Structure of pathological TDP-43 filaments from ALS with FTLD.

The abnormal aggregation of TAR DNA-binding protein 43 kDa (TDP-43) in neurons and glia is the defining pathological hallmark of the neurodegenerative disease amyotrophic lateral sclerosis (ALS) and multiple forms of frontotemporal lobar degeneration (FTLD)1,2. It is also common in other diseases, including Alzheimer's and Parkinson's. No disease-modifying therapies exist for these conditions and early diagnosis is not possible. The structures of pathological TDP-43 aggregates are unknown. Here we used cryo-electron microscopy to determine the structures of aggregated TDP-43 in the frontal and motor cortices of an individual who had ALS with FTLD and from the frontal cortex of a second individual with the same diagnosis. An identical amyloid-like filament structure comprising a single protofilament was found in both brain regions and individuals. The ordered filament core spans residues 282-360 in the TDP-43 low-complexity domain and adopts a previously undescribed double-spiral-shaped fold, which shows no similarity to those of TDP-43 filaments formed in vitro3,4. An abundance of glycine and neutral polar residues facilitates numerous turns and restricts ß-strand length, which results in an absence of ß-sheet stacking that is associated with cross-ß amyloid structure. An uneven distribution of residues gives rise to structurally and chemically distinct surfaces that face external densities and suggest possible ligand-binding sites. This work enhances our understanding of the molecular pathogenesis of ALS and FTLD and informs the development of diagnostic and therapeutic agents that target aggregated TDP-43.

Comparative Ultrastructural Analysis of Thalamocortical Innervation of the Primary Motor Cortex and Supplementary Motor Area in Control and MPTP-Treated Parkinsonian Monkeys.

The synaptic organization of thalamic inputs to motor cortices remains poorly understood in primates. Thus, we compared the regional and synaptic connections of vGluT2-positive thalamocortical glutamatergic terminals in the supplementary motor area (SMA) and the primary motor cortex (M1) between control and MPTP-treated parkinsonian monkeys. In controls, vGluT2-containing fibers and terminal-like profiles invaded layer II-III and Vb of M1 and SMA. A significant reduction of vGluT2 labeling was found in layer Vb, but not in layer II-III, of parkinsonian animals, suggesting a potential thalamic denervation of deep cortical layers in parkinsonism. There was a significant difference in the pattern of synaptic connectivity in layers II-III, but not in layer Vb, between M1 and SMA of control monkeys. However, this difference was abolished in parkinsonian animals. No major difference was found in the proportion of perforated versus macular post-synaptic densities at thalamocortical synapses between control and parkinsonian monkeys in both cortical regions, except for a slight increase in the prevalence of perforated axo-dendritic synapses in the SMA of parkinsonian monkeys. Our findings suggest that disruption of the thalamic innervation of M1 and SMA may underlie pathophysiological changes of the motor thalamocortical loop in the state of parkinsonism.

Exercise and microstructural changes in the motor cortex of older adults.

Exercise has been shown to counteract age-related volume decreases in the human brain, and in this imaging study, we ask whether the same holds true for the microstructure of the cortex. Healthy older adults (n = 47, 65-90 years old) either exercised three times a week on a stationary bike or maintained their usual physical routine over a 12-week period. Quantitative longitudinal relaxation rate (R1 ) magnetic resonance imaging (MRI) maps were made at baseline and after the 12-week intervention. R1 is commonly taken to reflect cortical myelin density. The change in R1 (ΔR1 ) was significantly increased in a region of interest (ROI) in the primary motor cortex containing motor outputs to the leg musculature in the exercise group relative to the control group (p = .04). The change in R1 in this ROI correlated with an increase in oxygen consumption at the first ventilatory threshold (VT1) (p = .04), a marker of improvement in submaximal aerobic performance. An exploratory analysis across the cortex suggested that the correlation was predominately confined to the leg representation in the motor cortex. This study suggests that microstructural declines in the cortex of older adults may be staved off by exercise.

