Live Rodent Brain Research Articles

Intravital 3D visualization and segmentation of murine neural networks at micron resolution

Optical coherence tomography (OCT) allows label-free, micron-scale 3D imaging of biological tissues’ fine structures with significant depth and large field-of-view. Here we introduce a novel OCT-based neuroimaging setting, accompanied by a feature segmentation algorithm, which enables rapid, accurate, and high-resolution in vivo imaging of 700 μm depth across the mouse cortex. Using a commercial OCT device, we demonstrate 3D reconstruction of microarchitectural elements through a cortical column. Our system is sensitive to structural and cellular changes at micron-scale resolution in vivo, such as those from injury or disease. Therefore, it can serve as a tool to visualize and quantify spatiotemporal brain elasticity patterns. This highly transformative and versatile platform allows accurate investigation of brain cellular architectural changes by quantifying features such as brain cell bodies’ density, volume, and average distance to the nearest cell. Hence, it may assist in longitudinal studies of microstructural tissue alteration in aging, injury, or disease in a living rodent brain.

Brain-Implantable Multifunctional Probe for Simultaneous Detection of Glutamate and GABA Neurotransmitters: Optimization and In Vivo Studies.

Imbalances in levels of glutamate (GLU) and gamma-aminobutyric acid (GABA) and their sub-second signaling dynamics occur in several brain disorders including traumatic brain injury, epilepsy, and Alzheimer’s disease. The present work reports on the optimization and in vivo testing of a silicon (Si) multifunctional biosensor probe for sub-second simultaneous real-time detection of GLU and GABA. The Si probe features four surface-functionalized platinum ultramicroelectrodes (UMEs) for detection of GLU and GABA, a sentinel site, and integrated microfluidics for in-situ calibration. Optimal enzyme concentrations, size-exclusion phenylenediamine layer and micro spotting conditions were systematically investigated. The measured GLU sensitivity for the GLU and GABA sites were as high as 219 ± 8 nA μM−1 cm−2 (n = 3). The measured GABA sensitivity was as high as 10 ± 1 nA μM−1 cm−2 (n = 3). Baseline recordings (n = 18) in live rats demonstrated a useful probe life of at least 11 days with GLU and GABA concentrations changing at the levels of 100′s and 1000′s of μM and with expected periodic bursts or fluctuations during walking, teeth grinding and other activities and with a clear difference in the peak amplitude of the sensor fluctuations between rest (low) and activity (higher), or when the rat was surprised (a reaction with no movement). Importantly, the probe could improve methods for large-scale monitoring of neurochemical activity and network function in disease and injury, in live rodent brain.

Multi-modal nonlinear optical and thermal imaging platform for label-free characterization of biological tissue

The ability to characterize the combined structural, functional, and thermal properties of biophysically dynamic samples is needed to address critical questions related to tissue structure, physiological dynamics, and disease progression. Towards this, we have developed an imaging platform that enables multiple nonlinear imaging modalities to be combined with thermal imaging on a common sample. Here we demonstrate label-free multimodal imaging of live cells, excised tissues, and live rodent brain models. While potential applications of this technology are wide-ranging, we expect it to be especially useful in addressing biomedical research questions aimed at the biomolecular and biophysical properties of tissue and their physiology.

CRISPR/Cas9-Mediated Gene Knockout in the Mouse Brain Using In Utero Electroporation.

This unit describes a highly efficient and rapid procedure for brain-specific disruption of genes in the developing mouse brain using pX330 plasmids expressing humanized Cas9 and single-guide RNAs (sgRNAs) against target genes. The pX330 plasmids are delivered into the rodent brain using in utero electroporation. Focusing on the Satb2 gene, which encodes an AT-rich DNA-binding transcription factor, we found that the introduction of pX330-Satb2 induced insertion/deletion (indel) mutations near the predicted cleavage site in the Satb2 gene, resulting in a dramatic reduction of Satb2 expression in post-mitotic neurons. Moreover, introduction of pX330-Satb2 induced abnormalities in axonal projection patterns, which was consistent with the phenotypes observed in Satb2 mutant mice. Thus, the procedure described here, combining the CRISPR/Cas9 system and in utero electroporation, is useful for knocking out genes of interest in the living rodent brain. © 2017 by John Wiley & Sons, Inc.

