Name: behavioral assessment of manual dexterity in non-human primates
Uploaded: 2011-11-11T13:00:00
Duration: 16 min
Description: Watch this Scientific Journal Video about Behavioral Assessment of Manual Dexterity in Non-Human Primates at JoVE.com

Författarna vill tacka Professor ME Schwab, Dr P. Freund, Dr A. Wyss, Dr S. Bashir, Dr A. Mir, Dr J. Bloch, Dr JF Brunet, Dr J. Aebischer, Dr A. Meszaros, Dr V. Goetschman för deras bidrag till tidigare experiment och analyser. Den experimentella uppställningar byggdes av André Gaillard, Bernard Aebischer och Laurent Monney. I djurhuset var apor stå under övervakning av professionella Skötaren: Josef Corpataux, Laurent Bossy och Jacques Maillard. Beteendemässiga tester och analys av data, samt histologi, genomfördes med mycket värdefulla bidrag laboratorietekniker: Véronique Moret (även webb-master), Françoise Tinguely, Christine Roulin, Monica Bennefeld, Christiane Marti och Georgette Fischer. Detta arbete stöddes av den schweiziska National Science Foundation, stipendier Nej 31-61857,00, från 310.000 till 110.005, 31003A-132.465 (EMR), 310.030-118.357, 31003A-104.061 (TW), 310.030-120.411 (ABS), PZ00P3_121646 (ES), Novartis Foundation, Nationellt centrum för kompetens inom forskning (NCCR) om &quot;Neural plasticitet och reparation&quot;.

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Neurosci. 29, 983-996 (2009).</li><li>Kaeser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+Unilateral+Motor+Cortex+Lesion+on+Ipsilesional+Hand's+Reach+and+Grasp+Performance+in+Monkeys:+Relationship+With+Recovery+in+the+Contralesional+Hand.">Effects of Unilateral Motor Cortex Lesion on Ipsilesional Hand's Reach and Grasp Performance in Monkeys: Relationship With Recovery in the Contralesional Hand.</a> J. Neurophysiol. 103, 1603-1645 (2010).</li><li>Kaeser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autologous+adult+cortical+cell+transplantation+enhances+functional+recovery+following+unilateral+lesion+of+motor+cortex+in+primates:+a+pilot+study.">Autologous adult cortical cell transplantation enhances functional recovery following unilateral lesion of motor cortex in primates: a pilot study.</a> Neurosurgery. 68, 1405-1417 (2011).</li><li>Bashir, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short-term+effects+of+unilateral+lesion+of+the+primary+motor+cortex+(M1)+on+Ipsilesional+hand+dexterity+in+adult+macaque+monkeys.">Short-term effects of unilateral lesion of the primary motor cortex (M1) on Ipsilesional hand dexterity in adult macaque monkeys.</a> Brain Structure and Function. , (2011).</li><li>Hamadjida, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+anti-Nogo-A+treatment+on+the+reorganization+of+callosal+connectivity+of+the+premotor+cortical+areas+following+unilateral+lesion+of+primary+motor+cortex+(M1)+in+adult+macaque+monkeys.">Influence of anti-Nogo-A treatment on the reorganization of callosal connectivity of the premotor cortical areas following unilateral lesion of primary motor cortex (M1) in adult macaque monkeys.</a> , (2011).</li><li>Jenny, A. 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Inga intressekonflikter deklareras.

