Publications by authors named "Thoralf Opitz"

27 Publications

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Complex effects of eslicarbazepine on inhibitory micro networks in chronic experimental epilepsy.

Epilepsia 2021 02 16;62(2):542-556. Epub 2021 Jan 16.

Medical Faculty, Institute for Experimental Epileptology and Cognition Research, University of Bonn, Bonn, Germany.

Objective: Many antiseizure drugs (ASDs) act on voltage-dependent sodium channels, and the molecular basis of these effects is well established. In contrast, how ASDs act on the level of neuronal networks is much less understood.

Methods: In the present study, we determined the effects of eslicarbazepine (S-Lic) on different types of inhibitory neurons, as well as inhibitory motifs. Experiments were performed in hippocampal slices from both sham-control and chronically epileptic pilocarpine-treated rats.

Results: We found that S-Lic causes an unexpected reduction of feed-forward inhibition in the CA1 region at high concentrations (300 µM), but not at lower concentrations (100 µM). Concurrently, 300 but not 100 μM S-Lic significantly reduced maximal firing rates in putative feed-forward interneurons located in the CA1 stratum radiatum of sham-control and epileptic animals. In contrast, feedback inhibition was not inhibited by S-Lic. Instead, application of S-Lic, in contrast to previous data for other drugs like carbamazepine (CBZ), resulted in a lasting potentiation of feedback inhibitory post-synaptic currents (IPSCs) only in epileptic and not in sham-control animals, which persisted after washout of S-Lic. We hypothesized that this plasticity of inhibition might rely on anti-Hebbian potentiation of excitatory feedback inputs onto oriens-lacunosum moleculare (OLM) interneurons, which is dependent on Ca -permeable α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. Indeed, we show that blocking Ca -permeable AMPA receptors completely prevents upmodulation of feedback inhibition.

Significance: These results suggest that S-Lic affects inhibitory circuits in the CA1 hippocampal region in unexpected ways. In addition, ASD actions may not be sufficiently explained by acute effects on their target channels, rather, it may be necessary to take plasticity of inhibitory circuits into account.
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http://dx.doi.org/10.1111/epi.16808DOI Listing
February 2021

CD8 T-Lymphocyte-Driven Limbic Encephalitis Results in Temporal Lobe Epilepsy.

Ann Neurol 2021 Apr 15;89(4):666-685. Epub 2021 Jan 15.

Section for Translational Epilepsy Research, Department of Neuropathology, University Hospital Bonn, Bonn, Germany.

Objective: Limbic encephalitis (LE) comprises a spectrum of inflammatory changes in affected brain structures including the presence of autoantibodies and lymphoid cells. However, the potential of distinct lymphocyte subsets alone to elicit key clinicopathological sequelae of LE potentially inducing temporal lobe epilepsy (TLE) with chronic spontaneous seizures and hippocampal sclerosis (HS) is unresolved.

Methods: Here, we scrutinized pathogenic consequences emerging from CD8 T cells targeting hippocampal neurons by recombinant adeno-associated virus-mediated expression of the model-autoantigen ovalbumin (OVA) in CA1 neurons of OT-I/RAG1 mice (termed "OVA-CD8 LE model").

Results: Viral-mediated antigen transfer caused dense CD8 T cell infiltrates confined to the hippocampal formation starting on day 5 after virus transduction. Flow cytometry indicated priming of CD8 T cells in brain-draining lymph nodes preceding hippocampal invasion. At the acute model stage, the inflammatory process was accompanied by frequent seizure activity and impairment of hippocampal memory skills. Magnetic resonance imaging scans at day 7 of the OVA-CD8 LE model revealed hippocampal edema and blood-brain barrier disruption that converted into atrophy until day 40. CD8 T cells specifically targeted OVA-expressing, SIINFEKL-H-2K -positive CA1 neurons and caused segmental apoptotic neurodegeneration, astrogliosis, and microglial activation. At the chronic model stage, mice exhibited spontaneous recurrent seizures and persisting memory deficits, and the sclerotic hippocampus was populated with CD8 T cells escorted by NK cells.

Interpretation: These data indicate that a CD8 T-cell-initiated attack of distinct hippocampal neurons is sufficient to induce LE converting into TLE-HS. Intriguingly, the role of CD8 T cells exceeds neurotoxic effects and points to their major pathogenic role in TLE following LE. ANN NEUROL 2021;89:666-685.
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http://dx.doi.org/10.1002/ana.26000DOI Listing
April 2021

Nonspecific Expression in Limited Excitatory Cell Populations in Interneuron-Targeting Cre-driver Lines Can Have Large Functional Effects.

Front Neural Circuits 2020 27;14:16. Epub 2020 Apr 27.

Institute for Experimental Epileptology and Cognition Research, University of Bonn, Bonn, Germany.

Transgenic Cre-recombinase expressing mouse lines are widely used to express fluorescent proteins and opto-/chemogenetic actuators, making them a cornerstone of modern neuroscience. The investigation of interneurons in particular has benefitted from the ability to genetically target specific cell types. However, the specificity of some Cre driver lines has been called into question. Here, we show that nonspecific expression in a subset of hippocampal neurons can have substantial nonspecific functional effects in a somatostatin-Cre (SST-Cre) mouse line. Nonspecific targeting of CA3 pyramidal cells caused large optogenetically evoked excitatory currents in remote brain regions. Similar, but less severe patterns of nonspecific expression were observed in a widely used SST-IRES-Cre line, when crossed with a reporter mouse line. Viral transduction on the other hand yielded more specific expression but still resulted in nonspecific expression in a minority of pyramidal layer cells. These results suggest that a careful analysis of specificity is mandatory before the use of Cre driver lines for opto- or chemogenetic manipulation approaches.
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http://dx.doi.org/10.3389/fncir.2020.00016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7197702PMC
April 2020

Partial sciatic nerve ligation leads to an upregulation of Ni-resistant T-type Ca currents in capsaicin-responsive nociceptive dorsal root ganglion neurons.

