Direct and indirect pathways for heterosynaptic interaction underlying developmental synapse elimination in the mouse cerebellum.

Developmental synapse elimination is crucial for shaping mature neural circuits. In the neonatal mouse cerebellum, Purkinje cells (PCs) receive excitatory synaptic inputs from multiple climbing fibers (CFs) and synapses from all but one CF are eliminated by around postnatal day 20. Heterosynaptic interaction between CFs and parallel fibers (PFs), the axons of cerebellar granule cells (GCs) forming excitatory synapses onto PCs and molecular layer interneurons (MLIs), is crucial for CF synapse elimination. However, mechanisms for this heterosynaptic interaction are largely unknown. Here we show that deletion of AMPA-type glutamate receptor functions in GCs impairs CF synapse elimination mediated by metabotropic glutamate receptor 1 (mGlu1) signaling in PCs. Furthermore, CF synapse elimination is impaired by deleting NMDA-type glutamate receptors from MLIs. We propose that PF activity is crucial for CF synapse elimination by directly activating mGlu1 in PCs and indirectly enhancing the inhibition of PCs through activating NMDA receptors in MLIs.

DNA hypomethylator phenotype reprograms glutamatergic network in receptor tyrosine kinase gene-mutated glioblastoma.

DNA methylation is crucial for chromatin structure and gene expression and its aberrancies, including the global "hypomethylator phenotype", are associated with cancer. Here we show that an underlying mechanism for this phenotype in the large proportion of the highly lethal brain tumor glioblastoma (GBM) carrying receptor tyrosine kinase gene mutations, involves the mechanistic target of rapamycin complex 2 (mTORC2), that is critical for growth factor signaling. In this scenario, mTORC2 suppresses the expression of the de novo DNA methyltransferase (DNMT3A) thereby inducing genome-wide DNA hypomethylation. Mechanistically, mTORC2 facilitates a redistribution of EZH2 histone methyltransferase into the promoter region of DNMT3A, and epigenetically represses the expression of DNA methyltransferase. Integrated analyses in both orthotopic mouse models and clinical GBM samples indicate that the DNA hypomethylator phenotype consistently reprograms a glutamate metabolism network, eventually driving GBM cell invasion and survival. These results nominate mTORC2 as a novel regulator of DNA hypomethylation in cancer and an exploitable target against cancer-promoting epigenetics.

CTCF loss induces giant lamellar bodies in Purkinje cell dendrites.

CCCTC-binding factor (CTCF) has a key role in higher-order chromatin architecture that is important for establishing and maintaining cell identity by controlling gene expression. In the mature cerebellum, CTCF is highly expressed in Purkinje cells (PCs) as compared with other cerebellar neurons. The cerebellum plays an important role in motor function by regulating PCs, which are the sole output neurons, and defects in PCs cause motor dysfunction. However, the role of CTCF in PCs has not yet been explored. Here we found that the absence of CTCF in mouse PCs led to progressive motor dysfunction and abnormal dendritic morphology in those cells, which included dendritic self-avoidance defects and a proximal shift in the climbing fibre innervation territory on PC dendrites. Furthermore, we found the peculiar lamellar structures known as "giant lamellar bodies" (GLBs), which have been reported in PCs of patients with Werdnig-Hoffman disease, 13q deletion syndrome, and Krabbe disease. GLBs are localized to PC dendrites and are assumed to be associated with neurodegeneration. They have been noted, however, only in case reports following autopsy, and reports of their existence have been very limited. Here we show that GLBs were reproducibly formed in PC dendrites of a mouse model in which CTCF was deleted. GLBs were not noted in PC dendrites at infancy but instead developed over time. In conjunction with GLB development in PC dendrites, the endoplasmic reticulum was almost absent around the nuclei, the mitochondria were markedly swollen and their cristae had decreased drastically, and almost all PCs eventually disappeared as severe motor deficits manifested. Our results revealed the important role of CTCF during normal development and in maintaining PCs and provide new insights into the molecular mechanism of GLB formation during neurodegenerative disease.

Spike firing attenuation of serotonin neurons in learned helplessness rats is reversed by ketamine.

