Peter Igelmund

Pharmacological and electrographic properties of epileptiform activity induced by elevated K2+ and lowered Ca2+ and Mg2+ concentration in rat hippocampal slices

We studied some of the physiological and pharmacological properties of an in vitro model of epileptic seizures induced by elevation of [K+]0 (to 8 mM and 10 mM) in combination with lowering of [Mg2+]0 (to 1.4 mM and 1.6 mM) and [Ca2+]0 (to 0.7 mM and 1 mM) in rat hippocampal slices. These concentrations correspond to the ionic constitution of the extracellular microenvironment during seizures in vivo. The resulting activity was rather variable in appearance. In area CA3 recurrent discharges were observed which resulted in seizure-like events with either clonic-like or tonic-cloniclike ictaform events in area CA1. With ion-sensitive electrodes, we measured the field potential and the changes in extracellular ion concentrations which accompany this activity. The recurrent discharges in area CA3 were accompanied by small fluctuations in [K+]0 and [Ca2+]0. The grouped clonic-like discharges in area CA1 were associated with moderate increases in [K+]0 and small decreases in [Ca2+]0 in the order of 2 mM and 0.2 mM, respectively. Large, negative field-potential shifts and increases in [K+]0 to 13 mM, as well as decreases in [Ca2+]0 by up to 0.4 mM, accompanied the tonic phase of ictaform events. The ictaform events were not blocked by D-2-aminophosphonovalerate (2-APV) but were sensitive to 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) alone and in combination with 2-APV and ketamine. In order to determine the pharmacological characteristics of the ictaform events we bath-applied most clinically employed anticonvulsants (carbamazepine, phenytoin, valproate, phenobarbital, ethosuximide, trimethadione) and some experimental anticonvulsants (losigamone, vinpocetine, and apovincaminic acid). Carbamazepine, phenytoin, valproate, and phenobarbital were effective at clinically relevant doses. The data suggest that the high-K+ model of epileptiform activity is a good model of focal convulsant activity.

Effect of Cardioactive Drugs on Action Potential Generation and Propagation in Embryonic Stem Cell-Derived Cardiomyocytes

Extracellular recordings of spontaneous electrical activity in contracting cardiac clusters differentiated from murine embryonic stem cells enable to study electrophysiological features of this in-vitro cardiac-like tissue as well as effects of pharmacological compounds on its chronotropy and electrical conduction. To test if the microelectrode array (MEA) system could serve as a basis for development of a pharmacological screening tool for cardioactive drugs, we used spontaneously beating outgrowths of three-dimensional ES cell aggregates (“embryoid bodies”, EBs) plated onto substrate-integrated MEAs. The effects of the L-type Ca2+ channel antagonist verapamil and Na+ and K+ channel blockers (tetrodotoxin, 4-aminopyridine, and sparfloxacin) on the deduced interrelated cardiac network function were investigated. Application of 10-6 M verapamil led to arrhythmic spiking with a burst-like pattern; at a higher concentration (10-5 M) the drug caused a sustained negative chronotropy up to complete stop of beating. In the presence of tetrodotoxin a conduction block was observed. Since modulation of K+ channel activity can cause anti- or proarrhythmic effects, the influence of K+ channel blockers, namely 4-aminopyridine and sparfloxacin, was investigated. 4-aminopyridine (2x10-3 M) significantly stabilized beating frequency, while the field potential duration (FPD) was concentration-dependently prolonged up to 2.7-fold. Sparfloxacin (3x10-6 M) stabilized the beating frequency as well. At a higher concentration of sparfloxacin (3x10-5 M), a significant prolongation of the spike duration was registered; application of the drug caused also early afterdepolarizations. The results demonstrate a suitability of the studied in-vitro cardiac cell model for pharmacological drug testing in cardiovascular research.

Action potential propagation failures in long-term recordings from embryonic stem cell-derived cardiomyocytes in tissue culture

Abstract Three-dimensional cell aggregates (embryoid bodies, EBs) containing clusters of spontaneously beating cardiomyocytes were derived from permanent mouse embryonic stem (ES) cells. Extracellular recordings of the population action potentials of cardiomyocyte clusters were made using permanently mounted silver wire electrodes and microelectrode arrays integrated into the bottom of the culture dish. These techniques allowed long-term recordings (for up to several weeks) from individual EBs under cell culture conditions. The normal electrical activity consisted of regular spiking with a frequency of 0.5–5 Hz. However, most EBs (87%) spontaneously developed temporary or persistent complex activity patterns because of intermittent block of action potential propagation at narrow pathways connecting larger beating areas. Similar propagation blocks could also be reversibly induced in regularly spiking EBs by nimodipine (NDP). In addition to a slowing of pacemaker activity, NDP (20–200 nM) induced a stepwise decrease of the action potential frequency at the recording site. Perforated patch-clamp recordings from enzymatically isolated ES-cell-derived cardiomyocytes showed that similar activity patterns do not occur at the single-cell level. We suggest that this novel approach may provide a useful tool for in vitro studies of chronotropy and phenomena of propagation failure similar to AV block.

