Inferior Cardiac Nerve Research Articles

Evidence for Cholinergic Collateral Projections between Sympathetic Neurons in the Murine Stellate Ganglia

Postganglionic neurons of the stellate ganglia (SG) are an integral component of sympathetic regulation of the heart. SG neurons receive preganglionic cholinergic inputs via T‐2 and send noradrenergic projections to the heart via the inferior cardiac nerve (iCN) to influence cardiac activity. SG neurons are thought to receive simple monosynaptic inputs from preganglionic neurons, but early studies have hinted that cholinergic collateral projections may be present in the SG. In culture, SG neurons form noradrenergic and cholinergic synapses between one another, suggesting that SG neurons express dual neurochemical phenotypes. This study tests the hypothesis that cholinergic collateral projections are present between sympathetic neurons of the intact SG in mice.Whole‐cell patch clamp recordings were made of intact SG neurons from male (N=8) and female (N=6) ChATTH+CreERT2/lox mice with and without tamoxifen treatment (N=7 ChAT KO mice and N=7 control mice, respectively). Tamoxifen treatment in ChATTH+CreERT2/lox mice induced the deletion of choline acetyltransferase (ChAT) in tyrosine hydroxylase positive (i.e. noradrenergic) cells. This strategy blocks putative acetylcholine synthesis in SG neurons while leaving preganglionic cholinergic synapses intact. To search for collaterals, the preganglionic nerve trunk, T‐2, was electrically stimulated to evoke excitatory postsynaptic currents (eEPSCs). The amplitude and jitter (i.e. variability in latency) of eEPSCs were examined. Additionally, the postganglionic nerve trunk, the iCN, was electrically stimulated to evoke retrograde action currents (rAC) and the presence of evoked synaptic currents was assessed. The amplitude and frequency of spontaneous EPSCs (sEPSCs) were also examined.ChAT KO significantly reduced the frequency of sEPSCs compared to control mice (1.21±0.559events/s vs. 0.13±0.048events/s, P<0.05 from Student’s unpaired t‐test, N=5‐6 cells) while the amplitude was unaffected (74.4±26.10pA vs. 39.4±10.46pA, P>0.05 from Student’s unpaired t‐test, N=5‐6 cells). This suggests that ChAT KO reduces the presynaptic activity of postganglionic noradrenergic neurons within the SG. In control mice, stimulation of T‐2 evoked high jitter eEPSCs, which was significantly reduced in ChAT KO mice (327.1±92.14μs vs. 39.2±15.69μs, P<0.05 from Student’s unpaired t‐test, N=5‐6 cells) Additionally, the proportion of neurons that exhibited evoked synaptic currents following stimulation of the iCN was significantly reduced in ChAT KO mice (3/4 cells vs. 1/9 cells, P<0.05 from χ² test).Chemical transmission at additional synapses accounts for the high jitter events observed in disynaptic pathways. High jitter responses following T‐2 stimulation and evoked synaptic responses after iCN stimulation in control neurons are both consistent with the collateral hypothesis. The loss of high jitter responses and evoked synaptic currents in ChAT KO mice also suggests these collateral projections are cholinergic. This suggests that collateral synaptic activity contributes to the increased frequency of sEPSCs in control mice. Understanding SG neurocircuitry is critical in deciphering changes in sympathetic activity in many pathophysiological conditions such as myocardial infarction and heart failure and may identify novel therapeutic targets for the treatment of autonomic imbalance.

