Neurochemical features of the autonomic neurons projecting to the cremaster muscle of the boar.

The cremaster muscle (CM) is a striated muscle showing some unusual features for ordinary striated muscles, in fact it receives, besides somatic innervation, a conspicuous autonomic sympathetic innervation. The autonomic neurons associated with the CM of 4 male intact pigs were typified combining the retrograde nontrans-synaptic fluorescent tracer Fast Blue (FB) and double labeling immunohistochemical methods. We collected the L4 sympathetic trunk ganglion (STG), that our preliminary studies proved to contain the highest number (575.5 ± 152.93; mean ± S.E.M., n = 4) of FB+ sympathetic neurons projecting to CM. About half of the CM projecting neurons of this ganglion were catecholaminergic and showed the colocalization of Tyrosine Hydroxylase (TH) with Neuropeptide Y (NPY), Leu-Enkephaline (LENK), Vasoactive Intestinal Polypeptide (VIP), Calcitonine Gene Related Peptide (CGRP), Substance P (SP), neuronal Nitric Oxyde Sinthase (n-NOS), and Vesicular Acetylcholine Transporter (VAChT). The noncatecholaminergic neurons were immunoreactive for all the other markers tested, even if in small percentages. The conspicuous and heterogeneous contribution of the sympathetic autonomic neurons to the muscle innervation is consistent with the hypothesis of a possible origin of the CM fibers by transdifferentiation of the smooth muscle-like gubernaculum mesenchyma into striated myotubes, suggesting that the cremaster myogenesis is independent from that of the abdominal muscles.

A combined acetylcholinesterase and immunohistochemical method for precise anatomical analysis of intrinsic cardiac neural structures.

A significant challenge when investigating autonomic neuroanatomy is being able to reliably obtain tissue that contains neuronal structures of interest. Currently, histochemical staining for acetylcholinesterase (AChE) remains the most feasible and reliable method to visualize intrinsic nerves and ganglia in whole organs. In order to precisely visualize and sample intrinsic cardiac nerves and ganglia for subsequent immunofluorescent labeling, we developed a modified histochemical AChE method using material from pig and sheep hearts. The method involves: (1) chemical prefixation of the whole heart, (2) short-term and weak histochemical staining for AChE in situ, (3) visual examination and extirpation of the stained neural structures from the whole heart, (4) freezing, embedding and cryostat sectioning of the tissue of interest, and (5) immunofluorescent labeling and microscopic analysis of neural structures. Firstly, our data demonstrate that this modified AChE protocol labeled intrinsic cardiac nerves as convincingly as our previously published data. Secondly, there was the added advantage that adrenergic, cholinergic and peptidergic neuropeptides, namely protein gene product 9.5 (PGP 9.5), neurofilament (NF), tyrosine hydroxylase (TH), vesicular monoamine transporter (VMAT2), neuronal nitric oxide synthase (nNOS), choline acetyltransferase (ChAT), calcitonin gene related peptide (CGRP), and substance P may be identified. Our method allows the precise sampling of neural structures including autonomic ganglia, intrinsic nerves and bundles of nerve fibers and even single neurons from the whole heart. This method saves time, effort and a substantial amount of antisera. Nonetheless, the proof of specific staining for many other autonomic neuronal markers has to be provided in subsequent studies.

Sensory and autonomic innervation of the cervical intervertebral disc in rats: the pathomechanics of chronic discogenic neck pain.

