Cell-Type-Specific Effects of Somatostatin on Synaptic Transmission in the Anterior Cingulate Cortex.

Inhibitory modulation of glutamatergic information processing is a prerequisite for proper network function. Among the many groups of interneurons (INs), somatostatin-expressing interneurons (SOM-INs) play an important role in the maintenance of physiological brain activity. We have previously shown that somatostatin (SOM) causes a reduction in pyramidal cell (PC) excitability. However, the mechanisms of action of the peptide on cortical synaptic circuits are still unclear. To understand the effects of the neuropeptide SOM on cortical synaptic circuits, we performed a detailed side-by-side comparison of its postsynaptic effects on PCs, SOM-INs, and layer 1 interneurons (L1-INs) in the anterior cingulate cortex of male and female mice and found that SOM produced pronounced postsynaptic effects in PCs while having little to no effect on either IN type. This comparison allowed us to link the observed postsynaptic effects to SOM-induced modulations of glutamatergic and GABAergic synaptic transmission and to trace the impact of the neuropeptide on the neuronal circuitry between these three cell types. We show here that SOM depresses glutamatergic synaptic transmission via a presynaptic mechanism while exerting a differential impact on GABAA receptor- and GABAB receptor-mediated transmission at the pre- and postsynaptic level resulting in a shift of inhibition in L2/3 PCs from L1-INs to SOM-INs. In summary, this study unravels a novel aspect by which SOM modulates synaptic signaling between PCs, L1-INs, and SOM-INs.

Development, Diversity, and Death of MGE-Derived Cortical Interneurons.

In the mammalian brain, cortical interneurons (INs) are a highly diverse group of cells. A key neurophysiological question concerns how each class of INs contributes to cortical circuit function and whether specific roles can be attributed to a selective cell type. To address this question, researchers are integrating knowledge derived from transcriptomic, histological, electrophysiological, developmental, and functional experiments to extensively characterise the different classes of INs. Our hope is that such knowledge permits the selective targeting of cell types for therapeutic endeavours. This review will focus on two of the main types of INs, namely the parvalbumin (PV+) or somatostatin (SOM+)-containing cells, and summarise the research to date on these classes.

Diversity and Function of Somatostatin-Expressing Interneurons in the Cerebral Cortex.

Inhibitory interneurons make up around 10-20% of the total neuron population in the cerebral cortex. A hallmark of inhibitory interneurons is their remarkable diversity in terms of morphology, synaptic connectivity, electrophysiological and neurochemical properties. It is generally understood that there are three distinct and non-overlapping interneuron classes in the mouse neocortex, namely, parvalbumin-expressing, 5-HT3A receptor-expressing and somatostatin-expressing interneuron classes. Each class is, in turn, composed of a multitude of subclasses, resulting in a growing number of interneuron classes and subclasses. In this review, I will focus on the diversity of somatostatin-expressing interneurons (SOM+ INs) in the cerebral cortex and elucidate their function in cortical circuits. I will then discuss pathological consequences of a malfunctioning of SOM+ INs in neurological disorders such as major depressive disorder, and present future avenues in SOM research and brain pathologies.

Reactive astrogliosis causes the development of spontaneous seizures.

