OG - P는 CONACyT S 그랜트 (CB - 2008-101476)과 FRABA (10분의 686)에 의해 지원되었다. AQ - H는 건강의 국립 연구소, 하워드 휴즈 의학 연구소의 로버트 우드 존슨 재단과 메릴랜드 줄기 세포 재단에 의해 지원.

<ol><li> 마우스 수술 전에 유리 바늘 만들기 : <ol class="lalpha"><li> micropipette의 풀러의 모세관 유리 튜브를 넣어. </li><li> 지역화된 영역의 유리 튜브를 부드럽게하는 유리 튜브의 가운데를 열. </li><li> 지역화된 영역의 유리 튜브의 직경의 감소를 일으킬 수있는 충분한 초기 거리하여 세로 축을 따라 유리관을 스트레치. </li><li> 그것이 휴식 때까지 유리 튜브를 스트레칭 유지. 이런 방식으로 두 개의 동일한 하나의 배럴의 바늘은 얻을 수 있습니다. </li></ol></li><li> microforge에 유리 바늘을 넣어. 30 ° 각도만큼 베벨 팁을하는 것입니다. </li><li> 각각의 바늘이 정확한 내경을 가지고있는 현미경으로 확인합니다. 친수성 약물의 대부분 30-50 μm의 내경은 (그림 1) 이상적인 제품입니다. </li><li> 유리 바늘의 넓은 끝에서 광유로 바늘을 입력합니다. 그걸로 모세관 튜브의 길이의 ~ 50 %에 도달할 때까지 미네랄 오일 (MSDS, 고양이. M7700) 모세관 현상에 의해 입력하자. 입어 높은 진공 바늘의 넓은 끝을 밀봉하는 플런저 주변 그리스 (다우 코닝, 고양이. 05054 - AB). </li><li> 바늘의 넓은 말까지 플런저를 넣고 기름의 작은 방울이 유리 바늘 끝을 들락날락 때까지 부드럽게 미네랄 오일을 밀어. </li><li> microinjector의 소유자에 유리 바늘을 고정합니다. </li><li> 2.5 % Avertin (2,2,2 - tribromoethanol + tert - 아밀 알코올, 1시 1분 W / V)와 마우스를 마취. Dosis g intraperitoneal 당 25 30μL. </li><li> 37 동물의 체온을 유지하는 stereotactic 장치 ° C.에 히터 패드를 넣어 </li><li> 장소 및 귀 바, 치아 홀더를 사용하여 stereotactic 장치에서 마우스 머리를 확보. </li><li> 두 번째 시간을 70 % 에탄올과 0.1 % Chlorhexidine 글루 콘 산 다음에 0.1 % Chlorhexidine 글루 콘 산 솔루션으로 마우스 머리를 청소하십시오. </li><li> Lamba 스컬 라인에 마우스 귀를에서 수술 블레이드 # 15 피부를 절개. </li><li> 그것을 건조하고 수술 현장에서 피부를 잡아 면봉 스왑으로 두개골을 폴란드어. </li><li> Bregma의 봉합의 정점에서 시점에 유리 바늘의 팁을 사용하십시오. 여기 주입기 (좌표 &quot;제로&quot;)의 초기 위치를 설정해야합니다. </li><li> &quot;X&quot;와 두개골을 통해 stereotactic 장치의 &quot;Y&quot;축을 이동하여 몰락하는 지점을 설정합니다. </li><li> 선택한 좌표에 매우 조심스럽게 구멍을 드릴. Microdrills 및 기타 금속 도구는 이전에 다음 ID 250에 소독을했다 ° 마른 유리 구슬 살균기와 함께 60 초 동안 C (콜 팔머, 고양이. 번호 EW - 10779-00). </li><li> 좋은 포셉 조심스럽게 뼈의 가장 깊은 층을 제거하고 duramatter 막을 (당신이 밖으로 누출 일부 대뇌 척수를 볼 수 있습니다) 휴식과 함께. </li><li> 그 바늘이 뚫고 구멍 갈 수 확인합니다. </li><li> 당신은 parafilm의 작은 조각에 주사하고 데려가고 싶다는 약물의 피펫 1 μl. </li><li> 두개골에 parafilm 놔. </li><li> 당신은 액체가 유리 바늘로 올라간다는 것을 현미경으로 확인하면서, microinjector 휠을 회전하여 약물을 빨아. </li><li> parafilm를 제거하고 제로 좌표를 다시 설정합니다. </li><li> 유리 바늘 끝이 부드럽게 뇌의 표면을 만지 제로에서 &quot;Z&quot;좌표를 설정할 때까지 홀더 아래, 선택한 좌표에 바늘을 이동합니다. </li><li> 선택 깊이에서 뇌 실질에 유리 바늘을 소개합니다. </li><li> 천천히 ~ 1 NL / 초의 속도로 약물을 주사. </li><li> 원하는 볼륨이 분사되면, 2 분 실질에 마약 확산하자. 그런 다음 부드럽게 천천히 바늘을 제거하는 진행합니다. </li><li> 접착제는 수술 상처와 stereotactic 장치에서 동물을 제거하고 마취 복구를 위해 깨끗한 침대를 포함하는 미리 예열 케이지에 동물을 넣어. </li><li> 이후 수술 진통제를 제공하기 위해 모든 동물은 24 H. 5 MG / kg 피하 Ketorolac 매 12 시간을 받았습니다 </li></ol> 대표 결과 : 이 프로토콜을 따라하면, 매우 정확하게 주사는 취득과 매우 좁은 바늘 트랙 뇌 손상을 최소화합니다. 이 방법의 대표적인 결과로, 우리는 흰 물질의 책자 1-4 demyelination를 생산 코퍼스의 callosum에 lysophosphatidyl 콜린 (lysolecithin)을 주입했다. 유리 바늘에 의해 생산되는 뇌 손상을 최소화하기 위해, 우리는 코퍼스의 callosum에 lysolecithin의 20 NL을 주입, 200 NL만큼 필요한 경우에는 높은 볼륨이 동일한 방법으로 주입 수 있습니다. Demyelination은 흰색 물질 책자 (그림 2)에서 myelin 기본 단백질 표현의 부재에 의해 감지됩니다. <img src="/files/ftp_upload/2082/2082fig1.jpg" alt="그림 1" /> 그림 1. 50 μm의 직경 유리 바늘. 이 짧은 거려 사이의 거리규모에 50 μm의 길이를 나타냅니다. <img src="/files/ftp_upload/2082/2082fig2.jpg" alt="그림 2" /> 코퍼스의 callosum 그림 2. Lysolecithin 분사. Demyelination은 myelin 기본 단백질 표현 (점선 영역) 없음으로 표시됩니다. 유리 바늘 트랙트 (화살표)의 작은 크기를합니다. 바 = 100 μm의

