Os autores gostaria de agradecer a graduação Daniel Korus por sua ajuda a executar o código Matlab e graduação Alex Boettcher por sua assistência na execução dos experimentos comportamentais. Este projeto é apoiado pela NSF IOS 1256799 de R.L.M e pelo Instituto Nacional sobre abuso de drogas do institutos nacionais da saúde sob prêmio número T32DA007234.

<ol>
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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+measurement+of+glutamate+release+in+the+brain+using+a+dual+enzyme-based+electrochemical+sensor.">Direct measurement of glutamate release in the brain using a dual enzyme-based electrochemical sensor.</a> Brain Res. 659 (1-2), 117-125 (1994).</li><li>Agnesi, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wireless+instantaneous+neurotransmitter+concentration+system-based+amperometric+detection+of+dopamine,+adenosine,+and+glutamate+for+intraoperative+neurochemical+monitoring.">Wireless instantaneous neurotransmitter concentration system-based amperometric detection of dopamine, adenosine, and glutamate for intraoperative neurochemical monitoring.</a> J Neurosurg. 111 (4), 701-711 (2009).</li><li>Erksine, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Solicitation+behavior+in+the+estrous+female+rat:+A+review.">Solicitation behavior in the estrous female rat: A review.</a> Horm Beh. 23 (4), 473-502 (1989).</li><li>Weiner, I., Gal, G., Rawlins, J. N., Feldon, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+involvement+of+the+shell+and+core+subterritories+of+the+nucleus+accumbens+in+latent+inhibition+and+amphetamine-induced+activity.">Differential involvement of the shell and core subterritories of the nucleus accumbens in latent inhibition and amphetamine-induced activity.</a> Behav Brain Res. 81 (1-2), 123-133 (1996).</li><li>Heidbreder, C., Feldon, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amphetamine-induced+neurochemical+and+locomotor+responses+are+expressed+differentially+across+the+anteroposterior+axis+of+the+core+and+shell+subterritories+of+the+nucleus+accumbens.">Amphetamine-induced neurochemical and locomotor responses are expressed differentially across the anteroposterior axis of the core and shell subterritories of the nucleus accumbens.</a> Synapse. 29 (4), 310-322 (1998).</li><li>Pierce, R. C., Kalivas, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amphetamine+produces+sensitized+increases+in+locomotion+and+extracellular+dopamine+preferentially+in+the+nucleus+accumbens+shell+of+rats+administered+repeated+cocaine.">Amphetamine produces sensitized increases in locomotion and extracellular dopamine preferentially in the nucleus accumbens shell of rats administered repeated cocaine.</a> J Pharmacol Exp Ther. 275 (2), 1019-1029 (1995).</li><li>Pfaff, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Drive: Molecular and physiological analyses of a simple reproductive behavior. , (1999).</li><li>Carter, C. 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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+microdialysis+study+of+ventral+striatal+dopamine+during+sexual+behavior+in+female+Syrian+hamsters.">A microdialysis study of ventral striatal dopamine during sexual behavior in female Syrian hamsters.</a> Beh Brain Res. 55 (2), 151-157 (1993).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> 4 Channel EEG/EMG/Biosensor Manual V005. , (2012).</li><li>Pellis, S. M., Pellis, V. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Play-fighting+differs+from+serious+play+fighting+in+both+target+of+attack+and+tactics+of+fighting+in+the+laboratory+rats+Rattus+norvegicus.">Play-fighting differs from serious play fighting in both target of attack and tactics of fighting in the laboratory rats Rattus norvegicus.</a> Aggress Behav. 13 (3), 227-242 (1987).</li><li>Pellis, S. M., Pellis, V. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+rates+of+attack,+defense+and+counterattack+during+the+developmental+decrease+in+play+fighting+by+male+and+female+rats.">Differential rates of attack, defense and counterattack during the developmental decrease in play fighting by male and female rats.</a> Dev Psychobiol. 23 (3), 215-231 (1990).</li><li>Pellis, S. M., Hastings, E., Shimizu, T., Kamitakahara, H., Komorowska, J., Forgie, M. L., Kolb, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+orbital+frontal+cortex+damage+on+the+modulation+of+defensive+responses+by+rats+in+playful+and+non-playful+social+contexts.">The effects of orbital frontal cortex damage on the modulation of defensive responses by rats in playful and non-playful social contexts.</a> Behav Neurosci. 120 (1), 72-84 (2006).</li><li>Wakabayashi, K. T., Kiyatkin, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+changes+in+extracellular+glutamate+induced+by+natural+arousing+stimuli+and+intravenous+cocaine+in+the+nucleus+accumbens+shell+and+core.">Rapid changes in extracellular glutamate induced by natural arousing stimuli and intravenous cocaine in the nucleus accumbens shell and core.</a> J Neurophysiol. 108 (1), 285-299 (2012).</li><li>Kapelsohn, K. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+methods+for+cutting,+mounting,+and+staining+tissue+for+neural+histology.">Improved methods for cutting, mounting, and staining tissue for neural histology.</a> Protoc Exch. , (2015).</li><li>Siegel, H. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+7:+Male+sexual+behavior.">Chapter 7: Male sexual behavior.</a> The Hamster: Reproduction and Behavior. , (1985).</li><li>Day, J. J., Carelli, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+nucleus+accumbens+and+pavlovian+reward+learning.">The nucleus accumbens and pavlovian reward learning.</a> Neuroscientist. 13 (2), 148-159 (2007).</li><li>Meisel, R. L., Mullins, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sexual+experience+in+female+rodents:+Cellular+mechanisms+and+functional+consequences.">Sexual experience in female rodents: Cellular mechanisms and functional consequences.</a> Brain Res. 1126 (1), 56-65 (2006).</li><li>Meredith, G. 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Os autores não têm nada para divulgar.

