This research was supported by grants from the German Research Foundation (DFG) to MCS (FOR 1581, STU 544/1-1) and OG (FOR 1581, SFB 874). The website of the DFG is http://www.dfg.de/en/index.jsp. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.The authors thank Thomas Seidenbecher for providing us with the gold-plating protocol as well as Tobias Otto for help with setting up the electrophysiological recording equipment.

<ol>
	<li>Bingman, V. P., Gasser, B. A., Colombo, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Responses+of+pigeon+(Columba+livia)+wulst+neurons+during+acquisition+and+reversal+of+a+visual+discrimination+task.">Responses of pigeon (Columba livia) wulst neurons during acquisition and reversal of a visual discrimination task.</a> Behav Neurosci. 122, 1139-1147 (2008).</li><li>Herry, C., Ciocchi, S., Senn, V., Demmou, L., M&#252;ller, C., L&#252;thi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Switching+on+and+off+fear+by+distinct+neuronal+circuits.">Switching on and off fear by distinct neuronal circuits.</a> Nature. 454, 600-606 (2008).</li><li>Quirk, G. J., Repa, C., LeDoux, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fear+conditioning+enhances+short-latency+auditory+responses+of+lateral+amygdala+neurons:+parallel+recordings+in+the+freely+behaving+rat.">Fear conditioning enhances short-latency auditory responses of lateral amygdala neurons: parallel recordings in the freely behaving rat.</a> Neuron. 15, 1029-1039 (1995).</li><li>Quirk, G. J., Armony, J. L., Ledoux, J. 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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=Neurons+in+medial+prefrontal+cortex+signal+memory+for+fear+extinction.">Neurons in medial prefrontal cortex signal memory for fear extinction.</a> Nature. 420, 713-717 (2002).</li><li>Hobin, J. A., Goosens, K. A., Maren, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Context-dependent+neuronal+activity+in+the+lateral+amygdala+represents+fear+memories+after+extinction.">Context-dependent neuronal activity in the lateral amygdala represents fear memories after extinction.</a> J Neurosci. 23, 8410-8416 (2003).</li><li>Maren, S., Hobin, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hippocampal+regulation+of+context-dependent+neuronal+activity+in+the+lateral+amygdala.">Hippocampal regulation of context-dependent neuronal activity in the lateral amygdala.</a> Learn Mem. 14, 318-324 (2007).</li><li>Knapska, E., 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=Differential+involvement+of+the+central+amygdala+in+appetitive+versus+aversive+learning.">Differential involvement of the central amygdala in appetitive versus aversive learning.</a> Learn Mem. 13, 192-200 (2006).</li><li>Harloe, J. P., Thorpe, A. J., Lichtman, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+endocannabinoid+regulation+of+extinction+in+appetitive+and+aversive+Barnes+maze+tasks.">Differential endocannabinoid regulation of extinction in appetitive and aversive Barnes maze tasks.</a> Learn Mem. 15, 806-809 (2008).</li><li>Gray, C. M., Maldonado, P. E., Wilson, M., McNaughton, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tetrodes+markedly+improve+the+reliability+and+yield+of+multiple+single-unit+isolation+from+multi-unit+recordings+in+cat+striate+cortex.">Tetrodes markedly improve the reliability and yield of multiple single-unit isolation from multi-unit recordings in cat striate cortex.</a> J Neurosci Methods. 63, 43-54 (1995).</li><li>Lewicki, M. 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The authors have nothing to disclose.

This protocol describes a complex behavioral task suitable for concurrent single-unit recordings. We have described the SIFC task for pigeons, but it can be easily adapted to rodents&#160;by requiring nose pokes or lever pressing rather than key pecks, and substituting visual by olfactory, auditory, or tactile stimuli.
Perhaps the most critical steps during the training procedure are 1) gradual reduction of reward probability and 2) increase in trial number. Regarding intermittent reinforcemen...

