Kölliker-Fuse Research Articles

The pontine Kölliker-Fuse nucleus is important for reduced post-inspiratory airflow elicited by stimulation of the ventral respiratory parafacial region.

Considering that the retrotrapezoid nucleus/respiratory parafacial region (RTN/pFRG) would be an important center in the central nervous system involved in the maintenance and modulation of respiratory activity, we hypothesized that neurons in this nucleus would also be involved in the postinspiratory phase of the respiratory cycle through a connection with the pontine Kölliker-Fuse (KF) region. Here we performed pharmacogenetic manipulation (AAV-hM3D(Gq)-mCherry or AAV-hM4D(Gi)-mCherry) in VGlut2-cre, Ai6 conscious mice to evaluate breathing parameters through whole body plethysmography under baseline conditions (normoxia: FiO2 = 0.21) or under hypercapnia or hypoxia challenges (FiCO2 = 0.07 or FiO2 = 0.08). Under normoxia, selective stimulation of RTN/pFRG resulted in a smaller increase in VE (1,272 ± 102.5, vs. RTN/pFRG stimulation: 1,878 ± 122.1 ml/kg/min), due to a smaller increase in VT (5.4 ± 0.35, vs. RTN/pFRG stimulation: 7.77 ± 0.21 ml/kg) without changing fR in a condition of KF inhibition. However, inhibition of the VGlut2 neurons in the KF did affect the TE1 produced by selective activation of RTN/pFRG (119.9 ± 2.53, vs. RTN/pFRG stimulation: 104 ± 2.46 ms). Both the hypercapnia and hypoxia ventilatory response were reduced after inhibition of VGlut2-expressing KF neurons. Therefore, consistent with anatomical projections RTN/pFRG neurons regulate lung ventilation by controlling all aspects of breathing, i.e breathing frequency, inspiration, postinspiration and active expiration. All the modulation seems to be dependent on the integrity of the glutamatergic neurons in the KF region.

Lateral parabrachial FoxP2 neurons regulate respiratory responses to hypercapnia

About half of the neurons in the parabrachial nucleus (PB) that are activated by CO2 are located in the external lateral (el) subnucleus, express calcitonin gene-related peptide (CGRP), and cause forebrain arousal. We report here, in male mice, that most of the remaining CO2-responsive neurons in the adjacent central lateral (PBcl) and Kölliker-Fuse (KF) PB subnuclei express the transcription factor FoxP2 and many of these neurons project to respiratory sites in the medulla. PBclFoxP2 neurons show increased intracellular calcium during wakefulness and REM sleep and in response to elevated CO2 during NREM sleep. Photo-activation of the PBclFoxP2 neurons increases respiration, whereas either photo-inhibition of PBclFoxP2 or genetic deletion of PB/KFFoxP2 neurons reduces the respiratory response to CO2 stimulation without preventing awakening. Thus, augmenting the PBcl/KFFoxP2 response to CO2 in patients with sleep apnea in combination with inhibition of the PBelCGRP neurons may avoid hypoventilation and minimize EEG arousals.

Interaction between Kölliker-Fuse/A7 and the parafacial respiratory region on the control of respiratory regulation

Respiration is regulated by various types of neurons located in the pontine-medullary regions. The Kölliker-Fuse (KF)/A7 noradrenergic neurons play a role in modulating the inspiratory cycle by influencing the respiratory output. These neurons are interconnected and may also project to brainstem and spinal cord, potentially involved in regulating the post-inspiratory phase. In the present study, we hypothesize that the parafacial (pF) neurons, in conjunction with adrenergic mechanisms originating from the KF/A7 region, may provide the neurophysiological basis for breathing modulation. We conducted experiments using urethane-anesthetized, vagotomized, and artificially ventilated male Wistar rats. Injection of L-glutamate into the KF/A7 region resulted in inhibition of inspiratory activity, and a prolonged and high-amplitude genioglossal activity (GGEMG). Blockade of the α1 adrenergic receptors (α1-AR) or the ionotropic glutamatergic receptors in the pF region decrease the activity of the GGEMG without affecting inspiratory cessation. In contrast, blockade of α2-AR in the pF region extended the duration of GG activity. Notably, the inspiratory and GGEMG activities induced by KF/A7 stimulation were completely blocked by bilateral blockade of glutamatergic receptors in the Bötzinger complex (BötC). While our study found a limited role for α1 and α2 adrenergic receptors at the pF level in modulating the breathing response to KF/A7 stimulation, it became evident that BötC neurons are responsible for the respiratory effects induced by KF/A7 stimulation.

Inhibitory synaptic transmission is impaired in the Kölliker-Fuse of male, but not female, Rett syndrome mice.