Diverse Morphology of Sympathetic Neuron Subpopulations in the Stellate Ganglia

Sympathetic nerves innervate the heart and changes in sympathetic efferent activity can modulate cardiac function. The post‐ganglionic sympathetic neurons that innervate the heart are located primarily in the stellate ganglia (SG), which also contains neurons that project to brown adipose tissue (BAT), among other target tissues. We aim to examine morphological changes in SG neurons after myocardial infarction, and characterize the specificity of neural plasticity within the ganglion. To interpret morphological changes induced by cardiac injury, we must first fully understand the normal morphology of distinct groups of SG neurons. To that end, we examined SG neurons following injections of retrograde tracers into cardiac tissue and/or intrascapular brown adipose tissue (BAT) in adult male and female C57BL/6J mice (8 males, 2 females, 28‐32 weeks, 24‐39 g; The Jackson Laboratory). Using cholera toxin B (CTb) tracers conjugated to two different fluorophores (1 mg/mL in 0.01 M PBS; Alexa Fluor 488 or Alexa Fluor 555), we assessed the number, dispersion, pre‐synaptic cholinergic inputs and morphology of BAT‐ and cardiac‐projecting SG neurons. We also examined the quality of the CTb tracer detection at varying times after BAT injection (7, 14 and 28 days; n=2 mice per time point). BAT‐projecting SG neuron visualization was optimal 7 days post‐injection compared to 14 and 28 days, and could be visualized directly or with antibodies against CTb. BAT‐projecting SG neurons were located in both left and right SG, predominately ipsilateral to the injection site and were widely dispersed. In contrast, cardiac‐projecting SG neurons were more localized near the base of the inferior cardiac nerve. In BAT‐ and cardiac‐injected mice (n=4), we found that SG neurons were not dual‐labeled, supporting the notion that sympathetic innervation to target tissues is specific. We found cardiac‐projecting SG neuron somas have more volume (mm3) and cardiac‐projecting cells are less abundant within the SG than BAT‐projecting neurons with our tracer‐labeling paradigm. We also discovered a positive correlation between number of primary dendrites per neuron and soma volume in cardiac‐projecting SG neurons, though not in BAT‐projecting SG neurons. Additionally, in both SG subpopulations, the number of cholinergic inputs marked with vesicular acetylcholine transporter (VAChT) on the cell body is positively correlated to soma volume. Tract tracing distinct subpopulations of neurons will allow us to characterize morphological changes after cardiac injury in specific cell groups. This approach will allow insights into whether mechanisms of plasticity are cell specific or occur globally within the SG.

Distinct morphology of cardiac- and brown adipose tissue-projecting neurons in the stellate ganglia of mice.

Sympathetic neurons that innervate the heart are located primarily in the stellate ganglia (SG), which also contains neurons that project to brown adipose tissue (BAT). These studies were designed to examine the morphology of these two populations (cardiac‐ and BAT‐projecting) and their target connectivity. We examined SG neurons in C57BL/6J mice following injections of the retrograde tracer cholera toxin B (CTb) conjugated to Alexa Fluor 488 and Alexa Fluor 555, into cardiac tissue and intrascapular BAT. BAT‐projecting SG neurons were widely dispersed in SG, while cardiac‐projecting SG neurons were localized primarily near the inferior cardiac nerve base. SG neurons were not dual‐labeled, suggesting that sympathetic innervation is specific to the heart and BAT, supporting the idea of “labeled lines” of efferents. Morphologically, cardiac‐projecting SG somata had more volume and were less abundant than BAT‐projecting neurons using our tracer‐labeling paradigm. We found a positive correlation between the number of primary dendrites per neuron and soma volume in cardiac‐projecting SG neurons, though not in BAT‐projecting neurons. In both SG subpopulations, the number of cholinergic inputs marked with vesicular acetylcholine transporter (VAChT) puncta contacting the soma was positively correlated to soma volume, suggesting scaling of inputs across a range of neuronal sizes. In separate studies using dual tracing from left and right BAT, we found that BAT‐projecting SG neurons were located predominately ipsilateral to the injection, but a small subset of SG neurons project bilaterally to BAT. This tracing approach will allow the assessment of cell‐specific mechanisms of plasticity within subpopulations of SG neurons.

Tragus Nerve Stimulation Suppresses Post-Infarction Ventricular Arrhythmia by Modulating Autonomic Activity and Heterogeneities of Cardiac Receptor Distribution.