Cerebrospinal and interstitial fluid transport via the glymphatic pathway modeled by optimal mass transport

The glymphatic pathway is a system which facilitates continuous cerebrospinal fluid (CSF) and interstitial fluid (ISF) exchange and plays a key role in removing waste products from the rodent brain. Dysfunction of the glymphatic pathway may be implicated in the pathophysiology of Alzheimer's disease. Intriguingly, the glymphatic system is most active during deep wave sleep general anesthesia. By using paramagnetic tracers administered into CSF of rodents, we previously showed the utility of MRI in characterizing a macroscopic whole brain view of glymphatic transport but we have yet to define and visualize the specific flow patterns. Here we have applied an alternative mathematical analysis approach to a dynamic time series of MRI images acquired every 4min over ∼3h in anesthetized rats, following administration of a small molecular weight paramagnetic tracer into the CSF reservoir of the cisterna magna. We use Optimal Mass Transport (OMT) to model the glymphatic flow vector field, and then analyze the flow to find the network of CSF-ISF flow channels. We use 3D visualization computational tools to visualize the OMT defined network of CSF-ISF flow channels in relation to anatomical and vascular key landmarks from the live rodent brain. The resulting OMT model of the glymphatic transport network agrees largely with the current understanding of the glymphatic transport patterns defined by dynamic contrast-enhanced MRI revealing key CSF transport pathways along the ventral surface of the brain with a trajectory towards the pineal gland, cerebellum, hypothalamus and olfactory bulb. In addition, the OMT analysis also revealed some interesting previously unnoticed behaviors regarding CSF transport involving parenchymal streamlines moving from ventral reservoirs towards the surface of the brain, olfactory bulb and large central veins.

Injectable electronics as a modern day ‘ship in a bottle’

Although the first wearable pacemakers were introduced in the late 1950s, bio-integrated electronics are as relevant today as they were 50 years ago, with modern devices offering the potential for either recording or modulating cellular behavior in three dimensions (3D).1 Such devices hold the promise for opening up exciting new avenues of treatment in a variety of ailments, from neurodegenerative disorders to traumatic spinal injuries.2 However, one key challenge in this field is designing devices that are minimally invasive, with researchers constantly pushing to overcome the challenges associated with incorporating hard planar semiconductors with soft pliable biological tissues.3 Among these concerns, flexibility, size and cytotoxicity all play a crucial role in determining how successful a device is. In their recent work ‘Syringe-injectable electronics’ Charles Lieber and his colleges at Harvard University have taken significant strides in confronting these challenges, exhibiting meshed electronic polymers that can be injected into silicone gels and directly into a live rodent's brain.4 This injectable approach offers several distinct benefits: First, it enables a precise positioning of these probes at a desirable target location. Second, it limits the device’s surgical profile, allowing it to be introduced in a minimally invasive fashion, helps promote long-term bio-integration and reduces glial cell response. Additionally, once injected, these structures are seen to unfurl, expanding into a mesh network with final lateral dimensions up to 30 times the diameter of the insertion needle. This emergent structure included an array of protruding metal nanowire electrodes, connected by polymer ribbons bisecting at 45°. This expansion allows the probes to form intimate contact with the surrounding tissue, enabling multiplexed mechanical strain and neural signaling recordings. We liken this style of design to a modern ‘ship in a bottle’, where a complex object emerges after passing through a narrow channel (Figure 1), enabling an expanded device design while remaining minimally disruptive to the native material. We believe that this approach offers substantial promise and suggests a variety of intriguing device designs. For instance, it should be possible to incorporate an origami/kirigami style folding element into the existing mesh design, allowing the controllable formation of 3D structures. Additionally, recent work has shown that silicon nanowires can be internalized by cellular systems.5 Such constructs could also be incorporated with the current design, acting as possible electronic/force probes and stimulators, enabling studies in previously difficult-to-access intracellular domains. Finally, one important next step is introducing wireless functionality to avoid intrusive electrical contacts. Taken together, this technology offers a promising new approach to bio-integration and helps enable a new generation of minimally invasive devices.

Optimal-mass-transfer-based estimation of glymphatic transport in living brain.

It was recently shown that the brain-wide cerebrospinal fluid (CSF) and interstitial fluid exchange system designated the 'glymphatic pathway' plays a key role in removing waste products from the brain, similarly to the lymphatic system in other body organs1,2. It is therefore important to study the flow patterns of glymphatic transport through the live brain in order to better understand its functionality in normal and pathological states. Unlike blood, the CSF does not flow rapidly through a network of dedicated vessels, but rather through para-vascular channels and brain parenchyma in a slower time-domain, and thus conventional fMRI or other blood-flow sensitive MRI sequences do not provide much useful information about the desired flow patterns. We have accordingly analyzed a series of MRI images, taken at different times, of the brain of a live rat, which was injected with a paramagnetic tracer into the CSF via the lumbar intrathecal space of the spine. Our goal is twofold: (a) find glymphatic (tracer) flow directions in the live rodent brain; and (b) provide a model of a (healthy) brain that will allow the prediction of tracer concentrations given initial conditions. We model the liquid flow through the brain by the diffusion equation. We then use the Optimal Mass Transfer (OMT) approach3 to derive the glymphatic flow vector field, and estimate the diffusion tensors by analyzing the (changes in the) flow. Simulations show that the resulting model successfully reproduces the dominant features of the experimental data.

