Cortical Complex Research Articles

Detecting Central Auditory Processing Disorders in Awake Mice.

Mice are increasingly used as models of human-acquired neurological or neurodevelopmental conditions, such as autism, schizophrenia, and Alzheimer's disease. All these conditions involve central auditory processing disorders, which have been little investigated despite their potential for providing interesting insights into the mechanisms behind such disorders. Alterations of the auditory steady-state response to 40 Hz click trains are associated with an imbalance between neuronal excitation and inhibition, a mechanism thought to be common to many neurological disorders. Here, we demonstrate the value of presenting click trains at various rates to mice with chronically implanted pins above the inferior colliculus and the auditory cortex for obtaining easy, reliable, and long-lasting access to subcortical and cortical complex auditory processing in awake mice. Using this protocol on a mutant mouse model of autism with a defect of the Shank3 gene, we show that the neural response is impaired at high click rates (above 60 Hz) and that this impairment is visible subcortically-two results that cannot be obtained with classical protocols for cortical EEG recordings in response to stimulation at 40 Hz. These results demonstrate the value and necessity of a more complete investigation of central auditory processing disorders in mouse models of neurological or neurodevelopmental disorders.

Parvalbumin neurons enhance temporal coding and reduce cortical noise in complex auditory scenes

Cortical representations supporting many cognitive abilities emerge from underlying circuits comprised of several different cell types. However, cell type-specific contributions to rate and timing-based cortical coding are not well-understood. Here, we investigated the role of parvalbumin neurons in cortical complex scene analysis. Many complex scenes contain sensory stimuli which are highly dynamic in time and compete with stimuli at other spatial locations. Parvalbumin neurons play a fundamental role in balancing excitation and inhibition in cortex and sculpting cortical temporal dynamics; yet their specific role in encoding complex scenes via timing-based coding, and the robustness of temporal representations to spatial competition, has not been investigated. Here, we address these questions in auditory cortex of mice using a cocktail party-like paradigm, integrating electrophysiology, optogenetic manipulations, and a family of spike-distance metrics, to dissect parvalbumin neurons’ contributions towards rate and timing-based coding. We find that suppressing parvalbumin neurons degrades cortical discrimination of dynamic sounds in a cocktail party-like setting via changes in rapid temporal modulations in rate and spike timing, and over a wide range of time-scales. Our findings suggest that parvalbumin neurons play a critical role in enhancing cortical temporal coding and reducing cortical noise, thereby improving representations of dynamic stimuli in complex scenes.

Sound-modulations of visual motion perception implicate the cortico-vestibular brain

A widely used example of the intricate (yet poorly understood) intertwining of multisensory signals in the brain is the audiovisual bounce inducing effect (ABE). This effect presents two identical objects moving along the azimuth with uniform motion and towards opposite directions. The perceptual interpretation of the motion is ambiguous and is modulated if a transient (sound) is presented in coincidence with the point of overlap of the two objects’ motion trajectories. This phenomenon has long been written-off to simple attentional or decision-making mechanisms, although the neurological underpinnings for the effect are not well understood. Using behavioural metrics concurrently with event-related fMRI, we show that sound-induced modulations of motion perception can be further modulated by changing motion dynamics of the visual targets. The phenomenon engages the posterior parietal cortex and the parieto-insular-vestibular cortical complex, with a close correspondence of activity in these regions with behaviour. These findings suggest that the insular cortex is engaged in deriving a probabilistic perceptual solution through the integration of multisensory data.

P 52 Short-term therapeutic effects of functional electrical stimulation on the somatosensory evoked potentials in the rehabilitation of the patients after the stroke: an interventional study

Objective: The aim was to investigate whether the short-term therapeutic effects of functional electrostimulation (FES) have a role in improving pathological latencies and/or amplitudes of somatosensory evoked potentials (SSEPs) caused by a cortical post-stroke lesion in acute patients with a foot drop. Methods: A randomized clinical sequential two-period crossover-design protocol was constructed and the study followed the PICO Consort criteria. 32 patients were divided in a two separate consecutive treatment periods as part of a four-week inpatient neurological rehabilitation. All patients received conventional rehabilitation therapy. Additionally, group A was treated with FES for two weeks and after this period Group A and Group B were switched. Results: The initial results indicated interaction between functional electrostimulation and the primary somatosensory cortical complex because we observed at this stage of the study that the cortical latency P40 of the intervention group remained invariable. Nevertheless, the control group indicated increase in P40 latency delay. Further data evaluation is necessary to answer the main objective of the study.

