effects of intraventricular 6-hydroxydopamine on the dopaminergic innervation of striatum:...

8 Brain Research, 376 (1986) 8-19 Elsevier

BRE 11774

Effects of Intraventricular 6-Hydroxydopamine on the Dopaminergic Innervation of Striatum: Histochemical and Neurochemical Analysis

SHAO-PII ONN, THEODORE W. BERGER, EDWARD M. STRICKER and MICHAEL J. ZIGMOND

Departments of Psychology, Biological Sciences and Psychiatry, and the Center for Neuroscience, University of Pittsburgh, PA 15260 (U.S.A.)

(Accepted October 29th, 1985)

Key words: dihydroxyphenylacetic acid - - dopamine - - 6-hydroxydopamine - - striatum - - tyrosine hydroxylase

The impact of intracerebroventricular administration of 6-hydroxydopamine (6-HDA) on the dopamine (DA)-containing nigrostriatal projection was determined by regional histochemical and biochemical analyses. One week postinjection, we observed that tyrosine hydroxylase (TH)-positive terminals were almost completely absent from the medial portion of striatum but gradually increased in density toward the lateral margin of this structure. A similar gradient was indicated by fluorescence histochemistry and biochemical analyses of DA. In contrast, the 6-HDA-induced changes in TH activity and in dihydroxyphenylacetic acid content were less severe and showed little or no medial-to-lateral gradient. These high levels of TH activity and DOPAC content, relative to local DA concen- trations, suggest an increase in the synthesis and release of DA from residual terminals that may serve to compensate for the brain damage. By 4 months postlesion, both histochemical and biochemical analyses indicated the presence of more DA terminals in striatum than there had been one week postlesion. This change was most markedly obvious in the medial striatum, which had been almost completely devoid of terminals at one week postlesion. Retrograde tracing experiments revealed that terminals appearing in the medial striatum at 4 months postlesion arise from the same region of the substantia nigra that innervates the medial striatum in the intact animal. Thus, no change in the topographic relation between substantia nigra and striatum occurred as a result of the lesion.

INTRODUCTION

6-Hydroxydopamine (6 -HDA) adminis tered into

cerebrospinal fluid by way of the lateral cerebroven-

tricles of rats produces pe rmanen t and widespread

degenerat ion of central dopaminergic terminals30.

Near- total destruct ion of these terminals leads to

gross behavioral dysfunctions including aphagia and

akinesia. However , such impai rments do not occur

when at least 5 - 1 0 % of the dopaminergic terminals

remain33. We have p roposed that this sparing of be-

havioral function is due in par t to neurochemical

compensat ions that take place in residual dopaminer -

gic terminals 2s. Such compensat ions are suggested by

the observat ion that after 6 - H D A adminis t ra t ion the

loss of d ihydroxyphenylacet ic acid ( D O P A C ) , the

principal metabol i te of dopamine (DA) , is much

smaller than the loss of DA32 (see also refs. 2, 13).

Likewise, the activity of tyrosine hydroxylase (TH),

the rate-l imiting enzyme in D A synthesis, is less af-

fected by the lesions than is D A 3e.

The precise relat ion be tween lesion size, behavior-

al deficit and D A turnover cannot be assessed by

studies of whole str iatum, however , because intra-

ventricularly adminis tered 6 - H D A produces a het-

erogeneous loss of dopaminergic terminals within stri-

atum, with the greatest degenera t ion occurring in

more medial areas e6. We therefore examined subre-

gions of the str iatum in b ra in -damaged rats. Specifi-

cally, we have used TH immunocytochemical tech-

niques to examine the effects of 6 -HDA- induced le-

sions on the distr ibution of D A terminals in striatum.

Then, by modifying existing procedures to permi t the

visualization of DA-conta in ing terminals so they

could be combined with biochemical analyses of al-

ternate sections from the same brains, we have exam-

ined the regional distr ibution of D A , T H activity and

D O P A C after brain damage. In a companion study22,

Correspondence: M.J. Zigmond, Psychobiology Program, 573 Crawford Hall, University of Pittsburgh, Pittsburgh, PA 15260, U.S.A.

we also have examined the consequences of intraventricular 6-HDA treatment on the electrophysiologic- al activity of neurons in these same subregions of striatum.

