Synaptic cell-adhesion molecule latrophilin-2 is differentially directed to dendritic domains of hippocampal neurons.

Hippocampal pyramidal cells possess elaborate dendritic arbors with distinct domains that are targeted with input-specific synaptic sites. This synaptic arrangement is facilitated by synaptic cell-adhesion molecules that act as recognition elements to connect presynaptic and postsynaptic neurons. In this study, we investigate the organization of the synaptic recognition molecule latrophilin-2 at the surface of pyramidal neurons classified by spatial positioning and action potential firing patterns. Surveying two hippocampal neurons that highly express latrophilin-2, late-bursting CA1 pyramidal cells and early-bursting subiculum pyramidal cells, we found the molecule to be differentially positioned on their respective dendritic compartments. Investigating this latrophilin-2 positioning at the synaptic level, we found that the molecule is not present within either the pre- or postsynaptic terminal but rather is tightly coupled to synapses at a perisynaptic location. Together these findings indicate that hippocampal latrophilin-2 distribution patterning is cell-type specific, and requires multiple postsynaptic neurons for its synaptic localization.

Patients with allergic asthma have lower risk of severe COVID-19 outcomes than patients with nonallergic asthma.

BACKGROUND: Although asthma does not appear to be a risk factor for severe coronavirus disease 2019 (COVID-19), outcomes could vary for patients with different asthma subtypes. The objective of this analysis was to compare COVID-19 outcomes in real-world cohorts in the United States among patients with asthma, with or without evidence of allergy. METHODS: In a retrospective analysis of the COVID-19 Optum electronic health record dataset (February 20, 2020-January 28, 2021), patients diagnosed with COVID-19 with a history of moderate-to-severe asthma were divided into 2 cohorts: those with evidence of allergic asthma and those without (nonallergic asthma). After 1:1 propensity score matching, in which covariates were balanced and potential bias was removed, COVID-19 outcomes were compared between cohorts. RESULTS: From a COVID-19 population of 591,198 patients, 1595 patients with allergic asthma and 8204 patients with nonallergic asthma were identified. After propensity score matching (n = 1578 per cohort), risk of death from any cause after COVID-19 diagnosis was significantly lower for patients with allergic vs nonallergic asthma (hazard ratio, 0.48; 95% CI 0.28-0.83; P = 0.0087), and a smaller proportion of patients with allergic vs nonallergic asthma was hospitalized within - 7 to + 30 days of COVID-19 diagnosis (13.8% [n = 217] vs 18.3% [n = 289]; P = 0.0005). Among hospitalized patients, there were no significant differences between patients with allergic or nonallergic asthma in need for intensive care unit admission, respiratory support, or COVID-19 treatment. CONCLUSIONS: Asthma subtype may influence outcomes after COVID-19; patients with allergic asthma are at lower risk for hospitalization/death than those with nonallergic asthma.

Contributions of Astrocyte and Neuronal Volume to CA1 Neuron Excitability Changes in Elevated Extracellular Potassium.

Rapid increases in cell volume reduce the size of the extracellular space (ECS) and are associated with elevated brain tissue excitability. We recently demonstrated that astrocytes, but not neurons, rapidly swell in elevated extracellular potassium (∧[K+] o ) up to 26 mM. However, effects of acute astrocyte volume fluctuations on neuronal excitability in ∧[K+] o have been difficult to evaluate due to direct effects on neuronal membrane potential and generation of action potentials. Here we set out to isolate volume-specific effects occurring in ∧[K+] o on CA1 pyramidal neurons in acute hippocampal slices by manipulating cell volume while recording neuronal glutamate currents in 10.5 mM [K+] o + tetrodotoxin (TTX) to prevent neuronal firing. Elevating [K+] o to 10.5 mM induced astrocyte swelling and produced significant increases in neuronal excitability in the form of mixed α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/N-methyl-D-aspartate (NMDA) receptor mEPSCs and NMDA receptor-dependent slow inward currents (SICs). Application of hyperosmolar artificial cerebrospinal fluid (ACSF) by addition of mannitol in the continued presence of 10.5 mM K+ forced shrinking of astrocytes and to a lesser extent neurons, which resisted swelling in ∧[K+] o . Cell shrinking and dilation of the ECS significantly dampened neuronal excitability in 10.5 mM K+. Subsequent removal of mannitol amplified effects on neuronal excitability and nearly doubled the volume increase in astrocytes, presumably due to continued glial uptake of K+ while mannitol was present. Slower, larger amplitude events mainly driven by NMDA receptors were abolished by mannitol-induced expansion of the ECS. Collectively, our findings suggest that cell volume regulation of the ECS in elevated [K+] o is driven predominantly by astrocytes, and that cell volume effects on neuronal excitability can be effectively isolated in elevated [K+] o conditions.

