Cortical layers are ubiquitous structures throughout neocortex1-4 that consist of highly recurrent local networks. In recent years, significant progress has been made in our understanding of differences in response properties of neurons in different cortical layers5-8, yet there is still a great deal left to learn about whether and how neuronal populations encode information in a laminar-specific manner.
Existing multi-electrode array techniques, although informative for measuring responses across many millimeters of cortical space along the cortical surface, are unsuitable to approach the issue of laminar cortical circuits. Here, we present our method for setting up and recording individual neurons and local field potentials (LFPs) across cortical layers of primary visual cortex (V1) utilizing multi-contact laminar electrodes (Figure 1; Plextrode U-Probe, Plexon Inc).
The methods included are recording device construction, identification of cortical layers, and identification of receptive fields of individual neurons. To identify cortical layers, we measure the evoked response potentials (ERPs) of the LFP time-series using full-field flashed stimuli. We then perform current-source density (CSD) analysis to identify the polarity inversion accompanied by the sink-source configuration at the base of layer 4 (the sink is inside layer 4, subsequently referred to as granular layer9-12). Current-source density is useful because it provides an index of the location, direction, and density of transmembrane current flow, allowing us to accurately position electrodes to record from all layers in a single penetration6, 11, 12.
21 Related JoVE Articles!
Developing Neuroimaging Phenotypes of the Default Mode Network in PTSD: Integrating the Resting State, Working Memory, and Structural Connectivity
Institutions: Alpert Medical School, Brown University, University of Georgia.
Complementary structural and functional neuroimaging techniques used to examine the Default Mode Network (DMN) could potentially improve assessments of psychiatric illness severity and provide added validity to the clinical diagnostic process. Recent neuroimaging research suggests that DMN processes may be disrupted in a number of stress-related psychiatric illnesses, such as posttraumatic stress disorder (PTSD).
Although specific DMN functions remain under investigation, it is generally thought to be involved in introspection and self-processing. In healthy individuals it exhibits greatest activity during periods of rest, with less activity, observed as deactivation, during cognitive tasks, e.g.
, working memory. This network consists of the medial prefrontal cortex, posterior cingulate cortex/precuneus, lateral parietal cortices and medial temporal regions.
Multiple functional and structural imaging approaches have been developed to study the DMN. These have unprecedented potential to further the understanding of the function and dysfunction of this network. Functional approaches, such as the evaluation of resting state connectivity and task-induced deactivation, have excellent potential to identify targeted neurocognitive and neuroaffective (functional) diagnostic markers and may indicate illness severity and prognosis with increased accuracy or specificity. Structural approaches, such as evaluation of morphometry and connectivity, may provide unique markers of etiology and long-term outcomes. Combined, functional and structural methods provide strong multimodal, complementary and synergistic approaches to develop valid DMN-based imaging phenotypes in stress-related psychiatric conditions. This protocol aims to integrate these methods to investigate DMN structure and function in PTSD, relating findings to illness severity and relevant clinical factors.
Medicine, Issue 89, default mode network, neuroimaging, functional magnetic resonance imaging, diffusion tensor imaging, structural connectivity, functional connectivity, posttraumatic stress disorder
Simultaneous Scalp Electroencephalography (EEG), Electromyography (EMG), and Whole-body Segmental Inertial Recording for Multi-modal Neural Decoding
Institutions: National Institutes of Health, University of Houston, University of Houston, University of Houston, University of Houston.
Recent studies support the involvement of supraspinal networks in control of bipedal human walking. Part of this evidence encompasses studies, including our previous work, demonstrating that gait kinematics and limb coordination during treadmill walking can be inferred from the scalp electroencephalogram (EEG) with reasonably high decoding accuracies. These results provide impetus for development of non-invasive brain-machine-interface (BMI) systems for use in restoration and/or augmentation of gait- a primary goal of rehabilitation research. To date, studies examining EEG decoding of activity during gait have been limited to treadmill walking in a controlled environment. However, to be practically viable a BMI system must be applicable for use in everyday locomotor tasks such as over ground walking and turning. Here, we present a novel protocol for non-invasive collection of brain activity (EEG), muscle activity (electromyography (EMG)), and whole-body kinematic data (head, torso, and limb trajectories) during both treadmill and over ground walking tasks. By collecting these data in the uncontrolled environment insight can be gained regarding the feasibility of decoding unconstrained gait and surface EMG from scalp EEG.
