Autonomic reinnervation and functional regeneration in autologous transplanted submandibular glands in patients with severe keratoconjunctivitis sicca.

Autologous submandibular gland (SMG) transplantation has been proved to ameliorate the discomforts in patients with severe keratoconjunctivitis sicca. The transplanted glands underwent a hypofunctional period and then restored secretion spontaneously. This study aims to investigate whether autonomic nerves reinnervate the grafts and contribute to the functional recovery, and further determine the origin of these nerves. Parts of the transplanted SMGs were collected from the epiphora patients, and a rabbit SMG transplantation model was established to fulfill the serial observation on the transplanted glands with time. The results showed that autonomic nerves distributed in the transplanted SMGs and parasympathetic ganglionic cells were observed in the stroma of the glands. Low-dense and unevenly distributed cholinergic axons, severe acinar atrophy and fibrosis were visible in the patients' glands 4-6 months post-transplantation, whereas the cholinergic axon density and acinar area were increased with time. The acinar area or the secretory flow rate of the transplanted glands was statistically correlated with the cholinergic axon density in the rabbit model, respectively. Meanwhile, large cholinergic nerve trunks were found to locate in the temporal fascia lower to the gland, and sympathetic plexus concomitant with the arteries was observed both in the adjacent fascia and in the stroma of the glands. In summary, the transplanted SMGs are reinnervated by autonomic nerves and the cholinergic nerves play a role in the morphological and functional restoration of the glands. Moreover, these autonomic nerves might originate from the auriculotemporal nerve and the sympathetic plexus around the supplying arteries.

[Establishment of pelvic nerve denervation modal in mice].

OBJECTIVE: To establishment and verify pelvic nerve denervation (PND) model in mice. METHODS: (1) Establishment of models. Seventy-two healthy male SPE class C57 mice with age of 7 weeks and body weight of (25±1) g were chosen. These 72 mice were randomly divided into PND group containing 36 mice and sham operation group containing 36 mice. Referring to the establishment method of PND rats, after anesthesia, a laparotomy was performed on the mouse with an abdominal median incision. Under the dissection microscope, the pelvic nerves behind and after each sides of the prostate gland were bluntly separated with cotton swabs and cut with a dissecting scissor. After the operation, the urination of mice was assisted twice every day. For the mice of sham operation group, the pelvic nerves were only exposed without cutting. (2) Detection of models. Colonic transit test was performed in 18 mice chosen randomly from each group to detect the colonic transit ratio (colored colon by methylene blue/ whole colon) and visceral sensitivity tests was performed in the rest mice to observe and record the changes of electromyogram. RESULTS: Three mice died of colonic transit test in each group. Uroschesis occurred in all the mice of PND group and needed bladder massage to assist the urination. Colonic transit test showed that the colonic transit ratios of sham operation group at postoperative day (POD) 1, 3 and 7 were (0.4950±0.3858)%, (0.6386±0.1293)% and (0.6470±0.1088)% without significant difference (F=0.3647, P=0.058), while in PND group, the colonic transit ratio at POD 7 [(0.6044±0.1768) %] was obviously higher than that both at POD 3[(0.3876±0.1364)%, P=0.022] and POD 1[(0.2542±0.0371)%, P=0.001], indicating a recovery trend of colonic transit function (F=9.143, P=0.004). Compared with the sham operation group, the colonic transit function in PND group decreased significantly at POD 1 and POD 3(both P<0.05), and at POD 7, there was no significant difference between two groups. Visceral sensitivity test showed that the visceral sensitivity of sham operation group at POD 1, 3 and 7 was 24.2808±9.5566, 33.6725±7.9548 and 43.9086±12.1875 with significant difference (F=5.722, P=0.014). The visceral sensitivity of PND group at POD 1, 3 and 7 was 11.7609±2.1049, 21.8415±8.1527 and 26.2310±4.2235 with significant difference as well (F=11.154, P=0.001). The visceral sensitivity at POD 3 and POD 7 was obviously higher than that at POD 1 (P=0.006, P<0.001), and there was no significant difference between POD 3 and POD 7 (P=0.183). Compared with sham operation group, the visceral sensitivity of PND group decreased significantly at POD 1, 3 and 7(all P<0.05). CONCLUSIONS: Denervation of pelvic nerves can obviously decrease the colonic transit function and the visceral sensitivity of mice, but these changes can recover over time, which suggests that the establishment of PND model in mice is successful.

Ontogeny of sensory and autonomic nerves in the developing mouse skeleton.