Även om den nuvarande beteende uppgifter har beaktats så långt i vårt laboratorium i samband med studier rörande skada av hals sladden eller skada på motoriska cortex i syfte att testa olika behandlingar (se 14,15,17 och <a href="http://www.unifr.ch/neuro/rouiller">http:/ / www.unifr.ch / neuro / rouiller</a> &gt; välj &quot;forskning&quot; i det översta fältet menyn, sedan&gt; &quot;motorsystem&quot;&gt; &quot;återhämtning efter lesion&quot;), kan de också ha en bredare tillämpning, som fingerfärdighet är också en aspekt att tänka i andra sjukdomar, såsom Parkinsons sjukdom (MPTP apor) eller vid sensorisk de-afferentation påverkar proprioception och / eller känsel (särskilt testet 2 i avsaknad av visuell kontroll). Den beteendemässiga tester som här föreslås är lämpliga att undersöka motorstyrning av distala rörelser forelimb, som deltar i fingerfärdighet. Specificiteten för testerna framgår avavsaknad av underskott (förutom ett par dagar) i händelse av en skada som inte försämrar relevanta komponenter i styrsystemet: ja, i händelse av en lesion placeras mer caudal än motoneurons kontrollera handen muskler, det fanns ingen underskott. Relevans av testet 1 kan uppskattas genom att jämföra på videosekvenser tas vid två tidpunkter efter skada återhämtningen kurva, som skiljer sig genom en förstärkning av funktionell återhämtning på 25%, eventuellt i förhållande till en cellterapi för behandling 17. Trots vissa inledande, relativt kort utbildning fasen i början (som varar i regel 2-3 månader), den beteendemässiga tester som här föreslås är relativt &quot;naturliga&quot; och okomplicerad, jämfört med komplexa (t.ex. villkorade) uppgifter som utbildning av apan Det kan ta nästan ett år eller mer. Den positiva förstärkningen är baserad på fast föda, vilket är mindre känslig på etisk synvinkel än vatten försakelser, som vanligtvis används i mer comPlex uppgifter 23. Det finns ingen anledning att beröva aporna från mat för att få stabila och konsekventa resultat. Den pellets som erhållits under de arbetsuppgifter som representerar det första tillgång till mat om dagen för beteende-sessionen (förutsatt att apan inte äta under den föregående natten, men extra mat ges till slutet av eftermiddagen föregående dag) . Det är viktigt att varje apa utför beteendemässiga session vid samma tidpunkt på dagen, samt respekterar samma ordningsföljd mellan de olika aporna bildar en grupp i huset rummet. Eftersom aporna är känsliga för yttre störande händelser bör beteendemässiga uppgifter ske i närvaro av bakgrundsmusik, maskering potentiella störande ljud från angränsande rum eller laboratorier. Det är viktigt att under hela av experimentet (från grundutbildning fram till den sista dagliga experimentella session, en period av flera månader om inte år), är en given apaplaceras dagligen under överinseende av samma försöksledaren. Den nuvarande beteende-tester används sedan flera år i vårt laboratorium för att kvantifiera fingerfärdighet, i viss mån jämförbara med andra tester av fingerfärdighet nyligen rapporterats i litteraturen 24-28. Det finns dock ett stort behov av att standardisera tester mellan olika laboratorier (för bättre jämförelse), vilket är ett preliminärt mål för denna rapport. På begäran, illustrerade den detaljerade egenskaper uppställningar här för test 1-4 kan tillhandahållas av korresponderande författare, för att reproducera dem. Utöver frågan om regenerativ medicin (återhämtning från skada i ryggmärgen eller cerebral cortex) kan den nuvarande paletten av tester vara lämpliga att behandla i normala icke-mänskliga primater utvecklingsfrågor (t.ex. tid under motoriska utveckling skicklig rörelser), för att undersöka lateralization aspekter (sidan preferens / dominans) och att dechiffrera evolutionära frågor av comparing motorn förmågor av olika arter av primater, inklusive människor. Observera dock att måtten på apparatur bör anpassas efter siffrorna &quot;storlek (tjocklek och längd) av primat arter, eftersom det kan påverka uppdraget prestanda. I föreliggande studie var de tester som gjorts på Macaca fascicularis apor, från 2,5 till 8 år gammal och väger mellan 2,5 och 8 kg. Längden på pekfingret (används först manipulera pellets) varierar från 32 till 35 mm, medan omkretsen av distala falangen (spets) av pekfingret var mellan 22 och 25 mm i apor som ingår i våra studier. Som testats i tidigare försök, samma gripa tester är lämpliga för Macaca mulatta också. Alla experiment utfördes i enlighet med Handbok för vård och användning av försöksdjur (1996) och godkända av lokala (schweizisk) veterinärmyndigheterna. Alla experimentella procedurer på apanS, liksom frihetsberövande förhållandena i djurhuset, beskrevs i detalj i de senaste rapporterna från vårt laboratorium: se referenser 12-18.

