Phenotyping of sigma-1 receptor knock-out rodents

Abstract

[eng] The sigma-1 receptor is a chaperone that is primarily expressed in the mitochondria-associated endoplasmic reticulum (MAM). It was cloned years ago from different tissues of various species and its structure has been recently elucidated.In 2003, our laboratory generated the first knock-out (KO) mouse for the sigma-1 receptor. These mice served to demonstrate the involvement of the sigma-1 receptor in acute and chronic pain. In addition to pain, the sigma-1 receptor has been implicated in other physiological processes and pathological conditions including depression and addiction. The sigma-1 receptor has also been shown to be associated with the regulation of other proteins, including dopamine (DAT) transporter. In this work, the generation of a sigma-1 receptor KO rat model is reported. The behavioural response of sigma-1 KO mice in two models of depression has been characterized, and the physiological and behavioural phenotyping of sigma-1 KO rats has been carried out, with special attention to the possible effects of the absence of the receptor in models of depression and addiction. By means of CRISPR / Cas9 technology, two KO strains for the sigma-1 receptor with deletions of 218bp and 7bp were obtained. Wild-type (WT) and the two KO strains showed no significant differences in the Irwin test, spontaneous locomotor activity, open field test, startle response, or pre-pulse inhibition. In contrast, the results obtained in response to mechanical or thermal stimulation led us to select the strain with a deletion of 218bp. Using this strain, WT (+/+), KO heterozygous (+/-) and KO homozygous (-/-) rats were phenotyped. No significant differences were found in growth or survival curves, nor in most of the assessed physiological or behavioural parameters. Regarding depression, no significant difference was found in the acute study after administration of fluoxetine (an antidepressant with sigma-1 affinity) or venlafaxine (without sigma-1 affinity) on the day of the test. This was an expected outcome, based on the literature and previous findings regarding the ineffectiveness of antidepressants in an acute treatment. In the sub-acute study, the tendency for lower immobility during training observed in mice was confirmed. On test days 1 and 7, neither fluvoxamine nor venlafaxine showed any efficacy in reducing immobility. In contrast, at day 14, the two antidepressants significantly reduced immobility only in KO rats. Given that there is no sigma-1 receptor in these rats and that both antidepressants showed activity, regardless of whether they had sigma-1 affinity, it seems that the efficacy may be due to some change in their action on SERT.Regarding locomotor activity, as a surrogate measure of addictive potential, the set of results seems to indicate a greater role of the sigma-1 receptor in the rearing activity, especially as stereotyped behaviour, rather than its merely exploratory activity, being the difference between the two genotypes mainly at high doses. There have been fewer differences in locomotor activity and, surprisingly, no differences after cocaine administration, that has a sigma-1 affinity. Differences between mobility and rearing can be assigned to the dopaminergic pathways involved, meso-limbic for mobility, and nigro-striatal for stereotypes.In conclusion, in rats, deletion of the gene encoding the sigma-1 receptor generates a viable phenotype very similar to the WT strain under normal conditions. Behavioural response under conditions of environmental stimulation and / or pharmacological treatment, reveals some differences between WT and KO, in both mice and rats. The absence of the receptor seems to lead to the adaptation of other proteins. The results further support the concept that the sigma-1 receptor may be involved in the development of depression and addiction and drugs acting on sigma-1 receptors could be useful in such pathologies.

[cat] The sigma-1 receptor is a chaperone that is primarily expressed in the mitochondria-associated endoplasmic reticulum (MAM). It was cloned years ago from different tissues of various species and its structure has been recently elucidated.In 2003, our laboratory generated the first knock-out (KO) mouse for the sigma-1 receptor. These mice served to demonstrate the involvement of the sigma-1 receptor in acute and chronic pain. In addition to pain, the sigma-1 receptor has been implicated in other physiological processes and pathological conditions including depression and addiction. The sigma-1 receptor has also been shown to be associated with the regulation of other proteins, including dopamine (DAT) transporter. In this work, the generation of a sigma-1 receptor KO rat model is reported. The behavioural response of sigma-1 KO mice in two models of depression has been characterized, and the physiological and behavioural phenotyping of sigma-1 KO rats has been carried out, with special attention to the possible effects of the absence of the receptor in models of depression and addiction. By means of CRISPR / Cas9 technology, two KO strains for the sigma-1 receptor with deletions of 218bp and 7bp were obtained. Wild-type (WT) and the two KO strains showed no significant differences in the Irwin test, spontaneous locomotor activity, open field test, startle response, or pre-pulse inhibition. In contrast, the results obtained in response to mechanical or thermal stimulation led us to select the strain with a deletion of 218bp. Using this strain, WT (+/+), KO heterozygous (+/-) and KO homozygous (-/-) rats were phenotyped. No significant differences were found in growth or survival curves, nor in most of the assessed physiological or behavioural parameters. Regarding depression, no significant difference was found in the acute study after administration of fluoxetine (an antidepressant with sigma-1 affinity) or venlafaxine (without sigma-1 affinity) on the day of the test. This was an expected outcome, based on the literature and previous findings regarding the ineffectiveness of antidepressants in an acute treatment. In the sub-acute study, the tendency for lower immobility during training observed in mice was confirmed. On test days 1 and 7, neither fluvoxamine nor venlafaxine showed any efficacy in reducing immobility. In contrast, at day 14, the two antidepressants significantly reduced immobility only in KO rats. Given that there is no sigma-1 receptor in these rats and that both antidepressants showed activity, regardless of whether they had sigma-1 affinity, it seems that the efficacy may be due to some change in their action on SERT.Regarding locomotor activity, as a surrogate measure of addictive potential, the set of results seems to indicate a greater role of the sigma-1 receptor in the rearing activity, especially as stereotyped behaviour, rather than its merely exploratory activity, being the difference between the two genotypes mainly at high doses. There have been fewer differences in locomotor activity and, surprisingly, no differences after cocaine administration, that has a sigma-1 affinity. Differences between mobility and rearing can be assigned to the dopaminergic pathways involved, meso-limbic for mobility, and nigro-striatal for stereotypes.In conclusion, in rats, deletion of the gene encoding the sigma-1 receptor generates a viable phenotype very similar to the WT strain under normal conditions. Behavioural response under conditions of environmental stimulation and / or pharmacological treatment, reveals some differences between WT and KO, in both mice and rats. The absence of the receptor seems to lead to the adaptation of other proteins. The results further support the concept that the sigma-1 receptor may be involved in the development of depression and addiction and drugs acting on sigma-1 receptors could be useful in such pathologies.