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Met-enkephalin concentrations in individual brain nuclei of ansa lenticularis and stria terminalis transected rats
Brain Research, 216 (1981) 203-209 © Elsevier/North-Holland Biomedical Press
203
Met-enkephalin concentrations in individual brain nuclei of ansa lenticularis and stria terminalis transected rats
M I K L 6 S PALKOVITS*, JACQUES EPELBAUM and C. GROS
1st Department of Anatomy, Semmelweis University Medical School, Budapest, Ttizoltd u. 58, H-1450 (Hungary), (.I.E.) U. 159 de Neuroendocrinologie de I'INSERM, 2 rue d'Alesia, Paris and ( C.G.) Unite de Radioimmunologie Analytique, Institut Pasteur, 25, rue de Docteur Roux, 75015 Paris (France) (Accepted February 26th, 1981)
Key words: Met-enkephalin - - radioimmunoassay - - hypothalamus - - limbic system - - ansa lenticularis - - stria terminalis
Recent radioimmuno3-5, 7 and immunocytochemica16,14-t6,1s, t9 studies have verified the presence of methionine (Met) and leucine (Leu) enkephalins in all major areas of the CNS. Their distribution is, however, uneven with a peak concentration in the globus pallidus. By means of immunocytochemistry Met-enkephalin containing perikarya were demonstrated in various CNS areas6,1z,14,t6,is. In the present study two well-defined, anatomically identified pathways were surgically interrupted and postoperation Met-enkephalin like immunoreactivity was measured by radioimmunoassay in the globus pallidus, bed nucleus of the stria terminalis (NIST) and some hypothalamic, amygdaloid and brain stem nuclei. CFY-strain, 200 -4- 5 g body weight male rats kept under standard conditions (24 4- 1 °C, ~70 ~o humidity, 12-12 h light-dark periods, standard rat-chow pellets, tap water ad libitum) were killed by decapatation. The brains were frozen on dry-ice, cut in a cryostat on --10 °C (section thickness 300 #m). Selected brain areas were removed by the 'punch-technique 't0. Tissue pellets were homogenized in 120 #10.1 N perchloric acid (PCA). Protein was determined in 5 and 10 #1 of the homogenate according to Lowry et al. 9. Met-enkephalin levels were determined by RIA as described by Gros et alA, using competition towards binding of 10 pM [t25I]Met-enkephalin to antibodies raised in rabbits against Met-enkephalin coupled to ovalbumin with carbodiimide. The sensitivity (lowest detection limit 0.1 nM and IC50 0.57 nM) and specificity (5 ~o of cross-reactivity with Leu-enkephalin and less than 0.1 ~o with peptide fragment from enkephalin molecule or with a- and fl-endorphin) of the assay allowed accurate estimation of the Met-enkephalin content in the extracts. Concentrations were expressed in ng/mg protein. * To whom correspondence should be addressed at: Lab. Clin. Sci. NIMH, NIH, Bd. 10, Rm. 2047, Bethesda, Md. 20205, U.S.A.
204
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< Fig. 1. Coronal sections of rat diencephalon with knife cuts. A: stria terminals transection. Drawings after K~Smgand Klippel s. B: ansa lenticularis is transected at the ventromedial border of the globus pallidus. Distances rostral to the mterauricular line are given in pm. Abbrevations: A, amygdala; Ah, anterior hypothalamlc nucleus; B, bed nucleus of the stria terminalis; C, corpus cailosum; CI, internal capsule; CP, caudate-putamen; F, fibria hippocampi; G, globus pallidus; Po, medial preoptic nucleus; S, stria terminalis.
