Recreating an introductory physiology unit in the Core Concepts form: helping students to think like a physiologist.

This article showcases the redesign of an introductory undergraduate vertebrate physiology unit at Murdoch University (BMS107) to promote student mastery of six Core Concepts of Physiology (Michael J, Cliff W, McFarland J, Modell H, Wright A, SpringerLink. The Core Concepts of Physiology: a New Paradigm for Teaching Physiology, 2017). Concepts were selected for their suitability in an introductory physiology unit and their ability to scaffold advanced physiology learning. Innovative curricular and pedagogical approaches were employed to 1) create a Core Concepts structure, 2) sell the Core Concepts approach to students, 3) foreground Core Concepts in learning materials, 4) actively engage students with Core Concepts, 5) revise, and 6) assess Core Concepts understanding. Median student marks and overall satisfaction with the unit were unaffected by the introduction of a Core Concepts approach. Notably, though, there was a 14% increase in student agreement with the statement "I received feedback that helped me to learn." The challenge of the Core Concepts approach was articulated by students, but these novice learners also recognized Core Concepts as a mechanism to focus their understanding of physiology and promote critical thinking. For teaching staff, a Core Concepts approach was a reinvigorating opportunity to apply their expertise to the teaching of introductory physiology. We propose that a strong Core Concepts emphasis, while challenging, is highly rewarding for staff and provides students with a "disciplinary passport" that better prepares them to progress in diverse courses and professions.NEW & NOTEWORTHY This article presents a "how-to" guide for redesigning an introductory physiology unit to emphasize the Core Concepts of Physiology. Detailed descriptions are provided of innovative, scalable, adjustments to content delivery, assessment, learning objectives, and activities. Staff reflections and student experience suggest a strong Core Concepts emphasis, while challenging, can promote critical thinking and develop an understanding of underlying chemical, physical and biological principles.

Unpacking and validating the "cell membrane" core concept of physiology by an Australian team.

A task force of physiology educators from 25 Australian universities generated an Australia-wide consensus on seven core concepts for physiology curricula. One adopted core concept was "cell membrane," defined as "Cell membranes determine what substances enter or leave the cell and its organelles. They are essential for cell signaling, transport, and other cellular functions." This concept was unpacked by a team of 3 Australian physiology educators into 4 themes and 33 subthemes arranged in a hierarchical structure up to 5 levels deep. The four themes related to defining the cell membrane, cell membrane structure, transport across cell membranes, and cell membrane potentials. Subsequently, 22 physiology educators with a broad range of teaching experience reviewed and assessed the 37 themes and subthemes for importance for students to understand and the level of difficulty for students on a 5-point Likert scale. The majority (28) of items evaluated were rated as either Essential or Important. Theme 2: cell membrane structure was rated as less important than the other three themes. Theme 4: membrane potential was rated most difficult, while theme 1: defining cell membranes was rated as the easiest. The importance of cell membranes as a key aspect of biomedical education received strong support from Australian educators. The unpacking of the themes and subthemes within the cell membrane core concept provides guidance in the development of curricula and should facilitate better identification of the more challenging aspects within this core concept and help inform the time and resources required to support student learning.NEW & NOTEWORTHY The "cell membrane" core concept was unpacked by a team of Australian physiology educators into a conceptual framework to provide guidance for students and educators. Key themes in the cell membrane core concept were cell membrane definition and structure, transport across cell membranes, and membrane potentials. Australian educators reviewing the framework identified cell membrane as an essential yet relatively simple core concept, suggesting that this is well-placed in foundational physiology courses across a diverse range of degrees.

But science is international! Finding time and space to encourage intercultural learning in a content-driven physiology unit.

