Cholera Toxin Beta Subunit Research Articles

Development and validation of a multiplex bead-based immunoassay for the simultaneous detection of fifteen pathogenic biological agents

In this study, a multiplex bead-based capture sandwich immunoassay (MIA) for the simultaneous detection of fifteen plant and bacterial Gram-positive and Gram-negative pathogenic biological agents (PBAs) was developed. This study included the selection of “sandwich” pairs of monoclonal antibodies, the development of an analytical protocol, and an evaluation of the specificity and sensitivity of detection. The working range, linearity, precision, and limits of detection of the developed assay were evaluated. The LLOQ of the multiplex assay was around 104 – 105 CFU/ml for bacteria and 0,4 ng/ml – 6,6 ng/ml for antigens. The assay can be used for the simultaneous detection of 15 PBAs, including Francisella tularensis, Brucella abortus, Bacillus anthracis, Legionella pneumophila, Staphylococcus aureus, Staphylococcus haemolyticus, Pseudomonas aeruginosa, and Salmonella typhimurium, as well as cholera toxin beta-subunit, viscumin, ricin, staphylococcal enterotoxins A and B, and botulinum toxins A and B.

Computational study to investigate Proteus mirabilis proteomes for multi-epitope vaccine construct design

Proteus mirabilis is a gram-negative bacterium particularly known for its unique swarming ability. The swarming gives the bacteria ability to enhance adherence to the catheter surface and epithelium cells of the urethra to cause catheter associated urinary tract infections. P. mirabilis has evolved resistant to antibiotics. Additionally, there is an approved vaccine against P. mirabilis, thus demanding for identification of new vaccine targets. This gram-negative bacterium consists of 19,502 core proteins, out of which 19,063 are redundant proteins and remaining 439 are non-redundant proteins. The non-redundant proteins have 21 proteins present on the cell surface out of which 11 proteins are virulent. Antigenicity analysis predicted only 2 proteins as antigenic (fimbrial biogenesis outer membrane usher protein and ligand-gated channel protein). Four and seven B-cells epitopes were predicted from the former and later proteins, respectively. The predicted B-cells epitopes were used for T- cells epitopes prediction. The predicted epitopes were linked to each other through GPGPG linkers and joined with cholera toxin beta subunit adjuvant. A multi-epitopes vaccine construct consisting of 226 residues was docked with MHC-I, MHC-II and TLR-4. The best docked complex in each case has binding energy of −714.6, −744.6 and −829.5 kcal/mol, respectively. Moreover, the docking results were validated through molecular dynamics simulation and binding free energies estimation. The net energy of −137.2 kcal/mol was calculated for vaccine-MHC-I complex, −133.39 kcal/mol for vaccine-MHC-II and −158.68 kcal/mol for vaccine-TLR-4 complex. The designed vaccine construct could provoke immune responses against targeted pathogen and may be used in experimental testing. Communicated by Ramaswamy H. Sarma

Spinal trigeminal subnucleus pars muralis: An interface for apneic reflex

Noxious stimulation of the nasal cavity elicits protective reflexes for the upper respiratory tract, including sneezing, mucus secretion, apnea and cardiovascular reflexes. Yet, no studies have shown the full circuit responsible for the apneic response upon noxious stimulation of the nasal mucosa. Here, we show that injection of cholera toxin beta subunit into the nasal mucosa leads to terminal labeling in the transition zone between the interpolaris and caudalis subnuclei of the trigeminal sensory complex, i.e. subnucleus muralis (SpVm). We also provide physiological evidence that ammonia vapor delivered into the nasal cavity induces an apneic reaction and activates some SpVm cells. Subnucleus muralis cells also respond to mechanical stimulation of the narial pad and the nasal mucosa. Yet, none of the muralis cells responded to vibrissa deflection. Electrolytic lesion of SpVm or unilateral transection of the anterior ethmoidal nerve prevent apnea induced by ammonia delivery to the nasal epithelium, demonstrating their crucial role in the apneic reflex. We also found direct projections from SpVm to the respiratory centers, particularly the preBötzinger complex (preBötC). Inhibition of inspiratory preBötC cells and increased firing of expiratory preBötC cells are associated with ammonia‐induced apnea.Anatomical and physiological data presented here support the hypothesis that nasal induced apnea may be mediated by direct projections from nasal innervated Subnucleus muralis neurons to the PreBötC complex.

