Remodeling of adult sensory axons in the superior cervical ganglion in response to exogenous nerve growth factor

Oxidative stress is involved in neuronal degeneration in cerebrovascular injury, neuropathology and aging. When rat CNS neurons were cultured in a high (50%) oxygen atmosphere, the neurons died. This high oxygen-induced cell death showed features of apoptotic cell death, characterized by DNA fragmentation, and was blocked by inhibitor of protein synthesis. We found that cystatin C and HuC mRNA, the products of which are an inhibitor of cysteine proteases and an RNA binding protein, respectively, were up-regulated in neurons cultured in the high oxygen atmosphere. In the present study, we focused on cystatin C. Cystatin C protein levels were also increased in neurons cultured in the high oxygen atmosphere. In situ hybridization with an RNA probe for rat cystatin C and immunocytochemistry with anti-human cystatin C antibody showed that microtubule-associated protein 2 (MAP2)-positive neurons expressed cystatin C mRNA and protein, respectively, in the high oxygen atmosphere. These results indicated that oxidative stress stimulates an increase in cystatin C expression in cultured neurons, and that cystatin C might have important roles in regulation of apoptosis elicited by oxidative stress.     

Sympathetic ingrowth to the trigeminal ganglion following intracerebroventricular infusion of nerve growth factor

The objective of the present study was to examine the remodeling of uninjured sympathetic axons in the adult rat trigeminal ganglion following a 2-week in vivo intracerebroventricular infusion of NGF. The accumulation of infused NGF in the trigeminal was assessed using ELISA and sympathetic fibers were localized immunohistochemically with an antibody to tyrosine hydroxylase (TH). In addition, high performance liquid chromatography coupled with electrochemical detection (HPLC-ECD) allowed for biochemical measurements of the catecholamines norepinephrine (NE) and dopamine (DA). Increased NGF protein in the trigeminal ganglion was paralleled by a significant increase in sympathetic fibers and pericellular plexuses (i.e. baskets) in the cell body regions. Some ganglia showed elevated NE following NGF infusion, yet the 88% increase in mean NE did not reach significance. Following bilateral removal of the sympathetic superior cervical ganglia (SCG), a significant reduction was observed in overall NE levels and in TH-immunoreactive (-ir) fibers in the cell body regions and peripheral branches, suggesting the SCG as the origin of the sympathetic ingrowth. However, mean DA levels as well as TH-ir fibers within the trigeminal central branch were unaffected by NGF infusion or removal of the SCG and likely resulted from intrinsic dopaminergic cell bodies. In conclusion, our data provide evidence that the increased availability of NGF in the young adult rat trigeminal ganglion observed following in vivo NGF infusion enhanced sympathetic associations with the sensory neurons in the trigeminal, supporting a role for NGF in the regulation of sympathosensory interactions.     

Quantitative analysis of the number and distribution of neurons richly innervated by GABA-immunoreactive axons in the rat superior cervical ganglion

The superior cervical ganglion of rats contains a considerable number of nerve fibers with GABA-like immunoreactivity which show a nonuniform distribution within the ganglion. The topography of these fibers has been analyzed by using antibodies raised against GABA-BSA-glutaraldehyde complexes. GABA-positive axons and axon varicosities accumulated around a subpopulation of principal ganglion cells forming basketlike patterns. These neurons richly innervated by GABA-positive axons (RIG-neurons) in turn were aggregated in patches with strong immunoreactivity. The size and packing density of the patches containing RIG-neurons and GABA-positive axons approaching them had rostral-to-caudal and medial-to-lateral gradients. Similar patterns were found in right and left ganglia. In five ganglia, a quantitative analysis revealed on average 1,344 RIG-neurons per ganglion representing about 5% of the total neuron population, with small variations (standard deviation 122) despite the highly variable shape of the ganglia. The distribution of RIG-neurons resembles that of neurons sending their axons into the internal carotid nerve. To check this possible correlation, HRP was injected into the eye and applied to the transected external carotid nerve. Double staining for the retrogradely transported peroxidase and GABA immunohistochemistry revealed that RIG-neurons formed a small subpopulation of retrogradely labelled neurons in both experiments. This suggests that RIG-neurons innervate various target organs. This conclusion is in agreement with the observation that RIG-neurons also exist in other sympathetic ganglia. Data presented suggest that sympathetic ganglion cells can be classified on the basis of non-uniform innervation patterns formed by axons that use different neurotransmitters.     

