The purpose of this study was to determine if endogenous tachykinins can cause bradycardia in the isolated perfused guinea pig heart through stimulation of cholinergic neurons. Capsaicin was used to stimulate release of tachykinins and calcitonin gene-related peptide (CGRP) from cardiac afferents. A bolus injection of 100 nmol capsaicin increased heart rate by 26 ± 7% from a baseline of 257 ± 14 beats/min (n = 6, P < 0.01). This positive chronotropic response was converted to a minor bradycardic effect in hearts with 1 μM CGRP (8-37) present to block CGRP receptors. The negative chronotropic response to capsaicin was markedly potentiated in another group of hearts with the further addition of 0.5 μM neostigmine to inhibit cholinesterases. In this group, capsaicin decreased heart rate by 30 ± 10% from a baseline of 214 ± 6 beats/min (n = 8, P < 0.05). This large bradycardic response to capsaicin was inhibited by 1) infusion of neurokinin A to desensitize tachykinin receptors or 2) treatment with 1 μM atropine to block muscarinic receptors. The latter observations implicate tachykinins and acetylcholine, respectively, as mediators of the bradycardia. These findings support the hypothesis that endogenous tachykinins could mediate axon reflexes to stimulate cholinergic neurons of the intrinsic cardiac ganglia.

Background Myocardial infarction (MI) induces remodeling in stellate ganglion neurons (SGNs). Objective We investigated whether infarct site has any impact on the laterality of morphologic changes or neuropeptide expression in stellate ganglia. Methods Yorkshire pigs underwent left circumflex coronary artery (LCX; n = 6) or right coronary artery (RCA; n = 6) occlusion to create left- and right-sided MI, respectively (control: n = 10). At 5 ± 1 weeks after MI, left and right stellate ganglia (LSG and RSG, respectively) were collected to determine neuronal size, as well as tyrosine hydroxylase (TH) and neuropeptide Y immunoreactivity. Results Compared with control, LCX and RCA MIs increased mean neuronal size in the LSG (451 ± 25 vs 650 ± 34 vs 577 ± 55 μm2, respectively; P =.0012) and RSG (433 ± 22 vs 646 ± 42 vs 530 ± 41 μm2, respectively; P =.002). TH immunoreactivity was present in the majority of SGNs. Both LCX and RCA MIs were associated with significant decreases in the percentage of TH-negative SGNs, from 2.58% ± 0.2% in controls to 1.26% ± 0.3% and 0.7% ± 0.3% in animals with LCX and RCA MI, respectively, for LSG (P =.001) and from 3.02% ± 0.4% in controls to 1.36% ± 0.3% and 0.68% ± 0.2% in LCX and RCA MI, respectively, for RSG (P =.002). Both TH-negative and TH-positive neurons increased in size after LCX and RCA MI. Neuropeptide Y immunoreactivity was also increased significantly by LCX and RCA MI in both ganglia. Conclusion Left- and right-sided MIs equally induced morphologic and neurochemical changes in LSG and RSG neurons, independent of infarct site. These data indicate that afferent signals transduced after MI result in bilateral changes and provide a rationale for bilateral interventions targeting the sympathetic chain for arrhythmia modulation.

Very little is known about the development of cardiac parasympathetic ganglia and cholinergic innervation of the mouse heart. Accordingly, we evaluated the growth of cholinergic neurons and nerve fibers in mouse hearts from embryonic day 18.5 (E18.5) through postnatal day 21(P21). Cholinergic perikarya and varicose nerve fibers were identified in paraffin sections immunostained for the vesicular acetylcholine transporter (VAChT). Satellite cells and Schwann cells in adjacent sections were identified by immunostaining for S100β calcium binding protein (S100) and brain-fatty acid binding protein (B-FABP). We found that cardiac ganglia had formed in close association to the atria and cholinergic innervation of the atrioventricular junction had already begun by E18.5. However, most cholinergic innervation of the heart, including the sinoatrial node, developed postnatally (P0.5-P21) along with a doubling of the cross-sectional area of cholinergic perikarya. Satellite cells were present throughout neonatal cardiac ganglia and expressed primarily B-FABP. As they became more mature at P21, satellite cells stained strongly for both B-FABP and S100. Satellite cells appeared to surround most cardiac parasympathetic neurons, even in neonatal hearts. Mature Schwann cells, identified by morphology and strong staining for S100, were already present at E18.5 in atrial regions that receive cholinergic innervation at later developmental times. The abundance and distribution of S100-positive Schwann cells increased postnatally along with nerve density. While S100 staining of cardiac Schwann cells was maintained in P21 and older mice, Schwann cells did not show B-FABP staining at these times. Parallel development of satellite cells and cholinergic perikarya in the cardiac ganglia and the increase in abundance of Schwann cells and varicose cholinergic nerve fibers in the atria suggest that neuronal-glial interactions could be important for development of the parasympathetic nervous system in the heart.

