Functional neuroanatomy of the noradrenergic locus coeruleus: its roles in the regulation of arousal and autonomic function part I: principles of functional organisation.

The locus coeruleus (LC) is the major noradrenergic nucleus of the brain, giving rise to fibres innervating extensive areas throughout the neuraxis. Recent advances in neuroscience have resulted in the unravelling of the neuronal circuits controlling a number of physiological functions in which the LC plays a central role. Two such functions are the regulation of arousal and autonomic activity, which are inseparably linked largely via the involvement of the LC. The LC is a major wakefulness-promoting nucleus, resulting from dense excitatory projections to the majority of the cerebral cortex, cholinergic neurones of the basal forebrain, cortically-projecting neurones of the thalamus, serotoninergic neurones of the dorsal raphe and cholinergic neurones of the pedunculopontine and laterodorsal tegmental nucleus, and substantial inhibitory projections to sleep-promoting GABAergic neurones of the basal forebrain and ventrolateral preoptic area. Activation of the LC thus results in the enhancement of alertness through the innervation of these varied nuclei. The importance of the LC in controlling autonomic function results from both direct projections to the spinal cord and projections to autonomic nuclei including the dorsal motor nucleus of the vagus, the nucleus ambiguus, the rostroventrolateral medulla, the Edinger-Westphal nucleus, the caudal raphe, the salivatory nuclei, the paraventricular nucleus, and the amygdala. LC activation produces an increase in sympathetic activity and a decrease in parasympathetic activity via these projections. Alterations in LC activity therefore result in complex patterns of neuronal activity throughout the brain, observed as changes in measures of arousal and autonomic function.

Functional neuroanatomy of the noradrenergic locus coeruleus: its roles in the regulation of arousal and autonomic function part II: physiological and pharmacological manipulations and pathological alterations of locus coeruleus activity in humans.

The locus coeruleus (LC), the major noradrenergic nucleus of the brain, gives rise to fibres innervating most structures of the neuraxis. Recent advances in neuroscience have helped to unravel the neuronal circuitry controlling a number of physiological functions in which the LC plays a central role. Two such functions are the regulation of arousal and autonomic activity, which are inseparably linked largely via the involvement of the LC. Alterations in LC activity due to physiological or pharmacological manipulations or pathological processes can lead to distinct patterns of change in arousal and autonomic function. Physiological manipulations considered here include the presentation of noxious or anxiety-provoking stimuli and extremes in ambient temperature. The modification of LC-controlled functions by drug administration is discussed in detail, including drugs which directly modify the activity of LC neurones (e.g., via autoreceptors, storage, reuptake) or have an indirect effect through modulating excitatory or inhibitory inputs. The early vulnerability of the LC to the ageing process and to neurodegenerative disease (Parkinson's and Alzheimer's diseases) is of considerable clinical significance. In general, physiological manipulations and the administration of stimulant drugs, alpha(2)-adrenoceptor antagonists and noradrenaline uptake inhibitors increase LC activity and thus cause heightened arousal and activation of the sympathetic nervous system. In contrast, the administration of sedative drugs, including alpha(2)-adrenoceptor agonists, and pathological changes in LC function in neurodegenerative disorders and ageing reduce LC activity and result in sedation and activation of the parasympathetic nervous system.

Arousal and the pupil: why diazepam-induced sedation is not accompanied by miosis.

