Increased diaphragm motor unit discharge frequencies during quiet breathing in people with chronic tetraplegia.

KEY POINTS: Respiratory muscle strength is compromised in people with tetraplegia, which may be compensated for by an increase in neural drive to the diaphragm. We found that the discharge frequencies of diaphragm motor units are higher in people with chronic tetraplegia compared with able-bodied people during quiet breathing. Furthermore, we found that the area of single motor unit potentials was increased in people with tetraplegia. These results suggest an increased motoneurone output to the diaphragm and remodelling of diaphragm motor units to maintain ventilation in tetraplegia. ABSTRACT: People with tetraplegia have reduced inspiratory muscle strength, ∼40% of able-bodied individuals. Paralysed or partially paralysed respiratory muscles as a result of tetraplegia compromise lung function, increase the incidence of respiratory infections and can cause dyspnoea. We hypothesised that reduced inspiratory muscle strength in tetraplegia may increase neural drive to the inspiratory muscles to maintain ventilation. We recorded the discharge properties of single motor units from the diaphragm in participants with chronic tetraplegia (8 males, 42-78 years, C3-C6 injury, AIS A-C) and able-bodied control participants (6 males matched for age and body mass index). In each group, 117 and 166 single motor units, respectively, were discriminated from recordings in the costal diaphragm using a monopolar electrode. A linear mixed-effects model analysis showed higher peak discharge frequencies of motor units during quiet breathing in tetraplegia (17.8 ± 4.9 Hz; mean ± SD) compared with controls (12.4 ± 2.2 Hz) (P < 0.001). There were no differences in tidal volume, inspiratory time or mean air flow between groups. Motor unit potentials in tetraplegia, compared with controls, were larger in amplitude (1.1 ± 0.7 mV and 0.5 ± 0.3 mV, respectively, P = 0.007) and area (1.83 ± 1.49 µV ms and 0.69 ± 0.52 µV ms, respectively, P = 0.003). The findings indicate that diaphragm motor unit remodelling is likely to have occurred in people with chronic tetraplegia and that there is an increase in diaphragm motor unit discharge rates during quiet breathing. These neural changes ensure that ventilation is maintained in people with chronic tetraplegia.

Discharge properties of human diaphragm motor units with ageing.

KEY POINTS: Ageing is associated with changes in the respiratory system including in the lungs, rib cage and muscles. Neural drive to the diaphragm, the principal inspiratory muscle, has been reported to increase during quiet breathing with ageing. We demonstrated that low-threshold motor units of the human diaphragm recruited during quiet breathing have similar discharge frequencies across age groups and shorter discharge times in older age. With ageing, motor unit action potential area increased. We propose that there are minimal functionally significant changes in the discharge properties of diaphragm motor units with ageing despite remodelling of the motor unit in the periphery. ABSTRACT: There are changes in the skeletal, pulmonary and respiratory neuromuscular systems with healthy ageing. During eupnoea, one study has shown relatively higher crural diaphragm electromyographic activity (EMG) in healthy older adults (>51 years) than in younger adults, but these measures may be affected by the normalisation process used. A more direct method to assess neural drive involves the measurement of discharge properties of motor units. Here, to assess age-related changes in neural drive to the diaphragm during eupnoea, EMG was recorded from the costal diaphragm using a monopolar needle electrode in participants from three age groups (n ≥ 7 each): older (65-80 years); middle-aged (43-55 years) and young (23-26 years). In each group, 154, 174 and 110 single motor units were discriminated, respectively. A mixed-effects linear model showed no significant differences between age groups for onset (group mean range 9.5-10.2 Hz), peak (14.1-15.0 Hz) or offset (7.8-8.5 Hz) discharge frequencies during eupnoea. The motor unit recruitment was delayed in the older group (by ∼15% of inspiratory time; p = 0.02 cf. middle-aged group) and had an earlier offset time (by ∼15% of inspiratory time; p = 0.04 cf. young group). However, the onset of multiunit activity was similar across groups, consistent with no global increase in neural drive to the diaphragm with ageing. The area of diaphragm motor unit potentials was ∼40% larger in the middle-aged and older groups (P < 0.02), which indicates axonal sprouting and re-innervation of muscle fibres associated with ageing, even in middle-aged participants.

