This research was supported by funds to FD. MM was supported by an IGERT Fellowship under National Science Foundation Grant No. 0903622. The authors wish to thank the Dolcos Lab members for assistance with data collection and preparation.

1. Amygdala
<ol>
	<li>Anterior Slices of the&#160;AMY
	<ol>
		<li>Identify the first slice of the AMY in which the limen insula initially appears, where the white matter connection between the frontal and temporal lobes is continuous and visible30. In the coronal view, use the angular bundle as the inferolateral border of the AMY.</li>
		<li>Locate the optic chiasm as a landmark for the appearance of the AMY. Use the axial and sagittal views to distinguish the AMY in its early slices from the surrounding unc...

Traditionally, manual segmentation has been considered the gold standard by many researchers. Nevertheless, a precise delineation of the individual structures has been complicated by the highly variable morphology of the MTL structures, and by the usually weak MRI contrasts of these structures against the surrounding neural tissue and non-neural areas. Historically, there have been conflicting descriptions in the literature for some MTL structures. In some cases of segmenting the PRC, for example, the collateral sulcus h...

A correction was made to <a href="/video/50991">A Comprehensive Protocol for Manual Segmentation of the Medial Temporal Lobe Structures</a>. Table 1 and its legend were updated. References 10 and 14 were also updated.
The references were updated from:
<ol start="10">
<li>Wager, T. D. &amp; Smith, E. E. Neuroimaging studies of working memory: a meta-analysis. Cognitive, Affective &amp; Behavioral Neuroscience. 3(4), 255-274 (2003).</li>
</ol>
<ol start="14">
<li>Scheltens, Ph, et al. Atrophyofmedialtemporallobeson MRIin 'probable' Alzheimer's disease and normal ageing: diagnostic value and neuropsychological correlates. Journal of Neurology, Neurosurgery, and Psychiatry. 55(10), 967-972, (1992).</li>
</ol>
to:
<ol start="10">
<li>Wager, T. D., Phan, K. L., Liberzon, I., &amp; Taylor, S. F. Valence, gender, and lateralization of functional brain anatomy in emotion: a meta-analysis of findings from neuroimaging. Neuroimage. 19 (3), 513-31, doi:10.1016/S1053-8119(03)00078-8 (2003).</li>
</ol>
<ol start="14">
<li>de Leon, M. J. et al. Imaging and CSF studies in the preclinical diagnosis of Alzheimer's disease. Annals of the New York Academy of Sciences. 1097, 114-145, doi:10.1196/annals.1379.012 (2007).</li>
</ol>
Table 1 had its legend updated from:
Table 1. Representative volumetric results of the bilateral AMY and the HC of a single subject, from manual tracing using the present protocol and automatic segmentation. Automatic segmentation has underestimated the volume of each of the four structures compared. Corrected volume was calculated as the ratio between Voxel volume and Intracranial volume (ICV). For this subject, ICV = 1446616.73 mm3.
to:
Table 1. Representative volumetric results of the bilateral AMY and the HC of a single subject, from manual tracing using the present protocol and automatic segmentation. Automatic segmentation has misestimated the volume of each of the four structures compared. Corrected volume was calculated as the ratio between Voxel volume and ICV. For this subject, ICV = 1599482.11 mm3. <a href="http://www.jove.com/files/ftp_upload/50991/50991table1.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

A correction was made to <a href="/video/50991">A Comprehensive Protocol for Manual Segmentation of the Medial Temporal Lobe Structures</a>. Table 1 and its legend were updated. References 10 and 14 were also updated.

Erratum: A Comprehensive Protocol for Manual Segmentation of the Medial Temporal Lobe Structures

