Antisense oligonucleotide strategies in neuropharmacology

TiPS - Fe~r~i~ry 1994 Wol. 251

In view of the facts that many antisense oligodeoxynucleotides are useful expe~mental tools showing high specificity, and some regulatory agencies now consider o~~godeox~u~~tides to be drugs, it is interesting to note the coolness with which the pha~acologi~al community has been receiving reports on these compounds. Only a limited number of pharmacologists have recognized the potential of antisense oligodeoxynucleotides as antiviral or anticancer agentsre3. This is somewhat of a paradox since in combating viruses and cancer, it is desirable in many instances to eliminate targeted protein(s) completely. On the other hand, neuropharmacologists, for exampte. would perhaps be most satisfied with certain degrees of inhibition, protein synthesis which indeed is now achievable. Much work has been devoted to producing nuclease-resistant ohgonucleotide analogues as it has been perceived, probably correctly, that unmodified oligodeoxynucleotid~s will never become pharmaceuticals’, However, many experimental settings (using cultured cells or injection into living brain) in which sera are not present, seem to allow the use of unmodified, or ‘first generation”, oligodeoxynucleotides. The basic idea of antisense or antigene approaches is to interfere with the information fiow from gene to protein, and to do so in a very specific manner. Briefly, reagents that bind to single- or double-stranded nucleic acids are potential candidates for therapeutics targeted at specific genes, either at the mRNA (antisense) or the double-stranded DNA (antigene) level. These mechanisms provide an opportun~~ to design true isotypically selective pharmacological tools, and should allow testing of many types of hypotheses in biomedicine. This brief review focuses on the experimental use of oligodeoxynucleotides as neuropha~a~ological tools, focusing on their appli0 19%EIwxp~ Scicwc Ltd QW - fi~~?~~~~~~.~

cation to the living brain, and makes comparisons with alternative approaches to manipulate gene or protein expression. Selection of optimal oligodeoxynucleotide targets The actions of oligodeoxynu~l~tides can be rationalized by traditional receptor theory. The affinity of oligodeoxynucleotides fi;r their receptors, which are other nucleic acids, results from hybridtzation interactions that depend upon hydrogen bonding and base stacking in the double helix that is formed. A minimum level of affinity is required for the desired interaction and this can be achieved with an oligodeoxynucieotide that is at least 15 nucleotides in length. [An oligodeoxynucleotide that consists of 11-15 nucleotides will bind to a single cellular RNA species (see Ref. I).] Qn the other hand, since specific mechanisms may govern cellular uptake (see below), it is desirable to limit the length to a maximum of 20-25 nucleotides. For example, oligodeoxynucleotides of 28 nucleotides have often been used@. In principle, several ohgodeoxynucleotides can be applied in combination. However, using moderately active oligodeoxynucleotides to target the same RNA species rather than using one, more active, oligodeoxynucteotide should perhaps be avoided, since many ohgodeoxynucleotides may contribute to nonspecific actions while adding little to total efficacy. Several computational proaches have been designedayo help predict antisense ohgodeoxynucleot~de efficacy. A socalled ‘Dscore’ (relating to duplex formation energy) was found to be a consistent predictor of activity”. Standard computer programs for optimizing oligodeoxynucleotide sequences for DNA probes or polymerase chain reaction primers may also be of use. It is imperative that every oligodeoxynucleotide sequence is checked

for similarities with other sequences present in gene databases. Our laboratory has avoided antisense oligodeoxyusing nucleotides with a GC: AT ratio exceeding ~~~~~~. When possible, we have selected sequences with terminai purines at both ends, and avoided sequences with single nucleotide repeats. Most of our antisense oligodeoxynucleotides have been designed to bind to the translation initiation codon or its immediate vicinity, perhaps resulting in translational arrest”*. A major advantage of the oligodeoxynucleotide antisense approach is that the facile synthesis and testing of a number of oligodeoxynucieotides allows for some ‘trial and error’, In this translated screening process, mRNA regions (including, but not necessarily limited to, regions near the AUG initiation codon) as well as 5”- (e.g. capping region) or 3’-untranslated mRNA regions can be targeted. The potency of antisense oiigodeoxynucleotides differs widely depending on the site selected, even for comparable analogues (for example, see Ref. 11). Thus, it is imperative to screen sites carefully and to construct thorough concentration-response curves. Choice of DNA analogue The replacement of the phosphodiester (PDE) linkage of the DNA backbone with neutral, achiral, nuclease resistant entities has been considered to be desirable for some time. medicinal chemists have also suggested various modifications of base and sugar entities. The next few years will presumably see the further development of new generations of o~igodeoxynucleotides with improved properties. New analogues that are already in use include 2’-modified oligodeoxynucleotides’*, oligodeoxynucleotides with 5’-cholesteryl moieties’3 and peptide nucleic acids”. Moreover, ribozyme techniques already offer interesting new possibilities for antisense research3. To date, we have used either unmodified oligodeoxynucleotides (PDE-oligodeoxynuc~eotides) or phosphorothioate analogues (S-oligodeoxynucleotides) and we feel confident that either analogue is useful for itt vitro or brain injection experiments; the

