Omeprazole affects the expression of serotonin-1A in the brain regions and alleviates anxiety in rat model of immobilization-induced stress.

Omeprazole, a drug of choice for the management of gastric hyperacidity, influences serotonergic neurotransmission in brain regions and its long-term use is known to cause stress-related behavioral deficits including anxiety. Aim of the current study was to explore the effects of omeprazole treatment on immobilization-induced anxiety in rats, specifically on the role of serotonin (5-HT). In view of the role of serotonin-1A (5-HT1A) autoreceptor in the availability of 5-HT in brain regions, mRNA expression of this autoreceptor was performed in raphe nuclei. Similarly, because of the role of hippocampal 5-HT neurotransmission in anxiety-like disorders, expression of the 5-HT1A heteroreceptors was determined in this region. We found that the treatment with omeprazole reduces anxiety-like behavior in rats, increases the expression of 5-HT1A autoreceptor in the raphe and decreases the hippocampal expression of 5-HT1A heteroreceptor. This suggests a role of 5-HT1A receptor types in omeprazole-induced behavioral changes. It also indicates a potential role of omeprazole in the management of serotonergic disorders.

Human corticospinal-motoneuronal output is reduced with 5-HT2 receptor antagonism.

Animal models indicate that serotonin (5-HT) release onto motoneurons facilitates motor output, particularly during strong motor activities. However, evidence for 5-HT effects during human movement are limited. This study examined how antagonism of the 5-HT2 receptor, which is a 5-HT receptor that promotes motoneuron excitability, affects human movement. Ten healthy participants (24.2 ± 1.9 yr) ingested 8 mg of cyproheptadine (competitive 5-HT2 antagonist) in a double-blinded, placebo-controlled, repeated-measures design. Transcranial magnetic stimulation (TMS) of the motor cortex was used to elicit motor evoked potentials (MEPs) from biceps brachii. First, stimulus-response curves (90%-160% active motor threshold) were obtained during very weak elbow flexions (10% of maximal). Second, to determine if 5-HT effects are scaled to the intensity of muscle contraction, TMS at a fixed intensity was applied during elbow flexions of 20%, 40%, 60%, 80%, and 100% of maximal. Cyproheptadine reduced the size of MEPs across the stimulus-response curves (P = 0.045). Notably, MEP amplitude was 22.3% smaller for the cyproheptadine condition for the strongest TMS intensity. In addition, cyproheptadine reduced maximal torque (P = 0.045), lengthened the biceps silent period during maximal elbow flexions (P = 0.037), and reduced superimposed twitch amplitude during moderate-intensity elbow flexions (P = 0.035). This study presents novel evidence that 5-HT2 receptors influence corticospinal-motoneuronal output, which was particularly evident when a large number of descending inputs to motoneurons were active. Although it is likely that antagonism of 5-HT2 receptors reduces motoneuron gain to ionotropic inputs, supraspinal mechanisms may have also contributed to the study findings.NEW & NOTEWORTHY Voluntary contractions and responses to magnetic stimulation of the motor cortex are dependent on serotonin activity in the central nervous system. 5-HT2 antagonism decreased evoked potential size to high-intensity stimulation, and reduced torque and lengthened inhibitory silent periods during maximal contractions. We provide novel evidence that 5-HT2 receptors are involved in muscle activation, where 5-HT effects are strongest when a large number of descending inputs activate motoneurons.

Neuronal mechanisms of adenosine A2A receptors in the loss of consciousness induced by propofol general anesthesia with functional magnetic resonance imaging.

