Molecular Brain Research, 13 (1992) 223-229 © 1992 Elsevier Science Publishers B.V. All rights reserved. 0169-328X/92/$05.00
223
BRESM 70403
Effects of reserpine on tyrosine hydroxylase mRNA levels in locus coeruleus and medullary A1 and A2 neurons analyzed by in situ hybridization histochemistry and quantitative image analysis methods Richard D. Hartman, Jiin-Jia Liaw, Ju-Ren He and Charles A. Barraclough Department of Physiology, School of Medicine, University of Maryland, Baltimore, MD 21201 (USA) and Center for Studies in Reproduction, Baltimore, MD (USA) (Accepted 5 November 1991 ) Key words: Locus coeruleus; A1 noradrenergic neuron; A2 noradrenergic neuron; Reserpine; Tyrosine hydroxylase mRNA
These studies examined the effects of reserpine on concentrations of norepinephrine (NE), dopamine (DA) and epinephrine (EPI) and on levels of tyrosine hydroxylase (TH) mRNA in locus coeruleus (LC) and medullary A1 and A2 neurons. Noradrenergic neurons in these regions first were identified by immunocytochemistry and, thereafter, by in situ hybridization histochemistry. Levels of TH mRNA were measured by quantitative image analysis methods. Changes in catecholamine concentrations in micropunches of these brain regions were analyzed by HPLC. Epinephrine was not detected in any of the nuclei examined. Twenty-four hours after reserpine treatment, NE concentrations declined in A1, A2 and LC neurons by 46, 69 and 34% respectively while DA declined only in the region of A2 neurons. This reserpine-induced depletion of NE was accompanied by a 2- to 3-fold increase in TH mRNA levels in LC and A1 neurons but no change in message levels occurred in A2 cells 24 h after reserpine. Forty eight hours later, message levels in A1 and LC neurons did not differ significantly from the elevated 24 h values but TH mRNA levels in A2 neurons now were significantly elevated compared to 24 h values. TH mRNA levels 72 h after reserpine did not differ from 48 h values in A1, A2 and LC neurons. Thus, TH gene expression in A1 neurons increases after reserpine treatment in a manner equivalent to that observed in LC, adrenal medulla and superior cervical ganglia. The reason why it required 48 h for TH mRNA to increase in A2 neurons remains unclear. INTRODUCTION Tyrosine hydroxylase (TH) is the rate-limiting enzyme in the synthesis of n o r e p i n e p h r i n e 2° and its activity and changes in gene expression of this enzyme have been studied extensively in adrenal chromaffin cells, superior cervical ganglia and, within the brain, in locus coeruleus n o r a d r e n e r g i c neurons. Stimuli such as cold stress or the administration of reserpine cause a 2- to 4-fold increase in T H activity 19'28'29'32'33. This effect is referred to as transsynaptic induction 14'29 as it can be abolished by interruption of preganglionic inputs to adrenal medulla or superior cervical ganglia. The rise in T H activity is entirely attributable to the synthesis of specific tyrosine hydroxylase p r o t e i n and not to activation of pre-existing enzyme molecules 14'24. R e s e r p i n e t r e a t m e n t also increases T H m R N A in adrenal medulla, sympathetic ganglia, locus coeruleus but not substantia nigra (dopaminergic neurons) 3'4'11'17'21'25'27. The observation that the increase in T H activity is p r e c e d e d by an increase in the level of T H m R N A suggests that the transsynaptic induction of T H is primarily the result of an increase in the intracellular concentration of T H m R N A 4'27.
In contrast to the numerous studies of changes in T H activity and T H m R N A levels in the p e r i p h e r a l noradrenergic system, similar studies of medullary A1 and A 2 noradrenergic neurons have not been forthcoming. The hypothalamus receives its major n o r e p i n e p h r i n e (NE) innervation from medullary A1 and A 2 neurons and, to a lesser extent, from the locus coeruleus 6'18 and a subpopulation of these noradrenergic cells contain estrogen receptors m13. Changes in hypothalamic N E secretion influence the release of numerous hypothalamic p e p t i d e h o r m o n e s which, in turn, regulate adenohypophyseal function 2'8'18. A s well, the m e d u l l a oblongata has an i m p o r t a n t role in regulating a variety of cardiovascular, gastrointestinal and respiratory functions. Presumably, integration of m a n y of the autonomic functions occurs via changes in activity in a variety of medullary neurotransmitter neurons including the A1 and A 2 cells groups. In spite of these i m p o r t a n t functions, surprisingly little is know about how T H activity or T H m R N A levels change in A1 and A 2 neurons in response to stimuli which regulate their activity. In the present studies, we evaluated the changes which occur in N E , d o p a m i n e ( D A ) and epinephrine (EPI) concentrations and in T H
Correspondence: C.A. Barraclough, Department of Physiology, School of Medicine, University of Maryland, Baltimore, MD 21201, USA.
