Este trabalho foi financiado pelo NIH / NIDDK (DK077106 para ECM).

<ol>
	<li>McNay, E. C., Fries, T. M., Gold, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decreases+in+rat+extracellular+hippocampal+glucose+concentration+associated+with+cognitive+demand+during+a+spatial+task.">Decreases in rat extracellular hippocampal glucose concentration associated with cognitive demand during a spatial task.</a> Proc. Natl. Acad. Sci. U.S.A. 97 (6), 2881 (2000).</li><li>McNay, E. C., Gold, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-related+differences+in+hippocampal+extracellular+fluid+glucose+concentration+during+behavioral+testing+and+following+systemic+glucose+administration.">Age-related differences in hippocampal extracellular fluid glucose concentration during behavioral testing and following systemic glucose administration.</a> J. Gerontol. A. Biol. Sci. Med. Sci. 56 (2), 66 (2001).</li><li>McNay, E. C., McCarty, R. C., Gold, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluctuations+in+brain+glucose+concentration+during+behavioral+testing:+dissociations+between+brain+areas+and+between+brain+and+blood.">Fluctuations in brain glucose concentration during behavioral testing: dissociations between brain areas and between brain and blood.</a> Neurobiol. Learn. Mem. 75 (3), 325 (2001).</li><li>McNay, E. C., Gold, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+glucose+concentrations+in+the+rat+hippocampus+measured+by+zero-net-flux:+effects+of+microdialysis+flow+rate.">Extracellular glucose concentrations in the rat hippocampus measured by zero-net-flux: effects of microdialysis flow rate.</a> 72 (2), 785 (1999).</li><li>McNay, E. C., Sherwin, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+recurrent+hypoglycemia+on+spatial+cognition+and+cognitive+metabolism+in+normal+and+diabetic+rats.">Effect of recurrent hypoglycemia on spatial cognition and cognitive metabolism in normal and diabetic rats.</a> Diabetes. 53 (2), 418 (2004).</li><li>McNay, E. C., 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=Hippocampal+memory+processes+are+modulated+by+insulin+and+high-fat-induced+insulin+resistance.">Hippocampal memory processes are modulated by insulin and high-fat-induced insulin resistance.</a> Neurobiology of Learning and Memory. 93 (4), 546 (2010).</li><li>McNay, E. C., McCarty, R. C., Gold, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluctuations+in+brain+glucose+concentration+during+behavioral+testing:+dissociations+between+brain+areas+and+between+brain+and+blood.">Fluctuations in brain glucose concentration during behavioral testing: dissociations between brain areas and between brain and blood.</a> Neurobiology of Learning &amp; Memory. 75 (3), 325 (2001).</li><li>McNay, E. C., Williamson, A., McCrimmon, R. J., Sherwin, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cognitive+and+neural+hippocampal+effects+of+long-term+moderate+recurrent+hypoglycemia.">Cognitive and neural hippocampal effects of long-term moderate recurrent hypoglycemia.</a> Diabetes. 55 (4), 1088 (2006).</li><li>McNay, E. C., Sherwin, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+artificial+cerebro-spinal+fluid+(aCSF)+to+artificial+extracellular+fluid+(aECF):+microdialysis+perfusate+composition+effects+on+in+vivo+brain+ECF+glucose+measurements.">From artificial cerebro-spinal fluid (aCSF) to artificial extracellular fluid (aECF): microdialysis perfusate composition effects on in vivo brain ECF glucose measurements.</a> Journal of Neuroscience Methods. 132 (1), 35 (2004).</li><li>McNay, E. C., 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=Hippocampal+memory+processes+are+modulated+by+insulin+and+high-fat-induced+insulin+resistance.">Hippocampal memory processes are modulated by insulin and high-fat-induced insulin resistance.</a> Neurobiology of Learning and Memory. 93 (4), 546 (2010).