Catecholamine and serotonin colocalization in projection neurons of the area postrema

doi:10.1016/0006-8993(87)91147-4

Brain Research

Volume 412, Issue 2, 2 June 1987, Pages 381-385

https://doi.org/10.1016/0006-8993(87)91147-4 Get rights and content

Abstract

A large population of the rat area postrema (AP) neurons which project to the parabrachial area (PBA) are serotonergic. Many AP neurons contain tyrosine hydroxylase (TH) and are presumed to be noradrenergic. However, it was not previously known whether TH-containing AP neurons also have projections to the PBA. TH-containing and serotonin (5-HT)-containing neurons have wide and overlapping distributions within the AP, and it was therefore possible that TH and 5-HT may be contained within the same AP neurons. In the present study immunohistochemical and retrograde axonal transport techniques were combined to determine whether TH-containing AP neurons project to the PBA and whether TH and 5-HT coexist in AP neurons. Adult male rats were given bilateral injections of the retrograde transport tracer, True blue (TB), into the PBA. After a 4-day survival period, vibratome sections of the caudal brainstem were processed for both TH and 5-HT immunohistochemistry. Examination of the sections revealed that over 25% of the 5-HT and over 30% of the TH-containing AP neurons were retrogradely labelled with TB. More surprising were our findings that many AP neurons displayed both TH and 5-HT immunoreactivity and that almost 40% of these double-labelled cells project to the PBA. Our results indicate that serotonin and noradrenaline coexist in a substantial proportion of the neurons of a major ascending viscerosensory pathway from the AP to the PBA.

