Morphine-induced receptor endocytosis in a novel knockin mouse reduces tolerance and dependence

Abstract Opioid drugs, such as morphine, are among the most effective analgesics available. However, their utility for the treatment of chronic pain is limited by side effects including tolerance and dependence. Morphine acts primarily through the mu-opioid receptor (MOP-R) , which is also a target of endogenous opioids. However, unlike endogenous ligands, morphine fails to promote substantial receptor endocytosis both in vitro, and in vivo. Receptor endocytosis serves at least two important functions in signal transduction. First, desensitization and endocytosis act as an "off" switch by uncoupling receptors from G protein. Second, endocytosis functions as an "on" switch, resensitizing receptors by recycling them to the plasma membrane. Thus, both the off and on function of the MOP-R are altered in response to morphine compared to endogenous ligands. To examine whether the low degree of endocytosis induced by morphine contributes to tolerance and dependence, we generated a knockin mouse that expresses a mutant MOP-R that undergoes morphine-induced endocytosis. Morphine remains an excellent antinociceptive agent in these mice. Importantly, these mice display substantially reduced antinociceptive tolerance and physical dependence. These data suggest that opioid drugs with a pharmacological profile similar to morphine but the ability to promote endocytosis could provide analgesia while having a reduced liability for promoting tolerance and dependence

Trends in nurse-administered procedural sedation and analgesia across cardiac catheterisation laboratories in Australia and New Zealand : results of an electronic survey

Background Knowledge of current trends in nurse-administered procedural sedation and analgesia (PSA) in the cardiac catheterisation laboratory (CCL) may provide important insights into how to improve safety and effectiveness of this practice. Objective To characterise current practice as well as education and competency standards regarding nurse-administered PSA in Australian and New Zealand CCLs. Design A quantitative, cross-sectional, descriptive survey design was used. Methods Data were collected using a web-based questionnaire on practice, educational standards and protocols related to nurse-administered PSA. Descriptive statistics were used to analyse data. Results A sample of 62 nurses, each from a different CCL, completed a questionnaire that focused on PSA practice. Over half of the estimated total number of CCLs in Australia and New Zealand was represented. Nurse-administered PSA was used in 94% (n = 58) of respondents CCLs. All respondents indicated that benzodiazepines, opioids or a combination of both is used for PSA (n = 58). One respondent indicated that propofol was also used. 20% (n = 12) indicated that deep sedation is purposefully induced for defibrillation threshold testing and cardioversion without a second medical practitioner present. Sedation monitoring practices vary considerably between institutions. 31% (n = 18) indicated that comprehensive education about PSA is provided. 45% (n = 26) indicated that nurses who administer PSA should undergo competency assessment. Conclusion By characterising nurse-administered PSA in Australian and New Zealand CCLs, a baseline for future studies has been established. Areas of particular importance to improve include protocols for patient monitoring and comprehensive PSA education for CCL nurses in Australia and New Zealand.

Nurse-administered procedural sedation and analgesia in the cardiac catheterisation laboratory : a mixed methods study