Inhibitory Projections in the Mouse Auditory Tectothalamic System.

The medial geniculate body (MGB) is the target of excitatory and inhibitory inputs from several neural sources. Among these, the inferior colliculus (IC) is an important nucleus in the midbrain that acts as a nexus for auditory projections, ascending and descending, throughout the rest of the central auditory system and provides both excitatory and inhibitory inputs to the MGB. In our study, we assessed the relative contribution from presumed excitatory and inhibitory IC neurons to the MGB in mice. Using retrograde tract tracing with cholera toxin beta subunit (CTβ)-Alexa Fluor 594 injected into the MGB of transgenic, vesicular GABA transporter (VGAT)-Venus mice, we quantitatively analyzed the projections from both the ipsilateral and contralateral IC to the MGB. Our results demonstrate inhibitory projections from both ICs to the MGB that likely play a significant role in shaping auditory processing. These results complement prior studies in other species, which suggest that the inhibitory tectothalamic pathway is important in the regulation of neuronal activity in the auditory forebrain.

Tau accumulation in the retina promotes early neuronal dysfunction and precedes brain pathology in a mouse model of Alzheimer\u2019s disease

BackgroundTau is an axon-enriched protein that binds to and stabilizes microtubules, and hence plays a crucial role in neuronal function. In Alzheimer’s disease (AD), pathological tau accumulation correlates with cognitive decline. Substantial visual deficits are found in individuals affected by AD including a preferential loss of retinal ganglion cells (RGCs), the neurons that convey visual information from the retina to the brain. At present, however, the mechanisms that underlie vision changes in these patients are poorly understood. Here, we asked whether tau plays a role in early retinal pathology and neuronal dysfunction in AD.MethodsAlterations in tau protein and gene expression, phosphorylation, and localization were investigated by western blots, qPCR, and immunohistochemistry in the retina and visual pathways of triple transgenic mice (3xTg) harboring mutations in the genes encoding presenilin 1 (PS1M146 V), amyloid precursor protein (APPSwe), and tau (MAPTP301L). Anterograde axonal transport was assessed by intraocular injection of the cholera toxin beta subunit followed by quantification of tracer accumulation in the contralateral superior colliculus. RGC survival was analyzed on whole-mounted retinas using cell-specific markers. Reduction of tau expression was achieved following intravitreal injection of targeted siRNA.ResultsOur data demonstrate an age-related increase in endogenous retinal tau characterized by epitope-specific hypo- and hyper-phosphorylation in 3xTg mice. Retinal tau accumulation was observed as early as three months of age, prior to the reported onset of behavioral deficits, and preceded tau aggregation in the brain. Intriguingly, tau build up occurred in RGC soma and dendrites, while tau in RGC axons in the optic nerve was depleted. Tau phosphorylation changes and missorting correlated with substantial defects in anterograde axonal transport that preceded RGC death. Importantly, targeted siRNA-mediated knockdown of endogenous tau improved anterograde transport along RGC axons.ConclusionsOur study reveals profound tau pathology in the visual system leading to early retinal neuron damage in a mouse model of AD. Importantly, we show that tau accumulation promotes anterograde axonal transport impairment in vivo, and identify this response as an early feature of neuronal dysfunction that precedes cell death in the AD retina. These findings provide the first proof-of-concept that a global strategy to reduce tau accumulation is beneficial to improve axonal transport and mitigate functional deficits in AD and tauopathies.

The ascending projections of the nuclei of the descending trigeminal tract (nTTD) in the zebra finch (Taeniopygia guttata).

In our traditional view of the avian somatosensory system, input from the beak and head reaches the telencephalon via a disynaptic pathway, involving projections from the principal sensory nucleus (PrV) directly to nucleus basorostralis (previously called nucleus basalis), whereas input from the rest of the body follows a trisynatic pathway similar to that in mammals, involving projections from the dorsal column nuclei to the thalamus, and thence to somatosensory wulst. However, the role of the nuclei of the descending trigeminal tract (nTTD) in this scenario is unclear, partly because their ascending projections have been examined in only one species, the mallard duck. Here we examine the ascending projections of the nTTD in the zebra finch, using in vivo injections of biotinylated dextran amine and verification of projections by means of retrograde transport of the beta subunit of cholera toxin. The results show a high degree of interconnectivity within the nTTD, and that these nuclei project to PrV. We also find a projection from nTTD to the contralateral thalamic nucleus uvaeformis, a multi-sensory nucleus connected to the song system. Furthermore, our finding of a projection from nTTD to the contralateral somatosensory thalamic nucleus dorsalis intermedius ventralis anterior (DIVA) is consistent with the well-known projection in mammals from nTTD to the ventrobasal thalamus, suggesting that the ascending trigeminal pathways in birds and mammals are more similar than previously thought.

