Pre- and post-synaptic roles of action potential activity in synapse elimination revealed by using ectopic neuromuscular junction formation by a foreign nerve.

The formation of the neuromuscular junction (nmj) is based on molecular cascades initiated by neural agrin as well as electrical activity in the neuromuscular structures. This review focuses on the latter factor, emphasizing the multiplicity of its mechanisms in the process of synapse elimination following initial polyneuronal innervation. Pre- and post-synaptic components of activity have in fact been identified through experiments on an adult model of nmj formation: ectopic reinnervation of the rat soleus muscle by the fibular nerve. Two activity-dependent elimination processes are thus compared: competition between distributed nmjs, which depends on evoked muscle impulse activity, and competition between axons converging on single nmjs, which instead depends on differences in the timing of impulses in the converging axons.

Genetic and pharmacological regulation of the endocannabinoid CB1 receptor in Duchenne muscular dystrophy.

The endocannabinoid system refers to a widespread signaling system and its alteration is implicated in a growing number of human diseases. However, the potential role of endocannabinoids in skeletal muscle disorders remains unknown. Here we report the role of the endocannabinoid CB1 receptors in Duchenne's muscular dystrophy. In murine and human models, CB1 transcripts show the highest degree of expression at disease onset, and then decline overtime. Similar changes are observed for PAX7, a key regulator of muscle stem cells. Bioinformatics and biochemical analysis reveal that PAX7 binds and upregulates the CB1 gene in dystrophic more than in healthy muscles. Rimonabant, an antagonist of CB1, promotes human satellite cell differentiation in vitro, increases the number of regenerated myofibers, and prevents locomotor impairment in dystrophic mice. In conclusion, our study uncovers a PAX7-CB1 cross talk potentially exacerbating DMD and highlights the role of CB1 receptors as target for potential therapies.

Activity-dependent vs. neurotrophic modulation of acetylcholine receptor expression: Evidence from rat soleus and extensor digitorum longus muscles confirms the exclusive role of activity.

Evoked electrical muscle activity suppresses the transcription of mRNAs for acetylcholine receptors in extrajunctional myonuclei. Muscle denervation or disuse releases such inhibition and extrajunctional receptors appear. However, in soleus muscles paralysed with nerve-applied tetrodotoxin, a restricted perijunctional region has been described where myonuclei remain inhibited, a finding attributed to nerve-derived trophic factor(s). Here, we reinvestigate extrajunctional acetylcholine receptor expression in soleus and extensor digitorum longus muscles up to 90 days after denervation or up to 20 days of disuse, to clarify the role of trophic factors, if any. The perijunctional region of soleus muscles strongly expressed acetylcholine receptors during the first 2-3 weeks of denervation. After 2-3 months, this expression had disappeared. No perijunctional expression was seen after paralysis by tetrodotoxin or botulinum toxin A. In contrast, the extensor digitorum longus never displayed suppressed perijunctional acetylcholine receptor expression after any treatment, suggesting that it is an intrinsic property of soleus muscles. Soleus denervation only transiently removed the suppression, and its presence in long-term denervated soleus muscles contradicts any contribution from nerve-derived trophic factor(s). In conclusion, our results confirm that evoked electrical activity is the physiological factor controlling the expression of acetylcholine receptors in the entire extrajunctional membrane of skeletal muscles.

Endocannabinoid-dependent disinhibition of orexinergic neurons: Electrophysiological evidence in leptin-knockout obese mice.

OBJECTIVES: In the ob/ob mouse model of obesity, chronic absence of leptin causes a significant increase of orexin (OX) production by hypothalamic neurons and excessive food intake. The altered OX level is linked to a dramatic increase of the inhibitory innervation of OX producing neurons (OX neurons) and the over expression of the endocannabinoid 2-arachidonoylglycerol (2-AG) by OX neurons of ob/ob mice. Little is known about the function of the excitatory synapses of OX neurons in ob/ob mice, and their modulation by 2-AG. In the present study, we fill this gap and provide the first evidence of the overall level of activation of OX neurons in the ob/ob mice. METHODS: We performed in vitro whole-cell patch-clamp recordings on OX neurons located in the perifornical area of the lateral hypothalamus in acute brain slices of wt and ob/ob mice. We identified OX neurons on the basis of their electrophysiological membrane properties, with 96% of concordance with immunohistochemisty. RESULTS: We found that OX neurons of ob/ob mice are innervated by less efficient and fewer excitatory synapses than wt mice. Consequently, ob/ob OX neurons show more negative resting membrane potential and lower action potential firing frequency than wt. The bath application of the cannabinoid type-1 receptor agonist WIN55,212-2, depresses both the excitatory and the inhibitory synapses in ob/ob animals, but only the excitatory synapses in wt animals. Finally, the physiologic release of 2-AG induces a prevalent depression of inhibition (disinhibition) of OX neurons in ob/ob animals but not in wt. CONCLUSIONS: In ob/ob mice, chronic absence of leptin induces a 2-AG mediated functional disinhibition of OX neurons. This helps explain the increase of OX production and, consequently, the excessive food intake of ob/ob mice.

