- Email: [email protected]
In vivo release of enkephalin from the globus pallidus
Neuroscience Letters, 24 ( 1981) 65-70
65
Elsevier/North-Holland Scientific Publishers Ltd.
IN VIVO R E L E A S E OF E N K E P H A L I N F R O M T H E G L O B U S P A L L I D U S
ALEJANDRO BAYON, WILLIAM J. SHOEMAKER*, LOURDES LUGO, RAANA AZAD*, NICHOLAS LING**, RENE R. DRUCKER-COL1Nand FLOYD E. BLOOM* Department o f Neuroscience, Centro de Investigaciones en Fisiologia Celular, Universidad Nacional Autonoma de Mexico, Apartado Postal 70-600, Mexico City 20 (Mexico)
and *,4. V. Davis Center f o r Behavioral Neurobiology and **The Laboratories f o r Neuroendocrinology, The Salk Institute, P.O. Box 85800, San Diego, CA 92138 (U.S.A.)
(Received November29th, 1980; Revised version received March 3rd, 1981;AcceptedMarch 16th, 1981)
Push-pull cannulae wereacutelypositioned through previouslyimplanted guidesin the globus pallidus of unanesthetized freely moving cats and rats. During slow-flow perfusions, enkephalin release was detected in resting conditions and increased more than 3-fold when both 50 mM K÷ and 1.8 mM Ca2÷ were present in the perfusing medium. Local perfusion with veratrine also enhanced enkephalin release. Furthermore, in vivo, electrical stimulation of the rat caudo-putamen enhanced enkephalin release in the pallidum. This latter finding is consistent with a functional strio-pallidal enkephalin-containing pathway previously postulated by immunohistochemical or lesion experiments.
The endogenous opiate peptides LeuS-enkephalin and MetS-enkephalin [9] are present in neuronal perikarya and nerve terminals in many regions of the central nervous system [8, 18]. In spite of the early observation of their chemical relationship to ~-lipotropin [9], the enkephalin-containing systems have been shown to exist separately from those containing the higher molecular weight endorphins [1, 4, 5, 17, 21]. More recently, the separation between enkephalins and other endorphins has been confirmed by demonstration of several possible enkephalin precursors unrelated to the lipotropin-derived endorphins [20]. The corpus striatum (c.s. = caudate, globus pallidus and putamen) possesses attributes of a model system to study the neurobiology of the enkephalins because it has one of the highest enkephalin concentrations in the brain [17, 23] and no chemically detectable endorphin [1, 5, 17, 21]. The globus pallidus is very rich in enkephalin fibers and terminals; cell bodies are found in the caudo-putamen [8, 18] and only a few of them are located on the pallidal border [3]. These advantages have been exploited only in part when studying the release (in vitro) of the enkephalins [2, 7, 10]. The use of an in vivo preparation to study the characteristics of the enkephalin release exploits the anatomical segregation of enkephalin-containing 03404-3940/81/0000-0000/$ 02.50 ©Elsevier/North-Holland Scientific Publishers Ltd.
