Galanin receptor subtypes in rodent models of mood disorders

Major depressive disorder (MDD) is a serious mental illness, with a complex etiology. It is a multifactorial disorder caused by a combination of genetic, environmental and epigenetic factors. Exposure to stressful life events seems to be a common, contributing cause of morbidity. Discoveries during the last 60 years have provided increasing evidence for a dysfunctional serotonergic and/or noradrenergic neurotransmission in MDD. However, other transmitter systems also appear to be involved, e.g. GABAergic, glutamatergic and cholinergic systems, as well as several neuropeptide systems. Since its discovery in the 1980s at Karolinska Institutet, the neuropeptide galanin has been implicated in a diversity of physiological processes including not only feeding and nociception, but also anxiety and stress. The main aim of this thesis was to investigate the role of galanin and galanin receptors in rodent models of mood disorders, and to evaluate the possible interaction between exposure to stress and galanin receptor subtype stimulation by measuring the expression of neuronal chemical markers of relevance for depression in serotonin/5-hydroxytryptamine (5-HT) and noradrenaline (NA) neurons. Several behavioral tests were employed including the forced swim test. The validity of this test was examined by the use of the 5-HT selective reuptake inhibitor fluoxetine, a widely used antidepressant. Galanin, given intracerebroventricularly (i.c.v.) to rats, increased immobility time in the forced swim test indicative of depression-like behavior. Co-infusion of the unspecific galanin receptor antagonist M35 blocked the effect of galanin. Importantly, M35 alone decreased immobility time. The galanin receptor 1 (GalR1) agonist M617 and the galanin receptor 2 (GalR2) antagonist M871 both increased immobility time in the FST, whereas, the GalR2 agonist M1896 caused a decrease. In situ hybridization studies performed after infusion of galanin (i.c.v.) and exposure to swim stress showed elevated levels of galanin mRNA in the locus coeruleus versus control animals. However, a significant increase of galanin mRNA levels was also observed in the locus coeruleus in saline- and fluoxetine-treated animals indicating that the stress of injection is adding to the stress of swimming. In contrast, in the dorsal raphe nucleus, the swim/injection procedure did not change the mRNA levels of galanin or tryptophan hydroxylase, indicating that 5-HT neurons are not as sensitive as the NA neurons in locus coeruleus to stress. The levels of 5-HT1A receptor mRNA were reduced in the dorsal raphe nucleus following galanin or the GalR2 agonist M1896 infusion. These results support an involvement of brain galanin systems in depression-like behavior in rodents, and galanin receptor subtypes appear to play differential roles in modulation of depression-like behavior. The action of galanin and galanin receptor ligands seems to modulate depression-like behavior in rodents partly via changes in expression of certain monoaminergic molecules relevant for depression. To further analyze the functional role of GalR2 in rodent models of depression, mice overexpressing this receptor (GalR2OE mice) under the platelet-derived growth factor B promoter were generated. These mice displayed decreased immobility time in the FST compared to the wild-type controls indicative of antidepressant-like behavior. However, anxiety-like behavior, emotional memory and locomotor activity measures did not differ between the genotypes. Overexpression of the GalR2 transcripts and receptor protein was detected in limbic areas such as subregions of the medial prefrontal cortex and subiculum. Thus, GalR2 in these areas may contribute to the depression-like behavior observed in these mice. The present thesis gives evidence for a role of the neuropeptide galanin and its receptors in rodent models of depression-like behavior and suggests that GalR1 antagonists, and GalR2 agonists may represent new candidates for the development of drugs for treatment of mood disorders

Electrostatic Domino Effect in the Shaker K Channel Turret

Voltage-gated K channels are regulated by extracellular divalent cations such as Mg2+ and Sr2+, either by screening of fixed negative surface charges, by binding directly or close to the voltage sensor, or by binding to the pore. Different K channels display different sensitivity to divalent cations. For instance, 20 mM MgCl2 shifts the conductance versus voltage curve, G(V), of the Kv1-type Shaker channel with 14 mV, while the G(V) of Kv2.1 is shifted only with 7 mV. This shift difference is paralleled with different working ranges. Kv1-type channels open at ∼−20 mV and Kv2.1 channel open at ∼+5 mV. The aim of this study was to identify critical residues for this Mg2+-induced G(V) shift by introducing Kv2.1 channel residues in the Shaker K channel. The K channels were expressed in Xenopus laevis oocytes and studied with the two-electrode voltage-clamp technique. We found that three neutral-to-positive amino-acid residue exchanges in the extracellular loops connecting transmembrane segments S5 and S6 transferred the Mg2+-shifting properties. The contributions of the three residues were additive, and thus independent of each other, with the contributions in the order 425 > 419 > 451. Charging 425 and 419 not only affect the Mg2+-induced G(V) shift with 5–6 mV, but also shifts the G(V) with 17 mV. Thus, a few strategically placed surface charges clearly modulate the channel's working range. Residue 425, located at some distance away from the voltage sensor, was shown to electrostatically affect residue K427, which in turn affects the voltage sensor S4—thus, an electrostatic domino effect