Cardiac sympathetic denervation in 6-OHDA-treated nonhuman primates.

Cardiac sympathetic neurodegeneration and dysautonomia affect patients with sporadic and familial Parkinson's disease (PD) and are currently proposed as prodromal signs of PD. We have recently developed a nonhuman primate model of cardiac dysautonomia by iv 6-hydroxydopamine (6-OHDA). Our in vivo findings included decreased cardiac uptake of a sympathetic radioligand and circulating catecholamines; here we report the postmortem characterization of the model. Ten adult rhesus monkeys (5-17 yrs old) were used in this study. Five animals received 6-OHDA (50 mg/kg i.v.) and five were age-matched controls. Three months post-neurotoxin the animals were euthanized; hearts and adrenal glands were processed for immunohistochemistry. Quantification of immunoreactivity (ir) of stainings was performed by an investigator blind to the treatment group using NIH ImageJ software (for cardiac bundles and adrenals, area above threshold and optical density) and MBF StereoInvestigator (for cardiac fibers, area fraction fractionator probe). Sympathetic cardiac nerve bundle analysis and fiber area density showed a significant reduction in global cardiac tyrosine hydroxylase-ir (TH; catecholaminergic marker) in 6-OHDA animals compared to controls. Quantification of protein gene protein 9.5 (pan-neuronal marker) positive cardiac fibers showed a significant deficit in 6-OHDA monkeys compared to controls and correlated with TH-ir fiber area. Semi-quantitative evaluation of human leukocyte antigen-ir (inflammatory marker) and nitrotyrosine-ir (oxidative stress marker) did not show significant changes 3 months post-neurotoxin. Cardiac nerve bundle α-synuclein-ir (presynaptic protein) was reduced (trend) in 6-OHDA treated monkeys; insoluble proteinase-K resistant α-synuclein (typical of PD pathology) was not observed. In the adrenal medulla, 6-OHDA monkeys had significantly reduced TH-ir and aminoacid decarboxylase-ir. Our results confirm that systemic 6-OHDA dosing to nonhuman primates induces cardiac sympathetic neurodegeneration and loss of catecholaminergic enzymes in the adrenal medulla, and suggests that this model can be used as a platform to evaluate disease-modifying strategies aiming to induce peripheral neuroprotection.

Small intensely fluorescent cells of the rat paracervical ganglion synthesize adrenaline, receive afferent innervation from postganglionic cholinergic neurones, and contain muscarinic receptors.

In the paracervical ganglion (PCG) of the rat, double-labelling immunofluorescence for catecholamine-synthesizing enzymes and HPLC measurement of catecholamine contents were first performed to evaluate whether intraganglionic small intensely fluorescent (SIF) cells are capable of synthesizing adrenaline. Immunolabelling for tyrosine hydroxylase (TH), dopamine beta-hydroxylase and phenylethanolamine-N-methyl transferase (PNMT) occurred in all SIF cells of the PCG, thus demonstrating the presence of all the enzymes required for adrenaline biosynthesis. Adrenaline levels were undetectable in the PCG but to test the hypothesis that PNMT is active in SIF cells, catecholamines were measured in ganglia of rats pretreated with pargyline, an inhibitor of the monoamine oxidase, the major enzyme involved in the catecholamine degradation. Pargyline treatment increased adrenaline levels in the PCG, thus demonstrating that SIF cells are capable of adrenaline synthesis. The undetectable levels of adrenaline in the PCG of untreated rats suggested a slow rate of biosynthesis of adrenaline in the ganglion. Furthermore, the use of double-labelling showed that SIF cells of the PCG were stained for muscarinic receptors and were approached by varicose ChAT-immunoreactive nerve fibres. Nerve fibres immunoreactive for ChAT were also observed associated with nerve cell bodies of ganglion neurones. Following deafferentation of the PCG, the ChAT-immunoreactive nerve fibres surrounding nerve cell bodies totally disappeared indicating their preganglionic origin, while those associated with SIF cells did not degenerate, which demonstrate that they derived from intraganglionic cholinergic neurones. Taken together, the results show that adrenaline may be a transmitter for SIF cells in the PCG and suggest that cholinergic neurones of the parasympathetic division of the PCG can modulate the SIF cell activity through the activation of muscarinic receptors.

