Density Of Sympathetic Fibers Research Articles

Paradoxical Changes of Cutaneous Microcirculation and Sympathetic Fibers of Rat Hind Limbs after Sciatic Nerve Compression.

Recent studies show evidence that surgical nerve decompression could improve cutaneous blood flow (CBF), which might benefit ulcer healing. However, the change of CBF and sympathetic fibers after nerve compression is poorly understood. In the current study, a unilateral sciatic nerve compression model was created in Sprague-Dawley rats. A laser Doppler imaging system was applied to assess the CBF of the regions below the ankles. Immunohistochemistry and transmission electron microscopy were used to investigate the histopathologic changes of sympathetic fibers in sciatic nerve samples. Laser Doppler imaging revealed decreased CBF of both the lesional limb and the contralesional limb, which occurred earlier in the lesional side, indicating an enhanced sympathetic tone on vasomotor function. Intraneural density of sympathetic fibers decreased on both sides and the ultrastructure of unmyelinated fibers of both sides degenerated in a nonsynchronized manner. The study revealed nonsynchronized reduced CBF of bilateral hind limbs with paradoxically degenerated and diminished sympathetic fibers in bilateral sciatic nerves after unilateral sciatic nerve compression. These results may validate the importance of and broaden the indications for surgical nerve decompression in preventing or treating foot ulcers.

Senescent Cell-Secreted Netrin-1 Modulates Aging-Related Disorders by Recruiting Sympathetic Fibers.

Cellular senescence is implicated in several lines of aging-related disorders. However, the potential molecular mechanisms by which cellular senescence modulates age-related pathologies remain largely unexplored. Herein, we report that the density of sympathetic fibers (SFs) is significantly elevated in naturally aged mouse tissues and human colon adenoma tissues compared to the SFs densities in the corresponding young mouse tissues and human non-lesion colon tissues. A dorsal root ganglion (DRG)-human diploid fibroblast coculture assay revealed that senescent cells promote the outgrowth of SFs, indicating that the senescent cells induce recruitment of SFs in vitro. Additionally, subcutaneous transplantation of 2BS fibroblasts in nude mice shows that transplanted senescent 2BS fibroblasts promote SFs infiltration. Intra-articular senolytic molecular injection can reduce SFs density and inhibit SFs infiltration caused by senescent cells in osteoarthritis (OA), suggesting senescent cells promote the infiltration of SFs in vivo in aged tissues. Notably, the elevated level of SFs contributes to impaired cognitive function in naturally aged mice, which can be reversed by treatment with propranolol hydrochloride, a non-selective β receptor blocker that inhibits sympathetic nerve activity (SNA) by blocking non-selective β receptors. Additionally, 6-hydroxydopamine (6-OHDA)-induced sympathectomy improved hepatic sympathetic overactivity mediated hepatic steatosis in high fat diet (HFD)-fed APOE knockout mice (APOE−/− mice) by reducing hepatic SNA. Taken together, this study concludes that senescent cell-secreted netrin-1 mediated SFs outgrowth and infiltration, which contributes to aging-related disorders, suggesting that clearing senescent cells or inhibiting SNA is a promising therapeutic strategy for improving sympathetic nervous system (SNS) hyperactivity-induced aging-related pathologies.

Senescent Cell-Secreted Netrin-1 Modulates Ageing-Related Disorders by Recruiting Sympathetic Fibres

