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Factors influencing potentials in normal and cretinous rats
308
ELECTROFNCEPHALOGRAPHYA N D CLINICALNEUROPHYSIOLOGY
FACTORS
INFLUENCING
NORMAL
AND
POTENTIALS
CRETINOUS
IN
RATS
P. B. BRADLEY,D.Sc., J. T. EAYBS,1 D.Sc. AND NEST M. RZCHARDS,M.Sc. Departments of Experimental Nearopharmacology and Anatomy, University of Birmingham (Great Britain) (Accepted for publication: January 16, 1964)
Thyroid deficiency arising early in life causes severe impairment to the growth and maturation of the central nervous system of the rat (Eayrs 1959, 1960). The electrophysiological implications of these changes and the accompanying metabolic deficiency have been studied in terms of the electroencephalogram (Bradley et al. 1960) and of the recruiting response (Bradley et al. 1961; Eayrs et al. 1962). These investigations have shown that the amplitude of the EEG in rats after neonatal thyroidectomy is reduced and that the onset of spindling, blocking to auditory stimulation, and photic driving is delayed. Furtl~rmore, the latency and duration of an evoked response are increased while its amplitude is decreased. Short term medication with I-triiodothyroninecan restore the temporal parameters to normal without increasing the amplitude, but thyroidectomy in the mature animal engenders an increase in latency and duration without a reduction in amplitude. It thus seems that the temporal aspects of electro. cortical activity, at least in so far as this response is concerned, are regulated by the metabolic state of the animal, andamplitude by maturational changes. It has been suggested that the reduction in the probability of axo-dendritic interaction, demonstrated histologically (Eayrs 1955), may be the factor involved. One possibility, which was not taken into account in the experiments cited above, is that the optimum parameters of stimulation might differ in the two classes of rat. This has now been investigated and the present paper reports the effects of varying the voltage and frequency of t Present address: Department of NeuroendocrinoloBy, Institute of Psychiatry, The Maudsley Hospital, London, S.E. 5.
stimulation upon the recruiting response in cretinoid rats and their normal litter mates. MATERIALSAND METHODS
Preparation of animals Records were made from twenty pairs of litter mate rats of the same sex, bred from the Birmingham strain. One rat of each pair was thyroidectomised ov~the day of birth by a single injectionof 120/,c xaxIcontainedin about 0.05 ml saline. The control animal received ~n identical quantity of the vehicle. The animals were used for electrical recording at 24--28 days of age. Recording procedures The rats were orientated in a stereotactic instrument (Cort and Harding 1954) modified for small animals, under tribromoethanol anaesthesia (200 mg/kg). The skull was exposed, dried and covered with a thin layer of dental acrylic cement, Burr holes were then drilled for insertion of recording electrodes in standard positions overlying the frontal, parietal and occipital cortex and a further burr hole was made, in a position computed to overlie the medial thalamic nuclei and 0.5 mm from the midline, to accommodate the stimulating electrode (Fig. I). The positioning of all these electrodes was adjusted to compensate for differences in the size of skull of normal and thyroidectomised rats, Recording electrodes consisted of varnished silver wire, 0.012" (0.3 ram) in diameter, with the varnish removed at one end to form a loop for contact with the cortical surface, soldered to flexible recording leads at the other. They were fixed into position ~vith acrylic cement. The stimulating electrode was of the bipolar type (Bradley and Key 1958) and w~s ~n_itNllyguided EIectroeneeph. din. Neurophysiol., 19641 17:308-313
CORTICAL POTENTIALSIN TIlE RAT
309
staining by the method of Kliiver and Barrera (1953).
Fig. 1 Relative pmitions of recording fnumbered) and stimulating (S) electrodes.
Analysis of records The photographed records were enlarged by projection through an "Aldis" projector on to graph paper, which was used to measure the components of the response analysed by Bradley et al. (1961), i.e., (a) the latency between the stimulus and the beginning of the negative wave; (b) the duration and (c) the amplitude of the negative wave. Similar measurement was made of the callosal response except that the amplitude and duration of the initial positive wave were estimated in place of the latency of the recruited potential. The data for each component of the evoked response were analysed statistically by plotting the several dimensions against the appropriate value of each parameter of stimulation and computing a linear regression for each rat. Each regression coefficient was taken as a measure of the effect of varying the conditions &stimulation and the mean of these coefficients was tested, by means of its standard error, for the statistical significance of any departure from z~ro. Differential effects of stimulation in the two classes of rat were assessed by calculating the regressions of the difference between the paired sets of data and testing for the significance of the slope and position of the computed line.
