The ventral midbrain supports a variety of functions through the heterogeneity of neurons. Dopaminergic and GABA neurons within this region are particularly susceptible targets of amphetamine-class psychostimulants such as methamphetamine. While this has been evidenced through single-neuron methods, it remains unclear whether and to what extent the local neuronal network is affected and if so, by which mechanisms. Both GABAergic and dopaminergic neurons were heavily featured within the primary ventral midbrain network model system. Using spontaneous calcium activity, our data suggest methamphetamine decreased total network output via a D2 receptor-dependent manner. Over culture duration, functional connectivity between neurons decreased significantly but was unaffected by methamphetamine. However, across culture duration, exposure to methamphetamine significantly altered changes in network assortativity. Here we have established primary ventral midbrain networks culture as a viable model system that reveals specific changes in network activity, connectivity, and topology modulation by methamphetamine. This network culture system enables control over the type and number of neurons that comprise a network and facilitates detection of emergent properties that arise from the specific organization. Thus, the multidimensional properties of methamphetamine can be unraveled, leading to a better understanding of its impact on the local network structure and function.

Dopaminergic neurons of the substantia nigra pars compacta (SNC) and ventral tegmental area (VTA) exhibit spontaneous firing activity. The dopaminergic neurons in these regions have been shown to exhibit differential sensitivity to neuronal loss and psychostimulants targeting dopamine transporter. However, it remains unclear whether these regional differences scale beyond individual neuronal activity to regional neuronal networks. Here, we used live-cell calcium imaging to show that network connectivity greatly differs between SNC and VTA regions with higher incidence of hub-like neurons in the VTA. Specifically, the frequency of hub-like neurons was significantly lower in SNC than in the adjacent VTA, consistent with the interpretation of a lower network resilience to SNC neuronal loss. We tested this hypothesis, in DAT-cre/loxP-GCaMP6f mice of either sex, when activity of an individual dopaminergic neuron is suppressed, through whole-cell patch clamp electrophysiology, in either SNC or VTA networks. Neuronal loss in the SNC increased network clustering, whereas the larger number of hub-neurons in the VTA overcompensated by decreasing network clustering in the VTA. We further show that network properties are regulatable via a dopamine transporter but not a D2 receptor dependent mechanism. Our results demonstrate novel regulatory mechanisms of functional network topology in dopaminergic brain regions.

In order to optimize outcomes in the face of uncertainty, one must recall past experiences and extrapolate to the future by assigning values to different choice outcomes. This behavior requires an interplay between memory and reward valuation, necessitating communication across many brain regions. At the anatomical nexus of this interplay is the perirhinal cortex (PRC). The PRC is densely connected to the amygdala and orbital frontal cortex, regions that have been implicated in reward-based decision making, as well as the hippocampus. Thus, the PRC could serve as a hub for integrating memory, reward, and prediction. The PRC's role in value-based decision making, however, has not been empirically examined. Therefore, we tested the role of the PRC in a spatial delay discounting task, which allows rats to choose between a 1-second delay for a small food reward and a variable delay for a large food reward, with the delay to the large reward increasing after choice of each large reward and decreasing after each small reward. The rat can therefore adjust the delay by consecutively choosing the same reward or stabilize the delay by alternating between sides. The latter has been shown to occur once the 'temporal cost' of the large reward is established and is a decision-making process termed 'exploitation'. When the PRC was bilaterally inactivated with the GABA(A) agonist muscimol, rats spent fewer trials successfully exploiting to maintain a fixed delay compared to the vehicle control condition. Moreover, PRC inactivation resulted in an increased number of vicarious trial and error (VTE) events at the choice point,

Dopaminergic neurons of the substantia nigra (SNC) and ventral tegmental area (VTA) exhibit spontaneous firing activity. The dopaminergic neurons in these regions have been shown to exhibit differential sensitivity to neuronal loss and psychostimulants targeting dopamine transporter. However, it remains unclear whether these regional differences scale beyond individual neuronal activity to regional neuronal networks. Here we utilized live-cell calcium imaging to show that network connectivity greatly differs between SNC and VTA regions with higher incidence of hub-like neurons in the VTA. Specifically, the frequency of hub-like neurons was significantly lower in SNC dopamine neurons than in the adjacent VTA, consistent with the interpretation of a lower network resilience to SNC neuronal loss. We tested this hypothesis when activity of an individual dopaminergic neuron is suppressed, through whole-cell patch clamp electrophysiology, in either SNC, or VTA networks. Neuronal loss in the SNC decreased network clustering, whereas the larger number of hub-neurons in the VTA overcompensated by increasing network clustering in the VTA. We further show that network properties are regulatable via a dopamine transporter but not a D2 receptor dependent mechanism. Our results demonstrate novel regulatory mechanisms of functional network topology in dopaminergic brain regions.

Dysregulation of dopamine neurotransmission profoundly affects motor, motivation and learning behaviors, and is often observed during the prodromal phase of Parkinson's disease (PD). However, the mechanism underlying these pathophysiological changes remains to be elucidated. Mutations in vacuolar protein sorting 35 (VPS35) and leucine-rich repeat kinase 2 (LRRK2) both lead to autosomal dominant PD, and VPS35 and LRRK2 may physically interact to govern the trafficking of synaptic cargos within the endo-lysosomal network in a kinase-dependent manner. To better understand the functional role of VPS35 and LRRK2 on dopamine physiology, we examined Vps35 haploinsufficient (Haplo) and Vps35 p.D620N knock-in (VKI) mice and how their behavior, dopamine kinetics and biochemistry are influenced by LRRK2 kinase inhibitors. We found Vps35 p.D620N significantly elevates LRRK2-mediated phosphorylation of Rab10, Rab12 and Rab29. In contrast, Vps35 haploinsufficiency reduces phosphorylation of Rab12. While striatal dopamine transporter (DAT) expression and function is similarly impaired in both VKI and Haplo mice, that physiology is normalized in VKI by treatment with the LRRK2 kinase inhibitor, MLi-2. As a corollary, VKI animals show a significant increase in amphetamine induced hyperlocomotion, compared to Haplo mice, that is also abolished by MLi-2. Taken together, these data show Vps35 p.D620N confers a gain-of-function with respect to LRRK2 kinase activation, and VPS35 and LRRK2 functionally interact to regulate DAT trafficking and striatal dopamine neurotransmission.