<ol>
	<li>Menn, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Origin+of+oligodendrocytes+in+the+subventricular+zone+of+the+adult+brain.">Origin of oligodendrocytes in the subventricular zone of the adult brain.</a> J Neurosci. 26, 7907-7918 (2006).</li><li>Gonzalez-Perez, O., Romero-Rodriguez, R., Soriano-Navarro, M., Garcia-Verdugo, J. M., Alvarez-Buylla, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidermal+Growth+Factor+Induces+the+Progeny+of+Subventricular+Zone+Type+B+Cells+to+Migrate+and+Differentiate+into+Oligodendrocytes.">Epidermal Growth Factor Induces the Progeny of Subventricular Zone Type B Cells to Migrate and Differentiate into Oligodendrocytes.</a> Stem Cells. 27, 2032-2043 (2009).</li><li>Hall, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Some+aspects+of+remyelination+after+demyelination+produced+by+the+intraneural+injection+of+lysophosphatidyl+choline.">Some aspects of remyelination after demyelination produced by the intraneural injection of lysophosphatidyl choline.</a> J Cell Sci. 13, 461-477 (1973).</li><li>Webster, G. R., Thompson, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Observations+on+the+presence+of+lysolecithin+in+nervous+tissues.">Observations on the presence of lysolecithin in nervous tissues.</a> Biochim Biophys Acta. 63, 38-45 (1962).</li><li>Doetsch, F., Caille, I., Lim, D. A., Garc&#237;a-Verdugo, J. M., Alvarez-Buylla, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subventricular+Zone+Astrocytes+Are+Neural+Stem+Cells+in+the+Adult+mammalian+Brain.">Subventricular Zone Astrocytes Are Neural Stem Cells in the Adult mammalian Brain.</a> Cell. 97, 1-20 (1999).</li><li>Doetsch, F., Petreanu, L., Caille, I., Garcia-Verdugo, J. M., Alvarez-Buylla, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EGF+converts+transit-amplifying+neurogenic+precursors+in+the+adult+brain+into+multipotent+stem+cells.">EGF converts transit-amplifying neurogenic precursors in the adult brain into multipotent stem cells.</a> Neuron. 36, 1021-1034 (2002).</li><li>Baraban, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduction+of+seizures+by+transplantation+of+cortical+GABAergic+interneuron+precursors+into+Kv1.1+mutant+mice.">Reduction of seizures by transplantation of cortical GABAergic interneuron precursors into Kv1.1 mutant mice.</a> Proc Natl Acad Sci U S A. 106, 15472-15477 (2009).</li><li>Richardson, R. M., Barbaro, N. M., Alvarez-Buylla, A., Baraban, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developing+cell+transplantation+for+temporal+lobe+epilepsy.">Developing cell transplantation for temporal lobe epilepsy.</a> Neurosurg Focus. 24, E17-E17 (2008).</li><li>Alvarez-Dolado, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cortical+inhibition+modified+by+embryonic+neural+precursors+grafted+into+the+postnatal+brain.">Cortical inhibition modified by embryonic neural precursors grafted into the postnatal brain.</a> J Neurosci. 26, 7380-7389 (2006).</li></ol>

