Brain Research, 58l (1992) 19-32 © 1992 Elsevier Scicnce Publishers B.V. All rights reserved. 0006-8993/92/$05.0(1

19

BRES 17722

Somatotopic organization of tyrosine hydroxylase expression in the rat locus coeruleus: long term effect of RU24722 L.I. Debure a, E. Moyse u, M. Fevre-Montange b, H. Hardin b, M.F. Belin b, C. Rousset a, J.F. P u j o l a a n d D. W e i s s m a n n ~ "Laboratoire de ,\europharmacologie Mol&ulaire. CERMEP, Lyon (France) and r'INSERM (JFgOIO CNRS URA 1105, F'acultd Ah'xis Carrel, Lyon (France) (Accepted 24 December 1991) Key words: Locus ceruleus, Tyrosine hydroxylase: RU24722: In situ hybridization: Sleeping cell

Tyrosine hydroxylase (TH) tissue concentration was determined by immunostaining of tissue sections directly transferred onto nitrocellulose membranes in the restricted region of the noradrenergic perikarya of the locus coeruleus (LC) along its postero-anterior axis. TH containing cells were systematically counted on adjacent post fixed sections stained by immunohistochemistry. The absolute quantity of TH was estimated in each section and was found to be linearly related to the number of TH immuno-positive cells found in the adjacent section. The ratio between these two parameters was thus used as an index of the celhdar concentration of TH in noradrenergic cells. In the LC of control rats, the TH cellular concentration was lower (-39%) in the anterior than in the posterior half of the structure. Three days after an injection of 20 mg/kg of RU24722, an eburnamine derivative known to increase the quantity of TH in the LC. increases in quantities of TH were flmnd in both portions of the LC. Moreover in the posterior LC the increase in the amount of TH resulted from a significant increase in the number of TH-immunopositive cells. In the anterior part, however, it was primarily the result of a significant increase in TH cellular concentration. Throughout the LC there was an increase in the cellular concentration of TH which was inversely proportional to the concentrations found in control animals. TH mRNA content was measured by a quantitative in situ hybridization in sections of both the posterior and anterior LC one day after a single injection of RU24722 at the same dose. The quantity of TH mRNA was significantly increased in both parts. The number of TH mRNA-expressing neurons also increased, especially in the anterior LC. Thus the effects at the level of TH protein and TH mRNA were strikingly parallel though increase ira TH protein occurred later than the increase ira the TH mRNA. These results suggest that ira the rat LC: (1) there is a significant population of ~sleeping cells" ira which TH expression is either inactivated or, at a low level of activation: (2) TH cellular concentration could exert a retroconlrol on its own expression in cells of the LC that contained TH and (3) TH expression appears to be regulated by different selective mechanisms in these two different subpopulations of noradrenergic cells within the L('. INTRODU('TI()N

f o u n d to p r e c e d e i n c r e a s e s in T H p r o t e i n c o n c e n t r a t i o n . T h e e x i s t e n c e o f a p h y s i o l o g i c a l c o n t r o l of T H g e n e ex-

Long lasting changes

in t y r o s i n e h y d r o x y l a s e

(tyrosine-3-monooxygenase,

EC

(TH)

1.14.16.2) activity a n d

concentration have been demonstrated

in n o r a d r e n e r g i c

n e u r o n s w i t h i n t h e locus c o e r u l e u s ( L C ) of r a t a n d m o u s e

p r e s s i o n specifically m o d u l a t i n g T H p r o t e i n c o n c e n t r a t i o n in L C n e u r o n s was t h e r e f o r e p o s t u l a t e d . RU24722

( 1 4 , 1 5 - d i h y d r o - 2 0 , 2 l - d i - n o r e b u r n a m i n e - 14-

ol) is a n e b u r n a m i n e

d e r i v a t i v e . It h a s b e e n p r e s e n t e d

in a v a r i e t y of p h y s i o l o g i c a l a n d p h a r m a c o l o g i c a l m o d -

as a p o t e n t b r a i n a c t i v a t o r w h i c h e x e r t s p o s i t i v e effects

els: e.g. (1) i n a c t i v a t i o n o f s e r o t o n i n e r g i c a f f e r e n t s 1°'1'' ae.ee,.es.39: (2) cold stress4°'5"'54; a n d (3) r e s e r p i n e a d m i n -

o n f u n c t i o n a l r e c o v e r y a f t e r c e r e b r a l i s c h e m i a 4. Its p h a r -

i s t r a t i o n tl'el'ev'37"3s'41-43"55

which

R U 2 4 7 2 2 at a d o s e of 10 m g / k g i.v. i n c r e a s e s t h e t u r n -

w e r e n o t o b s e r v e d in d o p a m i n e r g i c n e u r o n s of s u b s t a n -

o v e r of n o r a d r e n a l i n e in m o u s e brain4S: it i n c r e a s e s sin-

These

modifications,

macological

profile m a y b e s u m m m a r i z e d

tia n i g r a a n d v e n t r a l t e g m e n t a l a r e a , w e r e i n t e r p r e t e d to

gle-cell

b e t h e c o n s e q u e n c e of a s e l e c t i v e c o n t r o l o f T H p r o t e i n

(DOPAC)

concentration

a f t e r d r u g a d m i n i s t r a t i o n 3 5 : it s t i m u l a t e s b r a i n o r n i t h i n e

in n o r a d r e n e r g i c

perikarya,

via m e c h a -

firing

and

as follows:

3,4-dihydroxyphenylacetic

acid

c o n c e n t r a t i o n in t h e r a t L C as e a r l y as 10 m i n

n i s m s m o d u l a t i n g T H g e n e e x p r e s s i o n . I n d e e d in t h e s e

d e c a r b o x y l a s e activity" a n d it d o e s n o t affect m o n o a m i n e

m o d e l s , T H p r o t e i n q u a n t i t i e s a n d T H activity w e r e cor-

uptake and monoamine

related.

Furthermore,

after reserpine

o x i d a s e activity. R U 2 4 7 2 2 h a s

administration >

b e e n s h o w n to i n d u c e a l o n g l a s t i n g , d o s e - d e p e n d e n t

~,33 a n d cold s t r e s s 4°, a significant e l e v a t i o n in t h e ex-

c h a n g e in T H p r o t e i n c o n c e n t r a t i o n in t h e rat L C > . T h i s

p r e s s i o n o f t h e specific m R N A

l o n g t e r m effect o n T H p r o t e i n c o n c e n t r a t i o n was d e m -

encoding for TH

was

Uorres'pondence. D. Weissmann, CNRS UMR 105, CERMEP, 59 Bd Pinel, 69003 Lyon. France. Fax: (33) (78) 53.31.12.

