3D printable device for automated operant conditioning in the mouse

Operant conditioning is a classical paradigm and a standard technique used in experimental psychology in which animals learn to perform an action in order to achieve a reward. By using this paradigm, it is possible to extract learning curves and measure accurately reaction times. Both these measurements are proxy of cognitive capabilities and can be used to evaluate the effectiveness of therapeutic interventions in mouse models of disease. Here we describe a fully 3D printable device that is able to perform operant conditioning on freely moving mice, while performing real-time tracking of the animal position. We successfully trained 6 mice, showing stereotyped learning curves that are highly reproducible across mice and reaching more than 70% of accuracy after two days of conditioning. Different products for operant conditioning are commercially available, though most of them do not provide customizable features and are relatively expensive. This data demonstrate that this system is a valuable alternative to available state-of-the-art commercial devices, representing a good balance between performance, cost, and versatility in its use.Significance Statement 3D printing is a revolutionary technology that combines cost-effectiveness with an optimal trade off between standardization and customization. Here we show a device that performs operant conditioning in mice using largely 3D printed parts. This tool can be employed to test learning and memory in models of disease. We expect that the open design of the chamber will be useful for scientific teaching and research as well as for further improvements from the open hardware community

{MEYE}: Web-app for translational and real-time pupillometry

Pupil dynamics alterations have been found in patients affected by a variety of neuropsychiatric conditions, in- cluding autism. Studies in mouse models have used pupillometry for phenotypic assessment and as a proxy for arousal. Both in mice and humans, pupillometry is noninvasive and allows for longitudinal experiments sup- porting temporal specificity; however, its measure requires dedicated setups. Here, we introduce a convolu- tional neural network that performs online pupillometry in both mice and humans in a web app format. This solution dramatically simplifies the usage of the tool for the nonspecialist and nontechnical operators. Because a modern web browser is the only software requirement, this choice is of great interest given its easy deployment and setup time reduction. The tested model performances indicate that the tool is sensitive enough to detect both locomotor-induced and stimulus-evoked pupillary changes, and its output is compara- ble to state-of-the-art commercial devicesPupil dynamics alterations have been found in patients affected by a variety of neuropsychiatric conditions, including autism. Studies in mouse models have used pupillometry for phenotypic assessment and as a proxy for arousal. Both in mice and humans, pupillometry is noninvasive and allows for longitudinal experiments supporting temporal specificity; however, its measure requires dedicated setups. Here, we introduce a convolutional neural network that performs online pupillometry in both mice and humans in a web app format. This solution dramatically simplifies the usage of the tool for the nonspecialist and nontechnical operators. Because a modern web browser is the only software requirement, this choice is of great interest given its easy deployment and setup time reduction. The tested model performances indicate that the tool is sensitive enough to detect both locomotor-induced and stimulus-evoked pupillary changes, and its output is comparable to state-of-the-art commercial devices

Novel translational phenotypes and biomarkers for creatine transporter deficiency

Abstract Creatine transporter deficiency is a metabolic disorder characterized by intellectual disability, autistic-like behaviour and epilepsy. There is currently no cure for creatine transporter deficiency, and reliable biomarkers of translational value for monitoring disease progression and response to therapeutics are sorely lacking. Here, we found that mice lacking functional creatine transporter display a significant alteration of neural oscillations in the EEG and a severe epileptic phenotype that are recapitulated in patients with creatine transporter deficiency. In-depth examination of knockout mice for creatine transporter also revealed that a decrease in EEG theta power is predictive of the manifestation of spontaneous seizures, a frequency that is similarly affected in patients compared to healthy controls. In addition, knockout mice have a highly specific increase in haemodynamic responses in the cerebral cortex following sensory stimuli. Principal component and Random Forest analyses highlighted that these functional variables exhibit a high performance in discriminating between pathological and healthy phenotype. Overall, our findings identify novel, translational and non-invasive biomarkers for the analysis of brain function in creatine transporter deficiency, providing a very reliable protocol to longitudinally monitor the efficacy of potential therapeutic strategies in preclinical, and possibly clinical, studies

A Comprehensive Atlas of Perineuronal Net Distribution and Colocalization with Parvalbumin in the Adult Mouse Brain

Perineuronal nets (PNNs) surround specific neurons in the brain and are involved in various forms of plasticity and clinical conditions. However, our understanding of the PNN role in these phenomena is limited by the lack of highly quantitative maps of PNN distribution and association with specific cell types. Here, we present a comprehensive atlas of Wisteria Floribunda Agglutinin (WFA) positive PNNs and colocalization with parvalbumin (PV) cells for over 600 regions of the adult mouse brain. Data analysis shows that PV expression is a good predictor of PNN aggregation. In the cortex, PNNs are dramatically enriched in layer 4 of all primary sensory areas in correlation with thalamocortical input density, and their distribution mirrors intracortical connectivity patterns. Gene expression analysis identifies many PNN correlated genes. Strikingly, PNN anticorrelated transcripts are enriched in synaptic plasticity genes, generalizing PNN role as circuit stability factors

