This is the most robust and sensitive method for assessing olfactory sensitivity in marine fish as it records a purely sensory signal prior to any processing by the CNS. The main advantages of this technique are its sensitivity and that it can be easily transferred to a different species from a different environment independent of external salinity. To prepare the control water, collect one liter of charcoal-filtered seawater and use a pH probe to check the pH.
The pH should be around 8.2. If it is not, bubble the seawater with atmospheric air until a pH of 8.2 is reached. Next, use an alkalinity titrator to measure the alkalinity of the water, then measure the water temperature and salinity.
To prepare high carbon dioxide water, filter one liter of seawater through particulate inactivated charcoal filters before bubbling with carbon dioxide until the desired pH is reached. Use a pH probe to check the pH, which should be around 7.7, then use an alkalinity titrator to measure the alkalinity of the water and measure the water temperature and salinity. To determine the carbon dioxide pressure in both the control and high carbon dioxide water, open a software program designed to calculate carbon dioxide parameters in water and add the water salinity, temperature, pressure, total alkalinity, and pH values into the input window.
Then select the constants, scales, and units, and click process to determine the carbon dioxide pressure. After confirming an appropriate level of sedation by a lack of response to tail pinch, place the anesthetized fish onto a cushion support customized for the model and place an appropriately-sized silicon tube into the fish's mouth. The tube is connected to a submersible pump and a reservoir of anesthetic containing aerated seawater and water is pumped over the gills in an approximately 100 milliliter per minute per kilogram rate.
Next, insert an earthing pin into the flank muscle and connect the pin to the head stage of the amplifier. Cover the fish with damp cloth with only the head exposed and position the tube with the stimulus delivery system within the nostril. To expose the olfactory nerves, use a drill and a dissecting microscope to remove the skin and bone of the skull between the eyes.
When using a sea bream, use a circular saw to remove the part of the skull immediately above the eyes to just posterior to the eyes and use the drill to remove the bone between the eyes. Once sufficient bone has been cleared, use fine forceps to remove the fat and connective tissue overlying the olfactory nerves without damaging the nerves or vessels. Clean the electrodes prior to use by connecting them to the negative pole of a three volt DC source and placing the tip in physiological saline for 20 to 30 seconds.
A steady stream of small bubbles should be seen coming from the tip. Connect parylene-coated tungsten electrodes to the head stage of an alternating current AC preamplifier. Once the olfactory nerves have been exposed, use micro-manipulators to insert the electrodes into the appropriate olfactory nerve in a position that gives a maximum response to the standard and a minimal response to the blank.
Providing the dissection has gone smoothly, the critical step is getting the electrodes into the correct position. This can take time and involves a certain degree of trial and error. To obtain electrophysiological readings, use a solenoid operated three-way valve to set up a stimulus delivery system to allow the rapid switching from clean background water to stimulus containing water and connect the common outlet to the tube carrying water to the olfactory rosette.
Place one line in background seawater and the other in the test solution. Connect the valve driver to the trigger of an analog digital converter and configure the software to start recording at the trigger event and to continue for a predetermined period. To check the stability of the preparation, repeatedly record and measure the amplitude of the integrated response repeatedly with the standard allowing one minute to elapse between successive stimuli.
Next, record the olfactory nerve responses to amino acids in control seawater, and allow one minute to elapse between successive stimuli, then record the response to one times 10 to the negative third molar serine in a control water blank solution. Then record the olfactory nerve responses to amino acids in high carbon dioxide seawater. The response to a high carbon dioxide water blank solution and the response to one times 10 to the negative third molar serine in a control water blank solution.
Here, typical responses to positive and negative control stimuli recorded from the olfactory nerve of a sea bream are shown. Within about one second of stimulus application, a rapid increase in activity can be observed followed by a period of accommodation while the stimulus is still present and a return to baseline activity once the stimulus has ended. In contrast, a blank stimulus evokes little or no response.
In this typical concentration response curve, it can be observed that increasing concentrations of an odorant can evoke increasingly larger increases in the activity and therefore amplitude of the integrated responses. Plotting of the normalized data and corresponding linear regression allows estimation of the threshold of detection for the concentration responses of each animal or a group of animals. Sigmoidal concentration response curves can be plotted semi-logarithmically, allowing calculation of the odorant concentration that evokes at 50%maximal response.
It is important to keep the fish as healthy as possible. For example, make sure that the gills are adequately irrigated and that the olfactory epithelium is kept wet during the recording. One of our goals is to trace the neural pathways in the brain to where the sensory information is relayed.
