Inhibition of medullary raphe serotonergic neurons has age-dependent effects on the CO2 response in newborn piglets.

Medullary raphé serotonergic neurons are chemosensitive in culture and are situated adjacent to blood vessels in the brain stem. Selective lesioning of serotonergic raphé neurons decreases the ventilatory response to systemic CO2 in awake and sleeping adult rats. Abnormalities in the medullary serotonergic system, including the raphé, have been implicated in the sudden infant death syndrome (48). In this study, we ask whether serotonergic neurons in the medullary raphé and extra-raphé regions are involved in the CO2 response in unanesthetized newborn piglets, 3-16 days old. Whole body plethysmography was used to examine the ventilatory response to 5% CO2 before and during focal inhibition of serotonergic neurons by 8-hydroxy-2-di-n-propylaminotetralin (8-OH-DPAT), a 5-HT1A receptor agonist. 8-OH-DPAT (10 or 30 mM in artificial cerebrospinal fluid) decreased the CO2 response in wakefulness in an age-dependent manner, as revealed by a linear regression analysis that showed a significant negative correlation (P < 0.001) between the percent change in the CO2 response and piglet age. Younger piglets showed an exaggerated CO2 response. Control dialysis with artificial cerebrospinal fluid had no significant effect on the CO2 response. Additionally, 8-OH-DPAT increased blood pressure and decreased heart rate independent of age (P < 0.05). Finally, sleep cycling was disrupted by 8-OH-DPAT, such that piglets were awake more and asleep less (P < 0.05). Because of the fragmentary sleep data, it was not possible to examine the CO2 response in sleep. Inhibition of serotonergic medullary raphé and extra-raphé neurons decreases ventilatory CO2 sensitivity and alters cardiovascular variables and sleep cycling, which may contribute to the sudden infant death syndrome.     

Changes in RNA concentration in neurons and perineuronal glia of the caudal nucleus of the vagus nerve after a single maximal overload

By the method of quantitative cytophotometry, the content of ribonucleic acid in neurons and mantle gliocytes of the cat vagus caudal node has been studied after a single maximal overloading of 10 units. It has been stated that a decreased RNA content in the mantle gliocytes is less pronounced; that could be explained by their different nutritional conditions (owing to their various position towards the microvessels of the node), on the one hand, and by different functional loading experienced by the two types of cells under the effect of the extreme factor studied, on the other hand. RNA content in gliocytes is restored more quickly owing to its less decrease, and also owing to a more favourable protein synthesis in glycocytes--"for itself".     

Immunofluorescence localization of corticoliberin neurons in the brain of pigeons

With immunofluorescence techniques using one anti-rat or two different anti-ovine CRF, the localization of corticotropin-releasing factor (CRF) producing neurons was characterized in frozen sections of pigeon brain. Colchicine was administered intraventricularly at various day hours. The CRF neurons were localized in the telencephalon: lobus parolfactorius, nucleus (n.) accumbens, anterior commissure; in the diencephalon: n. dorso-medialis and lateralis thalami and in different structures of the hypothalamus: n. praeopticus periventricularis and medialis, paraventricularis, supraopticus medialis, lateralis, ectomamillaris and in the stratum cellulare externum. Concerning the hypothalamic localizations, results are discussed in the light of physiological studies on corticotropic regulations in pigeons. Additional populations of CRF neurons were also located in various brainstem areas substantia grisea centralis, locus caeruleus, n. tegmenti dorsalis, sensorius principalis nervi trigemini, vestibularis latetalis, solitarius, nervi hypoglossi, in the dorsal area of the n. pontis lateralis and in the n. paramedianus paragiganto--cellularis, raphes, nervi facialis, subcaeruleus and the area ventralis. These particular localizations may lead to the assumption that CRF might be involved in nervous regulations other than those related to the corticotropic function.     

Ventilatory response to hypercapnia and hypoxia after extensive lesion of medullary serotonergic neurons in newborn conscious piglets.

Acute inhibition of serotonergic (5-HT) neurons in the medullary raphé (MR) using a 5-HT(1A) receptor agonist had an age-dependent impact on the "CO(2) response" of piglets (33). Our present study explored the effect of chronic 5-HT neuron lesions in the MR and extra-raphé on the ventilatory response to hypercapnia and hypoxia in piglets, with possible implications on the role of 5-HT in the sudden infant death syndrome. We established four experimental groups. Group 1 (n = 11) did not undergo any treatment. Groups 2, 3, and 4 were injected with either vehicle or the neurotoxin 5,7-dihydroxytryptamine in the cisterna magna during the first week of life (group 2, n = 9; group 4, n = 11) or second week of life (group 3, n = 10). Ventilation was recorded in response to 5% CO(2) (all groups) and 12% O(2) (group 2) during wakefulness and sleep up to postnatal day 25. Surprisingly, the piglets did not reveal changes in their CO(2) sensitivity during early postnatal development. Overall, considerable lesions of 5-HT neurons (up to 65% decrease) in the MR and extra-raphé had no impact on the CO(2) response, regardless of injection time. Postlesion raphé plasticity could explain why we observed no effect. 5,7-Dihydroxytryptamine-treated males, however, did present a lower CO(2) response during sleep. Hypoxia significantly altered the frequency during sleep in lesioned piglets. Further studies are necessary to elucidate the role of plasticity, sex, and 5-HT abnormalities in sudden infant death syndrome.     