Evaluation of the Spectral Response of Functionalized Silk Inverse Opals as Colorimetric Immunosensors.

Regenerated silk fibroin is a high molecular weight protein obtained by purifying the cocoons of the domesticated silkworm, Bombyx mori. This report exploits the aqueous processing and tunable β sheet secondary structure of regenerated silk to produce nanostructures (i.e., inverse opals) that can be used as colorimetric immunosensors. Such sensors would enable direct detection of antigens by changes in reflectance spectra induced by binding events within the nanostructure. Silk inverse opals were prepared by solution casting and annealing in a humidified atmosphere to render the silk insoluble. Next, antigen sensing capabilities were imparted to silk through a three step synthesis: coupling of avidin to silk surfaces, coupling of biotin to antibodies, and lastly antibody attachment to silk through avidin-biotin interactions. Varying the antibody enables detection of different antigens, as demonstrated using different protein antigens: antibodies, red fluorescent protein, and the beta subunit of cholera toxin. Antigen binding to sensors induces a red shift in the opal reflectance spectra, while sensors not exposed to antigen showed either no shift or a slight blue shift. This work constitutes a first step for the design of biopolymer-based optical systems that could directly detect antigens using commercially available reagents and environmentally friendly chemistries.

Golgi fragmentation precedes neuromuscular denervation and is associated with endosome abnormalities in SOD1-ALS mouse motor neurons

BackgroundFragmentation of stacked cisterns of the Golgi apparatus into dispersed smaller elements is a feature associated with degeneration of neurons in amyotrophic lateral sclerosis (ALS) and some other neurodegenerative disorders. However, the role of Golgi fragmentation in motor neuron degeneration is not well understood.ResultsHere we use a SOD1-ALS mouse model (low-copy Gurney G93A-SOD1 mouse) to show that motor neurons with Golgi fragmentation are retrogradely labeled by intramuscularly injected CTB (beta subunit of cholera toxin), indicating that Golgi fragmentation precedes neuromuscular denervation and axon retraction. We further show that Golgi fragmentation may occur in the absence of and precede two other pathological markers, i.e. somatodendritic SOD1 inclusions, and the induction of ATF3 expression. In addition, we show that Golgi fragmentation is associated with an altered dendritic organization of the Golgi apparatus, does not depend on intact apoptotic machinery, and is facilitated in transgenic mice with impaired retrograde dynein-dependent transport (BICD2-N mice). A connection to altered dynein-dependent transport also is suggested by reduced expression of endosomal markers in neurons with Golgi fragmentation, which also occurs in neurons with impaired dynein function.ConclusionsTogether the data indicate that Golgi fragmentation is a very early event in the pathological cascade in ALS that is associated with altered organization of intracellular trafficking.

Astrocyte Reactivity: A Biomarker for Retinal Ganglion Cell Health in Retinal Neurodegeneration.

Retinal ganglion cell (RGC) loss in glaucoma is sectorial in nature and preceded by deficits in axonal transport. Neuroinflammation plays an important role in the pathophysiology of glaucoma in the retina, optic nerve and visual centers of the brain, where it similarly appears to be regulated spatially. In a murine model, we examined the spatial characteristics of astrocyte reactivity (migration/proliferation, hypertrophy and GFAP expression) in healthy retina, retina with two glaucoma-related risk factors (aging and genetic predisposition) and glaucomatous retina and established relationships between these reactivity indices and the spatial organization of astrocytes as well as RGC health. Astrocyte reactivity was quantified by morphological techniques and RGC health was determined by uptake and transport of the neural tracer cholera toxin beta subunit (CTB). We found that: (1) astrocyte reactivity occurs in microdomains throughout glaucomatous retina as well as retina with risk factors for glaucoma, (2) these astrocyte microdomains are primarily differentiated by the degree of retinal area covered by the astrocytes within them and (3) percent retinal area covered by astrocytes is highly predictive of RGC health. Our findings suggest that microdomains of astrocyte reactivity are biomarkers for functional decline of RGCs. Based on current and emerging imaging technologies, diagnostic assessment of astrocytes in the nerve fiber layer could succeed in translating axonal transport deficits to a feasible clinical application.

