Materials and methods b, BDNF and NT-3 by RT-PCR

J . Zhou et al. Brain Research 885 2000 182 –191 183 In our previous study [46], however, CORT applied 1 h tion. The hippocampus was carefully dissected in 95 before tetanus without causing any morphological modi- O 5 CO -saturated ice-cold artificial cerebrospinal fluid 2 2 fication also significantly inhibited both induction and ACSF. A transverse hippocampal slice with thickness of maintenance phases of LTP in vivo, suggesting the inhib- 400–450 mm was prepared using a vibroslice World itory effect may be mediated by a quick and direct Precision Instruments, USA and recovered in warmed, mechanism. 95 O 5 CO -saturated ACSF for at least 1 h as 2 2 Neurotrophins are traditionally identified as a family of described previously [43]. The slices were then transferred protein factors essential for neuronal survival and differen- in a submerged chamber perfused with warmed 348C tiation. However, a series of recent studies suggested a ACSF pH 7.4 containing in mM NaCl 124, KCl 3.0, novel role of neurotrophins in synaptic transmission and CaCl 2.5, MgCl 1.5, NaHCO 26, KH PO 1.25, 2 2 3 2 4 plasticity [4,22,24,39]. Long-term application of the BDNF glucose 10 and ascorbic acid 2. Field EPSP was evoked in and NT-3 was found to enhance synaptic transmission at CA1 stratum radiatum by stimulating the Schaffer colla- the developing neuromuscular junction in culture teral pathway with twisted bipolar nichrome electrodes and [21,23,40]. The acute effects of neurotrophins on neuronal recorded with ACSF-filled glass pipette ,5 MV using a activity and synaptic transmission were also observed in microelectrode amplifier MEZ-8201, Nihon Kohden, cultured neurons and in slices by a number of laboratories Japan. Test stimuli consisted of monophasic 100-ms [15,19,20]. Kang and Schuman [13] showed that acute pulses of constant current delivered by electronic application of BDNF and NT-3 enhanced basal excitatory stimulator SEN-3201, Nihon Kohden. Basal synaptic synaptic transmission in CA1 synapse of adult hippocam- transmission was examined by alternating, low-frequency pal slices. Tanaka [38] found BDNF did not affect basal stimulation at 0.033 Hz one stimuli for every 30 s of two excitatory synaptic transmission but rapidly reduced IPSC separate pathways via two stimulating electrodes at CA1 synapses in hippocampal slices. Meanwhile, BDNF positioned on both sides of the recording electrode. Only knock-out conferred deficit in generating LTP in mouse slices exhibiting EPSP of 2–3 mV in amplitude without hippocampal CA1 synapses and this defect can be rescued superimposed population spikes were used. Stimulus in- after prolonged incubation with BDNF-containing adeno- tensity was adjusted to produce an EPSP with |50 of virus [16]. Application of exogenous BDNF promotes LTP maximum amplitude. Tetanic stimulation, which consisted induction in neonatal hippocampal slices, where the endog- of 100 pulses at 100 Hz, was used to induce LTP. LTP was enous BDNF levels are low [9]. In contrast, treatment with thought to be induced and expressed when both EPSP TrkB-IgG, a fusion protein that scavenges endogenous amplitude and slope were potentiated more than 25 BDNF, reduces the magnitude of LTP in the adult hip- above the baseline for at least 30 min after tetanus. We also pocampus [14], where the endogenous BDNF levels are employed PPF to examine the synaptic strength presynapti- high. Moreover, BDNF perfused with mice hippocampal cally. A paired stimuli with interpulse intervals from 20 to slice for 3 h enhanced PPF, suggesting that BDNF-induced 50 ms was given to the Schaffer collateral pathway and the enhancement of synaptic efficacy may be somewhat in- evoked EPSPs were recorded. The ratios of evoked EPSP volved in a presynaptic mechanism [10]. slope and amplitude in the second stimuli to those in the In our present study, the effect of CORT on hippocam- first one were calculated to examine presynaptic facilita- pal synaptic transmission and its underlying mechanism tion during PPF. Both slopes and amplitudes of EPSP were involving the neurotrophin gene expression are explored. digitized 10 kHz, filtered at 1 kHz eight-pole Bessel We found that CORT application significantly inhibited filter, stored on magnetic media using an acquisition LTP, PPF as well as BDNF