Stable mossy fiber long-term potentiation requires calcium influx at the granule cell soma, protein synthesis, and microtubule-dependent axonal transport

Abstract

The synapses formed by the mossy fiber (MF) axons of hippocampal dentate gyrus granule neurons onto CA3 pyramidal neurons exhibit an intriguing form of experience-dependent synaptic plasticity that is induced and expressed presynaptically. In contrast to most other CNS synapses, long-term potentiation (LTP) at the MF-CA3 synapse is readily induced even during blockade of postsynaptic glutamate receptors. Furthermore, blocking voltage-gated Ca(2+) channels prevents MF-LTP, supporting an involvement of presynaptic Ca(2+) signaling via voltage-gated Ca(2+) channels in MF-LTP induction. We examined the contribution of activity in both dentate granule cell somata and MF terminals to MF-LTP. We found that the induction of stable MF-LTP requires tetanization-induced action potentials not only at MF boutons, but also at dentate granule cell somata. Similarly, blocking Ca(2+) influx via voltage-gated Ca(2+) channels only at the granule cell soma was sufficient to disrupt MF-LTP. Finally, blocking protein synthesis or blocking fast axonal transport mechanisms via disruption of axonal tubulin filaments resulted in decremental MF-LTP. Collectively, these data suggest that-in addition to Ca(2+) influx at the MF terminals-induction of MF synaptic plasticity requires action potential-dependent Ca(2+) signaling at granule cell somata, protein synthesis, and fast axonal transport along MFs. A parsimonious interpretation of these results is that somatic activity triggers protein synthesis at the soma; newly synthesized proteins are then transported to MF terminals, where they contribute to the stabilization of MF-LTP. Finally, the present data imply that synaptic plasticity at the MF-CA3 synapse can be affected by local modulation of somatic and presynaptic Ca(2+) channel activity.

Figure 1.

Activation of postsynaptic CA3 pyramidal cells is not necessary for mossy fiber long-term potentiation. A, Time course of the amplitude of the EPSPs recorded at the MF pathway. Tetanization (4 × 50 stimuli, 100 Hz, delivered at time point zero) induced an LTP of MF-EPSPs during the recording period of 2 h. Bath application of the selective mGluR-2 agonist DCG-IV (3 μm) dramatically reduced MF-EPSP amplitudes and verified that our recording had little contamination from other pathways. Insets show representative sample traces obtained at the time points indicated by the lowercase letters. B, Time course of MF-EPSP amplitudes with tetanization (filled circles) or without (empty circles) tetanization. Bath application of 25 μm CNQX (filled bar) during the tetanus dramatically reduced the amplitude of MF-EPSPs. After washout of CNQX, there was a large potentiation of MF-EPSPs present in the tetanized slices versus the untetanized control slices. C, Time course of the amplitude of the EPSPs recorded at the MF pathway. Tetanized slices (filled circles) were compared to untetanized controls (unfilled circles). Bath application of the selective mGluR1 and 5 antagonists MPEP (10 μm) and CPCCOEt (100 μm) along with CNQX (25 μm; filled bar) during the tetanus dramatically reduced the amplitude of MF-EPSPs. After washout of the blockers, there was a large potentiation of MF-EPSPs present in tetanized relative to untetanized control slices. D, Time course of the amplitude of the EPSPs recorded at the MF pathway. Tetanized (filled circles) slices were compared to untetanized control slices (unfilled circles). Bath application of the Ca²⁺ channel blocker Co²⁺ (filled bar) during the tetanus yielded no difference in the size of MF-EPSPs in tetanized versus untetanized controls. E, Summary of the amount of MF-LTP present in the results presented in A–D. Asterisks indicate significant (p < 0.05) differences in the amount of LTP displayed by tetanized (filled bars) versus untetanized control (open bars) slices.

Figure 2.

