Adrenergic agonist induces rhythmic firing in quiescent cardiac preganglionic neurons in nucleus ambiguous via activation of intrinsic membrane excitability.

Cholinergic vagal nerves projecting from neurons in the brain stem nucleus ambiguus (NAm) play a predominant role in cardiac parasympathetic pacemaking control. Central adrenergic signaling modulates the tone of this vagal output; however, the exact excitability mechanisms are not fully understood. We investigated responses of NAm neurons to adrenergic agonists using in vitro mouse brain stem slices. Preganglionic NAm neurons were identified by ChAT-tdTomato fluorescence in young adult transgenic mice, and their cardiac projection was confirmed by retrograde dye tracing. Juxtacellular recordings detected sparse or absent spontaneous action potentials (AP) in NAm neurons. However, bath application of epinephrine or norepinephrine strongly and reversibly activated most NAm neurons regardless of their basal firing rate. Epinephrine was more potent than norepinephrine, and this activation largely depends on α1-adrenoceptors. Interestingly, adrenergic activation of NAm neurons does not require an ionotropic synaptic mechanism, because postsynaptic excitatory or inhibitory receptor blockade did not occlude the excitatory effect, and bath-applied adrenergic agonists did not alter excitatory or inhibitory synaptic transmission. Instead, adrenergic agonists significantly elevated intrinsic membrane excitability to facilitate generation of recurrent action potentials. T-type calcium current and hyperpolarization-activated current are involved in this excitation pattern, although not required for spontaneous AP induction by epinephrine. In contrast, pharmacological blockade of persistent sodium current significantly inhibited the adrenergic effects. Our results demonstrate that central adrenergic signaling enhances the intrinsic excitability of NAm neurons and that persistent sodium current is required for this effect. This central balancing mechanism may counteract excessive peripheral cardiac excitation during increased sympathetic tone. NEW & NOTEWORTHY Cardiac preganglionic cholinergic neurons in the nucleus ambiguus (NAm) are responsible for slowing cardiac pacemaking. This study identified that adrenergic agonists can induce rhythmic action potentials in otherwise quiescent cholinergic NAm preganglionic neurons in brain stem slice preparation. The modulatory influence of adrenaline on central parasympathetic outflow may contribute to both physiological and deleterious cardiovascular regulation.

Neuropharmacological lesion localization in idiopathic Horner's syndrome in Golden Retrievers and dogs of other breeds.

OBJECTIVE: To investigate whether idiopathic Horner's syndrome (HS) in Golden Retrievers is an exclusively preganglionic disorder based on denervation hypersensitivity pharmacological testing with phenylephrine. ANIMALS STUDIED: Medical records of dogs presented with HS between 2000 and 2012. Dogs presented with additional ocular or systemic signs were excluded. PROCEDURES: Clinical data examined included age, sex, duration of clinical signs, ancillary diagnostic test results, and time to mydriasis on topical ocular application of 1% phenylephrine. Lesions were diagnosed as postganglionic (mydriasis within 20 min) or preganglionic (mydriasis between 20 and 45 min). RESULTS: Medical records of 21 dogs of nine different breeds were included. An etiopathogenesis for Horner's syndrome was determined in five dogs, none of which were Golden Retrievers. All diagnoses correlated with pharmacological lesion localization. Ten Golden Retrievers were included (eight male and two female) with a mean age of 8.5 years (range: 4-13). Lesion localization was diagnosed as postganglionic in eight (mean: 10 min [range: 6-18]) and preganglionic in two Golden Retrievers (20 and 24 min). All cases were unilateral and had completely resolved within 15 weeks (range: 11-20). Recurrence was not reported in any of the patients. CONCLUSIONS: Idiopathic postganglionic HS was diagnosed in eight of 10 Golden Retrievers contradicting previous reports of a purely preganglionic localization. Etiopathogenesis of canine idiopathic HS remains to be determined; nevertheless, a vascular etiology cannot be excluded. Future studies using magnetic resonance angiography may aid in clarifying the pathogenesis.

