Perineuronal nets in the rat medial prefrontal cortex alter hippocampal-prefrontal oscillations and reshape cocaine self-administration memories.

The medial prefrontal cortex (mPFC) is a major contributor to relapse to cocaine in humans and to reinstatement behavior in rodent models of cocaine use disorder. Output from the mPFC is modulated by parvalbumin (PV)-containing fast-spiking interneurons, the majority of which are surrounded by perineuronal nets (PNNs). Here we tested whether chondroitinase ABC (ABC)- mediated removal of PNNs prevented the acquisition or reconsolidation of a cocaine self-administration memory. ABC injections into the dorsal mPFC prior to training attenuated the acquisition of cocaine self-administration. Also, ABC given 3 days prior to but not 1 hr after memory reactivation blocked cue-induced reinstatement. However, reduced reinstatement was present only in rats given a novel reactivation contingency, suggesting that PNNs are required for the updating of a familiar memory. In naive rats, ABC injections into mPFC did not alter excitatory or inhibitory puncta on PV cells but reduced PV intensity. Whole-cell recordings revealed a greater inter-spike interval 1 hr after ABC, but not 3 days later. In vivo recordings from the mPFC and dorsal hippocampus (dHIP) during novel memory reactivation revealed that ABC in the mPFC prevented reward-associated increases in beta and gamma activity as well as phase-amplitude coupling between the dHIP and mPFC. Together, our findings show that PNN removal attenuates the acquisition of cocaine self-administration memories and disrupts reconsolidation of the original memory when combined with a novel reactivation session. Further, reduced dHIP/mPFC coupling after PNN removal may serve as a key biomarker for how to disrupt reconsolidation of cocaine memories and reduce relapse.

Ischemia-reperfusion myocardial infarction induces remodeling of left cardiac-projecting stellate ganglia neurons.

Neurons in the stellate ganglion (SG) provide sympathetic innervation to the heart, brown adipose tissue (BAT), and other organs. Sympathetic innervation to the heart becomes hyperactive following myocardial infarction (MI). The impact of MI on the morphology of cardiac sympathetic neurons is not known, but we hypothesized that MI would stimulate increased cell and dendritic tree size in cardiac neurons. In this study, we examined the effects of ischemia-reperfusion MI on sympathetic neurons using dual retrograde tracing methods to allow detailed characterization of cardiac- and BAT-projecting neurons. Different fluorescently conjugated cholera toxin subunit B (CTb) tracers were injected into the pericardium and the interscapular BAT pads, respectively. Experimental animals received a 45-min occlusion of the left anterior descending coronary artery and controls received sham surgery. One week later, hearts were collected for assessment of MI infarct and SGs were collected for morphological or electrophysiological analysis. Cardiac-projecting SG neurons from MI mice had smaller cell bodies and shorter dendritic trees compared with sham animals, specifically on the left side ipsilateral to the MI. BAT-projecting neurons were not altered by MI, demonstrating the subpopulation specificity of the response. The normal size and distribution differences between BAT- and cardiac-projecting stellate ganglion neurons were not altered by MI. Patch-clamp recordings from cardiac-projecting left SG neurons revealed increased spontaneous excitatory postsynaptic currents despite the decrease in cell and dendritic tree size. Thus, increased dendritic tree size does not contribute to the enhanced sympathetic neural activity seen after MI.NEW & NOTEWORTHY Myocardial infarction (MI) causes structural and functional changes specifically in stellate ganglion neurons that project to the heart, but not in cells that project to brown adipose fat tissue.

Remyelination protects neurons from DLK-mediated neurodegeneration.

Chronic demyelination is theorized to contribute to neurodegeneration and drive progressive disability in demyelinating diseases like multiple sclerosis. Here, we describe two genetic mouse models of inducible demyelination, one distinguished by effective remyelination, and the other by remyelination failure and persistent demyelination. By comparing these two models, we find that remyelination protects neurons from apoptosis, improves conduction, and promotes functional recovery. Chronic demyelination of neurons leads to activation of the mitogen-associated protein kinase (MAPK) stress pathway downstream of dual leucine zipper kinase (DLK), which ultimately induces the phosphorylation of c-Jun in the nucleus. Both pharmacological inhibition and CRISPR/Cas9-mediated disruption of DLK block c-Jun phosphorylation and the apoptosis of demyelinated neurons. These findings provide direct experimental evidence that remyelination is neuroprotective and identify DLK inhibition as a potential therapeutic strategy to protect chronically demyelinated neurons.

