Oxidative burden and mitochondrial dysfunction in a mouse model of Rett syndrome.

Rett syndrome is an X chromosome-linked neurodevelopmental disorder associated with cognitive impairment, motor dysfunction and breathing irregularities causing intermittent hypoxia. Evidence for impaired mitochondrial function is also accumulating. A subunit of complex III is among the potentially dys-regulated genes, the inner mitochondrial membrane is leaking protons, brain ATP levels seem reduced, and Rett patient blood samples confirm increased oxidative damage. We therefore screened for mitochondrial dysfunction and impaired redox balance. In hippocampal slices of a Rett mouse model (Mecp2(-/y)) we detected an increased FAD/NADH baseline-ratio indicating intensified oxidization. Cyanide-induced anoxia caused similar decreases in FAD/NADH ratio and mitochondrial membrane potential in both genotypes, but Mecp2(-/y) mitochondria seemed less polarized. Quantifying cytosolic redox balance with the genetically-encoded optical probe roGFP1 confirmed more oxidized baseline conditions, a more vulnerable redox-balance, and more intense responses of Mecp2(-/y) hippocampus to oxidative challenge and mitochondrial impairment. Trolox treatment improved the redox baseline of Mecp2(-/y) hippocampus and dampened its exaggerated responses to oxidative challenge. Microarray analysis of the hippocampal CA1 subfield did not detect alterations of key mitochondrial enzymes or scavenging systems. Yet, quantitative PCR confirmed a moderate upregulation of superoxide dismutase 1 in Mecp2(-/y) hippocampus, which might be a compensatory response to the increased oxidative burden. Since several receptors and ion-channels are redox-modulated, the mitochondrial and redox changes which already manifest in neonates could contribute to the hyperexcitability and diminished synaptic plasticity in MeCP2 deficiency. Therefore, targeting cellular redox balance might qualify as a potential pharmacotherapeutic approach to improve neuronal network function in Rett syndrome.

Computational modelling of 5-HT receptor-mediated reorganization of the brainstem respiratory network.

Brainstem respiratory neurons express the glycine α(3) receptor (Glyα(3) R), which is a target of modulation by several serotonin (5-HT) receptor agonists. Application of the 5-HT(1A) receptor (5-HT(1A) R) agonist 8-OH-DPAT was shown (i) to depress cellular cAMP, leading to dephosphorylation of Glyα(3) R and augmentation of postsynaptic inhibition of neurons expressing Glyα(3) R (Manzke et al., 2010) and (ii) to hyperpolarize respiratory neurons through 5-HT-activated potassium channels. These processes counteract opioid-induced depression and restore breathing from apnoeas often accompanying pharmacotherapy of pain. The effect is postulated to rely on the enhanced Glyα(3) R-mediated inhibition of inhibitory neurons causing disinhibition of their target neurons. To evaluate this proposal and investigate the neural mechanisms involved, an established computational model of the brainstem respiratory network (Smith et al., 2007), was extended by (i) incorporating distinct subpopulations of inhibitory neurons (glycinergic and GABAergic) and their synaptic interconnections within the Bötzinger and pre-Bötzinger complexes and (ii) assigning the 5-HT(1A) R-Glyα(3) R complex to some of these inhibitory neuron types in the network. The modified model was used to simulate the effects of 8-OH-DPAT on the respiratory pattern and was able to realistically reproduce a number of experimentally observed responses, including the shift in the onset of post-inspiratory activity to inspiration and conversion of the eupnoeic three-phase rhythmic pattern into a two-phase pattern lacking the post-inspiratory phase. The model shows how 5-HT(1A) R activation can produce a disinhibition of inspiratory neurons, leading to the recovery of respiratory rhythm from opioid-induced apnoeas.

The counteraction of opioid-induced ventilatory depression by the serotonin 1A-agonist 8-OH-DPAT does not antagonize antinociception in rats in situ and in vivo.