Urotensin II promotes vagal-mediated bradycardia by activating cardiac-projecting parasympathetic neurons of nucleus ambiguus.

Urotensin II (U-II) is a cyclic undecapeptide that regulates cardiovascular function at central and peripheral sites. The functional role of U-II nucleus ambiguus, a key site controlling cardiac tone, has not been established, despite the identification of U-II and its receptor at this level. We report here that U-II produces an increase in cytosolic Ca(2+) concentration in retrogradely labeled cardiac vagal neurons of nucleus ambiguus via two pathways: (i) Ca(2+) release from the endoplasmic reticulum via inositol 1,4,5-trisphosphate receptor; and (ii) Ca(2+) influx through P/Q-type Ca(2+) channels. In addition, U-II depolarizes cultured cardiac parasympathetic neurons. Microinjection of increasing concentrations of U-II into nucleus ambiguus elicits dose-dependent bradycardia in conscious rats, indicating the in vivo activation of the cholinergic pathway controlling the heart rate. Both the in vitro and in vivo effects were abolished by the urotensin receptor antagonist, urantide. Our findings suggest that, in addition, to the previously reported increase in sympathetic outflow, U-II activates cardiac vagal neurons of nucleus ambiguus, which may contribute to cardioprotection.

Differential activation of adrenal, renal, and lumbar sympathetic nerves following stimulation of the rostral ventrolateral medulla of the rat.

Under acute and chronic conditions, the sympathetic nervous system can be activated in a differential and even selective manner. Activation of the rostral ventrolateral medulla (RVLM) has been implicated in differential control of sympathetic outputs based on evidence primarily in the cat. Although several studies indicate that differential control of sympathetic outflow occurs in other species, only a few studies have addressed whether the RVLM is capable of producing varying patterns of sympathetic activation in the rat. Therefore, the purpose of the present study was to determine whether activation of the RVLM results in simultaneous and differential increases in preganglionic adrenal (pre-ASNA), renal (RSNA), and lumbar (LSNA) sympathetic nerve activities. In urethane-chloralose anesthetized rats, pre-ASNA, RSNA, and LSNA were recorded simultaneously in all animals. Microinjections of selected concentrations and volumes of glutamate increased pre-ASNA, RSNA, and LSNA concurrently and differentially. Pre-ASNA and RSNA (in most cases) exhibited greater increases compared with LSNA on a percentage basis. By varying the volume or location of the glutamate microinjections, we also identified individual examples of differential and selective activation of these nerves. Decreases in arterial pressure or bilateral blockade of RVLM GABA(A) receptors also revealed differential activation, with the latter having a 3- to 4-fold greater effect on sympathetic activity. Our data provide evidence that activation of the rat RVLM increases renal, lumbar, and preganglionic adrenal sympathetic nerve activities concurrently, differentially, and, in some cases, selectively.

Targeted ablation of cardiac sympathetic neurons reduces the susceptibility to ischemia-induced sustained ventricular tachycardia in conscious rats.

The Cardiac Arrhythmia Suppression Trial demonstrated that antiarrhythmic drugs not only fail to prevent sudden cardiac death, but actually increase overall mortality. These findings have been confirmed in additional trials. The "proarrhythmic" effects of most currently available antiarrhythmic drugs makes it essential that we investigate novel strategies for the prevention of sudden cardiac death. Targeted ablation of cardiac sympathetic neurons may become a therapeutic option by reducing sympathetic activity. Thus cholera toxin B subunit (CTB) conjugated to saporin (a ribosomal inactivating protein that binds to and inactivates ribosomes; CTB-SAP) was injected into both stellate ganglia to test the hypothesis that targeted ablation of cardiac sympathetic neurons reduces the susceptibility to ischemia-induced, sustained ventricular tachycardia in conscious rats. Rats were randomly divided into three groups: 1) control (no injection); 2) bilateral stellate ganglia injection of CTB; and 3) bilateral stellate ganglia injection of CTB-SAP. CTB-SAP rats had a reduced susceptibility to ischemia-induced, sustained ventricular tachycardia. Associated with the reduced susceptibility to ventricular arrhythmias were a reduced number of stained neurons in the stellate ganglia and spinal cord (segments T(1)-T(4)), as well as a reduced left ventricular norepinephrine content and sympathetic innervation density. Thus CTB-SAP retrogradely transported from the stellate ganglia is effective at ablating cardiac sympathetic neurons and reducing the susceptibility to ventricular arrhythmias.