</li><li>Benveniste, H., Drejer, J., Schousboe, A., Diemer, N. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regional+cerebral+glucose+phosphorylation+and+blood+flow+after+insertion+of+a+microdialysis+fiber+through+the+dorsal+hippocampus+in+the+rat.">Regional cerebral glucose phosphorylation and blood flow after insertion of a microdialysis fiber through the dorsal hippocampus in the rat.</a> Journal of Neurochemistry. 49 (3), 729 (1987).</li><li>Benveniste, H., Diemer, N. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+reactions+to+implantation+of+a+microdialysis+tube+in+the+rat+hippocampus.">Cellular reactions to implantation of a microdialysis tube in the rat hippocampus.</a> Acta Neuropathologica. 74 (3), 234 (1987).</li><li>McNay, E. C., Fries, T. M., Gold, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decreases+in+rat+extracellular+hippocampal+glucose+concentration+associated+with+cognitive+demand+during+a+spatial+task.">Decreases in rat extracellular hippocampal glucose concentration associated with cognitive demand during a spatial task.</a> Proceedings of the National Academy of Sciences of the United States of America. 97 (6), 2881 (2000).</li><li>McNay, E. C., 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=Hippocampal+memory+processes+are+modulated+by+insulin+and+high-fat-induced+insulin+resistance.">Hippocampal memory processes are modulated by insulin and high-fat-induced insulin resistance.</a> Neurobiol. Learn Mem. 93 (4), 546 (2010).</li><li>Lonnroth, P., Jansson, P. -. A., Smith, U. <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+method+allowing+characterization+of+intercellular+water+space+in+humans.">A microdialysis method allowing characterization of intercellular water space in humans.</a> American Journal of Physiology. 1987, E228 (1987).</li><li>McNay, E. C., Gold, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+glucose+concentrations+in+the+rat+hippocampus+measured+by+zero-net-flux:+effects+of+microdialysis+flow+rate.">Extracellular glucose concentrations in the rat hippocampus measured by zero-net-flux: effects of microdialysis flow rate.</a> 72 (2), 785 (1999).</li><li>Lalonde, R. o. b. e. r. t. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+neurobiological+basis+of+spontaneous+alternation.">The neurobiological basis of spontaneous alternation.</a> Neuroscience &amp; Biobehavioral Reviews. 26 (1), 91 (2002).</li><li>Richman, C., Dember, W., Kim, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spontaneous+alternation+behavior+in+animals:+A+review.">Spontaneous alternation behavior in animals: A review.</a> Current Psychology. 5 (4), 358 (1986).</li><li>McNay, E. C., Canal, C. E., Sherwin, R. S., Gold, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+memory+with+septal+injections+of+morphine+and+glucose:+effects+on+extracellular+glucose+levels+in+the+hippocampus.">Modulation of memory with septal injections of morphine and glucose: effects on extracellular glucose levels in the hippocampus.</a> Physiol. Behav. 87 (2), 298 (2006).</li><li>McNay, E. C., Gold, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Memory+modulation+across+neural+systems:+intra-amygdala+glucose+reverses+deficits+caused+by+intraseptal+morphine+on+a+spatial+task+but+not+on+an+aversive+task.">Memory modulation across neural systems: intra-amygdala glucose reverses deficits caused by intraseptal morphine on a spatial task but not on an aversive task.</a> Journal of Neuroscience. 18 (10), 3853 (1998).</li><li>Rex, A., Bert, B., Fink, H., Voigt, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stimulus-dependent+changes+of+extracellular+glucose+in+the+rat+hippocampus+determined+by+in+vivo+microdialysis.">Stimulus-dependent changes of extracellular glucose in the rat hippocampus determined by in vivo microdialysis.</a> Physiol. Behav. 98 (4), 467 (2009).</li></ol>

