Extracellular matrix (ECM) is a network of macromolecules which has two forms-perineuronal nets (PNNs) and a diffuse ECM (dECM)-both influence brain development, synapse formation, neuroplasticity, CNS injury and progression of neurodegenerative diseases. ECM remodeling can influence extrasynaptic transmission, mediated by diffusion of neuroactive substances in the extracellular space (ECS). In this study we analyzed how disrupted PNNs and dECM influence brain diffusibility. Two months after oral treatment of rats with 4-methylumbelliferone (4-MU), an inhibitor of hyaluronan (HA) synthesis, we found downregulated staining for PNNs, HA, chondroitin sulfate proteoglycans, and glial fibrillary acidic protein. These changes were enhanced after 4 and 6 months and were reversible after a normal diet. Morphometric analysis further indicated atrophy of astrocytes. Using real-time iontophoretic method dysregulation of ECM resulted in increased ECS volume fraction α in the somatosensory cortex by 35%, from α = 0.20 in control rats to α = 0.27 after the 4-MU diet. Diffusion-weighted magnetic resonance imaging revealed a decrease of mean diffusivity and fractional anisotropy (FA) in the cortex, hippocampus, thalamus, pallidum, and spinal cord. This study shows the increase in ECS volume, a loss of FA, and changes in astrocytes due to modulation of PNNs and dECM that could affect extrasynaptic transmission, cell-to-cell communication, and neural plasticity.

• Keywords
• extracellular diffusion, extracellular matrix, extracellular transmission, hyaluronan synthase, perineuronal nets, plasticity,
• MeSH
• Astrocytes drug effects   MeSH
• Extracellular Matrix * drug effects   metabolism   pathology   MeSH
• Extracellular Space * drug effects   metabolism   MeSH
• Glial Fibrillary Acidic Protein metabolism   MeSH
• Hymecromone pharmacology   MeSH
• Rats MeSH
• Hyaluronic Acid metabolism   MeSH
• Brain * drug effects   metabolism   MeSH
• Nerve Net * drug effects   pathology   MeSH
• Rats, Sprague-Dawley MeSH
• Rats, Wistar MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Male MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Glial Fibrillary Acidic Protein MeSH
• Hymecromone MeSH
• Hyaluronic Acid MeSH

Hapln4 is a link protein which stabilizes the binding between lecticans and hyaluronan in perineuronal nets (PNNs) in specific brain regions, including the medial nucleus of the trapezoid body (MNTB). The aim of this study was: (1) to reveal possible age-related alterations in the extracellular matrix composition in the MNTB and inferior colliculus, which was devoid of Hapln4 and served as a negative control, (2) to determine the impact of the Hapln4 deletion on the values of the ECS diffusion parameters in young and aged animals and (3) to verify that PNNs moderate age-related changes in the ECS diffusion, and that Hapln4-brevican complex is indispensable for the correct protective function of the PNNs. To achieve this, we evaluated the ECS diffusion parameters using the real-time iontophoretic method in the selected region in young adult (3 to 6-months-old) and aged (12 to 18-months-old) wild type and Hapln4 knock-out (KO) mice. The results were correlated with an immunohistochemical analysis of the ECM composition and astrocyte morphology. We report that the ECM composition is altered in the aged MNTB and aging is a critical point, revealing the effect of Hapln4 deficiency on the ECS diffusion. All of our findings support the hypothesis that the ECM changes in the MNTB of aged KO animals affect the ECS parameters indirectly, via morphological changes of astrocytes, which are in direct contact with synapses and can be influenced by the ongoing synaptic transmission altered by shifts in the ECM composition.

• Keywords
• Aging, Diffusion, Extracellular matrix, Extracellular space, Hapln4,
• MeSH
• Trapezoid Body metabolism   pathology   MeSH
• Diffusion * MeSH
• Extracellular Matrix Proteins deficiency   MeSH
• Extracellular Matrix metabolism   pathology   MeSH
• Extracellular Space metabolism   MeSH
• Mice, Inbred C57BL MeSH
• Mice, Knockout MeSH
• Mice MeSH
• Protein Deficiency metabolism   pathology   MeSH
• Organ Culture Techniques MeSH
• Peripheral Nerves metabolism   pathology   MeSH
• Nerve Tissue Proteins deficiency   MeSH
• Auditory Pathways metabolism   pathology   MeSH
• Aging metabolism   pathology   MeSH
• Animals MeSH
• Check Tag
• Male MeSH
• Mice MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Extracellular Matrix Proteins MeSH
• Hapln4 protein, mouse MeSH Browser
• Nerve Tissue Proteins MeSH

