Activity-Dependent Global Downscaling of Evoked Neurotransmitter Release across Glutamatergic Inputs in Drosophila.

Within mammalian brain circuits, activity-dependent synaptic adaptations, such as synaptic scaling, stabilize neuronal activity in the face of perturbations. Stability afforded through synaptic scaling involves uniform scaling of quantal amplitudes across all synaptic inputs formed on neurons, as well as on the postsynaptic side. It remains unclear whether activity-dependent uniform scaling also operates within peripheral circuits. We tested for such scaling in a Drosophila larval neuromuscular circuit, where the muscle receives synaptic inputs from different motoneurons. We used motoneuron-specific genetic manipulations to increase the activity of only one motoneuron and recordings of postsynaptic currents from inputs formed by the different motoneurons. We discovered an adaptation which caused uniform downscaling of evoked neurotransmitter release across all inputs through decreases in release probabilities. This "presynaptic downscaling" maintained the relative differences in neurotransmitter release across all inputs around a homeostatic set point, caused a compensatory decrease in synaptic drive to the muscle affording robust and stable muscle activity, and was induced within hours. Presynaptic downscaling was associated with an activity-dependent increase in Drosophila vesicular glutamate transporter expression. Activity-dependent uniform scaling can therefore manifest also on the presynaptic side to produce robust and stable circuit outputs. Within brain circuits, uniform downscaling on the postsynaptic side is implicated in sleep- and memory-related processes. Our results suggest that evaluation of such processes might be broadened to include uniform downscaling on the presynaptic side.SIGNIFICANCE STATEMENT To date, compensatory adaptations which stabilise target cell activity through activity-dependent global scaling have been observed only within central circuits, and on the postsynaptic side. Considering that maintenance of stable activity is imperative for the robust function of the nervous system as a whole, we tested whether activity-dependent global scaling could also manifest within peripheral circuits. We uncovered a compensatory adaptation which causes global scaling within a peripheral circuit and on the presynaptic side through uniform downscaling of evoked neurotransmitter release. Unlike in central circuits, uniform scaling maintains functionality over a wide, rather than a narrow, operational range, affording robust and stable activity. Activity-dependent global scaling therefore operates on both the presynaptic and postsynaptic sides to maintain target cell activity.

Defects in synaptic transmission at the neuromuscular junction precede motor deficits in a TDP-43Q331K transgenic mouse model of amyotrophic lateral sclerosis.

Transactive response DNA-binding protein-43 (TDP-43) is involved in gene regulation via the control of RNA transcription, splicing, and transport. TDP-43 is a major protein component of ubiquinated inclusions that are found in amyotrophic lateral sclerosis (ALS); however, the function of TDP-43 at the neuromuscular junction (NMJ) and its role in ALS pathogenesis is largely unknown. Here, we show that TDP-43Q331K mutation in mice resulted in impaired neurotransmission by age 3 mo, preceding deficits in motor function and motor neuron loss, which were observed from age 10 mo. These defects were in the effective fusion and release of synaptic vesicles within the motor nerve terminal and manifested in decreased quantal content and reduced probability of quantal release. We observed morphologic alterations that were associated with the TDP-43Q331K mutation, such as aberrant innervation patterns and the distribution of synaptic vesicle-related proteins, which is indicative of a failing NMJ undergoing synaptic remodeling. These findings support a growing acceptance that dysregulation of the NMJ function is a key early event in the pathology of ALS.-Chand, K. K., Lee, K. M., Lee, J. D., Qiu, H., Willis, E. F., Lavidis, N. A., Hilliard, M. A., Noakes, P. G. Defects in synaptic transmission at the neuromuscular junction precede motor deficits in a TDP-43Q331K transgenic mouse model of amyotrophic lateral sclerosis.

Climatic modulation of neurotransmitter release in amphibian neuromuscular junctions: role of dynorphin-A.