224 mRNA
levels in A 1 , A 2 a n d locus c o e r u l e u s n e u r o n s
f o l l o w i n g a single i n j e c t i o n o f r e s e r p i n e using H P L C a n d in situ h y b r i d i z a t i o n h i s t o c h e m i s t r y a n d q u a n t i t a t i v e image analysis m e t h o d o l o g i e s . T h e p u r p o s e o f t h e s e p r e l i m i n a r y s t u d i e s was to l e a r n if o t h e r c o m p o n e n t s o f t h e central n o r a d r e n e r g i c system r e s p o n d to reserpine-ind u c e d d e p l e t i o n o f n o r e p i n e p h r i n e by c h a n g e s in T H m R N A in a m a n n e r similar to t h a t p r e v i o u s l y o b s e r v e d in t h e a d r e n a l m e d u l l a , a n d s u p e r i o r cervical ganglia. MATERIALS AND METHODS Adult Sprague-Dawley male rats (200-224 g) (Zivic Miller, Allison Park, PA) were purchased and housed in a temperature- and light-controlled room for approximately one week before use. Three groups of rats were used for study: (1) untreated control animals; (2) vehicle-treated control rats; and (3) reserpine-treated rats. At the time of sacrifice, the animals were deeply anesthetized with ether, were perfused with cold saline via the common carotid artery and then were decapitated. All brains were rapidly removed and divided into rostral and caudal blocks at the level of the mammillary bodies. These two brain segments were rapidly frozen by emersion in isopentane cooled by dry ice. Thereafter, the brains were wrapped in parafilm and stored at -70°C until they were sectioned. The brain segments which contained the pons, midbrain and medulla were sectioned in a cryostat at -14°C. Alternate serial sections (12/~m) were taken through brain regions which contained the locus coeruleus and the medullary A1 and A2 cell groups and were mounted on two sets of gel-coated slides. One set of slides which contained the brain sections from untreated control rats was used for the immunohistochemical detection of TH enzyme. The second set of slides which contained brain sections from control and reserpine-treated rats was used for the in situ hybridization studies. All slides were kept frozen at -70°C until they were processed using either procedure.
lmmunohistochemistry Slides first were fixed in 4% formalin/PBS, placed in 0.5 M Trisbuffered saline (1.5% NaCI, pH 7.6) (TBS) and then in 3% normal goat serum (NGS) in TBS for 1 h. Thereafter, the sections were incubated for 24 h in TH antiserum (Boehringer-Mannheim, 1:25 diluted in 2% NGSFFBS) which was placed directly on the sections (100/A for A1 and A2 cells, 200/*1 for LC) and Parafilm coverslips then were placed over the sections. After incubation, the slides were rinsed in TBS and goat-anti-mouse antibody (1:20 in 2% NGS/ TBS) was placed directly on the sections for 1 h. Following a rinse in TBS, the sections were incubated in mouse peroxidase anti- peroxidase (PAP) (1:100 in 2% NGSFFBS) for 1 h. After a TBS rinse~ the sections were exposed to 0.01% hydrogen peroxide in diaminobenzidine tetrachloride (DAB-500 #g/ml in TBS) for 5-8 min. Thereafter, the slides were washed in several changes of TBS and distilled water, stained with Cresyl violet and then dehydrated through a series of alcohols (70-100%), cleared in xylene and coverslipped.
In situ hybridization histochemistry (ISHH) To study the effects of reserpine on TH mRNA levels in LC, A1 and A2 neurons, three groups of rats (n = 6/group) received a single injection of reserpine (10 mg/kg i.p.) at 10.00 h (0 time) and the animals were sacrificed 24, 48 and 72 h later. Two control groups were used, those in which no treatment was given and a second group in which only the vehicle (1% citric acid, 10% ethanol, 23% polyethylene glycol, 66% water) for reserpine was injected i.p. All control brains were obtained at the same time of day that drug-treated rats were sacrificed (10.00 h) (n = 5/group). Serial sections (12/*m) which contained LC, A1 and A2 neurons were
prepared as described above. These slides were stored at -70°C until all brains had been sectioned. ISHH was performed on all brain sections simultaneously as described previously23. The TH probe was a 48 base oligodeoxynucleotide complementary to rat TH cDNA (bases 1441-1488) described by Grima et al. 9. This probe was made on an Applied Biosynthesis 380A DNA synthesizer and purified by HPLC. It has been shown previously31 by Northern analysis of total RNA from hypothalamus that this probe hybridizes to an RNA species of 1.9 kb in agreement with the work by others9"16'30. The probe was 3' end-labeled by incubation (37°C, 5 min) with [3sS]dATP (1 uM; 1300 Ci/mmol; NEN, Boston, MA) and terminal deoxynucleotidyl transferase (50 U, Boehringer Mannheim) to a specific activity of about 7,000 Ci/mmol. Prehybridization treatment consisted of warming the brain sections to room temperature, after which they were fixed, for 5 min in 4% formaldehyde in 0.12 M sodium phosphate-buffered saline (PBS; pH 7.4), rinsed in 2 x SSC and then acetylated for 10 min in 0.25% acetic anhydride in 0.1 M triethanolamine hydrochloride0.9% NaC1 (pH 8.0). After rinsing with 2 x SSC, sections were dehydrated through an ethanol series (70-100%), delipidated in chloroform, rehydrated in 95% ethanol and air-dried. Labeled probe was applied in a saturating concentration to sections in 45 ld of hybridization buffer. This buffer contained 4 × SSC, 50% (v/v) formamide, 10% (w/v) dextran sulfate, 250/~g/ml yeast transfer RNA, 500/~g/ml sheared single-stranded salmon sperm DNA, 1 x Denhardt's solution and 50 mM fresh dithiothreitol. Hybridization proceeded under Parafilm coverslips, overnight, at 37°C in humid chambers. Sections were washed four times under stringent conditions (2 x SSC-50% formamide) at 40°C (approximately 19°C below Tin) for 15 min each, and then twice under less stringent conditions (1 × SSC) at 22°C for 30 rain each. After the last wash, slides were dipped briefly in water and then 70% and 95% ethanol and dried. Those slides which contained medullary A1 and A2 neurons were dipped in fresh Kodak NTB-3 nuclear-track emulsion, exposed for 10 days at 4°C, developed and the cell nuclei were lightly counterstained with Toluidine blue. Slides containing LC sections were placed against Kodak X-Omat film in X-ray film holders and exposed for 24 h. The optimal times of exposure of brain sections to either emulsion or film was previously established using 35S brain paste standards (emulsion and film) or 14C standards (film). As well, a series of test slides containing hybridized A1 and A2 neurons were developed at 6, 8, 10 and 12 days to measure background and the intensity of silver grains.