Cellular senescence is implicated in several lines of ageing-related disorders. However, the potential molecular mechanisms by which cellular senescence modulates age-related pathologies remain largely unexplored. Herein, we report that the density of sympathetic fibres (SFs) is significantly elevated in naturally aged mouse tissues and human colon adenoma tissues compared to the SF densities in the corresponding young mouse tissues and human non-lesion colon tissues. A dorsal root ganglion (DRG)-human diploid fibroblast coculture assay revealed that senescent cells promote the outgrowth of SFs, indicating that the senescent cell-induced recruitment of SFs contributes to an elevation in SFs density. Clearing senescent cells by senolytics (Dasatinib(D)+ Quercetin (Q)) improved osteoarthritis (OA), thereby reducing the SF infiltration in the superficial zone of articular cartilage. Mechanistically, senescent cells recruited SFs by secreting the axon guidance cue netrin-1. Notably, the elevated level of SFs contributes to impaired cognitive function in naturally aged mice, which can be reversed by treatment with propranolol hydrochloride, a non-selective β receptor blocker that inhibits sympathetic nerve activity (SNA) by blocking non-selective β receptors. Additionally, 6-hydroxydopamine (6-OHDA)-induced sympathectomy improved hepatic sympathetic overactivity mediated hepatic steatosis in high fat diet (HFD)-fed APOE knockout mice (APOE-/-mice) by reducing hepatic sympathetic nerve activity (SNA). We conclude that senescent cell-secreted netrin-1 mediates SFs outgrowth and leads to elevated SNA, which contributes to ageing-related disorders. This suggests that clearing senescent cells is a promising therapeutic strategy for improving sympathetic nervous system (SNS) hyperactivity-induced ageing-related pathologies. Funding Statement: Our work was supported by the National Natural Science Foundation of China (grant number 81771505 and 81773079), and the Fundamental Research Funds for the Higher Education of Tianjin (Grant Number 2018KJ079). Declaration of Interests: The authors stated that no potential conflicts of interest were disclosed. Ethics Approval Statement: All experiments involving the handling of mice were approved by the animal ethics committee of Peking University Health Science Centre. The human tissue samples were obtained with informed consent, and the study was approved by the Clinical Research Ethics Committee.

Abstract #4337 Introduction and validation of a new semi-automated method to determine sympathetic fiber density in target tissues

Abstract #4337 Introduction and validation of a new semi-automated method to determine sympathetic fiber density in target tissues

Introduction and validation of a new semi-automated method to determine sympathetic fiber density in target tissues.

In recent years, the role of sympathetic nervous fibers in chronic inflammation has become increasingly evident. At the onset of inflammation, sympathetic activity is increased in the affected tissue. However, sympathetic fibers are largely absent from chronically inflamed tissue. Apparently, there is a very dynamic relationship between sympathetic innervation and the immune system in areas of inflammation, and hence a rapid and easy method for quantification of nerve fiber density of target organs is of great value to answer potential research questions. Currently, nervous fiber densities are either determined by tedious manual counting, which is not suitable for high throughput approaches, or by expensive automated processes relying on specialized software and high-end microscopy equipment. Usually, tyrosine hydroxylase (TH) is used as the marker for sympathetic fibers. In order to overcome the current quantification bottleneck with a cost-efficient alternative, an automated process was established and compared to the classic manual approach of counting TH-positive sympathetic fibers. Since TH is not exclusively expressed on sympathetic fibers, but also in a number of catecholamine-producing cells, a prerequisite for automated determination of fiber densities is to reliably distinct between cells and fibers. Therefore, an additional staining using peripherin exclusively expressed in nervous fibers as a secondary marker was established. Using this novel approach, we studied the spleens from a syndecan-3 knockout (SDC3KO) mouse line, and demonstrated equal results on SNS fiber density for both manual and automated counts (Manual counts: wildtype: 22.57 +/- 11.72 fibers per mm2; ko: 31.95 +/- 18.85 fibers per mm2; p = 0.05; Automated counts: wildtype: 31.6 +/- 18.98 fibers per mm2; ko: 45.49 +/- 19.65 fibers per mm2; p = 0.02). In conclusion, this new and simple method can be used as a high-throughput approach to reliably and quickly estimate SNS nerve fiber density in target tissues.

