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The maturational and metabolic consequences of neonatal thyroidectomy upon the recruiting response in the rat
THE M A T U R A T I O N A L A N D METABOLIC CONSEQUENCES OF N E O N A T A L THYROIDECTOMY U P O N THE RECRUITING RESPONSE IN THE RAT P. B. BRADLEY, D.Sc., J. T. EAYRS, D.Sc. 1, A. GLASS, M.B., CH.B. ~ AND R. W. HEATH, M.Sc. 3 Departments of Anatomy and Experimental Psychiatry, University of Birmingham (Gr. Britain) (Received for publication: October 15, 1960)
gested grounds on which such changes might be related to the impaired growth of conducting elements within the cerebral cortex. Accordingly, in order further to elucidate the relationship between the structural and electrophysiological sequelae of neonatal thyroidectomy a study has been made of a better defined cortical response which would enable changes in temporal relations to be detected. The recruiting response, first described by Dempsey and Morison (1942) and analysed into functional components by Arduini and Terzuolo (1951) and by Li et al. (1956), was selected as being the most suitable for this purpose.
INTRODUCTION
The relation that the thyroid gland bears to the maturation and maintenance of function of the central nervous system has for a long time been well-known (for reviews see Eayrs 1959, 1960a) and continued investigation of the neuroendocrinological effects of thyroxine or its lack may be expected to shed further light upon the inter-relationship between the structure of the cerebral cortex during development and its function measured in terms of behaviour or electrical activity. Recent investigations have shown, for instance, that thyroid deficiency, arising during the early stages of maturation, gives rise to marked abnormalities in cerebral development prominent among which is a decrease in the extent and complexity of the cortical neuropil (Eayrs 1955) and a consequent reduction in the probability of interaction between neurones (Eayrs 1960b) associated with quantitatively observed behavioural impairment (Eayrs and Lishman 1953). The neurophysiological consequences attendant upon such changes were initially investigated by Bradley et al. (1960) who recorded the EEG of neonatally thyroidectomised rats and found a significant decrease in the amplitude of the spontaneous electrical activity coupled with an absence of the slow wave potentials normally associated with drowsiness and of response to rhythmic photic stimulation. These authors sug-
MATERIALS AND METHODS
General experimental procedure
1 Henry Head Research Fellow of the Royal Societyp London. Assisted by a grant from the Caroline Harrold Research Fund. 3 Supported by a grant from the Office of Scientific Research of the Air Research and Development Council, United States Air Force, Contract No. AF61 (514)-1184. 577
Seventy-four rats of the Birmingham albino strain were used, divided into 31 groups of littermates of the same sex in accordance with the procedures outlined below. The experiment was divided into the following four parts: Series A. Twelve littermate pairs were used, one rat of each pair being radiothyroidectomised on the day of birth (Goldberg and Chaikoff 1948) by the injection of 150 #C of 1311, its littermate receiving a similar dose of the vehicle. The recruiting response was recorded under very light anaesthesia with tribromoethanol at the age of 24-26 days when, as suggested by the previously cited work on the EEG of such rats, the effect of neonatal thyroidectomy was likely to be maximal. Series B. Seven littermate triads were used in this series. Two of the three rats were thyroidectomised at birth as for series A and the third acted as control. Beginning on the second day
578
P.D. BRADLEYeta].
before that scheduled for recording, one of the thyroidectomised animals was given a daily subcutaneous injection 1.5 #g of 1-triiodothyronine (T3) dissolved in 0.1 ml saline, the remaining thyroidectomised rat and the control receiving the vehicle alone. Records were taken as for series A with addition that, after the first record had been taken 3/tg of T3 was given intraperitoneally to each animal and further records were made at intervals of 15 and 30 rain after injection. Series C. This series of experiments was conducted to resolve doubts concerning the statistical validity of certain of the results obtained in series B. Using five littermate triads, the procedures of series B were repeated except that acute medication with T3 was not carried out in this instance. Series D. Six pairs of littermates were used. One rat of each pair was surgically thyroidectoraised during adult life; the other underwent mock operation. Records of the recruiting response were taken 28 days later.
Electrophysiological techniques For the experiments in series A - C an array of six spherically ended silver wire electrodes each of diameter 0.124" was sealed with Araldite into an artificial calvarium made from acrylic resin moulded to fit the skull of a 25-day old rat. Under tribromoethanol anaesthesia (200 mg/kg) the animal was placed in a stereotactic instrument (modified from that described by Cort and Harding 1953), the scalp reflected and a window cut in the fronto-parietal part of the skull symmetrically about the midline. The dura was then reflected on either side of the sagittal sinus and, after haemostasis had been secured, the electrode-bearing cap was sealed over the fenestration with dental cement so that the electrode tips came into contact with the cortical surface. This procedure was modified for the adult rats used in series D, for in such animals it is extremely hazardous to remove the skull without damage to the superior sagittal sinus. Accordingly an array of four ball-ended silver wire electrodes, insulated by PVC sleeving, was positioned stereotactically and the electrode tips brought into contact with the cortex through independently drilled bur-holes and secured with acrylic resin. A bipolar concentric type of stimulating
electrode (Bradley and Key 1958) of outer diameter 0.012" was inserted stereotactically through the bur-hole drilled behind the plate bearing the recording electrodes until its tip occupied a position previously computed to correspond with the co-ordinates of the dorsal limits of the nucleus medio-dorsalis (De Groot 1959). A careful search was then made, by racking the fine adjustment of the stereotactic instrument, for the position giving the maximal recruiting response on stimulation. The position occupied by the electrode tip was subsequently checked histologically (see below). The electrocorticogram of each rat was recorded from bilaterally placed pairs of electrodes on a six-channel Marconi EEG penrecorder and rectangular stimulating pulses of 4 msec width were applied at a frequency of 6/sec. The strength of stimulus (about l0 V) was always above threshold. The electrode pair giving the response of maximal amplitude was selected and the output fed into a Cossor oscilloscope from the penultimate stage of the appropriate channel of the pen-recorder. The oscilloscope was triggered by the stimulator and approximately six superimposed traces of the response were photographed (Kodak RP 30 paper). After five repetitions stimulation was discontinued for 10 sec and the procedure then repeated. Subsequently the region of the nucleus medio-dorsalis was searched with the stimulating electrode for a site giving a response of amplitude higher than that already recorded. If found records made from this new focus were substituted for those already taken.
A
Fig. l Record of recruiting response in the rat showing measurements taken for quantitative analysis: L latency; II. duration-rising phase; III. duration-falling phase; IV. total duration; V. amplitude.
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THYROIDECTOMY AND RECRUITING RESPONSE
Assessment of records Each exposure on the record was enlarged by projection with an epidiascope and the following measurements, confined to the characteristics of the surface-negative component of the response, were made (Fig. 1): 24
DAY OLD
Post-mortem examination After recording was completed the animal was killed by giving an overdose of anaesthetic, the recording electrode was withdrawn and a steel needle was stereotactically inserted into the brain to occupy the same co-ordinates. With this Li'rTERMATES
NED-NATAL THY ROIDECTOMY
NORMAL
ADULT
NORMAL
N E D - NATAL THYROIDECTOMY 2 DAYS M E D I C A T I O N
LITTERMATE S
ADULT THYROIDECTOMY
Fig. 2 Typical records of recruiting response illustrating the changes in character which result from variol~s treatments. Note that the scale of the calibration diagrams differs between records. Vertical line: 100/zV; horizontal line: 20 msec in each case.
