Optogenetic activation primes neurons for glutamate5...
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Introduction Optogenetic activation primes neurons for glutamate induced translation of the synaptic plasticity protein Arc Khadijah Mazhar, Kichan Kim, Darya Gonzalez, Kimberly Huber Department of Neuroscience, University of Texas Southwestern Medical Center, Dallas, Texas Activity-regulated cytoskeletal-associated protein (Arc/Arg3.1) is an immediate early gene and Arc induction is critical for consolidation of synaptic plasticity and memory. Brief novel experience has been shown to induce Arc mRNA, but not Arc protein, suggesting that Arc remains translationally suppressed. Stimulation of group 1/5 metabotropic glutamate receptors removes this inhibition, leading to production of dendritic Arc protein and long-term synaptic depression (mGluR-LTD). High frequency photostimulation can mimic novel experience to excite and promote Arc transcription in CA1 neurons expressing channelrhodopsin 2 (ChR2), a light-gated cation channel. Arc transcription serves to prime CA1 neurons for Arc translation following mGluR activation. Figure 1: Neuronal activity enhances mGluRinduced LTD mGluR activation induces translation and synthesis of Arc, which stimulates AMPA receptor endocytosis and long-term synaptic depression. Neuronal activity induced by theta burst photostimulation increases Arc mRNA, and primes neurons for rapid Arc induction after mGluR activation. Fragile X syndrome (FXS) is the most common inherited form of mental retardation and autism, and FMR1 knockout mice, which are unable to produce functional fragile X mental retardation protein (FMRP), can be studied as an animal model of FXS. In this study, we examine the effect on Arc induction after theta burst photostimulation (TBS) of CA1 neurons. Additionally, we analyze how this priming model is affected by FMR1 knockout. References Acknowledgements Methods Berndt, A., Schoenenberger, P., Mattis, J., Tye, K. M., Deisseroth, K., Hegemann, P., & Oertner, T. G. (2011). High-efficiency channelrhodopsins for fast neuronal stimulation at low light levels. Proceedings of the National Academy of Sciences of the United States of America, 108(18), 7595±7600. Bramham, C. R., Alme, M. N., Bittins, M., Kuipers, S. D., Nair, R. R., Pai % « Wibrand, K. (2010). The Arc of synaptic memory. Experimental Brain Research. Experimentelle Hirnforschung. Experimentation Cerebrale, 200(2), 125±140. Goold, C. P., & Nicoll, R. A. (2010). Single-Cell Optogenetic Excitation Drives Homeostatic Synaptic Depression. Neuron, 68(3), 512±528. Guzowski, John F. et al. (2006). Recent Behavioral History Modifies Coupling between Cell Activity and Arc Gene Transcription in Hippocampal CA1 Neurons. Proceedings of the National Academy of Sciences of the United States of America, 103.4 Niere, F., Wilkerson, J. R., & Huber, K. M. (2012). Evidence for an FMRP-mediated translational switch in mGluR-triggered Arc translation and LTD. The Journal of Neuroscience, 32(17), 5924±5936. Jakkamsetti, V., Tsai, N.-P., Gross, C., Molinaro, G., Collins, K. A., Nicoletti ) « +XEHU . 0 ([SHULHQFH-induced Arc/Arg3.1 primes CA1 pyramidal neurons for mGluR-dependent long-term synaptic depression. Neuron, 80 72±79 Mattis, J., Tye, K. M., Ferenczi ( $ 5DPDNULVKQDQ & 2¶6KHD ' - 3UDNDVK 5 « Deisseroth, K. (2012). Principles for applying optogenetic tools derived from direct comparative analysis of microbial opsins. Nature Methods, 9(2), 159±172. 2¶'RQQHOO, W.T., and Warren, S.T. (2002). A decade of molecular studies of fragile X syndrome. Annual Review Neuroscience, 25, 315±338. Park, S., Park, J. M., Kim, S., Kim, J.