Embora relativamente simples, alguns problemas podem surgir quando empregando esta técnica. Em primeiro lugar, o posicionamento estereotáxica das sondas deve ser preciso: ao contrário do microdialysis que as amostras de um raio mais amplo do meio extracelular em torno da sonda, esta técnica só permite a medição de um neurotransmissor que entra em contato direto com a sonda. Em segundo lugar, no caso da gravação de fibra de carbono, devido a pequena largura da fibra, pode ocorrer a ruptura, e a sonda deve ser int...

A capacidade de medir a liberação de neurotransmissores em animais comportamento acordados permite que os pesquisadores de vincular comportamentos específicos com padrões espaciais e temporais da neurotransmissão-uma poderosa ferramenta para investigar os mecanismos e circuitos subjacentes a ambos naturais e comportamentos operante em tempo real. Historicamente, o microdialysis tem sido empregado para medir substâncias eletricamente reativas e não reactivas no meio extracelular do cérebro1. Esta técnica usa um fluxo contínuo de uma solução aquosa de composição iônica semelhante ao fluido extracelular, através de uma sonda de microdialysis composta de uma haste pequena com uma dica de uma membrana de fibra oca semipermeable2. Após a inserção da sonda, neurotransmissores ou outros analitos de interesse podem atravessar a membrana semipermeável por difusão passiva, antes de ser coletados em intervalos para posterior análise por cromatografia líquida de alto desempenho (HPLC), um técnica de química analítica, comumente utilizada para separar, identificar e quantificar os componentes em uma mistura heterogênea de3.
Embora microdialysis é uma técnica sensível que pode ser usada para medir praticamente qualquer substância de interesse, a resolução temporal é baixa, com taxas de amostragem máxima da ordem de dezenas de minutos1,2minutos. A invenção de varredura rápida voltametria cíclica (FSCV), uma técnica que se baseia no potencial redox de espécies eletroativos, pode elucidar perto instantâneas concentrações do analito de interesse no fluido extracelular. Em breve (ver Robinson et al 4 para uma extensa revisão), um eletrodo é aplicado para levantar e abaixar a tensão de forma de onda triangular em um rápido tempo de escala4. Quando a tensão está no intervalo correto, o composto de interesse repetidamente é oxidado e reduzido. Esta oxidação e redução resulta em um movimento de elétrons que cria uma pequena corrente alternada. Varredura de taxas terá lugar na escala de subsegundos com oxidação e redução de compostos ocorrem em microssegundos. Subtraindo-se o plano de fundo atual criado pela sonda de corrente resultante, pode-se gerar uma tensão vs atual trama original para cada composto. Desde que a escala de tempo das oscilações da tensão é conhecida, esses dados podem ser usados para calcular uma parcela da corrente em função do tempo. Assim, a concentração relativa do composto pode ser determinada, enquanto o número de elétrons transferidos em cada oxidação e reação de redução é conhecida4.
Esta especificidade química e alta resolução temporal fazem FSCV uma técnica poderosa para a detecção de alteração química concentrações na vivo. No entanto, apesar dessas vantagens múltiplas, esta técnica requer vastos conhecimentos técnicos e equipamentos caros e instalação. Além disso, nonelectroactive neurotransmissores (e.g., glutamato) não podem ser medidos usando esta técnica. Felizmente, os avanços tecnológicos no campo da eletroquímica5, bem como a comercialização destas invenções, introduziu uma abordagem relativamente simples para medir os neurotransmissores não-eletroativos em animais comportamento acordados sem comprometer a técnica de precisão, um temporal conhecida como tecnologia biosensor enzimática. Esta técnica usa conversão enzimática do neurotransmissor nonelectroactive de interesse em dois substratos, um dos quais é eletroativos de peróxido de hidrogênio que é detectado como uma oxidação amperométrico atual gerada por um potencial aplicado5 . Biosensor comercialmente disponíveis sondas seletivamente (ver Figura 1) medem analitos de interesse, competitivamente, reduzindo a contribuição de interferentes endógenos. No caso de glutamato, a contribuição do ácido ascórbico interferent comuns (AA) é reduzida competitivamente à corrente medida pelo co localizando oxidase de AA na superfície do sensor, conversão de AA para não-eletroativos enzimática ativa dihydroascorbate e água. Além disso, uma camada de polímero Nafion carregada negativamente presente sob a camada de enzima exclui compostos aniônicos endógenos.
