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Local protein synthesis and GABAB receptors regulate the reversibility of long-term potentiation at murine hippocampal mossy fibre-CA3 synapses - PubMed

️Thu Jan 01 2004

Local protein synthesis and GABAB receptors regulate the reversibility of long-term potentiation at murine hippocampal mossy fibre-CA3 synapses

Chiung-Chun Huang et al. J Physiol. 2004.

Abstract

Reversal of long-term potentiation (LTP) by long trains of low-frequency stimulation is generally referred to as depotentiation. One of the intriguing aspects of depotentiation is that the magnitude of depotentiation is inversely proportional to the time lag of depotentiation stimulation following LTP induction. Although the mechanisms underlying depotentiation have been widely explored, the factors that regulate the susceptibility of LTP to depotentiation stimulation remain largely unclear. We now report that multiple trains of high-frequency stimulation provide immediate synaptic resistance to depotentiation stimulation at the mossy fibre-CA3 synapses. The synaptic resistance to depotentiation stimulation depends on the amount of synaptic stimulation used to induce LTP; it is prevented by protein synthesis inhibitors and is input specific. In contrast, neither the transection of mossy fibre axons near granule cell somata nor the application of RNA synthesis inhibitors influences synaptic resistance to depotentiation stimulation. We also provide evidence that the induction of depotentiation is regulated by GABA(B) receptors. Application of a GABA(B) receptor antagonist significantly promoted the synaptic resistance to depotentiation stimulation, whereas inhibition of GABA transport delayed the onset of this synaptic resistance. These results suggest that local protein synthesis is required for the development of synaptic resistance to depotentiation stimulation, whereas the activation of GABA(B) receptors promotes the susceptibility to depotentiation stimulation. These two factors may crucially regulate the reversal and stability of long-term information storage.

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Figures

**Figure 1. The reversal of long-term potentiation (LTP) by low-frequency stimulation (LFS) is activity and time dependent**
A, summary of experiments (n = 8) showing that LFS at 1 Hz for 15 min elicits long-term depression (LTD). B, summary of experiments showing that LFS given 10 min after two trains of 100 Hz high-frequency stimulation (HFS) almost completely reversed LTP (n = 8; •), whereas fEPSPs in slices that received HFS without LFS exhibited persistent potentiation (n = 7; ○). C, summary of experiments showing that four trains of 100 Hz HFS induced a stable LTP (n = 8; ○). Application of LFS at 10 min after HFS initially depressed fEPSPs that subsequently recovered to previously potentiated levels (n = 8; •). D, summary of experiments showing that LFS given 30 min after a single tetanus (25 Hz for 5 s) revealed a significant reversal of LTP (n = 6; •). Field EPSPs in slices that received HFS without LFS exhibited persistent potentiation (n = 7; ○). E, summary of experiments showing that LFS given 30 min after four trains of 100 Hz HFS did not reveal a significant reversal of LTP (n = 6; •). F, summary data comparing the effects of LFS given at 1, 3, 10 and 30 min after LTP induction by three different stimulation protocols. The magnitude of potentiation remaining was calculated at 40 min after the end of LFS. In A–F, each value is the mean ±
s.e.m.
of independent determinations in six to eight slices. The superimposed fEPSP in the inset of each graph illustrates respective recordings from example experiments taken at the time indicated by number. Upward arrows indicate application of HFS. The horizontal bars denote the period of delivery of 1 Hz LFS. The horizontal dotted lines indicate the average value of the normalized amplitude during the control period.

**Figure 2. The development of synaptic resistance to depotentiation stimulation is dependent on protein synthesis but not mRNA synthesis**
A, summary of experiments showing that preincubation of slices with the protein synthesis inhibitor anisomycin (20 μ
m
; 60 min) permitted persistent LFS-induced depotentiation (LFS-DEP) of four trains of 100 Hz LTP (n = 8; •) but had no effect on the induction of LTP (n = 8; ○). B, summary of experiments showing that preincubation of slices with another protein synthesis inhibitor, cycloheximide (60 μ
m
; 30–60 min), also permitted persistent LFS-DEP of four trains of 100 Hz LTP (n = 7; •) but had no effect on the induction of LTP (n = 8; ○). C, summary of experiments showing that preincubation of slices with transcriptional inhibitor, actinomycin-D (25 μ
m
; 30–60 min), did not affect the induction of either the synaptic resistance to LFS-DEP (n = 6; •) or LTP (n = 8; ○). D, summary histogram comparing the effects of different concentrations of anisomycin on the induction of four trains of 100 Hz LTP (filled columns) and LFS-DEP (open columns). E, summary histogram comparing the effects of different concentrations of cycloheximide on the induction of four trains of 100 Hz LTP (filled columns) and LFS-DEP (open columns). F, summary histogram comparing the effects of different concentrations of actinomycin-D on the induction of four trains of 100 Hz LTP (filled columns) and LFS-DEP (open columns). The magnitude of LTP was calculated at 50 min after HFS and the magnitude of LFS-DEP was calculated at 40 min after the end of LFS. In D–F the numbers in parentheses indicate the number of slices tested. Asterisks represent a significant difference compared with slices not receiving LFS.

