Sinus and atrioventricular nodal distribution of sympathetic fibers that contain neuropeptide Y.

Neuropeptide Y and norepinephrine are localized in sympathetic nerve terminals throughout the heart. We sought to determine the functional distribution of the neuropeptide Y-containing sympathetic fibers to the sinus and atrioventricular (AV) nodal regions. We recorded cycle length, AV interval, and arterial pressure in 14 anesthetized dogs. We assessed the release of neuropeptide Y from sympathetic nerve terminals by measuring the attenuation of the vagal effects on cycle length and AV interval that occurred after unilateral ansa subclavia stimulation. Three-minute trains of right or left ansa stimulation, each applied at frequencies of 2, 5, and 10 Hz, produced a frequency-dependent inhibition of the vagal effects on cycle length and AV interval. After right ansa stimulation (10 Hz), however, the percent inhibition of the vagal effects on cycle length was 21 +/- 5% greater (p less than 0.001) than the percent inhibition of the vagal effects on AV interval. Conversely, after left ansa stimulation (10 Hz), the percent inhibition of the vagal effects on AV interval was 54 +/- 7% greater (p less than 0.001) than the percent inhibition of the vagal effects on cycle length. The vagal stimulus characteristics (frequency or voltage) did not significantly alter the percent inhibition, nor did the percent inhibition depend on the vagus stimulated (right or left vagus). We conclude that most of the neuropeptide Y-containing sympathetic fibers at the sinus node originate in right-sided ganglia, whereas most of those at the AV node originate in left-sided ganglia.

Innervation of somatostatin synthesizing neurons by adrenergic, phenylethanolamine-N-methyltransferase (PNMT)-immunoreactive axons in the anterior periventricular nucleus of the rat hypothalamus.

The adrenergic innervation of somatostatin synthesizing neurons located in the anterior region of the rat hypothalamic periventricular nucleus was studied by means of a light and electron microscopic immunocytochemical double labelling technique. This region which is the source of hypophysiotrophic somatostatin immunoreactive (IR) neurons also receives a dense plexus of adrenergic axons as determined by immunocytochemistry of phenylethanolamine-N-methyltransferase (PNMT), the marker enzyme for the central adrenergic system. The simultaneous detection of PNMT and somatostatin antigens in hypothalamic sections of colchicine pretreated animals revealed a congruency in the distribution of the labelled elements and also close juxtaposition of PNMT-IR axons to somatostatin producing neurons. At the ultrastructural level, axo-somatic and axo-dendritic synaptic connections were found between PNMT-containing axons and somatostatin expressing neurons. These morphological findings support the view that the central adrenergic system might influence the production and secretion of growth hormone in the pituitary gland by a direct monosynaptic interaction with somatostatin synthesizing neurons.

Distribution of phenylethanolamine N-methyltransferase cell bodies, axons, and terminals in monkey brainstem: an immunohistochemical mapping study.

Adrenaline (epinephrine) is an important candidate transmitter in descending spinal control systems. To date intrinsic spinal adrenergic neurons have not been reported; thus adrenergic input is presumably derived from brainstem sites. In this regard, the localization of adrenergic neurons in the brainstem is an important consideration. Maps of adrenergic cell bodies and to a lesser extent axons and terminal fields have been made in various species, but not in monkeys. Thus, the present study concerns the organization of adrenergic systems in the brainstem of a monkey (Macaca fascicularis) immunohistochemically mapped by means of an antibody to the enzyme phenylethanolamine N-methyltransferase (PNMT). PNMT-immunostained cell bodies are distributed throughout the medulla in two principal locations. One concentration of labeled cells is in the dorsomedial medulla and includes the nucleus of the solitary tract (NTS), the dorsal motor nucleus of the vagus (X), and an area ventral to X in a region of the reticular formation (RF) known as the central nucleus dorsalis (CnD) of the medulla. A few scattered cells are observed in the periventricular gray just ventral to the IVth ventricle and on midline in the raphe. The second major concentration of PNMT-immunostained cells is located in the ventrolateral RF, lateral and dorsolateral to the inferior olive (IO), including some cells in the rostral part of the lateral reticular nucleus (LRN). Terminal fields are located in the NTS, X, area postrema (AP), and the floor of the IVth ventricle in the medulla and pons. A light terminal field is also observed in the raphe, particularly raphe pallidus (RP). A heavy terminal field is present in locus coeruleus (LC). Fibers labeled for PNMT form two major fiber tracts. One is in the dorsomedial RF extending as a well-organized bundle through the medulla, pons, and midbrain. A second tract is located on the ventrolateral edge of the medulla and caudal pons. Fibers in this tract appear to descend to the spinal cord. A comparison with maps of other catecholamine neurons in primates is discussed, confirming that the distribution of the adrenergic system in monkeys is similar to that described in the human.