TiPS - Fe~ruar~ 1994 /Vol. 251 choice depends on the experimental setting. While the Soligodeoxynucle0tide analogue, in which one of the non-bridging equivalent internucleotide oxygen molecules is replaced by sulphur, is the most active of the first generation anal0gues, it has significant limitations, notably related to sequence-independent effects’*t5. This drawback of S-oligodeoxy~ucleotides should be considered in relation to their possible higher cell binding and uptake compared with PDEoligodeoxynucleotidess6. Importantly, for S-oligodeoxynucleotides as well as for PDE-oligodeoxynucleotides (maybe less so for the latter), there may exist a rather narrow ‘window’ of effective concentration in vitro, above which the oligode0xynucleotides may cause nonspecific effects. The use of a PDE-oligodeoxynucleotide is advisable for initial in vitro experiments whenever sera can be omitted. For subsequent application into the living brain, either znalogue (Pan-oligodeoxynucleotide or S-oligodeoxynucleotide) may be useful (see below). Mode and time requirement of o~i~odeoxynucleotide action There is evidence that the mechanism of oligodeoxynucleotide uptake is predominantly fluid-phase endocytosis (pinocytosis’7), perhaps initiated by receptor-like recognition’*. However, this matter is complex and depends, for example, on oligodeoxynucleotide chain length, concentration and chemical class. Uptake may be enhanced by coadministration of cationic lipids”. The mechanisms of potential oligodeoxynucleotide interactions with target nucleic acids are complex (possibly reiating to many sites along the sequence of events Ieading from DNA to protein synthesis) and beyond the scope of this review. Briefly, according to the most popular theories the antisense 0ligodeoxynucleotides exert their effects by inducing translational arrest, or by providing substrates for RNase H activity (an enzyme that degrades the RNA strand of an RNA-DNA duplex). The S-o!igodeoxynucleotide analogue retains its negative charge and is therefore a substrate for RNase H (Refs 1-3). However, it is unknown whether RNase H

10’

lh

4h

24h

Fig. 1. Stabifity of an l&n~ P~~-ot~~e~~u~tide used to inhi&it syntftesis of the ne#ro~eptide Y Y, receptof in vitro and in viva in fresh rat CSF (Ref. 4). t~cubatioos were carried outat 37°C for the time periods indicatedand the P~E~~~~ffu~~t~e was tfteo analysed by 18% polyacryiamide gef electropborasis. The PRE-o&odeoxynucfeotlde #nce~tra~on in this in vitro ex~erimeni was 7U~~lgm/-’ of GSF, i.e. similar to the setting in vivo, assuming a rat GSF volume of U.Sml.

plays an important role in antigenmediated mRNA degradation in mammalian neurones. Indeed, an oligodeoxynucleotide targeted to the initiation region of the NMDA glutamate receptor did not significantly affect the mRNA while reducing the protein concentrations. Interestingly, it seems that various cell types may differ greatly with respect to oligodeoxynucieotide uptake as well as to oligodeoxynucleotide de~adation. It has been suggested that many neurones have a relatively low capacity to degrade oligodeoxynucleotides and that they are therefore amenable to treatment with either PDE-olligodeoxynucleotides or nuclease-stable analogues4*5~7*8. In contrast, other cell types such as HeLa cells2’ may have high degradative capacity. Moreover, PDE-oIigodeoxynucleotides can enter cultured neurones at a concentration of up to 5% in the media”. Taken together, the available data indicate that PDEo~ig0deoxynucleotide uptake and degradation conditions in highquality primary neuronal cultures may be quite favourable when compared, for example, with many popufar cell lines. It is important to consider the time factor of antisense oligodeoxynucleotide treatment. Obviously, when studying an inducible gene product, such as

