Dopamine Receptor Binding and Quantitative Autoradiographic Study

Abstract

The central nervous system (CNS) dopamine system plays an important role in mediating the reinforcing effects of drugs of abuse (1). In addition, dopamine receptors have been the principal target of drugs employed in the treatment of neuropsychiatric disorders such as schizophrenia and Parkinson’s disease. Until 1990, the dopamine receptor population in the brain and periphery was believed to consist of two subtypes, D₁ and D₂, which were distinguished by their pharmacology and coupling to signal transduction systems (for review see ref. 2). D₁ and D₂ receptors exhibit similar distributions in brain with the highest densities in the striatum (for review see ref. 3). A number of selective radioligands have been synthesized and extensively used to characterize the classical dopamine receptor subtypes. These radioligands include the D₁-selective ligands [³H]SCH 23390 and [¹²⁵I]SCH 23982. D₂ receptor-selective ligands include the antagonists [³H]spiperone, [³H]YM 09151-2, and [¹²⁵I]iodosulpiride and the agonists [³H]propylnorapomorphine and [³H]quinpirole.

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1 Introduction

The central nervous system (CNS) dopamine system plays an important role in mediating the reinforcing effects of drugs of abuse (1). In addition, dopamine receptors have been the principal target of drugs employed in the treatment of neuropsychiatric disorders such as schizophrenia and Parkinson’s disease. Until 1990, the dopamine receptor population in the brain and periphery was believed to consist of two subtypes, D₁ and D₂, which were distinguished by their pharmacology and coupling to signal transduction systems (for review see ref. 2). D₁ and D₂ receptors exhibit similar distributions in brain with the highest densities in the striatum (for review see ref. 3). A number of selective radioligands have been synthesized and extensively used to characterize the classical dopamine receptor subtypes. These radioligands include the D₁-selective ligands [³H]SCH 23390 and [¹²⁵I]SCH 23982. D₂ receptor-selective ligands include the antagonists [³H]spiperone, [³H]YM 09151-2, and [¹²⁵I]iodosulpiride and the agonists [³H]propylnorapomorphine and [³H]quinpirole.

Molecular cloning efforts have revealed additional dopamine receptor subtypes: the D₃ and D₄ subtypes, which have homology with D₂; and the D₅ which has homology with D₁ (4–6). These novel subtypes are expressed in roughly 10- to 100-fold lower density than the classical dopamine receptor subtypes. D₃ sites are preferentially localized in limbic brain regions such as the nucleus accumbens and islands of Calleja. D₄ sites are of greatest abundance in regions such as the frontal cortex. The distribution of D₅ receptors, as assessed by D₅ mRNA, appears to be similar to that of the D₁ receptors. Because of the pharmacological similarity of these sites with the D₁ and D₂ receptors, it is often not possible to distinguish between these sites in a heterogeneous tissue with currently available radioligands. As such the site labeled by any given radioligand in a such a tissue (i.e., brain) will, of course, be dependent on the relative affinities of that ligand for the related subtypes and the population of sites present in the tissue employed. Thus, although interactions with specific D₁- or D₂-like subtypes may be inferred, it is likely that sites labeled by these radioligands in brain tissue represent a mixture of the related subtypes. Several radioligands that appear to exhibit selectivity for the D₃ receptor have been synthesized including [³H]7-hydroxy-2-(di-n-propylamino)tetralin ([³H]7-OH-DPAT) and [³H]PD 128907. These compounds label similar populations of sites in rat brain which are generally consistent with that reported for the D₃ receptor (7–9).

This chapter outlines protocols for radioligand binding and autoradiographic assays for different subtypes of dopamine receptors in brain. The scope of the chapter is limited to the widely used, well established protocols for binding and autoradiographic assays that employ commercially available, tritiated and iodinated ligands. Radioligand binding assays, or “grind and bind” studies, can be used to measure receptor density and affinity in homogenized tissue samples. This method is rapid and highly quantitative; however, localization of binding sites is limited by the accuracy of dissection and any variation in binding within the brain region of interest will be obscured by homogenization. Receptor autoradiography, on the other hand, has a high degree of anatomical resolution. However, even when performed in a quantitative manner, the method is only semiquantitative compared to radioligand binding. Accordingly, receptor autoradiography is most suited to determining receptor localization and relative densities in various brain areas or between different treatment groups. In addition, receptor autoradiography can require extended periods of time for the exposure of autoradiograms.