Cyclocreatine treatment ameliorates the cognitive, autistic and epileptic phenotype in a mouse model of Creatine Transporter Deficiency

Creatine Transporter Deficiency (CTD) is an inborn error of metabolism presenting with intellectual disability, behavioral disturbances and epilepsy. There is currently no cure for this disorder. Here, we employed novel biomarkers for monitoring brain function, together with well-established behavioral readouts for CTD mice, to longitudinally study the therapeutic efficacy of cyclocreatine (cCr) at the preclinical level. Our results show that cCr treatment is able to partially correct hemodynamic responses and EEG abnormalities, improve cognitive deficits, revert autistic-like behaviors and protect against seizures. This study provides encouraging data to support the potential therapeutic benefit of cyclocreatine or other chemically modified lipophilic analogs of Cr

mGluR5 PAMs rescue cortical and behavioural defects in a mouse model of CDKL5 deficiency disorder

Cyclin-dependent kinase-like 5 (CDKL5) deficiency disorder (CDD) is a devastating rare neurodevelopmental disease without a cure, caused by mutations of the serine/threonine kinase CDKL5 highly expressed in the forebrain. CDD is characterized by early-onset seizures, severe intellectual disabilities, autistic-like traits, sensorimotor and cortical visual impairments (CVI). The lack of an effective therapeutic strategy for CDD urgently demands the identification of novel druggable targets potentially relevant for CDD pathophysiology. To this aim, we studied Class I metabotropic glutamate receptors 5 (mGluR5) because of their important role in the neuropathological signs produced by the lack of CDKL5 in-vivo, such as defective synaptogenesis, dendritic spines formation/maturation, synaptic transmission and plasticity. Importantly, mGluR5 function strictly depends on the correct expression of the postsynaptic protein Homer1bc that we previously found atypical in the cerebral cortex of Cdkl5-/y mice. In this study, we reveal that CDKL5 loss tampers with (i) the binding strength of Homer1bc-mGluR5 complexes, (ii) the synaptic localization of mGluR5 and (iii) the mGluR5-mediated enhancement of NMDA-induced neuronal responses. Importantly, we showed that the stimulation of mGluR5 activity by administering in mice specific positive-allosteric-modulators (PAMs), i.e., 3-Cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide (CDPPB) or RO6807794, corrected the synaptic, functional and behavioral defects shown by Cdkl5-/y mice. Notably, in the visual cortex of 2 CDD patients we found changes in synaptic organization that recapitulate those of mutant CDKL5 mice, including the reduced expression of mGluR5, suggesting that these receptors represent a promising therapeutic target for CDD

Expression of a Secretable, Cell-Penetrating CDKL5 Protein Enhances the Efficacy of Gene Therapy for CDKL5 Deficiency Disorder

Although delivery of a wild-type copy of the mutated gene to cells represents the most effective approach for a monogenic disease, proof-of-concept studies highlight significant efficacy caveats for treatment of brain disorders. Herein, we develop a cross-correction-based strategy to enhance the efficiency of a gene therapy for CDKL5 deficiency disorder, a severe neurodevelopmental disorder caused by CDKL5 gene mutations. We created a gene therapy vector that produces an Igk-TATk-CDKL5 fusion protein that can be secreted via constitutive secretory pathways and, due to the cell-penetration property of the TATk peptide, internalized by cells. We found that, although AAVPHP.B_Igk-TATk-CDKL5 and AAVPHP.B_CDKL5 vectors had similar brain infection efficiency, the AAVPHP.B_Igk-TATk-CDKL5 vector led to higher CDKL5 protein replacement due to secretion and penetration of the TATk-CDKL5 protein into the neighboring cells. Importantly, Cdkl5 KO mice treated with the AAVPHP.B_Igk-TATk-CDKL5 vector showed a behavioral and neuroanatomical improvement in comparison with vehicle or AAVPHP.B_CDKL5 vector-treated Cdkl5 KO mice. In conclusion, we provide the first evidence that a gene therapy based on a cross-correction approach is more effective at compensating Cdkl5-null brain defects than gene therapy based on the expression of the native CDKL5, opening avenues for the development of this innovative approach for other monogenic diseases

Cell-specific vulnerability to metabolic failure: the crucial role of parvalbumin expressing neurons in creatine transporter deficiency