STUDY DESIGN: An immunohistological analysis of the cervical intervertebral disc (IVD). OBJECTIVE: To investigate sensory and autonomic innervation of the rat cervical IVD. SUMMARY OF BACKGROUND DATA: Many clinicians are challenged with treating wide-ranging chronic neck pain. Several authors have reported that sympathetic nerves participate in chronic pain, and various sympathectomy procedures can effectively treat chronic pain. METHODS: The neuro-tracer Fluoro-gold (FG) was applied to the anterior surfaces of C5-C6 IVDs from 10 Sprague-Dawley rats to label the neurons of the innervating dorsal root ganglion (DRG), stellate ganglion (SG; sympathetic ganglion), and nodose ganglion (NG; parasympathetic ganglion). Seven days postsurgery, DRGs from level C1-C8, SG, and NG neurons were harvested, sectioned, and immunostained for calcitonin gene-related peptide (CGRP; a marker for peptide-containing neurons) and isolectin B4 (IB4; a marker for nonpeptide-containing neurons). The proportion of FG-labeled DRG neurons that were CGRP-immunoreactive (CGRP-IR), IB4-binding, and non-CGRP-IR and IB4-binding, and the proportion of FG-labeled SG neurons and NG neurons were calculated. RESULTS: FG-labeled neurons innervating the C5-C6 IVD were distributed throughout the C2-C8 DRGs. The proportions of FG-labeled DRG neurons that were CGRP-IR, IB4-binding, non-CGRP-IR and IB4-binding, as well as SG neurons, and NG neurons were 20.6%, 3.3%, 55.7%, 8.9%, and 11.5%, respectively. The proportion of CGRP-IR FG-labeled DRG neurons was significantly higher than the proportion of IB4-binding FG-labeled DRG neurons at each level (P < 0.05). CONCLUSION: The C5-C6 IVD was innervated multisegmentally from neurons of the C2-C8 DRG, SG, and NG. Overall, 79.6% of the nerve fibers innervating the IVD were sensory nerves and 20.4% were autonomic nerves. Furthermore, 23.9% of the nerve fibers innervating the IVD were afferent sensory pain-related nerves, 8.9% were efferent sympathetic nerves, and 11.5% were efferent parasympathetic nerves. These findings may explain the wide-ranging and chronic discogenic pain that occurs via the somatosensory and autonomic nervous system.

Immunohistochemical characteristics of the nerve fibres of sow retractor clitoridis muscle.

The occurrence of several biologically active neuropeptides (calcitonine gene-related peptide, leu-enkephaline, neuropeptide Y, substance P, and vasoactive intestinal peptide) or nitric oxide-synthesizing enzymes (neuronal nitric oxide synthase), tyrosine hydroxylase, vesicular acetylcholine transporter, and their co-localization with tyrosine hydroxylase were investigated by immunohistochemistry in the retractor clitoridis muscle of slaughtered sows. Single immunolabelling revealed that tyrosine hydroxylase and neuropeptide Y immunoreactive nerve fibres were the most numerous, followed by the neuronal nitric oxide synthase and calcitonine gene-related peptide immunoreactive ones, the vasoactive intestinal peptide, substance P and leu-enkephaline immunoreactive nerve fibres were few and vesicular acetylcholine transporter immunoreactivity were observed only in single fibres. Double immunolabelling revealed the only co-localization of tyrosyne hydroxylase with neuropeptide Y. The most reliable labelling of nerve fibres of the retractor clitoridis muscle was observed around blood vessels, followed by non-vascular smooth muscles. The present data indicate that the sow retractor clitoridis muscle receives nerve fibres that exhibit different chemical codes and, likely, differences in their chemical coding depend on the target-structure.

Increased nerve fibers in placental bed myometrium in women with preeclampsia.

Narrowing of the uterine spiral arterioles below the deciduomyometrial junction is 1 of the key pathophysiological changes in women with preeclampsia. The contribution of pelvic autonomic nerves to decidualization and impaired placentation in preeclampsia is not clear. Placental bed biopsies were obtained from 10 women with preeclampsia and 23 nornotensive women at caesarean section. We stained them with anti-S100 and CD34 antibodies to detect the presence of nerve fibers and blood vessels, respectively. We detected S100-immunoactive nerve fibers in the myometrium but not in the decidua in both groups of women. S100-immunoactive nerve fiber density in the placental bed myometrium was significantly increased in women with preeclampsia compared to normotensive women. There was no clear relationship between the densities of nerve fibers and CD34-positive blood vessels in these biopsies. These results suggest increased nerve fibers in the placental bed myometrium may play a role in the pathogenesis of the preeclampsia.

Division of autonomic nerves within the neurovascular bundles distally into corpora cavernosa and corpus spongiosum components: immunohistochemical confirmation with three-dimensional reconstruction.

