Wir danken Joachim Erber für die Einführung in die taktile Lernparadigma in Honigbienen. Diese Arbeit wurde durch den Exzellenzcluster CITEC 277, die im Rahmen der deutschen Exzellenzinitiative gefördert unterstützt.

Ein. Vorbereitung der Bienen <ol><li> Nektar oder Pollen Sammlerinnen im Feld entweder aus einer Saccharose Feeder oder direkt aus dem Flugloch gefangen, während der Rückkehr von einem Nahrungssuche Reise. Jede einzelne Biene wird in einen Glaskolben, der mit einem Schaum-Stopfen verschlossen ist und unmittelbar in das Labor für die weitere Handhabung getroffen eingefangen. </li><li> Im Labor werden die aufgenommenen Bienen kurz im Kühlschrank bei 4 ° C abgekühlt, bis sie erste Anzeichen von Immobilität zu zeigen. </li><li> Jede einzelne Biene immobilisiert ist in einem kleinen Metallrohres mit einem Klebestreifen zwischen Kopf und Thorax und über den Bauch befestigt. Es sollte darauf geachtet, dass der Rüssel und Antennen frei beweglich sind. </li><li> Malen Sie die Facettenaugen und Augenflecken der festen Biene mit weißer Farbe (zB lösemittelfreie Tipp-Ex) zur Vision zu verschließen. </li><li> Fügen Sie einen kleinen Tropfen geschmolzenen Wachs hinter dem Kopf der Biene um es dem Band zwischen Kopf und Thorax zu fixieren Kopfbewegungen während der Aufnahmen zu verhindern. </li><li> Markieren Sie jede einzelne Biene mit einer Nummer auf dem Band zur besseren Identifizierung und platzieren Sie den Schlauch mit dem festen Biene in einer feuchten Atmosphäre, um eine Dehydratation zu verhindern. </li><li> Führen Sie jede einzelne Biene für 5 sec mit Tröpfchen einer 30% Saccharose-Lösung mit einer Spritze vorgestellt und lassen Sie alle Bienen für 30 min erholen, bevor mit dem taktilen Klimaanlage Protokoll. </li></ol> 2. Tactile Conditioning <ol><li> Bevor Klimaanlage hat jede einzelne Biene für die proboscis extension response (PER) auf 30% Saccharose Stimulus zu den Antennen getestet werden. Wodurch die Spitze des Rüssels muss eine virtuelle Linie zwischen der geöffneten Mandibeln überqueren. Entsorgen Sie alle Bienen, nicht mit einem KGV der Saccharose Reiz reagieren. </li><li> Für taktile Konditionierung mit einem Messing cube (zB 3 x 5 mm) mit einer glatten oder einer gravierten Muster, zB horizontale oder vertikale Nuten bilden ein Gitter mit 150 um Wellenlänge, als die konditionierten Reiz (CS). Foder die unbedingte Reiz (US) mit einem 30% Saccharose-Lösung (Haushaltszucker, in Wasser verdünnt). </li><li> Das Messing Würfel (CS) in einer Halterung an einem Mikromanipulator platziert (zB Märzhäuser MM33) eine genaue Positionierung während der Konditionierung zu gewährleisten. Die USA die Biene mit einer Spritze mit einer 30% Saccharose-Lösung gefüllt präsentiert. </li><li> Die Konditionierung besteht aus fünf Paarungen der taktilen Reiz (CS) und Saccharose-Lösung (US) mit einem inter-trial-Intervall (ITI) von 5 min. Legen Sie eine einzelne Biene vor dem Mikromanipulator mit dem montierten taktilen Reiz (CS). Positionieren des CS langsam, so dass die Oberfläche des taktilen Reiz parallel zum Kopf der Biene (Abbildung 1 A und B) ist. Der Abstand zwischen dem Tier und dem taktilen Objekt sollte im Bereich des antennalen Ausladung der getesteten Biene sein, dh die Biene der Lage sein sollten, um die taktilen Reiz scannen in einer komfortablen Position with beide Antennen. In dem Beispiel in 1 gezeigt, war der Abstand von 3 mm. Lassen Sie die Biene scannen die taktilen Reiz (CS) für 5 Sekunden. Nach der ersten 3 sec, präsentieren ein Tröpfchen der 30% Sucrose (US)-Lösung mit einer Spritze unter den Rüssel. Verwenden Sie die Spitze der Spritze vorsichtig heben die Rüssel. Saccharose Stimulation unter den Rüssel wird entlocken die unbedingte PER 15. Lassen Sie die Biene, die Saccharose Belohnung lecken. Verwenden Sie eine Stoppuhr mit einem Alarmsignal, um die genauen Zeitintervallen während der Konditionierung zu halten. </li><li> Das konditionierte PER wird als Maß für den Lernerfolg einer Biene verwendet. Nach den ersten belohnt Paarungen starten Bienen zu CS Präsentation durch Erweiterung ihres Rüssels zu reagieren, was darauf hinweist, dass sie die bevorstehende Belohnung erwarten. Ein vollständig ausgefahrenen Rüssels