J Pain Res 2019 11;12:635-647. Epub 2019 Feb 11.

Department of Epileptology, University of Bonn Medical Center, Bonn, Germany,

Background: Neuropathic pain resulting from peripheral nerve lesions is a common medical condition, but current analgesics are often insufficient. The identification of key molecules involved in pathological pain processing is a prerequisite for the development of new analgesic drugs. Hyperexcitability of nociceptive DRG-neurons due to regulation of voltage-gated ion-channels is generally assumed to contribute strongly to neuropathic pain. There is increasing evidence, that T-type Ca-currents and in particular the Ca3.2 T-type-channel isoform play an important role in neuropathic pain, but experimental results are contradicting.

Purpose: To clarify the role of T-type Ca-channels and in particular the Ca3.2 T-type-channel isoform in neuropathic pain.

Methods: The effect of partial sciatic nerve ligation (PNL) on pain behavior and the properties of T-type-currents in nociceptive DRG-neurons was tested in wild-type and Ca3.2-deficient mice.

Results: In wild-type mice, PNL of the sciatic nerve caused neuropathic pain and an increase of T-type Ca-currents in capsaicin-responsive neurons, while capsaicin-unresponsive neurons were unaffected. Pharmacological experiments revealed that this upregulation was due to an increase of a Ni-resistant Ca-current component, inconsistent with Ca3.2 up-regulation. Moreover, following PNL Ca3.2-deficient mice showed neuropathic pain behavior and an increase of T-Type Ca-currents indistinguishable to that of PNL treated wild-type mice.

Conclusion: These data suggest that PNL induces an upregulation of T-Type Ca-currents in capsaicin-responsive DRG-neurons mediated by an increase of a Ni-insensitive current component (possibly Ca3.1 or Ca3.3). These findings provide relevance for the development of target specific analgesic drugs.
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http://dx.doi.org/10.2147/JPR.S138708DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6375107PMC
February 2019

Effects of eslicarbazepine on slow inactivation processes of sodium channels in dentate gyrus granule cells.

Epilepsia 2018 08 28;59(8):1492-1506. Epub 2018 Jun 28.

Institute of Experimental Epileptology and Cognition Research, University of Bonn, Bonn, Germany.

Objective: Pharmacoresistance is a problem affecting ∼30% of chronic epilepsy patients. An understanding of the mechanisms of pharmacoresistance requires a precise understanding of how antiepileptic drugs interact with their targets in control and epileptic tissue. Although the effects of (S)-licarbazepine (S-Lic) on sodium channel fast inactivation are well understood and have revealed maintained activity in epileptic tissue, it is not known how slow inactivation processes are affected by S-Lic in epilepsy.

Methods: We have used voltage clamp recordings in isolated dentate granule cells (DGCs) and cortical pyramidal neurons of control versus chronically epileptic rats (pilocarpine model of epilepsy) and in DGCs isolated from hippocampal specimens from temporal lobe epilepsy patients to examine S-Lic effects on sodium channel slow inactivation.

Results: S-Lic effects on entry into and recovery from slow inactivation were negligible, even at high concentrations of S-Lic (300 μmol/L). Much more pronounced S-Lic effects were observed on the voltage dependence of slow inactivation, with significant effects at 100 μmol/L S-Lic in DGCs from control and epileptic rats or temporal lobe epilepsy patients. For none of these effects of S-Lic could we observe significant differences either between sham-control and epileptic rats, or between human DGCs and the two animal groups. S-Lic was similarly effective in cortical pyramidal neurons from sham-control and epileptic rats. Finally, we show in expression systems that S-Lic effects on slow inactivation voltage dependence are only observed in Na 1.2 and Na 1.6 subunits, but not in Na 1.1 and Na 1.3 subunits.

Significance: From these data, we conclude that a major mechanism of action of S-Lic is an effect on slow inactivation, primarily through effects on slow inactivation voltage dependence of Na 1.2 and Na 1.6 channels. Second, we demonstrate that this main effect of S-Lic is maintained in both experimental and human epilepsy and applies to principal neurons of different brain areas.
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http://dx.doi.org/10.1111/epi.14504DOI Listing
August 2018

Polyamine Modulation of Anticonvulsant Drug Response: A Potential Mechanism Contributing to Pharmacoresistance in Chronic Epilepsy.

J Neurosci 2018 06 22;38(24):5596-5605. Epub 2018 May 22.

Institute for Experimental Epileptology and Cognition Research,

Despite the development of numerous novel anticonvulsant drugs, ∼30% of epilepsy patients remain refractory to antiepileptic drugs (AEDs). Many established and novel AEDs reduce hyperexcitability via voltage- and use-dependent inhibition of voltage-gated Na channels. For the widely used anticonvulsant carbamazepine (CBZ), use-dependent block of Na channels is significantly reduced both in experimental and human epilepsy. However, the molecular underpinnings of this potential cellular mechanism for pharmacoresistance have remained enigmatic.Here, we describe the mechanism that leads to the emergence of CBZ-resistant Na channels. We focused on the endogenous polyamine system, which powerfully modulates Na channels in a use-dependent manner. We had shown previously that the intracellular polyamine spermine is reduced in chronic epilepsy, resulting in increased persistent Na currents. Because spermine and CBZ both bind use-dependently in spatial proximity within the Na channel pore, we hypothesized that spermine loss might also be related to diminished CBZ response. Using the pilocarpine model of refractory epilepsy in male rats and whole-cell patch-clamp recordings, we first replicated the reduction of use-dependent block by CBZ in chronically epileptic animals. We then substituted intracellular spermine via the patch pipette in different concentrations. Under these conditions, we found that exogenous spermine significantly rescues use-dependent block of Na channels by CBZ. These findings indicate that an unexpected modulatory mechanism, depletion of intracellular polyamines, leads both to increased persistent Na currents and to diminished CBZ sensitivity of Na channels. These findings could lead to novel strategies for overcoming pharmacoresistant epilepsy that target the polyamine system. Pharmacoresistant epilepsy affects ∼18 million people worldwide, and intense efforts have therefore been undertaken to uncover the underlying molecular and cellular mechanisms. One of the key known candidate mechanisms of pharmacoresistance has been a loss of use-dependent Na channel block by the anticonvulsant carbamazepine (CBZ), both in human and experimental epilepsies. Despite intense scrutiny, the molecular mechanisms underlying this phenomenon have not been elucidated. We now show that a loss of intracellular spermine in chronic epilepsy is a major causative factor leading to the development of CBZ-resistant Na currents. This finding can be exploited both for the screening of anticonvulsants in expression systems, and for novel strategies to overcome pharmacoresistance that target the polyamine system.
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http://dx.doi.org/10.1523/JNEUROSCI.0640-18.2018DOI Listing
June 2018

