David González-Forero

Nitric Oxide-Directed Synaptic Remodeling in the Adult Mammal CNS

In adult mammals, learning, memory, and restoration of sensorimotor lost functions imply synaptic reorganization that requires diffusible messengers-mediated communication between presynaptic and postsynaptic structures. A candidate molecule to accomplish this function is the gaseous intercellular messenger nitric oxide (NO), which is involved in synaptogenesis and projection refinement during development; however, the role of NO in synaptic reorganization processes in adulthood remains to be established. In this work, we tested the hypothesis that this free radical is a mediator in the adult mammal CNS synaptic remodeling processes using a model of hypoglossal axonal injury recently developed by us. Axonal injury-induced disconnection of motoneurons from myocytes produces withdrawal of synaptic inputs to motoneurons and concomitant upregulation of the neuronal isoform of NO synthase (NOS-I). After recovery of the neuromuscular function, synaptic coverage is reestablished and NOS-I is downregulated. We also report, by using functional and morphological approaches, that chronic inhibition of the NO/cGMP pathway prevents synaptic withdrawal evoked by axon injury, despite the persistent muscle disconnection. After successful withdrawal of synaptic boutons, inhibition of NO synthesis, but not of cGMP, accelerated the recovery of synaptic coverage, although neuromuscular disconnection was maintained. Furthermore, protein S-nitrosylation was upregulated after nerve injury, and this effect was reversed by NOS-I inhibition. Our results suggest that during synaptic remodeling in the adult CNS, NO acts as a signal for synaptic detachment and inhibits synapse formation by cGMP-dependent and probably S-nitrosylation-mediated mechanisms, respectively. We also suggest a feasible role of NO in neurological disorders coursing with NOS-I upregulation.

Differential postnatal maturation of GABAA, glycine receptor, and mixed synaptic currents in renshaw cells and ventral spinal interneurons

Renshaw cells (RCs) receive excitatory inputs from motoneurons to which then they inhibit. The gain of this spinal recurrent inhibitory circuit is modulated by inhibitory synapses on RCs. Inhibitory synapses on RCs mature postnatally, developing unusually large postsynaptic gephyrin clusters that colocalize glycine and GABAA receptors. We hypothesized that these features potentiate inhibitory currents in RCs. Thus, we analyzed glycinergic and GABAergic “inhibitory” miniature postsynaptic currents (mPSCs) in neonatal [postnatal day 1 (P1) to P5] and mature (P9-P15) RCs and compared them to other ventral interneurons (non-RCs). Recorded neurons were Neurobiotin filled and identified as RCs or non-RCs using post hoc immunohistochemical criteria. Glycinergic, GABAergic, and mixed glycine/GABA mPSCs matured differently in RCs and non-RCs. In RCs, glycinergic and GABAA mPSC peak amplitudes increased 230 and 45%, respectively, from P1-P5 to P9-P15, whereas in non-RCs, glycinergic peak amplitudes changed little and GABAA amplitudes decreased. GABAA mPSCs were slower in RCs (P1-P5, τ = 58 ms; P9-P15, τ = 43 ms) compared with non-RCs (P1-P5, τ = 27 ms; P9-P15, τ = 14 ms). Thus, fast glycinergic currents dominated “mixed” mPSC peak amplitudes in mature RCs, and GABAA currents dominated their long decays. In non-RCs, GABAergic and mixed events had shorter durations, and their frequencies decreased with development. Functional maturation of inhibitory synapses on RCs correlates well with increased glycine receptor recruitment to large gephyrin patches, colocalization with α3/α5-containing GABAA receptors, and maintenance of GABA/glycine corelease. As a result, charge transfer in GABA, glycine, or mixed mPSCs was larger in mature RCs than in non-RCs, suggesting RCs receive potent inhibitory synapses.