BACKGROUND: Spontaneous breathing during mechanical ventilation is gaining increasing importance during intensive care but is depressed by narcotics, such as opioids. Serotonin 1A-receptor (5-HT(1A)-R) agonists have been shown to antagonize opioid-induced ventilatory depression, but both enhancement and attenuation of nociceptive reflexes have been found with different experimental models. To clarify contradictory findings, we simultaneously determined dose-response functions of the standard 5-HT(1A)-R-agonist 8-OH-DPAT and two different opioids for spontaneous ventilation and nociception. Two hypotheses were tested: 1) 8-OH-DPAT at a dose to stimulate spontaneous breathing does not activate nociceptive reflexes. 2) 8-OH-DPAT does not diminish opioid-induced antinociception. METHODS: (A) A dose-response relationship of 8-OH-DPAT, spontaneous phrenic nerve activity and a nociceptive C-fiber reflex (CFR) were established simultaneously in an in situ perfused, nonanesthetized, rat brainstem-spinal cord preparation. (B) Fentanyl was administered in situ to investigate the interaction with 8-OH-DPAT on phrenic nerve activity and nociceptive CFR. Additional experiments involved the selective 5-HT(1A)-R-antagonist WAY 100 635 to exclude effects of receptors other than 5-HT(1A)-R. (C) The effects of 8-OH-DPAT on spontaneous ventilation and nociceptive tail-flick reflex with and without morphine were verified in in vivo anesthetized rats. RESULTS: Low-dose 8-OH-DPAT (0.001 and 0.01 microM in situ, 0.1 microg/kg in vivo) enhanced nociceptive reflexes but did not activate spontaneous ventilation. On the contrary, high doses of 8-OH-DPAT (1 microM in situ and 10-100 microg/kg in vivo) stimulated ventilation, whereas nociceptive CFR amplitude in situ returned to baseline and tail-flick reflex was depressed in vivo. Opioid-induced ventilatory depression was antagonized by 8-OH-DPAT (1 microM in situ, and 10 microg/kg in vivo), whereas antinociception sustained. Selective 5-HT(1A)-R-antagonist WAY 100 635 (1 microM) prevented the effects of 8-OH-DPAT in situ. CONCLUSION: 5-HT(1A)-R-agonist 8-OH-DPAT activates spontaneous breathing without diminishing opioid-induced antinociception in rats.

Emergence of brain-derived neurotrophic factor-induced postsynaptic potentiation of NMDA currents during the postnatal maturation of the Kolliker-Fuse nucleus of rat.

The Kölliker-Fuse nucleus (KF) contributes essentially to respiratory pattern formation and adaptation of breathing to afferent information. Systems physiology suggests that these KF functions depend on NMDA receptors (NMDA-R). Recent investigations revealed postnatal changes in the modulation of glutamatergic neurotransmission by brain-derived neurotrophic factor (BDNF) in the KF. Therefore, we investigated postnatal changes in NMDA-R subunit composition and postsynaptic modulation of NMDA-R-mediated currents by BDNF in KF slice preparations derived from three age groups (neonatal: postnatal day (P) 1-5; intermediate: P6-13; juvenile: P14-21). Immunohistochemistry showed a developmental up-regulation of the NR2D subunit. This correlated with a developmental increase in decay time of NMDA currents and a decline of desensitization in response to repetitive exogenous NMDA applications. Thus, developmental up-regulation of the NR2D subunit, which reduces the Mg(2+) block of NMDA-R, causes these specific changes in NMDA current characteristics. This may determine the NMDA-R-dependent function of the mature KF in the control of respiratory phase transition. Subsequent experiments revealed that bath-application of BDNF progressively potentiated these repetitively evoked NMDA currents only in intermediate and juvenile age groups. Pharmacological inhibition of protein kinase C (PKC), as a downstream component of the BDNF-tyrosine kinase B receptor (trkB) signalling, prevented BDNF-induced potentiation of NMDA currents. BDNF-induced potentiation of NMDA currents in later developmental stages might be essential for synaptic plasticity during the adaptation of the breathing pattern in response to peripheral/central commands. The lack of plasticity in neonatal neurones strengthens the hypothesis that the respiratory network becomes permissive for activity-dependent plasticity with ongoing postnatal development.

