Neurotransmitter Receptor Localization: From Autoradiography to

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Neurotransmitter Receptor Localization: From Autoradiography to Imaging Mass Spectrometry Iván Manuel, Gabriel Barreda-Gómez, Estíbaliz González de San Román, Antonio Veloso, José A. Fernández, Teresa Giralt, and Rafael Rodriguez-Puertas ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/cn500281t • Publication Date (Web): 03 Feb 2015 Downloaded from http://pubs.acs.org on February 11, 2015

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ACS Chemical Neuroscience

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30th January 2015

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(Highlight in: Neurotransmitters and receptors)

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Neurotransmitter Receptor Localization:

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From Autoradiography to Imaging Mass Spectrometry

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Iván Manuel1§, Gabriel Barreda-Gómez3§, Estibaliz González de San Román1, Antonio

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Veloso2, José A. Fernández2, M. Teresa Giralt1, Rafael Rodríguez-Puertas1*

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§ Both authors contributed equally to this work.

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1

Department of Pharmacology, Faculty of Medicine and Odontology; 2Department of

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Physical Chemistry, Faculty of Science and Technology. University of the Basque

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Country (UPV/EHU). Leioa. 48940. Spain.

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3

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612, modulo 5, 48160 Derio, Spain.

IMG Pharma Biotech S.L. Parque Tecnológico de Zamudio. Astondo Bidea, ed. Kabi

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* Corresponding author

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ACS Paragon Plus Environment

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ABSTRACT: Autoradiography is used to determine the anatomical distribution of

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biological molecules in human tissue and experimental animal models. This method is

4

based on the analysis of the specific binding of radiolabeled compounds to locate

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neurotransmitter receptors or transporters in fresh frozen tissue slices. The anatomical

6

resolution obtained by the quantification of the radioligands has allowed the density of

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receptor proteins to be mapped over the last forty years. The data yielded by

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autoradiography identify the receptors at their specific microscopic localization in the

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tissues and also in their native microenvironment, the intact cell membrane.

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Furthermore, in functional autoradiography the effects of small molecules on the

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activity of G protein-coupled receptors are evaluated. More recently, autoradiography

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has been combined with membrane microarrays to improve the high throughput

13

screening of compounds. These technical advances have made autoradiography an

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essential analytical method for the progress of drug discovery. We include the future

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prospects and some preliminary results for imaging mass spectrometry (IMS) as a

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useful new method in pharmacodynamic and pharmacokinetic studies, complementing

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autoradiographic studies. IMS results could also be presented as density maps of

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molecules, proteins and metabolites in tissue sections, that can be identified, localized

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and quantified, with the advantage of avoiding any labeling of marker molecules. The

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limitations and future developments of these techniques are discussed here.

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Graphical Table of Contents

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Table of Contents

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. INTRODUCTION

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. LOCALIZATION OF NEUROTRANSMITTER RECEPTORS BY

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AUTORADIOGRAPHY

10

. EX VIVO AUTORADIOGRAPHY

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. IN VITRO AUTORADIOGRAPHY

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. FUNCTIONAL AUTORADIOGRAPHY FOR G PROTEIN-COUPLED

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RECEPTORS

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. AUTORADIOGRAPHY ON MEMBRANE MICROARRAYS

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. IMAGING MASS SPECTROMETRY (IMS)

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. CONCLUSION

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. INTRODUCTION

2 3

Autoradiography, as its name indicates, is based on the use of luminography to

4

generate images of slices of organs or tissues exposed to a radiosensitive film. The

5

development of this technique has gone from the first autoradiographic images obtained

6

from experimental animals after their treatment with radioisotopes ex vivo [1], to the

7

current images of the whole animal obtained by phosphor imager equipments [2].

8

Several variants of autoradiography have been developed, but all of them share the basis

9

of a detection method with spatial information about the distribution of radioactive

10

signals after being exposed to photosensitive films or to a photographic emulsion. The

11

sections are usually obtained either from animals treated with a radioligand (ex vivo) or

12

from tissue sections incubated with a radiolabeled compound in vitro [3-6].

13

Over the last few new techniques or variants which provide density maps of

14

anatomical images, but using non-radioactive detection methods, such as some mass

15

spectrometric methods have been developed [7-8]. These techniques, that avoid the use

16

of radioligands, are still limited by some technical problems, which mainly affect both

17

the type of compounds or biological substances that can be detected, and the image

18

resolution. These limitations, together with the standardization achieved by

19

quantification in autoradiography have made this technique an irreplaceable procedure

20

for the anatomical quantification of proteins such as membrane receptors or

21

neurotransmitter transporters, and are necessary for a multitude of pharmacodynamic

22

studies [9]. Radioactive labeling can also be used to determine the spatial profile of

23

different compounds that recognize molecules, such as DNA and RNA, by in situ

24

hybridization (e.g. specific sequences of nucleotides) or even to recognize proteins by

25

using immunohistochemistry. However, although these techniques could also be

26

considered as autoradiographic methods, the present work is focused on the application

27

of autoradiography in pharmacodynamic studies for drug discovery and the evaluation

28

of imaging mass spectrometry (IMS) as a new complementary method.

29 30

In particular, this review concerns the application of autoradiography techniques to the study of neurotransmitter receptors, an important class of drug targets.

31

The anatomical distribution of the myriad of subtypes of both super-families of

32

neurotransmitter receptors, ionotropic and G protein-coupled receptor [10-14], has been

33

characterized to a great extent thanks to the use of autoradiography. In relation to


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ionotropic receptors, the nicotinic for acetylcholine, ionotropic glutamate and purinergic

2

P2X receptors are examples of channels through which diverse cations flow, while other

3

receptors such as GABAA subtype for gamma-aminobutyric acid, are permeable for

4

anions [11-13]. Some second messengers, such as cyclic nucleotide-gated channels, also

5

activate some ion channels and can be identified by autoradiographic methods. The

6

other family of neurotransmitter receptors is constituted by the G protein-coupled

7

receptors (GPCR) [14,15]. The rhodopsin family is the most abundant and is composed

8

by monoamine, peptide and prostaglandin receptors, as well as odorant, visual and some

9

kinds of viral receptors. Therefore, the molecules that recognize the different subtypes

10

of GPCR are potential markers, when labeled by radioisotopes, to localize their

11

receptors by autoradiography. These molecules can be endogenous substances such as

12

neurotransmitters, but also synthetic compounds that bind to their specific binding sites

13

interacting with aminoacid residues usually located at the transmembrane domains, that

14

constitute the binding pocket. The intracellular loops of this family of receptors are

15

coupled to guanine nucleotide binding proteins (G protein) [16].

