Markus Cremer

Neurotransmitter Receptor Imbalances in Motor Cortex and Basal Ganglia in Hepatic Encephalopathy

Hepatic encephalopathy (HE) in chronic liver disease is characterized by neuropsychiatric and motor disturbances and associated with a net increase of inhibitory neurotransmission. Though many studies, mostly carried out in animal models, have linked dysfunctions of single neurotransmitter systems with the pathogenesis of HE, reports concerning neurotransmitter receptor alterations are controversial. Little is known about the situation in humans. We carried out a multireceptor assessment of HE-associated changes in neurotransmitter receptor densities and affinities in human post-mortem brain samples. Dissociation constants and densities of different binding sites for glutamate, GABA, acetylcholine, norepinephrine, serotonin, dopamine and adenosine were determined by in vitro binding assays and quantitative receptor autoradiography in the motor cortex and putamen of HE and control brains. HE cases do not build a homogeneous group, but differ concerning direction and intensity of binding site density divergences from control values. The acetylcholine M2 binding site dissociation constant was significantly higher in HE brains. Nicotinic acetylcholine and adenosine type 1 and 2A densities were significantly down-regulated in the putamen of HE brains. Our data suggest that neurotransmitter alterations are probably not the primary key factor responsible for the neuropsychiatric and motor disturbances associated with HE.

Preclinical Pharmacokinetic Studies of the Tritium Labelled D-Enantiomeric Peptide D3 Developed for the Treatment of Alzheimer´s Disease

Targeting toxic amyloid beta (Aβ) oligomers is currently a very attractive drug development strategy for treatment of Alzheimer´s disease. Using mirror-image phage display against Aβ1-42, we have previously identified the fully D-enantiomeric peptide D3, which is able to eliminate Aβ oligomers and has proven therapeutic potential in transgenic Alzheimer´s disease animal models. However, there is little information on the pharmacokinetic behaviour of D-enantiomeric peptides in general. Therefore, we conducted experiments with the tritium labelled D-peptide D3 (3H-D3) in mice with different administration routes to study its distribution in liver, kidney, brain, plasma and gastrointestinal tract, as well as its bioavailability by i.p. and p.o. administration. In addition, we investigated the metabolic stability in liver microsomes, mouse plasma, brain, liver and kidney homogenates, and estimated the plasma protein binding. Based on its high stability and long biological half-life, our pharmacokinetic results support the therapeutic potential of D-peptides in general, with D3 being a new promising drug candidate for Alzheimer´s disease treatment.

Cerebral kinetics of the dopamine D2 receptor ligand [123I]IBZM in mice

INTRODUCTION In vivo small animal imaging of the dopaminergic system is of great interest for basic and applied neurosciences, especially in transgenic mice. Small animal SPECT is particularly attractive because of its superior spatial resolution and tracer availability. We investigated the kinetics of the commercial dopamine D(2) receptor (DZR) ligand [(123)I]IBZM in mice as a prerequisite for an appropriate design of translational SPECT imaging between mice and humans. METHODS Cerebral kinetics of [(123)I]IBZM under isoflurane anaesthesia were assessed by autoradiography in mice sacrificed at 30, 60, 120 and 200 min after iv injection. To explore the possible effects of isoflurane anaesthesia, an additional mice group was only anaesthetized for 20 min before being sacrificed at 140 min (putative time of single-scan SPECT analysis). RESULTS Maximum [(123)I]IBZM uptake in the striatum (D(2)R-rich; 10.5+/-2.7 %ID/g) and cerebellum (D(2)R-devoid; 2.4+/-0.7 %ID/g) was observed at 30 min after injection. Thereafter, [(123)I]IBZM uptake decreased slowly in striatum and rapidly in the cerebellum (200 min: 5.3+/-1.9 and 0.4+/-0.2 %ID/g, respectively). The striatum-to-cerebellum (S/C) [(123)I]IBZM uptake ratio increased from 4.6+/-1.2 at 30 min to 11.6+/-2.6 at 120 min. The S/C ratio at 200 min was highly variable (17.8+/-10.1), possibly indicating pseudo-equilibration in some animals. In mice, which were only anaesthetized between 120 and 140 min, a higher S/C ratio of 17.0+/-5.1 was observed. CONCLUSIONS The present study suggests that [(123)I]IBZM is a suitable ligand for D(2)R-SPECT in mice. Although a single-scan analysis may be a pragmatic semi-quantitative approach, tracer kinetic analyses on dynamic SPECT data should be pursued. The interfering effects of isoflurane anaesthesia need to be considered.