Epilepsy is one of the most common chronic neurologic diseases, yet approximately one-third of affected patients do not respond to anticonvulsive drugs that target neurons or neuronal circuits. Reactive astrocytes are commonly found in putative epileptic foci and have been hypothesized to be disease contributors because they lose essential homeostatic capabilities. However, since brain pathology induces astrocytes to become reactive, it is difficult to distinguish whether astrogliosis is a cause or a consequence of epileptogenesis. We now present a mouse model of genetically induced, widespread chronic astrogliosis after conditional deletion of ß1-integrin (Itgß1). In these mice, astrogliosis occurs in the absence of other pathologies and without BBB breach or significant inflammation. Electroencephalography with simultaneous video recording revealed that these mice develop spontaneous seizures during the first six postnatal weeks of life and brain slices show neuronal hyperexcitability. This was not observed in mice with neuronal-targeted ß1-integrin deletion, supporting the hypothesis that astrogliosis is sufficient to induce epileptic seizures. Whole-cell patch-clamp recordings from astrocytes further suggest that the heightened excitability was associated with impaired astrocytic glutamate uptake. Moreover, the relative expression of the cation-chloride cotransporters (CCC) NKCC1 (Slc12a2) and KCC2 (Slc12a5), which are responsible for establishing the neuronal Cl(-) gradient that governs GABAergic inhibition were altered and the NKCC1 inhibitor bumetanide eliminated seizures in a subgroup of mice. These data suggest that a shift in the relative expression of neuronal NKCC1 and KCC2, similar to that observed in immature neurons during development, may contribute to astrogliosis-associated seizures.

Determination and compensation of series resistances during whole-cell patch-clamp recordings using an active bridge circuit and the phase-sensitive technique.

We present a technique which combines two methods in order to measure the series resistance (R S) during whole-cell patch-clamp recordings from excitable and non-excitable cells. R S is determined in the amplifier's current-clamp mode by means of an active bridge circuit. The correct neutralization of the electrode capacitance and the adjustment of the bridge circuit is achieved by the so-called phase-sensitive method: Short sine wave currents with frequencies between 3 and 7 kHz are injected into the cells. Complete capacitance neutralization is indicated by the disappearance of the phase lag between current and voltage, and correct bridge balance is indicated by a minimized voltage response to the sine wave current. The R S value determined in the current-clamp mode then provides the basis for R S compensation in the voltage-clamp recording mode. The accuracy of the procedure has been confirmed on single-compartment cell models where the error amounted to 2-3 %. Similar errors were observed during dual patch clamp recordings from single neocortical layer 5 pyramidal cells where one electrode was connected to the bridge amplifier and the other one to a time-sharing, single-electrode current- and voltage-clamp amplifier with negligible R S. The technique presented here allows R S compensation for up to 80-90 %, even in cells with low input resistances (e.g., astrocytes). In addition, the present study underlines the importance of correct R S compensation by showing that significant series resistances directly affect the determination of membrane conductances as well as the kinetic properties of spontaneous synaptic currents with small amplitudes.

Direct neuronal reprogramming of NDUFS4 patient cells identifies the unfolded protein response as a novel general reprogramming hurdle.

Mitochondria account for essential cellular pathways, from ATP production to nucleotide metabolism, and their deficits lead to neurological disorders and contribute to the onset of age-related diseases. Direct neuronal reprogramming aims at replacing neurons lost in such conditions, but very little is known about the impact of mitochondrial dysfunction on the direct reprogramming of human cells. Here, we explore the effects of mitochondrial dysfunction on the neuronal reprogramming of induced pluripotent stem cell (iPSC)-derived astrocytes carrying mutations in the NDUFS4 gene, important for Complex I and associated with Leigh syndrome. This led to the identification of the unfolded protein response as a major hurdle in the direct neuronal conversion of not only astrocytes and fibroblasts from patients but also control human astrocytes and fibroblasts. Its transient inhibition potently improves reprogramming by influencing the mitochondria-endoplasmic-reticulum-stress-mediated pathways. Taken together, disease modeling using patient cells unraveled novel general hurdles and ways to overcome these in human astrocyte-to-neuron reprogramming.

Pumilio2 and Staufen2 selectively balance the synaptic proteome.

