Learning from Principles of Evidence-Based Medicine to Optimize Nonclinical Research Practices.

Thousands of pharmacology experiments are performed each day, generating hundreds of drug discovery programs, scientific publications, grant submissions, and other efforts. Discussions of the low reproducibility and robustness of some of this research have led to myriad efforts to increase data quality and thus reliability. Across the scientific ecosystem, regardless of the extent of concerns, debate about solutions, and differences among goals and practices, scientists strive to provide reliable data to advance frontiers of knowledge. Here we share our experience of current practices in nonclinical neuroscience research across biopharma and academia, examining context-related factors and behaviors that influence ways of working and decision-making. Drawing parallels with the principles of evidence-based medicine, we discuss ways of improving transparency and consider how to better implement best research practices. We anticipate that a shared framework of scientific rigor, facilitated by training, enabling tools, and enhanced data sharing, will draw the conversation away from data unreliability or lack of reproducibility toward the more important discussion of how to generate data that advances knowledge and propels innovation.

Cellular plasticity induced by anti-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor encephalitis antibodies.

OBJECTIVE: Autoimmune-mediated anti-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) encephalitis is a severe but treatment-responsive disorder with prominent short-term memory loss and seizures. The mechanisms by which patient antibodies affect synapses and neurons leading to symptoms are poorly understood. METHODS: The effects of patient antibodies on cultures of live rat hippocampal neurons were determined with immunostaining, Western blot, and electrophysiological analyses. RESULTS: We show that patient antibodies cause a selective decrease in the total surface amount and synaptic localization of GluA1- and GluA2-containing AMPARs, regardless of receptor subunit binding specificity, through increased internalization and degradation of surface AMPAR clusters. In contrast, patient antibodies do not alter the density of excitatory synapses, N-methyl-D-aspartate receptor (NMDAR) clusters, or cell viability. Commercially available AMPAR antibodies directed against extracellular epitopes do not result in a loss of surface and synaptic receptor clusters, suggesting specific effects of patient antibodies. Whole-cell patch clamp recordings of spontaneous miniature postsynaptic currents show that patient antibodies decrease AMPAR-mediated currents, but not NMDAR-mediated currents. Interestingly, several functional properties of neurons are also altered: inhibitory synaptic currents and vesicular γ-aminobutyric acid transporter (vGAT) staining intensity decrease, whereas the intrinsic excitability of neurons and short-interval firing increase. INTERPRETATION: These results establish that antibodies from patients with anti-AMPAR encephalitis selectively eliminate surface and synaptic AMPARs, resulting in a homeostatic decrease in inhibitory synaptic transmission and increased intrinsic excitability, which may contribute to the memory deficits and epilepsy that are prominent in patients with this disorder.

Acute mechanisms underlying antibody effects in anti-N-methyl-D-aspartate receptor encephalitis.

OBJECTIVE: A severe but treatable form of immune-mediated encephalitis is associated with antibodies in serum and cerebrospinal fluid (CSF) against the GluN1 subunit of the N-methyl-D-aspartate receptor (NMDAR). Prolonged exposure of hippocampal neurons to antibodies from patients with anti-NMDAR encephalitis caused a reversible decrease in the synaptic localization and function of NMDARs. However, acute effects of the antibodies, fate of the internalized receptors, type of neurons affected, and whether neurons develop compensatory homeostatic mechanisms were unknown and are the focus of this study. METHODS: Dissociated hippocampal neuron cultures and rodent brain sections were used for immunocytochemical, physiological, and molecular studies. RESULTS: Patient antibodies bind to NMDARs throughout the rodent brain, and decrease NMDAR cluster density in both excitatory and inhibitory hippocampal neurons. They rapidly increase the internalization rate of surface NMDAR clusters, independent of receptor activity. This internalization likely accounts for the observed decrease in NMDAR-mediated currents, as no evidence of direct blockade was detected. Once internalized, antibody-bound NMDARs traffic through both recycling endosomes and lysosomes, similar to pharmacologically induced NMDAR endocytosis. The antibodies are responsible for receptor internalization, as their depletion from CSF abrogates these effects in hippocampal neurons. We find that although anti-NMDAR antibodies do not induce compensatory changes in glutamate receptor gene expression, they cause a decrease in inhibitory synapse density onto excitatory hippocampal neurons. INTERPRETATION: Our data support an antibody-mediated mechanism of disease pathogenesis driven by immunoglobulin-induced receptor internalization. Antibody-mediated downregulation of surface NMDARs engages homeostatic synaptic plasticity mechanisms, which may inadvertently contribute to disease progression.

