Denys V. Volgin

Postnatal development of serotonin 1B, 2 A and 2C receptors in brainstem motoneurons

The effects of serotonin (5‐HT) on motoneurons are mediated via multiple receptor subtypes. In hypoglossal (XII) motoneurons, the prototypic brainstem motoneurons whose functions change during the postnatal period, 5‐HT effects evolve from inhibitory to excitatory, probably in association with changes in receptor expression. We studied 5‐HT1B, 5‐HT2A and 5‐HT2C receptor mRNA in 414 dissociated XII motoneurons and 5‐HT2A protein in the XII, facial and spinal cervical (C2‐3) motor nuclei. The percentage of motoneurons expressing distinct mRNAs varied with the postnatal age (P3‐33 days) and receptor subtype. Initially, 5‐HT1B mRNA was present in 50–85% of cells, but on P14 its expression transiently decreased below 35%. 5‐HT2A mRNA was present in nearly all cells after P6, but in less than 65% on P3‐5. Normal and/or short splice variants of the 5‐HT2C mRNA were expressed in less than 20% of motoneurons on P3‐9, and in ∼ 35% thereafter. 5‐HT1B and 5‐HT2A mRNAs often were expressed in different cells during early and intermediate postnatal periods, whereas 5‐HT2C mRNA never occurred alone. The 5‐HT2A receptor protein level gradually increased through P15 in the XII and facial nuclei, with dendritic labelling appearing in XII motoneurons only after P12. In spinal motoneurons, both somatic and dendritic labelling was strongest on P5 and then decreased. The development of 5‐HT receptors in XII motoneurons may be related to changes in feeding behaviour, whereas different cues regulate 5‐HT receptor expression in upper spinal motoneurons.

Developmental changes in the orexin 2 receptor mRNA in hypoglossal motoneurons.

Hypothalamic orexin-containing neurons project to many CNS targets, including motoneurons. We assessed developmental changes in the expression of the orexin type 2 receptor (ORX2r) mRNA in hypoglossal (XII) motoneurons. Identified motoneurons were dissociated from 4- to 33-day-old rats and subjected to single-cell reverse transcription and PCR; nearly all contained the ORX2r mRNA. In 40 motoneurons studied using semi-nested PCR, and in another 39 subjected to quantitative, real-time PCR, the number of reverse-transcribed mRNA copies per cell was significantly higher around day 20 postnatally than at any other age. Thus, ORX may postsynaptically excite XII motoneurons, with the ORX2r mRNA production increased during the critical period for development of the rapid eye movement sleep and its disorder narcolepsy/cataplexy.

α1B receptors are the main postsynaptic mediators of adrenergic excitation in brainstem motoneurons, a single-cell RT-PCR study

Norepinephrine (NE) is an important modulator of brainstem motoneurons. It is released at high levels during wakefulness, whereas its reduced release during sleep may contribute to motor suppression, including upper airway hypotonia. To identify the receptors that mediate postsynaptic effects of NE in brainstem motoneurons of juvenile and adult rats, we determined the pattern of adrenoceptor mRNA expression and co-expression in retrogradely labeled and acutely dissociated hypoglossal (XII) motoneurons (n=121) using single-cell, real-time reverse transcription-polymerase chain reaction (RT-PCR). The alpha(1B) receptor mRNA was present in most motoneurons (33/39 or 85%). The remaining six adrenoceptor mRNA species investigated were consistently present in micropunches of tissue extracted from the XII nucleus, but were either rarely expressed in individual motoneurons (alpha(1A) mRNA in 15%, alpha(1D) in 14%, alpha(2B/C) in 2% of cells) or absent (alpha(2A), beta(1) and beta(2)). When present, the alpha(1A) and alpha(1D) mRNAs were co-expressed with alpha(1B) mRNA. The adrenoceptor mRNA expression profiles in dissociated locus coeruleus and inferior olive neurons were significantly different. We conclude that postsynaptic effects of NE in XII motoneurons are primarily mediated by alpha(1B) receptors; the effects ascribed to alpha(2) and/or beta adrenoceptors may be exerted presynaptically.