Propofol is the most common intravenous anesthetic agent for induction and maintenance of anesthesia, and has been used clinically for more than 30 years. However, the mechanism by which propofol induces loss of consciousness (LOC) remains largely unknown. The adenosine A2A receptor (A2A R) has been extensively proven to have an effect on physiological sleep. It is, therefore, important to investigate the role of A2A R in the induction of LOC using propofol. In the present study, the administration of the highly selective A2A R agonist (CGS21680) and antagonist (SCH58261) was utilized to investigate the function of A2A R under general anesthesia induced by propofol by means of animal behavior studies, resting-state magnetic resonance imaging and c-Fos immunofluorescence staining approaches. Our results show that CGS21680 significantly prolonged the duration of LOC induced by propofol, increased the c-Fos expression in nucleus accumbens (NAc) and suppressed the functional connectivity of NAc-dorsal raphe nucleus (DR) and NAc-cingulate cortex (CG). However, SCH58261 significantly shortened the duration of LOC induced by propofol, decreased the c-Fos expression in NAc, increased the c-Fos expression in DR, and elevated the functional connectivity of NAc-DR and NAc-CG. Collectively, our findings demonstrate the important roles played by A2A R in the LOC induced by propofol and suggest that the neural circuit between NAc-DR maybe controlled by A2A R in the mechanism of anesthesia induced by propofol.

Repeated but Not Single Administration of Ketamine Prolongs Increases of the Firing Activity of Norepinephrine and Dopamine Neurons.

BACKGROUND: Clinical studies have shown that the rapid antidepressant effect of the glutamate N-methyl-D-aspartate receptor antagonist ketamine generally disappears within 1 week but can be maintained by repeated administration. Preclinical studies showed that a single ketamine injection immediately increases the firing and burst activity of norepinephrine (NE) neurons, but not that of serotonin (5-HT) neurons. It also enhances the population activity of dopamine (DA) neurons. In the present study, we investigated whether such alterations of monoamine neuronal firing are still present 1 day after a single injection, and whether they can be maintained by repeated injections. METHODS: Rats received a single ketamine injection or 6 over 2 weeks and the firing activity of dorsal raphe nucleus 5-HT, locus coeruleus NE, and ventral tegmental area DA neurons was assessed. RESULTS: One day following a single injection of ketamine, there was no change in the firing activity of 5-HT, NE, or DA neurons. One day after repeated ketamine administration, however, there was a robust increase of the firing activity of NE neurons and an enhancement of burst and population activities of DA neurons, but still no change in firing parameters of 5-HT neurons. The increased activity of NE neurons was no longer present 3 days after the last injection, whereas that of DA neurons was still present. DA neurons were firing normally 7 days after repeated injections. CONCLUSION: These results imply that the enhanced activity of NE and DA neurons may play a significant role in the maintenance of the antidepressant action of ketamine.

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, 1 mM) or 8-OH-DPAT (5-HT1A agonist, 1 mM) 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.

Key role of the 5-HT1A receptor addressing protein Yif1B in serotonin neurotransmission and SSRI treatment.

Background: Altered function of serotonin receptor 1A (5-HT1AR) has been consistently implicated in anxiety, major depressive disorder and resistance to antidepressants. Mechanisms by which the function of 5-HT1AR (expressed as an autoreceptor in serotonergic raphe neurons and as a heteroreceptor in serotonin [5-HT] projection areas) is altered include regulation of its expression, but 5-HT1AR trafficking may also be involved. Methods: We investigated the consequences of the lack of Yif1B (the 5-HT1AR trafficking protein) on 5-HT neurotransmission in mice, and whether Yif1B expression might be affected under conditions known to alter 5-HT neurotransmission, such as anxious or depressive states or following treatment with fluoxetine (a selective serotonin reuptake inhibitor) in humans, monkeys and mice. Results: Compared with wild-type mice, Yif1B-knockout mice showed a significant decrease in the forebrain density of 5-HT projection fibres and a hypofunctionality of 5-HT1A autoreceptors expressed on raphe 5-HT neurons. In addition, social interaction was less in Yif1B-knockout mice, which did not respond to the antidepressant-like effect of acute fluoxetine injection. In wild-type mice, social defeat was associated with downregulated Yif1B mRNA in the prefrontal cortex, and chronic fluoxetine treatment increased Yif1B expression. The expression of Yif1B was also downregulated in the postmortem prefrontal cortex of people with major depressive disorder and upregulated after chronic treatment with a selective serotonin reuptake inhibitor in monkeys. Limitations: We found sex differences in Yif1B expression in humans and monkeys, but not in mice under the tested conditions. Conclusion: These data support the concept that Yif1B plays a critical role in 5-HT1AR functioning and brain 5-HT homeostasis. The opposite changes in its expression observed in anxious or depressive states and after therapeutic fluoxetine treatment suggest that Yif1B might be involved in vulnerability to anxiety and depression, and fluoxetine efficacy.

