This work was supported by a Grant-in-Aid for Scientific Research (B) and a Grant-in Aid for Scientific Research on Innovative Areas 'Systems Molecular Ethology' from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan. T.U. was the recipient of a Grant-Aid from the Japan Society for the Promotion of Science for Young Scientists.

1. Preparation of Single-cohort Colonies
<ol>
	<li>Prepare three honeybee colonies to create two single-cohort colonies and to obtain a sufficient number of newly emerged workers.
	<ol>
		<li>Check that some pupae in the capped peripheral cells in the combs have brown eyes and a pigmented cuticle by opening the capped combs using tweezers. If these pupae exist in peripheral comb cells, most of pupae in the whole combs will emerge in approximately 1-3 days.</li>
		<li>Subsequently, collect the combs containing these pupae after all adherent adult bees have been removed with a brush, and mix combs from the three colonies to minimize variability among colonies.</li>
	</ol>
	</li>
	<li>Place the collected combs in an empty hive box and incubate at 33 &#176;C under humid conditions (&#62;60% relative humidity). Collect approximately 6,000 newly emerged workers over 3 days (3,000 workers per colony) from the collected combs.</li>
	<li>Determine the quantity of workers based on the weight of five newly emerged workers randomly collected from unmanipulated colonies.
	<ol>
		<li>Weigh five newly emerged workers randomly collected from unmanipulated colonies using weighing machines. 
		Note: The weight of five newly emerged workers is generally 500-600 mg.</li>
		<li>Estimate the quantity of collected workers by dividing the weight of the collected workers by the average weight of five newly emerged workers.</li>
	</ol>
	</li>
	<li>Apply paint marks to the thoraces of approximately 1,800 workers (900 workers per colony) using water-based poster paint markers. 
	Note: Washable marker which is used to apply the paint is listed in the Table of Materials.</li>
	<li>Introduce a queen and approximately 3,000 of the 0-2 day old workers to a new hive box that contains two combs, one comb containing honey and pollen as preserved foods and another empty comb for egg-laying by the queen. Collect the comb containing honey and pollen from another unmanipulated colony after all adherent bees are removed. 
	Note: The use of nuclear hive box (small sized hive box) is recommended.</li>
	<li>Collect nurse bees and precocious foragers with paint marks 6 to 8 days after creating single-cohort colonies.
	<ol>
		<li>To collect nurse bees, pick up workers that put their heads into comb cells to take care of the brood. Use tweezers to collect the nurse bees from combs that contain many brood and adult workers. 
		Note: If HPGs of collected workers are developed when dissected as described in step 1.7, these workers are defined as nurse bees. Holding the thoraces by tweezers is recommended to pick up workers from the combs.</li>
		<li>To collect foragers, use an insect net to capture workers that return to their hives with pollen loads on their hind legs. 
		Note: If HPGs of captured workers appear shrunken when dissected as described in step 1.7, those workers are defined as foragers.</li>
	</ol>
	</li>
	<li>Classify the HPG into three groups, &#39;Developed&#39;, &#39;Intermediate&#39;, and &#39;Shrunken&#39; by the dissection under a stereomicroscope.
	<ol>
		<li>Anesthetize 10-15 bees in an insect cage in a refrigerator for 10-15 min until bees cannot fly or walk. Then, move bees onto ice and complete the anesthesia on ice for 5 min. 
		Note: It takes about total 15-20 min to achieve anesthesia. If the anesthesia time needs to be shortened, the number of bees in a cage should be reduced to 1-3 bees per cage.</li>
		<li>Dissect the head from the body with scissors and tweezers. Fix the head on dental wax placed on a Petri dish with pins. Insert two pins into the bases of the antennae to fix the head. Soak fixed heads in insect saline (130 mM NaCl, 5 mM KCl, 1 mM CaCl2).</li>
		<li>Remove the anterior aspect of the cuticle of the head using fine tweezers and a surgical knife under a binocular microscope. 
		Note: The HPGs can be easily found in the head after removal of cuticle. The HPG is a botryoid organ that exists around the brain in the head.</li>
		<li>Remove the tracheal tissue which is a white membranous tissue under the cuticle. Then, dissect the HPGs from the head by slowly pulling them with tweezers.</li>
		<li>Classify HPGs with large and circular acini as &#39;Developed&#39;. Classify HPGs with small and distorted acini as &#39;Shrunken&#39;. Classify HPGs corresponding to neither &#39;Developed&#39; nor &#39;Shrunken&#39; as &#39;Intermediate&#39;. 
		Note: Representative photographs that show these three classes of HPG states are indicated in Figure 2.</li>
		<li>Subject &#39;Developed&#39; and &#39;Shrunken&#39; HPGs to RNA extraction as &#39;nurse bee HPGs&#39; and &#39;forager HPGs&#39;, respectively, and compare gene expression in the HPGs between nurse bees and foragers (section 4).</li>
	</ol>
	</li>
</ol>
2. Injection of 20E
<ol>
	<li>Collect nurse bees from typical colonies as described in the procedure 1.6.1. 
	Note: HPGs cannot be examined in these bees as the bees will be reared.</li>
	<li>Anesthetize the bees at 4 &#176;C in a refrigerator (not on ice). 
	Note: It takes about 30 min to achieve anesthesia. If the anesthesia time is shortened, the number of bees in the cage should be reduced to 1-3 bees per cage.</li>
	<li>Using tweezers, immobilize the bees on dental wax placed on a Petri dish.</li>