Organization and dynamics of the cortical complexes controlling insulin secretion in β-cells.

ABSTRACTInsulin secretion in pancreatic β-cells is regulated by cortical complexes that are enriched at the sites of adhesion to extracellular matrix facing the vasculature. Many components of these complexes, including bassoon, RIM, ELKS and liprins, are shared with neuronal synapses. Here, we show that insulin secretion sites also contain the non-neuronal proteins LL5β (also known as PHLDB2) and KANK1, which, in migrating cells, organize exocytotic machinery in the vicinity of integrin-based adhesions. Depletion of LL5β or focal adhesion disassembly triggered by myosin II inhibition perturbed the clustering of secretory complexes and attenuated the first wave of insulin release. Although previous analyses in vitro and in neurons have suggested that secretory machinery might assemble through liquid–liquid phase separation, analysis of endogenously labeled ELKS in pancreatic islets indicated that its dynamics is inconsistent with such a scenario. Instead, fluorescence recovery after photobleaching and single-molecule imaging showed that ELKS turnover is driven by binding and unbinding to low-mobility scaffolds. Both the scaffold movements and ELKS exchange were stimulated by glucose treatment. Our findings help to explain how integrin-based adhesions control spatial organization of glucose-stimulated insulin release.

Aberrant Changes in Cortical Complexity in Right-Onset Versus Left-Onset Parkinson's Disease in Early-Stage.

There is increasing evidence to show that motor symptom lateralization in Parkinson’s disease (PD) is linked to non-motor features, progression, and prognosis of the disease. However, few studies have reported the difference in cortical complexity between patients with left-onset of PD (LPD) and right-onset of PD (RPD). This study aimed to investigate the differences in the cortical complexity between early-stage LPD and RPD. High-resolution T1-weighted magnetic resonance images of the brain were acquired in 24 patients with LPD, 34 patients with RPD, and 37 age- and sex-matched healthy controls (HCs). Cortical complexity including gyrification index, fractal dimension (FD), and sulcal depth was analyzed using surface-based morphometry via CAT12/SPM12. Familywise error (FWE) peak-level correction at p < 0.05 was performed for significance testing. In patients with RPD, we found decreased mean FD and mean sulcal depth in the banks of the left superior temporal sulcus (STS) compared with LPD and HCs. The mean FD in the left superior temporal gyrus (STG) was decreased in RPD compared with HCs. However, in patients with LPD, we did not identify significantly abnormal cortical complex change compared with HCs. Moreover, we observed that the mean FD in STG was negatively correlated with the 17-item Hamilton Depression Scale (HAMD) among the three groups. Our findings support the specific influence of asymmetrical motor symptoms in cortical complexity in early-stage PD and reveal that the banks of left STS and left STG might play a crucial role in RPD.

Multimodal Long-Term Predictors of Outcome in Out of Hospital Cardiac Arrest Patients Treated with Targeted Temperature Management at 36 °C.

Introduction: Early prediction of long-term outcomes in patients resuscitated after cardiac arrest (CA) is still challenging. Guidelines suggested a multimodal approach combining multiple predictors. We evaluated whether the combination of the electroencephalography (EEG) reactivity, somatosensory evoked potentials (SSEPs) cortical complex and Gray to White matter ratio (GWR) on brain computed tomography (CT) at different temperatures could predict survival and good outcome at hospital discharge and six months after the event. Methods: We performed a retrospective cohort study including consecutive adult, non-traumatic patients resuscitated from out-of-hospital CA who remained comatose on admission to our intensive care unit from 2013 to 2017. We acquired SSEPs and EEGs during the treatment at 36 °C and after rewarming at 37 °C, Gray to white matter ratio (GWR) was calculated on the brain computed tomography scan performed within six hours of the hospital admission. We primarily hypothesized that SSEP was associated with favor-able functional outcome at distance and secondarily that SSEP provides independent information from EEG and CT. Outcomes were evaluated using the Cerebral Performance Category (CPC) scale at six months from discharge. Results: Of 171 resuscitated patients, 75 were excluded due to missing data or uninterpretable neurophysiological findings. EEG reactivity at 37 °C has been shown the best single predictor of good out-come (AUC 0.803) while N20P25 was the best single predictor for survival at each time point. (AUC 0.775 at discharge and AUC 0.747 at six months follow up). The predictive value of a model including EEG reactivity, average GWR, and SSEP N20P25 amplitude was superior (AUC 0.841 for survival and 0.920 for good out-come) to any combination of two tests or any single test. Conclusions: Our study, in which life-sustaining treatments were never suspended, suggests SSEP cortical complex N20P25, after normothermia and off sedation, is a reliable predictor for survival at any time. When SSEP cortical complex N20P25 is added into a model with GWR average and EEG reactivity, the predictivity for good outcome and survival at distance is superior than each single test alone.

