Intrapericardial injections using a novel technique.

Intrapericardial injections have been proposed as a means to specifically treat diseases of the myocardium, pericardium, and coronary vasculature. The pericardial space is potential drug reservoir, allowing sustained delivery of drug to the target tissue. In this study we have demonstrated a novel method for pericardial delivery in the mouse.

Contributors to cognitive impairment in congestive heart failure: a pilot case-control study.

BACKGROUND: Cognitive impairment and heart failure are both serious health problems related to population ageing. Impaired cognitive function is an important but underrecognized complication of congestive heart failure (CHF). The aim of the study was to examine the sociodemographic, clinical, neuroimaging and biochemical parameters affecting cognition in CHF. METHODS: Thirty-one patients with CHF (left-ventricular ejection fraction < 40%) and 24 controls without CHF, all free of clinically significant cognitive impairment, participating in a case-control study were assessed using a cognitive battery (CAMCOG), a depression scale, 6-min-walk test, left-ventricular ejection fraction, semi-quantitative magnetic resonance imaging, and cortisol, aldosterone and renin concentrations. RESULTS: The CHF patients had lower CAMCOG scores than controls (93.5 +/- 6.1 vs 99.9 +/- 2.4, P < 0.001) and had significantly lower scores on visuospatial, executive function, visual memory and verbal learning tasks. Concentrations of renin and aldosterone were higher in patients with CHF (5.4 +/- 6.0 vs 0.8 +/- 0.7 mU/L, P < 0.001 and 598.2 +/- 306.2 vs 346.0 +/- 201.5, P= 0.003). Right medial temporal lobe atrophy was more prominent in CHF (P= 0.030). Left medial temporal lobe atrophy and deep white matter hyperintensities showed moderate association with cognitive scores in CHF, whereas functional capacity and biochemical parameters were fairly correlated to cognition. CONCLUSION: Congestive heart failure is associated with a pattern of generalized cognitive decline. Structural brain changes, functional capacity and biochemical parameters are associated with the cognitive performance of patients with CHF, but their contribution appears modest. The design of a definitive case-control study is described.

Coronary heart disease is associated with regional grey matter volume loss: implications for cognitive function and behaviour.

Coronary heart disease (CHD) has been associated with impaired cognition, but the mechanisms underlying these changes remain unclear. We designed this study to determine whether adults with CHD show regional brain losses of grey matter volume relative to controls. We used statistical parametric mapping (SPM5) to determine regional changes in grey matter volume of T(1)-weighted magnetic resonance images of 11 adults with prior history of myocardial infarction relative to seven healthy controls. All analyses were adjusted for total grey and white matter volume, age, sex and handedness. CHD participants showed a loss of grey matter volume in the left medial frontal lobe (including the cingulate), precentral and postcentral cortex, right temporal lobe and left middle temporal gyrus, and left precuneus and posterior cingulate. CHD is associated with loss of grey matter in various brain regions, including some that play a significant role in cognitive function and behaviour. The underlying causes of these regional brain changes remain to be determined.

Vasoconstrictor role for vasopressin in experimental heart failure in the rabbit.

Vasopressin's role as a vasoconstrictor in chronic heart failure, was examined in rabbits with adriamycin cardiomyopathic congestive heart failure. Chronic adriamycin treatment resulted in a decrease in cardiac output (829 +/- 38-610 +/- 36 ml/min, P less than 0.005) and blood pressure (83 +/- 2-76 +/- 3 mmHg, P less than 0.01), and an increase in peripheral resistance (8,377 +/- 381-10,170 +/- 657 dyn-s-cm-5, P less than 0.05). Plasma renin activity (4.7 +/- 0.6-10.9 +/- 2.8 ng angiotensin I/ml X h) and norepinephrine (0.7 +/- 0.1-1.3 +/- 0.2 pmol/ml, P less than 0.05) increased while plasma vasopressin levels did not change. Vasopressin infusion, however, produced significantly greater increases in peripheral resistance in animals with heart failure than in controls. Moreover, a specific vasopressin vascular antagonist reduced blood pressure (7 +/- 3%) and peripheral resistance (14 +/- 4%) and increased cardiac output (10 +/- 3%) in animals with heart failure but had no cardiovascular effects in normal rabbits. These results suggest that vascular sensitivity to vasopressin is increased in heart failure, and that it contributes significantly to the increased afterload in heart failure despite normal plasma levels. In this model of severe, chronic heart failure the sympathetic, renin-angiotensin, and vasopressin systems all appear to be activated.

