Delta-opioid receptor-mediated modulation of excitability of individual hippocampal neurons: mechanisms involved.

BACKGROUND: Delta-opioid receptor (DOR)-mediated modulation of hippocampal neural networks is involved in emotions, cognition, and in pathophysiology and treatment of mood disorders. In this study, we examined the effects of DOR agonist (SNC80) and antagonist (naltrindole) on the excitability of individual hippocampal neurons. METHODS: Primary neuronal cultures were prepared from hippocampi of newborn rats and cultivated in vitro for 8-14 days (DIV8-14). The effects of SNC80 naltrindole on evoked and spontaneous action potentials (APs) were measured at DIV8-9 and DIV13-14, respectively. RESULTS: SNC80 (100 µM) potentiated spontaneous AP firing and stimulated sodium current; naltrindole had opposite effects. The stimulatory effect of 100 µM of SNC80 was revoked by pre-administration of 1 µM of naltrindole. SNC80 and naltrindole induced similar inhibitory effects on the evoked AP firing and on the calcium current. Further, SNC80 inhibited both peak and sustained potassium currents. Naltrindole had no effect on potassium currents. CONCLUSION: We suggest that the effects of naltrindole and high concentration of SNC80 on the sodium currents are mediated via DORs and underlying the changes in spontaneous activity. The inhibitory effects of SNC80 on calcium and potassium currents might also be DOR-dependent; these currents might mediate SNC80 effect on the evoked AP firing. The inhibitory effects of naltrindole on calcium and of low doses of SNC80 on sodium currents might be however DOR independent. The behavioral effects of SNC80 and naltrindole, observed in previous studies, might be mediated, at least in part, via the modulatory effect of these ligands on the excitability of hippocampal neurons.

Maternal immune activation in rats attenuates the excitability of monoamine-secreting neurons in adult offspring in a sex-specific way.

Higher risk of depression and schizophrenia in descendants of mothers experienced acute infection during the pregnancy has been reported. Since monoamines are fundamental in mentioned psychopathologies, it is possible that maternal immune activation leads to impaired functioning of serotonin (5-HT), noradrenaline, and dopamine neurons in offspring. To test this hypothesis, we examined the effect of maternal immune activation by lipopolysaccharide (LPS) in rats on the excitability of monoamine-secreting neurons in the offspring. LPS was administered during days 15-19 of the gestation in the rising doses of 20-80 µg/kg; control dams received vehicle. During days 53-63 postpartum, rats were anesthetized and electrodes were inserted into the dorsal raphe nucleus, locus coeruleus, and ventral tegmental area for in vivo excitability assessment of 5-HT, noradrenaline, and dopamine neurons. Maternal immune activation suppressed the firing rate of 5-HT neurons in both sexes and stimulated the firing rate of dopamine neurons in males. Decrease in the firing rate of 5-HT neurons was accompanied with an increase, and increase in the firing rate of dopamine neurons with a decrease, in the density of spontaneously active cells. Maternal immune activation also decreased the variability of interspike intervals in 5-HT and dopamine neurons. It is possible that the alteration of excitability of 5-HT and dopamine neurons by maternal immune activation is involved in the psychopathologies induced by infectious disease during the pregnancy. Stimulation of dopamine excitability in males might be a compensatory mechanism secondary to the maternal immune challenge-induced suppression of 5-HT neurons.

Pre-gestational stress impacts excitability of hippocampal cells in vitro and is associated with neurobehavioral alterations during adulthood.

Chronic stress during pregnancy or even prior to gestation can negatively affect offspring´s neurobehavioural development. Several studies have shown, that offspring who had experienced excessive stress during gestation had higher rates of cognitive and mood disorders later during adolescence or in adulthood. Hippocampal neurons play a crucial role in the regulation of behavior, mainly in anxiety-related behaviors and spatial learning and memory. Recently, it has been shown, that excessive stress even prior to gestation could interfere with sensitive developmental processes in the brain and may affect hippocampal functioning with severe neurobehavioural consequences in later life. The aim of this work was to investigate the effects of pre-gestational stress of the rat dams on the hippocampal excitability of the pups right after the birth. Neurobehavioural consequences of pre-gestational stress were analyzed during adolescence (35-40 postnatal days) and in early adulthood (75-80 postnatal days). We have shown that even pre-gestational chronic maternal stress increased resting membrane potential, suppressed depolarization-activated action potential firing, and increased spontaneous activity of hippocampal cells from newborn offspring. Altered function of hippocampus was reflected at the behavioural level. Adolescent male offspring of dams exposed stress prior to conception showed hyperactivity-like behaviour in a new stressful environment and increased anxiety-like behaviour during adulthood compared to adult males from non-stress group. Together, this work suggests, that chronic stress even prior to gestation can interfere with functional brain development of the offspring and can cause long-term behavioural changes at the level of neurobehavioural adaptations.