Electrophysiological responses of sympathetic preganglionic neurons to ANG II and aldosterone.

The intermediolateral cell column (IML) of the spinal cord is an important area where sympathetic impulses propagate to peripheral sympathetic organs. ANG II and aldosterone are important components of the renin-angiotensin-aldosterone system (RAAS), which activate the sympathetic nervous system. Each is partly synthesized in the brain and plays a paracrine role in the regulation of blood pressure independently of RAAS in the periphery. Our purpose in the present study was to clarify the contributions of sympathetic preganglionic neurons in the IML (IML neurons) and the effects of ANG II and aldosterone on the sympathetic nervous system. To examine responses to ANG II and aldosterone, we intracellularly recorded 104 IML neurons using a whole cell patch-clamp technique in spinal cord slice preparations. IML neurons were classified into two types: silent and firing. Both neuron types were significantly depolarized by ANG II, and candesartan inhibited this depolarization. After pretreatment with TTX, firing neurons (but not silent neurons) were significantly depolarized by ANG II. Aldosterone significantly increased the number of excitatory postsynaptic potentials (EPSPs) in both neuron types, but this response disappeared after pretreatment with TTX. ANG II and aldosterone had no synergistic effects on the IML neurons. The silent neurons had large cell soma, and many more dendrites than the firing neurons. These results suggest that ANG II acts presynaptically and postsynaptically in IML neurons, while aldosterone acts mainly presynaptically. Thus, the physiological effects of these substances are likely to be transmitted via specific membrane receptors of IML and/or presynaptic neurons.

Effects of thoracic epidural anaesthesia on cardiac repolarization.

1. Prolongation of the QT interval is associated with a risk of life-threatening cardiac arrhythmias. In the present study, we examined whether reversible blockade of preganglionic sympathetic fibres of the heart, achieved by thoracic epidural anaesthesia, affects cardiac repolarization and thus confers an anti-arrhythmic effect. 2. Fifty-two men, aged between 28 and 65 years, were included in the study: 28 were patients scheduled for thoracic epidural anaesthesia (Group T) and 24 were patients scheduled for lumbar epidural anaesthesia (Group L). Epidural blockade was achieved with 0.5% isobaric bupivacaine solution. Measurements were taken from electrocardiogram fragments obtained before epidural anaesthesia and after detection of blockade (T1 or T8 segment sensory block in Groups T and L, respectively). Correction of the QT interval was calculated using Bazett's formula (QTcb), Fridericia's correction (QTcf) and the Framingham formula (QTcF). Transmural dispersion of repolarization (TDR) was determined using the Tpeak--Tend time, where Tpeak is the peak of the T wave and Tend is the end of the T wave. 3. Significant shortening of the QTcb interval and TDR was detected after blockade in Group T. However, there were no changes in the ECG parameters in Group L. 4. In conclusion, reversible blockade of preganglionic sympathetic fibres to the heart, achieved by thoracic epidural anaesthesia, results in a reduction in QTcb, as well as the TDR. These changes may explain the anti-arrhythmic action seen with central blockade.

Long-term depression in the superior cervical ganglion of the rat.

