The supramammillary region (SuM) is a posterior hypothalamic structure, known to regulate hippocampal theta oscillations and arousal. However, recent studies reported that the stimulation of SuM neurons with neuroactive chemicals, including substances of abuse, is reinforcing. We conducted experiments to elucidate how SuM neurons mediate such effects. Using optogenetics, we found that the excitation of SuM glutamatergic (GLU) neurons was reinforcing in mice; this effect was relayed by their projections to septal GLU neurons. SuM neurons were active during exploration and approach behavior and diminished activity during sucrose consumption. Consistently, inhibition of SuM neurons disrupted approach responses, but not sucrose consumption. Such functions are similar to those of mesolimbic dopamine neurons. Indeed, the stimulation of SuM-to-septum GLU neurons and septum-to-ventral tegmental area (VTA) GLU neurons activated mesolimbic dopamine neurons. We propose that the supramammillo-septo-VTA pathway regulates arousal that reinforces and energizes behavioral interaction with the environment.

Intracranial self-stimulation, in which an animal performs an operant response to receive regional brain electrical stimulation, is a widely used procedure to study motivated behavior. While local neuronal activity has long been measured immediately before or after the operant, imaging the whole brain in real-time remains a challenge. Herein we report a method that permits functional MRI (fMRI) of brain dynamics while mice are cued to perform an operant task: licking a spout to receive optogenetic stimulation to the medial prefrontal cortex (MPFC) during a cue ON, but not cue OFF. Licking during cue ON results in activation of a widely distributed network consistent with underlying MPFC projections, while licking during cue OFF (without optogenetic stimulation) leads to negative fMRI signal in brain regions involved in acute extinction. Noninvasive whole brain readout combined with circuit-specific neuromodulation opens an avenue for investigating adaptive behavior in both healthy and disease models.

18The supramammillary region (SuM) is a posterior hypothalamic structure, known to regulate 19 hippocampal theta oscillations and arousal. However, recent studies reported that the 20 stimulation of SuM neurons with neuroactive chemicals, including substances of abuse, is 21reinforcing. We conducted experiments to illuminate how SuM neurons mediate such effects. 22The excitation of SuM glutamatergic (GLU) neurons was reinforcing in mice; this effect was 23 relayed by the projections to septal GLU neurons. SuM neurons were active during exploration 24and approach behavior and diminished activity during sucrose consumption. Consistently, 25inhibition of SuM neurons disrupted approach responses, but not sucrose consumption. Such 26functions are similar to those of mesolimbic dopamine neurons. Indeed, the stimulation of SuM-27to-septum GLU neurons and septum-to-ventral tegmental area (VTA) GLU neurons activated 28 mesolimbic dopamine neurons. We propose that the supramammillo-septo-VTA pathway is 29 important in reinforcement and approach motivation and may play an important role in mood 30and substance-use disorders. 31 32 102The SuM is comprised of a heterogeneous population of neurons including GLU, DA and 103GABAergic neurons 3 , 9 , 28 , 29. To determine which type of neurons are responsible for the 104 reinforcing effect, we selectively expressed ChR2 in these three respective neuron subtypes 105 ( Fig. 1f-g; Fig. S2). To address site specificity, we prepared a second cohort of vGlut2-Cre 106 mice injected with a small volume (50nL, instead of 200nl) of the vector (n=6). Regardless of 107 injection volumes, both vGlut2-Cre groups quickly learned to press on the active lever with no 108 group difference ( Fig. 1h; a 2group x 2lever x 7session ANOVA, Fgroup(1,16)=0.29, p=0.59); thus, these 109 data were combined. By contrast, TH-Cre and vGat-Cre mice failed to significantly acquire 110 active-lever pressing (a 3group x 5session ANOVA; Fgroup(2,33)=5.79; Tukey HSD posthoc tests for 111 main group effects PvGlut2 vs Th=0.037, PvGlut2 vs vGat=0.016, PvGat vs Th=0.99). These results suggest 112 that the excitation of SuM GLU, but not DA or GABA, neurons is reinforcing. 113