Nitric Oxide Induces Pathological Synapse Loss by a Protein Kinase G-, Rho Kinase-Dependent Mechanism Preceded by Myosin Light Chain Phosphorylation

The molecular signaling that underpins synapse loss in neuropathological conditions remains unknown. Concomitant upregulation of the neuronal nitric oxide (NO) synthase (nNOS) in neurodegenerative processes places NO at the center of attention. We found that de novo nNOS expression was sufficient to induce synapse loss from motoneurons at adult and neonatal stages. In brainstem slices obtained from neonatal animals, this effect required prolonged activation of the soluble guanylyl cyclase (sGC)/protein kinase G (PKG) pathway and RhoA/Rho kinase (ROCK) signaling. Synapse elimination involved paracrine/retrograde action of NO. Furthermore, before bouton detachment, NO increased synapse myosin light chain phosphorylation (p-MLC), which is known to trigger actomyosin contraction and neurite retraction. NO-induced MLC phosphorylation was dependent on cGMP/PKG-ROCK signaling. In adulthood, motor nerve injury induced NO/cGMP-dependent synaptic stripping, strongly affecting ROCK-expressing synapses, and increased the percentage of p-MLC-expressing inputs before synapse destabilization. We propose that this molecular cascade could trigger synapse loss underlying early cognitive/motor deficits in several neuropathological states.

Regulation of Gephyrin Cluster Size and Inhibitory Synaptic Currents on Renshaw Cells by Motor Axon Excitatory Inputs

Renshaw cells receive a high density of inhibitory synapses characterized by large postsynaptic gephyrin clusters and mixed glycinergic/GABAergic inhibitory currents with large peak amplitudes and long decays. These properties appear adapted to increase inhibitory efficacy over Renshaw cells and mature postnatally by mechanisms that are unknown. We tested the hypothesis that heterosynaptic influences from excitatory motor axon inputs modulate the development of inhibitory synapses on Renshaw cells. Thus, tetanus (TeNT) and botulinum neurotoxin A (BoNT-A) were injected intramuscularly at postnatal day 5 (P5) to, respectively, elevate or reduce motor axon firing activity for ∼2 weeks. After TeNT injections, the average gephyrin cluster areas on Renshaw cells increased by 18.4% at P15 and 28.4% at P20 and decreased after BoNT-A injections by 17.7% at P15 and 19.9% at P20. The average size differences resulted from changes in the proportions of small and large gephyrin clusters. Whole-cell recordings in P9-P15 Renshaw cells after P5 TeNT injections showed increases in the peak amplitude of glycinergic miniature postsynaptic currents (mPSCs) and the fast component of mixed (glycinergic/GABAergic) mPSCs compared with controls (60.9% and 78.9%, respectively). GABAergic mPSCs increased in peak amplitude to a smaller extent (45.8%). However, because of the comparatively longer decays of synaptic GABAergic currents, total current transfer changes after TeNT were similar for synaptic glycine and GABAA receptors (56 vs 48.9% increases, respectively). We concluded that motor axon excitatory synaptic activity modulates the development of inhibitory synapse properties on Renshaw cells, influencing recruitment of postsynaptic gephyrin and glycine receptors and, to lesser extent, GABAA receptors.

Inhibition of resting potassium conductances by long-term activation of the NO/cGMP/protein kinase G pathway: a new mechanism regulating neuronal excitability.

Glutamate-induced excitotoxicity, the most common pathological mechanism leading to neuronal death, may occur even with normal levels of glutamate if it coincides with a persistent enhancement of neuronal excitability. Neurons expressing nitric oxide (NO) synthase (NOS-I), which is upregulated in many human chronic neurodegenerative diseases, are highly susceptible to neurodegeneration. We hypothesized that chronic production of NO in damaged neurons may increase their intrinsic excitability via modulation of resting or “leak” K+ currents. Peripheral XIIth nerve injury in adult rats induced de novo NOS-I expression and an increased incidence of low-threshold motor units, the latter being prevented by chronic inhibition of the neuronal NO/cGMP pathway. Accordingly, sustained synthesis of NO maintained an enhanced basal activity in injured motoneurons that was slowly reverted (over the course of 2–3 h) by NOS-I inhibitors. In slice preparations, persistent, but not acute, activation of the NO/cGMP pathway evoked a robust augment in motoneuron excitability independent of synaptic activity. Furthermore, chronic activation of the NO/cGMP pathway fully suppressed TWIK-related acid-sensitive K+ (TASK) currents through a protein kinase G (PKG)-dependent mechanism. Finally, we found evidence for the involvement of this long-term mechanism in regulating membrane excitability of motoneurons, because their pH-sensitive currents were drastically reduced by nerve injury. This NO/cGMP/PKG-mediated modulation of TASK conductances might represent a new pathological mechanism that leads to hyperexcitability and sensitizes neurons to excitotoxic damage. It could explain why de novo expression of NOS-I and/or its overexpression makes them susceptible to neurodegeneration under pathological conditions.