16

Autoradiography can also be used to quantify and localize the incorporation of

17

radioactively labeled GTP analogues in the α subunit of G protein. Thus, functional

18

autoradiography has allowed the localization of the activity or effects elicited by GPCR

19

agonists by quantifying the increase in the labeling with [35S]GTPγS. Figure 1

20

summarizes the main steps in the different autoradiography formats; in vitro, ex vivo

21

and functional autoradiography. For ex vivo autoradiography the radioligand is

22

administered directly to the live animal. After euthanize the animal the tissue is

23

dissected and cut and mounted on slides in a cryostat. However, the slides are only

24

incubated with the radioligand for in vitro autoradiography. Functional autoradiography

25

could be considered a subtype of in vitro autoradiography. The following steps are

26

common for all variants of autoradiography and include washing of the unlabeled

27

radioligand, and exposure to a radiosensitive plate such as a film to obtain the density

28

images that are then analyzed by computer software (Figure 1).

29

The study of the anatomical distribution of radioligand binding sites and the

30

activation of G proteins by specific neurotransmitter receptors, are the main in vitro

31

applications of autoradiography. The advantages and limitations of each procedure and

32

the present and possible future developments of the technique are discussed.

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1

.

LOCALIZATION

2

AUTORADIOGRAPHY

OF

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NEUROTRANSMITTER

RECEPTORS

BY

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The anatomical localization of neurotransmitter receptors was one of the first

5

and most widely used applications of autoradiography. It is common to perform the first

6

pharmacodynamic experiments for the pharmacological characterization of a compound

7

in cell membrane homogenates of a dissected tissue, since it is faster and more

8

economical. However, the microscopic information about the density of binding sites is

9

only obtained by the use of autoradiography in tissue sections, and its use is therefore

10

also necessary for small-sized regions that cannot be efficiently dissected.

11

Furthermore, GPCR genes represent up to 1% of the total genome of

12

mammalians. In the human genome, it is estimated that approximately 1,000 genes for

13

GPCR are expressed. The high number and diversity of receptors has made the GPCR

14

one of the principal targets for drugs, especially in the Central Nervous System (CNS)

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[17]. The development of novel high throughput screening (HTS) techniques is

16

allowing the pharmacological characterization of a large number of molecules

17

synthesized by the pharmaceutical industry. Autoradiography is also becoming a HTS

18

technique and its use is going towards non-radioactive methods.

19 20

Ex vivo autoradiography. The use of radioligands and the analysis of binding sites in

21

tissue membrane homogenates have allowed the discovery and pharmacological

22

characterization of multiple types of receptors for neurotransmitters and hormones for

23

many years. These experimental approaches presented a great limitation regarding the

24

anatomic information that can be obtained until the development of the whole body

25

autoradiography technique by Ullberg in 1954, based on previous studies in animals on

26

the administration of radioisotopes. He treated mice with

27

sections were exposed to a film [18]. Since then, this technique has been improved with

28

new protocols, equipment and image analysis software. Basically, the whole body

29

autoradiography technique consists of administering a molecule labeled with a

30

radioactive isotope (such as

31

whole body is quickly frozen; optionally it can be embedded in inert polymers, such as

32

carboxymethylcellulose. Sections around 30-60 µm are obtained in a heavy-duty

33

research grade cryostat for whole body sections (Leica CM 3600XP). These sections

34

can be mounted onto large slides or even on adhesive tape, then dehydrated and exposed

14

C, 3H,

125

35

S-penicillin and tissue

I) to an animal. After killing the animal, the


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to X-ray films or to phosphor-imaging plates, together with the corresponding

2

standards. The images are calibrated transforming the optical densities into radioactive

3

units per weight (usually nCuries per gram of tissue equivalent, nCi/g t.e.) and then the

4

optical density of the different tissues of interest is quantified using image analysis

5

software [6, 19].

6

The main advantage when comparing whole body ex vivo autoradiography to in

7

vitro autoradiography is that it enables the distribution of a drug in all organs and tissues

8

to be studied simultaneously. But both types of autoradiography enables the

9

microscopic localization of drug binding sites in discrete areas such as the layers of the

10

cerebral cortex that can not be clearly dissected to perform binding studies in tissue

11

homogenates. Then it is possible to obtain very valuable information of

12

pharmacokinetic parameters regarding the absorption, distribution and excretion of the

13

compounds. However, the pharmacokinetic processes can also modify the calculation of

14

the pharmacodynamic parameters in ex vivo autoradiography, such as the maximal

15

density of receptors (Bmax) that could be better estimated by in vitro experiments.

16

The exposure time of the sections to films depends not only on the activity of the

17

radioligand, but also on the density of the binding sites in the analyzed tissue slice. The

18

sophistication of the exposure methods with phosphor-imager systems permits the

19

images to be obtained within a few days, or even hours, in comparison with the weeks

20

or months that are necessary with beta radiation sensitive films. The exposure time is a

21

factor, which restricts the use of short-lived isotopes, such as

22

main limitation is the sectioning of the tissue within the short time window (a few

23

hours) before the counts decay away, especially with C-11 [20]. Radioisotopes with a

24

longer decay factor, such as

25

One of the limitations of ex vivo autoradiography is that the technique cannot

26

distinguish directly between specific and nonspecific binding sites. A limitation

27

common to all the methods based on the detection of radioligands, is that they involve

28

the modification of the native ligand structure that may change the affinity and

29

therefore, the selectivity of the original molecule. Moreover, in ex vivo autoradiography

30

the possible formation of metabolites, which can have different pharmacodynamic

31

properties from the original drug, also exists. Additionally, the radioactive

32

contamination of the equipment, organic material and the need of authorized labs and

33

workers to carry out the experiments, are important handicaps for the use of whole body

34

autoradiography. In this context, the section thickness is restricted by the

125

I, 3H,

35

S or

32

90

Y,

11

C or

18

F, but the

P are a better choice for in vivo studies.