The medial temporal lobe (MTL), a putative area of the highest level of integration of sensory information1, has been a frequent subject of targeted analyses. For example, the hippocampus and the associated parahippocampal areas have been extensively studied in memory research2-5. Also, the role of the amygdala has been frequently emphasized in research examining emotion processing and emotion-cognition interactions6-11. Recently, various MTL regions have also received attention in the emerging field of personality neuroscience, which links the structure and function of these and other brain regions to individual variation in personality traits12. Assessing the anatomy and function of the MTL structures can be important in facilitating diagnosis of degenerative diseases where specific structural and functional anomalies can occur in different MTL structures. For example, in Alzheimer&#8217;s disease (AD), significant atrophy of the entorhinal cortex and hippocampus can be observed13,14, and atrophy of the hippocampus can predict the transition from mild cognitive impairment to AD15. Automatic segmentation algorithms have recently become popular for segmenting cortical and subcortical structures, but as with any tool, these programs inevitably encounter errors in some cases. In such instances a researcher should be equipped with both the knowledge and guidelines to recognize the anatomical borders of the MTL structures. The tendency in the extant literature has been to target individual MTL subregions16-21, with many protocols tending to focus on hippocampus16-19.
Unlike most of the available published guidelines for MTL tracing, the present protocol provides a comprehensive set of guidelines that allow for clear localization of all MTL subregions. Tracing guidelines for the following MTL structures are described: the amygdala (AMY), the hippocampus (HC), the perirhinal cortex (PRC), the entorhinal cortex (ERC), and the parahippocampal cortex (PHC). The AMY and the HC are traced first, and are then followed by the parahippocampal gyrus (PHG) structures. Note that the generic term HC is used here to refer to the HC formation, which encompasses the HC proper, the subiculum, and the posterior segment of the uncus22-24. Also, note that the PHG can be divided into two segments, the anterior portion and the posterior portion. Within the anterior portion of the PHG, it can be further divided into the lateral and medial anterior PHG, whose cortical areas correspond to the PRC and the ERC, respectively. The PHC, the cortical area of the posterior portion of the PHG, corresponds to the parahippocampal cortex proper. For simplicity reasons, we will be using the terms PRC and ERC to refer to the lateral and medial anterior PHG, and PHC to refer to the posterior PHG. The segmentation for each structure begins with a rough localization of the anterior and posterior borders, along with other relevant landmarks, which is then followed by the actual tracing performed slice-by-slice in the coronal plane, in an anterior-posterior/rostro-caudal direction. In all cases, the sagittal and axial sections are closely monitored to assist the localization of anatomical boundaries and landmarks.
The need for such tracing guidelines is also illustrated in figures displaying possible differences between the output of automatic and manual segmentation protocols. The advantage of a protocol that describes all of the MTL structures in the current visual format is that variations in the anatomy (e.g., the collateral sulcus [CS] depth) that can affect border definitions can be described in context with the surrounding anatomy (e.g., the PRC and ERC medial and lateral borders vary in location depending on the depth of the CS25). This might not be clear or understandable to an inexperienced tracer or an experienced tracer who only traces single or separate structures, and to our knowledge, such a visually comprehensive guideline does not exist.
The present protocol is an explicit presentation of guidelines used for MTL tracing in a previous investigation identifying differential contributions from MTL subregions to the memory enhancing effect of emotion26, adapted to higher resolution brain images allowed by recent developments in structural magnetic resonance (MR) imaging. The tracing is illustrated on scans obtained from a healthy volunteer (female, aged 24), using a 3T MR scanner. Anatomical images were acquired as 3D MPRAGE (TR = 1,800 msec; TE = 2.26 msec; FOV = 256 x 256 mm; voxel size = 1 x 0.5 x 0.5 mm) with an acquisition angle parallel to AC-PC. If image data is acquired with a different acquisition angle, such as oblique orientation, the data should be regridded to a parallel or perpendicular orientation to AC-PC, such that anatomical landmark descriptions translate appropriately. The images were then translated to NIFTI format and input into segmentation software27 for manual tracing. Scan data used in the current protocol was collected as part of a study that was approved by the Institutional Review Board, and the volunteer provided written consent.
By drawing information from various separate tracing protocols for these structures18-22,25,28-31, as well as from anatomical analyses and atlases23,32-37, the present protocol presents a comprehensive set of guidelines that address inconsistencies in the extant literature. Complemented by the accompanying visual materials, this work is expected to promote clearer understanding of the MTL structures, and stir up interest of future research in adopting manual segmentation, either as a primary method of MTL tracing or as a supplementary method to automatic segmentation. By providing an accurate, intuitive, and convenient guide for understanding the MTL anatomy, this protocol will help researchers identify the location of all MTL subregions, relative to their neighboring structures, even when only some MTL structures are specifically targeted for analyses. This will not only increase localization accuracy but will also help tracers make informed decisions in cases of morphological variation, which is highly likely in the MTL. These guidelines can be applied to research involving structural and/or functional MRI investigations of the MTL, including volumetric analyses and brain anomaly detection, as well as localizing procedures for functional, anatomical, and tractographic analyses, in healthy groups. The present protocol could also be used to inform segmentation of MTL structures for patients (e.g., patients with atrophy), if the major anatomical landmarks are relatively preserved. Tracing clinical subjects&#8217; data can take additional time and effort, depending on the severity of atrophy and/or anatomical changes.
It is important to consider the distinction between gyri and cortices when defining ROI. Anatomically, gyrus here refers to both white matter and grey matter, while cortex refers to grey matter only. Depending on the intended use of the ROI, segmentations might include white matter or exclude it.
We recommend the tracing to be performed sequentially, substructure by substructure, one hemisphere at a time. Certain software packages27 allow for tracing borders outlined on one slice to be pasted onto subsequent slices, a feature that speeds up the process. It is always advisable to reference the opposing hemisphere as needed, in order to check for consistency across the two sides (e.g., in detecting anatomical landmarks). Alternatively, parallel tracing of the same structures within the two hemispheres can also be performed. Regardless of whether the tracing is sequential or parallel, once the process is complete, the tracers should double-check the end-result and make adjustments as needed, referencing both hemispheres and multiple plane views. Depending on the experience of the tracer and the resolution of the imaging data, manual segmentation of the MTL for healthy subject data can take from 8-10 hr or more, in the case of a novice tracer, to 3-4 hr, in the case of an experienced one.
<img alt="Figure 1" fo:content-width="4in" src="/files/ftp_upload/50991/50991fig1highres.jpg" width="400" /> 
Figure 1. A 3D overview of the MTL, traced using the present protocol. Structures shown here are the AMY (red), the HC (blue), the PRC (yellow), the ERC (pink), and the PHC (green).

