The authors would like to thank Isabelle Reus for establishment of tracing the electrode insertion side, Tobias Rosenbaum for LabView programming, Anneke Meyer for data analyzes and helpful discussions. We thank Randolf Menzel for discussion and practical help during early stage of electrode development. Furthermore we thank Brian Smith for postdoctoral association to MS-B. This work was supported by the Deutsche Forschungsgemeinschaft (DFG, SPP 1392, Ro1177/5-2) to WR.

1. Electrode Building (Figure 1)
<ol>
	<li>Production of an electrode adapter that fits the electrode interface board of commercial multi channel amplifier systems1,2,25.
	<ol>
		<li>Use a small Plexiglas plate glued to an 18 Pin connector base.</li>
		<li>Connect the base with 3&#160;short pieces of insulated wire to 3&#160;separate soldering lugs screwed onto the Plexiglas plate (Figure 1 A1-A3).</li>
		<li>Insert a groove into the Plexiglas plate in which a glass capillary can easily move and be held in place by a screw (Figure&#160;1 B1).</li>
		<li>Extend the glass capillary about 5 mm using a minutien pin.</li>
		<li>Attach the micro electrode wires along the minutien pin and the glass capillary to guarantee stabilization and support.</li>
	</ol>
	</li>
	<li>Multi-channel micro wire production (adopted from Ryuichy Okada32,33)
	<ol>
		<li>Span 3&#160;micro wires (polyurethane coated copper wire, 15 &#181;m diameter) in a way that they are placed next to each other (Figure&#160;1 B2).</li>
		<li>Use a 12 V soldering needle to spread a thin film of low melting dental wax (50 &#176;C) partially along the wires to glue them together (electrode tip) (Figure&#160;1 B3). Leave a few centimeters unglued (electrode end) as this section will be used later to connect the micro wires with the electrode adapter.</li>
	</ol>
	</li>
	<li>Connect the multi channel micro wire to the electrode adapter
	<ol>
		<li>Remove the glass capillary from the holder and attach it with the minutien pin to the electrode tip. Bring it into a position parallel to the micro-electrode (Figure1 B3).</li>
		<li>Glue the electrode tip to the minutien pin using low melting dental wax and cut the micro electrode at the tip, protruding 2-3 cm from the minutien pin and at the electrode end (small arrows Figure&#160;1 B3).</li>
		<li>Slip the capillary slightly back into the electrode adapter. Use the screw to fix it (Figure&#160;1 B4).</li>
		<li>Solder the loose ends of the three wires to the soldering lugs using a soldering gun with a temperature of around 360 &#176;C to ensure melting of the insulation (Figure&#160;1 B4). After soldering, ensure that there is adequate electrical contact (~300 kOhm).</li>
		<li>Mount one of the electrodes (master) to the electrode interface board of the headstage and fix the other multi channel electrode (slave) on a separate adapter. Connect the channels of the slave to the master electrode (Figure&#160;1 C1). Additionally, solder the reference as well as muscle electrodes to the master electrode base&#160;(Figure&#160;1 C2).</li>
	</ol>
	</li>
</ol>
2. Bee Preparation (Figure 2)
In these described experiments, the honeybee (Apis mellifera), which is an invertebrate animal and therefore does not require specific ethical permits for usage, is used.
<ol>
	<li>Catch honeybee foragers (A. mellifera) at the hive entrance in the morning as shown by others34,35.</li>
	<li>Chill the bees on crushed ice till immobilization (5 to 10 min) and fix one in a standard Plexiglas holder or metal tube in a way that the head is exposed (Figure&#160;2 A). For minimizing head movements use low-melting dental wax (~50 &#176;C) and fix the head carefully to the holder around the basis of the compound eyes and the neck.</li>
	<li>Use low melting wax to fixate the scapi of the antennae on the head capsule (Figure&#160;2 B) without touching the flagellum. The flagellum of the antenna needs to be pointed forward. Ensure that the bee can freely move its proboscis.</li>
	<li>Shave the head capsule to ensure an undisturbed view and access to the top of the head.</li>
	<li>Feed the bee with a 30% sucrose solution until saturation to ensure sufficient moistening of brain tissue and good viability of the animal (Figure 2 C).</li>
	<li>Make careful incisions vertically along the borders of the compound eyes and horizontally above the antennal bases as well as beneath the ocelli and remove the loosened piece of cuticle (Figure&#160;2 D).</li>
	<li>Carefully set aside hypopharyngeal glands and remove the trachea to ensure a clear view and access to the brain prior to electrode insertion (Figures 2E, 2F).</li>
</ol>
3. Electrode Insertion
In the example case illustrated in Figure &#160;2, one electrode is positioned aiming at the l-ALT, the other one aiming on the m-ALT2. Using particular landmarks, other target regions are possible as well, for example the AL and MB output regions1.
<ol>
	<li>Position the electrodes using micromanipulators at the region of interest (Figure&#160;2G and 3A). To target the m-ALT place the electrode between the AL and medially from the vertical lobe of the MB. Ensure the insertion site being above the branching point of the mediolateral ALT PNs. Protrude the electrode into the brain with a depth of about 180 &#181;m (Figure&#160;2E). For l-ALT PN recording place the electrode below the lateral protocerebrum (the LH) in the middle of an imaginary line between the lateral side of vertical lobe and the middle of the AL. Insert the electrode with a depth of about 300 &#181;m (Figure&#160;2E)</li>
	<li>Insert the reference (silver wire, about 25 &#181;m diameter) into the ipsilateral compound eye through a small cut in the cuticle. Insert another silver wire into the muscle projection region below the lateral ocelli. NOTE: If necessary the learning behavior of the bee can be monitored with high temporal precision by recording the muscle M17, which is involved in the proboscis extension response (PER) of the bee36 as described in25.</li>
	<li>To solidly anchor the electrodes within the brain and the head capsule, cover the entire space above the brain with two component silicon (Figure&#160;2H), which will prevent the brain from drying out. NOTE: the recordings can last for hours up to days, and the bees can for example be recorded during a classical conditioning procedure (Figure&#160;2H) or stimulated with a large panel of different odors.</li>
</ol>
4. Data Acquisition and Preprocessing
<ol>
	<li>Use proper acquisition software that meets the following requirements: sampling rate of min&#160;25 kHz; analogue1,25 or digital2 cross differentiation between the electrode channels; band pass filter from 300 Hz to 8,000 Hz to extract spike events.</li>
	<li>Use available spike sorting software to extract single unit activity, for example template matching techniques as included in the Spike2 software (Figure 3).</li>
	<li>For further analysis use the time stamps of the extracted units to compute single unit averaged odor responses (Figure 4) or to calculate population vectors for Principal Component Analysis (PCA) (Figure 5) using commercially available software. To further analyze single unit and population response latency please compare recent publications1,2,25.</li>
</ol>
5. Visualization of Relative Electrode Position (Figure 4)
<ol>
	<li>Dip the electrodes tips into a solution of either 5% Alexa&#160;hydrazide 568 or 5% Alexa hydrazide 488 which is dissolved in 0.5 M potassium chloride solution prior to the recording experiments.</li>
	<li>Remove the electrodes and the covering silicon carefully after the experiments, rinse the brain with bee Ringer solution, remove glands and trachea and insert tiny crystals of tetramethylrhodamin dextran or insert a 5% solution solved in 1.0 M potassium acetate into the AL to label the ALTs anterogradely. Perform the following steps in darkness.</li>
	<li>Allow the dye to be taken up and transported by the projection neurons along their axonal tracts (30-45 min) (Figure 4A), before washing the brain with bee Ringer&#160;solution three times for another 30-45 min.</li>
	<li>Chill the bee on ice until immobilization and carefully remove the brain from the head capsule. Fixate the brain by rinsing it in a 0.1 M PBS solution containing 4% formaldehyde and keep it overnight at 4 &#176;C.</li>
	<li>Wait at least 12 hr before washing the brain two times in 0.1 M PBS (10 min&#160;each).</li>
	<li>Wash the brain 3x&#160;for 20 min in 0.2% Triton X-100 diluted in 0.1 M PBS before dehydrating it in an ascending alcohol series (30%, 50%, 70%, 90%, 95%, 3x 100% ethanol, 20 min each step).</li>
	<li>Embed the dehydrated brain in Methylsalicylate on a microscope slide and seal it with a cover slide.</li>
	<li>Use a confocal laser scanning microscope and scan the brain as optical sections every 2-5 &#181;m using a Harmonic Compound Plan Apochromat objective (10X&#160;0.4 NA immersion). Excite the tissue using 568 nm wavelength for tetramethylrhodamin dextran and a wavelength of 488 nm for the electrode position.</li>
	<li>Reconstruct the stained brain structures and electrode paths from the image stacks in 3D with reconstruction software (e.g. AMIRA or Fiji) (Figure&#160;4).</li>
</ol>

