Medullary Brainstem Research Articles

Atlantoaxial subluxation and Down syndrome: A cross-sectional analysis.

Atlantoaxial subluxation (AAS) is a diagnosis describing misalignment of the C1 vertebra relative to C2. Excessive translation of this joint, located adjacent to the medullary brain stem, can lead to devastating neurological consequences. A higher prevalence of AAS within the Down syndrome (DS) population has been well-established. This study aims to establish a prevalence rate of DS in patients hospitalized for AAS and compare outcomes between AAS patients with and without DS. This study utilized the National Inpatient Sample (NIS) provided by the Healthcare Cost and Utilization Project (HCUP). In accordance with HCUP 2023 Clinical Classifications Software Refined files, data were queried using the International Classification of Diseases 10th Edition codes for DS and AAS. Demographics, comorbidities, hospital course, and outcomes were examined and compared using binary and linear multivariate regression. IBM SPSS software was used for data analysis. Of the 213,095 patients in the NIS database admitted between 2016 and 2020 with AAS as their primary diagnosis, 7.2% were DS patients. DS patients were significantly younger (26.56 ± 20.81 vs. 49.39 ± 27.63, P < 0.01), less likely to be female (33.30% vs. 52.10%), and had fewer comorbidities (diabetes mellitus, hypertension, and hyperlipidemia) than non-DS patients. There was no significant difference in likelihood to undergo surgical fusion between DS patients and non-DS patients with AAS. This large-scale study using NIS data determined that 7.2% of all patients admitted to hospitals for AAS are DS patients. The analysis of demographics, hospital course, and outcomes can influence the development of treatment protocols for AAS in the DS population.

Centrally derived gasotransmitter modulation of hypoglossal motoneuron activity

Airway obstruction during inspiration is a hallmark trait of sleep apnea (SA). In mice, genetic elimination of the CO producing enzyme, heme oxygenase-2 (HO-2), leads to a SA phenotype predominately associated with recurrent airway obstruction. This phenotype is mitigated by either inhibition or genetic ablation of the H2S producing enzyme, cystathionine g-lysase (CSE). While these observations indicate that mutual interactions between HO-2/CO and CSE/H2S are important for regulating airway clearance, the extent of contribution from these interaction in the central nervous system to airway tone are unclear. Here, we determine how HO-2/CO and CSE/H2S impact inspiratory output from the hypoglossal nucleus (XIIn) in the isolated rhythmic brainstem slice preparation. We hypothesize that these enzymes and their respective gasotransmitter products locally interact in the inspiratory circuit to modulate the input-output (I/O) relationship between preBötC and XIIn. Electrophysiological studies conducted in brainstem slices from mice (P5-12) showed that HO-2 dysregulation, using either in wildtype slices exposed to Cr (III) Mesoporphyrin IX chloride or in slices from HO-2 null mice, increased transmission failure from preBötC to XIIn and altered the I/O relationship between the rhythm generating network and XIIn. HO-2 dysregulation also increased subnetwork activity in the preBötC that was associated with a decrease in amplitude regularity. While many transmission failures to the XIIn were associated with preBötC subnetwork activity, simultaneous recordings from the preBötC, premotor area, and the XIIn revealed that preBötC subnetwork activity was reliably transmitted to the premotor field but not to the XIIn. Dual extracellular and intracellular recordings from the preBötC and individual XIIn neurons under disinhibited conditions revealed that HO-2 dysregulation reduced inspiratory drive currents received by XIIn neurons, decreased the intrinsic excitability of XIIn neurons while also reducing the amount of preBötC subnetwork activity. Additionally, targeted inhibition of CSE/H2S activity using L-PAG or by genetic elimination of CSE in HO-2 null mice mitigated these effects of HO-2 dysregulation. Thus, the mutual interactions HO-2/CSE and CSE/H2S in the medullary brainstem appear to modulate XIIn output by: (1) causing a E/I imbalance that promotes preBötC subnetwork activity; (2) suppressing inspiratory drive received in XIIn; and (3) suppressing intrinsic excitability of XIIn neurons. These findings may be key to understanding the action by which central gasotransmitters actions contribute to upper airway muscle regulation. This work was supported by NIH: P01-HL-44454; R01NS107421. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Clinical experience with an in-NICU magnetic resonance imaging system

