impact protein function and localization, largely via modulating membrane affi nity and protein stability (7)(8)(9). In contrast to the stable thioether linkage of S -prenylation, the thioester linkage of S -acylation confers a reversible and dynamic nature on this modifi cation, and many recent efforts are shedding light on how this modifi cation is regulated ( 8-11 ).There are a variety of methodologies to detect protein S -acylation/palmitoylation in intact cells. A well-established method involves incubating cells with 3 H-labeled palmitate, followed by autoradiography to visualize the degree of isotopic incorporation. However, this approach requires high levels of [ 3 H]palmitate (as many as several mCi per sample) and exposure times on the order of weeks ( 12, 13 ). More recent methods have cleverly circumvented these issues by using nonradioactive derivatives of palmitate, which can be enriched or detected via cycloaddition reactions ( "click chemistry") ( 14-17 ). Nonetheless, these "palmitate-centric" approaches are encumbered by i ) the need for radioactive or chemically modifi ed palmitate analogs; ii ) the likely bias for proteins that undergo rapid palmitate turnover versus proteins that are more stably palmitoylated; iii ) diffi culty in detecting individual S -acylated proteins or their specifi c sites of S -acylation; and iv ) the inability to detect proteins that are acylated with moieties other than palmitate (e.g., shorter, longer, or unsaturated lipid chains).Recently, a "cysteine-centric" approach to identify S -acylated proteins was introduced that uses the conversion of the protein thioester to a disulfi de-linked biotin ( 18, 19 ). This assay, known as acyl-biotin exchange (ABE), is readily Abstract Protein S -acylation is a major posttranslational modifi cation whereby a cysteine thiol is converted to a thioester. A prototype is S -palmitoylation (fatty acylation), in which a protein undergoes acylation with a hydrophobic 16 carbon lipid chain. Although this modifi cation is a well-recognized determinant of protein function and localization, current techniques to study cellular S -acylation are cumbersome and/or technically demanding. We recently described a simple and robust methodology to rapidly identify S -nitrosylation sites in proteins via resin-assisted capture (RAC) and provided an initial description of the applicability of the technique to S -acylated proteins (acyl-RAC). Here we expand on the acyl-RAC assay, coupled with mass spectrometry-based proteomics, to characterize both previously reported and novel sites of endogenous S -acylation. Acyl-RAC should therefore fi nd general applicability in studies of both global and individual protein S -acylation in mammalian cells. Supplementary key words acylation • H-Ras • lipid • palmitoylation • proteomicsProtein cysteine residues undergo a wide variety of chemical reactions owing to thiol nucleophilicity and redox reactivity. These reactions include S -nitrosylation ( 1, 2 ), S -prenylation ( 3, 4 ), and S -acylation ( 5, 6 ), wh...

Summary Brain-wide fluctuations in local field potential oscillations reflect emergent network-level signals that mediate behavior. Cracking the code whereby these oscillations coordinate in time and space (spatiotemporal dynamics) to represent complex behaviors would provide fundamental insights into how the brain signals emotional pathology. Using machine learning, we discover a spatiotemporal dynamic network that predicts the emergence of major depressive disorder (MDD)-related behavioral dysfunction in mice subjected to chronic social defeat stress. Activity patterns in this network originate in prefrontal cortex and ventral striatum, relay through amygdala and ventral tegmental area, and converge in ventral hippocampus. This network is increased by acute threat, and it is also enhanced in three independent models of MDD vulnerability. Finally, we demonstrate that this vulnerability network is biologically distinct from the networks that encode dysfunction after stress. Thus, these findings reveal a convergent mechanism through which MDD vulnerability is mediated in the brain.

Summary Circuits distributed across cortico-limbic brain regions compose the networks that mediate emotional behavior. The prefrontal cortex (PFC) regulates ultraslow (<1Hz) dynamics across these networks, and PFC dysfunction is implicated in stress-related illnesses including major depressive disorder (MDD). To uncover the mechanism whereby stress-induced changes in PFC circuitry alter emotional networks to yield pathology, we used a multi-disciplinary approach including in vivo recordings in mice and chronic social-defeat stress. Our network model, inferred using machine learning, linked stress-induced behavioral pathology to the capacity of PFC to synchronize amygdala and VTA activity. Direct stimulation of PFC-amygdala circuitry with DREADDs normalized PFC-dependent limbic synchrony in stress-susceptible animals and restored normal behavior. In addition to providing insights into MDD mechanisms, our findings demonstrate an interdisciplinary approach that can be used to identify the large-scale network changes that underlie complex emotional pathologies and the specific network nodes that can be used to develop targeted interventions.