The in vivo contribution of motor neuron TrkB receptors to mutant SOD1 motor neuron disease.

Brain-derived neurotrophic factor (BDNF) and its receptor tropomyosin-related kinase B (TrkB) are widely expressed in the vertebrate nervous system and play a central role in mature neuronal function. In vitro BDNF/TrkB signaling promotes neuronal survival and can help neurons resist toxic insults. Paradoxically, BDNF/TrkB signaling has also been shown, under certain in vitro circumstances, to render neurons vulnerable to insults. We show here that in vivo conditional deletion of TrkB from mature motor neurons attenuates mutant superoxide dismutase 1 (SOD1) toxicity. Mutant SOD1 mice lacking motor neuron TrkB live a month longer than controls and retain motor function for a longer period, particularly in the early phase of the disease. These effects are subserved by slowed motor neuron loss, persistence of neuromuscular junction integrity and reduced astrocytic and microglial reactivity within the spinal cord. These results suggest that manipulation of BDNF/TrkB signaling might have therapeutic efficacy in motor neuron diseases.

Genetic deletion of trkB.T1 increases neuromuscular function.

Neurotrophin-dependent activation of the tyrosine kinase receptor trkB.FL modulates neuromuscular synapse maintenance and function; however, it is unclear what role the alternative splice variant, truncated trkB (trkB.T1), may have in the peripheral neuromuscular axis. We examined this question in trkB.T1 null mice and demonstrate that in vivo neuromuscular performance and nerve-evoked muscle tension are significantly increased. In vitro assays indicated that the gain-in-function in trkB.T1(-/-) animals resulted specifically from an increased muscle contractility, and increased electrically evoked calcium release. In the trkB.T1 null muscle, we identified an increase in Akt activation in resting muscle as well as a significant increase in trkB.FL and Akt activation in response to contractile activity. On the basis of these findings, we conclude that the trkB signaling pathway might represent a novel target for intervention across diseases characterized by deficits in neuromuscular function.

Determinants of synaptic strength vary across an axon arbor.

We used synaptophysin-pHluorin expressed in hippocampal neurons to address how functional properties of terminals, namely, evoked release, total vesicle pool size, and release fraction, vary spatially across individual axon arbors. Consistent with previous reports, over short arbor distances (≈ 100 µm), evoked release was spatially heterogeneous when terminals contacted different postsynaptic dendrites or neurons. Regardless of the postsynaptic configuration, the evoked release and total vesicle pool size spatially covaried, suggesting that the fraction of synaptic vesicles available for release (release fraction) was similar over short distances. Evoked release and total vesicle pool size were highly correlated with the amount of NMDA receptors and PSD-95 in postsynaptic specialization. However, when individual axons were followed over longer distances (several hundred micrometers), a significant increase in evoked release was observed distally that was associated with an increased release fraction in distal terminals. The increase in distal release fraction can be accounted for by changes in individual vesicle release probability as well as readily releasable pool size. Our results suggest that for a single axon arbor, presynaptic strength indicated by evoked release over short distances is correlated with heterogeneity in total vesicle pool size, whereas over longer distances presynaptic strength is correlated with the spatial modulation of release fraction. Thus the mechanisms that determine synaptic strength differ depending on spatial scale.

Cellular and synaptic mechanisms of anti-NMDA receptor encephalitis.

