Biomedical subjects

Arndt Rolfs

Publications and source records attributed to Arndt Rolfs.

At least 19 recordsLinked to original sources

PubMed2007

2-DE profiling of GDNF overexpression-related proteome changes in differentiating ST14A rat progenitor cells.

Targeted differentiation of neural progenitor cells (NPCs) is a challenge for treatment of neurodegenerative diseases by cell replacement therapy and cell signalling manipulation. Here, we applied a proteome profiling approach to the rat striatal progenitor model cell line ST14A in order to elucidate cellular differentiation processes. Native cells and cells transfected with the glial cell line-derived neurotrophic factor (GDNF) gene were investigated at the proliferative state and at seven time points up to 72 h after induction of differentiation. 2-DE combined with MALDI-MS was used to create a reference 2-DE-map of 652 spots of which 164 were identified and assigned to 155 unique proteins. For identification of protein expression changes during cell differentiation, spot patterns of triplicate gels were matched to the 2-DE-map. Besides proteins that display expression changes in native cells, we also noted 43 protein-spots that were differentially regulated by GDNF overexpression in more than four time points of the experiment. The expression patterns of putative differentiation markers such as annexin 5 (ANXA5), glucosidase II beta subunit (GLU2B), phosphatidylethanolamine-binding protein 1 (PEBP1), myosin regulatory light chain 2-A (MLRA), NASCENT polypeptide-associated complex alpha (NACA), elongation factor 2 (EF2), peroxiredoxin-1 (PRDX1) and proliferating cell nuclear antigen (PCNA) were verified by Western blotting. The results reflect the large rearrangements of the proteome during the differentiation process of NPCs and their strong modification by neurotrophic factors like GDNF.

Animals↗

PubMed2006

Efferent connections of the parabigeminal nucleus to the amygdala and the superior colliculus in the rat: a double-labeling fluorescent retrograde tracing study.

The parabigeminal nucleus (Pbg) is a subcortical visual center that besides reciprocal connections with the superior colliculus (SC), also projects to the amygdala (Am). The Pbg-Am connection is part of a multineuronal pathway that conveys extrageniculostriate inputs of the retina to the Am, and it rapidly responds to the sources of threat before conscious detection. The present study demonstrates that Pbg projects bilaterally to Am and SC. The ipsilateral projections arise from separate cell populations, whilst the contralaterally projecting Pbg neurons emit branching axons that simultaneously innervate Am and SC.

Amygdala↗

PubMed2006

3-Hydroxy-3-methylglutaryl coenzyme A reductase inhibitor Atorvastatin mediated effects depend on the activation status of target cells in PLP-EAE.

The effect of Atorvastatin on transcriptional activity in murine experimental autoimmune encephalomyelitis (EAE) induced by PLP peptide 139-151 was analyzed by DNA microarray technique in lymph nodes and spinal cord at onset (10 days), height (20 days) and first remission (30 days) of disease. Fourteen genes were selectively influenced by Atorvastatin in EAE mice. They are mainly related to immune cell functions and regulation of cell-to-cell interaction. Interestingly, seven genes were also differentially regulated in CFA-injected control mice. But qualitative and quantitative differences to EAE mice argue for a dependency of statin effects on the activation status of target cells. Differential regulation of the newly detected candidate genes of statin effects COX-1 and HSP-105 and the previously known statin-responsive genes ICAM-1 and CD86 was confirmed by Western blot and immunohistochemistry. Flow cytometric analysis of lymph node cells revealed that the effect of Atorvastatin treatment in non-immunized healthy animals resembled the effect of immunization with PLP peptide regarding changes of T helper cells, activated B cells and macrophages. In EAE mice, these effects were partially reversed by Atorvastatin treatment. Monitoring of expression of the newly identified candidate genes and patterns of lymphocyte subpopulations might predict the responsiveness of multiple sclerosis patients to statin treatment.

Animals↗

PubMed2006

Repeat expansion in spinocerebellar ataxia type 17 alleles of the TATA-box binding protein gene: an evolutionary approach.

