This project was funded by ARC grant DP0986021 and NHMRC grant 585442. ABB is supported by an ARC Future Fellowship (FT140100452). JAP is supported by an iMQRES scholarship awarded by Macquarie University and by a DAAD-Doktorandenstipendium awarded by the German Academic Exchange Service. JMD is supported by CNRS and University Paul Sabatier.

<ol>
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The authors have nothing to disclose.

Die Methoden oben skizzierten ermöglichen eine einfache, effektive und robuste Behandlung von entweder freifliegenden oder spannte Honigbienen. Diese Verfahren sind kompatibel mit vielen experimentellen Paradigmen und biologischen Fragen (Tabelle 1). Alle der freifliegenden Methoden leicht nutzbar Bienen angewendet werden kann. Das Gegenteil ist weniger erfolgreich, da jedoch vorübergehende Zurückhaltung und invasive Behandlungsmethoden oft Bienenflugfähigkeit beeinträchtigen können. <p class=...

Seit Karl von Frisch ihre Tanzsprache aufgeklärt 1, Honigbienen sind eine populäre Studie Arten für die Forscher das Verhalten der Tiere und der Neurobiologie geblieben. In den letzten Jahren an der Schnittstelle zwischen diesen beiden Bereichen und mehreren anderen Disziplinen (zB Molekularbiologie, Genomik und Informatik) haben neben ihnen entstanden entstanden eine Vielzahl von neuen Disziplinen. Dies hat zu einer raschen Entwicklung neuer Theorien und Modelle für das Verständnis, wie das Verhalten von Aktivität führt innerhalb des Nervensystems. Aufgrund der einzigartigen Lebensstil, reiche Verhaltensrepertoire und Leichtigkeit der experimentellen und pharmakologische Manipulation, Bienen haben an der Spitze dieser Revolution blieb. Honigbienen werden verwendet neurobiologischen Grund Fragen zu untersuchen, wie die zugrunde liegenden Lernen und Gedächtnis 2,3, Entscheidung 4 zu machen, Riech 5 oder visuelle Verarbeitung 6. In den letzten Jahren die hon11, schlafen 12, Alterung 13 oder die zugrunde liegenden Mechanismen der Anästhesie 14 - ey Biene hat für die medizinische Forschung, wie die Auswirkungen von Suchtmitteln im Allgemeinen reserviert sogar bereits 7 für das Studium Themen als Modell verwendet. Im Gegensatz zu den klassischen genetischen Modellorganismen (zB D. melanogaster, C. elegans, M. musculus), gibt es nur sehr wenige genetische Werkzeuge zur Verfügung , für die Manipulation von neuronalen Funktionen in Honigbienen, obwohl dies derzeit 15 ändern. Stattdessen haben Studien Honigbiene in erster Linie auf pharmakologische Manipulationen verlassen. Dies war sehr erfolgreich; jedoch ist die Vielfalt der Bienenforschung, so dass eine Reihe von Methoden für die pharmakologische Verabreichung benötigt werden. Forschung mit Honigbienen befasst sich sehr unterschiedliche Fragen, wird von Forschern aus verschiedenen Disziplinen und Hintergründe untersucht und verwendet eine Vielzahl von experimentellen Ansätzen. Viele ReseBogen Fragen erfordern Bienen entweder frei fliegenden sein, frei in ihrer Kolonie interagieren, oder beides. Dies kann es den Überblick über einzelne Versuchstiere machen schwer zu halten und macht Zurückhaltung oder Kanülierung nicht machbar. Um die Vielfalt von Bienenforschung aufzunehmen, eine Vielzahl von Drug-Delivery-Methoden benötigt werden, so dass für eine robuste und flexible Verwaltung, während sichergestellt wird, dass die pharmakokinetischen und pharmakodynamischen Profile, Invasivität des Verfahrens und seine Zuverlässigkeit, das Paradigma in Frage entsprechen. Aufgrund dieser unterschiedlichen Bedürfnisse haben die meisten Forschungsgruppen ihre eigenen einzigartigen Wirkstoffverabreichung Methoden entwickelt. Bisher hat dies eine Stärke der Bienenforschungsgemeinschaft gewesen; für die Verabreichung desselben Arzneimittels in verschiedenen Umständen es ermöglicht die Entwicklung von Anordnungen von Methoden geführt. Unser Ziel ist es nicht ein einziges standardisiertes Verfahren für die pharmakologische Manipulationen von Bienen zu entwickeln, sondern Methoden zu betonen, dasshaben sich als besonders erfolgreich zu sein, und helfen den Forschern übernehmen diese. Wir diskutieren die grundlegenden Prinzipien, wie sie arbeiten, sowie ihre Vor- und Nachteile.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Sucrose</td>
