A ganglionic intestinointestinal reflex activated by acute noxious challenge.

To investigate noxious stimulation-responsive neural circuits that could influence the gut, we recorded from intestinally directed (efferent) nerve filaments dissected from mesenteric nerves close to the small intestine in anesthetized rats. These exhibited baseline multiunit activity that was almost unaffected by vagotomy (VagX) and reduced only slightly by cutting the splanchnic nerves. The activity was halved by hexamethonium (Hex) treatment. When an adjacent gut segment received an intraluminal stimulus 2,4,6-trinitrobenzenesulfonate (TNBS) in 30% ethanol, mesenteric efferent nerve activity increased for more than 1 h. The increased activity was almost unaffected by bilateral vagotomy or splanchnic nerve section, indicating a lack of central nervous involvement, but it was 60% reduced by hexamethonium. Spike sorting discriminated efferent single and predominantly single-unit spike trains that responded to TNBS, were unaffected by splachnectomy but were silenced by hexamethonium. After noxious stimulation of one segment, the adjacent segment showed no evidence of suppression of gut motility or vasoconstriction. We conclude that luminal application of a noxious stimulus to the small intestine activates an entirely peripheral, intestinointestinal reflex pathway. This pathway involves enteric intestinofugal neurons that excite postganglionic sympathetic neurons via a nicotinic synapse. We suggest that the final sympathetic efferent neurons that respond to a tissue damaging stimulus are distinct from vasoconstrictor, secretomotor, and motility inhibiting neurons.NEW & NOTEWORTHY An intraluminal noxious chemical stimulus applied to one segment of small intestine increased mesenteric efferent nerve activity to an adjacent segment. This was identified as a peripheral ganglionic reflex that did not require vagal or spinal connections. Hexamethonium blocked most, but not all, ongoing and reflex mesenteric efferent activity. The prevertebral sympathetic efferent neurons that are activated likely affect inflammatory and immune functions of other gut segments.

Characterization of neuromuscular transmission and projections of muscle motor neurons in the rat stomach.

The stomach is the primary reservoir of the gastrointestinal tract, where ingested content is broken down into small particles. Coordinated relaxation and contraction is essential for rhythmic motility and digestion, but how the muscle motor innervation is organized to provide appropriate graded regional control is not established. In this study, we recorded neuromuscular transmission to the circular muscle using intracellular microelectrodes to investigate the spread of the influence of intrinsic motor neurons. In addition, microanatomical investigations of neuronal projections and pharmacological analysis were conducted to investigate neuromuscular relationships. We found that inhibitory neurotransmission to the circular muscle is graded with stimulus strength and circumferential distance from the stimulation site. The influence of inhibitory neurons declined between 1 and 11 mm from the stimulation site. In the antrum, corpus, and fundus, the declines at 11 mm were about 20%, 30%, and 50%, respectively. Stimulation of inhibitory neurons elicited biphasic hyperpolarizing potentials often followed by prolonged depolarizing events in the distal stomach, but only hyperpolarizing events in the proximal stomach. Excitatory neurotransmission influence varied greatly between proximal stomach, where depolarizing events occurred, and distal stomach, where no direct electrical effects in the muscle were observed. Structural studies using microlesion surgeries confirmed a dominant circumferential projection. We conclude that motor neuron influences extend around the gastric circumference, that the effectiveness can be graded by the recruitment of different numbers of motor neuron nerve terminals to finely control gastric motility, and that the ways in which the neurons influence the muscle differ between anatomical regions.NEW & NOTEWORTHY This study provides a detailed mapping of nerve transmission to the circular muscle of the different anatomical regions of rat stomach. It shows that excitatory and inhibitory influences extend around the gastric circumference and that there is a summation of neural influence that allows for finely graded control of muscle tension and length. Nerve-mediated electrical events are qualitatively and quantitatively different between regions, for example, excitatory neurons have direct effects on fundus but not antral muscle.

Morphologies, dimensions and targets of gastric nitric oxide synthase neurons.