Internationalization of the curriculum is central to the strategic direction of many modern universities and has widespread benefits for student learning. However, these clear aspirations for internationalization of the curriculum have not been widely translated into more internationalized course content and teaching methods in the classroom, particularly in scientific disciplines. This study addressed one major challenge to promoting intercultural competence among undergraduate science students: finding time to scaffold such learning within the context of content-heavy, time-poor units. Small changes to enhance global and intercultural awareness were incorporated into existing assessments and teaching activities within a second-year biomedical physiology unit. Interventions were designed to start a conversation about global and intercultural perspectives on physiology, to embed the development of global awareness into the assessment and to promote cultural exchanges through peer interactions. In student surveys, 40% of domestic and 60% of international student respondents articulated specific learning about interactions in cross-cultural groups resulting from unit activities. Many students also identified specific examples of how cultural beliefs would impact on the place of biomedical physiology within the global community. In addition, staff observed more widespread benefits for student engagement and learning. It is concluded that a significant development of intercultural awareness and a more global perspective on scientific understanding can be supported among undergraduates with relatively modest, easy to implement adaptations to course content.

Postnatal development of intrinsic and synaptic properties transforms signaling in the layer 5 excitatory neural network of the visual cortex.

Information flow in neocortical circuits is regulated by two key parameters: intrinsic neuronal properties and the short-term activity-dependent plasticity of synaptic transmission. Using multineuronal whole-cell voltage recordings, we characterized the postnatal maturation of the electrophysiological properties and short-term plasticity of excitatory synaptic transmission between pairs of layer 5 (L5) pyramidal neurons (n = 158) in acute slices of rat visual cortex over the first postnatal month. We found that the intrinsic and synaptic properties of L5 pyramidal neurons develop in parallel. Before postnatal day 15 (P15), intrinsic electrophysiological properties were tuned to low-frequency operation, characterized by high apparent input resistance, a long membrane time constant, and prolonged somatic action potentials. Unitary excitatory synaptic potentials were of large amplitude (P11-P15; median, 514 µV), but showed pronounced use-dependent depression during prolonged regular and physiologically relevant presynaptic action potential firing patterns. In contrast, in mature animals we observed a developmental decline of the peak amplitude of unitary EPSPs (P25-P29; median, 175 µV) paralleled by a decrease in apparent input resistance, membrane time constant, and somatic action potential duration. Notably, synaptic signaling of complex action potential firing patterns was also transformed, with P25-P29 connections faithfully signaling action potential trains at frequencies up to 40 Hz (1st to 50th action potential ratio, 0.91 ± 0.12). Postnatal refinement of intrinsic properties and short-term plasticity therefore transforms the capacity of the L5 excitatory neural network of the visual cortex to generate and process patterns of action potential firing and contribute to network activity.

A Preclinical Study of Standard Versus Accelerated Transcranial Magnetic Stimulation for Depression in Adolescents.

Objective: Ongoing studies are focused on adapting transcranial magnetic stimulation (TMS) for the treatment of major depressive disorder in adolescent humans. Most protocols in adolescent humans to date have delivered daily 10 Hz prefrontal stimulation with mixed results. Novel TMS dosing strategies such as accelerated TMS have recently been considered. There are knowledge gaps related to the potential clinical and pragmatic advantages of accelerated TMS. This pilot study compared the behavioral effects of a standard daily and accelerated low-intensity TMS (LI-TMS) protocol in an adolescent murine model of depression. Methods: Male adolescent Sprague Dawley rats were placed in transparent plexiglass tubes for 2.5 hours daily for 13 days as part of a study to validate the chronic restraint stress (CRS) protocol. Rats subsequently received 10 minutes of active or sham 10 Hz LI-TMS daily for 2 weeks (standard) or three times daily for 1 week (accelerated). Behavior was assessed using the elevated plus maze and forced swim test (FST). Hippocampal neurogenesis was assessed by injection of the thymidine analogue 5-ethynyl-2'-deoxyuridine at the end of LI-TMS treatment (2 weeks standard, 1 week accelerated), followed by postmortem histological analysis. Results: There were no significant differences in behavioral outcomes among animals receiving once-daily sham or active LI-TMS treatment. However, animals treated with accelerated LI-TMS demonstrated significant improvements in behavioral outcomes compared with sham treatment. Specifically, animals receiving active accelerated treatment showed greater latency to the first immobility behavior (p < 0.05; active: 130 ± 46 seconds; sham: 54 ± 39 seconds) and increased climbing behaviors (p < 0.05; active: 16 ± 5; sham: 9 ± 5) during FST. There were no changes in hippocampal neurogenesis nor any evidence of cell death in histological sections. Conclusions: An accelerated LI-TMS protocol outperformed the standard (once-daily) protocol in adolescent male animals with depression-like behaviors induced by CRS and was not accompanied by any toxicity or tolerability concerns. These preliminary findings support the speculation that novel TMS dosing strategies should be studied in adolescent humans and will inform future clinical protocols.

