Differential effects of calcium entry blockers on pre- and postsynaptic influx of calcium in the rat hippocampus in vitro

A decrease in extracellular free Ca ([Ca²⁺]₀) in response to stimulation of Schaffer collaterals could be recorded in or near the stratum pyramidale even when synaptic transmission was completely blocked. Under the same conditions, alvear stimulation also evoked a decrease in [Ca²⁺]₀ at the same site. We attributed the former to influx of Ca²⁺ into presynaptic terminals and the latter to influx postsynaptic (pyramidal) cells. Both pre- and postsynaptic Ca²⁺ influx were completely blocked by Ni²⁺ (2.5 mM). Nifedipide (5–10 μM), verapamil (50–100 μM) and fendiline (100–200 μM) reduced the posysnaptic influx of Ca²⁺ but did not alter Ca²⁺ loss from the extracellular space into presynaptic terminals. The calcium channel activators, BAY-K 8644 and CGP 28,392, had no consistent effect on either pre- or postsynaptic influx. Occasional enhancement of both pre- and postsynaptic responses was seen. In most studies the agents were without effect and on occasions a reduction in both responses was seen. The results could indicate that Ca-channels at pre- and postsynaptic sites in CA₁ may be of different types.     

Loss of synapses in the entorhinal-dentate gyrus pathway following repeated induction of electroshock seizures in the rat

The goal of this study was to answer the question of whether repeated administration of electroconvulsive shock (ECS) seizures causes structural changes in the entorhinal-dentate projection system, whose neurons are known to be particularly vulnerable to seizure activity. Adult rats were administered six ECS seizures, the first five of which were spaced by 24-hr intervals, whereas the last two were only 2 hr apart. Stereological approaches were employed to compare the total neuronal and synaptic numbers in sham- and ECS-treated rats. Golgi-stained material was used to analyze dendritic arborizations of the dentate gyrus granule cells. Treatment with ECS produced loss of neurons in the entorhinal layer III and in the hilus of the dentate gyrus. The number of neurons in the entorhinal layer II, which provides the major source of dentate afferents, and in the granular layer of the dentate gyrus, known to receive entorhinal projections, remained unchanged. Despite this, the number of synapses established between the entorhinal layer II neurons and their targets, dentate granule cells, was reduced in ECS-treated rats. In addition, administration of ECS seizures produced atrophic changes in the dendritic arbors of dentate granule cells. The total volumes of entorhinal layers II, III, and V-VI were also found to be reduced in ECS-treated rats. By showing that treatment with ECS leads to partial disconnection of the entorhinal cortex and dentate gyrus, these findings shed new light on cellular processes that may underlie structural and functional brain changes induced by brief, generalized seizures.     

Beta-adrenergic receptor binding in different regions of rat brain after various intensities of electroshock: relationship to postictal EEG

Relationships among different intensities of electroshock treatment (EST), EEG-recordable primary after discharge (PAD), and beta-adrenergic binding in various brain regions were investigated in adult rats. Different intensities of EST (1-2, 6-8, 20-30 mamp) were applied through ear-clip electrodes for 7 consecutive days. A sigmoidal relationship was observed between the PAD and the applied current-intensity. 3H-dihydroalprenolol (DHA) was used as a ligand to evaluate beta-adrenergic receptor binding in membrane preparation of the following brain regions: Cerebral cortex, cerebellum, pons-medulla, and "midregion"-which consisted of the remainder tissue including thalamus, striatum, and midbrain. A significant decrease (28%) in binding was observed in cortex tissue after EST of 6-8 mamp but no changes were observed after either 1-2 mamp or 20-30 mamp. In "midregion," a significant increase (40%) in binding was observed after EST with the subconvulsive intensity (1-2 mamp) whereas no changes were seen at higher intensities. The results suggest that in cortex, the function relating beta-adrenergic binding to current intensities may not be monotonic. The increase in binding seen in the "midregion" after subconvulsive EST may not be associated with changes related to the clinical efficacy of EST.     