Physical training improves skeletal muscle metabolism in patients with chronic heart failure.

OBJECTIVES: This study investigated the effects of physical training on skeletal muscle metabolism in patients with chronic heart failure. BACKGROUND: Skeletal muscle metabolic abnormalities in patients with chronic heart failure have been associated with exercise intolerance. Muscle deconditioning is a possible mechanism for the intrinsic skeletal muscle metabolic changes seen in chronic heart failure. METHODS: We used phosphorus-31 nuclear magnetic resonance spectroscopy to study muscle metabolism during exercise in 12 patients with stable ischemic chronic heart failure undergoing 8 weeks of home-based bicycle exercise training in a randomized crossover controlled trial. Changes in muscle pH and concentrations of phosphocreatine and adenosine diphosphate (ADP) were measured in phosphorus-31 spectra of calf muscle obtained at rest, throughout incremental work load plantar flexion until exhaustion and during recovery from exercise. Results were compared with those in 15 age-matched control subjects who performed a single study only. RESULTS: Before training, phosphocreatine depletion, muscle acidification and the increase in ADP during the 1st 4 min of plantar flexion exercise were all increased (p < 0.04) compared with values in control subjects. Training produced an increase (p < 0.002) in incremental plantar flexion exercise tolerance. After training, phosphocreatine depletion and the increase in ADP during exercise were reduced significantly (p < 0.003) at all matched submaximal work loads and at peak exercise, although there was no significant change in the response of muscle pH to exercise. After training, changes in ADP were not significantly different from those in control subjects, although phosphocreatine depletion was still greater (p < 0.05) in trained patients than in control subjects. The phosphocreatine recovery half-time was significantly (p < 0.05) shorter after training, although there was no significant change in the half-time of adenosine diphosphate recovery. In untrained subjects, the initial rate of phosphocreatine resynthesis after exercise (a measure of the rate of oxidative adenosine triphosphate [ATP] synthesis) and the inferred maximal rate of mitochondrial ATP synthesis were reduced compared with rates in control subjects (p < 0.003) and both were significantly increased (p < 0.05) by training, so that they were not significantly different from values in control subjects. CONCLUSIONS: The reduction in phosphocreatine depletion and in the increase in ADP during exercise, and the enhanced rate of phosphocreatine resynthesis in recovery (which is independent of muscle mass) indicate that a substantial correction of the impaired oxidative capacity of skeletal muscle in chronic heart failure can be achieved by exercise training.

Hypotension is associated with diuretic resistance in severe chronic heart failure, independent of renal function.

BACKGROUND: Diuretic resistance and systemic hypotension are common in chronic heart failure (CHF), however, the two have not been associated. AIMS: Since blood pressure (BP) might be an important determinant of sodium excretion, we searched for an association between BP and diuretic dosage in severe CHF. METHODS: Our heart failure database was retrospectively reviewed for patients with severe left ventricular systolic dysfunction. The 54-patient cohort was divided on the basis of frusemide dosage (high-dose > or = 250 mg daily, n=26). RESULTS: Patients taking high-dose frusemide had higher serum creatinine, and lower systolic and diastolic BP. On logistic regression analysis, increased serum creatinine and reduced diastolic BP were independent predictors of the use of high-dose frusemide. Grouping these variables into tertiles, the odds ratio for the use of high-dose frusemide was 4.0 as diastolic BP decreased (p<0.01), and 6.8 as serum creatinine increased (p<0.001). CONCLUSIONS: We have found an association between hypotension and the use of high-dose frusemide in severe CHF, which is independent of renal function, and which may be an important physiologic mechanism of diuretic resistance in severe CHF.

