Receptor Binding Techniques UNIT 1.4

Initial interest in radioligand autoradiogra- ate assays. The ability to perform multiple as- phy of brain tissue was driven by the potential says in individual brains is an important asset value of this technique in studying the in vivo in the study of brain pathology, where subjects distribution of centrally acting —e.g., an- may have variable severity and locations of ticonvulsants and sedatives—and in evaluation abnormality. Autoradiographic screening of of their mechanisms of action and pharmaco- wide anatomic areas precludes the need to kinetics (Roth and Barlow, 1961). A variety of prospectively identify all areas of potential in- clinically useful agents had been discovered terest by gross dissection, as is necessary for empirically through behavioral screening, but homogenate tissue assays. without clear indication as to their biochemical Pharmaceutical development may be as- actions in the brain. Subsequently, it was rec- sisted both by in vitro and in vivo autora- ognized that many drugs interacted with neuro- diography. Although in vitro methods are pre- transmitter receptors or other minor protein ferred for the detailed characterization of bind- constituents by means of stereospecific, satu- ing-site , in vivo distribution rable binding. The in vivo distribution of radi- studies continue to be of use in determining the oligands was then applied to detailed mapping detailed biodistributions of new drugs, afford- of the binding sites in brain (Yamamura et al., ing important data on and 1974). It was later determined that in vitro regional distribution in preclinical animal stud- assays employing radioligands could be per- ies. By extension, emission tomographic imag- formed for the same purpose (Young and ing studies of distribution in the living Kuhar, 1979). The potential advantages of the human brain can provide important data on the in vitro design became immediately apparent— distribution and effects of novel agents both in e.g., the ability to directly control and measure controls and disease states. This information aspects of the binding reaction, the use of re- may become increasingly important in the duced amounts of radioligand, and the possi- search for more effective drugs and in attempts bility of employing radioligand binding to to better distinguish therapeutic effects from evaluate the properties of unlabeled drugs in unwanted side effects. competition assays. In vitro autoradiographic binding-site as- RADIOLIGAND SELECTION says are now important tools in contemporary and pharmacology, with Isotopic Label strengths and limitations that are often comple- Identification of candidate radioligands for mentary to those of homogenate binding as- autoradiography of binding sites first necessi- says. Performance of rapid kinetic association tates consideration of the possible radioiso- and dissociation assays and detailed multisite topes for their labeling. The most widely em- saturation or competition assays can be more ployed isotope, tritium (3H), has the advantages difficult by autoradiography than with the use of low cost, ease of safe handling and disposal, of tissue homogenates. This is due to temporal and a wide variety of approaches for introduc- limitations of intact-tissue kinetics and to limits ing label into ligands. Tritium is a neutron-rich on the number of assays that can be performed hydrogen isotope derived from nuclear fission on small structures represented in only a few reactor bombardment. It decays by the conver- tissue sections. Conversely, the very high sen- sion of a neutron to a proton with emission of sitivity and spatial resolution of autoradiogra- a β− particle and an antineutrino, thus increas- phy affords the ability to study structures too ing the number of protons in the nucleus and small to accurately dissect for homogenate as- resulting in an atom of helium. The process of says. The ability to combine binding-site β− emission results in a spectrum of emitted autoradiography with other anatomical imag- particle energies resulting from the differential ing procedures, including routine histological distribution of the transition energy between staining, histochemical staining, immunohisto- the antineutrino and the β− particle. The average chemistry, nucleic acid hybridization assays, β− energy is approximately one-third of the and some in vivo radioligand distribution as- maximum transition energy, and in the instance says, permits powerful and flexible multidisci- of 3H is 5.7 keV. Tritium is unique among the plinary protocols not possible with homogen- isotopes employed for autoradiography since Neuroanatomical Methods Contributed by Kirk A. Frey and Roger L. Albin 1.4.1 Current Protocols in Neuroscience (1997) 1.4.1-1.4.14 Copyright © 1997 by John Wiley & Sons, Inc. Table 1.4.1 Properties of Some Nuclides Useful in Autoradiography

Max. Avg. Max. tissue Avg. tissue Max. sp. act. Particles per Nuclide Emission energy energy Half-life range (mm) range (mm) (Ci/mol) transition (keV) (keV)

3H β− 18.6 5.7 0.008 0.004 12.3 years 2.9 × 104 1.00 14C β− 156 49 0.301 0.042 5370 years 6.7 × 101 1.00 18F β+ 635 250 2.440 0.637 1.83 hr 1.7 × 109 1.00 32P β− 1710 695 8.210 2.750 14.3 days 9.2 × 106 1.00 33P β− 250 77 0.637 0.092 25 days 5.1 × 106 1.00 35S β− 167 49 0.336 0.042 87 days 1.5 × 106 1.00 99mTc ICEa 120 120 0.195 0.195 6.01 hr 5.2 × 108 0.09 123IICEa 127 127 0.214 0.214 13.2 hr 2.4 × 108 0.14 125IAuEb 30 24c 0.019 0.012 60 days 2.2 × 106 0.20 131I β− 606 192 2.300 0.421 8.04 days 1.6 × 107 0.89 aInternal conversion electron decay. bAuger electron decay. cFor 125I (which exhibits Auger electron decay), the average energy is calculated from the abundance-weighted mean of electron energies resulting from ten distinct transition possibilities with energies ranging from 22 to 30 keV.

its average β− energy permits travel through The most commonly employed nuclide with only a few micrometers of tissue before its very high specific activity is iodine-125 (125I), absorption (Table 1.4.1). Typical frozen histo- obtainable at almost 100-fold higher activity logical tissue sections are much thicker than the per atom on the basis of its shorter half-life. average 3H β− particle path length; sections of However, additional features, including abun- 15 to 20 µm or greater are “infinitely thick” dance of charged particulate emissions per de- with respect to 3H emissions. Autoradiograms cay, effective film-exposure index per particle, of tritium-ligand distribution in most tissue and the number of nuclide atoms per ligand sections are thus insensitive to minor inconsis- additionally influence the relative tencies in sectioning precision. The advantage sensitivities of ligands labeled with 125I versus afforded by this insensitivity to tissue-section those labeled with 3H. Labeling of ligands with thickness is counterbalanced by the effects of 125I may be accomplished by replacement of tissue self-absorption and by the need for spe- stable 127I or by iodination of a parent ligand at cialized films and emulsions for detection of a position distant from its site of interaction the low-energy 3H β− emissions (see discussion with the binding site of interest. Iodine-bearing of Contact Autoradiography). ligands are often more challenging to employ As depicted in Table 1.4.1, radionuclides in vitro than their non-iodinated counterparts employed for autoradiography differ in mode because of the effects of increased molecular of decay as well as in physical half-life, each size and greater lipophilic character conferred of which affect the feasibility of their use in by iodine. These properties tend to slow the binding-site autoradiography. As a general kinetics of ligand diffusion, binding, and dis- guide, if binding sites are present at an average sociation—and also promote increased non- tissue concentration of 10 to 100 nM, a single specific tissue binding when assays employ tritium atom per ligand molecule will permit polar, saline-based buffers. The radionuclide autoradiographic imaging with exposure times decay scheme of 125I is more complicated than of 5 to 7 days and specialized tritium-sensitive that of 3H. It decays by electron capture, a X-ray films. Increasing the number of labeled process involving absorption of an inner-shell atoms per molecule or increasing the exposure electron (most often a K-shell electron) by the time will each independently lower the detec- nucleus, which results in conversion of a proton tion threshold. If the known target binding-site to a neutron with emission of a neutrino. The density is substantially lower, use of a shorter- orbital vacancy left by the captured electron is half-life nuclide at higher specific activity per then filled by an electron from a higher energy labeled atom may be necessary. shell, leaving the atom with excess energy. The Binding Techniques 1.4.2