Transgenic minipig model of Huntington's disease exhibiting gradually progressing neurodegeneration.

Recently developed therapeutic approaches for the treatment of Huntington's disease (HD) require preclinical testing in large animal models. The minipig is a suitable experimental animal because of its large gyrencephalic brain, body weight of 70-100 kg, long lifespan, and anatomical, physiological and metabolic resemblance to humans. The Libechov transgenic minipig model for HD (TgHD) has proven useful for proof of concept of developing new therapies. However, to evaluate the efficacy of different therapies on disease progression, a broader phenotypic characterization of the TgHD minipig is needed. In this study, we analyzed the brain tissues of TgHD minipigs at the age of 48 and 60-70 months, and compared them to wild-type animals. We were able to demonstrate not only an accumulation of different forms of mutant huntingtin (mHTT) in TgHD brain, but also pathological changes associated with cellular damage caused by mHTT. At 48 months, we detected pathological changes that included the demyelination of brain white matter, loss of function of striatal neurons in the putamen and activation of microglia. At 60-70 months, we found a clear marker of neurodegeneration: significant cell loss detected in the caudate nucleus, putamen and cortex. This was accompanied by clusters of structures accumulating in the neurites of some neurons, a sign of their degeneration that is also seen in Alzheimer's disease, and a significant activation of astrocytes. In summary, our data demonstrate age-dependent neuropathology with later onset of neurodegeneration in TgHD minipigs.

Voluntary wheel running promotes myelination in the motor cortex through Wnt signaling in mice.

Myelin of the central nervous system exhibits strong plasticity, and skill learning exercise promotes oligodendrogenesis and adaptive myelination. Increasing evidence shows that brain structures and functions are affected by physical activity. However, the impact of voluntary physical activity on central myelination and its underlying mechanism remains unclear. The present study aimed to investigate the effect of voluntary wheel running (VWR) on central oligodendrogenesis and adaptive myelination in mice. Adult C57BL/6 J mice were placed in running wheels and allowed for voluntary running 2 weeks. Myelin levels in the central nervous system were detected using western blotting, qRT-PCR, immunohistochemical staining, and electron microscopy. Oligodendrocyte precursor cells (OPCs) and oligodendrocytes (OLs) were detected using immunohistochemical staining and 5-bromo-2-deoxyuridine (BrdU) assays. Motor abilities of the animals were examined using open-field, rotarod running, and beam-walking behavioral paradigms. Vital molecules of Wnt signaling were detected, and the involvement of such molecules was verified using in vitro culture of OPCs. Our results showed that VWR significantly enhanced the myelination in the motor cortex. VWR promoted the proliferation and differentiation of OPCs, and the maturation of OLs. The VWR-regulated myelination was associated with the improved motor skill and decreased mRNA level of Wnt3a/9a, whereas stimulation of Wnt signaling pathway with Wnt3a or Wnt9a suppressed OPCs proliferation and differentiation in vitro. The present study demonstrated that physical activity is highly efficient at promoting myelination in the motor cortex, by enhancing the proliferation of OPCs and accelerating the generation of myelin, providing a step forward in understanding the beneficial effects of physical activity on central myelination and its underlying mechanism.

Posterior thalamic nucleus axon terminals have different structure and functional impact in the motor and somatosensory vibrissal cortices.

