Journal of Neuroseience Methods, 6 (1982) 59-73 59 Elsevier Biomedical Press

Quantitative receptor autoradiography using [3H]Ultrofilm: application to multiple

benzodiazepine receptors

James R. Unnerstall, Debra L. Niehoff, Michael J. Kuhar and J o s e M. P a l a c i o s 1

Departments of Neuroscience, Pharmacoloy3' and Experimental Therapeutics, and P.~Tchiatrv and the Behacioral Sciences, The Johns Hopkins Unit, ersi(v School of Medicine, Baltimore, MD 21205 ( U. S. A.

(Received September 25 th, 1981 ) (Revised version received November 16th, 1981 )

(Accepted December 18th, 1981 )

Key words: autoradiography - quantitation of drug and neurotransmitter receptors - radiohistochemistry - [3H]Ultrofilm - microdens i tomet ry- benzodiazepine receptors-- triazolopyridazines-- beta-carbolines

Radiohistochemical methods utilized to label drug and neurotransmitter receptors in vitro at the light microscopic level are quantitative. In this report, the response of tritium-sensitive sheet film ([~H]Ultro- film) was characterized under conditions used for the light microscopic localization of neurotransmitter receptors by in vitro autoradiographic techniques. Radioactive standards containing varying concentra- tions of tritium were prepared from brain tissue and exposed to [3H]Uhrofilm for varying lengths of time, and the response of the film was measured by microdensitometry. A In optical density versus In radioactivity plot provided a useful linear relationship. Relationships established between radioactivity concentrations, exposure times and optical density were utilized to establish appropriate exposure conditions for several [3H]ligands in brain sections. The accuracy and pharmacological relevance of these methods were tested by studying the regional distribution of multiple benzodiazepine (BZ) receptors, and by analyzing the inhibitory potency of the triazolopyridazine, CL218,872, and methyl-beta-carboline- carboxylate, two agents which discriminate between type 1 and type 2 BZ receptors. The results obtained compared favorably with results previously obtained in tissue homogenates by biochemical methods.

Overall, these results have practical implications. For example, quantitative autoradiography can be used to generateaccurate kinetic and pharmacological displacement curves for small tissue areas. Also. since the film response curve is not linear with exposure (radioactivity× time), autoradiographs of the same sections can have relatively different density patterns depending on the time of exposure.

Introduction

D r u g a n d n e u r o t r a n s m i t t e r r e c e p t o r s c a n b e l o c a l i z e d in b r a i n b y l i g h t m i c r o -

s c o p i c a u t o r a d i o g r a p h y f o l l o w i n g in v i v o a n d in v i t r o l a b e l i n g o f r e c e p t o r s ( Y o u n g et

] Present address: Sandoz Ltd., Preclinical Research, 360/604, CH-4002 Basel, Switzerland.

60

al., 1980). Generally. the procedures have involved either the mounting of thin sections of frozen, labeled tissue to slides coated with nuclear emulsion or thc apposition of emulsion-coated coverslips to labeled, mounted tissue (Kuhar, 1978a, b: Young and Kuhar, 1979a). It has been shown that these procedures call bc quantitative (Unnerstall et al., 1981).

Recently, the use of tritium-sensitive sheet film for receptor localization has been described. The advantages of the film have been discussed and include the ability to readily quantitate the autoradiograms by microdensitometry (Palacios et al., 1981: Penney et al., 1981: Rainbow et al., 1981). Proper utilization of the film requires an understanding of the response of the film under the conditions used for receptor localization. Accordingly, using radioactive standards prepared from brain tissue, we have studied the response characteristics of the film and applied this to the quantitative analysis of multiple benzodiazepine (BZ) receptors at the light micro- scopic level.

Materials and Methods

Preparation of standards for autoradiography Radioactive standards were prepared from brain tissue which was ground to a

paste. Brain paste provides reasonable standards because it has the same cutting. mounting and density characteristics as the brain tissue sections being studied.

