We thank C. Montojo, J. Reyes, and M. Armour for technical advice and assistance. This work was supported by US National Institutes of Health grant F31 NS063550 (to D.A.L.), a Basil O'Connor Starter Scholar Award and grants from the Klingenstein Fund and NARSAD (to S.B.). S.B. is a W.M. Keck Distinguished Young Scholar in Medical Research.

Animal Usage
Obtain approval from institutional Animal Care and Use Committee for standard care and use protocols. The current protocol was developed for focal irradiation studies on 5.5-10&#160;week&#160;old adult C57BL6/J mice, as previously described (Figure 2)2. However, other ages and small animal species (rats, hamsters, ground squirrels, etc.) can also be used, provided that effective anesthesia protocols and a radiographic reference atlas allowing identification of the region of interest (ROI) are available.
1. Calibration of CT-guided Radiological Platform
Perform film-based calibrations for radiation dose on the radiological platform in advance for each collimator size and type of X-ray tube filter used. In order to target an arc beam of irradiation at the desired target site, use the radiation dose-planning software49 available on most commercial radiation platforms to calculate required delivery time based on the radiation dose, the depth of the region of interest (ROI) based on CT scans of the mouse, and the rotation speed of the robotic platform; details on this are left out of this protocol as they differ between radiation platforms. For the purposes of visualizing the ventrobasal hypothalamus2 (Figure 3) with CT imaging, operate the X-ray tube with a 0.4-mm focal spot and beam energy of 100 kVp with 1 mm Al filtration. For focal irradiation of the HPZ, operate the X-ray tube at 225 kVp, 13 mA, and a 1-mm focal spot with filtration of 0.15 mm Cu, to deliver 10 Gy of radiation at a tissue depth of 0.6 cm over a treatment time of 4.6 min. Targeting between different ROIs will require calculating different parameters.
<ol>
	<li>If studying the effects of high-fat diet on neurogenesis in the median eminence as previously described2, obtain four-week-old female C57BL6/J mice from Jackson Mouse laboratories, and house four mice per cage. Switch food from normal chow to a high-fat diet at five weeks old. Allow mice to acclimate to new housing and food. Perform CFIR treatment at 5.5 weeks old. Transport subjects to the operation room that contains the radiological platform. Minimize stress levels during transfer. Prepare isoflurane gas anesthesia chamber. Then, add a single mouse into chamber. In parallel, prepare heating pad (low setting) for post-treatment.</li>
	<li>Once mouse is under and no longer responding to foot-pad compression, bring the subject to the radiological platform and place on the immobilization bed of the robotic stage. Place subject&#39;s mouth into the nose-cone anesthesia cup, and teeth into the bite guard (Figure 1D). Lay mouse flat on the immobilization bed and check to see if it continues to maintain unresponsiveness. If so, tape down mouse to the bed with gauze tape. While taping down the mouse to the bed, make sure the head is leveled to a horizontal plane. This can be determined by pulling up the ears and seeing if they are leveled. Once the mouse is in the correct position, close the lead protective shield.</li>
	<li>Acquire the computer-tomography scan using on-board software of the experimenter&#39;s radiological platform (this will differ between platforms), which will provide a three-dimensional anatomical structural scan of the mouse subject. Check to see if the mouse head is leveled to the horizontal plane. If not, repeat steps 1.2 - 1.3 until the subject&#39;s head is leveled.</li>
	<li>Identify ROI by CT image. Calculate distance from ROI to the surface of the skull using a 45&#176;&#160;angle to the horizontal plane as shown in Figure 3E.</li>
	<li>Using on-board software, take an X-ray of the mouse subject from above as shown in Figure 4A. Then, remove mouse from the radiological platform, put on heating pad, and monitor until active.</li>
	<li>From the coronal CT images, calculate the average ROI anatomical depth of at least three mice in order to determine delivery dosing. As an example from a previous study2 where 10 Gy of irradiation was administered to the ventrobasal hypothalamus, the depth of the ROI from the skull (from a 45&#176; angle) is 0.66 cm (See Figure 3E). Knowing that, the researchers used the dose planning software (DPS) installed on their radiological platform to calculate the appropriate rotation speed and length of treatment to achieve desired dosage to the ROI.</li>
	<li>After determining treatment duration and rotation speed of the robotic stage with the dose-planning software, measure dose distributions of the calculated parameters with GAFchromic radiation-sensitive films embedded in a water-equivalent plastic mock-mouse model. To do this, embed three GAFchromic radiation-sensitive films between four vertically stacked water-equivalent plastic blocks29 as shown in Figure 4B.</li>
	<li>Place mock mouse model containing GAFchromic films on robotic stage, and run the focal irradiation beam with the newly calculated parameters. For example, to target the ventrobasal hypothalamus, input the parameters of 0.15 mm Cu filter, 225 kVp, 13 mA, 1-mm diameter radiation beam setting, 45&#176; gantry angle, 1.3&#176;/sec rotation, and 4.6 min to achieve an ROI radiological dose of 10 Gy.</li>
	<li>Following irradiation, check films for pattern and intensity of radiation dosage. For a 360&#176; angle rotation with the parameters listed to target the ventrobasal hypothalamus, a dark ring in the film above the isocenter, a small crisp spot at the isocenter film, and a lighter ring in the film below the isocenter corresponding to the cone-beam administration of the irradiation will be observed (Figure 4B).</li>
	<li>Superimpose isocenter GAFchromic film over the X-rays of mouse subjects from which the parameters were calculated. The irradiated focal point at isocenter should overlap with the desired ROI region as shown in Figure 4C.</li>
</ol>
Alternative method: If difficulty in targeting brain ROI persists, use iodine contrast injected intrathecally to enhance visualization of ventricles under CT images. For the sake of brevity, this procedure is left out of this protocol, but is previously described35, 50. Iodine contrast will provide additional ventricular landmarks (Figure 5A).