Rett syndrome (RTT) is a severe neurodevelopmental disorder that mainly affects females due to silencing mutations in the X-linked MECP2 gene. One of the most troubling symptoms of RTT is breathing irregularity, including apneas, breath-holds, and hyperventilation. Mice with silencing mutations in Mecp2 exhibit breathing abnormalities similar to human patients and serve as useful models for studying mechanisms underlying breathing problems in RTT. Previous work implicated the pontine, respiratory-controlling Kölliker-Fuse (KF) in the breathing problems in RTT. The goal of this study was to test the hypothesis that inhibitory synaptic transmission is deficient in KF neurons from symptomatic male and female RTT mice. We performed whole cell voltage-clamp recordings from KF neurons in acute brain slices to examine spontaneous and electrically evoked inhibitory post-synaptic currents (IPSCs) in RTT mice and age- and sex-matched wild-type mice. The frequency of spontaneous IPSCs was reduced in KF neurons from male RTT mice but surprisingly not in female RTT mice. In addition, electrically evoked IPSCs were less reliable in KF neurons from male, but not female, RTT mice, which was positively correlated with paired-pulse facilitation, indicating decreased probability of release. KF neurons from male RTT mice were also more excitable and exhibited shorter-duration action potentials. Increased excitability of KF neurons from male mice was not explained by changes in axon initial segment length. These findings indicate impaired inhibitory neurotransmission and increased excitability of KF neurons in male but not female RTT mice and suggest that sex-dependent mechanisms contribute to breathing problems in RTT.NEW & NOTEWORTHY Kölliker-Fuse (KF) neurons in acute brain slices from male Rett syndrome (RTT) mice receive reduced inhibitory synaptic inputs compared with wild-type littermates. In female RTT mice, inhibitory transmission was not different in KF neurons compared with controls. The results from this study show that sex-specific alterations in synaptic transmission occur in the KF of RTT mice.

Opioid suppression of an excitatory pontomedullary respiratory circuit by convergent mechanisms.

Opioids depress breathing by inhibition of interconnected respiratory nuclei in the pons and medulla. Mu opioid receptor (MOR) agonists directly hyperpolarize a population of neurons in the dorsolateral pons, particularly the Kölliker-Fuse (KF) nucleus, that are key mediators of opioid-induced respiratory depression. However, the projection target and synaptic connections of MOR-expressing KF neurons are unknown. Here, we used retrograde labeling and brain slice electrophysiology to determine that MOR-expressing KF neurons project to respiratory nuclei in the ventrolateral medulla, including the preBötzinger complex (preBötC) and rostral ventral respiratory group (rVRG). These medullary-projecting, MOR-expressing dorsolateral pontine neurons express FoxP2 and are distinct from calcitonin gene-related peptide-expressing lateral parabrachial neurons. Furthermore, dorsolateral pontine neurons release glutamate onto excitatory preBötC and rVRG neurons via monosynaptic projections, which is inhibited by presynaptic opioid receptors. Surprisingly, the majority of excitatory preBötC and rVRG neurons receiving MOR-sensitive glutamatergic synaptic input from the dorsolateral pons are themselves hyperpolarized by opioids, suggesting a selective opioid-sensitive circuit from the KF to the ventrolateral medulla. Opioids inhibit this excitatory pontomedullary respiratory circuit by three distinct mechanisms-somatodendritic MORs on dorsolateral pontine and ventrolateral medullary neurons and presynaptic MORs on dorsolateral pontine neuron terminals in the ventrolateral medulla-all of which could contribute to opioid-induced respiratory depression.

Exploring sex differences in inhibitory synaptic transmission in the kolliker-fuse of rett syndrome mice

Rett Syndrome (RTT) is a rare neurodevelopmental disorder characterized by severe motor impairments including breathing problems. RTT is caused by mutations in the methyl CpG binding protein 2 (MeCP2) gene located on the X chromosome. Accordingly, girls and women account for the majority of RTT patients. The genetic nature of RTT lends itself to study in mice with loss of function mutations in the MeCP2 gene. Importantly, RTT mice display breathing irregularities similar to human patients. Insufficient inhibitory neurotransmission in the brainstem respiratory network is thought to contribute to the breathing problems in RTT. In the pontine Kölliker-Fuse (KF) area of RTT mice, there are fewer GABAergic neurites and injection of a GABA reuptake antagonist alleviates breathing irregularity in vivo. However, no one has examined the physiology of inhibitory synaptic transmission in the KF of RTT mice. In the present work, we tested the hypothesis that inhibitory synaptic transmission was deficient in the respiratory-controlling KF of MeCP2Bird mice. Although girls and women make up the majority of RTT patients, most studies examining synaptic physiology in the brainstem have been performed using male MeCP2-deficient mice. Hemizygous male MeCP2Bird mice lack MeCP2 protein and exhibit rapid onset of severe RTT symptoms which leads to short life spans. Due to partial X-inactivation, MeCP2 expression in heterozygous female MeCP2-deficient mice is mosaic, leading to variable symptom severity and delayed onset, which more closely resembles the human condition. In the present study, we examined inhibitory synaptic transmission in the KF of female heterozygous MeCP2Bird mice compared to male hemizygous MeCP2Bird mice and wild type littermates. Whole-cell voltage-clamp recordings from KF neurons contained in acute brain slices were used to measure inhibitory currents. Most notably, the frequency of spontaneous inhibitory postsynaptic currents is reduced in the KF of male, but not female, MeCP2Bird mice compared to wild type littermates. To better understand this sex difference in inhibitory neurotransmission, we assessed the influence of estrous cycle stage and MeCP2 expression status in the recorded neuron. The outcomes of this research will increase our understanding of synaptic function in respiratory control neurons in female Rett Syndrome mice. This research was supported by the International Rett Syndrome Foundation Basic Research Grant 3608. 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.