ObjectiveTo evaluate the utility of the 1 Tesla (1 T) Embrace (Aspect Imaging) neonatal magnetic resonance imaging (MRI) scanner in a level III NICU.Study designEmbrace brain MRI findings for 207 infants were reviewed, including 32 scans directly compared within 5 days with imaging on a 3 T Siemens Trio. Clinical MRI scan abnormalities were also compared to cranial ultrasound findings.ResultOf the 207 Embrace brain MRIs, 146 (70.5%) were obtained for clinical indications and 61 (29.5%) were research cases. Abnormal findings were found in 80 scans, most commonly hemorrhage and white matter injury. Notable findings included a stroke, medullary brainstem tumor, and polymicrogyria. In the 1 T versus 3 T comparison cohort, results were discordant in only one infant with punctate foci of susceptibility noted only on the 3 T scan.ConclusionThe Embrace MRI scans detected clinically relevant brain abnormalities and in a subset were clinically comparable to 3 T scans.

The Role of Arginine Vasopressin in Modulating Inspiratory Bursting Behavior in the Hypoglossal Motor Nucleus in Neonatal Mice

Arginine vasopressin (AVP) is classically known as a hormone, but it also acts as a neurotransmitter that binds V1a receptors in the central nervous system. V1a receptors are expressed at high levels early in postnatal life in cranial and spinal motoneurons. While AVP receptor expression levels decrease in some cranial motoneuron populations with increasing developmental age, AVP receptor mRNA levels are expressed at high levels in the hypoglossal (XII) motor nucleus in adult mice. Recordings from individual XII motoneurons demonstrated that AVP has excitatory effects on young XII motoneurons, although the signaling pathways and effector ion channels remain to be fully elucidated. Additionally, previous research demonstrated that GABAergic disinhibition within the paraventricular nucleus of adult male rats, which has the effect of promoting AVP release, led to stimulation of breathing and increased diaphragm and genioglossus (innervated by XII, primary tongue protruder) muscle activity. Whether AVP acts directly at XII motoneurons to potentiate inspiratory bursting is unknown. Our aim was to characterize the effects of AVP on inspiratory bursting behavior at XII motoneurons. To test our hypothesis that AVP will potentiate inspiratory bursting behavior, we utilized an in vitro medullary brainstem slice (600 µM) preparation of neonatal (postnatal day, P0-5) CD1 mouse brains of either sex that contains the nuclei for respiratory movement of the tongue. By superfusing the slice with warmed artificial cerebral spinal fluid, we were able to record inspiratory bursting activity using a suction electrode under baseline conditions and in response to local drug application. Local injections of AVP at 0.01 µM had little effect on inspiratory bursting amplitude (111% ± 3% of baseline, n=6), but at 0.1 µM or 1 µM, initial results suggest it increased inspiratory burst amplitude (134% ± 5% of baseline, n=13 and 130% ± 8% of baseline, n=7 respectively, p = 0.0889). The excitatory effect of AVP was significantly diminished using a V1a receptor antagonist, d(CH2)51Tyr(Me)2Arg8) Vasopressin (500nM) (Initial AVP increase in burst amplitude: 141% ± 8%; AVP after antagonist: 113% ± 4%; AVP after washout: 120% ± 3%, n=10, p = 0.0437). Vasopressin and oxytocin are closely related nonapeptides, and since the XII motor nucleus also expresses oxytocin receptors, we next tested whether the AVP effects could be attributed to activation of the oxytocin receptor. Our preliminary results suggest that the oxytocin receptor antagonist, L-371,257 (50nM), did attenuate the response of AVP (Initial AVP increase in burst amplitude: 144% ± 11%; AVP after antagonist: 114% ± 5%; AVP after Washout: 122% ± 6%, n=8, p = 0.0901). Lastly, we discovered that vasopressin-related potentiation of inspiratory burst amplitude has an age-dependent increase from P0-5 (regression fit: y=13.933x+12.401, R2=0.4411, p=0.000402). Our results indicate that vasopressin, acting at V1a, and possibly oxytocin, receptors, can potentiate XII inspiratory bursting behavior in neonatal mice. Whether vasopressin has a role in maintaining airway patency in vivo in adult animals will need to be addressed in future studies.