Learning new stimulus-response-outcome associations engages a wide range of neural plasticity processes. These processes are ultimately reflected in the changing spike output of individual neurons. Arguably, one of the most frequently employed learning paradigms is Pavlovian fear conditioning conducted with rodents. In this setting, the acquisition and extinction of a conditioned response take place within a few dozen trials2. The rapid development of conditioned fear can be advantageous because it allows running a large number of animals within a short time. Also, acquisition and extinction can be observed within a few tens of trials on a single day in naive animals3,4 or spread across 2&#160;to 3&#160;days2,5-8. However, the insights gained about the changes of neural activity during learning in these experiments do not necessarily apply outside the domain of fear conditioning. For example, goal-directed behavior driven by positive reinforcement is more adequately modeled by operant rather than Pavlovian conditioning procedures, and may in part depend on different neural substrates9,10. Also, fear conditioning develops so rapidly that neural responses to the CS can only be observed for a few dozen trials, placing severe limits on the analysis of changes of neural activity during learning.
Unfortunately, the acquisition and extinction of operant responding usually takes many days. This is detrimental for neurophysiological investigations, because it is notoriously difficult to record the activity of single cells over more than a few hours. Due to the high similarity of the waveforms of extracellularly recorded action potentials, it is problematic to claim that spikes recorded on one day are generated from the same cell as spikes with similar waveforms recorded on the next11,12, especially in areas with a high cell density such as the hippocampus.
To address these issues, we developed a novel behavioral paradigm utilizing 3&#160;learning conditions within one experimental session on a single day. This requires that the experimental animal is willing to perform hundreds of trials under varying conditions on a thin schedule of reinforcement. Homing pigeons (Columbia livia forma domestica) are classic model organisms in experimental psychology13-17. These birds are able to perform complex visual discriminations18, can flexibly adapt behavior to changing reinforcement contingencies19,20, and are uniquely avid workers, performing 1,000 trials with minimal amount of reinforcement. These characteristics make them especially suitable for the experiments described below.

<table border="0" cellpadding="0" cellspacing="0" style="width:1379px;" width="1377">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">Name of Material/ Equipment</td>
			<td style="width:305px;">Company</td>
			<td style="width:325px;">Catalog Number</td>
			<td style="width:471px;">Comments/Description</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:277px;">Resistance wire (for use as electrodes)</td>
			<td style="width:305px;">California Fine Wire, Grover Beach (CA), USA</td>
			<td></td>
			<td>Stablohm 675; formvar-coated nichrome wires (outer diameter 25 &#181;m)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:277px;">Microconnectors</td>
			<td style="width:305px;">Ginder Scientific, Nepean, Ontario, Canada</td>
			<td style="width:325px;"></td>
			<td>GS18PLG-220 (plug) &#38; GS18SKT-220 (socket to build headstage)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:277px;">Cannulae</td>
			<td style="width:305px;">Henke Sass Wolf, Tuttlingen, Germany</td>
			<td style="width:325px;"></td>
			<td>0.4x20mm/ 27Gx3/4&#34;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:277px;">Gold solution for plating</td>
			<td style="width:305px;">Neuralynx, Bozeman (MT), USA</td>
			<td style="width:325px;"></td>
			<td>SIFCO Process Gold Non-Cyanide, Code 5355</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:277px;">Solution for ultrasonic bath</td>
			<td style="width:305px;">Alconox, Inc., New York, USA</td>
			<td style="width:325px;">1304</td>
			<td>Tergazyme</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:277px;">Conductive glue</td>
			<td style="width:305px;">Henkel Loctite</td>
			<td></td>
			<td>LOCTITE 3888 Silver filled, conductive, adhesive</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:277px;">Stainless steel screws</td>
			<td style="width:305px;">J.I. Morris, Southbridge (MA), USA</td>
			<td style="width:325px;"></td>
			<td>F0CE125 self-tapping miniature screws, body length 1/8 inches</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:277px;">Light-curing dental cement</td>
			<td style="width:305px;">van der Ven Dental, Duisburg, Germany</td>
			<td style="width:325px;"></td>
			<td>Omniceram Evo Flow A2</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:277px;">Light-curing unit</td>
			<td style="width:305px;">van der Ven Dental, Duisburg, Germany</td>
			<td style="width:325px;"></td>
			<td>Jovident Excelled 215 Curing Light (wireless LED light curing unit)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:277px;">Filter amplifiers</td>
			<td style="width:305px;">npi electronic GmbH, Germany</td>
			<td>DPA-2FS</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:277px;">A/D converter</td>
			<td style="width:305px;">Cambridge Electronic Design, Cambridge, UK</td>
			<td>power 1401</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:277px;">Spike2 software</td>
			<td style="width:305px;">Cambridge Electronic Design, Cambridge, UK</td>
			<td style="width:325px;"></td>
			<td>Version 7.06a</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">Matlab</td>
			<td style="width:305px;">The Mathworks, Natick (MA), USA</td>
			<td style="width:325px;"></td>
			<td>R2012a</td>
		</tr>
	</tbody>
</table>

recording single neurons' action potentials from freely moving pigeons across three stages of learning

