TRANSECTION OF VARIOUS AFFERENT NERVOUS PATHWAYS TO THE HYPOTHALAMUS

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1 CORTICOTROPHIN RELEASE INDUCED BY SURGICAL TRAUMA AFTER TRANSECTION OF VARIOUS AFFERENT NERVOUS PATHWAYS TO THE HYPOTHALAMUS G. B. MAKARA, E. STARK, J. MARTON and T. M\l=E'\SZ\l=A'\ROS Institute of Experimental Medicine, Hungarian Academy of Sciences, Budapest VIII, Szigony u. 43, Hungary (Received 19 August 1971) SUMMARY Corticotrophin (ACTH) release induced by surgical trauma under pentobarbitone anaesthesia was studied in rats. The plasma corticosterone level was used as an index of 'rapid' ACTH release. One hour after surgical trauma the plasma corticosterone level had risen in rats with various cuts around the medial basal hypothalamus except in the group with lateral cuts. After stress no significant difference was found between the plasma levels of the controls and those of the rats with anterior, 'low' superior, 'low' anterosuperior, and 'short' posterior cuts. In contrast, in rats with 'high' superior, 'high' anterosuperior, 'long' posterior and lateral cuts the plasma corticosterone level was lower than in the appropriate sham-operated controls. It is suggested that the nerve fibres initiating ACTH release after surgical trauma ascend the spinal cord to the medulla and mid-brain whence the pathways pass forward in the region of the dorsal longitudinal fasciculus and/or the median forebrain bundle to the lateral hypothalamic area, and from there to the medial basal hypothalamus. INTRODUCTION Stressful stimuli triggering adrenocorticotrophin (ACTH) release have been tentatively classified as 'humoral' or 'neural', according to the nature of the route by which they activate the hypothalamo-hypophysial complex (Makara, Stark, Palkovits, Révész & Mihály, 1969Ò). Evidence derived from experiments on rats with deafferented hypothalamic islands suggests that stimuli such as ether and rubber band tourniquet, as well as large doses of formaldehyde, histamine and insulin, can be classified as 'humoral', for they trigger ACTH release in the absence of central nervous input to the hypothalamo-hypophysial complex (Matsuda, Duyck, Kendall & Greer, 1964; Feldman, Conforti, Chowers <& Davidson, 1970; Greer, Allen, Gibbs & Gullickson, 1970; Makara, Stark <& Palkovits, 1970&). On the other hand, the rapid ACTH release that normally follows small subcutaneous doses of formaldehyde or capsaicin, as well as audiogenic stimulation, bone fracture or surgical trauma, has

been shown to be dependent on the integrity of nervous pathways to the medial basal hypothalamus (Matsuda et al. 1964; Makara et al. 19696, 19706; Feldman et al. 1970; Greer et al.

2 been shown to be dependent on the integrity of nervous pathways to the medial basal hypothalamus (Matsuda et al. 1964; Makara et al , 19706; Feldman et al. 1970; Greer et al. 1970); these stimuli may thus be called 'neural'. Based on indirect evidence, Green (1969) made the suggestion that pathways coming from the thalamus, septum, amygdala and/or mid-brain reticular formation are equally likely to activate that part of the hypothalamus which is concerned with ACTH release. There is, however, little direct information on the location of such pathways. In the present study an attempt was made to locate the pathways which bring about the rapid, 'neural' ACTH release which follows surgical trauma. MATERIALS AND METHODS Male rats of the CFE strain, weighing g, were kept in an air-conditioned room ( C, % humidity) and given rat pellets and tap water ad libitum. They were handled daily for at least 1 week before being killed. Sectioning of the brain stem was performed in a stereotaxic apparatus under pentobarbitone anaesthesia (Mebubarbital, Rhone-Poulenc, Paris; 3-4 mg/100g, i.p.) in eight series of experiments as follows. Anterior cuts were begun at the posterior edge of the optic chiasma as described by Halász, Slusher & Gorski (1967). Superior and anterosuperior cuts were made with the rats placed in the head holder 10 nose down. The knife with a 1-8 mm long horizontal blade was placed in the mid-sagittal plane with the point 1 0 mm behind the bregma, lowered mm below the top of the skull, and rotated + 90 to right and left. Then the knife was moved 2-4 mm caudally and then rostrally, after each 90 rotation to right and left. This completed the superior cuts. At this point the knife was lowered on either side until it touched the base of the skull to make the anterosuperior cuts. The posterior cuts were made with the ear and tooth bars in the same horizontal plane, and the shaft of the knife 4-2 mm rostral to the lambda. To make 'short' posterior cuts a horizontal blade mm long was lowered in the sagittal plane, to 7-5 mm below the top of the skull, turned + 90 as before and lowered 1-5 mm on each side. To make the 'long' posterior cuts a blade mm long was lowered 6-0 mm below the lambda, rotated + 90 and lowered a further 3-0 mm. The procedure for making lesions in the posterior lower pole of the thalamus was similar to that for the ' long ' posterior cuts but after rotating the blade it was lowered a further 1-5 mm only. The lateral cuts were made with a 2-5 mm long horizontal blade in a parasagittal plane + 1 mm from the mid-line. It was lowered to the base of the skull, raised 2-5 mm, moved 2-0 mm caudally and lowered again till it touched the bone. After performing each of these cuts the knife was removed following, in reverse order, the cuts already made. All control animals were subjected to sham operations, which consisted of lowering the knife into the brain at the appropriate starting co-ordinates, and then removing it without making any lesion. Electrolytic lesions were made by a 5 ma anodic current passed through a mm thick glass-insulated platinum electrode, which was positioned according to the atlas of Szentágothai, Flerkó, Mess & Halász (1968). When making a lesion in the ventrobasal complex of the thalamus the electrode tip was positioned at 4,

