Name: generalized psychophysiological interaction (ppi) analysis of memory related connectivity in individuals at genetic risk for alzheimer's disease
Uploaded: 2017-11-14T12:30:00
Description: Watch this Scientific Journal Video about Generalized Psychophysiological Interaction PPI Analysis of Memory Related Connectivity in Individuals at Genetic Risk for Alzheimer's Disease at JoVE.com

This work was supported by the National Institute of Aging (grant number R01AG013308 to SYB, F31AG047041 to TMH). The authors used computational and storage services associated with the Hoffman2 Shared Cluster provided by UCLA Institute for Digital Research and Education's Research Technology Group.

The present study was performed in compliance with the UCLA Institutional Review Board (IRB) protocols and approved by the UCLA Human Subjects Protection Committee. All participants gave written informed consent in order to enroll in this study.
1. Participant Selection
<ol>
	<li>Obtain IRB approval to perform the study.</li>
	<li>Screen individuals aged 55 and older for cognitive decline using a standardized neuropsychological battery. Include tests of General Intelligence (Subtests of the ...

<ol>
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Early task-based fMRI studies were designed to uncover statistical relationships between particular cognitive processes or demands and changes in the BOLD signal relative to a baseline measurement. This traditional approach is useful for identifying specific regions in the brain where activity is modulated by an experimental task. In contrast, a PPI analysis is chiefly concerned with the modulation of functional connectivity, or synchrony of activity, that results from a task-induced cognitive process. PPI measures conte...

The term &#34;connectome&#34; was coined in 2005 marking a paradigm shift in neuroscience that continues to this day1. The brain is increasingly described in terms of functional networks, connectivity and interactions between and among regions on a large scale. Nevertheless, the delineation of regional functional specialization and associations between fMRI-measured activity and task demands are still valid and useful approaches. In light of the growing interest in connectomics, functional connectivity approaches to task fMRI analysis are growing in popularity. One approach to measuring functional connectivity changes dependent on task demands makes use of the concept of PPI. A PPI is the interaction of an active task phase or particular task demand (&#34;psycho&#34;) with the functional connectivity (&#34;physio&#34;) of a region of interest or &#34;seed&#34; in the brain. PPI differs from bivariate, correlation-based analysis of functional connectivity, which generally measures the degree of correlation between the activity in two regions without any constraints related to task demands.
The concept and framework of a PPI analysis was originally described by Friston and colleagues in 19972. The authors asserted that their approach was important because it would allow the investigation of connectivity to be more functionally specific and allow for inferences that activity in a distal seed could be modulating activity resulting from a task demand. In 2012, McLaren and colleagues added to this original framework and described a gPPI approach in which all task phases and their interactions are included in a single model3. This approach leads to results that are more sensitive and specific to the task phase and interaction being investigated. It is this updated gPPI approach that we employ in the present study (see step 6.2.2 in Protocol). The gPPI approach has now been cited in over 200 studies. For clarity, hereafter we use &#39;PPI&#39; to describe common features of both the standard and generalized version. &#39;gPPI&#39; will be used to discuss specific advances associated with the newer framework.
The overall goal of a PPI analysis is to understand how the demands of a cognitive task influence or modulate the functional connectivity of a seed region. A PPI analysis requires a strong a priori hypothesis. Activity in the seed region must be modulated by the task in order for the PPI approach to work effectively4. For example, in the present study, we based our seed selection on the strong evidence that hippocampal activity is modulated by the cognitive demands of a memory task. Using PPI, regions that are significantly more or less functionally connected to the hippocampus during specific task phases can be identified. In short, we ask the question, &#34;in which regions is activity more correlated with the seed during context A as compared with baseline?&#34; We can also ask the logical opposite (as it is important to understand the difference): &#34;in which regions is activity less correlated with the seed during context A as compared to baseline?&#34; When interpreting group differences in PPI effects, it is important to examine the data and whether positive or negative change in functional connectivity, or both, is driving group differences.
The PPI approach has been used to study dynamic task control hubs in healthy controls, how modulation of functional connectivity is related to cognitive performance in Alzheimer&#39;s disease (AD), intelligence in individuals with autism, motor network connectivity in individuals with Parkinson&#39;s disease, face processing in individuals with body dysmorphic disorder and anorexia, emotion regulation, memory, and many other specific questions related to connectivity5,6,7,8,9,10,11. In the present study, we compare changes in functional connectivity of subregions of the hippocampus during memory encoding and retrieval between a group of individuals at increased genetic risk for AD to a group without the risk factor12. The following describes the protocol that we used, applying the gPPI approach, to allow us to test if task-elicited changes in functional connectivity differ in association with the presence of APOE&#949;4, a genetic risk factor for AD.