<table border="0" cellpadding="0" cellspacing="0" width="836">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;">ITK-SNAP</td>
			<td>ITK-SNAP Team at University of Pennsylvania and University of Utah</td>
			<td>ITK-SNAP v2.2</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FSL</td>
			<td>Functional Magnetic Resonance Imaging of the Brain (FMRIB) Analysis Group</td>
			<td>FSL v4.1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">3T Siemens Trio MR Scanner</td>
			<td>Siemens</td>
			<td>3T Trio</td>
			<td></td>
		</tr>
	</tbody>
</table>

a comprehensive protocol for manual segmentation of the medial temporal lobe structures

The present paper describes a comprehensive protocol for manual tracing of the set of brain regions comprising the medial temporal lobe (MTL): amygdala, hippocampus, and the associated parahippocampal regions (perirhinal, entorhinal,&#160;and parahippocampal proper). Unlike most other tracing protocols available, typically focusing on certain MTL areas (e.g., amygdala and/or hippocampus), the integrative perspective adopted by the present tracing guidelines allows for clear localization of all MTL subregions. By integrating information from a variety of sources, including extant tracing protocols separately targeting various MTL structures, histological reports, and brain atlases, and with the complement of illustrative visual materials, the present protocol provides an accurate, intuitive, and convenient guide for understanding the MTL anatomy. The need for such tracing guidelines is also emphasized by illustrating possible differences between automatic and manual segmentation protocols. This knowledge can be applied toward research involving not only structural MRI investigations but also structural-functional colocalization and fMRI signal extraction from anatomically defined ROIs, in healthy and clinical groups alike.

Illustration of Possible Differences between Manual and Automatic Segmentation
A 3D model of the manual segmentation for the AMY, HC, PRC, ERC, and PHC is shown in&#160;Figure 1, and a sagittal section of the segmentation is shown in&#160;Figure 2. For the purpose of illustrating extreme possible differences between manual and automatic tracings, slices of the AMY from a representative subject with erroneous automated segmentation were juxtapo...

Watch this Scientific Journal Video about A Comprehensive Protocol for Manual Segmentation of the Medial Temporal Lobe Structures at JoVE.com

A Comprehensive Protocol for Manual Segmentation of the Medial Temporal Lobe Structures

The present work provides a comprehensive set of guidelines for manually tracing the medial temporal lobe (MTL) structures. This protocol can be applied to research involving structural and/or combined structural-functional magnetic resonance imaging (MRI) investigations of the MTL, in both healthy and clinical groups.

The present paper describes a comprehensive protocol for manual tracing of the set of brain regions comprising the medial temporal lobe (MTL): amygdala, ...

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20.1K ビュー.  University of Illinois Urbana-Champaign. The present work provides a comprehensive set of guidelines for manually tracing the medial temporal lobe (MTL) structures. This protocol can be applied to research involving structural and/or combined structural-functional magnetic resonance imaging (MRI) investigations of the MTL, in both healthy and clinical groups.

ビデオ: A Comprehensive Protocol for Manual Segmentation of the Medial Temporal Lobe Structures

The present work provides a comprehensive set of guidelines for manually tracing the medial temporal lobe (MTL) structures. This protocol can be applied to research involving structural and/or combined structural-functional magnetic resonance imaging (MRI) investigations of the MTL, in both healthy and clinical...

high-resolution in vivo manual segmentation protocol for human hippocampal subfields using 3t magnetic resonance imaging

High-resolution In Vivo Manual Segmentation Protocol for Human Hippocampal Subfields Using 3T Magnetic Resonance Imaging

The goal of this manuscript is to study the hippocampus and hippocampal subfields using MRI. The manuscript describes a protocol for segmenting the hippocampus and five hippocampal substructures: cornu ammonis (CA) 1, CA2/CA3, CA4/dentate gyrus, strata radiatum/lacunosum/moleculare, and subiculum.

High-resolution In Vivo Manual Segmentation Protocol for Human Hippocampal Subfields Using 3T Magnetic Resonance Imaging

manual segmentation of the human choroid plexus using brain mri

Author Spotlight: Bridging Gaps in Anatomy and Establishing a Foundation for Algorithmic Studies 

Despite the crucial role of the choroid plexus in the brain, neuroimaging studies of this structure are scarce due to the lack of reliable automated segmentation tools. The present protocol aims to ensure gold-standard manual segmentation of the choroid plexus that can inform future neuroimaging studies.

Manual Segmentation of the Human Choroid Plexus Using Brain MRI

segmentation of the choroid plexus using manual delineations

Segmentation of the Choroid Plexus Using Manual Delineations  

Enhancing Neuroimaging Analysis in Brain Disorders

https://cloudfront.jove.com/CDNSource/teasers/65341_b.jpg

systemic pilocarpine treatment: mouse model of temporal lobe epilepsy

30 minutes after the scopolamine and terbutaline administration, inject pilocarpine solution into each mouse. Immediately after pilocarpine injection, place the mice in an incubator with 28 to 30 degrees Celsius for observation. Next, monitor the behavior of the mice until status epilepticus or SE is induced. If limbic motor seizures that correspond to stage 3 or higher according to Racine&#39;s scale are detected, record the time and monitor the...