jederzeit während der 3 sec Zeitfenster Tastreizes Aufmachung und Präsentation vor Saccharose beobachtet wird als positive Reaktion gewertet. Keine Antwort gezählt negativ. Die occurrence des PER ist vom Experimentator vermerkt. Der Prozentsatz der Bienen, die die PER während CS Präsentation ist für jeden Versuch aufgetragen. </li></ol> 3. Kinematische Recordings <ol><li> Antennalen Bewegungen eines einzigen genutzt Biene mit einer digitalen Videokamera mit einem geeigneten Makro-Objektiv (zB Basler A602f-2 mit TECHSPEC VZM 200 ausgestattet, betrieben bei 50 fps über einen Firewire-Anschluss) aufgezeichnet. Die Kamera ist oberhalb des Tieres in einer Draufsicht (Figur 1 A) positioniert ist. In Abbildung 1 ist die räumliche Auflösung der Aufnahmen 0,02 mm / Pixel. </li><li> Kalibrieren Sie die Kamera durch die Aufnahme einzelner Bilder von einem 10 x 10 mm Schachbrett mit einer Kantenlänge von 1 mm aus verschiedenen Ausrichtungen unter dem Kamera-Objektiv. Die Kalibrierung kann mit der Kamera-Kalibrierung tool box Matlab 16 durchgeführt werden. </li><li> Legen Sie eine einzelne feste bee unterhalb der Kameralinse. Präsentieren des taktilen Reiz, der an einem Mikromanipulator, An das Tier. Gehen Sie in der gleichen Weise wie für die taktile Konditionierung, und notieren Sie die antennalen Bewegung, während die Biene ist das Scannen des Objekts. Es ist wichtig, dass die volle antennalen Arbeitsbereich und der Tastreizes sichtbar sind. Die Auswahl der Reizmuster getestet und Studie-Nummern hängen von der experimentellen Design. </li></ol> 4. Data Analysis <ol><li> Die Berechnung der Bildhintergrund wird in Matlab getan. Erstens hat der Median Grauwert über die Zeit, um für jedes Pixel berechnet werden. Statische Objekte, wie die festen Kopf der Biene und der taktilen Reiz, bilden den Bildhintergrund. Bewegte Objekte, wie die beiden Antennen, werden nicht mehr in den Hintergrund. </li><li> Jeder Rahmen des aufgenommenen Videos hat, sequentiell geladen werden, und die Differenz zwischen dem aktuellen Frame und dem Bildhintergrund muss berechnet werden. Das Ergebnis dieser Subtraktion betont diejenigen Teile des Bildes, die von Rahmen zu Rahmen bewegen. Identifikationeally, sind die Antennen der Biene die einzigen Bereiche von Nicht-Null-Werte (Abbildung 1C). </li><li> Zur weiteren Verarbeitung werden die beiden größten Flächen mit von Null angenommen Pixelwerte die Antennen sein. Zu den beiden Antennen eine binäre Maske zu erzeugen, die für jedes Pixel einen Wert 1, falls das Pixel gehört zu der Antenne und den Wert 0 ansonsten. Diese Maske dient als Grundlage für die Lokalisierung der antennalen Tipps später. Um eine vorläufige Maske für das gesamte Bild zu gewinnen die grau-Wert jedes Pixels des Differenzbilds mit einem vordefinierten Schwellenwert verglichen. Da wir kümmern uns um Geräusche später diese Schwelle gewählt wird recht gering zu sein. Die vorläufige Maske dann noch trägt zwei Arten von Fehlern: Erstens wegen Bildrauschen kleinen Regionen sind immer noch Teil der Maske. Zweitens könnten Bereiche, die an der Antenne gehören nicht unbedingt in vollem Umfang angeschlossen werden. Letzteres geschieht meist, wenn der Hintergrund hat ungefähr die gleiche wie die Antenne Lumineszenz. Um diese Artefakte zu eliminieren, StandardBildverarbeitung morphologische Operation angewendet werden, dh eine Kombination von Bild Erosion und Dilatation, gestützt auf die Standard-Matlab-Funktionen imerode und imdilate sind (siehe 17 S. 158-205 für weitere Erläuterungen). Nach dem Verfahren werden die Antennen Rauschunterdrückung Hypothesen werden noch innerhalb des gleichen Maske enthalten. Daher in einem nächsten Schritt die binäre Maske für disjunkte Bereiche mit dem Standard Matlab-Funktion bwlabel (siehe 17 S. 40-48 für weitere Details auf das Bild Segmentierungsalgorithmus) gebündelt. </li><li> Die Anzahl von Pixeln pro Cluster wird gezählt und die beiden größten Clustern ausgewählt. Der Schwerpunkt berechnet wird, um zwischen dem linken und rechten Antenne (1D) zu unterscheiden. </li><li> Die antennalen Spitze für jede Antenne als das Pixel in einem Cluster mit dem höchsten Wert in proximal-zu-distal (siehe 1D) definiert werden. </li></ol>

