Research ReportRegional preferences of AMPA receptor modulators determined through agonist binding autoradiography
Introduction
AMPA receptors are oligomeric proteins assembled from four homologous subunits 6, 15. Each of the subunits occurs in alternatively spliced isoforms termed `flip' and `flop' [21]and in several variants with single amino acid substitutions resulting from RNA editing [18]. Studies of recombinant receptors expressed in non-neuronal cell lines indicate that virtually any combination of subunits can form a functional receptor, including those that contain a single subunit type. The balance between subunits and between subunit variants changes with age [19]and differs across populations of neurons [8]but recurring patterns have been identified [9]. A number of reports have indicated that biophysical properties of neuronal AMPA receptors such as calcium permeability and desensitization kinetics correlate closely with those predicted from the subunit type prevalent in the respective cells [8]. Together these results indicate that different brain regions utilize AMPA receptors that are distinctly different with regard to composition and function.
The regional variations noted above raise the possibility that drugs directed at AMPA receptors will have regionally differentiated effects. Several classes of compounds modulate the receptors in a non-competitive manner, two of which, generally referred to as ampakines and benzothiadiazides, are of particular interest with regard to selective actions within the brain. These drugs enhance and prolong AMPA receptor currents and accordingly may have therapeutic applications in conditions in which glutamatergic transmission is subnormal. Studies with recombinantly expressed homomeric receptors indicate that cyclothiazide, one of the benzothiadiazides, has an approximately ten-fold higher affinity for the flip variants of most AMPA receptor subunits 13, 14, 20. Moreover, recent studies have shown that cyclothiazide enhances currents with a lower EC50 (and thus with higher affinity) in patches from field CA3, an area of the hippocampus enriched in flip variants, as compared to patches from the flop enriched area CA1 [2]. Ampakines, to the extent that they discriminate between subunits in binding [13]and physiological experiments (Arai et al., in preparation), have a modest preference for the flop variants.
Based on these observations, ampakines and benzothiadiazides would be expected to produce distinguishably different regional patterns of AMPA receptor modulation. However, most of the results summarized above were collected using extrasynaptic receptors and there is evidence that the synaptic environment profoundly modifies the binding properties of AMPA receptors [10]. Ligand autoradiography provides a means for testing if ampakines and benzothiadiazides act in a regionally differentiated fashion vis-à-vis their effects on AMPA receptors in situ. Several AMPA receptor modulators have been found to increase or reduce the binding of agonists like [3H]AMPA to membranes isolated from rat brain 3, 4, 11, 16. Moreover, while the direction and magnitude of the changes depend on the particulars of the assay conditions, the EC50 values were found to closely approximate those obtained in physiological tests including those involving synaptic responses. The present study used binding autoradiography with [3H]AMPA and the recently introduced compound [3H]fluorowillardiine [12]to test if the relative potencies of cyclothiazide and an ampakine vary across brain regions according to the balance of flip vs. flop subunits and as predicted from homomeric receptor studies.
Section snippets
Materials and methods
Two-month old Sprague–Dawley rats were anaesthetized with ether and killed by decapitation. The brains were quickly removed and dissected after a brief immersion in ice-cold phosphate-buffered saline. Coronal sections 15 μm in thickness were collected from the mid-part of the brain such that they included the rostral part of the hippocampus. Three or four sections were mounted together on a collagen-coated slide and stored at −20°C until use. After thawing, the sections were washed three times
Results
Cyclothiazide was shown earlier to reduce [3H]AMPA binding to brain membranes [16]and to homomeric AMPA receptors composed of subunits GluR1 to 4 [13]when assays were carried out in the presence of SCN−. A similar effect was observed in the present study using binding autoradiography as illustrated by the photographs on the left side of Fig. 1. A quantitative analysis of the effect of cyclothiazide over a concentration range of 10 to 500 μM is shown in Fig. 2. As expected for a non-competitive
Discussion
The EC50 values for cyclothiazide determined here are of the same order as those determined in rat brain membranes [11]and are comparable to the ∼10 μM affinities typically obtained in excised patch studies [22]. Similar conclusions apply for CX614; this drug enhanced EPSPs in hippocampal slices and AMPA receptor currents in patches from CA1 with EC50 values of 20–50 μM (Arai et al., in preparation), which is in the range of the values shown in Fig. 4. Thus, measuring the non-competitive
Acknowledgements
This research project was supported by grant CP-19982 from Cortex Pharmaceuticals, Irvine, and by grant 95-1-0304 from the Air Force Office for Scientific Research. The authors wish to thank Jackie Porter and Marla Lay for their secretarial assistance.
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