Superior Cervical Ganglion Research Articles

Analysis of Phosphoinositide-Dependence of Action Potential Firing in Sympathetic Neurons by Electrophysiological Recordings and Mathematical Modeling

Superior cervical ganglion (SCG) neurons play an important role for the innervation of the pineal gland. Due to their essential role in controlling the circadian rhythm, we analyzed action potential firing and its dependence on the rare phospholipid phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) in SCG neurons isolated from adult male rats by a combination of electrophysiological, biochemical, and mathematical approaches. Previously, we found action potential firing to be controlled by KCNQ2/3 and Maxi-K potassium channels, and a decrease in their activities by more than 80% was necessary to evoke action potential firing. Physiologically, action potential firing in SCG neurons is induced by activation of muscarinic acetylcholine receptors, which triggers activation of phospholipase C and hydrolysis of PI(4,5)P2. While KCNQ2/3 channel activity has been shown to be strongly dependent on PI(4,5)P2, a regulation of Maxi-K channel activity by phosphoinositides is under debate and has been speculated to be dependent on the subunit composition of Maxi-K channels. To analyze which Maxi-K subunits are expressed in SCGs, we isolated total RNA from SCGs and performed RT-PCR with oligonucleotides for both α- and β-subunits of Maxi-K channels. Our results showed expression of only the Maxi-K α-subunit. Expression of Maxi-K α-subunits in HEK293 cells together with either muscarinic acetylcholine receptors or a voltage-sensitive lipid phosphatase to transiently deplete PI(4,5)P2 showed no decrease in Maxi-K channel activity upon depletion of PI(4,5)P2. We incorporated our electrophysiological and biochemical results into a model of phosphoinositide metabolism of SCG neurons previously published by our group to develop a mathematical description of phosphoinositide metabolism and PI(4,5)P2-dependent ion channel activity in SCG neurons. In conclusion, our model allows for the first time a simulation of phosphoinositide metabolism and its regulation of action potential firing of SCG neurons.

Effects of autonomic denervations on the rhythms in axial length and choroidal thickness in chicks.

In chicks, axial length and choroidal thickness undergo circadian oscillations. The choroid is innervated by both branches of the autonomic nervous system, but their contribution(s) to these rhythms is unknown. We used two combination lesions to test this. For parasympathectomy, nerve VII was sectioned presynaptic to the pterygopalatine ganglia, and the ciliary post-ganglionics were cut (double lesion; n = 8). Triple lesions excised the sympathetic superior cervical ganglion as well (n = 8). Sham surgery was done in controls (n = 7). 8-14days later, axial dimensions were measured with ultrasonography at 4-h intervals over 24h. Rhythm parameters were assessed using a "best fit" function, and growth rates measured. Both types of lesions resulted in ultradian (> 1 cycle/24h) rhythms in choroidal thickness and axial length, and increased vitreous chamber growth (Exp-fellow: double: 69µm; triple: 104µm; p < 0.05). For double lesions, the frequency was 1.5 cycles/day for both rhythms; for triples the choroidal rhythm was 1.5 cycles/day, and the axial was 3 cycles/day. For double lesions, the amplitudes of both rhythms were larger than those ofsham surgery controls (axial: 107 vs 54µm; choroid: 124 vs 29µm, p < 0.05). These findings provide evidence for the involvement of abnormal ocular rhythms in the growth stimulation underlying myopia development.

The effect of ultrasound guided superior cervical sympathetic ganglion block to abducens nerve palsy caused by microvascular ischemia-a case report

The abducens nerve palsy is most likely caused by microvascular issue. Spontaneous recovery of vasculopathic abducens nerve palsies was common at 3 to 6 months. But recovery time was longer when many risk factors were present. Several patients had residual esotropia or abduction deficit. Cervical sympathetic block has an established use in treating patients with disorders related to cranial circulatory insufficiency. It causes a significant increase in cerebral blood flow 1,2. We report a case of a 67-year-old man with acute horizontal diplopia and right periocular pain. He had been diagnosed with right abducens nerve palsy caused by microvascular ischemia. We performed ultrasound-guided superior cervical sympathetic ganglion blocks. The patient was placed in a supine position, and his head was turned approximately 45 degrees to the left side. A transducer was transversely oriented over the right side of his neck, and the transverse process of C6 was located to the cricoid cartilage level. The transducer was moved in the cephalad direction along the neck to locate the common carotid bifurcation. After identifying the oval-shaped and hypoechoic structure between the longus capitis muscle and the starting point to branch off the common carotid bifurcation, a 23-gauge needle was introduced slowly from a lateral approach with the in-plane technique using ultrasound guidance. The needle was inserted until it approached the target site, and it was positioned immediately adjacent to the structure appearing to be the SCSG. We performed the SCSG block with 5 cc of 0.2 % ropivacaine, and then we identified Horner's sign on the same side after the injection. He received blocks in the same manner once every three or four days due. After four weeks, the symptoms had been completely resolved. We introduce ultrasound-guided superior cervical sympathetic ganglion blocks for management of abducens nerve palsy caused by microvascular ischemia, which could be an effective novel method to promote recovery from diplopia. We think that the recovery of ischemic demyelination of a portion of the nerve is greatly assisted by an improvement in the cerebral blood flow.

