5-HT1A Agonist Research Articles

GC–MS analysis of methylenedioxybenzyl analogues of the serotonin receptor agonists 25X-NBOMe drugs

In this study, the oxygenated benzyl aromatic ring of the classic NBOMe drugs such as 25B-NBOMe and 25I-NBOMe (with N-methoxybenzyl) were modified to contain the 2,3- and 3,4-methylenedioxy group in both phenethylamine and phenylisopropylamine molecular scaffolds. The aromatic ring of the phenethylamine core structural skeleton was held constant as the 2,5-dimethoxy substitution pattern with the 4-position group varied yielding the 4-bromo (25B), the 4-iodo (25I) as well as the 25H unsubstituted subsets. The 25H and 25B subsets for the phenylisopropylamine skeleton were also included and the resulting ten compounds were evaluated in GC–MS studies. The ten compounds were baseline resolved using a 30 m column containing a silarylene film phase (Rxi®-17Sil MS) eluting over a period of about eleven minutes. The 2,3-methylenedioxybenzyl substituted isomer eluted before the corresponding 3,4-methylenedioxybenzyl substituted regioisomer for each subset and the branched chain phenylisopropylamine analogues eluted before the phenethylamines having identical aromatic ring substituents.Initial ionization at the amine nitrogen followed by iminium cation formation (m/z 164 or m/z 178) via loss of the various substituted (H, Br, or I)-2,5-dimethoxybenzyl radicals allows the phenethylamines to be distinguished from the phenylisopropylamine analogues. The m/z 135 ion can form directly from the molecular radical cation and as the product ion of the iminium cation. A series of less prominent high mass ions in the halogenated dimethoxyphenyl substituted compounds occur via a common pathway yielding the halogenated dimethoxybenzyl cation and radical cation.

Differential modulation of active expiration during hypercapnia by the medullary raphe in unanesthetized rats.

Active expiration represents an important mechanism to improve ventilation in conditions of augmented ventilatory demand, such as hypercapnia. While a rostral ventromedullary region, the parafacial respiratory group (pFRG), has been identified as a conditional expiratory oscillator, little is known about how central chemosensitive sites contribute to modulate active expiration under hypercapnia. In this study, we investigated the influence of the medullary raphe in the emergence of phasic expiratory abdominal activity during hypercapnia in unanesthetized adult male rats, in a state-dependent manner. To do so, reverse microdialysis of muscimol (GABAA receptor agonist, 1mM) or 8-OH-DPAT (5-HT1A agonist, 1mM) was applied in the MR during sleep and wakefulness periods, both in normocapnic (room air) and hypercapnic conditions (7% CO2). Electromyography (EMG) of diaphragm and abdominal muscles was performed to measure inspiratory and expiratory motor outputs. We found that active expiration did not occur in room air exposure during wakefulness or sleep. However, hypercapnia did recruit active expiration, and differential effects were observed with the drug dialyses in the medullary raphe. Muscimol increased the diaphragm inspiratory motor output and also increased the amplitude and frequency of abdominal expiratory rhythmic activity during hypercapnia in wakefulness periods. On the other hand, the microdialysis of 8-OH-DPAT attenuated hypercapnia-induced active expiration in a state-dependent manner. Our data suggest that the medullary raphe can either inhibit or potentiate respiratory motor activity during hypercapnia, and the balance of these inhibitory or excitatory outputs may determine the expression of active expiration.

Lurasidone – pharmacodynamic and pharmacokinetic properties, clinical potential and interaction risk

Lurasidone is a novel second-generation antipsychotic approved for the treatment of schizophrenia and bipolar depression in adults. It displays high affinity for D2 and 5-HT2A and 5-HT7 receptors, moderate affinity for 5-HT1A and α2C-noradrenergic receptors, and negligible affinity for histamine H1 and muscarinic M1 receptors. It acts as potent D2, 5-HT2A and 5-HT7 antagonist and partial 5-HT1A agonist. Lurasidone taken orally is rapidly absorbed with the time to maximum concentration of 1-3 hours. Lurasidone should be taken with food (at least 350 kcal) due to limited absorption. The mean elimination half-life of lurasidone is 18.1–25.5 hours for doses 20–80 mg/day and 28.8–37.4 hours for doses of 120–160 mg/day. Steady-state is reached within 7 days. The drug is metabolised via CYP 3A4 and excreted mainly in faeces (67–80%) and with urine (about 8–19%). The use of lurasidone with strong inhibitors or inducers of CYP 3A4 (e.g. ketoconazole, erythromycin, or carbamazepine, respectively) is contraindicated. In the case of combined treatment of lurasidone and moderate inhibitors of CYP 3A4, the dose of lurasidone should be decreased to 40 mg/day. Lurasidone is an inhibitor of P-glycoprotein and could increase the level of digoxin and potentate the side effects risk of this drug. Pomelo, grapefruit, or a large amount of oranges should be avoided in the diet during treatment with lurasidone. Pharmacodynamic properties of lurasidone underlie its antipsychotic, antidepressant, precognitive, and sleep-awake rhythm normalising activity.

