Synaptic control of motoneuron excitability in rodents: from months to milliseconds.

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 alpha 1- and beta-adrenoceptor excitatory mechanisms and to a decreasing inhibitory mechanism mediated by alpha 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 diverse time scales emphasizes the active role that synaptic factors play in controlling MN excitability and shaping behaviour.

A method for activating neurons using endogenous synaptic waveforms.

Neuronal input-output functions are traditionally studied using rectangular or ramp waveforms of injected current. These waveforms are easy to produce and responses to them easy to quantify; thus they have been central to our understanding of the roles that membrane properties play in controlling repetitive firing. However, since smooth rectangular step and ramp waveforms lack the dynamic features of endogenous synaptic input, their use has the potential to underemphasize the importance of input patterns in controlling physiological patterns of neuronal output. To activate neurons with current waveforms that replicate natural synaptic input, we developed a method for acquiring, digitally manipulating and reinjecting endogenous synaptic currents. We demonstrate, by applying this technique to phrenic motoneurons (PMNs) in rhythmically-active in vitro preparations from neonatal rats, that stimulation of neurons with endogenous current waveforms produces responses that mimic those produced by spontaneous synaptic inputs. Acquired waveforms can be reinjected repeatedly to produce consistent responses, and can also be amplified or filtered prior to reinjection to yield a range of information including standard descriptors of firing behavior such as frequency/current plots. This technique provides a valuable tool for analysing characteristics of the synaptic waveform important in generating neuronal output and how synaptic factors interact with membrane properties to control repetitive firing.

Concurrent inhibition and excitation of phrenic motoneurons during inspiration: phase-specific control of excitability.

The movements that define behavior are controlled by motoneuron output, which depends on the excitability of motoneurons and the synaptic inputs they receive. Modulation of motoneuron excitability takes place over many time scales. To determine whether motoneuron excitability is specifically modulated during the active versus the quiescent phase of rhythmic behavior, we compared the input-output properties of phrenic motoneurons (PMNs) during inspiratory and expiratory phases of respiration. In neonatal rat brainstem-spinal cord preparations that generate rhythmic respiratory motor outflow, we blocked excitatory inspiratory synaptic drive to PMNs and then examined their phase-dependent responses to superthreshold current pulses. Pulses during inspiration elicited fewer action potentials compared with identical pulses during expiration. This reduced excitability arose from an inspiratory-phase inhibitory input that hyperpolarized PMNs in the absence of excitatory inspiratory inputs. Local application of bicuculline blocked this inhibition as well as the difference between inspiratory and expiratory firing. Correspondingly, bicuculline locally applied to the midcervical spinal cord enhanced fourth cervical nerve (C4) inspiratory burst amplitude. Strychnine had no effect on C4 output. Nicotinic receptor antagonists neither potentiated C4 output nor blocked its potentiation by bicuculline, further indicating that the inhibition is not from recurrent inhibitory pathways. We conclude that it is bulbospinal in origin. These data demonstrate that rapid changes in motoneuron excitability occur during behavior and suggest that integration of overlapping, opposing synaptic inputs to motoneurons is important in controlling motor outflow. Modulation of phasic inhibition may represent a means for regulating the transfer function of PMNs to suit behavioral demands.

Developmental modulation of mouse hypoglossal nerve inspiratory output in vitro by noradrenergic receptor agonists.