Neurons have the capacity to adapt to environmental stimuli, a phenomenon termed cellular plasticity. The underlying processes are controlled by a network of RNA-binding proteins (RBPs). Their precise impact, however, is largely unknown. To address this important question, we chose Pumilio2 (Pum2) and Staufen2 (Stau2), which both regulate synaptic transmission. Surprisingly, even though both RBPs dynamically interact with each other in neurons, their respective impact on the transcriptome and proteome is highly selective. Although Pum2 deficiency leads to reduced translation and protein expression, Stau2 depletion preferentially impacts RNA levels and increases protein abundance. Furthermore, we show that Pum2 activates expression of key GABAergic synaptic components, e.g., the GABAA receptor scaffold protein Gephyrin. Consequently, Pum2 depletion selectively reduced the amplitude of miniature inhibitory postsynaptic currents. Together, our data argue for an important role of RBPs to maintain proteostasis in order to control distinct aspects of synaptic transmission.

Activity-dependent regulation of neuronal apoptosis in neonatal mouse cerebral cortex.

A massive neuronal loss during early postnatal development has been well documented in the murine cerebral cortex, but the factors that drive cells into apoptosis are largely unknown. The role of neuronal activity in developmental apoptosis was studied in organotypic neocortical slice cultures of newborn mice. Multielectrode array and whole-cell patch-clamp recordings revealed spontaneous network activity characterized by synchronized burst discharges, which could be blocked by tetrodotoxin and ionotropic glutamate receptor antagonists. The identical neuropharmacological manipulations also caused a significant increase in the number of apoptotic neurons as early as 6 h after the start of drug treatment. Moreover, inhibition of the NMDA receptor subunit NR2A or NR2B induced a differential short-term versus delayed increase in the apoptosis rate, respectively. Activation of L-type, voltage-dependent calcium channels was neuroprotective and could prevent activity-dependent apoptosis during NMDA receptor blockade. Furthermore, this effect involved phosphorylation of cAMP response element-binding protein and activation of the tropomyosin-related kinase (Trk) receptors. Inhibition of electrical synapses and blockade of ionotropic gamma-aminobutyric acid receptors induced specific changes in spontaneous electrical activity patterns, which caused an increase in caspase-3-dependent cell death. Our results demonstrate that synchronized spontaneous network bursts activating ionotropic glutamate receptors promote neuronal survival in the neonatal mouse cerebral cortex.

Long-lasting actions of somatostatin on pyramidal cell excitability in the mouse cingulate cortex.

Many neurological diseases are related to disturbances of somatostatin- (SOM-) expressing interneurons in the cingulate cortex. Therefore, their role within the circuitry of the cingulate cortex needs to be investigated. We describe here the physiological time course of SOM effects onto pyramidal cell excitability and action potential discharge pattern. Furthermore, we show that the GRK2 inhibitor Gallein had no effect on the reduced SOM-induced response following repetitive SOM applications.

Gad1-promotor-driven GFP expression in non-GABAergic neurons of the nucleus endopiriformis in a transgenic mouse line.

Transgenic animals have become a widely used model to identify and study specific cell types in whole organs. Promotor-driven reporter gene labeling of the cells under investigation has promoted experimental efficacy to a large degree. However, rigorous assessment of transgene expression specificity in these animal models is highly recommended to validate cellular identity and to isolate potentially mislabeled cell populations. Here, we report on one such mislabeled neuron population in a widely used transgenic mouse line in which GABAergic somatostatin-expressing interneurons (SOMpos INs) are labeled by eGFP (so-called GIN mouse, FVB-Tg(GadGFP)45704Swn/J). These neurons represent a subpopulation of all SOMpos INs. However, we report here on GFP labeling of non-GABAergic neurons in the nucleus endopiriformis of this mouse line.

Glucocorticoids trigger Alzheimer disease-like pathobiochemistry in rat neuronal cells expressing human tau.