Cardiac autonomic neuropathy is a frequent complication of diabetes and often presents as impaired cholinergic regulation of heart rate. Some have assumed that diabetics have degeneration of cardiac cholinergic nerves, but basic knowledge on this topic is lacking. Accordingly, our goal was to evaluate the structure and function of cardiac cholinergic neurons and nerves in C57BL/6 mice with streptozotocin-induced diabetes. Electrocardiograms were obtained weekly from conscious control and diabetic mice for 16. weeks. Resting heart rate decreased in diabetic mice, but intrinsic heart rate was unchanged. Power spectral analysis of electrocardiograms revealed decreased high frequency and increased low frequency power in diabetic mice, suggesting a relative reduction of parasympathetic tone. Negative chronotropic responses to right vagal nerve stimulation were blunted in 16-week diabetic mice, but postjunctional sensitivity of isolated atria to muscarinic agonists was unchanged. Immunohistochemical analysis of hearts from diabetic and control mice showed no difference in abundance of cholinergic neurons, but cholinergic nerve density was increased at the sinoatrial node of diabetic mice (16. weeks: 14.9 ± 1.2% area for diabetics versus 8.9 ± 0.8% area for control, P< 0.01). We conclude that disruption of cholinergic function in diabetic mice cannot be attributed to a loss of cardiac cholinergic neurons and nerve fibers or altered cholinergic sensitivity of the atria. Instead, decreased responses to vagal stimulation might be caused by a defect of preganglionic cholinergic neurons and/or ganglionic neurotransmission. The increased density of cholinergic nerves observed at the sinoatrial node of diabetic mice might be a compensatory response.

Baroreflex sensitivity is impaired by diabetes mellitus. Previously, we found that diabetes induces a deficit of central mediation of baroreflex-mediated bradycardia. In this study, we assessed whether diabetes induces degeneration of the nucleus ambiguus (NA) and reduces heart rate (HR) responses to l-Glutamate (L-Glu) microinjection into the NA. FVB control and OVE26 diabetic mice (5-6 months) were anesthetized. Different doses of L-Glu (0.1-5 mM/l, 20 nl) were delivered into the left NA using a multi-channel injector. In other animals, the left vagus was electrically stimulated at 1-40 Hz (1 ms, 0.5 mA, 20 s). HR and mean arterial blood pressure (MAP) responses to L-Glu microinjections into the NA and to the electrical stimulation of the vagus were measured. The NA region was defined by tracer TMR-D injection into the ipsilateral nodose ganglion to retrogradely label vagal motoneurons in the NA. Brainstem slices at - 600, - 300, 0, + 300, and + 600 μm relative to the obex were processed using Nissl staining and the number of NA motoneurons was counted. Compared with FVB control, we found in OVE26 mice that: 1) HR responses to L-Glu injection into the NA at doses of 0.2-0.4 (mM/l, 20 nl) were attenuated (p < 0.05), but MAP responses were unchanged (p > 0.05). 2) HR responses to vagal stimulation were increased (p < 0.05). 3) The total number of NA (left and right) motoneurons was reduced (p < 0.05). Taken together, we concluded that diabetes reduces NA control of HR and induces degeneration of NA motoneurons. Degeneration of NA cardiac motoneurons may contribute to impairment of reflex-bradycardia in OVE26 diabetic mice.

