Persistent changes in spontaneous firing of Purkinje neurons triggered by the nitric oxide signaling cascade

Abstract

Many types of neurons fire spontaneously because of the activity of pacemaking ion channels. Although endogenous firing can serve as a persistent signal to downstream targets, little attention has been paid to factors that might modulate such intrinsic electrical activity. We tested for modulation of spontaneous firing of Purkinje neurons in cerebellar slices under conditions in which principal synaptic inputs were blocked. Loose-patch recordings from single neurons show that sustained (>40 min) increases in the spontaneous firing rate can be triggered by activation of the nitric oxide-cGMP signaling pathway. Inhibitors of soluble guanylate cyclase and protein kinase G block this modulation. Increases in firing rate are also observed after stimulation of parallel fibers but not in response to basket cell activity. These findings elucidate a novel role for the nitric oxide-cGMP signaling cascade in the brain. This mechanism could permit long-term adjustments in the baseline firing rate of endogenously active neurons in response to changes in afferent activity.

Fig. 1.

The nitric oxide donor NOR-4 causes an increase in baseline firing rate. A, Example traces taken at times −10 min, 5 min, and 35 min relative to the start of NOR-4 application (C). Calibration: 75 pA, 100 msec.B, Histograms of interspike intervals (ISIs) constructed from 10 min periods at −10 min (open bars) and 30 min (filled bars). Superimposed are Gaussian curves of the form $y={\text{Ae}}^{-\frac{(x-\overline{x}{)}^2}{2{SD}^2}}$where A is an amplitude factor, andx̄ is the mean. For the open bars,x̄ = 55 msec, SD = 2.9, and A = 0.137; for the filled bars, x̄ = 47 msec, SD = 2.7, and A = 0.145. χ² values for both fits are <2 × 10⁻⁴. C, Normalized, average firing rate versus time for six neurons. After a 10 min control period, 100 μm NOR-4 was added to the bath as indicated by the bar. On average, firing rates increased 11.4 ± 1.8% (at t = 17 min) and remained elevated for >50 min.

Fig. 2.

Membrane-permeant cGMP analogs also increase baseline firing rate. A, Example traces from one neuron in control and 35 min after application of 50 μm8p-CPT-cGMP. Calibration: 30 pA, 100 msec. B, Average, normalized firing rate versus time for 10 Purkinje neurons. After a 10 min control period, 50 μm 8p-CPT-cGMP was added at the indicated time. On average, the firing rate increased 17.7 ± 4.1%.

Fig. 3.

Summary data for 8p-CPT-cGMP, NOR-4, and control cells. A, Plot of the control firing rate for each cell measured over the 5 min period just before drug application versus the firing rate 7 min after the end of the experimental treatment. Each type of experiment is represented by different symbols (control, ●; NOR-4, ○; 8p-CPT-cGMP, ■; 8-Br-cGMP, ▵), and the error bars indicate SD. Thediagonal dotted line indicates no change in firing rate. The vertical distance between the marker and thedotted line represents the number of additional spikes every second. * denotes the example cell in Figure 1, and # indicates the example cell in Figure 2. Inset, Shown at a compressed scale to show the cells with higher firing rates.B, Average time course of four control cells. Although there is a small tendency toward increased firing rates, it is not significant and does not resemble the increases seen in the experimental conditions. C, Histogram shows average change in firing rate at t = 5–10 min after drug application compared with control (−5–0 min) under various conditions. NOR-4 increased firing rate (n = 6;p = 0.0015). ODQ (10 μm), an inhibitor of sGC activity, blocked the increase in firing rate when coapplied with NOR-4 (n = 4; p= 0.24). KT5823 (1 μm), an inhibitor of PKG activity, also blocked an increase in firing rate when coapplied with NOR (n = 6; p = 0.87).

Fig. 4.

Stimulation of parallel fibers or basket cells to generate endogenous NO. A, To maximize NO production from PFs, presynaptic fiber volleys were measured and stimulus intensity was adjusted before each experiment. Average fiber volley across all experiments was 3.0 ± 1.4 mV (range, 2–5 mV). Calibration: 3 mV, 0.5 msec. B, An example of one experiment. Five trains of stimuli (20 Hz for 5 sec, 6 sec intertrain interval) was delivered to the PFs at t = 0 min.C, Average results from seven control cells;D, eight NOS inhibitor cells. NOS inhibitors block an increase in firing rate. At t = 20 min, control cells tend toward higher firing rates, whereas cells in the presence of NOS inhibitors tend toward baseline firing rates. E, Paired recordings (1 whole-cell recording from a basket cell, 1 extracellular recording from a synaptically connected Purkinje neuron) were established in the absence of picrotoxin. Calibration: 20 mV, 75 pA, 100 msec. F, After addition of 100 μmpicrotoxin to block the fast synaptic inhibition, a presynaptic basket cell was stimulated at time t = 0 min. Little change in firing is visible in this example recording from the connected Purkinje neuron. G, On average, no significant increase could be detected in four pairs.