Effects of 2,5-Dimethoxy-4-Methylamphetamine (DOM) and 2-Piperazin-1-yl-Quinoline (Quipazine) on Fentanyl Versus Food Choice in Rhesus Monkeys.

There has been increasing interest in the potential therapeutic effects of drugs with agonist properties at serotonin 2A subtype (5-HT2A) receptors (e.g., psychedelics), including treatment of substance use disorders. Studying interactions between 5-HT2A receptor agonists and other drugs is important for understanding potential therapeutic effects as well as adverse interactions. Direct-acting 5-HT2A receptor agonists such as 2,5-dimethoxy-4-methylamphetamine (DOM) and 2-piperazin-1-yl-quinoline (quipazine) enhance some (e.g., antinociceptive) effects of opioids; however, it is unclear whether they alter the abuse-related effects of opioids. This study examined whether DOM and quipazine alter the reinforcing effects of fentanyl in rhesus monkeys (n = 6) responding under a food versus drug choice procedure. Responding on one lever delivered sucrose pellets and responding on the other lever delivered intravenous (i.v.) infusions. In one set of experiments, fentanyl (0.1-3.2 µg/kg/infusion) versus food choice sessions were preceded by noncontingent i.v. pretreatments with DOM (0032-0.32 mg/kg), quipazine (0.32-1.0 mg/kg), naltrexone (0.032 mg/kg), or heroin (0.1 mg/kg). In another set of experiments, fentanyl was available during choice sessions in combination with DOM (0.32-100 µg/kg/infusion) or quipazine (3.2-320 µg/kg/infusion) in varying dose ratios. Naltrexone decreased and heroin increased fentanyl choice, demonstrating sensitivity of responding to pharmacological manipulation. However, whether given as a pretreatment or made available in combination with fentanyl as a mixture, neither DOM nor quipazine significantly altered fentanyl choice. These results suggest that 5-HT2A receptor agonists do not enhance the reinforcing effects of opioids and, thus, will not likely enhance abuse potential. SIGNIFICANCE STATEMENT: Serotonin 2A subtype receptor agonists enhance some (e.g., antinociceptive) effects of opioids, suggesting they could be combined with opioids in some therapeutic contexts such as treating pain. However, it is unclear whether they also enhance adverse effects of opioids, including abuse. Results of this study indicate that serotonin 2A subtype receptor agonists do not reliably enhance opioid self-administration and, thus, are unlikely to enhance the abuse potential of opioids.

Acute intrathecal administration of quipazine elicits air-stepping behavior.

Serotonin plays a pivotal role in the initiation and modulation of locomotor behavior in the intact animal, as well as following spinal cord injury. Quipazine, a serotonin 2 receptor agonist, has been used successfully to initiate and restore motor behavior in rodents. Although evidence suggests that the effects of quipazine are spinally mediated, it is unclear whether intrathecal (IT) quipazine administration alone is enough to activate locomotor-like activity or whether additional stimulation is needed. Thus, the current study examined the effects of IT administration of quipazine in postnatal day 1 rats in two separate experiments. In experiment 1, quipazine (0.1, 0.3, or 1.0 mg/kg) was dissolved in saline and administered via IT injection to the thoracolumbar cord. There was no significant effect of drug on hindlimb alternating stepping. In experiment 2, quipazine (0.3 or 1.0 mg/kg) was dissolved in a polysorbate 80-saline solution (Tween 80) and administered via IT injection. Polysorbate 80 was used to disrupt the blood-brain barrier to facilitate absorption of quipazine. The injection was followed by tail pinch 5 minutes post-injection. A significant increase in the percentage of hindlimb alternating steps was found in subjects treated with 0.3 mg/kg quipazine, suggesting that IT quipazine when combined with sensory stimulation to the spinal cord, facilitates locomotor-like behavior. These findings indicate that dissolving the drug in polysorbate 80 rather than saline may heighten the effects of IT quipazine. Collectively, this study provides clarification on the role of quipazine in evoking spinally-mediated locomotor behavior.

