Interleukin 1beta modulates rat subfornical organ neurons as a result of activation of a non-selective cationic conductance

Abstract

The circumventricular organs (CVOs) are ideal locations at which circulating pyrogens may act to communicate with the CNS during an immune challenge. Their dense vasculature and fenestrated capillaries allow direct access of these pyrogens to CNS tissue without impediment of the blood-brain barrier (BBB). One such CVO, the subfornical organ (SFO), has been implicated as a site at which the circulating endogenous pyrogen interleukin 1beta (IL-1beta) acts to initiate the febrile response. This study was designed to determine the response of rat SFO neurons to IL-1beta (1 nM to 100 fM) using whole-cell current-clamp and voltage-damp techniques. We found that physiological(subseptic) concentrations of IL-1beta (1 pM, 500 fM, 100 fm) induced a transient depolarization in SFO neurons accompanied by a significant increase in spike frequency. In contrast,pharmacological (septic) concentrations of IL-1beta (1 nM) evoked a sustained hyperpolarization. While depolarizations in response to IL-1beta were abolished by treatment of cells with the IL-1 receptor antagonist (IL-1ra), hyperpolarizations were still observed. Voltage-clamp analysis revealed that the majority (85 %) of SFO neurons responding to IL-1beta with depolarization (29 of 34 cells) exhibited an electrophysiological profile characterized by a dominant delayed rectifier potassium current (DIK), a conductance that we also found to be reduced to 84.4 +/- 3.3 % of control by bath application of IL-1beta. In addition, using slow voltage ramps we demonstrated that IL-1beta activates a non-selective cationic current (INSC) with a reversal potential of -38.8 +/- 1.8 mV. These studies identify the cellular mechanisms through which IL-1beta can influence the excitability of SFO neurons and, as a consequence of such actions, initiate the febrile response to exogenous pyrogens.

Figure 1. Voltage-clamp recordings indicate the presence of two populations of SFO neurons

Cells were held at −70 mV and currents were elicited by 250 ms voltage steps from −70 to +30 mV in 10 mV increments. The trace on the lower left depicts a typical dominant I_A cell (DI_A) and the trace on the lower right a typical dominant I_K cell (DI_K). The pie chart displays the ratio of DI_A (60 %) to DI_K (40 %) cells recorded. The single traces show the −10 mV step, where the peak (*) to steady-state (○) ratio was determined. As indicated in the table, cells with a peak to steady-state ratio greater than 2 at this −10 mV step were classified as DI_A cells; those with a ratio less than 2 were classified as DI_K cells.

Figure 2. Current-clamp recordings during application of IL-1β

Current-clamp traces showing responses to 120 s application of IL-1β. A-C, transient depolarizations with recovery at low dose 500 fM, 1 pM and 10 pM IL-1β, respectively. D, transient depolarization followed by a sustained hyperpolarization at 100 pM IL-1β. E, sustained hyperpolarization at high dose 1 nM IL-1β. Dotted lines represent resting membrane potentials.

Figure 3. IL-1β influences membrane potential and input resistance

A, peak changes in membrane potential for neurons responding to IL-1β with a transient depolarization at low doses (100–500 fM), a transient depolarization followed by a sustained hyperpolarization at intermediate doses (1–100 pM), or a sustained hyperpolarization in response to a high dose (1 nM − note that very few cells demonstrated such hyperpolarization in isolation). Each bar represents the mean ±s.e.m. for group data with n values indicated in boxes. B, IL-1β also decreased input resistance in SFO neurons as illustrated in the V-I curves shown here, which were obtained from a single SFO neuron prior to (▪) and during (□) the depolarization observed in response to 10 pM IL-1β administration. C, histogram summarizing the effects of IL-1β on 8 responsive (depolarization) SFO neurons in which input resistance decreased from 815 ± 57 to 690 ± 57 MΩ during IL-1β application with a recovery to 800 ± 59 MΩ (**P < 0.005, paired t test, n = 8).

Figure 4. Current-clamp recordings during application of IL-1ra and IL-1β

A, current-clamp recording from an SFO neuron illustrating the lack of a depolarizing response to 100 pM IL-1β (grey bars) administered at the same time as bath application of IL-1ra (open bars). Notice that although the depolarization was not observed, a longer latency hyperpolarization did still occur in response to this dose of IL-1β. Hyperpolarizing pulses of 25 pA were given at 10 s intervals throughout this recording. Bi, current-clamp recordings from a second SFO neuron that showed no response to 120 s application of 500 fM IL-1β during IL-1ra treatment. ii, in contrast a second application of the same dose of IL-1β following wash-out of IL-1ra results in a depolarization. Dotted lines represent resting membrane potential.

Figure 5. Peak to steady-state ratios of IL-1β-responsive and non-responsive neurons

Peak to steady-state ratios of cells within the effective IL-1β depolarizing dose range (100 pM to 100 fM); 34 of 56 cells depolarized, 29 of which were classified as DI_K as they had a peak to steady-state ratio less than 2. The mean values ±s.e.m. for the responsive and non-responsive cell groups (▪) were 1.73 ± 0.17 and 2.75 ± 0.35, respectively (*P < 0.01). The inset depicts the percentage of depolarizing cells in the DI_K (78 %, 29 of 37 cells) and DI_A (26 %, 5 of 19 cells) cell populations.

Figure 6. Voltage-clamp recordings displaying activation of NSCC following IL-1β application

A, voltage-clamp recordings showing the currents produced by a 12 mV s⁻¹ depolarizing ramp. Shown here is the control current, the current elicited by 500 fM IL-1β (red) and the recovery current following ACSF wash. The difference current obtained by subtracting control currents from currents measured during IL-1β administration are displayed in the inset. B, average linear difference current obtained from the 9 cells (out of 15) that responded to 500 fM IL-1β during this slow ramp protocol. The slope of this current was 0.60 ± 0.12 pA mV⁻¹ and the reversal potential was −38.8 ± 1.8 mV.

Figure 7. Voltage-clamp recordings displaying a decrease in I_K following IL-1β application

Voltage-clamp step protocols (250 ms steps from −70 to +30 mV in the presence of TTX), with (as shown in A) or without a 500 ms prepulse at −30 mV to inactivate I_A, were used to permit isolation of I_K and I_A. A, the isolated I_K before (left) and after (right) 500 fM application of IL-1β, revealing a decrease in this current. Bi, overlaid control and IL-1β currents; C, I-V relationship of the 4 I_K cells that responded to 500 fM IL-1β (out of a total of 7 tested), illustrating that IL-1β caused a significant decrease in current in this subpopulation of SFO neurons in the +10 to +30 mV range (*P < 0.05). Bii, overlaid traces showing the complete lack of effect of IL-1β on the subtracted I_A.