- About us
- Scientific meetings
- Grants and funding
- Grant Making Policy
- Meetings grants
- Travel grants
- Departmental seminar scheme
- Outreach grants
- Public engagement grants
- Support for undergraduates
- Benevolent fund
- International grants
- Paton Prize Bursary
- Research grants
- Teaching grants
- Education and resources
- Higher education workshops
- Postgraduate / early career
- Techniques workshops
- Physiology jobs
- In vivo short courses
- Media galleries
- Rob Clarke Awards
- Press centre
- Public engagement
Journal of Physiology
RSS feed -- current issue.
Updated: 15 hours 49 min ago
Hydrogen sulphide (H2S) is a signalling molecule that appears to regulate diverse cell physiological process in several organs and systems including vascular and airway smooth muscle cell (SMC) contraction. Decreases in endogenous H2S synthesis have been associated with the development of cardiovascular diseases and asthma. Here we investigated the mechanism of airway SMC relaxation induced by H2S in small intrapulmonary airways using mouse lung slices and confocal and phase-contrast video microscopy. Exogenous H2S donor Na2S (100 m) reversibly inhibited Ca2+ release and airway contraction evoked by inositol-1,4,5-trisphosphate (InsP3) uncaging in airway SMCs. Similarly, InsP3-evoked Ca2+ release and contraction was inhibited by endogenous H2S precursor l-cysteine (10 mm) but not by l-serine (10 mm) or either amino acid in the presence of dl-propargylglycine (PPG). Consistent with the inhibition of Ca2+ release through InsP3 receptors (InsP3Rs), Na2S reversibly inhibited acetylcholine (ACh)-induced Ca2+ oscillations in airway SMCs. In addition, Na2S, the H2S donor GYY-4137, and l-cysteine caused relaxation of airways pre-contracted with either ACh or 5-hydroxytryptamine (5-HT). Na2S-induced airway relaxation was resistant to a guanylyl cyclase inhibitor (ODQ) and a protein kinase G inhibitor (Rp-8-pCPT-cGMPS). The effects of H2S on InsP3-evoked Ca2+ release and contraction as well as on the relaxation of agonist-contracted airways were mimicked by the thiol-reducing agent dithiothreitol (DTT, 10 mm) and inhibited by the oxidizing agent diamide (30 m). These studies indicate that H2S causes airway SMC relaxation by inhibiting Ca2+ release through InsP3Rs and consequent reduction of agonist-induced Ca2+ oscillations in SMCs. The results suggest a novel role for endogenously produced H2S that involves the modulation of InsP3-evoked Ca2+ release – a cell-signalling system of critical importance for many physiological and pathophysiological processes.
Altered skeletal muscle mitochondrial biogenesis but improved endurance capacity in trained OPA1-deficient mice
The role of OPA1, a GTPase dynamin protein mainly involved in the fusion of inner mitochondrial membranes, has been studied in many cell types, but only a few studies have been conducted on adult differentiated tissues such as cardiac or skeletal muscle cells. Yet OPA1 is highly expressed in these cells, and could play different roles, especially in response to an environmental stress like exercise. Endurance exercise increases energy demand in skeletal muscle and repeated activity induces mitochondrial biogenesis and activation of fusion–fission cycles for the synthesis of new mitochondria. But currently no study has clearly shown a link between mitochondrial dynamics and biogenesis. Using a mouse model of haploinsufficiency for the Opa1 gene (Opa1+/–), we therefore studied the impact of OPA1 deficiency on the adaptation ability of fast skeletal muscles to endurance exercise training. Our results show that, surprisingly, Opa1+/– mice were able to perform the same physical activity as control mice. However, the adaptation strategies of both strains after training differed: while in control mice mitochondrial biogenesis was increased as expected, in Opa1+/– mice this process was blunted. Instead, training in Opa1+/– mice led to an increase in endurance capacity, and a specific adaptive response involving a metabolic remodelling towards enhanced fatty acid utilization. In conclusion, OPA1 appears necessary for the normal adaptive response and mitochondrial biogenesis of skeletal muscle to training. This work opens new perspectives on the role of mitochondrial dynamics in skeletal muscle cells and during adaptation to stress.
