Ultrafast Two-Photon Imaging of a High-Gain Voltage Indicator in Awake Behaving Mice.

Optical interrogation of voltage in deep brain locations with cellular resolution would be immensely useful for understanding how neuronal circuits process information. Here, we report ASAP3, a genetically encoded voltage indicator with 51% fluorescence modulation by physiological voltages, submillisecond activation kinetics, and full responsivity under two-photon excitation. We also introduce an ultrafast local volume excitation (ULoVE) method for kilohertz-rate two-photon sampling in vivo with increased stability and sensitivity. Combining a soma-targeted ASAP3 variant and ULoVE, we show single-trial tracking of spikes and subthreshold events for minutes in deep locations, with subcellular resolution and with repeated sampling over days. In the visual cortex, we use soma-targeted ASAP3 to illustrate cell-type-dependent subthreshold modulation by locomotion. Thus, ASAP3 and ULoVE enable high-speed optical recording of electrical activity in genetically defined neurons at deep locations during awake behavior.

Influence of spatially segregated IP3-producing pathways on spike generation and transmitter release in Purkinje cell axons.

It has been known for a long time that inositol-trisphosphate (IP3) receptors are present in the axon of certain types of mammalian neurons, but their functional role has remained unexplored. Here we show that localized photolysis of IP3 induces spatially constrained calcium rises in Purkinje cell axons. Confocal immunohistology reveals that the axon initial segment (AIS), as well as terminals onto deep cerebellar cells, express specific subtypes of Gα/q and phospholipase C (PLC) molecules, together with the upstream purinergic receptor P2Y1. By contrast, intermediate parts of the axon express another set of Gα/q and PLC molecules, indicating two spatially segregated signaling cascades linked to IP3 generation. This prompted a search for distinct actions of IP3 in different parts of Purkinje cell axons. In the AIS, we found that local applications of the specific P2Y1R agonist MRS2365 led to calcium elevation, and that IP3 photolysis led to inhibition of action potential firing. In synaptic terminals on deep cerebellar nuclei neurons, we found that photolysis of both IP3 and ATP led to GABA release. We propose that axonal IP3 receptors can inhibit action potential firing and increase neurotransmitter release, and that these effects are likely controlled by purinergic receptors. Altogether our results suggest a rich and diverse functional role of IP3 receptors in axons of mammalian neurons.

CBX3 antagonizes IFNγ/STAT1/PD-L1 axis to modulate colon inflammation and CRC chemosensitivity.

As an important immune stimulator and modulator, IFNγ is crucial for gut homeostasis and its dysregulation links to diverse colon pathologies, such as colitis and colorectal cancer (CRC). Here, we demonstrated that the epigenetic regulator, CBX3 (also known as HP1γ) antagonizes IFNγ signaling in the colon epithelium by transcriptionally repressing two critical IFNγ-responsive genes: STAT1 and CD274 (encoding Programmed death-ligand 1, PD-L1). Accordingly, CBX3 deletion resulted in chronic mouse colon inflammation, accompanied by upregulated STAT1 and CD274 expressions. Chromatin immunoprecipitation indicated that CBX3 tethers to STAT1 and CD274 promoters to inhibit their expression. Reversely, IFNγ significantly reduces CBX3 binding to these promoters and primes gene expression. This antagonist effect between CBX3 and IFNγ on STAT1/PD-L1 expression was also observed in CRC. Strikingly, CBX3 deletion heightened CRC cells sensitivity to IFNγ, which ultimately enhanced their chemosensitivity under IFNγ stimulation in vitro with CRC cells and in vivo with a syngeneic mouse tumor model. Overall, this work reveals that by negatively tuning IFNγ-stimulated immune genes' transcription, CBX3 participates in modulating colon inflammatory response and CRC chemo-resistance.

Alpha E beta 7 integrin interaction with E-cadherin promotes antitumor CTL activity by triggering lytic granule polarization and exocytosis.