Behavior, Issue 77, Neuroscience, Neurobiology, Medicine, Anatomy, Physiology, Biomedical Engineering, Molecular Biology, Electroencephalography, EEG, Electromyography, EMG, electroencephalograph, gait, brain-computer interface, brain machine interface, neural decoding, over-ground walking, robotic gait, brain, imaging, clinical techniques
A Simple Stimulatory Device for Evoking Point-like Tactile Stimuli: A Searchlight for LFP to Spike Transitions
Institutions: National Research Council, National Research Council, University of Manchester.
Current neurophysiological research has the aim to develop methodologies to investigate the signal route from neuron to neuron, namely in the transitions from spikes to Local Field Potentials (LFPs) and from LFPs to spikes.
LFPs have a complex dependence on spike activity and their relation is still poorly understood1
. The elucidation of these signal relations would be helpful both for clinical diagnostics (e.g.
stimulation paradigms for Deep Brain Stimulation) and for a deeper comprehension of neural coding strategies in normal and pathological conditions (e.g.
epilepsy, Parkinson disease, chronic pain). To this aim, one has to solve technical issues related to stimulation devices, stimulation paradigms and computational analyses. Therefore, a custom-made stimulation device was developed in order to deliver stimuli well regulated in space and time that does not incur in mechanical resonance. Subsequently, as an exemplification, a set of reliable LFP-spike relationships was extracted.
The performance of the device was investigated by extracellular recordings, jointly spikes and LFP responses to the applied stimuli, from the rat Primary Somatosensory cortex. Then, by means of a multi-objective optimization strategy, a predictive model for spike occurrence based on LFPs was estimated.
The application of this paradigm shows that the device is adequately suited to deliver high frequency tactile stimulation, outperforming common piezoelectric actuators. As a proof of the efficacy of the device, the following results were presented: 1) the timing and reliability of LFP responses well match the spike responses, 2) LFPs are sensitive to the stimulation history and capture not only the average response but also the trial-to-trial fluctuations in the spike activity and, finally, 3) by using the LFP signal it is possible to estimate a range of predictive models that capture different aspects of the spike activity.
Neuroscience, Issue 85, LFP, spike, tactile stimulus, Multiobjective function, Neuron, somatosensory cortex
Correlating Behavioral Responses to fMRI Signals from Human Prefrontal Cortex: Examining Cognitive Processes Using Task Analysis
Institutions: Centre for Vision Research, York University, Centre for Vision Research, York University.
The aim of this methods paper is to describe how to implement a neuroimaging technique to examine complementary brain processes engaged by two similar tasks. Participants' behavior during task performance in an fMRI scanner can then be correlated to the brain activity using the blood-oxygen-level-dependent signal. We measure behavior to be able to sort correct trials, where the subject performed the task correctly and then be able to examine the brain signals related to correct performance. Conversely, if subjects do not perform the task correctly, and these trials are included in the same analysis with the correct trials we would introduce trials that were not only for correct performance. Thus, in many cases these errors can be used themselves to then correlate brain activity to them. We describe two complementary tasks that are used in our lab to examine the brain during suppression of an automatic responses: the stroop1
and anti-saccade tasks. The emotional stroop paradigm instructs participants to either report the superimposed emotional 'word' across the affective faces or the facial 'expressions' of the face stimuli1,2
. When the word and the facial expression refer to different emotions, a conflict between what must be said and what is automatically read occurs. The participant has to resolve the conflict between two simultaneously competing processes of word reading and facial expression. Our urge to read out a word leads to strong 'stimulus-response (SR)' associations; hence inhibiting these strong SR's is difficult and participants are prone to making errors. Overcoming this conflict and directing attention away from the face or the word requires the subject to inhibit bottom up processes which typically directs attention to the more salient stimulus. Similarly, in the anti-saccade task3,4,5,6
, where an instruction cue is used to direct only attention to a peripheral stimulus location but then the eye movement is made to the mirror opposite position. Yet again we measure behavior by recording the eye movements of participants which allows for the sorting of the behavioral responses into correct and error trials7
which then can be correlated to brain activity. Neuroimaging now allows researchers to measure different behaviors of correct and error trials that are indicative of different cognitive processes and pinpoint the different neural networks involved.
