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Fetal suprachiasmatic nucleus transplants: diurnal rhythm recovery of lesioned rats
Brain Research, 311 (1984) 353-357
353
Elsevier BRE 20404
Fetal suprachiasmatic nucleus transplants: diurnal rhythm recovery of lesioned rats RENI~ DRUCKER-COLIN, RAUL AGUILAR-ROBLERO, FERNANDO GARC[A-HERNANDEZ, FEDERICO FERNANDEZ-CANCINO and FEDERICO BERMUDEZ RATTONI Departamento de Neurociencias, Centro de lnvestigaciones en Fisiologia Celular, Universidad Nacional A ut6noma de MOxico, MOxico D,F. (MOxico)
(Accepted May 29th, 1984) Key words: suprachiasmatic nucleus - - fetal transplants-- diurnal rhythm
The diurnal rhythm organization of drinking behavior was determined prior to and after suprachiasmatic nucleus (SCN) lesions. Five weeks after SCN lesions which produced total loss of the diurnal rhythm, the anterior hypothalamus including the SCN of fetal rats was transplanted into the floor of the 3rd ventricle of the lesioned rats. Eight weeks after the graft, rats recovered their rhythm. The results show that grafts allow animals to recover lost functional properties due to lesions, and further support the notion that the SCN is a pacemaker for certain behaviors. The recovery from brain injury has recently been studied using the technique of transplanting neuronal tissue into the brains of neonataP, 5 as well as adult15, ~9 recipients. These and other studies have demonstrated that a variety of neuronal types survive and establish extensive and specific connections with the host tissue 3,12. Such studies, however, have mostly been interested in demonstrating neural connectivity between the transplanted tissue and the host. There are as yet comparatively fewer studies demonstrating functional development of the grafts. To date it has been shown that grafts of appropriate fetal brain areas, can correct polyuria and polydipsia of Brattleboro rats 7, and hypogonadism of gonadotropin-releasing hormone deficient mutant mice 8. In addition it has been reported that grafts can correct motor abnormalities after nigrostriatal lesions 4,17, promote recovery of a learned discrimination task 13, enhance sexual behavior 1 and produce close to normal recovery of electrophysiological responses 6,14. The present study was undertaken to determine whether transplanting a putative pacemaker, the suprachiasmatic nucleus (SCN), into lesioned adult rats can restore the diurnal rhythm of drinking.
Thirty adult male Wistar rats (200-210 g body weight at the start of the experiment) were used in this study. All rats were kept on a strict 12:12 h light dark (LD) cycle (08.00 h on; 20.00 h off) for a month prior to the start of the experiments. Drinking data were obtained from each rat by monitoring water spout contacts. Each contact, triggered a monostable circuit which generated a single pulse fed in turn on line to a PDP-11/34 computer. A continuous 48 h recording of drinking was thus obtained for each rat. They were then anesthetized with 35 mg/kg sodium pentobarbital in order to perform stereotaxic bilateral electrolytic lesions of the SCN. After a 4 day recovery period the rats were returned to the cages where drinking was again monitored once a week during 48 h for the next 5 weeks. Ten animals whose loss of drinking rhythm persisted during 5 weeks were utilized for transplant experiments. Seventeen day old fetuses were removed from the abdominal cavity of pregnant rats. The cerebrum of the fetuses was removed, and the anterior hypothalamic neurons which included the SCN were dissected under a microscope. This block of tissue was then stereotaxically placed with the aid of a can-
Correspondence: R. Drucker-Colin, Departamento de Neurociencias, Centro de Investigaciones en Fisiologia Celular, Universidad
Nacional Aut6noma de M6xico, Apartado Postal 70-600, M6xico D.F., M6xico. 0006-8993/84/$03.00 © 1984 Elsevier Science Publishers B.V.