Various T cell adhesion molecules and their cognate receptors on target cells promote T cell receptor (TCR)-mediated cell killing. In this report, we demonstrate that the interaction of epithelial cell marker E-cadherin with integrin alpha(E)(CD103)beta(7), often expressed by tumor-infiltrating lymphocytes (TILs), plays a major role in effective tumor cell lysis. Indeed, we found that although tumor-specific CD103(+) TIL-derived cytotoxic T lymphocyte (CTL) clones are able to kill E-cadherin(+)/intercellular adhesion molecule 1(-) autologous tumor cells, CD103(-) peripheral blood lymphocyte (PBL)-derived counterparts are inefficient. This cell killing is abrogated after treatment of the TIL clones with a blocking anti-CD103 monoclonal antibody or after targeting E-cadherin in the tumor using ribonucleic acid interference. Confocal microscopy analysis also demonstrated that alpha(E)beta(7) is recruited at the immunological synapse and that its interaction with E-cadherin is required for cytolytic granule polarization and subsequent exocytosis. Moreover, we report that the CD103(-) profile, frequently observed in PBL-derived CTL clones and associated with poor cytotoxicity against the cognate tumor, is up-regulated upon TCR engagement and transforming growth factor beta1 treatment, resulting in strong potentiation of antitumor lytic function. Thus, CD8(+)/CD103(+) tumor-reactive T lymphocytes infiltrating epithelial tumors most likely play a major role in antitumor cytotoxic response through alpha(E)beta(7)-E-cadherin interactions.

KCa1.1 channels contribute to optogenetically driven post-stimulation silencing in cerebellar molecular layer interneurons.

Using cell-attached recordings from molecular layer interneurons (MLI) of the cerebellar cortex of adult mice expressing channel rhodopsin 2, we show that wide-field optical activation induces an increase in firing rate during illumination and a firing pause when the illumination ends (post-stimulation silencing; PSS). Significant spike rate changes with respect to basal firing rate were observed for optical activations lasting 200 ms and 1 s as well as for 1 s long trains of 10 ms pulses at 50 Hz. For all conditions, the net effect of optical activation on the integrated spike rate is significantly reduced because of PSS. Three lines of evidence indicate that this PSS is due to intrinsic factors. Firstly, PSS is induced when the optical stimulation is restricted to a single MLI using a 405-nm laser delivering a diffraction-limited spot at the focal plane. Secondly, PSS is not affected by block of GABA-A or GABA-B receptors, ruling out synaptic interactions amongst MLIs. Thirdly, PSS is mimicked in whole-cell recording experiments by step depolarizations under current clamp. Activation of Ca-dependent K channels during the spike trains appears as a likely candidate to underlie PSS. Using immunocytochemistry, we find that one such channel type, KCa1.1, is present in the somato-dendritic and axonal compartments of MLIs. In cell-attached recordings, charybdotoxin and iberiotoxin significantly reduce the optically induced PSS, while TRAM-34 does not affect it, suggesting that KCa1.1 channels, but not KCa3.1 channels, contribute to PSS.

Aurora B is dispensable for megakaryocyte polyploidization, but contributes to the endomitotic process.

Polyploidization of megakaryocytes (MKs), the platelet precursors, occurs by endomitosis, a mitotic process that fails at late stages of cytokinesis. Expression and function of Aurora B kinase during endomitosis remain controversial. Here, we report that Aurora B is normally expressed during the human MK endomitotic process. Aurora B localized normally in the midzone or midbody during anaphase and telophase in low ploidy megakaryocytes and in up to 16N rare endomitotic MKs was observed. Aurora B was also functional during cytokinesis as attested by phosphorylation of both its activation site and MgcRacGAP, its main substrate. However, despite its activation, Aurora B did not prevent furrow regression. Inhibition of Aurora B by AZD1152-HQPA decreased cell cycle entry both in 2N to 4N and polyploid MKs and induced apoptosis mainly in 2N to 4N cells. In both MK classes, AZD1152-HQPA induced p53 activation and retinoblastoma hypophosphorylation. Resistance of polyploid MKs to apoptosis correlated to a high BclxL level. Aurora B inhibition did not impair MK polyploidization but profoundly modified the endomitotic process by inducing a mis-segregation of chromosomes and a mitotic failure in anaphase. This indicates that Aurora B is dispensable for MK polyploidization but is necessary to achieve a normal endomitotic process.

Cytopathic effects of the cytomegalovirus-encoded apoptosis inhibitory protein vMIA.

