Distinct kainate receptor phenotypes in immature and mature mouse cerebellar granule cells

Abstract

1. Although glutamate receptors have been shown to be involved in neuronal maturation, a developmental role for kainate-type receptors has not been described. In addition, the single-channel properties of native kainate receptors have not been studied in situ. We have characterized the electrophysiological properties of native kainate receptors of granule cell neurons at two distinct stages in postnatal development, using whole-cell and outside-out patch-clamp recordings in acute cerebellar slices. 2. Kainate-type currents were detected in both immature and mature granule cells. However, noise analysis showed that the apparent unitary conductance of kainate-type channels is significantly higher in proliferating than post-migratory granule cells. The conductance and rectification behaviour of the channels in immature granule cells indicate that they contain unedited GluR5 and GluR6 subunits and are likely to be calcium permeable. 3. Single-channel kainate-type currents were observed in outside-out patches from proliferating granule cells in the external germinal layer. The kinetic behaviour of kainate receptors in immature cells was complex. Openings to multiple conductance levels were observed, although our analysis indicates that the channels spend most of their open time in a 4 pS state.

Figure 3. Kainate receptors in immature granule cells have a larger apparent unitary conductance than those in mature cells

Aa, whole-cell current evoked by 10 μM kainate in an EGL cell (-60 mV). Scale, 20 pA, 5 s. b, portions of the record in a before (control) and during kainate at a larger gain and on a faster time scale. Scale, 10 pA, 50 ms. B, whole-cell current evoked by 10 μM kainate in an IGL cell (-60 mV). Note the smaller increase in kainate-evoked current noise compared with the current evoked in the EGL cell. C and D, power spectra of the currents in A and B, respectively. The data were fitted (continuous line) with the sum of two Lorentzian components (dashed lines). The half-power frequencies and γ_noise values obtained from the fits are indicated. Data were sampled at 9.4 kHz and low-pass filtered at 2 kHz. E, current-variance plot from a response to 10 μM kainate in an EGL cell. F, plot of P_open values measured in EGL and IGL cells. The numbers represent how many cells gave a P_open value below 0.05. In these cases, the P_open was poorly defined (although very low), and conductance measurements were not corrected. The γ_noise values were corrected for all cells that gave P_open > 0.05.

Figure 1. Kainate-evoked whole-cell currents from granule cells in acute cerebellar slices

Holding potential, -60 mV. Aa, in an EGL cell, 300 μM kainate did not elicit a detectable inward current in the absence of Con A (P9 mouse); b and c, in the presence of Con A (25 μM), 10 μM and 300 μM kainate evoked responses of similar amplitudes in another EGL cell (P9 mouse). Ba, kainate-type current in an IGL cell from a P19 mouse in the presence of Con A (25 μM) and GYKI 53655 (200 μM); b, in the absence of GYKI, 300 μM kainate produced a large AMPA-type current in the same cell. c, the AMPA receptor response to 300 μM kainate is blocked by GYKI with a delay. The size of the GYKI-resistant current is similar to that evoked by 10 μM kainate. All currents shown were fully reversible. Data were sampled at 9.4 kHz and low-pass filtered at 2 kHz.

Figure 2. Native kainate receptors in immature cerebellar granule cells show a high affinity for kainate and display inward rectification

A, concentration-response data for steady-state kainate-evoked currents in an EGL cell (with Con A, 25 μM). The results were fitted with Hill-type equations, either including (dashed line) or excluding (continuous line) the 300 μM data point. The EC₅₀ values obtained from the fits were similar (7 μM vs. 10 μM), suggesting that contamination of the currents by AMPA-type channels was minimal. B, current-voltage curve from voltage ramps (-60 to +60 mV) during a steady-state response to 10 μM kainate in an EGL cell. Despite the presence of potassium channel blockers, similar measurements could not be made for mature IGL cells due to residual potassium currents.

Figure 4. Single-channel currents through native kainate receptors

A, unitary currents in outside-out patches from two EGL cells (-100 mV, Con A). Data were sampled at 18.8 kHz and filtered at 1 kHz. B, analysis of the bottom trace in A gave a low-variance open point histogram with three visible peaks. The three-Gaussian fit to the results gave mean conductance values of 4, 9 and 15 pS (dashed lines in A), levels frequently observed in other portions of the record. C, low-variance open point histogram for 2.5 min of the recording during application of 500 nM domoate. The peak corresponds to a conductance of 4 pS. D, whole-cell recording at -100 mV from a Con A-treated IGL cell during application of 10 μM kainate in the presence of 200 μM GYKI 53655, 20 μM APV, 20 μM 7-chlorokynurenate and 10 μM bicuculline. Note the long duration of the channel openings (downwards).