Axonal projections of Renshaw cells in the thoracic spinal cord.

Renshaw cells are widely distributed in all segments of the spinal cord, but detailed morphological studies of these cells and their axonal branching patterns have only been made for lumbosacral segments. For these, a characteristic distribution of terminals was reported, including extensive collateralization within 1-2 mm of the soma, but then more restricted collaterals given off at intervals from the funicular axon. Previous authors have suggested that the projections close to the soma serve inhibition of motoneurons (known to be greatest for the motor nuclei providing the Renshaw cell excitation) but that the distant projections serve mainly the inhibition of other neurons. However, in thoracic segments, inhibition of motoneurons is known to occur over two to three segments (20-40 mm) from the presumed somatic locations of the Renshaw cells. Here, we report the first detailed morphological study of Renshaw cell axons outside the lumbosacral segments, which investigated whether this different distribution of motoneuron inhibition is reflected in a different pattern of Renshaw cell terminations. Four Renshaw cells in T7 or T8 segments were intracellularly labeled with neurobiotin in anesthetized cats and their axons traced for distances ≥6 mm from the somata. The only morphological difference detected within this distance in comparison with Renshaw cells in the lumbosacral cord was a minimal taper in the funicular axons, where in the lumbosacral cord this is pronounced. Patterns of termination were virtually identical to those in the lumbosacral segments, so we conclude that these patterns are unrelated to the pattern of motoneuronal inhibition.

Protein kinase G-dependent mechanisms modulate hypoglossal motoneuronal excitability and long-term facilitation.

Since protein kinase-dependent modulation of motoneuronal excitability contributes to adaptive changes in breathing, we hypothesized that cGMP-dependent pathways activating protein kinase G (PKG) modulate motoneuronal inspiratory drive currents and long-term plasticity. In a medullary slice preparation from neonatal rat (postnatal days 0-4) generating spontaneous respiratory-related rhythm, hypoglossal (XII) motoneuronal inspiratory drive currents and respiratory-related XII nerve activity were recorded. Focal application of a PKG activator, 8-bromoguanosine-3',5'-cyclomonophosphate (8-Br-cGMP), to voltage-clamped XII motoneurones decreased inspiratory drive currents. In the presence of tetrodotoxin (TTX), 8-Br-cGMP decreased the exogenous postsynaptic inward currents induced by focal application of AMPA. Intracellular dialysis of XII motoneurones with an inhibitory peptide to PKG (PKGI) increased endogenous inspiratory-drive currents and exogenous AMPA-induced currents. Application of 8-Br-cGMP with PKGI had no further effect on spontaneous or evoked currents, confirming that the observed effects were induced by PKG. However, PKG differentially increased longer-term plasticity. Three 3 min applications (separated by 5 min) of the α(1)-adrenergic agonist phenylephrine (PE) in combination with 8-Br-cGMP yielded greater in vitro long-term facilitation than PE alone. These data indicate the presence of a cGMP/PKG-dependent signalling pathway in XII motoneurones that modulates inspiratory drive currents and plasticity of XII motoneurones, possibly contributing to their adaptation during physiological challenges, such as sleep and exercise.

Episodic stimulation of alpha1-adrenoreceptors induces protein kinase C-dependent persistent changes in motoneuronal excitability.

In vitro long-term facilitation (ivLTF) is a novel form of activity-independent postsynaptic enhancement of AMPA receptor function in hypoglossal (XII) motoneurons that can be induced by intermittent activation of 5-HT2 receptors. In vivo respiratory long-term facilitation (LTF) is characterized by a persistent 5-HT2 receptor-dependent increase in respiratory motor output or ventilation after episodic exposures to hypoxia in adult rats. Here, we demonstrate that ivLTF can also be induced by episodic but not continuous stimulation of alpha1-adrenergic receptors that requires protein kinase C (PKC), but not PKA (protein kinase A), activation. Additionally, we show that in vivo respiratory LTF is also alpha1-adrenergic receptor dependent. We suggest that, in vivo, concurrent episodic activation of 5-HT2 and alpha1-adrenergic receptors is necessary to produce long-lasting changes in the excitability of respiratory motoneurons, possibly involving PKC activation via the G alpha(q)-PLC (phospholipase C) signaling pathway common to both receptor subtypes. Such plasticity of XII motor output may increase upper airway muscle (innervated by XII nerve) tone and improve the likelihood that airway patency will be maintained. Elucidating the mechanism underlying LTF can be of clinical importance to the patients suffering from sleep-disordered breathing.

Modulation of hypoglossal motoneuron excitability by intracellular signal transduction cascades.

