Regional Brain Distribution Research Articles

Molecular cloning and characterization of secretogranin II in the catfish Heteropneustes fossilis: Sex and seasonal brain regional variations and its gonadotropin regulation

Secretoneurin (SN) is a novel functional peptide derived from secretogranin II, a protein belonging to the class of chromogranins. Catfish Heteropneustes fossilis is an economically important species of the Asian subcontinent and holds an important place in reproductive physiology study. In the present study, the full-length cDNA encoding secretogranin IIb (SgIIb) was cloned from the brain of catfish H. fossilis. Sequence analysis showed that a 33 amino acid SN peptide (SNb) is present in SgIIb proprotein. The full-length sequence of SgIIb is 2912 bp with open–reading frame 1761 bp long. The 5'UTR is 681 bp long upstream and the 3'UTRis 470 bp long downstream. The ORF encodes 586 amino acid residues comprising signal peptide (24 amino acids) and SNb (33 amino acids), EM66 (66 amino acids). Catfish SgIIb showed 88% nucleotide homology and 90% protein identity with Ictalurus punctatus. Tissue expression showed that it is highly expressed in the brain among all tissues studied. In situ hybridization and quantitative real-time PCR showed significant brain regional distribution with highest transcript abundance in the pituitary. In ovary tissue SgIIb transcripts were localized in the granulosa layer. In the brain, hCG administration stimulated expression highly at 16 h. In ovary, hCG treatment under in vivo and in vitro condition decreased SgIIb expression at all doses. Thus, in the present study in situ localization of SgIIb mRNAs in different regions of brain and pituitary, and ovary suggest its putative role in regulating hypothalamic-pituitary-gonadal (HPG) axis and also suggest that the SgIIb expression pattern in the brain and ovary could be related to reproductive activity and may be involved in the neuroendocrine role of SgIIb in the catfish H. fossilis.

Quantitative Analysis of 18F-PF-06684511, a Novel PET Radioligand for Selective β-Secretase 1 Imaging, in Nonhuman Primate Brain.

β-secretase 1 (BACE1) is a key enzyme in the generation of β-amyloid, which is accumulated in the brain of Alzheimer disease patients. PF-06684511 was identified as a candidate PET ligand for imaging BACE1 in the brain and showed high specific binding in an initial assessment in a nonhuman primate (NHP) PET study using 18F-PF-06684511. In this effort, we aimed to quantitatively evaluate the regional brain distribution of 18F-PF-06684511 in NHPs under baseline and blocking conditions and to assess the target occupancy of BACE1 inhibitors. In addition, NHP whole-body PET measurements were performed to estimate the effective radiation dose. Methods: Initial brain PET measurements were performed at baseline and after oral administration of 5 mg/kg of LY2886721, a BACE1 inhibitor, in 2 cynomolgus monkeys. Kinetic analysis was performed with the radiometabolite-corrected plasma input function. In addition, a wide dose range of another BACE1 inhibitor, PF-06663195, was examined to investigate the relationship between the brain target occupancy and plasma concentration of the drug. Finally, the effective radiation dose of 18F-PF-06684511 was estimated on the basis of the whole-body PET measurements in NHPs. Results: Radiolabeling was accomplished successfully with an incorporation radiochemical yield of 4%-12% (decay-corrected) from 18F ion. The radiochemical purity was greater than 99%. The whole-brain uptake of 18F-PF-06684511 peaked (∼220% SUV) at approximately 20 min and decreased thereafter (∼100% SUV at 180 min). A 2-tissue-compartment model described the time-activity curves well. Pretreatment with LY2886721 reduced the total distribution volume of 18F-PF-06684511 by 48%-80% depending on the brain region, confirming its in vivo specificity. BACE1 occupancy of PF-06663195, estimated using the Lassen occupancy plot, showed a dose-dependent increase. The effective dose of 18F-PF-06684511 was 0.043 mSv/MBq for humans. Conclusion: 18F-PF-06684511 is the first successful PET radioligand for BACE1 brain imaging that demonstrates favorable in vivo binding and brain kinetics in NHPs.