EVERSUN: a phase 2 trial of alternating sunitinib and everolimus as first-line therapy for advanced renal cell carcinoma.

BACKGROUND: We hypothesised that alternating inhibitors of the vascular endothelial growth factor receptor (VEGFR) and mammalian target of rapamycin pathways would delay the development of resistance in advanced renal cell carcinoma (aRCC). PATIENTS AND METHODS: A single-arm, two-stage, multicentre, phase 2 trial to determine the activity, feasibility, and safety of 12-week cycles of sunitinib 50 mg daily 4 weeks on / 2 weeks off, alternating with everolimus 10 mg daily for 5 weeks on / 1 week off, until disease progression or prohibitive toxicity in favourable or intermediate-risk aRCC. The primary end point was proportion alive and progression-free at 6 months (PFS6m). The secondary end points were feasibility, tumour response, overall survival (OS), and adverse events (AEs). The correlative objective was to assess biomarkers and correlate with clinical outcome. RESULTS: We recruited 55 eligible participants from September 2010 to August 2012. DEMOGRAPHICS: mean age 61, 71% male, favourable risk 16%, intermediate risk 84%. Cycle 2 commenced within 14 weeks for 80% of participants; 64% received ≥22 weeks of alternating therapy; 78% received ≥22 weeks of any treatment. PFS6m was 29/55 (53%; 95% confidence interval [CI] 40% to 66%). Tumour response rate was 7/55 (13%; 95% CI 4% to 22%, all partial responses). After median follow-up of 20 months, 47 of 55 (86%) had progressed with a median progression-free survival of 8 months (95% CI 5-10), and 30 of 55 (55%) had died with a median OS of 17 months (95% CI 12-undefined). AEs were consistent with those expected for each single agent. No convincing prognostic biomarkers were identified. CONCLUSIONS: The EVERSUN regimen was feasible and safe, but its activity did not meet pre-specified values to warrant further research. This supports the current approach of continuing anti-VEGF therapy until progression or prohibitive toxicity before changing treatment. AUSTRALIAN NEW ZEALAND CLINICAL TRIALS REGISTRY: ACTRN12609000643279.

Incidence of hyperoxia in trauma patients receiving pre-hospital emergency anaesthesia: results of a 5-year retrospective analysis.

BACKGROUND: Previous studies have demonstrated an association between hyperoxia and increased mortality in various patient groups. Critically unwell and injured patients are routinely given high concentration oxygen in the pre-hospital phase of care. We aim to investigate the incidence of hyperoxia in major trauma patients receiving pre-hospital emergency anesthesia (PHEA) in the pre-hospital setting and determine factors that may help guide clinicians with pre-hospital oxygen administration in these patients. METHODS: A retrospective cohort study was performed of all patients who received PHEA by a single helicopter emergency medical service (HEMS) between 1 October 2014 and 1 May 2019 and who were subsequently transferred to one major trauma centre (MTC). Patient and treatment factors were collected from the electronic patient records of the HEMS service and the MTC. Hyperoxia was defined as a PaO2 > 16 kPA on the first arterial blood gas analysis upon arrival in the MTC. RESULTS: On arrival in the MTC, the majority of the patients (90/147, 61.2%) had severe hyperoxia, whereas 30 patients (20.4%) had mild hyperoxia and 26 patients (19.7%) had normoxia. Only 1 patient (0.7%) had hypoxia. The median PaO2 on the first arterial blood gas analysis (ABGA) after HEMS handover was 36.7 [IQR 18.5-52.2] kPa, with a range of 7.0-86.0 kPa. SpO2 pulse oximetry readings before handover were independently associated with the presence of hyperoxia. An SpO2 ≥ 97% was associated with a significantly increased odds of hyperoxia (OR 3.99 [1.58-10.08]), and had a sensitivity of 86.7% [79.1-92.4], a specificity of 37.9% [20.7-57.8], a positive predictive value of 84.5% [70.2-87.9] and a negative predictive value of 42.3% [27.4-58.7] for the presence of hyperoxemia. CONCLUSION: Trauma patients who have undergone PHEA often have profound hyperoxemia upon arrival at hospital. In the pre-hospital setting, where arterial blood gas analysis is not readily available a titrated approach to oxygen therapy should be considered to reduce the incidence of potentially harmful tissue hyperoxia.