Repeated exposure to chlorpyrifos leads to prolonged impairments of axonal transport in the living rodent brain

The toxicity of the class of chemicals known as the organophosphates (OP) is most commonly attributed to the inhibition of the enzyme acetylcholinesterase. However, there is significant evidence that this mechanism may not account for all of the deleterious neurologic and neurobehavioral symptoms of OP exposure, especially those associated with levels that produce no overt signs of acute toxicity. In the study described here we evaluated the effects of the commonly used OP-pesticide, chlorpyrifos (CPF) on axonal transport in the brains of living rats using manganese (Mn2+)-enhanced magnetic resonance imaging (MEMRI) of the optic nerve (ON) projections from the retina to the superior colliculus (SC). T1-weighted MEMRI scans were evaluated at 6 and 24h after intravitreal injection of Mn2+. As a positive control for axonal transport deficits, initial studies were conducted with the tropolone alkaloid colchicine administered by intravitreal injection. In subsequent studies both single and repeated exposures to CPF were evaluated for effects on axonal transport using MEMRI. As expected, intravitreal injection of colchicine (2.5μg) produced a robust decrease in transport of Mn2+ along the optic nerve (ON) and to the superior colliculus (SC) (as indicated by the reduced MEMRI contrast). A single subcutaneous (s.c.) injection of CPF (18.0mg/kg) was not associated with significant alterations in the transport of Mn2+. Conversely, 14-days of repeated s.c. exposure to CPF (18.0mg/kg/day) was associated with decreased transport of Mn2+ along the ONs and to the SC, an effect that was also present after a 30-day (CPF-free) washout period. These results indicate that repeated exposures to a commonly used pesticide, CPF can result in persistent alterations in axonal transport in the living mammalian brain. Given the fundamental importance of axonal transport to neuronal function, these observations may (at least in part) explain some of the long term neurological deficits that have been observed in humans who have been repeatedly exposed to doses of OPs not associated with acute toxicity.

Correction: Metabolic Profiling of Dividing Cells in Live Rodent Brain by Proton Magnetic Resonance Spectroscopy (1HMRS) and LCModel Analysis

Correction: Metabolic Profiling of Dividing Cells in Live Rodent Brain by Proton Magnetic Resonance Spectroscopy (1HMRS) and LCModel Analysis

Microfabricated solid-state ion-selective electrode probe for measuring potassium in the living rodent brain: Compatibility with DC-EEG recordings to study spreading depression

There is considerable need in neuroscience research to reliably measure concentrations of extracellular ions in the living brain as the dynamics of ion levels increasingly is considered to play a key role in the pathophysiology of many brain disorders. Unfortunately, most probes currently used for direct measurement of extracellular ion concentrations in living brain tissue in experimental animals have considerable limitations. They are custom-made glass capillary ion-selective microelectrodes that are fragile, time-consuming to prepare, and can practically only be used to measure at one depth and one location in the brain. We here present the design and fabrication of a novel type of miniaturized probe that can simultaneously measure, in the brain of a living animal, multiple parameters relevant to the neurological phenomenon of spreading depression (SD) at multiple depths and locations. SD is characterized by a slowly propagating wave of initial neuronal and glial cell depolarization that is followed by depression of activity. SD is accompanied by a massive redistribution of ions, including K+, between intracellular and extracellular compartments and is considered important in brain diseases such as stroke, traumatic brain injury, and migraine with aura. This work focuses on the functionalization of fabricated probes for measuring changes in extracellular K+ concentration (and its associated changes in neuronal activity) using an ion-selective potentiometric sensor. We show that the choice of conductive polymer and the method of electro-deposition is critical to obtain low cross-sensitivity for pH and O2. The sensor's response to K+ is approximately linear between 2 and 40mM with a voltage-response of 39mV/log[K+]. Proof-of-principle in vivo brain recordings were performed in the cortex of a wild-type mouse during induction of cortical SD. We reliably measured the theoretically expected rise and fall of brain tissue K+ levels during a locally induced cortical SD. We envisage that our probe can be of great use to reliably measure K+, without being affected by pH or O2 changes, in the living brain and be widely applicable in neuroscience research.