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Vorbereitung der Bienen: Sammeln und Fixieren die Bienen schnell getan werden sollte, um den Stress des Tieres gering zu halten. Stress hat einen Einfluss auf die PER-Reaktionsfähigkeit und könnte daher einen indirekten Einfluss auf die Lernleistung bei Bienen 19, 20. Die Belastung kann durch Platzieren der Glasfläschchen mit den Bienen direkt auf Eis unmittelbar nach der Entnahme, um sie schnell zu immobilisieren verringert werden. Es muss berücksichtigt werden, dass die Bienen mehr Zeit...

<table border="1"><tbody><tr><td> Name des Reagenz / Material </td><td> Firma </td><td> Catalog Number </td><td> Kommentare </td></tr><tr><td> Pollensammlerinnen, Apis mellifera L. </td><td></td><td></td><td> Private Nesselsucht </td></tr><tr><td> Glasfläschchen (22 x 45 mm) </td><td> Carl Roth GmbH &amp; Co </td><td> X655.1 </td><td></td></tr><tr><td> Messingrohre, 10 x 30 mm </td><td></td><td></td><td> Selbst erstellte </td></tr><tr><td> Messing Würfel, 3 x 5 mm </td><td></td><td></td><td> Selbst erstellte </td></tr><tr><td> Tipp-Ex (lösemittelfrei) </td><td> Tipp-Ex GmbH &amp; Co KG </td><td></td><td></td></tr><tr><td> Saccharose-Lösung (30%) </td><td></td><td></td><td> Haushalt suger in Wasser verdünnt </td></tr><tr><td> Ethanol (70%) </td><td> Carl Roth GmbH &amp; Co </td><td> 9065,3 &lt;/ Td&gt; <td></td></tr><tr><td> Spritze (1 ml) und Nadel (0,6 x 25) </td><td> Carl Roth GmbH &amp; Co </td><td> 59,1 </td><td></td></tr><tr><td> Stoppuhr </td><td> Carl Roth GmbH &amp; Co </td><td> L423.1 </td><td></td></tr><tr><td> Micromanipulator </td><td> Märzhäuser MM33 </td><td> 00-42-101-0000 </td><td></td></tr><tr><td> Digitale Videokamera </td><td> Basler A602f-2 </td><td></td><td></td></tr><tr><td> Makro-Objektiv für die Kamera </td><td> TECHSPEC VZM 200 </td><td></td><td></td></tr><tr><td> Matlab R2009b </td><td> The MathWorks </td><td></td><td></td></tr></tbody></table>

tactile conditioning and movement analysis of antennal sampling strategies in honey bees (apis mellifera l.)