c-Fos (Refs 22,23), less time for oligodeoxynu~leotide treatment is required than when attempting to affect a Constitutively active gene product. Thus, reduction of steady-state levels will depend upon turnover rate. For example, to achieve inhibition of neurotransmitter receptor synthesis, a minimum treatment time of two to five days seems necessarye-7a24. Qligode0xynucleotide reagent in vitro Micromolar concentrations of PDE-oligodeoxynucleotides are typically required for activity in many cell types, including neurones4,5,25-29, and astrocyte.?a vascular smooth muscle cellsh. As curves concentration-response may be steep, many oligodeoxynucleotide concentrations should be tested over a narrow range. In neurones maintained without sera, we have found that l-3 PM of PDE-oligodeoxynuclcotide effectively inhibits the synthesis of certain receptor proteins4ts, while times several concentrations higher may show nonspecific effects. Specifically, caution must be taken when neuronal cultures are exposed to 21Op~ concentrations for several days (other cell types may well tolerate higher serum When concentrations). cannot be omitted, S-oligodeoxynucleotides may be used (for

Fig. 2. Specific IabafMtg of &Is in rat brati, f Smtn after ;nlra~ereb~veotn~/ar mjection of b~otl~y!ated PDE-O@Odeoxyr?u&?otide. Sfainiq was cytopfasmic and particulate. and appeared to be ra~domtydistfi6uf~ w&in many types of cells. The iabefled motecute was prepared by inrtoducing biotinyfateddTin position eight of the 18-mer. NMDAR tAS/c (Ref. 5). P 3s with lateral ventricular cannulae received lOUt1g of the PDE-olig~eo~y~ucfeotide On 5% satinef. and were then perfused wrrth4% paraformatdehyde. Sections 30vm thrck ware processed with an avidfn-brofin kit (VectaStai@ Differen&at rntertefence contrast optics were used for light microscopy ( u4tX3).

example, s2e Refs 28, 29). PDEoiigodcoxynucieotide~ can be tested at higher, frequently replenished concentrations using regular or, better, heat-inactivated sera (which are reduced in, but not devoid of nucfease activities) with an appropriate mismatched control (see below). Most importantly, the ‘window’ of activity (that is, the defined interval between the effective and maximum tolerable oligodeoxynucleotide concentration) must be carefully determined in any given culture. Whenever possible, it is advisable to test for otigodeoxynudeotide efficacy itr zjifro before proceeding to an experiment in i+zlo. Oligod~ox~~~c~e~tide administration to rat CSF B&s injections As shown in Fig. I, an 1%mer PDE-oligodeoxynucleotide is essentially stable (for up to 24h) when incubated with rat CSF in zpifro. Moreover, an internally monobiotinylated PDE-oligodeox~ucleotide was found to pen-

etrate brain tissue and neurones, giving rise to specific, cellular staining following intracerebroventricular application in the rat (Fig. 2). Following repeated injections of 5@-100 ug of antisense PDE-oligodeoxynuc~eotides twice daily for at least two to three days, reductions in the levels of rat neuropeptide Y Yr receptor’ and rat glutamate NMDA receptor were elicited”; such changes in receptor numbers were accompanied by anxiolytic-like behaviour and a reduction in focal ischaemic infarctions, respectively. Unmodified ohgodeoxynucleotides are evidently also relatively stable in the intrathecal space. A very recent study directed I-5yg of an antisense ~~~-oligodeoxyintrathecally three nucleotide times, with 4S h intervals, to the o-opioid receptor in mice. This resulted in selective and reversible loss of h-opioid analgesia without affecting other opioid receptor subtypesr.

When osmotic minipumps were used to deliver antisense S-oligodeoxynucleotides to the NMDA receptor subunit NMDARl (Ref. 5) or the dopamine D2 receptor”” at a rate of 1 ttl h-’ for 72 h directly into the lateral ventricle of the rat brain, reductions in receptor concentrations were observed; total doses given were 5UOyg (Ref. 5) and 7201.18 (Ref. 24): respectively. Another study showed that osmotic minipumps can maintain micromolar S-oligudeoxynucleotide concentrations one for week”‘. In contrast with encouraging data using S-oligadeoxynuc~eotides, experiments have indicated that PDE-oligodeoxynucleotides may not be suitable for administration by such subcutaneously placed minipumps3’. Injection of oligodeoxynucleotide into brain parenchyma fn the case of site injections, some tissue damage can be expected; therefore, metabolically stable oligodeoxynucleotide analogues such as S-oligodeoxynucleotides may be preferable. However, higher doses of PDEoligodeoxynucleotide have also been successfully used in this contexts,““. Effective antisense