205 TABLE I Concentrations of Met-enkephalin (mean 4- S.E.M.) in certain forebrain nuclei following surgical transection of the stria terminalis
Number of animals m parentheses. Sham-operated
Stria terminalis transection Ipsilateral
Bed nucleus of the stria terminalis Central amygdaloid nucleus Suprachiasmatic nucleus Ventromedial nucleus
2.37 4- 0.27 1.53 4- 0.26 1.28 4- 0.20 1.52 4- 0.31
(7) (7) (7) (7)
1.70 4- 0.17 0.73 ± 0.22 0.45 4- 0.24 1.06 4- 0.13
Contralateral
(6) (7)* (7)* (7)
2.59 4- 0.54 0.62 4- 0.11 0.75 4- 0.21 1.29 4- 0.19
(7) (7)* (7) (7)
* P < 0.05. Transections o f the stria terminalis. Unilateral operation was carried out with a 1.5 m m wide glass knife introduced vertically at the level o f the bregma, 1.3 m m lateral to the midline. The cut was directed t h r o u g h the frontal cortex, corpus callosum and lateral ventricle. The stria terminalis was interrupted at its entering to the diencephalon, dorsal to the N I S T (Fig. 1A). Transections o f the ansa lenticularis. Unilateral operations were carried out by introducing a glass knife 0.5-2.0 m m behind the bregma on the side opposite to the planned transection. The ansa lenticularis was then transected below and behind the globus pallidus (Fig. 1B). The 'ansa lenticularis' transection is so called, because this cut transects only one o f the major fibers inputs to the globus pallidus, namely the ansa lenticularis. The ipsilateral striopallidal connections remained intact. Detailed description o f the knife cut and the dimensions o f the d a m a g e d areas have been published elsewhere 11.
One g r o u p o f the operated animals were decapitated 8 days after operations and brain samples (see Tables I and II) were taken as detailed above. The other group o f TABLE II Concentrations of Met-enkephalin (mean 4- S.E.M.) in certain brain areas following surgical transection of the ansa lenticularis
Number of animals in parentheses. Sham-operated
Ansa lenticularts transections Ipsilateral
Caudate-putamen Globus pallidus Ventromedial nucleus Median eminence Substantla nigra Midbrain central gray * P < 0.05.
0.75 4- 0.16 (7) 13.95 4- 4.08 (7) 1.52 4- 0.31 (7) 0.91 4- 0.08 (7) 0.34 4- 0.10 (7) 1.33 4- 0.16 (7)
Contralateral
1.20 -4- 0.31 (10) 0.50 4- 0.06 5.30 4- 1.62 (10)* 16.38 4- 6.01 2.59 4- 0.48 (9) 1.58 4- 0.22 1.07 4- 0.18 (10) 0.15 4- 0.08 (10) 0.22 4- 0.08 1.33 4- 0.65 (10)
(9) (10) (10) (10)
206 animals were sacrificed by the perfuslon of Karnovsky's fixative to control the ultrastructure of the globus patlidus on the second postoperatwe day. Blocks of the globus pallidus were embedded in Durcupan (Fluka), stained with uranyl acet,~te and lead citrate and viewed under a JEOL 100B electron microscope. Far the highest concentration was found m the globus pallidus (Table II). Relatively high concentrations were measured in the hypothalamlc nuclei except for the supraoptic where no detectable amount of this peptide occurred. In the amygdala the central nucleus contained the highest Met-enkephalin concentration. From the lower brain stem only a few samples were taken. In the midbrain central gray matter (1.33 0.16: mean + S.E.M.), dorsal raphe nucleus (0.97 ~: 0.23) and locus coeruleus (0.91 q- 0.70) moderate, in the substantia nigra low Met-enkephalin concentrations (0.34 ~_ 0.10) were measured. In the nucleus of the sohtary tract the second highest CNS Metenkephalin level (4.54 ~ 0.77) was found. With immunocytochemlstry Met-enkephalin containing perikarya and nerve terminal have been shown m this nucleus6,15,16.
