Behavioral correlates of the activity of serotonergic and non-serotonergic neurons in caudal raphe nuclei

We investigated the behavioral correlates of the activity of serotonergic and non-serotonergic neurons in the nucleus raphe pallidus (NRP) and nucleus raphe obscurus (NRO) of unanesthetized and unrestrained cats. The animals were implanted with electrodes for recording single unit activity, parietal oscillographic activity, and splenius, digastric and masseter electromyographic activities. They were tested along the waking-sleep cycle, during sensory stimulation and during drinking behavior. The discharge of the serotonergic neurons decreased progressively from quiet waking to slow wave sleep and to fast wave sleep. Ten different patterns of relative discharge across the three states were observed for the non-serotonergic neurons. Several non-serotonergic neurons showed cyclic discharge fluctuations related to respiration during one, two or all three states. While serotonergic neurons were usually unresponsive to the sensory stimuli used, many non-serotonergic neurons responded to these stimuli. Several non-serotonergic neurons showed a phasic relationship with splenius muscle activity during auditory stimulation. One serotonergic neuron showed a tonic relationship with digastric muscle activity during drinking behavior. A few non-serotonergic neurons exhibited a tonic relationship with digastric and/or masseter muscle activity during this behavior. Many non-serotonergic neurons exhibited a phasic relationship with these muscle activities, also during this behavior. These results suggest that the serotonergic neurons in the NRP and NRO constitute a relatively homogeneous population from a functional point of view, while the non-serotonergic neurons form groups with considerable functional specificity. The data support the idea that the NRP and NRO are implicated in the control of somatic motor output.

Frequent occurrence of nicotinic acetylcholine receptors in GABAergic neurons of the chick visual system

Double-labeling immunohistochemical methods were used to investigate the occurrence of the alpha8 and alpha5 nicotinic receptor subunits in presumptive GABAergic neurons of the chick nervous system. Nicotinic receptor immunoreactivity was often found in cells exhibiting GABA-like immunoreactivity, especially in the visual system. The alpha8 subunit appeared to be present in presumptive GABAergic cells of the ventral lateral geniculate nucleus, nucleus of the basal optic root of the accessory optic system, and the optic tectum, among several other structures. The alpha5 subunit was also found in GABA-positive neurons, as observed in the lentiform nucleus of the mesencephalon and other pretectal nuclei. The numbers of alpha8- and alpha5-positive neurons that were also GABA-positive represented high percentages of the total number of neurons containing nicotinic receptor labeling in several brain areas, which indicates that most of the alpha8 and alpha5 nicotinic receptor subunits are present in GABAergic cells. Taken together with data from other studies, our results indicate an important role of the nicotinic acetylcholine receptors in the functional organization of GABAergic circuits in the visual system.

The origin of cortical neurons

Neurons of the mammalian cerebral cortex comprise two broad classes: pyramidal neurons, which project to distant targets, and the inhibitory nonpyramidal cells, the cortical interneurons. Pyramidal neurons are generated in the germinal ventricular zone, which lines the lateral ventricles, and migrate along the processes of radial glial cells to their positions in the developing cortex in an `inside-out' sequence. The GABA-containing nonpyramidal cells originate for the most part in the ganglionic eminence, the primordium of the basal ganglia in the ventral telencephalon. These cells follow tangential migratory routes to enter the cortex and are in close association with the corticofugal axonal system. Once they enter the cortex, they move towards the ventricular zone, possibly to obtain positional information, before they migrate radially in the direction of the pial surface to take up their positions in the developing cortex. The mechanisms that guide interneurons throughout these long and complex migratory routes are currently under investigation.

Protective effects of centrally acting sympathomodulatory drugs on myocardial ischemia induced by sympathetic overactivity in rabbits