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cryomicrotome and this feature is important to define the use of the right standard scales

2

that are necessary for image calibration and, therefore, for the evaluation of the

3

radioligand concentration in each tissue or organ.

4

Microautoradiography is a "smaller variant" of quantitative whole body

5

autoradiography that was developed during the 1960s [21]. Microautoradiography

6

consists, as does ex vivo autoradiography, of the systemic administration of some

7

molecules labeled with radioisotopes (usually 3H,

8

dissected, as in ex vivo autoradiography, and frozen. Next, tissue is cut into 4 µm

9

sections. These cryosections are mounted on photographic emulsion-coated slides,

10

which are placed in a dark box in the presence of desiccants for a period of time that is

11

empirically determined. After the exposure period, the entire slices are developed

12

following a similar procedure to that of photographic films [21, 22]. This technique has

13

been used to identify the cellular targets for drugs or endogenous metabolites, and has

14

contributed to the characterization of new drugs providing valuable microscopic

15

information about the distribution of receptors [23, 24].

16 17

14

C,

35

S or

125

I). Then, the animal is

These techniques provide a high-resolution tool for pharmacological research to localize the ligand binding sites at cellular and subcellular resolution [25-27].

18 19

In vitro autoradiography. In vitro receptor autoradiography was one of the first

20

molecular techniques used to observe the localization of the receptors or transporters in

21

sections of biological samples, and consists of the incubation of tissue slices with the

22

radioligand. Autoradiography of tissue sections has similar experimental requirements

23

as those of membrane homogenates in relation to the properties of ligand-receptor

24

interactions (e.g. affinity, selectivity, nonspecific binding), incubation conditions

25

(temperature, pH, time) and in the pharmacological analysis of the results. During the

26

screening and development of drugs, it is necessary to pharmacologically characterize

27

their affinity by calculating parameters such as the dissociation constant (Kd) and the

28

maximal number of binding sites in a tissue (Bmax). In addition, in vitro differs from ex

29

vivo autoradiography in that there is the possibility of controlling other factors that may

30

modulate the specific binding of a compound to the target protein or to nonspecific

31

binding sites. Therefore, experimental factors such as the variations of pH, temperature

32

or incubation time can be specifically analyzed to obtain the optimal incubation

33

conditions for each radioligand.


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These radioligand binding techniques are useful in several scientific areas, but they

2

became especially important in biomedicine, where they have been employed for the

3

development of new molecules or drugs for therapeutic use. Moreover, autoradiography

4

has allowed mapping of the distribution of a large number of receptors, as well as

5

providing information about some aspects of their physiological role.

6

As was commented on above, receptor autoradiography is based on the analysis

7

of the binding of a radioligand to a receptor for this molecule. The in vitro assays are

8

relatively simple, although one must take into account that the peculiarities of each drug

9

or compound could produce nonspecific binding sites to the slide surface or to the

10

coating (gelatin etc.) or to lipids presents in the tissue. The nonspecific binding is

11

measured in a consecutive slice (on another slide), by incubating it with the radioligand

12

but in the presence of a displacing compound. The compound must to show a high

13

affinity and selectivity for the receptor and must be used at a concentration high enough

14

to displace or inhibit the radioligand binding. Figure 2 shows the images obtained in rat

15

brain in a representative in vitro autoradiography experiment with a neuropeptide

16

labeled with the [125I] isotope. The images in the lower row show the nonspecific

17

binding of the radioligand that can be observed in each of the slices at the same

18

intensity (Figure 2). Sometimes, the analyzed receptor protein has special requirements

19

to be labeled, e.g. presence of Na+. In general terms, the assay of autoradiography

20

requires: 1) pre-incubation of the tissue sections mounted on slides in the presence of an

21

adequate buffer for a period of time and at a temperature that facilitates the removal of

22

possible endogenous ligands; 2) incubation of the tissue in the presence of a radioligand

23

in the appropriate conditions of temperature, pH, presence of ions, solubility etc., to

24

produce the binding; 3) the washing of the excess of radioactive ligand, and immersion

25

of tissue in cold deionized water to remove salts; 4) exposure to either a photographic

26

film, a photographic emulsion or a phosphor-imager plate, after drying the slides in a

27

stream of cold air. It is common to obtain digitalized images that can be calibrated with

28

radioactive standards and analyzed by different software (ImageJ is one of the most

29

widely used) to quantify the parameters of interest by measuring the optical densities.

30

The results are usually expressed as fmol/mg or nCi/g of tissue equivalent.

31

The most common application of in vitro autoradiographic labeling of receptors

32

has been to produce quantitative maps of the anatomical distribution of receptor

33

proteins. Therefore, the information obtained from the assay contributes not only to the

34

anatomical and pharmacological characterization of the receptor, but also suggests its


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possible physiological roles. This technique allows the analysis of the receptor in

2

different physiological states and in different stages of development or aging, as well as

3

in different stages of a given disease such as Alzheimer’s [28,29]. It is also possible to

4

determine some changes in the density of receptors after administration of drugs [30].

5

This technique can be performed both, in postmortem tissue from animals and humans

6

[31]. It is also possible to determine the pharmacological parameters (Kd, Bmax and Ki)

7

of a compound in microscopic areas by carrying out experiments of saturation or kinetic

8

inhibition. Moreover, it is also possible to microscopically identify and characterize

9

different receptor subtypes using subtype-selective drugs in specific brain nuclei or

10

areas [32].

11

The in vitro assays present some clear advantages when compared to in vivo

12

experiments; they are simple, rapid and the radioactive material used can be handled

13

without generating large quantities of contaminated waste. Regardless of the type of

14

ligand used only the incubation of the slices in the presence of the radioligand with a

15

relatively low specific activity (few nCi) and volume (usually at nanomolar

16

concentrations) is required. In addition, the assays have a high reproducibility and

17

reliability index, since they are normalized and calibrated by the use of radioactive

18

standards as has been previously mentioned. Probably the most striking advantage is

19

being able to obtain high-resolution images of the spatial distribution of the binding

20

sites that are characteristic, for example, of a receptor subtype. These images can be

21

analyzed both qualitatively and quantitatively, but the anatomic resolution may be

22

limited by different experimental factors: the type and intensity of the emission energy

23

of the isotope, the distance between the radiation-emitting source and the emulsion or

24

the thickness and sensitivity of the emulsion. In addition, another disadvantage is the

25

sensitivity of this technique to pre- and postmortem phenomena, which have influence

26

on ligand binding, (e.g. asphyxia, drug treatments or the degree of tissue preservation).