관심 없음 충돌 선언하지 않습니다.

방법은 두뇌에 매우 정확한 장소에 약물이나 바이러스 벡터의 대부분을 제공하는 매우 유용합니다이 비디오에서 보여주었다. 이 기술의 주요 장점 중 일부는 표적 지점, 주사의 정확성 및 뇌 병변 및 요로 손상 1, 2, 5, 6의 작은 크기의 신뢰성입니다. 일단 기술이 mistargeting의 범위 표준화되어 있어야 50 μm의 또는 2, 1 이하. 세포 이식은 또한 더 넓은, 100-150 μm의 7-9, 바늘을 ?...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Capillary glass tube</td>
<td>&nbsp;</td>
<td>Wiretrol I</td>
<td>5-000-1001</td>
<td>&nbsp;</td>
<tr>
<td>Micropipette puller</td>
<td>&nbsp;</td>
<td>Kopf</td>
<td>Model 730</td>
<td>&nbsp;</td>
<tr>
<td>Microforge</td>
<td>&nbsp;</td>
<td>World Precision Instruments</td>
<td>Model 48000</td>
<td>&nbsp;</td>
<tr>
<td>Mineral oil</td>
<td>&nbsp;</td>
<td>MSDS</td>
<td>M7700</td>
<td>&nbsp;</td>
<tr>
<td>High-vacuum grease</td>
<td>&nbsp;</td>
<td>Dow Corning</td>
<td>05054-AB</td>
<td>&nbsp;</td>
<tr>
<td>Anesthesia: 2.5% Avertin</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>2,2,2-tribromoethanol + tert-amyl alcohol, 1:1 w/v</td>
</tr>
<tr>
<td>Heater pad</td>
<td>&nbsp;</td>
<td>Mastex</td>
<td>Model 900</td>
<td>&nbsp;</td>
<tr>
<td>Stereotactic device</td>
<td>&nbsp;</td>
<td>Kopf</td>
<td>Model Kopf-900</td>
<td>&nbsp;</td>
<tr>
<td>Surgical scalpel blade # 15</td>
<td>&nbsp;</td>
<td>Medi-Cut</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Micro driller</td>
<td>&nbsp;</td>
<td>Ideal Micro Drill</td>
<td>67-1000</td>
<td>&nbsp;</td>
<tr>
<td>Fine forceps</td>
<td>&nbsp;</td>
<td>Fine Scientific Tools</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>1-&mu;L Micropipette</td>
<td>&nbsp;</td>
<td>Rainin</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Parafilm M.</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Surgical microscope</td>
<td>&nbsp;</td>
<td>Zeiss</td>
<td>Vasiorkop with Contraves system</td>
<td>&nbsp;</td>
<tr>
<td>Microinjector</td>
<td>&nbsp;</td>
<td>Narishige</td>
<td>Model MO-10</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

targeting of deep brain structures with microinjections for delivery of drugs, viral vectors, or cell transplants