Transection of the stria terminalis. All 4 territories investigated showed an ipsilateral decrease m Met-enkephahn concentration. However, a significant fall of Metenkephalin levels occurred, only m the central amygdaloid (57 ~o) and suprachiasmatic (65 ~ ) nuclei. In the case of the former there was also a contralateral decrease comparable to the ipsilateral decrease (Table I). Both the NIST and the amygdala contain significant amounts of Met-enkephalin. Both regions were shown to contain Met-enkephalin perikarya e,17,19. No significant change has been found m the Met-enkephalin immunofluorescence of the central amygdalold nucleus after the transection of the stria termlnalis 17. There was, however, a substantml loss of Met-enkephahn m the NIST after strm termmahs transection or a lesion m the central amygdaloid nucleus 17. This indicated the existence of Met-enkephalin amygdalofugal fibers m the stria terminalis. In the present work only a 28 o//odecrease m the ipsilateral NIST was measured 8 days after unilateral transection of the stria termlnalis. In contrast to the report of Uhl et al. 17, we have found a substantial bilateral decrease in the central amygdaloid nucleus following the transection of the stria terminalis (Table 1). There was no difference between Metenkephahn concentrations of the two sides. The strm termmahs is known to contain a significant number of commissural fibers crossing in the anterior commissure 2. These were interrupted by the cut. The consequent decrease of Met-enkephalin suggests two alternatives: (1) in the stria termmahs amygdalopetal (afferent) Met-enkephahn axons are also present; (2) fibers of the stria terminalis convey input necessary for peptidesynthes~s m the Met-enkephahn cells of the central amygdalo~d nucleus. Following stria terminalis transection there was a substantml decrease of concentration in the suprachiasmatic nucleus, a moderate decrease occurred in the ventromedml nucleus. The changes were in both cases ipsilateral (Table I). The hypothalamic termination of the stria termmalis is known, and caudal to the NIST it is approvedly ipsilateral 2. Our results suggest that some of these fibers contain Met-enkephalin. Here too, the lack of amygdaloid neural input as an alternative explanation for the loss of Met-enkephalin must be reckoned with.
207 Transections o f the ansa lenticularis. The survival was 70 ~o. Necrotic territories were found exclusively in the close vicinity of the cut. In the globus pallidus whose caudal end was 0.2-0.5 mm dorsolaterally to the knife cut, several degenerated nerve terminals were seen under the electron microscope. Perikarya and larger dendrites appeared to be intact without any sign of retrograde degeneration. Eight days after the transection a significant (60 ~) decrease in Met-enkephahn concentration was encountered in the ipsilateral globus pallidus while practically no change was found contralaterally (Table II). In the substantia nigra Met-enkephalin level decreased by 58 ~o but due to originally very low values the biological significance of this finding is questionable. No change took place in the median eminence and midbrain central gray. A non-significant rise occurred in the caudate-putamen and hypothalamic ventromedial nucleus (Table II). In agreement with earlier radioimmuno studies3,5,7 very high Met-enkephalin levels were found in the globus pallidus exceeding with an order of magnitude the mean concentration of this peptide in the brain. This biochemical finding is consistent with immunocytochemical observations demonstrating a rich Met-enkephalin neuronal network in the globus pallidus1,13-15,18,19. However, Met-enkephalin containing perikarya were not seen there. This led to the assumption that Met-enkephalin fibers terminating in the globus pallidus originate from the neighbourmg caudate nucleus where Met-enkephalin perikarya are situated1, la. After surgical isolation of the globus pallidus from the caudate nucleus (as well as from other brain areas rostral, dorsal and lateral to the globus pallidus) a disappearance of Met-enkephalin-immunoreactive fibers from the globus pallidus was demonstrated by Cuello and Paxinos 1. Our findings suggest the contribution to the Met-enkephalin innervation of globus pallidus other territories in addition to the caudate-putamen. The postoperative decrease of Met-enkephalin level in the globus pallidus may be explained in 3 ways: (1) ascending Met-enkephalin fibers terminating in the globus pallidus were cut; (2) fibers were cut which affect pallidal Met-enkephalin synthesis; (3) postoperative ischaemia or retrograde degeneration affects Met-enkephalin containing pallidal element. (1) Fibers ascending to the globus pallidus may