20 onstrated to be i n d e p e n d e n t of the initial activation of the LC neurons 2°. Unlike reserpine, this drug is devoid of cellular toxicity. The aim of this study was to answer 3 main questions: (1) if we hypothesize a selective control of T H steady state expression in the rat LC, are the steady state parameters of the synthesis of this protein homogeneous or related to the recently demonstrated 24 somatotopic organization of the efferents leaving the LC? (2) Is the long lasting increase of T H protein concentration described in the LC 2° generated by an elevation of the T H concentration within noradrenergic perikarya, or by an increase in the n u m b e r of cells synthesizing T H protein? (3) Are the long term effects of RU24722 on the steady state concentration of T H protein related to variations in the concentration of T H m R N A ? The distribution of both T H protein quantity and the n u m b e r of T H immuno-positive cells were determined along the postero-anterior axis of the LC u n d e r normal conditions and 3 days after the administration of RU24722 when the maximal increase in T H protein concentration has been shown to occur z°. Q u a n t i t a t i o n of T H and the d e t e r m i n a t i o n of the n u m b e r of T H positive cells were performed using a quantitative i m m u n o c h e m ical method with a high anatomical resolution that was recently developed in our laboratory 52. F u r t h e r m o r e , we assessed the effects of RU24722 upon the distribution and content of T H m R N A within the LC at the same level of resolution, using an in situ hybridization approach. Long term increases in T H protein content in the LC due to reserpine treatment or to cold stress have been shown previously to result from increases in T H m R N A levels 6't1'23. In the case of reserpine treatment, the dose o p t i m u m was identical for both T H protein and m R N A responses in the LC. Maximal increases following either a single reserpine injection or the onset of exposure to cold, were reached after 2 days for T H m R N A vs. 3 - 4 days for T H protein content and activity. In the present model, RU24722 induced a peak in T H content in the LC and adrenal medulla 3 days after administration of 20 mg/kg rat 2°. Therefore, by analogy with the former paradigm, we carried out T H m R N A assessment 1 day after i.p. injection of RU24722 at the dose of 20 mg/kg rat. MATERIALS AND METHODS The protein assay Tissue preparation Fourteen OFA male rats (180-200 g; IFFA-CREDO, Lyon, France) were kept under a 12-12 h dark-light cycle at 25°C with food and water ad libitum: 7 were treated with a single intraperitoneal injection of RU24722 (synthesized by the CNRS Laboratory

of Chimie des substances naturelles, Dr. C. lhal. Gif sur Yvette, France) (20 mg/kg in 400 itl of HCI 0.04 N): 7 received 400 ,td of the vehicle. The animals were sacrificed 3 days after treatment and their brains were rapidly removed and frozen by immersion in isopentane at -30°C for 1 rain. Coronal serial sections (20 f~m of thickness) were cut with a cryomicrotome (MICROM) from the posterior extremity of the LC (interaural -1.04 mm) to its anterior extremity (interaural 0.36 mm)34. As previously reported 5"~sections were selected at 200 l~m intervals, apposed onto nitrocellulose filters (Millipore, HAHY) and kept at room temperature until further processing (2-10 h). The two adjacent sections were respectively immunostained for TH (according to a slightly modified method of Sternberger et al.~") and Nissl-stained with Cresyl violet. Tissue processing TH immunochemical and radioautographic detection after protein transfer. All incubations were carried out at room temperature. After 1 h incubation of the nitrocellulose filters in a Tris-HCl-buffered saline solution (TBS, Tris 50 raM, NaCI 150 mM, pH 7.4) containing 1% (w/v) bovine serum albumin (BSA, Boehringer Mannheim), the filters were incubated for 18 h in anti-TH monoclonal antibody (Boehringer) diluted 1/4,000 in the above buffer (assay) or just in buffer (blank). This concentration of antibody was selected as the lowest that saturated all the antigenic sites. After washing once in a solution of TBS-1% BSA (10 min), and twice in a solution of TBS (10 min), the filters were incubated in a TBS-1% BSA solution containing 125Ilabelled protein A (1/2,000 v/v; Amersham SA; 30 mCi/mg) for 2 h, rinsed once for 10 rain in TBS-I% BSA and twice for 10 min in TBS, then dried. The filters were then apposed against 3H sensitive hyperfilm (Amersham RPN 12) for 48 h. Immunocytochemical localization of TH positive cells. After overnight fixation of the tissue at 4°C in a phosphate buffered saline solution (sodium phosphate 50 raM, NaCI 75 mM, pH 7.4) containing 4% (w/v) of paraformaldehyde, the sections were washed 3 times (30 min) in a phosphate buffered saline solution (PBS, sodium phosphate 100 mM~ NaC1 150 mM, pH 7.4), and then incubated in a PBS solution containing 1% normal swine serum (NSS; Dako) for the saturation of non-specific sites. All incubations except the fixation and the incubation with the anti-TH serum were made at room temperature. After rinsing 3 times (30 min) in PBS-I% NSS, sections were.incubated for 48 h in a solution of PBS-1% NSS containing rabbit anti-TH serum (dilution: 1/10,000, Institut Jacques Boy). After washing 3 times (30 min) in PBS-1% NSS, sections were incubated for 2 h in a PBS-1% NSS solution containing swine anti-rabbit serum (dilution: 1/500, Dako). After washing 3 times (30 min) in PBS-1% NSS and incubating overnight in a solution of PBS-1% NSS containing rabbit peroxidase-anti-peroxidase (dilution: 1/500, Dako) the sections were then immersed for 10 min in a Tris-HCl buffered solution (50 mM, pH 7.6) containing 13.33% (w/v) of 3,3'-diaminobenzidine (Fluka), 0.001% (w/v) H202 (Merck), and 4 g/1 nickel ammonium sulfate (Carlo Erba). The sections were finally rinsed 3 times (30 min) in a TrisHC1 buffered solution (Tris 50 mM, pH 7,4), dehydrated-delipidated in graded ethanols and methy[cycl0hexane, and coverslipped with Permount for light microscopy. Quantitative radioautography An image analysis system (IMSTAR) was used to determine the optical density using a variable gray scale (Autorad softwareCNRS). The area of interest was defined at each anatomical level using sections stained for immunocytochemistry. The integration of these areas over density (determined by a previouscalibration procedure) yielded an integrated optical density (O.D~) measurement. Division by the total number of contributing picture elements (pixels) yielded the average O.D. per unit area. The contribution of the non-specific.bacKground to the density of each region was determined by measuring the O.D. at the same regior~ transferred onto nitrocellulose from an adjacent coronal section incubated

21

without anti-TH antibody. This background density was subtracted from the overall staining of the circumscribed region of interest to obtain staining specifically due to TH. These measures were performed every 200 ~m along the postero-anterior axis of the LC.

TH m R N A assay Animals and tissue preparation

A scale of 6 standard TH protein concentrations was prepared from a solution of rat adrenal glands homogenized (20% w/v) in a phosphate buffered solution (potassium phosphate 5 mM, pH 6.0) containing 0.2% of Triton X100, and diluted with rat cerebellum homogenized under the same conditions, to keep the total protein concentration equivalent. 0.5 pl of each of these 6 standards was directly deposed onto a nitrocellulose filter (Millipore, H A H Y ) and was incubated for the same time as the filters carrying tissue sections. A scale was systematically apposed against each 3H sensitive hyperfilm. Each standard concentration was calibrated into units of TH per milligram of tissue (UTH/mgt). One unit of TH was defined as the concentration of rat adrenal TH protein which elicited a maximum catalytic activity (Vmax of 2.2 pmol of 3,4-dihydroxyphenylalanine/min "in vitro', under conditions already described 2°. As the efficiency of protein transfer onto nitrocellulose was equivalent for the tissue brain sections and for the standard tissue homogenates (data not shown), there was no need to correct the TH tissue concentration found.

