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Temporal changes in tyrosine hydroxylase mRNA levels in A1, A2 and locus ceruleus neurons following electrical stimulation of A1 noradrenergic neurons
Molecular Brain Research, 13 (1992) 171-174 © 1992 Elsevier Science Publishers B.V. All rights reserved. 0169-328X/92/$05.00
171
BRESM 80118
Temporal changes in tyrosine hydroxylase mRNA levels in A1, A2 and locus ceruleus neurons following electrical stimulation of A1 noradrenergic neurons Jiin-Jia Liaw, Ju-Ren He and Charles A. Barraclough Center for Studies in Reproduction, Department of Physiology, School of Medicine, University of Maryland, Baltimore, MD 21201 (U.S.A.)
(Accepted 19 November 1991) Key words: Electrical stimulation; Tyrosine hydroxylase; mRNA; A1 neuron; A2 neuron; Locus ceruleus neuron; In situ hybridization
We examined the effects of electrical stimulation (ES) of right A1 noradrenergic cells on temporal changes in tyrosine hydroxylase (TH) mRNA levels in A1, A2 and locus ceruleus (LC) neurons by in situ hybridization histochemistry and quantitative image analysis methods. The stimulation parameters used previously have been shown to increase hypothalamic norepinephrine (NE) release. Within 1 h after heginning A1 stimulation, TH mRNA levels were significantly increased and they continued to rise to reach plateau by 6 h. TH message levels at 12 h were not difference from 6 h values. A1-ES did not affect TH mRNA levels in contralateral AI or in A2 or locus ceruleus neurons. These data suggest that changes in TH mRNA levels may serve as an index of increased A1 neuronal activity in circumstances when increases in hypothalamic NE secretion occur. In general, stimuli which activate the release of neuropeptides and neurotransmitters also increase transcription of the genes encoding these peptides or the rate limiting enzymes responsible for synthesis of the neurotransmitters. For example, stimuli which increase vasopressin, corticotropin-releasing factor, luteinizing hormone releasing hormone ( L H R H ) etc. also increase the m R N A s encoding these neuropeptides 1t'14'2°. Similarly, depletion of norepinephrine (NE) by reserpine is accompanied by a significant rise in levels of tyrosine hydroxylase (TH) m R N A in adrenal medulla, and superior cervical ganglia and this event depends upon an increase in transsynaptic stimulation 1. As well, increases in adrenal medullary neuropeptide Y m R N A occur following reserpine treatment and transection of the splanchnic nerves almost completely prevents this rise 12. Within the brain, electrical stimulation of the hippocampal dentate gyrus increases proenkephalin m R N A and decreases the levels of prodynorphin m R N A 8. Others have reported that medial forebrain bundle stimulation or D2 dopamine receptor activation increases preproenkephalin m R N A in rat striatum 2. The concept that emerges from these diverse observations is that changes in levels of m R N A s encoding neuropeptides or T H reflect changes in activity within these various systems. We have taken advantage of this information and have determined whether electrical stimulation (ES) of right
medullary A1 noradrenergic cells increases T H m R N A levels in these neurons. Previously, we have shown by indirect methods, that such stimuli evoke hypothalamic N E release 6 and, more recently, Herbison et al. 8 reported a significant rise in N E in microdialysed samples obtained from the medial preoptic area following A l ES. Accordingly, in the present study we examined the temporal changes which occur in T H message levels in right vs left A1 noradrenergic cells following ES of the right ventrolateral medulla. We also determined whether such stimuli affect T H m R N A levels in A2 or locus ceruleus neurons either as a consequence of non-specific current spread or via axonal projections from stimulated A1 neurons to these other noradrenergic cell groups. We reasoned that if A1-ES releases hypothalamic N E and it concurrently elevates T H m R N A levels, perhaps increases in T H m R N A could serve as one index of suspected increases in A1 neuronal activity under a variety of physiologichl conditions. Adult Sprague-Dawley male rats (200-224 g; Zivic Miller, Allison Park, PA) were purchased and housed in a temperature- and light-controlled r o o m for approximately one week before use. Previously, we showed that there are no diurnal variations in A1 T H m R N A levels in untreated rats during the times of day that the current studies were performed 1°. Accordingly, these controis were not repeated in the present study. Three
Correspondence: J.-J. Liaw, Center for Studies in Reproduction, Department of Physiology, School of Medicine, University of Maryland, Baltimore, MD 21201, U.S.A.
