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Figure 2. Impaired hippocampal LTP in CHI mice was improved by DCS. A) Sample traces of fEPSPs recorded in the CA1 region of hippocampal slices prepared 16 d post-injury from sham, CHIVehicle or CHIDCS mice at the time indicated in (B). The stimulus artifacts are truncated. B) The averaged time course of LTP in sham (n10 slices, 5 mice), CHIVehicle (n12 slices, 7 mice) and CHIDCS (n13 slices, 7 mice) mice. Initial fEPSPs slopes were measured, and the values were normalized in each experiment using the averaged slope value measured during the control period (time, 10 to 0 min). Tetanic stimulation (100 Hz, 1 s, 2 trains 10 s apart) was applied at time 0. C) paired pulse facilitation (PPF) of synaptic responses in the CA1 region was induced by delivering afferent fiber stimulation twice at an interstimulus interval of 50 msec at stimulus intensity of 50% of maximal response. The facilitation ratios were calculated by dividing the amplitude of the second fEPSPs by that of the first fEPSPs. Ten consecutive traces were averaged from each slice, and the averaged amplitude from all slices is presented as mean sem (sham, 4 mice, 12 slices; CHIVehicle, 5 mice, 13 slices; CHIDCS, 4 mice, 16 slices). There was a significant difference in PPF between the sham mice and CHIVehicle (sham 1.450.03; CHIPBS 0.840.01; P0.001, t test) or CHIDCS mice (sham 1.450.03; CHIDCS 0.930.02; P0.001, t test).

2B). The magnitude of LTP in sham slices was 35% 0.05 sem while in CHI slices we observed no potentia- tion (Fig. 2A,B traces 1,2 vs. 5,6). However, in CHIDCS slices the magnitude of potentiation recov-

DCS IMPROVES FUNCTIONAL RECOVERY AND LTP IN CHI MICE

ered to 19% 0.08. Thus, LTP that was blunted in CHI mice was partially recovered in DCS injected mice. Although the recovery in the magnitude of LTP in DCS injected mice is most likely due to an enhancement of NMDAR activity by DCS, it is also possible that a change in the presynaptic transmitter release probability may be involved in the modulation of LTP. We thus exam- ined the paired-pulse facilitation (PPF) to assess whether CHI may affect the presynaptic release mech- anisms. PPF induced at an interstimulus interval of 50 msec was significantly different between sham and CHIVehicle mice or sham and CHIDCS mice (Fig. 2C; sham 1.450.03; CHIVehicle 0.840.01; P

    • 0.001

      , CHIDCS mice (sham 1.450.03; CHIDCS

    • 0.93

      0.02; P0.001, t test), suggesting that presynaptic

release probability is affected by CHI but DCS has no effect on the alterations in presynaptic release proba- bility.

Immunohistochemistry of CA1 hippocampal area

To determine whether DCS affected neuronal or glial cell number or morphology, neuronal degeneration was assessed with FluoroJade B, glial activation with glial fibrillary acidic protein (GFAP) and microglia with lectin in animals sacrificed 16 d post CHI. CHI induced over the frontal cortex, did not lead to neurodegenra- tion at the CA1 area of the injured hippocampus when compared to the contralateral control side (data not shown). DCS treatment had also no effect on neuronal cell morphology either at the injured or contralateral control CA1 area, both in injured and sham operated animals. However, activated astrocytes were detected at the injured CA1 area in both groups of injured animals (Fig. 3C,D), and DCS did not induce any significant change either in their number or morphology. Acti- vated microglia cells were not detected at the CA1 area in all groups of animals (Fig. 3A,B). These results suggest that DCS-induced alterations did not involve cell death.

DCS ameliorates CHI-induced reduction of BDNF but not synaptophysin

Since stimulation of NMDAR was shown to increase release of brain-derived neurotrophic factor (BDNF) from hippocampal neurons in culture, and this trophic factor plays a critical role in long-term potentiation (LTP) and affects neuronal survival (44), we stained CA1 area taken from mice 16 d post CHI for BDNF (Fig. 4Ad, g). In addition, we stained the same area for synaptophysin, a major integral transmembrane pro- tein of synaptic vesicles, which serves as a functional marker of the brain (Fig. 4Ab, e, h) and the nuclear marker DAPI (Fig. 4Ac, f, i). Both BDNF expression and synaptophysin immunoreactive presynaptic but- tons were dramatically reduced in the injured CA1 areas in either group of animals. However, the number of BDNF positive cells was significantly higher following DCS treatment (Fig. 4Ag; P0.005 vs. vehicle-treated

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