The cardiac catheterisation laboratory (CCL) is a specialised medical radiology facility where both chronic-stable and life-threatening cardiovascular illness is evaluated and treated. Although there are many potential sources of discomfort and distress associated with procedures performed in the CCL, a general anaesthetic is not usually required. For this reason, an anaesthetist is not routinely assigned to the CCL. Instead, to manage pain, discomfort and anxiety during the procedure, nurses administer a combination of sedative and analgesic medications according to direction from the cardiologist performing the procedure. This practice is referred to as nurse-administered procedural sedation and analgesia (PSA). While anecdotal evidence suggested that nurse-administered PSA was commonly used in the CCL, it was clear from the limited information available that current nurse-led PSA administration and monitoring practices varied and that there was contention around some aspects of practice including the type of medications that were suitable to be used and the depth of sedation that could be safely induced without an anaesthetist present. The overall aim of the program of research presented in this thesis was to establish an evidence base for nurse-led sedation practices in the CCL context. A sequential mixed methods design was used over three phases. The objective of the first phase was to appraise the existing evidence for nurse-administered PSA in the CCL. Two studies were conducted. The first study was an integrative review of empirical research studies and clinical practice guidelines focused on nurse-administered PSA in the CCL as well as in other similar procedural settings. This was the first review to systematically appraise the available evidence supporting the use of nurse-administered PSA in the CCL. A major finding was that, overall, nurse-administered PSA in the CCL was generally deemed to be safe. However, it was concluded from the analysis of the studies and the guidelines that were included in the review, that the management of sedation in the CCL was impacted by a variety of contextual factors including local hospital policy, workforce constraints and cardiologists’ preferences for the type of sedation used. The second study in the first phase was conducted to identify a sedation scale that could be used to monitor level of sedation during nurse-administered PSA in the CCL. It involved a structured literature review and psychometric analysis of scale properties. However, only one scale was found that was developed specifically for the CCL, which had not undergone psychometric testing. Several weaknesses were identified in its item structure. Other sedation scales that were identified were developed for the ICU. Although these scales have demonstrated validity and reliability in the ICU, weaknesses in their item structure precluded their use in the CCL. As findings indicated that no existing sedation scale should be applied to practice in the CCL, recommendations for the development and psychometric testing of a new sedation scale were developed. The objective of the second phase of the program of research was to explore current practice. Three studies were conducted in this phase using both quantitative and qualitative research methods. The first was a qualitative explorative study of nurses’ perceptions of the issues and challenges associated with nurse-administered PSA in the CCL. Major themes emerged from analysis of the qualitative data regarding the lack of access to anaesthetists, the limitations of sedative medications, the barriers to effective patient monitoring and the impact that the increasing complexity of procedures has on patients' sedation requirements. The second study in Phase Two was a cross-sectional survey of nurse-administered PSA practice in Australian and New Zealand CCLs. This was the first study to quantify the frequency that nurse-administered PSA was used in the CCL setting and to characterise associated nursing practices. It was found that nearly all CCLs utilise nurse-administered PSA (94%). Of note, by characterising nurse-administered PSA in Australian and New Zealand CCLs, several strategies to improve practice, such as setting up protocols for patient monitoring and establishing comprehensive PSA education for CCL nurses, were identified. The third study in Phase Two was a matched case-control study of risk factors for impaired respiratory function during nurse-administered PSA in the CCL setting. Patients with acute illness were found to be nearly twice as likely to experience impaired respiratory function during nurse-administered PSA (OR=1.78; 95%CI=1.19-2.67; p=0.005). These significant findings can now be used to inform prospective studies investigating the effectiveness of interventions for impaired respiratory function during nurse-administered PSA in the CCL. The objective of the third and final phase of the program of research was to develop recommendations for practice. To achieve this objective, a synthesis of findings from the previous phases of the program of research informed a modified Delphi study, which was conducted to develop a set of clinical practice guidelines for nurse-administered PSA in the CCL. The clinical practice guidelines that were developed set current best practice standards for pre-procedural patient assessment and risk screening practices as well as the intra and post-procedural patient monitoring practices that nurses who administer PSA in the CCL should undertake in order to deliver safe, evidence-based and consistent care to the many patients who undergo procedures in this setting. In summary, the mixed methods approach that was used clearly enabled the research objectives to be comprehensively addressed in an informed sequential manner, and, as a consequence, this thesis has generated a substantial amount of new knowledge to inform and support nurse-led sedation practice in the CCL context. However, a limitation of the research to note is that the comprehensive appraisal of the evidence conducted, combined with the guideline development process, highlighted that there were numerous deficiencies in the evidence base. As such, rather than being based on high-level evidence, many of the recommendations for practice were produced by consensus. For this reason, further research is required in order to ascertain which specific practices result in the most optimal patient and health service outcomes. Therefore, along with necessary guideline implementation and evaluation projects, post-doctoral research is planned to follow up on the research gaps identified, which are planned to form part of a continuing program of research in this field.

The structure of Pavlovian fear conditioning in the amygdala

Do different brains forming a specific memory allocate the same groups of neurons to encode it? One way to test this question is to map neurons encoding the same memory and quantitatively compare their locations across individual brains. In a previous study, we used this strategy to uncover a common topography of neurons in the dorsolateral amygdala (LAd) that expressed a learning-induced and plasticity-related kinase (p42/44 mitogen-activated protein kinase; pMAPK), following auditory Pavlovian fear conditioning. In this series of experiments, we extend our initial findings to ask to what extent this functional topography depends upon intrinsic neuronal structure. We first showed that the majority (87 %) of pMAPK expression in the lateral amygdala was restricted to principal-type neurons. Next, we verified a neuroanatomical reference point for amygdala alignment using in vivo magnetic resonance imaging and in vitro morphometrics. We then determined that the topography of neurons encoding auditory fear conditioning was not exclusively governed by principal neuron cytoarchitecture. These data suggest that functional patterning of neurons undergoing plasticity in the amygdala following Pavlovian fear conditioning is specific to memory formation itself. Further, the spatial allocation of activated neurons in the LAd was specific to cued (auditory), but not contextual, fear conditioning. Spatial analyses conducted at another coronal plane revealed another spatial map unique to fear conditioning, providing additional evidence that the functional topography of fear memory storing cells in the LAd is non-random and stable. Overall, these data provide evidence for a spatial organizing principle governing the functional allocation of fear memory in the amygdala.

