Brain
Research
Bulkfin,
Vol.
26, pp. 413417.
0 Pergamon
Press plc, 1991. Printed in
0361-9230/91 $3.00 +
the U.S.A
.OO
Pineal Microdialysis in Freely Moving Rats TAKAHARU AZEKAWA, ATSUKO SANO, HIROYOSHI SEI, AKIRA YAMAMOTO,” KAZUHIRO A01 AND YUSUKE MORITA’ Department of Physiology and *Department of Anatomy, School of Medicine The University of Tokushima, Tokushima 770, Japan Received 24 August 1990
AZEXAWA, T., A. SANO, H. SEI, A. YAMAMOTO, K. A01 AND Y. MORITA. Pineal microdialysis in freely moving rats. BRAIN RES BULL 26(3) 413417, 1991. -We describe a surgical technique to implant the guide cannula for in vivo microdialysis in the rat pineal gland. This technique has the following features and advantages: (a) does not require ligation of the superior or transverse sinus, (b) minimizes bleeding from the dural veins, (c) does not disturb the sympathetic innervation originating from superior cervical ganglia, which is essential for pineal function. This new technique makes it possible to carry out chronic pineal microdialysis of freely moving rats. In vivo
Brain microdialysis
Pineal gland
M.D.,
Freely moving rat
gland without ligature and resection of the sinus. This technique, which minimizes bleeding and does not disturb the pineal sympathetic innervation, offers the accessibility of chronic in vivo microdialysis from freely moving rats.
majority of neuro- and biochemical studies of the pineal gland have been carried out by using cell or organ culture and tissue slice preparation (11,33) and by analyzing the chemical content of the homogenized tissue (16,35). Although these approaches continue to provide important information on pineal function, research on the dynamics of the pineal neuroendocrine process would be made possible by the development of an in vivo sampling technique. Brain microdialysis has recently been developed as a useful technique for in vivo monitoring of extracellular electroactive compounds, which are separated by high performance liquid chromatography (HPLC) and quantified with electrochemical detection (ED) (5, 3 1, 34). In vivo measurements in the rat pineal gland, on the other hand, are thought to be complicated and difficult (23) because the rat pineal gland is located just below the confluence of the superior sagittal sinus (SSS) and transverse sinus (TS). Many techniques involving the rat pineal gland, such as pinealectomy (10, 12, 15, 24) and insertion of electrodes (4, 6-9, 21-23, 2528), require ligation of SSS or TS, or piercing the electrode through the sinus. Such procedures change the local blood flow (23) and often cause fatal hemorrhaging from surrounding dural sinuses (12,24). It is also known that the bilateral sympathetic innervation from the superior cervical ganglia (SCGs) to the pineal is essential for its function (3, 20, 30). The method involving double ligature and resection of one of the transverse sinuses disturbs the pineal innervation, which is a condition similar to unilateral sympathectomy. We have developed a new surgical technique for implantation of the guide cannula for a microdialysis probe in the rat pineal THE
‘Requests for reprints should be addressed to Yusuke MO&, Tokushima 770, Japan.
Surgical technique
Melatonin
METHOD
The present animal research was conducted in precise accordance with “Guiding Principles for Care and Use of Animals in the Field of Physiological Sciences, the Physiological Society of Japan, 1988.” Surgical Operation and Implantation Male Wistar rats were kept for at least 14 days to acclimate to a 12-h (lights-on at 07:00, about 250 lux) photoperiodic cycle and controlled ambient temperature (23 f 2°C) and relative humidity (40-60%). Food and water were freely available. The animals, weighing 200-350 g wt. at the beginning of surgery, were deeply anesthetized with pentobarbital sodium (Nembutal 50 mg/ kg b.wt. IP). The animal’s head was shaved and positioned in a stereotaxic frame with the plane between the bregma and the lambda levelled to horizontal (Fig. 1). A longitudinal incision (approximately 2 cm) was made in the midline extending to the occipital ridge. The sagittal and lambdoid sutures were then exposed by scraping away the periosteum to the temporal bone attachments of the temporal muscles. As shown in Fig. 1 (a-b-c-d), a sequential opening in the skull, approximately 5 mm anteroposterior extent and approximately 5 mm mediolateral extent, was made with a dental burr drill (1.2 or 1.4 mm o.d.). The bone was
Department
413
of Physiology,
School of Medicine,
The University
of Tokushima,
414
AZEKAWA
E’I’ AL
away from the target point and oriented at approximately 45” angle to the frontal plane and at approximately 30” angle to the midsagittal plane (Fig. 2C), in order to avoid touching the sinus. The target point was penetrated by the dialysis probe extended I .5 mm beyond the tip of guide cannula. The surface of the cortex and the dura was protected by covering it with a layer of absorbable hemostat (Spongel, Yamanouchi Pharmaceutical. Tokyo. Japan). Two stainless steel screws were placed nearby on the skull to serve as anchors. After the operation area was cleaned and dried, the guide cannula with the stylet was mounted on the skull with dental cement. After surgery, each rat was housed in an individual cage for microdialysis in a chamber under the above conditions and was allowed to recover from surgery for two weeks prior to the initiation of the experiments. Each rat received an antibiotics therapy of sodium ampicillin (Pentrex. 10-50 mglkg b.wt. IP, Banyu Pharmaceutical, Tokyo, Japan) during the first three postoperative days.
