Decreasing dorsal cochlear nucleus activity ameliorates noise-induced tinnitus perception in mice.

BACKGROUND: The dorsal cochlear nucleus (DCN) is a region known to integrate somatosensory and auditory inputs and is identified as a potential key structure in the generation of phantom sound perception, especially noise-induced tinnitus. Yet, how altered homeostatic plasticity of the DCN induces and maintains the sensation of tinnitus is not clear. Here, we chemogenetically decrease activity of a subgroup of DCN neurons, Ca2+/Calmodulin kinase 2 α (CaMKII α)-positive DCN neurons, using Gi-coupled human M4 Designer Receptors Exclusively Activated by Designer Drugs (hM4Di DREADDs), to investigate their role in noise-induced tinnitus. RESULTS: Mice were exposed to loud noise (9-11kHz, 90dBSPL, 1h, followed by 2h of silence), and auditory brainstem responses (ABRs) and gap prepulse inhibition of acoustic startle (GPIAS) were recorded 2 days before and 2 weeks after noise exposure to identify animals with a significantly decreased inhibition of startle, indicating tinnitus but without permanent hearing loss. Neuronal activity of CaMKII α+ neurons expressing hM4Di in the DCN was lowered by administration of clozapine-N-oxide (CNO). We found that acutely decreasing firing rate of CaMKII α+ DCN units decrease tinnitus-like responses (p = 3e -3, n = 11 mice), compared to the control group that showed no improvement in GPIAS (control virus; CaMKII α-YFP + CNO, p = 0.696, n = 7 mice). Extracellular recordings confirmed CNO to decrease unit firing frequency of CaMKII α-hM4Di+ mice and alter best frequency and tuning width of response to sound. However, these effects were not seen if CNO had been previously administered during the noise exposure (n = 6 experimental and 6 control mice). CONCLUSION: We found that lowering DCN activity in mice displaying tinnitus-related behavior reduces tinnitus, but lowering DCN activity during noise exposure does not prevent noise-induced tinnitus. Our results suggest that CaMKII α-positive cells in the DCN are not crucial for tinnitus induction but play a significant role in maintaining tinnitus perception in mice.

Chrna2-Martinotti Cells Synchronize Layer 5 Type A Pyramidal Cells via Rebound Excitation.

Martinotti cells are the most prominent distal dendrite-targeting interneurons in the cortex, but their role in controlling pyramidal cell (PC) activity is largely unknown. Here, we show that the nicotinic acetylcholine receptor α2 subunit (Chrna2) specifically marks layer 5 (L5) Martinotti cells projecting to layer 1. Furthermore, we confirm that Chrna2-expressing Martinotti cells selectively target L5 thick-tufted type A PCs but not thin-tufted type B PCs. Using optogenetic activation and inhibition, we demonstrate how Chrna2-Martinotti cells robustly reset and synchronize type A PCs via slow rhythmic burst activity and rebound excitation. Moreover, using optical feedback inhibition, in which PC spikes controlled the firing of surrounding Chrna2-Martinotti cells, we found that neighboring PC spike trains became synchronized by Martinotti cell inhibition. Together, our results show that L5 Martinotti cells participate in defined cortical circuits and can synchronize PCs in a frequency-dependent manner. These findings suggest that Martinotti cells are pivotal for coordinated PC activity, which is involved in cortical information processing and cognitive control.

The expanding fireball of Nova Delphini 2013.

A classical nova occurs when material accreting onto the surface of a white dwarf in a close binary system ignites in a thermonuclear runaway. Complex structures observed in the ejecta at late stages could result from interactions with the companion during the common-envelope phase. Alternatively, the explosion could be intrinsically bipolar, resulting from a localized ignition on the surface of the white dwarf or as a consequence of rotational distortion. Studying the structure of novae during the earliest phases is challenging because of the high spatial resolution needed to measure their small sizes. Here we report near-infrared interferometric measurements of the angular size of Nova Delphini 2013, starting one day after the explosion and continuing with extensive time coverage during the first 43 days. Changes in the apparent expansion rate can be explained by an explosion model consisting of an optically thick core surrounded by a diffuse envelope. The optical depth of the ejected material changes as it expands. We detect an ellipticity in the light distribution, suggesting a prolate or bipolar structure that develops as early as the second day. Combining the angular expansion rate with radial velocity measurements, we derive a geometric distance to the nova of 4.54 ± 0.59 kiloparsecs from the Sun.

