Till Manzke

Differential modulation of Ih 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‐HT1A, 5‐HT4 and 5‐HT7 receptors, which couple to Gαi or Gαs proteins and operate on different intracellular signalling pathways. In the present paper we verify such differential serotonergic modulation for the hyperpolarization‐activated current Ih. Activation of 5‐HT1A receptors induced an augmentation of current‐induced hyperpolarization responses, while the responses declined after 5‐HT4 receptors were activated. The resting potential of neurons hyperpolarized (−2.3 ± 0.7 mV) after 5‐HT1A receptor activation, activation of 5‐HT4 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‐HT1A receptors reduced Ih and shifted the activation curve to a more negative voltage by −5 mV at half‐maximal activation. Activation of 5‐HT4 and 5‐HT7 receptors increased Ih and shifted the activation curve to the right by +5 mV. Specific activation of 5‐HT4 receptors by BIMU8 increased membrane conductance and showed an increase in Ih 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‐HT4A receptor, revealing receptor expression at the somata of the CA1 region. A similar expression pattern was found with a new antibody against 5‐HT7 receptors which reveals immunofluorescence staining on the cell bodies of pyramidal neurons.

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.

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-HT1AR 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-HT1AR activation stabilizes the rhythmicity of breathing during opiate medication of pain.

The potency of different serotonergic agonists in counteracting opioid evoked cardiorespiratory disturbances

Serotonin receptor (5-HTR) agonists that target 5-HT4(a)R and 5-HT1AR can reverse μ-opioid receptor (μ-OR)-evoked respiratory depression. Here, we have tested whether such rescuing by serotonin agonists also applies to the cardiovascular system. In working heart–brainstem preparations in situ, we have recorded phrenic nerve activity, thoracic sympathetic chain activity (SCA), vascular resistance and heart rate (HR) and in conscious rats, diaphragmatic electromyogram, arterial blood pressure (BP) and HR via radio-telemetry. In addition, the distribution of 5-HT4(a)R and 5-HT1AR in ponto-medullary cardiorespiratory networks was identified using histochemistry. Systemic administration of the μ-OR agonist fentanyl in situ decreased HR, vascular resistance, SCA and phrenic nerve activity. Subsequent application of the 5-HT1AR agonist 8-OH-DPAT further enhanced bradycardia, but partially compensated the decrease in vascular resistance, sympathetic activity and restored breathing. By contrast, the 5-HT4(a)R agonist RS67333 further decreased vascular resistance, HR and sympathetic activity, but partially rescued breathing. In conscious rats, administration of remifentanyl caused severe respiratory depression, a decrease in mean BP accompanied by pronounced bradyarrhythmia. 8-OH-DPAT restored breathing and prevented the bradyarrhythmia; however, BP and HR remained below baseline. In contrast, RS67333 further suppressed cardiovascular functions in vivo and only partially recovered breathing in some cases. The better recovery of μ-OR cardiorespiratory disturbance by 5-HT1AR than 5-HT4(a)R is supported by the finding that 5-HT1AR was more densely expressed in key brainstem nuclei for cardiorespiratory control compared with 5-HT4(a)R. We conclude that during treatment of severe pain, 5-HT1AR agonists may provide a useful tool to counteract opioid-mediated cardiorespiratory disturbances.

Emergence of brain-derived neurotrophic factor-induced postsynaptic potentiation of NMDA currents during the postnatal maturation of the Kölliker–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 Mg2+ 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.

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-HT1A-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-HT1A-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-HT1A-R-antagonist WAY 100 635 to exclude effects of receptors other than 5-HT1A-R

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

Brainstem respiratory neurons express the glycine α3 receptor (Glyα3R), which is a target of modulation by several serotonin (5‐HT) receptor agonists. Application of the 5‐HT1A receptor (5‐HT1AR) agonist 8‐OH‐DPAT was shown (i) to depress cellular cAMP, leading to dephosphorylation of Glyα3R and augmentation of postsynaptic inhibition of neurons expressing Glyα3R ( 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 Glyα3R‐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‐HT1AR‐Glyα3R 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‐HT1AR activation can produce a disinhibition of inspiratory neurons, leading to the recovery of respiratory rhythm from opioid‐induced apnoeas.

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-HT2A, 5-HT2B, and 5-HT2C receptors that are directed towards protein kinase C (PKC). In contrast to 5-HT2ARs, expression and function of 5-HT2BRs within the respiratory network are still unclear. 5-HT2BR 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-HT2ARs and 5-HT2BRs 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-HT2BR. 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.

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‐HT4(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‐HT4(a)R‐IR was most extensive in rats at an age of E18. The 5‐HT4(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‐HT4(a) and μ‐opioid receptors were strongly coexpressed in neurons of the pre‐BötC, whereas 5‐HT4(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‐HT4(a)R was localized predominantly within the NA and appeared only sporadically within the pre‐BötC. We conclude that 5‐HT4(a)Rs are important not only for neuromodulation of cellular excitability but also for respiratory network formation. J. Comp. Neurol. 506:775–790, 2008.

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.