Brainstem ADCYAP1+ neurons control multiple aspects of sickness behaviour.

Infections induce a set of pleiotropic responses in animals, including anorexia, adipsia, lethargy and changes in temperature, collectively termed sickness behaviours1. Although these responses have been shown to be adaptive, the underlying neural mechanisms have not been elucidated2-4. Here we use of a set of unbiased methodologies to show that a specific subpopulation of neurons in the brainstem can control the diverse responses to a bacterial endotoxin (lipopolysaccharide (LPS)) that potently induces sickness behaviour. Whole-brain activity mapping revealed that subsets of neurons in the nucleus of the solitary tract (NTS) and the area postrema (AP) acutely express FOS after LPS treatment, and we found that subsequent reactivation of these specific neurons in FOS2A-iCreERT2 (also known as TRAP2) mice replicates the behavioural and thermal component of sickness. In addition, inhibition of LPS-activated neurons diminished all of the behavioural responses to LPS. Single-nucleus RNA sequencing of the NTS-AP was used to identify LPS-activated neural populations, and we found that activation of ADCYAP1+ neurons in the NTS-AP fully recapitulates the responses elicited by LPS. Furthermore, inhibition of these neurons significantly diminished the anorexia, adipsia and locomotor cessation seen after LPS injection. Together these studies map the pleiotropic effects of LPS to a neural population that is both necessary and sufficient for canonical elements of the sickness response, thus establishing a critical link between the brain and the response to infection.

GDF15 mediates adiposity resistance through actions on GFRAL neurons in the hindbrain AP/NTS.

BACKGROUND: Elevated circulating levels of the divergent transforming growth factor-beta (TGFb) family cytokine, growth differentiation factor 15 (GDF15), acting through its CNS receptor, glial-derived neurotrophic factor receptor alpha-like (GFRAL), can cause anorexia and weight loss leading to anorexia/cachexia syndrome of cancer and other diseases. Preclinical studies suggest that administration of drugs based on recombinant GDF15 might be used to treat severe obesity. However, the role of the GDF15-GFRAL pathway in the physiological regulation of body weight and metabolism is unclear. The critical site of action of GFRAL in the CNS has also not been proven beyond doubt. To investigate these two aspects, we have inhibited the actions of GDF15 in mice started on high-fat diet (HFD). METHODS: The actions of GDF15 were inhibited using two methods: (1) Groups of 8 mice under HFD had their endogenous GDF15 neutralised by monoclonal antibody treatment, (2) Groups of 15 mice received AAV-shRNA to knockdown GFRAL at its hypothesised major sites of action, the hindbrain area postrema (AP) and the nucleus of the solitary tract (NTS). Metabolic measurements were determined during both experiments. CONCLUSIONS: Treating mice with monoclonal antibody to GDF15 shortly after commencing HFD results in more rapid gain of body weight, adiposity and hepatic lipid deposition than the control groups. This is accompanied by reduced glucose and insulin tolerance and greater expression of pro-inflammatory cytokines in adipose tissue. Localised AP and NTS shRNA-GFRAL knockdown in mice commencing HFD similarly caused an increase in body weight and adiposity. This effect was in proportion to the effectiveness of GFRAL knockdown, indicated by quantitative analysis of hindbrain GFRAL staining. We conclude that the GDF15-GFRAL axis plays an important role in resistance to obesity in HFD-fed mice and that the major site of action of GDF15 in the CNS is GFRAL-expressing neurons in the AP and NTS.

Morphological, histological and immunohistochemical study of the area postrema in the dog.

Circumventricular organs are specialized brain structures that are located mainly at the midsagittal line, around the third and fourth ventricles, often protruding into the lumen. They are positioned at the interface between the neuroparenchyma and the ventricular system of the brain. These highly vascularized nervous tissue structures differ from the brain parenchyma, as they lack a blood-brain barrier. Circumventricular organs have specialized sensory and secretory functions. It is essential for any pathologist who evaluates brain sections to have a solid knowledge of microscopic neuroanatomy and to recognize these numerous specialized structures within the nervous system as normal and not mistake them for pathological changes. The purpose of this study was to provide, for the first time, a detailed and complete histological description of the healthy canine area postrema and to determine its resemblance to that of other mammalian species. Anatomical dissections with routine histological and immunohistochemical techniques were carried out on ten canine brains. The cellular composition of area postrema proved to be largely comparable to that of other mammal species.

