Modulation of the excitability of cholinergic basal forebrain neurones by KATP channels

Abstract

The expression of ATP-sensitive K(+) (K(ATP)) channels by magnocellular cholinergic basal forebrain (BF) neurones was investigated in thin brain slice and dissociated cell culture preparations using a combination of whole-cell, perforated-patch and single-channel recording techniques. Greater than 95% of BF neurones expressed functional K(ATP) channels whose activation resulted in membrane hyperpolarization and a profound fall in excitability. The whole-cell K(ATP) conductance was 14.0 +/- 1.5 nS and had a reversal potential of -91.4 +/- 0.9 mV that shifted by 59.6 mV with a tenfold increase in [K(+)](o). I(KATP) was inhibited reversibly by tolbutamide (IC(50) of 34.1 microM) and irreversibly by glibenclamide (0.3-3 nM) and had a low affinity for [ATP](i) (67% reduction with 6 mm[MgATP](i)). Using perforated-patch recording, a small proportion of the conductance was found to be tonically active. This was weakly potentiated by diazoxide (0.1 mm extracellular glucose) but insensitive to pinacidil (< or =500 microM). Single-channel K(ATP) currents recorded in symmetrical 140 mm K(+)-containing solutions exhibited weak inward rectification with a mean conductance of 66.2 +/- 1.9 pS. Channel activity was inhibited by MgATP (>50 microM) and activated by MgADP (200 microM). The K(+) channels opener diazoxide (200-500 microM) increased channel opening probability (NP(o)) by 486 +/- 120% whereas pinacidil (500 microM) had no effect. In conclusion, the characteristics of the K(ATP) channels expressed by BF neurones are very similar to channels composed of SUR1 and Kir6.2 subunits. In the native cell, their affinity for ATP is close to the resting [ATP](i), potentially allowing them to be modulated by physiologically relevant changes in [ATP](i). The effect of these channels on the level of ascending cholinergic excitation of the cortex and hippocampus is discussed.

Figure 1. Characteristics of basal forebrain (BF) neurones from brain slice and dissociated cell culture preparations

A, recording from BF neurones in brain slice preparations (13-day-old rats). Ai illustrates the spike after-hyperpolarization (AHP) (upper panels), slow firing rate and pronounced A-type current-induced delay of spike initiation (lower panel) that are typical features of cholinergic basal forebrain neurones. The cholinergic nature of this cell was confirmed using single-cell RT-PCR for ChAT (see panel C, gel lane 1). Aii shows typical firing characteristics (high frequency discharge with no spike AHP) associated with non-cholinergic BF neurones. Again the non-cholinergic nature of the cell was confirmed using RT-PCR (see panel C, lane 2). B illustrates that the firing characteristics of cholinergic BF neurones are still maintained after prolonged periods in culture. The cell shown was from a 32-day-old culture and the recording was carried out using perforated patch recording. Upper, middle and lower panels, respectively, show the spike AHP, I–V relationship and slow rate of firing characteristic of cholinergic neurones in intact preparations. Again the cholinergic nature of the cells was confirmed by RT-PCR (see panel C, lane 4). C, composite gel showing the presence and absence of ChAT (323 bp) reaction product in 4 different BF neurones. Cholinergic BF neurones also express a Substance P-sensitive inward rectifier current (Yamaguchi et al. 1990). D shows that this current is also maintained when the cells are maintained in culture (17-day-old culture). From a holding potential of –70 mV voltage steps from –125 to –55 mV (160 ms duration; 5 mV increments) were imposed to generate an I–V relationship under control conditions (0.5 μm TTX present throughout) and in the presence of 600 nm substance P.

Figure 2

A, whole-cell recording of a basal forebrain (BF) neurone from the diagonal band region of a thin (250 μm) brain slice. Immediately upon breaking through to whole-cell (1 mm[MgATP]_i), resting potential (V_m) was –58 mV. Within less than a minute of cell dialysis commencing, V_m slowly began to hyperpolarize, reaching a maximum of –71 mV after approximately 15 min. During this period, excitability declined markedly (see Bi and ii). Subsequent application of glibenclamide (3 nm) reversed both the hyperpolarization and fall in excitability (Biii). In this particular cell, excitability in the presence of glibenclamide was slightly higher than under control conditions. At the end of the record shown in A (glibenclamide present), current was directly injected through the electrode in order to restore the membrane potential to that observed at the peak of the hyperpolarization. Biv shows that the increase in excitability observed in the presence of glibenclamide (Biii) was not simply the result of the change in V_m (compare Bii and Biv). Ci, perforated-patch recording from a BF neurone maintained in culture for 13 days (2 mm extracellular glucose). Under control conditions, V_m was –63 mV. Hyperpolarizing current steps (100 pA (100 ms)⁻¹) were applied at 0.5 Hz to monitor changes membrane resistance (R_in). Application of tolbutamide (100 μm) caused a membrane depolarization associated with a fall in R_in. Cii illustrates the direct effect of tolbutamide on R_in after the associated depolarization had been nulled by current injection.

Figure 3. Voltage and K⁺ dependence of the sulphonylurea-sensitive run-up current

A, shows the whole-cell membrane current in response to ramping the membrane potential (V_m) from –126 to –47 mV (ramp 80 mV s⁻¹) following current run-up and after subsequent application of glibenclamide (30 nm). B, I–V relationship of the glibenclamide-sensitive component of the current shown in A. C, examples of the extremes of rectification observed in the I–V relation of the sulphonylurea-sensitive from different cells. D, the reversal potentials of the sulphonylurea-sensitive current measured in 3, 6 and 10 mm[K⁺]_o were 91.4 ± 0.94, –74.5 ± 1.01 and –59.9 ± 1.17 mV (n = 5), respectively, slope 59.6 mV for a 10-fold change in [K⁺]_o.