	<li>Inject 1 &#181;l of 20E solution into the anterior aspect of the head using an injection tip made from a calibrated capillary micropipette using a glass needle puller.
	<ol>
		<li>Dissolve 20E in ethanol-insect saline (130 mM NaCl, 5 mM KCl, 1 mM CaCl2) mixture (1:4) at 2.5 &#181;g/&#181;l.</li>
		<li>Connect the injection tip to a rubber tube, and connect a yellow micropipette tip to the opposite side of the tube.</li>
		<li>Place 1 &#181;l of 20E solution on parafilm using a micropipette, hold the yellow micropipette tip in the mouth, and draw the solution into the injection tip.</li>
		<li>Insert the injection tip into the base of the antennae, and inject the solution by blowing through the yellow micropipette tip. 
		Note: The protocol shown in this step is the representative injection method, but the use of the automated injection machine is recommended for safety as necessary20.</li>
	</ol>
	</li>
	<li>Rear the injected bees in insect cages at 33-36 &#176;C (the ideal temperature is 35 &#176;C) under dark and humid conditions (&#62;60 % relative humidity) for 1 or 3 days. Supply honey-water mixture (1:1) to the bees. If needed, count surviving bees after injection.</li>
</ol>
3. Application of Methoprene
<ol>
	<li>Collect the combs that contain pupae from typical colonies after all adherent adult bees are removed with a brush. Incubate the combs at 33-36 &#176;C (the ideal temperature is 35 &#176;C) in humid conditions (&#62;60% relative humidity) for up to 1 day.</li>
	<li>Collect newly emerged bees from the collected combs and apply paint marks to their thoraces using poster paint as described in the protocol for preparing single-cohort colonies. Return the marked bees to their colonies.</li>
	<li>After 6 days, collect painted workers (6 day old) from the colonies. Pick up painted workers from the combs by holding their thoraces with tweezers.</li>
	<li>Anesthetize the bees at 4 &#176;C in a refrigerator (not on ice) as in the procedure 2.2. Using tweezers, immobilize the bees on dental wax placed on a Petri dish and apply 1-5 &#181;l of methoprene solution (50-250 &#181;g/&#181;l) dissolved in acetone to their heads using a micropipette. Use a filter tip that is resistant to acetone. 
	Note: Acetone may have the harmful effects on bees leading to death. If the high mortality of bees is observed, the reduction of the volume of acetone to 1 &#181;l at the minimum is recommended.</li>
	<li>Rear the bees in insect cages at 33-36 &#176;C (the ideal temperature is 35 &#176;C) for 7 days under dark and humid conditions (&#62;60% relative humidity). Supply honey-water mixture (1:1) to the bees. If needed, count surviving bees after topical application. 
	Note: In this article, surviving bees are counted 7 days after application (Table 3).</li>
</ol>
4. RNA Extraction and Quantitative RT-PCR
<ol>
	<li>Anesthetize bees and dissect HPGs as described in steps 1.7.1 to 1.7.4.</li>
	<li>Collect the dissected HPGs in a microcentrifuge tube on dry ice and store at -80 &#176;C until use.</li>
	<li>Add 400 &#181;l of the reagent for protein denaturation. 
	Note: This reagent is commercially available (see the Table of Materials).</li>
	<li>Homogenize the HPG tissues using a hand held electric mixer with a homogenization pestle that fits inside a microcentrifuge tube. Homogenize at a rotation speed of approximately 300 rpm for 1 min on ice to break and lyse tissue cells. 
	Note: The hand held electric mixer is commercially available (see the Table of Materials).</li>
	<li>Add 80 &#181;l chloroform and mix well. Incubate for 5 min at RT.</li>
	<li>Centrifuge the microcentrifuge tubes at 14,170 x g for 15 min at 4 &#176;C. Transfer the aqueous phase to a new tube. Add an equal volume of isopropanol and 1 &#181;l glycogen (5 mg/ml). Incubate at -20 &#176;C for at least 20 min.</li>
	<li>Centrifuge the microcentrifuge tubes at 14,170 x g for 10 min at 4 &#176;C and remove the supernatant.</li>
	<li>Add 400 &#181;l of 75% ethanol to the pellet and mix well, centrifuge the microcentrifuge tubes at 14,170 x g for 15 min at 4 &#176;C, and remove the supernatant. Repeat these procedures once more.</li>
	<li>Air-dry the pellet for 5-10 min and then dissolve it in 10 &#181;l RNase-free water. 
	Note: Complete drying of the pellet may cause insolubilization of the RNA pellet in water. Thus, the pellet which is slightly wet is appropriate for dissolving.</li>
	<li>Measure the RNA concentration using a spectrophotometer such as NanoDrop or similar.</li>
	<li>Treat 500 ng of total RNA with 2.0 U of DNase I by incubating at 37 &#176;C for 30 min to degrade any contaminating genomic DNA.</li>
	<li>Reverse-transcribe 200 ng of DNase I-treated RNA and perform real time PCR using commercially available kits (see the list in the Table of Materials and Reagents) and gene-specific primers (mrjp2; 5&#39;-AAATGGTCGCTCAAAATGACAGA-3&#39;, and 5&#39;-ATTCATCCTTTACAGGTTTGTTGC-3&#39;, Hbg3; 5&#39;-TACCTGGCTTCGTGTCAAC-3&#39; and 5&#39;-ATCTTCGGTTTCCCTAGAGAATG-3&#39;). Perform the PCR as follows: [95 &#176;C x 30 sec + (95&#176;C x 5 sec + 60&#176;C x 15 sec + 72 &#176;C x 20 sec) x 45-55 cycles].</li>
	<li>Normalize the amount of transcript with that of elongation factor 1&#945;-F2 (EF1&#945;&#727;F2) or ribosomal protein 49 (rp49)9,16,21,22. These genes are reliable control genes for analyzing gene expression in the HPGs using qRT-PCR.</li>
	<li>Perform two-tailed t-test to detect the significant differences between two experiment groups (nurse bees vs. precocious foragers, 20E-treated bees vs. control bees, and methoprene-treated bees vs. control bees) using statistical software. 
	Note: If F-test assumes the homogeneity of variance, Student&#39;s t-test can be used. If not, Welch&#39;s t-test can be used.</li>
</ol>