66 terminals in the pallidum, and their preserved functional connection with other brain regions. In this communication we present initial data on the in vivo enkephalin release from the globus pallidus, its stimulation by locally applied depolarizing agents and the enhanced release produced by electrical stimulation of the head of the caudate-putamen nucleus. The push-pull cannula perfusion technique used in these experiments is based on that previously described by Myers [14]. Briefly, stainless steel cannulae that serve as stereotaxic guides for the push-pull cannulae were chronically implanted in the skull of adult cats (2-2.5 kg) and albino rats (150-200 g) under sodium pentobarbital anesthesia. The lower end of the guides was temporarily blocked with short mandrels. Coordinates for globus pallidus were: A 11.5, L 9.5, H 0.5 in the cat [19]; and A 6.4, L 2.5, H 0.2 in the rat [11]. Some rats were also implanted with bipolar stimulating electrodes in the caudate nucleus (A 8.4, L 2.5, H 0.2). One week after surgery the mandrels were removed without anesthesia and concentric push-pull cannulae were inserted through the guides to reach the pallidal tissue. The outer (pull) cannula was made of 21-gauge thin wall steel tubing; the inner (push) cannula is a 27-gauge needle protruding 1.5 mm from the tip of the pull cannula. Each perfusing cannula was connected through polyethylene tubing to the infusion and withdrawal syringes positioned in a Harvard reciprocating pump; animals were freely moving throughout subsequent perfusion sessions. The perfusion of the unanesthetized-freely moving animals was started using sterile Krebs-Ringer bicarbonate buffer (127 mM NAC1/3.73 mM KC1/1.8 mM CAC12/1.18 mM KH2PO4/1.18 mM MgSO4/20 mM NaHCO3/D-glucose, 2 g/liter), containing bovine serum albumin (0.1°70) and bacitracin (30 ~g/ml). The flow rate was held constant at 23 ~l/min (both in the 'push' and in the 'pull' systems) and 15 min fractions were collected in the withdrawal syringe, which contained ice-cold 2 N acetic acid and a stirring bar to mix the acid with the perfusate. The well-known artefact caused by syringe manipulation at the pump [22] was minimized by using a 3-way stopcock that allowed smooth transitions to another syringe when the first emptied. In addition, the pump was reset every 15 min, exactly corresponding to the collection periods. The regularity of the collection and syringe changing procedures would result in any artefactual changes in enkephalin level to be present in all collection periods and could not account for the experimental results observed. The collected fractions were boiled for 15 min, lyophilized and resuspended in buffer for the LeuS-enkephalin radioimmunoassay [16]. The positions of the tips of the cannulae were verified by inspection under a dissection microscope of frontal sections of the brain. The samples were subjected to radioimmunoassay at two dilutions in duplicate. The LeuS-enkephalin assay was as described [17]. The minimum detectable amount of LeuS-enkephalin is approximately 1 pg; Metenkephalin cross-reacts in this assay to the extent of 3°70 on a weight basis. The peptides used as standards were prepared by solid phase synthesis [12]. Following the procedures described above, a K÷-stimulated, Ca2*-dependent
67
release of enkephalin was obtained from the globus pallidus o f freely moving cats (Fig. 1). In animals perfused for the first time we observed a 200-300070 fold increase in enkephalin release over the preceding control periods. This K +stimulated release was only partially reduced (to 74070) over control values when Ca 2+ was omitted from the perfusing medium; subsequent addition of Ca 2+
30"
2
~V
It"'
PERFUSION (N-3)
2nd PERFUSION (N-3)
Z
+Co ~ -Co J +Co ~ -
+Co = -Co ~ +Co
Z
.._ detection ,imit.
'~
60
30
15
'~
~_
W ~,
~
Z
"7 3,0
.
i
.
.
.
3 rd PERFUSION (N=3) 15
[
ILl
60 i20 TIME (rnln)
180
S
I--I
m
:z)
! 60 120 TIME (rain)
180
0
60 TIME
] 120 (min)
Fig. 1. In vivo release of enkephalin from the globus pallidus of the cat. Sequential p u s h - p u l l cannula perfusions were performed at one week intervals after chronic implantation of the guide cannulae. In all perfusions the flow rate was held constant at 23 # i / m i n and 15 rain fractions were collected on ice-cold 2 N acetic acid (the volume of tissue perfused was estimated with a blue-dextran solution as approximately 2 mm3). Samples were boiled, lyophilized, redissolved in buffer and assayed for Leu 5enkephalin immunoreactivity (l enkephalin unit is equivalent to the trace displacement produced by 1 pg o f Leu-enkephalin in the Leu-enkephalin radioimmunoassay). White bars correspond to low K + -resting release periods; 50 m M K + medium was perfused during the periods indicated by the hatched bars. During the second hour of perfusion Mg 2+ was doubled in the medium and Ca 2+ was omitted. Vertical lines represent the standard error of the mean; N is the number of bilaterally perfused cats. Enkephalin values lower than the m i n i m u m detectable a m o u n t are indicated by a discontinuous line on top of the bars. Fig. 2. In vivo release of enkephalin from the globus pallidus of the rat. Perfusion conditions are as indicated in Fig. 1. V = low potassium medium, containing veratrine (60 #g/ml); S = electrical stimulation o f the c a u d o - p u t a m e n using a coiled bipolar electrode: Square pulses (0.5 msec duration, applied voltage 10 V; average current 100 #A) were delivered at 40, 60 and 80 Hz each during a 5 min period. At each frequency the trains of stimulation lasted 30 sec and were followed by 30 sec resting periods. Enkephalin values are the average o f released data obtained from 3 rats.