The authors declare that they have no competing financial interests.

This article demonstrates the production and usage of custom designed multi channel micro wire-electrodes. The described electrodes are suitable for recording both single unit and population activity which is especially useful for latency measurements and other temporal response properties of different neurons and different neuropils within a single specimen (for details see1,2,25). Additionally we have shown how to permanently implement the micro&#160;wire electrodes to allow stable long-term recordings in behaving honeybees that last for hours up to days.
Extracellular multi-unit recordings became a favorable tool to achieve high temporal resolution combined with spatial information. In our case, these are either parallel neuronal tracts2 or two different neuropils1. Multiple neurons can be recorded and analyzed at the single neuron level in parallel and at high temporal resolution. Multi-unit recordings were first applied in mammals38 and later also in insects39-41. Substantial progress was achieved with the development and improvement of extracellular multi-channel recording techniques42,43. This, for example, includes the development of new electrodes44 or novel spike sorting and clustering algorithms45. General methods of extracellular multi&#8208;unit recording techniques are well described46-48. The self built electrodes shown in this video additionally can be adapted by adding more microwires per electrode or the micro wires can be twisted to gain measureable constant distances between the tips. Both procedures would, however, lead to decreasing flexibility and increasing thickness of the electrode.
Compared to silicon probes commonly used for extracellular recordings in much larger insects like the hawk moth, locust and cockroach40,49&#8211;51 the described micro&#160;wire-electrodes are smaller, flexible and can cope easily with potential brain movements and, thus, can be reliably used in small social insects like bees and ants that show a much broader behavioral repertoire. Most silicone probes have sharp shank like structures cutting axons and neural tissue along their insertion channel, while the described micro wires are round, flexible and smaller and are hence less harmful to the surrounding tissue which is a clear advantage if the goal is to study long-term plasticity in an intact and behaving animal. Another advantage of micro wire electrodes is their low cost production and the easy handling. Instead of carefully cleaning an expensive silicone probe the electrode wires are freshly cut prior to brain insertion and, therefore, lack problems of congestion. Furthermore it is possible to use more than one micro wire electrode in the same preparation either inserted in different neuropiles1 or tracts2 as we show here. This approach is especially favorable to analyze and compare temporal aspects like response latencies and interactions at different neural processing levels.
We are aware of the fact that an extracellular recorded signal does not reflect single cell activity per se. It is always a compound of voltage activity around the electrode tip. To pinpoint the source of the signal the difference of two neighboring micro&#160;wire channels within one electrode is always computed. Thus the source for the spike signals used to extract single unit activity were always very close to either one or the other electrode channel resulting in easily distinguishable spike waveforms. Signals from farther away, like muscle activity or activity of neighboring neuropils, reach both electrodes at the same time evoking comparable shapes and amplitudes and will be discarded by this procedure. Using the template matching technique of Spike2, we are very confident to obtain single unit activity, which is not the same, but very close to single neuron activity. However, the issue of spike sorting could be avoided by using intracellular recording techniques.
Single cell recordings with either sharp electrodes or patch pipettes allow indepth knowledge about physiological properties of a single neuron. However, due to the small size of insect neurons and their neurites (e.g., less than 1 &#181;m for honeybee PNs 52) only short term recordings are manageable. Furthermore, intra cellular recordings might be invasive and might harm the cell which is possibly another reason for the temporal limitations. In vivo intracellular recordings in insects rarely outlast one hour. A time window which was sufficient for the pioneering work of Martin Hammer53 who recorded intracellular from a single identified neuron, the ventral unpaired maxilar neuron #1 (VUMmx1). He could link its activity directly to the reward pathway. Juliane Mauelshagen54 registered intracellularly the activity of an identified mushroom body extrinsic neuron, the pedunculus extrinsic neuron#1 (PE1) during classical conditioning. The same neuron was in the focus of Menzel and Manz55 when they found LTP after electrical stimulation of Kenyon Cells. However, Okada and colleagues56 could use the intracellularly well characterized spiking pattern (double and triple spikes) for the identification of the PE1 during extracellular recordings. After all a combination of both methods, intracellular recordings from identified neurons and extracellular long term recordings might be a powerful tool for future investigations.
However, using sharp electrodes to record multiple cells (units) simultaneously at different processing levels over many hours up to days to analyze their temporal response relationships and/or even plastic changes is almost impossible.
With the first calcium imaging approaches in the honeybee57,58 using calcium-sensitive dyes the analysis of spatial patterns of odor responses were accessible59-62. However, in many cases the calcium sensitive dyes have to be introduced to the brain tissue via invasive manipulations that again limit the bee&#8217;s life span and intrinsic properties of the analyzed cells. This issue is overcome in other model organisms like the fruitfly using genetically introduced calcium sensors63,64. However, in general, calcium sensors may introduce other limitations as they can act as calcium buffers that likely influence the temporal properties of odor responses. Simultaneous intracellular recordings combined with calcium imaging or computational approaches can prove the proper temporal resolution of imaging processes65,66. However, the temporal resolution of the imaging process itself is rather limited. Optical acquisition systems usually use CCD-imaging with a temporal resolution of 5-20 Hz 67, although 2-Photon-Imaging might be able to acquire faster sequences68. However, increasing sampling rate always goes along with a loss in spatial resolution. Furthermore the calcium-sensitive dyes used in the honeybee undergo bleaching, which also reduces acquisition time69.
Compared to other physiological recording techniques in insects our flexible multi-channel micro-wire electrodes ensure long-time access to single unit and population neuronal activity in behaving bees.
We demonstrated how to use two of these electrodes at different processing stages in the same animal, which facilitates the analysis of temporal coding aspects between the different recording sites. Dependent on the research problem and the model insect the basic method of electrode building demonstrated here is easily extendable and/or can be adapted. For example it is conceivable to use more than the three single wires to produce multi-channel electrodes. In addition, the number of recording sites can be extended and observing temporal aspects of more than two tracts or neuropils is feasible. Our hope is that this method will inspire many scientists and will contribute positively to the understanding of sophisticated neuronal processing in small brains.