Orexin-A and Endocannabinoid Activation of the Descending Antinociceptive Pathway Underlies Altered Pain Perception in Leptin Signaling Deficiency.

Pain perception can become altered in individuals with eating disorders and obesity for reasons that have not been fully elucidated. We show that leptin deficiency in ob/ob mice, or leptin insensitivity in the arcuate nucleus of the hypothalamus in mice with high-fat diet (HFD)-induced obesity, are accompanied by elevated orexin-A (OX-A) levels and orexin receptor-1 (OX1-R)-dependent elevation of the levels of the endocannabinoid, 2-arachidonoylglycerol (2-AG), in the ventrolateral periaqueductal gray (vlPAG). In ob/ob mice, these alterations result in the following: (i) increased excitability of OX1-R-expressing vlPAG output neurons and subsequent increased OFF and decreased ON cell activity in the rostral ventromedial medulla, as assessed by patch clamp and in vivo electrophysiology; and (ii) analgesia, in both healthy and neuropathic mice. In HFD mice, instead, analgesia is only unmasked following leptin receptor antagonism. We propose that OX-A/endocannabinoid cross talk in the descending antinociceptive pathway might partly underlie increased pain thresholds in conditions associated with impaired leptin signaling.

Adult rat motor neurons do not re-establish electrical coupling during axonal regeneration and muscle reinnervation.

Gap junctions (GJs) between neurons are present in both the newborn and the adult nervous system, and although important roles have been suggested or demonstrated in a number of instances, in many other cases a full understanding of their physiological role is still missing. GJs are expressed in the rodent lumbar cord at birth and mediate both dye and electrical coupling between motor neurons. This expression has been proposed to mediate: (i) fast synchronization of motoneuronal spike activity, in turn linked to the process of refinement of neuromuscular connections, and (ii) slow synchronization of locomotor-like oscillatory activity. Soon after birth this coupling disappears. Since in the adult rat regeneration of motor fibers after peripheral nerve injury leads to a recapitulation of synaptic refinement at the target muscles, we tested whether GJs between motor neurons are transiently re-expressed. We found that in conditions of maximal responsiveness of lumbar motor neurons (such as no depression by anesthetics, decerebrate release of activity of subsets of motor neurons, use of temporal and spatial summation by antidromic and orthodromic stimulations, testing of large ensembles of motor neurons) no firing is observed in ventral root axons in response to antidromic spike invasion of nearby counterparts. We conclude that junctional coupling between motor neurons is not required for the refinement of neuromuscular innervation in the adult.

Neurotoxicity and synaptic plasticity impairment of N-acetylglucosamine polymers: implications for Alzheimer's disease.

We assessed whether polymers of N-acetylglucosamine (GlcNAc) have any pathogenetic role in Alzheimer's disease (AD). First, by using specific dyes, we found deposits of polymers of GlcNAc in sporadic but not in familial AD. We found that neurons and microglia exposed to GlcNAc and uridine diphosphate (UDP)-GlcNAc are able to form GlcNAc polymers, which display a significant neurotoxicity in vitro. Moreover, the exposure of organotypic hippocampal cultures to the same compounds led to synaptic impairment with decreased levels of syntaxin and synaptophysin. In addition, acute hippocampal slices treated with GlcNAc/UDP-GlcNAc showed a clear reduction of long-term potentiation of excitatory synapses. Finally, we demonstrated that microglial cells are able to phagocytose chitin particles and, when exposed to GlcNAc/UDP-GlcNAc, show cellular activation and intracellular deposition of GlcNAc polymers that are eventually released in the extracellular space. Taken together, our results indicate that both microglia and neurons produce GlcNAc polymers, which trigger neurotoxicity both directly and through microglia activation. GlcNAc polymer-driven neurotoxicity offers novel pathogenic insights in sporadic AD and new therapeutic options.

