Proc. Natl. Acad. Sci. USAVol. 81, pp. 1599-1603, March 1984Neurobiology

Autoradiographic visualization of angiotensin-converting enzyme inrat brain with [3H]captopril: Localization to a striatonigral pathway

(hypothalamus/circumventricular organs/dipeptidylcarboxypeptidase/ibotenic acid/colchicine)

STEPHEN M. STRITTMATTER, MATHEW M. S. Lo, JONATHAN A. JAVITCH, AND SOLOMON H. SNYDERDepartments of Neuroscience, Pharmacology and Experimental Therapeutics, Psychiatry and Behavioral Sciences, Johns Hopkins University School ofMedicine, 725 North Wolfe Street, Baltimore, Maryland 21205

Contributed by Solomon H. Snyder, November 29, 1983

ABSTRACT We have visualized angiotensin-convertingenzyme (ACE; dipeptidyl carboxypeptidase, peptidylpeptidehydrolase, EC 3.4.15.1) in rat brain by in vitro [3H~captoprilautoradiography. [3H]Captopril binding to brain slices dis-plays a high affinity (Kd = 1.8 x 10-9 M) and a pharmacologi-cal profile similar to that of ACE activity. Very high densitiesof [ H]captopril binding were found in the choroid plexus andthe subfornical organ. High densities were present in the cau-date putamen and substantia nigra, zona reticulata. Moderatelevels were found in the entopeduncular nucleus, globus palli-dus, and median eminence of the hypothalamus. Lower levelswere detectable in the supraoptic and paraventricular nuclei ofthe hypothalamus, the medial habenula, the median preopticarea, and the locus coeruleus. Injection of ibotenic acid or col-chicine into the caudate putamen decreased [3Hlcaptopril-as-sociated autoradiographic grains by 85% in the ipsilateral cau-date putamen and by >50% in the ipsilateral substantia nigra.Thus, ACE in the substantia nigra is located on presynapticterminals of axons originating from the caudate putamen, andACE in the caudate putamen is situated in neuronal perikaryaor at the terminals of striatal interneurons. The lack of effectof similar injections into the substantia nigra confirmed thatthe caudate putamen injections did not cause trans-synapticchanges. The presence of [3lH]captopril binding is consistentwith an ACE-mediated production of angiotensin II in somebrain regions. Although [31H]captopril autoradiography re-veals ACE in a striatonigral pathway, there is no evidence forangiotensin II involvement in such a neuronal pathway.

Angiotensin II (A-IT) is an octapeptide that increases bloodpressure peripherally by direct vasoconstriction and stimu-lates aldosterone release and, hence, salt reabsorption. Thecentral actions of A-IT include stimulation of drinking, in-creased salt appetite, increase of blood pressure, and releaseof several pituitary hormones (1). A-IT immunoreactivity (2,3) and A-TI receptor binding (4) have been identified in thecentral nervous system.

Angiotensin-converting enzyme (ACE; dipeptidyl car-boxypeptidase, peptidylpeptide hydrolase, EC 3.4.15.1) isthe dipeptidylcarboxypeptidase that produces circulating A-II by removing histidylleucine from angiotensin I. Captoprilis an extremely potent and selective ACE inhibitor that ishighly effective in treating hypertension (5). Recently, wedescribed the binding of [3H]captopril to ACE in membranefractions of the brain and in various peripheral tissues (6). Inthe present study, we have visualized ACE in the brain byautoradiographic analysis of [3H]captopril binding and com-pared its distribution to that of endogenous A-TI and A-IIreceptors.

