Simultaneous Long-term Recordings at Two Neuronal Processing Stages in Behaving Honeybees
PREPARAÇÃO DO INSTRUTOR
CONCEITOS
PROTOCOLO DO ALUNO
1. Electrode Building (Figure 1)
- Production of an electrode adapter that fits the electrode interface board of commercial multi channel amplifier systems1,2,25.
- Use a small Plexiglas plate glued to an 18 Pin connector base.
- Connect the base with 3 short pieces of insulated wire to 3 separate soldering lugs screwed onto the Plexiglas plate (Figure 1 A1-A3).
- Insert a groove into the Plexiglas plate in which a glass capillary can easily move and be held in place by a screw (Figure 1 B1).
- Extend the glass capillary about 5 mm using a minutien pin.
- Attach the micro electrode wires along the minutien pin and the glass capillary to guarantee stabilization and support.
- Multi-channel micro wire production (adopted from Ryuichy Okada32,33)
- Span 3 micro wires (polyurethane coated copper wire, 15 µm diameter) in a way that they are placed next to each other (Figure 1 B2).
- Use a 12 V soldering needle to spread a thin film of low melting dental wax (50 °C) partially along the wires to glue them together (electrode tip) (Figure 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.
- Connect the multi channel micro wire to the electrode adapter
- 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).
- 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 1 B3).
- Slip the capillary slightly back into the electrode adapter. Use the screw to fix it (Figure 1 B4).
- Solder the loose ends of the three wires to the soldering lugs using a soldering gun with a temperature of around 360 °C to ensure melting of the insulation (Figure 1 B4). After soldering, ensure that there is adequate electrical contact (~300 kOhm).
- 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 1 C1). Additionally, solder the reference as well as muscle electrodes to the master electrode base (Figure 1 C2).
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.
- Catch honeybee foragers (A. mellifera) at the hive entrance in the morning as shown by others34,35.
- 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 2 A). For minimizing head movements use low-melting dental wax (~50 °C) and fix the head carefully to the holder around the basis of the compound eyes and the neck.
- Use low melting wax to fixate the scapi of the antennae on the head capsule (Figure 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.
- Shave the head capsule to ensure an undisturbed view and access to the top of the head.
- 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).
- 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 2 D).
- 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).
3. Electrode Insertion
In the example case illustrated in Figure 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.
- Position the electrodes using micromanipulators at the region of interest (Figure 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 µm (Figure 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 µm (Figure 2E)
- Insert the reference (silver wire, about 25 µm 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.
- To solidly anchor the electrodes within the brain and the head capsule, cover the entire space above the brain with two component silicon (Figure 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 2H) or stimulated with a large panel of different odors.
4. Data Acquisition and Preprocessing
- Use proper acquisition software that meets the following requirements: sampling rate of min 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.
- Use available spike sorting software to extract single unit activity, for example template matching techniques as included in the Spike2 software (Figure 3).
- 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.
5. Visualization of Relative Electrode Position (Figure 4)
- Dip the electrodes tips into a solution of either 5% Alexa hydrazide 568 or 5% Alexa hydrazide 488 which is dissolved in 0.5 M potassium chloride solution prior to the recording experiments.
- 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.
- 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 solution three times for another 30-45 min.
- 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 °C.
- Wait at least 12 hr before washing the brain two times in 0.1 M PBS (10 min each).
- Wash the brain 3x 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).
- Embed the dehydrated brain in Methylsalicylate on a microscope slide and seal it with a cover slide.
- Use a confocal laser scanning microscope and scan the brain as optical sections every 2-5 µm using a Harmonic Compound Plan Apochromat objective (10X 0.4 NA immersion). Excite the tissue using 568 nm wavelength for tetramethylrhodamin dextran and a wavelength of 488 nm for the electrode position.
- Reconstruct the stained brain structures and electrode paths from the image stacks in 3D with reconstruction software (e.g. AMIRA or Fiji) (Figure 4).
