The ventrolateral periaqueductal gray (vlPAG) is a key structure in the descending pain modulatory circuit. Activation of the circuit occurs via disinhibition of GABAergic inputs onto vlPAG output neurons. In these studies, we tested the hypothesis that GABAergic inhibition is increased during persistent inflammation, dampening activation of the descending circuit from the vlPAG. Our results indicate that persistent inflammation induced by Complete Freund's adjuvant (CFA) modulates GABA signaling differently in male and female rats. CFA treatment results in increased presynaptic GABA release but decreased high-affinity tonic GABA A currents in female vlPAG neurons. These effects are not observed in males. The tonic currents in the vlPAG are dependent on GABA transporter activity and are modulated by agonists that activate GABA A receptors containing the ␦ subunit. The GABA A ␦ agonist THIP (gaboxadol) induced similar amplitude currents in naive and CFA-treated rats. In addition, a positive allosteric modulator of the GABA A ␦ subunit, DS2 (4-chloro-N-[2-(2-thienyl)imidazo[1,2-a]pyridin-3-yl]benzamide), increased tonic currents. These results indicate that GABA A ␦ receptors remain on the cell surface but are less active in CFA-treated female rats. In vivo behavior studies showed that morphine induced greater antinociception in CFA-treated females that was reversed with microinjections of DS2 directly into the vlPAG. DS2 did not affect morphine antinociception in naive or CFA-treated male rats. Together, these data indicate that sex-specific adaptations in GABA A receptor signaling modulate opioid analgesia in persistent inflammation. Antagonists of GABA A ␦ receptors may be a viable strategy for reducing pain associated with persistent inflammation, particularly in females.

Abstractα2δ proteins (Cacna2d1-4) are auxiliary subunits of voltage-dependent calcium channels that also drive synapse formation and maturation. Because cerebellar Purkinje cells (PCs) only express one isoform of this family, α2δ-2 (Cacna2d2), we used PCs as a model system to examine roles of α2δ in excitatory synaptic function in a Cacna2d2 knockout mouse. Whole-cell recordings of PCs from acute cerebellar slices revealed altered climbing fiber (CF)-evoked complex spike generation, as well as increased amplitude and faster decay of CF-evoked excitatory postsynaptic currents (EPSCs). CF terminals in the KO were localized more proximally on PC dendrites, as indicated by VGLUT2+ immunoreactive puncta, and computational modeling demonstrated that the increased EPSC amplitude can be partly attributed to the more proximal location of CF terminals. In addition, CFs in KO mice exhibited increased multivesicular transmission, corresponding to greater sustained responses during repetitive stimulation, despite a reduction in the measured probability of release. Electron microscopy demonstrated that mutant CF terminals had twice as many vesicle release sites, providing a morphologic explanation for the enhanced glutamate release. Though KO CFs evoked larger amplitude EPSCs, the charge transfer was the same as wildtype as a result of increased glutamate re-uptake, producing faster decay kinetics. Together, the larger, faster EPSCs in the KO explain the altered complex spike responses, which degrade information transfer from PCs and likely contribute to ataxia in Cacna2d2 KO mice. Our results also illustrate the multidimensional synaptic roles of α2δ proteins.Significance Statementα2δ proteins (Cacna2d1-4) regulate synaptic transmission and synaptogenesis, but co-expression of multiple α2δ isoforms has obscured a clear understanding of how various α2δ proteins control synaptic function. We focused on roles of the α2δ-2 protein (Cacna2d2), whose deletion causes cerebellar ataxia and epilepsy in mice and humans. Because cerebellar Purkinje cells only expresses this single isoform, we studied excitatory climbing fiber synaptic function onto Purkinje cells in Cacna2d2 knockout mice. Using optical and electrophysiological analysis, we provide a detailed description of the changes in Purkinje cells lacking α2δ-2, and provide a comprehensive mechanistic explanation for how functional synaptic phenotypes contribute to the altered cerebellar output.

␣2␦ proteins (Cacna2d1-4) are auxiliary subunits of voltage-dependent calcium channels that also drive synapse formation and maturation. Because cerebellar Purkinje cells (PCs) predominantly, if not exclusively, express one isoform of this family, ␣2␦-2 (Cacna2d2), we used PCs as a model system to examine roles of ␣2␦ in excitatory synaptic function in male and female Cacna2d2 knockout (KO) mice. Whole-cell recordings of PCs from acute cerebellar slices revealed altered climbing fiber (CF)-evoked complex spike generation, as well as increased amplitude and faster decay of CF-evoked EPSCs. CF terminals in the KO were localized more proximally on PC dendrites, as indicated by VGLUT2 ϩ immunoreactive puncta, and computational modeling demonstrated that the increased EPSC amplitude can be partly attributed to the more proximal location of CF terminals. In addition, CFs in KO mice exhibited increased multivesicular transmission, corresponding to greater sustained responses during repetitive stimulation, despite a reduction in the measured probability of release. Electron microscopy demonstrated that mutant CF terminals had twice as many vesicle release sites, providing a morphologic explanation for the enhanced glutamate release. Though KO CFs evoked larger amplitude EPSCs, the charge transfer was the same as wild-type as a result of increased glutamate reuptake, producing faster decay kinetics. Together, the larger, faster EPSCs in the KO explain the altered complex spike responses, which degrade information transfer from PCs and likely contribute to ataxia in Cacna2d2 KO mice. Our results also illustrate the multidimensional synaptic roles of ␣2␦ proteins.

BackgroundWe tested the hypothesis that αv-integrin and the human epidermal growth factor receptor type 2 (HER2) interact with each other in brain trophic metastatic breast cancer cells and influence their invasive phenotype.MethodsClones of MDA-MB231BR human breast cancer cells with stable knock down of αv-integrin in combination with high or low levels of HER2 were created. The interactions of these two proteins and their combined effect on cell migration and invasion were investigated in vitro and in vivo.ResultsKnockdown of αv-integrin in MDA-MB231BR clones altered the actin cytoskeleton and cell morphology. HER2 co-precipitated with αv-integrin in three breast cancer cell lines in vitro, suggesting they complex in cells. Knockdown of αv-integrin altered HER2 localization from its normal membrane position to a predominantly lysosomal localization. When αv-integrin expression was decreased by 69–93% in HER2-expressing cells, cellular motility was significantly reduced. Deficiency of both αv-integrin and HER2 decreased cellular migration and invasion by almost 90% compared to cells expressing both proteins (P<0.01). After intracerebral inoculation, cells expressing high levels of both αv-integrin and HER2 showed a diffusely infiltrative tumor phenotype, while cells deficient in αv-integrin and/or HER2 showed a compact tumor growth phenotype. In the αv-integrin positive/HER2 positive tumors, infiltrative growth was 57.2 ± 19% of tumor volume, compared to only 5.8 ± 6.1% infiltration in the double deficient tumor cells.Conclusionsαv-integrin interacts with HER2 in breast cancer cells and may regulate HER2 localization. The combined impacts of αv-integrin and HER2 influence the invasive phenotype of breast cancer cells. Targeting αv-integrin in HER2-positive breast cancer may slow growth and decrease infiltration in the normal brain.