Sagitial
suture
Microdialyis
1
FIG. 1. The rat head with a partly exposed skull placed in the stereotaxic frame under pentobarbital anesthesia (50 mg/kg b.wt. IP). The square “a-b-c-d” in the skull indicates the opening for stereotaxic implantation of the guide cannula for the microdialysis probe. For detailed methods, see the text.
carefully and gently planed to avoid causing hemorrhage from the sinuses below and meticulous attention was paid not to leave any fragments of the bone. The confluence of the sinus was exposed over the triangular space at the junction of the parietal and occipital lobes. With the tip of a 24-gauge needle or a No. 11 scalpel blade, a 1S-2.0-mm cut was made in the dura mater on the lateral edges of SSS, and approximately l-mm cuts were made laterally in both ends of the incision (Fig. 2A, e-f-g-h). The dura was then reflected to the lateral side (Fig. 2B). According to the atlas of Paxinos and Watson (19), the target point was fixed at the center of the pineal body: 8.3 mm posterior to bregma, 0 mm lateral to midsagittal plane, 2.0 mm ventral to skull surface. The guide cannula (0.7 mm o.d.) was stereotaxically placed 1.O mm
(A)
(B)
and HPLC-ED
Analysis
The sampling of pineal microdialysate was carried out as detailed in our previous paper (2). In brief, the microdialysis probe was constructed in our laboratory as a modification of Nakahara et al.‘s method (17) and was made by joining the Cuprophan dialysis fiber (5,000 mol.wt. cutoff, 0.2 mm i.d., 8 km thickness. 3 mm long; Nikkiso, Tokyo, Japan), leaving 1.5 mm available for diffusion. A representation of the microdialysis probe with the guide cannula is shown in Fig. 3. For microdialysis, the stylet was replaced by the dialysis probe and fixed with wax. The dialysis probe was continuously perfused via the PTFE tube (0.4 mm o.d., 0.1 mm i.d.) at a flowrate of 1.5 pl/min with Ringer’s solution (NaCl 147 mM, KC1 4 mM, CaCl, 4.5 mM, pH 6.5). Samples were collected every 19 min or 29 min via the PTFE tube in the 100 )*l loop of automatic injector (AS-IO, EICOM, Kyoto, Japan), which was on-line with the HPLC system. The sample loop was set to be retained in the load position during 19 min or 29 min and was automatically switched to the injection position for 1 min, after which the cycle was repeated. The rats were linked to the apparatus for assay with the two PTFE tubes coiled. These coiled tubes, which were less entangled, allowed the animal for unrestricted movement throughout in vivo microdialysis. The analytical conditions for the detection of melatonin were as detailed in our previous paper (2). Briefly, the analytical col-
(Cl
FIG. 2. Procedure for the implantation of the guide cannula. (A) Dural incisions are made in the dotted line “e-f-g-h” along the edge of superior sagittal sinus. (B) The dura is lifted and reflected to the lateral side. (C) The open arrow indicates the direction to implant the guide cannula, which is slowly inserted into the pineal gland through two different angles to avoid touching the sinus. See the text for details.
415
RAT PINEAL MICRODIALYSIS
A output 1 24-gauge stainless steel cannula bb
I
A
t
DISCUSSION
J 7: 19 mm
16mm
1.5 mm
2
/
22-gauge stainless steel cannula
Cuprophan dialysis hollow fiber 216Ilm0.0.
c Dialysis
probe>
FIG. 3. Schematic drawing of the guide cannula (A) and the microdialy-
sis probe (B). For details, see the text and the previous reports (2.15).