(a) The latency of the response (msec), i.e., the time elapsing between the arrival of the stimulus at the thalamus and the start of the surface-negative wave (the small initial surfacepositive deflection was frequently masked on the record and was, for this reason disregarded). (b) The duration of the rising and falling phases of the negative wave, and hence the total duration of the period of surface negativity (msec). (c) The amplitude of the potential (#V). Measurements were subjected to the appropriate statistical procedures for assessing the reliability of difference engendered by the different treatments applied. In series A and D the form of t test for paired observations was used to compare the responses of thyroidectomised and normal animals; the data derived from series B and C were subjected to analysis of variance, the estimate of variance in each case being used for the statistical comparison of the treatment means.
needle in position, the animal was perfused with formol saline and the head fixed by immersion for a further period. The brain was then removed and paraffin sections cut and stained by the method of Kltiver and Barrera (1953) in order to confirm the location of the tip of the stimulating electrode. RESULTS
Normal recruiting response (Tables I and llI) The normal recruiting response has not previously been described in the immature, or indeed in the adult rat. The response obtained in the normal 25 day old animal consisted of a small surface positive wave, which was inconstantly seen, followed by a large surface-negative potential which rose rapidly to a peak (Fig. 2). This surface-negative wave had an average latency of 11 msec and a duration of 32 msec of which 14 msec was occupied by the rising and 18 msec by the falling phase. The amplitude of the
P.D. BRADLEYet al.
580
response was f o u n d to be o f the o r d e r o f 1 5 0 / t V in the experiments o f series A a n d B, a l t h o u g h c o n s i d e r a b l y higher values were r e c o r d e d in later series. T h e rate o f i n c r e m e n t o f the potential was 11/zV/msec. T h e p a r a m e t e r s o f the response did n o t change during the course o f the experiment, there being no significant difference between m e a s u r e m e n t s ta k e n f r o m r e c o r d s m a d e during the first period o f stimulation a n d those m a d e
during the second (see materials and methods). N o r were there any differences in latency an d d u r a t i o n when m e a s u r e d b e t w e e n electrodes that were widely separated and electrode pairs that were closely set. T h e recruiting response in the adult was similar to that seen in the i m m a t u r e animal except that the initial surface positive wave a p p e a r e d m o r e frequently and was greatly accentuated.
TABLE I Effect of neonatal thyroidectomy on recruiting response (series A) Measurement
Class of rat Normal
Latency (msec) Duration-rising phase (msec) Duration-falling phase (msec) Duration-total (msec) Amplitude (/~V) Rate of increment (/~V/msec)1
Neonatally thyroidectomised
Difference 4Standard Error (SE)
P
10.71 14.32
20.21 22.22
9.50 ± 7.90 i
1.613 1.991
<0.001 0.01-0.001
17.56
26.94
9.37 ±
1.461
<0.001
31.89 145.67 10.89
49.16 82.52 4.01
17.27 ± 2 . 3 2 7 63.15 ± 31.021 6.88 ± 2 . 1 7 7
<0.001 0.1-0.05 0.01-0.001
These statistics are derived from the mean of the ratios: amplitude/duration - rising phase. TABLE I[ Effect of semi-acute administration of l-triiodothyronine on recruiting response (series B and C) Class of rat Measurement
Series
Normal (N)
Neonatally thyroidectomised (Tx)
Neonatally Differences between treatments thyroidectomised N-Tx N - T x M Tx-TxM medicated (TxM)
SE
Latency (msec)
B C
9.72 6.78
16.97 16.46
10.27 7.85
7.243 9.682
0.55 1.07
6.69a 8.61 e
~ 1.403 ~ 2.261
Duration-rising phase (msec)
B C
12.96 15.02
24.63 25.00
14.95 16.52
11.67 a 9.98 a
1.99 1.50
9.682 8.482
~ 2.265 -- 2.072
Duration-falling phase (msec)
B C
18.15 17.38
26.20 23.42
18.69 16.96
8.052 6.041
0.54 0.42
7.512 6.46 t
+ 1.833 = 2.389
Duration- total (msec)
B C
31.12 32.75
50.83 48.26
33.65 33.51
19.713 15.51'-'
2.53 0.76
17.183 14.75 ~
:/- 3.312 [ 4.318
Amplitude (/~V)
B C
39.1 144.12
37.9 156.32
1.2 12.2
~ 36.26 ~ 42.48
Levels of statistical significance: 1 p = 0.05-0.02 2 p = 0.01-0.001 a p = <0.001
143.7 276.7
104.5 132.6
105.8 120.4
THYROIDECTOMY AND RECRUITING RESPONSE
The duration of the temporal phases of the surface negative wave were strikingly similar in the adult to those in the immature rat, but the amplitude of the response was much higher (Fig. 2). The rate of increment was correspondingly increased. The response in the adult rat would appear to be qualitatively the same as the response described in other animals, including the monkey (Starzl and Whitiock 1952), but on the basis of the data available from the present experiment the latency to the start of the negative wave would appear to be shorter, and the amplitude considerably lower than in the cat (Dempsey and Morison 1942; Jasper 1949) and the rabbit (Kerr and O'Leary 1957).
Effect of neo-natal thyroidectomy on recruitment (Tables I and II) Tables I and II, which give the results of experiments in series A arid B show that neo-natal thyroidectomy resulted in a significant increase in both the latency and duration of the recruited negative potential (Fig. 2) but a reduction in its rate of increment. Latency was nearly double that seen in the normal animal and the other measurements of the order of 50 per cent greater, the measurements made during the two series of experiments showing a high degree of correlation. The amplitude of the response in both series was reduced, but owing to a high element of variance there remained some doubt as to whether the statistical probability of this finding warranted the conclusion that this effect could be attributed to the experimental treatment rather than to random valiation. The results of series C, however, would seem to put this beyond reasonable doubt for in addition to confirming the findings of series A and B as regards latency and duration the reduction in amplitude measured in this further group of rats showed a high degree of statistical significance (P -- 0.01-0.001).
Effect of thyroid medication on recruitment (Table
11) The administration of T3 to cretinoid rats for 2 days before recording was associated with a restoration of the latency and duration of the response but not of the amplitude (see also Fig. 2).
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Thus the latency of the spike and the time taken for both its rising and falling phases were all similar to those of the normal littermate, but differed from those of thyroidectomised individuals at a high level of statistical significance. The amplitude of the negative wave, however, remained at the level characteristic for the neonatally thyroidectomised individual and was significantly less than that recorded from the normal rat. Comparison of the data given in Table I1 shows that the results obtained during the experiments in series C (in which the procedures were repeated) were almost identical with those of the earlier series B. The mean amplitude of the records in the normal rat in the later experiment was, however, considerably greater than that found in rats studied earlier. No satisfactory explanation can be offered for this differ-
NORMAL
f40 120~ I001 80 60' 40 20
120 ~'I00
~ H y k N EO-NATAL ROD I ECTOMY
&8o
~ 6o
= 40 -, 20 a.
/;' g
< I20
OO8o
/Ak^,
/,;:,-"
NEO-NATAL
2
M O, AT,ON
40
2b 3'o 4:o Jo go 7'o Fig. 3 Stylised diagrams plotted from the estimated means for the three classes of rat showing the absence of consistent effect following the administration of T3 during the course of recording the recruiting response. Pre-administration record: Record 15 rain after giving T3: Record 30 rain after giving T3 :
P.D. BRADLEYet al.