MATERIALS AND METHODS

Animals Male Sprague-Dawley rats (Zivic-Miller Labora-

tories, Allison Park, PA), weighing 175-200 g on arrival, were housed in metal cages in a temperature- controlled (22 °C) room illuminated from 08.00 h to 20.00 h. The animals were allowed free access to Pu- rina Rodent Laboratory Chow and tap water, and were handled daily. One week after arrival, they were anesthetized with Equithesin (Abbott Labora- tories, North Chicago, IL) (2 ml/kg), and 6-HDA (6-hydroxydopamine hydrobromide, Regis Chemic- al Co.; 250 Bg free base) or vehicle solution (0.1% as- corbic acid in 0.9% NaCI) was infused into the lateral ventricles (10 B1 each hemisphere; 4/A/min). The animals had received desmethylimipramine (25 mg/kg, i.p.; Merrill-National Laboratories, Cincinnati, OH) 20-30 min earlier, to block the effects of the neuro- toxin on noradrenergic terminals 7.

TH immunocytochemistry Either one week (n = 20) or ca. 4 months (range,

15-20 weeks; n = 15) after surgery, rats were anesthetized with phenobarbital (60 mg/kg) and perfused via the left ventricle of the heart with 200 ml of chilled saline (0.15% sodium chloride) followed with 300 ml of 4% paraformaldehyde buffered with 0.1 M sodium acetate (pH 6.5), and finally with 500 ml of 4% paraformaldehyde buffered with 0.1 M sodium borate (pH 11) 5. The brain was removed rapidly from the skull and immersed overnight at 4 °C in the last perfusate, which had been adjusted to contain 15% sucrose. Frozen sections were cut with a freezing mi- crotome and collected in 0.1 M phosphate buffer (pH 7.4). Sections were incubated for 20 h at 4 °C in a TH-specific antiserum medium (1:1000 dilution with 1% normal goat serum; a gift from Dr. T. Joh) TM. This was usually followed by successive 30-60 min incu- bations with rabbit IgG (100x dilution; Sternberger- Meyer Immunocytochemicals, Jarrettsville, MD) and rabbit peroxidase anti-peroxidase (PAP) (120× dilution; Sternberger-Meyer Immunocytochemic-

als). The tissue was then treated with a freshly fil- tered solution of 33% diaminobenzidine (Sigma, St. Louis, MO) for 10 min at room temperature, with ad- dition of H202 after the first 2 min. At the end of treatment, sections were mounted on gelatinized slides, air-dried and cover-slipped with Permount. TH-positive terminals were visualized with a Leitz Orthoplan microscope.

In some cases (n = 10 at both i week and 4 months postlesion), sections were incubated with fluorescence-isothiocyanate (FITC)-labeled goat anti-rabbit lgG (100x dilution; Miles Lab, Elkhart, IN) in- stead of rabbit-IgG and PAP. The FITC-labeled TH terminals were localized by fluorescence microscopy (Zeiss E filter, with excitation/emission wavelengths of 450/535 nm).

DA histochemistry A method combining sucrose-potassium phos-

phate-glyoxylic acid (SPG) histochemistry and mi- cropunch techniques was established to permit histological visualization of DA-containing terminals and regional biochemical analyses from the same brain specimens. Animals were sacrificed by decapitation 1 week (n = 20) or 4 months (n = 17) after 6-HDA administration and their brains were removed and cut into slabs ca. 10 mm thick. The slabs were placed im- mediately on a precooled cryostat chuck with embed- ding material. The chuck with tissue then was re- turned to the cryostat (-15 to -20 °C) and cut into two thick sections with boundaries of ca. 8920-8380 pm and 7890-7190 pm, according to the atlas of K6nig and Klippe116. Thin (20 pm) tissue sections also were cut before, after and between these thick sections. The thick sections were placed on slides and set in the cryostat for later biochemical analysis, whereas the thin sections were collected on warm slides and processed for SPG histochemistry using the technique of de la Torre and Surgeon 8. DA-containing terminals were localized by fluorescence microscopy (Zeiss D filter, 425/490 nm).