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Todas as soluções utilizadas no microdiálise deve ser filtrada antes da utilização, utilizando um filtro de 0,2 | iM. Após a inserção da sonda na cânula guia, observar para confirmar que o fluxo é livre e coleta de amostra está ocorrendo. Se o fluxo pára após a inserção, a causa mais provável é a lesão da membrana causada por sonda cuidados insuficientes na inserção, e uma sonda de fresco tem de ser substituído. Como observado acima, uma vantagem chave para esses método...

<table border="1"><tbody><tr><td></td><td></td><td></td><td> A maioria dos reagentes são de qualidade padrão de laboratório e podem ser obtidos a partir de um fornecedor de escolha. Do mesmo modo, o equipamento tal como bombas de seringa e os tubos podem ser usados ​​a partir de qualquer um dos vários fabricantes. Itens específicos usados ​​aqui para que os detalhes são importantes incluem: </td></tr><tr><td> CMA 12 sondas de microdiálise </td><td> CMA microdiálise / </td><td> CMA-12-XXX </td><td> Estes estão disponíveis em vários comprimentos de membrana e cortes, indicados por códigos específicos no &#39;XXX. </td></tr><tr><td> Insulina humana (Humulin) </td><td> Eli Lilly </td><td> N / a </td><td></td></tr><tr><td> Giratória líquido </td><td> Instech </td><td> 375/D/22QM </td><td> Isto tem o torque rotativo específico muito baixo e o volume interno, bem como um nonrearevestimento de quartzo ctive. </td></tr></tbody></table>

hippocampal insulin microinjection and in vivo microdialysis during spatial memory testing

O metabolismo da glicose é um marcador útil para a actividade neural local, formando a base de métodos, tais como a imagiologia de ressonância 2-desoxiglicose e magnética funcional. No entanto, o uso de tais métodos em modelos animais e, portanto, requer anestesia ambos altera o estado do cérebro e impede medidas comportamentais. Um método alternativo é a utilização de microdiálise in vivo de tomar a medição contínua da concentração do fluido extracelular do cérebro de glucose, lactato, e afins metabolitos em animais acordados, sem restrições. Esta técnica é especialmente útil quando combinado com as tarefas destinadas a confiar em regiões específicas do cérebro e / ou manipulação farmacológica aguda, por exemplo, as medições do hipocampo durante uma tarefa espacial da memória de trabalho (alternação espontânea) mostram uma queda na glucose extracelular e aumento do lactato que são sugestivo de 1,2 glicólise aumentada, e administração de insulina intrahippocampal tanto melhora a memória e aumenta glicol hipocampoYsis 3. Substâncias como a insulina pode ser entregue ao hipocampo através da sonda de microdiálise mesmo utilizado para medir metabolitos. O uso de alternação espontânea, como uma medida da função do hipocampo foi concebido para evitar qualquer confundir motivadores de stress (por exemplo, choque nas patas), de retenção, ou recompensas (por exemplo, comida), todos os quais podem alterar o desempenho de tarefas e metabolismo, o que proporciona também uma tarefa medida da atividade motora que permite o controle de efeitos não específicos de tratamento. Combinados, esses métodos permitem a medição direta das variáveis ​​neuroquímicos e metabólicos que regulam o comportamento.

Ratos deve se recuperar rapidamente de cirurgia e estar alerta, móveis e ativo dentro de 30 minutos após o término da anestesia. O impacto da tampa da cânula deve ser mínima, se a cirurgia for realizada de forma limpa e cimento dental mínima é utilizada. Se qualquer sinal de infecção é observado durante o acompanhamento pós-operatório, ou o rato é de qualquer forma a mostrar sinais de desconforto ou constrangimento, rescindir imediatamente o experimento, o que deve ser extremamente raro. Durante a manipula?...

Watch this Scientific Journal Video about Hippocampal Insulin Microinjection and In vivo Microdialysis During Spatial Memory Testing at JoVE.com

Hippocampal Insulin Microinjection and In vivo Microdialysis During Spatial Memory Testing

Modulação da hippocampally dependente de memória espacial de trabalho por microinjeção intrahippocampal direta, acompanhado e seguido por<em&gt; In vivo</em&gt; Microdiálise para metabólitos em conscientes, animais se comportando.

Microinjeção insulina hipocampal e<em&gt; Em vivo</em&gt; Microdiálise Durante um teste de memória espacial

Glucose metabolism is a useful marker for local neural activity, forming the basis of methods such as 2-deoxyglucose and functional magnetic resonance ...

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JoVE Journal

Neuroscience

Hippocampal Insulin Microinjection and In vivo Microdialysis During Spatial Memory Testing

16.4K visualizações.  University at Albany. Modulação da hippocampally dependente de memória espacial de trabalho por microinjeção intrahippocampal direta, acompanhado e seguido por In vivo Microdiálise para metabólitos em conscientes, animais se comportando.