BACKGROUND: Detailed knowledge of the distribution and distal course of periprostatic nerves is essential to improve functional outcomes (erection and continence) after radical prostatectomy (RP). OBJECTIVE: To describe the location of nerve fibres within neurovascular bundles (NVBs) and around the prostate by three-dimensional (3D) computer-assisted anatomic dissection (CAAD) in human foetuses and adult cadavers. DESIGN, SETTING, AND PARTICIPANTS: Serial transverse sections of the pelvic portion were performed in seven human male foetuses and four male adult cadavers. Sections were treated by histologic coloration and neuronal immunolabelling of S100 protein. 3D pelvic reconstruction was achieved with digitised serial sections and WinSurf software. MEASUREMENTS: We evaluated the distribution of nerve fibres within the NVB qualitatively. The distribution of periprostatic nerves was also evaluated quantitatively in the adult specimens. RESULTS AND LIMITATIONS: Periprostatic nerve fibres were dispersed around the prostate on all sides with a significant percentage of these fibres present in the anterior and anterolateral sectors. At the prostate apex and the urethral levels, the NVBs have two divisions: cavernous nerves (CNs) and corpus spongiosum nerves (CSNs). The CNs were a continuation of the anterior and anterolateral fibres around the apex of the prostate, travelling towards the corpora cavernosa. The CSNs were a continuation of the posterolateral NVBs, and they eventually reached the corpus spongiosum. The limitations of this study were the small number of specimens available and the lack of functional information. CONCLUSIONS: The anterolateral position of CNs at the apex of the prostate and the autonomic innervation towards the corpus spongiosum via CSNs indicate possible ways to minimise the effect of prostate surgery on sexual function. The ideal dissection plane should probably include the preservation of the anterolateral tissues and fascias to avoid CN lesions. Anatomic knowledge gained from CAAD pertains directly to proper surgical technique and subsequent recovery of erectile function after RP.

Rationale of pelvic autonomic nerve preservation in rectal cancer surgery based on immunohistochemical study.

PURPOSE: Previous studies revealed that the incidence of cancer cell involvement along the pelvic autonomic nerves ranged from 4 to 14%. However, patients' profiles and methodologies differed among the studies. This study was conducted to clarify the incidence of cancer cell involvement in and around the pelvic autonomic nerves immunohistochemically. METHODS: Immunohistochemical staining was performed on pelvic autonomic nerve specimens resected from 17 patients with p-Stage I-III lower rectal cancers. Antibodies used were pan-cytokeratin (AE1/AE3) for staining cancer cells, S-100 for autonomic nerves, and D2-40 for lymphatic vessels. Lymphatic permeation around the pelvic autonomic nerves was defined as present when AE1/AE3-positive cells were detected in D2-40-stained lymphatic vessels. The presence of metastasis to the interstitial tissue or contaminants was also recorded. RESULTS: TNM staging was stage I in 1, stage II in 5, and stage III in 11 cases, respectively. No cases had lymphatic permeation or metastasis to the interstitial tissue in and around the pelvic autonomic nerves. Cancer cell contaminants were seen in four cases (23%). In three cases (18%), metastatic nodes were located at the root of the middle rectal artery, very close to the pelvic autonomic nerves. CONCLUSIONS: Cancer cell involvement was not seen in and around the pelvic autonomic nerves, suggesting that complete pelvic autonomic nerve preservation may be feasible, unless nerves are invaded by the tumor. In some cases, however, metastatic nodes were seen very close to the nerves. Meticulous lymph node dissection along the pelvic autonomic nerves is mandatory.

Neurochemistry of the paraventricular nucleus of the hypothalamus: implications for cardiovascular regulation.

The paraventricular nucleus of the hypothalamus (PVN) is an important site for autonomic and endocrine homeostasis. The PVN integrates specific afferent stimuli to produce an appropriate differential sympathetic output. The neural circuitry and some of the neurochemical substrates within this circuitry are discussed. The PVN has at least three neural circuits to alter sympathetic activity and cardiovascular regulation. These pathways innervate the vasculature and organs such as the heart, kidney and adrenal medulla. The basal level of sympathetic tone at any given time is dependent upon excitatory and inhibitory inputs. Under normal circumstances the sympathetic nervous system is tonically inhibited. This inhibition is dependent upon GABA and nitric oxide such that nitric oxide potentiates local GABAergic synaptic inputs onto the neurones in the PVN. Excitatory neurotransmitters such as glutamate and angiotensin II modify the tonic inhibitory activity. The neurotransmitters oxytocin, vasopressin and dopamine have been shown to affect cardiovascular function. These neurotransmitters are found in neurones of the PVN and within the spinal cord. Oxytocin and vasopressin terminal fibres are closely associated with sympathetic preganglionic neurones (SPNs). Sympathetic preganglionic neurones have been shown to express receptors for oxytocin, vasopressin and dopamine. Oxytocin causes cardioacceleratory and pressor effects that are greatest in the upper thoracic cord while vasopressin cause these effects but more significant in the lower thoracic cord. Dopaminergic effects on the cardiovascular system include inhibitory or excitatory actions attributed to a direct PVN influence or via interneuronal connections to sympathetic preganglionic neurones.