Developmental changes of serotonin 4(a) receptor expression in the rat pre-Bötzinger complex.

Serotonin receptors (5-HTRs) are known to be involved in the regulation of breathing behavior and to mediate neurotrophic actions that exert a significant function in network formation during development. We studied neuronal 5-HT(4(a))R-immunoreactivity (-IR) at developmental ages from E14 to P10. Within the pre-Bötzinger complex (pre-BötC), a part of the respiratory network important for rhythmogenesis, 5-HT(4(a))R-IR was most extensive in rats at an age of E18. The 5-HT(4(a))-IR was found predominantly in the neuropil, whereas somatic staining was sporadic at late embryonic (E18-E20) stages. At birth, we observed a dramatic change to a predominantly somatic staining, and neuropil staining was greatly reduced and disappeared at an age of P4. In all developmental stages, 5-HT(4(a)) and mu-opioid receptors were strongly coexpressed in neurons of the pre-BötC, whereas 5-HT(4(a))R expression was absent in neurons within the dorsal horn. Nestin, a marker for CNS progenitor cells, was used to obtain information about the degree of pre-BötC differentiation. Nestin-positive cells did not appear within the pre-BötC before age E20. At E16, nestin-expressing cells were absent in the nucleus ambiguus (NA) and its ventral periphery. The number of nestin-positive cells increased after birth within and outside the pre-BötC, the majority of cells being glial. Coexpression of nestin and 5-HT(4(a))R was localized predominantly within the NA and appeared only sporadically within the pre-BötC. We conclude that 5-HT(4(a))Rs are important not only for neuromodulation of cellular excitability but also for respiratory network formation.

Persistent Expression of Serotonin Receptor 5b Alters Breathing Behavior in Male MeCP2 Knockout Mice.

Mutations in the transcription factor methyl-CpG-binding protein 2 (MeCP2) cause the neurodevelopmental disorder Rett syndrome (RTT). Besides many other neurological problems, RTT patients show irregular breathing with recurrent apneas or breath-holdings. MeCP2-deficient mice, which recapitulate this breathing phenotype, show a dysregulated, persistent expression of G-protein-coupled serotonin receptor 5-ht5b (Htr5b) in the brainstem. To investigate whether the persistence of 5-ht5b expression is contributing to the respiratory phenotype, we crossbred MeCP2-deficient mice with 5-ht5b-deficient mice to generate double knockout mice (Mecp2-/y ;Htr5b-/-). To compare respiration between wild type (WT), Mecp2-/y and Mecp2-/y ;Htr5b-/- mice, we used unrestrained whole-body plethysmography. While the breathing of MeCP2-deficient male mice (Mecp2-/y ) at postnatal day 40 is characterized by a slow breathing rate and the occurrence of prolonged respiratory pauses, we found that in MeCP2-deficient mice, which also lacked the 5-ht5b receptor, the breathing rate and the number of pauses were indistinguishable from WT mice. To test for a potential mechanism, we also analyzed if the known coupling of 5-ht5b receptors to Gi proteins is altering second messenger signaling. Tissue cAMP levels in the medulla of Mecp2-/y mice were decreased as compared to WT mice. In contrast, cAMP levels in Mecp2-/y ;Htr5b-/- mice were indistinguishable from WT mice. Taken together, our data points towards a role of 5-ht5b receptors within the complex breathing phenotype of MeCP2-deficient mice.

The Serotonin Receptor Subtype 5b Specifically Interacts with Serotonin Receptor Subtype 1A.