Pharmacokinetics of GM1 Ganglioside Following Parenteral Administration

The pharmacokinetic parameters of monosialotetrahexosylganglioside (GM1) have been determined in healthy volunteers at 3 dose levels: 100, 200, 300 mg. Each dose was administered to separate groups of 12 volunteers. GM1 levels were determined in plasma, urine, and faeces by a method based on the property of the cholera toxin beta subunit to react specifically with GM1 ganglioside. A non-compartmental model was applied to determine standard pharmacokinetic parameters. The average AUC increased with dose (1002 +/- 121.2, 1306 +/- 146.1, 3155 +/- 121.6 micrograms mL-1 h after 100, 200, 300 mg, respectively). Plasma clearance was less than 3 mL min-1 and the distribution volume was close to the plasma volume (on average between 4.3 and 7.2 L). Mean residence time was about 43 h for all doses. GM1 was not detected in urine, while in faeces the amount of GM1 determined was similar to the baseline values obtained before dosing.

Cloning and expression of a cholera toxin beta subunit in Escherichia coli

Cholera is an acute infection of the intestine caused by theVibrio cholerae bacterium. This bacterium, a member of Vibrionaceae family, is a facultatively anaerobic, Gram-negative, non-spore-forming curved rod, about 1.4–2.6 µm long, and capable of both respiratory and fermentative metabolism. Cholera, characterized by numerous voluminous watery stools, is often accompanied by vomiting (leading to bicarbonate loss). One of the most important virulence factors of cholera is enterotoxin (ctxAB). The cholera toxin beta subunit (CTB) is a pentameric non-toxic portion of V. cholerae toxin, responsible for holotoxin binding to the GM1-ganglioside receptor that is present on most nucleated cells. When conjugated to autoantigens, CTB dramatically increases its tolerogenic potential after oral administration. In the present study, the CTB gene from a hypertoxigenic V. cholerae strain was amplified and cloned. We first amplified the CTB gene with specific primers and the Prime STAR enzyme. The amplified CTB gene was then cloned in pET-28 vector and introduced into Escherichia coli DH5α. pET plasmids containing the CTB gene plasmids were then extracted from the bacteria and used to transform an expression host (BL21). After culturing and induction of positive bacteria, SDS-PAGE and Western blotting for protein determination and verification were performed. We also quantified the level of recombinant CTB by ELISA. Our results show that, due to its rapid growth, an expression host such as E. coli can be useful for production of CTB protein. The ELISA results showed yields of recombinant protein of up to 1 mg/L culture. However, optimal conditions for expression in our study included choice of host strain, temperature used for culture, and concentration of antibiotic and inducer. Beta subunit has many important scientific applications. There is a great deal of interest in the use of CTB as an adjuvant for vaccines targeted for delivery to the mucosa-associated lymphatic tissues. The importance and multifunctional nature of Beta subunit of V. cholerae enterotoxin, e.g., in oral vaccine preparation, will make the use of prokaryotic systems (such as Escherichia coli) for production of this protein most advantageous.

Phenotype and function of raphe projections to the suprachiasmatic nucleus

The circadian clock, located in the suprachiasmatic nucleus (SCN), receives a major afferent from the median raphe nucleus (MRN). In the Syrian hamster, only about 50% of the cells giving rise to this afferent contain serotonin. There is mixed evidence as to whether the serotonergic portion of this projection is involved in non-photic phase shifting of circadian locomotor rhythms. In order to better characterize the non-serotonergic projections, we conducted retrograde tract tracing using the beta subunit of cholera toxin combined with multi-label immunohistochemistry. Similar to previous findings, almost half of the retrogradely labeled cells contained serotonin. Additionally, approximately 30% of the retrogradely labeled cells contained vesicular glutamate transporter 3 (VGLUT3), but not serotonin. Surprisingly, some dorsal raphe cholera toxin labeling was also noted, particularly in animals with central-SCN injections. To determine if the non-serotonergic projections were important for non-photic phase shifts elicited by MRN stimulation, the MRN was electrically stimulated in animals pretreated with SCN injection of either the serotonin neurotoxin 5,7-dihydroxytryptamine or vehicle control. Intact animals phase advanced to midday electrical stimulation of the raphe while lesioned animals did not. Together, these results show that although some of the non-serotonergic raphe projections to the SCN contain VGLUT3, it is the serotonergic raphe innervation of the SCN that is critical for non-photic phase shifting elicited by MRN stimulation.

