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 AODs1.</li>
 <li>Two AODs with large apertures (10 mm for a 40x water immersion objective with NA 0.8) deflect the laser beam in two dimensions.</li>
 <li>A reflective diffraction grating with 100 groves/mm is placed 13 cm behind the AODs to compensate the spatial dispersion introduced by AODs when using short laser pulses.</li>
 <li>The laser beam is directed with two relay telescopes into the camera port of an upright microscope.</li>
 <li>Irises are placed at regular intervals for alignment of the optical components.</li>
 <li>A dichroic beamsplitter in front of the objective transmits the infrared excitation light to the specimen and reflects the fluorescence light from the specimen onto a detector.</li>
 <li>Epi- and transfluorescence detectors (photomultipliers, PMTs) collect fluorescence signal through the objective and - if applicable - through the condenser.</li>
 <li>Colored glass filters (BG-39, 3-5 mm) are placed in front of the detectors to prevent excitation light reaching the detectors.</li>
 <li>The AOD deflection angles are controlled by a computer equipped with a digital-analog-converter board (156.25 kHz clock rate), which in turn drives voltage-controlled oscillators.</li>
 <li>The signal from the photomultipliers is relayed through a low-pass Butterworth filter (cut-off frequency of 100 kHz) and digitized by an analog-digital-converter (156.25 kHz clock rate) before being stored in a computer for analysis.</li>
 <li>Alignment and electrical noise are tested by recording the distribution of fluorescence signals with and without laser light, at low and high gain of the photomultipliers, as well as with and without indicator. The scanner is correctly setup and shielded when the width of the distribution of the fluorescence signals at high gain and with indicator is much larger than the widths of the other distributions of fluorescence signals (Figure 2).</li>
</ol>
2. Experimental Procedures
<ol>
 <li>Dithered random-access scanning relies on detection of intracellular increases in calcium. A large number of neurons can be stained using bolus injection of the ester form of a calcium indicator (for example Oregon Green 488 Bapta-1 AM) into neural tissue5.</li>
 <li>Several locations from each neuron soma are recorded, each for a short time ("dithering", 4 locations, 6.4 &mu;sec for each location = 25.6 &mu;sec recording time for each neuron in each cycle, Figure 3C). To select neurons of interest a full frame consisting of 256x256 pixels is acquired (Figure 3A). The center of each neuron soma to be recorded is selected manually within this image. The control software automatically adds three points at 2 &mu;m distance around this center.</li>
 <li>In each cycle, fluorescence signal is recorded from each of the 40 neurons (Figure 3B). This process is repeated for the entire duration of one recording (5 sec recording = 3,255 cycles, 1 cycle = 1.536 msec).</li>
</ol>
3. Online Software Ttools to Maximize Spike Detection Efficiency
<ol>
 <li>Spike detection from fluorescence somatic calcium signals relies on a high signal-to-noise (S/N) ratio of the somatic fluorescence calcium signals. A high S/N can be achieved by increasing excitation intensity. Excitation intensity, however, can only be increased to a certain limit because of photodamage. Spike detection is high within a very small window of excitation intensities only where fluorescence signals have a high S/N but only very little photodamage is observed3. In order to ensure that the recorded signals are within the window of high spike detection during recordings we monitor photon rate (see equation 3.2) and decline of baseline fluorescence using online analysis.</li>
 <li>Approximate photon rate per neuron is calculated from a short time window (100-200 ms) of baseline noise. Number of photons (N&lambda;) and photon rate (&lambda; = N&lambda; / &Delta;t) is calculated from the fluorescence values by fitting the distribution (&sigma;) of relative fluorescence changes with the following equation:</li>
</ol>
<img alt="Equation 1" src="/files/ftp_upload/4052/4052eq1.jpg" /> 
This equation represents the poisson distribution for photon shot noise with a change of variable to relative fluorescence change: &Delta;F/F = (G*N&lambda;(t)-G*N&lambda;,0)/G*N&lambda;,0 where G denotes the cumulative gain of photomultiplier and all other electronic components. Note that this equation does not correctly determine the number of detected photons for in vivo recordings because there are other sources of noise (movement artifacts), in addition to photon shot noise. Nevertheless this equation is useful for in vivo recordings to estimate the noise.
<ol start="3">
 <li>Baseline fluorescence is calculated from the same time window and plotted as a function of time or trials. The average decline of baseline is kept below 0.0002/sec by adjusting laser power because spike detection rapidly declines when exceeding this limit.</li>
 <li>Every 10-20 min the neuron somata positions are verified by again acquiring a full frame image. If required, recording locations are adjusted. Locations can be adjusted for all neurons at once, or for individual neurons.</li>
</ol>
4. Reconstruction of Spike Timings from Fluorescence Signals (Deconvolution)
<ol>
 <li>The fluorescence signals resulting from neural activity often summate in time because the decay of the calcium transients is long (several hundred millisec). A deconvolution method reconstructs spike and spike timings from fluorescence signals.</li>
 <li>To determine the most likely spike train underlying the recorded fluorescence signal, different models are compared. Here we used a genetic algorithm to determine the model - and thus the spike train and spike timings - with the maximum likelihood.</li>