umn was an Eicompak reversed-phase column (MA-ODS, 250 x 4.6 mm i.d. EICOM). The mobile phase consisted of phosphate buffer (0.1 M KH,PG4 and 0.05 M H,PO,, pH 3.1) containing 34% v/v methanol, 4 (LM EDTA and was delivered at 1 .O ml/min by an EP-10 pump (EICOM). The apparatus for electrochemical detection was an ECD-100 amperometric detector (EICOM) equipped with a WE-3G working electrode (EICOM) and an Ag/ AgCl reference electrode. The applied potential of the working was set at + 0.8.5V vs. the reference electrode. In this assay, basal levels of melatonin (mean?S.D.) in a 29-min dialysate sample were 12.5 r0.6 pg during the light period and 72.6248.8 pg during the dark period, as shown in the previous report (2). Histological Verification The identification of probe placement was performed as follows: after the termination of each experiment, the animal was
200 El60
decapitated or perfusated with 5% formalin under pentobarbital anesthesia. Then, its brain was carefully removed and stored in 10% formalin. After embedding the brain in paraffm or plastic, serial coronal sections were cut at 4- or 6-pm intervals and stained with toluidine blue, hematoxylin-eosin and azan. Location of the probe was histologically verified in each animal (Fig. 5).
-
Time of day FIG. 4. Pattern of the extracellular melatonin (MEL) levels over the tran-
sition from the light phase to the dark phase. The dark phase is indicated by the black bar just over the curves of melatonin levels.
To the best of our knowledge, there has been to date no recorded technique that has permitted successive in vivo sampling of pineal substances from an unanesthetized or freely moving rat. This is probably because of the specific location of the pineal gland just below the confluence of the sinuses. In order to monitor the changes of the pineal substances such as melatonin, serotonin, etc., it has been necessary to obtain pineal glands from many rats for each sampling, using the analysis of postmortem chemical contents in the homogenized pineal tissue. Such off-line measurements reflect a rough discrete point at a particular time and are dominated only by the intracellular content of the tissue. Contents of the pineal indoles released from the intracellular vesicles to the extracellular space and its more dynamic changes in response to external lighting or other conditions have not been well described. We have previously reported the basal levels of extracellular melatonin (2). In this paper, we report on the patterns of pineal extracellular melatonin levels over the transition from the light period to the dark period, obtained from freely moving rats by the use of this surgical technique (Fig. 4). The nocturnaI rise in extracellular melatonin levels corresponds with findings of studies that utilized homogenated-tissue analysis (1, 16, 30, 35). The important feature of this surgical technique is that it does not require ligature nor resection of the sinuses. We have noted a passing reference to this matter in a report by Hsieh and Ota (13). The current technique yields two major advantages to pineal research: 1) It holds bleeding and any change of blood flow to a minimum. Many previous studies indicated certain amounts of hemorrhage from SSS and TS caused by the ligating and resecting of the sinuses, because the pineal is located ventral to the center of venous drainage system of the brain. Furthermore, both ligating the sinus and piercing the electrode through the sinus change the local blood flow (23). In the new technique, incision of the dura mater sometimes causes bleeding from the dural veins flowing into the sinus, but this can readily be treated by gently putting a piece of absorbable hemostat on the bleeding site, with no significant impact on the placement of the probe. 2) It does not disturb the pineal sympathetic innervation from SCGs. The neural information from the environmental photoperiod reaches the pineal gland by way of postganglionic adrenergic fibers which bilaterally ascend to the carotid plexus and the tentorium cerebelli (14). Superior cervical ganglionectomy abolishes periodic melatonin secretion (20). Bowers and Zigmond (3) reported that the innervation from the bilateral SCGs interacts within the pineal gland. Based on the above findings, both ligating and resecting the transverse sinus modify various pineal metabolic processes and complicate the theoretical consideration of the experiments. One handicap of this technique is that the dialysis probe does not always penetrate the pineal gland for two reasons: fiit, this technique does not allow a visible approach to the pineal gland; and second, since the rat pineal gland lacks rigid attachment, the pineal often rotates along its anteroposterior axis (18). Figure 5 shows the coronal section of the pineal gland of a rat. Evidence of the placement of the probe was found in the compartment of
FIG. 5. Photomicrograph of a coronal section of the pineal gland (PI. The arrow shows the site of Implantation of the dialysis probe.