stereotactically into the approximate position for stimulation (nucleus mediodorsalis) and finally adjusted to give the optimum response by inspection of the oscilloscope trace. Stimulation frequencies of 2, 4, 6, 8, 12 and 16/see were applied at the voltage setting usually found to give the optimum response at 6/sec: i.e., 9.3 V for the normal and 11.3 V for the thyroidectomised animal. Voltages ranging from RESULTS 7-18.7 V were used at a frequency of 6/sec. [n the cretinous rats the durations of all Oscillograph records were made of the responses evoked by all these parameters of stimulation by temporal parameters of the recruited response photographing three frames, each containing six were longer than in normal litter mates at a high superimposed traces, for each parameter. A pulse level of statistical significance (P <0.001), but the effect of varying the strength and frequency of width of 2 msec was used throughout. During the course of these experiments the stimulation was slight. These findings are illuscallosal response was recorded in six pairs of trated in Fig. 2 and 3, in which the recruited animals. The same antero-posterior and lateral responses obtained under differing conditions coordinates were used as for the recruited re- of stimulation are set out as schematic diagrams, sponsc and, with the electrode t!p positioned in drawn to scale with origins at the start of the the corpus callosum, stimuli were delivered at a negative wave. The upright lines marking the start of the latent period and the end of the strength of i 1.0 V and a frequency of 2/sec. negative wave are the computed regressions of the latency and duration of the response plotted Histological examination T~,e position o;' tile tip of the stimulating against the varying parameter of stimulation. electrode was verified by preparing paraffin The trends in the records of both normal ~-nd sections cut coronally through the brain and cretinous animals (i.e., small reductions in ]atenElectroenceph. clin. Neurophysiol., 1964, 17:308-313
p.B. BRADLEY el a~
310
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.
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reduction in duration, with increase in frequency of stimulation) proved to be identical, but none approached statistical significance. The amplitude of the negative wave, on the other hand, was sensitive both to the strength and frequency of stimulation. This can be seen in Fig. 2 and 3 but is more clearly shown in Fig. 4 and 5. Fig. 4 shows that amplitude steadily increased, with a good linear relationship to the stimulating voltage, in both normal and cretinous rats, at a high level of statistical significance (P <0.001 in each case). Although this increase was somewhat less for the cretinous X Normal ® Cretin
~
0 10'2030
10 0 ~ 3 0 4 0 ~
Dumtkm (reset) Fig. 2
b.10.7±2.23
CinmiNs in chm©t©ristics of recruited response with incr~lsinB voltap of stimulation (see text). The statisti~
at tl~ top of ugh vertical line repmJent the regression
400 ~"
data plotted a~ the m u m for the two classes of rat,
.*¢ •
~ ,
A :007.,0.05
c~t~
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< loo
Chanses in mean amplitude of recruited reapome with
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C~..anlp~s in characteristics of r~_ruited response with increasin8 frequency of stimulation. The statistics h(,zdinl each vertical line are the relprasion coefficients of latency and duration on frequency. The data plotted are the m ~ n s for the two classes of rat,
cy and duration when the stimulating voltage was increased and a slight increase in latency, but
~ ~
,
, ....
,
2
4
6
8
.......16
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Frequency of stimulation (pulses/see)
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Eleetroenceph. din. Newophysiol., 1964,
17:308-313
'
CORTICAL POTENTIALSIN THE RAT
group, there was no evidence for a differential effect. On the other hand, the pattern of change to increase in the frequency of stimulation differed markedly between the two classes of rat. Fig. 5 shows, in the case of the cretinous animals, a steady decay in the amplitude of the response as a result of increase in the frequency of stimulation. A satisfactory linear relationship, having a statistically significant regression coefficient (P <0.001) is evident. By contrast, the relationship between frequency and amplitude in the normal individual was non.linear. A slight, but non-significant, increase in amplit~tde occurred when the frequency of stimulation was increased between 2-8/sec which was followed, at higher frequencies, by a highly significant (P <0.001) falling off in amplitude. Differences between normal and cretinous groups in the form of the callosal response were similar to those observed in the recruited potential. The duration of both positive and negative waves was extended and their amplitudes redueed (Fig. 6). @Cretin x Normal
\ t
/
l
/
tials evoked from normal and cretinous individ. uals and the effects of varying the parameters of stimulation. The former, which demonstrate an increase in the latency and duration of the response, amply confirm the earlier findings of Bradley et al. (1961) in rats of similar age and, indeed, the data for both. classes of animal show a remarkable corresponeence with those previously obtained. Furthermore, in both experiments, the amplitude was significanzly reduced as a result of neonatal thyroidectomy. The potentials recorded from both groups of rats in the present experiment, however, were greater than those observed previously, but this observation almost certainly reflects differences in the method of applying the recording electrodes to the cortical surface. The possible mechanisms underlying these changes and their relationship to metabolic and developmental factors have already been discussed (Bradley et al. 1961) and the additional finding that similar changes in latency, duration and amplitude occur in the callosal response reinforces the suggestion that they are, to a large measure, mediated intracortically rather than by way ofthalamic involvement.