Grina/TMBIM3 modulates voltage-gated CaV2.2 Ca2+ channels in a G-protein-like manner.

Grina/TMBIM3 is a poorly characterized transmembrane protein with a broad expression pattern in mammals and with a very ancient origin within eukaryotes. Although initially characterized as an NMDA-receptor associated subunit, there is increasing evidence that Grina/TMBIM3 is involved in the unfolded protein response and controls apoptosis via regulation of Ca2+ homeostasis. Here, we investigate a putative direct interaction of Grina/TMBIM3 with voltage gated Ca2+ channels, in particular with the CaV2.2 α1-subunit and describe its modulatory effects on the current through CaV2.2 N-type channels. Direct interaction was confirmed by co-immunoprecipitation studies and membrane localization was proven. Co-expression of Grina/TMBIM3 with CaV2.2 channels resulted in a significant decrease of the current amplitude and in a slowing of the kinetics of current activation. This effect was accompanied by a significant shift of the voltage dependencies of activation time constants towards more depolarized voltages. Application of a stimulus protocol including a strong depolarizing pulse relieved inhibition of current amplitude by Grina/TMBIM3. When Grina/TMBIM3 was present, inactivation by an action potential-like train of pulses was diminished. Both observations resemble mechanisms that are well-studied modulatory effects of G-protein ßγ subunits on CaV2 channels. The impact of Grina/TMBIM3 and G-protein ßγ subunits are rather comparable with respect to suppression of current amplitude and slowing of activation kinetics. Furthermore, both modulators had the same effect on current inactivation when evoked by an action potential-like train of pulses.

Correction to: Four novel interaction partners demonstrate diverse modulatory effects on voltage-gated CaV2.2 Ca2+ channels.

The article was originally published with one author missing. The name of the co-author Roman Moravcik was inadvertently omitted. His name and affiliation have now been added to the author list. The original article has been corrected.

Four novel interaction partners demonstrate diverse modulatory effects on voltage-gated CaV2.2 Ca2+ channels.

Voltage-gated Ca2+ channels are embedded in a network of protein interactions that are fundamental for channel function and modulation. Different strategies such as high-resolution quantitative MS analyses and yeast-two hybrid screens have been used to uncover these Ca2+ channel nanodomains. We applied the yeast split-ubiquitin system with its specific advantages to search for interaction partners of the CaV2.2 Ca2+ channel and identified four proteins: reticulon 1 (RTN1), member 1 of solute carrier family 38 (SLC38), prostaglandin D2 synthase (PTGDS) and transmembrane protein 223 (TMEM223). Interactions were verified using the yeast split-ubiquitin system and narrowed down to CaV2.2 domain IV. Colocalization studies using fluorescent constructs demonstrated defined regions of subcellular localization. Detailed electrophysiological studies revealed that coexpression of RTN1 modulated CaV2.2 channels only to a minor extent. SLC38 accelerated the cumulative current inactivation during a high-frequency train of brief depolarizing pulses. As neurons expressing CaV2.2 channels were exposed to high-frequency bursts under physiological conditions, observed regulation may have a negative modulatory effect on transmitter release. Coexpression of PTGDS significantly lowered the average current density and slowed the kinetics of cumulative current inactivation. Since the latter effect was not significant, it may only partly compensate the first one under physiological conditions. Expression of TMEM223 lowered the average current density, accelerated the kinetics of cumulative current inactivation and slowed the kinetics of recovery from inactivation. Therefore, TMEM223 and, to a lesser extent, PTGDS, may negatively modulate Ca2+ entry required for transmitter release and/or for dendritic plasticity under physiological conditions.

Role of individual S4 segments in gating of Cav3.1 T-type calcium channel by voltage.

Contributions of voltage sensing S4 segments in domains I - IV of CaV3.1 channel to channel activation were analyzed. Neutralization of the uppermost charge in individual S4 segments by exchange of arginine for cysteine was employed. Mutant channels with single exchange in domains I - IV, in two adjacent domains, and in all four domains were constructed and expressed in HEK 293 cells. Changes in maximal gating charge Qmax and the relation between Qmax and maximal conductance Gmax were evaluated. Qmax was the most affected by single mutation in domain I and by double mutations in domains I + II and I + IV. The ratio Gmax/Qmax proportional to opening probability of the channel was significantly decreased by the mutation in domain III and increased by mutations in domains I and II. In channels containing double mutations Gmax/Qmax ratio increased significantly when the mutation in domain I was included. Mutations in domains II and III zeroed each other. Mutation in domain IV prevented the decrease caused by the mutation in domain III. Neither ion current nor gating current was observed when channels with quadruple mutations were expressed. Immunocytochemistry analysis did not reveal the presence of channel protein in the cell membrane. Likely, quadruple mutation results in a structural change that affects the channel's trafficking mechanism. Altogether, S4 segments in domains I-IV of the CaV3.1 channel unequally contribute to channel gating by voltage. We suggest the most important role of the voltage sensor in the domain I and lesser roles of voltage sensors in domains II and III.