Lysophosphatidic acid depletes intracellular calcium stores different from those mediating capacitative calcium entry in C6 rat glioma cells

Lysophosphatidic acid (LPA) functions as an extracellular lipid mediator stimulating phospholipase C and affecting the structure of the cytoskeleton in several cell types. In rat glioma C6 cells, LPA mobilizes calcium from intracellular calcium stores and reverts morphological changes induced by elevated cytosolic cAMP-concentrations. Here we show that LPA-stimulation of C6 cells loaded with the calcium-sensitive fluorescent dye indo-1 results in calcium release from a subset of intracellular calcium stores that are not sensitive to the tumor promoter thapsigargin and do not overlap with calcium stores depleted during purinergic receptor stimulation with ATP. Furthermore, depletion of LPA-sensitive calcium stores does not induce capacitative calcium entry from the extracellular space into the cytosol to the same extent as ATP. These results indicate that inositol phosphate signaling induced by LPA or ATP may differ in kinetics or in spatial organisation within the cell. This may represent a possible explanation for the previous observation that only LPA, but not other calcium-mobilizing agonists, reverts cAMP-induced changes in the cytoskeletal organization in C6 cells. GLIA 19:67–73, 1997. © 1997 Wiley-Liss, Inc.     

Permeability of the blood-cerebrospinal fluid and blood-brain barriers to thyrotropin-releasing hormone

The permeability of the blood-cerebrospinal fluid (CSF) barrier to 3H-labelled thyrotropin-releasing hormone (TRH), was studied at the blood-tissue interface of the isolated perfused choroid plexus of the sheep, using a rapid (less than 30 s), single circulation paired-tracer dilution technique, in which D-[14C]mannitol serves as an extracellular marker. Arterio-venous loss of 14C radioactivity reflects the percentage of the D-mannitol dose that crosses the blood-CSF barrier using a non-specific pathway. This loss suggests that the choroidal epithelium is moderately leaky. Cellular uptake of TRH, estimated by directly comparing venous dilution profiles of [3H]TRH and D-[14C]mannitol was independent of this leakiness. The unidirectional transport of TRH could not be saturated with unlabelled TRH at a concentration as high as 10 mM, but was markedly reduced by 10 mM proline and by the inhibitor of amidase and aminopeptidase activity, bacitracin (2 mM). Permeability of the blood-brain barrier to [3H]TRH was studied in the adult rat, employing the intracarotid injection technique of Oldendorf in which [14C]butanol served as an 'internal standard'. Brain-uptake of 3H radioactivity corrected for residual vascular space indicated a low extraction from the blood of TRH during a 15 s period of exposure to the peptide. Self-inhibition of [3H]TRH uptake by unlabelled TRH (10 mM) could not be demonstrated, but L-proline (10 mM) and bacitracin (2 mM) strongly inhibited this uptake.(ABSTRACT TRUNCATED AT 250 WORDS)     

Electroconvulsive treatment: Effects on phospholipase C activity and GTP binding activity in rat brain

Summary The effect of electroconvulsive treatment (ECT) on activities of phospholipase C hydrolyzing phosphatidylinositol (PI-PLC) and phosphatidylinositol 4,5-bisphosphate (PIP₂-PLC) and guanosine-5-(3-O-thio)triphosphate (GTP S) binding activity were examined in membrane and cytosol fractions from four discrete areas (prefrontal cortex, hippocampus, striatum, and amygdala) of the rat brain. A single ECT resulted in an increase in cytosolic activities of PI-PLC in the prefrontal cortex and of PIP₂PLC in all 4 brain regions examined. There were no significant changes in either PI-PLC or PIP₂-PLC activity in membrane fractions after a single ECT. Repeated ECT caused regionally specific changes in PLC activities as follows: in the prefrontal cortex, both cytosolic PI-PLC and PIP₂-PLC and membranous PI-PLC activities were decreased; in the hippocampus, no changes in any PLC activities were seen; in the striatum, only membranous PI-PLC activities were increased; and, in the amygdala, cytosolic and membranous PI-PLC and cytosolic PIP₂-PLC activities were increased. The pattern of changes in GTP S binding activity following repeated ECT resembled those found in PLC activity as follows: in the prefrontal cortex, GTP S binding activities were significantly reduced in both membrane and cytosol; in the hippocampus, the activity was decreased in membrane; in the striatum, no changes in GTP S binding activity were seen in any fraction; and, in the amygdala, the activity was increased in cytosol. These findings suggest that ECT has complex effects on the G protein-phospholipase C system, possibly affecting neuronal signal transduction.     