354 nula on the floor of the 3rd v e n t r i c l e of the S C N le-
e m b e d d e d sections stained by the K l u v e r - B a r r e r a
s i o n e d rats. T h e t r a n s p l a n t e d rats w e r e a l l o w e d to re-
method.
c o v e r for a n o t h e r 4 days. and their d r i n k i n g b e h a v i o r
T h e d r i n k i n g d a t a w e r e a n a l y z e d as follows: w a t e r
again m o n i t o r e d o n c e a w e e k for 48 h. t h r o u g h o u t
b o t t l e contacts w e r e a u t o m a t i c a l l y r e g i s t e r e d by t h e
the f o l l o w i n g 8 w e e k s .
c o m p u t e r in 15 min t i m e bins. T h e s e d a t a w e r e t h e n
plotted over 48 h in o r d e r to visualize periods of maximal or minimal contacts. Per cent contacts during night vs day were also obtained (see Table I). To clar-
A t the end of the e x p e r i m e n t s rats w e r e sacrificed and p e r f u s e d with 10% f o r m a l i n , the brains r e m o v e d and histological analysis d o n e on 6 m m thick paraffin
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CYCLES / HR Fig. 1. This graph represents, on the left. computer print outs of the distribution of water spout contacts plotted as a histogram o[ 15 min time bins across 48 h of recordings for each of 6 rats. The return of circadian periodicity is evident 8 weeks after the graft. On the right, the spectral density of drinking time senes is shown. The ordinate gives arbitrary units, and the abscissa gives the cyeles/h. This means for example that a cycle of 0.2 represents a 5-h period, and 0.042 represents a 24 h period.
355 TABLE I Mean + S.D. percent water bottle contacts over day and night (n = 6) Control Day
Night
SCN lesion (5th week)
Graft
Day
1st week
10.9 89.1" 48.4 S.D. 5.7 5.2 19.1 * P < 0.001. Control vs SCN lesion. ** P < 0.02. Control vs graft 8th week.
Night
51.6 19.1
5th week
8th week
Day
Night
Day
Night
Day
Night
47.5 16.2
52.5 16.4
33.7 18.6
66.3 18.6
24.3 13.0
75.7** t3.0
ify temporal characteristics of the drinking rhythm, analysis of spectral density was employed. This involved the Fast Fourier transform of the autocorrelation function of time series 2, which allows visualization of the main components of the rhythm (see Fig. 2). A simple analysis of variance for repeated measures was also performed, with further comparisons using a paired t-test. After SCN lesions 20 of the rats recovered their drinking rhythm spontaneously. Fourteen of them recovered it after the first week, 4 after the second and two after the third. Histological analysis of these rats indicated that the lesions missed variable amounts of SCN. In the 10 remaining rats which did not recover their rhythm after 5 weeks, it was assumed that complete destruction of the SCN was accomplished. This assumption was borne out by the fact that 4 of the transplanted rats never recovered their rhythm even 8 weeks after the transplant. The histology of these rats showed necrosed grafts with an SCN which remained totally destroyed. The behavior of the 6 remaining grafted rats is illustrated in Fig. 1. During the pre-lesion control each rat had a distinct diurnal drinking rhythm. Following complete SCN destruction they were significantly disrupted up to the 5th week in which they were tested. After the transplant, animals slowly recovered their diurnal rhythm (see Table I), until by the 8th week it was very similar to their pre-lesion control. Analysis of variance with repeated measures showed a difference between treatments with an F4.5 = 9.38 P < 0.016. This latter result prompted a t-test to compare significance of treatments. This analysis yielded a non-significant difference between controls and transplant (t = 2.33, P < 0.10). while both controls and transplant (8th week) were significantly different