Mutations in the solute carrier family 6-member 8 (Slc6a8) gene, encoding the protein responsible for cellular creatine (Cr) uptake, cause Creatine Transporter Deficiency (CTD), an X-linked neurometabolic disorder presenting with intellectual disability, autistic-like features, and epilepsy. The pathological determinants of CTD are still poorly understood, hindering the development of therapies. In this study, we generated an extensive transcriptomic profile of CTD showing that Cr deficiency causes perturbations of gene expression in excitatory neurons, inhibitory cells, and oligodendrocytes which result in remodeling of circuit excitability and synaptic wiring. We also identified specific alterations of parvalbumin-expressing (PV+) interneurons, exhibiting a reduction in cellular and synaptic density, and a hypofunctional electrophysiological phenotype. Mice lacking Slc6a8 only in PV+ interneurons recapitulated numerous CTD features, including cognitive deterioration, impaired cortical processing and hyperexcitability of brain circuits, demonstrating that Cr deficit in PV+ interneurons is sufficient to determine the neurological phenotype of CTD. Moreover, a pharmacological treatment targeted to restore the efficiency of PV+ synapses significantly improved cortical activity in Slc6a8 knock-out animals. Altogether, these data demonstrate that Slc6a8 is critical for the normal function of PV+ interneurons and that impairment of these cells is central in the disease pathogenesis, suggesting a novel therapeutic venue for CTD.This work has been supported by grant GR-2017–02364378 funded by the Italian Ministry of Health and by Telethon grant GGP19177 to LB; Italian Ministry of Health, RC 2021; grant from Fondazione Cassa di Risparmio di Firenze “Human Brain Optical Mapping” to TP; grants from the Spanish Ministry of Science and Innovation (MICINN) co-financed by ERDF (grant no. RTI2018-102260-B-I00; Generalitat Valenciana, project no. PROMETEO/2020/007; and CSIC Interdisciplinary Thematic Platform (PTI +) NEURO-AGINGl + (PTI-NEURO-AGING +). C.M.N-I. was the recipient of a FPI fellowship from the MICINN. The Instituto de Neurociencias (UMH-CSIC) is a “Centre of Excellence Severo Ochoa” (grant no. SEV-2017–0723).Peer reviewe

Neuroplasticity in the adult visual cortex: role of Semaphorin 3A aggregation by perineuronal nets and development of a custom apparatus for longitudinal evaluation of visual cortical plasticity using Intrinsic Signal Optical Imaging