Current Protocols in Neuroscience atom then dissipates this energy by the emission corporated in an alkyl substituent (e.g., when of X rays or ejection of electrons. A given employing fluoromethyl or fluoroethyl substi- radionuclide will emit a mix of X rays and tutions for methyl or ethyl groups). The process electrons, depending on the energy to be dissi- of β+ emission involves the conversion of a pated and on the nuclear and electron compo- proton to a neutron with the emission of the β+ sition of the atom. The X rays are termed particle and a neutrino from the nucleus. In the “characteristic X rays.” The emitted electrons, instance of 18F, decay to stable 18O involves termed “Auger electrons,” are the principal emission of a β+ particle with maximum energy means of autoradiographic detection of 125I. of 635 keV, but as in the instance of β− decay, The energy of the Auger electron is charac- division of energy between the β+ particle and teristic of the difference in energy states of the neutrino results in a spectrum of emitted β+ initial electron transition as well as the energy energies averaging ∼250 keV. The emitted par- of the electron absorbing the transition energy. ticles are effective in exposing autoradiog- In the decay of 125I, Auger electrons of ten raphic film, although they are of such high individual energies within the range of 20 to 30 energy that anatomic resolution is considerably keV are emitted with a cumulative probability less than that typically achieved with either 3H of 20%. Thus, only one in five decaying 125I or 125I. An additional consideration is the ulti- atoms emit an Auger electron likely to interact mate fate of the β+ particle, since after loss of with autoradiographic film (see discussion of its initial kinetic energy it undergoes annihila- Basic Autoradiographic Principles). The ener- tion with an electron in the absorbing medium gies of these Auger emissions, however, are whereby the β+ particle and electron are con- sufficiently higher than those of 3H β− particles verted to 511-keV photons. These electromag- so that their tissue-penetration distances are netic radiations, indistinguishable in properties similar to typical frozen-section thicknesses. from γ rays and X rays, require the use of Also, the effective film exposure per particle is shielding in the laboratory to reduce exposure much greater, and traditional anti-scratch- of personnel during in vitro assays. Although coated X ray film can be used for autoradiog- the short half-life of 18F can present challenges raphy. Overall, autoradiographic detection sen- in tissue processing, this isotope has been suc- sitivity per 125I-decay particle may be 5- to cessfully employed for ex vivo detection of 10-fold higher than for 3H, and when this is ligand distributions. It would theoretically be combined with the differences in specific ac- possible to use 18F for in vitro assays as well if tivity and particle yield per decay, autoradiog- local cyclotron and chemistry facilities for ra- raphic detection may be enhanced by 50- to dioligand syntheses are available. 100-fold over 3H at the same tissue concentra- The nuclides technetium-99m (99mTc) and tions. iodine-123 (123I) can decay by emission of Other radionuclides have been employed internal conversion electrons with probabilities successfully for autoradiography involving the of 9% and 14%, respectively, per transition. emissions of β− particles, including 14C, 32P, 33P, These particles, like Auger electrons, are orbital 35S, and 131I. Of these nuclides, all but 14C have electrons ejected from the atom after absorbing specific activities adequate for routine autora- energy from a nuclear transition. In the instance diographic imaging of low-abundance binding of 99mTc, the transition is from an excited nu- sites such as receptors and clear energy state (a metastable excited state transporters. The emitted energies of these trac- denoted by the “m” in 99mTc) to a more stable ers are all high enough to penetrate typical nucleus of unchanged proton/neutron compo- frozen-section tissue thicknesses well; further- sition. In ∼89% of 99mTc to 99Tc transitions, more these isotopes are not subject to tissue energy is released as a γ ray of 140 keV. In one self-absorption concerns and do not require of several alternative transition routes, how- specialized films or emulsions for detection. ever, energy is ultimately released as an internal However, the range of ligands that can be la- conversion electron of 120 keV. In the case of beled with these nuclides is more limited than 123I, initial transition by electron capture results with 3H, based on their infrequent occurrence in conversion of a proton to a neutron, and in active drugs. ∼14% of transitions lead subsequently to the The β+ emitter 18F has proven useful in emission of an internal conversion electron of positron emission tomographic (PET) imaging 127 keV that is suitable for autoradiographic of neuroreceptors and other binding sites when imaging. Because 99mTc is a transition metal, it incorporated in ligands to replace stable 19F, can be incorporated by chelation or coordina- Neuroanatomical when substituted for OH groups, or when in- tion chemistry into organic ligands; however, Methods 1.4.3

Current Protocols in Neuroscience as in the instance of iodination, the resulting tion of the partitioning properties of new li- ligands generally have much larger molecular gands by extrapolation from measurements sizes than related parent drugs. The most com- made on related compounds (i.e., from the mon autoradiographic application of 99mTc is partitioning behavior of the parent molecular ex vivo imaging of cerebral perfusion on the structure and its substituents). Ligands with basis of in vivo accumulation of the tracers octanol:saline partition coefficients less than [99mTc]hexamethylpropyleneamine oxime unity are sufficiently polar that they do not cross (HMPAO) or [99mTc]N,N′′-1,2-ethylene-dibis- cell membranes in intact cells and are likely to L-cysteine diethyl ester hydrochloride (ECD). detect binding sites only in hydrophilic do- Radionuclides with complicated transition mains. Ligands with partition coefficients >10 schemes—e.g., 99mTc, 123I, 125I, and 131I—most are sufficiently lipophilic that they enter mem- often give rise to multiple forms of emitted branes readily, and are likely to detect binding energy, including γ rays employed in medical sites in both lipophilic and hydrophilic do- diagnostic imaging. While these radionuclides mains. Ligands with partition coefficients are useful in tissue autoradiographic assays, >1000 pose additional problems in in vitro this aspect necessitates consideration of addi- assays, which result from nonspecific binding. tional shielding during in vitro assays, which is These ligands, encountered frequently among not necessary with the use of pure, low-energy iodinated compounds, have very poor solubil- β− emitters such as 3H, 14C, 33P, and 35S. ity in saline-based buffers and tend to distribute avidly into tissue sections in binding assays. Pharmacological and Chemical The addition of carriers in the buffer (e.g., Ligand Profiles albumin) or reduction in buffer polarity by Ligands selected for in vitro autoradiog- isoosmotic substitution of sucrose for sodium raphic assays share many characteristics with chloride may be necessary to reduce nonspe- ligands employed for homogenate binding as- cific binding in some instances. says. The most useful ligands have high affinity for a well-defined population or populations of CHARACTERIZATION OF binding sites, exhibit relatively low nonsatur- LIGAND-BINDING ASSAYS able or nonspecific binding, and have kinetic properties facilitating convenient equilibrium- General Aspects of Ligand-Binding binding assay development. Generally speak- Autoradiography ing, antagonist ligands that do not distinguish Autoradiographic ligand assays share paral- different receptor states on the basis of effector lel aspects of experimental design with conven- coupling provide the best tools for quantifica- tional in vitro homogenate binding assays, de- tion of binding-site numbers. As in the instance scribed comprehensively elsewhere (Yama- of homogenate binding assays, ligand polarity mura et al., 1985, 1990). A tissue source must may influence the ability to interact with the be selected and preprocessed, tissue samples full tissue complement of binding sites. The use are incubated in the presence of labeled ligand of highly polar ligands, such as quaternary under carefully controlled conditions, the la- amines, may limit detection to receptors ex- beled tissue is washed to remove nonspecifi- posed on membrane surfaces, and, in intact cally bound ligand, and specifically bound li- cells, to cell-surface receptors. Lipophilic li- gand is quantified. Although there are some gands often detect additional binding sites pre- important distinctions between autoradiog- sumed to reside in more lipophilic domains. In raphic and homogenate studies, the princi- the example of binding to intact cells, these may ples—reversible bimolecular binding pro- include intracellular or internalized receptors. cesses as well as kinetic, saturation, and com- In homogenate assays and other broken-cell petition analyses—apply equally to both preparations, lipophilic ligands may still inter- designs. In the following sections, general as- act with a larger number of binding sites than pects and guidelines for performance of autora- polar ligands, suggesting that intramembra- diographic assays will be considered. nous receptor location or adjacent lipophilic membrane domains may be important. Ligand Tissue Preparation polarity/lipophilicity is most readily estimated Tissues suitable for homogenate binding as- on the basis of partitioning between aqueous says are usually acceptable in autoradiographic and organic phases. A widely employed system analyses as well. In addition to specimens ob- Receptor Binding uses octanol and saline phases. This system has tained at biopsy or necropsy of experimental Techniques been sufficiently studied to enable the estima- animals, human surgical resection and post- 1.4.4