A Freshening Breath of Insight

A Freshening Breath of Insight

Upper Motor Neuron Diseases and Laryngeal Presentations

The purpose of this manuscript is to review laryngeal manifestations of upper motor neuron disease. A review of the relevant neuroanatomy is performed as well as a description of common laryngeal signs of upper motor neuron disease and the disease entities that cause them. Upper motor neuron lesions must occur in the brainstem or in the bilateral cerebral hemisphere in order to cause signs and symptoms in the larynx. The presence of bilateral vocal fold paralysis or paresis with concurrent signs of myoclonus, tremor, rigidity, hyperactive gag reflex, and/or laryngospasm episodes should prompt an investigation for possible medullary brainstem or bilateral cerebral cortex pathology.

Therapeutic anti‐P2X4 receptor scFv antibody for chronic neuropathic pain

The P2X purinoceptor 4 (P2X4) plays a crucial role in signaling leading to chronic inflammatory and neuropathic pains, thus, the P2X4 receptor provides a new potential therapeutic target for their treatment. We have generated a panel of mouse single‐chain Fv‐antibodies (scFvs) recognizing extracellular peptide of P2X4 using cell‐free ribosome display, for use to pain‐signalling neurons. A mouse scFv library was generated, and ribosome display was applied for anti‐P2X4 scFv recombinant antibody selection. After three rounds of bio‐panning, a panel of recombinant antibodies was isolated followed by ELISA, cross reactivity analysis, western blotting and immunofluorescence staining. Anti‐P2X4 scFv clones with high specificity and affinity were successfully selected. Indirect ELISA assay revealed differential P2X4 peptide binding capability and specificity for three scFvs, with no reaction to the negative control anti‐Zika scFv 7‐2. The panning process was efficient in selecting clones of high affinity, as was evident from affinity differences of the scFvs: scFv95 and scFv12 had the highest and second highest affinities, respectively, while scFv103 had lower affinity. The highest affinity P2X4 receptor scFvs (12 and 95) were selected for in vivo behavior testing in the Foramen Rotundum Inflammatory Constriction Trigeminal Infraorbital Nerve injury (FRICT‐ION) Model. Single dose intraperitoneal injection (4 mg/kg, 100 μl) reversed mechanical allodynia and anxiety in mice with long standing neuropathic pain (8–10 weeks). Reversal to naïve baseline within one week was maintained for more than 10 weeks post‐treatment at experiment end. Continued reversal was maintained at baseline for &gt;20 weeks in a second cohort. The Western blot of the medullary brainstem provides evidence of brain penetration of both these scFv antibodies that reduce hypersensitivity and anxiety behaviors. We also performed whole‐cell patch clamp recordings from dissociated trigeminal ganglia neurons of nerve‐injured vs naïve mice and determined the effect of P2X4 receptor scFvs on neuronal excitability. These data provide support for pursuit of P2X4 receptor scFvs as translational therapy for pain relief.Support or Funding Information1. GRANT FUNDING NIH NIDCR R21 DE028096 2. Start‐up Funds to Dr. Durvasula 3. Startup Funds to Dr. Westlund

Considerable differences between auditory medulla, auditory midbrain, and hippocampal synapses during sustained high-frequency stimulation: Exceptional vesicle replenishment restricted to sound localization circuit