the pineal gland. Successful implantation of the dialysis probe in the pineal gland can be obtained prior to histological verification, based upon melatonin concentrations and their nocturnal increase. Melatonin is synthesized from serotonin by two steps: first, Nacetylation of serotonin to form N-acetylserotonin by N-acetyltransferase (NAT, E. C. 2.3.1.87) and its subsequent 0-methylation to form melatonin by hydroxy-0-methyl-transferase (HIOMT, E.C.2.1.1.4.). In the rat, both NAT and HIOMT are found in high concentration only in the pineal gland, and in substantially lower concentrations in the retina (29). Therefore, only the pineal gland synthesizes and releases melatonin in the vicinity of the dialysis probe. If melatonin in in vivo microdialysate is detectable, it may be presumed that the dialysis probe is implanted in the pineal gland. The stereotaxic coordinate by our technique is different from that by Patrickson and Smith (18), who studied the central innervation of the rat pineal gland. In fact, we failed to detect melatonin in in vivo microdialysis using Patrickson and Smith’s coordinate. This may be due to coordinate difference and placement of the
probe in the different rat strains utilized (between Wistar and Sprague-Dawley rats). The sizes or volumes in the pineal gland of laboratory rats differ with interstrain and interstock (32). In conclusion, we have reported here a new surgical technique for in vivo microdialysis in the pineal gland of the conscious rat and described its features and advantages. This technique will enable us to carry out in vivo pineal microdialysis in freely moving rats. In vivo pineal microdialysis promises to bring a new dimension to the field of research in pineal physiology as well as its neurochemistry and to give us further insight into the relationship between ongoing behavior and dynamic changes of pineal indoleamines. ACKNOWLEDGEMENTS
We thank Professor S. Yamamoto, Professor K. lshimura and Dr. T. Yoshimoto for their support of this work, Dr. M. Kihara and Ms. Mae F. Sakamoto for critical reading of the manuscript, and Ms. N. Kida for her expert secretarial assistance.
REFERENCES I. Axelrod,
2.
3.
4.
5. 6.
J. The pineal gland: A neurochemical transducer. Science 184:1341-1348; 1974. Azekawa, T.; Sano, A.; Aoi, K.; Sei, H.; Morita, Y. Concurrent on-line sampling of melatonin in pineal microdialysates from conscious rat and its analysis by high-performance liquid chromatography with electrochemical detection. J. Chromatogr. 530:47-55; 1990. Bowers, C. W.; Zigmond. R. E. The influence of the frequency and pattern of sympathetic nerve activity on serotonin N-acetyltransferase in the rat pineal gland. J. Physiol. (Land.) 330:279-296; 1982. Brooks, C. McC.; Ishikawa, T.; Koizumi, K. Autonomic system control of the pineal gland and the role of this complex in the integration of body function. Brain Res. 87:181-190; 1975. Church, W. H:; Justice, J. B., Jr. Rapid sampling and determination of extracellular dopamine in vivo. Anal. Chem. 59:712-716; 1987. Dafny, N.; McClung, R. Pineal body: Neuronal recording. Experientia 31:321-322; 1975.
7. Dafny , N. ; McClung, R. ; Strada, S J. Neurophysiological properties of the pineal body 1. Field potential. Life Sci. 16:61 I-620; 1975. 8. Dafny, N.; Burks, T. F. Opiate and endocrine interaction: Morphine effects on hypothalamus and pineal body. Neuroendocrinology 22: 72-88; 1976. 9. Dafny. N. Two photic pathways contribute to pineal evoked responses. Life Sci. 26:737-742; 1980. 10. Das Gupta, T. K.; Terz, J. Influence of pineal gland on the growth and spread of melanoma in the hamster. Cancer Res. 27: 1306-l 3 11; 1967. Il. Ducis, I.; DiStefano, V. Evidence for a serotonin uptake system in isolated bovine pinealocyte suspensions. Mol. Pharmacol. 18:438446; 1980. 12. Hoffman, R.; Reiter, R. J. Rapid pinealectomy in hamsters and small rodents. Anat. Rec. 153:19-22; 1965. 13. Hsieh, K. S.; Ota. M. Improved procedure of pinealectomy in rat.
RAT PINEAL
MICRODIALYSIS
Endocrinol. Jpn. 16:477-478; 1969. 14. Kappers, J. A. The development, topographical relations and innervation of the epiphysis cerebti in the albino rat. Z. Zellforsch. 52: 163-215; 1960. 15. Kuszak, J.; Rodin, M. A new technique of pinealectomy for adult rats. Experientia 33:283-284; 1977. 16. Mefford, I. N.; Chang, P.; Klein, D. C.; Namboodiri, M. A. A.; Sugden, D.; Barchas, J. Reciprocal day/night relationship between serotonin oxidation and N-acetylation products in the rat pineal gland. Endocrinology 113:1582-1586; 1983. 17. Nakahara, D.; Ozaki, N.; Nagatsu, T. A removable brain microdialysis probe unit for in vivo monitoring of neurochemical activity. Biogenie Amines 6:559-564; 1989. 18. Patrickson, J. W.; Smith, T. Innervation of the pineal gland in the rat: An HRP study. Exp. Neurol. 95:207-215; 1987. 19. Paxinos, G.; Watson, C. The rat brain in stereotaxic coordinates. New York: Academic Press; 1982. 20. Reiter, R. J.; Rudeen, P. K.; Banks, A. F.; Rollag, M. D. Acute effects of unilateral or bilateral superior cervical ganglionectomy on rat pineal N-acetyltransferase activity and melatonin content. Experientia 35:691692; 1979. 21. Reuss, S.; Vollrath, L. Electrophysiological properties of rat pinealocytes: Evidence for circadian rhythms. Exp. Brain Res. 55:455461; 1984. 22. Reuss, R.; Olcese, J.; Vollrath, L. Electrophysiological and endocrinological aspects of aging in the rat pineal gland. Neuroendocrinology 43:466-471; 1986. 23. Reuss, S. Electrical activity of the mammalian pineal gland. In: Reiter, R. J., ed. Pineal research reviews. vol. 5. New York: Alan R. Liss; 1987:153-189. 24. Rodin, A. E. The growth and spread of walker 256 carcinoma in pinealectomized rats. Cancer Res. 23:1545-1550; 1963. 25. Ronnekleiv, 0. K.; Kelly, M. J.; Wurrke, W. Single unit recordings in the rat pineal gland: Evidence for habenulo-pineal neural connections. Exp. Brain Res. 39:187-192; 1980.
