Mapping the Brain’s Connections to Midbrain Dopamine Neurons

LYNN SHI ’13

Figure 1: Experimental Design The initial injection at the ventral tegmental area (VTA) consists of AAV-Flex-TVA-mCherry and AAV- Flex-RG followed in two weeks by the injection of the modified rabies virus, SAD!G-GFP(EnvA). The secondary injection consists of AAV8-RG coupled with AAV5-tdTomato and is injected on the same day as the flex-AAVs. The site of the secondary injection is different for each treatment group (VP, Vst, and LH). The rabies virus label neurons directly presynaptic to the starter neurons in the VTA. At the site of the second injection, the rabies virus label neurons directly presynaptic a second time. This yields a map of di-synaptic connections to the VTA starter population via region of second injection (VP, Vst, LH). The starter cells are mCherry-positive and EGFP-positive. The transsynaptically labeled neurons are EGFP-positive.
The initial injection at the ventral tegmental area (VTA) consists of AAV-Flex-TVA-mCherry and AAV- Flex-RG followed in two weeks by the injection of the modified rabies virus, SAD!G-GFP(EnvA). The secondary injection consists of AAV8-RG coupled with AAV5-tdTomato and is injected on the same day as the flex-AAVs. The site of the secondary injection is different for each treatment group (VP, Vst, and LH). The rabies virus label neurons directly presynaptic to the starter neurons in the VTA. At the site of the second injection, the rabies virus label neurons directly presynaptic a second time. This yields a map of di-synaptic connections to the VTA starter population via region of second injection (VP, Vst, LH).
The starter cells are mCherry-positive and EGFP-positive. The transsynaptically labeled neurons are EGFP-positive.

To elaborate and expand our knowledge of the dopamine system, I identified the sources of inputs at the di-synaptic level.

The human brain contains billions of neurons organized into circuits that process specific kinds of information and give rise to behavior. Information encoded by these circuits hold the key to understanding how the brain works and offer the promise of clarifying the causes of neurological and psychiatric diseases. Establishing improved methods to understand how neural circuits are connected is a critical step toward understanding how neurons communicate.

In this project, I investigated the connectivity of the dopamine system in mice using rabies virus that have been modified to control which neuron groups are infected and the strength of that infection. The rabies virus has the special ability to travel backwards, or retrogradely, in the mammalian nervous system. Thus, the rabies-mediated retrograde tracing method harvests that special ability and allowed researchers to investigate the neurons which communicate with the neuronal population of interest, which in this case are the dopamine neurons. Dopamine neurons are localized in two adjacent but distinct structures, the ventral tegmental area (VTA) and the substantia nigra pars compacta (SNc). These neurons make diffuse connections with other neurons all over the brain and play pivotal roles in the brain, regulating motivation, movement control, and reward-seeking behavior. By looking at the inputs to dopamine neurons, we can evaluate how these neurons integrate information and compute signals to regulate behavior.

Recently, our laboratory had comprehensively identified the monosynaptic inputs, or neurons that make direct single-synaptic connections to dopamine neurons. To elaborate and expand our knowledge of the dopamine system, I identified the sources of inputs at the di-synaptic level, which are neurons that make indirect projections to dopamine neurons via regions that make monosynaptic inputs. I found that several brain regions, including the isocortex, olfactory area, cortical subplate, hypothalamus, thalamus, and hippocampal formation, project to midbrain dopamine neurons. This project demonstrates the utility and constraints of our methodology and contributes to our understanding of information transmission in the brain. Unraveling multi-synaptic input pathways for dopamine neurons provides a foundational knowledge in the regulation of dopamine neuron activity, and therefore a better neurobiological basis for the development of improved and targeted clinical interventions to improve the health of people and populations.

Figure 2. Visualization of Inputs to Dopamine Neurons in the VTA, part 1. (A) Monosynaptic inputs to VTA dopamine neurons (1-step inputs). (B) Mono- and di-synaptic inputs to VTA dopamine neurons via ventral striatum (2-step Vst). (C) Mono- and di-synaptic inputs to VTA dopamine neurons via ventral pallidum (2-step VP). (D) Mono- and di-synaptic inputs to VTA dopamine neurons via lateral hypothalamus (2-step LH). Scale bars represent 2000 μm.
Visualization of Inputs to Dopamine Neurons in the VTA, part 1.
(A) Monosynaptic inputs to VTA dopamine neurons (1-step inputs).
(B) Mono- and di-synaptic inputs to VTA dopamine neurons via ventral striatum (2-step Vst).
(C) Mono- and di-synaptic inputs to VTA dopamine neurons via ventral pallidum (2-step VP).
(D) Mono- and di-synaptic inputs to VTA dopamine neurons via lateral hypothalamus (2-step LH). Scale bars represent 2000 μm.

The writer can be reached at shi3@college.harvard.edu.