Comparison of Barrett Toric Calculations Using Measured and Predicted Posterior Corneal Astigmatism in Cataract Surgery Patients.

Aim: To compare residual astigmatism prediction errors across Barrett toric calculations using predicted posterior corneal astigmatism (PCA) and PCA measured using the IOL Master 700 with total keratometry (IOLM). Methods: A retrospective cohort study was undertaken on patients with corneal astigmatism and no other ocular comorbidities that underwent uneventful refractive femtosecond laser-assisted cataract surgery with toric IOL implantation between May 2019 and November 2019. Toric calculations were performed using the Barrett toric calculator and the following values: predicted PCA with anterior corneal measurements from Pentacam, IOLM standard keratometry (SK), OPD scan, and median measurements from these devices; predicted PCA with IOLM total keratometry (TK); and measured PCA with IOLM SK or IOLM TK. Residual astigmatism prediction error was calculated for each device and method of calculation at postoperative month 1 and 3 using the astigmatism double angle plot tool. Results: A total of 24 eyes, 10 with-the-rule (WTR), 10 against-the-rule (ATR) and 4 oblique astigmatism, from 24 patients were included in this study. PCA ranged from 0.00 to 0.67 D with a mean of 0.24 ± 0.15 D in all eyes. PCA was significantly greater in WTR eyes (0.32 D) compared to ATR eyes (0.16 D; p < 0.05). In ATR eyes, calculations made using IOLM SK and measured PCA had significantly lower total corneal astigmatism and toric IOL cylinder power compared to calculations made using Pentacam and IOLM TK (p < 0.05). No significant difference in mean absolute or centroid residual astigmatism prediction error was observed across devices or calculation methods. The percentage of eyes with absolute astigmatism prediction errors ≤0.5 D was not significantly different across groups. Conclusion: Barrett toric calculations using predicted PCA and PCA measured using IOLM produced comparable residual astigmatism prediction errors. The incorporation of median measurements did not significantly impact calculation accuracy.

VENOUS STASIS RETINOPATHY SECONDARY TO MYELIN OLIGODENDROCYTE GLYCOPROTEIN ANTIBODY-POSITIVE OPTIC NEURITIS.

PURPOSE: To report a case of a 38-year-old woman with venous stasis retinopathy secondary to myelin oligodendrocyte glycoprotein-IgG optic neuritis. METHODS: Observational case report. RESULTS: We report a unique case of venous stasis retinopathy secondary to myelin oligodendrocyte glycoprotein-IgG optic neuritis with significant optic disc edema, tortuous and dilated retinal venules, and retinal hemorrhages, which resolved promptly with high-dose corticosteroids. The retinal changes were likely secondary to severe inflammation of the optic nerve and optic nerve sheath, which exhibited significant postcontrast enhancement on magnetic resonance imaging. Despite aggressive treatment with high-dose corticosteroids and plasmapheresis, the patient had a significant generalized visual field defect at 6 months. CONCLUSION: Venous stasis retinopathy may be secondary to myelin oligodendrocyte glycoprotein-IgG optic neuritis due to reduced venous outflow from significant optic nerve edema. This may be a poor prognostic factor and a marker for more severe optic nerve inflammation.

Incremental effect of topical and oral moxifloxacin administration with surgical intracameral prophylaxis.