Amyloid precursor protein (APP) mis-processing and aberrant tau hyperphosphorylation are causally related to the pathogenesis and neurodegenerative processes that characterize Alzheimer's disease (AD). Abnormal APP metabolism leads to the generation of neurotoxic amyloid beta (Abeta), whereas tau hyperphosphorylation culminates in cytoskeletal disturbances, neuronal dysfunction and death. Many AD patients hypersecrete glucocorticoids (GC) while neuronal structure, function and survival are adversely influenced by elevated GC levels. We report here that a rat neuronal cell line (PC12) engineered to express the human ortholog of the tau protein (PC12-htau) becomes more vulnerable to the toxic effects of either Abeta or GC treatment. Importantly, APP metabolism in GC-treated PC12-htau cells is selectively shifted towards increased production of the pro-amyloidogenic peptide C99. Further, GC treatment results in hyperphosphorylation of human tau at AD-relevant sites, through the cyclin-dependent kinase 5 (E.C. 2.7.11.26) and GSK3 (E.C. 2.7.11.22) protein kinases. Pulse-chase experiments revealed that GC treatment increased the stability of tau protein rather than its de novo synthesis. GC treatment also induced accumulation of transiently expressed EGFP-tau in the neuronal perikarya. Together with previous evidence showing that Abeta can activate cyclin-dependent kinase 5 and GSK3, these results uncover a potential mechanism through which GC may contribute to AD neuropathology.

Two types of somatostatin-expressing GABAergic interneurons in the superficial layers of the mouse cingulate cortex.

Somatostatin-expressing (SOM+), inhibitory interneurons represent a heterogeneous group of cells and given their remarkable diversity, classification of SOM+ interneurons remains a challenging task. Electrophysiological, morphological and neurochemical classes of SOM+ interneurons have been proposed in the past but it remains unclear as to what extent these classes are congruent. We performed whole-cell patch-clamp recordings from 127 GFP-labeled SOM+ interneurons ('GIN') of the superficial cingulate cortex with subsequent biocytin-filling and immunocytochemical labeling. Principal component analysis followed by k-means clustering predicted two putative subtypes of SOM+ interneurons, which we designated as group I and group II GIN. A key finding of our study is the fact that these electrophysiologically and morphologically distinct groups of SOM+ interneurons can be correlated with two neurochemical subtypes of SOM+ interneurons described recently in our laboratory. In particular, all SOM+ interneurons expressing calbindin but no calretinin could be classified as group I GIN, whereas all but one neuropeptide Y- and calretinin-positive interneurons were found in group II.

Direct pericyte-to-neuron reprogramming via unfolding of a neural stem cell-like program.

Ectopic expression of defined transcription factors can force direct cell-fate conversion from one lineage to another in the absence of cell division. Several transcription factor cocktails have enabled successful reprogramming of various somatic cell types into induced neurons (iNs) of distinct neurotransmitter phenotype. However, the nature of the intermediate states that drive the reprogramming trajectory toward distinct iN types is largely unknown. Here we show that successful direct reprogramming of adult human brain pericytes into functional iNs by Ascl1 and Sox2 encompasses transient activation of a neural stem cell-like gene expression program that precedes bifurcation into distinct neuronal lineages. During this transient state, key signaling components relevant for neural induction and neural stem cell maintenance are regulated by and functionally contribute to iN reprogramming and maturation. Thus, Ascl1- and Sox2-mediated reprogramming into a broad spectrum of iN types involves the unfolding of a developmental program via neural stem cell-like intermediates.

Pumilio2-deficient mice show a predisposition for epilepsy.