Rodents extract information about nearby objects from the movement of their whiskers through dynamic computations that are carried out by a network of forebrain structures that includes the thalamus and the primary sensory (S1BF) and motor (M1wk) whisker cortices. The posterior nucleus (Po), a higher order thalamic nucleus, is a key hub of this network, receiving cortical and brainstem sensory inputs and innervating both motor and sensory whisker-related cortical areas. In a recent study in rats, we showed that Po inputs differently impact sensory processing in S1BF and M1wk. Here, in C57BL/6 mice, we measured Po synaptic bouton layer distribution and size, compared cortical unit response latencies to "in vivo" Po activation, and pharmacologically examined the glutamatergic receptor mechanisms involved. We found that, in S1BF, a large majority (56%) of Po axon varicosities are located in layer (L)5a and only 12% in L2-L4, whereas in M1wk this proportion is inverted to 18% and 55%, respectively. Light and electron microscopic measurements showed that Po synaptic boutons in M1wk layers 3-4 are significantly larger (~ 50%) than those in S1BF L5a. Electrical Po stimulation elicits different area-specific response patterns. In S1BF, responses show weak or no facilitation, and involve both ionotropic and metabotropic glutamate receptors, whereas in M1wk, unit responses exhibit facilitation to repetitive stimulation and involve ionotropic NMDA glutamate receptors. Because of the different laminar distribution of axon terminals, synaptic bouton size and receptor mechanisms, the impact of Po signals on M1wk and S1BF, although simultaneous, is likely to be markedly different.

PRRT2 deficiency induces paroxysmal kinesigenic dyskinesia by influencing synaptic function in the primary motor cortex of rats.

Proline-rich transmembrane protein 2 (PRRT2) was identified as the causative gene of paroxysmal kinesigenic choreoathetosis (PKC) as well as various other neurological diseases. However, the molecular mechanisms of how mutant PRRT2 leads to abnormal synaptic function and triggers PKC are still obscure. We generated a Prrt2 truncated mutant rat model which shows spontaneous PKC-like attacks with a relative low frequency as well as increased susceptibility to pentylenetetrazol (PTZ)-induced seizures. We demonstrate that PRRT2 is expressed on both pre- and post-synaptic membranes in the M1 cortex. PRRT2 negatively regulates SNARE complex assembly through interaction with SNAP25, STX1A, and VAMP2. In the M1 cortex of the rat model, release of amino acid neurotransmitters is increased. Protein levels of glutamate receptor subunit GRIA1 are significantly increased in PRRT2 mutant rats, while GABA receptor subunits GABRA1 are significantly reduced. Both frequency and amplitude of mEPSC are significantly increased, while amplitude of mIPSC is decreased and the ratio of mEPSC/mIPSC is significantly increased. The balance between excitatory and inhibitory neuronal activity is disrupted, which could lead to abnormal neuronal hyperexcitability. These results provide new insights into the function of PRRT2 in synaptic transmission and movement control, as well as the pathogenic mechanism underlying PKC.

Modulating Regional Motor Cortical Excitability with Noninvasive Brain Stimulation Results in Neurochemical Changes in Bilateral Motor Cortices.

Learning a novel motor skill is dependent both on regional changes within the primary motor cortex (M1) contralateral to the active hand and also on modulation between and within anatomically distant but functionally connected brain regions. Interregional changes are particularly important in functional recovery after stroke, when critical plastic changes underpinning behavioral improvements are observed in both ipsilesional and contralesional M1s. It is increasingly understood that reduction in GABA in the contralateral M1 is necessary to allow learning of a motor task. However, the physiological mechanisms underpinning plasticity within other brain regions, most importantly the ipsilateral M1, are not well understood. Here, we used concurrent two-voxel magnetic resonance spectroscopy to simultaneously quantify changes in neurochemicals within left and right M1s in healthy humans of both sexes in response to transcranial direct current stimulation (tDCS) applied to left M1. We demonstrated a decrease in GABA in both the stimulated (left) and nonstimulated (right) M1 after anodal tDCS, whereas a decrease in GABA was only observed in nonstimulated M1 after cathodal stimulation. This GABA decrease in the nonstimulated M1 during cathodal tDCS was negatively correlated with microstructure of M1:M1 callosal fibers, as quantified by diffusion MRI, suggesting that structural features of these fibers may mediate GABA decrease in the unstimulated region. We found no significant changes in glutamate. Together, these findings shed light on the interactions between the two major network nodes underpinning motor plasticity, offering a potential framework from which to optimize future interventions to improve motor function after stroke.SIGNIFICANCE STATEMENT Learning of new motor skills depends on modulation both within and between brain regions. Here, we use a novel two-voxel magnetic resonance spectroscopy approach to quantify GABA and glutamate changes concurrently within the left and right primary motor cortex (M1) during three commonly used transcranial direct current stimulation montages: anodal, cathodal, and bilateral. We also examined how the neurochemical changes in the unstimulated hemisphere were related to white matter microstructure between the two M1s. Our results provide insights into the neurochemical changes underlying motor plasticity and may therefore assist in the development of further adjunct therapies.