Previously, we described the dysregulation of serotonin (5-HT) receptor subtype 5b (5-ht5b) in a mouse model of Rett syndrome (RTT). 5-ht5b has not been extensively studied, so we set out to characterize it in more detail. Unlike common cell surface receptors, 5-ht5b displays no membrane expression, while receptor clusters are located in endosomes. This unusual subcellular localization is at least in part controlled by glycosylation of the N-terminus, with 5-ht5b possessing fewer glycosylation sites than related receptors. We analyzed whether the localization to endosomes has any functional relevance and found that 5-ht5b receptors can specifically interact with 5-HT1A receptors and retain them in endosomal compartments. This interaction reduces 5-HT1A surface expression and is mediated by interactions between the fourth and fifth trans-membrane domain (TMD). This possibly represents a mechanism by which 5-ht5b receptors regulate the activity of other 5-HT receptor.

Analysis of the Serotonergic System in a Mouse Model of Rett Syndrome Reveals Unusual Upregulation of Serotonin Receptor 5b.

Mutations in the transcription factor methyl-CpG-binding-protein 2 (MeCP2) cause a delayed-onset neurodevelopmental disorder known as Rett syndrome (RTT). Although alteration in serotonin levels have been reported in RTT patients, the molecular mechanisms underlying these defects are not well understood. Therefore, we chose to investigate the serotonergic system in hippocampus and brainstem of male Mecp2-/y knock-out mice in the B6.129P2(C)-Mecp2(tm1.1Bird) mouse model of RTT. The serotonergic system in mouse is comprised of 16 genes, whose mRNA expression profile was analyzed by quantitative RT-PCR. Mecp2-/y mice are an established animal model for RTT displaying most of the cognitive and physical impairments of human patients and the selected areas receive significant modulation through serotonin. Using anatomically and functional characterized areas, we found region-specific differential expression between wild type and Mecp2-/y mice at post-natal day 40. In brainstem, we found five genes to be dysregulated, while in hippocampus, two genes were dysregulated. The one gene dysregulated in both brain regions was dopamine decarboxylase, but of special interest is the serotonin receptor 5b (5-ht5b), which showed 75-fold dysregulation in brainstem of Mecp2-/y mice. This dysregulation was not due to upregulation, but due to failure of down-regulation in Mecp2-/y mice during development. Detailed analysis of 5-ht5b revealed a receptor that localizes to endosomes and interacts with Gαi proteins.

Serotonin receptors: guardians of stable breathing.

Disturbances of breathing arising from failures of the respiratory center are not uncommon. Among them, breath holding and apnea occur most frequently as consequences of pulmonary and cardiac diseases, hypoxia, head trauma, cerebral inflammatory processes, genetic defects, degenerative brain diseases, alcoholism, deep anesthesia and drug overdose. They are often life-threatening and fail to respond to existing pharmacotherapies. After extensive research, there is now a reliable basis for new strategies to treat respiratory disturbances by pharmacological manipulation of intracellular signaling pathways, particularly those involving the serotonin receptor family. Specific activation of these pathways effectively prevails respiratory disturbances and can be extended to treatment of life-threatening respiratory disorders in patients.

Distribution of serotonin 4(a) receptors in the juvenile rat brain and spinal cord.