BackgroundImbalanced cardiac autonomic control and cardiac receptors redistribution contribute to the arrhythmogenic substrate under the myocardial infarction (MI) condition. Stimulating the auricular branch of vagus nerve (AB-VNS) has been proven to reduce post-infarction ventricular arrhythmia (VAs), but its potential mechanisms were largely unknown.This study aimed to investigate whether long-term intermittent low-intensity AB-VNS could produce a protective effect on modulating autonomic activities and abnormal redistribution of autonomic nerve efferent receptors in a MI canine model.Material/MethodsTwelve healthy beagle dogs underwent ligation of the left anterior descending coronary artery to establish a MI model and were randomized into 2 groups: an AB-VNS group, (AB-VNS for 4 weeks) and a control group (sham stimulation for 4 weeks). Dynamic electrocardiogram recording, neural recording, catecholamine concentration, and histological studies were conducted subsequently.ResultsCompared to the control group, the AB-VNS group had significantly suppressed post-infarction VAs, reduced low frequency (LF) power and increased high frequency (HF) power. In the AB-VNS group, with the progression of reduced cardiac sympathetic activities and augmented cardiac parasympathetic activities, the catecholamine concentration in heart tissue declined in the peripheral infarction area and right ventricle (RV); tyrosine hydroxylase (TH)-positive neurons decreased in the inferior cardiac sympathetic nerve, and choline acetyltransferase (ChAT)-positive neurons increased in the cervical vagus nerve. Expression of TrkA and P75NGFR were reduced in the peripheral MI (peri-MI) and non-MI area with AB-VNS. The mRNA expression of adrenergic and nicotinic receptors (β1-AR, β3-AR, and CHRNA7) significantly declined in the peri-MI and non-MI area of the AB-VNS group.ConclusionsChronic intermittent low-intensity AB-VNS effectively suppressed post-infarction VAs by potentially rebalancing extracardiac intrathoracic autonomic activities, reducing excessive cardiac sympathetic denervation, and attenuating the heterogeneities of cardiac efferent nerve receptors distribution.

The sympathetic regulation of the heart.

Dr Murray Esler discusses the role of the sympathetic innervation to the heart with relevance to Takotsubo cardiomyopathy The spinal cells of origin for the sympathetic nerves of the heart lie in the intermediolateral cell column of the upper 3–4 thoracic segments of the spinal cord. These cardiac pre-ganglionic neurons relay primarily through the stellate (cervicothoracic) and thoracic sympathetic ganglia. The output of post-ganglionic fibres from these ganglia are grouped in the superior, middle and inferior cardiac nerves, to be distributed to the cardiac plexus of nerves on the surface of the heart, then to the individual anatomical components receiving sympathetic innervation. Intrinsic cardiac ganglia are enmeshed in the cardiac neural plexus, and participate in autonomic regulatory influences on the heart. Central reflex and behavioural sympathetic control of the heart resides in four hierarchical levels of the neuroaxis: cortex (insular cortex, anterior cingulate cortex); subcortical forebrain (hypothalamus, amygdala); brainstem (including rostral ventrolateral medulla, caudal ventrolateral medulla, nucleus tractus solitarius, periaqueductal gray, raphe nuclei); spinal level (pre-ganglionic sympathetic neurons of intermediolateral column). From some of these brain regions there are direct, monosynaptic relays to the spinal pre-ganglionic neurons. With behavioural stimuli, CNS inputs to sympathetic outflow from the insular cortex, hypothalamus, and amygdala are prominent. In many and diverse circumstances …

Autonomic modulation for the management of patients with chronic heart failure.

Dysregulation of the autonomic nervous system in heart failure (HF) has received considerable attention during the past 3 decades, largely because of the well-recognized association between increased sympathetic activity and the elaboration of biologically active molecules, collectively referred to as neurohormones, that help to maintain cardiovascular homeostasis through increased volume expansion, peripheral arterial vasoconstriction, and increased myocardial contractility. However, high and sustained levels of these biologically active molecules (eg, norepinephrine, angiotensin II, aldosterone) are overtly toxic to the heart and circulation.1 These and other insights have led to the clinical use of neurohormonal antagonists, such as angiotensin-converting enzyme inhibitors, angiotensin receptor blockers, aldosterone antagonists, and β-blockers to treat patients with HF with a reduced left ventricular ejection fraction (LVEF).1 The effectiveness of these pharmacological agents is predominantly because of their ability to directly antagonize the deleterious effects of excessive sympathetic and renin–angiotensin activation. However, the current guideline-directed medical therapy (GDMT) in patients with HF fails to completely restore normal autonomic balance disrupted as a part of HF pathophysiology. During the last decade, a novel approach has generated widespread interest: modulation of the autonomic nervous system as a result of either a one-time intervention (eg, denervation) or of ongoing active therapy (eg, electric stimulation) as a means of further diminishing the sympathovagal imbalance that develops in HF.2,3 Of note, therapeutic neuromodulation with device-based therapies, either with spinal cord stimulation (SCS) or vagal stimulation (VS), has been used safely in patients with chronic pain, epilepsy, and depression, since the 1980s. As noted above, HF with a reduced LVEF is associated with sustained activation of the sympathetic nervous system that is accompanied by a withdrawal of parasympathetic tone. Impaired arterial baroreflexes have been proposed as an important mechanism that contributes to the sympathovagal imbalance present in HF4 …

