Results Because of the target specificity of the BDNF effect (A.F. Schinder, B. Berninger, and M.-m. Poo, in prep), we have chosen to study pairs or triplets of hippocampal neurons consisting of glutamatergic neurons only. Excitatory postsynaptic currents were evoked by a brief step depolarization of a designated presynaptic neuron, with perforated patch recording ( Hamill et al. 1981; Horn and Marty 1988; Rae et al. 1991). Application of exogenous of BDNF (100 ng/ml) rapidly and markedly increased the amplitude of EPSCs in a subpopulation of these synaptic connections. In the case shown in Figure 1A, the synapse exhibited an average EPSC of ~80 pA during the control period (prior to BDNF treatment). Clear strengthening of synaptic efficacy was detected after 10 min in the presence of BDNF, and the EPSC amplitude reached a plateau value of ~140 pA after 20 min. In contrast, for the case shown in Figure 1B, in which the synapse showed initial EPSCs of ~1 nA, no effect of BDNF on synaptic transmission was observed, suggesting that the potentiation effect may depend on the initial synaptic strength. Results from all recordings (including those showing no BDNF effect) are summarized in Figure 2. The average EPSC amplitude at 20–30 min after the onset of BDNF application (100 ng/ml) was 134 ± 8% ( s.e.m.; n= 19) of the control values (prior to the exposure to BDNF). The effect of BDNF on synaptic transmission was dose dependent (Fig. 2): The average EPSC amplitude after exposure to 0 and 20 ng/ml of BDNF was 97 ± 6% ( n= 5) and 110 ± 6% ( n= 5) of the control values, respectively. | Figure 1Potentiation of glutamatergic transmission by BDNF depends on the initial synaptic strength. (A) Example of a recording of glutamatergic synaptic transmission with a low initial EPSC amplitude (83 pA) prior to exposure to BDNF. Both the designated pre- (more ...) |
To assess whether differences in the initial synaptic strength account for the variability in the response of these synapses to BDNF, we compared the degree of potentiation by BDNF for synaptic connections of different initial EPSC amplitudes. Marked synaptic potentiation was observed in most cases of initially weak synaptic connections, whereas synapses with high EPSC amplitudes were much less affected. For synapses with EPSC amplitudes of <400 pA, exposure to 100 ng/ml BDNF resulted in a potentiation of 158 ± 15% ( n= 7), whereas for those with amplitudes between 400 and 800 pA and >800 pA synaptic potentiation was 124 ± 7% ( n= 6) and 105 ± 7% ( n= 4), respectively. The correlation between the degree of potentiation and the initial EPSC amplitude is shown in Figure 3A ( r= −0.552, P= 0.0028, ANOVA). | Figure 3The degree of synaptic potentiation by BDNF correlates with initial synaptic strength, CV, and paired–pulse facilitation. (A) Relationship between synaptic potentiation and initial EPSC amplitude. Initial EPSC amplitude was calculated for 40 events (more ...) |
It was shown previously that BDNF-induced synaptic potentiation of glutamatergic synapses in hippocampal cultures ( Lessmann and Heumann 1998; A.F. Schinder, B. Berninger, M.-m. Poo, in prep.) and neuromuscular synapses in Xenopus cultures ( Boulanger and Poo 1999) is accompanied by a decrease in the coefficient of variation (CV) and a reduction in the paired-pulse facilitation. The CV is a measure of the fluctuation of the postsynaptic response which, according to classical quantal theory, is solely determined by presynaptic properties, that is, the release probability ( Pr) and the number of release sites ( N). Paired–pulse facilitation (or depression) of the postsynaptic response is commonly observed for the second of two stimulation pulses applied in close succession, and has been shown to be a function of Pr. A decrease in CV and paired–pulse facilitation therefore suggests that the effect of BDNF can be largely accounted for by an enhanced transmitter release, presumably due to either an increase in Pr and/or in N. This is also consistent with the finding of increased frequency of miniature EPSCs after BDNF treatment of hippocampal and neuromuscular synapses ( Lohof et al. 1993, Lessmann et al. 1994; Li et al. 1998 a, b). Because BDNF exerts its effect mainly by changing presynaptic release properties, it is reasonable to expect synapses with lower release capability to be more susceptible to potentiation by BDNF. We found that BDNF-induced synaptic potentiation significantly correlated with the initial CV (Fig. 3B; r= 0.74, P< 0.001, ANOVA) and the extent of paired–pulse facilitation (Fig. 3C; r= 0.586, P= 0.0042, ANOVA). Thus, synapses with low initial Pr and/or N are more responsive to BDNF than those synapses with a high initial Pr and/or N. To determine whether BDNF-induced synaptic potentiation may be masked at synapses with high initial Pr, we performed two manipulations to lower Pr. For these experiments we selected synapses with high initial amplitude, which typically show no potentiation induced by BDNF under control conditions ([Ca 2+] o= 3 m m, no adenosine). In the first set of experiments, after recording a baseline to determine the initial EPSC amplitude, we lowered [Ca 2+] o from 3 to 0.5 m m. As shown for a synapse with an initial EPSC amplitude of ~1050 pA (Fig. 4A), lowering [Ca 2+] o reduced the EPSC amplitude by 97%, indicating a drastic reduction in Pr. Although these conditions resulted in an initial EPSC amplitude of approximately −30 pA for the control period, subsequent BDNF treatment did not significantly enhance synaptic transmission. All experiments involving lowering of [Ca 2+] o are summarized in Figure 4B. On the average, EPSC amplitudes decreased from −2387 ± 1313 pA to −206 ± 199 pA, whereas CV increased from 0.033 ± 0.01 to 0.44 ± 0.10 and paired–pulse facilitation increased from 0.43 ± 0.01 to 1.73 ± 0.35. Despite the strong reduction in Pr, however, no potentiation by BDNF was revealed (94 ± 8 % of control period, n= 3). | Figure 4Lowering Pr at stronger synapses does not unmask response to BDNF. (A) Example for absence of BDNF effect after lowering Pr by reducing [Ca2+]o. Initial EPSC was ~−1050 pA and was reduced to −30 pA after (more ...) |
As an alternative way of reducing Pr, we applied adenosine (1–2 μ m), which is known to inhibit presynaptic Ca 2+ influx ( Wu and Saggau 1994). This treatment resulted in a decrease in EPSC amplitude (from −1111 ± 209 to −367 ± 110 pA), with CV increasing from 0.06 ± 0.01 to 0.18 ± 0.02 and paired–pulse facilitation increasing from 0.46 ± 0.04 to 0.96 ± 0.43, reflecting a strong reduction in Pr. As summarized in Figure 4B, no potentiation was observed in response to BDNF after treatment with adenosine (87 ± 5% of control period, n= 3). Taken together with the low [Ca 2+] o experiments, these results demonstrate that lowering Pr at initially strong synapses is not sufficient to allow synaptic potentiation by BDNF. Is the susceptibility to synaptic modification by BDNF a global property of the presynaptic neuron or a property of its individual presynaptic terminals? To address this question we recorded from triplets, in which a single presynaptic neuron projected divergent outputs to the two other neurons with substantially different synaptic efficacy. Results from two experiments are shown in Figure 5. In both cases, BDNF induced a higher degree of potentiation in the synaptic connection with initially weaker synaptic response. Thus, the size dependence of synaptic modification by BDNF is also found for divergent outputs from the same presynaptic neuron, suggesting that individual synaptic terminals may undergo independent synaptic modification. |
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