pass through the ansa lenticularis. Among the cell groups having connections w~th the ansa lenticularis the ventromedial nucleus and the substantia nigra but not the entopeduncular nucleus were found to contain Met-enkephalin perikarya6,18,~9. After the transection of the ansa lenticularis in the ipsilateral ventromedial nucleus an elevated Met-enkephalin level was detected (Table II). This can be interpreted as a perikaryonal accumulation due to the efferent fiber transection. (An alternative explanation is the impairment of inhibitory input to the ventromedial nucleus through the ansa lenticularis.) The existence of a neural link between the ventromedial nucleus and pallidum has been known for long (cf. 12). A part of these connections may be Met-enkephalin fibers. Besides the ansa lenticularls other fibers may also enter the globus pallidus from central and caudal which were cut together with the ansa lenticularis. Closely ventral to the globus pallidus (in the medial forebrain bundle) and caudal to it (mainly in the medial hypothalamus) several Met-enkephalin perikarya were described6, is. These areas may also supply Met-enkephalin axons or axon-collaterals to the globus pal-
208 lidus. Wamsley et ala s were able to trace Met-enkephalin immunopositive fibers from the central amygdaloid nucleus to the globus pallidus. These fibers also were cut together with the ansa lenticularis. (2) This alternative cannot be fully excluded. However, as enkephalin-synthetizing penkarya were not yet detected in the globus pallidus it seems that not the synthesis-inducing stimuli are missing. These axons may regulate enkephalin synthesis at the locus of cell bodies in the caudate-putamen, or alternatively may effect enkephahn release from caudate-putamen enkephalinergic neurons which project to the globus pallidus. (3) Nor can the third alternative be ruled out completely. Necrosis was seen only in the cut reaching the globus palhdus. In fact, the surgical cut closer to the globus pallidus resulted in a more effective decrease in the Met-enkephalin level of the globus pallidus than cuts more caudal to this nucleus. This can be explained also with a more complete denervation rather than with a direct damage to the nucleus. (It ~s noteworthy that the pallidus has a common blood supply with the caudate-putamen where no significant rise in Met-enkephalin was found after the transection of the ansa lentlculans.) Following ansa lenticularis transection an ipsilateral rise was observed also m the caudate-putamen. A substantial part of the ansa lenticularis is known to originate from the caudate-putamen (cf. ref. 1 1). It cannot be ruled out that following the transection the perikaryonal accumulation of Met-enkephalin would be responsible for the rise. This would imply that in the ansa lentlcularis Met-enkephalin fibers from the caudate-putamen are contained which do not terminate in the pallidum but run more caudally. This may be the reason of the decrease of Met-enkephalin in the substantia nigra after ansa lenticularis transection. However, changes in the low Metenkephalin levels of the substantia nigra do not permit far-reaching conclusions. Several biochemical pathways were verified by biochemical measurements following surgical interferences. However, owing to the nature of this approach results can be interpreted in alternative ways. Decision between them requires combined biochemical and immunocytochemical studies.
1 Cuello, A. C. and Paxinos, G., Evidence for a long leu-enkephalin stnopallidal pathway m rat brain, Nature (Lond.), 271 (1978) 178-180. 2 De Olmos, J S. and Ingram, W. R., The projection of the stria terminahs in the rat brain. An experimental study, J. comp. Neurol., 146 (1972) 303-334. 3 Dupont, A., Barden, N., Cusan, L., Merand, Y., Labne, F. and Vaudry, H, fl-endorphin and met-enkephalins: their distribution, modulation by estrogens and haloperidol, and role in neuroendocrine control, Fed. Proc., 39 (1980) 2544-2550. 4 Gros, C., Pradelles, P., Rouget, C., Bepoldin, O. and Dray, F., Radiolmmunoassay of meth~onmeand leucine-enkephalins in regions of rat brain and comparison with endorphins estimated by a radioreceptor assay, J. Neurochem., 31 (1978) 29-39. 5 Hong, J. S., Yang, H.-Y., Fratta, W. and Costa, E., Determination of methionine enkephahn m discrete regions of rat bram, Brain Research, 134 (1977) 383-386. 6 Johansson, O., H6kfelt, T., Elde, R. P., Schultzberg, M. and Teremus, L., Immunocytochem~cal distribution of enkephalin neurons, Advanc. Biochem. Psychopharmacol., 18 (1978) 51-70. 7 Kobayashi, R. M., Palkovits, M., Miller, R. J., Chang, K.-J. and Cuatrecasas, P., Brain enkephahn distribution is unaltered by hypopbysectomy,Life Sci., 22 (1978) 527-530.