Replication of human cytomegalovirus (CMV) requires the expression of the viral mitochondria-localized inhibitor of apoptosis (vMIA). vMIA inhibits apoptosis by recruiting Bax to mitochondria, resulting in its neutralization. We show that vMIA decreases cell size, reduces actin polymerization, and induces cell rounding. As compared with vMIA-expressing CMV, vMIA-deficient CMV, which replicates in fibroblasts expressing the adenoviral apoptosis suppressor E1B19K, induces less cytopathic effects. These vMIA effects can be separated from its cell death-inhibitory function because vMIA modulates cellular morphology in Bax-deficient cells. Expression of vMIA coincided with a reduction in the cellular adenosine triphosphate (ATP) level. vMIA selectively inhibited one component of the ATP synthasome, namely, the mitochondrial phosphate carrier. Exposure of cells to inhibitors of oxidative phosphorylation produced similar effects, such as an ATP level reduced by 30%, smaller cell size, and deficient actin polymerization. Similarly, knockdown of the phosphate carrier reduced cell size. Our data suggest that the cytopathic effect of CMV can be explained by vMIA effects on mitochondrial bioenergetics.

Gap junction communication between autologous endothelial and tumor cells induce cross-recognition and elimination by specific CTL.

Cellular interactions in the tumor stroma play a major role in cancer progression but can also induce tumor rejection. To explore the role of endothelial cells in these interactions, we used an in vitro three-dimensional collagen matrix model containing a cytotoxic T lymphocyte CTL clone (M4.48), autologous tumor cells (M4T), and an endothelial cell (M4E) line that are all derived from the same tumor. We demonstrate in this study that specific killing of the endothelial cells by the CTL clone required the autologous tumor cells and involved Ag cross-presentation. The formation of gap junctions between endothelial and tumor cells is required for antigenic peptide transfer to endothelial cells that are then recognized and eliminated by CTL. Our results indicate that gap junctions facilitate an effective CTL-mediated destruction of endothelial cells from the tumor microenvironment that may contribute to the control of tumor progression.

The cooperative induction of hypoxia-inducible factor-1 alpha and STAT3 during hypoxia induced an impairment of tumor susceptibility to CTL-mediated cell lysis.

Hypoxia is an essential component of tumor microenvironment. In this study, we investigated the influence of hypoxia (1% PO(2)) on CTL-mediated tumor cell lysis. We demonstrate that exposure of target tumor cells to hypoxia has an inhibitory effect on the CTL clone (Heu171)-induced autologous target cell lysis. Such inhibition correlates with hypoxia-inducible factor-1alpha (HIF-1alpha) induction but is not associated with an alteration of CTL reactivity as revealed by granzyme B polarization or morphological change. Western blot analysis indicates that although hypoxia had no effect on p53 accumulation, it induced the phosphorylation of STAT3 in tumor cells by a mechanism at least in part involving vascular endothelial growth factor secretion. We additionally show that a simultaneous nuclear translocation of HIF-1alpha and phospho-STAT3 was observed. Interestingly, gene silencing of STAT3 by small interfering RNA resulted in HIF-1alpha inhibition and a significant restoration of target cell susceptibility to CTL-induced killing under hypoxic conditions by a mechanism involving at least in part down-regulation of AKT phosphorylation. Moreover, knockdown of HIF-1alpha resulted in the restoration of target cell lysis under hypoxic conditions. This was further supported by DNA microarray analysis where STAT3 inhibition resulted in a partly reversal of the hypoxia-induced gene expression profile. The present study demonstrates that the concomitant hypoxic induction of phospho-STAT3 and HIF-1alpha are functionally linked to the alteration of non-small cell lung carcinoma target susceptibility to CTL-mediated killing. Considering the eminent functions of STAT3 and HIF-1alpha in the tumor microenvironment, their targeting may represent novel strategies for immunotherapeutic intervention.

Megakaryocyte endomitosis is a failure of late cytokinesis related to defects in the contractile ring and Rho/Rock signaling.

Megakaryocyte (MK) is the naturally polyploid cell that gives rise to platelets. Polyploidization occurs by endomitosis, which was a process considered to be an incomplete mitosis aborted in anaphase. Here, we used time-lapse confocal video microscopy to visualize the endomitotic process of primary human megakaryocytes. Our results show that the switch from mitosis to endomitosis corresponds to a late failure of cytokinesis accompanied by a backward movement of the 2 daughter cells. No abnormality was observed in the central spindle of endomitotic MKs. A furrow formation was present, but the contractile ring was abnormal because accumulation of nonmuscle myosin IIA was lacking. In addition, a defect in cell elongation was observed in dipolar endomitotic MKs during telophase. RhoA and F-actin were partially concentrated at the site of furrowing. Inhibition of the Rho/Rock pathway caused the disappearance of F-actin at midzone and increased MK ploidy level. This inhibition was associated with a more pronounced defect in furrow formation as well as in spindle elongation. Our results suggest that the late failure of cytokinesis responsible for the endomitotic process is related to a partial defect in the Rho/Rock pathway activation.

