Changes in steady-state levels of tryptophan hydroxylase protein in adult rat brain after neonatal 6-hydroxydopamine lesion

Neuroscience Vol. 67, No. 2, pp. 463-475, 1995

~ Pergamon

0306-4522(95)00064-X

Elsevier ScienceLtd Copyright © 1995IBRO Printed in Great Britain.All rights reserved 0306-4522/95 $9.50+ 0.00

CHANGES IN STEADY-STATE LEVELS OF TRYPTOPHAN HYDROXYLASE PROTEIN IN ADULT RAT BRAIN AFTER NEONATAL 6-HYDROXYDOPAMINE LESION S. RAISON,* D. W E I S S M A N N , * t C. ROUSSET,* J.-F. PUJOL* and L. DESCARRIES:~ *Laboratoire de Neuropharmacologie, UCB-CNRS UMR 105, CERMEP, 59 Bd Pinel, 69003 Lyon, France :~D6partement de pathologie et Centre de recherche en sciences neurologiques, Facult6 de m6decine, Universit6 de Montr6al, Montr6al, Qu6bec, Canada H3C 3J7 Abstract--A recently developed technique of immunoautoradiography on nitrocellulose transfers of serial frozen sections was used to determine tryptophan hydroxylase concentration in selected areas of the adult rat brain following neonatal 6-hydroxydopamine destruction of nigrostriatal dopamine neurons. Particular attention was paid to the neostriatum, known to be serotonin-hyperinnervated under these conditions, and to the nucleus raphe dorsalis, containing the cell bodies of origin for these nerve terminals. The hippocampus was also investigated as a territory of structurally intact serotonin innervation arising primarily from the nucleus raphe medianus. Tryptophan hydroxylase protein was measured at successive transverse levels across the entire caudorostral extent of all these regions. Similar measurements of tyrosine hydroxylase protein across the substantia nigra and the neostriatum verified the disappearance of the nigrostriatal dopamine neurons. The average tryptophan hydroxylase tissue concentration in the dorsal third of the serotonin-hyperinnervated neostriatum was up by 36% above control, i.e. significantly less than the number of its serotonin axon terminals or varicosities. This was therefore indicative of a lowering of the tryptophan hydroxylase protein content per serotonin ending. Interestingly, a tight correlation between the respective level-by-level concentrations of tryptophan hydroxylase and tyrosine hydroxylase protein in the control neostriatum allowed the prediction the tryptophan hydroxylase concentration after dopamine denervation with a serotonin hyperinnervation. Tryptophan hydroxylase concentration was also significantly reduced in both the nucleus raphe dorsalis and nucleus raphe medianus, notably at those raphe dorsalis levels known to give rise to the serotonin hyperinnervation of neostriatum. It is hypothesized that the lower steady-state level of tryptophan hydroxylase inside the terminals and cell bodies of hyperinnervating serotonin neurons was the result of a feedback inhibition of the synthesis of the enzyme by its end-product, presumably because of the increased amount of serotonin in these terminals.

Early destruction of the nigrostriatal dopamine (DA) neurons by intraventricular administration of 6-hydroxydopamine (6-OHDA) in neonatal rats leads to an excessive serotonin (5-HT) innervation (hyperinnervation) of the adult neostriatum. This 5-HT hyperinnervation accounts for a regional increase in neostriatal 5-HT content, 7'2~'24'41'42'59[3H]5-HT uptake into synaptosomes 37'59 and n u m b e r of 5-HTimmunostained axonal fibers and varicosities (terminals). 19'37'38'57 The supernumerary 5-HT endings presumably arise from cell bodies located in the nucleus raphe dorsalis (RD), since the rostral portion of this nucleus then exhibits an augmented n u m b e r of cells labeled by retrograde axonal transport after neostriatal injection of horseradish peroxidase. 7'57 A

tTo whom correspondence should be addressed. Abbreviations: BSA, bovine serum albumin; DA, dopamine;

5-HT, 5-hydroxytryptamine (serotonin); 6-OHDA, 6-hydroxydopamine; RD, raphe dorsalis; RM, raphe medianus; RP, raphe pontis; TBS, Tris-buffered saline; TH, tyrosine hydroxylase; TpOH, tryptophan hydroxylase.

recent quantitative autoradiographic study has shown that the n u m b e r of hyperinnervating 5-HT varicosities is 55% above normal in the whole of neostriatum and 80% in its dorsal third. 43 In terms of size, shape and frequency of synaptic contacts, these terminals do not look different from normal. ~9 However, a recent biochemical study has indicated that their 5-HT content per terminal is significantly elevated. 4j To further understand these adaptations of 5-HT neurons to the ontogenetic loss of DA, we have investigated the consequences of the neonatal nigrostriatal DA lesion on the tryptophan hydroxylase (TpOH) protein content of different brain regions. A recently developed technique of immunoautoradiography on nitrocellulose transfers of serial frozen sections TM was used to determine the section-bysection distribution, concentration and a m o u n t of T p O H protein, along the whole caudorostral extent of the 5-HT-hyperinnervated compared to normal neostriatum. The hippocampus was similarly investigated as a territory of presumed normal 5-HT

463

S. Raison et al.

464

i n n e r v a t i o n , a n d also the p o n t o m e s e n c e p h a l i c nuclei c o n t a i n i n g the cell bodies of origin for b o t h these 5-HT innervations. C o n c u r r e n t quantification o f tyrosine hydroxylase (TH) in the n e o s t r i a t u m a n d s u b s t a n t i a nigra m a d e it possible to verify the success of the nigrostriatal D A lesion. EXPERIMENTAL PROCEDURES

All experiments were conducted in accordance with the guidelines for care and use of laboratory animals issued by the French Minist6re de rAgriculture et de la For6t (87-848) and the European Economic Community (86-609).

Neonatal dopamine denervation According to a protocol described in detail previously, 29.59 three-day-old male OFA rat pups (Iffa-Credo) were anesthetized with ether and subjected to bilateral cerebroventricular administration of 6-OHDA (lesioned) or vehicle solution (sham-operated), 45 min after a subcutaneous dose of desmethylimipramine (25 mg/kg; Sigma) to protect noradrenaline neurons? ° Fifty micrograms (free base) of the cytotoxic drug (6-OHDA hydrochloride; Sigma), dissolved in 5/al of 0.9% NaC1 containing 0.1% ascorbic acid, were slowly instilled (3 min) into each lateral ventricle. The injections were made at the level of the bregma, 1.5 mm lateral to the sagittal suture and 3.3 mm below the skull surface.

Tissue sectioning and nitrocellulose transfer At three months of age, the rats were decapitated and their brains rapidly removed and frozen by immersion in isopentane at - 3 0 ° C for 1 rain. Transverse sections (20/am thick) were then cut with a Frigocut (Reichert), from the midbrain raphe nuclei (entire caudorostral extent: IA - 0 . 3 to + 1.9 mm in Paxinos and Watson's stereotaxic atlas), 5° substantia nigra (IA +2.7 to +4.1 mm), rostral hippocampus (IA +4.8 to + 7.2 mm) and rostral neostriatum (IA +8.1 to +10.9mm). At intervals of 200/am in the raphe nuclei and substantia nigra, and 400/am in the hippocampus and neostriatum, one or two adjacent sections were collected onto nitrocellulose filters as described previously TM and kept at room temperature until further processing. A third serial section was stained with Cresyl Violet for histological reference.