SOCS3 Deletion Promotes Optic Nerve Regeneration In Vivo

Axon regeneration failure accounts for permanent functional deficits following CNS injury in adult mammals. However, the underlying mechanisms remain elusive. In analyzing axon regeneration in different mutant mouse lines, we discovered that deletion of suppressor of cytokine signaling 3 (SOCS3) in adult retinal ganglion cells (RGCs) promotes robust regeneration of injured optic nerve axons. This regeneration-promoting effect is efficiently blocked in SOCS3-gp130 double-knockout mice, suggesting that SOCS3 deletion promotes axon regeneration via a gp130-dependent pathway. Consistently, a transient upregulation of ciliary neurotrophic factor (CNTF) was observed within the retina following optic nerve injury. Intravitreal application of CNTF further enhances axon regeneration from SOCS3-deleted RGCs. Together, our results suggest that compromised responsiveness to injury-induced growth factors in mature neurons contributes significantly to regeneration failure. Thus, developing strategies to modulate negative signaling regulators may be an efficient strategy of promoting axon regeneration after CNS injury.

Brainstem sites controlling the lower esophageal sphincter and crural diaphragm in the ferret: A neuroanatomical study

The lower esophageal sphincter (LES) and the crural diaphragm (CD) surrounding the esophagogastric junction are key components of the gastroesophageal reflex mechanism, which engages the vago-vagal brainstem circuitry. Although both components work in conjunction to prevent gastroesophageal reflux, little is known about the brain area(s) where this integration takes place. The aims of this study were to: (1) trace the brainstem circuitry associated with the CD and the LES, and (2) determine possible sites of convergence. Experiments were done in adult male ferrets. Under isoflurane anesthesia, recombinant strains of the transneuronal pseudorabies virus (PRV-151 or PRV-Bablu) or the monosynaptic retrograde tracer cholera toxin beta-subunit (CTb) were injected into either the CD or the LES. Following a survival period of 5–7 days, animals were euthanized, perfused and their brains removed for dual-labeling immunofluorescence processing. In animals injected with recombinants of PRV into the CD and the LES, distinct labeling was found in various brainstem nuclei including: area postrema, DMV, nucleus tractus solitarius (NTS), medial reticular formation (MRF) and nucleus ambiguous (NA). Double-labeled cells were only evident in the DMV, NTS and MRF. Injections of CTb into the CD or the LES resulted in retrograde labeling only in the DMV. These findings demonstrate the presence of a direct projection from the DMV to the CD. They further suggest that the neuronal connections responsible for CD or LES function are contained in circuitries that, though largely independent, may converge at the level of DMV, NTS and MRF.

Connections of cat auditory cortex: II. Commissural system.

The commissural projections between 13 areas of cat auditory cortex (AC) were studied using retrograde tracers. Areal and laminar origins were characterized as part of a larger study of thalamic input and cortical origins of projections to each area. Cholera toxin beta subunit (CTbeta) and cholera toxin beta subunit gold-conjugate (CTbetaG) were injected separately within an area or in different areas in an experiment. The areas were identified independently with SMI-32, which revealed differences in neurofilament immunoreactivity in layers III, V, and VI. Each area received convergent AC input from 3 to 6 (mean, 5) contralateral areas. Most of the projections (>75%) were homotopic and from topographically organized loci in the corresponding area. Heterotopic projections (>1 mm beyond the main homotopic projection) constituted approximately 25% of the input. Layers III and V contained >95% of the commissural neurons. Commissural projection neurons were clustered in all areas. Commissural divergence, assessed by double labeling, was less than 3% in each area. This sparse axonal branching is consistent with the essentially homotopic connectivity of the commissural system. The many heterotopic origins represent unexpected commissural influences converging on an area. Areas more dorsal on the cortical convexity have commissural projections originating in layers III and V; more ventral areas favor layer III at the expense of layer V, to its near-total exclusion in some instances. Some areas have almost entirely layer III origins (temporal cortex and area AII), whereas others have a predominantly layer V input (anterior auditory field) or dual contributions from layers III and V (the dorsal auditory zone). A topographic distribution of commissural cells of origin is consistent with the order observed in thalamocortical and corticocortical projections, and which characterizes all extrinsic projection systems (commissural, corticocortical, and thalamocortical) in all AC areas. Thus, laminar as well as areal differences in projection origin distinguish the auditory cortical commissural system.