Amphibian neuromuscular junctions (NMJs) become relatively more silent during the dry winter season in Australia. During the dry, calcium sensitivity is reduced, whereas calcium dependence remains unchanged. Endogenous opioid peptides play an important role in the regulation of the physiological functions of active and dormant vertebrates. Previous findings suggest that dynorphin-A is more potent than other opiates in decreasing evoked neurotransmission in amphibian NMJs. Dynorphin-A has been shown not to alter the amplitude or the frequency of miniature quantal neurotransmitter release. In the present study, we report that dynorphin-A exerted a more pronounced inhibitory effect on evoked neurotransmitter release during the dry (hibernating period) when compared with the wet (active period) season. Dynorphin-A increased the frequency and decreased the amplitude of miniature neurotransmitter release only at relatively high concentration during the dry season. In the present study, we propose that dynorphin-A suppresses evoked neurotransmitter release and thus contraction of skeletal muscles, while allowing subthreshold activation of the NMJ by miniature neurotransmission, thus preventing any significant neuromuscular remodeling. The inhibitory effect of dynorphin-A on evoked transmitter release is reduced by increasing the extracellular calcium concentration.

Inhibition of Fatty Acid Amide Hydrolase by PF-3845 Alleviates the Nitrergic and Proinflammatory Response in Rat Hippocampus Following Acute Stress.

BACKGROUND: Long-term exposure to stress has been demonstrated to cause neuroinflammation through a sustained overproduction of free radicals, including nitric oxide, via an increased inducible nitric oxide synthase activity. We previously demonstrated that inducible nitric oxide synthase activity and mRNA are significantly upregulated in the rat hippocampus following just 4 hours of restraint stress. Similar to nitric oxide, endocannabinoids are synthesized on demand, with preclinical observations suggesting that cannabinoid receptor agonists and endocannabinoid enhancers inhibit nitrergic activity. Specifically, previous work has shown that enhancement of endocannabinoids via inhibition of fatty acid amide hydrolase with PF-3845 reduced inducible nitric oxide synthase-expressing microglia following traumatic brain injury. However, this describes cannabinoid modulation following physical injury, and therefore the present study aimed to examine the effects of PF-3845 in the modulation of nitrergic and inflammatory-related genes within the hippocampus after acute stress exposure. METHODS: Following vehicle or PF-3845 injections (5 mg/kg; i.p.), male Wistar rats were exposed to 0 (control), 60, 240, or 360 minutes of restraint stress after which plasma and dorsal hippocampus were isolated for further biochemical and gene expression analysis. RESULTS: The results demonstrate that pretreatment with PF-3845 rapidly ameliorates plasma corticosterone release at 60 minutes of stress. An increase in endocannabinoid signalling also induces an overall attenuation in inducible nitric oxide synthase, tumor necrosis factor-alpha convertase, interleukin-6, cyclooxygenase-2, peroxisome proliferator-activated receptor gamma mRNA, and the transactivation potential of nuclear factor kappa-light-chain-enhancer of activated B cells in the hippocampus. CONCLUSIONS: These results suggest that enhanced endocannabinoid levels in the dorsal hippocampus have an overall antinitrosative and antiinflammatory effect following acute stress exposure.

Loss of laminin-α4 results in pre- and postsynaptic modifications at the neuromuscular junction.

Synaptic basal lamina such as laminin-421 (α4ß2γ1) mediate differentiation of the neuromuscular junction (NMJ). Laminins interact with their pre- or postsynaptic receptors to provide stability and alignment of the pre- to postsynaptic specializations. Knockout of the laminin-α4 gene (lama4) does not alter gross NMJ morphogenesis. However, mice deficient in laminin-α4 (lama4-/- mice) display disruptions in the alignment of the active zones and postsynaptic folds at the NMJ, although the physiological consequences of this loss have not been examined. The present study investigated the differences in neurotransmission during the early development and maturation of the NMJ in lama4-/- and wild-type mice. Lama4-/- NMJs demonstrated a decrease in miniature end-plate potential (EPP) frequency and increased amplitude of miniature EPPs and evoked EPPs. Binomial parameters analysis of neurotransmitter release revealed a decrease in quantal release, the result of a decrease in the number of active release sites, but not in the probability of transmitter release. Lama4-/- NMJs displayed higher levels of synaptic depression under high-frequency stimulation and altered facilitation, suggesting compromised delivery of synaptic vesicles. This idea is supported by our molecular investigations of lama4-/- NMJs, where we see altered distribution of Bassoon, a molecular component of active zones, presumably resulting from perturbed neurotransmission.-Chand, K. K., Lee, K. M., Lavidis, N. A., Noakes, P. G. Loss of laminin-α4 results in pre- and postsynaptic modifications at the neuromuscular junction.