OBJECTIVE: To determine how supplemental perioperative topical or oral moxifloxacin administration impacts anterior chamber (AC) antibiotic concentrations beyond those achieved by intracameral (IC) administration alone for postoperative endophthalmitis (POE) prophylaxis. DESIGN: Mathematical modeling. METHODS: The mathematical model developed by Arshinoff, Modabber, and Felfeli was adapted to calculate all reported data. A literature review of pharmacokinetic data for topical and oral moxifloxacin was used to inform the expansion of the model. RESULTS: Our previously constructed IC model yields a dose of moxifloxacin in the AC sufficient to confer bactericidal coverage against the most common POE pathogen, methicillin-sensitive Staphylococcus aureus (MSSA), for ∼40 hours postoperatively. Topical 0.5% moxifloxacin eye drops alone, administered every 4 or 6 hours, achieve an AC concentration just above or at the mutant prevention concentration (MPC) for MSSA, respectively, whereas 8-hour dosing produces levels generally below the MPC. Combining topical moxifloxacin with IC increases the AC concentration above IC alone only after 20 or more hours and maintains the AC concentration at, or just below, the MPC for MSSA for as long as the drops are continued. Combined perioperative oral moxifloxacin with IC increases AC levels over IC alone only after 16 hours and maintains the AC concentration above the MPC for MSSA for an additional 5 hours, owing to the systemic reservoir. CONCLUSIONS: The addition of topical or oral moxifloxacin supplemental to IC can extend the duration of bactericidal coverage for the most common, but not the most resistant POE-causing pathogens.

Pannexin-1 Deficiency Decreases Epileptic Activity in Mice.

OBJECTIVE: Pannexin-1 (Panx1) is suspected of having a critical role in modulating neuronal excitability and acute neurological insults. Herein, we assess the changes in behavioral and electrophysiological markers of excitability associated with Panx1 via three distinct models of epilepsy. Methods Control and Panx1 knockout C57Bl/6 mice of both sexes were monitored for their behavioral and electrographic responses to seizure-generating stimuli in three epilepsy models-(1) systemic injection of pentylenetetrazol, (2) acute electrical kindling of the hippocampus and (3) neocortical slice exposure to 4-aminopyridine. Phase-amplitude cross-frequency coupling was used to assess changes in an epileptogenic state resulting from Panx1 deletion. RESULTS: Seizure activity was suppressed in Panx1 knockouts and by application of Panx1 channel blockers, Brilliant Blue-FCF and probenecid, across all epilepsy models. In response to pentylenetetrazol, WT mice spent a greater proportion of time experiencing severe (stage 6) seizures as compared to Panx1-deficient mice. Following electrical stimulation of the hippocampal CA3 region, Panx1 knockouts had significantly shorter evoked afterdischarges and were resistant to kindling. In response to 4-aminopyridine, neocortical field recordings in slices of Panx1 knockout mice showed reduced instances of electrographic seizure-like events. Cross-frequency coupling analysis of these field potentials highlighted a reduced coupling of excitatory delta-gamma and delta-HF rhythms in the Panx1 knockout. SIGNIFICANCE: These results suggest that Panx1 plays a pivotal role in maintaining neuronal hyperexcitability in epilepsy models and that genetic or pharmacological targeting of Panx1 has anti-convulsant effects.

Homonymous hemi-macular atrophy of the ganglion cell-inner plexiform layer with preserved visual function.

Homonymous hemimacular thinning of the retinal ganglion cell-inner plexiform layer (GCIPL) on optical coherence tomography (OCT) in the absence of significant visual field defects may be identified in the workup of patients with visual complaints, but the causes of this finding remain unknown. We retrospectively reviewed 1425 consecutive patients referred for neuro-ophthalmic assessment who had high quality OCT scans and reliable Humphrey 24-2 SITA-Fast testing. A total of 7 patients, 3 females and 4 males, with a mean age of 39.4 ± 10.5 years that had homonymous thinning of the OCT macular-GCIPL without significant visual field defects were included in the study. Four patients had demyelinating disease and 3 patients had traumatic brain injury. Three patients with demyelinating disease had a documented prior homonymous visual field defect that resolved. The differential diagnosis of homonymous hemimacular thinning of the GCIPL without obvious visual field defects includes previous retrochiasmal demyelination and traumatic brain injury. OCT GCIPL provides a permanent objective way of documenting previous retrochiasmal disease including demyelination and may be helpful in establishing dissemination in time and space in patients being evaluated for multiple sclerosis.

Spontaneous Resolution of Dorsal Midbrain Syndrome Caused by a Pineal Cyst.