We recently described a severe, potentially lethal, but treatment-responsive encephalitis that associates with autoantibodies to the NMDA receptor (NMDAR) and results in behavioral symptoms similar to those obtained with models of genetic or pharmacologic attenuation of NMDAR function. Here, we demonstrate that patients' NMDAR antibodies cause a selective and reversible decrease in NMDAR surface density and synaptic localization that correlates with patients' antibody titers. The mechanism of this decrease is selective antibody-mediated capping and internalization of surface NMDARs, as Fab fragments prepared from patients' antibodies did not decrease surface receptor density, but subsequent cross-linking with anti-Fab antibodies recapitulated the decrease caused by intact patient NMDAR antibodies. Moreover, whole-cell patch-clamp recordings of miniature EPSCs in cultured rat hippocampal neurons showed that patients' antibodies specifically decreased synaptic NMDAR-mediated currents, without affecting AMPA receptor-mediated currents. In contrast to these profound effects on NMDARs, patients' antibodies did not alter the localization or expression of other glutamate receptors or synaptic proteins, number of synapses, dendritic spines, dendritic complexity, or cell survival. In addition, NMDAR density was dramatically reduced in the hippocampus of female Lewis rats infused with patients' antibodies, similar to the decrease observed in the hippocampus of autopsied patients. These studies establish the cellular mechanisms through which antibodies of patients with anti-NMDAR encephalitis cause a specific, titer-dependent, and reversible loss of NMDARs. The loss of this subtype of glutamate receptors eliminates NMDAR-mediated synaptic function, resulting in the learning, memory, and other behavioral deficits observed in patients with anti-NMDAR encephalitis.

mll ortholog containing functional domains of human MLL is expressed throughout the zebrafish lifespan and in haematopoietic tissues.

Infant leukaemia is an embryonal disease in which the underlying MLL translocations initiate in utero. Zebrafish offer unique potential to understand how MLL impacts haematopoiesis from the earliest embryonic timepoints and how translocations cause leukaemia as an embryonal process. In this study, a zebrafish mll cDNA syntenic to human MLL spanning the 5' to 3' UTRs, was cloned from embryos, and mll expression was characterized over the zebrafish lifespan. The protein encoded by the 35-exon ORF exhibited 46·4% overall identity to human MLL and 68-100% conservation in functional domains (AT-hooks, SNL, CXXC, PHD, bromodomain, FYRN, taspase1 sites, FYRC, SET). Maternally supplied transcripts were detected at 0-2 hpf. Strong ubiquitous early zygotic expression progressed to a cephalo-caudal gradient during later embryogenesis. mll was expressed in the intermediate cell mass (ICM) where primitive erythrocytes are produced and in the kidney where definitive haematopoiesis occurs in adults. mll exhibits high cross species conservation, is developmentally regulated in haematopoietic and other tissues and is expressed from the earliest embryonic timepoints throughout the zebrafish lifespan. Haematopoietic tissue expression validates using zebrafish for MLL haematopoiesis and leukaemia models.

Astrocyte secreted proteins selectively increase hippocampal GABAergic axon length, branching, and synaptogenesis.

Astrocytes modulate the formation and function of glutamatergic synapses in the CNS, but whether astrocytes modulate GABAergic synaptogenesis is unknown. We demonstrate that media conditioned by astrocytes, but not other cells, enhanced GABAergic but not glutamatergic axon length and branching, and increased the number and density of presynaptically active GABAergic synapses in dissociated hippocampal cultures. Candidate mechanisms and factors, such as activity, neurotrophins, and cholesterol were excluded as mediating these effects. While thrombospondins secreted by astrocytes are necessary and sufficient to increase hippocampal glutamatergic synaptogenesis, they do not mediate astrocyte effects on GABAergic synaptogenesis. We show that the factors in astrocyte conditioned media that selectively affect GABAergic neurons are proteins. Taken together, our results show that astrocytes increase glutamatergic and GABAergic synaptogenesis via different mechanisms and release one or more proteins with the novel functions of increasing GABAergic axon length, branching and synaptogenesis.

Mechanisms underlying autoimmune synaptic encephalitis leading to disorders of memory, behavior and cognition: insights from molecular, cellular and synaptic studies.

Recently, several novel, potentially lethal and treatment-responsive syndromes that affect hippocampal and cortical function have been shown to be associated with auto-antibodies against synaptic antigens, notably glutamate or GABA-B receptors. Patients with these auto-antibodies, sometimes associated with teratomas and other neoplasms, present with psychiatric symptoms, seizures, memory deficits and decreased levels of consciousness. These symptoms often improve dramatically after immunotherapy or tumor resection. Here we review studies of the cellular and synaptic effects of these antibodies in hippocampal neurons in vitro and preliminary work in rodent models. Our work suggests that patient antibodies lead to rapid and reversible removal of neurotransmitter receptors from synaptic sites, leading to changes in synaptic and circuit function that in turn are likely to lead to behavioral deficits. We also discuss several of the many questions raised by these and related disorders. Determining the mechanisms underlying these novel anti-neurotransmitter receptor encephalopathies will provide insights into the cellular and synaptic bases of the memory and cognitive deficits that are hallmarks of these disorders, and potentially suggest avenues for therapeutic intervention.