Long-term depression (LTD) is a use-dependent decrease in synaptic efficacy widely recognized as a form of synaptic plasticity related to cognitive function in the central nervous system. Such response has previously not been demonstrated in autonomic ganglia. In the isolated superior cervical ganglion (SCG) of the rat (superfused with Locke solution containing 100 microM choline), low-frequency stimulation (LFS, 3-5 Hz/15 min) of the preganglionic nerve produced a long-lasting (up to 3 h ), significant (20-40%) decrease in the amplitude of the extracellularly recorded postganglionic compound action potential. Pretreatment of ganglia with the 5-HT(3) receptor antagonist tropisetron (0.5 microM) completely prevented the induction of ganglionic LTD (gLTD). Treatment of ganglia with the 5-HT(3) receptor antagonist MDL 72222 (0.5 microM) during the maintenance phase of established gLTD (1 h after LFS) antagonized the LFS-induced depression. Inhibition of nitric oxide (NO) synthase with l-NOARG (20-50 microM), applied before or after LFS, failed to affect the expression of gLTD. Additionally, pretreatment with the protein synthesis inhibitor emetine (1 microM) totally prevented the expression of gLTD. However, inhibition of protein phosphatase with cantharidin (30 microM) did not interfere with the expression of gLTD. These results indicate the presence of LTD in the rat SCG and suggest that expression of gLTD involves activation of 5-HT(3) receptor.

Long-term stimulation of cardiac vagal preganglionic neurons reduces blood pressure in the spontaneously hypertensive rat.

BACKGROUND: Arterial hypertension is associated with autonomic nervous system dysfunction. Different interventional strategies have been implemented in recent years for the reduction of sympathetic activity in patients with hypertension. However, the therapeutic benefit of increasing vagal tone in hypertensive patients remains largely unexplored. OBJECTIVE: Here, we describe the effects of long-term activation of vagal neural pathways on arterial pressure, heart rate arterial pressure variability and spontaneous baroreflex sensitivity in spontaneously hypertensive rats (SHR) and normotensive Wistar rats. METHODS: Brainstem vagal preganglionic neurons residing in the dorsal vagal motor nucleus (DVMN) were targeted with a lentiviral vector to induce the expression of an artificial G(s) protein-coupled receptor termed designer receptors exclusively activated by designer drugs (DREADD-Gs). The transduced neurons were activated daily by systemic administration of otherwise inert ligand clozapine-n-oxide. Arterial pressure measurements were recorded in conscious freely moving animals after 21 consecutive days of DVMN stimulation. RESULTS: Resting arterial pressure was significantly lower in SHRs expressing DREADD-Gs in the DVMN, compared with control SHRs expressing enhanced green fluorescent protein. No changes in arterial pressure were detected in Wistar rats expressing DREADD-Gs compared with rats expressing enhanced green fluorescent protein in the DVMN. Pharmacogenetic activation of DREADD-Gs-expressing DVMN neurons in SHRs was accompanied with increased baroreflex sensitivity and a paradoxical decrease in cardio-vagal components of heart rate and systolic arterial pressure variability in SHRs. CONCLUSION: These results suggest that long-term activation of vagal parasympathetic pathways is beneficial in restoring autonomic balance in an animal model of neurogenic hypertension and might be an effective therapeutic approach for the management of hypertension.

A spinal muscarinic M2 receptor-GABAergic disinhibition pathway that modulates peripheral inflammation in mice.

Previous data from our laboratories using the mouse air pouch model demonstrated that intrathecal injection of the cholinomimetic drug, neostigmine, produces a significant peripheral anti-inflammatory effect through activation of spinal muscarinic type 2 receptors. This anti-inflammatory effect is mediated by activation of sympathetic preganglionic neurons and subsequent release of adrenomedullary catecholamines. It has been established that adrenomedullary catecholamine release is controlled by sympathetic preganglionic neurons and that these neurons are modulated by GABAergic inhibitory input. To further establish the neurochemical circuitry underlying spinally mediated anti-inflammation, the present study examined whether spinal muscarinic type 2 receptors are associated with this spinal GABAergic pathway. Intrathecal injection of the M(2) receptor agonist, arecaidine but-2-ynyl ester tosylate (ABET) dose-dependently suppressed zymosan-induced leukocyte migration into the air pouch and increased Fos (neuronal activation marker) expression in sympathetic preganglionic neurons of the T7-T11 spinal cord segments (which mainly project to the adrenal medulla), but not in sympathetic preganglionic neurons of the T1-T6 or T12-L2 segments. These effects of arecaidine but-2-ynyl ester tosylate were completely blocked by intrathecal pretreatment with baclofen (a GABA(B)R agonist) but not muscimol (a GABA(A)R agonist). Intrathecal saclofen (a GABA(B)R antagonist), but not bicuculline (a GABA(A)R antagonist), significantly reduced leukocyte migration and increased Fos expression in T7-T11 sympathetic preganglionic neurons. More importantly, this intrathecal saclofen-induced anti-inflammatory effect was completely blocked by adrenalectomy or systemic pretreatment with propranonol (a beta-adrenoceptor antagonist). Collectively, these novel findings suggest that activation of spinal muscarinic type 2 receptors suppress spinal GABA(B) receptor input and that this disinhibition mechanism ultimately leads to the release of adrenal catecholamines and a subsequent reduction in peripheral inflammation.