GABAB receptor positive allosteric modulators with different efficacies affect neuroadaptation to and self-administration of alcohol and cocaine.

Drugs of abuse induce widespread synaptic adaptations in the mesolimbic dopamine (DA) neurons. Such drug-induced neuroadaptations may constitute an initial cellular mechanism eventually leading to compulsive drug-seeking behavior. To evaluate the impact of GABAB receptors on addiction-related persistent neuroplasticity, we tested the ability of orthosteric agonist baclofen and two positive allosteric modulators (PAMs) of GABAB receptors to suppress neuroadaptations in the ventral tegmental area (VTA) and reward-related behaviors induced by ethanol and cocaine. A novel compound (S)-1-(5-fluoro-2,3-dihydro-1H-inden-2-yl)-4-methyl-6,7,8,9-tetrahydro-[1,2,4]triazolo[4,3-a]quinazolin-5(4H)-one (ORM-27669) was found to be a GABAB PAM of low efficacy as agonist, whereas the reference compound (R,S)-5,7-di-tert-butyl-3-hydroxy-3-trifluoromethyl-3H-benzofuran-2-one (rac-BHFF) had a different allosteric profile being a more potent PAM in the calcium-based assay and an agonist, coupled with potent PAM activity, in the [35 S] GTPγS binding assay in rat and human recombinant receptors. Using autoradiography, the high-efficacy rac-BHFF and the low-efficacy ORM-27669 potentiated the effects of baclofen on [35 S] GTPγS binding with identical brain regional distribution. Treatment of mice with baclofen, rac-BHFF, or ORM-27669 failed to induce glutamate receptor neuroplasticity in the VTA DA neurons. Pretreatment with rac-BHFF at non-sedative doses effectively reversed both ethanol- and cocaine-induced plasticity and attenuated cocaine i.v. self-administration and ethanol drinking. Pretreatment with ORM-27669 only reversed ethanol-induced neuroplasticity and attenuated ethanol drinking but had no effects on cocaine-induced neuroplasticity or self-administration. These findings encourage further investigation of GABAB receptor PAMs with different efficacies in addiction models to develop novel treatment strategies for drug addiction.

Expanding Brain-Computer Interfaces for Controlling Epilepsy Networks: Novel Thalamic Responsive Neurostimulation in Refractory Epilepsy.

Seizures have traditionally been considered hypersynchronous excitatory events and epilepsy has been separated into focal and generalized epilepsy based largely on the spatial distribution of brain regions involved at seizure onset. Epilepsy, however, is increasingly recognized as a complex network disorder that may be distributed and dynamic. Responsive neurostimulation (RNS) is a recent technology that utilizes intracranial electroencephalography (EEG) to detect seizures and delivers stimulation to cortical and subcortical brain structures for seizure control. RNS has particular significance in the clinical treatment of medically refractory epilepsy and brain–computer interfaces in epilepsy. Closed loop RNS represents an important step forward to understand and target nodes in the seizure network. The thalamus is a central network node within several functional networks and regulates input to the cortex; clinically, several thalamic nuclei are safe and feasible targets. We highlight the network theory of epilepsy, potential targets for neuromodulation in epilepsy and the first reported use of RNS as a first generation brain–computer interface to detect and stimulate the centromedian intralaminar thalamic nucleus in a patient with bilateral cortical onset of seizures. We propose that advances in network analysis and neuromodulatory techniques using brain–computer interfaces will significantly improve outcomes in patients with epilepsy. There are numerous avenues of future direction in brain–computer interface devices including multi-modal sensors, flexible electrode arrays, multi-site targeting, and wireless communication.

