SYNAPSE 6:351-357 (1990)
Tyrosine Hydroxylase and Galanin *A Levels in Locus Coeruleus Neurons Are Increased Following. Reserpine Administration v
MARK C. AUSTIN, SANDRA L. COTTINGHAM, STEVEN M. PAUL, AND JACQUELINE N. CRAWLEY Clinical Neuroscience Branch, National Institute of Mental Health, Bethesda, Maryland, 20892
KEY WORDS
Neuropeptide, Norepinephrine, Neuron, Coexistence, Synthesis, Depletion
ABSTRACT
The neuropeptide galanin coexists in 80-90% of the norepinephrinecontaining neurons in the locus coeruleus. In situ hybridization histochemistry was used to examine the effects of reserpine treatment or swim stress on tyrosine hydroxylase and galanin mRNA concentrations in locus coeruleus neurons. Reserpine administration significantly increased tyrosine hydroxylase and galanin mRNA levels in the locus coeruleus. The reserpine-induced increase in tyrosine hydroxylase mRNA was significantly correlated with the reserpine-induced increase in galanin mRNA. Three consecutive days of swim stress did not significantly alter either tyrosine hydroxylase or galanin mRNA concentrations in the locus coeruleus. These data suggest that both tyrosine hydroxylase and galanin gene expression in locus coeruleus neurons may be regulated by a reserpine-sensitive mechanism.
INTRODUCTION Galanin, a 29 amino acid residue pe tide originally isolated from orcine small intestine ( atemoto et al., 1983), is foun in the majority of norepinephrine (NE)containing neurons of the locus coeruleus (Melander et al., 1986; Holets et al., 1988). The functional consequences of this coexistence are unclear; however, it has been re orted that alanin (GAL) inhibits NE-induced cyclic P accumu ation in the cerebral cortex (Nishibori et al., 1988) and inhibits locus coeruleus neuronal firing in vitro (Seutin et al., 1989). Locus coeruleus (LC) neurons appear to be activated by stress, such as chronic cold exposure (Thoenen, 1970; Zigmond et al., 1974; Richard et al., 1988). This treatment, as well as treatment with the catecholaminedepleting drug, reserpine, increased tyrosine hydroxylase (TH) activity (Mueller et al., 1969; Reis et al., 1974, 1975; Renaud et al., 1979; Zigmond et al., 1974; Zigmond, 1979; Richard et al., 1988,1989) and TH mRNA levels (Mallet et al., 1983; Faucon Biguet et al., 1986; Berod et al., 1987; Labatut et al., 1988; Richard et al., 1988) in the locus coeruleus. Furthermore, reser ine administration has been previously reported to re uce both NE levels and neuropeptide Y immunoreactivity in neurons of the eripheral nervous system (Lundber et al., 1985), 5-rlydroxytryptamine, substance P, an$ thyrotropin-releasing hormone levels in ventral spinal cord neurons (Gilbert et al., 1981), and dopamine and neurotensin levels in prefrontal cortical neurons (Bean et al., 1989). These studies demonstrate that in regions of coexistenceboth monoamine and neuropeptide levels can be depleted by reserpine. The present study was designed to investigate
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whether two treatments thought to deplete NE in the LC, the relatively severe reserpine treatment and the relatively mild swim stress, would increase or decrease gene expression for both TH and GAL in LC neurons, and whether the direction of change would be the same for these coexisting neurotransmitters. In situ hybridization histochemistry was chosen to quantitate mRNA levels of TH and GAL, since the LC in the rat is a small, discrete, bilateral structure. MATERIALS AND METHODS Male Sprague-Dawley rats, 225-250 g, were group housed in a temperature- and humidity-controlled vivarium and provided with food and water ad libitum. The room was maintained on a 12 hour lightldark cycle, with lights on 7 am to 7 pm. In the first experiment, rats were injected with reserpine, either 2 or 10 mg/kg, i.p. (Serpasil; Ciba-Gei , Summit, NJ) or vehicle (0.05% ascorbic acid, 5% o yethylene glycol, distilled water) and returned to t eir home cage. Twent -four hours followin injection, rats were sacrificed b ecapitation, and the rains were rapidly removed an frozen on dry ice. In the second experiment, rats were individually laced into a round plexiglass tub filled with water 2 6 T ) and allowed to swim for 15 min. After the 15 min swim, the rat was removed, dried, and placed under a heat lam until dry. The rat was returned to its home cage, an the procedure repeated on the next 2 days. Rats were sacrificed by decapitation 24 h r after the third daily swim session and the brains rapidly removed
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Received December 7, 1989;accepted in revised form June 1,1990
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M.C. AUSTIN ET AL.
and frozen on dry ice. Coronal tissue sections (12 pm) were cut usin a cryostat and thaw-mounted coated slides. ections were chosen for in situ tion which corres onded to Figures 56-58 and Watson (1986f: Sections were fixed in 4% formaldeh de in 0.12 M sodium phos hate-buffered saline (PBS, p 7.4), rinsed twice in PB , and placed in 0.25% acetic anhydride in 0.1 M triethanolamine/0.9% NaC1, pH 8.0 for 10 min. Sections were then delipidated in a graded series of ethanol washes (70, 80, 95, and 100%) and chloroform. The rat TH oli onucleotide probe (Young et al., 19861, kindly provideI f by W.S. Young 111, NIMH, was com lementa to bases 1441-1488 of the rat TH 1985). The rat galanin oli mRNA (8rima et otide probe (Rokaeus et al., 19881, synthesizer by l eB.Martin, NIMH, was complementary to bases 115-153 of the mRNA encoding orcine pre rogalanin (Rokaeus and Brownstein, 19867. Oligonuc eotides were labeled with 35S dATP (New England Nuclear, Boston, MA) using terminal deoxynucleotidyltransferase (Boehringer Mannheim, West Germany) to a specific activity of 4,600-5,500 CYmmole. Brain sections were hybridized with 0.3-1.0 x lo6 cpdsection of TH or galanin robe in a buffer containing 50% formamide, 600 mM%aCl, 80 mM Tris-HC1(pH 7.5),4 mM EDTA, 0.2% sodium dode10%(wh)dextran placed in a humid incubator (37°C)for 16-20 hr. Sections were washed 4 times for 15 min each in 2~ SSC ( l x SSC: 0.15 M NaCV0.015 M sodium citrate, pH 7.2) 6 0 % formamide followed by two 30 min washes in l x SSC. All washes were done at 40°C. Slides were rinsed in distilled water, then in 70% ethanol, dried, and ex osed to X-ray film (XAR-5, Kodak) for 24-72 hours. xposure time was adjusted so that optical densities would be within the limits of saturation. Radioactive standards were made from serial dilutions of 35SdATP mixed with brain paste, sectioned, and placed on microscope slides which were coexposed to film with the tissue sections. After autoradio aphic analysis, slides were dipped in nuclear emugon (NTB 2, Kodak, diluted 1:l with distilled water) under safelight conditions and exposed for 1-3 weeks at 4°C. Slides were developed in D-19 developer (Kodak) for 2 min, rinsed in H 2 0 for 30 sec, fixed for 5 min, rinsed in H 0 for 15 min, and stained with thionin for histological iientification. Optical density measurements were determined using an image analysis system consisting of a li htbox (Northern Light) and camera (Sierra Scientificf urchased from Imaging Research, Apple Macintosh I1 and Image Inc. (Marlbaro, 1.23h software (W. Rasband, NIMH, Bethesda, MD) as previously described (Cottin ham et al., 1990). Optical density values quantitated rom the radioactive standards were entered with their corresponding d m values into a calibration table, and the relations ip between tissue radioactivity and optical density determined using the Image 1.23H software program. Using the Image 1.23h software, the domaycentral portion of each LC section was sampled usin a 7 x 7 mm square by two independent investigators. his region of the LC was chosen based on the report by Holets et al. (1988) showing that TH- and GAL-like immunoreactive neurons are more abundant in the dorsal and central ortions of the LC. Dpm values for each LC were derive by interpolation from the standard curve. Cellular mea-
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surements were performed by digitizing the brightfield image from a Zeiss microscope onto a computer screen via a video camera and the Image 1.23h software. Individual erikarya, as well as the nucleus and nucleolus of eac neuron were outlined and area of each structure was measured by the Image 1.23h software. Cellular measurements and swim stress data were analyzed usin a Student’s t-test. Reserpine treatment data were ana yzed using a one-way Analysis of Variance followed by a Scheffe’s F-test.
