Activation of trace amine-associated receptor 1 (TAAR1) transiently reduces alcohol drinking in socially housed mice.

Alcohol dependence is characterized by the abnormal release of dopamine in the brain reward-related areas. Trace amine-associated receptor 1 (TAAR1) is a G protein-coupled receptor that negatively regulates dopamine neurotransmission and thus is a promising target in the treatment of drug addiction. However, the role of TAAR1 in the regulation of alcohol abuse remains understudied. Here, we assessed the effect of TAAR1 activation on alcohol drinking behaviours of C57Bl/6J female mice housed in IntelliCages. The animals were administered with either vehicle or TAAR1 full selective agonist, RO5256390, and tested for alcohol consumption, alcohol preference and motivation for alcohol seeking. We found that mice with the highest preference for alcohol (high drinkers) in the RO5256390 group consumed less alcohol and had lower alcohol preference in comparison with high drinkers in the vehicle group, during 20 h of free alcohol access (FAA). We also found decreased alcohol consumption and alcohol preference comparing all animals in the RO5256390 to all animals in the vehicle group, during 20 h of FAA performed after the abstinence. These effects of RO5256390 lasted for the first 24 h after administration that roughly corresponded to the compound level in the brain, measured by mass spectrometry. Finally, we found that administration of RO5256390 may attenuate motivation for alcohol seeking. Taken together, our findings reveal that activation of TAAR1 may transiently reduce alcohol drinking; thus, TAAR1 is a promising target for the treatment of alcohol abuse and relapse.

Global brain c-Fos profiling reveals major functional brain networks rearrangements after alcohol reexposure.

Many fundamental questions on alcohol use disorder (AUD) are frequently difficult to address by examining a single brain structure, but should be viewed from the whole brain perspective. c-Fos is a marker of neuronal activation. Global brain c-Fos profiling in rodents represents a promising platform to study brain functional networks rearrangements in AUD. We used a mouse model of alcohol drinking in IntelliCage. We trained mice to voluntarily drink alcohol, next subjected them to withdrawal and alcohol reexposure. We have developed a dedicated image computational workflow to identify c-Fos-positive cells in three-dimensional images obtained after whole-brain optical clearing and imaging in the light-sheet microscope. We provide a complete list of 169 brain structures with annotated c-Fos expression. We analyzed functional networks, brain modularity and engram index. Brain c-Fos levels in animals reexposed to alcohol were different from both control and binge drinking animals. Structures involved in reward processing, decision making and characteristic for addictive behaviors, such as precommissural nucleus, nucleus Raphe, parts of colliculus and tecta stood out particularly. Alcohol reexposure leads to a massive change of brain modularity including a formation of numerous smaller functional modules grouping structures involved in addiction development. Binge drinking can lead to substantial functional remodeling in the brain. We provide a list of structures that can be used as a target in pharmacotherapy but also point to the networks and modules that can hold therapeutic potential demonstrated by a clinical trial in patients.

Experimental optimization of lensless digital holographic microscopy with rotating diffuser-based coherent noise reduction.

Laser-based lensless digital holographic microscopy (LDHM) is often spoiled by considerable coherent noise factor. We propose a novel LDHM method with significantly limited coherent artifacts, e.g., speckle noise and parasitic interference fringes. It is achieved by incorporating a rotating diffuser, which introduces partial spatial coherence and preserves high temporal coherence of laser light, crucial for credible in-line hologram reconstruction. We present the first implementation of the classical rotating diffuser concept in LDHM, significantly increasing the signal-to-noise ratio while preserving the straightforwardness and compactness of the LDHM imaging device. Prior to the introduction of the rotating diffusor, we performed LDHM experimental hardware optimization employing 4 light sources, 4 cameras, and 3 different optical magnifications (camera-sample distances). It was guided by the quantitative assessment of numerical amplitude/phase reconstruction of test targets, conducted upon standard deviation calculation (noise factor quantification), and resolution evaluation (information throughput quantification). Optimized rotating diffuser LDHM (RD-LDHM) method was successfully corroborated in technical test target imaging and examination of challenging biomedical sample (60 µm thick mouse brain tissue slice). Physical minimization of coherent noise (up to 50%) was positively verified, while preserving optimal spatial resolution of phase and amplitude imaging. Coherent noise removal, ensured by proposed RD-LDHM method, is especially important in biomedical inference, as speckles can falsely imitate valid biological features. Combining this favorable outcome with large field-of-view imaging can promote the use of reported RD-LDHM technique in high-throughput stain-free biomedical screening.