It is recognized that an imbalance of the autonomic nervous system is involved in the genesis of ventricular arrhythmia and sudden death during myocardial ischemia. In the present study we investigated the effects of clonidine and rilmenidine, two centrally acting sympathomodulatory drugs, on an experimental model of centrally induced sympathetic hyperactivity in pentobarbital-anesthetized New Zealand albino rabbits of either sex (2-3 kg, N = 89). We also compared the effects of clonidine and rilmenidine with those of propranolol, a ß-blocker, known to induce protective cardiovascular effects in patients with ischemic heart disease. Central sympathetic stimulation was achieved by intracerebroventricular injection of the excitatory amino acid L-glutamate (10 µmol), associated with inhibition of nitric oxide synthesis with L-NAME (40 mg/kg, iv). Glutamate triggered ventricular arrhythmia and persistent ST-segment shifts in the ECG, indicating myocardial ischemia. The intracisternal administration of clonidine (1 µg/kg) and rilmenidine (30 µg/kg) or of a nonhypotensive dose of rilmenidine (3 µg/kg) decreased the incidence of myocardial ischemia (25, 14 and 25%, respectively, versus 60% in controls) and reduced the mortality rate from 40% to 0.0, 0.0 and 12%, respectively. The total number of ventricular premature beats per minute fell from 30 ± 9 in the control group to 7 ± 3, 6 ± 3 and 2 ± 2, respectively. Intravenous administration of clonidine (10 µg/kg), rilmenidine (300 µg/kg) or propranolol (500 µg/kg) elicited similar protective effects. We conclude that clonidine and rilmenidine present cardioprotective effects of central origin, which can be reproduced by propranolol, a lipophilic ß-blocking agent.

Electrophysiological evidence for glial-subtype glutamate transporter functional expression in rat cerebellar granule neurons

A glutamate-sensitive inward current (Iglu) is described in rat cerebellar granule neurons and related to a glutamate transport mechanism. We examined the features of Iglu using the patch-clamp technique. In steady-state conditions the Iglu measured 8.14 ± 1.9 pA. Iglu was identified as a voltage-dependent inward current showing a strong rectification at positive potentials. L-Glutamate activated the inward current in a dose-dependent manner, with a half-maximal effect at about 18 µM and a maximum increase of 51.2 ± 4.4%. The inward current was blocked by the presence of dihydrokainate (0.5 mM), shown by others to readily block the GLT1 isoform. We thus speculate that Iglu could be attributed to the presence of a native glutamate transporter in cerebellar granule neurons.

Cardiovascular responses to microinjections of GABA or anesthetics into the rostral ventrolateral medulla of conscious and anesthetized rats

The rostral ventrolateral medulla (RVLM) contains neurons involved in tonic and reflex control of arterial pressure. We describe the effects of gamma-aminobutyric acid (GABA) and anesthetics injected into the RVLM of conscious and urethane (1.2 g/kg, iv) anesthetized Wistar rats (300-350 g). In conscious rats, bilateral microinjection of GABA (50 nmol/200 nl) induced a small but significant decrease in blood pressure (from 130 ± 3.6 to 110 ± 5.6 mmHg, N = 7). A similar response was observed with sodium pentobarbital microinjection (24 nmol/200 nl). However, in the same animals, the fall in blood pressure induced by GABA (from 121 ± 8.9 to 76 ± 8.8 mmHg, N = 7) or pentobarbital (from 118 ± 4.5 to 57 ± 11.3 mmHg, N = 6) was significantly increased after urethane anesthesia. In contrast, there was no difference between conscious (from 117 ± 4.1 to 92 ± 5.9 mmHg, N = 7) and anesthetized rats (from 123 ± 6.9 to 87 ± 8.7 mmHg, N = 7) when lidocaine (34 nmol/200 nl) was microinjected into the RVLM. The heart rate variations were not consistent and only eventually reached significance in conscious or anesthetized rats. The right position of pipettes was confirmed by histology and glutamate microinjection into the RVLM. These findings suggest that in conscious animals the RVLM, in association with the other sympathetic premotor neurons, is responsible for the maintenance of sympathetic vasomotor tone during bilateral RVLM inhibition. Activity of one or more of these premotor neurons outside the RVLM can compensate for the effects of RVLM inhibition. In addition, the effects of lidocaine suggest that fibers passing through the RVLM are involved in the maintenance of blood pressure in conscious animals during RVLM inhibition.

Evidence that urocortin is absent from neurons of the Edinger-Westphal nucleus in pigeons

The Edinger-Westphal nucleus (EWN) is a central preganglionic parasympathetic cell group that gives rise to cholinergic input to the ciliary ganglion, thereby regulating several neurovegetative ocular functions. Recently, the supposed presence of the neuropeptide urocortin (UCN) has been reported in EWN neurons in rodent brain. The purpose of the present study was to examine the distribution of UCN in avian brain and to investigate by immunohistochemical analysis the possible use of this substance as an EWN marker in a non-mammalian class of vertebrates. Brain tissue of pigeons was incubated with a specific antibody against UCN and the results showed labeling of many small neurons, forming a double wing in the dorsal mesodiencephalic transition area. Their size and shape, however, differed from those of EWN neurons, and they were preferentially located rostral to the EWN. Double-label experiments employing an antibody against the enzyme choline acetyltransferase (ChAT) showed that UCN is not localized to the cholinergic cells of the EWN and confirmed the rostral distributionof UCN never overlapping the ChAT+ EWN cells. Taken together, these results suggest that, at least in pigeons, the UCN+ population does not belong to the traditionally defined EWN.