Endogenous Rho-Kinase Signaling Maintains Synaptic Strength by Stabilizing the Size of the Readily Releasable Pool of Synaptic Vesicles

Rho-associated kinase (ROCK) regulates neural cell migration, proliferation and survival, dendritic spine morphology, and axon guidance and regeneration. There is, however, little information about whether ROCK modulates the electrical activity and information processing of neuronal circuits. At neonatal stage, ROCKα is expressed in hypoglossal motoneurons (HMNs) and in their afferent inputs, whereas ROCKβ is found in synaptic terminals on HMNs, but not in their somata. Inhibition of endogenous ROCK activity in neonatal rat brainstem slices failed to modulate intrinsic excitability of HMNs, but strongly attenuated the strength of their glutamatergic and GABAergic synaptic inputs. The mechanism acts presynaptically to reduce evoked neurotransmitter release. ROCK inhibition increased myosin light chain (MLC) phosphorylation, which is known to trigger actomyosin contraction, and reduced the number of synaptic vesicles docked to active zones in excitatory boutons. Functional and ultrastructural changes induced by ROCK inhibition were fully prevented/reverted by MLC kinase (MLCK) inhibition. Furthermore, ROCK inhibition drastically reduced the phosphorylated form of p21-associated kinase (PAK), which directly inhibits MLCK. We conclude that endogenous ROCK activity is necessary for the normal performance of motor output commands, because it maintains afferent synaptic strength, by stabilizing the size of the readily releasable pool of synaptic vesicles. The mechanism of action involves a tonic inhibition of MLCK, presumably through PAK phosphorylation. This mechanism might be present in adults since unilateral microinjection of ROCK or MLCK inhibitors into the hypoglossal nucleus reduced or increased, respectively, whole XIIth nerve activity.

Influence of afferent synaptic innervation on the discharge variability of cat abducens motoneurones

The discharge variability of abducens motoneurones was studied after blocking inhibitory synaptic inputs or both excitatory and inhibitory inputs by means of an intramuscular (lateral rectus) injection of either a low (0.5 ng kg−1) or a high dose (5 ng kg−1) of tetanus neurotoxin (TeNT), respectively. Motoneuronal firing increased after low‐dose TeNT. High‐dose treatment, however, produced a firing depression, and in some cells, a total lack of modulation in relation to eye movements. Firing became increasingly more regular with larger TeNT doses as shown by significant reductions in the coefficient of variation after low‐ and high‐dose treatments. Similarly, autocorrelation histograms of interspike intervals increased the number of resolvable peaks twofold in low‐dose‐treated motoneurones and sevenfold in high‐dose‐treated motoneurones. The plots of standard deviation versus the mean instantaneous firing frequency showed an upward deflexion with low firing frequencies. The upward deflexion occurred in controls at 39.9 ± 4.9 ms, an interval similar to the mean afterhyperpolarisation (AHP) duration (48.4 ± 8.8 ms). Low‐dose TeNT treatment shifted the deflexion point to 20.9 ± 3.9 ms, whereas the high dose increased it to 60.7 ± 6.1 ms, in spite of the fact that no differences in AHP parameters between groups were found. The density of synaptophysin‐immunoreactive boutons decreased by 14 % after the low‐dose treatment and 40.5 % after the high‐dose treatment, indicating that protracted synaptic blockade produces elimination of synaptic boutons. It is concluded that abducens motoneurone spike variability during spontaneous ocular fixations depends largely on the balance between inhibitory and excitatory synaptic innervation.