Tendon and skeletal muscle matrix gene expression and functional responses to immobilisation and rehabilitation in young males: effect of growth hormone administration
We examined the effect of growth hormone (GH) on connective tissue of tendon and skeletal muscle during immobilisation and re-training in humans. Young men (20–30 years; n = 20) were randomly assigned to daily recombinant human GH (rhGH) (33–50 g kg–1 day–1) or placebo (Plc), and had one leg immobilised for 2 weeks, followed by 6 weeks of strength training. The cross-sectional area (CSA), maximal muscle strength (maximal voluntary contraction, MVC) and biomechanical properties of the quadriceps muscle and patellar tendon were determined. Muscle and tendon biopsies were analysed for mRNA of collagen (COL1A1/3A1), insulin-like growth factors (IGF-1Ea/Ec), lysyl oxidase (LOX), matrix metalloproteases (MMP-2 and MMP-9), decorin and tenascin-C. Fibril morphology was analysed by transmission electron microscopy (TEM) to detect changes in the fibril diameter distribution. In muscle, CSA and MVC declined with immobilisation and recovered with rehabilitation similarly in both groups. Likewise, both groups showed increased IGF-1Ea/Ec and COL1A1/3A1 expression in muscle during re-training after immobilisation compared with baseline, and the increase was more pronounced when subjects received GH. The tendon CSA did not change during immobilisation, but increased in both groups during 6 weeks of rehabilitation (~14%). A decline in tendon stiffness after immobilisation was observed only in the Plc group, and an increase during 6 weeks of rehabilitation was observed only in the GH group. IGF-1Ea and COL1A1/3A1 mRNA increased with immobilisation in the GH group only, and LOX mRNA was higher in the GH group than in the Plc group after immobilisation. Both groups showed an increase in MMP-2 with immobilisation, whereas no changes in MMP-9, decorin and tenascin-C were observed. The tendon fibril diameter distribution remained unchanged in both groups. In conclusion, GH stimulates collagen expression in both skeletal muscle and tendon, abolishes the normal inactivity-related decline in tendon stiffness and LOX, and results in increased tendon CSA and stiffness during rehabilitation. GH has a matrix-stabilising effect during periods of inactivity and rehabilitation in humans.
Endogenous and maximal sarcoplasmic reticulum calcium content and calsequestrin expression in type I and type II human skeletal muscle fibres
The relationship between sarcoplasmic reticulum (SR) Ca2+ content and calsequestrin (CSQ) isoforms was investigated in human skeletal muscle. A fibre-lysing assay was used to quantify the endogenous Ca2+ content and maximal Ca2+ capacity of the SR in skinned segments of type I and type II fibres from vastus lateralis muscles of young healthy adults. Western blotting of individual fibres showed the great majority contained either all fast or all slow isoforms of myosin heavy chain (MHC), troponins C and I, tropomyosin and SERCA, and that the strontium sensitivity of the force response was closely indicative of the troponin C isoform present. The endogenous SR Ca2+ content was slightly lower in type I compared to type II fibres (0.76 ± 0.03 and 0.85 ± 0.02 mmol Ca2+ per litre of fibre, respectively), with virtually all of this Ca2+ evidently being in the SR, as it could be rapidly released with a caffeine-low [Mg2+] solution (only 0.08 ± 0.01 and <0.07 mmol l–1, respectively, remaining). The maximal Ca2+ content that could be reached with SR Ca2+ loading was 1.45 ± 0.04 and 1.79 ± 0.03 mmol l–1 in type I and type II fibres, respectively (P < 0.05). In non-lysed skinned fibres, where the SR remained functional, repeated cycles of caffeine-induced Ca2+ release and subsequent Ca2+ reloading similarly indicated that (i) maximal SR Ca2+ content was lower in type I fibres than in type II fibres (P < 0.05), and (ii) the endogenous Ca2+ content represented a greater percentage of maximal content in type I fibres compared to type II fibres (~59% and 41%, respectively, P < 0.05). Type II fibres were found on average to contain ~3–fold more CSQ1 and ~5–fold less CSQ2 than type I fibres (P < 0.001). The findings are consistent with the SR Ca2+ content characteristics in human type II fibres being primarily determined by the CSQ1 abundance, and in type I fibres by the combined amounts of both CSQ1 and CSQ2.