Oxytocin curbs calorie intake via food-specific increases in the activity of brain areas that process reward and establish cognitive control.

The hypothalamic neurohormone oxytocin decreases food intake via largely unexplored mechanisms. We investigated the central nervous mediation of oxytocin's hypophagic effect in comparison to its impact on the processing of generalized rewards. Fifteen fasted normal-weight, young men received intranasal oxytocin (24 IU) or placebo before functional magnetic resonance imaging (fMRI) measurements of brain activity during exposure to food stimuli and a monetary incentive delay task (MID). Subsequently, ad-libitum breakfast intake was assessed. Oxytocin compared to placebo increased activity in the ventromedial prefrontal cortex, supplementary motor area, anterior cingulate, and ventrolateral prefrontal cortices in response to high- vs. low-calorie food images in the fasted state, and reduced calorie intake by 12%. During anticipation of monetary rewards, oxytocin compared to placebo augmented striatal, orbitofrontal and insular activity without altering MID performance. We conclude that during the anticipation of generalized rewards, oxytocin stimulates dopaminergic reward-processing circuits. In contrast, oxytocin restrains food intake by enhancing the activity of brain regions that exert cognitive control, while concomitantly increasing the activity of structures that process food reward value. This pattern points towards a specific role of oxytocin in the regulation of eating behaviour in humans that might be of relevance for potential clinical applications.

Ultrastructural evidence for synaptic scaling across the wake/sleep cycle.

It is assumed that synaptic strengthening and weakening balance throughout learning to avoid runaway potentiation and memory interference. However, energetic and informational considerations suggest that potentiation should occur primarily during wake, when animals learn, and depression should occur during sleep. We measured 6920 synapses in mouse motor and sensory cortices using three-dimensional electron microscopy. The axon-spine interface (ASI) decreased ~18% after sleep compared with wake. This decrease was proportional to ASI size, which is indicative of scaling. Scaling was selective, sparing synapses that were large and lacked recycling endosomes. Similar scaling occurred for spine head volume, suggesting a distinction between weaker, more plastic synapses (~80%) and stronger, more stable synapses. These results support the hypothesis that a core function of sleep is to renormalize overall synaptic strength increased by wake.

An Ultrastructural Study of the Thalamic Input to Layer 4 of Primary Motor and Primary Somatosensory Cortex in the Mouse.

