Single-Nucleus Chromatin Accessibility Landscape Reveals Diversity in Regulatory Regions Across Distinct Adult Rat Cortex.

Rats have been widely used as an experimental organism in psychological, pharmacological, and behavioral studies by modeling human diseases such as neurological disorders. It is critical to identify and characterize cell fate determinants and their regulatory mechanisms in single-cell resolutions across rat brain regions. Here, we applied droplet-based single-nucleus assay for transposase-accessible chromatin using sequencing (snATAC-seq) to systematically profile the single-cell chromatin accessibility across four dissected brain areas in adult Sprague-Dawley (SD) rats with a total of 59,023 single nuclei and identified 16 distinct cell types. Interestingly, we found that different cortex regions exhibit diversity in both cellular compositions and gene regulatory regions. Several cell-type-specific transcription factors (TFs), including SPI1, KLF4, KLF6, and NEUROD2, have been shown to play important roles during the pathogenesis of various neurological diseases, such as Alzheimer's disease (AD), astrocytic gliomas, autism spectrum disorder (ASD), and intellectual disabilities. Therefore, our single-nucleus atlas of rat cortex could serve as an invaluable resource for dissecting the regulatory mechanisms underlying diverse cortex cell fates and further revealing the regulatory networks of neuropathogenesis.

Alpha-Lipoic Acid Attenuates Cadmium- and Lead-Induced Neurotoxicity by Inhibiting Both Endoplasmic-Reticulum Stress and Activation of Fas/FasL and Mitochondrial Apoptotic Pathways in Rat Cerebral Cortex.

Although many studies have reported toxic effects of cadmium (Cd) and lead (Pb) in the central nervous system, few studies have investigated the combined toxicity of Cd and Pb. The mechanisms by which these combined heavy metals induce toxicity, as well as effective means to exert neuroprotection from these agents, remain poorly understood. To investigate the protective effects of alpha-lipoic acid (α-LA) on Cd- and/or Pb-induced cortical damage in rats, 48 Sprague-Dawley rats were exposed to drinking water containing 50 mg/L of Cd and/or 300 mg/L of Pb for 12 weeks, in the presence or absence of α-LA co-treatment (50 mg/kg) via gavage. We observed that exposure to Cd and/or Pb decreased the brain weight/body weight ratio and increased Cd and/or Pb contents as well as ultrastructural damage to the cerebral cortex. Cd and/or Pb also induced endoplasmic-reticulum (ER) stress and activated Fas (CD95/APO-1)/Fas ligand (FasL) and mitochondrial apoptotic pathways. Furthermore, co-treatment of Cd and Pb further exacerbated part of these phenotypes than treatment of Cd or Pb alone. However, simultaneous supplementation with α-LA attenuated Cd and/or Pb-induced neurotoxicity by increasing the brain weight/body weight ratio, reducing Cd and/or Pb contents, ameliorating both nuclear/mitochondrial damage and ER stress, and attenuating activation of Fas/FasL and mitochondrial apoptotic pathways. Collectively, our results indicate that the accumulation of Cd and/or Pb causes cortical damage and that α-LA exerts protection against Cd- and/or Pb-induced neurotoxicity. These findings highlight that α-LA may be exploited for the treatment and prevention of Cd- and/or Pb-induced neurotoxicity.

Pulsatile flow drivers in brain parenchyma and perivascular spaces: a resistance network model study.

BACKGROUND: In animal models, dissolved compounds in the subarachnoid space and parenchyma have been found to preferentially transport through the cortex perivascular spaces (PVS) but the transport phenomena involved are unclear. METHODS: In this study two hydraulic network models were used to predict fluid motion produced by blood vessel pulsations and estimate the contribution made to solute transport in PVS and parenchyma. The effect of varying pulse amplitude and timing, PVS dimensions, and tissue hydraulic conductivity on fluid motion was investigated. RESULTS: Periodic vessel pulses resulted in oscillatory fluid motion in PVS and parenchyma but no net flow over time. For baseline parameters, PVS and parenchyma peak fluid velocity was on the order of 10 µm/s and 1 nm/s, with corresponding Peclet numbers below 103 and 10-1 respectively. Peak fluid velocity in the PVS and parenchyma tended to increase with increasing pulse amplitude and vessel size, and exhibited asymptotic relationships with hydraulic conductivity. CONCLUSIONS: Solute transport in parenchyma was predicted to be diffusion dominated, with a negligible contribution from convection. In the PVS, dispersion due to oscillating flow likely plays a significant role in PVS rapid transport observed in previous in vivo experiments. This dispersive effect could be more significant than convective solute transport from net flow that may exist in PVS and should be studied further.