Motoneuronal excitability is highly modulated by various inputs; however, comparatively little is known about postsynaptic signal transduction cascades that affect motoneuron excitability. In this review, we discuss the role of intracellular signaling cascades in the modulation of respiratory motoneuronal excitability. In particular, protein kinases and phosphatases dynamically and constitutively modulate respiratory-modulated inputs to XII motoneurons: (i) activation of protein kinase A (PKA) potentiates both excitatory and inhibitory drive currents; (ii) protein kinase G (PKG) depresses excitatory currents, and (iii) inhibition of protein phosphatases potentiates excitatory drive currents. We also describe a novel form of persistent plasticity (in vitro long-term facilitation; ivLTF) of motoneuronal output. ivLTF is induced by episodic activation of 5-HT(2) or alpha(1)-adrenoreceptors and is manifested as an increase in the amplitude of XII nerve output due to an increase in alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-mediated motoneuronal drive currents. Blockade of Group 1 metabotropic glutamate receptors or protein kinase C (PKC) prevents the induction of ivLTF.

Rostrocaudal distribution of motoneurones and variation in ventral horn area within a segment of the feline thoracic spinal cord.

Retrograde transport of horseradish peroxidase, applied to cut peripheral nerves, was used to determine the rostrocaudal distribution of motoneurones supplying different branches of the ventral ramus for a single mid- or caudal thoracic segment in the cat. The motoneurones occupied a length of spinal cord equal to the segmental length but displaced rostrally from the segment as defined by the dorsal roots, with the number of motoneurones per unit length of cord higher in the rostral part of a segment (close to the entry of the most rostral dorsal root) than in the caudal part. The cross-sectional area of the ventral horn showed a rostrocaudal variation that closely paralleled the motoneurone distribution. The ratio between the number of motoneurones per unit length in the caudal and rostral regions of a segment (0.70) was similar to the ratio previously reported for the strength of functional projections of expiratory bulbospinal neurones (0.63). This is consistent with the motoneurones being the main targets of the bulbospinal neurones.

Dynamic interactions of excitatory and inhibitory inputs in hypoglossal motoneurones: respiratory phasing and modulation by PKA.

The balance of excitation and inhibition converging upon a neurone is a principal determinant of neuronal output. We investigated the role of inhibition in shaping and gating inspiratory drive to hypoglossal (XII) motoneuronal activity. In neonatal rat medullary slices that generate a spontaneous respiratory rhythm, patch-clamp recordings were made from XII motoneurones, which were divided into three populations according to their inhibitory inputs: non-inhibited, inspiratory-inhibited and late-inspiratory-inhibited. In late-inspiratory-inhibited motoneurones, blockade of GABA(A) receptors with bicuculline abolished inspiratory-phased inhibition and increased the duration of inspiratory drive currents. In inspiratory-inhibited motoneurones, bicuculline abolished phasic inhibition, which frequently revealed excitatory inspiratory drive currents. In non-inhibited motoneurones, neither bicuculline nor strychnine markedly changed inspiratory drive currents. Inhibitory currents in XII motoneurones were potentiated by protein kinase A (PKA) activity. Intracellular dialysis of the catalytic subunit of PKA or bath application of the PKA activator Sp-cAMP significantly increased the amplitude of expiratory-phased IPSCs without any change in IPSP frequency. Inspiratory-phased inhibition in inspiratory-inhibited motoneurones was potentiated by Sp-cAMP. We conclude that inspiratory-phased inhibition is prevalent in neonatal XII motoneurones and plays an important role in shaping motoneuronal output. These inhibitory inputs are modulated by PKA, which also modulates excitatory inputs.

Dynamic modulation of inspiratory drive currents by protein kinase A and protein phosphatases in functionally active motoneurons.

Plasticity underlying adaptive, long-term changes in breathing behavior is hypothesized to be attributable to the modulation of respiratory motoneurons by intracellular second-messenger cascades. In quiescent preparations, protein kinases, including cAMP-dependent protein kinase A (PKA), potentiate glutamatergic inputs. However, the dynamic role of protein kinases or phosphatases in functionally active and behaviorally relevant preparations largely remains to be established. Rhythmic inspiratory drive to motoneurons innervating inspiratory muscles is mediated by the release of glutamate acting predominantly on AMPA receptors. In rhythmically active brainstem slices from neonatal rats, we investigated whether synaptic AMPA receptor function could be modulated by changes in intracellular PKA activity, affecting inspiratory drive in hypoglossal (XII) motoneurons. Intracellular perfusion of the catalytic subunit of PKA potentiated endogenous synaptic and (exogenously applied) AMPA-induced currents in XII motoneurons. Conversely, when a peptide inhibitor of PKA was perfused intracellularly, inspiratory drive currents were depressed. Intracellular perfusion with microcystin, a potent phosphatase 1 and 2a inhibitor, increased both endogenous and exogenous AMPA receptor-mediated currents, further supporting a role of phosphorylation in modulating motoneuronal excitability affecting behaviorally relevant synaptic inputs. These findings suggest that PKA is constitutively active in XII motoneurons in vitro. Thus, endogenous synaptic AMPA currents in XII motoneurons are influenced by phosphorylation, specifically by PKA, and dephosphorylation. The role of this modulation may be to keep the activity of motoneurons within a dynamic range that aids in responding to different physiological challenges affecting breathing, such as exercise, hypoxia, and sleep.