2. Determining Accuracy of Irradiation Beam
Further confirm CFIR beam accuracy by direct visualization of the radiation beam in tissue2, 35, 51. To do this, perform immunohistochemistry to detect &#947;H2AX 51, a histone protein and an early marker of DNA double strand breaks. Mouse subjects must be transcardially perfused and fixed within one hour of irradiation. Following irradiation, DNA repair rapidly ensues, and levels of &#978;H2Ax decrease significantly35.
<ol>
	<li>Repeat steps 1.1 - 1.3.</li>
	<li>After the target is identified on CT, the mouse subject is moved under robotic control to align this target with the radiation delivery beam. Input calculated parameters (rotation speed and length of treatment to achieve desired dosage) from step 1.6 into dose-planning software and begin treatment. Treatment is delivered with the gantry pointed to 45&#176; from the vertical while the mouse rotates about a vertically-oriented axis.</li>
	<li>Following irradiation, perform transcardial perfusion on mouse subjects. Perform perfusion within one hour52. Following perfusion, immersion fix brains in 4% PFA/PBS and gently rock overnight at 4 &#176;C.</li>
	<li>The following morning, wash 5 min in PBS 3x to remove paraformaldehyde fixative. Then, immerse in 30% sucrose in 1x PBS on a rocker at 4 &#176;C.</li>
	<li>Gently rock at 4 &#176;C (usually 12-16 hr), until brain sinks to bottom of tube. This serves as a cryoprotection step. Once brains sink, remove brain with a perforated spoon to prevent excess 30% sucrose/1x PBS transfer. Quickly embed in freezing medium on dry ice. Swirl freezing medium with pipette tip and align brain in plastic mold.</li>
	<li>Once completely frozen, transfer brain blocks to -20 &#176;C freezer for storage.</li>
	<li>On the day when immunohistochemistry will be performed, section coronally at 40 &#956;m thickness, and float into a 24-well plate containing PBS with a thin paintbrush. Sections should be kept in correct serial order for immunostaining to determine irradiation coverage of target region.</li>
	<li>Prewarm 0.01 M sodium citrate solution to 80 &#176;C in water bath in preparation for antigen retrieval step. Simultaneously, while the sodium citrate is warming, perform 5 min&#160;washes 3x in 0.01 M PBS.</li>
	<li>Once sodium citrate solution is at 80 &#176;C, submerge brain sections in the sodium citrate buffer solution. Leave in water bath at 80 &#176;C for 1 hr.</li>
	<li>Remove section and allow sodium citrate solution to reach room temperature, then perform three 5-minute washes in 0.01 M PBS.</li>
	<li>Block brain sections for 1 hr in PBS-Triton containing 5% normal goat serum.</li>
	<li>Incubate brain sections overnight in PBS-Triton containing 5% normal goat serum with 1:700 concentration of mouse anti-phospho-H2AX primary antibody at 4 &#176;C.</li>
	<li>The next day, perform 15 min&#160;washes 3x in 0.01 M PBS-Triton.</li>
	<li>Incubate brain sections PBS-Triton containing 5% normal goat serum with goat anti-mouse secondary antibody conjugated with 488 nm fluorphore at 1:500 concentration for 2 hr.</li>
	<li>Perform 15 min&#160;washes 3x in 0.01 M PBS-Triton.</li>
	<li>Perform 4&#39;,6-diamidino-2-phenylindole (DAPI) stain (1:5,000 in PBS) for 10 min to visualize nucleus. Then, wash brain sections for 5 min with PBS.</li>
	<li>Mount on electrostatically charged microscope slides by floating section in PBS. Wipe off excess PBS and allow slides to dry. Coverslip the slides using mounting medium and allow the slides to dry in the dark at room temperature overnight.</li>
	<li>Take pictures of serial coronal sections with fluorescent microscope. &#978;H2Ax immunostaining (green) indicates site of irradiation. DAPI (blue) is a nuclear stain (Figure 5B).</li>
</ol>
3. Preparation of Mouse Subjects for Focal Irradiation
Examine &#978;H2Ax immunostaining results. Once satisfied with the calibration and targeting of the irradiation beam, proceed with the experiment. At this point, the total time required to treat a mouse (from animal setup to completion of beam delivery) is approximately 10-15 min for a 10-Gy treatment with a 1-mm beam.
<ol>
	<li>Order four-week-old C57BL6/J female mice from Jackson Mouse laboratories. House four mice per cage, and switch food from normal chow to a high-fat diet at five weeks old. Ear punch mice to give them unique identifying marks. Monitor health of mice daily. Note: metallic markers cannot be used as they will result in streaking artifacts on the CT.</li>
	<li>Weigh mice the day before radiation or sham treatment. Split mice into two cohorts for radiation or sham treatment and ensure that there is no significant difference in weight between cohorts. On the day of treatment, when mice are six weeks old, reweigh all mice and record their mass. Gently transport both cohorts to the radiological platform. Take care to minimize stress levels.</li>
	<li>Prepare isoflurane gas anesthesia chamber. Anesthetize two mice, one in the predetermined irradiation group, and another in the sham control group. Prepare heating pad set in the low setting for postoperative treatment.</li>
	<li>Follow steps 1.2 - 1.4 for the mouse to receive irradiation. For the sham mouse, keep mouse in anesthesia chamber while treatment is going on. Make sure that anesthesia chamber is near the CFIR platform so any effects on ambient radiation are factored in. After the target is identified on CT, move mouse subject under robotic control to align this target with the radiation delivery beam. Input calculated parameters (rotation speed and length of treatment) into dose-planning software.</li>
	<li>Once irradiation treatment is complete, return both sham and irradiated mice to heating pad, and monitor until they wake up.</li>
	<li>Return both sham and irradiated mice to animal facility. Monitor every day. Weigh the mice every half week. To confirm irradiation of targeted hypothalamic proliferative zone, administer intraperitoneal injections of BrdU (50 mg/kg) three days post-treatment and examine neurogenesis between groups by colabeling of immunohistochemistry for BrdU and a neuronal marker one month following initial BrdU exposure (Figure 6)2.</li>
</ol>