Cognitively Impaired Old Mice Display Correlated Reduction in Cortical NMDA Receptor and Complex IV

Cognitive decline in older adults represents a major challenge since cognitive impairment is found in 10% of those ≥ 65 and 50% ≥ 85. Thus it is increasingly important to understand the impact of aging on cognitive health. We performed a battery of tests to assess cognition in 6 month-old (n=12) and 24 month-old (n=8) C57BL/6J mice, equivalent to 30 and 70 year old humans, respectively, and also assessed protein markers in cortex for mitochondrial health and cognition. We found that aged mice displayed fewer spontaneous alternations in the T maze test (p=0.034) and lower recognition of novel objects (p=0.022). In addition, aged mice showed prolonged escape time in the Barnes maze (p=0.035), all of which taken together suggest reduced capacity for learning and recall. Aged mice also exhibited diminished nest building (p<0.001), revealing an impaired functional capacity analogous to the instrumental activities of daily living (IADL) geriatric assessment. We found reduced mitochondrial complex IV expression in the cortices of aged mice concomitant with less expression of N-Methyl D-Aspartate (NMDA) receptor subunits 1, 2A and 2B. The cortices from old mice also exhibited greater expression of immature brain derived neurotrophic factor (pro-BDNF). The alterations in NMDA receptors and pro BDNF are consistent with memory impairment and greater neuronal cell death. Therefore, aged mice exhibit significantly reduced recall and learning ability alongside marked alterations in mitochondrial complex, NMDA receptor, and pro-BDNF expression. Studies are underway to assess whether these molecular changes are responsible for the cognitive declines with aging.

Force-Dependent Regulation of Talin-KANK1 Complex at Focal Adhesions.

KANK proteins mediate cross-talk between dynamic microtubules and integrin-based adhesions to the extracellular matrix. KANKs interact with the integrin/actin-binding protein talin and with several components of microtubule-stabilizing cortical complexes. Because of actomyosin contractility, the talin-KANK complex is likely under mechanical force, and its mechanical stability is expected to be a critical determinant of KANK recruitment to focal adhesions. Here, we quantified the lifetime of the complex of the talin rod domain R7 and the KN domain of KANK1 under shear-force geometry and found that it can withstand forces for seconds to minutes over a physiological force range up to 10 pN. Complex stability measurements combined with cell biological experiments suggest that shear-force stretching promotes KANK1 localization to the periphery of focal adhesions. These results indicate that the talin-KANK1 complex is mechanically strong, enabling it to support the cross-talk between microtubule and actin cytoskeleton at focal adhesions.

The Phase and Shift-Invariant Feature by Adaptive Independent Subspace Analysis for Cortical Complex Cells

It has previously been suggested that complex cells in primary visual cortex (V1) selectly respond to bars and edges at a particular location and orientation emerged from the natural image rather than random stimuli. In this paper, we proposed an adaptive independent subspace analysis (AISA) algorithm based on the maximum likelihood of super-gaussian distribution of the norm of the partially independent components involving difference to efficiently study the characteristics of complex cells. The multiple parameters are updated with different restrictions by contrast divergence (CD) method based on the super-Gaussian (sparse) distribution. In terms of the AISA results, the inside and outside subspace of energy correlation illustrate the properties of independence. Furthermore, AISA features, similar to the characteristics of complex cells in V1, have phase and shift-invariant, as well as frequency and orientation selectivity via the energy function of receptive fields (RFs) from tuning curve test with sinusoidal function. Compared with the principal component analysis (PCA) filters and other ICA filters, AISA filters are analogous to Gabor filters for edge detection and human visual system via varying the frequency and orientation representation.

Chronic Stress Causes Sex-Specific and Structure-Specific Alterations in Mitochondrial Respiratory Chain Activity in Rat Brain.