Colocalization of acetylcholinesterase and vasoactive intestinal peptide (VIP) in nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) positive neurons in the intralingual ganglia and perivascular nerve fibers around lingual arteries in the porcine, monkey and canine tongue.

Distribution of nitric oxide synthase in the intrinsic ganglia in the porcine, monkey and canine tongue was histologically investigated using the reduced nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) method, acetylcholinesterase histochemistry and vasoactive intestinal peptide (VIP) immunohistochemistry. The majority of intralingual ganglionic cells showed intense NADPH-d reactivity with positive acetylcholinesterase reaction or positive VIP immunohistochemistry. The NADPH-d positive, acetylcholinesterase-rich and the NADPH-d positive, VIP immunoreactive nerve fibers are particularly conspicuous around intralingual blood vessels. These fibers around the arteries in the tongue may be partly derived from the intralingual ganglion cells, because some bundles associated with these nerve cells were easily traced on the wall of blood vessels. The present study suggests the view that the three markers coexist in the axons and nerve terminals of these intralingual neurons.

Nitric oxide synthase and chemical coding in cat sympathetic postganglionic neurons.

Nitric oxide synthase-like immunoreactivity was found in a subpopulation of sympathetic postganglionic neurons in the cat stellate and lower lumbar ganglia. In the ganglia of other segments such cells were rare. Double staining for tyrosine hydroxylase-like immunoreactivity and nitric oxide synthase-like immunoreactivity or the reduced nicotinamide adenine dinucleotide phosphate diaphorase reaction indicated that nitric oxide synthase-like immunoreactivity and reduced nicotinamide adenine dinucleotide phosphate diaphorase reactivity was always co-localized and was confined to tyrosine hydroxylase-negative (presumably cholinergic) ganglion cells, and was present in most of them. The occurrence of nitric oxide synthase in two subpopulations of cholinergic postganglionic neurons was investigated in triple staining experiments. Presumptive sudomotor neurons have been previously defined as scattered cells containing calcitonin gene-related peptide-like immunoreactivity, usually accompanied by vasoactive intestinal peptide-like immunoreactivity: 99% of these contained nitric oxide synthase. Presumptive muscle vasodilator neurons have been previously identified as clumped cells with strong vasoactive intestinal peptide-like immunoreactivity but no calcitonin gene-related peptide-like immunoreactivity: 70% of these contained nitric oxide synthase. Sweat glands were found in the paw pad skin surrounded by varicose fibres showing calcitonin gene-related peptide-like immunoreactivity and vasoactive intestinal peptide-like immunoreactivity, confirming previous work. Such fibres also stained for nitric oxide synthase-like immunoreactivity and reduced nicotinamide adenine dinucleotide phosphate diaphorase reactivity, although their staining was relatively weaker than in the corresponding cell bodies. Varicose fibres with the same chemical coding were also found around all large and most medium and small arteries in the paw skin as well as around arteriovenous anastomoses. Fibres with the muscle vasodilator coding (vasoactive intestinal peptide-like immunoreactivity without calcitonin gene-related peptide-like immunoreactivity) were not seen in paw skin. These results suggest that nitric oxide may act as a co-transmitter (with acetylcholine, substance P, vasoactive intestinal peptide and calcitonin gene-related peptide) in sudomotor neurons and (with acetylcholine and vasoactive intestinal peptide) in vasodilator neurons. Collateral branches of sudomotor neurons may innervate skin vessels, and release vasodilator transmitters including nitric oxide to cause the vasodilatation which provides the fluid supply for sweat formation. Alternatively, separate vasodilator neurons to skin may share the same chemical code as sudomotor neurons.

Localization of NADPH diaphorase in bladder afferent and postganglionic efferent neurons of the rat.

NADPH diaphorase histochemistry was used in combination with axonal labelling techniques to determine if NADPH diaphorase is present in afferent and postganglionic efferent pathways to the urinary bladder of the rat. In the L6 and S1 dorsal root ganglia, 80.9 and 78.5%, respectively, of bladder afferent neurons labelled with fluorescent dyes were NADPH diaphorase positive. In the major pelvic ganglion (MPG), many non-labelled neurons and fibers were intensely stained for NADPH diaphorase. Intensely stained cells were clustered near the exit of the penile nerve although stained cells were also scattered throughout the ganglion. Only a small percentage (3.5%) of bladder postganglionic neurons in the MPG were NADPH diaphorase positive. Since NADPH diaphorase activity commonly reflects the presence of nitric oxide synthase, the present findings raise the possibility that nitric oxide may have a role as a neurotransmitter or neuromodulator in afferent pathways from the urinary bladder.