Crosstalk between mitochondria, calcium channels and actin cytoskeleton modulates noradrenergic activity of locus coeruleus neurons.

Locus coeruleus (LC) is the name of a group of large sized neurons located at the brain stem, which provides the main source of noradrenaline to the central nervous system, virtually, innervating the whole brain. All noradrenergic signalling provided by this nucleus is dependent on an intrinsic pacemaker process. Our study aims to understand how noradrenergic neurons finely tune their pacemaker processes and regulate their activities. Here we present that mitochondrial perturbation in the LC from mice, inhibits spontaneous firing by a hyperpolarizing response that involves Ca2+ entry via L-type Ca2+ channels and the actin cytoskeleton. We found that pharmacological perturbation of mitochondria from LC neurons using the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP), induced a dominant hyperpolarizing response when electrophysiological approaches were performed. Surprisingly, the CCCP-induced hyperpolarizing response was dependent on L-type Ca2+ channel-mediated Ca2+ entry, as it was inhibited by: the removal of extracellular Ca2+ ; the addition of Cd2+ ; nifedipine or nicardipine; but not by the intracellular dialysis with the Ca2+ chelator 1,2-Bis(2-Aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, the latter indicating that the response was not because of a global change in [Ca2+ ]c but does not exclude action at intracellular microdomains. Further to this, the incubation of slices with cytochalasin D, an agent that depolymerises the actin cytoskeleton, inhibited the hyperpolarizing response indicating an involvement of the actin cytoskeleton. The data are consistent with the hypothesis that there is a crosstalk between mitochondria and L-type Ca2+ channels leading to modulation of noradrenergic neuronal activity mediated by the actin cytoskeleton. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/.

Chrna2-OLM interneurons display different membrane properties and h-current magnitude depending on dorsoventral location.

The hippocampus is an extended structure displaying heterogeneous anatomical cell layers along its dorsoventral axis. It is known that dorsal and ventral regions show different integrity when it comes to functionality, innervation, gene expression, and pyramidal cell properties. Still, whether hippocampal interneurons exhibit different properties along the dorsoventral axis is not known. Here, we report electrophysiological properties of dorsal and ventral oriens lacunosum moleculare (OLM) cells from coronal sections of the Chrna2-cre mouse line. We found dorsal OLM cells to exhibit a significantly more depolarized resting membrane potential compared to ventral OLM cells, while action potential properties were similar between the two groups. We found ventral OLM cells to show a higher initial firing frequency in response to depolarizing current injections but also to exhibit a higher spike-frequency adaptation than dorsal OLM cells. Additionally, dorsal OLM cells displayed large membrane sags in response to negative current injections correlating with our results showing that dorsal OLM cells have more hyperpolarization-activated current (Ih ) compared to ventral OLM cells. Immunohistochemical examination indicates the h-current to correspond to hyperpolarization-activated cyclic nucleotide-gated subunit 2 (HCN2) channels. Computational studies suggest that Ih in OLM cells is essential for theta oscillations in hippocampal circuits, and here we found dorsal OLM cells to present a higher membrane resonance frequency than ventral OLM cells. Thus, our results highlight regional differences in membrane properties between dorsal and ventral OLM cells allowing this interneuron to differently participate in the generation of hippocampal theta rhythms depending on spatial location along the dorsoventral axis of the hippocampus.

Salicylate induces anxiety-like behavior and slow theta oscillation and abolishes the relationship between running speed and fast theta oscillation frequency.