MATERIALS AND METHODS[Prolyl-3,4-3H]-S-acetylcaptopril (New England Nuclear; 50Ci/mmol; 1 Ci = 37 GBq) was converted to [3H~captopril bytreatment with 0.1 M NaOH for 20 min at 23°C as described(6). Male Sprague-Dawley rats (150-200 g) were anesthe-tized with pentobarbital and perfused via the left ventricle ofthe heart with 0.9% NaCl/50 mM sodium phosphate, pH 7.5,and then with 50 mM sodium phosphate/0.3 M sucrose.Brains were removed, embedded in brain paste, and rapidlyfrozen at -70°C on chucks. Sections (8 ,um) were cut at-15°C and thaw-mounted on gelatin-coated slides. Theslides were dessicated and stored at -20°C. For autoradio-graphic studies, sections were incubated at 4°C for 5 min in50 mM Tris HCl, pH 7.9 (4°C)/100 mM NaCl/2 mg of bovineserum albumin per ml (Sigma, RIA grade) and then incubat-ed for 40 min at 4°C in the same buffer with [3H]captopril(standard concentration, 3 nM) and any inhibitors. Nonspe-cific binding was determined in the presence of 1 ,M capto-pril. After two consecutive 1-min washes in the same buffer,the slides were dipped in water and immediately dried undera stream of cold air. Autoradiograms were generated by ex-posing LKB Ultrofilm to the slides for 12 days at 4°C (7) orby apposition of emulsion-coated coverslips for 14 days at4°C (8). Tissue was stained after autoradiography with 0.1%toluidine blue. Density of autoradiograms on Ultrofilm wasquantified by microdensitometry and converted to fmol of[3H]captopril bound per mg of protein (7).Saturation analysis of binding used 0.22, 0.67, 2, 6, and 18

nM [3H]captopril. The highest level of binding in the serialsections of caudate putamen was quantified as describedabove. Total and nonspecific binding for each concentrationwere averaged from two sections from each of two brainsthat varied by less than 15%.For lesion studies, 4 ,ug of colchicine (Sigma), 15 ,ug of

ibotenic acid (Regis, Morton Grove, IL), or 8 ug of 6-hy-droxydopamine hydrobromide (Sigma) in 2 ,ul of 0.9% NaClwere injected over 1 min into the center of the left caudateputamen or the left substantia nigra using stereotaxic coordi-nates measured from the interaural line. The location of theneedle tip in an age-matched rat was confirmed by dissectionafter the injection of a dye. Coronal sections at the level ofthe caudate putamen and of the substantia nigra were ob-tained as described above either 7 days or 14 days after theinjections.For inhibition studies, caudate putamen sections were in-

cubated with 3 nM [3H]captopril and inhibitor concentra-tions that varied by factors of 10. The highest density ofbinding in the caudate putamen was determined by autoradi-ography and microdensitometry. Concentrations of inhibi-tors that produced 50% inhibition were determined graphi-

Abbreviations: ACE, angiotensin-converting enzyme; A-TI, angio-tensin II.

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cally and Ki values were calculated assuming competitive in-hibition and a Kd of 1.8 x 10-9 M for [3H]captopril. Theresults for each inhibition concentration were averages oftwo sections from each of two brains that varied by <20%.

RESULTS

Biochemical Properties of [3H]Captopril Binding to Slices ofthe Corpus Striatum. To ensure that [3H]captopril labelsACE in brain slices, we evaluated the pharmacological prop-erties of [3H]captopril binding to slices of rat corpus striatumby microdensitometric analysis of autoradiograms. As weobserved previously in homogenates of brain tissue (6),[3H]captopril binds saturably and with high affinity to stria-tal slices. Total binding with 3 nM [3H]captopril is about 100times greater than nonspecific binding measured in the pres-ence of 1 AM captopril (Fig. 1C). Specific binding, the differ-ence between total and nonspecific binding, begins to pla-teau at about 6 nM and reaches half-maximal values at about2 nM. Scatchard analysis reveals a single component of bind-ing (correlation coefficient of 0.99) with a dissociation con-stant (Kd) of 1.8 x 10-9 M and a maximal number of bindingsites (Bmax) of 1500 fmol per mg of protein. These valuesagree closely with those obtained in homogenate studies (6)and with the known potency of captopril as an inhibitor ofACE catalytic activity (5).The specificity of [3H]captopril binding was examined by

A

FIG. 1. [3H]Captopril binding to rat brain. (A) Toluidine bluestaining; (B) [3H]captopril autoradiography; (C) [3H]captopril auto-radiography in the presence of 1 ,uM captopril. Note the intense la-beling of the choroid plexus (Ch) in B. The caudate putamen (Ca)and the substantia nigra (SN) are also visualized by autoradiogra-phy. The streaks of decreased grain density in the caudate putamencorrespond to the location of white matter tracts. The unmarkedarrows indicate the band that appears to connect the caudate puta-men and substantia nigra.