Eff ect of a single asenapine treatment on Fos expression in the brain catecholamine-synthesizing neurons: impact of a chronic mild stress preconditioning.

OBJECTIVE: Fos protein expression in catecholamine-synthesizing neurons of the substantia nigra (SN) pars compacta (SNC, A8), pars reticulata (SNR, A9), and pars lateralis (SNL), the ventral tegmental area (VTA, A10), the locus coeruleus (LC, A6) and subcoeruleus (sLC), the ventrolateral pons (PON-A5), the nucleus of the solitary tract (NTS-A2), the area postrema (AP), and the ventrolateral medulla (VLM-A1) was quantitatively evaluated aft er a single administration of asenapine (ASE) (designated for schizophrenia treatment) in male Wistar rats preconditioned with a chronic unpredictable variable mild stress (CMS) for 21 days. Th e aim of the present study was to reveal whether a single ASE treatment may 1) activate Fos expression in the brain areas selected; 2) activate tyrosine hydroxylase (TH)-synthesizing cells displaying Fos presence; and 3) be modulated by CMS preconditioning. METHODS: Control (CON), ASE, CMS, and CMS+ASE groups were used. CMS included restraint, social isolation, crowding, swimming, and cold. Th e ASE and CMS+ASE groups received a single dose of ASE (0.3 mg/kg, s.c.) and CON and CMS saline (300 µl/rat, s.c.). The animals were sacrificed 90 min aft er the treatments. Fos protein and TH-labeled immunoreactive perikarya were analyzed on double labeled histological sections and enumerated on captured pictures using combined light and fluorescence microscope illumination. RESULTS: Saline or CMS alone did not promote Fos expression in any of the structures investigated. ASE alone or in combination with CMS elicited Fos expression in two parts of the SN (SNC, SNR) and the VTA. Aside from some cells in the central gray tegmental nuclei adjacent to LC, where a small number of Fos profiles occurred, none or negligible Fos occurrence was detected in the other structures investigated including the LC and sLC, PON-A5, NTS-A2, AP, and VLM-A1. CMS preconditioning did not infl uence the level of Fos induction in the SN and VTA elicited by ASE administration. Similarly, the ratio between the amount of free Fos and Fos colocalized with TH was not aff ected by stress preconditioning in the SNC, SNR, and the VTA. CONCLUSIONS: Th e present study provides an anatomical/functional knowledge about the nature of the acute ASE treatment on the catecholamine-synthesizing neurons activity in certain brain structures and their missing interplay with the CMS preconditioning.

Leptin influences the excitability of area postrema neurons.

The area postrema (AP) is a circumventricular organ with important roles in central autonomic regulation. This medullary structure has been shown to express the leptin receptor and has been suggested to have a role in modulating peripheral signals, indicating energy status. Using RT-PCR, we have confirmed the presence of mRNA for the leptin receptor, ObRb, in AP, and whole cell current-clamp recordings from dissociated AP neurons demonstrated that leptin influenced the excitability of 51% (42/82) of AP neurons. The majority of responsive neurons (62%) exhibited a depolarization (5.3 ± 0.7 mV), while the remaining affected cells (16/42) demonstrated hyperpolarizing effects (-5.96 ± 0.95 mV). Amylin was found to influence the same population of AP neurons. To elucidate the mechanism(s) of leptin and amylin actions in the AP, we used fluorescence resonance energy transfer (FRET) to determine the effect of these peptides on cAMP levels in single AP neurons. Leptin and amylin were found to elevate cAMP levels in the same dissociated AP neurons (leptin: % total FRET response 25.3 ± 4.9, n = 14; amylin: % total FRET response 21.7 ± 3.1, n = 13). When leptin and amylin were coapplied, % total FRET response rose to 53.0 ± 8.3 (n = 6). The demonstration that leptin and amylin influence a subpopulation of AP neurons and that these two signaling molecules have additive effects on single AP neurons to increase cAMP, supports a role for the AP as a central nervous system location at which these circulating signals may act through common intracellular signaling pathways to influence central control of energy balance.

