Hippocampal metaplasticity induced by deficiency in the extracellular matrix glycoprotein tenascin-R

Abstract

Predisposition of synapses to undergo plastic changes can be dynamically adjusted according to the history of synaptic activity (i.e., synapses are metaplastic). In search of a molecular mechanism underlying metaplasticity, we investigated mice deficient in the glycoprotein tenascin-R (TNR), based on the observations that this mutant exhibits elevated basal excitatory synaptic transmission and reduced perisomatic GABAergic inhibition. TNR is a major extracellular matrix glycoprotein of the CNS and carries the HNK-1 carbohydrate (human natural killer cell glycan), which has been identified as the functional epitope mediating regulation of GABAergic transmission via GABA(B) receptors. Here, we used patch-clamp recordings in hippocampal slices to determine the critical levels of postsynaptic neuron depolarization necessary for induction of long-term potentiation (LTP) at CA3-CA1 synapses and found that deficiency in TNR leads to a metaplastic increase in the threshold for induction of LTP. Reconstitution of slices from TNR-deficient mice with an HNK-1 glycomimetic or pharmacological treatment with either a GABA(A) receptor agonist, a GABA(B) receptor antagonist, an L-type voltage-dependent Ca2+ channel blocker, or an inhibitor of protein serine/threonine phosphatases restored LTP to the levels seen in wild-type mice. We propose that a chain of events initiated by reduced GABAergic transmission and proceeding via Ca2+ entry into cells and elevated activity of phosphatases mediates homeostatic adjustment of hippocampal plasticity in the absence of TNR. These data uncover a novel mechanism by which an extracellular matrix molecule and its associated carbohydrate provide conditions beneficial for induction of LTP in the CA1 region of the hippocampus.

Figure 1.

Synaptic changes induced by pairing at 0, −10, and −20 mV in CA1 pyramidal cells of TNR deficient mice. A–C, Pairing of 1 Hz stimulation (applied at time 0) of Schaffer collateral/commissural fibers with membrane depolarization to 0 mV (A), −10 mV (B), or −20 mV (C) induces changes in EPSCs in TNR+/+ and TNR−/− mice. Data show means + SEM of normalized current amplitudes; n indicates the number of slices and N indicates the number of mice. Panels above LTP profiles show EPSCs recorded before and 30 min after pairing in TNR+/+ and TNR−/− mice. Calibrations: A, 10 ms, 100 pA; B, C, 10 ms, 50 pA. D, The levels of potentiation or depression (above or below the baseline level) as a function of membrane voltage (Vm) and genotype.

Figure 2.

Reduction of excitatory synaptic transmission by induction of LTD does not normalize impaired LTP in the CA1 region of TNR−/− mice. A, Two trains of 1 Hz stimulation (marked by horizontal bars) induced similar LTD in TNR+/+ and TNR−/− mice. The levels of potentiation evoked by TBS of Schaffer collateral/commissural fibers (arrow) after induction of LTD (de-depression) were strikingly different between genotypes. Data show means + SEM; n indicates the number of slices and N indicates the number of mice. Insets show averaged fEPSPs recorded 10 min before and 50–60 min after the second train of 1 Hz stimulation (LTD) or 20–30 min after TBS (de-depression). B, LTP evoked by TBS of Schaffer collateral/commissural fibers (marked by arrow) in TNR+/+ and TNR−/− mice. Insets show fEPSPs recorded before and 30 min after TBS. C, Cumulative data show significant differences between TNR+/+ and TNR−/− mice in LTP (***p < 0.001) and de-depression (**p < 0.01). LTP in TNR+/+ mice was also significantly larger than de-depression in TNR−/− mice (⁺⁺⁺p < 0.001). Calibrations: A, 10 ms, 250 μV; B, 10 ms, 250 μV.

Figure 3.