Respiratory, metabolic and cardiac functions are altered by disinhibition of subregions of the medial prefrontal cortex
The prefrontal cortex (PFC) is referred to as the visceral motor cortex; however, little is known about whether this region influences respiratory or metabolic outflows. The aim of this study was to describe simultaneous changes in respiratory, metabolic and cardiovascular functions evoked by disinhibition of the medial PFC (mPFC) and adjacent lateral septal nucleus (LSN). In urethane-anaesthetized rats, bicuculline methiodide was microinjected (2 mm; GABA-A receptor antagonist) into 90 sites in the mPFC at 0.72–4.00 mm from bregma. Phrenic nerve amplitude and frequency, arterial pressure, heart rate, splanchnic and lumbar sympathetic nerve activities (SNA), expired CO2, and core and brown adipose tissue temperatures were measured. Novel findings included disturbances to respiratory rhythm evoked from all subregions of the mPFC. Injections into the cingulate cortex evoked reductions in central respiratory function exclusively, whereas in ventral sites, particularly the infralimbic region, increases in respiratory drive and frequency, and metabolic and cardiac outflows were evoked. Disinhibition of sites in surrounding regions revealed that the LSN could evoke cardiovascular changes accompanied by distinct oscillations in SNA, as well as increases in respiratory amplitude. We show that activation of neurons within the mPFC and LSN influence respiratory, metabolic and cardiac outflows in a site-dependent manner. This study has implications with respect to the altered PFC neuronal activity seen in stress-related and mental health disorders, and suggests how basic physiological systems may be affected.
Mitochondrial dysfunction and reactive oxygen species (ROS) have been implicated in the aetiology of skeletal muscle insulin resistance, although there is considerable controversy regarding these concepts. Mitochondrial function has been traditionally assessed in the presence of saturating ADP, but ATP turnover and the resultant ADP is thought to limit respiration in vivo. Therefore, we investigated the potential link between submaximal ADP-stimulated respiration rates, ROS generation and skeletal muscle insulin sensitivity in a model of type 2 diabetes mellitus, the ZDF rat. Utilizing permeabilized muscle fibres we observed that submaximal ADP-stimulated respiration rates (250–2000 m ADP) were lower in ZDF rats than in lean controls, which coincided with decreased adenine nucleotide translocase 2 (ANT2) protein content. This decrease in submaximal ADP-stimulated respiration occurred in the absence of a decrease in electron transport chain function. Treating ZDF rats with resveratrol improved skeletal muscle insulin resistance and this was associated with elevated submaximal ADP-stimulated respiration rates as well as an increase in ANT2 protein content. These results coincided with a greater ability of ADP to attenuate mitochondrial ROS emission and an improvement in cellular redox balance. Together, these data suggest that mitochondrial dysfunction is present in skeletal muscle insulin resistance when assessed at submaximal ADP concentrations and that ADP dynamics may influence skeletal muscle insulin sensitivity through alterations in the propensity for mitochondrial ROS emission.