Current Protocols in Neuroscience mortem samples are often sufficiently pre- age with good tissue preservation and mainte- served for autoradiography. In experimental nance of sectioning properties. Storage at animal studies, simple gross organ or tissue higher temperatures or repeated temperature dissection followed by freezing is preferred. cycling from −70°C to cryostat-sectioning tem- Perfusion fixation (UNIT 1.1), while sometimes perature may hasten loss of some tissue bio- affording improvements in histological, histo- chemical reactivities. If there is concern that chemical, or in situ nucleic acid hybridization aging or repeated temperature cycling may alter studies, should generally be avoided in tissues or degrade binding-site properties, this can be that are to be used in ligand-binding experi- explored directly in control tissues. ments. Even very mild aldehyde or other tis- Tissue is prepared typically for in vitro sue-fixation schemes may modify kinetic and autoradiographic binding assays by sectioning pharmacological properties of binding sites. If in a cryostat microtome (UNIT 1.1). Usual frozen- fixed tissues must be employed, it is important section thicknesses of 10 to 30 µm are accept- to conduct all initial characterizations of the able for autoradiographic purposes. It is impor- binding site(s) in similarly processed tissues. tant to bear in mind when selecting tissue-sec- Effects of postmortem tissue handling and de- tioning conditions that generation of 3H lay prior to dissection and freezing can theo- autoradiograms will not be appreciably af- retically influence the recovery of binding sites fected by section thicknesses within this range, and other biochemical tissue markers. These nor will section-to-section imprecision affect considerations are particularly important in as- the autoradiograms. However, direct scintilla- says of human postmortem tissues, where in- tion spectrometry assays of sections (see dis- tervals of many hours may pass before speci- cussion of Determination of Incubation and mens are collected. Potential postmortem arti- Washing Parameters) and autoradiographic facts may be reduced by careful matching of binding assays employing radionuclides with sample histories between experimental groups. higher particulate-energy emissions will each This approach, however, does not entirely guar- be sensitive both to the selected tissue thickness antee that important information and distinc- and to the precision of sectioning. In the in- tions have not been lost prior to tissue collection stance of 3H autoradiography, it is typical to and storage. An additional approach to this collect duplicate adjacent tissue sections to problem is to conduct controlled experiments guard against the intrusion of artifacts in proc- emulating human postmortem conditions and essing. With other radionuclides, triplicate sec- intervals in experimental animals, thereby per- tions should be considered, to reduce additional mitting direct assessment of possible differ- section-thickness error effects, unless the mi- ences between immediate and delayed tissue crotome precision is known to be excellent. dissection and freezing. Microtome precision can be assessed experi- After dissection, freezing of small speci- mentally by comparison of autoradiographic mens can be readily accomplished by covering densities of adjacent sections after incubation them in crushed dry ice. This method is appli- reactions with radionuclides of moderate to cable to tissue blocks of up to 3 to 5 g with good high penetration, or in ex vivo autoradiograms results. Blocks of considerably larger size re- after the accumulation of tracers labeled with quire more efficient heat transfer during freez- penetrating radionuclides. Motorized cryostat ing to avoid microscopic ice-crystal formation microtomes offer the benefit of improved sec- or macroscopic fissuring. Large tissue blocks tion-thickness precision as compared with should be frozen by intermittent, repetitive im- manual sectioning, but are considerably more mersion in isopentane chilled with dry ice (UNIT costly than comparable manually driven mod- 1.1). Blocks should be dipped for several sec- els. onds at a time, followed by withdrawal for Tissue sections for use in autoradiographic several seconds to permit equalization of tem- binding assays are usually mounted frozen on perature between the block surfaces and center. microscope slides, thawed, and dried. Slides Otherwise, the surfaces will freeze first, and should be prewashed and subbed to promote when the center subsequently freezes and ex- section adhesion during binding assays. Sub- pands the tissue may fracture. Frozen tissue bing solutions employing gelatin (UNIT 1.1) are blocks can be maintained for years if stored at generally inferior to those containing poly-L- −70°C and protected from desiccation. Coating lysine (see CPMB UNIT 14.1 and APPENDIX 1A in this the surfaces of tissue blocks with a frozen-sec- manual) for use in lengthy in vitro assays. High tion embedding medium and packaging in air- heat conditions should be avoided during sec- Neuroanatomical tight plastic freezer bags permits routine stor- tion drying to prevent binding-site denatura- Methods 1.4.5