L + 3, V2, and the current was passed for 30 s; for producing lesions in the centromedian and parafascicular nuclei, the current was passed for 10 s at position 4, L+l-5, V2. After the operations s.c. injections of 1-5 mg oxytetracycline were given daily for 7 days.

3 L + 3, V2, and the current was passed for 30 s; for producing lesions in the centromedian and parafascicular nuclei, the current was passed for 10 s at position 4, L+l-5, V2. After the operations s.c. injections of 1-5 mg oxytetracycline were given daily for 7 days. The rats were divided randomly into groups of equal mean body weight and were used 7-8 days after the operation. They were housed individually for about 18 h before the intraperitoneal injection of pentobarbitone (3 mg/100 g). Control animals were then left undisturbed until a blood sample was taken 80 min later. The remaining groups were subjected to the stress of unilateral sham-adrenalectomy 20 min after the injection of the anaesthetic; blood was withdrawn after a further 60 min. Blood was collected between and h under ether anaesthesia from the aorta within 2 min after removal from the cage. Plasma corticosterone concentration was used as an indicator of ACTH release; it was determined by the method of Guillemin, Clayton, Lipscomb & Smith (1959). At the end of each experiment, the animals were decapitated and a block of the brain stem including the hypothalamus was fixed in 10 % formaldehyde. Later it was embedded in paraffin wax, sectioned serially, and stained with haematoxylin-eosin or Luxol fast blue-cresyl violet. Only results from rats with histologically verified transections were used. The values of plasma corticosterone concentrations were subjected to logarithmic transformation, and one-way analysis of variance and paired comparisons were carried out separately for each experimental series as described by Scheffé (1959). RESULTS After the stereotaxic operations most of the rats recovered quickly, without any sign of disturbance. However, in the groups with ' long ' posterior and parasagittal cuts some of the animals were hyper-reactive and vicious during handling, and lost g weight in a week. Histological examination showed that the anterior cut extended in a semicircle behind the optic chiasma and severed all the neural connexions entering rostrally within mm of the mid-line (Plate, figs 1 and 2). The groups with superior and anterosuperior cuts (Plate, figs 3 and 4) were divided into sub groups with 'low' and 'high' deafferentations. The former lay usually at the mid-hypothalamic level, the latter between the upper pole of the hypothalamic ventromedial nuclei and the lower part of the thalamus. The superior cut extended about 1-8 mm on each side of the midline from the level of the optic chiasma to that of the intrapeduncular nuclei, and must have severed all vertical connexions between the medial basal hypothalamus and the thalamus. In addition to this, the anterosuperior combination also severed rostral connexions to the medial basal hypothalamus (MBH), leaving only the lateral and some caudal connexions intact. The posterior sections reached the base of the brain within or immediately behind the mamillary body. The 'short' ones (Plate, fig. 5) did not exceed the upper limit of the mamillary body; the 'long' ones (Plate, fig. 6) severed the nerve fibres passing within mm of the mid-line up to the lower posterior pole of the thalamus. The lateral sections (Plate, fig. 7) produced two sagittal cuts of about 2-5 mm length from the cortex to the anterior half of the hypothalamus as well as 2 mm long cuts in the same plane alongside the posterior hypothalamus but extending 2-5 mm from the base of the brain only.

