Nitric oxide is required for the induction and heterosynaptic spread of long-term potentiation in rat cerebellar slices

Abstract

1. In the cerebellar cortex, brief, 8 Hz activation of parallel fibres (PFs) induces a cyclic adenosine 3'5'-monophosphate (cAMP) and protein kinase A (PKA)-dependent form of long-term potentiation between PFs and Purkinje cells. 2. With 10 mM BAPTA in the recording pipette, potentiation evoked by raised frequency stimulation (RFS) to one of two, synaptically independent PF inputs to the same Purkinje cell did not remain input specific but consistently spread to synapses that did not receive RFS, up to the maximum distance tested of 168 microm. 3. LTP at activated and non-activated sites was accompanied by a decrease in paired pulse facilitation (PPF). The PKA inhibitor H-89 blocked both of these effects. Inhibition of nitric oxide synthase (NOS), either by 7-nitro-indazole (7-NI) or N (G)-nitro-L-arginine methyl ester (L-NAME), completely prevented heterosynaptic potentiation and associated reduction in PPF. LTP at distant synapses was selectively prevented by the nitric oxide scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO). Inhibition of soluble guanylate cyclase or protein kinase G had no effect on either pathway. 4. Synaptic potentiation at PF-PC synapses, induced by the adenylate cyclase activator forskolin, was also prevented by inhibition of NOS. Forskolin-induced increases in mEPSC frequency were similarly prevented by NOS inhibition and mimicked by the NO donor spermine NONOate. 5. These results are consistent with the notion that heterosynaptic potentiation is of pre-synaptic origin and dependent upon activation of cAMP/PKA and NO. Moreover, they suggest that cAMP/PKA activation stimulates NO production and this diffusible messenger facilitates pre-synaptic transmitter release at synapses within a radius of upwards of 150 microm, through a mechanism that does not involve cGMP.

Figure 1. RFS to one of two PF inputs induces PKA-dependent LTP in Purkinje cells that is associated with a decrease in the PPR in both pathways

A, once baseline responses to 0.2 Hz activation of two separate PF inputs to Purkinje cells were stable, one pathway, designated P₀ (•), was activated at a raised frequency of 8 Hz for 15 s at time 0 (RFS, arrow). The other pathway (P₁; ○) was not activated during this period. Stimulation to both pathways was then resumed at 0.2 Hz. With 10 mm BAPTA included in the patch pipette, RFS led to a long-lasting increase in synaptic responses in both pathways. B, LTP was accompanied by a reduction in the paired-pulse ratio in both pathways. Data are expressed as the percentage change in the ratio of the second pulse to the first compared to baseline levels. C, a single, representative example of EPSCs elicited by stimulation of P₀ (top) and P₁ (bottom) at times a and b indicated in panel A. The amplitudes of the first pulse in P₀ and P₁ were normalized to illustrate the accompanying reduction in the PPR (a + b). D, the means and standard errors of P₀ (▪) and P₁ normalized EPSCs (□) measured 20 min after RFS are shown under conditions of 10 mm intracellular BAPTA, BAPTA plus 0.2 μm extracellular H-89 and following cell hyperpolarization to -90 mV during RFS with 0.5 mm EGTA inside the recording pipette. E, t he associated changes in PPR measured at the same points in time are shown. In graphs (A, B, D and E), the means and s.e.m. of six experiments are shown. Asterisks indicate a statistical difference between test and control conditions (Mann-Whitney U test, P < 0.05).

Figure 2. P₀ and P₁ display pathway independence

A, parallel fibre pathways P₀ and P₁ to a single Purkinje cell were activated at 50 ms intervals and again in reverse order 150 ms later. When preceded by activation of the alternate pathway, P₀ and P₁ EPSC amplitudes remained similar to naïve responses, in this and in four other examples. B, the second response to P₁-P₀ stimulation (*) and P₀-P₁ stimulation (#) shown in A are superimposed on P₀-P₀ and P₁-P₁ responses, respectively. C, the ratios of the percentage level of potentiation compared to baseline levels observed in P₀ compared to that in P₁ are plotted against the separations between electrodes for a group of 15 cells. The dashed line represents the fitted linear regression.