Neuronal ER-plasma membrane junctions couple excitation to Ca2+-activated PKA signaling.

Junctions between the endoplasmic reticulum (ER) and the plasma membrane (PM) are specialized membrane contacts ubiquitous in eukaryotic cells. Concentration of intracellular signaling machinery near ER-PM junctions allows these domains to serve critical roles in lipid and Ca2+ signaling and homeostasis. Subcellular compartmentalization of protein kinase A (PKA) signaling also regulates essential cellular functions, however, no specific association between PKA and ER-PM junctional domains is known. Here, we show that in brain neurons type I PKA is directed to Kv2.1 channel-dependent ER-PM junctional domains via SPHKAP, a type I PKA-specific anchoring protein. SPHKAP association with type I PKA regulatory subunit RI and ER-resident VAP proteins results in the concentration of type I PKA between stacked ER cisternae associated with ER-PM junctions. This ER-associated PKA signalosome enables reciprocal regulation between PKA and Ca2+ signaling machinery to support Ca2+ influx and excitation-transcription coupling. These data reveal that neuronal ER-PM junctions support a receptor-independent form of PKA signaling driven by membrane depolarization and intracellular Ca2+, allowing conversion of information encoded in electrical signals into biochemical changes universally recognized throughout the cell.

Experimental design considerations for studies of human tear proteins.

PURPOSE: Human tears contain abundant, diverse sets of proteins that may serve as biomarkers of ocular surface health. There is a need for reproducible methods that consider multiple factors influencing the tear proteome, in addition to the variable of interest. Here we examined a workflow for proteomic analysis of tear proteins without the need to pool tear samples from multiple individuals, thus allowing for analyses based on individual factors, and increasing opportunities for protein biomarker discovery. METHODS: Tears were collected by Schirmer strip following topical ocular anesthetic application then individually stored at -80 °C prior to processing for proteomics. Tear proteins were extracted from Schirmer strips, digested using suspension trapping spin columns (S-Trap), and labeled with high multiplicity tandem mass tags (TMT). Peptide digests were then extensively fractionated by two-dimensional chromatography and analyzed by mass spectrometry to identify and measure changes in protein abundance in each sample. Analysis of select samples was performed to test protocols and to compare the impact of clinically relevant parameters. To facilitate comparison of separate TMT experiments, common pool samples were included in each TMT instrument run and internal reference scaling (IRS) was performed. RESULTS: Differences in subsets of tear proteins were noted for: geographic site of tear collection, contact lens use, and differences in tear fluid volume among individuals. CONCLUSION: These findings demonstrate that proteomic analysis of human tear proteins can be performed without the need to pool samples, and that development of analytic workflows must consider factors that may affect outcomes in studies focused on diverse clinical samples.

Ocular Pain after Refractive Surgery: Interim Analysis of Frequency and Risk Factors.