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			<td height="21" style="height:21px;width:307px;">3T manetic resonance imaging scanner</td>
			<td style="width:280px;">Siemens Medical Solutions</td>
			<td style="width:237px;">MAGNETOM Trio, A Tim System</td>
			<td style="width:267px;">3T MRI Scanner</td>
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			<td height="21" style="height:21px;width:307px;">FSL (FMRIB Software Library)</td>
			<td style="width:280px;">Oxford University</td>
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			<td>Functional Imaging Processing Software</td>
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			<td height="42" style="height:42px;width:307px;">AFNI (Analysis of Functional Neuroimaging)</td>
			<td style="width:280px;">National Institute of Mental Health, National Institutes of Health</td>
			<td style="width:237px;">Any version after May 2015</td>
			<td>Functional Imaging Processing Software</td>
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			<td height="21" style="height:21px;width:307px;">SPM8 (Statistical Parametric Mapping)</td>
			<td style="width:280px;">University College of London</td>
			<td style="width:237px;">SPM8</td>
			<td>Functional Imaging Processing Software</td>
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			<td height="21" style="height:21px;width:307px;">Matlab Software</td>
			<td style="width:280px;">The Mathworks, Inc</td>
			<td style="width:237px;">Version R2012a</td>
			<td>Computing Software</td>
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			<td height="21" style="height:21px;width:307px;">SDS Software</td>
			<td style="width:280px;">Applied Biosystems, Inc</td>
			<td>7900HT Fast Real-Time PCR System</td>
			<td>Real Time PCR</td>
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			<td height="21" style="height:21px;width:307px;">Taqman Assays</td>
			<td style="width:280px;">ThermoFisher Scientific</td>
			<td style="width:237px;">Specific to SNP</td>
			<td>SNP Genotyping</td>
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generalized psychophysiological interaction (ppi) analysis of memory related connectivity in individuals at genetic risk for alzheimer's disease

In neuroimaging, functional magnetic resonance imaging (fMRI) measures the blood-oxygenation-level dependent (BOLD) signal in the brain. The degree of correlation of the BOLD signal in spatially independent regions of the brain defines the functional connectivity of those regions. During a cognitive fMRI task, a psychophysiological interaction (PPI) analysis can be used to examine changes in the functional connectivity during specific contexts defined by the cognitive task. An example of such a task is one that engages the memory system, asking participants to learn pairs of unrelated words (encoding) and recall the second word in a pair when presented with the first word (retrieval). In the present study, we used this type of associative memory task and a generalized PPI (gPPI) analysis to compare changes in hippocampal connectivity in older adults who are carriers of the Alzheimer's disease (AD) genetic risk factor apolipoprotein-E epsilon-4 (APOE&#949;4). Specifically, we show that the functional connectivity of subregions of the hippocampus changes during encoding and retrieval, the two active phases of the associative memory task. Context-dependent changes in functional connectivity of the hippocampus were significantly different in carriers of APOE&#949;4 compared to non-carriers. PPI analyses make it possible to examine changes in functional connectivity, distinct from univariate main effects, and to compare these changes across groups. Thus, a PPI analysis may reveal complex task effects in specific cohorts that traditional univariate methods do not capture. PPI analyses cannot, however, determine directionality or causality between functionally connected regions. Nevertheless, PPI analyses provide powerful means for generating specific hypotheses regarding functional relationships, which can be tested using causal models. As the brain is increasingly described in terms of connectivity and networks, PPI is an important method for analyzing fMRI task data that is in line with the current conception of the human brain.

With two different active task phases (encoding and retrieval) and two seed regions (anterior and posterior hippocampus) there are four conditions to report results for each group. The within-group task activation maps (not shown here, see Harrison et al., 201612) show that the occipital lobe, auditory cortex, large regions of parietal lobe, frontal language areas, superior temporal gyrus, and caudate (more pronounced during retrieval) have significant BOL...