Amylin receptor components and the leptin receptor are co-expressed in single rat area postrema neurons.

Amylin is a pancreatic ß-cell hormone that acts as a satiating signal to inhibit food intake by binding to amylin receptors (AMYs) and activating a specific neuronal population in the area postrema (AP). AMYs are heterodimers that include a calcitonin receptor (CTR) subunit [CTR isoform a or b (CTRa or CTRb)] and a member of the receptor activity-modifying proteins (RAMPs). Here, we used single-cell quantitative polymerase chain reaction to assess co-expression of AMY subunits in AP neurons from rats that were injected with amylin or vehicle. Because amylin interacts synergistically with the adipokine leptin to reduce body weight, we also assessed the co-expression of AMY and the leptin receptor isoform b (LepRb) in amylin-activated AP neurons. Single cells were collected from Wistar rats and from transgenic Fos-GFP rats that express green fluorescent protein (GFP) under the control of the Fos promoter. We found that the mRNAs of CTRa, RAMP1, RAMP2 and RAMP3 were all co-expressed in single AP neurons. Moreover, most of the CTRa+ cells co-expressed more than one of the RAMPs. Amylin down-regulated RAMP1 and RAMP3 but not CTR mRNAs in AMY+ neurons, suggesting a possible negative feedback mechanism of amylin at its own primary receptors. Interestingly, amylin up-regulated RAMP2 mRNA. We also found that a high percentage of single cells that co-expressed all components of a functional AMY expressed LepRb mRNA. Thus, single AP cells expressed both AMY and LepRb, which formed a population of first-order neurons that presumably can be directly activated by amylin and, at least in part, also by leptin.

Presynaptically mediated effects of cholecystokinin-8 on the excitability of area postrema neurons in rat brain slices.

Cholecystokinin (CCK) is a well-known gut hormone that shows anorexigenic effects via action at peripheral and central receptors. CCK is also widely distributed throughout the mammalian brain and appears to function as a neurotransmitter and neuromodulator. The area postrema is one of the circumventricular organs, located on the dorsal surface of the medulla oblongata at the caudal end of the fourth ventricle. Blood vessels in the area postrema lack a blood brain barrier, offering specific central neural elements unique access to circulating substances. Immunohistochemical studies show CCK-A receptors in the area postrema, and we reported CCK-sensitive area postrema neurons. However, the receptive mechanism of CCK in area postrema neurons still remains unexplained. We investigated the responses of area postrema neurons to agonists and antagonists of CCK receptors using whole cell and perforated patch-clamp recordings in rat brain slices. The application of CCK-8 elicited excitatory responses, such as increases in the frequency of mEPSCs (miniature excitatory postsynaptic currents), a shift toward larger amplitude mEPSCs, and increases in the frequency of action potentials. These changes were found mostly in cells not displaying the hyperpolarization-activated cation current (Ih), except for small excitatory changes in a minority of Ih-positive neurons. Tonic inward currents or an inhibitory response to CCK-8 were never seen. Analysis of the amplitude of mEPSCs before and after the administration of CCK-8 indicated the responses mediated via the presynaptic receptors. The effect of CCK-8 was abolished in the presence of CNQX (AMPA type glutamate receptor antagonist). In the presence of lorglumide (a selective CCK-A receptor antagonist), CCK-8-induced excitatory responses were inhibited. No cells responded to the administration of non-sulfated CCK-8 (CCK-8NS, a selective CCK-B receptor agonist). We conclude that CCK-8 exerts its action via presynaptic CCK-A receptors to facilitate glutamate release onto Ih-negative area postrema cells.

Changes in pericytic expression of NG2 and PDGFRB and vascular permeability in the sensory circumventricular organs of adult mouse by osmotic stimulation.