Differential activation of the human costal and crural diaphragm during voluntary and involuntary breaths.

The diaphragm is the primary muscle that generates the negative intrathoracic pressure to drive inspiratory airflow. The diaphragm consists of two parts, the costal and crural portions, with different roles during inspiration in animals, particularly when the stimulus to breathe is increased. In this study, the neural drive to the costal and crural portions of the diaphragm was assessed in nine healthy participants [8 male, aged 32 ± 13 yr (mean ± SD)]. Inspiratory electromyographic activity (EMG) was recorded from the costal diaphragm by using an intramuscular electrode and from the crural diaphragm with a multipair gastroesophageal catheter. Participants performed voluntary augmented breaths at 120%, 140%, and 160% of their tidal volume and also underwent progressive hypercapnia to induce involuntary breathing. Irrespective of the task, the increase in crural activity (normalized to quiet breathing) was only ~60% of the increase in costal activity (slope: 0.56 ± 0.30, P < 0.001). The onset and peak timing of EMG activity was similar for the costal and crural diaphragm during quiet breathing. Thus, when stimulated by either a voluntary or involuntary drive to breathe above tidal volume, the neural drive to the diaphragm was greater to the costal than to the crural portion.NEW & NOTEWORTHY Simultaneous electromyographic recordings from the human costal and crural diaphragm during voluntary augmented breathing and involuntary rebreathing show that the increase in inspiratory crural diaphragm activity was ~60% of the increase in costal diaphragm activity. However costal to crural diaphragm activation did not differ between the two tasks. The dissociation in the amplitude of activation of the costal and crural diaphragm becomes apparent only as the drive to breathe increases above tidal breathing.

Abdominal Functional Electrical Stimulation to Augment Respiratory Function in Spinal Cord Injury.

Background: Functional electrical stimulation (FES) is the application of electrical pulses to a nerve to achieve a functional muscle contraction. Surface electrical stimulation of the nerves that innervate the abdominal muscles, termed abdominal FES, can cause the abdominal muscles to contract, even when paralysed after spinal cord injury. As the abdominal muscles are the major expiratory muscles, and commonly partially or completely paralysed in tetraplegia, abdominal FES offers a promising method of improving respiratory function for this patient group. Objective: The aim of the article is to provide readers with a better understanding of how abdominal FES can be used to improve the health of the spinal cord-injured population. Methods: A narrative review of the abdominal FES literature was performed. Results: Abdominal FES can achieve an immediate effective cough in patients with tetraplegia, while the repeated application over 6 weeks of abdominal FES can improve unassisted respiratory function. Ventilator duration and tracheostomy cannulation time can also be reduced with repeated abdominal FES. Conclusion: Abdominal FES is a noninvasive method to achieve functional improvements in cough and respiratory function in acute and chronically injured people with tetraplegia. Potential practical outcomes of this include reduced ventilation duration, assisted tracheostomy decannulation, and a reduction in respiratory complications. All of these outcomes can contribute to reduced morbidity and mortality, improved quality of life, and significant potential cost savings for local health care providers.

Riemannian Geometry Applied to Detection of Respiratory States From EEG Signals: The Basis for a Brain-Ventilator Interface.

GOAL: During mechanical ventilation, patient-ventilator disharmony is frequently observed and may result in increased breathing effort, compromising the patient's comfort and recovery. This circumstance requires clinical intervention and becomes challenging when verbal communication is difficult. In this study, we propose a brain-computer interface (BCI) to automatically and noninvasively detect patient-ventilator disharmony from electroencephalographic (EEG) signals: a brain-ventilator interface (BVI). METHODS: Our framework exploits the cortical activation provoked by the inspiratory compensation when the subject and the ventilator are desynchronized. Use of a one-class approach and Riemannian geometry of EEG covariance matrices allows effective classification of respiratory states. The BVI is validated on nine healthy subjects that performed different respiratory tasks that mimic a patient-ventilator disharmony. RESULTS: Classification performances, in terms of areas under receiver operating characteristic curves, are significantly improved using EEG signals compared to detection based on air flow. Reduction in the number of electrodes that can achieve discrimination can be often desirable (e.g., for portable BCI systems). By using an iterative channel selection technique, the common highest order ranking, we find that a reduced set of electrodes (n = 6) can slightly improve for an intrasubject configuration, and it still provides fairly good performances for a general intersubject setting. CONCLUSION: Results support the discriminant capacity of our approach to identify anomalous respiratory states, by learning from a training set containing only normal respiratory epochs. SIGNIFICANCE: The proposed framework opens the door to BVIs for monitoring patient's breathing comfort and adapting ventilator parameters to patient respiratory needs.