Salicylate intoxication is a cause of tinnitus in humans and it is often used to produce tinnitus-like perception in animal models. Here, we assess whether salicylate induces anxiety-like electrophysiological and behavioral signs. Using microwire electrode arrays, we recorded local field potential in the ventral and, in some experiments dorsal hippocampus, in an open field arena 1 hr after salicylate (300 mg/kg) injection. We found that animals treated with salicylate moved dramatically less than saline treated animals. Salicylate-treated animals showed a strong 4-6 Hz (type 2) oscillation in the ventral hippocampus (with smaller peaks in dorsal hippocampus electrodes). Coherence in the 4-6 Hz-theta band was low in the ventral and dorsal hippocampus when compared to movement-related theta coherence (7-10 Hz). Moreover, movement related theta oscillation frequency decreased and its dependency on running speed was abolished. Our results suggest that salicylate-induced theta is mostly restricted to the ventral hippocampus. Slow theta has been classically associated to anxiety-like behaviors. Here, we show that salicylate application can consistently generate low frequency theta in the ventral hippocampus. Tinnitus and anxiety show strong comorbidity and the increase in ventral hippocampus low frequency theta could be part of this association.

Chronic in vivo optogenetic stimulation modulates neuronal excitability, spine morphology, and Hebbian plasticity in the mouse hippocampus.

Prolonged increases in excitation can trigger cell-wide homeostatic responses in neurons, altering membrane channels, promoting morphological changes, and ultimately reducing synaptic weights. However, how synaptic downscaling interacts with classical forms of Hebbian plasticity is still unclear. In this study, we investigated whether chronic optogenetic stimulation of hippocampus CA1 pyramidal neurons in freely moving mice could (a) cause morphological changes reminiscent of homeostatic scaling, (b) modulate synaptic currents that might compensate for chronic excitation, and (c) lead to alterations in Hebbian plasticity. After 24 hr of stimulation with 15-ms blue light pulses every 90 s, dendritic spine density and area were reduced in the CA1 region of mice expressing channelrhodopsin-2 (ChR2) when compared to controls. This protocol also reduced the amplitude of mEPSCs for both the AMPA and NMDA components in ex vivo slices obtained from ChR2-expressing mice immediately after the end of stimulation. Finally, chronic stimulation impaired the induction of LTP and facilitated that of LTD in these slices. Our results indicate that neuronal responses to prolonged network excitation can modulate subsequent Hebbian plasticity in the hippocampus.

Farming characteristics and self-reported health outcomes of Irish farmers.

Background: Irish farmers represent a 'high-risk' group for non-communicable diseases, which, arguably, pose a greater occupational health challenge for farmers. To date, there has been little exploration of the farming characteristics associated with farmers' poor health outcomes. Aims: To examine the relationship between farming and male farmers' self-reported health outcomes and to compare the study findings to national health studies to explore which factors specifically are associated with Irish farmers' poorer health outcomes relative to the general population. Methods: This cross-sectional survey research used self-reported quantitative data on the health outcomes and health behaviours of male farmers from the South-East of Ireland. Data were entered into SPSS and descriptive and binary regression techniques were used for data analysis. Results: There were 314 participants (99% response rate). Age, full-time farming and dairy farming significantly impacted self-reported health outcomes and health behaviours. There was a high prevalence of self-reported arthritis compared with the national average of Irish males. 'Younger' farmers (<45 years) were more likely to engage in harmful health behaviours such as smoking and 'binge-drinking' one or more times per week. Conclusions: This study identified self-reported patterns of risky lifestyle behaviours among particular subgroups of Irish farmers for whom targeted health interventions are warranted. Interventions are particularly important for younger farmers who may see themselves as invincible and impregnable to ill-health.

A Respiration-Coupled Rhythm in the Rat Hippocampus Independent of Theta and Slow Oscillations.