Assessment of sigma-1 receptor occupancy in mice with non-radiolabelled FTC-146 as a tracer

The use of liquid chromatography coupled with mass spectrometry (LC-MS/MS) is advantageous in in-vivo receptor occupancy assays at pre-clinical drug developmental stages. Relatively, its application is effective in terms of high throughput, data reproducibility, sensitivity, and sample processing. In this perspective, we have evaluated the use of FTC-146 as a non-radiolabelled tracer to determine the sigma-1 receptor occupancy of test drugs in mice brain. Further, the brain and plasma exposures of test drug were determined at their corresponding occupancies. In this occupancy method, the optimized tracer treatment (sacrification) time after intravenous administration was 30 min. The tracer dose was 3 µg/kg and specific brain regions of interest were frontal cortex, pons and midbrain. Mice were pretreated orally with SA4503, fluspidine, haloperidol, and donepezil followed by tracer treatment. Among the test drugs, SA4503 was used as positive control group at its highest test dose (7 mg/kg, intraperitoneal). There was a dose-dependent decrease in brain regional FTC-146 binding in pretreated mice. From the occupancy curves of SA4503, fluspidine, haloperidol, and donepezil the effective dose (ED50) value ranges are 0.74–1.45, 0.09–0.11, 0.11–0.12, and 0.07–0.09 mg/kg, respectively. Their corresponding brain effective concentration (EC50) values are 74.3–132.5, 3.4–3.7, 122.5–139.5, and 8.8–11.0 ng/g and plasma EC50 values are 34.3–53.7, 0.08–0.10, 7.8–9.5, and 0.6–0.7 ng/mL. Brain regional distribution and binding inhibition upon pretreatment were comparable with data reported with labeled [18F]FTC-146. Drug exposures were simultaneously determined and correlated with sigma-1 occupancy from the same experiment. Wide category drugs can be assayed for sigma-1 receptor engagement and their correlation with exposures aid in clinical development.

2181 Age-related change in 5-HT6 receptor availability in healthy male volunteers measured with 11C-GSK215083 PET

OBJECTIVES/SPECIFIC AIMS: The serotonin receptor 6 (5-HT6) is a potential therapeutic target given its distribution in brain regions that are important in depression, anxiety, and cognition. This study sought to investigate the effects of age on 5-HT6 receptor availability using 11C GSK215083, a PET ligand with affinity for 5-HT6 in the striatum and 5-HT2A in the cortex. METHODS/STUDY POPULATION: In total, 28 healthy male subjects (age range: 23–52 years) were scanned with 11C-GSK215083 on the HR+PET scanner. Time-activity curves in regions-of-interest were fitted with multilinear analysis-1 method. Binding potentials (BPND) were calculated using cerebellum as the reference region and corrected for partial volume effects. RESULTS/ANTICIPATED RESULTS: In 5-HT6 rich areas, regional 11C-GSK215083 displayed a negative correlation between BPND and age in the caudate (r=−0.41, p=0.03) (14% change per decade), and putamen (r=−0.30, p=0.04) (11% change per decade), but not in the ventral striatum and pallidum. Negative correlation with age was also seen in cortical regions (r=−0.41, p=0.03) (7% change per decade), consistent with the literature on 5-HT2A availability. DISCUSSION/SIGNIFICANCE OF IMPACT: This is the first in vivo study in humans to examine the effect of age on 5-HT6 receptor availability. The study demonstrated a significant age-related decline in 5-HT6 availability (BPND) in the caudate and putamen.

Deuterium-substituted 2-(2'-((dimethylamino)methyl)-4'-[18 F](fluoropropoxy)phenylthio)benzenamine as a serotonin transporter imaging agent.