J.W. has a research funding and consultation agreements with Xstrahl, Inc.

CT-guided focal irradiation (CFIR) is a novel and complete system approach capable of delivering radiation fields to targets in small animals under robotic control using CT-guidance32. The capability of CFIR to deliver highly focused beams to small animal models provides new research opportunities to bridge laboratory research and clinical translation. This paper describes the CFIR approach for precise radiation delivery to specifically target a hypothalamic neural progenitor population. We demonstrate here ho...

Recent discoveries have demonstrated that the adult mammalian brain can undergo a remarkable degree of plasticity. Adult-born neurons are generated throughout adulthood in specialized niches of the mammalian brain1. What is the function of these adult-born neurons? And more so, do they play a role in physiology and behavior? Studies on this topic have traditionally focused on the subventricular zone of the lateral ventricles and the subgranular zone of the hippocampus; however, recent studies have characterized neurogenesis in other brain regions such as the mammalian hypothalamus2. Neurogenesis has been reported in the postnatal and adult hypothalamus2-10, and the function of these newborn hypothalamic neurons remains an active area of investigation.
The functional characterization of adult-born neurons remains a significant challenge for the neuroscience field in general. Selective inhibition of specific neural progenitor populations remains limited by the lack of available molecular markers that are unique to single neural progenitor populations11. Thus, selective deletion of adult-born neurons from these neural progenitors via genetic targeting remains difficult. Likewise, viral delivery to target adult-born neurons suffers from potential confounding variables such as introducing injury and inflammation into the environment12.
New radiological tools are emerging, however, that allow one to circumvent these confounds and investigate these questions through accurate and precise anatomical targeting in small animals. Ionizing radiation inhibits the birth and differentiation of new neurons, and allows a noninvasive method to target neural progenitor populations13-15. Recently, we described a germinal region of the mammalian hypothalamic median eminence (ME) that we termed the hypothalamic proliferative zone (HPZ)2. We found that when young adult female mice were given a high-fat diet (HFD), levels of neurogenesis in HFD-fed mice were substantially higher than their normal chow (NC) fed controls in this ME region2. To test whether adult neurogenesis within the hypothalamic ME regulates metabolism and weight, we sought to disrupt this process. The median eminence is a small unilateral structure at the base of the third ventricle from which regulatory hormones are released. In order to inhibit proliferation and subsequent neurogenesis without altering the other physiological functions of this brain region, we developed a noninvasive focal irradiation technique to selectively inhibit the birth of newly born adult neurons in the hypothalamic median eminence2.
A number of groups have employed radiation to suppress neurogenesis in canonical regions14-28. However, previous radiological approaches have generally targeted large areas, or often unintentionally also targeted multiple brain areas where neurogenesis has been reported, making it difficult to unambiguously associate any behavioral defects observed with defects in specific neural progenitor populations. The capability for more targeted irradiation is provided by radiological platforms that combine computer tomography-guided imaging with focal beam irradiation (CFIR) delivery to enable precise anatomical targeting29-36. Radiation beams as small as 0.5 mm in diameter are available to target specific neural progenitor populations35. This methodology allows us to target the hypothalamic ME and arrest proliferation and block neurogenesis in small animals. Following radiological treatment on these progenitor populations, physiological and behavioral tests can be performed to illuminate the potential function of adult-born cells. Focal targeting is especially important for our application since the pituitary gland is located close to the hypothalamic median eminence; irradiation of the pituitary may affect hormonal function and subsequently confound results.
The biological basis for the suppression of neurogenesis following irradiation still remains unclear. Previous radiation studies have relied on large area beams, and have concluded that the suppression of neurogenesis is mediated through an inflammatory response14, 37. As such it is unclear whether highly focal irradiation could suppress neurogenesis, since it does not evoke a substantial inflammatory response. However, recent studies by our group of the classic neurogenic region in the hippocampus have demonstrated that highly focal irradiation with a dose of 10 Gy can suppress neurogenesis for at least 4 weeks after irradiation35.
