Degenerative and inflammatory lesions in sympathetic ganglia: further morphological evidence for an autonomic neuropathy in AIDS.

There is accumulating evidence that autonomic dysfunction occurs in HIV infection. While many studies have demonstrated autonomic abnormalities on clinical basis, only one has studied the morphology of sympathetic ganglia. The superior sympathetic ganglia of 12 randomly selected AIDS patients and those of 6 controls were examined morphologically in order to determine the frequency and severity of their involvement. Although they had not been investigated for autonomic dysfunction, 5 had suffered from non-infectious diarrhoea, one showed bilateral ptosis and another had non-specified visual problems. All cases showed clusters, and perivascular mononuclear inflammatory cells, occasionally infiltrating vessel walls, some evidence of nerve cell degeneration, and proliferation of capsule cells. Immunostainings showed T lymphocytes and an increased number of macrophages. HIV antigens were detected in macrophages, in 6 cases (50%). This study provides further morphological support for the autonomic dysfunction in association with HIV infection. As for the mechanism of this dysfunction, it has been postulated a direct infection, the virus entering the ganglia through macrophages and acting as a reservoir for HIV, and an autoimmune pathogenesis. Since HIV antigens were not detected in 50% of the cases in this and in a previous study, despite the existence of morphological lesions, it is possible that, as in HIV-related sensory-motor peripheral neuropathies, an autoimmune mechanism may also play a role in the development of the autonomic lesions.

Complement regulatory proteins and selective vulnerability of neurons to lysis on exposure to acetylcholinesterase antibody.

Systemic injection of antibodies against acetylcholinesterase (AChE) induces complement-mediated destruction of preganglionic nerve terminals in paravertebral sympathetic ganglia, but spares other AChE-rich structures, such as nerve terminals in prevertebral sympathetic ganglia, parasympathetic ganglia, and the neuromuscular junction. This pattern of differing sensitivity to "AChE immunolesion" might be explained by a differing expression of proteins that serve to protect host cells from complement activation. Two major complement regulatory proteins in rats are Crry, which interferes with the assembly of C3 convertase, and CD59, which blocks formation of the terminal cytolytic membrane attack complex. The present study used immunohistochemistry to demonstrate an inverse relation between levels of CD59 and Crry expression and sensitivity to AChE immunolesion in several AChE-rich targets. Thus, the most sensitive structures, i.e., preganglionic nerve terminals in the adrenal gland and superior cervical ganglion (SCG), expressed undetectable levels of CD59 and Crry immunoreactivities. By contrast, AChE-rich, but antibody-resistant, cholinergic nerve terminals in the inferior mesenteric ganglia (IMG) and diaphragm muscle expressed significant amounts of CD59 and Crry. Such expression was functionally important because, after membrane-anchored CD59 was removed from explanted IMG with phosphatidylinositol phospholipase C, exposure to AChE antibody and complement caused greater immunolesion. It was concluded that differential expression of regulatory proteins in different parts of the nervous system influences regional vulnerability to complement mediated damage.

Characterization of novel neutralizing monoclonal antibodies specific to human neurturin.

Neurturin (NTN) a structural and functional relative of glial cell line-derived neurotrophic factor, was originally identified based on its ability to support the survival of sympathetic neurons in culture. Similar to glial cell line-derived neurotrophic factor (GDNF), Neurturin has been shown to bind to a high affinity glycosylphosphatidylinositol (GPI)-linked receptor (GFRalpha2) and induce phosphorylation of the tyrosine kinase receptor Ret, resulting in the activation of the mitogen activated protein kinase (MAPK) signalling pathway. A panel of six novel murine monoclonal antibodies (MAbs) specific to human Neurturin has been developed and characterized. Four of the MAbs tested inhibit, to varying degrees, binding of NTN to the GPI-linked GFRalpha2 receptor. Three MAbs cross-react with the murine homolog. These antibodies have been shown to be useful reagents for Western blotting, immunohistochemistry, and also for the development of a sensitive, quantitative enzyme-linked immunosorbent assay (ELISA) for human NTN. Novel, specific MAbs with varying epitope specificities and blocking activity will be valuable tools for both the in vitro and in vivo characterization of NTN and its relationship to the GFRalpha2 and Ret receptors.