뇌 실질에 Microinjections 마약, 바이러스성 벡터 또는 세포 이식을 전달하는 것이 중요 절차입니다. 주사 바늘는 궤도 도중에 생성되는 뇌 병변뿐만 아니라 두뇌가 작은뿐만 아니라 때로는 여러 개의 주사가 필요합니다 특히 마우스 두뇌의 주요 관심사입니다. 우리는 여기서 크게 뇌 손상을 감소하고 쥐 두뇌에 정확한 타겟팅을 수있는 50 μm의 루멘과 유리 모세관 바늘을 생산하는 방법을 보여줍니다. 이 방법은 작은 볼륨의 전달 (20-100 NL에서)는, 출혈 위험을 줄일 수 있도록하고, 뇌 실질 조직에 약물의 수동적 확산을 최소화합니다. 모세관 유리 튜브의 다른 크기를 사용하거나 바늘 루멘을 변경하여, 물질과 세포의 여러 종류의 주입 수 있습니다. 유리 모세관과 Microinjections는 분사 기술과 소형 설치류에 최소한의 피해 타겟팅 깊은 두뇌에 상당한 개선을 나타냅니다.

Watch this Scientific Journal Video about Targeting of Deep Brain Structures with Microinjections for Delivery of Drugs, Viral Vectors, or Cell Transplants at JoVE.com

Targeting of Deep Brain Structures with Microinjections for Delivery of Drugs, Viral Vectors, or Cell Transplants

이 문서에서는, 우리는 50 μm의 루멘과 유리 모세관 바늘을하는 방법을 보여줍니다. 이 기술은 크게, 뇌 손상을 감소 약물의 수동적 확산을 최소화하고 쥐 두뇌에 정확한 타겟팅을 수 있습니다.

마약, 바이러스성 벡터, 또는 세포 이식 전달 Microinjections 깊은 뇌 구조의 타겟팅

Microinjections into the brain parenchyma are important procedures to deliver drugs, viral vectors or cell transplants.  The brain lesion that an ...

targeting-deep-brain-structures-with-microinjections-for-delivery

Research

JoVE Journal

Neuroscience

12.5K 조회수.  University of Colima. 이 문서에서는, 우리는 50 μm의 루멘과 유리 모세관 바늘을하는 방법을 보여줍니다. 이 기술은 크게, 뇌 손상을 감소 약물의 수동적 확산을 최소화하고 쥐 두뇌에 정확한 타겟팅을 수 있습니다.

비디오: Targeting of Deep Brain Structures with Microinjections for Delivery of Drugs, Viral Vectors, or Cell Transplants

Scalable Fluidic Injector Arrays for Viral Targeting of Intact 3-D Brain Circuits

Our understanding of neural circuits--how they mediate the computations that subserve sensation, thought, emotion, and action, and how they are corrupted in neurological and psychiatric disorders--would be greatly facilitated by a technology for rapidly targeting genes to complex 3-dimensional neural circuits, enabling fast creation of "circuit-level transgenics." We have recently developed methods in which viruses encoding for light-sensitive proteins can sensitize specific cell types to millisecond-timescale activation and silencing in the intact brain. We here present the design and implementation of an injector array capable of delivering viruses (or other fluids) to dozens of defined points within the 3-dimensional structure of the brain (Figure. 1A, 1B). The injector array comprises one or more displacement pumps that each drive a set of syringes, each of which feeds into a polyimide/fused-silica capillary via a high-pressure-tolerant connector. The capillaries are sized, and then inserted into, desired locations specified by custom-milling a stereotactic positioning board, thus allowing viruses or other reagents to be delivered to the desired set of brain regions. To use the device, the surgeon first fills the fluidic subsystem entirely with oil, backfills the capillaries with the virus, inserts the device into the brain, and infuses reagents slowly (&lt;0.1 microliters/min). The parallel nature of the injector array facilitates rapid, accurate, and robust labeling of entire neural circuits with viral payloads such as optical sensitizers to enable light-activation and silencing of defined brain circuits. Along with other technologies, such as optical fiber arrays for light delivery to desired sets of brain regions, we hope to create a toolbox that enables the systematic probing of causal neural functions in the intact brain. This technology may not only open up such systematic approaches to circuit-focused neuroscience in mammals, and facilitate labeling of brain regions in large animals such as non-human primates, but may also open up a clinical translational path for cell-specific optical control prosthetics, whose precision may enable improved treatment of intractable brain disorders. Finally, such devices as described here may facilitate precisely-timed fluidic delivery of other payloads, such as stem cells and pharmacological agents, to 3-dimensional structures, in an easily user-customizable fashion.