Positron emission tomography imaging of serotonin transporter (SERT) is useful for studying brain diseases with altered serotonergic function. A deuterated imaging agent, ([18 F]2-((2-((bis(methyl-d3 )amino)methyl)-4-(3-fluoropropoxy-1,1,2,2,3,3-d6 )phenyl)thio)aniline, [18 F]D12FPBM, [18 F]1), was prepared as a new chemical entity. The deuterated agent, 1, showed excellent binding affinity to SERT; Ki was 0.086 nM, comparable with the undeuterated FPBM. In vivo biodistribution studies in rats with [18 F]1 showed good brain uptake (1.09% dose/g at 2min post injection) and high specific uptake into the hypothalamus (HY) as compared with cerebellum (CB) (HY/CB=7.55 at 120min), suggesting a specific localization to SERT binding sites. Regional brain distribution in rats provided clear indication that [18 F]1 concentrated in the hypothalamus, hippocampus, and striatum, areas with a high SERT density. Results indicate that very little D to H substitution effect was found; [18 F]FPBM and [18 F]1 showed very similar SERT binding. [18 F]1 might be an excellent candidate for SERT imaging.

Age-Related Change in 5-HT6 Receptor Availability in Healthy Male Volunteers Measured with 11C-GSK215083 PET.

Serotonin receptor 6 (5-hydroxytrypamine-6, or 5-HT6) is a potential therapeutic target given its distribution in brain regions that are important in depression, anxiety, and cognition. This study sought to investigate the effects of age on 5-HT6 receptor availability using 11C-GSK215083, a PET ligand with affinity for 5-HT6 in the striatum and 5-HT2A in the cortex. Methods: Twenty-eight healthy male volunteers (age range, 23-52 y) were scanned with 11C-GSK215083 PET. Time-activity curves in regions of interest were fitted using a multilinear analysis method. Nondisplaceable binding potential (BPND) was calculated using the cerebellum as the reference region and corrected for partial-volume effects. Results: In 5-HT6-rich areas, regional 11C-GSK215083 showed a negative correlation between BPND and age in the caudate (r = -0.41, P = 0.03) (14% change per decade) and putamen (r = -0.30, P = 0.04) (11% change per decade) but not in the ventral striatum or pallidum. A negative correlation with age was also seen in cortical regions (r = -0.41, P = 0.03) (7% change per decade), consistent with the literature on 5-HT2A availability. Conclusion: To our knowledge, this was the first in vivo study on humans to examine the effect of age on 5-HT6 receptor availability. The study demonstrated a significant age-related decline in 5-HT6 availability (BPND) in the caudate and putamen.

Synthesis and Evaluation of a New 18F-Labeled Radiotracer for Studying the GABAB Receptor in the Mouse Brain.

New GABAB agonists, fluoropyridyl ether analogues of baclofen, have been synthesized as potential PET radiotracers. The compound with highest inhibition binding affinity as well as greatest agonist response, ( R)-4-amino-3-(4-chloro-3-((2-fluoropyridin-4-yl)methoxy)phenyl)butanoic acid (1b), was radiolabeled with 18F with good radiochemical yield, high radiochemical purity, and high molar radioactivity. The regional brain distribution of the radiolabeled ( R)-4-amino-3-(4-chloro-3-((2-[18F]fluoropyridin-4-yl)methoxy)phenyl)butanoic acid, [18F]1b, was studied in CD-1 male mice. The study demonstrated that [18F]1b enters the mouse brain (1% ID/g tissue). The accumulation of [18F]1b in the mouse brain was inhibited (35%) by preinjection of GABAB agonist 1a, suggesting that the radiotracer brain uptake is partially mediated by GABAB receptors. The presented data demonstrate a feasibility of imaging of GABAB receptors in rodents and justify further development of GABAB PET tracers with improved specific binding and greater blood-brain barrier permeability.

Time-dependent regional brain distribution of methadone and naltrexone in the treatment of opioid addiction.