The traditional classification of primary motor cortex (M1) as an agranular area has been challenged recently when a functional layer 4 (L4) was reported in M1. L4 is the principal target for thalamic input in sensory areas, which raises the question of how thalamocortical synapses formed in M1 in the mouse compare with those in neighboring sensory cortex (S1). We identified thalamic boutons by their immunoreactivity for the vesicular glutamate transporter 2 (VGluT2) and performed unbiased disector counts from electron micrographs. We discovered that the thalamus contributed proportionately only half as many synapses to the local circuitry of L4 in M1 compared with S1. Furthermore, thalamic boutons in M1 targeted spiny dendrites exclusively, whereas ∼9% of synapses were formed with dendrites of smooth neurons in S1. VGluT2+ boutons in M1 were smaller and formed fewer synapses per bouton on average (1.3 vs 2.1) than those in S1, but VGluT2+ synapses in M1 were larger than in S1 (median postsynaptic density areas of 0.064 µm2 vs 0.042 µm2). In M1 and S1, thalamic synapses formed only a small fraction (12.1% and 17.2%, respectively) of all of the asymmetric synapses in L4. The functional role of the thalamic input to L4 in M1 has largely been neglected, but our data suggest that, as in S1, the thalamic input is amplified by the recurrent excitatory connections of the L4 circuits. The lack of direct thalamic input to inhibitory neurons in M1 may indicate temporal differences in the inhibitory gating in L4 of M1 versus S1.SIGNIFICANCE STATEMENT Classical interpretations of the function of primary motor cortex (M1) emphasize its lack of the granular layer 4 (L4) typical of sensory cortices. However, we show here that, like sensory cortex (S1), mouse M1 also has the canonical circuit motif of a core thalamic input to the middle cortical layer and that thalamocortical synapses form a small fraction (M1: 12%; S1: 17%) of all asymmetric synapses in L4 of both areas. Amplification of thalamic input by recurrent local circuits is thus likely to be a significant mechanism in both areas. Unlike M1, where thalamocortical boutons typically form a single synapse, thalamocortical boutons in S1 usually formed multiple synapses, which means they can be identified with high probability in the electron microscope without specific labeling.

Remodeling of Dendritic Spines in the Avian Vocal Motor Cortex Following Deafening Depends on the Basal Ganglia Circuit.

Deafening elicits a deterioration of learned vocalization, in both humans and songbirds. In songbirds, learned vocal plasticity has been shown to depend on the basal ganglia-cortical circuit, but the underlying cellular basis remains to be clarified. Using confocal imaging and electron microscopy, we examined the effect of deafening on dendritic spines in avian vocal motor cortex, the robust nucleus of the arcopallium (RA), and investigated the role of the basal ganglia circuit in motor cortex plasticity. We found rapid structural changes to RA dendritic spines in response to hearing loss, accompanied by learned song degradation. In particular, the morphological characters of RA spine synaptic contacts between 2 major pathways were altered differently. However, experimental disruption of the basal ganglia circuit, through lesions in song-specialized basal ganglia nucleus Area X, largely prevented both the observed changes to RA dendritic spines and the song deterioration after hearing loss. Our results provide cellular evidence to highlight a key role of the basal ganglia circuit in the motor cortical plasticity that underlies learned vocal plasticity.

Rescue of the Functional Alterations of Motor Cortical Circuits in Arginase Deficiency by Neonatal Gene Therapy.

UNLABELLED: Arginase 1 deficiency is a urea cycle disorder associated with hyperargininemia, spastic diplegia, loss of ambulation, intellectual disability, and seizures. To gain insight on how loss of arginase expression affects the excitability and synaptic connectivity of the cortical neurons in the developing brain, we used anatomical, ultrastructural, and electrophysiological techniques to determine how single-copy and double-copy arginase deletion affects cortical circuits in mice. We find that the loss of arginase 1 expression results in decreased dendritic complexity, decreased excitatory and inhibitory synapse numbers, decreased intrinsic excitability, and altered synaptic transmission in layer 5 motor cortical neurons. Hepatic arginase 1 gene therapy using adeno-associated virus rescued nearly all these abnormalities when administered to neonatal homozygous knock-out animals. Therefore, gene therapeutic strategies can reverse physiological and anatomical markers of arginase 1 deficiency and therefore may be of therapeutic benefit for the neurological disabilities in this syndrome. SIGNIFICANCE STATEMENT: These studies are one of the few investigations to try to understand the underlying neurological dysfunction that occurs in urea cycle disorders and the only to examine arginase deficiency. We have demonstrated by multiple modalities that, in murine layer 5 cortical neurons, a gradation of abnormalities exists based on the functional copy number of arginase: intrinsic excitability is altered, there is decreased density in asymmetrical and perisomatic synapses, and analysis of the dendritic complexity is lowest in the homozygous knock-out. With neonatal administration of adeno-associated virus expressing arginase, there is near-total recovery of the abnormalities in neurons and cortical circuits, supporting the concept that neonatal gene therapy may prevent the functional abnormalities that occur in arginase deficiency.