-e(
E ,q
311
\ 40
Duration (reset)
Fig. 6 Stylised diagram of the mean values for the callosal response in six pairs of normal and cretinous rats.
DISCUSSION
The results of this experimen t fall into two groups: the differences between recruited poten-
The Jack of any differential effect of varying the stimulus parameters, other than that relating frequency to amplitude, provides little support for the possibility that the altered response ofthe central nervous system of the cretin may be attributable to changes involving the optimum parameters of stimulation. In no instence did variation of either voltage or frequency restore the response in the cretinous individual to anything approaching that recorded from the normal. The results, which indicate that any changes induced by the experimental treatment are quantitative rather than qualitative, may be interpreted in terms of the reduced probability of axe-dendritic interaction shown to arise as a result of neonatal thyroidectomy (Eayrs 1955). If, as has been suggested (Li et al. 1956; Purpura and Grundfest 1956), the negative wave represents a summation of post-synaptic dendritic potentials a reduction in the number of participating elements would help to account for the smaller amplitude seen in the cretin. In these circumstances, n¢ differential response to an increase in the voltage of stimulation would be Electroenceph. din. NeurophysloL, 1964, 17:308-313
312
p.a. hADLEY et al.
anticipate~ since, in both normal and cretinous rats, the number of "units" activated might be expected to increase in direct relationship to the number of non.specificthalamo-corticai afferents coming within the expanding zone of depolarization generated in the vicinityof the stimulating electrode. Equally, in a system in which all stimulation is carried out at a supra-threshold level, opportunities for reducing temporal parameters by the process of facilitation are minimal and it is therefore perhaps not surprising that neither change of the voltage nor of the frequency of stimulation had any significant effect on latency or duration. A relationship between frequency of stimulation and amplitude similar to, though more complex than, that seen in the present experiment has been reported in the eat by Verzeano et al. (1953) who suggested that maximal amplitudes are attained when thalamic discharges synchronise with the natural frequencies of oscillation of self-reexciting cortie,al circuits. When such discharges fall out of phase and arrive during the refractory periods of the constituent elements of these circuits the number of units contributing to the response would be diminished and amplitude consequently reduced. The slower spontaneous activity occurring in hypothyroidism (Bertrand er ol. 1938; Hoagland 1936) might therefore account for the lower optimum frequency and earlier decay in amplitude seen in the recruited response of the cretinous rat. An alternative explanation, suggested by the finding that different projection systems can be activated from an identical focus by varying the parameters of stimulation (Livingston 1949; Nulsen et al. 1948), is that stimulation at higher frequencies gives rise to converge~tce effects or the activation of inhibitory systems. Yet a third possibility derives from the finding that synaptic transmission can be blocked by repetitive stimu. lotion (see Chang 1959), with the result that amplitude falls as the number of participating synapses is reduced. An earlier appearance of this effect in the cretinous individual wight be expected by reason of an increase in the time needed for repolarization due to an impaired metabolism; but the frequencies at which this effect has normally been obtained far exceed those used in the present experiment (e.g.,
Bishop and McLeod 19~4),although they may be much reduced in a polysyrmptic pathway (Forbes and Morison 1939; King et al. 1957). Such concepts, however, are clearly speculative and need further investigation. SUMMARY
I. The effects of varying the parameters of stimulation upon the recruited response have been studied in normal and cretinous rats. 2. Latency between stimulus and the beginning of the negative wave and the duration of the negative wave were both longer in the cretinous animal than in the normal. Increasing the voltage or frequency of stimulation had very little effect on these measures in either class of animal and such slight trends as were seen were similar in both instances. 3. The amplitude of the response at a fixed frequency of stimulation was significantly less in the cretinous rat than in the normal at all voltages. An increase in the voltage resulted in a comparable increase in amplitude in both classes of rat, without significant differential effect. 4. In the normal animal amplitude increased slightly with frequency over the range 2-6/sec but with further increase in the rate of stimula. tion it fell off sharply, The decay of amplitude in cretinous rats was observed over the whole range of frequencies studied (2=16/sec),
R~UM~. FACTEUI~ MODIFIANT L[~ POTENTIEI~ CHEZ LE RAT NORMAL ET LE RAT CII~TINO|DE
I, Les effetscaus6s sur la r~ponse recrutante
par la variation des param~tres de stimulation ont ~t6 6tudi6s chez le rat normal et ¢hez le rat cr~tinolde. 2. La latence entre le stimulus et le d~but de l'onde n~gative, alnsi que la dur,~ de celle-ci, ~taient plus longues chez I'animul cr~tino|de que chez l'animal normal. L'aceroissement du vol. tage ou de la fr~luenee de stimulation avalt tr~s peu d'effet sur ces mesures, chez l'un et rautre type d'animal, et toutes les 16g~res variations observ6es ~taient similalres clans les deux cos. 3. L'amplitude de |a r~ponse i une fr~tcluence de stimulation donn~ ~tait significativement moindre chez le rat cr~tinolde que chez le rat Hectroenceph. olin, Ne~ophysiol., 1964, 17:308--313
CORTICAL POTENTIAI~ IN THE RAT
313
normal pour tous les voltages. Un accroissement de voltage avait pour r~sultat un accroissement comparable d'amplitude chez les deux types de rat sans effet diff~rentiel significatif. 4. Chez l'animal normal, l'amplitude augmentait 16gi~rement pour la bande de fr6quences de 2 A 6/see, mais elle tombait brusquement lorsque la fr~luence de stimulation 6tait augmenter. Le dCLclinde ramplitude chez le rat cr~tinoide s'observait pour toute la gamme de fr6quences ¢hudi~s (2-16/sec).
