Gregory Funk

Muscarinic acetylcholine receptors enhance neonatal mouse hypoglossal motoneuron excitability in vitro

Publisher: American Physiological Society

Date: 10-2012

DOI: 10.1152/JAPPLPHYSIOL.00699.2011

Abstract: In brain stem slices from neonatal ( postnatal days 0–4) CD-1 mice, muscarinic ACh receptors (MAChRs) increased rhythmic inspiratory-related and tonic hypoglossal nerve discharge and depolarized single hypoglossal motoneurons (HMs) via an inward current without changing input resistance. These responses were blocked by the MAChR antagonist 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide (4-DAMP 100 nM). MAChRs shifted voltage-dependent activation of the hyperpolarization-activated cation current to more positive levels. MAChRs increased the HM repetitive firing rate and decreased rheobase, with both effects being blocked by 4-DAMP. Muscarinic agonists reduced the afterhyperpolarization of single action potentials (APs), suggesting that small-conductance Ca 2+ -dependent K + current inhibition increased the HM firing rate. Muscarinic agonists also reduced the AP litude and slowed its time course, suggesting that MAChRs inhibited voltage-gated Na + channels. To compare muscarinic excitation of single HMs to muscarinic excitatory effects on motor output in thicker brain stem slices requiring higher extracellular K + for rhythmic activity, we tested the effects of muscarinic agonists on single HM excitability in high-K + artificial cerebrospinal fluid (aCSF). In high-K + aCSF, muscarinic agonists still depolarized HMs and altered AP size and shape, as in standard aCSF, but did not increase the steady-state firing rate, decrease afterhyperpolarization, or alter threshold potential. These results indicate that the basic cellular response of HMs to muscarinic receptors is excitatory, via a number of distinct mechanisms, and that this excitatory response will be largely preserved in rhythmically active brain stem slices.

ATP sensitivity of preBötzinger complex neurones in neonatal rat in vitro: mechanism underlying a P2 receptor‐mediated increase in inspiratory frequency

Publisher: Wiley

Date: 29-02-2008

DOI: 10.1113/JPHYSIOL.2007.143024

Synaptic Control Of Motoneuron Excitability In Rodents: From Months To Milliseconds

Publisher: Wiley

Date: 04-01-2000

DOI: 10.1046/J.1440-1681.2000.03202.X

Abstract: 1. Motoneurons (MN) shape motor patterns by transforming inputs into action potential output. This transformation, excitability, is determined by an interaction between synaptic inputs and intrinsic membrane properties. Excitability is not static, but changes over multiple time scales. The purpose of the present paper is to review our recent data on synaptic factors important in the dynamic control of MN excitability over time scales ranging from weeks to milliseconds. 2. Developmental changes in modulation of MN excitability are well established. Noradrenergic potentiation of hypoglossal (XII) MN inspiratory activity in rhythmically active medullary slice preparations from rodents increases during the first two postnatal weeks. This is due to increasing α 1 ‐ and β‐adrenoceptor excitatory mechanisms and to a decreasing inhibitory mechanism mediated by α 2 ‐adrenoceptors. Over a similar period, ATP potentiation of XII inspiratory activity does not change. 3. Motoneuron excitability may also change on a faster time scale, such as between different behaviours or different phases of a behaviour. Examination of this has been confounded by the fact that excitatory synaptic drives underlying behaviour can obscure smaller concurrent changes in excitability. Using the rhythmically active neonatal rat brain‐stem–spinal cord preparation, we blocked excitatory inspiratory drive to phrenic MN (PMN) to reveal a reduction in PMN excitability specific to the inspiratory phase that: (i) arises from an inhibitory GABAergic input (ii) is not mediated by recurrent pathways and (iii) is proportional to and synchronous with the excitatory inspiratory input. We propose that the proportionality of the concurrent inhibitory and excitatory drives provides a means for phase‐ specific modulation of PMN gain. 4. Modulation across such erse time scales emphasizes the active role that synaptic factors play in controlling MN excitability and shaping behaviour.

