Timo Kirschstein

Impaired synaptic plasticity in a rat model of tuberous sclerosis

Tuberous sclerosis complex (TSC) is a common hereditary disorder caused by mutations in either the TSC1 or TSC2 genes, and characterized by severe epilepsy, cerebral hamartomas and mental retardation. We have used rats that are heterozygous for an autosomal‐dominant germline mutation in the TSC2 gene (TSC2+/– rats) to examine the consequences of TSC2 mutations for hippocampal synaptic plasticity. While basal synaptic transmission in the Schaffer collateral–CA1 synapse was not altered, paired‐pulse plasticity was significantly enhanced in TSC2+/– rats (interpulse intervals 20–200 ms). Moreover, TSC2+/– rats exhibited a marked reduction of different forms of synaptic plasticity. Long‐term potentiation (LTP) elicited following high‐frequency tetanization of Schaffer collaterals was significantly decreased from 1.45 ± 0.05‐fold potentiation to 1.15 ± 0.04 (measured after 60 min). This difference in LTP levels between TSC2+/– and wild‐type rats also persisted in the presence of the γ‐aminobutyric acid (GABA)A receptor antagonist bicuculline. In addition to changed LTP, the level of long‐term depression (LTD) elicited by different forms of low‐frequency stimulation was significantly less in TSC2+/– rats. These results suggest that TSC2 mutations may cause hippocampal synapses to lose much of their potential for activity‐dependent synaptic modification. An understanding of the underlying molecular pathways may suggest new therapeutic approaches aimed at inhibiting the development of the profound mental retardation in TSC.

Positive shifts of the GABAA receptor reversal potential due to altered chloride homeostasis is widespread after status epilepticus

Purpose:  γ‐Aminobutyric acid (GABA)ergic transmission plays an important role in the initiation of epileptic activity and the generation of ictal discharges. The functional alterations in the epileptiform hippocampus critically depend on GABAergic mechanisms and cation‐chloride cotransporters.

Localization of HCN1 Channels to Presynaptic Compartments: Novel Plasticity That May Contribute to Hippocampal Maturation

Increasing evidence supports roles for the current mediated by hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, Ih, in hippocampal maturation and specifically in the evolving changes of intrinsic properties as well as network responses of hippocampal neurons. Here, we describe a novel developmental plasticity of HCN channel expression in axonal and presynaptic compartments: HCN1 channels were localized to axon terminals of the perforant path (the major hippocampal afferent pathway) of immature rats, where they modulated synaptic efficacy. However, presynaptic expression and functions of the channels disappeared with maturation. This was a result of altered channel transport to the axons, because HCN1 mRNA and protein levels in entorhinal cortex neurons, where the perforant path axons originate, were stable through adulthood. Blocking action potential firing in vitro increased presynaptic expression of HCN1 channels in the perforant path, suggesting that network activity contributed to regulating this expression. These findings support a novel developmentally regulated axonal transport of functional ion channels and suggest a role for HCN1 channel-mediated presynaptic Ih in hippocampal maturation.

Enhanced NMDA receptor-dependent LTP in the epileptic CA1 area via upregulation of NR2B.

Impairment of synaptic plasticity such as long-term potentiation (LTP) is a common finding in various animal models of a number of neurodegenerative disorders. While cognitive deficits associated with these models are plausibly attributed to impaired plasticity, it is an intriguing question whether learning impairment correlates in general with compromised synaptic plasticity. In the present study, we have addressed this issue and discovered an enhancement of theta-burst stimulation-induced LTP at Schaffer collateral-CA1 synapses from chronically epileptic animals. The LTP enhancement was abolished by the NMDA receptor 2B (NR2B) blocker Ro 25-6981 (1μM) while it was preserved following application of the NR2A blocker NVP-AAM077 (50nM). Moreover, pharmacological characterization of intracellularly recorded excitatory postsynaptic potentials (EPSP) from CA1 pyramidal neurons indicated an increased NR2B/NR2A ratio in epileptic tissue, and NMDA receptor mediated excitatory postsynaptic currents showed significantly longer decay times. Quantitative reverse-transcriptase PCR confirmed the transcriptional up-regulation of NR2B-mRNA in chronically epileptic animals. To test the significance for epileptiform activity, recurrent epileptiform discharges (REDs) in the CA1 area induced by bath application of either high K(+) (8mM) plus gabazine (5μM) or 4-aminopyridine (50μM), were also characterized pharmacologically. While in control slices the presence of Ro 25-6981 had no effect on the RED frequency, NR2B inhibition significantly increased epileptic activity in tissue from epileptic animals. Our results demonstrate that CA1 synapses in chronically epileptic tissue can undergo an LTP enhancement due to an NR2B up-regulation in CA1 pyramidal neurons. On the network level, this up-regulation appears to be a compensatory process, since blockade of these receptors leaves the tissue more susceptible to hyperexcitability.