Image analysis Autoradiograms of brain sections containing A1 and A2 neurons were analyzed as described previously23. Briefly, tissue sections were matched anatomically between animals. The autoradiographic signal was quantitated in a Bioquant Image Analysis System IV (R&M Biometrics, Inc., Nashville, TN, USA). The image analysis system was carefully calibrated and checked before each slide was read so that light level and camera sensitivity were consistent for all measurements. Cells were considered specifically labeled if the grain density obtained was at least 10 x background. In these studies, grain density was consistently about 66 × 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 400 x magnification utilizing a neutral density filter as follows: The image analysis system was set for 'grain counting' (VCMTE Video Count) and an illumination threshold was established. A gray level threshold was chosen such that the silver grains were computer enhanced and measured but the histologically stained nuclei were below threshold. Due to unavoidable stacking of silver grains in autoradiographic emulsions, this measurement cannot properly be considered a 'grain count'. Therefore, 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 pixels0~m2 at 400 x magnification. A single background grain gave a reading of 4 (pixels). When film was analyzed using the Bioquant IV Image Analysis
225 System, light intensity was carefully calibrated for each film using afilm strip which contained tac 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.
Data analysis and statistics Approximately 120 A1 and 110 A2 neurons on each side of the medulla were analyzed in every rat in the control and reserpinetreated groups and calculations of each animal's mean for either side was calculated. Similarly, density of the hybridization signal in LC was measured in 8 individual sections/side/rat on the left and right sides of the brain. As no significant differences were found in the response to reserpine on the right versus the left side of the brain, these values were combined (approximately 240 A1 and 220 A2 cells/animal and 16 sections for LC) and expressed as a single mRNA data value for that animal. The means for individual animals in the control and experimental groups (n = 5-6/group) then were averaged to provide a single grand mean + S.E.M. for each group. Data were analyzed statistically by one-way ANOVA and Duncan's multiple range test for significant differences among groups. P < 0.05 was considered significant in all of the statistical tests.
Micropunch and H P L C methodology Micropunch-method. One group of rats (n = 5) received reserpine (10 mg/kg, i.p.) and was sacrificed 24 h later together with a second group of untreated control rats (n = 8). The brains were rapidly removed and divided into rostral and caudal blocks as described earlier. The caudal blocks were frozen on dry ice, mounted on microtome chucks and coronal sections (300 pm) of those brain regions which contained A1, A2 and LC neurons were made. Regions of the medulla previously identified by immunocytochemistry as containing A1 neurons (area between the retroambiguus N. and the lateral reticular N.; atlas coordinates = Bregma -14.30, and -14.60 mm) and A2 neurons (medial nucleus of the solitary tract and a portion of dorsal nucleus of the vagus; Bregma -14.30 and - 1 4 . 0 8 mm) were micropunched using a 21 gauge hypodermic needle (cannula lumen diameter = 500/zm). The region of the pons containing LC was identified and micropunches from the approximate middle of this nucleus were made (Bregma -9.68, and -9.80 ram). The stereotaxic coordinates listed above were obtained from the atlas of Paxinos and Watson 22. A single punch on the right side was taken from each of the two consecutive 300/~m frozen sections containing the AI, A2 or LC brain regions. The two micropunches from each brain region were placed in Eppendorf centrifuge tubes and stored frozen (-70°C) until the time of assay. H P L C method. Analyses of NE, DA and epinephrine (EPI) concentrations in all micropunches from a specific region were completed before a second region was analysed. Thus, all A1 tissue was examined and then all A2 tissue etc. such that control and experimental groups were included in each HPLC analysis on any single day. Mobile phase was composed of 5.93 g of NaH2PO 4, 105 mg octylsodium sulfate, 60 ml methanol, 11.94 mg EDTA, 1.2 ml diethylamine and 940 ml of HPLC quality water (pH adjusted to 3.47 with phosphoric acid). Various volumes of mobile phase were added to the centrifuge tubes which contained tissue punches (70 pl for A2, 50 pl for A1 and 200 pl for LC), they were briefly sonicated and 2 pl of ascorbate oxidase (100 U/ml, Sigma A- 2769) then was added. Thereafter, the samples were vortexed and centrifuged for 4 min in an IEC Centra-M centrifuge. Aliquots (20 B1) were removed and injected into the HPLC apparatus. Duplicate measurements were made of each sample. The remaining tissue pellets were resuspended in 50 pl of 1 N NaOH and stored at 4°C for not more than 1 week. These samples were measured for protein content by the Bradford dye binding micromethod5. Catecholamine concentrations were measured in micropunches using reversed-phase HPLC and an ESA Coulochem Model 5100A
Fig. 1. Immunocytochemical localization of tyrosine hydroxylasecontaining medullary A1 neurons located in the dorsal pole of the lateral reticular nucleus. Note the considerable intertwining of dendritic processes (400 x).
electrochemical detector set in a redox mode (detector 1 set at - 0 . 2 mV and detector 2 set at +0.25 mV). The stationary phase consisted of a C18 reversed-phase 'Microsorb-Short One' column (3 /~m spherical particles, C8-80-300-C3, Rainin). Concentrations of catechols in the samples were determined using a Hewlett Packard 3390 integrator to compare peak heights with those of standards for NE, DA and EPI. Sample values were adjusted for recovery based on measurements of an internal standard (isoproterenol) which had been added to all samples and standards. The minimal detectable levels of NE, DA and EPI were 2, 4 and 4 pg, respectively.
RESULTS
Immunocytochemistry T H - i m m u n o r e a c t i v e neurons were identified bilater-
TABLE I
Effects of reserpine on N E and DA concentrations in A1, A2 and LC neurons Control, n = 8; reserpine, n = 5.
Neurons
Treatment
Concentration (pg/l~g protein) NE
DA
A1
Control Reserpine
30 + 0.8 16.3 + 2.0*
6.8 + 0.4 7.4 + 2.1
A2
Control Reserpine
60.9 + 2.4 18.7 + 1.5"
23.8 _+ 2.8 13.4 + 2.7*
LC
Control Reserpine
148 _ 10 98 + 11.7"
35.9 + 5 30.2 + 4.3
* Significantly different from control (P < 0.05).