Honeybees as well as most other insects heavily rely on olfaction. Amongst others they use olfactory cues for orientation, mating, communication with conspecifics, and foraging. Their well elaborated olfactory system contributes to a rich repertoire of learning behaviors related to floral odor stimuli. These behaviors can be easily studied under controlled laboratory conditions (for review see3-5). Their &#8220;mini brains&#8221; (cp.6) with their relatively small numbers of neurons makes the honeybee a well suited model organism for studying olfactory coding and learning during monitoring of neural activity.
The olfactory system in insects as well as in mammals shows analogous organization to a great extent (for review see7,8). In honeybees about 80,000 receptor neurons9 situated in sensillae along the antennae10,11 translate the environmental odor stimulus into a neuronal signal. Axons from olfactory receptor neurons innervate the antennal lobe (AL), which has a glomerular organization comparable to the vertebrate olfactory bulb. The AL comprises about 164 glomeruli interconnected with each other by about 4,000 local interneurons (LN) (for review see12). Especially in the honeybee it has been shown recently that LNs provide patchy lateral connectivity and that different subpopulations possess elemental and configural olfactory coding properties13,14 . The AL was shown to be subdivided into a ventral and a dorsal hemi lobe giving rise to the medial and the lateral antennal lobe tract (m- and l-ALT; formerly termed m- and l-APT for medial- and lateral antennal lobe protocerebral tract15-17). Here a new tract terminology introduced by a recent effort for a unified nomenclature of the insect brain will be used18. Both ALTs (l- and m-ALT) combine either 410 (l-ALT) or 510 (m-ALT) uniglomerular projection neurons (PN), respectively15,16,19. PNs of both tracts have recently been shown to code odors in parallel2&#160;(for review see17,20), and both tracts synaptically form divergent connections with Kenyon Cells (KC), the mushroom body (MB) principal neurons. Each MB contains about 172,000 KCs21-23. The MBs are known to be involved in stimulus integration, learning, and memory formation. The axo dendrites of KCs form the peduncle (the mushroom&#180;s stem), which has two main output regions: the vertica &#160;or alpha- lobe and the horizontal &#160;or beta-lobe22,24. The output of the MB converges to only about 400 extrinsic neurons (EN)24. ENs responsible for olfactory information processing mostly innervate the ventral aspect of the vertical lobe22. Recently, it has been shown that ENs recorded at this area encode the odor reward association25.
Temporal aspects within the olfactory system of insects as well as vertebrates have become an important and significant aspect as a potential coding principle26-29. To be able to simultaneously record multiple neurons from different sites at high temporal resolution, we established double multi unit recording techniques using customized multi channel wire electrodes introduced to different target regions in the bee&#8217;s olfactory system. This approach enables us to analyze and compare temporal processing in the honeybee olfactory system at the level of single neurons and populations of neurons either between parallel olfactory pathways, the dual olfactory pathway2 or between different subsequent neuropils1. Recently with a similar experimental approach in the locust olfactory system30 using a different configuration of electrodes were able to analyze spatiotemporal coding mechanism for background-invariant odor recognition31. Thus, the established dual recordings enable gathering spatial information about simultaneous neuronal activity profiles.
Compared to the broader spatial sampling obtained from calcium imaging this method allows recording from two spots only. However, the advantage compared to calcium imaging techniques is the high temporal precision of action potential recordings, which cannot be provided by either conventional CCD imaging or 2-photon&#160;imaging acquisition. The extracellular electrodes described here are permanently implanted and fixed relative to the brain and head capsule avoiding electrode drift. This is a clear advantage compared to the usage of sharp intracellular electrodes. Another advantage compared to intracellular recordings and calcium imaging is the extended neural observation time ranging from many hours up to days. This is an important prerequisite to investigate neural correlates of learning and memory formation. Additional benefits of multi unit recordings are further outlined in the discussion section.
In this methodological overview the manufacturing procedure of custom design wire electrodes will be shown, adapted from32,33 and suited for long term multi unit recordings in the honeybee brain. Additionally, an example how these types of electrodes are permanently implanted at two different recording sites within the honeybee olfactory system to record simultaneously the l- and the m-ALT over long periods of time to allow many stimulation protocols is shown2. For verification of the recording positions an example and protocol for staining and post recording visualization of the recording sites is provided.