Known amounts of increasing concentrations of a non-volatile radioactive com- pound ([~H]ornithine, 33.6 Ci /mmol , 1 mCi /ml , New England Nuclear, NEN, Boston, MA) were thoroughly mixed with brain tissue ground to a paste. Pressure sensitive tape was used to form wells around brass microtome chucks (1 cm diame- ter). These wells were filled with tissue containing the varying concentrations of radioactivity. The tissue was then degassed in a vacuum dessicator and was stirred several times during this procedure with wooden applicators to facilitate the removal of air bubbles trapped in the paste. The stirring was continued until the radioactivity was uniformly distributed throughout the tissue and the tissue itself reached a thick, viscous consistency.

The blocks of tissue on brass chucks containing the various concentrations of radioactivity were frozen over dry-ice and placed in a Harris cryostat microtome (Harris Manufacturing, North Billerica, MA) for sectioning. Sections, 6 / ,m in thickness, were routinely taken from each of the chucks and thaw-mounted onto acid cleaned, subbed (chrome alum and gelatin) microscope slides. At intervals during the sectioning process, sections were taken from the knife and placed in cooled, tared scintillation vials. The vials were brought back to room temperature, the weight of the tissue was determined, the tissue was solubilized and radioactivity was de- termined. Counting efficiency (approximately 45%) was determined with the use of [3H]toluene as an internal standard. We determined that this overall procedure resulted in approximately a 65-75% loss of water from the tissue. Because moisture was lost from the tissue during the process, protein concentrations in the tissue sections from standards and whole brain were determined by the method of Lowry

61

et al. (195 l) using crystalline bovine serum albumin as standard. Protein concentra- tion in both standards and whole brain sections were determined to be 0.3 mg protein per 1.0 mg of tissue section which was partially dessicated as described. For the characterization of the response of the film, radioactivity concentrations were expressed per mg tissue dry weight, i.e. with about a 70% loss of water from the sections. In subsequent analyses of binding data, these concentrations were con- verted to d i s in tegra t ions /min /mg protein.

Preparation of autoradiograms Slides were affixed to mounting board with double-sided Scotch brand tape.

[)H]Ultrofilm (LKB Industries, Rockville, MD) was apposed to the slides in the dark and stored in 10)< 12 in. Wolf Slimline X-ray film cassettes at 4°C. For studying the response of the film to the standards, exposure times were varied from 1 to 6 weeks. After exposure, the film was removed from the cassettes in the dark and developed. The film was developed at 20°C for 5min with undiluted D19 Kodak developer. After the stop bath rinse, the film was immersed in Kodak rapid fixer for film at 20°C for 5 min. The film was rinsed in running water for 20 rain at 20°C, then rinsed in Photo-Flo solution and hung to dry.

Optical densities (ODs) were determined utilizing a G a m m a Scientific DR-2H microdensitometer system (Gamma Scientific, San Diego, CA) at 100 p,m resolution, and the relationship between OD and concentration of radioactivity for various exposure times was determined. In subsequent experiments, autoradiograms from labeled tissue sections were generated in the same way, and standards were exposed simultaneously with the tissue being studied. Conversion of ODs to molar quantities is described below.

Cholinergic muscarinic receptor autoradiography Six micron coronal sections from rat CNS were incubated with [3H]quinuclidinyl

benzilate ([3H]QNB) (32 Ci//mmol, Amersham, Arlington Heights, IL) to label muscarinic cholinergic receptors (Wamsley et al., 1980a). Serial sections were in- cubated for 2h at room temperature in phosphate-buffered saline with l nM [~H]QNB in the absence or presence of 10 ~'M atropine to assess non-specific binding. Sections were given 2-5 min washes in cold buffer. [3H]Uhrofilm was exposed to the dried labeled sections.