582
ence other than that the animals were larger and seemed to be more mature than those used for the first series of studies. Fig. 3, in which are illustrated stylised representations of the negative component of the recruiting response in the three groups of rats used, shows that acute medication with T3 proved to be without consistent or significant effect on either normal or thyroidectomised rats over a post-injection period of 15 or 30 rain. This finding applied equally to all criteria measured.
dorso-medialis. In spite of this scatter there was no apparent correlation between the positioning of the electrodes and the dimensions of any of the parameters recorded, either between individuals or between treatments. DISCUSSION
Three main findings emerge from these experiments. In the first place, the surface negative wave of the recruiting response in the neonatally thyroidectomised rat was quantita-
TABLE IIl Effect of adult thyroidectomy on recruiting response Class of rat
Measurement Latency (msec) Duration-rising phase
(msec) Duration-falling phase (msec) Duration-total (msec) Amplitude (/~V)
Difference !~ SE
P
Normal
Thyroidectomised
9.29 12.56
25.33 30.79
)6.04 ~_ 3.205 18.21 + 3.531
0.01-0.001 0.01-0.001
16.28
51.86
35.58 ± 7.335
0.01-O.001
28.84 312.84
82.65 241.04
53.81 ± 8.933 71.80 ± 55.776
Effect of thyroidectomy during adult life (Table III and Fig. 2) The effects of thyroidectomy during adult life were similar to those seen following neonatal thyreoprivus in so far as the latency and duration of the response were both increased. They differed in two respects: first, the relative increase in duration of the falling phase of the negative wave far exceeded that seen in the 25-day old neonatally thyroidectomised animal; and second the amplitude of the "spike" did not differ from that of the normal littermate.
Histological examination of cerebral tissues Inspection of histological preparations for the purpose of checking the final placement of the stimulatory electrode showed that the points of stimulation were located for the most part, as had been intended, in the region of the nucleus medio-dorsalis thalami and in the adjacent territory occupied by the fasciculus retroflexus and nucleus parafascicularis. Some electrode tracks were positioned rather more laterally, the tips of the electrodes being located in the nucleus ventralis thalami, partes anterior, medialis and
-<0.001 0.3-0.2
tively increased in latency and in the duration of both its rising and falling phases bul, at the same time, reduced in amplitude and rate of increment. Second, although acute medication with 1-triiodothyronine proved to have no consistent effect, administration of this hormone for 2 days restored the temporal relations of the response to normal without, however, affecting amplitude. And finally, in animals thyroidectomised during adult life, the recruiting response recorded after 28 days of hypothyroidism showed a retardation of the temporal components similar to that associated with neonatal thyroidectomy but little or no fall in amplitude. In order to link these observations into an ordered sequence or pattern it is necessary to examine them in relation to what is already known of the influence of the thyroid gland on the maturation of the nervous system, the effects of thyroid hormone on central nervous excitability and the mechanism of the recruiting response itself.
Effect of neonatal thyroidectomy The finding that the temporal relations of the several phases of the recruiting response are
THYROIDECTOMY AND RECRUITING RESPONSE
much increased by neonatal thyroidectomy while the amplitude is reduced can to some extent be explained in terms of the anatomy and physiology of the structures underlying its mediation. The period of latency to the start of the main surface negative wave of the response incorporates the small initial positive wave shown by Arduini and Terzuolo (1951) to be due primarily to activity in the sub-cortical afferent pathways from the thalamus. It might therefore be expected that the increase in latency seen in hypothyroidism might be caused by factors influencing primarily subcortical events. It is possible that thyroxine lack may affect the thalamo-cortical reverberatory circuit proposed by Verzeano et al. (1953) as the mechanism underlying the response, but consideration of the morphological relations of the structures involved, i.e., thalamus, diffuse thalamic projection system, and cortex, suggests that an increase in conduction time alone would be too small to explain the considerable delay. It is reasonable to postulate, however, that the impairment to both latency and duration may largely be attributable to alterations in the metabolic properties of synapses resulting from thyroid deficiency. If, as suggested by Li et al. (1956), the surface-negative phase of the response is attributable to dendritic potentials, then this factor, augmented by changes in the spatial relationships of synapses caused by the depletion in axonal density characteristic of cerebral tissues in the cretinoid animal (Eayrs 1959) could well give rise to the observed effects either by influencing the pattern of decremental conduction in dendrites (Clare and Bishop 1955, 1957; Lorente de N6 1960) or alternatively by acting upon axo-dendritic synapses and thus influencing the rate of summation of post-synaptic potentials (Purpura and Grundfest 1956; Purpura 1959). The fall in amplitude of the surface-negative wave following neonatal thyroidectomy conforms with the reduced amplitude of the spontaneous electrical activity of the brain reported by Bradley et al. (1960). It would seem that, again drawing on the work of Clare and Bishop (1957) and of Purpura and Grundfest (1956), an important factor underlying the fall in amplitude of the response would be a reduction in the numbers of the participating elements, i.e., in dendrites (Clare and Bishop) or in axo-dendritic synapses
583
(Purpura and Grundfest), rather than the metabolic influences playing on the individual synapses as would seem to be the case with the factors controlling the duration of this phase of the response. This interpretation is consistent with what is already known of the relative reduction in the probability of axodendritic interaction :in the imperfectly matured cortex of the neonatally thyroidectomised individual (Eayrs 1960b) and with the findings in relation to the effects of semi-acute medication, and of thyroidectomy in the adult animal. An alternative explanation of the findings is that the parameters of stimulation needed to elicit a response of maximal amplitude in the cretinoid individual differ from those in the normal. Although this possibility cannot entirely be excluded the negative effects of varying the stimulating voltage during the present experiment provided no supporting evidence.
Effects of semi-acute medication with triiodothyronine The observation that semi-acute medication restores the temporal relations of the recruiting response of neonatally thyroidectomised ani reals to normal while leaving the amplitude of the response unaffected implies that thyroidectomy early in life has a dual mode of action upon the electrophysiological functions of the central nervous system. It is thus tempting to infer that these may be respectively related to the metabolic and growth promoting effects of thyroid hormone, the former affecting the temporalcomponents of the response and the latter the amplitude. The possibility that the amelioration of effects on latency and duration by medication could be attributable to a restoration of cortical growth is, in the absence of any direct evidence, minimised by three arguments: first, that it seems unlikely that the period over which T3 was administered (2 days) could be sufficient to remedy the extreme impairment to growth known to be associated with neonatal thyroidectomy; second, ~it is difficult to see how an acceleration of growth could affect one group of changes without affecting another; and third, similar changes in latency and duration were observed following thyroidectomy in the adult at a time when cerebral growth was complete. The manner,
584
P.D. BRADLEYet al.