DA, DOPAC and TH activity assays Fluorescence microscopy provided guidance for

dissection of the thick sections. The dissection was accomplished while maintaining the slices at ca. -20 °C, using rectangularly shaped stainless steel punches (cross section, 0.7 mm × 1.7 mm) 23. With

10

the long axis of the punch held vertically, samples were taken from the medial, central and lateral portions of striatum. Samples were taken one week after intraventricular injection of either 6-HDA (n = 11-17; different sample n's for DA, DOPAC and TH) or its vehicle (n = 11-17), and 4 months after either 6-HDA (n = 8) or vehicle (n = 8). Right and left samples from each of the two thick sections from a given brain were combined and stored at -80 °C before the biochemical assays were performed. Tissue punches were homogenized in 250 #1 of 50 mM Tris- HC1 (pH 6) with a glass pestle homogenizer. Fifty/~1 aliquots of the homogenates were taken for measurement of DA and DOPAC. The aliquots were added to 100 #1 of 0.4 N HCIO 4 and then were centrifuged at 40,000 g for 15 min. The supernatants were removed, the catechols separated with alumina 3 followed by reverse-phase high performance liquid chromatography (HPLC) TM, and quantification carried out by electrochemical detection TM. The HPLC was carried out using a microBondapak C-18 column together with an elution buffer (pH 3.1) containing, per liter, 14.2 g of monochloroacetic acid, 1 ml of 0.1 M

Na2EDTA, 60 ml of methanol and 4 ml of 0.2 M octyl sulfate (Eastman Organics, Rochester, NY). 3,4-Di- hydroxybenzylamine was used as an internal standard. All samples yielded distinct peaks that were well above the background.

The remaining 200/~l of homogenate were assayed by a coupled decarboxylase method to measure soluble TH activity 1,~5. The homogenates were centri-

fuged at 40,000 g for 30 min. The supernatants were incubated in a 2 M Tris-acetate buffer (pH 5.7) in the presence of 75/~M of L-[1-14C]tyrosine (54 mCi/m-

tool, New England Nuclear, Boston, MA) and a satu- rating concentration (3 mM) of the cofactor, 6-meth- yi-5,6,7,8-tetrahydropterin-HC1 (6MPH4, Calbio- chem-Behring Corp., La Jolla, CA). An excess of ar- omatic amino acid decarboxylase then was added and the resulting 14CO2 was trapped and counted by liq-

uid scintillation spectroscopy. All sample counts were at least twice those obtained in the absence of tissue.

The DA and DOPAC contents were expressed as~ pg/~g of acid-insoluble protein and were quantified by the method of Lowry et al. 17. TH activity was expressed as pmol/min//~g of soluble protein assayed by the method of Bradford 6. The ratio of acid-insoluble

protein to acid-soluble protein was ca. 6.0.

Photometry A Leitz fluorescence microscope equipped with an

MPV2 photometer was used to quantify the fluorescence intensity of sections processed by the SPG or FITC methods. The SPG or FITC materials from control animals (n = 4 for SPG; n = 3 for FITC) or

SPG CONTROL

v c c

6-HDA (1 week post-injection)

CC

6-HDA (15 weeks pos t - in jec t ion)

CC

0.5 m m

Fig. 1. Glyoxylic acid-induced fluorescence (SPG method) in a representative coronal section of striatum at an anterior-posterior plane of ca. 8380/~m according to K6nig and KlippeP 6, presumably reflecting DA-containing terminals. The lateral ventricle (V) is shown on the left, the corpus callosum (CC) on the right. Upper panel, control striatum. Middle panel, striatum one week after the intraventricular administration of 6-hydroxydopamine (6-HDA). Lower panel, 4 months after 6-HDA.

11

animals injected with 6-HDA one week (n = 7 each

for SPG and FITC) or 4 months earlier (n = 4 each for SPG and FITC) were examined at 3 anterior- posterior planes of the striatum (3 adjacent sections/ plane). The fluorescence microscope was calibrated before each use, using a standardized, freshly prepared solution of FITC. The amount of background fluorescence in the corpus callosum was used as a ref- erence for calibration. At each of the anterior-posterior planes, a 0.8 mm diameter portion of the medial,

2-

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[ ] A8920 I ~

[ ] A8380p

AT190p

i 0 i 2

Z

0

MEDIAL CENTRAL LATERAL

Fig. 2. Photometric measurements of fluorescence in the medial, central and lateral aspects of the normal striatum determined at 3 antero-posterior planes. Each bar represents the mean + S.E.M. of samples from 3-4 animals. Upper panel, FITC-treated tissue, presumably reflecting the density of TH- positive terminals. Lower panel, SPG-treated tissue, presumably reflecting the density of DA-containing terminals.

central and lateral striatum was examined 3 times using a 5 s exposure and an averaged voltmeter read-

ing was determined.