 <li>In inhomogeneous populations of neurons, the spike-evoked calcium signal can vary between neurons. For unsupervised analysis of data sets we designed an algorithm that takes into account the variation of the spike-evoked calcium signal from neuron to neuron.</li>
 <li>To avoid a large number of false positive detections it is useful to constrict the allowed amplitude and decay time constant of the model of the spike-evoked calcium signal. The joint distribution of the amplitude and decay time constant of single-spike evoked calcium transients are recorded in a separate set of experiments from the same type of neurons under the same experimental conditions using simultaneous electrophysiological and optical recordings.</li>
 <li>To account for slow baseline changes and to reduce computational costs of deconvolving, longer recordings are divided into several shorter traces of 1-5 sec.</li>
 <li>For each neuron and each recording, the deconvolution algorithm may test a large number of models (up to 1,000,000 different models or more). To speed up deconvolution, one experiment is deconvolved on up to 10 different computers in parallel.</li>
 <li>After deconvolution, the spike data is analyzed and inspected. A peri-stimulus time histogram, spike probability, and firing rate (average spike per neurons) are calculated in an automated manner.</li>
</ol>
5. Representative Results
Successful spike detection hinges on a high signal-to-noise ratio of the recorded fluorescence somatic calcium signals. Simply using high excitation rates (high laser power) can result in an adverse impact of photoeffects on biological material (photodamage). In dithered random-access scanning photodamage manifests as decreases in baseline fluorescence and decreases of the spike-evoked calcium fluorescence signals. The decrease in the spike-evoked signal can quickly result in a failure to detect spikes. There is only a very small window of excitation intensity where spike detection from fluorescence signals is high. On the higher end this window is limited by photodamage, on the lower end the fluorescence signals have a low signal-to-noise ratio. For cortical neurons in acute slices we use laser power resulting in photon rates of about 400,000-1,500,000 photon/sec when recording at around 100 &mu;m below slice surface. When using a high-affinity indicator - here Oregon Green 488 BAPTA - 1 - this signal is sufficient to detect individual spikes. Figure 3E shows an example of a fluorescence signal recorded at very low excitation rate, one example of a recording within the detection window, and one at very high excitation rate.
Compared to other techniques to record neural activity with single-cell and single-spike resolution, dithered random-access scanning can record from a larger number of neurons from the same, local population, and is less invasive for example compared to tetrode/multielectrode recordings. Thus dithered random-access scanning can be used to record neural activity from many neurons to measure mutual information signaled by suprathreshold activity6 (Figure 4A), changes of neural activity in a population of neurons (cortical plasticity), and propagation of suprathreshold activity through populations of neurons14 (Figure 4B)
<img alt="Figure 1" src="/files/ftp_upload/4052/4052fig1.jpg" /> 
Figure 1. Optical design of the dithered-random access scanning setup.
<img alt="Figure 2" src="/files/ftp_upload/4052/4052fig2.jpg" /> 
Figure 2. Alignment and testing: distributions of fluorescence signals recorded under different conditions. A) No laser light and low photomultiplier gain, B) At higher PMT gain, but no laser light, the distribution is wider because of the photomultiplier dark current. C) With the laser on and recorded at high PMT gain. A difference between the distributions in shown in B and this distribution would indicate that excitation light reaches the PMT detectors. D) The distribution of fluorescence signals recorded at high gain from neuron somata. If no other noise source contributes, this distribution arises from photon shot-noise only.
<img alt="Figure 3" src="/files/ftp_upload/4052/4052fig3.jpg" /> 
Figure 3. A) Full frame fluorescence image to detect and select neuron somata positions, B) Scan path of one cycle, C) Illustration of the dithering principle; in each soma (circle) several locations are recorded before moving the beam to the next soma, D) Illustration of the output of the two D/A channels. For each neuron soma, fluorescence signal is recorded from 4 different spots in each soma (s1-s4). The location of each spot is given by its x and y position. The x and y positions for all spots and all neurons are sent to the digital-to-analog converter in a sequential manner. While the beam is moved between two neuron somata, no signal is acquired (blank). E) Examples of fluorescence signals. Note that each example shows response to one spike (as measured with electrophysiological cell-attached recording).
<img alt="Figure 4" src="/files/ftp_upload/4052/4052fig4.jpg" /> 
Figure 4. Studying cortical function using dithered random-access scanning. A) Measuring mutual information signaled by populations of neurons. Upper image shows photomicrograph of an acute brain slice and two stimulation pipettes placed in the same cortical column in layer 4(L4). Center graphs show neural responses for each repetition of a stimulus. Lower graph shows Shannon's mutual information signaled by the recorded population of neurons. B) Measuring propagation of suprathreshold spiking activity (signal propagation) between populations of cortical neurons. Upper graph illustrates experimental design, center image shows fluorescence image, dashed lines indicate barrel borders, lower graph shows detected spikes in response to electrical stimulation of thalamocortical fibers (triangles).