Correlation between the high-frequency content of the QRS on murine surface electrocardiogram and the sympathetic nerves density in left ventricle after myocardial infarction: Experimental study

Denervated post-infarct scar is arrhythmogenic. Our aim was to compare QRS frequency content in denervated and innervated left ventricular (LV) scar. In-vivo single lead ECG telemetry device was implanted in 17 heterozygous PTPσ (HET) and 7 lacking PTPσ (KO) transgenic mice. Myocardial infarction (MI) with reperfusion and sham surgery was performed. HET mice developed a denervated scar, whereas KO mice developed innervated scar. The power spectral density was used to assess the QRS frequency content. Denervated as compared to innervated post-MI scar was characterized by the higher relative contribution of 300–500 Hz (14 ± 1 vs. 9 ± 1%; P = 0.001) but reduced relative contribution of 200–300 Hz (86 ± 1 vs. 91 ± 1%; P = 0.001). Norepinephrine concentration in peri-infarct zone correlated with both 1–200 Hz (r = 0.75; P = 0.03) and 200–500 Hz QRS power (r = 0.73; P = 0.04). Sympathetic fiber density within the infarct correlated with 200–300/200–500 Hz QRS power ratio (r = 0.56; P = 0.005). Intracellular sigma peptide injections in post-MI HET mice restored the QRS power.

Neural control of white, beige and brown adipocytes.

Reports of brown-like adipocytes in traditionally white adipose tissue (WAT) depots occurred ~30 years ago, but interest in white adipocyte 'browning' only has gained attention more recently. We integrate some of what is known about the sympathetic nervous system (SNS) innervation of WAT and brown adipose tissue (BAT) with the few studies focusing on the sympathetic innervation of the so-called 'brite' or 'beige' adipocytes that appear when WAT sympathetic drive increases (for example, cold exposure and food deprivation). Only one brain site, the dorsomedial hypothalamic nucleus (DMH), selectively browns some (inguinal WAT (IWAT) and dorsomedial subcutaneous WAT), but not all WAT depots and only when DMH neuropeptide Y gene expression is knocked down, a browning effect is mediated by WAT SNS innervation. Other studies show that WAT sympathetic fiber density is correlated with the number of brown-like adipocytes (multilocular lipid droplets, uncoupling protein-1 immunoreactivity) at both warm and cold ambient temperatures. WAT and BAT have sensory innervation, the latter important for acute BAT cold-induced temperature increases, therefore suggesting the possible importance of sensory neural feedback from brite/beige cells for heat production. Only one report shows browned WAT capable of producing heat in vivo. Collectively, increases in WAT sympathetic drive and the phenotype of these stimulated adipocytes seems critical for the production of new and/or transdifferentiation of white to brite/beige adipocytes. Selective harnessing of WAT SNS drive to produce browning or selective browning independent of the SNS to counter increases in adiposity by increasing expenditure appears to be extremely challenging.

Neonatal testosterone exposure induces early development of follicular cysts followed by sympathetic ovarian hyperinnervation.

This study analysed the temporal association between ovarian cyst development induced by neonatal androgenisation and sympathetic innervation. Neonatal rats (postnatal Days 1 to 5) were treated with testosterone or dihydrotestosterone and the effects were evaluated at postnatal Days 20, 40, 90 or 180. Ovulation rate, number of cystic follicles and density of sympathetic fibres were analysed. The effects of surgical denervation or gonadotrophin stimulation were also assessed. Rats exposed to testosterone showed no oestrous cycle activity and did not ovulate, maintaining a polycystic ovarian morphology at all ages studied. Also, a significant increase in ovarian density of noradrenergic fibres was detected at postnatal Days 90 and 180. Sympathectomy was unable to re-establish ovarian activity; however, human chorionic gonadotrophin stimulation was enough to induce ovulation. The impact of dihydrotestosterone on ovarian function was less noticeable, showing the coexistence of corpora lutea and cystic structures without changes in sympathetic innervation. Our findings suggest that a remodelling of ovarian sympathetic innervation occurs as a response to modifications in the pattern of follicular growth induced by testosterone. A role of sympathetic innervation in the maintenance of the polycystic condition is suggested.