Preparation of tyrosine hydroxylase and tryptophan hydroxylase standards A microscale of TH protein standards were produced as described previously. ~70ne-microliter aliquots of rat adrenal gland homogenate diluted with increasing amounts of rat cerebellum homogenate (1:0 to 1:64) were directly deposited on a nitrocellulose filter (Millipore HAHY). TpOH protein standards were generated by thorough mixing of a defined number of tissue punches from frozen sections of the RD together with an increasing number of similar punches from cerebellum. The different dilutions of RD tissue (1:0 to 1:16) were collected into plastic tubes, frozen, cut as 20-#m-thick sections and transferred onto separate nitrocellulose filters.

Immunoautoradiography After saturation in Tris-HCl-buffered saline solution (TBS; 50 raM, pH 7.4) containing 1% bovine serum albumin (BSA; Boehringer) for I h, the nitrocellulose transfers (standards and tissue sections) were incubated for 18 h in a solution of TBS/1% BSA containing either TH mouse monoclonal antiserum (1:4000; Boehringer), TpOH sheep polyclonal antiserum (1:2000; gift of Dr M. Maitre), 14 or normal sheep or mouse serum (1:4000; Nordic) for blanks. These concentrations of antibodies have already been shown

to saturate all respective antigenic sites in rat brain tissue. 17'73 After a single wash in TBS/I% BSA (10min) and two washes in TBS (2 × 10 min), the filters were incubated for 2 h in TBS/I% BSA containing ~25I-labeled protein G (1:3000; Amersham, 12 mCi//~g) or 12SI-labeled protein A (1:2000; Amersham, 30/aCi/#g) and rinsed for 10rain in TBS/I% BSA, TBS and I0 x TBS before drying, The immunoradiolabeled filters were then apposed against 3Hsensitive Hyperfilm (Amersham RPN 12) for two to 10 days, depending on the region to be examined. Overexposure was carefully avoided.

Quantitative analysis of immunoautoradiographs The immunoautoradiographs were examined with the aid of a computerized image analysis system (IMSTAR). Sections from a given experiment were first set in anatomical register by reference to the adjacent Cresyl Violet sections. In regions of axon terminal labeling (neostriatum and hippocampus), single optical density readings of the whole area of interest were directly obtained from every section, and a background value, from nearby empty filters or adjacent section transfers incubated with normal sheep or mouse serum instead of specific antibody, was automatically subtracted from each reading. In the case of the TpOH labeling of the raphe, a threshold was initially set for each experiment, at a fixed level which eliminated the surrounding labeling from every section across this area. This highlighted zone corresponded to both labeled cell bodies and their proximal dendrites, thereafter designated "somata/dendrites". Two measurements were obtained from every section (Pronew Software-CNRS): (i) an average optical density per unit of surface for that portion of the image showing labeling above background and/or threshold, and (ii) the actual surface (mm 2) of this portion of the image. Each concentration of standard was calibrated into arbitrary units of TH or TpOH per milligram (mg) of tissue. One unit of TH (uTH) corresponded to the mean TH protein content in 10/ag of adrenal gland tissue from 40-day-old controls and 100 units of TpOH (uTpOH) were defined as the TpOH quantity in one RD of control rat. The densitometric measurements were converted into concentration of enzyme by reference to the respective standards.

Expression of results In the mesencephalic raphe, the sequential level-by-level measurement of TpOH protein was carried out on 12 transfer immunoautoradiographs per rat. In keeping with previously published maps of the respective 5-HT nerve cell body populations, ~6'2°'6~ the RD was visualized from IA - 0 . 3 to + 1.9 mm, the nucleus raphe medianus (RM) from IA +0.9 to + 1.9 mm and the nucleus raphe pontis (RP) from IA - 0 . 3 to +0.7 mm. Three different parameters were systematically determined at each caudorostral level across the raphe nuclei: (i) TpOH labeling volume (mm3), defined as the surface of labeling above threshold multiplied by the thickness 6f the section; (ii) TpOH tissue concentration (uTpOH per mg of tissue or mgt), I mgt being assumed to correspond to I ram3; (iii) TpOH protein quantity (uTpOH), calculated as the product of the tissue concentration multiplied by the volume. In the neostriatum, both TH and TpOH concentration were measured at eight consecutive caudorostral levels, within the dorsal sector depicted in Fig. 1, identified in Nissl-stained sections and outlined on a transparent overlay. In the substantia nigra zona compacta, TH was determined at eight levels across this entire region. In the hippocampus, TH was measured at seven levels across the dentate gyrus, and TpOH in a broader region including CA 1, CA3 and the dentate gyrus. The results at each level were expressed as the mean + S.E.M. for a 20-/am-thick section. The anatomical distribution of these results was statistically tested using a one-way analysis of variance (ANOVA I). Comparisons

TpOH after neonatal 6-OHDA lesion between sham-operated and lesioned rats were assessed with a two-way analysis of variance (ANOVA II). The two tested factors were the effect of the anatomical level (fl) and the effect of the 6-OHDA lesion (f2). In the tables, results are expressed as the mean + S.E.M. of the considered parameter. The volume and the TpOH protein content in the whole structure were calculated from the estimated values at each interval of 200 #m or 400/~m (the value in one interval was the value determined in a 20-/zm section multiplied by 10 or 20, respectively). Statistical significance between mean values was determined with a Student's t-test after verification of the homogeneity of variance. Linear regression was calculated by the least square method. RESULTS

Tyrosine hydroxylase decreases in the neostriatum and substantia nigra In transfers of the neostriatum and of the mesencephalic tegmentum (substantia nigra) prepared at three months (Fig. 2A-D), four of the six rats neonatally lesioned with 6-OHDA showed a quasitotal, bilateral absence of TH protein. This result was in keeping with abundant biochemical or immunocytochemical evidence for an almost complete disappearance of the nigrostriatal DA neurons in these rats, i.e. of the DA axon terminals (varicosities) throughout the neostriatum and DA cell bodies in the zona compacta of the substantia nigra} 9'25'37'57'59

Rostral

465

Accordingly, the mean TH protein concentrations respectively measured in the dorsal neostriatum (53.4+8.0uTH/mgt) and substantia nigra zona compacta of the controls (25.60 + 2.14 uTH/mgt) were markedly decreased after neonatal lesion (1.43 -I- 0.46 uTH/mgt and 0.19 __+0.08 uTH/mgt in the neostriatum and substantia nigra, respectively; P <0.001) (Fig. 2E, F). The caudorostral distribution of the TH protein appeared rather homogeneous throughout both regions in the controls, and was severely reduced at every level examined in the lesioned rats. Interestingly, as shown in Fig. 2A and C, the TH immunolabeling in the hippocampus was not affected by the lesion. The mean TH tissue concentration measured after the lesion (10.10___ 1.98uTH/mgt) was not significantly different from control (9.08 + 1.40 uTH/mgt).

Tryptophan hydroxylase content of dopamine-denerrated neostriatum As shown in Fig. 3A, the TpOH concentration measured at eight intervals across the caudorostral extent of the dorsal neostriatum was rather uniform in the sham-operated rats. In the neonatally lesioned rats, a significant increase was observed at each but the last of these anatomical levels (ANOVA II f2, P < 0.001). The overall increase amounted to 36% of mean control value (Fig. 3B; P < 0.05 by Student's t-test).

