An immunohistochemical analysis of SERT in the blood–brain barrier of the male rat brain

5-Hydroxytryptamine (5-HT) was originally discovered as a vasoconstrictor. 5-HT lowers blood pressure when administered peripherally to both normotensive and hypertensive male rats. Because the serotonin transporter (SERT) can function bidirectionally, we must consider whether 5-HT can be transported from the bloodstream to the central nervous system (CNS) in facilitating the fall in blood pressure. The blood–brain barrier (BBB) is a highly selective barrier that restricts movement of substances from the bloodstream to the CNS and vice versa, but the rat BBB has not been investigated in terms of SERT expression. This requires us to determine whether the BBB of the rat, the species in which we first observed a fall in blood pressure to infused 5-HT, expresses SERT. We hypothesized that SERT is present in the BBB of the male rat. To test this hypothesis, over 500 blood vessels were sampled from coronal slices of six male rat brains. Immunofluorescence of these coronal slices was used to determine whether SERT and RecA-1 (an endothelial cell marker) colocalized to the BBB. Blood vessels were considered to be capillaries if they were between 1.5 and 23 µm (intraluminal diameter). SERT was identified in the largest pial vessels of the BBB (mean ± SEM = 228.70 ± 18.71 µm, N = 9) and the smallest capillaries (mean ± SEM = 2.75 ± 0.12 µm, N = 369). SERT was not identified in the endothelium of blood vessels ranging from 20 to 135 µm (N = 45). The expression of SERT in the rat BBB means that 5-HT entry into the CNS must be considered a potential mechanism when investigating 5-HT-induced fall in blood pressure.     

Distribution of acetylated α-tubulin in brain

Summary We studied the solubility properties of brain acetylated -tubulin, as well as the localization of this tubulin in brain tissue. Endogenous unpolymerized tubulin and cytoskeletal tubulin were fractionated after brain Triton-solubilization. Using the immunoblotting technique, we found that acetylated -tubulin was recovered in the cytoskeletal fraction, and that most (92%) of the acetylated microtubules of this fraction were depolymerized by cold/Ca²⁺ treatment. In another set of experiments, axonal and soma-dendritic preparations were found to have equivalent amounts of acetylated -tubulin. By immunogold electron microscopy, we established that acetylated microtubules are widely distributed in dendrites of the central nervous system.     

Breakdown of the blood–brain barrier to proteins in white matter of the developing brain following systemic inflammation

Compromised blood–brain barrier permeability resulting from systemic inflammation has been implicated as a possible cause of brain damage in fetuses and newborns and may underlie white matter damage later in life. Rats at postnatal day (P) 0, P8 and P20 and opossums (Monodelphis domestica) at P15, P20, P35, P50 and P60 and adults of both species were injected intraperitoneally with 0.2–10 mg/kg body weight of 055:B5 lipopolysaccharide. An acute-phase response occurred in all animals. A change in the permeability of the blood–brain barrier to plasma proteins during a restricted period of postnatal development in both species was determined immunocytochemically by the presence of proteins surrounding cerebral blood vessels and in brain parenchyma. Blood vessels in white matter, but not grey matter, became transiently permeable to proteins between 10 and 24 h after lipopolysaccharide injection in P0 and P8 rats and P35–P60 opossums. Brains of Monodelphis younger than P35, rats older than P20 and adults of both species were not affected. Permeability of the blood–cerebrospinal fluid (CSF) barrier to proteins was not affected by systemic inflammation for at least 48 h after intraperitoneal injection of lipopolysaccharide. These results show that there is a restricted period in brain development when the blood–brain barrier, but not the blood–CSF barrier, to proteins is susceptible to systemic inflammation; this does not appear to be attributable to barrier immaturity but to its stage of development and only occurs in white matter.This work was supported by NIH grant number R01 NS043949-01A1.     

Effects of β-endorphin on brain serotonin metabolism

Human β-endorphin (15 μg) administered intracisternally increased concentrations of serotonin (5HT) and its metabolite, 5-hydroxyindoleacetic. acid (5-HIAA), in brain stem and hypothalamus and decreased 5-HIAA concentrations in hippocampus. These data are compatible with the hypothesis that β-endorphin increases 5HT turnover in brain stem and hypothalamus and decreases 5HT turnover in hippocampus. β-endorphin increased in brain stem and hypothalamus and decreased in hippocampus the rate of pargyline-induced decline of 5-HIAA. β-endorphin decreased the rate of pargyline-induced accumulation of 5HT in all these brain regions. The probenecid-induced accumulation of 5-HIAA in brain stem was decreased by β-endorphin. These data are compatible with the hypothesis that β-endorphin increases release of 5HT from neurons in brain stem and hypothalamus and decreases release of 5HT from neurons in hippocampus. The data require further a hypothesis that β-endorphin either decreases 5HT reuptake in these three brain regions or increases 5-HIAA egress from brain.     

