NSF IOB0718344 and BRAIN EAGER to WJM and Mary Gates Endowment for Students to CD.

All steps described below are in compliance with the animal use and regulation policies of the University of Washington and IACUC.
1. Advanced Preparations
<ol>
	<li>Prepare 5% agar in 100 mL of water and autoclave for 20 min at 120 &#176;C to dissolve the agar. Immediately remove the agar from the autoclave to prevent the solution from solidifying. Portion it into 5-mL petri dishes and cover them with lids. Place the dishes in a refrigerator.</li>
	<li>Make 1 L of artificial cerebrospinal f...

<ol>
	<li>Spitzer, N. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrical+activity+in+early+neuronal+development.">Electrical activity in early neuronal development.</a> Nature. 444 (7120), 707-712 (2006).</li><li>Easton, C. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+elimination+of+GABAergic+neurotransmission+reveals+two+distinct+pacemakers+for+spontaneous+waves+of+activity+in+the+developing+mouse+cortex.">Genetic elimination of GABAergic neurotransmission reveals two distinct pacemakers for spontaneous waves of activity in the developing mouse cortex.</a> J Neurosci. 34 (11), 3854-3863 (2014).</li><li>Adelsberger, H., Garaschuk, O., Konnerth, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cortical+calcium+waves+in+resting+newborn+mice.">Cortical calcium waves in resting newborn mice.</a> Nat Neurosci. 8 (8), 988-990 (2005).</li><li>Easton, C. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+calcium+signals+in+developing+cortical+interneurons+persist+despite+disorganization+of+cortex+by+Tbr1+KO.">Distinct calcium signals in developing cortical interneurons persist despite disorganization of cortex by Tbr1 KO.</a> Dev. Neurobiol. , (2015).</li><li>Bortone, D., Polleux, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=KCC2+Expression+Promotes+the+Termination+of+Cortical+Interneuron+Migration+in+a+Voltage-Sensitive+Calcium-Dependent+Manner.">KCC2 Expression Promotes the Termination of Cortical Interneuron Migration in a Voltage-Sensitive Calcium-Dependent Manner.</a> Neuron. 62 (1), 53-71 (2009).</li><li>Hanganu-Opatz, I. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Between+molecules+and+experience:+role+of+early+patterns+of+coordinated+activity+for+the+development+of+cortical+maps+and+sensory+abilities.">Between molecules and experience: role of early patterns of coordinated activity for the development of cortical maps and sensory abilities.</a> Brain Res. Rev. 64 (1), 160-176 (2010).</li><li>Lischalk, J. W., Easton, C. R., Moody, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bilaterally+propagating+waves+of+spontaneous+activity+arising+from+discrete+pacemakers+in+the+neonatal+mouse+cerebral+cortex.">Bilaterally propagating waves of spontaneous activity arising from discrete pacemakers in the neonatal mouse cerebral cortex.</a> Dev. Neurobiol. 69 (7), 407-414 (2009).</li><li>Barger, Z., Easton, C. R., Neuzil, K. E., Moody, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+network+activity+propagates+bidirectionally+between+hippocampus+and+cortex.">Early network activity propagates bidirectionally between hippocampus and cortex.</a> Dev. Neurobiol. 76 (6), 661-672 (2016).</li><li>Namiki, S., Norimoto, H., Kobayashi, C., Nakatani, K., Matsuki, N., Ikegaya, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Layer+III+Neurons+Control+Synchronized+Waves+in+the+Immature+Cerebral+Cortex.">Layer III Neurons Control Synchronized Waves in the Immature Cerebral Cortex.</a> J. Neurosci. 33 (3), 987-1001 (2013).</li><li>Conhaim, J., Easton, C. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+changes+in+propagation+patterns+and+transmitter+dependence+of+waves+of+spontaneous+activity+in+the+mouse+cerebral+cortex.">Developmental changes in propagation patterns and transmitter dependence of waves of spontaneous activity in the mouse cerebral cortex.</a> J. Physiol. 589 (Pt 10), 2529-2541 (2011).</li><li>Elias, L., Kriegstein, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organotypic+slice+culture+of+E18+rat+brains.">Organotypic slice culture of E18 rat brains.</a> J Vis Exp. (6), e235 (2007).</li><li>McCabe, A. K., Chisholm, S. L., Picken-Bahrey, H. L., Moody, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+self-regulating+nature+of+spontaneous+synchronized+activity+in+developing+mouse+cortical+neurones.">The self-regulating nature of spontaneous synchronized activity in developing mouse cortical neurones.</a> J Physiol. 577 (Pt 1), 155-167 (2006).</li><li>Ting, J. T., Daigle, T. L., Chen, Q., Feng, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+brain+slice+methods+for+adult+and+aging+animals:+application+of+targeted+patch+clamp+analysis+and+optogenetics.">Acute brain slice methods for adult and aging animals: application of targeted patch clamp analysis and optogenetics.</a> Methods Mol Biol. 1183, 221-242 (2014).</li><li>Buskila, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extending+the+viability+of+acute+brain+slices.">Extending the viability of acute brain slices.</a> Sci Rep. 4, 12-20 (2014).</li><li>Barnett, H. M., Gjorgjieva, J., Weir, K., Comfort, C., Fairhall, A. L., Moody, W. 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The combination of confocal imaging of fluorescent proteins to label individual cells with widefield epifluorescent calcium imaging uses important features of each technique. Epifluorescence imaging provides several benefits over other fluorescence imaging methods. Compared to confocal imaging, it allows for fast image acquisition at low light levels, as removal of out-of-focus light reduces the overall fluorescence signal. The use of high signal-to-noise fluorescent indicators (such as Fluo4 AM) allows for the resolutio...