Quipazine Elicits Swallowing in the Arterially Perfused Rat Preparation: A Role for Medullary Raphe Nuclei?

Pharmacological neuromodulation of swallowing may represent a promising therapeutic option to treat dysphagia. Previous studies suggested a serotonergic control of swallowing, but mechanisms remain poorly understood. Here, we investigated the effects of the serotonergic agonist quipazine on swallowing, using the arterially perfused working heart-brainstem (in situ) preparation in rats. Systemic injection of quipazine produced single swallows with motor patterns and swallow-breathing coordination similar to spontaneous swallows, and increased swallow rate with moderate changes in cardiorespiratory functions. Methysergide, a 5-HT2 receptor antagonist, blocked the excitatory effect of quipazine on swallowing, but had no effect on spontaneous swallow rate. Microinjections of quipazine in the nucleus of the solitary tract were without effect. In contrast, similar injections in caudal medullary raphe nuclei increased swallow rate without changes in cardiorespiratory parameters. Thus, quipazine may exert an excitatory effect on raphe neurons via stimulation of 5-HT2A receptors, leading to increased excitability of the swallowing network. In conclusion, we suggest that pharmacological stimulation of swallowing by quipazine in situ represents a valuable model for experimental studies. This work paves the way for future investigations on brainstem serotonergic modulation, and further identification of neural populations and mechanisms involved in swallowing and/or swallow-breathing interaction.

Electrophysiological biomarkers of neuromodulatory strategies to recover motor function after spinal cord injury.

The spinal cord contains the circuitry to control posture and locomotion after complete paralysis, and this circuitry can be enabled with epidural stimulation [electrical enabling motor control (eEmc)] and/or administration of pharmacological agents [pharmacological enabling motor control (fEmc)] when combined with motor training. We hypothesized that the characteristics of the spinally evoked potentials after chronic administration of both strychnine and quipazine under the influence of eEmc during standing and stepping can be used as biomarkers to predict successful motor performance. To test this hypothesis we trained rats to step bipedally for 7 wk after paralysis and characterized the motor potentials evoked in the soleus and tibialis anterior (TA) muscles with the rats in a non-weight-bearing position, standing and stepping. The middle responses (MRs) to spinally evoked stimuli were suppressed with either or both drugs when the rat was suspended, whereas the addition of either or both drugs resulted in an overall activation of the extensor muscles during stepping and/or standing and reduced the drag duration and cocontraction between the TA and soleus muscles during stepping. The administration of quipazine and strychnine in concert with eEmc and step training after injury resulted in larger-amplitude evoked potentials [MRs and late responses (LRs)] in flexors and extensors, with the LRs consisting of a more normal bursting pattern, i.e., randomly generated action potentials within the bursts. This pattern was linked to more successful standing and stepping. Thus it appears that selected features of the patterns of potentials evoked in specific muscles with stimulation can serve as effective biomarkers and predictors of motor performance.

On the Basicity of 8-Phenylsulfanyl Quipazine Derivatives: New Potential Serotonergic Agents.

A protonation state of serotonergic ligands plays a crucial role in their pharmacological activity. In this research, the basicity of 8-phenylsulfanyl quipazine derivatives as new potential serotonergic agents was studied. The most favorable protonation sites were determined in the gas and aqueous phases. In water, a solvation effect promoting the protonation of the N3 atom overcomes a positive charge delocalization phenomenon favoring a N1 atom protonation. The most stable conformations of neutral and protonated molecules in gas and water were found. It was demonstrated that a diprotonation reaction may occur. The most favorable among the diprotonated structures is the molecule with the N1 and N3 atoms protonated. A calculation of the pKa and pKa2 in water of a set of monosubstituted 8-phenylsulfanyl quipazine derivatives was performed using B3LYP/6-31G(d) and the SMD continuum solvation model. Enthalpic and entropic contributions to the pKa and pKa2 in gas and water were separated for a rationalization of a substituent effect on values of the pKa and pKa2. The relationship of the proton affinity and the solvation enthalpy in water with some reactivity descriptors, such as the Fukui function, the molecular electrostatic potential (MEP), and the global softness, was investigated. The order of the pKa values is the most controlled by the entropy. The diprotonation reaction, despite having an unfavorable enthalpy in water, is driven entropically. Final state effects in the diprotonated species were analyzed with the triadic formula. Results of a calculation of the theoretical basicity of the 8-phenylsulfanyl quipazines indicate that they should be monoprotonated on the N3 atom in the CNS environment. Diprotonation of the studied compounds may occur in very acidic body fluids such as the gastric juice.