Chronic restraint stress (CRS) induces a variety of changes in brain function, some of which are mediated by glucocorticoids. The response to stress occurs in a sex-specific way, and may include mitochondrial and synaptic alterations. The synapse is highly dependent on mitochondrial energy supply, and when mitochondria become dysfunctional, they orchestrate cell death. This study aimed to investigate the CRS effects on mitochondrial respiratory chain activity, as well as mitochondrial potential and mass in cell body and synapses using hippocampus, cortex and striatum of male and female rats. Rats were divided into non-stressed (control) and stressed group (CRS during 40days). Results showed that CRS increased complex I-III activity in hippocampus. We also observed an interaction between CRS and sex in the striatal complex II activity, since CRS induced a reduction in complex II activity in males, while in females this activity was increased. Also an interaction was observed between stress and sex in cortical complex IV activity, since CRS induced increased activity in females, while it was reduced in males. Glucocorticoid receptor (GR) content in cortex and hippocampus was sexually dimorphic, with female rats presenting higher levels compared to males. No changes were observed in GR content, mitochondrial potential or mass of animals submitted to CRS. It was concluded that CRS induced changes in respiratory chain complex activities, and some of these changes are sex-dependent: these activities are increased in the striatal mitochondria by CRS protocol mainly in females, while in males it is decreased.

Two populations of cytoplasmic dynein contribute to spindle positioning in C. elegans embryos.

The position of the mitotic spindle is tightly controlled in animal cells as it determines the plane and orientation of cell division. Contacts between cytoplasmic dynein and astral microtubules (MTs) at the cell cortex generate pulling forces that position the spindle. An evolutionarily conserved Gα-GPR-1/2Pins/LGN-LIN-5Mud/NuMA cortical complex interacts with dynein and is required for pulling force generation, but the dynamics of this process remain unclear. In this study, by fluorescently labeling endogenous proteins in Caenorhabditis elegans embryos, we show that dynein exists in two distinct cortical populations. One population directly depends on LIN-5, whereas the other is concentrated at MT plus ends and depends on end-binding (EB) proteins. Knockout mutants lacking all EBs are viable and fertile and display normal pulling forces and spindle positioning. However, EB protein-dependent dynein plus end tracking was found to contribute to force generation in embryos with a partially perturbed dynein function, indicating the existence of two mechanisms that together create a highly robust force-generating system.

Selective inhibition of metabotropic glutamate type 1 alpha receptor (mGluR1α) reduces cell proliferation and migration following status epilepticus in early development

The present study examined whether a single or multiple episode(s) of status epilepticus induced with kainic acid (KA) during the first 3 weeks of postnatal (P) development would aberrantly stimulate proliferation zones that alters migration to potentially injured areas and whether they would be blocked by selective Group I mGluR antagonists. mGluR1α (LY367385) and mGluR5 (MPEP) antagonists were administered 2h following KA-induced status epilepticus and animals were examined after 7days. Proliferating cells of the subventricular zone (SVZ), third ventricle, hippocampus, amygdala cortical complex were analyzed with the proliferative marker, Ki67; and two complementary retrograde dye tracers. Proliferation increased in extrahippocampal limbic structures when KA was administered on P13 or P20 which correlated with number of injured cells at the older age. LY367385 post-treatment caused striking decreases in proliferation in all limbic structures in the presence and absence of injury, whereas a reduction with MPEP was observed only within the amygdala cortical complex (Amg/ERcx) in the presence of multiple seizures (3×KA). After 3×KA and LY367385 post-treatments, diminished co-staining of dye tracers with Ki67 was observed within the Amg/ERcx despite high levels of progenitors marked by the retrograde tracers in this region. This indicates that not only was local proliferation within the SVZ and distant structures inhibited, but also that migration itself was reduced indirectly since there were less cells to migrate from the SVZ. Co-labeling with biomarkers provided evidence for neuronal differentiation suggesting potential aberrant integration may occur in distant locations, and that targeting of mGluR1α receptors may be a potential therapeutic strategy for future development.

Cortical Folding Pattern and its Consistency Induced by Biological Growth.

Cortical folding, characterized by convex gyri and concave sulci, has an intrinsic relationship to the brain’s functional organization. Understanding the mechanism of the brain’s convoluted patterns can provide useful clues into normal and pathological brain function. In this paper, the cortical folding phenomenon is interpreted both analytically and computationally, and, in some cases, the findings are validated with experimental observations. The living human brain is modeled as a soft structure with a growing outer cortex and inner core to investigate its developmental mechanism. Analytical interpretations of differential growth of the brain model provide preliminary insight into critical growth ratios for instability and crease formation of the developing brain. Since the analytical approach cannot predict the evolution of cortical complex convolution after instability, non-linear finite element models are employed to study the crease formation and secondary morphological folds of the developing brain. Results demonstrate that the growth ratio of the cortex to core of the brain, the initial thickness, and material properties of both cortex and core have great impacts on the morphological patterns of the developing brain. Lastly, we discuss why cortical folding is highly correlated and consistent by presenting an intriguing gyri-sulci formation comparison.