While the subject of learning has attracted immense interest from both behavioral and neural scientists, only relatively few investigators have observed single-neuron activity while animals are acquiring an operantly conditioned response, or when that response is extinguished. But even in these cases, observation periods usually encompass only a single stage of learning, i.e. acquisition or extinction, but not both (exceptions include protocols employing reversal learning; see Bingman&#160;et al.1 for an example). However, acquisition and extinction entail different learning mechanisms and are therefore expected to be accompanied by different types and/or loci of neural plasticity.
Accordingly, we developed a behavioral paradigm which institutes three stages of learning in a single behavioral session and which is well suited for the simultaneous recording of single neurons&#39; action potentials. Animals are trained on a single-interval forced choice task which requires mapping each of two possible choice responses to the presentation of different novel visual stimuli (acquisition). After having reached a predefined performance criterion, one of the two choice responses is no longer reinforced (extinction). Following a certain decrement in performance level, correct responses are reinforced again (reacquisition). By using a new set of stimuli in every session, animals can undergo the acquisition-extinction-reacquisition process repeatedly. Because all three stages of learning occur in a single behavioral session, the paradigm is ideal for the simultaneous observation of the spiking output of multiple single neurons. We use pigeons as model systems, but the task can easily be adapted to any other species capable of conditioned discrimination learning.

Behavior
Figure 4A shows the behavioral performance of an animal in one example session. The performance level of the animal reaches criterion for NS 2 within 180 trials (45 stimulus presentations) and is close to 100% for the NS 1 from the beginning. This strategy - first responding to the same key for both new stimuli, and then adjusting responses for one of the stimuli - is about as often observed as initial random responding to both NS. In this session, the NS 2 was randomly ...

Watch this Scientific Journal Video about Recording Single Neurons' Action Potentials from Freely Moving Pigeons Across Three Stages of Learning at JoVE.com

Recording Single Neurons&#039; Action Potentials from Freely Moving Pigeons Across Three Stages of Learning

Learning new stimulus-response associations engages a wide range of neural processes which are ultimately reflected in changing spike output of individual neurons. Here we describe a behavioral protocol allowing for the continuous registration of single-neuron activity while animals acquire, extinguish, and reacquire a conditioned response within a single experimental session.

Recording Single Neurons' Action Potentials from Freely Moving Pigeons Across Three Stages of Learning

While the subject of learning has attracted immense interest from both behavioral and neural scientists, only relatively few investigators have observed ...

recording-single-neurons-action-potentials-from-freely-moving-pigeons

Research

JoVE Journal

Neuroscience

11.9K Views.  Ruhr-University Bochum. Learning new stimulus-response associations engages a wide range of neural processes which are ultimately reflected in changing spike output of individual neurons. Here we describe a behavioral protocol allowing for the continuous registration of single-neuron activity while animals acquire, extinguish, and reacquire a conditioned response within a single experimental session.

Video: Recording Single Neurons' Action Potentials from Freely Moving Pigeons Across Three Stages of Learning