Horseradish peroxidase histochemistry Rats were lightly anesthetized with Equithesin and

horseradish peroxidase (HRP, 20 nl of a 10% solution, Sigma) was placed stereotaxically into the medial striatum (1.0 mm anterior to bregma, 1.8 mm lateral to the midline and 4.1 mm ventral from the dura) or the lateral striatum (3.8 mm lateral to the midline). The HRP injections were made either one week (medial striatum, n = 7) or 4 months (medial,

n = 6) after intraventricular injection of 6-HDA, and either 1 week (medial, n = 5; lateral, n = 5) or 4 months (medial, n = 4) after intraventricular injection of its vehicle. Twenty-four hours later rats were anesthetized with phenobarbital (60 mg/kg) and perfused via the left heart ventricle with 200 ml of chilled saline, followed with 1000 ml of 1% paraformaldehyde and 1.25% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) and finally with 800 ml of 10% sucrose in phosphate buffer. Then the brain was removed from the skull and immersed overnight at 4 °C in the last perfusate. Frozen sections (40 #m) were taken throughout the entire anterior-posterior ex-

tent of the substantia nigra (SN) and ventral tegmen- tal area (VTA) (ca. 40 sections per animal). Sections

were collected in phosphate buffer and tissue was processed using benzidine dihydrochloride (Sigma)

as the chromogen 19. The distribution and numbers of retrogradely labelled neurons within the area containing the SN and the VTA were determined under

dark field microscopy for all sections.

RESULTS

Normal striatum Using PAP immunocytochemistry, the density of

TH-positive terminals appeared to be evenly distributed across the coronal plane in the striatum of control rats, except that the density around the periventricular area was slightly higher than the density else- where. The FITC preparation showed a similar distribution of TH-positive terminals. Because these immunocytochemical techniques require brain perfusion, they are not compatible with biochemical analyses. Thus, the SPG histofluorescence technique,

12

Fig. 3. Cells in substantia nigra (SN) labelled with HRP injected into the medial (panels A-D) or lateral (panels E-H) aspects of striatum. Panels A and E, injection sites; panels B and F, low power photomicrographs of SN; panels C, D, G and H, higher power photomicrographs of the areas indicated in the preceding panels. Arrows indicate HRP-positive cells.

which does not require perfusion, was used to relate

the biochemical indices to the anatomical observations. This procedure also revealed a relatively ho-

mogeneous distribution of DA-containing terminals in the coronal plane (Fig. 1, upper panel) and a moder-

ate decline from rostral to caudal striatum.

Photometric techniques were used to quantify the amount of fluorescence present in specimens pre-

pared by the FITC and SPG procedures (Fig. 2). Measurements taken from FITC material at medial,

central and lateral regions at each of the anterior,

middle and posterior levels of the striatum were compared in a 3 × 3 analysis of variance. The results con-

firmed the apparent homogeneity of TH-positive ter-

minals in the coronal planes (F = 0.71, df = 2,18, n.s.), but also indicated a significant decline in fluo-

rescence intensity from the rostral to the caudal poles

of the striatum (F = 12.81, df = 2,18, P < 0.001). Photometric measurements taken from SPG material

were similar to the FITC data (Fig. 2). Visualization of HRP-positive cells in SN after ad-

ministration of the enzyme into normal striatum of

animals injected with vehicle i week earlier revealed

the topographic organization of the nigrostriatal projection that has been described previously 11. Specifi-

cally, the majority of retrogradely labeled neurons

(mean total per animal + S.E. = 358 + 13) were lo-

cated in the medial SN and VTA after injection of

HRP into medial striatum, and the majority of labeled neurons (total = 375 + 17) were located in the lat-

eral SN after injection into lateral striatum (Fig. 3). Biochemical analysis of the DA and DOPAC con-

tent of control striatum suggested a distribution of

dopaminergic terminals similar to the one seen histo-

logically. The concentration of the amine and its me- tabolite at the 3 anterior-posterior and medial-lateral levels of the striatum were compared using analysis

of variance procedures and were found to be homog-

eneous in the medial-lateral plane (for DA, F = 1.02, df = 3,27, n.s.; for DOPAC, F = 0.06, df = 3,27, n.s.), while declining in the rostral-caudal axis (for