Esta mesma configuração experimental biosensor pode medir eletroativos neurotransmissores como FSCV, mas em vez disso, ele emprega um potencial fixo gravação6. Em contraste com a tensão oscilante aplicada em FSCV, na gravação de um potencial fixo a tensão é mantida no redox potencial para o analito de interesse. Embora seja menos quimicamente seletiva do que FSCV como vários neurotransmissores podem ter o mesmo potencial, de redox em áreas do cérebro que esmagadoramente inclinação para um neurotransmissor, a natureza da curva-chave desta abordagem supera a falta de química especificidade.
A capacidade de medir tanto eletroativos e nonelectroactive lançamento do neurotransmissor em tempo quase real e vinculá-lo a eventos comportamentais específicos fornece uma oportunidade para examinar a liberação de neurotransmissor convergentes. Este manuscrito detalha o uso deste sistema para interrogar a neurotransmissão tanto dopamina e glutamato em resposta a recompensa natural em hamsters comportamento acordados. O objetivo deste trabalho é a detalhar o processo de medição esta liberação de neurotransmissor durante o comportamento sexual em hamsters femininos, com o objetivo de demonstrar a sua viabilidade para examinar outros comportamentos e paradigmas experimentais.
Os hamsters são um modelo ideal para uso em gravações de eletroquímicas Historicamente, os modelos de ratos e camundongos têm sido empregados no estudo do comportamento sexual. Estas espécies de roedores se envolver em uma sequência de imperatividade dinâmica, envolvendo inúmeros comportamentos de solicitação feminino que incluem lupulagem, arremessam e orelha mexer para atrair o macho de perseguir e, finalmente, montar o feminino7. A montagem pelo macho (com ou sem penetração vaginal) dura apenas alguns segundos, durante o qual a fêmea se engaja na sua postura de comportamento sexual (denominada lordose) também só por alguns segundos antes de retomar comportamentos ativo solicitação. Este padrão de comportamento, composto por altos níveis de atividade intercalados com breves períodos de imobilidade, é problemático para medir a neurotransmissão em comportamento de animais. Primeiro, pode haver artefatos de movimento em amperométrico gravações que estão relacionadas com a atividade neural. Em segundo lugar, a locomoção é associada com a liberação de neurotransmissores particulares em certas regiões do cérebro. Por exemplo, liberação de dopamina tem sido acoplada a atividade locomotora do striatum dorsal e ventral8,9, uma descoberta que formou a base para medições microdialysis de dopamina após psicoestimulantes Administração10. Porque os comportamentos de solicitação fêmea-típico em mosroedores de t envolvem altos níveis de atividade locomotora e são representados pela maior parte de um teste de comportamento sexual de 10 minutos, isso dificulta a alterações na neurotransmissão, atribuímos aos componentes explícitos de comportamento sexual que coletivamente durar apenas minutos.
Para analisar o perfil neuroquímico do comportamento sexual feminino, este laboratório procurou uma espécie em que há atividade locomotora mínima que acompanha o comportamento sexual. A sequência copulador no hamster sírio (Mesocricetus auratus) é ideal para gravações neuroquímicas devido à falta de comportamentos de solicitação normalmente visto em ratos e camundongos11. Como consequência, os hamsters fêmeas entrará e manter a postura de lordose para mais de 9 minutos a 10 minutos de sessão12de teste. Com a falta de estranhos movimentos de locomoção pelo sexo feminino, na vivo gravações eletroquímicas que podem ser associadas a componentes de interações sexuais com o macho podem ser obtidas.
Copuladores lutas em hamsters Após a introdução de um animal de estímulo masculino na câmara de teste, o macho irá inicialmente envolver anogenitais investigação (AI) da fêmea antes de montá-la (Figura 2). Em ordem para montar o macho, a fêmea deve assumir uma postura sexual receptiva, conhecida como lordose, no qual ela arcos de volta e desvia a cauda para que o macho de montagem pode ganhar acesso do pênis para a vagina dela. O macho vai montar a fêmea, abraçando seu traseiro com as duas patas (Figura 2B) e começar a empurrar na tentativa de ganhar penetração peniana (Figura 2C). O macho irá montar a fêmea (sem inserção), bem como intromit um número de vezes antes de finalmente conseguir a ejaculação. Esta sequência de montagens e intromissões levando a ejaculação é denominada um tal"imperatividade". Os machos terá várias lutas copulativo dentro de uma única sessão.