PURPOSE: To examine the frequency and risk factors for ocular pain after laser assisted in situ keratomileusis (LASIK) and photorefractive keratectomy (PRK). DESIGN: Prospective study of individuals undergoing refractive surgery at 2 different centers. PARTICIPANTS: One hundred nine individuals undergoing refractive surgery: 87% LASIK and 13% PRK. METHODS: Participants rated ocular pain on a numerical rating scale (NRS) of 0 to 10 before surgery and 1 day, 3 months, and 6 months after surgery. A clinical examination focused on ocular surface health was performed 3 and 6 months after surgery. Persistent ocular pain was defined as an NRS score of 3 or more at both 3 and 6 months after surgery (patients), and this group was compared with individuals with NRS scores of < 3 at both time points (control participants). MAIN OUTCOME MEASURES: Individuals with persistent ocular pain after refractive surgery. RESULTS: The 109 patients who underwent refractive surgery were followed up for 6 months after surgery. Mean age was 34 ± 8 years (range, 23-57 years); 62% self-identified as female, 81% as White, and 33% as Hispanic. Eight patients (7%) reported ocular pain (NRS score ≥ 3) before surgery, with the frequency of ocular pain increasing after surgery to 23% (n = 25) at 3 months and 24% (n = 26) at 6 months. Twelve patients (11%) reported an NRS score of 3 or more at both time points and constituted the persistent pain group. Factors that predicted persistent pain after surgery in a multivariable analysis were (1) ocular pain before surgery predicated persistent pain after surgery (odds ratio [OR], 1.87; 95% confidence interval [CI], 1.06-3.31), (2) symptom report of depression before surgery (Patient Health Questionnaire-9: OR, 1.3; 95% CI, 1.1-1.6; P = 0.01), (3) use of an oral antiallergy medication before surgery (OR, 13.6; 95% CI, 2.1-89.3; P = 0.007), and (4) pain intensity day 1 after surgery (OR, 1.6; 95% CI, 1.2-2.2; P = 0.005). There were no significant associations between ocular surface signs of tear dysfunction and ocular pain, P > 0.05 for all ocular surface signs. Most individuals (> 90%) were completely or somewhat satisfied with their vision at 3 and 6 months. CONCLUSIONS: Eleven percent of individuals reported persistent ocular pain after refractive surgery, with several preoperative and perioperative factors predicting pain after surgery. FINANCIAL DISCLOSURE(S): Proprietary or commercial disclosure may be found after the references.

Glial TGFß activity promotes neuron survival in peripheral nerves.

Maintaining long, energetically demanding axons throughout the life of an animal is a major challenge for the nervous system. Specialized glia ensheathe axons and support their function and integrity throughout life, but glial support mechanisms remain poorly defined. Here, we identified a collection of secreted and transmembrane molecules required in glia for long-term axon survival in vivo. We showed that the majority of components of the TGFß superfamily are required in glia for sensory neuron maintenance but not glial ensheathment of axons. In the absence of glial TGFß signaling, neurons undergo age-dependent degeneration that can be rescued either by genetic blockade of Wallerian degeneration or caspase-dependent death. Blockade of glial TGFß signaling results in increased ATP in glia that can be mimicked by enhancing glial mitochondrial biogenesis or suppressing glial monocarboxylate transporter function. We propose that glial TGFß signaling supports axon survival and suppresses neurodegeneration through promoting glial metabolic support of neurons.

Discoidin domain receptor regulates ensheathment, survival and caliber of peripheral axons.

Most invertebrate axons and small-caliber axons in mammalian peripheral nerves are unmyelinated but still ensheathed by glia. Here, we use Drosophila wrapping glia to study the development and function of non-myelinating axon ensheathment, which is poorly understood. Selective ablation of these glia from peripheral nerves severely impaired larval locomotor behavior. In an in vivo RNA interference screen to identify glial genes required for axon ensheathment, we identified the conserved receptor tyrosine kinase Discoidin domain receptor (Ddr). In larval peripheral nerves, loss of Ddr resulted in severely reduced ensheathment of axons and reduced axon caliber, and we found a strong dominant genetic interaction between Ddr and the type XV/XVIII collagen Multiplexin (Mp), suggesting that Ddr functions as a collagen receptor to drive axon wrapping. In adult nerves, loss of Ddr decreased long-term survival of sensory neurons and significantly reduced axon caliber without overtly affecting ensheathment. Our data establish essential roles for non-myelinating glia in nerve development, maintenance and function, and identify Ddr as a key regulator of axon-glia interactions during ensheathment and establishment of axon caliber.

Resveratrol increases tear production and ocular pain after corneal abrasion in male, but not female, rats using a photorefractive keratectomy model.