Ten OFA male rats (180-200 g) were purchased from IFFAC R E D O and kept under a 12-12 h dark-light cycle at 25°C with food and water ad libitum. One day before sacrifice, each animal received one intraperitoneal injection, either of 400 ~1 RU24722 at 20 mg/kg body weight (n = 5 ) or of vehicle alone (HCI 0.04 N, n = 5). Twenty-four hours later, animals were anesthetized with sodium pentobarbital (0.4 mg/kg b.wt.) and perfused through the ascending aorta with 2% paraformaldehyde in 0.1 M phosphate buffer at pH 7.4 (300 ml at 4°C, 50 ml/min). Brains were dissected out with sterile instruments, and immersed in the same fixative for one more hour at 4°C, further immersed in a sterile 15% sucrose, phosphate buffered solution at 4°C for 48 h, then snap-frozen in liquid isopentane at -45°C for 20 s, and stored at -80°C until use. Sixteen itm-thick coronal sections from the locus coeruleus of each rat were cut on a cryomicrotome (frigocut, Reichert) at -20°C and mounted onto slides which had been heat-sterilized and subbed into a filter-sterilized solution of 1% gelatin and 0.5% chromium-potassium sulfate. The sections were collected at two distinct anatomical levels of LC corresponding to interaural -0.64 mm and interaural -0.04 mm: accuracy of sampling was checked histologically during sectioning.

TH positive cell counting

In situ hybridization and radioautography

The image analysis system allowed the counting of the TH positive cells after immunocytochemical staining and observation under a light microscope (Reichert). A camera (Pulnix) adapted to the microscope transmitted the image to a high resolution monitor. Anagra software (P. Platzer-CNRS) was used to count the stained cells selected as a function of their O.D. and of the surface area of their perikarya. The operator determined: (1) a threshold of O.D. under which cells were not considered stained; and (2) typical positive cells from which the mean area of the cells was determined. Once cells were counted, their number was systematically corrected by the Abercrombie equation: N = n * t / (t + d), where "n' is the number of the counted cells, 't' the mean section thickness, 'd' the mean perikarya diameter of the counted cell, and 'N' the corrected cell number ~. Furthermore the software determined the total surface of the circumscribed region of interest (i.e. the surface of the structure that contained cells). In this study, the surface (S, mm 2) of the LC in each anatomical plane, was circumscribed to the limit of the TH positive cells detected by immunocytochemistry. It was thus possible to calculate for each section, the corresponding LC volume (V, mm3; where V = S * t) and cell density (D, cell/mm3; where D = N/S * 50, since 50 sections of 20 p m of thickness are required for a 1 mm cylinder).

A synthetic oligonucleotide probe corresponding to the bases 1233-1267 of the rat TH complementary D N A (cDNA) ~5 was synthesized by the phosphite/phosphotriester method on an automated D N A synthesizer (Applied Biosystems Inc.) and radiolabeled by 'tailing' the 3' end using terminal desoxynucleotidyl transferase 8. In a final volume of 10/A of buffer (100 mM potassium cacodylate, 2 mM cobalt chloride, 0.2 mM dithiothreitol), 2.5 pmol of the 35mers oligonucleotide were incubated with 20 pCi of [35S]desoxyadenosine triphosphate (dATP, 1220 Ci/mmol) and 22 units of terminal desoxynucleotidyl transferase for 1 h at 37°C. The labelled oligonucleotide was purified by cold ethanol precipitation. Specific activity of the labelled oligomer was 5.106 cpm/pmol. Sections were incubated for 1 h at room temperature in a prehybridization buffer containing 4 x SSC (1 x SSC = 0.15 M sodium chloride/0.015 M sodium citrate, pH 7.0) and 1 x Denhardt's (0.02% Ficoll, 0.02% polyvinyl pyrrolidone, 0.02% bovine serum albumin). The slides were then dehydrated and air-dried. The labelled probe diluted at 1 nM in hybridization buffer (50% deionized formamide, 3 x SSC, 1 x Denhardt's, 100 ~g/ml salmon sperm D N A , 125 pg/ml brockers yeast tRNA, 10 mM dithiothreitol, 0.8% sarcosyl) was applied to each slide, covered with a sterile coverslip and sealed with rubber cement. The slides were placed into a humid chamber and hybridization was allowed to proceed for 18 h at 38°C. At the end of hybridization, coverslips were carefully removed in 2 x SSC and the slides were then immersed into successive washing solutions of buffer 2 x SSC (2 h), 1 x SSC (1 h) and 0.5 x SSC (1 h). Sections were dehydrated in ethanol, air dried and apposed to r - m a x film (Amersham). Following development of the r - m a x films, tissue sections were coated with Ilford K-5 nuclear emulsion diluted in water (1/1) and exposed at 4°C for 10-20 days. The slides were developed with Kodak D-19, counterstained with Cresyl violet and coverslipped. In order to determine the concentration of the radioactive probe which ensures saturation of tissular TH m R N A , 3 series of 4 adjacent sections were hybridized in a preliminary experiment with increasing probe concentrations, ranging from 0.5 to 2 nM.

Calibration of radioautographic reaction

Expression of the results Values of 6 different parameters were systematically determined in each anatomical interval (200/~m of thickness) studied along the postero-anterior axis of the LC: (1) the TH immuno-positive cell number; (2) the volume occupied by these cells (mm3); (3) the density of TH positive cells (cells/mm3); (4) the TH tissue concentration (UTH/mgt) determined after radioautography; (5) the ratio of the TH tissue concentration to the TH immuno-positive cell density which was considered an index of the mean TH cellular concentration (UTH/celI); and (6) the TH protein quantity (UTH) estimated as the product of the TH tissue concentration and the LC volume (one mg of tissue was assumed to have a volume of 1 mm3). Results were expressed as the mean + S.E.M. (n = 7 rats) of each calculated parameter. Statistical analysis of the results was performed using a Student's t-test to compare values at each anatomical level and a one-way analysis of variance ( A N O V A I) where n is the number of sections analyzed for all animals in the considered region, to compare values of the total LC and of its posterior and anterior region. Linear regression was calculated by using the least square method (RS1 version 4 software, BBN).

