BubbleDrive, a low-volume incubation chamber for acute brain slices.

Acute brain slices are a common and useful preparation in experimental neuroscience. A wide range of incubation chambers for brain slices exists but only a few are designed with very low volumes of the bath solution in mind. Such chambers are necessary when high-cost chemicals are to be added to the solution or when small amounts of substances released by the slice are to be collected for analysis. The principal challenge in designing a very low-volume incubation chamber is maintaining good oxygenation and flow without mechanically disturbing or damaging the slices. We designed and validated BubbleDrive, a 3D-printed incubation chamber with a minimum volume of 1.5 mL which can hold up to three coronal mouse slices from one hemisphere. It employs the carbogen gas bubbles to drive the flow circulation in a consistent and reproducible manner, and without disturbing the brain slices. The BubbleDrive design and construction were successfully validated by comparison to a conventional large-volume incubation chamber in several experimental designs involving measurements of extracellular diffusion parameters, the electrophysiology of neuronal and astrocytic networks, and the effectiveness of slice incubation with hyaluronidase enzyme.

Brain extracellular space of the naked mole-rat expands and maintains normal diffusion under ischemic conditions.

Brain extracellular space (ECS) forms a conduit for diffusion, an essential mode of molecular transport between brain vasculature, neurons and glia. ECS volume is reduced under conditions of hypoxia and ischemia, contributing to impaired extracellular diffusion and consequent neuronal dysfunction and death. We investigated the ECS volume fraction and diffusion permeability of the African naked mole-rat (NM-R; Heterocephalus Glaber), a rodent with a remarkably high tolerance for hypoxia and ischemia. Real-Time Iontophoretic and Integrative Optical Imaging methods were used to evaluate diffusion transport in cortical slices under normoxic and ischemic conditions, and results were compared to values previously collected in rats. NM-R brains under normoxic conditions had a smaller ECS volume fraction than rats, and a correspondingly decreased diffusion permeability for macromolecules. Surprisingly, and in sharp contrast to rats, the NM-R ECS responded to ischemic conditions at the center of thick brain slices by expanding, rather than shrinking, and preserving diffusion permeabilities for small and large molecules. The NM-R thick slices also showed a blunted accumulation of ECS potassium compared to rats. The remarkable dynamic response of the NM-R ECS to ischemia likely demonstrates an adaptation for NM-R to maintain brain function in their extreme nest environment.

Rapid volume pulsation of the extracellular space coincides with epileptiform activity in mice and depends on the NBCe1 transporter.

KEY POINTS: Extracellular space (ECS) rapid volume pulsation (RVP) accompanying epileptiform activity is described for the first time. Such RVP occurs robustly in several in vitro and in vivo mouse models of epileptiform activity. In the in vitro 4-aminopyridine model of epileptiform activity, RVP depends on the activity of the electrogenic Na+ /HCO3- cotransporter (NBCe1). NBCe1 pharmacological inhibition suppresses RVP and epileptiform activity. Inhibition of changes in ECS volume may be a useful target in epilepsy patients who are resistant to current treatments. ​ ABSTRACT: The extracellular space (ECS) of the brain shrinks persistently by approximately 35% during epileptic seizures. Here we report the discovery of rapid volume pulsation (RVP), further transient drops in ECS volume which accompany events of epileptiform activity. These transient ECS contractions were observed in multiple mouse models of epileptiform activity both in vivo (bicuculline methiodide model) and in vitro (hyaluronan synthase 3 knock-out, picrotoxin, bicuculline and 4-aminopyridine models). By using the probe transients quantification (PTQ) method we show that individual pulses of RVP shrank the ECS by almost 15% in vivo. In the 4-aminopyridine in vitro model, the individual pulses of RVP shrank the ECS by more than 4%, and these transient changes were superimposed on a persistent ECS shrinkage of 36% measured with the real-time iontophoretic method. In this in vitro model, we investigated several channels and transporters that may be required for the generation of RVP and epileptiform activity. Pharmacological blockages of Na+ /K+ /2Cl- cotransporter type 1 (NKCC1), K+ /Cl- cotransporter (KCC2), the water channel aquaporin-4 (AQP4) and inwardly rectifying potassium channel 4.1 (Kir4.1) were ineffective in halting the RVP and the epileptiform activity. In contrast, pharmacological blockade of the electrogenic Na+ /HCO3- cotransporter (NBCe1) by 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS) eliminated both the RVP and the persistent ECS shrinkage. Importantly, this blocker also stopped the epileptiform activity. These results demonstrate that RVP is closely associated with epileptiform activity across several models of epileptiform activity and therefore the underlying mechanism could potentially represent a novel target for epilepsy management and treatment.