Recent studies have suggested that centrally generated motor commands contribute to the perception of position and movement at the wrist, but not at the elbow. Because the wrist and elbow experiments used different methods, this study was designed to resolve the discrepancy. Two methods were used to test both the elbow and wrist (20 subjects each). For the wrist, subjects sat with their right arm strapped to a device that restricted movement to the wrist. Before each test, voluntary contraction of wrist flexor or extensor muscles controlled for muscle spindle thixotropy. After relaxation, the wrist was moved to a test angle. Position was indicated either with a pointer, or by matching with the contralateral wrist, under two conditions: when the reference wrist was relaxed or when its muscles were contracted isometrically (30% maximum). The elbow experiment used the same design to measure position sense in the passive elbow and with elbow muscles contracting (30% maximum). At the wrist when using a pointer, muscle contraction altered significantly the perceived wrist angle in the direction of contraction by 7 deg [3 deg, 12 deg] (mean [95% confidence interval]) with a flexor contraction and 8 deg [4 deg, 12 deg] with an extensor contraction. Similarly, in the wrist matching task, there was a change of 13 deg [9 deg, 16 deg] with a flexor contraction and 4 deg [1 deg, 8 deg] with an extensor contraction. In contrast, contraction of elbow flexors or extensors did not alter significantly the perceived position of the elbow, compared with rest. The contribution of central commands to position sense differs between the elbow and the wrist.
Modelling genetic reorganization in the mouse spinal cord affecting left-right coordination during locomotion
The spinal neural circuit contains inhibitory (CINi) and excitatory (CINe) commissural interneurons with axons crossing the mid-line. Direction of these axons to the other side of the cord is controlled by axon guidance molecules, such as Netrin-1 and DCC. The cord also contains glutamatergic interneurons, whose axon guidance involves the EphA4 receptor. In EphA4 knockout (KO) and Netrin-1 KO mice, the normal left–right alternating pattern is replaced with a synchronized hopping gait, and the cord of DCC KO mice exhibits uncoordinated left and right oscillations. To investigate the effects of these genetic transformations, we used a computational model of the spinal circuits containing left and right rhythm-generating neuron populations (RGs), each with a subpopulation of EphA4-positive neurons, and CINi and CINe populations mediating mutual inhibition and excitation between the left and right RGs. In the EphA4 KO circuits, half of the EphA4-positive axons crossed the mid-line and excited the contralateral RG neurons. In the Netrin-1 KO model, the number of contralateral CINi projections was significantly reduced, while in the DCC KO model, the numbers of both CINi and CINe connections were reduced. In our simulations, the EphA4 and Netrin-1 KO circuits switched from the left–right alternating pattern to a synchronized hopping pattern, and the DCC KO network exhibited uncoordinated left–right activity. The amplification of inhibitory interactions re-established an alternating pattern in the EphA4 and DCC KO circuits, but not in the Netrin-1 KO network. The model reproduces the genetic transformations and provides insights into the organization of the spinal locomotor network.
Burst generation mediated by cholinergic input in terminal nerve-gonadotrophin releasing hormone neurones of the goldfish
Peptidergic neurones play a pivotal role in the neuromodulation of widespread areas in the nervous system. Generally, it has been accepted that the peptide release from these neurones is regulated by their firing activities. The terminal nerve (TN)-gonadotrophin releasing hormone (GnRH) neurones, which are one of the well-studied peptidergic neurones in vertebrate brains, are characterised by their spontaneous regular pacemaker activities, and GnRH has been suggested to modulate the sensory responsiveness of animals. Although many peptidergic neurones are known to exhibit burst firing activities when they release the peptides, TN-GnRH neurones show spontaneous burst firing activities only infrequently. Thus, it remains to be elucidated whether the TN-GnRH neurones show burst activities and, if so, how the mode switching between the regular pacemaking and bursting modes is regulated in these neurones. In this study, we found that only a single pulse electrical stimulation of the neuropil surrounding the TN-GnRH neurones reproducibly induces transient burst activities in TN-GnRH neurones. Our combined physiological and morphological data suggest that this phenomenon occurs following slow inhibitory postsynaptic potentials mediated by cholinergic terminals surrounding the TN-GnRH neurones. We also found that the activation of muscarinic acetylcholine receptors induces persistent opening of potassium channels, resulting in a long-lasting hyperpolarisation. This long hyperpolarisation induces sustained rebound depolarisation that has been suggested to be generated by a combination of persistent voltage-gated Na+ channels and low-voltage-activated Ca2+ channels. These new findings suggest a novel type of cholinergic regulation of burst activities in peptidergic neurones, which should contribute to the release of neuropeptides.