UNLABELLED: During slow-wave sleep and deep anesthesia, the rat hippocampus displays a slow oscillation (SO) that follows "up-and-down" state transitions in the neocortex. There has been recent debate as to whether this local field potential (LFP) rhythm reflects internal processing or entrains with respiratory inputs. To solve this issue, here we have concomitantly recorded respiration along with hippocampal, neocortical, and olfactory bulb (OB) LFPs in rats anesthetized with urethane. During the course of anesthesia, LFPs transitioned between activity states characterized by the emergence of different oscillations. By jointly analyzing multisite LFPs and respiratory cycles, we could distinguish three types of low-frequency hippocampal oscillations: (1) SO, which coupled to neocortical up-and-down transitions; (2) theta, which phase-reversed across hippocampal layers and was largest at the fissure; and (3) a low-frequency rhythm with largest amplitude in the dentate gyrus, which coupled to respiration-entrained oscillations in OB and to respiration itself. In contrast, neither theta nor SO coupled to respiration. The hippocampal respiration-coupled rhythm and SO had frequency <1.5 Hz, whereas theta tended to be faster (>3 Hz). Tracheotomy abolished hippocampal respiration-coupled rhythm, which was restored by rhythmic delivery of air puffs into the nasal cavity. These results solve the apparent contradictions among previous studies by demonstrating that the rat hippocampus produces multiple types of low-frequency oscillations. Because they synchronize with different brain circuits, however, we postulate that each activity pattern plays a unique role in information processing. SIGNIFICANCE STATEMENT: The rat hippocampus exhibits a large-amplitude slow oscillation (<1.5 Hz) during deep sleep and anesthesia. It is currently debated whether this rhythm reflects internal processing with the neocortex or entrainment by external inputs from rhythmic nasal respiration, which has similar frequency. Here we reconcile previous studies by showing that the hippocampus can actually produce two low-frequency rhythms at nearby frequencies: one that indeed couples to respiration and another that is coupled to the neocortex. We further show that the respiration-coupled rhythm differs from theta oscillations. The results support a role for brain oscillations in connecting distant brain regions, and posit the respiratory cycle as an important reference for neuronal communication between olfactory and memory networks.

Limiting glutamate transmission in a Vglut2-expressing subpopulation of the subthalamic nucleus is sufficient to cause hyperlocomotion.

The subthalamic nucleus (STN) is a key area of the basal ganglia circuitry regulating movement. We identified a subpopulation of neurons within this structure that coexpresses Vglut2 and Pitx2, and by conditional targeting of this subpopulation we reduced Vglut2 expression levels in the STN by 40%, leaving Pitx2 expression intact. This reduction diminished, yet did not eliminate, glutamatergic transmission in the substantia nigra pars reticulata and entopeduncular nucleus, two major targets of the STN. The knockout mice displayed hyperlocomotion and decreased latency in the initiation of movement while preserving normal gait and balance. Spatial cognition, social function, and level of impulsive choice also remained undisturbed. Furthermore, these mice showed reduced dopamine transporter binding and slower dopamine clearance in vivo, suggesting that Vglut2-expressing cells in the STN regulate dopaminergic transmission. Our results demonstrate that altering the contribution of a limited population within the STN is sufficient to achieve results similar to STN lesions and high-frequency stimulation, but with fewer side effects.

Interconnection and synchronization of neuronal populations in the mouse medial septum/diagonal band of Broca.

The medial septum/diagonal band of Broca (MS/DBB) is crucial for hippocampal theta rhythm generation (4-12 Hz). However, the mechanisms behind theta rhythmogenesis are still under debate. The MS/DBB consists, in its majority, of three neuronal populations that use acetylcholine, GABA, or glutamate as neurotransmitter. While the firing patterns of septal neurons enable the MS/DBB to generate rhythmic output critical for the generation of the hippocampal theta rhythm, the ability to synchronize these action potentials is dependent on the interconnectivity between the three major MS/DBB neuronal populations, yet little is known about intraseptal connections. Here we assessed the connectivity between pairs of MS/DBB neurons with paired patch-clamp recordings. We found that glutamatergic and GABAergic neurons provide intraseptal connections and produce sizable currents in MS/DBB postsynaptic cells. We also analyzed linear and nonlinear relationships between the action potentials fired by pairs of neurons belonging to various MS/DBB neuronal populations. Our results show that while the synchrony index for action potential firing was significantly higher in pairs of GABAergic neurons, coherence of action potential firing in the theta range was similarly low in all pairs analyzed. Recurrence analysis demonstrated that individual action potentials were more recurrent in cholinergic neurons than in other cell types. Implementing sparse connectivity in a computer model of the MS/DBB network reproduced our experimental data. We conclude that the interplay between the intrinsic membrane properties of different MS/DBB neuronal populations and the connectivity among these populations underlie the ability of the MS/DBB network to critically contribute to hippocampal theta rhythmogenesis.