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from individual neurons are critical for understanding how neural circuits function under natural conditions. Traditionally, these recordings have been performed 'blind', meaning the identity of the recorded cell is unknown at the start of the recording. Cellular identity can be subsequently determined via intracellular1, juxtacellular2 or loose-patch3 iontophoresis of dye, but these recordings cannot be pre-targeted to specific neurons in regions with functionally heterogeneous cell types. Fluorescent proteins can be expressed in a cell-type specific manner permitting visually-guided single-cell electrophysiology4-6. However, there are many model systems for which these genetic tools are not available. Even in genetically accessible model systems, the desired promoter may be unknown or genetically homogenous neurons may have varying projection patterns. Similarly, viral vectors have been used to label specific subgroups of projection neurons7, but use of this method is limited by toxicity and lack of trans-synaptic specificity. Thus, additional techniques that offer specific pre-visualization to record from identified single neurons in vivo are needed. Pre-visualization of the target neuron is particularly useful for challenging recording conditions, for which classical single-cell recordings are often prohibitively difficult8-11. The novel technique described in this paper uses retrograde transport of a fluorescent dye applied using tungsten needles to rapidly and selectively label a specific subset of cells within a particular brain region based on their unique axonal projections, thereby providing a visual cue to obtain targeted electrophysiological recordings from identified neurons in an intact circuit within a vertebrate CNS.
The most significant novel advancement of our method is the use of fluorescent labeling to target specific cell types in a non-genetically accessible model system. Weakly electric fish are an excellent model system for studying neural circuits in awake, behaving animals12. We utilized this technique to study sensory processing by "small cells" in the anterior exterolateral nucleus (ELa) of weakly electric mormyrid fish. "Small cells" are hypothesized to be time comparator neurons important for detecting submillisecond differences in the arrival times of presynaptic spikes13. However, anatomical features such as dense myelin, engulfing synapses, and small cell bodies have made it extremely difficult to record from these cells using traditional methods11, 14. Here we demonstrate that our novel method selectively labels these cells in 28% of preparations, allowing for reliable, robust recordings and characterization of responses to electrosensory stimulation.

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

Retrograde transport of fluorescent dye labels a sub-population of neurons based on anatomical projection. Labeled axons can be visually targeted in vivo, permitting extracellular recording from identified axons. This technique facilitates recording when neurons cannot be labeled through genetic manipulation or are difficult to isolate using 'blind' in vivo approaches.

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from ...

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.

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 ...

Recording EEG in Freely Moving Neonatal Rats Using a Novel Method

EEG is a useful method to detect electrical activity in the brain. Moreover, it is a widely used diagnostic tool for various neurological conditions, such as epilepsy and neurodegenerative disorders. However, it is technically difficult to obtain EEG recordings in neonates as it requires specialized handling and great care. Here, we present a novel method to record EEG in neonatal rat pups (P8-P15). We designed a simple and reliable electrode using computer pin loci; it can be easily implanted into the skull of a rat pup to record high-quality EEG signals in the normal and epileptic brain. Pups were given an intraperitoneal (i.p.) injection of the neurotoxin kainic acid (KA) to induce epileptic seizures. The surgical implantation performed in this procedure is less expensive than other EEG procedures for neonates. This method allows one to record high-quality and stable EEG signals for more than 1 week. Furthermore, this procedure can also be applied to adult rats and mice to study epilepsy or other neurological disorders.

Here, we introduce a novel technique designed to record electroencephalography (EEG) in freely moving neonatal epileptic pups and describe its procedures, features, and applications. This method allows one to record EEG for more than 1 week.

EEG is a useful method to detect electrical activity in the brain. Moreover, it is a widely used diagnostic tool for various neurological conditions, such ...

Simultaneous Recordings of Cortical Local Field Potentials, Electrocardiogram, Electromyogram, and Breathing Rhythm from a Freely Moving Rat

Monitoring the physiological dynamics of the brain and peripheral tissues is necessary for addressing a number of questions about how the brain controls body functions and internal organ rhythms when animals are exposed to emotional challenges and changes in their living environments. In general experiments, signals from different organs, such as the brain and the heart, are recorded by independent recording systems that require multiple recording devices and different procedures for processing the data files. This study describes a new method that can simultaneously monitor electrical biosignals, including tens of local field potentials in multiple brain regions, electrocardiograms that represent the cardiac rhythm, electromyograms that represent awake/sleep-related muscle contraction, and breathing signals, in a freely moving rat. The recording configuration of this method is based on a conventional micro-drive array for cortical local field potential recordings in which tens of electrodes are accommodated, and the signals obtained from these electrodes are integrated into a single electrical board mounted on the animal's head. Here, this recording system was improved so that signals from the peripheral organs are also transferred to an electrical interface board. In a single surgery, electrodes are first separately implanted into the appropriate body parts and the target brain areas. The open ends of all of these electrodes are then soldered to individual channels of the electrical board above the animal's head so that all of the signals can be integrated into the single electrical board. Connecting this board to a recording device allows for the collection of all of the signals into a single device, which reduces experimental costs and simplifies data processing, because all data can be handled in the same data file. This technique will aid the understanding of the neurophysiological correlates of the associations between central and peripheral organs.