NADPH-diaphorase staining in autonomic and somatic cranial ganglia of the rat.

Using NADPH-diaphorase staining as a marker for the enzyme nitric oxide synthase (NOS) we have investigated the possible sites of nitric oxide (NO) synthesis in a number of cranial ganglia in the rat. Intense staining was found in the majority of neurones in the sphenopalatine ganglion, suggesting a major role for NO in postganglionic parasympathetic systems in the head. In contrast the neurones of the superior cervical ganglion were not stained by this histochemical procedure but were enveloped by a mesh of intensely staining fibres. As preganglionic sympathetic neurones in the intermediolateral horn of the spinal cord stain for NADPH-diaphorase, our results would suggest that NO acts as a neurotransmitter between pre- and post-ganglionic sympathetic neurones.

Parasympathetic nerves in penile erectile tissue of the rat contain choline acetyltransferase.

Choline acetyltransferase (ChAT), a biochemical marker of cholinergic neurons, was measured in the erectile tissue of intact rats and in rats in which postganglionic fibers from the pelvic plexus were interrupted. ChAT activity in the denervated erectile tissue fell by 56% compared to control tissues. Acetylcholinesterase positive (AChE+) nerves also fell by about 48%. Penile neurons distal to the lesion probably account for the residual ChAT activity and remaining AChE+ nerve fibers in erectile tissue. These results indicate that acetylcholine is an important neurotransmitter in the regulation of penile erection.

Localization of monoamine oxidase (MAO) in the rat peripheral nervous system--existence of MAO-containing unmyelinated axons.

Using a new coupled peroxidation method modified by adding nickel ammonium sulfate, we demonstrated the localization of monoamine oxidase (MAO) in the rat peripheral nervous system. MAO was localized in the endothelial cells of endoneurial vessels, in Schwann cell cytoplasm encircling myelinated axons, and in some unmyelinated axons. The morphometric ratio of these MAO-containing axons to the total unmyelinated axons was 10-13%. These MAO-containing unmyelinated axons were assumed to coincide with postganglionic sympathetic noradrenergic ones passing in the sciatic nerve. Histochemical MAO staining may be utilized to identify the postganglionic sympathetic nerves in normal and pathological conditions at the electron microscopic level in particular.

Cholinesterase activity in the rat superior cervical ganglion: effect of denervation and axotomy.

Cholinesterase activity and its various forms in the superior cervical ganglion of the rat were studied after denervation, axotomy and double section. The response of this activity to various inhibitors was also studied. The specific activity of cholinesterase activity was 323 +/- 25 nmol/mg protein/min in homogenates of normal ganglia and the total enzyme activity was 78 +/- 5 nmol/ganglion/min. Axotomy produced approximately a 40% decrease in total activity in ganglia at 3 days compared to contralateral ganglia. Activity in the contralateral ganglia was also decreased by about 30% compared to normal ganglia. Decentralization alone for 3 days did not affect the specific or total cholinesterase activity. However, decentralization followed by axotomy 3 days later resulted in a greater loss in activity compared to axotomy and a significant change in the forms of cholinesterase. After chromatography on Sepharose 6B-100 columns, 3 forms (a, c, d) of cholinesterase activity could be separated from homogenates of single ganglia. The 3 forms had estimated molecular weights of 1,934,000, 129,000 and 59,000 daltons, respectively. A fourth form (b) presented as a shoulder and had an estimated molecular weight of 404,000 daltons. Decentralization or axotomy decreased forms a and d. A reduction in the proportion of form c was also observed in the axotomized ganglion. Decentralization followed by axomoty resulted in large decreases in forms a, b and c and complete loss of form d. Assay of the separated forms in the presence of the specific inhibitor of acetylcholinesterase, BW284C-51, indicated that the majority of the enzyme recovered after chromatography is acetylcholinesterase.

Regional distribution of tyrosine hydroxylase and dopamine beta-hydroxylase activities in guinea pig heart.

