Imaging neuromodulation in the brain

成瘾涉及大脑系统调节内部状态的动机，唤醒和奖励，以及

Addiction involves brain systems mediating internal states of motivation, arousal and reward, as well as emotions. Such internal states influence goal-directed behaviors and decision-making. A common feature of such internal states is their valence and their persistence: they can have a positive or negative valence, and can outlast their triggering stimulus for many minutes. However the neurobiological mechanisms that underlie the persistence of internal states, and their relationship to the encoding of valence, are poorly understood. Drosophila provides a tractable genetic model organism for studying how neuromodulators act on neural circuits to control persistent internal states that govern goal-directed behavior and decision-making. We have discovered that P1 interneurons, which control male courtship behavior, can when activated promote a persistent internal state of social arousal or motivation, which can last for minutes. In a publication supported by the base grant, we have obtained evidence of a link between P1 interneurons and neurons that respond to octopamine (OA), an insect homolog of norepinephrine (NE), which is known to facilitate psychostimulant self- administration in rodents. We have also identified a downstream target of P1 neurons, called pCd cells, which appear to play a key requisite role in determining the persistence of an internal state of social arousal. During the extension period, we will continue our studies of how P1 neurons promote a rewarding internal state, and the relationship of these mechanisms to the positive valence, or rewarding nature, of P1 stimulation. In the first 2 years, we will focus on pursuing Aims 3 and 4 of the base grant. These aims were: Aim 3) to test the hypothesis that P1 neuron activation is positively valenced and rewarding; Aim 4) to investigate neuromodulatory mechanisms involved in P1 reward learning. In unpublished experiments, we have discovered that activation of P1 neurons can produce a real-time place preference (RTPP), and that it can also serve as an unconditional stimulus (US) for conditioned olfactory preference (COP). Both of these findings indicate that P1 activation is positively valenced, and that it can be rewarding. We plan to investigate whether plasticity during COP occurs at or downstream of P1 neurons, and whether P1 neurons are necessary for expression of the COP (Aim 3). Preliminary experiments suggest that dopamine (DA) may play a role in modulating the effects effects of P1 stimulation. We will confirm and extend these findings, and also investigate the role(s) of other neuromodulators including biogenic amines such as octopamine (OA), which we have shown to modulate the effect of P1 stimulation to activate aSP2 neurons that control social behavior9. Furthermore, we will investigate whether mushroom body (MB) neurons involved in reward learning are also involved in P1-mediated odor conditioning (Aim 4). Given previous data, we expect to find a role for the MB, but precisely which subset of MB neurons are involved is not clear. In Merit Extension Aim 5, we will investigate the role of other neuromodulators in P1-induced persistent social arousal and reward learning. Candidate neuromodulatory targets of P1 neurons include serotonergic (Trh+) neurons, a subset of which is activated in response to P1 stimulation (preliminary results), and neuropeptide F (NPF), which has been implicated in reward in other contexts. We will approach this problem using functional connectomics, in which optogenetic activation of P1 neurons is combined with calcium imaging in populations containing putative neuromodulatory targets of these cells. Target neurons can be “filled” using photo-activatable GFP (PA-GFP), and their morphology used as a “search image” to identify specific genetic drivers that label that subset of cells. Using these drivers, activation and silencing of these neurons can be performed in the context of both P1-mediated reward (RTPP and COP assays), and persistent social arousal. We have successfully established this approach and used it to identify pCd neurons, which are persistently activated by P1 neurons and required for persistent social behaviors triggered by P1 activation. As a complementary approach, we will take advantage of recent advances that we have made in techniques for whole-mount fluorescent in situ hybridization (FISH) in the adult brain, which allow identification of candidate follower cells activated by optogenetic stimulation of P1 neurons using FISH probes for hr38, an immediate early gene (analogous to c-fos) in Drosophila. Double-label FISH can be performed using hr38 and probes for neurotransmitter biosynthetic enzymes or neuropeptides, to identify neuromodulators expressed in P1 targets. A fundamental question is whether the mechanism mediating P1-induced persistent activity is also involved in reward. To address this question, in Merit Extension Aim 6 we will investigate the role of pCd neurons in P1- mediated reward learning, using functional manipulations of these cells. Preliminary data suggest that persistent activation of pCd neurons by P1 cells is modulated by DA, and we will investigate how this modulatory influence is exerted. We anticipate that these experiments will yield general principles of how persistent internal reward states are encoded by brains, with potentially broad relevance across phylogeny.