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+recording+of+neuronal+spiking+activity+from+unbiased+populations+of+neurons+with+high+spike+detection+efficiency+and+high+temporal+precision.">Optical recording of neuronal spiking activity from unbiased populations of neurons with high spike detection efficiency and high temporal precision.</a> J. Neurophysiol. 104, 1812-1824 (2010).</li><li>Grewe, B. F., Langer, D., Kasper, H., Kampa, B. M., Helmchen, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-speed+in+vivo+calcium+imaging+reveals+neuronal+network+activity+with+near-millisecond+precision.">High-speed in vivo calcium imaging reveals neuronal network activity with near-millisecond precision.</a> Nat. 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No conflicts of interest declared.

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

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11.8K Views.  The University of Texas at Austin. 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 ...

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

Surgical Implantation of Chronic Neural Electrodes for Recording Single Unit Activity and Electrocorticographic Signals

The success of long-term electrophysiological recordings often depends on the quality of the implantation surgery. Here we provide useful information for surgeons who are learning the process of implanting electrode systems. We demonstrate the implantation procedure of both a penetrating and a surface electrode. The surgical process is described from start to finish, including detailed descriptions of each step throughout the procedure. It should also be noted that this video guide is focused towards procedures conducted in rodent models and other small animal models. Modifications of the described procedures are feasible for other animal models.

We provide useful information for surgeons who are learning the process of implanting chronic neural recording electrodes. Techniques for both penetrating and surface electrode systems are described in a rodent animal model.

The success of long-term electrophysiological recordings often depends on the quality of the implantation surgery.  Here we provide useful information for ...

Multi-unit Recording Methods to Characterize Neural Activity in the Locust (Schistocerca Americana) Olfactory Circuits

Detection and interpretation of olfactory cues are critical for the survival of many organisms. Remarkably, species across phyla have strikingly similar olfactory systems suggesting that the biological approach to chemical sensing has been optimized over evolutionary time1. In the insect olfactory system, odorants are transduced by olfactory receptor neurons (ORN) in the antenna, which convert chemical stimuli into trains of action potentials. Sensory input from the ORNs is then relayed to the antennal lobe (AL; a structure analogous to the vertebrate olfactory bulb). In the AL, neural representations for odors take the form of spatiotemporal firing patterns distributed across ensembles of principal neurons (PNs; also referred to as projection neurons)2,3. The AL output is subsequently processed by Kenyon cells (KCs) in the downstream mushroom body (MB), a structure associated with olfactory memory and learning4,5. Here, we present electrophysiological recording techniques to monitor odor-evoked neural responses in these olfactory circuits.
First, we present a single sensillum recording method to study odor-evoked responses at the level of populations of ORNs6,7. We discuss the use of saline filled sharpened glass pipettes as electrodes to extracellularly monitor ORN responses. Next, we present a method to extracellularly monitor PN responses using a commercial 16-channel electrode3. A similar approach using a custom-made 8-channel twisted wire tetrode is demonstrated for Kenyon cell recordings8. We provide details of our experimental setup and present representative recording traces for each of these techniques.

Multi-unit Recording Methods to Characterize Neural Activity in the Locust Schistocerca Americana Olfactory Circuits

We demonstrate variations of the extracellular multi-unit recording technique to characterize odor-evoked responses in the first three stages of the invertebrate olfactory pathway. These techniques can easily be adapted to examine ensemble activity in other neural systems as well.