The blood-brain barrier (BBB) is a barrier that prevents free access of blood-derived substances to the brain through the tight junctions and maintains a specialized brain environment. Circumventricular organs (CVOs) lack the typical BBB. The fenestrated vasculature of the sensory CVOs, including the organum vasculosum of the lamina terminalis (OVLT), subfornical organ (SFO) and area postrema (AP), allows parenchyma cells to sense a variety of blood-derived information, including osmotic ones. In the present study, we utilized immunohistochemistry to examine changes in the expression of NG2 and platelet-derived growth factor receptor beta (PDGFRB) in the OVLT, SFO and AP of adult mice during chronic osmotic stimulation. The expression of NG2 and PDGFRB was remarkably prominent in pericytes, although these angiogenesis-associated proteins are highly expressed at pericytes of developing immature vasculature. The chronic salt loading prominently increased the expression of NG2 in the OVLT and SFO and that of PDGFRB in the OVLT, SFO and AP. The vascular permeability of low-molecular-mass tracer fluorescein isothiocyanate was increased significantly by chronic salt loading in the OVLT and SFO but not AP. In conclusion, the present study demonstrates changes in pericyte expression of NG2 and PDGFRB and vascular permeability in the sensory CVOs by chronic osmotic stimulation, indicating active participation of the vascular system in osmotic homeostasis.

Tranexamic acid induces kaolin intake stimulating a pathway involving tachykinin neurokinin 1 receptors in rats.

Tranexamic acid suppresses post-partum haemorrhage and idiopathic menorrhagia through its anti-fibrinolytic action. Although it is clinically useful, it is associated with high risks of side effects such as emesis. Understanding the mechanisms underlying tranexamic acid-induced emesis is very important to explore appropriate anti-emetic drugs for the prevention and/or suppression of emesis. In this study, we examined the receptors involved in tranexamic acid-induced kaolin intake in rats, which reflects the drug's clinical emetogenic potential in humans. Further, we examined the brain regions activated by administration of tranexamic acid and elucidated pivotal pathways of tranexamic acid-induced kaolin intake. We examined the effects of ondansetron, a 5-hydroxytryptamine 3 receptor antagonist, domperidone, a dopamine 2 receptor antagonist, and aprepitant, a tachykinin neurokinin 1 (NK1) receptor antagonist, on tranexamic acid-induced kaolin intake in rats. Then, we determined the brain regions that showed increased numbers of c-Fos immunoreactive cells. Finally, we examined the effects of an antagonist(s) that reduced tranexamic acid-induced kaolin intake on the increase in c-Fos immunoreactive cells. Aprepitant significantly decreased tranexamic acid-induced kaolin intake. However, neither ondansetron nor domperidone decreased kaolin intake. Tranexamic acid significantly increased c-Fos immunoreactive cells by approximately 5.5-fold and 22-fold in the area postrema and nucleus of solitary tract, respectively. Aprepitant decreased the number of c-Fos immunoreactive cells in both areas. Tranexamic acid induced kaolin intake possibly via stimulation of tachykinin NK1 receptors in rats. The tachykinin NK1 receptor could be targeted to prevent and/or suppress emesis in patients receiving tranexamic acid.

ENaC-expressing neurons in the sensory circumventricular organs become c-Fos activated following systemic sodium changes.

The sensory circumventricular organs (CVOs) are specialized collections of neurons and glia that lie in the midline of the third and fourth ventricles of the brain, lack a blood-brain barrier, and function as chemosensors, sampling both the cerebrospinal fluid and plasma. These structures, which include the organum vasculosum of the lamina terminalis (OVLT), subfornical organ (SFO), and area postrema (AP), are sensitive to changes in sodium concentration but the cellular mechanisms involved remain unknown. Epithelial sodium channel (ENaC)-expressing neurons of the CVOs may be involved in this process. Here we demonstrate with immunohistochemical and in situ hybridization methods that ENaC-expressing neurons are densely concentrated in the sensory CVOs. These neurons become c-Fos activated, a marker for neuronal activity, after various manipulations of peripheral levels of sodium including systemic injections with hypertonic saline, dietary sodium deprivation, and sodium repletion after prolonged sodium deprivation. The increases seen c-Fos activity in the CVOs were correlated with parallel increases in plasma sodium levels. Since ENaCs play a central role in sodium reabsorption in kidney and other epithelia, we present a hypothesis here suggesting that these channels may also serve a related function in the CVOs. ENaCs could be a significant factor in modulating CVO neuronal activity by controlling the magnitude of sodium permeability in neurons. Hence, some of the same circulating hormones controlling ENaC expression in kidney, such as angiotensin II and atrial natriuretic peptide, may coordinate ENaC expression in sensory CVO neurons and could potentially orchestrate sodium appetite, osmoregulation, and vasomotor sympathetic drive.