68 restored the K+-stimulated release to initial values. A second perfusing session carried out 7 days later produced lower amounts of enkephalin both during resting and stimulated release; in a third perfusion enkephalin values in the perfusate were below the detection limit of the assay. Since there is no decrease within the first perfusing session, lasting as long as 8 h and with several cannula penetrations, the reduced amounts of enkephalin released in second or third perfusion sessions appear to be related to the long-term effects of tissue damage. Other methodological variables also affected the enkephalin levels measured in the perfusates. Increasing the flow rate to 51 #l/rain usually leads to fluctuating baseline enkephalin values during the course of a perfusing session. A similar observation was reported for the in vivo release of substance P, although it was also noted that much higher flow rates yield more consistent results [13]. Addition of the aminopeptidase inhibitor bacitracin to our perfusing media was a requisite to obtain enkephalin levels in the collected fractions well within the measurable range of our assay. We have previously reported its protective action during the in vitro release of enkephalins [10]. A requirement for protective agents has also been reported for the measurements of released substance P [13]. Finally, when the tip of the push-pull cannula was aimed at the border between the internal capsule and the globus pallidus, in order to avoid damage of the pallidal tissue, the enkephalin collected in the perfusate was, in most cases, below the sensitivity of the radioimmunoassay. This observation indicates the anatomical specificity of the perfusion. The above results were next extended using rats as experimental subjects. Both K ÷ (50 mM) and veratrine were shown to stimulate the enkephalin release from the globus pallidus. Furthermore, in rats implanted with a bipolar electrode in the caudate nucleus, the electrical stimulation of this area elicited an increased enkephalin release in the globus pallidus (Fig. 2). We have used stimulation frequencies from 40 to 80 Hz. Since these frequencies have been shown to lead (in the cat) to the blockade of EPSPs evoked from the caudate nucleus in the pallidum due to the onset of slow IPSPs [15] - the trains of stimulation were alternated with resting periods every 30 sec. Work is in progress to study the frequency independence of this enkephalin release. When the electrodes were located in septal or preoptic areas no significant stimulation of pallidal enkephalin release was observed (data not shown). In vivo enkephalin release from the pallidum cannot be easily compared to that measured in vitro because of the wide differences between the preparations. However the enkephalin recovered in vivo is about one order of magnitude less than that in vitro [2, 10], this suggests a higher stability of enkephalin stored in the nonsliced pallidal tissue. These results show that not only can the local perfusion of pallidal tissue with chemical depolarizing agents elicit a Ca 2+-dependent release of enkephalin from this region (rich in enkephalin fibers), but also that electrical stimulation of a separate but connected area, the neighboring caudate nucleus, produces a quantitatively -
69
similar effect. This observations is consistent with the existence of the strio-pallidal enkephalin-containing pathway postulated by Cuello and Paxinos on the basis of lesion experiments [6]. Altogether, the results of these studies on the release of enkephalin from the brain region richest in these opioid peptides strongly support the concept that the enkephalins play a role in neural communication. This Research was partially supported by NIDA 01785. Travel expenses for A.B. were covered in part by the Consejo Nacional de Ciencia y Tecnologia, Mexico. 