Comparison of Muscle Regeneration after BMSC-Conditioned Medium, Syngeneic, or Allogeneic BMSC Injection.

For many years optimal treatment for dysfunctional skeletal muscle characterized, for example, by impaired or limited regeneration, has been searched. Among the crucial factors enabling its development is finding the appropriate source of cells, which could participate in tissue reconstruction or serve as an immunomodulating agent (limiting immune response as well as fibrosis, that is, connective tissue formation), after transplantation to regenerating muscles. MSCs, including those derived from bone marrow, are considered for such applications in terms of their immunomodulatory properties, as their naive myogenic potential is rather limited. Injection of autologous (syngeneic) or allogeneic BMSCs has been or is currently being tested and compared in many potential clinical treatments. In the present study, we verified which approach, that is, the transplantation of either syngeneic or allogeneic BMSCs or the injection of BMSC-conditioned medium, would be the most beneficial for skeletal muscle regeneration. To properly assess the influence of the tested treatments on the inflammation, the experiments were carried out using immunocompetent mice, which allowed us to observe immune response. Combined analysis of muscle histology, immune cell infiltration, and levels of selected chemokines, cytokines, and growth factors important for muscle regeneration, showed that muscle injection with BMSC-conditioned medium is the most beneficial strategy, as it resulted in reduced inflammation and fibrosis development, together with enhanced new fiber formation, which may be related to, i.e., elevated level of IGF-1. In contrast, transplantation of allogeneic BMSCs to injured muscles resulted in a visible increase in the immune response, which hindered regeneration by promoting connective tissue formation. In comparison, syngeneic BMSC injection, although not detrimental to muscle regeneration, did not result in such significant improvement as CM injection.

The IntelliCage System: A Review of Its Utility as a Novel Behavioral Platform for a Rodent Model of Substance Use Disorder.

The use of animal models for substance use disorder (SUD) has made an important contribution in the investigation of the behavioral and molecular mechanisms underlying substance abuse and addiction. Here, we review a novel and comprehensive behavioral platform to characterize addiction-like traits in rodents using a fully automated learning system, the IntelliCage. This system simultaneously captures the basic behavioral navigation, reward preference, and aversion, as well as the multi-dimensional complex behaviors and cognitive functions of group-housed rodents. It can reliably capture and track locomotor and cognitive pattern alterations associated with the development of substance addiction. Thus, the IntelliCage learning system offers a potentially efficient, flexible, and sensitive tool for the high-throughput screening of the rodent SUD model.

The matrix metalloproteinase inhibitor marimastat inhibits seizures in a model of kainic acid-induced status epilepticus.

An intra-hippocampus injection of kainic acid serves as a model of status epilepticus and the subsequent development of temporal lobe epilepsy. Matrix metalloproteinase-9 (MMP-9) is an enzyme that controls remodeling of the extracellular milieu under physiological and pathological conditions. In response to brain insult, MMP-9 contributes to pathological synaptic plasticity that may play a role in the progression of an epileptic condition. Marimastat is a metalloproteinase inhibitor that was tested in clinical trials of cancer. The present study assessed whether marimastat can impair the development of epilepsy. The inhibitory efficacy of marimastat was initially tested in neuronal cultures in vitro. As a marker substrate, we used nectin-3. Next, we investigated the blood-brain barrier penetration of marimastat using mass spectrometry and evaluated the therapeutic potential of marimastat against seizure outcomes. We found that marimastat inhibited the cleavage of nectin-3 in hippocampal neuronal cell cultures. Marimastat penetrated the blood-brain barrier and exerted an inhibitory effect on metalloproteinase activity in the brain. Finally, marimastat decreased some seizure parameters, such as seizure score and number, but did not directly affect status epilepticus. The long-term effects of marimastat were evident up to 6 weeks after kainic acid administration, in which marimastat still inhibited seizure duration.

