Эта работа была поддержана Национальным институтом здоровья (NIH) гранты DA030396. Благодаря членам лаборатории Drenan за полезные обсуждения и критику рукописи. Особая благодарность Ми Ран Ким технической помощи и Джонатан Томас Ting для консультаций по вопросам взрослого кусочки мозга мыши.

1. Подготовка решения для мозга Подготовка Slice и электрофизиологии <ol><li> Решения для подготовки срезах мозга были описаны 3, 4. Подготовка N-метил-D-глюкамин (NMDG)-основанной резки и восстановления данных следующего состава (в мМ): 93 N-метил-D-глюкамин, 2,5 KCl, 1,2 NaH 2 4 PO, 30 NaHCO 3, 20 HEPES, 25 глюкозы, 5 Na + аскорбиновая кислота, 2 тиомочевины, 3 Na + пируват, 10 MgSO 4 • 7H 2 O, 0,5 CaCl 2 • 2H 2 O. Отрегулируйте до 300-310 мосм с NMDG. Отрегулируйте рН до 7.3-7.4 с 10 N HCl. <ol class="lalpha"><li> Растворите MgSO 4 • 7H 2 O и CaCl 2 • 2H 2 O в Millipore воды, чтобы сделать 2 М растворов каждого из них. </li><li> Взвесить другие компоненты в указанном порядке и добавить в мерную колбу частично заполненный Millipore воды при перемешивании до растворения. Как только все будет добавлен, введитемерную колбу с водой Millipore. </li><li> Измерить рН и корректировать с 10 N HCl. </li><li> Измерьте осмолярности и настроить NMDG если это необходимо. </li></ol></li><li> Подготовка HEPES проведения искусственной цереброспинальной жидкости (ACSF) следующего состава (в мМ): 92 NaCl, 2,5 KCl, 1,2 NaH 2 4 PO, 30 NaHCO 3, 20 HEPES, 25 глюкозы, 5 Na + аскорбиновая кислота, 2 тиомочевины, 3 Na + пируват, 2 MgSO 4 • 7H 2 O, 2 CaCl 2 • 2H 2 O. Отрегулируйте до 300-310 мосм с сахарозой. Отрегулируйте рН до 7.3-7.4 с 1 N HCl или NaOH. <ol class="lalpha"><li> Взвесить компоненты в указанном порядке и добавить в мерную колбу частично заполненный Millipore воды при перемешивании до растворения. Как только все будет добавлен, введите мерную колбу с водой Millipore. </li><li> Измерить рН и корректировать с NaOH или HCl, если необходимо. </li><li> Измерьте и отрегулируйте осмолярности с сахарозой, если необходимо. </li></ol></li><li> Подготовка стандартной записи ACSF решение следующего состава (в мМ): 124 NaCl, 2,5 KCl, 1,2 NaH 2 4 PO, 24 NaHCO 3, 12,5 глюкозы, 2 MgSO 4 • 7H 2 O, 2 CaCl 2 • 2H 2 O. Отрегулируйте до 300-310 мосм с сахарозой. Отрегулируйте рН до 7.3-7.4 с 1 N HCl или NaOH. </li></ol> 2. Подготовка Острые фрагменты мозга <ol><li> Мозг резки срез и восстановление выполняется, как описано ранее 3, 4. Bubble два блюда кристалл заполнен NMDG решение для восстановления с 95% кислорода / 5% CO 2 газ (карбогена), одно блюдо на льду, другая на 33 ° C. </li><li> Bubble одном кристалле блюдо заполнено HEPES проведения ACSF с карбогена при комнатной температуре. </li><li> Anesthetize взрослой мыши (в возрасте от 2 до 12 месяцев) с Na + фенобарбитала (200 мг / кг, внутрибрюшинно). Продолжайте процедуру, когда животное не имеет ответа на ноги-пинча. </li><li> Если необходимо, вырезать образец ткани хвоста для последующего genotypING животного. </li><li> Откройте грудную полость, подвергать сердце, и зажмите нисходящей аорты. Lacerate в правое предсердие. </li><li> Transcardially заливать животных с 5-10 мл 0-4 ° C NMDG-решения для восстановления. </li><li> Обезглавьте животного, удалить мозга и поместить его в 4 ° C NMDG-решения для восстановления в течение 1 мин. </li><li> На льду охлажденной металлической пластины, обрезать мозга в нужной ориентации (корональные, парасагиттальных, горизонтальная и т.д.), включают в сферу интересов. </li><li> Прикрепите блок с мозгом суперклей на сухой поверхности этапе вибрирующий среза (ДСК-Zero 1; Dosaka), погрузить мозг в блоке 4 ° C NMDG-решения для восстановления и постоянно пузыря решение с карбогена. </li><li> Вырежьте кусочки мозга сфере интересов. Толщина среза составляет 200-400 мкм, в зависимости от конкретной области мозга интересов, возраста животных, и эксперимент должны быть выполнены. </li><li> Сразу же после резки, инкубировать желаемого ломтики в carbogenated, 33 ° C NMDG восстановления solutiна ровно на 12 мин, затем переносить их на carbogenated, комнатной температуры HEPES проведение решение для начального периода 60 мин. После этого 60 мин инкубации срезы могут быть использованы для записи. Фрагменты сохраняются в HEPES проведение решение в течение дня, пока они используются для записи. </li></ol> 3. Patch Clamp Запись от нейронов в срезах мозга <ol><li> Потяните стандартных микропипетки патч с программируемым Flaming-Brown съемник (Саттер Р-97 или эквивалент вертикального съемник). Пипетки, которые являются оптимальными для гига-ом уплотнения образование, обычно имеют сопротивление 4-6 МОм </li><li> Трансфер один кусочек к записи камеры (RC-27L; Warner Instruments) на этапе прямого микроскопа (Nikon FN-1). Постоянно переливать (1,5 - 2,0 мл / мин) с ломтиком carbogenated, стандартная запись ACSF нагревают и выдерживают при 32 ° C. Кроме того, ломтики можно поддерживать при комнатной температуре. </li><li> Найдите и центр мозга прочих интересных использования10X цель воздуха (Nikon Plan Fluor 10X, NA 0,3; WD: 16 мм). </li><li> Визуализация отдельных нейронов использованием 40X ближней инфракрасной (ИК) погружение в воду цель (APO Nikon 40XW; NA: 0,80; WD: 3,5 мм), подключенный к ИК-дифференциального интерференционного контраста (DIC) Оптика и