We investigated the distributions and targets of nitrergic neurons in the rat stomach, using neuronal nitric oxide synthase (NOS) immunohistochemistry and nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase histochemistry. Nitrergic neurons comprised similar proportions of myenteric neurons, about 30%, in all gastric regions. Small numbers of nitrergic neurons occurred in submucosal ganglia. In total, there were ~ 125,000 neuronal nitric oxide synthase (nNOS) neurons in the stomach. The myenteric cell bodies had single axons, type I morphology and a wide range of sizes. Five targets were identified, the longitudinal, circular and oblique layers of the external muscle, the muscularis mucosae and arteries within the gastric wall. The circular and oblique muscle layers had nitrergic fibres throughout their thickness, while the longitudinal muscle was innervated at its inner surface by fibres of the tertiary plexus, a component of the myenteric plexus. There was a very dense innervation of the pyloric sphincter, adjacent to the duodenum. The muscle strands that run between mucosal glands rarely had closely associated nNOS nerve fibres. Both nNOS immunohistochemistry and NADPH histochemistry showed that nitrergic terminals did not provide baskets of terminals around myenteric neurons. Thus, the nitrergic neuron populations in the stomach supply the muscle layers and intramural arteries, but, unlike in the intestine, gastric interneurons do not express nNOS. The large numbers of nNOS neurons and the density of innervation of the circular muscle and pyloric sphincter suggest that there is a finely graded control of motor function in the stomach by the recruitment of different numbers of inhibitory motor neurons.

Organisation of the musculature of the rat stomach.

The strengths, directions and coupling of the movements of the stomach depend on the organisation of its musculature. Although the rat has been used as a model species to study gastric function, there is no detailed, quantitative study of the arrangement of the gastric muscles in rat. Here we provide a descriptive and quantitative account, and compare it with human gastric anatomy. The rat stomach has three components of the muscularis externa, a longitudinal coat, a circular coat and an internal oblique (sling) muscle in the region of the gastro-oesophageal junction. These layers are similar to human. Unlike human, the rat stomach is also equipped with paired muscular oesophago-pyloric ligaments that lie external to the longitudinal muscle. There is a prominent muscularis mucosae throughout the stomach and strands of smooth muscle occur in the mucosa, between the glands of the corpus and antrum. The striated muscle of the oesophageal wall reaches to the stomach, unlike the human, in which the wall of the distal oesophagus is smooth muscle. Thus, the continuity of gastric and oesophageal smooth muscle bundles, that occurs in human, does not occur in rat. Circular muscle bundles extend around the circumference of the stomach, in the fundus forming a cap of parallel muscle bundles. This arrangement favours co-ordinated circumferential contractions. Small bands of muscle make connections between the circular muscle bundles. This is consistent with a slower conduction of excitation orthogonal to the circular muscle bundles, across the corpus towards the distal antrum. The oblique muscle merged and became continuous with the circular muscle close to the gastro-oesophageal junction at the base of the fundus, and in the corpus, lateral to the lesser curvature. Quantitation of muscle thickness revealed gradients of thickness of both the longitudinal and circular muscle. This anatomical study provides essential data for interpreting gastric movements.

Neuronal regulation of the gut immune system and neuromodulation for treating inflammatory bowel disease.