Quantitatit,e analysis of multiple bencodiazepine receptors [ ~ H]Diazepam and [ 3 H]flunitrazepam ([ 3 H]FL U) uniformly label BZ receptors in

brain homogenates (Mohler and Okada, 1977; Squires and Braestrup. 1977; see Tallman et al., 1980 for additional references) and tissue sections (Young and Kuhar, 1979b: Young and Kuhar, 1980; Unnerstall et al., 1981). However. two pharmacologically, biochemically, functionally and anatomically distinct receptor types have been described (Squires et al., 1979; Klepner et al., 1979: Lippa et al.. 1979a, b, 1980; Sieghart and Karobath, 1980; Williams et al.. 1980: Young et al., 1981). A novel series of triazolopyridazine (TPZ) drugs can distinguish these two types of receptors. The high affinity sites for TPZ were designated as type 1

62

receptors, while those with low affinity for TPZ were designated as type 2 receptors (Lippa et al., 1979a, b, 1980). Another class of compounds, beta-carboline-carbox'~- late esters (B-CC), have been described which also apparently distinguish between these two types of BZ receptors (Braestrup et al., 1980; Nielsen and Braestrup, 1980: Braestrup et al., 1980a, b: O'Brien et al., 1981). These compounds have a higher affinity for the type 1 receptors than TPZ drugs, although the differences in affinitie~ of B-CCs for the two BZ sites is not as great as for the TPZs.

Two experiments were performed using drugs from these two classes of com- pounds to study the pharmacological relevance of [3H]FLU ,binding analyzed quantitatively by microdensitometry utilizing [3H]Ultrofilm, In the first experiment, the TPZ CL218,872 was utilized to study the anatomical distribution of type 1 and type 2 BZ receptors in the rat CNS. Sequential coronal CNS sections (6 #m) from 3 rats were incubated in 1 nM [3H]FLU (86,4 Ci /mmol , NEN) in the presence and absence of 200 nM of CL218-872. In the presence of CL218-872, it has been determined that the [3H]FLU bound to type 1 receptors will be preferentially displaced (Young et al., 1981: also see Table2). Blanks were determined in the presence of 1/~M clonazepam.

In a second series of experiments designed to study the kinetic parameters of TPZ and B-CC displacement of [3H]FLU, sagittal sections from 3 rats were incubated with 1 nM [3H]FLU in the presence of varying concentrations of CL218,872 (10 ~ to 10 ~ M) or methyl beta-carboline carboxylate (Me-beta-CC) (10 10 to 10 6 M), (Braestrup et al., 1981). [3H]Ultrofilm was exposed to these sections along with standards for 10 days as described above. Regional analysis of [3H]FLU binding was performed by microdensitometry, and OD was related to concentration of radioactivity in the various regions by use of the standard curve generated concom- itantly with the autoradiograms.

Molar quantities of ligand bound were determined by the following relationship:

mmol - OD A x ( D P M / m g protein) X ~(mm°l) × (1Ci) mg protein (OD)B (Ci)C (2.22 x 1012 DPM)

where factor A represents the OD over the region being studied, factor B represents the slope of the standard curve, and factor C is the specific activity of the ligandl Ligand concentrations per mg tissue dry weight were converted to concentrations per mg protein as described above.

Results and Discussion

Analysis and use of radioactive standards

Standards were prepared using brain tissue as described in Materials and Meth- ods. The concentration of radioactivity in the standards varied from 10 3 tO 10 6

D P M / m g tissue dry weight. Sections from these standards were exposed for varying amounts of time and the autoradiographic images were analyzed by microdensitome- try. An example of the autoradiographic image produced following 56 days of exposure is shown in Fig. 1.

63

Fig. 1. Autoradiographic image of a set of standards after 56-day exposure to [3H]Ultrofilm. Tissue sections were thaw-mounted on to subbed microscope slides as described in Materials and Methods. Each section contains a different amount of radioactivity per mg of dry tissue. A: 1.13× 105 DPM. B: 2.00X 104 DPM. C: 3.15X 104 DPM. D: 1.22)<106 DPM. E: 2.12x 103 DPM. F: 9.459<102 DPM. G: 7.45X102 DPM. H: 5.75×102 DPM. I: background. For receptor quantitation, these concentrations were converted into DPMs / m g protein as described in Materials and Methods, where 1 mg tissue -0.3 mg protein. The photo is a bright-field micrograph of the image on the Ultrofilm.