Seasonal factors influence quantal transmitter release and calcium dependence at amphibian neuromuscular junctions.

Amphibian neuromuscular junctions (NMJs) are composed of hundreds of neurotransmitter release sites that exhibit nonuniform transmitter release probabilities and demonstrated seasonal modulation. We examined whether recruitment of release sites is variable when the extracellular calcium concentration ([Ca2+]o) is increased in the wet and dry seasons. The amount of transmitter released from the entire nerve terminal increases by approximately the fourth power as [Ca2+]o is increased. Toad (Bufo marinus) NMJs were visualized using 3,3'-diethyloxardicarbocyanine iodide [DiOC2(5)] fluorescence, and focal loose patch extracellular recordings were used to record the end-plate currents (EPCs) from small groups of release sites. Quantal content (m̄e ), average probability of quantal release (pe ), and the number of active release sites (ne ) were determined for different [Ca2+]o Our results indicated that the recruitment of quantal release sites with increasing [Ca2+]o differs spatially (between different groups of release sites) and also temporally (in different seasons). These differences were reflected by the nonuniform alterations in pe and ne Most release site groups demonstrated an increase in both pe and ne when [Ca2+]o increased. In ~30% of release site groups examined, pe decreased while ne increased only during the active period (wet season). Although the dry season induced parallel right shift in the quantal release versus extracellular calcium concentration when compared with the wet season, the dependence of quantal content on [Ca2+]o was not changed. These results demonstrate the flexibility, reserve, and adaptive capacity of neuromuscular junctions in maintaining appropriate levels of neurotransmission.

Noninvasive assessment of altered activity following restraint in mice using an automated physiological monitoring system.

In the laboratory setting, typical endocrine and targeted behavioral tests are limited in their ability to provide a direct assessment of stress in animals housed in undisturbed conditions. We hypothesized that an automated phenotyping system would allow the detection of subtle stress-related behavioral changes well beyond the time-frames examined using conventional methods. In this study, we have utilized the TSE PhenoMaster system to continuously record basal behaviors and physiological parameters including activity, body weight, food intake and oxygen consumption in undisturbed and stressed C57Bl/6J male mice (n = 12/group), with a pharmacological intervention using the conventional anxiolytic, diazepam (5 mg kg-1 i.p.; n = 8/group). We observed significant 20-30% reductions in locomotor activity in the dark phase, with subtle reductions in light phase activity for up to 96 h following a single 2 h episode of restraint stress. A single administration of diazepam reduced plasma corticosterone concentrations by 30-35% during stress exposure when compared to mice treated with vehicle. This treatment did not result in significantly different locomotor activity compared to vehicle within the first 48 h following restraint stress. However, diazepam treatment facilitated restoration of locomotor activity at 72 and 96 h after restraint stress exposure in comparison to vehicle-treated mice. Hence, the use of an automated phenotyping system allows a real time assessment of basal behaviors and empirical metabolism following exposure to restraint stress and demonstrates major and subtle changes in activity persist for several days after stress exposure.

Assessing students' ability to critically evaluate evidence in an inquiry-based undergraduate laboratory course.