The variability and mutational changes of the CAG microsatellite in the TATA-box binding protein gene (TBP) were studied. We sequenced the microsatellite of the TBP gene of 25 unrelated individuals from northern Germany (10 SCA17 patients and 15 unaffected control individuals). In addition, the microsatellites were sequenced from individuals of 10 northern German families with at least one family member affected by SCA17. To study also the evolutionary history of this CAG/CAA microsatellite in nonhuman primates, the homologous regions were analysed from Pan troglodytes, Gorilla gorilla, Pongo pygmaeus, P. abellii, Hylobates lar, Nomascus leucogenys, Symphalangus syndactylus, Macaca mulatta, Papio hamadryas, Colobus polykomos and Callithrix jacchus. Three major conclusions were drawn: (i) Patterns of synonymous CAA interruptions in the microsatellite are characteristic and likely to result from selection for stabilizing the repetitive region; (ii) Interspecific comparisons indicate that SCA17 is likely to be a human trait. The most common allele in humans (37 repeats) is close to the threshold value upon which neurodegenerative changes can occur and may act as a repository for expanded, pathogenic alleles; (iii) The cassette-like structure of five out of 17 expanded alleles can be attributed to unequal crossing over. This can explain the rare and sporadic de novo generation of SCA17 alleles.

Animals↗

PubMed2006

2-DE proteome analysis of a proliferating and differentiating human neuronal stem cell line (ReNcell VM).

The proteome of a proliferating human stem cell line was analyzed and then utilized to detect stem cell differentiation-associated changes in the protein profile. The analysis was conducted with a stable human fetal midbrain stem cell line (ReNcell VM) that displays the properties of a neural stem cell. Therefore, acquisition of proteomic data should be representative of cultured human neural stem cells (hNSCs) in general. Here we present a 2-DE protein-map of this cell line with annotations of 402 spots representing 318 unique proteins identified by MS. The subsequent proteome profiling of differentiating cells of this stem cell line at days 0, 4 and 7 of differentiation revealed changes in the expression of 49 identified spots that could be annotated to 45 distinct proteins. This differentiation-associated expression pattern was validated by Western blot analysis for transgelin-2, proliferating cell nuclear antigen, as well as peroxiredoxin 1 and 4. The group of regulated proteins also included NudC, ubiquilin-1, STRAP, stress-70 protein, creatine kinase B, glial fibrillary acidic protein and vimentin. Our results reflect the large rearrangement of the proteome during the differentiation process of the stem cells to terminally differentiated neurons and offer the possibility for further characterization of specific targets driving the stem cell differentiation.

Blotting, Western↗

PubMed2006

Ih is maturing: implications for neuronal development.

Vast electrophysiological activity near resting potential, including rhythmic oscillatory activity, is a hallmark of many brain regions and a motor of the developing CNS. This activity is mediated and influenced by diverse receptor-operated and voltage-gated ion channels. In turn, these channels are modulated during the course of development by altering their density, distribution and properties. The hyperpolarization-activated and cyclic nucleotide-gated cation current, Ih, impacts on the resting membrane potential and is involved in the generation and modulation of neuronal oscillatory activity. Therefore, it is conceivable that Ih is well suited to govern the specific processes involved in activity-dependent neuronal development. Here, we review the evidence that maturation of Ih accounts, at least in part, for the control of membrane properties during neuronal development of various parts of the brain. The temporal and regional variations in Ih development might underlie the normal maturation of neuronal circuits and, consequently, the perturbations of this might account for some of the neuropathology of the brain. This review summarizes the evidence for the stage and localization dependence of Ih in CNS development with a focus on arborized cells with high dendritic Ih. Further, it outlines hypotheses on the contribution of Ih to neuronal and network maturation.

Animals↗

PubMed2006

Wnt signal pathways and neural stem cell differentiation.

Self-renewal, migration and differentiation of neural progenitor cells are controlled by a variety of pleiotropic signal molecules. Members of the morphogen family of Wnt molecules play a crucial role for developmental and repair mechanisms in the embryonic and adult nervous system. A strategy of disclosure of the role of different canonical (glycogen synthase kinase-3beta/beta-catenin-dependent) and noncanonical (Ca2+- and JNK-dependent) signal pathways for progenitor cell expansion and differentiations is illustrated at the example of the rat striatal progenitor cell line ST14A that is immortalized by stable retroviral transfection with a temperature-sensitive mutant of the SV40 large T antigen. A shift from permissive 33 degrees C to nonpermissive 39 degrees C leads to proliferation stop and start of differentiation into glial and neuronal cells. Investigation of expression of Wnts, Wnt receptors and Wnt-dependent signal pathway assay point to a stage-dependent involvement of canonical and noncanonical signaling in proliferation and differentiation of ST14A cells, whereby a mutual suppression of pathway activities is likely. Canonical Wnt molecules are not detected in proliferating and differentiating ST14A cells except Wnt2. The noncanonical Wnt molecules Wnt4, Wnt5a and Wnt11 are expressed in proliferating cells and increase during differentiation, whereas cellular beta-catenin decreases in the early phase and is restored in the late phase of differentiation. Accumulation of beta-catenin at the membrane in undifferentiated proliferating cells and its nuclear localization in nondividing undifferentiated cells under differentiation conditions argues for a distinct spatially regulated role of the molecule in the proliferation and early differentiation phase. Ca2+-dependent and JNK-dependent noncanonical Wnt signaling is not detected during differentiation of ST14A cells. Complete exploration of the role of Wnt pathways, for differentiation of the neural progenitor cells ST14A will require Wnt overexpression and exposure of ST14A cells to exogenous Wnts either with purified Wnts or by co-cultures with Wnt producers.