Allergic lung inflammation affects central noradrenergic control of cholinergic outflow to the airways in ferrets.

Brain stem noradrenergic cell groups mediating autonomic responses to stress project to airway-related vagal preganglionic neurons (AVPNs). In ferrets, their activation produces withdrawal of cholinergic outflow to the airways via release of norepinephrine and activation of alpha(2A)-adrenergic receptors (alpha(2A)-AR) expressed by AVPNs. In these studies, we examined the effects of allergen exposure of the airway (AE) with ovalbumin on noradrenergic transmission regulating the activity of AVPNs and, consequently, airway smooth muscle tone. Experiments were performed in vehicle control (Con) and AE ferrets. Microperfusion of an alpha(2A)-AR agonist (guanabenz) in close proximity to AVPNs elicited more pronounced effects in Con than AE ferrets, including a decrease in unit activity and reflexly evoked responses of putative AVPN neurons with a corresponding decrease in cholinergic outflow to the airways. Although no differences were found in the extent of noradrenergic innervation of the AVPNs, RT-PCR and Western blot studies demonstrated that AE and repeated exposure to antigen significantly reduced expression of alpha(2A)-ARs at message and protein levels. These findings indicate that, in an animal model of allergic asthma, sensitization and repeated challenges with a specific allergen diminish central inhibitory noradrenergic modulation of AVPNs, possibly via downregulation of alpha(2A)-AR expression by these neurons.

Modulation of GABAergic synaptic transmission by terminal nicotinic acetylcholine receptors in the central autonomic nucleus of the neonatal rat spinal cord.

Using patch clamp recordings from an in vitro spinal cord slice preparation of neonatal rats (9-15days old), we characterized the GABAergic synaptic transmission in sympathetic preganglionic neurones (SPN) of the central autonomic nucleus (CA) of lamina X. Local applications of isoguvacine (100microM), a selective agonist at GABA(A) receptors, induced in all cells tested a chloride current which was abolished by bicuculline, a competitive antagonist at GABA(A) receptors. In addition, 25% of the recorded cells displayed spontaneous tetrodotoxin-insensitive and bicuculline-sensitive chloride miniature inhibitory postsynaptic currents (mIPSCs). Acetylcholine (100microM) increased the frequency of GABAergic mIPSCs without affecting their amplitudes or their kinetic properties indicating a presynaptic site of action. The presynaptic effect of ACh was restricted to GABAergic neurones synapsing onto sympathetic preganglionic neurones. The facilitatory effect of ACh was abolished in the absence of external calcium or in the presence of 100microM cadmium added to the bath solution. Choline 10mM, an agonist at alpha7 nicotinic acetylcholine receptors (nAChRs) or muscarine (10microM), a muscarinic receptor agonist, did not reproduce the presynaptic effect of ACh. The presynaptic effect of ACh was blocked by 1microM of dihydro-beta-erythroidine (DHbetaE), an antagonist of non-alpha7 nAChRs but was insensitive to alpha7 nAChRs antagonists (strychnine, alpha-bungarotoxin and methyllycaconitine) or to the muscarinic receptor antagonist atropine (10microM). It was concluded that SPNs of the central autonomic nucleus displayed a functional GABAergic transmission which is facilitated by terminal non alpha7 nAChRs.