**Figure 3. Presynaptic local protein synthesis is required for the development of synaptic resistance to depotentiation stimulation**
A, summary of experiments showing that LFS given 10 min after two trains of 100 Hz HFS almost completely reversed LTP (n = 4; •), whereas EPSCs in slices that received HFS without LFS exhibited persistent potentiation (n = 6; ○). Whole-cell patch clamp recordings were made of CA3 pyramidal cells at a holding potential of −70 mV. B, summary of experiments showing that the synaptic resistance to LFS-DEP was not affected by postsynaptic injection of the cap analogue m⁷ GpppG. Whole-cell patch clamp recordings were made of CA3 pyramidal cells at a holding potential of −70 mV. When LFS was applied 10 min after four trains of 100 Hz HFS in CA3 pyramidal cells loaded with m⁷ GpppG (250 μ
m
) (n = 6; •), transient synaptic depression was seen, but EPSCs recovered to potentiated levels not significantly different those seen in control cells without m⁷ GpppG (n = 6; ○). C, summary of experiments showing that CA1 LTD induced by DHPG (50 μ
m
, 5 min) was prevented by postsynaptic injection of m⁷ GpppG (250 μ
m
) (n = 6; •). Whole-cell patch clamp recordings were made of CA1 pyramidal cells at a holding potential of −70 mV. D1, schematic diagram showing the incision (thick black line) made to transect mossy fibre axons from the granule cell somata and the positions of recording (Rec) and stimulation (Stim) electrodes. D2, bright-field image of a hippocampal slice after the isolation of mossy fibre axons from the granule cell somata. Arrows indicate the site of lesion. Scale bar, 500 μm. E1, schematic diagram showing a mossy fibre axon transection slice, where an electrical stimulus was delivered to the stratum lucidum of the CA3 region and the evoked antidromic population spikes were recorded from the granulosum of the dentate gyrus (DG). E2, absence of antidromic population spikes in stratum granulosum of the dentate gyrus when stimulation was applied at the stratum lucidum of the CA3 region in the mossy fibre axon transection slice, whereas an antidromic population spike was successfully evoked in control slices. F, summary of experiments showing that mossy fibre axon transection had no effect on either the synaptic resistance to LFS-DEP (n = 6; •) or the induction of LTP (n = 8; ○). G, summary of experiments showing that preincubation of mossy fibre axon transection slices with protein synthesis inhibitor anisomycin (20 μ
m
; 60 min) permitted persistent LFS-DEP of four trains of 100 Hz LTP (n = 6; •) but had no effect on the induction of LTP (n = 6; ○).

**Figure 4. Synaptic resistance to depotentiation stimulation is input specific**
A, a typical example shows that two stimulating electrodes were used to activate two independent groups of afferent inputs. Field EPSPs were evoked by paired stimulations applied at 30 ms intervals to the first and/or second afferents. Paired-pulse facilitation was present when stimuli were applied twice to the same afferent (homosynaptic facilitation) but not when the stimuli were applied to different afferents (no heterosynaptic facilitation). Having confirmed the independence of afferent inputs activated, four trains of 100 Hz HFS were applied to one pathway (Control) to induce synaptic resistance to LFS-DEP. This treatment had no significant effect on the induction of depotentiation at a second adjacent pathway (Test) when LFS was applied at 10 min after two trains of 100 Hz HFS. B, summary of data from six experiments performed as in A.