This study introduces a method for the simultaneous recording of local field potentials in the brain, electrocardiograms, electromyograms, and breathing signals of a freely moving rat. This technique, which reduces experimental costs and simplifies data analysis, will contribute to the understanding of the interactions between the brain and peripheral organs.

Monitoring the physiological dynamics of the brain and peripheral tissues is necessary for addressing a number of questions about how the brain controls ...

Noninvasive EEG Recordings from Freely Moving Piglets

The method allows the recording of high-quality electroencephalograms (EEGs) from freely moving piglets directly in the pigpen. We use a one-channel telemetric electroencephalography system in combination with standard self-adhesive hydrogel electrodes. The piglets are calmed down without the use of sedatives. After their release into the pigpen, the piglets behave normally&#8212;they drink and sleep in the same cycle as their siblings. Their sleep phases are used for the EEG recordings.

Here, we present a protocol to record telemetric electroencephalograms (EEGs) from freely moving piglets directly in the pigpen without the use of a sedative, making it possible to record typical EEG patterns during non-REM sleep, like spindle bursts.

The method allows the recording of high-quality electroencephalograms (EEGs) from freely moving piglets directly in the pigpen. We use a one-channel ...

Microdialysis of Excitatory Amino Acids During EEG Recordings in Freely Moving Rats

Microdialysis is a well-established neuroscience technique that correlates the changes of neurologically active substances diffusing into the brain interstitial space with the behavior and/or with the specific outcome of a pathology (e.g., seizures for epilepsy). When studying epilepsy, the microdialysis technique is often combined with short-term or even long-term video-electroencephalography (EEG) monitoring to assess spontaneous seizure frequency, severity, progression and clustering. The combined microdialysis-EEG is based on the use of several methods and instruments. Here, we performed in vivo microdialysis and continuous video-EEG recording to monitor glutamate and aspartate outflow over time, in different phases of the natural history of epilepsy in a rat model. This combined approach allows the pairing of changes in the neurotransmitter release with specific stages of the disease development and progression. The amino acid concentration in the dialysate was determined by liquid chromatography. Here, we describe the methods and outline the principal precautionary measures one should take during in vivo microdialysis-EEG, with particular attention to the stereotaxic surgery, basal and high potassium stimulation during microdialysis, depth electrode EEG recording and high-performance liquid chromatography analysis of aspartate and glutamate in the dialysate. This approach may be adapted to test a variety of drug or disease induced changes of the physiological concentrations of aspartate and glutamate in the brain. Depending on the availability of an appropriate analytical assay, it may be further used to test different soluble molecules when employing EEG recording at the same time.

Here, we describe a method for in vivo microdialysis to analyze aspartate and glutamate release in the ventral hippocampus of epileptic and non-epileptic rats, in combination with EEG recordings. Extracellular concentrations of aspartate and glutamate may be correlated with the different phases of the disease.

Microdialysis is a well-established neuroscience technique that correlates the changes of neurologically active substances diffusing into the brain ...

Hybrid Microdrive System with Recoverable Opto-Silicon Probe and Tetrode for Dual-Site High Density Recording in Freely Moving Mice

Multi-regional neural recordings can provide crucial information to understanding fine-timescale interactions between multiple brain regions. However, conventional microdrive designs often only allow use of one type of electrode to record from single or multiple regions, limiting the yield of single-unit or depth profile recordings. It also often limits the ability to combine electrode recordings with optogenetic tools to target pathway and/or cell type specific activity. Presented here is a hybrid microdrive array for freely moving mice to optimize yield and a description of its fabrication and reuse of the microdrive array. The current design employs nine tetrodes and one opto-silicon probe implanted in two different brain areas simultaneously in freely moving mice. The tetrodes and the opto-silicon probe are independently adjustable along the dorsoventral axis in the brain to maximize the yield of unit and oscillatory activities. This microdrive array also incorporates a set-up for light, mediating optogenetic manipulation to investigate the regional- or cell type-specific responses and functions of long-range neural circuits. In addition, the opto-silicon probe can be safely recovered and reused after each experiment. Because the microdrive array consists of 3D-printed parts, the design of microdrives can be easily modified to accommodate various settings. First described is the design of the microdrive array and how to attach the optical fiber to a silicon probe for optogenetics experiments, followed by fabrication of the tetrode bundle and implantation of the array into a mouse brain. The recording of local field potentials and unit spiking combined with optogenetic stimulation also demonstrate feasibility of the microdrive array system in freely moving mice.

