Cell-type specific expression of ATP-sensitive potassium channels in the rat hippocampus

Abstract

1. The distribution of ATP-sensitive K+ channels (KATP channels) was investigated in four cell types in hippocampal slices prepared from 10- to 13-day-old rats: CA1 pyramidal cells, interneurones of stratum radiatum in CA1, complex glial cells of the same area and granule cells of the dentate gyrus. The neuronal cell types were identified visually and characterized by the shapes and patterns of their action potentials and by neurobiotin labelling. 2. The patch-clamp technique was used to study the sensitivity of whole-cell currents to diazoxide (0.3 mM), a KATP channel opener, and to tolbutamide (0.5 mM) or glibenclamide (20 microM), two KATP channel inhibitors. The fraction of cells in which whole-cell currents were activated by diazoxide and inhibited by tolbutamide was 26% of pyramidal cells, 89 % of interneurones, 100% of glial cells and 89% of granule cells. The reversal potential of the diazoxide-induced current was at the K+ equilibrium potential and a similar current activated spontaneously when cells were dialysed with an ATP-free pipette solution. 3. Using the single-cell RT-PCR method, the presence of mRNA encoding KATP channel subunits (Kir6.1, Kir6.2, SUR1 and SUR2) was examined in CA1 pyramidal cells and interneurones. Subunit mRNA combinations that can result in functional KATP channels (Kir6.1 together with SUR1, Kir6.2 together with SUR1 or SUR2) were detected in only 17% of the pyramidal cells. On the other hand, KATP channels may be formed in 75% of the interneurones, mainly by the combination of Kir6.2 with SUR1 (58% of all interneurones). 4. The results of these combined analyses indicate that functional KATP channels are present in principal neurones, interneurones and glial cells of the rat hippocampus, but at highly different densities in the four cell types studied.

Figure 1. Morphological and electrophysiological characterization of CA1 pyramidal cells, interneurones and dentate granule cells

Top row, hippocampal slice stained with DAPI. The white frames enclose the regions in which the somata of CA1 pyramidal cells (A), interneurones from stratum radiatum CA1 (B) and dentate granule cells (C) were located. Second row, morphology of a pyramidal cell (A), an interneurone (B) and a granule cell (C). The cells were filled with neurobiotin, coupled to avidin-fluorescein, and imaged using a two-photon microscope. Axons are marked by white arrows. Note that the interneurone (B) is shown at a higher magnification and that the distal dendrites of this cell were cut during slicing. Third row, spontaneous action potentials recorded in the three cell types. Fourth row, trains of action potentials evoked by 400 ms current pulses of 50 pA, as indicated by the bars.

Figure 7. Presence of K_ATP channel subunit mRNA in adult rat brain

Upper panel, agarose gel electrophoresis of RT-PCR products from total RNA of adult rat brain stained with ethidium bromide. The amplification products of the K_ATP channel subunits Kir6.1, Kir6.2, SUR1 and SUR2 as well as their restriction analysis are indicated for the different lanes. The restriction fragments are visible as faint bands except the smaller restriction fragment of the Kir6.2 amplification product, which was too small to be detected in the gel. The penultimate lane shows a 547 bp amplification product of the NMDA receptor subunits NR2A-C. M, molecular weight marker pGEM (fragment size in bp). Lower panel, restriction analysis with fragment sizes of the multiplex PCR products encoding K_ATP channel subunits, the restriction enzymes used and their corresponding restriction fragments. Furthermore, the position of digestion relative to the translation start site of the coding sequence for each K_ATP channel subunit is given.

Figure 2. Response of a dentate granule cell to application of diazoxide and tolbutamide

Responses of whole-cell currents in a granule cell of the dentate gyrus to diazoxide (Diaz) and tolbutamide (Tol). A, currents recorded before, during and after 100 ms pulses from -70 to -60 mV at the times indicated by a-c in the experiment illustrated in B. Inset, protocol of voltage pulses to -80 and -60 mV. B, stationary currents at -80 and -60 mV recorded at 15 s intervals at various times after forming the whole-cell mode. Diazoxide (0.3 mM) and tolbutamide (0.5 mM) were applied twice each as indicated by the bars. The bars show the time during which the drugs were present in the bath.

Figure 3. Response of a CA1 interneurone to application of diazoxide and tolbutamide

Responses of whole-cell currents in an interneurone of the stratum radiatum of CA1 to diazoxide (0.3 mM) and tolbutamide (0.5 mM). A, currents recorded before, during and after 100 ms pulses from -70 to -60 mV at the times indicated by a-d in the experiment illustrated in B. B, stationary currents at -60 and -80 mV at various times after forming the whole-cell mode. The pipette solution contained no ATP in this experiment.

Figure 4. Response of a complex glial cell and a pyramidal cell to application of diazoxide and tolbutamide

Responses of stationary whole-cell currents at -60 and -80 mV to diazoxide (0.3 mM) and tolbutamide (0.5 mM) in a complex glial cell from stratum radiatum CA1 (A) and in a CA1 pyramidal cell (B) at various times after forming the whole-cell mode.

Figure 5. Reversal potential of the diazoxide-induced whole-cell current

Current-voltage relationships for an interneurone from the stratum radiatum of CA1 measured using voltage ramps of 2 s duration from +40 to -120 mV (voltage protocol in the upper inset). Each trace is the average of three ramps: a, control, before the application of diazoxide; b, diazoxide, in the presence of 0.3 mM diazoxide. b - a is the current induced by diazoxide. The lower inset shows the reversal potentials of the three traces at a higher resolution.

Figure 6. Amplitude of diazoxide-induced currents in different hippocampal cell types

Maximum currents induced by 0.3 mM diazoxide at a potential of -60 mV in four hippocampal cell types as indicated. The figure summarizes experiments performed with pipette solutions without (n= 17) or with 0.5 mM ATP (n= 57). The lower panel shows the proportion of cells that responded to diazoxide and to tolbutamide. The maximum currents of the responding cells were normalized to the cell capacitance and plotted in the upper panel. The columns and bars denote the means ±s.e.m.

Figure 8. K_ATP channel subunit mRNA in single hippocampal neurones

The cytoplasm of single cells was submitted to RT-multiplex PCR, and the reaction products were separated in agarose gel in the presence of the molecular weight marker pGEM (M) and stained with ethidium bromide. The scale to the left of each gel indicates the fragment size (in bp). The CA1 pyramidal cell (cell 67) expressed the SUR2 and NR2A-C subunit mRNAs, whereas the interneurone from stratum radiatum CA1 (cell 59) showed expression of Kir6.2 and SUR1 mRNA.