Neuroscience Techniques (SC/BIOL 4370 Fall 2024) PDF

11/29/2024 SC/BIOL 4370 – 3.0 [Fall 2024] Neurobiology Lecture 11: Neuroscience Techniques 1 1 1  11/29/2024 SCHEDULE 2 2...

11/29/2024 SC/BIOL 4370 – 3.0 [Fall 2024] Neurobiology Lecture 11: Neuroscience Techniques 1 1 1  11/29/2024 SCHEDULE 2 2 2  11/29/2024 Housekeeping Remember Written Assignment! Due: Midnight (12:00am) December 4th Submission link now posted on eClass Make sure to submit in Word format Final exam: Saturday, December 14th 7:00pm-10:00pm Similar format to midterm 3 Hours 100-110+ marks In-person 3 3  11/29/2024 Outline Understand techniques used to understand neurobiological questions Explore how changes in neurons and synapses reveal the nature of brain disorders Final take home messages 4 4 4  11/29/2024 MDGAs regulate Excitation/inhibition balance 5 5  11/29/2024 Level of resolution: Single cell analyses Can isolate and culture cells from transgenic models Can further manipulate these cells (genetically, pharmacologically) to determine manipulation effects Allows refined exploration of changes in how neurons form connections (synapses) Caveat: May not recapitulate how neurons behave in the intact CNS 6 6  11/29/2024 Overexpression of MDGA2 suppresses excitatory and inhibitory synapses in cultured neurons Does MDGA2 regulate synapse development in vivo? 7 7  11/29/2024 Level of resolution: Neural circuits Can look at brain tissue samples and assay for the density and size of distinct synapse subtypes Provides a “snapshot” of the distribution and quantity of synapses Closer (but not perfect) representation of in vivo state of synapses Caveat: Temporal resolution is minimal. -samples drawn reflect neural circuits at discreet developmental stage 8 8  11/29/2024 Reduction of MDGA2 increases excitatory synapse numbers in vivo Cell bodies (pyramidale) Dendrites (radiatum) By: Ina Ammendrup-Johnsen Are excitatory synapse proteins upregulated following reduction of MDGA2? 9 9  11/29/2024 Excitatory synaptic proteins are selectively upregulated in Mdga2+/- mouse hippocampus (NL1) By: Yuan Ge 10 10  11/29/2024 Components of excitatory synaptic transmission AMPARs: mediate basal transmission; trafficking increases in response to activity NMDARs: induce synaptic strength changes Mdga2+/+ AMPAR (GluA1) Scaffold (PSD-95) Mdga2+/- By: Hong Lu Does reduction of MDGA2 alter basal synaptic transmission in vivo? 11 11  11/29/2024 Level of resolution: Neuronal Function Can look at neurons in brain slices and how their synapses are functioning Provides direct assessment of change in core cellular characteristics and measures of synaptic inputs onto these cells Can span from single channel to whole cell analysis Caveat: Relatively isolated preparation, outside of normal context of intact brain -effects may reflect indirect changes in cellular function that result from manipulations 12 12  11/29/2024 Reduction of MDGA2 in vivo results in enhanced excitatory transmission Increased synapse number Increased basal strength Is evoked excitatory synaptic transmission increased in Mdga2+/- mouse hippocampus? 13 13  11/29/2024 Level of resolution: Neuronal Networks Can look at population-responses to understand how neurons are behaving at the network level Provides assessment of how network properties have changed (how neuronal populations respond0 Can span from single neuron to multiple cell scales Caveat: Hard to differentiate network from cell-autonomous effects -also, only sampling some isolated features of network function 14 14  11/29/2024 Evoked Excitatory transmission is elevated in Mdga2+/- hippocampus What components of excitatory synaptic transmission are upregulated in Mdga2+/- mice? 15 15  11/29/2024 AMPA receptor surface expression and synaptic transmission are increased when MDGA2 is reduced AMPAR AMPA/NMDA (Surface GluA1) NMDA AMPA Are cellular mechanisms of memory altered by Mdga2 reduction? 16 16  11/29/2024 Level of resolution: Neural Plasticity Can look at population-responses to understand how neurons are behaving at the network level Provides assessment of how network properties have changed (how neuronal populations respond0 Can span from single neuron to multiple cell scales Caveat: Hard to differentiate network from cell-autonomous effects -also, only sampling some isolated features of network function 17 17  11/29/2024 Mdga1-/- mice display increased Inhibitory synapses in the cell body layer Pyramidale Radiatum GABA synthesis enzyme (GAD65) Glutamate transporter (VGlut1) Cell body maker (DAPI) By: Ina Ammendrup-Johnsen Does MDGA1 regulate synapse density in vivo? 18 18  11/29/2024 Inhibitory synapse density is increased specifically in cell body layer of Mdga1-/- mice Pyramidale Radiatum By: Ina Ammendrup-Johnsen Is basal inhibitory transmission increased in Mdga1-/- mice? 19 19  11/29/2024 Functional inhibitory synapses are increased in Mdga1-/- mouse hippocampus Is evoked inhibitory transmission upregulated in Mdga1-/- mouse hippocampus? 