226 within the LC and were organized as a distinct compact group of cells. The location and organization of this nucleus was almost identical to that originally described by Dahlstrom and Fuxe 6.
Catecholamine concentrations: effects of reserpine. EPI was not detected in any of the brain regions examined. The effects of reserpine on NE and D A concentrations are shown in Table I. Twenty-four hours after reserpine treatment, NE had decreased by 46% in A1, 69% in A2 and 34% in LC neurons. D A concentrations declined by 44% but only in A2 neurons.
In situ hybridization histochemistry: effects of reserpine. Fig. 2. Immunocytochemical localization of tyrosine hydroxylasecontaining medullary A2 neurons located in the nucleus of the solitary tract. These multipolar neurons do not exhibit the dendritic arborizations observed in A1 cells (400×).
ally in the dorsal pole of the lateral reticular nucleus (A1) and dorsal to the central canal within the medial nucleus of the solitary tract (A2). Only a few scattered neurons were detected in the dorsal motor nucleus of the vagus (A2). These A1 and A2 medullary TH-immunoreactive neurons were found in regions originally described by Dahlstrom and Fuxe 6 using histofluorescent methods and are almost identical to locations provided by Kalia et al. 15 using immunocytochemical procedures to detect TH-containing neurons. The A1 cells usually appeared as a network of neurons with intertwining dendritic processes (Fig. 1). In contrast, A2 neurons appeared as large multipolar cells which lacked the characteristic dendritic arborizations observed in A1 cells. The A2 neurons were most densely packed in the nucleus of the solitary tract (Fig. 2). TH-immunoreactive neurons were readily detected
~°"
Medullary neurons containing T H m R N A were identified in the regions of the lateral reticular nucleus and in the nucleus of the solitary tract. These locations are identical to those in which we identified TH-containing neurons on adjacent sections using immunocytochemical procedures. Similarly, T H m R N A was intensely labeled in LC neurons. Because of the considerable overlap of grains within this structure, it was not possible to perform single cell analysis. Consequently, the density of the entire nucleus was measured on film following a 24 h exposure interval. As there were no significant differences in T H m R N A levels in A1, A2 or LC neurons in untreated or vehicletreated control rats sacrificed at 10.00 h at 0 time, 24, 48 or 72 h, the control values for each group of noradrenergic neurons were combined and expressed as a single control value in Figs. 5-7. Treatment of rats with reserpine resulted in a highly significant increase in the levels of T H m R N A in both A1 and LC noradrenergic neurons. The effects of reserpine on T H m R N A levels in A1 cells 24 h after reserpine can clearly be seen by comparing Figs. 3 and 4. When expressed as area of grains ~m2), T H m R N A levels had increased approximately 2-fold in A1 neurons by 24 h and these message levels remained elevated to 72 h (Fig. 5). As well, the density of T H m R N A in LC had increased by about 3-fold by 24 h and remained elevated to 72 h (Fig. 6). In contrast, T H m R N A levels in A2 neurons were not changed 24 h after reserpine but they were significantly elevated 48 and 72 h later. Quantitation of T H m R N A (area of grains (flm2)) in A2 neurons revealed an approximate 2- fold increase 48 h after reserpine with no additional increase nor decline at 72 h. DISCUSSION
/
Fig. 3. Tyrosine hydroxylase mRNA in A1 neurons of intact control rats (400×).
The location of T H enzyme and T H m R N A using immunocytochemical and in situ hybridization methods demonstrated that neurons containing this enzyme and
227 250
!i i!; 200
iO0 50 0 il ~
!i
i!!? ¸
CONTROL 24h 48h 72h RESERPINE
...........
iiiiii ¸ ii!iiiii i : i ¸
iiili
Fig. 4. Levels of tyrosine hydroxylase mRNA had increased significantly in A1 neurons 24 h after reserpine (400x).
its message are located in identical regions of the medulla and in the LC. In these studies, we attempted to distinguish those A2 neurons which were located exclusively in the medial nucleus of the solitary tract versus an occasional cell located in the dorsal m o t o r nucleus of the vagus. A2 cells located in other regions (e.g. obex, area postrema, etc.) that exhibited cluster of grains were not analyzed. Consequently, we believe that many of the T H m R N A containing A2 cells which we evaluated are noradrenergic neurons. This conclusion is supported by our observations that high concentrations of N E are contained in micropunches of this region. The increased levels of T H m R N A in LC after reserpine treatment agree closely with the observations of others who used somewhat different experimental paradigms (dosage, duration of treatment, etc). As well, different laboratories employed either radiolabelled synthetic oligodeoxynucleotide probes or riboprobes 3'4'17'21 and quantitated the levels of message by various types
Fig. 6. Quantitation of tyrosine hydroxylase mRNA (expressed as density) in the locus coeruleus showed that level of message had increased by about 3-fold 24 h following reserpine treatment and remained at these elevated levels to 72 h.