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RESULTS As previously reported, reserpine administration (2 or 10 mgkg) significantly increased TH mRNA levels in the LC compared to vehicle injected controls (Fig. 1A; treatment F, 13 = 21.62, P < .0002; Scheffe’sF-test P < .05 for vehicle vs. 2 mgkg, P < .005 for vehicle vs. 10
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Reserpine (mgkg, i.p.) Fig. 1. A The effect of reserpine on tyrosine hydroxylase mRNA levels in locus coeruleus neurons. B: The effect of reserpine on galanin mRNA levels in locus coeruleus neurons. Data are expressed as mean + S.E.M. d p d m g tissue. The number of ratdtreatment group is shown in parentheses. Multiple brain sections (2-4) were analyzed for each rat. *P < .05, **P < ,005 compared to the vehicle treatment group.
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TYROSINE HYDROXYLASE AND GALANIN mRNA
mgkg, P < .05 for 2 vs. 10 mgkg reser ine). Figure 1B shows that administration of 10 m&g of reserpine significantly increased GAL mRNA levels in LC neurons compared to controls (treatment F?, - 7.17, P < .012; Scheffe’s F-test P < .05 for v e h d e i s . 10 mgkg, NS for 2 vs. 10 mgkg reserpine). Although a trend may be a parent for 2 mgkg of reser ine to elevate GAL ml! NA levels, it was not signi icantly different from vehicle injected controls (P = .072). TH mRNA concentration was highly correlated with GAL mRNA concentration following reserpine or vehicle for the 11 animals in the three treatment groups (Fig. 2; P < .01). Autoradiograms obtained with the TH and GAL probes following photographic emulsion ex osure illustrate the increased silver ain densities of H and GAL mRNAs in LC perikarya ollowing reserpine treatment (Figs. 3,4). The area of the locus coeruleus (Figs. 3C,F) and size of individual neurons (Fig. 4B,D) were increased in the 10 mgkg reserpine group as compared to the control group. Soma area (vehicle; 1,037.8 & 41.7 sq mm, reserpine; 2,090.9 f 59.4 sq mm, P < .0001), nucleus area (vehicle; 286.0 9.9 sq mm, reserpine; 494.6 2 13.4 sq mm, P < .0001), and nucleolus area (vehicle; 73.0 f 2.4 sqmm, reserpine; 95.8 ? 2.2 sqmm, P < .0001)were all si ificantly increased by reserpine. E g u r e 5 shows that 3 consecutive days of swim stress did not significantly alter TH or GAL mRNA levels in
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LC neurons com ared to control values, 24 hr after the swim stress (TJ? t = 0.34, df = 7, NS; GAL: t = 1.42, df = 7, NS). DISCUSSION Previous biochemical studies using Northern blot techniques and in situ hybridization have documented that treatment with the monoamine-de leting drug, reserpine, significantly elevates TH mRN levels in the locus coeruleus (Mallet et al., 1983, Faucon Biguet et al., 1986, Berod et al., 1987). By employing in situ hybridization on tissue sections, we have confirmed and extended the previous reports by demonstratin a significant increase in TH mRNA levels in L neurons following both low and high acute doses of reserpine. The 123%increase reported herein a t 24 hr after acute reserpine (10 mgkg) is less than the 300% increase reported by Faucon Biguet et al. (1986) using the same dose of reserpine and time of sacrifice. Variability in reserpine effects on TH mRNA in LC neurons has been reported elsewhere, e. , Mallet et al. (1983) reported a 65% increase in TH miNA levels at 4 days following 10 mgkg reserpine, whereas Faucon Biguet et al. (1986) reported a 200% increase at 4 da s using the same dose of reser ine. In addition, in bot of these studies, TH mRNA evels were determined by Northern blot analysis rather than in situ h bridization. Only the 10 mgkg ose of reserpine significantly elevated GAL mRNA levels in LC neurons. The 40%
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Tyrosine Hydroxylase mRNA (dpdmg) Fig. 2. Significant correlation between the increase in tyrosine hydroxylase mRNA levels and the increase in galanin mRNA levels in locus coeruleus neurons followin vehicle or reserpine. Data are expressed as mean d p d m g tissue. 8pen circles represent 3 rats treated
with vehicle, shaded circles represent 4 rats treated with 2 m g k g reserpine, and solid circles represent 4 rats treated with 10 m g k g reserpine.
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M.C. AUSTIN ET AL.
