Voltage-gated calcium channels play a central role in regulating the electrical and biochemical properties of neurons and muscle cells. One of the ways in which calcium channels regulate long-lasting neuronal properties is by activating signaling pathways that control gene expression, but the mechanisms that link calcium channels to the nucleus are not well understood. We report that a C-terminal fragment of Ca(V)1.2, an L-type voltage-gated calcium channel (LTC), translocates to the nucleus and regulates transcription. We show that this calcium channel associated transcription regulator (CCAT) binds to a nuclear protein, associates with an endogenous promoter, and regulates the expression of a wide variety of endogenous genes important for neuronal signaling and excitability. The nuclear localization of CCAT is regulated both developmentally and by changes in intracellular calcium. These findings provide evidence that voltage-gated calcium channels can directly activate transcription and suggest a mechanism linking voltage-gated channels to the function and differentiation of excitable cells.

Apoptosis plays a critical role in many neurologic diseases, including stroke. Cytochrome c release and activation of various caspases are known to occur after focal and global ischemia. However, recent reports indicate that caspase-independent pathways may also be involved in ischemic damage. Apoptosis-inducing factor (AIF) is a novel flavoprotein that helps mediate caspase-independent apoptotic cell death. AIF translocates from mitochondria to nuclei where it induces caspase-independent DNA fragmentation. Bcl-2, a mitochondrial membrane protein, protects against apoptotic and necrotic death induced by different insults, including cerebral ischemia. In the present study, Western blots confirmed that AIF was normally confined to mitochondria but translocated to nuclei or cytosol 8, 24, and 48 hours after onset of ischemia. Overall, AIF protein levels also increased after stroke. Confocal microscopy further demonstrated that nuclear AIF translocation occurred in the peri-infarct region but not in the ischemic core where only some cytosolic AIF release was observed. Our data also suggest that AIF translocated into nuclei after cytochrome c was released into the cytosol. Bcl-2 transfection in the peri-infarct region blocked nuclear AIF translocation and improved cortical neuron survival.

Calcium imaging is a common technique that is useful for measuring calcium signals in cultured cells. Calcium imaging techniques take advantage of calcium indicator dyes, which are BAPTA-based organic molecules that change their spectral properties in response to the binding of Ca2+ ions. Calcium indicator dyes fall into two categories, ratio-metric dyes like Fura-2 and Indo-1 and single-wavelength dyes like Fluo-4. Ratio-metric dyes change either their excitation or their emission spectra in response to calcium, allowing the concentration of intracellular calcium to be determined from the ratio of fluorescence emission or excitation at distinct wavelengths. The main advantage of using ratio-metric dyes over single wavelength probes is that the ratio signal is independent of the dye concentration, illumination intensity, and optical path length allowing the concentration of intracellular calcium to be determined independently of these artifacts. One of the most common calcium indicators is Fura-2, which has an emission peak at 505 nM and changes its excitation peak from 340 nm to 380 nm in response to calcium binding. Here we describe the use of Fura-2 to measure intracellular calcium elevations in neurons and other excitable cells. Protocol Cell CultureCells can be grown using established techniques but must be plated on #1 glass coverslips coated with a cellular adhesive (like polylysine, polyornithine or laminin) to prevent the cells from detaching or moving during imaging experiments. SolutionsCalcium imaging experiments can be performed using a variety of physiological solutions including cell culture media. It is important, however, to make sure that the solutions are free of phenol red, which greatly increases the fluorescent background. We use Tyrodes solution, which is easily made and mimics cerebrospinal fluid, and we supplement it with 0.1% Bovine Serum Albumin. We use depolarization with 60-90 mM potassium chloride to activate voltage gated calcium channels and 1μM Thapsigargin (1 mM stock in DMSO) or 2μM Ionomycin (1 mM stock in DMSO) to activate store operated CRAC channels. It is often convenient to remove calcium from the extracellular solution to show that calcium elevations are due to calcium influx. When removing calcium it is necessary to maintain the total concentration of divalent cations (Mg 2 + and Ca 2 +) constant. When substituting potassium for sodium it is necessary to maintain the osmotic balance. Tyrodes solutions:Adjust pH to 7.4 with NaOH Loading of Fura-2 calcium dyeWe load cells with acetoxy-methyl-ester Fura-2 (Fura-2 AM), which diffuses across the cell membrane and is de-esterified by cellular esterases to yield Fura-2 free acid. The exact parameters for Fura-2 loading vary widely across cell types. We recommend testing various conditions by preparing several loading solutions containing a multiple concentrations of Fura-2 raging from 1-4 µM, incubating cells in the loading solution for a variety of times from 15 minutes to 2 hours and testing the loading at room tempe...