Activity-dependent downregulation of D-type K+ channel subunit Kv1.2 in rat hippocampal CA3 pyramidal neurons
The intrinsic excitability of neurons plays a critical role in the encoding of memory at Hebbian synapses and in the coupling of synaptic inputs to spike generation. It has not been studied whether somatic firing at a physiologically relevant frequency can induce intrinsic plasticity in hippocampal CA3 pyramidal cells (CA3-PCs). Here, we show that a conditioning train of 20 action potentials (APs) at 10 Hz causes a persistent reduction in the input conductance and an acceleration of the AP onset time in CA3-PCs, but not in CA1-PCs. Induction of such long-term potentiation of intrinsic excitability (LTP-IE) was accompanied by a reduction in the D-type K+ current, and was abolished by the inhibition of endocytosis or protein tyrosine kinase (PTK). Consistently, the CA3-PCs from Kv1.2 knock-out mice displayed no LTP-IE with the same conditioning. Furthermore, the induction of LTP-IE depended on the back-propagating APs (bAPs) and intact distal apical dendrites. These results indicate that LTP-IE is mediated by the internalization of Kv1.2 channels from the distal regions of apical dendrites, which is triggered by bAP-induced dendritic Ca2+ signalling and the consequent activation of PTK.
Comparison of Ca2+ transients and [Ca2+]i in the dendrites and boutons of non-fast-spiking GABAergic hippocampal interneurons using two-photon laser microscopy and high- and low-affinity dyes
Using two-photon laser microscopy, high- and low-affinity dyes and patch clamp electrophysiology, we successfully measured somatic stimulation-evoked Ca2+ transients simultaneously in the dendrites and axonal boutons of the same non-fast-spiking GABAergic interneurons in acute slice preparations obtained from hippocampal area CA1. The advantage of the acute preparation is that both neuronal connections and anatomy are maintained. Calculated as unperturbed values, the amplitudes of Ca2+ transients and changes in [Ca2+]i in response to somatic single or burst stimulation were much higher in boutons (428 nm/AP) than in dendrites (49 nm/AP), leading to the conclusion that the much greater influx of Ca2+ observed in terminals might be due to a higher density of N-type voltage-sensitive Ca2+ channels compared to the L-type channels present in dendrites. Whereas the decay of Ca2+ transients recorded in dendrites was primarily mono-exponential, the decay in boutons was bi-exponential, as indicated by an initial fast phase, followed by a much slower reduction in fluorescence intensity. The extrusion of Ca2+ was much faster in boutons than in dendrites. To avoid saturation effects and the flawed conversion of fluorescence measures of [Ca2+]i, we assessed the limits of [Ca2+] measurements (which ranged between 6 and 82% of the applied dye saturation) when high- and low-affinity dyes were applied at different concentrations. When two APs were delivered at a high frequency (>3 Hz) of stimulation, the low-affinity indicators OGB-6F (KD = 3.0 m) and OGB-5N (KD = 20 m) were able to accurately reflect the changes in F/F produced by the consecutive APs. There was no difference in the endogenous buffer capacity (E), which can shape Ca2+ signals, calculated in dendrites (E = 354) or boutons (E = 458).
Previously, the use of two-photon scanning microscopy to characterize Ca2+ transients at the individual bouton level has been primarily limited to glutamatergic terminals.
No previous study has attempted to record Ca2+ dynamics in individual boutons in response to somatic stimulation or to compare them with the Ca2+ dynamics recorded in the dendrites of the same GABAergic interneurons located in the CA1 area of the rat hippocampus. In addition, the endogenous buffer capacity that affects the decay of transients was also estimated.
This study was limited to interneurons that responded to somatic current stimulation at frequencies <60 Hz and that had cell bodies located in the stratum radiatum.