высокоскоростных инфракрасных устройств с зарядовой связью (CCD) видеокамеры (Hamamatsu C-7500). В ИК-DIC наблюдения, здоровые нейроны обладают гладкие, светло-серый плазматической мембраны. </li><li> Заполните микропипетки с подходящим внутриклеточных решение для записи. Для большинства приложений, мы используем следующие решения (в мм): 135 глюконат калия, 5 EGTA, 0,5 2 CaCl, 2 MgCl 2, 10 HEPES, 2 Mg-АТФ, ГТФ и 0,1 (рН до 7,25 с Tris базы, осмолярность скорректирована до 290 мосм с сахарозой). Создание цельноклеточной запись с визуализировать нейрона. </li></ol> 4. Местное применение лекарственных средств для нейронов в срезах <ol><li> Потяните стандартных микропипетки зажим патч как описано выше. Использованиепипетки съемник, таких как Саттер Р-97, который производит два одинаковых &quot;сестра&quot; Советы из того же куска стекла выгодно, когда несколько решений препарата будут использоваться на той же клетке. Совет размеров, что бы сопротивление ~ 5 МОм, если они были наполнены внутриклеточных решения описанных выше оптимальной. </li><li> Засыпка микропипетки с раствором препарата разводят в carbogenated, стандартный ACSF записи. Направьте наконечник вниз, и вылить препарат заполненные микропипетки, чтобы удалить пузырьки воздуха в ловушке в колонке решение. </li><li> Установите наркотиков заполненные микропипетки в пипетку держатель связанных с трубкой подходящей системы выброса давления, такие как Picospritzer III (General Valve Co.) Держатель пипетки должны быть установлены на микроманипулятора с таким же разрешением, как манипуляторы широко используются для исправления записи зажим (например, Sutter MP-285). Кроме того, манипулятор должен позволять диагональное движение в и из среза. Человекually извлечь давление от 3 до 4 раз в микропипетки для того, чтобы создать достаточное давление за колонной жидкости. </li><li> Использование микроманипулятора управления, снизить наркотиков пипетки в срезе при визуализации кончик при ИК-DIC оптики. В идеале, нужно визуально определить и в центре нейрона быть записан с, а затем позиционирования препарата пипетку рядом с ячейкой до снижения записи микропипетки в срез. Позиционирование препарата пипетку может вызвать движение ткани в непосредственной близости от записанной ячейки, которые могут нарушить записи, если препарат пипеткой перемещается в позицию после установления гигаом уплотнения. </li><li> Использование видео IR, позиционировать препарат микропипетки на верхней поверхности ткани срез. Выполнить одну выброса давления и монитор 1) вблизи острия для перемещения мусора, связанные с выбросом и 2) микропипетки совет для любых признаков того, что наконечник засорился или заблокированы. Если кончик заблокирован / забиты, убрать pipettе и заменить его на новый. </li><li> Подойдите к записанной ячейки по диагонали от верхнего среза так, что, когда в конечном положении, кончик наркотиков заполненной пипетки 1) в той же фокальной плоскости (или чуть ниже), записанный клетки и кончик электрода записи , и 2) 10 до 40 мкм от записали клетки. Если препарат заполненной пипетки выше записанного клетки, сила от давления выброса могут нарушить гигаом уплотнения. </li><li> Запись желаемый клеточный ответ, удерживая ячейки в режиме напряжения зажима или текущем режиме зажим. Мы регулярно записывать ацетилхолина и / или никотина вызвала сотовой токи, удерживая нейронов в режиме напряжения зажима. Несмотря на давление выброса может быть вызвано вручную при помощи Picospritzer III, для более воспроизводимых результатов желательно, чтобы вызвать давление выброса использованием системы сбора (Axon Digidata 1440 и pClamp 10,3) с цифровой TTL импульса. </li></ol> 5. Управлениенаркотиков заполненные микропипетки с пьезоэлектрическим переводчик <ol><li> Если возможно, установите наркотиков заполненные пипетки держателю одномерный пьезоэлектрических переводчик (например, Piezojena PA-100 или Burleigh ПЗМ-150), который способен принимать аналоговое входное напряжение (опять же, от приобретения системы), которые затем установлен на высокоточные микроманипулятора (Sutter MP-285). Пьезоэлектрические переводчик полезно, потому что движение наркотиков заполненные пипетки в и из среза, в том числе сроков и скорости въезда / выезда, может быть легко контролируется и воспроизводится от эксперимента к эксперименту. При изучении нАХР, десенсибилизирующее воздействие никотина утечки наркотиков заполненной пипетки, если они когда-нибудь произойдет, сводятся к минимуму, когда пипетки только переехали около клетки с пьезоэлектрическими переводчиком на данный момент давление выброса. </li><li> Использование ручного контролируемого потенциометра на пьезоэлектрический усилитель напряжения управления, набрать напряжениядо максимального значения. </li><li> Использование микроманипулятора, должность наркотиков заполненной пипетки в срезе, где пипетка будет извлечь его содержимое на записанную ячейку, и отказаться от пипетки вручную снижении напряжения до нуля на пьезоэлектрический усилитель управления напряжения. </li><li> Используя аналоговый сигнал от системы сбора записи, переместите наркотиков заполненные пипетки в срезе в течение 250 мс от 300 мс до давления выброса TTL импульса происходит. Уберите пипетки в течение 250 мс, начиная с 50 мс после окончания импульса TTL. </li></ol>