Comparison of intravenous and intraperitoneal [123I]IBZM injection for dopamine D2 receptor imaging in mice.

INTRODUCTION Intraperitoneal (IP) injection represents an attractive alternative route of radiotracer administration for small animal imaging, e.g., for longitudinal studies in transgenic mouse models. We explored the cerebral kinetics of the reversible dopamine D2 receptor ligand [(123)I]IBZM after IP injection in mice. METHODS Cerebral [(123)I]IBZM kinetics were assessed by ex vivo autoradiography in mice sacrificed between 30 and 200 min after IP or intravenous (IV) injection. The striatum-to-cerebellum (S/C) uptake ratio at 140 min was evaluated in wild-type mice and R6/2 transgenic mice (a Huntingtons disease model) in comparison with in vitro autoradiography using [(3)H]raclopride. RESULTS [(123)I]IBZM uptake was slower and lower after IP injection [maximum uptake in striatum 5.6% injected dose per gram (ID/g) at 60 min] than IV injection (10.5%ID/g at 30 min). Between 60 and 120 min, striatal (cerebellar) uptake after IP injection reached 63% (91%) of the uptake after IV injection. The S/C uptake ratio increased to 15.5 at 200 min after IP injection, which corresponds to 87% of the IV injection value (17.8). Consistent with in vitro [(3)H]raclopride autoradiography, the S/C ratio given by ex vivo [(123)I]IBZM autoradiography (140 min after IP injection) was significantly reduced in R6/2 mice. CONCLUSIONS Although IP injection resulted in slower kinetics, relevant measures of dopamine D2 receptor availability were comparable. Thus, IP injection represents a promising route of tracer administration for small animal [(123)I]IBZM SPECT. This should considerably simplify the implementation of longitudinal small animal neuroimaging studies, e.g., in transgenic mouse models.

In vivo imaging of rat brain A1 adenosine receptor occupancy by caffeine

Caffeine, the most commonly used central nervous system (CNS) stimulant, is a potent antagonist of A1 adenosine receptors (A1ARs). These receptors can now be quantified and imaged with PET and [18F]CPFPX [1, 2]. The figure shows PET images of cerebral A1ARs (see cross-wires) in a control rat (a) and in a rat after blockade of A1ARs with caffeine (b) (4 mg/kg body weight, corresponding to three to four cups of coffee in a human being). Sagittal views are displayed on the left and transverse views on the right. *, Injection site. An ECAT EXACT HR+ scanner was used to obtain the PET images.

Chapter 18 – High-Resolution Fiber and Fiber Tract Imaging Using Polarized Light Microscopy in the Human, Monkey, Rat, and Mouse Brain

Polarized light imaging (PLI) enables ultra-high resolution visualization of nerve fibers in postmortem brains by the birefringent property of myelin. Using the Jones calculus, 3D orientation of fibers can be determined in serial sections throughout the brain. We present examples of fiber architecture with an unprecedented spatial resolution both in the white matter and within the cerebral cortex of human, vervet monkey, and rodent brains. Crossing of fibers can be directly visualized without any assumptions or modeling, and fibers can be followed from the white matter into the most superficial layers of the cortex. Even extremely small fiber tracts are visible. Furthermore, the fiber architecture within the cerebral cortex provides a new approach for its parcellation into areas with distinct distribution, orientation, and density patterns of nerve fibers. PLI also provides the anatomical ground truth for the evaluation of results derived from diffusion weighted imaging.