The ontogeny of the noradrenergic receptor subtypes modulating hypoglossal (XII) nerve inspiratory output was characterized. Noradrenergic agents were locally applied over the XII nucleus of rhythmically active medullary slice preparations isolated from mice between zero and 13 days of age (P0-P13) and the effects on XII inspiratory burst amplitude quantified. The alpha1 receptor agonist phenylephrine (PE, 0.1-10 microM) produced a dose-dependent, prazosin-sensitive (0.1-10 microM) increase in XII nerve inspiratory burst amplitude. The magnitude of this potentiation increased steadily from a maximum of 15+/-8% in P0 mice to 134+/-4% in P12-P13 mice. The beta receptor agonist isoproterenol (0.01-1.0 mM) produced a prazosin-insensitive, propranolol-sensitive potentiation of XII nerve burst amplitude. The isoproterenol-mediated potentiation increased with development from 27+/-5% in P0-P1 slices, to 37+/-3% in P3 slices and 45+/-4% in P9-P10 slices. The alpha2 receptor agonist clonidine (1 mM) reduced XII nerve inspiratory burst amplitude in P0-P3 slices by 29+/-5%, but had no effect on output from P12-P13 slices. An alpha2 receptor-mediated inhibition of inspiratory activity in neonates (P0-P3) was further supported by a 19+/-3% reduction in XII nerve burst amplitude when norepinephrine (NE, 100 microM) was applied in the presence of prazosin (10 microM) and propranolol (100 microM). Results indicate that developmental increases in potentiating alpha1 and, to a lesser extent, beta receptor mechanisms combine with a developmentally decreasing inhibitory mechanism, most likely mediated by alpha2 receptors, to determine the ontogenetic time course by which NE modulates XII MN inspiratory activity.

Clonidine reduces hyperpolarization-activated inward current (Ih) in rat hypoglossal motoneurons.

We used intracellular recording techniques to investigate the actions of clonidine on hypoglossal motoneurons (HMs) in rat brainstem slices. Clonidine (10-100 microM) produced a small (2-6 mV), dose-dependent hyperpolarization in HMs, accompanied by an increase in peak input resistance (RN). It also slowed the time course of the depolarizing 'sag' of the voltage response to constant hyperpolarizing current steps. These effects were mimicked by the alpha2-adrenoceptor (alpha2-AR) agonist guanabenz, but not by the Ih-imidazoline receptor agonists moxonidine or rilmenidine. Recorded in single-electrode voltage clamp mode, clonidine decreased input conductance of HMs and reduced the amplitude of a hyperpolarization-activated inward current (Ih). Clonidine's effect on Ih was three-fold: it shifted the half-activation voltage (V1/2) in the hyperpolarizing direction (by 4.4 +/- 0.7 mV at a dose of 10 microM), decreased the maximal current (by approximately 20%), and slowed the time course of Ih activation at all voltage steps. At the most hyperpolarized potential steps, clonidine slowed activation of Ih dramatically, yielding a striking increase in the activation time constant. The alpha2-AR antagonists yohimbine and idazoxan reduced clonidine's effect on V1/2 and on the Ih activation time course, but neither blocked clonidine's reduction of the maximal current, nor its strong slowing of Ih activation at the most hyperpolarized steps. We were unable to mimic or occlude clonidine's actions with the adenylate cyclase inhibitor SQ 22536 nor with the non-specific protein kinase inhibitor H-7. We conclude that clonidine hyperpolarizes HMs via a reduction of the amount of Ih that is active at rest, and that the response is mediated in part by alpha2-ARs. Some effects of clonidine on these neurons do not appear to be receptor-mediated, and may be due to physical block by clonidine of Ih channels.

P2 receptor excitation of rodent hypoglossal motoneuron activity in vitro and in vivo: a molecular physiological analysis.

The role of P2 receptors in controlling hypoglossal motoneuron (XII MN) output was examined (1) electrophysiologically, via application of ATP to the hypoglossal nucleus of rhythmically active mouse medullary slices and anesthetized adult rats; (2) immunohistochemically, using an antiserum against the P2X2 receptor subunit; and (3) using PCR to identify expression of P2X2 receptor subunits in micropunches of tissue taken from the XII motor nucleus. Application of ATP to the hypoglossal nucleus of mouse medullary slices and anesthetized rats produced a suramin-sensitive excitation of hypoglossal nerve activity. Additional in vitro effects included potentiation of inspiratory hypoglossal nerve output via a suramin- and pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS)-sensitive mechanism, XII MN depolarization via activation of a suramin-sensitive inward current, decreased neuronal input resistance, and a slow-onset theophylline-sensitive reduction of inspiratory output likely resulting from hydrolysis of extracellular ATP to adenosine and activation of P1 receptors. Immunohistochemically, P2X2 receptors were detected in inspiratory XII MNs that were labeled with Lucifer yellow. These data, combined with identification of mRNA for three P2X2 receptor subunit isoforms within the hypoglossal nucleus (two of which have not been localized previously in brain) and the previous demonstration that P2X receptors are ubiquitously expressed in cranial and spinal motoneuron pools, support not only a role of P2 receptors in modulating inspiratory hypoglossal activity but a general role of P2 receptors in modulating motor outflow from the CNS.