4 The electrolytic lesions were large in size and destroyed more than 60 % of the ventrobasal complex of the thalamus (Plate, fig. 8) as well as the centromedian parafascicular nuclei. Table 1. Effect of surgical trauma on the plasma corticosterone level in rats with various cuts of the medial basal hypothalamus* (means ± s.e.m.) Expt no. 1 Preliminary operation Anterior cut 'Low' superior cut 'High' superior cut 'Low' anterosuperior cut 'High' anterosuperior cut ' Short ' posterior cut ' Long ' posterior out Parasagittal cut Plasma corticosterone (/ig/100 ml) Anaesthesia (12) ll-5±3-6(10) (10) (12) (13) (10) (9) (12) (4) (12) 4-6 ±0-8 (5) (9) Surgical trauma ± ± ± ' ; 6-5: 2(12)t l(12)t 2(13)t 7 (8)t 7(17)f 7(12)t 9 (7)t 7(13)tt 2(12)t l(16)t '3(10)t l(ll)tî 9 (5)t 4(10» * All rats were anaesthetized with 3 mg pentobarbitone/100 g, i.p. Blood was sampled 1 h after trauma, t Significantly different from the appropriate anaesthetized controls (P < 0-05). J Signifioantly different from appropriate sham-operated controls subjected to surgical trauma (P < 0-05). Number of rats given in parentheses. Table 2. Effect of surgical trauma on the plasma corticosterone level in rats with various lesions in the thalamus* (means ± s.e.m.) Expt no. Preliminary operation 7 Ventrobasal thalamic lesion Centromedian thalamic lesion 8 Lesion in the posteroventral pole of the thalamus Plasma corticosterone (,ug/100 ml) Anaesthesia (7) (4) (5) (6) (9) Surgical trauma (12)f (6) (5)t (9)t (15)t * AH rats were anaesthetized with 3 mg pentobarbitone/100 g, i.p. Blood was sampled 1 h after trauma, f Significantly different from appropriate anaesthetized controls (P < 0-05). Number of rats in paren theses. The plasma corticosterone values obtained in the different experiments are summarized in Tables 1 and 2. Comparing the appropriate sham-operated groups with those with transections, none of the sections or lesions caused a significant change in the plasma corticosterone level of the unstressed, anaesthetized rats. One hour after surgical trauma the plasma corticosterone level rose significantly in the rats with all the different sections and lesions except the group with lateral cuts (Tables 1 and 2). There was no significant difference between the plasma cortico-

sterone levels of the appropriate stressed sham-operated rats and that of the stressed groups with anterior, ' low ' superior, ' low ' anterosuperior, and ' short ' posterior cuts or that of the