Vídeo: Hippocampal Insulin Microinjection and In vivo Microdialysis During Spatial Memory Testing

Selective Viral Transduction of Adult-born Olfactory Neurons for Chronic in vivo Optogenetic Stimulation

Local interneurons are continuously regenerated in the olfactory bulb of adult rodents1-3. In this process, called adult neurogenesis, neural stem cells in the walls of the lateral ventricle give rise to neuroblasts that migrate for several millimeters along the rostral migratory stream (RMS) to reach and incorporate into the olfactory bulb. To study the different steps and the impact of adult-born neuron integration into preexisting olfactory circuits, it is necessary to selectively label and manipulate the activity of this specific population of neurons. The recent development of optogenetic technologies offers the opportunity to use light to precisely activate this specific cohort of neurons without affecting surrounding neurons4,5. Here, we present a series of procedures to virally express Channelrhodopsin2(ChR2)-YFP in a temporally restricted cohort of neuroblasts in the RMS before they reach the olfactory bulb and become adult-born neurons. In addition, we show how to implant and calibrate a miniature LED for chronic in vivo stimulation of ChR2-expressing neurons.

Selective Viral Transduction of Adult-born Olfactory Neurons for Chronic in vivo Optogenetic Stimulation

Adult-born neurons of the olfactory bulb can be optogenetically controlled using Channelrhodopsin2-expressing lentiviral injection in the rostral migratory stream and chronic photostimulation with an implanted miniature LED.

Transdução Viral seletiva de Adultos-nascido neurônios olfativos de crônica In vivo Estímulo Optogenetic

Local interneurons are continuously regenerated in the olfactory bulb of adult rodents1-3. In this process, called adult neurogenesis, neural stem cells ...

Neurociência

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 ...

Microdialysis of Ethanol During Operant Ethanol Self-administration and Ethanol Determination by Gas Chromatography

Operant self-administration methods are commonly used to study the behavioral and pharmacological effects of many drugs of abuse, including ethanol. However, ethanol is typically self-administered orally, rather than intravenously like many other drugs of abuse. The pharmacokinetics of orally administered drugs are more complex than intravenously administered drugs. Because understanding the relationship between the pharmacological and behavioral effects of ethanol requires knowledge of the time course of ethanol reaching the brain during and after drinking, we use in vivo microdialysis and gas chromatography with flame ionization detection to monitor brain dialysate ethanol concentrations over time. 
Combined microdialysis-behavioral experiments involve the use of several techniques. In this article, stereotaxic surgery, behavioral training and microdialysis, which can be adapted to test a multitude of self-administration and neurochemical centered hypotheses, are included only to illustrate how they relate to the subsequent phases of sample collection and dialysate ethanol analysis. Dialysate ethanol concentration analysis via gas chromatography with flame-ionization detection, which is specific to ethanol studies, is described in detail. Data produced by these methods reveal the pattern of ethanol reaching the brain during the self-administration procedure, and when paired with neurochemical analysis of the same dialysate samples, allows conclusions to be made regarding the pharmacological and behavioral effects of ethanol.

A method to determine the time course of ethanol concentration in the brains of rats during operant ethanol self-administration is described. Gas chromatography with flame ionization detection is used to quantify ethanol in the dialysate samples, because it has the sensitivity required for the small volumes that are generated.

Microdiálise de etanol durante a operante determinação auto-administração de etanol e etanol por cromatografia gasosa

Operant self-administration methods are commonly used to study the behavioral and pharmacological effects of many drugs of abuse, including ethanol.  ...