The amplitudes of the Ca2+ transients and changes in [Ca2+]i that occurred in response to somatic single or burst stimulation were much higher in boutons (428 nm/AP) than in dendrites (49 nm/AP). These data were calculated as unperturbed values, which excluded the modulatory effect of the dye's buffer capacity and the possible failure to reach equilibrium in dye concentrations in dendrites and boutons. Therefore, the higher density of Ca2+ channels expressed in boutons might account for the nine-fold difference in [Ca2+]i observed in boutons and dendrites.
Our results indicate that care should be taken when using high- and low-affinity dyes to measure [Ca2+]i in boutons to avoid saturation and the erroneous calculation of [Ca2+]i.
Stomatin-domain protein interactions with acid-sensing ion channels modulate nociceptor mechanosensitivity
Acid-sensing ion channels (ASICs) and their interaction partners of the stomatin family have all been implicated in sensory transduction. Single gene deletion of asic3, asic2, stomatin, or stoml3 all result in deficits in the mechanosensitivity of distinct cutaneous afferents in the mouse. Here, we generated asic3–/–:stomatin–/–, asic3–/–:stoml3–/– and asic2–/–:stomatin–/– double mutant mice to characterize the functional consequences of stomatin–ASIC protein interactions on sensory afferent mechanosensitivity. The absence of ASIC3 led to a clear increase in mechanosensitivity in rapidly adapting mechanoreceptors (RAMs) and a decrease in the mechanosensitivity in both A- and C-fibre nociceptors. The increased mechanosensitivity of RAMs could be accounted for by a loss of adaptation which could be mimicked by local application of APETx2 a toxin that specifically blocks ASIC3. There is a substantial loss of mechanosensitivity in stoml3–/– mice in which ~35% of the myelinated fibres lack a mechanosensitive receptive field and this phenotype was found to be identical in asic3–/–:stoml3–/– mutant mice. However, A-nociceptors showed much reduced mechanosensitivity in asic3–/–:stoml3–/– mutant mice compared to asic3–/– controls. Interestingly, in asic2–/–:stomatin–/– mutant mice many A-nociceptors completely lost their mechanosensitivity which was not observed in asic2–/– or stomatin–/– mice. Examination of stomatin–/–:stoml3–/– mutant mice indicated that a stomatin/STOML3 interaction is unlikely to account for the greater A-nociceptor deficits in double mutant mice. A key finding from these studies is that the loss of stomatin or STOML3 in asic3–/– or asic2–/– mutant mice markedly exacerbates deficits in the mechanosensitivity of nociceptors without affecting mechanoreceptor function.
Low voltage-activated calcium channels gate transmitter release at the dorsal root ganglion sandwich synapse
A subpopulation of dorsal root ganglion (DRG) neurons are intimately attached in pairs and separated solely by thin satellite glial cell membrane septa. Stimulation of one neuron leads to transglial activation of its pair by a bi-, purinergic/glutamatergic synaptic pathway, a transmission mechanism that we term sandwich synapse (SS) transmission. Release of ATP from the stimulated neuron can be attributed to a classical mechanism involving Ca2+ entry via voltage-gated calcium channels (CaV) but via an unknown channel type. Specific blockers and toxins ruled out CaV1, 2.1 and 2.2. Transmission was, however, blocked by a moderate depolarization (–50 mV) or low-concentration Ni2+ (0.1 mm). Transmission persisted using a voltage pulse to –40 mV from a holding potential of –80 mV, confirming the involvement of a low voltage-activated channel type and limiting the candidate channel type to either CaV3.2 or a subpopulation of inactivation- and Ni2+-sensitive CaV2.3 channels. Resistance of the neuron calcium current and SS transmission to SNX482 argue against the latter. Hence, we conclude that inter-somatic transmission at the DRG SS is gated by CaV3.2 type calcium channels. The use of CaV3 family channels to gate transmission has important implications for the biological function of the DRG SS as information transfer would be predicted to occur not only in response to action potentials but also to sub-threshold membrane voltage oscillations. Thus, the SS synapse may serve as a homeostatic signalling mechanism between select neurons in the DRG and could play a role in abnormal sensation such as neuropathic pain.