The ability to critically evaluate and use evidence from one's own work or from primary literature is invaluable to any researcher. These skills include the ability to identify strengths and weakness of primary literature, to gauge the impact of research findings on a field, to identify gaps in a field that require more research, and to contextualize findings within a field. This study developed a model to examine undergraduate science students' abilities to critically evaluate and use evidence through an analysis of laboratory reports from control and experimental groups in nonresearch-aligned and research-aligned inquiry-based laboratory classes, respectively, and contrasted these with published scientific research articles. The reports analyzed (n = 42) showed that students used evidence in a variety of ways, most often referring to literature indirectly, and least commonly highlighting limitations of literature. There were significant positive correlations between grade awarded and the use of references, evidence, and length, but there were no significant differences between control and experimental groups, so data were pooled. The use of evidence in scientific research articles (n = 7) was similar to student reports except that expert authors were more likely to refer to their own results and cite more references. Analysis showed that students, by the completion of the second year of their undergraduate degree, had expertise approaching that of published authors. These findings demonstrate that it is possible to provide valuable broad-scale undergraduate research experiences to all students in a cohort, giving them exposure to the methods and communication processes of research as well as an opportunity to hone their critical evaluation skills.

Loss of ß2-laminin alters calcium sensitivity and voltage-gated calcium channel maturation of neurotransmission at the neuromuscular junction.

KEY POINTS: Neuromuscular junctions from ß2-laminin-deficient mice exhibit lower levels of calcium sensitivity. Loss of ß2-laminin leads to a failure in switching from N- to P/Q-type voltage-gated calcium channel (VGCC)-mediated transmitter release that normally occurs with neuromuscular junction maturation. The motor nerve terminals from ß2-laminin-deficient mice fail to up-regulate the expression of P/Q-type VGCCs clusters and down-regulate N-type VGCCs clusters, as they mature. There is decreased co-localisation of presynaptic specialisations in ß2-laminin-deficient neuromuscular junctions as a consequence of lesser P/Q-type VGCC expression. These findings support the idea that ß2-laminin is critical in the organisation and maintenance of active zones at the neuromuscular junction via its interaction with P/Q-type VGCCs, which aid in stabilisation of the synapse. ß2-laminin is a key mediator in the differentiation and formation of the skeletal neuromuscular junction. Loss of ß2-laminin results in significant structural and functional aberrations such as decreased number of active zones and reduced spontaneous release of transmitter. In vitro ß2-laminin has been shown to bind directly to the pore forming subunit of P/Q-type voltage-gated calcium channels (VGCCs). Neurotransmission is initially mediated by N-type VGCCs, but by postnatal day 18 switches to P/Q-type VGCC dominance. The present study investigated the changes in neurotransmission during the switch from N- to P/Q-type VGCC-mediated transmitter release at ß2-laminin-deficient junctions. Analysis of the relationship between quantal content and extracellular calcium concentrations demonstrated a decrease in the calcium sensitivity, but no change in calcium dependence at ß2-laminin-deficient junctions. Electrophysiological studies on VGCC sub-types involved in transmitter release indicate N-type VGCCs remain the primary mediator of transmitter release at matured ß2-laminin-deficient junctions. Immunohistochemical analyses displayed irregularly shaped and immature ß2-laminin-deficient neuromuscular junctions when compared to matured wild-type junctions. ß2-laminin-deficient junctions also maintained the presence of N-type VGCC clustering within the presynaptic membrane, which supported the functional findings of the present study. We conclude that ß2-laminin is a key regulator in development of the NMJ, with its loss resulting in reduced transmitter release due to decreased calcium sensitivity stemming from a failure to switch from N- to P/Q-type VGCC-mediated synaptic transmission.

Syntaxin1A-mediated Resistance and Hypersensitivity to Isoflurane in Drosophila melanogaster.

BACKGROUND: Recent evidence suggests that general anesthetics activate endogenous sleep pathways, yet this mechanism cannot explain the entirety of general anesthesia. General anesthetics could disrupt synaptic release processes, as previous work in Caenorhabditis elegans and in vitro cell preparations suggested a role for the soluble NSF attachment protein receptor protein, syntaxin1A, in mediating resistance to several general anesthetics. The authors questioned whether the syntaxin1A-mediated effects found in these reductionist systems reflected a common anesthetic mechanism distinct from sleep-related processes. METHODS: Using the fruit fly model, Drosophila melanogaster, the authors investigated the relevance of syntaxin1A manipulations to general anesthesia. The authors used different behavioral and electrophysiological endpoints to test the effect of syntaxin1A mutations on sensitivity to isoflurane. RESULTS: The authors found two syntaxin1A mutations that confer opposite general anesthesia phenotypes: syxH3-C, a 14-amino acid deletion mutant, is resistant to isoflurane (n = 40 flies), and syxKARRAA, a strain with two amino acid substitutions, is hypersensitive to the drug (n = 40 flies). Crucially, these opposing effects are maintained across different behavioral endpoints and life stages. The authors determined the isoflurane sensitivity of syxH3-C at the larval neuromuscular junction to assess effects on synaptic release. The authors find that although isoflurane slightly attenuates synaptic release in wild-type animals (n = 8), syxH3-C preserves synaptic release in the presence of isoflurane (n = 8). CONCLUSION: The study results are evidence that volatile general anesthetics target synaptic release mechanisms; in addition to first activating sleep pathways, a major consequence of these drugs may be to decrease the efficacy of neurotransmission.