BACKGROUND: Pineal lesions are common causes of dorsal midbrain syndrome and typically require surgical intervention in symptomatic patients. We describe a unique case of spontaneous resolution of dorsal midbrain syndrome resulting from a pineal gland cyst. CASE DESCRIPTION: A 23-year-old woman developed a supranuclear upgaze palsy, convergence-retraction nystagmus, and light-near dissociation from a pineal gland cyst (1.0 × 1.3 × 1.2 cm) with mild mass effect on the posterior surface of the tectum. Seven days after symptom onset, she had complete, spontaneous resolution of her symptoms, and examination returned to normal. Repeat magnetic resonance imaging demonstrated an unchanged pineal cyst with new T2/fluid attenuated inversion recovery hyperintensity along the mesial surface of the left thalamus. CONCLUSIONS: Dorsal midbrain syndrome resulting from a pineal cyst may spontaneously improve even without a significant change in lesion size. This suggests that observation may be an appropriate initial management strategy.

Chronic chiasmal compression and persistent visual field defect without detectable changes in optical coherence tomography of the macular ganglion cell complex.

PURPOSE: Optical coherence tomography (OCT) of the retinal nerve fiber layer (RNFL) and macular ganglion cell complex (GCC) are important in the ophthalmological evaluation of patients with sellar masses. Changes in OCT of the RNFL and macular GCC often precede visual field changes in patients with chronic chiasmal compression. OCT of the macular GCC has been shown to have better correlation with visual function and allow for even earlier detection of compression of the anterior visual pathways. We present a case of a chronic visual field defect from a pituitary adenoma with largely normal OCT parameters and only subtle changes in OCT of the RNFL and no perceptible changes in OCT of the macular GCC. OBSERVATIONS: A 32-year-old man presented with a four-month history of decreased vision in his left eye and was found to have a monocular temporal visual field defect from a pituitary adenoma. OCT of the RNFL showed only a subtle change in that the nasal quadrant was mildly reduced and the optic nerve did not follow the ISNT rule. There was no asymmetry, deviation from normal parameters or differences in the nasal and temporal sextants on OCT of the macular GCC. This remained stable after testing two months later and a worsening visual field defect. He was found to have an elevated prolactinoma and after initiation of cabergoline, his visual field defect rapidly resolved within a few days. CONCLUSIONS AND IMPORTANCE: OCT RNFL and macular GCC may have parameters in the normal range in patients with chronic chiasmal compression, emphasizing the importance of both anatomical and psychophysical testing. OCT of the RNFL may show these changes earlier than OCT macular GCC and both should be performed for the pre-treatment evaluation of patients with sellar masses. Preserved RNFL and macular GCC thickness confer a good prognosis as demonstrated in this case with rapid resolution of visual changes after medical treatment.

Retinoid receptor-based signaling plays a role in voltage-dependent inhibition of invertebrate voltage-gated Ca2+ channels.

The retinoic acid receptor (RAR) and retinoid X receptor (RXR) mediate the cellular effects of retinoids (derivatives of vitamin A). Both RAR and RXR signaling events are implicated in hippocampal synaptic plasticity. Furthermore, retinoids can interact with calcium signaling during homeostatic plasticity. We recently provided evidence that retinoids attenuate calcium current (ICa) through neuronal voltage-gated calcium channels (VGCCs). We now examined the possibility that constitutive activity of neuronal RXR and/or RAR alters calcium influx via the VGCCs. We found that in neurons of the mollusk Lymnaea stagnalis, two different RXR antagonists (PA452 and HX531) had independent and opposing effects on ICa that were also time-dependent; whereas the RXR pan-antagonist PA452 enhanced ICa, HX531 reduced ICa Interestingly, this effect of HX531 occurred through voltage-dependent inhibition of VGCCs, a phenomenon known to influence neurotransmitter release from neurons. This inhibition appeared to be independent of G proteins and was largely restricted to Cav2 Ca2+ channels. Of note, an RAR pan-antagonist, LE540, also inhibited ICa but produced G protein-dependent, voltage-dependent inhibition of VGCCs. These findings provide evidence that retinoid receptors interact with G proteins in neurons and suggest mechanisms by which retinoids might affect synaptic calcium signaling.