Dysfunction of the intrinsic cardiac nervous system is implicated in the genesis of atrial and ventricular arrhythmias. While this system has been studied extensively in animal models, far less is known about the intrinsic cardiac nervous system of humans. This study was initiated to anatomically identify neurotransmitters associated with the right atrial ganglionated plexus (RAGP) of the human heart. Biopsies of epicardial fat containing a portion of the RAGP were collected from eight patients during cardiothoracic surgery and processed for immunofluorescent detection of specific neuronal markers. Colocalization of markers was evaluated by confocal microscopy. Most intrinsic cardiac neuronal somata displayed immunoreactivity for the cholinergic marker choline acetyltransferase and the nitrergic marker neuronal nitric oxide synthase. A subpopulation of intrinsic cardiac neurons also stained for noradrenergic markers. While most intrinsic cardiac neurons received cholinergic innervation evident as punctate immunostaining for the high affinity choline transporter, some lacked cholinergic inputs. Moreover, peptidergic, nitrergic, and noradrenergic nerves provided substantial innervation of intrinsic cardiac ganglia. These findings demonstrate that the human RAGP has a complex neurochemical anatomy, which includes the presence of a dual cholinergic/nitrergic phenotype for most of its neurons, the presence of noradrenergic markers in a subpopulation of neurons, and innervation by a host of neurochemically distinct nerves. The putative role of multiple neurotransmitters in controlling intrinsic cardiac neurons and mediating efferent signaling to the heart indicates the possibility of novel therapeutic targets for arrhythmia prevention.

Previously, we have shown that chronic intermittent hypoxia (CIH) impairs baroreflex control of heart rate and augments aortic baroreceptor afferent function. In the present study, we examined whether CIH induces structural changes of aortic afferent axons and terminals. Young-adult Fischer 344 (F344, 4 months old) rats were exposed to room air (RA) or CIH for 35-45 days. After 14-24 days of exposure, they received tracer DiI injection into the left nodose ganglion to anterogradely label vagal afferent nerves. After surgery, animals were returned to their cages to continue RA or CIH exposure. Twenty-one days after DiI injection, the animals were sacrificed and the aortic arch was examined using confocal microscopy. In both RA and CIH rats, we found that DiI-labeled vagal afferent axons entered the wall of the aortic arch, then fanned out and branched into large receptive fields with numerous terminals (flower-sprays, end-nets and free endings). Vagal afferent axons projected much more to the anterior wall than to the posterior wall. In general, the flower-sprays, end-nets and free endings were widely and similarly distributed in the aortic arch of both groups. However, several salient differences between RA and CIH rats were found. Compared to RA control, CIH rats appeared to have larger vagal afferent receptive fields. The CIH rats had many abnormal flower-sprays, end-nets, and free endings which were intermingled and diffused into "bush-like" structures. However, the total number of flower-sprays was comparable (P>0.05). Since there was a large variance of the size of flower-sprays, we only sampled the 10 largest flower-sprays from each animal. CIH substantially increased the size of large flower-sprays (P<0.01). Numerous free endings with enlarged varicosities were identified, resembling axonal sprouting structures. Taken together, our data indicate that CIH induces significant remodeling of afferent terminal structures in the aortic arch of F344 rats. We suggest that such an enlargement of vagal afferent terminals may contribute to altered aortic baroreceptor function following CIH.