172 groups of rats were used and 3 time periods were studied (09.00-10.00 h, 15.00-16.00 h, 21.00-22.00 h). All rats were anesthetized with chloral hydrate (450 mg/kg i.p.) between 09.00-10.00 h. The groups of rats used for study were: Group 1: these control rats received chloral hydrate but, thereafter, they were not subjected to any further experimental procedures (n = 6/time period). Group 2: these anesthetized, sham control rats (n = 6/time period) were placed in a stereotaxic instrument between 09.00-10.00 h, the scalp was incised, a burr hole was drilled through the calvarium and a coaxial electrode (MS 303/2, Plastic Products Co., Roanoke, VA) was inserted into the right medullary A1 region 6. No electrical current was delivered to these rats. Group 3: these anesthetized rats (n = 6/time period) differed from the sham controls described in Group 2 only in that biphasic rectangular pulses of 100/~A, 1 ms duration, 20 Hz were delivered through the electrode in 15 s on/off trains for 20 min using a specially designed stimulator. ES was performed between 09.00 h and 10.00 h. Control, sham-stimulated and electrically stimulated rats were sacrificed together at the same times of day. Rats were sacrificed 1, 6 or 12 h after beginning ES (0 time). At the time of sacrifice, all rats were deeply anesthetized with ether and decapitated. Their brains were rapidly removed and frozen by immersion in isopentane cooled by dry ice. Thereafter, the brains were stored at -70°C until they were sectioned. Frozen serial sections (12 pm) were taken through brain regions which contained the locus ceruleus (LC) and the medullary A1 and A2 cell groups and were mounted on gel-coated slides. All slides were kept frozen at -70°C until they were processed for in situ hybridization histochemistry (ISHH). ISHH was performed on all brain sections simultaneously as described previously 13'1s. The TH probe was a 48-base oligodeoxynucleotide complementary to rat TH cDNA 7 (bases 14411488) whose specificity has been confirmed in rat brain by Northern blot analysis and controls for positive chemography 19. The probe was 3"-end-labelled with [35S]dATP (1 ktM; 1300 Ci/mmol; NEN, Boston, MA) and terminal deoxynucleotidyl transferase (50 U, Boehringer Mannheim) to a specific activity of 7000 Ci/ mmol. Hybridization proceeded under Parafilm coverslips, overnight at 36°C in humid chambers. Those slides which contained medullary A1 and A2 neurons were dipped in fresh Kodak NTB-3 nucleartrack emulsion, exposed for 10 days at 4°C, developed and the cell nuclei were lightly stained with Toluidine blue. Slides containing LC neurons were placed against Kodak X-Omat film in X-ray film holders and exposed for 24 h. Autoradiograms of brain sections containing A1 and
A2 neurons were analyzed as described previously ~°'t3. Briefly, tissue sections were matched anatomically amongst animals. The autoradiographic signal was quantitated in a Bioquant Image Analysis System IV (R&M Biometrics, Inc., Nashville, TN). In these studies, grain density was consistently about 60x background. Grain clusters first were determined to be over cell bodies by identifying the lightly stained nucleus of each cell. These cells were analyzed at 400x utilizing a neutral density filter. We have expressed our data as the mean 'area of grains' by dividing the number of pixels above threshold (i.e. the 'video count' value) by the number of pixels/ #m 2 at 400X magnification. A single background grain gave a reading of 4 (pixels). Film was analyzed as described previously 1°'1s using the Bioquant IV Image Analysis System. Light intensity was carefully calibrated for each film using a film strip which contained 14C standards. The density of the film adjacent to the LC first was measured (background) and then the entire area containing LC was carefully circumscribed and the density of this region minus film background was calculated by the computer. For the analysis of A1 and A2 neurons (on the same sections), first TH mRNA levels in the right A1 and A2 cells were measured and then the left A1 and A2 cells were analyzed. Next the sham control cells were evaluated and finally, the A1-ES neurons were analyzed. Thus, a procedure was followed in which TH mRNA levels in A1 and A2 neurons in control, sham control and A1-ES rats were sequentially analyzed. A similar procedure was followed when TH mRNA levels (density) were determined in LC neurons. A total of 80 + 10 cells from the right and 80 _ 9 cells in the left A1 neurons were analyzed in each rat. A total of 55 + 5 A2 cells in the right and 55 _ 8 cells in the left side of the medulla were measured for TH mRNA levels in each animal. Similarly, density of the hybridization signal in 8 individual sections of the right and 8 sections of the left LC were analyzed for each rat. From the data generated, we calculated each animal's mean _ S.E.M. for the individual treatment group. All individual means for each animal in the control and experimental groups (Groups 1-3) then were averaged to provide a single grand mean + S.E.M. for each group. Multiple statistical tests were applied to determine the effects of sham stimulation and A1-ES on the right and left sides of the brain for each of the regions examined. To determine the effect of time after A1-ES on TH mRNA levels in controls vs ES rats, two-way A N O V A with repeated measures was applied. If a significant effect of time was detected, Duncan's multiple range test was used to evaluate which times were different from each other. A similar test was applied to compare the
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Fig. 1. Temporal changes in TH mRNA in right and left A1, A2 and locus ceruleus neurons following electrical stimulation on right A1 neurons. C, chloral hydrate anesthetized controls; S, sham stimulated controls; ES, electrically stimulated animals. *P < 0.05 vs C; **P < 0.05 vs 1 h ES values.