Neuroscience, Issue 64, fMRI, eyetracking, BOLD, attention, inhibition, Magnetic Resonance Imaging, MRI
Eye Movement Monitoring of Memory
Institutions: Rotman Research Institute, University of Toronto, University of Toronto.
Explicit (often verbal) reports are typically used to investigate memory (e.g. "Tell me what you remember about the person you saw at the bank yesterday."), however such reports can often be unreliable or sensitive to response bias 1
, and may be unobtainable in some participant populations. Furthermore, explicit reports only reveal when information has reached consciousness and cannot comment on when memories were accessed during processing, regardless of whether the information is subsequently accessed in a conscious manner. Eye movement monitoring (eye tracking) provides a tool by which memory can be probed without asking participants to comment on the contents of their memories, and access of such memories can be revealed on-line 2,3
. Video-based eye trackers (either head-mounted or remote) use a system of cameras and infrared markers to examine the pupil and corneal reflection in each eye as the participant views a display monitor. For head-mounted eye trackers, infrared markers are also used to determine head position to allow for head movement and more precise localization of eye position. Here, we demonstrate the use of a head-mounted eye tracking system to investigate memory performance in neurologically-intact and neurologically-impaired adults. Eye movement monitoring procedures begin with the placement of the eye tracker on the participant, and setup of the head and eye cameras. Calibration and validation procedures are conducted to ensure accuracy of eye position recording. Real-time recordings of X,Y-coordinate positions on the display monitor are then converted and used to describe periods of time in which the eye is static (i.e. fixations) versus in motion (i.e., saccades). Fixations and saccades are time-locked with respect to the onset/offset of a visual display or another external event (e.g. button press). Experimental manipulations are constructed to examine how and when patterns of fixations and saccades are altered through different types of prior experience. The influence of memory is revealed in the extent to which scanning patterns to new images differ from scanning patterns to images that have been previously studied 2, 4-5
. Memory can also be interrogated for its specificity; for instance, eye movement patterns that differ between an identical and an altered version of a previously studied image reveal the storage of the altered detail in memory 2-3, 6-8
. These indices of memory can be compared across participant populations, thereby providing a powerful tool by which to examine the organization of memory in healthy individuals, and the specific changes that occur to memory with neurological insult or decline 2-3, 8-10
Neuroscience, Issue 42, eye movement monitoring, eye tracking, memory, aging, amnesia, visual processing
In vivo Ca2+- Imaging of Mushroom Body Neurons During Olfactory Learning in the Honey Bee
Institutions: Freie Universität Berlin, Free University Berlin - Freie Universitaet Berlin.
The in vivo
and semi-in vivo
preparation for Calcium imaging has been developed in our lab by Joerges, Küttner and Galizia over ten years ago, to measure odor evoked activity in the antennal lobe1
. From then on, it has been continuously refined and applied to different neuropiles in the bee brain. Here, we describe the preparation currently used in the lab to measure activity in mushroom body neurons using a dextran coupled calcium-sensitive dye (Fura-2). We retrogradely stain mushroom body neurons by injecting dye into their axons or soma region. We focus on reducing the invasiveness, to achieve a preparation in which it is still possible to train the bee using PER conditioning. We are able to monitor and quantify the behavioral response by recording electro-myograms from the muscle which controls the PER (M17)2
After the physiological experiment the imaged structures are investigated in greater detail using confocal scanning microscopy to address the identity of the neurons.
Neuroscience, Issue 30, Calcium Imaging, Insects, Mushroom Body, PER Conditioning, Olfaction, Fura-2
Optical Recording of Suprathreshold Neural Activity with Single-cell and Single-spike Resolution
Institutions: The University of Texas at Austin.
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 (>97% spike detection efficiency), has a low rate of false positive spike detection (< 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
Neuroscience, Issue 67, functional calcium imaging, spatiotemporal patterns of activity, dithered random-access scanning
Implementing Dynamic Clamp with Synaptic and Artificial Conductances in Mouse Retinal Ganglion Cells
Institutions: University of Sydney , University of Western Sydney, University of Sydney .