Quantitative analysis of radioautographs Film radioautographic labelling was quantified by computerized densitometry using the image analysis system (IMSTAR; Gel software-CNRS). This package allowed us to measure the intensity of the specific hybridization signal integrated above the background densities of film and tissue as follows. On each radioautograph

22 analysed, a specifically labelled structure was delineated on the screen of the computer with a 'mouse'-coupled digitizing table and

the optical density unit (ODU) of labelling was measured within. The area thus defined was then translocated over a distinct region of the same section devoid of any specific hybridization signal, and the ODU of labelling measured herein. Automatic subtraction of the latter value from the former systematically yielded the ODU corresponding to the specifically hybridized probe. ODU per pixel reflected the mean TH mRNA concentration per unit area found in the LC in a section. It was converted in ODU per mm 3 of tissue by multiplying the surface of the pixel in mm 2 and the thickness of the section in mm. Emulsion-coated sections were analyzed with a light microscope (Reichert). The surface defined by the cluster of specifically hybridized neurons was measured on Cresyl violet stained radioautographs with an image analysis system (Autorad software) coupled to the microscope. Specifically labelled neurons of the pontine tegmentum were mapped by camera lucida and numerated (Anagra software, P. Platzer-CNRS). Numbers of TH mRNA-expressing neurons per 200 ,um interval were extrapolated with the Abercrombie formula, The volume occupied by TH mRNA positive cells of the LC per 200 pm interval was also extrapolated. The volume above was used to calculate an index of the absolute quantity of TH mRNA found in the interval by multiplying it by the mean ODU/mm 3. Results were expressed as the mean + S.E.M. (n = 51) of each calculated parameter. Statistical analysis of the results was performed using Student's t-test (RS1 4 software, BBN) where n is the number of sections analyzed for all animals in the considered anatomical interval, RESULTS

Distribution of TH immuno-positive cells in the tAi T H immunocytochemistry allowed the population of T H containing neurons in the whole LC of controls to be estimated as 1602 + 166 cells distributed in a volume of 0.1222 + 0.0042 mm 3 (Table 1): this c o r r e s p o n d e d to a density of 11763 + 831 cells/mm 3 (Table I). The distribution of the n u m b e r of the T H immunopositive cells was m e a s u r e d at 200/~m intervals along the p o s t e r o - a n t e r i o r LC axis and closely followed the variation in volume of the LC as strictly delimited by the TH positive p e r i k a r y a (Fig. 2A, B). M a r k e d differences were observed between the posterior and anterior LC with respect to the above p a r a m e t e r s (Fig. I C, F). The posterior region (800 p m length) contained 88% of the T H immuno-positive cells, which were distributed in 85% of the total volume of the LC. In the anterior region (800 p m length), the LC had a smaller cross-sectional area and contained only 12% of the T H immuno-positive cells in 15% of the total LC volume (Table I). The posteroanterior distribution of cell density was less pronounced (Fig. 2C, Table I), however the mean T H cell density of the anterior extremity of the LC (anatomical intervals 7 and 8) was 40% less than that of its posterior extremity (anatomical intervals 1 and 2) (P -< 0.05 significantly different using Student's t-test).

TH protein steady state within the locus coeruleus Calibration of the radioautographic reaction Radioautographic detection of TH protein and localization of TH immunocytochemical-positive cells Fig. 1A, D show T H protein stained by the immunochemical-radioautographic m e t h o d after transfer of sections from control animals onto nitrocellulose membranes. T H immuno-positive cells from adjacent sections are shown in Fig. 1B, E, in two anatomical planes corresponding to the posterior (left column) and the anterior (right column) regions of the L C (interaural coronal coordinate - 0 . 8 4 and 0.24 m m 34, respectively). A very high specific r a d i o a u t o g r a p h i c reaction was o b s e r v e d in the n o r a d r e n e r g i c nucleus in both regions of the LC. The intensity of the T H protein specific reaction on film corr e s p o n d e d closely to the T H immunostaining in adjacent sections. This superimposition provided a reliable anatomical control of the O . D . m e a s u r e m e n t and allowed the restriction of the measure to the LC region strictly corresponding to the n o r a d r e n e r g i c cell bodies (shown in Fig. 1C, F after a 12-fold enlargement of Fig 1B, E).

The specific O . D . per surface unit corresponding to each standard of the T H concentration scale was measured. This O.D./surface unit was directly proportional to the T H tissue concentration (y = 558 x + 8.5; n = 15; P -< 0.001). Thus it allowed the quantification of tile T H tissue concentration from the radioautographic reaction.

Distribution of TH quantity and TH concentration in the LC The O . D . m e a s u r e m e n t allowed us to estimate the T H protein concentration of the whole LC as 69.8 + 3 U T H / m g t and the total quantity of T H as 12.8 _+ 0.5 U T H (Table II). T H tissue concentration was approximatively 2-fold greater ( P _< 0.001) in the posterior 800 ~m. Ninety-two % of the total T H was contained in the posterior region (Fig. 3, Table II). A significant linear relationship was o b s e r v e d between

Fig. 1. Radioautograms obtained from 20 ~m rat brain coronal sections transferred onto nitrocellulose filters and incubated with anti-tyrosine hydroxylase (TH) antibody are shown in A and D; bar= 1.2 ram. A high specific reaction can be observed in (A) the caudal and in (D) the rostral (interaural coronal coordinates -0.84 and -0.24 mm, respectively) locus coeruleus (LC) regions 32. Adjacent sections treated for TH immunocytochemistry are shown below (B and E); bar = 1.2 mm. A 12.5-fold enlargement of the LC region from sections in B and E is shown in C and F; Bar = 0.096 mm,

23

24 TABLE I Comparison of the distribution of the mean of TH immunoreactive cell number, volume occupied by these cells and cell density in the tom/ LC and in its posterior and anterior parts in control and RU24722 treated rats TH immunopositive cells were counted and the volume of the LC, as strictly delimited by the labelled perikarya, was measured in the whole nucleus and in its posterior and anterior regions (each of 800 micrometers length). The density of TH positive cells was also calculated in each region. Results are expressed as the means _+ S.E.M., n = 7. Mean % of variation + S.E.M. of treated vs. control rats is given for each parameter in the considered region. Total

Posterior

TH + cells Controls RU24722 % of variation

1602 _+ 166 2049 4- 76 28 4- 5

1414 4- 165 1804 + 72 28 4- 5*

Volume (mm 3) Controls RU24722 % of variation

0.1222 4- 0.0042 0.1421 4- 0.0073 17 _+ 5

0.1035 4- 0.0037 0.1217 + 0.0053 18 4- 5

Cell density (cells/mm3) Controls RU24722 % of variation

11763 4- 831 12312 + 433 8 + 5

13161 + 1174 14437 + 496 10 4- 4

Anterior

188 4- 40 ~ 246 + 31 31 4- 17

0.0188 + 0.0021 t* 0.0204 4- 0.0027 9 + i4

10349 + 1362'* 10860 + 745 5 + 7

*P ~< 0.05, **P ~< 0.02, ***P ~< 0.01, ****P ~< levels of significance of difference between treated and control values using a oneway ANOVA; n is the number of sections obtained for all animals in the considered region (n = 51-56 in the total LC). *P ~< 0.02, **P ~< 0.001 levels of significance of difference between the values of the posterior and anterior regions of the LC using a one way ANOVA; n is the number of sections analyzed for all animals in the considered region (n = 23-28 in the posterior and anterior region).

