Time-course variations in tyrosine hydroxylase activity in the rat locus coeruleus after electrolytic destruction of the nuclei raphe dorsalis or raphe centralis

Brain Research, 108 (1976) 339-349

© ElsevierScientificPublishingCompany,Amsterdam- Printed in The Netherlands

339

TIME-COURSE VARIATIONS IN TYROSINE HYDROXYLASE ACTIVITY IN THE RAT LOCUS COERULEUS AFTER ELECTROLYTIC DESTRUCTION OF THE NUCLEI RAPHE DORSALIS OR RAPHE CENTRALIS

B. DOUGLAS LEWIS, BERNARD RENAUD*, MICHEL BUDA AND JEAN-FRANCOIS PUJOL

Ddpartement de Mddecine Expdrimentale, Universitd Claude Bernard, 8, Avenue Rockefeller, 69373 Lyon (France) (Accepted October 20th, 1975)

SUMMARY

Time-course variations in tyrosine hydroxylase activity were measured in the locus coeruleus of the albino rat after electrolytic coagulation of either the nucleus raphe dorsalis or the nucleus raphe centralis. Highly significant increases were measured at 4 days after lesioning of the raphe dorsalis (30.33 %) and the raphe centralis (81.55%), compared with control values, whereas the activity in groups A9 and A10 was unchanged at this time-point. In conjunction with other experimental evidences, an hypothesis is proposed that the catecholaminergic neurons located in the locus coeruleus are directly and/or indirectly controlled by the serotonin-containing neurons located in the anterior raphe system nuclei.

INTRODUCTION

A major distinction has been made between the indoleamine (IA)-containing neurons (principally serotonin, 5-HT) of the raphe system and the topographically proximate neurons of the dorsal-ascending catecholaminergic (CA) systems,9,11,~5. Moreover, there is evidence of inter-regulation phenomena between specific groups of CA and 5-HT neurons: electrolytic coagulations in the dorsal-ascending noradrenergic (NA) system have caused significant changes in IA metabolism in the cat2, 22 and in the rat zS. The drugs 6-hydroxydopamine (6-OHDA) 8, and a-methyl-p-tyrosine * Present address: Laboratoirede Pharmacodynamie,UER des SciencesPharmaceutiques,Universit6 Claude Bernard, 69373 Lyon Codex 2, France.

340

Fig. 1. Anatomic relationships between principal structures under study. RD, nucleus raphe dorsalis; RC, nucleus raphe centralis; A6, nucleus locus coeruleus. Both sagittal (upper right) and frontal sections are represented

(AMPT) 24 radically alter central IA metabolism and anatomic findings15,17 further support the concept of a CA neuron mediated control of IA metabolism. Additionally, there is evidence for regulation of the CA system by IA-containing neurons: both electrolytic and chemical lesions of these neurons greatly alter NA metabolism globally and in the dorsal NA system13, z3. Tyrosine hydroxylase (TH), the enzyme catalyzing the rate-limitmg step in NA biosynthesis, has been localized in the nucleus locus coeruleus (LC)2°; the LC has also been shown to contain the cell bodies of the dorsal-ascending NA fiber system7,14,16,z5 and also projections from the 5-HT-containing neurons of the anterior raphe system s. Having prospectively hypothesized that the destruction of either the nucleus raphe dorsalis (RD) or the nucleus raphe centralis z6 (RC) would induce a change in TH activity m the NA neurons of the LC, we thus measured TH actiwty in the LC and also in the dopamme (DA)-containing neurons of groups A9 and AI0 (see ref. 9) as a test of the specificity of raphe-LC inter-regulation. Fig. 1 schematically illustrates the anatomical relationships between the major structures in our experimental model. METHODS

Sixty-four male OFA (IFFA Credo supplied) rats (weighing 250-300 g at the time of the experiment) received lesions of either the RD (n ---- 22) or the RC (n --=-22) or served as sham-operated control animals (n = 20). They were maintained 8-12/ cage in a constant temperature (25 °C) room, received ad libitum water and pellet

341 TABLE I LESIONING PARAMETERS

All coordinates are gwen as positive values with respect to the corresponding DV (dorsal-ventral) and AP (anterio-posterior) planes of K6nig and Khppel 1~ and lesions were at medlo-lateral zero. Nucleus raphe dorsalis

Experimental descent at 45° (mm) Vertical equwalent at 90° (mm) Time (sec) DC current passed (mA)