Controlling and analyzing neural circuits in vivo would be facilitated by a technology for delivery of viruses and other reagents to desired 3-dimensional sets of brain regions. We demonstrate customized fluidic injector array fabrication, and delivery of virally-encoded optical sensitizers, enabling optical manipulation of complex brain circuits.

손상 3 - D 뇌 회로의 타겟팅 바이러스성을위한 확장 가능한 유체 인젝터 배열

Our understanding of neural circuits--how they mediate the computations that subserve sensation, thought, emotion, and action, and how they are corrupted ...

연구

신경과학

Intracranial Injection of Adeno-associated Viral Vectors

Intracranial injection of viral vectors engineered to express a fluorescent protein is a versatile labeling technique for visualization of specific subsets of cells in different brain regions both in vivo and in brain sections. Unlike the injection of fluorescent dyes, viral labeling offers targeting of individual cell types and is less expensive and time consuming than establishing transgenic mouse lines. In this technique, an adeno-associated viral (AAV) vector is injected intracranially using stereotaxic coordinates, a micropipette and an automated pump for precise delivery of AAV to the desired area with minimal damage to the surrounding tissue. Injection parameters can be tailored to individual experiments by adjusting the animal age at injection, injection location, volume of injection, rate of injection, AAV serotype and the promoter driving gene expression. Depending on the conditions chosen, virally-induced transgene expression can allow visualization of groups of cells, individual cells or fine cellular processes, down to the level of dendritic spines. The experiment shown here depicts an injection of double-stranded AAV expressing green fluorescent protein for the labeling of neurons and glia in the mouse primary visual cortex.

Here we present the intracranial injection of AAV vectors for fluorescent labeling of neurons and glia in the visual cortex.

Adeno - 관련 바이러스성 벡터의 Intracranial 분사

Intracranial injection of viral vectors engineered to express a fluorescent protein is a versatile labeling technique for visualization of specific ...

Deep Brain Stimulation with Simultaneous fMRI in Rodents

In order to visualize the global and downstream neuronal responses to deep brain stimulation (DBS) at various targets, we have developed a protocol for using blood oxygen level dependent (BOLD) functional magnetic resonance imaging (fMRI) to image rodents with simultaneous DBS. DBS fMRI presents a number of technical challenges, including accuracy of electrode implantation, MR artifacts created by the electrode, choice of anesthesia and paralytic to minimize any neuronal effects while simultaneously eliminating animal motion, and maintenance of physiological parameters, deviation from which can confound the BOLD signal.&#160;Our laboratory has developed a set of procedures that are capable of overcoming most of these possible issues. For electrical stimulation, a homemade tungsten bipolar microelectrode is used, inserted stereotactically at the stimulation site in the anesthetized subject. In preparation for imaging, rodents are fixed on a plastic headpiece and transferred to the magnet bore. For sedation and paralysis during scanning, a cocktail of dexmedetomidine and pancuronium is continuously infused, along with a minimal dose of isoflurane; this preparation minimizes the BOLD ceiling effect of volatile anesthetics. In this example experiment, stimulation of the subthalamic nucleus (STN) produces BOLD responses which are observed primarily in ipsilateral cortical regions, centered in motor cortex. Simultaneous DBS and fMRI allows the unambiguous modulation of neural circuits dependent on stimulation location and stimulation parameters, and permits observation of neuronal modulations free of regional bias. This technique may be used to explore the downstream effects of modulating neural circuitry at nearly any brain region, with implications for both experimental and clinical DBS.

