Neural development - One year on

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Improved methods for detection of β-galactosidase (lacZ) activity in hard tissue

The ß-galactosidase gene (lacZ) of Escherichia coli is widely used as a reporter gene. The expression of lacZ can be detected by enzyme-based histochemical staining using chromogenic substrates such as 5-bromo-4-chloro-3-indolyl-ß-D: -galactoside (X-gal). Because the enzymatic activity of lacZ is vulnerable to high temperatures and acid treatment for demineralization, detection of lacZ on paraffinized sections is difficult, especially for hard tissues, which require demineralization before sectioning in paraffin. To circumvent this problem, whole-mount X-gal staining before sectioning is performed. However, detection of lacZ activity in the center of larger portions of hard whole adult tissues is challenging. In this study, focusing on fixation procedures, we determined the conditions conducive to improved detection of lacZ activity in deeper areas of whole tissues. We used an annexin a5 (Anxa5)-lacZ reporter mouse model in which the Anxa5 expression in hard tissue is indicated by lacZ activity. We found that lacZ activity could be detected throughout the periodontal ligament of adult mice when fixed in 100% acetone, whereas it was not detected in the periodontal ligament around the root apex fixed in glutaraldehyde and paraformaldehyde. This staining could not be detected in wild-type mice. Acetone maintains the lacZ activity within 48 h of fixation at both 4°C and at room temperature. In conclusion, acetone is the optimal fixative to improve permeability for staining of lacZ activity in large volumes of adult hard tissues

Cell culture-based analysis of postsynaptic membrane assembly in muscle cells

We report a method for studying postsynaptic membrane assembly utilizing the replating of aneural cultures of differentiated skeletal muscle cells onto laminin-coated surfaces. A significant limitation to the current cell culturebased approaches has been their inability to recapitulate the multistage surface acetylcholine receptor (AChR) redistribution events that produce complex AChR clusters found at the intact neuromuscular junction (NMJ). By taking advantage of the ability of substrate laminin to induce advanced maturation of AChR aggregates on the surface of myotubes, we have developed a secondary-plating method that allows more precise analysis of the signaling events connecting substrate laminin stimulation to complex AChR cluster formation. We validate the utility of this method for biochemical and microscopy studies by demonstrating the roles of RhoGTPases in substrate laminin-induced complex cluster assembly

Satb1 Regulates Contactin 5 to Pattern Dendrites of a Mammalian Retinal Ganglion Cell

The size and shape of dendritic arbors are prime determinants of neuronal connectivity and function. We asked how ON-OFF direction-selective ganglion cells (ooDSGCs) in mouse retina acquire their bistratified dendrites, in which responses to light onset and light offset are segregated to distinct strata. We found that the transcriptional regulator Satb1 is selectively expressed by ooDSGCs. In Satb1 mutant mice, ooDSGC dendrites lack ON arbors, and the cells selectively lose ON responses. Satb1 regulates expression of a homophilic adhesion molecule, Contactin 5 (Cntn5). Both Cntn5 and its co-receptor Caspr4 are expressed not only by ooDSGCs, but also by interneurons that form a scaffold on which ooDSGC ON dendrites fasciculate. Removing Cntn5 from either ooDSGCs or interneurons partially phenocopies Satb1 mutants, demonstrating that Satb1-dependent Cntn5 expression in ooDSGCs leads to branch-specific homophilic interactions with interneurons. Thus, Satb1 directs formation of a morphologically and functionally specialized compartment within a complex dendritic arbor

Presynaptic partner selection during retinal circuit reassembly varies with timing of neuronal regeneration in vivo

Whether neurons can restore their original connectivity patterns during circuit repair is unclear. Taking advantage of the regenerative capacity of zebrafish retina, we show here the remarkable specificity by which surviving neurons reassemble their connectivity upon regeneration of their major input. H3 horizontal cells (HCs) normally avoid red and green cones, and prefer ultraviolet over blue cones. Upon ablation of the major (ultraviolet) input, H3 HCs do not immediately increase connectivity with other cone types. Instead, H3 dendrites retract and re-extend to contact new ultraviolet cones. But, if regeneration is delayed or absent, blue-cone synaptogenesis increases and ectopic synapses are made with red and green cones. Thus, cues directing synapse specificity can be maintained following input loss, but only within a limited time period. Further, we postulate that signals from the major input that shape the H3 HC's wiring pattern during development persist to restrict miswiring after damage

