Anterograde transneuronal tracing and genetic control with engineered yellow fever vaccine YFV-17D.

Transneuronal viruses are powerful tools for tracing neuronal circuits or delivering genes to specific neurons in the brain. While there are multiple retrograde viruses, few anterograde viruses are available. Further, available anterograde viruses often have limitations such as retrograde transport, high neuronal toxicity or weak signals. We developed an anterograde viral system based on a live attenuated vaccine for yellow fever-YFV-17D. Replication- or packaging-deficient mutants of YFV-17D can be reconstituted in the brain, leading to efficient synapse-specific and anterograde-only transneuronal spreading, which can be controlled to achieve either monosynaptic or polysynaptic tracing. Moreover, inducible transient replication of YFV-17D mutant is sufficient to induce permanent transneuronal genetic modifications without causing neuronal toxicity. The engineered YFV-17D systems can be used to express fluorescent markers, sensors or effectors in downstream neurons, thus providing versatile tools for mapping and functionally controlling neuronal circuits.

Differential vesicular sorting of AMPA and GABAA receptors.

In mature neurons AMPA receptors cluster at excitatory synapses primarily on dendritic spines, whereas GABAA receptors cluster at inhibitory synapses mainly on the soma and dendritic shafts. The molecular mechanisms underlying the precise sorting of these receptors remain unclear. By directly studying the constitutive exocytic vesicles of AMPA and GABAA receptors in vitro and in vivo, we demonstrate that they are initially sorted into different vesicles in the Golgi apparatus and inserted into distinct domains of the plasma membrane. These insertions are dependent on distinct Rab GTPases and SNARE complexes. The insertion of AMPA receptors requires SNAP25-syntaxin1A/B-VAMP2 complexes, whereas insertion of GABAA receptors relies on SNAP23-syntaxin1A/B-VAMP2 complexes. These SNARE complexes affect surface targeting of AMPA or GABAA receptors and synaptic transmission. Our studies reveal vesicular sorting mechanisms controlling the constitutive exocytosis of AMPA and GABAA receptors, which are critical for the regulation of excitatory and inhibitory responses in neurons.

Cyclophilin D over-expression increases mitochondrial complex III activity and accelerates supercomplex formation.

Cyclophilin D (CyPD), a mitochondrial matrix protein, has been widely studied for its role in mitochondrial-mediated cell death. Unexpectedly, we previously discovered that overexpression of CyPD in a stable cell line, increased mitochondrial membrane potentials and enhanced cell survival under conditions of oxidative stress. Here, we investigated the underlying mechanisms responsible for these findings. Spectrophotometric measurements in isolated mitochondria revealed that overexpression of CyPD in HEK293 cells increased respiratory chain activity, but only for Complex III (CIII). Acute treatment of mitochondria with the immumosupressant cyclosporine A did not affect CIII activity. Expression levels of the CIII subunits cytochrome b and Rieske-FeS were elevated in HEK293 cells overexpressing CyPD. However, CIII activity was still significantly higher compared to control mitochondria, even when normalized by protein expression. Blue native gel electrophoresis and Western blot assays revealed a molecular interaction of CyPD with CIII and increased levels of supercomplexes in mitochondrial protein extracts. Radiolabeled protein synthesis in mitochondria showed that CIII assembly and formation of supercomplexes containing CIII were significantly faster when CyPD was overexpressed. Taken together, these data indicate that CyPD regulates mitochondrial metabolism, and likely cell survival, by promoting more efficient electrons flow through the respiratory chain via increased supercomplex formation.

Excess of de novo deleterious mutations in genes associated with glutamatergic systems in nonsyndromic intellectual disability.