CPLA2α-/- sympathetic neurons exhibit increased membrane excitability and loss of N-Type Ca2+ current inhibition by M1 muscarinic receptor signaling.

Group IVa cytosolic phospholipase A2 (cPLA2α) mediates GPCR-stimulated arachidonic acid (AA) release from phosphatidylinositol 4,5-bisphosphate (PIP2) located in plasma membranes. We previously found in superior cervical ganglion (SCG) neurons that PLA2 activity is required for voltage-independent N-type Ca2+ (N-) current inhibition by M1 muscarinic receptors (M1Rs). These findings are at odds with an alternative model, previously observed for M-current inhibition, where PIP2 dissociation from channels and subsequent metabolism by phospholipase C suffices for current inhibition. To resolve cPLA2α’s importance, we have investigated its role in mediating voltage-independent N-current inhibition (~40%) that follows application of the muscarinic agonist oxotremorine-M (Oxo-M). Preincubation with different cPLA2α antagonists or dialyzing cPLA2α antibodies into cells minimized N-current inhibition by Oxo-M, whereas antibodies to Ca2+-independent PLA2 had no effect. Taking a genetic approach, we found that SCG neurons from cPLA2α-/- mice exhibited little N-current inhibition by Oxo-M, confirming a role for cPLA2α. In contrast, cPLA2α antibodies or the absence of cPLA2α had no effect on voltage-dependent N-current inhibition by M2/M4Rs or on M-current inhibition by M1Rs. These findings document divergent M1R signaling mediating M-current and voltage-independent N-current inhibition. Moreover, these differences suggest that cPLA2α acts locally to metabolize PIP2 intimately associated with N- but not M-channels. To determine cPLA2α’s functional importance more globally, we examined action potential firing of cPLA2α+/+ and cPLA2α-/- SCG neurons, and found decreased latency to first firing and interspike interval resulting in a doubling of firing frequency in cPLA2α-/- neurons. These unanticipated findings identify cPLA2α as a tonic regulator of neuronal membrane excitability.

Computing neurite outgrowth and arborization in superior cervical ganglion neurons

Dendrites are the primary site of synaptic activity in neurons and changes in synapses are often the first pathological stage in neurodegenerative diseases. Molecular studies of these changes rely on morphological analysis of the imaging of somas and dendritic arbors of cultured or primary neurons. As research on preventing or reversing synaptic degeneration develops, demands increase for user-friendly 2D neurite analyzers without undermining accuracy and reproducibility. The most common method of 2D neurite analysis is manual by using ImageJ. This method relies completely on the user’s ability to distinguish the shape and size of dendrites and trace morphology with a series of straight connected lines. Semi-automatic methods have also been developed, such as the NeuronJ plugin for ImageJ. These methods still rely on the user to identify the start and end of the dendrites, but automatically determine the shape, reducing the likelihood of user bias and speeding the process. Some automatic methods have been developed through image processing software, like ImagePro. These programs tend to be expensive, but have been shown to be fast and effective, limiting user interaction. In this study, we compare three methods of neurite analysis—ImageJ, NeuronJ, and ImagePro—in measuring the soma size, number of dendrites, and length of dendrites per cell of embryonic sympathetic rat neurons with BMP-7-induced dendritic growth. Our results indicate that ImageJ and NeuronJ measurements were of similar effectiveness and consistent throughout various images and multiple trials. NeuronJ required less user interaction in measuring the length of dendrites than the manual method and therefore, was faster and less labor intensive. Conversely, ImagePro tended to be inconsistent across images, overestimating both soma size and the number of dendrites per cell while underestimating the length of dendrites. Overall, NeuronJ, in conjunction with ImageJ, is the most reliable and efficient method of 2D neurite analysis tested in the present study.