Fig. 3. Darkfield photomicrographs revealing distribution of tyrosine hydroxylase (A,B,C) and galanin (D,E,F) mRNA in the locus coeruleus following injection of vehicle (A,D), 2 (B,E) or 10 (C,F) m g k g reserpine. 4V, fourth ventricle. Bar = 50 Fm.
increase in GAL mRNA levels is less than the observed increase in "H mRNA levels following the same dose of reserpine. '1 hese observations suggest that while both TH and GAL mRNA levels are increased by a high acute dose of reserpine, the induction of GAL gene expression ap ears to be less sensitive to reserpine treatment. $he increase in locus coeruleus volume and neuronal size that occurs following 10 m /kg reserpine is a novel observation. To the best of our nowledge, there are no ublished reports of LC neuronal volume increasing pollowin$ reserpine treatment. The mechanism by which a igh dose of reserpine would increase neuronal volume is unknown. Speculations include direct effects
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of increased intraneuronal biochemical activity roduced by reserpine, or a non-specific neurotoxic e fect produced by the high dose of this drug. Further experiments are planned to investigate this phenomenon. In terms of the present experiment, concentrations of mRNA would be artifactually reduced in the calculation of mRNA concentration per area in the enlarged locus coeruleus. The increases in TH and GAL mRNA observed after 10 mg/kg reserpine may therefore have been greater than that reported herein, if the LC neuronal size had remained normal. Studies have documented that chronically exposing rats to cold temperatures increases TH activity (Thoe-
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TYROSINE HYDROXYLASE AND GALANIN mRNA
Fig. 4. Brightfield photomicrogra hs of emulsion-coated autoradiograms demonstrating cellular localization of tyrosine hydroxylase (AJ) and galanin (C,D) mRNAs in locus coeruleus neurons following injection ofvehicle(A,C)or 10mgk of reserpine (B,D).Arrows illustrate neurons with dense silver grains correspondingto TH and GAL mRfiAs. Bar = 50 pm.
nen, 1970; Zigmond et al., 1974) and TH mRNA levels (Richard et al., 1988) in the LC. In the present study, 3 consecutive days of swim stress failed to produce a significant increase in TH or GAL mRNA concentrations in the LC 24 hr after the last stressor. This lack of effect may reflect the milder nature of the brief daily swim paradigm. After the daily swim session, the rats were allowed to recover until the next day, such that the sacrifice time point of 24 hr after the last swim session would be consistent with the time of sacrifice for the reserpine experiment. This swim stress aradigm differs from the cold stress experiments in w ich rats were constantly exposed to the stress (Zigmond et al., 1974; Richard et al., 1988).Thus, the swim stress procedure is considerabl less severe than the cold stress procedure. Alternative y, the time point of sacrifice, 24 hr after the last swim session, may allow small changes in mRNA levels induced by the swim stress to return t o baseline.
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The resent findings of in mR8A for TH and mechanisms which are sive. One possibility is that the increases in TH and GAL mRNA levels following reserpine are due to reductions in the enzymatic degradation of the mRNAs. Another more interesting possibility is that reserpine acts directly to deplete a opulation of vesicles in which galanin and norepinep rine may be costored. Su port for this argument was shown in a recent study y Bean et al. (1989)su gesting that a percentage of neurotensin is costored wit dopamine in reserpine-sensitive large vesicles in prefrontal cortical neurons. The fact that GAL gene expression appears less sensitive than TH gene expression to reserpine treatment may argue for the existence of different vesicular 001s for these neurotransmitters. Nerve terminal relpease of neuropeptides which are colocalized with classical neurotrans-
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depleted GAL terminal stores. Our finding that reserpine, but not 3 days of mild swim stress, increased TH and GAL mRNA in LC neurons would suggest that severe conditions are required to induce transcriptional activity for the synthesis of TH and GAL.
CONTROL 3-DAYSWIM
ACKNOWLEDGMENTS We wish to thank Dr. Marianne Schultzberg for her advice and assistance in preparing the photomicrographs, Dr. Brian Martin for synthesizing the alanin oligonucleotideprobe, Dr. Scott Young for rovi in the t rosine hydroxylase oligonucleotide prole, Mr. fohn Jvers for technical assistance in sectioning the LC, and Ms. Marie Potter for technical assistance in quantitating mRNA concentrations.
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REFERENCES
TH
GAL
Fig. 5. Lack of effect of 3 day swim stress on tyrosine hydroxylase (TH)and galanin (GAL)mRNA levels in locus coeruleus neurons. Data are expressed as mean + S.E.M. "dmg tissue. The number of rats/ treatment grou is shown in parent eses Multiple brain sections( 2 4 ) were analyzed &r each rat.
mitters often require more rolonged or higher frequencies of neuronal firing bartfai et al., 1988). Thus, low doses of reserpine could preferentially deplete the sub opulation of vesicles containing NE, whereas higher foses of reserpine could deplete both NE and the less sensitive NE and GAL storage vesicles. Nerve terminal depletion of these stora e compartments would then increase transcription or both of these neurotransmitters. Galanin is colocalized in 80-90% of the norepinephrine-containing neurons in the LC (Melander et al., 1986; Holets et al., 1988). In addition, alanin-immunoreactive neurons appear to be most a undant in the dorsal and central portions of the LC , and virtual1 all of these cells co-contain TH-immunoreactivity (Ho ets et al., 1988). The dorsal and central LC was chosen for quantitation in the present study. Therefore, it is unlike1 that the effects of reserpine on TH and GAL mR A levels in the present study are due to an action on a specific subpopulation of LC neurons, since the dorsal/ central LC is relatively homogeneous for neurons containing these two neurotransmitters. The increase in TH and GAL mRNA levels may be related to the frequency of neuronal firing. Reser ine ma act directly to increase LC neuronal firing (jitts an Marwah, 1987). In addition, the physiological state of the rat induced by high doses of reserpine may be a severe stressor which indirectly activates LC neurons. The prolonged increase in firin rate may then deplete terminal stores of NE and G , and thus induce an increase in transcription. Our finding that reserpine increased TH mRNA levels in a dose-de endent fashion, GAL m NA levels after only this higher dose of reserLC neuronal firing and thus