stereo:axle instrument. J. Physiol. (Land.), 1954, 123: 15P. EAYRS, J. T. The cerebral cortex of normal and hypothyroid rats. Acre. anat. (Basel), 19aS, 2.5: 160-283. EAYas, J. T. The status of the thyroid gland in relation to the development of the nervous system. Brit. J. Anita. Behav., 195'9, 7: 1-17. EA,~ts, J. T. Influence of the thyroid on the central nervous system. Brit. meal. Bull., 1960, 16: 122-127. EAYitS, J. T., GLASS,A. and BROADHUttSr,P. L. Thyroid function and central nervous activity. J. Endocr., 1962, 24: viii-ix. FORBES, A. and Moat.soN, B. R. Cortical response to sensory stimulation under deep barbiturate narcosis. J. Neurophysiol., 1939, 2: 112-228. REFERENCES HOAOLAND, H. Pacemakers of human brainwaves in BERTRAND,!., DELAY,J. et OUILLAIN,J. L'~:2ectro-enc~ tnorma2s and in general paretics Amer. J. PhysioL, pha2ogramme dans le myxoed6me. C. R. Soc. Biol. 1936, 116: 604-615. (Paris), 1938, 129: 395-398. gJSO, E. E., NAqUET,R. and MAGOUN,H. W. Alterations BmHov, P. O. and McLEoD, J. G. Nature of potentials in somatic afferent transmission through the thalamus associated with synaptic transmission in lateral by central mechanisms and barbiturates. J. Pharma. geniculate of cat. J. Neurophysiol., 1954, 17: 387-414. col. exp. Ther., 1957, 119: 48-63. BRADLEY,P. B., EAYRS,J. T., GLASS,A, and HEATEr,R. W. KL0vr~, H. and BARRERA,E. A. A method for the comThe maturational and metabolic consequences of bined staining of cells and fibres in the nervous system. neonatal thyroidectomy upon the recruiting response J. Neuropath. exp. Neurol., 1953, 12: 400-403. in the rat. Electroenceph. c/in. NeurophysloL, 1961, Lt, C. L., CULLEN,C. and JASPER,H. H. Laminar micro13: 577-586. electrode analysis of conical unspecific recruiting BRADLEY,P. B., EAYRS,J. T. and SCIIMALBACH,K. The responses and spontaneous rhythms. J. Nearophysiol., 194J6, 19: 131-143. electroencephedogram of normal and hypothyroid rats. Electroeneeph. din. NearophysioL, 1960, 12: LIVINGSTON, R. B. Cited by J. F. FULTON,Functional localization in the frontal lobes and cerebellum. 467-.477. BBADLEY,P. B. and KEy, B. J. Thc effect of drugs on Clarendon Press,Oxford, 1949. arousal rmpon~ produced by electrical stimulation NUIJEN, F. E., BLACK, S. P. W. and DRAKE, C. O. of the reticular formation of the brain, Eiectroenceph. Inhibition and facilitation of motor activity by the anterior cerebellum. Fed. Proc., 1948, 7: 86-87. elin. NeuPophyaiol., 1958, i0:97-120. CHANO0H. T. The evoked potentials, in J. FIELD,H. W. PURPURA,D. P. and GRuNomrr, H. Nature of dendritic potentials and synaptic mechanisms in cerebral MAOOUN and V. E. HALL (Eds,),Handbook ofphyslolo. cortex of cat, 7. Neurophy$1ol., 1956, 19: 573--595. ~y, See, L Anter. Physiol. Sot., Wuhinston, 1959, VERZEANO, M., LINDliLEV, D. B. and MAOOUN,H. W. I: 209-~.13. Nature of recruiting response. J, Neurophyaiol., 1953, COAT, J. H. and HARDINO, H. F. Inexpensive precision 16: 183-195,
Reference: BRADLEY,P. B., EAYP.q,J. T. and RICHARDS,N. M. Factors influencing potentia2s in normal and cretinous rats. Electroenceph. cltn. NeurophysioL, 1964, 17: 308-313.