The sympathetic and parasympathetic autonomic nervous systems regulate heart rate and myocardial contractility. Using sensitive radioisotopic assays, we examined the regional distribution of tyrosine hydroxylase and dopamine beta-hydroxylase activity, which are markers for sympathetic autonomic innervation, in specialized pacemaker and conduction tissue and in contractile heart tissue. Of the 20 regions of guinea pig heart examined, we find that the highest activities occur in the sinoatrial node and the right atrial appendage. Intermediate activity occurs in the left atrium, interatrial septum and right ventricular papillary muscle. Activities in the remainder of the heart are lower and rather uniform. Comparing the enzyme markers for the parasympathetic [17] and sympathetic system, we find that these systems have different distributions. The former, for example, is closely associated with the specialized pacemaker and conduction system including the sinoatrial and atrioventricular nodes and regions rich in Purkinje fibers. The sympathetic system, on the other hand, is associated with both contractile and conducting tissue.

On the mechanism of the uptake of horseradish peroxidase into the retrograde transport system of ligated postganglionic sympathetic nerves in vitro.

The mechanism of the uptake of horseradish peroxidase (HRP) by damaged post-ganglionic sympathetic axons was studied in vitro. HRP was applied to the damaged axons at the time of nerve injury or after a 3 hours or 17 hours delay. An interval of 3 hours or 17 hours between nerve injury and exposure to HRP had no effect on the localisation of the HRP in the damaged axons or on its retrograde transport to their perikarya. Evidence was found for the pinocytotic uptake of the enzyme by the damaged axons and its accumulation within those axons in elongated cisternae and larger rounded vesicles. In further experiments the damaged axons were treated with HRP at 0 degrees C and then washed in HRP-free medium. The tracer entered the axons in a diffuse form under these conditions but no pinocytotic uptake was observed. However, following 24 hours further incubation at 37 degrees C, HRP could not be found in the perikarya. Treatment at 0 degrees C did not produce any lasting damage to the retrograde transport mechanism. The results of these experiments are compatible with the involvement of pinocytosis in the uptake of HRP in a form suitable for retrograde transport.

Increase in choline acetyltransferase activity in surgically isolated postganglionic parasympathetic neurones of the urinary bladder of adult rats.

In the urinary bladder of adult male rats the choline acetyltransferase activity in postganglionic neurones isolated from the central nervous system was shown to increase markedly and rapidly following section of the contralateral postganglionic neurones. The enzyme activity of the operated bladder was 32% of the control 3 days postoperatively, while at the last observation, 25 days postoperatively, it was 86%. As judged from additional studies on totally denervated bladders and on totally decentralized bladders the increase found in the enzyme activity was not due to ingrowth of nerves from outside or to unspecific acetylcholine synthesis neither was it due to increase in bladder wall tension or to increase in tissue mass.

[Molecular forms of acetylcholinesterase in the chicken ciliary ganglion; changes after denervation, axotomy and double section]. / Formes moléculaires de l'acétylcholinestérase dans le ganglion ciliaire du Poulet; évolution après dénervation, axotomie et double section.

Four main molecular forms of acetylcholinesterase (AChE), with sedimentation coefficients of 5, 7.5, 11.5 and 20 S, are found in Chicken ciliary ganglion. After denervation, the loss in 11.5 and 20 S forms occuring in 48 hrs coincides with the disappearance of presynaptic structures. In contrast, axotomy induces an early and durable increase in 7.5 S form. From these results, it is inferred that 11.5 and 20 S forms are predominant in presynaptic structures and 7.5 S form is mainly postsynaptic. In addition, the effects observed after simultaneous denervation and axotomy show a reciprocal control between pre- and postsynaptic elements. Finally, a trans-synaptic effect is exerted on 20 S AChE in controlateral ganglion after preganglionic sections.

Choline acetyltransferase activity in postganglionic parasympathetic nerves after "pharmacological decentralization".

The ganglion blocking drug chlorisondamine given frequently and in gradually increasing doses over a period of time to adult rats causes the activity of choline acetyltransferase to fall in the postganglionic parasympathetic nerves of parotid glands. Such a "pharmacologically" decentralized gland was also found to have lost weight and to have developed a supersensitivity to chemical stimuli. All these phenomena are thought to be consequences of loss or reduction of secretory impulses from the central nervous system due to impaired ganglionic transmission.