The gut immune system in the healthy intestine is anti-inflammatory, but can move to a pro-inflammatory state when the gut is challenged by pathogens or in disease. The nervous system influences the level of inflammation through enteric neurons and extrinsic neural connections, particularly vagal and sympathetic innervation of the gastrointestinal tract, each of which exerts anti-inflammatory effects. Within the enteric nervous system (ENS), three neuron types that influence gut immune cells have been identified, intrinsic primary afferent neurons (IPANs), vasoactive intestinal peptide (VIP) neurons that project to the mucosa, and cholinergic neurons that influence macrophages in the external muscle layers. The enteric neuropeptides, calcitonin gene-related peptide (CGRP), tachykinins, and neuromedin U (NMU), which are contained in IPANs, and VIP produced by the mucosa innervating neurons, all influence immune cells, notably innate lymphoid cells (ILCs). ILC2 are stimulated by VIP to release IL-22, which promotes microbial defense and tissue repair. Enteric neurons are innervated by the vagus, and, in the large intestine, by the pelvic nerves. Vagal nerve stimulation reduces gut inflammation, which may be both by stimulation of efferent (motor) pathways to the ENS, and stimulation of afferent pathways that connect to integrating centers in the CNS. Efferent pathways from the CNS have their anti-inflammatory effects through either or both vagal efferent neurons and sympathetic pathways. The final neurons in sympathetic pathways reduce gut inflammation by the action of noradrenaline on ß2 adrenergic receptors expressed by immune cells. Activation of neural anti-inflammatory pathways is an attractive option to treat inflammatory bowel disease that is refractory to other treatments. Further investigation of the ways in which enteric reflexes, vagal pathways and sympathetic pathways integrate their effects to modulate the gut immune system and gut inflammation is needed to optimize neuromodulation therapy.

Autonomic neuromuscular junctions.

This review traces the history of the discovery and subsequent understanding of smooth muscle cells and their motor innervation. Smooth muscle tissue is made up of thousands of very small, individual, electrically connected, muscle cells. Each axon that enters a smooth muscle tissue branches extensively to form a terminal arbour that comes close to hundreds of smooth muscle cells. The branches of the terminal arbour are varicose, and each varicosity, of which there can be thousands, contains numerous transmitter storage vesicles. However, the probability of an individual varicosity releasing transmitter onto the adjacent muscle cells when an action potential passes is low. Many axons influence each muscle cell, some because they release transmitter close to the cell, and some because the events that they cause in other cells are electrically coupled to the cell under investigation. In tissues where this has been assessed, 20 or more axons can influence a single smooth muscle cell. We present a model of the innervation and influence of neurons on smooth muscle.

The identification of neuronal control pathways supplying effector tissues in the stomach.

The stomach acts as a buffer between the ingestion of food and its processing in the small intestine. It signals to the brain to modulate food intake and it in turn regulates the passage of a nutrient-rich fluid, containing partly digested food, into the duodenum. These processes need to be finely controlled, for example to restrict reflux into the esophagus and to transfer digesta to the duodenum at an appropriate rate. Thus, the efferent pathways that control gastric volume, gastric peristalsis and digestive juice production are critically important. We review these pathways with an emphasis on the identities of the final motor neurons and comparisons between species. The major types of motor neurons arising from gastric enteric ganglia are as follows: immunohistochemically distinguishable excitatory and inhibitory muscle motor neurons; four neuron types innervating mucosal effectors (parietal cells, chief cells, gastrin cells and somatostatin cells); and vasodilator neurons. Sympathetic efferent neurons innervate intramural arteries, myenteric ganglia and gastric muscle. Vagal efferent neurons with cell bodies in the brain stem do not directly innervate gastric effector tissues; they are pre-enteric neurons that innervate each type of gastric enteric motor neuron. The principal transmitters and co-transmitters of gastric motor neurons, as well as key immunohistochemical markers, are the same in rat, pig, human and other species.

A new algorithm for drift compensation in multi-unit recordings of action potentials in peripheral autonomic nerves over time.