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The authors have nothing to disclose.

Preparation of single-cohort colonies
Here we described the protocol used in our previous study16 to prepare single-cohort colonies for analysis of HPG physiology associated with the shift in the role of worker bees. Nurse bees and precocious foragers that satisfied the criteria described in procedures 1.6-1.7 and Figure 2 were observed in the single-cohort colonies (Table 1). The photographs in Figure 2 would be useful in classific...

The European honeybee, Apis mellifera, is a eusocial insect with a highly organized society1. Worker honeybees (labor caste) are engaged in various tasks to maintain colony activity, and these tasks change according to the worker honeybee&#39;s age after eclosion, which is referred to as age-related division of labor2-4. Young workers (&#60;13 days old) take care of the brood in the hive by secreting royal jelly (nurse bees), while older workers (&#62;15 days old) collect nectar and pollen outside of the hive (foragers)2-4. The physiology of the workers changes in association with this role shift. For example, the function of the hypopharyngeal glands (HPGs), paired exocrine glands located in the head, changes in association with the role shift from nursing to foraging2,5. Nurse bee HPGs mainly synthesize major royal jelly proteins, which are major components of bee milk. On the other hand, forager HPGs mainly synthesize carbohydrate-metabolizing enzymes, such as &#945;-glucosidase III, to process nectar into honey by converting sucrose into glucose and fructose. Our previous studies revealed that the expression of mrjp2, which encodes a major royal jelly protein, and Hbg3, which encodes &#945;-glucosidase III, changes during the role shift6-9.
To determine whether the physiological changes in the worker tissues, including the HPGs, is associated with the role shift or with the age of the workers, it would be useful to manipulate the population composition of a honeybee colony, such as to prepare single-cohort colonies in which workers of almost the same age perform different tasks10,11. Robinson et al. (1989) described a method for establishing a single-cohort colony10. Single-cohort colonies initially comprise a queen and 0-2 day old workers. Several days after establishing the colonies, workers of almost the same age assume different tasks. Some workers perform nursing tasks as in typical colonies, whereas other workers perform foraging tasks earlier than usual and are thus called precocious foragers. Gene expression comparisons between nurse bees and precocious foragers would provide useful information about the role-associated physiology of worker tissues12-16. Here, we describe a detailed protocol for preparing single-cohort colonies for analysis of the role- and/or age-associated physiology of HPGs16. We also briefly describe how to examine the gene expression of mrjp2 and Hbg3 by quantitative reverse transcription-polymerase chain reaction (RT-PCR) to evaluate HPG physiology.
In addition to the analysis of worker physiology in single-cohort colonies, examination of the endocrine system is important for analyzing the regulatory mechanisms of role-associated worker physiology. Juvenile hormone (JH), which is known as the &#39;status quo&#39; hormone in insect larvae, accelerates the shift in the role from nursing to foraging in worker honeybees11. Furthermore, ecdysone, which is known as the molting hormone during metamorphosis, might be involved in the role shift as genes encoding ecdysone signaling molecules are expressed in the mushroom bodies, a higher center of the worker brain17-19. Therefore, we also describe the detailed protocol used in our previous study16 to treat workers with 20E, which is an active form of ecdysone, and methoprene, a JH analogue, for analysis of the effect of the endocrine system on HPG physiology (expression of mrjp2 and Hbg3).