behavioral assessment of manual dexterity in non-human primates

Den corticospinal (CS) tarmkanalen är den anatomiska stöd av utsökta motoriska förmåga att skickligt manipulera små objekt, ett privilegium huvudsakligen av primater 1. I händelse av skada som påverkar CS projektionssystem till sitt ursprung (lesion av motoriska hjärnbarken områden) eller längs dess bana (cervikal sladd lesion), finns det en dramatisk förlust av fingerfärdighet (handen förlamning), vilket kan ses i vissa tetraplegisk eller hemiplegisk patienter . Även om det finns någon spontan funktionell återhämtning efter en sådan skada, är det fortfarande mycket begränsad i den vuxna. Olika terapeutiska strategier är för närvarande föreslagna (t.ex. cellterapi, neutralisering av hämmande axonal tillväxt molekyler, tillämpning av tillväxtfaktorer, etc), som oftast utvecklas hos gnagare. Men innan klinisk tillämpning är det ofta rekommenderas att testa genomförbarhet, effekt och säkerhet vid behandling i icke-mänskliga primater. Detta gäller särskilt när målet är att återställa fingerfärdighet efteren skada i centrala nervsystemet, som organisationen av motoriska system av gnagare är annorlunda än primater 1,2. Makak-apor illustreras här som en lämplig beteendevetenskaplig modell för att kvantifiera fingerfärdighet i primater, för att återspegla de underskotten till följd av skada på motor cortex eller livmoderhalscancer sladden till exempel mäta omfattningen av spontana funktionell återhämtning och när det sker behandling, utvärdera hur mycket det kan förbättra den funktionella återhämtningen. Den beteendemässiga Bedömningen av fingerfärdighet är baserat på fyra olika, kompletterande, sträcka ut och gripa manuella uppgifter (användning av Precision-greppet att greppa pellets), vilket kräver en grundutbildning för vuxna makak-apor. Beredningen av djuren visat samt positionering med avseende på beteende-set-up. Prestandan hos en typisk apa illustreras för varje uppgift. Insamling och analys av relevanta parametrar som speglar exakt handen manipuleringtion, liksom kontrollen av kraft, förklaras och demonstreras med representativa resultat. Dessa uppgifter läggs sedan i ett större sammanhang, som visar hur beteendemässiga data kan utnyttjas för att undersöka effekten av en ryggmärgen skada eller en skada på motor cortex och i vilken utsträckning en behandling kan öka den spontana funktionell återhämtning, genom att jämföra olika grupper av apor (som behandlas mot simulerad behandlas till exempel). Fördelar och begränsningar beteendetester diskuteras. Den nuvarande beteende ligger i linje med tidigare rapporter betonar relevansen av de icke-mänskliga primater modell i samband med nervsystemet 2,3.

Watch this Scientific Journal Video about Behavioral Assessment of Manual Dexterity in Non-Human Primates at JoVE.com

Behavioral Assessment of Manual Dexterity in Non-Human Primates

Som fingerfärdighet är ett privilegium huvudsakligen av primater, har beteendevetenskaplig uppgifter tagits fram i makak-apor. Fyra sträcka ut och gripa behärskning av uppgifter, som mäter förmågan handen manipulation och kraft, gör att upprätta funktionella återhämtningen efter en skada i centrala nervsystemet och för att testa effekten av en behandling.