Honigbienen (Apis mellifera L.) sind eusocial Insekten und bekannt für ihre komplexen Arbeitsteilung und assoziative Lernfähigkeit 1, 2 bekannt. Die Arbeitsbienen verbringen die erste Hälfte ihres Lebens in den dunklen Bienenstock, wo sie Pflege der Larven oder den Bau der regelmäßigen sechseckigen Waben für Lebensmittel (zB Pollen oder Nektar) und Brut 3. Die Antennen sind außergewöhnlich multisensorische Fühlern und spielen eine entscheidende Rolle in verschiedenen taktilen vermittelten Aufgaben 4, einschließlich hive Gebäude 5 und Mustererkennung 6. Später im Leben hinterlässt jedes einzelne Biene den Stock, um nach Nahrung zu suchen. Dann eine Biene muss lernen, profitable Nahrungsquellen zu diskriminieren, zu speichern ihre Lage und kommunizieren sie ihre Nestgefährten 7. Bienen verwenden verschiedene florale Signale wie Farben oder Gerüche 7, 8, sondern auch taktile Hinweise aus der petal Oberfläche 9 bis multisensorische Erinnerungen an die Nahrungsquelle zu bilden. Unter Laborbedingungen conditions, können Bienen in einem appetitive Lernparadigma geschult werden, um taktile Objekteigenschaften, wie Kanten oder Rillen mit ihren Antennen 10, 11, 12, 13 unterscheiden. Diese Lernparadigma ist eng mit dem klassischen olfaktorischen Konditionierung des Rüssels Verlängerung Reaktion (PER) in angeschirrt Bienen 14 verbunden. Der Vorteil der taktilen Lernparadigma im Labor ist die Möglichkeit der Kombination Verhaltensexperimente auf das Lernen mit verschiedenen physiologischen Messungen, einschließlich der Analyse der antennalen Bewegungsmuster.

In dem folgenden Experiment haben wir untersucht, wie taktile Lernen antennalen Probenahme Verhalten von Honigbienen beeinflusst. Hierzu überwachten wir die Bewegung der Spitze in antennalen naiven und konditionierten Bienen vor und während der Darstellung eines taktilen Reiz. Zuerst wurde die spontane antennalen Bewegung einer Gruppe von Pollensammlerinnen (N = 42) für 1 Minute aufgezeichnet. Die eine Hälfte der Bienen (N = 21) wurde dann durch Paarung fünf Mal eine taktile Reiz mit ei...

Watch this Scientific Journal Video about Tactile Conditioning And Movement Analysis Of Antennal Sampling Strategies In Honey Bees Apis mellifera L. at JoVE.com

Tactile Conditioning And Movement Analysis Of Antennal Sampling Strategies In Honey Bees Apis mellifera L.

In diesem Protokoll zeigen wir, wie Bedingung Honigbienen auf taktile Reize genutzt und die Einführung einer 2D-Motion-Capture-Technik zur Analyse der Kinematik feinskaligen antennalen Abtastmuster.

Tactile Conditioning und Bewegungsanalyse der antennalen Sampling Strategies In Honey Bees (<em&gt; Apis mellifera</em&gt; L.)

Honey bees (Apis mellifera L.) are eusocial insects and well known for their complex division of labor and associative learning capability1, 2. The worker ...

tactile-conditioning-movement-analysis-antennal-sampling-strategies

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Tactile Conditioning And Movement Analysis Of Antennal Sampling Strategies In Honey Bees (Apis mellifera L.)

10.5K Aufrufe.  Bielefeld University. In diesem Protokoll zeigen wir, wie Bedingung Honigbienen auf taktile Reize genutzt und die Einführung einer 2D-Motion-Capture-Technik zur Analyse der Kinematik feinskaligen antennalen Abtastmuster.

Serie: Tactile Conditioning And Movement Analysis Of Antennal Sampling Strategies In Honey Bees Apis mellifera L.

Behavioural Pharmacology in Classical Conditioning of the Proboscis Extension Response in Honeybees (Apis mellifera)

Honeybees (Apis mellifera) are well known for their communication and orientation skills and for their impressive learning capability1,2. Because the survival of a honeybee colony depends on the exploitation of food sources, forager bees learn and memorize variable flower sites as well as their profitability. Forager bees can be easily trained in natural settings where they forage at a feeding site and learn the related signals such as odor or color. Appetitive associative learning can also be studied under controlled conditions in the laboratory by conditioning the proboscis extension response (PER) of individually harnessed honeybees3,4. This learning paradigm enables the study of the neuronal and molecular mechanisms that underlie learning and memory formation in a simple and highly reliable way5-12. A behavioral pharmacology approach is used to study molecular mechanisms. Drugs are injected systemically to interfere with the function of specific molecules during or after learning and memory formation13-16.
Here we demonstrate how to train harnessed honeybees in PER conditioning and how to apply drugs systemically by injection into the bee flight muscle.

Behavioural Pharmacology in Classical Conditioning of the Proboscis Extension Response in Honeybees Apis mellifera

We demonstrate how to implement a behavioral pharmacology method in an appetitive olfactory conditioning paradigm in honeybees (Apis mellifera) by systemic application of drugs. This method allows investigation of the mechanisms underlying learning and memory formation in a simple and reliable way.