Glial TLR4 signaling does not contribute to opioid-induced depression of respiration

Publisher: American Physiological Society

Date: 15-10-2014

DOI: 10.1152/JAPPLPHYSIOL.00534.2014

Abstract: Opioids activate glia in the central nervous system in part by activating the toll-like receptor 4 (TLR4)/myeloid differentiation 2 (MD2) complex. TLR4/MD2-mediated activation of glia by opioids compromises their analgesic actions. Glial activation is also hypothesized as pivotal in opioid-mediated reward and tolerance and as a contributor to opioid-mediated respiratory depression. We tested the contribution of TLR4 to opioid-induced respiratory depression using rhythmically active medullary slices that contain the pre-Bötzinger Complex (preBötC, an important site of respiratory rhythm generation) and adult rats in vivo. Injection with DAMGO (μ-opioid receptor agonist 50 μM) or bath application of DAMGO (500 nM) or fentanyl (1 μM) slowed frequency recorded from XII nerves to 40%, 40%, or 50% of control, respectively. This DAMGO-mediated frequency inhibition was unaffected by preapplication of lipopolysaccharides from Rhodobacter sphaeroides (a TLR4 antagonist, 2,000 ng/ml) or (+)naloxone (1–10 μM, a TLR4-antagonist). Bath application of (−)naloxone (500 nM a TLR4 and μ-opioid antagonist), however, rapidly reversed the opioid-mediated frequency decrease. We also compared the opioid-induced respiratory depression in slices in vitro in the absence and presence of bath-applied minocycline (an inhibitor of microglial activation) and in slices prepared from mice injected (ip) 18 h earlier with minocycline or saline. Minocycline had no effect on respiratory depression in vitro. Finally, the respiratory depression evoked in anesthetized rats by tail vein infusion of fentanyl was unaffected by subsequent injection of (+)naloxone, but completely reversed by (−)naloxone. These data indicate that neither activation of microglia in preBötC nor TLR4/MD2-activation contribute to opioid-induced respiratory depression.

Prenatal nicotine exposure increases apnoea and reduces nicotinic potentiation of hypoglossal inspiratory output in mice

Publisher: Wiley

Date: 02-2002

DOI: 10.1113/JPHYSIOL.2001.012705

Abstract: We examined the effects of in utero nicotine exposure on postnatal development of breathing pattern and ventilatory responses to hypoxia (7.4 % O2) using whole-body plethysmography in mice at postnatal day 0 (P0), P3, P9, P19 and P42. Nicotine delayed early postnatal changes in breathing pattern. During normoxia, control and nicotine-exposed P0 mice exhibited a high frequency of apnoea (f(A)) which declined by P3 in control animals (from 6.7 +/- 0.7 to 2.2 +/- 0.7 min(-1)) but persisted in P3 nicotine-exposed animals (5.4 +/- 1.3 min(-1)). Hypoxia induced a rapid and sustained reduction in f(A) except in P0 nicotine-exposed animals where it fell initially and then increased throughout the hypoxic period. During recovery, f(A) increased above control levels in both groups at P0. By P3 this increase was reduced in control but persisted in nicotine-exposed animals. To examine the origin of differences in respiratory behaviour, we compared the activity of hypoglossal (XII) nerves and motoneurons in medullary slice preparations. The frequency and variability of the respiratory rhythm and the envelope of inspiratory activity in XII nerves and motoneurons were indistinguishable between control and nicotine-exposed animals. Activation of postsynaptic nicotine receptors caused an inward current in XII motoneurons that potentiated XII nerve burst litude by 25 +/- 5 % in control but only 14 +/- 3 % in nicotine-exposed animals. Increased apnoea following nicotine exposure does not appear to reflect changes in basal activity of rhythm or pattern-generating networks, but may result, in part, from reduced nicotinic modulation of XII motoneurons.