RATIONALE: There is a close relationship between arousal and pupil diameter, decrease in the level of arousal being accompanied by constriction of the pupil (miosis), probably reflecting the attenuation of sympathetic outflow as sedation sets in. Paradoxically, sedation induced by benzodiazepines is not accompanied by miosis. OBJECTIVE: The objective of this study was to examine the hypothesis that diazepam may attenuate both the sympathetic and the opposing parasympathetic outflow to the iris, which may mask the miosis. Dapiprazole (sympatholytic) and tropicamide (parasympatholytic) were applied topically, together with the cold pressor test (CPT), to manipulate the sympathetic/parasympathetic balance. MATERIALS AND METHODS: Sixteen healthy male volunteers participated in four weekly sessions according to a balanced double-blind protocol. Diazepam 10 mg (two sessions) and placebo (two sessions), associated with either 0.01% tropicamide or 0.5% dapiprazole eyedrops, were administered orally. Pupil diameter, light and darkness reflexes and pupillary sleepiness waves were recorded with infrared video pupillometry, alertness was measured by critical flicker fusion frequency (CFFF) and visual analogue scales (VAS), blood pressure and heart rate by conventional methods. CPT was applied after post-treatment testing. Data were analysed by analysis of variance, with multiple comparisons. RESULTS: Diazepam caused sedation (reduction in VAS alertness scores and CFFF, increase in sleepiness waves), dapiprazole had a sympatholytic and tropicamide a parasympatholytic effect on the pupil. Diazepam had no effect on pupil diameter and reflexes or their modifications by the antagonists. CPT increased pupil diameter, blood pressure and heart rate, and the increase only in systolic blood pressure was attenuated by diazepam. CONCLUSIONS: Diazepam-induced sedation is not accompanied by any change in either the sympathetic or parasympathetic influence on the iris.

Diazepam-induced disruption of classically-conditioned fear-potentiation of late-latency auditory evoked potentials is prevented by flumazenil given before, but not after, CS/US pairing.

Classical fear conditioning involves pairing a neutral conditional stimulus (CS) with an aversive unconditional stimulus (US). Subsequent presentation of the CS alone induces fear responses. Acquisition of conditioned fear is thought to involve learning of the CS/US association, followed by memory consolidation. Recently we reported that the N1/P2 auditory evoked potential was enhanced by fear conditioning in humans. Diazepam 10 mg, given before CS/US pairing, prevented subsequent expression of fear potentiation when the response was elicited, 1 week later, in the presence of the CS. In this experiment, we examined whether this effect of diazepam was caused by disruption of the formation of CS/US associations or by disruption of consolidation. The benzodiazepine antagonist flumazenil was used to block the effect of diazepam either during the association period or during subsequent consolidation. Forty-two male volunteers (18-35 years) participated in two sessions separated by 7 days. In Session One, they ingested diazepam 10 mg or placebo: 60 minutes later they received flumazenil 1 mg or saline intravenously (i.v.). Then they received 20 presentations of a light (CS), 50% of which terminated with electric shock (US). This was followed by a second infusion of flumazenil or saline. Subjects received placebo/saline/saline (Group 1), diazepam/saline/saline (Group 2), diazepam/flumazenil/saline (Group 3) and diazepam/saline/flumazenil (Group 4). In Session Two, the CS was presented without the US; 50% of CS presentations terminated with a sound pulse; an equal number of sound pulses were presented without the CS. Auditory evoked potentials were recorded at Cz. In Session Two, CS presentation enhanced the auditory N1/P2 potential in placebo-treated subjects (Group 1). This enhancement was prevented by diazepam (Group 2). Flumazenil reversed diazepam's effect on fear potentiation if it was administered before conditioning (Group 3), but not if it was administered afterwards (Group 4). The results confirm that diazepam prevents the acquisition of fear conditioning in humans, and suggest that it disrupts the formation of CS/US associations, rather than the consolidation of fear memory.

Comparison of pramipexole and amisulpride on alertness, autonomic and endocrine functions in healthy volunteers.