Behavioural Pharmacology in Classical Conditioning der Proboscis Erweiterung Reaktionszeit in Honigbienen ( Apis mellifera)

Honeybees (Apis mellifera) are well known for their communication and orientation skills and for their impressive learning capability1,2. Because the ...

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Automated Sholl Analysis of Digitized Neuronal Morphology at Multiple Scales

Neuronal morphology plays a significant role in determining how neurons function and communicate1-3. Specifically, it affects the ability of neurons to receive inputs from other cells2 and contributes to the propagation of action potentials4,5. The morphology of the neurites also affects how information is processed. The diversity of dendrite morphologies facilitate local and long range signaling and allow individual neurons or groups of neurons to carry out specialized functions within the neuronal network6,7. Alterations in dendrite morphology, including fragmentation of dendrites and changes in branching patterns, have been observed in a number of disease states, including Alzheimer's disease8, schizophrenia9,10, and mental retardation11. The ability to both understand the factors that shape dendrite morphologies and to identify changes in dendrite morphologies is essential in the understanding of nervous system function and dysfunction.
Neurite morphology is often analyzed by Sholl analysis and by counting the number of neurites and the number of branch tips. This analysis is generally applied to dendrites, but it can also be applied to axons. Performing this analysis by hand is both time consuming and inevitably introduces variability due to experimenter bias and inconsistency. The Bonfire program is a semi-automated approach to the analysis of dendrite and axon morphology that builds upon available open-source morphological analysis tools. Our program enables the detection of local changes in dendrite and axon branching behaviors by performing Sholl analysis on subregions of the neuritic arbor. For example, Sholl analysis is performed on both the neuron as a whole as well as on each subset of processes (primary, secondary, terminal, root, etc.) Dendrite and axon patterning is influenced by a number of intracellular and extracellular factors, many acting locally. Thus, the resulting arbor morphology is a result of specific processes acting on specific neurites, making it necessary to perform morphological analysis on a smaller scale in order to observe these local variations12.
The Bonfire program requires the use of two open-source analysis tools, the NeuronJ plugin to ImageJ and NeuronStudio. Neurons are traced in ImageJ, and NeuronStudio is used to define the connectivity between neurites. Bonfire contains a number of custom scripts written in MATLAB (MathWorks) that are used to convert the data into the appropriate format for further analysis, check for user errors, and ultimately perform Sholl analysis. Finally, data are exported into Excel for statistical analysis. A flow chart of the Bonfire program is shown in Figure 1.

We have developed a computer program to analyze neuronal morphology. In combination with two existing open source analysis tools, our program performs Sholl analysis and determines the number of neurites, branch points, and neurite tips. The analyses are performed so that local changes in neurite morphology can be observed.

Automatisierte Sholl Analyse digitalisierter neuronale Morphologie bei Multiple Scales

Neuronal morphology plays a significant role in determining how neurons function and communicate1-3.  Specifically, it affects the ability of neurons to ...

The Culture of Primary Motor and Sensory Neurons in Defined Media on Electrospun Poly-L-lactide Nanofiber Scaffolds

Electrospinning is a technique for producing micro- to nano-scale fibers. Fibers can be electrospun with varying degrees of alignment, from highly aligned to completely random. In addition, fibers can be spun from a variety of materials, including biodegradable polymers such as poly-L-lactic acid (PLLA). These characteristics make electrospun fibers suitable for a variety of scaffolding applications in tissue engineering. Our focus is on the use of aligned electrospun fibers for nerve regeneration. We have previously shown that aligned electrospun PLLA fibers direct the outgrowth of both primary sensory and motor neurons in vitro. We maintain that the use of a primary cell culture system is essential when evaluating biomaterials to model real neurons found in vivo as closely as possible. Here, we describe techniques used in our laboratory to electrospin fibrous scaffolds and culture dorsal root ganglia explants, as well as dissociated sensory and motor neurons, on electrospun scaffolds. However, the electrospinning and/or culture techniques presented here are easily adapted for use in other applications.

Aligned electrospun fibers direct the growth of neurons in vitro and are a potential component of nerve regeneration scaffolds. We describe a procedure for preparing electrospun fiber substrates and the serum-free culture of primary rat E15 sensory (DRG) and motor neurons. Visualization of neurons by immunocytochemistry is also included.

The Culture of Primary Motor und sensorischen Neuronen in definierten Medien auf Elektrogesponnene Poly-L-Lactid Nanofiber Gerüste

Electrospinning is a technique for producing micro- to nano-scale fibers. Fibers can be electrospun with varying degrees of alignment, from highly aligned ...