GABAergic and Glycinergic Synaptic Transmission in Respiratory‐Controlling Kolliker‐Fuse Neurons in Female and Male Rett Syndrome Mice

Rett Syndrome (RTT) is a neurodevelopmental disorder characterized by severe motor behavior disturbances including breathing problems. RTT is caused by silencing mutations in the methyl‐CpG binding protein 2 (MeCP2) gene located on the X chromosome. Accordingly, girls and women account for the majority of RTT patients. The genetic nature of RTT lends itself to study in mice with loss of function mutations in the MeCP2 gene. Importantly, RTT mice display breathing irregularities similar to human patients. Insufficient inhibitory neurotransmission in the brainstem respiratory network is thought to contribute to the breathing problems in RTT. In the pontine Kölliker‐Fuse (KF) area of RTT mice, there are fewer GABAergic axon terminals and injection of a GABA reuptake antagonist alleviates breathing problems in vivo. However, no one has examined the physiology of GABA or glycine synapses in the KF of RTT mice. In the present work, we tested the hypothesis that GABAergic and glycinergic synapses are deficient in the KF of RTT mice. We performed whole‐cell voltage clamp recordings from KF neurons contained in brain slices of MeCP2‐deficient RTT mice and wild type littermates. The frequency of spontaneous inhibitory postsynaptic currents (sIPSCs) in the KF neurons of male RTT mice was reduced compared to wild type (WT) littermates. In contrast, female RTT mice showed no change in sIPSC frequency in the KF compared to control, despite having breathing abnormalities. The amplitude of sIPSCs in the KF of female mice were smaller compared to WT littermates, whereas male RTT mice showed no change in amplitude compared to WT littermates. These results spotlight the importance of studying respiratory controlling KF neurons in both male and female RTT mice, and suggest that reductions in IPSC frequency and amplitude are not sufficient to explain the breathing irregularities observed in RTT in females and males, respectively.

Roles of glutamate and GABA of the Kölliker-Fuse nucleus in generating the cardiovascular chemoreflex.

The Kölliker-Fuse (KF) nucleus is a part of the parabrachial complex, located in the dorsolateral pons. It is involved in the chemoreflex-evoked cardiovascular and respiratory changes, but the role of GABA and glutamate in cardiovascular chemoreflex has not been shown yet. This study was performed to determine the role of GABA, glutamate, and their interaction in the KF, in cardiovascular chemoreflex in anesthetized rat. The antagonists were microinjected into the KF, and arterial pressure, heart rate, and single-unit responses were recorded simultaneously. The chemoreflex was evoked by i.v. injection of KCN, consisted of a short pressor followed by long bradycardia responses. Both responses were significantly attenuated by injection of a synaptic blocker (CoCl2) into the KF, confirming involvement of the KF in generating the reflex. Microinjection of AP5, an NMDA receptor antagonist, into the KF significantly attenuated the pressor and bradycardia responses, while blocking the AMPA receptors by CNQX had no significant effect. Blockade of GABAA receptors by bicuculline methiodide (BMI) potentiated both responses. Co-injection of BMI and CNQX potentiated the responses too. Co-injection of BMI and AP5 had no significant effect on the pressor response but significantly attenuated the bradycardia response. In conclusion, the KF plays a role in generating cardiovascular chemoreflex via its glutamate NMDA but not AMPA receptors. GABA inhibits both components of this reflex through GABAA receptors. There is an interaction between GABAA and NMDA receptors in regulating the bradycardia response of the reflex. Single-unit results were also presented which were correlated with and supported the homodynamic findings.

Effects of Different Systemic Opioid Doses on Subareas of the Ventral Respiratory Column