27

Other limiting factors are the lack of availability of a highly specific radioligand with

28

comparable chemical properties to the cold ligand, and the receptor must be present at a

29

sufficiently high density in the tissue.

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1

.

FUNCTIONAL

2

RECEPTORS

AUTORADIOGRAPHY

FOR

G

PROTEIN-COUPLED

3 4

Functional autoradiography is a method that measures the first step in a key

5

intracellular signal transduction cascade coupled to neurotransmitter receptor

6

stimulation. Unlike “classic” autoradiography, this type also provides information about

7

the functionality of the receptors in postmortem tissues together with the anatomical

8

localization of the G proteins activated by a GPCR, and therefore, also indicates the

9

density of the activated receptors. Functional autoradiography allows the calculation of

10

the half maximal effective concentration (EC50) and the efficacy, the latter usually being

11

expressed as the maximal effect. This method, alternatively known as autoradiography

12

of the binding of [35S]GTPγS stimulated by agonist, always consists of the incubation of

13

the slices with the same radioligand to label the activated G proteins, but in the presence

14

of different “cold” drugs. The classical methods used for measuring the activation of G

15

proteins analyze the GTPase activity [33-35]. However, these assays analyze the

16

GTPase activity by measuring GTP hydrolysis, which is an indirect activity that can be

17

altered by other factors independently of G protein activation [36]. The binding of the

18

non-hydrolysable GTP analogue, guanosine 5'-(γ-[35S] thiotriphosphate) or [35S]GTPγS

19

directly reflects the activation of the G protein alpha subunit by the receptor, measuring

20

the exchange of GDP for GTP (or [35S]GTPγS) in the G protein (Figure 3). The

21

[35S]GTPγS binding assay has been used in reconstituted systems of purified proteins

22

[37], in membrane homogenates [37-40], in cells transfected to over-express GPCR [41-

23

42], in immunoprecipitation assays [43] and in in vitro autoradiography [44].

24

The first evidence of functional receptor-G protein complex in frozen tissue

25

sections was described in the study of Zarbin et al., 1983, in which the authors showed

26

that the binding of some β2 adrenergic agonists decreased in the presence of GTP

27

analogues [4]. Later, it was demonstrated that GTP presence produced a decrease of

28

high affinity agonist binding sites by converting these sites into low affinity binding

29

sites [45]. Therefore, they showed that the two states of affinity of the GPCR were

30

preserved, and this provided additional evidence of the interaction between the receptors

31

and the G proteins in fresh or frozen preparations. GTP analogues, such as

32

[3H]Gpp(NH)p or [35S]GTPγS, or tritiated GTP itself ([3H]GTP), were used in the first

33

approaches. The first autoradiographic study of localization of guanine nucleotides


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Page 12 of 44

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binding sites was performed in rat brain using [3H]Gpp(NH)p [46]. However, the most

2

frequently used radioligand since then has been [35S]GTPγS. The first study using

3

[35S]GTPγS was to detect G protein activity in the compound eye of Drosophila [47].

4

Subsequently, this technique was adapted by other authors to rat brain cryosections [3,

5

48].

6

[35S]GTPγS binding assays provide valuable information about the functionality

7

or activity mediated by GPCR. Consequently, the pharmacological parameters that can

8

be calculated are efficiency, expressed as Emax, and potency, expressed as EC50. Emax

9

represents the highest rate of stimulation produced by a drug in a system or tissue area,

10

while the second parameter, EC50, represents the concentration of the drug that induces

11

half the maximal effect. Determination of the EC50 by functional autoradiography

12

requires the incubation of consecutive sections of tissue (instead of tubes used for

13

binding studies in membrane homogenates) at different concentrations of ligand, in a

14

similar way to the assays to calculate the affinity (Ka or Kd) of a receptor with a

15

radiolabeled drug.

16

The functional autoradiography allows elucidating the activity evoked by a drug

17

in the different discrete areas of a tissue section, and enables one to classify the

18

compounds as either agonists or antagonists in a specific tissue. The former compounds

19

increase the binding of [35S]GTPγS up to the Emax level (total agonists) or below (partial

20

agonists), while the antagonists do not produce binding. On the other hand, the inverse

21

agonists inhibit the basal or constitutive binding [49]. Some authors are questioning the

22

cause of the constitutive activity and suggest that as some endogenous ligands are not

23

correctly removed during the experimental procedures these could contribute to the

24

basal activity. In fact, it has been reported that much of this activity present in rat brain

25

is due to stimulation of adenosine receptors by endogenous adenosine itself [50].

26

The type of G protein to which the GPCR are coupled limits the characterization

27

of ligands by functional autoradiography; only the activity of receptors that are coupled

28

to the Gi/o family of proteins can be reliably detected. The reason for not detecting other

29

G protein activities (Gs or Gq/11) is poorly understood. The low densities of the receptors

30

coupled to other subtypes, or of the Gs or Gq/11 proteins themselves, or low coupling

31

efficiencies might account for this problem [51]. Functional autoradiography can be

32

combined with receptor autoradiography. The anatomical information provided by both

33

methods is often similar for the same GPCR subtype but sometimes there may be slight


12

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1

differences [52]. This could be because the receptors detected in a specific area are not

2

active or coupled to Gi/o proteins (Figure 4).

3

The expression or function of GPCR may be altered during development, aging

4

or by different diseases. For example, alterations to different GPCR densities, such as

5

muscarinic receptors, have been described in Alzheimer's disease [28]. Functional

6

autoradiography is used to evaluate if modifications to receptor densities during disease

7

states or following chronic drug treatments are compensated by a modulation of

8

receptor activity.