Accelerated low-intensity rTMS does not rescue anxiety behaviour or abnormal connectivity in young adult rats following chronic restraint stress.

Currently approved repetitive transcranial magnetic stimulation (rTMS) protocols for the treatment of major depressive disorder (MDD) involve once-daily (weekday) stimulation sessions, with 10 Hz or intermittent theta burst stimulation (iTBS) frequencies, over 4-6 weeks. Recently, accelerated treatment protocols (multiple daily stimulation sessions for 1-2 weeks) have been increasingly studied to optimize rTMS treatments. Accelerated protocols might confer unique advantages for adolescents and young adults but there are many knowledge gaps related to dosing in this age group. Off-label, clinical practice frequently outpaces solid evidence as rigorous clinical trials require substantial time and resources. Murine models present an opportunity for high throughput dose finding studies to focus subsequent clinical trials in humans. This project investigated the brain and behavioural effects of an accelerated low-intensity rTMS (LI-rTMS) protocol in a young adult rodent model of chronic restraint stress (CRS). Depression and anxiety-related behaviours were induced in young adult male Sprague Dawley rats using the CRS model, followed by the 3-times-daily delivery of 10 Hz LI-rTMS, for two weeks. Behaviour was assessed using the Elevated Plus Maze and Forced Swim Test, and functional, chemical, and structural brain changes measured using magnetic resonance imaging techniques. CRS induced an agitated depression-like phenotype but therapeutic effects from the accelerated protocol were not detected. Our findings suggest that the age of rodents may impact response to CRS and LI-rTMS. Future studies should also examine higher intensities of rTMS and accelerated theta burst protocols.

Pressure pain threshold and temporal summation in adults with episodic and persistent low back pain trajectories: a secondary analysis at baseline and after lumbar manipulation or sham.

BACKGROUND: People with chronic low back pain (LBP) typically have increased pain sensitivity compared to healthy controls, however its unknown if pain sensitivity differs based on LBP trajectory at baseline or after manual therapy interventions. We aimed to compare baseline pressure pain threshold (PPT) and temporal summation (TS) between people without LBP, with episodic LBP, and with persistent LBP, and to compare changes over time in PPT and TS after a lumbar spinal manipulation or sham manipulation in those with LBP. METHODS: Participants were aged 18-59, with or without LBP. Those with LBP were categorised as having either episodic or persistent LBP. PPT and TS were tested at baseline. LBP participants then received a lumbar spinal manipulation or sham, after which PPT and TS were re-tested three times over 30 min. Generalised linear mixed models were used to analyse data. RESULTS: One hundred participants (49 female) were included and analysed. There were 20 non-LBP participants (mean age 31 yrs), 23 episodic LBP (mean age 35 yrs), and 57 persistent LBP (mean age 37 yrs). There were no significant differences in PPT or TS between groups at baseline. There was a non-significant pattern of lower PPT (higher sensitivity) from the non-LBP group to the persistent LBP group at baseline, and high variability. Changes in PPT and TS after the interventions did not differ between the two LBP groups. DISCUSSION: We found no differences between people with no LBP, episodic LBP, or persistent LBP in baseline PPT or TS. Changes in PPT and TS following a lumbar manual therapy intervention do not appear to differ between LBP trajectories. TRIAL REGISTRATION: The trial was prospectively registered with ANZCTR (ACTRN12617001094369).