Serotonin 1A receptor density measured by F18-Mefway PET/CT in mesiotemporal cortex and raphe does not discriminate therapeutic response in patients with major depressive episode.

BACKGROUND: More than 50% of patients with major depressive episode (MDE) fail to respond to initial treatment with first line pharmacological therapy. Altered receptor and serotonin transporter function are considered to be associated with mental disorders. Our investigation aimed on the density of the HT1A receptor in mesiotemporal cortex (MTC) and raphe measured by F18-Mefway in patients with MDD. METHODS: Patients with untreated clinically suspected major depressive episode were recruited from June 2012 to May 2014. 49 patients were included into the study: 36 patients (73%) were identified as responders, whereas 13 (27%) were non-responders. Gender distribution was 26 men (56%) and 23 women (44%). For treatment, only a standard medication of a selective serotonin reuptake inhibitor (SSRI) with escitalopram in a range of 10-20 mg/day was permitted. Responders were defined by improvement of the MADRS>50%. Visually MTC had the highest uptake of F18-Mefway among all brain regions, an asymmetry could not be observed in any patient. An elliptical region was drawn over the amygdala and hippocampus area and a small circular region was drawn over the raphe nuclei. All data were calculated related to (unspecific) cerebellar uptake. RESULTS: The quotient of the right MTC was 5.00 [4.33; 5.50] in all patients, in responders 5.00 [4.00; 5.75] and in non-responders 5.00 [4.50; 5.50] (P=0.56). The quotient of the left MTC presented with a median level of 4.50 [4.50; 5.50] in all persons. The responders had 4.50 [4.50; 5.75] which was not statistically significant to the data of the non-responders with 5.00 [4.50; 5.50] at P=0.64. The raphe had a median quotient of 2.50 [2.00; 3.00] in all and the cohort of responders, whereas non-responders had 2.50 [2.00; 2.50] (P=0.61). Also the absolute values of SUV in the three brain regions were not statistically different between the cohorts. Additionally, we did not find any sex-related differences in our patient group. CONCLUSIONS: Serotonin 1A receptor density can be assessed efficiently by F18-Mefway and PET-CT in patients with MDE. The method can be estimated as a possible tool for clinical and academic investigation, marked tracer uptake can constantly be observed at MTC and the raphe. Anyhow, under conditions of real life in patient care, it is not possible to distinguish patients with a good prognosis who will respond to standard SSRI therapy from non-responders who would benefit from a different therapeutic approach starting earlier.

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.

Cocaine Selectively Reorganizes Excitatory Inputs to Substantia Nigra Pars Compacta Dopamine Neurons.

Substantia nigra pars compacta (SNc) dopamine neurons and their targets are involved in addiction and cue-induced relapse. However, afferents onto SNc dopamine neurons themselves appear insensitive to drugs of abuse, such as cocaine, when afferents are collectively stimulated electrically. This contrasts with ventral tegmental area (VTA) dopamine neurons, whose glutamate afferents react robustly to cocaine. We used an optogenetic strategy to isolate identified SNc inputs and determine whether cocaine sensitivity in the mouse SNc circuit is conferred at the level of three glutamate afferents: dorsal raphé nucleus (DR), pedunculopontine nucleus (PPN), and subthalamic nucleus (STN). We found that excitatory afferents to SNc dopamine neurons are sensitive to cocaine in an afferent-specific manner. A single exposure to cocaine in vivo led to PPN-innervated synapses reducing the AMPA-to-NMDA receptor-mediated current ratio. In contrast to work in the VTA, this was due to increased NMDA receptor function with no change in AMPA receptor function. STN synapses showed a decrease in calcium-permeable AMPA receptors after cocaine, but no change in the AMPA-to-NMDA ratio. Cocaine also increased the release probability at DR-innervated and STN-innervated synapses, quantified by decreases in paired-pulse ratios. However, release probability at PPN-innervated synapses remained unaffected. By examining identified inputs, our results demonstrate a functional distribution among excitatory SNc afferent nuclei in response to cocaine, and suggest a compelling architecture for differentiation and separate parsing of inputs within the nigrostriatal system.SIGNIFICANCE STATEMENT Prior studies have established that substantia nigra pars compacta (SNc) dopamine neurons are a key node in the circuitry that drives addiction and relapse, yet cocaine apparently has no effect on electrically stimulated excitatory inputs. Our study is the first to demonstrate the functional impact of a drug of abuse on synaptic mechanisms of identified afferents to the SNc. Optogenetic dissection of inputs originating from dorsal raphé, pedunculopontine, and subthalamic nuclei were tested for synaptic modifications following in vivo cocaine exposure. Our results demonstrate that cocaine differentially induces modifications to SNc synapses depending on input origin. This presents implications for understanding dopamine processing of motivated behavior; most critically, it indicates that dopamine neurons selectively modulate signal reception processed by afferent nuclei.

