Main The function of WAKE is conserved in mammalsWe previously identified the clock-output molecule WIDE AWAKE (WAKE) from a forward genetic screen in Drosophila 4 . WAKE modulates the activity of arousalpromoting clock neurons at night, in order to promote sleep onset and quality 4,5 . The mammalian proteome contains a single ortholog, mWAKE (also named ANKFN1/Nmf9), with 56% sequence similarity and which is enriched in the core region of the master circadian pacemaker suprachiasmatic nucleus (SCN) 4,6 ( Fig. 1a, Extended Data Fig. 1a). To investigate whether the function of WAKE is conserved in mice, we generated a putative null allele of mWAKE (mWAKE (-) ) by CRISPR/Cas9 insertion of 8 base pairs (containing a stop codon and generating a downstream frameshift) in exon 4, which is predicted to be in all splice isoforms of mWAKE ( Fig. 1b). As expected, mWAKE expression, as assessed by quantitative PCR and in situ hybridization (ISH), was markedly reduced in mWAKE (-/-) mice, likely due to nonsense-mediated decay (Fig. 1c, 1d). Given mWAKE expression in the SCN, we first examined locomotor circadian rhythms and found that mWAKE (-/-) mice exhibit a mild but non-significant decrease in circadian period length (Extended Data Fig. 1b, 1c). These results are similar to findings from fly wake mutants and mice bearing the Nmf9 mutation (a previously identified ENU-generated allele of mWAKE) 4,6 .Because we previously demonstrated that WAKE mediates circadian regulation of sleep timing and quality in fruit flies 4,5 , we next assessed sleep in mWAKE (-/-) mice via electroencephalography (EEG). Under light:dark (L:D) conditions, there was no difference in the amount of wakefulness, non-rapid eye movement (NREM), or REM sleep between mWAKE (-/-) mutants and wild-type (WT) littermate controls (Extended Data Fig. 1d). In constant darkness (D:D), there is a modest main effect of genotype on wakefulness (P<0.05) and NREM sleep (P<0.05), and a mild but significant decrease in REM sleep in mWAKE (-/-) mutants (Fig. 1e). Although the amount of wakefulness did not appreciably differ in mWAKE (-/-) mutants compared to controls, there was a change in the distribution of wakefulness at night; mutants spent more daily time in prolonged wake bouts, and some mutants exhibited dramatically long bouts of wakefulness (Extended Data Fig. 1e, 1f).

Sex differences in cocaine’s mechanisms of action and behavioral effects have been widely reported. However, little is known about how sex influences intracellular signaling cascades involved with drug-environment associations. We investigated whether ERK/CREB intracellular responses in the mesocorticolimbic circuitry underlying cocaine environmental associations are sexually dimorphic. We used a standard 4 day conditioned place preference (CPP) paradigm using 20mg/kg cocaine—a dose that induced CPP in male and female Fischer rats. In the nucleus accumbens (NAc) following CPP expression, cocaine treated animals showed increased phosphorylated ERK (pERK), phosphorylated CREB (pCREB) and ΔFosB protein levels. In the hippocampus (HIP) and caudate putamen (CPu), pERK and FosB/ΔFosB levels were also increased, respectively. Cocaine females had a larger change in HIP pERK and CPu ΔFosB levels than cocaine males; partly due to lower protein levels in saline female rats when compared to saline males. Prefrontal cortex (PfC) pCREB levels increased in cocaine males, but not females, whereas PfC pERK levels were increased in cocaine females, but not males. CPP scores were positively correlated to NAc pERK, HIP pERK and CPu FosB protein levels, suggesting that similar to males, the ERK/CREB intracellular pathway in mesocorticolimbic regions undergoes cocaine induced neuroplasticity in female rats. However, there seem to be intrinsic (basal) sexual dimorphisms in this pathway that may contribute to responses expressed after cocaine-CPP. Taken together, our results suggest that cellular responses associated with the expression of learned drug-environment associations may play an important role in sex differences in cocaine addiction and relapse.