Changes in pressure pain threshold and temporal summation in rapid responders and non-rapid responders after lumbar spinal manipulation and sham: A secondary analysis in adults with low back pain.

BACKGROUND: People with LBP who experience rapid improvement in symptoms after spinal manipulative therapy (SMT) are more likely to experience better longer-term outcomes compared to those who don't improve rapidly. It is unknown if short-term hypoalgesia after SMT could be a relevant finding in rapid responders. OBJECTIVES: We aimed to explore whether rapid responders had different short-term pressure pain threshold (PPT) and temporal summation (TS) outcomes after SMT and sham compared to non-rapid responders. METHODS: This was a planned secondary analysis of a randomised controlled trial that recruited 80 adults with LBP (42 females, mean age 37 yrs). PPT at the calf, lumbar spine, and shoulder and TS at the hands and feet were measured before and three times over 30 min after a lumbar SMT or sham manipulation. Participants were classified as rapid responders or non-rapid responders based on self-reported change in LBP over the following 24 h. RESULTS: Shoulder PPT transiently increased more in the rapid responders than non-rapid responders immediately post-intervention only (between-group difference in change from baseline = 0.29 kg/cm2, 95% CI 0.02-0.56, p = .0497). There were no differences in calf PPT, lumbar PPT, hand TS, or foot TS based on responder status. CONCLUSIONS: Hypoalgesia in shoulder PPT occurred transiently in the rapid responders compared to the non-rapid responders. This may or may not contribute to symptomatic improvement after SMT or sham in adults with LBP, and may be a spurious finding. Short-term changes in TS do not appear to be related to changes in LBP.

Validation of Chronic Restraint Stress Model in Young Adult Rats for the Study of Depression Using Longitudinal Multimodal MR Imaging.

Prior research suggests that the neurobiological underpinnings of depression include aberrant brain functional connectivity, neurometabolite levels, and hippocampal volume. Chronic restraint stress (CRS) depression model in rats has been shown to elicit behavioral, gene expression, protein, functional connectivity, and hippocampal volume changes similar to those in human depression. However, no study to date has examined the association between behavioral changes and brain changes within the same animals. This study specifically addressed the correlation between the outcomes of behavioral tests and multiple 9.4 T magnetic resonance imaging (MRI) modalities in the CRS model using data collected longitudinally in the same animals. CRS involved placing young adult male Sprague Dawley rats in individual transparent tubes for 2.5 h daily over 13 d. Elevated plus maze (EPM) and forced swim tests (FSTs) confirmed the presence of anxiety-like and depression-like behaviors, respectively, postrestraint. Resting-state functional MRI (rs-fMRI) data revealed hypoconnectivity within the salience and interoceptive networks and hyperconnectivity of several brain regions to the cingulate cortex. Proton magnetic resonance spectroscopy revealed decreased sensorimotor cortical glutamate (Glu), glutamine (Gln), and combined Glu-Gln (Glx) levels. Volumetric analysis of T2-weighted images revealed decreased hippocampal volume. Importantly, these changes parallel those found in human depression, suggesting that the CRS rodent model has utility for translational studies and novel intervention development for depression.

Frequency-specific effects of low-intensity rTMS can persist for up to 2 weeks post-stimulation: A longitudinal rs-fMRI/MRS study in rats.