In vitro evidence that sulfite impairs glutamatergic neurotransmission and inhibits glutathione metabolism-related enzymes in rat cerebral cortex.

Sulfite oxidase (SOX) deficiency is an inherited neurometabolic disorder biochemically characterized by tissue accumulation and high urinary excretion of sulfite and thiosulfate. Affected patients present severe neurological dysfunction accompanied by seizures, whose pathophysiology is poorly known. In the present study we evaluated the in vitro effects of sulfite and thiosulfate on important parameters of glutamatergic neurotransmission and redox homeostasis in rat cerebral cortex slices. We verified that sulfite, but not thiosulfate, significantly decreased glutamate uptake when cerebral cortex slices were exposed during 1h to these metabolites. We also observed that thiosulfate inhibited glutamine synthetase (GS) activity. A pronounced trend toward GS inhibition induced by sulfite was also found. Regarding redox homeostasis, sulfite, at the concentration of 10 µM, increased thiobarbituric acid-reactive substances and decreased glutathione concentrations after 1h of exposure. In contrast, thiosulfate did not alter these parameters. We also found that 500 µM sulfite increased sulfhydryl group content in rat cerebral cortex slices and increased GSH levels in a medium containing oxidized GSH (GSSG) and devoid of cortical slices, suggesting that sulfite reacts with disulfide bonds to generate sulfhydryl groups. Moreover, sulfite and thiosulfate did not alter the activities of glutathione peroxidase (GPx), glutathione reductase (GR), glutathione S-transferase (GST) and glucose-6-phosphate dehydrogenase (G6PDH) after 1h of incubation. However, sulfite inhibited the activities of GPx, GST and G6PDH when cortical slices were exposed for 3h to sulfite. We finally verified that sulfite did not induce cell death after 1h of incubation. Our data show that sulfite impairs glutamatergic neurotransmission and redox homeostasis in cerebral cortex. Therefore, it may be presumed that these pathomechanisms contribute, at least in part, to the seizures observed in patients affected by SOX deficiency.

Quantitative Autoradiography on Ontogenic Development of alpha-Adrenoceptor in the Rat Cerebral Cortex / 대한해부학회지

Adrenoceptors mediate response to catecholamines throughout the body. To investigate postnatal ontogenic development of alpha1- and alpha2- adrenoceptors in the rat cerebral cortex, in vitro autoradiography was done on frontal, parietal and temporal cortex in P0, P5, P10, P15, P20, P30 and adult animals. Binding sites for the alpha1-adrenergic receptor ligand, [3H]-prazosin, and the alpha2-adrenergic receptor ligand, [3H]-rauwolscine, were visualized by in vitro autoradiography, and anatomically localized by comparing the autoradiogram to Nissl-stained sections. Nonspecific binding was detected with unlabeled phentolamine (alpha1) and yohimbine (alpha2). There is uniform increase in alpha1- and alpha2- adrenoceptors from birth through first three or four postnatal weeks, followed by a decrease to adult level. Two alpha-adrenoceptors have very different ontogenic patterns of distribution during postnatal development. alpha1- adrenoceptors were expressed differentially in different cortical (frontal, temporal, parietal) regions and in different cortical layers (layers V, II-IV, VI) at same age. alpha2- adrenoceptor was expressed homogenously in throughout regions and layers. These findings may provide evidence that alpha1- adrenoceptors are involved in regulating cortical development or function more specifically than alpha2- adrenoceptors during postnatal development.

Effect of a Rostral Basal Forebrain Lesion on Neuropeptide Containing Neurons in the Rat Cerebral Cortex / 대한해부학회지

The present study was designed to elucidate the effects of rostral basal forebrain lesions on neuropeptide containing neurons in the cerebral cortex. Nine male Sprague-Dawley rats[250-300gm] received bilateral injections of ibotenic acid into the basal forebrain[A : +0.7mm, L : 2.3mm, D : 8.6mm] and additional five served as sham operated animals. Brains were removed at 8-14 days after lesioning and frozen coronal sections of 40 micrometer thickness were made. Immunohistochemical staining was performed against the somatostatin[SOM], neuropeptide Y[NPY], and vasoactive intestinal polypeptide[VIP]. No differences were observed in the number of the SOM-immunoreactive[SOM-ir] or NPY-ir neurons between the lesioned and the control groups. Density of the NPY-ir fibers also did not show any significant difference between the two groups. In contrast, the number of VIP-ir neurons in the frontal cortex was significantly reduced following the basal forebrain lesioning. These results suggest the functional relationship between the basal forebrain and the cortical VIP-ir neurons.