Galanin-like immunoreactivity in sympathetic and parasympathetic neurons of the toad Bufo marinus.

Immunoreactivity (IR) to galanin (GAL) was detected in a wide range of peripheral autonomic neurons in the toad Bufo marinus. Forty percent of adrenergic nerve cell bodies in paravertebral sympathetic ganglia had GAL-IR in addition to neuropeptide Y (NPY)-IR. Some of these neurons projected to systemic arteries. GAL-IR was localized in parasympathetic neurons supplying the heart, lung, pulmonary artery, bladder, rectum and tongue. Eighty-two percent of intracardiac vagal nerve cell bodies had both GAL-IR and somatostatin (SOM)-IR. GAL-IR and SOM-IR were also co-localized in cholinergic post-ganglionic vagal neurons supplying the lung musculature and the pulmonary artery, and in neurons intrinsic to the bladder. Many postganglionic glossopharyngeal neurons in the tongue contained both GAL-IR and vasoactive intestinal peptide (VIP)-IR. Therefore, in Bufo marinus, a GAL-like peptide, in combination with other peptides or with adrenaline or acetylcholine, may be involved in neurotransmission in several different functional classes of autonomic neurons.

Localization of adrenergic and neuropeptide tyrosine-containing nerves in the mammalian liver.

The distribution of adrenergic nerves in guinea pig and rat liver was studied by the immunolocalization of fibers containing tyrosine hydroxylase and dopamine beta-hydroxylase, enzymes involved in the synthesis of catecholamines. In both species, adrenergic fibers were identified within portal tracts, often in close proximity to hepatic artery branches. In guinea pig liver, but not rat liver, abundant intraacinar fibers were identified; fibers were also seen within the walls of terminal hepatic vein radicles and larger hepatic veins. The presence of peptidergic nerves containing the regulatory peptide neuropeptide tyrosine and the C-flanking peptide CPON was investigated by indirect immunofluorescence. The distribution of these nerves was similar to that of tyrosine hydroxylase- and dopamine beta-hydroxylase-positive nerves and showed the same species difference. The effector sympathetic nature of tyrosine hydroxylase- and neuropeptide tyrosine-positive fibers in rat liver was confirmed by chemical denervation studies using 6-hydroxydopamine.

Noradrenergic neurons with divergent projections to the motor trigeminal nucleus and the spinal cord: a double retrograde neuronal labeling study.