Tissue clearing-based method for unobstructed three-dimensional imaging of mouse penis with subcellular resolution.

Although mice are widely used to elucidate factors contributing to penile disorders and develop treatment options, quantification of tissue changes upon intervention is either limited to minuscule tissue volume (histology) or acquired with limited spatial resolution (MRI/CT). Thus, imaging method suitable for expeditious acquisition of the entire mouse penis with subcellular resolution is described that relies on both aqueous- (clear, unobstructed brain imaging cocktails and computational analysis) and solvent-based (fluorescence-preserving capability imaging of solvent-cleared organs) tissue optical clearing (TOC). The combined TOC approach allows to image mouse penis innervation and vasculature with unprecedented detail and, for the first time, reveals the three-dimensional structure of murine penis fibrocartilage.

Systematic Evaluation of Chemically Distinct Tissue Optical Clearing Techniques in Murine Lymph Nodes.

Activation of adaptive immunity is a complex process coordinated at multiple levels in both time and the three-dimensional context of reactive lymph nodes (LNs). Although microscopy-based visualization of its spatiotemporal dynamics unravels complexities of developing immune response, such approach is highly limited by light-obstructing nature of tissue components. Recently, tissue optical clearing (TOC) techniques were established to bypass this obstacle and now allow to image and quantify the entire murine organs with cellular resolution. However, the spectrum of TOC is represented by wide variety of chemically distinct methods, each having certain advantages and disadvantages that were unsatisfactorily compared for suitability to LNs clearing. In this study, we have systematically tested 13 typical TOC techniques and assessed their impact on a number of critical factors such as LN transparency, imaging depth, change in size, compatibility with proteinaceous fluorophores, immunostaining, H&E staining, and light-sheet fluorescence microscopy. Based on the detailed data specific to TOC process of murine LNs, we provide a reliable reference for most suitable methods in an application-dependent manner.

Psychosocial Stress Induces Schizophrenia-Like Behavior in Mice With Reduced MMP-9 Activity.

Understanding gene-environment interactions in the pathogenesis of schizophrenia remains a major research challenge. Matrix metalloproteinase-9 (MMP-9) has been previously implicated in the pathophysiology of schizophrenia. In the present study, adolescent Mmp-9 heterozygous mice, with a genetically lower level of MMP-9, were subjected to resident-intruder psychosocial stress for 3 weeks and then examined in behavioral tests that evaluated cognitive deficits and positive- and negative-like symptoms of schizophrenia. Cognitive and positive symptoms in unstressed Mmp-9 heterozygous mice were unaffected by stress exposure, whereas negative symptoms were manifested only after stress exposure. Interestingly, negative symptoms were ameliorated by treatment with the antipsychotic drug clozapine. We describe a novel gene × environment interaction mouse model of schizophrenia. Lower MMP-9 levels in the brain might be a risk factor for schizophrenia that, in combination with environmental factors (e.g., psychosocial stress), may evoke schizophrenia-like symptoms that are sensitive to antipsychotic treatment.

MMPs in learning and memory and neuropsychiatric disorders.