Photorefractive keratectomy (PRK) is an alternative to LASIK and can cause intense acute pain that is often not relieved by standard treatments. To assess potential therapeutics for this type of acute pain, appropriate preclinical models are needed. We describe a preclinical corneal abrasion rat model that simulates the initial stages of PRK surgery and demonstrates similar pain and tear dysfunction as seen clinically. We used both behavioral and homeostatic assays to determine the therapeutic potential of resveratrol on pain and tear production. Studies were conducted in male and female Sprague-Dawley rats. Heptanol was applied to one eye and the superficial corneal epithelium was removed, mimicking the abrasion used in PRK. Spontaneous pain was assessed with orbital tightening (OT) scores for 7 days. Topical resveratrol increased OT scores sex-specifically in abraded males, but not females, at 72 h and 1 week after abrasion. Resveratrol increased tear production in abraded males, with no effect in abraded females. There was no correlation between OT score at 1 week and tear production measurements, demonstrating no relationship between spontaneous ocular pain and tear dysfunction in this model. These findings demonstrate the usefulness of our corneal abrasion preclinical PRK model for the assessment of ocular pain therapeutics and indicate that topical resveratrol may not be useful for managing PRK-induced pain.

Distinct morphology of cardiac- and brown adipose tissue-projecting neurons in the stellate ganglia of mice.

Sympathetic neurons that innervate the heart are located primarily in the stellate ganglia (SG), which also contains neurons that project to brown adipose tissue (BAT). These studies were designed to examine the morphology of these two populations (cardiac- and BAT-projecting) and their target connectivity. We examined SG neurons in C57BL/6J mice following injections of the retrograde tracer cholera toxin B (CTb) conjugated to Alexa Fluor 488 and Alexa Fluor 555, into cardiac tissue and intrascapular BAT. BAT-projecting SG neurons were widely dispersed in SG, while cardiac-projecting SG neurons were localized primarily near the inferior cardiac nerve base. SG neurons were not dual-labeled, suggesting that sympathetic innervation is specific to the heart and BAT, supporting the idea of "labeled lines" of efferents. Morphologically, cardiac-projecting SG somata had more volume and were less abundant than BAT-projecting neurons using our tracer-labeling paradigm. We found a positive correlation between the number of primary dendrites per neuron and soma volume in cardiac-projecting SG neurons, though not in BAT-projecting neurons. In both SG subpopulations, the number of cholinergic inputs marked with vesicular acetylcholine transporter (VAChT) puncta contacting the soma was positively correlated to soma volume, suggesting scaling of inputs across a range of neuronal sizes. In separate studies using dual tracing from left and right BAT, we found that BAT-projecting SG neurons were located predominately ipsilateral to the injection, but a small subset of SG neurons project bilaterally to BAT. This tracing approach will allow the assessment of cell-specific mechanisms of plasticity within subpopulations of SG neurons.

Analysis of rod/cone gap junctions from the reconstruction of mouse photoreceptor terminals.

Electrical coupling, mediated by gap junctions, contributes to signal averaging, synchronization, and noise reduction in neuronal circuits. In addition, gap junctions may also provide alternative neuronal pathways. However, because they are small and especially difficult to image, gap junctions are often ignored in large-scale 3D reconstructions. Here, we reconstruct gap junctions between photoreceptors in the mouse retina using serial blockface-scanning electron microscopy, focused ion beam-scanning electron microscopy, and confocal microscopy for the gap junction protein Cx36. An exuberant spray of fine telodendria extends from each cone pedicle (including blue cones) to contact 40-50 nearby rod spherules at sites of Cx36 labeling, with approximately 50 Cx36 clusters per cone pedicle and 2-3 per rod spherule. We were unable to detect rod/rod or cone/cone coupling. Thus, rod/cone coupling accounts for nearly all gap junctions between photoreceptors. We estimate a mean of 86 Cx36 channels per rod/cone pair, which may provide a maximum conductance of ~1200 pS, if all gap junction channels were open. This is comparable to the maximum conductance previously measured between rod/cone pairs in the presence of a dopamine antagonist to activate Cx36, suggesting that the open probability of gap junction channels can approach 100% under certain conditions.