Nucleus raphe centralis

Descent 1

Descent 2

Descent 1

Descent 2

AP DV

5.0 5.7

6.0 5.6

2.6 2.9

3.7 3.1

AP DV

1.0 4.1

2.1 4.0

0.5 2.1

1.5 2.2

12 3

20 3

14 2

6 2

rat food and were adapted (for at least one month) to a 12 light-12 dark (lights on at 7:00 h) schedule. They were removed from their cages for only about 30 min during surgery. Lesions

Animals were lesioned under sodium pentobarbital (50 mg/kg, i.p.) anesthesia while held in a David K o p f Instruments model 900 stereotaxic instrument. A hole was drilled in the skull sufficiently large to allow passage of a coagulating electrode through the cerebellum (Rhodes Instruments model NEX-100 coaxial electrode used as a monopolar electrode with an outside shaft diameter of 0.5 m m and an exposed contact of 0.2 m m diameter × 0.5 m m long and mounted at 45 ° relative to the K6nig and Klippel lz horizontal-zero plane). A 45 ° descent angle was needed to avoid piercing the region of the confluence of the superor sagittal and transverse sinuses. Table I lists our lesioning parameters. Two highly localized lesions were made per nucleus at coordinates modified from the atlas of K6nig and Klippel 1~. The anode was attached to a wound edge. Sham-operated animals underwent identical surgery, including electrode descent (no current was passed), simultaneously with lesioned rats. Ninety per cent of the animals survived until the scheduled time of sacrifice without major neurologic signs or local infection. Biochemical determinations

Animals were sacrificed by rapid decapitation at 1, 4, 8 or 15 days after surgery. The brains were stereotaxically sectioned in the A5 plane of De G r o o t 10, removed, and quickfrozen on dry-ice. The time from decapitation to freezing was about 4 min. Sections of 500 # m were subsequently cut with a Leitz 1310 freezing microtome and thereafter maintained at - - 1 0 °C during microdissection of nuclei (see Results).

342

F~g. 2 Sites of tissue removal of the locus coeruleus and groups A9 and A 10 for biochemical analyses LC, nucleus locus coeruleus, CI, colhculus inferior; SG, substantla gnsea; GvH, nervus faclahs, OS, nucleus ohvarls superior, FP, tractus cort~cospmahs, C, cortex, LM, lemmscus medmhs; CC, crus cerebn; CM, format~o reticulans Nomenclature in th~s and subsequent figures ~s adapted from the atlases of Konig and Khppel lz and Palkowts and Jacobowltz 1~

TH activity was measured no more than 5 h after animal sacrifice according to the technique of Buda et al.6: tissue was first sonicated in 0.002 M KPO4 buffer (pH = 6.00) containing 0.2% Triton-X-100. A 50/A aliquot was then reacted by addition of 50 #1 of a solution (0.2 M KPO4 as buffer (pH ---- 5.50)) containing 114 n M DMPH4, 2600 units catalase, 300 n M FeSO4, 2,0 n M tyrosine, and 5 x 105 counts/min of L-[3,5-3H]tyrosine. The incubation time was 25 min. The LC and A9 group were dissected and maintained in their two separate portions for T H assay while the A10 group contributed one assay per rat. Fig. 2 shows the locations of tissue removal. Histochemistry and anatomic control In experiments where only the LC was dissected, the tissue block remaming after its removal, and containing the midbrain raphe groups, was cut directly into either 25 or 40/~m slices with an IEC model C T D cryostat. In the case where T H activity was assayed in the A9 and A10 groups as well, the 500 p m sections from which they had been dissected for assay were subsequently recut into 25 or 40 # m slices. All tissue samples were allowed to dry during 24 h and then stained with cresyl violet. Tissue scarring and obvious intense glial cell reaction were studied and each lesion was designed and quantified by superimposition over a pre-prepared map of the midbrain. This procedure was discontinued for the sham-operated animals when it became clear that an electrode descent alone lesioned no more than 1% of either nucleus. Statistical analysis A 't-test' of the difference between means was used to compare mean per cent control values ± S.D. (sham-operated animals) with the mean per cent deviation from control values ± S.D. in lesioned animals.