Neuropathic pain in experimental autoimmune neuritis is associated with altered electrophysiological properties of nociceptive DRG neurons.

Exp Neurol 2017 11 19;297:25-35. Epub 2017 Jul 19.

Department of Neurology, University of Bonn Medical Center, Sigmund Freud Straße 25, 53105 Bonn, Germany; Department of Epileptology, University of Bonn Medical Center, Sigmund Freud Straße 25, 53105 Bonn, Germany. Electronic address:

Guillain-Barré syndrome (GBS) is an acute, immune-mediated polyradiculoneuropathy characterized by rapidly progressive paresis and sensory disturbances. Moderate to severe and often intractable neuropathic pain is a common symptom of GBS, but its underlying mechanisms are unknown. Pathology of GBS is classically attributed to demyelination of large, myelinated peripheral fibers. However, there is increasing evidence that neuropathic pain in GBS is associated with impaired function of small, unmyelinated, nociceptive fibers. We therefore examined the functional properties of small DRG neurons, the somata of nociceptive fibers, in a rat model of GBS (experimental autoimmune neuritis=EAN). EAN rats developed behavioral signs of neuropathic pain. This was accompanied by a significant shortening of action potentials due to a more rapid repolarization and an increase in repetitive firing in a subgroup of capsaicin-responsive DRG neurons. Na current measurements revealed a significant increase of the fast TTX-sensitive current and a reduction of the persistent TTX-sensitive current component. These changes of Na currents may account for the significant decrease in AP duration leading to an overall increase in excitability and are therefore possibly directly linked to pathological pain behavior. Thus, like in other animal models of neuropathic and inflammatory pain, Na channels seem to be crucially involved in the pathology of GBS and may constitute promising targets for pain modulating pharmaceuticals.
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http://dx.doi.org/10.1016/j.expneurol.2017.07.011DOI Listing
November 2017

Activity of the anticonvulsant lacosamide in experimental and human epilepsy via selective effects on slow Na channel inactivation.

Epilepsia 2017 01 19;58(1):27-41. Epub 2016 Nov 19.

Department of Epileptology, Laboratory for Experimental Epileptology and Cognition Research, University of Bonn, Bonn, Germany.

Objective: In human epilepsy, pharmacoresistance to antiepileptic drug therapy is a major problem affecting ~30% of patients with epilepsy. Many classical antiepileptic drugs target voltage-gated sodium channels, and their potent activity in inhibiting high-frequency firing has been attributed to their strong use-dependent blocking action. In chronic epilepsy, a loss of use-dependent block has emerged as a potential cellular mechanism of pharmacoresistance for anticonvulsants acting on voltage-gated sodium channels. The anticonvulsant drug lacosamide (LCM) also targets sodium channels, but has been shown to preferentially affect sodium channel slow inactivation processes, in contrast to most other anticonvulsants.

Methods: We used whole-cell voltage clamp recordings in acutely isolated cells to investigate the effects of LCM on transient Na currents. Furthermore, we used whole-cell current clamp recordings to assess effects on repetitive action potential firing in hippocampal slices.

Results: We show here that LCM exerts its effects primarily via shifting the slow inactivation voltage dependence to more hyperpolarized potentials in hippocampal dentate granule cells from control and epileptic rats, and from patients with epilepsy. It is important to note that this activity of LCM was maintained in chronic experimental and human epilepsy. Furthermore, we demonstrate that the efficacy of LCM in inhibiting high-frequency firing is undiminished in chronic experimental and human epilepsy.

Significance: Taken together, these results show that LCM exhibits maintained efficacy in chronic epilepsy, in contrast to conventional use-dependent sodium channel blockers such as carbamazepine. They also establish that targeting slow inactivation may be a promising strategy for overcoming target mechanisms of pharmacoresistance.
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http://dx.doi.org/10.1111/epi.13602DOI Listing
January 2017

Downregulation of Spermine Augments Dendritic Persistent Sodium Currents and Synaptic Integration after Status Epilepticus.

J Neurosci 2015 Nov;35(46):15240-53

Laboratory for Experimental Epileptology and Cognition Research, Department of Epileptology, and Deutsches Zentrum für Neurodegenerative Erkrankungen e.V., 53175 Bonn, Germany

Unlabelled: Dendritic voltage-gated ion channels profoundly shape the integrative properties of neuronal dendrites. In epilepsy, numerous changes in dendritic ion channels have been described, all of them due to either their altered transcription or phosphorylation. In pilocarpine-treated chronically epileptic rats, we describe a novel mechanism that causes an increased proximal dendritic persistent Na(+) current (INaP). We demonstrate using a combination of electrophysiology and molecular approaches that the upregulation of dendritic INaP is due to a relief from polyamine-dependent inhibition. The polyamine deficit in hippocampal neurons is likely caused by an upregulation of the degrading enzyme spermidine/spermine acetyltransferase. Multiphoton glutamate uncaging experiments revealed that the increase in dendritic INaP causes augmented dendritic summation of excitatory inputs. These results establish a novel post-transcriptional modification of ion channels in chronic epilepsy and may provide a novel avenue for treatment of temporal lobe epilepsy.

