Norepinephrine effects on identified neurons of the rat dorsal motor nucleus of the vagus

The dorsal motor nucleus of the vagus (DMV) receives more noradrenergic terminals than any other medullary nucleus; few studies, however, have examined the effects of norepinephrine (NE) on DMV neurons. Using whole cell recordings in thin slices, we determined the effects of NE on identified gastric-projecting DMV neurons. Twenty-five percent of DMV neurons were unresponsive to NE, whereas the remaining 75% responded to NE with either an excitation (49%), an inhibition (26%), or an inhibition followed by an excitation (4%). Antrum/pylorus- and corpus-projecting neurons responded to NE with a similar percentage of excitatory (49 and 59%, respectively) and inhibitory (20% for both groups) responses. A lower percentage of excitatory (37%) and a higher percentage of inhibitory (36%) responses were, however, observed in fundus-projecting neurons. In all groups, pretreatment with prazosin or phenylephrine antagonized or mimicked the NE-induced excitation, respectively. Pretreatment with yohimbine or UK-14304 antagonized or mimicked the NE-induced inhibition, respectively. These data suggest that NE depolarization is mediated by alpha(1)-adrenoceptors, whereas NE hyperpolarization is mediated by alpha(2)-adrenoceptors. In 16 neurons depolarized by NE, amplitude of the action potential afterhyperpolarization (AHP) and its kinetics of decay (tau) were significantly reduced vs. control. No differences were found on the amplitude and tau of AHP in neurons hyperpolarized by NE. Using immunohistochemical techniques, we found that the distribution of tyrosine hydroxylase fibers within the DMV was significantly different within the mediolateral extent of DMV; however, distribution of cells responding to NE did not show a specific pattern of localization.     

Dorsal motor nucleus of the vagus: a site for evoking simultaneous changes in crural diaphragm activity, lower esophageal sphincter pressure, and fundus tone

The sphincter mechanism at the esophagogastric junction includes smooth muscle of the lower esophagus and skeletal muscle of the crural diaphragm (CD). Smooth muscle is known to be under the control of the dorsal motor nucleus of the vagus (DMV), while central nervous system (CNS) control of the CD is unknown. The main purposes of our study were to determine the CNS site that controls the CD and whether simultaneous changes in lower esophageal sphincter (LES) pressure and CD activity occur when this site is activated. Experiments were performed on anesthetized male ferrets whose LES pressure, CD activity, and fundus tone were monitored. To activate DMV neurons, L-glutamate was microinjected unilaterally into the DMV at three areas: intermediate, rostral, and caudal. Stimulation of the intermediate DMV decreased CD activity (-4.8 +/- 0.1 bursts/min and -0.3 +/- 0.01 mV) and LES pressure (-13.2 +/- 2.0 mmHg; n = 9). Stimulation of this brain site also produced an increase in fundus tone. Stimulation of the rostral DMV elicited increases in the activity of all three target organs (n = 5). Stimulation of the caudal DMV had no effect on the CD but did decrease both LES pressure and fundus tone (n = 5). All changes in LES pressure, fundus tone, and some DMV-induced changes in CD activity (i.e., bursts/min) were prevented by ipsilateral vagotomy. Our data indicate that simultaneous changes in activity of esophagogastric sphincters and fundus tone occur from rostral and intermediate areas of the DMV and that these changes are largely mediated by efferent vagus nerves.     

Spatial organization of neurons in the dorsal motor nucleus of the vagus synapsing with intragastric cholinergic and nitric oxide/VIP neurons in the rat