1 Bayon, A., Koda, L., Battenberg, E., Azad, R., Guillemin, R. and Bloom, F.E., Regional distribution of endorphin, methionine-enkephalin and leucine-enkephalin in the pigeon brain, Neurosci. Lett., 16 (1980) 75-80. 2 Bayon, A., Rossier, J., Mauss, A., Bloom, F.E., Iversen, L.L., Ling, N. and Guillemin, R., In vitro release of methionine-enkephalin and leucine-enkephalin from the rat globus pallidus, Proc. nat. Acad. Sci. (Wash.), 75 (1978) 3503-3506. 3 Bayon, A., Koda, L., Battenberg, E. and Bloom, F.E., Redistribution of endorphin and enkephalin immunoreactivity in the rat brain and pituitary after in vivo treatment with colchicine or cytochalasin B, Brain Res., 183 (1980) 103-111. 4 Bayon, A., Shoemaker, W.J., Bloom, F.E., Mauss, A. and Guillemin, R., Perinatal development of the endorphin-enkephalin-containing systems in the rat brain, Brain Res., 179 (1979) 93-101. 5 Bloom, F., Battenberg, E., Rossier, J., Ling, N. and Guillemin, R., Neurons containing B-endorphin in rat brain exist separately from those containing enkephalin: immunocytochemical studies, Proc. nat. Acad. Sci. (Wash.), 75 (1978) 1591-1595. 6 Cuello, A.C. and Paxinos, G., Evidence for a long LeuS-enkephalin strio-pallidal pathway in rat brain, Nature (Lond.), 271 (1978) 178-180. 7 Henderson, G., Hughes, J. and Kosterlitz, H.W., In vitro release of Leu- and Met-enkephalin from corpus striatum, Nature (Lond.), 271 (1978) 677-679. 8 H0kfelt, T., Elde, R., Johansson, O., Terenius, L. and Stein, L., The distribution of enkephalin immunoreactive cell bodies in the rat central nervous system, Neurosci. Lett., 5 (1977) 25-31. 9 Hughes, J., Smith, T.W., Kosterlitz, H.W., Fothergill, L.H., Morgan, B.A. and Morris, H., Identification of two related pentapeptides from the brain with potent opiate agonist activity, Nature (Lond.), 255 (1975) 577-579. 10 lversen, L.L., lversen, S.D., Bloom, F.E., Vargo, T. and Guillemin, R., Release of enkephalin from rat globus pallidus in vitro, Nature (Lond.), 271 (1978) 679-681. 11 KOnig, J.F.R. and Klippel, R.A., The Rat Brain, Krieger, Huntington, N.Y., 1967. 12 Ling, N., Solid phase synthesis of porcine/~-endorphin and ~,-endorphin, two hypothalamic-pituitary peptides with opiate activity, Biochem. biophys. Res. Commun., 74 (1977) 248-255. 13 Michelot, R., Leviel, V., Torrens, Y., Glowinski, J. and Cheramy, A., In vivo release of substance P in the cat substantia nigra, Neurosci. Lett., 15 (1979) 141-146. 14 Myers, R.D., An improved push-pull cannula system for perfusing an isolated region of the brain, Physiol. Behav., 5 (1970) 243-246. 15 Purpura, D.P., Physiological organization of the basal ganglia. In M.D. Yahr (Ed.), The Basal Ganglia, Raven Press, New York, 1976, pp. 91-114. 16 Rossier, J., Bayon, A., Vargo, T.M., Ling, N., Guiilemin, R. and Bloom, F.E., Radioimmunoassay of brain peptides. Evaluation of a methodology for the assay of/~-endorphin and enkephalin, Life Sci., 21 (1977) 847-852.
70 17 Rossier, J., Vargo, T.M., Minick, S., Ling, N., Bloom, F. and Guillemin, R., Regional dissociation of ~-endorphin and enkephalin contents in rat brain and pituitary, Proc. nat. Acad. Sci. (Wash.), 74 (1977) 5162-5165. 18 Sar, M., Stumpf, 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. 19 Snider, R.S. and Niemer, W.T., A Stereotaxic Atlas of the Cat Brain, University of Chicago Press, Chicago, 1970. 20 Stern, A.S., Lewis, R.V., Kimura, S., Rossier, J., Gerber, L.D., Brink, L., Stein, S. and Udenfriend, S., Isolation of the opioid heptapeptide Met-enkephalin-Arg6-Phe7 from bovine adrenal medullary granules and striatum, Proc. nat. Acad. Sci. (Wash.), 76 (1979) 6680-6683. 21 Watson, S.J., Akil, H., Richard, C.W. and Barchas, J.D., Evidence for two separate opiate neuronal systems, Nature (Lond.), 275 (1978) 226-228. 22 Yaksh, T.L. and Yamamura, H.I. Factors effecting performance of the push-pull cannula in brain, J. appl. Physiol., 37 (1974) 428-434. 23 Yang, H.-Y., Hong, J.S. and Costa, E., Regional distribution of Leu- and Met-enkephalin in rat brain, Neuropharmacology, 16 (1977) 303-307.