Figure 4. Kinetics of the whole-cell K_ATP channel current

I–V relationship (–127 to –57 mV in 10 mV increments) in the presence of (TTX, 0.5 μm) under (A) control conditions (V_h–87 mV) and (B) after addition of tolbutamide (100 μm). C, the tolbutamide-sensitive component of current exhibits no fast kinetic activation or inactivation. D, the I–V curve of the steady state (end of pulse) tolbutamide-sensitive current shown in B.

Figure 5

A, activation of the K_ATP conductance by diazoxide (200 μm) in a perforated-patched cell superfused with low extracellular glucose (0.1 mm) containing Krebs solution. hyperpolarizing current steps (100 pA (100 ms)⁻¹) were applied at 0.5 Hz to monitor changes membrane resistance (R_in). B illustrates the direct effect of diazoxide on R_in (*traces) after the associated depolarization had been nulled by current injection. C, histogram showing the sensitivity of whole-cell K_ATP run-up conductance to intracellular [ATP]. In each case, the K_ATP conductance was calculated by measuring the amplitude of the tolbutamide-sensitive current activated in response to stepping from –80 to –40 mV following full current run-up. A significant reduction in the amplitude of the run-up conductance was only observed with [ATP]_i of 6 mm.

Figure 6

A, inhibition of run-up current by glibenclamide. The K_ATP conductance was allowed to maximally activate under whole-cell recording conditions (0 mm[ATP]_i). Ordinate is membrane current (I_m) evoked by stepping from –80 to –40 mV (see inset) after run-up and in the presence of 0.3 nm glibenclamide. Inset shows membrane current in response to a 2 s step to –40 mV from V_h–80 mV followed by a 0.1 s step to –120 mV and ramp change in V_m to –40 mV (ramp 80 mV s⁻¹) before stepping back to V_h (protocol repeated every 20 s) at the points recording indicated by the asterisk. B, dose–response curve for the K_ATP current to tolbutamide. The curve was constructed from the mean IC₅₀ and slope values obtained from the individual cells. IC₅₀ and Hill slope values were 34.1 μm and 1.03, respectively. All points are mean ± s.e.m.

Figure 7. Simultaneous recording of single-channel and whole-cell K_ATP currents from a cholinergic basal forebrain neurone maintained in culture

Upper trace shows the whole cell current recorded from the cell after full activation of the K_ATP current. The cell was voltage clamped at a depolarized potential (–40 mV). The lower traces show single-channel activity from a cell-attached patch on the same cell (V_p+40 mV). Both the whole-cell and patch-electrodes were filled with 140 mm K⁺-containing solutions, whilst the bathing Krebs solution contained 3 mm K⁺. Under control conditions, an outward current was recorded by the whole-cell electrode and a high level of K_ATP channel activity was observed in the patch. Application of tolbutamide (100 μm) to the bathing solution reduced both the standing membrane current and single-channel activity. On washout the K_ATP current and channel activity both recovered. Note the occasional brief upward deflections from the zero current level are recording artefacts not channel openings.

Figure 8. Voltage-dependent rectification by K_ATP channels in the inside-out recording configuration

A, steady-state channel activity recorded in symmetrical 140 mm K⁺-containing solution for membrane potentials ranging between –80 and +80 mV (Note: the larger brief channel openings marked with an asterisk on the +80 mV record are clipped openings of a much larger unidentified channel). B, I–V curve for the single-channel currents shown in A. (Note: channel slope conductance was measured over the relatively linear region of the curve between –25 and –80 mV). C, rectification of single-channel currents as revealed by slowly ramping patch potential between –61 and +59 mV (ramp 67 mV s⁻¹). The patch contained at least two K_ATP channels.

Figure 9. K_ATP channel pharmacology (inside-out configuration in symmetrical 140 mm K⁺)

A, on excision there was an initial high level of channel activity which characteristically declined to a lower level (V_p+50 mV). Application of 0.5 mm MgATP inhibited all K_ATP channel activity (remaining small unidentified channel openings remain). On washout channel activity returned to a level similar to that observed immediately after excision but higher than that immediately prior to adding ATP (channel refreshment). Application of tolbutamide (100 μm) greatly reduced the frequency of channel openings. B, a second patch displaying increased channel opening in the presence of diazoxide (300 μm; V_p+80 mV). Again channel activity was greatly reduced by application of tolbutamide.

Figure 10

A, examination of the effect of acute changes in extracellular glucose levels on the excitability of cholinergic BF neurones. The illustrated recording was made from a cell maintained in culture for 6 days using the perforated-patch recording technique. Downward deflections are membrane voltage responses to current pulses delivered at 0.5 Hz (100 pA, 200 ms) used to monitor changes in input resistance. The threshold current required to evoke spike discharge was determined just prior to changing the extracellular glucose concentration (upward deflections). Insets show firing (200 ms duration pulses) and the threshold current under each condition. Note, prolonged current stimulation (0.5–1 s evoked a sustained slow rate of firing). B, the effect upon excitability of exposure to sodium azide (1 mm). Downward deflections are membrane voltage responses to 50 pA, 200 ms duration current pulses delivered at 1 Hz. On exposure to sodium azide membrane potential typically depolarized transiently before slowly hyperpolarizing. The hyperpolarization was associated with a fall in input resistance (R_in) and inhibited by tolbutamide confirming the involvement of K_ATP channels (data not shown). In the cell shown, R_in fell from 340 to 60 mΩ whilst the current required to evoke an action potential increased from 130 to 850 pA. Asterisk marks the region of the voltage trace where the change in membrane potential was nulled by direct current injection. From this it can be see that the majority of the fall in input resistance was the result of sodium azide-induced channel opening rather than as a secondary consequence of membrane hyperpolarization.