Opioid addiction is a serious public health concern with severe health and social implications; therefore, extensive therapeutic efforts are required to keep users drug free. The two main pharmacological interventions, in the treatment of addiction, involve management with methadone an mu (μ)-opioid agonist and treatment with naltrexone, μ-opioid, kappa (κ)-opioid and delta (δ)-opioid antagonist. MET and NAL are believed to help individuals to derive maximum benefit from treatment and undergo a full recovery. The aim of this study was to determine the localization and distribution of MET and NAL, over a 24-hour period in rodent brain, in order to investigate the differences in their respective regional brain distributions. This would provide a better understanding of the role of each individual drug in the treatment of addiction, especially NAL, whose efficacy is controversial. Tissue distribution was determined by using mass spectrometric imaging (MSI), in combination with quantification via liquid chromatography tandem mass spectrometry. MSI image analysis showed that MET was highly localized in the striatal and hippocampal regions, including the nucleus caudate, putamen and the upper cortex. NAL was distributed with high intensities in the mesocorticolimbic system including areas of the cortex, caudate putamen and ventral pallidum regions. Our results demonstrate that MET and NAL are highly localized in the brain regions with a high density of μ-receptors, the primary sites of heroin binding. These areas are strongly implicated in the development of addiction and are the major pathways that mediate brain stimulation during reward.

Real-Time Quaking-Induced Conversion Detection of Bovine Spongiform Encephalopathy Prions in a Subclinical Steer.

Bovine spongiform encephalopathy (BSE) belongs to a group of fatal prion diseases that result from the misfolding of the cellular prion protein (PrPC) into a pathogenic form (PrPSc) that accumulates in the brain. In vitro assays such as serial protein misfolding amplification and real-time quaking-induced conversion (RT-QuIC) allow assessment of the conversion of PrPC to PrPSc. RT-QuIC can be used for the detection of prions in a variety of biological tissues from humans and animals. However, there is no such comparison of RT-QuIC data between BSE positive and presymptomatic cattle. Further, the current study assesses prion distribution in multiple brain regions of clinically ill or subclinical animals. Here, we compare RT-QuIC reactions seeded with brain samples collected from experimentally inoculated cattle that were clinically ill or subclinically affected with BSE. The results demonstrate RT-QuIC seeding in various brain regions of an animal with subclinical BSE despite being determined negative by immunohistochemistry. Bioassay of the subclinical animal and RT-QuIC of brainstem from inoculated knockout (PRNP−/−) cattle were used to confirm infectivity in the subclinical animal and determine that RT-QuIC reactions were not the result of residual inoculum, respectively. These results confirm that RT-QuIC is a highly sensitive prion detection assay that can detect prions in a steer prior to the onset of clinical signs of BSE.

Skull and cerebrospinal fluid effects on microwave radiation propagation in human brain

The determination of microwave absorption distribution in the human brain is necessary for the detection of brain tumors using thermo-acoustic imaging and for removing them using hyperthermia treatment. In contrast to ionizing radiation, hyperthermia treatment can be applied to remove tumors inside the brain without the concern of including secondary malignancies, which typically form from the neuronal cells of the septum pellucidum. The aim of this study is to determine the microwave absorption distribution in an adult human brain and to study the effects of skull and cerebrospinal fluid on the propagation of microwave radiation inside the brain. To this end, we simulate the microwave absorption distribution in a realistic adult brain model (Colin 27) using the mesh-based Monte Carlo (MMC) method. This is because in spite of there being other numerical methods, the MMC does not require a large memory, even for complicated geometries, and its algorithm is simple and easy to implement with low computational cost. The brain model is constructed using high-resolution (1 mm isotropic voxel) and low noise magnetic resonance imaging (MRI) scans and its volume contains voxels, covering the brain completely. Using the MMC method, the radiative transport equation is solved and the absorbed microwave energy distribution in different brain regions is obtained without any fracture or anomaly. The simulation results show that the skull and cerebrospinal fluid guide the microwave radiation and suppress its penetration through deep brain compartments as a shielding factor. These results reveal that the MMC can be used to predict the amount of required energy to increase the temperature inside the tumour during hyperthermia treatment. Our results also show why a deep tumour inside an adult human brain cannot be efficiently treated using hyperthermia treatment. Finally, the accuracy of the presented numerical method is verified using the signal flow graph technique.