Palmitic acid methyl ester is a novel neuroprotective agent against cardiac arrest

We previously discovered that palmitic acid methyl ester (PAME) is a potent vasodilator first identified and released from the superior cervical ganglion and remain understudied. Thus, we investigated PAME's role in modulating cerebral blood flow (CBF) and neuroprotection after 6 min of cardiac arrest (model of global cerebral ischemia). Our results suggest that PAME can enhance CBF under normal physiological conditions, while administration of PAME (0.02 mg/kg) immediately after cardiopulmonary resuscitation can also enhance CBF in vivo. Additionally, functional learning and spatial memory assessments (via T-maze) 3 days after asphyxial cardiac arrest (ACA) suggest that PAME-treated rats have improved learning and memory recovery versus ACA alone. Furthermore, improved neuronal survival in the CA1 region of the hippocampus were observed in PAME-treated, ACA-induced rats. Altogether, our findings suggest that PAME can enhance CBF, alleviate neuronal cell death, and promote functional outcomes in the presence of ACA.

Localization of CGRP and VEGF mRNAs in the mouse superior cervical ganglion during pre- and postnatal development.

The neuropeptide calcitonin gene-related peptide (CGRP) mediates inflammation and head pain by influencing the functional vascular blood supply. CGRP is a well-characterized mediator of receptor-regulated neurotransmitter release. However, knowledge regarding the role of CGRP during the development of the superior cervical ganglion (SCG) is limited. In the present study, we observed the localization of CGRP and vascular endothelial growth factor (VEGFA) mRNAs during prenatal development at embryonic day 14.5 (E14.5), E17.5 and postnatal day 1 (P1) using in situ hybridization. The antisense probe for CGRP was detected by in situ hybridization at E14.5, E17.5, and P1, and the highest levels were detected at E17.5. In contrast, the antisense probe for VEGF-A was detected by in situ hybridization in gradually increasing intensity from E14.5 to P1. The differences in the expression of these two markers revealed specific characteristics related to CGRP concentration and release compared to those of VEGF-A during development. The correlation between CGRP and VEGF-A may influence functional stress and the vascular blood supply during prenatal and postnatal development.

Sympathetic Hyperactivity and Age Affect Segregation and Expression of Neurotransmitters

Sympathetic neurons of the rat superior cervical ganglion (SCG) can segregate their neurotransmitters and co-transmitters to separate varicosities of single axons. We have shown that transmitter segregation is a plastic phenomenon and that it is correlated with the strength of synaptic transmission. Here, we determined whether sympathetic dysfunction occurring in stress and hypertension was correlated with plastic changes of neurotransmitter segregation. We characterized the expression of the markers, L-glutamic acid decarboxylase of 67 kDa (GAD67) and vesicular acetylcholine (ACh) transporter (VAChT) in the SCG of cold stressed and spontaneously hypertensive rats (SHR). Considering that the SCG comprises a heterogeneous neuronal population, we explored whether the expression and segregation of neurotransmitters would also have an intraganglionic heterogeneous distribution in ganglia of stressed and hypertensive rats. Furthermore, since hypertension in SHR is detected around 8–10 weeks, we evaluated expression and segregation of ACh and GABA in adult hypertensive (12-week old (wo)) and young pre-hypertensive (6-wo) SHR. We found an increase in segregation of ACh and GABA with no change in transmitter expression in ganglia of stressed animals. In contrast, in SHR, there was an increase in GABA expression, although segregation did not vary. Segregation showed a caudo-rostral gradient in controls but not in the ganglia of stressed animals. GABA expression showed a rostro-caudal gradient in adult SHR, which was not present in young 6-wo rats. In young SHR, ACh increased and, unexpectedly, segregation of ACh and GABA was higher than in adults. Data suggest that ACh and GABA segregation increases in acute sympathetic hyperactivity like stress, but does not vary in chronic hyperactivity such as in hypertension. Changes in segregation are age-dependent and might be involved in the mechanisms underlying stress and hypertension.

Axons of Passage and Inputs to Superior Cervical Ganglion in Rat.