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	</colgroup>
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:329px;">Nembutal</td>
			<td style="width:183px;">Oak Pharmaceuticals Inc.</td>
			<td style="width:323px;">76478-501-50</td>
			<td style="width:504px;">Pentobarbital sodium injection, USP. This lab uses 8.5mg/100g body weight, injected intraperotineally.&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:329px;">Loxicom analgesic&#160;</td>
			<td style="width:183px;">Norbrook Laboratories&#160;</td>
			<td style="width:323px;">6451603670</td>
			<td style="width:504px;">NSAID antinflammatory and analgesic used for post-operative pain control. Generic: meloxicam.&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:329px;">Enroflox antibiotic&#160;</td>
			<td style="width:183px;">Norbrook Laboratories&#160;</td>
			<td style="width:323px;">5552915411</td>
			<td style="width:504px;">Fluoroquinolone antibiotic for post-operative infection prevention. Generic: Enrofloxacin.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Beuthanasia-D&#160;</td>
			<td style="width:183px;">Merck Animal Health</td>
			<td style="width:323px;">00061047305</td>
			<td style="width:504px;">Pentobarbital Sodium, Phenytoin Sodium euthanasia agent.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bone screws</td>
			<td>Pinnacle Technologies, Inc.</td>
			<td>8111-16</td>
			<td style="width:504px;">1/8&#34; bone screw (Pkg. of 16) used to affix skull cap to skull.&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Dental acrylic (Bosworth Duz-All)</td>
			<td>Bosworth&#160;</td>
			<td>166261C&#160;</td>
			<td style="width:504px;">Self curing dental acrylic is used in construction of a skull cap to affix cannula and head mount to skull.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">Hardware biosensor setup&#160;</td>
			<td>Pinnacle Technologies, Inc.</td>
			<td>8400-K2</td>
			<td style="width:504px;">Pinnacle offers complete hardware kits for new users of our tethered biosensor system for rats. Kits include a commutator, preamplifier, and data conditioning and acquisition system</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;">Base video computer package</td>
			<td>Pinnacle Technologies, Inc.</td>
			<td>9000-K1</td>
			<td style="width:504px;">The base computer package includes a preconfigured computer with ample hard disk storage, a high-definition monitor, a keyboard and mouse, an uninterruptible power supply, and all necessary cables.&#160;</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;">Video EQ700 EverFocus camera&#160; package</td>
			<td>Pinnacle Technologies, Inc.</td>
			<td>&#160;9000-K10&#160;</td>
			<td style="width:504px;">EQ700 night vision capable box camera with independent IR source was obtained as part of Pinnacle video computer package. Dome camera (9000-K9) and HD camera (9000-K11) options are also available.&#160;</td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;">Sirenia Acquisition software</td>
			<td>Pinnacle Technologies, Inc.</td>
			<td>Free--available to download from pinnaclet.com</td>
			<td style="width:504px;">Sirenia&#160;Acquisition provides a single platform for recording data from any Pinnacle hardware system. The software features synchronization of all data streams, user-configurable settings, data consolidation, and multiple export options. In addition, the software includes basic review and analysis modules for biosensor recordings. Sirenia&#160;delivers free ll-in-one software that is ideal for data acquisition and review.</td>
		</tr>
		<tr height="147">
			<td height="147" style="height:147px;">Tethered rat&#160;in vitro&#160;calibration kit</td>
			<td>Pinnacle Technologies, Inc.</td>
			<td>7000-K2-T-BAS&#160;</td>
			<td style="width:504px;">In order to relate the current changes measured by a biosensor to actual changes in analyte concentration, it is necessary to calibrate the biosensor prior to implantation into the animal. The process also confirms the integrity and selectivity of the sensors.&#160;Calibration kit includes 20 mL jacketed beaker (#7058), 1/2&#34; by 1/8&#34; magnetic stir bar (#7059), right angle clamp (#7056), 2 prong single-adjustment clamp (#7055), 4-channel calibration preamplifer (#7053), and calibration holder (#7051).&#160;</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">Stir plate&#160;</td>
			<td>Corning</td>
			<td>6795-410D</td>
			<td style="width:504px;">Corning digital Stirrer, 5&#34; x 7&#34;, 120 VAC used to spin magnetic stirrer in jacketed beaker during in vitro calibration of glutamate biosensors.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">Water bath capable of closed loop circulation</td>
			<td>PolyScience</td>
			<td>8006A11B</td>
			<td style="width:504px;">PolyScience 8006A11B 6L Standard Digital Heated Circulating Bath, 120VAC water bath was used with plastic tubing to heat jacketed beaker to physiological temperature.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Carbon fiber sensor with BASi rat cannulae</td>
			<td>Pinnacle Technology, Inc.</td>
			<td>7002-CFS</td>
			<td style="width:504px;">Carbon fiber electrode used for recording dopamine neurotransmission.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ag/AgCl reference electrode</td>
			<td>Pinnacle Technology, Inc.</td>
			<td>7065</td>
			<td style="width:504px;">Necessary for carbon fiber recordings.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Glutamate biosensors&#160;</td>
			<td>Pinnacle Technology, Inc.</td>
			<td>7001&#160;</td>
			<td style="width:504px;">Enzymatic biosensor probe used for recording glutamatergic neurotransmission.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BASi guide cannulae</td>
			<td>Pinnacle Technologies, Inc.</td>
			<td>7030&#160;</td>
			<td style="width:504px;">Guide cannulae implanted into brain region of interest to guide probe.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BASi cannula plastic headpiece for rats&#160;</td>
			<td>Pinnacle Technologies, Inc.</td>
			<td>7011&#160;</td>
			<td style="width:504px;">Headmount stabilizes probe and attaches to potentiostat.</td>
		</tr>
	</tbody>
</table>

measuring in vivo changes in extracellular neurotransmitters during naturally rewarding behaviors in female syrian hamsters