Double retrograde axonal tracing was combined with the indirect immunofluorescence antibody method to determine whether noradrenergic neurons have divergent projections to the motor nucleus of the trigeminal nerve and the spinal cord. Rhodamine-labeled microspheres were injected into the motor trigeminal nucleus and True Blue was deposited into lumbar segments of the spinal cord. After a 10-18-day survival period, brainstem sections were processed for immunofluorescence staining of noradrenergic neurons using antibodies to rat dopamine-beta-hydroxylase. Rhodamine-labeled noradrenergic neurons were observed ipsilaterally throughout the A5 and A7 groups; the contralateral A5 and A7 groups contained few rhodamine-labeled cells. A few rhodamine-labeled noradrenergic neurons were observed in the locus coeruleus and subcoeruleus. True Blue-labeled noradrenergic neurons were identified in the A5 and A7 groups, in the ventral part of the locus coeruleus and in the subcoeruleus. Double retrogradely labeled noradrenergic neurons were observed in the A5 and A7 groups but not in the locus coeruleus and subcoeruleus. Of the total number of rhodamine-labeled noradrenergic cells, a large percentage also contained True Blue: 54% in the caudal A5 group, 59% in the rostral A5 group, and 72% in the A7 group. Of the total number of True Blue-labeled noradrenergic neurons, the percentage of double retrogradely labeled cells was 33% in the caudal A5 group, 46% in the rostral A5 group, and 56% in the A7 group. The findings of this study provide the first anatomic evidence for the existence of a prominent population of noradrenergic cells in the A5 and A7 groups with divergent projections to the motor trigeminal nucleus and the spinal cord. We propose that this subpopulation of noradrenergic neurons in the A5 and A7 groups influences motoneurons at multiple levels of the neuraxis.

Multiple neuropeptides in nerves supplying mammalian lymph nodes: messenger candidates for sensory and autonomic neuroimmunomodulation?

By the use of light microscopic (LM) immunohistochemistry, the presence of peptides and of dopamine beta-hydroxylase (DBH) in nerves supplying mammalian (guinea pig, rat, cat, pig, mouse, human) lymph nodes were examined. In all species, lymph nodes of various somatic and visceral regions were found to contain nerve fibers which stained for neuropeptide Y (NPY), vasoactive intestinal polypeptide (VIP), peptide histidine isoleucine (PHI), substance P (SP), calcitonin gene-related peptide (CGRP) or DBH. SP- and CGRP-immunoreactive (ir) fibers completely overlapped and exhibited the widest distribution. They were present in perivascular, paravascular and many non-vascular fibers travelling in close contact with lymphoid cells. In contrast, NPY-ir fibers coincided with those staining for DBH, prevailed in perivascular plexus and only rarely branched off into lymphoid parenchyma. Alternate staining of adjacent sections revealed that SP/CGRP-ir fibers were different from NPY/DBH-ir fibers. The distribution of VIP-ir fibers was identical to that of PHI-ir fibers and partially overlapped with that of ir-NPY/DBH or ir-SP/CGRP fibers. We conclude that the NPY innervation of lymph nodes is sympathetic noradrenergic while nerves coding for co-existing SP and CGRP are most likely of sensory origin. The nerves containing co-existing VIP and PHI may be of heterogenous origin (sensory, cholinergic sympathetic, and/or parasympathetic). We suggest that these distinct sensory and autonomic peptidergic pathways linking the nervous system with the lymph nodes may play a differential role in bidirectional neuroimmunomodulation.

Immunohistochemical detection of GTP-binding regulatory protein (Go) in the autonomic nervous system including the enteric nervous system, superior cervical ganglion and adrenal medulla.

The localization of a GTP-binding regulatory protein, Go, in the autonomic nervous system including the enteric nervous system, superior cervical ganglion, and adrenal medulla, has been immunohistochemically examined by use of affinity-purified antibody against the alpha-subunit of Go. In the small intestine, dense Go-immunoreactive products were localized on the enteric nervous system, i.e. the myenteric plexus of Auerbach and the submucosal plexus of Meissner. In the superior cervical ganglion, presynaptic terminals were strongly immunoreactive to the Go antibody. The adrenal medulla was stained with this antibody, but the adrenal cortex was not immunoreactive to this antibody. Thus, the present study strongly suggests that Go is localized in the autonomic nervous system and plays its role in transmembrane signal transmission in this system.

Anatomic evidence for coexistence of cholinergic and adrenergic neurons in the developing human intestine: new aspects in the pathogenesis of developmental neuronal abnormalities.