Two opposite signaling outputs are driven by the KIR2DL1 receptor in human CD4+ T cells.

Inhibitory killer Ig-like receptors (KIR), expressed by human natural killer cells and effector memory CD8(+) T-cell subsets, bind HLA-C molecules and suppress cell activation through recruitment of the Src homology 2 domain-containing protein tyrosine phosphatase 1 (SHP-1). To further analyze the still largely unclear role of inhibitory KIR receptors on CD4(+) T cells, KIR2DL1 transfectants were obtained from a CD4(+) T-cell line and primary cells. Transfection of CD4(+) T cells with KIR2DL1 dramatically increased the T-cell receptor (TCR)-induced production of interleukin-2 independently of ligand binding but inhibited TCR-induced activation after ligation. KIR-mediated costimulation of TCR activation involves intact KIR2DL1-ITIM phosphorylation, SHP-2 recruitment, and PKC- phosphorylation. Synapses leading to activation were characterized by an increase in the recruitment of p-Tyr, SHP-2, and p-PKC-, but not of SHP-1. Interaction of KIR2DL1 with its ligand led to a strong synaptic accumulation of KIR2DL1 and the recruitment of SHP-1/2, inhibiting TCR-induced interleukin-2 production. KIR2DL1 may induce 2 opposite signaling outputs in CD4(+) T cells, depending on whether the KIR receptor is bound to its ligand. These data highlight unexpected aspects of the regulation of T cells by KIR2DL1 receptors, the therapeutic manipulation of which is currently being evaluated.

Synergism of type 1 metabotropic and ionotropic glutamate receptors in cerebellar molecular layer interneurons in vivo.

Type 1 metabotropic glutamate receptors (mGluR1s) are key elements in neuronal signaling. While their function is well documented in slices, requirements for their activation in vivo are poorly understood. We examine this question in adult mice in vivo using 2-photon imaging of cerebellar molecular layer interneurons (MLIs) expressing GCaMP. In anesthetized mice, parallel fiber activation evokes beam-like Cai rises in postsynaptic MLIs which depend on co-activation of mGluR1s and ionotropic glutamate receptors (iGluRs). In awake mice, blocking mGluR1 decreases Cai rises associated with locomotion. In vitro studies and freeze-fracture electron microscopy show that the iGluR-mGluR1 interaction is synergistic and favored by close association of the two classes of receptors. Altogether our results suggest that mGluR1s, acting in synergy with iGluRs, potently contribute to processing cerebellar neuronal signaling under physiological conditions.

Membrane-bound interleukin (IL)-15 on renal tumor cells rescues natural killer cells from IL-2 starvation-induced apoptosis.

Renal cell carcinoma primary tumors and lung metastases are infiltrated by activated natural killer (NK) cells. Interleukin (IL)-15, a major cytokine involved in cross-talk between accessory cells (dendritic cells and macrophages) and NK cells, is produced by epithelial renal cells. We show that renal cell carcinoma cells and normal renal cells express IL-15 mRNA and membrane-bound IL-15 (MbIL-15). These cells also express IL-15 receptor alpha (IL-15Ralpha). Silencing of IL-15Ralpha by specific small interfering RNA in renal cell carcinoma had no effect on MbIL-15 production, indicating that the cytokine is not cross-presented by IL-15Ralpha in renal cell carcinoma cells but anchored to the membrane. Furthermore, we show that MbIL-15 from renal cell carcinoma cells is functional and involved in rapid nuclear translocation of phosphorylated signal transducers and activators of transcription 3 in IL-2-starved NK cells. MbIL-15 on the target did not interfere with resting NK cell activation and target cell cytolysis but rescued NK cells from IL-2 starvation-induced apoptosis through contact-dependent interaction. Masking of MbIL-15 with soluble IL-15Ralpha molecules restored NK cell apoptosis. These findings suggest that IL-15 produced by renal tumor cells is involved in the maintenance of active NK cells at the tumor site.