The dorsal motor nucleus of the vagus (DMV) contains preganglionic neurons that control gastric motility and secretion. Stimulation of different parts of the DMV results in a decrease or an increase in gastric motor activities, suggesting a spatial organization of vagal preganglionic neurons in the DMV. Little is known about how these preganglionic neurons in the DMV synapse with different groups of intragastric motor neurons to mediate contraction or relaxation of the stomach. We used pharmacological and immunohistochemical methods to characterize intragastric neural pathways involved in mediating gastric contraction and relaxation in rats. Microinjections of L-glutamate (L-Glu) into the rostral or caudal DMV produced gastric contraction and relaxation, respectively, in a dose-related manner. Intravenous infusion of hexamethonium blocked these actions, suggesting mediation via preganglionic cholinergic pathways. Atropine inhibited gastric contraction by 85.5 +/- 4.5%. Gastric relaxation was reduced by intravenous administration of N(G)-nitro-L-arginine methyl ester (L-NAME; 52.5 +/- 11.9%) or VIP antagonist (56.3 +/- 14.9%). Combined administration of L-NAME and VIP antagonist inhibited gastric relaxation evoked by L-Glu (87.8 +/- 4.3%). Immunohistochemical studies demonstrated choline acetyltransferase immunoreactivity in response to L-Glu microinjection into the rostral DMV in 88% of c-Fos-positive intragastric myenteric neurons. Microinjection of L-Glu into the caudal DMV evoked expression of nitric oxide (NO) synthase and VIP immunoreactivity in 81 and 39%, respectively, of all c-Fos-positive intragastric myenteric neurons. These data indicate spatial organization of the DMV. Depending on the location, microinjection of L-Glu into the DMV may stimulate intragastric myenteric cholinergic neurons or NO/VIP neurons to mediate gastric contraction and relaxation.     

In vitro analysis of the effects of cholecystokinin on rat brain stem motoneurons

Using whole cell patch clamp in thin brain stem slices, we tested the effects of cholecystokinin (CCK) on identified gastric-projecting neurons of the rat dorsal motor nucleus of the vagus (DMV). Perfusion with the sulfated form of CCK octapeptide (CCK8s, 30 pM-300 nM, EC50 approximately 4 nM) induced a concentration-dependent inward current in 35 and 41% of corpus- and antrum/pylorus-projecting DMV neurons, respectively. Conversely, none of the fundus-projecting DMV neurons responded to perfusion with CCK8s. The CCK8s-induced inward current was accompanied by a 65 +/- 17% increase in membrane input resistance and reversed at 90 +/- 4 mV, indicating that the excitatory effects of CCK8s were mediated by the closure of a potassium conductance. Pretreatment with the synaptic blocker TTX (0.3-1 microM) reduced the CCK8s-induced current, suggesting that a portion of the CCK8s-induced current was mediated indirectly via an action on presynaptic neurons apposing the DMV membrane. Pretreatment with the selective CCK-A receptor antagonist lorglumide (0.3-3 microM) attenuated the CCK8s-induced inward current in a concentration-dependent manner, with a maximum inhibition of 69 +/- 12% obtained with 3 microM lorglumide. Conversely, pretreatment with the selective CCK-B antagonist triglumide did not attenuate the CCK8s-induced inward current; pretreatment with triglumide (3 microM) and lorglumide (1 microM) attenuated the CCK8s-induced current to the same extent as pretreatment with lorglumide alone. Immunohistochemical experiments showed that CCK-A receptors were localized on the membrane of 34, 65, and 60% of fundus-, corpus-, and antrum/pylorus-projecting DMV neurons, respectively. Our data indicate that CCK-A receptors are present on a subpopulation of gastric-projecting neurons and that their activation leads to excitation of the DMV membrane.     

Catecholaminergic microcircuitry controlling the output of airway-related vagal preganglionic neurons.

In this study, we have investigated the ultrastructure and function of the catecholaminergic circuitry modulating the output of airway-related vagal preganglionic neurons (AVPNs) in ferrets. Immunoelectron microscopy was employed to characterize the nature of catecholaminergic innervation of AVPN at the ultrastructural level. In addition, immunofluorescence was used to examine the expression of the alpha(2A)-adrenergic receptor (alpha(2A)-AR) on AVPNs, and norepinephrine release within the rostral nucleus ambiguous (rNA) was measured by using microdialysis. Physiological experiments were performed to determine the effects of stimulation of the noradrenergic locus coeruleus (LC) cell group on airway smooth muscle tone. The results showed that 1) catecholaminergic nerve endings terminate in the vicinity of identified AVPNs but very rarely form axosomatic or axodendritic synapses with the AVPNs that innervate the extrathoracic trachea; 2) AVPNs express the alpha(2A)-AR; 3) LC stimulation-induced norepinephrine release within the rNA region was associated with airway smooth muscle relaxation; and 4) blockade of alpha(2A)-AR on AVPNs diminished the inhibitory effects of LC stimulation on airway smooth muscle tone. It is concluded that a noradrenergic circuit originating within the LC is involved in the regulation of AVPN activity within the rNA, and stimulation of the LC dilates the airways by the release of norepinephrine and activation of alpha(2A)-AR expressed by AVPNs, mainly via volume transmission.     

