The temperature at which in vitro electrophysiological experiments are performed varies widely between laboratories. Recently it was shown that the production of cytokines within brain slices is dependent upon incubation temperature (Blond et al. 2001). The pro-inflammatory cytokine interleukin-1β (IL-1β) exerts a number of neuromodulatory effects in the CNS. Therefore the aim of our study was to investigate the temperature dependency of cytokine production and synaptic plasticity in slices of hippocampus.
Field excitatory postsynaptic potentials (fEPSPs) were recorded from the CA1 region of hippocampal slices prepared from C56BL/6 mice (21Ð35 days old; animals were killed according to UK legislation). Slices were incubated in normal ACSF either at room temperature (22Ð24 °C) or at 33Ð35 °C for at least 60 min in a holding chamber gassed with 95 % O2 and 5 % CO2. To measure cytokine production hippocampal slices were incubated at room temperature or at 35 °C for 3 h. Specific ELISAs determined immunoreactive IL-1β and IL-6 levels in tissue and bathing ACSF. In electrophysiology experiments the perfusing ACSF was either at room temperature or 34Ð35 °C. LTP was induced using a theta-burst stimulation protocol (5 trains of 15 bursts, each consisting of 4 pulses at 100 Hz with an inter-burst interval of 200 ms with the trains given at 0.1 Hz). IL-1β and IL-1ra were added to the perfusing medium for periods of 30 and 45 min, respectively. Data are expressed as means ± S.E.M. Statistical analysis was done using Student’s paired t test within groups and ANOVA between groups. P < 0.05 was taken to be significant.
Hippocampal slices incubated for 3 h at 35 °C had increased IL-1β levels when compared with slices incubated at room temperature (64 ± 14 and 8 ± 1 pg ml-1, respectively, P <0.01, n = 4). There was no difference in the amount of IL-6 in hippocampal slices incubated either at 35 °C or room temperature (17 ± 3 and 16 ± 3 pg ml-1, respectively).
The magnitude of LTP induced did not significantly differ at 60 min between the two temperatures. However, at 35 °C there was an initial peak 5Ð10 min after the tetanus had been given, which was not seen at room temperature (374 ± 51 % of control at 35 °C, n = 4 versus 172 ± 18 % at room temperature, n = 3, P < 0.05). Perfusion of IL-1ra (1000 and 2000 ng ml-1) reduced this initial peak and dose-dependently reduced the extent of the resulting LTP. Perfusion with IL-1β (10 ng ml-1) prior to the tetanus produced an initial increase in fEPSP slope, which was not different from under control conditions. However, at 60 min this potentiation had diminished such that the fEPSP was at approximately control values.
In conclusion incubation of hippocampal slices at physiological temperature (~37 °C) results in the specific production of IL-1β. This increase in endogenous IL-1β does not appear to affect the induction of LTP. However, IL-1ra significantly reduced the initial potentiation and also that at 60 min, suggesting that endogenous IL-1β may, in some way, contribute to LTP induction.
This research was supported by a MRC ROPA award.
All procedures accord with current UK legislation.