Selective decrease in protein intake following brain serotonin depletion.

The effect of reduced brain serotonin concentration achieved with drugs or raphé lesions on the control of protein intake and energy intake by young rats was examined. All rats were provided an oppurtunity to select from 15% and 55% casein diets provided simultaneously in two food cups. Systemic parachlorphenylalanine, central 5, 7-dihydroxytryptamine or mid-brain raphé lesions reduced by 20–30% the amount of protein consumed from these two diets over a two week study period. Across these same groups total energy intake was not different from the control groups. This decrease in the proportion of dietary energy selected as protein by the treated self-selecting rats was associated with the reduction observed in brain serotonin and its major metabolite, 5-hydroxyindole acetic acid at the completion of the feeding period. These findings support previous work implicating brain serotonin metabolism in the selective control of protein intake.     

Biosynthesis of serotonin in Raphé nuclei of rat brain: effect of p-chlorophenylalanine

The activity of tryptophan hydroxylase (EC 1.99.1.4) in the region of the raphé nuclei of rat brain was higher than that of any other brain area. The content of serotonin and the rate of serotonin synthesis were also highest in the raphé nuclei. Following the administration of p-chlorophenylalanine the injection of tryptophan and pargyline increased the content of serotonin in the region of the raphé nuclei of rat brain. The results suggest that the raphé nuclei retained the capacity to hydroxyl-late tryptophan to some extent after the injection of p-chlorophenylalanine.     

Ontogenesis of serotoninergic nuclei in the rat stem

The development of central serotoninergic neurons has been investigated with immunohistochemical techniques using the indirect peroxidase-antiperoxidase (PAP) method in 16-and 19-day-old rat embryos, in 1, 10 and 26 days old young and in adult animals. Immunoreactive neurons were present on embryonic day 16 in the subventricular area of the brain stem. First the countour of nucleus raphe dorsalis became distinct in the subventricular cell mass of the lower midbrain. In the ventral part of the tegmentum, cells were grouped along the midline in bilateral columns from which the nucleus centralis superior, the nucleus raphe pontis and the nuclei pontis differentiated. These nuclei were well defined in the newborn on either side of the midline, and the nucleus centralis superior and nucleus raphe pontis were fused on the midline in 10-day old rat. In the ventral part of the pons and medulla, a bilateral cell mass was also found along the midline. A number of immunoreactive cells moving off the midline constituted the nucleus raphe magnus which was formed on 19. embryonis day. Another contingent of immunoreactive cells remained at the midline and formed the nuclei raphe obscurus and pallidus. In newborn rat, these nuclei were well separated from the nucleus raphe magnus. They would later fuse on the midline, whereas the nucleus raphe magnus would remain a bilateral structure.     

Rat hypothalamic noradrenaline levels following midbrain raphé nuclei lesions

Electrolytic lesions of the median and dorsal raphé nuclei resulted in statistically significant reductions in rat hypothalamic noradrenaline which were observed 1 or 2 days after lesioning, while no changes were observed 7 or 14 days after lesioning. The short term (1-2 days) raphé nuclei lesions produced no changes in hypothalamic 5-hydroxytryptamine or a small reduction in 5-hydroxyindole acetic acid while the expected marked reductions were observed after 7 days. The reduction in hypothalamic noradrenaline observed after short term raphé nuclei lesions suggests the existence of a positive feedback loop between 5-hydroxytryptamine neurons and noradrenaline terminals in the hypothalamus.     

Evidence that GABA in the nucleus dorsalis raphé induces stimulation of locomotor activity and eating behavior

Recent electrophysiological studies have provided evidence that GABA controls inhibitorily the activity of the serotonin containing cell bodies in nucleus dorsalis raphé (NDR). The present investigation shows that local injection of baclofen or the GABA agonist muscimol (25–100 ng) into the NDR strongly increased locomotor activity and stimulated eating in satiated rats. These effects are antagonized by the GABA antagonists bicuculline or picrotoxin given systemically or locally. Muscimol injected in NDR also decreased serotonin and 5-hydroxyindole acetic acid in hypothalamus but not in striatum. These findings support a transmitter role of GABA in NDR and may be interpreted related to a decreased activity of serotonin.     