Double Immunohistochemical Study on the Postnatal Development of Somatostatin - and Neuropeptide Y - Immunoreactive Neurons in Rat Cerebral Cortex / 대한해부학회지

The postnatal development of somatostatin [SOM]- and neuropeptide Y[NPY]- immunoreactive[ir] neurons were examined in rat cerebral cortex considering their coexistence in cortical neurons. Using double immunohistochemical staining for SOM and NPY with diaminobenzidine and benzidine dihydrochloride as chromogens, we subdivided immunoreactive cells into double-labeled SOM/NPY-, SOM only-, and NPY only-ir neurons. Interestingly, SOM/NPY- and SOM only-ir neurons were detectable even at the day of birth, in contrast to NPY only-ir cells which first appeared in most cortices from two weeks of age. The morphological features of double-labeled SOM/NPY neurons were not identical to those SOM only- and NPY only-ir neurons. No apparent changes in the shape and size of single-labeled neurone occurred with age ; throughout their postnatal life they were round and ovoid, had a thin rim of perinuclear cytoplasm, and short processes. In contrast, the features of SOM/NPY-ir neurons were not consistent during postnatal life. By day P7, these neurons showed immature features ; they began to show more advanced neuronal characteristics by week P2, when they had a larger and more intensely-stained cytoplasm. In addition, their processes were longer, thicker and more complex than at earlier ages. At this age, SOM/NPY-ir somata were close to their maximum size. From week P4, they became smaller and were lightly labeled. SOM/NPY-ir somata were larger than SOM only- and NPY only-ir somata at and after two weeks of age. The present results showing different postnatal maturation patterns such as time of appearance and morphological features suggest that double-labeled SOM/NPY and single-labeled neurons might be different populations regulated by different mechanisms in their development, and with different functional properties during development.

Mechanisms of release of 3H Hydroxytryptamine evoked by hypoxia and hypoglycemia in rat cerebral cortex slices

Pathophysiology of brain ischemia is characterixed by a complex cascade of hemodynamic, electrophysiological and biochemical processes. It has been generally accepted that glutamate mediates the ischemic brain damage, excitotoxicity, and induce neurotramsmitter release from various brain tissues in ischemic milieu. In presen study, the mechanism for ischemia-induced [3HT]5-hydroxytryptamine(5-HT) from cerebral cortex slices of the rat was examined. Ischemia, deprivation of oxygen and glucose from Mg2+-free artificl cerebrospinal fluid, induced significiant release of [3H]5-HT(7.2+0.6% of total tissue content) from the tissues. This ischemia-induced release of [3H]5-HT from the slices was significiantly attenuated by TTX(1 yM), Mg2+(102mM). MK-801(10yM), ketamine(10yM), NMDA receptor antagonists, DNQX(30yM), a kainate/AMPA receptor antagonist, or carbetapentane(31yM), an inhibitor of glutamate release. Fluoxetine, a selective blocker for 5-HT transporter, inhibited the ischemia-induced release of [3H]5-HT. Omission of Ca2+ from incubation media potentiated ischemia-evoked [3H]5-HT release and the inhibitory effect of blockers for transporter. Dantrolene (30yM) and ryanodine(100 nM) and -conotoxinGVIA(100 nM), inhibitors of N-type Ca2+ channels, sifnificiantly attenuated the ischemia-induced release of [3H]5-HT, but verapamil(5 yM), an inhibitor of L-type Ca2+ channels, did not. Fluoxetine(100 nM), a relatively selective 5-HT transporter blocker, significiantly inhibited the ischemia-induced release of [3H]5-HT. Theses results suggest that glutamate is involned in ischemia-evoked [3H]5-HT release, and this release is achieved by Ca2+=dependent exocytosis and reverdsal of transporters, and can be modulated by various neuronal mechanisms.