A capacidade de medir a liberação de neurotransmissores em uma escala de tempo rápida permite que os padrões de neurotransmissão para ser ligado a comportamentos específicos ou manipulações; uma poderosa ferramenta na elucidação de mecanismos e circuitos subjacentes. Enquanto a técnica do microdialysis tem sido usada há décadas para medir praticamente qualquer analito de interesse no cérebro, esta técnica é limitada em resolução temporal. Alternativamente, voltametria cíclica de varredura rápida é temporalmente precisos e extremamente sensível; no entanto, porque esse método tecnicamente difícil depende do electroactivity do analito de interesse, é eliminada a possibilidade de detectar substâncias nonelectroactive (por exemplo, o glutamato neurotransmissor). Este documento detalha o uso de um sistema turn-key que combina potencial fixo amperometry e biosensing enzimático para medir os neurotransmissores tanto eletroativos e nonelectroactive com precisão temporal. O emparelhamento destas duas poderosas técnicas permite a medição de tônico e neurotransmissão fásicos com relativa facilidade e permite a gravação de vários neurotransmissores simultaneamente. O objetivo deste manuscrito é demonstrar o processo de medição de dopamina e glutamato neurotransmissão no vivo usando um comportamento naturalmente gratificante (ou seja, comportamento sexual) em hamsters femininos, com o objetivo de exibir a viabilidade técnica deste teste para examinar outros comportamentos e paradigmas experimentais.

Usando a eletroquímica e comportamental de codificação metodologia descrita acima, este laboratório começou a caracterizar ambos tônico e fásicas flutuações em dopamina e glutamato durante gravações no vivo de comportamento sexual. Devido a maneira temporalmente-precisa desta metodologia, nós pode caracterizar com mais precisão neurotransmissão durante o comportamento sexual; bem como atribuem alterações específicas nos padrões de lançamento de correspondentes ...

Watch this Scientific Journal Video about Measuring In Vivo Changes in Extracellular Neurotransmitters During Naturally Rewarding Behaviors in Female Syrian Hamsters at JoVE.com

Measuring In Vivo Changes in Extracellular Neurotransmitters During Naturally Rewarding Behaviors in Female Syrian Hamsters

Este documento detalha a utilização do potencial fixo amperométrico gravações usando eletrodos de fibra de carbono e tecnologia enzimática biosensor para medir a liberação de dopamina e glutamato com alta resolução temporal durante o comportamento natural de gratificante na hamster fêmea.

Medição In Vivo alterações em neurotransmissores extracelulares durante naturalmente recompensando comportamentos em Hamsters sírios femininos

The ability to measure neurotransmitter release on a rapid time scale allows patterns of neurotransmission to be linked to specific behaviors or ...

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Measuring In Vivo Changes in Extracellular Neurotransmitters During Naturally Rewarding Behaviors in Female Syrian Hamsters

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10.1K visualizações.  University of Minnesota. Este documento detalha a utilização do potencial fixo amperométrico gravações usando eletrodos de fibra de carbono e tecnologia enzimática biosensor para medir a liberação de dopamina e glutamato com alta resolução temporal durante o comportamento natural de gratificante na hamster fêmea.

Vídeo: Measuring In Vivo Changes in Extracellular Neurotransmitters During Naturally Rewarding Behaviors in Female Syrian Hamsters

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

Transfecção de células de camundongo por Ganglionares da Retina In vivo Eletroporação

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

Neurociência

In vivo Laser Axotomy in C. elegans

Neurons communicate with other cells via axons and dendrites, slender membrane extensions that contain pre- or post-synaptic specializations. If a neuron is damaged by injury or disease, it may regenerate. Cell-intrinsic and extrinsic factors influence the ability of a neuron to regenerate and restore function. Recently, the nematode C. elegans has emerged as an excellent model organism to identify genes and signaling pathways that influence the regeneration of neurons1-6. The main way to initiate neuronal regeneration in C. elegans is laser-mediated cutting, or axotomy. During axotomy, a fluorescently-labeled neuronal process is severed using high-energy pulses. Initially, neuronal regeneration in C. elegans was examined using an amplified femtosecond laser5. However, subsequent regeneration studies have shown that a conventional pulsed laser can be used to accurately sever neurons in vivo and elicit a similar regenerative response1,3,7.
We present a protocol for performing in vivo laser axotomy in the worm using a MicroPoint pulsed laser, a turnkey system that is readily available and that has been widely used for targeted cell ablation. We describe aligning the laser, mounting the worms, cutting specific neurons, and assessing subsequent regeneration. The system provides the ability to cut large numbers of neurons in multiple worms during one experiment. Thus, laser axotomy as described herein is an efficient system for initiating and analyzing the process of regeneration.

