Fluoxetine treatment abolishes the in vitro respiratory response to acidosis in neonatal mice

International audienceBACKGROUND: To secure pH homeostasis, the central respiratory network must permanently adapt its rhythmic motor drive to environment and behaviour. In neonates, it is commonly admitted that the retrotrapezoid/parafacial respiratory group of neurons of the ventral medulla plays the primary role in the respiratory response to acidosis, although the serotonergic system may also contribute to this response.METHODOLOGY/PRINCIPAL FINDINGS: Using en bloc medullary preparations from neonatal mice, we have shown for the first time that the respiratory response to acidosis is abolished after pre-treatment with the serotonin-transporter blocker fluoxetine (25-50 µM, 20 min), a commonly used antidepressant. Using mRNA in situ hybridization and immunohistology, we have also shown the expression of the serotonin transporter mRNA and serotonin-containing neurons in the vicinity of the RTN/pFRG of neonatal mice.CONCLUSIONS: These results reveal that the serotonergic system plays a pivotal role in pH homeostasis. Although obtained in vitro in neonatal mice, they suggest that drugs targeting the serotonergic system should be used with caution in infants, pregnant women and breastfeeding mothers

Glutamate presynaptic vesicular transporter and postsynaptic receptor levels correlate with spatial memory status in aging rat models

AbstractIn humans, memory capacities are generally affected with aging, even without any reported neurologic disorders. The mechanisms behind cognitive decline are not well understood. We studied here whether postsynaptic glutamate receptor and presynaptic vesicular glutamate transporters (VGLUTs) levels may change in the course of aging and be related to cognitive abilities using various age-impaired (AI) or age-unimpaired rat strains. Twenty-four-month-old Long-Evans (LE) rats with intact spatial memory maintained postsynaptic ionotropic glutamate receptor levels in the hippocampal-adjacent cortex similar to those of young animals. In contrast, AI rats showed significantly reduced expression of ionotropic glutamate receptor GluR2, NR2A and NR2B subunits. In AI LE rats, VGLUT1 and VGLUT2 levels were increased and negatively correlated with receptor levels as shown by principal component analysis and correlation matrices. We also investigated whether glutamatergic receptors and VGLUT levels were altered in the obesity-resistant LOU/C/Jall (LOU) rat strain which is characterized by intact memory despite aging. No difference was observed between 24-month-old LOU rats and their young counterparts. Taken together, the unaltered spatial memory performance of 24-month-old age-unimpaired LE and LOU rats suggests that intact coordination of the presynaptic and postsynaptic hippocampal-adjacent cortex glutamatergic networks may be important for successful cognitive aging. Accordingly, altered expression of presynaptic and postsynaptic glutamatergic components, such as in AI LE rats, could be considered a marker of age-related cognitive deficits

Axonal Segregation and Role of the Vesicular Glutamate Transporter VGLUT3 in Serotonin Neurons

International audienceA subset of monoamine neurons releases glutamate as a cotransmitter due to presence of the vesicular glutamate transporters VGLUT2 or VGLUT3. In addition to mediating vesicular loading of glutamate, it has been proposed that VGLUT3 enhances serotonin (5-HT) vesicular loading by the vesicular monoamine transporter (VMAT2) in 5-HT neurons. In dopamine (DA) neurons, glutamate appears to be released from specialized subsets of terminals and it may play a developmental role, promoting neuronal growth and survival. The hypothesis of a similar developmental role and axonal localization of glutamate co-release in 5-HT neurons has not been directly examined. Using postnatal mouse raphe neurons in culture, we first observed that in contrast to 5-HT itself, other phenotypic markers of 5-HT axon terminals such as the 5-HT reuptake transporter (SERT) show a more restricted localization in the axonal arborization. Interestingly, only a subset of SERT-and 5-HT-positive axonal varicosities expressed VGLUT3, with SERT and VGLUT3 being mostly segregated. Using VGLUT3 knockout mice, we found that deletion of this transporter leads to reduced survival of 5-HT neurons in vitro and also decreased the density of 5-HT-immunoreactivity in terminals in the dorsal striatum and dorsal part of the hippocampus in the intact brain. Our results demonstrate that raphe 5-HT neurons express SERT and VGLUT3 mainly in segregated axon terminals and that VGLUT3 regulates the vulnerability of these neurons and the neurochemical identity of their axonal domain, offering new perspectives on the functional connectivity of a cell population involved in anxiety disorders and depression