Parahippocampal latrophilin-2 (ADGRL2) expression controls topographical presubiculum to entorhinal cortex circuit connectivity.

Brain circuits are comprised of distinct interconnected neurons that are assembled by synaptic recognition molecules presented by defined pre- and post-synaptic neurons. This cell-cell recognition process is mediated by varying cellular adhesion molecules, including the latrophilin family of adhesion G-protein-coupled receptors. Focusing on parahippocampal circuitry, we find that latrophilin-2 (Lphn2; gene symbol ADGRL2) is specifically enriched in interconnected subregions of the medial entorhinal cortex (MEC), presubiculum (PrS), and parasubiculum (PaS). Retrograde viral tracing from the Lphn2-enriched region of the MEC reveals unique topographical patterning of inputs arising from the PrS and PaS that mirrors Lphn2 expression. Using a Lphn2 conditional knockout mouse model, we find that deletion of MEC Lphn2 expression selectively impairs retrograde viral labeling of inputs arising from the ipsilateral PrS. Combined with analysis of Lphn2 expression within the MEC, this study reveals Lphn2 to be selectively expressed by defined cell types and essential for MEC-PrS circuit connectivity.

Astrocyte-Selective Volume Increase in Elevated Extracellular Potassium Conditions Is Mediated by the Na+/K+ ATPase and Occurs Independently of Aquaporin 4.

Astrocytes and neurons have been shown to swell across a variety of different conditions, including increases in extracellular potassium concentration (^[K+]o). The mechanisms involved in the coupling of K+ influx to water movement into cells leading to cell swelling are not well understood and remain controversial. Here, we set out to determine the effects of ^[K+]o on rapid volume responses of hippocampal CA1 pyramidal neurons and stratum radiatum astrocytes using real-time confocal volume imaging. First, we found that elevating [K+]o within a physiological range (to 6.5 mM and 10.5 mM from a baseline of 2.5 mM), and even up to pathological levels (26 mM), produced dose-dependent increases in astrocyte volume, with absolutely no effect on neuronal volume. In the absence of compensating for addition of KCl by removal of an equal amount of NaCl, neurons actually shrank in ^[K+]o, while astrocytes continued to exhibit rapid volume increases. Astrocyte swelling in ^[K+]o was not dependent on neuronal firing, aquaporin 4, the inwardly rectifying potassium channel Kir 4.1, the sodium bicarbonate cotransporter NBCe1, , or the electroneutral cotransporter, sodium-potassium-chloride cotransporter type 1 (NKCC1), but was significantly attenuated in 1 mM barium chloride (BaCl2) and by the Na+/K+ ATPase inhibitor ouabain. Effects of 1 mM BaCl2 and ouabain applied together were not additive and, together with reports that BaCl2 can inhibit the NKA at high concentrations, suggests a prominent role for the astrocyte NKA in rapid astrocyte volume increases occurring in ^[K+]o. These findings carry important implications for understanding mechanisms of cellular edema, regulation of the brain extracellular space, and brain tissue excitability.

A Dose-Ranging Study of Epinephrine Hydrofluroalkane Metered-Dose Inhaler (Primatene® MIST) in Subjects with Intermittent or Mild-to-Moderate Persistent Asthma.

Background: Two sequential single-dose crossover dose-ranging studies were performed to evaluate the clinical efficacy and safety profile of epinephrine hydrofluroalkane (HFA) metered-dose inhaler (MDI) formulation at various doses in subjects with asthma. Methods: In these multicenter, multiarm, double-blinded, or evaluator-blinded studies, subjects were randomized to receive the epinephrine HFA (Primatene® MIST HFA) MDI medication at doses ranging from 90 to 440 µg/dose, as well as to a placebo (PLA) control and an active control of epinephrine CFC (chlorofluorocarbon) MDI (Primatene® MIST CFC) at 220 µg/inhalation. Results: Spirometry testing for FEV1 (Forced Expiratory Volume in one second) demonstrated statistically significant improvements over PLA for epinephrine HFA MDI at all doses above 125 µg, as the amount out of the actuator (i.e., mouthpiece). The efficacy results for epinephrine HFA MDI in the dose range of 125-250 µg were also comparable to epinephrine CFC MDI (220 µg/inh). Safety assessments demonstrated minimal safety concerns for all treatment groups. No notable safety differences were observed between the studied doses of epinephrine HFA MDI and the active control formulation of epinephrine CFC MDI. Conclusion: The findings indicate that epinephrine HFA MDI provided clinically significant bronchodilator efficacy with minimal safety concerns in a dose range of 125-250 µg. These findings confirmed the optimal treatment doses of 125-250 µg that were appropriate for use in longer term 12 and 26 week chronic dosing studies of epinephrine HFA MDI for patients with intermittent or mild to moderate persistent asthma. Clinical trials registration number: NCT01025648.