Protocols are outlined for saturation analysis of radioligand binding for D₁-like receptors using the antagonist ligand [³H]SCH 23390 and D₂-like receptors using the antagonist ligand [³H]spiperone (see Note 1 ). A protocol for the putatively selective labeling of D₃ sites using [³H]PD 128907 is also presented. For autoradiographic studies, protocols are outlined for D₁-like receptors using the antagonist ligand [³H]SCH 23390, D₂-like receptors using the antagonist ligand [¹²⁵I]iodosulpiride, and D₃ sites using agonist [³H]PD 128907. Procedures are generally similar for the respective assays; however, specific details, such as buffer composition, radioligand concentration, or incubation time may vary for each ligand. Ligand-specific details for each assay are presented at the relevant step of the protocol.

2 Materials

2.1 Radioligand Binding Assays

2.1.1 Membrane Homogenate Preparation

• 1. Brain tissue: Fresh or fresh-frozen and stored at −70°C. Caudate-putamen is most commonly used for D₁-like and D₂-like receptors, ventral striatum (nucleus accumbens and olfactory tubercles) for D₃.

• 2. Homogenizer (e.g., Brinkman Polytron, Tekmar Tissuemizer, or PRO).

• 3. Centrifuge tubes.

• 4. High-speed, refrigerated centrifuge.

• 5. Assay buffers. Store at 4°C for up to 2 wk.

  • a. [³H]SCH 23390: 50 mM Tris-HCl, 120 mM NaCl, 5 mM KCl, 2 mM CaCl₂, 1 mM MgCl₂, pH 7.4 at 23°C.

  • b. [³H]Spiperone: 50 mM Tris-HCl, 5 mM KCl, 2 mM CaCl₂, 1 mM MgCl₂, pH 7.4, at 23°C.

  • c. [³H]PD 128907: 50 mM Tris-HCl, 1 mM EDTA, pH 7.4, at 23°C.

2.1.2 Binding Assay

• 1. Radioligands:

  • a. D₁-like receptors: [³H]SCH 23390 (Amersham) (see Note 2 ).

  • b. D₂-like receptors: [³H]Spiperone (Amersham) (see Note 3 ).

  • c. D₃ receptors: [³H]PD 128907 (Amersham) (see Note 4 ).

• 2. Assay buffers. Store at 4°C for up to 2 wk.

  • a. [³H]SCH 23390: 50 mM Tris-HCl, 120 mM NaCl, 5 mM KCl, 2 mM CaCl₂, 1 mM MgCl₂, pH 7.4, at 23°C.

  • b. [³H]Spiperone: 50 mM Tris-HCl, 5 mM KCl, 2 mM CaCl₂, 1 mM MgCl₂, pH 7.4, at 23°C.

  • c. [³H]PD 128907: 50 mM Tris-HCl, 1 mM EDTA, pH 7.4, at 23°C.

• 3. Competing ligands (Sigma). A 1 mM stock may be prepared in ethanol and stored at −20°C.

  • a. [³H]SCH 23390: (+)-Butaclamol.

  • b. [³H]Spiperone: (+)-Butaclamol.

  • c. [³H]PD 128907: Spiperone.

• 4. Membrane homogenate.

• 5. 12×75 Polystyrene culture tubes.

• 6. Cell harvester (see Note 5 ).

• 7. Whatman GF/B filters.

• 8. Wash buffer: 50 mM Tris-HCl, pH 7.4, at 23°C. Store at 4°C.

• 9. Scintillation cocktail suitable for use with filters.

• 10. Scintillation vials.

• 11. Scintillation counter.

• 12. Protein assay kit.

• 13. Radioligand binding analysis software.

2.2 Receptor Autoradiography

2.2.1 Preparation of Slide Mounted Sections

• 1. Cryostat.

• 2. Chrome-alum-coated, precleaned, glass microscope slides (see Note 6 ).

• 3. Fresh-frozen rat brains. Stored at −70°C (see Note 7 ).

2.2.2 Labeling of Sections

• 1. Radioligands:

  • a. D₁-like receptors: [³H]SCH 23390 (Amersham).

  • b. D₂-like receptors: [¹²⁵I]Iodosulpiride (Amersham) (see Note 8 ).

  • c. D₃ receptors: [³H]PD 128907 (Amersham).

• 2. Assay buffer. Store at 4°C for up to 2 wk.