BACKGROUND: Evidence suggests that repetitive transcranial magnetic stimulation (rTMS), a non-invasive neuromodulation technique, alters resting brain activity. Despite anecdotal evidence that rTMS effects wear off, there are no reports of longitudinal studies, even in humans, mapping the therapeutic duration of rTMS effects. OBJECTIVE: Here, we investigated the longitudinal effects of repeated low-intensity rTMS (LI-rTMS) on healthy rodent resting-state networks (RSNs) using resting-state functional MRI (rs-fMRI) and on sensorimotor cortical neurometabolite levels using proton magnetic resonance spectroscopy (MRS). METHODS: Sprague-Dawley rats received 10 min LI-rTMS daily for 15 days (10 Hz or 1 Hz stimulation, n = 9 per group). MRI data were acquired at baseline, after seven days and after 14 days of daily stimulation and at two more timepoints up to three weeks post-cessation of daily stimulation. RESULTS: 10 Hz stimulation increased RSN connectivity and GABA, glutamine, and glutamate levels. 1 Hz stimulation had opposite but subtler effects, resulting in decreased RSN connectivity and glutamine levels. The induced changes decreased to baseline levels within seven days following stimulation cessation in the 10 Hz group but were sustained for at least 14 days in the 1 Hz group. CONCLUSION: Overall, our study provides evidence of long-term frequency-specific effects of LI-rTMS. Additionally, the transient connectivity changes following 10 Hz stimulation suggest that current treatment protocols involving this frequency may require ongoing "top-up" stimulation sessions to maintain therapeutic effects.

Manipulation-induced hypoalgesia in musculoskeletal pain populations: a systematic critical review and meta-analysis.

Background: Manipulation-induced hypoalgesia (MIH) represents reduced pain sensitivity following joint manipulation, and has been documented in various populations. It is unknown, however, whether MIH following high-velocity low-amplitude spinal manipulative therapy is a specific and clinically relevant treatment effect. Methods: This systematic critical review with meta-analysis investigated changes in quantitative sensory testing measures following high-velocity low-amplitude spinal manipulative therapy in musculoskeletal pain populations, in randomised controlled trials. Our objectives were to compare changes in quantitative sensory testing outcomes after spinal manipulative therapy vs. sham, control and active interventions, to estimate the magnitude of change over time, and to determine whether changes are systemic or not. Results: Fifteen studies were included. Thirteen measured pressure pain threshold, and four of these were sham-controlled. Change in pressure pain threshold after spinal manipulative therapy compared to sham revealed no significant difference. Pressure pain threshold increased significantly over time after spinal manipulative therapy (0.32 kg/cm2, CI 0.22-0.42), which occurred systemically. There were too few studies comparing to other interventions or for other types of quantitative sensory testing to make robust conclusions about these. Conclusions: We found that systemic MIH (for pressure pain threshold) does occur in musculoskeletal pain populations, though there was low quality evidence of no significant difference compared to sham manipulation. Future research should focus on the clinical relevance of MIH, and different types of quantitative sensory tests. Trial registration: Prospectively registered with PROSPERO (registration CRD42016041963).

Feasibility of using the Neuropen for temporal summation testing.

Aim: This pilot study aimed to assess the feasibility of measuring pinprick temporal summation (TS) with the Neuropen, a cheap and accessible device. Methods: Ten asymptomatic participants underwent TS testing using the Neuropen and answered a tolerability questionnaire. Results were compared against reference values, averaging three and five TS tests. Tolerability was assessed qualitatively. Results: The TS results were similar to reference values, with less overall variability. Averaging three versus five tests produced similar results with high correlation. The mean unpleasantness score was low. Conclusion: The Neuropen appears to be suitable for eliciting TS, using an average of three measurements, and has acceptable tolerability. This pilot study was used to inform the use of the Neuropen for measuring TS in subsequent clinical studies.

No difference in pressure pain threshold and temporal summation after lumbar spinal manipulation compared to sham: A randomised controlled trial in adults with low back pain.