Little is known about the genetics of nonsyndromic intellectual disability (NSID). We hypothesized that de novo mutations (DNMs) in synaptic genes explain an important fraction of sporadic NSID cases. In order to investigate this possibility, we sequenced 197 genes encoding glutamate receptors and a large subset of their known interacting proteins in 95 sporadic cases of NSID. We found 11 DNMs, including ten potentially deleterious mutations (three nonsense, two splicing, one frameshift, four missense) and one neutral mutation (silent) in eight different genes. Calculation of point-substitution DNM rates per functional and neutral site showed significant excess of functional DNMs compared to neutral ones. De novo truncating and/or splicing mutations in SYNGAP1, STXBP1, and SHANK3 were found in six patients and are likely to be pathogenic. De novo missense mutations were found in KIF1A, GRIN1, CACNG2, and EPB41L1. Functional studies showed that all these missense mutations affect protein function in cell culture systems, suggesting that they may be pathogenic. Sequencing these four genes in 50 additional sporadic cases of NSID identified a second DNM in GRIN1 (c.1679_1681dup/p.Ser560dup). This mutation also affects protein function, consistent with structural predictions. None of these mutations or any other DNMs were identified in these genes in 285 healthy controls. This study highlights the importance of the glutamate receptor complexes in NSID and further supports the role of DNMs in this disorder.

Examining a punishment-related brain circuit with miniature fluorescence microscopes and deep learning.

In humans experiencing substance use disorder (SUD), abstinence from drug use is often motivated by a desire to avoid some undesirable consequence of further use: health effects, legal ramifications, etc. This process can be experimentally modeled in rodents by training and subsequently punishing an operant response in a context-induced reinstatement procedure. Understanding the biobehavioral mechanisms underlying punishment learning is critical to understanding both abstinence and relapse in individuals with SUD. To date, most investigations into the neural mechanisms of context-induced reinstatement following punishment have utilized discrete loss-of-function manipulations that do not capture ongoing changes in neural circuitry related to punishment-induced behavior change. Here, we describe a two-pronged approach to analyzing the biobehavioral mechanisms of punishment learning using miniature fluorescence microscopes and deep learning algorithms. We review recent advancements in both techniques and consider a target neural circuit.

A modular, cost-effective, versatile, open-source operant box solution for long-term miniscope imaging, 3D tracking, and deep learning behavioral analysis.

In this procedure we have included an open-source method for a customized operant chamber optimized for long-term miniature microscope (miniscope) recordings. •The miniscope box is designed to function with custom or typical med-associates style accessories (e.g., houselights, levers, etc.).•The majority of parts can be directly purchased which minimizes the need for skilled and time-consuming labor.•We include designs and estimated pricing for a single box but it is recommended to build these in larger batches to efficiently utilize bulk ordering of certain components.

Identification of two functionally distinct endosomal recycling pathways for dopamine D2 receptor.

Dopamine D2 receptor (DRD2) is important for normal function of the brain reward circuit. Lower DRD2 function in the brain increases the risk for substance abuse, obesity, attention deficit/hyperactivity disorder, and depression. Moreover, DRD2 is the target of most antipsychotics currently in use. It is well known that dopamine-induced DRD2 endocytosis is important for its desensitization. However, it remains controversial whether DRD2 is recycled back to the plasma membrane or targeted for degradation following dopamine stimulation. Here, we used total internal reflection fluorescent microscopy (TIRFM) to image DRD2 with a superecliptic pHluorin tagged to its N terminus. With these technical advances, we were able to directly visualize vesicular insertion events of DRD2 in cultured mouse striatal medium spiny neurons. We showed that insertion of DRD2 occurs on neuronal somatic and dendritic surfaces. Lateral diffusion of DRD2 was observed following its insertion. Most importantly, using our new approach, we uncovered two functionally distinct recycling pathways for DRD2: a constitutive recycling pathway and a dopamine activity-dependent recycling pathway. We further demonstrated that Rab4 plays an important role in constitutive DRD2 recycling, while Rab11 is required for dopamine activity-dependent DRD2 recycling. Finally, we demonstrated that the two DRD2 recycling pathways play distinct roles in determining DRD2 function: the Rab4-sensitive constitutive DRD2 recycling pathway determines steady-state surface expression levels of DRD2, whereas the Rab11-sensitive dopamine activity-dependent DRD2 recycling pathway is important for functional resensitization of DRD2. Our findings underscore the significance of endosomal recycling in regulation of DRD2 function.