Effects on the growth of damaged ganglion cell axons after peripheral nerve transplantation in adult hamsters

After transplantation of autologous sciatic nerve segments into the retina of adult hamsters for 1-2 months, retrograde labelling with horseradish peroxidase demonstrated a population of ganglion cells situated peripheral to the graft. If an additional lesion was placed between the insertion of the graft and the optic disc at the same time as transplantation, in addition to labelled cells situated peripheral to the graft, retrograde labelling with horseradish peroxidase demonstrated a population of labelled neurons located between the graft and the optic disc which was not observed in animals without the additional lesion. Since the axons of this population of cells would have to turn around away from their normal course towards the optic disc and travel for about 1.5 mm in order to grow into the graft, it suggests that the peripheral nerve graft might play an active role in attracting and/or guiding damaged ganglion cell axons to grow into it.     

Sex differences in nerve growth factor levels in superior cervical ganglia and pineals

The current studies were undertaken to determine whether males or neonatally testosterone-treated rats of either gender have elevated endogenous levels of NGF in the SCG and one of its targets, the pineal gland. The ages studied were 5 days postnatal, which is at the peak of normal neuron death in the SCG but before a significant gender difference is present; 15 days, when normal neuron death is largely complete and males have more SCG neurons than females; and 30 days, when target innervation has matured.

At 5 days, while neuron death is occurring, but before there is a significant gender difference in neuron number in the SCG, pineal glands and SCGs of males had higher NGF content than those of females. The increased NGF in the ganglia of males at the time that these neurons are undergoing neuron death may play a role in the development of the sex difference in SCG neuron numbers. At 15 days, females had more NGF in their pineal glands and SCGs than did males, even though males have significantly more SCG neurons at this age than do females. This gender difference in the developmental course of NGF content could promote the survival of different populations of neurons in males and females. By 30 days, SCG and pineal NGF content of males was almost twice that of females. This is consistent with the presence of more neurons in the SCGs of males at this age.

Both the pineal gland and the SCG showed a loss of approximately 80% content of NGF during the first postnatal month. In males, this loss occurred between postnatal days 5 and 15, while in females, the drop in NGF content occurred between postnatal days 15 and 30. Treatment with testosterone from birth reduced NGF content in the SCGs of both males and females at 5 days of age. The depression of NGF levels by testosterone treatment may reflect a further acceleration of the developmental fall of NGC levels.     

Remodeling of motor nerve terminals in demyelinating axons of periaxin-null mice

Myelin formation around axons increases nerve conduction velocity and influences both the structure and function of the myelinated axon. In the peripheral nervous system, demyelinating forms of hereditary Charcot-Marie-Tooth (CMT) diseases cause reduced nerve conduction velocity initially and ultimately axonal degeneration. Several mouse models of CMT diseases have been generated, allowing the study of the consequences of disrupting Schwann cell function on peripheral nerve fibers. Nevertheless, the effect of demyelination at the level of the neuromuscular synapse has been largely overlooked. Here we show that in mice lacking functional Periaxin (Prx) genes, a model of a recessive type of CMT disease known as CMT4F, neuromuscular junctions (NMJs) develop profound morphological changes in the preterminal region of motor axons. These changes include extensive preterminal branches that originate in demyelinated regions of the nerve fiber and axonal swellings associated with residually-myelinated regions of the fiber. Using intracellular recording from muscle fibers we detected asynchronous failure of action potential transmission at high but not low stimulation frequencies, a phenomenon consistent with branch point failure. Taken together, our morphological and electrophysiological findings suggest that preterminal branching due to segmental demyelination near the neuromuscular synapse in Periaxin KO mice may underlie some characteristics of disabilities, including coordination deficits, present in this mouse model of CMT disease. These results reveal the importance of studying how demyelinating diseases might influence NMJ function and contribute to clinical disability.     