In vivo Laser Axotomy in C. elegans

A protocol to cut neurons in C. elegans with a MicroPoint pulsed laser is presented. We describe setting up the system, immobilizing worms, and severing labeled neurons. Advantages include a relatively low-cost system and the ability to sever neuronal processes or ablate cells in vivo.

In vivo Laser axotomia em C. elegans

Neurons communicate with other cells via axons and dendrites, slender membrane extensions that contain pre- or post-synaptic specializations. If a neuron ...

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from individual neurons are critical for understanding how neural circuits function under natural conditions. Traditionally, these recordings have been performed 'blind', meaning the identity of the recorded cell is unknown at the start of the recording. Cellular identity can be subsequently determined via intracellular1, juxtacellular2 or loose-patch3 iontophoresis of dye, but these recordings cannot be pre-targeted to specific neurons in regions with functionally heterogeneous cell types. Fluorescent proteins can be expressed in a cell-type specific manner permitting visually-guided single-cell electrophysiology4-6. However, there are many model systems for which these genetic tools are not available. Even in genetically accessible model systems, the desired promoter may be unknown or genetically homogenous neurons may have varying projection patterns. Similarly, viral vectors have been used to label specific subgroups of projection neurons7, but use of this method is limited by toxicity and lack of trans-synaptic specificity. Thus, additional techniques that offer specific pre-visualization to record from identified single neurons in vivo are needed. Pre-visualization of the target neuron is particularly useful for challenging recording conditions, for which classical single-cell recordings are often prohibitively difficult8-11. The novel technique described in this paper uses retrograde transport of a fluorescent dye applied using tungsten needles to rapidly and selectively label a specific subset of cells within a particular brain region based on their unique axonal projections, thereby providing a visual cue to obtain targeted electrophysiological recordings from identified neurons in an intact circuit within a vertebrate CNS.
The most significant novel advancement of our method is the use of fluorescent labeling to target specific cell types in a non-genetically accessible model system. Weakly electric fish are an excellent model system for studying neural circuits in awake, behaving animals12. We utilized this technique to study sensory processing by "small cells" in the anterior exterolateral nucleus (ELa) of weakly electric mormyrid fish. "Small cells" are hypothesized to be time comparator neurons important for detecting submillisecond differences in the arrival times of presynaptic spikes13. However, anatomical features such as dense myelin, engulfing synapses, and small cell bodies have made it extremely difficult to record from these cells using traditional methods11, 14. Here we demonstrate that our novel method selectively labels these cells in 28% of preparations, allowing for reliable, robust recordings and characterization of responses to electrosensory stimulation.

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

Retrograde transport of fluorescent dye labels a sub-population of neurons based on anatomical projection. Labeled axons can be visually targeted in vivo, permitting extracellular recording from identified axons. This technique facilitates recording when neurons cannot be labeled through genetic manipulation or are difficult to isolate using 'blind' in vivo approaches.

Labeling fluorescentes retrógrada Permite alvejado Extracelular Gravação Single-unidade dos neurônios identificados<em&gt; In vivo</em

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from ...

Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains technically challenging. One approach, Activation-Induced Manganese-enhanced MRI (AIM MRI), has been used successfully to map neuronal activity in rodents 1-5. In AIM MRI, Mn2+ acts a calcium analog and accumulates in depolarized neurons 6,7. Because Mn2+ shortens the T1 tissue property, regions of elevated neuronal activity will enhance in MRI. Furthermore, Mn2+ clears slowly from the activated regions; therefore, stimulation can be performed outside the magnet prior to imaging, enabling greater experimental flexibility. However, because Mn2+ does not readily cross the blood-brain barrier (BBB), the need to open the BBB has limited the use of AIM MRI, especially in mice.
One tool for opening the BBB is ultrasound. Though potentially damaging, if ultrasound is administered in combination with gas-filled microbubbles (i.e., ultrasound contrast agents), the acoustic pressure required for BBB opening is considerably lower. This combination of ultrasound and microbubbles can be used to reliably open the BBB without causing tissue damage 8-11.
Here, a method is presented for performing AIM MRI by using microbubbles and ultrasound to open the BBB. After an intravenous injection of perflutren microbubbles, an unfocused pulsed ultrasound beam is applied to the shaved mouse head for 3 minutes. For simplicity, we refer to this technique of BBB Opening with Microbubbles and UltraSound as BOMUS 12. Using BOMUS to open the BBB throughout both cerebral hemispheres, manganese is administered to the whole mouse brain. After experimental stimulation of the lightly sedated mice, AIM MRI is used to map the neuronal response.
To demonstrate this approach, herein BOMUS and AIM MRI are used to map unilateral mechanical stimulation of the vibrissae in lightly sedated mice 13. Because BOMUS can open the BBB throughout both hemispheres, the unstimulated side of the brain is used to control for nonspecific background stimulation. The resultant 3D activation map agrees well with published representations of the vibrissae regions of the barrel field cortex 14. The ultrasonic opening of the BBB is fast, noninvasive, and reversible; and thus this approach is suitable for high-throughput and/or longitudinal studies in awake mice.