Plasma membrane insertion of the AMPA receptor GluA2 subunit is regulated by NSF binding and Q/R editing of the ion pore.

The delivery of AMPA receptors to the plasma membrane is a critical step both for the synaptic delivery of these receptors and for the regulation of synaptic transmission. To directly visualize fusion events of transport vesicles containing the AMPA receptor GluA2 subunit with the plasma membrane we used pHluorin-tagged GluA2 subunits and total internal reflection fluorescence microscopy. We demonstrate that the plasma membrane insertion of GluA2 requires the NSF binding site within its intracellular cytoplasmic domain and that RNA editing of the Q/R site in the ion channel region plays a key role in GluA2 plasma membrane insertion. Finally, we show that plasma membrane insertion of heteromeric GluA2/3 receptors follows the same rules as homomeric GluA2 receptors. These results demonstrate that the plasma membrane delivery of GluA2 containing AMPA receptors is regulated by its unique structural elements.

GRIP1 and 2 regulate activity-dependent AMPA receptor recycling via exocyst complex interactions.

PSD-95/SAP90/DLG/ZO-1 (PDZ) domain-mediated protein-protein interactions play important roles in regulating AMPA receptor trafficking and neuronal plasticity. GRIP1 and GRIP2 are homologous multi-PDZ domain-containing proteins that bind to the C-termini of AMPA-R GluA2 and GluA3 subunits. Previous attempts to determine the cellular roles of GRIP1 and GRIP2 in neurons have been complicated by nonspecific reagents, and by the embryonic lethality of conventional GRIP1 KO mice. To circumvent these issues we developed a conditional targeted deletion strategy to knock out GRIP1 in postnatal neurons derived from GRIP2 KO mice. Loss of GRIP1 and 2 did not affect normal AMPA-R steady-state trafficking and endocytosis, but strikingly impaired activity-dependent AMPA-R recycling. This previously uncharacterized role for GRIP1 appears to be mediated by novel interactions with the cellular trafficking machinery via the exocyst protein complex. Indeed, disruption of GRIP1-exocyst binding caused a strikingly similar deficit in AMPA-R recycling. Together these findings reveal a previously unidentified role for AMPA-R-GRIP1-exocyst protein complexes in activity-dependent AMPA-R trafficking.

Jump-GRS: a multi-phase approach to structured pruning of neural networks for neural decoding.

Objective.Neural decoding, an important area of neural engineering, helps to link neural activity to behavior. Deep neural networks (DNNs), which are becoming increasingly popular in many application fields of machine learning, show promising performance in neural decoding compared to traditional neural decoding methods. Various neural decoding applications, such as brain computer interface applications, require both high decoding accuracy and real-time decoding speed. Pruning methods are used to produce compact DNN models for faster computational speed. Greedy inter-layer order with Random Selection (GRS) is a recently-designed structured pruning method that derives compact DNN models for calcium-imaging-based neural decoding. Although GRS has advantages in terms of detailed structure analysis and consideration of both learned information and model structure during the pruning process, the method is very computationally intensive, and is not feasible when large-scale DNN models need to be pruned within typical constraints on time and computational resources. Large-scale DNN models arise in neural decoding when large numbers of neurons are involved. In this paper, we build on GRS to develop a new structured pruning algorithm called jump GRS (JGRS) that is designed to efficiently compress large-scale DNN models.Approach.On top of GRS, JGRS implements a 'jump mechanism', which bypasses retraining intermediate models when model accuracy is relatively less sensitive to pruning operations. Design of the jump mechanism is motivated by identifying different phases of the structured pruning process, where retraining can be done infrequently in earlier phases without sacrificing accuracy. The jump mechanism helps to significantly speed up execution of the pruning process and greatly enhance its scalability. We compare the pruning performance and speed of JGRS and GRS with extensive experiments in the context of neural decoding.Main results.Our results demonstrate that JGRS provides significantly faster pruning speed compared to GRS, and at the same time, JGRS provides pruned models that are similarly compact as those generated by GRS.Significance.In our experiments, we demonstrate that JGRS achieves on average 9%-20% more compressed models compared to GRS with 2-8 times faster speed (less time required for pruning) across four different initial models on a relevant dataset for neural data analysis.