Matrix metalloproteinases (MMPs) are a group of over twenty proteases, operating chiefly extracellularly to cleave components of the extracellular matrix, cell adhesion molecules as well as cytokines and growth factors. By virtue of their expression and activity patterns in animal models and clinical investigations, as well as functional studies with gene knockouts and enzyme inhibitors, MMPs have been demonstrated to play a paramount role in many physiological and pathological processes in the brain. In particular, they have been shown to influence learning and memory processes, as well as major neuropsychiatric disorders such as schizophrenia, various kinds of addiction, epilepsy, fragile X syndrome, and depression. A possible link connecting all those conditions is either physiological or aberrant synaptic plasticity where some MMPs, e.g., MMP-9, have been demonstrated to contribute to the structural and functional reorganization of excitatory synapses that are located on dendritic spines. Another common theme linking the aforementioned pathological conditions is neuroinflammation and MMPs have also been shown to be important mediators of immune responses.

MMP-9 Contributes to Dendritic Spine Remodeling Following Traumatic Brain Injury.

Traumatic brain injury (TBI) occurs when a blow to the head causes brain damage. Apart from physical trauma, it causes a wide range of cognitive, behavioral, and emotional deficits including impairments in learning and memory. On neuronal level, TBI may lead to circuitry remodeling and in effect imbalance between excitatory and inhibitory neurotransmissions. Such change in brain homeostasis may often lead to brain disorders. The basic units of neuronal connectivity are dendritic spines that are tiny protrusions forming synapses between two cells in a network. Spines are dynamic structures that undergo morphological transformation throughout life. Their shape is strictly related to an on/off state of synapse and the strength of synaptic transmission. Matrix metalloproteinase-9 (MMP-9) is an extrasynaptically operating enzyme that plays a role in spine remodeling and has been reported to be activated upon TBI. The aim of the present study was to evaluate the influence of MMP-9 on dendritic spine density and morphology following controlled cortical impact (CCI) as animal model of TBI. We examined spine density and dendritic spine shape in the cerebral cortex and the hippocampus. CCI caused a marked decrease in spine density as well as spine shrinkage in the cerebral cortex ipsilateral to the injury, when compared to sham animals and contralateral side both 1 day and 1 week after the insult. Decreased spine density was also observed in the dentate gyrus of the hippocampus; however, in contrast to the cerebral cortex, spines in the DG became more filopodia-like. In mice lacking MMP-9, no effects of TBI on spine density and morphology were observed.

Elevation of MMP-9 Levels Promotes Epileptogenesis After Traumatic Brain Injury.

Posttraumatic epilepsy (PTE) is a recurrent seizure disorder that often develops secondary to traumatic brain injury (TBI) that is caused by an external mechanical force. Recent evidence shows that the brain extracellular matrix plays a major role in the remodeling of neuronal connections after injury. One of the proteases that is presumably responsible for this process is matrix metalloproteinase-9 (MMP-9). The levels of MMP-9 are elevated in rodent brain tissue and human blood samples after TBI. However, no studies have described the influence of MMP-9 on the development of PTE. The present study used controlled cortical impact (CCI) as a mouse model of TBI. We examined the detailed kinetics of MMP-9 levels for 1 month after TBI and observed two peaks after injury (30 min and 6 h after injury). We tested the hypothesis that high levels of MMP-9 predispose individuals to the development of PTE, and MMP-9 inhibition would protect against PTE. We used transgenic animals with either MMP-9 knockout or MMP-9 overexpression. MMP-9 overexpression increased the number of mice that exhibited TBI-induced spontaneous seizures, and MMP-9 knockout decreased the appearance of seizures. We also evaluated changes in responsiveness to a single dose of the chemoconvulsant pentylenetetrazol. MMP-9-overexpressing mice exhibited a significantly shorter latency between pentylenetetrazol administration and the first epileptiform spike. MMP-9 knockout mice exhibited the opposite response profile. Finally, we found that the occurrence of PTE was correlated with the size of the lesion after injury. Overall, our data emphasize the contribution of MMP-9 to TBI-induced structural and physiological alterations in brain circuitry that may lead to the development of PTE.

Optimized perfusion-based CUBIC protocol for the efficient whole-body clearing and imaging of rat organs.