Half of the cholinergic neurons of human and primate intrinsic cardiac ganglia (ICG) have a dual cholinergic/noradrenergic phenotype. Likewise, a large subpopulation of cholinergic neurons of the mouse heart expresses enzymes needed for synthesis of norepinephrine (NE), but they lack the vesicular monoamine transporter type 2 (VMAT2) required for catecholamine storage. In the present study, we determined the full scope of noradrenergic properties (i.e. synthetic enzymes and transporters) expressed by cholinergic neurons of mouse ICG, estimated the relative abundance of neurons expressing different elements of the noradrenergic phenotype, and evaluated the colocalization of cholinergic and noradrenergic markers in atrial nerve fibers. Stellate ganglia were used as a positive control for noradrenergic markers. Using fluorescence immunohistochemistry and confocal microscopy, we found that about 30% of cholinergic cell bodies contained tyrosine hydroxylase (TH), including the activated form that is phosphorylated at Ser-40 (pSer40 TH). Dopamine β-hydroxylase (DBH) and norepinephrine transporter (NET) were present in all cholinergic somata, indicating a wider capability for dopamine metabolism and catecholamine uptake. Yet, cholinergic somata lacked VMAT2, precluding the potential for NE storage and vesicular release. In contrast to cholinergic somata, cardiac nerve fibers rarely showed colocalization of cholinergic and noradrenergic markers. Instead, these labels were closely apposed but clearly distinct from each other. Since cholinergic somata expressed several noradrenergic proteins, we questioned whether these neurons might also contain trophic factor receptors typical of noradrenergic neurons. Indeed, we found that all cholinergic cell bodies of mouse ICG, like noradrenergic cell bodies of the stellate ganglia, contained both tropomyosin-related kinase A (TrkA) and p75 neurotrophin receptors. Collectively, these findings demonstrate that mouse intrinsic cardiac neurons (ICNs), like those of humans, have a complex neurochemical phenotype that goes beyond the classical view of cardiac parasympathetic neurons. They also suggest that neurotrophins and local NE synthesis might have important effects on neurons of the mouse ICG.

The presence of vasoactive intestinal polypeptide (VIP) has been analyzed in fibers and neurons within the guinea pig intrinsic cardiac ganglia and in fibers innervating cardiac tissues. In whole-mount preparations, VIP-immunoreactive (IR) fibers were present in about 70% of the cardiac ganglia. VIP was co-localized with neuronal nitric oxide synthase (nNOS) in fibers innervating the intrinsic ganglia but was not present in fibers immunoreactive for pituitary adenylate cyclase-activating polypeptide, choline acetyltransferase (ChAT), tyrosine hydroxylase, or substance P. A small number of the intrinsic ChAT-IR cardiac ganglia neurons (approximately 3%) exhibited VIP immunoreactivity. These few VIP-IR cardiac neurons also exhibited nNOS immunoreactivity. After explant culture for 72 h, the intraganglionic VIP-IR fibers degenerated, indicating that they were axons of neurons located outside the heart. In cardiac tissue sections, VIP-IR fibers were present primarily in the atria and in perivascular connective tissue, with the overall abundance being low. VIP-IR fibers were notably sparse in the sinus node and conducting system and generally absent in the ventricular myocardium. Virtually all VIP-IR fibers in tissue sections exhibited immunoreactivity to nNOS. A few VIP-IR fibers, primarily those located within the atrial myocardium, were immunoreactive for both nNOS and ChAT indicating they were derived from intrinsic cardiac neurons. We suggest that, in the guinea pig, the majority of intraganglionic and cardiac tissue VTP-IR fibers originate outside of the heart. These extrinsic VIP-IR fibers are also immunoreactive for nNOS and therefore most likely are a component of the afferent fibers derived from the vagal sensory ganglia.

This study was done to determine if pituitary adenylate cyclase-activating peptide (PACAP)-immunoreactive nerve fibers occur in cardiac muscle as well as intracardiac ganglia of rats and guinea pigs and to clarify the chronotropic actions of PACAP27 in the same species using isolated heart preparations. PACAP nerve fibers were not detected in atrial or ventricular muscle of rat or guinea pig but a few stained nerve fibers occurred in the atrioventricular bundle of the guinea pig. Stained nerve fibers were prominent in intracardiac ganglia of both species. PACAP27 caused a dose-dependent tachycardia in isolated rat hearts (+39 ± 3 beats/min with 1 nmol, n = 6). Positive and/or negative chronotropic responses were evoked by PACAP27 in guinea pig heart, depending on dose and prior exposure to the peptide. PACAP27 also caused arrhythmias in several guinea pig hearts. Treatment with atropine eliminated or prevented PACAP-evoked bradycardia and arrhythmias, implicating cholinergic neurons in these responses. Positive chronotropic responses to PACAP were unaffected by beta-adrenergic receptor blockade in either species, suggesting that tachycardia resulted from a direct action on the heart. These observations support the conclusion that endogenous PACAP could have a role in regulating parasympathetic input to the heart but through different mechanisms in rats versus guinea pigs. A direct positive chronotropic influence of endogenous PACAP is unlikely since atrial muscle lacks PACAP-immunoreactive nerve fibers.