Neurons can talk to each other in two ways: they can send chemical messengers across specialized junctions between two cells, or they can directly pass electrical signals to one another. This latter process is made possible by gap junctions, a system of channel-like structures which connect neighbouring cells and let ions move between them. In most neurons, gap junction channels are made from a specialized protein called connexin 36. Gap junctions are small, difficult to observe, and therefore often ignored by researchers studying neural circuits. In response, Ishibashi et al. focused on nerve cells in the mouse retina, in particular the cones (which detect color during the day) and the rods (which are essential for night vision). Gap junctions between rods and cones allow them to communicate; for example, they enable rod signals to directly activate cones. This provides an alternative route for rod signaling known as the 'secondary rod pathway', which seems to be open at night and switches to closed around dawn. Both rods and cones only produce connexin 36, so Ishibashi et al. labeled these proteins with fluorescent tags to pinpoint gap junctions. This showed that each cone makes around 50 gap junctions with nearby rods; however, gap junctions were not detected between cells of the same type. In addition, 3D reconstruction helped to establish the length of each gap junction. Further experiments showed that a typical rod was connected to a cone by about 80 connexin 36 channels. Finally, calculations revealed that the gap junction channels would all need to open to account for the level of electrical activity required for the secondary rod pathway. This suggests that gap junctions may be much more active and important than previously thought. The work by Ishibashi et al. provides a new understanding of the number, size and activity of gap junctions in the retina, potentially paving the way to prevent diseases where light-sensing cells degenerate and cause blindness.

Nicotinamide Riboside Alleviates Corneal and Somatic Hypersensitivity Induced by Paclitaxel in Male Rats.

Purpose: Patients receiving chemotherapy may experience ocular discomfort and dry eye-like symptoms; the latter may be neuropathic in nature. This study assessed corneal and somatic hypersensitivity in male rats treated with paclitaxel and whether it was relieved by nicotinamide riboside (NR). Methods: Corneal sensitivity to tactile and chemical stimulation, basal tear production, and sensitivity of the hindpaw to tactile and cool stimuli were assessed before and after paclitaxel in the absence and presence of sustained treatment with 500 mg/kg per os NR. Corneal nerve density and hindpaw intraepidermal nerve fiber (IENF) density were also examined. Results: Paclitaxel-treated rats developed corneal hypersensitivity to tactile stimuli, enhanced sensitivity to capsaicin but not hyperosmolar saline, and increased basal tear production. Corneal nerve density visualized with anti-ß-tubulin or calcitonin gene-related peptide (CGRP) was unaffected. Paclitaxel induced tactile and cool hypersensitivity of the hindpaw and a loss of nonpeptidergic hindpaw IENFs visualized with anti-protein gene product (PGP) 9.5 and CGRP. NR reversed tactile hypersensitivity of the cornea without suppressing tear production or chemosensitivity; it did not alter corneal afferent density. NR also reversed tactile and cool hypersensitivity of the hindpaw without reversing the loss of hindpaw IENFs. Conclusions: These findings suggest that paclitaxel may be a good translational model for chemotherapy-induced ocular discomfort and that NR may be useful for its relief. The ability of NR to relieve somatic tactile hypersensitivity independent of changes in sensory nerve innervation suggests that reversal of terminal arbor degeneration is not critical to the actions of NR.

Distinct roles for the Charcot-Marie-Tooth disease-causing endosomal regulators Mtmr5 and Mtmr13 in axon radial sorting and Schwann cell myelination.

The form of Charcot-Marie-Tooth type 4B (CMT4B) disease caused by mutations in myotubularin-related 5 (MTMR5; also called SET binding factor 1, SBF1) shows a spectrum of axonal and demyelinating nerve phenotypes. This contrasts with the CMT4B subtypes caused by MTMR2 or MTMR13 (SBF2) mutations, which are characterized by myelin outfoldings and classic demyelination. Thus, it is unclear whether MTMR5 plays an analogous or distinct role from that of its homolog, MTMR13, in the peripheral nervous system (PNS). MTMR5 and MTMR13 are pseudophosphatases predicted to regulate endosomal trafficking by activating Rab GTPases and binding to the phosphoinositide 3-phosphatase MTMR2. In the mouse PNS, Mtmr2 was required to maintain wild-type levels of Mtmr5 and Mtmr13, suggesting that these factors function in discrete protein complexes. Genetic elimination of both Mtmr5 and Mtmr13 in mice led to perinatal lethality, indicating that the two proteins have partially redundant functions during embryogenesis. Loss of Mtmr5 in mice did not cause CMT4B-like myelin outfoldings. However, adult Mtmr5-/- mouse nerves contained fewer myelinated axons than control nerves, likely as a result of axon radial sorting defects. Consistently, Mtmr5 levels were highest during axon radial sorting and fell sharply after postnatal day seven. Our findings suggest that Mtmr5 and Mtmr13 ensure proper axon radial sorting and Schwann cell myelination, respectively, perhaps through their direct interactions with Mtmr2. This study enhances our understanding of the non-redundant roles of the endosomal regulators MTMR5 and MTMR13 during normal peripheral nerve development and disease.