Reliable synaptic transmission is essential for interneuronal communication. Synaptic inputs to auditory brainstem neurons, particularly those involved in sound localization, are characterized by resilience during sustained activity and temporal precision in the sub-millisecond range. Both features are obtained by synchronous release of a high number of synaptic vesicles following a single action potential. Here, we compare transmission behavior of three heterogeneous types of inputs in the auditory midbrain and medulla. The first terminate in the central inferior colliculus (ICc) and are glutamatergic (activated from the lateral lemniscus, LL). The medullary inputs terminate in the lateral superior olive (LSO) and are glutamatergic (from the cochlear nuclear complex, CN) or glycinergic (from the medial nucleus of the trapezoid body, MNTB). LSO neurons are the first to integrate binaural information and compute interaural level differences, whereas ICc neurons receive information from almost all auditory brainstem nuclei and construct an initial auditory image used for reflexive behavior. We hypothesized that CN-LSO and MNTB-LSO inputs are more resilient to synaptic fatigue during sustained stimulation than LL-ICc inputs. To test the hypothesis, we performed whole-cell patch-clamp recordings in acute brainstem slices of juvenile mice. We investigated the synaptic performance during prolonged periods of high-frequency stimulation (60 s, up to 200 Hz) and assessed several features, e.g. depression, recovery, latency, temporal precision, quantal size and content, readily releasable pool size, release probability, and replenishment rate. Overall, LL-ICc inputs performed less robustly and temporally precisely than CN-LSO and MNTB-LSO inputs. When stimulated at ≥50 Hz, the former depressed completely within a few seconds. In contrast, CN-LSO and MNTB-LSO inputs transmitted faithfully up to 200 Hz, indicative of very efficient replenishment mechanisms. LSO inputs also displayed considerably lower latency jitter than LL-ICc inputs. The latter behaved similarly to two types of input in the hippocampus for which we performed a meta-analysis. Mechanistically, the high-fidelity behavior of LSO inputs, particularly MNTB-LSO synapses, is based on exceptional release properties not present at auditory midbrain or hippocampal inputs. We conclude that robustness and temporal precision are hallmarks of auditory synapses in the medullary brainstem. These key features are less eminent at higher stations, such as the ICc, and they are also absent outside the central auditory system, namely the hippocampal formation.

Upregulation of Neuronal VEGF in Experimental Cerebral Malaria Coincides with Disease Severity Biomarkers in Humans: A Novel Mechanism of Pathophysiology

Abstract Cerebral malaria (CM) is a deadly neurological complication of Plasmodium falciparum infection resulting in high mortality or severe cognitive deficit. Current therapeutics are highly ineffective, namely due to drug resistance and lack of mechanistic insight into pathophysiology. CM is characterized by VEGF upregulation, disruption of blood-brain barrier (BBB), vascular permeability, and severe edema detectable by MRI. Using small animal MRI and the established animal model of CM Plasmodium berghei ANKA, we recapitulate these features of human disease. Here we show that regions of brain permeability during CM as identified by T1-weighted gadolinium-enhanced and T2-weighted MRI coincide with VEGF mRNA upregulation. Regions of analysis include granular and glomerular cell layers of main olfactory bulb and superior medullary brainstem. We determined through RNA in situ hybridization that neurons are the major cell type responsible for the increased VEGF production during CM. Interestingly, we identified another cell type found to minimally upregulate VEGF in close proximity to these neurons. These VEGF expressing cells may play a role in support or induction of the neuronal response. Intriguingly, astrocytes show no VEGF upregulation during this pathology. Overall, these findings suggest a novel role for neuronal-VEGF in promoting CNS pathology, and a putative mechanism of BBB disruption and vascular permeability during CM.

Perception of Verticality and Vestibular Disorders of Balance and Falls.

Objective: To review current knowledge of the perception of verticality, its normal function and disorders. This is based on an integrative graviceptive input from the vertical semicircular canals and the otolith organs.Methods: The special focus is on human psychophysics, neurophysiological and imaging data on the adjustments of subjective visual vertical (SVV) and the subjective postural vertical. Furthermore, examples of mathematical modeling of specific vestibular cell functions for orientation in space in rodents and in patients are briefly presented.Results: Pathological tilts of the SVV in the roll plane are most sensitive and frequent clinical vestibular signs of unilateral lesions extending from the labyrinths via the brainstem and thalamus to the parieto-insular vestibular cortex. Due to crossings of ascending graviceptive fibers, peripheral vestibular and pontomedullary lesions cause ipsilateral tilts of the SVV; ponto-mesencephalic lesions cause contralateral tilts. In contrast, SVV tilts, which are measured in unilateral vestibular lesions at thalamic and cortical levels, have two different characteristic features: (i) they may be ipsi- or contralateral, and (ii) they are smaller than those found in lower brainstem or peripheral lesions. Motor signs such as head tilt and body lateropulsion, components of ocular tilt reaction, are typical for vestibular lesions of the peripheral vestibular organ and the pontomedullary brainstem (vestibular nucleus). They are less frequent in midbrain lesions (interstitial nucleus of Cajal) and rare in cortical lesions. Isolated body lateropulsion is chiefly found in caudal lateral medullary brainstem lesions. Vestibular function in the roll plane and its disorders can be mathematically modeled by an attractor model of angular head velocity cell and head direction cell function. Disorders manifesting with misperception of the body vertical are the pusher syndrome, the progressive supranuclear palsy, or the normal pressure hydrocephalus; they may affect roll and/or pitch plane.Conclusion: Clinical determinations of the SVV are easy and reliable. They indicate acute unilateral vestibular dysfunctions, the causative lesion of which extends from labyrinth to cortex. They allow precise topographical diagnosis of side and level in unilateral brainstem or peripheral vestibular disorders. SVV tilts may coincide with or differ from the perception of body vertical, e.g., in isolated body lateropulsion.