Rostral ventrolateral medullary but not medullary lateral tegmental field neurons mediate sympatho-sympathetic reflexes in cats

This study was designed to build on past work from this laboratory by testing the hypothesis that medullary lateral tegmental field (LTF) neurons play a critical role in mediating sympathoexcitatory responses to activation of sympathetic afferent fibers. We studied the effects of microinjection of N-methyl-d-aspartate (NMDA) or non-NMDA receptor antagonists or muscimol bilaterally into the LTF on the area under the curve of the computer-averaged sympathoexcitatory potential in the right inferior cardiac nerve elicited by short trains of stimuli applied to afferent fibers in the left inferior cardiac or left splanchnic nerve (CN, SN) of baroreceptor-denervated and vagotomized cats anesthetized with a mixture of diallylbarbiturate and urethane. In contrast to our hypothesis, sympathoexcitatory responses to stimulation of CN (n = 5-7) or SN (n = 4-7) afferent fibers were not significantly affected by these procedures. We then determined whether the rostral and caudal ventrolateral medulla (RVLM, CVLM) and nucleus tractus solitarius (NTS) were involved in mediating these reflexes. Blockade of non-NMDA, but not NMDA, receptors in the RVLM significantly reduced the area under the curve of the sympathoexcitatory responses to electrical stimulation of either CN (P = 0.0110; n = 6) or SN (P = 0.0131; n = 5) afferent fibers. Neither blockade of excitatory amino acid receptors nor chemical inactivation of CVLM or NTS significantly affected the responses. These data show that activation of non-NMDA receptors in the RVLM is a critical step in mediating the sympatho-sympathetic reflex.

Rostral ventrolateral medullary (RVLM) but not medullary lateral tegmental field (LTF) neurons are in the pathway mediating sympathoexcitatory (SE) responses elicited by activation of cardiac and splanchnic sympathetic afferents

The LTF is involved in mediating sympathetic responses to activation of baroreceptor, arterial chemoreceptor, and vagal afferents but not trigeminal or sciatic afferents (Orer et al, Am J Physiol 286: R451‐64, 2004). In the current study, we tested the hypothesis that the LTF is an element of the sympatho‐sympathetic reflex pathway. We studied the effects of blockade of NMDA or non‐NMDA receptors bilaterally in the LTF on the area under the curve of the computer‐averaged SE potential in the right inferior cardiac nerve elicited by trains of stimuli applied at 0.5 Hz to afferents in left inferior cardiac and splanchnic nerves (lCN, lSN) of Dial‐urethane anesthetized cats. SE responses to lCN stimulation were unchanged by microinjection of either D‐AP5 (101±5% of control; n = 5) or NBQX (98±8% of control; n = 5). Responses to lSN stimulation were similarly unaffected: 92 ± 4 (n = 6) and 90 ± 5 (n = 4) % of control after D‐AP5 and NBQX. Thus, LTF is not a part of these reflex pathways. However, microinjection of NBQX into RVLM significantly (P ≤ 0.05) reduced SE responses to stimulation of lCN (37± 11% of control; n = 6) and lSN (35 ± 11 % of control; n = 5). D‐AP5 microinjection into RVLM did not significantly effect SE responses to stimulation of lCN or lSN (79 ± 13 and 72 ± 17 % of control; n = 5 each). These data show that activation of non‐NMDA receptors in RVLM contribute to SE responses to activation of sympathetic afferents. (Supported by HL33266)

The posterior vermis of the cerebellum selectively inhibits 10-Hz sympathetic nerve discharge in anesthetized cats

We studied the changes in inferior cardiac sympathetic nerve discharge (SND) and mean arterial pressure (MAP) produced by aspiration or chemical inactivation (muscimol microinjection) of lobule IX (uvula) of the posterior vermis of the cerebellum in baroreceptor-denervated and baroreceptor-innervated cats anesthetized with urethane. Autospectral analysis was used to decompose SND into its frequency components. Special attention was paid to the question of whether the experimental procedures affected the rhythmic (10-Hz and cardiac-related) components of SND. Aspiration or chemical inactivation of lobule IX produced an approximately three-fold increase in the 10-Hz rhythmic component of SND (P < or = 0.05) in baroreceptor-denervated cats. Total power (0- to 20-Hz band) was unchanged. Despite the absence of a change in total power in SND, there was a statistically significant increase in MAP. In baroreceptor-innervated cats, neither aspiration nor chemical inactivation of the uvula caused a significant change in cardiac-related or total power in SND or MAP. These results are the first to demonstrate a role of cerebellar cortical neurons of the posterior vermis in regulating the frequency composition of naturally occurring SND. Specifically, these neurons selectively inhibit the 10-Hz rhythm-generating network in baroreceptor-denervated, urethane-anesthetized cats. The functional implications of these findings are discussed.