<table><tbody><tr><td>Paraffin oil</td><td>Fluka</td><td>76235</td><td></td></tr><tr><td>Odors</td><td>Sigma Aldrich&amp;nbsp;</td><td></td><td></td></tr><tr><td>PBS</td><td></td><td></td><td>pH 7.2</td></tr><tr><td>4% Formaldehyde</td><td>ThermoScientific</td><td>28908</td><td>Methanol free</td></tr><tr><td>Triton X</td><td>BioChemica</td><td>A1388</td><td></td></tr><tr><td>Methylsalicylate</td><td>Roth</td><td>4529.1</td><td></td></tr><tr><td>Tetramethylrhodamin dextran, 10,000 MW (Microruby)</td><td>Invitrogen</td><td>D7162</td><td>keep dark</td></tr><tr><td>Alexa 488 hydrazide</td><td>Invitrogen</td><td>A-10436</td><td>keep dark</td></tr><tr><td>Alexa 568 hydrazide</td><td>Invitrogen</td><td>A-10437</td><td>keep dark</td></tr><tr><td>Bee Ringer Solution</td><td>see Brill &lt;em&gt;et al.&lt;/em&gt;&lt;sup&gt;2&lt;/sup&gt;</td><td></td><td></td></tr><tr><td>Polyurethane-coated copper wire</td><td>Elektrisola</td><td>15 &amp;micro;m diameter &amp;amp; P155 insulation</td><td></td></tr><tr><td>Dental Wax&amp;nbsp;</td><td>Densply Detrey</td><td>64103015S1</td><td>moderate melting point</td></tr><tr><td>Dental Wax</td><td>Flexaponal&amp;nbsp;</td><td>124-202-00</td><td>low melting Wax</td></tr><tr><td>KWIK SIl</td><td>WPI</td><td>03L</td><td></td></tr><tr><td>18 Pin Socket</td><td>Conrad Electronic</td><td>189634-62</td><td></td></tr><tr><td>Hot melting glue</td><td>Conrad Electronic</td><td>827673</td><td></td></tr><tr><td>Soldering needle</td><td>Conrad Electronics</td><td>830283</td><td>12 V</td></tr><tr><td>Soldering terminal lug&amp;nbsp;</td><td>Conrad Electronic</td><td>531901</td><td></td></tr><tr><td>Glaselectrodes</td><td>WPI</td><td>1B100F-3</td><td></td></tr><tr><td>Minutien Pins</td><td>Fine Science Tools</td><td>26002-20</td><td>V2A 0.2 x 12 mm</td></tr><tr><td>Switchable headstage</td><td>Tucer Davis Technologies</td><td>SH16</td><td></td></tr><tr><td>Headstage connection module</td><td>NPI</td><td>INT-03M</td><td></td></tr><tr><td>Amplifier Module</td><td>NPI</td><td>PDA-2F</td><td></td></tr><tr><td>Data Acquisition boards</td><td>National Instruments</td><td>NI-6123, Ni-6143</td><td></td></tr><tr><td>Acquisition Software</td><td>National Instruments</td><td>Lab View 8.2</td><td>custom design</td></tr><tr><td>Spike-Sorting&amp;nbsp;</td><td>CED&amp;nbsp;</td><td>Spike 2 v7.11</td><td></td></tr><tr><td>Matlab</td><td>Mathworks</td><td>R2008B</td><td></td></tr><tr><td>Micromanipulator</td><td>Leitz</td><td></td><td>manual</td></tr><tr><td>AG-wires</td><td>WPI</td><td>AGT05100</td><td></td></tr><tr><td>Confocal laser scanning microscope</td><td>Leica</td><td>TCS SP2 AOBS</td><td></td></tr><tr><td>AMIRA</td><td>Mercury Computer Systems&amp;nbsp;</td><td>2/5/2000</td><td></td></tr></tbody></table>

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.