Animals suffering from uncontrollable stress sometimes show low effort to escape stress (learned helplessness). Changes in serotonin (5-hydroxytryptamine) signalling are thought to underlie this behaviour. Although the release of 5-hydroxytryptamine is triggered by the action potential firing of dorsal raphe nuclei 5-hydroxytryptamine neurons, the electrophysiological changes induced by uncontrollable stress are largely unclear. Herein, we examined electrophysiological differences among 5-hydroxytryptamine neurons in naïve rats, learned helplessness rats and rats resistant to inescapable stress (non-learned helplessness). Five-week-old male Sprague Dawley rats were exposed to inescapable foot shocks. After an avoidance test session, rats were classified as learned helplessness or non-learned helplessness. Activity-dependent 5-hydroxytryptamine release induced by the administration of high-potassium solution was slower in free-moving learned helplessness rats. Subthreshold electrophysiological properties of 5-hydroxytryptamine neurons were identical among the three rat groups, but the depolarization-induced spike firing was significantly attenuated in learned helplessness rats. To clarify the underlying mechanisms, potassium (K+) channels regulating the spike firing were initially examined using naïve rats. K+ channels sensitive to 500 µM tetraethylammonium caused rapid repolarization of the action potential and the small conductance calcium-activated K+ channels produced afterhyperpolarization. Additionally, dendrotoxin-I, a blocker of Kv1.1 (encoded by Kcna1), Kv1.2 (encoded by Kcna2) and Kv1.6 (encoded by Kcna6) voltage-dependent K+ channels, weakly enhanced the spike firing frequency during depolarizing current injections without changes in individual spike waveforms in naïve rats. We found that dendrotoxin-I significantly enhanced the spike firing of 5-hydroxytryptamine neurons in learned helplessness rats. Consequently, the difference in spike firing among the three rat groups was abolished in the presence of dendrotoxin-I. These results suggest that the upregulation of dendrotoxin-I-sensitive Kv1 channels underlies the firing attenuation of 5-hydroxytryptamine neurons in learned helplessness rats. We also found that the antidepressant ketamine facilitated the spike firing of 5-hydroxytryptamine neurons and abolished the firing difference between learned helplessness and non-learned helplessness by suppressing dendrotoxin-I-sensitive Kv1 channels. The dendrotoxin-I-sensitive Kv1 channel may be a potential target for developing drugs to control activity of 5-hydroxytryptamine neurons.

An Autism-Associated Neuroligin-3 Mutation Affects Developmental Synapse Elimination in the Cerebellum.

Neuroligin is a postsynaptic cell-adhesion molecule that is involved in synapse formation and maturation by interacting with presynaptic neurexin. Mutations in neuroligin genes, including the arginine to cystein substitution at the 451st amino acid residue (R451C) of neuroligin-3 (NLGN3), have been identified in patients with autism spectrum disorder (ASD). Functional magnetic resonance imaging and examination of post-mortem brain in ASD patients implicate alteration of cerebellar morphology and Purkinje cell (PC) loss. In the present study, we examined possible association between the R451C mutation in NLGN3 and synaptic development and function in the mouse cerebellum. In NLGN3-R451C mutant mice, the expression of NLGN3 protein in the cerebellum was reduced to about 10% of the level of wild-type mice. Elimination of redundant climbing fiber (CF) to PC synapses was impaired from postnatal day 10-15 (P10-15) in NLGN3-R451C mutant mice, but majority of PCs became mono-innervated as in wild-type mice after P16. In NLGN3-R451C mutant mice, selective strengthening of a single CF relative to the other CFs in each PC was impaired from P16, which persisted into juvenile stage. Furthermore, the inhibition to excitation (I/E) balance of synaptic inputs to PCs was elevated, and calcium transients in the soma induced by strong and weak CF inputs were reduced in NLGN3-R451C mutant mice. These results suggest that a single point mutation in NLGN3 significantly influences the synapse development and refinement in cerebellar circuitry, which might be related to the pathogenesis of ASD.

Tonic GABAergic Inhibition Is Essential for Nerve Injury-Induced Afferent Remodeling in the Somatosensory Thalamus and Ectopic Sensations.