Hippocampal and Cortical Pyramidal Neurons Swell in Parallel with Astrocytes during Acute Hypoosmolar Stress.

Normal nervous system function is critically dependent on the balance of water and ions in the extracellular space (ECS). Pathological reduction in brain interstitial osmolarity results in osmotically-driven flux of water into cells, causing cellular edema which reduces the ECS and increases neuronal excitability and risk of seizures. Astrocytes are widely considered to be particularly susceptible to cellular edema due to selective expression of the water channel aquaporin-4 (AQP4). The apparent resistance of pyramidal neurons to osmotic swelling has been attributed to lack of functional water channels. In this study we report rapid volume changes in CA1 pyramidal cells in hypoosmolar ACSF (hACSF) that are equivalent to volume changes in astrocytes across a variety of conditions. Astrocyte and neuronal swelling was significant within 1 min of exposure to 17 or 40% hACSF, was rapidly reversible upon return to normosmolar ACSF, and repeatable upon re-exposure to hACSF. Neuronal swelling was not an artifact of patch clamp, occurred deep in tissue, was similar at physiological vs. room temperature, and occurred in both juvenile and adult hippocampal slices. Neuronal swelling was neither inhibited by TTX, nor by antagonists of NMDA or AMPA receptors, suggesting that it was not occurring as a result of excitotoxicity. Surprisingly, genetic deletion of AQP4 did not inhibit, but rather augmented, astrocyte swelling in severe hypoosmolar conditions. Taken together, our results indicate that neurons are not osmoresistant as previously reported, and that osmotic swelling is driven by an AQP4-independent mechanism.

Turning down the volume: Astrocyte volume change in the generation and termination of epileptic seizures.

Approximately 1% of the global population suffers from epilepsy, a class of disorders characterized by recurrent and unpredictable seizures. Of these cases roughly one-third are refractory to current antiepileptic drugs, which typically target neuronal excitability directly. The events leading to seizure generation and epileptogenesis remain largely unknown, hindering development of new treatments. Some recent experimental models of epilepsy have provided compelling evidence that glial cells, especially astrocytes, could be central to seizure development. One of the proposed mechanisms for astrocyte involvement in seizures is astrocyte swelling, which may promote pathological neuronal firing and synchrony through reduction of the extracellular space and elevated glutamate concentrations. In this review, we discuss the common conditions under which astrocytes swell, the resultant effects on neural excitability, and how seizure development may ultimately be influenced by these effects.

Astrocyte calcium microdomains are inhibited by bafilomycin A1 and cannot be replicated by low-level Schaffer collateral stimulation in situ.

Astrocyte Gq GPCR and IP3 receptor-dependent Ca(2+) elevations occur spontaneously in situ and in vivo. These events vary considerably in size, often remaining confined to small territories of astrocyte processes called "microdomains" and sometimes propagating over longer distances that can include the soma. It has remained unclear whether these events are driven by constitutive (basal) GPCR signaling activity, neuronal action potential-dependent or quantal vesicular release, or some combination of these mechanisms. Here, we applied manipulations to increase or inhibit neuronal vesicular neurotransmitter release together with low-level stimulation of Schaffer collaterals in acute mouse hippocampal slices in an effort to determine the mechanisms underlying spontaneous astrocyte Ca(2+) events. We found no significant change in spontaneous microdomain astrocyte Ca(2+) elevations when neuronal action potentials were significantly enhanced or blocked. The astrocyte Ca(2+) activity was also not affected by inhibitors of group I mGluRs. However, blockade of miniature neurotransmitter release using Bafilomycin A1 significantly reduced the frequency of microdomain astrocyte Ca(2+) elevations. We then tested whether astrocyte Ca(2+) microdomains can be evoked by low intensity SC stimulation. Importantly, microdomains could not be reproduced even using single, low intensity pulses to the SCs at a minimum distance from the astrocyte. Evoked astrocyte Ca(2+) responses most often included the cell soma, were reduced by group I mGluR antagonists, and were larger in size compared to spontaneous Ca(2+) microdomains. Overall, our findings suggest that spontaneous microdomain astrocyte Ca(2+) elevations are not driven by neuronal action potentials but require quantal release of neurotransmitter which cannot be replicated by stimulation of Schaffer collaterals.