Systemic Pilocarpine Treatment: Mouse Model of Temporal Lobe Epilepsy

This video presents the procedure for inducing status epilepticus (SE) in mice by systemic administration of pilocarpine to model temporal lobe...

network analysis of the default mode network using functional connectivity mri in temporal lobe epilepsy

Network Analysis of the Default Mode Network Using Functional Connectivity MRI in Temporal Lobe Epilepsy

The Default Mode Network (DMN) in Temporal Lobe Epilepsy (TLE) is analyzed in the resting state of the brain using seed-based functional connectivity MRI...

using a bipolar electrode to create a temporal lobe epilepsy mouse model by electrical kindling of the amygdala

Using a Bipolar Electrode to Create a Temporal Lobe Epilepsy Mouse Model by Electrical Kindling of the Amygdala

The amygdala plays a key role in temporal lobe epilepsy, which originates in and propagates from this structure. This article provides a detailed description of the fabrication of deep brain electrodes with both recording and stimulating functions. It introduces a model of medial temporal lobe epilepsy originating from the...

the pilocarpine model of temporal lobe epilepsy and eeg monitoring using radiotelemetry system in mice

The Pilocarpine Model of Temporal Lobe Epilepsy and EEG Monitoring Using Radiotelemetry System in Mice

This manuscript describes a method of inducing status epilepticus by systemic pilocarpine injection and of monitoring spontaneous recurrent seizures in live animals using a wireless telemetry video and electroencephalogram system. This protocol can be utilized for studying the pathophysiologic mechanisms of chronic epilepsy, epileptogenesis, and acute...

extracting the cochlea from a human temporal bone: a cadaveric protocol

Extracting the Cochlea from a Human Temporal Bone: A Cadaveric Protocol

This article presents a reliable method for extracting the human cochlea from the cadaver temporal bone by drill-out while following distinct anatomical landmarks.

preparation and implantation of electrodes for electrically kindling vgat-cre mice to generate a model for temporal lobe epilepsy

Preparation and Implantation of Electrodes for Electrically Kindling VGAT-Cre Mice to Generate a Model for Temporal Lobe Epilepsy

This report describes the methods to generate a model of temporal lobe epilepsy based on the electrical kindling of transgenic VGAT-Cre mice. Kindled VGAT-Cre mice may be useful in determining what causes epilepsy and for screening novel...

network analysis of foramen ovale electrode recordings in drug-resistant temporal lobe epilepsy patients

Network Analysis of Foramen Ovale Electrode Recordings in Drug-resistant Temporal Lobe Epilepsy Patients

This protocol describes a procedure to track the evolution of mesial network measures in temporal lobe epilepsy (TLE) patients. It is based on the combination of intracranial recordings with a novel numerical technique for data analysis. Specifically, we present a protocol for network analyses of foramen ovale...

a protocol of manual tests to measure sensation and pain in humans

A Protocol of Manual Tests to Measure Sensation and Pain in Humans

The goal of this procedure is to demonstrate a battery of quantitative techniques for sensory and pain measurement in humans. The equipment and techniques described are commonly found in pain clinics or are easy to...

a protocol for comprehensive assessment of bulbar dysfunction in amyotrophic lateral sclerosis (als)

A Protocol for Comprehensive Assessment of Bulbar Dysfunction in Amyotrophic Lateral Sclerosis ALS

Objective assessments of the physiological mechanisms that support speech are needed to monitor disease onset and progression in persons with ALS and to quantify treatment effects in clinical trials. In this video, we present a comprehensive, instrumentation-based protocol for quantifying speech motor performance in clinical populations.

A Protocol for Comprehensive Assessment of Bulbar Dysfunction in Amyotrophic Lateral Sclerosis (ALS)

a label-free segmentation approach for intravital imaging of mammary tumor microenvironment

A Label-Free Segmentation Approach for Intravital Imaging of Mammary Tumor Microenvironment

The intravital imaging method described here utilizes collagen second harmonic generation and endogenous fluorescence from the metabolic co-factor NAD(P)H to non-invasively segment an unlabeled tumor microenvironment into tumor, stromal, and vascular compartments for in-depth analysis of 4D intravital...

a label-free technique for the spatio-temporal imaging of single cell secretions

A Label-free Technique for the Spatio-temporal Imaging of Single Cell Secretions

Inter-cellular communication is critical for controlling various physiological activities within and outside the cell. This paper describes a protocol for measuring the spatio-temporal nature of single cell secretions. To achieve this, a multidisciplinary approach is used which integrates label-free nanoplasmonic sensing with live cell...

a comprehensive high-efficiency protocol for isolation, culture, polarization, and glycolytic characterization of bone marrow-derived macrophages