What is the Source of the EEG

Neurons in the human cortex generally process their information by means of electrical signals and thus enable the electrical recording of their activity, the electroencephalogram (EEG). Due to their unique orientation with their long apical dendrites perpendicular to the cortical surface, large cortical pyramidal neurons in deep cortical layers play a major role in the generation of the EEG. Specific and non-specific thalamic nuclei, as well as distant cortical areas, terminate on these apical dendrites and form myriads of excitatory and inhibitory afferents. The release of excitatory and inhibitory neurotransmitters by these fibers activates specific postsynaptic receptors and generates excitatory and inhibitory postsynaptic potentials, respectively. By electrotonic spread of postsynaptic potentials along the apical dendrites and equivalent capacitive currents, they become electrical dipoles. Positive or negative deflections are generated by both excitatory and inhibitory afferents, depending on the location of these synapses on the apical dendrites. Negative (upward) deflections are due to superficial excitatory or deep inhibitory inputs, whereas positive (downward) deflections represent deep excitatory or superficial inhibitory inputs.

High-frequency magnetic stimulation induces long-term potentiation in rat hippocampal slices

Recent reports indicate that the exposure of brain tissues to transcranial magnetic stimulation induces persistent changes in neuronal activity and influences hippocampal synaptic plasticity. However, the modulation of synaptic efficiency by magnetic stimulation in vitro is still unclear. In the present study, we investigated whether high-frequency magnetic stimulation (HFMS) can induce long-term potentiation (LTP) in rat hippocampal slices in vitro. During baseline recording and after HFMS, field excitatory postsynaptic potentials (fEPSPs) were recorded within the CA1 stratum radiatum in response to electrical stimulation of the Schaffer collateral inputs. For LTP induction, HFMS was delivered through a circular coil positioned closely above the slices using two different paradigms (A: 10 trains of 20 pulses at 100 Hz with 1s intervals, 5 repetitions with 10s intervals; B: 3 trains of 100 pulses at 100 Hz with 20s intervals). The intensity of the magnetic stimulus was adjusted to 60-75 A/micros. After application of HFMS, electrically evoked CA1 fEPSPs were enhanced showing significant levels of LTP by both paradigms (A: 142+/-9% of baseline, n=6; B: 129+/-7%, n=8). Furthermore, HFMS-induced LTP induced by paradigm A was prevented by the presence of the selective N-methyl-D-aspartate receptor (NMDAR) blocker D-AP5 (50 microM) in the bath solution (95+/-6% of the baseline, n=6; p<0.01 compared to control condition without D-AP5). Further, the lack of changes in paired-pulse ratio and the afferent fiber volleys exclude presynaptic involvement in HFMS-induced LTP. In summary, we have demonstrated that HFMS can induce NMDAR-dependent LTP in the CA1 region in vitro.

Seizure frequency in pilocarpine‐treated rats is independent of circadian rhythm

Pilocarpine‐induced status epilepticus (SE) results in chronic spontaneous recurrent seizures resembling human temporal lobe epilepsy. In this and other experimental models, behaviorally monitored seizure frequency was suggested to vary in a circadian fashion, and to increase with time. We re‐addressed those hypotheses using continuous video–electroencephalography (EEG) telemetry in rats with SE at 30 days of age. In 11 chronically epileptic animals monitored up to 300 days after SE in a fixed 12 h light/dark cycle, we found that seizure frequency did not correlate with circadian rhythm.