Ganglion cells are the output neurons of the retina and their activity reflects the integration of multiple synaptic inputs arising from specific neural circuits. Patch clamp techniques, in voltage clamp and current clamp configurations, are commonly used to study the physiological properties of neurons and to characterize their synaptic inputs. Although the application of these techniques is highly informative, they pose various limitations. For example, it is difficult to quantify how the precise interactions of excitatory and inhibitory inputs determine response output. To address this issue, we used a modified current clamp technique, dynamic clamp, also called conductance clamp 1, 2, 3
and examined the impact of excitatory and inhibitory synaptic inputs on neuronal excitability. This technique requires the injection of current into the cell and is dependent on the real-time feedback of its membrane potential at that time. The injected current is calculated from predetermined excitatory and inhibitory synaptic conductances, their reversal potentials and the cell's instantaneous membrane potential. Details on the experimental procedures, patch clamping cells to achieve a whole-cell configuration and employment of the dynamic clamp technique are illustrated in this video article. Here, we show the responses of mouse retinal ganglion cells to various conductance waveforms obtained from physiological experiments in control conditions or in the presence of drugs. Furthermore, we show the use of artificial excitatory and inhibitory conductances generated using alpha functions to investigate the responses of the cells.
Neuroscience, Issue 75, Neurobiology, Biomedical Engineering, Anatomy, Physiology, Molecular Biology, Cellular Biology, Neurons, Retinal Neurons, Retinal Ganglion Cells, Eye, Retina, Neurosciences, retina, ganglion cells, synaptic conductance, artificial conductance, tetrodotoxin (TTX), patch clamp, dynamic clamp, conductance clamp, electrophysiology, mouse, animal model
Functional Imaging of Auditory Cortex in Adult Cats using High-field fMRI
Institutions: University of Western Ontario, University of Western Ontario, University of Western Ontario, University of Western Ontario, University of Western Ontario, University of Western Ontario, University of Western Ontario.
Current knowledge of sensory processing in the mammalian auditory system is mainly derived from electrophysiological studies in a variety of animal models, including monkeys, ferrets, bats, rodents, and cats. In order to draw suitable parallels between human and animal models of auditory function, it is important to establish a bridge between human functional imaging studies and animal electrophysiological studies. Functional magnetic resonance imaging (fMRI) is an established, minimally invasive method of measuring broad patterns of hemodynamic activity across different regions of the cerebral cortex. This technique is widely used to probe sensory function in the human brain, is a useful tool in linking studies of auditory processing in both humans and animals and has been successfully used to investigate auditory function in monkeys and rodents. The following protocol describes an experimental procedure for investigating auditory function in anesthetized adult cats by measuring stimulus-evoked hemodynamic changes in auditory cortex using fMRI. This method facilitates comparison of the hemodynamic responses across different models of auditory function thus leading to a better understanding of species-independent features of the mammalian auditory cortex.
Neuroscience, Issue 84, Central Nervous System, Ear, Animal Experimentation, Models, Animal, Functional Neuroimaging, Brain Mapping, Nervous System, Sense Organs, auditory cortex, BOLD signal change, hemodynamic response, hearing, acoustic stimuli
Cortical Source Analysis of High-Density EEG Recordings in Children
Institutions: UCL Institute of Child Health, University College London.
EEG is traditionally described as a neuroimaging technique with high temporal and low spatial resolution. Recent advances in biophysical modelling and signal processing make it possible to exploit information from other imaging modalities like structural MRI that provide high spatial resolution to overcome this constraint1
. This is especially useful for investigations that require high resolution in the temporal as well as spatial domain. In addition, due to the easy application and low cost of EEG recordings, EEG is often the method of choice when working with populations, such as young children, that do not tolerate functional MRI scans well. However, in order to investigate which neural substrates are involved, anatomical information from structural MRI is still needed. Most EEG analysis packages work with standard head models that are based on adult anatomy. The accuracy of these models when used for children is limited2
, because the composition and spatial configuration of head tissues changes dramatically over development3
In the present paper, we provide an overview of our recent work in utilizing head models based on individual structural MRI scans or age specific head models to reconstruct the cortical generators of high density EEG. This article describes how EEG recordings are acquired, processed, and analyzed with pediatric populations at the London Baby Lab, including laboratory setup, task design, EEG preprocessing, MRI processing, and EEG channel level and source analysis.