An organization of visual and auditory fear conditioning in the lateral amygdala

Pavlovian fear conditioning is an evolutionary conserved and extensively studied form of associative learning and memory. In mammals, the lateral amygdala (LA) is an essential locus for Pavlovian fear learning and memory. Despite significant progress unraveling the cellular mechanisms responsible for fear conditioning, very little is known about the anatomical organization of neurons encoding fear conditioning in the LA. One key question is how fear conditioning to different sensory stimuli is organized in LA neuronal ensembles. Here we show that Pavlovian fear conditioning, formed through either the auditory or visual sensory modality, activates a similar density of LA neurons expressing a learning-induced phosphorylated extracellular signal-regulated kinase (p-ERK1/2). While the size of the neuron population specific to either memory was similar, the anatomical distribution differed. Several discrete sites in the LA contained a small but significant number of p-ERK1/2-expressing neurons specific to either sensory modality. The sites were anatomically localized to different levels of the longitudinal plane and were independent of both memory strength and the relative size of the activated neuronal population, suggesting some portion of the memory trace for auditory and visually cued fear conditioning is allocated differently in the LA. Presenting the visual stimulus by itself did not activate the same p-ERK1/2 neuron density or pattern, confirming the novelty of light alone cannot account for the specific pattern of activated neurons after visual fear conditioning. Together, these findings reveal an anatomical distribution of visual and auditory fear conditioning at the level of neuronal ensembles in the LA.

A neuronal topography of visual and acoustic fear conditioning within the amygdala

The lateral amygdala (LA) receives information from auditory and visual sensory modalities, and uses this information to encode lasting memories that predict threat. One unresolved question about the amygdala is how multiple memories, derived from different sensory modalities, are organized at the level of neuronal ensembles. We previously showed that fear conditioning using an auditory conditioned stimulus (CS) was spatially allocated to a stable topography of neurons within the dorsolateral amygdala (LAd) (Bergstrom et al, 2011). Here, we asked how fear conditioning using a visual CS is topographically organized within the amygdala. To induce a lasting fear memory trace we paired either an auditory (2 khz, 55 dB, 20 s) or visual (1 Hz, 0.5 s on/0.5 s off, 35 lux, 20 s) CS with a mild foot shock unconditioned stimulus (0.6 mA, 0.5 s). To detect learning-induced plasticity in amygdala neurons, we used immunohistochemistry with an antibody for phosphorylated mitogen-activated protein kinase (pMAPK). Using a principal components analysis-based approach to extract and visualize spatial patterns, we uncovered two unique spatial patterns of activated neurons in the LA that were associated with auditory and visual fear conditioning. The first spatial pattern was specific to auditory cued fear conditioning and consisted of activated neurons topographically organized throughout the LAd and ventrolateral nuclei (LAvl) of the LA. The second spatial pattern overlapped for auditory and visual fear conditioning and was comprised of activated neurons located mainly within the LAvl. Overall, the density of pMAPK labeled cells throughout the LA was greatest in the auditory CS group, even though freezing in response to the visual and auditory CS was equivalent. There were no differences detected in the number of pMAPK activated neurons within the basal amygdala nuclei. Together, these results provide the first basic knowledge about the organizational structure of two different fear engrams within the amygdala and suggest they are dissociable at the level of neuronal ensembles within the LA

Neurobiological correlates of encoding strength of Pavlovian fear conditioned memory

Emotionally significant memories, especially those induced in conjunction with physical and mental trauma, are frequently retained for an individual’s lifetime. How these memories are organized and encoded within neural networks is a fundamental question. The lateral amygdala (LA) is a key nucleus for acquisition and maintenance of associative emotional memories. We used Pavlovian fear conditioning to study how ‘weaker’ and ‘stronger’ memories are encoded in neural networks of the LA. In Pavlovian fear conditioning a neutral stimulus, in this case a tone, is temporally paired with an aversive unconditioned stimulus (US), such as a foot shock. The previously neutral stimulus becomes a conditioned stimulus (CS) capable of eliciting defensive responses. We used time spent freezing when the CS is presented in a neutral context as a dependent variable measure of memory ‘strength’.

Biophysical correlates of intrinsic and stress-induced morphological variability in lateral amygdaloid neurons: A computational study

Morphological and physiological characteristics of neurons located in the dorsolateral and two ventral subdivisions of the lateral amygdala (LA) have been compared in order to differentiate their roles in the formation and storage of fear memories (Alphs et al, SfN abs 623.1, 2003). Briefly, in these populations, significant differences are observed in input resistance, membrane time constant, firing frequency, dendritic tortuosity, numbers of primary dendrites, dendritic segments and dendritic nodes...