9

Functional autoradiography is complementary to receptor autoradiography with

10

radiolabeled drugs and offers the possibility of quantifying an effect mediated by a

11

GPCR. This functional technique supplies information about GPCR that are active,

12

while conventional autoradiography shows the localization of all receptors,

13

independently of their activation. Therefore, functional autoradiography can help to

14

discriminate the agonistic or antagonistic actions of a drug in each anatomical area as it

15

is shown for the muscarinic receptors in Figure 4. Conventional autoradiography

16

provides information about receptor densities; i.e. the receptor protein that is

17

radioactively labeled by the agonist or antagonist compound. Conversely, functional

18

autoradiography localizes the densities of Gi/o proteins coupled to GPCR, allowing one

19

to observe, for example, functional alterations in pathological processes in which

20

receptor density may or may not be modified. The versatility of functional

21

autoradiography, which permits a great variety of different subtypes to be analyzed

22

simultaneously in a single experiment (consecutive slices), is also an advantage in

23

studies directed at comparing differences among GPCR subtypes, as the same

24

[35S]GTPγS radioligand is the responsible for the different signal maps.

25 26

. AUTORADIOGRAPHY ON MEMBRANE MICROARRAYS

27 28

Autoradiographic studies in tissue sections require highly qualified staff to

29

process the samples and to analyze the results. This fact makes its automation difficult

30

and therefore limits its use as a high throughput screening technology. On the other

31

hand, and as has been previously explained, studies in membrane homogenates showed

32

several limitations when used in drug discovery. One of the most important ones is that

33

a relatively large amount of sample is required. Thus, the autoradiography technique can

34

be combined with membrane microarrays to overcome these limitations. In a membrane


13


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Page 14 of 44

1

microarray each spot is made up of only nanograms of membrane and the microarray

2

analysis can be automated. In addition, a large number of tissues, cell types or proteins

3

can be tested simultaneously in a single experiment.

4

Microarray technology traditionally consists of immobilizing a set of molecules,

5

such as DNA [53], proteins [54], carbohydrates [55], drug-like compounds [56], cells

6

[57], tissues [58] or membrane homogenates [34], on a solid support, which is usually a

7

microscope slide. Depending on the type of molecule to be captured, the surface of the

8

support can be coated with nitrocellulose, aminopropylsilane, polylysine, avidin, or

9

metals (e.g. gold and silver) or with derivatized with reactive groups such as n-

10

hydroxysuccinimide esters, aldehydes, or epoxy groups.

11

In the case of membrane microarrays, the glass surfaces used to immobilize the

12

cell membranes are typically coated with aminopropylsilane [34]. Other chemical

13

treatments have also been applied such as surfaces coated with cholesterol [59] or

14

phosphate groups [35, 60]. The membrane microarrays are made of a collection of

15

different membrane homogenates, isolated from diverse tissues or organs of

16

experimental animal models or even from human samples, as well as from transgenic

17

cells that over-express specific proteins. These microarrays could be employed in drug

18

discovery by the incubation in the presence of candidate compounds to become

19

therapeutic drugs, which could be labeled fluorescently [34] or radioactively [36]. After

20

incubation with the drug, the unbound compound is washed and the microarrays are

21

exposed to films, phosphor-imager or scanners and then analyzed by using specific

22

software. An example is shown in Figure 5, combining radioligand binding with

23

immunodetection of the receptor protein. In general, there is good correlation between

24

the density of radiolabeled receptors (muscarinic) and the immunolabeled receptor

25

protein (M1 subtype), but it is possible to detect nonspecific binding sites for both the

26

radioligand and the antibody. Therefore, the combination of different detection methods

27

in consecutive arrays also constitutes a test for different markers (Figure 5).

28

Using GTPγS autoradiography, these microarrays could be used not only to

29

determine the specificity and selectivity of the drug, but also to predict the functional

30

response. This kind of functional autoradiography allows one to identify the

31

pharmacological profile of drugs that act through G protein-coupled receptors. In this

32

context, membrane microarrays have been validated for the study of the functional

33

activity of diverse GPCR such as muscarinic, opioid and cannabinoid receptors with

34

Europium-GTPγS [61], as well as with [35S]GTPγS [35]. Figure 6 shows the application


14

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1

of functional autoradiography in the microarrays of cell membranes to evaluate the

2

GTPγS activation induced by a specific agonist in a concentration dependent manner

3

and simultaneously in different tissues. The example shows the functional

4

autoradiography of CB1 receptors using a specific cannabinoid agonist, WIN55,212-2.

5

CB1 receptors are expressed at much lower densities in peripheral tissues, such as the

6

stomach, compared to the CNS, and this type of peripheral tissue was included as a

7

reference region or negative control. In fact, one of the most important advantages of

8

the cell membrane microarrays is that this technology permits the simultaneous study of

9

several samples, providing a huge amount of information about the primary (desired)

10

and secondary targets (undesired and responsible for adverse effects) of the tested

11

compound in a single miniaturized assay. Therefore, the membrane microarray

12

autoradiography is also useful for predicting drug toxicity. This type of microarrays

13

shows additional advantages such as reducing the quantity of samples, drugs, chemicals

14

and radioactive residues. On the contrary, the use of membrane homogenates of

15

different brain areas can mask the binding of discrete brain areas or subregions.

16

New and promising developments in autoradiography are currently being

17

adapted to high throughput screening of compounds in membrane microarrays

18

composed of multiple spots from different samples, spotted together on a single slide.

19

This technique combines the versatility of the radioligand binding in membrane

20

homogenates with the anatomical and visual information provided by the

21

autoradiography, reducing the number of samples and radioisotopes to a minimum.

22

Membrane microarray autoradiography could be used in the drug discovery process as a

23

first screening of the pharmacological properties of newly synthesized compounds.

24

Furthermore, functional autoradiography can also be applied to these arrays to obtain

25

valuable information about the possible pharmacological effects of a drug on multiple

26

types of tissues and cells.

27

The applicability of autoradiography to localize drug targets within a tissue is

28

frequently limited for different reasons, some of them concerning the restrictions and

29

official authorizations that are required in order to manipulate radioactive materials in a

30

laboratory. Another difficulty of autoradiography that is inherent to the radiolabeling of

31

molecules is that the chemical modifications that are necessary for the introduction of

32

the radioisotope, sometimes involve a change in the molecular structure of the native

33

ligand that can affect its pharmacological properties (affinity, selectivity). In addition,

34

the detected labeling shows the distribution of the radioisotopes independently of


15


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Page 16 of 44

1

possible metabolic processes or degradation of the compound. Therefore, it is almost

2

impossible to distinguish between the original molecule binding sites and the

3

contribution to the final signal of the possible metabolites. Furthermore, it can take from

4

several days to several weeks of exposure time to films to obtain autoradiographic

5

images of sufficient sensitivity for distribution studies.