oligodeoxynucleotide site injections have been placed in the nucleus accumbens”“, striatum’“, arcaate nucleus” and ventromedia~ hypothalamus?“. Appropriate controls Contruisfor SyPcificity of f&P oligodeoxyntrcleotide Most studies in the literature .lave involved the use of sense or scrambled analogues of the presumably active oligodeoxy~u~~eo~ tide molecules for controls. However, a more stringent control is a mjsmatched analogue maintaining the same base composition as the antisense molecule. For example, in a recent study we used an 18-mer with three mismatches and found that it lacked activity”. The decrease in affinity associated with a mismatched base pair is dependent on several factors’*2 and will on average be 500 times less than with the oligodeoxynucleotide (see Ref. 11). Thus, molecules oligodeoxynucleotide with one to four mismatches are recommended for use in every type of experiment. As pointed out above, quality pharmacological evamation calls concentrationfor thorough response curves using the active oligodeoxynucleotide(s). It is also important that the mismatched analogue or other control oligodeoxynucieotjdes are tested over an identical concentration range. An important control for specificity of the active oligodeoxynucleotide is to identify one or several non-overlapping antisense c’igodeoxynucleotides that induce similar inhibition of synthesis of the targeted protein”,‘. Cuntrol~~or the targeted~rote~~~ and nrRNA It is obviously also necessary to oligodeoxynucleotide rule out effects on proteins other than the targeted one. For example, when a specific neurotransmitter receptor subtype is the target, it is appropriate to assay for a related receptor subtype by iigand-binding techniques. As degradation of the corresponding mRNA by RNase H is considered a mandatory event by some, a quantitative analysis of the transcript (for example, solution hybridization, based on RNase protection, accompanied by northern blotting} may be most

TiPS - FebrrrnrJl 1994 [Vol. ?51 TABLE I. Comparison of antisense oligonucleotide inhibition (‘knockdown’) and ‘knockout’ approaches in experimental animals Advantages

Disadvantages -.-

Antlsense oflaonucleotlde Applicable to any stage of development

incomplete treatment (no effect if there is redundancy cr spare capaciiy)

A range of phenotypes can be created Product of

sequence-independent effects ~notabfy with S-oligodeoxynucleotides)

cloned gene from any species can be studied

The effect is reversible

narrow experimental and therapeutic windows continuous or repeated administration necessary

Low cost to the laboratory Little specialized equipment is required Allows for ‘trial and error’ May have therapeutic potential Homologous reoombi~ation knockout Complete disappearance of gene product

taborious (e.g. both gene alleles should be identified)

The effect is completely specific

limited access to some manipulated animals

No variability between animals

compensatory mechanisms might be operative only used in selected laboratories because of high costs possibility of lethal phenotype (no adulthood survival)

informative. mRN_4 should

An independent serve as control.

Controfsforyossible

toxicity

cultured cells, it is a good habit to assess the number of viable cells, as well as protein following oligodeoxycontent, nucleotide treatment (for example, see Ref. 5). Similarly, treatment in viva should be followed by analyses of gross or specific types of behaviour as well as analyses of the dissected tissue (protein content and histology). Reversibility of effect will also be an important reassurance of lack of toxicity. In

Problems and pitfalls Some potential problems associated with oligodeoxynucleotides are summarized in Table I. Apart from occasional nonspecific or toxic actions, the greatest drawback may be an incomplete effect of oligodeoxynucleotide treatment. This means that if a biological phenomenon with a great deal of spare capacity or redundancy is studied, where, for example, only a fraction of receptors have to be occupied to elicit a full response, treatment oligodeoxynucleotide may become functionally ‘silent’.