Vasopressin and angiotensin II contribute equally to the increased afterload in rabbits with heart failure.

STUDY OBJECTIVE: Vasopressin, like angiotensin, has both vasoconstrictor and fluid retaining properties and therefore may make an important contribution to the pathogenesis of low output congestive heart failure. The study aimed to examine the relative importance of the renin-angiotensin system and vasopressin in an animal model of heart failure. DESIGN: The acute haemodynamic effects of vasopressin receptor blockade with a selective antagonist, d(CH2)5DAVP (AVPA) (30 micrograms.kg-1) and angiotensin converting enzyme inhibition with captopril (1 mg.kg-1) were compared. The effect of combined blockade (ie, vasopressin receptor antagonist + angiotensin converting enzyme inhibitor) was also examined. EXPERIMENTAL MATERIAL: Rabbits, 2.5-3.5 kg, with doxorubicin induced cardiomyopathy and heart failure (n = 20) were used. There were 15 controls. MEASUREMENTS AND MAIN RESULTS: Both AVPA and captopril produced significant increases in cardiac output (11% and 13% respectively) and falls in peripheral vascular resistance (21% and 17% respectively). Inhibition of the two vasoconstrictor systems was additive and resulted in a fall in peripheral vascular resistance to levels found in normal animals. CONCLUSIONS: Vasopressin and angiotensin II make equal contributions to the raised peripheral vascular resistance observed in this model of heart failure. Vasopressin inhibition may be useful in the treatment of heart failure either alone or as an adjunct to angiotensin converting inhibition.

Adriamycin cardiomyopathy in the rabbit: an animal model of low output cardiac failure with activation of vasoconstrictor mechanisms.

Chronic administration of intravenous adriamycin (1 mg . kg-1 twice weekly for 8 weeks) to rabbits resulted in a cardiomyopathy which was similar to that occurring in patients with adriamycin cardiotoxicity. We studied systemic and renal haemodynamics and the activation of vasoconstrictor mechanisms reflected by changes in plasma renin activity (PRA), noradrenaline (NA) and vasopressin (AVP) levels during the development of heart failure in this animal model. By 8 weeks cardiac failure was clearly established. At postmortem all animals had dilated hearts, pleural and pericardial effusions, ascites and hepatic congestion. Heart weights were increased (8.1 +/- 0.7 g in treated animals n = 9 vs 6.0 +/- 0.2 g in controls n = 9 p less than 0.05). Cardiac output (measured by thermodilution) fell at 8 weeks from 799 +/- 61 ml . min-1 to 624 +/- 44 ml . min-1 (n = 6 p less than 0.05) with a parallel fall in mean blood pressure from 85 +/- 2 mmHg to 75 +/- 4 mmHg. Total peripheral resistance rose in four of the six rabbits. Renal blood flow fell from 108 +/- 4 ml . min-1 to 61 +/- 6 ml . min-1 (p less than 0.05) by 8 weeks. Renal vascular resistance increased in all animals. PRA increased from 5.1 +/- 0.5 ng AI . ml-1 . h-1 to 11.6 +/- 2.6 ng AI . ml-1 . h-1 by 4 weeks (p less than 0.05) and remained elevated thereafter.(ABSTRACT TRUNCATED AT 250 WORDS)

Altered c-fos in rostral medulla and spinal cord of spontaneously hypertensive rats.

