Optogenetic Modulation of Neural Circuits that
Underlie Reward Seeking
Garret D. Stuber, Jonathan P. Britt, and Antonello Bonci
The manifestation of complex neuropsychiatric disorders, such as drug and alcohol addiction, is thought to result from progressive
maladaptive alterations in neural circuit function. Clearly, repeated drug exposure alters a distributed network of neural circuit elements.
components that underlie addictive behaviors. The development of optogenetic strategies for selectively modulating the activity of
a level of precision that has been previously unobtainable. Here, we briefly review the main optogenetic studies that have contributed to
manifestation of addiction-related behaviors. Additional targeted manipulation of genetically defined neural populations in these brain
regions, as well as afferent and efferent structures, promises to delineate the cellular mechanisms and circuit components required for the
transition from natural goal-directed behavior to compulsive reward seeking despite negative consequences.
Key Words: Accumbens, addiction, behavior, dopamine, electro-
peutics for psychiatric diseases such as addiction. The basic neuro-
anatomical substrates required for reward-related behavior have
been identified, but the specific function of genetically defined
neurons and neural circuits remains unclear. To define the func-
tional connectivity between neurons and their role in modulating
complex behaviors, such as reward seeking, the ability to perturb
specific neural circuits on physiologically relevant time scales is
required. Given the complexity and high degree of interconnectiv-
ity within neural tissue, manipulating the activity of a single genet-
major obstacle to unraveling how functional connectivity within
tion, has circumvented many of the technical limitations of tradi-
tional techniques used in systems and behavioral neuroscience
research. A new level of mechanistic insight into the neural under-
pinnings of motivated behaviors is now possible.
Two highly interconnected brain regions play critical roles in
mediating reward-seeking behaviors, including those related to
bens (NAc). These brain regions are comprised of multiple, geneti-
cally distinct cell groups that integrate and convey reward-related
study neural circuit function. We then provide an overview of the
synaptic connectivity within the VTA and NAc and highlight how
he organizational complexity of the brain is both a source of
standing of neural systems and the development of thera-
within these regions.
Tools and Strategies for Optogenetic Manipulation of
Neural Circuits that Underlie Reward Processing
A number of optogenetic actuators are now available for both
excitation and inhibition of neural circuits for use both in vitro and
in vivo (1). To date, the most commonly used light-gated proteins
for activation of neural tissue are engineered mutants of channel-
rhodopsin-2 (ChR2) (2). Channelrhodopsin-2 mutants are typically
maximally activated by 450 nm to 500 nm light, which allows for
large inward flux of sodium ions and calcium ions at resting mem-
brane potentials. Brief pulses of light (typically 1–5 msec) result in
reliable and repeatable action potential generation in a variety of
neuronal subtypes over a large range of firing frequencies (3,4).
activate pathway-specific neurotransmitter release in brain slices
(3,5,6) or in behaving animals (3,7) to study the effects of afferent-
specific synaptic transmission. For optogenetic inhibition studies,
modified variants of both halorhodopsin (8–10) and archaerho-
dopsin (11,12) have been shown to reliably silence neural activity
both in vitro and in vivo. Transgenes coding for gated proteins to
modulate neural activity are typically introduced into neural tissue
via transgenic animals that express these proteins under cell type
brain nuclei. For delivery of light to neural tissue in vivo, optical
fibers coupled with high-powered light sources, such as lasers or
light-emitting diodes (LEDs), are used (15–17), providing a way to
restrict light delivery and thus optical modulation to specific brain
structures. Combined with genetic targeting approaches to selec-
tively express these light-activated proteins in genetically defined
neurons (4), use of these tools allows for selective modulation of
neural circuits that underlie reward-related behaviors.
Neuronal Populations in the VTA and Their Role in
Mediating Reward-Seeking Behavior
populations that are essential to the expression of motivated be-
haviors related to addiction (18,19). While the VTA is oftentimes
treated as a distinct neural structure, few anatomical markers dis-
pars compacta (SNc). Immunohistochemical and tracing studies
University of North Carolina Neuroscience Center, University of North
Carolina at Chapel Hill, Chapel Hill, North Carolina; Intramural Research
Program (JPB, AB), National Institute on Drug Abuse, Baltimore, Mary-
land; Department of Neurology (AB), University of California, San Fran-
cisco, California; and Solomon H. Snyder Neuroscience Institute (AB),
Johns Hopkins University School of Medicine, Baltimore, Maryland.