Important contribution of action potential firing at granule cell somata to mossy fiber long-term potentiation. A, Diagram of the experimental setup: We recorded antidromically stimulated MF-EPSPs by placing a recording electrode (a) and a stimulation electrode (b) in the CA3 stratum lucidum. TTX (10 μm) was focally applied via two patch pipettes (d) to the dentate gyrus (DG) while the antidromic population spike was monitored with a recording electrode (c). The large arrow indicates the direction of the ACSF flow in the recording chamber. B, Puff application of TTX (open bar) dramatically reduced the amplitude of the population spike in the DG, while omission of the puff application (filled bar) left the population spike amplitude unaffected. Asterisk indicates significant differences (p < 0.05). C, MF-EPSP amplitudes were unaffected by puff application of TTX (open bar) compared to recordings in which puff application was omitted (filled bar). D, Time course of the amplitude of the EPSPs recorded at the MF pathway. After the focal application (indicated by the arrows) of ACSF (filled circles) during the tetanus (time point zero), a robust MF-LTP could be measured. This MF-LTP was reduced by the puff application (indicated by two arrows) of TTX (open circles) to the DG. Insets show representative sample traces obtained at the time points indicated by the lowercase letters. E, Summary of amount of MF LTP displayed in the experiments presented in D. Asterisk indicates significant (p < 0.05) differences in the level of MF-LTP following focal TTX application (open bar) compared to recordings in which puff application was omitted (filled bar) 60 min after tetanization. F, Similar levels of MF post-tetanic potentiation were obtained with (open bar) and without (filled bar) an antecedent TTX application.

Figure 3.

Contribution of action potential firing at mossy fiber terminals to the induction of long-term potentiation. A, Diagram of the experimental setup: Recording of MF-EPSPs was performed by stimulation of dentate gyrus (DG) granule cell somata (a). A recording electrode was placed in the stratum lucidum of area CA3 (b). ACSF or Na⁺-free solution was focally applied to the CA3 stratum lucidum (c) near the site of the recording electrode at position b. To ensure that the focal applications were restricted to the CA3 region, we monitored PP-EPSPs in the DG (d and e). The large arrow illustrates the direction of the ACSF flow in the recording chamber. B, Puff application of Na⁺-free solution (open bar) or ACSF (filled bar) to the CA3 stratum lucidum had no impact on the amplitude of PP-EPSPs. C, Focal application of Na⁺-free solution (open bar) to the CA3 dramatically reduced the amplitudes of MF-EPSPs when compared to the application of ACSF (filled bar). D, Time course of the MF-EPSP amplitudes before and after tetanization. A robust MF-LTP could be established after puff application (indicated by the two arrows) of ACSF (filled circles), but was completely abolished by focal application of Na⁺-free solution (filled squares) during the tetanus. Focal application of Na⁺-free solution to untetanized controls (open squares) demonstrated that the effect of puff applying Na⁺-free solution was reversible within 1 h. Insets show representative sample traces obtained at the time points indicated by the lowercase letters. E, Summary of the magnitude of MF-LTP 60 min after the tetanus in the experiments presented in D. Asterisk indicates significant (p < 0.05) differences in the levels of LTP. There was a complete loss of MF-LTP (60 min after tetanus) following the application of Na⁺-free solution to the MF terminals (filled bar) compared to sham application of ACSF (open bar) and untetanized controls with focal application of ACSF (filled bar).

Figure 4.