BACKGROUND: Peripheral autonomic nerves control visceral organs and convey information regarding their functional states and are, therefore, potential targets for new therapeutic and diagnostic approaches. Conventionally recorded multi-unit nerve activity in vivo undergoes slow differential drift of signal and noise amplitudes, making accurate monitoring of nerve activity for more than tens of minutes problematic. NEW METHOD: We describe an on-line drift compensation algorithm that utilizes recursive least-squares to estimate the relative change in spike amplitude due to changes in the nerve-electrode interface over time. RESULTS: We tested and refined our approach using simulated data and in vivo recordings from nerves supplying the small intestine under control conditions and in response to gut inflammation over several hours. The algorithm is robust to changes in recording conditions and signal-to-noise ratio and applicable to both single and multi-unit recordings. In uncompensated records, drift prevented "spike families" and single units from being discriminated accurately over hours. After rescaling, these were successfully tracked throughout recordings (up to 3 h). COMPARISON WITH EXISTING METHODS: Existing methods are subjective or compensate for drift using spatial information and spike shape data which is not practical in multi-unit peripheral nerve recordings. In contrast, this method is objective and applicable to data from a single differential multi-unit recording. In comparisons using simulated data the algorithm performed as well as or better than existing methods. CONCLUSIONS: Results suggest our drift compensation algorithm is widely applicable and robust, though conservative, when differentiating prolonged responses from drift in signal. Extracellular nerve recordings; drift compensation; chronic nerve recordings; closed-loop; multi-unit activity; spike discrimination; recursive least squares; real-time.

Distribution and co-expression patterns of specific cell markers of enteroendocrine cells in pig gastric epithelium.

Although the pig is an accepted model species for human digestive physiology, no previous study of the pig gastric mucosa and gastric enteroendocrine cells has investigated the parallels between pig and human. In this study, we have investigated markers for each of the classes of gastric endocrine cells, gastrin, ghrelin, somatostatin, 5-hydroxytryptamine, histidine decarboxylase, and PYY cells in pig stomach. The lining of the proximal stomach consisted of a collar of stratified squamous epithelium surrounded by gastric cardiac glands in the fundus. This differs considerably from human that has only a narrow band of cardiac glands at its entrance, surrounded by a fundic mucosa consisting of oxyntic glands. However, the linings of the corpus and antrum are similar in pig and human. Likewise, the endocrine cell types are similar and similarly distributed in the two species. As in human, gastrin cells were almost exclusively in the antrum, ghrelin cells were most abundant in the oxyntic mucosa and PYY cells were rare. In the pig, 70% of enterochromaffin-like (ECL) cells in the antrum and 95% in the fundus contained 5-hydroxytryptamine (5-HT), higher proportions than in human. Unlike the enteroendocrine of the small intestine, most gastric enteroendocrine cells (EEC) did not contain colocalised hormones. This is similar to human and other species. We conclude that the pig stomach has substantial similarity to human, except that the pig has a protective lining at its entrance that may reflect the difference between a pig diet with hard abrasive components and the soft foods consumed by humans.

Distributions and relationships of chemically defined enteroendocrine cells in the rat gastric mucosa.

This paper provides quantitative data on the distributions of enteroendocrine cells (EEC), defined by the hormones they contain, patterns of colocalisation between hormones and EEC relations to nerve fibres in the rat gastric mucosa. The rat stomach has three mucosal types: non-glandular stratified squamous epithelium of the fundus and esophageal groove, a region of oxyntic glands in the corpus, and pyloric glands of the antrum and pylorus. Ghrelin and histamine were both contained in closed cells, not contacting the lumen, and were most numerous in the corpus. Gastrin cells were confined to the antrum, and 5-hydroxytryptamine (5-HT) and somatostatin cells were more frequent in the antrum than the corpus. Most somatostatin cells had basal processes that in the antrum commonly contacted gastrin cells. Peptide YY (PYY) cells were rare and mainly in the antrum. The only numerous colocalisations were 5-HT and histamine, PYY and gastrin and gastrin and histamine in the antrum, but each of these populations was small. Peptide-containing nerve fibres were found in the mucosa. One of the most common types was vasoactive intestinal peptide (VIP) fibres. High-resolution analysis showed that ghrelin cells were closely and selectively approached by VIP fibres. In contrast, gastrin cells were not selectively innervated by VIP or CGRP fibres. The study indicates that there are distinct populations of gastric EEC and selective innervation of ghrelin cells. It also shows that, in contrast to EEC of the small intestine, the majority of EEC within the stomach contained only a single hormone.