Current and emerging potential for magnetoencephalography in pediatric epilepsy

Magnetoencephalography (MEG) is a noninvasive neuroimaging tool that is increasingly becoming useful for presurgical delineation of epileptogenic zones and eloquent cortex in both lesional and non-lesional pediatric cases. During the past 10 yrs, the use of MEG in pediatric epilepsy research has increased. This paper starts with a review of the use of MEG in pediatric epilepsy. We then describe the protocol used for epilepsy patients at the pediatric MEG facility in Boston Children’s Hospital and present two case studies of intractable epilepsy obtained in our laboratory -cortical dysplasia and tuberous sclerosis complex -to illustrate our methodology in localizing epileptiform generators. In both cases, we are able to localize generators of interictal spikes in the irritative zone just outside the lesion. We also present results on localization of the somatosensory cortex based on our pediatric MEG system to illustrate the utility of MEG for identification of the eloquent cortex. We complete this review by considering advantages and limitations of MEG in children with epilepsy, its future developments and research applications. Application of MEG in pediatric epilepsy will accelerate during the coming years as different types of pediatric whole-head MEG systems and more advanced data analysis methods become available to the researchers and clinicians. These advances will lead to greater use of MEG as a complement to clinical electroencephalography, with improved noninvasive delineation of the epileptogenic zone.

Learning Slowness in a Sparse Model of Invariant Feature Detection.

Primary visual cortical complex cells are thought to serve as invariant feature detectors and to provide input to higher cortical areas. We propose a single model for learning the connectivity required by complex cells that integrates two factors that have been hypothesized to play a role in the development of invariant feature detectors: temporal slowness and sparsity. This model, the generative adaptive subspace self-organizing map (GASSOM), extends Kohonen's adaptive subspace self-organizing map (ASSOM) with a generative model of the input. Each observation is assumed to be generated by one among many nodes in the network, each being associated with a different subspace in the space of all observations. The generating nodes evolve according to a first-order Markov chain and generate inputs that lie close to the associated subspace. This model differs from prior approaches in that temporal slowness is not an externally imposed criterion to be maximized during learning but, rather, an emergent property of the model structure as it seeks a good model of the input statistics. Unlike the ASSOM, the GASSOM does not require an explicit segmentation of the input training vectors into separate episodes. This enables us to apply this model to an unlabeled naturalistic image sequence generated by a realistic eye movement model. We show that the emergence of temporal slowness within the model improves the invariance of feature detectors trained on this input.

Auditory evoked cortical potentials with competing noise in children with auditory figure ground deficit

Objective: Listening in real life with noise competition is a great challenge for a considerable number of schoolchildren with auditory processing disorders. These children usually have difficulty in auditory figure ground (AFG) ability. Diagnosis and follow-up of those children is mainly conducted using psychophysical tests that may not be sufficient considering the possible learning effect of these tests or the testing difficulties at a young age. The aim of this study was to investigate a proposed objective tool for the diagnosis of an AFG deficit. Method: The cortical P1-N1 complex with and without ipsilateral competing noise (ICN) was measured for 15 schoolchildren demonstrating an AFG deficit psychophysically. The study group's results were compared to those of 15 age-matched children with normal peripheral and central hearing who comprised the control group (I), and eight age-matched children demonstrating auditory processing disorder(s) but not including an AFG deficit, as a further control group (II). Results: The P1-N1 complex was reproducible in all groups with distortion of the morphology and depression of the amplitude when recorded with an ICN. The impact of an ICN was significantly more pronounced on the study group’s recordings than those of the other groups. All cortical measurements were sensitive to diagnose an AFG deficit, while only the threshold of the P1-N1 complex in ICN was specific to it. Conclusion: The P1-N1 complex threshold with an ICN is a useful tool to diagnose an AFG deficit. It can be considered a physiological correlate of the psychophysical SPIN test.

