Highly unstable heterogeneous representations in VIP interneurons of the anterior cingulate cortex.

A hallmark of the anterior cingulate cortex (ACC) is its functional heterogeneity. Functional and imaging studies revealed its importance in the encoding of anxiety-related and social stimuli, but it is unknown how microcircuits within the ACC encode these distinct stimuli. One type of inhibitory interneuron, which is positive for vasoactive intestinal peptide (VIP), is known to modulate the activity of pyramidal cells in local microcircuits, but it is unknown whether VIP cells in the ACC (VIPACC) are engaged by particular contexts or stimuli. Additionally, recent studies demonstrated that neuronal representations in other cortical areas can change over time at the level of the individual neuron. However, it is not known whether stimulus representations in the ACC remain stable over time. Using in vivo Ca2+ imaging and miniscopes in freely behaving mice to monitor neuronal activity with cellular resolution, we identified individual VIPACC that preferentially activated to distinct stimuli across diverse tasks. Importantly, although the population-level activity of the VIPACC remained stable across trials, the stimulus-selectivity of individual interneurons changed rapidly. These findings demonstrate marked functional heterogeneity and instability within interneuron populations in the ACC. This work contributes to our understanding of how the cortex encodes information across diverse contexts and provides insight into the complexity of neural processes involved in anxiety and social behavior.

Increased expression of schizophrenia-associated gene C4 leads to hypoconnectivity of prefrontal cortex and reduced social interaction.

Schizophrenia is a severe mental disorder with an unclear pathophysiology. Increased expression of the immune gene C4 has been linked to a greater risk of developing schizophrenia; however, it is not known whether C4 plays a causative role in this brain disorder. Using confocal imaging and whole-cell electrophysiology, we demonstrate that overexpression of C4 in mouse prefrontal cortex neurons leads to perturbations in dendritic spine development and hypoconnectivity, which mirror neuropathologies found in schizophrenia patients. We find evidence that microglia-mediated synaptic engulfment is enhanced with increased expression of C4. We also show that C4-dependent circuit dysfunction in the frontal cortex leads to decreased social interactions in juvenile and adult mice. These results demonstrate that increased expression of the schizophrenia-associated gene C4 causes aberrant circuit wiring in the developing prefrontal cortex and leads to deficits in juvenile and adult social behavior, suggesting that altered C4 expression contributes directly to schizophrenia pathogenesis.

Determinants of Cation Permeation and Drug Sensitivity in Predicted Transmembrane Helix 9 and Adjoining Exofacial Re-entrant Loop 5 of Na+/H+ Exchanger NHE1.

Mammalian Na(+)/H(+) exchangers (NHEs) regulate numerous physiological processes and are involved in the pathogenesis of several diseases, including tissue ischemia and reperfusion injuries, cardiac hypertrophy and failure, and cancer progression. Hence, NHEs are being targeted for pharmaceutical-based clinical therapies, but pertinent information regarding the structural elements involved in cation translocation and drug binding remains incomplete. Molecular manipulations of the prototypical NHE1 isoform have implicated several predicted membrane-spanning (M) helices, most notably M4, M9, and M11, as important determinants of cation permeation and drug sensitivity. Here, we have used substituted-cysteine accessibility mutagenesis and thiol-modifying methanethiosulfonate (MTS) reagents to further probe the involvement of evolutionarily conserved sites within M9 (residues 342-363) and the adjacent exofacial re-entrant loop 5 between M9 and M10 (EL5; residues 364-415) of a cysteine-less variant of rat NHE1 on its kinetic and pharmacological properties. MTS treatment significantly reduced the activity of mutants containing substitutions within M9 (H353C, S355C, and G356C) and EL5 (G403C and S405C). In the absence of MTS, mutants S355C, G403C, and S405C showed modest to significant decreases in their apparent affinities for Na(+) o and/or H(+) i. In addition, mutations Y370C and E395C within EL5, whereas failing to confer sensitivity to MTS, nevertheless, reduced the affinity for Na(+) o, but not for H(+) i. The Y370C mutant also exhibited higher affinity for ethylisopropylamiloride, a competitive antagonist of Na(+) o transport. Collectively, these results further implicate helix M9 and EL5 of NHE1 as important elements involved in cation transport and inhibitor sensitivity, which may inform rational drug design.

Activation of AMP-activated protein kinase regulates hippocampal neuronal pH by recruiting Na(+)/H(+) exchanger NHE5 to the cell surface.

Strict regulation of intra- and extracellular pH is an important determinant of nervous system function as many voltage-, ligand-, and H(+)-gated cationic channels are exquisitely sensitive to transient fluctuations in pH elicited by neural activity and pathophysiologic events such as hypoxia-ischemia and seizures. Multiple Na(+)/H(+) exchangers (NHEs) are implicated in maintenance of neural pH homeostasis. However, aside from the ubiquitous NHE1 isoform, their relative contributions are poorly understood. NHE5 is of particular interest as it is preferentially expressed in brain relative to other tissues. In hippocampal neurons, NHE5 regulates steady-state cytoplasmic pH, but intriguingly the bulk of the transporter is stored in intracellular vesicles. Here, we show that NHE5 is a direct target for phosphorylation by the AMP-activated protein kinase (AMPK), a key sensor and regulator of cellular energy homeostasis in response to metabolic stresses. In NHE5-transfected non-neuronal cells, activation of AMPK by the AMP mimetic AICAR or by antimycin A, which blocks aerobic respiration and causes acidification, increased cell surface accumulation and activity of NHE5, and elevated intracellular pH. These effects were effectively blocked by the AMPK antagonist compound C, the NHE inhibitor HOE694, and mutation of a predicted AMPK recognition motif in the NHE5 C terminus. This regulatory pathway was also functional in primary hippocampal neurons, where AMPK activation of NHE5 protected the cells from sustained antimycin A-induced acidification. These data reveal a unique role for AMPK and NHE5 in regulating the pH homeostasis of hippocampal neurons during metabolic stress.