Animals↗

PubMed2006

2-DE proteomic profiling of neuronal stem cells.

Proteomics has become a powerful tool in neuroscience studies. Although numerous human neural stem cells are available for research purposes since many years, there exists only limited information on proteomic data from stable neural stem cell lines. Profiling and functional proteome studies of neuronal stem cells will help to describe the protein inventory as well as protein activity and interactions, subcellular localization and posttranslational modifications. The proteomic analysis of neuronal differentiation processes will elucidate the complex events leading to the generation of different phenotypes via distinctive developmental programs that control self-renewal, differentiation, and plasticity. Using the ReNcell VM197 model, a cell line derived from human fetal ventral mesencephalon stem cells, we studied the protein inventory of the stem cells by 2-DE gel electrophoresis and mass spectrometric protein identification and constructed a 2-DE protein map consisting of more than 400 identified protein spots. This proteome reference database constitutes the basis for further investigations of differential protein expression during differentiation. A profiling of the neuronal differentiation-associated changes displayed the large rearrangement of the proteome during this process, and the proteomic techniques proved to be a valuable tool for the elucidation of neuronal differentiation process and for target protein screening.

Animals↗

PubMed2005

Prevalence of Fabry disease in patients with cryptogenic stroke: a prospective study.

BACKGROUND: Strokes are an important cause of morbidity and mortality in young adults. However, in most cases the cause of the stroke remains unclear. Anderson-Fabry disease is an X-linked recessive lysosomal storage disease resulting from deficient alpha-galactosidase and causes an endothelial vasculopathy followed by cerebral ischaemia. To determine the importance of Fabry disease in young people with stroke, we measured the frequency of unrecognised Fabry disease in a cohort of acute stroke patients. METHODS: Between February, 2001, and December, 2004, 721 German adults aged 18 to 55 years suffering from acute cryptogenic stroke were screened for Fabry disease. The plasma alpha-galactosidase activity in men was measured followed by sequencing of the entire alpha-GAL gene in those with low enzyme activity. By contrast, the entire alpha-GAL gene was genetically screened for mutations in women even if enzyme activity was normal. FINDINGS: 21 of 432 (4.9%) male stroke patients and seven of 289 (2.4%) women had a biologically significant mutation within the alpha-GAL gene. The mean age at onset of symptomatic cerebrovascular disease was 38.4 years (SD 13.0) in the male stroke patients and 40.3 years (13.1) in the female group. The higher frequency of infarctions in the vertebrobasilar area correlated with more pronounced changes in the vertebrobasilar vessels like dolichoectatic pathology (42.9%vs 6.8%). INTERPRETATION: We have shown a high frequency of Fabry disease in a cohort of patients with cryptogenic stroke, which corresponds to about 1.2% in young stroke patients. Fabry disease must be considered in all cases of unexplained stroke in young patients, especially in those with the combination of infarction in the vertebrobasilar artery system and proteinuria.

Adult↗

PubMed2005

Functional significance of HCN2/3-mediated I(h) in striatal cells at early developmental stages.