SNC80 and naltrindole modulate voltage-dependent sodium, potassium and calcium channels via a putatively delta opioid receptor-independent mechanism.

SNC80 was designed as a highly selective nonpeptide delta opioid receptor (DOR) agonist. Antidepressant-like and antinociceptive effects of this compound were demonstrated in animal models. Naltrindole was synthetized as a highly selective DOR antagonist. Its antitussive and antinociceptive effects were reported. Observed effects of SNC80 and naltrindole may be accompanied by changes in neuronal excitability including modulation of voltage-dependent ion channels. We investigated possible DOR-independent modulation of neuronal sodium, calcium and potassium currents by both agents. NG108-15 cells lacking expression of DOR protein were used as model of neuronal cells. Cells were differentiated into neuronal phenotype by exposure to dibutyryl cyclic-AMP (dbcAMP). Lack of DORs expression in NG108-15 cells and the presence of DOR expression in brain and neuronal cultures were demonstrated by Western blot analysis. Both SNC80 and naltrindole exerted low to moderate modulatory effects on voltage-dependent ion currents. SNC80 weakly inhibited sodium current, potentiated calcium current, and did not act on potassium channels. Naltrindole inhibited sodium current, did not act on calcium current and inhibited potassium current at a high concentration. Such effects should be taken into account when these compounds are used for investigation of DOR-mediated signaling pathways.

Fibrotic scar model and TGF-ß1 differently modulate action potential firing and voltage-dependent ion currents in hippocampal neurons in primary culture.

Traumatic injury of the central nervous system is accompanied by various functional and morphological changes. Animal models of traumatic brain injury are commonly used to investigate changes in behaviour, morphology, in the expression of various proteins around the site of the injury, or the expression of diagnostically important biomarkers. Excitability of a single neuron at, or close to, the site of injury was rarely investigated. Several in vitro models were developed which allow such investigation. In the present work, we employed a fibrotic scar model according to Kimura-Kuroda and coauthors to analyse altered excitability of rat hippocampal neurons under the conditions mimicking traumatic brain injury. Hippocampal neurons from newborn rats were cultured either on a fibrotic scar model or in the presence of TGF-ß1, a cytokine secreted at a brain injury site that may have both neuroprotective and neurodegenerative function. Fibrotic scar facilitated ability of neonatal hippocampal neurons to fire action potential series by increasing the density of voltage activated sodium and potassium currents. Chondroitin sulphate proteoglycans played substantial role in these effects, as proven by their full reversion after administration of Chondroitinase ABC. In contrast, TGF-ß1 did not contribute to them. An application of TGF-ß1 itself attenuated generation of action potentials, inhibited sodium current and potentiated potassium currents. Main alteration of electrophysiological parameters of neonatal hippocampal neurons caused by a fibrotic scar model is enhanced excitability. TGF-ß1 may have predominantly neuroprotective role in injured rat hippocampus.

Effect of Physical Exercise and Acute Escitalopram on the Excitability of Brain Monoamine Neurons: In Vivo Electrophysiological Study in Rats.

Background: The antidepressant effect of physical exercise has been reported in several clinical and animal studies. Since serotonin, norepinephrine, and dopamine play a central role in depression, it is possible that the beneficial effects of physical exercise are mediated via monoamine pathways. This study investigates the effects of voluntary wheel running on the excitability of monoamine neurons. Materials and Methods: Male Sprague-Dawley rats were used in the study. Voluntary wheel running (VWR) rats were housed in individual cages with free access to a running wheel, while control animals were housed in standard laboratory cages. After three weeks, the rats were anesthetized, and in vivo electrophysiological recordings were taken from dorsal raphe nucleus serotonin neurons, locus coeruleus norepinephrine neurons, and ventral tegmental dopamine neurons. Results: VWR stimulated activity in serotonin, but not in norepinephrine or dopamine neurons. Subsequently, acute administration of the selective serotonin reuptake inhibitor escitalopram in control rats led to complete suppression of serotonin neurons; this suppression was reversed by subsequent administration of selective antagonist of serotonin-1A receptors, WAY100135. Escitalopram induced only partial inhibition of serotonin neurons in the VWR rats while WAY100135 increased the firing activity of serotonin neurons above the baseline value. Conclusions: The beneficial effect of physical exercise on mood is mediated, at least in part, via activation of serotonin neurons. Physical exercise can potentiate the response to selective serotonin reuptake inhibitors by increasing the basal firing activity and diminishing selective serotonin reuptake inhibitor-induced inhibition of serotonin neurons.