Neuromodulation of motor-evoked potentials during stepping in spinal rats.

The rat spinal cord isolated from supraspinal control via a complete low- to midthoracic spinal cord transection produces locomotor-like patterns in the hindlimbs when facilitated pharmacologically and/or by epidural electrical stimulation. To evaluate the role of epidural electrical stimulation in enabling motor control (eEmc) for locomotion and posture, we recorded potentials evoked by epidural spinal cord stimulation in selected hindlimb muscles during stepping and standing in adult spinal rats. We hypothesized that the temporal details of the phase-dependent modulation of these evoked potentials in selected hindlimb muscles while performing a motor task in the unanesthetized state would be predictive of the potential of the spinal circuitries to generate stepping. To test this hypothesis, we characterized soleus and tibialis anterior (TA) muscle responses as middle response (MR; 4-6 ms) or late responses (LRs; >7 ms) during stepping with eEmc. We then compared these responses to the stepping parameters with and without a serotoninergic agonist (quipazine) or a glycinergic blocker (strychnine). Quipazine inhibited the MRs induced by eEmc during nonweight-bearing standing but facilitated locomotion and increased the amplitude and number of LRs induced by eEmc during stepping. Strychnine facilitated stepping and reorganized the LRs pattern in the soleus. The LRs in the TA remained relatively stable at varying loads and speeds during locomotion, whereas the LRs in the soleus were strongly modulated by both of these variables. These data suggest that LRs facilitated electrically and/or pharmacologically are not time-locked to the stimulation pulse but are highly correlated to the stepping patterns of spinal rats.

Removal of supraspinal input reveals a difference in the flexor and extensor monosynaptic reflex response to quipazine independent of motoneuron excitation.

The purpose of this study was to determine if quipazine, a serotonergic agonist, differentially modulates flexor and extensor motor output. This was achieved by examining the monosynaptic reflex (MSR) of the tibial (extensor) and peroneal (flexor) nerves, by determining the basic and rhythmic properties of extensor and flexor motoneurons, and by recording extracellular Ia field potentials of the tibial and peroneal nerves in the in vivo adult decerebrate rat in both spinal intact and acute spinalized preparations. In the spinal intact preparation, the tibial and peroneal MSR amplitude significantly increased compared with baseline in response to quipazine, with no difference between nerves (P < 0.05). In the spinalized preparation, the MSR was significantly increased in both the tibial and peroneal nerves with the latter increasing more than the former (5.7 vs. 3.6 times; P < 0.05). Intracellular motoneuron experiments demonstrated that rheobase decreased, while input resistance, afterhyperpolarization amplitude, and the firing rate at a given current injection increased in motoneurons following quipazine administration with no differences between extensor and flexor motoneurons. Both the tibial and peroneal nerve extracellular Ia field potentials increased with the peroneal demonstrating a significantly greater increase (7 vs. 38%; P < 0.05) following quipazine. It is concluded that in the spinal intact preparation quipazine does not have a differential effect on flexor or extensor motor output. However, in the acute spinalized preparation, quipazine preferentially affects the flexor MSR compared with the extensor MSR, likely due to the removal of a descending tonic inhibition on flexor Ia afferents.

Neonatal +-methamphetamine exposure in rats alters adult locomotor responses to dopamine D1 and D2 agonists and to a glutamate NMDA receptor antagonist, but not to serotonin agonists.