Restoration of TBS-induced LTP in the CA1 region of TNR-deficient mice by systemic injection of the GABA_A receptor agonist muscimol (1 mg/kg). A, Scheme of experiments shown in B–D. Note that for washout of muscimol, slices were in the perfusion chamber for 1 h before recording of stimulus–response curve (SRC) and LTP. B, C, LTP evoked by TBS of Schaffer collateral/commissural fibers (marked by arrow) 24 h after injection of ACSF (B) or muscimol (C) into TNR+/+ and TNR−/− mice. Data show means + SEM; n indicates the number of slices and N indicates the number of mice. Insets show fEPSPs recorded before and 30 min after TBS. Calibrations: 10 ms, 250 μV. D, Cumulative data show that the difference in LTP between ASCF injected TNR+/+ and TNR−/− mice (*p < 0.05) is abolished after treatment with muscimol (⁺⁺p < 0.01, significant difference between ACSF and muscimol injected TNR−/− mice). Seven days after muscimol injection into TNR−/− mice, the level of LTP is not different from that in ACSF-injected TNR−/− mice (*p < 0.05, significantly different from TNR+/+ mice).

Figure 4.

Restoration of LTP in the CA1 region of TNR-deficient mice by preincubation of slices with the GABA_A receptor agonists muscimol (1 μm) and zolpidem (0.3 μm), the GABA_B receptor antagonist CGP54626A (0.2 μm), and the HNK-1 peptidomimetic (100 μg/ml). A, Scheme of experiments shown in B–D. Note that drugs were applied for 2 h, followed by 1 h of washout before recording of stimulus–response curve (SRC) and LTP. B, LTP evoked by TBS of Schaffer collateral/commissural fibers (arrow) after a 2 h preincubation of slices with zolpidem in ACSF. Data show means + SEM; n indicates the number of slices and N indicates the number of mice. Insets show fEPSPs recorded before and 30 min after TBS. Calibration: 10 ms, 250 μV. C, Pairing of 1 Hz stimulation (arrows) of Schaffer collateral/commissural fibers with postsynaptic pyramidal cell depolarization to −10 mV, but not to −20 mV, induces LTP of postsynaptic currents in TNR+/+ and TNR−/− mice. D, Cumulative data show that the difference in TBS-induced potentiation of fEPSP between TNR+/+ and TNR−/− (*p < 0.05; **p < 0.01; ***p < 0.001) is abolished after treatment with muscimol, zolpidem, CGP54626A, and HNK-1 peptidomimetic (⁺⁺p < 0.01; ⁺⁺⁺p < 0.001, comparison with untreated slices of the same genotype; ^#p < 0.05, comparison between HNK-1 peptidomimetic and scrambled HNK-1 peptidomimetic, for control). The preincubation of slices with scrambled HNK-1 peptidomimetic does not affect LTP. Nicotine (1 μm) reduces LTP in TNR+/+ mice but does not eliminate the difference between genotypes.

Figure 5.

Restoration of impaired LTP in the CA1 region of TNR-deficient mice by preincubation of slices with the L-type Ca²⁺ channel blocker nifedipine (1 μm) and inhibitor of protein phosphatases calyculin A (1 μm). A, B, LTP evoked by TBS of Schaffer collateral/commissural fibers (arrow) after 2 h preincubation of slices with nifedipine (A) or calyculin A (B) in ACSF. Data show means + SEM; n indicates the number of slices and N indicates the number of mice. Insets show fEPSPs recorded before and 30 min after TBS. Calibrations: 10 ms, 250 μV. C, Cumulative data show that the difference in LTP between TNR+/+ and TNR−/− mice (***p < 0.001; **p < 0.01) is not abrogated after treatment with APV (50 μm) and MCPG (200 μm), as antagonists of NMDA and metabotropic glutamate receptors, respectively, whereas treatments with nifedipine and calyculin A elevated levels of LTP in TNR−/− (⁺p < 0.05 and ⁺⁺⁺p < 0.001, comparison with untreated slices of TNR−/− mice).

Figure 6.

Normalization of basal synaptic transmission in the CA1 region of TNR-deficient mice by pretreatment of slices with the blocker of L-type Ca²⁺ channels nifedipine (1 μm) or the inhibitor of protein phosphatases calyculin A (1 μm). A, B, Stimulus–response curves in TNR−/− mice are above those in TNR+/+ mice in control untreated slices (A) and after treatment with the NMDA receptor antagonist APV (50 μm) (B) (*p < 0.05; **p < 0.01). C, D, There is no difference between genotypes after treatment with nifedipine (C) or calyculin A (D).

Figure 7.

Scheme of suggested chain of events involved in metaplasticity in TNR−/− mice.