Rapid and bi-directional regulation of AMPA receptor phosphorylation and trafficking by JNK.

Jun N-terminal kinases (JNKs) are implicated in various neuropathological conditions. However, physiological roles for JNKs in neurons remain largely unknown, despite the high expression level of JNKs in brain. Here, using bioinformatic and biochemical approaches, we identify the AMPA receptor GluR2L and GluR4 subunits as novel physiological JNK substrates in vitro, in heterologous cells and in neurons. Consistent with this finding, GluR2L and GluR4 associate with specific JNK signaling components in the brain. Moreover, the modulation of the novel JNK sites in GluR2L and GluR4 is dynamic and bi-directional, such that phosphorylation and de-phosphorylation are triggered within minutes following decreases and increases in neuronal activity, respectively. Using live-imaging techniques to address the functional consequence of these activity-dependent changes we demonstrate that the novel JNK site in GluR2L controls reinsertion of internalized GluR2L back to the cell surface following NMDA treatment, without affecting basal GluR2L trafficking. Taken together, our results demonstrate that JNK directly regulates AMPA-R trafficking following changes in neuronal activity in a rapid and bi-directional manner.

Dysbindin-1, BDNF, and GABAergic Transmission in Schizophrenia.

Schizophrenia is a psychiatric disorder characterized by hallucinations, anhedonia, disordered thinking, and cognitive impairments. Both genetic and environmental factors contribute to schizophrenia. Dysbindin-1 (DTNBP1) and brain-derived neurotrophic factor (BDNF) are both genetic factors associated with schizophrenia. Mice lacking Dtnbp1 showed behavioral deficits similar to human patients suffering from schizophrenia. DTNBP1 plays important functions in synapse formation and maintenance, receptor trafficking, and neurotransmitter release. DTNBP1 is co-assembled with 7 other proteins into a large protein complex, known as the biogenesis of lysosome-related organelles complex-1 (BLOC-1). Large dense-core vesicles (LDCVs) are involved in the secretion of hormones and neuropeptides, including BDNF. BDNF plays important roles in neuronal development, survival, and synaptic plasticity. BDNF is also critical in maintaining GABAergic inhibitory transmission in the brain. Two studies independently showed that DTNBP1 mediated activity-dependent BDNF secretion to maintain inhibitory transmission. Imbalance of excitatory and inhibitory neural activities is thought to contribute to schizophrenia. In this mini-review, we will discuss a potential pathogenetic mechanism for schizophrenia involving DTNBP1, BDNF, and inhibitory transmission. We will also discuss how these processes are interrelated and associated with a higher risk of schizophrenia development.

GRIN lens applications for studying neurobiology of substance use disorder.

Substance use disorder (SUD) is associated with severe health and social consequences. Continued drug use results in alterations of circuits within the mesolimbic dopamine system. It is critical to observe longitudinal impacts of SUD on neural activity in vivo to identify SUD predispositions, develop pharmaceuticals to prevent overdose, and help people suffering from SUD. However, implicated SUD associated areas are buried in deep brain which makes in vivo observation of neural activity challenging. The gradient index (GRIN) lens can probe these regions in mice and rats. In this short communications review, we will discuss how the GRIN lens can be coupled with other technologies such as miniaturized microscopes, fiberscopes, fMRI, and optogenetics to fully explore in vivo SUD research. Particularly, GRIN lens allows in vivo observation of deep brain regions implicated in SUD, differentiation of genetically distinct neurons, examination of individual cells longitudinally, correlation of neuronal patters with SUD behavior, and manipulation of neural circuits.