Combinatorial effects of Alpha- and Gamma-Protocadherins on neuronal survival and dendritic self-avoidance

The clustered Protocadherins (Pcdhs) comprise 58 cadherin-related proteins encoded by three tandemly-arrayed gene clusters, Pcdh-alpha, -beta, and --gamma (Pcdha, Pcdhb, Pcdhg). Pcdh isoforms from different clusters are combinatorially expressed in neurons. They form multimers that interact homophilically, and mediate a variety of developmental processes, including neuronal survival, synaptic maintenance, axonal tiling and dendritic self-avoidance. Most studies have analyzed clusters individually. Here, we assess functional interactions between Pcdha and Pcdhg clusters. To circumvent neonatal lethality associated with deletion of Pcdhgs, we used Crispr-Cas9 genome editing in mice to combine a constitutive Pcdha mutant allele with a conditional Pcdhg allele. We analyzed roles of Pcdhas and Pcdhgs in the retina and cerebellum from mice (both sexes) lacking one or both clusters. In retina, Pcdhgs are essential for survival of inner retinal neurons and dendrite self-avoidance of starburst amacrine cells, while Pcdhas are dispensable for both processes. Deletion of both Pcdha and Pcdhg clusters led to far more dramatic defects in survival and self-avoidance than Pcdhg deletion alone. Comparisons of an allelic series of mutants support the conclusion that Pcdhas and Pcdhgs function together in a dose-dependent and cell-type specific manner to provide a critical threshold of Pcdh activity. In the cerebellum, Pcdhas and Pcdhgs also act synergistically to mediate self-avoidance of Purkinje cell dendrites, with modest but significant defects in either single mutant and dramatic defects in the double mutant. Together, our results demonstrate complex patterns of redundancy between Pcdh clusters and the importance of Pcdh cluster diversity in postnatal CNS development

Competition-based model of pheromone component ratio detection in the moth

For some moth species, especially those closely interrelated and sympatric, recognizing a specific pheromone component concentration ratio is essential for males to successfully locate conspecific females. We propose and determine the properties of a minimalist competition-based feed-forward neuronal model capable of detecting a certain ratio of pheromone components independently of overall concentration. This model represents an elementary recognition unit for the ratio of binary mixtures which we propose is entirely contained in the macroglomerular complex (MGC) of the male moth. A set of such units, along with projection neurons (PNs), can provide the input to higher brain centres. We found that (1) accuracy is mainly achieved by maintaining a certain ratio of connection strengths between olfactory receptor neurons (ORN) and local neurons (LN), much less by properties of the interconnections between the competing LNs proper. An exception to this rule is that it is beneficial if connections between generalist LNs (i.e. excited by either pheromone component) and specialist LNs (i.e. excited by one component only) have the same strength as the reciprocal specialist to generalist connections. (2) successful ratio recognition is achieved using latency-to-first-spike in the LN populations which, in contrast to expectations with a population rate code, leads to a broadening of responses for higher overall concentrations consistent with experimental observations. (3) when longer durations of the competition between LNs were observed it did not lead to higher recognition accuracy