DA, F = 16.22, df = 3,27, P < 0.001; for DOPAC, F = 12.17, df = 3,27, P < 0.001). In contrast, there was a relatively uniform distribution of TH activity in

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Fig. 4. The concentration of dopamine and dihydroxyphenylacetic acid (DOPAC) and the activity of tyrosine hydroxylase (TH) in the medial, central and lateral aspects of the normal striatum determined at 3 antero-posterior planes. Each bar represents the mean + S.E.M. of samples taken from 4 animals.

both the medial-lateral (F = 3.01, df = 2,18, n.s.)

and rostral-caudal planes (F = 0.06, df = 2,18, n.s.)

(Fig. 4).

Effects of 6-HDA one week postlesion When examined one week after intraventricular

6 -HDA administration, residual TH-positive termi-

13

nals in striatum appeared to be distributed heteroge- neously, with the medial periventricular area most af-

fected and the lateral area least affected (Fig. 5, upper panel). Photometric analysis of FITC material in-

dicated that about 10% of the fluorescence observed

in control animals remained in the medial striatum of 6-HDA-lesioned animals. However , measurement

of an area of medial striatum with no visible fluores-

cent terminals suggested that virtually all of this sig-

nal was autofluorescent and did not represent TH-

containing terminals, a conclusion supported by sub- sequent biochemical assays of D A content (see be-

low). In contrast to the absence of TH-related fluo-

rescence in the medial striatum, about 40% of control fluorescence remained in the lateral area, most of

which could be attributed to the presence of TH-containing terminals (Fig. 6). Analyses of variance con-

firmed the medial-lateral gradient of TH-containing

terminals (F = 6.65, df = 2,18, P < 0.01). This gradi-

ent was similar at all three levels of the striatum (F =

0.28, df = 2,18, n.s.). A medial-to-lateral gradient was observed when

DA-containing terminals were visualized using SPG

histofluorescence (Fig. 1, middle panel) which paral- leled that for TH-positive terminals. Again, few re-

sidual terminals were visible adjacent to the ventricle, whereas many more could be seen in the lateral

striatum. Photometry of SPG-prepared tissue showed a similar trend; ca. 2% and 15% of control

fluorescence remained in the medial and lateral

areas, respectively (Fig. 6). Terminal loss was associated with a marked disap-

pearance of TH-positive cells in the SN. Considera-

ble loss could be detected by two weeks. However, several additional weeks were required before cell

loss was complete (data not shown). When H R P was administered into medial striatum

one week after 6 -HDA administration, the location

of the cells to which H R P was transported was similar to that seen in normal animals (majority contained

within the medial SN and VTA), though the number

of such cells was reduced to 2% of control (total per

animal = 7 + 2) (Fig. 7, panels A - D ) . Biochemical assay of striatal D A content indicated

a gradient similar to that observed histochemically

(medial vs lateral striatum; t = 5.46, df = 32, P < 0.002). In the medial area of striatum, D A levels

were reduced to 2% of control whereas in the lateral

14

F I T C 6-HDA (l week post-injection)

V CC

6-HDA (15 weeks post-injection)

v c c

0.5 m m

Fig. 5. Tyrosine hydroxylase-positive terminals in a representative section of striatum. The lateral ventricle (V) is shown on the left, the corpus callosum (CC) on the right. Upper panel, striatum one week after the intraventricular administration of 6-hydroxydopamine; lower panel, 4 months postlesion.