Noninvasive vagus nerve stimulation alters neural response and physiological autonomic tone to noxious thermal challenge.

The mechanisms by which noninvasive vagal nerve stimulation (nVNS) affect central and peripheral neural circuits that subserve pain and autonomic physiology are not clear, and thus remain an area of intense investigation. Effects of nVNS vs sham stimulation on subject responses to five noxious thermal stimuli (applied to left lower extremity), were measured in 30 healthy subjects (n = 15 sham and n = 15 nVNS), with fMRI and physiological galvanic skin response (GSR). With repeated noxious thermal stimuli a group × time analysis showed a significantly (p < .001) decreased response with nVNS in bilateral primary and secondary somatosensory cortices (SI and SII), left dorsoposterior insular cortex, bilateral paracentral lobule, bilateral medial dorsal thalamus, right anterior cingulate cortex, and right orbitofrontal cortex. A group × time × GSR analysis showed a significantly decreased response in the nVNS group (p < .0005) bilaterally in SI, lower and mid medullary brainstem, and inferior occipital cortex. Finally, nVNS treatment showed decreased activity in pronociceptive brainstem nuclei (e.g. the reticular nucleus and rostral ventromedial medulla) and key autonomic integration nuclei (e.g. the rostroventrolateral medulla, nucleus ambiguous, and dorsal motor nucleus of the vagus nerve). In aggregate, noninvasive vagal nerve stimulation reduced the physiological response to noxious thermal stimuli and impacted neural circuits important for pain processing and autonomic output.

Abstract #54: Imaging Medullary Brainstem Response to Respiratory-Gated Auricular Vagal Afferent Nerve Stimulation (RAVANS) at Ultrahigh-Field (7T)

Abstract #54: Imaging Medullary Brainstem Response to Respiratory-Gated Auricular Vagal Afferent Nerve Stimulation (RAVANS) at Ultrahigh-Field (7T)

D-serine released by astrocytes in brainstem regulates breathing response to CO2 levels

Central chemoreception is essential for adjusting breathing to physiological demands, and for maintaining CO2 and pH homeostasis in the brain. CO2-induced ATP release from brainstem astrocytes stimulates breathing. NMDA receptor (NMDAR) antagonism reduces the CO2-induced hyperventilation by unknown mechanisms. Here we show that astrocytes in the mouse caudal medullary brainstem can synthesize, store, and release d-serine, an agonist for the glycine-binding site of the NMDAR, in response to elevated CO2 levels. We show that systemic and raphe nucleus d-serine administration to awake, unrestrained mice increases the respiratory frequency. Application of d-serine to brainstem slices also increases respiratory frequency, which was prevented by NMDAR blockade. Inhibition of d-serine synthesis, enzymatic degradation of d-serine, or the sodium fluoroacetate-induced impairment of astrocyte functions decrease the basal respiratory frequency and the CO2-induced respiratory response in vivo and in vitro. Our findings suggest that astrocytic release of d-serine may account for the glutamatergic contribution to central chemoreception.

Chronic Intermittent Hypoxia Differentially Impacts Different States of Inspiratory Activity at the Level of the preBötzinger Complex.