5 sterone levels of the appropriate stressed sham-operated rats and that of the stressed groups with anterior, ' low ' superior, ' low ' anterosuperior, and ' short ' posterior cuts or that of the groups with electrolytic or surgical lesions in the thalamus. However, 1 h after surgical trauma, the plasma corticosterone level was not as high in the groups with 'high' superior, 'high' anterosuperior, and 'long' posterior sections as in the appropriate sham-operated groups. The rise produced by surgical trauma in experiment 5 was small; however, a similar increase was found in two additional experiments in which different survival times were used, suggesting that some path ways spared by this section were involved in activating the MBH to release ACTH. DISCUSSION We have previously shown that surgical trauma releases ACTH through neural pathways, since release is prevented by transecting the anterior, lateral and superior neural connexions of the MBH (Makara et al ). In the present experiments neither the anterior, the 'short' posterior, nor the anterosuperior sections alone prevented the ACTH release caused by surgical trauma. This strongly suggests that neural connexions between the medial and lateral hypothalamus represent a major route by which sensory information elicited by surgical trauma reaches the hypo thalamo-hypophysial complex. Parasagittal section which severed, amongst others, the connexions between the lateral hypothalamus and the MBH prevented the ACTH release which normally occurs 1 h after surgical trauma ; this provides further support for this suggestion. The experiments of Greer et al. (1970) with traumatic stress of a leg fracture also suggest that in rats the lateral connexions to the MBH are responsible for release of ACTH. Transection of anterior, or posterior basal as well as the vertical connexions of the MBH at mid-hypothalamic level failed to change significantly the response to surgical trauma, which indicates that afférents from these directions are not in dispensable for this response; they may, however, have some contributory role. By contrast, the anterior connexions of the MBH are important in the control of ovula tion (Halász, 1969; Köves <fe Halász, 1969) as well as in maintaining the circadian rhythm of ACTH release (Halász et al. 1967). The observation that the ' high ' superior and anterosuperior sections significantly decreased the ACTH release evoked by surgical trauma, whereas their ' low ' counter parts failed to do so, was unexpected. Since some of these deafferentations were accompanied by lesions of small to moderate size in the thalamus, this observation might be thought to indicate a thalamic relay in the pathway. However, electrolytic or surgical lesions in various posterior thalamic regions, known to be involved in responses to nociceptive stimulation (Mitchell & Kaelber, 1966), did not interfere the idea with the response to surgical trauma (see Table 2), which would not support of a thalamic relay in the pathways involved. All the 'high' superior and antero superior sections, and the long, posterior section which also diminished the release of ACTH after surgical trauma, appeared to have a common point of action in that they all damaged pathways near the lateral or superior border of the mammillary body. It therefore seems likely that these pathways are involved in release of ACTH. The ' long ' posterior section which greatly reduced the ACTH release induced by

6 surgical trauma severed most direct pathways connecting medullary and mid-brain structures with the hypothalamus. It transected the mammillary peduncles and the medial portion of the median forebrain bundle as well as the diffuse fibres from the region of the dorsal longitudinal fascicle. On the other hand, the 'low' posterior deafferentation, which severed the mammillary peduncles only, failed to change the response. This suggests that the fibres travelling in the region ofthe dorsal longitudinal fascicle and/or the median forebrain bundle are essential for the release of ACTH after surgical trauma. The effects of the various hypothalamic sections on the pituitary-adrenal response to surgical trauma are compatible with several possible arrangements of the pathways involved, but the simplest is the following: the nerve fibres leading to release of ACTH ascend the spinal cord, probably in the region of the lateral spinothalamic tract (Makara, Stark «fe Mihály, 1969, 1970 ; Gibbs, 1969); but, as they pass forward, the pathways ascend in the region of the dorsal longitudinal fascicle and/or the median forebrain bundle to the lateral hypothalamic area, whence the information is trans mitted to the cells producing corticotrophin releasing factor in the medial basal hypothalamus. This conclusion refers only to the rapid 'neural' phase of ACTH release which follows surgical trauma within 1 h. In contrast to this rapid phase, the ACTH release seen 2-4 h after the onset of trauma is not prevented by transection of all anterior, lateral and superior afférents to the MBH (Stark, Makara, Palkovits & Mihály, 1970). Moreover, more severe trauma of the same kind may rapidly activate not only 'neural' but also 'humoral' pathways triggering ACTH release. Such a phenomenon has been shown to occur using formaldehyde injections as a stimulus: ACTH release normally produced by a small subcutaneous dose was prevented by transection of the nerves supplying the site of injection or by 'deafferentation' of the MBH, but a large dose of the same substance stimulated ACTH release after denervation of the site of injection or hypothalamic deafferentation (Makara, Stark «fe Mihály, 1967, 1970 ; Makara et al ; Stark et al. 1970). REFERENCES Feldman, S., Conforti, N., Chowers, I. «fe Davidson, J. M. (1970). Pituitary adrenal activation in rats with medial basal hypothalamic islands. Acta endocr., Copenh. 63, Gibbs, F. P. (1969). Central nervous system lesions that block release of ACTH caused by traumatic stress. Am. J. Physiol. 217, Green, J. D. (1969). Neural pathways to the hypophysis : anatomical and functional. In The hypothalamus, pp Eds W. Haymaker, E. Anderson «fe W. J. H. Nauta. Springfield, III.: C. C. Thomas. Greer,.., Allen, C. F., Gibbs, F. P. «fe Gullickson, C. (1970). Pathways at the hypothalamic leve through which traumatic stress activates ACTH secretion. Endocrinology 86, Guillemin, R., Clayton, G. W., Lipscomb, H. S. «fe Smith, J. D. (1959). Fluorometric measurement of rat plasma and adrenal corticosterone concentration. J. Lab. clin. Med. 53, Halász,. (1969). The endocrine effects of isolation of the hypothalamus from the rest of the brain. In Frontiers in neuroendocrinology, pp Eds W. F. Ganong «fe L. Martini. Oxford University Press. Halász,., Slusher,.. <& Gorski, R.. (1967). Adrenocorticotropic hormone secretion in rats after partial or total deafferentation of the medial basal hypothalamus. Neuroendocrinology 2, Köves,. «fe Halász,. (1969). Data on the location of the neural structures indispensable for the occurrence of ovarian compensatory hypertrophy. Neuroendocrinology i, Makara, G. B., Stark, E. «fe Mihály,. (1967). Site at which formalin and capsaicin act to stimulate corticotrophin secretion. Can. J. Physiol. 45, Makara, G. B., Stark, E. «fe Mihály,. (1969 ). Corticotrophin release induced by injection of formalin in rats with hemisection of the spinal cord. Acta physiol. hung. 35,