of blot analyses. In the present study, we used a 48 base oligodeoxynucleotide probe which previous studies have shown to be specific for T H m R N A in brain 9'16'3°'31. O u r in situ hybridization immunocytochemistry method coupled with the use of quantitative image analysis allowed us to measure changes in T H m R N A in individual A1 and A2 neurons. Twenty-four hours after reserpine, a significant rise in T H m R N A had occurred in A1 and LC neurons and these high levels were still present 72 h later. In contrast, no change in T H m R N A was detected in A2 neurons 24 h after drug treatment but by 48 h, message levels were significantly increased and remained elevated at 72 h. These differences were due to an increase in the number of grains/cell and not to an increase in the number of cells expressing detectable message. Others have performed temporal studies on the effects of reserpine on T H m R N A levels in LC and adrenal medulla following a single injection of reserpine. Tank et al. 22 observed that the relative levels of adrenal medullary T H m R N A were maximally increased by 12 h after reserpine or cold stress. In contrast, Bignet et al. 4
500
I
&-" 400
30o
z
i50
200
,,
100
:iiI/ c~
,,
223
BRESM 70403
Effects of reserpine on tyrosine hydroxylase mRNA levels in locus coeruleus and medullary A1 and A2 neurons analyzed by in situ hybridization histochemistry and quantitative image analysis methods Richard D. Hartman, Jiin-Jia Liaw, Ju-Ren He and Charles A. Barraclough Department of Physiology, School of Medicine, University of Maryland, Baltimore, MD 21201 (USA) and Center for Studies in Reproduction, Baltimore, MD (USA) (Accepted 5 November 1991 ) Key words: Locus coeruleus; A1 noradrenergic neuron; A2 noradrenergic neuron; Reserpine; Tyrosine hydroxylase mRNA
These studies examined the effects of reserpine on concentrations of norepinephrine (NE), dopamine (DA) and epinephrine (EPI) and on levels of tyrosine hydroxylase (TH) mRNA in locus coeruleus (LC) and medullary A1 and A2 neurons. Noradrenergic neurons in these regions first were identified by immunocytochemistry and, thereafter, by in situ hybridization histochemistry. Levels of TH mRNA were measured by quantitative image analysis methods. Changes in catecholamine concentrations in micropunches of these brain regions were analyzed by HPLC. Epinephrine was not detected in any of the nuclei examined. Twenty-four hours after reserpine treatment, NE concentrations declined in A1, A2 and LC neurons by 46, 69 and 34% respectively while DA declined only in the region of A2 neurons. This reserpine-induced depletion of NE was accompanied by a 2- to 3-fold increase in TH mRNA levels in LC and A1 neurons but no change in message levels occurred in A2 cells 24 h after reserpine. Forty eight hours later, message levels in A1 and LC neurons did not differ significantly from the elevated 24 h values but TH mRNA levels in A2 neurons now were significantly elevated compared to 24 h values. TH mRNA levels 72 h after reserpine did not differ from 48 h values in A1, A2 and LC neurons. Thus, TH gene expression in A1 neurons increases after reserpine treatment in a manner equivalent to that observed in LC, adrenal medulla and superior cervical ganglia. The reason why it required 48 h for TH mRNA to increase in A2 neurons remains unclear. INTRODUCTION Tyrosine hydroxylase (TH) is the rate-limiting enzyme in the synthesis of n o r e p i n e p h r i n e 2° and its activity and changes in gene expression of this enzyme have been studied extensively in adrenal chromaffin cells, superior cervical ganglia and, within the brain, in locus coeruleus n o r a d r e n e r g i c neurons. Stimuli such as cold stress or the administration of reserpine cause a 2- to 4-fold increase in T H activity 19'28'29'32'33. This effect is referred to as transsynaptic induction 14'29 as it can be abolished by interruption of preganglionic inputs to adrenal medulla or superior cervical ganglia. The rise in T H activity is entirely attributable to the synthesis of specific tyrosine hydroxylase p r o t e i n and not to activation of pre-existing enzyme molecules 14'24. R e s e r p i n e t r e a t m e n t also increases T H m R N A in adrenal medulla, sympathetic ganglia, locus coeruleus but not substantia nigra (dopaminergic neurons) 3'4'11'17'21'25'27. The observation that the increase in T H activity is p r e c e d e d by an increase in the level of T H m R N A suggests that the transsynaptic induction of T H is primarily the result of an increase in the intracellular concentration of T H m R N A 4'27.
In contrast to the numerous studies of changes in T H activity and T H m R N A levels in the p e r i p h e r a l noradrenergic system, similar studies of medullary A1 and A 2 noradrenergic neurons have not been forthcoming. The hypothalamus receives its major n o r e p i n e p h r i n e (NE) innervation from medullary A1 and A 2 neurons and, to a lesser extent, from the locus coeruleus 6'18 and a subpopulation of these noradrenergic cells contain estrogen receptors m13. Changes in hypothalamic N E secretion influence the release of numerous hypothalamic p e p t i d e h o r m o n e s which, in turn, regulate adenohypophyseal function 2'8'18. A s well, the m e d u l l a oblongata has an i m p o r t a n t role in regulating a variety of cardiovascular, gastrointestinal and respiratory functions. Presumably, integration of m a n y of the autonomic functions occurs via changes in activity in a variety of medullary neurotransmitter neurons including the A1 and A 2 cells groups. In spite of these i m p o r t a n t functions, surprisingly little is know about how T H activity or T H m R N A levels change in A1 and A 2 neurons in response to stimuli which regulate their activity. In the present studies, we evaluated the changes which occur in N E , d o p a m i n e ( D A ) and epinephrine (EPI) concentrations and in T H
Correspondence: C.A. Barraclough, Department of Physiology, School of Medicine, University of Maryland, Baltimore, MD 21201, USA.