Correlation between neostriatal tyrosine hydroxylase and tryptophan hydroxylase concentrations Owing to the number of measurements, it was possible to seek a correlation between the two parameters, TH and TpOH protein tissue concentration, in control and DA-denervated neostriatum. As indicated by the solid line in Fig. 4, in sham-operated rats there was a significant correlation between TH and TpOH tissue concentrations measured individually from adjacent section transfers at each anatomical level (36 values). Although there was no such correlation after DA denervation, the corresponding equation allowed the prediction of a TpOH concentration of 3.17 uTpOH/mgt at null TH concentration that was nearly the mean TpOH concentration measured experimentally in the neonatally lesioned rats, 3.20 + 0.28 uTpOH/mgt (Fig. 3B). An almost identical relationship was found between the two parameters (dotted line in Fig. 4) when all values were used in the calculation (lesioned and sham-operated rats: 68 values).

~J

Tryptophan hydroxylase content of hippocarnpus

Caudal IA-8.1 mm

;

4;0

IA-10.9 mm 800

1 2 0 0 1600 LEVEL

2o'00 2,~oo 2~O0~m

Fig. 1. Schematic representation of the dorsal neostriatal area (hatched) from which TH and TpOH protein measurements were obtained at every 400-/tin caudorostral interval (1-8) between transverse planes IA +8.1 and + 10.9 mm) °

TpOH measurements were obtained from the hippocampus as representative of a territory of 5-HT innervation originating primarily from the R M . 3'9'51'71 In the dorsal sector examined, TpOH concentration was found to be in the same range as in the dorsal neostriatum, and rather uniformly distributed along

466

S. Raison et al.

NEOSTRIATUM

SUBSTANTIA NIGRA A

B

C

D

I CO

r

a LU

I

k E

r..m._, t "ml'l 'l°' F

60 40

20

0

60

"

40

En rLrLn 1

2

3

4

5

LEVEL

6

~

r

I"

:!:

Z

I"

20

I 0 7

8

1

2

3

4

5

6

7

LEVEL

Fig. 2. Transfer immunoa.utoradiographs of TH protein (A-D) and corresponding measurements of TH concentration (E, F) in the substantia nigra and the neostriatum of sham-operated (n = 5) and neonatally lesioned rats (n = 4) (A and C are level 6 in E; B and D are level 4 in F). In C and D, note the striking bilateral decreases in TH reactivity reflecting the disappearance of DA nerve cell bodies in the substantia nigra zona compacta and ventral tegmental area (C), and of their axon terminals across the neostriatum (D). In contrast, a slight labeling of the hippocampal area (A, C), presumably representing its noradrenaline innervation, is spared by the neonatal 6-OHDA lesion. E and F document the dramatic decrease in mean TH tissue concentration (_+S.E.M.) at every anatomical level along the caudorostral extent of both regions (ANOVA II f2, P < 0.001). The lack of black bars indicates undetectable levels. H, hippocampus; NS, neostriatum; SNC, substantia nigra compacta; SNR, substantia nigra reticulata; VTA, ventral tegrnental area. Scale bar = 200/~m (A-D).

8

TpOH after neonatal 6-OHDA lesion

A

467

B 4 E

:3

3

"T" I

2

TpOH Concentration ( U/mgt )

w

0

1

2

3

4

5

6

7

sham

2.36 + 0.12

lesioned

3.20 + 0.28 *

8

LEVEL Fig. 3. (A) Level-by-levelcomparison of the TpOH concentration (means + S.E.M.) in the neostriatum of lesioned (n = 4) versus sham-operated (n = 5) rats; the difference is highly significant (ANOVA II f2, P < 0.001). As indicated in B, the mean increase (+36%) in TpOH concentration is also significant (P < 0.05 by Student's t-test). the caudorostral axis (Fig. 5A, B). In the lesioned group, a slight increase in TpOH concentration was found at most levels (ANOVA II f2, P <0'05; Fig. 5A), but the mean concentration was not significantly different between the two groups (Fig. 5B).

Tryptophan hydroxylase content of mesencephalic raphe nuclei As shown in Fig. 6, the above-threshold labeling reflecting the presence of TpOH protein was visualized along the whole caudorostral extent of the pontomesencephalic raphe nuclei RP, RM and RD (levels 1-12). At each transverse level, the TpOH protein transfers reproduced the known topographic features of the respective 5-HT cell body groups. 2°'6L72 In sham-operated rats, the distribution of the volume occupied by the TpOH-labeled somata/dendrites, the TpOH tissue concentration and the amount of TpOH protein at the different caudorostral levels were highly characteristic of each nucleus (open bars in Fig. 8A~2). These caudorostral distributions were heterogeneous in the RM and RD (ANOVA I,

P < 0.001), but not in the RP. A large fraction of the volume defined by the TpOH labeling (80%) and most of the content in TpOH protein (90%) were found in the five rostralmost levels across the RD (levels 8-12). The highest values were those at level 10, containing the RM and RD (see also Fig. 7E). The mean value for every parameter (volume, tissue concentration, amount) was different between the three nuclei (Table 1). The RD showed the highest value for each parameter, followed by the RM and the RP. In the RD of lesioned rats, there was a considerable reduction of the extent and intensity of TpOH labeling (Fig. 7A-F). The corresponding digitized images not only showed a reduction of the surface of the anatomical area exhibiting above-threshold labeling, but an actual disappearance of this labeling within portions of that area at certain levels. The three TpOH parameters continued to show a heterogeneous distribution (black bars in Fig. 8A-C; ANOVA I, P < 0-001). At every level across the RD, there was a significant decrease in the volume 0 •

sham lesioned

4

•

E 3

F-

.............

...............................

•I

Q_

°o

o Ooo

......

2

I

I

I

I

I

I

I

0

10

20

30

40

50

60

70

[ TH ] ( U/mgt ) Fig. 4. Correlation between TpOH (Y) and the TH (X) tissue concentrations measured from adjacent sections at every neostriatal level, in sham-operated (open circles) and lesioned rats (filled circles), in the left cerebral hemisphere. Note that the same level of correlation was found for the analysis of the sham-operated sample alone (solid line; 36 values from five rats; Y = -0.015X + 3.17; P < 0.05) as opposed to the entire sample (dashed line; 68 values from nine rats; Y = -0.015X + 3.22; P < 0.001). lxS(" 6 7 2

II

S. Raison et al.

468

B

A 4

TpOH Concentration ( U/mgt )

3 '-i-

2

sham

2.78 + 0.18

1

lesioned

3.30 :t: 0.36

0 1

2

3

4

5

6

7

LEVEL Fig. 5. Level-by-level(A) and mean TpOH concentrations (B) in the hippocampus (CA1, CA3 and dentate gyrus) of neonatally lesioned (n = 4) versus sham-operated (n = 5) rats (ANOVA II f2, P < 0.05).

