Low-cost reversible tandem lens mesoscope for brain imaging in rodents.

Significance: The development of imaging systems that are cost-efficient and modular is essential for modern neuroscience research. Aim: In the current study, we designed, developed, and characterized a low-cost reversible tandem lens mesoscope for brain imaging in rodents. Approach: Using readily available components, we assembled a robust imaging system that is highly efficient and cost-effective. We developed a mesoscope that offers high-resolution structural and functional imaging with cost-effective lenses and CMOS camera. Results: The reversible tandem lens configuration of the mesoscope offers two fields of view (FOVs), which can be achieved by swapping the objective and imaging lenses. The large FOV configuration of 12.6×10.5 mm provides a spatial resolution up to 4.92 µm, and the small FOV configuration of 6×5 mm provides a resolution of up to 2.46 µm. We demonstrate the efficiency of our system for imaging neuronal calcium activity in both rat and mouse brains in vivo. Conclusions: The careful selection of the mesoscope components ensured its compactness, portability, and versatility, meaning that different types of samples and sample holders can be easily accommodated, enabling a range of different experiments both in vivo and in vitro. The custom-built reversible FOV mesoscope is cost-effective and was developed for under US$10,000 with excellent performance.

Recording plasticity in neuronal activity in the rodent intrinsic cardiac nervous system using calcium imaging techniques.

The intrinsic cardiac nervous system (ICNS) is composed of interconnected clusters of neurons called ganglionated plexi (GP) which play a major role in controlling heart rate and rhythm. The function of these neurons is particularly important due to their involvement in cardiac arrhythmias such as atrial fibrillation (AF), and previous work has shown that plasticity in GP neural networks could underpin aberrant activity patterns that drive AF. As research in this field increases, developing new techniques to visualize the complex interactions and plasticity in this GP network is essential. In this study we have developed a calcium imaging method enabling the simultaneous recording of plasticity in neuronal activity from multiple neurons in intact atrial GP networks. Calcium imaging was performed with Cal-520 AM labeling in aged spontaneously hypertensive rats (SHRs), which display both spontaneous and induced AF, and age-matched Wistar Kyoto (WKY) controls to determine the relationship between chronic hypertension, arrhythmia and GP calcium dynamics. Our data show that SHR GPs have significantly larger calcium responses to cholinergic stimulation compared to WKY controls, as determined by both higher amplitude and longer duration calcium responses. Responses were significantly but not fully blocked by hexamethonium, indicating multiple cholinergic receptor subtypes are involved in the calcium response. Given that SHRs are susceptible to cardiac arrhythmias, our data provide evidence for a potential link between arrhythmia and plasticity in calcium dynamics that occur not only in cardiomyocytes but also in the GP neurons of the heart.

Auditory processing in rodent models of autism: a systematic review.

Autism is a complex condition with many traits, including differences in auditory sensitivity. Studies in human autism are plagued by the difficulty of controlling for aetiology, whereas studies in individual rodent models cannot represent the full spectrum of human autism. This systematic review compares results in auditory studies across a wide range of established rodent models of autism to mimic the wide range of aetiologies in the human population. A search was conducted in the PubMed and Web of Science databases to find primary research articles in mouse or rat models of autism which investigate central auditory processing. A total of 88 studies were included. These used non-invasive measures of auditory function, such as auditory brainstem response recordings, cortical event-related potentials, electroencephalography, and behavioural tests, which are translatable to human studies. They also included invasive measures, such as electrophysiology and histology, which shed insight on the origins of the phenotypes found in the non-invasive studies. The most consistent results across these studies were increased latency of the N1 peak of event-related potentials, decreased power and coherence of gamma activity in the auditory cortex, and increased auditory startle responses to high sound levels. Invasive studies indicated loss of subcortical inhibitory neurons, hyperactivity in the lateral superior olive and auditory thalamus, and reduced specificity of responses in the auditory cortex. This review compares the auditory phenotypes across rodent models and highlights those that mimic findings in human studies, providing a framework and avenues for future studies to inform understanding of the auditory system in autism.

The Role of Zinc and NMDA Receptors in Autism Spectrum Disorders.