Significance Statement: In this paper, we describe a novel mechanism that causes increased dendritic persistent Na(+) current. We demonstrate using a combination of electrophysiology and molecular approaches that the upregulation of persistent Na(+) currents is due to a relief from polyamine-dependent inhibition. The polyamine deficit in hippocampal neurons is likely caused by an upregulation of the degrading enzyme spermidine/spermine acetyltransferase. Multiphoton glutamate uncaging experiments revealed that the increase in dendritic persistent Na current causes augmented dendritic summation of excitatory inputs. We believe that these results establish a novel post-transcriptional modification of ion channels in chronic epilepsy.
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http://dx.doi.org/10.1523/JNEUROSCI.0493-15.2015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6605494PMC
November 2015

Zinc regulates a key transcriptional pathway for epileptogenesis via metal-regulatory transcription factor 1.

Nat Commun 2015 Oct 26;6:8688. Epub 2015 Oct 26.

Section for Translational Epilepsy Research, Department of Neuropathology, University of Bonn Medical Center, Bonn 53105, Germany.

Temporal lobe epilepsy (TLE) is the most common focal seizure disorder in adults. In many patients, transient brain insults, including status epilepticus (SE), are followed by a latent period of epileptogenesis, preceding the emergence of clinical seizures. In experimental animals, transcriptional upregulation of CaV3.2 T-type Ca(2+)-channels, resulting in an increased propensity for burst discharges of hippocampal neurons, is an important trigger for epileptogenesis. Here we provide evidence that the metal-regulatory transcription factor 1 (MTF1) mediates the increase of CaV3.2 mRNA and intrinsic excitability consequent to a rise in intracellular Zn(2+) that is associated with SE. Adeno-associated viral (rAAV) transfer of MTF1 into murine hippocampi leads to increased CaV3.2 mRNA. Conversely, rAAV-mediated expression of a dominant-negative MTF1 abolishes SE-induced CaV3.2 mRNA upregulation and attenuates epileptogenesis. Finally, data from resected human hippocampi surgically treated for pharmacoresistant TLE support the Zn(2+)-MTF1-CaV3.2 cascade, thus providing new vistas for preventing and treating TLE.
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http://dx.doi.org/10.1038/ncomms9688DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4846312PMC
October 2015

RIM3γ and RIM4γ are key regulators of neuronal arborization.

J Neurosci 2013 Jan;33(2):824-39

Department of Neuropathology, Department of Epileptology, Laboratory for Experimental Epileptology and Cognition Research, University of Bonn, 53105 Bonn, Germany.

The large isoforms of the Rab3 interacting molecule (RIM) family, RIM1α/β and RIM2α/β, have been shown to be centrally involved in mediating presynaptic active zone function. The RIM protein family contains two additional small isoforms, RIM3γ and RIM4γ, which are composed only of the RIM-specific C-terminal C2B domain and varying N-terminal sequences and whose function remains to be elucidated. Here, we report that both, RIM3γ and RIM4γ, play an essential role for the development of neuronal arborization and of dendritic spines independent of synaptic function. γ-RIM knock-down in rat primary neuronal cultures and in vivo resulted in a drastic reduction in the complexity of neuronal arborization, affecting both axonal and dendritic outgrowth, independent of the time point of γ-RIM downregulation during dendrite development. Rescue experiments revealed that the phenotype is caused by a function common to both γ-RIMs. These findings indicate that γ-RIMs are involved in cell biological functions distinct from the regulation of synaptic vesicle exocytosis and play a role in the molecular mechanisms controlling the establishment of dendritic complexity and axonal outgrowth.
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http://dx.doi.org/10.1523/JNEUROSCI.2229-12.2013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6704911PMC
January 2013

Loss of β1 accessory Na+ channel subunits causes failure of carbamazepine, but not of lacosamide, in blocking high-frequency firing via differential effects on persistent Na+ currents.

Epilepsia 2012 Nov 27;53(11):1959-67. Epub 2012 Sep 27.

Department of Epileptology, University of Bonn, Sigmund-Freud-Strasse 25, Bonn, Germany

Purpose:   In chronic epilepsy, a substantial proportion of up to 30% of patients remain refractory to antiepileptic drugs (AEDs). An understanding of the mechanisms of pharmacoresistance requires precise knowledge of how AEDs interact with their targets. Many commonly used AEDs act on the transient and/or the persistent components of the voltage-gated Na(+) current (I(NaT) and I(NaP) , respectively). Lacosamide (LCM) is a novel AED with a unique mode of action in that it selectively enhances slow inactivation of fast transient Na(+) channels. Given that functional loss of accessory Na(+) channel subunits is a feature of a number of neurologic disorders, including epilepsy, we examined the effects of LCM versus carbamazepine (CBZ) on the persistent Na(+) current (I(NaP) ), in the presence and absence of accessory subunits within the channel complex.

Methods:   Using patch-clamp recordings in intact hippocampal CA1 neurons of Scn1b null mice, I(NaP) was recorded using slow voltage ramps. Application of 100 μm CBZ or 300 μm LCM reduced the maximal I(NaP) conductance in both wild-type and control mice.

Key Findings:   As shown previously by our group in Scn1b null mice, CBZ induced a paradoxical increase of I(NaP) conductance in the subthreshold voltage range, resulting in an ineffective block of repetitive firing in Scn1b null neurons. In contrast, LCM did not exhibit such a paradoxical increase, and accordingly maintained efficacy in blocking repetitive firing in Scn1b null mice.

Significance:   These results suggest that the novel anticonvulsant LCM maintains activity in the presence of impaired Na(+) channel β(1) subunit expression and thus may offer an improved efficacy profile compared with CBZ in diseases associated with an impaired expression of β sub-units as observed in epilepsy.
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http://dx.doi.org/10.1111/j.1528-1167.2012.03675.xDOI Listing
November 2012

The presynaptic active zone protein RIM1α controls epileptogenesis following status epilepticus.

J Neurosci 2012 Sep;32(36):12384-95

Department of Neuropathology, University of Bonn, 53105 Bonn, Germany.