Electrophysiological evidence for the presence of NR2C subunits of N-methyl-D-aspartate receptors in rat neurons of the nucleus tractus solitarius

The nucleus tractus solitarius (NTS) plays an important role in the control of autonomic reflex functions. Glutamate, acting on N-methyl-D-aspartate (NMDA) and non-NMDA ionotropic receptors, is the major neurotransmitter in this nucleus, and the relative contribution of each receptor to signal transmission is unclear. We have examined NMDA excitatory postsynaptic currents (NMDA-EPSCs) in the subpostremal NTS using the whole cell patch clamp technique on a transverse brainstem slice preparation. The NMDA-EPSCs were evoked by stimulation of the solitary tract over a range of membrane potentials. The NMDA-EPSCs, isolated pharmacologically, presented the characteristic outward rectification and were completely blocked by 50 µM DL-2-amino-5-phosphonopentanoic acid. The I-V relationship of the NMDA response shows that current, with a mean (± SEM) amplitude of -41.2 ± 5.5 pA, is present even at a holding potential of -60 mV, suggesting that the NMDA receptors are weakly blocked by extracellular Mg2+ at near resting membrane potentials. This weak block can also be inferred from the value of 0.67 ± 0.17 for parameter delta obtained from a fit of the Woodhull equation to the I-V relationship. The maximal inward current measured on the I-V relationship was at -38.7 ± 4.2 mV. The decay phase of the NMDA currents was fitted with one exponential function with a decay time constant of 239 ± 51 and 418 ± 80 ms at a holding potential of -60 and +50 mV, respectively, which became slower with depolarization (e-fold per 145 mV). The biophysical properties of the NMDA receptors observed in the present study suggest that these receptors in the NTS contain NR2C subunits and may contribute to the synaptic signal integration.

Quantitative evidence for neurofilament heavy subunit aggregation in motor neurons of spinal cords of patients with amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS), a neurodegenerative disease of unknown etiology, affects motor neurons leading to atrophy of skeletal muscles, paralysis and death. There is evidence for the accumulation of neurofilaments (NF) in motor neurons of the spinal cord in ALS cases. NF are major structural elements of the neuronal cytoskeleton. They play an important role in cell architecture and differentiation and in the determination and maintenance of fiber caliber. They are composed of three different polypeptides: light (NF-L), medium (NF-M) and heavy (NF-H) subunits. In the present study, we performed a morphological and quantitative immunohistochemical analysis to evaluate the accumulation of NF and the presence of each subunit in control and ALS cases. Spinal cords from patients without neurological disease and from ALS patients were obtained at autopsy. In all ALS cases there was a marked loss of motor neurons, besides atrophic neurons and preserved neurons with cytoplasmic inclusions, and extensive gliosis. In control cases, the immunoreaction in the cytoplasm of neurons was weak for phosphorylated NF-H, strong for NF-M and weak for NF-L. In ALS cases, anterior horn neurons showed intense immunoreactivity in focal regions of neuronal perikarya for all subunits, although the difference in the integrated optical density was statistically significant only for NF-H. Furthermore, we also observed dilated axons (spheroids), which were immunopositive for NF-H but negative for NF-M and NF-L. In conclusion, we present qualitative and quantitative evidence of NF-H subunit accumulation in neuronal perikarya and spheroids, which suggests a possible role of this subunit in the pathogenesis of ALS.

Functional mapping of the cardiorespiratory effects of dorsal and median raphe nuclei in the rat