Imaging the time-integrated cerebral metabolic activity with subcellular resolution through nanometer-scale detection of biosynthetic products deriving from (13)C-glucose.

Glucose is the primary source of energy for the brain but also an important source of building blocks for proteins, lipids, and nucleic acids. Little is known about the use of glucose for biosynthesis in tissues at the cellular level. We demonstrate that local cerebral metabolic activity can be mapped in mouse brain tissue by quantitatively imaging the biosynthetic products deriving from [U-(13)C]glucose metabolism using a combination of in situ electron microscopy and secondary ion mass-spectroscopy (NanoSIMS). Images of the (13)C-label incorporated into cerebral ultrastructure with ca. 100 nm resolution allowed us to determine the timescale on which the metabolic products of glucose are incorporated into different cells, their sub-compartments and organelles. These were mapped in astrocytes and neurons in the different layers of the motor cortex. We see evidence for high metabolic activity in neurons via the nucleus (13)C enrichment. We observe that in all the major cell compartments, such as e.g. nucleus and Golgi apparatus, neurons incorporate substantially higher concentrations of (13)C-label than astrocytes.

Experience with the "good" limb induces aberrant synaptic plasticity in the perilesion cortex after stroke.

Following unilateral stroke, the contralateral (paretic) body side is often severely impaired, and individuals naturally learn to rely more on the nonparetic body side, which involves learning new skills with it. Such compensatory hyper-reliance on the "good" body side, however, can limit functional improvements of the paretic side. In rats, motor skill training with the nonparetic forelimb (NPT) following a unilateral infarct lessens the efficacy of rehabilitative training, and reduces neuronal activation in perilesion motor cortex. However, the underlying mechanisms remain unclear. In the present study, we investigated how forelimb movement representations and synaptic restructuring in perilesion motor cortex respond to NPT and their relationship with behavioral outcomes. Forelimb representations were diminished as a result of NPT, as revealed with intracortical microstimulation mapping. Using transmission electron microscopy and stereological analyses, we found that densities of axodendritic synapses, especially axo-spinous synapses, as well as multiple synaptic boutons were increased in the perilesion cortex by NPT. The synaptic density was negatively correlated with the functional outcome of the paretic limb, as revealed in reaching performance. Furthermore, in animals with NPT, there was dissociation between astrocytic morphological features and axo-spinous synaptic density in perilesion motor cortex, compared with controls. These findings demonstrate that skill learning with the nonparetic limb following unilateral brain damage results in aberrant synaptogenesis, potentially of transcallosal projections, and this seems to hamper the functionality of the perilesion motor cortex and the paretic forelimb.

Histological-MRI correlation in the primary motor cortex of patients with amyotrophic lateral sclerosis.

PURPOSE: To establish the relationship between ALS histopathology and quantitative MRI metrics. MATERIALS AND METHODS: ALS patients (N = 8) in advanced stages of the disease were enrolled and, immediately after death, the brain of each patient was removed. Freshly excised ALS tissue was imaged at 3.0 Tesla with T1 and T2 mapping protocols and subsequently stained with astrocyte, myelin, and neuronal markers. Measures of ALS histological stains were compared with the internal control (primary visual cortex) and longitudinal parametric maps. RESULTS: Post-mortem T1 -weighted images demonstrate diminished contrast between gray and white matter and alterations in T1 relaxation within the primary motor cortex. An increase in astrocyte number and reactivity as well as evident neuronal loss, a decrease in axonal density, and unraveling of the myelin sheaths in subcortical white matter were found in the ALS primary motor cortex exhibiting significant T1 relaxation and contrast changes. CONCLUSION: This study provides a histopathological basis for differences in MR T1 contrast and relaxation seen in the ALS brain.