Current Protocols in Neuroscience tion. After complete drying, slide-mounted sec- determine critical assay parameters to maxi- tions may generally be stored at −70°C for up mize its separation from nonspecific binding. to several weeks without loss of binding activ- Preliminary studies to determine optimal incu- ity. If a particular binding site is known to be bation and post-incubation washing conditions fragile or is of unknown stability, effects of are often performed without the use of autora- post-sectioning storage should be investigated diography for radioligand measurement. In this directly in control experiments. instance, it is common to conduct binding stud- ies identically to autoradiographic assays with Detection of Specific Binding the exception that after post-incubation wash- Perhaps the most critical aspect of any li- ing, sections are wiped from the microscope gand-binding assay is the detection and defini- slide with glass fiber filter paper and assayed tion of saturable, specific binding. In the devel- by liquid scintillation counting or by γ count- opment of new assays, considerable attention ing, depending on the radionuclide involved. In should be focused on this aspect; when adopt- this way, rapid results can be obtained and ing assays from the published literature, it is multiple potential assay modifications and re- equally important to review results of prior finements tested in logical sequence. However, assay-development reports and give strong a series of tissue sections bearing reproducible consideration to replicating critical methodo- numbers of binding sites is necessary for these logical experiments prior to use of the assay. assays. Even though section-thickness vari- Autoradiographic assays are comparable to ation may have no effect on the autoradiog- homogenate binding experiments in the funda- raphic detection of 3H, it is critical in these mentals of defining specific ligand binding. direct tissue assays. Specific binding is by definition saturable, thus, As in the case of homogenate radioligand- a minimum criterion is that the radioligand binding assays, aspects of the basic binding demonstrate a component of binding that does incubation—including choice of buffer system, not increase linearly with free ligand concen- pH, time, temperature, and duration of incuba- tration. This is most easily demonstrated by the tion—should be explored to maximize specific effect of adding unlabeled ligand to the assay. binding. Often, important initial estimates of More precise pharmacological characterization these quantities can be derived from prior in of specific binding is achievable when the ra- vitro homogenate binding assays. It is generally dioligand and the unlabeled competitor are advisable in autoradiographic assays, however, chemically dissimilar, minimizing the likeli- to employ incubation buffers that are isotonic, hood of multiple sites of overlapping tissue so as to avoid osmotic stresses on tissue sec- interaction. It is helpful to begin the search for tions. Hypotonic buffers are particularly prob- specific binding of a new radioligand with lematic if tissue-section adhesion is marginal, knowledge of one or more unlabeled competi- and their use may result in tissue fragmentation tors of the ligand at the desired binding site as and losses from the slide. well as the inhibitory equilibrium binding con- An advantage of autoradiographic binding stant (Ki) of each. It is best to employ an assays over homogenate designs is the ability unlabeled competitor at concentrations not to sample the binding incubation buffer directly above 1000 × Ki, resulting in occupancy of and analyze it during the course of the assay. In 99.9% of binding sites. Use of unnecessarily the early phases of assay development, direct higher inhibitor concentrations increases the measures of free ligand concentration and chro- possibility of detecting unwanted, secondary matography of the radioligand after tissue ex- sites rather than the binding site of interest. In posure can assist with detection of unantici- the sections to follow, it is assumed that a pated technical artifacts. If ligand depletion definition of nonspecific radioligand binding resulting from metabolism is noted, a lower has been formulated and that its basis is the use incubation temperature should be considered. of a competitive inhibitor at an appropriate If ligand depletion resulting from nonspecific concentration relative to its Ki, the radioligand binding is noted, alteration of the buffer prop- concentration, and the radioligand’s equilib- erties to promote ligand solubility may help. If rium binding constant (Kd). depletion is due to specific binding, the volume of incubation buffer per tissue section must be Determination of Incubation and increased. Washing Parameters An initial parameter for assay optimization Receptor Binding After establishing an initial definition of is the post-incubation washing time that best Techniques specific radioligand binding, it is important to distinguishes specific from nonspecific bind- 1.4.6

Current Protocols in Neuroscience ing. Comparable tissue sections are usually ing level can be safely used in saturation analy- incubated in solutions containing labeled radi- ses. In practice, equilibration-time determina- oligand with and without unlabeled competitor tions are straightforward in design, involving to block specific binding. Sections are then incubations of tissue with radioligand for pro- assayed for radioligand activity after varying gressively increasing durations, followed by post-incubation washing times, including an routine post-incubation washing and assay of initial measurement without washing (i.e., a bound tracer. A caveat, however, is the possible “zero wash time,” or t = 0 sample). These initial loss of intact binding sites in tissue sections sections are processed by very brief (1 to 2 sec) during prolonged incubations. This may arise, dipping in buffer lacking both the ligand and in theory, because of solubilization of some competitor to remove binding assay buffer from sites into the binding buffer. Alternatively, the slide surface. This step is important for losses of high-affinity ligand recognition sites assessing the possibility of loss of labeled sites may reflect proteolytic degradation or dissocia- during prolonged washing. Varying wash times tion of multiple proteins or cofactors that are are assessed, comparing total, specific and non- required together for ligand binding. If ligand- specific bindings to the initial t = 0 estimates. binding sites are lost over the interval of the Optimal post-incubation washing procedures assay, apparent equilibration may take place, should reduce nonspecific binding to a mini- but the data will actually reflect competition mum, but not promote loss of any specific between increasing ligand binding and decreas- binding detected in t = 0 samples (Frey and ing total binding capacity (Bmax). This aspect Howland, 1992). Prolonged washing proce- of ligand equilibrium can be controlled by con- dures that enhance specific over nonspecific ducting parallel assays at the lowest ligand binding, but at the expense of lost specific concentration of interest and at a higher, satu- binding, run the risk of preferentially revealing rating concentration where binding equili- a subset of binding sites. Unless the nature of brates more rapidly. The low-concentration specific binding that remains versus that poten- data are used to assess equilibration rate, while tially lost in these processes is understood, lack of decrease in binding over time at the high experimental results may be biased and results concentration will exclude the possibility of of studies employing distinct radioligands tar- binding-site losses during incubation. Desir- geting the same site(s) may be discordant. able assay characteristics include incubation Estimates of specific-to-nonspecific label- times sufficiently short that no detectable losses ing of tissues derived from whole-section as- of binding sites occur, but which readily permit says may actually provide minimum values in equilibration of sub-Kd ligand concentrations. comparison with autoradiographic assays Ligands that do not readily equilibrate at low when 3H-labeled ligands are employed. This concentrations may, nevertheless, be useful for phenomenon is due to the likelihood that wash- assays of regional Bmax distribution alone. ing is most effective at the section surface, and However, it is recommended that use of a near- most protracted at the section-slide interface saturating ligand concentration for this purpose where the diffusion distance to reach the buffer be used to guard against effects of possible solution is greatest. Thus, the limited tissue- unrecognized affinity differences that cannot depth penetration of 3H β− particles may result be explored directly because of protracted in autoradiographic images of the most favor- equilibration times at low ligand concentration. able specific-to-nonspecific binding strata. The appropriate tissue incubation time to Saturation Studies achieve equilibrium in the radioligand assay In addition to regional mapping of ligand- should be determined prior to conducting satu- binding sites, assays to determine the pharma- ration assays or other pharmacological charac- cological properties of saturable binding sites terizations. Since the forward ligand-receptor can be conducted by quantitative autoradiogra- binding rate is a second-order process, it is phy (Penney et al., 1981). In particular, satura- sensitive to ligand and receptor concentration tion binding assays are indispensible for evalu- effects. The time required to achieve equilib- ation of new radioligands in autoradiography. rium is, therefore, a function of radioligand Although in vitro homogenate binding assays concentration, and will be longest at the lowest are often initial sources for selection of ligands ligand concentration studied. Thus, it is useful for autoradiographic use, it is uncommon that to have initial estimates of radioligand Kd (see kinetic association and dissociation binding UNIT 7.5) so that equilibration time for ligand rates or the equilibrium Neuroanatomical concentrations below the half-maximal bind- are identical in tissue homogenates and intact Methods 1.4.7