Hyperpolarization-activated cAMP-gated cation currents (I(h)) were recently linked to pre- and postnatal developmental processes in several brain regions, including the ventral telencephalon. To evaluate the role of I(h) in striatal development, we used short-term cultured cells from the lateral ganglionic eminence at embryonic day 14 (E14) and postnatal days 1-3 (P1-3) as well as the embryonic striatal progenitor cell line ST14A. Western blot analysis of the I(h) underlying subunit proteins HCN1-4 revealed strong HCN2 expression in proliferating ST14A cells and weak expression in postmitotic ST14A cells and in cells from the developing brain. We also found HCN3 expression only in ST14A cells at both proliferative and nonproliferative stages but not in short-term cultured striatal cells. In all cases, HCN1 and HCN4 transcripts were below the detection level. Despite the selective protein expression, RT-PCR analysis showed stable expression of HCN2-4 but not HCN1 mRNA in all short-term-cultured striatal cells and in the ST14A cell line. Consistent with the strong protein expression, an I(h) was recorded with features of an HCN2-mediated current in ST14A cells at the proliferative stage and in short-term-cultured E14 cells. Of particular importance is that we detected no currents upon hyperpolarization in the ST14A cells at the nonproliferative stage when only HCN3 protein was present. These results suggest the potential importance of ST14A cells in defining the molecular mechanisms regulating I(h) expression and function.

Animals↗

PubMed2005

Identification of quantitative trait loci controlling cortical motor evoked potentials in experimental autoimmune encephalomyelitis: correlation with incidence, onset and severity of disease.

Experimental autoimmune encephalomyelitis (EAE) is a polygenic chronic inflammatory demyelinating disease of the nervous system, commonly used as an animal model of multiple sclerosis. Previous studies have identified multiple quantitative trait loci (QTLs) controlling different aspects of disease pathogenesis. However, direct genetic control of cortical motor evoked potentials (cMEPs) as a straightforward measure of extent of demyelination or synaptic block has not been investigated earlier. Here, we examined the genetic control of different traits of EAE in a F2 intercross population generated from the EAE susceptible SJL/J (SJL) and the EAE resistant C57BL/10.S (B10.S) mouse strains involving 400 animals. The genotypes of 150 microsatellite markers were determined in each animal and correlated to phenotypic data of onset and severity of disease, cell infiltration and cMEPs. Nine QTLs were identified. Three sex-linked QTLs mapped to chromosomes 2, 10 and 18 linked to disease severity in females, whereas QTLs on chromosomes 1, 8 and 15 linked to the latency of the cMEPs. QTLs affecting T-lymphocyte, B-lymphocyte and microglia infiltration mapped on chromosomes 8 and 15. The cMEP-associated QTLs correlated with incidence, onset or severity of disease, e.g. QTL on chromosome 8, 32-48 cM (EAE 31) (LOD 6.9, P<0.001), associated to cMEP latencies in non-immunized mice and correlated with disease onset and EAE 32 on chromosome 15 linked to cMEP latencies 15 days post-immunization and correlated with disease severity. Additionally, applying tissue microarray technology, we identified QTLs associated to microglia and lymphocytes infiltration on chromosomes 8 and 15, which are different from the QTLs controlling cMEP latencies. There were no alterations in the morphological appearance of the myelin sheaths. Our findings suggest a possible role of myelin composition and/or synaptic transmission in susceptibility to EAE.

Animals↗

PubMed2005

Dopamine exerts no acute effects on Kv1.3 in activated encephalitogenic T cells.

Apart from a central function in the extrapyramidal motor system, dopamine has been suggested to play a role in neuroimmune interactions. Particularly in diseases of the central nervous system, such as multiple sclerosis, alterations in dopamine homeostasis might have immunological consequences. We investigated potential effects of dopamine stabilized by ascorbic acid on specifically activated encephalitogenic T cells at the peak of activation. Those cells exhibited an upregulation of voltage-sensitive K+ channels which play a role in many neurotransmitter responses of lymphocytes and fulfilled a prerequisite to respond to dopamine, i.e. stable expression of mRNA for dopamine receptors DRD1, DRD2 and DRD3. However, whole-cell and perforated whole-cell recordings revealed no change in voltage-sensitive K+ currents. Moreover, T cell proliferation was not changed in the presence of dopamine. Previously reported dopamine effects on T cells may be explained by a comparatively lower activation of the cells under investigation, suggesting an activation dependence of dopamine effects that may not be mediated by K+ channels. Alternatively, the occurrence of dopamine degradation products under unprotected conditions may account for the changes reported. Nevertheless, care should be taken when using the dopamine-protecting anti-oxidant ascorbic acid, since we found that it markedly inhibited both K+ currents and lymphocyte proliferation at higher concentrations.

Adenosine Triphosphate↗

PubMed2005

Representation of individual gene expression in completely pooled mRNA samples.