a rea they were r educed to 17% (Table I). ~ o.3 z

D O P A C con ten t and T H act ivi ty also w e r e re- (/3

duced by 6 - H D A . H o w e v e r , bo th indices s h o w e d a ~ _ o z

smal le r decrease than did D A con t en t (Fig. 8, u p p e r ~ o.i

panel ) , resul t ing in an e leva t ion of the ra t ios T H / D A V-

and D O P A C / D A above control . T h e increases in ,7 o

these rat ios were inverse ly p r o p o r t i o n a l to the loss of

D A . Thus , T H / D A was inc reased severa l - fo ld in the z i.o i , i

media l aspect of s t r ia tum and gradua l ly dec reased to

cont ro l levels toward the la tera l po r t i on of the struc- ~ ~ o5

ture (Fig. 9). A similar re la t ion was seen b e t w e e n ~ ~ ~9

D O P A C / D A and D A con ten t (da ta no t shown) . 0_ (O o

Effects o f 6 -HDA four months postlesion

Severa l months af ter 6 - H D A admin is t ra t ion , the re

was an increase in the n u m b e r o f de tec t ab le TH-pos i -

t ive te rmina ls in s t r ia tum re la t ive to the n u m b e r ob-

se rved one w e e k post les ion. This a p p e a r e d to be t rue

t h ro u g hou t the s t ructure but it was par t icu lar ly

I WEEK POST-6HDA

. . . . . . . . i i ..... [ ] A 8 9 2 0 # [ ] A 8380~- • A T I 9 0 ~

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


Fig. 6. Photometric analysis of fluorescence induced by FITC (upper) and SPG (lower) processing in medial, central and lateral striatum one week after intraventricular administration of 6-hydroxydopamine. Each bar represents mean + S.E.M. The dotted lines represent the level of fluorescence in an area of medial striatum containing no visible terminals and which may reflect autofluorescence.

TABLE I

Dopamine content (pg/l~g protein) in regions of striatum one week and four months after the intraventricular administration of 6-hydroxydopamine

Each value is the mean + S.E.M. of samples taken from 17 animals (one week postoperative) or 8 animals (4 months postoperative). In parentheses the dopamine content in samples from lesioned animals are expressed as a percentage of control values.

Area 1 week postoperative 4 months postoperative

Sham 6-HDA Sham 6-HDA

Medial 114.7 + 3.8 2.7 + 0.3 (2.4) 122.0 + 10.7 10.0 + 1.2 (8.2) Central 116.3 + 6.7 10.3 + 1.4 (9.0) 126.4 + 15.0 26.0 + 4.4 (20.6) Lateral 113.1 + 6.7 19.7 + 3.1 (17.2) 127.2 + 13.8 37.8 + 4.6 (29.7)

15

l mm

Fig. 7. Cells in substantia nigra (SN) labelled with HRP injected into the medial striatum one week (panels A-D) or 4 months (panels E-H) after intraventricular administration of 6-hydroxydopamine. Panels A and E. injection sites; panels B and F, low power photomicrographs of SN: panels C, D, G, and H, higher power photomicrographs of the areas indicated in the preceding panels. Arrows in G indicate HRP-positive cells•

50

40

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0 I--

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20 I0 0

E~DDA CONTENT I DOPAC CONTENT I WEEK POST-LESION I TH ACTIVITY I

4 ~ ~ M O N T H S POST-LESION


Fig. 8. The concentration of dopamine and dihydroxyphenylacetic acid (DOPAC) and the activity of tyrosine hydroxylase (TH) in the medial, central and lateral aspects of the striatum one week and 4 months after intraventricular administration of 6-hydroxydopamine. Data (mean + S.E.M.) are expressed as a percent of control values. Control values for DA were 114.7 + 3.3 pg/~g protein (1 week), 125.2 + 7.1 pg//~g (4 months); for DOPAC, 15.1 + 2.6 pg//xg protein (1 week), 16.5 + 1.1 pg//~g (4 months); for TH activity, 0.884 + 0.034 pmol/min/~g protein (1 week), 0.852 + 0.096 pmol/min//~g (4 months).

marked in the medial , per iventr icular area (Fig. 5,

lower panel) . Comparab le findings were obta ined

when DA-conta in ing terminals were examined (Fig.

1, lower panel) . Photometr ic analysis confirmed the

impression of an increase in f luorescence throughout

the str iatum between one week and 4 months postle-

sion (Fig. 10). For statistical comparisons, pho tome-

tric measurements taken through the media l - la tera l

planes were combined for each of the a n t e r i o r -pos -

ter ior striatal levels for each of the lesion groups

(1 week and 4 months) . Results showed that in-

creased fluorescence at 4 months post lesion occurred

at media l (t = 17.39, df = 15, P < 0.002) and lateral (t

= 9.82, df = 15, P < 0.002) striatal locations.