Central Administration of Insulin Combined With Resistin Reduces Renal Sympathetic Nerve Activity in Rats Fed a High Fat Diet.

Insulin receptors are widely distributed in the central nervous system and their activation by insulin elicits renal sympatho-excitatory effects. Resistin, an adipokine, promotes resistance to the metabolic effects of insulin. Resistin also induces increases in renal sympathetic nerve activity (RSNA) by acting in the brain, but whether it can influence insulin's actions on RSNA is unknown. In the present study we investigated, in male Sprague-Dawley rats (7-8 weeks of age), the effects of central administration of insulin combined with resistin on RSNA following a normal diet (ND) and a high fat diet (HFD) (22% fat), since HFD can reportedly attenuate insulin's actions. RSNA, mean arterial pressure (MAP) and heart rate (HR) responses were monitored and recorded before and for 180 min after intracerebroventricular injection of saline (control) (n = 5 HFD and ND), resistin (7 µg; n = 4 ND, n = 5 HFD), insulin (500 mU; n = 6 ND, n = 5 HFD), and the combination of both resistin and insulin (n = 7 ND, n = 5 HFD). The key finding of the present study was that when resistin and insulin were combined there was no increase in RSNA induced in rats fed a normal diet or the high fat diet. This contrasted with the sympatho-excitatory RSNA effects of the hormones when each was administered alone in rats fed the ND and the HFD.

Ginsenosides Act As Positive Modulators of P2X4 Receptors.

We investigated the selectivity of protopanaxadiol ginsenosides from Panax ginseng acting as positive allosteric modulators on P2X receptors. ATP-induced responses were measured in stable cell lines overexpressing human P2X4 using a YOPRO-1 dye uptake assay, intracellular calcium measurements, and whole-cell patch-clamp recordings. Ginsenosides CK and Rd were demonstrated to enhance ATP responses at P2X4 by ∼twofold, similar to potentiation by the known positive modulator ivermectin. Investigations into the role of P2X4 in mediating a cytotoxic effect showed that only P2X7 expression in HEK-293 cells induces cell death in response to high concentrations of ATP, and that ginsenosides can enhance this process. Generation of a P2X7-deficient clone of BV-2 microglial cells using CRISPR/Cas9 gene editing enabled an investigation of endogenous P2X4 in a microglial cell line. Compared with parental BV-2 cells, P2X7-deficient BV-2 cells showed minor potentiation of ATP responses by ginsenosides, and insensitivity to ATP- or ATP+ ginsenoside-induced cell death, indicating a primary role for P2X7 receptors in both of these effects. Computational docking to a homology model of human P2X4, based on the open state of zfP2X4, yielded evidence of a putative ginsenoside binding site in P2X4 in the central vestibule region of the large ectodomain.

Relationships of endocrine cells to each other and to other cell types in the human gastric fundus and corpus.

Gastric endocrine cell hormones contribute to the control of the stomach and to signalling to the brain. In other gut regions, enteroendocrine cells (EECs) exhibit extensive patterns of colocalisation of hormones. In the current study, we characterise EECs in the human gastric fundus and corpus. We utilise immunohistochemistry to investigate EECs with antibodies to ghrelin, serotonin (5-HT), somatostatin, peptide YY (PYY), glucagon-like peptide 1, calbindin, gastrin and pancreastatin, the latter as a marker of enterochromaffin-like (ECL) cells. EECs were mainly located in regions of the gastric glands populated by parietal cells. Gastrin cells were absent and PYY cells were very rare. Except for about 25% of 5-HT cells being a subpopulation of ECL cells marked by pancreastatin, colocalisation of hormones in gastric EECs was infrequent. Ghrelin cells were distributed throughout the fundus and corpus; most were basally located in the glands, often very close to parietal cells and were closed cells i.e., not in contact with the lumen. A small proportion had long processes located close to the base of the mucosal epithelium. The 5-HT cells were of at least three types: small, round, closed cells; cells with multiple, often very long, processes; and a subgroup of ECL cells. Processes were in contact with their surrounding cells, including parietal cells. Mast cells had very weak or no 5-HT immunoreactivity. Somatostatin cells were a closed type with long processes. In conclusion, four major chemically defined EEC types occurred in the human oxyntic mucosa. Within each group were cells with distinct morphologies and relationships to other mucosal cells.