corresponding to the TpOH protein labeling (Fig. 8A; ANOVA II f2, P < 0.01), of the TpOH tissue concentration (Fig. 8B; ANOVA II f2, P <0.05) and of the amount of TpOH protein (Fig. 8C; ANOVA II f2, P < 0.01). Even if the mean values for the RD were not significantly different between sham-operated and lesioned rats (Table 1), some levels across this nucleus exhibited a visible decrease in the extent and intensity of TpOH labeling (levels 1, 2 and 8-12 in Fig. 8; see also Fig. 7). In the RM there was also a significant decrease ( - 3 0 % ) of the volume from which TpOH protein was detected (Fig. 8A; ANOVA II f2, P < 0.001), and of the actual amount ( - 2 8 % ) of TpOH protein (Fig. 8C; ANOVA II f2, P < 0.01). This was most apparent at level 8, where respective mean decreases of - 6 8 % (Fig. 8A) and - 7 0 % (Fig. 8B) were measured for these two parameters. In the RP, none of the three TpOH parameters showed any significant difference from control (Fig. 8A-C, Table 1). DISCUSSION

Main findings In brief, major changes in biosynthetic enzyme content were associated with the neonatal destruction of nigrostriatal DA neurons and ensuing 5-HT hyperinnervation of adult rat neostriatum: (i) a profound reduction of the TH content in both neostriatum and substantia nigra zona compacta reflected the almost total disappearance of DA neurons from these regions; (ii) a moderate elevation of the neostriatal TpOH content was not as high as expected from the increased number of 5-HT axonal varicosities, indicating a reduced amount of the enzyme per hyperinnervating terminal. Additional observations included: (iii) a tight correlation between the level-bylevel TH and TpOH concentrations in the DAdenervated and 5-HT-hyperinnervated, as well as control, neostriatum; (iv) a slight increase of the TpOH content in the hippocampus; (v) a localized reduction of the TpOH content in the rostral RD and

RM, at anatomical levels corresponding to those of 5-HT cell bodies projecting to neostriatum and hippocampus.

Tyrosine hydroxylase and tryptophan hydroxylase content of normal tissue In sham-operated rats, the distribution of TH protein appeared rather uniform throughout the dorsal neostriatum. The mean TH tissue concentration in the neostriatum as a whole was about twice that in the substantia nigra zona compacta, a ratio equivalent to that derived from previous in vitro estimates of the enzyme activity in these two regions. 34'54 In the hippocampus, TH was also uniformly distributed throughout the sector examined (dentate gyrus), at an average concentration about six times less than that in neostriatum. This latter ratio was consistent with previous measurements of the respective catecholamine contents in these two regions. 1n49 However, considering the much higher number of DA terminals in neostriatum22versus that of DA plus noradrenaline terminals in hippocampus47 (ratio of 100: 1), it must be concluded that TH and its end-products are present in much lower amounts (and concentration) in the DA terminals of neostriatum versus the DA and/or noradrenaline terminals of hippocampus. TpOH was also found to be rather evenly distributed throughout the caudorostral extent of neostriatum and of hippocampus, with a similar average concentration of these two regions. This was in keeping with earlier determinations of 5-HT content, ~2 of regional TpOH concentration23 and of TpOH activity in t~it)o, 66 which had also shown comparable values in these two regions. Recent estimates of 5-HT innervation density have, however, indicated a higher average number of 5-HT terminals per mm 3 of tissue in the neostriatum43 than in the hippocampus. 46 As the mean size of these 5-HT axon terminals is comparable, 4s'58 it may therefore be assumed that both the amount and the concentration of TpOH protein per 5-HT terminal are considerably lower in the neostriatum than hippocampus.

TpOH after neonatal 6-OHDA lesion In this and an earlier study, 73 the level-by-level distribution of T p O H protein in the pons and mesencephalon was found to closely mimic the topograph-

•

iiiiiii~iiI ,~, ~i,~~

,,

~

L

~

......

469

ical features of the 5-HT somata/dendrites regrouped in the RD, R M and RP. 2°'61'63 The T p O H content in the ventral component ( R M + RP) of this 5-HT

¸

',

Fig. 6. Transfer immunoautoradiographs of TpOH protein at successive caudorostral levels, 1-12, across the mesencephalic raphe nuclei of a sham-operated rat (each level represents the transfer of a 20-/~m-thick section taken at each 200-/~m interval). The different raphe nuclei are designated by their abbreviation. Scale bar = 200/~m.

470

S. Raison et al.

SHAM

LESIONED

content in 5-hydroxytryptophan 66 and T p O H activity in vitroJ 3"32 In neostriatum, the average T p O H concentration was approximately 5% of that in the RD, at least when expressed per mg of tissue. This proportion was equivalent to the corresponding ratios for 5-hydroxytryptophan content (3.3%) and T p O H activity in vivo (5.1%), 66 whereas 5-HT content is approximately two to five times lower in neostriatum than the RD. 37'42The similarity in the ratios of T p O H protein and T p O H activity in neostriatum (terminal) versus R D (cell bodies of origin 3°'4°'6°'62"63"69) would seem to indicate that comparable proportions of active and inactive forms of the enzymatic protein are present within both these neuronal compartments.

Dopamine denervation in neostriatum and substantia nigra The neonatal treatment with 6 - O H D A resulted in a severe and permanent loss of D A nigrostriatal neurons, as repeatedly demonstrated by earlier studies. 7"19"21'25"38"41'42'57"59 In the present experiments, this lesion was evidenced by the drastic decrease in neostriatal and nigral T H protein content at the age of three months. T H protein content was unaltered in the hippocampus, as already described for the D A and noradrenaline contents of this region? 8 This confirmed that neonatal pretreatment with desipramine had efficiently protected the dorsal noradrenaline system from the cytotoxic effects of 6 - O H D A . 1°'3L35 It also indicated that, if the D A innervation of the dentate gyrus arises partly from the zona compacta of the substantia nigra 65'68 (also see Refs 33 and 55), it represents a very minor proportion of the catecholamine terminals in this part of the brain.47. 65,68

Serotonin hyperinnervation in neostriatum i

T p O H protein concentration in the DA-denervated and 5-HT-hyperinnervated dorsal neostriatum was uniformly and moderately elevated ( + 3 6 % ) . This Table 1. Volume of tryptophan ing, tryptophan hydroxylase tryptophan hydroxylase protein of 6-hydroxydopamine-lesioned

Fig. 7. Transfer immunoautoradiographs of TpOH protein at different caudorostral levels across the raphe nuclei of representative sham-operated (left) and 6-OHDA-lesioned rats (right). A and B correspond to caudal level 2, C and D to middle level 8, and E and F to rostral level 10 in Fig. 6. Note the dramatic decreases of TpOH protein staining in the lesioned rat, particularly in the RD and RP at level 2 (A, B), and RD and RM at levels 8 (C, D) and 10 (E, F). Scale bar = 50/~ m. neuron population represented about one-third of that in its dorsal component (RD), in keeping with the respective numbers of 5-HT cell bodies in these various nuclei. 2°'36'44 Similar ratios between R D and R M have been determined for their respective

Volume (mm 3) RD Sham Lesioned RP Sham Lesioned RM Sham Lesioned

hydroxylase protein staintissue concentration and content in the raphe nuclei versus sham-operated rats

TpOH tissue TpOH protein concentration content (uTpOH/mgt) (uTpOH)

2.07 + 0.20 1.73+0.10

44.93 + 2.06 41.60+0.97

0.14 + 0.01 0.11 +0.01

29.93+ 1.97 30.12__+0.80

0.48+0.02 36.39+ 1.77 0.29 + 0.02** 36.09 + 1.98

108.12 + 12.89 82.13+4.90 4.66 4- 0.49 3.85_.+0.29 18.54+ 1.12 11.42_ 1.03"*

Means+S.E.M. from four 6-OHDA-lesioned and five sham-operated rats; uTpOH/mgt, arbitry units of TpOH per milligram of tissue. **P < 0.01 compared to shamoperated, by Student's t-test.