To interrogate the function of adult-born hypothalamic neurons in the median eminence, we use a precision radiation device capable of delivering CT imaging in combination with small-diameter radiation beams to inhibit ME neurogenesis. Using an X-ray tube attached to a gantry that rotates over a range of 360&#176;, we deliver arc-beam micro irradiation beam with the use of a robotically controlled specimen stage that allows rotation of an animal subject during radiation treatment (Figure 1). A high-resolution X-ray detector is used to acquire images when the gantry is in the horizontal position33. For this study, CT images were reconstructed with an isotropic voxel size of 0.20 mm. On-board CT imaging allowed the identification of a target while the animal is in the treatment position. The target was localized using the CT navigation dose-planning software, which was included with our commercially available radiological platform. After localizing our ROI by CT imaging, the animal was moved to the appropriate treatment position by the robotic specimen stage that has four degrees of freedom (X, Y, Z, &#952;). Through a combination of gantry and robot stage angles, beams can be delivered from nearly any direction relative to the animal, and stereotactic arc-like treatments are possible29. For these and all other imaging studies, the mice were positioned in an immobilization device that allows delivery of anesthetic isoflurane gas while restricting movement. The immobilization bed is CT compatible, and connects to the robotic specimen stage34.
We expect that CFIR will provide conceptual advances in a number of research areas. Although we use radiological targeting of the hypothalamic median eminence as proof of principle of this technique, CFIR can be used to target any region of the body of any small model organism in principle. In the neurosciences, for example, we envision this technique could be used to evaluate the function of actively proliferative progenitor populations that have been suggested to exist in other circumventricular organs, such as the area postrema38, 39, subfornical organ40, and the pituitary41. Longstanding controversies regarding the functional role of adult neurogenesis and identifying a causal role in behavior can also now be better addressed. In songbird, this technique might address the role of adult neurogenesis in maintaining the robust and seasonal behavior of birdsong42, which has been hampered by the ability to selectively inhibit neurogenesis in specific brain regions. Understanding this robust behavioral model might shed new insight into the role of adult neurogenesis in regulating other sexually dimorphic behaviors. Alternatively, in the metabolic field, CFIR might be used to explore aspects of the role of hepatocyte proliferation and its role in metabolism and energy balance. The possibility for conceptual advance in multiple research disciplines is enhanced by the introduction of this technique.
In this paper, we demonstrate the capabilities of CFIR for precision anatomical targeting of a focal irradiation beam. Although we initially developed this small animal radiation research platform (SARRP) for our studies, other similar devices are now commercially available that can accomplish similar CT-guided focal irradiation43, 44. Hence, we generalize this CFIR protocol with steps required for all research platforms rather than those specific for SARRP. The advantages of CFIR over previous radiological approaches to inhibit neurogenesis are that this technique allows three-dimensional volumetric image guidance for localization and targeting of the dose, conformal dose minimizes exposure to nontargeted brain regions, and high precision beam geometry allows for conformal dose distribution with sharp beam boundaries. We outline how to use CT-guided imaging to target the dose to a specific anatomical region, and upon doing so, how to visualize the radiation dose distribution directly in tissue using immunohistochemical staining for &#947;-H2AX, a marker of DNA double-stranded breaks35, 45-48. The use of this approach for selective irradiation of neurogenic niches may have significant implications in revealing the functional role of new adult-born neurons on physiology and disease.

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>SARRP research platform</td>
			<td>Xstrahl</td>
			<td>RS225A</td>
			<td><a href="http://www.xstrahl.com/xstrahlrs225.htm" target="_blank">http://www.xstrahl.com/xstrahlrs225.htm</a></td>
		</tr>
		<tr>
			<td>SARRP irradiation bunker</td>
			<td>Xstrahl</td>
			<td></td>
			<td>Optional, but radiation exposure should be contained with alternative lead shielding</td>
		</tr>
		<tr>
			<td>GAF chromic film</td>
			<td>IPS</td>
			<td>GAFchromic ETB2</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse phantom</td>
			<td>Gammex</td>
			<td>457</td>
			<td>Purchase 0.5 cm x 30 cm x 30 cm solid water slabs from Gammex and cut to desired size.</td>
		</tr>
		<tr>
			<td>Mouse anti-phospho-histone H2AX Ser139 antibody</td>
			<td>Millipore, Inc.</td>
			<td>05-636</td>
			<td>clone JBW301</td>
		</tr>
		<tr>
			<td>High-fat rodent diet</td>
			<td>Research Diets</td>
			<td>D12492i</td>
			<td>60% of the calories as fat, food should be irradiated</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Baxter Healthcare Corporation</td>
			<td>10019-360-40</td>
			<td></td>
		</tr>
		<tr>
			<td>0.01 M Sodium citrate</td>
			<td>Fisher Scientific</td>
			<td></td>
			<td>1.471 g of sodium citrate dissolved in 500 ml deionized water</td>
		</tr>
		<tr>
			<td>Superfrost Plus slides</td>
			<td>Fisher Scientific</td>
			<td>12-550-15</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Fisher Scientific</td>
			<td></td>
			<td>nuclear counterstain</td>
		</tr>
		<tr>
			<td>Mounting medium</td>
			<td>Fisher Scientific</td>
			<td></td>
			<td>Vectashield or Gelvatol is preferred</td>
		</tr>
	</tbody>
</table>