Null mutations for exon III and exon IV of the p75 neurotrophin receptor gene enhance sympathetic sprouting in response to elevated levels of nerve growth factor in transgenic mice

Under normal conditions, expression of the p75 neurotrophin receptor (p75NTR) by sympathetic neurons can increase the affinity of the signaling receptor, trkA, to target-derived nerve growth factor (NGF) at distal axons. We have previously reported that sprouting of sympathetic axons into NGF-rich target tissues is enhanced when p75NTR expression is perturbed, leading to the postulate that p75NTR may restrain sympathetic sprouting in response to elevated NGF levels. These observations were made using mice having a null mutation of the third p75NTR exon, a line that may express a hypomorphic form of this receptor. Since mice carrying a null mutation of the fourth p75NTR exon may not express a similar splice variant, we sought to determine whether these animals possess the same phenotype of enhanced sympathetic sprouting in response to elevated levels of NGF. Both lines of transgenic mice lacking p75NTR displayed similar degrees of sympathetic axonal sprouting into the cerebellum and trigeminal ganglia, two target tissues having elevated levels of NGF protein. Furthermore, the densities of sympathetic axons in both targets were significantly greater than those observed in age-matched NGF transgenic siblings expressing full-length p75NTR. Our new findings provide a comparative analysis of the phenotype in two independent mutations of the same neurotrophin receptor, revealing that p75NTR plays an important role in restricting sympathetic sprouting in response to higher NGF levels.     

Morphological response of axotomized septal neurons to nerve growth factor

Septal efferent fibers from the neurons in the medial septal nucleus are destroyed by fimbria-fornix aspirative lesion. In the present study we used quantitative morphometric techniques to evaluate the response of these axotomized septal neurons to a constant infusion of nerve growth factor (NGF). By 2 weeks following the lesion, approximately 75% of the cholinergic neurons had degenerated in the untreated rats. The remaining cholinergic neurons showed few signs of the effect of the lesion when stained for a polyclonal antibody to ChAT and examined in 40-micron-thick sections. In 1-micron-thick sections the remaining ChAT-immunoreactive (IR) neurons also appeared no different from the intact ChAT neurons. However, non-ChAT-IR neurons had a shrunken nucleus, while all other morphometric parameters appeared normal. NGF infusion protected most of the ChAT-IR neurons from degenerating. The saved neurons had the same parameters as the undamaged ChAT-IR neurons when examined in either 40-micron- or 1-micron-thick sections. In addition, the shrunken appearance of the non-ChAT-IR neurons' nuclei was avoided by the NGF infusions. Enlarged ChAT-IR processes were evident in the dorsolateral quadrant of the septum following damage to the fimbria-fornix. NGF-infusions prevented the formation of these processes. Instead, in the treated animals the dorsal lateral quadrant contained a dense plexus of fine ChAT-IR varicosities. Taken together these results demonstrate that NGF not only can protect the cholinergic neurons from axotomy-induced degeneration but can also cause the saved neurons to maintain the same morphometric appearance as intact ChAT-IR neurons.(ABSTRACT TRUNCATED AT 250 WORDS)     

Gamma-aminobutyric acid-containing sympathetic preganglionic neurons in rat thoracic spinal cord send their axons to the superior cervical ganglion