RATIONALE: In a previous study in healthy volunteers, the anti-Parkinsonian drug pramipexole caused sedation and pupil dilatation, consistent with the stimulation of inhibitory D(2)/D(3) autoreceptors on the ventral tegmental area dopaminergic neurones. The sedation may be related to the removal of the dopaminergic excitation of the locus coeruleus (via the meso-coerulear pathway), whereas the pupil dilatation may be due to the removal of the dopaminergic excitation of the Edinger-Westphal nucleus (via a putative meso-pupillomotor pathway). OBJECTIVES: We investigated the hypothesis that amisulpride, a D(2)/D(3) receptor antagonist, would have effects opposite to those of pramipexole on alertness, pupillary and endocrine functions. MATERIALS AND METHODS: Pramipexole (0.5 mg), amisulpride (50 mg), and their combination were administered to 16 healthy males in a balanced, cross-over, double-blind design. Tests included measures of alertness (Pupillographic Sleepiness Test, critical flicker fusion frequency, visual analogue scales), pupillary functions (resting pupil diameter, light and darkness reflex responses), non-pupillary autonomic functions (heart rate, blood pressure, salivation, core temperature), and endocrine functions [blood concentrations of prolactin, growth hormone (GH) and thyroid stimulating hormone (TSH)]. Data were analysed by ANOVA. RESULTS: Pramipexole reduced alertness and pupillary light reflex response amplitude, tended to reduce core temperature, reduced prolactin levels and increased GH levels. Amisulpride reduced pupil diameter, increased the amplitude of the light reflex response and prolactin and TSH levels. CONCLUSIONS: The opposite effects of pramipexole and amisulpride on alertness, pupillary function and pituitary hormone levels are consistent with their interactions with inhibitory D(2)/D(3) receptors on VTA neurones and in the tuberoinfundibular system.

Comparison of pramipexole and modafinil on arousal, autonomic, and endocrine functions in healthy volunteers.

The noradrenergic locus coeruleus is a major wakefulness-promoting nucleus of the brain, which is also involved in the regulation of autonomic and endocrine functions. The activity of the locus coeruleus is believed to be tonically enhanced by a mesocoerulear dopaminergic pathway arising from the ventral tegmental area of the midbrain. Both modafinil, a wakefulness-promoting drug, and pramipexole, a D(2)/D(3)receptor agonist with sedative properties, may act on this pathway, with modafinil increasing and pramipexole decreasing locus coeruleus activity. The aim of this study was to compare the two drugs on alertness, autonomic and endocrine functions in healthy volunteers. Pramipexole (0.5mg), modafinil (200mg), and their combination were administered to 16 healthy males in a double-blind, placebo-controlled design. Methods included tests of alertness (pupillographic sleepiness test, critical flicker fusion frequency, visual analogue scales), autonomic functions (resting pupil diameter, light and darkness reflex responses, heart rate, blood pressure, salivation, core temperature), and endocrine functions (blood concentrations of prolactin, growth hormone, and thyroid stimulating hormone). Data were analysed by ANOVA. Pramipexole reduced alertness, caused pupil dilatation, increased heart rate, reduced prolactin and thyroid stimulating hormone, and increased growth hormone level. Modafinil caused small increases in blood pressure and core temperature, and reduced prolactin levels. The sedative effect of pramipexole and the autonomic effects of modafinil are consistent with altered activity in the mesocoerulear pathway; the pupil dilatation following pramipexole suggests reduced dopaminergic excitation of the Edinger-Westphal nucleus.

Why patients with Alzheimer's disease may show increased sensitivity to tropicamide eye drops: role of locus coeruleus.

RATIONALE: Patients suffering from Alzheimer's disease (AD) may show increased sensitivity to tropicamide, a muscarinic cholinoceptor antagonist. AD is associated with a severe loss of noradrenergic neurones in the locus coeruleus (LC), which can be "switched off" experimentally by the alpha(2)-adrenoceptor agonist clonidine. The possibility arises that increased pupillary sensitivity to tropicamide in AD may be due to diminished LC activity. OBJECTIVE: To examine the hypothesis that clonidine may potentiate tropicamide-evoked mydriasis. MATERIALS AND METHODS: Sixteen healthy male volunteers participated in two experimental sessions (0.2 mg clonidine or placebo) conducted 1 week apart. In each session tropicamide (0.01% 10 microl x 2) was applied to the left eye and artificial tear (10 microl x 2) was applied to the right eye. Pupillary functions (resting pupil diameter and light and darkness reflexes), alertness and non-pupillary autonomic functions (blood pressure, heart rate, core temperature and salivary output) were measured. Data were analysed by ANOVA, with multiple comparisons. RESULTS: Tropicamide increased resting pupil diameter, velocity and amplitude of the darkness reflex response, and decreased recovery time of the light reflex response. Clonidine affected all these pupillary measures in the opposite direction with the exception of the recovery time. The mydriatic response to tropicamide was potentiated by pre-treatment with clonidine. Clonidine reduced critical flicker fusion frequency, subjective alertness, blood pressure, salivation and temperature. CONCLUSIONS: The potentiation of tropicamide-evoked pupil dilatation by clonidine may be due to the abolition of the increase in parasympathetically mediated pupil constriction due to reduced LC activity.