The vesicular glutamate transporter VGLUT3 contributes to protection against neonatal hypoxic stress

International audienceKey points Hypoxic stress is an important cause of morbidity and mortality in neonates. We examined the role of VGLUT3, an atypical transporter of glutamate present in serotonergic neurons involved in breathing and heat production, in the response to hypoxia. The respiratory responses to chemical stimuli and the turnover of serotonin in the brainstem were impaired in newborn mice lacking VGLUT3. Under cold conditions, metabolic rate, body temperature, baseline breathing and the ventilatory response to hypoxia were disrupted. Thus, VGLUT3 expression is required for optimal response to hypoxic stress in neonates. Abstract Neonates respond to hypoxia initially by increasing ventilation, and then by markedly decreasing both ventilation (hypoxic ventilatory decline) and oxygen consumption (hypoxic hypometabolism). This latter process, which vanishes with age, reflects a tight coupling between ventilatory and thermogenic responses to hypoxia. The neurological substrate of hypoxic hypometabolism is unclear, but it is known to be centrally mediated, with a strong involvement of the 5-hydroxytryptamine (5-HT, serotonin) system. To clarify this issue, we investigated the possible role of VGLUT3, the third subtype of vesicular glutamate transporter. VGLUT3 contributes to glutamate signalling by 5-HT neurons, facilitates 5-HT transmission and is expressed in strategic regions for respiratory and thermogenic control. We therefore assumed that VGLUT3 might significantly contribute to the response to hypoxia. To test this possibility, we analysed this response in newborn mice lacking VGLUT3 using anatomical, biochemical, electrophysiological and integrative physiology approaches. We found that the lack of VGLUT3 did not affect the histological organization of brainstem respiratory networks or respiratory activity under basal conditions. However, it impaired respiratory responses to 5-HT and anoxia, showing a marked alteration of central respiratory control. These impairments were associated with altered 5-HT turnover at the brainstem level. Furthermore, under cold conditions, the lack of VGLUT3 disrupted the metabolic rate, body temperature, baseline breathing and the ventilatory response to hypoxia. We conclude that VGLUT3 expression is dispensable under basal conditions but is required for optimal response to hypoxic stress in neonates

Acidosis increases the phrenic burst frequency of en bloc medullary preparations.

<p>A – Schematic presentation of the <i>en bloc</i> preparation of neonatal mice (A1) and example of raw and integrated phrenic bursts (bottom and top traces, respectively) produced by the isolated respiratory rhythm generator (RRG) on the C4 ventral roots of <i>en bloc</i> preparations (A2). B – Columns of the histogram show the mean (and SEM) phrenic burst frequency (PBf; expressed as % of the control PBf) measured every min (one column  = 1 min) in 12 <i>en bloc</i> preparations when the control aCSF(7.4) (white columns) superfusing the preparations was replaced by aCSF(7.1) for 5 min (black columns). Note that acidosis significantly increased the PBf (asterisks indicate a p<0.05 statistical difference). C- As in B but aCSF(7.1) application for 10 min to 6 other preparations. Note that the PBf reached a plateau from the 5^th to the 10^th min of aCSF(7.1) application.</p

SERT mRNA and 5-HT-containing neurons in the vicinity of the RTN/pFRG of neonatal mice.

<p>A: Two serial coronal sections passing through the RTN/PFRG area of a neonatal mouse show SERT mRNA expression (A1) and Cresyl Violet staining (A2). Anatomical limits drawn from A2 Cresyl Violet section have been superimposed on A1 section (dotted line). Note SERT mRNA is expressed in three areas, the median Raphe Magnus (Rm), the para-pyramidal group (ppy) lateral to the pyramid tract (py) and a lateral spot (arrow) located in a medio-ventral position from the facial motor nucleus (n7), as defined from Cresyl Violet staining (in A2). B- 5-HT neurons in the RTN/pFRG area of a neonatal mouse. Note 5-HT neurons in the median raphe magnus (Rm), the parapyramidal group (ppy, doted circle) lateral to the pyramidal tract (py) and the lateral spot (arrow in B1) in the medio-ventral location of the facial nucleus (n7). In B2 and B3, note the superficial location of some 5-HT neurons (arrows). B3 is an enlargement of B1.</p

Fluoxetine and serotonin pre-treatments abolish the increase of phrenic burst frequency induced by acidosis.