Current Protocols in Neuroscience tissue sections. Some differences may relate to ing similar receptor types and numbers. Suc- a higher degree of compartmentalization of cess of detailed multisite competition assays is sites in intact sections, limiting ligand diffusion enhanced if large, relatively invariant brain re- initially to a single dimension (perpendicular gions are selected (e.g., cerebral or cerebellar to the section face). The lack of vigorous tissue cortical regions, striatum, or major thalamic disruption in autoradiographic assays may ad- nuclei). In order to guard against unanticipated ditionally promote maintained association of anatomic receptor gradients, repetition of the effector coupling of other proteins with the total binding condition (absence of competitor) binding sites of interest, modifying the confor- should be performed at regular anatomic inter- mation and characteristics of ligand binding. vals throughout a series of sections. If the total Autoradiographic ligand-binding saturation binding does not change over the anatomic analyses should be designed and conducted in extent of the sections, differences may be safely parallel with in vitro homogenate assays. A ascribed to effects of the competitor. If subtle range of ligand concentrations is employed in gradients in total binding are detected, it may incubations of slides bearing a series of adja- be possible to interpolate between the control cent tissue sections. At each concentration, or sections and use an adjusted Bmax for each at regular, less frequent intervals, assessment anatomic level. of nonspecific binding is made in additional sections. A 100-fold range of ligand concentra- QUANTIFICATION OF LIGAND tions centered on the Kd constitutes an ideal BINDING selection for estimation of both affinity and binding capacity. As detailed previously, it is Basic Autoradiographic Principles important to verify technical aspects of the Quantitative receptor autoradiography is a assay conditions so that artifacts do not result logical extension of qualitative autoradiog- in erroneous saturation curves. It is important raphic techniques developed in the 1950s and to verify by direct assay of the binding buffer 1960s for localization of radioactive tracers in that ligand depletion has not occurred during tissue specimens (see Rogers, 1979, for re- the incubation. It is also important that suffi- view). All these techniques are based on the cient time be allowed to permit equilibration of apposition of a photographic film or emulsion ligand and binding sites. Each of these consid- to materials containing a radioactive source. erations is more important the lower the ligand The passage of charged particulate emissions concentrations used, and failure to properly such as electrons, β−, and β+ particles through design assays will result in an underestimation the silver halide crystals of photographic emul- of binding. These effects result in curvilinear sions results in the conversion of silver ions to Rosenthall (Scatchard) plots, mimicking ef- metallic silver. During development, these me- fects of cooperativity at interacting binding tallic silver ions catalyze the transformation of sites. whole silver halide crystals into metallic silver, with consequent opacification of the emulsion. Competition Assays While electromagnetic radiation in the form of Competition assays often require six or γ rays, X rays, or annihilation photons is also more distinct incubation conditions per binding capable of exposing X-ray film, direct interac- site to assess binding affinity and capacity. In tions between these radiations and photo- instances of multiple receptor subtypes that are graphic emulsions are much less efficient than distinguished by the competing ligand, as many those involving charged particulate emissions. as 15 to 20 parallel conditions may be neces- Thus, little if any contribution to tissue autora- sary. Homogenate binding studies are adapted diography with the radionuclides discussed to these designs, permitting a large number of previously is attributable to γ- or X-ray emis- individual samples with highly similar tissue sions. and binding-site compositions. In autoradiog- Numerous factors influence the response of raphic assays, however, anatomic gradients and autoradiographic emulsions to a radioactive variability from section to section make iden- source. These include the type of ionizing ra- tification of truly identical tissue samples dif- diation emitted by the source, the energy of the ficult. As a result, there is a methodological ionizing particles, the thickness of the speci- trade-off between the detail of the experimental men containing the radioactive source, the dis- design (number of replicate sections per condi- tribution of radioactivity within the specimen, Receptor Binding tion and number of conditions) and the ability the distance from the specimen to the emulsion, Techniques to select sufficient adjacent tissue sections bear- the thickness of the emulsion, and the density 1.4.8

Current Protocols in Neuroscience of silver halide crystals within the emulsion. In In film-contact autoradiography applica- quantitative receptor autoradiography, many of tions, the radionuclide used has a marked influ- these issues are not a major concern as long as ence on the choice of film and subsequent data experiments are performed in a standardized analysis. By far the most common radionuclide manner. is the β− emitter 3H, on the basis of its pre- viously-discussed favorable properties (see Contact Autoradiography discussion of Isotopic Label). The relatively The ligand–binding site interaction and the low energy of its β− emissions means that these radionuclide employed impose technical con- emissions travel shorter distances both through straints in autoradiographic binding studies. tissue sections and within film emulsions, ne- The reversible binding of most radioligands cessitating specialized autoradiographic tech- means that exposure to aqueous solutions after niques. Typical photographic and autoradiog- completion of the binding assay may result in raphic films are protected by a gelatinous “anti- unwanted losses in both the amount and scratch” layer overlaying the silver halide anatomic localization of bound ligand. This matrix, which is designed to reduce mechanical means that the traditional method for detection damage as well as chemographic and contact- and high-resolution localization of labeled induced silver reductions. In the instance of 3H, macromolecules (proteins labeled by amino these protective layers are too thick for β− acid incorporation or nucleic acids labeled by particles to effectively penetrate them, and the incorporation of labeled nucleotides)—i.e., resulting autoradiographic sensitivity is ex- dipping slide-mounted specimens directly in tremely low. Thus, specialized autoradiog- liquid photographic emulsions, may result in raphic films lacking the protective antiscratch migration or loss of label in binding-site autora- layer are necessary for contact autoradiogra- diography. As a result, methods have been de- phy. These films are more costly than conven- veloped to allow dry contact between labeled tional X-ray or other photographic films useful tissue sections and autoradiographic emulsions in autoradiography of higher-energy emitters, (Roth and Stumpf, 1969). In the first technique, and must be handled with great care to prevent which is an extension of the traditional emul- mechanical damage and exposure of the emul- sion-dipping method, acid-washed coverslips sion. Additional consideration must be given to may be coated with an appropriate nuclear the possibility of direct chemical interactions emulsion. Coverslips are dried, affixed to mi- between these emulsions and the section and croscope slides bearing the labeled tissue sam- slide, as is also possible with liquid-emulsion ple fixed at one end with contact cement, and and dipped-coverslip autoradiography. While then fastened with a removable clip at the op- specialized tritium-sensitive films will cer- posite end, apposing the emulsion to the sec- tainly work with 14C, 35S, and 32P, use of such tions. After exposure, the clips are removed and film in detection of the higher-energy emis- the coverslips are gently raised from the tissue sions of these isotopes is unnecessary. Conven- opposite the side of permanent attachment, to tional X-ray films or finer-grained mammogra- permit development and fixation of the emul- phy films work well for these radioisotopes; the sion. finer-grained emulsions afford optimal The more popular and more readily quanti- anatomic resolution with 14C and 35S, but at the fiable autoradiographic method is to appose expense of longer exposure times. tissue sections to an appropriate acetate-backed film. Using double-sided tape or contact ce- Film Densitometry and Radioligand ment, sections on microscope slides or Quantification coverslips are mounted on paper or cardboard Data analysis by film densitometry has be- and placed in a rigid, light-tight cassette. Film come straightforward through the availability is placed immediately on top of the sections and of commercial computer-assisted densitomet- the cassette sealed. The cassettes are stored in ric systems. The relative ease of quantitative a cool, dry environment for an appropriate densitometry conceals some potential pitfalls period, and then the film is removed and devel- in the analysis of autoradiographic data. It is oped. In this design, technical aspects of autora- important to recall that the relationship between diogram generation are simplified; however, incident-radiation exposure and optical density the structural registration of the image with on developed film is not linear over an indefi- corresponding tissue sections is less precise nite range of exposures. At higher levels of than with the emulsion-dipping or coverslip- exposure, optical density reaches a ceiling (film Neuroanatomical apposition techniques. saturation) beyond which it no longer reflects Methods 1.4.9