This protocol describes the construction of a hybrid microdrive array that allows implantation of nine independently adjustable tetrodes and one adjustable opto-silicon probe in two brain regions in freely moving mice. Also demonstrated is a method for safely recovering and reusing the opto-silicon probe for multiple purposes.

Multi-regional neural recordings can provide crucial information to understanding fine-timescale interactions between multiple brain regions. However, ...

Multi-Fiber Photometry to Record Neural Activity in Freely-Moving Animals

Recording the activity of a group of neurons in a freely-moving animal is a challenging undertaking. Moreover, as the brain is dissected into smaller and smaller functional subgroups, it becomes paramount to record from projections and/or genetically-defined subpopulations of neurons. Fiber photometry is an accessible and powerful approach that can overcome these challenges. By combining optical and genetic methodologies, neural activity can be measured in deep brain structures by expressing genetically-encoded calcium indicators, which translate neural activity into an optical signal that can be easily measured. The current protocol details the components of a multi-fiber photometry system, how to access deep brain structures to deliver and collect light, a method to account for motion artifacts, and how to process and analyze fluorescent signals. The protocol details experimental considerations when performing single and dual color imaging, from either single or multiple implanted optic fibers.

This protocol details how to implement and perform multi-fiber photometry recordings, how to correct for calcium-independent artifacts, and important considerations for dual-color photometry imaging.

Recording the activity of a group of neurons in a freely-moving animal is a challenging undertaking. Moreover, as the brain is dissected into smaller and ...

Wireless Electrophysiological Recording of Neurons by Movable Tetrodes in Freely Swimming Fish

The neural mechanisms governing fish behavior remain mostly unknown, although fish constitute the majority of all vertebrates. The ability to record brain activity from freely moving fish would advance research on the neural basis of fish behavior considerably. Moreover, precise control of the recording location in the brain is critical to studying coordinated neural activity across regions in fish brain. Here, we present a technique that records wirelessly from the brain of freely swimming fish while controlling for the depth of the recording location. The system is based on a neural logger associated with a novel water-compatible implant that can adjust the recording location by microdrive-controlled tetrodes. The capabilities of the system are illustrated through recordings from the telencephalon of goldfish.

A novel wireless technique for recording extracellular neural signals from the brain of freely swimming goldfish is presented. The recording device is composed of two tetrodes, a microdrive, a neural data logger, and a waterproof case. All parts are custom-made except for the data logger and its connector.

The neural mechanisms governing fish behavior remain mostly unknown, although fish constitute the majority of all vertebrates. The ability to record brain ...

Single-Cell Electroporation across Different Organotypic Slice Culture of Mouse Hippocampal Excitatory and Class-Specific Inhibitory Neurons

Electroporation has established itself as a critical method for transferring specific genes into cells to understand their function. Here, we describe a single-cell electroporation technique that maximizes the efficiency (~80%) of in vitro gene transfection in excitatory and class-specific inhibitory neurons in mouse organotypic hippocampal slice culture. Using large glass electrodes, tetrodotoxin-containing artificial cerebrospinal fluid and mild electrical pulses, we delivered a gene of interest into cultured hippocampal CA1 pyramidal neurons and inhibitory interneurons. Moreover, electroporation could be carried out in cultured hippocampal slices up to 21 days in vitro with no reduction in transfection efficiency, allowing for the study of varying slice culture developmental stages. With interest growing in examining the molecular functions of genes across a diverse range of cell types, our method demonstrates a reliable and straightforward approach to in vitro gene transfection in mouse brain tissue that can be performed with existing electrophysiology equipment and techniques.

Presented here is a protocol for single-cell electroporation that can deliver genes in both excitatory and inhibitory neurons across a range of in vitro hippocampal slice culture ages. Our approach provides precise and efficient expression of genes in individual cells, which can be used to examine cell-autonomous and intercellular functions.

Electroporation has established itself as a critical method for transferring specific genes into cells to understand their function. Here, we describe a ...