Behavioral Bedömning av fingerfärdig i icke-mänskliga primater

The corticospinal (CS) tract is the anatomical support of the exquisite motor ability to skillfully manipulate small objects, a prerogative mainly of ...

behavioral-assessment-of-manual-dexterity-in-non-human-primates

Research

JoVE Journal

Neuroscience

A General Method for Evaluating Incubation of Sucrose Craving in Rats

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and relapse to food taking 1. Food craving conceptualized in this way is akin to drug craving in response to drug-paired cues. A rich literature has been developed around understanding the behavioral and neurobiological determinants of drug craving; we and others have been focusing recently on translating techniques from basic addiction research to better understand addiction-like behaviors related to food 2-4.
As done in previous studies of drug craving, we examine sucrose craving behavior by utilizing a rat model of relapse. In this model, rats self-administer either drug or food in sessions over several days. In a session, lever responding delivers the reward along with a tone+light stimulus. Craving behavior is then operationally defined as responding in a subsequent session where the reward is not available. Rats will reliably respond for the tone+light stimulus, likely due to its acquired conditioned reinforcing properties 5. This behavior is sometimes referred to as sucrose seeking or cue reactivity. In the present discussion we will use the term "sucrose craving" to subsume both of these constructs.
In the past decade, we have focused on how the length of time following reward self-administration influences reward craving. Interestingly, rats increase responding for the reward-paired cue over the course of several weeks of a period of forced-abstinence. This "incubation of craving" is observed in rats that have self-administered either food or drugs of abuse 4,6. This time-dependent increase in craving we have identified in the animal model may have great potential relevance to human drug and food addiction behaviors.
Here we present a protocol for assessing incubation of sucrose craving in rats. Variants of the procedure will be indicated where craving is assessed as responding for a discrete sucrose-paired cue following extinction of lever pressing within the sucrose self-administration context (Extinction without cues) or as responding for sucrose-paired cues in a general extinction context (Extinction with cues).

Responding for food or drug-paired cues increases over the course of a period of abstinence and this may relate to an increased susceptibility to relapse behaviors. Here we detail a procedure for evaluating this "incubation of craving" in rats that have self-administered sucrose.

En allmän metod för att utvärdera Inkubation av Sackaros Craving på råttor

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and ...

Recording and Analysis of Circadian Rhythms in Running-wheel Activity in Rodents

When rodents have free access to a running wheel in their home cage, voluntary use of this wheel will depend on the time of day1-5. Nocturnal rodents, including rats, hamsters, and mice, are active during the night and relatively inactive during the day. Many other behavioral and physiological measures also exhibit daily rhythms, but in rodents, running-wheel activity serves as a particularly reliable and convenient measure of the output of the master circadian clock, the suprachiasmatic nucleus (SCN) of the hypothalamus. In general, through a process called entrainment, the daily pattern of running-wheel activity will naturally align with the environmental light-dark cycle (LD cycle; e.g. 12 hr-light:12 hr-dark). However circadian rhythms are endogenously generated patterns in behavior that exhibit a ~24 hr period, and persist in constant darkness. Thus, in the absence of an LD cycle, the recording and analysis of running-wheel activity can be used to determine the subjective time-of-day. Because these rhythms are directed by the circadian clock the subjective time-of-day is referred to as the circadian time (CT). In contrast, when an LD cycle is present, the time-of-day that is determined by the environmental LD cycle is called the zeitgeber time (ZT).
Although circadian rhythms in running-wheel activity are typically linked to the SCN clock6-8, circadian oscillators in many other regions of the brain and body9-14 could also be involved in the regulation of daily activity rhythms. For instance, daily rhythms in food-anticipatory activity do not require the SCN15,16 and instead, are correlated with changes in the activity of extra-SCN oscillators17-20. Thus, running-wheel activity recordings can provide important behavioral information not only about the output of the master SCN clock, but also on the activity of extra-SCN oscillators. Below we describe the equipment and methods used to record, analyze and display circadian locomotor activity rhythms in laboratory rodents.

Circadian rhythms in voluntary wheel-running activity in mammals are tightly coupled to the molecular oscillations of a master clock in the brain. As such, these daily rhythms in behavior can be used to study the influence of genetic, pharmacological, and environmental factors on the functioning of this circadian clock.