Serotonin 5-HT4 Receptor Agonists Improve Facilitation of Contextual Fear Extinction in an MPTP-Induced Mouse Model of Parkinson's Disease.

Previously, we found that 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced Parkinson's disease (PD) model mice (PD mice) showed facilitation of hippocampal memory extinction via reduced cyclic adenosine monophosphate (cAMP)/cAMP-dependent response element-binding protein (CREB) signaling, which may cause cognitive impairment in PD. Serotonergic neurons in the median raphe nucleus (MnRN) project to the hippocampus, and functional abnormalities have been reported. In the present study, we investigated the effects of the serotonin 5-HT4 receptor (5-HT4R) agonists prucalopride and velusetrag on the facilitation of memory extinction observed in PD mice. Both 5-HT4R agonists restored facilitation of contextual fear extinction in PD mice by stimulating the cAMP/CREB pathway in the dentate gyrus of the hippocampus. A retrograde fluorogold-tracer study showed that γ-aminobutyric acid-ergic (GABAergic) neurons in the reticular part of the substantia nigra (SNr), but not dopaminergic (DAergic) neurons in the substantia nigra pars compacta (SNpc), projected to serotonergic neurons in the MnRN, which are known to project their nerve terminals to the hippocampus. It is possible that the degeneration of the SNpc DAergic neurons in PD mice affects the SNr GABAergic neurons, and thereafter, the serotonergic neurons in the MnRN, resulting in hippocampal dysfunction. These findings suggest that 5HT4R agonists could be potentially useful as therapeutic drugs for treating cognitive deficits in PD.

Phrenic long-term depression evoked by intermittent hypercapnia is modulated by serotonergic and adrenergic receptors in raphe nuclei.