209 8 KOnig, J. F. R. and Klippel, R. A., The Rat Brain: A Stereotaxic Atlas of the Forebrain and Lower Parts of the Brain Stem, Williams and Wilkins, Baltimore, 1963. 9 Lowry, O. H., Rosebrough, N. Y., Farr, A. L. and Randall, R. J., Protein measurement with the Foline phenol reagent, J. biol. Chem., 193 (1951) 265-275. 10 Palkovits, M., Isolated removal of hypothalamic or other brain nuclei of the rat, Brain Research, 59 (1973) 449--450. 11 Palkovlts, M., Mroz, E. A., Brownstein, M. J. and Leeman, S. E., Descending substance P-containing pathway: a component of the ansa lenticularis, Brain Research, 156 (1978) 124-128. 12 Palkovits, M. and Z/tborszky, L., Neural connections of the hypothalamus, In P. J. Morgane and J. Panksepp (Eds.), Handbook of the Hypothalamus, 1Iol. 1., Marcel Dekker, New York, 1979, pp. 379-509. 13 Plckel, V. M., Sumal, K. K., Beckley, S. C., Miller, R. J. and Reis, D. J., Immunocytochemical localization of enkephalin in the neostriatum of rat brain" a light and electron microscopic study, J. comp. Neurol., 189 (1980) 721-740. 14 Sar, M., Strumpf, W. E., Miller, R. J., Chang, K.-J. and Cuatrecasas, P., Immunohistochemical localization of enkephalin in rat brain and spinal cord, J. comp. Neurol., 182 (1978) 17-38. 15 Simantov, R., Kuhar, M. J., Pasternak, G. W. and Snyder, S. H., The regional distribution of a morphine-like factor enkephalin in monkey brain, Brain Research, 106 (1976) 189-197. 16 Uhl, G. R., Goodman, R. R., Kuhar, M. J., Childers, S. R. and Snyder, S. H., lmmunohistochemical mapping of enkephalin-containing cell bodies, fibers and nerve terminals in the brain stem of the rat, Brain Research, 166 (1979) 75-94. 17 Uhl, G. R., Kuhar, M. J. and Snyder, S. H., Enkephalln-containing pathways: amygdaloid efferents in the stria termlnahs, Brain Research, 149 (1978) 223-228. 18 Wamsley, J. K., Young, W. S. III. and Kuhar, M. J., Immunohistochemical localization of enkephalin in rat forebrain, Brain Research, 190 (1980) 153-174. 19 Watson, S., Akil, H., Sullivan, S. and Barchas, J., Immunocytochemical localization ol methionine enkephalin: preliminary observations, Life Sci., 21 (1977) 733-738.
203
Met-enkephalin concentrations in individual brain nuclei of ansa lenticularis and stria terminalis transected rats
M I K L 6 S PALKOVITS*, JACQUES EPELBAUM and C. GROS
1st Department of Anatomy, Semmelweis University Medical School, Budapest, Ttizoltd u. 58, H-1450 (Hungary), (.I.E.) U. 159 de Neuroendocrinologie de I'INSERM, 2 rue d'Alesia, Paris and ( C.G.) Unite de Radioimmunologie Analytique, Institut Pasteur, 25, rue de Docteur Roux, 75015 Paris (France) (Accepted February 26th, 1981)
Key words: Met-enkephalin - - radioimmunoassay - - hypothalamus - - limbic system - - ansa lenticularis - - stria terminalis
Recent radioimmuno3-5, 7 and immunocytochemica16,14-t6,1s, t9 studies have verified the presence of methionine (Met) and leucine (Leu) enkephalins in all major areas of the CNS. Their distribution is, however, uneven with a peak concentration in the globus pallidus. By means of immunocytochemistry Met-enkephalin containing perikarya were demonstrated in various CNS areas6,1z,14,t6,is. In the present study two well-defined, anatomically identified pathways were surgically interrupted and postoperation Met-enkephalin like immunoreactivity was measured by radioimmunoassay in the globus pallidus, bed nucleus of the stria terminalis (NIST) and some hypothalamic, amygdaloid and brain stem nuclei. CFY-strain, 200 -4- 5 g body weight male rats kept under standard conditions (24 4- 1 °C, ~70 ~o humidity, 12-12 h light-dark periods, standard rat-chow pellets, tap water ad libitum) were killed by decapatation. The brains were frozen on dry-ice, cut in a cryostat on --10 °C (section thickness 300 #m). Selected brain areas were removed by the 'punch-technique 't0. Tissue pellets were homogenized in 120 #10.1 N perchloric acid (PCA). Protein was determined in 5 and 10 #1 of the homogenate according to Lowry et al. 9. Met-enkephalin levels were determined by RIA as described by Gros et alA, using competition towards binding of 10 pM [t25I]Met-enkephalin to antibodies raised in rabbits against Met-enkephalin coupled to ovalbumin with carbodiimide. The sensitivity (lowest detection limit 0.1 nM and IC50 0.57 nM) and specificity (5 ~o of cross-reactivity with Leu-enkephalin and less than 0.1 ~o with peptide fragment from enkephalin molecule or with a- and fl-endorphin) of the assay allowed accurate estimation of the Met-enkephalin content in the extracts. Concentrations were expressed in ng/mg protein. * To whom correspondence should be addressed at: Lab. Clin. Sci. NIMH, NIH, Bd. 10, Rm. 2047, Bethesda, Md. 20205, U.S.A.
