Astrocytes regulate heterogeneity of presynaptic strengths in hippocampal networks.

Dendrites are neuronal structures specialized for receiving and processing information through their many synaptic inputs. How input strengths are modified across dendrites in ways that are crucial for synaptic integration and plasticity remains unclear. We examined in single hippocampal neurons the mechanism of heterosynaptic interactions and the heterogeneity of synaptic strengths of pyramidal cell inputs. Heterosynaptic presynaptic plasticity that counterbalances input strengths requires N-methyl-d-aspartate receptors (NMDARs) and astrocytes. Importantly, this mechanism is shared with the mechanism for maintaining highly heterogeneous basal presynaptic strengths, which requires astrocyte Ca(2+) signaling involving NMDAR activation, astrocyte membrane depolarization, and L-type Ca(2+) channels. Intracellular infusion of NMDARs or Ca(2+)-channel blockers into astrocytes, conditionally ablating the GluN1 NMDAR subunit, or optogenetically hyperpolarizing astrocytes with archaerhodopsin promotes homogenization of convergent presynaptic inputs. Our findings support the presence of an astrocyte-dependent cellular mechanism that enhances the heterogeneity of presynaptic strengths of convergent connections, which may help boost the computational power of dendrites.

Motoneurons derived from induced pluripotent stem cells develop mature phenotypes typical of endogenous spinal motoneurons.

Induced pluripotent cell-derived motoneurons (iPSCMNs) are sought for use in cell replacement therapies and treatment strategies for motoneuron diseases such as amyotrophic lateral sclerosis (ALS). However, much remains unknown about the physiological properties of iPSCMNs and how they compare with endogenous spinal motoneurons or embryonic stem cell-derived motoneurons (ESCMNs). In the present study, we first used a proteomic approach and compared protein expression profiles between iPSCMNs and ESCMNs to show that <4% of the proteins identified were differentially regulated. Like ESCs, we found that mouse iPSCs treated with retinoic acid and a smoothened agonist differentiated into motoneurons expressing the LIM homeodomain protein Lhx3. When transplanted into the neural tube of developing chick embryos, iPSCMNs selectively targeted muscles normally innervated by Lhx3 motoneurons. In vitro studies showed that iPSCMNs form anatomically mature and functional neuromuscular junctions (NMJs) when cocultured with chick myofibers for several weeks. Electrophysiologically, iPSCMNs developed passive membrane and firing characteristic typical of postnatal motoneurons after several weeks in culture. Finally, iPSCMNs grafted into transected mouse tibial nerve projected axons to denervated gastrocnemius muscle fibers, where they formed functional NMJs, restored contractile force. and attenuated denervation atrophy. Together, iPSCMNs possess many of the same cellular and physiological characteristics as ESCMNs and endogenous spinal motoneurons. These results further justify using iPSCMNs as a source of motoneurons for cell replacement therapies and to study motoneuron diseases such as ALS.

Presynaptic NCAM is required for motor neurons to functionally expand their peripheral field of innervation in partially denervated muscles.

The function of neural cell adhesion molecule (NCAM) expression in motor neurons during axonal sprouting and compensatory reinnervation was explored by partially denervating soleus muscles in mice lacking presynaptic NCAM (Hb9(cre)NCAM(flx)). In agreement with previous studies, the contractile force of muscles in wild-type (NCAM(+/+)) mice recovered completely 2 weeks after 75% of the motor innervation was removed because motor unit size increased by 2.5 times. In contrast, similarly denervated muscles in Hb9(cre)NCAM(flx) mice failed to recover the force lost due to the partial denervation because motor unit size did not change. Anatomical analysis indicated that 50% of soleus end plates were completely denervated 1-4 weeks post-partial denervation in Hb9(cre)NCAM(flx) mice, while another 25% were partially reinnervated. Synaptic vesicles (SVs) remained at extrasynaptic regions in Hb9(cre)NCAM(flx) mice rather than being distributed, as occurs normally, to newly reinnervated neuromuscular junctions (NMJs). Electrophysiological analysis revealed two populations of NMJs in partially denervated Hb9(cre)NCAM(flx) soleus muscles, one with high (mature) quantal content, and another with low (immature) quantal content. Extrasynaptic SVs in Hb9(cre)NCAM(flx) sprouts were associated with L-type voltage-dependent calcium channel (L-VDCC) immunoreactivity and maintained an immature, L-VDCC-dependent recycling phenotype. Moreover, acute nifedipine treatment potentiated neurotransmission at newly sprouted NMJs, while chronic intraperitoneal treatment with nifedipine during a period of synaptic consolidation enhanced functional motor unit expansion in the absence of presynaptic NCAM. We propose that presynaptic NCAM bridges a critical link between the SV cycle and the functional expansion of synaptic territory through the regulation of L-VDCCs.