A role for solute carrier family 10 member 4, or vesicular aminergic-associated transporter, in structural remodelling and transmitter release at the mouse neuromuscular junction.

The solute carrier and presynaptic vesicle protein solute carrier family 10 member 4, or vesicular aminergic-associated transporter (VAAT), was recently proven to have a modulatory role in central cholinergic signalling. It is currently unknown whether VAAT also affects peripheral cholinergic synapses. Here we demonstrated a regulatory role for the presynaptic vesicle protein VAAT in neuromuscular junction (NMJ) development and function. NMJs lacking VAAT had fewer branch points, whereas endplates showed an increased number of islands. Whereas the amplitude of spontaneous miniature endplate potentials in VAAT-deficient NMJs was decreased, the amplitude of evoked endplate potentials and the size of the readily releasable pool of vesicles were both increased. Moreover, VAAT-deficient NMJs displayed aberrant short-term synaptic plasticity with enhanced synaptic depression in response to high-frequency stimulation. Finally, the transcript levels of cholinergic receptor subunits in VAAT-deficient muscles were increased, indicating a compensatory postsynaptic sensitization. Our results suggested that VAAT modulates NMJ transmission efficiency and, as such, may represent a novel target for treatment of disorders affecting motor neurons.

Remote surveillance after colorectal cancer surgery: an effective alternative to standard clinic-based follow-up.

AIM: Most colorectal cancer recurrences are asymptomatic and are detected through routine postoperative clinic surveillance programmes with associated investigations. However, attendance at these clinics has a financial cost and may be associated with an increase in patient anxiety and dissatisfaction. The results of a remote follow-up system developed for selected patients are reported. METHOD: A remote surveillance programme has been in place in our institution for over 9 years. Patients having elective and emergency treatment for colorectal cancer were enrolled. The timeliness of the investigation, detection of local recurrence and distant metastases and overall 5-year survival rates were determined. A cost review and patient satisfaction survey were performed. RESULTS: The programme was suitable for over 900 patients who had received surgery for colorectal cancer between 2004 and 2012, representing some 50% of the total number of patients treated in this period. Of these, 811 (90%) had investigations carried out on time. Five-year survival rates were comparable with national data. Cost-minimization analysis demonstrated a financial saving of 63% and a 75% reduction in clinic appointments. High levels of overall patient satisfaction (97%) were noted with the programme. CONCLUSION: A remote surveillance system after colorectal cancer surgery is a safe and cost-effective alternative to traditional clinic-based follow up and has high patient satisfaction.

On high-frequency field oscillations (>100 Hz) and the spectral leakage of spiking activity.

Recent reports converge to the idea that high-frequency oscillations in local field potentials (LFPs) represent multiunit activity. In particular, the amplitude of LFP activity above 100 Hz-commonly referred to as "high-gamma" or "epsilon" band-was found to correlate with firing rate. However, other studies suggest the existence of true LFP oscillations at this frequency range that are different from the well established ripple oscillations. Using multisite recordings of the hippocampus of freely moving rats, we show here that high-frequency LFP oscillations can represent either the spectral leakage of spiking activity or a genuine rhythm, depending on recording location. Both spike-leaked, spurious activity and true fast oscillations couple to theta phase; however, the two phenomena can be clearly distinguished by other key features, such as preferred coupling phase and spectral signatures. Our results argue against the idea that all high-frequency LFP activity stems from spike contamination and suggest avoiding defining brain rhythms solely based on frequency range.

Automatic tracking of cells for video microscopy in patch clamp experiments.