204
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< Fig. 1. Coronal sections of rat diencephalon with knife cuts. A: stria terminals transection. Drawings after K~Smgand Klippel s. B: ansa lenticularis is transected at the ventromedial border of the globus pallidus. Distances rostral to the mterauricular line are given in pm. Abbrevations: A, amygdala; Ah, anterior hypothalamlc nucleus; B, bed nucleus of the stria terminalis; C, corpus cailosum; CI, internal capsule; CP, caudate-putamen; F, fibria hippocampi; G, globus pallidus; Po, medial preoptic nucleus; S, stria terminalis.
205 TABLE I Concentrations of Met-enkephalin (mean 4- S.E.M.) in certain forebrain nuclei following surgical transection of the stria terminalis
Number of animals m parentheses. Sham-operated
Stria terminalis transection Ipsilateral
Bed nucleus of the stria terminalis Central amygdaloid nucleus Suprachiasmatic nucleus Ventromedial nucleus
2.37 4- 0.27 1.53 4- 0.26 1.28 4- 0.20 1.52 4- 0.31
(7) (7) (7) (7)
1.70 4- 0.17 0.73 ± 0.22 0.45 4- 0.24 1.06 4- 0.13
Contralateral
(6) (7)* (7)* (7)
2.59 4- 0.54 0.62 4- 0.11 0.75 4- 0.21 1.29 4- 0.19
(7) (7)* (7) (7)
* P < 0.05. Transections o f the stria terminalis. Unilateral operation was carried out with a 1.5 m m wide glass knife introduced vertically at the level o f the bregma, 1.3 m m lateral to the midline. The cut was directed t h r o u g h the frontal cortex, corpus callosum and lateral ventricle. The stria terminalis was interrupted at its entering to the diencephalon, dorsal to the N I S T (Fig. 1A). Transections o f the ansa lenticularis. Unilateral operations were carried out by introducing a glass knife 0.5-2.0 m m behind the bregma on the side opposite to the planned transection. The ansa lenticularis was then transected below and behind the globus pallidus (Fig. 1B). The 'ansa lenticularis' transection is so called, because this cut transects only one o f the major fibers inputs to the globus pallidus, namely the ansa lenticularis. The ipsilateral striopallidal connections remained intact. Detailed description o f the knife cut and the dimensions o f the d a m a g e d areas have been published elsewhere 11.
One g r o u p o f the operated animals were decapitated 8 days after operations and brain samples (see Tables I and II) were taken as detailed above. The other group o f TABLE II Concentrations of Met-enkephalin (mean 4- S.E.M.) in certain brain areas following surgical transection of the ansa lenticularis
Number of animals in parentheses. Sham-operated
Ansa lenticularts transections Ipsilateral
Caudate-putamen Globus pallidus Ventromedial nucleus Median eminence Substantla nigra Midbrain central gray * P < 0.05.
