1H, 13C, and 15N backbone chemical shift assignments of coronavirus-2 non-structural protein Nsp10.

The international Covid19-NMR consortium aims at the comprehensive spectroscopic characterization of SARS-CoV-2 RNA elements and proteins and will provide NMR chemical shift assignments of the molecular components of this virus. The SARS-CoV-2 genome encodes approximately 30 different proteins. Four of these proteins are involved in forming the viral envelope or in the packaging of the RNA genome and are therefore called structural proteins. The other proteins fulfill a variety of functions during the viral life cycle and comprise the so-called non-structural proteins (nsps). Here, we report the near-complete NMR resonance assignment for the backbone chemical shifts of the non-structural protein 10 (nsp10). Nsp10 is part of the viral replication-transcription complex (RTC). It aids in synthesizing and modifying the genomic and subgenomic RNAs. Via its interaction with nsp14, it ensures transcriptional fidelity of the RNA-dependent RNA polymerase, and through its stimulation of the methyltransferase activity of nsp16, it aids in synthesizing the RNA cap structures which protect the viral RNAs from being recognized by the innate immune system. Both of these functions can be potentially targeted by drugs. Our data will aid in performing additional NMR-based characterizations, and provide a basis for the identification of possible small molecule ligands interfering with nsp10 exerting its essential role in viral replication.

1H, 13C, and 15N backbone chemical shift assignments of the apo and the ADP-ribose bound forms of the macrodomain of SARS-CoV-2 non-structural protein 3b.

The SARS-CoV-2 genome encodes for approximately 30 proteins. Within the international project COVID19-NMR, we distribute the spectroscopic analysis of the viral proteins and RNA. Here, we report NMR chemical shift assignments for the protein Nsp3b, a domain of Nsp3. The 217-kDa large Nsp3 protein contains multiple structurally independent, yet functionally related domains including the viral papain-like protease and Nsp3b, a macrodomain (MD). In general, the MDs of SARS-CoV and MERS-CoV were suggested to play a key role in viral replication by modulating the immune response of the host. The MDs are structurally conserved. They most likely remove ADP-ribose, a common posttranslational modification, from protein side chains. This de-ADP ribosylating function has potentially evolved to protect the virus from the anti-viral ADP-ribosylation catalyzed by poly-ADP-ribose polymerases (PARPs), which in turn are triggered by pathogen-associated sensing of the host immune system. This renders the SARS-CoV-2 Nsp3b a highly relevant drug target in the viral replication process. We here report the near-complete NMR backbone resonance assignment (1H, 13C, 15N) of the putative Nsp3b MD in its apo form and in complex with ADP-ribose. Furthermore, we derive the secondary structure of Nsp3b in solution. In addition, 15N-relaxation data suggest an ordered, rigid core of the MD structure. These data will provide a basis for NMR investigations targeted at obtaining small-molecule inhibitors interfering with the catalytic activity of Nsp3b.

Relaxin-3/RXFP3 signalling in mouse hypothalamus: no effect of RXFP3 activation on corticosterone, despite reduced presynaptic excitatory input onto paraventricular CRH neurons in vitro.