Impaired d-Serine-Mediated Cotransmission Mediates Cognitive Dysfunction in Epilepsy

The modulation of synaptic plasticity by NMDA receptor (NMDAR)-mediated processes is essential for many forms of learning and memory. Activation of NMDARs by glutamate requires the binding of a coagonist to a regulatory site of the receptor. In many forebrain regions, this coagonist is d-serine. Here, we show that experimental epilepsy in rats is associated with a reduction in the CNS levels of d-serine, which leads to a desaturation of the coagonist binding site of synaptic and extrasynaptic NMDARs. In addition, the subunit composition of synaptic NMDARs changes in chronic epilepsy. The desaturation of NMDARs causes a deficit in hippocampal long-term potentiation, which can be rescued with exogenously supplied d-serine. Importantly, exogenous d-serine improves spatial learning in epileptic animals. These results strongly suggest that d-serine deficiency is important in the amnestic symptoms of temporal lobe epilepsy. Our results point to a possible clinical utility of d-serine to alleviate these disease manifestations.

Network excitability in a model of chronic temporal lobe epilepsy critically depends on SK channel-mediated AHP currents.

Hippocampal CA1 pyramidal neurons generate an after-hyperpolarization (AHP) whose medium component is thought to be generated by small-conductance Ca(2+)-activated K(+) channels (SK channels). Neuronal excitability is increased in epilepsy, and the AHP in turn is fundamentally involved in regulation of cellular excitability. We therefore investigated the involvement of the SK channel-mediated AHP in controlling cell and network excitability in the pilocarpine model epilepsy. Both acutely isolated CA1 pyramidal cells and isolated hippocampal slices were investigated in terms of the impact of SK channel-mediated AHP on hyperexcitability. Our findings show that pilocarpine-treated chronically epileptic rats exhibit significantly reduced SK channel-mediated hyperpolarizing outward current which was accompanied by a significant decrease in the somatic AHP. Paradoxically, inhibiting SK channels strongly exacerbated 0-Mg(2+)-induced epileptiform activity in slices from pilocarpine-treated animals, while having a significantly smaller effect in control tissue. This suggests that in chronically epileptic tissue, network excitability very critically depends on the remaining SK-channel mediated AHP. Additional real-time RT-PCR and semiquantitative Western blot experiments revealed that both the SK2 channel transcript and protein were significantly downregulated in the epileptic CA1 region. We conclude that SK2 channels are down-regulated in chronic epilepsy underlying the impaired SK channel function in CA1 pyramidal cells, and a further reduction of the remaining critical mass of SK channels results in an acute network decompensation.

High K+-induced contraction requires depolarization-induced Ca2+ release from internal stores in rat gut smooth muscle

AbstractAim:Depolarization-induced contraction of smooth muscle is thought to be mediated by Ca2+ influx through voltage-gated L-type Ca2+ channels. We describe a novel contraction mechanism that is independent of Ca2+ entry.Methods:Pharmacological experiments were carried out on isolated rat gut longitudinal smooth muscle preparations, measuring isometric contraction strength upon high K+-induced depolarization.Results:Treatment with verapamil, which presumably leads to a conformational change in the channel, completely abolished K+-induced contraction, while residual contraction still occurred when Ca2+ entry was blocked with Cd2+. These results were further confirmed by measuring intracellular Ca2+ transients using Fura-2. Co-application of Cd2+ and the ryanodine receptor blocker DHBP further reduced contraction, albeit incompletely. Additional blockage of either phospholipase C (U 73122) or inositol 1,4,5-trisphophate (IP3) receptors (2-APB) abolished most contractions, while sole application of these blockers and Cd2+ (without parallel ryanodine receptor manipulation) also resulted in incomplete contraction block.Conclusion:We conclude that there are parallel mechanisms of depolarization-induced smooth muscle contraction via (a) Ca2+ entry and (b) Ca2+ entry-independent, depolarization-induced Ca2+-release through ryanodine receptors and IP3, with the latter being dependent on phospholipase C activation.