Whole-organ and whole-body optical tissue clearing methods allowing imaging in 3 dimensions are an area of profound research interest. Originally developed to study nervous tissue, they have been successfully applied to all murine organs, yet clearing and imaging of rat peripheral organs is less advanced. Here, a modification of CUBIC clearing protocol is presented. It provides a rapid and simple approach to clear the entire adult rat organism and thus all organs within as little as 4 days. Upgraded perfusion-based rat CUBIC protocol preserves both anatomical structure of organs and signal from proteinaceous fluorophores, and furthermore is compatible with antibody staining. Finally, it enables also volumetric cells analyses and is tailored for staining of calcium deposits within unsectioned soft tissues.

[Getting an insight into the brain - new optical clearing techniques and imaging using light-sheet microscope]. / Zajrzec w glab mózgu - nowe techniki oczyszczania optycznego i obrazowania z zastosowaniem mikroskopu arkusza swiatla.

One of the biggest challenges in neuroscience is to understand how brain operates. For this, it would be the best to image the whole brain with at least cellular resolution, preserving the three-dimensional structure in order to capture the connections between different areas. Most currently available high-resolution imaging techniques are based on preparing thin brain sections that are next photographed one by one and subsequently bigger structures are reconstructed. These techniques are laborious and create artifacts. Recent optical clearing methods allow to obtain literally transparent brains that can be imaged using light-sheet microscope. The present review summarizes the most popular optical clearing techniques, describing their different mechanisms and comparing advantages and disadvantages of different approaches, and presents the principle of light-sheet microscopy and its use in imaging. Finally, it gives examples of application of optical tissue clearing and light-sheet imaging in neuroscience and beyond it.

Matrix Metalloproteinase-9 and Synaptic Plasticity in the Central Amygdala in Control of Alcohol-Seeking Behavior.

BACKGROUND: Dysfunction of the glutamatergic system has been implicated in alcohol addiction; however, the molecular underpinnings of this phenomenon are still poorly understood. In the current study we have investigated the possible function of matrix metalloproteinase-9 (MMP-9) in alcohol addiction because this protein has recently emerged as an important regulator of excitatory synaptic plasticity. METHODS: For long-term studies of alcohol drinking in mice we used IntelliCages. Dendritic spines were analyzed using Diolistic staining with DiI. Whole-cell patch clamp was used to assess silent synapses. Motivation for alcohol in human subjects was assessed on the basis of a Semi-Structured Assessment for the Genetics of Alcoholism interview. RESULTS: Mice devoid of MMP-9 (MMP-9 knockout) drank as much alcohol as wild-type animals; however, they were impaired in alcohol seeking during the motivation test and withdrawal. The deficit could be rescued by overexpression of exogenous MMP-9 in the central nucleus of the amygdala (CeA). Furthermore, the impaired alcohol seeking was associated with structural alterations of dendritic spines in the CeA and, moreover, whole-cell patch clamp analysis of the basal amygdala to CeA projections showed that alcohol consumption and withdrawal were associated with generation of silent synapses. These plastic changes were impaired in MMP-9 knockout mice. Finally, C/T polymorphism of MMP-9 gene at position -1562, which upregulates MMP-9 expression, correlated with increased motivation for alcohol in alcoholics. CONCLUSIONS: In aggregate, our results indicate a novel mechanism of alcohol craving that involves MMP-9-dependent synaptic plasticity in CeA.

Light-sheet microscopy imaging of a whole cleared rat brain with Thy1-GFP transgene.

Whole-brain imaging with light-sheet fluorescence microscopy and optically cleared tissue is a new, rapidly developing research field. Whereas successful attempts to clear and image mouse brain have been reported, a similar result for rats has proven difficult to achieve. Herein, we report on creating novel transgenic rat harboring fluorescent reporter GFP under control of neuronal gene promoter. We then present data on clearing the rat brain, showing that FluoClearBABB was found superior over passive CLARITY and CUBIC methods. Finally, we demonstrate efficient imaging of the rat brain using light-sheet fluorescence microscopy.