A Comprehensive High-Efficiency Protocol for Isolation, Culture, Polarization, and Glycolytic Characterization of Bone Marrow-Derived Macrophages

This protocol provides detailed and comprehensive methods for the isolation, culture, polarization, and measurement of the glycolytic metabolic state of live bone marrow-derived macrophages (BMDMs). This paper provides step-by-step instructions with realistic visual illustrations for workflow and glycolytic assessment of BMDMs in...

simulating the mechanics of lens accommodation via a manual lens stretcher

Simulating the Mechanics of Lens Accommodation via a Manual Lens Stretcher

We present an efficient method of studying lens accommodation by using a manual lens stretcher. The protocol mimics physiological accommodation by pulling the zonules connected around the lens capsule, thereby, stretching the...

experimental protocol to determine the chloride threshold value for corrosion in samples taken from reinforced concrete structures

Experimental Protocol to Determine the Chloride Threshold Value for Corrosion in Samples Taken from Reinforced Concrete Structures

We propose a method to measure a parameter that is highly relevant for corrosion assessments or predictions of reinforced concrete structures, with the main advantage of permitting testing of samples from engineering structures. This ensures real conditions at the steel-concrete interface, which are crucial to avoid artifacts of laboratory-made...

The NeuroStar TMS Device: Conducting the FDA Approved Protocol for Treatment of Depression

The Neuronetics NeuroStar Transcranial Magnetic Stimulation (TMS) System is a class II medical device that produces brief duration, pulsed magnetic fields. These rapidly alternating fields induce electrical currents within localized, targeted regions of the cortex which are associated with various physiological and functional brain changes.1,2,3
In 2007, O'Reardon et al., utilizing the NeuroStar device, published the results of an industry-sponsored, multisite, randomized, sham-stimulation controlled clinical trial in which 301 patients with major depression, who had previously failed to respond to at least one adequate antidepressant treatment trial, underwent either active or sham TMS over the left dorsolateral prefrontal cortex (DLPFC). The patients, who were medication-free at the time of the study, received TMS five times per week over 4-6 weeks.4
The results demonstrated that a sub-population of patients (those who were relatively less resistant to medication, having failed not more than two good pharmacologic trials) showed a statistically significant improvement on the Montgomery-Asberg Depression Scale (MADRS), the Hamilton Depression Rating Scale (HAMD), and various other outcome measures. In October 2008, supported by these and other similar results5,6,7, Neuronetics obtained the first and only Food and Drug Administration (FDA) approval for the clinical treatment of a specific form of medication-refractory depression using a TMS Therapy device (FDA approval K061053).
In this paper, we will explore the specified FDA approved NeuroStar depression treatment protocol (to be administered only under prescription and by a licensed medical profession in either an in- or outpatient setting).

In this article, we examine the methodology and considerations relevant to the FDA approved depression treatment protocol using the Neuronetics NeuroStar TMS device.

The Neuronetics NeuroStar Transcranial Magnetic Stimulation (TMS) System is a class II medical device that produces brief duration, pulsed magnetic ...

Calcium Imaging of Odor-evoked Responses in the Drosophila Antennal Lobe

The antennal lobe is the primary olfactory center in the insect brain and represents the anatomical and functional equivalent of the vertebrate olfactory bulb1-5. Olfactory information in the external world is transmitted to the antennal lobe by olfactory sensory neurons (OSNs), which segregate to distinct regions of neuropil called glomeruli according to the specific olfactory receptor they express. Here, OSN axons synapse with both local interneurons (LNs), whose processes can innervate many different glomeruli, and projection neurons (PNs), which convey olfactory information to higher olfactory brain regions.
Optical imaging of the activity of OSNs, LNs and PNs in the antennal lobe - traditionally using synthetic calcium indicators (e.g. calcium green, FURA-2) or voltage-sensitive dyes (e.g. RH414) - has long been an important technique to understand how olfactory stimuli are represented as spatial and temporal patterns of glomerular activity in many species of insects6-10. Development of genetically-encoded neural activity reporters, such as the fluorescent calcium indicators G-CaMP11,12 and Cameleon13, the bioluminescent calcium indicator GFP-aequorin14,15, or a reporter of synaptic transmission, synapto-pHluorin16 has made the olfactory system of the fruitfly, Drosophila melanogaster, particularly accessible to neurophysiological imaging, complementing its comprehensively-described molecular, electrophysiological and neuroanatomical properties2,4,17. These reporters can be selectively expressed via binary transcriptional control systems (e.g. GAL4/UAS18, LexA/LexAop19,20, Q system21) in defined populations of neurons within the olfactory circuitry to dissect with high spatial and temporal resolution how odor-evoked neural activity is represented, modulated and transformed22-24.
Here we describe the preparation and analysis methods to measure odor-evoked responses in the Drosophila antennal lobe using G-CaMP25-27. The animal preparation is minimally invasive and can be adapted to imaging using wide-field fluorescence, confocal and two-photon microscopes.

Calcium Imaging of Odor-evoked Responses in the Drosophila Antennal Lobe

We describe an established technique to measure and analyze odor-evoked calcium responses in the antennal lobe of living Drosophila melanogaster.