Designing microarray experiments, scientists are often confronted with the question of pooling due to financial constraints, but discussion of the validity of pooling tends toward a sub-pooling recommendation. Since complete pooling protocols can be considered part of sub-pooling designs, gene expression data from three complete pooling experiments were analyzed. Data from complete pooled versus individual mRNA samples of rat brain tissue were compared to answer the question whether the pooled sample represents individual samples in small-sized experiments. Our analytic approach provided clear results concerning the Affymetrix MAS 5.0 signal and detection call parameters. Despite a strong similarity of arrays within experimental groups, the individual signals were evidently not appropriately represented in the pooled sample, with slightly more than half of all the genes considered. Our analysis reveals problems in cases of small complete pooling designs with less than six subjects pooled.

Animals↗

PubMed2004

Neuromodulation by a cytokine: interferon-beta differentially augments neocortical neuronal activity and excitability.

The immunomodulatory cytokine interferon-beta (IFN-beta) is used in the treatment of autoimmune diseases such as multiple sclerosis. However, the effect of IFN-beta on neuronal functions is currently unknown. Intracellular recordings were conducted on somatosensory neurons of neocortical layers 2/3 and 5 exposed to IFN-beta. The excitability of neurons was increased by IFN-beta (10-10,000 U/ml) in two kinetically distinct, putatively independent manners. First IFN-beta reversibly influenced the subthreshold membrane response by raising the membrane resistance R(M) 2.5-fold and the membrane time constant tau 1.7-fold dose-dependently. The effect required permanent exposure to IFN-beta and was reduced in magnitude if the extracellular K+ was lowered. However, the membrane response to IFN-beta in the subthreshold range was prevented by ZD7288 (a specific blocker of I(h)) but not by Ni2+, carbachol, or bicuculline, pointing to a dependence on an intact I(h). Second, IFN-beta enhanced the rate of action potential firing. This effect was observed to develop for >1 h when the cell was exposed to IFN-beta for 5 min or >5 min and showed no reversibility (< or =210 min). Current-discharge (F-I) curves revealed a shift (prevented by bicuculline) as well as an increase in slope (prevented by carbachol and Ni2+). Layer specificity was not observed with any of the described effects. In conclusion, IFN-beta influences the neuronal excitability in neocortical pyramidal neurons in vitro, especially under conditions of slightly increased extracellular K+. Our blocker experiments indicate that changes in various ionic conductances with different voltage dependencies cause different IFN-beta influences on sub- and suprathreshold behavior, suggesting a more general intracellular process induced by IFN-beta.

Action Potentials↗

PubMed2004

Overexpression of glial cell line-derived neurotrophic factor induces genes regulating migration and differentiation of neuronal progenitor cells.

The glial cell line-derived neurotrophic factor (GDNF) is involved in the development and maintenance of neural tissues. Mutations in components of its signaling pathway lead to severe migration deficits of neuronal crest stem cells, tumor formation, or ablation of the urinary system. In animal models of Parkinson's disease, GDNF has been recognized to be neuroprotective and to improve motor function when delivered into the cerebral ventricles or into the substantia nigra. Here, we characterize the network of 43 genes induced by GDNF overproduction of neuronal progenitor cells (ST14A), which mainly regulate migration and differentiation of neuronal progenitor cells. GDNF down-regulates doublecortin, Paf-ah1b (Lis1), dynamin, and alpha-tubulin, which are involved in neocortical lamination and cytoskeletal reorganization. Axonal guidance depends on cell-surface molecules and extracellular matrix proteins. Laminin, Mpl3, Alcam, Bin1, Id1, Id2, Id3, neuregulin1, the ephrinB2-receptor, neuritin, focal adhesion kinase (FAK), Tc10, Pdpk1, clusterin, GTP-cyclooxygenase1, and follistatin are genes up-regulated by GDNF overexpression. Moreover, we found four key enzymes of the cholesterol-synthesis pathway to be down-regulated leading to decreased farnesyl-pyrophospate production. Many proteins are anchored by farnesyl-derivates at the cell membrane. The identification of these GDNF-regulated genes may open new opportunities for directly influencing differentiation and developmental processes of neurons.

Animals↗

PubMed2004

Wnt-5a expression in the rat neuronal progenitor cell line ST14A.