gene expression in rat system PClab 2.0 Beijing PClab Instruments, Beijing, hippocampal CA1 synapses while perfusion with BDNF China and analyzed off-line. CORT was added directly rescued the CORT-induced impairment of PPF, suggesting into the ACSF and perfused the rat hippocampal slice for 3 that the inhibitory effect of CORT on hippocampal synap- h before delivery of tetanus to the Schaffer collateral tic plasticity may be closely related to BDNF gene pathway. PPF is believed to be established when both the expression. Our experimental results provide further in- EPSP slope and amplitude in the second response is at sight in understanding endangerment of glucocorticoids on least 15 above the first one. the hippocampus. 2.2. Determination of the expression of mRNA for NGF-

2. Materials and methods b, BDNF and NT-3 by RT-PCR

2.1. Electrophysiological recording After termination of electrophysiological experiments, the CA1 region in rat hippocampal slice was carefully Male 9-week-old Sprague–Dawley rats Animal Center dissected under a dissecting microscope and quickly put of Beijing Institute of Pharmacology and Toxicology were into liquid nitrogen for semi-quantitative analysis of the anesthetized with ethyl ether and sacrificed by decapita- expression of neurotrophin mRNA, which included NGF- 184 J b, BDNF as well as NT-3 in the present study. RT-PCR 3. Results analysis was conducted according to the standard ex- perimental protocols in our laboratory [42]. Briefly, total 3.1. CORT treatment had no effect on basal RNA was firstly extracted from the dissected rat hip- hippocampal synaptic transmission pocampal CA1 tissues by the RNAzol reagents Life Technologies, USA. Then, reverse-transcription was im- Single low-frequency stimulation was delivered to the plemented with a total reaction volume of 12.5 ml. Schaffer collateral pathway to evoke the field EPSP. 26 Approximately 1 mg of total RNA was reverse-tran- Incubation of hippocampal slice with CORT 10 M and 25 scribed into cDNA at 458C for 30 min with a mixture of 10 M for 3 h had no effect on both the slopes and 50 mM Tris–HCl pH 8.3, 5 mM MgCl , 5 mM DTT, amplitudes of evoked EPSPs recorded in rat hippocampal 2 50 mM KCl, 0.5 mM each dNTP Boehringer, Mann- CA1 dendrites Fig. 1, P.0.05. heim, 0.25 mg of oligodT12–18 primers Pharmacia, Piscataway, NJ and 5 U avian myeloblastosis virus 3.2. CORT treatment inhibited the induction and AMV reverse transcriptase Life Technologies. Ac- maintenance of LTP in CA 1 synapses of the cording to the published sequence of cDNA encoding rat hippocampus NGF-b, BDNF and NT-3 and with the assistance of PCR primers design software, the primers used for PCR Before induction of LTP, we pre-screened the hippocam- amplification were designed and synthesized as follows: pal slices using the PPF approach. Only those slices NGF-b, 59-CCAAGGACGCAGCTTTCTAT-39 sense, showing potentiated response in the second stimuli during 59-CTCCGGTGAGTCCTGTTGAA-39 antisense; PPF were used while the hippocampal slices displaying BDNF, 59-CTGGATGAGGACCAGAAGGT-39 sense, paired-pulse depression PPD were discarded. In our 59-TATCCATAGTAAGGGCCCGA-39 antisense; NT-3, present study, high frequency stimulation 100 Hz3100 59-TTGATCCAGGCGGATATCTT-39 sense, pulses was used to induce LTP in hippocampal CA1 59-TGTCAATGGCTGAGGACTTG-39 antisense. Ampli- synapses. After delivery of tetanus, both the slope and fication with primers for constitutively expressed b-actin amplitude of EPSP were instantly facilitated with the served as an internal control as published elsewhere [42]. maximum increases of 70 and 90 above the baseline, PCR was performed in a total volume of 25 ml containing respectively, and this potentiation was persistent during the 1-ml aliquots of RT products, 0.5 mM of each sense and experimental period Fig. 2, indicating that LTP was antisense primers, 125 mM each dNTP, 13 PCR buffer 50 induced and expressed in hippocampal slices. Though mM KCl, 1.5 mM MgCl and 10 mM Tris–HCl, pH 9.0 tetanus also induced great increases of both slope and 2 and 1.25 U of Taq DNA Polymerase Life Technologies. amplitude of EPSP evoked in CORT-treated hippocampal After determining the linear amplification ranges, the slices, this promotion cannot persist within 60 min, thermal cycle profiles used were: 132 min at 948C, 30 suggesting only