**Figure 5. GABA_B receptor inhibition promotes the development of synaptic resistance to depotentiation stimulation**
A, summary of experiments showing that in slices treated with GABA_B receptor antagonist SCH50911 (20 μ
m
) the application of LFS at 10 min after two trains of 100 Hz HFS initially depressed fEPSPs that subsequently recovered to previously potentiated level (n = 8; •). B, summary of experiments showing that in contrast to control slices (n = 6; ○), a 5 min application of DCG-IV (3 μ
m
) at 10 min after two trains of 100 Hz HFS failed to elicit a persistent depotentiation in SCH50911-treated slices (n = 7; •). C, summary of experiments showing that SCH50911 treatment did not significantly affect the induction of LFS-DEP of two trains of 100 Hz LTP when LFS was applied at 3 min after LTP induction (n = 6). D, summary of experiments showing that SCH50911 treatment had no effect on the induction of DCG-IV-DEP of two trains of 100 Hz LTP when DCG-IV was applied at 3 min after LTP induction (n = 6). E, summary of experiments showing that with 25 Hz/125 pulse stimulation of mossy fibre pathways, the fEPSP amplitude showed a higher potentiation in SCH50911-treated slices (n = 7; •) than in control slices (n = 6; ○). F, summary of experiments showing that in slices treated with the GABA_B receptor antagonist SCH50911 (20 μ
m
), application of LFS at 10 min after 25 Hz/125 pulse stimulation did not produce a persistent depotentiation (n = 6; •). G, summary of experiments showing that treatment of slices with baclofen (0.2 μ
m
) permitted persistent LFS-DEP of four trains of 100 Hz LTP (n = 5; •) but had no effect on the induction of LTP (n = 5; ○). H, summary of experiments showing that treatment of slices with baclofen also permitted persistent DCG-IV-DEP of four trains of 100 Hz LTP (n = 5).

**Figure 6. An increase of the ratio of extracellular Ca²⁺ and Mg²⁺ concentration promotes the development of synaptic resistance to depotentiation stimulation**
A, summary of experiments showing that bath application of SCH50911 (20 μ
m
) caused a significant increase in the amplitude of mossy fibre fEPSPs (n = 5). B, summary of experiments showing that an increase in the extracellular Ca²⁺/Mg²⁺ concentration ratio from 4 m
m
/4 m
m
to 4 m
m
/2 m
m
mimicked the effect of SCH50911 on baseline mossy fibre transmission (n = 10). C, summary of experiments showing that in 4 m
m
Ca²⁺ and 2 m
m
Mg²⁺ aCSF, application of LFS at 10 min after two trains of 100 Hz HFS initially depressed fEPSPs that subsequently recovered to previously potentiated level (n = 5; •). Field EPSPs in slices that received HFS without LFS exhibited persistent potentiation (n = 5; ○).

**Figure 7. GABA transport inhibitors delayed the development of synaptic resistance to depotentiation stimulation**
A, summary of experiments showing that treatment of slices with the GABA transport inhibitor SKF89976A (50 μ
m
) permitted persistent LFS-DEP of four trains of 100 Hz LTP (n = 6; •) but had no effect on the induction of LTP (n = 6; ○). B, summary of experiments showing that a 5 min application of DCG-IV (3 μ
m
) at 10 min after four trains of 100 Hz HFS failed to elicit a persistent depotentiation in control slices (n = 6; ○). In contrast, treatment of slices with SKF89976A (50 μ
m
) permitted persistent DCG-IV-DEP of four trains of 100 Hz LTP (n = 6; •). C, summary of experiments showing that treatment of slices with another GABA transport inhibitor, nipecotic acid (500 μ
m
), permitted persistent LFS-DEP of four trains of 100 Hz LTP (n = 6; •). Field EPSPs in slices that received HFS without LFS exhibited persistent potentiation (n = 6; ○). D, summary of experiments showing that treatment of slices with nipecotic acid (500 μ
m
) also permitted persistent DCG-IV-DEP of four trains of 100 Hz LTP (n = 6).

**Figure 8. Stimulus interruption for 10 min immediately after the induction of LTP promotes the development of synaptic resistance to depotentiation stimulation**
A, a typical example showing that stimulus interruption for 10 min, commencing 20 s after HFS, prevented the induction of LFS-DEP when LFS was applied 10 min after two trains of 100 Hz HFS. B, summary of data from six experiments performed as in A. C, a typical example showing that stimulus interruption for 10 min commencing 20 s after HFS prevented the induction of DCG-IV-DEP (3 μ
m
for 5 min) when LFS was applied 10 min after two trains of 100 Hz HFS. D, summary of data from six experiments performed as in C.

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Local protein synthesis and GABAB receptors regulate the reversibility of long-term potentiation at murine hippocampal mossy fibre-CA3 synapses - PubMed