BACKGROUND: Bone innervation is implicated in bone modeling and remodeling. This study investigates skeletal nerve development in embryonic and newborn mice, focusing on sensory and autonomic nerves and their temporal occurrence. MATERIALS AND METHODS: The ontogeny of innervation and angiogenesis in the hindlimb skeleton of mice was studied from embryonic day (E) 15 to postnatal day (P) 20. Neuronal tissue was immunohistochemically labeled for detection of growth associated protein 43 (GAP-43), protein gene product 9.5 (PGP 9.5), calcitonin gene-related peptide (CGRP), tyrosine hydroxylase (TH), and neuropeptide Y (NPY). Vascular endothelium was labeled for platelet endothelium cell adhesion molecule-1 (PECAM-1). Morphology was evaluated with hematoxylin and eosin staining. RESULTS: GAP-43, PGP 9.5, CGRP, and PECAM-1 were all present at E 15, adjacent to areas with high osteogenic and chondrogenic activity. In the primary ossification centers, GAP-43 was found at E 15, PGP 9.5 at E 17, CGRP at E 19, and NPY at P 4. The same time lag in appearance was observed in the secondary ossification centers. The covering capillary network was initially dense, but became mature and sparse from P 12 onwards. CONCLUSION: A functional nerve supply co-localized with a rich capillary network is seen early in the developing mouse skeleton, especially in areas with high osteogenic activity. Sensory innervation occurs prior to partus, while autonomic innervation (revealed by the presence of NPY and TH) is established post partum. The findings indicate a time-related development of nerves with different qualities, according to skeletal development.

Detailed comparative anatomy of the extrinsic cardiac nerve plexus and postnatal reorganization of the cardiac position and innervation in the great apes: orangutans, gorillas, and chimpanzees.

To speculate how the extrinsic cardiac nerve plexus (ECNP) evolves phyletically and ontogenetically within the primate lineage, we conducted a comparative anatomical study of the ECNP, including an imaging examination in the great apes using 20 sides from 11 bodies from three species and a range of postnatal stages from newborns to mature adults. Although the position of the middle cervical ganglion (MG) in the great apes tended to be relatively lower than that in humans, the morphology of the ECNP in adult great apes was almost consistent with that in adult humans but essentially different from that in the lesser apes or gibbons. Therefore, the well-argued anatomical question of when did the MG acquire communicating branches with the spinal cervical nerves and appear constantly in all sympathetic cardiac nerves during primate evolution is clearly considered to be after the great apes and gibbons split. Moreover, a horizontal four-chambered heart and a lifted cardiac apex with a relatively large volume in newborn great apes rapidly changed its position downward, as seen in humans during postnatal growth and was associated with a reduction in the hepatic volume by imaging diagnosis and gross anatomy. In addition, our observation using a range of postnatal stages exhibits that two sympathetic ganglia, the middle cervical and cervicothoracic ganglia, differed between the early and later postnatal stages.

The developing left superior cervical ganglion of Pacas (Agouti paca).

In this study the main question investigated was the number and size of both binucleate and mononucleate superior cervical ganglion (SCG) neurons and, whether post-natal development would affect these parameters. Twenty left SCGs from 20 male pacas were used. Four different ages were investigated, that is newborn (4 days), young (45 days), adult (2 years), and aged animals (7 years). By using design-based stereological methods, that is the Cavalieri principle and a physical disector combined with serial sectioning, the total volume of ganglion and total number of mononucleate and binucleate neurons were estimated. Furthermore, the mean perikaryal (somal) volume of mononucleate and binucleate neurons was estimated using the vertical nucleator. The main findings of this study were a 154% increase in the SCG volume, a 95% increase in the total number of mononucleate SCG neurons and a 50% increase in the total volume of SCG neurons. In conclusion, apart from neuron number, different adaptive mechanisms may coexist in the autonomic nervous system to guarantee a functional homeostasis during ageing, which is not always associated with neuron losses.

5-HT2A receptors are concentrated in regions of the human infant medulla involved in respiratory and autonomic control.