H R P adminis tered into medial s tr iatum resul ted in

the labelling of significantly more cells in S N - V T A

(total per animal = 47 + 9) than had been de tec ted

one week pos topera t ive (t = 4.67, df = 11, P <

0.002) (Fig. 7, panels E - H ) . These cells were in the

same medial S N - V T A locat ion as cells revealed by

comparable H R P injections in control animals and in

6 -HDA- t r ea t ed rats one week postlesion.

16

2 0 6 5

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%

o

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Fig. 9. Tyrosine hydroxylase (TH) activity in regions of stria- turn, after intraventricular administration of 6-hydroxydopamine, as a function of dopamine depletion. Enzyme activity is expressed relative to dopamine content to reflect activity per remaining terminal. Values were obtained one week (opened symbols) and 4 months (filled symbols) postlesion from the medial (©, 0), central (A, A), and lateral ([~, . ) aspects of the striatum. The true control values for TH/DA were 66.5 + 5.3 x 10 .4 (1 week), 72.4 + 6.7 × 10 -4 (4 months). The mean of percentage of control _+ the standard deviation is indicated by the shaded area.

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MEDIAL C E N T R A L L A T E R A L

Fig. 10. Photometric analysis of fluorescence induced by FITC (upper) and SPG (lower) processing in medial, central and lateral striatum 4 months after intraventricular administration of 6-hydroxydopamine. The dotted lines, taken from Fig. 6, represent the level of fluorescence that is present in the absence of any visible terminals and which may reflect autofluorescence.

Measurement of regional D A content fur ther sug-

gested an increase toward normal of the dopaminer -

gic innervat ion of striatum. In the medial area D A

levels were 8% of control , a 3.5-fold increase over

D A levels observed one week after 6 - H D A (t = 7.94,

df = 22, P < 0.002); in the lateral area, D A levels

were 30% of control (t = 3.20, df = 22, P < 0.01), a

1.7-fold increase (Table I). Lit t le or no increases

were observed in D O P A C levels (medial str iatum:

t = 1.53, df = 22, n.s.; lateral str iatum: t = 1.75, df =

22, n.s.) or in T H activity (medial str iatum: t = 3.56,

df = 13, P < 0.01; la teral str iatum: t = 1.09, df = 13,

n.s.) (Fig. 8, lower panel) . Thus, the rat ios D O P A C /

D A and TH act iv i ty /DA were less e levated at 4

months than at one week post lesion (Fig. 9).

DISCUSSION

One of two approaches has usually been taken in

studying the distr ibution of catecholaminergic termi-

nals in brain. First , brain tissue has been analyzed

biochemically. Init ially such studies involved the

analysis of large tissue samples by bioassay31, but

over the years this approach has been ref ined

through the use of microdissect ion techniques and

the deve lopment of highly sensitive biochemical

analyses 23. The deve lopment of histochemical fluo-

rescence techniques 10, fol lowed by immunocyto-

chemical methods24, 27, p rovided the second approach

to determining terminal distr ibution. Al though quan-

tification is often absent in these histological studies

or is l imited to a subject ive rat ing scale, photomet r ic

analysis of histochemical observat ions has been used

by some investigators in recent years4, 9,25.

In this repor t we have combined histochemistry

and pho tomet ry with biochemical analyses of the

same brains. This was possible owing to the devel-

opment by de la Torre and Surgeon8 of a procedure

that does not require in vivo perfusion for visualizing

DA-conta in ing terminals. Using this technique, sec-

tions from unperfused brains were processed for his-

tochemical visualization of D A terminals and the in-

format ion obta ined was used to guide the dissection

of a l ternate sections for biochemical analysis. These

procedures were used to examine the impact of intra-

ventr icular 6 - H D A t rea tment on the dopaminergic

innervat ion of rat striatum. The high correla t ion be-

tween histochemical and biochemical da ta in the

presen,t results supports the feasibility of such an inte- grative approach.

When examined in the coronal plane, the distribution of DA- and TH-positive terminals appeared to be distributed homogeneously within normal striatum. This subjective impression was supported by photometric analysis of the histological material and by biochemical measurements of DA and DOPAC contents, and of TH activity. In contrast, heterogeneity was observed along the rostrocaudal axis; a significantly smaller number of DA terminals was present in the more caudal regions of striatum. These obser-

vations are consistent with those of Tassin and co- workers 29, who examined the distribution of DA and

of high affinity DA uptake sites in small punches of striatum.