To ensure operation of synaptic transmission within an appropriate dynamic range, neurons have evolved mechanisms of activity-dependent plasticity, including changes in presynaptic efficacy. The multidomain protein RIM1α is an integral component of the cytomatrix at the presynaptic active zone and has emerged as key mediator of presynaptically expressed forms of synaptic plasticity. We have therefore addressed the role of RIM1α in aberrant cellular plasticity and structural reorganization after an episode of synchronous neuronal activity pharmacologically induced in vivo [status epilepticus (SE)]. Post-SE, all animals developed spontaneous seizure events, but their frequency was dramatically increased in RIM1α-deficient mice (RIM1α(-/-)). We found that in wild-type mice (RIM1α(+/+)) SE caused an increase in paired-pulse facilitation in the CA1 region of the hippocampus to the level observed in RIM1α(-/-) mice before SE. In contrast, this form of short-term plasticity was not further enhanced in RIM1α-deficient mice after SE. Intriguingly, RIM1α(-/-) mice showed a unique pattern of selective hilar cell loss (i.e., endfolium sclerosis), which so far has not been observed in a genetic epilepsy animal model, as well as less severe astrogliosis and attenuated mossy fiber sprouting. These findings indicate that the decrease in release probability and altered short- and long-term plasticity as present in RIM1α(-/-) mice result in the formation of a hyperexcitable network but act in part neuroprotectively with regard to neuropathological alterations associated with epileptogenesis. In summary, our results suggest that presynaptic plasticity and proper function of RIM1α play an important part in a neuron's adaptive response to aberrant electrical activity.
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http://dx.doi.org/10.1523/JNEUROSCI.0223-12.2012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6621253PMC
September 2012

Direct conversion of fibroblasts into stably expandable neural stem cells.

Cell Stem Cell 2012 Apr 22;10(4):473-9. Epub 2012 Mar 22.

Stem Cell Engineering Group at the Institute of Reconstructive Neurobiology, University of Bonn, Germany.

Recent advances have suggested that direct induction of neural stem cells (NSCs) could provide an alternative to derivation from somatic tissues or pluripotent cells. Here we show direct derivation of stably expandable NSCs from mouse fibroblasts through a curtailed version of reprogramming to pluripotency. By constitutively inducing Sox2, Klf4, and c-Myc while strictly limiting Oct4 activity to the initial phase of reprogramming, we generated neurosphere-like colonies that could be expanded for more than 50 passages and do not depend on sustained expression of the reprogramming factors. These induced neural stem cells (iNSCs) uniformly display morphological and molecular features of NSCs, such as the expression of Nestin, Pax6, and Olig2, and have a genome-wide transcriptional profile similar to that of brain-derived NSCs. Moreover, iNSCs can differentiate into neurons, astrocytes, and oligodendrocytes. Our results demonstrate that functional NSCs can be generated from somatic cells by factor-driven induction.
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http://dx.doi.org/10.1016/j.stem.2012.03.003DOI Listing
April 2012

An increase in persistent sodium current contributes to intrinsic neuronal bursting after status epilepticus.

J Neurophysiol 2011 Jan 27;105(1):117-29. Epub 2010 Oct 27.

Department of Medical Neurobiology, Hebrew University-Hadassah School of Medicine, P.O. Box 12272, Jerusalem 91121, Israel.

Brain damage causes multiple changes in synaptic function and intrinsic properties of surviving neurons, leading to the development of chronic epilepsy. In the widely used pilocarpine-status epilepticus (SE) rat model of temporal lobe epilepsy (TLE), a major alteration is the marked increase in the fraction of intrinsically bursting CA1 pyramidal cells. Here we have differentiated between two types of bursting phenotypes: 1) bursting in response to threshold-straddling excitatory current pulses (low-threshold bursting) and 2) bursting only in response to suprathreshold stimuli (high-threshold bursting). Low-threshold bursting prevailed in 46.5% of SE-experienced neurons sampled 1-4 wk after pilocarpine-SE, but was rarely seen in control neurons (1.9%). As previously shown, it appeared to be driven predominantly by a T-type Ca(2+) current (I(CaT)) in the apical dendrites. After blocking low-threshold bursting with Ni(2+), the same neurons still manifested a high-threshold bursting phenotype. Another 40.1% of SE-experienced neurons displayed only a high-threshold bursting phenotype and the remaining 13.4% of these neurons were nonbursters. Altogether, high-threshold bursting prevailed in 86.6% of SE-experienced neurons, but only in 33.0% of control neurons. Several lines of evidence indicated that high-threshold bursting is driven by persistent Na(+) current (I(NaP)) at or near the soma. Congruently, I(NaP) was 1.5-fold larger in SE-experienced versus control neurons. We conclude that an increase in I(NaP), conjointly with an increase in I(CaT), strongly contributes to the predominance of bursting phenotypes in CA1 pyramidal cells early after pilocarpine-SE and thus likely plays a role in the development of a chronic epileptic condition in this TLE model.
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http://dx.doi.org/10.1152/jn.00184.2010DOI Listing
January 2011

Stable mossy fiber long-term potentiation requires calcium influx at the granule cell soma, protein synthesis, and microtubule-dependent axonal transport.

J Neurosci 2010 Sep;30(39):12996-3004

Department of Epileptology, University of Bonn, D-53105 Bonn, Germany.

