Presynaptic role of cGMP-dependent protein kinase during long-lasting potentiation

Abstract

Previous research has suggested that cGMP-dependent protein kinases (cGKs) may play a role in long-term potentiation in hippocampus, but their site of action has been unknown. We examined this question at synapses between pairs of hippocampal neurons in dissociated cell culture. Injection of a specific peptide inhibitor of cGK into the presynaptic but not the postsynaptic neuron blocked long-lasting potentiation induced by tetanic stimulation of the presynaptic neuron. As controls, injection of a scrambled peptide or a peptide inhibitor of cAMP-dependent protein kinase into either neuron did not block potentiation. Conversely, injection of the alpha isozyme of cGK type I into the presynaptic but not the postsynaptic neuron produced activity-dependent potentiation that did not require NMDA receptor activation. Evidence from Western blots, reverse transcription-PCR, activity assays, and immunocytochemistry indicates that endogenous cGK type I is present in the neurons, including presynaptic terminals. These results support the idea that cGK plays an important presynaptic role during the induction of long-lasting potentiation in hippocampal neurons.

Fig. 1.

Presynaptic injection of cGKi blocks potentiation by tetanic stimulation. A, Experimental arrangement. B₁, Example of potentiation by tetanic stimulation of the presynaptic neuron after injection of cGKi into the postsynaptic neuron. EPSCs were produced in the postsynaptic neuron by step depolarization sufficient to elicit an inward current in the presynaptic neuron once every 10 sec. The current in the presynaptic neuron has had leakage subtracted. Both recordings are a.c.-coupled. Sample traces are shown before (Pre) and 25 min after tetanic stimulation of the presynaptic neuron (three 50 Hz, 2 sec trains of depolarizations at 20 sec intervals) during brief perfusion with Mg²⁺-free solution. Four successive traces are superimposed at each time period. The dashed line shows the average Pre value.B₂, Example of block of potentiation by injection of cGKi into the presynaptic neuron. C, Average potentiation produced by tetanic stimulation (a; filled inverted triangles) and slight rundown of the EPSC in test-alone controls (b; open inverted triangles). Injection of cGKi into the postsynaptic neuron reduced but did not block potentiation by tetanic stimulation (c; filled triangles), whereas injection of cGKi into the presynaptic neuron completely blocked the potentiation (d; filled circles). Injection of cGKi alone into either the postsynaptic neuron (e; open triangles) or the presynaptic neuron (f;open circles) had no effect on the baseline EPSC. EPSC amplitude has been normalized to the average value during the 10 min before training (% of Pre) in each experiment. Average Pre values in picoamperes were as follows: (a) 137 ± 33,n = 21; (b) 157 ± 86, n = 15; (c) 123 ± 39, n = 22; (d) 110 ± 43,n = 11; (e) 325 ± 191, n= 12; and (f) 178 ± 108, n = 13; not significantly different by a one-way ANOVA. Tetanic stimulation (3 arrows) occurred at time 0. The horizontal bar shows the time during which the bath solution was briefly changed to one with 0 Mg²⁺. The inhibitor was present throughout the experiment. Each point represents the average of 20 successive trials, and the numbers at 25 min indicate the n at that point. Some experiments were terminated earlier if the electrode seal was lost or the input resistance changed in either the presynaptic or postsynaptic cell. Thepoints indicate the means, the error bars indicate SEM, and asterisks indicate a significant difference from both the Pre level (dashed line) and the nontetanized controls.

Fig. 2.

Injection of ScGKi or cAKi does not block potentiation. A, Experimental arrangement.B₁, Example of potentiation by tetanic stimulation after injection of cAKi into the postsynaptic neuron.B₂, Example of potentiation by tetanic stimulation after injection of cAKi into the presynaptic neuron.C, Average potentiation by tetanic stimulation after injection of ScGKi into the presynaptic neuron (a; filled inverted triangles). Tetanic stimulation also still produced potentiation after injection of cAKi into either the postsynaptic neuron (b; filled triangles) or the presynaptic neuron (c; filled circles). Injection of ScGKi alone into the presynaptic neuron (d; open inverted triangles) or injection of cAKi alone into either the postsynaptic neuron (e;open triangles) or the presynaptic neuron (f;open circles) did not have an effect on EPSC amplitude. Average Pre values in picoamperes were as follows: (a) 116 ± 46,n = 8; (b) 60 ± 24, n = 13; (c) 90 ± 24, n = 19; (d) 83 ± 30,n = 13; (e) 130 ± 44, n = 7; and (f) 136 ± 49, n = 15; not significantly different by a one-way ANOVA.

