Bipn140 Lect 15 Figs Nov10

Bipn140 Lect 15 Figs Nov10 - BIPN 140: Cellular...

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Unformatted text preview: BIPN 140: Cellular Neurobiology LECTURE #15: Synapse Formtion [Website:] INSTRUCTORS Nicholas C. Spitzer ( Darwin K. Berg ( ANNOUNCEMENTS 1) Final Exam: Friday, Dec 10, 7-10 pm, 107 Solis (here). 2) Review Session: Thurs, Dec 9, 7-9 pm, 3500 Pacific Hall. (Maybe also 6-7 pm, 2130 Bonner Hall?) 3) One time only: moved office hour to Wed, 11-12 noon, 11/24. FIG: Ultrastructural image of an NMJ active zone. Synapse from a frog sartorius neuromuscular junction showing vesicles clustered in the active zone, some docked at the membrane (arrows). (from Heuser, 1977) Box 7Bb,c Dendritic Spines Box 7Bd/e Dendritic Spines Fig 23.10: Number and Pattern of Synapses on Muscle Fig 23.11: Synapse Elimination in Muscle Box 23B: The Role of Agrin in Muscle Innervation Model: Roles of Agrin & ACh in NMJ Postsynaptic Development ACh represses synthesis of extrasynaptic nAChRs and disperses nAChR clusters. Agrin represses nAChR loss locally (at nerve contact) to promote synapse formation. Neuregulin increases muscle nAChR synthesis but also acts on Schwann cells to promote their contribution. (Kummer, Misgeld, & Sanes, 2006) Fig 23.7a: Molecular Mechanisms Involved in Synapse Formation Fig 23.7b: Molecular Mechanisms Involved in Synapse Formation Fig 23.7c: Molecular Mechanisms Involved in Synapse Formation Fig 23.8a,b: Potential Molecular Mediators of Synapse Identity Fig 23.8c: Potential Molecular Mediators of Synapse Identity Activity-Dependent Regulation of Inhibitory Synapse Development by Npas4 by Y. Lin, B.L. Bloodgood, J.L. Hauser, A.D. Lapan, A.C. Koon, T.-K. Kim, L.S. Hu, A.N. Malik, M.E. Greenberg Nature 455:1198-1205 (2008) BACKGROUND Much is known about the regulation of glutamate synapse formation, but GABA synapse formation is likely to be different because GABA synapses are destined to be inhibitory and are located on dendritic shafts instead of spines. This manuscript started with DNA microarrays to identify the candidate regulators of GABAergic synapse formation. Npas4 was chosen because: (a) it was a transcription factor, (b) it’s expression was increased by depolarization in a calcium-dependent manner, and (c) it was in the right places at the right time. These features seemed compatible with mechanisms that induce GABAergic synapses in an activity-dependent manner. Experiments were done to test the role of Npas4. EXPERIMENTS 1) Use Western blots to quantify the amount of Npas4 in cultures depolarized by KCl, and determine if calcium is necessary (± calcium blockers). 2) Test the requirement for Npas4 by using RNAi in cell culture to knock it down in neurons and then quantify GABAergic synapses by immunostaining for the presynaptic marker GAD65 and the postsynaptic marker GABAA- 2. 3) Use RNAi to test the requirement for Npas4 in slice culture, measuring mIPSC frequency and amplitude as indicators of GABAergic synapse abundance and strength. Fig. 1a-c: KCl-induced depolarization of neurons induces Npas4 in a calcium-dependent manner as seen by Western blots; the induction is transient. Hippocampal cultures were treated with the indicated conditions and then extracted and analyzed by Western blots to quantify the amounts of several transcription factors. (a) immunostaining showing Npas4 increased preferentialy by KCl. (b) Quantification showing that only KCl increases Npas4 and requires calcium to do so (blocked by EGTA) whereas other transcription factors (c-fos & p-CREB) respond to multiple conditions. (c) The KCl-induced increase in Npas4 is transient. Fig. 2b,d: Npas4 RNAi knocks Npas4 down in cell culture and reduces the number of GABAergic synapses seen by co-staining for presynaptic GAD65 and postsynaptic GABAA- 2. Transfecting hippocampal cultures with an RNAi knocking down Npas4 levels in the neurons reduced the number of GABAergic synapses seen by co-staining for the two markers, compared to cultures transfected with a control RNAi. (a) Images. (b) Quantification. [Vector control: no RNAi; Npas4-RNAi: construct specifically directed against the Npas4 mRNA; Control-RNAi: construct with a scrambled sequence so that it would target nothing intentionally.] Fig. 3a-c: Npas4 RNAi reduces functional GABAergic synapses in slice culture seen by decreases in mIPSC frequency and amplitude. Transfecting hippocampal slice cultures with Npas4 RNAi reduces mIPSC frequency (increased inter-event interval) and decreases mean mIPSC amplitude. Overexpressing Npas4, on the other hand, has the opposite effect (Npas4-minigene). (a) Recordings. (b) Quantification. (c) Mean values. TAKEHOME GABAergic synapse formation is regulated by activity in a calcium-dependent manner at the transcriptional level by Npas4. Other results in the paper show that Npas4 acts similarly in vivo and does not alter excitatory synapses. The bottomline is that Npas4 expression can alter the excitatory/inhibitory balance in neural circuits. ...
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This note was uploaded on 12/09/2010 for the course BIPN BIPN 140 taught by Professor Spitzer during the Spring '07 term at UCSD.

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