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Physiol. 587, 345-361 (2009).</li><li>Endo, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nicotinic+acetylcholine+receptor+subtypes+involved+in+facilitation+of+GABAergic+inhibition+in+mouse+superficial+superior+colliculus.">Nicotinic acetylcholine receptor subtypes involved in facilitation of GABAergic inhibition in mouse superficial superior colliculus.</a> J. Neurophysiol. 94, 3893-3902 (2005).</li><li>Bouzat, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+insights+into+the+structural+bases+of+activation+of+Cys-loop+receptors.">New insights into the structural bases of activation of Cys-loop receptors.</a> J. Physiol. Paris. , (2011).</li></ol>

Метод, представленный в этой статье в целом полезны для изучения лигандами ионных каналов функцию в подготовке среза мозга. Тем не менее, существует ряд факторов, которые существенно влияют на качество и воспроизводимость экспериментальных данных, которые являются результатом исполь...

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 </tr>
 <tr>
 <td>N-Methyl D-glucamine</td>
 <td>Sigma</td>
 <td>M2004</td>
 </tr>
 <tr>
 <td>KCl</td>
 <td>Sigma</td>
 <td>P3911</td>
 </tr>
 <tr>
 <td>NaH2PO4</td>
 <td>Sigma</td>
 <td>S9638</td>
 </tr>
 <tr>
 <td>NaHCO3</td>
 <td>Sigma</td>
 <td>S6014</td>
 </tr>
 <tr>
 <td>HEPES</td>
 <td>Sigma</td>
 <td>H3375</td>
 </tr>
 <tr>
 <td>glucose</td>
 <td>Sigma</td>
 <td>G5767</td>
 </tr>
 <tr>
 <td>Na+ ascorbate</td>
 <td>Sigma</td>
 <td>A4034</td>
 </tr>
 <tr>
 <td>thiourea</td>
 <td>Sigma</td>
 <td>T8656</td>
 </tr>
 <tr>
 <td>Na+ pyruvate</td>
 <td>Sigma</td>
 <td>P2256</td>
 </tr>
 <tr>
 <td>MgSO4&bull;7H2O</td>
 <td>Sigma</td>
 <td>230391</td>
 </tr>
 <tr>
 <td>CaCl2&bull;2H20</td>
 <td>Sigma</td>
 <td>223506</td>
 </tr>
 <tr>
 <td>NaCl</td>
 <td>Sigma</td>
 <td>S9625</td>
 </tr>
 <tr>
 <td>Na+ pentobarbital</td>
 <td>Vortech Pharmaceuticals</td>
 <td>76351315</td>
 </tr>
 <tr>
 <td>potassium gluconate</td>
 <td>Sigma</td>
 <td>G4500</td>
 </tr>
 <tr>
 <td>EGTA</td>
 <td>Sigma</td>
 <td>E3889</td>
 </tr>
 <tr>
 <td>Mg-ATP</td>
 <td>Sigma</td>
 <td>A9187 </td>
 </tr>
 <tr>
 <td>GTP</td>
 <td>Sigma</td>
 <td>G8877 </td>
 </tr>
 <tr>
 <td>DSK-Zero 1 Vibrating slicer</td>
 <td>Ted Pella, Inc.</td>
 <td></td>
 </tr>
 <tr>
 <td>P-97 Flaming/Brown micropipette puller</td>
 <td>Sutter</td>
 <td></td>
 </tr>
 <tr>
 <td>RC-27 Recording chamber</td>
 <td>Warner</td>
 <td></td>
 </tr>
 <tr>
 <td>TC-344B Perfusion heater controller</td>
 <td>Warner</td>
 <td>640101</td>
 </tr>
 <tr>
 <td>SH-27B Solution heater</td>
 <td>Warner</td>
 <td>640102</td>
 </tr>
 <tr>
 <td>Nikon FN-1</td>
 <td>Nikon</td>
 <td></td>
 </tr>
 <tr>
 <td>C-7500 CCD Video camera</td>
 <td>Hamamatsu</td>
 <td></td>
 </tr>
 <tr>
 <td>Picospritzer III</td>
 <td>General Valve Co.</td>
 <td></td>
 </tr>
 <tr>
 <td>MP-285 Micromanipulator </td>
 <td>Sutter </td>
 <td></td>
 </tr>
 <tr>
 <td>PA-100 Piezoelectric translator </td>
 <td>piezosystem jena, Inc.</td>
 <td></td>
 </tr>
 <tr>
 <td>12V40 piezo amplifier</td>