evaluating the potency of various ACE inhibitors to decreasebinding levels in striatal brain slices. MK-422, theactive diacid of enalapril, and N-(l(S)-carboxy-3-phenyl-propyl)-L-lysyl-L-proline, the lysyl analogue of MK-422,are potent ACE inhibitors (9) and potent in decreasing[3H]captopril binding with Ki values of 7.5 and 15 x 10-9 M,respectively. Teprotide, a somewhat weaker ACE inhibitordisplays a Ki value of 235 x 10-9 M. By contrast, thiorphan,an extremely potent inhibitor of enkephalinase A (10), an en-zyme also called endopeptidase 24.11 (EC 3.4.24.11) (11), isquite weak at [3H]captopril binding sites, failing to give 50%inhibition in concentrations as high as 100 ,uM. The chelatingagent EDTA, which inhibits ACE activity, also decreases[3H]captopril binding to negligible levels at 1 mM. The selec-tivity of these agents and the similarity of their potencies at[3H]captopril binding sites to their influences on ACE cata-lytic activity indicates that the sites visualized in the corpusstriatum by [3H]captopril autoradiography represent ACE.

Autoradiographic Localization of [ HICaptopril Sites in RatBrain. Table 1 summarizes the distribution of specific[3H]captopril binding obtained by autoradiography and mi-crodensitometry. The densest localization of [ H]captoprilbinding sites occurs in the choroid plexus throughout thebrain (Figs. 1 and 2A). The choroid plexus is labeled in thelateral ventricles, the third ventricle, and the fourth ventri-cle.Almost as densely labeled as the choroid plexus is the sub-

Table 1. Distribution of ACE, A-II receptors, and A-II[3H]Captopril

Region binding* ACEt A-IIRt A-h1

Choroid plexus +++++ +++++ 0 0Subfornical organ +++++ +++++ ++++ 0Caudate putamen ++++ ++++ 0 +Substantia nigra,

zona reticulata ++++ +++ 0 +Globus pallidus +++ +++ 0 0Entopeduncular n. + + + ND 0 0HypothalamusMedian eminence +++ + + + + ++++Supraoptic n. ++ + ++ PerikaryaParaventricular n.,

magnocellular + + ++++ PerikaryaSuprachiasmatic

n. 0 0 +++ +Periventricular n. 0 0 ++++ +

Medial habenula + +++ ++ 0Locus coeruleus + ++ ++ ++Median preoptic

area + 0 ++ ++Spinal cord 0 ND 0 ++++Hippocampus 0(+)$ 0 0 +Cerebellum 0(+)$ + 0 0Neocortex 0 0 0 +

ND, not determined; n, nucleus.*Data from microdensitometry after autoradiography with 3 nM[3H]captopril. + + + + +, >1501 fmol per mg of protein; + + + +,1500-701 fmol per mg of protein; + + +, 700-301 fmol per mg ofprotein; + +, 300-151 fmol per mg of protein; +, 150-51 fmol permg of protein; 0, < 50 fmol per mg of protein.

tData from refs. 12 and 13. + + + + +, >200 pmol per ,g of proteinper hr; + + + +, 200-101 pmol per ug of protein per hr; + + +, 101-50 pmol per ,g of protein per hr; + +, 50-21 pmol per ug of proteinper hr; +, 20-11 pmol per ,ug of protein per hr; 0, 10 pmol per ,g ofprotein per hr.*A-I1 receptors, data from ref. 4. + + + +, Very high; + + +, high;+ +, moderate to high; +, low to moderate; 0, low or very low.§Data from refs. 2 and 3. + + + +, Most intense or high; + +, moder-ate or low-moderate; +, widely scattered or scattered; 0, none.1A low level of labeling was observed in a few sections.

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FIG. 2. [3H]Captopril autoradiography of the choroid plexus, subfornical organ, entopeduncular nucleus, median eminence, and supraopticnucleus of the hypothalamus. (A) The subfornical organ (SFO) and the adjacent choroid plexus (Ch) are intensely labeled in this dark-field viewobtained using an emulsion-coated coverslip. The surrounding tissue exhibits few silver grains. (x 80.) (B) [3H]Captopril labeling of the entope-duncular nucleus (EP) and median eminence (ME) are shown in this low-power coronal section. (x 2.) Intense staining of the choroid plexus andmoderate staining of the tail of the caudate putamen are also visible. (C) Bright field reveals toluidine blue staining. The supraoptic nucleus ofthe hypothalamus (SO) is apparent as are several blood vessels, indicated with arrows. (x80.) (D) Observation of the same field as in Fig. 3Cunder dark-field conditions illustrates the presence of silver grains produced -by [3H]captopril in an emulsion-coated coverslip. The supraopticnucleus of the hypothalamus (SO) and blood vessels seen in Fig. 3C are labeled by [3H]captopril.

