An investigation to determine whether BDNF is a neurotrophin-specific modulator of LTP at the MF-CA3 pathway.

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Preliminary Studies:

BDNF dramatically enhances mossy fiber field potentials and LTP. fEPSP recordings were performed in order to determinate whether BDNF is a neurotrophin-specific modulator of LTP at the MF-CA3 pathway. As seen in Figure 1A, Depict an hippocampal slice where fEPSP recordings were obtained from the stratum lucidum of area CA3 via micropipette recording whereas the stimulation were applied via bipolar electrode at the Dentate Gyrus (DG) area. During the experiment the tissues were perfused with ACSF and BIC (Bicuculline, GABAA Blocker) whereas BDNF (200ng/ml) was administered directly to the CA3 region via the recording micropipette. Although LTP at the MF-CA3 hippocampal pathway is an NMDA receptor independent form of synaptic plasticity (Barea Edwin and Martínez Joe, Jr.et. al.2004), the tissues were also perfused with AP5 (NMDA blocker) during the baseline and 2 minutes prior to the test. In the presence of ACSF, HFS elicited an increase in synaptic plasticity in comparison with the baseline (Fig. 1B, black dots). In the presence of BDNF, HFS elicits a large potentiation of the field potential (Fig. 1B, red dots), when compared to ACSF (Fig 1B, black dots). Figure 1C, depict average traces showing that HFS potentiates the peak of the mossy fiber fEPSP. By other side, individually each traces shows the average taken prior to HFS (baseline) and 2 hrs after HFS with their respective drug treatment. The red trace depicts the average of BDNF in combination with HFS, showing the dramatic enhancement of mossy fiber LTP (Fig. 1C). However, the black trace depicts the average of ACSF in combination with HFS, showing the increase in synaptic plasticity in comparison with the baseline (Fig 1C). Our results suggest that BDNF plays an essential role in synaptic plasticity and LTP at the mossy fiber synapse through TrkB receptors. BDNF dramatically increases LTP at the MF-CA3 hippocampal pathway compared to all other groups (p<0.001 One Way ANOVA).

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K252A (trkB antagonist) reversed the significant increase of LTP at MF-CA3 hippocampal pathway elicited by BDNF. To confirm whether BDNF’s enhancements in LTP at the MF-CA3 pathway is directly via activation of TrkB receptors, the tissues were perfused with ACSF, BIC, and K252A (a trkB receptor blocker) in the presence of BDNF (200ng/ml) directly to the CA3 region via the recording micropipette. The tissues were also perfused with AP5 during the baseline and 2 minutes prior to the test (Fig. 2A). BDNF with HFS in the presence of K252A (200ng/ml) depressed mossy fiber field potentials. Thus, we demonstrated that the significant increase of LTP MF-CA3 hippocampal pathway with the activation of BDNF to trkB receptor was reversed by the K252a (Fig. 2A, red dots). ACSF and BIC was perfused in combination of HFS, this elicited an increase in synaptic plasticity in comparison with the baseline. Here, ACSF was used as a control to compare the fEPSP between the experiments such as BDNF in the presence of K252a and ACSF (Fig. 2A, black dots). Figure 2B, depicts average traces showing that HFS potentiates the peak of the mossy fiber fEPSP. By other side, individually each traces shows the average taken prior to HFS (baseline) and 2hrs after HFS with their respective drug treatment. The red trace depicts the average of BDNF in combination with HFS in the presence of K252a, showing depressed MF field potential (Fig. 2B). However, the black trace depicts the average of ACSF in combination with HFS, showing the increase in synaptic plasticity in comparison with the baseline (Fig. 2B). Our results suggest that the activation of BDNF ligand to trkB receptor is necessary in order to elicit a significant potentiation of the fEPSP at MF-CA3 hippocampal pathway. BDNF with HFS in the presence of K252a reversed and depressed the dramatically increase mossy fiber field potentials (p<0.001 One Way ANOVA).