Endogenous activation of nicotinic receptors underlies sympathetic tone generation in neonatal rat spinal cord in vitro.

Without the brainstem, thoracic spinal cords of neonatal rats in vitro spontaneously generate tonic sympathetic nerve discharge (SND) in the splanchnic nerves. Activation of nicotinic receptors in cords is known to alter a repertoire of neurotransmitter releases to sympathetic preganglionic neurons (SPNs). Using in vitro nerve-cord preparations, we investigated whether endogenous nicotinic receptor activity is essential for SND genesis. Application of mecamylamine, an open-channel nicotinic receptor blocker, reduced SND in a progressive manner. Exogenous activation of nicotinic receptors by application of various nicotinic agonists generally excited SND at low agonistic concentrations. At higher concentrations, however, agonists induced biphasic responses characterized by an initial excitation followed by prolonged SND suppression. Whether ionotropic glutamate, GABA(A), or glycine receptors are downstream signals of nicotinic receptor activation was explored by pretreatment of cords with selective antagonists. The initial excitation of SND persisted in the presence of ionotropic glutamate receptor antagonists. In contrast, the SND suppression was partially reversed by glycine or GABA(A) receptor antagonists. Incubation of the cord in a low Ca(2+)/high Mg(2+) bath solution to block Ca(2+)-dependent synaptic transmission did not affect SND excitation induced by nicotinic agonists, confirming direct activation of postsynaptic nicotinic receptors on SPNs. In conclusion, the endogenous activity of nicotinic receptors is essential for SND genesis in the thoracic spinal cord. Nicotinic activation of glycinergic and GABAergic interneurons may provide a recurrent inhibition of SPNs for homeostatic regulation of sympathetic outflow.

Ablation of vagal preganglionic neurons innervating the extra-thoracic trachea affects ventilatory responses to hypercapnia and hypoxia.

This study tested the hypothesis that during hypercapnia or hypoxia, airway-related vagal preganglionic neurons (AVPNs) of the nucleus ambiguus (NA) release acetylcholine (ACh), which in a paracrine fashion, activates ACh receptors expressed by inspiratory rhythm generating cells. AVPNs in the NA were ablated by injecting a saporin- (SA) cholera toxin b subunit (CTb-SA) conjugate into the extra-thoracic trachea (n=6). Control animals were injected with free CTb (n=6). In CTb treated rats, baseline ventilation and ventilatory responses to hypercapnia (5 and 12% CO(2) in O(2)) or hypoxia (8% O(2) in N(2)) were similar (p>0.05) prior to and 5 days after injection. CTb-SA injected rats maintained rhythmic breathing patterns 5 days post injection, however, tachypneic responses to hypercapnia or hypoxia were significantly reduced. The number of choline acetyltransferase (ChAT) immunoreactive cells in the NA was much lower (p<0.05) in CTb-SA rats as compared to animals receiving CTb only. These results suggest that AVPNs participate in the respiratory frequency response to hypercapnia or hypoxia.

Prenatal nicotine exposure alters central cardiorespiratory responses to hypoxia in rats: implications for sudden infant death syndrome.

Maternal cigarette smoking and prenatal nicotine exposure are the highest risk factors for sudden infant death syndrome (SIDS). During hypoxia, respiratory frequency and heart rate transiently increase and subsequently decrease. These biphasic cardiorespiratory responses normally serve to prolong survival during hypoxia by reducing the metabolic demands of cardiac and respiratory muscles. However, exaggerated responses to hypoxia may be life threatening and have been implicated in SIDS. Heart rate is primarily determined by the activity of brainstem preganglionic cardioinhibitory vagal neurons (CVNs) in the nucleus ambiguus. We developed an in vitro rat brainstem slice preparation that maintains rhythmic inspiratory-related activity and contains fluorescently labeled CVNs. Synaptic inputs to CVNs were examined using patch-clamp electrophysiological techniques. Hypoxia evoked a biphasic change in the frequency of both GABAergic and glycinergic IPSCs in CVNs, comprised of an initial increase followed by a decrease in IPSC frequency. Prenatal exposure to nicotine changed the GABAergic response to hypoxia from a biphasic response to a precipitous decrease in spontaneous GABAergic IPSC frequency. This study establishes a likely neurochemical mechanism for the heart rate response to hypoxia and a link between prenatal nicotine exposure and an exaggerated bradycardia during hypoxia that may contribute to SIDS.