effects of ES vs controls or sham controls on TH m R N A levels. P < 0.05 was considered significant in all of the statistical tests. The temporal changes in TH m R N A levels in A1, A2 and LC neurons following A1-ES are illustrated in Fig. 1. As our earlier studies did not detect any diurnal variation in TH m R N A levels in intact male rats throughout the hours of the day used in the present study, these controls were not repeated in the present study. Group 1: TH message levels in control rats (C) anesthetized between 09.00 h and 10.00 h did not vary throughout the
12 h of the study. Group 2: In these rats, the stress of sham stimulation (S) did not affect message levels in A1 neurons at any of the times studied. Group 3: unlike the lack of response in the two former groups, within 1 h following the beginning of ES (or 40 min after ending ES) of A1 neurons in the right ventrolateral medulla, TH m R N A in these cells had increased significantly (P < 0.05) above control values and these levels continued to rise significantly to 6 h before reaching a plateau. By 12 h TH m R N A values were not significantly different from those obtained at 6 h in the stimulated group. In contrast, TH message levels in the left A1 neurons of these stimulated rats were not significantly different from control values. In contrast to the responses obtained in A1 neurons, TH message levels did not differ in A2 or LC neurons throughout the day in rats anesthetized with chloral hydrate between 09.00 h and 10.00 h nor were message levels changed in these neurons in rats receiving sham or electrical stimulation of the right A1 noradrenergic cell group. Medullary A1 and A2 neurons, and to a lesser extent LC neurons, are the major contributors of NE to the hypothalamus 3. Thus, increases in activity within one or another of these noradrenergic cell groups could result in increased NE secretion in specific regions of the hypothalamus which contain various peptidergic neurons. The release of many of these neuropeptides has been attributed to an increase in hypothalamic NE secretion. These include preovulatory L H R H surges ~T, the castration-induced rise in L H R H release 5, stress-induced corticotropin-releasing hormone (CRF) secretion 16, increases in vasopressin secretion 4 etc. Whether such increases in hypothalamic NE are due solely to modulation of presynaptic NE release by local opiate and G A B A neurons or to increases in NE neuronal activity or to both events is not known. In the present study we examined the changes which occur in TH m R N A following ES of the right A1 noradrenergic cell group. Within 40 min after the end of ES, TH m R N A levels were significantly elevated above control values and message levels continued to rise over the next 5 h to reach plateau values 6 h after beginning ES. The electrical stimuli used in these studies previously have been shown to increase NE release in the medial preoptic nucleus 8, the site of L H R H perikarya, and such stimuli evoke the release of L H R H 6. Taken together, the data suggest that stimuli which augment hypothalamic NE secretion also increase levels of TH m R N A in these A1 neurons. There are many other instances in which either extrinsic stimuli or increases in transsynaptic axonal traffic have been shown to increase transcription of genes encoding neuropeptides or TH (see introduction for references). Thus, we
174 do not believe it is unreasonable to suggest that a rise in
neurons in the nucleus paragigantocellularis located in
T H m R N A levels in A1 neurons, under circumstances
the rostral ventrolateral medulla (C1, C3 cells) ~5. Seem-
in which hypothalamic N E secretion also is elevated,
ingly, if non-specific current spread to C1 and C3 adren-
could serve as an index of increased activity within these
ergic neurons had occurred, changes in T H m R N A lev-
A1 neurons. It should be noted that A1 neurons have
els in LC would have been detected.
diverse roles in regulating not only hypothalamic peptide
In summary, these data provide evidence that in-
release but also blood pressure. Thus changes in T H
creases in hypothalamic N E secretion are accompanied
m R N A levels in these neurons without concomitant ev-
by a rise in T H m R N A levels within A1 neurons. Per-
idence of an elevated release in hypothalamic N E might
haps this observation may prove useful in the future for
be misleading. The observation that T H message levels increased
establishing the site of origin of noradrenergic signals which activate hypothalamic peptidergic neurons.