'Seeing through a glass darkly': casting light on imidazoline 'I' sites.

Although imidazoline sites have been the subject of research for several years, there is still controversy about their structure, diversity and physiology. The I1 site is thought to exist principally as a binding site and is widely purported to play a role in controlling systemic blood pressure, although this is still unclear. The majority of I2 sites are widely accepted as being allosteric sites on monoamine oxidase; however, even with selective ligands, their exact function remains to be determined. A putative I3 site modulates insulin secretion and could represent the first functional site to be pharmacologically defined with selective agonists and antagonists. The structure and relevance of the proposed endogenous ligand 'clonidine-displacing substance' remains elusive. A potential candidate for this substance is agmatine; however, although it is capable of displacing bound clonidine from imidazoline sites, it lacks the functionality ascribed to the clonidine-displacing substance. In this review, Richard M. Eglen and colleagues assess our knowledge of imidazoline sites in the light of recent data.

[3H](-)Baclofen: an improved ligand for GABAB sites.

[3H](-)Baclofen, the radiolabelled form of the active isomer of baclofen, has been used as a ligand for GABAB (gamma-aminobutyric acid) receptors on synaptic membranes from whole brain of rat. The pharmacological profile for displacement of this ligand was observed to be identical with that for the racemic ligand [3H](+/-)baclofen and [3H]GABA under conditions where GABAB, but not GABAA sites, were labelled. The displaceable (specific) portion of membrane-bound [3H](-)baclofen was 47.5 +/- 2.3% of the total which was twice that obtained previously with [3H](+/-)baclofen. Two binding components were observed with affinities of 19 and 304 nM and binding capacities of 0.37 and 1.58 pmol/mg protein respectively. It is suggested that [3H](-)baclofen is an improvement over the labelled racemic form and binds to the same sites. It should provide a more reliable tool for studying GABAB receptors.

Evidence that SL75102 is an agonist at GABAb as well as GABAa receptors.

The compound SL75102 ([alpha(4-chlorophenyl)5-fluoro 2-hydroxy benzilidene-amino]-4-butanoate sodium) is a GABA-mimetic at bicuculline-sensitive and -insensitive receptors. It depolarized rat isolated superior cervical ganglia in a dose-dependent manner (relative potency = 0.101 +/- 0.013; GABA = 1). Bicuculline methobromide (13 microM) antagonized this action of SL75102 and shifted the log dose-response curve to the same extent as the GABA curve. The evoked release of [3H]noradrenaline from rat isolated atria was also reduced by SL75102 in a GABA-like manner. SL75102 also displaced [3H]GABA and [3H]baclofen specifically bound to divalent cation dependent GABAB sites on rat synaptic membranes (relative potency approximately 0.1 GABA throughout). The butyramide from which SL75102 can be formed within the body (SL76002) was much less active at GABAB sites (less than 0.02 atria, 0.001 binding, GABA = 1). It is suggested that in addition to any direct action of SL76002 itself the products of SL76002 metabolism, SL75102 and GABA may exert effects via baclofen-sensitive GABAB as well as GABAA sites in mammalian brain.

5-Isothiocyanato-2-benzofuranyl-2-imidazoline (BU99006) an irreversible imidazoline(2) binding site ligand: in vitro and in vivo characterisation in rat brain.