In the last 20 years there have been many studies investigating the distribution of 5-HT4-receptors in the brain of different species. Most studies are methodically based on RT-PCR or in situ hybridization and have analysed the receptors at the mRNA level. Furthermore there have been some autoradiographic studies using specific 5-HT4-receptor antagonists like [(3)H]GR113808, [(3)H]BIMU-1 or [(125)I]SB207710. This study investigates the topographical distribution of the 5-HT4(a)-receptor in the juvenile rat brain and spinal cord, which is important for neuromodulation of cellular excitability and could be involved in various developmental processes of the central nervous system. We analysed the 5-HT4(a)-receptor at protein level with a monospecific polyclonal antibody by using an immunohistochemical staining. We saw an intensive staining in some areas of the cortex, in the olfactory bulb, in most areas of the cerebellum, in hippocampal areas like the dentate gyrus and in several different areas of the brainstem, especially in the motor nuclei. Overall we have shown comparable results in accordance with the results of other studies investigating the distribution of 5-HT4(a)-receptors. Some areas like the islands of Calleja, the preoptic nucleus or the medial habenular nucleus showed a lower intensity of 5-HT4(a)-receptors in comparison with the results of other studies. As a novel result we found a higher intensity of 5-HT4(a)-receptor in several brain areas associated with motor function than was shown by other studies, especially in the motor cortex, in different areas of the cerebellum, in the red nucleus, in the motor nuclei of the brainstem or in the ventral horn cells of the spinal cord. We conclude that the 5-HT4(a)-receptor may play a more prominent role in the modulation of motor cortico-ponto-cerebellar, cortico-spinal, rubro-spinal, vestibulo-spinal and cortico-nuclear tracts during juvenile development.

Chronic fluoxetine treatment changes S100B expression during postnatal rat brain development.

BACKGROUND: Fluoxetine, a selective serotonin reuptake inhibitor, is approved for treatment of childhood depression. In rats, fluoxetine influences neuronal development, but it is unclear whether it also influences glia development. S100B is a glia-derived calcium-binding protein, which may influence the development of serotonergic fibers and, vice versa, serotonin may influence the expression of S100B. OBJECTIVES: The purpose of this study was to investigate whether fluoxetine treatment influences the expression of S100B during postnatal development, and whether potential changes are regionally dependent upon the time frame of drug administration. METHODS: S100B gene expression and S100B protein expression in three different brain regions (frontal cortex, hippocampus, and striatum) were studied by real-time polymerase chain reaction (PCR) and immunohistochemistry, respectively. First, a short-term effect, 24 hours after a 14 day fluoxetine treatment (5 mg/kg/bw s.c.) of rats either from postnatal day (PD) 1 to 15, 21 to 35, or 50 to 64, was investigated. Then, the same treatment was used to analyze S100B gene and protein levels at PD 90 (long-term effect). RESULTS: At PD 90, a significant increase of gene and protein expression was observed in all regions if rats were treated during PDs 21-35, whereas treatment during other periods had no long-term effects. A short-term effect 24 hours after fluoxetine treatment was found for almost all development stages and regions, demonstrated by a significant increase of S100B. CONCLUSIONS: These results support recent research indicating a highly drug-sensitive period (i.e., periadolescence) of rat brain development. Therefore, further clinical studies should be performed to clarify whether such a sensitive period also exists in children.

Expression and function of serotonin 2A and 2B receptors in the mammalian respiratory network.

Neurons of the respiratory network in the lower brainstem express a variety of serotonin receptors (5-HTRs) that act primarily through adenylyl cyclase. However, there is one receptor family including 5-HT(2A), 5-HT(2B), and 5-HT(2C) receptors that are directed towards protein kinase C (PKC). In contrast to 5-HT(2A)Rs, expression and function of 5-HT(2B)Rs within the respiratory network are still unclear. 5-HT(2B)R utilizes a Gq-mediated signaling cascade involving calcium and leading to activation of phospholipase C and IP3/DAG pathways. Based on previous studies, this signal pathway appears to mediate excitatory actions on respiration. In the present study, we analyzed receptor expression in pontine and medullary regions of the respiratory network both at the transcriptional and translational level using quantitative RT-PCR and self-made as well as commercially available antibodies, respectively. In addition we measured effects of selective agonists and antagonists for 5-HT(2A)Rs and 5-HT(2B)Rs given intra-arterially on phrenic nerve discharges in juvenile rats using the perfused brainstem preparation. The drugs caused significant changes in discharge activity. Co-administration of both agonists revealed a dominance of the 5-HT(2B)R. Given the nature of the signaling pathways, we investigated whether intracellular calcium may explain effects observed in the respiratory network. Taken together, the results of this study suggest a significant role of both receptors in respiratory network modulation.