Relaxin-3/RXFP3 signalling is proposed to be involved in the neuromodulatory control of arousal- and stress-related neural circuits. Furthermore, previous studies in rats have led to the proposal that relaxin-3/RXFP3 signalling is associated with activation of the hypothalamic-pituitary-adrenal axis, but direct evidence for RXFP3-related actions on the activity of hypothalamic corticotropin-releasing hormone (CRH) neurons is lacking. In this study, we investigated characteristics of the relaxin-3/RXFP3 system in mouse hypothalamus. Administration of an RXFP3 agonist (RXFP3-A2) intra-cerebroventricularly or directly into the paraventricular nucleus of hypothalamus (PVN) of C57BL/6J mice did not alter corticosterone levels. Similarly, there were no differences between serum corticosterone levels in Rxfp3 knockout (C57BL/6JRXFP3TM1) and wild-type mice at baseline and after stress, despite detection of the predicted stress-induced increases in serum corticosterone. We examined the nature of the relaxin-3 innervation of PVN in wild-type mice and in Crh-IRES-Cre;Ai14 mice that co-express the tdTomato fluorophore in CRH neurons, identifying abundant relaxin-3 fibres in the peri-PVN region, but only sparse fibres associated with densely packed CRH neurons. In whole-cell voltage-clamp recordings of tdTomato-positive CRH neurons in these mice, we observed a reduction in sEPSC frequency following local application of RXFP3-A2, consistent with an activation of RXFP3 on presynaptic glutamatergic afferents in the PVN region. These studies clarify the relationship between relaxin-3/RXFP3 inputs and CRH neurons in mouse PVN, with implications for the interpretation of current and previous in vivo studies and future investigations of this stress-related signalling network in normal and transgenic mice, under normal and pathological conditions.

Embedded synaptic feedback in the neuroendocrine stress axis.

Neural regulation of blood glucocorticoid levels is critical for defence of homeostasis during physiological or psychoemotional challenges. In mammals, this function is carried out by the neuroendocrine stress axis, coordinated by parvocellular neuroendocrine cells (PNCs) of the paraventricular hypothalamic nucleus. Feedback regulation of PNCs by glucocorticoids provides complex experience-dependent shaping of neuroendocrine responses. We review recent evidence for metaplastic actions of glucocorticoids as 'circuit breakers' at synapses directly regulating PNC excitability and explore how such mechanisms may serve as substrates for stress adaptation.

Physiological regulation of magnocellular neurosecretory cell activity: integration of intrinsic, local and afferent mechanisms.

The hypothalamic supraoptic and paraventricular nuclei contain magnocellular neurosecretory cells (MNCs) that project to the posterior pituitary gland where they secrete either oxytocin or vasopressin (the antidiuretic hormone) into the circulation. Oxytocin is important for delivery at birth and is essential for milk ejection during suckling. Vasopressin primarily promotes water reabsorption in the kidney to maintain body fluid balance, but also increases vasoconstriction. The profile of oxytocin and vasopressin secretion is principally determined by the pattern of action potentials initiated at the cell bodies. Although it has long been known that the activity of MNCs depends upon afferent inputs that relay information on reproductive, osmotic and cardiovascular status, it has recently become clear that activity depends critically on local regulation by glial cells, as well as intrinsic regulation by the MNCs themselves. Here, we provide an overview of recent advances in our understanding of how intrinsic and local extrinsic mechanisms integrate with afferent inputs to generate appropriate physiological regulation of oxytocin and vasopressin MNC activity.

The intricate link between glucocorticoids and endocannabinoids at stress-relevant synapses in the hypothalamus.

The relationship between glucocorticoids and endocannabinoids at hypothalamic synapses in the presence of stress is particularly complex. Under conditions of acute stress, glucocorticoids trigger the synthesis of endocannabinoids, which through activation of type I cannabinoid receptors (CB1Rs), inhibit stress-relevant neurons in the paraventricular nucleus of the hypothalamus (PVN). Through this signaling mechanism, endocannabinoids constrain the activity of the hypothalamic-pituitary-adrenal axis. However, following chronic or repeated stress, the ability of endocannabinoids to modulate synaptic activity is compromised because of a functional down-regulation in CB1Rs. Here we examine recent findings that highlight important aspects of endocannabinoid signaling in response to stress in the PVN and the dorsomedial hypothalamus (DMH), two hypothalamic nuclei that play integral roles in regulating the neuroendocrine and autonomic responses to stress.

Glial regulation of neuronal function: from synapse to systems physiology.