Depth of anaesthesia monitoring during procedural sedation and analgesia: A systematic review protocol

Background Procedural sedation and analgesia (PSA) is used to attenuate the pain and distress that may otherwise be experienced during diagnostic and interventional medical or dental procedures. As the risk of adverse events increases with the depth of sedation induced, frequent monitoring of level of consciousness is recommended. Level of consciousness is usually monitored during PSA with clinical observation. Processed electroencephalogram-based depth of anaesthesia (DoA) monitoring devices provide an alternative method to monitor level of consciousness that can be used in addition to clinical observation. However, there is uncertainty as to whether their routine use in PSA would be justified. Rigorous evaluation of the clinical benefits of DoA monitors during PSA, including comprehensive syntheses of the available evidence, is therefore required. One potential clinical benefit of using DoA monitoring during PSA is that the technology could improve patient safety by reducing sedation-related adverse events, such as death or permanent neurological disability. We hypothesise that earlier identification of lapses into deeper than intended levels of sedation using DoA monitoring leads to more effective titration of sedative and analgesic medications, and results in a reduction in the risk of adverse events caused by the consequences of over-sedation, such as hypoxaemia. The primary objective of this review is to determine whether using DoA monitoring during PSA in the hospital setting improves patient safety by reducing the risk of hypoxaemia (defined as an arterial partial pressure of oxygen below 60 mmHg or percentage of haemoglobin that is saturated with oxygen [SpO2] less than 90 %). Other potential clinical benefits of using DoA monitoring devices during sedation will be assessed as secondary outcomes. Methods/design Electronic databases will be systematically searched for randomized controlled trials comparing the use of depth of anaesthesia monitoring devices with clinical observation of level of consciousness during PSA. Language restrictions will not be imposed. Screening, study selection and data extraction will be performed by two independent reviewers. Disagreements will be resolved by discussion. Meta-analyses will be performed if suitable. Discussion This review will synthesise the evidence on an important potential clinical benefit of DoA monitoring during PSA within hospital settings.

Altered formalin-induced pain and Fos induction in the periaqueductal grey of preadolescent rats following neonatal LPS exposure

Animal and human studies have demonstrated that early pain experiences can produce alterations in the nociceptive systems later in life including increased sensitivity to mechanical, thermal, and chemical stimuli. However, less is known about the impact of neonatal immune challenge on future responses to noxious stimuli and the reactivity of neural substrates involved in analgesia. Here we demonstrate that rats exposed to Lipopolysaccharide (LPS; 0.05 mg/kg IP, Salmonella enteritidis) during postnatal day (PND) 3 and 5 displayed enhanced formalin-induced flinching but not licking following formalin injection at PND 22. This LPS-induced hyperalgesia was accompanied by distinct recruitment of supra-spinal regions involved in analgesia as indicated by significantly attenuated Fos-protein induction in the rostral dorsal periaqueductal grey (DPAG) as well as rostral and caudal axes of the ventrolateral PAG (VLPAG). Formalin injections were associated with increased Fos-protein labelling in lateral habenula (LHb) as compared to medial habenula (MHb), however the intensity of this labelling did not differ as a result of neonatal immune challenge. These data highlight the importance of neonatal immune priming in programming inflammatory pain sensitivity later in development and highlight the PAG as a possible mediator of this process

The mechanical strength of phosphates under friction-induced cross-linking

Purpose: In the present study, we consider mechanical properties of phosphate glasses under high temperatureinduced and under friction-induced cross-linking, which enhance the modulus of elasticity. Design/methodology/approach: Two nanomechanical properties are evaluated, the first parameter is the modulus of elasticity (E) (or Young's modulus) and the second parameter is the hardness (H). Zinc meta-, pyro - and orthophosphates were recognized as amorphous-colloidal nanoparticles were synthesized under laboratory conditions and showed antiwear properties in engine oil. Findings: Young's modulus of the phosphate glasses formed under high temperature was in the 60-89 GPa range. For phosphate tribofilm formed under friction hardness and the Young's modulus were in the range of 2-10 GPa and 40-215 GPa, respectively. The degree of cross-linking during friction is provided by internal pressure of about 600 MPa and temperature close to 1000°C enhancing mechanical properties by factor of 3 (see Fig 1). Research limitations/implications: The addition of iron or aluminum ions to phosphate glasses under high temperature - and friction-induced amorphization of zinc metaphosphate and pyrophosphate tends to provide more cross-linking and mechanically stronger structures. Iron and aluminum (FeO4 or AlO4 units), incorporated into phosphate structure as network formers, contribute to the anion network bonding by converting the P=O bonds into bridging oxygen. Future work should consider on development of new of materials prepared by solgel processes, eg., zinc (II)-silicic acid. Originality/value: This paper analyses the friction pressure-induced and temperature–induced the two factors lead phosphate tribofilm glasses to chemically advanced glass structures, which may enhance the wear inhibition. Adding the coordinating ions alters the pressure at which cross-linking occurs and increases the antiwear properties of the surface material significantly.