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.     

Mediation of vagal cardioinhibitory responses by glutamatergic receptors in the caudal medulla of turtles

Our previous studies showed that electrical stimulation of the nuclei ambiguous (NA) or dorsomotor nuclei of the vagus (DMV) complex in the brain stem of spontaneously breathing pond turtles (Cyclemys fiavomarginata), anesthetized with chloralose (4 mg/100 g) and urethane (40 mg/100 g), produced a marked slowing or even cessation of the heart rate, and resulted in an immediate fall of blood pressure. Results of the present study further demonstrated that the cardioinhibitory responses could also be elicited by microinjection of monosodium glutamate (0.2-20 nl, 50 mM) into the NA/DMV complex in turtles. A two-barrel glass micropipette held in a manipulator was connected to a pneumatic pressure pump for microinjection. The glutamate-induced cardioinhibitory responses could be significantly reduced in a dose-dependent manner by pretreatment with AP-5 (a NMDA receptor antagonist, at 1-8 nmole) or CNQX (a non-NMDA receptor antagonist; at 0.1-0.8 nmole) 20 min before glutamate administration. Histochemical verification by injecting horseradish peroxidase into the cervical vagus nerves revealed that retrogradely labeled glutamatergic neurons in the NA/DMV complex were observed. These results suggest that glutamatergic receptors in the caudal medulla may mediate vagal cardioinhibitory responses in the turtle.     

Catecholaminergic neurons in rat dorsal motor nucleus of vagus project selectively to gastric corpus

Nitric oxide synthase-immunoreactive (NOS-IR) neurons in the rat caudal dorsal motor nucleus of the vagus (DMV) project selectively to the gastric fundus and may be involved in vagal reflexes controlling gastric distension. This study aimed to identify the gastric projections of tyrosine hydroxylase-immunoreactive (TH-IR) DMV neurons, whether such neurons colocalize NOS-IR, and if they are activated after esophageal distension. Gastric-projecting neurons were identified after injection of retrograde tracers into the muscle wall of the gastric fundus, corpus, or antrum/pylorus before removal and processing of the brain stems for TH- and NOS-IR. A significantly higher proportion of corpus- compared with fundus- and antrum/pylorus-projecting neurons were TH-IR (14% compared with 4% and 2%, respectively, P < 0.05). Colocalization of NOS- and TH-IR was never observed in gastric-projecting neurons. In rats tested for c-Fos activation after intermittent esophageal balloon distension, no colocalization with TH-IR was observed in DMV neurons. These findings suggest that TH-IR neurons in the caudal DMV project mainly to the gastric corpus, constitute a subpopulation distinct from that of nitrergic vagal neurons, and are not activated on esophageal distension.     

Expression of tyrosine hydroxylase in vasopressinergic neurons of the supraoptic nucleus in rat ontogenesis and its modulation by noradrenergic afferents

An attempt is made to find out, at what stage of ontogenesis an expression of gene and synthesis of tyrosine hydroxylase (TH) is started, and whether noradrenergic afferents participate in regulation of these processes. The study is carried out on rats at the 21st embryonal day (E21), P3 and P13 with use of quantitative and semi-quantitative immunocytochemistry and hybridization in situ. Animals of all ages were subjected to a salt load, in some cases on the background of introduction of and α1-adrenoreceptor inhibitor, prazozine. According to the obtained data, the TH expression in SON neurons in response to the salt load begins at P3. The number of VP-ergic neurons expressing TH during the salt load is 3-fold reduced from P3 to P13. Taking into account that the innervation of VP-ergic SON neurons is realized for this period of development, we formulated a hypothesis that the TH expression is inhibited by noradrenergic afferents. According to the obtained data, TH is not expressed in osmotically stimulated VP-ergic neurons on the background of prazozine injection at E21; however, this combined effect results in increased TH expression at P3 and P13. At P13, i.e., in animals with a more developed afferent innervation, the amount of TH-immunoreactive neurons is three times lower, than at P3. Thus, in ontogenesis of rats, VP-ergic neurons begin to respond to osmotic stimulation by inclusion of the TH gene expression and its synthesis at the neonatal period, the both processes being under the inhibitory control of noradrenergic afferents mediated through α1-adrenoreceptors.     