Wheat germ agglutinin-horseradish peroxidase was injected into the entire (0.8 μL) or partial (rostral or caudal, 0.1-0.3 μL) superior cervical ganglion (SCG) of the rat (male Sprague-Dawley, N = 35) to examine the distribution of neurons in the middle (MCG) and inferior (ICG) cervical ganglion that send axons bypass the SCG. Whole-mounts of the SCG, cervical sympathetic trunk (CST), MCG, ICG, and sections of the brainstem and spinal cord were prepared. With entire SCG tracer injection, neurons were labeled evenly in the MCG (left: 258, right: 121), ICG (left: 848, right: 681), and CST (up to 770). Some neurons grouped in a single bulge just rostral to the MCG, which we termed as the "premiddle cervical ganglion" (pMCG). The left pMCG (120) is larger and has more neurons than the right pMCG (82). Centrally, neurons were labeled in lamina IX of cervical segments (C1: 18%, C2: 46%, C3: 33%, C4: 3%), intermediate zone of thoracic segments (T1: 31%, T2: 35%, T3: 27%, T4: 7%), and intermediate reticular nuclei (96%) and perifacial zone (4%) of brainstem. The rostral and caudal SCG injection selectively labeled neurons mainly in brainstem, C1-C2 and in T1-T2, respectively. Before projecting to their peripheral targets, many neurons in pMCG, MCG and ICG run rostrally within the CST rather than segmentally through the closest rami, from the level of SCG or above. Neurons in pMCG and MCG may have similar or complementary function and those in brainstem may be involved in the vestibulo-autonomic interaction. Anat Rec, 301:1906-1916, 2018. © 2018 Wiley Periodicals, Inc.

P7C3-A20 neuroprotection is independent of Wallerian degeneration in primary neuronal culture.

The antiapoptotic, neuroprotective compound P7C3-A20 reduces neurological deficits when administered to murine in-vivo models of traumatic brain injury. P7C3-A20 is thought to exert its activity through small-molecule activation of the enzyme nicotinamide phosphoribosyltransferase. This enzyme converts nicotinamide to nicotinamide mononucleotide, the precursor to nicotinamide adenine dinucleotide synthesis. Alterations to this bioenergetic pathway have been shown to induce Wallerian degeneration (WD) of the distal neurite following injury. This study aimed to establish whether P7C3-A20, through induction of nicotinamide phosphoribosyltransferase activity, would affect the rate of WD. The model systems used were dissociated primary cortical neurons, dissociated superior cervical ganglion neurons and superior cervical ganglion explants. P7C3-A20 failed to show any protection against WD induced by neurite transection or vincristine administration. Furthermore, there was a concentration-dependent neurotoxicity. These findings are important in understanding the mechanism by which P7C3-A20 mediates its effects – a key step before moving to human clinical trials.

Low levels of NMNAT2 compromise axon development and survival.

Nicotinamide mononucleotide adenylyltransferase 2 (NMNAT2) is an endogenous axon maintenance factor that preserves axon health by blocking Wallerian-like axon degeneration. Mice lacking NMNAT2 die at birth with severe axon defects in both the peripheral nervous system and central nervous system so the complete absence of NMNAT2 in humans is likely to be similarly harmful but probably rare. However, there is evidence of widespread natural variation in human NMNAT2 mRNA expression so it is important to establish whether reduced levels of NMNAT2 have consequences that impact health. While mice that express reduced levels of NMNAT2, either those heterozygous for a silenced Nmnat2 allele or compound heterozygous for one silenced and one partially silenced Nmnat2 allele, remain overtly normal into old age, we now report that Nmnat2 compound heterozygote mice present with early and age-dependent peripheral nerve axon defects. Compound heterozygote mice already have reduced numbers of myelinated sensory axons at 1.5months and lose more axons, likely motor axons, between 18 and 24months and, crucially, these changes correlate with early temperature insensitivity and a later-onset decline in motor performance. Slower neurite outgrowth and increased sensitivity to axonal stress are also evident in primary cultures of Nmnat2 compound heterozygote superior cervical ganglion neurons. These data reveal that reducing NMNAT2 levels below a particular threshold compromises the development of peripheral axons and increases their vulnerability to stresses. We discuss the implications for human neurological phenotypes where axons are longer and have to be maintained over a much longer lifespan.

Differential response of pineal microglia to surgical versus pharmacological stimuli.