BackgroundThe Parabrachial Nucleus (PBN)/Kölliker‐Fuse (KF) complex are important subareas of the ventral respiratory column (VRC) that regulate inspiratory on‐ ( 1) and off‐switch ( 2). We have shown in vagus‐intact rabbits that respiratory rate depression caused by clinical opioid concentrations was in part mediated by the PBN ( 3), and that systemic opioid effects on respiratory phase timing could be partially reversed in the preBötzinger complex (preBC) albeit without reversing respiratory rate depression ( 4). The present study was to determine how much the combination of local microinjections of naloxone into the KF, PBN and preBC could reverse respiratory depression from intravenous remifentanil (REMI).MethodsThe study was approved by the local Animal Care Committee and conformed to NIH standards. Adult New Zealand White rabbits (3–4kg) were anesthetized, tracheotomized, ventilated, decerebrated and vagotomized. Phrenic nerve activity was recorded from the c5 rootlet, time averaged and used to calculate inspiratory and expiratory duration and peak phrenic activity (PPA). Gridwise localized pressure microinjections of α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA, 50 μM, 70 nl) utilizing a multibarrel glass pipette into the VRC caudal to the inferior collicle identified the PBN where AMPA injection caused significant tachypnea and the KF where AMPA caused transient bradypnea and a decrease in PPA. The tachypneic response to AMPA injection in the medulla was used to identify the preBC area. After functional identification of the PBN, KF and preBC areas, we injected a bolus of 10mcg REMI IV, which resulted in apnea. On return of the breathing pattern, a REMI infusion was started to achieve a steady‐state respiratory rate depression of 50% of control rate. After reaching steady‐state, we injected naloxone (1mM, 700nl) into the bilateral KF, PBN and preBC. We then repeated the REMI bolus injection with the apneic dose of 10mcg. In a subset of animals, the 10 mcg REMI bolus was repeated after naloxone injection into the KF/PBN and after injection into the preBC. Residual respiratory depression was reversed at the end of the protocol with IV naloxone (300μM). Drug effects were compared with one‐way RM ANOVA. Data are expressed as mean±SD.ResultsIn 11 animals, IV REMI depressed respiratory rate from 39±7 to 18±6 breaths per minute (BPM). Naloxone injection recovered respiratory rate to 20±9 in the KF (p=0.36), to 26±9 after additional injection into the PBN (p<0.001), and to 28±7 bpm after injection into the preBC area (p=0.54). The IV REMI bolus caused apnea >30s at control but only depressed respiratory rate to 12±6 bpm after naloxone injection into the KF/PBN (p<0.001) and to 16±7 after additional naloxone reversal in the preBC area (p=0.21, n=5).ConclusionRespiratory rate depression from IV opioids is mostly mediated by the PBN, however, higher opioid doses may also affect other brainstem areas.Support or Funding InformationSupported by NIH R01GM112960701A1 (Dr. Stucke) and VA merit grant I01BX000721 (Dr. Zuperku)Figure 1

Defining Opioidergic, Glutamatergic Pontine Respiratory Patterned Neurons

Opioid overdose deaths have rapidly increased largely due to illicitly manufactured fentanyl. The main cause of death from opioid overdose is respiratory depression. Opioid‐induced respiratory depression is characterized by decreases in respiratory rate, chemoreceptor reflexes and impairment of the upper airway. Pharmacological inhibition of the Kölliker‐Fuse (KF) nucleus produces strikingly similar impairments to breathing as opioids, thus warranted investigation. Using the arterially perfused preparation, we have previously found that application of CTAP, a selective mu opioid receptor antagonist, directly into the KF prevents fentanyl induced apnea. Although the KF is sufficient to restore phasic motor output, it did not restore eupneic 3‐phase respiratory patterning. To determine the identity of KF neurons modulated by opioids, we performed extracellular recordings before, during and after fentanyl administration. Respiratory phase was determined by phrenic and vagal motor output. A subpopulation of inspiratory and post‐inspiratory pattered neurons stopped firing upon fentanyl administration, whereas, a variety of respiratory patterned neurons continued to fire in the presence of fentanyl, albeit some with changes in frequency. Although many medullary glutamatergic respiratory populations have been identified, the firing pattern of pontine respiratory modulated glutamatergic neurons remain unknown. In separate experiments, a promoter specific optogenetic approach was used with extracellular recording to identify Ca2+/Calmodulin‐dependent protein kinase II (CaMKIIa) expressing, presumably glutamatergic, neurons. These results suggest that KF neurons are differentially impacted by opioids and during overdose may promote aberrant glutamatergic transmission to downstream respiratory nuclei.Support or Funding InformationThis work was supported by NIH R01 DA047976.

Role of the Kölliker-Fuse nucleus in cardiovascular responses to hypoxia and baroreceptor activation in anesthetized rats.

Introduction: Parabrachial Kölliker-Fuse (KF) complex, located in dorsolateral part of the pons, is involved in the respiratory control, however, its role in the baroreflex and chemoreflex responses has not been established yet. This study was performed to test the contribution of the KF to chemoreflex and baroreflex and the effect of microinjection of a reversible synaptic blocker (Cocl2) into the KF in urethane anesthetized rats. Methods: Activation of chemoreflex was induced by systemic hypoxia caused by N2 breathing for 30 seconds "hypoxic- hypoxia methods" and baroreflex was evoked by intravenous injection (i.v.) of phenylephrine (Phe, 20 µg /kg/0.05–0.1 mL). N2 induced generalized vasodilatation followed by tachycardia reflex and Phe evoked vasoconstriction followed by bradycardia. Results: Microinjection of Cocl2 (5 mM/100 nL/side) produced no significant changes in the Phe-induced hypertension and bradycardia, whereas the cardiovascular effect of N2 was significantly attenuated by the injection of CoCl2 to the KF. Conclusion: The KF played no significant role in the baroreflex, but could account for cardiovascular chemoreflex in urethane anesthetized rats.

