Potentiation of [3H]MK-801 binding in rat cortex and hippocampus in vivo by d-serine and the glycine transporter-1 inhibitor, NFPS C.J.Langmead, K.R.Starr, J.M.Watson, C.Scott & H.J. Herdon Psychiatry CEDD, GlaxoSmithKline, Harlow, Essex, UK.

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MK-801 is an activity dependent, non-competitive antagonist of the NMDA receptor complex (Price et al., 1988). The NMDA receptor has been successfully labelled in vivo in the mouse brain using [³H]MK-801 (Price et al., 1988; Murray et al., 2000) and [³H]TCP (Maurice & Vignon, 1990) to determine receptor occupancy.

In this study we have used [³H]MK-801 to label NMDA receptors in the rat cortex and hippocampus in vivo. We have measured the ability of D-serine and a glycine transporter-1 inhibitor, NFPS (Herdon et al., 2001), to modulate this binding and hence NMDA receptor activity.

Studies were conducted in compliance with the Home Office Guidance on the operation of the UK Animals (Scientific Procedures) Act 1986. Animals were housed in a temperature controlled environment (20 ±1 degC) on a 12h light:dark cycle with lights on at 7.00am. Food and water was available ad libitum.

CD male rats (150-200g) were dosed intraperitoneally with either vehicle, MK-801 (3 mg kg^-1), D-serine (30 mg kg ^-1) or NFPS (30 mg kg^-1) 15 or 225 (NFPS) minutes prior to an i.v. bolus dose of approximately 100 µCi kg^-1 [³H]MK-801. Rats were sacrificed 15 minutes later and the cortex and hippocampus rapidly dissected out.

Tissues were weighed and homogenised in 10ml ice-cold 5mM Tris-acetate buffer (pH 7). Triplicate 500µl aliquots of each sample were filtered through Whatman GF/B filters and washed with 4 x 1ml washes of ice-cold buffer. Triplicate 500ml aliquots of homogenate of each sample were also reserved and levels of radioactivity in the filter and homogenate samples determined by liquid scintillation counting.

% accumulation of [³H]MK-801 was determined using filter dpm as a percentage of homogenate dpm. Total accumulation was determined as that in the filter fraction of the vehicle group; non-specific accumulation was defined as that in the filter fraction of animals treated with MK-801 (3mg kg^-1, i.p.). Results are expressed as % specific accumulation.

In the vehicle group, mean % specific accumulation of [³H]MK-801 was 32.3 ± 1.5 % and 31.5 ± 0.2 % in hippocampus and cortex respectively. These values are reduced to 10.1 ± 1.8 % and 8.4 ± 1.8 % respectively after pre-treatment with MK-801 giving ~70% specific binding in both cortex and hippocampus.

D-serine (30mg kg^-1, i.p.) significantly potentiated specific accumulation of [³H]MK-801 to 40.8 ± 3.1 % and 38.5 ± 1.6 % in hippocampus and cortex respectively (p < 0.01, one-way ANOVA with post-hoc T-test). These represent increases of 26% and 22% over vehicle. NFPS (30mg kg^-1, i.p.) also potentiated specific accumulation of [³H]MK-801 to 37.9 ± 2.7 % and 40.3 ± 1.3 % in hippocampus and cortex respectively, though the effect was only significant in cortex (p < 0.01; p = 0.13 in hippocampus). These data represent increases of 17% and 28% over vehicle.

These results support the use of [³H]MK-801 binding in the assessment of NMDA function in the rat brain and show that both D-serine and NFPS can increase [³H]MK-801 binding in vivo, suggesting that direct or indirect stimulation of the glycine binding site of the NMDA receptor can potentiate NMDA function in vivo.

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