El procedimiento de cálculo de la teoría T sobre la incertidumbre

After making a dorsal preparation, the MdN are severed to prevent movement of the mandibles. This allows for the mouthparts to be extended to allow for tastant delivery. The mouthparts are propped on a glass slide, and the glass slide is fixed with wax to the base of the tube that holds the bee. A tastant delivery system (Bioscience tools, PC-16 controller driven by Manostat Compulab 3) then pumps the tastant solution onto the mouthparts. This tastant system was set up to supply a 2 second flow of distilled water, followed by a 3 second flow of tastant solution, and finally a 3 second wash with distilled water. This was repeated for as many stimulations as required.

4.3.5 Spike sorting

The same technique was used as described in Chapter 2 for spike sorting the multi-channel tungsten recordings. The only difference here was that three or four multi-channels of

recording were used for the sorting procedure. Spike waveforms from all channels that exhibited prominent peaks of action potentials were used.

4.4 Results

4.4.1 Physiological preparation

The axons of GRNs project towards the sub-esophageal zone (SEZ) via the MxN, which in insects like the bumble bee can be accessed from either the dorsal or ventral sides of the head. For a ventral preparation, the head was tilted up and fixed in place by building a column of wax on either side of the head just under the eyes. To prevent the mandibles from closing on the mouthparts, incisions are then made a few millimeters into the cuticle below the mandibles to cut the muscles which control their movement.

The mouthparts are then pushed forwards along the elbow joint between the cardo and the base of the cardo, which exposes the membranous floor of the proboscis fossa (figure 4.2 a). The nerves from the SEZ leading towards the mouthparts are slack, which allow for the mouthparts to be moved forwards without damaging the nerves. The mouthparts are then kept in place by threading them through plastic tubing, and fixing the plastic tubing to the top of the head using wax. A wax dome is then built around the head capsule and is filled with a few drops of Manduca physiological saline [221]. Next, an incision is made in the proboscis fossa and the cardo are cut at the base on both sides. The thin layer of trachea that is underneath the fossa is then removed, which exposes part of the SEZ and the mouthpart nerves (figure 4.2 b).

Figure. 4.2: Ventral dissection. a) Ventral view of the bee head, anterior end facing the bottom of the page. Pushing the mouthparts forward exposes the proboscis fossa. Making an incision in the fossa exposes the sub-esophageal zone (SEZ) (b) Md, mandibles; Prm, prementum; LbN, labial nerve; MxN, maxillary nerve; MdN, mandibular nerve. Figure 4.1 a obtained from Snodgrass 1956

While the ventral preparation provides a way to reach the nerves and SEZ with minimal dissection, it was associated with several complications. First, the animals show a high tendency of either dying (4 out of 7 preparations died), as indicated by the absence of spiking activity from extracellular recordings made from the SEZ and the absence of any physical movement from the bee. The remaining 3 bees did not exhibit any response to tastant stimulation. Second, it was difficult to de-sheath the nerve and SEZ due to the fact that the working area for using forceps is very small in a ventral preparation. Finally, it was difficult to get the esophagus to stop

generating movement, even if a muscle-dissolving enzyme such as collagenase is used.

Approaching the SEZ from the dorsal side of the head overcomes these complications.

The dorsal head capsule of B. impatiens measures approximately 2.5 x 3.0 x 3.5 mm (anterior-posterior x dorso-ventral x lateral ; see figure 1.2 a for orientation). To make a dorsal preparation, the head is fixed in place with drops of wax on the sides of the head. A wax dome is then built around the head to hold saline. The antennae are then gently threaded through plastic tubing to prevent any injury, and the tubing was fixed to the wax dome. The resulting setup is shown in figure 4.3 a.

Incisions are made in the head capsule as shown in figure 4.3 a, and the antennae are gently pulled to the sides, making sure not sever the antennal nerves. This serves to pull apart the antennal lobes by their antennal nerves, which allows better access to the SEZ. It additionally allows for measuring responses of SEZ neurons that receive input from antennal olfactory neurons. Next, the fat bodies and tracheae are removed, which exposes the esophagus (figure 4.3 b). An incision is made in the esophagus and it is gently pulled towards the back of the head capsule. Severing the esophagus prevents the pumping movement of the gut which leads to an

unstable preparation. Care has to be taken at this stage as it is easy to damage the mouthpart nerves, which lie just under the esophagus. Further, the forceps have to keep the esophagus pinched while making the incision, otherwise digestive fluids can flow into the brain which causes immediate damage to the neurons. Another pair of forceps should be used to tease apart the trachea connecting the ventral side of the esophagus to the nerves and the SEZ. A drop of wax is used to fix the esophagus to the wax dome. At this stage, the brain should be quite dry which makes it easy to observe the three pairs of mouthpart nerves (figure 4.2 c). The

preparation can now be used to access the mouthpart nerves for staining, or can be readied for electrophysiological recording. The entire dorsal preparation takes approximately 45 minutes.

Figure 4.3 (legend on next page)

Figure 4.3 : Dorsal preparation. a) The bee is held in a tube with the head sticking out.

Antennae (Ant.) are threaded through silicone tubing and fixed to the wax dome built around the head. Incisions are made in the head capsule as indicated by dotted lines, which allow the cuticle at the base of the antennae to be pulled to the sides (arrows). b) View of the brain immediately after pulling apart the antennae. An incision (dashed blue line) is made in the esophagus (Eso, outlined by dashed yellow lines) and it is pulled towards the back of the head capsule (arrow). c) View of the dorsal dissection after moving the esophagus. SEZ:

sub-esophageal zone (anterior surface outlined in orange), MdN: mandibular nerve, MxN: maxillary nerve, LbN: labial nerve, AL: antennal lobe (outlined in pink).