the quantity of the TH protein and the number of TH

c o n c e n t r a t i o n . I n t h e t o t a l L C it w a s e s t i m a t e d as 0.0064

i m m u n o - p o s i t i v e cells p e r s e c t i o n (y = 0.0072 x + 0.1907;

+ 0.0006 U T H / c e l l (Table II). T h e T H c e l l u l a r c o n c e n -

n = 50, P _< 0.0001; Fig. 4). A t e a c h i n t e r v a l a l o n g t h e

t r a t i o n was also h e t e r o g e n e o u s l y d i s t r i b u t e d a l o n g t h e

p o s t e r o - a n t e r i o r axis o f t h e L C , t h e r a t i o o f t h e T H tis-

p o s t e r o - a n t e r i o r axis o f t h e L C , b e i n g 1.6-fold g r e a t e r ( P

sue c o n c e n t r a t i o n a n d t h e T H i m m u n o - p o s i t i v e cell d e n -

_ 0.001) in t h e p o s t e r i o r r e g i o n t h a n in t h e a n t e r i o r .

sity w a s c o n s i d e r e d as a n i n d e x o f t h e m e a n T H c e l l u l a r

TABLE II Comparison o f the distribution of the mean of TH tissue and cellular concentration and TH quantity in the total LC and in its posterior and anterior parts in control and RU24722 treated rats The TH tissue concentration was measured, the TH cellular concentration and the TH quantity were calculated in the whole nucleus, in its posterior and in its anterior region. Results are expressed in the same manner as those of Table I. Total UTH/mgt Controls RU24722 % of variation

69.7 + 30.1 91.9 4- 4.3 31.8 4- 6.1"***

UTH/Cell Controls RU24722 % of variation

0.0064 + 0.0006 0.0079 + 0.0006 23.6 + 8.7**

UTH Controls RU24722 % of variation

12.8 + 0.5 19.8 + 1.3 55 + 10.3"

Posterior

92.4 + 3.1 120 4- 4.7 29.8 + 5.1"***

0.0078 + 0.0010 0.0088 + 0.0006 12.6 + 7.1

11.8 + 1.0 17.9 _+ 1.2 52.1 + 9.8****

Anterior

43.9 + 4.1 ~ 63.8 + 4.4 45.5 4- 10.0"***

0.0047 + 0.0005 t~ 0.0068 + 0.0006 44.4 _+ 13.5"**

1.0 + 0.3 +÷ 1.9 + 0.3 88.7 + 25.8**

25

Long-term effect of RU24722 on TH distribution T h r e e days after a single injection of RU24722 (20 mg/kg) the relative variations in the studied p a r a m e t e r s (the m e a n percentage of increases vs. the m e a n control corresponding values) were d e t e r m i n e d in the LC. Post e r o - a n t e r i o r distribution of these variations is shown in Figs. 5 and 6. Significant increases in T H quantity and T H tissue concentration were o b s e r v e d in the whole LC. The cellular concentration of T H was almost exclusively increased in the anterior L C (Fig. 6 and Table II). M o r e over, in the anatomical intervals in which T H tissue concentration was significantly increased (Fig. 6), a linear inverse correlation (y = -13537 x + 124; n = 6, P

I

50-

03

e.-

E.o E~

10"

°i

-10 -30

1

2

3

4

5

6

7

8

C

90 6

5

•o

4 F-. 2D

°

03o~ E~ E c

~

ql

0

3

i

•

2

.-

7o- l 50 30

10" i -10 i

-30 " 1 0

I

~

O

•

n

•

200

400 Cells

600

2

3

4

5

postero-anterior

/

'

-

1

u

8

0

Fig. 4. Correlation between the quantities of TH and the number of TH immuno-positive cells. Absolute values of TH protein quantity were calculated at each 200 ,um interval along the postero-anterior axis. The corresponding number of TH positive cells was estimated from postfixed adjacent sections after immunocytochemical detection. (y = 0.0072 x + 0.1907: n = 50, r2 = 0.788, P -< 0.0001.)

6

7

8

axis

Fig. 5. Postero-anterior distribution of changes in: (A) the number of TH immunoreactive cells; (B) the volume occupied by these cells; and (C) the cell density from interaural coronal coordinates -1.04 mm to 0.36 mm 32, after the RU24722 injection (20 mg/kg), Each bar represents the mean percentage of variation + S.E.M. (n = 7) of the above parameters in the different anatomical planes, vs. mean control values in the same intervals. (* P _< 0.(35; ** P 0.02 significantly different from control values using Studenffs t-test.)

27 4-_ 166 cells) found in these postfixed sections was similar to that previously reported 4v after immunocytochemistry performed on sections of brains fixed by intracardiac perfusion with paraformaldehyde. The majority of TH-immunopositive cells were found in the posterior part of the LC as has already been reported ~3. As expected, the TH protein quantity was proportional to the number of TH immuno-positive cells along the posteroanterior axis of the LC. This relation yielded an estimation of the mean cellular concentration of 0.0072 UTH/ cell (Fig. 4) in the whole LC, which is quite comparable to that of 0.0064 UTH/cell (Table II) obtained from values calculated at each anatomical interval of each control rat. These results allow the total TH level of the whole noradrenergic cell population of the LC (1602 _+

80 70

o

60

~

4o

I-- m "t"

30 20 10 0

E

5o

1

2

3

4

5

120

6

7

8

ii

B

lOO _

~

o

g ~

O

80

60

•

200. -20 2

3

2°°(F 17511

i

i

4

5

p 6

7

= 8

"'T"

166 cells) to be estimated as 11.5 UTH. This result is quite similar to the 12.8 UTH (Table II) obtained by summing the mean values found at each anatomical interval of the control rats. This close linear relationship supports the interpretation that under our conditions of measurement (area of measure restricted to the limits of the presence of immunostained cell bodies) the source of the transferred TH is mainly the noradrenergic cell bodies and proximal dendrites. These dendrites nearest to the TH positive cell bodies, which certainly contained the TH protein, can not be discriminated from them within the area of measurement, but they are probably a minor source of transferred TH protein in this area. Moreover previous results obtained from a study of the TH protein distribution in subcellular fractions of the LC tissue ~4 have indicated that the synaptosomal fraction contained only 2% of total TH protein. Thus we can consider that the ratio of TH tissue concentration to the density of TH immuno-positive cells is an accurate estimation of the mean cellular concentration of TH protein (found at each anatomical interval). The TH protein tissue concentration exhibited a well-marked postero-anterior gradient. The anterior region of the structure had a smaller TH tissue concentration (-52%) than the posterior. This reflects a lower mean cellular concentration of TH protein (-40%). Thus the steady state conditions of TH expression appear to be different between these two regions of the same nucleus. This could explain the different responses of the two regions to pharmacological stimulation. Three days after a single injection of RU24722 a significant increase in the level of TH protein was found throughout the LC. This increase was similar to that already described when TH protein was analyzed in dissected LC by a quantitative Western blot technique 2". However, the results presented here show that the relative increase in the level of TH observed in the total

c

:.