343

A

B

C

Fig. 3. Frontal sections (40/~m, stained with cresyl violet) from a control animal. Major landmarks are indicated as an aid in interpreting the extent of lesions shown in Fig. 4. sg, substantia[grisea; ntd, nucleus tegrnenti dorsalis Gudden; tim, fasciculus longitudinalis medlalis; RP, nucleus raphe pontis; P, tractus corticospinalis; n5, nucleus originis nervi trigemini; ac, aqueductus cerebri; RD, nucleus raphe dorsalis; RC, nucleus raphe centralis; n4, nucleus n. trochlearis; pcs, pedunculus cerebellaris superior; tts, tractus tectospmahs; ew3, nucleus Edinger-Westphal n. oculomotoni; n3, nucleus III; R, nucleus ruber; L, nucleus llnearis; IP, nucleus interpeduncularis; lm, lemniscus mediahs.

RESULTS Anatomic analysis Of 44 lesioned rats, 7 were rejected with unacceptable lesions, thus leaving 19 rats lesioned in the RD and 18 in the RC. Fig. 3 is a series of 40/am frontal sections from a control animal and serves as an anatomic reference for locating lesioned sites. Fig. 4 is a series of identically prepared sections including the full extent of the two lesions. Sections 1-5 show the typical posterior-anterior evolution of a RD lesion in a single animal and 6-10 likewise illustrate a RC lesion. Animals with deviations in lesion size of more than 20 ~o from these illustrated cases (in any of the' stereotaxic planes) were eliminated from the study. Furthermore, also unacceptable were animals with lesions surpassing the appropriate nuclear boundaries by greater than 10 ~o of the total nuclear area. The extent of tissue destruction is best determined by direct examination of Fig. 4. It can be seen that the size and location of the lesion varied with that of the nucleus. Section 1 shows that the ventral half of the posterior extremity of the RD was not lesioned. This was typically the case and mapping analysis revealed that at least 80 ~ of the RD was destroyed in all RD-lesioned animals. Sections 7-9 show that the dorsal tip of the RC was not destroyed in this animal. There were often such slight deviations from total RC destruction and mapping analysis revealed that at least 90 ~ of the RC was destroyed in all RC-lesioned animals. Biochemistry The mean absolute control T H activity 5: S.D. in the LC was 270.33 4- 99.04

344

J0

_J

F~g 4. Frontal sections, 40/~m, from rats lesioned m either the nucleus raphe d o r s a h s or the nucleus raphe centrahs Each series advances from posterior to anterior (left to r~ght) a n d c o m e s f r o m the s a m e a m m a l . Sections were stained w~th cresyl wolet a n d the ghal cell reaction at each les~oned s~te ~s visible as a dark m a s s in these Illustrations T h e distance represented in the a n t e r l o - p o s t e n o r plane ~s noted and the n u m b e r s refer to textual remarks

345

12g

IN U ,J Cp .Ig ,iJ

75

C

o-

.r I,n

Q gll

25, c

10 J~ O

.-g

u

i I DAY

4 DAYS

8 DAYS

15 DAYS

Fig. 5. Relative tyrosine hydroxylase activity in the locus coeruleus at various times after lesioning of the raphe dorsalis (solid bar) or the raphe centralis (dotted bar). Changes in activity are normalized to control values (horizontal zero line) and S.D.s are expressed for lesioned as well as control (shamoperated) animals. The number of observations is indicated in parenthesis.

pmoles DOPA formed/h/structure (n = 40) and there was no significant difference between the amount of protein from sham-operated and lesioned animals. Our principal findings in Fig. 5 are expressed as the percentage of the mean absolute T H activity in the LC of the appropriate control group (pmoles DOPA formed/h/structure) i S.D. Significant changes from control values were present at the 4-day point as follows: RD lesion + 30.33 -q- 15.20 ~ (P ~< 0.001, t ---- 7.74, n ---- 38) and RC lesion q- 81.55 -t- 31.01 ~ (P ~< 0.001, t = 11.75, n = 38). Furthermore, the change in enzyme activity 4 days after a RD lesion was significantly different from that at 1 day (P ~< 0.001, t =- 4.01, n = 22) and from that at 8 days (P ~< 0.001,

346

125

100

75 < Z c

~

E

50

2s ~

z

o

iii

~

;

25-

LC

A9

AI0

Fig. 6. Relatwe tyrosine hydroxylase activity in the locus coeruleus and in groups A9 and A10 at 4 days after lessoning of either the raphe dorsahs (solid bar) or the raphe centralis (dotted bar). The activity in the LC is included for comparison purposes. Further details are as in Fig. 5 t = 4.03, n = 24) after lesioning. The change in enzyme activity 4 days after a R C lesion was likewise different from that at 1 day (P ~ 0.001, t ---- 5.81, n ---- 19) and at 8 days (P ~< 0.001, t = 8.25, n = 26) after lesioning. Finally, a comparison o f the two separate lesions at the 4-day point yielded an equally significant difference (P ~< 0.001, t = 5.93, n -- 32). T H activity in groups A9 and A10 was not significantly changed f r o m control values at 4 days after either R D or R C lesions (Fig. 6). DISCUSSION

