Figure 1.
Layer III medial entorhinal cortex (LIII mEC) pyramidal neurons.
(A) Schematic representation of the entorhinal-hippocampal combined slice used in this study with the recording electrode (Recording) in LIII mEC while stimulating (Stimulation in LI mEC) the input from the lateral entorhinal cortex. (B) Electrophysiological and morphological properties of a typical LIII mEC pyramidal neuron. (C) In situ hybridization of GluK2 subunit of kainate receptor in the mEC. Data adapted from the Allen Atlas, Allen Institute of Brain Science.
Figure 2.
Kainate (KA) induced changes in whole-cell holding current and activation threshold of kainate receptors (KARs) on LIII mEC pyramidal neurons.
(A, B) Time course data for KA (100 nM, 300 nM, 1 µM and 3 µM) induced concentration dependent changes in the whole-cell holding current which is antagonized by NBQX (25 µM). (A) Single experiment with voltage-clamp transients corresponding to baseline, 300 nM, 3 µM and NBQX. (B) Group data (n = 6). (C) Time course data from a single experiment for two different KA concentrations (300 nM and 1 µM) with subsequent application of GYKI (20 µM) and NBQX (25 µM). (D, E) Time course data for determining the activation threshold of kainate receptors on LIII mEC pyramidal neurons. The change in the whole-cell holding current by bath application of 300 nM of KA was reversible and following treatment with GYKI (20 µM), 300 nM KA was applied for a second time. Holding current decreased to the same amplitude indicating that at a concentration of 300 nM KA, no AMPA receptors are activated. (D) Single experiment. (E) Group data (n = 4). (F) While there is no effect of GYKI (20 µM) on holding current at 300 nM KA (p = 0.344; n = 4) there is a significant effect at 1 µM KA (p<0.05; n = 4).
Figure 3.
Genetic deletion studies to determine the role of GluK1 and GluK2 subunits in the KAR mediated current on the LIII mEC pyramidal neurons.
(A, B) By bath applying 300 nM KA, the holding current in the GluK1 KO did not change significantly when compared to the wild-type mice (p = 0.181; n = 4). However, no change in holding current was observed for GluK2 KO (n = 9) upon bath applying 300 nM KA indicating that the GluK2 is the predominant KAR subunit responsible for mediating the observed KAR current in these neurons. (A) Single experiment. (B) Group data.
Figure 4.
IV curve and characterization of kainate receptors on LIII mEC pyramidal neurons using photolytic uncaging of glutamate.
(A) Photolytic uncaging of glutamate at the cell soma elicited inward currents, which were reduced to 34.32% (±2.05%; p<0.01; n = 5) of the baseline value in the presence of GYKI (20 µM). (B) At 20 µM of GYKI no residual AMPA current is seen as there is no potentiation of the resultant EPSC upon application of AMPAR desensitization blocker CTZ (10 µM; p = 0.956; n = 4). However, at a higher concentration of CTZ (100 µM), the effect of GYKI is antagonized (p<0.01; n = 6). In the presence of GYKI, APV and Gabazine, the holding membrane potential was changed in steps of 20 mV from −60 mV to 40 mV and at each step, 5 responses (5 laser flashes with a inter-stimulus interval of 30 seconds) were recorded by uncaging glutamate over the cell soma. (C) The peak current for each individual cell (n = 7) is plotted against the membrane potential along with the corresponding superimposed current traces (inset). (D) Group data (n = 7). A linear relationship between voltage and current, both at negative and positive potentials suggested the KARs on LIII mEC pyramidal neurons to be mostly of the Ca2+ impermeable edited form.
Figure 5.
Pathway specific activation of synaptic KARs in LIII mEC pyramidal neurons.
(A, B) Recorded LIII mEC pyramidal neurons were held in the whole-cell mode while stimulating the afferent pathway LI mEC. (A, lower panel) In the presence of GYKI (20 µM), no synaptically evoked EPSCKA is detected (n = 8) as seen in an example trace from a single experiment. (B) High frequency stimulations (5 pulses at 25 Hz, 10 pulses at 25 Hz, 5 pulses at 200 Hz and 10 pulses at 200 Hz) were performed in the presence of GYKI. There was no detectable EPSCKA under these stimulation conditions as well indicating the absence of synaptic KARs upon stimulation of this pathway. (C) To determine, whether any other input pathway would yield a significantly higher proportion of synaptic KAR mediated current, we stimulated at the border of LII–III mEC. In the presence of GYKI, an EPSCKA was observed (10.24%±1.1% of baseline; n = 5) as seen in an example trace from a single experiment (C, lower panel). Thus by stimulating a different pathway, a EPSCKA could be evoked on LIII mEC pyramidal neurons, suggesting the existence of synaptic KARs in a pathway specific manner.
Figure 6.
Role of GluK2 in network synchrony.
(A1) Rhythmic postsynaptic currents were recorded from WT mice (n = 7) following the application of 300 nM KA which had a frequency content of about 10–12 Hz. (A2) In comparison such a synchronised increase in spontaneous postsynaptic currents was absent in the GluK2 KO mice (n = 7). (B1, B2) Local field potential recordings within the superficial layers of the entorhinal cortex were done in an interphase chamber to record KA-induced gamma oscillations. (B1) Low concentrations of KA (300 nM) induced robust oscillations (n = 8 slices). Power spectra analysis revealed a major peak frequency of 40 Hz. The KA-induced gamma oscillations were blocked by the KAR/AMPAR-antagonist NBQX. (B2) Gamma oscillations were completely abolished in the GluK2 KO mice (n = 7 slices).