Amylin and GLP-1 target different populations of area postrema neurons that are both modulated by nutrient stimuli.

The area postrema mediates the hypophagic effect of the pancreatic hormone amylin and is also sensitive to glucagon-like peptide 1 (GLP-1). Protein seems to modulate amylin responsiveness because amylin seems to produce a stronger hypophagic effect and a stronger c-Fos expression when protein is absent from the diet. Accordingly, amylin induces a stronger c-Fos expression in the AP when injected in fasted compared to ad libitum fed rats. Here we tested the hypothesis that diet-derived protein attenuates the amylin dependent suppression of feeding and AP activation using isocaloric diets that differed in their protein content. Moreover, we investigated whether peripheral amino acid injection attenuates amylin-induced c-Fos expression in fasted rats. Since recent evidence suggests that GLP-1 may also reduce eating via the AP we tested whether 24 h fasting also increases neuronal AP responsiveness to GLP-1 similar to the fasting-induced increase in amylin responsiveness. Finally, we used the calcitonin receptor (CTR) as an immunohistochemical marker for amylin-receptive AP neurons to investigate whether amylin's target neurons differ from GLP-1 responsive AP neurons. We also dissociated amylin responsive cells from neurons implicated in other AP-mediated functions such as aversion or blood pressure regulation. For this purpose, we conducted c-Fos/CTR double staining after LiCl or angiotensin II treatment, respectively. Amylin (5 µg/kg s.c.) was more effective to reduce the intake of a 1% vs. an 8% or 18% protein diet and to induce c-Fos expression in the AP in rats receiving 1% vs. 18% protein diet. Increased protein intake was associated with increased blood amino acid levels. Peripheral injection of amino acids (1 g/kg i.p.) attenuated the amylin-induced AP activation in 24 h fasted rats. Similar to amylin, GLP-1 (100 µg/kg i.p.) elicited a significant c-Fos response only in fasted but not in ad libitum fed rats. However, in contrast to a high co-localization of amylin-induced c-Fos and CTR (68%), no c-Fos/CTR co-localization occurred after treatment with GLP-1 or the GLP-1R agonist exendin 4 (2 µg/kg ip). Similarly, LiCl (76 mg/kg ip) or AngII (50 µg/kg sc) led to c-Fos expression only in CTR negative AP neurons. In conclusion, our findings support a protein-dependent modulation of behavioral and neuronal amylin responsiveness under equicaloric feeding conditions. Amino acids might contribute to the inhibitory effect of diet-derived protein to reduce amylin-induced neuronal AP activation. Neuronal AP responsiveness to GLP-1 is also increased in the fasted state suggesting that diet-derived nutrients may also interfere with AP-mediated GLP-1 effects. Nevertheless, the primary target neurons for amylin appear to be distinct from cells targeted by GLP-1 and by stimuli producing aversion (LiCl) or contributing to blood pressure regulation (AngII) via the AP. Since amylin and GLP-1 analogs are targets for the treatment of obesity, the nutrient-dependent modulation of AP responsiveness might entail implications for such therapeutic approaches.

Electrophysiologically identified presynaptic mechanisms underlying amylinergic modulation of area postrema neuronal excitability in rat brain slices.