Address correspondence to Garret D. Stuber, Ph.D., University of North
Carolina at Chapel Hill, Departments of Psychiatry and Cell and Molecu-
Hill, NC 27599-7250; E-mail: firstname.lastname@example.org.
Received Aug 29, 2011; revised Nov 4, 2011; accepted Nov 8, 2011.
BIOL PSYCHIATRY 2012;71:1061–1067
© 2012 Society of Biological Psychiatry
have suggested that SNc is a relatively homogenous population of
neurons, the majority of which are dopamine (DA)ergic (90%) and
project to the dorsal striatum (20,21). The VTA, on the other hand,
contains a mixture of DAergic (?65%), gamma-aminobutyric acid
(GABA)ergic (?30%), and glutamatergic neurons (?5%) (22) that
project throughout the forebrain to structures including the
amygdala, prefrontal cortex, and NAc (20). Importantly, VTA neu-
rons that project to the NAc are a heterogeneous population of
both DAergic and GABAergic neurons (21,23,24). Within the VTA,
GABAergic neurons also are thought to form inhibitory contacts
onto at least some DAergic projection neurons (25). Thus, VTA
GABAergic neurons, as well as GABAergic input from the posterior
segment of the VTA, also known as the rostromedial tegmental
nucleus (26,27), may play an important role in regulating DAergic
likely components of distinct neural circuits incorporating neurons
from their afferent and efferent structures, which may act to medi-
ate specific aspects of motivated behavioral processing (28).
The pioneering optogenetic studies in this field introduced the
gic neurons using viral delivery methods to examine the role of
these specific VTA neurons in reward-seeking behavior (4,5,29,30).
While the actions of DA were known to play an important role in
activation of DAergic neurons alone could modulate reward-re-
VTA DAergic neurons led to the formation of a conditioned place
preference (CPP) to the associated environment (4). Importantly,
DAergic stimulation frequencies that led to the development of a
release in the NAc, suggesting that only stimulation frequencies
that lead to detectable changes in DA release can induce associa-
tive learning. In addition, it has been demonstrated that direct
optical activitation of these neurons can reinforce operant behav-
ioral responding (31) and also facilitate development of positive
reinforcment (30). Taken together, these studies demonstrate that
ioral conditioning and reinforce behavioral responding in the ab-
sence of any additional reward.
Optogenetic strategies have also been employed in brain slice
experiments to examine the possibility of neurotransmitter co-re-
lease. Neurons in the medial VTA co-express tyrosine hyrodylase
and the vesicular glutamate transporter-2 (VGluT2), indicating that
ever, it was not possible to selectively stimulate DAergic fibers
originating from VTA neurons to determine whether they co-re-
lease dopamine and other small-molecule neurotransmitters. Op-
able glutamate-mediated excitatory postsynaptic currents (5,33)
that were not present in mice that lack VGluT2 in DAergic neurons
atal regions despite optogenetic stimulation of DAergic fibers pro-
ducing detectable DA release (5). These studies suggest that mid-
brain DAergic neurons that project to the ventral, but not dorsal,
striatum can co-release glutamate as a neurotransmitter. Interest-
vesicular glutamate transporter-3 (34). These results suggest that
there may be major species-specific differences in the neurotrans-
mitter content of DAergic neurons (the optogenetic studies were
performed in mice, whereas the electron micrscopy study was per-
formed in rats). However, it is worth noting that cultured DAergic
neurons from rats also co-release glutamate (35,36) and that elec-
trical stimulation of the VTA results in glutamate-mediated excit-
atory postsynaptic responses in the prefrontal cortex of rats (37),
suggesting that DAergic neurons in species other than mice also
co-release glutamate. It is possible that different axonal fibers that
glutmate but not both. In addition, transcriptional suppression of
studies are required, these ideas could account for the discrepan-
cies between the optogenetic studies demonatrating DA/gluta-
mate co-release and the electron microscopy data showing that
axonal fibers in the striatum do not co-express TH and vesicular
glutamate transporter isoforms.