Sympathetic nerve innervation in cervical posterior longitudinal ligament as a potential causative factor in cervical spondylosis with sympathetic symptoms and preliminary evidence

Sympathetic symptoms associated with cervical disorders, such as vertigo, headache, dizziness, etc., are common clinical disorders bewildering both clinicians and patients. In our clinical practice we observed that sympathetic symptoms associated with cervical disorders were apparently relieved in some patients after undergoing routine anterior cervical decompression and fusion plus posterior longitudinal ligament (PLL) resection. This study was designed to investigate the sympathetic nerve innervations in the cervical PLL and its potential correlation with cervical sympathetic symptoms such as vertigo. MethodIn animal research, cervical PLLs of 9 adult rabbits were harvested and stained with sucrose–phosphate–glyoxylic acid (SPG), which is a specific fluorescence staining method for sympathetic postganglionic fibers. In human research, cervical PLL of 8 patients of cervical spondylosis with sympathetic symptoms were harvested during surgery and stained with SPG. All sections were observed under fluorescence microscope. Sympathetic symptoms were evaluated using the sympathetic symptom 20-point score preoperatively and at 1week, 2-month, and 6-month postoperatively. ResultsIn rabbit specimens, a large number of sympathetic postganglionic fibers were distributed in the cervical PLL of every segment. The density of sympathetic fibers distributed in the intervertebral portion of PLL was more than that in the vertebral portion. Compared with deep layer section, the nerve fibers in the superficial PLL layer section were thicker and more densely populated. Existence of sympathetic postganglionic fibers was also confirmed in human specimens. Those nerve fibers were mostly short and isolated in areatus form, with non-interwoven branches. The mean sympathetic symptoms score decreased significantly from 6.6±2.6 before surgery to 2.0±1.9 at 6months postoperatively after anterior cervical decompression and fusion with PLL removed. ConclusionAccording to the experimental result and clinical practice, we hypothesized that sympathetic nerve fibers distributed in PLL may represent a pathologic basis of stimulation induced by cervical vertebral degenerative changes and thus are susceptible to being a potential causative factor in cervical spondylosis with sympathetic symptoms.

Post-infarct remodeling of cardiac sympathetic nerves

Myocardial infarction (MI) leads to pathological changes in the cardiac sympathetic innervation. The distribution and density of sympathetic fibers are altered, along with regional changes in neurotransmitter synthesis. The resulting spatial heterogeneity of sympathetic transmission is implicated in the development of arrhythmias and sudden cardiac death. Despite the clinical importance of sympathetic dysfunction, the underlying causes are not well understood. We have investigated the molecular basis for these changes in sympathetic function, and have identified roles for gp130 inflammatory cytokines and neurotrophins/proneurotrophins in regulating region-specific denervation, hyperinnervation, and altered neurotransmitter production in the heart after ischemia-reperfusion.

Sympathetic Fiber Sprouting in Chronically Compressed Dorsal Root Ganglia Without Peripheral Axotomy

Sympathetic axonal sprouting in axotomized dorsal root ganglia (DRG) has been shown to be a major phenomenon implicated in neuropathic pain. However, it is not known whether sympathetic sprouting can occur in pathologic ganglia without peripheral axotomy. We thus examined presence and density of sympathetic axonal sprouting within DRG of rats subjected to a persistent compressive injury by inserting a stainless steel metal rod into L(4) and L(5) lumbar intervertebral foramen. Sympathetic axons were identified by immunohistochemical staining with anti-tyrosine hydroxylase antibodies. Results indicate that progressive increase in sympathetic axonal sprouting occurred in the bilateral DRGs between postoperative days 2 and 28. The sympathetic fiber density was greater on the lesion side than the contralateral side. In conclusion, chronic compressive injury of the DRG results in sympathetic sprouting in the non-axotomized ganglion and may partially contribute to the development and maintenance of certain pathological pain states.

Sympathetic Fiber Sprouting in Chronically Compressed Dorsal Root Ganglia Without Peripheral Axotomy

Sympathetic axonal sprouting in axotomized dorsal root ganglia (DRG) has been shown to be a major phenomenon implicated in neuropathic pain. However, it is not known whether sympathetic sprouting can occur in pathologic ganglia without peripheral axotomy. We thus examined presence and density of sympathetic axonal sprouting within DRG of rats subjected to a persistent compressive injury by inserting a stainless steel metal rod into L4 and L5 lumbar intervertebral foramen. Sympathetic axons were identified by immunohistochemical staining with anti-tyrosine hydroxylase antibodies. Results indicate that progressive increase in sympathetic axonal sprouting occurred in the bilateral DRGs between postoperative days 2 and 28. The sympathetic fiber density was greater on the lesion side than the contralateral side. In conclusion, chronic compressive injury of the DRG results in sympathetic sprouting in the non-axotomized ganglion and may partially contribute to the development and maintenance of certain pathological pain states.