Neurons immunoreactive for Fos, the protein product of the immediate early gene c-fos, have been compared in the rostral ventral medulla and spinal cord of conscious normotensive Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR) after baroreceptor unloading. Hypotension induced by a 60-minute intravenous infusion of sodium nitroprusside reduced baroreceptor activity; controls received intravenous saline. In WKY, 474 +/- 56 (n=6) Fos-positive neurons were identified in the rostral ventral medulla after nitroprusside infusion, a fivefold increase from controls; 50% of the tyrosine hydroxylase-containing neurons in the rostral ventral medulla were activated by this hypotension. Sympathetic preganglionic neurons, mainly sympathoadrenal neurons, were Fos positive after nitroprusside, but Fos-positive sympathetic preganglionic neurons were not observed in control WKY. In SHR, Fos immunoreactivity in the rostral ventral medulla was elevated in the control group compared with the WKY controls (236 +/- 31 and 93 +/- 15, respectively, n=6 for both). Nitroprusside hypotension did not further increase Fos immunoreactivity in the rostral ventral medulla, although the number of Fos-positive spinal sympathetic neurons increased. Our results have identified different neuronal activities between WKY and SHR in sites that are critical to sympathetic outflow. In WKY, nitroprusside effects are consistent with an activation of rostral ventral medulla neurons, including bulbospinal neurons, that are normally inhibited by baroreceptor activity. In SHR, basal nerve activity is increased, so even at rest, rostral ventral medulla neurons and sympathetic preganglionic neurons, mainly sympathoadrenal neurons, are Fos immunoreactive. These activated neurons are likely to contribute to the elevated blood pressure in this rat strain.

Close appositions between tyrosine hydroxylase immunoreactive boutons and respiratory neurons in the rat ventrolateral medulla.

The extent of the adrenergic input to respiratory neurons in the ventrolateral medulla oblongata of rats was assessed by using a combination of intracellular recording, dye filling, and immunohistochemistry. Twenty-two neurons that displayed a pronounced respiration-related modulation of their membrane potential, and could not be antidromically activated by electrical stimulation of the superior laryngeal, vagus, or facial nerves, were labelled by intracellular injection of biocytin. Three types of respiration-related neurons were labelled: small neurons located in the Bötzinger complex between 0.5 and 1.0 mm caudal to the facial nucleus; medium-sized neurons located in the ventral respiratory group 1.0 to 2.0 mm caudal to the facial nucleus; and large motoneurons located within the nucleus ambiguus 0.5 to 2.0 mm caudal to the facial nucleus. Small Bötzinger neurons [length = 22 +/- 5 microns, width = 13 +/- 3 microns, area = 222 +/- 79 microns2; (mean +/- SD, n = 5)] had membrane potentials of -15 to -27 mV during the recording period. Four of five of these cells had profuse axonal terminations between 50 microns caudal and 450 microns rostral to their somata, suggesting that they may form part of local networks responsible for generating respiratory activity. Medium-sized ventral respiratory group neurons (length = 26 +/- 5 microns, width = 18 +/- 4 microns, area = 377 +/- 141 microns2; n = 5) were found in the vicinity of the nucleus ambiguus dorsal to the lateral reticular nucleus. Three of five of these neurons had an axon that crossed the midline and travelled caudally. One axon had a collateral with varicosities close to its soma. The somata of motoneurons (length = 29 +/- 6 microns, width = 21 +/- 4 microns, area = 485 +/- 142 microns2; n = 12) were located within the nucleus ambiguus, and had axons that could be traced to exist points from the medulla. Tyrosine hydroxylase immunoreactive cells and their terminal fibres within the medulla were localised by immunocytochemistry. Small Bötzinger neurons received the largest number of close appositions from tyrosine hydroxylase immunoreactive boutons (13 +/- 2 appositions/neuron; n = 5). Medium-sized ventral respiratory group neurons received fewer appositions (8 +/- 4 appositions/neuron; n = 5). Most motoneurons (n = 10) received few appositions from tyrosine hydroxylase immunoreactive boutons, while two received none. The average number was 3 +/- 3 appositions/neuron (n = 12).(ABSTRACT TRUNCATED AT 400 WORDS)

Bötzinger neurons project towards bulbospinal neurons in the rostral ventrolateral medulla of the rat.