 <td>piezosystem jena, Inc.</td>
 <td></td>
 </tr>
 <tr>
 <td>Axopatch 200B</td>
 <td>Molecular Devices Corp.</td>
 <td></td>
 </tr>
 <tr>
 <td>Digidata 1440A</td>
 <td>Molecular Devices Corp.</td>
 <td></td>
 </tr>
 </tbody>
 </table>

local application of drugs to study nicotinic acetylcholine receptor function in mouse brain slices

Tobacco use leads to numerous health problems, including cancer, heart disease, emphysema, and stroke. Addiction to cigarette smoking is a prevalent neuropsychiatric disorder that stems from the biophysical and cellular actions of nicotine on nicotinic acetylcholine receptors (nAChRs) throughout the central nervous system. Understanding the various nAChR subtypes that exist in brain areas relevant to nicotine addiction is a major priority.
Experiments that employ electrophysiology techniques such as whole-cell patch clamp or two-electrode voltage clamp recordings are useful for pharmacological characterization of nAChRs of interest. Cells expressing nAChRs, such as mammalian tissue culture cells or Xenopus laevis oocytes, are physically isolated and are therefore easily studied using the tools of modern pharmacology. Much progress has been made using these techniques, particularly when the target receptor was already known and ectopic expression was easily achieved. Often, however, it is necessary to study nAChRs in their native environment: in neurons within brain slices acutely harvested from laboratory mice or rats. For example, mice expressing "hypersensitive" nAChR subunits such as &alpha;4 L9&prime;A mice 1 and &alpha;6 L9&prime;S mice 2, allow for unambiguous identification of neurons based on their functional expression of a specific nAChR subunit. Although whole-cell patch clamp recordings from neurons in brain slices is routinely done by the skilled electrophysiologist, it is challenging to locally apply drugs such as acetylcholine or nicotine to the recorded cell within a brain slice. Dilution of drugs into the superfusate (bath application) is not rapidly reversible, and U-tube systems are not easily adapted to work with brain slices.
In this paper, we describe a method for rapidly applying nAChR-activating drugs to neurons recorded in adult mouse brain slices. Standard whole-cell recordings are made from neurons in slices, and a second micropipette filled with a drug of interest is maneuvered into position near the recorded cell. An injection of pressurized air or inert nitrogen into the drug-filled pipette causes a small amount of drug solution to be ejected from the pipette onto the recorded cell. Using this method, nAChR-mediated currents are able to be resolved with millisecond accuracy. Drug application times can easily be varied, and the drug-filled pipette can be retracted and replaced with a new pipette, allowing for concentration-response curves to be created for a single neuron. Although described in the context of nAChR neurobiology, this technique should be useful for studying many types of ligand-gated ion channels or receptors in neurons from brain slices.