Microglial cells are one of the interstitial elements of the pineal gland (PG). We recently reported the pattern of microglia colonization and activation, and microglia-Pax6+ cell interactions during normal pineal ontogeny. Here, we describe the dynamics of microglia-Pax6+ cell associations and interactions after surgical or pharmacological manipulation. In adult rats, the superior cervical ganglia (SCG) were exposed, and either bilaterally excised (SCGx) or decentralized (SCGd). In the SCGx PGs, the density of Iba1+ microglia increased after surgery and returned to sham baseline levels 13 days later. Pineal microglia also responded to SCGd, a more subtle denervation. The number of clustered Iba1+ /PCNA+ /ED1+ microglia was higher 4 days after both surgeries compared to the sham-operated group. However, the number of Pax6+ /PCNA- cells and the percentage of Pax6+ cells contacted by and/or phagocytosed by microglia increased significantly only after SCGx. Separate groups of rats were treated with either bacterial lipopolysaccharides (LPS) or doxycycline (DOX) to activate or inhibit pineal microglia, respectively. Peripheral LPS administration caused an increase in the number of clustered Iba1+ /PCNA+ /ED1+ microglial cells, and in the percentage of Pax6+ cells associated with and/or engulfed by microglia. In the LPS-treated PGs, we also noted an increase in the number of PCNA+ cells that were Iba1- within the microglial cell clusters. The density of Pax6+ cells did not change after LPS treatment. DOX administration did not influence the parameters analyzed. These data suggest that pineal microglia are highly receptive cells capable of rapidly responding in a differential manner to surgical and pharmacological stimuli.

“Polytox” synthetic cathinone abuse: A potential role for organic cation transporter 3 in combined cathinone-induced efflux

Synthetic cathinone derivatives are a new class of psychoactive substances (NPS), also known as “bath salts”, designed to exert psychostimulant effects resembling those of well-known psychostimulants, such as cocaine and 3,4-methylenedioxymethamphetamine (MDMA, “ecstasy”). As major constituents of bath salts, the cathinone derivatives 3,4-methylenedioxypyrovalerone (MDPV) and 4-methylmethcathinone (mephedrone), have received considerable media attention. MDPV and mephedrone interfere with the function of the high affinity transporters for dopamine (DAT), norepinephrine (NET) and serotonin (SERT), resulting in increased extracellular levels of these monoamines, though their mechanism of action differs. MDPV acts as a non-transported inhibitor of DAT, NET and SERT, whereas mephedrone promotes transporter-mediated release in an amphetamine-like fashion. MDPV and mephedrone are often taken together, creating a conundrum in as much as non-transported inhibitors, like MDPV, prevent mephedrone-induced reverse transport via DAT, NET and SERT. Here we provide evidence supporting a role for organic cation transporter 3 (OCT3) in the actions of mephedrone, which may account for its ability to enhance effects of MDPV. We show that mephedrone can induce substrate efflux via OCT3 in the presence of MDPV. Real-time recordings of the fluorescent OCT3 substrate (4-(4-dimethylamino)styryl)-N-methylpyridinium (ASP+) and radiotracer-flux studies using [3H]1-methyl-4-phenyl-pyridinium (MPP+), demonstrated that OCT3 is MDPV-insensitive when expressed in human embryonic kidney (HEK293) cells. Ex vivo experiments performed in cultured superior cervical ganglia (SCG) cells, rich in NET and OCT3, revealed that mephedrone induces [3H]MPP+ release in an OCT3-dependent manner when NET is fully occupied with MDPV. These results extend our recent findings that OCT3 is key in the mechanism of action of amphetamine-induced substrate release. OCT3 likewise appears to be a mechanism through which mephedrone can induce release of monoamines, thereby accounting for the paradoxically more potent psychostimulant effects of MDPV taken together with mephedrone, and greater risk for deleterious side effects.

Modulation of Ion Channels in the Superior Cervical Ganglion Neurons by Myocardial Ischemia and Fluvastatin Treatment.

Background: The superior cervical ganglion (SCG) of the autonomic nervous system plays an important role in different cardiovascular diseases. In this study, we investigated the effects of ischemia and fluvastatin treatment on the ion channel characteristics of SCG neurons in a rabbit myocardial ischemia (MI) model.Methods: MI was induced by abdominal subcutaneous injections of isoproterenol (ISO). The properties of the delayed rectifier potassium channel current (IK), sodium channel current (INa), and action potential (APs) on isolated SCG neurons in the control, MI-7d, MI-14d, fluvastatin-7d (fluvastatin pretreated 14 days and treated 7 days after ISO-induced MI), and fluvastatin-14d (fluvastatin pretreated 14 days and treated 14 days after ISO-induced MI) groups were studied. In addition, the RNA expressions of KCNQ3 and SCN9A in the SCG tissue were determined by performing real-time PCR. Intracellular calcium concentration was monitored using laser scanning confocal microscopy.Results: Compared with the control group, the current amplitude of IK and INa were increased in the MI-7d and MI-14d groups. KCNQ3 RNA (corresponding to channel proteins of IK) expression and SCN9A RNA (corresponding to channel proteins of INa) expression were also increased in MI groups. Activation and inactivation curves for INa in the two MI groups shifted negatively compared with the control group. These changes were reversed by fluvastatin treatment. Intracellular calcium concentration in SCG neurons was not altered significantly by MI or fluvastatin treatment. By contrast, increased AP amplitude and shortened APD90 were observed in the MI-7d and MI-14d groups. These changes were reversed in the fluvastatin-treated MI group.Conclusion: Fluvastatin treatment partly reversed the characteristics of SCG neurons in MI. The ion channel of SCG neurons could be one of the potential targets of fluvastatin in treating coronary heart diseases.