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Bartfai, T., Iverfeldt, K., Fisone, G., and Serozo, P. (1988)Re lation of the release of coexisting neurotransmitters. Annu. Rev. Pyarmacol. Toxicol., 28:285-310. Bean, A.J., Adrian, T.E.,Modlin, I.M., and Roth, R.H. (1989)Dopamine and neurotensin storage in colocalized and noncolocalized neuronal populations. J . Pharmacol. Exp. Ther., 249:681487. Berod A,, Faucon Biguet, N., Dumas, S.,Bloch, B., and Mallet J. (1987) Modulation of tyrosine hydroxylase gene expression in the central nervous system visualized by in situ hybridization. Proc. Natl. Acad. Sci. USA, 84:1699-1703. Cottingham, S.L., Pickar, D., Shirnotake, T.K., Montpied, P., Paul, S.M., and Crawley, J.N., (1990) Tyrosine hydroxylase and cholecystokinin mRNA levels in the substantia nigra, ventral tegmental area and locus ceruleus are unaffected b acute and chronic haloperido1 administration. Cell. Mol. NeurobioT, 10:41-50. Faucon Biguet, N., Buda, M., Lamouroux,A., Sarnolyk, D., and Mallet, J. (1986) Time course of the changes of TH mRNA in rat brain and adrenal medulla after a single injection of reserpine. EMBO J. 5:287-291. Gilbert, R.F.T., Bennett, G.W., Marsden, C.A., andEmson, P.C. (1981) The effects of 5-hydroxytryptamine-depletingdrugs on peptides in the ventral spinal cord. Eur. J. Pharmacol., 76:203-210. Grima, B., Lamourom, A., Blanot, F., Faucon Biguet, N., and Mallet,J. (1985)Comuletecoding seauence of rat tvrosine hvdroxvlase mRNA. " Proc. Natl. kcad. Sci. GSA: 82:61 -621." Holets, V.R., Hokfelt, T., Rokaeus, ., Terenius, L., and Goldstein, M. (1988)Locus coeruleus neurons in the rat containinmeuroueutideY. tyrosine hydroxylaseor galanin and their efferent iro'ectibn's to the spinal cord, cerebral cortex and hypothalamus. keuroscience, 242393-906. Labatut, R., Buda, M., and Berod, A. (1988) Long-term changes in rat brain tyrosine hydrox lase followingreserpine treatment: a quantitative immunohistocgemical analysis. J. Neurochem., 50:13751380. Lundberli, J.M., Saria, A., Franco-Cereceda, A., Hokfelt, T., Terenius, L., an Goldstein, M. (1985) Differential effects of reserpine and 6-hydroxydopamineon neuropeptide Y (NPY) and noradrenaline in peripheral neurons. Naunyn Schmiedebergs Arch. Pharmacol., 328:331-340. Mallet, J., Faucon Biguet, N., Buda, M., Lamourom, A,, and Samolyk, D. (1983) Detection and regulation of the tyrosine h droxylase mRNA levels in rat adrenal medulla and brain tissues. d l d Spring Harbor S p uant. Biol., 48:305- 08. Melander, Ha . elt, T., Rokaeus, ., Cuello, A.C., Oertel, W.H., Verhofstad, A., and Goldstein, M. (1986) Coexistence of galaninlike immunoreactivity with catecholamines, 5-hydroxytryptamine, GABA and neuropeptides in the rat CNS. J. Neurosci., 6:3640-3654. Mueller, R.A., Thoenen, H., and Axelrod, J. (1969)Increase in tyrosine hydroxylase activity after reserpine administration. J. Pharmacol. Exp. Ther., 169:7479. Nishibori, M., Oishi, R., Itoh, Y., and Sacki, K. (1988)Galanin inhibits noradrenaline-induced accumulation of cyclic AMP in the rat cerebral cortex. J. Neurochem.. 51:1953-1955. Paxinos, G., and Watson, C. (1986) The Rat Brain in Stereotaxic Coordinates.Academic Press, New York. Pitts, D.K., and Manvah, J. (1987) Electrophysiological actions of cocaine on noradrenergic neurons in rat lo& ceruleus. J . Pharmacol. Ex Ther., 240:345-351. Reis, D . f ; Joh, T.H., Ross, R.A., and Pickel, V.M. (1974) Reser ine selectivelv increases tvrosine hvdroxvlase and douamine-B-hvzoxylase enzyme protein in central noridrenergic nehons. Bra& Res. 81:380-386.
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TYROSINE HYDROXYLASE AND GALANIN mRNA Reis, D.J., Joh, T.H., and Ross, R.A. (1975) Effects of reserpine on activities and amounts of tyrosine hydroxylase and dopaminehydroxylase in catecholamine neuronal systems in rat brain. J . harmacol. Exp. Ther., 193:775-784. Renaud, B., Joh, T.H., Snyder, D.W., and Reis, D.J. (1979) Induction and delayed activation of tyrosine hydroxylase in noradrenergic neurons of A1 and A2 groups of medulla oblongata of rat. Brain Res., 1761169-174. Richard, F., Faucon Biguet, N., Labatut, R., Rollet, D., Mallet, J., and Buda, M. (1988)Modulation of tyrosine hydroxylase ene expression in rat brain and adrenal by exposure to cold. J. aeurosci. Res., 20:32-37. Richard, F., Labatut, R., Weissmann,,D.,. Scarna, H., Buda, M., and Pujol, J.F. (1989) Further characterization of the tyrosine hydroxylase induction elicited by reserpine in the rat locus coeruleus and adrenals. Neurochem. Int., 14:199-205. Rokaeus, A,, and Brownstein, M.J. (1986) Construction of a porcine adrenal medullary cDNA library and nucleotide se uence analysis of two clones encoding a galanin Drecursor. Proc. NaA. Acad. Sci. USA, 83:6287-6291. Rokaeus, A., Young, W.S. 111, and Meze E (1988) Galanin coexists with vasopressin in the normal rat &pothalamus and galanin’s
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s thesis is increased in the Brattleboro (diabetes insipidus) rat. gurosci. Lett., 90:45-50. Seutin, V., Verbanck, P., Massotte, L., and Dresse, A. (1989) Galanin decreases the activity of locus coeruleus neurons in vitro. Eur. J. Pharmacol., 164:373- 76. Tatemoto K., Rokaeus, ., Jornvall, H., McDonald, T.J., and Mutt, V. (1983)dalanin-a novel biologicallyactive peptide from porcine intestine. FEBS Lett., 164:124-128. Thoenen, H. (1970)Induction oftyrosine hydroxylase in peripheral and central adrenergic neurones by cold-exposure of rats. Nature, 228:861-862. Young, W.S. 111, Bonner, T.I., and Brann, M.R. (1986) Mesence halic neurons repdate the expryion of neuropeptide mRNAs in t i e rat forebrain. roc Natl Acad Sci USA, 83:9827-9831. Zigmond, R.E. (1979) Tyrosine hydroxylase activity in noradrenergic neurons of the locus coeruleus after reserpine administration: seguential increase in cell bodies and nerve terminals. J. Neurochem., 2:23-29. Zi ond, R.E., Schon, F., and Iversen, L.L. (1974) Increased tyrosine gdroxylase activity in the locus coeruleus of rat brain stem after reserpine treatment and cold stress. Brain Res., 70:547-552.