5-Isothiocyanato-2-benzofuranyl-2-imidazoline (BU99006) is an irreversible ligand based on the highly selective I(2) binding site ligand 2BFI. In competition binding assays it has been shown to have high affinity and selectivity for the I(2) binding site and to irreversibly inhibit the binding of [(3)H]2BFI. In this present study we have sought to confirm and expand on these findings both in vitro and in vivo. In vitro pre-incubation of rat whole brain membranes with BU99006 (10 microM) was shown to reduce the specific binding of [(3)H]2BFI to 10% of the control values, an effect not seen using 2BFI or BU224. Pre-treatment of rat whole brain membranes by BU99006, or by the alpha(2)-adrenoceptor antagonists RX821002 or rauwolscine had no effect on the specific binding of [(3)H]RX821002. In vivo pre-treatment of rats with BU99006 (15 mg x kg(-1), i.v.) caused a substantial loss of [(3)H]2BFI specific binding in subsequent in vitro saturation analysis and autoradiography; this loss was shown to be dose dependent. These data indicate that BU99006 is selectively and irreversibly affecting I(2) binding sites both in vitro and in vivo and that it represents an invaluable tool in the further understanding of the I(2) binding site.

GABA receptor multiplicity. Visualization of different receptor types in the mammalian CNS.

Receptors for GABA in the mammalian brain are not homogeneous. A clear separation exists between receptors which recognize the antagonist bicuculline and a population which does not. These two classes have been designated GABAA and GABAB sites respectively. Within the GABAA category there may also be many subtypes which exhibit subtle pharmacological differences. Numerous centrally-active agents influence GABAA site function generally via an allosteric interaction. By comparison very few substances currently available interact with GABAB sites. The GABAA and GABAB sites show many contrasting characteristics not least of which is their distribution pattern within the rat brain. Autoradiographic analysis has indicated that although both receptors may be present within many regions, in some areas only one type is present. For example, GABAA sites only are present in the lamina molecularis of the olfactory bulb and granule cell layer of the cerebellum whereas GABAB sites are present in the interpeduncular nucleus without any evidence of GABAA sites. In the spinal cord GABAB sites are unevenly distributed with high densities in laminae I-IV. GABAA sites are more uniformly distributed throughout the dorsal and ventral horns. In conclusion, there is now good evidence for multiple GABA receptors and the way is open to determine the functional significance of the GABAB receptor in relation to the now classical GABAA site.

Imidazoline(2) (I(2)) binding site- and alpha(2)-adrenoceptor-mediated modulation of central noradrenergic and HPA axis function in control rats and chronically stressed rats with adjuvant-induced arthritis.

The aim of this study was to investigate imidazoline(2) (I(2)) binding site- and alpha(2)-adrenoceptor-mediated control of central noradrenergic and HPA axis activity in control rats and chronically stressed rats with adjuvant-induced arthritis (AA). Basal levels of extracellular nonadrenaline (NA) in the region of the hypothalamic paraventricular nucleus (PVN) of AA rats were significantly greater than controls. Both the I(2) binding site selective ligand BU224 (10 mg kg(-1) i.p.) and the alpha(2)-adrenoceptor antagonist RX821002 (2.5 mg kg(-1) i.p.) significantly elevated extracellular levels of NA in the PVN region and plasma corticosterone (CORT) in a rapid and transient manner in both control and AA rats. The noradrenergic response of AA rats to BU224 was significantly enhanced compared with drug treated controls. There was a significant correlation between extracellular NA in the PVN region and plasma CORT following BU224 and RX821002. In conclusion, central noradrenergic and HPA axis activity in control and chronically stressed AA rats appear to be under the control of both I(2) binding sites and alpha(2)-adrenoceptors. Increased basal levels of extracellular NA in the PVN region of AA rats suggests increased noradrenergic activity in these animals which is modulated to a greater extent by I(2) binding sites than by alpha(2)-adrenoceptors.

Localization of high affinity [3H]glibenclamide binding sites within the substantia nigra zona reticulata of the rat brain.