BACKGROUND: Changes in quantitative sensory tests have been observed after spinal manipulative therapy (SMT), particularly in pressure pain thresholds (PPT) and temporal summation (TS). However, a recent systematic review comparing SMT to sham found no significant difference in PPT in patients with musculoskeletal pain. The sham-controlled studies were generally low quality, and conclusions about other quantitative sensory tests could not be made. OBJECTIVES: We aimed to perform a sham-controlled study with the specific objective of investigating changes in PPT and TS short-term after lumbar SMT compared to sham manipulation in people with low back pain. METHODS: This was a double-blind randomised controlled trial comparing high-velocity low-amplitude lumbar SMT against sham manipulation in participants with low back pain. Primary outcome measures were PPT at the calf, lumbar spine and shoulder, and TS at the hands and feet. These were measured at baseline, then immediately, 15 min and 30 min post-intervention. RESULTS: Eighty participants (42 females) were included in the analyses (mean age 37 years), with 40 participants allocated to each intervention group. Significant between-group differences were only observed for calf PPT, which could be explained by a decrease in PPT (increased sensitivity) after SMT and an increase after sham. Feet TS decreased significantly over time after both SMT and sham, and any other changes over time were inconsistent. CONCLUSIONS: Our results suggest that lumbar SMT does not have a short-term hypoalgesic effect, as measured with PPT and TS, when compared to sham manipulation in people with low back pain.

Repetitive low intensity magnetic field stimulation in a neuronal cell line: a metabolomics study.

Low intensity repetitive magnetic stimulation of neural tissue modulates neuronal excitability and has promising therapeutic potential in the treatment of neurological disorders. However, the underpinning cellular and biochemical mechanisms remain poorly understood. This study investigates the behavioural effects of low intensity repetitive magnetic stimulation (LI-rMS) at a cellular and biochemical level. We delivered LI-rMS (10 mT) at 1 Hz and 10 Hz to B50 rat neuroblastoma cells in vitro for 10 minutes and measured levels of selected metabolites immediately after stimulation. LI-rMS at both frequencies depleted selected tricarboxylic acid (TCA) cycle metabolites without affecting the main energy supplies. Furthermore, LI-rMS effects were frequency-specific with 1 Hz stimulation having stronger effects than 10 Hz. The observed depletion of metabolites suggested that higher spontaneous activity may have led to an increase in GABA release. Although the absence of organised neural circuits and other cellular contributors (e.g., excitatory neurons and glia) in the B50 cell line limits the degree to which our results can be extrapolated to the human brain, the changes we describe provide novel insights into how LI-rMS modulates neural tissue.

Combined rTMS/fMRI Studies: An Overlooked Resource in Animal Models.

Repetitive transcranial magnetic stimulation (rTMS) is a non-invasive neuromodulation technique, which has brain network-level effects in healthy individuals and is also used to treat many neurological and psychiatric conditions in which brain connectivity is believed to be abnormal. Despite the fact that rTMS is being used in a clinical setting and animal studies are increasingly identifying potential cellular and molecular mechanisms, little is known about how these mechanisms relate to clinical changes. This knowledge gap is amplified by non-overlapping approaches used in preclinical and clinical rTMS studies: preclinical studies are mostly invasive, using cellular and molecular approaches, while clinical studies are non-invasive, including functional magnetic resonance imaging (fMRI), TMS electroencephalography (EEG), positron emission tomography (PET), and behavioral measures. A non-invasive method is therefore needed in rodents to link our understanding of cellular and molecular changes to functional connectivity changes that are clinically relevant. fMRI is the technique of choice for examining both short and long term functional connectivity changes in large-scale networks and is becoming increasingly popular in animal research because of its high translatability, but, to date, there have been no reports of animal rTMS studies using this technique. This review summarizes the main studies combining different rTMS protocols with fMRI in humans, in both healthy and patient populations, providing a foundation for the design of equivalent studies in animals. We discuss the challenges of combining these two methods in animals and highlight considerations important for acquiring clinically-relevant information from combined rTMS/fMRI studies in animals. We believe that combining rTMS and fMRI in animal models will generate new knowledge in the following ways: functional connectivity changes can be explored in greater detail through complementary invasive procedures, clarifying mechanism and improving the therapeutic application of rTMS, as well as improving interpretation of fMRI data. And, in a more general context, a robust comparative approach will refine the use of animal models of specific neuropsychiatric conditions.

Resting-state fMRI study of brain activation using low-intensity repetitive transcranial magnetic stimulation in rats.