Inhibition by melatonin of dopamine release from rat hypothalamus: regulation of calcium entry

The [Ca²⁺] dependency of dopamine release evoked by electrical field stimulation of hypothalamic tissue from female rats at the estrous stage was assessed in the presence of melatonin. At concentrations of 10⁻⁹–10⁻⁶ M, melatonin inhibited the [Ca²⁺]-dependent dopamine release.Melatonin reduced tissue uptake of ⁴⁵Ca²⁺ during stimulation either by an electrical field or by elevated K⁺ concentration. About 90% of this inhibition by melatonin of ⁴⁵Ca²⁺ uptake, as well as of dopamine release, was not observed in the presence of the calcium ionophore A23187. Hence, the inhibitory effect on dopamine release by melatonin may stem from the reduction of calcium entry into the presynaptic nerve endings.     

Synaptic sodium spikes trigger long-lasting depolarizations and slow calcium entry in rat olfactory bulb granule cells

In the mammalian olfactory bulb, axonless granule cells mediate self- and lateral inhibitory interactions between mitral/tufted cells via reciprocal dendrodendritic synapses. Synaptic output from granule cells occurs on both fast and slow timescales, allowing for multiple granule cell functions during olfactory processing. We find that granule cell sodium action potentials evoked by synaptic activation of the sensory input via mitral/tufted cells are followed by a long-lasting depolarization that is not observed after current-evoked action potentials or large excitatory postsynaptic potentials in the same cell. Using two-photon imaging in acute rat brain slices, we demonstrate that this prolonged electrical response is paralleled by an unusual, long-lasting postsynaptic calcium signal. We find that this slow synaptic Ca²⁺ signal requires sequential activation of NMDA receptors, a nonselective cation conductance I_CAN and T-type voltage-dependent Ca²⁺ channels. Remarkably, T-type Ca²⁺ channels are of critical importance for the 'globalization' of Ca²⁺ transients. In individual active spines, the local synaptic Ca²⁺ signal summates at least linearly with the global spike-mediated Ca²⁺ signal. We suggest that this robust slow synaptic Ca²⁺ signal triggers dendritic transmitter release and thus contributes to slow synaptic output of the granule cell. Therefore, the synaptic sodium spike signal could represent a special adaptation of granule cells to the wide range of temporal requirements for their dendritic output. Our findings demonstrate with respect to neuronal communication in general that action potentials evoked by somatic current injection may lack some of the information content of 'true' synaptically evoked spikes.     

Blocking of the receptor-stimulated calcium entry into human platelets by verapamil and nicardipine

Verapamil (ED₅₀=3×10⁻⁶ M) and nicardipine (ED₅₀=10⁻⁶ M) inhibited the platelet activating factor (PAF)-induced increase of free cytosolic calcium concentration ([Ca²⁺]_i) in quin2-loaded human platelets. In a Ca-free medium containing 5 mM BaCl₂, PAF stimulated the inflow of Ba²⁺ ions which is completely abolished by verapamil and nicardipine. Simultaneous determination of quin2 fluorescence and ⁴⁵Ca absorption showed that the action of verapamil is accounted for by blocking of the Ca²⁺ entry. Nicardipine suppresses also Ca²⁺ mobilization from intracellular stores. The effects of verapamil and nicardipine are not competitive with respect to PAF.The blockers reduce the [Ca²⁺]_i increase induced by ADP, vasopressin, and PGH₂ analogue U46619.     

Effects of postnatal electroshock convulsions on epileptogenesis of the amygdala and hippocampus in adult rats

Adult rats which received repeated electroshock convulsions (ESCs) during postnatal development underwent electrical kindling of the amygdala (AM) and hippocampus (HIPP). Completion of kindling, especially AM kindling, was significantly faster in the groups with ESCs from the neonatal to late infantile age than in the control group. It is discussed that especially the late infantile age in the postnatal development was supposed to play an important role in the faster kindling brought about by postnatal convulsions and that the faster kindling was due in part to abbreviation of the partial seizure process. However, the neonatal ESC group showed significantly delayed completion of HIPP kindling.     