Classically, glia have been regarded as non-excitable cells that provide nourishment and physical scaffolding for neurones. However, it is now generally accepted that glia are active participants in brain function that can modulate neuronal communication via several mechanisms. Investigations of anatomical plasticity in the magnocellular neuroendocrine system of the hypothalamic paraventricular and supraoptic nuclei led the way in the development of much of our understanding of glial regulation of neuronal activity. In this review, we provide an overview of glial regulation of magnocellular neurone activity from a historical perspective of the development of our knowledge of the morphological changes that are evident in the paraventricular and supraoptic nuclei. We also focus on recent data from the authors' laboratories presented at the 9th World Congress on Neurohypophysial Hormones that have contributed to our understanding of the multiple mechanisms by which glia modulate the activity of neurones, including: gliotransmitter modulation of synaptic transmission; trans-synaptic modulation by glial neurotransmitter transporter regulation of neurotransmitter spillover; and glial neurotransmitter transporter modulation of excitability by regulation of ambient neurotransmitter levels and their action on extrasynaptic receptors. The magnocellular neuroendocrine system secretes oxytocin and vasopressin from the posterior pituitary gland to control birth, lactation and body fluid balance, and we finally speculate as to whether glial regulation of individual magnocellular neurones might co-ordinate population activity to respond appropriately to altered physiological circumstances.

Metabotropic glutamate receptors: gatekeepers of homeostasis.

The capacity to appropriately respond to physiological challenges or perturbations in homeostasis is a requisite for survival. It is becoming increasingly clear that long-lasting alterations in synaptic efficacy are a fundamental mechanism for modifying neuroendocrine and autonomic output. We review recent advances in our understanding of plasticity at glutamate synapses onto magnocellular neurones (MNCs) in the paraventricular and supraoptic nuclei of the hypothalamus, with a focus on the contributions of metabotropic glutamate receptors (mGluRs) to long-lasting modifications in synaptic efficacy. Special attention is paid to the role of presynaptic mGluRs as gatekeepers for metaplasticity and regulation of body fluid homeostasis. The work highlighted here provides insight into the synaptic mechanisms that couple MNC activity to physiological states.

Comparison of renal allograft fibrosis after transplantation from heart-beating and non-heart-beating donors.

BACKGROUND: Renal transplants from non-heart-beating donors (NHBDs) yield acceptable function and allograft survival rates in the medium term. However, the long-term results are less certain and there is a paucity of information relating to the development of chronic allograft nephropathy. The aim of this study was to compare allograft fibrosis in kidneys transplanted from NHBDs and conventional heart-beating donors (HBDs). METHODS: A series of 37 NHBD and 75 HBD renal transplants were studied. Protocol renal transplant biopsies were performed at 6 and 12 months after transplantation. Biopsy sections were stained with Sirius red to demonstrate interstitial extracellular matrix. Renal allograft fibrosis was quantified using a computerized image analysis system. RESULTS: The mean first warm ischaemia time for kidneys from NHBDs was 24 min. A significant delay in graft function occurred in eight of 75 recipients in the HBD group and 31 of 37 in the NHBD group (P < 0.001). There were no significant differences in the level of allograft fibrosis between the two groups at any time point. CONCLUSION: Despite high rates of delayed graft function secondary to a prolonged warm ischaemia time, NHBD kidneys do not appear to be more susceptible to the development of renal allograft fibrosis. This study supports the growing body of evidence that kidneys from NHBDs are an acceptable alternative to those from HBDs.

A comparison of traditional open, minimal-incision donor nephrectomy and laparoscopic donor nephrectomy.

Laparoscopic donor nephrectomy (LDN) and minimal-incision donor nephrectomy (MILD) are less invasive procedures than the traditional open donor nephrectomy approach (ODN). This study compares donor and recipient outcome following those three different procedures. Sixty consecutive donor nephrectomies were studied (n = 20 in each group). Intra-operative variables, analgesic requirements, donor recovery, donor/recipient complications and allograft function were recorded prospectively. Operating and first warm ischaemia times were longer for LDN than for ODN and MILD (232+/-35 vs 121+/-24 vs 147+/-27 min, P < 0.001; 4+/-1 vs 2+/-2 vs 2+/-1 min, P < 0.01). Postoperative morphine requirements were significantly higher after ODN than after MILD and LDN (182+/-113 vs 86+/-48 vs 71+/-45 mg; P < 0.0001). There was no episode of delayed graft function in this study. Donors returned to work quicker after LDN than after ODN and MILD (6+/-2 vs 11+/-5 vs 10+/-7; P = 0.055). Donor and recipient complication rates and recipient allograft function were comparable. We concluded that MILD and LDN reduce postoperative pain and allow a faster recovery without compromising recipient outcome.