Epilepsy is a neurological disease that is caused by abnormal hypersynchronous activities of neuronal ensembles leading to recurrent and spontaneous seizures in human patients. Enhanced neuronal excitability and a high level of synchrony between neurons seem to trigger these spontaneous seizures. The molecular mechanisms, however, regarding the development of neuronal hyperexcitability and maintenance of epilepsy are still poorly understood. Here, we show that pumilio RNA-binding family member 2 (Pumilio2; Pum2) plays a role in the regulation of excitability in hippocampal neurons of weaned and 5-month-old male mice. Almost complete deficiency of Pum2 in adult Pum2 gene-trap mice (Pum2 GT) causes misregulation of genes involved in neuronal excitability control. Interestingly, this finding is accompanied by the development of spontaneous epileptic seizures in Pum2 GT mice. Furthermore, we detect an age-dependent increase in Scn1a (Nav1.1) and Scn8a (Nav1.6) mRNA levels together with a decrease in Scn2a (Nav1.2) transcript levels in weaned Pum2 GT that is absent in older mice. Moreover, field recordings of CA1 pyramidal neurons show a tendency towards a reduced paired-pulse inhibition after stimulation of the Schaffer-collateral-commissural pathway in Pum2 GT mice, indicating a predisposition to the development of spontaneous seizures at later stages. With the onset of spontaneous seizures at the age of 5 months, we detect increased protein levels of Nav1.1 and Nav1.2 as well as decreased protein levels of Nav1.6 in those mice. In addition, GABA receptor subunit alpha-2 (Gabra2) mRNA levels are increased in weaned and adult mice. Furthermore, we observe an enhanced GABRA2 protein level in the dendritic field of the CA1 subregion in the Pum2 GT hippocampus. We conclude that altered expression levels of known epileptic risk factors such as Nav1.1, Nav1.2, Nav1.6 and GABRA2 result in enhanced seizure susceptibility and manifestation of epilepsy in the hippocampus. Thus, our results argue for a role of Pum2 in epileptogenesis and the maintenance of epilepsy.

Immunocytochemical heterogeneity of somatostatin-expressing GABAergic interneurons in layers II and III of the mouse cingulate cortex: A combined immunofluorescence/design-based stereologic study.

Many neurological diseases including major depression and schizophrenia manifest as dysfunction of the GABAergic system within the cingulate cortex. However, relatively little is known about the properties of GABAergic interneurons in the cingulate cortex. Therefore, we investigated the neurochemical properties of GABAergic interneurons in the cingulate cortex of FVB-Tg(GadGFP)45704Swn/J mice expressing green fluorescent protein (GFP) in a subset of GABAergic interneurons (GFP-expressing inhibitory interneurons [GINs]) by means of immunocytochemical and design-based stereologic techniques. We found that GINs represent around 12% of all GABAergic interneurons in the cingulate cortex. In contrast to other neocortical areas, GINs were only found in cortical layers II and III. More than 98% of GINs coexpressed the neuropeptide somatostatin (SOM), but only 50% of all SOM + neurons were GINs. By analyzing the expression of calretinin (CR), calbindin (CB), parvalbumin, and various neuropeptides, we identified several distinct GIN subgroups. In particular, we observed coexpression of SOM with CR and CB. In addition, we found neuropeptide Y expression almost exclusively in those GINs that coexpressed SOM and CR. Thus, with respect to the expression of calcium-binding proteins and neuropeptides, GINs are surprisingly heterogeneous in the mouse cingulate cortex, and the minority of GINs express only one marker protein or peptide. Furthermore, our observation of overlap between the SOM + and CR + interneuron population was in contrast to earlier findings of non-overlapping SOM + and CR + interneuron populations in the human cortex. This might indicate that findings in mouse models of neuropsychiatric diseases may not be directly transferred to human patients. J. Comp. Neurol. 524:2281-2299, 2016. © 2015 Wiley Periodicals, Inc.

Corticosteroids: way upstream.

Studies into the mechanisms of corticosteroid action continue to be a rich bed of research, spanning the fields of neuroscience and endocrinology through to immunology and metabolism. However, the vast literature generated, in particular with respect to corticosteroid actions in the brain, tends to be contentious, with some aspects suffering from loose definitions, poorly-defined models, and appropriate dissection kits. Here, rather than presenting a comprehensive review of the subject, we aim to present a critique of key concepts that have emerged over the years so as to stimulate new thoughts in the field by identifying apparent shortcomings. This article will draw on experience and knowledge derived from studies of the neural actions of other steroid hormones, in particular estrogens, not only because there are many parallels but also because 'learning from differences' can be a fruitful approach. The core purpose of this review is to consider the mechanisms through which corticosteroids might act rapidly to alter neural signaling.