Detection and interpretation of olfactory cues are critical for the survival  of many organisms. Remarkably, species across phyla have strikingly similar ...

Optogenetic Perturbation of Neural Activity with Laser Illumination in Semi-intact Drosophila Larvae in Motion

Drosophila larval locomotion is a splendid model system in developmental and physiological neuroscience, by virtue of the genetic accessibility of the underlying neuronal components in the circuits1-6. Application of optogenetics7,8 in the larval neural circuit allows us to manipulate neuronal activity in spatially and temporally patterned ways9-13. Typically, specimens are broadly illuminated with a mercury lamp or LED, so specificity of the target neurons is controlled by binary gene expression systems such as the Gal4-UAS system14,15. In this work, to improve the spatial resolution to "sub-genetic resolution", we locally illuminated a subset of neurons in the ventral nerve cord using lasers implemented in a conventional confocal microscope. While monitoring the motion of the body wall of the semi-intact larvae, we interactively activated or inhibited neural activity with channelrhodopsin16,17 or halorhodopsin18-20, respectively. By spatially and temporally restricted illumination of the neural tissue, we can manipulate the activity of specific neurons in the circuit at a specific phase of behavior. This method is useful for studying the relationship between the activities of a local neural assembly in the ventral nerve cord and the spatiotemporal pattern of motor output.

Optogenetic Perturbation of Neural Activity with Laser Illumination in Semi-intact Drosophila Larvae in Motion

Here we describe a protocol for optogenetic manipulation of motoneuronal activity while monitoring changes in motor output (muscle contraction) in semi-intact Drosophila larvae using lasers within a conventional confocal microscope. This technique enables researchers to achieve local perturbation of neural activity in a few neuromeres to elucidate the dynamics of motor circuits.

Drosophila larval locomotion is a splendid model system in developmental and physiological neuroscience, by virtue of the genetic accessibility of the ...

Micron-scale Resolution Optical Tomography of Entire Mouse Brains with Confocal Light Sheet Microscopy

Understanding the architecture of mammalian brain at single-cell resolution is one of the key issues of neuroscience. However, mapping neuronal soma and projections throughout the whole brain is still challenging for imaging and data management technologies. Indeed, macroscopic volumes need to be reconstructed with high resolution and contrast in a reasonable time, producing datasets in the TeraByte range. We recently demonstrated an optical method (confocal light sheet microscopy, CLSM) capable of obtaining micron-scale reconstruction of entire mouse brains labeled with enhanced green fluorescent protein (EGFP). Combining light sheet illumination and confocal detection, CLSM allows deep imaging inside macroscopic cleared specimens with high contrast and speed. Here we describe the complete experimental pipeline to obtain comprehensive and human-readable images of entire mouse brains labeled with fluorescent proteins. The clearing and the mounting procedures are described, together with the steps to perform an optical tomography on its whole volume by acquiring many parallel adjacent stacks. We showed the usage of open-source custom-made software tools enabling stitching of the multiple stacks and multi-resolution data navigation. Finally, we illustrated some example of brain maps: the cerebellum from an L7-GFP transgenic mouse, in which all Purkinje cells are selectively labeled, and the whole brain from a thy1-GFP-M mouse, characterized by a random sparse neuronal labeling.

In this article we describe the full experimental procedure to reconstruct, with high resolution, the fine brain anatomy of fluorescently labeled mouse brains. The described protocol includes sample preparation and clearing, specimen mounting for imaging, data post-processing and multi-scale visualization.

Understanding the architecture of mammalian  brain at single-cell resolution is one of the key issues of neuroscience.  However, mapping neuronal soma and ...

Neural Activity Propagation in an Unfolded Hippocampal Preparation with a Penetrating Micro-electrode Array

This protocol describes a method for preparing a new in vitro flat hippocampus preparation combined with a micro-machined array to map neural activity in the hippocampus. The transverse hippocampal slice preparation is the most common tissue preparation to study hippocampus electrophysiology. A longitudinal hippocampal slice was also developed in order to investigate longitudinal connections in the hippocampus. The intact mouse hippocampus can also be maintained in vitro because its thickness allows adequate oxygen diffusion. However, these three preparations do not provide direct access to neural propagation since some of the tissue is either missing or folded. The unfolded intact hippocampus provides both transverse and longitudinal connections in a flat configuration for direct access to the tissue to analyze the full extent of signal propagation in the hippocampus in vitro. In order to effectively monitor the neural activity from the cell layer, a custom made penetrating micro-electrode array (PMEA) was fabricated and applied to the unfolded hippocampus. The PMEA with 64 electrodes of 200 &#181;m in height could record neural activity deep inside the mouse hippocampus. The unique combination of an unfolded hippocampal preparation and the PMEA provides a new in-vitro tool to study the speed and direction of propagation of neural activity in the two-dimensional CA1-CA3 regions of the hippocampus with a high signal to noise ratio.