224 mRNA
levels in A 1 , A 2 a n d locus c o e r u l e u s n e u r o n s
f o l l o w i n g a single i n j e c t i o n o f r e s e r p i n e using H P L C a n d in situ h y b r i d i z a t i o n h i s t o c h e m i s t r y a n d q u a n t i t a t i v e image analysis m e t h o d o l o g i e s . T h e p u r p o s e o f t h e s e p r e l i m i n a r y s t u d i e s was to l e a r n if o t h e r c o m p o n e n t s o f t h e central n o r a d r e n e r g i c system r e s p o n d to reserpine-ind u c e d d e p l e t i o n o f n o r e p i n e p h r i n e by c h a n g e s in T H m R N A in a m a n n e r similar to t h a t p r e v i o u s l y o b s e r v e d in t h e a d r e n a l m e d u l l a , a n d s u p e r i o r cervical ganglia. MATERIALS AND METHODS Adult Sprague-Dawley male rats (200-224 g) (Zivic Miller, Allison Park, PA) were purchased and housed in a temperature- and light-controlled room for approximately one week before use. Three groups of rats were used for study: (1) untreated control animals; (2) vehicle-treated control rats; and (3) reserpine-treated rats. At the time of sacrifice, the animals were deeply anesthetized with ether, were perfused with cold saline via the common carotid artery and then were decapitated. All brains were rapidly removed and divided into rostral and caudal blocks at the level of the mammillary bodies. These two brain segments were rapidly frozen by emersion in isopentane cooled by dry ice. Thereafter, the brains were wrapped in parafilm and stored at -70°C until they were sectioned. The brain segments which contained the pons, midbrain and medulla were sectioned in a cryostat at -14°C. Alternate serial sections (12/~m) were taken through brain regions which contained the locus coeruleus and the medullary A1 and A2 cell groups and were mounted on two sets of gel-coated slides. One set of slides which contained the brain sections from untreated control rats was used for the immunohistochemical detection of TH enzyme. The second set of slides which contained brain sections from control and reserpine-treated rats was used for the in situ hybridization studies. All slides were kept frozen at -70°C until they were processed using either procedure.
lmmunohistochemistry Slides first were fixed in 4% formalin/PBS, placed in 0.5 M Trisbuffered saline (1.5% NaCI, pH 7.6) (TBS) and then in 3% normal goat serum (NGS) in TBS for 1 h. Thereafter, the sections were incubated for 24 h in TH antiserum (Boehringer-Mannheim, 1:25 diluted in 2% NGSFFBS) which was placed directly on the sections (100/A for A1 and A2 cells, 200/*1 for LC) and Parafilm coverslips then were placed over the sections. After incubation, the slides were rinsed in TBS and goat-anti-mouse antibody (1:20 in 2% NGS/ TBS) was placed directly on the sections for 1 h. Following a rinse in TBS, the sections were incubated in mouse peroxidase anti- peroxidase (PAP) (1:100 in 2% NGSFFBS) for 1 h. After a TBS rinse~ the sections were exposed to 0.01% hydrogen peroxide in diaminobenzidine tetrachloride (DAB-500 #g/ml in TBS) for 5-8 min. Thereafter, the slides were washed in several changes of TBS and distilled water, stained with Cresyl violet and then dehydrated through a series of alcohols (70-100%), cleared in xylene and coverslipped.
In situ hybridization histochemistry (ISHH) To study the effects of reserpine on TH mRNA levels in LC, A1 and A2 neurons, three groups of rats (n = 6/group) received a single injection of reserpine (10 mg/kg i.p.) at 10.00 h (0 time) and the animals were sacrificed 24, 48 and 72 h later. Two control groups were used, those in which no treatment was given and a second group in which only the vehicle (1% citric acid, 10% ethanol, 23% polyethylene glycol, 66% water) for reserpine was injected i.p. All control brains were obtained at the same time of day that drug-treated rats were sacrificed (10.00 h) (n = 5/group). Serial sections (12/*m) which contained LC, A1 and A2 neurons were
prepared as described above. These slides were stored at -70°C until all brains had been sectioned. ISHH was performed on all brain sections simultaneously as described previously23. The TH probe was a 48 base oligodeoxynucleotide complementary to rat TH cDNA (bases 1441-1488) described by Grima et al. 9. This probe was made on an Applied Biosynthesis 380A DNA synthesizer and purified by HPLC. It has been shown previously31 by Northern analysis of total RNA from hypothalamus that this probe hybridizes to an RNA species of 1.9 kb in agreement with the work by others9"16'30. The probe was 3' end-labeled by incubation (37°C, 5 min) with [3sS]dATP (1 uM; 1300 Ci/mmol; NEN, Boston, MA) and terminal deoxynucleotidyl transferase (50 U, Boehringer Mannheim) to a specific activity of about 7,000 Ci/mmol. Prehybridization treatment consisted of warming the brain sections to room temperature, after which they were fixed, for 5 min in 4% formaldehyde in 0.12 M sodium phosphate-buffered saline (PBS; pH 7.4), rinsed in 2 x SSC and then acetylated for 10 min in 0.25% acetic anhydride in 0.1 M triethanolamine hydrochloride0.9% NaC1 (pH 8.0). After rinsing with 2 x SSC, sections were dehydrated through an ethanol series (70-100%), delipidated in chloroform, rehydrated in 95% ethanol and air-dried. Labeled probe was applied in a saturating concentration to sections in 45 ld of hybridization buffer. This buffer contained 4 × SSC, 50% (v/v) formamide, 10% (w/v) dextran sulfate, 250/~g/ml yeast transfer RNA, 500/~g/ml sheared single-stranded salmon sperm DNA, 1 x Denhardt's solution and 50 mM fresh dithiothreitol. Hybridization proceeded under Parafilm coverslips, overnight, at 37°C in humid chambers. Sections were washed four times under stringent conditions (2 x SSC-50% formamide) at 40°C (approximately 19°C below Tin) for 15 min each, and then twice under less stringent conditions (1 × SSC) at 22°C for 30 rain each. After the last wash, slides were dipped briefly in water and then 70% and 95% ethanol and dried. Those slides which contained medullary A1 and A2 neurons were dipped in fresh Kodak NTB-3 nuclear-track emulsion, exposed for 10 days at 4°C, developed and the cell nuclei were lightly counterstained with Toluidine blue. Slides containing LC sections were placed against Kodak X-Omat film in X-ray film holders and exposed for 24 h. The optimal times of exposure of brain sections to either emulsion or film was previously established using 35S brain paste standards (emulsion and film) or 14C standards (film). As well, a series of test slides containing hybridized A1 and A2 neurons were developed at 6, 8, 10 and 12 days to measure background and the intensity of silver grains.
