. G. and E. E. Coons. A behavioural measure of homosynaptic and heterosynaptic temporal summation in the self-stimulation systems of rats. Science 169: 785-787, 1970. 20. Wauquier, A., W. Melis, L. K. C. Desmedt and B. Sadowski. In: Brain-Stimulation Reward, edited by A. Wauquier and E. T. Rolls. Amsterdam: North-Holland Publishing Company, 1976, pp. 427-430. 21. Wetzel, M. C. Strength-duration effects measured behaviourally with self-stimulation. Communs behav. Biol. 6:31-36, 1971. 22. Wetzel, M. C. New evidence concerning refractory period in self-stimulation neurons. Physiol. Behav. 8: 397-402, 1972. 23. Yeomans, J. S. Quantitative measurement of neural poststimulation excitability with behavioural methods. Physiol. Behay. IS: 593-602, 1975.
Behavioural Measurements of Excitability Changes in Reward Sites in the Dog: A Frequency Dependent Effect A. W A U Q U I E R 1 A N D B. S A D O W S K I 2
Department of Pharmacology, Janssen Pharmaceutica Research Laboratories, B-2340 Beerse, Belgium and 2Polish Academy of Sciences, Laboratory of Applied Physiology, 00-730 Warsaw, Poland (Received 6 March 1978) WAUQUIER, A AND B. SADOWSKI. Behavioural measurements of excitability changes in reward sites of the dog: A frequency dependent effect. PHYSIOL. BEHAV. 21(2) 165-168, 1978.--The excitability changes of a heterogenous stimulation field, in self-stimulating dogs, were measured by a pulse-pair technique. Using such a technique behaviourally, results in a mixed measure of axonal and synaptic events. Excitability estimates, at various C-T intervals, vary systematically depending on the frequency (and/or current) used in a single placement. Using a frequency-threshold method allows a quantitative comparison of neuronal populations.
Neuronal excitability
Hypothalamus
Preopticarea
THE CLASSICAL method of measuring the refractory period in homogenous axons involves the application of a stimulatory pulse pair, the f'trst pulse being a conditioning or C-pulse, the second a test or T-pulse. The interval between the C- and T-pulses is then systematically varied. At relatively large C-T intervals both the C- and the T-pulse will elicit a neuronal response. At shorter intervals the T-pulse will fail to elicit a second response, thus giving an estimate of the refractoriness of the neuron. Increasing the intensity of the T-pulse in the relative refractory period will cause the axons to respond, but not if the T-pulse falls in the earlier absolute refractory period. If one assumes a correspondence between underlying neuronal events and overt behaviour, then it is possible by measuring changes in the behaviour, to infer the characteristics of neuronal events. One such application has been the measurement of the absolute refractory period of neurons sustaining lever-pressing for brain-stimulation reward (intracranial self-stimulation, ICS), by varying the C-T pulse interval of brain-stimulation [3, 6, 7, 9, 11, 13, 16, 17, 21, 22]. It is obvious that experimental and interpretational difficulties will arise in the transition of a method from homogenous axons to heterogenous stimulation fields composed of cell bodies, axons, dendrites, synapses and extracellular fluid. One problem relates to intensity; changes in current density in the stimulation field may recruit different numbers and unknown varieties of neuronal elements (spatial summation). Since single pulse stimulation does not result in behaviour another difficulty arises with regard to the frequency used; the higher the frequency the more likely it becomes that postsynaptic neurons will fire (temporal summation) [2, 4, 8, 15, 18]. Thus, true refractoriness may be masked by postsynaptic events.
Septum
Self-stimulation
Nevertheless an absolute refractory period has been reported for self-stimulation in the rat, but also for midbrain pain-systems [1, 5, 12] and stimulus bound-feeding [4]. Controversy has arisen however as to whether ICS is mediated by neurones which have different refractory periods [13, 16, 20] and whether the motivating and reinforcing properties of ICS might be separated at the neuronal level as proposed by Deutsch [6]. Previous experiments [15,20] have shown that ICS in the dog can be obtained from essentially the same placements as in the rat; in addition, the dog has a larger and more easily discernable behavioral repertoire. Most previous studies on refractory period have been confined to the rat and Wetzel [21] has questioned the validity of these studies after finding that multiple factors controlled the response in cats. We have therefore attempted a more rigorous examination of refractory period in the dog paying particular attention to the possible influence of stimulus frequency changes. METHOD
Adult male Labrador dogs aged 1 year at the time of surgery and bred in our laboratories were housed in pairs, in large cages, in which water was constantly available; standard food pellets were delivered once per day. For electrode implantation dogs were premeditated with 10 ml Hypnorm (10 mg fluanisone (R 2028) and 0.315 mg fentanyl dihydrogen citrate and 0.2 mg fentanyl base (R 4263) per ml) SC followed by 5 ml Nembutal (pentobarbital sodium, 60 mg/ml) IV. They were then intubated and artificially ventilated from a N20 blender (mark 4, Bird). If required, the dogs received an additional 2 ml of Nembutal to ensure sufficient anesthesia during surgery. After surgery the dogs were placed in a recovery room and treated daily with Dicastrepton 1500 IM for at least 5 days.