Intermittent hypercapnia evokes prolonged depression of phrenic nerve activity (phrenic long-term depression, pLTD). This study was undertaken to investigate the role of 5-HT and α2-adrenergic receptors in the initiation of pLTD. Adult male urethane-anesthetized, vagotomized, paralyzed, and mechanically ventilated Sprague-Dawley rats were exposed to a protocol of acute intermittent hypercapnia (AIHc; 5 episodes of 15% CO2 in air, each episode lasting 3 min). The experimental group received microinjection of the selective 5-HT1A receptor agonist 8-hydroxy-2-(dipropylamino)tetralin hydrobromide (8-OH-DPAT), the broad-spectrum 5-HT antagonist methysergide, or the α2-adrenergic antagonist yohimbine, whereas the control group received microinjection of 0.9% saline into the caudal raphe region. Peak phrenic nerve activity (pPNA) and burst frequency ( f) were analyzed during baseline (T0), during 5 hypercapnic episodes (THc1-THc5), and at 15, 30, and 60 min after the end of the last hypercapnic episode. In the control group, pPNA decreased 60 min after the end of the last hypercapnic episode compared with baseline values, i.e., pLTD developed ( P = 0.023). In the 8-OH-DPAT group, pPNA significantly decreased at T15, T30, and T60 compared with baseline values, i.e., pLTD developed ( P = 0.01). In the methysergide and yohimbine groups, AIHc did not evoke significant changes of the pPNA at T15, T30, and T60 compared with baseline values. In conclusion, activation of 5-HT1A receptors accentuated induction of pLTD, whereas blockade of α2-adrenergic receptors prevented development of pLTD following AIHc in anesthetized rats. These results suggest that chemical modulation of 5-HT and α2-adrenergic receptors in raphe nuclei affects hypercapnia-induced pLTD, offering important insights in understanding the mechanisms involved in development of respiratory plasticity. NEW & NOTEWORTHY Hypercapnia is a concomitant feature of many breathing disorders, including obstructive sleep apnea. In this study, acute intermittent hypercapnia evoked development of phrenic long-term depression (pLTD) 60 min after the last hypercapnic episode that was preserved if the selective 5-HT1A receptor agonist 8-hydroxy-2-(dipropylamino)tetralin hydrobromide was microinjected in the caudal raphe region before the hypercapnic stimulus. This study highlights that both 5-HT and adrenergic receptor activation is needed for induction of pLTD in urethane-anesthetized rats following intermittent hypercapnia exposure.

Functional Connectivity of the Raphe Nuclei: Link to Tobacco Withdrawal in Smokers.

Background: Although nicotine alters serotonergic neurochemistry, clinical trials of serotonergic medications for smoking cessation have provided mixed results. Understanding the role of serotonergic dysfunction in tobacco use disorder may advance development of novel pharmacotherapies. Methods: Functional magnetic resonance imaging was used to measure resting-state functional connectivity of the raphe nuclei as an indicator of serotonergic function. Connectivity of the dorsal and median raphe nuclei was compared between 18 young smokers (briefly abstinent, ~40 minutes post-smoking) and 19 young nonsmokers (16-21 years old); connectivity was also examined in a separate sample of overnight-abstinent smokers (18-25 years old), before and after smoking the first cigarette of the day. Relationships between connectivity of the raphe nuclei with psychological withdrawal and craving were tested in smokers. Results: Connectivity of the median raphe nucleus with the right hippocampal complex was weaker in smokers than in nonsmokers and was negatively correlated with psychological withdrawal in smokers. In overnight-abstinent smokers, smoking increased connectivity of the median raphe nucleus with the right hippocampal complex, and the increase was positively correlated with the decrease in psychological withdrawal. Conclusions: Relief of withdrawal due to smoking is potentially linked to the serotonergic pathway that includes the median raphe nucleus and hippocampal complex. These results suggest that serotonergic medications may be especially beneficial for smokers who endorse strong psychological withdrawal during abstinence from smoking.

Partial Raphe Dysfunction in Neurotransmission Is Sufficient to Increase Mortality after Anoxic Exposures in Mice at a Critical Period in Postnatal Development.