Retinoic acid inhibits neuronal voltage-gated calcium channels.

Retinoic acid is the active metabolite of vitamin A and regulates several important cellular processes by activating retinoic acid receptors (RAR) and retinoid X receptors (RXR). These receptors generally act as transcription factors, though non-genomic actions of both retinoic acid and the receptors have also been reported. One such nongenomic effect includes the modulation of Ca2+ levels during homeostatic synaptic plasticity in the hippocampus. Retinoic acid can thus affect Ca2+ signaling and can potentially control both synaptic plasticity and neuronal firing. However, whether retinoic acid can regulate voltage-gated Ca2+ channels (either via genomic or nongenomic actions), which are fundamental to these processes, has not yet been studied in detail. Here we demonstrate the effects of retinoic acid on the biophysical properties of voltage-gated Ca2+ channels in cultured invertebrate motorneurons. Overnight exposure to physiological concentrations of retinoic acid significantly inhibited the voltage-gated Ca2+ current (ICa) in an isomer-dependent manner. Specifically, all-trans retinoic acid (atRA), but not 9-cis RA (9cRA), depolarized the voltage of half-maximal activation of ICa. AtRA also reduced the rate of channel activation and delayed recovery from inactivation. We provide evidence that both L-type and non-L-type voltage-gated Ca2+ channels are affected by atRA, as both nifedipine-sensitive and nifedipine-resistant ICa were inhibited in these neurons. These effects of retinoic acid are thought to be at least partially mediated by the retinoid receptors, as treatment of the neurons with synthetic RAR and RXR agonists produced a similar inhibition of ICa.

Divergent neuroendocrine responses to localized and systemic inflammation.

The sympathetic nervous system (SNS) is part of an integrative network that functions to restore homeostasis following injury and infection. The SNS can provide negative feedback control over inflammation through the secretion of catecholamines from postganglionic sympathetic neurons and adrenal chromaffin cells (ACCs). Central autonomic structures receive information regarding the inflammatory status of the body and reflexively modulate SNS activity. However, inflammation and infection can also directly regulate SNS function by peripheral actions on postganglionic cells. The present review discusses how inflammation activates autonomic reflex pathways and compares the effect of localized and systemic inflammation on ACCs and postganglionic sympathetic neurons. Systemic inflammation significantly enhanced catecholamine secretion through an increase in Ca(2+) release from the endoplasmic reticulum. In contrast, acute and chronic GI inflammation reduced voltage-gated Ca(2+) current. Thus it appears that the mechanisms underlying the effects of peripheral and systemic inflammation neuroendocrine function converge on the modulation of intracellular Ca(2+) signaling.

Neural regulation of gastrointestinal inflammation: role of the sympathetic nervous system.

The sympathetic innervation of the gastrointestinal (GI) tract regulates motility, secretion and blood flow by inhibiting the activity of the enteric nervous system (ENS) and direct vasoconstrictor innervation of the gut microvasculature. In addition to these well-established roles, there is evidence that the sympathetic nervous system (SNS) can modulate GI inflammation. Postganglionic sympathetic neurons innervate lymphoid tissues and immune cells within the GI tract. Furthermore, innate and adaptive immune cells express receptors for sympathetic neurotransmitters. Activation of these receptors can affect a variety of important immune cell functions, including cytokine release and differentiation of helper T lymphocyte subsets. This review will consider the neuroanatomical evidence of GI immune cell innervation by sympathetic axons, the effects of blocking or enhancing SNS activity on GI inflammation, and the converse modulation of sympathetic neuroanatomy and function by GI inflammation.

Endotoxemia enhances catecholamine secretion from male mouse adrenal chromaffin cells through an increase in Ca(2+) release from the endoplasmic reticulum.