A prolonged reduction in central neural respiratory activity elicits a form of plasticity known as inactivity-induced phrenic motor facilitation (iPMF), a ‘rebound' increase in phrenic burst amplitude apparent once respiratory neural activity is restored. iPMF requires atypical protein kinase C (aPKC) activity within spinal segments containing the phrenic motor nucleus to stabilize an early transient increase in phrenic burst amplitude and to form long-lasting iPMF following reduced respiratory neural activity. Upstream signal(s) leading to spinal aPKC activation are unknown. We tested the hypothesis that spinal tumour necrosis factor- (TNF) is necessary for iPMF via an aPKC-dependent mechanism. Anaesthetized, ventilated rats were exposed to a 30 min neural apnoea; upon resumption of respiratory neural activity, a prolonged increase in phrenic burst amplitude (42 ± 9% baseline; P < 0.05) was apparent, indicating long-lasting iPMF. Pretreatment with recombinant human soluble TNF receptor 1 (sTNFR1) in the intrathecal space at the level of the phrenic motor nucleus prior to neural apnoea blocked long-lasting iPMF (2 ± 8% baseline; P > 0.05). Intrathecal TNF without neural apnoea was sufficient to elicit long-lasting phrenic motor facilitation (pMF; 62 ± 7% baseline; P < 0.05). Similar to iPMF, TNF-induced pMF required spinal aPKC activity, as intrathecal delivery of a -pseudosubstrate inhibitory peptide (PKC-PS) 35 min following intrathecal TNF arrested TNF-induced pMF (28 ± 8% baseline; P < 0.05). These data demonstrate that: (1) spinal TNF is necessary for iPMF; and (2) spinal TNF is sufficient to elicit pMF via a similar aPKC-dependent mechanism. These data are consistent with the hypothesis that reduced respiratory neural activity elicits iPMF via a TNF-dependent increase in spinal aPKC activity.
Modulation of the autonomic nervous system and behaviour by acute glial cell Gq protein-coupled receptor activation in vivo
Glial fibrillary acidic protein (GFAP)-expressing cells (GFAP+ glial cells) are the predominant cell type in the central and peripheral nervous systems. Our understanding of the role of GFAP+ glial cells and their signalling systems in vivo is limited due to our inability to manipulate these cells and their receptors in a cell type-specific and non-invasive manner. To circumvent this limitation, we developed a transgenic mouse line (GFAP-hM3Dq mice) that expresses an engineered Gq protein-coupled receptor (Gq-GPCR) known as hM3Dq DREADD (designer receptor exclusively activated by designer drug) selectively in GFAP+ glial cells. The hM3Dq receptor is activated solely by a pharmacologically inert, but bioavailable, ligand (clozapine-N-oxide; CNO), while being non-responsive to endogenous GPCR ligands. In GFAP-hM3Dq mice, CNO administration increased heart rate, blood pressure and saliva formation, as well as decreased body temperature, parameters that are controlled by the autonomic nervous system (ANS). Additionally, changes in activity-related behaviour and motor coordination were observed following CNO administration. Genetically blocking inositol 1,4,5-trisphosphate (IP3)-dependent Ca2+ increases in astrocytes failed to interfere with CNO-mediated changes in ANS function, locomotor activity or motor coordination. Our findings reveal an unexpectedly broad role of GFAP+ glial cells in modulating complex physiology and behaviour in vivo and suggest that these effects are not dependent on IP3-dependent increases in astrocytic Ca2+.