Gamma-aminobutyric acid (GABA)-containing fibers have been observed in the rat superior cervical ganglion (SCG) and, to a lesser extent, in the stellate ganglion (STG). The aim of present study is to clarify the source of these fibers. No cell body showed mRNAs for glutamic acid decarboxylases (GADs) or immunoreactivity for GAD of 67 kDa (GAD67) in the cervical sympathetic chain. Thus, GABA-containing fibers in the ganglia are suggested to be of extraganglionic origin. GAD67-immunoreactive fibers were found not in the dorsal roots or ganglia, but in the ventral roots, so GABA-containing fibers in the sympathetic ganglia were considered to originate from the spinal cord. Furthermore, almost all GAD67-immunoreactive fibers in the sympathetic ganglia showed immunoreactivity for vesicular acetylcholine transporter, suggesting that GABA was utilized by some cholinergic preganglionic neurons. This was confirmed by the following results. 1) After injection of Sindbis/palGFP virus into the intermediolateral nucleus, some anterogradely labeled fibers in the SCG were immunopositive for GAD67. 2) After injection of fluorogold into the SCG, some retrogradely labeled neurons in the thoracic spinal cord were positive for GAD67 mRNA. 3) When the ventral roots of the eighth cervical to the fourth thoracic segments were cut, almost all GAD67- and GABA-immunoreactive fibers disappeared from the ipsilateral SCG and STG, suggesting that the vast majority of GABA-containing fibers in those ganglia were of spinal origin. Thus, the present findings strongly indicate that some sympathetic preganglionic neurons are not only cholinergic but also GABAegic.     

Metabolic activation of specific postsynaptic elements in superior cervical ganglion by antidromic stimulation of external carotid nerve

Using the rat superior cervical ganglion in vivo as a model of the central nervous system, we have sought to determine whether postsynaptic elements as well as terminal processes are metabolically activated during impulse activity. The rate of glucose utilization in the ganglion was found to be stimulated by antidromic stimulation of the external carotid nerve. This stimulation was frequency-dependent and was confined only to the region in the caudal portion of the ganglion corresponding to the location of the cell bodies of origin of the external carotid nerve.     

Lack of dystrophin leads to the selective loss of superior cervical ganglion neurons projecting to muscular targets in genetically dystrophic mdx mice

Autonomic imbalance is a pathological aspect of Duchenne muscular dystrophy. Here, we show that the sympathetic superior cervical ganglion (SCG) of mdx mice, which lack dystrophin (Dp427), has 36% fewer neurons than that of wild-type animals. Cell loss occurs around P10 and affects those neurons innervating muscular targets (heart and iris), which, differently from the submandibular gland (non-muscular target), are precociously damaged by the lack of Dp427. In addition, although we reveal altered axonal defasciculation in the submandibular gland and reduced terminal sprouting in all SCG target organs, poor adrenergic innervation is observed only in the heart and iris. These alterations, detected as early as P5, when neuronal loss has not yet occurred, suggest that in mdx mice the absence of Dp427 directly impairs the axonal growth and terminal sprouting of sympathetic neurons. However, when these intrinsic alterations combine with structural and/or functional damages of muscular targets, neuronal death occurs.     

Fos induction by nerve growth factor in the adult rat brain

The distribution of Fos, the protein product of the immediate early gene c-fos, was studied with immunocytochemistry in the adult male rat brain after nerve growth factor (NGF) administration. NGF was injected in the lateral cerebral ventricle through a previously implanted cannula. The total number of Fos-immunoreactive (ir) neurons in the brain was 2–3 times higher after NGF administration than in control animals (untreated or injected with cytochrome c). With respect to control rats, in the NGF-treated cases Fos-ir cells were more numerous in the anterior olfactory nucleus, in the medial prefrontal and anterior cingulate cortices, in the basal forebrain, in the preoptic and ventromedial nuclei of the hypothalamus, as well as interior hypothalamic area, in the thalamic midline nuclei, and in some brainstem structures, such as the parabrachial nucleus. The relative quantitative increase of Fos-ir neurons varied in the different structures. In addition, Fos-ir neurons were evident after NGF administration in areas devoid of immunopositive cells in control animals. These included: frontoparietal and occipital cortical fields, the hypothalamic arcuate nucleus, and many brainstem structures, such as the dorsal nucleus of the lateral lemniscus, posterodorsal tegmental, medial and lateral vestibular, ventral cochlear, and prepositus hypoglossal nuclei. These findings demonstrate that the intracerebroventricular administration of NGF can induce c-fos expression in neurons in vivo. The distribution of Fos-ir neurons indicates that NGF can induce activation of functionally and chemically hetergeneous neuronal subsets in the brain.     