The Na(+)/H (+) exchanger NHE5 is sorted to discrete intracellular vesicles in the central and peripheral nervous systems.

The pH milieu of the central and peripheral nervous systems is an important determinant of neuronal excitability, function, and survival. In mammals, neural acid-base homeostasis is coordinately regulated by ion transporters belonging to the Na(+)/H(+) exchanger (NHE) and bicarbonate transporter gene families. However, the relative contributions of individual isoforms within the respective families are not fully understood. This report focuses on the NHE family, specifically the plasma membrane-type NHE5 which is preferentially transcribed in brain, but the distribution of the native protein has not been extensively characterized. To this end, we generated a rabbit polyclonal antibody that specifically recognizes NHE5. In both central (cortex, hippocampus) and peripheral (superior cervical ganglia, SCG) nervous tissue of mice, NHE5 immunostaining was punctate and highly concentrated in the somas and to lesser amounts in the dendrites of neurons. Very little signal was detected in axons. Similarly, in primary cultures of differentiated SCG neurons, NHE5 localized predominantly to vesicles in the somatodendritic compartment, though some immunostaining was also evident in punctate vesicles along the axons. NHE5 was also detected predominantly in intracellular vesicles of cultured SCG glial cells. Dual immunolabeling of SCG neurons showed that NHE5 did not colocalize with markers for early endosomes (EEA1) or synaptic vesicles (synaptophysin), but did partially colocalize with the transferrin receptor, a marker of recycling endosomes. Collectively, these data suggest that NHE5 partitions into a unique vesicular pool in neurons that shares some characteristics of recycling endosomes where it may serve as an important regulated store of functional transporters required to maintain cytoplasmic pH homeostasis.

International test results for objective lens quality, resolution, spectral accuracy and spectral separation for confocal laser scanning microscopes.

As part of an ongoing effort to increase image reproducibility and fidelity in addition to improving cross-instrument consistency, we have proposed using four separate instrument quality tests to augment the ones we have previously reported. These four tests assessed the following areas: (1) objective lens quality, (2) resolution, (3) accuracy of the wavelength information from spectral detectors, and (4) the accuracy and quality of spectral separation algorithms. Data were received from 55 laboratories located in 18 countries. The largest source of errors across all tests was user error which could be subdivided between failure to follow provided protocols and improper use of the microscope. This truly emphasizes the importance of proper rigorous training and diligence in performing confocal microscopy experiments and equipment evaluations. It should be noted that there was no discernible difference in quality between confocal microscope manufactures. These tests, as well as others previously reported, will help assess the quality of confocal microscopy equipment and will provide a means to track equipment performance over time. From 62 to 97% of the data sets sent in passed the various tests demonstrating the usefulness and appropriateness of these tests as part of a larger performance testing regiment.

CK2 phosphorylation of an acidic Ser/Thr di-isoleucine motif in the Na+/H+ exchanger NHE5 isoform promotes association with beta-arrestin2 and endocytosis.

Internalization of the Na(+)/H(+) exchanger NHE5 into recycling endosomes is enhanced by the endocytic adaptor proteins ß-arrestin1 and -2, best known for their preferential recognition of ligand-activated G protein-coupled receptors (GPCRs). However, the mechanism underlying their atypical association with non-GPCRs, such as NHE5, is unknown. In this study, we identified a highly acidic, serine/threonine-rich, di-isoleucine motif (amino acids 697-723) in the cytoplasmic C terminus of NHE5 that is recognized by ß-arrestin2. Gross deletions of this site decreased the state of phosphorylation of NHE5 as well as its binding and responsiveness to ß-arrestin2 in intact cells. More refined in vitro analyses showed that this site was robustly phosphorylated by the acidotropic protein kinase CK2, whereas other kinases, such as CK1 or the GPCR kinase GRK2, were considerably less potent. Simultaneous mutation of five Ser/Thr residues within 702-714 to Ala ((702)ST/AA(714)) abolished phosphorylation and binding of ß-arrestin2. In transfected cells, the CK2 catalytic α subunit formed a complex with NHE5 and decreased wild-type but not (702)ST/AA(714) NHE5 activity, further supporting a regulatory role for this kinase. The rate of internalization of (702)ST/AA(714) was also diminished and relatively insensitive to overexpression of ß-arrestin2. However, unlike in vitro, this mutant retained its ability to form a complex with ß-arrestin2 despite its lack of responsiveness. Additional mutations of two di-isoleucine-based motifs (I697A/L698A and I722A/I723A) that immediately flank the acidic cluster, either separately or together, were required to disrupt their association. These data demonstrate that discrete elements of an elaborate sorting signal in NHE5 contribute to ß-arrestin2 binding and trafficking along the recycling endosomal pathway.

Measuring and interpreting point spread functions to determine confocal microscope resolution and ensure quality control.

This protocol outlines a procedure for collecting and analyzing point spread functions (PSFs). It describes how to prepare fluorescent microsphere samples, set up a confocal microscope to properly collect 3D confocal image data of the microspheres and perform PSF measurements. The analysis of the PSF is used to determine the resolution of the microscope and to identify any problems with the quality of the microscope's images. The PSF geometry is used as an indicator to identify problems with the objective lens, confocal laser scanning components and other relay optics. Identification of possible causes of PSF abnormalities and solutions to improve microscope performance are provided. The microsphere sample preparation requires 2-3 h plus an overnight drying period. The microscope setup requires 2 h (1 h for laser warm up), whereas collecting and analyzing the PSF images require an additional 2-3 h.