417
26. Schapiro, S.; Salas, M. Effects of age, light and sympathetic innervation on electrical activity of the rat pineal gland. Brain Res. 28: 47-55; 1971. 27. Semm, P.; Schneider, T.; Vollrath, L. Effects of an earth-strength magnetic field on electrical activity of pineal cells. Nature 288:607608; 1980. 28. Semm, P.; Demaine, C.; Vollrath, L. Electrical responses of pineal cells to melatonin and putative transmitters; Evidence for circadian changes in sensitivity. Exp. Brain Res. 43:361-370; 1981. 29. Sugden, D. Melatonin biosynthesis in the mammalian pineal gland. Experientia 45:922-932; 1989. 30. Tamarkin, L.; Baird, C. J.; Almedia, 0. F. X. Melatonin: A coordinating signal for mammalian reproduction? Science 227:714-720; 1985. 31. Ungerstedt, U. Measurement of neurotransmitter release by intracranial dialysis. In: Marsden, C. A., ed. Measurement of neurotransmitter release in viva. New York: Wiley and Sons Ltd.; 1984:81-105. 32. Vollrath, L.; Vollrath, I.; Olcese, J. Interstrain and interstock differences of pineal gland volumes in male laboratory rats. Med. Sci. Res. 15:1391; 1987. 33. Walker, R. F.; Aloyo, V. J. Molecular mechanisms controlling norepinephtine-mediated release of serotonin from rat pineal gland. Molecular mechanism of neuronal responsiveness. In: Ehrlich, Y. H.; Lenox, R. H.; Komecki, E.; Berry, W. O., eds. Molecular mechanism of neuronal responsiveness. New York: Plenum Press; 1987: 223-236. 34. Westerink, B. H. C.; Damsma, G.; Rollema, H.; De Varies, J. B.; Horn, A. S. Scope and limitations of in vivo brain dialysis: A comparison of its application to various neurotransmitter systems. Life Sci. 41:1763-1776; 1987. 35. Wilkinson, M.; Arendt, J.; Bradtke, J.; De Zieger, D. Determination of a dark-induced increase of pineal N-acetyltransferase activity and simultaneous radioimmunoassay of melatonin in pineal, serum and pituitary tissue of the male rat. J. Endocrinol. 72:243-244; 1977.
Research
Bulkfin,
Vol.
26, pp. 413417.
0 Pergamon
Press plc, 1991. Printed in
0361-9230/91 $3.00 +
the U.S.A
.OO
Pineal Microdialysis in Freely Moving Rats TAKAHARU AZEKAWA, ATSUKO SANO, HIROYOSHI SEI, AKIRA YAMAMOTO,” KAZUHIRO A01 AND YUSUKE MORITA’ Department of Physiology and *Department of Anatomy, School of Medicine The University of Tokushima, Tokushima 770, Japan Received 24 August 1990
AZEXAWA, T., A. SANO, H. SEI, A. YAMAMOTO, K. A01 AND Y. MORITA. Pineal microdialysis in freely moving rats. BRAIN RES BULL 26(3) 413417, 1991. -We describe a surgical technique to implant the guide cannula for in vivo microdialysis in the rat pineal gland. This technique has the following features and advantages: (a) does not require ligation of the superior or transverse sinus, (b) minimizes bleeding from the dural veins, (c) does not disturb the sympathetic innervation originating from superior cervical ganglia, which is essential for pineal function. This new technique makes it possible to carry out chronic pineal microdialysis of freely moving rats. In vivo
Brain microdialysis
Pineal gland
M.D.,
Freely moving rat
gland without ligature and resection of the sinus. This technique, which minimizes bleeding and does not disturb the pineal sympathetic innervation, offers the accessibility of chronic in vivo microdialysis from freely moving rats.