Metabolic Profiling of Dividing Cells in Live Rodent Brain by Proton Magnetic Resonance Spectroscopy (1HMRS) and LCModel Analysis

RationaleDividing cells can be detected in the live brain by positron emission tomography or optical imaging. Here we apply proton magnetic resonance spectroscopy (1HMRS) and a widely used spectral fitting algorithm to characterize the effect of increased neurogenesis after electroconvulsive shock in the live rodent brain via spectral signatures representing mobile lipids resonating at ∼1.30 ppm. In addition, we also apply the same 1HMRS methodology to metabolically profile glioblastomas with actively dividing cells growing in RCAS-PDGF mice.Methods 1HMRS metabolic profiles were acquired on a 9.4T MRI instrument in combination with LCModel spectral analysis of: 1) rat brains before and after ECS or sham treatments and 2) RCAS-PDGF mice with glioblastomas and wild-type controls. Quantified 1HMRS data were compared to post-mortem histology.ResultsDividing cells in the rat hippocampus increased ∼3-fold after ECS compared to sham treatment. Quantification of hippocampal metabolites revealed significant decreases in N-acetyl-aspartate but no evidence of an elevated signal at ∼1.3 ppm (Lip13a+Lip13b) in the ECS compared to the sham group. In RCAS-PDGF mice a high density (22%) of dividing cells characterized glioblastomas. Nile Red staining revealed a small fraction (3%) of dying cells with intracellular lipid droplets in the tumors of RCAS-PDGF mice. Concentrations of NAA were lower, whereas lactate and Lip13a+Lip13b were found to be significantly higher in glioblastomas of RCAS-PDGF mice, when compared to normal brain tissue in the control mice.ConclusionsMetabolic profiling using 1HMRS in combination with LCModel analysis did not reveal correlation between Lip13a+Lip13b spectral signatures and an increase in neurogenesis in adult rat hippocampus after ECS. However, increases in Lip13a+Lip13b were evident in glioblastomas suggesting that a higher density of actively dividing cells and/or the presence of lipid droplets is necessary for LCModel to reveal mobile lipids.

In vivo voxel based morphometry: Detection of increased hippocampal volume and decreased glutamate levels in exercising mice

Voluntary exercise has tremendous effects on adult hippocampal plasticity and metabolism and thus sculpts the hippocampal structure of mammals. High-field (1)H magnetic resonance (MR) investigations at 9.4 T of metabolic and structural changes can be performed non-invasively in the living rodent brain. Numerous molecular and cellular mechanisms mediating the effects of exercise on brain plasticity and behavior have been detected in vitro. However, in vivo attempts have been rare. In this work a method for voxel based morphometry (VBM) was developed with automatic tissue segmentation in mice using a 9.4 T animal scanner equipped with a (1)H-cryogenic coil. The thus increased signal to noise ratio enabled the acquisition of high resolution T2-weighted images of the mouse brain in vivo and the creation of group specific tissue class maps for the segmentation and normalization with SPM. The method was used together with hippocampal single voxel (1)H MR spectroscopy to assess the structural and metabolic differences in the mouse brain due to voluntary wheel running. A specific increase of hippocampal volume with a concomitant decrease of hippocampal glutamate levels in voluntary running mice was observed. An inverse correlation of hippocampal gray matter volume and glutamate concentration indicates a possible implication of the glutamatergic system for hippocampal volume.

Simultaneous visualization of multiple neuronal properties with single-cell resolution in the living rodent brain

To understand the fine-scale structures and functional properties of individual neurons in vivo, we developed and validated a rapid genetic technique that enables simultaneous investigation of multiple neuronal properties with single-cell resolution in the living rodent brain. Our technique PASME (promoter-assisted sparse-neuron multiple-gene labeling using in uteroelectroporation) targets specific small subsets of sparse pyramidal neurons in layer 2/3, layer 5 of the cerebral cortex and in the hippocampus with multiple fluorescent reporter proteins such as postsynaptic PSD-95-GFP and GFP-gephyrin. The technique is also applicable for targeting independently individual neurons and their presynaptic inputs derived from surrounding neurons. Targeting sparse layer 2/3 neurons, we uncovered a novel subpopulation of layer 2/3 neurons in the mouse cerebral cortex. This technique, broadly applicable for probing and manipulating neurons with single-cell resolution in vivo, should provide a robust means to uncover the basic mechanisms employed by the brain, especially when combined with in vivo two-photon laser-scanning microscopy and/or optogenetic technologies.