only in the right A1 neurons suggests that the electrical stimuli which we delivered through coaxial electrodes remained localized within this region. No changes in message levels were observed in contralateral A1 neurons nor in A2 or LC neurons. T h e major afferent projections to the rat LC have been reported to originate in
1 Black, J.B., Chikaraishi, D.M. and Lewis, E.J., Trans-synaptic increase in RNA coding for tyrosine hydroxylase in rat sympathetic ganglion, Brain Research, 339 (1985) 151-153. 2 Bannon, M.J., Kelland, M. and Chiodo, L.A., Medial forebrain bundle stimulation or D-2 dopamine receptor activation increases proenkephalin mRNA in rat striatum, J. Neurochem., 52 (1989) 859-862. 3 Dahlstrom, A. and Fuxe, K., Evidence for tthe existence of monoamine-containing neurons in the central nervous system. I. Demonstration of monoamines in the cell bodies of brainstem neurons, Acta Physiol. Scand. Suppl., 232, 62 (1964) 1-55. 4 Day, T.A., Fergerson, A.V. and Renaud, L.P., Facilitatory influence of noradrenergic afferents on the excitability of rat paraventricular nucleus neurosecretory cells, J. Physiol., 355 (1984) 232-249. 5 DePaolo, L.V., McCann, S.M. and Negro-Vilar, A., A sex difference in the activation of hypothalamic catecholaminergic and luteinizing hormone-releasing hormone peptidergic neurons after acute castration, Endocrinology, 110 (1982) 531-539. 6 Gitler, M.S. and Barraclough, C.A., Stimulation of the medullary A1 noradrenergic system augments luteinizing hormone release induced by medial preoptic nucleus stimulation, Neuroendocrinology, 48 (1988) 351-359. 7 Grima, B., Lamouroux, A., Blanot, F., Bequet, N.F. and Mallet, J., Complete coding sequence of rat tyrosine hydroxylase mRNA, Proc. Natl. Acad. Sci. U.S.A., 82 (1985) 617-621. 8 Herbison, A.E., Heavens, R.P. and Dyer, R.G., Oestrogen modulation of excitatory A1 noradrenergic input to rat medial preoptic gamma aminobutyric acid neurons demonstrated by microdialysis, Neuroendocrinology, 52 (1990) 161-168. 9 Higuchi, H., Iwasa, A., Yoshida, H. and Miki, N., Long lasting increase in neuropeptide Y gene expression in rat adrenal gland with reserpine treatment: positive regulation of transsynaptic activation and membrane depolarization, Mol. Pharmacol., 38 (1990) 614-623. 10 Liaw, J.-J., He, J.-R., Hartman, R.D. and Barraclough, C.A., Changes in tyrosine hydroxylase mRNA levels in medullary AI and A2 neurons and locus coeruleus following castration and estrogen replacement in rats, Mol. Brain Res., 13 (1992). ll Lightman, J.L. and Young III, W.S. Vasopressin, oxytocin, dynorphin, enkephalin and corticotropin releasing factor mRNA
This work was supported in part by NIH Grant HD-02138 to C.A.B. and by a Special Research Initiative Support and Graduate Research Assistantship award from the UMAB Designated Research Initative Fund.
in the rat, J. Physiol., 394 (1987) 23-39. 12 Morris, B.J., Feasey, K.J., ten Bruggencate, G., Herz, A. and Hollt, V., Electrical stimulation in vivo increases the expression of proenkephalin mRNA and decreases the expression of prodynorphin mRNA in rat hippocampal granule cells, Proc. Natl. Acad. Sci. U.S.A., 85 (1988) 3226-3230. 13 Petersen, S.L., Cheuk, C., Hartman, R.D. and Barraclough, C.A., Medial preoptic implants of the antiestrogen Keoxifene affect luteinizing hormone-releasing hormone mRNA levels, median eminence luteinizing hormone-releasing hormone concentrations and luteinizing hormone release in ovariectomized estrogen-treated rats, J. Neuroendocrinol., 1 (1989) 279-283. 14 Petersen, S.L., McCrone, S., Keller, M. and Gardner, E., Rapid increase in LHRH mRNA following NMDA, Endocrinology, 129 (1991) 1679-1681. 15 Pieribone, V.A., Bohn, M.C. and Astron-Jones, G., Adrenergic and non-adrenergic afferents to the locus coeruleus from the C1 and C3 adrenergic cell groups, Neurosci. Lett., 85 (1988) 297-303. 16 Plotsky, P., Cunningham, E.T. and Widmaier, E.P., Catecholaminergic modulation of corticotropin-releasing factor and adrenocorticotropin secretion, Endocrine Rev., 10 (1989) 437455. 17 Rance, N., Wise, P.M., Selmanoff, M.K. and Barraclough, C.A., Catecholamine turnover rates in discrete hypothalamic areas and associated changes in median eminence luteinizing hormone releasing hormone and serum gonadotropins on proestrous and diestrous day 1, Endocrinology, 108 (1981) 17951802. 18 Selmanoff, M., Shu, C., Hartman, R.D., Barraclough, C.A. and Petersen, S.L., Tyrosine hydroxylase and POMC mRNA in the arcuate region are increased by castration and hyperprolactinemia, Mol. Brain Res., 10 (1990) 277-281. 19 Young, M.J., Warden, M. and Mezey, E., Tyrosine hydroxylase mRNA is increased by hyperosmotic stimuli in the paraventricular and supraoptic nuclei, Neuroendocrinology, 46 (1987) 439-444. 20 Young III. W.S. and Zoeller, R.T., Neuroendocrine gene expression in the hypothalamus: in situ hybridization histochemical studies, Cell Mol. Neurobiol., 7 (1987) 353-366.