majority of neuro- and biochemical studies of the pineal gland have been carried out by using cell or organ culture and tissue slice preparation (11,33) and by analyzing the chemical content of the homogenized tissue (16,35). Although these approaches continue to provide important information on pineal function, research on the dynamics of the pineal neuroendocrine process would be made possible by the development of an in vivo sampling technique. Brain microdialysis has recently been developed as a useful technique for in vivo monitoring of extracellular electroactive compounds, which are separated by high performance liquid chromatography (HPLC) and quantified with electrochemical detection (ED) (5, 3 1, 34). In vivo measurements in the rat pineal gland, on the other hand, are thought to be complicated and difficult (23) because the rat pineal gland is located just below the confluence of the superior sagittal sinus (SSS) and transverse sinus (TS). Many techniques involving the rat pineal gland, such as pinealectomy (10, 12, 15, 24) and insertion of electrodes (4, 6-9, 21-23, 2528), require ligation of SSS or TS, or piercing the electrode through the sinus. Such procedures change the local blood flow (23) and often cause fatal hemorrhaging from surrounding dural sinuses (12,24). It is also known that the bilateral sympathetic innervation from the superior cervical ganglia (SCGs) to the pineal is essential for its function (3, 20, 30). The method involving double ligature and resection of one of the transverse sinuses disturbs the pineal innervation, which is a condition similar to unilateral sympathectomy. We have developed a new surgical technique for implantation of the guide cannula for a microdialysis probe in the rat pineal THE
‘Requests for reprints should be addressed to Yusuke MO&, Tokushima 770, Japan.
Surgical technique
Melatonin
METHOD
The present animal research was conducted in precise accordance with “Guiding Principles for Care and Use of Animals in the Field of Physiological Sciences, the Physiological Society of Japan, 1988.” Surgical Operation and Implantation Male Wistar rats were kept for at least 14 days to acclimate to a 12-h (lights-on at 07:00, about 250 lux) photoperiodic cycle and controlled ambient temperature (23 f 2°C) and relative humidity (40-60%). Food and water were freely available. The animals, weighing 200-350 g wt. at the beginning of surgery, were deeply anesthetized with pentobarbital sodium (Nembutal 50 mg/ kg b.wt. IP). The animal’s head was shaved and positioned in a stereotaxic frame with the plane between the bregma and the lambda levelled to horizontal (Fig. 1). A longitudinal incision (approximately 2 cm) was made in the midline extending to the occipital ridge. The sagittal and lambdoid sutures were then exposed by scraping away the periosteum to the temporal bone attachments of the temporal muscles. As shown in Fig. 1 (a-b-c-d), a sequential opening in the skull, approximately 5 mm anteroposterior extent and approximately 5 mm mediolateral extent, was made with a dental burr drill (1.2 or 1.4 mm o.d.). The bone was
Department
413
of Physiology,
School of Medicine,
The University
of Tokushima,
414
AZEKAWA
E’I’ AL
away from the target point and oriented at approximately 45” angle to the frontal plane and at approximately 30” angle to the midsagittal plane (Fig. 2C), in order to avoid touching the sinus. The target point was penetrated by the dialysis probe extended I .5 mm beyond the tip of guide cannula. The surface of the cortex and the dura was protected by covering it with a layer of absorbable hemostat (Spongel, Yamanouchi Pharmaceutical. Tokyo. Japan). Two stainless steel screws were placed nearby on the skull to serve as anchors. After the operation area was cleaned and dried, the guide cannula with the stylet was mounted on the skull with dental cement. After surgery, each rat was housed in an individual cage for microdialysis in a chamber under the above conditions and was allowed to recover from surgery for two weeks prior to the initiation of the experiments. Each rat received an antibiotics therapy of sodium ampicillin (Pentrex. 10-50 mglkg b.wt. IP, Banyu Pharmaceutical, Tokyo, Japan) during the first three postoperative days.
