We use preference/avoidance olfactory tests, multi-sensory integration tests and appetitive/aversive conditioning experiments. This allows us to investigate the evolution of learning and behavioural control in vertebrates. It also provides us with useful sensory stimuli for functional neurobiology and physiology experiments. The picture on the right shows a fire-bellied toad attending to stimuli displayed on a computer screen, as part of a discrimination learning experiment.
Tract tracing of brain pathways and intracellular labelling of single/few neurons is 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 left 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 right shows the reconstruction of a cluster of thalamic neurons in the fire-bellied toad. We are now equipped with the software Neurolucida (MBF Bioscience), which allows reconstruction of neurons in 3D-like fashion.
Methods that indirectly measure brain activity are used in the lab. In amphibians, these methods enable the study of the whole brain in one experiment. Immunolabeling against c-Fos, a useful marker of the effects of various stimuli on the nervous system, can be performed to detect the Fos protein. In collaboration with the Heyland lab, we are also developing the method of in situ hybridization to detect c-fos mRNA, as well as other neuronal molecules. These experiments will help determine which brain regions are engaged by the behaviourally relevant sensory cues and conditioning procedures established in our behavioural studies, as well as the neurotransmitters and modulators involved. The micrograph on the top left shows c-Fos labelling in the salamander raphe median obtained after treatment with a courtship pheromone. The paired micrographs below show c-fos mRNA signal (antisense) and control (sense) in the preoptic area of the fire-bellied toad.
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. 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.