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 uncus. Follow the white matter tract around the AMY in the axial view to exclude the entorhinal area32.</li>
		<li>Moving posteriorly, identify the first slice in which the anterior commissure is continuous throughout both hemispheres20, where the AMY is visible in its typical shape. Trace the AMY counter-clockwise using the entorhinal sulcus as the superomedial border, the imaginary line from the fundus of the semianular sulcus along the white matter to the inferior tip of the AMY as the inferomedial border, the temporal stem as the lateral border, and back to the entorhinal sulcus to complete the tracing31.</li>
	</ol>
	</li>
	<li>Posterior Slices of the AMY
	<ol>
		<li>Note that at this level, both the AMY and HC are visible in the same slice(s).</li>
		<li>Still in the coronal view, identify the last slice of the AMY where the structure is superior to the medial extension of the temporal horn of lateral ventricle38 (or the alveus if the ventricle is not present) and lateral to the uncinate gyrus, the protuberance of the head of the HC. Check the sagittal and axial views for accurate and consistent tracing.</li>
		<li>Draw an imaginary line from the fundus of the inferior circular sulcus of the insula to the optic tract as the superior border of the AMY31, which also differentiates it from the grey matter of the globus pallidus and the putamen.</li>
		<li>Trace along the semilunar gyrus as the superomedial border and exclude the uncinate gyrus32. Use the inferior horn of the lateral ventricle and the temporal stem for the lateral delineation.</li>
	</ol>
	</li>
	<li>Consecutive Slices of the AMY in Anterior-posterior Direction
	<ol>
		<li>Systematically trace the AMY slice-by-slice using the relevant guidelines above. At the anterior portion of the AMY, use the same boundaries as for the anterior-most slice; conversely, at the posterior portion of the AMY, use the same boundaries as for the posterior-most slice.</li>
		<li>Continue to employ the axial and sagittal views to help define and further refine the AMY borders.</li>
	</ol>
	</li>
</ol>
2. Hippocampus
<ol>
	<li>Localizing the HC
	<ol>
		<li>Begin tracing the HC when the temporal horn of the lateral ventricle appears along the inferolateral border of the AMY. If the temporal horn of the lateral ventricle is already present on previous slices, note that the onset of the HC is then indicated by the temporal horn of the lateral ventricle enlarging and stretching superolaterally.</li>
		<li>End the tracing of the HC with its last appearance inferomedial to the trigone of the lateral ventricle31. Always utilize the alternative views to help localize the HC and its borders.</li>
	</ol>
	</li>
	<li>Border Definitions of the HC
	<ol>
		<li>Delineate the lateral HC against the temporal horn. In cases where the temporal horn of the lateral ventricle is not perceivable enough, exclude one row of voxels from the segmentation to denote it.</li>
		<li>Inferiorly, use the angular bundle (or its imaginary extension) to the ventricular cavity to separate the HC from the PHG. Use the alveus along with the fimbria as the superior border. Trace the HC using the same definitions throughout.</li>
		<li>Additionally, include the subiculum into the segmentation such that it medially borders the white matter bundle&#160;of the PHG, superiorly aligns with the curve of the uncus, and extends primarily horizontally from the HC39. Tracing posteriorly, maintain these definitions until the calcarine sulcus intervenes.</li>
	</ol>
	</li>
	<li>Noting Divisions of the&#160;HC
	<ol>
		<li>Note that the HC can be divided into three segments: head, body, and tail.</li>
		<li>Use the appearance of the uncal apex to mark the transition from the HC head to the HC body, and the rapid ascending and expansion in size, which typically coincides with the appearance of the crus of the fornix, to signify the appearance of the HC tail23,30,40.</li>
	</ol>
	</li>
	<li>Pay special attention when tracing the following structures.
	<ol>
		<li>Include the posterior uncus in the segmentation.</li>
		<li>Omit the choroid plexus above the alveus from the segmentation on the coronal slices, although this may not be possible on lower-resolution images.</li>
		<li>Refer to the two alternative views to avoid the inclusion of the tail of the caudate and the pulvinar at the superior aspect of the HC tail.</li>
		<li>Avoid inclusion of the fascicular gyrus by noting its emergence at the level of the crus of the fornix, where initially it is separated from the hippocampal tail by the fasciola cinerea and more posteriorly becomes the grey matter superior to the calcarine sulcus32.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 2" fo:content-width="4in" src="/files/ftp_upload/50991/50991fig2highres.jpg" width="400" /> 