			<td>Sigma-Aldrich</td>
			<td>S8501</td>
			<td>Any supplier will do</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>S7653</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>P9333&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium Chloride hexahydrate</td>
			<td>Sigma-Aldrich</td>
			<td>M2670</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Chloride dihydrate</td>
			<td>Sigma-Aldrich</td>
			<td>C8106</td>
			<td></td>
		</tr>
		<tr>
			<td>Dextrose monohydrate</td>
			<td>Sigma-Aldrich</td>
			<td>49159</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffer Saline (PBS)</td>
			<td>Sigma-Aldrich</td>
			<td>P4417</td>
			<td></td>
		</tr>
		<tr>
			<td>Protection Wax</td>
			<td>Dentaurum</td>
			<td>124-305-00</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr>
			<td>dimethylformamide</td>
			<td>Sigma-Aldrich</td>
			<td>D4551</td>
			<td></td>
		</tr>
		<tr>
			<td>95% Ethanol</td>
			<td>Sigma-Aldrich</td>
			<td>493511</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass capillary</td>
			<td>WPI</td>
			<td>1B100F-3</td>
			<td></td>
		</tr>
		<tr>
			<td>23 G NanoFil needle</td>
			<td>WPI</td>
			<td>NF33BV-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Very fine forsceps</td>
			<td>Dumont</td>
			<td>0208-55-PO</td>
			<td></td>
		</tr>
		<tr>
			<td>Electrode puller</td>
			<td>SRI</td>
			<td>2001</td>
			<td></td>
		</tr>
		<tr>
			<td>FemtoJet Microinjector</td>
			<td>Eppendorf</td>
			<td>5247 000.01</td>
			<td></td>
		</tr>
		<tr>
			<td>Eicosane</td>
			<td>Sigma-Aldrich</td>
			<td>219274</td>
			<td></td>
		</tr>
		<tr>
			<td>manual micromanipulator</td>
			<td>Brinkmann Instrumentenbau</td>
			<td>MM-33</td>
			<td></td>
		</tr>
		<tr>
			<td>electronic micromanipulator</td>
			<td>Luigs &#38; Neumann Feinmechanik + Elektortechnik</td>
			<td>Junior unit XYZ</td>
			<td></td>
		</tr>
		<tr>
			<td>stereomicroscope</td>
			<td>Leica</td>
			<td>M80</td>
			<td></td>
		</tr>
		<tr>
			<td>soldering iron</td>
			<td>Weller</td>
			<td>WESD51</td>
			<td></td>
		</tr>
		<tr>
			<td>Dextran, Alexa Fluor 546, 10000 MW</td>
			<td>ThermoFisher Scientific</td>
			<td>D-22911</td>
			<td></td>
		</tr>
		<tr>
			<td>Dextran, Alexa Fluor 568, 10000 MW</td>
			<td>ThermoFisher Scientific</td>
			<td>D-22912</td>
			<td></td>
		</tr>
		<tr>
			<td>small Petri dish</td>
			<td>Sigma-Aldrich</td>
			<td>P5481</td>
			<td></td>
		</tr>
		<tr>
			<td>mineral oil</td>
			<td>Sigma-Aldrich</td>
			<td>M5904</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL Centrifuge tube</td>
			<td>ThermoFisher Scientific</td>
			<td>339652</td>
			<td></td>
		</tr>
		<tr>
			<td>forceps</td>
			<td>Australian Entomological Supplies</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Blade holder and breaker</td>
			<td>Australian Entomological Supplies</td>
			<td>E130</td>
			<td></td>
		</tr>
		<tr>
			<td>Feather double edged razor blade</td>
			<td>ThermoFisher Scientific</td>
			<td>50-949-135</td>
			<td></td>
		</tr>
		<tr>
			<td>Nichrome wire</td>
			<td></td>
			<td></td>
			<td>Any supplier will do</td>
		</tr>
		<tr>
			<td>Electrical wires</td>
			<td></td>
			<td></td>
			<td>Any supplier will do</td>
		</tr>
		<tr>
			<td>Model paint</td>
			<td>Tamiya USA</td>
			<td>Depends on colour</td>
			<td></td>
		</tr>
		<tr>
			<td>Repeating dispenser</td>
			<td>Hamilton company</td>
			<td>PB-600-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass syringe</td>
			<td>WPI</td>
			<td>NANOFIL</td>
			<td></td>
		</tr>
		<tr>
			<td>flourescence viewing system</td>
			<td>Nightsea</td>
			<td>SFR-GR</td>
			<td></td>
		</tr>
		<tr>
			<td>graticule</td>
			<td>ProSciTech</td>
			<td>S8014-24</td>
			<td></td>
		</tr>
		<tr>
			<td>microcapillary with holder</td>
			<td>Drummond</td>
			<td>1-000-0010</td>
			<td></td>
		</tr>
		<tr>
			<td>Liquid silicone</td>
			<td></td>
			<td></td>
			<td>Any supplier will do</td>
		</tr>
		<tr>
			<td>Thermocouple</td>
			<td>Digitech</td>
			<td>QM-1324</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