Neonatal exposure to (+)-methamphetamine (Meth) results in long-term behavioural abnormalities but its developmental mechanisms are unknown. In a series of experiments, rats were treated from post-natal days (PD) 11-20 (stage that approximates human development from the second to third trimester) with Meth or saline and assessed using locomotor activity as the readout following pharmacological challenge doses with dopamine, serotonin and glutamate agonists or antagonists during adulthood. Exposure to Meth early in life resulted in an exaggerated adult locomotor hyperactivity response to the dopamine D1 agonist SKF-82958 at multiple doses, a high dose only under-response activating effect of the D2 agonist quinpirole, and an exaggerated under-response to the activating effect of the N-methyl-d-aspartic acid (NMDA) receptor antagonist, MK-801. No change in locomotor response was seen following challenge with the 5-HT releaser p-chloroamphetamine or the 5-HT2/3 receptor agonist, quipazine. These are the first data to show that PD 11-20 Meth exposure induces long-lasting alterations to dopamine D1, D2 and glutamate NMDA receptor function and may suggest how developmental Meth exposure leads to many of its long-term adverse effects.

The upright posture improves plantar stepping and alters responses to serotonergic drugs in spinal rats.

Recent studies on the restoration of locomotion after spinal cord injury have employed robotic means of positioning rats above a treadmill such that the animals are held in an upright posture and engage in bipedal locomotor activity. However, the impact of the upright posture alone, which alters hindlimb loading, an important variable in locomotor control, has not been examined. Here we compared the locomotor capabilities of chronic spinal rats when placed in the horizontal and upright postures. Hindlimb locomotor movements induced by exteroceptive stimulation (tail pinching) were monitored with video and EMG recordings. We found that the upright posture alone significantly improved plantar stepping. Locomotor trials using anaesthesia of the paws and air stepping demonstrated that the cutaneous receptors of the paws are responsible for the improved plantar stepping observed when the animals are placed in the upright posture.We also tested the effectiveness of serotonergic drugs that facilitate locomotor activity in spinal rats in both the horizontal and upright postures. Quipazine and (±)-8-hydroxy-2-(dipropylamino)tetralin hydrobromide (8-OH-DPAT) improved locomotion in the horizontal posture but in the upright posture either interfered with or had no effect on plantar walking. Combined treatment with quipazine and 8-OH-DPAT at lower doses dramatically improved locomotor activity in both postures and mitigated the need to activate the locomotor CPG with exteroceptive stimulation. Our results suggest that afferent input from the paw facilitates the spinal CPG for locomotion. These potent effects of afferent input from the paw should be taken into account when interpreting the results obtained with rats in an upright posture and when designing interventions for restoration of locomotion after spinal cord injury.

Mass-spectrometric studies of new 6-nitroquipazines-serotonin transporter inhibitors.

Six synthesized 6-nitroquipazine derivatives were examined by electron ionization (EI) and electrospray ionization (ESI) mass spectrometry in positive and negative ion mode. The compounds exhibit high affinity for the serotonin transporter (SERT) and belong to a new class of SERT inhibitors. The EI mass spectra registered in negative ion mode showed prominent molecular ions for all the compounds studied. All EI mass spectra and all ESI mass spectra showed similar fragmentation pathways of molecular ions, but the pathways differed between EI and ESI. The differences were explained with the aid of theoretical evaluation of the stability of the respective radical ions (EI MS) and protonated ions (ESI MS).

Motor deficits and recovery in rats with unilateral spinal cord hemisection mimic the Brown-Sequard syndrome.