Figure 2. A representative sagittal slice of the MTL traced using the present protocol, showing its actual position in the brain, and the relative positions among its major structures, i.e., the AMY (red), the HC (blue),&#160;the PRC (yellow), the ERC (pink),&#160;and the PHC (green).
3. Parahippocampal Gyrus
<ol>
	<li>Noting Divisions of the PHG
	<ol>
		<li>Note that the PHG can be divided into two main segments: the anterior PHG (i.e., the PRC and ERC), and the posterior PHG (i.e., the PHC).</li>
		<li>Note that in the anterior segment, the PRC appears earlier than the ERC, and flanks it laterally through its entire course.</li>
		<li>After the ERC disappears, note that the PRC subsumes its place on the PHG and continues on for 3 mm.</li>
		<li>Beyond this segment, trace the posterior PHG, where the PHC takes over the width of the PHG until its end30.</li>
	</ol>
	</li>
	<li>Anterior Slices of the PHG
	<ol>
		<li>Define the first slice of the PRC with the appearance of the CS25,34. Before the onset of the ERC, trace the PRC from the medial edge of the lateral bank of the CS to the lateral fundus of the gyrus of Schwalbe, or that of the medial one if two gyri of Schwalbe are present, or the midpoint of the dorsal temporopolar surface in the absence of this gyrus25,34.</li>
		<li>Start to trace the ERC 5 mm anterior to the limen insula35,36.</li>
		<li>Continue tracing the ERC using the fundus of the medial temporopolar sulcus as the superior end35, and the fundus of the semiannular sulcus after the AMY appears, or the point where the imaginary extension of the angular bundle meets the ventricular cavity if the semiannular sulcus is indistinguishable25. Note that it extends inferiorly to meet the ventricular cavity or the pial surface directly.</li>
		<li>The border between the PRC and the ERC may vary from slice to slice.&#160;
		<ol>
			<li>When the CS is deep (&#8805; 1.5 cm), trace the PRC from the medial edge of the medial bank of this sulcus, to the midpoint of its lateral bank25&#160;.</li>
			<li>In cases with a regular CS (depth of 1&#8211;1.5 cm), trace the PRC as the area from the midpoint of the medial bank of the collateral sulcus to the medial end of the lateral bank of the sulcus25.</li>
			<li>With a shallow CS (&#60; 1 cm), trace the PRC from the fundus of this sulcus to the midpoint of the crown of the fusiform gyrus25 .</li>
		</ol>
		</li>
		<li>When the CS is interrupted, usually at the level of the uncal apex, by a small gyrus emerging from its fundus, trace the PRC to the fundus of the lateral sulcus25. Include or exclude white matter according to the goal for the ROI.</li>
		<li>Trace the ERC until 1.5 mm posterior to the uncal apex, or the end of the gyrus intralimbicus37 .</li>
		<li>Extend the tracing of the PRC medially to occupy the place of the ERC after its termination, where the definitions for the latter continue to apply until 4.5 mm posterior to the uncal apex, or the end of the gyrus intralimbicus37. The PRC is then substituted by the PHC25,30.</li>
	</ol>
	</li>
	<li>Posterior Slices of the PHG
	<ol>
		<li>Start to trace the PHC on the slice posterior to the end of the PRC, until 4 mm posterior to the end of the HC tail32. Alternative definitions from the literature are described in the Discussion section. Again, include or exclude white matter depending on the goal.</li>
		<li>Delineate the PHC using the same method described in the posterior portion of the PRC after the disappearance of the ERC. Also use the white matter of the cingulum as the superior border once it appears. Continue tracing in this fashion until the appearance of the calcarine sulcus, which restricts the PHC superomedially to the inferior edge of the sulcus30.</li>
		<li>Should a mini-sulcus appear before the emergence of the calcarine sulcus, include it in the segmentation, but be cautious in differentiating it from the calcarine sulcus.</li>
	</ol>
	</li>
</ol>

The authors have no conflicts of interest to declare.

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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20.2K Views.  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.

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

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 ...

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

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.

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.

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 ...