The preBötzinger complex (preBötC) is a medullary brainstem network crucially involved in the generation of different inspiratory rhythms. In the isolated brainstem slice, the preBötC reconfigures to produce different rhythms that we refer to as “fictive eupnea” under baseline conditions (i.e., carbogen), and “fictive gasping” in hypoxia. We recently demonstrated that fictive eupnea is irregular following exposure to chronic intermittent hypoxia (CIH). However, it is unknown how CIH impacts fictive gasping. To address this, brain slices containing the preBötC were prepared from control and CIH exposed mice. Electrophysiological recordings of rhythmogenesis were obtained during the perihypoxic interval. We examined how CIH affects various dynamic aspects of the rhythm characterized by: (1) the irregularity score (IrS), to assess burst-to-variability; (2) the fluctuation value (χ), to quantify the gain of oscillations throughout the time series; and (3) Sample Entropy (sENT), to characterize the pattern/structure of oscillations in the time series. In baseline conditions, CIH increased IrS of amplitude (0.21 ± 0.2) and χ of amplitude (0.34 ± 0.02) but did not affect sENT of amplitude. This indicated that CIH increased burst-to-burst irregularity and the gain of amplitude fluctuations but did not affect the overall pattern/structure of amplitude oscillations. During the transition to hypoxia, 33% of control rhythms whereas 64% of CIH-exposed rhythms showed no doubling of period, suggesting that the probability for stable rhythmogenesis during the transition to hypoxia was greater following CIH. While 29% of control rhythms maintained rhythmicity throughout hypoxia, all slices from CIH exposed mice exhibited rhythms throughout the hypoxic interval. During hypoxia, differences in χ for amplitude were no longer observed between groups. To test the contribution of the persistent sodium current, we examined how riluzole influenced rhythmogenesis following CIH. In networks exposed to CIH, riluzole reduced the IrS of amplitude (-24 ± 14%) yet increased IrS of period (+49 ± 17%). Our data indicate that CIH affects the preBötC, in a manner dependent on the state of the oxygenation. Along with known changes that CIH has on peripheral sensory organs, the effects of CIH on the preBötC may have important implications for sleep apnea, a condition characterized by rapid transitions between normoxia and hypoxia.

Phox2b‐negative Parafacial Neurons Simultaneously Regulate Active Expiration and Airway Patency Under Hypercapnia in in situ Preparations of Rats

Hypercapnia produces active expiration in rats and the recruitment of late‐expiratory (late‐E) neurons located in the parafacial Respiratory Group (pFRG) of the ventral medullary brainstem. We tested the hypothesis that hypercapnia produces active expiration and concomitant cranial respiratory motor responses controlling the oropharyngeal and upper airway patency by pFRG late‐E neurons disinhibition, but not via synaptic excitation. Phrenic nerve, abdominal nerve (AbN), cranial respiratory motor nerves, subglottal pressure, and medullary and spinal neurons/motoneurons were recorded in in situ preparations of juvenile rats. Hypercapnia evoked AbN active expiration (n=12), exaggerated late‐E discharges in cranial respiratory motor outflows (n=12), and glottal dilatation (n=6) via late‐E synaptic excitation of abdominal (n=8), hypoglossal (n=6) and laryngeal (n=12) motoneurons. Simultaneous rhythmic late‐E activity in previously silent Phox2b‐negative pFRG neurons was also evoked by hypercapnia (n=12). In addition, hypercapnia‐evoked AbN active expiration, neural and neuronal late‐E activities were eliminated by pFRG inhibition (n=18), but not after blockade of synaptic excitation (n=9). On the other hand, pFRG inhibition did not affect either hypercapnia‐induced inspiratory increases in respiratory motor outflows (n=12) or CO2 sensitivity of the more medial Phox2b‐positive neurons in the retrotrapezoid nucleus (RTN; n=8), despite elimination of AbN active expiration. Our data suggest that neither RTN Phox2b‐positive nor other CO2‐sensitive brainstem neurons activate Phox2b‐negative pFRG late‐E neurons under hypercapnia to produce AbN active expiration and concomitant cranial motor respiratory responses controlling the oropharyngeal and upper airway patency. Hypercapnia seems to produce pFRG late‐E neuronal disinhibition in in situ preparations of juvenile rats to activate abdominal, hypoglossal and laryngeal motoneurons.Support or Funding InformationFAPESP: SÃO PAULO RESEARCH FOUNDATION

Non-chemosensitive parafacial neurons simultaneously regulate active expiration and airway patency under hypercapnia in rats.