Amylin, which is co-secreted together with insulin by pancreatic beta cells, is considered to be an important peptide hormone involved in the control of feeding behavior and energy homeostasis. Although the area postrema has been implicated to be a primary target of amylin, there are no studies of the mechanisms by which amylin may alter the excitability of area postrema neurons. To investigate the mechanism for amylinergic modulation of neuronal excitability, we performed perforated patch-clamp recordings from area postrema neurons in rat brainstem slices. Amylin-induced changes in excitatory responses, such as increases in the frequency of mEPSCs (miniature excitatory postsynaptic currents) and changes in the amplitude distribution of mEPSCs, were found in cells not displaying the hyperpolarization-activated cation current (I(h)). Area postrema cells displaying I(h) did not respond to amylin application. Inhibitory responses to amylin were never encountered. Bath application of CNQX (AMPA type glutamate receptor antagonist) abolished the effects of amylin. Depolarization of cells during amylin application was sufficient at 1 µM to cause action potential discharge by responding cells. We conclude that amylin receptors are located mostly on presynaptic glutamatergic terminals connecting to the area postrema neurons not displaying I(h) and amylin concentrations can increase glutamate release enough to cause cell firing. Modulation of amylinergic activity may offer a novel target to influence food intake and obesity.

The viral mimetic polyinosinic:polycytidylic acid (poly I:C) induces cellular responses in primary cultures from rat brain sites with an incomplete blood-brain barrier.

Primary microcultures of the organum vasculosum laminae terminalis (OVLT) and the area postrema (AP), brain sites with an incomplete blood-brain barrier, were established from topographically excised rat pup tissue, with cellular identification by marker protein-specific immunocytochemistry. Employing the ratio calcium imaging technique, we showed for the first time that polyinosinic:polycytidylic acid (poly I:C) can induce calcium signalling in single OVLT and AP cells. Poly I:C stimulation caused fast, transient rises in intracellular calcium in about 5% of neurons and astrocytes and some microglial cells. Frequently, the responses of astrocytes and microglial cells showed a shorter onset-latency compared to neurons. In addition, exposure to poly I:C led to a time dependent release of bioactive tumour necrosis factor (TNF) and interleukin-6 (IL-6) into the supernatants of OVLT and AP cultures. The demonstration of direct cellular responses of OVLT- and AP-intrinsic cells to stimulations with poly I:C is in agreement with the discovered existence of Toll-like receptor 3 (TLR3), the cognate receptor for poly I:C, in the brain.

Oxytocin-immunoreactive innervation of identified neurons in the rat dorsal vagal complex.

BACKGROUND: Oxytocin (OXT) has been implicated in reproduction and social interactions and in the control of digestion and blood pressure. OXT-immunoreactive axons occur in the dorsal vagal complex (DVC; nucleus tractus solitarius, NTS, dorsal motor nucleus of the vagus, DMV, and area postrema, AP), which contains neurons that regulate autonomic homeostasis. The aim of the present work is to provide a systematic investigation of the OXT-immunoreactive innervation of dorsal motor nucleus of the vagus (DMV) neurons involved in the control of gastrointestinal (GI) function. METHODS: We studied DMV neurons identified by (i) prior injection of retrograde tracers in the stomach, ileum, or cervical vagus or (ii) induction of c-fos expression by glucoprivation with 2-deoxyglucose. Another subgroup of DMV neurons was identified electrophysiologically by stimulation of the cervical vagus and then juxtacellularly labeled with biotinamide. We used two- or three-color immunoperoxidase labeling for studies at the light microscopic level. KEY RESULTS: Close appositions from OXT-immunoreactive varicosities were found on the cell bodies, dendrites, and axons of DMV neurons that projected to the GI tract and that responded to 2-deoxyglucose and juxtacellularly labeled DMV neurons. Double staining for OXT and choline acetyltransferase revealed that OXT innervation was heavier in the caudal and lateral DMV than in other regions. OXT-immunoreactive varicosities also closely apposed a small subset of tyrosine hydroxylase-immunoreactive NTS and DMV neurons. CONCLUSIONS & INFERENCES: Our results provide the first anatomical evidence for direct OXT-immunoreactive innervation of GI-related neurons in the DMV.

Chemical phenotypes of P2X2 purinoreceptor immunoreactive cell bodies in the area postrema.