Current Protocols in Neuroscience additional incident-radiation exposure. Even 14C standards have been used to control for before saturation is reached, the relationship exposures arising from 18F (Olds et al., 1985), between exposure and film optical density de- 35S, 99mTc (Taylor et al., 1992), 125I, and 123I. viates significantly from linearity, with reduced On the basis of these experiences, it may be resolution of differences in incident-radiation predicted that extension of this method of intensity. It is important to prepare and analyze standardization to other nuclides listed in Table autoradiograms under conditions in which 1.4.1 should also succeed. there is a strong relationship between incident- Another important consideration in the in- radiation exposure and developed-film optical terpretation of [3H]ligand autoradiography is density. This precaution necessitates careful that differential absorption of emissions within attention to the duration of film exposure and tissue, known as tissue quenching, may alter uniformity of development conditions. imaging results. In brain tissue, lipid-rich white A related problem is that of radioactivity matter absorbs 3H emissions to a greater extent standards. To perform truly quantitative recep- than gray matter. This absorption differential tor autoradiography, tissue sections must be gives rise to apparent ligand-concentration dif- coexposed together with standards that provide ferences between brain regions that may in part an external reference for the relationship be- reflect differing gray versus white–matter com- tween radiation exposure and film optical den- positions. This effect may confer as much as a sity. Originally, investigators prepared their 2-fold difference in the autoradiographic image own standards by labeling brain paste homo- exposures of comparably labeled gray and genates (Freygang and Sokoloff, 1958; Pan et white–matter structures (Geary and Wooten, al., 1983) or plastics with known amounts of 1985). Differential effects of 3H quenching may radioisotopes (Reivich et al., 1969; Pan et al., be advantageous for anatomic localization of 1983). Plastic standards are now commercially binding signals through contrast-enhancing ef- available and widely used; they include exam- fects. However, in between-group experimen- ples of several radioisotopes used in receptor tal comparisons, alterations in tissue composi- autoradiography. Standards containing short- tion rather than binding-site numbers may con- lived radioisotopes must be replenished and found autoradiogaphic measures. recalibrated frequently. This is true even for 3H Three approaches have been employed to standards if they are to be used over a period of address the 3H quenching problem. One design years. Standards for 14C have the advantage of is to determine the relative changes in a refer- not requiring recalibration because of the very ence binding site (a “control” site known to be slow rate of decay, and can be cross-calibrated experimentally unaffected) in comparison to against other radioisotopes to provide a univer- the binding site of interest. If both sites appar- sal set of standards. This concept, however, has ently change by the same fraction between been challenged by evidence that 14C and 3H experimental groups, altered tissue composi- plastic standards may not always produce par- tion is the most likely explanation. Some allel optical-density changes. In the authors’ groups have developed procedures to eliminate experience, this may occur when film reciproc- lipids from dried tissue sections, thus, eliminat- ity is violated in either of two ways. If the ing the major source of regional quenching intensity of one, but not both, of the exposures differences (Happe and Murrin, 1990). Unfor- has saturated, the exposures will no longer vary tunately, most delipidation procedures result in proportionately. In the instance of 14C and 3H, denaturation or loss of ligand-binding sites. most film emulsions permit 14C access to The final approach to evaluating and cor- deeper levels, resulting in higher maximal op- recting for differential quenching effects is to tical density and later saturation than with 3H. make direct measures of attenuation of a dif- Additionally, if exposure times are very pro- fusely introduced label in parallel sections to tracted (more than several months), fading of those in the binding assays. For example, ter- the latent images resulting from 14C and 3H may minal in vitro labeling of proteins followed by occur at distinct rates that are due to differing their autoradiography has been employed suc- degrees of lattice distortion produced by depo- cessfully for analysis and correction of tissue sition of high versus low β− particle energies. quenching effects (Smith et al., 1995). Quench- Good correlations between relative 14C and 3H ing is not a significant source of bias or error in exposures are obtained if films are exposed for typical 20-µm-thick tissue sections when la- less than 3 months time and if care is taken to beled with the higher-energy-emitting radionu- 3 Receptor Binding avoid saturation of the H exposure. In addition, clides. Techniques 1.4.10

Current Protocols in Neuroscience ANALYSES OF tions can be processed for histochemistry or AUTORADIOGRAPHIC DATA other techniques that may reveal boundaries or tissue distinctions not appreciated with conven- Anatomic Distribution tional histology. Interpretation of autoradiograms requires the ability to identify reliably the correct re- Saturation and Competition Isotherms gions of interest. Regional anatomy can be Analyses of binding curves to estimate af- identified in several complementary ways. The finities and densities of sites are identical to the most direct of these is use of the autoradiogram post-processing analyses conducted on data itself. With many ligands, there is sufficient from in vitro homogenate binding studies. As detail and contrast in the images to establish mentioned previously, care should be taken to regional boundaries, even for small regions. assure that radioligand Kd estimates used to Fiber tracts typically express low levels of sy- make estimations of unlabeled ligand Ki values naptic binding sites, and in the instance of are appropriate to intact tissue sections (see tritium-labeled ligands, the higher lipid levels discussion of Detection of Specific Binding). of fiber tracts will further reduce emulsion Ideally, all important binding parameters would exposure (see discussion of quenching under be determined in each individual brain and Film Densitometry and Radioligand Quantita- region studied. This is often impractical, how- tion). Consequently, structures such as the cor- ever, and Kd values determined in repre- pus callosum, the deep cerebellar white matter, sentative samples of an experimental group the internal capsule, and and other myelinated must often be extrapolated to regional mapping fiber tracts are identified readily and may serve and unlabeled ligand competition experiments as anatomic landmarks for structural identifi- performed in other brains. Computer-assisted cation. Many binding sites also have regional analyses (Munson and Rodbard, 1980) are of gray-matter distribution patterns that permit particular value when multiple binding-site es- ready identification of selected regions. For timations are undertaken to assure that unbi- example, the striatal complex expresses ased parameter estimates are made and to per- uniquely high levels of several neurotransmit- mit proper statistical comparisons of alternative ter receptors, including dopamine D1 and D2 binding models. With typical autoradiographic receptors and adenosine A2 receptors, while measurement variances and difficulties in as- cannabinoid receptors are expressed at very suring stable anatomic receptor expression high levels within the globus pallidus (Herken- throughout a large series of adjacent tissue ham et al., 1990). Autoradiograms of binding sections, it is unusual to identify and separate sites with more widespread patterns of expres- more than two sites in most binding experi- sion—e.g., GABAA (Pan et al., 1983), excita- ments (Frey and Howland, 1992). tory amino acid (Greenamyre et al., 1985), or muscarinic cholinergic receptors (Wamsley et Combination with Other Anatomic al., 1980)—often contain sufficient regional Methods variation in expression levels to permit accurate Receptor autoradiography can be combined identification of regions. with almost any neuroanatomical method for Conventional histology can also be used to which tissue preparation is compatible with guide interpretation of autoradiograms. Sec- receptor studies, using alternate sections de- tions exposed to film can be processed sub- rived from the same blocks of tissue for indi- sequently for Nissl staining (see discussion of vidual assays. Unfixed, frozen tissue sections Combination with Other Anatomic Methods) can be used in a wide variety of neuroanatomic and then used for regional anatomic identifica- methods. In addition, some comparison meas- tion. Some image-processing systems (e.g., the ures can be made on the same tissue sections MCID System family from Imaging Research; used for receptor autoradiography. Routine his- see SUPPLIERS APPENDIX) support parallel process- tology with Nissl staining is readily accom- ing and analysis of multiple images, allowing plished on sections used for receptor binding superposition of Nissl-stained sections and after film exposure, by post-fixing the sections autoradiograms and direct transposition of over paraformaldehyde vapors. Sections are structural boundaries from the histologic to the placed in a tightly sealed container in a rack or binding images. Sections immediately adjacent on a platform suspended above paraformalde- to those chosen for autoradiography can alter- hyde powder. The sections are exposed to para- natively be used for histology. In addition to formaldehyde vapors by warming of the cham- Neuroanatomical conventional histological tissue staining, sec- ber for a minimum of 48 hr, and then stained Methods 1.4.11

