Postsynaptic response to stimulation of the Schaffer collaterals with properties similar to those of synaptosomal aspartate release

Abstract

Aspartate satisfies all the criteria normally required for identification of a CNS neurotransmitter. Nevertheless, little electrophysiological evidence supports the existence of aspartate transmission. In studies with rat hippocampal synaptosomes, chemically evoked aspartate release differed from glutamate release in its relative sensitivity to increased Ca(2+) concentration outside the presynaptic active zones, inefficient coupling to P/Q-type Ca(2+) channels, sensitivity to KB-R7943, and resistance to native Clostridial toxins. We took advantage of these differences to search for a potential aspartate-mediated response at Schaffer collateral synapses in organotypic hippocampal slice cultures. The slice cultures were pretreated with botulinum neurotoxin C (BoNT/C) to eliminate most of the glutamate release so that an expectedly smaller aspartate-like component of the compound EPSC could be detected by whole cell patch clamp recording. In control cultures, NMDA receptor activation accounted for only 18% of the evoked EPSC and an NR2B-selective antagonist reduced the NMDA receptor-mediated component by only 20%. Block of P/Q-type Ca(2+) channels essentially eliminated the response and 0.1 muM KB-R7943 had no significant effect. In BoNT/C-pretreated cultures, however, NMDA receptor activation accounted for 77% of the evoked EPSC and an NR2B-selective antagonist reduced the NMDA receptor-mediated component by 57%. Block of P/Q-type Ca(2+) channels reduced the response by only 28%, but 0.1 muM KB-R7943 reduced it by 45%. These results suggest that part of the Schaffer collateral synaptic response has pharmacological properties similar to those of synaptosomal aspartate release and may therefore be mediated at least partly by released aspartate.

Fig. 1

Pretreatment of hippocampal slice cultures with BoNT/C increased the fraction of the Schaffer collateral compound EPSC mediated by NMDA receptors and the fraction of the NMDA component mediated by NR2B-containing receptors. Top: representative recordings. Slice cultures were exposed to 50 nM BoNT/C for 3 h at 37°C before recordings began. Recordings were made from CA1 pyramidal cells at 32°C and a holding potential of +40 mV. All solutions contained 30 μM bicuculline. Ten stimuli were applied to the Schaffer collaterals at a frequency of 30 Hz. Traces shown are the averaged responses to 10 such trains. Vertical deflections are stimulus artifacts. The NMDA component of the EPSC was isolated pharmacologically by adding 10 μM NBQX to the superfusion medium. The difference between recordings made in the presence and absence of 1 μM RO 25-6981 represents the portion of the NMDA component mediated by NR2B-containing receptors. The RO 25-6981-resistant response (equivalent to the NR2A-mediated EPSC) was abolished by 50 μM D-AP5. Bottom: grouped data from 6 control cultures and 9 cultures pretreated with BoNT/C. Values are means ± SEM. *Significantly different from control at P <0.001 (Student’s t-test).

Fig. 2

KB-R7943 (0.1 μM) reduced the size of the synaptic NMDA current only after pretreatment of the hippocampal slice culture with BoNT/C. Top: representative recordings. Recordings were made as described in figure 1 with a superfusion medium that contained 30 μM bicuculline and 10 μM NBQX. Bottom: grouped data from 7 control cultures and 8 cultures pretreated with BoNT/C. Values are means ± SEM. *Significantly different from control at P = 0.004.

Fig. 3

Aga IVA (1 μM) inhibited synaptic transmission less effectively when the hippocampal slice culture was pretreated with BoNT/C. Top: representative recordings. In control cultures, aga IVA essentially abolished Schaffer collateral transmission, both the AMPA/kainate and NMDA components. However, the NMDA receptor-mediated EPSC was reduced by only 28% when the culture was pretreated with BoNT/C. See figure 1 for other details. Bottom: grouped data from 5 control cultures and 5 cultures pretreated with BoNT/C. Values are means ± SEM. *Significantly different from control at P <0.001 (Student’s t-test).