Hypoglossal premotor neurons of the intermediate medullary reticular region express cholinergic markers

The inspiratory drive to hypoglossal (XII) motoneurons originates in the caudal medullary intermediate reticular (IRt) region. This drive is mainly glutamatergic, but little is known about the neurochemical features of IRt XII premotor neurons. Prompted by the evidence that XII motoneuronal activity is controlled by both muscarinic (M) and nicotinic cholinergic inputs and that the IRt region contains cells that express choline acetyltransferase (ChAT), a marker of cholinergic neurons, we investigated whether some IRt XII premotor neurons are cholinergic. In seven rats, we applied single-cell reverse transcription-polymerase chain reaction to acutely dissociated IRt neurons retrogradely labeled from the XII nucleus. We found that over half (21/37) of such neurons expressed mRNA for ChAT and one-third (13/37) also had M2 receptor mRNA. In contrast, among the IRt neurons not retrogradely labeled, only 4 of 29 expressed ChAT mRNA (P < 0.0008) and only 3 of 29 expressed M2 receptor mRNA (P < 0.04). The distributions of other cholinergic receptor mRNAs (M1, M3, M4, M5, and nicotinic alpha4-subunit) did not differ between IRt XII premotor neurons and unlabeled IRt neurons. In an additional three rats with retrograde tracers injected into the XII nucleus and ChAT immunohistochemistry, 5-11% of IRt XII premotor neurons located at, and caudal to, the area postrema were ChAT positive, and 27-48% of ChAT-positive caudal IRt neurons were retrogradely labeled from the XII nucleus. Thus the pre- and postsynaptic cholinergic effects previously described in XII motoneurons may originate, at least in part, in medullary IRt neurons.

Developmental profiles of neurotransmitter receptors in respiratory motor nuclei

We discuss the time course of postnatal development of selected neurotransmitter receptors in motoneurons that innervate respiratory pump and accessory respiratory muscles, with emphasis on other than classic respiratory signals as important regulatory factors. Functions of those brainstem motoneurons that innervate the pharynx and larynx change more dramatically during early postnatal development than those of spinal respiratory motoneurons. Possibly in relation to this difference, the time course of postnatal expression of distinct receptors for serotonin differ between the hypoglossal (XII) and phrenic motoneurons. In rats, distinct developmental patterns include a decline or increase that extends over the first 3-4 postnatal weeks, a rapid increase during the first 2 weeks, or a transient decline on postnatal days 11-14. The latter period coincides with major changes in many transmitters in brainstem respiratory regions that may be related to a brain-wide reconfiguration of sensorymotor processing resulting from eye and ear opening and beginning of a switch from suckling to mature forms of food seeking and processing. Such rapid neurochemical changes may impart increased vulnerability on the respiratory system. We also consider rapid eye movement sleep as a state during which some brain functions may revert to conditions typical of perinatal period. In addition to normal developmental processes, changes in the expression or function of neurotransmitter receptors may occur in respiratory motoneurons in response to injury, perinatal stress, or disease conditions that increase the load on respiratory muscles or alter the normal levels and patterns of oxygen delivery.

Chronic intermittent hypoxia alters hypothalamic transcription of genes involved in metabolic regulation

Epidemiological studies show that the obstructive sleep apnea syndrome (OSAS) is strongly associated with obesity, hypertension and diabetes, the three conditions characteristic of the metabolic syndrome. Since metabolic disorders usually involve altered homeostatic mechanisms both centrally and peripherally, it is likely that so it is in OSAS, but the underlying mechanisms remain largely unknown. We used an established rodent model to test whether chronic intermittent hypoxia (CIH) similar to that experienced by OSAS patients leads to distinct and relevant for metabolic regulation transcriptional changes in the posterior hypothalamus. Using quantitative reverse transcription-polymerase chain reaction, we found that rats exposed to CIH for 35 days (n=9) had twice higher levels of the adrenergic alpha2A receptor mRNA than the rats simultaneously submitted to a matching sham treatment (n=9). The mRNA levels of three members of the family of signal transducers and activators of transcription, STAT1, STAT3 and STAT5b, were also increased 2-4 times. The increases occurred only in the perifornical region, whereas no changes were detected in the ventromedial region comprising the ventromedial and arcuate nuclei or the dorsomedial region comprising the dorsomedial and paraventricular nuclei. These results show that, at least at the transcriptional level, CIH exerts a distinct and regionally selective central effect on the expression of selected mRNAs involved in metabolic regulation through adrenergic, leptinergic and inflammatory pathways.

Activation of HIF-1α mRNA by hypoxia and iron chelator in isolated rat carotid body

Abstract The hypoxia inducible factor-1α (HIF-1α) protein level is increased by hypoxia and iron chelator (ciclopirox olamine) in isolated rat carotid body (CB) and glomus cells. Reverse transcription and polymerase chain reaction (RT-PCR) are performed to test whether this increase is caused, at least in part, by increased HIF-1α gene transcription. HIF-1α mRNA levels dose-dependently increased and decreased in the rat CBs incubated for 1 h in a medium saturated with O 2 levels that were varied around nominally normoxic level of 21% in the 0–95% range. The iron chelator, ciclopirox olamine (5 μM), stimulated HIF-1α mRNA production under normoxic condition. Thus, in the CB, the main systemic O 2 -sensing organ, HIF-1α transcription is regulated by O 2 supply around the normoxic level; this may contribute to cellular and organismal adaptations to chronic changes in ambient O 2 .