therefore, in which such a dichotomy of effect could arise remains to be considered. (a) Relationship between medication and temporal aspects of response. The effect of thyroxine upon the excitability of the central nervous system is not unknown, and it has been shown that hypothyroidism is associated with an increase in the electro-shock seizure threshold of rats (Timiras and Woodbury 1956) whereas administration of thyroxine causes a decrease in this threshold. Furthermore earlier work has demonstrated that thyroid hormone influences temporal relations in the spontaneous activity of the human EEG, administration of thyroxine giving rise to an increase in the frequency of the alpha rhythm (Lindsley and Rubinstein 1937; Ross and Schwab 1939; Rubin et al. 1937) while in hypothyroid states this is reduced (Bertrand et al. 1938). Hoagland (1936) postulated that the frequency of the alpha rhythm was directly proportional to the rate of respiration of neurones. It might be expected therefore that factors similar to those which influence the excitability of the cortex and the frequency of its spontaneous activity might also affect the temporal relations of the recruiting response. One possible way in which the short term administration of exogenous thyroid hormone could, in the face of a decreased probability of synaptic transmission, compensate for the increased latency of the response in the hypothyroid state is by lowering synaptic thresholds in such a manner that corticopetal stimuli arriving by way of a reduced number of synapses are as effective as the larger number normally available. Alternatively, the increased latency and duration of the response in hypothyroidism could primarily be due to an increase in the threshold of the participating synapses and to a depression of neuronal metabolism due to lack of circulating thyroxine. Treatment with T3 would, in such a case, be expected to be followed by a return to normality. (b) Absence of relationship between medication and amplitude. The findings that the fall in the amplitude of the surface negative wave in the neonatally thyroidectomised rat is unaffected by short-term medication with T3 and that little change in amplitude occurs when thyroidectomy is performed in the mature individual tend to
support the suggestion, made earlier, that an impairment to cortical growth is the underlying factor of the many changes known to occur (for review, see Eayrs 1959, 1960a). The most likely to be implicated would seem to be a reduction in the growth of neuropil which results in a decrease in the probability of axo-dendritic interaction so severe as to be unlikely to respond to brief (or even to long) periods of replacement therapy. It is therefore of interest that analogous results have been obtained where synaptic activity has been reduced by pharmacological means. Clare and Bishop (1957), for instance, found that increasing concentrations of topically applied strychnine caused a diminution in the amplitude of the surface-negative wave of the recruiting response and attributed their results to a gradual decrease in the number of units active. Purpura and Grundfest (1956) likewise attributed the reduced amplitude of the surfacenegative dendritic potential after topical application of d-tubocurarine to a progressively increasing synaptic blockade. Thus a decrease in the number of synapses developmentally induced by neonatal thyroidectomy and a physiological reduction by synaptic blocking agents would both appear to lead to the same result, in the one case a potentially irreversible and in the other a reversible, decrease in amplitude of the recruiting response. Conversely, the marked increase in the probability of axo-dendritic interaction associated with the process of normal growth (Eayrs and Goodhead 1959) may be invoked to explain the greater amplitude of the normal adult record by comparison with that of the 25-day old animal. Effect of acute medication with triiodothyronine The failure of acute administration of T3 to influence the recruiting response in neonatally thyroidectomised rats where semi-acute medication succeeded is not without precedent. Woodbury et al. (1952) for instance found that the electro-shock seizure threshold was depressed only after 14 days medication with thyroxine, and an increase in alpha frequency in the human EEG observed by Rubin et al. (1937) did not appear until 2 or 3 days after medication had been begun. Possibly this lack of an acute response to T3 may be explained by the inability of the hormone to
THYROIDECTOMY AND RECRUITING RESPONSE
penetrate the haemato-encephalic barrier during the course of the experiment although, by contrast, recent work has shown that the intraperitoneal administration of thyrotrophic hormone had an inhibiting action on thalamic activity within 30-40 rain of its injection (Milcou et al. 1960). An alternative possibility is that T3 acts as a precursor whose metabolism requires more time than was available under the conditions of the present experiment. Conchlsions
The results of this experiment are thus consistent with the hypothesis that two factors, one metabolic and the other developmental, are implicated in the changes in the recruiting response which arise after neonatal thyroidectomy. The changed temporal aspects of the response are largely the outcome of the metabolic effect and are reversible when T3 is given for 2 days before recording. The fall in amplitude of the surfacenegative wave, on the other hand, reflects a developmental impairment and is therefore not reversed by short-term medication. This conclusion is supported by the finding that, when thyroidectomy is delayed until cerebral development is complete, only those changes attributed to metabolic factors arise. Although the mechanisms underlying the changes in the recruiting response remain open to conjecture it would seem, in the light of present knowledge, that the changes associated with hypothyroidism are largely mediated through the anatomical distribution and physiological properties of cortical axo-dendritic synapses. The increase in the latency and duration of the response may be attributed to alterations in the transmitting properties of synapses brought about by the metabolic consequences of thyroidectomy and the fall in amplitude to a reduction in the number of participating synapses and availability ofpostsynaptic potentials for summation. SUMMARY
1. The recruiting response has been studied in the rat with special reference to the effects of thyroidectomy and subsequent replacement therapy. 2. Neonatal thyroidectomy is associated in later life with an increase in the latency and
585
duration of the surface-negative component of the response and a reduction in its amplitude. 3. Medication with 1-triiodothyronine for 2 days prior to recording restores latency and duration to within normal range but is without effect on the amplitude of the response. Acute administration of this hormone during the process of recording is without significant effect on any parameter. 4. Thyroidectomy during adult life results in an increase in both the latency and duration of the negative wave, but does not alter the amplitude of the response. 5. These findings are discussed in the light of structural and metabolic differences between the brains of rats made hypothyroid during the course of cerebral development and after such development is complete. It is thought that they conform well with the view that the amplitude of the recruiting response may reflect the envelope of post-synaptic dendritic potentials whose number is significantly reduced in the neo-nalally thyroidectomised individual whereas its latency and duration, while not necessarily independent of structure, are more readily regulated by metabolic factors. REFERENCES ARDUINI, A. and TERZUOLO, C. Cortical and subcortical
components in the recruiting response. E[ectroenceph. din. NeurophysioL, 1951, 3:189 196. BERTRAND, I., DELAY, J. et GUILLAIN, J. L'61ectroenc6-
phalogramme dins le myxoed~me. C.R. Soc. Biol. (Paris), 1938, 129: 395-398. BRADLEY, P. B., EAYRS, J. T. and SCHMALBACH, K. The
electroencephalogram of normal and hypothyroid rats. Electroenceph. clin. Neurophysiol., 1960, 12:467 477. BRADLEY, P. B. and KEY, B. J. The effect of drugs on