Copyright © 1978 Brain Research Publications Inc.--0031-9384/78/0801-0165502.00/0
166
WAUQUIER AND SADOWSKI
The stainless steel electrodes consisted of a needle (0.5 mm dia.) with a wire inserted so as to protrude 1 mm beneath the point. All electrodes were insulated except for I/2 mm at the tip. The dogs were implanted with 3 bipolar electrodes in the anterior basal forebrain (lateral preoptic, nucleus accumbens, lateral septum) and 3 bipolar electrodes in the posterior forebrain (lateral and posterior hypothalamus, mammillary bodies). The coordinates were determined according the stereotaxic atlas of Dua Sharma et al. [9] and corrected for individual skull dimensions (Sadowski et al., in preparation). An uninsulatedjeweller's screw in the frontal bone served as an indifferent electrode for monopolar stimulation. Dogs were screened for self-stimulation 3--4 weeks after surgery, in a large cage equipped with a lever. The wires connecting the electrodes were attached to a stimulator (type ST stimulator, Janssen Scientific Instruments) via a large swivel device permitting the dog to move relatively freely. At least 2 out of 6-7 electrodes sustained self-stimulation. A total of eight electrodes in five dogs were selected. The stimulation parameters used during training consisted of a half second train of biphasic rectangular 1 msec pulses equally spaced and given at a frequency of 200 pulses per second (pps). The stimulation current varied between 100 and 500/~A, but was kept constant in each individual dog. After the initial training period of 2 weeks, the dogs were given further training in a 'Pavlov-stand' in which they were partially restrained but had access to a stainless steel 5 × 10 cm lever placed in front of them at a height of 20--25 cm from the floor. Lever-pressing delivered monophasic (negative going) pulses of constant current with C-pulses at an interval of 10 msec and T-pulses at 5 msec from the C-pulse (i.e. 200 pps frequency) through a monopolar electrode. A session consisted of fixed 3 min stimulation periods (trials) interspaced with intervals of 1 min or more. At the start of each trial the dogs were primed once at the given set of parameters used in the trial. If the dog continued leverpressing at a regular rate over the 3 min and persisted to press the lever during 5 to 10 sec after the stimulation was cut off, then the parameters used were considered to have reached a self-stimulation criterion. Three minute trials were selected since a too short period of lever-pressing might also reflect anticipation. Similary, the interval between trials had to be sufficiently long to minimize carry-over effects. RESULTS AND DISCUSSION
Frequency-Intensity The initial experiment sought to determine the optimal frequency-intensity combination for measuring refractory period. Monophasic negative going 0.2 msec pulses were given in a 0.5 see train at different frequencies (range 30-300 pps) and the current determined for which the dogs would fulfill criterion. In accordance with the results of Rolls [14] in the rat, increasing the frequency beyond 100-150 pps did not require a simultaneous increase in the intensity to fulfill the criterion. At high frequencies (e.g. 200 pps) the absolute refractory period could not be measured since further changes in C-T interval did not change the intensity n e e d e d to meet criterion.
0,2 r e s e t .
"T interval msec. rval : 30rnsec
20. 110 I00. 9080. 70605040. 302010Or--4l
is
,
,
,
,
,
,
,
,
I I .......
02 04 06 o8 10 1~ 1.4 16 C-T
interval
,
25
i
s0
/
is
in msec
FIG. 1. Number (mean _+ SE of 7 electrodes) of lever-pressing for brain-stimulation with pulse-pairs obtained during 3 min periods in dogs, as a function of intrapair intervals between pulses (C-T), expressed as a percentage of response obtained with a C-T interval of 15 msec (C-C interval was kept constant at 30 msec). measure the absolute refractory period. The standard frequency chosen was 67 pps, which means that from the beginning of one C-pulse to the beginning of the next there was an interval of 30 msec and between the end of the C- and the beginning of the T-pulse an interval of 15 msec (reference parameters) (Fig. 1). The pulse width chosen was 0.2 msec. These reference parameters gave effective ICS without resorting to excessive stimulatory currents (i.e. currents above 1 mA). Various C-T intervals between 0.2 and 2.5 msec and always including the values 0.2, 0.6 and 1.0 msec were then given in a random order, keeping the C-C interval constant at 30 msec, to determine whether one or two absolute refractory periods might be found. The same criterion of ICS was used as in the first experiment. One priming stimulus was given before each trial and the 3 min period commenced when the dog gave the first lever-press. The results of this experiment are shown in Fig. 1. With C-T intervals below 1 msec there was a low rate of responding, the lowest point being reached at C-T intervals of 0.61.0 msec depending on the electrode. Above I msec responding began to increase towards normal levels, which might be judged as the relative refractory period. The increase in responding at 1 msec and sometimes seen around C-T intervals of 2 msec might reflect a summation phenomena. The lowest C-T interval found (0.6 msec) should then be the refractory period, a finding which confirms Szabo's [17] results obtained in the rat.