Characterization of [123I]FP-CIT binding to the dopamine transporter in the striatum of tree shrews by quantitative in vitro autoradiography.

Aim of this study was to quantify the binding of [123I]FP‐CIT in striatum of healthy tree shrews. [123I]FP‐CIT is widely used in clinical SPECT imaging to reveal nigrostriatal degeneration in aid of the diagnosis of clinically uncertain parkinsonian syndromes. Despite its wide clinical use, the saturation binding parameters of [123I]FP‐CIT for the dopamine transporter (DAT) have not yet been determined in any mammalian brain. Tree shrews are genetically and neuroanatomically more similar to humans than are rodents and might therefore be a valuable animal model for research of neurological disorders involving brain dopamine.

Fast, quantitative in situ hybridization of rare mRNAs using 14C-standards and phosphorus imaging.

The use of radiolabelled probes for in situ hybridization (ISH) bears the advantage of high sensitivity and quantifiability. The crucial disadvantages are laborious hybridization protocols, exposition of hybridized sections to film for up to several weeks and the time consuming need to prepare tissue standards with relatively short-lived isotopes like (33)P or (35)S for each experiment. The quantification of rare mRNAs like those encoding for subunits of neurotransmitter receptors is therefore a challenge in ISH. Here, we describe a method for fast, quantitative in situ hybridization (qISH) of mRNAs using (33)P-labelled oligonucleotides together with (14)C-polymer standards (Microscales, Amersham Biosciences) and a phosphorus imaging system (BAS 5000 BioImage Analyzer, Raytest-Fuji). It enables a complete analysis of rare mRNAs by ISH. The preparation of short-lived (33)P-standards for each experiment was replaced by co-exposition and calibration of long-lived (14)C-standards together with (33)P-labelled brain paste standards. The use of a phosphorus imaging system allowed a reduction of exposition time following hybridization from several weeks to a few hours or days. We used this approach as an example for applications to quantify the expression of GluR1 and GluR2 subunit mRNAs of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor in the hippocampus of untreated rats, and after intraperitoneal application of the organo-arsenic compound dimethyl arsenic acid.

Acetylcholine Neurotransmitter Receptor Densities in the Striatum of Hemiparkinsonian Rats Following Botulinum Neurotoxin-A Injection

Cholinergic neurotransmission has a pivotal function in the caudate-putamen, and is highly associated with the pathophysiology of Parkinsons disease. Here, we investigated long-term changes in the densities of the muscarinic receptor subtypes M1, M2, M3 (mAchRs) and the nicotinic receptor subtype α4β2 (nAchRs) in the striatum of the 6-OHDA-induced hemiparkinsonian (hemi-PD) rat model using quantitative in vitro receptor autoradiography. Hemi-PD rats exhibited an ipsilateral decrease in striatal mAchR densities between 6 and 16%. Moreover, a massive and constant decrease in striatal nAchR density by 57% was found. A second goal of the study was to disclose receptor-related mechanisms for the positive motor effect of intrastriatally injected Botulinum neurotoxin-A (BoNT-A) in hemi-PD rats in the apomorphine rotation test. Therefore, the effect of intrastriatally injected BoNT-A in control and hemi-PD rats on mAchR and nAchR densities was analyzed and compared to control animals or vehicle-injected hemi-PD rats. BoNT-A administration slightly reduced interhemispheric differences of mAchR and nAchR densities in hemi-PD rats. Importantly, the BoNT-A effect on striatal nAchRs significantly correlated with behavioral testing after apomorphine application. This study gives novel insights of 6-OHDA-induced effects on striatal mAchR and nAchR densities, and partly explains the therapeutic effect of BoNT-A in hemi-PD rats on a cellular level.