Differential organization of excitatory and inhibitory synapses within the rat dorsal vagal complex

The dorsal motor nucleus of the vagus (DMV) is pivotal in the regulation of upper gastrointestinal functions, including motility and both gastric and pancreatic secretion. DMV neurons receive robust GABA- and glutamatergic inputs. Microinjection of the GABA(A) antagonist bicuculline (BIC) into the DMV increases pancreatic secretion and gastric motility, whereas the glutamatergic antagonist kynurenic acid (KYN) is ineffective unless preceded by microinjection of BIC. We used whole cell patch-clamp recordings with the aim of unveiling the brain stem neurocircuitry that uses tonic GABA- and glutamatergic synapses to control the activity of DMV neurons in a brain stem slice preparation. Perfusion with BIC altered the firing frequency of 71% of DMV neurons, increasing firing frequency in 80% of the responsive neurons and decreasing firing frequency in 20%. Addition of KYN to the perfusate either decreased (52%) or increased (25%) the firing frequency of BIC-sensitive neurons. When KYN was applied first, the firing rate was decreased in 43% and increased in 21% of the neurons; further perfusion with BIC had no additional effect in the majority of neurons. Our results indicate that there are several permutations in the arrangements of GABA- and glutamatergic inputs controlling the activity of DMV neurons. Our data support the concept of brain stem neuronal circuitry that may be wired in a finely tuned organ- or function-specific manner that permits precise and discrete modulation of the vagal motor output to the gastrointestinal tract.     

Altered setting of the pituitary-thyroid ensemble in hypocretin-deficient narcoleptic men

Hypo- or hyperthyroidism is associated with autonomic disorders. We studied Fos expression in the medullary dorsal motor nucleus of the vagus (DMV), nucleus tractus solitarii (NTS), and area postrema (AP) in four groups of rats with different thyroid states induced by a combination of drinking water and daily intraperitoneal injection for 1-4 wk: 1) tap water and vehicle; 2) 0.1% propylthiouracil (PTU) and vehicle; 3) PTU and thyroxine (T4; 2 microg/100 g); and 4) tap water and T4 (10 microg/100 g). The numbers of Fos immunoreactive (IR) positive neurons in the DMV, NTS, and AP were low in euthyroid rats but significantly higher in the 4-wk duration in hypothyroid rats, which were prevented by simultaneous T4 replacement. Hyperthyroidism had no effect on Fos expression in these areas. There were significant negative correlations between T4 levels and the numbers of Fos-IR-positive neurons in the DMV (r = -0.6388, P < 0.008), NTS (r = -0.6741, P < 0.003), and AP (r = -0.5622, P < 0.004). Double staining showed that Fos immunoreactivity in the DMV of hypothyroid rats was mostly localized in choline acetyltransferase-containing neurons. Thyroid hormone receptors alpha1 and beta2 were localized in the observed nuclei. These results indicate that thyroid hormone influences the DMV/NTS/AP neuronal activity, which may contribute to the vagal-related visceral disorders observed in hypothyroidism.     

Alpha(1)-adrenoceptor stimulation directly induces growth of vascular wall in vivo

Previous studies suggesting that norepinephrine is directly trophic for the vascular wall have been confounded by concomitant hemodynamic disturbances. Herein, a microcatheter connected to an osmotic minipump was implanted adjacent to the rat carotid for 2-wk perivascular suffusion of agents at systemic levels ~1,000 times below the threshold for altering arterial pressure. Norepinephrine decreased lumen and adventitial areas and circumference by 10, 14, and 5%, respectively (all P < 0.05); a nonsubtype-specific alpha(1)-adrenoceptor (AR) antagonist had no effect. When begun at the time of balloon injury, 2-wk norepinephrine increased lumen loss by 45%, increased neointimal area by 64% and collagen content by 33%, and reduced vessel circumference by 5% (all P < 0.05). alpha(1)-AR antagonists decreased neointimal area by 33% (all P < 0.05). alpha(1)A-AR antagonist reduced lumen loss by 70%, neointimal area by 54%, circumference decline by 84%, and adventitial thickening by 87% (all P < 0.05), whereas alpha(1B)-, alpha(1D)-, alpha(2)- and beta-AR antagonists were without effect. These are the first in vivo studies demonstrating that norepinephrine is directly trophic for the vascular wall and augments injury-induced intimal lesion growth.     