Current Protocols in Neuroscience with 0.5% cresyl violet in the conventional Chapter 14 and UNIT 1.3 in this manual). Use of manner (see UNIT 1.2). This method produces this technique together with receptor autora- sections with excellent regional histology and diography on alternate sections allows comple- surprisingly good preservation of perikaryal mentary evaluations of regional mRNA and cellular morphology. The quality of these sec- protein expressions; the ligand-binding assay tions is more than adequate to establish regional provides the index of gene expression. boundaries and to confirm the presence of ex- Tissue preparations for receptor autoradiog- perimental lesions or cannula placements. raphy and immunohistochemistry are not gen- Another technique that can be readily com- erally compatible. Fixation necessary for the bined with receptor autoradiography and that optimal visualization of many antigens often can employ either identical or alternate sections makes receptor autoradiography impossible. is [14C]2-deoxyglucose (2DG) autoradiogra- There are, however, some exceptions. Some phy of cerebral glucose metabolism (Sokoloff proteins, such as the reactive astrocyte marker et al., 1977). The same tissue sections utilized glial fibrillary acidic protein (GFAP; see UNIT for 2DG studies can be processed for receptor 3.5), can be visualized by immunohistochemis- autoradiography after the metabolic autora- try following alcohol or acetone post-fixation diograms are initially obtained, or, more often, of fresh-frozen sections (also see UNIT 1.2). Fur- adjacent sections from [14C]2DG-labeled ther experimentation may expand the range of brains can be processed for ligand binding and neuroanatomical methods compatible with re- autoradiograms exposed in parallel. The latter ceptor autoradiography. design is favored, since rapid desiccation of tissue sections on a slide warmer enhances the Three-Dimensional Reconstructions anatomic resolution of the 2DG method, but An emerging technique, developed initially may diminish the recovery of binding sites. In for analysis of image data from positron emis- any event, it is important to incorporate addi- sion tomography (PET) of blood flow in the tional prewashes into the receptor autoradiog- human brain, involves the analysis of binding- raphy protocol to completely remove the site distribution data throughout the entire brain [14C]2DG from the tissue prior to binding as- volume at high resolution (Frey et al., 1996). says with lower-energy radionuclides. This approach has been explored for its exten- A number of additional techniques can be sion to experimental-animal autoradiographic applied to alternate sections harvested during imaging, with encouraging preliminary results cryostat sectioning. Many histochemical tech- after reconstructions of the entire brain from niques can be applied to fresh-frozen tissue autoradiograms at regular intervals (Kim et al., sections. While somewhat lacking in cellular 1994). Screening of the entire neuroaxis with- resolution, histochemistry on fresh-frozen sec- out the need for predefined regions of interest tions can give valuable information about re- or preconceptions regarding regional bounda- gional anatomy. Some investigators, for exam- ries may permit detection of unanticipated focal ple, have used cytochrome oxidase histochem- changes or of significant gradients within larger istry on fresh-frozen sections to estimate the regions, which are not evident in qualitative extent of experimental lesions (Gonzalez and examinations of autoradiograms. Conversely, Garrosa, 1991). It is also possible to quantitate these parallel analyses of large numbers of the intensity of many histochemical reaction independent regions (voxels) require conserva- products with densitometric methods analo- tive statistical adjustments against false-posi- gous to those used for receptor autoradiograms. tive results that may limit detection sensitivity. The absence of fixation eliminates a confound- Experimental designs that employ limited ing variable for measurement of reaction-prod- numbers of a priori regional hypotheses, but uct density and can allow at least semiquanti- which additionally include omnibus whole- tative estimation of enzyme activity. Some in- brain survey techniques, may afford the great- vestigators have developed methods to produce est overall utility. enzyme activity standards that can be processed in parallel with fresh-frozen tissue sections to CONCLUSION yield a densitometric equivalent of using solu- Ligand-binding autoradiography is a pow- tion histochemistry to estimate enzyme activity erful technique with many strengths and advan- (Biegon and Wolff, 1985). tages. Unlike most neuroanatomic techniques, Another useful neuroanatomical method it is readily quantifiable and provides numerical Receptor Binding that can be employed in fresh-frozen sections data that can be subjected to parametric statis- Techniques is in situ mRNA hybridization (see CPMB tical analyses. In addition to providing maps of 1.4.12

Current Protocols in Neuroscience binding-site distribution, it can also yield data epilepsy (Burdette et al., 1995). Receptor about binding-site pharmacology and regula- autoradiography in human postmortem tissue tion that is often comparable to that obtained can be used to investigate the pharmacology of from homogenate binding techniques with less human binding sites, evaluate the fidelity of anatomical resolution. While receptor autora- animal models of human disease states, and diography does not typically provide cellular- guide the selection of candidate probes for PET level resolution, it can provide excellent reso- or single photon emission computed tomogra- lution on a brain-regional basis; with careful phy (SPECT) studies of the living human brain. technique and selection of ligands it can distin- Finally, the general method of receptor guish and quantify small nuclear regions and autoradiography can be applied to almost any subregions. Binding-site autoradiography is ligand-binding-site interaction with suitable li- also very efficient with respect to numbers of gands and binding kinetics. Originally devel- experimental subjects needed in assays. oped for analysis of neurotransmitter receptor Autoradiography often permits analyses of populations, ligand-binding autoradiography small brain structures that would require pool- is now employed routinely to study plasma ing of tissue from several subjects to obtain membrane neurotransmitter reuptake trans- sufficient tissue for homogenate assays. Fur- porters, synaptic vesicular neurotransmitter thermore, it is technically easier to screen the transporters, receptor–G-protein coupling, and entire neuroaxis in autoradiographic assays biochemical second-messenger and ion-chan- than to dissect and analyze a comparable num- nel modulating effects of , as ber of discrete regions by homogenate binding well as enzyme expressions. The advantages of methods. Tissue harvested for receptor autora- high anatomic resolution and robust quantifi- diography may also be used for routine histol- ability ensure that the autoradiographic ap- ogy, in situ mRNA hybridization, and histo- proach will continue to provide valuable tools chemical techniques—allowing use of tissues for future neuroscientific research. for multiple complementary analyses. Autoradiography provides unique informa- LITERATURE CITED tion about the regional distribution of binding Biegon, A. and Wolff, M. 1985. Quantitative histo- sites and may allow identification of otherwise chemistry of acetylcholinesterase in rat and hu- unsuspected anatomical boundaries. The exist- man brain postmortem. J. Neurosci. Methods 16:39-45. ence of striatal “patches” or “striosomes,” for example, was documented first with µ-opiate Burdette, D.E., Sakurai, S.Y., Henry, T.R., Ross, D.A., Pennell, P.B., Frey, K.A., Sackellares, J.C., receptor autoradiography (Herkenham and and Albin, R.L. 1995. Temporal lobe ben- Pert, 1981). Many other neuroanatomical zodiazepine binding in unilateral mesial tempo- boundaries and features are characterized by ral lobe epilepsy. Neurology 45:934-941. unique patterns of expression of receptors and Frey, K.A. and Howland, M.M. 1992. Quantitative other binding sites. The quantifiability of li- autoradiography of muscarinic cholinergic re- gand-binding autoradiography also permits the ceptor binding in the rat brain: Distinction of documentation of more subtle changes such as receptor subtypes in antagonist competition as- says. J. Pharmacol. Exp. Ther. 263:1391-1400. subregional gradients of binding-site expres- sion that may not be qualitatively obvious. Frey, K.A., Minoshima, S., Koeppe, R.A., Kilbourn, M.R., Berger, K.L., and Kuhl, D.E. 1996. Stereo- The association of specific populations of taxic summation analysis of human cerebral ben- binding sites with specific populations of neu- zodiazepine binding maps. J. Cereb. Blood Flow rons or nerve terminals allows use of receptor Metab. 16:409-417. autoradiography as a quantitative measure of Freygang, W.H. and Sokoloff, L. 1958. Quantitative the integrity of these neuronal elements. Ligand measurement of regional circulation in the cen- binding to the dopamine transporter or vesicu- tral nervous system by the use of radioactive inert lar monoamine transporter, for example, have gas. Adv. Biol. Med. Phys. 6:263-279. become standard techniques for assessment of Geary, W.A. and Wooten, G.F. 1985. Regional tri- the integrity of nigrostriatal dopaminergic ter- tium quenching in quantitative autoradiography of the central nervous system. Brain Res. minals (Vander Borght et al., 1995). Receptor 336:334-336. autoradiography is an established tool in ex- Gonzalez, L.F. and Garrosa, M. 1991. Quantitative amination of normal human neuroanatomy and histochemistry of cytochrome oxidase in rat in the characterization of neuropsychiatric pa- brain. Neuroscience 123:251-253. thologies. Benzodiazepine receptor autora- Greenamyre, J.T., Young, A.B., and Penney, J.B. diography, for example, has been used to quan- 1985. Quantitative autoradiographic distribution Neuroanatomical tify neuronal losses in human temporal lobe Methods 1.4.13