arousal responses produced by electrical stimulation of the reticular formation of the brain. Electroenceph. clin. Neurophysiol., 1958, I0:97-110. CLARE, M. C. and BISHOP,G. H. Dendritic circuits: the properties of cortical paths involving dendrites. Amer. J. Psychiat., 1955, 111: 818-825. CLARE, M. C. and BISHOP, G. H. Action of strychnine on recruiting response of dendrites of cat corte~:. J. Neurophysiol., 1957, 2: 195-215. CORT, J. H. and HARDING, H. F. Inexpensive precision stereotaxic instrument. J. Physiol. (Lond.), 1953, 123: 15P. DE GROOT,J. The rat forebrain in stereotaxic coordinates. Proe. kon. ned. Akad. Wet., 1959, 52: No. 4. DEMPSEY, E. W. and MORISON,R. S. The production of
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rhythmically recurrent cortical potentials after localised thalamic stimulation. ,4met. J. Physiol., 1942, 135: 293-300. EAYRS, J. T. The cerebral cortex of normal and hypothyroid rats. Acta anat. (Basel), 1955, 25: 160-183. EAYRS, J. T. The status of thyroid hormone in relation to the development of the nervous systems. Brit. J. Anita. Behav., 1959, 7: 1-17. EAYRS, J. Z. Influence of the thyroid on the central nervous system. Brit. reed. Bull., 1960, 16: 122--127. EAYRS, J. T. Functional correlates of modified cortical structure. In: D. B. TOWER and J. P. SCHADr~ (Editors), Structure and function o f the cerebral cortex. Elsevier, Amsterdam, 1960, pp. 43-50. EAYRS, J. T. and GOODHEAD, B. Postnatal development of the cerebral cortex in the rat. J. ,4nat. (Lond.), 1959, 93: 385-402. EAYRS, J. T. and LISHMAN, W. A. The maturation of behaviour in hypothyroidism and starvation. Brit. J. .4him. Behav., 1953, 3: 17-24. GOLDBERG, R. C. and CHAIKOFE, I. L. A simplified procedure for thyroidectomy of the new-born rat without concomitant parathyroidectomy. Endocrinology, 1948, 45: 64-70. HOAGLAND, H. Pacemakers of human brain-waves. Amer. J. Physiol., 1936, 116: 604-615. JASPER, H. H. Diffuse projection systems: the integrative action of the thalamic reticular system. Electroenceph. clin. Neurophysiol., 1949, 1 : 405-420. KERR, F. W. L. and O'LEARY, J. The thalamic source of cortical recruiting in the rodent. Electroenceph. clin. Neurophysiol., 1957, 9: 461-476. KLiiVER, H. and BARRERA, E. A method for the combined staining of cells and fibres in the nervous system. J. Neuropath. exp. Neurol., 1953, 12: 400~403. LI, C. L., CULLEN,C. and JASPER,H. H. Laminar microelectrode analysis of cortical unspecific responses and spontaneous rhythms. J. Neurophysiol., 1956, 19: 131-143. LINDSLEY, D. B. and RUB~NSTEIN, B. B. Relation between
brain potentials and some other physiological variables. Proc. Soc. exp. Biol. (N. Y.), 1937, 35: 558-563. LORENTE DE NG, R. Decremental conduction in peripheral nerve and its application to the interpretation of summation and inhibition in the central nervous system. In : O. B. TOWER and J. P. SCHADI~(Editors), Structure and function o f the cerebral cortex. Elsevier, Amsterdam, 1960, pp. 278-281. MILcOU, S. M., DEMETRESCU,M. and NAULESCU-CATORGI, A. The thyrotropin action upon the ascendant activating systems in cat. Electroenceph. clin. Neurophysiol., 1960, 12: 749. PURPURA, D. P. Nature of electro-cortical potentials and synaptic organisations in cerebral and cerebellar cortex. In: C. C. PFEIFFER and J. R. SMYTHIES (Editors), International Reviews o f Neurobiology, I. Academic Press, New York, 1959, pp. 47 163. PURPURA, D. P. and GRUNDFEST, H. Nature of cortical dendritic activity. Trans. Amer. neurol. Ass., 1956, 81: 163-165. Ross, D. A. and SCHWAB, R. The cortical alpha rhythm in thyroid disorders. Endocrinology, 1939, 25:75-79. RUalN, M. A., COHEN, L. H. and HOAGLAND, H. The effect of artificially raised metabolic rate on the electroencephalogram of schizophrenic patients. Emtocrinology, 1937, 21:536 540. STARZL, T. E. and WHITLOCK, D. G. Diffuse thalamic projection system in the monkey. J. Neurophysiol., 1952, 15: 449-468. TIMIRAS, P. S. and WOODBURY, D. M. Effect of thyroid activity on brain function and brain electrolyte distribution in rats. Endocrinology, 1956, 58:181 192. VERZEANO, M., L~NDSLEY, D. B. and MAGOUN, H. W. Nature of recruiting response. J. Neurophysiol., 1953, 16: 183-195. WOODBURY,D. M., HURLEY,R. E., LEwis, N. G., MeARTHUR, M. W., COPELAND, V. W., KIRSCHVINK, J. F. and GOODMAN,L. S. Effects of thyroxine, thyroidectomy and 6-N-propyl-2-thiouracil on brain function. J. Pharmacol. exp. Ther., 1952, 106: 331-340.
Reference: BRADLEY, P. B., EAYRS, J. T., GLASS, A. and HEATH, R. W. The maturational and metabolic consequences of neonatal thyroidectomy upon the recruiting response in the rat. Electroenceph. clin. Neurophysiol., 1961, 13: 577-586.
gested grounds on which such changes might be related to the impaired growth of conducting elements within the cerebral cortex. Accordingly, in order further to elucidate the relationship between the structural and electrophysiological sequelae of neonatal thyroidectomy a study has been made of a better defined cortical response which would enable changes in temporal relations to be detected. The recruiting response, first described by Dempsey and Morison (1942) and analysed into functional components by Arduini and Terzuolo (1951) and by Li et al. (1956), was selected as being the most suitable for this purpose.
INTRODUCTION
The relation that the thyroid gland bears to the maturation and maintenance of function of the central nervous system has for a long time been well-known (for reviews see Eayrs 1959, 1960a) and continued investigation of the neuroendocrinological effects of thyroxine or its lack may be expected to shed further light upon the inter-relationship between the structure of the cerebral cortex during development and its function measured in terms of behaviour or electrical activity. Recent investigations have shown, for instance, that thyroid deficiency, arising during the early stages of maturation, gives rise to marked abnormalities in cerebral development prominent among which is a decrease in the extent and complexity of the cortical neuropil (Eayrs 1955) and a consequent reduction in the probability of interaction between neurones (Eayrs 1960b) associated with quantitatively observed behavioural impairment (Eayrs and Lishman 1953). The neurophysiological consequences attendant upon such changes were initially investigated by Bradley et al. (1960) who recorded the EEG of neonatally thyroidectomised rats and found a significant decrease in the amplitude of the spontaneous electrical activity coupled with an absence of the slow wave potentials normally associated with drowsiness and of response to rhythmic photic stimulation. These authors sug-
MATERIALS AND METHODS
General experimental procedure
1 Henry Head Research Fellow of the Royal Societyp London. Assisted by a grant from the Caroline Harrold Research Fund. 3 Supported by a grant from the Office of Scientific Research of the Air Research and Development Council, United States Air Force, Contract No. AF61 (514)-1184. 577
Seventy-four rats of the Birmingham albino strain were used, divided into 31 groups of littermates of the same sex in accordance with the procedures outlined below. The experiment was divided into the following four parts: Series A. Twelve littermate pairs were used, one rat of each pair being radiothyroidectomised on the day of birth (Goldberg and Chaikoff 1948) by the injection of 150 #C of 1311, its littermate receiving a similar dose of the vehicle. The recruiting response was recorded under very light anaesthesia with tribromoethanol at the age of 24-26 days when, as suggested by the previously cited work on the EEG of such rats, the effect of neonatal thyroidectomy was likely to be maximal. Series B. Seven littermate triads were used in this series. Two of the three rats were thyroidectomised at birth as for series A and the third acted as control. Beginning on the second day
578
P.D. BRADLEYeta].
before that scheduled for recording, one of the thyroidectomised animals was given a daily subcutaneous injection 1.5 #g of 1-triiodothyronine (T3) dissolved in 0.1 ml saline, the remaining thyroidectomised rat and the control receiving the vehicle alone. Records were taken as for series A with addition that, after the first record had been taken 3/tg of T3 was given intraperitoneally to each animal and further records were made at intervals of 15 and 30 rain after injection. Series C. This series of experiments was conducted to resolve doubts concerning the statistical validity of certain of the results obtained in series B. Using five littermate triads, the procedures of series B were repeated except that acute medication with T3 was not carried out in this instance. Series D. Six pairs of littermates were used. One rat of each pair was surgically thyroidectoraised during adult life; the other underwent mock operation. Records of the recruiting response were taken 28 days later.