Subcellular localization of D2 receptors in the murine substantia nigra.

G-protein-coupled D2 autoreceptors expressed on dopamine neurons (D2Rs) inhibit transmitter release and cell firing at axonal endings and somatodendritic compartments. Mechanistic details of somatodendritic dopamine release remain unresolved, partly due to insufficient information on the subcellular distribution of D2Rs. Previous studies localizing D2Rs have been hindered by a dearth of antibodies validated for specificity in D2R knockout animals and have been limited by the small sampling areas imaged by electron microscopy. This study utilized sub-diffraction fluorescence microscopy and electron microscopy to examine D2 receptors in a superecliptic pHlourin GFP (SEP) epitope-tagged D2 receptor knockin mouse. Incubating live slices with an anti-SEP antibody achieved the selective labeling of plasma membrane-associated receptors for immunofluorescent imaging over a large area of the substantia nigra pars compacta (SNc). SEP-D2Rs appeared as puncta-like structures along the surface of dendrites and soma of dopamine neurons visualized by antibodies to tyrosine hydroxylase (TH). TH-associated SEP-D2Rs displayed a cell surface density of 0.66 puncta/µm2, which corresponds to an average frequency of 1 punctum every 1.50 µm. Separate ultrastructural experiments using silver-enhanced immunogold revealed that membrane-bound particles represented 28% of total D2Rs in putative dopamine cells within the SNc. Structures immediately adjacent to dendritic membrane gold particles were unmyelinated axons or axon varicosities (40%), astrocytes (19%), other dendrites (7%), or profiles unidentified (34%) in single sections. Some apposed profiles also expressed D2Rs. Fluorescent and ultrastructural analyses also provided the first visualization of membrane D2Rs at the axon initial segment, a compartment critical for action potential generation. The punctate appearance of anti-SEP staining indicates there is a population of D2Rs organized in discrete signaling sites along the plasma membrane, and for the first time, a quantitative estimate of spatial frequency is provided.

Sex differences in the expression of the endocannabinoid system within V1M cortex and PAG of Sprague Dawley rats.

BACKGROUND: Several chronic pain disorders, such as migraine and fibromyalgia, have an increased prevalence in the female population. The underlying mechanisms of this sex-biased prevalence have yet to be thoroughly documented, but could be related to endogenous differences in neuromodulators in pain networks, including the endocannabinoid system. The cellular endocannabinoid system comprises the endogenous lipid signals 2-AG (2-arachidonoylglycerol) and AEA (anandamide); the enzymes that synthesize and degrade them; and the cannabinoid receptors. The relative prevalence of different components of the endocannabinoid system in specific brain regions may alter responses to endogenous and exogenous ligands. METHODS: Brain tissue from naïve male and estrous staged female Sprague Dawley rats was harvested from V1M cortex, periaqueductal gray, trigeminal nerve, and trigeminal nucleus caudalis. Tissue was analyzed for relative levels of endocannabinoid enzymes, ligands, and receptors via mass spectrometry, unlabeled quantitative proteomic analysis, and immunohistochemistry. RESULTS: Mass spectrometry revealed significant differences in 2-AG and AEA concentrations between males and females, as well as between female estrous cycle stages. Specifically, 2-AG concentration was lower within female PAG as compared to male PAG (*p = 0.0077); female 2-AG concentration within the PAG did not demonstrate estrous stage dependence. Immunohistochemistry followed by proteomics confirmed the prevalence of 2-AG-endocannabinoid system enzymes in the female PAG. CONCLUSIONS: Our results suggest that sex differences exist in the endocannabinoid system in two CNS regions relevant to cortical spreading depression (V1M cortex) and descending modulatory networks in pain/anxiety (PAG). These basal differences in endogenous endocannabinoid mechanisms may facilitate the development of chronic pain conditions and may also underlie sex differences in response to therapeutic intervention.