BACKGROUND: Visualisation of neurons labeled with fluorescent proteins or compounds generally require exposure to intense light for a relatively long period of time, often leading to bleaching of the fluorescent probe and photodamage of the tissue. Here we created a technique to drastically shorten light exposure and improve the targeting of fluorescent labeled cells that is specially useful for patch-clamp recordings. We applied image tracking and mask overlay to reduce the time of fluorescence exposure and minimise mistakes when identifying neurons. METHODS: Neurons are first identified according to visual criteria (e.g. fluorescence protein expression, shape, viability etc.) and a transmission microscopy image Differential Interference Contrast (DIC) or Dodt contrast containing the cell used as a reference for the tracking algorithm. A fluorescence image can also be acquired later to be used as a mask (that can be overlaid on the target during live transmission video). As patch-clamp experiments require translating the microscope stage, we used pattern matching to track reference neurons in order to move the fluorescence mask to match the new position of the objective in relation to the sample. For the image processing we used the Open Source Computer Vision (OpenCV) library, including the Speeded-Up Robust Features (SURF) for tracking cells. The dataset of images (n = 720) was analyzed under normal conditions of acquisition and with influence of noise (defocusing and brightness). RESULTS: We validated the method in dissociated neuronal cultures and fresh brain slices expressing Enhanced Yellow Fluorescent Protein (eYFP) or Tandem Dimer Tomato (tdTomato) proteins, which considerably decreased the exposure to fluorescence excitation, thereby minimising photodamage. We also show that the neuron tracking can be used in differential interference contrast or Dodt contrast microscopy. CONCLUSION: The techniques of digital image processing used in this work are an important addition to the set of microscopy tools used in modern electrophysiology, specially in experiments with neuron cultures and brain slices.

Unraveling the role of Slc10a4 in auditory processing and sensory motor gating: Implications for neuropsychiatric disorders?

BACKGROUND: Psychiatric disorders, such as schizophrenia, are complex and challenging to study, partly due to the lack of suitable animal models. However, the absence of the Slc10a4 gene, which codes for a monoaminergic and cholinergic associated vesicular transporter protein, in knockout mice (Slc10a4-/-), leads to the accumulation of extracellular dopamine. A major challenge for studying schizophrenia is the lack of suitable animal models that accurately represent the disorder. We sought to overcome this challenge by using Slc10a4-/- mice as a potential model, considering their altered dopamine levels. This makes them a potential animal model for schizophrenia, a disorder known to be associated with altered dopamine signaling in the brain. METHODS: The locomotion, auditory sensory filtering and prepulse inhibition (PPI) of Slc10a4-/- mice were quantified and compared to wildtype (WT) littermates. Intrahippocampal electrodes were used to record auditory event-related potentials (aERPs) for quantifying sensory filtering in response to paired-clicks. The channel above aERPs phase reversal was chosen for reliably comparing results between animals, and aERPs amplitude and latency of click responses were quantified. WT and Slc10a4-/- mice were also administered subanesthetic doses of ketamine to provoke psychomimetic behavior. RESULTS: Baseline locomotion during auditory stimulation was similar between Slc10a4-/- mice and WT littermates. In WT animals, normal auditory processing was observed after i.p saline injections, and it was maintained under the influence of 5 mg/kg ketamine, but disrupted by 20 mg/kg ketamine. On the other hand, Slc10a4-/- mice did not show significant differences between N40 S1 and S2 amplitude responses in saline or low dose ketamine treatment. Auditory gating was considered preserved since the second N40 peak was consistently suppressed, but with increased latency. The P80 component showed higher amplitude, with shorter S2 latency under saline and 5 mg/kg ketamine treatment in Slc10a4-/- mice, which was not observed in WT littermates. Prepulse inhibition was also decreased in Slc10a4-/- mice when the longer interstimulus interval of 100 ms was applied, compared to WT littermates. CONCLUSION: The Slc10a4-/- mice responses indicate that cholinergic and monoaminergic systems participate in the PPI magnitude, in the temporal coding (response latency) of the auditory sensory gating component N40, and in the amplitude of aERPs P80 component. These results suggest that Slc10a4-/- mice can be considered as potential models for neuropsychiatric conditions.

5-MeO-DMT induces sleep-like LFP spectral signatures in the hippocampus and prefrontal cortex of awake rats.