These and other data provided the basis for the [sH]Ultrofilm response curves (Fig. 2a, b) showing the relationship between OD and exposure. Exposure is a measure of the amount of energy which impinges upon the film for a given period of

iO0. r.:]

9 0 -

80-

70-

~ 60- z •

~, 50. , . . ~ 40-

30-

20 - " / "

10-

,& 2~o 3~o 4~o r~o ,~o ~o 8~o EXPOSURE (DPM x DAYS/MG TISSUE )II IO "$

120, b !//t-~----

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LOG EXPOSURE (DPM x DAYS IMG TISSUE)

Fig. 2. a: response curve (optical density as a function of exposure) of [3H]Ultrofihn to varying concentrations of tritium in tissue sections exposed under conditions identical to those used for quantitative autoradiography of neurotransmitter receptors. Each point represents the mean of 6-10 densitometry readings using the Gamma Scientific DR-2H system from autoradiograms generated from tissue sections containing 10 different concentrations of radioactivity (9.,15 × 10- 2 to 1.22 × 106 D P M / m g tissue dry weight) exposed for 6 different lengths of time (1-6 weeks). The plot shown is a hyperbolic

65

TABLE 1

ESTIMATED EXPOSURE TIMES FOR TISSUE SECTIONS LABELED WITH SEVERAL TRITI- ATED LIGANDS UNDER STANDARD AUTORADIOGRAPHIC CONDITIONS

cpm/sl ide refers to radioactivity specifically bound to two mounted 10 /*m coronal sections (approxi- mately 1 mg tissue dry weight) which was wiped from the slides with glass fiber filters and counted using standard technique at 45-50% counting efficiency following standardized incubation procedures (for example, see Young and Kuhar, 1979a and Wamsley et al., 1980a). Estimates of exposure time were based on biochemical determinations listed in the fourth column. It was further assumed, based on previous experience, thal radioactivity would be concentrated to approximately half the area of the tissue, rather than being uniformly distributed over the surface. An exposure value of 2.5 × l0 s DPM ~< days /mg tissue was used in these determinations, which corresponds to an optical density of approximately 4(1 units (see Fig. 2).

Ligand (receptor) Specific Ligand cpm/sl ide activity concentration (Ci/mmol) (nM)

Exposure time (days)

Estimated Actual

[ 3 H]quinuclidinyl benzilate (muscarinic cholinergic) 30-40 1 14000 16000 3- 4 5

[ 3 Hlflu nitrazepam (benzodiazepine) 80-90 1 4500- 6500 9 l0 10

[ ~ H]lysergic acid diethylamide (serotonin) 25-35 5 2500- 3000 20 25 3(I

[ 3 H]dihydromorphine

(opiate, p) 70-80 4 2000 2500 25- 30 35

[ 3 H] para-aminoclonidine (alpha-2 adrenergic) 45-55 1 750 1 200 48- 56 56

[3H]muscimol(GABA) 10 20 5 600 1000 60- 65 70

[3H]kainic acid (kainate) 2 6 15 100- 300 120 130 112

time. These results agree well with those described by Ehn and Larsson (1979). The relationship between OD and exposure is not linear and can be approximately described as a rectangular hyperbola (Fig. 2a). Other investigators using carbon-14 standards with X-ray film have used a cubic function to describe the relationship (Goochee et al., 1980). When OD is plotted as a function of the log of exposure, one generates the so-called characteristic curve of the film (Fig. 2b). Since the slope of

function where:

OD = exposure r 2 _ 0.57. (0.0047 >(exposure) + 4548.9 '

Optical density values greater than 100 are not shown in this graph; however, the determination of this function utilized all the data points shown in b. b: characteristic curve of [~ H]Uhrofilm. Optical density is plotted as a function of log exposure. The plot shown is a power function, where OD=0.0017 * exposure °~°'~, r 2 0.94. The dashed line represents the saturation limits of the film. See text for details.

66

!