Cervical incomplete spinal cord injuries often lead to severe and persistent impairments of sensorimotor functions and are clinically the most frequent type of spinal cord injury. Understanding the motor impairments and the possible functional recovery of upper and lower extremities is of great importance. Animal models investigating motor dysfunction following cervical spinal cord injury are rare. We analysed the differential spontaneous recovery of fore- and hindlimb locomotion by detailed kinematic analysis in adult rats with unilateral C4/C5 hemisection, a lesion that leads to the Brown-Séquard syndrome in humans. The results showed disproportionately better performance of hindlimb compared with forelimb locomotion; hindlimb locomotion showed substantial recovery, whereas the ipsilesional forelimb remained in a very poor functional state. Such a differential motor recovery pattern is also known to occur in monkeys and in humans after similar spinal cord lesions. On the lesioned side, cortico-, rubro-, vestibulo- and reticulospinal tracts and the important modulatory serotonergic, dopaminergic and noradrenergic fibre systems were interrupted by the lesion. In an attempt to facilitate locomotion, different monoaminergic agonists were injected intrathecally. Injections of specific serotonergic and noradrenergic agonists in the chronic phase after the spinal cord lesion revealed remarkable, although mostly functionally negative, modulations of particular parameters of hindlimb locomotion. In contrast, forelimb locomotion was mostly unresponsive to these agonists. These results, therefore, show fundamental differences between fore- and hindlimb spinal motor circuitries and their functional dependence on remaining descending inputs and exogenous spinal excitation. Understanding these differences may help to develop future therapeutic strategies to improve upper and lower limb function in patients with incomplete cervical spinal cord injuries.

Why variability facilitates spinal learning.

Spinal Wistar Hannover rats trained to step bipedally on a treadmill with manual assistance of the hindlimbs have been shown to improve their stepping ability. Given the improvement in motor performance with practice and the ability of the spinal cord circuitry to learn to step more effectively when the mode of training allows variability, we examined why this intrinsic variability is an important factor. Intramuscular EMG electrodes were implanted to monitor and compare the patterns of activation of flexor (tibialis anterior) and extensor (soleus) muscles associated with a fixed-trajectory and assist-as-needed (AAN) step training paradigms in rats after a complete midthoracic (T8-T9) spinal cord transection. Both methods involved a robotic arm attached to each ankle of the rat to provide guidance during stepping. The fixed trajectory allowed little variance between steps, and the AAN provided guidance only when the ankle deviated a specified distance from the programmed trajectory. We hypothesized that an AAN paradigm would impose fewer disruptions of the control strategies intrinsic to the spinal locomotor circuitry compared with a fixed trajectory. Intrathecal injections of quipazine were given to each rat to facilitate stepping. Analysis confirmed that there were more corrections within a fixed-trajectory step cycle and consequently there was less coactivation of agonist and antagonist muscles during the AAN paradigm. These data suggest that some critical level of variation in the specific circuitry activated and the resulting kinematics reflect a fundamental feature of the neural control mechanisms even in a highly repetitive motor task.

Cholesterol and lipid phases influence the interactions between serotonin receptor agonists and lipid bilayers.

Solid state NMR techniques have been used to investigate the effect that two serotonin receptor 1a agonists (quipazine and LY-165,163) have on the phase behavior of, and interactions within, cholesterol/phosphocholine lipid bilayers. The presence of agonist, and particularly LY-165,163, appears to widen the phase transitions, an effect that is much more pronounced in the presence of cholesterol. It was found that both agonists locate close to the cholesterol, and their interactions with the lipids are modulated by the lipid phases. As the membrane condenses into mixed liquid-ordered/disordered phases, quipazine is pushed up toward the surface of the bilayer, whereas LY-165,163 moves deeper into the lipid chain region. In light of our results, we discuss the role of lipid/drug interactions on drug efficacy.

Psychedelic-like Properties of Quipazine and Its Structural Analogues in Mice.

Known classic psychedelic serotonin 2A receptor (5-HT2AR) agonists retain a tryptamine or phenethylamine at their structural core. However, activation of the 5-HT2AR can be elicited by drugs lacking these fundamental scaffolds. Such is the case of the N-substituted piperazine quipazine. Here, we show that quipazine bound to and activated 5-HT2AR as measured by [3H]ketanserin binding displacement, Ca2+ mobilization, and accumulation of the canonical Gq/11 signaling pathway mediator inositol monophosphate (IP1) in vitro and in vivo. Additionally, quipazine induced via 5-HT2AR an expression pattern of immediate early genes (IEG) in the mouse somatosensory cortex consistent with that of classic psychedelics. In the mouse head-twitch response (HTR) model of psychedelic-like action, quipazine produced a lasting effect with high maximal responses during the peak effect that were successfully blocked by the 5-HT2AR antagonist M100907 and absent in 5-HT2AR knockout (KO) mice. The acute effect of quipazine on HTR appeared to be unaffected by serotonin depletion and was independent from 5-HT3R activation. Interestingly, some of these features were shared by its deaza bioisostere 2-NP, but not by other closely related piperazine congeners, suggesting that quipazine might represent a distinct cluster within the family of psychoactive piperazines. Together, our results add to the mounting evidence that quipazine's profile matches that of classic psychedelic 5-HT2AR agonists at cellular signaling and behavioral pharmacology levels.