Intraventricular administration of 6-HDA ap-

peared to produce widespread loss of DA terminals throughout striatum. The effects were not distributed

homogeneously, however. Visual inspection of the histochemical material suggested that extensive loss of DA-positive terminals had occurred in the medial

portions of striatum, whereas progressively larger numbers of terminals remained present in more lateral areas. This was confirmed by photometric analysis of the histological samples and by biochemical assay of DA content.

We have observed previously that after intraventricular 6-HDA, gross behavioral dysfunctions such as akinesia and sensory neglect occur only with D A depletions in striatum of 95% or more. On the basis of that observation, we had concluded that normal striatal function could be maintained by 5% of the nigrostriatal dopaminergic neurons28,33. However, the

heterogeneous distribution of the DA terminals that remain after 6-HDA treatment, revealed by the present work, suggests that the number of DA-containing terminals that must be destroyed to precipitate a par- ticular dysfunction can only be determined by an examination of the striatal region involved in mediating that function. Thus, the true critical percentage may be significantly different from our previous estimate.

Partial recovery of the dopaminergic innervation

of striatum appeared to occur during the several months after 6-HDA treatment. Evidence for this change was obtained by histochemical analyses and was supported subsequently by biochemical measurements. It is not possible from these studies to de-

17

termine whether this additional innervation is part of

an active response to the injury or a normal devel- opmental process. In either case, the growth was not associated with an alteration in the topographic relation between SN and striatum. Thus, the growth could not be explained in terms of sprouting from the lateral portions of striatum into the extensively denervated medial area.

Although the 6-HDA treatment produced marked decreases both in DOPAC content and in TH activity, these latter measures were always less affected by the lesion than were DA levels. We have sug-

gested previously that such relative increases in DOPAC content and in TH activity are an indication of increased synthesis and release of DA from residual dopaminergic terminals, and represent an adaptive compensatory response to the extensive destruction of DA-containing neurons 34. However, two distinc- tions may be drawn between the present biochemical results and those that we have observed previously. First, there was a clear medial-to-lateral heterogeneity in the ratios of DOPAC content and TH activity to DA levels, which was highest in areas where DA concentration was lowest. Second, the maximal relative increases in DOPAC content (1600% of control) and in TH activity (500% of control) that were observed in the present study were much greater than those re- ported previously (an average of 300%32). These

large effects, revealed by microdissection of striatum and regional analysis of extensively denervated areas, had been obscured in assays of whole striatum.

The observations of heterogeneity in the ratios DOPAC/DA and TH/DA are reminiscent of previous findings from this laboratory that after the administration of 6-HDA along the medial forebrain bundle, the ratio of TH activity to norepinephrine level was elevated in hippocampus but not in cerebellum, where the projections from locus coeruleus remained intact 12. Taken together with the present results, such findings suggest that separate catecholamine-containing neurons from a given cluster of cell bodies can function rather independently of one an- other.

In conclusion, an examination of monoaminergic systems that combines biochemical and histological analyses of the same brains appears to be feasible. Using this approach we have observed a heterogeneous impact of 6-HDA on striatum that may have im-

18

portant implications for interpreting the relationship

between the size and location of a brain lesion and

the induced behavioral dysfunction. Moreover, we

have documented regional differences in the extent

of the compensatory changes that occur in response

to the injury that suggest considerable local control

over these events.

ACKNOWLEDGEMENTS

We thank Drs. Christine Gall, Nick Brecha, Har-

vey Karten, Robert Moore and Julia Bassett ,for as-

sistance in establishing the histochemical proce-

dures, Dr. Tong Joh for providing the TH antibody,

the Mellon Institute Graphics Depar tment for prepa-

ration of the figures, and Kendall Stanley for prepa-

ration of the manuscript. This research was sup-

ported in part by U.S.P.H.S. grants MH00058,

MH000343, MH30915, and NS19608. Preliminary

reports of portions of this work were presented at the

Twelfth and Four teenth Annua l Meetings of the So-

ciety for Neuroscience 2°'2~.

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