A combination of plant-derived odors reduces corticosterone and oxidative indicators of stress.

In this study, we measured typical stress markers in addition to oxidative status and reduced glutathione in erythrocytes, and plasma lipid peroxidation of restraint-stressed animals exposed to a combination of plant-derived odors (0.03% Z-3-hexen-1-ol, 0.03% E-2-hexenal, and 0.015% α-pinene in triethyl citrate). Male Wistar rats aged 6-7 weeks postnatal were exposed to vehicle (triethyl citrate, n = 12), plant-derived odors (n = 12), or 1% propionic acid odor (n = 12) under control or stress conditions, and blood samples were collected. Restraint stress increased plasma glucose and plasma corticosterone concentrations by approximately 10% (P < 0.01) and 125% (P < 0.001), respectively, in vehicle-exposed animals. Similar increases were observed in animals exposed to a 1% propionic acid odor, indicating the novelty of odor exposure does not alter stress responsiveness. There was also an increase of approximately 15% in both erythrocytic oxidative status (P < 0.001) and plasma lipid peroxidation (P < 0.05), and a decrease of approximately the same magnitude in reduced glutathione (P < 0.05) in restrained animals with vehicle exposure. There were no differences observed between control and stress treatment with plant-derived odor exposure in any of the measured parameters. It was concluded that exposure to plant-derived odors reduce corticosterone, glucose, and redox responses elicited by psychological stress.

Reactive nitrogen species contribute to the rapid onset of redox changes induced by acute immobilization stress in rats.

Acute stress leads to the rapid secretion of glucocorticoids, which accelerates cellular metabolism, resulting in increased reactive oxygen and nitrogen species generation. Although the nitrergic system has been implicated in numerous stress-related diseases, the time course and extent of nitrosative changes during acute stress have not been characterized. Outbred male Wistar rats were randomly allocated into control (n = 9) or 120 min acute immobilization stress (n = 9) groups. Serial blood samples were collected at 0 (baseline), 60, 90, and 120 min. Plasma corticosterone concentrations increased by approximately 350% at 60, 90, and 120 (p < 0.001) min of stress. The production of nitric oxide, measured as the benzotriazole form of 4-amino-5-methylamino-2',7'-difluorofluorescein, increased during stress exposure by approximately 5%, 10%, and 15% at 60 (p < 0.05), 90 (p < 0.01) and 120 (p < 0.001) min, respectively, compared to controls. Nitric oxide metabolism, measured as the stable metabolites nitrite and nitrate, showed a 40-60% increase at 60, 90, and 120 (p < 0.001) min of stress. The oxidative status of 2',7'-dichlorofluorescein in plasma was significantly elevated at 60 (p < 0.01), 90, and 120 (p < 0.001) min. A delayed decrease of approximately 25% in the glutathione redox ratio at 120 min (p < 0.001) also indicates stress-induced cellular oxidative stress. The peroxidation of plasma lipids increased by approximately 10% at 90 (p < 0.05) and 15% at 120 (p < 0.001) min, indicative of oxidative damage. It was concluded that a single episode of stress causes early and marked changes of both oxidative and nitrosative status sufficient to induce oxidative damage in peripheral tissues.

Complement C5a Receptor Signaling Alters Stress Responsiveness and Modulates Microglia Following Chronic Stress Exposure.