&#34;The present protocol allows simultaneous recordings at two different processing stages within individual honeybees and additionally allows to test underlying mechanisms of learning and memory via e.g., PER conditioning within restrained honeybees.&#34;&#160;This is a prerequisite for analyzing temporal aspects of neuronal processing. The method is easily adaptable for different scientific approaches to unravel the neuronal network of the bee&#8217;s olfactory system. For example this method is used (i) to analyze temporal processing of PNs within the dual olfactory pathway of the honeybee, the l- and m-ALT PNs (Figure&#160;5). In Figure 5A one example of an l-ALT PN simultaneously recorded with a PN of the m-ALT is given as ten trial average and illustrates their response strength and latency in respect to five different odor concentrations as color coded heat plot. In an average of seven bees with 11 l- and 13 m-ALT PNs (Figures 5B, 5C) illustrates that both the response strength as well as the response latency, to a certain extent, reflects odorant concentration. Thereby PNs in this example increase their response strength while with increasing odorant concentration their response latency declined (Figures 5B, 5C). This result is rather limited and only valid for the analyzed odorant, but is still consistent with recent computational models of the AL37. Whether odor concentration coding in the bee&#8217;s AL underlies non linear computations or underlies other coding properties still needs to be analyzed in future. Furthermore the method can be used (ii) to compare temporal aspects in the population activity at two subsequent processing stages, the AL- and MB-output (Figure&#160;6). Principal component analysis (PCA) illustrates that odor computation is prolonged and outlast the entire odor presentation at the PN level whereas in ENs only the odor on&#160;and off set were represented in the population activity (Figure&#160;6). Thereby the EN population reached their maximal activity already at a time point when the PN activity is still developing (cp. Movie 1).
<img alt="Figure 1" fo:content-width="4in" src="/files/ftp_upload/51750/51750fig1highres.jpg" width="400" /> 
Figure 1.&#160;Manufacturing three-channel micro-wire electrodes.&#160;A1) The endings of three wires are soldered to an IC pin connector in Positions 11, 13 and 16. A2) Four soldering lugs are screwed onto a Plexiglas plastic base plate; a minutien pin is inserted into the tip of a glass capillary which is then attached to the plastic plate. A3) The plastic plate is glued on top of an IC pin connector and the free end of each wire is soldered to one of the top soldering lugs. B1) To equip the electrode holder with the fine copper wires, the capillary has to be taken out once more. B2) The base of the holder is then fixed in a custom made aligning device. Three copper micro wires are aligned along the groove and fixed with adhesive tape at each end. B3) The parallel micro wires are glued together with dental wax and the capillary is put back in place (long arrows); the glued micro wires are then attached to the capillary with dental wax and its ends are cut off (short arrows). B4) The three loose ends of the copper wires are soldered to the three top soldering lugs thus bringing them into electrical contact with the IC pin connector. C1) Two fully assembled electrodes can be connected to each other for use with a single headstage. C2) For this purpose the pins of one electrode (left, slave) are connected via insulated wires to the other electrode (right, master), which is connected to the head stage; the headstage connected electrode can also collect input from a muscle (M17) and reference electrode (Ref).
<img alt="Figure 2" fo:content-width="5in" src="/files/ftp_upload/51750/51750fig2highres.jpg" width="500" /> 
Figure 2.&#160;Preparation and permanent electrode insertion into the bee brain.&#160;A) A bee is inserted into a Plexiglas holder after immobilization on ice. Antennae with Flagellum (FL) and Scapus (SC) are indicated. B) The head and antennae are fixed using dental wax. C) The head capsule is shaved and the bee is fed with sugar water. D) The head capsule is opened. E) After removing glands and trachea from the top of the brain, the different neuropiles and major landmarks can easily be distinguished. The trajectories of the ALTs are indicated together with a mark where the electrodes are inserted. (MB: Mushroom body, AL: Antennal lobe, OL: Optical lobe, &#945;: Alpha lobe or vertical lobe, ALT: antennal lobe tract, E1: electrode insertion side for m-ALT recordings, E2: electrode insertion side for l-ALT recordings). F) Reference (Ref) and muscle electrodes (M17) are inserted into the head capsule through little holes in the cuticle or the compound eye. G) The wire electrodes are inserted into the brain at the appropriate sites. H) After fixing the electrodes in place using two component silicon, the bee still shows PER and can be conditioned (e.g., using sugar water).
<img alt="Figure 3" fo:content-width="5in" src="/files/ftp_upload/51750/51750fig3highres.jpg" width="500" /> 
Figure 3.&#160;Extracellular recording at two neural tracts and single unit extraction (spike sorting). A) Schematic drawing of the experimental setup. The bee is fixated in a plexiglas holder. Odor stimulation is provided via a glass tube. Two electrode shanks are recording from the exposed bee brain. B) Simultaneous recordings from the l- and m-ALT PNs (green and purple traces) showing excitatory responses on both tracts to a 500 msec odor stimulation of honey in water solution at a concentration of 1:100 at 33 &#176;C. Each plotted line represents the differentiated channels as color-coded label from the electrode soldering lugs in A. Bar: 50 &#181;V. C) After spike sorting procedures single action potentials are sorted and color coded. Overlay of the sorted units illustrates the separation of the waveforms. D) Spike interval histogram indicates the separation quality of the sorted units as proof of adequate spike sorting. Note there is no spike within the unit&#8217;s refractory period. E) Two views (E1, E2) from a 3D clustering of the sorted unit with principal component analysis which indicates the distance of the sorted units to each other. Circles indicate the 2.5 fold Mahalanobis distance which resembles the SD in space and indicates a significant differentiation of the clusters in the principal component space F) Color coded units depict the action potentials visible in a magnification of three channels from one tract recording. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51750/51750fig3highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" fo:content-width="5in" src="/files/ftp_upload/51750/51750fig4highres.jpg" width="500" /> 
Figure 4.&#160;Post-recording visualization and 3D-reconstruction of the recording position.&#160;A) Projection view of ortho-slices along the z-axes with maximum intensity alignment of an antero&#160;and retrograde backfilling of the uniglomerular projection neurons projecting from the AL to the MB and LH. The intracellular tracer Microruby (tetramethylrhodamin dextran) was inserted into the AL after the recording experiments. Stained glomeruli inside the AL proof proper PN staining. B) Projection view of ortho slices along the z -axes with maximum intensity alignment from a staining of the two electrodes with Alexa hydrazide 488 indicating the electrode placement for the m-ALT (E1, arrow) and l-ALT (E2, arrow). The tracer Alexa Hydrazide 488 migrates to the electrode surrounding tissue and stains the electrode insertion site. Note, the prominent staining in the AL is a superficial artefact. C) 3D Reconstructions of the stained target cells (PNs) and the electrode insertion site from A,B (right side) together with a schematic overview of the honeybee olfactory system (left side) with the indication of l- and m-ALT trajectories. Note, only uniglomerular PN tracts are shown. AN: antennal nerve, AL: antennal lobe, LH: lateral horn, MB: mushroom body, E1,E2: electrode insertion sites, m-ALT: medial antennal lobe tract, l-ALT: lateral antennal lobe tract, c: caudal, r: rostral, m: medial, l: lateral. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51750/51750fig4highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" fo:content-width="3in" src="/files/ftp_upload/51750/51750fig5highres.jpg" width="300" /> 
Figure 5.&#160;Example of odor concentration coding within the dual olfactory pathway. A) Heat plots illustrate the response of a single l-ALT (green) and single m-ALT PN (purple) acquired simultaneously from an individual honeybee in response to the odorant hexanal at increasing odor concentrations (from 1: 10-6 to 1:100). Each line is an average of ten trial stimulation. The firing rate is shown as relative intensity change which is the firing rate in relation to the subtracted spontaneous activity. B) Population response latency of 11 l- and 13 m-ALT PNs from 7 recorded bees. In each bee the PNs from both tracts were recorded simultaneously. The population response latency shows a decreasing latency with increasing odor concentration in PNs from both tracts. The odor response onset at the antennae at 99 msec was recorded via electroantennograms and is subtracted from the PN response latencies. Note that at the lowest concentrations responses are too weak for latency measurements and, therefore, were excluded. C) Population response firing rate from same PNs as in B. With increasing odor concentration the response strength is increasing. The l-ALT shows a stronger response strength. In B,C the mean and SD are given.
<img alt="Figure 6" fo:content-width="5in" src="/files/ftp_upload/51750/51750fig6highres.jpg" width="500" /> 
Figure 6.&#160;Comparing population activity at two subsequent processing stages along the honeybee&#8217;s olfactory pathway.&#160;Data were recorded in 20 animals which were stimulated with 1-hexanol and 2-octanone. A) Each line represents the false color coded mean firing rate of one projection neuron (PN) calculated across 10 odor repetitions of 1-hexanol. Odor presentation starts at time 0 and lasted three seconds. B) Shows the same as in A) but for mushroom body extrinsic neurons (EN). The matrix shown in A) can be seen as a PN population vector during odor stimulation with 1-hexanol. We calculated the same kind of population vector during odor stimulation with 2-octanone and used both vectors in a principal component analysis (PCA) keeping the temporal dimension. C)&#160;The first three principal components (PC1, 2 and 3) were plotted against each other to illustrate the odor separation in the PN ensemble activity at the antennal lobe output. The time before odor onset is marked in black. Activity during three seconds of stimulation with 1-hexanol is shown in blue. The activity during stimulation with 2-octanol is shown in red. Furthermore, we show 1.5 seconds (post) of the activity after odor of set of 1-hexanol (light-blue) and 2-octanonen (pink). Note that at the PN ensemble level, both odors evoking very distinct trajectories settle in a &#8220;fixed point&#8221; which outlasts the whole odor stimulation period. Only after odor offset the trajectories move back to the baseline activity without odor stimulation. D) The same analysis was done at the EN ensemble level representing the activity at the mushroom body output. Compared to the PN activity odors evoke a less distinct trajectory. Furthermore a &#8220;fixed point&#8221; is not observable. The initially odor induced trajectories intermingle with baseline activity although the odor is still present. Only the odor offset evoked an additional trajectory.
<a href="/files/ftp_upload/51750/Movie_R3_Brill_et_al._JoVE.avi" target="_blank">Movie 1.</a> Time resolved evaluation of an odor induced trajectory after principal component analysis of a PN-population vector (left) and an EN population vector (right; cp. Figure&#160;6). The upper parts include the first three principal components (PC1, 2 and 3) plotted against each other. The lower panels illustrate the evaluation of PC1, 2 and 3 over time. Odor stimulation is marked by the gray bar. All panels were synchronized. Note that the EN population activity starts slightly before the PN population activity,&#160;a phenomenon which seems to be contraintuitive but can be explained by the connectivity and properties of the involved layers, which is discussed earlier1.