In order to interpret the interrelation of cholinergic and adrenergic myenteric neural elements in the developing human, histochemical methods were used to demonstrate cholinergic and adrenergic activity independently in consecutive cryostat sections of the esophagus, ileocecal region, and colon of fetuses of 9 to 22 weeks ovulation age. At least some of the neural cells remained plastic, with respect to their transmitter choice, and showed both cholinergic and adrenergic function. These cells were shown to be present first in the ileocecal region (11 weeks) then in the esophagus (12 weeks) and lastly in the colon (14 weeks). Our findings support the hypothesis of a dual gradient of maturation of enteric neurons. The choice of transmitters is influenced by a "microenvironmental" factor, which may also arrest the maturation or cause the death of neurons.

Adrenergic and non-adrenergic neurons in the C1 and C3 areas project to locus coeruleus: a fluorescent double labeling study.

Following iontophoretic injections of the retrograde tracer Fluoro-gold into the rat locus coeruleus (LC), retrogradely labeled neurons were seen predominantly in the area of C1 adrenergic neurons in the ventrolateral medulla (nucleus paragigantocellularis; PGi) and in the area of C3 adrenergic neurons in the dorsomedial medulla (nucleus prepositus hypoglossi; PrH). Subsequent immunofluorescence for phenylethanolamine-N-methyltransferase indicated that adrenergic and non-adrenergic LC projecting neurons in both areas are interdigitated, and that 21% of LC afferent neurons in the PGi are adrenergic while only 4% of LC afferent neurons in the area of PrH are adrenergic.

Distribution and origin of vasoactive intestinal polypeptide (VIP)-immunoreactive, acetylcholinesterase-positive and adrenergic nerves of the cerebral arteries in the bent-winged bat (Mammalia: Chiroptera).

The overall distribution and origins of vasoactive intestinal polypeptide (VIP)-immunoreactive (IR), acetylcholinesterase (AChE)-positive and adrenergic nerves in the walls of the cerebral arteries were investigated in the bent-winged bat. VIP-IR and AChE-positive nerves innervating the bat cerebral vasculature appear to arise mainly from VIP-IR and AChE-positive cell bodies within microganglia found in the nerve bundle accompanying the sympathetic nerve bundle within the tympanic cavity. These microganglia, as well as the nerve bundle containing them, do not emit catecholamine fluorescence, suggesting that they are of the cranial parasympathetic outflow, probably the facial or glossopharyngeal one. The axons from VIP-IR and AChE-positive microganglia run intermingled with sympathetic adrenergic nerves in the same thick fiber bundles, and reach the cranial cavity through the carotid canal. In addition, some of the VIP-IR fibers innervating the vertebro-basilar system, at least the basilar artery, originate from VIP-IR nerve cells located in the wall of this artery. The supply of VIP-IR fibers to the bat major cerebral arteries is the richest among mammals in that it is much greater in the vertebro-basilar system than in the internal carotid system: plexuses of VIP-IR nerves are particularly dense along the walls from the posterior ramus to posterior cerebral and basilar arteries. Small pial and intracerebral arteries of the vertebro-basilar system, especially those of the posterior cerebral artery which supply most parts of the diencephalon and cerebrum, are also richly innervated by peripheral VIP-IR fibers. This pattern corresponds well with the innervation pattern of adrenergic and AChE-positive nerves.

Measurement of intracellular Ca2+ in the bullfrog sympathetic ganglion cells using fura-2 fluorescence.

The intracellular free Ca2+ concentration ([Ca2+]i) of the bullfrog sympathetic ganglion cell was measured with fura-2 fluorescence under various conditions, and compared with changes in membrane potential recorded with an intracellular electrode. The [Ca2+]i was 109 nM on average under the resting condition and increased by raising the extracellular K+, stimulating repetitively the pre- or post-ganglionic nerve, or by applying acetylcholine or muscarine. Since all these procedures depolarized the cell membrane, most of the rise in [Ca2+]i could be the result of opening of voltage-dependent Ca2+ channels. However, Ca2+ entries through nicotinic acetylcholine receptor channels and the channel activated by the muscarinic acetylcholine receptor were also indicated by considering the threshold for the opening of voltage-dependent Ca2+ channels (for both entries) or a limited number of the cells showing the latter response.