Sagitial
suture
Microdialyis
1
FIG. 1. The rat head with a partly exposed skull placed in the stereotaxic frame under pentobarbital anesthesia (50 mg/kg b.wt. IP). The square “a-b-c-d” in the skull indicates the opening for stereotaxic implantation of the guide cannula for the microdialysis probe. For detailed methods, see the text.
carefully and gently planed to avoid causing hemorrhage from the sinuses below and meticulous attention was paid not to leave any fragments of the bone. The confluence of the sinus was exposed over the triangular space at the junction of the parietal and occipital lobes. With the tip of a 24-gauge needle or a No. 11 scalpel blade, a 1S-2.0-mm cut was made in the dura mater on the lateral edges of SSS, and approximately l-mm cuts were made laterally in both ends of the incision (Fig. 2A, e-f-g-h). The dura was then reflected to the lateral side (Fig. 2B). According to the atlas of Paxinos and Watson (19), the target point was fixed at the center of the pineal body: 8.3 mm posterior to bregma, 0 mm lateral to midsagittal plane, 2.0 mm ventral to skull surface. The guide cannula (0.7 mm o.d.) was stereotaxically placed 1.O mm
(A)
(B)
and HPLC-ED
Analysis
The sampling of pineal microdialysate was carried out as detailed in our previous paper (2). In brief, the microdialysis probe was constructed in our laboratory as a modification of Nakahara et al.‘s method (17) and was made by joining the Cuprophan dialysis fiber (5,000 mol.wt. cutoff, 0.2 mm i.d., 8 km thickness. 3 mm long; Nikkiso, Tokyo, Japan), leaving 1.5 mm available for diffusion. A representation of the microdialysis probe with the guide cannula is shown in Fig. 3. For microdialysis, the stylet was replaced by the dialysis probe and fixed with wax. The dialysis probe was continuously perfused via the PTFE tube (0.4 mm o.d., 0.1 mm i.d.) at a flowrate of 1.5 pl/min with Ringer’s solution (NaCl 147 mM, KC1 4 mM, CaCl, 4.5 mM, pH 6.5). Samples were collected every 19 min or 29 min via the PTFE tube in the 100 )*l loop of automatic injector (AS-IO, EICOM, Kyoto, Japan), which was on-line with the HPLC system. The sample loop was set to be retained in the load position during 19 min or 29 min and was automatically switched to the injection position for 1 min, after which the cycle was repeated. The rats were linked to the apparatus for assay with the two PTFE tubes coiled. These coiled tubes, which were less entangled, allowed the animal for unrestricted movement throughout in vivo microdialysis. The analytical conditions for the detection of melatonin were as detailed in our previous paper (2). Briefly, the analytical col-
(Cl
FIG. 2. Procedure for the implantation of the guide cannula. (A) Dural incisions are made in the dotted line “e-f-g-h” along the edge of superior sagittal sinus. (B) The dura is lifted and reflected to the lateral side. (C) The open arrow indicates the direction to implant the guide cannula, which is slowly inserted into the pineal gland through two different angles to avoid touching the sinus. See the text for details.
415
RAT PINEAL MICRODIALYSIS
A output 1 24-gauge stainless steel cannula bb
I
A
t
DISCUSSION
J 7: 19 mm
16mm
1.5 mm
2
/
22-gauge stainless steel cannula
Cuprophan dialysis hollow fiber 216Ilm0.0.
c Dialysis
probe>
FIG. 3. Schematic drawing of the guide cannula (A) and the microdialy-
sis probe (B). For details, see the text and the previous reports (2.15).
umn was an Eicompak reversed-phase column (MA-ODS, 250 x 4.6 mm i.d. EICOM). The mobile phase consisted of phosphate buffer (0.1 M KH,PG4 and 0.05 M H,PO,, pH 3.1) containing 34% v/v methanol, 4 (LM EDTA and was delivered at 1 .O ml/min by an EP-10 pump (EICOM). The apparatus for electrochemical detection was an ECD-100 amperometric detector (EICOM) equipped with a WE-3G working electrode (EICOM) and an Ag/ AgCl reference electrode. The applied potential of the working was set at + 0.8.5V vs. the reference electrode. In this assay, basal levels of melatonin (mean?S.D.) in a 29-min dialysate sample were 12.5 r0.6 pg during the light period and 72.6248.8 pg during the dark period, as shown in the previous report (2). Histological Verification The identification of probe placement was performed as follows: after the termination of each experiment, the animal was
200 El60
decapitated or perfusated with 5% formalin under pentobarbital anesthesia. Then, its brain was carefully removed and stored in 10% formalin. After embedding the brain in paraffm or plastic, serial coronal sections were cut at 4- or 6-pm intervals and stained with toluidine blue, hematoxylin-eosin and azan. Location of the probe was histologically verified in each animal (Fig. 5).
-
Time of day FIG. 4. Pattern of the extracellular melatonin (MEL) levels over the tran-
sition from the light phase to the dark phase. The dark phase is indicated by the black bar just over the curves of melatonin levels.