<table border="0" cellpadding="0" cellspacing="0" width="769">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">UNIPOSCA</td>
			<td style="width:120px;">Mitsubishi pencil</td>
			<td style="width:91px;">PC-5M</td>
			<td style="width:327px;">Marker pen for the application of marks to bees&#160;&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">20-hydroxyecdysone</td>
			<td>Sigma Aldrich</td>
			<td>H5142</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Methoprene</td>
			<td>Sigma Aldrich</td>
			<td>33375</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Breeding case insect</td>
			<td>IRIS OHYAMA</td>
			<td>CP-SS</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Electromotion mixier&#160;</td>
			<td>ISO</td>
			<td>23M-R25</td>
			<td>homogenization of tissue</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TRIZol Reagent</td>
			<td>Invitrogen</td>
			<td>15596-026</td>
			<td>the reagent for total RNA extraction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DNase I&#160;</td>
			<td>Takara</td>
			<td>2270A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PrimeScript RT reagent kit</td>
			<td>Takara</td>
			<td>RR037A</td>
			<td>the reagent for reverse transcription</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SYBR Premix ExTaq II</td>
			<td>Takara</td>
			<td>RR820A</td>
			<td>the reagent for real-time PCR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LightCycle 1.2 Instrument</td>
			<td>Roche</td>
			<td>12011468001</td>
			<td>the instrument for real-time PCR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LightCycle Capillaries (20&#956;l)</td>
			<td>Roche</td>
			<td>4929292001</td>
			<td>the material for real-time PCR</td>
		</tr>
	</tbody>
</table>

preparation of single-cohort colonies and hormone treatment of worker honeybees to analyze physiology associated with role and/or endocrine system

Honeybee workers are engaged in various tasks related to maintaining colony activity. The tasks of the workers change according to their age (age-related division of labor). Young workers are engaged in nursing the brood (nurse bees), while older workers are engaged in foraging for nectar and pollen (foragers). The physiology of the workers changes in association with this role shift. For example, the main function of the hypopharyngeal glands (HPGs) changes from the secretion of major royal jelly proteins (MRJPs) to the secretion of carbohydrate-metabolizing enzymes. Because worker tasks change as the workers age in typical colonies, it is difficult to discriminate the physiological changes that occur with aging from those that occur with the role shift. To study the physiological changes in worker tissues, including the HPGs, in association with the role shift, it would be useful to manipulate the honeybee colony population by preparing single-cohort colonies in which workers of almost the same age perform different tasks. Here we describe a detailed protocol for preparing single-cohort colonies for this analysis. Six to eight days after single-cohort colony preparation, precocious foragers that perform foraging tasks earlier than usual appear in the colony. Representative results indicated role-associated changes in HPG gene expression, suggesting role-associated HPG function. In addition to manipulating the colony population, analysis of the endocrine system is important for investigating role-associated physiology. Here, we also describe a detailed protocol for treating workers with 20-hydroxyecdysone (20E), an active form of ecdysone, and methoprene, a juvenile hormone analogue. The survival rate of treated bees was sufficient to examine gene expression in the HPGs. Gene expression changes were observed in response to 20E- and/or methoprene-treatment, suggesting that hormone treatments induce physiological changes of the HPGs. The protocol for hormone treatment described here is appropriate for examining hormonal effects on worker physiology.

An overview of the protocol for preparing single-cohort colonies is illustrated in Figure 1A. Time-course of experiments from preparing single-cohort colonies to sample collection is shown in Figure 1B. Workers that satisfied the behavioral criteria for nursing behavior or foraging behavior were collected from single-cohort colonies, and HPG development was estimated in these workers. Table 1 shows the classification of HPG development in...