Dedicated C-fiber vagal sensory afferent pathways to the paraventricular nucleus of the hypothalamus.

The nucleus of the solitary tract (NTS) receives viscerosensory information from the vagus nerve to regulate diverse homeostatic reflex functions. The NTS projects to a wide network of other brain regions, including the paraventricular nucleus of the hypothalamus (PVN). Here we examined the synaptic characteristics of primary afferent pathways to PVN-projecting NTS neurons in rat brainstem slices.Expression of the Transient Receptor Potential Vanilloid receptor (TRPV1+ ) distinguishes C-fiber afferents within the solitary tract (ST) from A-fibers (TRPV1-). We used resiniferatoxin (RTX), a TRPV1 agonist, to differentiate the two. The variability in the latency (jitter) of evoked excitatory postsynaptic currents (ST-EPSCs) distinguished monosynaptic from polysynaptic ST-EPSCs. Rhodamine injected into PVN was retrogradely transported to identify PVN-projecting NTS neurons within brainstem slices. Graded shocks to the ST elicited all-or-none EPSCs in rhodamine-positive NTS neurons with latencies that had either low jitter (<200 µs - monosynaptic), high jitter (>200 µs - polysynaptic inputs) or both. RTX blocked ST-evoked TRPV1 + EPSCs whether mono- or polysynaptic. Most PVN-projecting NTS neurons (17/21 neurons) had at least one input polysynaptically connected to the ST. Compared to unlabeled NTS neurons, PVN-projecting NTS neurons were more likely to receive indirect inputs and be higher order. Surprisingly, sEPSC rates for PVN-projecting neurons were double that of unlabeled NTS neurons. The ST synaptic responses for PVN-projecting NTS neurons were either all TRPV1+ or all TRPV1-, including neurons that received both direct and indirect inputs. Overall, PVN-projecting NTS neurons received direct and indirect vagal afferent information with strict segregation regarding TRPV1 expression.

Program evaluation of trauma-informed yoga for vulnerable populations.

BACKGROUND: Trauma is highly prevalent among vulnerable populations, including those who are incarcerated, in treatment for substance use, or seeking mental health services. Trauma-informed yoga seeks to create a safer yoga practice for individuals with a trauma history and may improve emotional and physical wellbeing. Thus, we conducted an evaluation of a trauma-informed yoga program to gain insight into participant experiences. METHODS: Trauma-informed yoga classes were led by trained volunteers and held in three sectors that work with vulnerable populations: corrections and reentry, substance use treatment and recovery, and community and mental health. Data were collected via anonymous survey using a retrospective pre-post design. The survey instrument captured reasons for student participation and perceived effects of yoga on emotional and physical wellbeing. RESULTS: Students were motivated to participate in yoga classes by expectations of physical, mental, and spiritual benefit. Students reported perceived improvements in emotional and physical wellbeing and greater use of self-regulation skills after starting yoga. CONCLUSION: Our findings suggest trauma-informed yoga is perceived as beneficial by vulnerable individuals, especially those in the correctional system or recovering from substance use. Our results support the value of offering trauma-informed yoga in institutionalized and community settings. Improvements in emotional and physical wellbeing warrant formal study.

Diurnal changes in perineuronal nets and parvalbumin neurons in the rat medial prefrontal cortex.