from SCN lesions (t = 10,10, P < 0.001 and t = 3.30, P < 0.02, respectively). The power spectral analysis of drinking (Fig. 1) demonstrates a clear 24 h rhythm since the largest peaks coincide at around 0.042 cycles/h (1/24 h frequency). The SCN lesions eliminate the coincidence of this peak at such time and shows the appearance of several ultradian rhythms. Eight weeks after the transplant there is a return to the 24 h rhythm. The fact that the peaks are not as high reflects the fact that recovery is not absolute. All transplanted rats were examined histologically •at the end of the experiments. The lesions completely destroyed the SCN, from the caudal border of the supraoptic nucleus (5780) 11 to caudal third of the anterior commissure (6860) 11. The necrotic area was localized in the midline extending about 1.4 mm on the lateral plane, and from the ventral third of the III ventricle to the dorsal border of the optic chiasm. Other areas damaged by the lesion include: the ventromedial area of the anterior hypothalamic nucleus, the periventricular preoptic nucleus, the preoptic nucleus (pars suprachiasmatica) and the tractus infundibularis. In the 4 rats with no drinking rhythm recovery, it was evident that the graft had deteriorated. The tissue was necrotic, macrophages were abundant and there was a substantial dilatation of the 3rd ventricle. In these animals the SCN destruction was still clearly evident. On the other hand in the recovered rats the transplant appeared healthy, and although there was some distortion of the 3rd ventricle (Fig. 2), this did not seem to affect their behavior which was similar to normals. Although histological examination of stained sections indicates that healthy normal neurons are present in the transplanted tissue (Fig. 2), connectivity is not proven. Such connectivity is hinted in Fig. 2 where the transplanted tissue though
356
Fig. 2. A shows representative coronal sections of area of transplant at 3 different levels according to K6nig and KlippeP 1. Arrows indicate area where the graft was located. B and C are different magnifications of A, while D is from a different rat. A x 25. B x 100. C × 400, D × 250. CO, optic chiasm; III, third ventricle; FMP, fascicutus medialis prosencephali; ha. anterior hypothalamus. (Publisher's reduction factor 0.9.) clearly within t h e 3rd v e n t r i c l e a p p e a r s to h a v e established bridges w i t h t h e n e i g h b o r i n g p a r e n c h y m a ,
T h e results o f this study s h o w that the c i r c a d i a n r h y t h m of rats which is d i s r u p t e d by S C N lesions can
357 be reconstituted by transplanting homologous fetal neuronal tissue. The results strongly suggest that the recuperation of the drinking rhythm is p r o d u c e d as a result of the transplanted tissue, since those animals in which the graft necrosed, the rhythm loss persisted even after 13 weeks. Since the diurnal rhythm organization of drinking was relatively slow in recovering, it is conceivable that its reorganization d e p e n d e d on a certain degree of neural reconnection, which took weeks to develop. Some support for this notion comes from experiments showing that in animals whose brain is devoid of all connections with the SCN (hypothalamic 'island') rhythmic multiple unit activity persists only in the SCN 9. In this respect the SCN transplant is similar to the optic lobe transplant in cockroach 16, but different from those of other organisms such as the silk moth TM, the fruit flyl0 and the sparrow20, whose transplanted tissue appears to control rhythmicity by release of a h o r m o n e without need of any neural connections.