Calcium Imaging of Odor-evoked Responses in the Drosophila Antennal Lobe

The antennal lobe is the primary olfactory center in the insect brain and represents the anatomical and functional equivalent of the vertebrate olfactory bulb1-5. Olfactory information in the external world is transmitted to the antennal lobe by olfactory sensory neurons (OSNs), which segregate to distinct regions of neuropil called glomeruli according to the specific olfactory receptor they express. Here, OSN axons synapse with both local interneurons (LNs), whose processes can innervate many different glomeruli, and projection neurons (PNs), which convey olfactory information to higher olfactory brain regions.
Optical imaging of the activity of OSNs, LNs and PNs in the antennal lobe - traditionally using synthetic calcium indicators (e.g. calcium green, FURA-2) or voltage-sensitive dyes (e.g. RH414) - has long been an important technique to understand how olfactory stimuli are represented as spatial and temporal patterns of glomerular activity in many species of insects6-10. Development of genetically-encoded neural activity reporters, such as the fluorescent calcium indicators G-CaMP11,12 and Cameleon13, the bioluminescent calcium indicator GFP-aequorin14,15, or a reporter of synaptic transmission, synapto-pHluorin16 has made the olfactory system of the fruitfly, Drosophila melanogaster, particularly accessible to neurophysiological imaging, complementing its comprehensively-described molecular, electrophysiological and neuroanatomical properties2,4,17. These reporters can be selectively expressed via binary transcriptional control systems (e.g. GAL4/UAS18, LexA/LexAop19,20, Q system21) in defined populations of neurons within the olfactory circuitry to dissect with high spatial and temporal resolution how odor-evoked neural activity is represented, modulated and transformed22-24.
Here we describe the preparation and analysis methods to measure odor-evoked responses in the Drosophila antennal lobe using G-CaMP25-27. The animal preparation is minimally invasive and can be adapted to imaging using wide-field fluorescence, confocal and two-photon microscopes.

Calcium Imaging of Odor-evoked Responses in the Drosophila Antennal Lobe

We describe an established technique to measure and analyze odor-evoked calcium responses in the antennal lobe of living Drosophila melanogaster.

Calcium Imaging von geruchs-evozierte Potentiale in der Drosophila Antennallobus

The antennal lobe is the primary olfactory center in the insect brain and represents the anatomical and functional equivalent of the vertebrate olfactory ...

Electrophysiological Measurements and Analysis of Nociception in Human Infants

Pain is an unpleasant sensory and emotional experience. Since infants cannot verbally report their experiences, current methods of pain assessment are based on behavioural and physiological body reactions, such as crying, body movements or changes in facial expression. While these measures demonstrate that infants mount a response following noxious stimulation, they are limited: they are based on activation of subcortical somatic and autonomic motor pathways that may not be reliably linked to central sensory processing in the brain. Knowledge of how the central nervous system responds to noxious events could provide an insight to how nociceptive information and pain is processed in newborns.
The heel lancing procedure used to extract blood from hospitalised infants offers a unique opportunity to study pain in infancy. In this video we describe how electroencephalography (EEG) and electromyography (EMG) time-locked to this procedure can be used to investigate nociceptive activity in the brain and spinal cord.
This integrative approach to the measurement of infant pain has the potential to pave the way for an effective and sensitive clinical measurement tool.

The assessment and treatment of pain in infants is difficult because infants cannot verbally report their experience. In this video we describe quantitative electrophysiological methods and analysis techniques that can be used to measure the response to noxious events from the infant nervous system.

Elektrophysiologische Messungen und Analyse von Nozizeption in Human Infants

Pain is an unpleasant sensory and emotional experience. Since infants cannot verbally report their experiences, current methods of pain assessment are ...

Rapid Determination of the Thermal Nociceptive Threshold in Diabetic Rats

Painful diabetic neuropathy (PDN) is characterized by hyperalgesia i.e., increased sensitivity to noxious stimulus, and allodynia i.e., hypersensitivity to normally innocuous stimuli1. Hyperalgesia and allodynia have been studied in many different rodent models of diabetes mellitus2. However, as stated by B&ouml;lcskei et al, determination of "pain" in animal models is challenging due to its subjective nature3. Moreover, the traditional methods used to determine behavioral responses to noxious thermal stimuli usually lack reproducibility and pharmacological sensitivity3. For instance, by using the hot-plate method of Ankier4, flinch, withdrawal and/or licking of either hind- and/or fore-paws is quantified as reflex latencies at constant high thermal stimuli (52-55 &deg;C). However, animals that are hyperalgesic to thermal stimulus do not reproducibly show differences in reflex latencies using those supra-threshold temperatures3,5. As the recently described method of B&ouml;lcskei et al.6, the procedures described here allows for the rapid, sensitive and reproducible determination of thermal nociceptive thresholds (TNTs) in mice and rats. The method uses slowly increasing thermal stimulus applied mostly to the skin of mouse/rat plantar surface. The method is particularly sensitive to study anti-nociception during hyperalgesic states such as PDN. The procedures described bellow are based on the ones published in detail by Alm&aacute;si et al 5 and B&ouml;lcskei et al 3. The procedures described here have been approved the Laboratory Animal Care and Use Committee (LACUC), Wright State University.