Novel solid phase-based ELISA assays contribute to an improved detection of anti-HLA antibodies and to an increased reliability of pre- and post-transplant crossmatching.

Antibodies directed against HLA antigens of a given organ donor represent the dominating reason for hyper-acute or acute allograft rejections. In order to select recipients without donor-specific antibodies, a standard crossmatch (CM) procedure, the complement-dependent cytotoxicity assay (CDC), was developed. This functional assay strongly depends on the availability of isolated vital lymphocytes of a given donor. However, the requirements of the donor's material may often not be fulfilled, so that the detection of the antibodies directed against HLA molecules is either impaired or becomes completely impossible. To circumvent the disadvantages of the CDC procedure, enzyme-linked immunosorbent assay (ELISA)-based and other solid phase-based ELISA-related techniques have been designed to reliably detect anti-HLA antibodies in recipients. Due to the obvious advantages of these novel technologies, when compared with the classical CDC assay, there is an urgent need to implement them as complementary methods or even as a substitution for the conventional CDC crossmatch that is currently being applied by all tissue typing laboratories.

Serotonin receptor 1A-modulated phosphorylation of glycine receptor α3 controls breathing in mice.

Rhythmic breathing movements originate from a dispersed neuronal network in the medulla and pons. Here, we demonstrate that rhythmic activity of this respiratory network is affected by the phosphorylation status of the inhibitory glycine receptor α3 subtype (GlyRα3), which controls glutamatergic and glycinergic neuronal discharges, subject to serotonergic modulation. Serotonin receptor type 1A-specific (5-HTR1A-specific) modulation directly induced dephosphorylation of GlyRα3 receptors, which augmented inhibitory glycine-activated chloride currents in HEK293 cells coexpressing 5-HTR1A and GlyRα3. The 5-HTR1A-GlyRα3 signaling pathway was distinct from opioid receptor signaling and efficiently counteracted opioid-induced depression of breathing and consequential apnea in mice. Paradoxically, this rescue of breathing originated from enhanced glycinergic synaptic inhibition of glutamatergic and glycinergic neurons and caused disinhibition of their target neurons. Together, these effects changed respiratory phase alternations and ensured rhythmic breathing in vivo. GlyRα3-deficient mice had an irregular respiratory rhythm under baseline conditions, and systemic 5-HTR1A activation failed to remedy opioid-induced respiratory depression in these mice. Delineation of this 5-HTR1A-GlyRα3 signaling pathway offers a mechanistic basis for pharmacological treatment of opioid-induced apnea and other breathing disturbances caused by disorders of inhibitory synaptic transmission, such as hyperekplexia, hypoxia/ischemia, and brainstem infarction.

Development of 5-HT transporter density and long-term effects of methylphenidate in an animal model of ADHD.

Although stimulants as the treatment of choice are widely prescribed in ADHD, little is known about their long-term neurobiological effects. Hence, for the first time the present study examined the long-term effects of chronic methylphenidate (MPH) administration on striatal 5-hydroxytryptamine transporter (5-HTT) densities in an animal model of ADHD. First, it compared the normal development of striatal 5-HTT densities of spontaneously hypertensive rats (SHR) as an animal model of ADHD and Wistar Kyoto (WKY) rats as controls; binding of the highly selective ligand of 5-HTT [(3)H]paroxetine was determined on membrane preparations of the striatum of SHR and WKY rats on postnatal days 25, 50, and 90, i.e. from the time of weaning until adulthood. Second, the long-term effect of chronic administration of 2 mg/kg per day MPH at two different developmental stages (days 25-39 or 50-64) on the striatal 5-HTT density was examined in both rat strains at day 90. Long-term effects of MPH treatment on striatal 5-HTT density in adulthood could be ruled out in both healthy (WKY) and "ADHD" rats (SHR). But a higher striatal 5-HTT density in older SHR versus WKY rats might indicate ADHD specific changes in the 5-HT system that needs further investigation not only in animals.