Synaptic Structural Modification Following Changes in Activity Induced By Tetanus Neurotoxin in Cat Abducens Neurons

A low or a high dose of tetanus neurotoxin (TeNT) injected in the lateral rectus muscle of the cat causes respectively, functional block of inhibitory synapses only or of both inhibitory and excitatory synapses simultaneously in abducens neurons (González‐Forero et al. [2003] J. Neurophysiol. 89:1878–1890). As a consequence, neuronal firing activity increases (at low dose) or decreases (at high dose). We investigated possible structural modifications of inhibitory synapses in response to these activity alterations induced by TeNT. We used immunofluorescence against postsynaptic (gephyrin) and presynaptic (vesicular γ‐aminobutyric acid [GABA] transporter [VGAT]) markers of inhibitory synapses in combination with cell type markers for abducens motoneurons (calcitonin gene‐related peptide or choline acetyltransferase) or internuclear neurons (calretinin). Seven days after high‐dose treatment, the number of gephyrin‐immunoreactive (IR) clusters per 100 μm of membrane perimeter was reduced on the soma of abducens motoneurons by 55.3% and by 60.1% on internuclear neurons. Proximal dendritic gephyrin‐IR clusters were also significantly altered but to a lesser degree. Partial synaptic re‐establishment was observed 15 days post injection, and complete recovery occurred after 42 days. Coverage by VGAT‐IR terminals was reduced in parallel. In contrast, a low dose of TeNT caused no structural alterations. With electron microscopy we estimated that overall synaptic coverage was reduced by 40% in both types of neurons after a high dose of TeNT. However, F‐type terminals with postsynaptic gephyrin were preferentially lost. Thus, the ratio between F and S terminals diminished from 1.28 to 0.39 on motoneurons and from 1.26 to 0.47 on internuclear neurons. These results suggest that the maintenance of proximal inhibitory synaptic organization on central neurons is best related to neuronal activity and not to the level of inhibitory synaptic function, which was equally blocked by the high or low dose of TeNT. J. Comp. Neurol. 471:201–218, 2004.

Reversible deafferentation of abducens motoneurons and internuclear neurons with tetanus neurotoxin.

Tetanus neurotoxin (TeNT) is a blocker of synaptic vesicle exocytosis in central synapses with preferential affinity for inhibitory neurotransmission. Following its intramuscular injection, TeNT is retrogradely and trans-synaptically transported towards the premotor terminals. Therefore, we have used TeNT as a tool to study the consequences of functional deafferentation on motoneurons following its peripheral administration. For this, we injected the toxin into the lateral rectus muscle at doses of 5 or 0.5 ng/kg and recorded the discharge activity of abducens motoneurons and internuclear neurons in the alert cat. Our results showed that: (i) TeNT blocked selectively the afferent inhibitory signals on abducens neurons only when used at a low dose, whereas both excitatory and inhibitory synaptic drive was lost after the high dose treatment; (ii) all effects were reversible within one month; and (iii) strikingly, the internuclear neurons of the abducens nucleus showed similar discharge alterations to the motoneurons, suggesting a TeNT action on shared common afferences.

Correlation Between CGRP Immunoreactivity and Firing Activity in Cat Abducens Motoneurons

A relationship between motoneuron activity and calcitonin gene‐related peptide (CGRP) expression was previously suggested based on indirect inferences. We show here a positive correlation between CGRP immunoreactivity and firing activity in an experimental model that used tetanus neurotoxin (TeNT) to alter basal firing levels. A low dose (0.5 ng/kg) of TeNT injected in the lateral rectus muscle raised the basal firing rate of ipsilateral abducens motoneurons, estimated as the firing rate at straight‐ahead gaze (F0); the firing rate returned to control values after 2 weeks. In contrast, a high dose (5 ng/kg) of TeNT decreased basal firing, which recovered slowly over a 7‐week period. Expression of CGRP immunoreactivity by abducens motoneurons, preferentially related to βCGRP gene expression, was analyzed during these periods of altered firing activity. The number of CGRP‐immunofluorescent abducens motoneurons increased to ∼120% by 7 days after low‐dose TeNT, to include all available motoneurons in the nucleus. In addition, the average CGRP immunofluorescence optical density inside motoneurons almost doubled after 4 days and returned toward control values in the following 2 weeks. In contrast, a high‐dose injection of TeNT reduced the number of CGRP‐immunofluorescent motoneurons to 5.4% of control 7 days post injection, and the number returned to 77.6% after 42 days. CGRP immunofluorescence intensity inside motoneurons was also reduced. Regression analysis of F0 values with either the number of CGRP‐immunolabeled motoneurons, their average immunofluorescence intensity, or both factors combined resulted in positive correlations with regression coefficients of 0.87 or higher. Therefore, CGRP expression and firing activity in abducens motoneurons are positively correlated. J. Comp. Neurol. 451:201–212, 2002.