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Preparation of Single-cohort Colonies and Hormone Treatment of Worker Honeybees to Analyze Physiology Associated with Role and/or Endocrine System

Here we describe our detailed protocol for the preparation of single-cohort honeybee colonies&#160;&#8211; a useful tool for analyzing the role-associated worker physiology. We also describe detailed protocols for treating workers with juvenile hormone and ecdysone to evaluate the involvement of these hormones in the regulation of worker behavior and/or physiology.

Honeybee workers are engaged in various tasks related to maintaining colony activity. The tasks of the workers change according to their age (age-related ...

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15.7K Views.  The University of Tokyo. Here we describe our detailed protocol for the preparation of single-cohort honeybee colonies – a useful tool for analyzing the role-associated worker physiology. We also describe detailed protocols for treating workers with juvenile hormone and ecdysone to evaluate the involvement of these hormones in the regulation of worker behavior and/or physiology.

Video: Preparation of Single-cohort Colonies and Hormone Treatment of Worker Honeybees to Analyze Physiology Associated with Role and/or Endocrine System

Preparation and Culture of Chicken Auditory Brainstem Slices

The chicken auditory brainstem is a well-established model system that has been widely used to study the anatomy and physiology of auditory processing at discreet periods of development 1-4 as well as mechanisms for temporal coding in the central nervous system 5-7.
Here we present a method to prepare chicken auditory brainstem slices that can be used for acute experimental procedures or to culture organotypic slices for long-term experimental manipulations.
The chicken auditory brainstem is composed of nucleus angularis, magnocellularis, laminaris and superior olive. These nuclei are responsible for binaural sound processing and single coronal slice preparations preserve the entire circuitry. Ultimately, organotypic slice cultures can provide the opportunity to manipulate several developmental parameters such as synaptic activity, expression of pre and postsynaptic components, expression of aspects controlling excitability and differential gene expression
This approach can be used to broaden general knowledge about neural circuit development, refinement and maturation.

The chicken auditory brainstem is comprised of nuclei responsible for binaural sound processing. A single coronal slice preparation maintains the entire circuitry while the cultured approach provides a unique preparation to study the development of neuronal structure and auditory function at the molecular, cellular and network levels.

The chicken auditory brainstem is a well-established model system that has been widely used to study the anatomy and physiology of auditory processing at ...

Preparation of Acute Subventricular Zone Slices for Calcium Imaging

The subventricular zone (SVZ) is one of the two neurogenic zones in the postnatal brain. The SVZ contains densely packed cells, including neural progenitor cells with astrocytic features (called SVZ astrocytes), neuroblasts, and intermediate progenitor cells. Neuroblasts born in the SVZ tangentially migrate a great distance to the olfactory bulb, where they differentiate into interneurons. Intercellular signaling through adhesion molecules and diffusible signals play important roles in controlling neurogenesis. Many of these signals trigger intercellular calcium activity that transmits information inside and between cells. Calcium activity is thus reflective of the activity of extracellular signals and is an optimal way to understand functional intercellular signaling among SVZ cells.
Calcium activity has been studied in many other regions and cell types, including mature astrocytes and neurons. However, the traditional method to load cells with calcium indicator dye (i.e. bath loading) was not efficient at loading all SVZ cell types. Indeed, the cellular density in the SVZ precludes dye diffusion inside the tissue. In addition, preparing sagittal slices will better preserve the three-dimensional arrangement of SVZ cells, particularly the stream of neuroblast migration on the rostral-caudal axis.
Here, we describe methods to prepare sagittal sections containing the SVZ, the loading of SVZ cells with calcium indicator dye, and the acquisition of calcium activity with time-lapse movies. We used Fluo-4 AM dye for loading SVZ astrocytes using pressure application inside the tissue. Calcium activity was recorded using a scanning confocal microscope allowing a precise resolution for distinguishing individual cells. Our approach is applicable to other neurogenic zones including the adult hippocampal subgranular zone and embryonic neurogenic zones. In addition, other types of dyes can be applied using the described method.

A method to load subventricular zone (SVZ) cells with calcium indicator dyes for recording calcium activity is described. The postnatal SVZ contains tightly packed cells including neural progenitor cells and neuroblasts. Rather than using bath loading we injected the dye by pressure inside the tissue allowing better dye diffusion.

The subventricular zone (SVZ) is one of the two neurogenic zones in the postnatal brain. The SVZ contains densely packed cells, including neural ...