Excitatory Afferent Projections to the VTA and Their
Role in Reward-Related Behaviors
Since direct optogenetic stimulation of VTA DAergic neurons
sufficient to modulate reward-related behaviors, an important line
of research that remains largely unexplored is determining how
specific VTA afferents modulate the activity of both DAergic and
non-DAergic neurons in the VTA. Both excitatory and inhibitory
neurons within the VTA. The heterogeneity of these inputs is such
that electrical stimulation cannot be used to activate specific pre-
VTA dopamine (DA)ergic neurons project to forebrain targets such as the
basolateral amygdale (BLA), medial prefrontal cortex (mPFC), and nucleus
accumbens (NAc). These neurons received excitatory synaptic inputs from
the lateral hypothalamus, mPFC, and pedunculopontine tegmental nucleus/
laterodorsal tegmental nucleus. Inhibitory inputs to the VTA DAergic neu-
rons likely arise from extended amygdala output structures. Ventral teg-
mental area gamma-aminobutyric acid (GABA)ergic neurons target
neighboring DAergic neurons as well as projecting to the mPFC and NAc.
These neurons are thought to receive excitatory inputs from the lateral
habenula and inhibitory inputs from the NAc. RMTg, rostromedial tegmen-
tal nucleus. (Illustrations by R.D. Weaver.)
1062 BIOL PSYCHIATRY 2012;71:1061–1067
G.D. Stuber et al.
synaptic fibers. Therefore, future studies will have to rely on affer-
aptic function in the VTA. Optogenetic stimulation of presynaptic
of afferent-specific inputs (3,7,38). Given that exposure to many
drugs and natural rewards can alter excitatory synaptic function
should provide a framework for better understanding the synaptic
function within the VTA.
The lateral hypothalamus (LH) is thought to send the largest
subcortical glutamatergic projection to the VTA/substantia nigra
(Sn) (46). Electrical stimulation of the LH predominately increases
potential waveforms (47). In contrast, neurons that show short-
duration waveforms are generally suppressed by LH stimulation
to the VTA are glutamatergic (47) and may also co-release the
neurons show increased activity as indexed by c-fos following co-
caine or morphine CPP (49,50), and VTA neurons show increases in
c-fos expression following LH stimulation (51). In addition, LH self-
stimulation leads to large increases in NAc DA release (52), further
demonstrating an important role of this pathway in the activation
of brain reward circuits, as well as reinforcing behavioral respond-
source of excitatory drive to the VTA. Future studies in which there
is afferent-specific optogenetic stimulation of LH afferents to the
VTA will illuminate the synaptic connectivity between these re-
gions, as well as their role in reward-related behaviors.
Another major source of glutamatergic input to the VTA comes
from a long-range projection from the medial prefrontal cortex
(mPFC), which is thought to target both DAergic and non-DAergic
neurons (46,53,54). Stimulation of the mPFC leads to an increase in
extracellular glutamate in the VTA (55), activates DAergic and non-
DAergic neurons (56–58), and elevates DA release in the forebrain
(59,60). Interestingly, an electron microscopy study by Carr and
Sesack (53) showed that mPFC afferents to the VTA form synapses
onto mPFC-projecting, but not NAc-projecting, DAergic neurons.
Medial prefrontal cortex afferents also formed synapses onto VTA
GABAergic neurons that project to the NAc but not those that
the VTA is needed to corroborate these findings.
The lateral habenula (LHb) is another major excitatory input to
GABAergic neurons there (65). Interestingly, electrophysiological re-
tation, while DAergic neurons in the midbrain show excitatory re-
in the NAc (67). These studies suggest that LHb neurons send a
direct glutamatergic projection to predominantly GABAergic neu-
rons in the VTA/Sn that can inhibit DAergic neuronal activity.
rons and increases DA release in the NAc (71). While the PPTg/LDT
plays an important role in driving drug-seeking behavior (72), how
the synaptic connectivity between the PPTg/LDT and the VTA con-
trols reward-related behaviors remains unknown.