Acute serotonin 2A receptor activation impairs behavioral flexibility in mice

Serotonin 2A (5-HT2A) receptors are the primary site of action of hallucinogenic drugs and the target of atypical antipsychotics. 5-HT2A receptors are also implicated in executive function, including behavioral flexibility. Previous studies showed that 5-HT2A receptor blockade improved behavioral flexibility in rodent models related to autism spectrum disorder and schizophrenia. The current study instead was conducted to examine the impact of acute 5-HT2A receptor activation on behavior flexibility in the control C57BL/6 J strain. Because of the therapeutic potential of serotonergic hallucinogens and the unknown impact of many of these compounds on cognition, the present study examined how the 5-HT2A/2C agonist 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) and the more selective 5-HT2A agonist 25CN-NBOH impacted behavioral flexibility in C57BL/6 J mice. Male mice were tested on a probabilistic spatial discrimination and reversal learning task after an intraperitoneal injection of vehicle, 2.5 mg/kg DOI, 1.0 mg/kg 25CN-NBOH, 1.0 mg/kg of the 5-HT2C receptor antagonist SER-082 or combined treatment with SER-082 (1.0 mg/kg) and 2.5 mg/kg DOI before testing of probabilistic reversal learning. All groups demonstrated comparable performance on the initial spatial discrimination, i.e. similar trials to criterion. DOI alone did not impair reversal learning, whereas 25CN-NBOH increased the number of trials to criterion during reversal learning. Because 5-HT2A and 5-HT2C receptors have been shown to functionally antagonize each other in several behavioral paradigms, we also tested whether blockade of 5-HT2C receptors would unmask 5-HT2A receptor activation by DOI and impair reversal learning. Mice treated with SER-082 in combination with DOI required significantly more trials to reach criterion. In an additional experiment, a dose response experiment with 25CN-NBOH revealed that the 1.0 mg/kg dose tested in reversal learning did not affect locomotor activity. Together, these findings indicate that activation of 5-HT2A receptors impairs probabilistic reversal learning and that 5-HT2A and 5-HT2C receptors exert opposing effects on behavioral flexibility in male mice.

Developmental programming of appetite and growth in male rats increases hypothalamic serotonin (5-HT)5A receptor expression and sensitivity.

Though it is well established that neonatal nutrition plays a major role in lifelong offspring health, the mechanisms underpinning this have not been well defined. Early postnatal accelerated growth resulting from maternal nutritional status is associated with increased appetite and body weight. Likewise, slow growth correlates with decreased appetite and body weight. Food consumption and food-seeking behaviour are directly modulated by central serotonergic (5-hydroxytryptamine, 5-HT) pathways. This study examined the effect of a rat maternal postnatal low protein (PLP) diet on 5-HT receptor mediated food intake in offspring. Microarray analyses, in situ hybridization or laser capture microdissection of the ARC followed by RT-PCR were used to identify genes up- or down-regulated in the arcuate nucleus of the hypothalamus (ARC) of 3-month-old male PLP rats. Third ventricle cannulation was used to identify altered sensitivity to serotonin receptor agonists and antagonists with respect to food intake. Male PLP offspring consumed less food and had lower growth rates up to 3 months of age compared with Control offspring from dams fed a normal diet. In total, 97 genes were upregulated including the 5-HT5A receptor (5-HT5AR) and 149 downregulated genes in PLP rats compared with Controls. The former obesity medication fenfluramine and the 5-HT receptor agonist 5-Carboxamidotryptamine (5-CT) significantly suppressed food intake in both groups, but the PLP offspring were more sensitive to d-fenfluramine and 5-CT compared with Controls. The effect of 5-CT was antagonized by the 5-HT5AR antagonist SB699551. 5-CT also reduced NPY-induced hyperphagia in both Control and PLP rats but was more effective in PLP offspring. Postnatal low protein programming of growth in rats enhances the central effects of serotonin on appetite by increasing hypothalamic 5-HT5AR expression and sensitivity. These findings provide insight into the possible mechanisms through which a maternal low protein diet during lactation programs reduced growth and appetite in offspring.