TpOH after neonatal 6-OHDA lesion

471

A 4 3 2 O

I O

E

2 3 4 5 6 7 8 9 101112

v IX)

E "6 >

1.5

RP

1.0

!RM ~

t

0.5 0

I 2 3 4 5 6 7 8 9 101112 LEVEL

80o

B

60

1 c

RD

40

RD

-r o

~ -rQ '-"

2

~-0

1 2 3 4 s 6 7 8 s101112

I 40 ~

RP

,,I

RM 3_

|

20 0

~

I 2 3 4 S 6 7 8 9 101112 LEVEL

2 3 4 5 6 7 8 9 101112 0.6

"r"

0.4 f

I--

0.2 O

RP

RM

~1

I 2 3 4 5 6 7 8 9 10111 LEVEL

Fig. 8. Level-by-level measurement of the volume of TpOH protein staining (A), TpOH tissue concentration (B) and TpOH protein content (C) in the mesencephalic raphe nuclei (RD, RP and RM) of lesioned (black bars, n = 4) versus sham-operated (open bars, n = 5) rats. See Fig. 6 for anatomical landmarks.

increase was obviously associated with the increased number of 5-HT fibers and varicosities previously documented in this model (see references in Introduction). Measurements of endogenous 5-HT in the dorsal half of the 5-HT-hyperinnervated neostriatum are not currently available from the literature, but should be of the order of two- to three-fold the normal content according to more global estim a t e s . 21'24"38"41'42"59 In a recent quantitative autoradiographic study by Mrini et al., 43 counts of the number of varicosities labeled by uptake and storage of [3H]5-HT at three rostrocaudal levels of the 5-HThyperinnervated neostriatum yielded values of 9.06 × 10 6 varicosities per mm 3 for its dorsal half, compared to 5.13 x 10 6 in the normal. A previous electron microscopic examination of these 5-HThyperinnervating varicosities has indicated that their actual size is not different from normal. 2° The present

results therefore demonstrate that, in spite of the overall increase in TpOH protein content measured in the dorsal neostriatum of the neonatally lesioned rats, the TpOH content of these hyperinnervating 5-HT terminals is reduced by 23% compared to normal. Numerous studies have insisted on the fact that, in the present model, 5-HT hyperinnervation is more pronounced in the rostral than the caudal half of neostriatum. 7'21'43"57'59The above counts of [3H]5-HTlabeled varicosities indeed demonstrated a disappearance if not a reversal of the rostrocaudally increasing gradient of 5-HT innervation density normally prevailing in this brain region. This might explain why the level-by-level TpOH protein measurements then showed a homogeneous distribution throughout the caudorostral extent of the neostriatum. A recent study of DA and 5-HT metabolism in the neostriatum of these rats has indicated that the

472

S. Raison et al.

regional increases in 5-HT content measured one and denervated and 5-HT-hyperinnervated neostrithree months after the lesion respectively corre- atum, 2L52'53 which might reflect interregulations sponded to 70% and 80% increases in 5-HT content having taken place quite early during ontogenic per 5-HT terminal.4~ That study also suggested that development. Structural interrelationships could this elevation in 5-HT content per terminal resulted provide for 5-HT-DA interactions within the RD of from a reduction of 5-HT release, possibly conse- lesioned rats, as postulated previously in normal quent to an increased number of neostriatal 5-HTIB rats. 26 DA cell bodies residing in the rostral part of autoreceptors. Previous autoradiographic ligand the RD might be of interest in this regard. ~8'45'64An binding studies have indeed demonstrated significant increased amount of DA in the RD after 6-OHDA elevations in the number of these 5-HT receptors in neonatal lesion42 suggests that these neurons are the neostriatum of neonatally lesioned rats examined spared by the neonatal lesion. Ultrastructural studies as adults? 2 Under such conditions, the increase in after double immunocytochemical labeling have endogenous 5-HT within the hyperinnervating 5-HT shown that they make dendrodendritic appositions terminals might be conveyed to the somata/dendrites with their 5-HT counterparts (Beaudet, personal by retrograde axonal transport of the amine ~'2 and communication). It must also be pointed out that an could lead to some inhibition of TpOH synthesis. 27~39 ectopic DA neoinnervation develops in the neonatally lesioned substantia nigra depopulated of its DA Correlation between the tyrosine hydroxylase and somata/dendrites. 25 It is not excluded that these abertryptophan hydroxylase contents in the dopaminerant DA terminals might influence 5-HT cell bodies denervated and serotonin-hyperinnervated as well as projecting to the neostriatum through an effect on normal neostriatum their axon collaterals within the substantia nigra. When measured on adjacent transfers, TpOH and Tryptophan hydroxylase content in hippocampus TH protein contents within the dorsal neostriatum were tightly correlated in control rats. This correIn the absence of any biochemical or immunocytolation allowed us to predict that, in severely lesioned chemical evidence of 5-HT hyperinnervation in the animals, the zero TH value would be associated with hippocampus of neonatally lesioned rats (Ref. 38 a TpOH value of 3.17 uTpOH/mgt. The actual and Descarries, unpublished observations), the levelmeasurement in lesioned rats was 3.20 uTpOH/mgt, by-level measurements showed a moderate but suggesting that, even after the lesion, a cellular statistically significant increase in TpOH tissue coninteraction between the two monoamine systems centration by comparison with control. However, maintained an equilibrium between the respective the mean increase in TpOH concentration for this enzymatic levels. whole region ( + 18%) represented only half of that Functional interactions between 5-HT and DA measured in the 5-HT-hyperinnervated neostriatum have already been demonstrated in normal rat neo- and did not reach statistical significance. As no striatum, where 5-HT has been shown to induce changes in hippocampal TH content were detected, changes in DA release both in vitro 8 and in I)it)o. 5'6 this increase presumably reflected some secondary Such 5-HT-DA interactions could depend on axoax- changes in the expression or addressing of the TpOH onic appositions or preferential proximity between protein at the level of its cell bodies of origin, namely the two kinds of terminals within the neostriatum the RD and R M . 3'9'51'71 itself, or else 5-HT-DA interrelationships taking Tryptophan hydroxylase content in mesencephalic place at the level of the substantia nigra, since the raphe nuclei zona compacta receives a relatively dense 5-HT innervation arising, at least in part, from axon collaterals One of the most striking observations in the preof RD neurons projecting to the neostriatum. ~s'67 In sent study was the considerable decrease in the TpOH a recent study, Hashiguti et al. 2s have provided labeling volume within the raphe nuclei of the neonabiochemical evidence for an interaction between the tally 6-OHDA-lesioned rats. This reduced size of the neostriatal activity of TH and TpOH in vivo. These somato/dendritic compartment, exhibiting a deauthors have demonstrated an inhibitory effect of tectable level of the enzyme, was the main factor 5-hydroxytryptophan upon the hydroxylation of responsible for the decreased TpOH protein content tyrosine, as well as a reduction of TpOH activity measured in the RD ( - 2 5 % , on average). Indeed, induced by L-DOPA administration. Similar re- TpOH concentration, as directly estimated by densitductions of TpOH activity upon L-DOPA adminis- ometry, appeared to be only slightly altered. tration have already been documented in brain The level-by-level measurement of the TpOH prohomogenates, and were then shown to be associated tein allowed us to localize the major decrease to the with a concomitant decrease of 5-HT synthesis. 56 five rostralmost levels of the RD, which are precisely In lesioned rats, early destruction of the DA system those levels containing the 5-HT cell bodies projectpresumably led to numerous, as yet unsuspected, ing to the neostriatum. 7'3°'57'62Since 5-HT cells procellular adaptations which could somehow affect jecting to the neostriatum have been identified in the rapheostriatal 5-HT neurons. 5-HT and DA receptor lateral as well as midline subgroups of the rostral changes have been found to occur within the DARD, at least after 5-HT hyperinnervation,7's7 these