Neurochemistry of bulbospinal presympathetic neurons of the medulla oblongata.

This review focuses on presympathetic neurons in the medulla oblongata including the adrenergic cell groups C1-C3 in the rostral ventrolateral medulla and the serotonergic, GABAergic and glycinergic neurons in the ventromedial medulla. The phenotypes of these neurons including colocalized neuropeptides (e.g., neuropeptide Y, enkephalin, thyrotropin-releasing hormone, substance P) as well as their relative anatomical location are considered in relation to predicting their function in control of sympathetic outflow, in particular the sympathetic outflows controlling blood pressure and thermoregulation. Several explanations are considered for how the neuroeffectors coexisting in these neurons might be functioning, although their exact purpose remains unknown. Although there is abundant data on potential neurotransmitters and neuropeptides contained in the presympathetic neurons, we are still unable to predict function and physiology based solely on the phenotype of these neurons.

Anatomy of synaptic circuits controlling the activity of sympathetic preganglionic neurons.

Sympathetic preganglionic neurons (SPN) are critical links in the sympathetic neural circuitry that controls every organ in the body. All sympathetic outflow to the periphery comes from SPN, which send their axons from thoracic and upper lumbar spinal segments to innervate post-ganglionic neurons in sympathetic ganglia and chromaffin cells in the adrenal medulla. Despite over 30 years of study, we still do not have a sufficiently detailed understanding of the synaptic circuits through which these important neurons receive information from other central sites. We know that there is direct synaptic input to SPN from both supraspinal and intraspinal neurons, but not sensory neurons. Ultrastructural studies support functional evidence that amino acids are the primary fast-acting transmitters controlling SPN activity and indicate that an amino acid transmitter occurs in every synaptic input to an SPN. In addition, axons that synapse on SPN contain neuropeptides and monoamines, which would co-exist with and be released with the amino acids. Receptors and transporters for transmitters have also been localized in SPN inputs. Light and electron microscopic observations suggest that there are qualitative and/or quantitative differences in the neurochemical types and origins of axons, which provide synaptic input to SPN that supply different targets or have different functions. However, more research is required before it can be confirmed that SPN receive projection- or function-specific patterns of innervation. This information is likely to be important if we are to understand how the central nervous system differentially regulates sympathetic outflow to different target tissues.

Innervation and neurotransmitter localization in the lung of the Nile bichir Polypterus bichir bichir.

Anatomical and functional studies of the autonomic innervation in the lung of dipnoan fishes and the bichirs are lacking. The present immunohistochemical studies demonstrated the presence of nerve fibers in the muscle layers of the lung of the bichir, Polypterus bichir bichir, and identified the immunoreactive elements of this innervation. Tyrosine hydroxylase, acetylcholinesterase, and peptide immunoreactivity was detected in the intramural nerve fibers. Extensive innervation was present in the submucosa where adenylatecyclase/activating polypeptide 38, substance P, P(2)X(2), and 5-hydroxytryptamine (5-HT)-immunoreactive nerve fibers mainly supplied blood vessels. A collection of monopolar neurons located in the submucosal and the muscular layers of the glottis expressed a variety of various transmitters. These neurons may be homologous to ganglion cells in the branchial and pharyngeal rami of the vagus in fishes. Nerves containing 5-HT and P(2)X(2) receptor immunoreactivity projected to the lung epithelium. Associated with neuroepithelial cells in mucociliated epithelium, were neuronal nitric oxide synthase-immunopositive axons. The physiological function of this innervation is not known. The present study shows that the pattern of autonomic innervation of the bichir lung may by similar in its elements to that in tetrapods.

Metastasis-associated S100A4 (Mts1) protein is expressed in subpopulations of sensory and autonomic neurons and in Schwann cells of the adult rat.