In our experiments, we routinely record from dopamine (DA)-producing neurons of the ventral tegmental area (VTA) and substantia nigra pars compacta (SNc). In voltage-clamp mode, pressure application of acetylcholine or nicotine to these cells will typically result in a rapid, inward cation current that reaches peak within 100-200 msec (Figure 1A-B). Decay of the current is largely dictated by diffusion of the drug from the site of action, and whether enzymes in the slice are present to metabolize the dru...

Watch this Scientific Journal Video about Local Application of Drugs to Study Nicotinic Acetylcholine Receptor Function in Mouse Brain Slices at JoVE.com

Local Application of Drugs to Study Nicotinic Acetylcholine Receptor Function in Mouse Brain Slices

В этой статье мы описываем полезный метод для изучения лигандами ионных каналов функции в нейронах остро изолированных кусочков мозга. Этот метод включает в себя использование наркотиков заполненные микропипетки для местного применения препаратов на нейроны, записанные с помощью стандартных методов зажим патч.

Местное применение наркотиков по изучению никотиновых рецепторов ацетилхолина функции в срезах мозга мыши

Tobacco use leads to numerous health problems, including cancer, heart disease, emphysema, and stroke. Addiction to cigarette smoking is a prevalent ...

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Drugs Acting on Autonomic Nervous System: Cholinergic Agonists and Antagonists Agents

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19.2K Views.  Purdue University. В этой статье мы описываем полезный метод для изучения лигандами ионных каналов функции в нейронах остро изолированных кусочков мозга. Этот метод включает в себя использование наркотиков заполненные микропипетки для местного применения препаратов на нейро...

Video: Local Application of Drugs to Study Nicotinic Acetylcholine Receptor Function in Mouse Brain Slices

Vibrodissociation of Neurons from Rodent Brain Slices to Study Synaptic Transmission and Image Presynaptic Terminals

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of interest. This allows for examination of synaptic transmission under conditions where the extracellular and postsynaptic intracellular environments can be well controlled. A vibration-based technique without the use of proteases, known as vibrodissociation, is the most popular technique for mechanical isolation. A micropipette, with the tip fire-polished to the shape of a small ball, is placed into a brain slice made from a P1-P21 rodent. The micropipette is vibrated parallel to the slice surface and lowered through the slice thickness resulting in the liberation of isolated neurons. The isolated neurons are ready for study within a few minutes of vibrodissociation. This technique has advantages over the use of primary neuronal cultures, brain slices and enzymatically isolated neurons including: rapid production of viable, relatively mature neurons suitable for electrophysiological and imaging studies; superior control of the extracellular environment free from the influence of neighboring cells; suitability for well-controlled pharmacological experiments using rapid drug application and total cell superfusion; and improved space-clamp in whole-cell recordings relative to neurons in slice or cell culture preparations. This preparation can be used to examine synaptic physiology, pharmacology, modulation and plasticity. Real-time imaging of both pre- and postsynaptic elements in the living cells and boutons is also possible using vibrodissociated neurons. Characterization of the molecular constituents of pre- and postsynaptic elements can also be achieved with immunological and imaging-based approaches.

This report demonstrates a technique for mechanical isolation of individual viable neurons retaining attached presynaptic boutons. Vibrodissociated neurons have the advantages of rapid production, excellent pharmacological control and improved space-clamp without influence from neighboring cells. This method can be used for imaging of synaptic elements and patch-clamp recording.

Vibrodissociation нейронов из кусочков мозга грызунов по изучению синаптической передачи изображения и пресинаптические терминалы

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of ...

Imaging Serotonergic Fibers in the Mouse Spinal Cord Using the CLARITY/CUBIC Technique

Long descending fibers to the spinal cord are essential for locomotion, pain perception, and other behaviors. The fiber termination pattern in the spinal cord of the majority of these fiber systems have not been thoroughly investigated in any species. Serotonergic fibers, which project to the spinal cord, have been studied in rats and opossums on histological sections and their functional significance has been deduced based on their fiber termination pattern in the spinal cord. With the development of CLARITY and CUBIC techniques, it is possible to investigate this fiber system and its distribution in the spinal cord, which is likely to reveal previously unknown features of serotonergic supraspinal pathways. Here, we provide a detailed protocol for imaging the serotonergic fibers in the mouse spinal cord using the combined CLARITY and CUBIC techniques. The method involves perfusion of a mouse with a hydrogel solution and clarification of the tissue with a combination of clearing reagents. Spinal cord tissue was cleared in just under two weeks, and the subsequent immunofluorescent staining against serotonin was completed in less than ten days. With a multi-photon fluorescent microscope, the tissue was scanned and a 3D image was reconstructed using Osirix software.