Integrity of White Matter is Compromised in Mice with Hyaluronan Deficiency.

Brain white matter is the means of efficient signal propagation in brain and its dysfunction is associated with many neurological disorders. We studied the effect of hyaluronan deficiency on the integrity of myelin in murine corpus callosum. Conditional knockout mice lacking the hyaluronan synthase 2 were compared with control mice. Ultrastructural analysis by electron microscopy revealed a higher proportion of myelin lamellae intruding into axons of knockout mice, along with significantly slimmer axons (excluding myelin sheath thickness), lower g-ratios, and frequent loosening of the myelin wrappings, even though the myelin thickness was similar across the genotypes. Analysis of extracellular diffusion of a small marker molecule tetramethylammonium (74 MW) in brain slices prepared from corpus callosum showed that the extracellular space volume increased significantly in the knockout animals. Despite this vastly enlarged volume, extracellular diffusion rates were significantly reduced, indicating that the compromised myelin wrappings expose more complex geometric structure than the healthy ones. This finding was confirmed in vivo by diffusion-weighted magnetic resonance imaging. Magnetic resonance spectroscopy suggested that water was released from within the myelin sheaths. Our results indicate that hyaluronan is essential for the correct formation of tight myelin wrappings around the axons in white matter.

Time-Resolved Integrative Optical Imaging of Diffusion during Spreading Depression.

An improved version of the integrative optical imaging method has been developed that substantially increases the time resolution of diffusion measurements. We present a theory for time-resolved integrative optical imaging that incorporates a time-dependent effective diffusion coefficient in homogeneous anisotropic media and a time-dependent nonspecific linear clearance. The method was applied to measure the very fast changes in extracellular diffusion that occur during spreading depression in rat hippocampal slices. We were able to achieve a time resolution of approximately 1 s, an improvement of at least 10 times compared to the standard methods for extracellular diffusion measurement. We have found that diffusion of a small fluorescent extracellular marker (MW 3000) completely stopped during the maximum direct current shift associated with the spreading depression wave, then gradually resumed over several minutes afterward. The effect of spreading depression on extracellular space is much larger than previously estimated by other methods with lower time resolution.

Anomalous extracellular diffusion in rat cerebellum.

Extracellular space (ECS) is a major channel transporting biologically active molecules and drugs in the brain. Diffusion-mediated transport of these substances is hindered by the ECS structure but the microscopic basis of this hindrance is not fully understood. One hypothesis proposes that the hindrance originates in large part from the presence of dead-space (DS) microdomains that can transiently retain diffusing molecules. Because previous theoretical and modeling work reported an initial period of anomalous diffusion in similar environments, we expected that brain regions densely populated by DS microdomains would exhibit anomalous extracellular diffusion. Specifically, we targeted granular layers (GL) of rat and turtle cerebella that are populated with large and geometrically complex glomeruli. The integrative optical imaging (IOI) method was employed to evaluate diffusion of fluorophore-labeled dextran (MW 3000) in GL, and the IOI data analysis was adapted to quantify the anomalous diffusion exponent dw from the IOI records. Diffusion was significantly anomalous in rat GL, where dw reached 4.8. In the geometrically simpler turtle GL, dw was elevated but not robustly anomalous (dw = 2.6). The experimental work was complemented by numerical Monte Carlo simulations of anomalous ECS diffusion in several three-dimensional tissue models containing glomeruli-like structures. It demonstrated that both the duration of transiently anomalous diffusion and the anomalous exponent depend on the size of model glomeruli and the degree of their wrapping. In conclusion, we have found anomalous extracellular diffusion in the GL of rat cerebellum. This finding lends support to the DS microdomain hypothesis. Transiently anomalous diffusion also has a profound effect on the spatiotemporal distribution of molecules released into the ECS, especially at diffusion distances on the order of a few cell diameters, speeding up short-range diffusion-mediated signals in less permeable structures.