The rat substantia nigra zona reticulata contains a high density of binding sites for glibenclamide, an adenosine triphosphate-sensitive potassium channel inhibitor, but the precise location of glibenclamide binding sites within this area has not previously been examined. By combining neurochemical lesion and autoradiographical studies we have shown that high affinity [3H]glibenclamide binding sites are located on striatonigral terminals. Unilateral injections of 6-hydroxydopamine into the medial forebrain bundle or of quinolinic acid into the striatum were performed in anaesthetized adult rats to lesion the nigrostriatal and striatonigral pathways respectively. Autoradiography was performed on coronal sections of midbrain with [3H]glibenclamide, [3H]YM-09151-2 (dopamine D2 receptor antagonist) and [3H]SCH 23390 (dopamine D1 receptor antagonist) at three rostrocaudal levels of the substantia nigra. Under the conditions of the incubation [3H]glibenclamide binds primarily to the high affinity site. Following the 6-hydroxydopamine nigrostriatal lesion, D2 receptor binding was reduced (by up to 67%) on the lesioned side at all three levels of the substantia nigra whereas D1 receptor and glibenclamide binding were not significantly affected. In contrast, following striatonigral pathway lesion with quinolinic acid D2 receptor binding was unchanged on the lesioned side, but both D1 receptor and glibenclamide binding were reduced at all three levels (by up to 85% and 63% in the area of maximum lesion, respectively). In adjacent sections, the pattern of D1 binding loss was closely paralleled by the loss of glibenclamide binding. These results demonstrate that the high affinity glibenclamide binding sites of the substantia nigra zona reticulata are, at least in part, located on the terminals of striatonigral projection neurons.

GABAA and GABAB receptor site distribution in the rat central nervous system.

An autoradiographic procedure has been used to determine the quantitative distributions of gamma-aminobutyric acid (GABAA and GABAB) receptor subtypes in rat brain. Although the concentrations of both receptor binding sites were similar in some brain regions GABAA sites generally outnumbered GABAB sites. The highest concentration of GABAA sites were detected in the frontal cortex, the granule cell layer of the cerebellum, the olfactory bulb and the thalamic medial geniculate. The highest concentration of GABAB sites occurred in the molecular layer of the cerebellum, the interpeduncular nucleus, frontal cortex, anterior olfactory nucleus and thalamic nuclei. In addition the globus pallidus, temporal cortex, lateral posterior thalamus, superior colliculus, pontine nucleus, raphe magnus, spinal trigeminal tract and substantia gelatinosa contained significantly more GABAB sites than GABAA sites. The physiological and pharmacological significance of this heterogeneity has yet to be determined.

Quantitative autoradiography of [3H]-MK-801 binding sites in mammalian brain.

1. An in vitro receptor autoradiography procedure is described for visualizing binding sites for the excitatory amino acid antagonist radiolabelled MK-801, in rat and gerbil brain sections. 2. Ten micron sections were labelled by incubation at room temperature for 20 min in 30 nM [3H]-MK-801. This was followed by 2 rinses for 20 s in fresh buffer solution. Specifically bound ligand determined with 100 microM unlabelled MK-801 amounted to 55-60% of total. 3. Phencyclidine, (+/-)-SKF 10047, ketamine and 2-aminophosphonovaleric acid (APV) (all 100 microM) prevented the specific binding of [3H]-MK-801. L-Glutamate and N-methyl D-aspartate (NMDA) (100 microM) had no effect. However, L-glutamate prevented the inhibition by APV. 4. The highest concentrations of [3H]-MK-801 binding sites occurred in the hippocampal formation, cerebral cortex, olfactory bulb and thalamus. Very low levels were detected in the brain stem and cerebellum. 5. The distribution of [3H]-MK-801 binding sites was comparable to that of NMDA sites and phencyclidine sites (labelled with [3H]-TCP) but not with high-affinity sigma sites labelled with [3H]-3-PPP. 6. The density of [3H]-MK-801 binding sites in the gerbil hippocampus was examined 1, 2, 6 and 22 days after unilateral carotid artery occlusion for 10 min. Only at 6 and 22 days was the binding reduced (by 36% and 46% respectively) in the CA1 region whereas a significant neuronal loss was apparent at day 2. In CA2 a decrease in binding was only evident at day 22. 7. These results indicate that binding sites for [3H]-MK-801 can be detected in mammalian brain sections by receptor autoradiography. Their distribution supports an association with the NMDA receptor complex and the loss in the hippocampus after carotid artery occlusion indicates their presence on pyramidal cells is vulnerable to ischaemic insult.

Characterization and autoradiographical localization of non-adrenoceptor idazoxan binding sites in the rat brain.