NMDAR-dependent presynaptic homeostasis in adult hippocampus: Synapse growth and cross-modal inhibitory plasticity.

Homeostatic plasticity (HP) encompasses a suite of compensatory physiological processes that counteract neuronal perturbations, enabling brain resilience. Currently, we lack a complete description of the homeostatic processes that operate within the mammalian brain. Here, we demonstrate that acute, partial AMPAR-specific antagonism induces potentiation of presynaptic neurotransmitter release in adult hippocampus, a form of compensatory plasticity that is consistent with the expression of presynaptic homeostatic plasticity (PHP) documented at peripheral synapses. We show that this compensatory plasticity can be induced within minutes, requires postsynaptic NMDARs, and is expressed via correlated increases in dendritic spine volume, active zone area, and docked vesicle number. Further, simultaneous postsynaptic genetic reduction of GluA1, GluA2, and GluA3 in triple heterozygous knockouts induces potentiation of presynaptic release. Finally, induction of compensatory plasticity at excitatory synapses induces a parallel, NMDAR-dependent potentiation of inhibitory transmission, a cross-modal effect consistent with the anti-epileptic activity of AMPAR-specific antagonists used in humans.

Astrocyte GluN2C NMDA receptors control basal synaptic strengths of hippocampal CA1 pyramidal neurons in the stratum radiatum.

Experience-dependent plasticity is a key feature of brain synapses for which neuronal N-Methyl-D-Aspartate receptors (NMDARs) play a major role, from developmental circuit refinement to learning and memory. Astrocytes also express NMDARs, although their exact function has remained controversial. Here, we identify in mouse hippocampus, a circuit function for GluN2C NMDAR, a subtype highly expressed in astrocytes, in layer-specific tuning of synaptic strengths in CA1 pyramidal neurons. Interfering with astrocyte NMDAR or GluN2C NMDAR activity reduces the range of presynaptic strength distribution specifically in the stratum radiatum inputs without an appreciable change in the mean presynaptic strength. Mathematical modeling shows that narrowing of the width of presynaptic release probability distribution compromises the expression of long-term synaptic plasticity. Our findings suggest a novel feedback signaling system that uses astrocyte GluN2C NMDARs to adjust basal synaptic weight distribution of Schaffer collateral inputs, which in turn impacts computations performed by the CA1 pyramidal neuron.

Neural cell adhesion molecule is required for stability of reinnervated neuromuscular junctions.