We have developed an in vitro unfolded hippocampus which preserves CA1-CA3 array of neurons. Combined with the penetrating micro-electrode array, neural activity can be monitored in both the longitudinal and transverse orientations. This method provides advantages over hippocampal slice preparations as the propagation in the entire hippocampus can be recorded simultaneously.

This protocol describes a method for preparing a new in vitro flat hippocampus preparation combined with a micro-machined array to map neural activity in ...

Extracellular Recording of Neuronal Activity Combined with Microiontophoretic Application of Neuroactive Substances in Awake Mice

Differences in the activity of neurotransmitters and neuromodulators, and consequently different neural responses, can be found between anesthetized and awake animals. Therefore, methods allowing the manipulation of synaptic systems in awake animals are required in order to determine the contribution of synaptic inputs to neuronal processing unaffected by anesthetics. Here, we present methodology for the construction of electrodes to simultaneously record extracellular neural activity and release multiple neuroactive substances at the vicinity of the recording sites in awake mice. By combining these procedures, we performed microiontophoretic injections of gabazine to selectively block GABAA receptors in neurons of the inferior colliculus of head-restrained mice. Gabazine successfully modified neural response properties such as the frequency response area and stimulus-specific adaptation. Thus, we demonstrate that our methods are suitable for recording single-unit activity and for dissecting the role of specific neurotransmitter receptors in auditory processing.
The main limitation of the described procedure is the relatively short recording time (~3 hr), which is determined by the level of habituation of the animal to the recording sessions. On the other hand, multiple recording sessions can be performed in the same animal. The advantage of this technique over other experimental procedures used to manipulate the level of neurotransmission or neuromodulation (such as systemic injections or the use of optogenetic models), is that the drug effect is confined to the local synaptic inputs to the target neuron. In addition, the custom-manufacture of electrodes allows adjustment of specific parameters according to the neural structure and type of neuron of interest (such as the tip resistance for improving the signal-to-noise ratio of the recordings).

We present methods for the construction of electrodes to simultaneously record extracellular neural activity and release multiple neuroactive substances at the vicinity of the recording sites in awake mice. This technique allows the detailed analysis of putative local synaptic inputs to the neuron of interest.

Differences in the activity of neurotransmitters and neuromodulators, and consequently different neural responses, can be found between anesthetized and ...

Fabrication of High Contact-Density, Flat-Interface Nerve Electrodes for Recording and Stimulation Applications

Many attempts have been made to manufacture multi-contact nerve cuff electrodes that are safe, robust and reliable for long term neuroprosthetic applications. This protocol describes a fabrication technique of a modified cylindrical nerve cuff electrode to meet these criteria. Minimum computer-aided design and manufacturing (CAD and CAM) skills are necessary to consistently produce cuffs with high precision (contact placement 0.51 &#177; 0.04 mm) and various cuff sizes. The precision in spatially distributing the contacts and the ability to retain a predefined geometry accomplished with this design are two criteria essential to optimize the cuff&#39;s interface for selective recording and stimulation. The presented design also maximizes the flexibility in the longitudinal direction while maintaining sufficient rigidity in the transverse direction to reshape the nerve by using materials with different elasticities. The expansion of the cuff&#39;s cross sectional area as a result of increasing the pressure inside the cuff was observed to be 25% at 67 mm Hg. This test demonstrates the flexibility of the cuff and its response to nerve swelling post-implant. The stability of the contacts&#39; interface and recording quality were also examined with contacts&#39; impedance and signal-to-noise ratio metrics from a chronically implanted cuff (7.5 months), and observed to be 2.55 &#177; 0.25 k&#8486; and 5.10 &#177; 0.81 dB respectively.

This article provides a detailed description on the fabrication process of a high contact-density flat interface nerve electrode (FINE). This electrode is optimized for recording and stimulating neural activity selectively within peripheral nerves.