The serotonergic (5-HT) system in the human medulla oblongata is well-recognized to play an important role in the regulation of respiratory and autonomic function. In this study, using both immunocytochemistry (n=5) and tissue section autoradiography with the radioligand (125)I-1-(2,5-dimethoxy-4-iodo-phenyl)2-aminopropane (n=7), we examine the normative development and distribution of the 5-HT(2A) receptor in the human medulla during the last part of gestation and first postnatal year when dramatic changes are known to occur in respiratory and autonomic control, in part mediated by the 5-HT(2A) receptor. High 5-HT(2A) receptor binding was observed in the dorsal motor nucleus of the vagus (preganglionic parasympathetic output) and hypoglossal nucleus (airway patency); intermediate binding was present in the nucleus of the solitary tract (visceral sensory input), gigantocellularis, intermediate reticular zone, and paragigantocellularis lateralis. Negligible binding was present in the raphé obscurus and arcuate nucleus. The pattern of 5-HT(2A) immunoreactivity paralleled that of binding density. By 15 gestational weeks, the relative distribution of the 5-HT(2A) receptor was similar to that in infancy. In all nuclei sampled, 5-HT(2A) receptor binding increased with age, with significant increases in the hypoglossal nucleus (p=0.027), principal inferior olive (p=0.044), and medial accessory olive (0.038). Thus, 5-HT(2A) receptors are concentrated in regions involved in autonomic and respiratory control in the human infant medulla, and their developmental profile changes over the first year of life in the hypoglossal nucleus critical to airway patency and the inferior olivary complex essential to cerebellar function.

Asymmetric post-natal development of superior cervical ganglion of paca (Agouti paca).

Functional asymmetry has been reported in sympathetic ganglia. Although there are few studies reporting on body side-related morphoquantitative changes in sympathetic ganglion neurons, none of them have used design-based stereological methods to address this issue during post-natal development. We therefore aimed at detecting possible asymmetry-related effects on the quantitative structure of the superior cervical ganglion (SCG) from pacas during ageing, using very precise design-based stereological methods. Forty (twenty left and twenty right) SCG from twenty male pacas were studied at four different ages, i.e. newborn, young, adult and aged animals. By using design-based stereological methods the total volume of ganglion and the total number of mononucleate and binucleate neurons were estimated. Furthermore, the mean perikaryal volume of mononucleate and binucleate neurons was estimated, using the vertical nucleator. The main findings of this study were: (1) the right SCG from aged pacas has more mononucleate and binucleate neurons than the left SCG in all other combinations of body side and animal age, showing the effect of the interaction between asymmetry (right side) and animal age, and (2) right SCG neurons (mono and binucleate) are bigger than the left SCG neurons (mono and binucleate), irrespective of the animal age. This shows, therefore, the exclusive effect of asymmetry (right side). At the time of writing there is still no conclusive explanation for some SCG quantitative changes exclusively assigned to asymmetry (right side) and those assigned to the interaction between asymmetry (right side) and senescence in pacas. We therefore suggest that forthcoming studies should focus on the functional consequences of SCG structural asymmetry during post-natal development. Another interesting investigation would be to examine the interaction between ganglia and their innervation targets using anterograde and retrograde neurotracers. Would differences in the size of target organs explain ganglia structural asymmetry?

Increased amygdala activation is related to heart rate during emotion processing in adolescent subjects.

Emotions have been conceptualized as representations of bodily responses to a stimulus that critically involves the autonomic nervous system (ANS). An association between amygdala activation and ANS activity has been shown in adults. However, to date, no studies have demonstrated this association in adolescents. Examining the interaction between the ANS and amygdala in healthy adolescents may provide information about age-related changes in the association between amygdala activation and ANS measures. Therefore, the aim of this study was to examine the relationship between amygdala activation and heart rate in normal adolescents. Eighteen 12- to 17-year old adolescents participated. Heart rate data was collected during functional magnetic resonance imaging while subjects performed a facial expression matching task that reliably activates the amygdala. Adolescents showed significant amygdala activation for all facial expressions relative to the shape-matching, control task. Moreover, the degree of activation in the right amygdala for Fearful faces was significantly correlated with heart rate (Spearman's rho=0.55, p=0.018, two-tailed). This study shows that amygdala activity is related to heart rate in healthy adolescents. Thus, similar to adults, adolescents show a coupling between processing emotional events and adjusting the ANS accordingly. Furthermore, this study confirms previous adolescent studies showing amygdala activation to Fearful, Angry, and Happy faces. Finally, the results of the present study lay the foundation for future research to investigate whether adolescents with mood or anxiety disorders show an altered coupling between processing emotionally salient events and ANS activity.

Neurotrophins in murine viscera: a dynamic pattern from birth to adulthood.