Hebb-based rules of neural plasticity: are they ubiquitously important for the refinement of synaptic connections in development?

Neuronal death and suppression of functional synaptic inputs are well-known regressive events characterizing PNS and CNS development. In the CNS, participation of activity in synapse elimination has been known ever since the pioneering studies of Hubel and Wiesel, but only recently has a Hebb-based mechanism of spike synchrony versus asynchrony received unequivocal experimental support in the visual system. At the neuromuscular junction (NMJ), where synapse elimination was discovered, the specific function of the "timing of activity" was addressed by only one group of studies and did not receive widespread attention. Here we critically review the latest NMJ investigation advocating an "activity-independent" mechanism for synapse elimination and contrast it with an equally recent study demonstrating a key role for spike timing. Finally, we highlight how the striking similarities between the two mentioned studies on spike timing (visual system and NMJ) establish conclusively its role in the development of the nervous system in general.

The timing of activity is a regulatory signal during development of neural connections.

In PNS and CNS remarkable rearrangements occur soon after the connections are laid down in the course of embryonic life. These processes clearly follow the period of developmental cell death and mostly take place during the very beginning of postnatal life. They consist in changes of the peripheral fields of neurons, marked by elimination of many inputs, while others undergo further maturation and strengthening. Along the efforts to uncover the signals that regulate development, it turned out that while the initial construction of the circuits is heavily based on chemical cues, the subsequent rearrangement is markedly influence by activity. Here we describe experiments testing the influence on developmental plasticity of a particular aspect of activity, the timing of nerve impulses in the competing inputs. Two recent investigations are reviewed, indicating strikingly similar developmental features in quite different systems, neuromuscular and visual. A sharp contrast between the effects of synchrony and asynchrony emerges, indicating that Hebb-related activity rules are important not only for learning but also for development.

Obesity-driven synaptic remodeling affects endocannabinoid control of orexinergic neurons.

Acute or chronic alterations in energy status alter the balance between excitatory and inhibitory synaptic transmission and associated synaptic plasticity to allow for the adaptation of energy metabolism to new homeostatic requirements. The impact of such changes on endocannabinoid and cannabinoid receptor type 1 (CB1)-mediated modulation of synaptic transmission and strength is not known, despite the fact that this signaling system is an important target for the development of new drugs against obesity. We investigated whether CB1-expressing excitatory vs. inhibitory inputs to orexin-A-containing neurons in the lateral hypothalamus are altered in obesity and how this modifies endocannabinoid control of these neurons. In lean mice, these inputs are mostly excitatory. By confocal and ultrastructural microscopic analyses, we observed that in leptin-knockout (ob/ob) obese mice, and in mice with diet-induced obesity, orexinergic neurons receive predominantly inhibitory CB1-expressing inputs and overexpress the biosynthetic enzyme for the endocannabinoid 2-arachidonoylglycerol, which retrogradely inhibits synaptic transmission at CB1-expressing axon terminals. Patch-clamp recordings also showed increased CB1-sensitive inhibitory innervation of orexinergic neurons in ob/ob mice. These alterations are reversed by leptin administration, partly through activation of the mammalian target of rapamycin pathway in neuropeptide-Y-ergic neurons of the arcuate nucleus, and are accompanied by CB1-mediated enhancement of orexinergic innervation of target brain areas. We propose that enhanced inhibitory control of orexin-A neurons, and their CB1-mediated disinhibition, are a consequence of leptin signaling impairment in the arcuate nucleus. We also provide initial evidence of the participation of this phenomenon in hyperphagia and hormonal dysregulation in obesity.

Spike timing plays a key role in synapse elimination at the neuromuscular junction.

Nerve impulse activity produces both developmental and adult plastic changes in neural networks. For development, however, its precise role and the mechanisms involved remain elusive. Using the classic model of synapse competition and elimination at newly formed neuromuscular junctions, we asked whether spike timing is the instructive signal at inputs competing for synaptic space. Using a rat strain whose soleus muscle is innervated by two nerves, we chronically evoked different temporal spike patterns in the two nerves during synapse formation in the adult. We found that asynchronous activity imposed upon the two nerves promotes synapse elimination, provided that their relative spikes are separated by 25 ms or more; remarkably, this elimination occurs even though an equal number of spikes were evoked in the competing axons. On the other hand, when spikes are separated by 20 ms or less, activity is perceived as synchronous, and elimination is prevented. Thus, in development, as in adult plasticity, precise spike timing plays an instructive role in synaptic modification.