Fig. 3. Autoradiographic images of muscarinic-cholinergic receptors in the rat forebrain following incubation with I nM [3H]QNB and exposure to [3H]Ultrofilm for (A) 5 days and (B) 21 days. In panel A, note the uniformly low grain densities over thalamic and hypothalamic regions, while optimal contrast has been obtained in the cortex (V, lamina V) and caudate-putamen (cp). In panel B, the cortex and caudate-putamen is now overexposed, while higher grain densities can be easily discriminated and quanti tated in thalamic regions such as the mediolateral (tml) and nucleus rhomboidus (rh). These photographic dark-field images were produced directly from the autoradiographic images generated with [3H]Ultrofim using a photographic enlarger under identical conditions and are thus negative images. Bright areas, then, correspond to regions of high receptor densities. The two autoradiographs were produced from the same section, and correspond to level A4380 ~ m of K/Snig and Klippel (1963). B a r = 1000 ~m.

67

the character is t ic curve as a funct ion of exposure gives a measure of pho tog raph ic contras t , this re la t ionship provides useful in fo rmat ion for op t imiz ing exposure

cond i t ions for au to rad iog raph ic exper iments . At O D s of less than 20 units, the change in s lope as a funct ion of exposure, i.e. contras t , is low. Thus, d i sc r imina t ion be tween dif ferent label ing in di f ferent b ra in regions is difficult . At ODs between 20 and 100 units, con t ras t is high, and it becomes relat ively easy to d i sc r imina te small d i f ferences in concen t ra t ions of radioact iv i ty . At ODs greater than 100 units, the f i lm rap id ly saturates, and no useful da t a can be ob ta ined . We have uti l ized this i n fo rma t ion to es t imate exposure t imes to p roduce op t imal cont ras t for au to rad io - g rams using previously s t andard ized incuba t ion condi t ions . This da ta is p resented in Tab le 1, a long with ac tual exposure t imes that we have found to be useful. In many s i tua t ions it seems useful to a im for an average O D of about 40-60 , which, in general , will avoid p rob lems of loss of cont ras t and subsequent errors in quan t i t a t ion at ei ther low or sa tura t ing opt ica l densit ies.

These d a t a indicate that it might be des i rable to expose labe led tissue sect ions for vary ing lengths of t ime in o rder to op t imize the pho tog raph ic contras t , and thus, increase the accuracy of quan t i t a t ion in regions of high or low receptor densi ty. F o r example , high densi t ies of muscar in ic chol inergic receptors are loca ted in the s t r i a tum and cerebra l cor tex ( K u h a r and Y a m a m u r a , 1976; Rot te r et al., 1979; Wa ms ley et al., 1980b; Y a m a m u r a and Snyder , 1974). When labeled with [3H]QNB, op t ima l O D s for these regions are achieved af ter 4 - 5 days of exposure. However , in o rde r to ob ta in accura te de te rmina t ions of receptor densi t ies in regions where there

5

4

c~

c~ 3

2 -

I

i /

6 8 I0 In standard radioactivity

Fig. 4. A ln-In plot of OD vs radioactivity (dpm/0.3 mg prot.) in standards. [3H]Ultrofilm was exposed to the standards for 10 days. Each point represents the average of 6-10 optical density readings taken from autoradiograms generated from triplicate standards exposed to the film at the same time as the tissue being studied (see Fig. 1). These curves are prepared for each film utilized for the quantitation of neurotransmitter receptors in brain sections, and the film is re-calibrated each time densitometry readings are taken from the film to control for day to day fluctuations in the performance of the densitometer. Dashed lines represent the standard error of estimate of x from y.

68

are lower concentrations of bound ligand, such as in the thalamus or medulla, il would be necessary to increase the exposure time to approximately 2-3 weeks. Under these conditions, ODs in the striatum or cortex would be near saturation (Fig. 3).