Many attempts have been made to manufacture multi-contact nerve cuff electrodes that are safe, robust and reliable for long term neuroprosthetic ...

Single-cell Resolution Fluorescence Live Imaging of Drosophila Circadian Clocks in Larval Brain Culture

The circadian pacemaker circuit orchestrates rhythmic behavioral and physiological outputs coordinated with environmental cues, such as day/night cycles. The molecular clock within each pacemaker neuron generates circadian rhythms in gene expression, which underlie the rhythmic neuronal functions essential to the operation of the circuit. Investigation of the properties of the individual molecular oscillators in different subclasses of pacemaker neurons and their interaction with neuronal signaling yields a better understanding of the circadian pacemaker circuit. Here, we present a time-lapse fluorescent microscopy approach developed to monitor the molecular clockwork in clock neurons of cultured Drosophila larval brain. This method allows the multi-day recording of the rhythms of genetically encoded fluorescent circadian reporters at single-cell resolution. This setup can be combined with pharmacological manipulations to closely analyze real-time response of the molecular clock to various compounds. Beyond circadian rhythms, this multipurpose method in combination with powerful Drosophila genetic techniques offers the possibility to study diverse neuronal or molecular processes in live brain tissue.

Single-cell Resolution Fluorescence Live Imaging of Drosophila Circadian Clocks in Larval Brain Culture

The goal of this protocol is to establish ex vivo Drosophila larval brain culture optimized to monitor circadian molecular rhythms with long-term fluorescence time-lapse imaging. The application of this method to pharmacological assays is also discussed.

The circadian pacemaker circuit orchestrates rhythmic behavioral and physiological outputs coordinated with environmental cues, such as day/night cycles. ...

Analyzing Neural Activity and Connectivity Using Intracranial EEG Data with SPM Software

Measuring neural activity and connectivity associated with cognitive functions at high spatial and temporal resolutions is an important goal in cognitive neuroscience. Intracranial electroencephalography (EEG) can directly record electrical neural activity and has the unique potential to accomplish this goal. Traditionally, averaging analysis has been applied to analyze intracranial EEG data; however, several new techniques are available for depicting neural activity and intra- and inter-regional connectivity. Here, we introduce two analytical protocols we recently applied to analyze intracranial EEG data using the Statistical Parametric Mapping (SPM) software: time-frequency SPM analysis for neural activity and dynamic causal modeling of induced responses for intra- and inter-regional connectivity. We report our analysis of intracranial EEG data during the observation of faces as representative results. The results revealed that the inferior occipital gyrus (IOG) showed gamma-band activity at very early stages (110 ms) in response to faces, and both the IOG and amygdala showed rapid intra- and inter-regional connectivity using various types of oscillations. These analytical protocols have the potential to identify the neural mechanisms underlying cognitive functions with high spatial and temporal profiles.

We present two analytical protocols that can be used to analyze intracranial electroencephalography data using the Statistical Parametric Mapping (SPM) software: time-frequency statistical parametric mapping analysis for neural activity, and dynamic causal modeling of induced responses for intra- and inter-regional connectivity.

Measuring neural activity and connectivity associated with cognitive functions at high spatial and temporal resolutions is an important goal in cognitive ...

Wide-field Single-photon Optical Recording in Brain Slices Using Voltage-sensitive Dye

Wide-field single photon voltage-sensitive dye (VSD) imaging of brain slice preparations is a useful tool to assess the functional connectivity in neural circuits. Due to the fractional change in the light signal, it has been difficult to use this method as a quantitative assay. This article describes special optics and slice handling systems, which render this technique stable and reliable. The present article demonstrates the slice handling, staining, and recording of the VSD-stained hippocampal slices in detail. The system maintains physiological conditions for a long time, with good staining, and prevents mechanical movements of the slice during the recordings. Moreover, it enables staining of slices with a small amount of the dye. The optics achieve high numerical aperture at low magnification, which allows recording of the VSD signal at the maximum frame rate of 10 kHz, with 100 pixel x 100-pixel spatial resolution. Due to the high frame rate and spatial resolution, this technique allows application of the post-recording filters that provide sufficient signal-to-noise ratio to assess the changes in neural circuits.

We introduce a reproducible and stable optical recording method for brain slices using voltage-sensitive dye. The article describes voltage-sensitive dye staining and recording of optical signals using conventional hippocampal slice preparations.

Wide-field single photon voltage-sensitive dye (VSD) imaging of brain slice preparations is a useful tool to assess the functional connectivity in neural ...