There is growing evidence that target-derived neurotrophins regulate the function of visceral neurons after birth. However, the postnatal profile of neurotrophin supply from internal organs is poorly described. In this study, we compared neurotrophin concentrations in lysates of murine peripheral target tissues (lung, heart, liver, colon, spleen, thymus, kidney and urinary bladder) at different time points after birth. In most organs, there was a decrease of neurotrophin concentrations in the first weeks after birth. In contrast, there were characteristic increases of specific neurotrophins during adolescence or adulthood. These increases were found for nerve growth factor (NGF) in the heart, thymus, kidney and liver, for brain-derived neurotrophic factor (BDNF) in the lung, and for neurotrophin-3 (NT-3) in the colon. In conclusion, we show that neurotrophins display a very differential and dynamic profile in internal organs after birth.

Extreme stretch growth of integrated axons.

Large animals can undergo enormous growth during development, suggesting that axons in nerves and white matter tracts rapidly expand as well. Because integrated axons have no growth cones to extend from, it has been postulated that mechanical forces may stimulate axon elongation matching the growth of the animal. However, this distinct form of rapid and sustained growth of integrated axons has never been demonstrated. Here, we used a microstepper motor system to evaluate the effects of escalating rates of stretch on integrated axon tracts over days to weeks in culture. We found that axon tracts could be stretch grown at rates of 8 mm/d and reach lengths of 10 cm without disconnection. Despite dynamic and long-term elongation, stretched axons increased in caliber by 35%, while the morphology and density of cytoskeletal constituents and organelles were maintained. These data provide the first evidence that mechanical stimuli can induce extreme "stretch growth" of integrated axon tracts, far exceeding any previously observed limits of axon growth.

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.

Sema3A and neuropilin-1 expression and distribution in rat white adipose tissue.

Semaphorins are cell surface and/or soluble signals that exert an inhibitory control on axon guidance. Sema3A, the vertebrate-secreted semaphorin, binds to neuropilin-1, which together with plexins constitutes the functional receptor. To verify whether Sema3A is produced by white adipocytes and, in that case, to detect its targets in white adipose tissue, we studied the cell production and tissue distribution of Sema3A and neuropilin-1 in rat retroperitoneal and epididymal adipose depots. Sema3A and neuropilin-1 were detected in these depots by Western blotting. The immunohistochemical results showed that Sema3A is produced in, and possibly secreted by, smooth muscle cells of arteries and white adipocytes. Accordingly, neuropilin-1 was found on perivascular and parenchymal nerves. Such a pattern of distribution is in line with a role for secreted Sema3A in the growth and plasticity of white adipose tissue nerves. Indeed, after fasting, when white adipocytes are believed to be overstimulated by noradrenaline and rearrangement of the parenchymal nerve supply may occur, adipocytic expression of Sema3A is reduced. Finally, the presence of neuropilin-1 in some white adipocytes raises the interesting possibility that Sema3A also exerts an autocrine-paracrine role on these cells.

Maturation of postsynaptic nicotinic structures on autonomic neurons requires innervation but not cholinergic transmission.

Postsynaptic development at the neuromuscular junction depends on nicotinic transmission and secreted components from the presynaptic motor nerve terminal. Similarly, secreted components and synaptic activity are both thought to guide development of glutamatergic synapses in the CNS. Nicotinic synapses on chick ciliary neurons are structurally complex: a large presynaptic calyx engulfs the postsynaptic neuron and overlays a series of discrete mats of receptor-rich somatic spines tightly interwoven and folded against the soma. We used fluorescence imaging of alpha 7-containing nicotinic receptors and the spine constituent drebrin to monitor postsynaptic development. The results show that surgical disruption of the preganglionic input or removal of the ganglionic synaptic target tissue after synapses form in the ganglion does not disrupt the receptor-rich spine mats. Similarly, removal of the target tissue even prior to synapse formation in the ganglion does not prevent subsequent formation of the receptor clusters and associated spine constituents. Postsynaptic development is arrested, however, if normal innervation is prevented by ablating the preganglionic neurons prior to synapse formation. In this case the neurons express reduced levels of nicotinic receptors and cytoskeletal components and organize them only into early-stage clusters. Even low levels of residual innervation, however, can restore much of the normal postsynaptic receptor patterns. Chronic pharmacological blockade of cholinergic synaptic activity fails to replicate the effects of ablating the preganglionic nucleus. The results indicate that ciliary neurons are programmed to express postsynaptic components and can initiate clustering of alpha 7-containing receptors but need presynaptic guidance for maturation of the postsynaptic structure.

Effects of the head-down tilt posture on postnatal development of the aortic baroreflex in the rabbit.