neuropharmacological manipulation of restrained and free-flying honey bees, apis mellifera

Honigbienen zeigen erstaunliche Lernfähigkeiten und fortschrittliche Sozialverhalten und Kommunikation. Außerdem ihr Gehirn ist klein, leicht zu visualisieren und zu untersuchen. Aus diesem Grund haben die Bienen schon lange ein beliebtes Modell unter Neurobiologie und Neuroethologen für die neuronalen Grundlagen der sozialen und natürlichen Verhalten zu studieren. Es ist jedoch wichtig, dass die experimentellen Techniken Bienen zu studieren interferieren nicht mit dem Verhalten untersucht. Aus diesem Grund war es notwendig, eine Reihe von Techniken für die pharmakologische Manipulation von Honigbienen zu entwickeln. In diesem Beitrag zeigen wir Methoden zur Behandlung von zurückhaltender oder frei fliegenden Honigbienen mit einem breiten Spektrum von pharmakologischen Wirkstoffen. Dazu gehören sowohl nicht-invasive Methoden wie orale und topische Behandlungen, sowie mehr invasive Methoden, die entweder systemisch oder lokalisierte Art und Weise für präzise Medikamentenabgabe zu ermöglichen. Schließlich diskutieren wir die Vor- und Nachteile der einzelnen Methoden und beschreibengemeinsame Hürden und wie man sie am besten überwinden. Wir schließen mit einer Diskussion über die Bedeutung der experimentellen Methode auf die biologischen Fragen der Anpassung und nicht umgekehrt.

Eine Auswahl von repräsentativen Ergebnissen für die oben beschriebenen Verfahren gezeigt sind, in erster Linie zu zeigen, dass die Verfahren pharmakologische Mittel erlauben das Gehirn und beeinflussen honey bee Verhalten zu erreichen. Spezifische Auswirkungen auf die Gehirnprozesse können leicht folgende Thorax Injektion erhalten werden. Da pharmakologische Wirkst...

Watch this Scientific Journal Video about Neuropharmacological Manipulation of Restrained and Free-flying Honey Bees, Apis mellifera at JoVE.com

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

Dieses Manuskript beschreibt mehrere Protokolle für pharmakologische Mittel zur Verabreichung von Honigbienen, einschließlich einfacher nicht-invasive Methoden zur freifliegenden Bienen, sowie mehr invasive Varianten, die eine präzise lokalisierte Behandlung von zurückhaltender Bienen ermöglichen.

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 ...

neuropharmacological-manipulation-restrained-free-flying-honey-bees

Research

JoVE Journal

Neuroscience

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

9.8K Aufrufe.  Volda University College.  Dieses Manuskript beschreibt mehrere Protokolle für pharmakologische Mittel zur Verabreichung von Honigbienen, einschließlich einfacher nicht-invasive Methoden zur freifliegenden Bienen, sowie mehr invasive Varianten, die eine präzise lokalisierte Behandlung von zurückhaltender Bienen ermöglichen. 