171
BRESM 80118
Temporal changes in tyrosine hydroxylase mRNA levels in A1, A2 and locus ceruleus neurons following electrical stimulation of A1 noradrenergic neurons Jiin-Jia Liaw, Ju-Ren He and Charles A. Barraclough Center for Studies in Reproduction, Department of Physiology, School of Medicine, University of Maryland, Baltimore, MD 21201 (U.S.A.)
(Accepted 19 November 1991) Key words: Electrical stimulation; Tyrosine hydroxylase; mRNA; A1 neuron; A2 neuron; Locus ceruleus neuron; In situ hybridization
We examined the effects of electrical stimulation (ES) of right A1 noradrenergic cells on temporal changes in tyrosine hydroxylase (TH) mRNA levels in A1, A2 and locus ceruleus (LC) neurons by in situ hybridization histochemistry and quantitative image analysis methods. The stimulation parameters used previously have been shown to increase hypothalamic norepinephrine (NE) release. Within 1 h after heginning A1 stimulation, TH mRNA levels were significantly increased and they continued to rise to reach plateau by 6 h. TH message levels at 12 h were not difference from 6 h values. A1-ES did not affect TH mRNA levels in contralateral AI or in A2 or locus ceruleus neurons. These data suggest that changes in TH mRNA levels may serve as an index of increased A1 neuronal activity in circumstances when increases in hypothalamic NE secretion occur. In general, stimuli which activate the release of neuropeptides and neurotransmitters also increase transcription of the genes encoding these peptides or the rate limiting enzymes responsible for synthesis of the neurotransmitters. For example, stimuli which increase vasopressin, corticotropin-releasing factor, luteinizing hormone releasing hormone ( L H R H ) etc. also increase the m R N A s encoding these neuropeptides 1t'14'2°. Similarly, depletion of norepinephrine (NE) by reserpine is accompanied by a significant rise in levels of tyrosine hydroxylase (TH) m R N A in adrenal medulla, and superior cervical ganglia and this event depends upon an increase in transsynaptic stimulation 1. As well, increases in adrenal medullary neuropeptide Y m R N A occur following reserpine treatment and transection of the splanchnic nerves almost completely prevents this rise 12. Within the brain, electrical stimulation of the hippocampal dentate gyrus increases proenkephalin m R N A and decreases the levels of prodynorphin m R N A 8. Others have reported that medial forebrain bundle stimulation or D2 dopamine receptor activation increases preproenkephalin m R N A in rat striatum 2. The concept that emerges from these diverse observations is that changes in levels of m R N A s encoding neuropeptides or T H reflect changes in activity within these various systems. We have taken advantage of this information and have determined whether electrical stimulation (ES) of right
medullary A1 noradrenergic cells increases T H m R N A levels in these neurons. Previously, we have shown by indirect methods, that such stimuli evoke hypothalamic N E release 6 and, more recently, Herbison et al. 8 reported a significant rise in N E in microdialysed samples obtained from the medial preoptic area following A l ES. Accordingly, in the present study we examined the temporal changes which occur in T H message levels in right vs left A1 noradrenergic cells following ES of the right ventrolateral medulla. We also determined whether such stimuli affect T H m R N A levels in A2 or locus ceruleus neurons either as a consequence of non-specific current spread or via axonal projections from stimulated A1 neurons to these other noradrenergic cell groups. We reasoned that if A1-ES releases hypothalamic N E and it concurrently elevates T H m R N A levels, perhaps increases in T H m R N A could serve as one index of suspected increases in A1 neuronal activity under a variety of physiologichl conditions. Adult Sprague-Dawley male rats (200-224 g; Zivic Miller, Allison Park, PA) were purchased and housed in a temperature- and light-controlled r o o m for approximately one week before use. Previously, we showed that there are no diurnal variations in A1 T H m R N A levels in untreated rats during the times of day that the current studies were performed 1°. Accordingly, these controis were not repeated in the present study. Three
Correspondence: J.-J. Liaw, Center for Studies in Reproduction, Department of Physiology, School of Medicine, University of Maryland, Baltimore, MD 21201, U.S.A.