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 Optogenetic Stimulation of the Rodent Central Nervous System

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution has been a long sought tool for neuroscientists in the systems-level search for the neural circuitry governing complex behavioral states. Optogenetics is a cutting-edge tool that is revolutionizing the field of neuroscience and represents one of the first systematic approaches to enable causal testing regarding the relation between neural signaling events and behavior. By combining optical and genetic approaches, neural signaling can be bi-directionally controlled through expression of light-sensitive ion channels (opsins) in mammalian cells. The current protocol describes delivery of specific wavelengths of light to opsin-expressing cells in deep brain structures of awake, freely-moving rodents for neural circuit modulation. Theoretical principles of light transmission as an experimental consideration are discussed in the context of performing in vivo optogenetic stimulation. The protocol details the design and construction of both simple and complex laser configurations and describes tethering strategies to permit simultaneous stimulation of multiple animals for high-throughput behavioral testing.

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

Optogenetics has become a powerful tool for use in behavioral neuroscience experiments. This protocol offers a step-by-step guide to the design and set-up of laser systems, and provides a full protocol for carrying out multiple and simultaneous in vivo optogenetic stimulations compatible with most rodent behavioral testing paradigms.

In vivo Optogenetic estimulação do sistema nervoso central de roedores

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution ...

High-resolution In Vivo Manual Segmentation Protocol for Human Hippocampal Subfields Using 3T Magnetic Resonance Imaging

The human hippocampus has been broadly studied in the context of memory and normal brain function and its role in different neuropsychiatric disorders has been heavily studied. While many imaging studies treat the hippocampus as a single unitary neuroanatomical structure, it is, in fact, composed of several subfields that have a complex three-dimensional geometry. As such, it is known that these subfields perform specialized functions and are differentially affected through the course of different disease states. Magnetic resonance (MR) imaging can be used as a powerful tool to interrogate the morphology of the hippocampus and its subfields. Many groups use advanced imaging software and hardware (&#62;3T) to image the subfields; however this type of technology may not be readily available in most research and clinical imaging centers. To address this need, this manuscript provides a detailed step-by-step protocol for segmenting the full anterior-posterior length of the hippocampus and its subfields: cornu ammonis (CA) 1, CA2/CA3, CA4/dentate gyrus (DG), strata radiatum/lacunosum/moleculare (SR/SL/SM), and subiculum. This protocol has been applied to five subjects (3F, 2M; age 29-57, avg. 37). Protocol reliability is assessed by resegmenting either the right or left hippocampus of each subject and computing the overlap using the Dice&#39;s kappa metric. Mean Dice&#39;s kappa (range) across the five subjects are: whole hippocampus, 0.91 (0.90-0.92); CA1, 0.78 (0.77-0.79); CA2/CA3, 0.64 (0.56-0.73); CA4/dentate gyrus, 0.83 (0.81-0.85); strata radiatum/lacunosum/moleculare, 0.71 (0.68-0.73); and subiculum 0.75 (0.72-0.78). The segmentation protocol presented here provides other laboratories with a reliable method to study the hippocampus and hippocampal subfields in vivo using commonly available MR tools.

High-resolution In Vivo Manual Segmentation Protocol for Human Hippocampal Subfields Using 3T Magnetic Resonance Imaging

The goal of this manuscript is to study the hippocampus and hippocampal subfields using MRI. The manuscript describes a protocol for segmenting the hippocampus and five hippocampal substructures: cornu ammonis (CA) 1, CA2/CA3, CA4/dentate gyrus, strata radiatum/lacunosum/moleculare, and subiculum.

Alta resolução<em&gt; In Vivo</em&gt; Manual Protocolo Segmentação para hipocampo humano subcampos Utilizando Ressonância Magnética 3T

The human hippocampus has been broadly studied in the context of memory and normal brain function and its role in different neuropsychiatric disorders has ...

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 ...

Immunohistochemical Visualization of Hippocampal Neuron Activity After Spatial Learning in a Mouse Model of Neurodevelopmental Disorders

Induction of phosphorylated extracellular-regulated kinase (pERK) is a reliable molecular readout of learning-dependent neuronal activation. Here, we describe a pERK immunohistochemistry protocol to study the profile of hippocampal neuron activation following exposure to a spatial learning task in a mouse model characterized by cognitive deficits of neurodevelopmental origin. Specifically, we used pERK immunostaining to study neuronal activation following Morris water maze (MWM, a classical hippocampal-dependent learning task) in Engrailed-2 knockout (En2-/-) mice, a model of autism spectrum disorders (ASD). As compared to wild-type (WT) controls, En2-/- mice showed significant spatial learning deficits in the MWM. After MWM, significant differences in the number of pERK-positive neurons were detected in specific hippocampal subfields of En2-/- mice, as compared to WT animals. Thus, our protocol can robustly detect differences in pERK-positive neurons associated to hippocampal-dependent learning impairment in a mouse model of ASD. More generally, our protocol can be applied to investigate the profile of hippocampal neuron activation in both genetic or pharmacological mouse models characterized by cognitive deficits.