Effect of β-phenylethylamine on dopaminergic systems of the rat brain

The time course of changes in the dopamine concentration in dopaminergic neurons of the nigro-neostriatal and mesolimbic systems of the rat brain during 1 h after intraperitoneal injection of -phenylethylamine (100 mg/kg) was studied by quantitative fluorescence-histochemical analysis. The results showed that -phenylethylamine causes a marked fall in the dopamine level in neurons of dopaminergic systems of the brain. The dopamine level in the bodies of dopaminergic neurons changes more than in their axon terminals. The fall in the dopamine concentration in the dopaminergic systems of the brain during the first hour is irregular in character: in the terminals between 10 and 30 min and in the bodies between 30 and 45 min there is actually a temporary increase in the dopamine concentration. The rise in the dopamine concentration in the terminals coincides with a sharp fall in the dopamine level in the neuron bodies, and conversely, the fall in the dopamine concentration in the terminals after 30 min is accompanied by some increase in the dopamine concentration in the neuron bodies. The results suggest that the increase in motor activity described in the literature in animals after injection of -phenylethylamine is connected with its action on catecholaminergic, especially dopaminergic, brain systems.Institute of Biophysics, Academy of Sciences of the USSR, Pushchino-on-Oka. Translated from Neirofiziologiya, Vol. 11, No. 6, pp. 578–584, November–December, 1979.     

What brain signals are suitable for feedback controzl of deep brain stimulation in Parkinson's disease?

Feedback control of deep brain stimulation (DBS) in Parkinson's disease has great potential to improve efficacy, reduce side effects, and decrease the cost of treatment. In this, the timing and intensity of stimulation are titrated according to biomarkers that capture current clinical state. Stimulation may be at standard high frequency or intelligently patterned to directly modify specific pathological rhythms. The search for and validation of appropriate feedback signals are therefore crucial. Signals recorded from the DBS electrode currently appear to be the most promising source of feedback. In particular, beta-frequency band oscillations in the local field potential recorded at the stimulation target may capture variation in bradykinesia and rigidity across patients, but this remains to be confirmed within patients. Biomarkers that reliably reflect other impairments, such as tremor, also need to be established. Finally, whether brain signals are causally important needs to be established before stimulation can be specifically patterned rather than delivered at empirically defined high frequency.     

Biosynthesis of serotonin in Raphé nuclei of rat brain: effect of p-chlorophenylalanine

The activity of tryptophan hydroxylase (EC 1.99.1.4) in the region of the raphé nuclei of rat brain was higher than that of any other brain area. The content of serotonin and the rate of serotonin synthesis were also highest in the raphé nuclei. Following the administration of p-chlorophenylalanine the injection of tryptophan and pargyline increased the content of serotonin in the region of the raphé nuclei of rat brain. The results suggest that the raphé nuclei retained the capacity to hydroxyl-late tryptophan to some extent after the injection of p-chlorophenylalanine.     

Isolation and characterization of bovine brain myelin distribution of 5′-nucleotidase

Myelin was isolated from bovine brain by several published procedures and modifications of these procedures. High activity of the myelin marker (2,3-cyclic nucleotide 3-phosphohydrolase) and low activity of contaminants markers in white matter homogenates in respect to cerebral cortex showed the white matter to be better than the cerebral cortex or the whole brain for myelin isolation. A procedure is described for the preparation of purified myelin from bovine white matter which yielded a content of protein (40%), myelin marker (51%), and 5-nucleotidase (25%) in purified myelin higher than by any used method. Acetylcholinesterase or succinate dehydrogenase was lower than 7% of its activity in the white matter homogenate, and monoamine oxidase and NADPH: cytochrome c reductase were not recovered in myelin fraction. Morphologically, myelin fraction was shown to mainly consist of multilamellar membranes of different sizes. Sodium dodecyl sulphate polyacrylamide gel electrophoresis of myelin fraction showed a characteristic protein pattern of myelin. When our procedure was applied to frozen white matter, lower protein (32%) and myelin marker (34%) and similar 5-nucleotidase activity (24%) were recovered in myelin, increasing its recovery in denser fractions of white matter.     