The Kölliker‐Fuse contains opioid‐sensitive inspiratory neurons and contributes to fentanyl‐induced apnea

Opioid overdose deaths have rapidly increased largely due to illicitly manufactured fentanyl. The cause of death from opioid overdose is respiratory depression. Opioid‐induced respiratory depression is characterized by decreases in respiratory rate, chemoreceptor reflexes and impairment of the upper airway. The Kölliker‐Fuse (KF) regulates upper airway coordination and modulates respiratory rate. Opioid agonists in the KF reduce respiratory rate and cause apneusis. The influence of the KF during systemic fentanyl‐induced respiratory depression was investigated using the in situ arterially perfused working heart‐brainstem preparation of rat. Application of fentanyl to the preparation resulted in dose‐dependent slowing of rate, apneusis consisting of low‐amplitude phrenic nerve bursts and loss of post‐inspiratory vagal output, and eventually apnea with occasional low amplitude phrenic bursts. Subsequent application of CTAP, a mu opioid receptor antagonist, directly into the KF increased the frequency of low amplitude phrenic nerve bursting to a rate similar to baseline, with no change in vagal output. A similar pattern emerged with pre‐application of the mu opioid antagonist CTAP into the KF prior to systemic fentanyl administration. In separate experiments, KF neurons were recorded extracellularly before and after fentanyl administration. Inspiratory neurons stopped firing upon fentanyl administration, whereas, expiratory neurons continued to fire in the presence of fentanyl. These results suggest that opioid‐sensitive inspiratory neurons in KF may initiate low amplitude phrenic bursts and reverse fentanyl‐induced apnea.Support or Funding InformationFunded by NIH Grant DA038069.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The Parabrachial Nucleus and Kölliker‐Fuse Nucleus contribute jointly to inspiratory and expiratory phase duration

The Parabrachial Nucleus (PBN)/ Kölliker‐Fuse (KF) complex are important subareas of the ventral respiratory column (VRC) that regulate inspiratory (1) and expiratory (2) phase off‐switch. Our goal was to determine whether the effects on phase duration of these two adjacent areas were specific for each area and independent of each other. The study was approved by the local Animal Care Committee and conformed to NIH standards. Adult New Zealand White rabbits (3–4 kg) were anesthetized, tracheotomized, ventilated, decerebrated and vagotomized. Phrenic nerve activity was recorded from the c5 rootlet, time averaged and used to calculate inspiratory and expiratory durations. Gridwise localized pressure microinjections of α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA, 50 μM, 70 nl) utilizing a multibarrel glass pipette with a tip diameter of 20μm into the VRC caudal to the inferior collicle identified two areas with distinctly different pattern changes: An area (on average 1mm caudal from inferior collicle, 2mm lateral from midline, 7 mm ventral to the dorsal surface) where AMPA injection caused significant tachypnea with increased peak phrenic activity (PPA) was identified as the PBN, and a second area (on average 1mm caudal, 0.5–1mm lateral and 2mm ventral to the PBN) where AMPA caused transient bradypnea and a decrease in PPA was identified as the KF. In ten animals, injection of the glutamate receptor antagonists (2,3‐dihydroxy‐6‐nitro‐7‐sulfamoyl‐benzo[f]quinoxaline) (NBQX, 1mM, 700nl) and (2R)‐amino‐5‐phosphonovaleric acid (AP5, 5mM, 700nl) into the KF area increased inspiratory (TI) and expiratory (TE) duration significantly from 0.8±0.1 to 8.7±3 sec (p=0.006, 2‐way ANOVA, factors: location, drug; mean±SE) and 1.2±0.1 to 8.5±2.7 sec (p=0.011), resp. Respiratory rate (RR) decreased from 32±3 bpm to 9±2 bpm (p<0.001) and peak phrenic activity (PPA) decreased from 100% to 89±3% (p=0.028). Subsequent glutamatergic block in the PBN caused an additional increase in TI to 18.8±3.4 sec (p<0.001) and TE to 23.5±3 sec (p<0.001). Respiratory rate was decreased to 2±1 bpm (p=0.009) without further change in PPA (83±6%, p=0.463). In six animals block of glutamatergic excitation first in the PBN and then in the KF resulted in similar overall effects (TI 0.8±0.1 to 20.9±6.1sec, TE 1.1±0.2 to 18.3±5.5sec, RR 36±6 to 5±3bpm, PPA 100 to 85±7%). While in some animals KF injections had greater effects on TI and PBN injections greater effects on TE, these differences were minor. Generally, the increases in TI and TE tracked closely suggesting that both, the KF and PBN contribute significantly to the inspiratory and expiratory off‐switch mechanism.Support or Funding InformationR01GM112960‐01A1 (Dr. Stucke) and VA merit grant I01BX000721 (Dr. Zuperku)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The Impact of Tauopathy on Ultrasonic Vocalization in a Mouse Model of Frontotemporal Dementia