Knockdown the P2X3 receptor in the stellate ganglia of rats relieved the diabetic cardiac autonomic neuropathy

Diabetic cardiac autonomic neuropathy (DCAN) is a common and serious complication of diabetes mellitus (DM), is manifested by nerve fiber injury in the sympathetic and parasympathetic nerve of the autonomic nervous system, and causes hypertension, cardiac arrhythmias, silent myocardial infarction, and sudden death. Our previous study observed that P2X3 receptor in superior cervical ganglia in rat was associated with sympathetic neuropathy caused by myocardial ischemia. However, whether the P2X3 receptor is involved in the diabetic cardiac autonomic neuropathy and the underlying mechanisms remain unclear. The aim of this research was explored the effect of P2X3 short hairpin RNA (shRNA) on information transmission of sympathetic nerve induced by DCAN. Sprague-Dawley (SD) male rats were randomly divided into four groups: Control, DM, DM treated with P2X3 shRNA and DM treated with scramble shRNA. Blood pressure, heart rate and heart rate variability were measured in each group. The expression of P2X3 in stellate ganglion (SG) was detected by immunohistochemistry, western blotting and QPCR. Results showed that P2X3 shRNA alleviated blood pressure and heart rate, improved heart rate variability, decreased the up-regulated expression levels of P2X3, interleukin-1beta and tumor necrosis factor alpha in stellate ganglion (SG) of diabetic rats. P2X3 shRNA also reduced the incremental concentration of serum epinephrine and the phosphorylation level of extracellular regulated protein kinases1/2 in diabetic rats. These results indicated that P2X3 shRNA could decrease sympathetic activity via inhibiting P2X3 receptor in the SG to alleviate DCAN.

Butyrate, a Gut Bacterial Metabolite, Lowers Intraocular Pressure in Normotensive But Not in Hypertensive Rats.

Accumulating evidence supports a role of gut bacterial metabolites in arterial blood pressure (BP) regulation. Since the mechanisms controlling intraocular pressure (IOP) and BP overlap, we hypothesized that butyrate, a bacterial metabolite, might affect both IOP and BP. IOP, BP, and heart rate (HR) were measured at baseline and after the intraperitoneal administration of either butyrate or a vehicle (0.9% NaCl) in anaesthetized normotensive Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR). At baseline, there was no significant difference between WKY and SHR in IOP and HR, whereas BP was significantly higher in SHR. In WKY administration of butyrate produced a significant decrease in IOP and BP, which lasted throughout the experiment. IOP response to butyrate was similar between sham WKY and WKY, which had undergone unilateral superior cervical ganglion excision. In contrast, in SHR butyrate treatment did not affect IOP and produced only a transient decrease in BP. WKY showed no significant changes in HR whereas SHR exhibited an increase in HR. Butyrate, a gut bacterial metabolite, lowers IOP in normotensive but not in hypertensive rats. The effect is independent of ocular sympathetic innervation. Given that changes in BP followed a different pattern than changes in IOP it seems that butyrate lowers IOP independently of BP changes.

196 Enhancing Neuromodulation-Induced Analgesia Through Personalized Neuro-Visual Stimulation

OBJECTIVE: The ascending pharyngeal artery (APA), a branch of the external carotid artery (ECA), supplies the lower cranial nerves, superior cervical ganglion, and nasopharyngeal structures. The APA can also supply blood to various intracranial lesions. We studied the anatomy of the APA in the context of its neurosurgical and endovascular relevance. METHODS: The cervical origin, branching pattern, and course of the APA were studied in 20 human cadaveric craniocervical sides. The diameter of the APA, the extension of its main trunk, and the distance of its origin from the common carotid artery bifurcation were measured. The relationships between the APA and surrounding structures were also observed. RESULTS: In 80% of the specimens, the APA originated from the ECA. It originated from its medial wall in 56% and from the posterior wall in 44%. The APA originated from the internal carotid artery, common carotid artery bifurcation, occipital artery, and a trunk common to the lingual and facial arteries in 5% each. The APA was usually the third branch of the ECA (40%). The mean distance from the origin of the APA to the common carotid artery bifurcation was 15.3 mm (range, 0-32; standard deviation, ± 8.3 mm). The APA was frequently the second smallest branch of the ECA (caliber, 1.54 mm; range, 1.1-2.1; standard deviation, ± 0.25 mm). CONCLUSION: The APA is an important channel for supplying neural structures of the posterior fossa. Knowledge of its anatomy, variants, and anastomotic channels is essential in the treatment of lesions supplied by its branches and to avoid complications related to its inadvertent injury.