1 Arendash, G. and Gorski, R., Enhancement of sexual behavior in female rats by neonatal transplantation of brain tissue from males, Science, 217 (1982) 1276-1278. 2 Bendat, J. S. and Piersol, A. G., Measurement and Analysis of Random Noise, Wiley, New York, 1966. 3 Bj6rklund, A. and Stenevi, U., Regeneration of monoaminergic and cholinergic neurons in mammalian central nervous system, Physiol. Rev., 59 (1979) 62-100. 4 Bj6rklund, A., Dunnett, S. B., Stenevi, U., Lewis, M. E. and Iversen, S. D., Reinnervation of the denervated striatum by substantia nigra transplants: functional consequences as revealed by pharmacological and sensorimotor testing, Brain Research, 199 (1980) 307-333. 5 Das, G. D., Transplantation of embryonic neural tissue in the mammalian brain. I. Growth and differentiation of neuroblasts from various regions of the embryonic brain in the cerebellum of neonate rats, Life Sci., 4 (1974) 93-124. 6 Dunnett, S. B,, Gage, F. H., Bj6rklund, A., Stenevi, U., Low, W. C. and Iversen, S. D., Hippocampal deafferentation: transplant derived reinnervation and functional recovery, Scand. J. Physiol., Suppl. 1 (1982) 104-111. 7 Gash. D., Sladek, J. R. and Sladek, C., Functional development of grafted vasopressin neurons, Sc&nce, 210 (1980) 1367-1369. 8 Gibson, M. J., Krieger, D. T., Perlow, M, J., Davies, T. F., Zimmerman, E. A., Ferin, M. and Charlton, N. M., Hypothalamic brain transplants reverse hypogonadism in male mutant mice with gonadotropin-releasing hormone deficiency, Trans. Ass. Amer. Phys., 95 (1982) 188-195. 9 Inouye, S. T. and Kawamura, H., Persistence of circadian rhythmicity in a mammalian hypothalamic 'island' containing the suprachiasmatic nucleus, Proc. nat. Acad. Sci. U.S.A., 76 (1979) 5962-5966. 10 Handler, A. M. and Komopka, R. J., Transplantation of a circadian pacemaker in Drosophilia, Nature (Lond.), 279
A l t h o u g h from our experiments the possibility of neurosecretory influences cannot be ruled out. It should be p o i n t e d out that both the lesion or the graft did not a p p e a r to p r o d u c e any i m p a i r m e n t of homeostatic mechanisms. Thus, although we did not measure the actual amount of water intake, the number of water spout contacts r e m a i n e d the same throughout a given 24 h day, only the distribution was modified. These experiments give further support to the notion that the SCN is a p a c e m a k e r for certain rhythms in rats 9, and in addition gives new evidence that transplanted tissue of fetal origin is capable of restoring functional losses of behavior in lesioned adult rats. This study was partially s u p p o r t e d by a G r a n t from the Fundaci6n Ricardo Z e v a d a to Ren6 D r u c k e r - C o l/n. W e wish to thank Dr. Jorge A r a u z for valuable collaboration during the initial aspects of this work.
(1979) 236-238. 11 K/~nig,J. F. and Klippel, R. A., The Rat Brain: A Stereotaxic Atlas of the Forebrain and Lower Parts and the Brain Stem. Williams and Wilkins, Baltimore, MD, 1963.
12 Kromer, L. F., Bj6rklund, A. and Stenevi, U., Intracephalic embryonic neural implants in the adult rat brain. I. Growth and mature organization of brainstem cerebellar and hippocampal implants, J. comp. Neurol., 218 (1983) 433-459. 13 Labbe, R., Firl, A., Mufson, E. J. and Stein, D. G., Fetal brain transplants: reduction of cognitive deficits in rats with frontal cortex lesions, Science, 221 (1983) 470-472. 14 Low, W. C., Lewis, P. R., Bunch, S., Dunnett, S. B., Thomas S. R., Iversen, S. D., Bj~rklund, A. and Stenevi, U., Neural transplants of embryonic septal nuclei into adult rats with septohippocampal lesions: the recovery of function, Nature (Lond.), 300 (1982) 260-262. 15 Lund, R. D. and Hauschka, S. D., Transplanted neural tissue develops connections with host brain, Science, 193 (1976) 582-584. 16 Page, T. L., Transplantation of the cockroach circadian pacemaker, Science, 216 (1982) 73-75. 17 Perlow, M. J., Freed, W. J., Hoffer, B. J., Seiger, A., OIson, L. and Wyatt, R. J., Brain grafts reduce motor abnormalities produced by destruction of the nigrostriatal dopamine system, Science, 204 (1979) 643-647. 18 Truman, J. W. and Riddiford, L. M., Neuroendocrine control of ecdysis in silkmoth, Science, 167 (1970) 1624-1626. 19 Stenevi, U., Bjorklund, A. and Svendgaard, N., Transplantation of central and peripheral monoamine neurons to the adult rat brain: techniques and conditions for survival, Brain Research, 114 (1976) 1-20. 20 Zimmerman, N. H. and Menaker, M., The pineal gland. A pacemaker within the circadian system of house sparrow, Proc. nat. Acad. Sci. U.S.A., 76 (1979) 999-1003.