Absence of Cx43 selectively from osteocytes enhances responsiveness to mechanical force in mice.

The osteocyte network is crucial for the response of bone to mechanical force. Within this network, connexin43 (Cx43) is thought to mediate the communication of osteocytes and osteoblasts among themselves and the exchange of small molecules with the extracellular milieu. Despite recent advances in understanding Cx43 role for the response of bone cells to mechanical stimulation, the contribution of Cx43 specifically in osteocytes to mechanotransduction in vivo is not well-known. We examined the anabolic response to ulnar axial loading of mice lacking Cx43 in osteocytes (Cx43(ΔOt)). Loading induced a greater increase in periosteal bone formation rate in Cx43(ΔOt) mice compared to control littermates, resulting from higher mineralizing surface and enhanced mineral apposition rate. Expression of ß-catenin protein, a molecule implicated in mechanotransduction, was higher in bones from Cx43(ΔOt) mice, compared to littermate controls. In addition, MLO-Y4 osteocytic cells knocked-down for Cx43 exhibited higher ß-catenin protein expression and enhanced response to mechanical stimulation. These findings suggest that osteocytes lacking Cx43 are "primed" to respond to mechanical stimulation and that absence of Cx43 in osteocytes unleashes bone formation, by a mechanism that might involve accumulation of ß-catenin.

Activation of protein kinase D1 in mast cells in response to innate, adaptive, and growth factor signals.

Little is known about the serine/threonine kinase protein kinase D (PKD)1 in mast cells. We sought to define ligands that activate PKD1 in mast cells and to begin to address the contributions of this enzyme to mast cell activation induced by diverse agonists. Mouse bone marrow-derived mast cells (BMMC) contained both PKD1 mRNA and immunoreactive PKD1 protein. Activation of BMMC through TLR2, Kit, or FcepsilonRI with Pam(3)CSK(4) (palmitoyl-3-cysteine-serine-lysine-4), stem cell factor (SCF), and cross-linked IgE, respectively, induced activation of PKD1, as determined by immunochemical detection of autophosphorylation. Activation of PKD1 was inhibited by the combined PKD1 and protein kinase C (PKC) inhibitor Gö 6976 but not by broad-spectrum PKC inhibitors, including bisindolylmaleimide (Bim) I. Pam(3)CSK(4) and SCF also induced phosphorylation of heat shock protein 27, a known substrate of PKD1, which was also inhibited by Gö 6976 but not Bim I in BMMC. This pattern also extended to activation-induced increases in mRNA encoding the chemokine CCL2 (MCP-1) and release of the protein. In contrast, both pharmacologic agents inhibited exocytosis of beta-hexosaminidase induced by SCF or cross-linked IgE. Our findings establish that stimuli representing innate, adaptive, and growth factor pathways activate PKD1 in mast cells. In contrast with certain other cell types, activation of PKD1 in BMMC is largely independent of PKC activation. Furthermore, our findings also indicate that PKD1 preferentially influences transcription-dependent production of CCL2, whereas PKC predominantly regulates the rapid exocytosis of preformed secretory granule mediators.

Occupational asthma: a case of Baker's asthma.

Asthma is one of the most prominent respiratory diseases worldwide. It is defined by airflow limitation and/or airway hyperresponsiveness and can be exacerbated by a number of environmental allergens. When allergic asthma exacerbations are attributed to stimuli in a particular work environment, then occupational asthma must be considered. Incidence estimates vary, but in 1999 the Surveillance of Work-Related and Occupational Respiratory Disease in the United Kingdom estimated 4293 incident cases of occupational respiratory disease, an increase of 1427 cases over the previous year. Occupational asthma represented 26% of these cases. Baker's asthma is one of the most frequently reported types of occupational asthma in several countries. Diagniostic steps include thorough history, careful exam, and demonstration of functional reversible airflow obstruction. Treatment modalities used for occupational asthma are similar to those used in the management of other forms of asthma, with particular attention to reducing the level of exposure to the inciting allergen.