5-methoxy-N,N-dimethyltryptamine (5-MeO-DMT) is a potent classical psychedelic known to induce changes in locomotion, behaviour, and sleep in rodents. However, there is limited knowledge regarding its acute neurophysiological effects. Local field potentials (LFPs) are commonly used as a proxy for neural activity, but previous studies investigating psychedelics have been hindered by confounding effects of behavioural changes and anaesthesia, which alter these signals. To address this gap, we investigated acute LFP changes in the hippocampus (HP) and medial prefrontal cortex (mPFC) of freely behaving rats, following 5-MeO-DMT administration. 5-MeO-DMT led to an increase of delta power and a decrease of theta power in the HP LFPs, which could not be accounted for by changes in locomotion. Furthermore, we observed a dose-dependent reduction in slow (20-50 Hz) and mid (50-100 Hz) gamma power, as well as in theta phase modulation, even after controlling for the effects of speed and theta power. State map analysis of the spectral profile of waking behaviour induced by 5-MeO-DMT revealed similarities to electrophysiological states observed during slow-wave sleep (SWS) and rapid-eye-movement (REM) sleep. Our findings suggest that the psychoactive effects of classical psychedelics are associated with the integration of waking behaviours with sleep-like spectral patterns in LFPs.

Challenges and opportunities in achieving effective regulatory T cell therapy in autoimmune liver disease.

Autoimmune liver diseases (AILD) include autoimmune hepatitis (AIH), primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC). These immune-mediated liver diseases involve a break down in peripheral self-tolerance with largely unknown aetiology. Regulatory T cells (Treg) are crucial in maintaining immunological tolerance. Hence, Treg immunotherapy is an attractive therapeutic option in AILD. Currently, AILD do not have a curative treatment option and patients take life-long immunosuppression or bile acids to control hepatic or biliary inflammation. Clinical investigations using good manufacturing practice (GMP) Treg in autoimmune liver disease have thus far demonstrated that Treg therapy is safe and that Treg migrate to inflamed liver tissue. For Treg immunotherapy to achieve efficacy in AILD, Treg must be retained within the liver and maintain their suppressive phenotype to dampen ongoing immune responses to hepatocytes and biliary epithelium. Therefore, therapeutic Treg subsets should be selected for tissue residency markers and maximal functionality. Optimisation of dosing regime and understanding longevity of Treg in vivo are critical to successful Treg therapy. It is also essential to consider combination therapy options to complement infused Treg, for instance low-dose interleukin-2 (IL-2) to support pre-existing and infused Treg survival and suppressive function. Understanding the hepatic microenvironment in both early- and late-stage AILD presents significant opportunity to better tailor Treg therapy in different patient groups. Modification of a hostile microenvironment to a more favourable one either prior to or during Treg therapy could enhance the efficacy and longevity of infused GMP-Treg. Applying recent technology to discovery of autoantigen responses in AILD, T cell receptor (TCR) sequencing and use of chimeric antigen receptor (CAR) technology represents the next frontier for disease-specific CAR-Treg therapies. Consideration of all these aspects in future trials and discovery research would position GMP Treg immunotherapy as a viable personalised-medicine treatment option for effective control of autoimmune liver diseases.

Laser Capture Microdissection Optimization for High-Quality RNA in Mouse Brain Tissue.

Laser Capture Microdissection (LCM) is a method that allows one to select and dissect well-defined structures, specific cell subpopulations, or even single cells from different types of tissue for subsequent extraction of DNA, RNA, or proteins. Its precision allows the dissection of specific groups of cells, avoiding unwanted cells. However, despite its efficiency, several steps can affect the sample RNA integrity. RNA instability represents a challenge in the LCM method, and low RNA integrity can introduce biases, as different transcripts often have different degradation rates. Here we describe an optimized protocol to provide good-concentration and high-quality RNA from specific structures: dentate gyrus and CA1 in the hippocampus, basolateral amygdala, and anterior cingulate cortex of mouse brain tissue. However, the protocol is applicable to other areas of interest. © 2022 Wiley Periodicals LLC. Basic Protocol: Laser capture microdissection of mouse brain tissue.