-A., Shepherd, J. D., Smith-+LFNV & / « :RUOH\ 3 ) (ORQJDWLRQ )DFWRU and Fragile X Mental Retardation Protein Control the Dynamic Translation of Arc/Arg3.1 Essential for mGluR-LTD. Neuron, 59(1), 70±83. Plath N. et al. . Arc/Arg3.1 is essential for the consolidation of synaptic plasticity and memories. (2006). Neuron, 52, 437± 444. I would like to express my deepest gratitude to Dr. Kimberly Huber, along with all of the members of her lab, for giving me the opportunity to conduct research with them. I would especially like to thank Dr. Kichan Kim for his continuous mentorship and support. I would also like to thank Deborah McGill, Vanessa Powell, Dr. Nancy Street, and Dr. Dean Sherry for providing the Green Fellowship, and Dr. Theodore Price and Dr. Uma Srikanth for their support. Figure 3: Total and Synaptic Arc levels in WT and FMR1KO neurons Total Arc protein is not affected with FMR1KO. Synaptic Arc is increased in FMR1KO neurons. While total Arc protein is unaffected by FMR1 KO in CA1 neurons, dendritic Arc is increased when its translationally suppressive binding protein, FMRP, is absent. This suggests that differences in local Arc protein expression account for the differences in mGluR-LTD development in FMR1 KO neurons. CA1 neurons exhibit Arc induction after treatment with an mGluR agonist, and induction is enhanced when the cells are exposed to one event of neuronal stimulation (1 TBS), suggesting that this event primes CA1 neurons for mGluR-LTD. This enhancement does not occur when these cells are repeatedly stimulated (R TBS), or are FMRP deficient. We hypothesize that repeated stimulation activates mGluRs, resulting in high levels of local Arc translation and expression that cannot be further increased with the addition of an mGluR agonist. FMR1 KO neurons, lacking a key Arc translation inhibition mechanism, cannot be induced to increase Arc production with mGluR stimulation as they express high levels of dendritic Arc in a basal state. Single or repeated stimulation of FMR1KO neurons increases Arc protein expression, suggesting that the priming mechanism for Arc induction is intact. Experiments were performed on CA1-enriched dissociated neuronal cultures, established from hippocampi extracted from P0 mice. Cells were transfected with ChR2 through a lenti- viral vector 1 day after culture plating. After 11 days of growth, cultures were treated with either 1 theta burst (1 TBS) or repeated theta bursts (R TBS) through LED photostimulation, or no theta bursts (No TBS). Figure 2: ChR2 Transfection and Theta Burst Photostimulation (TBS) Protocol 2 ms 10 ms 2 3 4 5 6 7 8 9 10 200 ms 1 2,300 ms B A C D 30 s 1 Theta Burst 2 3 4 5 6 7 8 9 30 min Repeated Theta Bursts Western blot Immunocytochemistry Lentiviral Transfection (ChR2 variant) Figure 4: Immunocytochemistry analysis of WT CA1 neurons Dendritic and somatic Arc are induced with DHPG, an mGluR agonist, in cells exposed to 1 or no TBS. Dendritic and somatic Arc in cells exposed to R TBS without DHPG treatment are higher than in cells exposed to 1 or no TBS without DHPG treatment. There is no difference in levels of BIII-tubulin, a neuronal marker, across treatments. Control refers to the no TBS, no DHPG condition. No TBS 1 TBS R TBS Vehicle 100uM DHPG Merged Arc BIII-Tubulin 100uM DHPG: - + - + - + No TBS 1 TBS R TBS Figure 5: Immunocytochemistry analysis of FMR1 KO CA1 neurons There is no difference in dendritic or somatic Arc with DHPG treatment within any of the TBS exposure conditions. Somatic and dendritic Arc in cells exposed to R TBS are higher than in cells exposed to 1 or no TBS. Somatic Arc in cells exposed to 1 TBS is higher than in cells exposed to no TBS. There is no difference in BIII- tubulin levels across treatments. Control refers to the no TBS, no DHPG condition. No TBS 1 TBS R TBS Vehicle 100uM DHPG Merged Arc BIII-Tubulin 100uM DHPG: - + - + - + No TBS 1 TBS R TBS Fmr1 KO Arc Synapsin WT Synaptosomal Protein ** Fmr1 KO Arc Gapdh WT Total Protein Results After 4 hours from the start of TBS, the cultures were treated with 100uM (RS)-3,5- dihydroxyphenylglycine (DHPG), an mGluR agonist. For Western Blotting assays, crude cell extracts were centrifuged for 10 min. at 