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Tyrosine Hydroxylase and Galanin *A Levels in Locus Coeruleus Neurons Are Increased Following. Reserpine Administration v
MARK C. AUSTIN, SANDRA L. COTTINGHAM, STEVEN M. PAUL, AND JACQUELINE N. CRAWLEY Clinical Neuroscience Branch, National Institute of Mental Health, Bethesda, Maryland, 20892
KEY WORDS
Neuropeptide, Norepinephrine, Neuron, Coexistence, Synthesis, Depletion
ABSTRACT
The neuropeptide galanin coexists in 80-90% of the norepinephrinecontaining neurons in the locus coeruleus. In situ hybridization histochemistry was used to examine the effects of reserpine treatment or swim stress on tyrosine hydroxylase and galanin mRNA concentrations in locus coeruleus neurons. Reserpine administration significantly increased tyrosine hydroxylase and galanin mRNA levels in the locus coeruleus. The reserpine-induced increase in tyrosine hydroxylase mRNA was significantly correlated with the reserpine-induced increase in galanin mRNA. Three consecutive days of swim stress did not significantly alter either tyrosine hydroxylase or galanin mRNA concentrations in the locus coeruleus. These data suggest that both tyrosine hydroxylase and galanin gene expression in locus coeruleus neurons may be regulated by a reserpine-sensitive mechanism.
INTRODUCTION Galanin, a 29 amino acid residue pe tide originally isolated from orcine small intestine ( atemoto et al., 1983), is foun in the majority of norepinephrine (NE)containing neurons of the locus coeruleus (Melander et al., 1986; Holets et al., 1988). The functional consequences of this coexistence are unclear; however, it has been re orted that alanin (GAL) inhibits NE-induced cyclic P accumu ation in the cerebral cortex (Nishibori et al., 1988) and inhibits locus coeruleus neuronal firing in vitro (Seutin et al., 1989). Locus coeruleus (LC) neurons appear to be activated by stress, such as chronic cold exposure (Thoenen, 1970; Zigmond et al., 1974; Richard et al., 1988). This treatment, as well as treatment with the catecholaminedepleting drug, reserpine, increased tyrosine hydroxylase (TH) activity (Mueller et al., 1969; Reis et al., 1974, 1975; Renaud et al., 1979; Zigmond et al., 1974; Zigmond, 1979; Richard et al., 1988,1989) and TH mRNA levels (Mallet et al., 1983; Faucon Biguet et al., 1986; Berod et al., 1987; Labatut et al., 1988; Richard et al., 1988) in the locus coeruleus. Furthermore, reser ine administration has been previously reported to re uce both NE levels and neuropeptide Y immunoreactivity in neurons of the eripheral nervous system (Lundber et al., 1985), 5-rlydroxytryptamine, substance P, an$ thyrotropin-releasing hormone levels in ventral spinal cord neurons (Gilbert et al., 1981), and dopamine and neurotensin levels in prefrontal cortical neurons (Bean et al., 1989). These studies demonstrate that in regions of coexistenceboth monoamine and neuropeptide levels can be depleted by reserpine. The present study was designed to investigate
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PUBLISHED 1990 WILEY-LISS. INC
whether two treatments thought to deplete NE in the LC, the relatively severe reserpine treatment and the relatively mild swim stress, would increase or decrease gene expression for both TH and GAL in LC neurons, and whether the direction of change would be the same for these coexisting neurotransmitters. In situ hybridization histochemistry was chosen to quantitate mRNA levels of TH and GAL, since the LC in the rat is a small, discrete, bilateral structure. MATERIALS AND METHODS Male Sprague-Dawley rats, 225-250 g, were group housed in a temperature- and humidity-controlled vivarium and provided with food and water ad libitum. The room was maintained on a 12 hour lightldark cycle, with lights on 7 am to 7 pm. In the first experiment, rats were injected with reserpine, either 2 or 10 mg/kg, i.p. (Serpasil; Ciba-Gei , Summit, NJ) or vehicle (0.05% ascorbic acid, 5% o yethylene glycol, distilled water) and returned to t eir home cage. Twent -four hours followin injection, rats were sacrificed b ecapitation, and the rains were rapidly removed an frozen on dry ice. In the second experiment, rats were individually laced into a round plexiglass tub filled with water 2 6 T ) and allowed to swim for 15 min. After the 15 min swim, the rat was removed, dried, and placed under a heat lam until dry. The rat was returned to its home cage, an the procedure repeated on the next 2 days. Rats were sacrificed by decapitation 24 h r after the third daily swim session and the brains rapidly removed
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Received December 7, 1989;accepted in revised form June 1,1990
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M.C. AUSTIN ET AL.