Mass spectrometric and computational analysis of cytokine-induced alterations in the astrocyte secretome.

The roles of astrocytes in the CNS have been expanding beyond the long held view of providing passive, supportive functions. Recent evidence has identified roles in neuronal development, extracellular matrix maintenance, and response to inflammatory challenges. Therefore, insights into astrocyte secretion are critically important for understanding physiological responses and pathological mechanisms in CNS diseases. Primary astrocyte cultures were treated with inflammatory cytokines for either a short (1 day) or sustained (7 days) exposure. Increased interleukin-6 secretion, nitric oxide production, cyclooxygenase-2 activation, and nerve growth factor (NGF) secretion confirmed the astrocytic response to cytokine treatment. MS/MS analysis, computational prediction algorithms, and functional classification were used to compare the astrocyte protein secretome from control and cytokine-exposed cultures. In total, 169 secreted proteins were identified, including both classically and nonconventionally secreted proteins that comprised components of the extracellular matrix and enzymes involved in processing of glycoproteins and glycosaminoglycans. Twelve proteins were detected exclusively in the secretome from cytokine-treated astrocytes, including matrix metalloproteinase-3 (MMP-3) and members of the chemokine ligand family. This compilation of secreted proteins provides a framework for identifying factors that influence the biochemical environment of the nervous system, regulate development, construct extracellular matrices, and coordinate the nervous system response to inflammation.

Heterogeneity in synaptic vesicle release at neuromuscular synapses of mice expressing synaptopHluorin.

Mammalian neuromuscular junctions are useful model synapses to study the relationship between synaptic structure and function, although these have rarely been studied together at the same synapses. To do this, we generated transgenic lines of mice in which the thy1.2 promoter drives expression of synaptopHluorin (spH) as a means of optically measuring synaptic vesicle distribution and release. SpH is colocalized with other synaptic vesicle proteins in presynaptic terminals and does not alter normal synaptic function. Nerve stimulation leads to readily detectable and reproducible fluorescence changes in motor axon terminals that vary with stimulus frequency and, when compared with electrophysiological recordings, are reliable indicators of neurotransmitter release. Measurements of fluorescence intensity changes reveal a surprising amount of heterogeneity in synaptic vesicle release throughout individual presynaptic motor axon terminals. Some discrete terminal regions consistently displayed a greater rate and extent of release than others, regardless of stimulation frequency. The amount of release at a particular site is highly correlated to the relative abundance of synaptic vesicles there, indicating that a relatively constant fraction of the total vesicular pool, approximately 30%, is released in response to activity. These studies reveal previously unknown relationships between synaptic structure and function at mammalian neuromuscular junctions and demonstrate the usefulness of spH expressing mice as a tool for studying neuromuscular synapses in adults, as well as during development and diseases that affect neuromuscular synaptic function.

In vivo imaging of preferential motor axon outgrowth to and synaptogenesis at prepatterned acetylcholine receptor clusters in embryonic zebrafish skeletal muscle.

Little is known about the spatial and temporal dynamics of presynaptic and postsynaptic specializations that culminate in synaptogenesis. Here, we imaged presynaptic vesicle clusters in motor axons and postsynaptic acetylcholine receptor (AChR) clusters in embryonic zebrafish to study the earliest events in synaptogenesis in vivo. Prepatterned AChR clusters are present on muscle fibers in advance of motor axon outgrowth from the spinal cord. Motor axon growth cones and filopodia are selectively extended toward and contact prepatterned AChR clusters, followed by the rapid clustering of presynaptic vesicles and insertion of additional AChRs, hallmarks of synaptogenesis. All initially formed neuromuscular synapses contain AChRs that were inserted into the membrane at the time the prepattern is present. Examination of embryos in which AChRs were blocked or clustering is absent showed that neither receptor activity or receptor protein is required for these events to occur. Thus, during initial synaptogenesis, postsynaptic differentiation precedes presynaptic differentiation, and prepatterned neurotransmitter clusters mark sites destined for synapse formation.