Spontaneous physiological activity regulates brain development via the actions of calcium, which serves as a second messenger to affect processes such as neurogenesis and cellular migration1. Physiological activity may elicit changes in cellular calcium levels in different ways. For example, during the first postnatal week of mouse development, neurons of the cerebral cortex exhibit spontaneous action potential firing, which propagates as a wave through a large subset of cortical neurons2,3. Calcium influx occurs through voltage and ligand-gated ion channels and triggers further calcium release from internal stores. At the same time, neurons of the same network may exhibit other types of calcium activity. For example, individual neurons may exhibit calcium spikes, which are not synchronous across multiple cells and are driven largely by L-type calcium channels4,5. Such channels may be activated by subthreshold spontaneous depolarizations resultant from stochastic ion channel opening or ligand binding. Different types of calcium signaling may elicit distinct effects on development. Waves have been proposed to play roles in patterning cortical connections4,6, while L-type calcium channels are important for neuronal migration5.
Calcium imaging techniques are used to describe the activity of calcium in vivo or in brain slice preparations. Imaging calcium dynamics rather than electrical signals in development has several benefits. Measurements of the properties of the calcium signal, such as the frequency and duration of calcium events or the correlation of activity in one cell to the activity of another cell, inform experiments that examine how calcium activity affects development. Importantly, calcium imaging allows for the simultaneous measurement of the activity of the hundreds of cells in a neural network. Additionally, calcium spikes may be used as a proxy for electrical activity, as population calcium events in the developing neocortex correlate 1:1 with simultaneously recorded local field potentials2. Brain slices have been useful for examining spontaneous activity in cortical development, although synchronous wave activity can be difficult to record in slices due to its dependence on exceptional slice health. Nonetheless, calcium imaging in brain slice studies has shown that cortical waves initiate in discrete pacemaker regions7,8,9 and are driven by two neurotransmitter systems2,10. Recent technical innovations have allowed the measurement of calcium dynamics during wave activity in individual genetically-labeled inhibitory interneurons4.
The preparation described here is similar to that of Elias and Kriegstein, 200711. However, the recording of physiological activity from individual cells requires increased attention to slice health during the culturing and imaging process. These methods allow for activity generation with more physiological levels of potassium and magnesium than are often used in experiments on brain slices. Increasing potassium may cause regions of slices that do not normally initiate wave activity to become pacemakers and can change the duration and number of wave events, in part by increasing the participation of NMDA(n-methyl-D-aspartate)-type glutamate receptors. While the anatomy of the slice may change in culture over time, short-term cultures preserve slice anatomy while allowing for the recovery of slice viability. The addition of antibiotics to the culture medium could be predicted to affect the network, but is had previously been shown that ion channel properties develop normally in culture12 and that inhibitory interneurons terminate their migration in culture on a normal timeline4. While special cutting solutions13 and recovery procedures14 may be used to improve the viability of acute slices, short-term cultures provide the best opportunity for cell recovery after slicing, allowing for the clearance of dead tissue at the surface of the slice, which might impair the imaging of deeper, healthier cells. Cultured slices are necessary to study early postnatal activity in the cortex of genetic mutants that do not survive past birth4,2. Chronic pharmacological manipulations can be conducted to study the effects of activity on network development12.
This work also describes methods of calcium imaging in organotypic brain slices. Brain slices have been used to measure population activity propagating across large regions of the cortex2,8,10, as well as single-cell activity when higher microscope magnifications are used4,9. Propagation patterns are measured with low-magnification microscope objectives, whereas single-cell activity is measured with higher-powered objectives. The methods presented here focus on higher-magnification imaging experiments that use spinning-disk confocal microscopy for the acquisition of confocal images of transgenically labeled cell populations, as well as and wide-field epifluorescence imaging of calcium activity. This system allows for the fast acquisition of calcium recordings with a high signal-to-noise ratio and a low bleaching rate, and it is more cost effective than laser-scanning confocal systems. These techniques allow for the analysis of cellular-level studies of the mechanisms and functions of spontaneous physiological activity in development by enabling the simultaneous measurement of calcium transients in genetically labeled and non-labeled neuronal subpopulations.

