Gabrb3 endothelial cell-specific knockout mice display abnormal blood flow, hypertension, and behavioral dysfunction

Abstract

Our recent studies uncovered a novel GABA signaling pathway in embryonic forebrain endothelial cells that works independently from neuronal GABA signaling and revealed that disruptions in endothelial GABAA receptor-GABA signaling from early embryonic stages can directly contribute to the origin of psychiatric disorders. In the GABAA receptor β3 subunit endothelial cell conditional knockout (Gabrb3ECKO) mice, the β3 subunit is deleted selectively from endothelial cells, therefore endothelial GABAA receptors become inactivated and dysfunctional. There is a reduction in vessel densities and increased vessel morphology in the Gabrb3ECKO telencephalon that persists in the adult neocortex. Gabrb3ECKO mice show behavioral deficits such as impaired reciprocal social interactions, communication deficits, heightened anxiety, and depression. Here, we characterize the functional changes in Gabrb3ECKO mice by evaluating cortical blood flow, examine the consequences of loss of endothelial Gabrb3 on cardiac tissue, and define more in-depth altered behaviors. Red blood cell velocity and blood flow were increased in the cortical microcirculation of the Gabrb3ECKO mice. The Gabrb3ECKO mice had a reduction in vessel densities in the heart, similar to the brain; exhibited wavy, myocardial fibers, with elongated ‘worm-like’ nuclei in their cardiac histology, and developed hypertension. Additional alterations in behavioral function were observed in the Gabrb3ECKO mice such as increased spontaneous exploratory activity and rearing in an open field, reduced short term memory, decreased ambulatory activity in CLAMS testing, and altered prepulse inhibition to startle, an important biomarker of psychiatric diseases such as schizophrenia. Our results imply that vascular Gabrb3 is a key player in the brain as well as the heart, and its loss in both organs can lead to concurrent development of psychiatric and cardiac dysfunction.

GABAA and GABAB Receptors Mediate GABA-Induced Intracellular Ca2+ Signals in Human Brain Microvascular Endothelial Cells

“…The evidence, which is further illustrated below, that the endothelial Ca 2+ response to GABA is triggered by both GABA A and GABA B receptors could explain the peculiar profile of the dose–response relationship obtained in hCMEC/D3 cells. These preliminary observations confirmed that intracellular Ca 2+ signaling could be instrumental for GABA to control endothelial cell functions [ 17 , 20 ]. A novel GABA biosensor based upon a dual-enzyme immobilization approach recently showed that, during neuronal activity, GABA concentration may transiently raise up to ≈80 µM in the cortex [ 47 ].…”

“…Based upon their expression levels, GABA A receptors in hCMEC/D3 cells are predicted to incorporate the α1, β1, and γ1 subunits. Surprisingly, human cerebrovascular endothelial cells do not present the GABA A receptor β3 subunit, which is a crucial component of mouse GABA A receptors [ 17 , 20 , 21 ]. The expression of the GABA B receptor has hitherto been suggested only by an early study reporting on baclofen-induced nitric oxide (NO) release and collagen constriction in mouse cortical microvascular endothelial cells [ 19 ].…”

“…GABA is emerging as a crucial mediator of neuro-to-vascular communication at the NVU [ 68 ]; mouse cerebrovascular endothelial cells express ionotropic GABA A receptors that perceive GABA released during neuronal activity from inhibitory interneurons and trigger a signaling pathway that finely controls cerebral angiogenesis [ 17 ] and CBF [ 20 ]. It has been suggested that GABA activates GABA A receptors to evoke intracellular Ca 2+ signals [ 17 , 20 ], which could, in turn, drive endothelial cell proliferation and angiogenesis [ 25 , 26 , 69 ] and recruit endothelial nitric oxide synthase (eNOS) to produce NO, i.e., the most important vaso-relaxing mediator in the brain [ 27 , 28 , 31 , 68 ]. Nevertheless, GABA A receptors are ionotropic receptors that are permeable to Cl − and, therefore, are not expected to directly raise the [Ca 2+ ] i during GABAergic signaling [ 6 ].…”

“…Although the metabotropic GABA B receptors are known to induce intracellular Ca 2+ signals both within [ 9 , 10 , 11 ] and outside [ 24 , 64 , 71 , 72 ] the NVU, the increase in the [Ca 2+ ] i whereby GABA regulates multiple endothelium-dependent functions in brain microvessels is mediated by the ionotropic GABA A receptors [ 17 , 20 ]. GABA A receptor activation by the selective agonist muscimol elicits an inward Cl − current in mouse cerebrovascular endothelial cells, thereby elevating the [Ca 2+ ] i [ 17 ].…”

“…Mouse brain microvascular endothelial cells are endowed with the α1, α2, α6, β1, β2, β3, γ1, γ2 and γ3 subunits of GABA A receptors [ 18 ], while only functional evidence has been documented in favor of GABA B receptor expression [ 19 ]. The role played by endothelial GABA A receptors at the NVU has gathered growing interest upon the discovery that they control cerebral angiogenesis [ 17 ] and guide the radial migration of GABA interneurons [ 16 ] during embryonic development in mice, and regulate cerebral blood flow (CBF) and prevent neurological deficits in the adult [ 20 ]. The signaling pathways that are activated downstream of GABA A receptors in cerebrovascular endothelial cells are yet to be fully unraveled [ 21 ].…”