To the best of our knowledge, there has been to date no recorded technique that has permitted successive in vivo sampling of pineal substances from an unanesthetized or freely moving rat. This is probably because of the specific location of the pineal gland just below the confluence of the sinuses. In order to monitor the changes of the pineal substances such as melatonin, serotonin, etc., it has been necessary to obtain pineal glands from many rats for each sampling, using the analysis of postmortem chemical contents in the homogenized pineal tissue. Such off-line measurements reflect a rough discrete point at a particular time and are dominated only by the intracellular content of the tissue. Contents of the pineal indoles released from the intracellular vesicles to the extracellular space and its more dynamic changes in response to external lighting or other conditions have not been well described. We have previously reported the basal levels of extracellular melatonin (2). In this paper, we report on the patterns of pineal extracellular melatonin levels over the transition from the light period to the dark period, obtained from freely moving rats by the use of this surgical technique (Fig. 4). The nocturnaI rise in extracellular melatonin levels corresponds with findings of studies that utilized homogenated-tissue analysis (1, 16, 30, 35). The important feature of this surgical technique is that it does not require ligature nor resection of the sinuses. We have noted a passing reference to this matter in a report by Hsieh and Ota (13). The current technique yields two major advantages to pineal research: 1) It holds bleeding and any change of blood flow to a minimum. Many previous studies indicated certain amounts of hemorrhage from SSS and TS caused by the ligating and resecting of the sinuses, because the pineal is located ventral to the center of venous drainage system of the brain. Furthermore, both ligating the sinus and piercing the electrode through the sinus change the local blood flow (23). In the new technique, incision of the dura mater sometimes causes bleeding from the dural veins flowing into the sinus, but this can readily be treated by gently putting a piece of absorbable hemostat on the bleeding site, with no significant impact on the placement of the probe. 2) It does not disturb the pineal sympathetic innervation from SCGs. The neural information from the environmental photoperiod reaches the pineal gland by way of postganglionic adrenergic fibers which bilaterally ascend to the carotid plexus and the tentorium cerebelli (14). Superior cervical ganglionectomy abolishes periodic melatonin secretion (20). Bowers and Zigmond (3) reported that the innervation from the bilateral SCGs interacts within the pineal gland. Based on the above findings, both ligating and resecting the transverse sinus modify various pineal metabolic processes and complicate the theoretical consideration of the experiments. One handicap of this technique is that the dialysis probe does not always penetrate the pineal gland for two reasons: fiit, this technique does not allow a visible approach to the pineal gland; and second, since the rat pineal gland lacks rigid attachment, the pineal often rotates along its anteroposterior axis (18). Figure 5 shows the coronal section of the pineal gland of a rat. Evidence of the placement of the probe was found in the compartment of
FIG. 5. Photomicrograph of a coronal section of the pineal gland (PI. The arrow shows the site of Implantation of the dialysis probe.
the pineal gland. Successful implantation of the dialysis probe in the pineal gland can be obtained prior to histological verification, based upon melatonin concentrations and their nocturnal increase. Melatonin is synthesized from serotonin by two steps: first, Nacetylation of serotonin to form N-acetylserotonin by N-acetyltransferase (NAT, E. C. 2.3.1.87) and its subsequent 0-methylation to form melatonin by hydroxy-0-methyl-transferase (HIOMT, E.C.2.1.1.4.). In the rat, both NAT and HIOMT are found in high concentration only in the pineal gland, and in substantially lower concentrations in the retina (29). Therefore, only the pineal gland synthesizes and releases melatonin in the vicinity of the dialysis probe. If melatonin in in vivo microdialysate is detectable, it may be presumed that the dialysis probe is implanted in the pineal gland. The stereotaxic coordinate by our technique is different from that by Patrickson and Smith (18), who studied the central innervation of the rat pineal gland. In fact, we failed to detect melatonin in in vivo microdialysis using Patrickson and Smith’s coordinate. This may be due to coordinate difference and placement of the
probe in the different rat strains utilized (between Wistar and Sprague-Dawley rats). The sizes or volumes in the pineal gland of laboratory rats differ with interstrain and interstock (32). In conclusion, we have reported here a new surgical technique for in vivo microdialysis in the pineal gland of the conscious rat and described its features and advantages. This technique will enable us to carry out in vivo pineal microdialysis in freely moving rats. In vivo pineal microdialysis promises to bring a new dimension to the field of research in pineal physiology as well as its neurochemistry and to give us further insight into the relationship between ongoing behavior and dynamic changes of pineal indoleamines. ACKNOWLEDGEMENTS
We thank Professor S. Yamamoto, Professor K. lshimura and Dr. T. Yoshimoto for their support of this work, Dr. M. Kihara and Ms. Mae F. Sakamoto for critical reading of the manuscript, and Ms. N. Kida for her expert secretarial assistance.
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