20 20  11/29/2024 Evoked perisomatic inhibition is upregulated in Mdga1-/- mouse CA1 Pyramidale Radiatum Is E/I balance shifted within the hippocampus network? 21 21  11/29/2024 Network inhibition is increased in Mdga1-/- hippocampus Does reduction of MDGA1 impair LTP? 22 22  11/29/2024 Level of resolution: Neural Plasticity Altered synaptic plasticity may be harbinger for deficits consistent with brain disorders Provides assessment of how neural networks respond to changes in neural activity associated with cognition Most brain regions are amenable to these tests Caveat: Synaptic plasticity changes can present in the absence of behavioral indicators of neuropathologies 23 23  11/29/2024 Long-term potentiation is reduced in Mdga1-/- mouse hippocampus Early-LTP Are learning and memory impaired in Mdga1-/- mice? 24 24  11/29/2024 Loss of MDGA1 impairs learning and memory By: Tohru Yomamoto Are hippocampal circuits lacking MDGA1 resistant to excitation? 25 25  11/29/2024 Level of resolution: Functional changes indicative of neurological disorders Can apply treatments that allow assessment of susceptibility to neurodevelopmental/neurological disorders Provides assessment of whether transgenic models are resistant (or not) to insults that mimic brain disorders Gene-linkage studies can point us in the right direction Caveat: Mice are not human! May be alternative reasons for phenotypes 26 26  11/29/2024 Reduction of Mdga1 confers resistance to cell excitation Pyramidale Mg2+ +Mg2+ 0Mg2+ 27 27  11/29/2024 Reduction of Mdga1 confers resistance to cell excitation High K+ Low K+ Event Amplitude Event Frequency 28 28  11/29/2024 Summary 29 29  11/29/2024 Conclusions MDGA2 regulates excitatory synapse strength and function, whereas MDGA1 regulates inhibitory synapses Disrupted E/I balance following loss of MDGAs compromises neural circuits involved in synaptic plasticity and cognitive processing Neurodevelopmental disorder phenotypes emerge in mice lacking either isoform of MDGA 30 30  11/29/2024 MDGA2 peak expression coincides with the peak of synaptic development By: Tohru Yomamoto Therefore, reduction of Mdga2 will result in early autism endophenotypes, shifts in E/I balance, and compromised synaptic plasticity. 31 31  11/29/2024 Determine if LTD/synaptic pruning are altered in Mdga2+/- mice Penzes et al., 2011; Nature Neuroscience Brain overgrowth is one of the earliest signs of ASD Postmortem analysis of spine density suggests pruning deficits in ASD Most consistent finding in ASD model mice is dysregulation of LTD WE hypothesize that NMDA receptor-dependent LTD, which plays a significant role in synaptic pruning, is altered during the peak period of synaptic pruning (P21-P44). 32 32  11/29/2024 Determine if LTD/synaptic pruning are altered in Mdga2+/- mice LTD appears to be impaired in Mdga2+/- mice, consistent with an inability to suppress synapse development. 33 33  11/29/2024 Rescuing synaptic function: Determine the molecular mechanisms contributing to altered long-term potentiation Enhanced Early-LTP Impaired L-LTP 34 34  11/29/2024 Future directions: 3A) Targeted reversal of molecular pathologies observed in Mdga2+/- mice 1) Selective reduction of MAGUK family proteins (PSD-95) Cell membrane penetrating peptide Protein binding domain CMA targeting motif Target protein Cell-permeable Protein Lysosome Degradation Fan et al., 2014; Nat Neurosci 35 35  11/29/2024 Future Futuredirections: directions:Targeted reversal 3A) Targeted of molecular reversal pathologies of molecular pathologies observed following reduction +/- observed in Mdga2 mice of MDGA2 3) Small molecule targeted to neuroligin-neurexin binding interface Neuroligin-1 (NL1) Presynaptic Neurexin Presynaptic (Nrx1β) Ca2+ Small molecule Postsynaptic Drug Postsynaptic Interfere with Ca2+ binding as well 36 36  11/29/2024 Final Notes Take home messages: 1) Experience shapes your brain (and, by definition, you) literally! -Precautionary: Be wary of what you expose yourself to. -Prescriptive: Positive, enriching experiences lay the foundation for optimized brain function 2) Take care of your CNS -Sleep, exercise and nutrition are essential for brain health 3) Take care of others -Pro-cognitive effects social interactions, caretaking and externalized focus are well-established 37 37 37  11/29/2024 Final Notes Thank you all for your incredible effort-it was a lot of fun! It was a pleasure getting to know all of you and I wish you the best in your respective journeys. I hope you move forward with a greater appreciation and understanding of how the brain functions, and this knowledge not only enriches your perspective on the mind, but also provides you with a more profound insight into yourselves! 38 38 38  11/29/2024 Fin 39 39 39  11/29/2024 40 40