A rapid genetic strategy for multiple-gene, single-neuron labeling in the living rodent brain

A rapid genetic strategy for multiple-gene, single-neuron labeling in the living rodent brain

The metabolomic profile during isoflurane anesthesia differs from propofol anesthesia in the live rodent brain

Development of noninvasive techniques to discover new biomarkers in the live brain is important to further understand the underlying metabolic pathways of significance for processes such as anesthesia-induced apoptosis and cognitive dysfunction observed in the undeveloped brain. We used in vivo proton magnetic resonance spectroscopy and two different signal processing approaches to test the hypothesis that volatile (isoflurane) and intravenous (propofol) anesthetics at equipotent doses produce distinct metabolomic profiles in the hippocampus and parietal cortex of the live rodent. For both brain regions, prolonged isoflurane anesthesia was characterized by higher levels of lactate (Lac) and glutamate compared with long-lasting propofol. In contrast, propofol anesthesia was characterized by very low concentrations of Lac ([lac]) as well as glucose. Quantitative analysis revealed that the [lac] was fivefold higher with isoflurane compared with propofol anesthesia and independent of [lac] in blood. The metabolomic profiling further demonstrated that for both brain regions, Lac was the most important metabolite for the observed differences, suggesting activation of distinct metabolic pathways that may impact mechanisms of action, background cellular functions, and possible agent-specific neurotoxicity.

Dimebon®: A Clinically Promising Drug for Alzheimer's Disease, Regulates Amyloid-Beta Metabolism in Cultured Cells, In Isolated Nerve Terminals, and In the Interstitial Fluid of the Living Rodent Brain

Dimebon®: A Clinically Promising Drug for Alzheimer's Disease, Regulates Amyloid-Beta Metabolism in Cultured Cells, In Isolated Nerve Terminals, and In the Interstitial Fluid of the Living Rodent Brain

Optical coherence tomography guided neurosurgical procedures in small rodents

The delivery of therapeutic agents directly to targets deep within the brain is becoming an important tool in the treatment of a variety of neurological disorders. Currently, the standard method to accomplish this is by using stereotactic procedures. While this existing method is adequate for many experimental situations, it is essentially a blind procedure that cannot provide real-time feedback on whether the actual location deviated from the intended location or whether the therapeutic agent was actually delivered. Here we describe an optical guidance technique that is designed to work in conjunction with existing stereotactic procedures to provide the needed real-time feedback for therapeutic delivery in live animals. This real-time feedback is enabled by a technology called catheter-based optical coherence tomography (OCT). In this study we show that OCT can provide real-time position feedback based on microanatomic landmarks from the live rodent brain. We show that OCT can provide the necessary guidance to perform microsurgery such as the selective transection of the Schaffer collateral inputs to the CA1 region of the hippocampus with minimal perturbation of overlying structures. We also show that OCT allows visual monitoring of the successful delivery of viral vectors to specific subregions of the hippocampus.

Imaging Microglia in Live Brain Slices and Slice Cultures

INTRODUCTIONThis protocol describes methods for fluorescence labeling and time-lapse confocal imaging of microglia in acutely prepared tissue slices from developing and adult animals, and to slice cultures derived from early post-natal day 7 (<PND7) animals. The techniques permit real-time analyses of microglial structure and dynamic remodeling in live rodent brain tissues.

Imaging the Peripheral Benzodiazepine Receptor Response in Central Nervous System Demyelination and Remyelination

We used a rodent model of cuprizone-induced demyelination to examine the peripheral benzodiazepine receptor (PBR) response during remyelination. C57BL/6J mice were fed a 0.2% cuprizone-containing or control diet for 3 weeks and then removed to allow for remyelination. Quantitative autoradiography of 3H-(R)-PK11195 binding to PBR in the corpus callosum showed increased levels at 3 weeks of demyelination and gradually decreased as a function of remyelination. PBR levels were associated with the degree of remyelination and activation of microglia and astrocytes. However, the temporal pattern suggests that the PBR signal during the late stages of remyelination was primarily associated with astrocytes. We also used small-animal positron-emission tomography (PET) imaging to determine if this technique could be used to monitor PBR levels in the brain of living mice. The results indicate that 11C-(R)-PK11195 levels are significantly elevated in the mouse brain during cuprizone-induced demyelination and normalize at a time in which remyelination is complete. These findings support the notion that PBR is a sensitive marker for the visualization and quantification of brain injury and recovery. Further, the in vivo imaging of the PBR response is now possible in the living rodent brain.