<p><b>A</b> - Columns of the histogram show the mean (and SEM) PBf (expressed as % of the control PBf) measured every min (one column  = 1 min) in 10 preparations when the control aCSF(7.4) (white columns) superfusing the preparations was first replaced by aCSF(7.4) containing fluoxetine (10–25 µM) for 20 min to block the serotonin transporter SERT (grey columns) and thereafter by aCSF(7.1) containing the same amount of fluoxetine for 5 min (black columns). Note that pre-treatment with fluoxetine did not significantly increase the PBf but abolished the PBf increase under acidosis. <b>B</b> - PBf changes (expressed as % of control) every min (one column) but for 14 preparations superfused with control aCSF(7.4) (white columns), aCSF(7.4) containing serotonin, 5-HT (1–5 µM) for 20 min (grey columns) and thereafter aCSF(7.1) containing the same amount of 5-HT for 5 min (black columns). Note that pre-treatment with 5-HT significantly increased the PBf by about 50% and, similarly to fluoxetine treatment, abolished the PBf increase under acidosis.</p

Targeted Inactivation of Mapk4 in Mice Reveals Specific Nonredundant Functions of Erk3/Erk4 Subfamily Mitogen-Activated Protein Kinases ▿

Erk4 and Erk3 are atypical members of the mitogen-activated protein (MAP) kinase family. The high sequence identity of Erk4 and Erk3 proteins and the similar organization of their genes imply that the two protein kinases are paralogs. Recently, we have shown that Erk3 function is essential for neonatal survival and critical for the establishment of fetal growth potential and pulmonary function. To investigate the specific functions of Erk4, we have generated mice with a targeted disruption of the Mapk4 gene. We show that Erk4-deficient mice are viable and fertile and exhibit no gross morphological or physiological anomalies. Loss of Erk4 is not compensated by changes in Erk3 expression or activity during embryogenesis or in adult tissues. We further demonstrate that additional loss of Erk4 does not exacerbate the fetal growth restriction and pulmonary immaturity phenotypes of Erk3−/− mice and does not compromise the viability of Erk3+/− neonates. Interestingly, behavioral phenotyping revealed that Erk4-deficient mice manifest depression-like behavior in the forced-swimming test. Our analysis indicates that the MAP kinase Erk4 is dispensable for mouse embryonic development and reveals that Erk3 and Erk4 have acquired specialized functions through evolutionary diversification

Unexpected contributions of striatal projection neurons coexpressing dopamine D1 and D2 receptors in balancing motor control

Abstract The central function of the striatum and its dopaminergic (DA) afferents in motor control and the integration of cognitive and emotional processes is commonly explained by the two striatal efferent pathways characterized by striatal projection neurons (SPNs) expressing DA D1 receptors and D2 receptors (D1-SPNs and D2-SPNs), without regard to SPNs coexpressing both receptors (D1/D2-SPNs). We developed an approach that enables the targeting of these hybrid SPNs and demonstrated that although these SPNs are less abundant, they play a major role in guiding the motor function of the other two main populations. D1/D2-SPNs project exclusively to the external globus pallidus (GPe) and have specific electrophysiological features with distinctive integration of DA signals. Optogenetic stimulation and loss-of-function experiments indicated that D1/D2-SPNs potentiate the prokinetic and antikinetic functions of D1-SPNs and D2-SPNs, respectively, and restrain the integrated motor response to psychostimulants. Overall, our findings demonstrate the essential role of this third unacknowledged population of D1/D2 coexpressing neurons, which orchestrates the fine-tuning of DA regulation in the thalamo-cortico-striatal loops. One-Sentence Summary D1/D2 SPNs modulate the motor function of both D1- and D2-SPN