Medullary lateral tegmental field: an important source of basal sympathetic nerve discharge in the cat

We used blockade of excitatory amino acid (EAA) neurotransmission in the medullary lateral tegmental field (LTF) and rostral ventrolateral medulla (RVLM) to assess the roles of these regions in the control of inferior cardiac sympathetic nerve discharge (SND) and mean arterial pressure (MAP) in urethan-anesthetized, baroreceptor-denervated cats. Bilateral microinjection of a non-N-methyl-D-aspartate (NMDA)-receptor antagonist [1,2,3, 4-tetrahydro-6-nitro-2,3-dioxobenzo-[f]quinoxaline-7-sulfonamide (NBQX)] into the LTF significantly decreased SND to 46 +/- 4% of control (as demonstrated with power-density spectral analysis) and MAP by 16 +/- 6 mmHg. In contrast, bilateral microinjection of an NMDA-receptor antagonist [D(-)-2-amino-5-phosphonopentanoic acid (D-AP5)] into the LTF did not decrease SND or MAP. These results demonstrate that the LTF is an important synaptic relay in the pathway responsible for basal SND in the cat. Bilateral microinjection of NBQX or D-AP5 into the RVLM significantly decreased power in SND to 48 +/- 5 or 61 +/- 5% of control, respectively, and reduced MAP by 15 +/- 2 or 8 +/- 4 mmHg, respectively. These data indicate that EAA-mediated synaptic drive to RVLM-spinal sympathoexcitatory neurons accounts for a significant component of their basal activity.     

Excitatory amino acid signal transduction in the hippocampus: role of noradrenergic afferents and nitric oxide in cGMP increases in vivo.

Previous studies have demonstrated that excitatory amino acid (EAA)-dependent increases in cerebellar cGMP are dependent upon the prior activation of nitric oxide (NO) synthase. Additionally, the actions of NMDA, but not kainate or quisqualate, in elevating cerebellar cGMP have been shown to be dependent upon intact noradrenergic innervation of the cerebellum. In the current study we extended these observations to the hippocampus and again found that EAA-dependent increases in hippocampal cGMP also involve prior formation of NO. And as in the case of the cerebellum, NMDA-dependent increases in hippocampal cGMP involve prior release of norepinephrine which in turn apparently activates an alpha 1-adrenergic receptor to elicit cGMP increases. In toto, these data suggest that a key role of NMDA receptors in these brain regions is to presynaptically regulate the release of norepinephrine, thereby modulating the tone of this monoaminergic system. This may be a general principle which needs experimentation in other terminal fields of noradrenergic pathways.     

Presynaptic regulation of cardiac sympathetic function in hypoxic guinea pigs

In the normal heart, presynaptic cholinergic muscarinic and alpha 2-adrenergic mechanisms modify the fractional rate constant for norepinephrine (NE) synthesis (kNE), an index of sympathetic neural function. To evaluate presynaptic regulation of kNE, conscious guinea pigs subjected to normoxia and then hypoxia (n = 7-8 in each group) were pretreated with 1) vehicle; 2) a cholinergic muscarinic antagonist, methyl atropine; 3) an alpha 2-antagonist, yohimbine; or 4) a combination of the two. An increase of kNE was determined from incorporation of radiolabeled tyrosine into NE in a control period (arterial PO2 130 +/- 1.7 Torr, PCO2 36 +/- 0.5 Torr) and during a hypoxic state (PO2 49.6 +/- 1.0 Torr, PCO2 36 +/- 0.5 Torr). Hypoxia activated kNE in the atrioventricular node and right ventricular moderator band in vehicle-treated animals (P less than 0.05). Sympathetic activation was more general, however, because alpha 2-presynaptic influence acted to limit kNE in all tissues tested (P less than 0.05) except muscle, spleen, and posterior left ventricle. Cholinergic muscarinic presynaptic restraint on kNE was detected during hypoxia only in the left atrial appendage and lung (P less than 0.05). These data indicate that hypoxia increases kNE in the heart, but restraint by cholinergic muscarinic and alpha 2-adrenergic presynaptic mechanisms limits increases in neurotransmitter synthesis and noradrenergic activation regionally.     