Decreasing sympathetic sprouting in pathologic sensory ganglia: a new mechanism for treating neuropathic pain using lidocaine

Lidocaine brings relief to those suffering from certain neuropathic pain syndromes in humans and in animal models. Evidence suggests that some neuropathic pain behaviors are closely associated with extensive sprouting of noradrenergic sympathetic fibers in the dorsal root ganglia (DRG). Using immunohistochemistry, we examined lidocaine's effects on abnormal sprouting of sympathetic fibers in two animal models: rats with unilateral spinal nerve ligation (SNL) and rats with complete sciatic nerve transection (CSNT). For the first time, we have demonstrated that systemic lidocaine beginning at the time of surgery via an implanted osmotic pump remarkably reduces sympathetic sprouting (2–3 fold) (e.g. the density of sympathetic fibers and the number of DRG neurons surrounded by sympathetic fibers) in axotomized DRGs in SNL rats. The effects of systemic lidocaine lasted more than 7 days after the termination of lidocaine administration. Similar results were obtained after topical application of lidocaine to the nerve trunk to block abnormal discharges originating in the neuroma in CSNT rats. Results strongly suggest that sympathetic sprouting in pathologic DRG may be associated with abnormal spontaneous activity originating in the DRG or the injured axons (e.g. neuroma). This finding provides new insight into the mechanisms underlying sympathetic sprouting and increases our current understanding of the prolonged therapeutic effects of lidocaine on neuropathic pain syndromes.

Sympathetic sprouting in the dorsal root ganglion after spinal nerve ligation: evidence of regenerative collateral sprouting

It is well documented that there is an increase in the number of sympathetic fibers within the dorsal root ganglion (DRG) after a peripheral nerve injury. The present study examined the numbers and distribution of sympathetic fibers in the DRG and their sprouting routes by utilizing various surgical manipulations and retrograde tracing and immunohistochemical staining methods in spinal nerve-ligated neuropathic rats. The appearance of many double immunostained fibers with antibodies to tyrosine hydroxylase (TH) and growth associated protein-43 (GAP-43) in the L5 DRG 1 week after L5 spinal nerve ligation, indicated sprouting of sympathetic fibers. The confined location of early sprouting sympathetic fibers in the distal half of the L5 DRG confirmed that sprouting fibers come primarily from the injured spinal nerve. A second cut proximal to the previously ligated L5 spinal nerve — a process which would transect the regenerating sympathetic fibers extending from the injury site — did not change the density of sympathetic fibers in the L5 DRG. When retrograde tracers (fast blue and diamidino yellow) were injected into the L5 spinal nerve and DRG, respectively, the number of double-labeled sympathetic postganglionic neurons was greatly increased after spinal nerve ligation, suggesting the increased number of sympathetic neurons projecting to both the spinal nerve and DRG. All these results indicate that many sympathetic fibers in the DRG are regenerating branches that are sprouting from the proximal part of the injured spinal nerve (regenerative collateral sprouting).

Comparison of sympathetic sprouting in sensory ganglia in three animal models of neuropathic pain

Sympathetic postganglionic fibers sprout in the dorsal root ganglion (DRG) after peripheral nerve injury. Therefore, one possible contributing factor of sympathetic dependency of neuropathic pain is the extent of sympathetic sprouting in the DRG after peripheral nerve injury. The present study compared the extent of sympathetic sprouting in the DRG as well as in the injured peripheral nerve in three rat neuropathic pain models: (1) the chronic constriction injury model (CCI); (2) the partial sciatic nerve ligation injury model (PSI); and (3) the segmental spinal nerve ligation injury model (SSI). All three methods of peripheral nerve injury produced behavioral signs of ongoing and evoked pain with some differences in the magnitude of each pain component. The density of sympathetic fibers in the DRG was significantly higher at all examined postoperative times than controls in the SSI model, while it was somewhat higher than controls only at the last examined postoperative time (20 weeks) in the CCI and PSI models. Therefore, data suggest that, although sympathetic changes in the DRG may contribute to neuropathic pain syndromes in the SSI model, other mechanisms seem to be more important in the CCI and PSI models at early times following peripheral nerve injury.