Sudden infant death syndrome (SIDS) cases often have abnormalities of the brainstem raphe serotonergic (5-HT) system. We hypothesize that raphe dysfunction contributes to a failure to autoresuscitate from multiple hypoxic events, leading to SIDS. We studied autoresuscitation in two transgenic mouse models in which exocytic neurotransmitter release was impaired via conditional expression of the light chain from tetanus toxin (tox) in raphe neurons expressing serotonergic bacterial artificial chromosome drivers Pet1 or Slc6a4. These used recombinase drivers targeted different portions of medullary raphe serotonergic, tryptophan hydroxylase 2 (Tph2)(+) neurons by postnatal day (P) 5 through P12: approximately one-third in triple transgenic Pet1::Flpe, hßactin::cre, RC::PFtox mice; approximately three-fourths inSlc6a4::cre, RC::Ptox mice; with the first model capturing a near equal number of Pet1(+),Tph2(+) versus Pet1(+),Tph2(low or negative) raphe cells. At P5, P8, and P12, "silenced" mice and controls were exposed to five, ∼37 s bouts of anoxia. Mortality was 5-10 times greater in "silenced" pups compared with controls at P5 and P8 (p = 0.001) but not P12, with cumulative survival not differing between experimental transgenic models. "Silenced" pups that eventually died took longer to initiate gasping (p = 0.0001), recover heart rate (p = 0.0001), and recover eupneic breathing (p = 0.011) during the initial anoxic challenges. Variability indices for baseline breathing distinguished "silenced" from controls but did not predict mortality. We conclude that dysfunction of even a portion of the raphe, as observed in many SIDS cases, can impair ability to autoresuscitate at critical periods in postnatal development and that baseline indices of breathing variability can identify mice at risk. SIGNIFICANCE STATEMENT: Many sudden infant death syndrome (SIDS) cases exhibit a partial (∼26%) brainstem serotonin deficiency. Using recombinase drivers, we targeted different fractions of serotonergic and raphe neurons in mice for tetanus toxin light chain expression, which prevented vesicular neurotransmitter release. In one model, approximately one-third of medullary Tph2(+) neurons are silenced by postnatal (P) days 5 and 12, along with some Pet1(+),Tph2(low or negative) raphe cells; in the other, approximately three-fourths of medullary Tph2(+) neurons, also with some Tph2(low or negative) cells. Both models demonstrated excessive mortality to anoxia (a postulated SIDS stressor) at P5 and P8. We demonstrated fatal vulnerability to anoxic stress at a specific time in postnatal life induced by a partial defect in raphe function. This models features of SIDS.

Early life diets with prebiotics and bioactive milk fractions attenuate the impact of stress on learned helplessness behaviours and alter gene expression within neural circuits important for stress resistance.

Manipulating gut microbes may improve mental health. Prebiotics are indigestible compounds that increase the growth and activity of health-promoting microorganisms, yet few studies have examined how prebiotics affect CNS function. Using an acute inescapable stressor known to produce learned helplessness behaviours such as failure to escape and exaggerated fear, we tested whether early life supplementation of a blend of two prebiotics, galactooligosaccharide (GOS) and polydextrose (PDX), and the glycoprotein lactoferrin (LAC) would attenuate behavioural and biological responses to stress later in life. Juvenile, male F344 rats were fed diets containing either GOS and PDX alone, LAC alone, or GOS, PDX and LAC. All diets altered gut bacteria, while diets containing GOS and PDX increased Lactobacillus spp. After 4 weeks, rats were exposed to inescapable stress, and either immediately killed for blood and tissues, or assessed for learned helplessness 24 h later. Diets did not attenuate stress effects on spleen weight, corticosterone and blood glucose; however, all diets differentially attenuated stress-induced learned helplessness. Notably, in situ hybridization revealed that all diets reduced stress-evoked cfos mRNA in the dorsal raphe nucleus (DRN), a structure important for learned helplessness behaviours. In addition, GOS, PDX and LAC diet attenuated stress-evoked decreases in mRNA for the 5-HT1A autoreceptor in the DRN and increased basal BDNF mRNA within the prefrontal cortex. These data suggest early life diets containing prebiotics and/or LAC promote behavioural stress resistance and uniquely modulate gene expression in corresponding circuits.

Relative contribution of the dorsal raphe nucleus and ventrolateral periaqueductal gray to morphine antinociception and tolerance in the rat.

The dorsal raphe nucleus (DRN) is embedded in the ventral part of the caudal periaqueductal gray (PAG). Electrical or chemical activation of neurons throughout this region produces antinociception. The objective of this manuscript is to determine whether the ventrolateral PAG and DRN are distinct antinociceptive systems. This hypothesis was tested by determining the antinociceptive potency of microinjecting morphine into each structure (Experiment 1), creating a map of effective microinjection sites that produce antinociception (Experiment 2) and comparing the development of antinociceptive tolerance to repeated microinjections of morphine into the ventrolateral PAG and DRN (Experiment 3). Morphine was more potent following cumulative injections (1.0, 2.2, 4.6 & 10 µg/0.2 µL) into the ventrolateral PAG (D50  = 3.3 µg) compared to the lateral (4.3 µg) or medial DRN (5.8 µg). Antinociception occurred following 94% of the morphine injections into the ventrolateral PAG, whereas only 68.3% and 78.3% of the injections into the lateral and medial aspects of the DRN produced antinociception. Repeated microinjections of morphine into the ventrolateral PAG produced tolerance as indicated by a 528% difference in potency between morphine and saline pretreated rats. In contrast, relatively small changes in potency occurred following repeated microinjections of morphine into the lateral and medial aspects of the DRN (107% and 49%, respectively). These data indicate that the ventrolateral PAG and DRN are distinct antinociceptive structures. Antinociception is greater with injections into the ventrolateral PAG compared to the DRN, but this antinociception disappears rapidly because of the development of tolerance.