Isolation of various forms of sterol beta-D-glucoside from the seed of Cycas circinalis: neurotoxicity and implications for ALS-parkinsonism dementia complex.

The factors responsible for ALS-parkinsonism dementia complex (ALS-PDC), the unique neurological disorder of Guam, remain unresolved, but identification of causal factors could lead to clues for related neurodegenerative disorders elsewhere. Earlier studies focused on the consumption and toxicity of the seed of Cycas circinalis, a traditional staple of the indigenous diet, but found no convincing evidence for toxin-linked neurodegeneration. We have reassessed the issue in a series of in vitro bioassays designed to isolate non-water soluble compounds from washed cycad flour and have identified three sterol beta-d-glucosides as potential neurotoxins. These compounds give depolarizing field potentials in cortical slices, induce alterations in the activity of specific protein kinases, and cause release of glutamate. They are also highly toxic, leading to release of lactate dehydrogenase (LDH). Theaglycone form, however, is non-toxic. NMDA receptor antagonists block the actions of the sterol glucosides, but do not compete for binding to the NMDA receptor. The most probable mechanism leading to cell death may involve glutamate neuro/excitotoxicity. Mice fed cycad seed flour containing the isolated sterol glucosides show behavioral and neuropathological outcomes, including increased TdT-mediated biotin-dUTP nick-end labelling (TUNEL) positivity in various CNS regions. Astrocytes in culture showed increased caspase-3 labeling after exposure to sterol glucosides. The present results support the hypothesis that cycad consumption may be an important factor in the etiology of ALS-PDC and further suggest that some sterol glucosides may be involved in other neurodegenerative disorders.

Cognitive-based measures screening for depression in the medically ill: the DMI-10 and the DMI-18.

OBJECTIVE: We suggest that the identification of depression in the medically ill (DMI) might be improved by focussing on cognitive features. METHOD: We recruited 302 patients to complete our provisional cognitive-based measure. Subsets also completed one of two comparator screening measures, either the Hospital and Anxiety Depression Scale (HADS) or the Beck Depression Inventory for Primary Care (BDI-PC). One hundred and sixty patients were then assessed by a psychiatrist who estimated whether they were 'clinically depressed' and who also administered a standardized interview for depression (the CIDI). RESULTS: Analyses identified items discriminating clinically depressed and non-depressed individuals, allowing development of brief (10-item) and extended (18-item) measures. The two new measures were compared with the HADS and the BDI-PC in discriminating depressed and non-depressed medically ill patients. CONCLUSION: A cognitive construct-based approach to assessing depression in the medically ill appears strongly supported. We provide brief (DMI-10) and extended (DMI-18) measures that appear to have utility as screening instruments. Consideration of the discriminating items may also assist clinical decision making.

Statistical model relating CA3 burst probability to recovery from burst-induced depression at recurrent collateral synapses.

When neuronal excitability is increased in area CA3 of the hippocampus in vitro, the pyramidal cells generate periodic bursts of action potentials that are synchronized across the network. We have previously provided evidence that synaptic depression at the excitatory recurrent collateral synapses in the CA3 network terminates each population burst so that the next burst cannot begin until these synapses have recovered. These findings raise the possibility that burst timing can be described in terms of the probability of recovery of this population of synapses. Here we demonstrate that when neuronal excitability is changed in the CA3 network, the mean and variance of the interburst interval change in a manner that is consistent with a timing mechanism comprised of a pool of exponentially relaxing pacemakers. The relaxation time constant of these pacemakers is the same as the time constant describing the recovery from activity-dependent depression of recurrent collateral synapses. Recovery was estimated from the rate of spontaneous transmitter release versus time elapsed since the last CA3 burst. Pharmacological and long-term alterations of synaptic strength and network excitability affected CA3 burst timing as predicted by the cumulative binomial distribution if the burst pace-maker consists of a pool of recovering recurrent synapses. These findings indicate that the recovery of a pool of synapses from burst-induced depression is a sufficient explanation for burst timing in the in vitro CA3 neuronal network. These findings also demonstrate how information regarding the nature of a pacemaker can be derived from the temporal pattern of synchronous network activity. This information could also be extracted from less accessible networks such as those generating interictal epileptiform discharges in vivo.

