Synaptic depression in the CA1 region of freely behaving mice is highly dependent on afferent stimulation parameters

Abstract

Persistent synaptic plasticity has been subjected to intense study in the decades since it was first described. Occurring in the form of long-term potentiation (LTP) and long-term depression (LTD), it shares many cellular and molecular properties with hippocampus-dependent forms of persistent memory. Recent reports of both LTP and LTD occurring endogenously under specific learning conditions provide further support that these forms of synaptic plasticity may comprise the cellular correlates of memory. Most studies of synaptic plasticity are performed using in vitro or in vivo preparations where patterned electrical stimulation of afferent fibers is implemented to induce changes in synaptic strength. This strategy has proven very effective in inducing LTP, even under in vivo conditions. LTD in vivo has proven more elusive: although LTD occurs endogenously under specific learning conditions in both rats and mice, its induction has not been successfully demonstrated with afferent electrical stimulation alone. In this study we screened a large spectrum of protocols that are known to induce LTD either in hippocampal slices or in the intact rat hippocampus, to clarify if LTD can be induced by sole afferent stimulation in the mouse CA1 region in vivo. Low frequency stimulation at 1, 2, 3, 5, 7, or 10 Hz given in the range of 100 through 1800 pulses produced, at best, short-term depression (STD) that lasted for up to 60 min. Varying the administration pattern of the stimuli (e.g., 900 pulses given twice at 5 min intervals), or changing the stimulation intensity did not improve the persistency of synaptic depression. LTD that lasts for at least 24 h occurs under learning conditions in mice. We conclude that a coincidence of factors, such as afferent activity together with neuromodulatory inputs, play a decisive role in the enablement of LTD under more naturalistic (e.g., learning) conditions.

Keywords: LTD; STD; hippocampus; in vivo; murine.

Figure 1

Nissl-stained microphotographs showing the hippocampus electrode positions. Photographs of hippocampal slices following the Nissl staining procedure. The white arrows point approximately to the final position of the recording electrode in the CA1 stratum radiatum dendritic layer on the left photograph and to the final position of the stimulating electrode in the Schaffer collaterals on the right photograph respectively.

Figure 2

Low-frequency stimulation at 1 Hz did not induce synaptic depression at the mouse Schaffer collateral–CA1 synapses in vivo. (A) When no external patterned stimulation was applied, the mice (n = 18) exhibited stable synaptic transmission in response to test-pulse stimulation (0.025 Hz) throughout the entire course of the recording (1500 min). (B) Low-frequency stimulation (LFS) at 1 Hz, 900 stimuli, a stimulation protocol used to induce robust long-term depression (LTD) in freely behaving rats and mouse hippocampal slices, failed to induce any form of detectable synaptic plasticity (ANOVA, p = 0.95011) at the CA1 synapses in freely behaving mice (n = 7). (C) Another commonly applied LFS protocol which consists of 450 pairs of stimuli given at 1 Hz was also incapable of inducing any significantly detectable changes (ANOVA, p = 0.07712) in synaptic efficacy in freely behaving mice (n = 6). Insets: analog traces illustrating the Schaffer collateral–CA1 field potentials at pre-stimulation, 5 min and 24 h (1440 min). Vertical scale bar corresponds to 2 mV and horizontal scale bar corresponds to 5 ms.

Figure 3

Low-frequency stimulation at 2 Hz did not induce synaptic depression but initiated slow-onset potentiation at higher number of stimuli. (A) Low-frequency stimulation (LFS) applied at 2 Hz for 300 pulses did not result in a significant change (ANOVA, p = 0.42910) in synaptic efficacy at the CA1 synapses in freely behaving mice (n = 8). (B) Increasing the number of stimuli to 1800 at 2 Hz led to a significant slow-onset potentiation (ANOVA, p < 0.0001; n = 6). Insets: analog traces illustrating the Schaffer collateral–CA1 field potentials at pre-stimulation, 5 min and 24 h (1440 min). Vertical scale bar corresponds to 2 mV and horizontal scale bar corresponds to 5 ms.

Figure 4

Low-frequency stimulation at 3 Hz given for 200 or 300 times induced robust short-term depression. (A) Low-frequency stimulation (LFS) given at 3 Hz for 200 stimuli (ANOVA, p < 0.05; n = 6) and (B) 300 stimuli (ANOVA, p < 0.05; n = 9) both induced robust and significant short-term depression (STD). Increasing the number of stimuli by 100 from 200 to 300 at 3 Hz resulted in an enhancement in the initial synaptic depression from 28.52 ± 12.28% to 12.46 ± 5.35% whilst post-hoc analysis revealed an increase in the duration of significant synaptic depression from 15 min to 30 min post-stimulation. Insets: analog traces illustrating the Schaffer collateral–CA1 field potentials at pre-stimulation, 5 min and 24 h (1440 min). Vertical scale bar corresponds to 2 mV and horizontal scale bar corresponds to 5 ms.