functional interrogation of adult hypothalamic neurogenesis with focal radiological inhibition

The functional characterization of adult-born neurons remains a significant challenge. Approaches to inhibit adult neurogenesis via invasive viral delivery or transgenic animals have potential confounds that make interpretation of results from these studies difficult. New radiological tools are emerging, however, that allow one to noninvasively investigate the function of select groups of adult-born neurons through accurate and precise anatomical targeting in small animals. Focal ionizing radiation inhibits the birth and differentiation of new neurons, and allows targeting of specific neural progenitor regions. In order to illuminate the potential functional role that adult hypothalamic neurogenesis plays in the regulation of physiological processes, we developed a noninvasive focal irradiation technique to selectively inhibit the birth of adult-born neurons in the hypothalamic median eminence. We describe a method for Computer tomography-guided focal irradiation (CFIR) delivery to enable precise and accurate anatomical targeting in small animals. CFIR uses three-dimensional volumetric image guidance for localization and targeting of the radiation dose, minimizes radiation exposure to nontargeted brain regions, and allows for conformal dose distribution with sharp beam boundaries. This protocol allows one to ask questions regarding the function of adult-born neurons, but also opens areas to questions in areas of radiobiology, tumor biology, and immunology. These radiological tools will facilitate the translation of discoveries at the bench to the bedside.

Assessing CT-guided Targeting and Accuracy
The mechanical calibration of the system is critical for ensuring that beams from various angles all intersect in a single point. Calibration was accomplished with a semi-automatic imaging-based method, where end-to-end alignment accuracy has been measured to be 0.2 mm29. This accuracy is highly critical as the volume of the hypothalamic median eminence structure is small2. To test this calibration, we measured dose distributions wi...

Watch this Scientific Journal Video about Functional Interrogation of Adult Hypothalamic Neurogenesis with Focal Radiological Inhibition at JoVE.com

Functional Interrogation of Adult Hypothalamic Neurogenesis with Focal Radiological Inhibition

The function of adult-born mammalian neurons remains an active area of investigation. Ionizing radiation inhibits the birth of new neurons. Using computer tomography-guided focal irradiation (CFIR), three-dimensional anatomical targeting of specific neural progenitor populations can now be used to assess the functional role of adult neurogenesis.

The functional characterization of adult-born neurons remains a significant challenge. Approaches to inhibit adult neurogenesis via invasive viral ...

functional-interrogation-adult-hypothalamic-neurogenesis-with-focal

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12.1K Views.  California Institute of Technology. The function of adult-born mammalian neurons remains an active area of investigation. Ionizing radiation inhibits the birth of new neurons. Using computer tomography-guided focal irradiation (CFIR), three-dimensional anatomical targeting of specific neural progenitor populations can now be used to assess the functional role of adult neurogenesis.

Video: Functional Interrogation of Adult Hypothalamic Neurogenesis with Focal Radiological Inhibition

Functional Mapping with Simultaneous MEG and EEG

We use magnetoencephalography (MEG) and electroencephalography (EEG) to locate and determine the temporal evolution in brain areas involved in the processing of simple sensory stimuli. We will use somatosensory stimuli to locate the hand somatosensory areas, auditory stimuli to locate the auditory cortices, visual stimuli in four quadrants of the visual field to locate the early visual areas. These type of experiments are used for functional mapping in epileptic and brain tumor patients to locate eloquent cortices. In basic neuroscience similar experimental protocols are used to study the orchestration of cortical activity. The acquisition protocol includes quality assurance procedures, subject preparation for the combined MEG/EEG study, and acquisition of evoked-response data with somatosensory, auditory, and visual stimuli. We also demonstrate analysis of the data using the equivalent current dipole model and cortically-constrained minimum-norm estimates. Anatomical MRI data are employed in the analysis for visualization and for deriving boundaries of tissue boundaries for forward modeling and cortical location and orientation constraints for the minimum-norm estimates.

We use magnetoencephalography (MEG) and electroencephalography (EEG) to map brain areas involved in the processing of simple sensory stimuli.

We use magnetoencephalography (MEG) and electroencephalography (EEG) to locate and determine the temporal evolution in brain areas involved in the ...