Alcohol preference and consumption are controlled by the caudal linear nucleus in alcohol-preferring rats.

The neuroanatomical and neurochemical basis of alcohol drinking has been extensively studied, but the neural circuitry mediating alcohol reinforcement has not been fully delineated. In the present experiments, we used both neuroimaging and pharmacological tools to identify neural systems associated with alcohol preference and high voluntary alcohol drinking in alcohol-preferring AA (Alko Alcohol) rats. First, we compared the basal brain activity of AA rats with that of heterogeneous Wistar rats with manganese-enhanced magnetic resonance imaging (MEMRI). Briefly, alcohol-naïve rats were implanted with subcutaneous osmotic minipumps delivering 120 mg/kg MnCl2 over a 7-day period, and were then imaged using a three-dimensional rapid acquisition-relaxation enhanced pulse sequence. MEMRI analysis revealed that the most conspicuous subcortical activation difference was located in the caudal linear nucleus of raphe (CLi), with AA rats displaying significantly lower T1 signal in this region compared to Wistar rats. However, following long-term alcohol drinking, CLi activity was increased in AA rats. In the second experiment, the CLi was targeted with pharmacological tools. AA rats trained to drink 10% alcohol during 2-h sessions were implanted with guide cannulas aimed at the CLi and were given injections of the GABAA receptor agonist muscimol into the CLi before drinking sessions. Muscimol dose-dependently increased alcohol drinking, and co-administration of the gamma aminobutyric acid (GABA)A antagonist bicuculline blocked muscimol's effect. These findings suggest that the mediocaudal region of the ventral tegmental area, particularly the CLi, is important for the propensity for high alcohol drinking and controls alcohol reward via GABAergic transmission.

Ontogeny of SERT Expression and Antidepressant-like Response to Escitalopram in Wild-Type and SERT Mutant Mice.

Depression is a disabling affective disorder for which the majority of patients are not effectively treated. This problem is exacerbated in children and adolescents for whom only two antidepressants are approved, both of which are selective serotonin reuptake inhibitor (SSRIs). Unfortunately SSRIs are often less effective in juveniles than in adults; however, the mechanism(s) underlying age-dependent responses to SSRIs is unknown. To this end, we compared the antidepressant-like response to the SSRI escitalopram using the tail suspension test and saturation binding of [(3)H]citalopram to the serotonin transporter (SERT), the primary target of SSRIs, in juvenile [postnatal day (P)21], adolescent (P28), and adult (P90) wild-type (SERT+/+) mice. In addition, to model individuals carrying low-expressing SERT variants, we studied mice with reduced SERT expression (SERT+/-) or lacking SERT (SERT-/-). Maximal antidepressant-like effects were less in P21 mice relative to P90 mice. This was especially apparent in SERT+/- mice. However, the potency for escitalopram to produce antidepressant-like effects in SERT+/+ and SERT+/- mice was greater in P21 and P28 mice than in adults. SERT expression increased with age in terminal regions and decreased with age in cell body regions. Binding affinity values did not change as a function of age or genotype. As expected, in SERT-/- mice escitalopram produced no behavioral effects, and there was no specific [(3)H]citalopram binding. These data reveal age- and genotype-dependent shifts in the dose-response for escitalopram to produce antidepressant-like effects, which vary with SERT expression, and may contribute to the limited therapeutic response to SSRIs in juveniles and adolescents.