ELECTROFNCEPHALOGRAPHYA N D CLINICALNEUROPHYSIOLOGY
FACTORS
INFLUENCING
NORMAL
AND
POTENTIALS
CRETINOUS
IN
RATS
P. B. BRADLEY,D.Sc., J. T. EAYBS,1 D.Sc. AND NEST M. RZCHARDS,M.Sc. Departments of Experimental Nearopharmacology and Anatomy, University of Birmingham (Great Britain) (Accepted for publication: January 16, 1964)
Thyroid deficiency arising early in life causes severe impairment to the growth and maturation of the central nervous system of the rat (Eayrs 1959, 1960). The electrophysiological implications of these changes and the accompanying metabolic deficiency have been studied in terms of the electroencephalogram (Bradley et al. 1960) and of the recruiting response (Bradley et al. 1961; Eayrs et al. 1962). These investigations have shown that the amplitude of the EEG in rats after neonatal thyroidectomy is reduced and that the onset of spindling, blocking to auditory stimulation, and photic driving is delayed. Furtl~rmore, the latency and duration of an evoked response are increased while its amplitude is decreased. Short term medication with I-triiodothyroninecan restore the temporal parameters to normal without increasing the amplitude, but thyroidectomy in the mature animal engenders an increase in latency and duration without a reduction in amplitude. It thus seems that the temporal aspects of electro. cortical activity, at least in so far as this response is concerned, are regulated by the metabolic state of the animal, andamplitude by maturational changes. It has been suggested that the reduction in the probability of axo-dendritic interaction, demonstrated histologically (Eayrs 1955), may be the factor involved. One possibility, which was not taken into account in the experiments cited above, is that the optimum parameters of stimulation might differ in the two classes of rat. This has now been investigated and the present paper reports the effects of varying the voltage and frequency of t Present address: Department of NeuroendocrinoloBy, Institute of Psychiatry, The Maudsley Hospital, London, S.E. 5.
stimulation upon the recruiting response in cretinoid rats and their normal litter mates. MATERIALSAND METHODS
Preparation of animals Records were made from twenty pairs of litter mate rats of the same sex, bred from the Birmingham strain. One rat of each pair was thyroidectomised ov~the day of birth by a single injectionof 120/,c xaxIcontainedin about 0.05 ml saline. The control animal received ~n identical quantity of the vehicle. The animals were used for electrical recording at 24--28 days of age. Recording procedures The rats were orientated in a stereotactic instrument (Cort and Harding 1954) modified for small animals, under tribromoethanol anaesthesia (200 mg/kg). The skull was exposed, dried and covered with a thin layer of dental acrylic cement, Burr holes were then drilled for insertion of recording electrodes in standard positions overlying the frontal, parietal and occipital cortex and a further burr hole was made, in a position computed to overlie the medial thalamic nuclei and 0.5 mm from the midline, to accommodate the stimulating electrode (Fig. I). The positioning of all these electrodes was adjusted to compensate for differences in the size of skull of normal and thyroidectomised rats, Recording electrodes consisted of varnished silver wire, 0.012" (0.3 ram) in diameter, with the varnish removed at one end to form a loop for contact with the cortical surface, soldered to flexible recording leads at the other. They were fixed into position ~vith acrylic cement. The stimulating electrode was of the bipolar type (Bradley and Key 1958) and w~s ~n_itNllyguided EIectroeneeph. din. Neurophysiol., 19641 17:308-313
CORTICAL POTENTIALSIN TIlE RAT
309
staining by the method of Kliiver and Barrera (1953).
Fig. 1 Relative pmitions of recording fnumbered) and stimulating (S) electrodes.
Analysis of records The photographed records were enlarged by projection through an "Aldis" projector on to graph paper, which was used to measure the components of the response analysed by Bradley et al. (1961), i.e., (a) the latency between the stimulus and the beginning of the negative wave; (b) the duration and (c) the amplitude of the negative wave. Similar measurement was made of the callosal response except that the amplitude and duration of the initial positive wave were estimated in place of the latency of the recruited potential. The data for each component of the evoked response were analysed statistically by plotting the several dimensions against the appropriate value of each parameter of stimulation and computing a linear regression for each rat. Each regression coefficient was taken as a measure of the effect of varying the conditions &stimulation and the mean of these coefficients was tested, by means of its standard error, for the statistical significance of any departure from z~ro. Differential effects of stimulation in the two classes of rat were assessed by calculating the regressions of the difference between the paired sets of data and testing for the significance of the slope and position of the computed line.