In the last decade, burst suppression has been increasingly studied by many to examine whether it is a mechanism leading to postoperative cognitive impairment. Despite a lack of consensus across trials, the current state of research suggests that electroencephalogram (EEG) burst suppression, duration and EEG emergence trajectory may predict postoperative delirium (POD). A mini literature review regarding evidence about burst suppression impact and susceptibilities was conducted, resulting in conflicting studies. Primarily, studies have used different algorithm values to replace visual burst suppression examination, although many studies have since emerged showing that algorithms underestimate burst suppression duration. As these methods may not be interchangeable with visual analysis of raw data, it is a potential factor for the current heterogeneity between data. Even though additional research trials incorporating the use of raw EEG data are necessary, the data currently show that monitoring with commercial intraoperative EEG machines that use EEG indices to estimate burst suppression may help physicians identify burst suppression and guide anesthetic titration during surgery. These modifications in anesthetics could lead to preventing unfavorable outcomes. Furthermore, some studies suggest that brain age, baseline impairment, and certain medications are risk factors for burst suppression and postoperative delirium. These patient characteristics, in conjunction with intraoperative EEG monitoring, could be used for individualized patient care. Future studies on the feasibility of raw EEG monitoring, new technologies for anesthetic monitoring and titration, and patient-associated risk factors are crucial to our continued understanding of burst suppression and postoperative delirium.

BACKGROUND: Ketamine is typically used by anesthesiologists as an adjunct for general anesthesia and as a nonopioid analgesic. It has been explored for prevention of postoperative delirium, although results have been contradictory. In this study, we investigated the association of ketamine with postoperative delirium and specific encephalographic signatures. Furthermore, we examined these associations in the context of baseline neurocognition as measured by a validated assessment. METHODS: We conducted a prospective observational study from January 2019 to December 2020. Ninety-eight patients aged ≥65 years and undergoing spine surgery scheduled for ≥3 hours were included in the study. All participants who completed the University of California San Francisco (UCSF) Brain Health Assessment preoperatively and postoperatively were assessed with the confusion assessment method for intensive care unit (CAM-ICU) and/or the Nursing Delirium Screening Scale (NuDESC). Patients had frontal electroencephalogram (EEG) recordings (SedLine Root, Masimo, Corp) quantitatively analyzed. We used 60 seconds of artifact-free EEG (without burst suppression) extracted from the middle of the maintenance period to calculate the normalized power spectral density (PSD). Comparisons were made between those who did or did not receive ketamine and according to results from neurocognitive assessments. RESULTS: Ninety-eight patients (of a total of 155, enrolled and consented) had EEG of sufficient quality for analysis (42 women). Overall, we found a significant increase in the EEG power in the moderate frequency range (10-20 Hz) in patients that received ketamine. When the patients were divided by their preoperative cognitive status, this result in the ketamine group only held true for the cognitively normal patients. Patients that were cognitively impaired at baseline did not demonstrate a significant change in EEG characteristics based on ketamine administration, but impaired patients that received ketamine had a significantly higher rate of postoperative delirium (52% ketamine versus 20% no ketamine) (odds ratio [OR], 4.36; confidence interval [CI], 1.02-18.22; P = .048). In patients determined to be preoperatively cognitively normal, the incidence of postoperative delirium was not significantly associated with ketamine administration (19% ketamine versus 17% no ketamine) (OR, 1.10; CI, 0.30-4.04; P = .5833). CONCLUSIONS: Ketamine-related changes in EEG are observed in a heterogeneous group of patients receiving spine surgery. This result was driven primarily by the effect of ketamine on cognitively normal patients and not observed in patients that were cognitively impaired at baseline. Furthermore, patients who were cognitively impaired at baseline and who had received ketamine were more likely to develop postoperative delirium, suggesting that cognitive vulnerability might be predicted by the lack of a neurophysiologic response to ketamine. (Anesth Analg 2022;135:683-92) KEY POINTS• Question: How does ketamine affect the electroencephalog...