Inactivation of nucleus incertus impairs passive avoidance learning and long term potentiation of the population spike in the perforant path-dentate gyrus evoked field potentials in rats.

Involvement of brainstem nucleus incertus (NI) in hippocampal theta rhythm suggests that this structure might play a role in hippocampal-dependent learning and memory. In the present study we aimed to address if NI is involved in an avoidance learning task as well as dentate gyrus (DG) short-term and long-term potentiation. Lidocaine was injected into the NI to transiently inactivate the nucleus, and control rats received saline. Role of NI was studied in passive avoidance learning (PAL) in 3 memory phases of acquisition, consolidation and retrieval. Levels of hippocampal phosphorylated p70 were also assessed in rats involved in PAL. Perforant path-DG short-term synaptic plasticity was studied upon NI inactivation before the paired-pulse stimulation, and also before or after tetanic stimulation in freely moving rats. It was found that NI inactivation delayed learning and impaired retention in the PAL task, with decreased levels of phosphorylated p70 in the respective groups. However, short-term plasticity was not affected by NI inactivation. But long term potentiation (LTP) of DG population spike was poorly induced with NI inactivation compared to the saline group, and it had no effect on population excitatory post-synaptic potential. Furthermore, when NI was inactivated after the induction of LTP, there was no difference between the saline and lidocaine groups. These observations suggest that NI has a role in PAL task, and its inactivation does not change the perforant path-DG granule cell synaptic input but decreases the excitability of the DG granule cells. Further studies should elucidate direct and indirect paths through which NI might influence hippocampal activity.

Presynaptic gating of excitation in the dorsal raphe nucleus by GABA.

The dorsal raphe nucleus (DR) controls forebrain serotonin neurotransmission to influence emotional states. GABA neurotransmission in the DR has been implicated in regulating sleep/wake states and influencing anxiety and aggression. To gain insight into how GABA regulates DR activity, we analyzed the organization of both GABA and glutamate axons in the rat DR using a high-resolution immunofluorescence technique, array tomography, as well as EM. This analysis revealed that a third or more of GABA-containing axons are organized in synaptic triads with a glutamatergic axon and a common postsynaptic target. Electrophysiological recordings showed that GABA has the capacity to presynaptically gate glutamate release in the DR through a combination of GABA-A and GABA-B receptor-mediated effects. Thus, GABA-glutamate synaptic triads are a common feature of the network architecture of the DR with the potential to regulate excitation of the nucleus.

Antidepressant Potential of Chlorogenic Acid-Enriched Extract from Eucommia ulmoides Oliver Bark with Neuron Protection and Promotion of Serotonin Release through Enhancing Synapsin I Expression.

Eucommia ulmoides Oliver (E. ulmoides) is a traditional Chinese medicine with many beneficial effects, used as a tonic medicine in China and other countries. Chlorogenic acid (CGA) is an important compound in E. ulmoides with neuroprotective, cognition improvement and other pharmacological effects. However, it is unknown whether chlorogenic acid-enriched Eucommia ulmoides Oliver bark has antidepressant potential through neuron protection, serotonin release promotion and penetration of blood-cerebrospinal fluid barrier. In the present study, we demonstrated that CGA could stimulate axon and dendrite growth and promote serotonin release through enhancing synapsin I expression in the cells of fetal rat raphe neurons in vitro. More importantly, CGA-enriched extract of E. ulmoides (EUWE) at 200 and 400 mg/kg/day orally administered for 7 days showed antidepressant-like effects in the tail suspension test of KM mice. Furthermore, we also found CGA could be detected in the the cerebrospinal fluid of the rats orally treated with EUWE and reach the level of pharmacological effect for neuroprotection by UHPLC-ESI-MS/MS. The findings indicate CGA is able to cross the blood-cerebrospinal fluid barrier to exhibit its neuron protection and promotion of serotonin release through enhancing synapsin I expression. This is the first report of the effect of CGA on promoting 5-HT release through enhancing synapsin I expression and CGA-enriched EUWE has antidepressant-like effect in vivo. EUWE may be developed as the natural drugs for the treatment of depression.