Serotonin targets inhibitory synapses to induce modulation of network functions.

The cellular effects of serotonin (5-HT), a neuromodulator with widespread influences in the central nervous system, have been investigated. Despite detailed knowledge about the molecular biology of cellular signalling, it is not possible to anticipate the responses of neuronal networks to a global action of 5-HT. Heterogeneous expression of various subtypes of serotonin receptors (5-HTR) in a variety of neurons differently equipped with cell-specific transmitter receptors and ion channel assemblies can provoke diverse cellular reactions resulting in various forms of network adjustment and, hence, motor behaviour. Using the respiratory network as a model for reciprocal synaptic inhibition, we demonstrate that 5-HT(1A)R modulation primarily affects inhibition through glycinergic synapses. Potentiation of glycinergic inhibition of both excitatory and inhibitory neurons induces a functional reorganization of the network leading to a characteristic change of motor output. The changes in network operation are robust and help to overcome opiate-induced respiratory depression. Hence, 5-HT(1A)R activation stabilizes the rhythmicity of breathing during opiate medication of pain.

Differential modulation of I(h) by 5-HT receptors in mouse CA1 hippocampal neurons.

CA1 pyramidal neurons of the hippocampus express various types of serotonin (5-HT) receptors, such as 5-HT(1A), 5-HT(4) and 5-HT(7) receptors, which couple to Galpha(i) or Galpha(s) proteins and operate on different intracellular signalling pathways. In the present paper we verify such differential serotonergic modulation for the hyperpolarization-activated current I(h). Activation of 5-HT(1A) receptors induced an augmentation of current-induced hyperpolarization responses, while the responses declined after 5-HT(4) receptors were activated. The resting potential of neurons hyperpolarized (-2.3 +/- 0.7 mV) after 5-HT(1A) receptor activation, activation of 5-HT(4) receptors depolarized neurons (+3.3 +/- 1.4 mV). Direct activation of adenylyl cyclase (AC) by forskolin also produced a depolarization. In voltage clamp, the Ih current was identified by its characteristic voltage- and time-dependency and by blockade with CsCl or ZD7288. Activation of 5-HT(1A) receptors reduced I(h) and shifted the activation curve to a more negative voltage by -5 mV at half-maximal activation. Activation of 5-HT(4) and 5-HT(7) receptors increased I(h) and shifted the activation curve to the right by +5 mV. Specific activation of 5-HT(4) receptors by BIMU8 increased membrane conductance and showed an increase in I(h) in a subset of cells, but did not induce a significant alteration in the activation curve. In order to verify spatial differences, we applied BIMU8 selectively to the soma and to the dendrites. Only somatic application induced receptor activation. These data are confirmed by immunofluorescence stainings with an antibody against the 5-HT(4) receptor, revealing receptor expression at the somata of the CA1 region. A similar expression pattern was found with a new antibody against 5-HT(7) receptors which reveals immunofluorescence staining on the cell bodies of pyramidal neurons.

5-HT4(a) receptors avert opioid-induced breathing depression without loss of analgesia.

Opiates are widely used analgesics in anesthesiology, but they have serious adverse effects such as depression of breathing. This is caused by direct inhibition of rhythm-generating respiratory neurons in the Pre-Boetzinger complex (PBC) of the brainstem. We report that serotonin 4(a) [5-HT4(a)] receptors are strongly expressed in respiratory PBC neurons and that their selective activation protects spontaneous respiratory activity. Treatment of rats with a 5-HT4 receptor-specific agonist overcame fentanyl-induced respiratory depression and reestablished stable respiratory rhythm without loss of fentanyl's analgesic effect. These findings imply the prospect of a fine-tuned recovery from opioid-induced respiratory depression, through adjustment of intracellular adenosine 3',5'-monophosphate levels through the convergent signaling pathways in neurons.