Peripheral nerve injury induces functional and structural remodeling of neural circuits along the somatosensory pathways, forming the basis for somatotopic reorganization and ectopic sensations, such as referred phantom pain. However, the mechanisms underlying that remodeling remain largely unknown. Whisker sensory nerve injury drives functional remodeling in the somatosensory thalamus: the number of afferent inputs to each thalamic neuron increases from one to many. Here, we report that extrasynaptic γ-aminobutyric acid-type A receptor (GABAAR)-mediated tonic inhibition is necessary for that remodeling. Extrasynaptic GABAAR currents were potentiated rapidly after nerve injury in advance of remodeling. Pharmacological activation of the thalamic extrasynaptic GABAARs in intact mice induced similar remodeling. Notably, conditional deletion of extrasynaptic GABAARs in the thalamus rescued both the injury-induced remodeling and the ectopic mechanical hypersensitivity. Together, our results reveal a molecular basis for injury-induced remodeling of neural circuits and may provide a new pharmacological target for referred phantom sensations after peripheral nerve injury.

Hyperactivation of mTORC1 disrupts cellular homeostasis in cerebellar Purkinje cells.

Mammalian target of rapamycin (mTOR) is a central regulator of cellular metabolism. The importance of mTORC1 signaling in neuronal development and functions has been highlighted by its strong relationship with many neurological and neuropsychiatric diseases. Previous studies demonstrated that hyperactivation of mTORC1 in forebrain recapitulates tuberous sclerosis and neurodegeneration. In the mouse cerebellum, Purkinje cell-specific knockout of Tsc1/2 has been implicated in autistic-like behaviors. However, since TSC1/2 activity does not always correlate with clinical manifestations as evident in some cases of tuberous sclerosis, the intriguing possibility is raised that phenotypes observed in Tsc1/2 knockout mice cannot be attributable solely to mTORC1 hyperactivation. Here we generated transgenic mice in which mTORC1 signaling is directly hyperactivated in Purkinje cells. The transgenic mice exhibited impaired synapse elimination of climbing fibers and motor discoordination without affecting social behaviors. Furthermore, mTORC1 hyperactivation induced prominent apoptosis of Purkinje cells, accompanied with dysregulated cellular homeostasis including cell enlargement, increased mitochondrial respiratory activity, and activation of pseudohypoxic response. These findings suggest the different contributions between hyperactivated mTORC1 and Tsc1/2 knockout in social behaviors, and reveal the perturbations of cellular homeostasis by hyperactivated mTORC1 as possible underlying mechanisms of neuronal dysfunctions and death in tuberous sclerosis and neurodegenerative diseases.

Microglia permit climbing fiber elimination by promoting GABAergic inhibition in the developing cerebellum.

Circuit refinement during postnatal development is finely regulated by neuron-neuron interactions. Recent studies suggest participation of microglia in this process but it is unclear how microglia cooperatively act with neuronal mechanisms. To examine roles of microglia, we ablate microglia by microglia-selective deletion of colony-stimulating factor 1 receptor (Csf1r) by crossing floxed-Csf1r and Iba1-iCre mice (Csf1r-cKO). In Csf1r-cKO mice, refinement of climbing fiber (CF) to Purkinje cell (PC) innervation after postnatal day 10 (P10)-P12 is severely impaired. However, there is no clear morphological evidence suggesting massive engulfment of CFs by microglia. In Csf1r-cKO mice, inhibitory synaptic transmission is impaired and CF elimination is restored by diazepam, which suggests that impairment of CF elimination is caused by a defect of GABAergic inhibition on PCs, a prerequisite for CF elimination. These results indicate that microglia primarily promote GABAergic inhibition and secondarily facilitate the mechanism for CF elimination inherent in PCs.

CAPS1 RNA Editing Promotes Dense Core Vesicle Exocytosis.

Calcium-dependent activator protein for secretion 1 (CAPS1) plays a distinct role in the priming step of dense core vesicle (DCV) exocytosis. CAPS1 pre-mRNA is known to undergo adenosine-to-inosine RNA editing in its coding region, which results in a glutamate-to-glycine conversion at a site in its C-terminal region. However, the physiological significance of CAPS1 RNA editing remains elusive. Here, we created mutant mice in which edited CAPS1 was solely expressed. These mice were lean due to increased energy expenditure caused by physical hyperactivity. Electrophysiological and biochemical analyses demonstrated that the exocytosis of DCVs was upregulated in the chromaffin cells and neurons of these mice. Furthermore, we showed that edited CAPS1 bound preferentially to the activated form of syntaxin-1A, a component of the exocytotic fusion complex. These findings suggest that RNA editing regulates DCV exocytosis in vivo, affecting physical activity.