Altered IFNgamma signaling and preserved susceptibility to activated natural killer cell-mediated lysis of BCR/ABL targets.

Previous studies have shown that BCR/ABL oncogene, the molecular counterpart of the Ph1 chromosome, could represent a privileged target to natural killer (NK) cells. In the present study, we showed that activated peripheral NK cells killed high-level BCR/ABL transfectant UT-7/9 derived from the pluripotent hematopoietic cell line UT-7 with a high efficiency. To further define the mechanisms controlling BCR/ABL target susceptibility to NK-mediated lysis, we studied the effect of IFNgamma, a key cytokine secreted by activated NK cells, on the lysis of these targets. Treatment of UT-7, UT-7/neo, and low BCR/ABL transfectant UT-7/E8 cells with IFNgamma resulted in a dramatic induction of human leukocyte antigen class I (HLA-I) molecules and subsequently in their reduced susceptibility to NK-mediated cytolysis likely as a consequence of inhibitory NK receptors engagement. In contrast, such treatment neither affected HLA-I expression on transfectants expressing high level of BCR/ABL (UT-7/9) nor modulated their lysis by NK cells. Our data further show that the high-level BCR/ABL in UT-7/9 cells display an altered IFNgamma signaling, as evidenced by a decrease in IFN regulatory factor-1 (IRF-1) and signal transducers and activators of transcription (STAT) 1 induction and activation in response to IFNgamma, whereas this pathway is normal in UT-7 and UT-7/E8 cells. A decreased HLA-I induction and nuclear phospho-STAT1 nuclear translocation were also observed in blasts from most chronic myelogenous leukemia patients in response to IFNgamma. These results outline the crucial role of IFNgamma in the control of target cell susceptibility to lysis by activated NK cells and indicate that the altered response to IFNgamma in BCR/ABL targets may preserve these cells from the cytokine-induced negative regulatory effect on their susceptibility to NK-mediated lysis.

Opposite effects of estrogen receptors alpha and beta on MCF-7 sensitivity to the cytotoxic action of TNF and p53 activity.

We have investigated the effect of estrogen on p53 cellular location and its influence on tumor cell susceptibility to tumor necrosis factor (TNF)-mediated cytotoxic action. For this purpose, we have used the TNF-sensitive human breast adenocarcinoma MCF-7 and its derivative, the TNF-resistant 1001 clone. Our data indicate that although estrogen receptor (ER)alpha is present in both cell lines, estrogen treatment (1x10(-8) M) has an influence only on the MCF-7 cells and protects these cells from the TNF cytotoxicity. This protective effect is associated with translocation of p53 from the nucleus to the cytoplasm in p53 wild-type MCF-7 and not in p53-mutated 1001 cells. The translocation of p53 in MCF-7 cells results in a decrease in its transcriptional activity, as revealed by diminished p21(WAF1/CIP1) induction and an altered ratio of Bax and Bcl-2 proteins. The estrogen-induced effects are reversed by the selective estrogen inhibitor 182, 780 (1x10(-6) M). Interestingly, transient transfection of MCF-7 cells with ERbeta but not ERalpha cDNA encoding plasmid results in retention of p53 in the nucleus, a subsequent potentiation of its transcriptional activity, and in an increased MCF-7 sensitivity to TNF. The estrogen effects on p53 location and transcriptional activity may involve the mdm2 protein since both events were reversed following MCF-7 transfection with plasmid encoding the ARF cDNA. These studies suggest that estrogen-induced MCF-7 cell survival in the presence of TNF requires a transcriptionally active p53 and, more importantly, indicate that introduction of ERbeta can attenuate the estrogen effects on the p53 protein location, its transcriptional activity and also results in a potentiation of cell sensitivity to TNF-mediated cell death.

Prion protein prevents human breast carcinoma cell line from tumor necrosis factor alpha-induced cell death.

To define genetic determinants of tumor cell resistance to the cytotoxic action of tumor necrosis factor alpha (TNF), we have applied cDNA microarrays to a human breast carcinoma TNF-sensitive MCF7 cell line and its established TNF-resistant clone. Of a total of 5760 samples of cDNA examined, 3.6% were found to be differentially expressed in TNF-resistant 1001 cells as compared with TNF-sensitive MCF7 cells. On the basis of available literature data, the striking finding is the association of some differentially expressed genes involved in the phosphatidylinositol-3-kinase/Akt signaling pathway. More notably, we found that the PRNP gene coding for the cellular prion protein (PrP(c)), was 17-fold overexpressed in the 1001 cell line as compared with the MCF7 cell line. This differential expression was confirmed at the cell surface by immunostaining that indicated that PrP(c) is overexpressed at both mRNA and protein levels in the TNF-resistant derivative. Using recombinant adenoviruses expressing the human PrP(c,) our data demonstrate that PrP(c) overexpression converted TNF-sensitive MCF7 cells into TNF-resistant cells, at least in part, by a mechanism involving alteration of cytochrome c release from mitochondria and nuclear condensation.