Developmental downregulation of P2X₃ receptors in motoneurons of the compact formation of the nucleus ambiguus

Publisher: Wiley

Date: 08-2005

DOI: 10.1111/J.1460-9568.2005.04261.X

Abstract: Motoneurons of the compact ision of the nucleus ambiguus (cNA) are the final output neurons of the swallowing pattern generator. Thus, their normal function is critical to neonatal survival. To explore the role of purinergic signaling in modulating the excitability of these motoneurons during development, immunohistochemical and whole-cell recording techniques were used to characterize expression patterns of ionotropic P2X receptors and the effects of ATP on cNA motoneurons. Medullary slices containing the cNA were prepared from neonatal (P0-4) and juvenile (P15-21) rats. In neonatal cNA motoneurons, local application of 1 mM ATP produced a large (-133 +/- 17 pA n = 78), desensitizing, inward current that was mimicked by 1 mM alpha,beta meATP and 2meSATP, and inhibited by the P2 antagonist, PPADS (5 microM), and the P2X3 antagonist, A-317481 (0.1-1 mM). In juvenile cNA motoneurons, 1 mM ATP produced negligible currents, while 10 mM ATP produced small (-59 +/- 14 pA n = 42), primarily non-desensitizing currents. Immunohistochemistry demonstrated that in the neonate, the expression of P2X3 was robust, P2X2 and P2X5 moderate, P2X4 and P2X6 weak, and P2X1 absent. In the juvenile cNA, only low levels of P2X5 and P2X6 labeling were detected. These data indicate that P2X receptors in cNA motoneurons are profoundly downregulated during the first two postnatal weeks, and suggest a role for the purinoceptor system, particularly P2X3 receptors, in the control of esophageal motor networks during early postnatal periods.

Receptor subtype-specific modulation by dopamine of glutamatergic responses in striatal medium spiny neurons

Publisher: Elsevier BV

Date: 2003

DOI: 10.1016/S0006-8993(02)03757-5

Abstract: The output of GABAergic medium-sized spiny neurons in the dorsal striatum is controlled in part by glutamatergic input from the neocortex and the thalamus, and dopaminergic input from ventral midbrain. We acutely isolated these neurons from juvenile (P14-24) rats to study the consequences of the interaction between glutamate and dopamine for neuronal excitability. Single-cell RT-PCR analysis was used to identify the expression patterns of dopamine receptors. D1 and D2 dopamine receptor mRNA was detected in 11/22 and 3/22 of isolated neurons, respectively. Receptor mRNA co-expression was detected in 1/22 cells tested. Whole-cell voltage cl recording (V(h)=-70 mV) was combined with local or bath application of dopaminergic and glutamatergic agonists to explore dopamine receptor modulation of glutamatergic excitation. Glutamate-evoked inward currents (5 microM, Mg(2+)-free, 1 microM glycine) were attenuated by dopamine (5 microM) to 83.2+/-3.6% (n=31). NMDA-evoked (20 microM), APV-sensitive currents were attenuated by dopamine to 80.9+/-4.5% (n=24). NMDA-induced responses were also attenuated by the D1 receptor agonist SKF 38393 (1 microM n=28), while the D2/3 receptor agonist quinpirole (10 microM) had no effect. The currents evoked by application of AMPA (5 microM) displayed a steady rundown. Application of dopamine abolished or significantly reduced the rundown in the cells tested (n=17). A similar effect was observed after the application of SKF 38393 (1 microM), while quinpirole (10 microM) had no significant effect. Our results provide direct evidence for modulation by dopamine of glutamatergic responses of striatal medium spiny neurons, and demonstrate that the effects of this neuromodulator are receptor subtype specific. Disruption of this modulatory effect is likely to contribute to movement disorders associated with Parkinson's disease.