In vitro synaptic reconsolidation in amygdala slices prepared from rat brains

The retrieval of consolidated fear memory causes it to be labile or deconsolidated, and the deconsolidated fear memory is reconsolidated over time in a protein synthesis-dependent manner. We have recently developed an ex vivo model where during fear memory deconsolidation and reconsolidation the synaptic state can be monitored at thalamic input synapses onto the lateral amygdala (T-LA synapses), a storage site for auditory fear memory. In this ex vivo model, the deconsolidation and reconsolidation processes of auditory fear memory in the intact brain were prevented following brain slicing; therefore, we could monitor the synaptic state for memory deconsolidation and reconsolidation at the time of brain slicing. However, why the synaptic reconsolidation process stopped after brain slicing in the ex vivo model is not known. One possibility is that brain slicing severs neuromodulatory innervations, which are required for memory reconsolidation, from other brain regions (e.g., noradrenergic innervation). In the present study, we supplemented amygdala slices with exogenous norepinephrine as a substitute for the severed noradrenergic innervations. DHPG (a group I metabotropic glutamate receptor agonist)-induced depotentiation (mGluRI-depotentiation), a marker for consolidated synapses, was observed following norepinephrine application to slices prepared immediately after tone presentation (fear memory retrieval) to rats that had been pre-conditioned to a tone paired with a shock. These results suggest that noradrenergic activation initiates synaptic reconsolidation. In contrast, mGluRI-depotentiation was absent following norepinephrine application to slices that were prepared immediately after the tone presentation (no fear memory retrieval) to rats when a tone and a shock were unpaired, ruling out the possibility that noradrenergic activation somehow facilitates a subsequent synaptic depression induced by DHPG irrespective of synaptic reconsolidation. Furthermore, the restored mGluRI-depotentiation following application of exogenous norepinephrine was dependent on de novo protein synthesis, as is memory reconsolidation. Thus, our findings suggest that T-LA synapses from acute slice preparations can undergo a reconsolidation process, thereby providing an optimal preparation to study a fear memory reconsolidation process in vitro.     

Cerebrovascular smooth muscle culture. II. Characterization of adrenergic receptors linked to adenylate cyclase

Cultured and propagated smooth muscle cells contain adenylate cyclase (AC) responsive to catecholamines and their analogues. Isoproterenol and zinterol were the most effective stimulants of AC activity with EC50 = 8.5 X 10(-8)M. They were followed by epinephrine, phenylephrine and norepinephrine (EC50 = 7.5 X 10(-7)M, 6.5 X 10(-6)M and 4 X 10(-6)M, respectively). When the selective antagonists for beta 1 and beta 2 receptors (beta 1-type practolol and atenolol, beta 1/beta 2-type propranolol and beta 2-type butoxamine) were tested against isoproterenol, epinephrine and norepinephrine stimulation of AC activity, the beta 1 in contrast to beta 2 antagonists were found ineffective. The alpha-blockers (phentolamine alpha 1/alpha 2-type antagonists) and yohimbine (alpha 2-type antagonist) alone or in the presence of propranolol did not significantly inhibit the catecholamine-induced enhancement of cAMP formation. On the other hand, prazosine (alpha 1-type antagonist) blocked the stimulatory effect of epinephrine and norepinephrine on AC system. Similarly, the alpha 2-agonist, clonidine, did not affect the catecholamines' stimulated AC activity while alpha 1 agonist, phenylephrine, induced an additive enhancement of norepinephrine production of cAMP. The findings of beta-2- and alpha-1-type adrenergic receptors in the cultured cerebrovascular smooth muscle provide additional support for the implicated involvement of adrenergic innervation in the regulation of cerebral blood flow and/or systemic blood pressure.     