Here, we describe a rapid reliable and simple procedure to determine the lowest temperature at which rats or mice show nocifensive behavior, i.e. the thermal nociceptive threshold (TNT). This method applies a slowly increasing thermal stimulus allowing precise and reproducible estimation of TNTs with minimum, if any, stress to the animals.

Schnelle Bestimmung der Wärmeleistung und nozizeptiven Grenzwerts bei diabetischen Ratten

Painful diabetic neuropathy (PDN) is characterized by hyperalgesia i.e., increased sensitivity to noxious stimulus, and allodynia i.e., hypersensitivity ...

RNAi-mediated Double Gene Knockdown and Gustatory Perception Measurement in Honey Bees (Apis mellifera)

This video demonstrates novel techniques of RNA interference (RNAi) which downregulate two genes simultaneously in honey bees using double-stranded RNA (dsRNA) injections. It also presents a protocol of proboscis extension response (PER) assay for measuring gustatory perception.	
	RNAi-mediated gene knockdown is an effective technique downregulating target gene expression. This technique is usually used for single gene manipulation, but it has limitations to detect interactions and joint effects between genes. In the first part of this video, we present two strategies to simultaneously knock down two genes (called double gene knockdown). We show both strategies are able to effectively suppress two genes, vitellogenin (vg) and ultraspiracle (usp), which are in a regulatory feedback loop. This double gene knockdown approach can be used to dissect interrelationships between genes and can be readily applied in different insect species.	
	The second part of this video is a demonstration of proboscis extension response (PER) assay in honey bees after the treatment of double gene knockdown. The PER assay is a standard test for measuring gustatory perception in honey bees, which is a key predictor for how fast a honey bee's behavioral maturation is. Greater gustatory perception of nest bees indicates increased behavioral development which is often associated with an earlier age at onset of foraging and foraging specialization in pollen. In addition, PER assay can be applied to identify metabolic states of satiation or hunger in honey bees. Finally, PER assay combined with pairing different odor stimuli for conditioning the bees is also widely used for learning and memory studies in honey bees.

RNAi-mediated Double Gene Knockdown and Gustatory Perception Measurement in Honey Bees Apis mellifera

In this protocol, we describe two strategies that simultaneously suppress two genes (double gene knockdown) in honey bees. Then we present how to use the proboscis extension response (PER) assay to study the effect of double gene knockdown on honey bee gustatory perception.

RNAi-vermittelte Gene Knockdown Doppelzimmer und Gustatorisch Perception Measurement in Honey Bees (<em&gt; Apis mellifera</em&gt;)

This video demonstrates novel techniques of RNA  interference (RNAi) which downregulate two genes simultaneously in honey bees  using double-stranded RNA ...

Neuropharmacological Manipulation of Restrained and Free-flying Honey Bees, Apis mellifera

Honey bees demonstrate astonishing learning abilities and advanced social behavior and communication. In addition, their brain is small, easy to visualize and to study. Therefore, bees have long been a favored model amongst neurobiologists and neuroethologists for studying the neural basis of social and natural behavior. It is important, however, that the experimental techniques used to study bees do not interfere with the behaviors being studied. Because of this, it has been necessary to develop a range of techniques for pharmacological manipulation of honey bees. In this paper we demonstrate methods for treating restrained or free-flying honey bees with a wide range of pharmacological agents. These include both noninvasive methods such as oral and topical treatments, as well as more invasive methods that allow for precise drug delivery in either systemic or localized fashion. Finally, we discuss the advantages and disadvantages of each method and&#160;describe common hurdles and how to best overcome them. We conclude with a discussion on the importance of adapting the experimental method to the biological questions rather than the other way around.

Neuropharmacological Manipulation of Restrained and Free-flying Honey Bees, Apis mellifera

This manuscript describes several protocols for administering pharmacological agents to honey bees, including simple noninvasive methods for free-flying bees, as well as more invasive variants that allow precise localized treatment of restrained bees.