Normal and injury‐induced sympathetic innervation of rat dorsal root ganglia increases with age

In rats, partial injury to a peripheral nerve often leads to sympathetically maintained pain (SMP). In humans, this condition is especially apparent in the elderly. Nerve injury also causes perivascular sympathetic axons to sprout into the dorsal root ganglion (DRG), forming a possible anatomical substrate for SMP. Here, we describe the effects of chronic sciatic nerve constriction injury (CCI) in young (3 months) and old (16 months) rats on neuropathic pain behavior and on sympathetic sprouting in DRG. Behavioral tests assessed changes in thermal allodynia and hyperalgesia and in mechanical allodynia. We found that 1) sympathetic innervation of the DRG increased naturally with age, forming pericellular baskets mainly around large DRG neurons, and that sympathetic fibers were often associated with myelinated sensory axons; 2) sympathetic fiber density following CCI was also greater in old than in young rats; and 3) in old rats, thermal allodynia was less pronounced than in young rats, whereas thermal hyperalgesia and mechanical allodynia were more pronounced. These results highlight the possibility that sympathetic sprouting in the DRG is responsible for the sympathetic generation or maintenance of pain, especially in the elderly. J. Comp. Neurol. 394:38–47, 1998. © 1998 Wiley-Liss, Inc.

Normal and injury-induced sympathetic innervation of rat dorsal root ganglia increases with age

In rats, partial injury to a peripheral nerve often leads to sympathetically maintained pain (SMP). In humans, this condition is especially apparent in the elderly. Nerve injury also causes perivascular sympathetic axons to sprout into the dorsal root ganglion (DRG), forming a possible anatomical substrate for SMP. Here, we describe the effects of chronic sciatic nerve constriction injury (CCI) in young (3 months) and old (16 months) rats on neuropathic pain behavior and on sympathetic sprouting in DRG. Behavioral tests assessed changes in thermal allodynia and hyperalgesia and in mechanical allodynia. We found that 1) sympathetic innervation of the DRG increased naturally with age, forming pericellular baskets mainly around large DRG neurons, and that sympathetic fibers were often associated with myelinated sensory axons; 2) sympathetic fiber density following CCI was also greater in old than in young rats; and 3) in old rats, thermal allodynia was less pronounced than in young rats, whereas thermal hyperalgesia and mechanical allodynia were more pronounced. These results highlight the possibility that sympathetic sprouting in the DRG is responsible for the sympathetic generation or maintenance of pain, especially in the elderly.

Sympathetic sprouting in the dorsal root ganglia of the injured peripheral nerve in a rat neuropathic pain model.

The extent of the sprouting of sympathetic postganglionic fibers in the dorsal root ganglion (DRG) and the peripheral nerves was examined in neuropathic rats at different postoperative times. After the L5 and L6 spinal nerves were ligated on one side, three different pain behavior tests (representing mechanical allodynia, cold allodynia, ongoing pain exacerbated by cold stress) were performed at various time intervals. The sympathetic postganglionic fibers were visualized by immunostaining with antibodies to tyrosine hydroxylase (TH). In the neuropathic rats, all three pain behaviors were fully developed within 3 days after the surgery, maintained up to 2 weeks, and then started to decline gradually afterward. At 20 weeks after neuropathic surgery, pain behaviors were reduced significantly compared to the peak response, but were still higher than the presurgery levels. Sympathectomy, performed 4 days after neuropathic surgery, almost completely abolished the signs of mechanical allodynia and ongoing pain behaviors, and it reduced the behaviors of cold allodynia to approximately half. The numerical density of sympathetic fibers in the DRG of an injured segment was significantly higher at 1, 4, and 20 weeks after neuropathic surgery as compared to the normal, suggesting that there is sprouting of sympathetic fibers in the DRG after peripheral nerve injury. Sprouting of sympathetic fibers in the DRG was extensive as early as 2 days after the spinal nerve ligation, and the sprouted fibers were almost completely eliminated after sympathectomy. The data suggest that sympathetic innervation of the DRG may play an important role in the development and maintenance of sympathetically maintained neuropathic pain.