Registrering och analys av dygnsrytmen i Running-wheel aktivitet hos gnagare

When rodents have free access to a running wheel in their home cage, voluntary use of this wheel will depend on the time of day1-5. Nocturnal rodents, ...

Assessment of Dendritic Arborization in the Dentate Gyrus of the Hippocampal Region in Mice

Dendritic arborization has been shown to be a reliable marker for examination of structural and functional integrity of neurons. Indeed, the complexity and extent of dendritic arborization correlates well with the synaptic plasticity in these cells. A reliable method for assessment of dendritic arborization is needed to characterize the deleterious effects of neurological disorders on these structures and to determine the effects of therapeutic interventions. However, quantification of these structures has proven to be a formidable task given their complex and dynamic nature. Fortunately, sophisticated imaging techniques can be paired with conventional staining methods to assess the state of dendritic arborization, providing a more reliable and expeditious means of assessment. Below is an example of how these imaging techniques were paired with staining methods to characterize the dendritic arborization in wild type mice. These complementary imaging methods can be used to qualitatively and quantitatively assess dendritic arborization that span a rather wide area within the hippocampal region.

We describe two methods for visualization and quantification of dendritic arborization in the hippocampus of mouse models: real-time and extended depth of field imaging. While the former method allows sophisticated topographical tracing and quantification of the extent of branching, the latter allows speedy visualization of the dendritic tree.

Bedömning av dendritiska arborization i gyrus dentatus i hippocampus regionen hos möss

Dendritic arborization has been shown to be a reliable marker for examination of structural and functional integrity of neurons. Indeed, the complexity ...

Quantification of Filamentous Actin (F-actin) Puncta in Rat Cortical Neurons

Filamentous actin protein (F-actin) plays a major role in spinogenesis, synaptic plasticity, and synaptic stability. Changes in dendritic F-actin rich structures suggest alterations in synaptic integrity and connectivity. Here we provide a detailed protocol for culturing primary rat cortical neurons, Phalloidin staining for F-actin puncta, and subsequent quantification techniques. First, the frontal cortex of E18 rat embryos are dissociated into low-density cell culture, then the neurons grown in vitro for at least 12-14 days. Following experimental treatment, the cortical neurons are stained with AlexaFluor 488 Phalloidin (to label the dendritic F-actin puncta) and microtubule-associated protein 2 (MAP2; to validate the neuronal cells and dendritic integrity). Finally, specialized software is used to analyze and quantify randomly selected neuronal dendrites. F-actin rich structures are identified on second order dendritic branches (length range 25-75 &#181;m) with continuous MAP2 immunofluorescence. The protocol presented here will be a useful method for investigating changes in dendritic synapse structures subsequent to experimental treatments.

Quantification of Filamentous Actin F-actin Puncta in Rat Cortical Neurons

Filamentous actin (F-actin) plays an important role in spinogenesis, synaptic plasticity, and synaptic stability. Quantification of F-actin puncta is therefore a useful tool to study the integrity of synaptic structures. This protocol describes the procedures of quantifying F-actin puncta labeled with Phalloidin in low-density primary cortical neuronal cultures.

Kvantifiering av fintrådiga aktin (F-aktin) puncta i Rat kortikala Neuroner

Filamentous actin protein (F-actin) plays a major role in spinogenesis, synaptic plasticity, and synaptic stability. Changes in dendritic F-actin rich ...