NMDA-type glutamate receptors are critical for synaptic plasticity in the central nervous system. Their unique properties and age-dependent arrangement of subunit types underpin their role as a coincidence detector of pre- and postsynaptic activity during brain development and maturation. NMDAR function is highly modulated by zinc, which is co-released with glutamate and concentrates in postsynaptic spines. Both NMDARs and zinc have been strongly linked to autism spectrum disorders (ASDs), suggesting that NMDARs are an important player in the beneficial effects observed with zinc in both animal models and children with ASDs. Significant evidence is emerging that these beneficial effects occur via zinc-dependent regulation of SHANK proteins, which form the backbone of the postsynaptic density. For example, dietary zinc supplementation enhances SHANK2 or SHANK3 synaptic recruitment and rescues NMDAR deficits and hypofunction in Shank3ex13-16-/- and Tbr1+/- ASD mice. Across multiple studies, synaptic changes occur in parallel with a reversal of ASD-associated behaviours, highlighting the zinc-dependent regulation of NMDARs and glutamatergic synapses as therapeutic targets for severe forms of ASDs, either pre- or postnatally. The data from rodent models set a strong foundation for future translational studies in human cells and people affected by ASDs.

Somatostatin interneurons restrict cell recruitment to retinally driven spontaneous activity in the developing cortex.

During early development, before the eyes open, synaptic refinement of sensory networks depends on activity generated by developing neurons themselves. In the mouse visual system, retinal cells spontaneously depolarize and recruit downstream neurons to bursts of activity, where the number of recruited cells determines the resolution of synaptic retinotopic refinement. Here we show that during the second post-natal week in mouse visual cortex, somatostatin (SST)-expressing interneurons control the recruitment of cells to retinally driven spontaneous activity. Suppressing SST interneurons increases cell participation and allows events to spread farther along the cortex. During the same developmental period, a second type of high-participation, retina-independent event occurs. During these events, cells receive such large excitatory charge that inhibition is overwhelmed and large parts of the cortex participate in each burst. These results reveal a role of SST interneurons in restricting retinally driven activity in the visual cortex, which may contribute to the refinement of retinotopy.

Shankopathies in the Developing Brain in Autism Spectrum Disorders.

The SHANK family of proteins play critical structural and functional roles in the postsynaptic density (PSD) at excitatory glutamatergic synapses. Through their multidomain structure they form a structural platform across the PSD for protein-protein interactions, as well as recruiting protein complexes to strengthen excitatory synaptic transmission. Mutations in SHANKs reflect their importance to synapse development and plasticity. This is evident in autism spectrum disorder (ASD), a neurodevelopmental disorder resulting in behavioural changes including repetitive behaviours, lack of sociability, sensory issues, learning, and language impairments. Human genetic studies have revealed ASD mutations commonly occur in SHANKs. Rodent models expressing these mutations display ASD behavioural impairments, and a subset of these deficits are rescued by reintroduction of Shank in adult animals, suggesting that lack of SHANK during key developmental periods can lead to permanent changes in the brain's wiring. Here we explore the differences in synaptic function and plasticity from development onward in rodent Shank ASD models. To date the most explored brain regions, relate to the behavioural changes observed, e.g., the striatum, hippocampus, sensory, and prefrontal cortex. In addition, less-studied regions including the hypothalamus, cerebellum, and peripheral nervous system are also affected. Synaptic phenotypes include weakened but also strengthened synaptic function, with NMDA receptors commonly affected, as well as changes in the balance of excitation and inhibition especially in cortical brain circuits. The effects of shankopathies in activity-dependent brain wiring is an important target for therapeutic intervention. We therefore highlight areas of research consensus and identify remaining questions and challenges.

Disruption of Critical Period Plasticity in a Mouse Model of Neurofibromatosis Type 1.