5-HT1A receptor-mediated attenuation of heat hyperalgesia and mechanical allodynia by chrysin in mice with experimental mononeuropathy

BackgroundPersistent neuropathic pain poses a health problem, for which effective therapy or antidote is in dire need. This work aimed to investigate the pain-relieving effect of chrysin, a natural flavonoid...

An Ex Vivo Study to Evaluate the Effect of Tegaserod on Platelet Activation and Aggregation

Tegaserod, an orally active, potent 5-hydroxytryptamine-4 serotonin receptor agonist, was previously indicated for irritable bowel syndrome but was voluntarily withdrawn due to potential cardiovascular side effects. In vitro studies suggested that tegaserod increased platelet aggregation, but these results were not reproduced or were inconclusive. We sought to assess ex vivo effects of tegaserod on platelet aggregation. In this double-blind, placebo-controlled, crossover study, we randomized a majority of healthy patients with no history of cardiovascular risk factors (n = 21) to receive tegaserod or matching placebo for 7 + 2 days followed by a 7- to 10-day washout period, and then patients were crossed over to the other study drug for the next 7 + 2 days. Unstimulated and agonist-stimulated platelet aggregation; P-selectin expression; serum thromboxane (Tx)B2 and urinary 11-dehydro (11-dh) TxB2; and tegaserod and M29.0 concentrations were serially assessed. There was no significant difference in percentage change in unstimulated or adenosine diphosphate (ADP)- and ADP + serotonin-, collagen- and thrombin receptor activating peptide-induced maximum platelet aggregation and in platelet P-selectin expression in the presence of tegaserod at any time point when compared to placebo. Similarly, there was no significant difference in percentage change in serum TxB2 or urinary 11-dhTxB2 levels between placebo and tegaserod. No new or unexpected findings were observed in evaluations of safety or pharmacokinetic parameters. This comprehensive pharmacodynamic study, by employing established markers used in prior investigations, which have been considered by the Food and Drug Administration to indicate drug-related platelet effects, does not demonstrate any influence of tegaserod treatment on platelet function.

The Role of Serotonin in Singultus: A Review.

The use of dopamine receptor blockers for chronic singultus treatment is based—at least partially—on circular thinking: chlorpromazine is FDA-approved for hiccups, chlorpromazine is a neuroleptic, neuroleptics are dopamine receptor blockers, and therefore hiccup is due to dopaminergic dysfunction. Chlorpromazine interacts with high affinity with a multitude of receptors and ion channels. This promiscuity is the basis for many of the therapeutic effects and adverse drug reactions of this drug. While an involvement of dopamine is certain, it is by no means clear that dopaminergic dysfunction is the hallmark of singultus. The common denominator of most remedies for transient hiccup is their ability to activate the vagus nerve. Both afferent and efferent vagal activity and the central integration of the Xth cranial nerve function are modulated, inter alia, via serotonergic mechanisms; beneficial (therapeutic) effects for hiccup are to be expected from serotonin (5-HT) receptor subtype ligands that enhance vagal activity. Taken together, it appears that the ability to increase vagus output is mainly associated with 5-HT1A, 5-HT3, and 5-HT7 agonists and with 5-HT2C antagonists. The plausibility of the serotonergic singultus hypothesis is examined against available pharmacokinetic, pharmacodynamic, and clinical data for a number of drugs.

Biodiversity of β-Carboline Profile of Banisteriopsis caapi and Ayahuasca, a Plant and a Brew with Neuropharmacological Potential.