Enhancements in mossy fiber long-term potentiation (LTP) are neurotrophin-specific. Around the Central Nervous System (CNS) exists different kinds of neurotrophins. In addition to BDNF is evident the existence of NGF (binds to trkA receptor) and NT3 (binds to trkC receptor). In order to verify whether the observed enhancements in MF LTP are specific to BDNF, we studied the role of the neurotrophins NGF and NT3 on LTP MF-CA3 (Fig. 3). During the experiment the tissues were perfused with ACSF and BIC (Bicuculline, GABAA Blocker). The tissues were also perfused with AP5 during the baseline and 2 minutes prior to the test (Fig. 3A & Fig. 3C). Individually NGF (200ng/ml) and NT3 (200ng/ml) were directly administered to the CA3 region via the recording micropipette (Fig. 3A & Fig 3C). NGF in combination with HFS elicited an increase in synaptic plasticity in comparison with the baseline (Fig 3A, red dots) similar to ACSF in combination with HFS (Fig 3A, black dots). NT3 in combination with HFS elicited an increase in synaptic plasticity in comparison with the baseline (Fig 3C, red dots) similar to ACSF in combination with HFS (Fig 3C, black dots).

By other side, individually each traces shows the average taken prior to HFS (baseline) and 2hrs after HFS with their respective drug treatment (Fig. 3B & Fig. 3D). Red trace depicts the average of NGF in combination with HFS, showing the increase in synaptic plasticity in comparison with the baseline (Fig. 3B). Red trace depicts the average of NT3 in combination with HFS, showing the increase in synaptic plasticity in comparison with the baseline (Fig. 3D). However, the black traces depicts the average of ACSF showing the increase in synaptic plasticity in comparison with the baseline (Fig. 3B & Fig. 3D). No enhancements of LTP were found following administration of either NT3 or NGF, indicating that the enhancements in mossy fiber LTP are BDNF-specific (p>0.001, ANOVA). Our results indicate that BDNF plays an essential role and neurotrophin specific in synaptic plasticity and LTP at the mossy fiber synapse.

Naloxone (µ-Opioid antagonist) disrupts LTP MF-CA3 hippocampal pathway elicited by BDNF. Previous studies demonstrated that LTP MF-CA3 hippocampal pathway is an NMDA receptor-independent form of synaptic plasticity (Harris and Cotman et. al. 1996; Derrick et. al. 1991; Williams Stephen and Johnston Daniel et. al. 1996; Barea Edwin and Martínez Joe Jr. et. al.2004) but this pathway requires the activation of MOR (µ-Opioid receptor) and opioid peptide release (Braham et. al 1988; Derrick, Barea E, Martinez Joe Jr. et. al.1991). The MF-CA3 hippocampal pathway of the rat hippocampus is sensitive to opioid antagonist. Naloxone is an µ-Opioid receptor antagonist shown to block LTP in lateral perforant path to dentate gyrus in vivo (Braham, C et. al. 1988). Naloxone also blocks LTP of mossy fiber in guinea pig hippocampus in vitro (Martin M.R. et. al. 1992), and MF LTP induction at rat hippocampus (Escobar M.L., Martinez J et. al.1997). The MF-CA3 hippocampal pathway contains a high density of opioid receptors (Crain et. al. 1981; Mc Lean et. al. 1987; Mansour et. al. 1995). Previous studies suggest that opioids play an important role in LTP induction (Jaffe and Johnston et. al. 1990; Derrick et. al. 1991). In order to verify the role of Naloxone in combination with BDNF on potentiation of LTP at MF-CA3 hippocampal pathway, we bath perfused our tissue with Naloxone (10µM) in vitro in the presence of BDNF directly administered to CA3 (Fig.4A). fEPSP recordings were obtained in Stratum Lucidum of CA3 area, and a reduction in MF LTP was observed (Fig.4A, black square). Our results suggest that BDNF- mediated enhancement appears to work via interaction with MOR. This further suggests that BDNF-mediated enhancement occurs via an opioid-dependent mechanism at the MF-CA3 synapse.

Summary of fEPSP Peak Amplitude at the MF-CA3 hippocampal pathway. This is a comparison of % fEPSP Peak Amplitude response for each of the drugs administered at the MF-CA3 hippocampal pathway (Fig 5). BDNF administration results in a robust enhancement of MF LTP with the combination of BDNF + HFS. These results further confirm that BDNF plays an important role in MF LTP and hippocampal excitability. The observed BDNF-mediated potentiation was blocked by a trkB blocker, and was not induced by other neurotrophins. In addition, co-application of BDNF with the opioid antagonist Naloxone resulted in decreased MF-LTP. Individually, in each experiment we confirmed with the perfusion of AP5 that MF LTP does not depend on the activation of NMDA receptors.

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