Abstract
Purpose::
The discovery that D-serine acts as an endogenous coagonist to NMDA receptors has resulted in numerous attempts to manipulate its level in vitro. One of the most common methods for lowering D-serine levels has been the addition of D-amino acid oxidase (DAAO) to the tissue of interest. Several problems with this enzyme have recently been articulated including a possible cross reaction with glycine, which can also act as a coagonist of the NMDA receptor, and questions as to the level of purity of available DAAO. We investigated the utility of D-serine deaminase (DsdA), a bacterial derived enzyme that specifically degrades D-serine (expressed in E coli by Dr Herman Wolosker). The results from electrophysiological and capillary electrophoresis (CE) experiments suggest that DsdA is the preferred enzyme for examining the role of D-serine in the retina.
Methods::
DsdA’s utility was investigated in electrophysiological recordings, both extracellular and whole-cell patch clamp, and in CE measurements of D-serine levels. In a retinal eyecup preparation, the proximal negative response (PNR) was measured using sharp, beveled microelectrodes. In isolated retinas, we used whole-cell recordings to measure light responses of retinal ganglion cells. In both preparations, we compared the light responses in control Ringer solution to those following the addition of DsdA to the perfusion solution. We used capillary electrophoresis to analyze Ringer solutions containing D-serine and glycine to determine the efficacy of D-serine degradation by DsdA versus DAAO.
Results::
Capillary electrophoresis results show a rapid elimination of D-serine in Ringer samples following exposure to DsdA. No D-serine was detected even under the shortest exposure time; while, the addition of DAAO shows a slow but continual drop in D-serine levels. Electrophysiological results show a rapid, reversible reduction in light responses measured both by the PNR and through whole-cell voltage clamp of retinal ganglion cells. The PNR amplitude was reduced 16.3 ± 7.3%. In the ganglion cell recordings, both the on and off responses to the light stimulus were decreased following DsdA perfusion. The ON response amplitude decreased 28.8 ±13.1% and the total charge decreased 33.0 ± 22.4 %. The OFF response decreased 32.0 ± 17.3% and 23.8 ± 13.6% (amplitude, charge).
Conclusions::
Our results indicate a rapid, specific degradation of D-serine by the enzyme DsdA that unequivocally establishes D-serine as an endogenous NMDA receptor coagonist in the retina. Its rate and specificity make DsdA the method of choice for D-serine degradation in the retina.
Keywords: retina: proximal (bipolar, amacrine, and ganglion cells) • excitatory amino acid receptors • glia