Genetically encoded dopamine sensors based on green fluorescent protein (GFP) enable high-resolution imaging of dopamine dynamics in behaving animals. However, these GFP-based variants cannot be readily combined with commonly used optical sensors and actuators, due to spectral overlap. We therefore engineered red-shifted variants of dopamine sensors called RdLight1, based on mApple. RdLight1 can be combined with GFP-based sensors with minimal interference, and shows high photostability permitting prolonged continuous imaging. We demonstrate the utility of RdLight1 for receptor-specific pharmacological analysis in cell culture, simultaneous assessment of dopamine release and cell type-specific neuronal activity, and simultaneous subsecond monitoring of multiple neurotransmitters in freely behaving rats. Dual-color photometry revealed that dopamine release in the nucleus accumbens evoked by reward-predictive cues is accompanied by a rapid suppression of glutamate release. By enabling multiplexed imaging of dopamine with other circuit components
in vivo
, RdLight1 opens avenues for understanding many aspects of dopamine biology.
Down syndrome (DS) is a genetic disorder that causes cognitive impairment. The staggering effects associated with an extra copy of human chromosome 21 (HSA21) complicates mechanistic understanding of DS pathophysiology. We examined the neuron-astrocyte interplay in a fully recapitulated HSA21 trisomy cellular model differentiated from DS-patient-derived induced pluripotent stem cells (iPSCs). By combining calcium imaging with genetic approaches, we discovered the functional defects of DS astroglia and their effects on neuronal excitability. Compared with control isogenic astroglia, DS astroglia exhibited more-frequent spontaneous calcium fluctuations, which reduced the excitability of co-cultured neurons. Furthermore, suppressed neuronal activity could be rescued by abolishing astrocytic spontaneous calcium activity either chemically by blocking adenosine-mediated signaling or genetically by knockdown of inositol triphosphate (IP) receptors or S100B, a calcium binding protein coded on HSA21. Our results suggest a mechanism by which DS alters the function of astrocytes, which subsequently disturbs neuronal excitability.
Following repeated opioid use, some dependent individuals experience persistent cognitive deficits that contribute to relapse of drug-taking behaviors, and one component of this response may be mediated by the endogenous dynorphin/kappa opioid system in neocortex. In C57BL/6 male mice, we find that acute morphine withdrawal evokes dynorphin release in the medial prefrontal cortex (PFC) and disrupts cognitive function by activation of local kappa opioid receptors (KORs). Immunohistochemical analyses using a phospho-KOR antibody confirmed that both withdrawal-induced and optically evoked dynorphin release activated KOR in PFC. Using a genetically encoded sensor based on inert KOR (kLight1.2a), we revealed the in vivo dynamics of endogenous dynorphin release in the PFC. Local activation of KOR in PFC produced multi-phasic disruptions of memory processing in an operant-delayed alternation behavioral task, which manifest as reductions in response number and accuracy during early and late phases of an operant session. Local pretreatment in PFC with the selective KOR antagonist norbinaltorphimine (norBNI) blocked the disruptive effect of systemic KOR activation during both early and late phases of the session. The early, but not late phase disruption was blocked by viral excision of PFC KORs, suggesting an anatomically dissociable contribution of pre-and postsynaptic KORs. Naloxone-precipitated withdrawal in morphine-dependent mice or optical stimulation of pdyn Cre neurons using Channelrhodopsin-2 disrupted delayed alternation performance, and the dynorphin-induced effect was blocked by local norBNI. Our findings describe a mechanism for control of cortical function during opioid dependence and suggest that KOR antagonism could promote abstinence.
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