Rat hypothalamic noradrenaline levels following midbrain raphé nuclei lesions

• 1.1. Electrolytic lesions of the median and dorsal raphé nuclei resulted in statistically significant reductions in rat hypothalamic noradrenaline which were observed 1 or 2 days after lesioning, while no changes were observed 7 or 14 days after lesioning.
• 2.2. The short term (1–2 days) raphé nuclei lesions produced no changes in hypothalamic 5-hydroxytryptamine or a small reduction in 5-hydroxyindole acetic acid while the expected marked reductions were observed after 7 days.
• 3.3. The reduction in hypothalamic noradrenaline observed after short term raphé nuclei lesions suggests the existence of a positive feedback loop between 5-hydroxytryptamine neurons and noradrenaline terminals in the hypothalamus.

     

Preliminary observations on the distribution of serotoninergic and cerebellar-projecting neurons of the raphe nuclei of the brain stem

Cerebellar projection from raphe nuclei were investigated in rabbit by using retrograde transport of HRP and serotonergic mapping by direct fluorescence. A close topographical correlation between the HRP labeled cells and the serotonergic neurons has been observed. The current study has demonstrated the presence of paramedian and lateral cells whose cytoarchitecture is identical with midline cells of many raphe nuclei. All of the raphe nuclei except the linear nuclei, contained serotonergic perikarya. The midline and paramedian portions of the nuclei raphe obscurus, pallidus, magnus, and nucleus raphe dorsalis contained principally serotonergic neurons; the lateral portions of the medullary raphe nuclei and the nuclei raphe pontis and centralis superior contained a significant number of non-fluorescent cells. In these regions, fluorescent sections often revealed the size, shape, and orientation of the perikarya and dendrites; further verification of cytoarchitectural characteristics of these neurons depended heavily upon these clues.     

Molecular cloning and functional identification of mouse vesicular glutamate transporter 3 and its expression in subsets of novel excitatory neurons

We have cloned and functionally characterized a third isoform of a vesicular glutamate transporter (VGLUT3) expressed on synaptic vesicles that identifies a distinct glutamatergic system in the brain that is partly and selectively promiscuous with cholinergic and serotoninergic transmission. Transport activity was specific for glutamate, was H(+)-dependent, was stimulated by Cl(-) ion, and was inhibited by Rose Bengal and trypan blue. Northern analysis revealed higher mRNA levels in early postnatal development than in adult brain. Restricted patterns of mRNA expression were observed in presumed interneurons in cortex and hippocampus, and projection systems were observed in the lateral and ventrolateral hypothalamic nuclei, limbic system, and brainstem. Double in situ hybridization histochemistry for vesicular acetylcholine transporter identified VGLUT3 neurons in the striatum as cholinergic interneurons, whereas VGLUT3 mRNA and protein were absent from all other cholinergic cell groups. In the brainstem VGLUT3 mRNA was concentrated in mesopontine raphé nuclei. VGLUT3 immunoreactivity was present throughout the brain in a diffuse system of thick and thin beaded varicose fibers much less abundant than, and strictly separated from, VGLUT1 or VGLUT2 synapses. Co-existence of VGLUT3 in VMAT2-positive and tyrosine hydroxylase -negative varicosities only in the cerebral cortex and hippocampus and in subsets of tryptophan hydroxylase-positive cell bodies and processes in differentiating primary raphé neurons in vitro indicates selective and target-specific expression of the glutamatergic/serotoninergic synaptic phenotype.     

Neuronal-vascular relationships in the raphe nuclei, locus coeruleus, and substantia nigra in primates.

A fluorescence histochemical and electron microscopic study of the monoaminergic cell groups in the squirrel monkey and Rhesus monkey brains has revealed the direct apposition of blood vessels to perikarya and dendrites of monoaminergic neurons. Capillaries and small arterioles or venules, ranging from 8-50 microns in diameter, showed perikarya and dendrites abutting the basement membrane without evidence of glial interposition. This neuronal-vascular relationship was present in 20% to 30% of the small vessels in the serotonergic nuclei raphe dorsalis and centralis superior and in the noradrenergic locus coeruleus. Such contacts were clearly present but observed less frequently in the dopaminergic substantia nigra pars compacta and in the serotonergic nuclei raphe obscurus, pallidus, magnus, and pontis. We postulate that monoamine-containing neurons apposed to blood vessels in certain regions of the brain may be influenced directly by hormones or other substances in blood.     