Imaging Calcium Responses in GFP-tagged Neurons of Hypothalamic Mouse Brain Slices

Despite an enormous increase in our knowledge about the mechanisms underlying the encoding of information in the brain, a central question concerning the precise molecular steps as well as the activity of specific neurons in multi-functional nuclei of brain areas such as the hypothalamus remain. This problem includes identification of the molecular components involved in the regulation of various neurohormone signal transduction cascades. Elevations of intracellular Ca2+ play an important role in regulating the sensitivity of neurons, both at the level of signal transduction and at synaptic sites.
New tools have emerged to help identify neurons in the myriad of brain neurons by expressing green fluorescent protein (GFP) under the control of a particular promoter. To monitor both spatially and temporally stimulus-induced Ca2+ responses in GFP-tagged neurons, a non-green fluorescent Ca2+ indicator dye needs to be used. In addition, confocal microscopy is a favorite method of imaging individual neurons in tissue slices due to its ability to visualize neurons in distinct planes of depth within the tissue and to limit out-of-focus fluorescence. The ratiometric Ca2+ indicator fura-2 has been used in combination with GFP-tagged neurons1. However, the dye is excited by ultraviolet (UV) light. The cost of the laser and the limited optical penetration depth of UV light hindered its use in many laboratories. Moreover, GFP fluorescence may interfere with the fura-2 signals2. Therefore, we decided to use a red fluorescent Ca2+ indicator dye. The huge Stokes shift of fura-red permits multicolor analysis of the red fluorescence in combination with GFP using a single excitation wavelength. We had previously good results using fura-red in combination with GFP-tagged olfactory neurons3. The protocols for olfactory tissue slices seemed to work equally well in hypothalamic neurons4. Fura-red based Ca2+ imaging was also successfully combined with GFP-tagged pancreatic &beta;-cells and GFP-tagged receptors expressed in HEK cells5,6. A little quirk of fura-red is that its fluorescence intensity at 650 nm decreases once the indicator binds calcium7. Therefore, the fluorescence of resting neurons with low Ca2+ concentration has relatively high intensity. It should be noted, that other red Ca2+-indicator dyes exist or are currently being developed, that might give better or improved results in different neurons and brain areas.

In this protocol, we update recent progress in imaging Ca2+ signals of GFP-tagged neurons in brain tissue slices using a red fluorescent Ca2+ indicator dye.

Despite an enormous increase in our knowledge about the mechanisms underlying the encoding of information in the brain, a central question concerning the ...

Heat-Induced Antigen Retrieval: An Effective Method to Detect and Identify Progenitor Cell Types during Adult Hippocampal Neurogenesis

Traditional methods of immunohistochemistry (IHC) following tissue fixation allow visualization of various cell types. These typically proceed with the application of antibodies to bind antigens and identify cells with characteristics that are a function of the inherent biology and development. Adult hippocampal neurogenesis is a sequential process wherein a quiescent neural stem cell can become activated and proceed through stages of proliferation, differentiation, maturation and functional integration. Each phase is distinct with a characteristic morphology and upregulation of genes. Identification of these phases is important to understand the regulatory mechanisms at play and any alterations in this process that underlie the pathophysiology of debilitating disorders. Our heat-induced antigen retrieval approach improves the intensity of the signal that is detected and allows correct identification of the progenitor cell type. As discussed in this paper, it especially allows us to circumvent current problems in detection of certain progenitor cell types.

During adult hippocampal neurogenesis, a distinct set of genetic markers are expressed when a quiescent neural stem cell sequentially progresses and develops into a functionally integrated neuron in the circuit. Using heat-induced antigen retrieval, progenitor cell types that are otherwise difficult to detect are identified with improved effectiveness.

Traditional  methods of immunohistochemistry (IHC) following tissue fixation allow visualization  of various cell types. These typically proceed with the ...

Methods for the Modulation and Analysis of NF-&#954;B-dependent Adult Neurogenesis

The hippocampus plays a pivotal role in the formation and consolidation of episodic memories, and in spatial orientation. Historically, the adult hippocampus has been viewed as a very static anatomical region of the mammalian brain. However, recent findings have demonstrated that the dentate gyrus of the hippocampus is an area of tremendous plasticity in adults, involving not only modifications of existing neuronal circuits, but also adult neurogenesis. This plasticity is regulated by complex transcriptional networks, in which the transcription factor NF-&#954;B plays a prominent role. To study and manipulate adult neurogenesis, a transgenic mouse model for forebrain-specific neuronal inhibition of NF-&#954;B activity can be used.
In this study, methods are described for the analysis of NF-&#954;B-dependent neurogenesis, including its structural aspects, neuronal apoptosis and progenitor proliferation, and cognitive significance, which was specifically assessed via a dentate gyrus (DG)-dependent behavioral test, the spatial pattern separation-Barnes maze (SPS-BM). The SPS-BM protocol could be simply adapted for use with other transgenic animal models designed to assess the influence of particular genes on adult hippocampal neurogenesis. Furthermore, SPS-BM could be used in other experimental settings aimed at investigating and manipulating DG-dependent learning, for example, using pharmacological agents.

Methods for the Modulation and Analysis of NF-&amp;#954;B-dependent Adult Neurogenesis

Methods for the manipulation and analysis of NF-&#954;B-dependent adult hippocampal neurogenesis are described. A detailed protocol is presented for a dentate gyrus-dependent behavioral test (termed the spatial pattern separation-Barnes maze) for the investigation of cognitive outcome in mice. This technique should also help enable investigations in other experimental settings.