Fig. 4 shows a very useful plot for translating ODs to radioactivity and ultimately to molar quantities of ligand bound (see Materials and Methods). After a 10-day

Fig. 5. Bright-field photographic images of autoradiograms generated by 1 nM ['~H]FLU exposed to [3H]Ultrofilm for 10 days. Serial sections were incubated in the absence (A) and presence (B) of 200 nM CL218,872. In these photographs, dark regions represent areas of high receptor densities. Note the dramatic decrease in binding in lamina IV of the cortex (IV) and the substantia nigra, pars reticulata (SNR) in the presence of CL218,872. These regions represent areas of primarily type I BZ receptors (Table 2). On the other hand, very little decrease in binding is seen in the molecular layer of the dentate gyrus (dgm) or the superficial layer of the superior colliculus (sgs). The sections correspond to level A1760 ~m of KOnig and Klippel (1963). Bar= 1000 ~m.

69

e x p o s u r e of f i lm to s t a n d a r d s , a I n - I n t r a n s f o r m a t i o n y ie lds a l i n e a r p lo t (coeff i -

c i e n t of d e t e r m i n a t i o n , 0 . 9 6 - 0 . 9 9 in seve ra l e x p e r i m e n t s ) . T h e s e p l o t s were used in

t he f o l l o w i n g e x p e r i m e n t s w i t h B Z r e c e p t o r s .

The study of multiple benzodiazepine receptors

In o r d e r to f u r t h e r s t u d y the v a l i d i t y of th i s a p p r o a c h in q u a n t i t a t i n g n e u r o t r a n s -

m i t t e r r e c e p t o r b i n d i n g u s i n g r a d i o h i s t o c h e m i c a l t e c h n i q u e s , we a p p l i e d these p r o c e -

d u r e s to the s t u d y of m u l t i p l e b e n z o d i a z e p i n e r e c e p t o r s . Severa l p r e v i o u s s t u d i e s

h a v e s h o w n the e x i s t e n c e of at l eas t two types of b e n z o d i a z e p i n e r e c e p t o r s . A

p r e v i o u s a u t o r a d i o g r a p h i c s t u d y h a s d e f i n e d the c o n d i t i o n s w h e r e b y the se two

s u b t y p e s of r e c e p t o r s c a n be d i s c r i m i n a t e d u s i n g the T P Z d r u g C L 2 1 8 , 8 7 2 ( Y o u n g et

al., 1981). S ince the T P Z d r u g s h a v e a r e l a t ive ly h i g h a f f i n i t y for o n e s u b t y p e of

r e c e p t o r , a n a p p r o p r i a t e c o n c e n t r a t i o n of th i s d r u g c a n be i n c l u d e d in l a b e l i n g

e x p e r i m e n t s to se l ec t ive ly i n h i b i t [3HIFI_,U b i n d i n g to a t ype 1 s u b t y p e of r e c e p t o r .

T h e u t i l i z a t i o n of the f i lm p r o v i d e d resu l t s i d e n t i c a l to t h o s e f o u n d ea r l i e r u s i n g

TABLE 2

REGIONAL DISTRIBUTION OF TYPE-1 AND TYPE-2 BZ RECEPTORS AS DETERMINED BY QUANTITATIVE AUTORADIOGRAPHY

Serial sections were incubated with l nM [3H]FLU+-200 nM CL218,872L Binding is expressed as fmol/mg protein--S.E,M. (n=3). See text for details.

Region Total specific + CL218,872 ~ Decrease binding

I, Primarily type 1 receptors Substantia nigra, pars reticulata 517.4 + 33.3 88.1 + t2.7 83.0 Globus pallidus 296.2 + 45.0 70.2 ± 5.7 76.3 Cerebellum, molecular layer 355.3 + 21.3 89.7 + 5.3 74.8 Cortex, lamina IV 564.2 + 17.7 152.4 + 14.3 73.0

II. Mixed receptor population Inferior colliculus 686.0 + 17.7 341.3 + 23.3 50.2 Olfactory tubercle 530.3 + 59.3 288.0÷ 6.0 45.7 Floor IV ventricle 410.7 + 35.3 247.7 +- 19.3 40.2 Medial thalamus 339.3 + 48.3 229.3 + 26.0 32.4 Superior colliculus, superficial layer 557.7 ÷ 50.0 401.8 + 8.0 28.0 Amygdala, postero-lateral 465.7 + 30.0 362.0 ± 12.3 22.3 Hippocampus, CA 1 372.3" 49.0 297.0 + 18.3 20.2