TpOH after neonatal 6-OHDA lesion reductions in the extent of the somato/dendritic compartment expressing T p O H protein were all the more significant. In the present study, it could not be determined whether the decreased volume occupied by the T p O H protein reflected a diminution in the number of TpOH-expressing somata in the RD, or only of the amount of T p O H protein contained in these somata and/or their proximal and entangled dendrites. Until now, there have been no indications of any 5-HT cell body loss after neonatal 6 - O H D A lesion. 38 Further immunohistochemical studies will be needed to correlate the number of TpOH-positive cells with the amount of T p O H protein within the RD. In any event, such a lowering of the somato/ dendritic level of T p O H was highly suggestive of a resetting of the regulation of the enzymatic protein at an abnormal steady-state level, insufficient to provide the expanded terminal compartment with its usual complement of axonally transported protein. The decrease in T p O H content measured at the two caudalmost levels of the R D could be somehow related to the modifications of the enzyme content in the hippocampus, since the 5-HT innervation of this brain region arises at least in part from the caudal RD. 3°'33'69 As the R D has been shown to project densely to the R M and RP, 7° and some R M fibers have been demonstrated to innervate both the R D 4'9w and RP, 97L 5-HT interconnections between these

473

nuclei might also be responsible for secondary changes in the RM upon influences originally confined to the RD.

CONCLUSIONS

It seems likely that a redistribution of the T p O H protein between the somato/dendritic and terminal field compartments of 5-HT neurons occurs in the present model of neonatal D A denervation followed by 5-HT hyperinnervation of the neostriatum. The mechanisms which lead to the resetting of T p O H protein expression at a reduced level in the somata/ dendrites as well as axon terminals of these neurons remain to be determined. The augmented 5-HT content inside the neostriatal 5-HT terminals after neonatal D A lesion 4~ may be detected by the cell bodies after retrograde axonal transport of 5-HT 1'2 and could represent the signal inhibiting T p O H synthesis. 27'39 In situ hybridization experiments are currently in progress to substantiate this possibility. This work was supported by grants from Universit6 Claude Bernard (Lyon I), the Centre National de la Recherche Scientifique (CNRS-UMR 105), the Minist6re de la Recherche et de l'Enseignement (MRE 92024) and the Minist6re des Affaires internationales du Qu6bec (Cooperation France-Quebec: Recherche M~dicale). The authors are also grateful to Sylvia Garcia and Giovanni Battista Filosi for technical assistance. Acknowledgements

REFERENCES

1. Araneda S., Bobillier P., Buda M. and Pujol J. F. (1980) Retrograde axonal transport following injection of [3H]-serotonin in the olfactory bulb. I. Biochemical study. Brain Res. 196, 405-415. 2. Araneda S., Gamrani H., Font C., Calas A., Pujol J. F. and Bobillier P. (1980) Retrograde axonal transport tbllowing injection of [3H]-serotonin into the olfactory bulb. II. Radioautographic study. Brain Res. 196, 417-427. 3. Azmitia E. C. and Segal M. (1978) An autoradiographic analysis of the differential ascending projections of the dorsal and median raphe nuclei in the rat. J. comp. Neurol. 179, 641 668. 4. Behzadi G., Kal6n P., Parvopassu F. and Wiklund L. (1990) Afferents to the median raphe nucleus of the rat: retrograde cholera toxin and wheat germ conjugated horseradish peroxidase tracing, and selective D-[3H]aspartate labelling of possible excitatory amino acid inputs. Neuroscience 37, 77 100. 5. Benloucif S. and Galloway M. P. (1991) Facilitation of dopamine release in vivo by serotonin agonists: studies with microdiatysis. Eur. J. Pharmac. 200, 1-8. 6. Benloucif S., Keegan M. J. and Galloway M. P. (1993) Serotonin-facilitated dopamine release in vivo: pharmacological characterization. J. Pharmac. exp. Ther. 265, 373-377. 7. Berger T. W., Kaul S., Stricker E. M. and Zigmond M. (1985) Hyperinnervation of the striatum by dorsal raphe afferents after dopamine-depleting brain lesions in neonatal rats. Brain Res. 336, 354-358. 8. Blandina P., Goldfarb J , Craddock-Royal B. and Green J. P. (1989) Release of endogenous dopamine by stimulation of 5-hydroxytryptamine-3 receptors in rat striatum. J. Pharmac. exp. Ther. 251, 803-809. 9. Bobillier P., Seguin S., Degueurce A., Lewis B. D. and Pujol J. F. (1979) The efferent connections of the nucleus raphe centralis superior in the rat as revealed by radioautography. Brain Res. 166, 1-8. 10. Breese G. R. and Taylor T. D. (1971) Depletion of brain noradrenaline and dopamine by 6-hydroxydopamine. Br. J. Pharmac. 42, 88 99. 11. Brownstein M., Saavedra J. M. and Palkovits M. (1974) Norepinephrine and dopamine in the limbic system of the rat. Brain Res. 79, 431-436. 12. Brownstein M. J. and Palkovits M. (1984) Catecholamines, serotonin, acetylcholine, and 7-aminobutyric acid in the rat brain: biochemical studies. In Handbook o f Chemical Neuroanatomy, Classical Transmitters in the CNS, Part I (eds Bj6rklund A. and H6kfelt T.), Vol. 2, pp. 23 53. Elsevier, Amsterdam. 13. Brownstein M. J., Palkovits M., Saavedra J. M. and Kizer J. S. (1975) Tryptophan hydroxylase in the rat brain. Brain Res. 97, 163-166. 14. Cash C., Vayer P., Mandel P. and Maitre M. (1985) Tryptophan-5-hydroxylase. Rapid purification from whole rate brain and production of specific antiserum. Eur. J. Biochem. 149, 239 245. 15. Corvaja N., Doucet G. and Bolam J. P. (1993) Ultrastructure and synaptic targets of the raphe-nigral projection in the rat. Neuroscience 55, 417-427. 16. Dahlstr6m A. and Fuxe K. (1964) Evidence for the existence of monoamine-containing neurons in the central nervous system--I. Demonstration of monoamines in the cell bodies of brain stem neurons. Acta physiol, scand. 62, 5 55.