Simultaneous Long-term Recordings at Two Neuronal Processing Stages in Behaving Honeybees
Learning Objectives
"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." 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’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 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’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 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 and off set were represented in the population activity (Figure 6). Thereby the EN population reached their maximal activity already at a time point when the PN activity is still developing (cp. Movie 1).
Figure 1. Manufacturing three-channel micro-wire electrodes. 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).
Figure 2. Preparation and permanent electrode insertion into the bee brain. 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, α: 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).
Figure 3. 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 °C. Each plotted line represents the differentiated channels as color-coded label from the electrode soldering lugs in A. Bar: 50 µV. 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’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. Please click here to view a larger version of this figure.
Figure 4. Post-recording visualization and 3D-reconstruction of the recording position. A) Projection view of ortho-slices along the z-axes with maximum intensity alignment of an antero 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. Please click here to view a larger version of this figure.
Figure 5. 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.
Figure 6. Comparing population activity at two subsequent processing stages along the honeybee’s olfactory pathway. 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) 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 “fixed point” 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 “fixed point” 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.
Movie 1. 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 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, a phenomenon which seems to be contraintuitive but can be explained by the connectivity and properties of the involved layers, which is discussed earlier1.
List of Materials
| Paraffin oil | Fluka | 76235 | |
| Odors | Sigma Aldrich | ||
| PBS | pH 7.2 | ||
| 4% Formaldehyde | ThermoScientific | 28908 | Methanol free |
| Triton X | BioChemica | A1388 | |
| Methylsalicylate | Roth | 4529.1 | |
| Tetramethylrhodamin dextran, 10,000 MW (Microruby) | Invitrogen | D7162 | keep dark |
| Alexa 488 hydrazide | Invitrogen | A-10436 | keep dark |
| Alexa 568 hydrazide | Invitrogen | A-10437 | keep dark |
| Bee Ringer Solution | see 2 | ||
| Polyurethane-coated copper wire | Elektrisola | 15µm diameter & P155 insulation | |
| Dental Wax | Densply Detrey | 64103015S1 | moderate melting point |
| Dental Wax | Flexaponal | 124-202-00 | low-melting Wax |
| KWIK SIl | WPI | 03L | |
| 18 Pin Socket | Conrad Electronic | 189634-62 | |
| Hot melting glue | Conrad Electronic | 827673 | |
| soldering needle | Conrad Electronics | 830283 | 12 V |
| Soldering terminal lug | Conrad Electronic | 531901 | |
| Glaselectrodes | WPI | 1B100F-3 | |
| Minutien Pins | Fine Science Tools | 26002-20 | V2A 0.2 x 12 mm |
| switchable headstage | Tucer Davis Technologies | SH16 | |
| Headstage connection module | NPI | INT-03M | |
| Amplifier Module | NPI | PDA-2F | |
| Data Acquisition boards | National Instruments | NI-6123, Ni-6143 | |
| Acquisition Software | National Instruments | Lab View 8.2 | custom design |
| Spike-Sorting | CED | Spike 2 v7.11 | |
| Matlab | Mathworks | R2008B | |
| Micromanipulator | Leitz | manual | |
| AG-wires | WPI | AGT05100 | |
| Confocal laser scanning microscope | Leica | TCS SP2 AOBS | |
| AMIRA | Mercury Computer Systems | 2/5/2000 |
Preparação do Laboratório
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. 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 channel wire electrodes is demonstrated. Pairwise differential amplification of the micro 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 term recordings over many hours up to days, which is a clear advantage compared to conventional extra and intracellular in vivo recording techniques.
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. 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 channel wire electrodes is demonstrated. Pairwise differential amplification of the micro 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 term recordings over many hours up to days, which is a clear advantage compared to conventional extra and intracellular in vivo recording techniques.
Procedimento
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. 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 channel wire electrodes is demonstrated. Pairwise differential amplification of the micro 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 term recordings over many hours up to days, which is a clear advantage compared to conventional extra and intracellular in vivo recording techniques.