To clarify the effects of the head-down tilt (HDT) posture on the postnatal development of the aortic baroreflex, we raised rabbits from 3-4 weeks of age for 34-36 d in this posture, which simulates the headward shift of body fluid in space, and examined the structural and functional characteristics of the aortic nerves and baroreflex responses. The rabbits were divided into 3 different groups: 20 degrees HDT, environmental control (EC), and vivarium control (VIV). Left aortic nerve activity (ANA) at basal arterial pressure was defined as 100%, and the maximum ANA at maximal increase in arterial pressure was 265+/-103% (mean+/-SD, n=5) in the HDT, 482+/-170% (n=4) in the EC, and 677+/-491% (n=4) in the VIV groups. The minimum ANA at maximal decrease in arterial pressure was 67.3+/-13.8% in the HDT, 40.1+/-10.2% in the EC, and 38.6+/-13.8% in the VIV groups. The maximal change of ANA in response to maximal change in arterial pressure in the HDT group was significantly less than that of either the EC or VIV group (p<0.05). The average baroreflex gain in the HDT group was significantly less than in the VIV group (p<0.05). The number of unmyelinated fibers in the left aortic nerve in the HDT group was significantly less than that of each control group (p<0.05). Thus the development of the aortic baroreflex system is apparently depressed by the HDT posture, suggesting that similar consequences will be observed in mammals that develop in space.

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.

Vasoactive intestinal polypeptide and calcitonin gene-related peptide in the developing rat heart atria.

Vasoactive intestinal polypeptide-like immunoreactivity (VIP-LI) and calcitonin gene-related peptide (CGRP)-LI concentrations were determined in the developing rat heart atria using radioimmunoassay. Peptide levels were analysed on postnatal days 1, 10, 25, 45, 60, and 85 (P1-P85) separately in the right (RA) and left atria (LA). No sex differences were revealed at any age examined. VIP-LI has been already detected in both atria at P1 in concentrations comparable to values at P10. In the RA, VIP-LI levels increased significantly between days P10 and P25, remained high at P45 and then declined. In the LA, VIP-LI concentrations did not differ from those in the RA on days P1, P10, P25, and P45. However, regional differences were found at P60 and P85, when the peptide levels were significantly higher in the LA than in the RA. The postnatal changes in CGRP-LI concentrations were comparable in both atria with similar values at P1 and P85. After birth, CGRP levels decreased gradually till P45, then they increased till P60 and declined again at P85. The results demonstrate that there is an asymmetry in the postnatal development of the atrial VIP-LI and CGRP-LI concentrations. VIP-LI levels reached their maximum at P25, whereas CGRP-LI levels at P60. Relatively high peptide concentrations in neonatal atria and their variations during development might be related to diverse trophic functions of VIP and CGRP.

Postnatal maturation of neuroepithelial bodies and carotid body innervation: a quantitative investigation in the rabbit.

The intrapulmonary airways contain oxygen-sensitive chemoreceptors which may be analogous to the arterial chemoreceptors: the neuroepithelial bodies (NEB). While the NEB are prominent in the neonatal lung, physiological studies indicate that the carotid bodies are still relatively inactive at birth. This points to an unequal degree of development of both during the early neonatal period. As a reflexogenic chemoreceptor function depends on a well-developed innervation, we undertook a comparative investigation of the development of the NEB and the carotid body glomus cell innervation. Two morphological aspects of the innervation of NEB and carotid body glomus cells were quantified in rabbits of different age groups. The total sectional area of intracorpuscular and intraglomerular nerve endings per NEB or glomus cell group, respectively, was measured and the area percentage of mitochondria and synaptic vesicles was determined. In the NEB, no significant difference in total sectional area of the nerve endings between the age groups was observed, while in the carotid body there was a significant increase in the adult age group. In addition, the area percentage of mitochondria and synaptic vesicles of the nerve endings did not change significantly with age in the NEB, while in the carotid body these increased and decreased, respectively, with age. These observations point to a shift from morphologically efferent nerve endings, rich in synaptic vesicles, to morphologically afferent nerve endings, rich in mitochondria. Our interpretation of these findings is that, at birth, the NEB innervation is more mature than the carotid body glomus cell innervation and that the latter matures at a later time than the former. These findings support the theory that the NEB may act as complementary chemoreceptors to the carotid body during the early postnatal period.

Contribution of the ascending cholinergic pathways in the production of ultrasonic vocalization in the rat.