353
Elsevier BRE 20404
Fetal suprachiasmatic nucleus transplants: diurnal rhythm recovery of lesioned rats RENI~ DRUCKER-COLIN, RAUL AGUILAR-ROBLERO, FERNANDO GARC[A-HERNANDEZ, FEDERICO FERNANDEZ-CANCINO and FEDERICO BERMUDEZ RATTONI Departamento de Neurociencias, Centro de lnvestigaciones en Fisiologia Celular, Universidad Nacional A ut6noma de MOxico, MOxico D,F. (MOxico)
(Accepted May 29th, 1984) Key words: suprachiasmatic nucleus - - fetal transplants-- diurnal rhythm
The diurnal rhythm organization of drinking behavior was determined prior to and after suprachiasmatic nucleus (SCN) lesions. Five weeks after SCN lesions which produced total loss of the diurnal rhythm, the anterior hypothalamus including the SCN of fetal rats was transplanted into the floor of the 3rd ventricle of the lesioned rats. Eight weeks after the graft, rats recovered their rhythm. The results show that grafts allow animals to recover lost functional properties due to lesions, and further support the notion that the SCN is a pacemaker for certain behaviors. The recovery from brain injury has recently been studied using the technique of transplanting neuronal tissue into the brains of neonataP, 5 as well as adult15, ~9 recipients. These and other studies have demonstrated that a variety of neuronal types survive and establish extensive and specific connections with the host tissue 3,12. Such studies, however, have mostly been interested in demonstrating neural connectivity between the transplanted tissue and the host. There are as yet comparatively fewer studies demonstrating functional development of the grafts. To date it has been shown that grafts of appropriate fetal brain areas, can correct polyuria and polydipsia of Brattleboro rats 7, and hypogonadism of gonadotropin-releasing hormone deficient mutant mice 8. In addition it has been reported that grafts can correct motor abnormalities after nigrostriatal lesions 4,17, promote recovery of a learned discrimination task 13, enhance sexual behavior 1 and produce close to normal recovery of electrophysiological responses 6,14. The present study was undertaken to determine whether transplanting a putative pacemaker, the suprachiasmatic nucleus (SCN), into lesioned adult rats can restore the diurnal rhythm of drinking.
Thirty adult male Wistar rats (200-210 g body weight at the start of the experiment) were used in this study. All rats were kept on a strict 12:12 h light dark (LD) cycle (08.00 h on; 20.00 h off) for a month prior to the start of the experiments. Drinking data were obtained from each rat by monitoring water spout contacts. Each contact, triggered a monostable circuit which generated a single pulse fed in turn on line to a PDP-11/34 computer. A continuous 48 h recording of drinking was thus obtained for each rat. They were then anesthetized with 35 mg/kg sodium pentobarbital in order to perform stereotaxic bilateral electrolytic lesions of the SCN. After a 4 day recovery period the rats were returned to the cages where drinking was again monitored once a week during 48 h for the next 5 weeks. Ten animals whose loss of drinking rhythm persisted during 5 weeks were utilized for transplant experiments. Seventeen day old fetuses were removed from the abdominal cavity of pregnant rats. The cerebrum of the fetuses was removed, and the anterior hypothalamic neurons which included the SCN were dissected under a microscope. This block of tissue was then stereotaxically placed with the aid of a can-
Correspondence: R. Drucker-Colin, Departamento de Neurociencias, Centro de Investigaciones en Fisiologia Celular, Universidad
Nacional Aut6noma de M6xico, Apartado Postal 70-600, M6xico D.F., M6xico. 0006-8993/84/$03.00 © 1984 Elsevier Science Publishers B.V.