Novel autonomic neurotransmitters and intestinal function.

A wide variety of substances, including amines and peptides, have been detected within the complex neuronal pathways of the enteric nervous system using immunohistochemical techniques. In this article we have discussed some of the more recent data on the effects of these substances on intestinal activity. We have also commented on the many difficulties associated with ascribing neurotransmitter status to individual compounds. The technique of immunoblockade of neurogenic functional responses has been used in an attempt to identify some of the putative neurotransmitter substances. The search for selective antagonists continues.

Phosphorylated extracellular signal-regulated kinase 1/2 immunoreactivity identifies a novel subpopulation of sympathetic preganglionic neurons.

Distinct chemical codes are thought to reflect functional specificity in sympathetic preganglionic neurons (SPN). Although a number of chemical candidates have been identified including neurotransmitter-related, calcium-binding and other proteins, signal transduction proteins have been largely neglected. Not only might these chemicals allow discrimination of functionally unique chemical signatures, but they may also identify activated neurons. Immunoreactivity (ir) to phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2) was differentially located within the thoracic spinal cord depending upon which of three forms of killing was used: the only exception to this was the intermediolateral cell column (IML) which was consistently, densely labeled. The presence or absence of p-ERK1/2 in SPN (n=17,541) within the IML of the thoraco-lumbar spinal cord was determined in seven rats. SPN were identified on the basis of their location, size and that they contained choline acetyltransferase ir. On average, 58% of SPN contained p-ERK1/2, however, more SPN in both the upper (72%; C8-T4) and lower (78%; T11-L3) thoraco-lumbar spinal cord contained p-ERK1/2-ir than the middle thoracic region (47%; T4-T10). p-ERK1/2-ir was also examined in SPN (n=1895) innervating the adrenal medulla (identified by retrograde tracing using cholera toxin B subunit) combined with localization of neuronal nitric oxide synthase (nNOS) in three rats. On average, 64% of adrenal SPN contain p-ERK1/2-ir, and it was confirmed that all adrenal SPN contain nNOS-ir. It appears that p-ERK1/2-ir SPN, described in this study, have tonically activated receptors that are coupled to intracellular signal transduction pathways that lead to the phosphorylation of ERK1/2.

Cocaine- and amphetamine-regulated transcript peptide potentiates spinal glutamatergic sympathoexcitation in anesthetized rats.

Cocaine- and amphetamine-regulated transcript (CART) is widely expressed in the rat central nervous system, notably in areas involved in control of autonomic and neuroendocrine functions. The aim of this study was to evaluate the effects of CART peptide fragment 55-102, referred to herein as CARTp, by intrathecal injection on blood pressure (BP) and heart rate (HR) before and after intrathecal glutamate in urethane-anesthetized male Sprague-Dawley rats. CARTp (0.1-10 nmol) administered intrathecally caused no or a small, statistically insignificant increase of blood pressure and heart rate, except at the concentration of 10 nmol, which caused a significant increase of blood pressure and heart rate. Intrathecal glutamate (0.1-10 nmol) produced a dose-dependent increase in arterial pressure and heart rate. Pretreatment with CARTp dose-dependently potentiated the pressor effects of glutamate (1 nmol), which by itself elicited a moderate increase of blood pressure and heart rate. Further, CARTp significantly potentiated the tachycardic effect of glutamate at 1 and 5 nmol, but attenuated the response at 10 nmol. The effect of CARTp was long-lasting, as it enhanced glutamatergic responses up to 90 min after administration. Prior injection of CARTp antiserum (1:500) but not normal rabbit serum nullified the potentiating effect of CARTp on glutamatergic responses. The result suggests that CARTp, whose immunoreactivity is detectable in sympathetic preganglionic neurons as well as in fibers projecting into the intermediolateral cell column, augments spinal sympathetic outflow elicited by glutamate at lower concentrations and may directly excite neurons in the intermediolateral cell column at higher concentrations.