Complement-mediated lesion of sympathetic ganglia in vitro with acetylcholinesterase antibodies.

When administered to rats, antibodies against acetylcholinesterase (AChE) selectively destroy presynaptic inputs to sympathetic ganglia. To investigate the mechanism of this immunolesion, we created an in vitro system in which relevant components could be manipulated. Freshly dissected rat superior cervical ganglia (SCG) were incubated 15-20 h at 37 degrees C in fresh human serum (a potent source of complement) with continuous oxygenation. More than 96% of neurons in six control ganglia retained synaptic inputs, as defined by action potentials or excitatory postsynaptic potentials (EPSP) upon stimulation of the preganglionic trunk. However, when anti-AChE antibodies were present (0.16 mg/ml), none of 61 neurons from six incubated ganglia showed synaptic responses although membrane potential and input resistance remained normal. Staining for AChE and synaptophysin (a synaptic vesicle marker) was also disrupted in ganglia exposed to AChE antibodies in complement-sufficient serum. When complement was eliminated by substituting serum that was heat-inactivated or deficient in C3, synaptic input was retained in 60-90% of neurons incubated with AChE antibodies. Choline acetyltransferase activity (ChAT), an enzymatic marker of cholinergic cytoplasm in sympathetic ganglia, was largely lost after incubation with AChE antibodies and serum. However, incubation with AChE antibodies in heat-inactivated serum, or serum that was deficient in C3 or C8, caused no measurable loss of ganglionic ChAT activity. These findings strongly implicate the complement cascade in the destruction of preganglionic sympathetic terminals that follows binding of AChE antibodies.

Cultured sympathetic neurons express functional interleukin-1 receptors.

Since recent work has identified an apoptotic pathway in sympathetic neurons that is mediated by autocrine interleukin-1 (IL-1), we investigated whether cultured sympathetic neurons possess functional IL-1 receptors. Cultured sympathetic neurons express levels of IL-1RI and IL-1RAcP mRNAs consistent with signal transduction. Neurons stimulated with IL-1 demonstrate enhanced p65 NF-kappaB nuclear translocation and enhanced NF-kappaB DNA binding activity, with at least p65 and p50 subunits participating in the DNA binding activity. RNA differential display identified several neuronal mRNAs regulated by IL-1, including a member of the reticulon family. We conclude that IL-1 stimulates a potential component of a neuronal secretory pathway.

Selective disruption of neurotransmission by acetylcholinesterase antibodies in sympathetic ganglia examined with intracellular microelectrodes.

Antibodies to acetylcholinesterase (AChE) induce adrenergic dysfunction in rats by selective, complement-mediated destruction of preganglionic sympathetic nerve terminals. To analyze this phenomenon at the neuronal level, monoclonal antibodies to AChE (1.6 mg) were injected via the tail vein, and superior cervical ganglia (SCG) or inferior mesenteric ganglia (IMG) were studied in vitro. In control SCG, all impaled neurons generated action potentials during direct injection of depolarizing current or indirect stimulation through the preganglionic nerve. Current injection remained effective in ganglia from treated rats, but preganglionic stimulation was greatly impaired: at 12 h and 3 d, less than 10% of the neurons responded, even to a maximal stimulus (150 V); at 9 d, only 25% responded. By contrast, in IMG, synaptic transmission was much less affected by antibody exposure: 60% or more of examined neurons responded to preganglionic stimulation. Differences in antibody access did not explain differing sensitivities of SCG and IMG since immunohistochemistry showed rapid accumulation of IgG deposits in both ganglia. These results are believed to reflect widespread but subtotal preganglionic sympathectomy by AChE antibodies. Current information indicates that paravertebral ganglia are all antibody-sensitive, but some prevertebral ganglia are resistant, suggesting immunochemical differences between them.

Antigen-induced synaptic plasticity in sympathetic ganglia from actively and passively sensitized guinea-pigs.