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Making predictions about future rewards or pun-1 ishments is fundamental to adaptive behavior. 2 These processes are influenced by prior experi-3 ence. For example, prior exposure to aversive 4 stimuli or stressors changes behavioral responses 5 to negative-and positive-value predictive cues.6Here, we demonstrate a role for medial pre-7 frontal cortex (mPFC) neurons projecting to the 8 paraventricular nucleus of the thalamus (PVT; 9 mPFC→PVT) in this process. We found that a 10 history of punishments negatively biased behav-11 ioral responses to motivationally-relevant stim-12 uli in mice and that this negative bias was asso-13 ciated with hyperactivity in mPFC→PVT neu-14 rons during exposure to those cues. Further-15 more, artificially mimicking this hyperactive re-16 sponse with selective optogenetic excitation of 17 the same pathway recapitulated the punishment-18 induced negative behavioral bias. Together, our 19 results highlight how information flow within the 20 mPFC→PVT circuit is critical for making pre-21 dictions about imminent motivationally-relevant 22 outcomes as a function of prior experience. 23 vated by multiple forms of stressors (Chastrette et al., 75 1991; Sharp et al., 1991; Cullinan et al., 1995; Bubser 76 and Deutch, 1999; Spencer et al., 2004) and coordinate 77 behavioral responses to stress (Hsu et al., 2014; Do-78 Monte et al., 2015; Penzo et al., 2015; Zhu et al., 2016; 79 Do-Monte et al., 2017; Beas et al., 2018). On the other 80 hand, under conditions of opposing emotional valence, 81 PVT plays a role in multiple forms of stimulus-reward 82 learning and PVT neurons have been reported to show 83 reward-modulated responses (Schiltz et al., 2005; Igel-84 strom et al. , 2010; Martin-Fardon and Boutrel, 2012; 85 James and Dayas, 2013; Browning et al., 2014; Haight 86 and Flagel, 2014; Li et al., 2016; Choi et al., 2019). 87Activity in mPFC neurons projecting to the PVT also 88 suppresses both the acquisition and expression of condi-89 tioned reward seeking (Otis et al., 2017). 90Taken together, these studies place the mPFC to PVT 91 projection in a unique position to integrate information 92 about positive and negative motivationally-relevant cues 93 and translate it into adaptive behavioral responses. How 94 these projection-specific prefrontal neurons regulate be-95 115 phase, four odor cues (A, B, C and D, counterbalanced) 116 were presented. Odor A predicted an appetitive sweet 117 solution (3 µl of 5% sucrose water). Odor B predicted 118 an aversive bitter solution (3 µl of 10 mM denatonium 119 water). Odor C was associated with no reinforcement. 120 Odor D predicted a punishment (an unavoidable air puff 121 delivered to the mouse's right eye) in mice assigned to 122 the air puff group and was associated with no reinforce-123 ment in mice assigned to the no air puff group. Each 124 behavioral trial began with an odor (1 s; conditioned 125 stimulus, CS), followed by a 1-s delay and an outcome 126 (unconditioned stimulus, US). Mice showed essentially 127 binary respo...

The midbrain dorsal raphe (DR) and ventral tegmental area (VTA) contain two of the brains main ascending neuromodulatory transmitters: serotonin and dopamine. We studied the pathway from DR to VTA using single-cell RNA sequencing, anatomical tracing, and electrophysiology and behavior in mice. Single-cell sequencing confirmed a differential distribution of dopamine cell types between medial and lateral aspects of the VTA. This molecular diversity included differential expression of a subset of glutamatergic and serotonergic receptors. Anatomical data showed that distinct serotonergic and glutamatergic populations of DR neurons project to distinct medial-lateral locations in VTA. Physiological data showed that serotonergic neurons are positioned to excite putative dopaminergic neurons in lateral VTA on short timescales (within trial), and inhibit them on long timescales (on the next trial). Our results reveal precise anatomical specificity of DR projections to VTA, and suggest a functional role for serotonergic modulation of dopaminergic function across multiple timescales.

Making predictions about future rewards or punishments is fundamental to adaptive behavior. These processes are influenced by prior experience. For example, prior exposure to aversive stimuli or stressors changes behavioral responses to negative- and positive-value predictive cues. Here, we demonstrate a role for medial prefrontal cortex (mPFC) neurons projecting to the paraventricular nucleus of the thalamus (PVT; mPFC→PVT) in this process. We found that a history of aversive stimuli negatively biased behavioral responses to motivationally relevant cues in mice and that this negative bias was associated with hyperactivity in mPFC→PVT neurons during exposure to those cues. Furthermore, artificially mimicking this hyperactive response with selective optogenetic excitation of the same pathway recapitulated the negative behavioral bias induced by aversive stimuli, whereas optogenetic inactivation of mPFC→PVT neurons prevented the development of the negative bias. Together, our results highlight how information flow within the mPFC→PVT circuit is critical for making predictions about motivationally-relevant outcomes as a function of prior experience.

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