An extremely important feature of Drosophila that makes it useful for studying mechanisms of PD is its well-characterized and relatively simple dopamine neuron system. All of the Drosophila dopamine-producing neurons have been identified, and their development has been traced throughout the fly life cycle of this organism. Thus, investigators can readily identify genetic perturbations affecting the number, morphology, or locations of dopamine neurons in Drosophila.
During embryonic and larval brain development, dopa-mine is expressed in approximately eighty cells. Many of these cells are grouped together into three symmetrical clusters in the two lobes of the brain, with the remaining dopamine neurons distributed singly along the length of the ventral ganglion. These dopamine neurons are retained in the central nervous system of adult flies and are primarily grouped together into six major clusters. These six clusters are arranged symmetrically about the midline with the neuronal cell bodies residing at the periphery of the brain and their axons projecting toward the center (Nassel and Elekes 1992). As shown in Figure 1A, on the anterior side of the brain, there is a small cluster of approximately five dopamine neurons, designated the protocerebral anterior lateral (PAL), and a larger cluster of approximately sixty dopamine neurons with characteristically small cell bodies, designated the protocerebral anterior medial (PAM). Four additional clusters of dopamine neurons are on the posterior side of the brain: two medial clusters, designated the proto-cerebral posterior medial (PPM) 2 and 3, and two lateral clusters, named the protocerebral posterior lateral (PPL) 1 and 2. The PPM2/3 and PPL2 clusters typically have five to eight neurons while the PPL1 cluster contains approximately twelve neurons (Figure 1B). In addition to these clusters, there are also a small number of dopamine neurons that are not arranged into clusters, such as the protocerebral posterior medial 1 (PPM1), the deutocerebral 1 (D1), and a number of ventral unpaired medial (VUM) neurons.
To date, most studies of dopamine neuron integrity in Drosophila models of PD have used antiserum against tyro-sine hydroxylase (TH), an enzyme required for dopamine biosynthesis, to image dopamine neurons. However, several different immunocytochemical methods have been utilized to conduct these imaging studies. Most of the studies conducted to date have used thick sections of paraffin-embedded CNS samples to analyze dopamine neuron integrity. More recently, confocal microscopy of whole mount brain samples has been applied to image the CNS dopamine neurons. One potential advantage of the confocal microscopic imaging approach is that it provides more detailed analysis of these neurons in intact brains. This method allows a better visualization of the three-dimensional arrangement of the neurons across the whole brain and might facilitate studies of more subtle aspects of dopamine neuron dysfunction, such as axonal projection and synaptic defects (for example see Figure 1C).
In addition to the spatial distribution of dopamine neurons in Drosophila, researchers have also studied the functional effects of dopamine depletion and dopamine neuron perturbation. Genetic or pharmacological depletion of dopamine in Drosophila results in a variety of characteristic phenotypes. Mutations affecting the Dopa decarboxylase gene result in decreased learning ability, while mutations in the TH-encoding gene pale cause a dose-dependent loss of general locomotor ability (Tempel et al., 1984, Pendleton et al. 2002a). Systemically administering chemical inhibitors of TH synthesis, such as 3-iodo-tyrosine, caused developmental delay, decreased fertility, and inhibition of a simple learning paradigm (Neckameyer 1996, 1998). However, it is unclear from these studies whether these phenotypes result from loss of dopamine signaling in the nervous system or from a non-neuronal requirement for dopamine. The Drosophila pale gene encodes two alternatively spliced isoforms of TH; one isoform is neu-ronally expressed while the other is expressed in the developing mesoderm and required for cuticle hardening and pigmentation (Friggi-Grelin 2003b).
To address the specific roles of dopamine neurons in the Drosophila nervous system, authors of a recent report used the GAL4/UAS system to express tetanus toxin in TH-expressing neurons to block dopamine neuron signaling (Friggi-Grelin 2003a). Tetanus toxin cleaves the synaptic vesicle protein synaptobrevin, and this cleavage was previously shown to block evoked neurotransmitter release in Drosophila. Drosophila expressing tetanus toxin in the pattern of TH are viable, display normal locomotion, and have a wild-type appearance. The only phenotype displayed by these flies is a hyperexcitable response to a startle stimulus. Vigorous tapping of vials containing flies expressing tetanus toxin in dopamine neurons causes the flies to fall and whirl erratically, failing to immediately right themselves. This result suggests that one of the functions of dopamine signaling in the Drosophila nervous system is to negatively control excitability behavior. This phenotype provides a useful reference point for the behavioral analysis of Drosophila models of PD.
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