short-term potentiation STP was gener- cycles for neurotrophin gene amplification with each cycle ated Fig. 2. After calculation, we found that CORT 26 25 containing 1 min at 958C, 1 min at 558C, 1.5 min at 728C. incubation 10 M and 10 M with rat hippocampal Eight-ml aliquots of the PCR products 402 bp for NGF-b, slices for 3 h significantly attenuated the tetanus-facilitated 453 bp for BDNF, 393 bp for NT-3 and 462 bp for increments of both EPSP slopes and amplitudes Fig. 2, b-actin were electrophoresed on a 1.9 agarose gel, P,0.05, or P,0.01. In addition, LTP in 90 9 10 of stained by ethidium bromide and photographed under UV hippocampal slices were successfully induced after the light. The intensity of the PCR products generated by the pre-screening step Table 1. After CORT treatment, only b-actin and neurotrophins were quantitated by a computer- 30 of hippocampal slices 3 10 developed LTP while assisted, linear scanning densitometric analysis of the 70 7 10 hippocampal slices displayed short-term photograph in a reflectance mode Kodak 120, Kodak potentiation after tetanus. After statistics, we found CORT- Digital Science, USA. A ratio of neurotrophins to b-actin treated hippocampal slices showed a great decrease of PCR product units was used to calculate the relative successful rate for generating LTP P,0.05, or P,0.01, amount of neurotrophin mRNA expression in rat hip- Table 1 in comparison to control. pocampal CA1 cells. 3.3. CORT treatment inhibited PPF in rat hippocampal CA 1 synapses 2.3. Statistical analysis Paired pulses at different frequencies from 20 to 50 Hz Data are presented as the means6S.E. standard error. were employed to induce the PPF and the ratios of EPSP Statistical significance was determined by ANOVA, fol- slope or amplitude evoked by the second stimuli to that by lowed by Duncan analysis as a post hoc test. A non- the first one were figured out. In the present study, the parametric Mann–Whitney U-test was applied alternatively ratios showed a frequency-dependent change and paired- when indicated. Differences were considered significant at pulse stimulation at 25 Hz produced the maximum ratios a P value of less than 0.05. with increases of both EPSP slope and amplitude more J . Zhou et al. Brain Research 885 2000 182 –191 185 26 25 Fig. 1. CORT 10 M and 10 M treatment for 3 h had no effect on basal hippocampal synaptic transmission. Single low-frequency stimulation was delivered to rat hippocampal Schaffer collateral pathway to evoke the field EPSP. The amplitudes and slopes of EPSP were measured every 30 s before and during application of CORT to a submerged chamber black bar, up to 3 h. Each point represents the average amplitude and slope of 10 consecutive EPSPs 5-min interval normalized to the average of the EPSPs acquired before CORT administration in control 20 min baseline; n56. A Two sets of 25 traces represent the evoked EPSPs in control upper and CORT 10 M-treated lower hippocampal slice before t50 min and 60, 120 and 180 min after CORT treatment. The calibration scale stands for 15 ms of horizontal bar and 2 mV of vertical bar. B Two figures showing the changes of both slopes and amplitudes of EPSPs recorded in rat hippocampal CA1 dentrites after perfusion with CORT-treated ACSF for 3 h. No significance P.0.05 was observed during the experiments. than 55 above the first response. The ratios showed a sion between control and CORT-treated hippocampal frequency-dependent decrease Fig. 3 when interpulse slices. The ratios of neurotrophin gene expressions to those frequencies from 25 to 50 Hz were alternated. PPF was of b-actin were obtained. It showed that mRNA expression successfully established in 80 8 10 of hippocampal of neurotrophins, including NGF, BDNF as well as NT-3, 25 26 slices in control while CORT 10 M and 10 M expressed abundantly in rat hippocampal CA1 cells Fig. 26 25 application reduced the successful rate to 20 2 10 and 4. However, CORT 10 M and 10 M incubation 10 1 10, respectively, significantly smaller than those with hippocampal slice for 3 h dose-dependently dimin- 26 25 in control Table 1. In addition, CORT 10 M and 10 ished the mRNA expression of NGF-b and BDNF with the M application for 3 h markedly decreased the ratios maximum decrease of 40 and 60, respectively. Anyway, during PPF evoked at interpulse frequencies from 25 to 50 no effect on NT-3 mRNA expression was found in the Hz Fig. 3, P,0.05 or P,0.01. present study Fig. 4, P,0.05, or P,0.01. 