and frozen on dry ice. Coronal tissue sections (12 pm) were cut usin a cryostat and thaw-mounted coated slides. ections were chosen for in situ tion which corres onded to Figures 56-58 and Watson (1986f: Sections were fixed in 4% formaldeh de in 0.12 M sodium phos hate-buffered saline (PBS, p 7.4), rinsed twice in PB , and placed in 0.25% acetic anhydride in 0.1 M triethanolamine/0.9% NaC1, pH 8.0 for 10 min. Sections were then delipidated in a graded series of ethanol washes (70, 80, 95, and 100%) and chloroform. The rat TH oli onucleotide probe (Young et al., 19861, kindly provideI f by W.S. Young 111, NIMH, was com lementa to bases 1441-1488 of the rat TH 1985). The rat galanin oli mRNA (8rima et otide probe (Rokaeus et al., 19881, synthesizer by l eB.Martin, NIMH, was complementary to bases 115-153 of the mRNA encoding orcine pre rogalanin (Rokaeus and Brownstein, 19867. Oligonuc eotides were labeled with 35S dATP (New England Nuclear, Boston, MA) using terminal deoxynucleotidyltransferase (Boehringer Mannheim, West Germany) to a specific activity of 4,600-5,500 CYmmole. Brain sections were hybridized with 0.3-1.0 x lo6 cpdsection of TH or galanin robe in a buffer containing 50% formamide, 600 mM%aCl, 80 mM Tris-HC1(pH 7.5),4 mM EDTA, 0.2% sodium dode10%(wh)dextran placed in a humid incubator (37°C)for 16-20 hr. Sections were washed 4 times for 15 min each in 2~ SSC ( l x SSC: 0.15 M NaCV0.015 M sodium citrate, pH 7.2) 6 0 % formamide followed by two 30 min washes in l x SSC. All washes were done at 40°C. Slides were rinsed in distilled water, then in 70% ethanol, dried, and ex osed to X-ray film (XAR-5, Kodak) for 24-72 hours. xposure time was adjusted so that optical densities would be within the limits of saturation. Radioactive standards were made from serial dilutions of 35SdATP mixed with brain paste, sectioned, and placed on microscope slides which were coexposed to film with the tissue sections. After autoradio aphic analysis, slides were dipped in nuclear emugon (NTB 2, Kodak, diluted 1:l with distilled water) under safelight conditions and exposed for 1-3 weeks at 4°C. Slides were developed in D-19 developer (Kodak) for 2 min, rinsed in H 2 0 for 30 sec, fixed for 5 min, rinsed in H 0 for 15 min, and stained with thionin for histological iientification. Optical density measurements were determined using an image analysis system consisting of a li htbox (Northern Light) and camera (Sierra Scientificf urchased from Imaging Research, Apple Macintosh I1 and Image Inc. (Marlbaro, 1.23h software (W. Rasband, NIMH, Bethesda, MD) as previously described (Cottin ham et al., 1990). Optical density values quantitated rom the radioactive standards were entered with their corresponding d m values into a calibration table, and the relations ip between tissue radioactivity and optical density determined using the Image 1.23H software program. Using the Image 1.23h software, the domaycentral portion of each LC section was sampled usin a 7 x 7 mm square by two independent investigators. his region of the LC was chosen based on the report by Holets et al. (1988) showing that TH- and GAL-like immunoreactive neurons are more abundant in the dorsal and central ortions of the LC. Dpm values for each LC were derive by interpolation from the standard curve. Cellular mea-
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surements were performed by digitizing the brightfield image from a Zeiss microscope onto a computer screen via a video camera and the Image 1.23h software. Individual erikarya, as well as the nucleus and nucleolus of eac neuron were outlined and area of each structure was measured by the Image 1.23h software. Cellular measurements and swim stress data were analyzed usin a Student’s t-test. Reserpine treatment data were ana yzed using a one-way Analysis of Variance followed by a Scheffe’s F-test.
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RESULTS As previously reported, reserpine administration (2 or 10 mgkg) significantly increased TH mRNA levels in the LC compared to vehicle injected controls (Fig. 1A; treatment F, 13 = 21.62, P < .0002; Scheffe’sF-test P < .05 for vehicle vs. 2 mgkg, P < .005 for vehicle vs. 10
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Reserpine (mgkg, i.p.) Fig. 1. A The effect of reserpine on tyrosine hydroxylase mRNA levels in locus coeruleus neurons. B: The effect of reserpine on galanin mRNA levels in locus coeruleus neurons. Data are expressed as mean + S.E.M. d p d m g tissue. The number of ratdtreatment group is shown in parentheses. Multiple brain sections (2-4) were analyzed for each rat. *P < .05, **P < ,005 compared to the vehicle treatment group.
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TYROSINE HYDROXYLASE AND GALANIN mRNA
mgkg, P < .05 for 2 vs. 10 mgkg reser ine). Figure 1B shows that administration of 10 m&g of reserpine significantly increased GAL mRNA levels in LC neurons compared to controls (treatment F?, - 7.17, P < .012; Scheffe’s F-test P < .05 for v e h d e i s . 10 mgkg, NS for 2 vs. 10 mgkg reserpine). Although a trend may be a parent for 2 mgkg of reser ine to elevate GAL ml! NA levels, it was not signi icantly different from vehicle injected controls (P = .072). TH mRNA concentration was highly correlated with GAL mRNA concentration following reserpine or vehicle for the 11 animals in the three treatment groups (Fig. 2; P < .01). Autoradiograms obtained with the TH and GAL probes following photographic emulsion ex osure illustrate the increased silver ain densities of H and GAL mRNAs in LC perikarya ollowing reserpine treatment (Figs. 3,4). The area of the locus coeruleus (Figs. 3C,F) and size of individual neurons (Fig. 4B,D) were increased in the 10 mgkg reserpine group as compared to the control group. Soma area (vehicle; 1,037.8 & 41.7 sq mm, reserpine; 2,090.9 f 59.4 sq mm, P < .0001), nucleus area (vehicle; 286.0 9.9 sq mm, reserpine; 494.6 2 13.4 sq mm, P < .0001), and nucleolus area (vehicle; 73.0 f 2.4 sqmm, reserpine; 95.8 ? 2.2 sqmm, P < .0001)were all si ificantly increased by reserpine. E g u r e 5 shows that 3 consecutive days of swim stress did not significantly alter TH or GAL mRNA levels in
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LC neurons com ared to control values, 24 hr after the swim stress (TJ? t = 0.34, df = 7, NS; GAL: t = 1.42, df = 7, NS). DISCUSSION Previous biochemical studies using Northern blot techniques and in situ hybridization have documented that treatment with the monoamine-de leting drug, reserpine, significantly elevates TH mRN levels in the locus coeruleus (Mallet et al., 1983, Faucon Biguet et al., 1986, Berod et al., 1987). By employing in situ hybridization on tissue sections, we have confirmed and extended the previous reports by demonstratin a significant increase in TH mRNA levels in L neurons following both low and high acute doses of reserpine. The 123%increase reported herein a t 24 hr after acute reserpine (10 mgkg) is less than the 300% increase reported by Faucon Biguet et al. (1986) using the same dose of reserpine and time of sacrifice. Variability in reserpine effects on TH mRNA in LC neurons has been reported elsewhere, e. , Mallet et al. (1983) reported a 65% increase in TH miNA levels at 4 days following 10 mgkg reserpine, whereas Faucon Biguet et al. (1986) reported a 200% increase at 4 da s using the same dose of reser ine. In addition, in bot of these studies, TH mRNA evels were determined by Northern blot analysis rather than in situ h bridization. Only the 10 mgkg ose of reserpine significantly elevated GAL mRNA levels in LC neurons. The 40%
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Tyrosine Hydroxylase mRNA (dpdmg) Fig. 2. Significant correlation between the increase in tyrosine hydroxylase mRNA levels and the increase in galanin mRNA levels in locus coeruleus neurons followin vehicle or reserpine. Data are expressed as mean d p d m g tissue. 8pen circles represent 3 rats treated
with vehicle, shaded circles represent 4 rats treated with 2 m g k g reserpine, and solid circles represent 4 rats treated with 10 m g k g reserpine.
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M.C. AUSTIN ET AL.