Psychological stress contributes to the onset and exacerbation of nearly all neuropsychiatric disorders. Individual differences in stress-regulatory circuits can therefore dramatically affect vulnerability to these illnesses. Here we identify neural circuit mechanisms underlying individual differences in vulnerability to stress using a murine model of chronic social defeat stress. In chronically stressed mice, we find that the degree of prefrontal cortex (PFC) control of amygdala activity predicts stress-susceptibility in individual mice. Critically, we also find that individual differences in PFC activation (i.e. reactivity) during exposure to an aggressor mouse predict the emergence stress-induced behavioral deficits in stress naïve mice. Finally, we show that naturally occurring differences in PFC reactivity directly correspond to the intrinsic firing rate of PFC neurons. This demonstrates that naturally occurring differences in PFC function underlie individual differences in vulnerability to stress, raising the hypothesis that PFC modulation may prevent stress-induced psychiatric disorders.

Transcranial magnetic stimulation and deep brain stimulation have emerged as therapeutic modalities for treatment refractory depression; however, little remains known regarding the circuitry that mediates the therapeutic effect of these approaches. Here we show that direct optogenetic stimulation of prefrontal cortex (PFC) descending projection neurons in mice engineered to express Chr2 in layer V pyramidal neurons (Thy1–Chr2 mice) models an antidepressant-like effect in mice subjected to a forced-swim test. Furthermore, we show that this PFC stimulation induces a long-lasting suppression of anxiety-like behavior (but not conditioned social avoidance) in socially stressed Thy1–Chr2 mice: an effect that is observed >10 d after the last stimulation. Finally, we use optogenetic stimulation and multicircuit recording techniques concurrently in Thy1–Chr2 mice to demonstrate that activation of cortical projection neurons entrains neural oscillatory activity and drives synchrony across limbic brain areas that regulate affect. Importantly, these neural oscillatory changes directly correlate with the temporally precise activation and suppression of limbic unit activity. Together, our findings show that the direct activation of cortical projection systems is sufficient to modulate activity across networks underlying affective regulation. They also suggest that optogenetic stimulation of cortical projection systems may serve as a viable therapeutic strategy for treating affective disorders.

A hydrophobic core is a widely accepted determinant of protein stability. However, regulatory proteins undergoing ligand-induced conformational switching may expose interior residues to solvent and cannot afford to be extremely rigid. Optimizing the energetic balance between stability and binding is challenging. The addition of five interdomain residues to rat and Paramecium calmodulin N-domain fragments (residues 1-75) increased their thermostability by 9 degrees C and lowered their calcium affinity by a factor of 4. This demonstrates that the flexible linker regulates functional properties as well as tethering the neighboring domains and that protein stability may be increased markedly by minor modifications of the C-terminus. The sensitivity of this domain to few and conservative variations in helices A and D (D2E, S17A, T70S and M71L) is demonstrated by the rat CaM fragments having lower stability and higher calcium affinity than fragments of the same length derived from Paramecium CaM.

Background The prefrontal cortex (PFC) plays a critical role in regulating emotional behaviors, and dysfunction of PFC-dependent networks has been broadly implicated in mediating stress-induced behavioral disorders including major depressive disorder (MDD). Methods Here we acquire multi-circuit in vivo activity from eight cortical and limbic brain regions as mice are subjected to the tail suspension test (TST) and an open field test (OFT). We use a linear decoder to determine whether cellular responses across each of the cortical and limbic areas signal movement during the TST and OFT. We then perform repeat behavioral testing to identify which brain areas show cellular adaptations that signal the increase in immobility induced by repeat TST exposure. Results The increase in immobility observed during repeat TST exposure is linked to a selective functional upregulation of cellular activity in infralimbic cortex (IL) and medial dorsal thalamic (Thal), and an increase in the spatiotemporal dynamic interaction between these structures. Inducing this spatiotemporal dynamic using “closed-loop” optogenetic stimulation is sufficient to increase movement in the TST in stress-naïve mice, while stimulating above the carrier frequency of this circuit suppressed movement. This demonstrates that the adaptations in IL-Thal circuitry observed after stress reflect a compensatory mechanism whereby the brain drives neural systems to counterbalance stress effects. Conclusion Our findings provide evidence that targeting endogenous spatiotemporal dynamics is a potential therapeutic approach for treating stress-induced behavioral disorders, and that dynamics are a critical axis of manipulation for causal optogenetic studies.

Mentoring is a developmental experience intended to increase the willingness to learn and establish credibility while building positive relationships through networking. In this commentary, we focus on intentional mentoring for underrepresented mentees, including individuals that belong to minority racial, ethnic, and gender identity groups in Science, Technology, Engineering, Mathematics, and Medicine (STEMM) fields. Intentional mentoring is the superpower action necessary for developing harmony and comprehending the purpose and value of the mentor/mentee relationship. Regardless of a mentor's career stage, we believe the strategies discussed may be used to create a supportive and constructive mentorship environment; thereby improving the retention rates of underrepresented mentees within the scientific community.