A miniature fluorescence microscope for multi-plane imaging.

Miniature fluorescence microscopes are becoming an increasingly established tool to investigate neural circuits in freely moving animals. In this work we present a lightweight one-photon microscope capable of imaging at different focal depths. The focal plane can be changed dynamically by modulating the pulse width of the control signal to a variable focus liquid lens, which is synchronized to the image sensor to enable changing focal plane between frames. The system was tested by imaging GCaMP7f expressing neurons in the mouse medial prefrontal cortex (mPFC) in vivo during open field test. Results showed that with the proposed design it is possible to image neurons across an axial scan of ~ 60 µm, resulting in a ~ 40% increase of total neurons imaged compared to single plane imaging.

Characterization of operant social interaction in rats: effects of access duration, effort, peer familiarity, housing conditions, and choice between social interaction vs. food or remifentanil.

RATIONALE AND OBJECTIVE: Social factors play a critical role in drug addiction. We recently showed that rats will abstain from methamphetamine, cocaine, heroin, and remifentanil self-administration when given a choice between the addictive drug and operant social interaction. Here, we further characterized operant social interaction by determining the effects of access duration, effort, peer familiarity, and housing conditions. We also determined choice between social interaction vs. palatable food or remifentanil. METHODS: We first trained single-housed male and female rats to lever-press for social interaction with a sex- and age-matched peer. Next, we determined effects of access duration (3.75 to 240 s), effort (increasing fixed-ratio schedule requirements or progressive ratio schedule), peer familiarity (familiar vs. unfamiliar), and housing conditions (single vs. paired housing) on social self-administration. We also determined choice between social interaction vs. palatable food pellets or intravenous remifentanil (0, 1, 10 µg/kg/infusion). RESULTS: Increasing access duration to a peer decreased social self-administration under fixed ratio but not progressive ratio schedule; the rats showed similar preference for short vs. long access duration. Social self-administration under different fixed ratio requirements was higher in single-housed than in paired-housed rats and higher for a familiar vs. unfamiliar partner in single-housed but not paired-housed rats. Response rates of food-sated rats under increasing fixed-ratio requirements were higher for palatable food than for social interaction. The rats strongly preferred palatable food over social interaction and showed dose-dependent preference for social interaction vs. remifentanil. CONCLUSIONS: We identified parameters influencing the reinforcing effects of operant social interaction and introduce a choice procedure sensitive to remifentanil self-administration dose.

Using deep learning to study emotional behavior in rodent models.

Quantifying emotional aspects of animal behavior (e.g., anxiety, social interactions, reward, and stress responses) is a major focus of neuroscience research. Because manual scoring of emotion-related behaviors is time-consuming and subjective, classical methods rely on easily quantified measures such as lever pressing or time spent in different zones of an apparatus (e.g., open vs. closed arms of an elevated plus maze). Recent advancements have made it easier to extract pose information from videos, and multiple approaches for extracting nuanced information about behavioral states from pose estimation data have been proposed. These include supervised, unsupervised, and self-supervised approaches, employing a variety of different model types. Representations of behavioral states derived from these methods can be correlated with recordings of neural activity to increase the scope of connections that can be drawn between the brain and behavior. In this mini review, we will discuss how deep learning techniques can be used in behavioral experiments and how different model architectures and training paradigms influence the type of representation that can be obtained.

Aberrant neural activity in prefrontal pyramidal neurons lacking TDP-43 precedes neuron loss.