Bioelectric neuromodulation for gastrointestinal disorders: effectiveness and mechanisms.

The gastrointestinal tract has extensive, surgically accessible nerve connections with the central nervous system. This provides the opportunity to exploit rapidly advancing methods of nerve stimulation to treat gastrointestinal disorders. Bioelectric neuromodulation technology has considerably advanced in the past decade, but sacral nerve stimulation for faecal incontinence currently remains the only neuromodulation protocol in general use for a gastrointestinal disorder. Treatment of other conditions, such as IBD, obesity, nausea and gastroparesis, has had variable success. That nerves modulate inflammation in the intestine is well established, but the anti-inflammatory effects of vagal nerve stimulation have only recently been discovered, and positive effects of this approach were seen in only some patients with Crohn's disease in a single trial. Pulses of high-frequency current applied to the vagus nerve have been used to block signalling from the stomach to the brain to reduce appetite with variable outcomes. Bioelectric neuromodulation has also been investigated for postoperative ileus, gastroparesis symptoms and constipation in animal models and some clinical trials. The clinical success of this bioelectric neuromodulation therapy might be enhanced through better knowledge of the targeted nerve pathways and their physiological and pathophysiological roles, optimizing stimulation protocols and determining which patients benefit most from this therapy.

High Fat Diet Decreases Neuronal Activation in the Brain Induced by Resistin and Leptin.

Resistin and leptin are adipokines which act in the brain to regulate metabolic and cardiovascular functions which in some instances are similar, suggesting activation of some common brain pathways. High-fat feeding can reduce the number of activated neurons observed following the central administration of leptin in animals, but the effects on resistin are unknown. The present work compared the distribution of neurons in the brain that are activated by centrally administered resistin, or leptin alone, and, in combination, in rats fed a high fat (HFD) compared to a normal chow diet (ND). Immunohistochemistry for the protein, Fos, was used as a marker of activated neurons. The key findings are (i) following resistin or leptin, either alone or combined, in rats fed the HFD, there were no significant increases in the number of activated neurons in the paraventricular and arcuate nuclei, and in the lateral hypothalamic area (LHA). This contrasted with observations in rats fed a normal chow diet; (ii) in the OVLT and MnPO of HFD rats there were significantly less activated neurons compared to ND following the combined administration of resistin and leptin; (iii) In the PAG, RVMM, and NTS of HFD rats there were significantly less activated neurons compared to ND following resistin. The results suggest that the sensitivity to resistin in the brain was reduced in rats fed a HFD. This has similarities with leptin but there were instances where there was reduced sensitivity to resistin with no significant effects following leptin. This suggests diet influences neuronal effects of resistin.

Store-Operated Ca2+ Entry (SOCE) and Purinergic Receptor-Mediated Ca2+ Homeostasis in Murine bv2 Microglia Cells: Early Cellular Responses to ATP-Mediated Microglia Activation.