Behavior, Issue 88, EEG, electroencephalogram, development, source analysis, pediatric, minimum-norm estimation, cognitive neuroscience, event-related potentials
Automated, Quantitative Cognitive/Behavioral Screening of Mice: For Genetics, Pharmacology, Animal Cognition and Undergraduate Instruction
Institutions: Rutgers University, Koç University, New York University, Fairfield University.
We describe a high-throughput, high-volume, fully automated, live-in 24/7 behavioral testing system for assessing the effects of genetic and pharmacological manipulations on basic mechanisms of cognition and learning in mice. A standard polypropylene mouse housing tub is connected through an acrylic tube to a standard commercial mouse test box. The test box has 3 hoppers, 2 of which are connected to pellet feeders. All are internally illuminable with an LED and monitored for head entries by infrared (IR) beams. Mice live in the environment, which eliminates handling during screening. They obtain their food during two or more daily feeding periods by performing in operant (instrumental) and Pavlovian (classical) protocols, for which we have written protocol-control software and quasi-real-time data analysis and graphing software. The data analysis and graphing routines are written in a MATLAB-based language created to simplify greatly the analysis of large time-stamped behavioral and physiological event records and to preserve a full data trail from raw data through all intermediate analyses to the published graphs and statistics within a single data structure. The data-analysis code harvests the data several times a day and subjects it to statistical and graphical analyses, which are automatically stored in the "cloud" and on in-lab computers. Thus, the progress of individual mice is visualized and quantified daily. The data-analysis code talks to the protocol-control code, permitting the automated advance from protocol to protocol of individual subjects. The behavioral protocols implemented are matching, autoshaping, timed hopper-switching, risk assessment in timed hopper-switching, impulsivity measurement, and the circadian anticipation of food availability. Open-source protocol-control and data-analysis code makes the addition of new protocols simple. Eight test environments fit in a 48 in x 24 in x 78 in cabinet; two such cabinets (16 environments) may be controlled by one computer.
Behavior, Issue 84, genetics, cognitive mechanisms, behavioral screening, learning, memory, timing
Extracellularly Identifying Motor Neurons for a Muscle Motor Pool in Aplysia californica
Institutions: Case Western Reserve University , Case Western Reserve University , Case Western Reserve University .
In animals with large identified neurons (e.g.
mollusks), analysis of motor pools is done using intracellular techniques1,2,3,4
. Recently, we developed a technique to extracellularly stimulate and record individual neurons in Aplysia californica5
. We now describe a protocol for using this technique to uniquely identify and characterize motor neurons within a motor pool.
This extracellular technique has advantages. First, extracellular electrodes can stimulate and record neurons through the sheath5
, so it does not need to be removed. Thus, neurons will be healthier in extracellular experiments than in intracellular ones. Second, if ganglia are rotated by appropriate pinning of the sheath, extracellular electrodes can access neurons on both sides of the ganglion, which makes it easier and more efficient to identify multiple neurons in the same preparation. Third, extracellular electrodes do not need to penetrate cells, and thus can be easily moved back and forth among neurons, causing less damage to them. This is especially useful when one tries to record multiple neurons during repeating motor patterns that may only persist for minutes. Fourth, extracellular electrodes are more flexible than intracellular ones during muscle movements. Intracellular electrodes may pull out and damage neurons during muscle contractions. In contrast, since extracellular electrodes are gently pressed onto the sheath above neurons, they usually stay above the same neuron during muscle contractions, and thus can be used in more intact preparations.
To uniquely identify motor neurons for a motor pool (in particular, the I1/I3 muscle in Aplysia
) using extracellular electrodes, one can use features that do not require intracellular measurements as criteria: soma size and location, axonal projection, and muscle innervation4,6,7
. For the particular motor pool used to illustrate the technique, we recorded from buccal nerves 2 and 3 to measure axonal projections, and measured the contraction forces of the I1/I3 muscle to determine the pattern of muscle innervation for the individual motor neurons.