III. Primarily type 2 receptors Ventro-medial hypothalamus 346.0 + 40.0 286.0 ÷ 49.3 17.3 Caudate-putamen 125.1 :~ 12.3 107.7 + 14.7 13.9 Dentate gyrus molecular layer 451.7 + 23.9 425.0 ~ 30.6 5.9 Accumbens 282.0 +- 2.3 270.7 ~ 9.3 4.0

Under these conditions, assuming the [3 H]FLU uniformly occupies 40% of all BZ receptors (Unnerstall et al., 1981), 7.2% of type 1 receptors will be labeled with [3H]FLU in the presence of 200 nM CL218,872, while 35.9% of type 2 receptors will be labeled at this concentration of the TPZ, given Kjs for CL218,872 of 25 nM and 1000 nM for the type 1 and type 2 sites respectively (Lippa et al., 1980j.

70

n u c l e a r e m u l s i o n s a n d the c o v e r s l i p t e c h n i q u e ( Y o u n g et al., 1981) (Fig . 5 a n d

T a b l e 2). In o r d e r to tes t f u r t h e r the u s e f u l n e s s of t he f i lm for q u a n t i t a t i n g a u t o r a -

d i o g r a m s a n d d e t e r m i n i n g p h a r m a c o l o g i c a l p a r a m e t e r s , we p r e p a r e d a ser ies of

a u t o r a d i o g r a m s f r o m b r a i n t i s sue w h e r e [ ~ H ] F L U b i n d i n g was d i s p l a c e d by severa l ,

i n c r e a s i n g c o n c e n t r a t i o n s o f C L 2 1 8 , 8 7 2 a n d M e - b e t a - C C . T h e d i s p l a c e m e n t c u r v e s

d e t e r m i n e d f r o m th e a u t o r a d i o g r a p h s a re c o m p a r a b l e to t h o s e c u r v e s m e a s u r e d in

' ° °

.

LL 0 k- I00. z • o w ht

• 0

50"

[CL 21 . . . .

lO'm I0 -o lO'-e lO-t lO- I lO-S DRUG CONCENTRATION (M)

Fig. 6. Displacement curves of 1 nM [3H]FLU binding in tissue sections by Me-beta-CC (upper) and CL218,872 (lower) as determined by microdensitometry following exposure of the labeled sections to [3H]Ultrofilm for 10 days. Optical densities were converted to fmol/mg protein with the use of standard autoradiograms generated concomitantly with the labeled sections (see Materials and Methods and Figs. 1 and 4). IC50 values and Hill coefficients (nil) were determined for each compound in the molecular layer of the cerebellum (x-x), CAt of the hippocampus, stratum oriens ( 0 O) and the molecular layer of the dentate gyrus (© O). Curves were fit by the equation: % radioligand displaced= 100× [D] P/{[D] P+ [ICso]e}, where [D] is the concentration of the displacing drug and P is the Hill coefficient (McPherson and Summers, 1981). Upper curves: by utilizing quantitative autoradiographic techniques, IC5o values and Hill coefficients for Me-B-CC were obtained in these regions: cerebellum, ICs0 = 3.2 nM, n H = 1.17; hippocampus, IC50 = 14 nM, n H =0.72; dentate gyrus, IC5o = 15 nM, n H :0.81. In tissue homogenates under similar conditions (Braestrup et al., 1980; Nielsen and Braestrup, 1981), similar values were obtained for Me-B-CC: cerebellum, ICs0 =2.8 nM, n H =0.95; hippocampus, IC50 13 riM, nH =0.75. Lower curves: utilizing the TPZ C12t8,872, the following IC50 values and Hill coefficients were obtained by quantitative autoradiography: cerebellum, IC5o =90 nM, n H = 1.15; hippocampus, IC5o = 1.2 #M, n H =0.61; dentate gyrus, ICso =2.9 #M, n H = 1.11. Comparable values have been obtained utilizing tissue homogenates under similar conditions (Klepner et al., 1979): cerebellum, ICs0 = 37.4 nM, n H =0.9; hippocampus, IC5o =330 riM, n H =&6: Each point represents the mean of 6 measurements, 2 from each of 3 animals.