Purines such as adenosine 5'-triphosphate (ATP) act as extracellular messengers through specific purinergic receptors. Three different classes of purinergic receptors have been identified and termed P1, P2X, and P2Y. The purinergic receptor subunit P2X2 is a ligand-gated ion channel that is widely expressed by neurons in the CNS. In the brainstem medulla oblongata, the ionotropic P2X2 receptor (P2X2R) is enriched in the area postrema (AP). Two different antisera to P2X2R were used to determine the chemical nature of P2X2R immunoreactive cell bodies in the rat AP, an area lacking a blood-brain barrier. Subcellularly, P2X2R immunoreactivity was located to the periphery of individual cell bodies. The majority of P2X2R-immunoreactive cells were shown to contain tyrosine hydroxylase (TH) (63.5 ± 7.7%) and dopamine ß-hydroxylase (61.5 ± 5.1%). Phenylethanolamine N-methyltransferase (PNMT)-containing cells were not detected in the AP, supporting a noradrenergic nature of P2X2R cells in the AP. There were no P2X2R-immunoreactive cells in the AP that contained the GABA-synthesizing enzyme glutamic acid decarboxylase 65. Only single vesicular glutamate transporter 2-immunoreactive cell bodies that were not P2X2R-positive were demonstrated in the AP. Some P2X2R-positive cells in the AP were immunoreactive for the neuropeptides substance P and pituitary adenylate cyclase-activating polypeptide, whereas dynorphin-, enkephalin-, or cholecystokinin-positive cells were not P2X2R-immunoreactive. Presence of P2X2R in a majority of noradrenergic cells of the AP implies that ATP may have a regulatory action on neuronal noradrenaline release from the AP, a circumventricular organ with a strategic position enabling interactions between circulating substances and the central nervous system.

Serotonergic inputs to FoxP2 neurons of the pre-locus coeruleus and parabrachial nuclei that project to the ventral tegmental area.

The present study demonstrates that serotonin (5-hydroxytryptamine, 5-HT)-containing axons project to two sets of neurons in the dorsolateral pons that have been implicated in salt appetite regulation. These two neuronal groups are the pre-locus coeruleus (pre-LC) and a region in the parabrachial nucleus termed the external lateral-inner subdivision (PBel-inner). Neurons in both regions constitutively express the transcription factor Forkhead protein2 (FoxP2), and become c-Fos activated after prolonged sodium depletion. They send extensive projections to the midbrain and forebrain, including a strong projection to the ventral tegmental area (VTA)-a reward processing site. The retrograde neuronal tracer cholera toxin ß-subunit (CTb) was injected into the VTA region; this was done to label the cell bodies of the pre-LC and PBel-inner neurons. After 1 week, the rats were killed and their brainstems processed by a triple-color immunofluorescence procedure. The purpose was to determine whether the CTb-labeled pre-LC and PBel-inner neurons, which also had FoxP2 immunoreactive nuclei, received close contacts from 5-HT axons. Neurons with these properties were found in both sites. Since the origin of this 5-HT input was unknown, a second set of experiments was carried out in which CTb was injected into the pre-LC or lateral PB. One week later, the rats were perfused and the brainstems from these animals were analyzed for the presence of neurons that co-contained CTb and tryptophan hydroxylase (synthetic enzyme for 5-HT) immunoreactivity. Co-labeled neurons were found mainly in the area postrema and to a lesser degree, in the dorsal raphe nucleus. We propose that the 5-HT inputs to the pre-LC and PBel-inner may modulate the salt appetite-related functions that influence the reward system.

The serotonergic system in fish.

Neurons using serotonin (5-HT) as neurotransmitter and/or modulator have been identified in the central nervous system in representatives from all vertebrate clades, including jawless, cartilaginous and ray-finned fishes. The aim of this review is to summarize our current knowledge about the anatomical organization of the central serotonergic system in fishes. Furthermore, selected key functions of 5-HT will be described. The main focus will be the adult brain of teleosts, in particular zebrafish, which is increasingly used as a model organism. It is used to answer not only genetic and developmental biology questions, but also issues concerning physiology, behavior and the underlying neuronal networks. The many evolutionary conserved features of zebrafish combined with the ever increasing number of genetic tools and its practical advantages promise great possibilities to increase our understanding of the serotonergic system. Further, comparative studies including several vertebrate species will provide us with interesting insights into the evolution of this important neurotransmitter system.