65
Elsevier/North-Holland Scientific Publishers Ltd.
IN VIVO R E L E A S E OF E N K E P H A L I N F R O M T H E G L O B U S P A L L I D U S
ALEJANDRO BAYON, WILLIAM J. SHOEMAKER*, LOURDES LUGO, RAANA AZAD*, NICHOLAS LING**, RENE R. DRUCKER-COL1Nand FLOYD E. BLOOM* Department o f Neuroscience, Centro de Investigaciones en Fisiologia Celular, Universidad Nacional Autonoma de Mexico, Apartado Postal 70-600, Mexico City 20 (Mexico)
and *,4. V. Davis Center f o r Behavioral Neurobiology and **The Laboratories f o r Neuroendocrinology, The Salk Institute, P.O. Box 85800, San Diego, CA 92138 (U.S.A.)
(Received November29th, 1980; Revised version received March 3rd, 1981;AcceptedMarch 16th, 1981)
Push-pull cannulae wereacutelypositioned through previouslyimplanted guidesin the globus pallidus of unanesthetized freely moving cats and rats. During slow-flow perfusions, enkephalin release was detected in resting conditions and increased more than 3-fold when both 50 mM K÷ and 1.8 mM Ca2÷ were present in the perfusing medium. Local perfusion with veratrine also enhanced enkephalin release. Furthermore, in vivo, electrical stimulation of the rat caudo-putamen enhanced enkephalin release in the pallidum. This latter finding is consistent with a functional strio-pallidal enkephalin-containing pathway previously postulated by immunohistochemical or lesion experiments.
The endogenous opiate peptides LeuS-enkephalin and MetS-enkephalin [9] are present in neuronal perikarya and nerve terminals in many regions of the central nervous system [8, 18]. In spite of the early observation of their chemical relationship to ~-lipotropin [9], the enkephalin-containing systems have been shown to exist separately from those containing the higher molecular weight endorphins [1, 4, 5, 17, 21]. More recently, the separation between enkephalins and other endorphins has been confirmed by demonstration of several possible enkephalin precursors unrelated to the lipotropin-derived endorphins [20]. The corpus striatum (c.s. = caudate, globus pallidus and putamen) possesses attributes of a model system to study the neurobiology of the enkephalins because it has one of the highest enkephalin concentrations in the brain [17, 23] and no chemically detectable endorphin [1, 5, 17, 21]. The globus pallidus is very rich in enkephalin fibers and terminals; cell bodies are found in the caudo-putamen [8, 18] and only a few of them are located on the pallidal border [3]. These advantages have been exploited only in part when studying the release (in vitro) of the enkephalins [2, 7, 10]. The use of an in vivo preparation to study the characteristics of the enkephalin release exploits the anatomical segregation of enkephalin-containing 03404-3940/81/0000-0000/$ 02.50 ©Elsevier/North-Holland Scientific Publishers Ltd.
66 terminals in the pallidum, and their preserved functional connection with other brain regions. In this communication we present initial data on the in vivo enkephalin release from the globus pallidus, its stimulation by locally applied depolarizing agents and the enhanced release produced by electrical stimulation of the head of the caudate-putamen nucleus. The push-pull cannula perfusion technique used in these experiments is based on that previously described by Myers [14]. Briefly, stainless steel cannulae that serve as stereotaxic guides for the push-pull cannulae were chronically implanted in the skull of adult cats (2-2.5 kg) and albino rats (150-200 g) under sodium pentobarbital anesthesia. The lower end of the guides was temporarily blocked with short mandrels. Coordinates for globus pallidus were: A 11.5, L 9.5, H 0.5 in the cat [19]; and A 6.4, L 2.5, H 0.2 in the rat [11]. Some rats were also implanted with bipolar stimulating electrodes in the caudate nucleus (A 8.4, L 2.5, H 0.2). One week after surgery the mandrels were removed without anesthesia and concentric push-pull cannulae were inserted through the guides to reach the pallidal tissue. The outer (pull) cannula was made of 21-gauge thin wall steel tubing; the inner (push) cannula is a 27-gauge needle protruding 1.5 mm from the tip of the pull cannula. Each perfusing cannula was connected through polyethylene tubing to the infusion and withdrawal syringes