S100A4 (Mts1) is a member of a family of calcium-binding proteins of the EF-hand type, which are widely expressed in the nervous system, where they appear to be involved in the regulation of neuron survival, plasticity, and response to injury or disease. S100A4 has previously been demonstrated in astrocytes of the white matter and rostral migratory stream of the adult rat. After injury, S100A4 is markedly up-regulated in affected central nervous white matter areas as well as in the periventricular area and rostral migratory stream. Here, we show that S100A4 is expressed in a subpopulation of dorsal root, trigeminal, geniculate, and nodose ganglion cells; in a subpopulation of postganglionic sympathetic and parasympathetic neurons; in chromaffin cells of the adrenal medulla; and in satellite and Schwann cells. In dorsal root ganglia, S100A4-positive cells appear to constitute a subpopulation of small ganglion neurons, a few of which coexpressed calcitonin gene-related peptide (CGRP) and Griffonia simplicifolia agglutinin (GSA) isolectin B4 (B4). S100A4 protein appears to be transported from dorsal root ganglia to the spinal cord, where it is deposited in the tract of Lissauer. After peripheral nerve or dorsal root injury, a few S100A4-positive cells coexpress CGRP, GSA, or galanin. Peripheral nerve or dorsal root injury induces a marked up-regulation of S100A4 expression in satellite cells in the ganglion and in Schwann cells at the injury site and in the distal stump. This pattern of distribution partially overlaps that of the previously studied S100B and S100A6 proteins, indicating a possible functional cooperation between these proteins. The presence of S100A4 in sensory neurons, including their processes in the central nervous system, suggests that S100A4 is involved in propagation of sensory impulses in specific fiber types.

Dual viral transneuronal tracing of central autonomic circuits involved in the innervation of the two kidneys in rat.

The neural control of renal function is exerted by the central nervous system via sympathetic innervation of the kidneys. To determine the extent to which the control of the two kidneys is provided by the same brain neurons, the central circuitry involved in the innervation of both kidneys was characterized in individual rats by dual viral transneuronal tracing using isogenic recombinant strains (PRV-152 and BaBlu) of pseudorabies virus. Prior to dual tracing, the neuroinvasive properties of PRV-152 and BaBlu were characterized by conducting parametric studies, using the two kidneys as an anatomical model, and comparing the pattern of infection with that obtained following injection of the parental strain, PRV-Bartha, into the left kidney. Once the optimal concentrations of virus required to obtain equivalent infection were established, PRV-152 and BaBlu were injected into the left and right kidney, respectively, in the same rats. Immunocytochemical localization of viral reporter proteins at different postinoculation times allowed us to determine the sequence of infection in the brain, as well as to quantify dual- and single-labeled neurons in each infected area. Neurons that influence autonomic outflow to one or both kidneys coexist in all brain areas involved in the control of the sympathetic outflow to the kidneys at every hierarchical level of the circuit. The proportions of dual-infected neurons with respect to the number of total infected neurons varied across regions, but they were maintained at different survival times. The pattern of infection suggests that the activity of each kidney is controlled independently by organ-specific neurons, whereas the functional coordination of the two kidneys results from neurons that collaterize to modulate the sympathetic outflow to both organs. The advantages of using an anatomical symmetrical system, such as the two kidneys, as an experimental approach to characterize PRV recombinants in general are also discussed.

Motor, sensory and autonomic nerve terminals containing NAP-22 immunoreactivity in the rat muscle.

Neuron-enriched acidic protein having a molecular mass of 22 kDa, NAP-22, is a Ca(2+)-dependent calmodulin-binding protein and is phosphorylated with protein kinase C (PKC). This protein is localized to the biological membrane via myristoylation and found in the membrane fraction of the brain and in the synaptic vesicle fraction. Recent studies showed that NAP-22 is localized in the membrane raft domain in a cholesterol-dependent manner and suggest a role for NAP-22 in maturation and/or maintenance of nerve terminals by controlling cholesterol-dependent membrane dynamics. The present study revealed the immunohistochemical distribution of NAP-22 in the peripheral nerves in rat muscles. In all examined muscles, nerve terminals in the motor endplates showed NAP-22 immunoreactivity associated with the membranes of synaptic vesicles and nerve terminals. In the muscle spindles, annulospiral endings, which made spirals around the intrafusal muscles, showed intense NAP-22 immunoreactivity. Autonomic nerve fibers around the intramuscular blood vessels also showed the immunoreactivity for NAP-22. NAP-22 immunoreactivity in these peripheral nerves was observed from birth to adulthood (100 days after birth). Though growth-associated protein-43 (GAP-43) immunoreactivity in these nerves was observed from birth, this immunoreactivity decreased from 20 days after birth. These findings suggest that NAP-22 is distributed and regulates functions in the motor, sensory and autonomic nerve terminals in the peripheral nervous system.