Developmental modulation of glutamatergic inspiratory drive to hypoglossal motoneurons.

Proper function of hypoglossal motoneurons (XII MNs) innervating tongue muscles is critical for respiratory control of the airway. Morphological and electrophysiological properties of XII MNs change during postnatal development, as do modulatory systems. Despite these changes, the system producing respiratory movements must remain fully functional throughout life. Modulatory systems have therefore received considerable attention since coordination of their development with a developing neuromuscular system may be critical for maintenance of continuous, efficient breathing. Developmental modulation of XII inspiratory activity by three transmitter systems is examined. Thyrotropin-releasing hormone (TRH) mediates an increase in MN input resistance (RN) in juvenile but not neonate MNs, and this likely underlies the developmental increase in TRH potentiation of inspiratory activity. Norepinephrine (NE) potentiation of inspiratory activity, which in the neonate is produced in part by an alpha 1-mediated increase in RN, also increases postnatally. Effects of purinergic transmission on XII inspiratory activity remain constant during the first 2 weeks of postnatal development. Adenosine-triphosphate (ATP) produces tonic excitation and inspiratory potentiation that likely result from activation of postsynaptic P2 receptors. A secondary inhibitory effect likely results from hydrolysis of ATP to adenosine and activation of presynaptic A1 adenosine receptors. The functional relevance of these postnatal changes is discussed.

Actions of norepinephrine on rat hypoglossal motoneurons.

1. We used conventional intracellular recording techniques in 400-microns-thick slices from the brain stems of juvenile rats to investigate the action of norepinephrine (NE) on subthreshold and firing properties of hypoglossal motoneurons (HMs). 2. In recordings in current-clamp mode, 50 or 100 microM NE elicited a reversible depolarization accompanied by an increase in input resistance (RN) in all HMs tested (n = 74). In recordings in single-electrode voltage-clamp mode, NE induced a reversible inward current (INE) accompanied by a reduction in input conductance. The average reversal potential for INE was -104 mV. The NE responses could be elicited in a Ca(2+)-free solution containing tetrodotoxin, indicating that they were postsynaptic. 3. The NE response could be blocked by the alpha-adrenoceptor antagonist prazosin, but not by the beta-adrenoceptor antagonist propranolol, and could be mimicked by the alpha 1-adrenoceptor agonist phenylephrine but not by the alpha 2-adrenoceptor agonist UK 14,304 or by the beta-adrenoceptor agonist isoproterenol when alpha-adrenoceptors were blocked. 4. Substitution of barium for calcium in the perfusion solution blocked the increase in RN in response to NE without completely blocking the depolarization. Replacement of sodium chloride with choline chloride in the barium-substituted perfusion solution blocked the remaining depolarization. 5. The neuropeptide thyrotropin-releasing hormone (TRH), which also depolarizes and increases the RN of HMs, occluded the response of HMs to NE. 6. NE altered HM firing properties in three ways: it always lowered the minimum amount of injected current needed to elicit repetitive firing, it increased the slope of the firing frequency versus injected current relation in 8 of 14 cells tested, and it increased the delay from the onset of the depolarizing current pulse to the first evoked spike in all cells tested. 7. We conclude that NE acts directly on alpha 1-adrenoceptors to increase the excitability of HMs. It does this by reducing a barium-sensitive resting potassium current and activating a barium-insensitive inward current carried primarily by sodium ions. A portion of the intracellular pathway for these actions is shared by TRH. In addition, there is evidence that NE alters HM firing patterns by affecting currents that are activated following depolarization.