Hyaluronan deficiency due to Has3 knock-out causes altered neuronal activity and seizures via reduction in brain extracellular space.

Hyaluronan (HA), a large anionic polysaccharide (glycosaminoglycan), is a major constituent of the extracellular matrix of the adult brain. To address its function, we examined the neurophysiology of knock-out mice deficient in hyaluronan synthase (Has) genes. Here we report that these Has mutant mice are prone to epileptic seizures, and that in Has3(-/-) mice, this phenotype is likely derived from a reduction in the size of the brain extracellular space (ECS). Among the three Has knock-out models, namely Has3(-/-), Has1(-/-), and Has2(CKO), the seizures were most prevalent in Has3(-/-) mice, which also showed the greatest HA reduction in the hippocampus. Electrophysiology in Has3(-/-) brain slices demonstrated spontaneous epileptiform activity in CA1 pyramidal neurons, while histological analysis revealed an increase in cell packing in the CA1 stratum pyramidale. Imaging of the diffusion of a fluorescent marker revealed that the transit of molecules through the ECS of this layer was reduced. Quantitative analysis of ECS by the real-time iontophoretic method demonstrated that ECS volume was selectively reduced in the stratum pyramidale by ∼ 40% in Has3(-/-) mice. Finally, osmotic manipulation experiments in brain slices from Has3(-/-) and wild-type mice provided evidence for a causal link between ECS volume and epileptiform activity. Our results provide the first direct evidence for the physiological role of HA in the regulation of ECS volume, and suggest that HA-based preservation of ECS volume may offer a novel avenue for development of antiepileptogenic treatments.

Extracellular diffusion in laminar brain structures exemplified by hippocampus.

Numerous brain structures are composed of distinct layers and such stratification has a profound effect on extracellular diffusion transport in these structures. We have derived a more general form of diffusion equation incorporating inhomogeneities in both the extracellular volume fraction (α) and diffusion permeability (θ). A numerical solution of this equation for a special case of layered environment was employed to analyze diffusion in the CA1 region of hippocampus where stratum pyramidale occupied by the bodies of principal neurons is flanked by stratum radiatum and stratum oriens. Extracellular diffusion in the CA1 region was measured in vitro by real-time iontophoretic and real-time pressure methods, and numerical analysis found that stratum pyramidale had a significantly smaller extracellular volume fraction (α=0.127) and lower diffusion permeability (θ=0.327) than the other two layers (α=0.218, θ=0.447). Stratum pyramidale thus functioned as a diffusion barrier for molecules attempting to cross it. We also demonstrate that unless the detailed properties of all layers are taken into account when diffusion experiments are interpreted, the extracted apparent parameters of the extracellular space lose their physical meaning and capacity to describe any individual layer. Such apparent parameters depend on diffusion distance and direction, giving a false impression of microscopic anisotropy and non-Gaussian behavior. This finding has implications for all diffusion mediated physiological processes as well as for other diffusion methods including integrative optical imaging and diffusion-weighted magnetic resonance imaging.

Murine diffusion imaging using snapshot interleaved EPI acquisition at 7T.

Diffusion tensor imaging (DTI) is a powerful magnetic resonance imaging tool for quantitative assessment of white matter micro structure. The majority of DTI methods employ Echo Planar Imaging (EPI) because it is insensitive to motion. However, EPI suffers from distortions and signal losses induced by inhomogeneities in magnetic field susceptibility. This is particularly accentuated in murine imaging at very high magnetic fields. The purpose of this study is to demonstrate that a Snapshot Interleaved EPI acquisition block combined with a stimulated echo module for diffusion sensitization can be successfully used to obtain high quality DTI of a mouse brain at 7T. This technique preserves the EPI speed but reduces its susceptibility artifacts and signal losses. Signal to noise ratio is also reduced but remains higher than in the DTI acquisitions based on a fast low angle shot technique. In vivo results using this new approach are presented along with a full description of the methodology.

The effect of the combination of reducing and oxidising agents on the viscoelastic properties of dough and sensory characteristics of buns.