Hypercapnia or parafacial respiratory group (pFRG) disinhibition at normocapnia evokes active expiration in rats by recruitment of pFRG late-expiratory (late-E) neurons. We show that hypercapnia simultaneously evoked active expiration and exaggerated glottal dilatation by late-E synaptic excitation of abdominal, hypoglossal and laryngeal motoneurons. Simultaneous rhythmic expiratory activity in previously silent pFRG late-E neurons, which did not express the marker of ventral medullary CO2 -sensitive neurons (transcription factor Phox2b), was also evoked by hypercapnia. Hypercapnia-evoked active expiration, neural and neuronal late-E activities were eliminated by pFRG inhibition, but not after blockade of synaptic excitation. Hypercapnia produces disinhibition of non-chemosensitive pFRG late-E neurons to evoke active expiration and concomitant cranial motor respiratory responses controlling the oropharyngeal and upper airway patency. Hypercapnia produces active expiration in rats and the recruitment of late-expiratory (late-E) neurons located in the parafacial respiratory group (pFRG) of the ventral medullary brainstem. We tested the hypothesis that hypercapnia produces active expiration and concomitant cranial respiratory motor responses controlling the oropharyngeal and upper airway patency by disinhibition of pFRG late-E neurons, but not via synaptic excitation. Phrenic nerve, abdominal nerve (AbN), cranial respiratory motor nerves, subglottal pressure, and medullary and spinal neurons/motoneurons were recorded in in situ preparations of juvenile rats. Hypercapnia evoked AbN active expiration, exaggerated late-E discharges in cranial respiratory motor outflows, and glottal dilatation via late-E synaptic excitation of abdominal, hypoglossal and laryngeal motoneurons. Simultaneous rhythmic late-E activity in previously silent pFRG neurons, which did not express the marker of ventral medullary CO2 -sensitive neurons (transcription factor Phox2b), was also evoked by hypercapnia. In addition, hypercapnia-evoked AbN active expiration, neural and neuronal late-E activities were eliminated by pFRG inhibition, but not after blockade of synaptic excitation. On the other hand, pFRG inhibition did not affect either hypercapnia-induced inspiratory increases in respiratory motor outflows or CO2 sensitivity of the more medial Phox2b-positive neurons in the retrotrapezoid nucleus (RTN). Our data suggest that neither RTN Phox2b-positive nor other CO2 -sensitive brainstem neurons activate Phox2b-negative pFRG late-E neurons under hypercapnia to produce AbN active expiration and concomitant cranial motor respiratory responses controlling the oropharyngeal and upper airway patency. Hypercapnia produces disinhibition of non-chemosensitive pFRG late-E neurons in in situ preparations of juvenile rats to activate abdominal, hypoglossal and laryngeal motoneurons.

The anatomical, cellular and synaptic basis of motor atonia during rapid eye movement sleep.

Rapid eye movement (REM) sleep is a recurring part of the sleep-wake cycle characterized by fast, desynchronized rhythms in the electroencephalogram (EEG), hippocampal theta activity, rapid eye movements, autonomic activation and loss of postural muscle tone (atonia). The brain circuitry governing REM sleep is located in the pontine and medullary brainstem and includes ascending and descending projections that regulate the EEG and motor components of REM sleep. The descending signal for postural muscle atonia during REM sleep is thought to originate from glutamatergic neurons of the sublaterodorsal nucleus (SLD), which in turn activate glycinergic pre-motor neurons in the spinal cord and/or ventromedial medulla to inhibit motor neurons. Despite work over the past two decades on many neurotransmitter systems that regulate the SLD, gaps remain in our knowledge of the synaptic basis by which SLD REM neurons are regulated and in turn produce REM sleep atonia. Elucidating the anatomical, cellular and synaptic basis of REM sleep atonia control is a critical step for treating many sleep-related disorders including obstructive sleep apnoea (apnea), REM sleep behaviour disorder (RBD) and narcolepsy with cataplexy.

Vessel diameter measurements at the medullary brainstem in vivo as an index of trigeminal activity

Activity within the CNS can be quantified by a variety of methods. Here, we present an indirect method utilizing the neuro-vascular coupling via a continuous measurement of the vessel diameter. In anaesthetized rats, induced neuronal activity in the trigeminal system could be detected via arterial diameter measurements at the back of the medullary brainstem. Building upon the previously described diameter plugin, in images aligned to compensate for ventilation and heart-rate associated movement, automated processing allowed an evaluation immediately after acquisition. Electrical stimulation of facial trigeminal areas or the cranial dura mater caused vasodilatation of arteries in the dorsal medullary brainstem, which was abolished after application of the CGRP receptor antagonist olcegepant. No change in diameter was observed in corresponding veins. Intravenous infusion of sodium sulfide, which induces nitroxyl formation, also induced vasodilatation of brainstem-supplying arteries. Both experiments show a functional role of CGRP in the trigeminal nuclear brainstem complex. The presented method allows estimating central activity at the spinal level by vascular responses.