172 groups of rats were used and 3 time periods were studied (09.00-10.00 h, 15.00-16.00 h, 21.00-22.00 h). All rats were anesthetized with chloral hydrate (450 mg/kg i.p.) between 09.00-10.00 h. The groups of rats used for study were: Group 1: these control rats received chloral hydrate but, thereafter, they were not subjected to any further experimental procedures (n = 6/time period). Group 2: these anesthetized, sham control rats (n = 6/time period) were placed in a stereotaxic instrument between 09.00-10.00 h, the scalp was incised, a burr hole was drilled through the calvarium and a coaxial electrode (MS 303/2, Plastic Products Co., Roanoke, VA) was inserted into the right medullary A1 region 6. No electrical current was delivered to these rats. Group 3: these anesthetized rats (n = 6/time period) differed from the sham controls described in Group 2 only in that biphasic rectangular pulses of 100/~A, 1 ms duration, 20 Hz were delivered through the electrode in 15 s on/off trains for 20 min using a specially designed stimulator. ES was performed between 09.00 h and 10.00 h. Control, sham-stimulated and electrically stimulated rats were sacrificed together at the same times of day. Rats were sacrificed 1, 6 or 12 h after beginning ES (0 time). At the time of sacrifice, all rats were deeply anesthetized with ether and decapitated. Their brains were rapidly removed and frozen by immersion in isopentane cooled by dry ice. Thereafter, the brains were stored at -70°C until they were sectioned. Frozen serial sections (12 pm) were taken through brain regions which contained the locus ceruleus (LC) and the medullary A1 and A2 cell groups and were mounted on gel-coated slides. All slides were kept frozen at -70°C until they were processed for in situ hybridization histochemistry (ISHH). ISHH was performed on all brain sections simultaneously as described previously 13'1s. The TH probe was a 48-base oligodeoxynucleotide complementary to rat TH cDNA 7 (bases 14411488) whose specificity has been confirmed in rat brain by Northern blot analysis and controls for positive chemography 19. The probe was 3"-end-labelled with [35S]dATP (1 ktM; 1300 Ci/mmol; NEN, Boston, MA) and terminal deoxynucleotidyl transferase (50 U, Boehringer Mannheim) to a specific activity of 7000 Ci/ mmol. Hybridization proceeded under Parafilm coverslips, overnight at 36°C in humid chambers. Those slides which contained medullary A1 and A2 neurons were dipped in fresh Kodak NTB-3 nucleartrack emulsion, exposed for 10 days at 4°C, developed and the cell nuclei were lightly stained with Toluidine blue. Slides containing LC neurons were placed against Kodak X-Omat film in X-ray film holders and exposed for 24 h. Autoradiograms of brain sections containing A1 and
A2 neurons were analyzed as described previously ~°'t3. Briefly, tissue sections were matched anatomically amongst animals. The autoradiographic signal was quantitated in a Bioquant Image Analysis System IV (R&M Biometrics, Inc., Nashville, TN). In these studies, grain density was consistently about 60x background. Grain clusters first were determined to be over cell bodies by identifying the lightly stained nucleus of each cell. These cells were analyzed at 400x utilizing a neutral density filter. We have expressed our data as the mean 'area of grains' by dividing the number of pixels above threshold (i.e. the 'video count' value) by the number of pixels/ #m 2 at 400X magnification. A single background grain gave a reading of 4 (pixels). Film was analyzed as described previously 1°'1s using the Bioquant IV Image Analysis System. Light intensity was carefully calibrated for each film using a film strip which contained 14C standards. The density of the film adjacent to the LC first was measured (background) and then the entire area containing LC was carefully circumscribed and the density of this region minus film background was calculated by the computer. For the analysis of A1 and A2 neurons (on the same sections), first TH mRNA levels in the right A1 and A2 cells were measured and then the left A1 and A2 cells were analyzed. Next the sham control cells were evaluated and finally, the A1-ES neurons were analyzed. Thus, a procedure was followed in which TH mRNA levels in A1 and A2 neurons in control, sham control and A1-ES rats were sequentially analyzed. A similar procedure was followed when TH mRNA levels (density) were determined in LC neurons. A total of 80 + 10 cells from the right and 80 _ 9 cells in the left A1 neurons were analyzed in each rat. A total of 55 + 5 A2 cells in the right and 55 _ 8 cells in the left side of the medulla were measured for TH mRNA levels in each animal. Similarly, density of the hybridization signal in 8 individual sections of the right and 8 sections of the left LC were analyzed for each rat. From the data generated, we calculated each animal's mean _ S.E.M. for the individual treatment group. All individual means for each animal in the control and experimental groups (Groups 1-3) then were averaged to provide a single grand mean + S.E.M. for each group. Multiple statistical tests were applied to determine the effects of sham stimulation and A1-ES on the right and left sides of the brain for each of the regions examined. To determine the effect of time after A1-ES on TH mRNA levels in controls vs ES rats, two-way A N O V A with repeated measures was applied. If a significant effect of time was detected, Duncan's multiple range test was used to evaluate which times were different from each other. A similar test was applied to compare the
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Fig. 1. Temporal changes in TH mRNA in right and left A1, A2 and locus ceruleus neurons following electrical stimulation on right A1 neurons. C, chloral hydrate anesthetized controls; S, sham stimulated controls; ES, electrically stimulated animals. *P < 0.05 vs C; **P < 0.05 vs 1 h ES values.