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

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 ...

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Nanyang Technological University

Research

JoVE Journal

Neuroscience

12.9K Views.  University of Würzburg. 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.

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

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.

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

Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster

To study neuronal networks in terms of their function in behavior, we must analyze how neurons operate when each behavioral pattern is generated. Thus, simultaneous recordings of neuronal activity and behavior are essential to correlate brain activity to behavior. For such behavioral analyses, the fruit fly, Drosophila melanogaster, allows us to incorporate genetically encoded calcium indicators such as GCaMP1, to monitor neuronal activity, and to use sophisticated genetic manipulations for optogenetic or thermogenetic techniques to specifically activate identified neurons2-5. Use of a thermogenetic technique has led us to find critical neurons for feeding behavior (Flood et al., under revision). As a main part of feeding behavior, a Drosophila adult extends its proboscis for feeding6 (proboscis extension response; PER), responding to a sweet stimulus from sensory cells on its proboscis or tarsi. Combining the protocol for PER7 with a calcium imaging technique8 using GCaMP3.01, 9, I have established an experimental system, where we can monitor activity of neurons in the feeding center &#x2013; the suboesophageal ganglion (SOG), simultaneously with behavioral observation of the proboscis. I have designed an apparatus ("Fly brain Live Imaging and Electrophysiology Stage": "FLIES") to accommodate a Drosophila adult, allowing its proboscis to freely move while its brain is exposed to the bath for Ca2+ imaging through a water immersion lens. The FLIES is also appropriate for many types of live experiments on fly brains such as electrophysiological recording or time lapse imaging of synaptic morphology. Because the results from live imaging can be directly correlated with the simultaneous PER behavior, this methodology can provide an excellent experimental system to study information processing of neuronal networks, and how this cellular activity is coupled to plastic processes and memory.

Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster

The fruit fly, Drosophila melanogaster, extends its proboscis for feeding, responding to a sugar stimulus from its proboscis or tarsus. I have combined observations of the proboscis extension response (PER) with a calcium imaging technique, allowing us to monitor the activity of neurons in the brain, simultaneously with behavioral observation.

To study neuronal networks in terms of their function in behavior, we must analyze how neurons operate when each behavioral pattern is generated.  Thus, ...