The antennal lobe is the primary olfactory center in the insect brain and represents the anatomical and functional equivalent of the vertebrate olfactory ...

Preparation of Parasagittal Slices for the Investigation of Dorsal-ventral Organization of the Rodent Medial Entorhinal Cortex

Computation in the brain relies on neurons responding appropriately to their synaptic inputs. Neurons differ in their complement and distribution of membrane ion channels that determine how they respond to synaptic inputs. However, the relationship between these cellular properties and neuronal function in behaving animals is not well understood. One approach to this problem is to investigate topographically organized neural circuits in which the position of individual neurons maps onto information they encode or computations they carry out1. Experiments using this approach suggest principles for tuning of synaptic responses underlying information encoding in sensory and cognitive circuits2,3.
The topographical organization of spatial representations along the dorsal-ventral axis of the medial entorhinal cortex (MEC) provides an opportunity to establish relationships between cellular mechanisms and computations important for spatial cognition. Neurons in layer II of the rodent MEC encode location using grid-like firing fields4-6. For neurons found at dorsal positions in the MEC the distance between the individual firing fields that form a grid is on the order of 30 cm, whereas for neurons at progressively more ventral positions this distance increases to greater than 1 m. Several studies have revealed cellular properties of neurons in layer II of the MEC that, like the spacing between grid firing fields, also differ according to their dorsal-ventral position, suggesting that these cellular properties are important for spatial computation2,7-10.
Here we describe procedures for preparation and electrophysiological recording from brain slices that maintain the dorsal-ventral extent of the MEC enabling investigation of the topographical organization of biophysical and anatomical properties of MEC neurons. The dorsal-ventral position of identified neurons relative to anatomical landmarks is difficult to establish accurately with protocols that use horizontal slices of MEC7,8,11,12, as it is difficult to establish reference points for the exact dorsal-ventral location of the slice. The procedures we describe enable accurate and consistent measurement of location of recorded cells along the dorsal-ventral axis of the MEC as well as visualization of molecular gradients2,10. The procedures have been developed for use with adult mice (&gt; 28 days) and have been successfully employed with mice up to 1.5 years old. With adjustments they could be used with younger mice or other rodent species. A standardized system of preparation and measurement will aid systematic investigation of the cellular and microcircuit properties of this area.

We describe procedures for preparation and electrophysiological recording from brain slices that maintain the dorsal-ventral axis of the medial entorhinal cortex (MEC). Because neural encoding of location follows a dorsal-ventral organization within the MEC, these procedures facilitate investigation of cellular mechanisms important for navigation and memory.

Computation in the brain relies on neurons responding appropriately to their synaptic inputs. Neurons differ in their complement and distribution of ...

Manual Drainage of the Zebrafish Embryonic Brain Ventricles

Cerebrospinal fluid (CSF) is a protein rich fluid contained within the brain ventricles. It is present during early vertebrate embryonic development and persists throughout life. Adult CSF is thought to cushion the brain, remove waste, and carry secreted molecules1,2. In the adult and older embryo, the majority of CSF is made by the choroid plexus, a series of highly vascularized secretory regions located adjacent to the brain ventricles3-5. In zebrafish, the choroid plexus is fully formed at 144 hours post fertilization (hpf)6. Prior to this, in both zebrafish and other vertebrate embryos including mouse, a significant amount of embryonic CSF (eCSF) is present . These data and studies in chick suggest that the neuroepithelium is secretory early in development and may be the major source of eCSF prior to choroid plexus development7.
eCSF contains about three times more protein than adult CSF, suggesting that it may have an important role during development8,9. Studies in chick and mouse demonstrate that secreted factors in the eCSF, fluid pressure, or a combination of these, are important for neurogenesis, gene expression, cell proliferation, and cell survival in the neuroepithelium10-20. Proteomic analyses of human, rat, mouse, and chick eCSF have identified many proteins that may be necessary for CSF function. These include extracellular matrix components, apolipoproteins, osmotic pressure regulating proteins, and proteins involved in cell death and proliferation21-24. However, the complex functions of the eCSF are largely unknown.
We have developed a method for removing eCSF from zebrafish brain ventricles, thus allowing for identification of eCSF components and for analysis of the eCSF requirement during development. Although more eCSF can be collected from other vertebrate systems with larger embryos, eCSF can be collected from the earliest stages of zebrafish development, and under genetic or environmental conditions that lead to abnormal brain ventricle volume or morphology. Removal and collection of eCSF allows for mass spectrometric analysis, investigation of eCSF function, and reintroduction of select factors into the ventricles to assay their function. Thus the accessibility of the early zebrafish embryo allows for detailed analysis of eCSF function during development.

We present a method to collect cerebrospinal fluid (CSF) and to create a system which lacks CSF within the embryonic zebrafish brain ventricular system. This allows for further examination of CSF composition and its requirement during embryonic brain development.

Cerebrospinal fluid  (CSF) is a protein rich fluid contained within the brain ventricles. It is  present during early vertebrate embryonic development and ...