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Generalized Psychophysiological Interaction PPI Analysis of Memory Related Connectivity in Individuals at Genetic Risk for Alzheimer&#039;s Disease

This manuscript describes how to implement a psychophysiological interaction analysis to reveal task-dependent changes in functional connectivity between a selected seed region and voxels in other regions of the brain. Psychophysiological interaction analysis is a popular method to examine task effects on brain connectivity, distinct from traditional univariate activation effects.

Generalized Psychophysiological Interaction (PPI) Analysis of Memory Related Connectivity in Individuals at Genetic Risk for Alzheimer's Disease

In neuroimaging, functional magnetic resonance imaging (fMRI) measures the blood-oxygenation-level dependent (BOLD) signal in the brain. The degree of ...

The overall goal of this analysis is to identify memory-related context-dependent changes to functional connectivity between regions of the hippocampus and the rest of the brain. This method can help answer key questions in the field of cognitive neuroscience such as how does functional connectivity change in response to specific cognitive demands within an experimental task. The main advantage of this technique is that it allows researchers to test specific hypotheses regarding the functional connectivity of key brain regions during a cognitive task.For this experiment, include individual aged 55 and older with cognitive decline who have been genotyped for the Alzheimer's disease risk allele apolipoprotein E epsilon four prior to the experiment, screen subjects for MRI safety and obtain informed consent. Use a three-tesla MRI system to acquire whole brain imaging data. For functional imaging, collect axial slices using an echo-planar imaging sequence while running an unrelated words-associative memory task.To facilitate registration of the functional images, also acquire axial slices of T2-weighted coplanar structural images. For high-resolution structural imaging, collect axial slices using a 3D T1-weighted sequence. Once imaging is complete for all participants, set up preprocessing steps and the first level general linear model using FSL FMRI expert analysis tool, or FEAT, for the first participant.In the Data tab, click on Select 4D data and navigate to the motion corrected and brain extracted file. Set the TR to match that of the functional sequence and use the default high pass filter. Now, in the Pre-stats tab, click none under motion correction and uncheck BET brain extraction.Enter five millimeters to set the full width half maximum Gaussian kernel for spatial smoothing. Then, click Full model setup and create the task timing files denoting the onset and offset of the task phases. Add these to the GLM by choosing three-column format and navigating to the relevant text file.Include one for the encoding phase of the task and one for the retrieval phase. For convolution, choose the Double-Gamma HRF option. Next, use the output of the MCFLIRT tool to create six single-column text files that describe the motion correction performed at each volume within the data set.Select Full model setup and add the parameters and their temporal derivatives as explanatory variables, or EVs, in the GLM. For each motion EV, choose Custom for basic shape, none for convolution and temporal filling. Now, navigate to the Stats tab in the software and select the output of the FSL motion outliers tool under the Add additional confound EVs option.Now, in the Registration tab, check Expanded functional image and Main structural image for a two-step registration. Select the participants coplanar T2-weighted structural scan for the first step to register the functional to the structural data. Choose six degrees of freedom in the second dropdown box.For the next step, register the T2-weighted image to the high-resolution T1-weighted MP-RAGE by selecting boundary based registration from the dropdown box. Finally, register the high-resolution structural data to the standard MNI 152 template selecting 12 degrees of freedom and a linear transformation. Before setting up the psychophysiological interaction model, first load the preprocess data in FSL FEAT software.Choose the denoised image as the input file. In the Pre-stats tabs, set motion correction and brain extraction to None. Do not perform temporal filtering or spatial smoothing.Then, in the Stats tab, select Full model setup and in the EVs tab, add all the variables from the first-level modeling including motion correction, the confound matrix from FSL motion outliers and task timing. Include an EV for the physiological time course from the seed as a covariate of no interest. Next, create the PPI terms by choosing Interaction in the Basic shape menu, and select the seed time course EV and one task EV.For the Make zero option, choose Center for the task variables and Mean for the seed time course EV.Now, in the Contrasts and F-tests tab, model the following specific effects by entering one in the corresponding EV cells.Encoding task phase, retrieval task phase, seed time course, PPI of seed and encoding and PPI of seed and retrieval. Lastly, enter negative one to model negative PPIs for each task phase. Use statistical parametric mapping software tools to run group level comparisons.Begin by selecting Specify second level, then select Two-sample T-test under Design. Navigate to the directory with the parameter estimate images for group one and select them. Then, add the images for group two and run this comparison by clicking on the Play button.Now, return to the main window. Select Estimate and navigate to the SPM. mat file created in the previous step to run the model estimation.Next, under the Results tab, select Define a new contrast. Choose T-contrast and enter one negative one in the Contrast box for APOE-4 carriers greater than APOE-4 non-carriers, then click OK.Finally, run group comparison contrasts as seen here. Choose None for Apply masking, and then manually set the voxel-level threshold and the cluster size minimum according to output from AFNI's 3dClustSim software.Enter negative one-one for APOE-4 non-carriers greater than APOE-4 carriers. Within group generalized psychophysiological interaction analyses revealed significant decreases in functional connectivity in APOE-4 carriers, green, for both task conditions and hippocampal sub-regions. In APOE-4 non-carriers, red, significant decreases in functional connectivity were only observed with posterior hippocampus during encoding.During retrievals, significant differences between APOE-4 carriers and non-carriers were found in the left supramarginal gyrus, dark blue, the right supramarginal angular junction, orange, as well in the right precuneus, purple. The peak coordinate for each cluster is reported in MNI space. Here, contrasts of parameter estimates from each cluster are plotted by group.The red lines indicate zero and highlight that carriers have decreased functional connectivity to anterior hippocampus in these regions during retrieval. The band within the boxes represents the median, while the upper and lower edges of the boxes represent the first and third quartiles respectively. After its development, this technique paved the way for functional neuroimagers to explore dynamic task-related connectivity in humans.This includes both healthy and patient cohorts as well as individuals at increased genetic risk for disease as we describe here. After watching this video, you should have a good understanding of how to use a PPI analysis to test for context-dependent functional connectivity changes between your seed region of interest and the rest of the brain.