Figure 3. RFS-induced LTP requires NOS activity

A, inhibition of NOS with 7-NI in the extracellular perfusate prevented LTP in pathways P₀ and P₁. B, the associated mean changes in PPR over time are shown. C, a comparison of P₀ and P₁ responses recorded under standard control conditions of 10 mm intracellular BAPTA and in the presence of 7-NI and l-NAME. Application of 5 μm 7-NI to the bathing medium 5 min after RFS had no effect on the extent of LTP in either P₀ or P₁. D, the associated changes in PPR are shown. The means and s.e.m. of 6 (7-NI), 5 (l-NAME) and 4 (7-NI application 5 min after RFS) cells are shown. Asterisks indicate a statistical difference between test and control conditions (Mann-Whitney U test, *P < 0.05; **P < 0.01).

Figure 4. RFS induces input-specific LTP in the presence of the NO scavenger cPTIO

With 10 mm BAPTA in the patch pipette and 30 μm cPTIO in the extracellular perfusate, RFS to pathway P₀ led to an input-specific, long-lasting potentiation of P₀ synaptic responses (A) and an input-specific reduction in the paired pulse ratio (B). Examples of representative P₀ and P₁ EPSCs taken at times a and b indicated in panel A are shown below (C. D), the means and s.e.m. of P₀ (▪) and P₁ normalized EPSCs (□) measured 20 min after RFS are shown under control conditions (10 mm intracellular BAPTA) and with cPTIO in the perfusate together with changes in the PPR (E). In graphs (A, B, D and E), the means and s.e.m. of six experiments are shown. Asterisks indicate a statistical difference between P₀ and P₁ pathways (Wilcoxon signed-rank test, P < 0.01).

Figure 5. RFS-induced LTP does not require guanylate cyclase

Inhibition of guanylate cyclase with 5 μm extracellular ODQ did not prevent LTP (A) or the associated changes in PPR (B) in either P₀ or P₁. Similarly, neither intracellular ODQ (5 μm) nor the PKG inhibitor KT5823 (500 nm) prevented LTP or the decrease in PPR (D). Potentiation (C) and the reduction in PPR (D) was prevented in both pathways when the NMDA receptor antagonist AP5 (50 μm) was present in the perfusate. In all cases, the means and s.e.m. of six cells are shown.

Figure 6. Forskolin induces LTP

A, 10 min bath application of 10 μm forskolin led to a sustained potentiation of P₀ and P₁ responses. B, inhibition of NOS with extracellular 7-NI prevented forskolin-induced LTP. C, a comparison of P₀ and P₁ responses recorded in the presence of forskolin and additionally with 7-NI. Asterisks indicate statistical difference between test and control conditions (Mann-Whitney U test): **P < 0.01, *P < 0.05. D, the associated changes in normalized PPR are illustrated. The means and s.e.m. of six cells are shown.

Figure 7. Application of NO results in LTP

A, 10 min bath application of the NO donor spermine NONOate induced an increase of responses in both pathways. B, illustration of the associated change in PPR over time. C, a comparison of P₀ and P₁ responses measured 20 min after RFS under control conditions of 10 mm BAPTA and 20 min after NONOate application in the absence and the presence of H-89.

Figure 8. NO donor-induced potentiation does not require PKA

In the presence of 0.2 μm H-89, 15 s RFS (arrow) failed to induce potentiation. Subsequent application of 100 μm spermine NONOate (horizontal bar) produced a potentiation of P₀ and P₁ responses.

Figure 9. Forskolin increases mEPSC frequency via NOS and NO activities

A, changes in mEPSC frequency over time expressed as a percentage of baseline levels under control conditions of 10 mm intracellular BAPTA. The effects of application of 10 μm forskolin and 100 μm NONOate are shown. B, the frequency distributions of mEPSCs over two 4 min periods, prior to (□) and 15 min after application of forskolin (▪) in absence (left) and presence (centre) of 7-NI and the frequency distributions of mEPSCs recorded prior to and 15 min after application of 10 μm spermine NONOate (right) are shown. Inset are shown cumulative probability curves. C, means and s.e.m. of mEPSC frequency recorded under standard conditions, in the presence of forskolin (Forsk), in the additional presence of 7-NI, and after NONOate application. Asterisks represent a statistical difference (P < 0.01) between control and test conditions (Kolmogorov-Smirnov test).