Enhanced epinephrine secretion from adrenal chromaffin cells (ACCs) is an important homeostatic response to severe systemic inflammation during sepsis. Evidence suggests that increased activation of ACCs by preganglionic sympathetic neurons and direct alterations in ACC function contribute to this response. However, the direct effects of sepsis on ACC function have yet to be characterized. We hypothesized that sepsis enhances epinephrine secretion from ACCs by increasing intracellular Ca(2+) signaling. Plasma epinephrine concentration was increased 5-fold in the lipopolysaccharide-induced endotoxemia model of sepsis compared with saline-treated control mice. Endotoxemia significantly enhanced stimulus-evoked epinephrine secretion from isolated ACCs in vitro. Carbon fiber amperometry revealed an increase in the number of secretory events during endotoxemia, without significant changes in spike amplitude, half-width, or quantal content. ACCs isolated up to 12 hours after the induction of endotoxemia exhibited larger stimulus-evoked Ca(2+) transients compared with controls. Similarly, ACCs from cecal ligation and puncture mice also exhibited enhanced Ca(2+) signaling. Although sepsis did not significantly affect ACC excitability or voltage-gated Ca(2+) currents, a 2-fold increase in caffeine (10 mM)-stimulated Ca(2+) transients was observed during endotoxemia. Depletion of endoplasmic reticulum Ca(2+) stores using cyclopiazonic acid (10 µM) abolished the effects of endotoxemia on catecholamine secretion from ACCs. These findings suggest that sepsis directly enhances catecholamine secretion from ACCs through an increase in Ca(2+) release from the endoplasmic reticulum. These alterations in ACC function are likely to amplify the effects of increased preganglionic sympathetic neuron activity to further enhance epinephrine levels during sepsis.

A distinct boundary between the higher brain's susceptibility to ischemia and the lower brain's resistance.

Higher brain regions are more susceptible to global ischemia than the brainstem, but is there a gradual increase in vulnerability in the caudal-rostral direction or is there a discrete boundary? We examined the interface between `higher` thalamus and the hypothalamus the using live brain slices where variation in blood flow is not a factor. Whole-cell current clamp recording of 18 thalamic neurons in response to 10 min O2/glucose deprivation (OGD) revealed a rapid anoxic depolarization (AD) from which thalamic neurons do not recover. Newly acquired neurons could not be patched following AD, confirming significant regional thalamic injury. Coinciding with AD, light transmittance (LT) imaging during whole-cell recording showed an elevated LT front that initiated in midline thalamus and that propagated into adjacent hypothalamus. However, hypothalamic neurons patched in paraventricular nucleus (PVN, n= 8 magnocellular and 12 parvocellular neurons) and suprachiasmatic nucleus (SCN, n= 18) only slowly depolarized as AD passed through these regions. And with return to control aCSF, hypothalamic neurons repolarized and recovered their input resistance and action potential amplitude. Moreover, newly acquired hypothalamic neurons could be readily patched following exposure to OGD, with resting parameters similar to neurons not previously exposed to OGD. Thalamic susceptibility and hypothalamic resilience were also observed following ouabain exposure which blocks the Na(+)/K(+) pump, evoking depolarization similar to OGD in all neuronal types tested. Finally, brief exposure to elevated [K(+)]o caused spreading depression (SD, a milder, AD-like event) only in thalamic neurons so SD generation is regionally correlated with strong AD. Therefore the thalamus-hypothalamus interface represents a discrete boundary where neuronal vulnerability to ischemia is high in thalamus (like more rostral neocortex, striatum, hippocampus). In contrast hypothalamic neurons are comparatively resistant, generating weaker and recoverable anoxic depolarization similar to brainstem neurons, possibly the result of a Na/K pump that better functions during ischemia.

Toll-like receptor 4 activation reduces adrenal chromaffin cell excitability through a nuclear factor-κB-dependent pathway.

