Probes for CRE

The ventrolateral periaqueductal gray (vlPAG) constitutes a major descending pain modulatory system and is a crucial site for opioid-induced analgesia. A number of previous studies have demonstrated that glutamate and GABA play critical opposing roles in nociceptive processing in the vlPAG. It has been suggested that glutamatergic neurotransmission exerts antinociceptive effects, whereas GABAergic neurotransmission exert pro-nociceptive effects on pain transmission, through descending pathways. The inability to exclusively manipulate subpopulations of neurons in the PAG has prevented direct testing of this hypothesis. Here we demonstrate the different contributions of genetically-defined glutamatergic and GABAergic vlPAG neurons in nociceptive processing by employing cell type-specific chemogenetic approaches in mice. Global chemogenetic manipulation of vlPAG neuronal activity suggests that vlPAG neural circuits exert tonic suppression of nociception, consistent with previous pharmacological and electrophysiological studies. However, selective modulation of GABAergic or glutamatergic neurons demonstrates an inverse regulation of nociceptive behaviors by these cell populations. Selective chemogenetic activation of glutamatergic neurons, or inhibition of GABAergic neurons, in vlPAG suppresses nociception. In contrast, inhibition of glutamatergic neurons, or activation of GABAergic neurons, in vlPAG facilitates nociception. Our findings provide direct experimental support for a model in which excitatory and inhibitory neurons in the PAG bidirectionally modulate nociception.

Significance Statement The PAG is a midbrain region critical for the modulation of pain. However, the roles played by the distinct cell types within the PAG in nociceptive processing are poorly understood. This work addresses the divergent roles of glutamatergic and GABAergic PAG neuronal subpopulations in nociceptive processing. We demonstrate that activation of glutamatergic neurons or inhibition of GABAergic neurons suppresses nociception. Whereas inhibition of glutamatergic neuronal activity or activation of GABAergic neuronal activity potentiates nociception. This report identifies distinct roles for these neuronal populations in modulating nociceptive processing.

Abstract

BACKGROUND:

Transforming growth factor beta (TGFB) superfamily signaling is implicated in the development of sex cord-stromal tumors, a category of poorly defined gonadal tumors. The aim of this study was to determine potential effects of dysregulated TGFB signaling in the ovary using Cre recombinase driven by growth differentiation factor 9 (Gdf9) promoter known to be expressed in oocytes.

METHODS:

A mouse model containing constitutively active TGFBR1 (TGFBR1CA) using Gdf9-iCre (termed TGFBR1-CAG9Cre) was generated. Hematoxylin and eosin (H & E) staining, follicle counting, and immunohistochemistry and immunofluorescence analyses using antibodies directed to Ki67, forkhead box L2 (FOXL2), forkhead box O1 (FOXO1), inhibin alpha (INHA), and SRY (sex determining region Y)-box 9 were performed to determine the characteristics of the TGFBR1-CAG9Cre ovary. Terminal deoxynucleotidyl transferase (TdT) labeling of 3'-OH ends of DNA fragments, real-time PCR, and western blotting were used to examine apoptosis, select gene expression, and TGFBR1 activation. RNAscope in situ hybridization was used to localize the expression of GLI-Kruppel family member GLI1 (Gli1) in ovarian tumortissues.

RESULTS:

TGFBR1-CAG9Cre females were sterile. Sustained activation of TGFBR1 led to altered granulosa cell proliferation evidenced by high expression of Ki67. At an early age, these mice demonstrated follicular defects and development of ovarian granulosa cell tumors, which were immunoreactive for granulosa cell markers including FOXL2, FOXO1, and INHA. Further histochemical and molecular analyses provided evidence of overactivation of TGFBR1 in the granulosa cell compartment during ovarian pathogenesis in TGFBR1-CAG9Cre mice, along with upregulation of Gli1 and Gli2 and downregulation of Tgfbr3 in ovarian tumor tissues.

CONCLUSIONS:

These results reinforce the role of constitutively active TGFBR1 in promoting ovarian tumorigenesis in mice. The mouse model created in this study may be further exploited to define the cellular and molecular mechanisms of TGFB/activin downstream signaling in granulosa cell tumor development. Future studies are needed to test whether activation of TGFB/activin signaling contributes to the development of human granulosa cell tumors.