Neuropharmakologische Manipulation von Verhaltene und Free-Fliegen-Honig-Bienen,<em&gt; Apis mellifera</em

Honey bees demonstrate astonishing learning abilities and advanced social behavior and communication. In addition, their brain is small, easy to visualize ...

The Use of Trace Eyeblink Classical Conditioning to Assess Hippocampal Dysfunction in a Rat Model of Fetal Alcohol Spectrum Disorders

Neonatal rats were administered a relatively high concentration of ethyl alcohol (11.9% v/v) during postnatal days 4-9, a time when the fetal brain undergoes rapid organizational change and is similar to accelerated brain changes that occur during the third trimester in humans. This model of fetal alcohol spectrum disorders (FASDs) produces severe brain damage, mimicking the amount and pattern of binge-drinking that occurs in some pregnant alcoholic mothers. We describe the use of trace eyeblink classical conditioning (ECC), a higher-order variant of associative learning, to assess long-term hippocampal dysfunction that is typically seen in alcohol-exposed adult offspring. At 90 days of age, rodents were surgically prepared with recording and stimulating electrodes, which measured electromyographic (EMG) blink activity from the left eyelid muscle and delivered mild shock posterior to the left eye, respectively. After a 5 day recovery period, they underwent 6 sessions of trace ECC to determine associative learning differences between alcohol-exposed and control rats. Trace ECC is one of many possible ECC procedures that can be easily modified using the same equipment and software, so that different neural systems can be assessed. ECC procedures in general, can be used as diagnostic tools for detecting neural pathology in different brain systems and different conditions that insult the brain.

Trace eyeblink classical conditioning (ECC) was used to assess hippocampal-dependent associative learning in adult rats that were administered a high concentration (11.9% v/v) of alcohol during early neonatal brain development. In general, ECC procedures are sound diagnostic tools for detecting brain dysfunction across many psychological and biomedical settings.

Die Verwendung von Trace Eyeblink klassische Konditionierung hippocampale Funktionsstörung in einem Rattenmodell der Fetal Alcohol Spectrum Disorders bewerten

Neonatal rats were administered a relatively high concentration of ethyl alcohol (11.9% v/v) during postnatal days 4-9, a time when the fetal brain ...

Drosophila Courtship Conditioning As a Measure of Learning and Memory

Many insights into the molecular mechanisms underlying learning and memory have been elucidated through the use of simple behavioral assays in model organisms such as the fruit fly, Drosophila melanogaster. Drosophila is useful for understanding the basic neurobiology underlying cognitive deficits resulting from mutations in genes associated with human cognitive disorders, such as intellectual disability (ID) and autism. This work describes a methodology for testing learning and memory using a classic paradigm in Drosophila known as courtship conditioning. Male flies court females using a distinct pattern of easily recognizable behaviors. Premated females are not receptive to mating and will reject the male&#39;s copulation attempts. In response to this rejection, male flies reduce their courtship behavior. This learned reduction in courtship behavior is measured over time, serving as an indicator of learning and memory. The basic numerical output of this assay is the courtship index (CI), which is defined as the percentage of time that a male spends courting during a 10 min interval. The learning index (LI) is the relative reduction of CI in flies that have been exposed to a premated female compared to na&#239;ve flies with no previous social encounters. For the statistical comparison of LIs between genotypes, a randomization test with bootstrapping is used. To illustrate how the assay can be used to address the role of a gene relating to learning and memory, the pan-neuronal knockdown of Dihydroxyacetone phosphate acyltransferase (Dhap-at) was characterized here. The human ortholog of Dhap-at, glyceronephosphate O-acyltransferase (GNPT), is involved in rhizomelic chondrodysplasia punctata type 2, an autosomal-recessive syndrome characterized by severe ID. Using the courtship conditioning assay, it was determined that Dhap-at is required for long-term memory, but not for short-term memory. This result serves as a basis for further investigation of the underlying molecular mechanisms.

Drosophila Courtship Conditioning As a Measure of Learning and Memory

This protocol describes a Drosophila learning and memory assay called courtship conditioning. This classic assay is based on a reduction of male courtship behavior after sexual rejection by a non-receptive premated female. This natural form of behavioral plasticity can be used to test learning, short-term memory, and long-term memory.

Drosophila Courtship Conditioning Als Maß für Lernen und Gedächtnis

Many insights into the molecular mechanisms underlying learning and memory have been elucidated through the use of simple behavioral assays in model ...