The hippocampus plays a pivotal role in the formation and consolidation of episodic memories, and in spatial orientation. Historically, the adult ...

Functional Imaging of Auditory Cortex in Adult Cats using High-field fMRI

Current knowledge of sensory processing in the mammalian auditory system is mainly derived from electrophysiological studies in a variety of animal models, including monkeys, ferrets, bats, rodents, and cats. In order to draw suitable parallels between human and animal models of auditory function, it is important to establish a bridge between human functional imaging studies and animal electrophysiological studies. Functional magnetic resonance imaging (fMRI) is an established, minimally invasive method of measuring broad patterns of hemodynamic activity across different regions of the cerebral cortex. This technique is widely used to probe sensory function in the human brain, is a useful tool in linking studies of auditory processing in both humans and animals and has been successfully used to investigate auditory function in monkeys and rodents. The following protocol describes an experimental procedure for investigating auditory function in anesthetized adult cats by measuring stimulus-evoked hemodynamic changes in auditory cortex using fMRI. This method facilitates comparison of the hemodynamic responses across different models of auditory function thus leading to a better understanding of species-independent features of the mammalian auditory cortex.

Functional studies of the auditory system in mammals have traditionally been conducted using spatially-focused techniques such as electrophysiological recordings. The following protocol describes a method of visualizing large-scale patterns of evoked hemodynamic activity in the cat auditory cortex using functional magnetic resonance imaging.

Current knowledge of sensory processing in the mammalian auditory system is mainly derived from electrophysiological studies in a variety of animal ...

Stab Wound Injury of the Zebrafish Adult Telencephalon: A Method to Investigate Vertebrate Brain Neurogenesis and Regeneration

Adult zebrafish have an amazing capacity to regenerate their central nervous system after injury. To investigate the cellular response and the molecular mechanisms involved in zebrafish adult central nervous system (CNS) regeneration and repair, we developed a zebrafish model of adult telencephalic injury.
In this approach, we manually generate an injury by pushing an insulin syringe needle into the zebrafish adult telencephalon. At different post injury days, fish are sacrificed, their brains are dissected out and stained by immunohistochemistry and/or in situ hybridization (ISH) with appropriate markers to observe cell proliferation, gliogenesis, and neurogenesis. The contralateral unlesioned hemisphere serves as an internal control. This method combined for example with RNA deep sequencing can help to screen for new genes with a role in zebrafish adult telencephalon neurogenesis, regeneration, and repair.

To shed light on the cellular and molecular mechanisms of zebrafish adult neurogenesis and regeneration, we developed a protocol for invasive surgery causing mechanical injuries in the zebrafish adult telencephalon and subsequent monitoring of changes in the stabbed hemisphere by immunohistochemistry or in situ hybridization.

Adult zebrafish have an amazing capacity to regenerate their central nervous system after injury. To investigate the cellular response and the molecular ...

Multi-photon Intracellular Sodium Imaging Combined with UV-mediated Focal Uncaging of Glutamate in CA1 Pyramidal Neurons

Multi-photon fluorescence microscopy has enabled the analysis of morphological and physiological parameters of brain cells in the intact tissue with high spatial and temporal resolution. Combined with electrophysiology, it is widely used to study activity-related calcium signals in small subcellular compartments such as dendrites and dendritic spines. In addition to calcium transients, synaptic activity also induces postsynaptic sodium signals, the properties of which are only marginally understood. Here, we describe a method for combined whole-cell patch-clamp and multi-photon sodium imaging in cellular micro domains of central neurons. Furthermore, we introduce a modified procedure for ultra-violet (UV)-light-induced uncaging of glutamate, which allows reliable and focal activation of glutamate receptors in the tissue. To this end, whole-cell recordings were performed on Cornu Ammonis subdivision 1 (CA1) pyramidal neurons in acute tissue slices of the mouse hippocampus. Neurons were filled with the sodium-sensitive fluorescent dye SBFI through the patch-pipette, and multi-photon excitation of SBFI enabled the visualization of dendrites and adjacent spines. To establish UV-induced focal uncaging, several parameters including light intensity, volume affected by the UV uncaging beam, positioning of the beam as well as concentration of the caged compound were tested and optimized. Our results show that local perfusion with caged glutamate (MNI-Glutamate) and its focal UV-uncaging result in inward currents and sodium transients in dendrites and spines. Time course and amplitude of both inward currents and sodium signals correlate with the duration of the uncaging pulse. Furthermore, our results show that intracellular sodium signals are blocked in the presence of blockers for ionotropic glutamate receptors, demonstrating that they are mediated by sodium influx though this pathway. In summary, our method provides a reliable tool for the investigation of intracellular sodium signals induced by focal receptor activation in intact brain tissue.

We describe the combination of focal UV-induced photo-activation of neuro-active compounds with whole-cell patch-clamp and multi-photon imaging of intracellular sodium transients in dendrites and spines of hippocampal neurons in acute tissue slices of the mouse brain.