We describe an immunohistochemistry protocol to study the profile of hippocampal neuron activation following exposure to a spatial learning task in a mouse model characterized by cognitive deficits of neurodevelopmental origin. This protocol can be applied to both genetic or pharmacological mouse models characterized by cognitive deficits.

A visualização imuno-histoquímica de Hippocampal Neuron Atividade Após Spatial Aprendizagem em um rato modelo de desordens do desenvolvimento neurológico

Induction of phosphorylated extracellular-regulated kinase (pERK) is a reliable molecular readout of learning-dependent neuronal activation. Here, we ...

Assessing Spatial Learning and Memory in Small Squamate Reptiles

Clinical research has leveraged a variety of paradigms to assess cognitive decline, commonly targeting spatial learning and memory abilities. However, interest in the cognitive processes of nonmodel species, typically within an ecological context, has also become an emerging field of study. In particular, interest in the cognitive processes in reptiles is growing although experimental studies on reptilian cognition are sparse. The few reptilian studies that have experimentally tested for spatial learning and memory have used rodent paradigms modified for use in reptiles. However, ecologically important aspects of the physiology and behavior of this taxonomic group must be taken into account when testing for spatially based cognition. Here, we describe modifications of the dry land Barnes maze and associated testing protocol that can improve performance when probing for spatial learning and memory ability in small squamate reptiles. The described paradigm and procedures were successfully used with male side-blotched lizards (Uta stansburiana), demonstrating that spatial learning and memory can be assessed in this taxonomic group with an ecologically relevant apparatus and protocol.

This paper describes a modification of the Barnes maze, a standard rodent paradigm used to assess spatial memory and learning, for use in small squamate reptiles.

Avaliando a aprendizagem espacial e memória em pequenas Squamata Répteis

Clinical research has leveraged a variety of paradigms to assess cognitive decline, commonly targeting spatial learning and memory abilities. However, ...

An In Ovo Model for Testing Insulin-mimetic Compounds

Elevated blood glucose levels in type 2 diabetes mellitus (T2DM), a complex and multifactorial metabolic disease, are caused by insulin resistance and &#946;-cell failure. Various strategies, including the injection of insulin or the usage of insulin-sensitizing drugs, were pursued to treat T2DM or at least reduce the symptoms. In addition, the application of herbal compounds has attracted increasing attention. Thus, it is necessary to find efficient test systems to identify and characterize insulin-mimetic compounds. Here we developed a modified chick embryo model, which enables testing of synthetic compounds and herbal extracts with insulin-mimetic properties. Using a fluorescence microscopy-based primary screen, which quantifies the translocation of Glucose transporter 4 (Glut4) to the plasma membrane, we were able to identify compounds, mainly herbal extracts, which lead to an increase of intracellular glucose concentrations in adipocytes. However, the efficacy of these substances requires further verification in a living organism. Thus, we used an in-ovo approach to identify their blood glucose-reducing properties. The approval by an ethics committee is not needed since the use of chicken embryos during the first two-thirds of embryonic development is not considered an animal experiment. Here, the application of this model is described in detail.

An In Ovo Model for Testing Insulin-mimetic Compounds

In this study, we describe an in-ovo system as a promising tool to test insulin-mimetic compounds in a living organism. The main advantages include adequate throughput rates and acceptable costs, which enable the identification of phytochemicals with insulin-mimetic properties.

Um modelo em Ovo para testar compostos insulino-mimética

Elevated blood glucose levels in type 2 diabetes mellitus (T2DM), a complex and multifactorial metabolic disease, are caused by insulin resistance and ...