Relationship between the afferent innervation pattern and the spontaneous and aversive stimulus driven activity of individual VTA dopaminergic neurons.

Ventral tegmental area (VTA) dopaminergic (DA) neurons have been implicated in several brain functions such as attention, memory, behavioral reinforcement and motivation. Dysfunction of these neurons has been associated with depression, schizophrenia, attention deficit hyperactivity disorder and addictive behaviors among others. Anatomically, VTA DA neurons have been shown to send projections mainly to telencephalic and diencephalic areas of the brain, with little or no collateralization and to receive its most abundant input from ventral basal ganglia structures. Because of the above-mentioned multiplicity of normal and also disease states, understanding the biological bases that determine VTA DA neurons firing behavior is fundamental. Among several other underlying factors controlling their activity, we are interested in studying the organization of their dendritic tree and the inputs it receives, how many there are and how they are distributed onto the different compartments of the somatodendritic tree. To accomplish the study, we recorded and labelled single VTA neurons in vivo in anesthetized mice and subsequently immunohistochemically identified their neurochemical phenotype by tyrosine hydroxylase. Next, we labeled inhibitory and excitatory postsynaptic puncta to identify putative synaptic contacts on individual neurons, which were three dimensionally reconstructed. Then, postsynaptic profiles were counted and located on the different neuronal compartments. Next, we studied the morphological and geometrical properties of the reconstructed neurons and compared them with three-dimensionally reconstructed substantia nigra compacta (SNc) DA neurons. Finally, we correlated spontaneous and aversive stimulus response electrophysiological recordings with morphological and synaptic contacts distribution data. From morphological analyses we found that when considering the entire dendritic domain, the dendrites of ventral VTA neurons are smaller and simpler than dorsal VTA neurons and, independently of their location, both groups exhibited a dorso-medial and ventro-lateral preferential location of their dendrites which is significantly different from SNc DA neurons. Furthermore, we describe that they have large dendritic trees, encompassing a large proportion of the VTA are and they overlap significantly more than adjacent substantia nigra DA neurons. These last two properties partly explain why only overlapping input topologies have been described in the VTA; if the neuronal trees are so big and overlapping, incoming inputs, even if they were very well localized in the VTA could stablish contact with neuronal cell bodies that might be actually far away. Concerning input number and distribution, we found that VTA DA neurons received an average of 1001 putative postsynaptic puncta (PSP) of which 55.6% were glutamatergic and 44.4% GABAergic and regarding their distribution we found significantly more excitatory than inhibitory PSP in distal dendrites and in higher order dendrites. Electrophysiologically we found a wide range of values in spontaneous activity (firing rate, spike train irregularity, bursting activity and spike duration were analyzed) which is in agreement with what has been described in the literature for VTA DA neurons. We found that none of these variables related to the overall PSP number, density or distribution. After analyzing aversive stimulus-driven responses in our sample of 21 VTA DA neurons, we found 15 neurons inhibited by the aversive stimulus, 4 neurons were activated and 2 neurons were unresponsive. Activated neurons were significantly more medially located than inhibited neurons. In 12 reconstructed and further analyzed neurons there was, also, no significant difference in the total number, density or distribution of excitatory or inhibitory PSP between these groups. However, when considering only the inhibited neurons we found a correlation, that neurons with a higher density of inhibitory PSP also had a greater inhibition in response to the aversive stimulus. This result is consistent with what has been described for substantia nigra compacta DA neurons where the extent of inhibition was related to the amount of GABA inputs. These results support the notion that different responses might be related to differences in organization of synaptic inputs onto individual neurons. In summary, in this thesis we identify two morphologically different groups of VTA DA neurons, describe the location and overlapping of their dendritic trees that is significantly different from SNc DA neurons. We also find that they receive more excitatory synaptic contacts on their distal dendrites and last but not least, describe a correlation between the degree of inhibition by an aversive stimulus and the density of inhibitory synaptic contacts. These results not only help us discover the morphological features of VTA DA neurons and improve our knowledge of a system that has been implicated in a great number of normal and disease states, but also contribute to decoding learning of avoidance behaviors by describing how, in VTA DA neurons, aversive stimulus inhibition relates to the amount of inhibitory inputs.