Localization of vasoactive intestinal peptide- and peptide histidine isoleucine amide-like immunoreactivities in the rat superior cervical ganglion and its nerve trunks

Electrical stimulation of the preganglionic cervical sympathetic trunk causes an increase in dopa synthesis in the postganglionic neurons in the superior cervical ganglion (SCG). This transsynaptic biochemical effect can be blocked only partially by cholinergic antagonists, suggesting the involvement of a noncholinergic preganglionic sympathetic neurotransmitter(s). A survey of a large number of possible candidates for this neurotransmitter revealed that, in addition to cholinergic agonists, only a small group of peptides (all members of the secretin-glucagon family) stimulated dopa synthesis in the SCG. The effective peptides included vasoactive intestinal peptide (VIP), peptide histidine isoleucine amide (PHI), and secretin. Consequently we looked for the presence of immunoreactivities for these three peptides in the SCG. VIP- and PHI-like immunoreactive fibers were found in the SCG and in its major pre- and postganglionic nerve trunks. The distributions of the two immunoreactivities were very similar. Immunoreactive fibers were seen both singly and in bundles. In some instances, fibers were found apposed to neuronal cell bodies in the ganglion, and occasionally dense plexuses of fibers were found surrounding the neurons. In addition, punctate immunoreactive profiles were found apposed to the neurons in what appeared to be terminal fields. A small number of immunoreactive neuronal cell bodies were also seen in the ganglion. In a few instances, it was possible to establish, in serial sections, that the same cell body was immunostained with both VIP and PHI antisera. No secretin like-immunoreactive fibers or cells were observed. The presence of VIP-like and PHI-like-immunoreactive fibers in the cervical sympathetic trunk and in the SCG strengthens the possibility that these peptides, or a related molecule(s), serve as preganglionic neurotransmitters in this ganglion.     

Muscarinic receptor binding and oxidative enzyme activities in the adult rat superior cervical ganglion: effects of 6-hydroxydopamine and nerve growth factor

Administration of 6-hydroxydopamine to adult rats results in changes in the superior cervical ganglion similar to those noted after axotomy; namely, a decrease in muscarinic receptor binding and increases in activities of the oxidative enzymes of the pentose phosphate pathway. These changes were either prevented or attenuated markedly by the systemic administration of nerve growth factor. Administration of nerve growth factor alone did not significantly increase N-methylscopolamine binding in the ganglion or reduce the activities of the oxidative enzymes. Explants of the ganglion maintained in serum-free medium over a period of 3 days demonstrated increases in oxidative enzyme activity and a decrease in N-methylscopolamine binding. Addition of 20 nM nerve growth factor to the culture medium prevented the decline in N-methylscopolamine binding in ganglion explants. The increases in oxidative enzyme activities were unaltered. Addition of high amounts of nerve growth factor, 200 nM, resulted in a significant increase in tyrosine hydroxylase activity but no further increase in N-methylscopolamine binding in ganglion explants. Glucocorticoids added to the culture medium did not affect the muscarinic binding or enzyme activities. Thus, decreases in muscarinic binding activity which occur in the superior cervical ganglion after axotomy or 6-hydroxydopamine treatment may be explained by a loss of nerve growth factor supplied to the ganglion. Increases in the oxidative enzymes of the pentose phosphate pathway that occur in the ganglion after axonal injury appear to involve additional factors.     