stereotactically into the approximate position for stimulation (nucleus mediodorsalis) and finally adjusted to give the optimum response by inspection of the oscilloscope trace. Stimulation frequencies of 2, 4, 6, 8, 12 and 16/see were applied at the voltage setting usually found to give the optimum response at 6/sec: i.e., 9.3 V for the normal and 11.3 V for the thyroidectomised animal. Voltages ranging from RESULTS 7-18.7 V were used at a frequency of 6/sec. [n the cretinous rats the durations of all Oscillograph records were made of the responses evoked by all these parameters of stimulation by temporal parameters of the recruited response photographing three frames, each containing six were longer than in normal litter mates at a high superimposed traces, for each parameter. A pulse level of statistical significance (P <0.001), but the effect of varying the strength and frequency of width of 2 msec was used throughout. During the course of these experiments the stimulation was slight. These findings are illuscallosal response was recorded in six pairs of trated in Fig. 2 and 3, in which the recruited animals. The same antero-posterior and lateral responses obtained under differing conditions coordinates were used as for the recruited re- of stimulation are set out as schematic diagrams, sponsc and, with the electrode t!p positioned in drawn to scale with origins at the start of the the corpus callosum, stimuli were delivered at a negative wave. The upright lines marking the start of the latent period and the end of the strength of i 1.0 V and a frequency of 2/sec. negative wave are the computed regressions of the latency and duration of the response plotted Histological examination T~,e position o;' tile tip of the stimulating against the varying parameter of stimulation. electrode was verified by preparing paraffin The trends in the records of both normal ~-nd sections cut coronally through the brain and cretinous animals (i.e., small reductions in ]atenElectroenceph. clin. Neurophysiol., 1964, 17:308-313
p.B. BRADLEY el a~
310
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A
GC)6,02~
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.
-ODI
t
reduction in duration, with increase in frequency of stimulation) proved to be identical, but none approached statistical significance. The amplitude of the negative wave, on the other hand, was sensitive both to the strength and frequency of stimulation. This can be seen in Fig. 2 and 3 but is more clearly shown in Fig. 4 and 5. Fig. 4 shows that amplitude steadily increased, with a good linear relationship to the stimulating voltage, in both normal and cretinous rats, at a high level of statistical significance (P <0.001 in each case). Although this increase was somewhat less for the cretinous X Normal ® Cretin
~
0 10'2030
10 0 ~ 3 0 4 0 ~
Dumtkm (reset) Fig. 2
b.10.7±2.23
CinmiNs in chm©t©ristics of recruited response with incr~lsinB voltap of stimulation (see text). The statisti~
at tl~ top of ugh vertical line repmJent the regression
400 ~"
data plotted a~ the m u m for the two classes of rat,
.*¢ •
~ ,
A :007.,0.05
c~t~
"088
< loo
Chanses in mean amplitude of recruited reapome with
x N~
C~..anlp~s in characteristics of r~_ruited response with increasin8 frequency of stimulation. The statistics h(,zdinl each vertical line are the relprasion coefficients of latency and duration on frequency. The data plotted are the m ~ n s for the two classes of rat,
cy and duration when the stimulating voltage was increased and a slight increase in latency, but
~ ~
,
, ....
,
2
4
6
8
.......16
12
Frequency of stimulation (pulses/see)
s
Chanses in mean amplitude of recruited response with increasein freqencuy of stimulation. . . o
Eleetroenceph. din. Newophysiol., 1964,
17:308-313
'
CORTICAL POTENTIALSIN THE RAT
group, there was no evidence for a differential effect. On the other hand, the pattern of change to increase in the frequency of stimulation differed markedly between the two classes of rat. Fig. 5 shows, in the case of the cretinous animals, a steady decay in the amplitude of the response as a result of increase in the frequency of stimulation. A satisfactory linear relationship, having a statistically significant regression coefficient (P <0.001) is evident. By contrast, the relationship between frequency and amplitude in the normal individual was non.linear. A slight, but non-significant, increase in amplit~tde occurred when the frequency of stimulation was increased between 2-8/sec which was followed, at higher frequencies, by a highly significant (P <0.001) falling off in amplitude. Differences between normal and cretinous groups in the form of the callosal response were similar to those observed in the recruited potential. The duration of both positive and negative waves was extended and their amplitudes redueed (Fig. 6). @Cretin x Normal
\ t
/
l
/
tials evoked from normal and cretinous individ. uals and the effects of varying the parameters of stimulation. The former, which demonstrate an increase in the latency and duration of the response, amply confirm the earlier findings of Bradley et al. (1961) in rats of similar age and, indeed, the data for both. classes of animal show a remarkable corresponeence with those previously obtained. Furthermore, in both experiments, the amplitude was significanzly reduced as a result of neonatal thyroidectomy. The potentials recorded from both groups of rats in the present experiment, however, were greater than those observed previously, but this observation almost certainly reflects differences in the method of applying the recording electrodes to the cortical surface. The possible mechanisms underlying these changes and their relationship to metabolic and developmental factors have already been discussed (Bradley et al. 1961) and the additional finding that similar changes in latency, duration and amplitude occur in the callosal response reinforces the suggestion that they are, to a large measure, mediated intracortically rather than by way ofthalamic involvement.