Electrophysiological techniques For the experiments in series A - C an array of six spherically ended silver wire electrodes each of diameter 0.124" was sealed with Araldite into an artificial calvarium made from acrylic resin moulded to fit the skull of a 25-day old rat. Under tribromoethanol anaesthesia (200 mg/kg) the animal was placed in a stereotactic instrument (modified from that described by Cort and Harding 1953), the scalp reflected and a window cut in the fronto-parietal part of the skull symmetrically about the midline. The dura was then reflected on either side of the sagittal sinus and, after haemostasis had been secured, the electrode-bearing cap was sealed over the fenestration with dental cement so that the electrode tips came into contact with the cortical surface. This procedure was modified for the adult rats used in series D, for in such animals it is extremely hazardous to remove the skull without damage to the superior sagittal sinus. Accordingly an array of four ball-ended silver wire electrodes, insulated by PVC sleeving, was positioned stereotactically and the electrode tips brought into contact with the cortex through independently drilled bur-holes and secured with acrylic resin. A bipolar concentric type of stimulating
electrode (Bradley and Key 1958) of outer diameter 0.012" was inserted stereotactically through the bur-hole drilled behind the plate bearing the recording electrodes until its tip occupied a position previously computed to correspond with the co-ordinates of the dorsal limits of the nucleus medio-dorsalis (De Groot 1959). A careful search was then made, by racking the fine adjustment of the stereotactic instrument, for the position giving the maximal recruiting response on stimulation. The position occupied by the electrode tip was subsequently checked histologically (see below). The electrocorticogram of each rat was recorded from bilaterally placed pairs of electrodes on a six-channel Marconi EEG penrecorder and rectangular stimulating pulses of 4 msec width were applied at a frequency of 6/sec. The strength of stimulus (about l0 V) was always above threshold. The electrode pair giving the response of maximal amplitude was selected and the output fed into a Cossor oscilloscope from the penultimate stage of the appropriate channel of the pen-recorder. The oscilloscope was triggered by the stimulator and approximately six superimposed traces of the response were photographed (Kodak RP 30 paper). After five repetitions stimulation was discontinued for 10 sec and the procedure then repeated. Subsequently the region of the nucleus medio-dorsalis was searched with the stimulating electrode for a site giving a response of amplitude higher than that already recorded. If found records made from this new focus were substituted for those already taken.
A
Fig. l Record of recruiting response in the rat showing measurements taken for quantitative analysis: L latency; II. duration-rising phase; III. duration-falling phase; IV. total duration; V. amplitude.
579
THYROIDECTOMY AND RECRUITING RESPONSE
Assessment of records Each exposure on the record was enlarged by projection with an epidiascope and the following measurements, confined to the characteristics of the surface-negative component of the response, were made (Fig. 1): 24
DAY OLD
Post-mortem examination After recording was completed the animal was killed by giving an overdose of anaesthetic, the recording electrode was withdrawn and a steel needle was stereotactically inserted into the brain to occupy the same co-ordinates. With this Li'rTERMATES
NED-NATAL THY ROIDECTOMY
NORMAL
ADULT
NORMAL
N E D - NATAL THYROIDECTOMY 2 DAYS M E D I C A T I O N
LITTERMATE S
ADULT THYROIDECTOMY
Fig. 2 Typical records of recruiting response illustrating the changes in character which result from variol~s treatments. Note that the scale of the calibration diagrams differs between records. Vertical line: 100/zV; horizontal line: 20 msec in each case.
(a) The latency of the response (msec), i.e., the time elapsing between the arrival of the stimulus at the thalamus and the start of the surface-negative wave (the small initial surfacepositive deflection was frequently masked on the record and was, for this reason disregarded). (b) The duration of the rising and falling phases of the negative wave, and hence the total duration of the period of surface negativity (msec). (c) The amplitude of the potential (#V). Measurements were subjected to the appropriate statistical procedures for assessing the reliability of difference engendered by the different treatments applied. In series A and D the form of t test for paired observations was used to compare the responses of thyroidectomised and normal animals; the data derived from series B and C were subjected to analysis of variance, the estimate of variance in each case being used for the statistical comparison of the treatment means.
needle in position, the animal was perfused with formol saline and the head fixed by immersion for a further period. The brain was then removed and paraffin sections cut and stained by the method of Kltiver and Barrera (1953) in order to confirm the location of the tip of the stimulating electrode. RESULTS
Normal recruiting response (Tables I and llI) The normal recruiting response has not previously been described in the immature, or indeed in the adult rat. The response obtained in the normal 25 day old animal consisted of a small surface positive wave, which was inconstantly seen, followed by a large surface-negative potential which rose rapidly to a peak (Fig. 2). This surface-negative wave had an average latency of 11 msec and a duration of 32 msec of which 14 msec was occupied by the rising and 18 msec by the falling phase. The amplitude of the
P.D. BRADLEYet al.
580
response was f o u n d to be o f the o r d e r o f 1 5 0 / t V in the experiments o f series A a n d B, a l t h o u g h c o n s i d e r a b l y higher values were r e c o r d e d in later series. T h e rate o f i n c r e m e n t o f the potential was 11/zV/msec. T h e p a r a m e t e r s o f the response did n o t change during the course o f the experiment, there being no significant difference between m e a s u r e m e n t s ta k e n f r o m r e c o r d s m a d e during the first period o f stimulation a n d those m a d e
during the second (see materials and methods). N o r were there any differences in latency an d d u r a t i o n when m e a s u r e d b e t w e e n electrodes that were widely separated and electrode pairs that were closely set. T h e recruiting response in the adult was similar to that seen in the i m m a t u r e animal except that the initial surface positive wave a p p e a r e d m o r e frequently and was greatly accentuated.
TABLE I Effect of neonatal thyroidectomy on recruiting response (series A) Measurement
Class of rat Normal
Latency (msec) Duration-rising phase (msec) Duration-falling phase (msec) Duration-total (msec) Amplitude (/~V) Rate of increment (/~V/msec)1
Neonatally thyroidectomised
Difference 4Standard Error (SE)
P
10.71 14.32
20.21 22.22
9.50 ± 7.90 i
1.613 1.991
<0.001 0.01-0.001
17.56
26.94
9.37 ±
1.461
<0.001
31.89 145.67 10.89
49.16 82.52 4.01
17.27 ± 2 . 3 2 7 63.15 ± 31.021 6.88 ± 2 . 1 7 7
<0.001 0.1-0.05 0.01-0.001
These statistics are derived from the mean of the ratios: amplitude/duration - rising phase. TABLE I[ Effect of semi-acute administration of l-triiodothyronine on recruiting response (series B and C) Class of rat Measurement
Series
Normal (N)
Neonatally thyroidectomised (Tx)
Neonatally Differences between treatments thyroidectomised N-Tx N - T x M Tx-TxM medicated (TxM)
SE
Latency (msec)
B C
9.72 6.78
16.97 16.46
10.27 7.85
7.243 9.682
0.55 1.07
6.69a 8.61 e
~ 1.403 ~ 2.261
Duration-rising phase (msec)
B C
12.96 15.02
24.63 25.00
14.95 16.52
11.67 a 9.98 a
1.99 1.50
9.682 8.482
~ 2.265 -- 2.072
Duration-falling phase (msec)
B C
18.15 17.38
26.20 23.42
18.69 16.96
8.052 6.041
0.54 0.42
7.512 6.46 t
+ 1.833 = 2.389
Duration- total (msec)
B C
31.12 32.75
50.83 48.26
33.65 33.51
19.713 15.51'-'
2.53 0.76
17.183 14.75 ~
:/- 3.312 [ 4.318
Amplitude (/~V)
B C
39.1 144.12
37.9 156.32
1.2 12.2
~ 36.26 ~ 42.48
Levels of statistical significance: 1 p = 0.05-0.02 2 p = 0.01-0.001 a p = <0.001
143.7 276.7
104.5 132.6
105.8 120.4
THYROIDECTOMY AND RECRUITING RESPONSE
The duration of the temporal phases of the surface negative wave were strikingly similar in the adult to those in the immature rat, but the amplitude of the response was much higher (Fig. 2). The rate of increment was correspondingly increased. The response in the adult rat would appear to be qualitatively the same as the response described in other animals, including the monkey (Starzl and Whitiock 1952), but on the basis of the data available from the present experiment the latency to the start of the negative wave would appear to be shorter, and the amplitude considerably lower than in the cat (Dempsey and Morison 1942; Jasper 1949) and the rabbit (Kerr and O'Leary 1957).