effects of ES vs controls or sham controls on TH m R N A levels. P < 0.05 was considered significant in all of the statistical tests. The temporal changes in TH m R N A levels in A1, A2 and LC neurons following A1-ES are illustrated in Fig. 1. As our earlier studies did not detect any diurnal variation in TH m R N A levels in intact male rats throughout the hours of the day used in the present study, these controls were not repeated in the present study. Group 1: TH message levels in control rats (C) anesthetized between 09.00 h and 10.00 h did not vary throughout the
12 h of the study. Group 2: In these rats, the stress of sham stimulation (S) did not affect message levels in A1 neurons at any of the times studied. Group 3: unlike the lack of response in the two former groups, within 1 h following the beginning of ES (or 40 min after ending ES) of A1 neurons in the right ventrolateral medulla, TH m R N A in these cells had increased significantly (P < 0.05) above control values and these levels continued to rise significantly to 6 h before reaching a plateau. By 12 h TH m R N A values were not significantly different from those obtained at 6 h in the stimulated group. In contrast, TH message levels in the left A1 neurons of these stimulated rats were not significantly different from control values. In contrast to the responses obtained in A1 neurons, TH message levels did not differ in A2 or LC neurons throughout the day in rats anesthetized with chloral hydrate between 09.00 h and 10.00 h nor were message levels changed in these neurons in rats receiving sham or electrical stimulation of the right A1 noradrenergic cell group. Medullary A1 and A2 neurons, and to a lesser extent LC neurons, are the major contributors of NE to the hypothalamus 3. Thus, increases in activity within one or another of these noradrenergic cell groups could result in increased NE secretion in specific regions of the hypothalamus which contain various peptidergic neurons. The release of many of these neuropeptides has been attributed to an increase in hypothalamic NE secretion. These include preovulatory L H R H surges ~T, the castration-induced rise in L H R H release 5, stress-induced corticotropin-releasing hormone (CRF) secretion 16, increases in vasopressin secretion 4 etc. Whether such increases in hypothalamic NE are due solely to modulation of presynaptic NE release by local opiate and G A B A neurons or to increases in NE neuronal activity or to both events is not known. In the present study we examined the changes which occur in TH m R N A following ES of the right A1 noradrenergic cell group. Within 40 min after the end of ES, TH m R N A levels were significantly elevated above control values and message levels continued to rise over the next 5 h to reach plateau values 6 h after beginning ES. The electrical stimuli used in these studies previously have been shown to increase NE release in the medial preoptic nucleus 8, the site of L H R H perikarya, and such stimuli evoke the release of L H R H 6. Taken together, the data suggest that stimuli which augment hypothalamic NE secretion also increase levels of TH m R N A in these A1 neurons. There are many other instances in which either extrinsic stimuli or increases in transsynaptic axonal traffic have been shown to increase transcription of genes encoding neuropeptides or TH (see introduction for references). Thus, we
174 do not believe it is unreasonable to suggest that a rise in
neurons in the nucleus paragigantocellularis located in
T H m R N A levels in A1 neurons, under circumstances
the rostral ventrolateral medulla (C1, C3 cells) ~5. Seem-
in which hypothalamic N E secretion also is elevated,
ingly, if non-specific current spread to C1 and C3 adren-
could serve as an index of increased activity within these
ergic neurons had occurred, changes in T H m R N A lev-
A1 neurons. It should be noted that A1 neurons have
els in LC would have been detected.
diverse roles in regulating not only hypothalamic peptide
In summary, these data provide evidence that in-
release but also blood pressure. Thus changes in T H
creases in hypothalamic N E secretion are accompanied
m R N A levels in these neurons without concomitant ev-
by a rise in T H m R N A levels within A1 neurons. Per-
idence of an elevated release in hypothalamic N E might
haps this observation may prove useful in the future for
be misleading. The observation that T H message levels increased
establishing the site of origin of noradrenergic signals which activate hypothalamic peptidergic neurons.
