Benet J. Pardini

Organization of the sympathetic postganglionic innervation of the rat heart

The origins and organization of cardiac sympathetic postganglionic nerves in the rat were identified in the present investigation. The retrograde tracer, Diamidino Yellow, was injected into the right or left ventricles to label somata in the sympathetic chain. Analysis of all sympathetic ganglia from superior cervical ganglion through the 10th thoracic ganglion indicated that the postganglionic innervation of the rat cardiac ventricles originates bilaterally. The majority of these somata were located in the middle and inferior cervical ganglia (middle cervical-stellate ganglion complex) (approximately 92% of all labelled cells), with lesser contributions from the superior cervical and 4th through 6th thoracic ganglia. To confirm and further quantitate these findings, the middle cervical-stellate ganglion complex was removed (MC-S ganglionectomy) bilaterally or ipsilaterally from the left or right sides, and regional cardiac norepinephrine concentration (left and right atrial appendages and left and right ventricles) was analysed 7 or 28 days later. At both times after bilateral MC-S ganglionectomy, regional cardiac norepinephrine was reduced by 89% to 100%, indicating the removal of almost all cardiac noradrenergic cells of origin and possibly fibers of passage. The results of unilateral MC-S ganglionectomy experiments indicated that the atrial appendages and the left ventricle receive bilateral innervation from the middle cervical-stellate ganglion complex. However, the left middle cervical-stellate ganglion complex appears to contribute a majority of the norepinephrine to the right ventricle. Furthermore, between 7 and 28 days after contralateral MC-S ganglionectomy, atrial appendages, but not ventricles, display significant recovery of norepinephrine content. The present data demonstrate: (1) a bilateral locus of origin of cardiac sympathetic postganglionic neurons, limited longitudinally to cervical through mid-thoracic ganglia, and (2) the ability of the cardiac postganglionic innervation to regenerate after partial denervation. These results demonstrate anatomical evidence for significant bilateral integration of cardiac sympathetic activity at the level of the sympathetic ganglion in the rat.

Location, distribution and projections of intracardiac ganglion cells in the rat

Physiological studies indicate that cardiac parasympathetic nerves may act selectively at discrete cardiac sites. To determine anatomical sites at which selective integration of cardiac nerve activity may occur, the present study identified and described the location, distribution, and projections of intracardiac ganglion cells in the rat. The estimated 3992 ganglion cells per rat heart were located in 4 distinct groups, all above the atrioventricular groove: (1) between the superior vena cava and aorta (2.5% of total), (2) in the region of the superior interatrial septum (49.9%), (3) posterior to the left atrium (24.0%), and (4) posterior to the inferior interatrial septum and right atrium (23.5%). Only a few ganglion cells were located subepicardially within the infolding of the dorsal interatrial septum. Retrogradely transported fluorescent tracers injected into the left or right ventricles demonstrated that different groups of ganglion cells projected to discrete or selective regions of the heart. Projections to the left ventricle originate only from ganglion cells located posterior to the interatrial septum and the left atrium. In the rat, intracardiac ganglion cells, confined to 4 atrial regions, appear to have discrete sites of termination within the heart. It is proposed that selective activation of different intracardiac ganglion cell groups may elicit specific regional changes in cardiac parasympathetic nerve activity.

Innervation patterns of the middle cervical--stellate ganglion complex in the rat.

The present experiments were designed to clarify the distribution of innervation of the middle and inferior cervical ganglia in the rat (middle cervical-stellate ganglion complex), the sympathetic ganglia which give rise to virtually all cardiac sympathetic nerves. Seven or 28 days after middle cervical-stellate ganglionectomy (surgical sympathectomy) norepinephrine content was measured in 9 peripheral areas including both the left and right atria and ventricles of the heart. The results were also compared to chemical sympathectomy produced with 6-hydroxydopamine. Seven or 28 days after surgical sympathectomy norepinephrine concentrations were reduced in all cardiac regions by at least 94%. Norepinephrine concentration in sub-diaphragmatic (spleen), but not supra-diaphragmatic (left intrascapular fat, left forelimb muscle), non-cardiac organs was preserved at control levels. 6-Hydroxydopamine treatment significantly reduced the norepinephrine concentration in all of the cardiac and non-cardiac tissues. The present evidence indicates that the middle cervical-stellate ganglion complex in the rat projects to a rather limited number of peripheral organs. Additionally, surgical sympathectomy produces more selective cardiac sympathectomy than 6-hydroxydopamine.