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Furthermore, a large number of projections from the AP and NTS to the LPB are noradrenergic [67] or serotoninergic [68]. Interestingly, about 20% of AP noradrenergic neurons contain also 5-HT, and about 40% of these show projections to the parabrachial nucleus [69]. Our recent immunohistochemical studies indicate that neurons of these phenotypes are specifically activated by amylin.
Amylin is secreted by pancreatic beta-cells and is believed to be a physiological signal of satiation. Amylin's effect on eating has been shown to be mediated via a direct action at the area postrema (AP) via amylin receptors that are heterodimers of the calcitonin receptor core protein with a receptor activity modifying protein. Peripheral amylin leads to accumulation of cyclic guanosine monophosphate, phosphorylated extracellular-signal regulated kinase 1/2 and c-Fos protein in AP neurons. The particular amylin-activated AP neurons mediating its anorexigenic action seem to be noradrenergic. The central pathways mediating amylin's effects have been characterized by lesioning and tracing studies, identifying important connections from the AP to the nucleus of the solitary tract and lateral parabrachial nucleus. Amylin was shown to interact, probably at the brainstem, with other signals involved in the short term control of food intake, namely cholecystokinin, glucagon-like peptide 1 and peptide YY. Amylin also interacts with the adiposity signal leptin; this interaction, which is thought to involve the hypothalamus, may have important implications for the development of new and improved hormonal obesity treatments.
In conclusion, amylin actions on food intake seem to reside primarily within the brainstem, and the associated mechanisms are starting to be unraveled.
The paper represents an invited review by a symposium, award winner or keynote speaker at the Society for the Study of Ingestive Behavior [SSIB] Annual Meeting in Portland, July 2009.
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Aldosterone-sensitive neurons in the nucleus tractus solitarius (NTS) become activated during sodium depletion and could be key neural elements regulating sodium intake. The afferent inputs to these neurons have not yet been defined, but one source may be neurons in the area postrema, a neighboring circumventricular organ that innervates the NTS and exerts a powerful inhibitory influence on sodium appetite [Contreras RJ, Stetson PW (1981) Changes in salt intake after lesions of the area postrema and the nucleus of the solitary tract in rats. Brain Res 211:355–366]. After an anterograde axonal tracer was injected into the area postrema in rats, sections through the NTS were immunolabeled for the enzyme 11-β-hydroxysteroid dehydrogenase type 2 (HSD2), a marker for aldosterone-sensitive neurons, and examined by confocal microscopy. We found that some of the aldosterone-sensitive neurons received close appositions from processes originating in the area postrema, suggesting that input to the HSD2 neurons could be involved in the inhibition of sodium appetite by this site. Axonal varicosities originating from the area postrema also made close appositions with other neurons in the medial NTS, including the neurotensin-immunoreactive neurons in the dorsomedial NTS. Besides these projections, a dense field of neurotensinergic axon terminals overlapped the distribution of the HSD2 neurons. Neurotensin-immunoreactive axon terminals were identified in close apposition to the dendrites and cell bodies of some HSD2 neurons, as well as unlabeled neurons lying in the same zone within the medial NTS. A local microcircuit involving the area postrema, HSD2 neurons, and neurotensinergic neurons may play a major role in the regulation of sodium appetite.
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Therefore, the enhancement of 1.8% NaCl intake during FURO+CAP treatment did not result from further unloading of volume receptors. The LPBN receives many fibers from AP/mNTS (Norgren, 1981; Shapiro and Miselis, 1985) and serotonergic, adrenergic and noradrenergic neurons are present in the projection from AP/NTS to the LPBN (Lança and van der Kooy, 1985; Miceli et al., 1987; Herbert and Flugge, 1995; Talley et al., 1996). It is possible that injections of moxonidine into the LPBN, perhaps interacting with 5-HT or CCK, acts by removing inhibitory signals from cardiovascular receptors running through the AP/mNTS, thus reinforcing the effects of mild hypotension on the circuits subserving sodium intake as already suggested for the blockade of the LPBN serotonergic and CCKergic mechanisms (Menani et al., 1996, 2000; Menani and Johnson, 1998).
Water and NaCl intake is strongly inhibited by the activation of α₂-adrenergic receptors with clonidine or moxonidine (α₂-adrenergic/imidazoline agonists) injected peripherally or into the forebrain and by serotonin and cholecystokinin in the lateral parabrachial nucleus (LPBN). Considering that α₂-adrenergic receptors exist in the LPBN and the similar origin of serotonergic and adrenergic afferent pathways to the LPBN, in this study we investigated the effects of bilateral injections of moxonidine alone or combined with RX 821002 (α₂-adrenergic antagonist) into the LPBN on 1.8% NaCl and water intake induced by the treatment with s.c. furosemide (10 mg/kg)+captopril (5 mg/kg). Additionally, we investigated if moxonidine into the LPBN would modify furosemide+captopril-induced c-fos expression in the forebrain. Male Holtzman rats with cannulas implanted bilaterally in the LPBN were used. Contrary to forebrain injections, bilateral LPBN injections of moxonidine (0.1, 0.5 and 1 nmol/0.2 μl) strongly increased furosemide+captopril-induced 1.8% NaCl intake (16.6±2.7, 44.5±3.2 and 44.5±4.3 ml/2 h, respectively, vs. vehicle: 6.9±1.5 ml/2 h). Only the high dose of moxonidine increased water intake (23.3±3.8 ml/2 h, vs. vehicle: 12.1±2.6 ml/2 h). Prior injections of RX 821002 (10 and 20 nmol/0.2 μl) abolished the effect of moxonidine (0.5 nmol) on 1.8% NaCl intake. Moxonidine into the LPBN did not modify furosemide+captopril-induced c-fos expression in forebrain areas related to the control of fluid-electrolyte balance. The results show that the activation of LPBN α₂-adrenergic receptors enhances furosemide+captopril-induced 1.8% NaCl and water intake. This enhancement was not related to prior alteration in the activity of forebrain areas as suggested by c-fos expression. Previous and present results indicate opposite roles for α₂-adrenergic receptors in the control of sodium and water intake according to their distribution in the rat brain.