Sympathetic nerve activity often fluctuates with the respiratory cycle, but the central neurons that impose this respiratory modulation have not been conclusively identified. In the present study, we used intracellular recording and dye-filling to identify expiratory neurons in the Bötzinger complex. Our aim was to see if Bötzinger neurons project towards putative cardiovascular neurons in the rostral ventrolateral medulla. In the first series of experiments, histochemistry and immunohistochemistry were used to reveal the labelled Bötzinger neurons and neurons immunoreactive for tyrosine hydroxylase. Two out of four Bötzinger neurons had axon varicosities that were closely apposed to tyrosine hydroxylase-immunoreactive neurons with cell bodies located within 0.6 mm caudal to the facial nucleus (three and five close appositions, respectively). In a second series of studies, rats were injected with cholera toxin B into the intermediolateral cell column of the spinal cord 4-7 days before the electrophysiological recording. Eight of the fourteen labelled Bötzinger neurons had a direct projection towards cholera toxin B-labelled neurons in the rostral ventrolateral medulla. Close appositions were found on both somata and proximal dendrites (5 +/- 2 close appositions/neuron, n = 8). The present study supports the idea that a direct projection from Bötzinger neurons to presympathetic neurons in the rostral medulla plays a role in the respiratory modulation of sympathetic nerve activity.

Bulbospinal sympatho-excitatory neurons in the rat caudal raphe.

OBJECTIVES: To explore the rat caudal raphe nuclei for neurons that respond to activation of baroreceptor nerves and that have a spinal axon, and to compare the behavioural properties of barosensitive bulbospinal neurons in the rat caudal raphe with the properties of barosensitive bulbospinal neurons in the rostral ventrolateral medulla. DESIGN: Extracellular unit recordings were obtained from an area extending up to 1.0 mm caudally from the caudal edge of the facial nucleus. Two sites were explored: the rostral ventrolateral medulla and the midline. MATERIALS AND METHODS: Single-unit recordings were made in anaesthetized (75 mg/kg chloral hydrate and 30 mg/kg sodium pentobarbitone then 3-6 mg intravenously as required), immobilized (2 mg pancuronium as required) Sprague-Dawley rats. Central respiratory drive was recorded from phrenic nerve discharge. The barosensitivity of single units was assessed by R-wave triggered histograms and by histograms of their responses to aortic nerve stimulation or to intravenous injection of phenylephrine. Nociceptors were activated by a brief pinch of the tail. RESULTS: Eleven spontaneously active units in the midline that were inhibited by baroreceptor stimulation and had a spinal axon were studied. Respiratory modulation was present and was predominantly inspiratory. Barosensitive neurons in the rostral ventrolateral medulla were activated by nociceptive inputs; midline barosensitive neurons were not. CONCLUSIONS: The behavioural characteristics of midline neurons differ from those of the bulbospinal barosensitive neurons in the rostral ventrolateral medulla, indicating that raphe spinal neurons have different sets of afferent inputs and may subserve to a distinct physiological role. The present paper is the first report of bulbospinal neurons in the rat caudal raphe that are inhibited by activation of arterial baroreceptors.

Lacidipine, hydrochlorothiazide and their combination in systolic hypertension in the elderly.