Alterations in Cardiac Parasympathetic Indices in STZ-Induced Diabetic Rats

Autonomic neuropathy involving parasympathetic innervation is a complication of diabetes mellitus. Biochemical and morphological indices of the parasympathetic innervation of the heart were investigated in rats after diabetes mellitus was induced with streptozocin (STZ). Choline acetyltransferase (CAT) activity was used as a biochemical marker for parasympathetic innervation. Total CAT activity within the hearts of diabetic rats was unchanged after 1 and 2 wk of diabetes and was significantly reduced after 4, 8, and 12 wk. Morphological changes within the cardiac portion of the parasympathetic innervation were assessed at 8 wk when CAT activity was decreased. In diabetic rats, there was a reduction in both cardiac ganglion cell size and number. In contrast, in insulin-treated STZ-induced diabetic rats, ganglion cells were similar in size and number to those in a control group given 3-O-methylglucose to prevent induction of diabetes mellitus by STZ. Thus, diabetes mellitus is associated with alterations in cardiac parasympathetic innervation in rats, and supplemental insulin protects against these changes. These alterations may contribute to impaired parasympathetic neural control of the heart in diabetes mellitus.

Sites at which neuropeptide Y modulates parasympathetic control of heart rate in guinea pigs and rats

Immunohistological evidence indicates that neuropeptide Y (NPY) is present in the cardiac innervation of numerous species. The present experiments determined if NPY influences in vivo parasympathetic control of heart rate in guinea pigs and rats by either pre- or postganglionic mechanisms or by an interaction at muscarinic receptors at the sino-atrial node. Urethane-anesthetized animals were prepared with arterial and venous catheters, and ECG leads. The cervical vagi were sectioned and propranolol was administered to minimize reflex changes in heart rate. Methacholine injection, carbachol injection, or electrical stimulation of the peripheral end of the vagus nerve was performed to activate the neuroeffector site, intracardiac ganglion cells, or preganglionic neurons, respectively. All three trials were performed before, during, and after NPY infusion. No differences in methacholine- or carbachol-induced bradycardia were observed between control and NPY groups in either species. NPY infusion inhibited vagal-mediated bradycardia in guinea pigs and in rats. However, NPY inhibited vagal-mediated bradycardia at a lower dose in guinea pigs (1 microgram/kg/min) than in rats (4 micrograms/kg/min). These data indicate that NPY modulates cardiac vagal preganglionic, but not postganglionic nerve function or neuroeffector sites at the sino-atrial node, in guinea pigs and rats. Furthermore, due to the different effective dosages, NPY may play a greater modulatory role in guinea pigs than in rats.

Vagus Nerve Stimulation Alters Regional Acetylcholine Turnover in Rat Heart

The turnover of neurotransmitter is a direct measure of neuronal function, varying with the impulse activity of the nerve. It is not known if vagal stimulation increases acetylcholine release uniformly throughout the heart, or if modification of neural signals occurs between the vagal nerve trunks and postganglionic synaptic terminals. The rate constant of acetylcholine turnover was measured in conduction and contractile regions of heart by quantifying the incorporation of [3H]choline into acetylcholine after labeling of the blood choline pool in urethane-anesthetized rats during two levels of vagal activity. Choline and acetylcholine were assayed by high pressure liquid chromatography with electrochemical detection of post-column enzymic reaction product, peroxide. The specific activities of choline and acetylcholine in the tissues at sacrifice were used to calculate the fractional turnover rates in cardiac regions. Supramaximal bilateral vagal stimulation for 20 minutes decreased heart rate (P < 0.05), while mean arterial blood pressure remained constant. The rate constants for acetylcholine turnover in right atrial regions containing the sinoatrial node, left atrial tissues, and interatrial septum doubled from control values during vagal stimulation. In contrast, the fractional rate constants of acetylcholine turnover did not change in the right and left ventricles during vagal stimulation. We interpret these results to indicate general activation of postganglionic parasympathetic fibers to the atria and selective modulation of postganglionic parasympathetic neural function to the ventricles.