474

S. Raison et al.

17. Debure L. I., Moyse E., Fevre-Montange M., Hardin H., Belin M. F., Rousset C., Pujol J. F. and Weissmann D. (1992) Somatotopic organization of tyrosine hydroxylase expression in the rat locus coeruleus: long term effect of RU24722. Brain Res. 581, 19-32. 18. Descarries L., Berthelet F., Garcia S. and Beaudet A. (1986) Dopaminergic projection from nucleus raphe dorsalis to neostriatum in the rat. J. comp. Neurol. 249, 511 520. 19. Descarries L., Soghomonian J. J., Garcia S., Doucet G. and Bruno J. P. (1992) Ultrastructural analysis of the serotonin hyperinnervation in adult rat neostriatum following neonatal dopamine denervation with 6-hydroxydopamine. Brain Res. 569, 1-13. 20. Descarries L., Watkins K. C., Garcia S. and Beaudet A. (1982) The serotonin neurons in nucleus raphe dorsalis of the adult rat. A light and electron microscope radioautographic study. J. comp. Neurol. 207, 239-254. 21. Dewar K. M., Soghomonian J. J., Bruno J. P., Descarries L. and Reader T. A. (1990) Elevation of dopamine D: but not Dt receptors in adult rat neostriatum after neonatal 6-hydroxydopamine denervation. Brain Res. 536, 287-296. 22. Doucet G., Descarries L. and Garcia S. (1986) Quantification of dopamine innervation in adult rat neostriatum. Neuroscience 19, 427-445. 23. Ehret M., Gobaille S., Cash C. D., Mandel P. and Maitre M. (1987) Regional distribution in rat brain of tryptophan hydroxylase apoenzyme determined by enzyme-linked immunoassay. Neurosci. Lett. 73, 71 76. 24. El Mansari M., Radja F., Ferron A., Reader T. A., Molina-Holgado E. and Descarries L. (1994) Hypersensitivity to serotonin and its agonists in serotonin-hyperinnervated neostriatum after neonatal dopamine denervation. Eur. J. Pharmac. 261, 17l 178. 25. Fernandes Xavier F. G., Doucet G., Geffard M. and Descarries L. (1994) Dopamine neoinnervation in the substantia nigra and hyperinnervation in the interpeduncular nucleus of adult rat following neonatal cerebroventricular administration of 6-hydroxydopamine. Neuroscience 59, 77-87. 26. Ferr6 S. and Artigas F. (1993) Dopamine D 2 receptor-mediated regulation of serotonin extracellular concentration in the dorsal raphe nucleus of freely moving rats. J. Neurochem. 61, 772 775. 27. Hamon M., Bourgoin S. and Glowinski J. (1973) Feedback regulation of 5-HT synthesis in rat striatal slices. J. Neurochem. 20, 1727 1745. 28. Hashiguti H., Nakahara D., Maruyama W., Naoi M. and Ikeda T. (1993) Simultaneous determination of in vivo hydroxylation of tyrosine and tryptophan in rat striatum by microdialysis-HPLC: relationship between dopamine and serotonin biosynthesis. J. neural Transm. 93, 213--223. 29. Jackson D., Bruno J. P., Stachowiak M. K. and Zigmond M. J. (1988) Inhibition of striatal acetylcholine release by serotonin and dopamine after the intracerebral administration of 6-hydroxydopamine to neonatal rats. Brain Res. 457, 267 273. 30. Jacobs B. L., Foote S. L. and Bloom F. E. (1978) Differential projections of neurons within the dorsal raphe nucleus of the rat: a horseradish peroxidase (HRP) study. Brain Res. 147, 149-153. 31. Jonsson G. (1983) Chemical lesioning techniques: monoamine neurotoxins, In Handbook of Chemical Neuroanatomy, Methods in Chemical Neuroanatomy (eds Bj6rklund A. and H6kfelt T.), Vol. 1, pp. 463 507. Elsevier, Amsterdam. 32. Kan J. P., Buda M. and Pujol J. F. (1975) Tryptophan-5-hydroxylase activity in the raphe system of the rat brain stem. Brain Res. 93, 353 357. 33. K6hler C. and Steinbusch H. (1982) Identification of serotonin and non-serotonin-containing neurons of the midbrain raphe projecting to the entorhinal area and the hippocampal formation. A combined immunohistochemical and fluorescent retrograde tracing study in the rat brain. Neuroscience 7, 951-975. 34. Lewander T., Joh T. H. and Reis D. J. (1977) Tyrosine hydroxyalse: delayed activation in central noradrenergic neurons and induction in adrenal medulla elicited by stimulation of central cholinergic receptors. J. Pharmac. exp. Ther. 200, 523-534. 35. Lidbrink P. and Jonsson G. (1975) On the specificity of 6-hydroxydopamine induced degeneration of central noradrenaline neurons after intracerebral injection. Neurosci. Lett. I, 35 39. 36. Lorez H. P., Saner A. and Richards J. G. (1978) Evidence against a neurotoxic action of halogenated amphetamines on serotoninergic B9 cells. A morphometric fluorescence histochemical study. Brain Res. 146, 188 194, 37. Luthman J., Botioli B., Tsutsumi T., Verhofstad A. and Jonsson G. (1987) Sprouting of striatal serotonin nerve terminals following selective lesions of nigro-striatal dopamine neurons in neonatal rat. Brain Res. Bull. 19, 269 274. 38. Luthman J., Brodin B., Sundstr6m E. and Wiehager B. (1990) Studies on brain monoamine and neuropeptide systems after neonatal intracerebroventricular 6-hydroxydopamine treatment. Int. J. devl Neurosci. 8, 549 560. 39. Macon J. B., Sokoloff L. and Glowinski J. (1971) Feedback control of rat brain 5-hydroxytryptamine synthesis. J. Neurochem. 18, 323 331. 40. Miller J. J., Richardson T. L., Fibiger H. C. and McLennan H. (1975) Anatomical and electrophysiological identification of a projection from the mesencephalic raphe to the caudate-putamen in the rat. Brain Res. 97, 133 138. 41. Molina-Holgado E., Dewar K. M., Descarries L. and Reader T. A. (1994) Altered dopamine and serotonin metabolism in the dopamine-denervated and serotonin-hyperinnervated neostriatum of adult rat after neonatal 6-hydroxydopamine. J. Pharmac. exp. Ther. 270, 713-721. 42. Molina-Holgado E., Dewar K. M., Grondin L., Van Gelder N. M. and Reader T. A. (1993) Changes of amino acid and monoamine levels after neonatal 6-hydroxydopamine denervation in rat basal ganglia, substantia nigra, and raphe nuclei. J. Neurosci. Res. 35, 409-418. 43. Mrini A., Soucy J. P., Lafaille F., Lemoine P. and Descarries L. (1995) Quantification of the serotonin hyperinnervation in adult rat neostriatum after neonatal 6-hydroxydopamine lesion of nigral dopamine neurons. Brain Res. 669, 303 308. 44. Nygren L. G. and Olson L. (1977) Intracisternal neurotoxins and monoamine neurons innervating the spinal cord: acute and chronic effects on cell counts and nerve terminal densities. Histochemistry 52, 281 306. 45. Ochi J. and Shimizu K. (1978) Occurrence of dopamine-containing neurons in the midbrain raphe nuclei of the rat. Neurosci. Lett. 8, 317 320. 46. Oleskevich S. and Descarries L. (1990) Quantified distribution of the serotonin innervation in adult rat hippocampus. Neuroscience 34, 19 33. 47. Oleskevich S., Descarries L. and Lacaille J. C, (1989) Quantified distribution of the noradrenaline innervation in the hippocampus of adult rat. J. Neurosci. 9, 3803 3815.