800 rcf to yield a pellet containing the total protein sample, and a supernatant that was further centrifuged for 15 min. at 9200 rcf to obtain the synaptosomal protein sample. For Immunocytochemistry assays, cells were fixed with 4% PFA for 15 min. and permeabilized with 0.2% Triton X-100 for 10 min. Arc staining was quantified by subtracting background fluorescence intensity from fluorescence intensity of the dendrites. Conclusion Arc mRNA Neuronal activity (Theta burst stimulation) mGluR1/5 nucleus soma spine dendrite AMPAR mGluR1/5 mGluR activation mGluR activation Longterm synaptic depression (LTD) No stimulation ENHANCED Long term synaptic depression (LTD) Synaptic strength Synaptic strength Synaptic strength Synaptic strength Figure 6: Model for differences in Arc Induction with TBS or FMR1 KO FMRP FMRP Activated mGluR DHPG Arc Arc Arc translation ▲ FMRP mGluR FMRP FMRP FMRP mGluR FMRP FMRP Arc Activated mGluR DHPG Arc translation ▲ WT No TBS FMRP FMRP Arc Activated mGluR Arc FMRP FMRP Activated mGluR DHPG Arc Arc No further Arc translation Arc Arc Arc Arc mGluR DHPG Activated mGluR No further Arc translation Arc transcription ▲ Basal DHPG WT 1 TBS Basal DHPG WT Repeated TBS Basal DHPG FMR1 KO Basal DHPG Figure 7: Model of activity dependent Arc induction and longterm depression FMRP promotes transport and inhibits translation of Arc mRNA. Stimulation of mGluRs leads to dephosphorylation of FMRP, allowing synthesis of Arc protein. Arc induces endocytosis of AMPA glutamate receptors (AMPAR), leading to long-term synaptic depression. NUCLEUS SOMA DENDRITE FMRP Arc mGluR P P Ribosome AMPAR ChR2 Theta Burst Stimulation DHPG Arc mRNA Arc Arc transcription ▲ Arc translation ▲ Theta Burst Stimulation 1 TBS R TBS
Transcript of Optogenetic activation primes neurons for glutamate5...
Introduction
Optogenetic activation primes neurons for glutamate-induced translation of the synaptic plasticity protein Arc
Khadijah Mazhar, Kichan Kim, Darya Gonzalez, Kimberly HuberDepartment of Neuroscience, University of Texas Southwestern Medical Center, Dallas, Texas
Activity-regulated cytoskeletal-associated protein (Arc/Arg3.1) is an immediate early gene and Arc induction is critical for consolidation of synaptic plasticity and memory. Brief novel experience has been shown to induce Arc mRNA, but not Arc protein, suggesting that Arc remains translationally suppressed. Stimulation of group 1/5 metabotropic glutamate receptors removes this inhibition, leading to production of dendritic Arc protein and long-term synaptic depression (mGluR-LTD). High frequency photostimulation can mimic novel experience to excite and promote Arc transcription in CA1 neurons expressing channelrhodopsin 2 (ChR2), a light-gated cation channel. Arc transcription serves to prime CA1 neurons for Arc translation following mGluR activation.
Figure 1: Neuronal activity enhances mGluR-induced LTDmGluR activation induces translation and synthesis of Arc, which stimulates AMPA receptor endocytosis and long-term synaptic depression. Neuronal activity induced by theta burst photostimulation increases Arc mRNA, and primes neurons for rapid Arc induction after mGluR activation.
Fragile X syndrome (FXS) is the most common inherited form of mental retardation and autism, and FMR1 knockout mice, which are unable to produce functional fragile X mental retardation protein (FMRP), can be studied as an animal model of FXS. In this study, we examine the effect on Arc induction after theta burst photostimulation (TBS) of CA1 neurons. Additionally, we analyze how this priming model is affected by FMR1 knockout.
References
Acknowledgements
Methods
Berndt, A., Schoenenberger, P., Mattis, J., Tye, K. M., Deisseroth, K., Hegemann, P., & Oertner, T. G. (2011). High-efficiency channelrhodopsins for fast neuronal stimulation at low light levels. Proceedings of the National Academy of Sciences of the United States of America, 108(18), 7595 7600.