Autoantibodies to Agrin in Myasthenia Gravis Patients

To determine if patients with myasthenia gravis (MG) have antibodies to agrin, a proteoglycan released by motor neurons and is critical for neuromuscular junction (NMJ) formation, we collected serum samples from 93 patients with MG with known status of antibodies to acetylcholine receptor (AChR), muscle specific kinase (MuSK) and lipoprotein-related 4 (LRP4) and samples from control subjects (healthy individuals and individuals with other diseases). Sera were assayed for antibodies to agrin. We found antibodies to agrin in 7 serum samples of MG patients. None of the 25 healthy controls and none of the 55 control neurological patients had agrin antibodies. Two of the four triple negative MG patients (i.e., no detectable AChR, MuSK or LRP4 antibodies, AChR-/MuSK-/LRP4-) had antibodies against agrin. In addition, agrin antibodies were detected in 5 out of 83 AChR+/MuSK-/LRP4- patients but were not found in the 6 patients with MuSK antibodies (AChR-/MuSK+/LRP4-). Sera from MG patients with agrin antibodies were able to recognize recombinant agrin in conditioned media and in transfected HEK293 cells. These sera also inhibited the agrin-induced MuSK phosphorylation and AChR clustering in muscle cells. Together, these observations indicate that agrin is another autoantigen in patients with MG and agrin autoantibodies may be pathogenic through inhibition of agrin/LRP4/MuSK signaling at the NMJ

Motor Unit Abnormalities in Dystonia musculorum Mice

Dystonia musculorum (dt) is a mouse inherited sensory neuropathy caused by mutations in the dystonin gene. While the primary pathology lies in the sensory neurons of dt mice, the overt movement disorder suggests motor neurons may also be affected. Here, we report on the contribution of motor neurons to the pathology in dt27J mice. Phenotypic dt27J mice display reduced alpha motor neuron cell number and eccentric alpha motor nuclei in the ventral horn of the lumbar L1 spinal cord region. A dramatic reduction in the total number of motor axons in the ventral root of postnatal day 15 dt27J mice was also evident. Moreover, analysis of the trigeminal nerve of the brainstem showed a 2.4 fold increase in number of degenerating neurons coupled with a decrease in motor neuron number relative to wild type. Aberrant phosphorylation of neurofilaments in the perikaryon region and axonal swellings within the pre-synaptic terminal region of motor neurons were observed. Furthermore, neuromuscular junction staining of dt27J mouse extensor digitorum longus and tibialis anterior muscle fibers showed immature endplates and a significant decrease in axon branching compared to wild type littermates. Muscle atrophy was also observed in dt27J muscle. Ultrastructure analysis revealed amyelinated motor axons in the ventral root of the spinal nerve, suggesting a possible defect in Schwann cells. Finally, behavioral analysis identified defective motor function in dt27J mice. This study reveals neuromuscular defects that likely contribute to the dt27J pathology and identifies a critical role for dystonin outside of sensory neurons

Functional Refinement in the Projection from Ventral Cochlear Nucleus to Lateral Superior Olive Precedes Hearing Onset in Rat

Principal neurons of the lateral superior olive (LSO) compute the interaural intensity differences necessary for localizing high-frequency sounds. To perform this computation, the LSO requires precisely tuned, converging excitatory and inhibitory inputs that are driven by the two ears and that are matched for stimulus frequency. In rodents, the inhibitory inputs, which arise from the medial nucleus of the trapezoid body (MNTB), undergo extensive functional refinement during the first postnatal week. Similar functional refinement of the ascending excitatory pathway, which arises in the anteroventral cochlear nucleus (AVCN), has been assumed but has not been well studied. Using whole-cell voltage clamp in acute brainstem slices of neonatal rats, we examined developmental changes in input strength and pre- and post-synaptic properties of the VCN-LSO pathway. A key question was whether functional refinement in one of the two major input pathways might precede and then guide refinement in the opposite pathway. We find that elimination and strengthening of VCN inputs to the LSO occurs over a similar period to that seen for the ascending inhibitory (MNTB-LSO) pathway. During this period, the fractional contribution provided by NMDA receptors (NMDARs) declines while the contribution from AMPA receptors (AMPARs) increases. In the NMDAR-mediated response, GluN2B-containing NMDARs predominate in the first postnatal week and decline sharply thereafter. Finally, the progressive decrease in paired-pulse depression between birth and hearing onset allows these synapses to follow progressively higher frequencies. Our data are consistent with a model in which the excitatory and inhibitory projections to LSO are functionally refined in parallel during the first postnatal week, and they further suggest that GluN2B-containing NMDARs may mediate early refinement in the VCN-LSO pathway