Effect of neo-natal thyroidectomy on recruitment (Tables I and II) Tables I and II, which give the results of experiments in series A arid B show that neo-natal thyroidectomy resulted in a significant increase in both the latency and duration of the recruited negative potential (Fig. 2) but a reduction in its rate of increment. Latency was nearly double that seen in the normal animal and the other measurements of the order of 50 per cent greater, the measurements made during the two series of experiments showing a high degree of correlation. The amplitude of the response in both series was reduced, but owing to a high element of variance there remained some doubt as to whether the statistical probability of this finding warranted the conclusion that this effect could be attributed to the experimental treatment rather than to random valiation. The results of series C, however, would seem to put this beyond reasonable doubt for in addition to confirming the findings of series A and B as regards latency and duration the reduction in amplitude measured in this further group of rats showed a high degree of statistical significance (P -- 0.01-0.001).
Effect of thyroid medication on recruitment (Table
11) The administration of T3 to cretinoid rats for 2 days before recording was associated with a restoration of the latency and duration of the response but not of the amplitude (see also Fig. 2).
581
Thus the latency of the spike and the time taken for both its rising and falling phases were all similar to those of the normal littermate, but differed from those of thyroidectomised individuals at a high level of statistical significance. The amplitude of the negative wave, however, remained at the level characteristic for the neonatally thyroidectomised individual and was significantly less than that recorded from the normal rat. Comparison of the data given in Table I1 shows that the results obtained during the experiments in series C (in which the procedures were repeated) were almost identical with those of the earlier series B. The mean amplitude of the records in the normal rat in the later experiment was, however, considerably greater than that found in rats studied earlier. No satisfactory explanation can be offered for this differ-
NORMAL
f40 120~ I001 80 60' 40 20
120 ~'I00
~ H y k N EO-NATAL ROD I ECTOMY
&8o
~ 6o
= 40 -, 20 a.
/;' g
< I20
OO8o
/Ak^,
/,;:,-"
NEO-NATAL
2
M O, AT,ON
40
2b 3'o 4:o Jo go 7'o Fig. 3 Stylised diagrams plotted from the estimated means for the three classes of rat showing the absence of consistent effect following the administration of T3 during the course of recording the recruiting response. Pre-administration record: Record 15 rain after giving T3: Record 30 rain after giving T3 :
P.D. BRADLEYet al.
582
ence other than that the animals were larger and seemed to be more mature than those used for the first series of studies. Fig. 3, in which are illustrated stylised representations of the negative component of the recruiting response in the three groups of rats used, shows that acute medication with T3 proved to be without consistent or significant effect on either normal or thyroidectomised rats over a post-injection period of 15 or 30 rain. This finding applied equally to all criteria measured.
dorso-medialis. In spite of this scatter there was no apparent correlation between the positioning of the electrodes and the dimensions of any of the parameters recorded, either between individuals or between treatments. DISCUSSION
Three main findings emerge from these experiments. In the first place, the surface negative wave of the recruiting response in the neonatally thyroidectomised rat was quantita-
TABLE IIl Effect of adult thyroidectomy on recruiting response Class of rat
Measurement Latency (msec) Duration-rising phase
(msec) Duration-falling phase (msec) Duration-total (msec) Amplitude (/~V)
Difference !~ SE
P
Normal
Thyroidectomised
9.29 12.56
25.33 30.79
)6.04 ~_ 3.205 18.21 + 3.531
0.01-0.001 0.01-0.001
16.28
51.86
35.58 ± 7.335
0.01-O.001
28.84 312.84
82.65 241.04
53.81 ± 8.933 71.80 ± 55.776
Effect of thyroidectomy during adult life (Table III and Fig. 2) The effects of thyroidectomy during adult life were similar to those seen following neonatal thyreoprivus in so far as the latency and duration of the response were both increased. They differed in two respects: first, the relative increase in duration of the falling phase of the negative wave far exceeded that seen in the 25-day old neonatally thyroidectomised animal; and second the amplitude of the "spike" did not differ from that of the normal littermate.
Histological examination of cerebral tissues Inspection of histological preparations for the purpose of checking the final placement of the stimulatory electrode showed that the points of stimulation were located for the most part, as had been intended, in the region of the nucleus medio-dorsalis thalami and in the adjacent territory occupied by the fasciculus retroflexus and nucleus parafascicularis. Some electrode tracks were positioned rather more laterally, the tips of the electrodes being located in the nucleus ventralis thalami, partes anterior, medialis and
-<0.001 0.3-0.2
tively increased in latency and in the duration of both its rising and falling phases bul, at the same time, reduced in amplitude and rate of increment. Second, although acute medication with 1-triiodothyronine proved to have no consistent effect, administration of this hormone for 2 days restored the temporal relations of the response to normal without, however, affecting amplitude. And finally, in animals thyroidectomised during adult life, the recruiting response recorded after 28 days of hypothyroidism showed a retardation of the temporal components similar to that associated with neonatal thyroidectomy but little or no fall in amplitude. In order to link these observations into an ordered sequence or pattern it is necessary to examine them in relation to what is already known of the influence of the thyroid gland on the maturation of the nervous system, the effects of thyroid hormone on central nervous excitability and the mechanism of the recruiting response itself.
Effect of neonatal thyroidectomy The finding that the temporal relations of the several phases of the recruiting response are
THYROIDECTOMY AND RECRUITING RESPONSE
much increased by neonatal thyroidectomy while the amplitude is reduced can to some extent be explained in terms of the anatomy and physiology of the structures underlying its mediation. The period of latency to the start of the main surface negative wave of the response incorporates the small initial positive wave shown by Arduini and Terzuolo (1951) to be due primarily to activity in the sub-cortical afferent pathways from the thalamus. It might therefore be expected that the increase in latency seen in hypothyroidism might be caused by factors influencing primarily subcortical events. It is possible that thyroxine lack may affect the thalamo-cortical reverberatory circuit proposed by Verzeano et al. (1953) as the mechanism underlying the response, but consideration of the morphological relations of the structures involved, i.e., thalamus, diffuse thalamic projection system, and cortex, suggests that an increase in conduction time alone would be too small to explain the considerable delay. It is reasonable to postulate, however, that the impairment to both latency and duration may largely be attributable to alterations in the metabolic properties of synapses resulting from thyroid deficiency. If, as suggested by Li et al. (1956), the surface-negative phase of the response is attributable to dendritic potentials, then this factor, augmented by changes in the spatial relationships of synapses caused by the depletion in axonal density characteristic of cerebral tissues in the cretinoid animal (Eayrs 1959) could well give rise to the observed effects either by influencing the pattern of decremental conduction in dendrites (Clare and Bishop 1955, 1957; Lorente de N6 1960) or alternatively by acting upon axo-dendritic synapses and thus influencing the rate of summation of post-synaptic potentials (Purpura and Grundfest 1956; Purpura 1959). The fall in amplitude of the surface-negative wave following neonatal thyroidectomy conforms with the reduced amplitude of the spontaneous electrical activity of the brain reported by Bradley et al. (1960). It would seem that, again drawing on the work of Clare and Bishop (1957) and of Purpura and Grundfest (1956), an important factor underlying the fall in amplitude of the response would be a reduction in the numbers of the participating elements, i.e., in dendrites (Clare and Bishop) or in axo-dendritic synapses
583
(Purpura and Grundfest), rather than the metabolic influences playing on the individual synapses as would seem to be the case with the factors controlling the duration of this phase of the response. This interpretation is consistent with what is already known of the relative reduction in the probability of axodendritic interaction :in the imperfectly matured cortex of the neonatally thyroidectomised individual (Eayrs 1960b) and with the findings in relation to the effects of semi-acute medication, and of thyroidectomy in the adult animal. An alternative explanation of the findings is that the parameters of stimulation needed to elicit a response of maximal amplitude in the cretinoid individual differ from those in the normal. Although this possibility cannot entirely be excluded the negative effects of varying the stimulating voltage during the present experiment provided no supporting evidence.