During development, the brain goes through defined temporal windows of enhanced plasticity called Critical Periods. One of the hallmarks of the closure of these phases is the activity-dependent organization of the extra-cellular matrix (ECM) in densely organized structures called peri-neuronal nets (PNNs). The PNNs are composed of extracellular matrix molecules, including hyaluronic acid, link proteins (e.g., Crtl1) and chondroitin sulfate proteoglycans (CSPGs) and they shroud preferentially cortical parvalbumin positive (PV+) fast spiking interneurons, a subclass of inhibitory GABAergic neurons. The causal relationship between the organization of the ECM in these structures and the restriction of plasticity is emerging as very clear from a wide set of experimental evidences in both mice and rats. For example, mice lacking Crtl1, a key component for PNN stability, retain cortical plasticity into adulthood; furthermore, strikingly, the enzymatic digestion of CSPGs though intracortical injections of Chondroitinase ABC reactivates plasticity even in adult rats. Despite this growing set of data addressing the role of PNNs in neuroplasticity, it is still unclear which are the precise molecular mechanisms mediating their restrictive role towards plasticity. Semaphorin 3A (Sema3A) is a secreted protein that exhibits a chemorepulsive role for axonal growth. Recently it has been showed that Sema3A has a high biochemical affinity towards different classes of CSPGs (e.g., aggrecan, versican) and in histological preparations, it is strongly enriched around PNN-positive neurons. Intriguingly, both in mice lacking the link protein Crtl1 and in rats intracortically injected with Chondroitinase ABC, Sema3A does not concentrate around neurons and remains diffuse, suggesting that the inhibitory function of PNNs could be due to their competence in binding and accumulating repulsive soluble proteins present in ECM and presenting them in high concentrations to the neurons they enfold. In order to address this hypothesis, we interfered with the extracellular activity of Sema3A through the injection, in the visual cortex of adult rats, of adeno-associated viruses (AAV) that drive the expression of the “receptor body” neuropilin1-Fc. This protein contains a binding domain for Sema3A, but is soluble and present in the extracellular space, thus being unable to activate the intracellular signaling pathway that Sema3a would initiate. As a result, the receptor body competes with the transmembrane receptor for the binding of Sema3A and strongly reduces the relevance of its signaling pathway thanks to the strong concentration imbalance favoring the soluble and inactive form. After 3 weeks from the injection we tested whether the simple inhibition of Sema3A reactivates plasticity in adult rats by using the classical experimental paradigm of Monocular Deprivation (MD) to elicit plastic phenomena in the visual cortex. Normally, the binocular part of the rat’s visual cortex receives inputs from both eyes, but is driven more strongly by the contralateral eye, a property called Ocular Dominance (OD; i.e., the dominance of the contralateral inputs on the ipsilateral ones). By perturbing the cortex through the suture of the contralateral eye, and thus reducing the amount of information flowing through the dominant pathway, a plastic process called Ocular Dominance Shift can be elicited. The cortex responds to the manipulation favoring the inputs from the open, ipsilateral eye and/or progressively ignoring the silent contralateral eye. This phenomena is possible if and only if the cortex is still plastic (e.g., in young animals, but not in adult ones) and thus can be used to precisely assess the presence of plasticity in the visual cortex. After 7 days of monocular deprivation, we used in-vivo electrophysiology to measure the amplitude of visually evoked potentials (VEPs), low frequencies potentials that are generated in the visual cortex in response of a stimulus. By comparing the amplitude of VEPs generated by the stimulation of the ipsilateral and the contralateral eye, we showed that the inhibition of Sema3A pathway reactivates ocular dominance plasticity in the adult rat visual cortex. To better understand the relationship between Sema3A and PNNs, we examined their localization by immunohistochemical (IHC) staining in slices of the visual cortex of dark reared rats. Dark rearing is a condition known to dramatically delay the formation of PNNs and to prolong the Critical Period for Ocular Dominance Plasticity. We found that Sema3A positive cells are significantly rarer in dark reared animals, supporting the hypothesis that PNNs are essential to bind and accumulate soluble molecules to create local spots of high concentration. Even if interfering with the function of Semaphorin-3A can reactivate Ocular Dominance Plasticity in adult animals, it is still unknown whether the shift in Ocular dominance observed following MD is due to a decrease of the responses from the contralateral eye, an increase of the responses from the ipsilateral eye, or both. The importance of these data resides in the fact that these two types of plasticity are well studied in young animals and are known to be based on different physiological mechanisms. Understanding how Sema3A regulates neuroplasticity is therefore important to comprehend whether the disruption of its signaling pathway engages one, the other, or both types of plastic changes. Unfortunately, this kind of data is quite hard to obtain because it requires longitudinal measures of cortical responses in the same animal, while in our lab, VEPs recording is mainly an acute procedure thus yielding only cross-sectional measures. In order to be able to address this important question, we developed both the hardware and the software of a custom apparatus for Intrinsic Signal Optical Imaging, and we performed several steps of testing, with the goal of being able to record cortical evoked responses chronically and with extremely low invasiveness. “Intrinsic signals” are optical signals that can be used to measure neural cortical activity. They are characterized by a relatively slow (i.e., with a time constant in the order of seconds) decrease in light reflectance that is more evident at 630nm. They are generated in response of neural activity by a combination of a local increase in blood volume due to neurovascular coupling and by an increase in the proportion of deoxygenated Hemoglobin, which has slightly different optical properties from the oxygenated form. Importantly, given their physiological nature, these signals are detectable in naïve animals without using any external dye or genetically encoded indicator, so they are great candidate for low-invasiveness studies. The hardware for imaging is composed by a standard optical microscope, a low-noise camera (CMOS sensor), and a custom imaging chamber to accommodate the animal during the procedure. The imaging chamber has been designed and 3D printed specifically to accommodate two automated eye shutters, and a heating pad, all remotely controlled by an Arduino microcontroller. The software has been written in MATLAB and comprises scripts to control a “stimulator” computer that generates the visual stimuli and a “recording” computer that acquires images from the camera. In addition, we developed specific algorithm and Graphical User Interfaces (GUIs) for further data processing. Specifically, we implemented algorithms for image visualization and filtering, timeline visualization, movie visualization, artifact rejection and comparison of ipsilateral and contralateral recordings. Our goal was to use this setup for chronic imaging in adult animals to understand the physiological effects of interfering with the ECM, with the least possible amount of invasiveness, potentially through the intact skull. We performed several steps of testing to validate our apparatus: first, we addressed the possibility to record through the intact skull in young mice; second, we were able to detect the shift in Ocular Dominance produced by MD in young mice; third, we showed the feasibility of chronic recordings through the intact skull in adult mice, a challenging procedure, given the thickness and opacity of the adult skull. In the future, we plan to extend this technique also in rats to have a wider spectrum of possibility of investigation, and to better elucidate how the ECM regulates plasticity