Area postrema projects to FoxP2 neurons of the pre-locus coeruleus and parabrachial nuclei: brainstem sites implicated in sodium appetite regulation.

The area postrema (AP) is a circumventricular organ located in the dorsal midline of the medulla. It functions as a chemosensor for blood-borne peptides and solutes, and converts this information into neural signals that are transmitted to the nucleus tractus solitarius (NTS) and parabrachial nucleus (PB). One of its NTS targets in the rat is the aldosterone-sensitive neurons which contain the enzyme 11 ß-hydroxysteroid dehydrogenase type 2 (HSD2). The HSD2 neurons are part of a central network involved in sodium appetite regulation, and they innervate numerous brain sites including the pre-locus coeruleus (pre-LC) and PB external lateral-inner (PBel-inner) cell groups of the dorsolateral pons. Both pontine cell groups express the transcription factor FoxP2 and become c-Fos activated following sodium depletion. Because the AP is a component in this network, we wanted to determine whether it also projects to the same sites as the HSD2 neurons. By using a combination of anterograde axonal and retrograde cell body tract-tracing techniques in individual rats, we show that the AP projects to FoxP2 immunoreactive neurons in the pre-LC and PBel-inner. Thus, the AP sends a direct projection to both the first-order medullary (HSD2 neurons of the NTS) and the second-order dorsolateral pontine neurons (pre-LC and PB-el inner neurons). All three sites transmit information related to systemic sodium depletion to forebrain sites and are part of the central neural circuitry that regulates the complex behavior of sodium appetite.

Sensory circumventricular organs in health and disease.

Circumventricular organs (CVOs) are specialized brain structures located around the third and fourth ventricles. They differ from the rest of the brain parenchyma in that they are highly vascularised areas that lack a blood-brain barrier. These neurohaemal organs are classified as "sensory", when they contain neurons that can receive chemical inputs from the bloodstream. This review focuses on the sensory CVOs to describe their unique structure, and their functional roles in the maintenance of body fluid homeostasis and cardiovascular regulation, and in the generation of central acute immune and febrile responses. In doing so, the main neural connections to visceral regulatory centres such as the hypothalamus, the medulla oblongata and the endocrine hypothalamic-pituitary axis, as well as some of the relevant chemical substances involved, are described. The CVOs are vulnerable to circulating pathogens and can be portals for their entry in the brain. This review highlights recent investigations that show that the CVOs and related structures are involved in pathological conditions such as sepsis, stress, trypanosomiasis, autoimmune encephalitis, systemic amyloidosis and prion infections, while detailed information on their role in other neurodegenerative diseases such as Alzheimer's disease or multiple sclerosis is lacking. It is concluded that studies of the CVOs and related structures may help in the early diagnosis and treatment of such disorders.

The human area postrema and other nuclei related to the emetic reflex express cAMP phosphodiesterases 4B and 4D.

Phosphodiesterase 4 (PDE4) inhibitors, i.e. rolipram, are being extensively investigated as therapeutic agents in several diseases. Emesis is one of the most common side effects of PDE4 inhibitors. Given the fact that the area postrema is considered the chemoreceptor trigger zone for vomiting, the present study investigates the regional distribution and cellular localization of the four gene transcripts of the PDE4 subfamily (PDE4A, PDE4B, PDE4C and PDE4D) in human brainstem. In situ hybridization histochemistry was used to locate the mRNA distribution of the four PDE4 subfamilies in the area postrema and related nuclei of human postmortem brainstem. We have found that in the brainstem PDE4B and PDE4D mRNA expression is abundant and distributed not only in neuronal cells, but also in glial cells, and on blood vessels. The hybridization signals for PDE4B and PDE4D mRNAs in the area postrema were stronger than those in any other nuclei in the brainstem. They were also found in vomiting-related nuclei such as the nucleus of the solitary tract and the dorsal vagal motor nucleus. These findings suggest that cAMP signaling modification in the area postrema could mediate the emetic effects of PDE4 inhibitors in human brainstem.