Alterations in synaptic efficacy induced by antigen challenge to isolated superior cervical ganglia (SCG) were monitored by measuring the magnitude of the postganglionic compound action potential (CAP) elicited by electrical stimulation of the cervical sympathetic nerve trunk. Antigen-induced changes in the CAP were measured in SCG removed from actively and from passively sensitized guinea-pigs. Additionally, some SCG were sensitized in vitro by incubating naive ganglia 24 h in serum obtained from actively sensitized animals. Histamine released from SCG upon specific antigenic challenge was measured to assess the effectiveness of the two forms of sensitization. Challenging SCG isolated from passively or actively sensitized animals with the sensitizing antigen, ovalbumin (OVA), produced a sustained potentiation of the CAP lasting longer than 30 min (antigen-induced long-term potentiation, A-LTP) and a net increase in histamine release. Neither the magnitude nor duration of A-LTP induced by passive sensitization differed significantly (p < 0.05) from results after active sensitization. The existence of A-LTP in SCG following passive sensitization indicates that the afferent limb of the immune system is not required for the development of this phenomenon and that the immune cells and the mediators responsible for A-LTP are resident to sympathetic ganglia.

Calretinin-containing preganglionic nerve terminals in the rat superior cervical ganglion surround neurons projecting to the submandibular salivary gland.

The distribution and targets of calretinin-immunoreactive preganglionic nerve terminals in the superior cervical ganglion of the rat were examined using immunohistochemistry and retrograde neuronal tracing. Calretinin-immunoreactive nerve terminals were found throughout the ganglion, forming distinct pericellular baskets around a sub-population of postganglionic neurons. The targets of postganglionic neurons surrounded by calretinin-immunoreactive nerve terminals were determined after injection of tracer into the submandibular salivary gland, the extra-orbital lacrimal gland, the thyroid gland, the anterior chamber of the eye or the skin of the forehead. Only when tracer was injected into the submandibular gland were neurons labelled that were surrounded by calretinin-immunoreactive nerve terminals. When immunohistochemistry using antisera to neuropeptide Y was combined with retrograde tracing, only submandibular gland projecting neurons lacking neuropeptide Y were surrounded by calretinin-immunoreactive terminals. When retrograde neuronal tracer was injected into the superior cervical ganglion, a proportion of retrogradely-labelled neurons in the upper thoracic spinal cord showed relatively weak calretinin-immunoreactivity. All calretinin-immunoreactive terminals in the superior cervical ganglion disappeared following section of the sympathetic chain distal to the superior cervical ganglion. Thus, calretinin is present in a population of preganglionic neurons projecting exclusively to neuropeptide Y non-immunoreactive (presumably secretomotor) neurons innervating the submandibular salivary gland of the rat.

Antigen-induced long-term potentiation of nicotinic synaptic transmission in the superior cervical ganglion of the guinea pig.

1. Recordings of evoked postganglionic compound action potentials (CAPs) evoked by preganglionic stimulation were obtained from guinea pig superior cervical ganglia (SCGs) in vitro to study the effects of specific antigen challenge on ganglionic synaptic transmission. SCGs were removed from guinea pigs actively sensitized to ovalbumin. 2. Exposing SCGs from sensitized animals to the sensitizing antigen (0.01-10 micrograms/ml) for 5 min produced a sustained increase in the magnitude of the evoked CAP unaccompanied by a change in the preganglionic volley. Nonsensitizing antigens were ineffective. Also ineffective were antigens applied to nonsensitized SCG. This persistent antigen-induced increase in synaptic transmission was designated antigen-induced long-term potentiation (LTP) (A-LTP) because its duration (> 30 min) greatly outlasted posttetanic potentiation (PTP) in this ganglion. 3. A-LTP and neurogenic LTP (N-LTP) were observed to coexist in the same ganglion; the presence of one form of synaptic plasticity did not preclude the development of the other. Both phenomena were influenced by presynaptic factors: prolonged (2 h, 40 Hz) repetitive presynaptic stimulation abolished A-LTP or N-LTP but did not affect PTP. 4. By contrast to N-LTP, which requires a brief presynaptic tetanus, A-LTP could be triggered over a wide range of presynaptic stimulation (0.016-3 Hz) or even in the absence of presynaptic stimulation. 5. The amplitude and duration of A-LTP were not significantly affected by 1) H1, H2, or H3 histamine receptor antagonists added before or after antigen challenge; 2) the presence of saturating concentrations of histamine (100-300 microM); 3) the presence of specific or nonspecific lipoxygenase inhibitors or a selective cyclooxygenase inhibitor; or 4) blockade of alpha- or beta-adrenergic receptors, 5-HT3 receptors, muscarinic receptors, or glutamate receptors, or inhibition of acetylcholinesterase or protein synthesis. 6. Our results indicate that specific antigen challenge of isolated sympathetic ganglia activates resident mast cells to release substances that initiate a novel form of synaptic plasticity, an activity-independent and long-lasting increase in synaptic efficacy.