Slowly inactivating potassium conductance (I(D)): a potential target for stroke therapy.

BACKGROUND AND PURPOSE: Excessive accumulation of extracellular glutamate results in the death of most, but not all, neurons in the central nervous system. Understanding the unique properties of cells that can withstand this excitotoxic challenge may identify specific targets for novel stroke therapies. METHODS: A combination of in vivo methods for analysis of excitotoxic cell death after activation of N-methyl-D-aspartate (NMDA) receptors and in vitro patch-clamp analysis of specific conductances in hypothalamic slices and dissociated cells has been used to assess the roles of specific potassium conductances in delayed cell death after NMDA receptor activation. RESULTS: We report that a specific D-type potassium conductance (I(D)), necessary for the rapid repolarization of the membrane after a strong depolarization, serves such a protective purpose in magnocellular neurons of the paraventricular nucleus. Manipulations that inhibit this current (4-aminopyridine or angiotensin II) increase neuronal excitability and augment cell death after NMDA receptor activation. In addition, this protection is not observed in magnocellular neurons of spontaneously hypertensive rats, and intriguingly it can be reestablished by blocking angiotensin II receptors in these animals. CONCLUSIONS: These observations provide a persuasive experimental explanation for the unexpected finding that therapeutic treatments for hypertension that block central as well as peripheral angiotensin type 1 receptors reduce the severity and occurrence of stroke.

Screening for depression in the medically ill: the suggested utility of a cognitive-based approach.

OBJECTIVE: There is a need for a valid measure of depression in the medically ill, and one that is independent of medical illness characteristics. As yet, there is no such widely accepted measure. We thus report on the early development of such a measure using cognitive constructs that define depressive mood state nuances. METHOD: We studied 67 patients with a significant medical illness, verbally administering a set of 81 provisional items. Sample members also alternatively completed one of two comparison measures: the Hospital Anxiety and Depression Scale (HADS) or the Beck Depression Inventory for Primary Care (BDI-PC). A psychiatrist interviewed a subset to determine severity of any depression and whether subjects met formalized caseness criteria for depression. The Composite International Diagnostic Interview (CIDI) was also administered during interviews to assess agreement with psychiatrist judgements about caseness. RESULTS: A 16-item measure with high internal consistency was derived, with validation analyses suggesting it was distinctly superior to the HADS and somewhat superior to the BDI-PC measure. CONCLUSIONS: A cognitive-based approach (as used by both our measure and the BDI-PC) to screen for depression in medically ill groups appears to have distinct utility in identifying depressed patients, and in avoiding confounding influences of physical symptoms.

Methionine sulfoximine shows excitotoxic actions in rat cortical slices.