Facilitation of baroreflex-induced bradycardia by stimulation of specific hypothalamic sites in the rat

Hypothalamic stimulation generally inhibits baroreflex-induced bradycardia. However, we have noted discrete areas of the rat hypothalamus which facilitate reflex bradycardia. The effects of hypothalamic stimulation on baroreflex-induced changes in heart rate were investigated in urethane-anesthetized rats (1.2 g/kg, i.p.; n = 6) instrumented with femoral arterial and venous catheters. Bipolar electrodes (250 micron diameter) were implanted stereotaxically in the hypothalamus. Baroreflex-induced bradycardia was elicited by phenylephrine (PE) injection (8-20 micrograms/kg). Responses to stimulation (STIM) (50-150 microA, 80 Hz, 0.5 ms), PE, and Stim + PE were studied for 1 min. In the ventral medial and anterior hypothalamus, STIM caused transient increases in blood pressure and no changes in heart rate. Peak blood pressure was lower during STIM + PE than during PE (144 +/- 5 vs 164 +/- 3 mm Hg; P less than 0.05). However, STIM + PE resulted in a lower heart rate compared to PE (194 +/- 22 22 vs 270 +/- 17 bpm; P less than 0.05). At 1 min, the heart rate in STIM + PE rats remained lower than in PE rats (205 +/- 37 vs 319 +/- 16 bpm; P less than 0.05). Atropine administration indicated that the facilitation was primarily parasympathetic in nature. These results identify specific hypothalamic regions which facilitate baroreflex-induced bradycardia by parasympathetic mechanisms.

Overlap of segmental populations and axon collaterals in the thoracic sympathetic system of the cat

Sympathetic preganglionic axons are known to branch in the sympathetic chain, i.e. extraspinally. The possibility that the axons may branch within the spinal cord (intraspinally) and exit through different roots has not been well examined. The purpose of this study was to determine if intraspinal collaterals exist in the distribution of preganglionic neurons innervating the stellate ganglion of the cat using the double-labeled retrograde fluorescent dye technique (Diamidino Yellow and Fast Blue). The right stellate ganglion of pentobarbital-anesthetized cats was isolated; the sympathetic chain was ligated in two places just caudal to the T2 white ramus and then sectioned between the ligatures. One fluorescent tracer was injected into the stellate ganglion; the complementary-colored tracer was injected into the sympathetic chain below the cut. After a 6-day survival time, frozen serial 40-micron sections of spinal cord segments T1-T4 were cut and mounted. The longitudinal distribution of dyes: indicated a segmental preganglionic organization and overlapped approximately 700 micron. However, no sympathetic preganglionic neurons were double-labeled with both dyes. Additional experiments demonstrated a small number of extraspinal preganglionic axon collaterals.

Norepinephrine release from guinea pig cardiac sympathetic nerves is insensitive to ryanodine under physiological conditions.

The activation of neurotransmitter release in nerve cells appears to be primarily dependent upon influx of extracellular Ca2+, most of which is thought to cross nerve terminal membranes through N-type Ca2+ channels. Events in skeletal and cardiac muscle, in contrast, are regulated to a greater extent by intracellular Ca2+ exchange between cytosol and intracellular organelles such as sarcoplasmic reticulum. It is not known to what extent corresponding intracellular organelles, i.e. endoplasmic reticulum (ER), contribute to cytosolic Ca2+ transients and norepinephrine (NE) release from cardiac sympathetic nerves. Heart rate and NE release were measured in isolated perfused guinea pig hearts during 1-min stimulations (5 V, 4 Hz, 2 ms) of the right stellate ganglia prior to (S1), during the administration of (S2), and after (S3) the removal of ryanodine (1 microM) from the perfusate. Ryanodine is a selective modulator of caffeine-sensitive Ca2+ stores in ER. Baseline heart rates decreased significantly in the presence of ryanodine, documenting its physiological effect on cardiac cells. However, there was no detectable effect of ryanodine on nerve-stimulated increase in heart rate or NE release. These results indicate that the ryanodine-sensitive intracellular Ca2+ stores do not play a major role in cardiac sympathetic neurotransmission.

Location of cardiac vagal preganglionic cell bodies in the bullfrog

The bullfrog, Rana catesbeiana, provides a relatively simple model for study of the cardiac innervation. The present study located the origin of cardiac vagal preganglionic cell bodies in the frog with the retrograde tracer, horseradish peroxidase (HRP). HRP was injected subepicardially over the surface of atrial regions and along the major vessels exiting the heart. labeled cell bodies in the brainstem were confined to the nucleus of the glossopharyngeal-vagal complex, from approximately 1.5 mm caudal to 1.5 mm rostral to the calamus scriptorius (obex). Brainstem neurons were labeled ipsilateral to the intact vagus nerve when unilateral vagotomy was performed at the time of HRP injection. No labeled somata were identified in the spinal cord. Comparison of the cardiac pool of neurons with the population of neurons labeled after injection of HRP directly into the cervical vagus nerve demonstrated that cardiac preganglionic neurons are localized to the caudal region of the vagal neuron pool and that there are no dimensional differences between the two populations in terms of major and minor diameters.