Identification of Dopamine D1-Alpha Receptor Within Rodent Nucleus Accumbens by an Innovative RNA In Situ Detection Technology

In the central nervous system, the D1-alpha subtype receptor (Drd1&#945;) is the most abundant dopamine (DA) receptor, which plays a vital role in regulating neuronal growth and development. However, the mechanisms underlying Drd1&#945; receptor abnormalities mediating behavioral responses and modulating working memory function are still unclear. Using a novel RNA in situ hybridization assay, the current study identified dopamine Drd1&#945; receptor and tyrosine hydroxylase (TH) RNA expression from DA-related circuitry in the nucleus accumbens (NAc) area and substantia nigra region (SNR), respectively. Drd1&#945; expression in the NAc shows a &#34;discrete dot&#34; staining pattern. Clear sex differences in Drd1&#945; expression were observed. In contrast, TH shows a &#34;clustered&#34; staining pattern. Regarding TH expression, female rats displayed a higher signal expression per cell relative to male animals. The methods presented here provide a novel in situ hybridization technique for investigating changes in dopamine system dysfunction during the progression of central nervous system diseases.

Identification of Dopamine D1-Alpha Receptor Within Rodent Nucleus Accumbens by an Innovative RNA In Situ Detection Technology

Identification of dopamine D1-alpha receptor in the nucleus accumbens is critical for clarifying D1 receptor dysfunction during a central nervous system disease. We performed a novel RNA in situ hybridization assay to visualize single RNA molecules in a specific brain area.

Identifiering av dopamin D1-Alpha Receptor inom gnagare Nucleus Accumbens genom en innovativ RNA In Situ Detection-teknik

In the central nervous system, the D1-alpha subtype receptor (Drd1&#945;) is the most abundant dopamine (DA) receptor, which plays a vital role in ...

The Power of Interstimulus Interval for the Assessment of Temporal Processing in Rodents

Temporal processing deficits have been implicated as a potential elemental dimension of higher-level cognitive processes, commonly observed in neurocognitive disorders. Despite the popularization of prepulse inhibition (PPI) in recent years, many current protocols promote using a percent of control measure, thereby precluding the assessment of temporal processing. The present study used cross-modal PPI and gap prepulse inhibition (gap-PPI) to demonstrate the benefits of employing a range of interstimulus intervals (ISIs) to delineate effects of sensory modality, psychostimulant exposure, and age. Assessment of sensory modality, psychostimulant exposure, and age reveals the utility of an approach varying the interstimulus interval (ISI) to establish the shape of the ISI function, including increases (sharper curve inflections) or decreases (flattening of the response amplitude curve) in startle amplitude. Additionally, shifts in peak response inhibition, suggestive of a differential sensitivity to the manipulation of ISI, are often revealed. Thus, the systematic manipulation of ISI affords a critical opportunity to evaluate temporal processing, which may reveal the underlying neural mechanisms involved in neurocognitive disorders.

Temporal processing, a preattentive process, may underlie deficits in higher-level cognitive processes, including attention, commonly observed in neurocognitive disorders. Using prepulse inhibition as an exemplar paradigm, we present a protocol for manipulating interstimulus interval (ISI) to establish the shape of the ISI function to provide an assessment of temporal processing.

Kraften i Interstimulus intervall för bedömning av Temporal bearbetning i gnagare

Temporal processing deficits have been implicated as a potential elemental dimension of higher-level cognitive processes, commonly observed in ...

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.

Bedömning av långvarig depression induktion hos vuxna cerebellär skivor

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

Ballistic Labeling of Pyramidal Neurons in Brain Slices and in Primary Cell Culture

It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of pyramidal neurons and dendritic spines, a ballistic labeling technique can be utilized. In the present protocol, pyramidal neurons are labeled with DilC18(3) dye and analyzed using neuronal reconstruction software to assess neuronal morphology and dendritic spines. To investigate neuronal structure, dendritic branching analysis and Sholl analysis are performed, allowing researchers to draw inferences about dendritic branching complexity and neuronal arbor complexity, respectively. The evaluation of dendritic spines is conducted using an automatic assisted classification algorithm integral to the reconstruction software, which classifies spines into four categories (i.e., thin, mushroom, stubby, filopodia). Furthermore, an additional three parameters (i.e., length, head diameter, and volume) are also chosen to assess alterations in dendritic spine morphology. To validate the potential of wide application of the ballistic labeling technique, pyramidal neurons from in vitro cell culture were successfully labeled. Overall, the ballistic labeling method is unique and useful for visualizing neurons in different brain regions in rats, which in combination with sophisticated reconstruction software, allows researchers to elucidate the possible mechanisms underlying neurocognitive dysfunction.