Hypothermia-induced tyrosine phosphorylation of SIRPα in the brain

Signal regulatory protein α (SIRPα) is a neuronal membrane protein that undergoes tyrosine phosphorylation in the brain of mice in response to forced swim (FS) stress in cold water, and this response is implicated in regulation of depression-like behavior in the FS test. We now show that subjection of mice to the FS in warm (37 °C) water does not induce the tyrosine phosphorylation of SIRPα in the brain. The rectal temperature (T(rec) ) of mice was reduced to 27° to 30 °C by performance of the FS for 10 min in cold water, whereas it was not affected by the same treatment in warm water. The level of tyrosine phosphorylation of SIRPα in the brain was increased by administration of ethanol or picrotoxin, starvation, or cooling after anesthesia, all of which also induced hypothermia. Furthermore, the tyrosine phosphorylation of SIRPα in cultured hippocampal neurons was induced by lowering the temperature of the culture medium. CD47, a ligand of SIRPα, as well as Src family kinases or SH2 domain-containing protein phosphatase 2 (Shp2), might be important for the basal and the hypothermia-induced tyrosine phosphorylation of SIRPα. Hypothermia is therefore likely an important determinant of both the behavioral immobility and tyrosine phosphorylation of SIRPα observed in the FS test.     

In vitro palmitoylation of native bovine brain G_oα

The native Goα was purified from bovine brain cortex and palmitoylated in vitro. The in vitro palmitoylation site was the same as that in vivo. The internal palmitoylation of purified native Goα was found to be largely maintained. The apparent palmitoylation ratio was significantly increased after the Goα was treated with DTT. The GTPγS binding characteristic of Goα was not influenced by palmitoylation, however, the affinity for LUVs was increased dramatically. The in vitro palmitoylation model of Goα provides a better basis for studying the functional role of G protein palmitoylation in signal transduction.     

Role of Cl− channels in primary brain tumour

There is tight interplay between Ca²⁺ and Cl⁻ flux that can influence brain tumour proliferation, migration and invasion. Glioma is the predominant malignant primary brain tumour, accounting for ˜80% of all cases. Voltage-gated Cl⁻ channel family (ClC) proteins and Cl⁻ intracellular channel (CLIC) proteins are drastically overexpressed in glioma, and are associated with enhanced cell proliferation, migration and invasion. Ca²⁺ also plays fundamental roles in the phenomenon. Ca²⁺-activated Cl⁻ channels (CaCC) such as TMEM16A and bestrophin-1 are involved in glioma formation and assist Ca²⁺ movement from intracellular stores to the plasma membrane. Additionally, the transient receptor protein (TRP) channel TRPC1 can induce activation of ClC-3 by increasing intracellular Ca²⁺concentrations and activating Ca²⁺/calmodulin-dependent protein kinase II (CaMKII). Therefore, Ca²⁺ and Cl⁻currents can concurrently mediate brain tumour cellular functions. Glioma also expresses volume regulated anion channels (VRACs), which are responsible for the swelling-induced Cl⁻ current, I_Cl,swell. This current enables glioma cells to perform regulatory volume decrease (RVD) as a survivability mechanism in response to hypoxic conditions within the tumour microenvironment. RVD can also be exploited by glioma for invasion and migration. Effective treatment for glioma is challenging, which can be in part due to prolonged chemotherapy leading to mutations in genes associated with multi-drug resistances (MRP1, Bcl-2, and ABC family). Thus, a potential therapeutic strategy for treatment of glioma can be through the inhibition of selected Cl⁻ channels.     

Alpha synuclein is transported into and out of the brain by the blood–brain barrier

Alpha-synuclein (α-Syn), a small protein with multiple physiological and pathological functions, is one of the dominant proteins found in Lewy Bodies, a pathological hallmark of Lewy body disorders, including Parkinson's disease (PD). More recently, α-Syn has been found in body fluids, including blood and cerebrospinal fluid, and is likely produced by both peripheral tissues and the central nervous system. Exchange of α-Syn between the brain and peripheral tissues could have important pathophysiologic and therapeutic implications. However, little is known about the ability of α-Syn to cross the blood–brain barrier (BBB). Here, we found that radioactively labeled α-Syn crossed the BBB in both the brain-to-blood and the blood-to-brain directions at rates consistent with saturable mechanisms. Low-density lipoprotein receptor-related protein-1 (LRP-1), but not p-glycoprotein, may be involved in α-Syn efflux and lipopolysaccharide (LPS)-induced inflammation could increase α-Syn uptake by the brain by disrupting the BBB.     