Single-cell RT-PCR gene expression profiling of acutely dissociated and immunocytochemically identified central neurons

Identification of neurons for single-cell mRNA profiling is difficult when cells of interest are located in heterogeneous brain regions. We developed a protocol in which acutely dissociated neurons are immunocytochemically labeled prior to single-cell reverse transcription-polymerase chain reaction (RT-PCR). We tested the protocol on hypothalamic melanin-concentrating hormone (MCH) and prepro-orexin (PPO) neurons, which are similarly distributed but functionally different. Cells dissociated from the perifornical region of the posterior hypothalamus of juvenile or adult rats were incubated with anti-MCH or anti-PPO primary antibodies, followed by washout and incubation with fluorescein-tagged secondary antibodies. Individual labeled cells were subjected to RT-PCR with primers for PPO and MCH. MCH mRNA was detected in 26 out of the 38 successfully reverse-transcribed cells identified as MCH-containing, and 28 cells out of the 42 identified as PPO-containing expressed PPO mRNA. No cell expressed both mRNAs. Most MCH neurons tested (five out of six) expressed the adrenergic alpha2A receptor mRNA, whereas it was absent from all seven PPO neurons tested. Neither PPO (n = 11) nor MCH (n = 6) cells expressed the type 2 orexin receptor mRNA. Thus, the method allows, with at least 66% confidence, immunocytochemical cell identification prior to mRNA studies of single neurons located in heterogeneous brain regions.

Antagonism of orexin 1 receptors eliminates motor hyperactivity and improves homing response acquisition in juvenile rats exposed to alcohol during early postnatal period

Consequences of prenatal alcohol exposure (AE) include motor hyperactivity, disrupted sleep and cognitive deficits. Hypothalamic orexin (ORX)-synthesizing neurons are important for the maintenance of vigilance and regulation of motor activity but their hyperactivity may contribute to anxiety disorders. Using a rat model, we tested whether ORX plays a role in behavioral consequences of prenatal AE. Male rat pups received 2.625 g/kg of alcohol (AE group) intragastrically twice daily on postnatal days (PD)4-9, a developmental period equivalent to the third trimester of human pregnancy. Control pups were sham-intubated (S group). On PD12-14, they received daily injections of either the ORX-1 receptor antagonist, SB-334867 (SB; 20mg/kg, i.p.) or vehicle (V) during the lights-off period. On PD16, they were subjected to the homing response (HR) test. On PD17, their motor activity was monitored in a novel environment. The percentage of tests in which HR acquisition was not achieved and the number of trials needed to reach the shortest HR latency were higher, whereas the percentage of successful trials was lower, in AE-V than in S-V rats (p = 0.0009-0.03). In contrast, these measures were not significantly different between AE-SB and either S-SB or S-V rats. Motor activity in AE-V rats was significantly higher than in S-V (p = 0.003), S-SB (p = 0.007) or AE-SB (p = 0.02) rats, with no difference between S-SB and AE-SB group. Our findings suggest that excessive activity of ORX neurons contributes to motor hyperactivity and impaired HR acquisition following perinatal AE and that these symptoms may be alleviated by systemic antagonism of ORX-1 receptors.

Arginylation of Myosin Heavy Chain Regulates Skeletal Muscle Strength

Protein arginylation is a posttranslational modification with an emerging global role in the regulation of actin cytoskeleton. To test the role of arginylation in the skeletal muscle, we generated a mouse model with Ate1 deletion driven by the skeletal muscle-specific creatine kinase (Ckmm) promoter. Ckmm-Ate1 mice were viable and outwardly normal; however, their skeletal muscle strength was significantly reduced in comparison to controls. Mass spectrometry of isolated skeletal myofibrils showed a limited set of proteins, including myosin heavy chain, arginylated on specific sites. Atomic force microscopy measurements of contractile strength in individual myofibrils and isolated myosin filaments from these mice showed a significant reduction of contractile forces, which, in the case of myosin filaments, could be fully rescued by rearginylation with purified Ate1. Our results demonstrate that arginylation regulates force production in muscle and exerts a direct effect on muscle strength through arginylation of myosin.