Tauopathy features in many neurodegenerative diseases, including Alzheimer's disease and frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP‐17), where aggregation of misfolded and hyperphosphorylated tau impairs cytoplasmic function and axonal transport, and ultimately leads to cell death. Common complications in patients with FTDP‐17 include upper airway dysfunctions such as swallowing and voice disorders and mutism, and in previous work we demonstrated that upper airway and swallowing complications also occur in a mouse model of FTDP‐17 carrying the P301L mutation. Because of these complications, we investigated: 1) Whether Tau‐P301L transgenic mice also have impaired vocalization, owing to vocalization requiring expiratory airflow through the larynx; and 2) The underlying pathology in Tau‐P301L mice linking irregular vocal patterns to tauopathy.Male Tau‐P301L mice on an FVB/N background interacted with a female wildtype (WT) mouse in estrous for 5 minutes, during which ultrasonic vocalizations (USV) were recorded (UltraSoundGate condensor microphone, Avisoft Berlin, Germany). USV recordings were obtained from Tau‐P301L and wild‐type mice aged 3, 5, 7 and 9 months. In parallel, social interactivity was also monitored. While a trend for a lower number and duration of USV and a higher frequency of USV was observed in Tau‐P301L mice, USV in both Tau‐P301L and WT mice were diverse, with the large variance in the number, duration and frequency of vocalizations in Tau‐P301L and WT mice indicating no statistical difference between groups. However, in each age group, the number and duration of USV in a subpopulation of Tau‐P301L mice was lower than and outside the range of WT mice. In these Tau‐P301L mice, 80% of calls were less than 45 msec duration (vs 55% in WT), while 20% of calls were greater than 45 msec duration (vs 45% in WT). The same subpopulation of Tau‐P301L mice displayed a decreased complexity (frequency modulation) and repertoire (call types) of USV compared to WT. These changes to USV in a subpopulation of Tau‐P301L mice were already present in mice aged 3 months, and the parameters measured deteriorated with age. Immunohistochemical analysis identified presence of anti‐human PHF‐Tau (clone AT8, ThermoScientific) and anti‐tau phosphoserine 199/202 (Millipore) immunoreactivity in cell bodies of upper airway and vocalization‐related brain nuclei, including the Periaqueductal Gray (PAG), Nucleus Ambiguus and Kölliker Fuse (KF) nuclei. Further, compared to WT, 40% fewer forkhead box protein P2 (FOXP2) immunoreactive cell bodies were present in the KF of 9‐month‐old Tau‐P301L mice, while the number of FOXP2 immunoreactive cells in the PAG was similar.Our data indicate: 1) an early onset of vocalization disorder in a subpopulation of Tau‐P301L mice; 2) a deterioration in USV that progresses as Tau‐P301L mice age; and 3) presence of tauopathy and neurodegeneration in brain regions that regulate the upper airways and vocalization. Our results also provide a base for examining whether vocalization/voice disturbances are a potential ‘early’ detection diagnostic for dementia.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Desensitization and Tolerance of Mu Opioid Receptors on Pontine Kölliker-Fuse Neurons.

Acute desensitization of mu opioid receptors is thought to be an initial step in the development of tolerance to opioids. Given the resistance of the respiratory system to develop tolerance, desensitization of neurons in the Kölliker-Fuse (KF), a key area in the respiratory circuit, was examined. The activation of G protein-coupled inwardly rectifying potassium current was measured using whole-cell voltage-clamp recordings from KF and locus coeruleus (LC) neurons contained in acute rat brain slices. A saturating concentration of the opioid agonist [Met5]-enkephalin (ME) caused significantly less desensitization in KF neurons compared with LC neurons. In contrast to LC, desensitization in KF neurons was not enhanced by activation of protein kinase C or in slices from morphine-treated rats. Cellular tolerance to ME and morphine was also lacking in KF neurons from morphine-treated rats. The lack of cellular tolerance in KF neurons correlates with the relative lack of tolerance to the respiratory depressant effect of opioids.