This protocol describes a standard method for simultaneous functional magnetic resonance imaging and deep brain stimulation in the rodent. The combined use of these experimental tools allows for the exploration of global downstream activity in response to electrical stimulation at virtually any brain target.

설치류에 동시 fMRI를 깊은 뇌 자극

In order to visualize the global and downstream neuronal responses to deep brain stimulation (DBS) at various targets, we have developed a protocol for ...

Non-restraining EEG Radiotelemetry: Epidural and Deep Intracerebral Stereotaxic EEG Electrode Placement

Implantable EEG radiotelemetry is of central relevance in the neurological characterization of transgenic mouse models of neuropsychiatric and neurodegenerative diseases as well as epilepsies. This powerful technique does not only provide valuable insights into the underlying pathophysiological mechanisms, i.e., the etiopathogenesis of CNS related diseases, it also facilitates the development of new translational, i.e., therapeutic approaches. Whereas competing techniques that make use of recorder systems used in jackets or tethered systems suffer from their unphysiological restraining to semi-restraining character, radiotelemetric EEG recordings overcome these disadvantages. Technically, implantable EEG radiotelemetry allows for precise and highly sensitive measurement of epidural and deep, intracerebral EEGs under various physiological and pathophysiological conditions. First, we present a detailed protocol of a straight forward, successful, quick and efficient technique for epidural (surface) EEG recordings resulting in high-quality electrocorticograms. Second, we demonstrate how to implant deep, intracerebral EEG electrodes, e.g., in the hippocampus (electrohippocampogram). For both approaches, a computerized 3D stereotaxic electrode implantation system is used. The radiofrequency transmitter itself is implanted into a subcutaneous pouch in both mice and rats. Special attention also has to be paid to pre-, peri- and postoperative treatment of the experimental animals. Preoperative preparation of mice and rats, suitable anesthesia as well as postoperative treatment and pain management are described in detail.

Non-restraining EEG radiotelemetry is a valuable methodological approach to record in vivo long-term electroencephalograms from freely moving rodents. This detailed protocol describes stereotaxic epidural and deep intracerebral electrode placement in different brain regions in order to obtain reliable recordings of CNS rhythmicity and CNS related behavioral stages.

비 금지 EEG Radiotelemetry : 경막 외 및 깊은 뇌내 정위 EEG 전극 위치

Implantable EEG radiotelemetry is of central relevance in the neurological characterization of transgenic mouse models of neuropsychiatric and ...

Personalized Needles for Microinjections in the Rodent Brain

Microinjections have been used for a long time for the delivery of drugs or toxins within specific brain areas and, more recently, they have been used to deliver gene or cell therapy products. Unfortunately, current microinjection techniques use steel or glass needles that are suboptimal for multiple reasons: in particular, steel needles may cause tissue damage, and glass needles may bend when lowered deeply into the brain, missing the target region. In this article, we describe a protocol to prepare and use quartz needles that combine a number of useful features. These needles do not produce detectable tissue damage and, being very rigid, ensure reliable delivery in the desired brain region even when using deep coordinates. Moreover, it is possible to personalize the design of the needle by making multiple holes of the desired diameter. Multiple holes facilitate the injection of large amounts of solution within a larger area, whereas large holes facilitate the injection of cells. In addition, these quartz needles can be cleaned and re-used, such that the procedure becomes cost-effective.

We describe here a protocol for microinjection in the rodent brain that uses quartz needles. These needles do not produce detectable tissue damage and ensure reliable delivery even in deep regions. Moreover, they can be adapted to research needs by personalized designs and can be re-used.

쥐 두뇌에 있는 Microinjections에 대 한 개인화 된 바늘

Microinjections have been used for a long time for the delivery of drugs or toxins within specific brain areas and, more recently, they have been used to ...