Evidence for coexistence of serotonin and noradrenaline in sympathetic nerves supplying brain vessels of guinea pig.

Nerve fibers containing 5-hydroxytryptamine (5-HT) were demonstrated immunohistochemically in the wall of pial vessels associated with the circle of Willis in the guinea pig. The fibers formed a network structure which was more dense in the rostral part of the arterial circle and its branches than in the caudal part. The 5-HT immunoreactive fibers disappeared in all arteries studied after bilateral superior cervical ganglionectomy, and unilateral ganglionectomy eliminated the 5-HT immunoreactivity in the ipsilateral part of the middle cerebral, posterior cerebral and superior cerebellar arteries. Decentralization of the superior cervical ganglion had no effect on the perivascular nerve plexus. Subsequent staining with dopamine-beta-hydroxylase (DBH) antiserum following elution of the first antibody revealed that 5-HT was present in the noradrenergic nerve fibers. Small intensive fluorescent cells with positive immunoreaction for 5-HT and DBH, respectively, were located in clusters within the ganglion, which showed no immunohistochemical evidence for the presence of serotonergic neurons. It is concluded that 5-HT is probably not synthesized in truly serotonergic fibers but rather taken up and stored together with noradrenaline in cerebrovascular sympathetic nerves originating in the superior cervical ganglia.

Immunocytochemical evidence for substance P and serotonin input to medullary bulbospinal adrenergic neurons.

Using two-color immunoperoxidase staining combined with the retrograde transport of horseradish peroxidase injected into the rostral thoracic spinal cord, substance P-immunoreactive (SPI) and serotonin-immunoreactive (5HTI) varicosities have been observed in contiguity with medullary bulbospinal phenylethanolamine N-methyl transferase-immunoreactive (PNMTI) neurons of the C1, C2, and C3 cell groups. Since PNMTI terminals in the spinal cord are concentrated among sympathetic preganglionic neurons (SPGN) in the intermediolateral cell column, the close anatomical associations shown in the present study indicate that substance P- and serotonin-containing pathways in the medulla likely affect activity of SPGN via adrenergic bulbospinal neurons.

Innervation of radicular and extraparenchymal arteries of spinal cord. Histochemical and immunohistochemical study in primate.

The perivascular innervation of extraparenchymal arteries of spinal cord and the radicular arteries was examined using histochemical and immunohistochemical technics in monkey. The radicular and the extraparenchymal arteries of spinal cord were found to be invested with adrenergic, neuropeptide Y, vasoactive intestinal peptide, substance P and calcitonin gene-related peptide containing nerve fibres. The pattern of arrangement of fibres differed among the various fibre types. SP- and CGRP-containing fibres were less in density as compared to other nerve plexus. There was no difference in density of an individual type of nerve fibre in arteries of different cord segments or between the radicular arteries from different levels. The study reveals the existence of a comprehensive perivascular adrenergic and peptidergic innervation of spinal cord arterial system, with a possible role in neurogenic regulation of spinal cord circulation.

A developmentally regulated isoform of 150,000 molecular weight neurofilament protein specifically expressed in autonomic and small sensory neurons.