positioned in a Harvard reciprocating pump; animals were freely moving throughout subsequent perfusion sessions. The perfusion of the unanesthetized-freely moving animals was started using sterile Krebs-Ringer bicarbonate buffer (127 mM NAC1/3.73 mM KC1/1.8 mM CAC12/1.18 mM KH2PO4/1.18 mM MgSO4/20 mM NaHCO3/D-glucose, 2 g/liter), containing bovine serum albumin (0.1°70) and bacitracin (30 ~g/ml). The flow rate was held constant at 23 ~l/min (both in the 'push' and in the 'pull' systems) and 15 min fractions were collected in the withdrawal syringe, which contained ice-cold 2 N acetic acid and a stirring bar to mix the acid with the perfusate. The well-known artefact caused by syringe manipulation at the pump [22] was minimized by using a 3-way stopcock that allowed smooth transitions to another syringe when the first emptied. In addition, the pump was reset every 15 min, exactly corresponding to the collection periods. The regularity of the collection and syringe changing procedures would result in any artefactual changes in enkephalin level to be present in all collection periods and could not account for the experimental results observed. The collected fractions were boiled for 15 min, lyophilized and resuspended in buffer for the LeuS-enkephalin radioimmunoassay [16]. The positions of the tips of the cannulae were verified by inspection under a dissection microscope of frontal sections of the brain. The samples were subjected to radioimmunoassay at two dilutions in duplicate. The LeuS-enkephalin assay was as described [17]. The minimum detectable amount of LeuS-enkephalin is approximately 1 pg; Metenkephalin cross-reacts in this assay to the extent of 3°70 on a weight basis. The peptides used as standards were prepared by solid phase synthesis [12]. Following the procedures described above, a K÷-stimulated, Ca2*-dependent
67
release of enkephalin was obtained from the globus pallidus o f freely moving cats (Fig. 1). In animals perfused for the first time we observed a 200-300070 fold increase in enkephalin release over the preceding control periods. This K +stimulated release was only partially reduced (to 74070) over control values when Ca 2+ was omitted from the perfusing medium; subsequent addition of Ca 2+
30"
2
~V
It"'
PERFUSION (N-3)
2nd PERFUSION (N-3)
Z
+Co ~ -Co J +Co ~ -
+Co = -Co ~ +Co
Z
.._ detection ,imit.
'~
60
30
15
'~
~_
W ~,
~
Z
"7 3,0
.
i
.
.
.
3 rd PERFUSION (N=3) 15
[
ILl
60 i20 TIME (rnln)
180
S
I--I
m
:z)
! 60 120 TIME (rain)
180
0
60 TIME
] 120 (min)
Fig. 1. In vivo release of enkephalin from the globus pallidus of the cat. Sequential p u s h - p u l l cannula perfusions were performed at one week intervals after chronic implantation of the guide cannulae. In all perfusions the flow rate was held constant at 23 # i / m i n and 15 rain fractions were collected on ice-cold 2 N acetic acid (the volume of tissue perfused was estimated with a blue-dextran solution as approximately 2 mm3). Samples were boiled, lyophilized, redissolved in buffer and assayed for Leu 5enkephalin immunoreactivity (l enkephalin unit is equivalent to the trace displacement produced by 1 pg o f Leu-enkephalin in the Leu-enkephalin radioimmunoassay). White bars correspond to low K + -resting release periods; 50 m M K + medium was perfused during the periods indicated by the hatched bars. During the second hour of perfusion Mg 2+ was doubled in the medium and Ca 2+ was omitted. Vertical lines represent the standard error of the mean; N is the number of bilaterally perfused cats. Enkephalin values lower than the m i n i m u m detectable a m o u n t are indicated by a discontinuous line on top of the bars. Fig. 2. In vivo release of enkephalin from the globus pallidus of the rat. Perfusion conditions are as indicated in Fig. 1. V = low potassium medium, containing veratrine (60 #g/ml); S = electrical stimulation o f the c a u d o - p u t a m e n using a coiled bipolar electrode: Square pulses (0.5 msec duration, applied voltage 10 V; average current 100 #A) were delivered at 40, 60 and 80 Hz each during a 5 min period. At each frequency the trains of stimulation lasted 30 sec and were followed by 30 sec resting periods. Enkephalin values are the average o f released data obtained from 3 rats.