354 nula on the floor of the 3rd v e n t r i c l e of the S C N le-
e m b e d d e d sections stained by the K l u v e r - B a r r e r a
s i o n e d rats. T h e t r a n s p l a n t e d rats w e r e a l l o w e d to re-
method.
c o v e r for a n o t h e r 4 days. and their d r i n k i n g b e h a v i o r
T h e d r i n k i n g d a t a w e r e a n a l y z e d as follows: w a t e r
again m o n i t o r e d o n c e a w e e k for 48 h. t h r o u g h o u t
b o t t l e contacts w e r e a u t o m a t i c a l l y r e g i s t e r e d by t h e
the f o l l o w i n g 8 w e e k s .
c o m p u t e r in 15 min t i m e bins. T h e s e d a t a w e r e t h e n
plotted over 48 h in o r d e r to visualize periods of maximal or minimal contacts. Per cent contacts during night vs day were also obtained (see Table I). To clar-
A t the end of the e x p e r i m e n t s rats w e r e sacrificed and p e r f u s e d with 10% f o r m a l i n , the brains r e m o v e d and histological analysis d o n e on 6 m m thick paraffin
lib
.I,
CONTROL
.4
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•
32
.
24
.16 i
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.8
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SCN LESION
.4
SCN L E S I O N
I
.2
.16
4
08
•
0 0 8TH 1
VEER AFTER
GRAFT
.4 •
3 2
•
24
2 3
.2
.4
8TH WEEK AFTER GRAFT
.16
4 •
08
5 @
0 .2
.4
.6
.8
CYCLES / HR Fig. 1. This graph represents, on the left. computer print outs of the distribution of water spout contacts plotted as a histogram o[ 15 min time bins across 48 h of recordings for each of 6 rats. The return of circadian periodicity is evident 8 weeks after the graft. On the right, the spectral density of drinking time senes is shown. The ordinate gives arbitrary units, and the abscissa gives the cyeles/h. This means for example that a cycle of 0.2 represents a 5-h period, and 0.042 represents a 24 h period.
355 TABLE I Mean + S.D. percent water bottle contacts over day and night (n = 6) Control Day
Night
SCN lesion (5th week)
Graft
Day
1st week
10.9 89.1" 48.4 S.D. 5.7 5.2 19.1 * P < 0.001. Control vs SCN lesion. ** P < 0.02. Control vs graft 8th week.
Night
51.6 19.1
5th week
8th week
Day
Night
Day
Night
Day
Night
47.5 16.2
52.5 16.4
33.7 18.6
66.3 18.6
24.3 13.0
75.7** t3.0
ify temporal characteristics of the drinking rhythm, analysis of spectral density was employed. This involved the Fast Fourier transform of the autocorrelation function of time series 2, which allows visualization of the main components of the rhythm (see Fig. 2). A simple analysis of variance for repeated measures was also performed, with further comparisons using a paired t-test. After SCN lesions 20 of the rats recovered their drinking rhythm spontaneously. Fourteen of them recovered it after the first week, 4 after the second and two after the third. Histological analysis of these rats indicated that the lesions missed variable amounts of SCN. In the 10 remaining rats which did not recover their rhythm after 5 weeks, it was assumed that complete destruction of the SCN was accomplished. This assumption was borne out by the fact that 4 of the transplanted rats never recovered their rhythm even 8 weeks after the transplant. The histology of these rats showed necrosed grafts with an SCN which remained totally destroyed. The behavior of the 6 remaining grafted rats is illustrated in Fig. 1. During the pre-lesion control each rat had a distinct diurnal drinking rhythm. Following complete SCN destruction they were significantly disrupted up to the 5th week in which they were tested. After the transplant, animals slowly recovered their diurnal rhythm (see Table I), until by the 8th week it was very similar to their pre-lesion control. Analysis of variance with repeated measures showed a difference between treatments with an F4.5 = 9.38 P < 0.016. This latter result prompted a t-test to compare significance of treatments. This analysis yielded a non-significant difference between controls and transplant (t = 2.33, P < 0.10). while both controls and transplant (8th week) were significantly different