Repetitive transcranial magnetic stimulation (rTMS) is a non-invasive neuromodulation technique used to treat many neuropsychiatric conditions. However, the mechanisms underlying its mode of action are still unclear. This is the first rodent study using resting-state functional MRI (rs-fMRI) to examine low-intensity (LI) rTMS effects, in an effort to provide a direct means of comparison between rodent and human studies. Using anaesthetised Sprague-Dawley rats, rs-fMRI data were acquired before and after control or LI-rTMS at 1 Hz, 10 Hz, continuous theta burst stimulation (cTBS) or biomimetic high-frequency stimulation (BHFS). Independent component analysis revealed LI-rTMS-induced changes in the resting-state networks (RSN): (i) in the somatosensory cortex, the synchrony of resting activity decreased ipsilaterally following 10 Hz and bilaterally following 1 Hz stimulation and BHFS, and increased ipsilaterally following cTBS; (ii) the motor cortex showed bilateral changes following 1 Hz and 10 Hz stimulation, a contralateral decrease in synchrony following BHFS, and an ipsilateral increase following cTBS; and (iii) hippocampal synchrony decreased ipsilaterally following 10 Hz, and bilaterally following 1 Hz stimulation and BHFS. The present findings demonstrate that LI-rTMS modulates functional links within the rat RSN with frequency-specific outcomes, and the observed changes are similar to those described in humans following rTMS.

Modulation of synaptic vesicle exocytosis in muscle-dependent long-term depression at the amphibian neuromuscular junction.

We have labeled recycling synaptic vesicles at the somatic Bufo marinus neuromuscular junction with the styryl dye FM2-10 and provide direct evidence for refractoriness of exocytosis associated with a muscle activity-dependent form of long-term depression (LTD) at this synapse. FM2-10 dye unloading experiments demonstrated that the rate of vesicle exocytosis from the release ready pool (RRP) of vesicles was more than halved in the LTD (induced by 20 min of low frequency stimulation). Recovery from LTD, observed as a partial recovery of nerve-evoked muscle twitch amplitude, was accompanied by partial recovery of the refractoriness of RRP exocytosis. Unexpectedly, paired pulse plasticity, another routinely used indicator of presynaptic forms of synaptic plasticity, was unchanged in the LTD. We conclude that the LTD induces refractoriness of the neuromuscular vesicle release machinery downstream of presynaptic calcium entry.

Postsynaptic production of nitric oxide implicated in long-term depression at the mature amphibian (Bufo marinus) neuromuscular junction.

We report here evidence for endogenous NO signalling in long-term (>1 h) synaptic depression at the neuromuscular junction induced by 20 min of 1 Hz nerve stimulation. Synaptic depression was characterized by a 46% reduction in the end-plate potential (EPP) amplitude and a 21% decrease in miniature EPP (MEPP) frequency, but no change to MEPP amplitude, indicating a reduction in evoked quantal release. Both the membrane-impermeant NO scavenger cPTIO and the NOS inhibitor L-NAME blocked depression, suggesting that it is induced by NO originating from a source outside the terminal. The depression was dependent on activation of muscle-type, but not neuronal-type, nAChRs and was still observed when Ca2+ release from the sarcoplasmic reticulum and muscle contraction were blocked with dantrolene. These data suggest that the depression depends on transmission, but not muscle contraction. The calcineurin inhibitors cyclosporin A and FK506, as well as ODQ, an inhibitor of NO-sensitive soluble guanylyl cyclase, Rp-8-pCPT-cGMPS, an inhibitor of cGMP-dependent protein kinase, and the calmodulin antagonist phenoxybenzamine also blocked depression. We propose that low frequency synaptic transmission leads to production of NO at the synapse and depression of transmitter release via a cGMP-dependent mechanism. The NO could be generated either directly from the muscle, or possibly from the Schwann cell in response to an unidentified muscle-derived messenger. We showed that the long-lasting depression of transmitter release was due to sustained activity of the NO signalling pathway, and suggest dephosphorylation of NOS by calcineurin as the basis for continued NO production.