To maintain nutrient homeostasis the central nervous system integrates signals that promote or inhibit eating. The supply of vital amino acids is tuned by adjusting food intake according to its dietary protein content. We hypothesized that this effect is based on the sensing of individual amino acids as a signal to control food intake. Here, we show that food intake was most potently reduced by oral l-arginine (Arg), l-lysine (Lys) and l-glutamic acid (Glu) compared to all other 17 proteogenic amino acids in rats. These three amino acids induced neuronal activity in the area postrema and the nucleus of the solitary tract. Surgical lesion of the area postrema abolished the anorectic response to Arg and Glu, whereas vagal afferent lesion prevented the response to Lys. These three amino acids also provoked gastric distension by differentially altering gastric secretion and/or emptying. Importantly, these peripheral mechanical vagal stimuli were dissociated from the amino acids' effect on food intake. Thus, Arg, Lys and Glu had a selective impact on food processing and intake suggesting them as direct sensory input to assess dietary protein content and quality in vivo. Overall, this study reveals novel amino acid-specific mechanisms for the control of food intake and of gastrointestinal function.
Orexin neurons are indispensable for prostaglandin E2-induced fever and defence against environmental cooling in mice
We recently showed using prepro-orexin knockout (ORX-KO) mice and orexin neuron-ablated (ORX-AB) mice that orexin neurons in the hypothalamus, but not orexin peptides per se, are indispensable for stress-induced thermogenesis. To examine whether orexin neurons are more generally involved in central thermoregulatory mechanisms, we applied other forms of thermogenic perturbations, including brain prostaglandin E2 (PGE2) injections which mimic inflammatory fever and environmental cold exposure, to ORX-KO mice, ORX-AB mice and their wild-type (WT) litter mates. ORX-AB mice, but not ORX-KO mice, exhibited a blunted PGE2-induced fever and intolerance to cold (5°C) exposure, and these findings were similar to the results previously obtained with stress-induced thermogenesis. PGE2-induced shivering was also attenuated in ORX-AB mice. Both mutants responded similarly to environmental heating (39°C). In WT and ORX-KO mice, the administration of PGE2 and cold exposure activated orexin neurons, as revealed by increased levels of expression of c-fos. Injection of retrograde tracer into the medullary raphe nucleus revealed direct and indirect projection from the orexin neurons, of which the latter seemed to be preserved in the ORX-AB mice. In addition, we found that glutamate receptor antagonists (d-(–)-2-amino-5-phosphonopentanoic acid and 6-cyano-7-nitroquinoxaline-2,3-dione) but not orexin receptor antagonists (SB334867 and OX2 29) successfully inhibited PGE2-induced fever in WT mice. These results suggest that orexin neurons are important in general thermogenic processes, and their importance is not restricted to stress-induced thermogenesis. In addition, these results indicate the possible involvement of glutamate in orexin neurons implicated in PGE2-induced fever.
Three-dimensional organization of local excitatory and inhibitory inputs to neurons in laminae III-IV of the spinal dorsal horn
Laser scanning photostimulation was used to map the distribution of the synaptic input zones (sites that give local synaptic inputs) for dorsal horn laminae III–IV neurons, in parasagittal and transverse slices of the rat lumbar spinal cord, and examine how these inputs differed for neurons of different morphologies. All neurons received local excitatory and inhibitory synaptic inputs from within laminae III–IV, while a subset of neurons also received excitatory input from the superficial laminae, especially lamina IIi, as well as the II/III border region. Two anatomical properties were found to be predictive of the dorsoventral position of a neuron's input zone relative to its soma: (1) both excitatory and inhibitory input zones were more dorsal for neurons with longer dorsal dendrites, and (2) excitatory, but not inhibitory, input zones were more dorsal (relative to the soma) for more ventral neurons, with the transition between the dorsal input zones of laminae III–IV neurons and the ventral input zones of lamina II neurons occurring at the II/III border. The observed morphophysiological correlations support the idea that interlaminar connectivity is mediated via translaminar dendritic extensions and that, more generally, local connectivity within the dorsal horn is governed by rules relating the position of a neuron's soma and dendrites to the position of the local presynaptic neurons from which it receives inputs, which are specific to the axis and direction (dorsal vs. ventral), whether the input is excitatory or inhibitory, and the laminar position of the postsynaptic neuron.