However, in many instances pharmacologists may well prefer antisense oligodeoxynucleotide strategies, which, because they are associated with reversible changes (for example, see Refs 5,7), might perhaps be referred to as ‘knockdown’. Table I lists some advantages and disadvantages of knockout (homologous recombination) and knockdown (antisense inhibitions. Viral vectors for gene transfer will undoubtedly prove to be useful for biologists seeking to overexpress genes or to disrupt gene function (for example, by antisense RNA) in uivo. Potential problems recombinant when applying viruses to the experimental animal include: (1) laboratory safety; (Zf lack of effect if viral receptors (for example, for adenovirus) are absent; (3) presence of antibodies to the virus: (4) activation of second messengers by way of .Jiral receptors; and (5) nonspecific effects on host cell genetic machinery. Finally, antisense RNA techniques offer interesting possibilities to manipulate various genes specifically; these techniques were recently reviewed3. q

Advantages over other techniques Immunologists and developmental biologists have largely developed the powerful technique of homologous recombination in which a selected gene is disrupted (‘knocked out’). This is usually accomplished by homologous recombination between a targeted gene and DNA introduced into mouse embryonic stem cells33.

0

El

The approach of first testing the oligodeoxynucleotide efficacy in vitro followed by administration in vivo is advised whenever passible. Stringent mismatched control oligodeoxynucleotides should be used. It is expected that the described in uivo approach will be taken by many investigators and that some will be unsuccessful,

perhaps because much of their protein of interest is expressed in amounts far greater than necessary for function. However, when the oligodeoxynucleotide approach is successful, the road to obtaining conclusive exyerimental data or rational drug design may well be short. Oligodeoxynucleotides are perhaps especially useful for the receptor researcher in cases when a conventional inhibitor or antagonist is unavailable or shows limited selectivity. Note added in proof At the 1993 Society for Neuroscience meeting, a number of presentations described the use of antisense oIigodeoxynucleotides to inhibit gene expression in the living brain. CLAES WAHLESTEDT

Acknowledgements Research carried out in our laboratory was in large part supported by US Public Service Grants (DA 06805 and HL 18974). I thank my collaborators (see references) for their many helpful suggestions. F. Yee and V. M. Pickel are gratefully acknowledged for their participation in the work shown in the figures. References

TiPS - February 1994 /Vol. 251 6119-621

11 Crooke, 5. T. (1993) 12 hlonla,

Murray. J. A H.. and Crockett. N (1992) ,n .%rwr,w RN.4 attd ,7N.Z (Murray, I. A. H., cd.), pp. l--19, Wiley-Liss Wahlestedt, C., Pith, E. M., Koob. G. F.. Yee, F. and Heilig, M. (1993) Siwcc 259. 528-531 Wahlestedt, C. cl nl. (1993) N~twr 363, 260-263 Erlinge, D., Edvinsson, L., Brunkwall. J., Yee. F. and Wahlestedt, C. (1993) Ew. 1. Pham1nr0l. 240. n-80 Chien, C-C., K. M., Standifer, Wahlestedt, C., Brown. G. P. and Pastemak, C. W. Nearof~ (in press) Wahlestedt, C., Akabayashi. A, Alexander, J. T. and Leibowitz. S. F. (1994) MO/. Rrwr Rcs. 21. 55-61 Stull, R. A., Taylor, L. A. and Szoka. F. C.. Jr (1992) Nrtcleic Acids Rcs. 20. 3501-350s 10 Liebhaber, S. A., Cash, F. and Eshleman, S. S. (1992) /. Mol. Brnl. 226,

13 l-l

15 16

17 18 19

20

FASER

1. 7.533-539 B. P. et nl. (1993) 1. Birl. Chew.

268. 14514-14522 Krleg, A. M. ct RI.(1993) Proc. N&l Acnd. Q-i. USA 90, 1048-1052 Nielsen, P. E.. Egholm, M., Berg, R. H. and Buchardt, 0. (1991) Srrtv~cr 254, 1497-1500 Morvan, F. et of. (1993) /. Med. Chm. 36, 28s-287 Zhao, X., Matson, S., Herrera, C. J., Fisher, E., Yu, H. and Krieg, A. M. (1993) Atrtisctlse Kes. Dcu. 3, 5%66 Stein, C. A. ct nl. (1993) Biochen~is~ry 32, 4855-l861 Yakubov, L. A. et ~1. (1989) Proc. Nafl Acnd. Ser. USA 86, 6454-6458 Bennett, C. F., Chiang, M. Y., Chan, H., Shoemaker. J. E. ani Mirabelli, C. K. (1992) Mol. Plrnrt~rncol. 41, 1023-1033 Hoke, G. D. cf ~1. (1991) Nrdclcic Acids