Microglia activation is a neuroinflammatory response to parenchymal damage with release of intracellular metabolites, e.g., purines, and signaling molecules from damaged cells. Extracellular purines can elicit Ca2+-mediated microglia activation involving P2X/P2Y receptors with metabotropic (P2Y) and ionotropic (P2X) cell signaling in target cells. Such microglia activation results in increased phagocytic activity, activation of their inflammasome and release of cytokines to sustain neuroinflammatory (so-called M1/M2 polarization). ATP-induced activation of ionotropic P2X4 and P2X7 receptors differentially induces receptor-operated Ca2+ entry (ROCE). Although store-operated Ca2+ entry (SOCE) was identified to modulate ROCE in primary microglia, its existence and role in one of the most common murine microglia cell line, BV2, is unknown. To dissect SOCE from ROCE in BV2 cells, we applied high-resolution multiphoton Ca2+ imaging. After depleting internal Ca2+ stores, SOCE was clearly detectable. High ATP concentrations (1 mM) elicited sustained increases in intracellular [Ca2+]i whereas lower concentrations (≤100 µM) also induced Ca2+ oscillations. These differential responses were assigned to P2X7 and P2X4 activation, respectively. Pharmacologically inhibiting P2Y and P2X responses did not affect SOCE, and in fact, P2Y-responses were barely detectable in BV2 cells. STIM1S content was significantly upregulated by 1 mM ATP. As P2X-mediated Ca2+ oscillations were rare events in single cells, we implemented a high-content screening approach that allows to record Ca2+ signal patterns from a large number of individual cells at lower optical resolution. Using automated classifier analysis, several drugs (minocycline, U73122, U73343, wortmannin, LY294002, AZ10606120) were tested on their profile to act on Ca2+ oscillations (P2X4) and sustained [Ca2+]i increases. We demonstrate specific drug effects on purinergic Ca2+ pathways and provide new pharmacological insights into Ca2+ oscillations in BV2 cells. For example, minocycline inhibits both P2X7- and P2X4-mediated Ca2+-responses, and this may explain its anti-inflammatory action in neuroinflammatory disease. As a technical result, our novel automated bio-screening approach provides a biomedical engineering platform to allow high-content drug library screens to study neuro-inflammation in vitro.

Central leptin and resistin combined elicit enhanced central effects on renal sympathetic nerve activity.

NEW FINDINGS: What is the central question of this study? Leptin and resistin act centrally to increase renal sympathetic nerve activity (RSNA). We investigated whether a combination of resistin and leptin could induce a greater response than either alone. We also used Fos protein to quantify the number of activated neurons in the brain. What is the main finding and its importance? A combination of leptin and resistin induced a greater increase in RSNA than either hormone alone. This was correlated with a greater number of activated neurons in the arcuate nucleus than with either hormone alone. Leptin and resistin act centrally to increase renal sympathetic nerve activity (RSNA). We investigated whether a combination of resistin and leptin could induce a greater response than either alone. Mean arterial pressure, heart rate and RSNA were recorded before and for 3 h after intracerebroventricular saline (control; n = 5), leptin (7 µg; n = 5), resistin (7 µg; n = 4) and leptin administered 15 min after resistin (n = 6). Leptin alone and resistin alone significantly increased RSNA (74 ± 17 and 50 ± 14%, respectively; P < 0.0001 compared with saline). When leptin and resistin were combined, there was a significantly greater increase in RSNA (163 ± 23%) compared with either hormone alone (P < 0.0001). Maximal responses of mean arterial pressure and heart rate were not significantly different between groups. We also used Fos protein to quantify the number of activated neurons in the brain. Compared with controls, there were significant increases in numbers of Fos-positive neurons in the arcuate and hypothalamic paraventricular nuclei when leptin or resistin was administered alone or when they were combined, and in the lamina terminalis when leptin and resistin were combined. Only in the arcuate nucleus was the increase significantly greater compared with either hormone alone. The findings show that a combination of leptin and resistin induces a greater RSNA increase and a greater number of activated neurons in the arcuate nucleus than with either hormone alone. Given that leptin makes an important contribution to the elevated RSNA observed in obese and overweight conditions, the increased concentrations of leptin and resistin may mean that the contribution of leptin to the elevated RSNA in those conditions is enhanced.

Central Administration of Insulin and Leptin Together Enhance Renal Sympathetic Nerve Activity and Fos Production in the Arcuate Nucleus.