from SCN lesions (t = 10,10, P < 0.001 and t = 3.30, P < 0.02, respectively). The power spectral analysis of drinking (Fig. 1) demonstrates a clear 24 h rhythm since the largest peaks coincide at around 0.042 cycles/h (1/24 h frequency). The SCN lesions eliminate the coincidence of this peak at such time and shows the appearance of several ultradian rhythms. Eight weeks after the transplant there is a return to the 24 h rhythm. The fact that the peaks are not as high reflects the fact that recovery is not absolute. All transplanted rats were examined histologically •at the end of the experiments. The lesions completely destroyed the SCN, from the caudal border of the supraoptic nucleus (5780) 11 to caudal third of the anterior commissure (6860) 11. The necrotic area was localized in the midline extending about 1.4 mm on the lateral plane, and from the ventral third of the III ventricle to the dorsal border of the optic chiasm. Other areas damaged by the lesion include: the ventromedial area of the anterior hypothalamic nucleus, the periventricular preoptic nucleus, the preoptic nucleus (pars suprachiasmatica) and the tractus infundibularis. In the 4 rats with no drinking rhythm recovery, it was evident that the graft had deteriorated. The tissue was necrotic, macrophages were abundant and there was a substantial dilatation of the 3rd ventricle. In these animals the SCN destruction was still clearly evident. On the other hand in the recovered rats the transplant appeared healthy, and although there was some distortion of the 3rd ventricle (Fig. 2), this did not seem to affect their behavior which was similar to normals. Although histological examination of stained sections indicates that healthy normal neurons are present in the transplanted tissue (Fig. 2), connectivity is not proven. Such connectivity is hinted in Fig. 2 where the transplanted tissue though
356
Fig. 2. A shows representative coronal sections of area of transplant at 3 different levels according to K6nig and KlippeP 1. Arrows indicate area where the graft was located. B and C are different magnifications of A, while D is from a different rat. A x 25. B x 100. C × 400, D × 250. CO, optic chiasm; III, third ventricle; FMP, fascicutus medialis prosencephali; ha. anterior hypothalamus. (Publisher's reduction factor 0.9.) clearly within t h e 3rd v e n t r i c l e a p p e a r s to h a v e established bridges w i t h t h e n e i g h b o r i n g p a r e n c h y m a ,
T h e results o f this study s h o w that the c i r c a d i a n r h y t h m of rats which is d i s r u p t e d by S C N lesions can
357 be reconstituted by transplanting homologous fetal neuronal tissue. The results strongly suggest that the recuperation of the drinking rhythm is p r o d u c e d as a result of the transplanted tissue, since those animals in which the graft necrosed, the rhythm loss persisted even after 13 weeks. Since the diurnal rhythm organization of drinking was relatively slow in recovering, it is conceivable that its reorganization d e p e n d e d on a certain degree of neural reconnection, which took weeks to develop. Some support for this notion comes from experiments showing that in animals whose brain is devoid of all connections with the SCN (hypothalamic 'island') rhythmic multiple unit activity persists only in the SCN 9. In this respect the SCN transplant is similar to the optic lobe transplant in cockroach 16, but different from those of other organisms such as the silk moth TM, the fruit flyl0 and the sparrow20, whose transplanted tissue appears to control rhythmicity by release of a h o r m o n e without need of any neural connections.