Activation of afferents to the ventral tegmental area in response to acute amphetamine: a double‐labelling study

The ventral tegmental area (VTA), primary source of the mesocorticolimbic dopaminergic system, is regarded as a critical site for initiation of behavioural sensitization to psychostimulants. The present study was undertaken to identify the neural pathways converging on the VTA that are potentially implicated in this process. Rats were sensitized by a single exposure to amphetamine (5 mg/kg, s.c.). The distribution of VTA-projecting neurons activated by amphetamine was examined by combining retrograde transport of the cholera toxin beta subunit (CTb), injected into the VTA, with immunodetection of Fos. The quantitative analysis of CTb-Fos double labelling demonstrates that amphetamine induced a rapid activation of Fos in a large number of brain areas projecting to the VTA. More than half of the CTb-Fos double-labelled neurons were located in the prefrontal cortex, lateral preoptic area-lateral hypothalamus, pontomesencephalic tegmentum, dorsal raphe nucleus, ventral pallidum and nucleus accumbens. In addition, scattered CTb-Fos double-labelled cells were observed in many other VTA afferent structures, such as claustrum, lateral septum, diagonal band-magnocellular preoptic nucleus, deep mesencephalic nucleus, oral part of pontine reticular nucleus and dorsomedial tegmental area. This suggests that systemic amphetamine activates a wide population of neurons projecting to the VTA that may be important for the modulation of neurobehavioural plasticity produced by this psychostimulant.

Origin of Catecholamine Afferents to Rat Rostral Ventrolateral Medulla (RVLM)

The RVLM plays an important role in the central control of cardiovascular function. Although previous studies have reported that catecholamine neurons in A1, A5, area postrema, and the locus coeruleus innervate rat RVLM, the contributions of each of these cell groups to this innervation are not known. We addressed this question using retrograde tracing and fluorescence immunocytochemistry. Adult male rats were killed seven days after injection of the beta subunit of cholera toxin (CTb) into RVLM and processed for dual localization of CTb and tyrosine hydroxylase (TH). CTb labeled neurons were present in all areas known to project to RVLM, including neurons in the A1 area, the dorsal vagal complex (DVC), the contralateral RVLM, and the A5 area. Dual localization of CTb and TH demonstrated that the commissural projection to the contralateral RVLM arises principally from non-catecholamine neurons. Additionally, labeling in A1, A5, and the DVC demonstrated that RVLM afferents arise from only a subset of catecholamine neurons in each region. Additionally, retrograde labeling of non-catecholaminergic neurons was also observed in A1, A2, and A5 areas. These data suggest functional heterogeneity of the catecholamine cell groups. (RRO18604, HL55786, HL76312)

Alterations in the Brain–Gut Axis Underlying Visceral Chemosensitivity in Nippostrongylus brasiliensis-Infected Mice