Inhibitory Afferents to the VTA/SNc and Their Role in
rons in the VTA (73). Furthermore, some non-DAergic neurons that
receive inhibitory inputs from the NAc were shown to project back
to the NAc (73). This study elegantlydemonstratedtheprecisefunc-
such as those from the central nucleus of the amygdala and the bed
electrical stimulation of the bed nucleus of the stria terminalus
produces both excitatory and inhibitory responses in the VTA (75),
neural activity in vivo and during behavioral tasks should be ex-
Neuronal Populations in the NAc and Their Role in
The NAc integrates reward-related information conveyed by
dopamine and GABAergic inputs from the midbrain with glutama-
tergic inputs from regions throughout the brain. There is a fairly
consistent cellular architecture throughout the dorsal and ventral
known as medium spiny neurons (MSNs), make up more than 90%
of the local neurons in the NAc and are GABAergic (80). Striatal
interneurons are a mix of both cholinergic and GABAergic neurons
(81). Medium spiny neurons are typically classified into two main
groups based on the type of opioid peptides they release (dynor-
phin or enkephalin) and the dopamine receptors they express (D1
or D2), as well as their projection targets (direct or indirect to the
midbrain) (78,82) (Figure 2). However, this strict division between
striatum (83,84). It is also unclear if this division is as steadfast in
species other than mice, where transgenic animals have provided
Optogenetic investigations utilizing ChR2 in acute brain slices
have demonstrated that direct pathway MSNs in the NAc specifi-
cally target GABAergic neurons in the VTA (; see above). This
target specificity has also been observed in direct pathway MSNs of
the dorsal striatum, which only innervate GABAergic neurons of the
substantia nigra pars reticulata (86). Neither of these optogenetic in-
vestigations found evidence for direct innervation of midbrain DA
neurons by MSNs. Indirect pathway MSNs from the dorsal and ven-
the ventral pallidum, respectively (87). As with the direct pathway
target specific GABAergic neurons (86). In addition, striatal MSNs
form inhibitory synapses onto each other, as well as local cholin-
ergic, but not fast-spiking, GABAergic interneurons (86). The intri-
G.D. Stuber et al.
BIOL PSYCHIATRY 2012;71:1061–1067 1063
The distinct projection targets of the two main types of MSNs
can produce a bidirectional regulation of behavior (78,88). Recent
optogenetic investigations have provided additional support for
this hypothesis. When ChR2 was expressed selectively in either D1
medial striatum by viral delivery, basal locomotion could be in-
the idea that the dorsal striatum has a critical role in the bidirec-
tional control of motor behavior, selective activation of these dis-
was not possible without optogenetic manipulations.
Optogenetic investigations, specifically in the NAc, have ex-
plored whether the activation or inhibition of specific cell types
study in which ChR2 was expressed in D1R- or D2R-expressing
MSNs in naive mice, optogenetic activation of these two neuronal
However, when the mice were treated with multiple injections of
cocaine that could reliably produce behavioral sensitization, opto-
genetic activation of NAc D1R-expressing MSNs became sufficient
to enhance locomotion (90). This demonstrates that repeated ex-
posure to cocaine might alter the functional activity of NAc D1R-
expressing MSNs so that effects on locomotion become evident
for the increased locomotion observed in behavioral sensitization,
although this idea remains to be tested.
The optical activation of D1R- or D2R-expressing NAc MSNs on
stimulated at 10 Hz (90). However, activation of D1R-expressing
cocaine to produce a conditioned place preference (90). Similar
effects were observed with an indiscriminate ChR2-mediated acti-
sure diminished the development of a conditioned place prefer-
ence (90). These opposing effects are another example of how the
two types of MSNs can exert bidirectional control over behavior,
specifically on the ability of cocaine to induce a conditioned place
Both the light-activated chloride pump halorhodopsin 3.0 and
ChR2 have been introduced into NAc cholinergic interneurons by
light produced gross behavioral effects (91). These manipulations
in their own right were also ineffective in eliciting a place prefer-
ence (91). Inhibiting these neurons during cocaine exposure, how-
activating NAc D2R-expressing MSNs during cocaine exposure
increased the firing rate in the majority of neighboring cells (91).
activation of all NAc neurons with ChR2 augments cocaine reward
(90), but perhaps cholinergic interneuron inhibition preferentially
activates D2R-expressing MSNs.
Taken together, these experiments confirm that the two main
populations of MSNs exert opposing effects on behavior. In the
NAc, optogenetic manipulations to specific cell types affect the
development of cocaine-induced conditioned place preference.
of optogenetic manipulations to NAc GABAergic interneurons, it is
likely that they also influence cocaine-related behaviors. Overall,
the behaviors examined with optogenetic techniques to date (lo-
comotion, behavioral sensitization, and conditioned place prefer-
ence) are indirect metrics of reward seeking. As optogenetic tech-
niques become more widely adopted, it will be interesting to see
ing behaviors such as drug self-administration.
Inputs to the NAc and Their Role in Reward-Related
Both cortical and subcortical structures innervate the NAc and
(92). This location of DA synapses is consistent with the neuro-
modulatory role of DA. That is, the extent to which ongoing gluta-
matergic activity can influence the firing of MSNs is probably de-
pendent on local DA release (79,93). Importantly, as described
above, VTA DA neurons also release glutamate onto NAc MSNs
(5,33). In addition to the DA and glutamate projections from the
VTA, there is evidence of a GABAergic projection from the VTA to
neurons induce behavioral effects, how selective activation of the
VTA GABAergic projections can influence behavior is not known.