Calcitonin gene-related peptide-like immunoreactivity in spinal cord and superior cervical ganglion of the Djungarian hamster (Phodopus sungorus).

The indirect immunofluorescent method was employed to investigate the distribution of calcitonin gene-related peptide-like immunoreactivity (CGRP-LI) in the spinal cord and superior cervical ganglion of the Djungarian hamster Phodopus sungorus. In cross-sections of the spinal cord, immunoreactive fibres and terminals were found in laminae 1 and 2 in high density, in the dorsolateral (Lissauer's) tract, in ventral and lateral horns, and in the area surrounding the central canal. A few CGRP-LI perikarya were seen in the ventral but not the dorsal horn. CGRP-LI was further observed in preganglionic sympathetic neurons which were labelled by retrograde axonal transport of fluoro-gold (FG) following injection of the substance unilaterally into the superior cervical ganglion. Preganglionic sympathetic neurons (PSN) were localized ipsilateral to the injection site mainly in the intermediolateral nucleus and the lateral funiculus of the upper thoracic segments. Most PSN exhibited CGRP-LI. Immunoreactive PSN were not seen contralaterally to the site of FG application nor in animals that did not receive injections. When the preganglionic fibres were ligated 4 days before perfusion, CGRP-LI cell bodies were found in preganglionic sympathetic neurons similar to the situation seen upon FG treatment. In the superior cervical ganglia of untreated hamsters, immunoreactive fibres were seen to enter the ganglion in which they terminated at non-immunoreactive principal ganglion cells. The present study, the first in a hamster species, describes the widespread distribution of CGRP in the spinal cord of P. sungorus and supports the view that considerable interspecies differences exist in occurrence and location of this neuropeptide.

Development of substance P and neurokinin A immunoreactivity in ganglia supplying nerves to the submandibular glands of the rat.

Developing submandibular, trigeminal and superior cervical ganglia, which provide innervation to the submandibular glands, were studied for substance P (SP)- and neurokinin A (NKA)-immunoreactive (IR) ganglion cells and nerve fibres in rat. These ganglia were examined by using an indirect immunofluorescence technique at daily intervals from the 16th day in utero (i.u.) until birth, and subsequently on the 2nd, 5th, 7th, 12th, 16th, 30th, 42nd postnatal day and in the adult (3 months). In the submandibular ganglion SP- and NKA-IR cells and fibres first appeared in considerable numbers on the 19th day i.u. (in one sample out of five on the 18th day i.u.), when more than 90% of the ganglion cells were immunoreactive to SP and NKA. The number stayed at more than 90% to the 7th postnatal day and then slowly decreased to the levels of adult animals (18% SP, 17% NKA). The first SP- and NKA-IR ganglion cells and fibres appeared in the trigeminal ganglion on the 18th day i.u. when they represented 7% (SP) and 4% (NKA) of the ganglion cells. The number of SP- and NKA-IR cells increased steadily, reaching a maximum at the time of birth when 68% (SP) and 74% (NKA) of the ganglion cells were immunoreactive. Thereafter they began to decrease toward the level of an adult rat (10% SP, 11% NKA). In the superior cervical ganglion only a few SP- and NKA-IR ganglion cells were detected from the 19th day i.u. to the fifth postnatal day. Positive ganglion cells were also occasionally found in the nerve trunks outside the superior cervical ganglion.(ABSTRACT TRUNCATED AT 250 WORDS)