Perineuronal nets (PNNs) surrounding fast-spiking, parvalbumin (PV) interneurons provide excitatory:inhibitory balance, which is impaired in several disorders associated with altered diurnal rhythms, yet few studies have examined diurnal rhythms of PNNs or PV cells. We measured the intensity and number of PV cells and PNNs labeled with Wisteria floribunda agglutinin (WFA) and also the oxidative stress marker 8-oxo-deoxyguanosine (8-oxo-dG) in rat prelimbic medial prefrontal cortex (mPFC) at Zeitgeber times (ZT) ZT0 (lights-on, inactive phase), ZT6 (mid-inactive phase), ZT12 (lights-off, active phase), and ZT18 (mid-active phase). Relative to ZT0, the intensities of PNN and PV labeling were increased in the dark (active) phase compared with the light (inactive) phase. The intensity of 8-oxo-dG was decreased from ZT0 at all times (ZT6,12,18). We also measured GAD 65/67 and vGLUT1 puncta apposed to PV cells with and without PNNs. There were more excitatory puncta on PV cells with PNNs at ZT18 vs. ZT6, but no changes in PV cells without PNNs and no changes in inhibitory puncta. Whole-cell slice recordings in fast-spiking (PV) cells with PNNs showed an increased ratio of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor:N-methyl-D-aspartate receptor (AMPA: NMDA) at ZT18 vs. ZT6. The number of PV cells and PV/PNN cells containing orthodenticle homeobox 2 (OTX2), which maintains PNNs, showed a strong trend toward an increase from ZT6 to ZT18. Diurnal fluctuations in PNNs and PV cells are expected to alter cortical excitatory:inhibitory balance and provide new insights into treatments for diseases impacted by disturbances in sleep and circadian rhythms.

Cocaine memory reactivation induces functional adaptations within parvalbumin interneurons in the rat medial prefrontal cortex.

Substance use disorder is a complex disease created in part by maladaptive learning and memory mechanisms following repeated drug use. Exposure to drug-associated stimuli engages prefrontal cortex circuits, and dysfunction of the medial prefrontal cortex (mPFC) is thought to underlie drug-seeking behaviors. Growing evidence supports a role for parvalbumin containing fast-spiking interneurons (FSI) in modulating prefrontal cortical microcircuit activity by influencing the balance of excitation and inhibition, which can influence learning and memory processes. Most parvalbumin FSIs within layer V of the prelimbic mPFC are surrounded by specialized extracellular matrix structures called perineuronal nets (PNN). Previous work by our group found that cocaine exposure altered PNN-surrounded FSI function, and pharmacological removal of PNNs reduced cocaine-seeking behavior. However, the role of FSIs and associated constituents (parvalbumin and PNNs) in cocaine-related memories was not previously explored and is still unknown. Here, we found that reactivation of a cocaine conditioned place preference memory produced changes in cortical PNN-surrounded parvalbumin FSIs, including decreased parvalbumin intensity, increased parvalbumin cell axis diameter, decreased intrinsic excitability, and increased excitatory synaptic input. Further investigation of intrinsic properties revealed changes in the interspike interval, membrane capacitance, and afterhyperpolarization recovery time. Changes in these specific properties suggest an increase in potassium-mediated currents, which was validated with additional electrophysiological analysis. Collectively, our results indicate that cocaine memory reactivation induces functional adaptations in PNN-surrounded parvalbumin neurons, which likely alters cortical output to promote cocaine-seeking behavior.

PRC2 Acts as a Critical Timer That Drives Oligodendrocyte Fate over Astrocyte Identity by Repressing the Notch Pathway.

PRC2 creates the repressive mark histone H3 Lys27 trimethylation. Although PRC2 is involved in various biological processes, its role in glial development remains ambiguous. Here, we show that PRC2 is required for oligodendrocyte (OL) differentiation and myelination, but not for OL precursor formation. PRC2-deficient OL lineage cells differentiate into OL precursors, but they fail to trigger the molecular program for myelination, highlighting that PRC2 is essential for directing the differentiation timing of OL precursors. PRC2 null OL lineage cells aberrantly induce Notch pathway genes and acquire astrocytic features. The repression of the Notch pathway restores the myelination program and inhibits abnormal astrocytic differentiation in the PRC2-deficient OL lineage, indicating that Notch is a major target of PRC2. Altogether, our studies propose a specific action of PRC2 as a novel gatekeeper that determines the glial fate choice and the timing of OL lineage progression and myelination by impinging on the Notch pathway.