Satoshi Ikemoto, Ph.D. - US grants

Amphetamines are known to enhance alertness, mood, motivation and motor performance. Chronic use of amphetamines is associated with addiction and mental conditions including mood, psychotic, obsessive and compulsive disorders. These known effects of amphetamines may suggest that amphetamine administration alters motivational and cognitive processes that integrate sensory information for action. To study effects of damphetamine on such integrative processes, we employed sensation seeking of unconditioned visual stimuli (VS). Specifically, our procedure required rats to lever-press to obtain a flash of light, which is weakly reinforcing in rats. Because amphetamines effects on drug seeking and mental conditions can be exacerbated by stress, we also examined effects of stress induced by chronic food-restriction and its interaction with amphetamine on VS seeking. We found that in chronically food-restricted (FR) rats (85-90% of their original weights), intraperitoneal (IP) injections of 1 mg/kg amphetamine markedly increased lever-pressing reinforced by VS, while the 0.3-mg/kg dose had no reliable effect, and the 3-mg/kg dose reliably decreased VS seeking. Locomotor activity of FR rats during these tests was affected somewhat differently than VS seeking: The 1-mg/kg dose increased locomotor activity, and the 3-mg/kg dose increased it even more. Unlike FR rats, rats that were given ad libitum (AD) access to food did not reliably increase VS seeking when treated with these doses of amphetamine, while significantly increasing locomotor activity. We also examine effects of the length of food restriction on amphetamine-induced VS seeking. Food deprivation of 24 hours or FR of 3 or 7 days with amphetamine injections (1mg/kg) did not increase VS seeking , while a 2 week FR significantly increased VS seeking, suggesting that chronic food restriction is needed for amphetamine to increase VS seeking. We also examined effects of repeated injections (8 times over 2 weeks) of amphetamine (1 mg/kg, IP) followed by a 3-week incubation. When challenged with 0.1-1 mg/kg amphetamine after incubation, prior amphetamine exposure did not have clear effects on VS seeking in FR or AD rats, but had sensitizing effects on locomotor activity. However, the whole procedure consisting of repeated injections (either saline or amphetamine) followed by a 3-week incubation made FR rats more sensitive to the effects of amphetamine on VS seeking. While FR rats were not different from AD rats in VS seeking without amphetamine, FR rats with prior saline- or amphetamineexposure significantly increased VS seeking with treatments of 0.3 or 1 mg/kg amphetamine. In summary, these results suggest that food restriction is an importnat factor in amphetamine-induced VS seeking, but not in locomotor activity. Neural mechanisms that amphetamine modulates to facilitate VS seeking may be substantially different from those of locomotor activity.

Our major aim of this project is to determine detailed neural connections involved in drug reinforcement. Behavioral studies indicate that the mesolimbic dopamine system is functionally heterogeneous. The medial portion of the ventral striatum, which includes the medial accumbens shell and medial olfactory tubercle, is more responsible for primary reinforcing effects of psychomotor stimulants than the lateral ventral striatum, which includes the accumbens core, ventral shell, and lateral tubercle. In the dopamine cell-body region, the posterior VTA and the central linear nucleus raphe are more responsible than the anterior VTA for the reinforcing effects of a number of drugs including opiates and cholinergic agents. Based on these behavioral findings, we hypothesize that the reward-trigger zones in the ventral striatum (i.e., the medial accumbens shell and medial olfactory tubercle) receives projections from the reward-trigger zones in the ventral midbrain (i.e., the posterior VTA and central linear nucleus). When the retrograde tracer Fluorogold (FG) was deposited into the medial ventral striatum (the medial shell or medial tubercle), FG-filled cells were found largely in the posteromedial compartment of the VTA and the central linear nucleus. On the other hand, when FG was deposited into the lateral ventral striatum (the core, lateral shell, or lateral tubercle), FG-filled cells were found mostly in the lateral portion of the VTA. Our results suggest that the reward-trigger zones in the medial ventral striatum receive input from the reward-trigger zones in the posteromedial ventral midbrain.