Simultaneous Long-term Recordings at Two Neuronal Processing Stages in Behaving Honeybees

In both mammals and insects neuronal information is processed in different higher and lower order brain centers. These centers are coupled via convergent and divergent anatomical connections including feed forward and feedback wiring. Furthermore, information of the same origin is partially sent via parallel pathways to different and sometimes into the same brain areas. To understand the evolutionary benefits as well as the computational advantages of these wiring strategies and especially their temporal dependencies on each other, it is necessary to have simultaneous access to single neurons of different tracts or neuropiles in the same preparation at high temporal resolution. Here we concentrate on honeybees by demonstrating a unique extracellular long term access to record multi unit activity at two subsequent neuropiles1, the antennal lobe (AL), the first olfactory processing stage and the mushroom body (MB), a higher order integration center involved in learning and memory formation, or two parallel neuronal tracts2 connecting the AL with the MB.&#160;The latter was chosen as an example and will be described in full. In the supporting video the construction and permanent insertion of flexible multi&#160;channel wire electrodes is demonstrated. Pairwise differential amplification of the micro&#160;wire electrode channels drastically reduces the noise and verifies that the source of the signal is closely related to the position of the electrode tip. The mechanical flexibility of the used wire electrodes allows stable invasive long&#160;term recordings over many hours up to days, which is a clear advantage compared to conventional extra&#160;and intracellular in vivo recording techniques.

Simultaneous extracellular long term recordings from two different brain neuropiles or two different anatomical tracts were established in honeybees. These recordings allow the investigation of temporal aspects of neuronal processing across different brain areas at the single neuron as well as at the ensemble level in a behaving animal.

In both mammals and insects neuronal information is processed in different higher and lower order brain centers. These centers are coupled via convergent ...

Live Imaging of Nicotine Induced Calcium Signaling and Neurotransmitter Release Along Ventral Hippocampal Axons

Sustained enhancement of axonal signaling and increased neurotransmitter release by the activation of pre-synaptic nicotinic acetylcholine receptors (nAChRs) is an important mechanism for neuromodulation by acetylcholine (ACh). The difficulty with access to probing the signaling mechanisms within intact axons and at nerve terminals both in vitro and in vivo has limited progress in the study of the pre-synaptic components of synaptic plasticity. Here we introduce a gene-chimeric preparation of ventral hippocampal (vHipp)&#8211;accumbens (nAcc) circuit in vitro that allows direct live imaging to analyze both the pre- and post-synaptic components of transmission while selectively varying the genetic profile of the pre- vs post-synaptic neurons. We demonstrate that projections from vHipp microslices, as pre-synaptic axonal input, form multiple, reliable glutamatergic synapses with post-synaptic targets, the dispersed neurons from nAcc. The pre-synaptic localization of various subtypes of nAChRs are detected and the pre-synaptic nicotinic signaling mediated synaptic transmission are monitored by concurrent electrophysiological recording and live cell imaging. This preparation also provides an informative approach to study the pre- and post-synaptic mechanisms of glutamatergic synaptic plasticity in vitro.

We developed a gene-chimeric preparation of ventral hippocampal &#8211; accumbens circuit in vitro that allows direct live imaging to analyze presynaptic mechanisms of nicotinic acetylcholine receptors (nAChRs) mediated synaptic transmission. This preparation also provides an informative approach to study the pre- and post-synaptic mechanisms of synaptic plasticity.

Sustained enhancement of axonal signaling and increased neurotransmitter release by the activation of pre-synaptic nicotinic acetylcholine receptors ...

Utilizing Combined Methodologies to Define the Role of Plasma Membrane Delivery During Axon Branching and Neuronal Morphogenesis

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular guidance cues like netrin-1 and results in dramatic increases to the surface area of the axonal plasma membrane. Netrin-1-dependent axon branching likely involves temporal and spatial control of plasma membrane expansion, the components of which are supplied through exocytic vesicle fusion. These fusion events are preceded by formation of SNARE complexes, comprising a v-SNARE, such as VAMP2 (vesicle-associated membrane protein 2), and plasma membrane t-SNAREs, syntaxin-1 and SNAP25 (synaptosomal-associated protein 25). Detailed herein isa multi-pronged approach used to examine the role of SNARE mediated exocytosis in axon branching. The strength of the combined approach is data acquisition at a range of spatial and temporal resolutions, spanning from the dynamics of single vesicle fusion events in individual neurons to SNARE complex formation and axon branching in populations of cultured neurons. This protocol takes advantage of established biochemical approaches to assay levels of endogenous SNARE complexes and Total Internal Reflection Fluorescence (TIRF) microscopy of cortical neurons expressing VAMP2 tagged with a pH-sensitive GFP (VAMP2-pHlourin) to identify netrin-1 dependent changes in exocytic activity in individual neurons. To elucidate the timing of netrin-1-dependent branching, time-lapse differential interference contrast (DIC) microscopy of single neurons over the order of hours is utilized. Fixed cell immunofluorescence paired with botulinum neurotoxins that cleave SNARE machinery and block exocytosis demonstrates that netrin-1 dependent axon branching requires SNARE-mediated exocytic activity.