  • a. [³H]SCH 23390: 50 mM Tris-HCl, 120 mM NaCl, 5 mM KCl, 2 mM CaCl₂, 1 mM MgCl₂, pH 7.4, at 23°C.

  • b. [¹²⁵I]Iodosulpiride: 50 mM Tris-HCl, 120 mM NaCl, 5 mM KCl, 2 mM CaCl₂, 1 mM MgCl2, pH 7.4, at 23°C.

  • c. [³H]PD 128907: 50 mM Tris-HCl, 1 mM EDTA, pH 7.4, at 23°C.

• 3. Competing ligands (Sigma). A 1 mM stock may be prepared in ethanol and stored at −20°C.

  • a. [³H]SCH 23390: (+)-Butaclamol.

  • b. [¹²⁵I]Iodosulpiride: (+)-Butaclamol.

  • c. [³H]PD 128907: Spiperone.

• 4. Coplin jars or plastic slide mailers (Thomas Scientific)—depending on the number of slides to be run.

• 5. ³H-Hyperfilm (Amersham) (see Note 9 ).

• 6. X-ray cassettes.

• 7. [³H] or [¹²⁵I]methylmethacrylate autoradiography standards (Amersham).

• 8. Dark room.

• 9. Developing trays.

• 10. Developer and fixer suitable for X-ray film such as Kodak D-19 developer and rapid fixer.

• 11. Plastic sheet protectors.

• 12. Image analysis system.

3 Methods

The methods described in the following subheadings outline procedures for assay of dopamine receptors in brain tissue by (1) radioligand binding and (2) quantitative receptor autoradiography. Procedures for preparation of membrane homogenates and slide-mounted brain sections for use in the respective assays are also presented.

3.1 Radioligand Binding Assays

A protocol is outlined for the determination of receptor density and affinity using a 10-point saturation analysis design. If analysis of a larger or smaller number of data points is desired (i.e., triplicates, multiple samples, etc.), the quantity of each reagent required should be scaled up or down accordingly.

3.1.1 Preparation of Membrane Homogenates

These assays typically use brain regions in which the receptor of interest is expressed in greatest abundance. Accordingly, caudate-putamen is most commonly used for assay of D₁-like and D₂-like receptors, whereas ventral striatum (nucleus accumbens and olfactory tubercle) is used for D₃ receptors.

• 1. Dissect a sufficient quantity of brain tissue for assay to be performed (see Note 10 ). A 10-point saturation analysis with [³H]SCH 23390 or [³H]spiperone will require roughly 35 mg of caudate tissue. A 10-point saturation analysis with [³H]PD 128907 will require roughly 220 mg of ventral striatal tissue.

• 2. Homogenize tissue in 20 volumes (w/v) of the appropriate assay buffer for 10 s using a homogenizer (see Note 11 ).

• 3. Centrifuge the homogenate for 15 min at 48,000g at 4°C, then discard supernatant and resuspend the pellet in 20 volumes (w/v) of assay buffer using the homogenizer.

• 4. Centrifuge the homogenate for 15 min at 48,000g at 4°C, then discard the supernatant.

• 5. Final pellets may be used immediately or stored at −70°C for up to 1 mo for future use.

3.1.2 Assay Protocol

• 1. Using a homogenizer, resuspend the final pellet from membrane preparation in assay buffer to yield the desired concentration. Keep the membrane homgenates on ice while preparing other reagents.

  [³H]SCH 23390: 3 mg original wet weight/mL.

  [³H]Spiperone: 3 mg original wet weight/mL.

  [³H]PD 128907: 20 mg original wet weight/mL.

• 2. Prepare working solutions of radioligand. The assay is designed to test a range of concentrations spanning from roughly 10-fold above to 10-fold below the K _D for the respective radioligand. Working solutions are prepared at five times the desired final concentration to yield the desired final concentration after the addition of all reagents. For each assay, prepare 2 mL of radioligand in the respective assay buffer at the concentration shown below.

  [³H]SCH 23390: 15 nM.

  [³H]spiperone: 5 nM.

  [³H]PD 128907: 15 nM.

  Serially dilute the radioligand (1.2 mL of radioligand+800 μL of buffer) to produce 10 concentrations ( Table 1 ). Count duplicate aliquots of each dilution to allow determination of actual concentration.

• 3. Prepare 3 mL of the appropriate competing ligand (5 μM) in assay buffer.