Ayahuasca is a psychoactive infusion with a large pharmacological application normally prepared with Banisteriopsis caapi, which contains the monoamine oxidase inhibitors β-carbolines, and Psichotria virids, which contains the serotonin receptor agonist N,N dimethyltryptamine (DMT). The objectives of this study were to investigate the chemical profile of B. caapi and of ayahuasca collected in various Brazilian regions. In total, 176 plant lianas, of which 159 B. caapi and 33 ayahuasca samples were analyzed. Dried liana samples were powdered, extracted with methanol, diluted, and analyzed by LC-MS/MS. Ayahuasca samples were diluted and analyzed. Mean concentrations in B. caapi were 4.79 mg/g harmine, 0.451 mg/g harmaline, and 2.18 mg/g tetrahydroharmine (THH), with a high variability among the samples (RSD from 78.9 to 170%). Native B. caapi samples showed significantly higher harmine concentrations than cultivated ones, and samples from the Federal District/Goiás had higher THH content than those collected in the State of Acre. The other Malpighiaceae samples did not contain β-carbolines, except for one D. pubipetala sample. Concentrations in ayahuasca samples ranged from 0.109 to 7.11 mg/mL harmine, 0.012 to 0.945 mg/mL harmaline, 0.09 to 3.05 mg/mL THH, and 0.10 to 3.12 mg/mL DMT. The analysis of paired ayahuasca/B. caapi confirmed that harmine is reduced to harmaline and to THH during the brew preparation. This is the largest study conducted with Malpighiaceae samples and showed a large variability in the main β-carbolines present in B. caapi. This biodiversity is a challenge for standardization of the material used in ethnopharmacological studies of B. caapi and ayahuasca.

Inflammation induces developmentally regulated sumatriptan inhibition of spinal synaptic transmission.

While triptans are used to treat migraine, there is evidence that they also reduce inflammation-induced pain at the spinal level. The cellular mechanisms underlying this spinal enhancement are unknown. We examined whether inflammation alters sumatriptan modulation of synaptic transmission in the rat spinal dorsal horn. Three to four days following intraplantar injection of complete Freund's adjuvant (CFA) or saline, whole cell recordings of evoked glutamatergic EPSCs were made from lumbar lamina I-II dorsal horn neurons in rat spinal slices KEY RESULTS: In 2- to 3-week-old animals, sumatriptan reduced the amplitude of evoked EPSCs and this was greater in slices from CFA, compared to saline-injected rats. In CFA-injected animals, sumatriptan increased the paired pulse ratio of evoked EPSCs and reduced the rate of spontaneous miniature EPSCs. The 5-HT1B and 5-HT1D agonists CP9 3129 and PNU109291 both inhibited evoked EPSCs in CFA but not saline-injected rats. By contrast, the 5-HT1A agonist R(+)-8-OH-DPAT inhibited evoked EPSCs in saline but not CFA-injected rats. In CFA-injected rats, the sumatriptan-induced inhibition of evoked EPSCs was reduced by the 5-HT1B and 5-HT1D antagonists NAS181 and BRL-15572. Intriguingly, the difference in sumatriptan inhibition between CFA and saline-injected animals was only observed in animals less than 4 weeks old. These findings indicate that inflammation induces a developmentally regulated 5-HT1B/1D presynaptic inhibition of excitatory transmission into the rat superficial dorsal horn. Thus, triptans could potentially act as spinal analgesic agents for inflammatory pain in the juvenile setting.

Sub-Acute Treatment of Curcumin Derivative J147 Ameliorates Depression-Like Behavior Through 5-HT1A-Mediated cAMP Signaling.

BackgroundMajor depressive disorder (MDD) is a severe mental disorder related to the deficiency of monoamine neurotransmitters, particularly to abnormalities of 5-HT (5-hydroxytryptamine, serotonin) and its receptors. Our previous study suggested that acute treatment with a novel curcumin derivative J147 exhibited antidepressant-like effects by increasing brain derived neurotrophic factor (BDNF) level in the hippocampus of mice. The present study expanded upon our previous findings and investigated the antidepressant-like effects of sub-acute treatment of J147 for 3 days in male ICR mice and its possible relevancy to 5-HT1A and 5-HT1B receptors and downstream cAMP-BDNF signaling.MethodsJ147 at doses of 1, 3, and 9 mg/kg (via gavage) was administered for 3 days, and the anti-immobility time in the forced swimming and tail suspension tests (FST and TST) was recorded. The radioligand binding assay was used to determine the affinity of J147 to 5-HT1A and 5-HT1B receptor. Moreover, 5-HT1A or 5-HT1B agonist or its antagonist was used to determine which 5-HT receptor subtype is involved in the antidepressant-like effects of J147. The downstream signaling molecules such as cAMP, PKA, pCREB, and BDNF were also measured to determine the mechanism of action.ResultsThe results demonstrated that sub-acute treatment of J147 remarkably decreased the immobility time in both the FST and TST in a dose-dependent manner. J147 displayed high affinity in vitro to 5-HT1A receptor prepared from mice cortical tissue and was less potent at 5-HT1B receptor. These effects of J147 were blocked by pretreatment with a 5-HT1A antagonist NAD-299 and enhanced by a 5-HT1A agonist 8-OH-DPAT. However, 5-HT1B receptor antagonist NAS-181 did not appreciably alter the effects of J147 on depression-like behaviors. Moreover, pretreatment with NAD-299 blocked J147-induced increases in cAMP, PKA, pCREB, and BDNF expression in the hippocampus, while 8-OH-DPAT enhanced the effects of J147 on these proteins’ expression.ConclusionThe results suggest that J147 induces rapid antidepressant-like effects during a 3-day treatment period without inducing drug tolerance. These effects might be mediated by 5-HT1A-dependent cAMP/PKA/pCREB/BDNF signaling.