The synapses formed by the mossy fiber (MF) axons of hippocampal dentate gyrus granule neurons onto CA3 pyramidal neurons exhibit an intriguing form of experience-dependent synaptic plasticity that is induced and expressed presynaptically. In contrast to most other CNS synapses, long-term potentiation (LTP) at the MF-CA3 synapse is readily induced even during blockade of postsynaptic glutamate receptors. Furthermore, blocking voltage-gated Ca(2+) channels prevents MF-LTP, supporting an involvement of presynaptic Ca(2+) signaling via voltage-gated Ca(2+) channels in MF-LTP induction. We examined the contribution of activity in both dentate granule cell somata and MF terminals to MF-LTP. We found that the induction of stable MF-LTP requires tetanization-induced action potentials not only at MF boutons, but also at dentate granule cell somata. Similarly, blocking Ca(2+) influx via voltage-gated Ca(2+) channels only at the granule cell soma was sufficient to disrupt MF-LTP. Finally, blocking protein synthesis or blocking fast axonal transport mechanisms via disruption of axonal tubulin filaments resulted in decremental MF-LTP. Collectively, these data suggest that-in addition to Ca(2+) influx at the MF terminals-induction of MF synaptic plasticity requires action potential-dependent Ca(2+) signaling at granule cell somata, protein synthesis, and fast axonal transport along MFs. A parsimonious interpretation of these results is that somatic activity triggers protein synthesis at the soma; newly synthesized proteins are then transported to MF terminals, where they contribute to the stabilization of MF-LTP. Finally, the present data imply that synaptic plasticity at the MF-CA3 synapse can be affected by local modulation of somatic and presynaptic Ca(2+) channel activity.
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http://dx.doi.org/10.1523/JNEUROSCI.1847-10.2010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6633504PMC
September 2010

Efficacy loss of the anticonvulsant carbamazepine in mice lacking sodium channel beta subunits via paradoxical effects on persistent sodium currents.

J Neurosci 2010 Jun;30(25):8489-501

Department of Epileptology, University of Bonn Medical Center, 53105 Bonn, Germany.

Neuronal excitability is critically determined by the properties of voltage-gated Na(+) currents. Fast transient Na(+) currents (I(NaT)) mediate the fast upstroke of action potentials, whereas low-voltage-activated persistent Na(+) currents (I(NaP)) contribute to subthreshold excitation. Na(+) channels are composed of a pore-forming alpha subunit and beta subunits, which modify the biophysical properties of alpha subunits. We have examined the idea that the presence of beta subunits also modifies the pharmacological properties of the Na(+) channel complex using mice lacking either the beta(1) (Scn1b) or beta(2) (Scn2b) subunit. Classical effects of the anticonvulsant carbamazepine (CBZ), such as the use-dependent reduction of I(NaT) and effects on I(NaT) voltage dependence of inactivation, were unaltered in mice lacking beta subunits. Surprisingly, CBZ induced a small but significant shift of the voltage dependence of activation of I(NaT) and I(NaP) to more hyperpolarized potentials. This novel CBZ effect on I(NaP) was strongly enhanced in Scn1b null mice, leading to a pronounced increase of I(NaP) within the subthreshold potential range, in particular at low CBZ concentrations of 10-30 microm. A combination of current-clamp and computational modeling studies revealed that this effect causes a complete loss of CBZ efficacy in reducing repetitive firing. Thus, beta subunits modify not only the biophysical but also the pharmacological properties of Na(+) channels, in particular with respect to I(NaP). Consequently, altered expression of beta subunits in other neurological disorders may cause altered neuronal sensitivity to drugs targeting Na(+) channels.
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http://dx.doi.org/10.1523/JNEUROSCI.1534-10.2010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6634624PMC
June 2010

Inhibition of notch signaling in human embryonic stem cell-derived neural stem cells delays G1/S phase transition and accelerates neuronal differentiation in vitro and in vivo.

Stem Cells 2010 May;28(5):955-64

Institute of Reconstructive Neurobiology, LIFE & BRAIN Center, University of Bonn and Hertie Foundation, Bonn, Germany.

The controlled in vitro differentiation of human embryonic stem cells (hESCs) and other pluripotent stem cells provides interesting prospects for generating large numbers of human neurons for a variety of biomedical applications. A major bottleneck associated with this approach is the long time required for hESC-derived neural cells to give rise to mature neuronal progeny. In the developing vertebrate nervous system, Notch signaling represents a key regulator of neural stem cell (NSC) maintenance. Here, we set out to explore whether this signaling pathway can be exploited to modulate the differentiation of hESC-derived NSCs (hESNSCs). We assessed the expression of Notch pathway components in hESNSCs and demonstrate that Notch signaling is active under self-renewing culture conditions. Inhibition of Notch activity by the gamma-secretase inhibitor N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) in hESNSCs affects the expression of human homologues of known targets of Notch and of several cell cycle regulators. Furthermore, DAPT-mediated Notch inhibition delays G1/S-phase transition and commits hESNSCs to neurogenesis. Combined with growth factor withdrawal, inhibition of Notch signaling results in a marked acceleration of differentiation, thereby shortening the time required for the generation of electrophysiologically active hESNSC-derived neurons. This effect can be exploited for neural cell transplantation, where transient Notch inhibition before grafting suffices to promote the onset of neuronal differentiation of hESNSCs in the host tissue. Thus, interference with Notch signaling provides a tool for controlling human NSC differentiation both in vitro and in vivo.
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http://dx.doi.org/10.1002/stem.408DOI Listing
May 2010

A rosette-type, self-renewing human ES cell-derived neural stem cell with potential for in vitro instruction and synaptic integration.

Proc Natl Acad Sci U S A 2009 Mar 13;106(9):3225-30. Epub 2009 Feb 13.

Institute of Reconstructive Neurobiology, Life and Brain Center, University of Bonn and Hertie Foundation, D-53127 Bonn, Germany.

An intriguing question in human embryonic stem cell (hESC) biology is whether these pluripotent cells can give rise to stably expandable somatic stem cells, which are still amenable to extrinsic fate instruction. Here, we present a pure population of long-term self-renewing rosette-type hESC-derived neural stem cells (lt-hESNSCs), which exhibit extensive self-renewal, clonogenicity, and stable neurogenesis. Although lt-hESNSCs show a restricted expression of regional transcription factors, they retain responsiveness to instructive cues promoting the induction of distinct subpopulations, such as ventral midbrain and spinal cord fates. Using lt-hESNSCs as a donor source for neural transplantation, we provide direct evidence that hESC-derived neurons can establish synaptic connectivity with the mammalian nervous system. Combining long-term stability, maintenance of rosette-properties and phenotypic plasticity, lt-hESNSCs may serve as useful tool to study mechanisms of human NSC self-renewal, lineage segregation, and functional in vivo integration.
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http://dx.doi.org/10.1073/pnas.0808387106DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2651316PMC
March 2009

Transcriptional upregulation of Cav3.2 mediates epileptogenesis in the pilocarpine model of epilepsy.