We present a protocol to label and analyze pyramidal neurons, which is critical for evaluating potential morphological alterations in neurons and dendritic spines that may underlie neurochemical and behavioral abnormalities.

Ballistisk märkning av pyramidal neuroner i hjärnan skivor och i primär cell kultur

It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of ...

A Hydrophobic Tissue Clearing Method for Rat Brain Tissue

Hydrophobic tissue clearing methods are easily adjustable, fast, and low-cost procedures that allows for the study of a molecule of interest in unaltered tissue samples. Traditional immunolabeling procedures require cutting the sample into thin sections, which restricts the ability to label and examine intact structures. However, if brain tissue can remain intact during processing, structures and circuits can remain intact for the analysis. Previously established clearing methods take significant time to completely clear the tissue, and the harsh chemicals can often damage sensitive antibodies. The iDISCO method quickly and completely clears tissue, is compatible with many antibodies, and requires no special lab equipment. This technique was initially validated for the use in mice tissue, but the current protocol adapts this method to image hemispheres of control and transgenic rat brains. In addition to this, the present protocol also makes several adjustments to preexisting protocol to provide clearer images with less background staining. Antibodies for Iba-1 and tyrosine hydroxylase were validated in the HIV-1 transgenic rat and in F344/N control rats using the present hydrophobic tissue clearing method. The brain is an interwoven network, where structures work together more often than separately of one another. Analyzing the brain as a whole system as opposed to a combination of individual pieces is the greatest benefit of this whole brain clearing method.

Here we present a hydrophobic tissue clearing method that allows for the viewing of target molecules as part of intact brain structures. This technique has now been validated for F344/N control and HIV-1 transgenic rats of both sexes.

En hydrofobisk vävnadsrensningsmetod för råtta hjärnvävnad

Hydrophobic tissue clearing methods are easily adjustable, fast, and low-cost procedures that allows for the study of a molecule of interest in unaltered ...

A Rat Model of EcoHIV Brain Infection

It has been well studied that the EcoHIV infected mouse model is of significant utility in investigating HIV associated neurological complications. Establishment of the EcoHIV infected rat model for studies of drug abuse and neurocognitive disorders, would be beneficial in the study of neuroHIV and HIV-1 associated neurocognitive disorders (HAND). In the present study, we demonstrate the successful creation of a rat model of active HIV infection using chimeric HIV (EcoHIV). First, the lentiviral construct of EcoHIV was packaged in cultured 293 FT cells for 48 hours. Then, the conditional medium was concentrated and titered. Next, we performed bilateral stereotaxic injections of the EcoHIV-EGFP into F344/N rat brain tissue. One week after infection, EGFP fluorescence signals were detected in the infected brain tissue, indicating that EcoHIV successfully induces an active HIV infection in rats. In addition, immunostaining for the microglial cell marker, Iba1, was performed. The results indicated that microglia were the predominant cell type harboring EcoHIV. Furthermore, EcoHIV rats exhibited alterations in temporal processing, a potential underlying neurobehavioral mechanism of HAND as well as synaptic dysfunction eight weeks after infection. Collectively, the present study extends the EcoHIV model of HIV-1 infection to the rat offering a valuable biological system to study HIV-1 viral reservoirs in the brain as well as HAND and associated comorbidities such as drug abuse.

Here, we present a protocol to establish a new rat model of active HIV infection using chimeric HIV (EcoHIV), which is critical for enhancing our understanding of HIV-1 viral reservoirs in the brain and offering a system to study HIV-associated neurocognitive disorders and associated comorbidities (i.e., drug abuse).

En råttmodell av EcoHIV hjärninfektion

It has been well studied that the EcoHIV infected mouse model is of significant utility in investigating HIV associated neurological complications. ...