It has been well documented that cholinergic stimulation of the mediobasal forebrain structures induces 20-30 kHz ultrasonic vocalization in adult rats. If the cholinergic system plays a triggering role for ultrasonic vocalization, the question arises as to where the source of the cholinergic fibres, which innervate the mediobasal forebrain and induce vocalization, is located. In the present study, the role of the ascending cholinergic projection from the ponto-mesencephalic cholinergic nuclei to the mediobasal hypothalamic-preoptic region in production of 22 kHz calls was investigated. Cholinergic neurons were stimulated by local injection of L-glutamate and eventual vocalization was recorded by a S200 bat detector and analyzed sonographically. Intracerebral injection of L-glutamate into the laterodorsal tegmental nucleus induced short latency, 20-30 kHz ultrasonic calls. Sound frequency (pitch) and single call duration of the L-glutamate-induced vocalization did not differ from those obtained by cholinergic stimulation of the mediobasal hypothalamic-preoptic region with carbachol. However, L-glutamate stimulation of the laterodorsal tegmental nucleus was ineffective or less effective in 70% of responses, when the terminal fields in the mediobasal hypothalamic-preoptic region were pretreated with scopolamine, a muscarinic antagonist. The results demonstrate that the ascending cholinergic projection from the laterodorsal tegmental nucleus plays a triggering role for 20-20 kHz vocalization in adult rats.

Autonomic nervous system as a model of neuronal aging: the role of target tissues and neurotrophic factors.

The aim of this study was to determine the role of target tissues and neurotrophic factors in the growth and atrophy of autonomic neurons during development and aging. Using quantitative neuroanatomical techniques, it is shown that, although axonal and dendritic growth is apparent throughout postnatal development, different patterns of growth are found in autonomic neurons innervating different target tissues. For example, sympathetic neurons innervating the submandibular gland continue to grow well into maturity, but those innervating the iris cease net growth early in postnatal development. Similarly, although neuronal atrophy was observed in aged autonomic ganglia, this was not a general phenomenon but was specific to neurons innervating particular target tissues. Sympathetic neurons innervating the middle cerebral artery showed significant axonal and dendritic atrophy in old age, whereas neurons innervating the iris were morphologically unchanged. The trophic influence of peripheral target tissues on their innervating neurons has been shown to decline in old age possibly as a result of decreased availability of target-derived neurotrophic factors such as nerve growth factor (NGF) [Gavazzi et al. (1992) Neuroreport, 3:717-720]. Therefore, in an attempt to reverse neuronal atrophy where it occurred, NGF was infused via miniosmotic pumps over the peripheral axons of aged neurons. NGF induced increases in soma size, dendritic length and axonal arborization. However, in contrast to young adult neurons, no increase in the number of dendritic branch points or primary dendrites was observed, suggesting that some aspects of neuronal plasticity are impaired in old age. In sum, these results show a range of age- and target-specific differences in the axonal and dendritic morphology of autonomic neurons that may result from differing trophic interactions with their target tissues.

Target-specific differences in the dendritic morphology and neuropeptide content of neurons in the rat SCG during development and aging.

Our purpose in this work was to investigate the role of target tissues in the regulation of dendritic morphology from sympathetic neurons during development and aging. Neurons were retrogradely labeled from three targets, the iris, the submandibular gland (SMG), and the middle cerebral artery (MCA). They were then fixed and intracellularly injected to demonstrate their dendritic arborizations. Dendritic geometry varied quantitatively in sympathetic neurons innervating different target tissues at all stages of development. Neurons innervating the iris had the largest cell bodies and most extensive dendritic arborizations, whereas the vasomotor neurons were the smallest. The number of primary dendrites, however, did not vary significantly between the different neuronal populations. The growth of dendritic arborizations during postnatal development and their atrophy in old age were not concordant in the different neuron populations we studied. Neurons innervating the MCA and the iris ceased dendritic growth early in postnatal development, whereas the dendritic complexity of neurons supplying the SMG increased well into adulthood. By contrast, dendritic atrophy was seen in aged MCA- and SMG-projecting neurons but not in those innervating the iris, suggesting, with other evidence, correlated and distinct patterns of growth and atrophy in axons and dendrites of mature sympathetic neurons projecting to different targets. Swollen dendrites and protuberances on cell soma were a prominent feature of aged neurons. In addition to the target-specific variation in neuronal morphology, we observed diversity in neurotransmitter phenotype. For example, neuropeptide Y was expressed in iridial but not SMG-projecting neurons. These results show a range of age- and target-specific differences in the dendritic morphology and neuropeptide content of sympathetic neurons that may be a result of differing trophic interactions with their target tissues.