120 -

~

.

100 -

~

loo 75

.

• • T * _ 2 " ~ ~ l x _

"~

0

80-

•

I I-.

40 20

1

2

3

4

postero-anterior

5

6

7

8

axis

0 -20

0.000 Fig. 6. The p e r c e n t a g e d i s t r i b u t i o n of the v a r i a t i o n s of: ( A ) the T H tissue c o n c e n t r a t i o n : (B) the T H cellular c o n c e n t r a t i o n ; and (C) the q u a n t i t y of T H a l o n g the p o s t e r o - a n t e r i o r axis of the LC, after the R U 2 4 7 2 2 injection (20 m g / k g ) . R e s u l t s are e x p r e s s e d in the s a m e m a n n e r as those in Fig. 5. * P _< (/.05; ** P _< 0.02; *** P -< 0.01; **** P _< 0,001 significantly d i f f e r e n t from control v a l u e s nsing S t u d e n t ' s t-test.

0.;02 0.;04 UTH

0.;06 / Cell

0.;08 0.010

Fig. 7. R e l a t i o n s h i p b e t w e e n the m e a n p e r c e n t a g e v a r i a t i o n in the T H cellular c o n c e n t r a t i o n after R U 2 4 7 2 2 injection vs. m e a n control values, at e a c h a n a t o m i c a l i n t e r v a l w h e r e the T H tissue conc e n t r a t i o n i n c r e a s e d after the t r e a t m e n t . (y = - 1 3 5 3 7 x + 124; n = 6, r 2 = 0.734, P -< 0.03.)

28

Fig. 8. Subregional localization of the hybridization signal in emulsion-coated radioautographs of LC (under fight microscope) at (a) the anterior and (b) posterior levels, in sham animal, c: shows neuronal localization of the radioautographi¢ labelling at ~ magnification within LC. Note the uniform distribution of silver grains both over the nucleus and the cytoplasm of each neuron. IV, fourth ventricule; mv, mesencephalic nucleus of the fifth cranial nerve; Bar A and B =100/~m; C = 25/tm.

L C was larger than the augmentation of the T H cellular concentration. In fact, changes in T H quantity resulted from b o t h an augmentation of the cellular concentration of T H in cells previously synthesizing the enzyme and an increase in the n u m b e r of cells expressing T H . M o r e detailed analysis r e v e a l e d that these two different mecha-

nisms accounted for the augmentation in the quantity of T H , respectively, in the two anatomical subdivisions of the LC. Thus increases in the quantity o f T H protein in the posterior region of the structure resulted mainly from an increase in the n u m b e r of T H immuno-positive cells whereas in the anterior region, the primary effect

29 TABLE III Comparison of the distribution of the mean number of TH mRNA containing cells, volume occupied by these cells and cell density at a posterior and at an anterior level of the LC in control and RU24722 treated rats

TH mRNA containing cells were counted and the volume strictly delimited by the labelled cell bodies was measured at the chosen posterior and anterior levels (each of 200 micrometers length). The density of TH mRNA positive cells was also calculated at the two considered intervals. Mean % of variation + S.E.M. of treated vs. control rats is given for each parameter at the two anatomical levels. Posterior

Anterior

TH RNA + cells Controls RU24722 % of variation

597+42 794 + 66 33+11"

Volume (mm 3) Controls RU24722 % of variation

0.0230+0.0007 0.0281+0.0014 22+6**

0.0048+0.0003 0.0063+0.0005 32+10"

25877+1553 28723_+2588 11+10

10421_+834 17090+1772 64+17"*

Cell density (cell/mm 3) Controls RU24722 % of variation

49+4 117_+17 138+34"*

*P ~< 0.05, **P ~< 0.02, ***P ~< 0.01, ****P ~< 0.001 levels of significance of difference between treated and control values using a Student's t-test; n is the number of sections analyzed for all animals at the considered level (n = 5-6).

was a p p a r e n t l y an a u g m e n t a t i o n of T H cellular concentration. A n increase in the n u m b e r of T H positive cells was also o b s e r v e d in the anterior LC, however it did not

TABLE IV Comparison of the distribution of the mean TH m R N A tissue concentration (ODU/mm 3) and of the mean total quantity of TH mRNA present in the tissue (ODU) at a posterior and anterior level of the LC in control and RU24722 treated rats

Mean ODU/mm 3 which is a measure of the mean TH mRNA tissue concentration, was calculated at the two levels. Mean ODU was also calculated, it reflected the mean quantity of TH mRNA found in these intervals (each of 200 micrometers length). Results are expressed in the same manner as those of Table III. Posterior

TH mRNA tissue concentration (ODU/mm 3) Controls 20325_+1219.5 RU24722 27642+1423 % of variation 36+7****

Anterior

8010+1201 11054+1121 38+14"

TH mRNA quantity (ODU) Controls

RU24722 % of variation

467.5_+33

776+47 66+10"**

38.4___5.8

72.6+11.5 89_+30*

reach statistical significance. Thus the injection of RU24722 revealed the existence of 'sleeping cells' in which T H expression appears to be inactive or, at such a low level of activity, that it is beneath the threshold of detection by immunocytochemistry. In the rat LC, these cells a p p e a r to be localized mainly at the p e r i p h e r y of the structure since (1) the density of the T H immunopositive cells did not significantly increase; and (2) the volume of the T H positive nucleus was significantly higher after t r e a t m e n t with RU24722. Several questions arise from this observation. (1) D o e s the awakening of the sleeping cells result from the activation of the transcriptional mechanism? (2) W h a t other properties characterize these cells before the putative activation of the T H gene? (3) W h a t is the functional importance of such cellular plasticity? A fluctuating level of T H expression has already been observed in another population of catecholaminergic neurons that s e e m e d to be d e p e n d e n t on h o r m o n a l regulation 7. Thus it is reasonable to hypothesize that a 'fluctuating state' of the degree of T H gene expression may be a m o r e general p r o p e r t y which is precisely controlled in each population or subpoputation of catecholaminergic cells. Relative changes in tissue and cellular concentration of T H protein occurred mainly in the anterior part of the structure. The cells of this portion exhibited a significantly lower initial concentration of T H . I n d e e d , the greater the cellular concentration of T H found in steady state conditions the lower the amplitude of increase in T H concentration that resulted from the pharmacological induction process in L C cells. This observation may indicate that the T H protein concentration in cells could exert a retrocontrol on its own synthesis. Studies are in progress to elucidate whether this control could involve transcriptional and/or post-transcriptional mechanisms even if modifications of T H protein d e g r a d a t i o n cannot be totally rejected. H o w e v e r , if the increase in T H concentration o b s e r v e d after the t r e a t m e n t was due to a decrease of the d e g r a d a t i o n of the protein, it might be expected that this increase would be m o r e p r o n o u n c e d in cells exhibiting the largest T H cellular concentration, and this is not the case. The in situ hybridization approach allowed both visualization of TH-expressing neurons and semi-quantitative assessment of T H m R N A at anatomically well-defined levels of the LC. The synthetic oligonucleotide p r o b e used in the present study had been selected by H a n et al. 17 from the c o m p l e t e sequence of the T H gene 15. This oligonucleotide was shown to hybridize specifically with T H m R N A in the b r a i n : , yielding the same labelling p a t t e r n as the c D N A encoding the entire T H m R N A s 5. The distribution of label o b t a i n e d with both