Fig. 3. Darkfield photomicrographs revealing distribution of tyrosine hydroxylase (A,B,C) and galanin (D,E,F) mRNA in the locus coeruleus following injection of vehicle (A,D), 2 (B,E) or 10 (C,F) m g k g reserpine. 4V, fourth ventricle. Bar = 50 Fm.
increase in GAL mRNA levels is less than the observed increase in "H mRNA levels following the same dose of reserpine. '1 hese observations suggest that while both TH and GAL mRNA levels are increased by a high acute dose of reserpine, the induction of GAL gene expression ap ears to be less sensitive to reserpine treatment. $he increase in locus coeruleus volume and neuronal size that occurs following 10 m /kg reserpine is a novel observation. To the best of our nowledge, there are no ublished reports of LC neuronal volume increasing pollowin$ reserpine treatment. The mechanism by which a igh dose of reserpine would increase neuronal volume is unknown. Speculations include direct effects
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of increased intraneuronal biochemical activity roduced by reserpine, or a non-specific neurotoxic e fect produced by the high dose of this drug. Further experiments are planned to investigate this phenomenon. In terms of the present experiment, concentrations of mRNA would be artifactually reduced in the calculation of mRNA concentration per area in the enlarged locus coeruleus. The increases in TH and GAL mRNA observed after 10 mg/kg reserpine may therefore have been greater than that reported herein, if the LC neuronal size had remained normal. Studies have documented that chronically exposing rats to cold temperatures increases TH activity (Thoe-
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Fig. 4. Brightfield photomicrogra hs of emulsion-coated autoradiograms demonstrating cellular localization of tyrosine hydroxylase (AJ) and galanin (C,D) mRNAs in locus coeruleus neurons following injection ofvehicle(A,C)or 10mgk of reserpine (B,D).Arrows illustrate neurons with dense silver grains correspondingto TH and GAL mRfiAs. Bar = 50 pm.
nen, 1970; Zigmond et al., 1974) and TH mRNA levels (Richard et al., 1988) in the LC. In the present study, 3 consecutive days of swim stress failed to produce a significant increase in TH or GAL mRNA concentrations in the LC 24 hr after the last stressor. This lack of effect may reflect the milder nature of the brief daily swim paradigm. After the daily swim session, the rats were allowed to recover until the next day, such that the sacrifice time point of 24 hr after the last swim session would be consistent with the time of sacrifice for the reserpine experiment. This swim stress aradigm differs from the cold stress experiments in w ich rats were constantly exposed to the stress (Zigmond et al., 1974; Richard et al., 1988).Thus, the swim stress procedure is considerabl less severe than the cold stress procedure. Alternative y, the time point of sacrifice, 24 hr after the last swim session, may allow small changes in mRNA levels induced by the swim stress to return t o baseline.
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The resent findings of in mR8A for TH and mechanisms which are sive. One possibility is that the increases in TH and GAL mRNA levels following reserpine are due to reductions in the enzymatic degradation of the mRNAs. Another more interesting possibility is that reserpine acts directly to deplete a opulation of vesicles in which galanin and norepinep rine may be costored. Su port for this argument was shown in a recent study y Bean et al. (1989)su gesting that a percentage of neurotensin is costored wit dopamine in reserpine-sensitive large vesicles in prefrontal cortical neurons. The fact that GAL gene expression appears less sensitive than TH gene expression to reserpine treatment may argue for the existence of different vesicular 001s for these neurotransmitters. Nerve terminal relpease of neuropeptides which are colocalized with classical neurotrans-
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M.C. AUSTl'N ET AL.
depleted GAL terminal stores. Our finding that reserpine, but not 3 days of mild swim stress, increased TH and GAL mRNA in LC neurons would suggest that severe conditions are required to induce transcriptional activity for the synthesis of TH and GAL.
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ACKNOWLEDGMENTS We wish to thank Dr. Marianne Schultzberg for her advice and assistance in preparing the photomicrographs, Dr. Brian Martin for synthesizing the alanin oligonucleotideprobe, Dr. Scott Young for rovi in the t rosine hydroxylase oligonucleotide prole, Mr. fohn Jvers for technical assistance in sectioning the LC, and Ms. Marie Potter for technical assistance in quantitating mRNA concentrations.
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REFERENCES
TH
GAL
Fig. 5. Lack of effect of 3 day swim stress on tyrosine hydroxylase (TH)and galanin (GAL)mRNA levels in locus coeruleus neurons. Data are expressed as mean + S.E.M. "dmg tissue. The number of rats/ treatment grou is shown in parent eses Multiple brain sections( 2 4 ) were analyzed &r each rat.
mitters often require more rolonged or higher frequencies of neuronal firing bartfai et al., 1988). Thus, low doses of reserpine could preferentially deplete the sub opulation of vesicles containing NE, whereas higher foses of reserpine could deplete both NE and the less sensitive NE and GAL storage vesicles. Nerve terminal depletion of these stora e compartments would then increase transcription or both of these neurotransmitters. Galanin is colocalized in 80-90% of the norepinephrine-containing neurons in the LC (Melander et al., 1986; Holets et al., 1988). In addition, alanin-immunoreactive neurons appear to be most a undant in the dorsal and central portions of the LC , and virtual1 all of these cells co-contain TH-immunoreactivity (Ho ets et al., 1988). The dorsal and central LC was chosen for quantitation in the present study. Therefore, it is unlike1 that the effects of reserpine on TH and GAL mR A levels in the present study are due to an action on a specific subpopulation of LC neurons, since the dorsal/ central LC is relatively homogeneous for neurons containing these two neurotransmitters. The increase in TH and GAL mRNA levels may be related to the frequency of neuronal firing. Reser ine ma act directly to increase LC neuronal firing (jitts an Marwah, 1987). In addition, the physiological state of the rat induced by high doses of reserpine may be a severe stressor which indirectly activates LC neurons. The prolonged increase in firin rate may then deplete terminal stores of NE and G , and thus induce an increase in transcription. Our finding that reserpine increased TH mRNA levels in a dose-de endent fashion, GAL m NA levels after only this higher dose of reserLC neuronal firing and thus