Effects of changes in extracellular potassium, magnesium and calcium concentration on synaptic transmission in area CA1 and the dentate gyrus of rat hippocampal slices

The dependence of stimulus-induced synaptic potentials on changes of extracellular ionic concentrations of potassium ([K+]o 3, 5, 8 mM), magnesium ([Mg2+]o 2, 4, 8 mM) and calcium [Ca2+]o (2 mM and continuous lowering by washing with Ca2+-free solutions) was investigated in area CA1 and dentate gyrus of rat hippocampal slices. Field potentials (fps), [K+]o and [Ca2+]o were measured with double-barreled ion selective/reference microelectrodes. Paired pulse stimulation (interval 50-ms) was applied either to the lateral perforant path or to the Schaffer collaterals. Elevation of [K+]o from 5 to 8 mM and of [Mg2+]o from 2 to 8 mM depressed the rise of excitatory postsynaptic potentials, as well as the amplitude of population spikes. With elevation of [K+]o, the effect was stronger in the dentate gyrus, while with elevation of [Mg2+]o, the reduction was more pronounced in area CA1. During washout of Ca2+, synaptic potentials became reduced and finally depressed. The [Ca2+]o at which synaptic transmission was blocked increased with higher [Mg2+]o and decreased with a change of [K+]o from 3 to 5 mM, whereas with an elevation of [K+]o from 5 to 8 mM, it rose in area CA1 but was reduced in dentate gyrus. All ionic changes also affected frequency habituation and potentiation in paired pulse experimentes. In dentate gyrus, frequency habituation was reversed to frequency potentiation with moderate lowering of [Ca2+]o and with elevation of [Mg2+]o and [K+]o. In contrast, in area CA1 frequency potentiation was reduced upon elevation of [K+]o.

Electrical stimulation of paralyzed vibrissal muscles reduces endplate reinnervation and does not promote motor recovery after facial nerve repair in rats.

The outcome of peripheral nerve injuries requiring surgical repair is poor. Recent work has suggested that electrical stimulation (ES) of denervated muscles could be beneficial. Here we tested whether ES has a positive influence on functional recovery after injury and surgical repair of the facial nerve. Outcomes at 2 months were compared to animals receiving sham stimulation (SS). Starting on the first day after end-to-end suture (facial-facial anastomosis), electrical stimulation (square 0.1 ms pulses at 5 Hz at an ex tempore established threshold amplitude of between 3.0 and 5.0V) was delivered to the vibrissal muscles for 5 min a day, 3 times a week. Restoration of vibrissal motor performance following ES or SS was evaluated using the video-based motion analysis and correlated with the degree of collateral axonal branching at the lesion site, the number of motor endplates in the target musculature and the quality of their reinnervation, i.e. the degree of mono- versus poly-innervation. Neither protocol reduced collateral branching. ES did not improve functional outcome, but rather reduced the number of innervated motor endplates to approximately one-fifth of normal values and failed to reduce the proportion of poly-innervated motor endplates. We conclude that ES is not beneficial for recovery of whisker function after facial nerve repair in rats.

Effects of T-type, L-type, N-type, P-type, and Q-type calcium channel blockers on stimulus-induced pre- and postsynaptic calcium fluxes in rat hippocampal slices.

The contribution of T-, L-, N-, P-, and Q-type Ca2+ channels to pre-and postsynaptic Ca2+ entry during stimulus-induced high neuronal activity in area CA1 of rat hippocampal slices was investigated by measuring the effect of specific blockers on stimulus-induced decreases in extracellular Ca2+ concentration ([Ca2+]0). [Ca2+]0 was measured with ion-selective electrodes in stratum radiatum (SR) and stratum pyramidale (SP), while Ca2+ entry into neurons was induced with stimulus trains (20 Hz for 10 s) alternately delivered to SR and the alveus, respectively. The [Ca2+]0 decreases recorded in SR in response to SR stimulation represented mainly presynaptic Ca2+ entry (Capre), while [Ca2+]0 decreases recorded in SP in response to alvear stimulation were predominantly based on postsynaptic Ca2+ entry (Capost). Ethosuximide and trimethadione were ineffective m concentrations up to 1 mM. At 10 mM, they reduced Capost and, much less, also Capre Nimodipine (25 μM) reduced Capost and, to a minor extent, Capre. ω-Agatoxin IVA (0.4–1 μM) and ω-conotoxin MVIIC (1 μM) also reduced both Capre and Capost, but with a stronger action on Capre. ω-Conotoxin GVIA (3–8 μM) reduced Capost without effect on Capre. We conclude that during stimulus-induced, high-frequency neuronal activity Capost is carried by P/Q-, N-, and L-type channels and probably a further channel type different from these channels. Capre includes at least P/Q-and possibly L-type channels. N-type channels did not contribute to Capre in our experiments. Since ethosuximide and trimethadione were only effective in high concentrations, their action may be unspecific. Thus, T-type channels do not seem to play a major part in Ca2+ entry in this situation.