Calcium-PS-dependent protein kinase C and surfactant protein A in isolated fetal rabbit type II alveolar cells and surfactant-related material.

The fetal lung secretes significant quantities of surfactant during late gestation to prepare for initiation of respiration at birth. However, the mechanism by which this occurs has not been determined. Since Ca2+-phosphatidylserine (PS)-dependent protein kinase C has been implicated in surfactant secretion in adult lung, the present study was done to determine whether this enzyme is also involved in the initiation of surfactant release from fetal type II cells. Type II cells isolated from gestational day-24 fetal rabbits were used. Cells were prelabelled with [32P] and [3H]choline and exposed to 4beta phorbol ester (10(-5) M) for 2 h. Secretion product and subcellular fractions were isolated by removing the culture medium, mixing with homogenate from adult rabbit lung, and subfractionating by centrifugation on a sucrose gradient. Samples of secretion product were also prepared for electron microscopy. Ca2+-PS-dependent protein kinase C was also assayed in some samples, and an add-back technique was used to determine whether enzyme activity in the intracellularly stored surfactant fraction was due to contamination. The results showed that material released by fetal type II cells after exposure to phorbol ester coprecipitated with adult rabbit lung lamellar bodies and microsomes. Morphologically, a range of forms, including lamellar-body-like structures, was detected. The released material originated largely from the lamellar body compartment of the fetal type II cells and displayed immunoreactivity with antibody to surfactant protein A (SP-A) at 35 and 70 kDa apparent molecular mass. Assay of protein kinase C in fetal type II cells showed that exposure to conditioned medium, which induces differentiation, increased activity. Incubation with phorbol ester induced translocation of activity to the microsomal fraction. Add-back assays suggested that protein kinase C activation by treatment with phorbol ester induced translocation of enzyme activity to the lamellar body fraction; none was detected prior to treatment. These results support a role for Ca2+-PS-dependent protein kinase C in initiation of surfactant release by interaction with the developing lamellar body compartment in fetal type II cells.

Ca(+2)-phosphatidylserine-dependent protein kinase C activity in fetal, neonatal and adult rabbit lung and isolated lamellar bodies.

Evidence indicates that Ca(+2)-phosphatidylserine-dependent protein kinase C (PKC) is involved in regulation of surfactant secretion. This study was done to examine PKC activity in lung as surfactant synthesis and secretion is initiated, and at birth and to compare these enzyme levels with those in the adult lung. NZW rabbits were used. Fetal and adult lungs were fractionated into subcellular compartments including a lamellar body fraction, which represents intracellular surfactant. The time course for microsomal enzyme activity was compared between 24th gestational day and adult rabbit lung. The reactivity appeared similar in both fractions. PKC specific activity displayed a prominent peak between the 27th and 30th gestational days in total homogenate and lamellar bodies. Specific activity was also high in nuclear, mitochondrial and microsomal fractions the day prior to birth. Adult levels were similar or higher. Total PKC activity was high during late gestation but declined sharply the day prior to birth. A marked increase was present on the first postnatal day. In contrast lamellar bodies displayed a peak in activity between the 27th and 30th gestational days followed by a decline to adult levels. Delipidation of lamellar body fraction indicated that the high enzyme activity in this fraction on the 27th gestational day was not artifactual. The changes observed in PKC in fetal, neonatal and adult lung indicate this enzyme activity changes in lung during the period of onset of surfactant synthesis and secretion during late gestation and may be associated with lamellar bodies, in 27th gestational day fetal lung.

