We thank Dr. Randy Chitwood for critically reading the manuscript. This work was supported by the Whitehall Foundation and the Alfred P. Sloan Foundation grants to HJK.

1. Optical Setup (Figure 1)
<ol>
 <li>For two-photon excitation an infrared pulsed laser system with femtosecond pulses is used. A high laser output power (in some cases &gt;2W at 890 nm wavelength) is required to offset the large losses introduced by the optical components of the system.</li>
 <li>A prechirper system consisting of two prisms imparts a negative group velocity dispersion (GVD) onto the laser pulses prior to the acousto-optical deflectors (AODs) to compensate for the temporal dispersion introduced by the...

<ol>
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Methods. 7, 399-405 (2010).</li><li>Stosiek, C., Garaschuk, O., Holthoff, K., 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=In+vivo+two-photon+calcium+imaging+of+neuronal+networks.">In vivo two-photon calcium imaging of neuronal networks.</a> Proc. Natl. Acad. Sci. U.S.A. 100, 7319-7324 (2003).</li><li>Pita-Almenar, J. D., Ranganathan, G. N., Koester, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+cortical+plasticity+on+information+signaled+by+populations+of+neurons+in+the+cerebral+cortex.">Impact of cortical plasticity on information signaled by populations of neurons in the cerebral cortex.</a> J. Neurophysiol. 106, 1118-1124 (2011).</li><li>Kerr, J. 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Dithered random-access scanning indirectly detects suprathreshold spiking activity from the increases in intracellular somatic calcium associated with each spike in a neuron somata. The increases in intracellular calcium are detected by fluorescent calcium dyes. The limitations of dithered random-access scanning arise largely from of the limited signal-to-noise ratio of the calcium fluorescence signals. The signal-to-noise ratio is in turn limited by photodamage, which does not allow using high excitation rates. Because ...

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments (optional)</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Optical components are listed in order, starting from the laser</td>
		</tr>
		<tr>
			<td>Titan:Sapphire Laser</td>
			<td>Coherent Inc.</td>
			<td>Chameleon Ultra 2</td>
			<td>High power output recommended (&gt;2W at 900 nm)</td>
		</tr>
		<tr>
			<td>Achromatic lens f = 30 mm</td>
			<td>Thor labs</td>
			<td>AC254-030-B</td>
			<td>Anti-reflection (AR) coating for 650-1050 nm</td>
		</tr>
		<tr>
			<td>Achromatic lens f = 100 mm</td>
			<td>Thor labs</td>
			<td>AC254-100-B</td>
			<td>AR 650-1050 nm</td>
		</tr>
		<tr>
			<td>lens f = 75 mm</td>
			<td>Thor labs</td>
			<td>LA1608-B</td>
			<td>AR 650-1050 nm</td>
		</tr>
		<tr>
			<td>lens f = 175 mm</td>
			<td>Thor labs</td>
			<td>LA1229-B</td>
			<td>AR 650-1050 nm</td>
		</tr>
		<tr>
			<td>Achromatic lens f = 300 mm</td>
			<td>Thor labs</td>
			<td>AC254-300-B</td>
			<td>AR 650-1050 nm</td>
		</tr>
		<tr>
			<td>Achromatic lens f = 100 mm</td>
			<td>Thor labs</td>
			<td>AC254-100-B</td>
			<td>AR 650-1050 nm</td>
		</tr>
		<tr>
			<td>Achromatic lens f = 100 mm</td>
			<td>Thor labs</td>
			<td>AC254-100-B</td>
			<td>AR 650-1050 nm</td>
		</tr>
		<tr>
			<td>Acousto-optical deflectors</td>
			<td>Intraaction Corp</td>
			<td>ATD 6510CD2</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Reflective diffraction grating</td>
			<td>Newport</td>
			<td>53-011R</td>
			<td>100 grooves/mm for AODs with 65 MHz bandwidth and scan angle of 45 mrad</td>
		</tr>
		<tr>
			<td>21.6 mm Brewster prisms</td>
			<td>Lambda Research Optics Inc.</td>
			<td>IBP21.6SF10</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Colored Glass</td>
			<td>Schott</td>
			<td>BG-39</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Dichroic mirror</td>
			<td>Chroma Technology Corp</td>
			<td>Z532RDC</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Photomultiplier modules</td>
			<td>Hamamatsu</td>
			<td>H9305-03</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>DAC-ADC board</td>
			<td>National Instruments</td>
			<td>PCI-6115</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Oregon Green 488 Bapta-1 AM</td>
			<td>Invitrogen</td>
			<td>O-6807</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

optical recording of suprathreshold neural activity with single-cell and single-spike resolution

Signaling of information in the vertebrate central nervous system is often carried by populations of neurons rather than individual neurons. Also propagation of suprathreshold spiking activity involves populations of neurons. Empirical studies addressing cortical function directly thus require recordings from populations of neurons with high resolution. Here we describe an optical method and a deconvolution algorithm to record neural activity from up to 100 neurons with single-cell and single-spike resolution. This method relies on detection of the transient increases in intracellular somatic calcium concentration associated with suprathreshold electrical spikes (action potentials) in cortical neurons. High temporal resolution of the optical recordings is achieved by a fast random-access scanning technique using acousto-optical deflectors (AODs)1. Two-photon excitation of the calcium-sensitive dye results in high spatial resolution in opaque brain tissue2. Reconstruction of spikes from the fluorescence calcium recordings is achieved by a maximum-likelihood method. Simultaneous electrophysiological and optical recordings indicate that our method reliably detects spikes (&gt;97% spike detection efficiency), has a low rate of false positive spike detection (&lt; 0.003 spikes/sec), and a high temporal precision (about 3 msec) 3. This optical method of spike detection can be used to record neural activity in vitro and in anesthetized animals in vivo3,4.

Watch this Scientific Journal Video about Optical Recording of Suprathreshold Neural Activity with Single-cell and Single-spike Resolution at JoVE.com

Optical Recording of Suprathreshold Neural Activity with Single-cell and Single-spike Resolution

Understanding the function of the vertebrate central nervous system requires recordings from many neurons because cortical function arises on the level of populations of neurons. Here we describe an optical method to record suprathreshold neural activity with single-cell and single-spike resolution, dithered random-access scanning. This method records somatic fluorescence calcium signals from up to 100 neurons with high temporal resolution. A maximum-likelihood algorithm deconvolves the underlying suprathreshold neural activity from the somatic fluorescence calcium signals. This method reliably detects spikes with high detection efficiency and a low rate of false positives and can be used to study neural populations in vitro and in vivo.

Signaling of information in the vertebrate central nervous system is often carried by populations of neurons rather than individual neurons. Also ...

optical-recording-suprathreshold-neural-activity-with-single-cell

Research

JoVE Journal

Neuroscience

11.8K visualizações.  The University of Texas at Austin. A compreensão da função do sistema nervoso central, dos vertebrados requer gravações de muitos neurónios porque a função cortical surge no nível de populações de neurónios. Aqui nós descrevemos um método óptico para registrar a atividade neural supraliminares com resolução de uma única célula e um único ponto, pontilhado de acesso aleatório de digitalização. Este método registra somáticas fluorescência sinais de cálcio de até 100 neurônios com...