Mutant alpha-actinin-4 promotes tumorigenicity and regulates cell motility of a human lung carcinoma.

The precise role of alpha-actinin-4 encoding gene (ACTN4) is not very well understood. It has been reported to elicit tumor suppressor activity and to regulate cellular motility. To further assess the function of human ACTN4, we studied a lung carcinoma cell line expressing a mutated alpha-actinin-4, which is recognized as a tumor antigen by autologous CD8(+) cytotoxic T lymphocytes (CTL). Confocal immunofluorescence microscopy indicated that, while wild-type (WT) alpha-actinin-4 stains into actin cytoskeleton and cell surface ruffles, the mutated protein is only dispersed in the cytoplasm of the lung carcinoma cells. This loss of association with the cell surface did not appear to correlate with a decrease in in vitro alpha-actinin-4 crosslinking to filamentous (F)-actin. Interestingly, experiments using cell lines stably expressing ACTN4 demonstrated that as opposed to WT gene, mutant ACTN4 was unable to inhibit tumor cell growth in vitro and in vivo. Moreover, the expression of mutant alpha-actinin-4 resulted in the loss of tumor cell capacity to migrate. The identification of an inactivating mutation in ACTN4 emphasizes its role as a tumor suppressor gene and underlines the involvement of cytoskeleton alteration in tumor development and metastasis.

Impact of single-site axonal GABAergic synaptic events on cerebellar interneuron activity.

Axonal ionotropic receptors are present in a variety of neuronal types, and their function has largely been associated with the modulation of axonal activity and synaptic release. It is usually assumed that activation of axonal GABA(A)Rs comes from spillover, but in cerebellar molecular layer interneurons (MLIs) the GABA source is different: in these cells, GABA release activates presynaptic GABA(A) autoreceptors (autoRs) together with postsynaptic targets, producing an autoR-mediated synaptic event. The frequency of presynaptic, autoR-mediated miniature currents is twice that of their somatodendritic counterparts, suggesting that autoR-mediated responses have an important effect on interneuron activity. Here, we used local Ca(2+) photolysis in MLI axons of juvenile rats to evoke GABA release from individual varicosities to study the activation of axonal autoRs in single release sites. Our data show that single-site autoR conductances are similar to postsynaptic dendritic conductances. In conditions of high [Cl(-)](i), autoR-mediated conductances range from 1 to 5 nS; this corresponds to ∼30-150 GABA(A) channels per presynaptic varicosity, a value close to the number of channels in postsynaptic densities. Voltage responses produced by the activation of autoRs in single varicosities are amplified by a Na(v)-dependent mechanism and propagate along the axon with a length constant of 91 µm. Immunolabeling determination of synapse location shows that on average, one third of the synapses produce autoR-mediated signals that are large enough to reach the axon initial segment. Finally, we show that single-site activation of presynaptic GABA(A) autoRs leads to an increase in MLI excitability and thus conveys a strong feedback signal that contributes to spiking activity.

An excitatory GABA loop operating in vivo.

While it has been proposed that the conventional inhibitory neurotransmitter GABA can be excitatory in the mammalian brain, much remains to be learned concerning the circumstances and the cellular mechanisms governing potential excitatory GABA action. Using a combination of optogenetics and two-photon calcium imaging in vivo, we find that activation of chloride-permeable GABAA receptors in parallel fibers (PFs) of the cerebellar molecular layer of adult mice causes parallel fiber excitation. Stimulation of PFs at submaximal stimulus intensities leads to GABA release from molecular layer interneurons (MLIs), thus creating a positive feedback loop that enhances excitation near the center of an activated PF bundle. Our results imply that elevated chloride concentration can occur in specific intracellular compartments of mature mammalian neurons and suggest an excitatory role for GABAA receptors in the cerebellar cortex of adult mice.