The dorsal (DRN) and median (MRN) raphe nuclei are important sources of serotonergic innervation to the forebrain, projecting to sites involved in cardiovascular regulation. These nuclei have been mapped using electrical stimulation, which has the limitation of stimulating fibers of passage. The present study maps these areas with chemical stimulation, investigating their influence on cardiorespiratory parameters. Urethane-anesthetized (1.2 g/kg, iv) male Wistar rats (280-300 g) were instrumented for pulsatile and mean blood pressure (MBP), heart rate, renal nerve activity, and respiratory frequency recordings. Microinjections of L-glutamate (0.18 M, 50-100 nl with 1% Pontamine Sky Blue) were performed within the DRN or the MRN with glass micropipettes. At the end of the experiments the sites of microinjection were identified. The majority of sites within the MRN (86.1%) and DRN (85.4%) evoked pressor responses when stimulated (DRN: deltaMBP = +14.7 ± 1.2; MRN: deltaMBP = +13.6 ± 1.3 mmHg). The changes in renal nerve activity and respiratory rate caused by L-glutamate were +45 ± 11 and +42 ± 9% (DRN; P < 0.05%), +40 ± 10 and +29 ± 7% (MRN, P < 0.05), respectively. No significant changes were observed in saline-microinjected animals. This study shows that: a) the blood pressure increases previously observed by electrical stimulation within the raphe are due to activation of local neurons, b) this pressor effect is due to sympathoexcitation because the stimulation increased renal sympathetic activity but did not produce tachycardia, and c) the stimulation of cell bodies in these nuclei also increases the respiratory rate.

Sympathetic activation in rats with L-NAME-induced hypertension

We evaluated the hemodynamic pattern and the contribution of the sympathetic nervous system in conscious and anesthetized (1.4 g/kg urethane, iv) Wistar rats with L-NAME-induced hypertension (20 mg/kg daily). The basal hemodynamic profile was similar for hypertensive animals, conscious (N = 12) or anesthetized (N = 12) treated with L-NAME for 2 or 7 days: increase of total peripheral resistance associated with a decrease of cardiac output (CO) compared to normotensive animals, conscious (N = 14) or anesthetized (N = 14). Sympathetic blockade with hexamethonium essentially caused a decrease in total peripheral resistance in hypertensive animals (conscious, 2 days: from (means ± SEM) 2.47 ± 0.08 to 2.14 ± 0.07; conscious, 7 days: from 2.85 ± 0.13 to 2.07 ± 0.33; anesthetized, 2 days: from 3.00 ± 0.09 to 1.83 ± 0.25 and anesthetized, 7 days: from 3.56 ± 0.11 to 1.53 ± 0.10 mmHg mL-1 min-1) with no change in CO in either group. However, in the normotensive group a fall in CO (conscious: from 125 ± 4.5 to 96 ± 4; anesthetized: from 118 ± 1.5 to 104 ± 5.5 mL/min) was observed. The responses after hexamethonium were more prominent in the hypertensive anesthetized group. However, no difference was observed between conscious and anesthetized normotensive rats in response to sympathetic blockade. The present study shows that the vasoconstriction in response to L-NAME was mediated by the sympathetic drive. The sympathetic tone plays an important role in the initiation and maintenance of hypertension.

Modifications of the sympathetic skin response in workers chronically exposed to lead

The long-term effects of low-level lead intoxication are not known. The sympathetic skin response (SSR) was evaluated in a group of 60 former workers of a primary lead smelter, located in Santo Amaro, BA, Brazil. The individuals participating in the study were submitted to a clinical-epidemiological evaluation including questions related to potential risk factors for intoxication, complaints related to peripheral nervous system (PNS) involvement, neurological clinical examination, and also to electromyography and nerve conduction studies and SSR evaluation. The sample consisted of 57 men and 3 women aged 34 to 69 years (mean ± SD: 46.8 ± 6.9). The neurophysiologic evaluation showed the presence of lumbosacral radiculopathy in one of the individuals (1.7%), axonal sensorimotor polyneuropathy in 2 (3.3%), and carpal tunnel syndrome in 6 (10%). SSR was abnormal or absent in 12 cases, representing 20% of the sample. More than half of the subjects (53.3%) reported a history of acute abdominal pain requiring hospitalization during the period of work at the plant. A history of acute palsy of radial and peroneal nerves was reported by about 16.7 and 8.3% of the individuals, respectively. Mean SSR amplitude did not differ significantly between patients presenting or not the various characteristics in the current neurological situation, except for diaphoresis. The results suggest that chronic lead intoxication induces PNS damage, particularly affecting unmyelinated small fibers. Further systematic study is needed to more precisely define the role of lead in inducing PNS injury.