Nicotinic acetylcholine receptor distribution in relation to spinal neurotransmission pathways.

Neuronal nicotinic receptors (nAChR) are pentameric assemblies of subunits of a gene family where specified combinations of alpha and beta subunits form functional receptors. To extend our understanding of the role of spinal nAChR in the processing of sensory stimuli and regulation of autonomic and motor responses, we initiated investigations to localize nAChR subunit expression within discrete spinal regions and cell types. High-affinity epibatidine binding was present in the superficial dorsal and ventral horns, the mediolateral and central canal regions. RT-PCR identified transcripts for alpha3, alpha4, alpha5, beta2, and beta4 in both spinal cord parenchyma and dorsal root ganglia (DRG). Our affinity-purified antibodies against alpha3, alpha4, alpha5, beta2, and beta4 subunits identified specific protein bands of appropriate molecular mass (preadsorbed with the respective antigens) in specific tissues and cells that express nicotinic receptors, including the spinal cord and DRG neurons. Having established the absence of crossreactivity with related subunits, specific fluorescence labeling of nerve terminals and cell bodies was achieved and correlated with the distribution of defined marker proteins and nicotinic receptor binding sites determined autoradiographically. Our findings indicate that alpha3, alpha4, alpha5, beta2, and beta4 subunits are all expressed on primary afferents (IB4-positive terminals) in the spinal cord. The predominant presynaptic (synaptophysin colocalization) labeling is in the superficial layer of the dorsal horn. These receptor subunits, except for beta4, are also present in postsynaptic autonomic (anti-bNOS-positive) and somatic motor neurons (anti-VAChT-positive). The alpha3, alpha5, and beta2 subunits showed additional staining in glial (anti-GFAP-positive) cells. These studies reveal a dense and distinguishable distribution of nAChR subunits in the spinal cord and point toward future therapeutic targeting for specific spinal actions.

Autonomic innervation of the human cardiac conduction system: changes from infancy to senility--an immunohistochemical and histochemical analysis.

In order to study the changes in the pattern of autonomic innervation of the human cardiac conduction system in relation to age, the innervation of the conduction system of 24 human hearts (the age of the individuals ranged from newborn to 80 years), freshly obtained at autopsy, was evaluated by a combination of immunofluorescence and histochemical techniques. The pattern of distribution and density of nerves exhibiting immunoreactivity against protein gene product 9.5 (PGP), a general neural marker, dopamine beta-hydroxylase (DBH) and tyrosine hydroxylase (TH), indicators for presumptive sympathetic neural tissue, and those demonstrating positive acetylcholinesterase (AChE) activity, were studied. All these nerves showed a similar pattern of distribution and developmental changes. The density of innervation, assessed semiquantitatively, was highest in the sinus node, and exhibited a decreasing gradient through the atrioventricular node, penetrating and branching bundle, to the bundle branches. Other than a paucity of those showing AChE activity, nerves were present in substantial quantities in infancy. They then increased in density to a maximum in childhood, at which time the adult pattern was achieved and then gradually decreased in density in the elders to a level similar to or slightly less than that in infancy. In contrast, only scattered AChE-positive nerves were found in the sinus and atrioventricular nodes, but were absent from the bundle branches of the infant heart, whereas these conduction tissues themselves possessing a substantial amount of pseudocholinesterase. During maturation into adulthood, however, the conduction tissues gradually lost their content of pseudocholinesterase but acquired a rich supply of AChE-positive nerves, comparable in density to those of DBH and TH nerves. The decline in density of AChE-positive nerves in the conduction tissues in the elders was also similar to those of DBH and TH nerves. Our findings of initial sympathetic dominance in the neural supply to the human cardiac conduction system in infancy, and its gradual transition into a sympathetic and parasympathetic codominance in adulthood, correlate well with the physiologic alterations known to occur in cardiac rate during postnatal development. The finding of reduction in density of innervation of the conduction tissue with ageing is also in agreement with clinical and electrophysiological findings such as age-associated reduction in cardiac response to parasympathetic stimulation. Finally, our findings also support the hypothesis that, in addition to the para-arterial route, the parafascicular route of extension along the conduction tissue constitutes another pathway for the innervation of the conduction system of the human heart during development.

