Synaptic and extrasynaptic localization of brain‐derived neurotrophic factor and the tyrosine kinase B receptor in cultured hippocampal neurons

Abstract

Brain-derived neurotrophic factor (BDNF) regulates synapses, but the distribution of BDNF and its receptor TrkB relative to the location of glutamatergic and γ-aminobutyric acidergic (GABAergic) synapses is presently unknown. Immunocytochemistry was performed in primary hippocampal neuron cultures to determine whether BDNF and TrkB are preferentially localized to excitatory or inhibitory markers at 7, 14, and 21 days in vitro (DIV). Glutamatergic sites were localized with vesicular glutamate transporter type 1 (VGLUT1) as presynaptic marker and the NR1 subunit of the NMDA receptor and the GluR1 subunit of the AMPA receptor as receptor markers. GABAergic sites were labeled with the 65-kDa isoform of glutamic acid decarboxylase (GAD-65) as presynaptic marker and the γ2 subunit of the GABA A receptor as receptor marker. During development, <30% of BDNF punctae and TrkB clusters were localized to glutamatergic and GABAergic markers. Because their rates of colocalization did not change from 7 to 21 DIV, this study details the distribution of BDNF and TrkB at 14 DIV. BDNF was preferentially colocalized with glutamatergic markers VGLUT1 and NR1 (~30% each). TrkB was also relatively highly colocalized with VGLUT1 and NR1 (~20% each) but was additionally highly colocalized with GABAergic markers GAD-65 (~20%) and γ2 (~30%). NR1 clusters colocalized with BDNF puncta and TrkB clusters were mostly extrasynaptic, as were γ2 clusters colocalized with TrkB clusters. These results show that, whereas most BDNF and TrkB protein is extrasynaptic, BDNF is preferentially associated with excitatory markers and that TrkB is associated equally with excitatory and inhibitory markers. Indexing termsneurotrophins; glutamate; GABA; synaptogenesis Brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family, modulates many aspects of synaptic structure and function. For instance, BDNF modulates axonal and dendritic branching. It increased branching and complexity of optic axon terminal arbors in Xenopus (Cohen-Cory and Fraser, 1995) and in chick (Inoue and Sanes, 1997) and increased the length and complexity of apical and basal dendrites in Xenopus retina (Lom and CohenCory, 1999) and in mammalian visual cortex (McAllister et al., 1995). BDNF also modulates synaptic efficacy. It acutely potentiated excitatory transmission in the neuromuscular junction (Lohof et al., 1993) and hippocampus (Lessmann et al., 1994;Kang and Schuman, 1995;Levine et al., 1995;Messaoudi et al., 1998;Li et al., 1998) (Tanaka et al., 1997;Frerking et al., 1998;Brunig et al., 2001;Cheng and Yeh, 2003), and elicited action potentials in the hippocampus, cortex, and cerebellum by activating the tetrodotoxin-insensitive sodium channel Na v 1.9 (Kafitz et al., 1999;Blum et al., 2002). Furthermore, BDNF influences synaptic maturation. It increased the density of both excitatory and inhibitory synapses in the hippocampus (Vicario-Abejon et al., 1998;Martinez et al., 1998) and promoted activity-dependent inhibitory synaptogenesis (Seil and DrakeBaum...

Activity-dependent scaling of GABAergic synapse strength is regulated by brain-derived neurotrophic factor

“…2). Similar to the descriptions of GAD-65 immunoreactivity in these cultures provided in detail in the past (Swanwick et al, 2004), large discrete GAD-65 puncta were distributed widely throughout the neuronal processes of control pyramidal neurons (Figs. 2A,C).…”

BDNF-induced presynaptic facilitation of GABAergic transmission in the hippocampus of young adults is dependent of TrkB and adenosine A2A receptors

“…Although BDNF is only expressed in pyramidal cells, but not interneurons [45,46], it is generally accepted that interneurons do express TrkB receptors [45,47,48]. Increase in synaptic activity will favor the release of BDNF [49] and the formation of adenosine from released ATP [50], with consequent activation of TrkB receptors and adenosine A 2A Rs [2].…”

The Dynamic Distribution of TrkB Receptors before, during, and after Synapse Formation between Cortical Neurons

“…TrkB protein is often reported as very densely packed and diffuse throughout dendrites and somas (Fryer et al, 1996;Yan et al, 1997;Kryl et al, 1999), but recent reports suggest that TrkB is present in distinct puncta either throughout the dendrites (Swanwick et al, 2004) or specifically in the dendritic membrane (Elmariah et al, 2004). The presence of TrkB in axons is also debated, with some reporting TrkB in axons in both developing and adult neurons (Yan et al, 1997;Kryl et al, 1999;Swanwick et al, 2004), whereas others find TrkB present in axonal initial segments but mostly absent from axons and growth cones (Drake et al, 1999;Elmariah et al, 2004). TrkB is also found in dendritic spines and postsynaptic densities of excitatory synapses, but it is unclear whether TrkB is enriched there (Wu et al, 1996;Drake et al, 1999).…”

“…TrkB is also found in dendritic spines and postsynaptic densities of excitatory synapses, but it is unclear whether TrkB is enriched there (Wu et al, 1996;Drake et al, 1999). Although TrkB can be found both presynaptically and postsynaptically in excitatory synapses in mature (Drake et al, 1999;Aoki et al, 2000) and developing (Swanwick et al, 2004;Petralia et al, 2005) neurons, the localization of TrkB in axons and dendrites relative to presynaptic and postsynaptic proteins before, during, and after synapse formation remains unknown. There is also almost nothing known about the distribution of TrkB receptors in the plasma membranes of axons and dendrites during development, where they must be present to be functional.…”