Proteomic measures of gamma oscillations

Abstract

Background: Gamma oscillations serve complex processes, and the first stage of their generation is the reticular activating system (RAS), which mediates the gamma-activity states of waking and paradoxical sleep. We studied whether the pedunculopontine nucleus (PPN), part of the RAS in which every cell manifests intrinsic gamma oscillations, undergoes changes resulting in distinctive protein expression. New method: We previously found that a histone deacetylation inhibitor, trichostatin A (TSA), acutely (30 min) blocked these oscillations. We developed a proteomic method for sampling stimulated and unstimulated PPN and determining protein expression in 1 mm punches of tissue from brain slices subjected to various treatments. Results: We compared brain slices exposed for 30 min to TSA (unstimulated), to the cholinergic agonist carbachol (CAR), known to induce PPN gamma oscillations, or exposed to both TSA þ CAR. Comparison with existing methods: Label-free proteomics provides an unbiased and sensitive method to detect protein changes in the PPN. Our approach is superior to antibody-based methods that can lack specificity and can only be done for known targets. Proteomics methods like these have been leveraged to study molecular pathways in numerous systems and disease states. Conclusions: Significant protein changes were seen in two functions essential to the physiology of the PPN: cytoskeletal and intracellular [Ca 2þ ] regulation proteins. TSA decreased, while CAR increased, and TSA þ CAR had intermediate effects, on expression of these proteins. These results support the feasibility of the methods developed for determining proteomic changes in small samples of tissue participating in the most complex of brain processes.

Gamma oscillations in the pedunculopontine nucleus are regulated by F-actin: neuroepigenetic implications

“…We previously described proteomic analysis following in vitro preincubation with CAR or CAR ϩ TSA (2). We observed significant changes particularly in several proteins related to F-actin cytoskeleton and intracellular [Ca 2ϩ ] regulation in the PPN (2). Other authors have also described fast actions of the HDAC inhibitors SAHA and MS275 on the acetylation of several proteins related to the cytoskeleton (5).…”

“…The recent results of our group showed that 1) acute in vitro exposure to the histone deacetylation inhibitor trichostatin A (TSA) led to lower activation of high threshold, voltage-dependent Ca 2ϩ channel-mediated intrinsic membrane oscillations, specifically in the gamma-band range, but not lower frequency oscillations; 2) preincubation with TSA led to a similar decrease specifically in gamma-band oscillations; and 3) a significant reduction in calcium currents was elicited by TSA (11,34). These changes in PPN rhythmicity could be explained by decreased deacetylation of histones (i.e., changes in gene expression) and/or changes in the deacetylation of other protein targets like CaMKII and F-actin (2,5). Indeed, proteomic analysis of PPN tissue samples after preincubation (30 -60 min, in vitro) with carbachol (CAR) or CAR ϩ TSA showed significant protein changes related to F-actin cytoskeleton and intracellular Ca 2ϩ concentration ([Ca 2ϩ ]) regulation (2).…”

“…Slices were acutely incubated in either CAR or CAR ϩ TSA-modified aCSF solution from a minimum of 30 min and a maximum of 60 min. Incubation times followed acute periods of incubation previously reported by our group (2,34). No transcriptiontranslation inhibitor was used during these experiments.…”

“…These changes in PPN rhythmicity could be explained by decreased deacetylation of histones (i.e., changes in gene expression) and/or changes in the deacetylation of other protein targets like CaMKII and F-actin (2,5). Indeed, proteomic analysis of PPN tissue samples after preincubation (30 -60 min, in vitro) with carbachol (CAR) or CAR ϩ TSA showed significant protein changes related to F-actin cytoskeleton and intracellular Ca 2ϩ concentration ([Ca 2ϩ ]) regulation (2).…”

“…Proteomic analysis of PPN tissue samples showed significant protein changes related to F-actin cytoskeleton after acute inhibition of histone deacetylases (2). This study was designed to determine if PPN intrinsic gamma oscillations that are involved in arousal are affected by F-actin polymerization.…”