-e(
E ,q
311
\ 40
Duration (reset)
Fig. 6 Stylised diagram of the mean values for the callosal response in six pairs of normal and cretinous rats.
DISCUSSION
The results of this experimen t fall into two groups: the differences between recruited poten-
The Jack of any differential effect of varying the stimulus parameters, other than that relating frequency to amplitude, provides little support for the possibility that the altered response ofthe central nervous system of the cretin may be attributable to changes involving the optimum parameters of stimulation. In no instence did variation of either voltage or frequency restore the response in the cretinous individual to anything approaching that recorded from the normal. The results, which indicate that any changes induced by the experimental treatment are quantitative rather than qualitative, may be interpreted in terms of the reduced probability of axe-dendritic interaction shown to arise as a result of neonatal thyroidectomy (Eayrs 1955). If, as has been suggested (Li et al. 1956; Purpura and Grundfest 1956), the negative wave represents a summation of post-synaptic dendritic potentials a reduction in the number of participating elements would help to account for the smaller amplitude seen in the cretin. In these circumstances, n¢ differential response to an increase in the voltage of stimulation would be Electroenceph. din. NeurophysloL, 1964, 17:308-313
312
p.a. hADLEY et al.
anticipate~ since, in both normal and cretinous rats, the number of "units" activated might be expected to increase in direct relationship to the number of non.specificthalamo-corticai afferents coming within the expanding zone of depolarization generated in the vicinityof the stimulating electrode. Equally, in a system in which all stimulation is carried out at a supra-threshold level, opportunities for reducing temporal parameters by the process of facilitation are minimal and it is therefore perhaps not surprising that neither change of the voltage nor of the frequency of stimulation had any significant effect on latency or duration. A relationship between frequency of stimulation and amplitude similar to, though more complex than, that seen in the present experiment has been reported in the eat by Verzeano et al. (1953) who suggested that maximal amplitudes are attained when thalamic discharges synchronise with the natural frequencies of oscillation of self-reexciting cortie,al circuits. When such discharges fall out of phase and arrive during the refractory periods of the constituent elements of these circuits the number of units contributing to the response would be diminished and amplitude consequently reduced. The slower spontaneous activity occurring in hypothyroidism (Bertrand er ol. 1938; Hoagland 1936) might therefore account for the lower optimum frequency and earlier decay in amplitude seen in the recruited response of the cretinous rat. An alternative explanation, suggested by the finding that different projection systems can be activated from an identical focus by varying the parameters of stimulation (Livingston 1949; Nulsen et al. 1948), is that stimulation at higher frequencies gives rise to converge~tce effects or the activation of inhibitory systems. Yet a third possibility derives from the finding that synaptic transmission can be blocked by repetitive stimu. lotion (see Chang 1959), with the result that amplitude falls as the number of participating synapses is reduced. An earlier appearance of this effect in the cretinous individual wight be expected by reason of an increase in the time needed for repolarization due to an impaired metabolism; but the frequencies at which this effect has normally been obtained far exceed those used in the present experiment (e.g.,
Bishop and McLeod 19~4),although they may be much reduced in a polysyrmptic pathway (Forbes and Morison 1939; King et al. 1957). Such concepts, however, are clearly speculative and need further investigation. SUMMARY
I. The effects of varying the parameters of stimulation upon the recruited response have been studied in normal and cretinous rats. 2. Latency between stimulus and the beginning of the negative wave and the duration of the negative wave were both longer in the cretinous animal than in the normal. Increasing the voltage or frequency of stimulation had very little effect on these measures in either class of animal and such slight trends as were seen were similar in both instances. 3. The amplitude of the response at a fixed frequency of stimulation was significantly less in the cretinous rat than in the normal at all voltages. An increase in the voltage resulted in a comparable increase in amplitude in both classes of rat, without significant differential effect. 4. In the normal animal amplitude increased slightly with frequency over the range 2-6/sec but with further increase in the rate of stimula. tion it fell off sharply, The decay of amplitude in cretinous rats was observed over the whole range of frequencies studied (2=16/sec),
R~UM~. FACTEUI~ MODIFIANT L[~ POTENTIEI~ CHEZ LE RAT NORMAL ET LE RAT CII~TINO|DE
I, Les effetscaus6s sur la r~ponse recrutante
par la variation des param~tres de stimulation ont ~t6 6tudi6s chez le rat normal et ¢hez le rat cr~tinolde. 2. La latence entre le stimulus et le d~but de l'onde n~gative, alnsi que la dur,~ de celle-ci, ~taient plus longues chez I'animul cr~tino|de que chez l'animal normal. L'aceroissement du vol. tage ou de la fr~luenee de stimulation avalt tr~s peu d'effet sur ces mesures, chez l'un et rautre type d'animal, et toutes les 16g~res variations observ6es ~taient similalres clans les deux cos. 3. L'amplitude de |a r~ponse i une fr~tcluence de stimulation donn~ ~tait significativement moindre chez le rat cr~tinolde que chez le rat Hectroenceph. olin, Ne~ophysiol., 1964, 17:308--313
CORTICAL POTENTIAI~ IN THE RAT
313
normal pour tous les voltages. Un accroissement de voltage avait pour r~sultat un accroissement comparable d'amplitude chez les deux types de rat sans effet diff~rentiel significatif. 4. Chez l'animal normal, l'amplitude augmentait 16gi~rement pour la bande de fr6quences de 2 A 6/see, mais elle tombait brusquement lorsque la fr~luence de stimulation 6tait augmenter. Le dCLclinde ramplitude chez le rat cr~tinoide s'observait pour toute la gamme de fr6quences ¢hudi~s (2-16/sec).