Developmental presence and disappearance of postsynaptically silent synapses on dendritic spines of rat layer 2/3 pyramidal neurons.

Silent synapses are synapses whose activation evokes NMDA-type glutamate receptor (NMDAR) but not AMPA-type glutamate receptor (AMPAR) mediated currents. Silent synapses are prominent early in postnatal development and are thought to play a role in the activity- and sensory-dependent refinement of neuronal circuits. The mechanisms that account for their silent nature have been controversial, and both presynaptic and postsynaptic mechanisms have been proposed. Here, we use two-photon laser uncaging of glutamate to directly activate glutamate receptors and measure AMPAR- and NMDAR-dependent currents on individual dendritic spines of rat somatosensory cortical layer 2/3 pyramidal neurons. We find that dendritic spines lacking functional surface AMPARs are commonly found before postnatal day 12 (P12) but are absent in older animals. Furthermore, AMPAR-lacking spines are contacted by release-competent presynaptic terminals. After P12, the AMPAR/NMDAR current ratio at individual spines continues to increase, consistent with continued addition of AMPARs to postsynaptic terminals. Our results confirm the existence of postsynaptically silent synapses and demonstrate that the morphology of the spine is not strongly predictive of its AMPAR content.

Synapse formation and elimination: role of activity studied in different models of adult muscle reinnervation.

Synapse competition and elimination are a general developmental process both in central and in peripheral nervous systems that is strongly activity dependent. Some common features regulate synapse competition, and one of these is an application to development of the Hebb's postulate of learning: repeated coincident spike activity in competing presynaptic inputs on the same target cell inhibits competition, whereas noncoincident activity promotes weakening of some of the inputs and ultimately their elimination. Here we report experiments that indicate that the development of muscle innervation (initial polyneuronal innervation and subsequent synapse elimination) follows the Hebb's paradigm. We utilized two different models of muscle reinnervation in the adult rat: 1) we crushed nerves going to soleus or extensor digitorum longus muscles, to activate regeneration of the presynaptic component of the neuromuscular junctions (NMJ), or 2) we injected the soleus muscle with Marcaine (a myotoxic agent) to activate regeneration of the postsynaptic component, the muscle fiber. A condition of transient polyneuronal innervation occurs during NMJ regeneration in both cases, although the two models differ insofar as the relative strength of the competing inputs is concerned. During the period of competition (a few days or weeks, in Marcaine or crush experiments, respectively), we imposed a synchronous firing pattern on the competing inputs by stimulating motor axons distal to a chronic conduction block and demonstrated that this procedure strongly inhibits synapse elimination, with respect to control muscles in which regeneration occurs under natural impulse activity of motoneurons.

Activity-dependent synaptic competition at mammalian neuromuscular junctions.

Synapse elimination is a widespread developmental process in the peripheral and central nervous system that brings about refinement of neural connections through epigenetic mechanisms. Here we describe recent advances concerning the role of the pattern of motoneuronal firing, synchronous or asynchronous, in neuromuscular synapse elimination.

Perinatal switch from synchronous to asynchronous activity of motoneurons: link with synapse elimination.

Synaptic competition is a basic feature of developing neural connections. To shed light on its dependence on the activity pattern of competing inputs, we investigated in vivo rat motoneuronal firing during late embryonic and early neonatal life, when synapse elimination occurs in muscle. Electromyographic recordings with floating microelectrodes from tibialis anterior and soleus muscles revealed that action potentials of motoneurons belonging to the same pool have high temporal correlation. The very tight linkage, a few tens of milliseconds, corresponds to the narrow time windows of published paradigms of activity-dependent synaptic plasticity. A striking change occurs, however, soon after birth when motoneuronal firing switches to the adult uncorrelated type. The switch precedes the onset of synapse elimination, whose time course was determined with confocal microscopy. Interestingly, the soleus muscle, whose motoneurons switch to desynchronized activity later than those of the tibialis anterior muscle, also exhibits delayed synapse elimination. Our findings support a developmental model in which synchronous activity first favors polyneuronal innervation, whereas an asynchronous one subsequently promotes synapse elimination.