We demonstrate the complete process of first identifying motor neurons using muscle innervation, then characterizing their timing during motor patterns, creating a simplified diagnostic method for rapid identification. The simplified and more rapid diagnostic method is superior for more intact preparations, e.g.
in the suspended buccal mass preparation8
or in vivo9
. This process can also be applied in other motor pools10,11,12
or in other animal systems2,3,13,14
Neuroscience, Issue 73, Physiology, Biomedical Engineering, Anatomy, Behavior, Neurobiology, Animal, Neurosciences, Neurophysiology, Electrophysiology, Aplysia, Aplysia californica, California sea slug, invertebrate, feeding, buccal mass, ganglia, motor neurons, neurons, extracellular stimulation and recordings, extracellular electrodes, animal model
Perceptual and Category Processing of the Uncanny Valley Hypothesis' Dimension of Human Likeness: Some Methodological Issues
Institutions: University of Zurich.
Mori's Uncanny Valley Hypothesis1,2
proposes that the perception of humanlike characters such as robots and, by extension, avatars (computer-generated characters) can evoke negative or positive affect (valence) depending on the object's degree of visual and behavioral realism along a dimension of human likeness
) (Figure 1
). But studies of affective valence of subjective responses to variously realistic non-human characters have produced inconsistent findings 3, 4, 5, 6
. One of a number of reasons for this is that human likeness is not perceived as the hypothesis assumes. While the DHL can be defined following Mori's description as a smooth linear change in the degree of physical humanlike similarity, subjective perception of objects along the DHL can be understood in terms of the psychological effects of categorical perception (CP) 7
. Further behavioral and neuroimaging investigations of category processing and CP along the DHL and of the potential influence of the dimension's underlying category structure on affective experience are needed. This protocol therefore focuses on the DHL and allows examination of CP. Based on the protocol presented in the video as an example, issues surrounding the methodology in the protocol and the use in "uncanny" research of stimuli drawn from morph continua to represent the DHL are discussed in the article that accompanies the video. The use of neuroimaging and morph stimuli to represent the DHL in order to disentangle brain regions neurally responsive to physical human-like similarity from those responsive to category change and category processing is briefly illustrated.
Behavior, Issue 76, Neuroscience, Neurobiology, Molecular Biology, Psychology, Neuropsychology, uncanny valley, functional magnetic resonance imaging, fMRI, categorical perception, virtual reality, avatar, human likeness, Mori, uncanny valley hypothesis, perception, magnetic resonance imaging, MRI, imaging, clinical techniques
A Proboscis Extension Response Protocol for Investigating Behavioral Plasticity in Insects: Application to Basic, Biomedical, and Agricultural Research
Institutions: Arizona State University.
Insects modify their responses to stimuli through experience of associating those stimuli with events important for survival (e.g.
, food, mates, threats). There are several behavioral mechanisms through which an insect learns salient associations and relates them to these events. It is important to understand this behavioral plasticity for programs aimed toward assisting insects that are beneficial for agriculture. This understanding can also be used for discovering solutions to biomedical and agricultural problems created by insects that act as disease vectors and pests. The Proboscis Extension Response (PER) conditioning protocol was developed for honey bees (Apis mellifera
) over 50 years ago to study how they perceive and learn about floral odors, which signal the nectar and pollen resources a colony needs for survival. The PER procedure provides a robust and easy-to-employ framework for studying several different ecologically relevant mechanisms of behavioral plasticity. It is easily adaptable for use with several other insect species and other behavioral reflexes. These protocols can be readily employed in conjunction with various means for monitoring neural activity in the CNS via electrophysiology or bioimaging, or for manipulating targeted neuromodulatory pathways. It is a robust assay for rapidly detecting sub-lethal effects on behavior caused by environmental stressors, toxins or pesticides.
We show how the PER protocol is straightforward to implement using two procedures. One is suitable as a laboratory exercise for students or for quick assays of the effect of an experimental treatment. The other provides more thorough control of variables, which is important for studies of behavioral conditioning. We show how several measures for the behavioral response ranging from binary yes/no to more continuous variable like latency and duration of proboscis extension can be used to test hypotheses. And, we discuss some pitfalls that researchers commonly encounter when they use the procedure for the first time.