Neurofibromatosis type 1 (NF1) is a common monogenic neurodevelopmental disorder associated with physical and cognitive problems. The cognitive issues are thought to arise from increased release of the neurotransmitter GABA. Modulating the signaling pathways causing increased GABA release in a mouse model of NF1 reverts deficits in hippocampal learning. However, clinical trials based on these approaches have so far been unsuccessful. We therefore used a combination of slice electrophysiology, in vivo two-photon calcium imaging, and optical imaging of intrinsic signal in a mouse model of NF1 to investigate whether cortical development is affected in NF1, possibly causing lifelong consequences that cannot be rescued by reducing inhibition later in life. We find that, in NF1 mice of both sexes, inhibition increases strongly during the development of the visual cortex and remains high. While this increase in cortical inhibition does not affect spontaneous cortical activity patterns during early cortical development, the critical period for ocular dominance plasticity is shortened in NF1 mice due to its early closure but unaltered onset. Notably, after environmental enrichment, differences in inhibitory innervation and ocular dominance plasticity between NF1 mice and WT littermates disappear. These results provide the first evidence for critical period dysregulation in NF1 and suggest that treatments aimed at normalizing levels of inhibition will need to start at early stages of development.SIGNIFICANCE STATEMENT Neurofibromatosis type 1 is associated with cognitive problems for which no treatment is currently available. This study shows that, in a mouse model of neurofibromatosis type 1, cortical inhibition is increased during development and critical period regulation is disturbed. Rearing the mice in an environment that stimulates cognitive function overcomes these deficits. These results uncover critical period dysregulation as a novel mechanism in the pathogenesis of neurofibromatosis type 1. This suggests that targeting the affected signaling pathways in neurofibromatosis type 1 for the treatment of cognitive disabilities may have to start at a much younger age than has so far been tested in clinical trials.

Hippocampal Deficits in Amyloid-ß-Related Rodent Models of Alzheimer's Disease.

Alzheimer's disease (AD) is a progressive neurodegenerative disease that is the most common cause of dementia. Symptoms of AD include memory loss, disorientation, mood and behavior changes, confusion, unfounded suspicions, and eventually, difficulty speaking, swallowing, and walking. These symptoms are caused by neuronal degeneration and cell loss that begins in the hippocampus, and later in disease progression spreading to the rest of the brain. While there are some medications that alleviate initial symptoms, there are currently no treatments that stop disease progression. Hippocampal deficits in amyloid-ß-related rodent models of AD have revealed synaptic, behavioral and circuit-level defects. These changes in synaptic function, plasticity, neuronal excitability, brain connectivity, and excitation/inhibition imbalance all have profound effects on circuit function, which in turn could exacerbate disease progression. Despite, the wealth of studies on AD pathology we don't yet have a complete understanding of hippocampal deficits in AD. With the increasing development of in vivo recording techniques in awake and freely moving animals, future studies will extend our current knowledge of the mechanisms underpinning how hippocampal function is altered in AD, and aid in progression of treatment strategies that prevent and/or delay AD symptoms.

The cellular and molecular basis of in vivo synaptic plasticity in rodents.

Plasticity within the neuronal networks of the brain underlies the ability to learn and retain new information. The initial discovery of synaptic plasticity occurred by measuring synaptic strength in vivo, applying external stimulation and observing an increase in synaptic strength termed long-term potentiation (LTP). Many of the molecular pathways involved in LTP and other forms of synaptic plasticity were subsequently uncovered in vitro. Over the last few decades, technological advances in recording and imaging in live animals have seen many of these molecular mechanisms confirmed in vivo, including structural changes both pre- and postsynaptically, changes in synaptic strength, and changes in neuronal excitability. A well-studied aspect of neuronal plasticity is the capacity of the brain to adapt to its environment, gained by comparing the brains of deprived and experienced animals in vivo, and in direct response to sensory stimuli. Multiple in vivo studies have also strongly linked plastic changes to memory by interfering with the expression of plasticity and by manipulating memory engrams. Plasticity in vivo also occurs in the absence of any form of external stimulation, i.e., during spontaneous network activity occurring with brain development. However, there is still much to learn about how plasticity is induced during natural learning and how this is altered in neurological disorders.

Spontaneous Activity Patterns Are Altered in the Developing Visual Cortex of the Fmr1 Knockout Mouse.

Fragile X syndrome (FXS) is the most prevalent inherited cause of autism and is accompanied by behavioral and sensory deficits. Errors in the wiring of the brain during early development likely contribute to these deficits, but the underlying mechanisms are unclear. Spontaneous activity patterns, which are required for fine-tuning neuronal networks before the senses become active, are perturbed in rodent models of FXS. Here, we investigated spontaneous network activity patterns in the developing visual cortex of the Fmr1 knockout mouse using in vivo calcium imaging during the second postnatal week, before eye opening. We found that while the frequency, mean amplitude and duration of spontaneous network events were unchanged in the knockout mouse, pair-wise correlations between neurons were increased compared to wild type littermate controls. Further analysis revealed that interneuronal correlations were not generally increased, rather that low-synchronization events occurred relatively less frequently than high-synchronization events. Low-, but not high-, synchronization events have been associated with retinal inputs previously. Since we found that spontaneous retinal waves were normal in the knockout, our results suggest that peripherally driven activity is underrepresented in the Fmr1 KO visual cortex. Therefore, we propose that central gating of retinal inputs may be affected in FXS and that peripherally and centrally driven activity patterns are already unbalanced before eye opening in this disorder.