1 Arendash, G. and Gorski, R., Enhancement of sexual behavior in female rats by neonatal transplantation of brain tissue from males, Science, 217 (1982) 1276-1278. 2 Bendat, J. S. and Piersol, A. G., Measurement and Analysis of Random Noise, Wiley, New York, 1966. 3 Bj6rklund, A. and Stenevi, U., Regeneration of monoaminergic and cholinergic neurons in mammalian central nervous system, Physiol. Rev., 59 (1979) 62-100. 4 Bj6rklund, A., Dunnett, S. B., Stenevi, U., Lewis, M. E. and Iversen, S. D., Reinnervation of the denervated striatum by substantia nigra transplants: functional consequences as revealed by pharmacological and sensorimotor testing, Brain Research, 199 (1980) 307-333. 5 Das, G. D., Transplantation of embryonic neural tissue in the mammalian brain. I. Growth and differentiation of neuroblasts from various regions of the embryonic brain in the cerebellum of neonate rats, Life Sci., 4 (1974) 93-124. 6 Dunnett, S. B,, Gage, F. H., Bj6rklund, A., Stenevi, U., Low, W. C. and Iversen, S. D., Hippocampal deafferentation: transplant derived reinnervation and functional recovery, Scand. J. Physiol., Suppl. 1 (1982) 104-111. 7 Gash. D., Sladek, J. R. and Sladek, C., Functional development of grafted vasopressin neurons, Sc&nce, 210 (1980) 1367-1369. 8 Gibson, M. J., Krieger, D. T., Perlow, M, J., Davies, T. F., Zimmerman, E. A., Ferin, M. and Charlton, N. M., Hypothalamic brain transplants reverse hypogonadism in male mutant mice with gonadotropin-releasing hormone deficiency, Trans. Ass. Amer. Phys., 95 (1982) 188-195. 9 Inouye, S. T. and Kawamura, H., Persistence of circadian rhythmicity in a mammalian hypothalamic 'island' containing the suprachiasmatic nucleus, Proc. nat. Acad. Sci. U.S.A., 76 (1979) 5962-5966. 10 Handler, A. M. and Komopka, R. J., Transplantation of a circadian pacemaker in Drosophilia, Nature (Lond.), 279
A l t h o u g h from our experiments the possibility of neurosecretory influences cannot be ruled out. It should be p o i n t e d out that both the lesion or the graft did not a p p e a r to p r o d u c e any i m p a i r m e n t of homeostatic mechanisms. Thus, although we did not measure the actual amount of water intake, the number of water spout contacts r e m a i n e d the same throughout a given 24 h day, only the distribution was modified. These experiments give further support to the notion that the SCN is a p a c e m a k e r for certain rhythms in rats 9, and in addition gives new evidence that transplanted tissue of fetal origin is capable of restoring functional losses of behavior in lesioned adult rats. This study was partially s u p p o r t e d by a G r a n t from the Fundaci6n Ricardo Z e v a d a to Ren6 D r u c k e r - C o l/n. W e wish to thank Dr. Jorge A r a u z for valuable collaboration during the initial aspects of this work.
(1979) 236-238. 11 K/~nig,J. F. and Klippel, R. A., The Rat Brain: A Stereotaxic Atlas of the Forebrain and Lower Parts and the Brain Stem. Williams and Wilkins, Baltimore, MD, 1963.
12 Kromer, L. F., Bj6rklund, A. and Stenevi, U., Intracephalic embryonic neural implants in the adult rat brain. I. Growth and mature organization of brainstem cerebellar and hippocampal implants, J. comp. Neurol., 218 (1983) 433-459. 13 Labbe, R., Firl, A., Mufson, E. J. and Stein, D. G., Fetal brain transplants: reduction of cognitive deficits in rats with frontal cortex lesions, Science, 221 (1983) 470-472. 14 Low, W. C., Lewis, P. R., Bunch, S., Dunnett, S. B., Thomas S. R., Iversen, S. D., Bj~rklund, A. and Stenevi, U., Neural transplants of embryonic septal nuclei into adult rats with septohippocampal lesions: the recovery of function, Nature (Lond.), 300 (1982) 260-262. 15 Lund, R. D. and Hauschka, S. D., Transplanted neural tissue develops connections with host brain, Science, 193 (1976) 582-584. 16 Page, T. L., Transplantation of the cockroach circadian pacemaker, Science, 216 (1982) 73-75. 17 Perlow, M. J., Freed, W. J., Hoffer, B. J., Seiger, A., OIson, L. and Wyatt, R. J., Brain grafts reduce motor abnormalities produced by destruction of the nigrostriatal dopamine system, Science, 204 (1979) 643-647. 18 Truman, J. W. and Riddiford, L. M., Neuroendocrine control of ecdysis in silkmoth, Science, 167 (1970) 1624-1626. 19 Stenevi, U., Bjorklund, A. and Svendgaard, N., Transplantation of central and peripheral monoamine neurons to the adult rat brain: techniques and conditions for survival, Brain Research, 114 (1976) 1-20. 20 Zimmerman, N. H. and Menaker, M., The pineal gland. A pacemaker within the circadian system of house sparrow, Proc. nat. Acad. Sci. U.S.A., 76 (1979) 999-1003.