The dynamic characteristics of the anterior cingulate cortex in resting-state fMRI of patients with depression

BackgroundThe anterior cingulate cortex (ACC) is part of the limbic system of the brain. It is a bridge between attentional and emotional processing, which is responsible for the integration of visceral, attentional, and affective information. Lesioning of the ACC, which produces striking changes, is used to treat major depression disorder (MDD). Moreover, the brain dynamically integrates and coordinates functions of its different subparts to realize its cognitive capability. Hence, the spatio-temporal community distribution of the ACC is necessary to completely understand MDD. MethodsFirst, community structure detection was used to reveal the community distribution of brain regions. Thereafter, the flexibility, i.e., the frequency of community assignment changes of the ACC in such a community, and the module allegiance matrix (MAM) between the ACC and other brain regions, were analyzed. ResultsOur analysis demonstrated significant differences in the distribution of community assignment and flexibility of the ACC in MDD, compared to healthy controls (HC). The results also showed that the pairwise values of the MAMs between the ACC and the amygdala, insula, precuneus, and thalamus were significantly lower in patients with MDD compared to those in HC. LimitationsThe data collected is subject to patient-specific noise because (1) the medication effect varies from patient to patient, and (2) with most fMRI studies, the thoughts of the participants during imaging are difficult to control. ConclusionACC exhibits abnormal flexibility in community structures in MDD. The pairwise abnormal entries in the MAM for the ACC with four other brain regions, i.e., amygdala, insula, precuneus, and thalamus, quantified the role played by the ACC in MDD.

Quantification of Transporter and Receptor Proteins in Dog Brain Capillaries and Choroid Plexus: Relevance for the Distribution in Brain and CSF of Selected BCRP and P-gp Substrates.

Transporters at the blood-brain barrier (BBB) and the blood-cerebrospinal fluid barrier (BCSFB) play a pivotal role as gatekeepers for efflux or uptake of endogenous and exogenous molecules. The protein expression of a number of them has already been determined in the brains of rodents, nonhuman primates, and humans using quantitative targeted absolute proteomics (QTAP). The dog is an important animal model for drug discovery and development, especially for safety evaluations. The purpose of the present study was to clarify the relevance of the transporter protein expression for drug distribution in the dog brain and CSF. We used QTAP to examine the protein expression of 17 selected transporters and receptors at the dog BBB and BCSFB. For the first time, we directly linked the expression of two efflux transporters, P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP), to regional brain and CSF distribution using specific substrates. Two cocktails, each containing one P-gp substrate (quinidine or apafant) and one BCRP substrate (dantrolene or daidzein) were infused intravenously prior to collection of the brain. Transporter expression varied only slightly between the capillaries of different brain regions and did not result in region-specific distribution of the investigated substrates. There were, however, distinct differences between brain capillaries and choroid plexus. Largest differences were observed for BCRP and P-gp: both were highly expressed in brain capillaries, but no BCRP and only low amounts of P-gp were detected in the choroid plexus. Kp,uu,brain and Kp,uu,CSF of both P-gp substrates were indicative of drug efflux. Also, Kp,uu,brain for the BCRP substrates was low. In contrast, Kp,uu,CSF for both BCRP substrates was close to unity, resulting in Kp,uu,CSF/Kp,uu,brain ratios of 7 and 8, respectively. We conclude that the drug transporter expression profiles differ between the BBB and BCSFB in dogs, that there are species differences in the expression profiles, and that CSF is not a suitable surrogate for unbound brain concentrations of BCRP substrates in dogs.

Initial evaluation of a 99mTc-cyclopentadienyltricarbonyl technetium complex as a potential 5-HT1A receptor imaging agent

• We develop a new 99mTc-labeled for 5-HT1A receptor imaging agent. • A N-phenylferrocenecarboxamide labeled with technetium-99 m ( 99m Tc- 2 )has been successfully prepared in good yield. • 99mTc labeled complex was able to bind specifically to rat hippocampus. • Regional brain distribution study showed a clear correlation between distribution of radioactivity and distribution of 5-HT1A receptors in the brain. • Blocking study confirmed that 99m Tc-2 had specific binding to the 5-HT1A receptor.