Background: Accumulating evidence underscores the pivotal role of heightened inflammation in the pathophysiology of stress-related diseases, but the underlying mechanisms remain elusive. The complement system, a key effector of the innate immune system, produces the C5-cleaved activation product C5a upon activation, initiating inflammatory responses through the canonical C5a receptor 1 (C5aR1). While C5aR1 is expressed in stress-responsive brain regions, its role in stress responsiveness remains unknown. Methods: To investigate C5a-C5aR1 signaling in stress responses, mice underwent acute and chronic stress paradigms. Circulating C5a levels and messenger RNA expression of C5aR1 in the hippocampus and adrenal gland were measured. C5aR1-deficient mice were used to elucidate the effects of disrupted C5a-C5aR1 signaling across behavioral, hormonal, metabolic, and inflammation parameters. Results: Chronic restraint stress elevated circulating C5a levels while reducing C5aR1 messenger RNA expression in the hippocampus and adrenal gland. Notably, the absence of C5aR1 signaling enhanced adrenal sensitivity to adrenocorticotropic hormone, concurrently reducing pituitary adrenocorticotropic hormone production and enhancing the response to acute stress. C5aR1-deficient mice exhibited attenuated reductions in locomotor activity and body weight under chronic stress. Additionally, these mice displayed increased glucocorticoid receptor sensitivity and disrupted glucose and insulin homeostasis. Chronic stress induced an increase in C5aR1-expressing microglia in the hippocampus, a response mitigated in C5aR1-deficient mice. Conclusions: C5a-C5aR1 signaling emerges as a key metabolic regulator during stress, suggesting that complement activation and dysfunctional C5aR1 signaling may contribute to neuroinflammatory phenotypes in stress-related disorders. The results advocate for further exploration of complement C5aR1 as a potential therapeutic target for stress-related conditions.

How the immune system, particularly the complement system, influences responses to stress has not been fully clear. In this study, we focus on C5a-C5aR1 signaling, a part of the immune system, and found that it significantly affects stress-related reactions in mice. In chronic stress, we observed increased inflammation, altered hormonal responses, and disrupted metabolic regulation. Mice lacking C5aR1 showed reduced stress-induced behavioral changes, indicating that this receptor may play a vital role in modulating the stress response. Understanding these immune mechanisms sheds light on stress-related disorders and may open avenues for therapeutic interventions.

Dynamin inhibition blocks botulinum neurotoxin type A endocytosis in neurons and delays botulism.

The botulinum neurotoxins (BoNTs) are di-chain bacterial proteins responsible for the paralytic disease botulism. Following binding to the plasma membrane of cholinergic motor nerve terminals, BoNTs are internalized into an endocytic compartment. Although several endocytic pathways have been characterized in neurons, the molecular mechanism underpinning the uptake of BoNTs at the presynaptic nerve terminal is still unclear. Here, a recombinant BoNT/A heavy chain binding domain (Hc) was used to unravel the internalization pathway by fluorescence and electron microscopy. BoNT/A-Hc initially enters cultured hippocampal neurons in an activity-dependent manner into synaptic vesicles and clathrin-coated vesicles before also entering endosomal structures and multivesicular bodies. We found that inhibiting dynamin with the novel potent Dynasore analog, Dyngo-4a(TM), was sufficient to abolish BoNT/A-Hc internalization and BoNT/A-induced SNAP25 cleavage in hippocampal neurons. Dyngo-4a also interfered with BoNT/A-Hc internalization into motor nerve terminals. Furthermore, Dyngo-4a afforded protection against BoNT/A-induced paralysis at the rat hemidiaphragm. A significant delay of >30% in the onset of botulism was observed in mice injected with Dyngo-4a. Dynamin inhibition therefore provides a therapeutic avenue for the treatment of botulism and other diseases caused by pathogens sharing dynamin-dependent uptake mechanisms.

Sustained synaptic-vesicle recycling by bulk endocytosis contributes to the maintenance of high-rate neurotransmitter release stimulated by glycerotoxin.