Effect of exercise training and carvedilol treatment on cardiac function and structure in mice with sympathetic hyperactivity-induced heart failure

The present investigation was undertaken to study the effect of β-blockers and exercise training on cardiac structure and function, respectively, as well as overall functional capacity in a genetic model of sympathetic hyperactivity-induced heart failure in mice (α2A/α2CArKO). α2A/α2CArKO and their wild-type controls were studied for 2 months, from 3 to 5 months of age. Mice were randomly assigned to control (N = 45), carvedilol-treated (N = 29) or exercise-trained (N = 33) groups. Eight weeks of carvedilol treatment (38 mg/kg per day by gavage) or exercise training (swimming sessions of 60 min, 5 days/week) were performed. Exercise capacity was estimated using a graded treadmill protocol and HR was measured by tail cuff. Fractional shortening was evaluated by echocardiography. Cardiac structure and gastrocnemius capillary density were evaluated by light microscopy. At 3 months of age, no significant difference in fractional shortening or exercise capacity was observed between wild-type and α2A/α2CArKO mice. At 5 months of age, all α2A/α2CArKO mice displayed exercise intolerance and baseline tachycardia associated with reduced fractional shortening and gastrocnemius capillary rarefaction. In addition, α2A/ α2CArKO mice presented cardiac myocyte hypertrophy and ventricular fibrosis. Exercise training and carvedilol similarly improved fractional shortening in α2A/α2CArKO mice. The effect of exercise training was mainly associated with improved exercise tolerance and increased gastrocnemius capillary density while β-blocker therapy reduced cardiac myocyte dimension and ventricular collagen to wild-type control levels. Taken together, these data provide direct evidence for the respective beneficial effects of exercise training and carvedilol in α2A/α2CArKO mice preventing cardiac dysfunction. The different mechanisms associated with beneficial effects of exercise training and carvedilol suggest future studies associating both therapies.

Increased sympathetic activity in normotensive offspring of malignant hypertensive parents compared to offspring of normotensive parents

Malignant hypertension seems to be the consequence of very high blood pressure. Furthermore, an increase in sympathetic and renin-angiotensin system activity is considered to be the main mechanisms producing malignant hypertension. In the present study, 10 offspring of malignant hypertensive (OMH) parents (age 28 ± 5 years, 7 males, 3 females, 2 white and 8 non-white) and 10 offspring of normotensive (ONT) parents (age 28 ± 6 years, 2 males, 8 females, 3 white and 7 non-white) were evaluated. The OMH group had significantly higher (P < 0.05) casual blood pressure (125 ± 10/81 ± 5 mmHg) compared with ONT (99 ± 13/67 ± 5 mmHg). The increase in blood pressure was greater in OMH (Δ SBP = 17 ± 2 vs Δ SBP = 9 ± 1 mmHg in ONT) during cold pressor testing, but they had a lower increase in heart rate (Δ HR = 13 ± 2 vs Δ HR = 20 ± 3 bpm in ONT) during isometric exercise (handgrip test). Sympathetic activity, measured by microneurography, was significantly higher (P < 0.05) before exercise in OMH (17 ± 6 vs 11 ± 4 burst/min in ONT) and exhibited a greater increase (Δ = 18 ± 10 vs Δ = 8 ± 3 burst/min in ONT) during isometric exercise. This study showed increased sympathetic activity in OMH before exercise and a greater response during isometric exercise, suggesting an autonomic abnormality before exercise and a greater sympathetic response to physical stress in OMH compared to ONT.

The inhibitory role of sympathetic nervous system in the Ca2+-dependent proteolysis of skeletal muscle

Mammalian cells contain several proteolytic systems to carry out the degradative processes and complex regulatory mechanisms to prevent excessive protein breakdown. Among these systems, the Ca2+-activated proteolytic system involves the cysteine proteases denoted calpains, and their inhibitor, calpastatin. Despite the rapid progress in molecular research on calpains and calpastatin, the physiological role and regulatory mechanisms of these proteins remain obscure. Interest in the adrenergic effect on Ca2+-dependent proteolysis has been stimulated by the finding that the administration of β2-agonists induces muscle hypertrophy and prevents the loss of muscle mass in a variety of pathologic conditions in which calpains are activated. This review summarizes evidence indicating that the sympathetic nervous system produces anabolic, protein-sparing effects on skeletal muscle protein metabolism. Studies are reviewed, which indicate that epinephrine secreted by the adrenal medulla and norepinephrine released from adrenergic terminals have inhibitory effects on Ca2+-dependent protein degradation, mainly in oxidative muscles, by increasing calpastatin levels. Evidence is also presented that this antiproteolytic effect, which occurs under both basal conditions and in stress situations, seems to be mediated by β2- and β3-adrenoceptors and cAMP-dependent pathways. The understanding of the precise mechanisms by which catecholamines promote muscle anabolic effects may have therapeutic value for the treatment of muscle-wasting conditions and may enhance muscle growth in farm species for economic and nutritional purposes.