cAMP-dependent long-term potentiation of nitric oxide release from cerebellar parallel fibers in rats

Abstract

Nitric Oxide (NO) is released from parallel fibers (PFs) after PF stimulation. NO-cGMP signaling is essential for long-term depression (LTD) in cerebellar PF-Purkinje cell synapses, which also exhibit presynaptic long-term potentiation (LTP) after tetanic PF stimulation. This LTP is dependent on cAMP but not NO-cGMP signaling. In this study, we analyzed long-term changes of NO release from PFs in rat cerebellar slices using electrochemical NO probes. Repetitive PF stimulation at 10 Hz for 2 sec elicited a transient increase in NO concentration (2.2 +/- 0.1 nM; mean +/- SEM; n = 116). This NO release exhibited long-term potentiation (LTPNO) by 36 +/- 3% (n = 15) after tetanic PF stimulation. Induction of LTPNO was not affected by Glu receptor antagonists. NO release from PFs was also potentiated by L-Arg (ARG) (100 microM), forskolin (50 microM), and 8-bromo-cAMP (Br-cAMP) (1 mM) but not by 1,9-dideoxyforskolin (50 microM), a biologically inactive analog of forskolin. The potentiation induced by forskolin was significantly suppressed by H89 (10 microM), a blocker of cAMP-dependent protein kinase. The potentiation induced by forskolin, but not that induced by Arg, interfered with LTPNO. H89 (10 microM) and KT5720 (1 microM), another blocker of cAMP-dependent protein kinase, but not KT5823 (300 nM), a blocker of cGMP-dependent protein kinase, significantly suppressed LTPNO. These data indicate that neural NO release is under activity-dependent control, just as synaptic transmitter release is. LTPNO might play a role in cross talk between presynaptic and postsynaptic plasticity by facilitating NO-cGMP-dependent postsynaptic LTD after induction of cAMP-dependent presynaptic LTP and LTPNO.

Fig. 1.

NO release from PFs in coronal cerebellar slices.A, Schema showing the experimental setup.B, NO release elicited by repetitive PF stimulation (10 Hz for 2 sec) and recorded under different extracellular Ca²⁺ concentrations (0–3.0 mm) in a slice. C, Amplitude of NO release elicited by repetitive PF stimulation at 2 min intervals and blockade of NO release by 10 μm NA. Inset shows superimposed originaltraces recorded before (a) and during (b) NA application.

Fig. 2.

LTP_NO elicited by TS.A, Amplitude of NO release recorded before and after TS.Inset shows superimposed traces of NO release recorded at the initiation of recording (a), immediately before initiation of TS (b), immediately after cessation of TS (c), and 30 min after cessation of TS (d).B,a, Time course of relative NO release before and after TS (filled circles) or in the absence of TS (open circles).B,b, Averaged PF volley potentials elicited by single pulse stimulation immediately before initiation and 30 min after cessation of TS (asterisk).B,c, Averaged PF volley potentials elicited by 10 Hz train pulses immediately before initiation and 30 min after cessation of TS (asterisk).C, Changes in NO release (hatched bars) and field EPSPs (filled bars). Gradual potentiation during the initial 50 min of the recording (Gradual), TS-induced potentiation (TS), the reduction caused by the change in stimulus intensity from 500 to 400 μA (Stimulus), and the reduction caused by the change in extracellular Ca²⁺concentration from 2.4 to 1.8 mm(Ca2+) are shown. The amplitude of each change was normalized by the amplitude of NO release or field EPSPs recorded before the change occurred. Each bar anderror bar represent the absolute value of the mean ± SEM of five experiments. Except for the experiment in which extracellular Ca²⁺ concentration was decreased, NO release and field EPSPs were simultaneously recorded in the same slice, as shown by the inset.

Fig. 3.

Effects of Glu blockers on LTP_NO.A, Amplitude of NO release recorded before, during, and after application of CNQX (10 μm) and TS.Insets show superimposed traces recorded immediately before application of CNQX (a) or TS (b) and 30 min after cessation of TS (c). B, Superimposedtraces recorded before and 30 min after TS (asterisk), which was applied to the slice in the presence of 50 μm APV. C,Traces recorded before and 30 min after TS (asterisk) applied in the presence of 500 μm MCPG. D, Amplitude of control LTP_NO and LTP_NO elicited in the presence of 10 μm CNQX, 50 μm APV, 500 μmMCPG, or 10 μm CNQX plus 500 μm MCPG. The mean ± SEM are shown.

Fig. 4.

LTP_NO in the presence of excessive Arg. A, Dependence of the amplitude of NO release on Arg concentration in the perfusing media. Data represent the mean ± SEM of five experiments. The values are normalized by that recorded in the perfusing medium not containing Arg. Inset shows superimposed traces recorded in a slice in perfusing media containing 0–100 μm Arg. B, Changes in amplitude of NO release elicited by 100 μm Arg application and TS. Inset shows superimposedtraces recorded immediately before Arg application (a), immediately before initiation (b), and 30 min after cessation of TS (c). C, Time courses of LTP_NO in the presence (open circles) or absence of 100 μm Arg (filled circles).

Fig. 5.

Potentiation of NO release induced by forskolin (FSK) and a cAMP analog. A, Potentiation of NO release induced by 50 μmforskolin (filled circles).Inset shows superposed traces recorded before application of forskolin (a), in the presence of forskolin (b), and 30 min after the cessation of forskolin application (c). Forskolin-induced potentiation in the presence of 10 μmH89 is also shown (open circles). In this experiment, slices were incubated with 10 μm H89 for at least 2 hr before and throughout the recording. B, Potentiation of NO release induced by forskolin and TS (filled circles). Inset shows tracesrecorded before application of forskolin (a), before initiation of TS (b), and 30 min after the cessation of forskolin application and TS (c). Changes in NO release elicited by application of 1,9-dideoxyforskolin (50 μm, DideoxyFSK) are also shown (open circles). C, Potentiation of NO release induced by 1 mm Br-cAMP (horizontal bar). D, Effect of 1 mm Br-cGMP (horizontal bar) on NO release.

Fig. 6.

Blockade of LTP_NO and gradual potentiation of NO release by H89 or KT5720. A, Time course of LTP_NO recorded in the presence of 10 μm H89 (open circles), 1 μmKT5720 (open circles), or 300 nm KT5823 (filled circles). B, Time courses of gradual potentiation of NO release induced by test stimuli in the absence (filled circles) or presence of 10 μm H89 (open circles). Slices were incubated with 10 μm H89, 1 μm KT5720, or 300 nm KT5823 for at least 2 hr before and throughout the recording.