Application of neuroanatomical and immunocytochemical techniques for the detection of neurotransmitters in identified neurons in vertebrate And invertebrate tissues.

Abstract

The techniques that have become available to the neurobiologist have over the past 35 years transformed the ways in which the nervous system can be investigated both in terms of connectivity and chemical make up. When given a specific neurobiological question concerning the synaptic interactions or the neurochemistry of the neuronal populations, careful consideration has to made of the techniques that are applied to obtain meaningful results. Two specific biological questions are addressed and the techniques that were applied are discussed. Firstly, to investigate whether the reduction of brain- derived neurotrophic factor (BDNF) in the granule cells of the stargazer mutant mouse compromises the phenotype of the cerebellar neurons. The levels of γ- aminobutyric acid (GABA) and glutamate neurotransmitter expressed in cerebellar neurons and the phenotype of the synaptic contacts were compared in stargazer and wild-type controls using electron microscopy (EM) and quantitative post-embedding EM immunogold labelling. Secondly, the flight motor system of the locust represents a model preparation for the investigation of neuromodulation. The monosynaptic connections between the locust forewing stretch receptor (fSR) and the first basal motorneuron (BA1) is part of a sensory pathway involved in flight and by combining in vivo horseradish peroxidase (HRP) backfills of the fSR with post- embedding EM immunogold labelling the proportion and location of inputs to the fSR were analysed. This and other data show the majority of inputs to the fSR are from either glutamatergic or GABAergic neurons, but not all. Electrophysiological studies have shown that the fSR/BA1 synapse is also modulated by biogenic amines. ICC fluorescent methods have established that the neuropile region is octopamine immunoreactive (IR). To identify these octopamine-IR processes at the EM level two different techniques were used; an immunogold cryosectioning method and a pre-embedding immunogold silver intensification technique.