Contribution of Ca²⁺ influx at granule cell somata to the induction of mossy fiber long-term potentiation. A, Diagram of the experimental setup: We focally applied Co²⁺ (5 mm) to the dentate gyrus (DG) granule cell layer (a). We also elicited and recorded PP-EPSPs (c and b, respectively) to confirm the effectiveness of those applications. MF-EPSPs were obtained with a stimulation electrode (d) and a recording electrode (e) placed within the stratum lucidum of the CA3 area. The large arrow indicates the direction of the ACSF flow in the chamber. B, Placement of the pipettes a without puff application (filled bar) had no influence on PP-EPSPs, but the puff application of Co²⁺ (open bar) dramatically reduced the magnitude of PP-EPSPs. C, MF-EPSPs were left unaffected by puff applications of Co²⁺ (open bar) versus recordings in which no puff application was performed (filled bar) in the DG. D, Time course of the amplitude of the EPSPs recorded at the MF pathway. Recordings in which no puff application was performed (filled circles) during the tetanus (time point zero) produced a stable LTP. By contrast, the levels of MF-LTP were strongly reduced by a focal application of Co²⁺ (open circles). Insets show representative sample traces from the time points indicated by the lowercase letters. E, The magnitude of MF-LTP was strongly reduced 60 min following the tetanus in slices that received Co²⁺ (open bar) versus those that did not (filled bar) receive puff applications to the DG granule cells. Asterisks indicate significant differences (p < 0.05). F, Quantification of the MF PTP from the experiments illustrated in D showed that there were equivalent levels of PTP following tetanization in slices that received Co²⁺ puff applications (open bar) versus those that did not (filled bar).

Figure 5.

Contribution of Ca²⁺ influx at mossy fiber terminals to the induction of long-term potentiation. A, Diagram of the experimental setup: Co²⁺ (5 mm) was focally applied to MF synapses (a). MF-EPSPs were obtained with a stimulation electrode (b) and a recording electrode (c) that were placed in the CA3 stratum lucidum. To confirm that the Co²⁺ applications had no influence on Ca²⁺ influx at the dentate gyrus (DG) granule cell somata, we also elicited and recorded PP-EPSPs (d and e, respectively). B, Focal application of Co²⁺ did not reduce the amplitude of PP-EPSPs, while MF-EPSPs were strongly reduced, confirming that the focal application of Co²⁺ was restricted to the CA3. Representative traces are shown in the insets. C, Time course of average MF-EPSP amplitudes of tetanized (filled circles) and untetanized (open circles) slices after Co²⁺ application (time point indicated by black arrows). Tetanization was performed at time point zero. Although the effects of focal Co²⁺ applications (two arrows) were not completely reversible within the 1 h after tetanus, the comparison of tetanized and untetanized slices revealed an absence of MF-LTP following synaptic puff applications of Co²⁺. Insets show representative sample traces from the time points indicated by the lowercase letters. D, Focal application of Ca²⁺-free solution. Application was done via the field potential recording electrode (indicated in A, see Materials and Methods). Time course of average MF-EPSP amplitudes in tetanized (filled circles) and untetanized (open circles) slices. E, Summary of the amount of MF-LTP in the experiments illustrated in C. Tetanization of slices that received synaptic applications of Co²⁺ or Ca²⁺-free ACSF (filled bar) failed to produce any MF-LTP relative to untetanized (open bars) slices.

Figure 6.

Disrupting fast axonal transport or protein synthesis impairs the maintenance of mossy fiber long-term potentiation. A, Nocodazole disrupts microtubule structures in acute slices. Top, Representative SH signal from a hippocampal slice of a 27-d-old mouse. The image is a mosaic of z-projected image stacks. Bottom, Enlarged view of the infrapyramidal MF bundle. B, Time course of the SH signal in the area shown in the lower part of A. Dashed line indicates the beginning of the nocodazole (25 μm) application. C, Disruption of microtubules with 25 μm nocodazole impairs stable expression of MF-LTP. Time course of MF-EPSPs after tetanization is shown for slices preincubated for at least 1 h with nocodazole (open circles) and untreated control slices (filled circles). Insets show sample traces obtained at the time points indicated by the lowercase letters. D, Inhibition of protein synthesis with 25 μm emetine impairs stable MF-LTP. Time course of MF-EPSPs after tetanization is shown for slices preincubated for at least 1 h with emetine (open circles) and untreated control slices (filled circles). E, Quantification of the results presented in C and D revealed a significant reduction of MF-LTP following treatment with nocodazole or emetine. Asterisks indicate significant (p < 0.05) differences in the amount of MF-LTP 60 min after tetanization.