Different levels of immunoreactivity for synaptosomal-associated protein of 25 kDa in vasoconstrictor and vasodilator axons of guinea-pigs.

Immunoreactivity (IR) for synaptosomal-associated protein of 25 kDa (SNAP-25) was examined in axons of autonomic vasoconstrictor and vasodilator neurons innervating the lingual and uterine arteries of guinea-pigs. Polyacrylamide gel electrophoresis and immunoblotting of protein extracts demonstrated a SNAP-25-IR band at 25 kDa in both arteries. Quantitative confocal microscopy demonstrated significantly higher levels of SNAP-25-IR in varicosities with IR for vasoactive intestinal peptide (VIP) than in adjacent axons with IR for tyrosine hydroxylase (TH). Levels of SNAP-25-IR in TH-IR axons, relative to adjacent VIP-IR axons, were significantly higher in the lingual artery than the uterine artery. These differences in IR for SNAP-25, a protein considered essential for calcium-dependent exocytosis of neurotransmitters, raise the possibility that mechanisms of transmitter release may vary between different classes of autonomic neurons.

Changes in peptidergic nerves in the atrioventricular valves of streptozotocin-induced diabetic rats: a confocal microscopy study.

Several previous studies have described the distribution of neuropeptide Y (NPY)-like and calcitonin gene related peptide (CGRP)-like immunoreactive nerve fibres in the atrioventricular valves of humans and various animals. It has been suggested that peptide-containing nerve fibres might have motor or sensory roles in valvular function. Although there is evidence that diabetic changes occur in the sympathetic (preganglionic and postganglionic), parasympathetic (vagal) and peptidergic nerves of rats, the changes of peptide-containing nerve fibres in the atrioventricular valves of the diabetic rat have not been studied. The distribution, relative density and staining intensity of NPY-like and CGRP-like immunoreactive nerve fibres in the mitral and tricuspid valves were studied in whole mount preparations using confocal microscopy with a computer-assisted image analysis system. Streptozotocin-induced diabetic and control rats were sacrificed at 12 and 24 months. The nerve staining intensity within the tricuspid valve was greater than the mitral valve in both control (P < 0.01) and diabetic (P < 0.001) rats. Nerve density in the anterior leaflet was greater than the posterior leaflet of the mitral valve. However, the anterior leaflet of the mitral and tricuspid valves showed a decreased number of nerve fibres, followed by drastic reduction in the staining intensities for both the peptides studied (P < 0.001) in the long-term diabetic rat. The decrease in the number of nerve fibres that follow the mechanical interruption of nerves raises the possibility that cycles of degeneration may occur. It is suggested that these peptide-containing nerve fibres in the atrioventricular valves may be involved in valvular dysfunction in the diabetic state.

Natriuretic peptide immunoreactivity in nerve structures and Purkinje fibres of human, pig and sheep hearts.

Atrial natriuretic peptide is a well-described peptide in cardiac Purkinje fibres and has been shown to interfere with the autonomic regulation in the heart of various species, including man. Recently, we detected immunoreactivity for the peptide in intracardial ganglionic cells and nerve fibre varicosities of bovine hearts, by the use of a modified immunostaining technique that induced an improved detection of natriuretic peptides. These findings raised the question as to whether natriuretic peptides are detectable in these tissues in man and other species. The conduction system from human, pig and sheep hearts was dissected processed with antisera against atrial natriuretic peptide and the closely related brain natriuretic peptide. Immunostaining for the brain natriuretic peptide was detected in some Purkinje fibres in all of these species. Interestingly, in pig, sheep and human hearts, some ganglionic cells and nerve fibres showed atrial natriuretic peptide immunoreactivity, particularly in the soma of human ganglionic cells. This is the first study showing immunoreactivity for the atrial natriuretic peptide in nerve structures and for the brain natriuretic peptide in Purkinje fibres of the human heart. The results give a morphological correlate for the documented effects of atrial natriuretic peptide on the heart autonomic nervous system and for the presumable effects of brain natriuretic peptide in the conduction system of man.