Comparative Anatomy and Evolution of the Cardiac Innervation in New World Monkeys (Platyrrhini, E. Geoffroy, 1812)

The morphology of the autonomic cardiac nervous system (ACNS) was examined in 24 sides of 12 New World monkeys (Platyrrhini) of all four families to document the morphology systematically and to study the evolutionary changes of the ACNS in this primate lineage. We report the following: (1) Although several trivial intra- and inter-specific variations are present, a family-dependent morphology of the ACNS does not exist in New World monkeys. (2) The sympathetic ganglia in New World monkeys consist of the superior cervical, the middle cervical, and the cervicothoracic which is composed of the inferior cervical and first and second thoracic, and the thoracic ganglia starting with the third thoracic. The general cardiac nervous system is the sympathetic middle and inferior cardiac nerves and all parasympathetic vagal cardiac branches. (3) The morphology of the ACNS in the New World monkeys is almost consistent regardless of the number of vertebrae, the cardiac position and deviation (axis), and the great arterial branching pattern of the aortic arch, and it is very similar to that in the Old World monkeys, with only one difference: the superior cervical ganglion in the New World monkeys tends to be relatively smaller, higher, and provides a narrower contribution to the spinal nerves than in the Old World monkeys. The ACNS morphology exhibits significant evolutionary changes within the primate lineage from New and Old World monkeys to humans. The comparative morphology within the lineage is concordant with the phylogeny, suggesting that the primate ACNS preserves its evolutionary history in close alignment with phylogeny.

A Selective Inhibitory Effect of the Posterior Cerebellar Vermis on 10‐Hz Sympathetic Nerve Discharge

We compared the changes in inferior cardiac sympathetic nerve discharge (SND) and mean arterial pressure (MAP) produced by aspiration of portions of lobule IX and in some cases lobule X of the posterior cerebellar vermis with those produced by chemical inactivation (muscimol microinjection) or electrical stimulation of the same regions. The experiments were performed on 13 baroreceptor‐denervated and vagotomized cats anesthetized with urethane. Autospectral analysis was used to decompose SND into its frequency components. Special attention was paid to the question of whether the experimental procedures differentially affected the rhythmic and aperiodic components of SND. Aspiration or chemical inactivation with muscimol of lobule IX produced an approximately three‐fold increase in the 10‐Hz rhythmic component of SND (P = 0.05). In contrast, wideband SND at lower frequencies (= 6 Hz) was either slightly reduced or unaffected. Total power (0‐ to 20‐Hz band) was unchanged. Despite this, there was a statistically significant increase in MAP. High frequency (50‐Hz) electrical stimulation of depressor sites in lobule IX of the posterior vermis selectively and significantly reduced 10‐Hz power in SND. The lower frequency components of SND were unchanged. These results demonstrate for the first time a role of cerebellar cortical neurons of the posterior vermis in regulating the frequency composition of naturally occurring SND. The functional implications of these findings are discussed.(Supported by NIH HL33266.)

Role of serotonergic input to the ventrolateral medulla in expression of the 10-Hz sympathetic nerve rhythm

We studied the changes in inferior cardiac sympathetic nerve discharge (SND) produced by unilateral microinjections of 5-hydroxytryptamine (5-HT) receptor agonists and antagonists into the ventrolateral medulla (VLM) of urethane-anesthetized, baroreceptor-denervated cats. Microinjection of the 5-HT2 receptor antagonist LY-53857 (10 mM) into either the rostral or caudal VLM significantly reduced (P < or = 0.05) the 10-Hz rhythmic component of basal SND without affecting its lower-frequency, aperiodic component. The selective depression of 10-Hz power was accompanied by a statistically significant decrease in mean arterial pressure (MAP). Microinjection of LY-53857 into the VLM also attenuated the increase in 10-Hz power that followed tetanic stimulation of depressor sites in the caudal medullary raphé nuclei. Microinjection of the 5-HT2 receptor agonist 1-(2,5-dimethoxy-4-iodophenyl)2-amino-propane (DOI; 10 microM) into the VLM selectively enhanced 10-Hz SND, and intravenous DOI (1 mg/kg) partially reversed the reduction in 10-Hz SND produced by 5-HT2 receptor blockade in the VLM. Microinjection of the 5-HT1A receptor agonist, 8-hydroxy-2-(di-n-propylamino)tetralin (8-OHDPAT; 10 mM), into either the rostral or caudal VLM also selectively attenuated 10-Hz SND and significantly reduced MAP. The reduction in 10-Hz SND produced by 8-OHDPAT was partially reversed by intravenous WAY-100635 (1 mg/kg), which selectively blocks 5-HT1A receptors. These results support the view that serotonergic inputs to the VLM play an important role in expression of the 10-Hz rhythm in SND.