30 types of probes has been correlated with the distribution of TH-immunoreactive neurons in the brain ~. In situ hybridization with the oligonucleotide radiolabelled with the 35S gave a satisfactory resolution at the cellular level and avoided the long exposure times that are required with tritium-labeled probes 51. Film radioautography was used for quantification of the hybridization signal at the regional level. The specific hybridization signal in the LC being saturated at 1 nM concentration of probe, was already found for in situ hybridization of T H m R N A with a complementary R N A probe (cRNA) 3z. The specific labelling density could be used as an index of the tissular concentration of T H m R N A . In control animals, the distribution of specifically hybridized neurons at the two levels of the LC examined was very similar to the distribution of TH-immunoreactive neurons found. Indeed, morphometry-coupled densitometry showed that the number and spatial density of T H mRNA-containing neurons and the volume of the LC were very similar to the values obtained with preparations processed for T H immunocytochemistry at the same anatomical levels. In particular, the ratio between the posterior and anterior LC for each of the above 3 parameters was comparable to the corresponding ratio calculated from T H protein assessments. Following RU24722 treatment, the distribution of T H m R N A in the pontine tegmentum was altered in a way that was similar in many aspects to the modifications of T H protein contents. Thus, the number of cells containing T H m R N A was increased by at least as much as the number of T H immunoreactive cells at corresponding levels. The volume of the LC defined by the regional extension of the specific hybridization signal at the two levels assessed also increased by a similar amount as the volume of the LC defined by the regional extension of T H immunoreactivity. Finally, tissue concentrations of T H m R N A vs. T H protein contents indicate that the induction of T H protein in LC by RU24722 resulted from a rise in T H m R N A levels. In particular, the increases of the number of TH-expressing cells and LC volume appear to result from the appearance of T H m R N A in neurons located at the periphery of the LC which did not contain T H m R N A at detectable levels in control rats, as observed for T H protein. The present in situ hybridization approach thus confirmed the existence of a population of 'sleeping cells' which are induced to express T H after RU24722 treatment, in or around the LC. However, the alterations in T H m R N A and T H protein in the LC induced by RU24722 differ from each other in a few features. Thus, the number and density of T H m R N A containing cells in anterior LC increased much more than the number and density of T H immu-

noreactive cells, and than tile concentration ol I H m R N A in this region. This might b~, duc {o putatively slower kinetics of translation of the induced protein m the 'sleeping cells' as compared to L(? neurons that express TH in control rats. Thus some of tile cells that contain TH m R N A may not express T H protein at all or perhaps only after a kruger time delay. Thus analysis of the same cells 24 h later might yield a different ratio between TH m R N A and TH protein. The cytoplasmic concentration of T H may also regulate post-transcriptional events such as translation, as has been indicated recently for tryptophan-hydroxylase in the raphe nuclei 5~. Interestingly, similar discrepancies between alterations of T H m R N A and TH protein content were found in the adrenals and/or the LC in other models of TH induction. The increase of T H m R N A was thus 1.3-3-fold higher than the increase of T H protein in the rat superior cervical ganglion following preganglionic nerve stimulation 12, in rat brain and adrenals by exposure to cold 3' 4o in rat brain, superior cervical ganglion and adrenals after a single injection of reserpine :'6'tj'33. in rat sympathetic neurons undergoing prolonged depolarization by KC1 or veratridine in vitro ls~5, and in rat pheochromocytoma PC12 cells under glucocorticoid treatment 2:~'24. However, in most of these paradigms, it remains to be determined whether the increases in T H m R N A are caused by an increase in the transcription of the T H gene, an increase in the stability of the TH m R N A or an alteration in post-transcriptional processing. TH induction by reserpine was actually shown to result from an increase of gene transcription, since the increases in T H activity, protein and m R N A were totally blocked by transcription inhibitors -~". In the case of TH induction by glucocorticoids, the increase of the TH gene transcription rate was also directly demonstrated by nuclear transcript run-on assay 24. In all other models, as in the case of RU24722 injection, genomic activation remains to be directly assessed. For instance the induction of T H by N G F in cultured sympathetic neurons is preceded by an increase in TH m R N A which was proven to be insensitive to R N A synthesis inhibitors. This induction therefore resulted from some post-transcriptional control 3~'3642, Nonetheless, all the models of TH induction cited above display features common to the two cases of established T H gene activation, i.e., increases in TH protein lag behind the increases in TH m R N A and the ratio between T H m R N A and T H protein increases. These features were precisely those recorded in the present study for the effect of RU24722 on TH in the LC. as well as a spreading of these effects all over the LC. These observations suggest that a stimulation of m R N A synthesis by RU24722 occurs rather than an inhibition of R N A catabolism.

31 Finally, the study of TH mRNA

i n d i c a t e s t h a t t h e in-

In the present report,

we h a v e c o n f i r m e d a n d ex-

c r e a s e in T H p r o t e i n c o n c e n t r a t i o n w a s m o s t p r o b a b l y

p a n d e d t h e c h a r a c t e r i z a t i o n of a n e w e x a m p l e of T H in-

due to a stimulation of TH

s y n t h e s i s . It also

d u c t i o n , t h e m e c h a n i s m of w h i c h r e m a i n s to b e eluci-

c o n f i r m e d t h e e x i s t e n c e o f ' s l e e p i n g n e u r o n s ' in t h e L C ,

d a t e d . T h e m e c h a n i s m a n d p h y s i o l o g i c a l s i g n i f i c a n c e of

w h i c h d o n o t n o r m a l l y e x p r e s s T H , at a d e t e c t a b l e level,

t h e e m e r g e n c e of t h e e x p r e s s i o n o f n o r a d r e n e r g i c p h e -

a n d t h a t t h e s e c a n b e ' a w a k e n e d ' u n d e r c o m p e t e n t stim-

n o t y p e in ' s l e e p i n g cells' o f t h e L C m u s t n o w b e inves-

u l u s ( i ) . T h e s a m e p h e n o m e n o n h a s b e e n d e s c r i b e d in t h e

tigated.

mRNA

hypothalamic arcuate and periventricular nuclei, but not in t h e z o n a i n c e r t a , a f t e r t r e a t m e n t

w i t h g o n a d a l ste-

r o i d s 29"44, a n d in o l f a c t o r y p e r i g l o m e r u l a r s t r u c t u r e s o f t h e f o r e b r a i n u n d e r T H - i n d u c t i v e s e n s o r y i n p u t s 16. S i n c e these regions are dopaminergic and not noradrenergic, this n e u r o c h e m i c a l

plasticity m i g h t b e l i n k e d to s o m e

fundamental property of the TH gene.