  [³H]SCH 23390: (+)-Butaclamol.

  [³H]Spiperone: (+)-Butaclamol.

  [³H]PD 128907: Spiperone.

• 4. To perform the binding assay, assemble the following reactions in duplicate 12×75 mm polystyrene culture tubes ( Fig. 1 ): Total binding tubes:

  μL of assay buffer

  100 μL of radioligand (concentration 1–10)

  100 μL of assay buffer Nonspecific binding tubes:

  50 μL of assay buffer

  100 μL of radioligand (concentration 1–10)

  100 μL of competing ligand Then, add 250 μL of membrane homogenate to all tubes. Gently vortex-mix all assay tubes. Incubate at 23°C to attain steady-state binding.

  [³H]SCH 23390: 90 min.

  [³H]Spiperone: 90 min.

  [³H]PD 128907: 3 h.

• 5. Terminate reaction by rapid filtration over Whatman GF/B filters using a cell harvester. Wash filters three times with 3 mL of ice-cold wash buffer.

• 6. Place filters in scintillation vials, add cocktail, and count in a scintillation counter (see Note 12 ).

• 7. Determine protein concentration of membrane homogenate.

Table 1

Fig. 1.

Schematic of the layout for a 10-point saturation radioligand binding experiment.

3.1.3 Analysis of Radioligand Binding Data (see Note 13 )

The first step in the analysis of binding data is to determine the mean of the duplicate values obtained from the scintillation counter. If the scintillation counter expresses these data as counts per minute (cpm), convert the data to disintegrations per minute (dpm) using the following equation:

The next step is to determine the exact concentration of each concentration of radioligand used using the following equation:

Data can then be analyzed using any of a variety of computer programs for analysis of radioligand binding data such as LIGAND, EBDA, etc. Alternatively, data may be transformed an analyzed manually.

For manual analysis, determine the amount of specific binding at each concentration (see Note 14 ):

Binding data at each concentration of radioligand can then be plotted to generate a saturation curve ( Fig. 2 A).

Fig. 2.

Representative data from a 10-point saturation radioligand binding experiment (see Table 1 ). Data shown are for [³H]PD 128907 binding in rat ventral striatal membranes. Saturation data, expressed in dpm, is shown in (A). A Rosenthal (Scatchard) plot of the same data is shown in (B). Results of this experiment, as analyzed by LIGAND, were K _D=0.21 nM, B _max=23 fmol/mg of protein.

Data are then transformed and plotted in a Rosenthal (Scatchard) plot ( Fig. 2 B). First, convert specific binding counts into moles bound using the following equation:

This value is divided by the quantity of protein per tube to yield the concentration of specific binding expressed as moles per milligram of protein. Because this concentration will be quite low, data may be converted into more convenient units such as picomoles or femtomoles. This value is then divided by the concentration of radioligand to yield specific binding/total (B/T) (see Note 15 ). Finally, specific binding (moles permilligram of protein) is plotted vs B/T with specific binding on the x-axis and B/T on the y-axis. The density of binding sites (B _max) is indicated at the x-intercept. The affinity, or K _D value, is equal to −1/slope.

Representative analysis of radioligand binding is shown in Table 2 and Fig. 2 .

Table 2 Representative Analysis of Data from a 10-Point Saturation Radioligand Binding Experiment

3.2 Receptor Autoradiography

3.2.1 Preparation of Slide-Mounted Brain Sections

Using a cryostat, cut 10–20-μm sections containing the brain areas of interest (see Note 16 ). Regardless of the section thickness chosen, it is essential that the sections be of uniform thickness. Thaw-mount sections onto gelatin-chrome alum-coated glass microscope slides. Mount one or more sections near the bottom edge of the slide, away from the frosted end ( Fig. 3 ). Because you will need a total binding and a nonspecific binding slide for each data point, two slides with adjacent sections should be collected for each level of the brain to be analyzed. Allow the sections to dry at room temperature. Slide-mounted sections can then be stored at −70°C for months.

Fig. 3.

Incubation of slide-mounted brain sections in radioligand solution. Note the placement of sections at the lower edge of the slide and that the radioligand solution is of sufficient depth to completely cover the sections.