Identification of Olfactory Volatiles using Gas Chromatography-Multi-unit Recordings (GCMR) in the Insect Antennal Lobe

All organisms inhabit a world full of sensory stimuli that determine their behavioral and physiological response to their environment. Olfaction is especially important in insects, which use their olfactory systems to respond to, and discriminate amongst, complex odor stimuli. These odors elicit behaviors that mediate processes such as reproduction and habitat selection1-3. Additionally, chemical sensing by insects mediates behaviors that are highly significant for agriculture and human health, including pollination4-6, herbivory of food crops7, and transmission of disease8,9. Identification of olfactory signals and their role in insect behavior is thus important for understanding both ecological processes and human food resources and well-being.
	To date, the identification of volatiles that drive insect behavior has been difficult and often tedious. Current techniques include gas chromatography-coupled electroantennogram recording (GC-EAG), and gas chromatography-coupled single sensillum recordings (GC-SSR)10-12. These techniques proved to be vital in the identification of bioactive compounds. We have developed a method that uses gas chromatography coupled to multi-channel electrophysiological recordings (termed 'GCMR') from neurons in the antennal lobe (AL; the insect's primary olfactory center)13,14. This state-of-the-art technique allows us to probe how odor information is represented in the insect brain. Moreover, because neural responses to odors at this level of olfactory processing are highly sensitive owing to the degree of convergence of the antenna's receptor neurons into AL neurons, AL recordings will allow the detection of active constituents of natural odors efficiently and with high sensitivity. Here we describe GCMR and give an example of its use.
	Several general steps are involved in the detection of bioactive volatiles and insect response. Volatiles first need to be collected from sources of interest (in this example we use flowers from the genus Mimulus (Phyrmaceae)) and characterized as needed using standard GC-MS techniques14-16. Insects are prepared for study using minimal dissection, after which a recording electrode is inserted into the antennal lobe and multi-channel neural recording begins. Post-processing of the neural data then reveals which particular odorants cause significant neural responses by the insect nervous system.
	Although the example we present here is specific to pollination studies, GCMR can be expanded to a wide range of study organisms and volatile sources. For instance, this method can be used in the identification of odorants attracting or repelling vector insects and crop pests. Moreover, GCMR can also be used to identify attractants for beneficial insects, such as pollinators. The technique may be expanded to non-insect subjects as well.

Identification of Olfactory Volatiles using Gas Chromatography-Multi-unit Recordings GCMR in the Insect Antennal Lobe

Olfactory cues mediate many different behaviors in insects, and are often complex mixtures comprised of tens to hundreds of volatile compounds. Using gas chromatography with multi-channel recording in the insect antennal lobe, we describe a method for the identification of bioactive compounds.

All  organisms inhabit a world full of sensory stimuli that determine their  behavioral and physiological response to their environment. Olfaction is ...

Multi-unit Recording Methods to Characterize Neural Activity in the Locust (Schistocerca Americana) Olfactory Circuits

Detection and interpretation of olfactory cues are critical for the survival of many organisms. Remarkably, species across phyla have strikingly similar olfactory systems suggesting that the biological approach to chemical sensing has been optimized over evolutionary time1. In the insect olfactory system, odorants are transduced by olfactory receptor neurons (ORN) in the antenna, which convert chemical stimuli into trains of action potentials. Sensory input from the ORNs is then relayed to the antennal lobe (AL; a structure analogous to the vertebrate olfactory bulb). In the AL, neural representations for odors take the form of spatiotemporal firing patterns distributed across ensembles of principal neurons (PNs; also referred to as projection neurons)2,3. The AL output is subsequently processed by Kenyon cells (KCs) in the downstream mushroom body (MB), a structure associated with olfactory memory and learning4,5. Here, we present electrophysiological recording techniques to monitor odor-evoked neural responses in these olfactory circuits.
First, we present a single sensillum recording method to study odor-evoked responses at the level of populations of ORNs6,7. We discuss the use of saline filled sharpened glass pipettes as electrodes to extracellularly monitor ORN responses. Next, we present a method to extracellularly monitor PN responses using a commercial 16-channel electrode3. A similar approach using a custom-made 8-channel twisted wire tetrode is demonstrated for Kenyon cell recordings8. We provide details of our experimental setup and present representative recording traces for each of these techniques.

Multi-unit Recording Methods to Characterize Neural Activity in the Locust Schistocerca Americana Olfactory Circuits

We demonstrate variations of the extracellular multi-unit recording technique to characterize odor-evoked responses in the first three stages of the invertebrate olfactory pathway. These techniques can easily be adapted to examine ensemble activity in other neural systems as well.

Detection and interpretation of olfactory cues are critical for the survival  of many organisms. Remarkably, species across phyla have strikingly similar ...

In vivo Ca2+- Imaging of Mushroom Body Neurons During Olfactory Learning in the Honey Bee

The in vivo and semi-in vivo preparation for Calcium imaging has been developed in our lab by Joerges, K&uuml;ttner and Galizia over ten years ago, to measure odor evoked activity in the antennal lobe1. From then on, it has been continuously refined and applied to different neuropiles in the bee brain. Here, we describe the preparation currently used in the lab to measure activity in mushroom body neurons using a dextran coupled calcium-sensitive dye (Fura-2). We retrogradely stain mushroom body neurons by injecting dye into their axons or soma region. We focus on reducing the invasiveness, to achieve a preparation in which it is still possible to train the bee using PER conditioning. We are able to monitor and quantify the behavioral response by recording electro-myograms from the muscle which controls the PER (M17)2.
After the physiological experiment the imaged structures are investigated in greater detail using confocal scanning microscopy to address the identity of the neurons.

In vivo Ca2+- Imaging of Mushroom Body Neurons During Olfactory Learning in the Honey Bee

Bees can be conditioned in an appetitive olfactory learning paradigm (PER-conditioning). Using odors as stimuli, we established a method in which behavior is recorded while simultaneously Calcium Imaging is used to measure odor evoked activity in mushroom body neurons in vivo.

The in vivo and semi-in vivo preparation for Calcium imaging has been developed in our lab by Joerges, K&uuml;ttner and Galizia over ten years ago, to ...

Biology

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.

