Modulation of the hippocampal propensity to non- synaptic epileptiform synchronization in low-calcium model of epilepsy.

The CA3 and CAI regions are the main stages of the "three-synaptic pathway", which plays a role in the generation of hyper-synchronous events in the hippocampus. Under certain experimental conditions, this brain structure might support pathological epileptiform synchronization that is independent of active chemical synaptic transmission. In present work, we estimated the conditions that would facilitate non- synaptic synchronization of the hippocampus. Non-synaptic epileptiform activity was induced in hippocampal slices by the omission calcium ions from the extracellular milieu. The propensity of hippocampal regions to nonsynaptic interactions was estimated by measuring the delay time neededfor the development of low-Ca²âº discharges in the CA3 and CAI. Next, an increase of neuronal excitability was induced by the pre- incubation ofhippocampal slices in 4-aminopyridine (4-AP) and by the reduction ofextracellular osmolarity. Pre-incubation of hippbcampal slices with 4-AP under normal osmotic conditions resulted in decreased latency for non-synaptic discharges in the CA3, but not in the CAl. However hypo-osmotic conditions caused increased excitability of the CA3 region, which resulted in decreased delay time for nonsynaptic discharges and this level of cellular excitability was not further enhanced by the pre-incubation with 4-AR.

Surface charge impact in nonsynaptic model of epilepsy in rat hippocampus.

Decreasing of surface charge screening near voltage-gated ion channels via reduction of extracellular cation divalent ions provide potent mechanism of altering cellular excitability and seizure threshold. Spontaneous field potentials were recorded from horizontal brain slices of young Wistar rats (postnatal day 10-12). Extracellular registrations wereobtained from CA1 and CA3 area of hippocampus. For induction of nonsynaptic epileptiform activity slices were perfused with artificial cerebrospinal fluid with omitted Ca2+and Mg2+ ions. Effect of different Mg2+ concentration (1, 2, and 3mmol/l) on initial stage of nonsynaptic epileptiform discharges was studied. Our results suggest that the change in Mg2+ concentration dramatically affects the probability of induction of low-Ca2+seizure-like activity (SLA), providing evidence that Mg2+ can alter cerebral excitability by affecting the surface charge and supporting the idea that surface charge could be a pharmacological target for anti-epileptic treatment.

Modulation of 4-aminopyridine-induced neuronal activity and local pO(2)in rat hippocampal slices by changing the flow rate of the superfusion medium.

The brain slice preparation is the most frequently used tool for testing of pharmacological agents on the neuronal excitability. However in the absence of blood circulation in vitro, the tissue oxygenation strongly depends on the experimental conditions. It is well established that both hypoxia as well as hyperoxia can modulate the neuronal network activity. Thereby changes in tissue oxygen level during experiment may affect the final result. In the present study we investigated the effect of oxygenation on seizure susceptibility in the hippocampal slice preparation using 4-aminopyridine (4-AP) model of ictogenesis in inmature rats. We found that changing the medium perfusion rate in the range of 1-5 ml/min greatly affects the tissue oxygenation, amplitude and frequency of 4-AP-induced synchronous neuronal activity. The decrease in the flow rate as well as substitution of the oxygen in the extracellular medium with nitrogen causes a strong reduction of 4-AP-induced synchronous neuronal discharges. Our results demonstrate a significant linear correlation between the power of 4-AP-induced neuronal activity and the oxygen level in slice tissue. Also we demonstrated that the presence of medium flow is a necessary condition to support the constant level of the slice oxygenation. These data suggest that the oxygen supply of the brain slice strongly depends on experimental protocol and could modulate in vitro neuronal network excitability which should be taken into consideration when planning epilepsy-related studies.

[EFFECT OF NEONATAL SEIZURES ON THE SYNAPTIC PLASTICITY OF RAT SOMATOSENSORY CORTEX].