A genuine layer 4 in motor cortex with prototypical synaptic circuit connectivity.

The motor cortex (M1) is classically considered an agranular area, lacking a distinct layer 4 (L4). Here, we tested the idea that M1, despite lacking a cytoarchitecturally visible L4, nevertheless possesses its equivalent in the form of excitatory neurons with input-output circuits like those of the L4 neurons in sensory areas. Consistent with this idea, we found that neurons located in a thin laminar zone at the L3/5A border in the forelimb area of mouse M1 have multiple L4-like synaptic connections: excitatory input from thalamus, largely unidirectional excitatory outputs to L2/3 pyramidal neurons, and relatively weak long-range corticocortical inputs and outputs. M1-L4 neurons were electrophysiologically diverse but morphologically uniform, with pyramidal-type dendritic arbors and locally ramifying axons, including branches extending into L2/3. Our findings therefore identify pyramidal neurons in M1 with the expected prototypical circuit properties of excitatory L4 neurons, and question the traditional assumption that motor cortex lacks this layer.

[Neuronal populations: sources of the corticothalamic projections to the partially deafferented ventrolateral thalamic nucleus].

The features of distribution and morphological structure of the motor cortex neuronal populations projecting to the cerebellar-recipient ventrolateral nucleus of the thalamus after its partial deafferentation were studied in adult cats. The partial deafferentation of the ventrolateral nucleus was evoked by preliminary (three months) electrolytic destruction of the contralateral interpositus nucleus of the cerebellum. The method of retrograde axonal transport with local introductions of the marker was used. All labeled neurons were presented by populations of non-pyramidal neurons and small and medium-sized pyramids, which were distributed in the deep cortical layers: in a lower layer division of V and mostly in layer VI. The labeled neurons were observed in cortical fields 4γ and field 6αß. The data obtained showed no structural reorganization of cortical projections to the deafferented ventrolateral nucleus of the thalamus. It is assumed that this is due to the high degree of specialization of the studied system, triggering the motor program. Neuroplastic changes manifested in the abnormal transformation of proximal portions of dendrites and presence of a large number of "paired" pyramidal neurons compared to intact animals.

Compromised blood-brain barrier competence in remote brain areas in ischemic stroke rats at the chronic stage.

Stroke is a life-threatening disease leading to long-term disability in stroke survivors. Cerebral functional insufficiency in chronic stroke might be due to pathological changes in brain areas remote from the initial ischemic lesion, i.e., diaschisis. Previously, we showed that the damaged blood-brain barrier (BBB) was involved in subacute diaschisis. The present study investigated BBB competence in chronic diaschisis by using a transient middle cerebral artery occlusion (tMCAO) rat model. Our results demonstrated significant BBB damage mostly in the ipsilateral striatum and motor cortex in rats at 30 days after tMCAO. The BBB alterations were also determined in the contralateral hemisphere via ultrastructural and immunohistochemical analyses. Major BBB pathological changes in contralateral remote striatum and motor cortex areas included 1) vacuolated endothelial cells containing large autophagosomes, 2) degenerated pericytes displaying mitochondria with cristae disruption, 3) degenerated astrocytes and perivascular edema, 4) Evans blue extravasation, and 5) appearance of parenchymal astrogliosis. Discrete analyses of striatal and motor cortex areas revealed significantly higher autophagosome accumulation in capillaries of ventral striatum and astrogliosis in dorsal striatum in both cerebral hemispheres. These widespread microvascular alterations in ipsilateral and contralateral brain hemispheres suggest persistent and/or continued BBB damage in chronic ischemia. The pathological changes in remote brain areas likely indicate chronic ischemic diaschisis, which should be considered in the development of treatment strategies for stroke.