Methionine sulfoximine (MSO) is a rare amino acid. It occurs in nature or as a by-product of some forms of food processing. A notable example of the latter was a former method for bleaching wheat flour, using nitrogen trichloride, the "agene process," in use for most of the first 50 years of this century. "Agenized" flour was found to be responsible for various neurological disorders in animals, and MSO was identified as the toxic factor. The agene process was subsequently discontinued in the United States and the United Kingdom circa 1950. MSO inhibits the synthesis of both glutathione and glutamine, and it is possible that its actions on the nervous system arise from alterations in the amount or distribution of these molecules. Structurally, MSO resembles glutamate, an observation that has also raised the possibility that it might have more direct glutamate-like actions on neurons. In the present investigation, we report excitatory and toxic actions of MSO in an in vitro preparation of adult rat cortex. Field potential recordings in this preparation show that MSO application evokes a sustained depolarization, which can be blocked by the N-methyl-D-aspartate (NMDA) antagonist L-(+)-2-amino-5-phosphonovalerate (AP5). However, competition assays using MSO on [3H]CGP-39653 (DL-(E)-2-amino-4-propyl-1-phosphono-3-pentenoate) binding in rat cortical homogenates show only 20% displacement of total binding, suggesting that MSO is acting indirectly, perhaps by releasing glutamate. To investigate this possibility, we measured glutamate release during MSO application. Time course and dose-response experiments with MSO showed significant [3H]glutamate release, which was partially attenuated by AP5. To assess cellular toxicity, we measured lactate dehydrogenase (LDH) release from cortical sections exposed to MSO. MSO treatment led to a rapid increase in LDH activity, which could be blocked by AP5. These data suggest that MSO acts by increasing glutamate release, which then activates NMDA receptors, leading to excitotoxic cell death. These data suggest the possibility that MSO in processed flour had excitotoxic actions that may have been contributing factors to some human neuronal disorders.

Glutathione and signal transduction in the mammalian CNS.

The tripeptide glutathione (GSH) has been thoroughly investigated in relation to its role as antioxidant and free radical scavenger. In recent years, novel actions of GSH in the nervous system have also been described, suggesting that GSH may serve additionally both as a neuromodulator and as a neurotransmitter. In the present article, we describe our studies to explore further a potential role of GSH as neuromodulator/neurotransmitter. These studies have used a combination of methods, including radioligand binding, synaptic release and uptake assays, and electrophysiological recording. We report here the characteristics of GSH binding sites, the interrelationship of GSH with the NMDA receptor, and the effects of GSH on neural activity. Our results demonstrate that GSH binds via its gamma-glutamyl moiety to ionotropic glutamate receptors. At micromolar concentrations GSH displaces excitatory agonists, acting to halt their physiological actions on target neurons. At millimolar concentrations, GSH, acting through its free cysteinyl thiol group, modulates the redox site of NMDA receptors. As such modulation has been shown to increase NMDA receptor channel currents, this action may play a significant role in normal and abnormal synaptic activity. In addition, GSH in the nanomolar to micromolar range binds to at least two populations of binding sites that appear to be distinct from all known excitatory amino acid receptor subtypes. GSH bound to these sites is not displaceable by glutamatergic agonists or antagonists. These binding sites, which we believe to be distinct receptor populations, appear to recognize the cysteinyl moiety of the GSH molecule. Like NMDA receptors, the GSH binding sites possess a coagonist site(s) for allosteric modulation. Furthermore, they appear to be linked to sodium ionophores, an interpretation supported by field potential recordings in rat cerebral cortex that reveal a dose-dependent depolarization to applied GSH that is blocked by the absence of sodium but not by lowering calcium or by NMDA or (S)-2-amino-3-hydroxy-5-methyl-4-isoxazolepropionate antagonists. The present data support a reevaluation of the role of GSH in the nervous system in which GSH may be involved both directly and indirectly in synaptic transmission. A full accounting of the actions of GSH may lead to more comprehensive understanding of synaptic function in normal and disease states.

Reciprocal interactions between CA3 network activity and strength of recurrent collateral synapses.

In hippocampal slices, synchronous CA3 network activity induced persistent strengthening of active positive-feedback synapses. This altered network operation by increasing probability of future synchronous network activation. Long-term depression of synaptic strength induced by partial blockade of NMDA receptors during synchronous network activity reversed changes in probability of spontaneous network activation. These results suggest that specific network activity patterns selectively alter strength of active synapses. Stable, reversible alterations in network activity can also be effected by corresponding alterations in synaptic strength. These findings confirm the Hebb memory model at the neural-network level and suggest new therapies for pathological patterns of network activity in epilepsy.