generalized-psychophysiological-interaction-ppi-analysis-memory

Research

JoVE Journal

Neuroscience

Preparation of Oligomeric &beta;-amyloid1-42 and Induction of Synaptic Plasticity Impairment on Hippocampal Slices

Impairment of synaptic connections is likely to underlie the subtle amnesic changes occurring at the early stages of Alzheimer s Disease (AD). &beta;-amyloid (A&beta;), a peptide produced in high amounts in AD, is known to reduce Long-Term Potentiation (LTP), a cellular correlate of learning and memory. Indeed, LTP impairment caused by A&beta; is a useful experimental paradigm for studying synaptic dysfunctions in AD models and for screening drugs capable of mitigating or reverting such synaptic impairments. Studies have shown that A&beta; produces the LTP disruption preferentially via its oligomeric form. Here we provide a detailed protocol for impairing LTP by perfusion of oligomerized synthetic A&beta;1-42 peptide onto acute hippocampal slices. In this video, we outline a step-by-step procedure for the preparation of oligomeric A&beta;1-42. Then, we follow an individual experiment in which LTP is reduced in hippocampal slices exposed to oligomerized A&beta;1-42 compared to slices in a control experiment where no A&beta;1-42 exposure had occurred.

Preparation of Oligomeric &amp;#946;-amyloid1-42 and Induction of Synaptic Plasticity Impairment on Hippocampal Slices

One feature of Alzheimer's Disease is the elevation of A&beta;1-42 peptide. Here we provide a protocol for preparing synthetic A&beta;1-42 oligomers, which impairs hippocampal Long-Term Potentiation, a cellular correlate of memory. This procedure is useful for investigating mechanisms of A&beta;-induced pathology and drug screening.

Impairment of synaptic connections is likely to underlie the subtle amnesic changes occurring at the early stages of Alzheimer s Disease (AD). ...

Detection of Neuritic Plaques in Alzheimer's Disease Mouse Model

Alzheimer's disease (AD) is the most common neurodegenerative disorder leading to dementia. Neuritic plaque formation is one of the pathological hallmarks of Alzheimer's disease. The central component of neuritic plaques is a small filamentous protein called amyloid &beta; protein (A&beta;)1, which is derived from sequential proteolytic cleavage of the beta-amyloid precursor protein (APP) by &beta;-secretase and &gamma;-secretase. The amyloid hypothesis entails that A&gamma;-containing plaques as the underlying toxic mechanism in AD pathology2. The postmortem analysis of the presence of neuritic plaque confirms the diagnosis of AD. To further our understanding of A&gamma; neurobiology in AD pathogenesis, various mouse strains expressing AD-related mutations in the human APP genes were generated. Depending on the severity of the disease, these mice will develop neuritic plaques at different ages. These mice serve as invaluable tools for studying the pathogenesis and drug development that could affect the APP processing pathway and neuritic plaque formation. In this protocol, we employ an immunohistochemical method for specific detection of neuritic plaques in AD model mice. We will specifically discuss the preparation from extracting the half brain, paraformaldehyde fixation, cryosectioning, and two methods to detect neurotic plaques in AD transgenic mice: immunohistochemical detection using the ABC and DAB method and fluorescent detection using thiofalvin S staining method.