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

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 ...

Forschung

Neurowissenschaften

Mechanical Manipulation of Neurons to Control Axonal Development

Cell manipulations and extension of neuronal axons can be accomplished with calibrated glass micro-fibers capable of measuring and applying forces in the 10-1000 &mu;dyne range1,2. Force measurements are obtained through observation of the Hookean bending of the glass needles, which are calibrated by a direct and empirical method3. Equipment requirements and procedures for fabricating, calibrating, treating, and using the needles on cells are fully described. The force regimes previously used and different cell types to which these techniques have been applied demonstrate the flexibility of the methodology and are given as examples for future investigation4-6. The technical advantages are the continuous 'visualization' of the forces produced by the manipulations and the ability to directly intervene in a variety of cellular events. These include direct stimulation and regulation of axonal growth and retraction7; as well as detachment and mechanical measurements on any type of cultured cell8.

Application and direct measurements of forces on neurons in the 2-1000 microdyne range are achieved with high precision using calibrated glass needles. This methodology can be used to control and measure several aspects of axonal development, including axonal initiation, axonal tension, velocity of axonal elongation, and force vectors.

Mechanische Manipulation von Neuronen zu Axonal Entwicklungssteuerung

Cell manipulations and extension of neuronal axons can be accomplished with calibrated glass micro-fibers capable of measuring and applying forces in the ...

Lentivirus-mediated Genetic Manipulation and Visualization of Olfactory Sensory Neurons in vivo

Development of a precise olfactory circuit relies on accurate projection of olfactory sensory neuron (OSN) axons to their synaptic 
targets in the olfactory bulb (OB). The molecular mechanisms of OSN axon growth and targeting are not well understood. Manipulating gene expression and subsequent 
visualizing of single OSN axons and their terminal arbor morphology have thus far been challenging. To study gene function at the single cell level within a specified 
time frame, we developed a lentiviral based technique to manipulate gene expression in OSNs in vivo. Lentiviral particles are delivered to OSNs by microinjection 
into the olfactory epithelium (OE). Expression cassettes are then permanently integrated into the genome of transduced OSNs. Green fluorescent protein expression 
identifies infected OSNs and outlines their entire morphology, including the axon terminal arbor. Due to the short turnaround time between microinjection and 
reporter detection, gene function studies can be focused within a very narrow period of development. With this method, we have detected GFP expression within as 
few as three days and as long as three months following injection. We have achieved both over-expression and shRNA mediated knock-down by lentiviral microinjection. 
This method provides detailed morphologies of OSN cell bodies and axons at the single cell level in vivo, and thus allows characterization of candidate gene function 
during olfactory development.

Lentivirus-mediated Genetic Manipulation and Visualization of Olfactory Sensory Neurons in vivo

We present a lentiviral technique for genetic manipulation and visualization of single olfactory sensory neuron axon and its terminal arborization in vivo.

Lentivirus-vermittelte Gen-Manipulation und Visualisierung von Riechzellen In vivo</em

Development of a precise olfactory circuit relies on accurate projection of olfactory sensory neuron (OSN) axons to their synaptic 
targets in the ...

Tactile Conditioning And Movement Analysis Of Antennal Sampling Strategies In Honey Bees (Apis mellifera 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 bees spend the first half of their life inside the dark hive, where they are nursing the larvae or building the regular hexagonal combs for food (e.g. pollen or nectar) and brood3. The antennae are extraordinary multisensory feelers and play a pivotal role in various tactile mediated tasks4, including hive building5 and pattern recognition6. Later in life, each single bee leaves the hive to forage for food. Then a bee has to learn to discriminate profitable food sources, memorize their location, and communicate it to its nest mates7. Bees use different floral signals like colors or odors7, 8, but also tactile cues from the petal surface9 to form multisensory memories of the food source. Under laboratory conditions, bees can be trained in an appetitive learning paradigm to discriminate tactile object features, such as edges or grooves with their antennae10, 11, 12, 13. This learning paradigm is closely related to the classical olfactory conditioning of the proboscis extension response (PER) in harnessed bees14. The advantage of the tactile learning paradigm in the laboratory is the possibility of combining behavioral experiments on learning with various physiological measurements, including the analysis of the antennal movement pattern.

