Behavioural studies

We will use preference/avoidance olfactory tests, multi-sensory integration tests and appetitive/aversive conditioning experiments in order to find useful sensory stimuli for functional neurobiology experiments. The behavioural studies are of great interest in themselves especially as regards the distinction of innate versus learned control of behaviour in amphibians.

Descriptive neuroanatomy

Tract tracing of brain pathways and intracellular labelling of single/few neurons will be performed in in vitro brain preparations. The detection of tracer substances on brain tissue can be done using either light or fluorescence microscopy. The micrograph on the right shows the processes of a mitral cell contained within one brain section of the salamander accessory olfactory bulb labelled by intracellular injection of biocytin. The figure on the left shows the reconstruction of a cluster of thalamic neurons in the fire-bellied toad.

Functional neuroanatomy

Two methods that indirectly measure brain activity will be used. In amphibians, these methods enable the study of the whole brain in one experiment. Immunolabeling against c-Fos (left side picture), a useful marker of the effects of various stimuli on the nervous system, will be performed as well as thallium autometallography (right side picture). The latter method takes advantage of the permeability of potassium channels and/or the neuronal Na+/K+pump to thallium ions. After thallium injection, thallium ions are taken into active neurons at a faster rate and can be fixed in their current state after a treatment. The localization of thallium is then visualized using a silver stain procedure. These experiments will help determine which brain regions are engaged by the behaviourally relevant sensory cues and the conditioning procedures established in our behavioural studies. The two micrographs show functional labelling in the salamander raphe median obtained after similar treatment with a courtship pheromone using two different methods: on the left, c-Fos immunolabelling and on the right, thallium autometallography. Note that the micrograph on the right was taken at twice the magnification as the one on the left.

Electrophysiology

The above neuroanatomical experiments will determine the brain regions of interest for physiological investigations. Mechanisms of sensory integration in regions critical for behaviour will be investigated first in the in vitro brain preparation by artificial sensory stimulation, which is accomplished by stimulation of sensory nerves and brain regions that relay sensory information. This preparation enables simultaneous recording of intracellular and evoked potential responses, which reflect the activity of single neurons and of populations of neurons, respectively. In vivo extracellular electrophysiology will then be used in immobilized animals to investigate more realistic sensory processing using behaviourally relevant sensory cues. The use of multi-neuron extracellular recording will enable the study of information processing along sensory pathways. Here, the vomeronasal pathway of salamander will be of particular interest because it displays few stations between the receptor neurons and the regions of the brain involved in behavioural control.