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The observation that Fos expression in these nuclei, except the AP, was abolished or strongly diminished by vagotomy supports this view. Moreover, the significant Fos expression in the AP, but not the NTS, induced by secretin after vagotomy suggests that NTS activation by secretin is not mediated through the known AP projections to the NTS (Cunningham et al., 1994; Miceli et al., 1987; Shapiro and Miselis, 1985; van der Kooy and Koda, 1983). The high level of interconnectivity between the PBel, LC and CeA has been ascribed to augment the integrative processing of visceral sensory inputs and the relay of information in several autonomic regulatory mechanisms that elicit visceral reflex (Curtis et al., 2002).
I.v. injection of secretin activates neurons in brain areas controlling autonomic function and emotion. Peripheral administration of secretin inhibits gastric functions through a central mechanism that is mediated by vagal dependent pathways. We investigated whether the vagus nerve is involved in i.p. injection of secretin-induced brain neuronal activation in conscious rats as monitored by Fos immunohistochemistry. Secretin (40 or 100 μg/kg, i.p., 90 min) induced a dose-related increase in the number of Fos positive neurons in the central nucleus of the amygdala (CeA), and a plateau Fos response in the area postrema (AP), nucleus tractus solitarii (NTS), locus coeruleus (LC), Barrington's nucleus (Bar), external lateral subnucleus of parabrachial nucleus (PBel) and arcuate nucleus, and at 100 μg/kg, in the dorsal motor nucleus of the vagus (DMV) compared with i.p. injection of vehicle. Double immunohistochemistry showed that secretin (40 μg/kg, i.p.) activates tyrosine hydroxylase neurons in the NTS. Subdiaphragmatic vagotomy (7 days) abolished Fos expression-induced by i.p. secretin (40 μg/kg) in the NTS, DMV, LC, Bar, PBel and CeA, while a significant rise in the AP was maintained. In contrast, s.c. capsaicin (10 days) did not influence the Fos induction in the above nuclei. Reverse transcription polymerase chain reaction (RT-PCR) and quantitative real-time PCR showed that secretin receptor mRNA is expressed in the nodose ganglia and levels were higher in the right compared with the left ganglion. These results indicate that peripheral secretin activates catecholaminergic NTS neurons as well as neurons in medullary, pontine and limbic nuclei regulating autonomic functions and emotion through vagal-dependent capsaicin-resistant pathways. Secretin injected i.p. may signal to the brain by interacting with secretin receptors on vagal afferent as well as on AP neurons outside the blood–brain barrier.
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The parabrachial nucleus (PBN) surrounds the brachium conjunctivum in the dorsolateral pons. Although composed of numerous subnuclei, the PBN is typically organized into medial and lateral subdivisions according to their location relative to the brachium. In rodents, the medial PBN is part of the central gustatory system, whereas the lateral PBN is a component of the visceral sensory system. Lesions of the PBN disrupt conditioned taste aversion, a critically important learning mechanism that prevents the repeated ingestion of toxic food. Relevant neurobehavioral literature is reviewed to elucidate the role of the PBN in taste aversion learning.
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Serotonin modulates a variety of neural processes, and is present in a subpopulation of neurons in the raphe nuclei. To study their electrophysiological properties, cells from the mesopontine raphe nuclei of the neonatal rat were dissociated and grown for up to 10 weeks in microcultures. Approximately one third of the neurons were identified as serotonergic based on the presence of serotonin immunoreactivity, tryptophan hydroxylase immunoreactivity, or a high affinity monoamine transporter. About 5% of cultured raphe neurons contained tyrosine hydroxylase immunoreactivity, while 25% contained GABA immunoreactivity. However, no neurons contained both serotonin and tyrosine hydroxylase staining, and less than 1% displayed both serotonin and GABA immunoreactivities. Cultured serotonergic neurons did not exhibit pacemaker firing in the presence ofα₁ adrenergic receptor agonists such as phenylephrine or norepinephrine. Approximately one third were hyperpolarized by serotonin or the selective serotonin_1A receptor agonist,(±)-8-hydroxy-2-(di-N-propylamino)tetralin. Virtually all serotonergic neurons responded to application of glutamate, kainate, N-methyl-d-aspartate, GABA, and glycine. Depolarizing and hyperpolarizing synaptic potentials blocked by glutamate or GABA_A receptor antagonists were frequently observed in both serotonergic and non-serotonergic raphe neurons. Slow inhibitory postsynaptic potentials were evoked by activating single presynaptic serotonergic neurons with a brief intracellular current pulse. The slow inhibitory synaptic potential had a mean latency to onset of35 ± 5 ms, a duration of 0.8–2.6 s, and was inhibited by the serotonin_1A autoreceptor antagonists, (−)propranolol and spiperone. The rising and falling phases of the inhibitory potential could be fit by single exponential functions with mean time constants of53 ± 8ms and504 ± 78ms, respectively. Serotonin_1A receptor-mediated autoinhibition was observed in microcultures containing a solitary serotonergic neuron, and thus constituted synaptic serotonin release, responsiveness, and re-uptake by a single vertebrate neuron.
In summary, histochemical and electrophysiological evidence was obtained for catecholaminergic, GABAergic, and glutamatergic non-serotonergic raphe neurons in culture, many of which formed functional synaptic connections with neighboring cells. Additionally, cultured mesopontine serotonergic neurons expressed many of the cytochemical markers, neurotransmitter receptors, and synaptic functions observed in such cells in vivo, but the proportion of neurons sensitive to serotonergic and adrenergic agonists was significantly less than that reported in vivo. For the first time, the kinetics and pharmacology of serotonergic synaptic transmission by a single vertebrate serotonergic raphe neuron were determined, and were found to resemble those observed after extracellular stimulation of populations of raphe neurons in slices and in vivo.

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Catecholamine and serotonin colocalization in projection neurons of the area postrema

Abstract

Life Sci.

Brain Research

Neuroscience

Neuroscience

Brain Research

Immunocytochemical localization of catecholamine synthesizing enzymes and neuropeptides in area postrema and medial nucleus tractus solitarius

J. Comp. Neurol.

Effects of intrathecally administered methyseride and yohimbine on microstimulation-produced antinociception in the rat

Brain Research

Neuronal and vascular ultrastructure of the AP in the rat

J. Comp. Neurol.

The use of glyoxylic acid for the fluorescence histochemical demonstration of peripheral stores of noradrenaline and 5-hysdroxytryptamine in whole mounts

Histochemistry

Cellular localization of monoamines in the area postrema of certain mammals

J. Comp. Neurol.

Central projections of gustatory nerves in the rat

J. Comp. Neurol.