Spontaneous Activity Drives Local Synaptic Plasticity In Vivo.

Spontaneous activity fine-tunes neuronal connections in the developing brain. To explore the underlying synaptic plasticity mechanisms, we monitored naturally occurring changes in spontaneous activity at individual synapses with whole-cell patch-clamp recordings and simultaneous calcium imaging in the mouse visual cortex in vivo. Analyzing activity changes across large populations of synapses revealed a simple and efficient local plasticity rule: synapses that exhibit low synchronicity with nearby neighbors (<12 µm) become depressed in their transmission frequency. Asynchronous electrical stimulation of individual synapses in hippocampal slices showed that this is due to a decrease in synaptic transmission efficiency. Accordingly, experimentally increasing local synchronicity, by stimulating synapses in response to spontaneous activity at neighboring synapses, stabilized synaptic transmission. Finally, blockade of the high-affinity proBDNF receptor p75(NTR) prevented the depression of asynchronously stimulated synapses. Thus, spontaneous activity drives local synaptic plasticity at individual synapses in an "out-of-sync, lose-your-link" fashion through proBDNF/p75(NTR) signaling to refine neuronal connectivity. VIDEO ABSTRACT.

Autism-associated mutations in ProSAP2/Shank3 impair synaptic transmission and neurexin-neuroligin-mediated transsynaptic signaling.

Mutations in several postsynaptic proteins have recently been implicated in the molecular pathogenesis of autism and autism spectrum disorders (ASDs), including Neuroligins, Neurexins, and members of the ProSAP/Shank family, thereby suggesting that these genetic forms of autism may share common synaptic mechanisms. Initial studies of ASD-associated mutations in ProSAP2/Shank3 support a role for this protein in glutamate receptor function and spine morphology, but these synaptic phenotypes are not universally penetrant, indicating that other core facets of ProSAP2/Shank3 function must underlie synaptic deficits in patients with ASDs. In the present study, we have examined whether the ability of ProSAP2/Shank3 to interact with the cytoplasmic tail of Neuroligins functions to coordinate pre/postsynaptic signaling through the Neurexin-Neuroligin signaling complex in hippocampal neurons of Rattus norvegicus. Indeed, we find that synaptic levels of ProSAP2/Shank3 regulate AMPA and NMDA receptor-mediated synaptic transmission and induce widespread changes in the levels of presynaptic and postsynaptic proteins via Neurexin-Neuroligin transsynaptic signaling. ASD-associated mutations in ProSAP2/Shank3 disrupt not only postsynaptic AMPA and NMDA receptor signaling but also interfere with the ability of ProSAP2/Shank3 to signal across the synapse to alter presynaptic structure and function. These data indicate that ASD-associated mutations in a subset of synaptic proteins may target core cellular pathways that coordinate the functional matching and maturation of excitatory synapses in the CNS.

4D super-resolution microscopy with conventional fluorophores and single wavelength excitation in optically thick cells and tissues.

BACKGROUND: Optical super-resolution imaging of fluorescently stained biological samples is rapidly becoming an important tool to investigate protein distribution at the molecular scale. It is therefore important to develop practical super-resolution methods that allow capturing the full three-dimensional nature of biological systems and also can visualize multiple protein species in the same sample. METHODOLOGY/PRINCIPAL FINDINGS: We show that the use of a combination of conventional near-infrared dyes, such as Alexa 647, Alexa 680 and Alexa 750, all excited with a 671 nm diode laser, enables 3D multi-colour super-resolution imaging of complex biological samples. Optically thick samples, including human tissue sections, cardiac rat myocytes and densely grown neuronal cultures were imaged with lateral resolutions of ∼15 nm (std. dev.) while reducing marker cross-talk to <1%. Using astigmatism an axial resolution of ∼65 nm (std. dev.) was routinely achieved. The number of marker species that can be distinguished depends on the mean photon number of single molecule events. With the typical photon yields from Alexa 680 of ∼2000 up to 5 markers may in principle be resolved with <2% crosstalk. CONCLUSIONS/SIGNIFICANCE: Our approach is based entirely on the use of conventional, commercially available markers and requires only a single laser. It provides a very straightforward way to investigate biological samples at the nanometre scale and should help establish practical 4D super-resolution microscopy as a routine research tool in many laboratories.