J Neurosci 2008 Dec;28(49):13341-53

Departments of Neuropathology, University of Bonn Medical Center, University of Bonn, Bonn, Germany.

In both humans and animals, an insult to the brain can lead, after a variable latent period, to the appearance of spontaneous epileptic seizures that persist for life. The underlying processes, collectively referred to as epileptogenesis, include multiple structural and functional neuronal alterations. We have identified the T-type Ca(2+) channel Ca(v)3.2 as a central player in epileptogenesis. We show that a transient and selective upregulation of Ca(v)3.2 subunits on the mRNA and protein levels after status epilepticus causes an increase in cellular T-type Ca(2+) currents and a transitional increase in intrinsic burst firing. These functional changes are absent in mice lacking Ca(v)3.2 subunits. Intriguingly, the development of neuropathological hallmarks of chronic epilepsy, such as subfield-specific neuron loss in the hippocampal formation and mossy fiber sprouting, was virtually completely absent in Ca(v)3.2(-/-) mice. In addition, the appearance of spontaneous seizures was dramatically reduced in these mice. Together, these data establish transcriptional induction of Ca(v)3.2 as a critical step in epileptogenesis and neuronal vulnerability.
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http://dx.doi.org/10.1523/JNEUROSCI.1421-08.2008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6671595PMC
December 2008

Adult germ line stem cells as a source of functional neurons and glia.

Stem Cells 2008 Sep 17;26(9):2434-43. Epub 2008 Jul 17.

Institute of Reconstructive Neurobiology, University of Bonn LIFE & BRAIN Center, University of Bonn and Hertie Foundation, Bonn, Germany.

The derivation of autologous pluripotent cells has become a central goal in translational stem cell research. Although somatic cell nuclear transfer and transcription factor-based reprogramming enable the generation of pluripotent cells from adult tissue, both methodologies depend on complex epigenetic alterations. Recent data suggest that the adult germ line may represent an alternative and natural source of pluripotent stem cells. Multipotent adult germ line stem cells (maGSCs) with properties similar to those of embryonic stem cells have been derived from mouse spermatogonial stem cells. These cells exhibit extensive self-renewal, expression of pluripotency markers, and differentiation into derivatives of all three germ layers. Here we report the derivation of multipotent neural and glial precursors as well as adherently proliferating neural stem cells from maGSCs. Characterization of maGSC-derived neurons revealed segregation into GABAergic, glutamatergic, serotonergic, and tyrosine hydroxylase-positive phenotypes. On a functional level, maGSC-derived neurons generate spontaneously active functional networks, which use both glutamatergic and GABAergic synaptic transmission and engage in synchronized oscillatory activity. maGSC-derived oligodendrocytes undergo full maturation and ensheathe host axons in myelin-deficient tissue. Our data suggest that neural stem and precursor cells derived from maGSCs could provide a versatile and potentially autologous source of functional neurons and glia.
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http://dx.doi.org/10.1634/stemcells.2008-0163DOI Listing
September 2008

Lineage selection of functional and cryopreservable human embryonic stem cell-derived neurons.

Stem Cells 2008 Jul 17;26(7):1705-12. Epub 2008 Apr 17.

Institute of Reconstructive Neurobiology, Life & Brain Center, University of Bonn, Sigmund-Freud-Strasse 25, D-53105 Bonn, Germany.

A major prerequisite for the biomedical application of human embryonic stem cells (hESC) is the derivation of defined and homogeneous somatic cell types. Here we present a human doublecortin (DCX) promoter-based lineage-selection strategy for the generation of purified hESC-derived immature neurons. After transfection of hESC-derived neural precursors with a DCX-enhanced green fluorescent protein construct, fluorescence-activated cell sorting enables the enrichment of immature human neurons at purities of up to 95%. Selected neurons undergo functional maturation and are able to establish synaptic connections. Considering that the applicability of purified hESC-derived neurons would largely benefit from an efficient cryopreservation technique, we set out to devise defined freezing conditions involving caspase inhibition, which yield post-thaw recovery rates of up to 83%. Combined with our lineage-selection procedure this cryopreservation technique enables the generation of human neurons in a ready-to-use format for a large variety of biomedical applications.
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http://dx.doi.org/10.1634/stemcells.2008-0007DOI Listing
July 2008

Electrophysiological evaluation of engrafted stem cell-derived neurons.

Nat Protoc 2007 ;2(7):1603-13

Institute of Reconstructive Neurobiology, Life & Brain Center, University of Bonn and Hertie Foundation, Bonn, Germany.

Recent advances in the neural stem cell field have provided a wealth of methods for generating large amounts of purified neuronal precursor cells. It has become a question of paramount importance to determine whether these cells integrate and interact with established neural circuitry after engraftment. In principle, neurons have to fulfill three basic functions: receive incoming signals via synapses, compute and forward processed information to other neurons or effector cells. It is anticipated that functionally integrating stem cell-derived donor neurons perform accordingly. Here we provide protocols for the efficient electrophysiological evaluation of engrafted cells and highlight current limitations thereof.
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http://dx.doi.org/10.1038/nprot.2007.230DOI Listing
August 2007

Impaired hippocampal long-term potentiation in microtubule-associated protein 1B-deficient mice.

J Neurosci Res 2005 Oct;82(1):83-92

Skirball Institute, New York University School of Medicine, New York, USA.