Localization of TrkC to Schwann cells and effects of neurotrophin-3 signaling at neuromuscular synapses.

Neurotrophins and their receptors, the Trks, are differentially expressed among the cell types that make up neuromuscular and other synapses, but the function and directionality of neurotrophin signaling at synapses are poorly understood. Here we demonstrate, via immunostaining, Western blotting, and RT-PCR analyses, that TrkC, the receptor for neurotrophin-3 (NT3), is expressed by mouse perisynaptic and myelinating Schwann cells from birth through adulthood and is unaltered after denervation. Analyses of transgenic mice in which the NT3 coding sequence is replaced by lacZ showed that NT3 is expressed in motor neurons and Schwann cells during perinatal development, but not in adult mice. In muscle, NT3 is expressed by intrafusal muscle fibers within spindles, as has been previously reported. Surprisingly, NT3 is also expressed in extrafusal muscle fibers during perinatal life and in adults. Genetic approaches were used to explore the roles of NT3 and TrkC signaling at neuromuscular synapses. Overexpression of NT3 in muscle fibers during development resulted in an increased number of perisynaptic Schwann cells at neuromuscular synapses, without altering synaptic size, suggesting that muscle-derived NT3 might act as a mitogen or trophic factor for Schwann cells. Conditional deletion of NT3 from motor neurons did not alter the number of Schwann cells or other aspects of neuromuscular synaptic structure, suggesting that motor-neuron-derived NT3 is not required for normal development of perisynaptic Schwann cells or synapses. Together, these results demonstrate that NT3 expression is developmentally regulated in skeletal muscle and may modulate the number of Schwann cells at neuromuscular synapses.

Astrocytes regulate inhibitory synapse formation via Trk-mediated modulation of postsynaptic GABAA receptors.

Astrocytes promote the formation and function of excitatory synapses in the CNS. However, whether and how astrocytes modulate inhibitory synaptogenesis are essentially unknown. We asked whether astrocytes regulate the formation of inhibitory synapses between hippocampal neurons during maturation in vitro. Neuronal coculture with astrocytes or treatment with astrocyte-conditioned medium (ACM) increased the number of inhibitory presynaptic terminals, the frequency of miniature IPSCs, and the number and synaptic localization of GABA(A) receptor (GABA(A)R) clusters during the first 10 d in vitro. We asked whether neurotrophins, which are potent modulators of inhibitory synaptic structure and function, mediate the effects of astrocytes on inhibitory synapses. ACM from BDNF- or tyrosine receptor kinase B (TrkB)-deficient astrocytes increased inhibitory presynaptic terminals and postsynaptic GABA(A)R clusters in wild-type neurons, suggesting that BDNF and TrkB expression in astrocytes is not required for these effects. In contrast, although the increase in the number of inhibitory presynaptic terminals persisted, no increase was observed in postsynaptic GABA(A)R clusters after ACM treatment of hippocampal neurons lacking BDNF or TrkB. These results suggest that neurons, not astrocytes, are the relevant source of BDNF and are the site of TrkB activation required for postsynaptic GABA(A)R modulation. These data also suggest that astrocytes may modulate postsynaptic development indirectly by stimulating Trk signaling between neurons. Together, these data show that astrocytes modulate inhibitory synapse formation via distinct presynaptic and postsynaptic mechanisms.

Postsynaptic TrkB-mediated signaling modulates excitatory and inhibitory neurotransmitter receptor clustering at hippocampal synapses.