Diffusion in the extracellular space (ECS) of the brain is constrained by the volume fraction and the tortuosity and a modified diffusion equation represents the transport behavior of many molecules in the brain. Deviations from the equation reveal loss of molecules across the blood-brain barrier, through cellular uptake, binding, or other mechanisms. Early diffusion measurements used radiolabeled sucrose and other tracers. Presently, the real-time iontophoresis (RTI) method is employed for small ions and the integrative optical imaging (IOI) method for fluorescent macromolecules, including dextrans or proteins. Theoretical models and simulations of the ECS have explored the influence of ECS geometry, effects of dead-space microdomains, extracellular matrix, and interaction of macromolecules with ECS channels. Extensive experimental studies with the RTI method employing the cation tetramethylammonium (TMA) in normal brain tissue show that the volume fraction of the ECS typically is approximately 20% and the tortuosity is approximately 1.6 (i.e., free diffusion coefficient of TMA is reduced by 2.6), although there are regional variations. These parameters change during development and aging. Diffusion properties have been characterized in several interventions, including brain stimulation, osmotic challenge, and knockout of extracellular matrix components. Measurements have also been made during ischemia, in models of Alzheimer's and Parkinson's diseases, and in human gliomas. Overall, these studies improve our conception of ECS structure and the roles of glia and extracellular matrix in modulating the ECS microenvironment. Knowledge of ECS diffusion properties is valuable in contexts ranging from understanding extrasynaptic volume transmission to the development of paradigms for drug delivery to the brain.

• MeSH
• Diffusion MeSH
• Extracellular Space chemistry   diagnostic imaging   physiology   MeSH
• Quaternary Ammonium Compounds MeSH
• Humans MeSH
• Brain Chemistry physiology   MeSH
• Brain cytology   physiology   MeSH
• Neuroglia physiology   MeSH
• Neurons physiology   MeSH
• Radionuclide Imaging MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• Quaternary Ammonium Compounds MeSH
• tetramethylammonium MeSH Browser

Diffusion parameters of the extracellular space (ECS) are changed in many brain pathologies, disturbing synaptic as well as extrasynaptic "volume" transmission, which is based on the diffusion of neuroactive substances in the ECS. Amyloid deposition, neuronal loss, and disturbed synaptic transmission are considered to be the main causes of Alzheimer's disease dementia. We studied diffusion parameters in the cerebral cortex of transgenic APP23 mice, which develop a pathology similar to Alzheimer's disease. The real-time tetramethylammonium (TMA) method and diffusion-weighted MRI were used to measure the ECS volume fraction (alpha = ECS volume/total tissue volume) and the apparent diffusion coefficients (ADCs) of TMA (ADC(TMA)), diffusing exclusively in the ECS and of water (ADC(W)). Measurements were performed in vivo in 6-, 8-, and 17- to 25-month-old hemizygous APP23 male and female mice and age-matched controls. In all 6- to 8-month-old APP23 mice, the mean ECS volume fraction, ADC(TMA), and ADC(W) were not significantly different from age-matched controls (alpha = 0.20 +/- 0.01; ADC(TMA), 580 +/- 16 microm(2).s(-1); ADC(W), 618 +/- 19 microm(2).s(-1)). Aging in 17- to 25-month-old controls was accompanied by a decrease in ECS volume fraction and ADC(W), significantly greater in females than in males, but no changes in ADC(TMA). ECS volume fraction increased (0.22 +/- 0.01) and ADC(TMA) decreased (560 +/- 7 microm(2).s(-1)) in aged APP23 mice. The impaired navigation observed in these animals in the Morris water maze correlated with their plaque load, which was twice as high in females (20%) as in males (10%) and may, together with changed ECS diffusion properties, account for the impaired extrasynaptic transmission and spatial cognition observed in old transgenic females.

• MeSH
• Alzheimer Disease etiology   MeSH
• Amyloid beta-Protein Precursor genetics   physiology   MeSH
• Diffusion MeSH
• Extracellular Space metabolism   MeSH
• Quaternary Ammonium Compounds metabolism   MeSH
• Magnetic Resonance Imaging MeSH
• Disease Models, Animal * MeSH
• Mice, Transgenic MeSH
• Mice MeSH
• Aging pathology   MeSH
• Animals MeSH
• Check Tag
• Male MeSH
• Mice MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Amyloid beta-Protein Precursor MeSH
• Quaternary Ammonium Compounds MeSH
• tetramethylammonium MeSH Browser