Retrograde axonal transport of rabies virus is unaffected by interferon treatment but blocked by emetine locally in axons.

Neuroinvasive viruses, such as alpha herpesviruses (αHV) and rabies virus (RABV), initially infect peripheral tissues, followed by invasion of the innervating axon termini. Virus particles must undergo long distance retrograde axonal transport to reach the neuron cell bodies in the peripheral or central nervous system (PNS/CNS). How virus particles hijack the axonal transport machinery and how PNS axons respond to and regulate infection are questions of significant interest. To track individual virus particles, we constructed a recombinant RABV expressing a P-mCherry fusion protein, derived from the virulent CVS-N2c strain. We studied retrograde RABV transport in the presence or absence of interferons (IFN) or protein synthesis inhibitors, both of which were reported previously to restrict axonal transport of αHV particles. Using neurons from rodent superior cervical ganglia grown in tri-chambers, we showed that axonal exposure to type I or type II IFN did not alter retrograde axonal transport of RABV. However, exposure of axons to emetine, a translation elongation inhibitor, blocked axonal RABV transport by a mechanism that was not dependent on protein synthesis inhibition. The minority of RABV particles that still moved retrograde in axons in the presence of emetine, moved with slower velocities and traveled shorter distances. Emetine’s effect was specific to RABV, as transport of cellular vesicles was unchanged. These findings extend our understanding of how neuroinvasion is regulated in axons and point toward a role for emetine as an inhibitory modulator of RABV axonal transport.

Differences in autonomic innervation to the vertebrobasilar arteries in spontaneously hypertensive and Wistar rats.

Essential hypertension is associated with hyperactivity of the sympathetic nervous system and hypoperfusion of the brainstem area controlling arterial pressure. Sympathetic and parasympathetic innervation of vertebrobasilar arteries may regulate blood perfusion to the brainstem. We examined the autonomic innervation of these arteries in pre-hypertensive (PHSH) and hypertensive spontaneously hypertensive (SH) rats relative to age-matched Wistar rats. Our main findings were: (1) an unexpected decrease in noradrenergic sympathetic innervation in PHSH and SH compared to Wistar rats despite elevated sympathetic drive in PHSH rats; (2) a dramatic deficit in cholinergic and peptidergic parasympathetic innervation in PHSH and SH compared to Wistar rats; and (3) denervation of sympathetic fibres did not alter vertebrobasilar artery morphology or arterial pressure. Our results support a compromised vasodilatory capacity in PHSH and SH rats compared to Wistar rats, which may explain their hypoperfused brainstem. Neurogenic hypertension may result from brainstem hypoperfusion. We previously found remodelling (decreased lumen, increased wall thickness) in vertebrobasilar arteries of juvenile, pre-hypertensive spontaneously hypertensive (PHSH) and adult spontaneously hypertensive (SH) rats compared to age-matched normotensive rats. We tested the hypothesis that there would be a greater density of sympathetic to parasympathetic innervation of vertebrobasilar arteries in SH versus Wistar rats irrespective of the stage of development and that sympathetic denervation (ablation of the superior cervical ganglia bilaterally) would reverse the remodelling and lower blood pressure. Contrary to our hypothesis, immunohistochemistry revealed a decrease in the innervation density of noradrenergic sympathetic fibres in adult SH rats (P<0.01) compared to Wistar rats. Unexpectedly, there was a 65% deficit in parasympathetic fibres, as assessed by both vesicular acetylcholine transporter (α-VAChT) and vasoactive intestinal peptide (α-VIP) immunofluorescence (P<0.002) in PHSH rats compared to age-matched Wistar rats. Although the neural activity of the internal cervical sympathetic branch, which innervates the vertebrobasilar arteries, was higher in PHSH relative to Wistar rats, its denervation had no effect on the vertebrobasilar artery morphology or persistent effect on arterial pressure in SH rats. Our neuroanatomic and functional data do not support a role for sympathetic nerves in remodelling of the vertebrobasilar artery wall in PHSH or SH rats. The remodelling of vertebrobasilar arteries and the elevated activity in the internal cervical sympathetic nerve coupled with their reduced parasympathetic innervation suggests a compromised vasodilatory capacity in PHSH and SH rats that could explain their brainstem hypoperfusion.