Matrix metalloproteinase-9 involvement in the structural plasticity of dendritic spines.

Dendritic spines are the locus for excitatory synaptic transmission in the brain and thus play a major role in neuronal plasticity. The ability to alter synaptic connections includes volumetric changes in dendritic spines that are driven by scaffolds created by the extracellular matrix (ECM). Here, we review the effects of the proteolytic activity of ECM proteases in physiological and pathological structural plasticity. We use matrix metalloproteinase-9 (MMP-9) as an example of an ECM modifier that has recently emerged as a key molecule in regulating the morphology and dysmorphology of dendritic spines that underlie synaptic plasticity and neurological disorders, respectively. We summarize the influence of MMP-9 on the dynamic remodeling of the ECM via the cleavage of extracellular substrates. We discuss its role in the formation, modification, and maintenance of dendritic spines in learning and memory. Finally, we review research that implicates MMP-9 in aberrant synaptic plasticity and spine dysmorphology in neurological disorders, with a focus on morphological abnormalities of dendritic protrusions that are associated with epilepsy.

Expression of Ttyh1, a member of the Tweety family in neurons in vitro and in vivo and its potential role in brain pathology.

We have previously shown that Ttyh1 mRNA is expressed in neurons and its expression is up-regulated in the brain during epileptogenesis and epilepsy. In this study, we aimed to elucidate the role of Ttyh1 in neurons. We found widespread expression of Ttyh1 protein in neurons in vivo and in vitro. Ttyh1 immunoreactivity in vitro was frequently found in invaginations of dendritic spines; however, Ttyh1, seldom co-localized with synaptic markers in vivo. Silencing Ttyh1 expression with siRNA in hippocampal cultures resulted in alterations of MAP2 distribution along neurites causing it to appear in the form of chains of beads. Over-expression of Ttyh1 caused intense neuritogenesis and the formation of numerous filopodia-like protrusions. Similar protrusions were also produced in SH-SY5Y neuroblastoma cells over-expressing Ttyh1. Using a biotin-streptavidin pull-down assay and mass spectrometry, we identified proteins that can form complexes with Ttyh1 in the brain. Ttyh1 binding proteins are often expressed in the endoplasmic reticulum or the Golgi apparatus or are localized at synapses. Finally, we found increased expression of Ttyh1 in the inner molecular layer of the dentate gyrus in an animal model of epilepsy. On the basis of our findings, we propose Ttyh1 involvement in brain pathology.

Cloning of expressed sequence tags (ESTs) representing putative epileptogenesis-related genes and the localization of their expression in the normal brain.

The goal of this study was to clone and sequence selected expressed sequence tags (ESTs) which mRNA is upregulated in a rat model of epilepsy based on our previous work [17] and to determine the localization of their mRNA expression in the normal brain. Six ESTs that had not been assigned to any gene at the time of the inception of our experiments were chosen for analysis. Radioactive in situ hybridization revealed that the expression of four transcripts (AA955087, AA875438, AA899079, AA819660) was clearly localized to hippocampal neurons and was also detected in the cortex. Two transcripts, AA819523 and AA819766, displayed a uniform expression pattern. 5'RACE cloning and sequencing allowed for the annotation of five ESTs to known or predicted genes: sorting nexin-2 (SNX2), tweety homolog 1 (Ttyh1), selenoprotein T (SelT), transmembrane protein 204 (Tmem204) and methyl-CpG binding domain protein 3 (Mbd3). According to the results of our combined non-radioactive in situ hybridization and immunohistochemistry with cell-specific markers, mRNA coding for Ttyh1, Tmem204 and Mbd3 was expressed in neurons. The potential role of the studied genes in normal brain or in brain pathology remains elusive and requires further study because little is known about these genes in general.