1. In rat whole brain homogenates, saturation analysis revealed that both [3H]-idazoxan and [3H]-RX821002, a selective alpha 2-adrenoceptor ligand, bound with high affinity to an apparent single population of sites. However, the Bmax for [3H]-idazoxan was significantly (P less than 0.01) greater than that for [3H]-RX821002. 2. In competition studies, (-)-adrenaline displaced 3 nM [3H]-idazoxan binding with an affinity consistent with [3H]-idazoxan labelling alpha 2-adrenoceptors. However, this displacement was incomplete since 23.68 +/- 1.11% of specific [3H]-idazoxan binding remained in the presence of an excess concentration (100 microM) of (-)-adrenaline. In contrast, unlabelled idazoxan promoted a complete displacement of [3H]-idazoxan binding with a Hill slope close to unity and an affinity comparable with its KD determined in saturation studies. 3. Displacement of [3H]-idazoxan binding by the alpha 2-adrenoceptor antagonists yohimbine, RX821002 (2-(2-methoxy-1,4-benzodioxan-2-yl)-2-imidazoline) and RX811059 (2-(2-ethoxy-1,4-benzodioxan-2-yl)-2-imidazoline) was more complex, with Hill slopes considerably less than unity, and best described by a two-site model of interaction comprising a high and low affinity component. The proportion of sites with high affinity for each antagonist was similar (60-80%). 4. The rank order of antagonist potency for the high affinity component in each displacement curve (RX821002 greater than RX811059 greater than yohimbine) is similar to that determined against the binding of [3H]-RX821002 to rat brain, suggesting that these components reflect the inhibition of [3H]-idazoxan binding to alpha 2-adrenoceptors.The remaining component in each displacement curve exhibiting low affinity towards these antagonists is attributable to the displacement of [3H]-idazoxin from a non-adrenoceptor idazoxan binding site (NAIBS) since a comparable amount of [3H]-idazoxan binding was not displaced by an excess concentration of (-)-adrenaline.5. The displacement of [3H]-idazoxan binding by RX801023 (6-fluoro-(2-(1,4-benzodioxan-2-yl)-2-imidazoline) was also best described by a model assuming a two site interaction with 20.07 +/- 3.11% of the sites labelled displaying high affinity for RX801023. The Ki of RX801023 for the remainder of the sites labelled was similar to its Ki versus [3H]-RX821002, indicating that this drug displays improved affinity and NAIBS/z2-adrenoceptor selectivity compared with idazoxan.6. In autoradiographical studies, the distribution of 5 nM [3H]-idazoxan binding to sections of rat whole brain was consistent with that reported from previous studies and resembled the distribution ofM2-adrenoceptors. However, when sections of brain were coincubated with concentrations of alpha2-adrenoceptor agonists or antagonists predicted to saturate alpha2-adrenoceptors, there remained distinct areas of binding corresponding to discrete brain nuclei. This remaining binding was however displaced by unlabelled idazoxan (3 microM) or RX801023 (3 microM) indicative of the labelling of NAIBS.7. Quantitative autoradiography of NAIBS revealed several brain nuclei which contained higher densities of these sites than alpha2-adrenoceptors, notably the area postrema, interpeduncular nucleus,arcuate nucleus, ependyma and pineal gland.

Characteristics of GABAB receptor binding sites on rat whole brain synaptic membranes.