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

In this protocol we show how to condition harnessed honey bees to tactile stimuli and introduce a 2D motion capture technique for analyzing the kinematics of fine-scale antennal sampling pattern.

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 ...

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

Genetic modification of specific regions of the developing mammalian brain is a very powerful experimental approach. However, generating novel mouse mutants is often frustratingly slow. It has been shown that access to the mouse brain developing in utero with reasonable post-operatory survival is possible. Still, results with this procedure have been reported almost exclusively for the most superficial and easily accessible part of the developing brain, i.e. the cortex. The thalamus, a narrower and more medial region, has proven more difficult to target. Transfection into deeper nuclei, especially those of the hypothalamus, is perhaps the most challenging and therefore very few results have been reported. Here we demonstrate a procedure to target the entire hypothalamic neuroepithelium or part of it (hypothalamic regions) for transfection through electroporation. The keys to our approach are longer narcosis times, injection in the third ventricle, and appropriate kind and positioning of the electrodes. Additionally, we show results of targeting and subsequent histological analysis of the most recessed hypothalamic nucleus, the mammillary body.

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

Despite the functional and medical importance of the hypothalamus, in utero genetic manipulation of its development has rarely been attempted. We show a detailed procedure for in utero electroporation into the mouse hypothalamus and show representative results of total and partial (regional) hypothalamic transfection.

Genetische Manipulation der Maus entwickeln Hypothalamus durch<em&gt; In utero</em&gt; Elektroporation

Genetic  modification of specific regions of the developing mammalian brain is a very  powerful experimental approach. However, generating novel mouse ...

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 ...

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow for an in-depth characterization to identify pathways involved in these events. Cerebellar granule neurons (CGNs) that are derived from the developing cerebellum are an ideal model system that allows for morphological analyses. Here, we describe a method of how to genetically manipulate CGNs and how to study axono- and dendritogenesis of individual neurons. With this method the effects of RNA interference, overexpression or small molecules can be compared to control neurons. In addition, the rodent cerebellar cortex is an easily accessible in vivo system owing to its predominant postnatal development. We also present an in vivo electroporation technique to genetically manipulate the developing cerebella and describe subsequent cerebellar analyses to assess neuronal morphology and migration.

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Neuronal morphogenesis and migration are crucial events underlying proper brain development. Here, we describe methods to genetically manipulate cultured cerebellar granule neurons and the developing cerebellum for the assessment of morphology and migratory characteristics of neurons.

Genetische Manipulation von Kleinhirnkörnerzellen<em&gt; In-vitro-</em&gt; Und<em&gt; In Vivo</em&gt;, Um neuronale Morphologie und Migration Studieren

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow ...

Juxtacellular Monitoring and Localization of Single Neurons within Sub-cortical Brain Structures of Alert, Head-restrained Rats

There are a variety of techniques to monitor extracellular activity of single neuronal units. However, monitoring this activity from deep brain structures in behaving animals remains a technical challenge, especially if the structures must be targeted stereotaxically. This protocol describes convenient surgical and electrophysiological techniques that maintain the animal&#8217;s head in the stereotaxic plane and unambiguously isolate the spiking activity of single neurons. The protocol combines head restraint of alert rodents, juxtacellular monitoring with micropipette electrodes, and iontophoretic dye injection to identify the neuron location in post-hoc histology. While each of these techniques is in itself well-established, the protocol focuses on the specifics of their combined use in a single experiment. These neurophysiological and neuroanatomical techniques are combined with behavioral monitoring. In the present example, the combined techniques are used to determine how self-generated vibrissa movements are encoded in the activity of neurons within the somatosensory thalamus. More generally, it is straightforward to adapt this protocol to monitor neuronal activity in conjunction with a variety of behavioral tasks in rats, mice, and other animals. Critically, the combination of these methods allows the experimenter to directly relate anatomically-identified neurophysiological signals to behavior.

