W.W. Blessing

Loss of brainstem serotonin- and substance P-containing neurons in Parkinson's disease.

Using postmortem immunohistochemical analysis, we have identified degeneration of several different neuronal cell groups in the brainstem of patients dying with idiopathic Parkinsons disease. We report the first chemically identified loss of presumed serotonin neurons in the median raphe nucleus of the pons and of substance P-containing preganglionic neurons in the dorsal motor vagal nucleus. This evidence is concordant with other evidence that the primary neuropathological process is not confined either to a single pathway or to neurons containing a particular transmitter. Rather it appears that Parkinsons disease affects several classes of neurons in localized areas of the brainstem.

Cell groups in the lower brain stem of the rabbit projecting to the spinal cord, with special reference to catecholamine-containing neurons

Two groups of experiments were carried out in rabbits. In the first groups, the distribution of cell bodies within the pons and medulla projecting ipsilaterally and contralaterally to the thoracic or lumbar spinal cord was studied using the horseradish peroxidase (HRP)/tetramethylbenzidine (TMB) procedure. In the second group, both a previously described double-labeling technique and a new modification of it were used to determine the location of catecholamine (CA)-fluorescent pontomedullary cells projecting to the spinal cord. The results demonstrate that the catecholamine (probably norepinephrine)-containing neurons which innervate the thoracic spinal cord are confirmed almost exclusively to the pons where they were found within the A5, A7 and subcoeruleus groups, as well as the ventral portion of the principal part of the locus coeruleus and the more caudal locus coeruleus, including the A4 cell group. Within the medulla oblongata no doubly labeled A2 cells were observed and the few double labeled A1 cells which were observed were confined to the rostral portion of this group. A dense group of HRP-positive but non-fluorescent cells was found rostral to the A1 area in the ventrolateral reticular formation. These cells, which correspond in position to PNMT-containing cells in the rat, appear to project to both thoracic and lumbar segments of the spinal cord. In contrast, spinally projecting neurons within the nucleus tractus solitarius originated from different subnuclei according to their segmental destination. New information about the organization of medial reticulospinal and vestibulospinal pathways was also obtained.

Multiple-system atrophy: a new α-synuclein disease?

Multiple-system atrophy is a neurodegenerative disease that involves various combinations of parkinsonism, ataxia, corticospinal motor signs, and postural hypotension or urinary incontinence (Shy-Drager syndrome). The pathological hallmark of the disease is the presence of glial and neuronal cytoplasmic inclusions, shown by modified Bielschowsky silver impregnation and, to a limited extent, with antisera to ubiquitin or B-crystallin. We show that glial cytoplasmic inclusions and degenerating neurites in multiple-system atrophy are strongly immunopositive for -synuclein, a presynaptic protein present in brainstem and cortical Lewy bodies and related neurites in idiopathic Parkinson’s disease and dementia with Lewy bodies, and, in a mutated form, in some cases of familial Parkinson’s disease. Antiserum against human -synuclein (residues 111–131) was raised in a rabbit. Paraffin-embedded sections (5 m) and frozen sections (50 m) from different brain regions were immunostained with the -synuclein antibody (1 in 1000–2000 dilution) and the avidin-biotin-peroxidase procedure. Immunostaining was not seen with preadsorbed or preimmune serum or in the absence of the primary antiserum. Brains from people with multiple-system atrophy (n=8) were obtained from the South Australian Brain Bank. The clinical diagnoses before death had included Parkinson’s disease, multiple-system atrophy with olivopontocerebellar involvement, and multiple-system atrophy with autonomic symptoms. The neuropathological diagnosis of multiplesystem atrophy was made by the demonstration of glial cytoplasmic inclusions with the modified Bielschowsky silver stain and immunostaining for ubiquitin and B-crystallin. We assessed the same regions in 12 brains judged to be normal, five from people who had had motor-neuron disease, and ten from people with idiopathic Parkinson’s disease, all also from the brain bank. Sections from brainstem, basal ganglia, cerebellar and cerebral cortices in the multiple-system-atrophy brains all showed strong immunostaining of glial cytoplasmic inclusions, mainly in white matter (figure). Pronounced -synuclein immunoreactivity was also seen in degenerating neurites (figure) and in the cytoplasmic and intranuclear inclusions of neurons in the putamen and pontine nuclei. In normal brains and those from people with motor-neuron disease, no abnormal -synuclein-containing structures were seen; immunostaining for -synuclein was limited to the normal background pattern, which shows the presence of the protein in nerve terminals. In idiopathic Parkinson’s disease, Lewy bodies and degenerating neurites were positive for synuclein, as shown previously, but no immunopositive glial cytoplasmic inclusions were seen. We used confocal microscopy and double-staining for synuclein (1 in 500) and ubiquitin (monoclonal antibody 1510, Chemicon, 1 in 200) to assess the colocalisation of these two markers in glial cytoplasmic inclusions and degenerating neurites in multiple-system atrophy. Small numbers of synuclein-positive degenerating neurites and inclusions were ubiquitin-positive, but -synuclein immunoreactivity was clearly more sensitive than ubiquitin immunoreactivity as a neuropathological marker for the lesions of multiple-system atrophy (figure). The pronounced increase in the number of multiplesystem-atrophy inclusions and neurites shown with the synuclein antibody (compared with the ubiquitin antibody) contrasts with the number in Parkinson’s disease and dementia with Lewy bodies, disorders in which the two antibodies show similar neuropathological abnormalities. Immunoreactivity for -synuclein occurs in plaque-associated neurites in Alzheimer’s disease, but is absent from neuronal and glial inclusions in Alzheimer’s disease and other neurodegenerative disorders. Antibodies to -synuclein