Early experience improves neural discrimination/recognition of natural complex sounds

In natural environments, behaviorally relevant complex sounds are often produced with “speaker” variability and contaminated with environmental noises. To efficiently discriminate/recognize natural complex sounds, the auditory system has to tune to defining acoustic features and filter out random, meaningless features. In humans, for example, efficient speech recognition is achieved by enhancing perceptual contrast for native speech sounds, as well as reducing perceptual contrast for non-native speech sounds. The neural mechanisms underlying this perceptual transformation are still not well understood. We exposed juvenile rats to heterospecific vocalizations recorded in a natural environment, and subsequently examined their cortical complex sound representations. Cortical neurons became more responsive to dynamic and complex features of the complex sounds. In addition, more neurons were involved in representing the whole set of complex sounds, but fewer neurons actually responded to each individual sound. Cortical responses to different renderings of the same song motif were more similar, and responses to sounds of different motifs became more distinctive, indicating that cortical neurons were more selective to the defining features of the experienced sounds. These effects lead to better neural discrimination/recognition of the experienced complex sounds.

Deficiency in Mitochondrial Complex I Activity Due toNdufs6Gene Trap Insertion Induces Renal Disease

Defects in the activity of enzyme complexes of the mitochondrial respiratory chain are thought to be responsible for several disorders, including renal impairment. Gene mutations that result in complex I deficiency are the most common oxidative phosphorylation disorders in humans. To determine whether an abnormality in mitochondrial complex I per se is associated with development of renal disease, mice with a knockdown of the complex I gene, Ndufs6 were studied. Ndufs6 mice had a partial renal cortical complex I deficiency; Ndufs6gt/gt, 32% activity and Ndufs6gt/+, 83% activity compared with wild-type mice. Both Ndufs6gt/+ and Ndufs6gt/gt mice exhibited hallmarks of renal disease, including albuminuria, urinary excretion of kidney injury molecule-1 (Kim-1), renal fibrosis, and changes in glomerular volume, with decreased capacity to generate mitochondrial ATP and superoxide from substrates oxidized via complex I. However, more advanced renal defects in Ndufs6gt/gt mice were observed in the context of a disruption in the inner mitochondrial electrochemical potential, 3-nitrotyrosine-modified mitochondrial proteins, increased urinary excretion of 15-isoprostane F2t, and up-regulation of antioxidant defence. Juvenile Ndufs6gt/gt mice also exhibited signs of early renal impairment with increased urinary Kim-1 excretion and elevated circulating cystatin C. We have identified renal impairment in a mouse model of partial complex I deficiency, suggesting that even modest deficits in mitochondrial respiratory chain function may act as risk factors for chronic kidney disease. These studies identify for the first time that complex I deficiency as the result of interruption of Ndufs6 is an independent cause of renal impairment.

An ordered pathway for the assembly of fungal ESCRT-containing ambient pH signalling complexes at the plasma membrane.

The fungal pal/RIM signalling pathway, which regulates gene expression in response to environmental pH involves, in addition to dedicated proteins, several components of ESCRT complexes, which suggested that pH signalling proteins assemble on endosomal platforms. In Aspergillus nidulans, dedicated Pal proteins include the plasma membrane receptor PalH and its coupled arrestin, PalF, which becomes ubiquitylated in alkaline pH conditions, and three potentially endosomal ESCRT-III associates, including Vps32 interactors PalA and PalC and Vps24 interactor calpain-like PalB. We studied the subcellular locations at which signalling takes place after activating the pathway by shifting ambient pH to alkalinity. Rather than localising to endosomes, Vps32 interactors PalA and PalC transiently colocalise at alkaline-pH-induced cortical structures in a PalH-, Vps23- and Vps32-dependent but Vps27-independent manner. These cortical structures are much more stable when Vps4 is deficient, indicating that their half-life depends on ESCRT-III disassembly. Pull-down studies revealed that Vps23 interacts strongly with PalF, but co-immunoprecipitates exclusively with ubiquitylated PalF forms from extracts. We demonstrate that Vps23–GFP, expressed at physiological levels, is also recruited to cortical structures, very conspicuous in vps27Δ cells in which the prominent signal of Vps23–GFP on endosomes is eliminated, in a PalF- and alkaline pH-dependent manner. Dual-channel epifluorescence microscopy showed that PalC arrives at cortical complexes before PalA. As PalC recruitment is PalA independent and PalA recruitment is PalC dependent but PalB independent, these data complete the participation order of Pal proteins in the pathway and strongly support a model in which pH signalling takes place in ESCRT-containing, plasma-membrane-associated, rather than endosome-associated, complexes.