3.2.2 Receptor Autoradiography

• 1. Remove slides to be assayed from the freezer. Remember that you will need pairs of slides with adjacent sections to allow you to determine total binding and a nonspecific binding. Allow slides to dry thoroughly at room temperature (at least 15 min) (see Note 17 ). While slides are drying, designate and label slides to indicate which slide of each pair will be used for total and nonspecific binding using a pencil or solvent-proof marking pen. It is often helpful to include several extra pairs of slides in the assay to use to generate “test films” for evaluating the adequacy of exposure of the autoradiograms.

• 2. Prepare radioligand solutions. First determine how much radioligand solution you will need. This depends on the size and number of incubation vessels you plan to use (i.e., Coplin jars or slide mailers), which, in turn, depends on the number slides to be incubated. It is generally best to determine empirically the volume required by measuring the amount of solution required to completely cover the your slide-mounted sections in an incubation vessel filled with slides

  Protein conc.: 245 μg/tube
  	A	A	D	E	F	G
  Radioligand conc. (nM)	Total (dpm)	Nonspecific (dpm)	Specific (dpm)	Specific (mol)	Specific (fmol/mg of protein)	B/T
  1.96	4317	3060	1257	4.84E-15	19.75	10.08
  1.20	3100	1824	1276	4.91E-15	20.05	16.66
  0.71	2173	1012	1161	4.47E-15	18.24	25.53
  0.44	1635	615	1020	3.93E-15	16.03	36.09
  0.26	1207	397	810	3.12E-15	12.73	48.51
  0.16	805	241	564	2.17E-15	8.86	54.20
  0.10	607	241	564	1.73E-15	7.06	70.84
  0.06	434	104	330	1.27E-15	5.19	89.56
  0.04	296	82	214	8.24E-16	3.36	90.42
  0.02	190	58	132	5.08E-16	2.07	96.21

  ( Fig. 3 ). Remember that you will need enough radioligand solution for incubating both total binding and nonspecific binding slides. For example, if you plan to assay 14 pairs of slides and you need 30 mL of radioligand solution to cover your sections completely, you will need to make 60 mL of radioligand solution. This will allow enough solution for two Coplin jars. One, containing only radioligand, will generate total binding sections. Competing ligand will be added to the second Coplin jar to generate nonspecific binding sections. Prepare the radioligand in the respective assay buffer. The concentrations of radioligand used in these are designed to minimize exposure time and are therefore near-saturating for the tritiated ligands.

  [³H]SCH 23390: 0.7 nM.

  [¹²⁵I]Iodosulpiride: 0.3 nM.

  [³H]PD 128907: 0.7 nM. Transfer half of the radioligand solution into the Coplin jar to be used for total binding. Then add competing ligand to the remaining radioligand solution to produce a final concentration of 1 μM.

  [³H]SCH 23390: (+)-Butaclamol.

  [¹²⁵I]Iodosulpiride: (+)-butaclamol.

  [³H]PD 128907: Spiperone. Transfer this solution into the Coplin jar to be used for nonspecific binding.

• 3. Count an aliquot of the radioligand solution to allow determination of the exact concentration.

• 4. Place slides in the respective Coplin jars and incubate at room temperature for 2 h. This is sufficient time for the system to reach steady state. If large numbers of slides are to be assayed, multiple Coplin jars may be used and/or multiple batches of slides may be run. If multiple batches of slides are assayed, it is wise to count the radioligand binding solution between each batch to ascertain that the concentration of radioligand is not depleted after multiple incubations.

• 5. Terminate the binding reaction by dipping the slides in ice-cold assay buffer. This is followed by two wash incubations in ice-cold assay buffer followed by a dip in ice-cold dH₂O. Washes can be accomplished by either transferring slides from one wash vessel to the next or changing the buffer in the Coplin jar containing the slides. Wash times for the two wash incubations for each radioligand are as follows (see Note 18 ):

  [³H]SCH 23390: 2×5 min.

  [¹²⁵I]Iodosulpiride: 2×5 min.

  [³H]PD 128907: 2×2 min.

• 6. Blot slides and allow them to dry. Drying can be accomplished by standing slides in the dividers that come in boxes of 20-mL scintillation vials set on a piece of absorbent bench paper. Alternatively, slides can be aspirated using a Pasteur pipet attached to a vacuum source. Once dry, slides can be placed in slide boxes and allowed to dry thoroughly (at least overnight). Drying can be augmented by blowing slides with cool air using a fan, blower etc.