A technique is described for broadly opening the blood-brain barrier in the mouse using microbubbles and ultrasound. Using this technique, manganese can be administered to the mouse brain. Because manganese is an MRI contrast agent that accumulates in depolarized neurons, this approach enables imaging of neuronal activity.

Neuroimagem Funcional Usando rompimento da barreira hemato-encefálica ultra-sônico e Manganês-RM

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains ...

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

Dois registros eletrofisiológicos de sinapticamente evocadas Respostas astroglial e neuronal em fatias hipocampais agudas

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety of brain disorders1,2. To better understand the mechanisms by which brain circuits react to an animal's experience requires the ability to monitor the experience-dependent molecular changes in a given set of neurons, over a prolonged period of time, in the live animal. While experience and associated neural activity is known to trigger gene expression changes in neurons1,2, most of the methods to detect such changes do not allow repeated observation of the same neurons over multiple days or do not have sufficient resolution to observe individual neurons3,4. Here, we describe a method that combines in vivo two-photon microscopy with a genetically encoded fluorescent reporter to track experience-dependent gene expression changes in individual cortical neurons over the course of day-to-day experience. 
One of the well-established experience-dependent genes is Activity-regulated cytoskeletal associated protein (Arc)5,6. The transcription of Arc is rapidly and highly induced by intensified neuronal activity3, and its protein product regulates the endocytosis of glutamate receptors and long-term synaptic plasticity7. The expression of Arc has been widely used as a molecular marker to map neuronal circuits involved in specific behaviors3. In most of those studies, Arc expression was detected by in situ hybridization or immunohistochemistry in fixed brain sections. Although those methods revealed that the expression of Arc was localized to a subset of excitatory neurons after behavioral experience, how the cellular patterns of Arc expression might change with multiple episodes of repeated or distinctive experiences over days was not investigated. 
In vivo two-photon microscopy offers a powerful way to examine experience-dependent cellular changes in the living brain8,9. To enable the examination of Arc expression in live neurons by two-photon microscopy, we previously generated a knock-in mouse line in which a GFP reporter is placed under the control of the endogenous Arc promoter10. This protocol describes the surgical preparations and imaging procedures for tracking experience-dependent Arc-GFP expression patterns in neuronal ensembles in the live animal. In this method, chronic cranial windows were first implanted in Arc-GFP mice over the cortical regions of interest. Those animals were then repeatedly imaged by two-photon microscopy after desired behavioral paradigms over the course of several days. This method may be generally applicable to animals carrying other fluorescent reporters of experience-dependent molecular changes4.

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

Experience-dependent molecular changes in neurons are essential for the brain's ability to adapt in response to behavioral challenges. An in vivo two-photon imaging method is described here that allows the tracking of such molecular changes in individual cortical neurons through genetically encoded reporters.

Vivo em dois fótons de imagem de experiência dependentes de alterações moleculares em neurônios corticais

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety ...

Ex Vivo Preparations of the Intact Vomeronasal Organ and Accessory Olfactory Bulb

The mouse accessory olfactory system (AOS) is a specialized sensory pathway for detecting nonvolatile social odors, pheromones, and kairomones. The first neural circuit in the AOS pathway, called the accessory olfactory bulb (AOB), plays an important role in establishing sex-typical behaviors such as territorial aggression and mating. This small (&#60;1 mm3) circuit possesses the capacity to distinguish unique behavioral states, such as sex, strain, and stress from chemosensory cues in the secretions and excretions of conspecifics. While the compact organization of this system presents unique opportunities for recording from large portions of the circuit simultaneously, investigation of sensory processing in the AOB remains challenging, largely due to its experimentally disadvantageous location in the brain. Here, we demonstrate a multi-stage dissection that removes the intact AOB inside a single hemisphere of the anterior mouse skull, leaving connections to both the peripheral vomeronasal sensory neurons (VSNs) and local neuronal circuitry intact. The procedure exposes the AOB surface to direct visual inspection, facilitating electrophysiological and optical recordings from AOB circuit elements in the absence of anesthetics. Upon inserting a thin cannula into the vomeronasal organ (VNO), which houses the VSNs, one can directly expose the periphery to social odors and pheromones while recording downstream activity in the AOB. This procedure enables controlled inquiries into AOS information processing, which can shed light on mechanisms linking pheromone exposure to changes in behavior.

Ex Vivo Preparations of the Intact Vomeronasal Organ and Accessory Olfactory Bulb

The mouse accessory olfactory bulb (AOB) has been difficult to study in the context of sensory coding. Here, we demonstrate a dissection that produces an ex vivo preparation in which AOB neurons remain functionally connected to their peripheral inputs, facilitating research into information processing of mouse pheromones and kairomones.