Identification of Stress‐Activated Inputs to the Dorsomedial Hypothalamus

Acute psychological stress elicits a typical response characterized in the short term by elevated sympathetic nerve activity and increases in heart rate and blood pressure. While the primary neural circuitry that underpins these responses is still uncertain, the dorsomedial hypothalamus (DMH) is known to play a major role.1 However, the antecedent inputs responsible for DMH activation in response to stress are unknown. In the present study, retrograde tracing was combined with Fos immunohistochemistry following air jet stress (AJS) to identify neurons that both project to the DMH and are activated by stress.Sprague Dawley rats received a unilateral injection of 15–30 nl of retrograde tracer choleratoxin B subunit‐AlexaFluor 555 into the DMH. One week later rats were exposed to a 15 minute AJS protocol (n=4), while controls (n=2) were left undisturbed. Following perfusion, brain sections were processed for Fos immunohistochemistry and photographed. The distribution of retrogradely and/or Fos‐labeled neurons was mapped using a rat brain atlas.2 The number of labeled cells in key regions was counted. Data are expressed as mean ± SE cells per section.Retrogradely labeled neurons were found throughout the brainstem and forebrain (Table 1). The majority of labeled cells were located in the forebrain, in particular in the medial prefrontal cortex, lateral septal nucleus, claustrum and amygdala. Following AJS, Fos‐positive neurons were located in many, but not all, of the regions in which retrogradely labeled neurons were found. There was a significant increase in the number of Fos‐positive neurons in the Kolliker Fuse nucleus (KF) in the pons, the dorsomedial, dorsolateral and ventrolateral midbrain periaqueductal gray (PAG), amygdala, the cingulate cortex, lateral septal nucleus and the claustrum. Double‐labeled neurons were found in the brainstem in the KF and ventrolateral PAG, and in the forebrain in the amygdala, cingulate cortex, lateral septal nucleus and claustrum. However, overall there were very few double‐labeled neurons seen throughout the brain.Neurons that both project to the DMH and are activated by AJS were identified in several brain regions, particularly in the forebrain. The number of such neurons was very small, suggesting that these neurons may play a minor role in stress‐induced activation of the DMH. An alternate hypothesis is that recruitment of DMH in response to stress may be dependent upon disinhibition. Number of labeled cells in key nuclei Region CTb Fos CTb + Fos RVLM 0.7±0.4 9.2±5.1 0.0±0 RVMM 0.3±0.2 6.1±4 0.0±0 PBC 27.6±14.2 14.4±5.9 1.5±0.8 KF 20.2±14.8 11.0±4.0* 0.6±0.4* dmPAG 4.4±2.1 7.3±1* 0.2±0.2 dIPAG 4.2±1.5 10.7±0.6* 0.5±0.4 IPAG 8.6±2.7 19.9±2.4 0.3±0.1 vIPAG 12.0±1.6 18.4±3.2* 1.0±0.5* Amy 124.4±22.7 55.9±18.3* 2.4±0.7* Cingular Cor 88.8±32.1 17.0±0.3* 3.2±0.8* Lat SN 177.9±62.1 38.5±5.5* 4.9±1.8* Claustrum 148.3±55.2 35.8±11* 2.1±0.4* mPFC 192.0±79 42.4±21.1 3.3±0.8 Values are mean ± SE

Inhibition of the pontine Kölliker-Fuse nucleus reduces genioglossal activity elicited by stimulation of the retrotrapezoid chemoreceptor neurons

The Kölliker-Fuse (KF) region, located in the dorsolateral pons, projects to several brainstem areas involved in respiratory regulation, including the chemoreceptor neurons within the retrotrapezoid nucleus (RTN). Several lines of evidence indicate that the pontine KF region plays an important role in the control of the upper airways for the maintenance of appropriate airflow to and from the lungs. Specifically, we hypothesized that the KF region is involved in mediating the response of the hypoglossal motor activity to central respiratory chemoreflex activation and to stimulation of the chemoreceptor neurons within the RTN region. To test this hypothesis, we combined immunohistochemistry and physiological experiments. We found that in the KF, the majority of biotinylated dextran amine (BDA)-labeled axonal varicosities contained detectable levels of vesicular glutamate transporter-2 (VGLUT2), but few contained glutamic acid decarboxylase-67 (GAD67). The majority of the RTN neurons that were FluorGold (FG)-immunoreactive (i.e., projected to the KF) contained hypercapnia-induced Fos, but did not express tyrosine hydroxylase. In urethane-anesthetized sino-aortic denervated and vagotomized male Wistar rats, hypercapnia (10% CO2) or N-methyl-d-aspartate (NMDA) injection (0.1mM) in the RTN increased diaphragm (DiaEMG) and genioglossus muscle (GGEMG) activities and elicited abdominal (AbdEMG) activity. Bilateral injection of muscimol (GABA-A agonist; 2mM) into the KF region reduced the increase in DiaEMG and GGEMG produced by hypercapnia or NMDA into the RTN. Our data suggest that activation of chemoreceptor neurons in the RTN produces a significant increase in the genioglossus muscle activity and the excitatory pathway is dependent on the neurons located in the dorsolateral pontine KF region.

Inhibitory control of active expiration by the Kölliker‐Fuse in rats

Breathing is critically dependent on synchronized activity generated by neural respiratory centres within the pons and medulla. When respiratory drive is elevated, active expiration (AE) is observed, typically expressed by abdominal muscle activity at the end of the expiratory phase (late‐E). It is currently unknown what role the dorsolateral pons, specifically the Kolliker‐Fuse (KF), plays in generating the AE breathing pattern. Using the decerebrated in situ working heart‐brainstem preparation of juvenile male Holtzman rats (60–70 g), motor output from the vagus, phrenic and abdominal nerves were recorded during baseline conditions. The preparations were then exposed to hypercapnic conditions (8% CO2) to recruit AE. Hypercapnia resulted in a significant increase in the amplitude of abdominal late‐E activity (Δ: 118±30%, P<0.05), with little change to the other recorded parameters. During hypercapnia, the pharmacological inhibition of the KF, with bilateral microinjections of (50–75 nL) isoguvacine (10 mM), caused a significant decrease in breathing frequency (Δ: −5±2 cpm, P<0.05) due to increased inspiratory time (0.86±0.1 vs 1.37±0.1 s, P<0.05). Interestingly, while there was no change to the overall expiratory time, the duration (1.86±0.21 vs 1.47±0.13 s, P<0.05) and amplitude (3.6±0.4 vs 0.7±0.2 μV, P<0.05) of post inspiratory (post‐I) vagal activity decreased while the onset of the abdominal late‐E bursts occurred significantly earlier (1.3±0.1 vs 2.4±0.2 s, P<0.05). No changes were observed in the abdominal late‐E burst amplitude after KF inhibition. These findings suggest that the KF plays a relevant inhibitory role in coordinating the transition between post‐I activity and AE during hypercapnia.Support or Funding InformationFinancial support: NSERC, FAPESP (2013/17251‐6) and NIH/NCCIH (1R01AT008632‐01).