Light microscopy techniques coupled with biochemical assays elucidate the involvement of SNARE-mediated exocytosis in netrin-dependent axon branching. This combination of techniques permits identification of molecular mechanisms controlling axon branching and cell shape change.

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular ...

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have validated the utility of this method for enhancing neuronal preservation and overall brain slice viability. The implementation of this technique by early adopters has facilitated detailed investigations into brain function using diverse experimental applications and spanning a wide range of animal ages, brain regions, and cell types. Steps are outlined for carrying out the protective recovery brain slice technique using an optimized NMDG artificial cerebrospinal fluid (aCSF) media formulation and enhanced procedure to reliably obtain healthy brain slices for patch clamp electrophysiology. With this updated approach, a substantial improvement is observed in the speed and reliability of gigaohm seal formation during targeted patch clamp recording experiments while maintaining excellent neuronal preservation, thereby facilitating challenging experimental applications. Representative results are provided from multi-neuron patch clamp recording experiments to assay synaptic connectivity in neocortical brain slices prepared from young adult transgenic mice and mature adult human neurosurgical specimens. Furthermore, the optimized NMDG protective recovery method of brain slicing is compatible with both juvenile and adult animals, thus resolving a limitation of the original methodology. In summary, a single media formulation and brain slicing procedure can be implemented across various species and ages to achieve excellent viability and tissue preservation.

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

This protocol demonstrates the implementation of an optimized N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. A single media formulation is used to reliably obtain healthy brain slices from animals of any age and for diverse experimental applications.

This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have ...

Video Imaging and Spatiotemporal Maps to Analyze Gastrointestinal Motility in Mice

The enteric nervous system (ENS) plays an important role in regulating gastrointestinal (GI) motility and can function independently of the central nervous system. Changes in ENS function are a major cause of GI symptoms and disease and may contribute to GI symptoms reported in neuropsychiatric disorders including autism. It is well established that isolated colon segments generate spontaneous, rhythmic contractions known as Colonic Migrating Motor Complexes (CMMCs). A procedure to analyze the enteric neural regulation of CMMCs in ex vivo preparations of mouse colon is described. The colon is dissected from the animal and flushed to remove fecal content prior to being cannulated in an organ bath. Data is acquired via a video camera positioned above the organ bath and converted to high-resolution spatiotemporal maps via an in-house software package. Using this technique, baseline contractile patterns and pharmacological effects on ENS function in colon segments can be compared over 3-4 hr. In addition, propagation length and speed of CMMCs can be recorded as well as changes in gut diameter and contraction frequency. This technique is useful for characterizing gastrointestinal motility patterns in transgenic mouse models (and in other species including rat and guinea pig). In this way, pharmacologically induced changes in CMMCs are recorded in wild type mice and in the Neuroligin-3R451C mouse model of autism. Furthermore, this technique can be applied to other regions of the GI tract including the duodenum, jejunum and ileum and at different developmental ages in mice.

This article describes a video imaging technique and high-resolution spatiotemporal mapping to identify changes in the neural regulation of colonic motility in adult mice. Subtle effects on gastrointestinal (GI) function can be detected using this approach in isolated tissue preparations to advance our understanding of GI disease.

The enteric nervous system (ENS) plays an important role in regulating gastrointestinal (GI) motility and can function independently of the central ...

Methods and Tips for Intravenous Administration of Adeno-associated Virus to Rats and Evaluation of Central Nervous System Transduction

Adeno-associated virus (AAV) vectors are a key reagent in the neurosciences for clustered regularly interspaced short palindromic repeats (CRISPR), optogenetics, cre-lox targeting, etc. The purpose of this manuscript is to aid the investigator attempting expansive central nervous system (CNS) gene transfer in the rat via tail vein injection of AAV. Wide-scale expression is relevant for conditions with widespread pathology, and a rat model is significant due to its greater size and physiologic similarities to humans compared to mice. In this example application, a wide-scale neuronal transduction is used to mimic a neurodegenerative disease that affects the entire spinal cord, amyotrophic lateral sclerosis (ALS). The efficient wide-scale CNS transduction can also be used to deliver therapeutic protein factors in pre-clinical studies. After a post-injection expression interval of several weeks, the effects of the transduction are evaluated. For a green fluorescent protein (GFP) control vector, the amount of GFP in the cerebellum is estimated quickly and reliably by a basic imaging program. For motor disease phenotypes that are induced by the ALS related protein transactive response DNA-binding protein of 43 kDa (TDP-43), the deficits are scored by escape reflex and rotarod. Beyond disease modeling and gene therapy, there are diverse potential applications for the wide-scale gene targeting described here. The expanded use of this method will aid in expediting hypothesis testing in the neurosciences and neurogenetics.