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Bartfai, T., Iverfeldt, K., Fisone, G., and Serozo, P. (1988)Re lation of the release of coexisting neurotransmitters. Annu. Rev. Pyarmacol. Toxicol., 28:285-310. Bean, A.J., Adrian, T.E.,Modlin, I.M., and Roth, R.H. (1989)Dopamine and neurotensin storage in colocalized and noncolocalized neuronal populations. J . Pharmacol. Exp. Ther., 249:681487. Berod A,, Faucon Biguet, N., Dumas, S.,Bloch, B., and Mallet J. (1987) Modulation of tyrosine hydroxylase gene expression in the central nervous system visualized by in situ hybridization. Proc. Natl. Acad. Sci. USA, 84:1699-1703. Cottingham, S.L., Pickar, D., Shirnotake, T.K., Montpied, P., Paul, S.M., and Crawley, J.N., (1990) Tyrosine hydroxylase and cholecystokinin mRNA levels in the substantia nigra, ventral tegmental area and locus ceruleus are unaffected b acute and chronic haloperido1 administration. Cell. Mol. NeurobioT, 10:41-50. Faucon Biguet, N., Buda, M., Lamouroux,A., Sarnolyk, D., and Mallet, J. (1986) Time course of the changes of TH mRNA in rat brain and adrenal medulla after a single injection of reserpine. EMBO J. 5:287-291. Gilbert, R.F.T., Bennett, G.W., Marsden, C.A., andEmson, P.C. (1981) The effects of 5-hydroxytryptamine-depletingdrugs on peptides in the ventral spinal cord. Eur. J. Pharmacol., 76:203-210. Grima, B., Lamourom, A., Blanot, F., Faucon Biguet, N., and Mallet,J. (1985)Comuletecoding seauence of rat tvrosine hvdroxvlase mRNA. " Proc. Natl. kcad. Sci. GSA: 82:61 -621." Holets, V.R., Hokfelt, T., Rokaeus, ., Terenius, L., and Goldstein, M. (1988)Locus coeruleus neurons in the rat containinmeuroueutideY. tyrosine hydroxylaseor galanin and their efferent iro'ectibn's to the spinal cord, cerebral cortex and hypothalamus. keuroscience, 242393-906. Labatut, R., Buda, M., and Berod, A. (1988) Long-term changes in rat brain tyrosine hydrox lase followingreserpine treatment: a quantitative immunohistocgemical analysis. J. Neurochem., 50:13751380. Lundberli, J.M., Saria, A., Franco-Cereceda, A., Hokfelt, T., Terenius, L., an Goldstein, M. (1985) Differential effects of reserpine and 6-hydroxydopamineon neuropeptide Y (NPY) and noradrenaline in peripheral neurons. Naunyn Schmiedebergs Arch. Pharmacol., 328:331-340. Mallet, J., Faucon Biguet, N., Buda, M., Lamourom, A,, and Samolyk, D. (1983) Detection and regulation of the tyrosine h droxylase mRNA levels in rat adrenal medulla and brain tissues. d l d Spring Harbor S p uant. Biol., 48:305- 08. Melander, Ha . elt, T., Rokaeus, ., Cuello, A.C., Oertel, W.H., Verhofstad, A., and Goldstein, M. (1986) Coexistence of galaninlike immunoreactivity with catecholamines, 5-hydroxytryptamine, GABA and neuropeptides in the rat CNS. J. Neurosci., 6:3640-3654. Mueller, R.A., Thoenen, H., and Axelrod, J. (1969)Increase in tyrosine hydroxylase activity after reserpine administration. J. Pharmacol. Exp. Ther., 169:7479. Nishibori, M., Oishi, R., Itoh, Y., and Sacki, K. (1988)Galanin inhibits noradrenaline-induced accumulation of cyclic AMP in the rat cerebral cortex. J. Neurochem.. 51:1953-1955. Paxinos, G., and Watson, C. (1986) The Rat Brain in Stereotaxic Coordinates.Academic Press, New York. Pitts, D.K., and Manvah, J. (1987) Electrophysiological actions of cocaine on noradrenergic neurons in rat lo& ceruleus. J . Pharmacol. Ex Ther., 240:345-351. Reis, D . f ; Joh, T.H., Ross, R.A., and Pickel, V.M. (1974) Reser ine selectivelv increases tvrosine hvdroxvlase and douamine-B-hvzoxylase enzyme protein in central noridrenergic nehons. Bra& Res. 81:380-386.
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TYROSINE HYDROXYLASE AND GALANIN mRNA Reis, D.J., Joh, T.H., and Ross, R.A. (1975) Effects of reserpine on activities and amounts of tyrosine hydroxylase and dopaminehydroxylase in catecholamine neuronal systems in rat brain. J . harmacol. Exp. Ther., 193:775-784. Renaud, B., Joh, T.H., Snyder, D.W., and Reis, D.J. (1979) Induction and delayed activation of tyrosine hydroxylase in noradrenergic neurons of A1 and A2 groups of medulla oblongata of rat. Brain Res., 1761169-174. Richard, F., Faucon Biguet, N., Labatut, R., Rollet, D., Mallet, J., and Buda, M. (1988)Modulation of tyrosine hydroxylase ene expression in rat brain and adrenal by exposure to cold. J. aeurosci. Res., 20:32-37. Richard, F., Labatut, R., Weissmann,,D.,. Scarna, H., Buda, M., and Pujol, J.F. (1989) Further characterization of the tyrosine hydroxylase induction elicited by reserpine in the rat locus coeruleus and adrenals. Neurochem. Int., 14:199-205. Rokaeus, A,, and Brownstein, M.J. (1986) Construction of a porcine adrenal medullary cDNA library and nucleotide se uence analysis of two clones encoding a galanin Drecursor. Proc. NaA. Acad. Sci. USA, 83:6287-6291. Rokaeus, A., Young, W.S. 111, and Meze E (1988) Galanin coexists with vasopressin in the normal rat &pothalamus and galanin’s
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s thesis is increased in the Brattleboro (diabetes insipidus) rat. gurosci. Lett., 90:45-50. Seutin, V., Verbanck, P., Massotte, L., and Dresse, A. (1989) Galanin decreases the activity of locus coeruleus neurons in vitro. Eur. J. Pharmacol., 164:373- 76. Tatemoto K., Rokaeus, ., Jornvall, H., McDonald, T.J., and Mutt, V. (1983)dalanin-a novel biologicallyactive peptide from porcine intestine. FEBS Lett., 164:124-128. Thoenen, H. (1970)Induction oftyrosine hydroxylase in peripheral and central adrenergic neurones by cold-exposure of rats. Nature, 228:861-862. Young, W.S. 111, Bonner, T.I., and Brann, M.R. (1986) Mesence halic neurons repdate the expryion of neuropeptide mRNAs in t i e rat forebrain. roc Natl Acad Sci USA, 83:9827-9831. Zigmond, R.E. (1979) Tyrosine hydroxylase activity in noradrenergic neurons of the locus coeruleus after reserpine administration: seguential increase in cell bodies and nerve terminals. J. Neurochem., 2:23-29. Zi ond, R.E., Schon, F., and Iversen, L.L. (1974) Increased tyrosine gdroxylase activity in the locus coeruleus of rat brain stem after reserpine treatment and cold stress. Brain Res., 70:547-552.
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