Partial purification of γ-glutamyltransferase from human brain microvessels

Two forms of gamma-glutamyltransferase from human brain cortex microvessels were partially purified by gel permeation and ion-exchange and group-affinity chromatography. The specific activity of the purified preparations was 320-fold (detergent form) and 830-fold (proteolytic form) higher than that of the enzyme in the brain cortex homogenate. The relative molecular mass of the proteolytic form of the enzyme was about 90,000 as determined by gel permeation chromatography. The major part of the enzyme (about 80%) was absorbed on Con A-Sepharose 4B. The pH optima for transfer reactions with -glutamyl-4-nitroanilide as donor and glycylglycine andl-cystine as acceptors were in the range of 8.2 to 9.0. The studied enzyme was inhibited by a mixture ofl-serine and borate and by bromcresol green.     

The role of α-synuclein in brain lipid metabolism: a downstream impact on brain inflammatory response

α-Synuclein (Snca) is an abundant small cytosolic protein (140 amino acids) that is expressed in the brain, although its physiological role is poorly defined. Consistent with its ubiquitous distribution in the brain, we and others have established a role for Snca in brain lipid metabolism and downstream events such as neuroinflammation. In astrocytes, Snca is important for fatty acid uptake and trafficking, where its deletion decreases 16:0 and 20:4n-6 uptake and alters targeting to specific lipid pools. Although Snca has no impact on 22:6n-3 uptake into astrocytes, it is important for its targeting to lipid pools. Similar results for fatty acid uptake from the plasma are seen in studies using whole mice coupled with steady-state kinetic modeling. We demonstrate in gene-ablated mice a significant reduction in the incorporation rate of 20:4n-6 into brain phospholipid pools due to reduced recycling of 20:4n-6 through the ER-localized long-chain acyl-CoA synthetases (Acsl). This reduction results in a compensatory increase in the incorporation rate of 22:6n-3 into brain phospholipids. Snca is also important for brain and astrocyte cholesterol metabolism, where its deletion results in an elevation of cholesterol and cholesteryl esters. This increase may be due to the interaction of Snca with membrane-bound enzymes involved in lipid metabolism such as Acsl. Snca is critical in modulating brain prostanoid formation and microglial activities. In the absence of Snca, microglia are basally activated and demonstrate increased proinflammatory cytokine secretion. Thus, Snca, through its modulation of brain lipid metabolism, has a critical role in brain inflammatory responses.     

Rate of utilization of glucose and `compartmentation'' of α-oxoglutarate and glutamate in rat brain

1. The rate of incorporation of ¹⁴C into pyruvate, α-oxoglutarate, lactate and glucose of rat tissues was measured after the subcutaneous injection of uniformly labelled glucose. 2. In rat brain the specific radioactivities of lactate and glucose were similar to that of alanine. In liver the specific radioactivity of glucose was considerably higher than that of lactate or alanine. 3. The specific radioactivities of α-oxo acids of rat brain were lower than those of corresponding amino acids, alanine and glutamate. These findings have been explained in relation to metabolic compartments in vivo. 4. The approximate estimated rate of glucose utilization in rat brain in vivo is 0·96μmole/g. of brain/min.     

Development of the brain’s organization of working memory in young schoolchildren

In 7–8 and 9–10-years old children, we studied event-related potentials (ERPs) during paired comparison of non-verbalizable visuospatial stimuli presented at an interval of 1.5–1.8 s. Age-related differences were found in the involvement of various cortical areas in the formation and retention of a short-term memory trace of the reference stimulus and during comparison of the short-term trace with the test stimulus presented. In both age groups, working memory was associated with an elevation of the amplitude of the sensory-specific N1 component in the visual cortical areas. Age-related differences in the processing of sensory-specific characteristics of a stimulus were the greatest in the ERPs to the test stimulus: at the age of 9–10, the N1 component amplitude was significantly increased in all caudal leads and, in the occipital and inferior temporal leads, this component was preceded by P1 component. At this age, we observed the early involvement of the inferior frontal cortex, which was not observed at the age of seven. The increase in positivity over that area was observed in the interval of 100–200 ms. Substantial differences between age groups were found in the late ERP component corresponding to cognitive processes. At the age of 7–8, the presentation of both the reference and test stimuli causes the increase in the amplitude of the slow positive complex (SPC) in the caudal liads with the maximum enhancement found in the interval of 300–800 ms in the parietal leads. At the age of 9–10, the SPC increase, much like in adults, was observed in ERP to the test stimulus only. At this age, adult-like specific changes in the late phases of ERPs were observed in the fronto-central regions at the different stages of working memory. They are the increases in the negative N400 wave in the ERP to the reference stimulus and the SPC to the test stimulus. These data show that, at the age of 9–10, the functional organization of working memory of the adult type is formed; however, the extent to which the frontal cortex, and its dorsal regions in particular, is involved into working memory processes does not meet yet a definitive level.