Reinnervation of denervated iris by transplanted sympathetic ganglia: Effect of neuronal age

Neuronal outgrowth in vivo is aggressive postnatally, but is diminished with increasing age. This may be attributable to intrinsic features of the neuron or its interaction with other components of the developing organism. The purpose of this study was to determine if there is an age-dependent reduction in the intrinsic ability of sympathetic neurons to initiate fiber outgrowth. Superior cervical ganglia from donor rats aged 3–4, 11–12, 27–28 and 45–46 days were removed and transplanted to the anterior chamber of the sympathectomized eye of host rats 85–89 days of age. Ganglia with host irides were removed at 3, 6 and 10 days post-transplant and whole mounts were analysed using catecholamine histofluorescence for maximum sympathetic fiber density, length and initial rate of outgrowth. Fluorescent fibers were present in host irides of donors of all ages and at all post-transplant times. However, maximum fiber density was less for the 3–4 day-old donor ganglia (e.g. 43–71% of 11–46-day-old donor ganglia at 600 μm, 10 days post-transplant). Maximum fiber length was also less in the youngest group (e.g. 35–49% of 11–46-day-old donor ganglia, 10 days post-transplant). Further, the initial rate of outgrowth was decreased for the 3–4-day-old donor ganglia (128±46 μm/day for the 3–4-day-old ganglia vs 253±48 μm/day for the 11–12-day-old ganglia, 307±35 μm/day for the 27–28-day-old ganglia and 260±22 μm/day for the 45–46-day-old ganglia). We conclude that the capacity for neuronal outgrowth is limited in the early postnatal period, but rapidly attains the mature level by the second postnatal week. Therefore, diminished outgrowth in mature rats does not appear to be due to a decrease in the intrinsic ability of sympathetic nerves to initiate fiber outgrowth, but is attributable to other factors.

Disruption of target interactions prevents the development of enkephalin immunoreactivity in sympathetic neurons

We compared the development of enkephalin (Enk) expression in normal rats and rats in which target contact was transiently disrupted with 6-hydroxydopamine (6-OHDA). During the first 3 postnatal weeks, there was a striking increase in Enk immunoreactivity (-IR) in superior cervical ganglia (SCG) assayed by radioimmunoassay (RIA). This increase was correlated with the appearance of Enk-IR in postganglionic neurons. In the caudal region of the SCG, the proportion of Enk-IR neurons and their immunoreactivity increased until one-third of the neurons possessed Enk-IR between postnatal days (P) 14 and 21. After P21, the number of Enk-IR neurons and their immunofluorescence decreased. By 6 weeks, only occasional neurons possessed moderate Enk-IR. The increases in Enk-IR were correlated with increased ganglionic proenkephalin A mRNA detected by in situ hybridization. The decrease in IR after P21 was not, however, paralleled by a comparable decrease in proenkephalin A mRNA. To determine whether interactions between SCG neurons and their target tissues influence Enk expression, we disrupted them by treating neonatal rats with a single dose of 6-OHDA at P0. This treatment transiently reduced sympathetic fiber density in the submandibular gland, one target of Enk-IR neurons, over 90%. Two weeks later, the fiber density in glands of treated animals was not different from control. Following 6-OHDA, the concentration of Enk-IR in SCG extracts and the number of Enk-IR neurons and their immunofluorescence intensity failed to increase. SCG from treated rats also contained fewer neurons with proenkephalin A mRNA. In contrast, the content of neuropeptide Y (NPY) and the proportion of NPY-IR neurons were not decreased by 6-OHDA treatment. Our results indicate that the developmental history of Enk expression differs from that of other neuropeptides in rat sympathetic ganglia, suggesting that distinct mechanisms regulate the expression of individual neuropeptides. Further, they provide evidence that target contact during a critical period is important for the induction of Enk.