24 25

26 27 28 29 30 31 32

Res. 19. 5742-5748

21 Caceres, A. and Kosik, K. S. (1990) N~ttrre 343, 461-463 22 Chiasson, 8. J., Hooper, M. L., Murphy,

Gution is needed in interpreting data from dopamine 03 receptor studies

The dopamine D3 receptor: Chinese hamsters or Chin&e The cloning of the gene for a novel dopamine receptor, the D3 receptori, provided welcome impetus for research into the dopamine hypothesis of schizophrenia at a time when mental illness was at the forefront of public opinion. However, the initial hope that the receptor might be implicated in the aetiology of psychosis, or at least play a role in its amelioration by neuroleptic drugs, has yet to be realized. In an attempt to link D3 receptors with mental illness, molecular biologists held in great importance the results of mRNA distribution studies’, and eagerly reported that D3 receptors were preferentially distributed in the limbic dopamine system of rats. Most subsequent distribution studies have confirmed that the highest densities of D3 receptors are in limbic areas, but they have also shown that the receptors are present in the striatum&s. Consequently, the D.1receptor may offer no advantage over D2 re-

23

ceptors, since drugs acting at D3 receptors may not be free from motor side-effects associated with actions in the nigrostriatal dopamine system. Progress in this area requires an understanding of the function of D3 receptors, which in turn requires the discovery of new selective drugs. The biggest challenge in the development of new drugs has been to obtain separation between affinities at D3 and D2 receptors expressed in Chinese hamster ovary recombinant cell lines. Each of the groups working in the field has reported that known agonists at the D2 receptor, including dopamine, have selectivity for D3 receptors in binding assays. They have also demonstrated poor G protein coupling of D3 receptors (in recombinant cell lines’ and rat brain3e4). These two observations are closely linked: binding conditions can be manipulated to favour low-affinity agonist binding states of G protein-coupled

33

I’. R. and Robertson, H. A. (1992) Ew. /. Pharmncof. 227, 451-453 Heilig, M., Engel, J. A. and SSderpalm, B. (1993) Ertr. J. Pltnmincol. 236,339-340 Zhang, M. and Creese, I. (1993) N~rrroxi. Lgf. 161. 223-226 Ferreira, A.; Niclas, J., Vale, R. D., Banker, C. and Kosik, K. S. (1992) 1. Cell Blol. 117, 595-606 Vanderklish, P. ef nl. (1992) Syttnpsc 12, 333-337 Holopainen, I. and Wojcik, W. J. (1993) J. Plwrnmcol. Exp. Titer. 264, 423-430 Lallier, T. and Bronner-Fraser, M. (1993) Sciertce 259. 692-695 Osen-Sand, A. rf nl. (1993) Nofrrre 364, 445-448 Yu, A. C., Lee, Y. L. and Eng, L. F. (1993) I. Newosri. Rrs. 34, 295-303 Whitesell, L. et of. (1993) Proc. Nntl Acnd. Sci. USA 90, 4665-4669 Pollio, G.. Xue, P., Zanisi, M., Nicolin, A. and Maggi, A. (1993) Mol. Brain Rrs. 19,135-139 Koller, 8. H. and Smithies, 0. (1992) Amrc. Rev. Inrtnro~ol. 10, 705-730

receptors such as D2 receptors; however, since D3 receptors are poorly coupled to begin with, these manipulations do not reduce their affinities for the ligands. In this way, it is possible to enhance the selectivity of a D2 receptor agonist for D3 receptors in binding assays, simply by changing the assay conditions. The most l*ecent addition to the DJ receptor toolkit is 7-OH-DPAT, a dopamine receptor agonisP. In cell-line binding studies, 7-OHDPAT has between 40 and 80 times greater affinity for the D3 receptor than for the Dz receptor5e7. The greatest affinity separation is achieved under conditions that favour low-affinity agonist binding to D2 receptors, but that have no effect on affinities at D3 receptors. This ability to manipulate ligand selectivity is extremely useful for studies of receptor distribution; indeed, tritiated 7-OHDPAT has been used to provide the best assessment of D3 receptor distribution in the rat brain to date5. However, in studies of receptor function in viuo, it is not possible to manipulate conditions. On the strength of binding data, behavioural scientists are now using 7-OH-DPAT to implicate D3 receptors in certain types of behaviour in rats8s9. It is difficult to know whether these claims can be justified, since they rely on a pharmacological profile determined in a system that is completely nonphysiological and purposefully