Ex vivo preparações da Intacta Vomeronasal Órgão e acessórios bulbo olfatório

The mouse accessory olfactory system (AOS) is a specialized sensory pathway for detecting nonvolatile social odors, pheromones, and kairomones. The first ...

A Simple and Inexpensive Method for Determining Cold Sensitivity and Adaptation in Mice

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life. The cold plantar assay allows the objective and inexpensive assessment of cold sensitivity in mice, and can quantify both analgesia and hypersensitivity. Mice are acclimated on a glass plate, and a compressed dry ice pellet is held against the glass surface underneath the hindpaw. The latency to withdrawal from the cooling glass is used as a measure of cold sensitivity.
Cold sensation is also important for survival in regions with seasonal temperature shifts, and in order to maintain sensitivity animals must be able to adjust their thermal response thresholds to match the ambient temperature. The Cold Plantar Assay (CPA) also allows the study of adaptation to changes in ambient temperature by testing the cold sensitivity of mice at temperatures ranging from 30 &#176;C to 5 &#176;C. Mice are acclimated as described above, but the glass plate is cooled to the desired starting temperature using aluminum boxes (or aluminum foil packets) filled with hot water, wet ice, or dry ice. The temperature of the plate is measured at the center using a filament T-type thermocouple probe. Once the plate has reached the desired starting temperature, the animals are tested as described above.
This assay allows testing of mice at temperatures ranging from innocuous to noxious. The CPA yields unambiguous and consistent behavioral responses in uninjured mice and can be used to quantify both hypersensitivity and analgesia. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

The Cold Plantar Assay (CPA) measures cold responsiveness between 30 &#176;C and 5 &#176;C, and can also measure cold adaptation. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

Um método simples e barato para a determinação da sensibilidade Fria e Adaptação no Mice

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life.  The cold ...

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise from extra-retinal sources. Ex vivo ERG provides an efficient method to dissect the function of retinal cells directly from an intact isolated retina of animals or donor eyes. In addition, ex vivo ERG can be used to test the efficacy and safety of potential therapeutic agents on retina tissue from animals or humans. We show here how commercially available in vivo ERG systems can be used to conduct ex vivo ERG recordings from isolated mouse retinas. We combine the light stimulation, electronic and heating units of a standard in vivo system with custom-designed specimen holder, gravity-controlled perfusion system and electromagnetic noise shielding to record low-noise ex vivo ERG signals simultaneously from two retinas with the acquisition software included in commercial in vivo systems. Further, we demonstrate how to use this method in combination with pharmacological treatments that remove specific ERG components in order to dissect the function of certain retinal cell types.

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

Ex vivo ERG can be used to record electrical activity of retinal cells directly from isolated intact retinas of animals or humans. We demonstrate here how common in vivo ERG systems can be adapted for ex vivo ERG recordings in order to dissect the electrical activity of retinal cells.

Simultâneo<em&gt; Ex vivo</em&gt; Teste Funcional de Dois retinas por<em&gt; In vivo</em&gt; Sistema Eletrorretinograma

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise ...

Construction of Cell-based Neurotransmitter Fluorescent Engineered Reporters (CNiFERs) for Optical Detection of Neurotransmitters In Vivo

Cell-based neurotransmitter fluorescent engineered reporters (CNiFERs) provide a new tool for neuroscientists to optically detect the release of neurotransmitters in the brain in vivo. A specific CNiFER is created from a human embryonic kidney cell that stably expresses a specific G protein-coupled receptor, which couples to Gq/11 G proteins, and a FRET-based Ca2+-detector, TN-XXL. Activation of the receptor leads to an increase in the FRET signal. CNiFERs have nM sensitivity and a temporal response of seconds because a CNiFER clone utilizes the native receptor for a particular neurotransmitter, e.g., D2R for dopamine. CNiFERs are directly implanted into the brain, enabling them to sense neurotransmitter release with a spatial resolution of less than one hundred &#181;m, making them ideal to measure volume transmission in vivo. CNiFERs can also be used to screen other drugs for potential cross-reactivity in vivo. We recently expanded the family of CNiFERs to include GPCRs that couple to Gi/o G proteins. CNiFERs are available for detecting acetylcholine (ACh), dopamine (DA) and norepinephrine (NE). Given that any GPCR can be used to create a novel CNiFER and that there are approximately 800 GPCRs in the human genome, we describe here the general procedure to design, realize, and test any type of CNiFER.

Construction of Cell-based Neurotransmitter Fluorescent Engineered Reporters CNiFERs for Optical Detection of Neurotransmitters In Vivo

We present a protocol to create cell-based neurotransmitter fluorescent engineered reporters (CNiFERs) for the optical detection of volumetric neurotransmitter release.

Construção de-base celular Neurotransmissores fluorescentes Engineered Repórteres (CNiFERs) para detecção óptica de neurotransmissores<em&gt; In Vivo</em