Figure 5

The extent of short-term depression induced by low-frequency stimulation at 3 Hz depends on the number of stimuli and the stimulation intensity. (A) Further increasing the number of stimuli to 900 times at 3 Hz resulted in an STD that was depressed (ANOVA, p = 0.16118; n = 14) for 60 min post-stimulation. At 900 stimuli initial synaptic depression was, however, considerably ameliorated (58.64 ± 5.74%) compared to at 200 and 300 stimuli and a late-onset potentiation developed after approximately 60 min. (B) Decreasing the stimulation intensity used for 3 Hz 900 stimuli LFS to 60% of the maximum inducible fEPSP which was aimed at circumventing the late-onset synaptic potentiation failed to induce any significant (ANOVA, p = 0.70365; n = 6) form of synaptic plasticity at the CA1 synapses. Insets: analog traces illustrating the Schaffer collateral–CA1 field potentials at pre-stimulation, 5 min and 24 h (1440 min). Vertical scale bar corresponds to 2 mV and horizontal scale bar corresponds to 5 ms.

Figure 6

Varying the pattern of stimulation or increasing the number of stimuli at 3 Hz did not enhance but ameliorated synaptic depression. (A) Application of 900 stimuli in 3 trains of 300 stimuli (5 min inter-train interval) led to an initial depression of 79.08 ± 8.59% which was significantly depressed for 10 min post-stimulation and a late-onset potentiation that was significant between 150 min to 180 min (ANOVA, p = 0.54620; n = 6). (B) Two trains of 900 stimuli given at 3 Hz induced a synaptic response of similar profile. A small initial synaptic depression (81.12 ± 6.31%) appeared immediately after low-frequency stimulation (LFS) which gradually developed into a significant late-onset potentiation between 90 min to 225 min (ANOVA, p < 0.05; n = 7). (C) LFS given at 3 Hz for 1200 pulses did not induce any synaptic depression but resulted in robust slow-onset potentiation (ANOVA p < 0.01; n = 6). Insets: analog traces illustrating the Schaffer collateral–CA1 field potentials at pre-stimulation, 5 min and 24 h (1440 min). Vertical scale bar corresponds to 2 mV and horizontal scale bar corresponds to 5 ms.

Figure 7

Stimulation at 5 Hz resulted in varying degrees of synaptic depression depending on stimulation the number of stimuli administered. (A) Low-frequency stimulation (LFS) at 5 Hz with 300 stimuli induced short-term depression (STD) with an initial synaptic depression of 50.91 ± 6.10% that was significantly depressed for 15 min post-stimulation (ANOVA, p = 0.41773; n = 5). (B) Increasing the number of stimuli at 5 Hz to 900 pulses resulted in an attenuation of the initial synaptic depression (78.17 ± 12.38%) and reduction in the persistence of the synaptic depression to 10 min (ANOVA, p = 0.25328; n = 7). Insets: analog traces illustrating the Schaffer collateral–CA1 field potentials at pre-stimulation, 5 min and 24 h (1440 min). Vertical scale bar corresponds to 2 mV and horizontal scale bar corresponds to 5 ms.

Figure 8

Stimulation at 7 Hz resulted in varying degrees of synaptic depression depending on the number of stimuli administered. (A) Low-frequency stimulation (LFS) at 7 Hz elicited a small initial depression of 70.06 ± 5.05% that was significant for up to 10 min post-stimulation when applied for 100 pulses (ANOVA, p = 0.27804; n = 7). (B) Increasing the number of stimuli to 300 pulses at 7 Hz led to an enhancement of the initial depression (14.63 ± 3.81%) as well as the persistence of the depression (ANOVA, p = 0.55473; n = 8). Insets: analog traces illustrating the Schaffer collateral–CA1 field potentials at pre-stimulation, 5 min and 24 h (1440 min). Vertical scale bar corresponds to 2 mV and horizontal scale bar corresponds to 5 ms.

Figure 9

Stimulation at 10 Hz resulted in varying degrees of synaptic depression depending on the number of stimuli administered. (A) Low-frequency stimulation (LFS) given at 10 Hz for 100 pulses induced robust STD with an initial synaptic depression of 22.55 ± 4.39% and a significant depression that lasted 15 min post-LFS (ANOVA, p < 0.01; n = 15). (B) Increasing the number of stimuli at 10 Hz to 300 pulses strongly ameliorated the STD resulting in an initial depression of 71.02 ± 7.82% which persisted for 5 min post-stimulation (ANOVA, p = 0.71830). Insets: analog traces illustrating the Schaffer collateral–CA1 field potentials at pre-stimulation, 5 min and 24 h (1440 min). Vertical scale bar corresponds to 2 mV and horizontal scale bar corresponds to 5 ms.