Transplantation of cells derived from embryonic stem cells is currently under investigation as a promising strategy to restore functional deficits in neurodegenerative diseases, e.g. Parkinson's disease. To generate cells suitable for transplantation, a neuronal progenitor cell line (ST14A) was derived from embryonic day 14 rat striatum by stable retroviral transfection of the temperature-sensitive SV40 large T antigen and genetically modified by transfection with constructs of the neurotrophic factors ciliary neurotrophic factor (CNTF) [CNTF-ST14A] and glial cell line-derived neurotrophic factor (GDNF) [GDNF-ST14A], respectively. In order to investigate the capacity of these cells to regulate neuronal growth and physiological differentiation, e.g. remodeling of axons and synaptogenesis, we analyzed the expression of molecules which control the cell fate during embryonic development. For the first time, we found endogenous Wnt-5a, a regulator molecule that can induce dopaminergic phenotype, by RT-PCR, Western blot and flow cytometry in the neuronal progenitor cell line ST14A and its derivatives CNTF-ST14A and GDNF-ST14A. The protein was transiently upregulated at the differentiation-inducing non-permissive temperature of 39 degrees C and it was also secreted into the culture medium. Our findings are based on in vitro investigation of artificially immortalized cell lines. However, they raise the possibility that neuronal progenitor cells that might be used to treat neurodegenerative diseases express Wnt-5a, thus promoting their potential for dopaminergic differentiation.

Animals↗

PubMed2004

Gene-expression profiling of the early stages of MOG-induced EAE proves EAE-resistance as an active process.

Experimental autoimmune encephalomyelitis (EAE) induced by myelin oligodendrocyte glycoprotein (MOG) is a well-established animal model of multiple sclerosis (MS) in rodents. It reflects the wide spectrum of disease pathology and serves as a valuable tool for studying the pathogenesis and for testing new therapies of MS. In order to identify genes responsible for resistance to and modulation of the disease, we compared the mRNA expression profile of more than 12,000 genes by DNA microarray technique in lymph nodes of the highly EAE-susceptible mouse strain C57Bl/6 (B6) and the resistant strain C57Bl/10.S (B10). The disease onset in B6 mice was day 15. We identified 84 genes that were up-regulated more than two-fold in B10 mice compared to vehicle-treated controls, whereas only two genes were up-regulated in B6 mice after 7 and 15 days post-immunization (p.i.), respectively. We were able to match five up-regulated genes in B10 mice to known quantitative trait loci (QTLs), which control for EAE susceptibility. Only 17, respectively 5, genes were down-regulated at both time points in B10 and B6 mice. Tests for immunoreactivity to MOG (T cell proliferation and interferon-gamma (IFN-gamma) secretion) revealed no stronger immune response in B6 compared to B10 mice supporting the hypothesis of an immunosuppressive effect as a target to prevent EAE in the B10 mice. We conclude that resistance to EAE (and possibly to MS) is an active process mediated by multiple genes up-regulated in peripheral lymphatic organs of resistant animals. Thus, monitoring of the expression of these new candidate genes may serve as a tool for the disease progression and the pharmaceutical treatment.

Animals↗

PubMed2004

An impaired neocortical Ih is associated with enhanced excitability and absence epilepsy.

Neuronal subthreshold excitability and firing behaviour are markedly influenced by the activation and deactivation of the somato-dendritic hyperpolarization-activated cation current (Ih). Here, we evaluated possible contributions of Ih to hyperexcitability in an animal model of absence seizures (WAG/Rij rats). We investigated pyramidal neurons of the somatosensory neocortex, the site of generation of spike-wave discharges. Ih-mediated functions in neurons from WAG/Rij rats, Wistar rats (sharing the same genetic background with WAG/Rij, but less epilepsy-prone) and ACI rats (an inbred strain, virtually free of seizures) were compared. We complemented whole-cell recordings from layer 2-3 pyramidal neurons with immunohistochemistry, Western blot and RT-PCR analysis of the h-channel subunits HCN1-4. The fast component of Ih activation in WAG/Rij neurons was significantly reduced (50% reduction in the h-current density) and four times slower than in neurons from nonepileptic Wistar or ACI rats. The results showing decreases in currents corresponded to a 34% reduction in HCN1 protein in the WAG/Rij compared to the Wistar neocortex, but HCN1 mRNA showed stable expression. The other three Ih subunit mRNAs and proteins (HCN2-4) were not affected. The alterations in Ih magnitude and kinetics of gating in WAG/Rij neurons may contribute to augmented excitatory postsynaptic potentials, the increase in their temporal summation and the facilitation of burst firing of these neurons because each of these effects could be mimicked by the selective Ih antagonist ZD 7288. We suggest that the deficit in Ih-mediated functions may contribute to the development and onset of spontaneously occurring hyperexcitability in a rat model of absence seizures.

2-Amino-5-phosphonovalerate↗