Visceral hypersensitivity, a hallmark of irritable bowel syndrome, is generally considered to be mechanosensitive in nature and mediated via spinal afferents. Both stress and inflammation are implicated in visceral hypersensitivity, but the underlying molecular mechanisms of visceral hypersensitivity are unknown. Mice were infected with Nippostrongylus brasiliensis (Nb) larvae, exposed to environmental stress and the following separate studies performed 3-4 weeks later. Mesenteric afferent nerve activity was recorded in response to either ramp balloon distention (60 mm Hg), or to an intraluminal perfusion of hydrochloric acid (50 mmol/L), or to octreotide administration (2 micromol/L). Intraperitoneal injection of cholera toxin B-488 identified neurons projecting to the abdominal viscera. Fluorescent neurons in dorsal root and nodose ganglia were isolated using laser-capture microdissection. RNA was hybridized to Affymetrix Mouse whole genome arrays for analysis to evaluate the effects of stress and infection. In mice previously infected with Nb, there was no change in intestinal afferent mechanosensitivity, but there was an increase in chemosensitive responses to intraluminal hydrochloric acid when compared with control animals. Gene expression profiles in vagal but not spinal visceral sensory neurons were significantly altered in stressed Nb-infected mice. Decreased afferent responses to somatostatin receptor 2 stimulation correlated with lower expression of vagal somatostatin receptor 2 in stressed Nb-infected mice, confirming a link between molecular data and functional sequelae. Alterations in the intestinal brain-gut axis, in chemosensitivity but not mechanosensitivity, and through vagal rather than spinal pathways, are implicated in stress-induced postinflammatory visceral hypersensitivity.

Aldosterone‐sensitive neurons of the nucleus of the solitary tract: Multisynaptic pathway to the nucleus accumbens

The nucleus accumbens (NAc) is part of a forebrain system implicated in reward, motivation, and learning. NAc neurons become activated during various ingestive activities, including salt intake. A subset of neurons within the nucleus tractus solitarius (NTS) shows c-Fos activation during prolonged sodium deprivation in rats. These neurons express mineralocorticoid receptors and the enzyme 11-beta-hydroxysteroid dehydrogenase type 2 (HSD2), which makes them selectively sensitive to aldosterone-an adrenal hormone that modulates sodium appetite. Here we tested whether these neurons project multisynaptically to the core or shell subregions of the NAc. Pseudorabies virus (PRV)-a retrograde transneuronal tracer-was injected into the NAc in rats and after 3-4 days PRV-infected HSD2 neurons were identified. PRV injections into the NAc core yielded greater numbers of PRV-labeled HSD2 neurons than did comparable injections into the NAc shell. Transneuronal labeling was also found in brainstem sites that receive direct projections from HSD2 neurons, namely, lateral parabrachial and prelocus coeruleus nuclei. In other experiments a retrograde neural tracer (cholera toxin beta-subunit) was injected into the NAc. Extensive retrograde labeling was found in the midline thalamus and frontal cortical regions, but no cells were labeled in the NTS or parabrachial region. These findings indicate that the HSD2 neurons project via a multisynaptic pathway to the NAc, which may be relayed sequentially through two sites: the dorsolateral pons and the paraventricular thalamic nucleus. HSD2 neurons may be part of an ascending pathway involved in the salt-seeking behavior of sodium-depleted rats.

Long ascending propriospinal projections from lumbosacral to upper cervical spinal cord in the rat

The retrograde tracer cholera toxin beta-subunit (CTB) was used to trace long ascending propriospinal projections from neurons in the lumbosacral spinal cord to the upper cervical (C3) gray matter in adult male Sprague–Dawley rats. Following large 0.5 μl CTB injections restricted mainly to the upper cervical ventral horn ( n = 5), there were many lumbosacral CTB-positive neurons (14–17/section) in the intermediate gray and ventral horn (dorsal lamina VIII, medial VII extending into X) contralaterally, with fewer at corresponding ipsilateral locations. Labeled cells (4–8/section) were also observed in contralateral laminae IV–VI and the lateral spinal nucleus, with fewer ipsilaterally. Few labeled cells (< 2/section) were observed in superficial laminae I–II. Smaller (0.15 μl) microinjections of CTB restricted to the upper cervical ventral gray matter labeled cells in contralateral laminae VII–VIII (~ 6–9/section) with fewer ipsilaterally. There were relatively fewer (< 2/section) in the intermediate dorsal horn and very few (< 1/section) in lamina I. Larger (0.5 μl) CTB injections encompassing the C3 dorsal and ventral gray matter on one side labeled significantly more CTB-positive neurons (> 6/section) in contralateral lamina I compared to ventral horn injections. These results suggest direct projections from ventromedially located neurons of lumbar and sacral segments to the contralateral ventral gray matter of upper cervical segments, as well as from neurons in the intermediate but not superficial dorsal horn. They further suggest that some lumbosacral superficial dorsal horn neurons project to the upper cervical dorsal horn. These propriospinal projections may be involved in coordinating head and neck movements during locomotion or stimulus-evoked motor responses.