• 7. Once dry, arrange slides in X-ray cassettes. Include appropriate methylmethacrylate radioactivity standards on each film. In the dark room, apply ³H-Hyperfilm to the sections with the emulsion (dull) side directly against the sections. Exposure of autoradiograms with each radioligand will take roughly the following amount of time:

  [³H]SCH 23390: 3 wk.

  [¹²⁵I]Iodosulpiride: 48 h.

  [³H]PD 128907: 12 wk. This time may vary depending on variations in specific activity, actual radioligand concentration, tissue handling, experimental treatment, brain region to be examined, and so forth. Accordingly, it is often useful to also expose several “test films,” which expose a pair of slides and can be developed at various times, to determine the adequacy of exposure before developing the films with the actual experimental sections. It is equally important not to overexpose the films (see Note 19 ).

• 8. When exposure is adequate, develop films according to the manufacturer’s instructions. Note that because the emulsion on ³H-Hyperfilm has no coating, the film must be developed by hand. It will be damaged by automated X-ray film processing machines. It also scratches very easily. Therefore, it is essential that it be developed with the emulsion side up. Care should also be exercised when handling the film. After drying, store films in plastic sheet protectors to prevent scratching.

• 9. Capture images using a video-based densitometry image analysis system such as NIH Image, MCID, and so forth. To quantify autoradiograms, use the radioactivity standards to generate a standard curve to describe the relationship between optical density (OD) and radioactivity (using the values supplied by the manufacturer for nanoCuries per milligram of tissue equivalent). Images from each film can then be calibrated according to the standards included on that film. Sample the brain regions of interest from the total binding sections and the adjacent sections used for nonspecific binding. Specific binding in that brain area can then be calculated using the following equation:

  

  The density of ligand binding can then be calculated:

  

4 Notes

• 1. Saturation analysis represents only one type of radioligand binding experiment. It is used to determine receptor affinity and density. For detailed descriptions of a variety of types of receptor binding experiments see Bylund and Yamamura (10) and Levant (11).

• 2. Although [³H]SCH 23390 exhibits high selectivity for D₁ receptors over D₂, this ligand also possesses some affinity for the 5-hydroxytryptamine₂ (5-HT₂) receptor. Because caudate expresses very low density of 5-HT₂ receptors, the contribution of the sites is negligible in membranes prepared from that brain area. If other brain areas or tissues are used, 5-HT₂ receptors may be blocked in the presence of 40 μM ketanserin.

• 3. Although the [³H]spiperone binding is the most widely used assay for the D₂-like sites, this assay is somewhat limited by the fact that it utilizes a radioligand with high affinity but low specific activity. In striatal membranes, the ligand also exhibits relatively high nonspecific binding. As such, assays using [³H]spiperone must use relatively low concentrations of membrane homogenate to avoid ligand depletion. As a result, the magnitude of specific binding in terms of dpm obtained with [³H]spiperone is striatal membranes is relatively low compared to many other radioligand binding assays. It should also be noted that while [³H]spiperone exhibits high selectivity for D₂ receptors over D₁, this ligand also possesses relatively high affinity for the 5-HT₂ receptor. As striatum expresses very low density of 5-HT₂ receptors, the contribution of the sites is negligible in membranes prepared from that brain area. If other brain areas or tissues are used, 5-HT₂ receptors may be blocked in the presence of 40 μM ketanserin. Interaction of [³H]spiperone with an acceptor site, the spirodecanone site, has also been reported (12).

• 4. Obtaining selective labeling of the D₃ site with this, or other, radioligands, appears to be dependent on the use of assay conditions that disfavor agonist binding at the D₂ site. The greatest D₂/D₃ selectivity for these ligands has been obtained in the absence of Mg²⁺ and the presence of EDTA (7,9) in concordance with previous studies indicating that the high affinity agonist state of D₂-like receptors is not favored in the absence of Mg²⁺ (13). Because selective visualization of the putative D₃ receptor appears to require in vitro assay conditions that disfavor D₂ binding, interpretation of these results must consider that these conditions may also affect the binding or functional properties of the D₃ site.

• 5. Other types of filtering apparatus may be used. However, for best results, it is desirable that all binding reactions be subjected to uniform filtering conditions. Accordingly, a cell harvester produces the most satisfactory results.