Multi-photon fluorescence microscopy has enabled the analysis of morphological and physiological parameters of brain cells in the intact tissue with high ...

Immunohistochemistry and Multiple Labeling with Antibodies from the Same Host Species to Study Adult Hippocampal Neurogenesis

Adult neurogenesis is a highly regulated, multi-stage process in which new neurons are generated from an activated neural stem cell via increasingly committed intermediate progenitor subtypes. Each of these subtypes expresses a set of specific molecular markers that, together with specific morphological criteria, can be used for their identification. Typically, immunofluorescent techniques are applied involving subtype-specific antibodies in combination with exo- or endogenous proliferation markers. We herein describe immunolabeling methods for the detection and quantification of all stages of adult hippocampal neurogenesis. These comprise the application of thymidine analogs, transcardial perfusion, tissue processing, heat-induced epitope retrieval, ABC immunohistochemistry, multiple indirect immunofluorescence, confocal microscopy and cell quantification. Furthermore we present&#160;a sequential multiple immunofluorescence protocol which circumvents problems usually arising from the need of using primary antibodies raised in the same host species. It allows an accurate identification of all hippocampal progenitor subtypes together with a proliferation marker within a single section. These techniques are a powerful tool to study the regulation of different progenitor subtypes in parallel, their involvement in brain pathologies and their role in specific brain functions.

This video article illustrates a comprehensive protocol to detect and quantify all stages of adult hippocampal neurogenesis within the same tissue section. We elaborated a method to overcome the limitations of indirect multiple immunofluorescence that arise when suitable antibodies from different host species are unavailable.

Adult neurogenesis is a highly regulated, multi-stage process in which new neurons are generated from an activated neural stem cell via increasingly ...

Assessing Primary Neurogenesis in Xenopus Embryos Using Immunostaining

Primary neurogenesis is a dynamic and complex process during embryonic development that sets up the initial layout of the central nervous system. During this process, a portion of neural stem cells undergo differentiation and give rise to the first populations of differentiated primary neurons within the nascent central nervous system. Several vertebrate model organisms have been used to explore the mechanisms of neural cell fate specification, patterning, and differentiation. Among these is the African clawed frog, Xenopus, which provides a powerful system for investigating the molecular and cellular mechanisms responsible for primary neurogenesis due to its rapid and accessible development and ease of embryological and molecular manipulations. Here, we present a convenient and rapid method to observe the different populations of neuronal cells within Xenopus central nervous system. Using antibody staining and immunofluorescence on sections of Xenopus embryos, we are able to observe the locations of neural stem cells and differentiated primary neurons during primary neurogenesis.

Assessing Primary Neurogenesis in Xenopus Embryos Using Immunostaining

This article presents a convenient and rapid method for visualizing different neuronal cell populations in the central nervous system of Xenopus embryos using immunofluorescent staining on sections.

Primary neurogenesis is a dynamic and complex process during embryonic development that sets up the initial layout of the central nervous system. During ...

Online Transcranial Magnetic Stimulation Protocol for Measuring Cortical Physiology Associated with Response Inhibition

We describe the development of a reproducible, child-friendly motor response inhibition task suitable for online Transcranial Magnetic Stimulation (TMS) characterization of primary motor cortex (M1) excitability and inhibition. Motor response inhibition prevents unwanted actions and is abnormal in several neuropsychiatric conditions. TMS is a non-invasive technology that can quantify M1 excitability and inhibition using single- and paired-pulse protocols and can be precisely timed to study cortical physiology with high temporal resolution. We modified the original Slater-Hammel (S-H) stop signal task to create a &#34;racecar&#34; version with TMS pulses time-locked to intra-trial events. This task is self-paced, with each trial initiating after a button push to move the racecar towards the 800 ms target. GO trials require a finger-lift to stop the racecar just before this target. Interspersed randomly are STOP trials (25%) during which the dynamically adjusted stop signal prompts subjects to prevent finger-lift. For GO trials, TMS pulses were delivered at 650 ms after trial onset; whereas, for STOP trials, the TMS pulses occurred 150 ms after the stop signal. The timings of the TMS pulses were decided based on electroencephalography (EEG) studies showing event-related changes in these time ranges during stop signal tasks. This task was studied in 3 blocks at two study sites (n=38) and we recorded behavioral performance and event-related motor-evoked potentials (MEP). Regression modelling was used to analyze MEP amplitudes using age as a covariate with multiple independent variables (sex, study site, block, TMS pulse condition [single- vs. paired-pulse], trial condition [GO, successful STOP, failed STOP]). The analysis showed that TMS pulse condition (p&#60;0.0001) and its interaction with trial condition (p=0.009) were significant. Future applications for this online S-H/TMS paradigm include the addition of simultaneous EEG acquisition to measure TMS-evoked EEG potentials. A potential limitation is that in children, the TMS pulse sound could&#160;affect behavioral task performance.

We describe an experimental procedure to quantify excitability and inhibition of primary motor cortex during a motor response inhibition task by using Transcranial Magnetic Stimulation throughout the course of a Stop Signal Task.

We describe the development of a reproducible, child-friendly motor response inhibition task suitable for online Transcranial Magnetic Stimulation (TMS) ...