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Tricyclic antidepressants, specifically amitriptyline (ATL) and desipramine (DES) are currently used to treat depression and chronic pain of various origins, in which NMDA receptor dysfunctions play an important role. The effect of therapeutic ATL concentrations on calcium-dependent desensitization of NMDA receptors, driven by the level of free calcium in the cytoplasm, is well documented. In addition, in cardiomyocytes, ATL and DES can cause calcium release into the cytoplasm from intracellular stores by opening inositol-3-phosphate receptor (IP3R) and/or ryanodine receptors (RyR) channels. This aspect of ATL and DES effects on neurons remains poorly understood. We studied the dependence of calcium responses to DES and ATL on the activation of IP3R and RyR in the endoplasmic reticulum (ER) and mitochondria of rat neocortical neurons in primary culture. Short-term (30 s) paired (at a 5-min interval) applications of 200 µM DES or 200 µM ATL elicited similar-magnitude calcium responses in cortical neurons. The use of RyR and IP3R antagonists showed that responses to ATL are blocked by the IP3R antagonist 2-APB (100 µM), while responses to DES are blocked by the RyR antagonist ryanodine (100 nM). Since the intracellular distribution of RyR and IP3R is non-homogenous, it can be assumed that DES and ATL stimulate calcium release from different calcium stores located either in different segments of the ER or in the ER and mitochondria. In addition, ATL and DES, being channel blockers of NMDA receptors, inhibited calcium entry from the extracellular space via activated NMDA receptors. Considering high DES and ATL concentrations (>100 µM) required for the stimulation of calcium release in neurons, it seems unlikely that such effects appear during their therapeutic action. However, the revealed specificity of DES and ATL for RyR and IP3R, respectively, can be used as a tool for experimental purposes. [ABSTRACT FROM AUTHOR]

Abstract: The analysis of neuronal viability of cerebellar neurons in primary culture during long-lasting (240 min) treatment with glutamate revealed that forskolin (1 μM), an adenylate cyclase activator, prevents apoptosis and necrosis of cells, thus exhibiting neuroprotective properties. As the cytotoxic action of glutamate causes an increase of the intracellular Ca2+ concentration, we further studied forskolin influence on glutamate-evoked Ca2+ responses of neurons. A short-time pretreatment with 1 μM forskolin significantly decreases Ca2+ entry into neurons during glutamate action. The obtained results demonstrate an important role of adenylate cyclase and cAMP in regulation of the Ca2+ entry into neurons and intracellular signaling pathways preventing neuronal death of cerebellar neurons in excytotoxic stress. [ABSTRACT FROM AUTHOR]

Homocysteine, a sulfur-containing amino acid, exhibits neurotoxic effects and is involved in the pathogenesis of several major neurodegenerative disorders. In contrast to well studied excitoxicity of glutamate, the mechanism of homocysteine neurotoxicity is not clearly understood. By using whole-cell patch-clamp, calcium imaging (fluo-3) and measurements of mitochondrial membrane potential (rhodamine 123) we studied transmembrane currents, calcium signals and changes in mitochondrial membrane potential induced by homocysteine versus responses induced by NMDA and glutamate in cultured rat cortical neurons. L-homocysteine (50 µM) induced inward currents that could be completely blocked by the selective antagonist of NMDA receptors - AP-5. In contrast to NMDA-induced currents, homocysteine-induced currents had a smaller steady-state amplitude. Comparison of calcium responses to homocysteine, NMDA or glutamate demonstrated that in all cortical neurons homocysteine elicited short, oscillatory-type calcium responses, whereas NMDA or glutamate induced sustained increase of intracellular calcium. Analysis of mitochondrial changes demonstrated that in contrast to NMDA homocysteine did not cause a drop of mitochondrial membrane potential at the early stages of action. However, after its long-term action, as in the case of NMDA and glutamate, the changes in mitochondrial membrane potential were comparable with the full drop of respiratory chain induced by protonophore FCCP. Our data suggest that in cultured rat cortical neuron homocysteine at the first stages of action induces neurotoxic effects through activation of NMDA-type ionotropic glutamate receptors with strong calcium influx through the channels of these receptors. The long-term action of homocysteine may lead to mitochondrial disfuction and appears as a drop of mitochondrial membrane potential.

The effect of CyPPA, a positive modulator of small conductance calcium-activated potassium channels of type 3 and 2 (SK3/SK2), and of NS309, an activator of intermediate and small conductance calcium-activated potassium channels (IK/SK), on the activity of cerebellar Purkinje cells was studied in 2-month-old male mice. The use of 1 mM of CyPPA has led to a decrease of simple spike firing frequency in the discharge of Purkinje cells by 25%, on average, during 1 h after application. At the same time, application of 100 μM of NS309 has promoted a decrease in simple spike firing frequency by 47 %, on average, during 1 h after the beginning of the action. The obtained results confirm the hypothesis that SK channels participate in regulation of simple spike firing frequency in the discharge of Purkinje cells and are responsible for restriction of signal frequency. The effect of NS309 on simple spike firing frequency was more pronounced; therefore, the IK/SK channels may be suggested to play the cardinal role in regulation of spike activity of Purkinje cells. Since increasing simple spike frequency in the discharge of Purkinje cells is observed at many disturbances of motor activity, in particular, at spinocerebellar ataxia, it can be suggested that the studied compounds or substances of similar action are of interest as potential medicinal agents.

The generation features of postsynaptic currents were studied in primary culture of cortical neurons at 7-20 days in vitro (DIV). The use of specific blockers of postsynaptic ion channels after 10 DIV revealed all types of electrical activity found in adult cortex including miniature inhibitory (mIPSCs), excitatory (mEPSCs) and spontaneous giant excitatory currents and spikes. The frequency of mEPSCs increased exponentially from 7 to 20 DIV doubling every 2.2 days in parallel with changes in action potentials generation. The mEPSCs generated by NMDA and AMPA or by only AMPA receptor activation were found. The inhibition of NMDA receptors by magnesium ions or AP5 were shown to modulate the frequency and amplitude of mEPSCs, which differ primary culture from brain slices possibly because of the lack of glial control of synaptic transmission.

A contribution of necrosis and apoptotis as well as the particular apoptosis pathways in neuro-degeneration induced by glutamate and selective glutamate receptor agonists, NMDA and kainate, were studied. In experiments on primary neuron cultures of 7 days in vitro from embryonic rat cortex, the necrosis and apoptosis were recognized using vital fluorescence acridine orange and ethidium bromide staining. Immunostaining was used to visualize apoptotic peptides such as P53, Cas-3 and AIF. Death of neurons occurred by both necrosis and apoptosis following 240 min 3 mM glutamate, 30 microM NMDA and 30 microM kainate exposure. Quantities of necrotic neurons in the presence of NMDA and kainate were substantially reduced when compared to the glutamate action. The glutamate effects were realized through predominant activation of AMPA- and kainate receptors, since it could be greatly suppressed by 30 microM CNQX. AIF but not Cas-3, was found in a large amount of neurons when apoptosis was evoked by the selective NMDA receptor activation. On the contrary, during apoptosis induced by glutamate and kainate, many cells contained Cas-3 in nuclei rather than the AIF. The data suggest that apoptosis induced by the NMDA receptor activation develops through the caspase-3-independent pathway that involves direct AIF accumulation in nuclei. The AMPA/kainate receptor mediated apoptosis includes the caspase-3-dependent mechanism.