Fig. 3.

Cultured hippocampal neurons contain cGK type I.A, Western blot demonstration of endogenous cGK type I in cultured hippocampal cells. Standards of recombinant cGK I and cGK II (76 and 86 kDa, respectively) purified from Sf9 cells were used for identifying cGKs in samples (25 μg of protein) from hippocampal cultures or microdissected hippocampus. Both cultured hippocampal cells and microdissected hippocampus contained a protein that migrated like cGK I (A₁). The cGK I signals were blocked by antibody (Ab) preabsorption, indicating that they are specific. Microdissected hippocampus also contained a protein that migrated like cGK II, but cultured hippocampal cells did not (A₂). B, In agreement with the Western blot analysis, the RT-PCR technique detected cGK I but not cGK II mRNA in samples from cultured hippocampal cells. RT-PCR products obtained using oligonucleotide primers specific for cGK I and cGK II were analyzed using 1.5% agarose gels and ethidium bromide staining. DNA molecular weight standards (Marker) and the PCR product for cGK I (378 kb; arrow) are indicated. A cGK II product of expected size, 725 kb, was not detected.C, cGMP-dependent phosphorylation of an endogenous substrate protein, VASP. Hippocampal cultures were incubated in serum-free media with or without 8-pCPT-cGMP, and samples (30 μg of protein) were analyzed by Western blotting using a monoclonal antibody that recognized VASP phosphorylated on Ser-239 (which is preferentially phosphorylated by cGKs) as a 46 kDa protein. VASP-P was identified by comparison with a standard prepared from human platelets (left). cGK I was also assayed in the same samples, as described for A₁. D, Immunocytochemical localization of cGK type I in cultured hippocampal neurons. Cultured hippocampal cells showed positive immunoreactivity for cGK type I (green) compared with control cells treated with preimmune serum. The same cells that displayed positive labeling for cGK type I also showed positive labeling for MAP2 (red), a specific neuronal marker located in cell bodies and dendrites, confirming that the cGK I-labeled cells were neurons (left). They also showed positive labeling for synaptophysin (red), a presynaptic vesicle-associated protein, indicating that the localization of cGK type I includes presynaptic terminals (middle). Double-labeled structures appear yellow or orange. Scale bar, 10 μm.

Fig. 4.

Presynaptic injection of cGK type I produces activity-dependent long-lasting potentiation. A, Experimental arrangement. B₁, Example of long-lasting potentiation produced by injection of the α isozyme of cGK type I into the presynaptic neuron paired with weak tetanic stimulation (50 Hz, 0.5 sec) of that neuron in the presence of 50 μm APV. B₂, Example of lack of potentiation by injection of cGK type I into the postsynaptic neuron paired with weak tetanic stimulation.C, Average potentiation by presynaptic injection of cGK type I paired with weak tetanic stimulation (a; filled circles). The weak tetanus (arrow) occurred at time 0. The horizontal bar shows the time during which cGK type I was injected into the neuron. The cultures were perfused with APV throughout the experiment. There was no potentiation after either postsynaptic injection of cGK type I paired with weak tetanic stimulation (b; filled triangles) or weak tetanic stimulation alone (c; filled inverted triangles). Injection of cGK type I alone into the presynaptic neuron (d;open circles) or the postsynaptic neuron (e; open triangles) did not have an affect on EPSC amplitude. Average Pre values in picoamperes were as follows: (a) 43 ± 9,n = 15; (b) 173 ± 44, n = 11; (c) 22 ± 4, n = 8; (d) 153 ± 53,n = 11; and (e) 178 ± 92,n = 8.