Supraspinal projections are important for pain perception and other behaviors, and serotonergic fibers are one of these fiber systems. The present study focused on the application of the combined CLARITY/CUBIC protocol to the mouse spinal cord in order to investigate the termination of these serotonergic fibers.

Визуализация Серотонинергической волокон в спинном мозге мышей Использование Четкость / КУБИЧЕСКИЙ Техника

Long descending fibers to the spinal cord are essential for locomotion, pain perception, and other behaviors. The fiber termination pattern in the spinal ...

HSV-Mediated Transgene Expression of Chimeric Constructs to Study Behavioral Function of GPCR Heteromers in Mice

The heteromeric receptor complex between 5-HT2A and mGlu2 has been implicated in some of the behavioral phenotypes in mouse models of psychosis1,2. Consequently, investigation of structural details of the interaction between 5-HT2A and mGlu2 affecting schizophrenia-related behaviors represents a powerful translational tool. As previously shown, the head-twitch response (HTR) in mice is elicited by hallucinogenic drugs and this behavioral response is absent in 5-HT2A knockout (KO) mice3,4. Additionally, by conditionally expressing the 5-HT2A receptor only in cortex, it was demonstrated that 5-HT2A receptor-dependent signaling pathways on cortical pyramidal neurons are sufficient to elicit head-twitch behavior in response to hallucinogenic drugs3. Finally, it has been shown that the head-twitch behavioral response induced by the hallucinogens DOI and lysergic acid diethylamide (LSD) is significantly decreased in mGlu2-KO mice5. These findings suggest that mGlu2 is at least in part necessary for the 5-HT2A receptor-dependent psychosis-like behavioral effects induced by LSD-like drugs. However, this does not provide evidence as to whether the 5-HT2A-mGlu2 receptor complex is necessary for this behavioral phenotype. To address this question, herpes simplex virus (HSV) constructs to express either mGlu2 or mGlu2&#916;TM4N (mGlu2/mGlu3 chimeric construct that does not form the 5-HT2A-mGlu2 receptor complex) in the frontal cortex of mGlu2-KO mice were used to examine whether this GPCR heteromeric complex is needed for the behavioral effects induced by LSD-like drugs6.

This article describes how to inject viral vectors into the mouse frontal cortex to test behavioral assays that require GPCR heteromeric formation.

HSV-опосредованной Transgene экспрессия химерных конструктов для изучения поведенческой Функция GPCR гетеромеров у мышей

The heteromeric receptor complex between 5-HT2A and mGlu2 has been implicated in some of the behavioral phenotypes in mouse models of psychosis1,2.  ...

Application of Automated Image-guided Patch Clamp for the Study of Neurons in Brain Slices

Whole-cell patch clamp is the gold-standard method to measure the electrical properties of single cells. However, the in vitro patch clamp remains a challenging and low-throughput technique due to its complexity and high reliance on user operation and control. This manuscript demonstrates an image-guided automatic patch clamp system for in vitro whole-cell patch clamp experiments in acute brain slices. Our system implements a computer vision-based algorithm to detect fluorescently labeled cells and to target them for fully automatic patching using a micromanipulator and internal pipette pressure control. The entire process is highly automated, with minimal requirements for human intervention. Real-time experimental information, including electrical resistance and internal pipette pressure, are documented electronically for future analysis and for optimization to different cell types. Although our system is described in the context of acute brain slice recordings, it can also be applied to the automated image-guided patch clamp of dissociated neurons, organotypic slice cultures, and other non-neuronal cell types.

This protocol describes how to conduct automatic image-guided patch-clamp experiments using a system recently developed for standard in vitro electrophysiology equipment.

Применение автоматизированного патч-зажима для изображений для изучения нейронов в средах мозга

Whole-cell patch clamp is the gold-standard method to measure the electrical properties of single cells. However, the in vitro patch clamp remains a ...

Whole-cell Currents Induced by Puff Application of GABA in Brain Slices

Pharmacological administration is commonly used when conducting whole-cell patch-clamp recording in brain slices. One of the best methods of drug application during electrophysiological recording is the puff technique, which can be used to study the effect of pharmacological reagents on neuronal activities in brain slices. The greatest advantage of puff application is that the drug concentration around the recording site increases rapidly, thus preventing desensitization of membrane receptors. Successful use of puff application involves careful attention to the following elements: the concentration of the drug, the parameters of the puff micropipette, the distance between the tip of a puff micropipette and the neuron recorded, and the duration and pressure driving the puff (pounds per square inch, psi). This article describes a step-by-step procedure for recording whole-cell currents induced by puffing gamma-aminobutyric acid (GABA) onto a neuron of a prefrontal cortical slice. Notably, the same procedure can be applied with minor modifications to other brain areas such as the hippocampus and the striatum, and to different preparations, such as cell cultures.