Modeling of respiratory network: to sigh or not to sigh

The Pre-Botzinger Complex (PreBotC), a medullary brainstem region, is essential for generating breathing. Isolated in a transverse slice preparation, the preBotC continues to generate under normal conditions eupneic and at a lower frequency also sighs. The mechanisms generating the periodicity of fictive sighs as well as its intrinsic nature are largely unknown. Based the experimental observations such as 1) has the biphasic shape characterized as eupneic breath interrupted by augmentation with a second peak of activity, 2) the biphasic shape persists after blockage of inhibitory synapses, 3) the network is heterogeneous, we model PreBotC network that reproduces both types of activities. The model contains three types of neurons: intrinsically tonic spiking, intrinsic bursting neurons and intrinsic quiescent. Neurons within the network are sparsely connected through randomly distributed excitatory synapses. In this model we propose that the mechanism of generation is based slow oscillations generated by glia. Moreover, the model predicts that depending the modularity state of the system, the network exhibits sighs, does not exhibit sighs and shows only eupneic activity, or shows bi-stable activity meaning that sighs can be triggered between on and off states by short perturbation. The bistability depends slow calcium oscillations. The network model predicts that the presence of IP3-like channels is necessary to produce sigh behavior and to mimic transitions between activity states caused by neuromodulators. We analyze the model dynamics and describe dose-response curve to neuromodulator such as norepinephrine. Theoretical data are similar to experimental data from in vitro brain slice preparations and in vivo anesthetized mice.

P37. Effects of acetyl-dl-leucine on the cerebral activation pattern in cerebellar ataxia (FDG-PET study)

Introduction A recent case series study showed significant improvements of symptoms in patients with cerebellar ataxia on medication with the amino acid acetyl-dl-leucine (Tanganil) (Strupp et al., 2013). In analogy to electrophysiological studies in animals it was hypothesized that acetyl-DL-leucine modulates the activity of central vestibular neurons, preferably in the cerebellum, by normalizing the membrane potential of depolarized or hyperpolarized neurons and thereby improves central compensation. However, the mode of action of acetyl-dl-leucine and the site of central compensation remained speculative. To deepen our insights in the central compensatory processes induced by acetyl-dl-leucine we performed a FDG-PET study in cerebellar ataxic patients off and on treatment. Methods 21 patients with cerebellar ataxia of different etiologies were scanned by resting state FDG-PET twice, (A) off treatment (PET 1), and a second time (B) ⩾3months on treatment with 5g/day acetyl-dl-leucine (PET 2; n =18). At both time points the patients underwent a detailed neurological examination and measurements of standardized ataxia scales (SARA, SCAFI). Group subtraction analyses (PET 1 vs. PET 2; responders vs. non-responders) as well as statistical group comparisons with age- and gender-matched healthy controls were calculated with statistical parametric mapping (SPM). Results The contrast on vs. off treatment (PET 2 vs. PET 1) mainly showed signal differences in the mesencephalon (midbrain integration centers) as well as in the lateral medullary brainstem (vestibular nucleus region), thalami and midinsular regions bilaterally. The contrast PET 1 vs. PET 2 revealed signal differences in the posterior insula as well as in primary and secondary visual areas (including MT/V5) bilaterally. Restricting the analyses to treatment-responders ( n =11), PET 2 vs. PET 1 showed similar signal areas in the central midbrain and dorsal medullary brainstem bilaterally that were lacking in the corresponding contrast of the non-responders ( n =6). In the direct comparison of responders vs. non-responders PET 2 signal differences were located in both caudate nuclei, the superior temporal gyrus and hippocampus. The signal in responders – mainly in the posterior insula and the medial temporal gyrus – was lower compared to non-responders. In all contrasts cerebellar signal changes were sparse. Conclusions Central compensatory processes induced by acetyl-dl-leucine in cerebellar ataxia seem to involve mainly centers in the medulla (vestibular nuclei), midbrain (vestibular integration centers), thalami, basal ganglia and insular regions rather than neurons in the cerebellum, the primary site of dysfunction in cerebellar ataxia syndromes.