stereo:axle instrument. J. Physiol. (Land.), 1954, 123: 15P. EAYRS, J. T. The cerebral cortex of normal and hypothyroid rats. Acre. anat. (Basel), 19aS, 2.5: 160-283. EAYas, J. T. The status of the thyroid gland in relation to the development of the nervous system. Brit. J. Anita. Behav., 195'9, 7: 1-17. EA,~ts, J. T. Influence of the thyroid on the central nervous system. Brit. meal. Bull., 1960, 16: 122-127. EAYitS, J. T., GLASS,A. and BROADHUttSr,P. L. Thyroid function and central nervous activity. J. Endocr., 1962, 24: viii-ix. FORBES, A. and Moat.soN, B. R. Cortical response to sensory stimulation under deep barbiturate narcosis. J. Neurophysiol., 1939, 2: 112-228. REFERENCES HOAOLAND, H. Pacemakers of human brainwaves in BERTRAND,!., DELAY,J. et OUILLAIN,J. L'~:2ectro-enc~ tnorma2s and in general paretics Amer. J. PhysioL, pha2ogramme dans le myxoed6me. C. R. Soc. Biol. 1936, 116: 604-615. (Paris), 1938, 129: 395-398. gJSO, E. E., NAqUET,R. and MAGOUN,H. W. Alterations BmHov, P. O. and McLEoD, J. G. Nature of potentials in somatic afferent transmission through the thalamus associated with synaptic transmission in lateral by central mechanisms and barbiturates. J. Pharma. geniculate of cat. J. Neurophysiol., 1954, 17: 387-414. col. exp. Ther., 1957, 119: 48-63. BRADLEY,P. B., EAYRS,J. T., GLASS,A, and HEATEr,R. W. KL0vr~, H. and BARRERA,E. A. A method for the comThe maturational and metabolic consequences of bined staining of cells and fibres in the nervous system. neonatal thyroidectomy upon the recruiting response J. Neuropath. exp. Neurol., 1953, 12: 400-403. in the rat. Electroenceph. c/in. NeurophysloL, 1961, Lt, C. L., CULLEN,C. and JASPER,H. H. Laminar micro13: 577-586. electrode analysis of conical unspecific recruiting BRADLEY,P. B., EAYRS,J. T. and SCIIMALBACH,K. The responses and spontaneous rhythms. J. Nearophysiol., 194J6, 19: 131-143. electroencephedogram of normal and hypothyroid rats. Electroeneeph. din. NearophysioL, 1960, 12: LIVINGSTON, R. B. Cited by J. F. FULTON,Functional localization in the frontal lobes and cerebellum. 467-.477. BBADLEY,P. B. and KEy, B. J. Thc effect of drugs on Clarendon Press,Oxford, 1949. arousal rmpon~ produced by electrical stimulation NUIJEN, F. E., BLACK, S. P. W. and DRAKE, C. O. of the reticular formation of the brain, Eiectroenceph. Inhibition and facilitation of motor activity by the anterior cerebellum. Fed. Proc., 1948, 7: 86-87. elin. NeuPophyaiol., 1958, i0:97-120. CHANO0H. T. The evoked potentials, in J. FIELD,H. W. PURPURA,D. P. and GRuNomrr, H. Nature of dendritic potentials and synaptic mechanisms in cerebral MAOOUN and V. E. HALL (Eds,),Handbook ofphyslolo. cortex of cat, 7. Neurophy$1ol., 1956, 19: 573--595. ~y, See, L Anter. Physiol. Sot., Wuhinston, 1959, VERZEANO, M., LINDliLEV, D. B. and MAOOUN,H. W. I: 209-~.13. Nature of recruiting response. J, Neurophyaiol., 1953, COAT, J. H. and HARDINO, H. F. Inexpensive precision 16: 183-195,
Reference: BRADLEY,P. B., EAYP.q,J. T. and RICHARDS,N. M. Factors influencing potentia2s in normal and cretinous rats. Electroenceph. cltn. NeurophysioL, 1964, 17: 308-313.