Lysergic acid diethylamide (LSD) and lisuride: differentiation of their neuropharmacological actions.

The nonhallucinogenic ergot derivative lisuride exerts many pharmacological effects that are similar to those of its hallucinogenic congener, lysergic acid diethylamide (LSD). Animals trained to discriminate between the presence of one drug and the other can be used to differentiate the actions of these compounds on a neuronal level. The discriminative stimulus effect of LSD (the LSD cue) is similar to that of the serotonin agonist quipazine, whereas the lisuride cue is similar to that of the dopamine agonist apomorphine. These data support the hypothesis that serotonin is intricately involved in the hallucinogenic effects of LSD.

Effects of serotonergic agents on survival and hemolymph composition of the larval mosquito Aedes aegypti (Diptera: Culicidae, L.) in vivo: does serotonin regulate hemolymph acid-base homeostasis?

The role of serotonin in the regulation of larval Aedes aegypti hemolymph composition was investigated in vivo using two reuptake inhibitors (SSRIs), alaproclate HCl and 6-nitroquipazine maleate, and the receptor antagonist methiothepin mesylate. Larvae were placed in media differing in pH and salinity in the presence and absence of serotonergic agents. The toxicity of each agent was strongly influenced by ambient pH. For each agent, toxicity was negligible in acidic media, intermediate in neutral media and greatest in alkaline media. By contrast, toxicity of all agents was independent of salinity. No effects on mass-specific body water or hemolymph volume were observed whereas hemolymph osmotic pressure, Na(+) concentrations and pH differed significantly among treatments. 6-nitroquipazine caused a decrease in Na(+) from 115+/-1.7 to 103+/-0.9 mmol l(-1), and alaproclate caused alkalosis of the hemolymph from pH 7.55+/-0.026 to pH 7.72+/-0.044. Methiothepin decreased hemolymph osmotic pressure from 329+/-9.9 to 304+/-8.8 and showed the greatest overall toxicity. Control larvae excreted net base in pH 4 media (1.4 micromol g(-1) h(-1)) and net acid in pH 7 (1.2 micromol g(-1) h(-1)) and pH 11 (5.1 micromol g(-1) h(-1)) media. In pH 4 media, alaproclate and methiothepin caused a shift to net H(+) excretion (1.1 and 1.5 micromol g(-1) h(1), respectively) whereas these agents did not influence acid excretion rates in pH 7 or pH 11 media. The hypothesis that serotonin is involved in hemolymph acid-base balance is discussed.

Synthesis and preliminary screening of novel N-{2-[4-(substituted)piperazin-1-yl]-2-oxoethyl}acetamides as potential atypical antipsychotic agents.

A series of N-{2-[4-(substituted)piperazin-1-yl]-2-oxoethyl}acetamides were synthesized as prospective novel atypical antipsychotic agents. Microwave irradiation of acetyl glycine (I) with substituted piperazines in the presence of DCC in DMF for about 3-5 min gave the titled compounds (P:1-7). All the synthesized compounds were screened for their in vivo pharmacological activity in Swiss albino mice. D(2) antagonism studies were performed using the climbing mouse assay model and 5-HT(2A) antagonism studies were performed using quipazine induced head twitches in mice. Among the synthesized compounds P4 was found to be the most active compound.

Serotonin receptor agonist quipazine promotes proliferation and apoptosis of human hepatocyte strain of L-02 strain.