Studies examining the etiology of motoneuron diseases usually focus on motoneuron death as the defining pathophysiology of the disease. However, impaired neuromuscular transmission and synapse withdrawal often precede cell death, raising the possibility that abnormalities in synaptic function contribute to disease onset. Although little is known about the mechanisms maintaining the synaptic integrity of neuromuscular junctions (NMJs), Drosophila studies suggest that Fasciclin II plays an important role. Inspired by these studies we used a reinnervation model of synaptogenesis to analyze neuromuscular function in mice lacking neural cell adhesion molecule (NCAM), the Fasciclin II vertebrate homolog. Our results showed that the recovery of contractile force was the same in wild-type and NCAM-/- mice at 1 month after nerve injury, indicating that endplates were appropriately reformed. This normality was only transient because the contractile force and myofiber number decreased at 3 months after injury in NCAM-/- mice. Both declined further 3 months later. Myofibers degenerated, not because motoneurons died but because synapses were withdrawn. Although neurotransmission was initially normal at reinnervated NCAM-/- NMJs, it was significantly compromised 3 months later. Interestingly, the selective ablation of NCAM from motoneurons, or muscle fibers, did not mimic the deficits observed in reinnervated NCAM-/- mice. Taken together, these results indicate that NCAM is required to maintain normal synaptic function at reinnervated NMJs, although its loss pre-synaptically or post-synaptically is not sufficient to induce synaptic destabilization. Consideration is given to the role of NCAM in terminal Schwann cells for maintaining synaptic integrity and how NCAM dysfunction may contribute to motoneuron disorders.

A stem-cell based bioassay to critically assess the pathology of dysfunctional neuromuscular junctions.

Pluripotent stem cells can be directed to differentiate into motor neurons and assessed for functionality in vitro. An emerging application of this technique is to model genetically inherited diseases in differentiated motor neurons and to screen for new therapeutic targets. The neuromuscular junction (NMJ) is essential to the functionality of motor neurons and its dysfunction is a primary hallmark of motor neuron disease. However, mature NMJs that possess the functional and morphological characteristics of those formed in vivo have so far not been obtained in vitro. Here we describe the generation and analysis of mature NMJs formed between embryonic stem cell-derived motor neurons (ESCMNs) and primary myotubes. We compared the formation and maturation of NMJs generated by wild-type (NCAM+/+) ESCMNs to those generated by neural cell adhesion molecule null (NCAM-/-) ESCMNs in order to definitively test the sensitivity of this assay to identify synaptic pathology. We find that co-cultures using NCAM-/- ESCMNs replicate key in vivo NCAM-/- phenotypes and reveal that NCAM influences neuromuscular synaptogenesis by controlling the mode of synaptic vesicle endocytosis. Further, we could improve synapse formation and function in NCAM-/- co-cultures by chronic treatment with nifedipine, which blocks an immature synaptic vesicle recycling pathway. Together, our results demonstrate that this ESCMN/myofiber co-culture system is a highly sensitive bioassay for examining molecules postulated to regulate synaptic function and for screening therapeutics that will improve the function of compromised NMJs.

Generation of motor neurons from pluripotent stem cells.

Alpha motor neurons (also known as lower or skeletal motor neurons) have been studied extensively for over 100 years. Motor neurons control the contraction of skeletal muscles and thus are the final common pathway in the nervous system responsible for motor behavior. Muscles become paralyzed when their innervating motor neurons die because of injury or disease. Motor neuron diseases (MNDs), such as Amyotrophic Lateral Sclerosis, progressively destroy motor neurons until those inflicted succumb to the illness due to respiratory failure. One strategy being explored to study and treat muscle paralysis due to motor neuron loss involves deriving surrogate motor neurons from pluripotent stem cells. Guided by decades of research on the development of the spinal cord, recent advances in neurobiology have shown that functional motor neurons can be derived from mouse and human embryonic stem (ES) cells. Furthermore, ES cell-derived motor neurons restore motor behavior when transplanted into animal models of motor dysfunction. The recent discovery that mouse and human motor neurons can be derived from induced pluripotent stem (iPS) cells (i.e., somatic cells converted to pluripotency) has set the stage for the development of patient-specific therapies designed to treat movement disorders. Indeed, there is now hope within the scientific community that motor neurons derived from pluripotent stem cells will be used to treat MNDs through cell transplantation and/or to screen molecules that will prevent motor neuron death. In this chapter, we review the journey that led to the generation of motor neurons from ES and iPS cells, how stem cell-derived motor neurons have been used to treat/study motor dysfunction, and where the technology will likely lead to in the future.