Identification of Olfactory Volatiles using Gas Chromatography-Multi-unit Recordings (GCMR) in the Insect Antennal Lobe

All organisms inhabit a world full of sensory stimuli that determine their behavioral and physiological response to their environment. Olfaction is especially important in insects, which use their olfactory systems to respond to, and discriminate amongst, complex odor stimuli. These odors elicit behaviors that mediate processes such as reproduction and habitat selection1-3. Additionally, chemical sensing by insects mediates behaviors that are highly significant for agriculture and human health, including pollination4-6, herbivory of food crops7, and transmission of disease8,9. Identification of olfactory signals and their role in insect behavior is thus important for understanding both ecological processes and human food resources and well-being.
	To date, the identification of volatiles that drive insect behavior has been difficult and often tedious. Current techniques include gas chromatography-coupled electroantennogram recording (GC-EAG), and gas chromatography-coupled single sensillum recordings (GC-SSR)10-12. These techniques proved to be vital in the identification of bioactive compounds. We have developed a method that uses gas chromatography coupled to multi-channel electrophysiological recordings (termed 'GCMR') from neurons in the antennal lobe (AL; the insect's primary olfactory center)13,14. This state-of-the-art technique allows us to probe how odor information is represented in the insect brain. Moreover, because neural responses to odors at this level of olfactory processing are highly sensitive owing to the degree of convergence of the antenna's receptor neurons into AL neurons, AL recordings will allow the detection of active constituents of natural odors efficiently and with high sensitivity. Here we describe GCMR and give an example of its use.
	Several general steps are involved in the detection of bioactive volatiles and insect response. Volatiles first need to be collected from sources of interest (in this example we use flowers from the genus Mimulus (Phyrmaceae)) and characterized as needed using standard GC-MS techniques14-16. Insects are prepared for study using minimal dissection, after which a recording electrode is inserted into the antennal lobe and multi-channel neural recording begins. Post-processing of the neural data then reveals which particular odorants cause significant neural responses by the insect nervous system.
	Although the example we present here is specific to pollination studies, GCMR can be expanded to a wide range of study organisms and volatile sources. For instance, this method can be used in the identification of odorants attracting or repelling vector insects and crop pests. Moreover, GCMR can also be used to identify attractants for beneficial insects, such as pollinators. The technique may be expanded to non-insect subjects as well.

Identification of Olfactory Volatiles using Gas Chromatography-Multi-unit Recordings GCMR in the Insect Antennal Lobe

Olfactory cues mediate many different behaviors in insects, and are often complex mixtures comprised of tens to hundreds of volatile compounds. Using gas chromatography with multi-channel recording in the insect antennal lobe, we describe a method for the identification of bioactive compounds.

All  organisms inhabit a world full of sensory stimuli that determine their  behavioral and physiological response to their environment. Olfaction is ...

The human hippocampus has been broadly studied in the context of memory and normal brain function and its role in different neuropsychiatric disorders has been heavily studied. While many imaging studies treat the hippocampus as a single unitary neuroanatomical structure, it is, in fact, composed of several subfields that have a complex three-dimensional geometry. As such, it is known that these subfields perform specialized functions and are differentially affected through the course of different disease states. Magnetic resonance (MR) imaging can be used as a powerful tool to interrogate the morphology of the hippocampus and its subfields. Many groups use advanced imaging software and hardware (&#62;3T) to image the subfields; however this type of technology may not be readily available in most research and clinical imaging centers. To address this need, this manuscript provides a detailed step-by-step protocol for segmenting the full anterior-posterior length of the hippocampus and its subfields: cornu ammonis (CA) 1, CA2/CA3, CA4/dentate gyrus (DG), strata radiatum/lacunosum/moleculare (SR/SL/SM), and subiculum. This protocol has been applied to five subjects (3F, 2M; age 29-57, avg. 37). Protocol reliability is assessed by resegmenting either the right or left hippocampus of each subject and computing the overlap using the Dice&#39;s kappa metric. Mean Dice&#39;s kappa (range) across the five subjects are: whole hippocampus, 0.91 (0.90-0.92); CA1, 0.78 (0.77-0.79); CA2/CA3, 0.64 (0.56-0.73); CA4/dentate gyrus, 0.83 (0.81-0.85); strata radiatum/lacunosum/moleculare, 0.71 (0.68-0.73); and subiculum 0.75 (0.72-0.78). The segmentation protocol presented here provides other laboratories with a reliable method to study the hippocampus and hippocampal subfields in vivo using commonly available MR tools.

The goal of this manuscript is to study the hippocampus and hippocampal subfields using MRI. The manuscript describes a protocol for segmenting the hippocampus and five hippocampal substructures: cornu ammonis (CA) 1, CA2/CA3, CA4/dentate gyrus, strata radiatum/lacunosum/moleculare, and subiculum.

The human hippocampus has been broadly studied in the context of memory and normal brain function and its role in different neuropsychiatric disorders has ...