6

The above mentioned concerns related to the use of autoradiography have

7

increased the interest in the development of other techniques which avoid the use of

8

radiolabeled compounds.

9 10 11

. IMAGING MASS SPECTROMETRY (IMS)

12 13

Over the last few years mass spectrometry (MS) has emerged as a potent

14

technique of analytical chemistry with pharmacological applications [62]. Moreover,

15

some variations of the method such as matrix-assisted laser desorption-ionization

16

(MALDI) [63], secondary ion mass spectrometry (SIMS) [64] or desorption

17

electrospray ionization (DESI) [65], allow the identification of molecules by their

18

molecular weight, thereby avoiding the use of labeled compounds, as required other

19

drug screening techniques. MS permits the simultaneous detection and identification of

20

the compound or drug of interest and its metabolites. Simultaneously, hundreds of other

21

molecules present in the tissue can be detected. The mass and the fragmentation profile

22

are intrinsic characteristics of each molecule that help to match a peak with a particular

23

compound. During the last decade, MALDI, SIMS and other techniques have been

24

adapted in order to obtain maps of distribution of molecules in tissue sections (usually

25

fresh frozen). These methods are known as imaging mass spectrometry (IMS)

26

techniques and have been applied to determine the distribution of peptides and

27

metabolites in tissue slices, as well as lipids and administered drugs and their

28

metabolites [6, 66-70]. IMS emerged from the studies on desorption/ionization of

29

organic substances from surfaces [71] and on the identification of intact microorganisms

30

using MS [72-74]. Those initial studies were essential to the further development of the

31

first images analyzing the distribution of proteins and peptides desorbed directly from a

32

tissue [75-77]. At present, IMS is rapidly entering new biomedical fields, for example

33

research into novel methods for scanning tumor markers in tissue microarrays [78].


16

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1

Briefly, the IMS technique starts with the covering of a frozen tissue section

2

(10–20 µm), usually placed on a slide, with a suitable chemical matrix. Then, it is

3

introduced into the mass spectrometer under high vacuum. The mass spectrometer is

4

programmed to automatically scan the surface of the tissue, and the spectra obtained at

5

each coordinate of the grid are saved for further analysis. These spectra are then aligned

6

by maximizing the correlation with the overall averaged spectrum, normalized using the

7

total ion current, or any other suitable algorithm, and exported to computer software that

8

creates the images of each peak. A color-coded image can be created by different image

9

analysis software. The results can be similar to the autoradiograms regarding the spatial

10

resolution and density map distribution (Figure 7).

11

The main advantage resides in its ability to draw the distribution of many

12

substances and metabolites in a single experiment, allowing the identification of a high

13

amount of molecules by their mass/fragmentation information in m/z databases, and

14

more importantly, without any previous labeling with fluorescent probes or radioactive

15

isotopes. In addition, the time required to obtain the results from a single section in a

16

MALDI-IMS experiment can be as low as a few hours, while autoradiography requires

17

many hours or even days or months, depending on which phosphor imaging equipment

18

is used, the type and activity of the radioisotope, or if tritium and films are used.

19

However, the main problems currently faced by the IMS techniques are related to the

20

limited detection of some molecules, such as drugs. The localization of the binding sites

21

of a drug requires a technique able to detect them at nanomolar or femtomolar

22

concentrations. Therefore, the use of IMS to accurately detect and quantify drugs in

23

tissue sections is still less efficient than that of autoradiography, although it is rapidly

24

approaching its level of accuracy. Nevertheless, the previously mentioned features of

25

this technique suggest that IMS will replace autoradiography over the next years,

26

although further development of the equipment, experimental protocols and analysis

27

methods is required in order to overcome its current limitations.

28

In a preliminary study, we tried to detect the in vitro localization of the binding

29

sites for different drugs such as the cannabinoid compounds WIN55,212-2 or AM251 in

30

rodent brain sections (Figure 7). This was done by incubating the slices in a buffer

31

supplemented with the drug in a range of concentrations that would be able to saturate

32

the target receptors, mimicking the protocol used for autoradiography. We expected that

33

saturating the receptor binding sites would allow us to detect drugs in specific regions

34

of the brain where the receptors are located. The ionization of the drug was previously


17


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Page 18 of 44

1

tested using the same chemical matrix, the 2-mercaptobenzothiazole (MBT). In addition

2

the possible ion suppression of the compound was discarded because we also were able

3

to detect both cannabinoid compounds when deposited together with the MBT matrix

4

directly over the sections. The analysis of the obtained IMS data in these preliminary

5

experiments indicated that in the case of slices incubated with WIN55,212-2 (10-6M), an

6

intense peak was detected in the mass range of the cannabinoid compound (m/z

7

548,5508) with a similar distribution to that of the CB1 receptor. The mass could

8

correspond to the MBT + WIN55,212-2+H+, but after a further analysis of a consecutive

9

slice incubated in the same buffer, but without WIN55,212-2, we obtained the same

10

peak but with a lower intensity. Therefore, we concluded that it did not correspond to

11

the WIN55,212-2 compound and it could be a biomolecule present or formed in vitro in

12

the tissue (Figure 7). However, another limitation of the IMS technique is to assign the

13

obtained m/z values with specific molecules that depend on the robustness of the

14

databases. To the best of our knowledge there are not specific databases for CNS

15

compounds. Nevertheless, we could identify this peak by comparing the results in

16

different databases with a ceramide (d18:0/18:1), which was also described to be up-

17

regulated in rodent models of brain injury [79]. That is, the addition of WIN55,212-2 to

18

the buffer would activate CB1 receptors and induce the production of ceramide. To

19

validate this possibility it could be interesting to do MS/MS fragmentation or try to

20

make the experimental ionization conditions even more restrictive. However, both

21

experimental approaches are complicated to be applied for discrete areas in tissue

22

sections. The IMS technique is evolving rapidly and the accuracy of molecule databases

23

is continuously improving. Furthermore, the physiological information can also help to

24

identify the molecules. Interestingly, cannabinoid activation by the CB1 receptor could

25

induce the sphingomyelin hydrolysis producing ceramides, at least in glioma and glial

26

cells [80,81]. Further future developments and experiments will provide us how useful

27

IMS is to obtain pharmacological information similar to that obtained by the

28

autoradiography of radioligands.