OBJECTIVE: To compare with placebo the efficacies of once-daily administrations of lacidipine and hydrochlorothiazide separately and in combination to elderly patients with systolic hypertension. DESIGN AND METHODS: Nineteen elderly subjects (five men and 14 women, median age 71 years, range 62-79 years) participated in the study, which had a randomized double-blind crossover design. For each subject there were four treatment phases, each of duration 4 weeks. The initial treatments in each phase were 2 mg lacidipine once a day and 25 mg hydrochlorothiazide once a day, separately and in combination, and placebo. Doses of each agent could be doubled after 2 weeks in each phase if the patient's goal systolic blood pressure had not been achieved. The numbers of subjects administered the higher dose of each treatment were 13 for placebo, 14 for lacidipine, 11 for hydrochlorothiazide and eight for lacidipine plus hydrochlorothiazide. RESULTS: End-of-phase mean clinic blood pressures were 164/85 mmHg with placebo, 159/82 mmHg with lacidipine, 157/84 mmHg with hydrochlorothiazide and 152/82 mmHg with lacidipine plus hydrochlorothiazide. Systolic blood pressure was significantly reduced during all active treatment phases compared with placebo and that for the lacidipine plus hydrochlorothiazide phase was also significantly less than those for both of the other active treatment phases. There was no difference between sitting and standing blood pressure for any phase. Factorial analysis of the main effects of treatment indicated that the effects of lacidipine and hydrochlorothiazide on clinic blood pressure were additive and also that heart rate was higher when hydrochlorothiazide had been administered. Ambulatory blood pressure monitoring confirmed the pattern of the responses of blood pressure and showed that administration of hydrochlorothiazide had a significantly greater effect on systolic blood pressure and a longer duration of action than did administration of lacidipine. There was no difference in the frequency of adverse effects among any of the phases. CONCLUSIONS: In treating elderly systolic hypertensives the diuretic hydrochlorothiazide is a more effective antihypertensive agent with a longer duration of action than is the calcium channel antagonist lacidipine. In combination the effects of these two drugs on blood pressure are additive.

Evidence for increased in vivo Na(+)-H+ antiporter activity and an altered skeletal muscle contractile response in the spontaneously hypertensive rat.

We have assessed the in vivo activity of the Na(+)-H+ antiporter skeletal muscle in spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) controls using phosphorus (31P) nuclear magnetic resonance spectroscopy to measure changes in cytosolic acid concentrations during isometric contraction. During contraction there was a small rate of rise in skeletal muscle cytosolic acid concentration to a smaller maximum concentration in SHR. This difference in acid response was removed by amiloride and was not attributable to differences in cell buffering or the rate of production of lactic acid, suggesting that the difference in acid response in SHR skeletal muscle is due to increased in vivo Na(+)-H+ antiporter activity. Amiloride reduced resting muscle glycogen concentration and increased muscle lactate concentration in the SHR. This could be related to altered in vivo calcium metabolism. The maximum tension produced by skeletal muscle during contraction in SHR was less than in WKY rats, and relaxation between twitches was significantly greater, consistent with the finding of increased vascular smooth muscle relaxation in essential hypertension. Since increased Na(+)-H+ antiporter activity occurs in association with increased relaxation of both skeletal and vascular smooth muscle, these data are not consistent with a relationship between increased Na(+)-H+ antiporter activity and increased maximal muscle tension development. However, they show that increase Na(+)-H+ antiporter activity is associated with increased muscle relaxation.

Neurokinin-1 receptor-immunoreactive sympathetic preganglionic neurons: target specificity and ultrastructure.