REFERENCES 1 Abercrombie, M., Estimation of nuclear population from microtome sections, Anat. Rec., 94 (1946) 239-247. 2 Austin, M.C., Cottingham, S.L., Paul, S.M. and Crawley, J.N., Tyrosine hydroxylase and galanin mRNA levels in locus coeruleus neurons are increased following reserpine administration, Synapse, 6 (199(I) 351-357. 3 Baruchin, A., Weisberg, E.P., Miner, L.L., Ennis, D., Nisenbaum, L.K., Naylor, E., Stricker, E.M., Zigmond, M.J. and Kaplan, B.B., Effects of cold exposure on rat adrenal tyrosine hydroxylase: an analysis of RNA, protein, enzyme activity and cofactor level, J. Neurochem., 54 (1990) 1769-1775. 4 Barzaghi, F., Dragonetti, M. and Boissier, J.R., Effect of a new eburnamine derivative (RU24722) on EEG recovery in the conscious gerbil after cerebral ischemia, Drug. Dev. Res., 2 (1982) 533-541. 5 Berod, A., Faucon Biguet, N., Dumas, S., Bloch, B. and Mallet, J., Modulation of tyrosine hydroxylase gene expression in the central nervous system visualized by in situ hybridization, Proc. Natl. Acad. Sci. USA, 84 (1987) 1699-1703. 6 Black, I.B., Chikaraishi, D.M. and Lewis, E.J., Transsynaptic increase in RNA coding for tyrosine hydroxylase in a rat sympathetic ganglion, Brain Res., 339 (1985) 151-153. 7 Brawer, J., Bertley, J. and Beaudet, A., Testosterone inhibition of tyrosine hydroxylase expression in the hypothalamic arcuate nucleus, Neurosci. Lett., 67 (1986) 313-318. 8 Collins, M.L. and Hunsaker, W.R., Improved hybridization assays employing tailed oligonucleotide probes. A direct comparison with 5' end labeled oligonucleotide probes and nick-translated plasmid probes, Anal. Biochem., 151 (1985) 211-224. 9 Cousin, M.A., Lando, D., Gueniau, C. and Worcel, M., In vivo effect of RU24722 and drugs used for treatment of senile cerebral insufficiency on rat brain ornithine decarboxylase, J. Pharmacol., 16 (I985) 31-43. 10 Crespi, F., Buda, M., McRae-Degueurce, A. and Pujol, J.E, Alteration of tyrosine hydroxylase in the locus coeruleus after administration of p-chlorophenylalanine, Brain Res., 191 (1980) 501-509. 11 Faucon-Biguet, N., Buda, M., Lamouroux, A., Samolyk, D. and Mallet, J., Time-course of the changes of TH mRNA in rat brain and adrenal medulla after a single injection of reserpine, E M B O J., 5 (1986) 287-291. 12 Faucon-Biguet, N., Rittenhouse, A.R., Mallet, J. and Zigmond, R.E., Preganglionic nerve stimulation increases mRNA levels for tyrosine hydroxylase in the rat superior cervical ganglion, Neurosci. Lett., 104 (1989) 189-194. 13 Gagne, C., Moyse, E., Kocher, L., Bour, H. and Pujol, J.F., Light microscopic localization of somatostatin binding sites in

Acknowledgements. This work was supported by grants from CNRS (UMR 105), INSERM (CJF 90-10), Universite Claude Bernard LYON I, DRET (n ° 508850 and 508933) and Servier Laboratories. We are very grateful to Dr. C. Thal for the synthesis of RU24722, and to Dr. E. Derrington and Dr. D. Marcel for their valuable help in the preparation of the manuscript.

the locus coeruleus of the rat, Brain Res., 530 (1990) I96-204. 14 Gillon, J.Y., Labatut, R., Renaud, B. and Pujol, J.F., Subcellular distribution of tyrosine hydroxylase in some catecholaminergic rat brain areas determined by a quantitative immunoblot assay, J. Neurochem., 52 (1989) 677 683. 15 Grima, B., Lamouroux, A., Blanot, F.. Faucon Biguet, N. and Mallet, J., Complete coding sequence of rat tyrosine hydroxylase mRNA, Proc. Natl. Acad. Sci. USA, 82 (1985) 617-621. i6 Guthrie, K.M. and Leon, M., Induction of tyrosine hydroxylase expression in rat forebrain neurons, Brain Res., 497 (1989) 117-131. 17 Han, V.K.M., Snowaert, J., Towle, A.C., Lund, P.K. and Lauder, J.M., Cellular localization of tyrosine hydroxylase mRNA and its regulation in the rat adrenal medulla and brain by in situ hybridization with an oligonucleotide probe, J. Neurosei. Res., 17 (1987) 11-18. 18 Hefti, F., Gnahn, H., Schwab, M.E. and Thoenen, H., Induction of tyrosine hydroxylase by nerve growth factor and by elevated K + concentrations in cultures of dissociated sympathetic neurons, J. Neurosci., 2 (1982) 1554-1556. 19 Keane, P.E., Degueurce, A., Renaud, B., Crespi, E and Pujol, J.F., Alteration of tyrosine hydroxylase and dopamine-flhydroxylase activity in the locus coeruleus after 5,6-dihydroxytryptamine, Neurosci. Lett., 8 (1978) 143-15(/. 20 Labatut, R., Richard, F., Milne, B., Quintin, k., Lecestre, D. and Pujol, J.F., Long term effects of RU24722 on tyrosine hydroxylase of the rat brain, J. Neurochen*., 5l (1988a) 13671374. 21 Labatut, R., Buda. M. and Berod, A., Long term change in rat brain tyrosine hydroxylase following reserpine treatment: a quantitative immunochemical analysis, J. Neurochem., 50 (1988b) 1375-1378. 22 Lewis, B.D., Renaud, B., Buda, M. and Pujol, J.E, Time course variations in tyrosine hydroxylase activity in the rat locus coeruleus after electrolytic destruction of the nuclei raphe dorsalis or raphe centralis, Brain Res., 108 (1976) 339-349. 23 Lewis, E.J., Tank, W., Weiner, N. and Chikaraishi, D.M., Regulation of tyrosine hydroxylase mRNA by glucocorticoid and cyclic AMP in a rat pheochromocytoma cell line, J. Biol. Chem., 258 (1983) 14632-14637. 24 Lewis, E.J., Harrington, C.A. and Chikaraishi, D.M., Transcriptional regulation of the tyrosine hydroxylase gene by glucocorticoid and cyclic AMP, Proc. Natl. Aead. Sci. USA, 84 (1987) 3550-3554. 25 Loughlin, S.E., Foote, S.L. and Bloom, F.E., Efferent projections of nucleus locus coeruleus: topographic organization of cells of origin demonstrated by three-dimensional reconstruction, Neuroscience, 18 (1986) 291-306. 26 McRae-Degueurce, A. and Pujol, J.F., Correlation between the

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