Detection of Neuritic Plaques in Alzheimer&#039;s Disease Mouse Model

One of the pathological characteristics of AD is the formation of Amyloid &#x03B2; protein positive neuritic plaques. In this protocol we describe two methods to detect neuritic plaques in transgenic AD model mice: immunohistochemical detection using the ABC and DAB method and fluorescent detection using thioflavin S staining method.

Alzheimer's disease (AD) is the most common neurodegenerative disorder leading to dementia. Neuritic plaque formation is one of the pathological hallmarks ...

Morris Water Maze Test for Learning and Memory Deficits in Alzheimer&#39;s Disease Model Mice

The Morris Water Maze (MWM) was first established by neuroscientist Richard G. Morris in 1981 in order to test hippocampal-dependent learning, including acquisition of spatial memoryand long-term spatial memory 1. The MWM is a relatively simple procedure typically consisting of six day trials, the main advantage being the differentiation between the spatial (hidden-platform) and non-spatial (visible platform) conditions 2-4. In addition, the MWM testing environment reduces odor trail interference 5. This has led the task to be used extensively in the study of the neurobiology and neuropharmacology of spatial learning and memory. The MWM plays an important role in the validation of rodent models for neurocognitive disorders such as Alzheimer&#x2019;s Disease 6, 7. In this protocol we discussed the typical procedure of MWM for testing learning and memory and data analysis commonly used in Alzheimer&#x2019;s disease transgenic model mice.

Morris Water Maze Test for Learning and Memory Deficits in Alzheimer&#039;s Disease Model Mice

The Morris Water Maze is a behavioral task to test hippocampal-dependent learning and memory. It has been widely used in the study of neurobiology, neuropharmacology and neurocognitive disorders in rodent models.

The Morris Water Maze (MWM) was first established by neuroscientist Richard G. Morris in 1981 in order to test hippocampal-dependent learning, including ...

Morphological Analysis of Drosophila Larval Peripheral Sensory Neuron Dendrites and Axons Using Genetic Mosaics

Nervous system development requires the correct specification of neuron position and identity, followed by accurate neuron class-specific dendritic development and axonal wiring. Recently the dendritic arborization (DA) sensory neurons of the Drosophila larval peripheral nervous system (PNS) have become powerful genetic models in which to elucidate both general and class-specific mechanisms of neuron differentiation.
There are four main DA neuron classes (I-IV)1. They are named in order of increasing dendrite arbor complexity, and have class-specific differences in the genetic control of their differentiation2-10. The DA sensory system is a practical model to investigate the molecular mechanisms behind the control of dendritic morphology11-13 because: 1) it can take advantage of the powerful genetic tools available in the fruit fly, 2) the DA neuron dendrite arbor spreads out in only 2 dimensions beneath an optically clear larval cuticle making it easy to visualize with high resolution in vivo, 3) the class-specific diversity in dendritic morphology facilitates a comparative analysis to find key elements controlling the formation of simple vs. highly branched dendritic trees, and 4) dendritic arbor stereotypical shapes of different DA neurons facilitate morphometric statistical analyses.
DA neuron activity modifies the output of a larval locomotion central pattern generator14-16. The different DA neuron classes have distinct sensory modalities, and their activation elicits different behavioral responses14,16-20. Furthermore different classes send axonal projections stereotypically into the Drosophila larval central nervous system in the ventral nerve cord (VNC)21. These projections terminate with topographic representations of both DA neuron sensory modality and the position in the body wall of the dendritic field7,22,23. Hence examination of DA axonal projections can be used to elucidate mechanisms underlying topographic mapping7,22,23, as well as the wiring of a simple circuit modulating larval locomotion14-17.
We present here a practical guide to generate and analyze genetic mosaics24 marking DA neurons via MARCM (Mosaic Analysis with a Repressible Cell Marker)1,10,25 and Flp-out22,26,27 techniques (summarized in Fig. 1).