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the molecular and cellular basis of higher brain functions. A whole-mount preparation of adult brains with well-preserved morphology is critical for such whole brain-based studies, but can be technically challenging and time-consuming. This protocol describes an easy-to-learn, one-step dissection approach of an adult fly head in less than 10 s, while keeping the intact brain attached to the rest of the body to facilitate subsequent processing steps. The procedure helps remove most of the eye and tracheal tissues normally associated with the brain that can interfere with the later imaging step, and also places less demand on the quality of the dissecting forceps. Additionally, we describe a simple method that allows convenient flipping of the mounted brain samples on a coverslip, which is important for imaging both sides of the brains with similar signal intensity and quality. As an example of the protocol, we present an analysis of dopaminergic (DA) neurons in adult brains of WT (w1118) flies. The high efficacy of the dissection method makes it particularly useful for large-scale adult brain-based studies in Drosophila.

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

The adult Drosophila brain is a valuable system for studying neuronal circuitry, higher brain functions, and complex disorders. An efficient method to dissect whole brain tissue from the small fly head will facilitate brain-based studies. Here we describe a simple, one-step dissection protocol of adult brains with well-preserved morphology.

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the ...

Exposure of the Pig CNS for Histological Analysis: A Manual for Decapitation, Skull Opening, and Brain Removal

Pigs have become increasingly popular in large-animal translational neuroscience research as an economically and ethically feasible substitute to non-human primates. The large brain size of the pig allows the use of conventional clinical brain imagers and the direct use and testing of neurosurgical procedures and equipment from the human clinic. Further macroscopic and histological analysis, however, requires postmortem exposure of the pig central nervous system (CNS) and subsequent brain removal. This is not an easy task, as the pig CNS is encapsulated by a thick, bony skull and spinal column. The goal of this paper and instructional video is to describe how to expose and remove the postmortem pig brain and the pituitary gland in an intact state, suitable for subsequent macroscopic and histological analysis.

The goal of this paper and instructional video is to describe how to expose and remove the postmortem pig brain and pituitary gland in an intact state, suitable for subsequent macroscopic and histological analysis.

Pigs have become increasingly popular in large-animal translational neuroscience research as an economically and ethically feasible substitute to ...

The Power of Interstimulus Interval for the Assessment of Temporal Processing in Rodents

Temporal processing deficits have been implicated as a potential elemental dimension of higher-level cognitive processes, commonly observed in neurocognitive disorders. Despite the popularization of prepulse inhibition (PPI) in recent years, many current protocols promote using a percent of control measure, thereby precluding the assessment of temporal processing. The present study used cross-modal PPI and gap prepulse inhibition (gap-PPI) to demonstrate the benefits of employing a range of interstimulus intervals (ISIs) to delineate effects of sensory modality, psychostimulant exposure, and age. Assessment of sensory modality, psychostimulant exposure, and age reveals the utility of an approach varying the interstimulus interval (ISI) to establish the shape of the ISI function, including increases (sharper curve inflections) or decreases (flattening of the response amplitude curve) in startle amplitude. Additionally, shifts in peak response inhibition, suggestive of a differential sensitivity to the manipulation of ISI, are often revealed. Thus, the systematic manipulation of ISI affords a critical opportunity to evaluate temporal processing, which may reveal the underlying neural mechanisms involved in neurocognitive disorders.

Temporal processing, a preattentive process, may underlie deficits in higher-level cognitive processes, including attention, commonly observed in neurocognitive disorders. Using prepulse inhibition as an exemplar paradigm, we present a protocol for manipulating interstimulus interval (ISI) to establish the shape of the ISI function to provide an assessment of temporal processing.

Temporal processing deficits have been implicated as a potential elemental dimension of higher-level cognitive processes, commonly observed in ...

Stochastic Noise Application for the Assessment of Medial Vestibular Nucleus Neuron Sensitivity In Vitro

Galvanic vestibular stimulation (GVS) has been shown to improve balance measures in individuals with balance or vestibular impairments. This is proposed to be due to the stochastic resonance (SR) phenomenon, which is defined as application of a low-level/subthreshold stimulus to a non-linear system to increase detection of weaker signals. However, it is still unknown how SR exhibits its positive effects on human balance. This is one of the first demonstrations of the effects of sinusoidal and stochastic noise on individual neurons. Using whole-cell patch clamp electrophysiology, sinusoidal and stochastic noise can be applied directly to individual neurons in the medial vestibular nucleus (MVN) of C57BL/6 mice. Here we demonstrate how to determine the threshold of MVN neurons in order to ensure the sinusoidal and stochastic stimuli are subthreshold and from this, determine the effects that each type of noise has on MVN neuronal gain. We show that subthreshold sinusoidal and stochastic noise can modulate the sensitivity of individual neurons in the MVN without affecting basal firing rates.

Galvanic vestibular stimulation in humans exhibits improvements in vestibular function. However, it is unknown how these effects occur. Here, we describe how to apply sinusoidal and stochastic electrical noise and evaluate appropriate stimulus amplitudes in individual medial vestibular nucleus neurons in the C57BL/6 mouse.

Galvanic vestibular stimulation (GVS) has been shown to improve balance measures in individuals with balance or vestibular impairments. This is proposed ...