Substance P is involved in cardiovascular control at the spinal cord level, where it acts through neurokinin-1 receptors. In this study we used immunocytochemistry and retrograde tracing to investigate the presence of the neurokinin-1 receptor and its ultrastructural localization in rat sympathetic preganglionic neurons that project to the superior cervical ganglion or the adrenal medulla. Immunofluorescence for the neurokinin-1 receptor outlined the somatic and dendritic surfaces of neurons in autonomic subnuclei of spinal cord segments T1-T12, whereas immunofluorescence for the tracer, cholera toxin B subunit, filled retrogradely labelled cells. There was a significant difference in the proportion of neurokinin-1 receptor-immunoreactive sympathetic preganglionic neurons supplying the superior cervical ganglion and the adrenal medulla. Thirty-eight percent of the neurons that projected to the superior cervical ganglion were immunoreactive for the neurokinin-1 receptor compared to 70% of neurons innervating the adrenal medulla. Of neurons projecting to the superior cervical ganglion, significantly different proportions showed neurokinin-1 receptor immunoreactivity in spinal cord segment T1 (15%) versus segments T2 T6 (45%). At the ultrastructural level, neurokinin-1 receptor staining occurred predominantly on the inner leaflets of the plasma membranes of retrogradely labelled sympathetic preganglionic neurons. Deposits of intracellular label were often observed in dendrites and in the rough endoplasmic reticulum and Golgi apparatus of cell bodies. Neurokinin-1 receptor immunoreactivity was present at many, but not all, synapses as well as at non-synaptic sites, and occurred at synapses with substance P-positive as well as substance P-negative nerve fibres. Only 37% of the substance P synapses occurred on neurokinin-1-immunoreactive neurons in the intermediolateral cell column. These results show that presence of the neurokinin-1 receptor in sympathetic preganglionic neurons is related to their target. The ultrastructural localization of the receptor suggests that sympathetic preganglionic neurons may be affected (i) by substance P released at neurokinin-1 receptor-immunoreactive synapses, (ii) by other tachykinins (e.g., neurokinin A), which co-localize in substance P fibres in the intermediolateral cell column, acting through other neurokinin receptors, and (iii) by substance P that diffuses to neurokinin-1 receptors from distant sites.

Retrogradely transported CTB-saporin kills sympathetic preganglionic neurons.

Aiming to ablate sympathetic preganglionic neurons (SPN) innervating a defined target, we injected saporin conjugated to cholera toxin B subunit (CTB) unilaterally into the superior cervical ganglion of rats. In spinal cord segments T1-T3, the numbers of cholinergic neurons in the intermediolateral cell column ipsilateral and contralateral to the injected ganglion were significantly different by 3 days post-injection. By day 14, 77% of ipsilateral cholinergic neurons had disappeared. A higher percentage of neurons were killed in T1-T2 than in T3. Comparing SPN counts from CTB-saporin injected rats and counts from rats receiving unconjugated CTB into the superior cervical ganglion indicated that 84% of SPN supplying the ganglion had died by 14 days. Retrogradely transported CTB-saporin kills sympathetic preganglionic neurons and may also eliminate other types of neurons that transport CTB.

c-fos identifies GABA-synthesizing barosensitive neurons in caudal ventrolateral medulla.

Hypertension in the conscious rat, elicited by i.v. infusion of phenylephrine, evoked expression of the immediate early gene c-fos in discrete groups of brain stem neurons. Fos-immunoreactive neurons were located in the caudal ventrolateral medulla (CVLM); others were located in the nucleus of the tractus solitarius (NTS). Because of their sensitivity to alterations in arterial pressure, these neurons are likely to subserve the arterial baroreceptor reflex. The aim of this study was to identify the brain stem projections and the neurotransmitter content of the barosensitive CVLM neurons using neuronal tracing and immunohistochemistry. Some of the barosensitive CVLM neurons projected directly to the rostral ventrolateral medulla (RVLM), and many contained the GABA synthesizing enzyme, glutamic acid decarboxylase (GAD). Other CVLM neurons, containing markers of glutamate or catecholamine synthesis, were insensitive to baroreceptor stimulation. This study delineates neuronal pathways acting in the arterial baroreceptor reflex and identifies precisely GABA-synthesizing CVLM neurons as the source of inhibitory input to the RVLM.

Amino acid neurotransmitters in hypertension.