<table border="0" cellpadding="0" cellspacing="0" width="669">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Inverted Microscope</td>
			<td style="width:129px;">Olympus</td>
			<td style="width:157px;">IX81</td>
			<td style="width:167px;">With DSU&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Camera</td>
			<td style="width:129px;">Hamamatsu</td>
			<td>Orca-flash2.8 CMOS</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Light Source</td>
			<td style="width:129px;">Sutter</td>
			<td style="width:157px;">Lambda XL</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">40x Objective</td>
			<td style="width:129px;">Olympus</td>
			<td style="width:157px;">Uapo N 340 40x water</td>
			<td>NA 1.15, WD 0.25mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Calcium indicator - Fluo-4 AM</td>
			<td style="width:129px;">ThermoFisher</td>
			<td style="width:157px;">F14201</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Cre dependent tdTomato mouse</td>
			<td style="width:129px;">Jackson Labs</td>
			<td>007914&#160;</td>
			<td>Ai14 mouse from Allen Institute for Brain Science</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dlx5/6 Cre mice&#160;</td>
			<td style="width:129px;">Jackson Labs</td>
			<td>008199&#160;</td>
			<td>Labels inhibitory interneurons. Use a different Cre driver to label different cell types</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sterilizing Filter</td>
			<td style="width:129px;">Corning</td>
			<td>09-761-112</td>
			<td>500mL, 0.22 micron bottle-top filter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Millicell Cell Culture Inserts</td>
			<td style="width:129px;">EMD Millipore</td>
			<td>PICM0RG50</td>
			<td>30mm diameter, 0.4 micron</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:216px;">slice resting chamber</td>
			<td style="width:129px;">Warner Instruments</td>
			<td style="width:157px;">BSC-PC</td>
			<td>Prechamber meant to rest slices before imaging</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Neorbasal-A Medium</td>
			<td style="width:129px;">ThermoFisher</td>
			<td style="width:157px;">10888022</td>
			<td>cell culture basal medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">NIS Elements AR</td>
			<td style="width:129px;">Nikon</td>
			<td style="width:157px;"></td>
			<td>image acquisition software</td>
		</tr>
	</tbody>
</table>

measuring the calcium dynamics of individual, genetically-labeled neurons of the developing mouse neocortex

Spontaneous activity in the developing mammalian cortex is necessary for proper network formation. Such activity may be intrinsic to individual cells or driven by network interactions, and different types of activity may affect distinct components of development. A striking feature of cortical development is the propagating waves of activity that cause simultaneous action potential firing in neurons across broad cortical regions. Waves have been proposed to play roles in patterning connections, such as those between the cortex and thalamus, as well as in placing inhibitory interneurons into the correct cortical layers. Calcium signaling induced by waves is likely to mediate these effects on development. Calcium imaging techniques in brain slice preparations may be used to visualize wave activity propagating between brain structures and to examine the contribution of individual cells to population activity. Slices have an advantage over dissociated cultures because of the ability to examine cellular activity in a setting with preserved network features, such as cortical layering. However, slice preparation for the physiological examination of developing cells can be difficult. The slicing process reduces network connectivity and injures cells. High potassium ringer solutions are often necessary to produce the synchronous activity that is normally present in vivo. This work describes a set of methods for brain slice preparation that allow for the measurement of the physiological patterns of synchronous activity without increasing potassium by using short-term organotypic slice cultures to increase cell health. Methods to identify genetically-labeled neuronal subpopulations in the cortical plate of these slices while conducting calcium imaging of heterogeneous neurons in the cortical network are presented. An overview of the slice preparation and imaging techniques of the developing cortex, which are useful for assaying both single-cell and population-level activity patterns, are presented. These methods may be adapted to many different neuronal subtypes and anatomical regions.

Figure 1 shows components of the raw data that may be used to determine the activity of individual, transgenically labeled cells. To measure activity, regions of interest (ROIs) are drawn onto cell bodies in a confocal image of RFP-labeled cells (Figure 1A). These ROIs are then applied to the calcium record associated with the confocal image. In Figure 1B shows a single image from the calcium record ...

This protocol describes brain preparation and calcium imaging procedures for the measurement of calcium dynamics in heterogeneous cortical networks with neuronal subtypes that are genetically-labeled with red fluorescent protein.

Measuring the Calcium Dynamics of Individual, Genetically-labeled Neurons of the Developing Mouse Neocortex

Spontaneous activity in the developing mammalian cortex is necessary for proper network formation. Such activity may be intrinsic to individual cells or ...

Measuring the Calcium Dynamics of Individual, Genetically-labeled Neurons of the Developing Mouse Neocortex

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