Somatic action potentials are sufficient for late-phase LTP-related cell signaling

Abstract

A question of critical importance confronting neuroscientists today is how biochemical signals initiated at a synapse are conveyed to the nucleus. This problem is particularly relevant to the generation of the late phases of long-term potentiation (LTP). Here we provide evidence that some signaling pathways previously associated with late-LTP can be activated in hippocampal CA1 neurons without synaptic activity; somatic action potentials, induced by backfiring the cells, were found to be sufficient for phosphorylation of extracellular signal-regulated kinase-1/2 and cAMP response element-binding protein, as well as for induction of zif268. Furthermore, such antidromic stimulation was adequate to rescue "tagged" synapses (early-LTP) from decay. These results show that a synapse-to-nucleus signal is not necessary for late-phase LTP-associated signaling cascades in the regulation of gene expression.

Figure 1

Schematic diagram outlining two contrasting scenarios for LTP-related nuclear signaling. (A1) Synapse-to-nucleus signal (①): Activation of NMDA receptors that induces LTP creates a “synaptic tag” (for retrieval of gene product, or synthesis of protein from dendritic RNA), and (②) initiates a synapse-to-nucleus signal (black arrow) that triggers nuclear RNA synthesis and (③) distribution of protein or RNA throughout the neuron (blue arrow). (A2) Soma-to-nucleus signal (①). Activation of NMDA receptors and creation of the “tag” proceed as in (A1); (②) the induction of the gene product, in contrast to the case in A1, is induced by a rise in somatic and/or nuclear calcium by action potentials, possibly requiring activation of L-type calcium channels (③). The gene product is distributed in the same way as in A1. Note that the tag is essential for conferring synapse specificity of LTP. (B) Diagram showing how hippocampal CA1 neurons were activated nonsynaptically. Neurons were backfired through their own axons in the alveus, the white-matter structure lining the outside surface of CA1. Most neurons (red) in a restricted region of CA1, albeit not all of them (black), would be antidromically activated. LTP experiments could still be performed in the traditional way, synaptically, by stimulating the stratum radiatum.

Figure 2

Action potentials are sufficient to induce the phosphorylation of ERK1/2. (A) Antidromic stimulation (theta-burst) from the alveus induced staining for pERK in a restricted region of CA1 (n = 9). 6-Cyano-7-nitroquinoxaline-2,3-dione (20 μM), 2-amino-5-phosphonovaleric acid (50 mμM), and α-methyl-4-carboxyphenylglycine (300 μM) were used to block a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, NMDA, and mGluR receptors, respectively, which could have been activated through axon collaterals. Immunoreactive cells areas appear dark. Higher magnification of the indicated areas (Left, stimulated; Right, unstimulated) are shown to illustrate that pERK staining was limited to proximal dendrites and the cell bodies. (B) Effect of 20 μM nifedipine on antidromically stimulated phospho-ERK staining (n = 8).

Figure 3

Action potentials are sufficient to induce the phosphorylation of CREB. (A) Antidromic stimulation (theta-burst) induces pCREB-like immunoreactivity in the stimulated region of CA1. Kynurenic acid (3 mM) was present during the stimulation to block activity at collaterals (n = 10). (B). Effect of 20 μM nifedipine on antidromically stimulated pCREB staining (n = 8).

Figure 4

Action potentials are sufficient to induce the expression of Zif268. (A) Antidromic stimulation (theta-burst) induces Zif268-like immunoreactivity in the stimulated region of CA1. Kynurenic acid (3 mM) was present during the stimulation (n = 9). (B) Effect of 20 μM nifedipine on antidromically stimulated Zif268 staining (n = 4).

Figure 5

Action potentials are sufficient to rescue early-LTP from decay. (A) TBS (20–40 pulses in 100-Hz bursts) induces early-LTP, which decays to baseline levels by 3 h (n = 14). (B) Antidromic stimulation (3 × 2 theta-burst) rescues early-LTP from decay (n = 5). Induction was not significantly different between early-LTP and early-LTP rescues. (C) Example of rescue experiment. Traces (Inset) are the averages of 10 individual sweeps at the indicated times during the baseline period and at 7.5 h; scale bars represent 0.2 mV and 5 mS. (D) In two cases, 3 mM kynurenic acid was present during the stimulation to block activation of synapses from axon collaterals. One example experiment is shown. EPSP, excitatory postsynaptic potential