Neuroscience, Issue 91, PER, conditioning, honey bee, olfaction, olfactory processing, learning, memory, toxin assay
Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates
Institutions: University of California Riverside, University of California Riverside, University of California Riverside.
Close to two decades of research has established that astrocytes in situ
and in vivo
express numerous G protein-coupled receptors (GPCRs) that can be stimulated by neuronally-released transmitter. However, the ability of astrocytic receptors to exhibit plasticity in response to changes in neuronal activity has received little attention. Here we describe a model system that can be used to globally scale up or down astrocytic group I metabotropic glutamate receptors (mGluRs) in acute brain slices. Included are methods on how to prepare parasagittal hippocampal slices, construct chambers suitable for long-term slice incubation, bidirectionally manipulate neuronal action potential frequency, load astrocytes and astrocyte processes with fluorescent Ca2+
indicator, and measure changes in astrocytic Gq GPCR activity by recording spontaneous and evoked astrocyte Ca2+
events using confocal microscopy. In essence, a “calcium roadmap” is provided for how to measure plasticity of astrocytic Gq GPCRs. Applications of the technique for study of astrocytes are discussed. Having an understanding of how astrocytic receptor signaling is affected by changes in neuronal activity has important implications for both normal synaptic function as well as processes underlying neurological disorders and neurodegenerative disease.
Neuroscience, Issue 85, astrocyte, plasticity, mGluRs, neuronal Firing, electrophysiology, Gq GPCRs, Bolus-loading, calcium, microdomains, acute slices, Hippocampus, mouse
Simultaneous Long-term Recordings at Two Neuronal Processing Stages in Behaving Honeybees
Institutions: University of Würzburg.
In both mammals and insects neuronal information is processed in different higher and lower order brain centers. These centers are coupled via convergent and divergent anatomical connections including feed forward and feedback wiring. Furthermore, information of the same origin is partially sent via parallel pathways to different and sometimes into the same brain areas. To understand the evolutionary benefits as well as the computational advantages of these wiring strategies and especially their temporal dependencies on each other, it is necessary to have simultaneous access to single neurons of different tracts or neuropiles in the same preparation at high temporal resolution. Here we concentrate on honeybees by demonstrating a unique extracellular long term access to record multi unit activity at two subsequent neuropiles1
, the antennal lobe (AL), the first olfactory processing stage and the mushroom body (MB), a higher order integration center involved in learning and memory formation, or two parallel neuronal tracts2
connecting the AL with the MB. The latter was chosen as an example and will be described in full. In the supporting video the construction and permanent insertion of flexible multi channel wire electrodes is demonstrated. Pairwise differential amplification of the micro wire electrode channels drastically reduces the noise and verifies that the source of the signal is closely related to the position of the electrode tip. The mechanical flexibility of the used wire electrodes allows stable invasive long term recordings over many hours up to days, which is a clear advantage compared to conventional extra and intracellular in vivo
Neuroscience, Issue 89, honeybee brain, olfaction, extracellular long term recordings, double recordings, differential wire electrodes, single unit, multi-unit recordings
Recording Single Neurons' Action Potentials from Freely Moving Pigeons Across Three Stages of Learning
Institutions: Ruhr-University Bochum.
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 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' 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.
Neuroscience, Issue 88, pigeon, single unit recording, learning, memory, extinction, spike sorting, operant conditioning, reward, electrophysiology, animal cognition, model species
Functional Mapping with Simultaneous MEG and EEG
Institutions: MGH - Massachusetts General Hospital.
We use magnetoencephalography (MEG) and electroencephalography (EEG) to locate and determine the temporal evolution in brain areas involved in the processing of simple sensory stimuli. We will use somatosensory stimuli to locate the hand somatosensory areas, auditory stimuli to locate the auditory cortices, visual stimuli in four quadrants of the visual field to locate the early visual areas. These type of experiments are used for functional mapping in epileptic and brain tumor patients to locate eloquent cortices. In basic neuroscience similar experimental protocols are used to study the orchestration of cortical activity. The acquisition protocol includes quality assurance procedures, subject preparation for the combined MEG/EEG study, and acquisition of evoked-response data with somatosensory, auditory, and visual stimuli. We also demonstrate analysis of the data using the equivalent current dipole model and cortically-constrained minimum-norm estimates. Anatomical MRI data are employed in the analysis for visualization and for deriving boundaries of tissue boundaries for forward modeling and cortical location and orientation constraints for the minimum-norm estimates.