Distribution of GABAergic Neurons in the Nuclei of the Pneumotaxic Center in the Early Postnatal Period in Health and Prenatal Insufficiency of the Serotoninergic System in Rats

The aim of the present work was to study the distribution of GABAergic neurons in structures of the pneumotaxic center (medial subnucleus of the parabrachial complex and the Kolliker–Fuse nucleus) in health and in conditions of serotoninergic system insufficiency during the prenatal period of development in Wistar rats. Endogenous serotonin content was decreased in fetuses by inhibition of tryptophan hydroxylase with parachlorophenylalanine, which was given to females on day 16 of pregnancy. Specimens from the pons from experimental and control (intact) rat pups were investigated at early postnatal (days 5, 10, and 12) and juvenile (day 20) ages. Groups of 5–6 experimental and control rat pups were used at each time point. GABAergic neurons were detected using antibodies to its synthetic enzyme glutamate decarboxylase (GAD67). The results showed that the Kolliker–Fuse nucleus contained a population of GABAergic neurons during the early periods of postnatal development, the numbers of cells in this population persisting to juvenile age. The medial subnucleus of the parabrachial complex contained an insignificant number of GABAergic neurons at the early time points, with a slight increase by juvenile age, though neurons continued to give weak immune reactions. Serotonin deficiency in structures of the pneumotaxic center led to decreases in the number of GABAergic neurons and decreases in the numbers of GABAergic synapses and their accumulations. The decrease in serotonin during the prenatal period could induce impairments of inhibitory afferentation of the nuclei of the pneumotaxic center and changes in local inhibitory GABAergic networks in its nuclei, which could result in impairments to inhibitory processes in the structures of this center.

μ opioid receptor activation hyperpolarizes respiratory-controlling Kölliker-Fuse neurons and suppresses post-inspiratory drive.

In addition to reductions in respiratory rate, opioids also cause aspiration and difficulty swallowing, indicating impairment of the upper airways. The Kölliker-Fuse (KF) maintains upper airway patency and a normal respiratory pattern. In this study, activation of μ opioid receptors in the KF reduced respiratory frequency and tidal volume in anaesthetized rats. Nerve recordings in an in situ preparation showed that activation of μ opioid receptors in the KF eliminated the post-inspiration phase of the respiratory cycle. In brain slices, μ opioid agonists hyperpolarized a distinct population (61%) of KF neurons by activation of an inwardly rectifying potassium conductance. These results suggest that KF neurons that are hyperpolarized by opioids could contribute to opioid-induced respiratory disturbances, particularly the impairment of upper airways. Opioid-induced respiratory effects include aspiration and difficulty swallowing, suggesting impairment of the upper airways. The pontine Kölliker-Fuse nucleus (KF) controls upper airway patency and regulates respiration, in particular the inspiratory/expiratory phase transition. Given the importance of the KF in coordinating respiratory pattern, the mechanisms of μ opioid receptor activation in this nucleus were investigated at the systems and cellular level. In anaesthetized, vagi-intact rats, injection of opioid agonists DAMGO or [Met(5) ]enkephalin (ME) into the KF reduced respiratory frequency and amplitude. The μ opioid agonist DAMGO applied directly into the KF of the in situ arterially perfused working heart-brainstem preparation of rat resulted in robust apneusis (lengthened low amplitude inspiration due to loss of post-inspiratory drive) that was rapidly reversed by the opioid antagonist naloxone. In brain slice preparations, activation of μ opioid receptors on KF neurons hyperpolarized a distinct population (61%) of neurons. As expected, the opioid-induced hyperpolarization reduced the excitability of the neuron in response to either current injection or local application of glutamate. In voltage-clamp recordings the outward current produced by the opioid agonist ME was concentration dependent, reversed at the potassium equilibrium potential and was blocked by BaCl2 , characteristics of a G protein-coupled inwardly rectifying potassium (GIRK) conductance. The clinically used drug morphine produced an outward current in KF neurons with similar potency to morphine-mediated currents in locus coeruleus brain slice preparations. Thus, the population of KF neurons that are hyperpolarized by μ opioid agonists are likely mediators of the opioid-induced loss of post-inspiration and induction of apneusis.