There is considerable interest in the central actions of insulin and leptin. Both induce sympatho-excitation. This study (i) investigated whether centrally administered leptin and insulin together elicits greater increases in renal sympathetic nerve activity (RSNA), mean arterial pressure (MAP) and heart rate (HR) than when given alone, and (ii) quantified the number of activated neurons in brain regions influencing SNA, to identify potential central sites of interaction. In anesthetised (urethane 1.4-1.6 g/kg iv) male Sprague-Dawley rats, RSNA, MAP, and HR were recorded following intracerebroventricular (ICV) saline (control; n = 5), leptin (7 µg; n = 5), insulin (500 mU; n = 4) and the combination of leptin and insulin; (n = 4). Following leptin or insulin alone, RSNA was significantly increased (74 and 62% respectively). MAP responses were not significantly different between the groups. Insulin alone significantly increased HR. Leptin alone also increased HR but it was significantly less than following insulin alone (P < 0.005). When leptin and insulin were combined, the RSNA increase (124%) was significantly greater than the response to either alone. There were no differences between the groups in MAP responses, however, the increase in HR induced by insulin was attenuated by leptin. Of the brain regions examined, only in the arcuate nucleus did leptin and insulin together increase the number of Fos-positive cell nuclei significantly more than leptin or insulin alone. In the lamina terminalis and rostroventrolateral medulla, leptin and insulin together increased Fos, but the effect was not greater than leptin alone. The results suggest that when central leptin and insulin levels are elevated, the sympatho-excitatory response in RSNA will be greater. The arcuate nucleus may be a common site of cardiovascular integration.

Resistin, an Adipokine with Non-Generalized Actions on Sympathetic Nerve Activity.

The World Health Organization has called obesity a global epidemic. There is a strong association between body weight gain and blood pressure. A major determinant of blood pressure is the level of activity in sympathetic nerves innervating cardiovascular organs. A characteristic of obesity, in both humans and in animal models, is an increase in sympathetic nerve activity to the skeletal muscle vasculature and to the kidneys. Obesity is now recognized as a chronic, low level inflammatory condition, and pro-inflammatory cytokines are elevated including those produced by adipose tissue. The most well-known adipokine released from fat tissue is leptin. The adipokine, resistin, is also released from adipose tissue. Resistin can act in the central nervous system to influence the sympathetic nerve activity. Here, we review the effects of resistin on sympathetic nerve activity and compare them with leptin. We build an argument that resistin and leptin may have complex interactions. Firstly, they may augment each other as both are excitatory on sympathetic nerves innervating cardiovascular organs; In contrast, they could antagonize each other's actions on brown adipose tissue, a key metabolic organ. These interactions may be important in conditions in which leptin and resistin are elevated, such as in obesity.

Development of non-viral vehicles for targeted gene transfer into microglia via the integrin receptor CD11b.

Microglial activation is a central event in neurodegeneration. Novel technologies are sought for that specifically manipulate microglial function in order to delineate their role in onset and progression of neuropathologies. We investigated for the first time whether non-viral gene delivery based on polyethyleneglycol-polyethyleneimine conjugated to the monoclonal anti-CD11b antibody OX42 ("OX42-immunogene") could be used to specifically target microglia. We first conducted immunofluorescence studies with the OX42 antibody and identified its microglial integrin receptor CD11b as a potential target for receptor-mediated gene transfer based on its cellular specificity in mixed glia culture and in vivo and found that the OX42 antibody is rapidly internalized and trafficked to acidic organelles in absence of activation of the respiratory burst. We then performed transfection experiments with the OX42-immunogene in vitro and in rat brain showing that the OX42-immunogene although internalized was degraded intracellularly and did not cause substantial gene expression in microglia. Investigation of specific barriers to microglial gene transfer revealed that aggregated OX42-immunogene polyplexes stimulated the respiratory burst that likely involved Fcγ-receptors. Transfections in the presence of the endosomolytic agent chloroquine improved transfection efficiency indicating that endosomal escape may be limited. This study identifies CD11b as an entry point for antibody-mediated gene transfer into microglia and takes important steps toward the further development of OX42-immunogenes.