Microtubule-associated protein (MAP)1B-heterozygous (MAP1B+/-) mice are deficient in the expression of MAP1B in the hippocampus, cerebellum, and olfactory cortex. Although MAP1B+/- mice showed half the normal levels of MAP1B protein, they had no measurable amounts of phosphorylated MAP1B. High-frequency theta burst stimulation of Schaffer collateral-CA1 axons in hippocampal slices from MAP1B+/- mice elicited long-term potentiation (LTP) that decayed rapidly to baseline, in contrast to the non-decremental LTP exhibited by age-matched wild-type slices. A separate group of MAP1B+/- and wild-type slices was examined for a longer time course of 3 hr post-tetanus in response to multiple high-frequency stimulus trains that induced saturated LTP. MAP1B+/- slices showed marked reductions in both immediate post-tetanic potentiation and LTP that decayed much more rapidly than that in wild-type slices. The induction of LTP was associated with a rapid dephosphorylation of MAP1B within 5-15 min post-tetanus, suggesting that the normal expression of MAP1B and conversion to a dephosphorylated state may be a cellular mediator of cytoskeletal alterations necessary for long-term activity-dependent synaptic plasticity.
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http://dx.doi.org/10.1002/jnr.20624DOI Listing
October 2005

Activation of early silent synapses by spontaneous synchronous network activity limits the range of neocortical connections.

J Neurosci 2005 May;25(18):4605-15

Department of Developmental Physiology, Otto-von-Guericke University, Institute of Physiology, 39120 Magdeburg, Germany.

During the early development of neocortical networks, many glutamatergic synapses lack AMPA receptors and are physiologically silent. We show in neocortical cultures that spontaneous synchronous network activity is able to convert silent synapses to active synapses by the incorporation of AMPA receptors into synaptic complexes throughout the network within a few minutes. To test the effect of synaptic activation on the connectivity of neuronal populations, we created separated neuronal networks that could innervate each other. We allowed outgrowing axons to invade the neighboring network either before or after the onset of synchronous network activity. In the first case, both subnetworks connected to each other and synchronized their activity, whereas in the second case, axonal connections failed to form and network activity did not synchronize between compartments. We conclude that early spontaneous synchronous network activity triggers a global AMPAfication of immature synapses, which in turn prevents later-arriving axons from forming afferent connections. This activity-dependent process may set the range of corticocortical connections during early network development before experience-dependent mechanisms begin elaborating the mature layout of the neocortical connections and modules.
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http://dx.doi.org/10.1523/JNEUROSCI.3803-04.2005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6725027PMC
May 2005

Irreversible loss of a subpopulation of cortical interneurons in the absence of glutamatergic network activity.

Eur J Neurosci 2004 Jun;19(11):2931-43

Otto-von-Guericke Universität, Medizinische Fakultät, Institut für Physiologie, 39120 Magdeburg, Germany.

In the cerebral cortex of mammals, gamma-aminobutyric acid (GABA)ergic neurons represent 15-25% of all neurons, depending on the species and area being examined. Because converging evidence suggests that activity may play an important role in the neuritic maturation and synaptic function of GABAergic neurons, it is feasible that activity plays a role in the regulation of the proportion of GABAergic neurons. Here we provide direct evidence that early in cortical development activity blockade may deplete the network of a subpopulation of GABA immunoreactive neurons characterized by their small size and late generation in vitro. In a period of time coinciding with the emergence of synchronous network activity, the survival and morphological differentiation of GABAergic neurons was influenced by long-term blockade of synaptic activity. While GABA(A) receptor antagonists had a minor promoting effect on interneuronal survival during the second week in vitro, antagonists of ionotropic glutamate receptors strongly impaired survival and differentiation of immature GABAergic interneurons. Interneuronal loss was more severe when N-methyl-D-aspartate receptors were blocked than after blockade of alpha-amino-3-hydroxy-5-methylisoxazole-4-proprionic acid (AMPA)/kainate receptors. The decrease in the density of GABAergic neurons was irreversible, but could be prevented by the simultaneous addition of brain-derived neurotrophic factor (BDNF). These results suggest that there is a narrow time window during neocortical development when glutamatergic activity, and specially NMDA receptor stimulation, is crucial to assure survival and maturation of a subpopulation of late developing GABAergic interneurons.
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http://dx.doi.org/10.1111/j.0953-816X.2004.03403.xDOI Listing
June 2004

Spontaneous development of synchronous oscillatory activity during maturation of cortical networks in vitro.

J Neurophysiol 2002 Nov;88(5):2196-206

Department of Developmental Physiology, Institute for Physiology, Otto-von-Guericke University, 39120 Magdeburg, Germany.

Recent studies have focused attention on mechanisms of spontaneous large-scale wavelike activity during early development of the neocortex. In this study, we describe and characterize synchronous neuronal activity that occurs in cultured cortical networks naturally without pharmacological intervention. The synchronous activity that can be detected by means of Fluo-3 fluorescence imaging starts to develop at the beginning of the second week in culture and eventually includes the entire neuronal population about 1 wk later. A synchronous increase of [Ca(2+)](i) in the neuronal population is associated with a burst of action potentials riding on a long-lasting depolarization recorded in a single cell. It is suggested that this depolarization results directly from synaptic current, which was comprised of at least three different components mediated by AMPA, N-methyl-D-aspartate (NMDA), and GABA(A) receptors. We never observed a gradually depolarizing pacemaker potential and found no evidence for a change of excitability during inter-burst periods. However, we found evidence for a period of synaptic depression after bursts. Network excitability recovers gradually over seconds from this depression that can explain the episodic nature of spontaneous network activity. Using pharmacological manipulation to investigate the propagation of activity in the network, we show that synchronous network activity depends on both glutamatergic and GABA(A)ergic neurotransmission during a brief period. Reversal potential of GABA(A) receptor-mediated current was found to be significantly more positive than resting membrane potential both at 1 and 2 wk in culture, suggesting depolarizing action of GABA. However, in cultures older than 2 wk, inhibition of GABA(A) receptors does not result in block of synchronous network activity but in modulation of burst width and frequency.
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http://dx.doi.org/10.1152/jn.00316.2002DOI Listing
November 2002