Image analysis Autoradiograms of brain sections containing A1 and A2 neurons were analyzed as described previously23. Briefly, tissue sections were matched anatomically between animals. The autoradiographic signal was quantitated in a Bioquant Image Analysis System IV (R&M Biometrics, Inc., Nashville, TN, USA). The image analysis system was carefully calibrated and checked before each slide was read so that light level and camera sensitivity were consistent for all measurements. Cells were considered specifically labeled if the grain density obtained was at least 10 x background. In these studies, grain density was consistently about 66 × 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 400 x magnification utilizing a neutral density filter as follows: The image analysis system was set for 'grain counting' (VCMTE Video Count) and an illumination threshold was established. A gray level threshold was chosen such that the silver grains were computer enhanced and measured but the histologically stained nuclei were below threshold. Due to unavoidable stacking of silver grains in autoradiographic emulsions, this measurement cannot properly be considered a 'grain count'. Therefore, 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 pixels0~m2 at 400 x magnification. A single background grain gave a reading of 4 (pixels). When film was analyzed using the Bioquant IV Image Analysis
225 System, light intensity was carefully calibrated for each film using afilm strip which contained tac 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.
Data analysis and statistics Approximately 120 A1 and 110 A2 neurons on each side of the medulla were analyzed in every rat in the control and reserpinetreated groups and calculations of each animal's mean for either side was calculated. Similarly, density of the hybridization signal in LC was measured in 8 individual sections/side/rat on the left and right sides of the brain. As no significant differences were found in the response to reserpine on the right versus the left side of the brain, these values were combined (approximately 240 A1 and 220 A2 cells/animal and 16 sections for LC) and expressed as a single mRNA data value for that animal. The means for individual animals in the control and experimental groups (n = 5-6/group) then were averaged to provide a single grand mean + S.E.M. for each group. Data were analyzed statistically by one-way ANOVA and Duncan's multiple range test for significant differences among groups. P < 0.05 was considered significant in all of the statistical tests.
Micropunch and H P L C methodology Micropunch-method. One group of rats (n = 5) received reserpine (10 mg/kg, i.p.) and was sacrificed 24 h later together with a second group of untreated control rats (n = 8). The brains were rapidly removed and divided into rostral and caudal blocks as described earlier. The caudal blocks were frozen on dry ice, mounted on microtome chucks and coronal sections (300 pm) of those brain regions which contained A1, A2 and LC neurons were made. Regions of the medulla previously identified by immunocytochemistry as containing A1 neurons (area between the retroambiguus N. and the lateral reticular N.; atlas coordinates = Bregma -14.30, and -14.60 mm) and A2 neurons (medial nucleus of the solitary tract and a portion of dorsal nucleus of the vagus; Bregma -14.30 and - 1 4 . 0 8 mm) were micropunched using a 21 gauge hypodermic needle (cannula lumen diameter = 500/zm). The region of the pons containing LC was identified and micropunches from the approximate middle of this nucleus were made (Bregma -9.68, and -9.80 ram). The stereotaxic coordinates listed above were obtained from the atlas of Paxinos and Watson 22. A single punch on the right side was taken from each of the two consecutive 300/~m frozen sections containing the AI, A2 or LC brain regions. The two micropunches from each brain region were placed in Eppendorf centrifuge tubes and stored frozen (-70°C) until the time of assay. H P L C method. Analyses of NE, DA and epinephrine (EPI) concentrations in all micropunches from a specific region were completed before a second region was analysed. Thus, all A1 tissue was examined and then all A2 tissue etc. such that control and experimental groups were included in each HPLC analysis on any single day. Mobile phase was composed of 5.93 g of NaH2PO 4, 105 mg octylsodium sulfate, 60 ml methanol, 11.94 mg EDTA, 1.2 ml diethylamine and 940 ml of HPLC quality water (pH adjusted to 3.47 with phosphoric acid). Various volumes of mobile phase were added to the centrifuge tubes which contained tissue punches (70 pl for A2, 50 pl for A1 and 200 pl for LC), they were briefly sonicated and 2 pl of ascorbate oxidase (100 U/ml, Sigma A- 2769) then was added. Thereafter, the samples were vortexed and centrifuged for 4 min in an IEC Centra-M centrifuge. Aliquots (20 B1) were removed and injected into the HPLC apparatus. Duplicate measurements were made of each sample. The remaining tissue pellets were resuspended in 50 pl of 1 N NaOH and stored at 4°C for not more than 1 week. These samples were measured for protein content by the Bradford dye binding micromethod5. Catecholamine concentrations were measured in micropunches using reversed-phase HPLC and an ESA Coulochem Model 5100A
Fig. 1. Immunocytochemical localization of tyrosine hydroxylasecontaining medullary A1 neurons located in the dorsal pole of the lateral reticular nucleus. Note the considerable intertwining of dendritic processes (400 x).
electrochemical detector set in a redox mode (detector 1 set at - 0 . 2 mV and detector 2 set at +0.25 mV). The stationary phase consisted of a C18 reversed-phase 'Microsorb-Short One' column (3 /~m spherical particles, C8-80-300-C3, Rainin). Concentrations of catechols in the samples were determined using a Hewlett Packard 3390 integrator to compare peak heights with those of standards for NE, DA and EPI. Sample values were adjusted for recovery based on measurements of an internal standard (isoproterenol) which had been added to all samples and standards. The minimal detectable levels of NE, DA and EPI were 2, 4 and 4 pg, respectively.
RESULTS
Immunocytochemistry T H - i m m u n o r e a c t i v e neurons were identified bilater-
TABLE I
Effects of reserpine on N E and DA concentrations in A1, A2 and LC neurons Control, n = 8; reserpine, n = 5.
Neurons
Treatment
Concentration (pg/l~g protein) NE
DA
A1
Control Reserpine
30 + 0.8 16.3 + 2.0*
6.8 + 0.4 7.4 + 2.1
A2
Control Reserpine
60.9 + 2.4 18.7 + 1.5"
23.8 _+ 2.8 13.4 + 2.7*
LC
Control Reserpine
148 _ 10 98 + 11.7"
35.9 + 5 30.2 + 4.3
* Significantly different from control (P < 0.05).