Using an experimental model of neonatal recurrent seizures we investigated the influence of epileptic seizures in the various forms of synaptic plasticity in neurons of the somatosensory cortex. We found that early seizures do not affect the post-tetanic potentiation of the amplitude of the postsynaptic potentials and the depression of postsynaptic potentials during high-frequency stimulation. However they result in the chronic increase of the long-term potentiation of synaptic transmission. These changes of synaptic plasticity may affect the processing of the sensory information in patients with a history of recurrent seizures during early development.

Thrombin modulates persistent sodium current in CA1 pyramidal neurons of young and adult rat hippocampus.

Serine protease thrombin, a key factor of blood coagulation, participates in many neuronal processes important for normal brain functioning and during pathological conditions involving abnormal neuronal synchronization, neurodegeneration and inflammation. Our previous study on CA3 pyramidal neurons showed that application ofthrombin through the activation of specific protease-activated receptor 1 (PAR1) produces a significant hyperpolarizing shift of the activation of the TTX-sensitive persistent voltage-gated Na+ current (I(Nap)) thereby affecting membrane potential and seizure threshold at the network level. It was shown that PAR1 is also expressed in CA1 area of hippocampus and can be implicated in neuronal damage in this area after status epilepticus. The aim of the present study was to evaluate the effect of thrombin on I(NaP) in CA1 pyramidal neurons from adult and young rats. Using whole cell patch-clamp technique we demonstrate that thrombin application results in the hyperpolarization shift of I(NaP) activation as well as increase in the I(NaP) amplitude in both age groups. We have found that I(NaP) in pyramidal neurons of hippocampal CA 1 region is more vulnerable to the thrombin action than I(NaP) in pyramidal neurons of hippocampal CA3 region. We have also found that the immature hippocampus is more sensitive to thrombin action which emphasizes the contribution of thrombin-dependent pathway to the regulation of neuronal activity in immature brain.

Anaesthetic and postanaesthetic effect of isoflurane on the multiple-unit activity of the immature rat hippocampus.

We investigated anesthetic and postanaesthetic effect of isoflurane on the multi-unit activity (MUA) in the CA3 region of immature rat hippocampus. MUA amplitude did not significantly change during application of isoflurane. On the other hand MUA frequency significantly decreased during the anesthesia. After isoflurane discontinuation two phases of MUA frequency recovery were observed: initial rapid increase followed by a slower recovery to the control level. Comparison of recovering period of the receptor mediated systems and spontaneous field activity from isoflurane anesthesia is discussed.

The effect of neuraminidase blocker on gabazine-induced seizures in rat hippocampus.

Concentration of neuraminidase (NEU), an enzyme which cleaves negatively charged sialic acids from carbohydrate moieties of the cellular membrane, could vary depending on physiological conditions. Multiple evidences suggest that fluctuations of NEU extracellular concentrations can influence neuronal activity. In the present study we examined the effect of down regulation of endogenous NEU activity on seizure-like activity (SLA) induced by gabazine (specific blocker of inhibitory synaptic transmission) in the hippocampal CA1 pyramidal region of cultured slices. We show that in slices pretreated with the blocker of endogenous NEU, N-acetyl-2,3-dehydro-2-deoxyneuraminic acid (NADNA), duration of synchronous oscillations induced by gabazine was considerably increased comparatively to control untreated slices. This study adds further information that changes in the level of NEU activity is an important factor, which can affect neuronal network excitability.

Developmental changes in the expression of low-voltage-activated Ca2+ channels in rat visual cortical neurones.