0.75 4- 0.16 (7) 13.95 4- 4.08 (7) 1.52 4- 0.31 (7) 0.91 4- 0.08 (7) 0.34 4- 0.10 (7) 1.33 4- 0.16 (7)
Contralateral
1.20 -4- 0.31 (10) 0.50 4- 0.06 5.30 4- 1.62 (10)* 16.38 4- 6.01 2.59 4- 0.48 (9) 1.58 4- 0.22 1.07 4- 0.18 (10) 0.15 4- 0.08 (10) 0.22 4- 0.08 1.33 4- 0.65 (10)
(9) (10) (10) (10)
206 animals were sacrificed by the perfuslon of Karnovsky's fixative to control the ultrastructure of the globus patlidus on the second postoperatwe day. Blocks of the globus pallidus were embedded in Durcupan (Fluka), stained with uranyl acet,~te and lead citrate and viewed under a JEOL 100B electron microscope. Far the highest concentration was found m the globus pallidus (Table II). Relatively high concentrations were measured in the hypothalamlc nuclei except for the supraoptic where no detectable amount of this peptide occurred. In the amygdala the central nucleus contained the highest Met-enkephalin concentration. From the lower brain stem only a few samples were taken. In the midbrain central gray matter (1.33 0.16: mean + S.E.M.), dorsal raphe nucleus (0.97 ~: 0.23) and locus coeruleus (0.91 q- 0.70) moderate, in the substantia nigra low Met-enkephalin concentrations (0.34 ~_ 0.10) were measured. In the nucleus of the sohtary tract the second highest CNS Metenkephalin level (4.54 ~ 0.77) was found. With immunocytochemlstry Met-enkephalin containing perikarya and nerve terminal have been shown m this nucleus6,15,16.
Transection of the stria terminalis. All 4 territories investigated showed an ipsilateral decrease m Met-enkephahn concentration. However, a significant fall of Metenkephalin levels occurred, only m the central amygdaloid (57 ~o) and suprachiasmatic (65 ~ ) nuclei. In the case of the former there was also a contralateral decrease comparable to the ipsilateral decrease (Table I). Both the NIST and the amygdala contain significant amounts of Met-enkephalin. Both regions were shown to contain Met-enkephalin perikarya e,17,19. No significant change has been found m the Met-enkephalin immunofluorescence of the central amygdalold nucleus after the transection of the stria termlnalis 17. There was, however, a substantml loss of Met-enkephahn m the NIST after strm termmahs transection or a lesion m the central amygdaloid nucleus 17. This indicated the existence of Met-enkephalin amygdalofugal fibers m the stria terminalis. In the present work only a 28 o//odecrease m the ipsilateral NIST was measured 8 days after unilateral transection of the stria termlnalis. In contrast to the report of Uhl et al. 17, we have found a substantial bilateral decrease in the central amygdaloid nucleus following the transection of the stria terminalis (Table 1). There was no difference between Metenkephahn concentrations of the two sides. The strm termmahs is known to contain a significant number of commissural fibers crossing in the anterior commissure 2. These were interrupted by the cut. The consequent decrease of Met-enkephalin suggests two alternatives: (1) in the stria termmahs amygdalopetal (afferent) Met-enkephahn axons are also present; (2) fibers of the stria terminalis convey input necessary for peptidesynthes~s m the Met-enkephahn cells of the central amygdalo~d nucleus. Following stria terminalis transection there was a substantml decrease of concentration in the suprachiasmatic nucleus, a moderate decrease occurred in the ventromedml nucleus. The changes were in both cases ipsilateral (Table I). The hypothalamic termination of the stria termmalis is known, and caudal to the NIST it is approvedly ipsilateral 2. Our results suggest that some of these fibers contain Met-enkephalin. Here too, the lack of amygdaloid neural input as an alternative explanation for the loss of Met-enkephalin must be reckoned with.