Bramham, C. R., Alme, M. N., Bittins, M., Kuipers, S. D., Nair, R. R., Pai Wibrand, K. (2010). The Arc of synaptic memory. Experimental Brain Research. Experimentelle Hirnforschung. Experimentation Cerebrale, 200(2), 125 140.
Goold, C. P., & Nicoll, R. A. (2010). Single-Cell Optogenetic Excitation Drives Homeostatic Synaptic Depression. Neuron, 68(3), 512 528.
Guzowski, John F. et al. (2006). Recent Behavioral History Modifies Coupling between Cell Activity and Arc Gene Transcription in Hippocampal CA1 Neurons. Proceedings of the National Academy of Sciences of the United States of America, 103.4
Niere, F., Wilkerson, J. R., & Huber, K. M. (2012). Evidence for an FMRP-mediated translational switch in mGluR-triggered Arc translation and LTD. The Journal of Neuroscience, 32(17), 5924 5936.
Jakkamsetti, V., Tsai, N.-P., Gross, C., Molinaro, G., Collins, K. A., Nicoletti -induced Arc/Arg3.1 primes CA1 pyramidal neurons for mGluR-dependent long-term synaptic depression. Neuron, 80 72 79
Mattis, J., Tye, K. M., Ferenczi Deisseroth, K. (2012). Principles for applying optogenetic tools derived from direct comparative analysis of microbial opsins. Nature Methods, 9(2), 159 172.
, W.T., and Warren, S.T. (2002). A decade of molecular studies of fragile X syndrome. Annual Review Neuroscience, 25, 315 338.
Park, S., Park, J. M., Kim, S., Kim, J.-A., Shepherd, J. D., Smith-and Fragile X Mental Retardation Protein Control the Dynamic Translation of Arc/Arg3.1 Essential for mGluR-LTD. Neuron, 59(1), 70 83.
Plath N. et al. . Arc/Arg3.1 is essential for the consolidation of synaptic plasticity and memories. (2006). Neuron, 52, 437444.
I would like to express my deepest gratitude to Dr. Kimberly Huber, along with all of the members of her lab, for giving me the opportunity to conduct research with them. I would especially like to thank Dr. Kichan Kim for his continuous mentorship and support.I would also like to thank Deborah McGill, Vanessa Powell, Dr. Nancy Street, and Dr. Dean Sherry for providing the Green Fellowship, and Dr. Theodore Price and Dr. Uma Srikanth for their support.
Figure 3: Total and Synaptic Arc levels in WT and FMR1KO neuronsTotal Arc protein is not affected with FMR1KO. Synaptic Arc is increased in FMR1KO neurons.
While total Arc protein is unaffected by FMR1 KO in CA1 neurons, dendritic Arc is increased when its translationally suppressive binding protein, FMRP, is absent. This suggests that differences in local Arc protein expression account for the differences in mGluR-LTD development in FMR1 KO neurons. CA1 neurons exhibit Arc induction after treatment with an mGluR agonist, and induction is enhanced when the cells are exposed to one event of neuronal stimulation (1 TBS), suggesting that this event primes CA1 neurons for mGluR-LTD. This enhancement does not occur when these cells are repeatedly stimulated (R TBS), or are FMRP deficient. We hypothesize that repeated stimulation activates mGluRs, resulting in high levels of local Arc translation and expression that cannot be further increased with the addition of an mGluRagonist.FMR1 KO neurons, lacking a key Arc translation inhibition mechanism, cannot be induced to increase Arc production with mGluR stimulation as they express high levels of dendritic Arc in a basal state. Single or repeated stimulation of FMR1KO neurons increases Arc protein expression, suggesting that the priming mechanism for Arc induction is intact.
Experiments were performed on CA1-enriched dissociated neuronal cultures, established from hippocampi extracted from P0 mice. Cells were transfected with ChR2 through a lenti-viral vector 1 day after culture plating. After 11 days of growth, cultures were treated with either 1 theta burst (1 TBS) or repeated theta bursts (R TBS) through LED photostimulation, or no theta bursts (No TBS).