We describe the puff technique, by which pharmacological reagents can be administered during whole-cell patch-clamp recording, and highlight various aspects of the features that are crucial for its success.

Поклеточного течения индуцированных слоеного применение ГАМК в мозге ломтиками

Pharmacological administration is commonly used when conducting whole-cell patch-clamp recording in brain slices. One of the best methods of drug ...

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

Despite&#160;numerous studies that attempt to develop reliable animal models&#160;which reflecting the primary processes underlying neurodegeneration, very few have been widely accepted. Here, we propose&#160;a new procedure&#160;adapted from the well-known ex vivo brain slice technique, which offers a closer in vivo-like scenario than in vitro preparations, for investigating the early events triggering cell degeneration, as observed in Alzheimer&#39;s disease (AD). This variation consists of simple and easily reproducible steps, which enable preservation of the anatomical cytoarchitecture of the selected brain region and its local functionality in a physiological milieu. Different anatomical areas can be obtained from the same brain, providing the opportunity to perform multiple experiments with the treatments in question in a site-, dose-, and time-dependent manner. Potential limitations&#160;which could affect the outcomes related to this methodology are related to the conservation of the tissue, i.e., the maintenance of its anatomical integrity during the slicing and incubation steps and the section thickness, which can influence the biochemical and immunohistochemical analysis. This approach can be employed for different purposes, such as exploring molecular mechanisms involved in physiological or pathological conditions, drug screening, or dose-response assays. Finally, this protocol could also reduce the number of animals employed in behavioral studies. The application reported here has been recently described and tested for the first time on ex vivo rat brain slices containing the basal forebrain (BF), which is one of the cerebral regions primarily affected in AD. Specifically, it has been demonstrated that the administration of a toxic peptide derived from the C-terminus of acetylcholinesterase (AChE) could prompt an AD-like profile, triggering, along the antero-posterior axis of the BF, a differential expression of proteins altered in AD, such as the alpha7 nicotinic receptor (&#945;7-nAChR), phosphorylated Tau (p-Tau), and amyloid beta (A&#946;).

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

We present a method which can&#160;provide further insights into the early events underlying neurodegeneration and based on the established ex-vivo brain technique, combining the advantages of in vivo and in vitro experiments. Moreover, it represents a unique opportunity for direct comparison of treated and untreated group in the same anatomical plane.

Альтернативный подход к изучению основных событий в нейродегенеративные, используя срезы мозга крысы Ex Vivo

Despite&#160;numerous studies that attempt to develop reliable animal models&#160;which reflecting the primary processes underlying neurodegeneration, ...

Probing Nicotinic Acetylcholine Receptor Function in Mouse Brain Slices via Laser Flash Photolysis of Photoactivatable Nicotine

Acetylcholine (ACh) acts through receptors to modulate a variety of neuronal processes, but it has been challenging to link ACh receptor function with subcellular location within cells where this function is carried out. To study the subcellular location of nicotinic ACh receptors (nAChRs) in native brain tissue, an optical method was developed for precise release of nicotine at discrete locations near neuronal membranes during electrophysiological recordings. Patch-clamped neurons in brain slices are filled with dye to visualize their morphology during 2-photon laser scanning microscopy, and nicotine uncaging is executed with a light flash by focusing a 405 nm laser beam near one or more cellular membranes. Cellular current deflections are measured, and a high-resolution three-dimensional (3D) image of the recorded neuron is made to allow reconciliation of nAChR responses with cellular morphology. This method allows for detailed analysis of nAChR functional distribution in complex tissue preparations, promising to enhance the understanding of cholinergic neurotransmission.

This article presents a method for studying nicotinic acetylcholine receptors (nAChRs) in mouse brain slices by nicotine uncaging. When coupled with simultaneous patch clamp recording and 2-photon laser scanning microscopy, nicotine uncaging connects nicotinic receptor function with cellular morphology, providing a deeper understanding of cholinergic neurobiology.

Зондирование никотиновых ацетилхолиновых рецепторов функции срезы мозга мыши через лазерной вспышки фотолиза Photoactivatable никотина

Acetylcholine (ACh) acts through receptors to modulate a variety of neuronal processes, but it has been challenging to link ACh receptor function with ...