• 6. Buy precleaned slides or wash slides in soap and water followed by an ethanol dip to remove all dust and oil. Place in slide racks. To sub (gelatin-coat) slides, prepare a subbing solution containing 5 g of porcine gelatin (300 bloom) and 0.5 g of Chrome-Alum (chromium potassium sulfate) in 1 L of dH₂0. The actual amount of subbing solution required will depend on the size of the histology dishes to be used for treating the slides. Heat while stirring until gelatin is dissolved. Do not boil. Filter and allow to cool. Dip slides into subbing solution several times to ensure complete coating. Drain excess. Allow coating to dry. Protect slides from dust. Store at room temperature. Alternatively, charged slides (Fisher Plus) or poly-l-lysine-coated slides may be used.

• 7. After the animal is euthanized, remove the brain carefully. To maintain the shape and structural integrity of the brain, snap-freeze the brain by swirling it in an isopentane (2-methylbutane), dry-ice bath. When the brain is frozen, it will be opaque white in appearance. Some cracking of the brain along the longitudinal fissure may occur if the isopentane is too cold. If severe, the brain may split in half but will still be usable. Such cracking can be avoided with practice and experience. Liquid nitrogen is not recommended because it will tend to cause the brain to shatter.

• 8. [¹²⁵I]Iodosulpiride produces selective labeling of D₂-like receptors (14). Iodinated ligands have the advantage of producing relatively high-resolution autoradiograms in a short period of time. If a tritiated ligand is desired, 0.7 nM [³H]spiperone may be used in the presence of 40 μM ketanserin (15). The agonist [³H]quinpirole is also useful for autoradiographic localization of D₂-like sites (16).

• 9. Although some films, such as Kodak Biomax MS, are sensitive to tritium, in our experience, ³H-Hyperfilm is the only film that produces satisfactory autoradiograms with low-energy β-emitters for use in quantitative analysis of receptor binding in specific brain areas.

• 10. Brain regions may be identified using any of a variety of brain atlases such as the rat brain atlas of Paxinos and Watson (17).

• 11. Using a Polytron, homogenization at a setting of 6 out of 10 is sufficient.

• 12. Before counting, allow sufficient time for the cocktail to saturate the filters and for the mixture of radiolabeled membranes and scintillation cocktail to reach steady state. The amount of time required will depend on the cocktail used and can be determined experimentally.

• 13. In striatal membranes, saturation analysis with [³H]SCH 23390 should yield a K _D value of 0.1–0.5 nM and a B _max value of 700–1000 fmol/mg of protein (18–21). K _D values for [3H]spiperone in striatal membranes are most commonly reported to be 0.05–0.3 nM (21). A B _max value of approx 300–500 fmol/mg of protein should be anticipated (22,23). K _D values for [³H]PD 128907 in ventral striatal membranes are approx 0.3 nM. A B _max value of approx 20–40 fmol/mg of protein should be anticipated (24).

• 14. Computer packages for analysis of radioligand binding data provide unbiased analysis of untransformed data. Accordingly, use of such a package is highly recommended. However, it is often useful to analyze the data manually as well, thus allowing for verification of the computer output.

• 15. Binding in a saturation analysis is most appropriately expressed as a function of free radioligand. However, because binding in these assays at the K _D represents <10% of total counts added, depletion of free radioligand is negligible. Hence, for simplicity of calculation, the use of total radioligand, rather than free radioligand will generate a reasonable estimate of K _D and B _max values in manual calculations.

• 16. Temperatures for cutting brain tissue will vary depending on the instrument used. Typically, temperatures in the range of −11 to −20°C work well. Key points for successful cutting of brain tissue include having the tissue at the proper temperature. This will require moving the brain from storage at −70°C to the cryostat about 30 min before cutting. A sharp microtome knife is essential. Proper anti-roll plate adjustment is also important. Sections may be collected on either chilled or room temperature slide as preferred by the individual.

• 17. It is critical that the sections dry thoroughly or pieces of the sections will come off the slides during incubation.

• 18. Wash times were determined experimentally to maximize specific binding and minimize nonspecific binding.

• 19. The greater the number of counts bound and with high-energy isotopes, the greater the risk of overexposure. Accordingly, particular care should be taken with [¹²⁵I]iodosulpiride to avoid overexposure. On the other risk of overexposure is relatively low with tritiated ligands, particularly [³H]PD 128907. As such, one might err on extending the duration of exposure to ensure that sufficient signal is generated.

Fig. 4.

Representative autoradiograms. Sections shown represent (A) [³H]SCH 23390 binding, (B) [¹²⁵I]iodosulpiride binding, and (C) [³H]128907 binding in coronal sections of rat brain.