The relationship of free and conjugated catecholamines in plasma and cerebrospinal fluid in cerebral and meningeal disease

Summary The concentration of free and conjugated norepinephrine (NE), epinephrine (E) and dopamine (DA) were measured by a modified radioenzymatic assay in the plasma and in the cerebrospinal fluid (CSF) of 45 patients with normal and in 21 patients with disturbed blood-CSF barriers. In patients with an undisturbed blood-CSF barrier the free NE and E in CSF were 128±45 ng/l and 27±20 ng/l (mean values±S.E.), respectively, and represented about 50 % of the average plasma values. Mean DA was not different in plasma (47±22 ng/l) and in CSF (41±19 ng/l). Both in plasma and in CSF, considerable higher free catecholamine (CA) levels were measured in patients with dysfunction of the blood-CSF barrier. In one patient with bacterial meningitis twofold higher concentrations of free NE and DA in CSF as compared with plasma were detectable. Sulfate conjugates of catecholamines are predominant in plasma and CSF.The contribution of conjugated CA to total CA in plasma from patients with normal blood-CSF barrier averaged 69.7 %, 63.1 % and 98.1 % for NE, E and DA, respectively and was significantly lower in the CSF (p<0.001). In patients with disturbed blood-CSF barrier, the increases of conjugated CA were more pronounced in CSF than in plasma. Further, the contribution of conjugated NE and E to total NE and E in CSF was not only increased in patients with bacterial meningitis, but also in patients with renal insufficiency compared to the control patients (p<0.02 and p<0.001 resp.). Free and conjugated NE, E and DA in the plasma and CSF were related significantly (p<0.01 resp.) with stronger correlation for conjugated CA (p<0.001 resp.). These results together with findings in the literature, suggest that there is little or no rostral-caudal gradient in CSF CA conjugate concentrations and that even in patients with intact blood-CSF barrier plasma conjugated CA concentrations influence those in CSF. Thus only free CA levels in CSF may reflect the central adrenergic activity.     

Influence of aminophylline on the anticonvulsive action of gabapentin in the mouse maximal electroshock seizure threshold model

Summary Accumulating evidence indicates that aminophylline [theophylline₂ · ethylenediamine] markedly attenuates the anticonvulsant action of conventional antiepileptic drugs in experimental animal models of epilepsy and evokes severe seizure activity in patients treated with this methylxanthine. The objective of this study was to determine the influence of acute (single) and chronic (twice daily for 14 consecutive days) treatments with aminophylline on the anticonvulsant potential of gabapentin (a second-generation antiepileptic drug) in the mouse maximal electroshock seizure threshold model. Additionally, the effects of acute and chronic administration of aminophylline on the adverse effect potential of gabapentin in terms of motor coordination impairment were assessed in the chimney test. To evaluate pharmacokinetic characteristics of interaction between drugs, total brain concentrations of gabapentin and theophylline were estimated with high-pressure liquid chromatography and fluorescence polarization immunoassay, respectively. Results indicated that gabapentin (at doses of 75 and 100 mg/kg, i.p.) increased the threshold for electroconvulsions in mice. Aminophylline in non-convulsive doses of 50 and 100 mg/kg (i.p.), both in acute and chronic experiments, did not attenuate the anticonvulsant potential of gabapentin in the maximal electroshock seizure threshold test in mice. Similarly, aminophylline at a dose of 100 mg/kg had no impact on the adverse effect potential of gabapentin in the chimney test. Pharmacokinetic evaluation of total brain concentrations of gabapentin and theophylline revealed no significant changes in total brain concentrations of the drugs after both, acute and chronic applications of aminophylline in combination with gabapentin. The data show that aminophylline did not alter the ability of gabapentin to protect mice against seizures induced by electroconvulsive shock. The observed interaction between gabapentin and aminophylline in both acute and chronic experiments was pharmacodynamic in nature.     