063 Flibanserin: Enhanced Pharmacovigilance Study to Assess and Analyze the Risks of Adverse Events of Special Interest

Flibanserin, a 5-HT1A agonist and 5-HT2A antagonist, is approved in the US and Canada for the treatment of acquired, generalized hypoactive sexual desire disorder (HSDD) in premenopausal women. Flibanserin is subjected to an Enhanced Pharmacovigilance Plan (EPVP) in the USA only

Effects of Microinjections of a Serotonin Receptor (5-HT2A/C) Agonist and Antagonist into the Amygdala in Rats on Anxiety Behavior and Conditioned Reflex Fear

The role of 5-HT2A/C receptors in the amygdala in the acquisition, testing, and extinction of conditioned reflex fear and on anxiety behavior was studied. Microinjections of a receptor antagonist (ketanserin, 0.5 μg/0.5 μl) into the basolateral nucleus of the amygdala decreased signs of conditioned reflex fear in the form of freezing and prevented reacquisition, but had no effect on anxiety behavior in rats. Administration of an agonist (DOI, 1 μg/0.5 μl) led to an increase in movement activity, panic-like behavior, and decreased anxiety, with reductions in the signs of fear in the form of freezing. Both the agonist and the antagonist of 5-HT2A/C receptors were able to accelerate fear extinction in rats with prolonged freezing, which does not extinguish easily. These results provide evidence that 5-HT2A/C receptors in the amygdala play a significant role in the occurrence and persistence of conditioned reflex fear apparent as freezing.

036 Two-Arm, Prospective, Open-Label, Pilot Study of Flibanserin versus Flibanserin and Sex Therapy in Pre- and Postmenopausal Women with HSDD

Hypoactive Sexual Desire Disorder (HSDD) is the most common sexual dysfunction in women, having both biologic and psychologic components. Flibanserin, a multifunctional serotonin agonist and antagonist, was the first FDA-approved medication indicated for the safe and effective treatment of HSDD in premenopausal women. Flibanserin was studied in postmenopausal women with HSDD with similar safety and efficacy data as in premenopausal women. In our biopsychosocial model of multi-disciplinary sexual healthcare, new patients undergo both psychological and medical assessments routinely, however the benefits of concomitant sex therapy with pharmacological treatment of HSDD have not been adequately studied.

Molecular mechanistic vision on binding interaction of triptan drug, a serotonin (5-HT1) agonist with human serum albumin through multispectral and computational assessments

The triptan drug such as eletriptan in combination with hydrochloride (ETP) is a 5-HT1 receptor agonist used to treat the migraine headache. Human serum albumin (HSA), the fundamental serum protein, executes various functions, that includes transporting and binding of many ligands. HSA binding interaction with ETP is elucidated from molecular docking in composite with fluorescence (emission, 3D and synchronous), UV-vis and FT-IR spectroscopy at 296, 304 and 312 K (pH = 7.40). ETP after interaction modified the HSA secondary structure and its micro-environments. Energy transfer and thermodynamic parameters were evaluated. Various quenching and binding constants were computed for formed ETP-HSA complex. The dominant interactive forces for ETP and HSA binding are hydrogen bonds join up with van der Waals extent possibly at site III (IB). The presence of Ca2+, Co2+, Na+, Mg2+ and Fe3+ ions significantly affected binding ability of ETP towards HSA. The essentialness of this investigation is beneficial in life sciences, medicinal chemistry, pharmaceutical industry and clinical medicine.