3.4. CORT treatment reduced the expression of mRNA 3.5. BDNF co-applied with CORT antagonized the for NGF and BDNF in CA 1 region of rat hippocampal CORT-induced reduction of EPSP slope and amplitude slice ratios during PPF Semi-quantitative RT-PCR experiment was employed to Paired-pulse stimulation at a frequency of 50 Hz was determine the difference in neurotrophin mRNA expres- used to examine the effect of co-applied BDNF on CORT 186 J 26 25 Fig. 2. CORT 10 M and 10 M treatment inhibited the induction and maintenance of long-term potentiation LTP induced by high frequency stimulation HFS in rat hippocampal slice. A tetanus containing 100 testing stimulations at 100 Hz was used to induce LTP. A Two sets of traces showing the time-course changes of EPSP slope and amplitude during the LTP generation in CA1 dentrites of rat hippocampal slice. Four traces in both 25 control and CORT 10 M-treated slices displayed changes of EPSP slope and amplitude before t50 min and after tetanus t51, 30, 60 min delivery to the Schaffer collateral pathway. The calibration scale indicates 15 ms in horizontal bar and 2 mV in vertical bar. B Two graphs showing inhibition of both 26 25 induction and maintenance of LTP in hippocampal CA1 synapses after CORT 10 M and 10 M was applied to ACSF and perfused the hippocampal slice for 3 h. The amplitudes and slopes of EPSP were measured every 30 s before and during application of CORT to a submerged chamber. Each point represents the average amplitude and slope of 10 consecutive EPSPs 5-min interval normalized to the average of the EPSPs acquired before tetanus. n58–9, means6S.E., P,0.05, P,0.01, compared with control. 25 10 M-induced PPF impairment in hippocampal slices. stimuli during PPF with more than 45 decreases of ratios 25 CORT 10 M incubation with rat hippocampal slice for of both EPSP slope and amplitude compared with those in 3 h significantly impaired the response to the second control Fig. 5, P,0.05 or P,0.01. BDNF, at the final Table 1 a CORT application for 3 h reduced the generation probability of LTP and PPF in rat hippocampal CA1 synapses CORT Hippocampal slices induced Hippocampal slices induced PPF concentration LTP total hippocampal slice at 25 Hz total hippocampal slice M tested tested 9 10 8 10 26 10 4 10 2 10 25 10 4 10 1 10 a LTP is thought to be induced and expressed when EPSP amplitude and slope were potentiated more than 25 above the baseline for at least 30 min after tetanus while PPF at 25 Hz is believed to be established when both the EPSP slope and amplitude in the second response is at least 15 above the first one. Means6S.E., P,0.01, compared with control. J . Zhou et al. Brain Research 885 2000 182 –191 187 26 25 Fig. 3. CORT 10 M and 10 M incubation with rat hippocampal slice for 3 h inhibited the paired-pulse facilitation PPF. A Four sets of traces showing the changes of EPSP slopes and amplitudes during generation of paired-pulse facilitation PPF at different interpulse intervals from 50 ms|20 ms. a. Paired-pulse facilitation was induced with interpulse interval of 50 ms. b. Paired-pulse facilitation was induced with interpulse interval of 40 ms. The calibration scale for a and b indicates 25 ms in horizontal bar and 2 mV in vertical bar. c. Paired-pulse facilitation was induced with interpulse interval of 25 ms. d. Paired-pulse facilitation was induced with interpulse inverval of 20 ms. The calibration curve for c and d indicates 10 ms in horizontal bar and 2 26 25 mV in vertical bar. B CORT 10 M and 10 M treatment for 3 h significantly reduced the ratios of the second evoked EPSP slope and amplitude to the first one during PPF. n58–9, means6S.E., P,0.05, P,0.01, compared with that in control. 29 concentration of 10 M, applied half an hour before in vivo [7,32]. Our previous study [46] found that acute CORT addition to ACSF could antagonize these decreases application of CORT in single dose duplicated this effect, and basically rescued the PPF impairment Fig. 5. suggesting that elevated circulating CORT level might be responsible for the deficit of LTP generation in stressed animals. Our present results showed that CORT incubation

4. Discussion with rat hippocampal slice for 3 h significantly affected the