Glycerotoxin (GLTx), a large neurotoxin isolated from the venom of the sea worm Glycera convoluta, promotes a long-lasting increase in spontaneous neurotransmitter release at the peripheral and central synapses by selective activation of Ca(v)2.2 channels. We found that GLTx stimulates the very high frequency, long-lasting (more than 10 hours) spontaneous release of acetylcholine by promoting nerve terminal Ca(2+) oscillations sensitive to the inhibitor omega-conotoxin GVIA at the amphibian neuromuscular junction. Although an estimate of the number of synaptic vesicles undergoing exocytosis largely exceeds the number of vesicles present in the motor nerve terminal, ultrastructural examination of GLTx-treated synapses revealed no significant change in the number of synaptic vesicles. However, we did detect the appearance of large pre-synaptic cisternae suggestive of bulk endocytosis. Using a combination of styryl dyes, photoconversion and horseradish peroxidase (HRP)-labeling electron microscopy, we demonstrate that GLTx upregulates presynaptic-vesicle recycling, which is likely to emanate from the limiting membrane of these large cisternae. Similar synaptic-vesicle recycling through bulk endocytosis also occurs from nerve terminals stimulated by high potassium. Our results suggest that this process might therefore contribute significantly to synaptic recycling under sustained levels of synaptic stimulation.

Repeated acute stress modulates hepatic inflammation and markers of macrophage polarisation in the rat.

Bidirectional communication between the neuroendocrine stress and immune systems permits classically anti-inflammatory glucocorticoids to exert pro-inflammatory effects in specific cells and tissues. Liver macrophages/Kupffer cells play a crucial role in initiating inflammatory cascades mediated by the release of pro-inflammatory cytokines following tissue injury. However, the effects of repeated acute psychological stress on hepatic inflammatory phenotype and macrophage activation state remains poorly understood. We have utilised a model of repeated acute stress in rodents to observe the changes in hepatic inflammatory phenotype, including anti-inflammatory vitamin D status, in addition to examining markers of classically and alternatively-activated macrophages. Male Wistar rats were subjected to control conditions or 6 h of restraint stress applied for 1 or 3 days (n = 8 per group) after which plasma concentrations of stress hormone, enzymes associated with liver damage, and vitamin D status were examined, in addition to hepatic expression of pro- and anti-inflammatory markers. Stress increased glucocorticoids and active vitamin D levels in addition to expression of glucocorticoid alpha/beta receptor, whilst changes in circulating hepatic enzymes indicated sustained liver damage. A pro-inflammatory response was observed in liver tissues following stress, and inducible nitric oxide synthase being observed within hepatic macrophage/Kupffer cells. Together, this suggests that stress preferentially induces a pro-inflammatory response in the liver.

Size-dependent dendritic maladaptations of hypoglossal motor neurons in SOD1G93A mice.

The total motor neuron (MN) somato-dendritic surface area is correlated with motor unit type. MNs with smaller surface areas innervate slow (S) and fast fatigue-resistant (FR) motor units, while MNs with larger surface areas innervate fast fatigue-intermediate (FInt) and fast fatigable (FF) motor units. Differences in MN surface area (equivalent to membrane capacitance) underpin the intrinsic excitability of MNs and are consistent with the orderly recruitment of motor units (S > FR > FInt > FF) via the Size Principle. In amyotrophic lateral sclerosis (ALS), large MNs controlling FInt and FF motor units exhibit earlier denervation and death, compared to smaller and more resilient MNs of type S and FR motor units that are spared until late in ALS. Abnormal dendritic morphologies in MNs precede neuronal death in human ALS and in rodent models. We employed Golgi-Cox methods to investigate somal size-dependent changes in the dendritic morphology of hypoglossal MNs in wildtype and SOD1G93A mice (a model of ALS), at postnatal (P) day ~30 (pre-symptomatic), ~P60 (onset), and ~P120 (mid-disease) stages. In wildtype hypoglossal MNs, increased MN somal size correlated with increased dendritic length and spines in a linear fashion. By contrast, in SOD1G93A mice, significant deviations from this linear correlation were restricted to the larger vulnerable MNs at pre-symptomatic (maladaptive) and mid-disease (degenerative) stages. These findings are consistent with excitability changes observed in ALS patients and in rodent models. Our results suggest that intrinsic or synaptic increases in MN excitability are likely to contribute to ALS pathogenesis, not compensate for it.