Exploring Deep Space - Uncovering the Anatomy of Periventricular Structures to Reveal the Lateral Ventricles of the Human Brain

Anatomy students are typically provided with two-dimensional (2D) sections and images when studying cerebral ventricular anatomy and students find this challenging. Because the ventricles are negative spaces located deep within the brain, the only way to understand their anatomy is by appreciating their boundaries formed by related structures. Looking at a 2D representation of these spaces, in any of the cardinal planes, will not enable visualisation of all of the structures that form the boundaries of the ventricles. Thus, using 2D sections alone requires students to compute their own mental image of the 3D ventricular spaces. The aim of this study was to develop a reproducible method for dissecting the human brain to create an educational resource to enhance student understanding of the intricate relationships between the ventricles and periventricular structures. To achieve this, we created a video resource that features a step-by-step guide using a fiber dissection method to reveal the lateral and third ventricles together with the closely related limbic system and basal ganglia structures. One of the advantages of this method is that it enables delineation of the white matter tracts that are difficult to distinguish using other dissection techniques. This video is accompanied by a written protocol that provides a systematic description of the process to aid in the reproduction of the brain dissection. This package offers a valuable anatomy teaching resource for educators and students alike. By following these instructions educators can create teaching resources and students can be guided to produce their own brain dissection as a hands-on practical activity. We recommend that this video guide be incorporated into neuroanatomy teaching to enhance student understanding of the morphology and clinical relevance of the ventricles.

This paper demonstrates the effective use of a fiber dissection method to reveal the superficial white matter tracts and periventricular structures of the human brain, in three-dimensional space, to aid student comprehension of ventricular morphology.

깊은 공간-인간 두뇌의 측면 심 공개 뇌 구조 해부학 적 탐구

Anatomy students are typically provided with two-dimensional (2D) sections and images when studying cerebral ventricular anatomy and students find this ...

Quantitative Cell Biology of Neurodegeneration in Drosophila Through Unbiased Analysis of Fluorescently Tagged Proteins Using ImageJ

With the rising prevalence of neurodegenerative diseases, it is increasingly important to understand the underlying pathophysiology that leads to neuronal dysfunction and loss. Fluorescence-based imaging tools and technologies enable unprecedented analysis of subcellular neurobiological processes, yet there is still a need for unbiased, reproducible, and accessible approaches for extracting quantifiable data from imaging studies. We have developed a simple and adaptable workflow to extract quantitative data from fluorescence-based imaging studies using Drosophila models of neurodegeneration. Specifically, we describe an easy-to-follow, semi-automated approach using Fiji/ImageJ to analyze two cellular processes: first, we quantify protein aggregate content and profile in the Drosophila optic lobe using fluorescent-tagged mutant huntingtin proteins; and second, we assess autophagy-lysosome flux in the Drosophila visual system with ratiometric-based quantification of a tandem fluorescent reporter of autophagy. Importantly, the protocol outlined here includes a semi-automated segmentation step to ensure all fluorescent structures are analyzed to minimize selection bias and to increase resolution of subtle comparisons. This approach can be extended for the analysis of other cell biological structures and processes implicated in neurodegeneration, such as proteinaceous puncta (stress granules and synaptic complexes), as well as membrane-bound compartments (mitochondria and membrane trafficking vesicles). This method provides a standardized, yet adaptable reference point for image analysis and quantification, and could facilitate reliability and reproducibility across the field, and ultimately enhance mechanistic understanding of neurodegeneration.

Quantitative Cell Biology of Neurodegeneration in Drosophila Through Unbiased Analysis of Fluorescently Tagged Proteins Using ImageJ

We have developed a simple and adaptable workflow to extract quantitative data from fluorescence-imaging-based cell biological studies of protein aggregation and autophagic flux in the central nervous system of Drosophila models of neurodegeneration.

ImageJ를 사용 하 여 붙일 태그가 단백질의 편견된 분석 통해 초파리 에서 Neurodegeneration의 양적 세포 생물학

With the rising prevalence of neurodegenerative diseases, it is increasingly important to understand the underlying pathophysiology that leads to neuronal ...