1 Saturable binding of (+/-)-[3H]-baclofen and [3H]-gamma- aminobutyric acid ([3H]-GABA) to rat brain crude synaptic membranes has been examined by means of a centrifugation assay. 2 The binding of [3H]-baclofen could be detected in fresh or previously frozen tissue and was dependent on the presence of physiological concentrations of Ca2+ or Mg2+ although a lower affinity Na+ -dependent component could also be observed. Both components probably reflect binding to receptor recognition sites. 3 The saturable portion of bound [3H]-baclofen formed 20.3 +/- 6.9% of total bound ligand. This could be displaced by GABA (IC50 = 0.04 microM), (-)-baclofen (0.04 microM) and to a much lesser extent by (+)-baclofen (33 microM). Isoguvacine, piperidine-4-sulphonic acid and bicuculline methobromide were inactive (up to 100 microM) and muscimol was only weakly active (IC50 = 12.3 microM). 4 Saturable binding of [3H]-GABA increased on adding CaCl2 or MgSO4 (up to 2.5 mM and 5.0 mM respectively) to the Tris-HCl incubation solution. This binding (GABAB site binding) was additional to the bicuculline-sensitive binding of GABA (GABAA site binding) and could be completely displaced by (-)-baclofen (IC50 = 0.13 microM). 5 Increasing the Ca2+ concentration (0 to 2.5 mM) increased the binding capacity of the membranes without changing their affinity for the ligand. 6 The binding of [3H]-GABA to GABAB sites could be demonstrated in fresh as well as previously frozen membranes with a doubling of the affinity being produced by freezing. Further incubation with the non-ionic detergent Triton-X-100 (0.05% v/v) reduced the binding capacity by 50%. 7 The pharmacological profile of displacers of [3H]-GABA from GABAB sites correlated well with that for [3H]-baclofen displacement. A correlation with data previously obtained in isolated preparations of rat atria and mouse vas deferens was also apparent. 8 It is concluded that [3H]-baclofen or [3H]-GABA are both ligands for the same bicuculline-insensitive, divalent cation-dependent binding sites in the rat brain.

Behavioural and physiological effects induced by an infusion of antisense to alpha(2D)-adrenoceptors in the rat.

1. The aim of this study was to investigate the behavioural and physiological effects of an i.c.v. infusion of antisense oligonucleotide to the alpha(2D)-adrenoceptor subtype. Behavioural and physiological parameters were monitored for 2 days before the infusion, throughout the 3-day infusion period and for 3 days following the end of the infusion. 2. The antisense infusion resulted in a significant increase in behavioural activity characterized by increased locomotion and grooming scores. Behavioural activity scores of rats treated with antisense to alpha(2D)-adrenoceptors were significantly higher than those of rats treated with vehicle (H(2)O) or the mismatch toxicity control on day 4 and day 5 and, significantly higher than vehicle controls on day 6. 3. Body weight gain was significantly reduced in the antisense-treated rats at the end of the study compared to the vehicle (34%) and mismatch groups (30%), although daily food and water intakes were not significantly different at any time point. 4. Pupil diameters of rats infused with antisense to alpha(2D)-adrenoceptors were significantly greater than those of animals treated either with vehicle or mismatch oligonucleotide on day 5 of the study. On day 6, the pupil diameters of these animals were still significantly greater than the mismatch group. 5. In conclusion, an i.c.v. infusion of antisense to the alpha(2D)-adrenoceptor induced behavioural activation in rats, increased pupil diameter and reduced total weight gain. These effects were specific to the antisense-treated group and were fully reversed post-infusion.

Autoradiographical and behavioural effects of a chronic infusion of antisense to the alpha2D-adrenoceptor in the rat.

1. The aims of this study were, firstly to use receptor autoradiography to investigate the effect of antisense oligonucleotides to the alpha2D-adrenoceptor on receptor binding and, secondly to measure behavioural and physiological parameters to determine whether the chronic antisense infusion had any effect on alpha2-adrenoceptor function in vivo. 2. A 3 day infusion of antisense to the alpha2D-adrenoceptor significantly reduced specific [3H]-RX821002 binding in the septum (20 - 30%) and anterior hypothalamic area (20 - 30%). beta-Adrenoceptor expression was unaffected in those brain areas examined, indicating the antisense knockdown was specific to the alpha2-adrenoceptors. 3. On the second day of the infusion, the hypothermic response to UK 14,304 was significantly attenuated in the antisense-treated group compared with both vehicle and mismatch controls. The effect was fully reversible and a similar decrease in body temperature was observed in all the treatment groups 4 days after the end of infusion. 4. During the second day of the infusion, the effects of UK 14,304 on behaviour were reduced in the antisense-treated rats, but were not significantly lower than those of the vehicle and mismatch, UK 14, 304 controls. These trends were not observed 4 days after the end of the infusion. 5. In conclusion, antisense has been shown to selectively knockdown alpha2-adrenoceptor expression in specific brain areas. The consequence of this knockdown is a significant attenuation of UK 14,304-induced hypothermia and a reduction in its sedative actions. These changes were fully reversed 4 days after the end of the infusion.