Noradrenergic and dopaminergic projections to the medial preoptic area of the rat. A combined horseradish peroxidase/catecholamine fluorescence study

The medial preoptic area (MPOA) contains substantial quantities of noradrenaline (NA) and dopamine (DA) 2°, both of which appear to be contained within terminal fibres 19. These fibres are thought to originate from the discrete NA and DA cell groups which lie within the brain stem, caudal to the MPOA1, 3. As there is evidence that MPOA NA and DA participate in the regulation of a number of physiological functions3-~,lS, 21, it has become important to determine which of the NA and DA cell groups of the brain stem supply the MPOA. The introduction of the horseradish peroxidase (HRP) retrograde transport technique 1° has facilitated the mapping of neuronal pathways. The usefulness of combining the HRP technique with a procedure for identifying the transmitter-type of the HRP-labelled neurones was soon recognized ~1, and one group has studied the origins of MPOA NA fibres by combining the HRP technique with monoamine oxidase (MAO) staining 16. Although this is a useful approach, MAO is a relatively non-specific marker of catecholamine (CA) cells 1~. The recent introduction of a technique which enables the localization of retrogradely transported HRP and of specific CA-fluorescence within the same nerve cell 2 has significantly aided the mapping of NA and DA projections in the central nervous system. The present study employs this new combined procedure to localize both the NA and DA neurons which innervate the MPOA. Male Porton rats (200-300 g) were anaesthetized with sodium pentobarbital and HRP (10 70, Sigma type VI, 10-20 nl) was injected unilaterally into the MPOA through a glass micropipette (tip diameter 100/~m). Preliminary investigations indicated that this quantity of HRP was the smallest which would allow sufficient retrograde

Hypertension, bradycardia, and pulmonary edema in the conscious rabbit after brainstem lesions coinciding with the A1 group of catecholamine neurons.

We studied the effects of lesions in the ventrolateral medulla, in a region coinciding with the cell bodies of the Al group of catecholamine neurons. After bilateral electrolytic lesions at three contiguous rostrocaudal levels (obex and at 1 and 2 mm caudal to the obex), mean arterial pressure increased by 40 mm Hg in the conscious rabbit. This rise in pressure was associated with increased resistance in the distal aortic vascular bed and with profound bradycardia. Many lesioned animals developed respiratory distress in the first few postoperative hours and died with hemorrhagic pulmonary edema. In surviving rabbits, the distal aortic resistance remained raised throughout the 2-week observation period, but the blood pressure and heart rate returned to preoperative levels within 2 hours and then remained normal. Bilateral electrolytic lesions restricted to the level of the obex or to a level 1 mm caudal to tbe obex also caused transient hypertension and bradycardia, but most of the animals survived this more restricted damage and did not develop pulmonary edema. Micro-injections of kainic acid, a neurotoiin that specifically damages cell bodies, also caused transient hypertension and bradycardia, and after larger doses the rabbits died with acute pulmonary edema. Injections of 6- hydroxydopamine caused similar changes in pressure and heart rate, but doses necessary to destroy the Al cells caused nonspecific histological damage of an extent similar to that produced by electrolytic lesions. Sham-lesioned animals and those with control lesions in adjacent sites did not develop these cardiovascular changes. These experiments suggest that the persistent increase in peripheral resistance after lesions of the ventrolateral medulla results from destruction of neurons that normally act to inhibit sympathetic vasoconstrictor tone. It is our hypothesis that the increase in vascular resistance after the lesions results from destruction of the Al catecholamine cells within this ventrolateral region.

Raphe magnus/pallidus neurons regulate tail but not mesenteric arterial blood flow in rats.

In urethane-anesthetized rats with body temperature maintained at 39-40 degrees C, electrical stimulation of raphe magnus/pallidus/parapyramidal region within 0.5 mm of the ventral medullary surface reduced arterial blood flow to the tail cutaneous bed (measured with a chronically implanted Doppler ultrasonic flowmeter) from 28+/-5 to 6+/-1 cm/s (P<0.01), without changing mesenteric arterial blood flow, and with only small, variable changes in arterial pressure. Injection of bicuculline (50 pmol in 50 nl) at the same site reduced tail flow from 19+/-2 to 3+/-1 cm/s (P<0.01), again without significantly changing mesenteric flow, but with a moderate increase in arterial pressure. When the rat was cooled to reduce basal tail blood flow, injection of muscimol (1 nmol in 100 nl) or GABA (100 nmol in 100 nl) into the raphe site restored tail blood flow to 93+/-4% of the pre-cooling level. These recordings are the first reported direct measurements of rat tail blood flow changes elicited by alteration of neuronal function in the brainstem. The rostral medullary raphe controls the tail cutaneous vascular bed in a relatively selective manner. Our findings add to evidence that raphe magnus/pallidus/parapyramidal neurons are involved in regulating cutaneous blood flow in response to changes in body temperature in the rat.