Methods for a wide-scale central nervous system gene delivery in the rat are covered. In this example, the purpose is to mimic a disease that affects the entire spinal cord. The widespread transduction can be used to deliver a therapeutic protein to the CNS from a one-time, peripheral administration.

Adeno-associated virus (AAV) vectors are a key reagent in the neurosciences for clustered regularly interspaced short palindromic repeats (CRISPR), ...

Online Transcranial Magnetic Stimulation Protocol for Measuring Cortical Physiology Associated with Response Inhibition

We describe the development of a reproducible, child-friendly motor response inhibition task suitable for online Transcranial Magnetic Stimulation (TMS) characterization of primary motor cortex (M1) excitability and inhibition. Motor response inhibition prevents unwanted actions and is abnormal in several neuropsychiatric conditions. TMS is a non-invasive technology that can quantify M1 excitability and inhibition using single- and paired-pulse protocols and can be precisely timed to study cortical physiology with high temporal resolution. We modified the original Slater-Hammel (S-H) stop signal task to create a &#34;racecar&#34; version with TMS pulses time-locked to intra-trial events. This task is self-paced, with each trial initiating after a button push to move the racecar towards the 800 ms target. GO trials require a finger-lift to stop the racecar just before this target. Interspersed randomly are STOP trials (25%) during which the dynamically adjusted stop signal prompts subjects to prevent finger-lift. For GO trials, TMS pulses were delivered at 650 ms after trial onset; whereas, for STOP trials, the TMS pulses occurred 150 ms after the stop signal. The timings of the TMS pulses were decided based on electroencephalography (EEG) studies showing event-related changes in these time ranges during stop signal tasks. This task was studied in 3 blocks at two study sites (n=38) and we recorded behavioral performance and event-related motor-evoked potentials (MEP). Regression modelling was used to analyze MEP amplitudes using age as a covariate with multiple independent variables (sex, study site, block, TMS pulse condition [single- vs. paired-pulse], trial condition [GO, successful STOP, failed STOP]). The analysis showed that TMS pulse condition (p&#60;0.0001) and its interaction with trial condition (p=0.009) were significant. Future applications for this online S-H/TMS paradigm include the addition of simultaneous EEG acquisition to measure TMS-evoked EEG potentials. A potential limitation is that in children, the TMS pulse sound could&#160;affect behavioral task performance.

We describe an experimental procedure to quantify excitability and inhibition of primary motor cortex during a motor response inhibition task by using Transcranial Magnetic Stimulation throughout the course of a Stop Signal Task.

We describe the development of a reproducible, child-friendly motor response inhibition task suitable for online Transcranial Magnetic Stimulation (TMS) ...

Convection Enhanced Delivery of Optogenetic Adeno-associated Viral Vector to the Cortex of Rhesus Macaque Under Guidance of Online MRI Images

In non-human primate (NHP) optogenetics, infecting large cortical areas with viral vectors is often a difficult and time-consuming task. Here, we demonstrate the use of magnetic resonance (MR)-guided convection enhanced delivery (CED) of optogenetic viral vectors into primary somatosensory (S1) and motor (M1) cortices of macaques to obtain efficient, widespread cortical expression of light-sensitive ion channels. Adeno-associated viral (AAV) vectors encoding the red-shifted opsin C1V1 fused to yellow fluorescent protein (EYFP) were injected into the cortex of rhesus macaques under MR-guided CED. Three months post-infusion, epifluorescent imaging confirmed large regions of optogenetic expression (&#62;130 mm2) in M1 and S1 in two macaques. Furthermore, we were able to record reliable light-evoked electrophysiology responses from the expressing areas using micro-electrocorticographic arrays. Later histological analysis and immunostaining against the reporter revealed widespread and dense optogenetic expression in M1 and S1 corresponding to the distribution indicated by epifluorescent imaging. This technique enables us to obtain expression across large areas of the cortex within a shorter period of time with minimal damage compared to the traditional techniques and can be an optimal approach for optogenetic viral delivery in large animals such as NHPs. This approach demonstrates great potential for network-level manipulation of neural circuits with cell-type specificity in animal models evolutionarily close to humans.&#160;

Here, we demonstrate magnetic resonance (MR)-guided convection enhanced delivery (CED) of viral vectors into the cortex as an efficient and simplified approach for achieving optogenetic expression across large cortical areas in the macaque brain.