The anti-inflammatory effect of peripheral bee venom stimulation is mediated by central muscarinic type 2 receptors and activation of sympathetic preganglionic neurons.

The anti-inflammatory effect (AI) induced by peripheral injection of diluted bee venom (dBV) involves activation of spinal cord circuits and is mediated by catecholamine release from adrenal medulla, but the precise neuronal mechanisms involved are not fully understood. In a recent study, we demonstrated that an increase in spinal acetylcholine is involved in mediating the anti-inflammatory effect of dBV and that this mediation also involves adrenomedullary activation. The present study utilized the mouse air pouch inflammation model to evaluate the involvement of spinal acetylcholine receptors and sympathetic preganglionic neurons (SPNs) in dBV's anti-inflammatory effect (dBVAI). Intrathecal (IT) pretreatment with atropine (muscarinic cholinergic antagonist) but not hexamethonium (nicotinic cholinergic antagonist) significantly suppressed dBVAI on zymosan-evoked leukocyte migration. Subsequent experiments showed that IT pretreatment with methoctramine (a muscarinic receptor type 2; M(2) antagonist), but not pirenzepine (an M(1) antagonist) or 4-DAMP (an M(3) antagonist), suppressed the dBVAI. In addition, dBV stimulation specifically increased Fos expression in SPNs of the T7-T11, but not the T1-T6 or T12-L2 spinal cord segments, in animals with zymosan-induced inflammation. Moreover, IT methoctramine pretreatment suppressed this dBV-induced Fos expression specifically in SPNs of T7-T11 level. Peripheral sympathetic denervation using 6-hydroxydopamine (6-OHDA) treatment (which spares sympathetic adrenal medullary innervation) did not alter dBVAI. Collectively these results indicate that dBV stimulation leads to spinal cord acetylcholine release that in turn acts on spinal M(2) receptors, which via a hypothesized disinhibition mechanism activates SPNs that project to the adrenal medulla. This activation ultimately leads to the release of adrenal catecholamines that contribute to dBVAI.

Hydralazine administration activates sympathetic preganglionic neurons whose activity mobilizes glucose and increases cardiovascular function.

Hypotensive drugs have been used to identify central neurons that mediate compensatory baroreceptor reflex responses. Such drugs also increase blood glucose. Our aim was to identify the neurochemical phenotypes of sympathetic preganglionic neurons (SPN) and adrenal chromaffin cells activated following hydralazine (HDZ; 10mg/kg) administration in rats, and utilize this and SPN target organ destination to ascribe their function as cardiovascular or glucose regulating. Blood glucose was measured and adrenal chromaffin cell activation was assessed using c-Fos immunoreactivity (-ir) and phosphorylation of tyrosine hydroxylase, respectively. The activation and neurochemical phenotype of SPN innervating the adrenal glands and celiac ganglia were determined using the retrograde tracer cholera toxin B subunit, in combination with in situ hybridization and immunohistochemistry. Blood glucose was elevated at multiple time points following HDZ administration but little evidence of chromaffin cell activation was seen suggesting non-adrenal mechanisms contribute to the sustained hyperglycemia. 16±0.1% of T4-T11 SPN contained c-Fos and of these: 24.3±1.4% projected to adrenal glands and 29±5.5% projected to celiac ganglia with the rest innervating other targets. 62.8±1.4% of SPN innervating adrenal glands were activated and 29.9±3.3% expressed PPE mRNA whereas 53.2±8.6% of SPN innervating celiac ganglia were activated and 31.2±8.8% expressed PPE mRNA. CART-ir SPN innervating each target were also activated and did not co-express PPE mRNA. Neurochemical coding reveals that HDZ administration activates both PPE+SPN, whose activity increase glucose mobilization causing hyperglycemia, as well as CART+SPN whose activity drive vasomotor responses mediated by baroreceptor unloading to raise vascular tone and heart rate.