207 Transections o f the ansa lenticularis. The survival was 70 ~o. Necrotic territories were found exclusively in the close vicinity of the cut. In the globus pallidus whose caudal end was 0.2-0.5 mm dorsolaterally to the knife cut, several degenerated nerve terminals were seen under the electron microscope. Perikarya and larger dendrites appeared to be intact without any sign of retrograde degeneration. Eight days after the transection a significant (60 ~) decrease in Met-enkephahn concentration was encountered in the ipsilateral globus pallidus while practically no change was found contralaterally (Table II). In the substantia nigra Met-enkephalin level decreased by 58 ~o but due to originally very low values the biological significance of this finding is questionable. No change took place in the median eminence and midbrain central gray. A non-significant rise occurred in the caudate-putamen and hypothalamic ventromedial nucleus (Table II). In agreement with earlier radioimmuno studies3,5,7 very high Met-enkephalin levels were found in the globus pallidus exceeding with an order of magnitude the mean concentration of this peptide in the brain. This biochemical finding is consistent with immunocytochemical observations demonstrating a rich Met-enkephalin neuronal network in the globus pallidus1,13-15,18,19. However, Met-enkephalin containing perikarya were not seen there. This led to the assumption that Met-enkephalin fibers terminating in the globus pallidus originate from the neighbourmg caudate nucleus where Met-enkephalin perikarya are situated1, la. After surgical isolation of the globus pallidus from the caudate nucleus (as well as from other brain areas rostral, dorsal and lateral to the globus pallidus) a disappearance of Met-enkephalin-immunoreactive fibers from the globus pallidus was demonstrated by Cuello and Paxinos 1. Our findings suggest the contribution to the Met-enkephalin innervation of globus pallidus other territories in addition to the caudate-putamen. The postoperative decrease of Met-enkephalin level in the globus pallidus may be explained in 3 ways: (1) ascending Met-enkephalin fibers terminating in the globus pallidus were cut; (2) fibers were cut which affect pallidal Met-enkephalin synthesis; (3) postoperative ischaemia or retrograde degeneration affects Met-enkephalin containing pallidal element. (1) Fibers ascending to the globus pallidus may pass through the ansa lenticularis. Among the cell groups having connections w~th the ansa lenticularis the ventromedial nucleus and the substantia nigra but not the entopeduncular nucleus were found to contain Met-enkephalin perikarya6,18,~9. After the transection of the ansa lenticularis in the ipsilateral ventromedial nucleus an elevated Met-enkephalin level was detected (Table II). This can be interpreted as a perikaryonal accumulation due to the efferent fiber transection. (An alternative explanation is the impairment of inhibitory input to the ventromedial nucleus through the ansa lenticularis.) The existence of a neural link between the ventromedial nucleus and pallidum has been known for long (cf. 12). A part of these connections may be Met-enkephalin fibers. Besides the ansa lenticularls other fibers may also enter the globus pallidus from central and caudal which were cut together with the ansa lenticularis. Closely ventral to the globus pallidus (in the medial forebrain bundle) and caudal to it (mainly in the medial hypothalamus) several Met-enkephalin perikarya were described6, is. These areas may also supply Met-enkephalin axons or axon-collaterals to the globus pal-
208 lidus. Wamsley et ala s were able to trace Met-enkephalin immunopositive fibers from the central amygdaloid nucleus to the globus pallidus. These fibers also were cut together with the ansa lenticularis. (2) This alternative cannot be fully excluded. However, as enkephalin-synthetizing penkarya were not yet detected in the globus pallidus it seems that not the synthesis-inducing stimuli are missing. These axons may regulate enkephalin synthesis at the locus of cell bodies in the caudate-putamen, or alternatively may effect enkephahn release from caudate-putamen enkephalinergic neurons which project to the globus pallidus. (3) Nor can the third alternative be ruled out completely. Necrosis was seen only in the cut reaching the globus palhdus. In fact, the surgical cut closer to the globus pallidus resulted in a more effective decrease in the Met-enkephalin level of the globus pallidus than cuts more caudal to this nucleus. This can be explained also with a more complete denervation rather than with a direct damage to the nucleus. (It ~s noteworthy that the pallidus has a common blood supply with the caudate-putamen where no significant rise in Met-enkephalin was found after the transection of the ansa lentlculans.) Following ansa lenticularis transection an ipsilateral rise was observed also m the caudate-putamen. A substantial part of the ansa lenticularis is known to originate from the caudate-putamen (cf. ref. 1 1). It cannot be ruled out that following the transection the perikaryonal accumulation of Met-enkephalin would be responsible for the rise. This would imply that in the ansa lentlcularis Met-enkephalin fibers from the caudate-putamen are contained which do not terminate in the pallidum but run more caudally. This may be the reason of the decrease of Met-enkephalin in the substantia nigra after ansa lenticularis transection. However, changes in the low Metenkephalin levels of the substantia nigra do not permit far-reaching conclusions. Several biochemical pathways were verified by biochemical measurements following surgical interferences. However, owing to the nature of this approach results can be interpreted in alternative ways. Decision between them requires combined biochemical and immunocytochemical studies.
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