Differential baroreflex control of sympathetic drive by angiotensin II in the nucleus tractus solitarii

Microinjection of angiotensin II into the nucleus tractus solitarii attenuates the baroreceptor reflex-mediated bradycardia by inhibiting both vagal and cardiac sympathetic components. However, it is not known whether the baroreflex modulation of other sympathetic outputs (i.e., noncardiac) also are inhibited by exogenous angiotensin II (ANG II) in nucleus tractus solitarii (NTS). In this study, we determined whether there was a difference in the baroreflex sensitivity of sympathetic outflows at the thoracic and lumbar levels of the sympathetic chain following exogenous delivery of ANG II into the NTS. Experiments were performed in two types of in situ arterially perfused decerebrate rat preparations. Sympathetic nerve activity was recorded from the inferior cardiac nerve, the midthoracic sympathetic chain, or the lower thoracic-lumbar sympathetic chain. Increases in perfusion pressure produced a reflex bradycardia and sympathoinhibition. Microinjection of ANG II (500 fmol) into the NTS attenuated the reflex bradycardia (57% attenuation, P < 0.01) and sympathoinhibition of both the inferior cardiac nerve (26% attenuation, P < 0.05) and midthoracic sympathetic chain (37% attenuation, P < 0.05) but not the lower thoracic-lumbar chain (P = 0.56). We conclude that ANG II in the nucleus tractus solitarii selectively inhibits baroreflex responses in specific sympathetic outflows, possibly dependent on the target organ innervated.

Reflex Sympathetic Activity After Intravenous Administration of Midazolam in Anesthetized Cats

Although intrathecal midazolam has been reported to produce antinociceptive effects mediated by gamma-aminobutyric acid type A-benzodiazepine receptor complexes in the spinal cord, the effects of systemic midazolam on nociception remain unclear. We performed this study to examine the effects of IV-administered midazolam on somatosympathetic Adelta and C reflex discharges in brain-intact cats and decerebrate cats (with transection at midbrain level). Somatosympathetic Adelta and C reflexes were elicited in the inferior cardiac sympathetic nerve by electrical stimulation of myelinated (Adelta) and unmyelinated (C) afferent fibers of the superficial peroneal nerve in 28 mature cats. After control somatosympathetic reflex responses were obtained, midazolam was administered IV to four groups of randomly allocated cats as follows: brain-intact cats at a dose of 0.03 mg/kg, brain-intact cats at a dose of 0.1 mg/kg, brain-intact cats at a dose of 0.5 mg/kg, and decerebrate cats at a dose of 0.1 mg/kg. C reflex discharges were significantly augmented at the dose of 0.03 mg/kg and significantly depressed at the dose of 0.1 and 0.5 mg/kg in brain-intact cats. C reflex discharges were also significantly depressed at the dose of 0.1 mg/kg in decerebrate cats. We have demonstrated that IV midazolam produces dose-related effects on somatosympathetic reflex discharges. The clinical implication of these findings is that the effect of midazolam on nociception depends on its dosage. It also appears that the infra-midbrain region plays a major role in mediating the depressive effects of midazolam on somatosympathetic C reflex discharges.

