Name: functional interrogation of adult hypothalamic neurogenesis with focal radiological inhibition
Uploaded: 2013-11-14T22:30:00
Description: Watch this Scientific Journal Video about Functional Interrogation of Adult Hypothalamic Neurogenesis with Focal Radiological Inhibition at JoVE.com

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 iden...

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 ...

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