Scientific questions on brain reward systems were prompted by the discovery that animals learn to lever-press for electrical stimulation of certain brain areas, a phenomenon known as intracranial self-stimulation. Since then, numerous studies using anatomy, pharmacology and electrophysiology methods have established dopamine neurons in the ventral tegmental area (VTA projecting to the nucleus accumbens (NAc) as a key reward substrate. Recent studies using optogenetics confirmed that animals learn to self-stimulate VTA dopamine neurons, suggesting that excitation of dopamine neurons is sufficient in inducing reward. The question remains how excitation of VTA dopamine neurons influences downstream brain areas. We sought to identify firing patterns that encode dopamine neuron-mediated reward in the NAc. We injected the AAV-ChR2 viruses and implanted optical fibers in the VTA area of TH::Cre mice, a procedure that allowed us to specifically activate dopamine neurons through optical stimulation; a bundle of 8 tetrodes (32 wires) was implanted in the NAc shell for neural activity recording. We found that VTA optical stimulation in freely-behaving mice evoked fast excitatory local field potential (LFP) responses in the NAc, and the amplitudes of this LFP correlated well with the animals self-stimulation rates. Consistent with the LFP activity, 35% of the recorded NAc neurons showed fast phasic excitations, suggesting an excitatory input to the NAc from VTA dopamine neurons. We also recorded neurons that showed phasic inhibitions (17%). To determine whether these firing pattern changes were mediated by dopamine, mice were systemically injected with the dopamine D1 receptor antagonist SCH 23390. Although the antagonist decreased most basal firing in the NAc, it did not abolish optical stimulation-evoked neural responses, suggesting that transmitters other than dopamine were released by VTA dopamine neurons. In light of recent in vitro studies showing that dopamine neurons can also release glutamate and GABA to depolarize or hyperpolarize post-synaptic neurons, our results may be explained by excited dopamine neurons releasing glutamate or GABA. VTA dopamine neurons may employ multiple neural transmitters for inducing reward.

Motivational capacity to interact with the environment is fundamental for everyday healthy living. Motivated behaviors ultimately manifest through reward and aversion processes, where animals must approach positive rewarding stimuli and avoid negative aversive stimuli to survive. The neural mechanisms mediating these basic processes are not yet fully understood. However, technological advancements made in the past decade now allows us to investigate previously understudied brain regions involved with these processes. One such understudied region, the supramammillary nucleus (SuM), is a small posterior hypothalamic nucleus that provides dense projections throughout the cerebrum. Past research on SuM has focused on its role in arousal, learning, and memory. Our lab previously found that pharmacological stimulation of SuM neurons can reinforce behavior. In the current study, we designed experiments to further our understanding of the role the SuM and its related circuitry plays in reward and motivation. We first confirmed stimulation of SuM neurons is rewarding using a self-stimulation procedure with optogenetics involving channelrhodopsin-2 (ChR2) in wild-type (C57/BL7) mice. Mice with ChR2 and optic fibers in SuM quickly learned to respond on a lever reinforced by photostimulation and switch responding when lever assignments are reversed. Mice do not reliably self-stimulate when optic fibers are placed in areas adjacent to SuM, ie. in the mammillary bodies or ventral tegmental area. Next, using a Cre-dependent ChR2 and vGlut2-Cre, vGat-Cre or Th-Cre mice, we show this rewarding effect of SuM neuron stimulation is likely mediated by glutamatergic neurons, but not dopaminergic or GABAergic neurons. Then using optogenetic terminal-stimulation we dissect which glutamatergic projections from the SuM mediate self-stimulation behavior. Mice learned to respond for the stimulation of SuM glutamatergic neurons terminating in the septal area, but not terminals in the paraventricular thalamic nucleus (PVT), ventral subiculum, or diagonal band of Broca. In addition, mice show real-time place preference for activation of the SuM to septum circuit, and real-time place aversion for activation of the SuM to PVT circuit, indicating bivalent affective processes driven by SuM circuitry. To investigate the role of SuM neurons in food (sucrose) taking and seeking behavior, we conducted single-unit in-vivo electrophysiology experiments in mice seeking natural rewards. Most SuM neurons change their firing rates as a function of sucrose seeking, taking or both. Our results implicate the SuM and its downstream targets in motivational processes. As this circuitry is somewhat non-canonical in terms of classical reward circuits, we feel it warrants future research into its role in psychiatric disorders such as depression and addiction.

BACKGROUND: Studies using continuous access self-administration procedures showed that cocaine seeking progressively increases during abstinence (incubation of cocaine craving). Recently, studies using intermittent access self-administration procedures showed increased motivation to self-administer and seek cocaine. Here, we examined whether intermittent access cocaine self-administration would potentiate incubation of cocaine craving in male and female rats, and the role of estrous cycles in incubation. METHODS: In Experiment 1, male (n=52) and female (n=47) rats self-administered cocaine either continuously (8-h/d) or intermittently (5-min ON, 25-min OFF x 16) for 12 days, followed by relapse tests after 2 or 29 abstinence days. In Experiment 2-3, female rats self-administered cocaine intermittently for 6 (n=16), 12 (n=32), or 18 (n=16) sessions, followed by relapse tests after 2 or 29 abstinence days. In Experiment 3, the estrus cycle was measured using a vaginal smear test. RESULTS: Incubation of cocaine craving was observed in both sexes after either intermittent or continuous access drug self-administration. Independent of access condition and abstinence day, cocaine seeking was higher in females than in males. In both sexes, cocaine seeking on both abstinence days was higher after intermittent drug access than after continuous drug access. In females, incubation of craving after intermittent drug access occurred during the estrous phase, but not the non-estrous phases; this effect was independent of the training duration. CONCLUSION: In both sexes, intermittent cocaine access caused time-independent increases in drug seeking during abstinence. In females, the time-dependent increase in drug seeking (incubation) is dependent on the estrus cycle phase.