Synaptic integration of newly generated neurons in rat dissociated hippocampal cultures.

In the dentate gyrus of the hippocampus new neurons are born from precursor cells throughout development and into adulthood. These newborn neurons hold significant potential for self-repair of brain damage caused by neurodegenerative disease. However, the mechanism by which newborn neurons integrate into the brain is not understood due to a lack of knowledge of the molecular and functional characteristics of the synapses formed by newborn neurons. Here we report that dissociated hippocampal cultures continue to produce new granule cells in vitro that fire action potentials and become synaptically integrated into the existing network of mature hippocampal neurons. Quantification of the expression of synaptic proteins at newborn and mature granule cell synapses revealed synapse development onto newborn neurons occurs sequentially with initial synaptic contacts evident from 6 days after cell birth. These data also showed that the dendrites of newborn neurons have a high density of Piccolo and Bassoon puncta on them and therefore have a high potential to be integrated into the neuronal network through new synaptic connections. Electrophysiological recordings from newborn neurons reveal these synapses are functional within 10 days of cell birth. GABAergic input synapses were found to mature faster in newborn neurons than glutamatergic synapses where sequential recruitment of postsynaptic glutamate receptors occurred. Group I metabotropic glutamate receptors (mGluR1/5) were present at higher levels compared with ionotropic glutamate receptors (NMDA and AMPA receptors), suggesting that metabotropic and ionotropic receptors play differential roles at glutamatergic synapses in the integration and the maturation of newborn neurons. These data show that dissociated hippocampal cultures can provide a useful model system in which to study the integration of newborn neurons into existing neuronal circuits to increase our understanding of how the function of newborn neuron synapses could contribute to restoring damaged neuronal networks.

Synaptic SAP97 isoforms regulate AMPA receptor dynamics and access to presynaptic glutamate.

The synaptic insertion of GluR1-containing AMPA-type glutamate receptors (AMPARs) is critical for synaptic plasticity. However, mechanisms responsible for GluR1 insertion and retention at the synapse are unclear. The synapse-associated protein SAP97 directly binds GluR1 and participates in its forward trafficking from the Golgi network to the plasma membrane. Whether SAP97 also plays a role in scaffolding GluR1 at the postsynaptic membrane is controversial, attributable to its expression as a collection of alternatively spliced isoforms with ill-defined spatial and temporal distributions. In the present study, we have used live imaging and electrophysiology to demonstrate that two postsynaptic, N-terminal isoforms of SAP97 directly modulate the levels, dynamics, and function of synaptic GluR1-containing AMPARs. Specifically, the unique N-terminal domains confer distinct subsynaptic localizations onto SAP97, targeting the palmitoylated alpha-isoform to the postsynaptic density (PSD) and the L27 domain-containing beta-isoform primarily to non-PSD, perisynaptic regions. Consequently, alpha- and betaSAP97 differentially influence the subsynaptic localization and dynamics of AMPARs by creating binding sites for GluR1-containing receptors within their respective subdomains. These results indicate that N-terminal splicing of SAP97 can control synaptic strength by regulating the distribution of AMPARs and, hence, their responsiveness to presynaptically released glutamate.

Plasticity-dependent changes in metabotropic glutamate receptor expression at excitatory hippocampal synapses.

Group I metabotropic glutamate receptors, mGluR1 and mGluR5, modulate NMDA receptor-mediated synaptic transmission and plasticity and mediate mGluR-dependent plasticity. Here we report that the synaptic expression of mGluRs can be regulated by NMDA receptor-dependent synaptic plasticity, but that this is dependent on the subtype of mGluR. Silent synapses, but not active synapses, were found to lack Group I mGluRs showing that mGluRs must be inserted into synapses after they are unsilenced. The induction of LTP resulted in an increased synaptic expression of mGluR1 in an NMDA receptor-dependent manner. mGluR1 is internalized from synapses via NMDA receptor-dependent LTD. Interestingly we found no evidence for the regulation of mGluR5 by NMDA receptor-dependent plasticity. This regulation of Group I mGluRs will determine the ability of synapses to undergo mGluR-dependent modulation of synaptic transmission and plasticity, providing a mechanism for metaplasticity and state-dependent plasticity at hippocampal synapses.