Role of ventrolateral medulla in generating the 10-Hz rhythm in sympathetic nerve discharge

We recorded changes in right inferior cardiac and either left inferior cardiac or left vertebral sympathetic nerve discharge (SND) produced by unilateral microinjections of GABA-A and excitatory amino acid (EAA) receptor antagonists into the ventrolateral medulla (VLM) of urethane-anesthetized, baroreceptor-denervated cats. Unilateral microinjections of GABA-A receptor antagonists, SR-95531 or bicuculline, into single tracks in VLM anywhere between 1 and 5 mm rostral to the obex eliminated or markedly reduced 10-Hz power in SND on both sides of the body. Low-frequency components (<6 Hz) of SND were unaffected. Complete blockade of the 10-Hz rhythm occurred with a dose of SR-95531 as low as 6.25 pmol in a 50-nl volume. Unilateral microinjections of the nonselective EAA receptor antagonist, kynurenate (KYN; 7.5 nmol), into the caudal or rostral VLM significantly reduced, but did not eliminate, 10-Hz SND ipsilateral to the injection sites, while 10-Hz SND contralateral to the injection sites was not significantly changed. These observations suggest that 1) GABAergic transmission in VLM is critical for generation of the 10-Hz rhythm, 2) the caudal and rostral portions of VLM act together to generate the 10-Hz rhythm, and 3) 10-Hz rhythm generation depends, at least in part, on tonic or phasic excitatory drive to GABAergic interneurons in caudal VLM and presympathetic neurons in rostral VLM. The data also suggest that pathways interconnecting the two halves of the brain stem play an important role in promoting 10-Hz rhythm generation.

Hierarchical recruitment of the sympathetic and parasympathetic limbs of the baroreflex in normotensive and spontaneously hypertensive rats

The arterial baroreflex acts to buffer acute changes in blood pressure by reciprocal modulation of sympathetic and parasympathetic activity that controls the heart and vasculature. We have examined the baroreflex pressure-function curves for changes in heart rate and non-cardiac sympathetic nerve activity (SNA, thoracic chain T8-12) in artificially perfused in situ rat preparations. We found that the non-cardiac SNA baroreflex is active over a lower range of pressures than the cardiac baroreflex (threshold 66 +/- 1 mmHg versus 82 +/- 5 mmHg and mid-point 77 +/- 3 versus 87 +/- 4 mmHg, respectively, P < 0.05, n = 6). This can manifest as a complete dissociation of the baroreflex limbs at low pressures. This difference between the cardiac and non-cardiac SNA baroreflex is also seen in end-organ sympathetic outflows (adrenal and renal nerves). Recordings of the cardiac vagal (parasympathetic) and the inferior cardiac (sympathetic) nerves identify the cardiac parasympathetic baroreflex component as being active over a higher range of pressures. This difference in the operating range of the baroreflex-function curves is exaggerated in the spontaneously hypertensive rat where the cardiac component has selectively reset by 20-25 mmHg to a higher pressure range (threshold of 104 +/- 4 mmHg and mid-point 113 +/- 4, n = 6). The difference in the pressure-function curves for the cardiac versus the vascular baroreflex indicates that there is a hierarchical recruitment of the output limbs of the baroreflex with a sympathetic predominance at lower arterial pressures.

Role of GABA in generating the 10‐Hz rhythm in sympathetic nerve discharge

The 10-Hz rhythm in SND of the cat is dependent on neurons located in the caudal and rostral ventral lateral medulla (VLM) and medullary raphe (Barman and Gebber, J Biol Rhythms 15: –379, 2000). We sought to determine whether GABA-mediated synaptic transmission in these regions is involved in 10-Hz rhythm generation. For this purpose, we recorded the discharges of the left and right inferior cardiac or the left vertebral and right inferior cardiac postganglionic sympathetic nerves in baroreceptor-denervated cats anesthetized with urethane. Unilateral microinjection of GABA-A receptor antagonists (50 nl of 1- mM solution of either SR-95531 or bicuculline) into the VLM eliminated the 10-Hz rhythm bilaterally. This was the case for injections made in single or multiple tracks in the VLM between the obex and 6 mm rostral. Recovery occurred within 3 h, and 10-Hz SND was unchanged after microinjection of vehicle (phosphate buffered saline). In contrast, the 10-Hz rhythm in SND was enhanced by multiple microinjections of the GABA antagonists into the raphe from 0 to 6 mm rostral to the obex. These results indicate that GABA-mediated synaptic inhibition in the VLM is critical for 10-Hz rhythm generation. Moreover, the data support the view that networks of neurons located bilaterally throughout the VLM act as a unit to generate the 10-Hz rhythm in SND. (Supported by NIH grant HL33266.)