The adrenal medulla contains fenestrated capillaries that allow catecholamines and neuropeptides secreted by adrenal chromaffin cells (ACCs) to readily access the circulation. These capillaries may also allow bacterial products to enter the adrenal medulla and interact with ACCs during infection. One potential mediator of this interaction is toll-like receptor 4 (TLR-4), a pattern-recognition receptor that detects lipopolysaccharide (LPS) from Gram-negative bacteria. Evidence suggests that excitable cells can express TLR-4 and that LPS can modulate important neuronal and endocrine functions. The present study was therefore performed to test the hypothesis that TLR-4 activation by LPS affects ACC excitability and secretory output. RT-PCR revealed that TLR-4, cluster of differentiation 14, myeloid differentiation protein-2, and myeloid-derived factor 88 are expressed within mouse adrenal medullae. TLR-4 immunoreactivity was observed within all tyrosine hydroxylase immunoreactive ACCs. Incubation of isolated ACCs in LPS dose dependently hyperpolarized the resting membrane potential and enhanced large conductance (BK) Ca(2+)-activated K(+) currents. LPS (10 µg/ml) also increased rheobase, decreased the number of action potentials fired at rheobase, and reduced the percentage of ACCs exhibiting spontaneous and anodal break action potentials. Although catecholamine release was unaltered, LPS significantly reduced high-K(+)-stimulated neuropeptide Y release from isolated ACCs. LPS did not alter the excitability of ACCs from TLR-4(-/-) mice. Inhibition of nuclear factor-κB signaling with SC-514 (20 µm) abolished the effects of LPS on ACC excitability. Our findings suggest that LPS acts at TLR-4 to reduce ACC excitability and neuropeptide Y release through an nuclear factor-κB-dependent pathway.

The roles of purinergic signaling during gastrointestinal inflammation.

Extracellular purines play important roles as neurotransmitters and paracrine mediators in the gastrointestinal (GI) tract. Inflammation of the GI tract causes marked changes in the release and extracellular catabolism of purines, and can modulate purinoceptor expression and/or signaling. The functional consequences of this include suppression of the purinergic component of inhibitory neuromuscular and neurovascular transmission, increased release of purines from immune and epithelial cells, loss of enteric neurons to damage through P2X(7) purinoceptors, and enhanced activation of pain fibres. The purinergic system represents an important target for drug therapies that may improve GI inflammation and its consequences.

Altered adrenal chromaffin cell function during experimental colitis.

The sympathetic nervous system regulates visceral function through the release of catecholamines and cotransmitters from postganglionic sympathetic neurons and adrenal chromaffin cells (ACCs). Previous studies have shown that norepinephrine secretion is decreased during experimental colitis due to the inhibition of voltage-gated Ca(2+) current (I(Ca)) in postganglionic sympathetic neurons. The present study examined whether colonic inflammation causes a similar impairment in depolarization-induced Ca(2+) influx in ACCs using the dextran sulfate sodium (DSS) model of acute colitis in mice. Alterations in ACC function during colitis were assessed using fura 2-acetoxymethyl ester Ca(2+) imaging techniques and perforated patch-clamp electrophysiology. In ACCs isolated from mice with DSS-induced acute colitis, the high-K(+)-stimulated increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) was significantly reduced to 74% of the response of ACCs from control mice. Acute colitis caused a 10-mV hyperpolarization of ACC resting membrane potential, without a significant effect on cellular excitability. Delayed-rectifier K(+) and voltage-gated Na(+) current densities were significantly enhanced in ACCs from mice with DSS-induced acute colitis, with peak current densities of 154 and 144% that of controls, respectively. Importantly, acute colitis significantly inhibited I(Ca) in ACCs between -25 and +20 mV. Peak I(Ca) density in ACCs from mice with DSS-induced acute colitis was 61% that of controls. High-K(+)-induced increases in [Ca(2+)](i) were also reduced in ACCs from mice with 2,4,6-trinitrobenzene sulfonic acid-induced acute colitis and DSS-induced chronic colitis to 68 and 78% of the control responses, respectively. Our results suggest that, during colitis, voltage-dependent Ca(2+) influx is impaired in ACCs. Given the importance of Ca(2+) signaling in exocytosis, these alterations may decrease systemic catecholamine levels, which could play an important role in inflammatory bowel disease. This is the first demonstration of aberrant ACC function during experimental colitis.