Ionic Basis for Membrane Potential Resonance in Neurons of the Inferior Olive.

Some neurons have the ability to enhance output voltage to input current with a preferred frequency, which is called resonance. Resonance is thought to be a basis for membrane potential oscillation. Although ion channels responsible for resonance have been reported, the precise mechanisms by which these channels work remain poorly understood. We have found that resonance is reduced but clearly present in the inferior olivary neurons of Cav3.1 T-type voltage-dependent Ca(2+) channel knockout (KO) mice. The activation of Cav3.1 channels is strongly membrane potential dependent, but less frequency dependent. Residual resonance in Cav3.1 KO mice is abolished by a hyper-polarization-activated cyclic nucleotide-gated (HCN) channel blocker, ZD7288, and is partially suppressed by voltage-dependent K(+) channel blockers. Resonance is inhibited by ZD7288 in wild-type mice and impaired in HCN1 KO mice, suggesting that the HCN1 channel is essential for resonance. The ZD7288-sensitive current is nearly sinusoidal and strongly frequency dependent. These results suggest that Cav3.1 and HCN1 channels act as amplifying and resonating conductances, respectively.

A CDC42EP4/septin-based perisynaptic glial scaffold facilitates glutamate clearance.

The small GTPase-effector proteins CDC42EP1-5/BORG1-5 interact reciprocally with CDC42 or the septin cytoskeleton. Here we show that, in the cerebellum, CDC42EP4 is exclusively expressed in Bergmann glia and localizes beneath specific membrane domains enwrapping dendritic spines of Purkinje cells. CDC42EP4 forms complexes with septin hetero-oligomers, which interact with a subset of glutamate transporter GLAST/EAAT1. In Cdc42ep4(-/-) mice, GLAST is dissociated from septins and is delocalized away from the parallel fibre-Purkinje cell synapses. The excitatory postsynaptic current exhibits a protracted decay time constant, reduced sensitivity to a competitive inhibitor of the AMPA-type glutamate receptors (γDGG) and excessive baseline inward current in response to a subthreshold dose of a nonselective inhibitor of the glutamate transporters/EAAT1-5 (DL-TBOA). Insufficient glutamate-buffering/clearance capacity in these mice manifests as motor coordination/learning defects, which are aggravated with subthreshold DL-TBOA. We propose that the CDC42EP4/septin-based glial scaffold facilitates perisynaptic localization of GLAST and optimizes the efficiency of glutamate-buffering and clearance.

Cerebellar plasticity and motor learning deficits in a copy-number variation mouse model of autism.

A common feature of autism spectrum disorder (ASD) is the impairment of motor control and learning, occurring in a majority of children with autism, consistent with perturbation in cerebellar function. Here we report alterations in motor behaviour and cerebellar synaptic plasticity in a mouse model (patDp/+) for the human 15q11-13 duplication, one of the most frequently observed genetic aberrations in autism. These mice show ASD-resembling social behaviour deficits. We find that in patDp/+ mice delay eyeblink conditioning--a form of cerebellum-dependent motor learning--is impaired, and observe deregulation of a putative cellular mechanism for motor learning, long-term depression (LTD) at parallel fibre-Purkinje cell synapses. Moreover, developmental elimination of surplus climbing fibres--a model for activity-dependent synaptic pruning--is impaired. These findings point to deficits in synaptic plasticity and pruning as potential causes for motor problems and abnormal circuit development in autism.

Functional motifs composed of morphologically homologous neurons repeated in the hindbrain segments.

Segmental organization along the neuraxis is a prominent feature of the CNS in vertebrates. In a wide range of fishes, hindbrain segments contain orderly arranged reticulospinal neurons (RSNs). Individual RSNs in goldfish and zebrafish hindbrain are morphologically identified. RSNs sharing similar morphological features are called segmental homologs and repeated in adjacent segments. However, little is known about functional relationships among segmental homologs. Here we investigated the electrophysiological connectivity between the Mauthner cell (M-cell), a pair of giant RSNs in segment 4 (r4) that are known to trigger fast escape behavior, and different series of homologous RSNs in r4-r6. Paired intracellular recordings in adult goldfish revealed unidirectional connections from the M-cell to RSNs. The connectivity was similar in morphological homologs. A single M-cell spike produced IPSPs in dorsally located RSNs (MiD cells) on the ipsilateral side and excitatory postsynaptic depolarization on the contralateral side, except for MiD2cm cells. The inhibitory or excitatory potentials effectively suppressed or enhanced target RSNs spiking, respectively. In contrast to the lateralized effects on MiD cells, single M-cell spiking elicited equally strong depolarizations on bilateral RSNs located ventrally (MiV cells), and the depolarization was high enough for MiV cells to burst. Therefore, the morphological homology of repeated RSNs in r4-r6 and their functional M-cell connectivity were closely correlated, suggesting that each functional connection works as a functional motif during the M-cell-initiated escape.