Morphology sense organs and regeneration of the forelegs whips of the whip spider heterophrynus elaphus arachnida amblypygi

In the whip spider Heterophrynus elaphus the first pair of legs is specialized to serve sensory functions. The morphology of these “whips” and the sensory organs of their tarsi and tibiae are described using scanning and transmission electron microscopy. The tarsus is normally subdivided into 74 segments and bears 7 types of sensory hairs: bristles, club sensilla, two types of porous sensilla, two types of rod sensilla, and leaflike hairs. In addition there are modified claws, 3 kinds of slit sense organs, a “pit organ,” a “plate organ,” and probably a joint receptor. The tibia is usually subdivided into 33 segments. In addition to bristles the tibia bears 7 trichobothria at constant locations and a lyriform slit sense organ. The functional and systematic implications of these findings are discussed.

Manual stimulation, but not acute electrical stimulation prior to reconstructive surgery, improves functional recovery after facial nerve injury in rats.

UNLABELLED The outcome of peripheral nerve injuries requiring surgical repair is poor. Recent work suggested that electrical stimulation (ES) of the proximal nerve stump to produce repeated discharges of the parent motoneurons for one hour could be a beneficial therapy if delivered immediately prior to reconstructive surgery of mixed peripheral nerves. PURPOSE We tested whether ES has a positive influence on functional recovery after repair of a purely motor nerve, the facial nerve. METHODS Electrical stimulation (20 Hz) was delivered to the proximal nerve stump of the transected facial nerve for 1 hour prior to nerve reconstruction by end-to-end suture (facial-facial anastomosis, FFA). For manual stimulation (MS), animals received daily rhythmic stroking of the whisker pads. Restoration of vibrissal motor performance following ES or MS was evaluated using video-based motion analysis. We also assessed the degree of collateral axonal branching at the lesion site, by counting motoneuronal perikarya after triple retrograde labeling, and estimated the quality of motor end-plate reinnervation in the target musculature. Outcomes at 4 months were compared to animals receiving sham stimulation (SS) or MS. RESULTS Neither protocol reduced the degree of collateral sprouting. ES did not improve functional outcome and failed to reduce the proportion of polyinnervated motor end-plates. By contrast, MS restored normal whisking function and reduced polyinnervation. CONCLUSION Whereas acute ES is not beneficial for facial nerve repair, MS provides long-term benefits.

THE GIANT FIBER SYSTEM IN THE FORELEGS (WHIPS) OF THE WHIP SPIDER HETEROPHRYNUS ELAPHUS POCOCK (ARACHNIDA:AMBLYPYGI)

SummaryThe front legs of the whip spider H. elaphus are strongly modified to serve sensory functions. They contain several afferent nerve fibers which are so large that their action potentials can be recorded externally through the cuticle. In recordings from the tarsus 7 different types of afferent spikes were identified; 6 additional types of afferent spikes were discriminated in recordings from the tibia and femur. Most of the recorded potentials could be attributed to identifiable neurons serving different functions. These neurons include giant interneurons and giant fibers from diverse mechanoreceptors such as slit sense organs, trichobothria, and a joint receptor. In the present report these neurons are characterized using electrophysiological and histological methods. Their functions are discussed in the context of the animals behavior.

Histamine enhances synaptic transmission in hippocampal slices from hibernating and warm-acclimated Turkish hamsters

The effects of histamine on synaptic transmission were studied at 37 degrees C and 22 degrees C with extracellular recordings of stimulus-induced population action potentials in area CA1 of hippocampal slices prepared from hibernating (HTH) and warm-acclimated Turkish hamsters (WTH) and rats. In rat slices, application of 50 microM histamine had no effects on population spikes and field excitatory postsynaptic potentials (EPSPs). In HTH as well as WTH slices, 50 microM histamine generally increased the population spike amplitude. The slope of the field EPSP was unchanged. At 37 degrees C, the sensitivity for histamine was significantly higher in HTH slices than in WTH slices. At 22 degrees C, the effects of histamine were less pronounced in HTH as well as WTH slices. Hibernation-related improvement of sensitivity for histamine is interpreted as supporting hippocampal function during arousal from hibernation.