Stimulation and maintenance by nerve growth factor of phenylethanolamine-N-methyltransferase in superior cervical ganglia of adult rats

Treatment of newborn rats with nerve growth factor (NGF) results in a striking increase in phenylethanolamine-N-methyltransferase (PNMT) in the superior cervical ganglia. Between one and three weeks of age there is normally a 10-fold decrease in PNMT activity in ganglia of infant rats. NGF treatment maintains the PNMT in ganglia at levels 10-fold greater than in untreated controls, but the levels of enzyme in the ganglion show the same magnitude of decrease with age. Epinephrine levels are markedly increased in ganglia of NGF-treated rats younger than one week of age, but at older ages the levels of the catecholamine are only slightly greater than the controls. Dexamethasone is less effective than NGF in increasing the levels of PNMT in ganglia of infant rats and, unlike NGF, becomes ineffective by 44 days of age. These results suggest that there may be two types of PNMT-containing cells in ganglia of newborn rats.     

Evaluation of sympathetic skin response in Parkinson's disease

There is no clear definition on the role of sympathetic skin response (SSR) in the evaluation of patients with Parkinson's disease (PD). We recorded the SSR of the palms of 64 controls and 46 patients with PD to electrical stimulation of the median nerve at the wrist. We analyzed onset latency and peak-to-peak amplitude. A study of parasympathetic function (R–R interval analysis) was also undertaken. We found that patients with PD had more absent SSRs than controls. The mean amplitude of the SSR was significantly reduced in both lower and upper limbs of PD patients in comparison with control subjects (p<0.001). The onset latency was longer in the lower limbs of these patients in respect to the control group (p<0.003). There was a significant inverse correlation between SSR amplitudes and age, severity and late onset of the disease. There was no association of these parameters with dysautonomic symptoms or R–R interval variation. In conclusion, there is a significant association between altered SSR and PD and an inverse correlation in this group of patients between SSR values and older age, greater severity and later onset of disease. Therefore, the study of SSR may provide valuable information on cholinergic sympathetic function in patients with PD.     

Pre- and postnatal development of the small intensely fluorescent cells in the rat superior cervical ganglion

A fluorescence microscopical study has been performed in the pre- and postnatal development of the sympathetic superior cervical ganglion of Sprague-Dawley rat. Ganglia from 10.5- to 21.5-day-old embryos and newborn to 90-day-old postnatal rats were freeze-dried and the catecholamine-containing cells were demonstrated by formaldehyde-induced fluorescence.The first catecholamine-containing cells appeared on day 11.5 of gestation. The ganglia of 11.5- to 12.5-day-old embryos contained a continuous range of cells showing weak to bright fluorescence intensities. In the ganglia of 13.5- to 14.5-day-old embryos few solitary cells or small groups of cells showing bright fluorescence were discernible among weakly fluorescent developing principal nerve cells. The cell numbers of both types markedly increased during the prenatal period while the mean diameter of the brightly fluorescent cells significantly decreased. In the late prenatal ganglia clusters were observed in which a large brightly fluorescent cell was surrounded by other intensely fluorescent cells of various sizes.There were about 300 small intensely fluorescent (SIF) cells in the ganglia of newborn rats. This number decreased by about 50% during postnatal week 1 and again increased during postnatal week 4 reaching the adult value of about 500 cells/ganglion. During the postnatal development the SIF cells formed clusters, the largest of which contained several hundred cells.Occurrence of large intensely fluorescent cells during a limited period suggests that this cell type represents a transitional form. It is possible that in the rat the primitive sympathetic cells continue their development along two lines: some cells remain weakly fluorescent and give rise to the principal nerve cells, and others accumulate catecholamine(s) and are transformed, possibly after mitotic divisions, into smaller brightly fluorescent cells which mature into SIF cells late postnatally.