Neurons in the area postrema are the only catecholamine-synthesizing cells in the medulla or pons with projections to the rostral ventrolateral medulla (C1-area) in the rabbit

We have identified, in the rabbit medulla and pons, neurons which project to the C1-region of the rostral ventrolateral medulla. By combining tyrosine hydroxylase immunohistochemistry with retrograde transport of Fluoro-Gold we determined whether any of the retrogradely labelled neurons synthesize catecholamines. The only doubly labelled cells were located in the area postrema. No other group of catecholamine-synthesizing neurons in either the medulla or the pons was found to project to the C1-area of the rostral ventrolateral medulla. Pharmacological agents which lower arterial pressure by stimulating adrenoceptors in the rostral ventrolateral medulla may act on receptors which are not innervated by catecholamine-synthesizing perikarya located outside the C1-region.

Lower brainstem pathways regulating sympathetically mediated changes in cutaneous blood flow

Abstract1. When the individual is alerted by painful or salient stimuli, there is a vigorous sympathetically mediated constriction of the cutaneous vascular bed. We investigated central pathways mediating this response using chronically implanted Doppler ultrasonic probes to measure cutaneous blood flow in the rabbit ear pinna and in the rat tail.2. Blockade of neuronal function in the amygdala prevents cutaneous vasoconstriction elicited by salient stimuli, but does not prevent the response to painful stimuli. Blockade of neuronal function in raphe magnus/pallidus and the parapyramidal region in anesthetized rabbits prevents cutaneous vasoconstriction elicited by painful stimuli. A similar region of the medullary raphe regulates tail artery vasoconstriction in rats. Inhibition of neuronal function in this region reverses cutaneous vasoconstriction induced by cooling the animal.3. Bulbospinal presympathetic neurons in the rostral medullary raphe region appear to regulate cutaneous blood flow responses occurring as part of the response to painful or dangerous environmental events and as part of the regulation of body temperature.

Regional blood flow and nociceptive stimuli in rabbits: patterning by medullary raphe, not ventrolateral medulla

1 Regional blood flow was measured with Doppler ultrasonic probes in anaesthetized rabbits. We used focal microinjections of pharmacological agents to investigate medullary pathways mediating ear pinna vasoconstriction elicited by electrical stimulation of the spinal tract of the trigeminal nerve or by pinching the lip, and pathways mediating mesenteric vasoconstriction elicited by electrical stimulation of the afferent abdominal vagus nerve. 2 Bilateral injection of kynurenate into the rostral ventrolateral medulla reduced arterial pressure and prevented the mesenteric vasoconstriction and the rise in arterial pressure elicited by abdominal vagal stimulation. However, kynurenate did not prevent ear pinna vasoconstriction or the fall in pressure elicited by trigeminal tract stimulation. Similar injections of muscimol also failed to prevent the trigeminally elicited cardiovascular changes. 3 Injections of kynurenate into the raphe‐parapyramidal area did not diminish trigeminally elicited ear vasoconstriction or the depressor response. However, injections of muscimol substantially reduced or abolished the trigeminally elicited ear vasoconstriction, without affecting the depressor response. Raphe‐parapyramidal muscimol injections also entirely abolished ear vasoconstriction elicited by pinching the rabbits lip. 4 The trigeminal depressor response does not depend on either the rostral ventrolateral medulla or the raphe‐parapyramidal region. 5 Mesenteric vasoconstriction elicited by stimulation of the afferent abdominal vagus nerve is mediated via the rostral ventrolateral medulla, but ear vasoconstriction elicited by lip pinch or by stimulation of the trigeminal tract is mediated by the raphe‐parapyramidal region. Our study is the first to suggest a brainstem pathway mediating cutaneous vasoconstriction elicited by nociceptive stimulation.

Neuropeptide Y injected into the supraoptic nucleus causes secretion of vasopressin in the unanesthetized rat.

Injection of neuropeptide Y (NPY, 0.01-1.0 nmol in 0.25 microliter vehicle) into the supraoptic nucleus of unanesthetized rats increased plasma vasopressin, measured by radioimmunoassay, to a maximum of 90 +/- 18 ng/liter. Injections of vehicle or somatostatin did not increase plasma vasopressin, nor did injections of NPY into the amygdala. Double-labelling immunohistochemical studies demonstrated that fibers containing NPY-like immunoreactivity form a close association with vasopressin immunoreactive perikarya in the supraoptic nucleus. It appears that NPY may directly excite vasopressin-containing neurons causing secretion of vasopressin.