Glutamatergic inputs to the NAc come primarily from the
amygdala, prefrontal cortex (PFC), hippocampus, and thalamus
(87). These afferents are heterogeneously distributed, but specific
clusters or ensembles of cells tend to have a convergence of affer-
MSNs receive inputs from several afferent structures, including the
amygdala, PFC, and hippocampus (3,96). This synaptic arrange-
BLA / Hippocampus
aminobutyric acid (GABA)ergic medium spiny neurons (MSNs) project to
either the ventral pallidum (VP; D2 receptor [d2r] expressing) or to the
ventral tegmental area (VTA)/substantia nigra (D1 receptor [d1r] express-
ing). These projection neurons receive excitatory inputs from the medial
prefrontal cortex, basolateral amygdala (BLA), hippocampus, and thalamus
and dopaminergic input from the VTA. Both cholinergic and fast-spiking
GABAergic interneurons synapse onto MSNs, to modulate their activity. In
addition, MSNs synapse locally with each other and likely onto cholinergic
interneurons but not fast-spiking interneurons. (Illustrations by R.D.
1064 BIOL PSYCHIATRY 2012;71:1061–1067
G.D. Stuber et al.
axons innervating the striatum target both D1R- and D2R-express-
ing MSNs and single MSNs receive inputs from both cortical and
nature of the information flow into the ventral striatum.
Optogenetic examinations of afferents to the NAc have ex-
plored amygdala and PFC inputs (3). Channelrhodopsin-2 was in-
dulin-dependent protein kinase II promoter, which is preferentially
expressed in glutamatergic projection neurons originating from the
control reward-seeking behavior was then examined by determin-
ing whether mice would engage in self-stimulation behavior to
selectively activate either the BLA-to-NAc or mPFC-to-NAc path-
ways (3). Interestingly, activation of glutamatergic afferents from
the BLA readily supported self-stimulation behavior, while the
cific glutamatergic afferents to the NAc can promote reward-seek-
ing behavior. In addition, optogenetically silencing BLA-to-NAc af-
ferents disrupted the formation of cue-reward associations,
implying that both DA release from VTA-to-NAc fibers and gluta-
mate release from BLA inputs cooperatively regulate direct-path-
way neuronal output to facilitate reward seeking.
Corticostriatal projections, especially those to the NAc, are im-
portant for generating and maintaining goal-directed behaviors,
such as drug self-administration, but also for responding to a
changing environment (98). It has been hypothesized that re-
peated exposure to drugs of abuse results in a downregulation of
trol drug-taking behavior (99), although this has not yet been ex-
plored with optogenetic manipulations. Future research should
address this role of the mPFC inputs and the behavioral impact of
activating hippocampal or thalamic inputs to the NAc.
Conclusions and Future Directions
already helped establish and refute many hypotheses that were
previously untestable with traditional techniques, studies that will
ral circuits that underlie reward-seeking behavior are yet to come.
Most of the studies reviewed have utilized ChR2 for optogenetic
stimulation of defined neuronal populations. While these studies
have certainly provided groundbreaking findings, many of these
that optogenetic methodologies allow for—transient activation or
behaviorally relevant events. In addition, while optogenetic stimu-
behavior. Loss of function experiments utilizing optogenetic inhi-
bition of neural circuits have lagged behind the adoption of ChR2-
mediated stimulation, partially due to technical limitations that
have been recently overcome (73). These future studies will likely
play a more prominent role in determining the role of specific
connections between neurons in mediating motivated behavioral
responses. Finally, a plethora of in vivo electrophysiological and
neural activity and reward-seeking behavior. As optogenetic strat-
egies for neural circuit manipulation are refined, these techniques
to monitor neural activity will certainly become fully integrated
with optogenetic circuit manipulation to assess how neuronal ac-
optogenetic strategies for perturbation of neural function, used in
conjunction with sophisticated behavioral paradigms to study re-
ward seeking, will likely greatly enhance our understanding of the
circuitry that mediates behavior.
GDS was supported by grants from National Alliance for Research
on Schizophrenia and Depression, Alcoholic Beverage Medical Re-
search Foundation, The Foundation of Hope, Whitehall Foundation,
National Institute on Drug Abuse (DA029325), and startup funds pro-
A/S. Drs. Bonci and Britt report no biomedical financial interests or
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