Activation of N-methyl-D-aspartate receptors evokes calcium spikes in the dendrites of rat hypothalamic paraventricular nucleus neurons.

Activation of dendritic voltage-dependent calcium (Ca2+) conductances in neuroendocrine cells of the hypothalamus may underlie previously documented Ca2+ spikes in these cells. The present study, in which whole-cell recordings were obtained from paraventricular nucleus neurons in a hypothalamic slice preparation, addresses this issue by directly activating dendritic N-methyl-D-aspartate receptors in the presence of tetrodotoxin. Application of tetrodotoxin abolished spontaneous action potentials in all paraventricular nucleus neurons tested (n = 27). Following tetrodotoxin, spikes were evoked by depolarizing current pulses, in an all-or-none fashion in the majority of cells (n = 20). Removal of extracellular Ca2+ (n = 6) or addition of 500 microM CdCl2 (n = 4) abolished the spikes in response to pulses. Repetitive spiking activity (in tetrodotoxin) was also observed following N-methyl-D-aspartate agonist application in 75% of the cells tested (n = 15). The spikes, underscored by a slow membrane depolarization, were abolished by the administration of CdCl2 (n = 4). N-Methyl-D-aspartate agonist elicited a slow inward current in cells voltage-clamped at -60 mV (n = 5). Additionally, larger amplitude, transient inward currents were observed near the onset of the response. The activation threshold to elicit spikes following N-methyl-D-aspartate agonist application was significantly more negative (-54.6+/-3.6 mV) than the potential at which spikes were initiated as a result of depolarizing current injection (-32.3+/-1.8 mV; Student's t-test: P < 0.0001). In contrast to this, Na+ spikes in control solution had an invariable threshold (-49.6+/-0.7 mV vs -51.5+/-1.2 mV; P > 0.05), regardless of the stimulus used to initiate the spikes. These observations suggest that direct activation of N-methyl-D-aspartate receptors located on the dendrites of paraventricular nucleus neurons triggers Ca2+ spikes. Although the precise function of these spikes is unclear, previous data reporting dendritic neuropeptide release in the paraventricular nucleus raise the possibility that dendritically initiated spikes may serve as a local signal to trigger such release.

Premedication with clonidine does not attenuate suppression of certain lymphocyte subsets after surgery.

UNLABELLED: Sixty-four patients undergoing elective major surgery were randomly assigned into a double-blinded, placebo-controlled, clinical trial to test the hypothesis that premedication with clonidine would attenuate postoperative reductions in circulating lymphocytes. The treatment group (n = 28) received a clonidine skin patch (0.3 mg/d) and a 0.6-mg oral loading dose 60-90 min before surgery. The control group (n = 36) received placebo patches and pills. Absolute blood levels of the following lymphocyte subsets were measured before induction of a standardized general anesthetic (baseline) and the morning after surgery: CD2, CD3, CD4, CD8, CD20, CD56, and the CD4:CD8 ratio. Significant decreases in lymphocyte subsets CD2, CD3, and CD4 were found in both groups; CD56 was significantly decreased only in the placebo group. However, the extent of lymphocyte depletion from baseline to Postoperative Day 1 between the clonidine and placebo groups was not different. Plasma concentrations of epinephrine, norepinephrine, and cortisol were measured from blood samples drawn at 8:00 AM on Postoperative Day 1. Plasma norepinephrine levels were significantly lower among patients who received clonidine. However, no significant differences were found in plasma epinephrine or cortisol levels between the clonidine and placebo groups. With a clinical dose, clonidine did not prevent postoperative lymphocyte depletion. alpha2-Agonists may not suppress adrenocortical stress responses sufficiently to prevent postoperative immune suppression. IMPLICATIONS: Lymphocyte (white blood cell) counts often decrease after major surgery. We hypothesized that clonidine would reduce hormonal stress and blunt reductions in lymphocytes after major surgery. In a randomized trial, we found no differences from placebo in cortisol levels or lymphocyte changes. Lymphocyte levels did not predict infectious complications.