Hepatic Homeostasis of Metal Ions Following Acute Repeated Stress Exposure in Rats.

Essential metals such as copper, iron, and zinc are cofactors in various biological processes including oxygen utilisation, cell growth, and biomolecular synthesis. The homeostasis of these essential metals is carefully controlled through a system of protein transporters involved in the uptake, storage, and secretion. Some metal ions can be transformed by processes including reduction/oxidation (redox) reactions, and correspondingly, the breakdown of metal ion homeostasis can lead to formation of reactive oxygen and nitrogen species. We have previously demonstrated rapid biochemical responses to stress involving alterations in the redox state to generate free radicals and the resultant oxidative stress. However, the effects of stress on redox-active metals including iron and copper and redox-inert zinc have not been well characterised. Therefore, this study aims to examine the changes in these essential metals following exposure to short-term repeated stress, and to further elucidate the alterations in metal homeostasis through expression analysis of different metal transporters. Outbred male Wistar rats were exposed to unrestrained (control), 1 day, or 3 days of 6 h restraint stress (n = 8 per group). After the respective stress treatment, blood and liver samples were collected for the analysis of biometal concentrations and relative gene expression of metal transporter and binding proteins. Exposure to repeated restraint stress was highly effective in causing hepatic redox imbalance. Stress was also shown to induce hepatic metal redistribution, while modulating the mRNA levels of key metal transporters. Overall, this study is the first to characterise the gene expression profile of metal homeostasis following stress and provide insight into the changes occurring prior to the onset of chronic stress conditions.

What are Neurotransmitter Release Sites and Do They Interact?

It has long been known that each neuron in both the central and peripheral nervous system has a large number of active zones. Nonetheless, how active zones are regulated to maintain a homeostatic release state and response to the constantly changing environment remains poorly understood. Due to its relatively simple structure and easy accessibility, the neuromuscular synapse (NM-synapse) continues to be used as a model synapse to examine the basic nature of synaptic neurotransmission. In the NM-synapse, quantal neurotransmitter release can occur spontaneously or triggered by invading nerve impulses. Past research has indicated that some active zones tend to be involved more with spontaneous quantal release than evoked quantal release. Furthermore, evoked quantal release has been shown to be highly non-uniform between active zones along nerve terminal branches. How these large numbers of active zones along the same nerve terminal are functionally correlated remains unclear. This review starts with the basic features of quantal neurotransmitter release, then progresses to the current knowledge on how the active zones interact with each other along the same nerve terminal.

Proportional Downscaling of Glutamatergic Release Sites by the General Anesthetic Propofol at Drosophila Motor Nerve Terminals.

Propofol is the most common general anesthetic used for surgery in humans, yet its complete mechanism of action remains elusive. In addition to potentiating inhibitory synapses in the brain, propofol also impairs excitatory neurotransmission. We use electrophysiological recordings from individual glutamatergic boutons in male and female larval Drosophila melanogaster motor nerve terminals to characterize this effect. We recorded from two bouton types, which have distinct presynaptic physiology and different average numbers of release sites or active zones. We show that a clinically relevant dose of propofol (3 µm) impairs neurotransmitter release similarly at both bouton types by decreasing the number of active release sites by half, without affecting release probability. In contrast, an analog of propofol has no effect on glutamate release. Coexpressing a truncated syntaxin1A protein in presynaptic boutons completely blocked this effect of propofol. Overexpressing wild-type syntaxin1A in boutons also conferred a level of resistance by increasing the number of active release sites to a physiological ceiling set by the number of active zones or T-bars, and in this way counteracting the effect of propofol. These results point to the presynaptic release machinery as a target for the general anesthetic. Proportionally equivalent effects of propofol on the number of active release sites across the different bouton types suggests that glutamatergic circuits that involve smaller boutons with fewer release sites may be more vulnerable to the presynaptic effects of the drug.