fornical organ (Fig. 2A). [3H]Captopril binding is present inhigh density throughout the anatomical extent of the subfor-nical organ, adjacent to the equally dense labeling of the cho-roid plexus. The area postrema was not examined.

Outside the subfornical organ, the neuronal areas with thegreatest levels of [3H]captopril binding are the corpus stria-tum and substantia nigra (Table 1; Fig. 1). Extremely dense[3H]captopril-associated silver grains are apparent through-out the caudate putamen. Streaks of grain-free zones reflectwhite matter tracks passing through the caudate putamen.There is some variation in grain density in the caudate puta-men with highest levels being anterior and lateral. Gray mat-ter regions of the corpus striatum examined under high mag-nification show a-homogeneous distribution of silver grains,as opposed to the localized binding to blood vessels ob-served in Fig. 2 C and D. A lesser degree of [3H]captoprilbinding occurs in the globus pallidus, about one-half that ofthe highest levels in the caudate putamen.

Autoradiographic grains can be observed in a band thatappears to connect the caudate putamen and the zona reticu-lata of the substantia nigra (Fig. 1). An enlargement of thisband is detectable in the area of the entopeduncular nucleus(Fig. 2B).Within the substantia nigra, grains occur throughout the

zona reticulata with negligible grain density in the zona com-pacta. At some levels of the substantia nigra, variations ingrain density are apparent within the zona reticulata (Fig. 3).A small zone of increased grain density occurs in the mostdorsal portion of the zona reticulata. Examination at highmagnification reveals a homogenous distribution of silvergrains in the substantia nigra, zona reticulata, as in the graymatter of the corpus striatum.

In some brain sections [3H]captopril labels blood vessels(Fig. 2 C and D). It is not possible to determine whether thegrains are overlying endothelium or muscle layer. Only a mi-

nority of all blood vessel profiles observed are labeled by[3H]captopril.

Several other brain areas display substantial [3H]capto-pril-associated grains, although with levels lower than theareas mentioned above. Within the hypothalamus, grains arehighly localized to the median eminence (Fig. 2B), the su-praoptic nucleus (Fig. 2 C and D), and the paraventricularnucleus (not shown). Negligible levels are present elsewherein the hypothalamus.

In the thalamic area, the highest grain densities occur inthe medial habenula with undetectable levels in the lateralhabenula. No portion of the thalamus itself possesses detect-able concentrations of grains.The median preoptic area and the locus coeruleus have

grain densities just above the level of detection. Ependymalcells lining the ventricles exhibit low to moderate grain den-sity.The selectivity of [3H]captopril binding localization is ap-

parent in the many parts of the central nervous systems thatdisplay few if any grains. Negligible levels of binding occurthroughout the cerebral cortex, cerebellum, most of thebrain stem, spinal cord, and hipjpocampus (Table 1).

Effects of Brain Lesions on ['HICaptopril Binding in theCaudate Putamen and Substantia Nigra. To explore the pos-sibility of a descending striatonigral localization of [3H]cap-topril sites, we injected colchicine or ibotenic acid unilateral-ly into the caudate putamen (Fig. 4). Colchicine destroyscells by interfering with microtubular function. Ibotenic acidselectively destroys neuronal cells intrinsic to the site of in-jection (14). By day 7 after these injections, a marked deple-tion of [3H]captopril-associated grains is readily apparent inthe caudate putamen at the site of injection (Fig. 4; Table 2).Grain density in the ipsilateral substantia nigra is markedlydepleted 14 days after the injections into the corpus striatum,while only partial depletion is apparent after 7 days. The de-