P2 receptors modulate respiratory rhythm but do not contribute to central CO2 sensitivity in vitro

Publisher: Elsevier BV

Date: 08-2004

DOI: 10.1016/J.RESP.2004.04.007

P2Y₁Receptor Modulation of the Pre-Bötzinger Complex Inspiratory Rhythm Generating NetworkIn Vitro

Publisher: Society for Neuroscience

Date: 31-01-2019

DOI: 10.1523/JNEUROSCI.3948-06.2007

Abstract: ATP is released during hypoxia from the ventrolateral medulla (VLM) and activates purinergic P2 receptors (P2Rs) at unknown loci to offset the secondary hypoxic depression of breathing. In this study, we used rhythmically active medullary slices from neonatal rat to map, in relation to anatomical and molecular markers of the pre-Bötzinger complex (preBötC) (a proposed site of rhythm generation), the effects of ATP on respiratory rhythm and identify the P2R subtypes responsible for these actions. Unilateral microinjections of ATP in a three-dimensional grid within the VLM revealed a “hotspot” where ATP (0.1 m m ) evoked a rapid 2.2 ± 0.1-fold increase in inspiratory frequency followed by a brief reduction to 0.83 ± 0.02 of baseline. The hotspot was identified as the preBötC based on histology, overlap of injection sites with NK1R immunolabeling, and potentiation or inhibition of respiratory frequency by SP ([Sar 9 -Met(O 2 ) 11 ]-substance P) or DAMGO ([ d -Ala 2 , N -MePhe 4 ,Gly-ol 5 ]-enkephalin), respectively. The relative potency of P2R agonists [2MeSADP (2-methylthioadenosine 5′-diphosphate) ≈ 2MeSATP (2-methylthioadenosine 5′-triphosphate) ≈ ATPγs (adenosine 5′-[γ-thio]triphosphate tetralithium salt) ≈ ATP ≫ UTP ≈ αβmeATP (α,β-methylene-adenosine 5′-triphosphate)] and attenuation of the ATP response by MRS2179 (2′-deoxy- N 6 -methyladenosine-3′,5′-bisphosphate) (P2Y 1 antagonist) indicate that the excitation is mediated by P2Y 1 Rs. The post-ATP inhibition, which was never observed in response to ATPγs, is dependent on ATP hydrolysis. These data establish in neonatal rats that respiratory rhythm generating networks in the preBötC are exquisitely sensitive to P2Y 1 R activation, and suggest a role for P2Y 1 Rs in respiratory motor control, particularly in the P2R excitation of rhythm that occurs during hypoxia.

Effects of muscarinic acetylcholine receptor activation on membrane currents and intracellular messengers in medium spiny neurones of the rat striatum

Publisher: Wiley

Date: 09-2004

DOI: 10.1111/J.1460-9568.2004.03576.X

Cholinergic Modulation Of Respiratory Brain‐Stem Neurons And Its Function In Sleep–Wake State Determination

Publisher: Wiley

Date: 04-01-2000

DOI: 10.1046/J.1440-1681.2000.03192.X

Abstract: 1. Shifts in behavioural state are controlled by reciprocal changes in discharge of cholinergic and aminergic groups of brain‐stem ontine neurons. During rapid eye movement (REM) sleep, cholinergic neurons are most active and aminergic neurons are least active. 2. Significant changes occur in the central control of breathing during REM sleep respiration rate increases in frequency and variability, brain‐stem respiratory neuron discharge is generally enhanced and the outputs of some respiratory motor neuron pools are depressed. 3. Hypoglossal motor neurons (HM) control tongue movement and their depression during REM sleep has been implicated in obstructive sleep apnoea. The cellular basis of HM depression has been investigated in vitro and may be due to enhanced activation of cholinergic receptors or decreased activation of aminergic receptors. 4. In vitro preparations that show respiratory rhythmogenesis possess advantages for the investigation of the neurochemical basis of state‐dependent changes in respiration. Cholinergic changes in respiratory modulation of HM recorded in rhythmic brain‐stem slices from mice depend on the site of activation of cholinergic receptors.

The University Of Auckland

Location: New Zealand

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University Of Alberta

Location: Canada

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University Of British Columbia

Location: Canada

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