71

biochemical binding experiments in tissue homogenates and generate equivalent ICs0 values (Klepner et al., 1979; Braestrup et al., 1980; Nielsen and Braestrup, 1981) (Fig. 6). These studies indicate the reliability of the autoradiographic techniques when comparing them to biochemical binding studies.

Conclusion

This study demonstrates the ease, reliability and pharmacological relevance of the quantitative analysis of drug and neurotransmitter receptors utilizing standardized radiohistochemical methods with [3H]Ultrofilm. Data obtained by these techniques are amenable to analysis not only by manual microdensitometry, but also by scanning microdensitometry, photometry (Rogers, 1979), and as we have previously shown (Palacios et al., 1981), computer-assisted image analysis. Calibration curves generated by standards containing known concentrations of radioactivity exposed to the film simultaneously with the tissue being studied allows for the transformation of OD, transmittance or reflectance measurements into molar quantities of bound ligand.

We have previously suggested (Unnerstall et al., 1981) certain issues that need to be considered in the light microscopic quantitation of ligand binding. These not only include careful control and analysis of the biochemical characteristics of the labeling process and demonstration of the pharmacological relevance of the binding but also understanding of the physical properties of the interaction of radioactivity with nuclear emulsions. This study highlights further problems which must be addressed, including exposure times and the range of ODs being utilized in making densitomet- ric measurements.

While the use of [3H]Ultrofilm has definite advantages over the use of the 'coverslip-method' for quantitation (Palacios et al., 1981), there are several disad- vantages. First, there is the loss of anatomical register with the tissue being studied. Second, there is a loss of anatomical resolution. While [3H]Ultrofilm has greater sensitivity than the Kodak NTB-3 nuclear emulsion used in previous autoradio- graphic studies because of the high silver to gelatin ratio, the grain size of [3H]Ultrofilm (1.8m0.3 /~m) is nearly 10 times greater than that of the NTB-3 emulsion which results in a concomitant loss of anatomical resolution (Rogers, 1979). Resolution is further limited by the apparatus used to measure OD. While reliable grain density measurements can be made using coverslips up to a resolution of 10 /xm, microdensitometric techniques allow resolution of no more than 25 50 /~m before individual grains are being resolved. Photometric techniques can allow greater resolution (Rogers, 1979), but use of this method must be balanced by consideration of the anatomical resolution of the autoradiograph itself.

Acknowledgements

The authors wish to acknowledge Dr. D.H. Horst of Hoffman-LaRoche, Nutley, N J, for the gift of methyl-beta-carboline carboxylate and CL218,872 and Dr. E.D.

72

L o n d o n o f the G e r o n t o l o g y R e s e a r c h Uni t , N1H, B a l t i m o r e C i ty H o s p i t a l , for the

use of t he i r d e n s i t o m e t r y e q u i p m e n t . T h e a u t h o r s g r a t e f u l l y a c k n o w l e d g e the t e c h m -

cal a s s i s t a n c e o f Mrs . T h e r e s a K o p a j t i c , Mrs . R o b e r t a P r o c t o r a n d Mrs . N a o m i

T a y l o r , a n d the c ler ica l a s s i s t a n c e of Ms. D a r l e n e W e i m e r a n d Mrs . M a r y F lu tka .

T h i s r e s e a r c h was s u p p o r t e d b v R e s e a r c h ( } r a n t s M H 2 5 0 5 1 , N S 1 5 0 8 0 , D A 0 0 2 6 6 ,

R e s e a r c h C a r e e r D e v e l o p m e n t A w a r d T y p e 1I, M H 0 0 0 5 3 to M.J .K . , p o s t d o c t o r a l

f e l l o w s h i p T W 0 2 5 8 3 to J . M . P . a n d N a t i o n a l Sc i ence F o u n d a t i o n p r e d o c t o r a l fello~v-

s h i p s to J . R . U . a n d D . L . N .

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