Flow Cytometry of Synaptoneurosomes (FCS) Reveals Increased Ribosomal S6 and Calcineurin Proteins in Activated Medial Prefrontal Cortex to Nucleus Accumbens Synapses.

Learning and behavior activate cue-specific patterns of sparsely distributed cells and synapses called ensembles that undergo memory-encoding engram alterations. While Fos is often used to label selectively activated cell bodies and identify neuronal ensembles, there is no comparable endogenous marker to label activated synapses and identify synaptic ensembles. For the purpose of identifying candidate synaptic activity markers, we optimized a flow cytometry of synaptoneurosome (FCS) procedure for assessing protein alterations in activated synapses from male and female rats. After injecting yellow fluorescent protein (YFP)-expressing adeno-associated virus into medial prefrontal cortex (mPFC) to label terminals in nucleus accumbens (NAc) of rats, we injected 20 mg/kg cocaine in a novel context (cocaine+novelty) to activate synapses, and prepared NAc synaptoneurosomes 0-60 min following injections. For FCS, we used commercially available antibodies to label presynaptic and postsynaptic markers synaptophysin and PSD-95 as well as candidate markers of synaptic activity [activity-regulated cytoskeleton protein (Arc), CaMKII and phospho-CaMKII, ribosomal protein S6 (S6) and phospho-S6, and calcineurin and phospho-calcineurin] in YFP-labeled synaptoneurosomes. Cocaine+novelty increased the percentage of S6-positive synaptoneurosomes at 5-60 min and calcineurin-positive synaptoneurosomes at 5-10 min. Electron microscopy verified that S6 and calcineurin levels in synaptoneurosomes were increased 10 min after cocaine+novelty. Pretreatment with the anesthetic chloral hydrate blocked cocaine+novelty-induced S6 and calcineurin increases in synaptoneurosomes, and novel context exposure alone (without cocaine) increased S6, both of which indicate that these increases were due to neural activity per se. Overall, FCS can be used to study protein alterations in activated synapses coming from specifically labeled mPFC projections to NAc.SIGNIFICANCE STATEMENT Memories are formed during learning and are stored in the brain by long-lasting molecular and cellular alterations called engrams formed within specific patterns of cue-activated neurons called neuronal ensembles. While Fos has been used to identify activated ensemble neurons and the engrams within them, we have not had a similar marker for activated synapses that can be used to identify synaptic engrams. Here we developed a procedure for high-throughput in-line analysis of flow cytometry of synaptoneurosome (FCS) and found that ribosomal S6 protein and calcineurin were increased in activated mPFC-NAc synapses. FCS can be used to study protein alterations in activated synapses within specifically labeled circuits.

Prelimbic cortex is a common brain area activated during cue-induced reinstatement of cocaine and heroin seeking in a polydrug self-administration rat model.

Many preclinical studies examined cue-induced relapse to heroin and cocaine seeking in animal models, but most of these studies examined only one drug at a time. In human addicts, however, polydrug use of cocaine and heroin is common. We used a polydrug self-administration relapse model in rats to determine similarities and differences in brain areas activated during cue-induced reinstatement of heroin and cocaine seeking. We trained rats to lever press for cocaine (1.0 mg/kg per infusion, 3-hr/day, 18 day) or heroin (0.03 mg/kg per infusion) on alternating days (9 day for each drug); drug infusions were paired with either intermittent or continuous light cue. Next, the rats underwent extinction training followed by tests for cue-induced reinstatement where they were exposed to either heroin- or cocaine-associated cues. We observed cue-selective reinstatement of drug seeking: the heroin cue selectively reinstated heroin seeking and the cocaine cue selectively reinstated cocaine seeking. We used Fos immunohistochemistry to assess cue-induced neuronal activation in different subregions of the medial prefrontal cortex, dorsal striatum, nucleus accumbens, and amygdala. Fos expression results indicated that only the prelimbic cortex (PL) was activated by both heroin and cocaine cues; in contrast, no significant cue-induced neuronal activation was observed in other brain areas. RNA in situ hybridization indicated that the proportion of glutamatergic and GABAergic markers in PL Fos-expressing cells was similar for the heroin and cocaine cue-activated neurons. Overall, the results indicate that PL may be a common brain area involved in both heroin and cocaine seeking during polydrug use.

Recent advances in cardiovascular risk assessment: The added value of non-invasive anatomic imaging.

In 2022, multiple original research studies were conducted highlighting the utility of coronary artery calcium (CAC) imaging in young individuals and provided further evidence for the role of CAC to improve atherosclerotic cardiovascular disease (ASCVD) risk assessment. Mean calcium density was shown to be a more reliable predictor than peak density in risk assessment. Additionally, in light of the ACC/AHA/Multispecialty Chest Pain Guideline's recent elevation of coronary computed tomography angiography (CCTA) to a Class I (level of evidence A) recommendation as an index diagnostic test for acute or stable chest pain, several studies support the utility of CCTA and guided future directions. This review summarizes recent studies that highlight the role of non-invasive imaging in enhancing ASCVD risk assessment across different populations.

The correlation between intensive care unit attending physician continuity of care with financial and clinical outcomes.

PURPOSE: "Attending rotations" on intensive care unit (ICU) services have been in place in most teaching hospitals for decades. However, the ideal frequency of patient care handoffs is unknown. Frequent attending physician handoffs could result in delays in care and other complications, while too few handoffs can lead to provider burnout and exhaustion. Therefore, we sought to determine the correlation between frequency of attending shifts with ICU charges, 30-day readmission rates, and mortality rates. METHODS: We performed a retrospective cohort study at a large, urban, academic community hospital in Baltimore, MD. We included patients admitted into the cardiac or medical ICUs between September 1, 2012, and December 10, 2015. We tracked the number of attending shifts for each patient and correlated shifts with financial outcomes as a primary measure. RESULTS: For any given ICU length of stay, we found no distinct association between handoff frequency and charges, 30-day readmission rates, or mortality rates. CONCLUSIONS: Despite frequent handoffs in care, there was no objective evidence of care compromise or differences in cost. Further validation of these observations in a larger cohort is justified.