only in the right A1 neurons suggests that the electrical stimuli which we delivered through coaxial electrodes remained localized within this region. No changes in message levels were observed in contralateral A1 neurons nor in A2 or LC neurons. T h e major afferent projections to the rat LC have been reported to originate in
1 Black, J.B., Chikaraishi, D.M. and Lewis, E.J., Trans-synaptic increase in RNA coding for tyrosine hydroxylase in rat sympathetic ganglion, Brain Research, 339 (1985) 151-153. 2 Bannon, M.J., Kelland, M. and Chiodo, L.A., Medial forebrain bundle stimulation or D-2 dopamine receptor activation increases proenkephalin mRNA in rat striatum, J. Neurochem., 52 (1989) 859-862. 3 Dahlstrom, A. and Fuxe, K., Evidence for tthe existence of monoamine-containing neurons in the central nervous system. I. Demonstration of monoamines in the cell bodies of brainstem neurons, Acta Physiol. Scand. Suppl., 232, 62 (1964) 1-55. 4 Day, T.A., Fergerson, A.V. and Renaud, L.P., Facilitatory influence of noradrenergic afferents on the excitability of rat paraventricular nucleus neurosecretory cells, J. Physiol., 355 (1984) 232-249. 5 DePaolo, L.V., McCann, S.M. and Negro-Vilar, A., A sex difference in the activation of hypothalamic catecholaminergic and luteinizing hormone-releasing hormone peptidergic neurons after acute castration, Endocrinology, 110 (1982) 531-539. 6 Gitler, M.S. and Barraclough, C.A., Stimulation of the medullary A1 noradrenergic system augments luteinizing hormone release induced by medial preoptic nucleus stimulation, Neuroendocrinology, 48 (1988) 351-359. 7 Grima, B., Lamouroux, A., Blanot, F., Bequet, N.F. and Mallet, J., Complete coding sequence of rat tyrosine hydroxylase mRNA, Proc. Natl. Acad. Sci. U.S.A., 82 (1985) 617-621. 8 Herbison, A.E., Heavens, R.P. and Dyer, R.G., Oestrogen modulation of excitatory A1 noradrenergic input to rat medial preoptic gamma aminobutyric acid neurons demonstrated by microdialysis, Neuroendocrinology, 52 (1990) 161-168. 9 Higuchi, H., Iwasa, A., Yoshida, H. and Miki, N., Long lasting increase in neuropeptide Y gene expression in rat adrenal gland with reserpine treatment: positive regulation of transsynaptic activation and membrane depolarization, Mol. Pharmacol., 38 (1990) 614-623. 10 Liaw, J.-J., He, J.-R., Hartman, R.D. and Barraclough, C.A., Changes in tyrosine hydroxylase mRNA levels in medullary AI and A2 neurons and locus coeruleus following castration and estrogen replacement in rats, Mol. Brain Res., 13 (1992). ll Lightman, J.L. and Young III, W.S. Vasopressin, oxytocin, dynorphin, enkephalin and corticotropin releasing factor mRNA
This work was supported in part by NIH Grant HD-02138 to C.A.B. and by a Special Research Initiative Support and Graduate Research Assistantship award from the UMAB Designated Research Initative Fund.
in the rat, J. Physiol., 394 (1987) 23-39. 12 Morris, B.J., Feasey, K.J., ten Bruggencate, G., Herz, A. and Hollt, V., Electrical stimulation in vivo increases the expression of proenkephalin mRNA and decreases the expression of prodynorphin mRNA in rat hippocampal granule cells, Proc. Natl. Acad. Sci. U.S.A., 85 (1988) 3226-3230. 13 Petersen, S.L., Cheuk, C., Hartman, R.D. and Barraclough, C.A., Medial preoptic implants of the antiestrogen Keoxifene affect luteinizing hormone-releasing hormone mRNA levels, median eminence luteinizing hormone-releasing hormone concentrations and luteinizing hormone release in ovariectomized estrogen-treated rats, J. Neuroendocrinol., 1 (1989) 279-283. 14 Petersen, S.L., McCrone, S., Keller, M. and Gardner, E., Rapid increase in LHRH mRNA following NMDA, Endocrinology, 129 (1991) 1679-1681. 15 Pieribone, V.A., Bohn, M.C. and Astron-Jones, G., Adrenergic and non-adrenergic afferents to the locus coeruleus from the C1 and C3 adrenergic cell groups, Neurosci. Lett., 85 (1988) 297-303. 16 Plotsky, P., Cunningham, E.T. and Widmaier, E.P., Catecholaminergic modulation of corticotropin-releasing factor and adrenocorticotropin secretion, Endocrine Rev., 10 (1989) 437455. 17 Rance, N., Wise, P.M., Selmanoff, M.K. and Barraclough, C.A., Catecholamine turnover rates in discrete hypothalamic areas and associated changes in median eminence luteinizing hormone releasing hormone and serum gonadotropins on proestrous and diestrous day 1, Endocrinology, 108 (1981) 17951802. 18 Selmanoff, M., Shu, C., Hartman, R.D., Barraclough, C.A. and Petersen, S.L., Tyrosine hydroxylase and POMC mRNA in the arcuate region are increased by castration and hyperprolactinemia, Mol. Brain Res., 10 (1990) 277-281. 19 Young, M.J., Warden, M. and Mezey, E., Tyrosine hydroxylase mRNA is increased by hyperosmotic stimuli in the paraventricular and supraoptic nuclei, Neuroendocrinology, 46 (1987) 439-444. 20 Young III. W.S. and Zoeller, R.T., Neuroendocrine gene expression in the hypothalamus: in situ hybridization histochemical studies, Cell Mol. Neurobiol., 7 (1987) 353-366.