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FIG. 3. The effect of substantia nigra injections on [3Hlcaptoprilautoradiography. Ibotenic acid (A and B), colchicine (C and D), or 6-hydroxydopamine (E and F) was injected into the left substantia ni-gra. After 14 days, autoradiography was done. Note the equality oflabeling between the left, lesioned side and the right, control side inthe caudate putamen sections (A, C, and E) and in the substantianigra sections (B, D, and F). The variation in the density of labelingwithin one substantia nigra, which can be observed in B, D, and F ispresent whether or not an injection has been made into the left sub-stantia nigra. The intensely labeled structure in A, C, and E is thechoroid plexus.

pletion of binding throughout the substantia nigra is at leastas great as that shown in Table 2, which reports the maxi-mum density found at any one point in the substantia nigra as

opposed to the average density. [3H]Captopril-associatedgrains are not affected in the choroid plexus closely adjacent

I

FIG. 4. The effect of caudate putamen lesions on [3H]captoprilautoradiography. Ibotenic acid (A and B) or colchicine (C and D)were injected into the caudate putamen. After 14 days, autoradiog-raphy was carried out. The arrows indicate the location of the le-sion. Note the decrease in [3Hlcaptopril-associated grains on theleft, lesioned side in the caudate putamen in A and C and in thesubstantia nigra in B and D. The right, control side is unaffected.The choroid plexus is the intensely labeled structure inA and C; it isunaffected by the lesions. There is a degeneration in the size of thecaudate putamen and an enlargement of the lateral ventricle in A andC on the lesioned side.

Table 2. Effect of lesions on [3H]captopril binding

[3H]Captopril binding, fmolper mg of protein

Caudate putamen Substantia nigraControl Lesion Control Lesion

Drug Day side side side side

Caudate putamenlesions

Ibotenic acid 7 706 85 875 68014 695 128 646 375

Colchicine 7 638 85 841 53614 833 85 782 306

Substantia nigralesions

Ibotenic acid 7 926 859 604 75814 808 800 629 646

Colchicine 7 850 850 790 79914 800 808 620 629

6-OH-Dopamine 14 960 1054 791 706

Autoradiography was carried out with 3 nM [3H]captopril and fol-lowed by microdensitometry. The values are the average from twosections of each of two rats that varied by <20%. From most sec-tions, the highest density observed in the indicated brain region isreported. However, in the caudate putamen sections from brainsinjected in the caudate putamen, binding was measured in the centerof the obvious lesions (Fig. 4) and in a corresponding position on thecontrol side.

to the injected caudate putamen, thus indicating the selectiv-ity of the colchicine and ibotenic acid injections. Injectionsof saline alone have no effect on ACE levels after 7 days(data not shown).We also carried out unilateral injections into the substantia

nigra of colchicine, ibotenic acid, or 6-hydroxydopamine. 6-Hydroxydopamine injections in the substantia nigra selec-tively destroy dopamine-containing cells (15). None of thesethree treatments alters [3H]captopril binding levels in eitherthe substantia nigra or the caudate putamen (Fig. 3; Table 2).The effectiveness of these nigral lesions to destroy the ni-grostriatal dopamine system was tested in adjacent sectionsfrom the same brains. All three lesions caused a unilateraldepletion of [3H]mazindol binding in the ipsilateral caudateputamen (unpublished observations). [3H]Mazindol labelsneuronal dopamine uptake sites (16).There appears to be some somatotopic relationship of the

[3H]captopril binding sites in the caudate putamen and sub-stantia nigra (Fig. 4). Thus, injections that cause a loss of[3H]captopril binding in the more medial portions of the cau-date putamen are associated with a greater loss of [3H]capto-pril binding in more medial portions of the substantia nigra.

DISCUSSION[3H]Captopril autoradiography reveals ACE in specific brainregions (Table 1). The results agree with localizations ob-tained in microdissection studies measuring ACE catalyticactivity (Table 1) (12, 13). However, autoradiographic analy-sis has permitted a more complete localization of the en-zyme. Thus, it has been possible to visualize ACE in theentopeduncular nucleus, in a band apparently connecting thecorpus striatum and substantia nigra, and in the gray matterof the caudate putamen. Immunohistochemical techniqueshave also been used to localize ACE in the brain. Antibodydirected against rabbit lung ACE detected ACE in the cho-roid plexus, the subfornical organ, and scattered blood ves-sels of the rat brain (3, 17, 18). This antibody does not detectthe prominent striatonigral ACE system. Antibody to humankidney ACE did stain the substantia nigra and globus palli-dus but revealed little ACE in the caudate putamen of rat or

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human brain (19). Other regions, such as the neocortex, hip-pocampus, and hypothalamus, which show much lower lev-els of ACE by [3H]captopril autoradiography or by measure-ment of catalytic activity, were stained by this antibody.