Brief fasting decreases protein synthesis in the brain of adult rats

The influence of starvation on protein synthesis in the adult rat brain was studied in vivo by an intravenous injection of a flooding dose of unlabeled valine including a tracer dose ofL-[3,4(n)-³H]valine. Brief starvation (24 hours) induced a 20% decline in fractional and absolute rates of brain protein synthesis. This decline resulted from a 20% decrease in the efficiency of protein synthesis (g protein synthesized per day per g RNA) whereas the capacity for protein synthesis (g RNA per mg protein) was maintained. Prolonged starvation (5 days) was marked by no further significant changes in the fractional rate, absolute rate and efficiency of protein synthesis, whereas the capacity for protein synthesis cecreased slightly. The relative contribution of brain to wholebody body protein synthesis increased during fasting, and neither the protein nor the RNA brain content did change during the experiment. These results clearly indicate that brain proteins are spared in response to brief and prolonged food deprivation, and that brain protein synthesis is very sensitive to short-term fasting.     

Effect of fixation in Carnoy's solution on nucleic acid and protein concentration in the rabbit superior cervical sympathetic ganglion]

By means of biochemical techniques, the stability of RNA, DNA and protein contents has been demonstrated in the rabbit superior cervical sympathetic node fixed in Carnoy's solution for 2 hours at 4 degrees C. A 2 hour fixation in Carnoy's fluid at room temperature, results, however, in a considerable loss of RNA (21%). The loss of DNA and protein did not exceed 2% of the total amount of the fresh ganglion tissue. Fixation at a higher temperature (37%) increased the loss percentage protein and of nucleic acids: 3.1, 5.5 and 42%, for protein, DNA and RNA, resp.     

Distribution of reticulospinal neurons in the chicken by retrograde transport of WGA-HRP

To determine the distribution of reticulospinal (RS) neurons in the chicken, WGA-HRP was injected into the cervical or lumbosacral enlargement either unilaterally or bilaterally. The brainstem reticular nuclei sent largely descending fibers to both the spinal enlargements. The mesencephalon (medial and lateral mesencephalic reticular formation) and the rostral pons (nucleus reticularis [n.r.] pontis oralis) project mainly to the cervical enlargement. RS neurons were mainly distributed from the pontomedullary junction to the rostral medulla including n. r. pontis caudalis and pars gigantocellularis, n. r. gigantocellularis, n. r. parvocellularis, n. r. paragigantocellularis, and n. r. subtrigeminalis. It is suggested that the majority of these neurons send axons at least as far as the lumbosacral enlargement. In the lower medulla, RS neurons were distributed in the dorsal and ventral parts of the central nucleus of the medulla.     

Distribution of serotonin immunoreactive neurons in the brainstem of the hamster, guinea pig, rabbit, and rat

Immunohistochemical techniques were employed to study the distribution of serotonin (5-HT) immunoreactive neurons in the brainstem of the hamster, guinea pig, rabbit and rat. 5-HT neurons were principally found to be concentrated in the midline raphe nuclei, particularly, the raphe pallidus, raphe obscurus, raphe magnus, raphe median, raphe pontis and raphe dorsalis nuclei. Characteristically, these cell bodies are displayed in bands or wing-like patterns which extend laterally from the raphe into reticular formations. The formations often appear to blend with the catecholamine system. They are particularly evident in the brainstems of the rabbit and hamster which contain wider and more lateral extensions of the serotonergic (5-HT) neurons than those observed in the brainstems of the rat and guinea pig. The widespread distribution of 5-HT immunoreacted cell bodies in the brainstem shows that there are significant prospects of 5-HT in neuronal activities.     

Total protein content and concentration in the neurons of the "mirror" epileptiform focus in the rat brain at the late stages of its existence]

The experimental lesion in rat brain was produced by the implantation of cobaltogelatin rod into the right parietalis anterior area. A quantitative measurement of total protein was made by microdensitometer in neurons controlateral to cobalt lesion at 112-115 days after cobalt implantation. In the experimental animals, protein content decreased in neurons of lamina III and V area parietalis anterior and the nucleus lateralis thalami by 6, 60 and 53%, resp. It has been concluded that different type neurons have different protein changes in response to paroxyzmal activity at late stages of epileptogenic mirror focus.     

Hormonal induction of glutamine synthetase in cultures of embryonic retina cells: requirement for neuron-glia contact interactions

Cortisol induces glutamine synthetase (GS) in gliocytes of chick embryo neural retina. Using adherent cultures of retina cells we have demonstrated that responsiveness of the gliocytes to GS induction by the hormone requires contact with neurons. GS is not inducible in high-density cultures depleted of neurons and consisting only of gliocytes. In neuron-containing cultures, induced GS was detected immunohistochemically only in those gliocytes that were closely juxtaposed with clusters of neurons. Unlike the induction of GS, the expression of carbonic anhydrase-C (which does not require cortisol) persisted in these glia cells also in the absence of neurons. The nature and role of glia-neuron interactions in the hormonal induction of GS are briefly discussed.