The role of the cytoskeleton in capacitaftive calcium entry in myenteric glia

Capacitative calcium entry (CCE) is the process by which intracellular calcium is replenished from the external milieu upon depletion of intracellular stores. CCE is thought to participate in chemotaxis, proliferation and cell signalling. A physical interaction between intracellular stores and the plasma membrane is postulated to regulate CCE. We hypothesized that cytoskeletal disruption alters this interaction, inhibiting CCE in enteric glia. Cultured myenteric glia from neonatal guinea-pigs were treated with cytochalasin D (10 micro mol L-1), a microfilament disrupting agent, nocodazole (20 micro mol L-1), a microtubule disrupting agent, or vehicle (dimethyl sulphoxide). Intracellular calcium changes were measured using fura-2 microfluorimetry. To evaluate the rate of cation re-entry, barium was substituted for calcium because barium is not sequestered internally. Cytochalasin D-treated glia had diminished CCE responses (57 +/- 3 nmol L-1) compared with controls (97 +/- 7 nmol L-1) as did nocodazole-treated glia (30 +/- 2 nmol L-1) vs controls (77 +/- 6 nmol L-1). The proportion of cells demonstrating CCE abolition was greater in the cytochalasin (50 +/- 8%) and nocodazole-treated (89 +/- 2%) groups compared with controls (21 +/- 2%, 40 +/- 9%, respectively). Cytochalasin D and nocodazole treatment diminished the rate of cation re-entry based on diminished barium entry in treated vs control cells. From this study, we conclude that disruption of cytoskeletal elements diminishes calcium influx essential to calcium store repletion in myenteric glia.     

Long-term observations on calcium accumulation in postischemic gerbil brain

We studied delayed postischemic calcium accumulation and neuronal damage in the gerbil brain, using 45Ca autoradiography as a marker for detection of injured tissue and light microscopy. Transient cerebral ischemia was induced for 15 min. Sham-operated gerbils showed no abnormal calcium accumulation and neuronal damage throughout the brain. At 2 and 7 days following 15 min of ischemia, marked calcium accumulation and mild to severe neuronal damage were found in the selectively vulnerable areas such as neocortex, striatum, hippocampus and thalamus, and brainstem such as medial geniculate body, substantia nigra and inferior colliculus. After 1-2 months of recirculation, the calcium accumulation was not recognized in the brainstem. But, the accumulation was still detectable in the striatum, the hippocampus and the thalamus. Morphological study showed that marked proliferation of glia cells was rapid in the inferior colliculus and was relatively slow in the striatum and the hippocampus, although these structures were severely damaged after ischemia. The result suggests that the speed of restoration of injured tissue and the mechanisms for the damage after cerebral ischemia may be different between the selectively vulnerable areas and the brainstem. Furthermore, they suggest that 45Ca autoradiographic technique may provide a useful approach for diagnosis of the restoration of injured tissue at chronic stage following cerebral ischemia.     

Amiloride enhances the anticonvulsant action of various antiepileptic drugs in the mouse maximal electroshock seizure model

Accumulating evidence indicates that amiloride (a potassium-sparing diuretic) exerts the anticonvulsant action in various in vivo and in vitro experiments. Therefore, the objective of this study was to assess the influence of amiloride on the protective action of numerous conventional and second-generation antiepileptic drugs [AEDs: carbamazepine (CBZ), lamotrigine (LTG), oxcarbazepine (OXC), phenobarbital (PB), topiramate (TPM), and valproate (VPA)] against maximal electroshock (MES)-induced seizures in mice. Results indicate that amiloride [up to 100 mg/kg, intraperitoneally (i.p.), at 30, 60, and 120 min before the test] neither altered the threshold for electroconvulsions, nor protected the animals against MES-induced seizures in mice. Moreover, amiloride (75 and 100 mg/kg, i.p., 120 min prior to the test) significantly enhanced the anticonvulsant effects of all studied AEDs, except for LTG, by reducing their ED₅₀ values in the MES test. In contrast, amiloride at 50 mg/kg (i.p.) had no significant effect on the antielectroshock action of the tested AEDs in mice. Estimation of total brain AED concentrations revealed that amiloride (75 mg/kg) significantly increased total brain concentrations of CBZ, OXC, and PB, but not those of LTG, TPM, and VPA in mice. In conclusion, one can ascertain that the potentiation of the antiseizure action of TPM and VPA by amiloride in the MES test and lack of any pharmacokinetic interactions between drugs, make the combinations of amiloride with TPM and VPA of pivotal importance for epileptic patients.     