Distribution and characteristics of Ca+2-phosphatidylserine-dependent protein kinase C in subcellular fractions and lamellar bodies of adult rabbit lung.

Pulmonary surfactant prevents lung collapse at minimal alveolar diameter. Since surfactant acts extracellularly, secretion is vitally important in regulating the alveolar surfactant levels. Studies with phorbol esters which stimulate protein kinase C (PKC) activity suggest PKC is involved in regulating surfactant secretion. This study was done to characterize PKC activity in adult rabbit lung fractions. Lungs were removed, homogenized and subcellular fractions prepared by centrifugation on a discontinuous sucrose gradient. Calcium-phosphatidylserine-dependent PKC activity was assayed in fractions in the presence of 4 microM phorbol 12-myristate 13-acetate, 1mM EDTA, 8 mole% phosphatidylserine and 1mM Ca2+ by measuring the transfer of 32P from [gamma-32P]ATP to protein. Concurrent assays were done without Ca+2 or PS. Ca+2-PS dependent PKC activity was defined as the difference between the two. Select fractions were incubated with PKC inhibitors sangivamycin, acridine orange or 9-aminoacridine and activity measured. The results showed the majority of the PKC activity was in the cytosolic fraction (87%, specific activity, 142 pmoles/min/mg) but the lamellar bodies also appeared to contain a small amount of PKC activity (approximately 4.0%, 151 pmoles/min/mg). PKC inhibitors were used to examine the characteristics of the enzyme in the microsomal and lamellar body fractions. Sangivamycin was the most potent inhibitor. Some differences in the inhibition characteristics between the lamellar body and microsomal fractions were observed. However using an add-back approach with the lamellar body fraction, indicated that the small quantity of activity in this fraction be attributed to contamination by microsomes. These results indicate that PKC is active in adult rabbit lung subcellular compartments but is probably not associated with the intracellular surfactant storage organelles.

Lead concentrations in human bones from the Canadian population.

Stable lead was determined in post-mortem samples of human bones from three Canadian cities. All age groups and both sexes were represented. The cities selected for investigation were Winnipeg, Montreal and Charlottetown. No significant difference was found between the locations, although levels tended to be higher for Montreal. Mean lead concentrations (micrograms Pb/g ash) were 8.98 +/- 1.17, 11.11 +/- 1.74 and 8.47 +/- 1.06 for Winnipeg, Montreal and Charlottetown, respectively. Corresponding geometric means were 6.21, 7.88 and 6.71, respectively. Individual values ranged from 0.45 to 240.07 micrograms Pb/g ash. Concentrations were highest in the greater than 20-year age group, indicating increased body burden with age. An increase in lead concentration was observed for the ages 1-11 years with a decrease for the 12-19-year age group. Differences in concentrations between the sexes were not significant. Higher than average concentrations were observed in samples obtained from Winnipeg for the period 1976-1980, particularly in the 1-4-year age group. A similar pattern was observed in the air lead concentrations, suggesting a possible correlation between the concentrations of lead in the air and in bones at Winnipeg.

A case of accidental inorganic mercury poisoning.

Mercuric chloride was accidentally ingested by a nineteen-month old boy. He exhibited severe symptoms of inorganic mercury poisoning including acute renal failure. The blood mercury level at the time of admission to hospital was 1920 ng/mL. Following emergency hemodialysis, BAL (2, 3-dimercaptopropanol) therapy and penicillamine treatment, blood levels fell to 500 ng Hg/mL and urine production restarted six days after exposure. Urine mercury reached a high of 2349 ng/mL but rapidly decreased to less than 100 ng/mL within eight days after resumption of voiding. The patient was discharged from hospital a month after admission and follow-up examinations have indicated no permanent renal damage. Blood, hair, and urine samples collected 19 months after the exposure showed normal mercury levels (blood, 6 ng Hg/mL; urine, 7 ng Hg/mL; and hair 500-900 ng Hg/g).