A decerebrate, artificially-perfused in situ preparation of rat: Utility for the study of autonomic and nociceptive processing

By extending established technology, we have developed a relatively simple in situ preparation from juvenile rat that overcomes some technical restrictions present in vivo (e.g. need for anaesthesia, mechanical instability for intracellular recording and control over the extracellular milieu). The in situ preparation is decerebrate and artificially perfused via the left ventricle with a colloid containing solution. It exhibits an eupneic pattern of respiratory motor activity and demonstrates numerous somatic and visceral reflexes including those evoked by stimulation of the tail, hindlimbs, bladder, baroreceptors and peripheral chemoreceptors. We have employed this preparation to allow recordings from multiple sympathetic motor outflows such as thoracic and lumbar chain, inferior cardiac, splanchnic, renal and adrenal nerves. We show that the sympathetic motor discharge shows strong respiratory modulation and exhibits pronounced reflex modulation indicating intact communication between the periphery, the brainstem and the spinal cord. Further, we have made extracellular and whole cell recordings from neurones in the spinal cord, demonstrating good mechanical stability. The decerebrate, artificially-perfused rat (DAPR) provides a powerful methodology with which to study peripheral and central control of the autonomic nervous system with many of the benefits of an in vitro environment.

Dynamic changes in baroreceptor-sympathetic coupling during the respiratory cycle

In urethane-anesthetized, paralyzed, and artificially ventilated cats, we observed an unusual form of “phase walk” of the cardiac-related burst of inferior cardiac postganglionic sympathetic nerve discharge (SND) relative to the systolic phase of the arterial pulse (AP) and thus pulse-synchronous baroreceptor nerve activity. Unlike classic phase walk ascribable to weakened coupling (desynchronization) of two oscillators, AP-SND phase walk was characterized by epochs of progressive, bidirectional changes in the angle of strong coupling (AP-SND coherence values, >0.9) of these signals that recurred on the time scale of the respiratory cycle and whose range was approximately one third of the period of the heart beat. AP-SND phase walk was linked to two respiratory variables (central respiratory drive and vagal lung inflation afferent activity) as demonstrated by the following observations. First, in five normocapnic cats (end-tidal CO 2, 4.3 ± 0.2%) with intact vagus nerves and three vagotomized cats, AP-SND phase walk was characterized by a progressive heart-beat-to-heart-beat decrease in the lag of SND relative to the AP during the inspiratory phase of phrenic nerve activity and an increase in the lag during the expiratory phase. Second, in three cats with intact vagus nerves that were hyperventilated (end tidal CO 2, 1.6 ± 0.4%) to phrenic nerve quiescence, the lag of the cardiac-related burst of SND relative to the AP increased during lung inflation and decreased during lung deflation. Additional experimentation revealed that AP-SND phase walk is attributable to respiratory-induced changes in the frequency of the centrally generated sympathetic nerve rhythm rather than heart rate. Moreover, the data demonstrate that the frequency and amplitude of the sympathetic oscillation are independently controlled by the above mentioned respiratory parameters.

The autonomic nervous system of the human heart with special reference to its origin, course, and peripheral distribution

A submacroscopic anatomical investigation of the entire autonomic cardiac nervous system, from origin to peripheral distribution, was performed by examining 36 sides of 18 adult human cadavers under a stereomicroscope. The following new results and points of discussion were obtained: (1) The superior cervical, the middle cervical, the vertebral, and the cervicothoracic (stellate) ganglia, composed of the inferior cervical and 1st thoracic ganglia, were mostly consistent among the specimens. (2) The superior, middle, and inferior cardiac nerves innervated the heart by simply following the descent of the great arteries. In contrast, the thoracic cardiac nerve in the posterior mediastinum followed a complex course because of the long distance to the middle mediastinum. (3) The actual course of the right thoracic cardiac nerve differed from that of the previous descriptions in that it ascended obliquely or ran transversely to the vertebrae, regardless of the intercostal vessels. Regarding the right thoracic cardiac nerve, two descending courses were observed: the descent of the right thoracic cardiac nerve via the azygos vein and right venous porta, and the descent of the recurrent right thoracic cardiac nerve via the aorta. (4) The cranial cardiac nerve and branch tended to distribute into the heart medially, and the caudal cardiac nerve and branch tended to distribute into the heart laterally. (5) The mixing positions (cardiac plexus) of the sympathetic cardiac nerve and the vagal cardiac branch, as well as the definitive morphology of brachial arteries with the recurrent laryngeal nerves, tended to differ on both sides. These new and detailed anatomical descriptions of the human autonomic cardiac nervous system may provide important clues regarding the morphogenesis of autonomic cardiac nerves in addition to contributing to the improvement of cardiac surgery.