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

A Proboscis Extension Response Protocol for Investigating Behavioral Plasticity in Insects: Application to Basic, Biomedical, and Agricultural Research

Insects modify their responses to stimuli through experience of associating those stimuli with events important for survival (e.g., food, mates, threats). There are several behavioral mechanisms through which an insect learns salient associations and relates them to these events. It is important to understand this behavioral plasticity for programs aimed toward assisting insects that are beneficial for agriculture. This understanding can also be used for discovering solutions to biomedical and agricultural problems created by insects that act as disease vectors and pests. The Proboscis Extension Response (PER) conditioning protocol was developed for honey bees (Apis mellifera) over 50 years ago to study how they perceive and learn about floral odors, which signal the nectar and pollen resources a colony needs for survival. The PER procedure provides a robust and easy-to-employ framework for studying several different ecologically relevant mechanisms of behavioral plasticity. It is easily adaptable for use with several other insect species and other behavioral reflexes. These protocols can be readily employed in conjunction with various means for monitoring neural activity in the CNS via electrophysiology or bioimaging, or for manipulating targeted neuromodulatory pathways. It is a robust assay for rapidly detecting sub-lethal effects on behavior caused by environmental stressors, toxins or pesticides.
We show how the PER protocol is straightforward to implement using two procedures. One is suitable as a laboratory exercise for students or for quick assays of the effect of an experimental treatment. The other provides more thorough control of variables, which is important for studies of behavioral conditioning. We show how several measures for the behavioral response ranging from binary yes/no to more continuous variable like latency and duration of proboscis extension can be used to test hypotheses. And, we discuss some pitfalls that researchers commonly encounter when they use the procedure for the first time.

The Proboscis Extension Response (PER) conditioning protocol, developed for the honey bee (Apis mellifera), provides an ecologically-relevant and easily quantifiable means for studying several different mechanisms of learning in many insect species.

Insects modify their responses to stimuli through experience of associating those stimuli with events important for survival (e.g., food, mates, threats). ...

Simultaneous Monitoring of Wireless Electrophysiology and Memory Behavioral Test as a Tool to Study Hippocampal Neurogenesis

Brainwaves amplitude obtained from electroencephalography (EEG) has been well-recognized as a basis for cognitive capacity, memory, and learning on animals and humans. Adult neurogenesis mechanism is also linked to memory and learning improvement. Traditionally, researchers used to assess learning and memory parameters in rodent models by behavioral tasks. Therefore, the simultaneous monitoring of behavioral changes and EEG is particularly interesting in correlating data between brain activity and task-related behaviors. However, most of the equipment required to perform both studies are either complex, expensive, or uses a wired setup network that hinders the natural animals&#8217; movement. In this study, EEG was recorded with a wireless electrophysiology device along with the execution of a novel object recognition task (NORT). The animal&#8217;s behavior was monitored simultaneously by a video tracking system. Both recordings were analyzed offline by their timestamps which were synched to link EEG signals with the animal&#8217;s actions. Subjects consist of adult Wistar rats after medium-term environmental enrichment treatment. Six skull screw electrodes were fixed in pairs on both hemispheres over frontal, central, and parietal regions and were referenced to an electrode located posterior of the nasal bone. NORT protocol consists of exposing the animal to two identical objects for 10 min. After 2 h and 24 h, one of the objects was replaced with a novel one. Exploration time for each object was monitored by a behavioral tracking software (BTS) and EEG data recording. The analysis of the EEG synced with behavioral data consists of estimations of alpha and beta relative band power and comparisons between novel object recognition versus familiar object exploration, between three experimental stages. In this manuscript, we have discussed electrodes manufacturing process, epidural electrodes implantation surgery, environmental enrichment protocol, NORT protocol, BTS setup, EEG &#8211; BTS coupling for simultaneous monitoring in real-time, and EEG data analysis based on automatic events detection.

The protocol presented here provides information on the simultaneous electroencephalography (EEG) and behavioral assessment in real-time. We have discussed all steps involved in this protocol as an attractive solution for researchers in many fields of neuroscience, particularly in learning and memory areas.

Brainwaves amplitude obtained from electroencephalography (EEG) has been well-recognized as a basis for cognitive capacity, memory, and learning on ...

Behavior

Recording Single Neurons' Action Potentials from Freely Moving Pigeons Across Three Stages of Learning

While the subject of learning has attracted immense interest from both behavioral and neural scientists, only relatively few investigators have observed single-neuron activity while animals are acquiring an operantly conditioned response, or when that response is extinguished. But even in these cases, observation periods usually encompass only a single stage of learning, i.e. acquisition or extinction, but not both (exceptions include protocols employing reversal learning; see Bingman&#160;et al.1 for an example). However, acquisition and extinction entail different learning mechanisms and are therefore expected to be accompanied by different types and/or loci of neural plasticity.
Accordingly, we developed a behavioral paradigm which institutes three stages of learning in a single behavioral session and which is well suited for the simultaneous recording of single neurons&#39; action potentials. Animals are trained on a single-interval forced choice task which requires mapping each of two possible choice responses to the presentation of different novel visual stimuli (acquisition). After having reached a predefined performance criterion, one of the two choice responses is no longer reinforced (extinction). Following a certain decrement in performance level, correct responses are reinforced again (reacquisition). By using a new set of stimuli in every session, animals can undergo the acquisition-extinction-reacquisition process repeatedly. Because all three stages of learning occur in a single behavioral session, the paradigm is ideal for the simultaneous observation of the spiking output of multiple single neurons. We use pigeons as model systems, but the task can easily be adapted to any other species capable of conditioned discrimination learning.

Learning new stimulus-response associations engages a wide range of neural processes which are ultimately reflected in changing spike output of individual neurons. Here we describe a behavioral protocol allowing for the continuous registration of single-neuron activity while animals acquire, extinguish, and reacquire a conditioned response within a single experimental session.

While the subject of learning has attracted immense interest from both behavioral and neural scientists, only relatively few investigators have observed ...

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.

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

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

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Chapters in this video

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

Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster

Identification of Olfactory Volatiles using Gas Chromatography-Multi-unit Recordings (GCMR) in the Insect Antennal Lobe

Multi-unit Recording Methods to Characterize Neural Activity in the Locust (Schistocerca Americana) Olfactory Circuits

In vivo Ca²⁺- Imaging of Mushroom Body Neurons During Olfactory Learning in the Honey Bee

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

A Proboscis Extension Response Protocol for Investigating Behavioral Plasticity in Insects: Application to Basic, Biomedical, and Agricultural Research

Simultaneous Monitoring of Wireless Electrophysiology and Memory Behavioral Test as a Tool to Study Hippocampal Neurogenesis

Recording Single Neurons' Action Potentials from Freely Moving Pigeons Across Three Stages of Learning

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