Effects of semi-acute medication with triiodothyronine The observation that semi-acute medication restores the temporal relations of the recruiting response of neonatally thyroidectomised ani reals to normal while leaving the amplitude of the response unaffected implies that thyroidectomy early in life has a dual mode of action upon the electrophysiological functions of the central nervous system. It is thus tempting to infer that these may be respectively related to the metabolic and growth promoting effects of thyroid hormone, the former affecting the temporalcomponents of the response and the latter the amplitude. The possibility that the amelioration of effects on latency and duration by medication could be attributable to a restoration of cortical growth is, in the absence of any direct evidence, minimised by three arguments: first, that it seems unlikely that the period over which T3 was administered (2 days) could be sufficient to remedy the extreme impairment to growth known to be associated with neonatal thyroidectomy; second, ~it is difficult to see how an acceleration of growth could affect one group of changes without affecting another; and third, similar changes in latency and duration were observed following thyroidectomy in the adult at a time when cerebral growth was complete. The manner,
584
P.D. BRADLEYet al.
therefore, in which such a dichotomy of effect could arise remains to be considered. (a) Relationship between medication and temporal aspects of response. The effect of thyroxine upon the excitability of the central nervous system is not unknown, and it has been shown that hypothyroidism is associated with an increase in the electro-shock seizure threshold of rats (Timiras and Woodbury 1956) whereas administration of thyroxine causes a decrease in this threshold. Furthermore earlier work has demonstrated that thyroid hormone influences temporal relations in the spontaneous activity of the human EEG, administration of thyroxine giving rise to an increase in the frequency of the alpha rhythm (Lindsley and Rubinstein 1937; Ross and Schwab 1939; Rubin et al. 1937) while in hypothyroid states this is reduced (Bertrand et al. 1938). Hoagland (1936) postulated that the frequency of the alpha rhythm was directly proportional to the rate of respiration of neurones. It might be expected therefore that factors similar to those which influence the excitability of the cortex and the frequency of its spontaneous activity might also affect the temporal relations of the recruiting response. One possible way in which the short term administration of exogenous thyroid hormone could, in the face of a decreased probability of synaptic transmission, compensate for the increased latency of the response in the hypothyroid state is by lowering synaptic thresholds in such a manner that corticopetal stimuli arriving by way of a reduced number of synapses are as effective as the larger number normally available. Alternatively, the increased latency and duration of the response in hypothyroidism could primarily be due to an increase in the threshold of the participating synapses and to a depression of neuronal metabolism due to lack of circulating thyroxine. Treatment with T3 would, in such a case, be expected to be followed by a return to normality. (b) Absence of relationship between medication and amplitude. The findings that the fall in the amplitude of the surface negative wave in the neonatally thyroidectomised rat is unaffected by short-term medication with T3 and that little change in amplitude occurs when thyroidectomy is performed in the mature individual tend to
support the suggestion, made earlier, that an impairment to cortical growth is the underlying factor of the many changes known to occur (for review, see Eayrs 1959, 1960a). The most likely to be implicated would seem to be a reduction in the growth of neuropil which results in a decrease in the probability of axo-dendritic interaction so severe as to be unlikely to respond to brief (or even to long) periods of replacement therapy. It is therefore of interest that analogous results have been obtained where synaptic activity has been reduced by pharmacological means. Clare and Bishop (1957), for instance, found that increasing concentrations of topically applied strychnine caused a diminution in the amplitude of the surface-negative wave of the recruiting response and attributed their results to a gradual decrease in the number of units active. Purpura and Grundfest (1956) likewise attributed the reduced amplitude of the surfacenegative dendritic potential after topical application of d-tubocurarine to a progressively increasing synaptic blockade. Thus a decrease in the number of synapses developmentally induced by neonatal thyroidectomy and a physiological reduction by synaptic blocking agents would both appear to lead to the same result, in the one case a potentially irreversible and in the other a reversible, decrease in amplitude of the recruiting response. Conversely, the marked increase in the probability of axo-dendritic interaction associated with the process of normal growth (Eayrs and Goodhead 1959) may be invoked to explain the greater amplitude of the normal adult record by comparison with that of the 25-day old animal. Effect of acute medication with triiodothyronine The failure of acute administration of T3 to influence the recruiting response in neonatally thyroidectomised rats where semi-acute medication succeeded is not without precedent. Woodbury et al. (1952) for instance found that the electro-shock seizure threshold was depressed only after 14 days medication with thyroxine, and an increase in alpha frequency in the human EEG observed by Rubin et al. (1937) did not appear until 2 or 3 days after medication had been begun. Possibly this lack of an acute response to T3 may be explained by the inability of the hormone to
THYROIDECTOMY AND RECRUITING RESPONSE
penetrate the haemato-encephalic barrier during the course of the experiment although, by contrast, recent work has shown that the intraperitoneal administration of thyrotrophic hormone had an inhibiting action on thalamic activity within 30-40 rain of its injection (Milcou et al. 1960). An alternative possibility is that T3 acts as a precursor whose metabolism requires more time than was available under the conditions of the present experiment. Conchlsions
The results of this experiment are thus consistent with the hypothesis that two factors, one metabolic and the other developmental, are implicated in the changes in the recruiting response which arise after neonatal thyroidectomy. The changed temporal aspects of the response are largely the outcome of the metabolic effect and are reversible when T3 is given for 2 days before recording. The fall in amplitude of the surfacenegative wave, on the other hand, reflects a developmental impairment and is therefore not reversed by short-term medication. This conclusion is supported by the finding that, when thyroidectomy is delayed until cerebral development is complete, only those changes attributed to metabolic factors arise. Although the mechanisms underlying the changes in the recruiting response remain open to conjecture it would seem, in the light of present knowledge, that the changes associated with hypothyroidism are largely mediated through the anatomical distribution and physiological properties of cortical axo-dendritic synapses. The increase in the latency and duration of the response may be attributed to alterations in the transmitting properties of synapses brought about by the metabolic consequences of thyroidectomy and the fall in amplitude to a reduction in the number of participating synapses and availability ofpostsynaptic potentials for summation. SUMMARY
1. The recruiting response has been studied in the rat with special reference to the effects of thyroidectomy and subsequent replacement therapy. 2. Neonatal thyroidectomy is associated in later life with an increase in the latency and
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duration of the surface-negative component of the response and a reduction in its amplitude. 3. Medication with 1-triiodothyronine for 2 days prior to recording restores latency and duration to within normal range but is without effect on the amplitude of the response. Acute administration of this hormone during the process of recording is without significant effect on any parameter. 4. Thyroidectomy during adult life results in an increase in both the latency and duration of the negative wave, but does not alter the amplitude of the response. 5. These findings are discussed in the light of structural and metabolic differences between the brains of rats made hypothyroid during the course of cerebral development and after such development is complete. It is thought that they conform well with the view that the amplitude of the recruiting response may reflect the envelope of post-synaptic dendritic potentials whose number is significantly reduced in the neo-nalally thyroidectomised individual whereas its latency and duration, while not necessarily independent of structure, are more readily regulated by metabolic factors. REFERENCES ARDUINI, A. and TERZUOLO, C. Cortical and subcortical
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