Serotonergic treatment normalizes midbrain dopaminergic neuron increase after periaqueductal gray stimulation.

Electrical stimulation of the dorsolateral periaqueductal gray (dlPAG) in rats has been shown to elicit panic-like behaviour and can be a useful as an unconditioned stimulus for modelling anticipatory fear and agoraphobia in a contextual fear conditioning paradigm. In this study, we further analysed our previous data on the effects of escitalopram (a selective serotonin reuptake inhibitor, SSRI) and buspirone (a 5-HT1A receptor partial agonist) on dlPAG-induced anticipatory fear behaviour in a rat model using freezing as a measure. We then attempted to unravel some of the interactions with dopamine signalling using tyrosine hydroxylase (TH) immunohistochemistry to probe the effects on dopaminergic neurons. We showed that acute treatment of escitalopram, but not buspirone, was effective in reducing anticipatory freezing behaviour, while chronic administrations of both drugs were effective. We found that the dlPAG stimulation induced increase number of dopaminergic neurons in the ventral tegmental area (VTA) which was reversed in both chronic buspirone and escitalopram groups. We further found a strong positive correlation between the number of dopaminergic neurons and freezing in the VTA and showed positive correlations between dopaminergic neurons in the VTA and substantia nigra pars compacta (SNpc) in escitalopram and buspirone groups, respectively. Overall, we showed that chronic treatment with an SSRI and a 5-HT1A agonist reduced anticipatory freezing behaviour which seems to be associated, through correlative studies, with a reversal of dlPAG stimulation induced increase in number of dopaminergic neurons in the VTA and/or SNpc.

The Serotonin Neurotransmitter Modulates Virulence of Enteric Pathogens

The gut-brain axis is crucial to microbial-host interactions. The neurotransmitter serotonin is primarily synthesized in the gastrointestinal (GI) tract, where it is secreted into the lumen and subsequently removed by the serotonin transporter, SERT. Here, we show that serotonin decreases virulence gene expression by enterohemorrhagic E.coli (EHEC) and Citrobacter rodentium, a murine model for EHEC. The membrane-bound histidine sensor kinase, CpxA, is a bacterial serotonin receptor. Serotonin induces dephosphorylation of CpxA, which inactivates the transcriptional factor CpxR controlling expression of virulence genes, notably those within the locus of enterocyte effacement (LEE). Increasing intestinal serotonin by genetically or pharmacologically inhibiting SERT decreases LEE expression and reduces C.rodentium loads. Conversely, inhibiting serotonin synthesis increases pathogenesis and decreases host survival. As other enteric bacteria contain CpxA, this signal exploitation may be engaged by other pathogens. Additionally, repurposing serotonin agonists to inhibit CpxA may represent a potential therapeutic intervention for enteric bacteria.

Restoration of Motor Functions in Spinal Rats by Electrical Stimulation of the Spinal Cord and Locomotor Training

The recovery of motor functions in chronic spinal rats with locomotor training on a treadmill combined with electrical stimulation of the spinal cord was studied. Training to a bipedal gait on a moving band with the body weight supported and use of either subcutaneous or epidural electrical stimulation (40 Hz) was performed for five days per week for 20–30 min. The dynamics of changes in locomotor capacity using subcutaneous and epidural electrical stimulation and their actions on the neural structures of the spinal cord could be similar. After three weeks of motor rehabilitation, a single stimulation at a frequency of 1 Hz evoked reflex monosynaptic potentials in the hindlimb muscles, while simultaneous rhythmic stimulation of two loci in the spinal cord at a frequency of 40 Hz initiated locomotor-like activity on the moving band of the treadmill. A more marked rhythm was seen after nine weeks of training, which coincided with the appearance of polysynaptic spinal reflexes. Administration of the serotonin receptor agonist quipazine enhanced polysynaptic activity in reflex responses and improved locomotion. Use of noninvasive subcutaneous stimulation in combination with locomotor training was found to be an effective method of activating neural locomotor networks to an extent comparable to that obtained with invasive epidural stimulation.