Clinical and experimental evidence of sympathetic neural dysfunction during inflammatory bowel disease.

1. Inflammatory bowel diseases (IBD) alter the function of the enteric nervous system and the sensory innervation of the gastrointestinal (GI) tract. Less is known about whether IBD also affects the sympathetic nervous system (SNS). Given the importance of the SNS in regulating GI function and possibly immune system activation, the present review examines the evidence of sympathetic dysfunction during IBD and its possible consequences. 2. Sympathetic axons within the GI tract innervate several cell types, including vascular myocytes, enteric neurons and immune cells. The major neurotransmitters released from sympathetic varicosities are noradrenaline, neuropeptide Y and ATP or a related purine. 3. Clinical studies of IBD patients have provided evidence of an association between IBD and axonal or demyelinating neuropathy. Assays of autonomic function suggest that ulcerative colitis and Crohn's disease, the two major forms of IBD, have contrasting effects on sympathetic neural activity. 4. Animal models of IBD have been used to examine the effects of these diseases on sympathetic neurophysiology. A decrease in the release of noradrenaline from sympathetic varicosities in inflamed and uninflamed regions of the GI tract has consistently been reported. Recent findings suggest that the decrease in neurotransmitter release may be due to inhibition of N-type voltage-gated Ca(2+) current in post-ganglionic sympathetic neurons. 5. Interest in the role of the SNS in IBD is rapidly increasing. However, much work needs to be done to enhance understanding of how SNS function is altered during IBD and what contribution, if any, these changes make to pathogenesis.

Tumour necrosis factor alpha activates nuclear factor kappaB signalling to reduce N-type voltage-gated Ca2+ current in postganglionic sympathetic neurons.

Inflammation has profound effects on the innervation of affected tissues, including altered neuronal excitability and neurotransmitter release. As Ca(2+) influx through voltage-gated Ca(2+) channels (VGCCs) is a critical determinant of excitation-secretion coupling in nerve terminals, the aim of this study was to characterize the effect of overnight incubation in the inflammatory mediator tumour necrosis factor alpha (TNFalpha; 1 nM) on VGCCs in dissociated neurons from mouse superior mesenteric ganglia (SMG). Voltage-gated Ca(2+) currents (I(Ca)) were measured using the perforated patch clamp technique and the VGCC subtypes present in SMG neurons were estimated based on inhibition by selective VGCC blockers: omega-conotoxin GVIA (300 nM; N-type), nifedipine (10 microM; L-type), and omega-conotoxin MVIIC (300 nM; N-, P/Q-type). We used intracellular Ca(2+) imaging with Fura-2 AM to compare Ca(2+) influx during depolarizations in control and TNFalpha-treated neurons. TNF receptor and VGCC mRNA expression were measured using PCR, and channel alpha subunit (CaV2.2) was localized with immunohistochemistry. Incubation in TNFalpha significantly decreased I(Ca) amplitude and depolarization-induced Ca(2+) influx. The reduction in I(Ca) was limited to omega-conotoxin GVIA-sensitive N-type Ca(2+) channels. Depletion of glial cells by incubation in cytosine arabinoside (5 microM) did not affect I(Ca) inhibition by TNFalpha. Preincubation of neurons with SC-514 (20 microM) or BAY 11-7082 (1 microM), which both inhibit nuclear factor kappaB signalling, prevented the reduction in I(Ca) by TNFalpha. Inhibition of N-type VGCCs following TNFalpha incubation was associated with a decrease in CaV2.2 mRNA and reduced membrane localization of CaV2.2 immunoreactivity. These data suggest that TNFalpha inhibits I(Ca) in SMG neurons and identify a novel role for NF-kappaB in the regulation of neurotransmitter release during inflammatory conditions with elevated circulating TNFalpha, such as Crohn's disease and Guillain-Barré syndrome.