Agmatine enhances the anticonvulsant action of phenobarbital and valproate in the mouse maximal electroshock seizure model

Accumulating evidence indicates that agmatine (AGM—an endogenous neuromodulator/neurotransmitter in the brain) exerts the anticonvulsant action in various in vivo experiments. Therefore, the aim of this study was to assess the influence of AGM on the protective action of numerous conventional and newer antiepileptic drugs [carbamazepine (CBZ), lamotrigine (LTG), oxcarbazepine (OXC), phenobarbital (PB), phenytoin (PHT), topiramate (TPM) and valproate (VPA)] in the mouse maximal electroshock seizure (MES) model. Results indicate that AGM (up to 100 mg/kg, i.p., 45 min before the test) neither altered the threshold for electroconvulsions nor protected the animals against MES-induced seizures in mice. Moreover, AGM (100 mg/kg, i.p.) significantly enhanced the anticonvulsant effects of PB and VPA in the MES test by reducing their ED₅₀ values from 22.54 to 16.82 mg/kg (P < 0.01) for PB, and from 256.1 to 210.6 mg/kg (P < 0.05) for VPA, respectively. In contrast, AGM at 100 mg/kg (i.p.) had no significant effect on the antielectroshock action of the remaining drugs tested (CBZ, LTG, OXC, PHT, and TPM) in mice. Estimation of total brain PB and VPA concentrations revealed that the observed interactions between AGM and PB or VPA in the MES test were pharmacodynamic in nature because neither total brain PB, nor total brain VPA concentrations were altered after i.p. administration of AGM at 100 mg/kg. Moreover, none of the examined combinations of AGM (100 mg/kg) with CBZ, LTG, OXC, PB, PHT, TPM, and VPA (at their ED₅₀ values from the MES test) affected motor coordination in the chimney test, long-term memory in the passive avoidance task, and muscular strength in the grip-strength test in mice, indicating no acute adverse effects in animals. In conclusion, one can ascertain that the selective potentiation of the antielectroshock action of PB and VPA by AGM, lack of any pharmacokinetic interactions between drugs and no acute adverse effects, make the combinations of AGM with PB or VPA of pivotal importance for epileptic patients. It seems that modulation of AGM concentration in the brain may occur favorable in further clinical practice. The results of this study were presented in part at the “11th Congress of the European Federation of Neurological Societies” Brussels, Belgium, 25–28 August, 2007 [abstract in 2007, Eur J Neurol 14(Suppl 1):210].     

Bay K 8644 stimulation of calcium entry and endogenous dopamine release in rat striatal synaptosomes antagonized by nimodipine

The calcium channel agonist Bay K 8644 (0.1 – 100 nM) significantly increased the fast-phase entry of calcium and release of endogenous dopamine from rat striatal synaptosomes partially depolarized with 15 mM KCl. This increase was completely blocked by 10 nM nimodipine which had no inhibitory effect on calcium influx and dopamine release in the absence of Bay K 8644. Bay K 8644's agonist effect was attenuated with higher KCl concentrations. These findings suggest that Bay K 8644, in combination with partial KCl depolarization, may expose brain synaptosomal calcium channels which are sensitive to nanomolar concentrations of dihydropyridine calcium channel blockers.     

Cerebrospinal fluid from amyotrophic lateral sclerosis has no effect on intracellular free calcium in cultured cortical neurons

The data from the literature regarding the presence of a neurotoxic factor in amyotrophic lateral sclerosis (ALS) plasma or cerebrospinal fluid (CSF) remain controversial. As a new approach to this question, we have studied the effect of CSF from ALS patients on the temporal dynamics of the intracellular free calcium concentration ([Ca²⁺]_i) of murine cortical neurons in cultures using Fura-2 fluorescence videomicroscopy and single-cell imaging. CSF from seven ALS patients and controls was added at dilutions up to 20% to cortical neuronal cultures. The in vitro inhibition of CSF on [³H]kainic acid binding showed that the CSF did not contain any substances other than glutamate itself in larger amounts. At the concentrations used, the CSF did not have any effect on [Ca²⁺]_i or on the neuronal responsiveness as defined by the ability of the cells to respond with a transient increase in [Ca²⁺]_i to depolarization induced by KCl. The disturbance of the intracellular calcium homeostasis is one of the key mechanisms of action of excitotoxic compounds mediating delayed neuronal cell death by stimulation of glutamate receptor subtypes. In this study, CSF from ALS patients did not induce immediate rises in [Ca²⁺]_i or disturbances of the intracellular calcium homeostasis when measured over a period of 2 h.