0148 Serotonergic Dorsal Raphe Neurons Regulate Hypercapnia Induced Arousal Through 5HT2A Receptors on the Parabrachial Neurons

Abstract Introduction Serotoninergic dorsal raphe neurons (DRSert) are CO2 responsive, and mice lacking serotonin have impaired arousal to CO2. We showed that the neurons in external lateral parabrachial nucleus containing calcitonin gene related peptide (PBelCGRP), are required for CO2-arousal. PBelCGRP neurons also receive serotoninergic innervation from the DRSert. 5HT2A agonist restores CO2 responsiveness in mice lacking serotonin, suggesting that DRSert may modulate CO2 arousal by acting on 5HT2A receptors possibly on the PBel neurons. Methods We used serotonin transporter (Sert)-Cre mice to optogenetically inhibit DRSert neurons and their terminals in the PBel. We injected AAV-FLEX-ArchT into the DR and implanted an optical fiber just above it in one set of Sert-Cre mice and bilaterally in the PBel in another set. All mice were instrumented for sleep and optogenetics and were tested for EEG arousals to 10% CO2. Latencies of arousal were compared with optogenetic inhibition of either the DR neurons or their terminals in the PBel with a 593nm laser light. We further tested whether a 5HT2A agonist (TCB-2) can reverse blockade of CO2 arousal in mice where DRSert terminals in PBel were inhibited. Finally, TCB-2 was injected in mice with PBelCGRP deletions and arousal latency to CO2 was compared. Results Compared to the control (Laser-OFF) condition, arousal latency to CO2 was significantly increased by photoinhibition of either the DRSert neurons (n=6; latency- 40.9 ± 6.4 vs. 13.81± 0.69 sec; F3, 17= 11.5; P< 0.001) or their terminals in PBel (n=8; latency-34.9 ± 2.3 sec vs. 16.62 ± 0.97sec, F1, 14= 56.9; P< 0.001). This was reversed by the 5HT2A agonist TCB-2 (5mg/kg), as it reduced the latency to CO2 arousal in mice with photoinhibition of terminals in PBel from 35.48 ± 7.31 sec to 16.24 ± 1.06 sec (F3, 9= 8.05; P= 0.006), but had no effect in mice with PBelCGRP neurons deletions. Conclusion The serotonin system modulate CO2-arousals by the DRSert input to the PBel. TCB-2 reversed the effect of inhibition of DRSert terminals in the PBel, but not in mice with PBelCGRP deletions, suggests that DRSert modulate PBelCGRP neurons through 5HT2a receptors. Support NIH- 2P01 HL095491 and NS112175

Validation of canine uterine and testicular arteries for the functional characterisation of receptor-mediated contraction as a replacement for laboratory animal tissues in teaching.

Teaching practicals for receptor physiology/pharmacology in medical and veterinary schools have involved the use of in vitro experiments using tissues from laboratory animals, which have been killed for isolated vascular strip or ring preparations. However, the use of scavenged tissues has been advocated to reduce animal use. Utilising discarded tissues from routine surgical procedures, such as canine neutering, has not previously been investigated. Canine testicular and uterine tissues (discarded tissues) were obtained from routine neutering procedures performed by the veterinary team at a local animal neutering clinic for stray dogs. Rings of uterine and testicular artery were dissected and mounted on a Mulvany-Halpern wire myograph in order to characterize the adrenergic and serotonergic receptors mediating vasoconstriction. Cumulative contractile concentration-response curves were constructed for the alpha adrenoceptor agonists epinephrine (α1 and α2 receptors), phenylephrine (α1 selective) and UK14304 (α2 selective). Pre-treatment with the α1-selective antagonist, prazosin, was also investigated. The response to serotonin (5-HT) receptor agonists were also investigated, including 5-HT (acting at both 5-HT1 and 5-HT2 receptors), 5-carboxamidotryptamine (5-CT; 5-HT1 selective) and α-methyl 5-HT (5-HT2 selective). A contractile response was observed in both canine uterine and testicular arteries to epinephrine and phenylephrine, and prazosin caused a dose-dependent parallel rightward shift in the phenylephrine dose-response curve (pA2 values of 7.97 and 8.39, respectively). UK14304 caused a contractile response in canine testicular arteries but very little appreciable contractile response in uterine arteries. The maximum responses produced by the uterine arteries to 5-HT was significantly lower than those of the testicular arteries. In the testicular artery, the 5-HT2 receptor selective agonist, α-methyl 5-HT, produced a similar contractile response to 5-HT but the administration of 5-CT failed to produce a response in either the testicular or uterine artery segments. These results validate the use of discarded tissue from routine canine neutering procedures as a useful source of vascular tissue for pharmacological teaching, for characterizing alpha and 5-HT receptor contractile responses.