Imaging neuromodulation in the brain

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 In unpublished experiments, we have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-