Radiosynthesis and in-vivo evaluation of [125I]IBT: a single-photon emission computed tomography radiotracer for α7-nicotinic acetylcholine receptor imaging.

The aim of this study was to develop several novel α7-nicotinic acetylcholine receptor (nAChR) radioligands for the early diagnosis and treatment of Alzheimer's disease. The study reported six compounds and studied the in-vitro receptor binding affinity, and selected the I-labeled IBT with good characteristics as a novel radioligand suitable for studying α7-nAChRs. After verifying the stability of the radiotracer [I]IBT, the biodistribution in vivo and regional brain biodistribution studies were carried out in mice. Blocking studies with methyllycaconitine citrate and nicotine were carried out under control and blocking conditions, and the metabolic stability was assessed in vivo in the plasma, brain, and liver. The results of our study suggested that [I]IBT had affinity for α7-nAChRs. The in-vivo evaluation in mice of [I]IBT showed a high brain/blood ratio and excellent metabolic stability. The regional brain distribution studies and the blocking studies showed that it had favorable selectivity and specificity and was a potential α7-nAChRs radioligand.

Відмінності вмісту металотіонеїну у мозку гризунів за умов постнатального розвитку та кадмієвої інтоксикації

Introduction. Metal-binding metallothionein genes are found in a vast population of organisms. These proteins are non enzymatic and very rich in cysteine residues. The various metallothionein isoforms in different brain regions arguably change over time.The aim of the study – evaluating the distribution of metallothionein in different brain regions of gerbils and Wistar rats at different stages of postnatal development (PND), under standard and low dose Cd-induced conditions.Materials and Methods. 18 Mongolian gerbils and 36 Wistar rats were divided into 6 groups (n=6), by age and condition of experiment: groups 1, 2, 3, 4 – 1, 30, 90 and 180-days old were exposed to standard conditions; group 5 and 6 – 180-days old+0.1 or 1.0 µgµg Cd2+ per animal everyday for 36 days. The metallothionein content in the hippocampus, cerebellum, and thalamus were detected by the ELISA.Results and Discussion. Obtained data was shown the dynamic of metallothionein distribution in different brain regions of gerbils and Wistar rats depending on the stage of postnatal development and functional capacities. The content of metallothionein in the hippocampus continually decreased in both animal types but the cerebella metallothionein distribution pattern was different from that of the hippocampus, but identical in both rodents, rising from day one before decreasing on day 30. The low levels of metallothionein under the influence of Cd were proportional to the doses administered.Conclusions. The level of metallothionein in the brain varies depending on the stage of development of the functional capacities of the brain parts. The significant down-regulation of metallothionein in the investigated brain regions under Cd influence suggests that a decrease in metallothionein levels depends on the dose of Cd and on the time necessary for its accumulation.

Influence of peptide transporter 2 (PEPT2) on the distribution of cefadroxil in mouse brain: A microdialysis study

Peptide transporter 2 (PEPT2) is a high-affinity low-capacity transporter belonging to the proton-coupled oligopeptide transporter family. Although many aspects of PEPT2 structure-function are known, including its localization in choroid plexus and neurons, its regional activity in brain, especially extracellular fluid (ECF), is uncertain. In this study, the pharmacokinetics and regional brain distribution of cefadroxil, a β-lactam antibiotic and PEPT2 substrate, were investigated in wildtype and Pept2 null mice using in vivo intracerebral microdialysis. Cefadroxil was infused intravenously over 4h at 0.15mg/min/kg, and samples obtained from plasma, brain ECF, cerebrospinal fluid (CSF) and brain tissue. A permeability-surface area experiment was also performed in which 0.15mg/min/kg cefadroxil was infused intravenously for 10min, and samples obtained from plasma and brain tissues. Our results showed that PEPT2 ablation significantly increased the brain ECF and CSF levels of cefadroxil (2- to 2.5-fold). In contrast, there were no significant differences between wildtype and Pept2 null mice in the amount of cefadroxil in brain cells. The unbound volume of distribution of cefadroxil in brain was 60% lower in Pept2 null mice indicating an uptake function for PEPT2 in brain cells. Finally, PEPT2 did not affect the influx clearance of cefadroxil, thereby, ruling out differences between the two genotypes in drug entry across the blood-brain barriers. These findings demonstrate, for the first time, the impact of PEPT2 on brain ECF as well as the known role of PEPT2 in removing peptide-like drugs, such as cefadroxil, from the CSF to blood.