68 restored the K+-stimulated release to initial values. A second perfusing session carried out 7 days later produced lower amounts of enkephalin both during resting and stimulated release; in a third perfusion enkephalin values in the perfusate were below the detection limit of the assay. Since there is no decrease within the first perfusing session, lasting as long as 8 h and with several cannula penetrations, the reduced amounts of enkephalin released in second or third perfusion sessions appear to be related to the long-term effects of tissue damage. Other methodological variables also affected the enkephalin levels measured in the perfusates. Increasing the flow rate to 51 #l/rain usually leads to fluctuating baseline enkephalin values during the course of a perfusing session. A similar observation was reported for the in vivo release of substance P, although it was also noted that much higher flow rates yield more consistent results [13]. Addition of the aminopeptidase inhibitor bacitracin to our perfusing media was a requisite to obtain enkephalin levels in the collected fractions well within the measurable range of our assay. We have previously reported its protective action during the in vitro release of enkephalins [10]. A requirement for protective agents has also been reported for the measurements of released substance P [13]. Finally, when the tip of the push-pull cannula was aimed at the border between the internal capsule and the globus pallidus, in order to avoid damage of the pallidal tissue, the enkephalin collected in the perfusate was, in most cases, below the sensitivity of the radioimmunoassay. This observation indicates the anatomical specificity of the perfusion. The above results were next extended using rats as experimental subjects. Both K ÷ (50 mM) and veratrine were shown to stimulate the enkephalin release from the globus pallidus. Furthermore, in rats implanted with a bipolar electrode in the caudate nucleus, the electrical stimulation of this area elicited an increased enkephalin release in the globus pallidus (Fig. 2). We have used stimulation frequencies from 40 to 80 Hz. Since these frequencies have been shown to lead (in the cat) to the blockade of EPSPs evoked from the caudate nucleus in the pallidum due to the onset of slow IPSPs [15] - the trains of stimulation were alternated with resting periods every 30 sec. Work is in progress to study the frequency independence of this enkephalin release. When the electrodes were located in septal or preoptic areas no significant stimulation of pallidal enkephalin release was observed (data not shown). In vivo enkephalin release from the pallidum cannot be easily compared to that measured in vitro because of the wide differences between the preparations. However the enkephalin recovered in vivo is about one order of magnitude less than that in vitro [2, 10], this suggests a higher stability of enkephalin stored in the nonsliced pallidal tissue. These results show that not only can the local perfusion of pallidal tissue with chemical depolarizing agents elicit a Ca 2+-dependent release of enkephalin from this region (rich in enkephalin fibers), but also that electrical stimulation of a separate but connected area, the neighboring caudate nucleus, produces a quantitatively -
69
similar effect. This observations is consistent with the existence of the strio-pallidal enkephalin-containing pathway postulated by Cuello and Paxinos on the basis of lesion experiments [6]. Altogether, the results of these studies on the release of enkephalin from the brain region richest in these opioid peptides strongly support the concept that the enkephalins play a role in neural communication. This Research was partially supported by NIDA 01785. Travel expenses for A.B. were covered in part by the Consejo Nacional de Ciencia y Tecnologia, Mexico. 