Morphological Analysis of Drosophila Larval Peripheral Sensory Neuron Dendrites and Axons Using Genetic Mosaics

The dendritic arborization sensory neurons of the Drosophila larval peripheral nervous system are useful models to elucidate both general and neuron class-specific mechanisms of neuron differentiation. We present a practical guide to generate and analyze dendritic arborization neuron genetic mosaics.

Nervous system development requires the correct specification of neuron position and identity, followed by accurate neuron class-specific dendritic ...

T-maze Forced Alternation and Left-right Discrimination Tasks for Assessing Working and Reference Memory in Mice

Forced alternation and left-right discrimination tasks using the T-maze have been widely used to assess working and reference memory, respectively, in rodents. In our laboratory, we evaluated the two types of memory in more than 30 strains of genetically engineered mice using the automated version of this apparatus. Here, we present the modified T-maze apparatus operated by a computer with a video-tracking system and our protocols in a movie format. The T-maze apparatus consists of runways partitioned off by sliding doors that can automatically open downward, each with a start box, a T-shaped alley, two boxes with automatic pellet dispensers at one side of the box, and two L-shaped alleys. Each L-shaped alley is connected to the start box so that mice can return to the start box, which excludes the effects of experimenter handling on mouse behavior. This apparatus also has an advantage that in vivo microdialysis, in vivo electrophysiology, and optogenetics techniques can be performed during T-maze performance because the doors are designed to go down into the floor. In this movie article, we describe T-maze tasks using the automated apparatus and the T-maze performance of &alpha;-CaMKII+/- mice, which are reported to show working memory deficits in the eight-arm radial maze task. Our data indicated that &alpha;-CaMKII+/- mice showed a working memory deficit, but no impairment of reference memory, and are consistent with previous findings using the eight-arm radial maze task, which supports the validity of our protocol. In addition, our data indicate that mutants tended to exhibit reversal learning deficits, suggesting that &alpha;-CaMKII deficiency causes reduced behavioral flexibility. Thus, the T-maze test using the modified automatic apparatus is useful for assessing working and reference memory and behavioral flexibility in mice.

This article presents the protocol of T-maze tests using a modified automated apparatus for assessing the learning and memory functions in mice.

Forced alternation and left-right discrimination tasks using the T-maze have been widely used to assess working and reference memory, respectively, in ...

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 ...

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex 6-8. Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions.
Visualization and manipulation of neurons in primary cultures have contributed to a better understanding of the processes involved in dendrite, spine and synapse development. However neurons growing in vitro are not exposed to all the physiological cues that can affect dendrite and/or spine formation and maintenance during normal development. Our knowledge of dendrite and spine structures in vivo in wild-type or mutant mice comes mostly from observations using the Golgi-Cox method 9. However, Golgi staining is considered to be unpredictable. Indeed, groups of nerve cells and fiber tracts are labeled randomly, with particular areas often appearing completely stained while adjacent areas are devoid of staining. Recent studies have shown that IUE of fluorescent constructs represents an attractive alternative method to study dendrites, spines as well as synapses in mutant / wild-type mice 10-11 (Figure 1A). Moreover in comparison to the generation of mouse knockouts, IUE represents a rapid approach to perform gain and loss of function studies in specific population of cells during a specific time window. In addition, IUE has been successfully used with inducible gene expression or inducible RNAi approaches to refine the temporal control over the expression of a gene or shRNA 12. These advantages of IUE have thus opened new dimensions to study the effect of gene expression/suppression on dendrites and spines not only in specific cerebral structures (Figure 1B) but also at a specific time point of development (Figure 1C).
Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development. Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells. 
In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines.

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

This article describes in detail a protocol to electroporate in utero the cerebral cortex and the hippocampus at E14.5 in mice. We also show that this is a valuable method to study dendrites and spines in these two cerebral regions.