Production, Purification, and Quality Control for Adeno-associated Virus-based Vectors

Gene delivery tools based on adeno-associated viruses (AAVs) are a popular choice for the delivery of transgenes to the central nervous system (CNS), including gene therapy applications. AAV vectors are non-replicating, able to infect both dividing and non-dividing cells&#160;and provide long-term transgene expression. Importantly, some serotypes, such as the newly described PHP.B, can cross the blood-brain-barrier (BBB) in animal models, following systemic delivery. AAV vectors can be efficiently produced in the laboratory. However, robust and reproducible protocols are required to obtain AAV vectors with sufficient purity levels and titer values high enough for in vivo applications. This protocol describes an efficient and reproducible strategy for AAV vector production, based on an iodixanol gradient purification strategy. The iodixanol purification method is suitable for obtaining batches of high-titer AAV vectors of high purity, when compared to other purification methods. Furthermore, the protocol is generally faster than other methods currently described. In addition, a quantitative polymerase chain reaction (qPCR)-based strategy is described for a fast and accurate determination of the vector titer, as well as a silver staining method to determine the purity of the vector batch. Finally, representative results of gene delivery to the CNS, following systemic administration of AAV-PHP.B, are presented. Such results should be possible in all labs using the protocols described in this article.

Here, we describe an efficient and reproducible strategy to produce, titer, and quality-control batches of adeno-associated virus vectors. It allows the user to obtain a vector preparation with high-titer&#160;(&#8805;1 x 1013 vector genomes/mL) and a high purity, ready for in vitro or in vivo use.

생산, 정화, 및 Adeno 관련 바이러스 기반 벡터에 대 한 품질 관리

Gene delivery tools based on adeno-associated viruses (AAVs) are a popular choice for the delivery of transgenes to the central nervous system (CNS), ...

Three-Dimensional Shape Modeling and Analysis of Brain Structures

Statistical shape analysis of brain structures has been used to investigate the association between their structural changes and pathological processes. We have developed a software package for accurate and robust shape modeling and group-wise analysis. Here, we introduce an pipeline for the shape analysis, from individual 3D shape modeling to quantitative group shape analysis. We also describe the pre-processing and segmentation steps using open software packages. This practical guide would help researchers save time and effort in 3D shape analysis on brain structures.

We introduce a semi-automatic protocol for shape analysis on brain structures, including image segmentation using open software, and further group-wise shape analysis using an automated modeling package. Here, we demonstrate each step of the 3D shape analysis protocol with hippocampal segmentation from brain MR images.

뇌 구조의 3차원 형상 모델링 및 분석

Statistical shape analysis of brain structures has been used to investigate the association between their structural changes and pathological processes. ...

In Vivo Direct Reprogramming of Resident Glial Cells into Interneurons by Intracerebral Injection of Viral Vectors

Converting resident glia in the brain into functional and subtype-specific neurons in vivo provides a step forward towards the development of alternative cell replacement therapies while also creating tools to study cell fate in situ. To date, it has been possible to obtain neurons via in vivo reprogramming, but the precise phenotype of these neurons or how they mature has not been analyzed in detail. In this protocol, we describe a more efficient conversion and cell-specific identification of the in vivo reprogrammed neurons, using an AAV-based viral vector system. We also provide a protocol for functional assessment of the reprogrammed cells&#8217; neuronal maturation. By injecting flip-excision (FLEX) vectors, containing the reprogramming and synapsin-driven reporter genes&#160;to specific cell types in the brain that serve as the target for cell reprogramming. This technique allows for the easy identification of newly reprogrammed neurons. Results show that the obtained reprogrammed neurons functionally mature over time, receive synaptic contacts and show electrophysiological properties of different types of interneurons. Using the transcription factors Ascl1, Lmx1a and Nurr1, the majority of the reprogrammed cells have properties of fast-spiking, parvalbumin-containing interneurons.

This protocol aims at generating directly reprogrammed interneurons in vivo, using an AAV-based viral system in the brain and a FLEX synapsin-driven GFP reporter, which allows for cell identification and further analysis in vivo.

바이러스 벡터의 intracerebral 주입에 의해 인터뉴런으로 상주 신경교 세포의 생체 내 직접 재프로그래밍

Converting resident glia in the brain into functional and subtype-specific neurons in vivo provides a step forward towards the development of alternative ...