TpOH after neonatal 6-OHDA lesion

475

48. Oleskevich S., Descarries L., Watkins K. C., S6gu~la P. and Daszuta A. (1991) Ultrastructural features of the serotonin innervation in adult rat hippocampus: an immunocytochemical description in single and serial thin sections. Neuroscience 42, 777 791. 49. O'Shea L., Saari M., Pappas B. A., Ings R. and Stange K. (1983) Neonatal 6-hydroxydopamine attenuates the neural and behavioral effects of enriched rearing in the rat. Eur. J. Pharmac. 92, 4347. 50. Paxinos G. and Watson C. (1986) The Rat Brain in Stereotaxic Coordinates, 2nd edn. Academic Press, Tokyo. 51. Priestley J. V., Somogyi P. and Cuello A. C. (1981) Neurotransmitter-specific projection neurons related by combining PAP immunohistochemistry with retrograde transport of HRP. Brain Res. 220, 231 -240. 52. Radja F., Descarries L., Dewar K. M. and Reader T. A. (1993a) Serotonin 5-HT~ and 5-HT 2 receptors in adult rat brain after neonatal destruction of nigrostriatal dopamine neurons: a quantitative autoradiographic study. Brain Res. 606, 273-285. 53. Radja F., E1 Mansari M., Soghomonian J. J., Dewar K. M., Ferron A., Reader T. A. and Descarries L. (1993b) Changes of D 1 and D~ receptors in adult rat neostriatum after neonatal dopamine denervation: quantitative data from ligand binding, in situ hybridization and iontophoresis. Neuroscience 57, 635-648. 54. Reis D. J., Gilad G., Pickel V. M. and Joh T. H. (1978) Reversible changes in the activities and amounts of tyrosine hydroxylase in dopamine neurons of the substantia nigra in response to axonal injury as studied by the immunochemical and immunocytochemical methods. Brain Res. 144, 325 342. 55. Reymann K., Pohle W., Mfiller-Welde P. and Ott T. (1983) Dopaminergic innervation of the hippocampus: evidence for midbrain raphe neurons as the site of origin. Biomed. biochem. Acta 42, 1247 1255. 56. Roberge A. G. and Poirer L. J. (1973) Effect of chronically administered L-DOPA on DOPA/5HTP decarboxylase and tyrosine and tryptophan hydroxylases in cat brain. J. neural Transm. 34, 171- 185. 57. Snyder A. M., Zigmond M. J. and Lund R. D. (1986) Sprouting of serotoninergic afferents into striatum after dopamine-depleting lesions in infant rats: a retrograde transport and immunocytochemical study. J. comp. NeuroL 245, 274 28 I. 58. Soghomonian J. J., Descarries L. and Watkins K. C. (1989) Serotonin innervation in adult rat neostriatum. 1I. Ultrastructural features: a radioautographic and immunocytochemical study. Brain Res. 481, 67 86. 59. Stachowiak M. K., Bruno J. P., Snyder A. M., Stricker E. M. and Zigmond M. J. (1984) Apparent sprouting of striatal serotonergic terminals after dopamine-depleting brain lesions in neonatal rats. Brain Res. 291, 164 167. 60. Steinbusch H. W. M. (1981) Distribution of serotonin-immunoreactivity in the central nervous system of the rat cell bodies and terminals. Neuroscience 6, 557-618. 61. Steinbusch H. W. M. (1984) Serotonin-immunoreactive neurons and their projections in the CNS. In Handbook of Chemical Neuroanatomy, Classical Transmitters and Transmitter Receptors in the CNS, Part II (eds Bj6rklund A., H6kfelt T. and Kuhar M. J.), Vol. 3, pp. 68 118. Elsevier, Amsterdam. 62. Steinbusch H. W. M., Van Der Kooy D., Verhofstad A. A. J. and Pellegrino A. (1980) Serotonergic and non-serotonergic projections from the nucleus raphe dorsalis to the caudate putamen complex in the rat, studied by a combined immunofluorescence and fluorescent retrograde axonal labeling technique. Neurosci. Lett. 19, 137 142. 63. Steinbusch H. W. M., Niewenhuys R., Verhofstad A. A. J. and Van Der Kooy D. (1981) The nucleus raphe dorsalis of the rat and its projection upon the caudatoputamen. A combined cytoarchitectonic, immunohistochemical and retrograde transport study. J. Physiol., Paris 77, 157-174. 64. Stratford T. R. and Wirtshafter D. (1990) Ascending dopaminergic projections from the dorsal raphe nucleus in the rat. Brain Res. 511, 173 176. 65. Swanson L. W. (1982) The projections of the ventral tegmental area and adjacent regions: a combined fluorescent retrograde tracer and immunofluorescence study in the rat. Brain Res. Bull. 9, 321 353. 66. Tappaz M. and Pujol J. F. (1980) Estimation of the rate of tryptophan hydroxylation in t,ivo: a sensitive microassay in discrete rate brain nuclei. J. Neurochem. 34, 933 940. 67. Van der Kooy D. and Hattori T. (1980) Dorsal raphe cells with collateral projections to the caudat~putamen and substantia nigra: a fluorescent retrograde double labeling study in the rat. Brain Res. 186, 1 7. 68. Verney C., Baulac M., Berger B., Alvarez C., Vigny A. and Helle K. B. (1985) Morphological evidence for a dopaminergic terminal field in the hippocampal formation of young and adult rat. Neuroscience 14, 1039 1052. 69. Vertes R, P. (1991) A PHA-L analysis of ascending projections of the dorsal raphe nucleus in the rat. J. comp. Neurol. 313, 643-668. 70. Vertes R. P. and Kocsis B. (1994) Projections of the dorsal raphe nucleus to the brainstem: PHA-L analysis in the rat. J. comp. Neurol. 340, 11 26. 71. Vertes R. P. and Martin G. F. (1988) Autoradiographic analysis of ascending projections from the pontine and mesencephalic reticular formation and the median raphe nucleus in the rat. J. comp. Neurol. 275, 511 541. 72. Weissmann D., Belin M. F., Aguera M., Meunier C., Maitre M., Cash C. D., Ehret M., Mandel P. and Pujol J. F. (1987) lmmunohistochemistry of tryptophan hydroxylase in the rat brain. Neuroscience 23, 291 304. 73. Weissmann D., Chamba G., Debure L., Rousset C., Richard F., Maitre M. and Pujol J. F. (1990) Variation of tryptophan-5-hydroxylase concentration in the rat raphe dorsalis nucleus after p-chlorophenylalanine administration. II. Anatomical distribution of tryptophan-5-hydroxylase protein and regional variation of its turnover rate. Brain Res. 536, 46 55. 74. Weissmann D., Labatut R. Richard F., Rousset C. and Pujol J. F. (1989) Direct transfer into nitrocellulose and quantitative radioautographic anatomical determination of brain tyrosine hydroxylase protein concentration. J. Neurochem. 53, 793 799. (Accepted 20 January 1995)