Current Protocols in Neuroscience 3 of L-[ H]glutamate binding sites in the rat central by age and cognitive status. Neurobiol. Aging nervous system. J. Neurosci. 4:2133-2144. 16:161-173. Happe, H.K. and Murrin, L.C. 1990. Tritium quench Sokoloff, L., Reivich, M., Kennedy, C., Des Rosiers, in autoradiography during postnatal develop- M.H., Patlak, C.S., Pettigrew, K.D., Sakurada, ment of rat forebrain. Brain Res. 525:28-35. O., and Shinohara, M. 1977. The [14C]deoxyglu- Herkenham, M. and Pert, C.B. 1981. Mosaic distri- cose method for the measurement of local cere- bution of opiate receptors, parafascicular projec- bral glucose utilization: Theory, procedure and tions and acetylcholinesterase in rat striatum. normal values in the conscious and anesthetized Nature 291:415-418. albino rat. J. Neurochem. 28:897-916. Herkenham, M., Lynn, A.B., Little, M.D., Johnson, Taylor, S.F., Frey, K.A., Baldwin, R.M., Papadopou- M.R., Melvin, L.S., De Kosta, B.R., and Rice, los, S.M., Petry, N.A., Rogers, W.L., McBride, K.C. 1990. Cannabinoid receptor localization in B.J., Kerr, J.M., and Kuhl, D.E. 1992. Techne- brain. Proc. Natl. Acad. Sci. U.S.A. 87:1932- tium-99m-N1-(2-mercapto-2-methylpropyl)- 1936. N2-(2-propargylthio-2-methylpropyl)-1,2-ben zenediamine (T691): Preclinical studies of a po- Kim, B., Frey, K.A., Mukhopadhyay, S., Ross, B.D., tential new tracer of regional cerebral perfusion. and Meyer, C.R. 1994. Three-dimensional regis- J. Nucl. Med. 33:1836-1842. tration of MRI and volumetric autoradiography. Radiology 193:363. Vander Borght, T.M., Sima, A.A.F., Kilbourn, M.R., Desmond, T.J., Kuhl, D.E., and Frey, K.A. 1995. Munson, P.J. and Rodbard, D. 1980. Ligand: A [3H]Methoxytetrabenazine: A high specific ac- versatile computerized approach for charac- tivity ligand for estimating monoaminergic terization of ligand-binding systems. Anal. Bio- neuronal integrity. Neuroscience 68:955-962. chem. 107:220-239. Wamsley, J.K., Zarbin, M.A., Birdsall, N.J.M., and Olds, J.L., Frey, K.A., Ehrenkaufer, R.L., and Kuhar, M.J. 1980. Muscarinic cholinergic recep- Agranoff, B.W. 1985. A sequential double-label tors: Autoradiographic localization of high and autoradiographic method that quantifies altered low affinity binding sites. Brain Res. rates of regional glucose metabolism. Brain Res. 200:1-12. 361:217-224. Yamamura, H.I., Kuhar, M.J., and Snyder, S.H. Pan, H.S., Frey, K.A., Young, A.B., and Penney, J.B. 3 1974. In vivo identification of muscarinic cholin- Jr. 1983. Changes in [ H]muscimol binding in ergic receptor binding in rat brain. Brain Res. substantia nigra, entopeduncular nucleus, globus 80:170-176. pallidus, and thalamus after striatal lesions as demonstrated by quantitative receptor autora- Yamamura, H.I., Enna, S.J., and Kuhar, M.J. 1985. diography. J. Neurosci. 3:1189-1198. Neurotransmitter Receptor Binding, 2nd ed. Ra- ven Press, New York. Penney, J.B., Pan, H.S., Young, A.B., Frey, K.A., and Dauth, G.W. 1981. Quantitative autoradiography Yamamura, H.I., Enna, S.J., and Kuhar, M.J. 1990. of [3H]muscimol binding in rat brain. Science Methods in Neurotransmitter Receptor Analysis. 214:1036-1038. Raven Press, New York. Young, W.S. III and Kuhar, M.J. 1979. A new Reivich, M., Jehle, J., Sokoloff, L., and Kety, S.S. 3 1969. Measurement of regional cerebral blood method for receptor autoradiography: H-opioid flow with antipyrine-14C in awake cats. J. Appl. receptor labeling in mounted tissue sections. Physiol. 27:296-300. Brain Res. 179:255-270. Rogers, A.W. 1979. Techniques of Autoradiography. 3rd ed. Elsevier/North-Holland Biomedical Contributed by Kirk A. Frey Press, New York. University of Michigan Roth, L.J. and Barlow, C.F. 1961. Drugs in the brain. Ann Arbor, Michigan Science 134:22-31. Roger L. Albin Roth, L.J. and Stumpf, W.E. 1969. Autoradiography University of Michigan and Ann Arbor of Diffusible Substances. Academic Press, San Veterans’ Administration Medical Center Diego. Ann Arbor, Michigan Smith, T.D., Gallagher, M., and Leslie, F.M. 1995. Cholinergic binding sites in rat brain: Analysis

Receptor Binding Techniques 1.4.14

Current Protocols in Neuroscience