BACKGROUND: The effects of mixtures of reducing and oxidising agents on the rheological characteristics and quality of wheat flour dough and the sensory characteristics of buns were studied. RESULTS: In chemical analyses, no differences between control dough and doughs with individual mixtures of additives were found. Rheological measurements showed that increasing addition of L-ascorbic acid + L-tryptophan mixture to the dough increased its tenacity and decreased its extensibility. Increasing addition of L-ascorbic acid + L-threonine mixture also led to an increase in dough tenacity but to a slight increase in dough extensibility. Increasing addition of L-ascorbic acid + inactivated dry yeast mixture to the dough resulted in a decrease in tenacity and an increase in extensibility. Two other additive mixtures, L-ascorbic acid + L-cysteine hydrochloride monohydrate and L-cysteine p.a. + inactivated dry yeast, reduced both the tenacity and extensibility of the dough. In sensory analyses, differences in dryness, pliability, sensation when swallowing, quality and texture of buns with additive mixtures were found in comparison with control buns, but gumminess did not change significantly with increasing amount of additive mixtures. CONCLUSION: The additive mixtures tested had a positive influence on bakery products.

Blood pressure and white matter integrity in geriatric depression.

BACKGROUND: Cerebrovascular disease may increase vulnerability to geriatric depression, a syndrome often accompanied by frontal-subcortical lesions. High blood pressure is a risk factor for cerebrovascular disease and white matter changes. This study examined whether and in which brain regions blood pressure is associated with compromised white matter integrity in elderly depressed patients. METHODS: We studied the association between blood pressure and white matter integrity assessed by diffusion tensor imaging (fractional anisotropy, FA) in 41 older patients with major depression. Correlations between FA and blood pressure, after controlling for age, were examined with a voxelwise analysis. RESULTS: Significant associations between FA and blood pressure were detected throughout the anterior cingulate and in multiple frontostriatal and frontotemporal regions. LIMITATIONS: This study did not employ a healthy control group. Moreover, the relatively small sample size precluded a comparison of patients with and without hypertension. CONCLUSIONS: Compromised frontal-striatal white matter integrity may be the anatomical background through which blood pressure confers vulnerability to depression.

Characterizing molecular probes for diffusion measurements in the brain.

Brain diffusion properties are at present most commonly evaluated by magnetic resonance (MR) diffusion imaging. MR cannot easily distinguish between the extracellular and intracellular signal components, but the older technique of real-time iontophoresis (RTI) detects exclusively extracellular diffusion. Interpretation of the MR results would therefore benefit from auxiliary RTI measurements. This requires a molecular probe detectable by both techniques. Our aim was to specify a minimum set of requirements that such a diffusion probe should fulfill and apply it to two candidate probes: the cation tetramethylammonium (TMA(+)), used routinely in the RTI experiments, and the anion hexafluoroantimonate (SbF(6)(-)). Desirable characteristics of a molecular diffusion probe include predictable diffusion properties, stability, minimum interaction with cellular physiology, very slow penetration into the cells, and sufficiently strong and selective MR and RTI signals. These properties were evaluated using preparations of rat neocortical slices under normal and ischemic conditions, as well as solutions and agarose gel. While both molecules can be detected by MR and RTI, neither proved an ideal candidate. TMA(+) was very stable but it penetrated into the cells and accumulated there within tens of minutes. SbF(6)(-) did not enter the cells as readily but it was not stable, particularly in ischemic tissue and at higher temperatures. Its presence also resulted in a decreased extracellular volume. These probe properties help to interpret previously published MR data on TMA(+) diffusion and might play a role in other diffusion experiments obtained with them.

Diffusion of flexible random-coil dextran polymers measured in anisotropic brain extracellular space by integrative optical imaging.