Tyrosine receptor kinase B (TrkB)-mediated signaling modulates synaptic structure and strength in hippocampal and other neurons, but the underlying mechanisms are poorly understood. Full-length and truncated TrkB are diffusely distributed throughout the dendrites and soma of rat hippocampal neurons grown in vitro. Manipulation of TrkB-mediated signaling resulted in dramatic changes in the number and synaptic localization of postsynaptic NMDA receptor (NMDAR) and GABA(A) receptor (GABA(A)R) clusters. BDNF treatment resulted in an increase in the number of NMDAR and GABA(A)R clusters and increased the proportion of clusters apposed to presynaptic terminals. Downregulation of TrkB signaling resulted in a decrease in receptor cluster number and synaptic localization. Examination of the time course of the effects of BDNF on receptor clusters showed that the increase in GABA(A)R clusters preceded the increase in NMDAR clusters by at least 12 hr. Moreover, the TrkB-mediated effects on NMDAR clusters were dependent on GABA(A)R activation. Although TTX, APV, and CNQX treatment had no effect, blockade of GABA(A)Rs with bicuculline abolished the BDNF-mediated increase in NMDAR cluster number and synaptic localization. In contrast, application of exogenous GABA prevented the decrease in NMDAR clusters induced by BDNF scavenging. Together, these results suggest that TrkB-mediated signaling modulates the clustering of postsynaptic GABA(A)Rs and that receptor activity is required for a subsequent upregulation of NMDAR clusters. Therefore, TrkB-mediated effects on postsynaptic neurotransmitter clusters may be part of a mechanism that balances inhibitory and excitatory synaptic transmission in developing neural circuits.

Failure of brain-derived neurotrophic factor-dependent neuron survival in mouse trisomy 16.

The neurotrophin, brain derived neurotrophic factor (BDNF), exerts multiple effects on the development and maintenance of the nervous system, including regulating synaptic plasticity and promoting neuron survival. Here we report the selective failure of BDNF-dependent survival in cultured hippocampal neurons from the trisomy 16 (Ts16) mouse, an animal model of Down syndrome. This failure is accompanied by overexpression of a truncated, kinase-deficient isoform (T1) of the BDNF receptor tyrosine receptor kinase B (trkB). Adenovirus-mediated introduction of exogenous full-length trkB into Ts16 neurons fully restored BDNF-dependent survival, whereas exogenous truncated trkB expression in normal, euploid neurons reproduced the Ts16 BDNF signaling failure. Thus, the failure of Ts16 neurons to respond to BDNF is caused by dysregulation of trkB isoform expression. Such a neurotrophin signaling defect could contribute to developmental and degenerative disorders of the nervous system.

Neurobeachin is essential for neuromuscular synaptic transmission.

We report a random disruption in the mouse genome that resulted in lethal paralysis in homozygous newborns. The disruption blocked expression of neurobeachin, a protein containing a BEACH (beige and Chediak-Higashi) domain implicated in synaptic vesicle trafficking and an AKAP (A-kinase anchor protein) domain linked to localization of cAMP-dependent protein kinase activity. nbea-null mice demonstrated a complete block of evoked synaptic transmission at neuromuscular junctions, whereas nerve conduction, synaptic structure, and spontaneous synaptic vesicle release were completely normal. These findings support an essential role for neurobeachin in evoked neurotransmitter release at neuromuscular junctions and suggest that it plays an important role in synaptic transmission.

Dysmyelinated lower motor neurons retract and regenerate dysfunctional synaptic terminals.

Axonal degeneration is the major cause of permanent neurological disability in individuals with inherited diseases of myelin. Axonal and neuronal changes that precede axonal degeneration, however, are not well characterized. We show here that dysmyelinated lower motor neurons retract and regenerate dysfunctional presynaptic terminals, leading to severe neurological disability before axonal degeneration. In addition, dysmyelination led to a decreased synaptic quantal content, an indicator of synaptic dysfunction. The amplitude and rise time of miniature endplate potentials were also increased, but these changes were primarily consistent with an increase in the passive membrane properties of the transgenic muscle fibers. Maintenance of synaptic connections should be considered as a therapeutic target for slowing progression of neurological disability in primary diseases of myelin.

Activity-dependent synaptic plasticity: insights from neuromuscular junctions.

Experience-dependent editing shapes synaptic connections throughout the developing nervous system, but the underlying cellular mechanisms remain poorly understood. A useful model synapse for addressing these mechanisms is the neuromuscular junction, the connection between spinal motor neurons and skeletal muscle fibers. Here the authors review current ideas about the role of activity in editing neuromuscular synaptic connections. A variety of new tools are being used to address some unanswered questions in vivo and in vitro. Understanding activity-dependent plasticity at developing neuromuscular synapses may reveal how neural circuits in the central nervous system are altered by experience throughout life.