Refractory Period Classically Determined Using an intensity approximately twice the threshold value and the corresponding frequency we attempted to
Frequency Dependend Refractory Periods If the absolute refractory period of axons is measured by
B E H A V I O U R A L L Y M E A S U R E D E X C I T A B I L I T Y C H A N G E S IN T H E DOG
F [pps) 67--i
C-C Irnsec) 30.
71-1
28
77q
26
S3-I
2&
91-4
22
100-
20
111-
18-q
125134-
16152
I
:00-]
°/o
.too
V/
-9o 8o~
.7o~ -so i;_
o
-30 ~ -2o .~ -lO ; -0
r--~#,'
15
,
,
,
167
,
,
II
,
~
I1.~
0.2 0.6 0.91.01.11.2 1.8 SP C-T interval in msec ( C - C = 3 0 m s e c ]
1 0°011
}
'01 30-.I
FIG. 2. Decrease of C-T interval or increase in frequency at different C-T intervals to reach criterion of self-stimulation. SP or single pulse represents stimulation with the T-pulse omitted, o--o: electrode in mammillary body; o--o: electrode in posterior hypothalamus.
the above method (for instance at a C-T interval of 0.6 msec) it is logical that only a doubling of frequency, i.e. halving the C-C interval, keeping the T-pulse in the absolute refractory period, would restore self-stimulation to criterion this assumes that the T-pulse does not induce neuronal activity in the absolute refractory period. This postulate was tested using a C-C interval of 30 msec and C-T intervals of 0.2, 0.6 or 1.0 msec and up to 2.5 msec. At these C-T intervals, the C-C interval was gradually reduced by steps of 1 msec until sell-stimulation returned to the reference criterion. As shown in Fig. 2 at any given C-T interval the C-C interval did not have to be halved in order to restore self-stimulation to the criterion. However, if the T-pulse is omitted ('single pulse condition'), the C-C interval must be shortened from 30 msec to 15 msec in order to restore ICS to criterion. This method not only provides another technique for measuring excitability changes but in addition proves that the T-pulse does have a function in recruiting neuronal activity. The slope of recovery when the C-T intervals are gradually increased may be a measure of the homogeneity of the stimulation field. A homogenous field would recover at a rapid rate, as most neurones would begin firing again once the absolute refractory period was past. On the other hand, a heterogenous field would recover slowly, as increasing C-T intervals would act differentially on the various neuronal elements (see also Yeomans [23]).
T-pulse Effectiveness With the parameters chosen in the last experiment (C-C interval 30 msec, 67 pps) the C-C interval had to be shortened to about 50% in order to reach the reference criterion. We then investigated whether the T-pulse effectiveness depends on the original C-C pulse intervals (or frequencies) used (Yeomans [23]). The intensity which fulfilled criterion of ICS was measured for different frequencies with the T-pulse occurring equidistant between the C-pulses (C-C intervals of 60, 50, 40, 30, 20, 10, 8 msec). At these parameters, shortening the C-T interval to 0.6 msec failed to produce responding, except in the high frequency range (/>150 pps). Increasing frequency
c f o,,r 0 /o °,'0 FIG. 3. Contribution of T-pulse: percentage increase in frequency (or shortening C-C interval) at different frequencies of pulse pair stimulations with a C-T interval of 0.6 msec; calculations according to Yeomans [23]. Electrode in lateral hypothalamus.
by shortening C-C interval again restored ICS. The amount by which the frequency had to be increased was dependent on the original C-C interval used. The contribution of the T-pulse was calculated for each C-C interval from the Yeomans' formula [23], i.e. relative effectiviness of the T-pulse is equal to (number of equidistant pulses in a single pulse condition/number of C-pulses in a C-T condition) -1, multiplied by 100 to express the values in percentages. The calculated contribution of the T-pulse are displayed in Fig. 3. At low frequencies, the T-pulses contributed about 30% to the C-pulses and this rose to 100% at 200 pulses per second. CONCLUSIONS The present experiments are evidence that using a pulsepair technique in behavioural measurements results in the measurement of a mixture of neuronal events. At least three hypotheses can be advanced to account for this. During the refractory period the T-pulse could initially have no depolarizing effect on the neurons but may cause the accumulation of electrical charge on the axons. Secondly the T-pulse may cause recruitment from dendrites [17,23]. Thirdly, C-T pulse summation may occur at the periphery of the stimulation field, where the C-pulse only produces subthreshold excitation. Local potential summation is dependent on the size of the stimulation field; our use of 0.2 msec pulses instead of 0.1 msec pulses would increase the excitation of the sub-liminal fringe. Additional experiments carried out by the second author have indeed shown that 'refractoriness' is better seen using 0.1 msec rather than 0.2 msec pulses which suggests that local potential summation overcomes refractoriness, although this may depend on the placement of the electrode.
168 Since pulse-pair synaptic and axonal sizes and h e n c e the ICS. The technique
WAUQUIER AND SADOWSKI m e a s u r e m e n t s p r o d u c e a mixture of events, it is ditticult to evaluate axonal types o f neuronal systems operating in described in the present p a p e r allows a
quantitative c o m p a r i s o n of the excitability of neuronal populations, which may differ depending on the structure stimulated.
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