Neurofilament heterogeneity has been demonstrated using a monoclonal antibody (CH1) specific for the 150,000 molecular weight neurofilament subunit. In the peripheral nervous system of adult rats CH1 stained selectively sympathetic and parasympathetic neurons and a subpopulation of small neurons in the sensory dorsal root ganglia. Somatic motor neurons and large neurons in dorsal root ganglia were completely unreactive. In contrast, the anti-neurofilament antibody iC8, directed against the 150,000 molecular weight subunit, labelled all peripheral nervous system neurons. The immunostaining pattern with both antibodies was unchanged by phosphatase treatment. These data indicate that two antigenically distinct variants of the 150,000 molecular weight neurofilament subunit exist in somatic and autonomic neurons of adult animals. In addition, the phosphatase treatment suggests that the antigen recognized by CH1 is not masked by phosphorylation. In contrast, all neurons were labelled by this antibody in the peripheral nervous system of newborn rats. It is suggested that CH1 identifies a fetal 150,000 molecular weight neurofilament polypeptide isoform whose expression is prevented by the growth of somatic neurons and is selectively maintained in autonomic and small sensory neurons.

Immunohistochemical evidence for the adrenergic medullary longitudinal bundle as a major ascending pathway to the hypothalamus.

Three weeks after unilateral electrolytic lesion of the longitudinal axon bundle in the medulla oblongata of the rat, we observed a decrease in the number of phenylethanolamine-N-methyltransferase (PNMT)-immunoreactive (IR) nerve fibers in virtually all the regions of the diencephalon ipsilaterally to the lesion, especially in the dorsomedial nucleus and the paraventricular nucleus of the hypothalamus. These results indicate that the hypothalamic PNMT-IR terminal-like fibers originate in the ipsilateral medulla oblongata presumptive adrenaline-containing (Ad) neurons especially through ascending projections provided in majority by the longitudinal axon bundle. Further, no PNMT-IR cell bodies were detected in the hypothalamus even after colchicine treatment.

Intrathecal 6-hydroxydopamine or cervical spinal hemisection reduces norepinephrine content, but not the density of alpha 2-adrenoceptors, in the cat lumbar spinal enlargement.

The effect of the intrathecal administration of the catecholaminergic neurotoxin 6-hydroxydopamine, or of hemisection of the spinal cord at the Cl level, on the density of alpha 2-adrenoceptors and on the norepinephrine, dopamine, and serotonin content in the cat lumbar spinal enlargement was determined 2, 7 or 21 days after performance of each type of lesion. The intrathecal administration of 6-hydroxydopamine produced a time-dependent reduction of norepinephrine content in the cat lumbar spinal enlargement (95% reduction at 21 days) without significantly altering the serotonin content in this same tissue of the same cats. The dopamine content of the dorsal horn was not changed significantly, whereas ventral horn dopamine content was depleted after intrathecal 6-hydroxydopamine. alpha 2-Adrenoceptor binding site density was not significantly different from control either 2 or 21 days after 6-hydroxydopamine, but was increased significantly (50%) over the control density 7 days after 6-hydroxydopamine. Hemisection of the cervical spinal cord produced a bilateral 40-60% reduction of norepinephrine content in both the dorsal and ventral horns of the cat lumbar spinal enlargement 7 and 21 days later. Cervical hemisection did not significantly alter the alpha 2-adrenoceptor binding site density in these same cats either 2, 7, or 21 days after performance of the lesion. It is concluded that alpha 2-adrenoceptors located on the terminals of descending noradrenergic or other spinopetal fibers do not represent a significant fraction of the total population of alpha 2-adrenoceptors present in the dorsal or ventral cat lumbar enlargement.

Neurochemical evidence for two types of presynaptic alpha 2-adrenoceptors.

Neurochemical and pharmacological evidence has been obtained that noradrenergic varicosities (in mouse and rat vas deferens) and cholinergic varicosities (in the Auerbach's plexus) contain heterogenous alpha 2-adrenoceptors through which the release of [3H]noradrenaline and [3H]acetylcholine can be modulated. The quantitative data also support the hypothesis that different noradrenaline and xylazine sensitive alpha 2-adrenoceptors are present prejunctionally in the vas deferens and Auerbach's plexus preparations. Prazosin, although it has a presynaptic inhibitory effect on alpha 2-adrenoceptors of noradrenergic axon terminals, has no effect on cholinergic axon terminals. These data suggest that there are two different types of alpha 2-adrenoceptors at the presynaptic axon terminals.