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

Stereotaxic Infusion of Oligomeric Amyloid-beta into the Mouse Hippocampus

Alzheimer&#8217;s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the over-production of amyloid-beta and the deposition of amyloid-beta plaques in brain regions of the afflicted individuals. Throughout the years scientists have generated numerous Alzheimer&#8217;s disease mouse models that attempt to replicate the amyloid-beta pathology. Unfortunately, the mouse models only selectively mimic the disease features. Neuronal death, a prominent effect in the brains of Alzheimer&#8217;s disease patients, is noticeably lacking in these mice. Hence, we and others have employed a method of directly infusing soluble oligomeric species of amyloid-beta - forms of amyloid-beta that have been proven to be most toxic to neurons - stereotaxically into the brain. In this report we utilize male C57BL/6J mice to document this surgical technique of increasing amyloid-beta levels in a select brain region. The infusion target is the dentate gyrus of the hippocampus because this brain structure, along with the basal forebrain that is connected by the cholinergic circuit, represents one of the areas of degeneration in the disease. The results of elevating amyloid-beta in the dentate gyrus via stereotaxic infusion reveal increases in neuron loss in the dentate gyrus within 1 week, while there is a concomitant increase in cell death and cholinergic neuron loss in the vertical limb of the diagonal band of Broca of the basal forebrain. These effects are observed up to 2 weeks. Our data suggests that the current amyloid-beta infusion model provides an alternative mouse model to address region specific neuron death in a short-term basis. The advantage of this model is that amyloid-beta can be elevated in a spatial and temporal manner.

Here, we present a protocol for direct stereotaxic brain infusion of amyloid-beta. This methodology provides an alternative in vivo mouse model to address the short-term effects of amyloid-beta on brain neurons.

Alzheimer&#8217;s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the ...

Peering at Brain Polysomes with Atomic Force Microscopy

The translational machinery, i.e., the polysome or polyribosome, is one of the biggest and most complex cytoplasmic machineries in cells. Polysomes, formed by ribosomes, mRNAs, several proteins and non-coding RNAs, represent integrated platforms where translational controls take place. However, while the ribosome has been widely studied, the organization of polysomes is still lacking comprehensive understanding. Thus much effort is required in order to elucidate polysome organization and any novel mechanism of translational control that may be embedded. Atomic force microscopy (AFM) is a type of scanning probe microscopy that allows the acquisition of 3D images at nanoscale resolution. Compared to electron microscopy (EM) techniques, one of the main advantages of AFM is that it can acquire thousands of images both in air and in solution, enabling the sample to be maintained under near physiological conditions without any need for staining and fixing procedures. Here, a detailed protocol for the accurate purification of polysomes from mouse brain and their deposition on mica substrates is described. This protocol enables polysome imaging in air and liquid with AFM and their reconstruction as three-dimensional objects. Complementary to cryo-electron microscopy (cryo-EM), the proposed method can be conveniently used for systematically analyzing polysomes and studying their organization.

While ribosome structure has been extensively characterized, the organization of polysomes is still understudied. To overcome this lack of knowledge, we present here a detailed preparation protocol for accurate imaging of mammalian polysomes by atomic force microscopy (AFM) in air and liquid.

The translational machinery, i.e., the polysome or polyribosome, is one of the biggest and most complex cytoplasmic machineries in cells. Polysomes, ...

In Vivo Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal

An increasing number of super-resolution microscopy techniques are helping to uncover the mechanisms that govern the nanoscale cellular world. Single-molecule imaging is gaining momentum as it provides exceptional access to the visualization of individual molecules in living cells. Here, we describe a technique that we developed to perform single-particle tracking photo-activated localization microscopy (sptPALM) in Drosophila larvae. Synaptic communication relies on key presynaptic proteins that act by docking, priming, and promoting the fusion of neurotransmitter-containing vesicles with the plasma membrane. A range of protein-protein and protein-lipid interactions tightly regulates these processes and the presynaptic proteins therefore exhibit changes in mobility associated with each of these key events. Investigating how mobility of these proteins correlates with their physiological function in an intact live animal is essential to understanding their precise mechanism of action. Extracting protein mobility with high resolution in vivo requires overcoming limitations such as optical transparency, accessibility, and penetration depth. We describe how photoconvertible fluorescent proteins tagged to the presynaptic protein Syntaxin-1A can be visualized via slight oblique illumination and tracked at the motor nerve terminal or along the motor neuron axon of the third instar Drosophila larva.

In Vivo Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal

Here we illustrate how single molecule photo-activated localization microscopy can be carried out on the motor nerve terminal of a live Drosophila melanogaster third instar larva.