Role of the medullary lateral tegmental field in reflex-mediated sympathoexcitation in cats

We tested the hypothesis that blockade of N-methyl-D-aspartate (NMDA) and non-NMDA receptors on medullary lateral tegmental field (LTF) neurons would reduce the sympathoexcitatory responses elicited by electrical stimulation of vagal, trigeminal, and sciatic afferents, posterior hypothalamus, and midbrain periaqueductal gray as well as by activation of arterial chemoreceptors with intravenous NaCN. Bilateral microinjection of a non-NMDA receptor antagonist into LTF of urethane-anesthetized cats significantly decreased vagal afferent-evoked excitatory responses in inferior cardiac and vertebral nerves to 29 +/- 8 and 24 +/- 6% of control (n = 7), respectively. Likewise, blockade of non-NMDA receptors significantly reduced chemoreceptor reflex-induced increases in inferior cardiac (from 210 +/- 22 to 129 +/- 13% of control; n = 4) and vertebral nerves (from 253 +/- 41 to 154 +/- 20% of control; n = 7) and mean arterial pressure (from 39 +/- 7 to 21 +/- 5 mmHg; n = 8). Microinjection of muscimol, but not an NMDA receptor antagonist, caused similar attenuation of these excitatory responses. Sympathoexcitatory responses to the other stimuli were not attenuated by microinjection of a non-NMDA receptor antagonist or muscimol into LTF. In fact, excitatory responses elicited by stimulation of trigeminal, and in some cases sciatic, afferents were enhanced. These data reveal two new roles for the LTF in control of sympathetic nerve activity in cats. One, LTF neurons are involved in mediating sympathoexcitation elicited by activation of vagal afferents and arterial chemoreceptors, primarily via activation of non-NMDA receptors. Two, non-NMDA receptor-mediated activation of other LTF neurons tonically suppresses transmission in trigeminal-sympathetic and sciatic-sympathetic reflex pathways.     

Norepinephrine reduces ω-conotoxin-sensitive Ca2+ currents in renal afferent neurons in rats

Sympathetic efferent and peptidergic afferent renal nerves likely influence hypertensive and inflammatory kidney disease. Our recent investigation with confocal microscopy revealed that in the kidney sympathetic nerve endings are colocalized with afferent nerve fibers (Ditting T, Tiegs G, Rodionova K, Reeh PW, Neuhuber W, Freisinger W, Veelken R. Am J Physiol Renal Physiol 297: F1427-F1434, 2009; Veelken R, Vogel EM, Hilgers K, Amman K, Hartner A, Sass G, Neuhuber W, Tiegs G. J Am Soc Nephrol 19: 1371-1378, 2008). However, it is not known whether renal afferent nerves are influenced by sympathetic nerve activity. We tested the hypothesis that norepinephrine (NE) influences voltage-gated Ca(2+) channel currents in cultured renal dorsal root ganglion (DRG) neurons, i.e., the first-order neuron of the renal afferent pathway. DRG neurons (T11-L2) retrogradely labeled from the kidney and subsequently cultured, were investigated by whole-cell patch clamp. Voltage-gated calcium channels (VGCC) were investigated by voltage ramps (-100 to +80 mV, 300 ms, every 20 s). NE and appropriate adrenergic receptor antagonists were administered by microperfusion. NE (20 μM) reduced VGCC-mediated currents by 10.4 ± 3.0% (P < 0.01). This reduction was abolished by the α-adrenoreceptor inhibitor phentolamine and the α(2)-adrenoceptor antagonist yohimbine. The β-adrenoreceptor antagonist propranolol and the α(1)-adrenoceptor antagonist prazosin had no effect. The inhibitory effect of NE was abolished when N-type currents were blocked by ω-conotoxin GVIA, but was unaffected by other specific Ca(2+) channel inhibitors (ω-agatoxin IVA; nimodipine). Confocal microscopy revealed sympathetic innervation of DRGs and confirmed colocalization of afferent and efferent fibers within in the kidney. Hence NE released from intrarenal sympathetic nerve endings, or sympathetic fibers within the DRGs, or even circulating catecholamines, may influence the activity of peptidergic afferent nerve fibers through N-type Ca(2+) channels via an α(2)-adrenoceptor-dependent mechanism. However, the exact site and the functional role of this interaction remains to be elucidated.