O u r principal findings showed T H activity in the L C to have been augmented 4 days after electrolytic destruction o f either the R D or the RC, but that the per

347 centage of this elevation was different for each lesion. This disparity in elevation points to the highly selective nature of the lesions and attests to their functional independence; it is important to realize that the distance between the ventral extremity of the RD and the dorsal extremity of the RC was only 800/~m and that histological control showed no overlap of one lesion onto the adjacent nucleus. Given that the lesions were anatomically specific to each nucleus, were tissue responses to lesioning likewise specific? This appears to be the case because (a) there were non-equal physiological responses to tissue destruction in two extremely proximate areas and (b) the systematically related DA neurons (as represented by groups A9 and A10) were not affected as were the CA neurons in the LC. There are at least several possible explanations for our observed TH activity increase in the LC. There might have been a retrograde accumulation of TH after terminal destruction. The observed rise of TH activity in the LC after CA terminal destruction by 6-OHDA (refs. 5 and 21) would support such an hypothesis and we might therefore explain our results as having been due to a retrograde accumulation of TH in the LC after CA terminal destruction in the RD and RC. However, mitigating against this possibility is the anatomical evidence that only a very small number of the total fibers emanating from the LC connect with these raphe nucleiS,9,11,25. This fact is hard to rectify with the 50 ~ difference in effect of the two lesions. If indeed such a retrograde accumulation existed, it was only of minimal import and could not account for our results. TH activity might also have been affected either by the destruction of cell bodies in the lesioned nuclei having projections on the LC, or from the cutting of axons projecting on the LC and passing through the two nuclei, or both. Our electrolytic lesion study alone cannot resolve this question but may be interpreted conjointly with other evidence, such as experiments in which whole classes of cell types are selectively destroyed: 5,6-dihydroxytryptamine, which destroys 5-HT neurons, caused an increase in TH activity in the LC which was maximal between 4 and 6 days after administration2.~, and an increase in whole brain NA at 10-12 days after administration1. There are direct axonal connections between the anterior raphe nuclei and the LC (ref. 8), and 5-HT has been found in the LC (ref. 18). Viewing this together with our experimental results, it seems likely that there is a control, direct or indirect, of TH activity in the LC by 5-HT fibers originating in the anterior raphe system, and that perhaps the different influence of our two lesions on the LC is due to the destruction of separate and different raphe fiber systems. It is also conceivable that our lesion of the RD destroyed some portion of the RC axons projecting on the LC. The RD and RC are located in the same mediolateral and anterio-posterior planes and the RD is just dorsal to the RC. This topographical relationship, and recent work in our laboratory4, suggest that ascending fibers from the RC pass through or near the RD. But once again, however, the extremely disparate effects of the two lesions on the LC probably means that this latter situation could only partially account for our observations. Direct and indirect evidencela,22 suggest that there is an increased turnover of NA in the NA neurons of the LC after electrolytic destruction of portions of the

348 anterior raphe system. Our results indicate that alteratton of TH achvity m the LC might be one of the mechamsms responsible for such increased NA turnover. Finally, we may explain the time-course of decrease of TH activity between 8 and 15 days after lesionmg by suggesting that either a new steady-state of enzyme synthesis was reached m the LC or that there was a recovery of function in the damaged t~ssues at this time. Conclusion Viewing the results of this experiment in conjunction with the current physiologlc and anatomic evidence, we suggest that the function of catecholaminergic neurons in the locus coeruleus is directly and/or indirectly controlled by the serotoninergic neurons located in the nuclei raphe dorsalis and raphe centralis, and more importantly by those in the raphe centralis. The mechanism of this control is unique to this system, and is not the same, for example, as that controlling the activity of dopaminergic neurons in the groups A9 and A10. The precise nature of this mechanism remains to be elucidated. ACKNOWLEDGEMENTS

The extremely high quality and constant technical aid provided by Mss. Denise Rollet and Anne Mermet was essential in the completion of this research. The authors are very grateful for this help. B.D.L. was supported by a French Government Study Fellowship administered by the Centre National des Oeuvres Universitaires et Scolaires (No. A.74.4344). This work was further supported by INSERM (U52), CNRS (LA 162) and DRME (74-232).

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