This protocol describes the design and surgical implantation of a head-restraining mechanism to monitor neuronal activity in sub-cortical brain structures in alert rats. It delineates procedures to isolate single neurons in the juxtacellular configuration and to efficiently identify their anatomical locations.

Juxtacellular Überwachung und Lokalisierung von einzelnen Neuronen im Sub-kortikalen Hirnstrukturen der Alarm, Head-zurückhaltende Ratten

There are a variety of techniques to monitor extracellular activity of single neuronal units. However, monitoring this activity from deep brain structures ...

Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be stimulated by neuronally-released transmitter. However, the ability of astrocytic receptors to exhibit plasticity in response to changes in neuronal activity has received little attention. Here we describe a model system that can be used to globally scale up or down astrocytic group I metabotropic glutamate receptors (mGluRs) in acute brain slices. Included are methods on how to prepare parasagittal hippocampal slices, construct chambers suitable for long-term slice incubation, bidirectionally manipulate neuronal action potential frequency, load astrocytes and astrocyte processes with fluorescent Ca2+ indicator, and measure changes in astrocytic Gq GPCR activity by recording spontaneous and evoked astrocyte Ca2+ events using confocal microscopy. In essence, a &#8220;calcium roadmap&#8221; is provided for how to measure plasticity of astrocytic Gq GPCRs. Applications of the technique for study of astrocytes are discussed. Having an understanding of how astrocytic receptor signaling is affected by changes in neuronal activity has important implications for both normal synaptic function as well as processes underlying neurological disorders and neurodegenerative disease.

Here we describe an adaptation of protocols used to induce homeostatic plasticity in neurons for the study of plasticity of astrocytic G protein-coupled receptors. Recently used to examine changes in astrocytic group I mGluRs in juvenile mice, the method can be applied to measure scaling of various astrocytic GPCRs, in tissue from adult mice in situ and in vivo, and to gain a better appreciation of the sensitivity of astrocytic receptors to changes in neuronal activity.

Induktion von Plastizität Astrozytäre Rezeptoren durch Manipulation der neuronalen Feuerraten

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be ...

A Novel Behavioral Assay to Investigate Gustatory Responses of Individual, Freely-moving Bumble Bees (Bombus terrestris)

Generalist pollinators like the buff-tailed bumble bee, Bombus terrestris, encounter both nutrients and toxins in the floral nectar they collect from flowering plants. Only a few studies have described the gustatory responses of bees toward toxins in food, and these experiments have mainly used the proboscis extension response on restrained honey bees. Here, a new behavioral assay is presented for measuring the feeding responses of freely-moving, individual worker bumble bees to nutrients and toxins. This assay measures the amount of solution ingested by each bumble bee and identifies how tastants in food influence the microstructure of the feeding behavior.
The solutions are presented in a microcapillary tube to individual bumble bees that have been previously starved for 2-4 hr. The behavior is captured on digital video. The fine structure of the feeding behavior is analyzed by continuously scoring the position of the proboscis (mouthparts) from video recordings using event logging software. The position of the proboscis is defined by three different behavioral categories: (1) proboscis is extended and in contact with the solution, (2) proboscis is extended but not in contact with the solution and (3) proboscis is stowed under the head. Furthermore the speed of the proboscis retracting away from the solution is also estimated.
In the present assay the volume of solution consumed, the number of feeding bouts, the duration of the feeding bouts and the speed of the proboscis retraction after the first contact is used to evaluate the phagostimulatory or the deterrent activity of the compounds tested.
This new taste assay will allow researchers to measure how compounds found in nectar influence the feeding behavior of bees and will also be useful to pollination biologists, toxicologists and neuroethologists studying the bumble bee&#39;s taste system.

A Novel Behavioral Assay to Investigate Gustatory Responses of Individual, Freely-moving Bumble Bees Bombus terrestris

A novel behavioral assay is described for investigating the short term gustatory responses of the mouthparts of freely-moving bumble bees (Bombus terrestris) toward nutrients and toxins in solution.

A Novel Verhaltenstest Gustatory Antworten einzelner, frei beweglichen Bumble Bees zur Untersuchung (<em&gt; Bombus terrestris</em&gt;)

Generalist pollinators like the buff-tailed bumble bee, Bombus terrestris, encounter both nutrients and toxins in the floral nectar they collect from ...