29 30

. CONCLUSIONS

31 32

The incubation of tissue sections with radioligands and the subsequent analysis

33

of the specific binding sites by image analysis systems has been a very useful and

34

unique technique to obtain autoradiograms that indicate the distribution of the targets


18

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1

for drugs, commonly neurotransmitter receptors and transporters. During the last decade

2

the technique has been adapted in order to obtain information related to the activity of

3

the Gi/o proteins coupled receptors, i.e. to quantify the effect, with anatomical

4

resolution, that a drug is able to induce in specific areas of a tissue. This adaptation has

5

allowed the study of the effect of compounds acting through GPCR without labeling

6

them and only [35S]GTPγS is required. New and promising developments of

7

autoradiography are currently being applied to high throughput screening of compounds

8

in membrane microarrays composed of multiple spots from different tissues from

9

experimental animals or humans and cells in cultures spotted together on a single slide.

10

The autoradiography of cell membrane arrays combines the versatility of the

11

radioligand binding in membrane homogenates with the anatomical and visual

12

information provided by autoradiography, reducing the quantities of samples and

13

radioisotopes to a minimum. Membrane array autoradiography will accelerate the drug

14

discovery process principally as a first screening of the pharmacological properties of

15

newly synthesized compounds. Furthermore, functional autoradiography can also be

16

applied to these arrays providing valuable information about the possible

17

pharmacological effects of a drug on multiple types of tissue and cells.

18

Additionally, autoradiography is facing some restrictions that are inherent to the use of

19

radioactive isotopes, and some companies that developed reagents and materials for

20

autoradiography have tried to substitute them with fluorescent or luminescent labels

21

with relative success, since reaching the quantification in the femtomolar range of the

22

radioisotopes is complicated. Obtaining high resolution is one of the major challenges

23

facing IMS if it is to substitute autoradiography in the future. Also, the identification of

24

compounds without any labeling, just by their exact molecular mass and ionization, is

25

necessary for the success of IMS. It is not an easy task, but important research carried

26

out by multidisciplinary teams of pharmacologists, biochemists, synthetic chemists and

27

physical chemists are leading to the development of new mass spectrometers based on

28

MALDI and SIMS methods, and to new ways of ionizing the molecules so they can be

29

specifically recognized in the detectors. The future of autoradiography probably lies in

30

IMS and drug discovery departments should begin to take this into consideration and

31

become familiar with this technique. However, it is necessary to improve the method for

32

the detection of femtomolar concentrations of drugs in both tissue sections and in

33

membrane microarrays. Some of the factors precluding the use of IMS for

34

neurotransmitter receptor localization include avoiding the ion suppression of the


19


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Page 20 of 44

1

compound and the right assignment of the obtained m/z values to specific molecules

2

that depend on the accuracy and specificity of large databases. Specific databases for the

3

CNS could also improve the interpretation of IMS results related to molecules involved

4

in neurotransmission. The assignment could also be validated by combining different

5

methods such as MS/MS fragmentation coupled to HPLC and by increasing the

6

selectivity of the experimental ionization conditions e.g. developing selective matrices

7

for each type or chemical family of molecules. The application of IMS to different

8

experimental models can provide physiological information, which is helpful not only

9

for the identification of the molecules but also for understanding their modulation.

10

Future developments in the IMS technique could lead to it being able to fulfill

11

most of the screening requirements for the initial pharmacological characterization of

12

new drug candidates. Until that occurs, autoradiographic techniques will continue to be

13

used for their initial purpose, which is that of anatomically localizing drug targets.

14 15

KEY WORDS

16

Autoradiography, microarrays, [35S]GTPγS, MALDI, Imaging mass spectrometry

17

(IMS).

18 19

KEY CONCEPTS

20

- Bmax.

21

The concept of Bmax is a measure of the density of receptor molecules in a tissue and in

22

the techniques of receptor binding is equivalent to the bound compound when all the

23

receptors are occupied by a saturating concentration of the radioactive ligand. Receptor

24

binding studies are used to calculate the affinity (determined by Kd) of drugs for a

25

receptor as well as the maximal density of binding sites (Bmax). It is usually determined

26

in a saturation experiment, but it can also be calculated for a non-saturating

27

concentration (L) of the radioligand by the occupancy theory when the dissociation

28

equilibrium constant of a drug for the receptor (Kd) is previously known, using the

29

equation: Bound = Bmax x [L] /Kd + [L].

30 31

- G protein activation cycle.

32

When GPCR are activated, they induce the exchange of GDP for GTP in the α subunit

33

of the G protein that is dissociated to interact with intracellular or membrane effectors


20

Page 21 of 44

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1

(e.g. enzymes and ion channels). The activation cycle is completed when the GTPase of

2

α subunit hydrolyses GTP into GDP and restores the initial inactive conformation as

3

well as its affinity for βγ complex. In the presence of the non-hydrolysable analogue of

4

GTP, guanosine 5'-(γ-[35S] thiotriphosphate), ([35S]GTPγS), the cycle is stopped and

5

thus allows one to quantify the activity of G proteins in basal and activated states. The

6

binding of [35S]GTPγS, indicates the activation of the G protein α subunit by the

7

receptor when it is activated by an agonist.

8 9

- Emax.

10

Emax is the maximal effect or response elicited by a drug. The EC50 for in vitro

11

experiments (ED50 for in vivo) is the dose of agonist that produces 50% of the maximal

12

response.

13 14

. ACKNOWLEDGEMENTS

15

Supported by grants from the Basque Government IT584-13 and Spanish Government,

16

Ministry for Health, I.S.C.III PI 10/01202- co-funded by European Research

17

Development Fund.

18 19

. AUTHOR INFORMATION

20

The authors declare no competing financial interest.

21 22 23

. REFERENCES

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70. Trim, P.J., Djidja, M.C., Atkinson, S.J., Oakes, K., Cole, L.M., Anderson, D.M.,

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74. Krishnamurthy, T., Ross, P.L., and Rajamani, U. (1996) Detection of pathogenic

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flight mass spectrometry. Rapid Commun. Mass. Spectrom. 10(8); 883-8.