Spike timing-dependent selective strengthening of single climbing fibre inputs to Purkinje cells during cerebellar development.

Shaping functional neural circuits in developing brain involves activity-dependent refinement of early-formed redundant synapses. In the developing cerebellum, a one-to-one connection between a climbing fibre (CF) and a Purkinje cell (PC) is established by selective strengthening of a single CF followed by elimination of surplus CFs. Here we investigate developmental changes in CF-mediated responses in PCs by using in vivo whole-cell recordings and two-photon Ca(2+) imaging. We show that each neonatal PC receives temporally clustered inputs from multiple CFs and temporal integration of these inputs is required to induce burst spiking and Ca(2+) rise in PCs. Importantly, a single CF input closest to PC's spike output is selectively strengthened during postnatal development. This spike timing-dependent selective strengthening is much less prominent in PC-selective P/Q-type voltage-dependent Ca(2+) channel knockout mice. Thus, spike timing- and Ca(2+)-dependent plasticity appears to underlie the selection of a single 'winner' CF and the establishment of mature CF-PC connections.

Calcium-dependent regulation of climbing fibre synapse elimination during postnatal cerebellar development.

Functional neural circuit formation during postnatal development involves massive elimination of early-formed redundant synapses and strengthening of necessary synaptic connections. In the cerebellum, one-to-one connection from a climbing fibre (CF) to a Purkinje cell (PC) is established through four distinct phases: (1) strengthening of a single CF among multiple CFs in each PC at postnatal age P3-P7 days, (2) translocation of a single strengthened CF to PC dendrites from around P9, (3) early-phase (P7 to around P11) and (4) late-phase (around P12-P17) elimination of weak CF synapses from PC somata. Mice with PC-selective deletion of the P/Q-type voltage-dependent Ca(2+) channel (VDCC) exhibit severe defects in strengthening of single CFs, dendritic translocation of single CFs and CF elimination from P7. In contrast, mice with a mutation of a single allele for the GABA synthesizing enzyme GAD67 show selective impairment of CF elimination from P10. Electrophysiological and Ca(2+) imaging data suggest that GABAA receptor-mediated inhibition onto PC somata from putative basket cells influences CF-induced Ca(2+) transients and regulates elimination of redundant CF synapses from PC somata at P10-P16. Thus, regulation of Ca(2+) influx to PCs through VDCCs is crucial for the four phases of CF synapse elimination during postnatal development.

GABAergic inhibition regulates developmental synapse elimination in the cerebellum.

Functional neural circuit formation during development involves massive elimination of redundant synapses. In the cerebellum, one-to-one connection from excitatory climbing fiber (CF) to Purkinje cell (PC) is established by elimination of early-formed surplus CFs. This process depends on glutamatergic excitatory inputs, but contribution of GABAergic transmission remains unclear. Here, we demonstrate impaired CF synapse elimination in mouse models with diminished GABAergic transmission by mutation of a single allele for the GABA synthesizing enzyme GAD67, by conditional deletion of GAD67 from PCs and GABAergic interneurons or by pharmacological inhibition of cerebellar GAD activity. The impaired CF synapse elimination was rescued by enhancing GABA(A) receptor sensitivity in the cerebellum by locally applied diazepam. Our electrophysiological and Ca2+ imaging data suggest that GABA(A) receptor-mediated inhibition onto the PC soma from molecular layer interneurons influences CF-induced Ca2+ transients in the soma and regulates CF synapse elimination from postnatal day 10 (P10) to around P16.

A pilot study on effectiveness of music therapy in hospice in Japan.