1 Bayon, A., Koda, L., Battenberg, E., Azad, R., Guillemin, R. and Bloom, F.E., Regional distribution of endorphin, methionine-enkephalin and leucine-enkephalin in the pigeon brain, Neurosci. Lett., 16 (1980) 75-80. 2 Bayon, A., Rossier, J., Mauss, A., Bloom, F.E., Iversen, L.L., Ling, N. and Guillemin, R., In vitro release of methionine-enkephalin and leucine-enkephalin from the rat globus pallidus, Proc. nat. Acad. Sci. (Wash.), 75 (1978) 3503-3506. 3 Bayon, A., Koda, L., Battenberg, E. and Bloom, F.E., Redistribution of endorphin and enkephalin immunoreactivity in the rat brain and pituitary after in vivo treatment with colchicine or cytochalasin B, Brain Res., 183 (1980) 103-111. 4 Bayon, A., Shoemaker, W.J., Bloom, F.E., Mauss, A. and Guillemin, R., Perinatal development of the endorphin-enkephalin-containing systems in the rat brain, Brain Res., 179 (1979) 93-101. 5 Bloom, F., Battenberg, E., Rossier, J., Ling, N. and Guillemin, R., Neurons containing B-endorphin in rat brain exist separately from those containing enkephalin: immunocytochemical studies, Proc. nat. Acad. Sci. (Wash.), 75 (1978) 1591-1595. 6 Cuello, A.C. and Paxinos, G., Evidence for a long LeuS-enkephalin strio-pallidal pathway in rat brain, Nature (Lond.), 271 (1978) 178-180. 7 Henderson, G., Hughes, J. and Kosterlitz, H.W., In vitro release of Leu- and Met-enkephalin from corpus striatum, Nature (Lond.), 271 (1978) 677-679. 8 H0kfelt, T., Elde, R., Johansson, O., Terenius, L. and Stein, L., The distribution of enkephalin immunoreactive cell bodies in the rat central nervous system, Neurosci. Lett., 5 (1977) 25-31. 9 Hughes, J., Smith, T.W., Kosterlitz, H.W., Fothergill, L.H., Morgan, B.A. and Morris, H., Identification of two related pentapeptides from the brain with potent opiate agonist activity, Nature (Lond.), 255 (1975) 577-579. 10 lversen, L.L., lversen, S.D., Bloom, F.E., Vargo, T. and Guillemin, R., Release of enkephalin from rat globus pallidus in vitro, Nature (Lond.), 271 (1978) 679-681. 11 KOnig, J.F.R. and Klippel, R.A., The Rat Brain, Krieger, Huntington, N.Y., 1967. 12 Ling, N., Solid phase synthesis of porcine/~-endorphin and ~,-endorphin, two hypothalamic-pituitary peptides with opiate activity, Biochem. biophys. Res. Commun., 74 (1977) 248-255. 13 Michelot, R., Leviel, V., Torrens, Y., Glowinski, J. and Cheramy, A., In vivo release of substance P in the cat substantia nigra, Neurosci. Lett., 15 (1979) 141-146. 14 Myers, R.D., An improved push-pull cannula system for perfusing an isolated region of the brain, Physiol. Behav., 5 (1970) 243-246. 15 Purpura, D.P., Physiological organization of the basal ganglia. In M.D. Yahr (Ed.), The Basal Ganglia, Raven Press, New York, 1976, pp. 91-114. 16 Rossier, J., Bayon, A., Vargo, T.M., Ling, N., Guiilemin, R. and Bloom, F.E., Radioimmunoassay of brain peptides. Evaluation of a methodology for the assay of/~-endorphin and enkephalin, Life Sci., 21 (1977) 847-852.
70 17 Rossier, J., Vargo, T.M., Minick, S., Ling, N., Bloom, F. and Guillemin, R., Regional dissociation of ~-endorphin and enkephalin contents in rat brain and pituitary, Proc. nat. Acad. Sci. (Wash.), 74 (1977) 5162-5165. 18 Sar, M., Stumpf, 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. 19 Snider, R.S. and Niemer, W.T., A Stereotaxic Atlas of the Cat Brain, University of Chicago Press, Chicago, 1970. 20 Stern, A.S., Lewis, R.V., Kimura, S., Rossier, J., Gerber, L.D., Brink, L., Stein, S. and Udenfriend, S., Isolation of the opioid heptapeptide Met-enkephalin-Arg6-Phe7 from bovine adrenal medullary granules and striatum, Proc. nat. Acad. Sci. (Wash.), 76 (1979) 6680-6683. 21 Watson, S.J., Akil, H., Richard, C.W. and Barchas, J.D., Evidence for two separate opiate neuronal systems, Nature (Lond.), 275 (1978) 226-228. 22 Yaksh, T.L. and Yamamura, H.I. Factors effecting performance of the push-pull cannula in brain, J. appl. Physiol., 37 (1974) 428-434. 23 Yang, H.-Y., Hong, J.S. and Costa, E., Regional distribution of Leu- and Met-enkephalin in rat brain, Neuropharmacology, 16 (1977) 303-307.