226 within the LC and were organized as a distinct compact group of cells. The location and organization of this nucleus was almost identical to that originally described by Dahlstrom and Fuxe 6.
Catecholamine concentrations: effects of reserpine. EPI was not detected in any of the brain regions examined. The effects of reserpine on NE and D A concentrations are shown in Table I. Twenty-four hours after reserpine treatment, NE had decreased by 46% in A1, 69% in A2 and 34% in LC neurons. D A concentrations declined by 44% but only in A2 neurons.
In situ hybridization histochemistry: effects of reserpine. Fig. 2. Immunocytochemical localization of tyrosine hydroxylasecontaining medullary A2 neurons located in the nucleus of the solitary tract. These multipolar neurons do not exhibit the dendritic arborizations observed in A1 cells (400×).
ally in the dorsal pole of the lateral reticular nucleus (A1) and dorsal to the central canal within the medial nucleus of the solitary tract (A2). Only a few scattered neurons were detected in the dorsal motor nucleus of the vagus (A2). These A1 and A2 medullary TH-immunoreactive neurons were found in regions originally described by Dahlstrom and Fuxe 6 using histofluorescent methods and are almost identical to locations provided by Kalia et al. 15 using immunocytochemical procedures to detect TH-containing neurons. The A1 cells usually appeared as a network of neurons with intertwining dendritic processes (Fig. 1). In contrast, A2 neurons appeared as large multipolar cells which lacked the characteristic dendritic arborizations observed in A1 cells. The A2 neurons were most densely packed in the nucleus of the solitary tract (Fig. 2). TH-immunoreactive neurons were readily detected
~°"
Medullary neurons containing T H m R N A were identified in the regions of the lateral reticular nucleus and in the nucleus of the solitary tract. These locations are identical to those in which we identified TH-containing neurons on adjacent sections using immunocytochemical procedures. Similarly, T H m R N A was intensely labeled in LC neurons. Because of the considerable overlap of grains within this structure, it was not possible to perform single cell analysis. Consequently, the density of the entire nucleus was measured on film following a 24 h exposure interval. As there were no significant differences in T H m R N A levels in A1, A2 or LC neurons in untreated or vehicletreated control rats sacrificed at 10.00 h at 0 time, 24, 48 or 72 h, the control values for each group of noradrenergic neurons were combined and expressed as a single control value in Figs. 5-7. Treatment of rats with reserpine resulted in a highly significant increase in the levels of T H m R N A in both A1 and LC noradrenergic neurons. The effects of reserpine on T H m R N A levels in A1 cells 24 h after reserpine can clearly be seen by comparing Figs. 3 and 4. When expressed as area of grains ~m2), T H m R N A levels had increased approximately 2-fold in A1 neurons by 24 h and these message levels remained elevated to 72 h (Fig. 5). As well, the density of T H m R N A in LC had increased by about 3-fold by 24 h and remained elevated to 72 h (Fig. 6). In contrast, T H m R N A levels in A2 neurons were not changed 24 h after reserpine but they were significantly elevated 48 and 72 h later. Quantitation of T H m R N A (area of grains (flm2)) in A2 neurons revealed an approximate 2- fold increase 48 h after reserpine with no additional increase nor decline at 72 h. DISCUSSION
/
Fig. 3. Tyrosine hydroxylase mRNA in A1 neurons of intact control rats (400×).
The location of T H enzyme and T H m R N A using immunocytochemical and in situ hybridization methods demonstrated that neurons containing this enzyme and
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Fig. 4. Levels of tyrosine hydroxylase mRNA had increased significantly in A1 neurons 24 h after reserpine (400x).
its message are located in identical regions of the medulla and in the LC. In these studies, we attempted to distinguish those A2 neurons which were located exclusively in the medial nucleus of the solitary tract versus an occasional cell located in the dorsal m o t o r nucleus of the vagus. A2 cells located in other regions (e.g. obex, area postrema, etc.) that exhibited cluster of grains were not analyzed. Consequently, we believe that many of the T H m R N A containing A2 cells which we evaluated are noradrenergic neurons. This conclusion is supported by our observations that high concentrations of N E are contained in micropunches of this region. The increased levels of T H m R N A in LC after reserpine treatment agree closely with the observations of others who used somewhat different experimental paradigms (dosage, duration of treatment, etc). As well, different laboratories employed either radiolabelled synthetic oligodeoxynucleotide probes or riboprobes 3'4'17'21 and quantitated the levels of message by various types
Fig. 6. Quantitation of tyrosine hydroxylase mRNA (expressed as density) in the locus coeruleus showed that level of message had increased by about 3-fold 24 h following reserpine treatment and remained at these elevated levels to 72 h.
of blot analyses. In the present study, we used a 48 base oligodeoxynucleotide probe which previous studies have shown to be specific for T H m R N A in brain 9'16'3°'31. O u r in situ hybridization immunocytochemistry method coupled with the use of quantitative image analysis allowed us to measure changes in T H m R N A in individual A1 and A2 neurons. Twenty-four hours after reserpine, a significant rise in T H m R N A had occurred in A1 and LC neurons and these high levels were still present 72 h later. In contrast, no change in T H m R N A was detected in A2 neurons 24 h after drug treatment but by 48 h, message levels were significantly increased and remained elevated at 72 h. These differences were due to an increase in the number of grains/cell and not to an increase in the number of cells expressing detectable message. Others have performed temporal studies on the effects of reserpine on T H m R N A levels in LC and adrenal medulla following a single injection of reserpine. Tank et al. 22 observed that the relative levels of adrenal medullary T H m R N A were maximally increased by 12 h after reserpine or cold stress. In contrast, Bignet et al. 4
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