In some regions in which [3H]captopril reveals the pres-ence of ACE, the role of the enzyme appears to be the pro-duction of A-II. However, the caudate putamen, the sub-stantia nigra, zona reticulata, the entopeduncular nucleus,and the globus pallidus exhibit high concentrations of[3H]captopril binding with no evidence for A-TI receptors orendogenous A-IT. Striatal lesions carried out using ibotenicacid or colchicine show that ACE in the substantia nigra ispresent on presynaptic terminals of axons originating in theipsilateral caudate putamen and that the ACE of the caudateputamen is present in cell bodies projecting to the substantianigra or is associated with striatal interneurons. Our autora-diographic studies agree with earlier studies showing thatstriatal ibotenate lesions deplete ACE catalytic activity inthe caudate putamen (20, 21) and in the substantia nigra (22).Moreover, in Huntington's disease, the massive destructionof the corpus striatum is associated with a 75% depletion ofACE activity in the substantia nigra (22). The absence of ef-fects on ACE after substantia nigra lesions shows that thenigral changes observed with striatal lesions are not due totrans-synaptic effects.Because ACE is a dipeptidylcarboxypeptidase with wide

substrate specificity (23), the enzyme may act on a substrateother than angiotensin I to either convert an inactive precur-sor into a biologically active neuropeptide or to destroysome neuropeptide. The finding that ACE is contained in adescending striatonigral pathway indicates that such a hypo-thetical neuropeptide may also occur in this pathway. Sub-stance P immunoreactive projections exist from the striatumto the zona reticulata of the substantia nigra, the globus palli-dus, and the entopeduncular nucleus (24). The fact that sub-stance P contains a carboxyl-terminal amide had suggestedthat it might not be a substrate for ACE. However, we haverecently shown that substance P is readily cleaved by pureACE (unpublished results). Of course, the striatonigral en-dogenous substrate of ACE may be a heretofore unknownneuropeptide.Whereas ACE in the striatonigral system appears unrelat-

ed to endogenous angiotensin, the ACE activity of otherbrain regions may be involved with A-TI production. ACE inthe choroid plexus may regulate the A-TI content of the ven-tricles (25) from which the octapeptide can exert its dipso-genic and hypertensive effects on the circumventricular or-gans. The subfornical organ and the median preoptic area,where A-IT regulates drinking behavior (1), contain A-TI re-ceptors and [3H]captopril binding, suggesting the formationof A-TI at these sites. A-TI may regulate antidiuretic hormonedisposition in the median eminence, the supraoptic nucleus,and the paraventricular nucleus of the hypothalamus wherethe peptides have been colocalized to the same neurons (26).The medial habenula and locus coeruleus contain both[3H]captopril binding and A-TI receptors. Several areas con-tain little ACE but significant levels of A-II receptors-i.e.,the lateral olfactory tract and its nuclei, the organum vascu-losum of the lamina terminalis, the subthalamic nucleus, andthe suprachiasmatic and periventricular nuclei of the hypo-thalamus (4). Brain regions with high A-TI levels but no ACEinclude the central nucleus of the amygdala, the bed nucleusof the stria terminalis, and parts of the spinal cord (2, 3).The use of [3H]captopril binding to characterize ACE in

homogenates (6) and in brain slices of autoradiographic stud-ies provides a powerful extension of receptor-binding tech-niques to membrane-associated enzymes. [3H]Captoprilautoradiography can also be used to localize ACE in otherparts of the body. The two tissues with highest ACE activity

in the body are the lungs and testes. Recently, we have visu-alized [3H]captopril binding sites associated with ACE in themale reproductive system of the rat (unpublished observa-tions). ACE is highly concentrated in seminiferous tubules ofthe testes and in the epithelial surface and lumen of the epi-didymis.

This work was supported by U.S. Public Health Service GrantsMH-18501, DA-00266, NS-16375, and RSA Award DA-00074 toS.H.S., Training Grant GM-07309 to S.M.S. and J.A.J., and a grantof the McKnight Foundation.

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