7 Journal of Endocrinology, Vol. 53, No. 3 Plate (Facing p. 395)

Makara, G. B., Stark, E. «fe Mihály,. (1970 ). Corticotrophin release induced by traumatic stress in rats with unilateral spinal cord lesion. Acta physiol. hung. 38, 199-203. Makara, G. B., Stark, E. «fe Palkovits, M.

8 Makara, G. B., Stark, E. «fe Mihály,. (1970 ). Corticotrophin release induced by traumatic stress in rats with unilateral spinal cord lesion. Acta physiol. hung. 38, Makara, G. B., Stark, E. «fe Palkovits, M. (19706). Afferent pathways of stressful stimuli: corticotrophin release after hypothalamic deafferentation. J. Endocr. 47, Makara, G. B., Stark, E., Palkovits, M., Révész, T. «fe Mihály,. (19696). Afferent pathways of stressful stimuli: corticotrophin release after partial deafferentation of the medial basal hypothalamus. J. Endocr. 44, Matsuda, K., Duyck, C, Kendall, J. W. «fe Greer, M. A. (1964). Pathways by whioh traumatic stress and ether induoe increased ACTH release in the rat. Endocrinology 74, Mitchell, C. L. «fe Kaelber, W. W. (1966). Effect of medial thalamic lesions on responses elicited by tooth pulp stimulation. Am. J. Physiol. 210, Soheffé, H. (1959). The analysis of variance. New York: Wiley. Stark, E., Makara, G. B., Palkovits, M. «fe Mihály,. (1970). Afferent pathways of stressful stimuli: their dependence on strength and the time elapsed after the onset of stimulation. Acta physiol. hung. 38, Szentágothai, J., Flerkó,., Mess,. «fe Halász,. (1968). Hypotlialamic control of the anterior pituitary. An experimental-morphological study. Budapest: Akademiai Kiadó. DESCRIPTION OF PLATE Sections through the brains of rats showing positions of lesions. Fig. 1. Anterior cut in the medial basal hypothalamus (MBH); sagittal section. ( X 18 ) Fig. 2. Anterior cut in the MBH, horizontal section. ( X 25.) Fig. 3. Superior cut, sagittal section through the MBH. ( 20.) Fig. 4. Anterosuperior cut, sagittal section of the MBH. ( X 20. ) Fig. 5. 'Short' posterior cut, sagittal section. ( 18.) Fig. 6. 'Long' posterior cut, sagittal section. ( 18.) Fig. 7. Lateral cuts, coronal section of the MBH. ( 32.) Fig. 8. Electrolytic lesion in the ventrobasal complex of the thalamus, coronal section. ( X 8.) Sections in figs 1-6 were stained with Luxol fast blue cresyl violet. Sections in figs 7-8 were stained with haematoxylin-eosin. Abbreviations: ARC, arcuate nucleus; CA, anterior commissure; CHO, optic chiasma: CM, mammillary body; H, hippocampus; PV, paraventrioular nucleus; ST, pituitary stalk; T, thalamus ; V, third ventricle ; VM, ventromedial nucleus. The arrows point to the cuts or lesions.

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