Mislocalization of TAR DNA binding protein 43 kDa (TARDBP, or TDP-43) is a principal pathological hallmark identified in cases of neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). As an RNA binding protein, TDP-43 serves in the nuclear compartment to repress non-conserved cryptic exons to ensure the normal transcriptome. Multiple lines of evidence from animal models and human studies support the view that loss of TDP-43 leads to neuron loss, independent of its cytosolic aggregation. However, the underlying pathogenic pathways driven by the loss-of-function mechanism are still poorly defined. We employed a genetic approach to determine the impact of TDP-43 loss in pyramidal neurons of the prefrontal cortex (PFC). Using a custom-built miniscope imaging system, we performed repetitive in vivo calcium imaging from freely behaving mice for up to 7 months. By comparing calcium activity in PFC pyramidal neurons between TDP-43 depleted and TDP-43 intact mice, we demonstrated remarkably increased numbers of pyramidal neurons exhibiting hyperactive calcium activity after short-term TDP-43 depletion, followed by rapid activity declines prior to neuron loss. Our results suggest aberrant neural activity driven by loss of TDP-43 as the pathogenic pathway at early stage in ALS and FTD.

Striatal direct pathway neurons play leading roles in accelerating rotarod motor skill learning.

Dorsal striatum is important for movement control and motor skill learning. However, it remains unclear how the spatially and temporally distributed striatal medium spiny neuron (MSN) activity in the direct and indirect pathways (D1 and D2 MSNs, respectively) encodes motor skill learning. Combining miniature fluorescence microscopy with an accelerating rotarod procedure, we identified two distinct MSN subpopulations involved in accelerating rotarod learning. In both D1 and D2 MSNs, we observed neurons that displayed activity tuned to acceleration during early stages of trials, as well as movement speed during late stages of trials. We found a distinct evolution trajectory for early-stage neurons during motor skill learning, with the evolution of D1 MSNs correlating strongly with performance improvement. Importantly, optogenetic inhibition of the early-stage neural activity in D1 MSNs, but not D2 MSNs, impaired accelerating rotarod learning. Together, this study provides insight into striatal D1 and D2 MSNs encoding motor skill learning.

Detailed mapping of behavior reveals the formation of prelimbic neural ensembles across operant learning.

The prelimbic cortex (PrL) is involved in the organization of operant behaviors, but the relationship between longitudinal PrL neural activity and operant learning and performance is unknown. Here, we developed deep behavior mapping (DBM) to identify behavioral microstates in video recordings. We combined DBM with longitudinal calcium imaging to quantify behavioral tuning in PrL neurons as mice learned an operant task. We found that a subset of PrL neurons were strongly tuned to highly specific behavioral microstates, both task and non-task related. Overlapping neural ensembles were tiled across consecutive microstates in the response-reinforcer sequence, forming a continuous map. As mice learned the operant task, weakly tuned neurons were recruited into new ensembles, with a bias toward behaviors similar to their initial tuning. In summary, our data suggest that the PrL contains neural ensembles that jointly encode a map of behavioral states that is fine grained, is continuous, and grows during operant learning.

Circuit Investigation of Social Interaction and Substance Use Disorder Using Miniscopes.

Substance use disorder (SUD) is comorbid with devastating health issues, social withdrawal, and isolation. Successful clinical treatments for SUD have used social interventions. Neurons can encode drug cues, and drug cues can trigger relapse. It is important to study how the activity in circuits and embedded cell types that encode drug cues develop in SUD. Exploring shared neurobiology between social interaction (SI) and SUD may explain why humans with access to social treatments still experience relapse. However, circuitry remains poorly characterized due to technical challenges in studying the complicated nature of SI and SUD. To understand the neural correlates of SI and SUD, it is important to: (1) identify cell types and circuits associated with SI and SUD, (2) record and manipulate neural activity encoding drug and social rewards over time, (3) monitor unrestrained animal behavior that allows reliable drug self-administration (SA) and SI. Miniaturized fluorescence microscopes (miniscopes) are ideally suited to meet these requirements. They can be used with gradient index (GRIN) lenses to image from deep brain structures implicated in SUD. Miniscopes can be combined with genetically encoded reporters to extract cell-type specific information. In this mini-review, we explore how miniscopes can be leveraged to uncover neural components of SI and SUD and advance potential therapeutic interventions.