1. The functional properties of low-voltage-activated (LVA) Ca2+ channels were studied in pyramidal neurones from different rat visual cortical layers in order to investigate changes in their properties during early postnatal development. Ca2+ currents were recorded in brain slices using the whole-cell patch-clamp technique in rats from three age groups: 2, 3 and 12 days old (postnatal day (P) 2, P3 and P12). 2. It was demonstrated that LVA Ca2+ currents are present in neurones from superficial (I-II) and deep (V-VI) visual cortex layers of P2 and P3 rats. No LVA Ca2+ currents were observed in neurones from the middle (III-IV) layers of these rats. The LVA Ca2+ currents observed in P2 and P3 neurones from both superficial and deep layers could be completely blocked by nifedipine (100 microM) and were insensitive to Ni2+ (25 microM). 3. The density of LVA Ca2+ currents decreased rapidly during the early stages of postnatal development, while the density of high-voltage-activated (HVA) Ca2+ currents progressively increased up to the twelfth postnatal day. No LVA Ca2+ currents were found in P12 neurones from any of the layers. Only HVA Ca2+ currents with high sensitivity to F- applied through the patch pipette were observed. 4. The kinetics of LVA Ca2+ currents could be well approximated by the m2h Hodgkin-Huxley equation with an inactivation time constant of 24 +/- 6 ms. The steady-state inactivation curve fitted by a Boltzmann function had the following parameters: membrane potential at half-inactivation, -86.9 mV; steepness coefficient,3.4 mV. 5. It is concluded that, in visual cortical neurones, LVA Ca2+ channels are expressed only in the neurones of deep and superficial layers over a short period during the earliest postnatal stages. These channels are nifedipine sensitive and similar in functional properties to those in the laterodorsal (LD) thalamic nucleus. However, the cortical neurones do not express another ('slow') type of LVA Ca2+ channel, which is permanently present in LD thalamic neurones after the second postnatal week, indicating that the developmental time course of cortical and thalamic cells is different.

Two types of low-voltage-activated Ca2+ channels in neurones of rat laterodorsal thalamic nucleus.

1. The pharmacological and kinetic properties of two types of low-voltage-activated (LVA) Ca2+ currents were studied in thalamocortical neurones of the laterodorsal (LD) thalamic nucleus during early postnatal development. The whole-cell patch-clamp technique was used on brain slices from rats of three age groups: 12, 14 and 17 days old (postnatal day (P) 12, P14 and P17). 2. In P12 neurones, the population of LVA Ca2+ channels was homogeneous. LVA Ca2+ current elicited by depolarizing voltage steps from a holding potential more negative than -70 mV was sensitive to nifedipine (Kd = 2.6 microM). This current reached a maximum at about -55 mV and had a fast monoexponential decay with a time constant, tau h,f, of 32.3 +/- 4.0 ms. 3. The population of LVA Ca2+ channels in P14 and P17 neurones was found to be heterogeneous. A subpopulation of nifedipine-insensitive LVA Ca2+ channels was observed. The current-voltage curve of the Ca2+ current had a characteristic hump with two peaks at about -65 and -55 mV. As well as the fast component (designated IT,f), the decay of the LVA current also included a slow component (designated IT,s), with inactivation time constants (tau h,s) of 54.2 +/- 4.5 and 68.6 +/- 3.17 ms for P14 and P17 neurones, respectively. 4. The kinetics of both components could be well approximated by the m2h Hodgkin-Huxley equation. No significant difference in activation kinetics was observed. The activation time constants for the fast (tau m,f) and slow (tau m,s) components were 6.3 +/- 1.0 and 7.3 +/- 1.5 ms, respectively. 5. La3+ at a concentration of 1 microM effectively blocked the IT,f component but Ni2+ (25 microM) completely eliminated the IT,s component. 6. Steady-state inactivation curves of both components could be best fitted by a Boltzmann function with membrane potential values at half-maximal inactivation of -85.5 and -98.1 mV for the fast and slow components, respectively. 7. It was concluded that two different subtypes of LVA Ca2+ channel are present in LD neurones. Only the fast type is well expressed at the earliest postnatal stage (P12). The slow type could be found at the end of the second week (P14). The amplitude of the slow current increased progressively up to P17, obviously coinciding with dendritic expansion as judged by progressive increase of the membrane capacitance of the corresponding neurones. This property appears to differentiate neurones of the associative nuclei from neurones of other thalamic nuclei.