Figure 2: ChR2 Transfection and Theta Burst Photostimulation (TBS) Protocol
2 ms
10 ms 2 3 4 5 6 7 8 9 10200 ms
1
2,300 ms
BA
C D30 s
1 Theta Burst
2 3 4 5 6 7 8 930 min
Repeated Theta Bursts
Western blotImmunocytochemistry
LentiviralTransfection
(ChR2 variant)
Figure 4: Immunocytochemistry analysis of WT CA1 neuronsDendritic and somatic Arc are induced with DHPG, an mGluR agonist, in cells exposed to 1 or no TBS. Dendritic and somatic Arc in cells exposed to R TBS without DHPG treatment are higher than in cells exposed to 1 or no TBS without DHPG treatment. There is no difference in levels of BIII-tubulin, a neuronal marker, across treatments. Control refers to the no TBS, no DHPG condition.
No TBS 1 TBS R TBS
Vehicle
100uM DHPG
Merged
Arc
BIII-Tubulin
100uM DHPG: - + - + - +No TBS 1 TBS R TBS
Figure 5: Immunocytochemistry analysis of FMR1 KO CA1 neuronsThere is no difference in dendritic or somatic Arc with DHPG treatment within any of the TBS exposure conditions. Somatic and dendritic Arc in cells exposed to R TBS are higher than in cells exposed to 1 or no TBS. Somatic Arc in cells exposed to 1 TBS is higher than in cells exposed to no TBS. There is no difference in BIII-tubulin levels across treatments. Control refers to the no TBS, no DHPG condition.
No TBS 1 TBS R TBS
Vehicle
100uM DHPG
Merged
Arc
BIII-Tubulin
100uM DHPG: - + - + - +No TBS 1 TBS R TBSFmr1 KO
Arc
Synapsin
WTSynaptosomalProtein
**
Fmr1 KO
Arc
Gapdh
WTTotal Protein
Results
After 4 hours from the start of TBS, the cultures were treated with 100uM (RS)-3,5-dihydroxyphenylglycine (DHPG), an mGluR agonist.For Western Blotting assays, crude cell extracts were centrifuged for 10 min. at 800 rcfto yield a pellet containing the total protein sample, and a supernatant that was further centrifuged for 15 min. at 9200 rcf to obtain the synaptosomal protein sample.For Immunocytochemistry assays, cells were fixed with 4% PFA for 15 min. and permeabilized with 0.2% Triton X-100 for 10 min. Arc staining was quantified by subtracting background fluorescence intensity from fluorescence intensity of the dendrites.
Conclusion
Arc mRNA
Neuronal activity(Theta burst stimulation)
mGluR1/5
nucleus
somaspine
dendrite
AMPAR
mGluR1/5
mGluRactivation
mGluRactivation
Long-‐term synaptic
depression (LTD)
No stimulation
ENHANCED Long-‐term synaptic
depression (LTD)
Synaptic strength
Synaptic strength
Synaptic strength
Synaptic strength
Figure 6: Model for differences in Arc Induction with TBS or FMR1 KO
FMRP FMRP
ActivatedmGluR
DHPG
Arc
Arc
Arc translation ▲
FMRP
mGluR
FMRP
FMRP FMRP
mGluR
FMRPFMRP
Arc
ActivatedmGluR
DHPG
Arc translation ▲
WT No TBS
FMRP FMRP
Arc
ActivatedmGluR
Arc
FMRP FMRP
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DHPG
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No further Arc translation
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Arc
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ArcmGluR
DHPG
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No further Arc translationArc transcription ▲
Basal DHPG
WT 1 TBSBasal DHPG
WT Repeated TBSBasal DHPG
FMR1 KOBasal DHPG
Figure 7: Model of activity-dependent Arc induction and long-term depressionFMRP promotes transport and inhibits translation of Arc mRNA. Stimulation of mGluRsleads to dephosphorylation of FMRP, allowing synthesis of Arc protein. Arc induces endocytosis of AMPA glutamate receptors (AMPAR), leading to long-term synaptic depression.
NUCLEUS
SOMA DENDRITE
FMRP
Arc
mGluR
PP
Ribosome
AMPAR
ChR2
Theta Burst Stimulation
DHPG
Arc mRNA
Arc
Arc transcription ▲Arc translation ▲
Theta Burst Stimulation
1 TBS
R TBS