This study aims at determining the effectiveness of music therapy in a hospice setting. We employed the salivary cortisol level, which is widely used to measure stress level, as an objective and physical indicator and the Mood Inventory, which measures mood change, as the subjective and psychological indicators. Though many preceding studies have demonstrated that listening to music lowers cortisol levels and reduces stress, no study seems to have included hospice patients. This study measured, with the consent of 10 hospice inpatients, their salivary cortisol levels. Individual interviews, according to the Mood Inventory, were conducted before and after a small-group session. Since all the participants had terminal cancer, the 40-minute live session of songs of seasons and the participants' requests was given in a mostly passive manner considering their physical strength. Results showed significant lowering of salivary cortisol levels after the therapy session. As for the parameters of mood, refreshment was significantly increased. Though fatigue remained unchanged, anxiety and depression decreased while the score for excitement tended to increase. Thus, it was indicated that music therapy in a hospice setting reduces the stress level of patients and thereby plays a positive role in improving patients' quality of life.

GABAergic activation of an inwardly rectifying K+ current in mouse cerebellar Purkinje cells.

Cerebellar Purkinje cells integrate motor information conveyed by excitatory synaptic inputs from parallel and climbing fibres. Purkinje cells abundantly express B-type G-protein-coupled gamma-aminobutyric acid receptors (GABABR) that are assumed to mediate major responses, including postsynaptic modulation of the synaptic inputs. However, the identity and function of effectors operated by GABABR are not fully elucidated. Here we characterized an inwardly rectifying current activated by baclofen (Ibacl), a GABABR agonist, in cultured mouse Purkinje cells using a ruptured-patch whole-cell technique. Ibacl is operated by GABABR via Gi/o-proteins, as it is not inducible in pertussis-toxin-pretreated cells. Ibacl is carried by K+ because its reversal potential shifts with the equilibrium potential of K+. Ibacl is blocked by 10(-3) M Ba2+ or Cs+, and 10(-8) M tertiapin-Q. Upon the onset and offset of a hyperpolarizing step, Ibacl is activated and deactivated, respectively, with double-exponential time courses (time constants, <1 ms and 30-80 ms). Based on similarities in the above properties, G-protein-coupled inwardly rectifying K+ (GIRK) channels are thought to be responsible for Ibacl. Perforated-patch recordings from cultured Purkinje cells demonstrate that Ibacl hyperpolarizes the resting potential and the peak level achieved by glutamate-evoked potentials initiated in the dendrites. Moreover, cell-attached recordings from Purkinje cells in cerebellar slices demonstrate that Ibacl impedes spontaneous firing. Therefore, Ibacl may reduce the postsynaptic and intrinsic excitability of Purkinje cells under physiological conditions. These findings give a new insight into the role of GABABR signalling in cerebellar information processing.

Common sensory inputs and differential excitability of segmentally homologous reticulospinal neurons in the hindbrain.

In the hindbrain of zebrafish and goldfish, reticulospinal (RS) neurons are arranged in seven segments, with segmental homologs in adjacent segments. The Mauthner cell (M-cell) in the fourth segment (r4) is known to trigger fast escape behavior. Its serial homologs, MiD2cm in r5 and MiD3cm in r6, are predicted to contribute to this behavior, which can be evoked by head-tap stimuli. However, little is known about their input-output properties. Therefore, we studied afferent projections from the auditory posterior eighth nerve (pVIIIn) and firing properties of MiD2cm and MiD3cm for comparison with the M-cell in adult goldfish. Labeling of RS neurons and the pVIIIn afferents with fluorescent tracers showed that the pVIIIn projected to r4-r6. Tone burst and electrical stimulation of the pVIIIn evoked EPSPs in the M-cell, MiD2cm, and MiD3cm. Stepwise depolarization typically elicited a single spike at the onset in the M-cell but repetitive spiking in MiD2cm and MiD3cm. This atypical property of the M-cell was mediated by dendrotoxin-I (DTX-I)-sensitive voltage-gated potassium channels together with recurrent inhibition, because combined application of DTX-I, strychnine, and bicuculline led to continuous repetitive firing in M-cells. The M-cell but not MiD2cm or MiD3cm expressed Kv1.2, a DTX-I-sensitive potassium channel subunit. Thus, the M-cell and its segmental homologs may sense common auditory information but send different outputs to the spinal circuits to control adaptive escape behavior.