LA RISPECIFICAZIONE DEL TRASMETTITORE E DEL RECETTORE È UNA NUOVA FORMA DI PLASTICITÀ DEL SISTEMA NERVOSO NELL’ADULTO

After some words on the scientific role of Professor Paolo Mantegazza atthe University of Milan (4, 5, 6), I briefly illustrate some studies related to the occurrence of neurotransmitter and receptor re-specification in the adult animals. The greatdiscoveries of the early twentieth century on neuronal communication have established that the majority of communication between nerve cells occurs through a special structure, the synapse, allowing the one-way transfer of information between twocells through the release of a neurotransmitter from the presynaptic cell and its recognition by receptors localized in the postsynaptic cell. According to H. Dale axiom (9) each neuron could be identified on the basis of the neurotransmitter released and theinnervated cell by the type of receptors expressed; then neurons could be classified asexcitatory if they release acetylcholine, glutamate or other transmitters, or inhibitory ifthey release GABA or glycine. However, in recent years many studies have shown that, especially during development, a neuron could release and co-release several neuro-transmitters, sometimes even simultaneously, changing its classification from excitatory to inhibitory and vice versa(7). This researches opened a new field of study onsynaptic plasticity: the neurotransmitter and receptor re-specification. Our group, together with Prof. Mantegazza, tried to “force” it through experiments of denervation and heterologous re-innervation in the autonomic nervous system and at the neuromuscular junction. In a first series of experiments we studied the regenerative capabilities of the peripheral nervous system in three experimental models: a) re-innervation of the denervated superior cervical ganglion (SCG) (14, 15, 22) by cholinergicefferent vagal fibers, b) re-innervation of peripheral effectors smooth muscles (nicti-tating membrane) by the cholinergic preganglionic fibers; c) re-innervation in an in vivo transplant model of peripheral organs by the SCG. In these researches we haveestablished: 1) that a sympathetic ganglion could be re-innervated by vagal fibersforming normal ganglionic synapses, but with a strong reshaping, in vivo, of the cen-tral neural circuits so that sympathetic stimuli occurred through a vagal excitation; 2) preganglionic cholinergic fibers innervate the smooth muscle of the nictitating mem-brane releasing catecholamines instead of acetylcholine; 3) that in an in vivo model ofSCG transplant together with iris or adrenal medulla fragments, the SCG was able todistinguish between organs that required a postsynaptic innervation, iris, which wasinnervated, and organs that require a presynaptic innervation, the adrenal medulla,that was not innervated. We were then in the presence, even in the adult animal, of anew nervous plasticity with re-specification the neurotransmitter. These resultsdemonstrate that heterologous innervation could “force” plasticity in adult peripheralnervous system, alters the biological properties of neurons, upsets central neuronal circuits, but continues to maintain in experimental transplants basic rules of innervation between neurons and peripheral organs. Thirty years later, the group of prof. Brunelliin Brescia (23), along with pharmacologists and physiologists, had highlighted the pos-sibility of re-innervate striated muscles in a functional way with nerve fibers derivedfrom the red nucleus of the vestibular complex. The interest was, once again, in thefact that the re-innervating fibers were of glutamatergic type, and not cholinergic likethose of normal motor neurons, and that neuromuscular transmission was transformedfrom nicotinic cholinergic in glutamatergic. A new type of plasticity: the receptor re-specification had occurred also in this experimenal model. In close cooperationbetween our Milan and the Brescia group we could reconfirm with more appropriateexperiments that the re-innervation occurred; that neuromuscular junction had a glutamatergic transmission; that new re-innervating fibers made synapses at the same sitesof the previous neuromuscular junctions; that the new fibers release glutamate; andthat muscle cells expressed new glutamate receptors (24). Once again we were in thepresence of an extraordinary phenomenon of synaptic plasticity, in this case a receptorre-specification, and again with a strong impact on the central nervous system circuits.These experiences, along with many others now available in the literature, show thatthe adult peripheral nervous system, both autonomous and musculoskeletal, has aplasticity unthinkable before and open a field of great interest aiming at the understanding how neuronal specificity is regulated and at the investigation of non-canonical, but perhaps functional, re-innervation experiments in transplants and in post-traumatic surgery.