Studying Brain Function in Children Using Magnetoencephalography

Magnetoencephalography (MEG) is a non-invasive neuroimaging technique which directly measures magnetic fields produced by the electrical activity of the human brain. MEG is quiet and less likely to induce claustrophobia compared with magnetic resonance imaging (MRI). It is therefore a promising tool for investigating brain function in young children. However, analysis of MEG data from pediatric populations is often complicated by head movement artefacts which arise as a consequence of the requirement for a spatially-fixed sensor array that is not affixed to the child's head. Minimizing head movements during MEG sessions can be particularly challenging as young children are often unable to remain still during experimental tasks. The protocol presented here aims to reduce head movement artefacts during pediatric MEG scanning. Prior to visiting the MEG laboratory, families are provided with resources that explain the MEG system and the experimental procedures in simple, accessible language. An MEG familiarization session is conducted during which children are acquainted with both the researchers and the MEG procedures. They are then trained to keep their head still whilst lying inside an MEG simulator. To help children feel at ease in the novel MEG environment, all of the procedures are explained through the narrative of a space mission. To minimize head movement due to restlessness, children are trained and assessed using fun and engaging experimental paradigms. In addition, children's residual head movement artefacts are compensated for during the data acquisition session using a real-time head movement tracking system. Implementing these child-friendly procedures is important for improving data quality, minimizing participant attrition rates in longitudinal studies, and ensuring that families have a positive research experience.

This article introduces a child-friendly research protocol designed to improve data quality by reducing head movement during pediatric magnetoencephalography (MEG). We familiarize families with the MEG environment, train children to remain still using an MEG simulator, and correct for residual head movement artefacts using a real-time head movement detection system.

Изучение функций мозга у детей с помощью Магнитоэнцефалография

Magnetoencephalography (MEG) is a non-invasive neuroimaging technique which directly measures magnetic fields produced by the electrical activity of the ...

Chronic Implantation of Whole-cortical Electrocorticographic Array in the Common Marmoset

Electrocorticography (ECoG) allows the monitoring of electrical field potentials from the cerebral cortex with high spatiotemporal resolution. Recent development of thin, flexible ECoG electrodes has enabled conduction of stable recordings of large-scale cortical activity. We have developed a whole-cortical ECoG array for the common marmoset. The array continuously covers almost the entire lateral surface of cortical hemisphere, from the occipital pole to the temporal and frontal poles, and it captures whole-cortical neural activity in one shot. This protocol describes a chronic implantation procedure of the array in the epidural space of the marmoset brain. Marmosets have two advantages regarding ECoG recordings, one being the homologous organization of anatomical structures in humans and macaques, including frontal, parietal, and temporal complexes. The other advantage is that the marmoset brain is lissencephalic and contains a large number of complexes, which are more difficult to access in macaques with ECoG, that are exposed to the brain surface.These features allow direct access to most cortical areas beneath the surface of the brain. This system provides an opportunity to investigate global cortical information processing with high resolutions at a sub-millisecond order in time and millimeter order in space.

We have developed a whole-cortical electrocorticographic array for the common marmoset that continuously covers almost the entire lateral surface of cortex, from the occipital pole to the temporal and frontal poles. This protocol describes a chronic implantation procedure of the array in the epidural space of the marmoset brain.

Хронический имплантации целом корковых Electrocorticographic массива в общей игрунка

Electrocorticography (ECoG) allows the monitoring of electrical field potentials from the cerebral cortex with high spatiotemporal resolution. Recent ...

Assessment of Long-term Depression Induction in Adult Cerebellar Slices

Synaptic plasticity provides a mechanism for learning and memory. For cerebellar motor learning, long-term depression (LTD) of synaptic transmissions from parallel fibers (PF) to Purkinje cells (PC) is considered the basis for motor learning, and deficiencies of both LTD and motor learning are observed in various gene-manipulated animals. Common motor learning sets, such as adaptation of the optokinetic reflex (OKR), the vestibular-ocular reflex (VOR), and rotarod test were used for evaluation of motor learning ability. However, results obtained from the GluA2-carboxy terminus modified knock-in mice demonstrated normal adaptation of the VOR and the OKR, despite lacking PF-LTD. In that report, induction of LTD was only attempted using one type of stimulation protocol at room temperature. Thus, conditions to induce cerebellar LTD were explored in the same knock-in mutants using various protocols at near physiological temperature. Finally, we found stimulation protocols, by which LTD could be induced in these gene-manipulated mice. In this study, a set of protocols are proposed to evaluate LTD-induction, which will more accurately allow examination of the causal relationship between LTD and motor learning. In conclusion, experimental conditions are crucial when evaluating LTD in gene-manipulated mice.

In some gene-manipulated animals, using a single protocol may fail to induce LTD in cerebellar Purkinje cells, and there may be a discrepancy between LTD and motor learning. Multiple protocols are necessary to assess LTD-induction in gene-manipulated animals. Standard protocols are shown.

Оценка долгосрочной депрессии индукции в взрослых церебеллярной ломтики

Synaptic plasticity provides a mechanism for learning and memory. For cerebellar motor learning, long-term depression (LTD) of synaptic transmissions from ...

Местное применение наркотиков по изучению никотиновых рецепторов ацетилхолина функции в срезах мозга мыши

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