There is compelling evidence for the participation of excitatory and inhibitory amino acids in the neural regulation of blood pressure in the normotensive rat. This is most clearly evident in the neural pathways which form the baroreceptor reflex arc. Excitatory amino acids are contained in baroreceptor afferents, neurons in the nucleus tractus solitarius (NTS) and neurons in the rostral ventrolateral medulla (RVLM). Inhibitory neurons in the caudal ventrolateral medulla (CVLM) contain gamma-aminobutyric acid. Electrophysiological and pharmacological evidence indicates that amino acid neurotransmitters are critically important to the normal function of these integrative sites in the baroreceptor reflex. Spontaneously hypertensive rats (SHR) differ from Wistar Kyoto (WKY) controls in their responses to stimulation, inhibition or lesions of neurons in the baroreceptor arc. One week after baroreceptor denervation, blood pressure is elevated in WKY but not in SHR. Stimulation of the CVLM results in a greater fall in pressure in SHR than WKY, whereas injection of tetrodotoxin into the CVLM results in a smaller increase in pressure in SHR. Blockade of glutamate receptors in the spinal cord attenuates the response to stimulation of the RVLM in both SHR and WKY, but reduces resting blood pressure in SHR only. These experiments suggest that altered activity in amino acid pathways contributes to the pathogenesis of hypertension in SHR.

Tracer-toxins: cholera toxin B-saporin as a model.

We have shown previously that retrogradely-transported cholera toxin B (CTB)-saporin has eliminated sympathetic preganglionic neurons by 7 days after injection (Llewellyn-Smith, I.J., Martin, C.L., Arnolda, L.F., Minson, J.B., 1999. NeuroReport 10, 307). To ascertain whether this tracer-toxin can kill other types of neurons that transport CTB retrogradely with a similar time course, we injected CTB-saporin into the facial nerves of rats and allowed them to survive for 7 days. Facial motoneurons were counted ipsilateral and contralateral to the injected nerves in sections of perfused medulla processed to reveal immunoreactivity for choline acetyltransferase (ChAT). There was a statistically significant decrease in the number of ChAT-immunoreactive neurons ipsilateral to the injected nerve in three out of nine rats. Inadequate injections were probably the reason that most rats showed no decrease in motoneurons numbers after treatment with CTB-saporin, since the staining intensity and numbers of facial motoneurons that showed CTB-immunoreactivity varied markedly between rats after retrograde tracing with unconjugated CTB. These results show that CTB-saporin can eliminate motoneurons as well as sympathetic preganglionic neurons, indicate that protocols for the injection of tracer-toxins should be optimized to ensure maximum neuronal death and support our contention that CTB-saporin should kill any central neuron that expresses GM1 ganglioside, the membrane component to which CTB binds.

Intracellular recording from sympathetic preganglionic neurons in cat lumbar spinal cord.

Sympathetic preganglionic neurons (SPN) are responsible for the control of many autonomic targets including the heart and blood vessels. Previous intracellular studies have examined the morphology of SPN in the thoracic spinal cord, but there are no intracellular studies of SPN in the lumbar spinal cord. In this study we identified lumbar SPN using intracellular recording and dye-filling so that we could study their entire soma-dendritic tree, as well as their axons. At the same time, axonal conduction velocity was measured, and any evidence of an input in phase with phrenic nerve discharge was noted. Intracellular recordings were made from SPN in the L3 (n = 125) and T3 (n = 17) segments of the cat spinal cord. Axonal conduction velocities ranged from 0.6-8.4 m/s. In 85 lumbar SPN, the recordings lasted long enough to assess respiratory-related modulation. A respiratory-related modulation of the membrane potential was seen in 7 of these 85 neurons. All 7 respiratory-related neurons had a conduction velocity of 2.0 m/s or less, while none of the SPN with conduction velocities of more than 2.0 m/s had a respiratory rhythmicity. Histological analysis of 50 biocytin-filled SPN, including 3 with a respiratory-related modulation of their membrane potential, revealed that they occurred mostly in the principal part of the intermediolateral cell column and tended to be elongated in the rostro-caudal direction. Dendrites ramified in the intermediolateral cell column, the dorsolateral white matter and the ventral and medial gray matter. Axons arose either from cell bodies or from primary dendrites and did not bifurcate or have varicose intraspinal collaterals. This is the first report of the morphology of intracellularly filled SPN in the lumbar spinal cord.