JoVE neuroscience, Issue 40, neuroscience, brain, MEG, EEG, functional imaging
A Fully Automated and Highly Versatile System for Testing Multi-cognitive Functions and Recording Neuronal Activities in Rodents
Institutions: The Neurosciences Institute, San Diego, CA.
We have developed a fully automated system for operant behavior testing and neuronal activity recording by which multiple cognitive brain functions can be investigated in a single task sequence. The unique feature of this system is a custom-made, acoustically transparent chamber that eliminates many of the issues associated with auditory cue control in most commercially available chambers. The ease with which operant devices can be added or replaced makes this system quite versatile, allowing for the implementation of a variety of auditory, visual, and olfactory behavioral tasks. Automation of the system allows fine temporal (10 ms) control and precise time-stamping of each event in a predesigned behavioral sequence. When combined with a multi-channel electrophysiology recording system, multiple cognitive brain functions, such as motivation, attention, decision-making, patience, and rewards, can be examined sequentially or independently.
Neuroscience, Issue 63, auditory behavioral task, acoustic chamber, cognition test, multi-channel recording, electrophysiology, attention, motivation, decision, patience, rat, two-alternative choice pitch discrimination task, behavior
Measuring Exocytosis in Neurons Using FM Labeling
The ability to measure the kinetics of vesicle release can help provide insight into some of the basics of neurotransmission. Here we used real-time imaging of vesicles labeled with FM dye to monitor the rate of presynaptic vesicle release. FM4-64 is a red fluorescent amphiphilic styryl dye that embeds into the membranes of synaptic vesicles as endocytosis is stimulated. Lipophilic interactions cause the dye to greatly increase in fluorescence, thus emitting a bright signal when associated with vesicles and a nominal one when in the extracellular fluid. After a wash step is used to help remove external dye within the plasma membrane, the remaining FM is concentrated within the vesicles and is then expelled when exocytosis is induced by another round of electrical stimulation. The rate of vesicles release is measured from the resulting decrease in fluorescence. Since FM dye can be applied external and transiently, it is a useful tool for determining rates of exocytosis in neuronal cultures, especially when comparing the rates between transfected synapses and neighboring control boutons.
Neuroscience, Issue 1, neuron, imaging, exocytosis
Cross-Modal Multivariate Pattern Analysis
Institutions: University of Southern California.
Multivariate pattern analysis (MVPA) is an increasingly popular method of analyzing functional magnetic resonance imaging (fMRI) data1-4
. Typically, the method is used to identify a subject's perceptual experience from neural activity in certain regions of the brain. For instance, it has been employed to predict the orientation of visual gratings a subject perceives from activity in early visual cortices5
or, analogously, the content of speech from activity in early auditory cortices6
Here, we present an extension of the classical MVPA paradigm, according to which perceptual stimuli are not predicted within, but across sensory systems. Specifically, the method we describe addresses the question of whether stimuli that evoke memory associations in modalities other than the one through which they are presented induce content-specific activity patterns in the sensory cortices of those other modalities. For instance, seeing a muted video clip of a glass vase shattering on the ground automatically triggers in most observers an auditory image of the associated sound; is the experience of this image in the "mind's ear" correlated with a specific neural activity pattern in early auditory cortices? Furthermore, is this activity pattern distinct from the pattern that could be observed if the subject were, instead, watching a video clip of a howling dog?
In two previous studies7,8
, we were able to predict sound- and touch-implying video clips based on neural activity in early auditory and somatosensory cortices, respectively. Our results are in line with a neuroarchitectural framework proposed by Damasio9,10
, according to which the experience of mental images that are based on memories - such as hearing the shattering sound of a vase in the "mind's ear" upon seeing the corresponding video clip - is supported by the re-construction of content-specific neural activity patterns in early sensory cortices.
Neuroscience, Issue 57, perception, sensory, cross-modal, top-down, mental imagery, fMRI, MRI, neuroimaging, multivariate pattern analysis, MVPA