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78. Djidja, M.C., Claude, E., Snel, M.F., Francese, S., Scriven, P., Carolan, V., Clench,

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79. Hankin, J.A., Farias, S.E., Barkley, R.M., Heidenreich, K., Frey, L.C., Hamazaki,

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81. Sanchez, C., Galve-Roperh, I., Canova, C., Brachet, P., and Guzman, M. (1998)

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Delta9-tetrahydrocannabinol induces apoptosis in C6 glioma cells. FEBS Lett. 436;

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6-10.

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Page 30 of 44

1

2 3

Figure 1. Schematic diagram that summarizes the main steps in the different

4

autoradiography formats: in vitro, ex vivo and functional autoradiography. For ex vivo

5

autoradiography the radioligand is administered directly to the live animal. Then, the

6

tissue is dissected and cut and mounted on slides in a cryostat. The slides are incubated

7

afterwards with the radioligand only in the case of the in vitro autoradiography.

8

Functional autoradiography is a subtype of the in vitro autoradiography. The following

9

steps are common for all variants of autoradiography and include washing of the

10

unlabeled radioligand, and exposure to a radiosensitive plate such as a radioisotope-

11

sensitive film to obtain the density images that are later analyzed by computer software.

12


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1 2

Figure 2. Example of in vitro autoradiography images. The applicability to identify the

3

receptors for neuropeptides is usually limited by the availability of specific compounds

4

that are susceptible to be labeled with [3H]. It is common to use the endogenous

5

neuropeptide labeled with the [125I] isotope. The image shows the binding of [125I]-

6

galanin (0.1 nM) to coronal sections of Sprague Dawley rats. Note the specific binding

7

(A) in areas of the olfactory bulb, lateral olfactory tract, mesencephalic nuclei and

8

cognitive pathways (ventral hippocampus, entorhinal cortex and medial septum). The

9

nonspecific binding (B) was defined in the presence of a large mass excess of unlabeled

10

galanin (1µM).

11


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Page 32 of 44

1

2 3

Figure 3. The binding of the non-hydrolysable GTP analogue, guanosine 5'-(γ-[35S]

4

thiotriphosphate) or [35S]GTPγS directly reflects the activation of the G protein α

5

subunit by the receptor when it is activated by an agonist compound by measuring the

6

exchange of GDP for GTP (or [35S]GTPγS) in the G protein. The G protein is a trimer

7

that is dissociated, following its activation, to a βγ dimer and the α monomer bound to

8

GTP, both are able to activate intracellular effectors such as the adenylyl cyclase. The

9

GTP bound to the α subunit is then hydrolyzed to GDP and the trimeric G protein forms

10

again.

11 12


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1

2 3

Figure 4. Example of functional autoradiography. [35S]GTPγS binding to mouse sagittal

4

sections in the absence (basal binding, A) or in the presence of 100 µM carbachol

5

(cholinergic muscarinic agonist) (B) and 100 µM carbachol with 10 µM atropine

6

(cholinergic muscarinic antagonist) (C). As is shown, the effect induced by carbachol is

7

blocked in the presence of the specific muscarinic antagonist, atropine. The nonspecific

8

binding (D) was defined in the presence of 10 µM of unlabeled GTPγS.


33


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Page 34 of 44

1

2 3

Figure 5. Examples of the information provided by autoradiography of membrane

4

microarrays and the combination with other methods such as immunohistochemistry.

5

[3H]-NMS (upper left) and anti-M1 antibody (lower left) binding to cell membrane

6

microarrays made of homogenates isolated from different tissues and organs of

7

Sprague-Dawley rats and from cells that over-express a specific GPCR The cell

8

membranes were printed on slides activated with phosphate groups [60]. In the lower

9

right corner, the density of receptors quantified by autoradiography showed a good

10

correlation with that obtained by immunochemistry (r = 0.8947; p < 0.0001). [3H]-

11

NMS is a non-selective antagonist of muscarinic acetylcholine receptors and the used

12

antibody is designed to the specific recognition of the muscarinic M1 subtype.

13

However, in some tissues such as cerebellum (line 4 of the first square), the expression

14

measured by the M1 antibody is higher than the radioligand binding. The combination

15

of different detection methods in consecutive arrays also constitutes a test for different

16

markers. Scale bar = 1 mm.

17


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1 2

Figure 6. Information provided by the combination of functional autoradiography with

3

membrane microarrays. [35S]GTPγS autoradiographic images obtained from cell

4

membrane microarrays incubated with increasing concentrations of WIN55,212-2, that

5

demonstrate the functional activation of GPCR in these microarrays (WO/2007/012688)

6

CB1 receptors are expressed at much lower densities in peripheral tissues, such as the

7

stomach, compared to the CNS. Therefore, the cell membranes isolated from stomach

8

tissue could be considered here as a negative control. Each array (from the top to the

9

bottom) consists of four replicates of homogenates isolated from: rat frontal cortex (■),

10

cells that over-express the cannabinoid CB1 receptor (●), rat cerebellum (▼) and rat

11

stomach (♦). The cell membranes were printed on slides activated with phosphate

12

groups [60]. The graph shows the concentration-response curves obtained to represent

13

the percentages of stimulation over the [35S]GTPγS basal binding induce by increasing

14

concentrations of WIN55,212-2 quantified from the autoradiographic images shown

15

above. Scale bar = 1 mm.

16 ACS Paragon Plus Environment

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Page 36 of 44

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3 4

Figure 7. A and B show MALDI-IMS images representing the distribution of Cer

5

(d18:1/18:0) species collected in sagittal sections of rat brain incubated with (A)

6

WIN55,212-2 (10-6 M) and (B) without the drug. The conditions used to acquire the

7

MALDI spectra were as following: 10 shots per pixel, 20 µJ laser energy, spatial

8

resolution of 100 µm and 100,000 mass resolution. C is an image of CB1 receptor

9

autoradiography obtained in the presence of [3H]CP55940, showing the areas where the

10

CB1 receptor is located in rat CNS. The distribution of the [3H]CP55940 binding sites is

11

similar to the distribution obtained for the m/z 548.5408 peak by IMS.

12 13


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209x170mm (300 x 300 DPI)



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209x113mm (300 x 300 DPI)


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


74x14mm (300 x 300 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

209x134mm (300 x 300 DPI)


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


39x23mm (300 x 300 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

353x368mm (72 x 72 DPI)


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


234x224mm (72 x 72 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

199x99mm (300 x 300 DPI)


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Neurotransmitter Receptor Localization: From Autoradiography to

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