There are a limited number of methods available to quantify the extracellular diffusion of macromolecules in an anisotropic brain region, e.g., an area containing numerous aligned fibers where diffusion is faster along the fibers than across. We applied the integrative optical imaging method to measure diffusion of the fluorophore Alexa Fluor 488 (molecular weight (MW) 547) and fluorophore-labeled flexible random-coil dextran polymers (dex3, MW 3000; dex75, MW 75,000; dex282, MW 282,000; dex525, MW 525,000) in the extracellular space (ECS) of the anisotropic molecular layer of the isolated turtle cerebellum. For all molecules, two-dimensional images acquired an elliptical shape with major and minor axes oriented along and across, respectively, the unmyelinated parallel fibers. The effective diffusion coefficients, D*(major) and D*(minor), decreased with molecular size. The diffusion anisotropy ratio (DAR = D*(major)/D*(minor)) increased for Alexa Fluor 488 through dex75 but then unexpectedly reached a plateau. We argue that dex282 and dex525 approach the ECS width and deform to diffuse. In support of this concept, scaling theory shows the diffusion behavior of dex282 and dex525 to be consistent with transition to a reptation regime, and estimates the average ECS width at approximately 31 nm. These findings have implications for the interstitial transport of molecules and drugs, and for modeling neurotransmitter diffusion during ectopic release and spillover.

Macromolecular white matter abnormalities in geriatric depression: a magnetization transfer imaging study.

OBJECTIVE: Geriatric depression consists of complex and heterogeneous behaviors unlikely to be caused by a single brain lesion. However, abnormalities in specific brain structures and their interconnections may confer vulnerability to the development of late-life depression. The objective of this study was to identify subtle white matter abnormalities in late-life depression. DESIGN: The authors used magnetization transfer ratio (MTR) imaging, a technique that is thought primarily to reflect myelin integrity, to examine the hypothesis that individuals with late-life depression would exhibit white matter abnormalities in frontostriatal and limbic regions. SETTING: The study was conducted in a university-based, geriatric psychiatry clinic. PARTICIPANTS: Fifty-five older patients with major depression and 24 elderly comparison subjects were assessed. MEASUREMENT: Voxel-based analysis of MTR data were conducted with a general linear model using age as a covariate. RESULTS: Relative to comparison subjects, patients demonstrated lower MTR in multiple left hemisphere frontostriatal and limbic regions, including white matter lateral to the lentiform nuclei, dorsolateral and dorsomedial prefrontal, dorsal anterior cingulate, subcallosal, periamygdalar, insular, and posterior cingulate regions. Depressed patients had lower MTR in additional left hemisphere locales including the thalamus, splenium of the corpus callosum, inferior parietal, precuneus, and middle occipital white matter regions. CONCLUSION: These findings suggest that geriatric depression may be characterized by reduced myelin integrity in specific aspects of frontostriatal and limbic networks, and complement diffusion tensor studies of geriatric depression that indicate decreased organization of white matter fibers in specific frontal and temporal regions.

Dynamic relationship between neurostimulation and N-acetylaspartate metabolism in the human visual cortex: evidence that NAA functions as a molecular water pump during visual stimulation.

N-acetyl-l-aspartic acid (NAA), an amino acid synthesized and stored primarily in neurons in the brain, has been proposed to be a molecular water pump (MWP) whose function is to rapidly remove water from neurons against a water gradient. In this communication, we describe the results of a functional (1)H proton magnetic resonance spectroscopy (fMRS) study, and provide evidence that in the human visual cortex, over a 10-min period of visual stimulation, there are stimulation-induced graded changes in the NAA MRS signal from that of a preceding 10-min baseline period with a decline in the NAA signal of 13.1% by the end of the 10-min stimulation period. Upon cessation of visual stimulation, the NAA signal gradually increases during a 10-min recovery period and once again approaches the baseline level. Because the NAA MRS signal reflects the NAA concentration, these changes indicate rapid focal changes in its concentration, and transient changes in its intercompartmental metabolism. These include its rates of synthesis and efflux from neurons and its hydrolysis by oligodendrocytes. During stimulation, the apparent rate of NAA efflux and hydrolysis increased 14.2 times, from 0.55 to 7.8 micromol g(-1) h(-1). During recovery, the apparent rate of synthesis increased 13.3 times, from 0.55 to 7.3 micromol g(-1) h(-1). The decline in the NAA signal during stimulation suggests that a rapid increase in the rate of NAA-obligated water release to extracellular fluid (ECF) is the initial and seminal event in response to neurostimulation. It is concluded that the NAA metabolic cycle in the visual cortex is intimately linked to rates of neuronal signaling, and that the functional cycle of NAA is associated with its release to ECF, thus supporting the hypothesis that an important function of the NAA metabolic cycle is that of an efflux MWP.

White-matter integrity predicts stroop performance in patients with geriatric depression.