BACKGROUND: Liver disease is commonly seen in the clinic and its pathological characteristic is combined hepatocellular death and apoptosis. Promoting hepatocyte regeneration is one of the main methods of treating liver disease. Serotonin (5-HT) is an important compound which participates in various life process, and 95% of it is carried by platelets in the blood. A recent finding showed that platelet-derived serotonin is the key factor in liver regeneration, which fails without serotonin. This study aimed to investigate the effects of quipazine, a selective 5-HT receptor agonist, on proliferation and apoptosis in the human hepatocyte strain L-02. METHODS: L-02 cells were cultured in medium with 5-HT and quipazine, and samples were collected at 24, 48, and 72 hours. The methyl thiazolyl tetrazolium (MTT) method was used to test viability, flow cytometry to assess the cell cycle, the Annexin-V/PI method to evaluate apoptosis, and immunohistochemistry to detect proliferating cell nuclear antigen (PCNA). RESULTS: Compared with the control group, the viability of L-02 cells was improved in the 10, 50, and 250 microg/ml quipazine groups (P<0.05); the percentage of S-phase and PCNA-positive cells were increased in the 2, 10, 50, and 250 microg/ml quipazine groups (P>0.05); and no difference in the percentage of apoptotic cells was found between the 50 microg/ml quipazine and control groups (P>0.05). CONCLUSION: Quipazine improves proliferation of a human hepatocyte strain in vitro, and this is not based on the inhibition of apoptosis.

The Serotonergic Raphe Promote Sleep in Zebrafish and Mice.

The role of serotonin (5-HT) in sleep is controversial: early studies suggested a sleep-promoting role, but eventually the paradigm shifted toward a wake-promoting function for the serotonergic raphe. Here, we provide evidence from zebrafish and mice that the raphe are critical for the initiation and maintenance of sleep. In zebrafish, genetic ablation of 5-HT production by the raphe reduces sleep, sleep depth, and the homeostatic response to sleep deprivation. Pharmacological inhibition or ablation of the raphe reduces sleep, while optogenetic stimulation increases sleep. Similarly, in mice, ablation of the raphe increases wakefulness and impairs the homeostatic response to sleep deprivation, whereas tonic optogenetic stimulation at a rate similar to baseline activity induces sleep. Interestingly, burst optogenetic stimulation induces wakefulness in accordance with previously described burst activity of the raphe during arousing stimuli. These results indicate that the serotonergic system promotes sleep in both diurnal zebrafish and nocturnal rodents. VIDEO ABSTRACT.

Role of spinal 5-HT2 receptor subtypes in quipazine-induced hindlimb movements after a low-thoracic spinal cord transection.

A role of serotonin receptors (5-HTRs) in spinal rhythmogenesis has been proposed several years ago based mainly upon data showing that bath-applied 5-HT could elicit locomotor-like rhythms in in vitro isolated spinal cord preparations. Such a role was partially confirmed in vivo after revealing that systemically administered 5-HTR(2) agonists, such as quipazine, could induce some locomotor-like movements (LM) in completely spinal cord-transected (Tx) rodents. However, given the limited binding selectivity of currently available 5-HTR(2) agonists, it has remained difficult to determine clearly if one receptor subtype is specifically associated with LM induction. In situ hybridization, data using tissues from L1-L2 spinal cord segments, where critical locomotor network elements have been identified in mice, revealed greater 5-HTR(2A) mRNA levels in low-thoracic Tx than non-Tx animals. This expression level remained elevated for several days, specifically in the lateral intermediate zone, where peak values were detected at 1 week post-Tx and returned to normal at 3 weeks post-Tx. Behavioral and kinematic analyses revealed quipazine-induced LM in 1-week Tx mice either non-pretreated or pretreated with selective 5-HTR(2B) and/or 5-HTR(2C) antagonists. In contrast, LM completely failed to be induced by quipazine in animals pretreated with selective 5-HTR(2A) antagonists. Altogether, these results provide strong evidence suggesting that 5-HTR(2A) are specifically associated with spinal locomotor network activation and LM generation induced by quipazine in Tx animals. These findings may contribute to design drug treatments aimed at promoting locomotor function recovery in chronic spinal cord-injured patients.