Evaluation of TSPO PET imaging, a marker of glial activation, to study the neuroimmune footprints of morphine exposure and withdrawal

IntroductionA growing area of research suggests that neuroimmunity may impact the pharmacology of opioids. Microglia is a key component of the brain immunity. Preclinical and clinical studies have demonstrated that microglial modulators may improve morphine-induced analgesia and prevent the development of tolerance and dependence. Positron emission tomography (PET) using translocator protein 18kDa (TSPO) radioligand is a clinically validated strategy for the non-invasive detection of microglial activation. We hypothesized that TSPO PET imaging may be used to study the neuroimmune component of opioid tolerance and withdrawal. MethodsHealthy rats (n=6 in each group) received either saline or escalating doses of morphine (10–40mg/kg) on five days to achieve tolerance and a withdrawal syndrome after morphine discontinuation. MicroPET imaging with [18F]DPA-714 was performed 60h after morphine withdrawal. Kinetic modeling was performed to estimate [18F]DPA-714 volume of distribution (VT) in several brain regions using dynamic PET images and corresponding metabolite-corrected input functions. Immunohistochemistry (IHC) experiments on striatal brain slices were performed to assess the expression of glial markers (Iba1, GFAP and CD68) during 14days after morphine discontinuation. ResultsThe baseline binding of [18F]DPA-714 to the brain (VT=0.086±0.009mLcm−3) was not increased by morphine exposure and withdrawal (VT=0.079±0.010mLcm−3) indicating the absence of TSPO overexpression, even at the regional level. Accordingly, expression of glial markers did not increase after morphine discontinuation. ConclusionsMorphine tolerance and withdrawal did not detectably activate microglia and had no impact on [18F]DPA-714 brain kinetics in vivo.

Tau imaging with [18 F]THK-5351 in progressive supranuclear palsy.

Visualization of pathogenic protein aggregates is crucial to elucidate pathomechanisms and to make an accurate diagnosis in many neurodegenerative conditions. Aggregates of the microtubule-binding protein, tau, are one of the most important pathogenic molecules in neurodegenerative disorders. Progressive supranuclear palsy (PSP) is characterized by the deposition of tau proteins in some specific area such as the basal ganglia and brainstem. We tried to detect tau lesions in the brains of living patients with PSP with a novel positron emission tomography (PET) tracer, [18 F]THK-5351, which we have recently developed. Paraffin-embedded brain sections of the patients with PSP were used for autoradiography with [3 H]THK-5351 and immunohistochemistry. Nine healthy controls, 13 patients with Alzheimer's disease and three patients with PSP participated in this PET study with [18 F]THK-5351. To detect amyloid-β deposition, PET imaging with Pittsburgh compound B was also performed. Autoradiography in the brain sections of patients with PSP demonstrated [3 H]THK-5351 binding to tau deposits with a high selectivity. Although patients with PSP exhibited no remarkable [18 F]THK-5351 retention in the temporal cortex, significantly higher tracer retention was observed in the globus pallidus and midbrain. In contrast, amyloid imaging with Pittsburgh compound B showed no remarkable accumulation in the cerebral cortex of PSP. We conclude that [18 F]THK-5351 PET can potentially be used to detect the regional brain distribution of tau lesions in PSP, thereby facilitating the differential diagnosis of neurodegenerative disorders associated with tau protein.