BACKGROUND: This study tested the hypothesis that microstructural white matter abnormalities in frontostriatal-limbic tracts are associated with poor response inhibition on the Stroop task in depressed elders. METHOD: Fifty-one elders with major depression participated in a 12-week escitalopram trial. Diffusion tensor imaging was used to determine fractional anisotropy (FA) in white matter regions. Executive function (response inhibition) was assessed with the Stroop task. Voxelwise correlational analysis was used to examine the relationship between Stroop performance and fractional anisotropy. RESULTS: Significant associations between FA and Stroop color word interference were evident in multiple frontostriatal-limbic regions, including white matter lateral to the anterior and posterior cingulate cortex and white matter in prefrontal, insular, and parahippocampal regions. CONCLUSIONS: These findings suggest that microstructural white matter abnormalities of frontostriatal-limbic networks are associated with executive dysfunction of late-life depression. This observation provides the rationale for examination of specific frontostriatal-limbic pathways in the pathophysiology of geriatric depression.

Principles and limitations of NMR diffusion measurements.

Diffusion spectroscopy, imaging and particularly diffusion tensor imaging have become popular thanks to their numerous clinical and research applications which span from brain stroke evaluation to fiber tracking. With a few exceptions, these methods are rooted in the classic Stejskal-Tanner formula for the diffusion-attenuated signal, usually obtained by solving the Bloch-Torrey partial differential equations. Here we derive the Stejskal-Tanner formula in the simplest possible manner, avoiding integrals and differential equations. This approach makes it easy to understand the origin of the diffusion signal attenuation, the effects of various diffusion sequence parameters, and also the numerous important pitfalls, which are discussed in the last section.

Correction of eddy-current distortions in diffusion tensor images using the known directions and strengths of diffusion gradients.

PURPOSE: To correct eddy-current artifacts in diffusion tensor (DT) images without the need to obtain auxiliary scans for the sole purpose of correction. MATERIALS AND METHODS: DT images are susceptible to distortions caused by eddy currents induced by large diffusion gradients. We propose a new postacquisition correction algorithm that does not require any auxiliary reference scans. It also avoids the problematic procedure of cross-correlating images with significantly different contrasts. A linear model is used to describe the dependence of distortion parameters (translation, scaling, and shear) on the diffusion gradients. The model is solved numerically to provide an individual correction for every diffusion-weighted (DW) image. RESULTS: The assumptions of the linear model were successfully verified in a series of experiments on a silicon oil phantom. The correction obtained for this phantom was compared with correction obtained by a previously published method. The algorithm was then shown to markedly reduce eddy-current distortions in DT images from human subjects. CONCLUSION: The proposed algorithm can accurately correct eddy-current artifacts in DT images. Its principal advantages are that only images with comparable signals and contrasts are cross-correlated, and no additional scans are required.

Interleaved snapshot echo planar imaging of mouse brain at 7.0 T.

Single-shot echo planar imaging (EPI) of a mouse brain at high field is very challenging. Large susceptibility-induced gradients affect much of the brain volume, causing severe image deformations and signal loss. Segmented EPI and other conventional multi-shot approaches alleviate these problems but suffer from lower temporal resolution and motion artifacts. We demonstrate that interleaved snapshot EPI represents a simple and robust alternative approach and one that is particularly suitable for high-field T2*-weighted functional imaging of a mouse brain. Similarly to segmented multi-shot techniques, it significantly reduces the susceptibility-related artifacts. At the same time, it preserves the high temporal resolution and the snapshot capability of a conventional EPI by acquiring entire image within a single TR period. We discuss implementation details of the interleaved snapshot EPI sequence and the trade-offs involved between the imaging efficiency, the number of interleaved excitation-acquisition blocks and the artifact reduction. To document the sequence utility, murine brain in vivo imaging with the interleaved snapshot EPI method was compared with a conventional EPI. We found that at least five interleaved blocks were necessary to restore the signal in most cortical areas. We also show that a standard global shimming procedure provides sufficient homogeneity for multi-slice interleaved snapshot EPI acquisition. In contrast, the conventional EPI of comparable image quality would be limited to a single slice with highly optimized local shim. Finally, an in vitro comparison with turbo FLASH acquisition shows the interleaved snapshot EPI to have superior time resolution and signal-to-noise ratio.