Peer Wulff

Impaired renal Na+ retention in the sgk1-knockout mouse

The serum- and glucocorticoid-regulated kinase (sgk1) is induced by mineralocorticoids and, in turn, upregulates heterologously expressed renal epithelial Na(+) channel (ENaC) activity in Xenopus oocytes. Accordingly, Sgk1 is considered to mediate the mineralocorticoid stimulation of renal ENaC activity and antinatriuresis. Here we show that at standard NaCl intake, renal water and electrolyte excretion is indistinguishable in sgk1-knockout (sgk1(-/-)) mice and wild-type (sgk1(+/+)) mice. In contrast, dietary NaCl restriction reveals an impaired ability of sgk1(-/-) mice to adequately decrease Na(+) excretion despite increases in plasma aldosterone levels and proximal-tubular Na(+) and fluid reabsorption, as well as decreases in blood pressure and glomerular filtration rate.

Exploitation of KESTREL to identify NDRG family members as physiological substrates for SGK1 and GSK3

We detected a protein in rabbit skeletal muscle extracts that was phosphorylated rapidly by SGK1 (serum- and glucocorticoid-induced kinase 1), but not by protein kinase Ba, and identified it as NDRG2 (N-myc downstream-regulated gene 2). SGK1 phosphorylated NDRG2 at Thr330, Ser332 and Thr348 in vitro. All three residues were phosphorylated in skeletal muscle from wild-type mice, but not from mice that do not express SGK1. SGK1 also phosphorylated the related NDRG1 isoform at Thr328, Ser330 and Thr346 (equivalent to Thr330, Ser332 and Thr348 of NDRG2), as well as Thr356 and Thr366. Residues Thr346, Thr356 and Thr366 are located within identical decapeptide sequences GTRSRSHTSE, repeated three times in NDRG1. These threonines were phosphorylated in NDRG1 in the liver, lung, spleen and skeletal muscle of wild-type mice, but not in SGK1-/- mice. Knock-down of SGK1 in HeLa cells using small interfering RNA also suppressed phosphorylation of the threonine residues in the repeat region of NDRG1. The phosphorylation of NDRG1 by SGK1 transformed it into an excellent substrate for GSK3 (glycogen synthase kinase 3), which could then phosphorylate Ser342, Ser352 and Ser362 in the repeat region. Incubation of HeLa cells with the specific GSK3 inhibitor CT 99021 increased the electrophoretic mobility of NDRG1 in HeLa cells, demonstrating that this protein is phosphorylated by GSK3 in cells. Our results identify NDRG1 and NDRG2 as physiological substrates for SGK1, and demonstrate that phosphorylation of NDRG1 by SGK1 primes it for phosphorylation by GSK3.

Hippocampal theta rhythm and its coupling with gamma oscillations require fast inhibition onto parvalbumin-positive interneurons

Hippocampal theta (5–10 Hz) and gamma (35–85 Hz) oscillations depend on an inhibitory network of GABAergic interneurons. However, the lack of methods for direct and cell-type-specific interference with inhibition has prevented better insights that help link synaptic and cellular properties with network function. Here, we generated genetically modified mice (PV-Δγ2) in which synaptic inhibition was ablated in parvalbumin-positive (PV+) interneurons. Hippocampal local field potential and unit recordings in the CA1 area of freely behaving mice revealed that theta rhythm was strongly reduced in these mice. The characteristic coupling of theta and gamma oscillations was strongly altered in PV-Δγ2 mice more than could be accounted for by the reduction in theta rhythm only. Surprisingly, gamma oscillations were not altered. These data indicate that synaptic inhibition onto PV+ interneurons is indispensable for theta- and its coupling to gamma oscillations but not for rhythmic gamma-activity in the hippocampus. Similar alterations in rhythmic activity were obtained in a computational hippocampal network model mimicking the genetic modification, suggesting that intrahippocampal networks might contribute to these effects.

Synaptic inhibition of Purkinje cells mediates consolidation of vestibulo-cerebellar motor learning

Although feedforward inhibition onto Purkinje cells was first documented 40 years ago, we understand little of how inhibitory interneurons contribute to cerebellar function in behaving animals. Using a mouse line (PC-Δγ2) in which GABAA receptor–mediated synaptic inhibition is selectively removed from Purkinje cells, we examined how feedforward inhibition from molecular layer interneurons regulates adaptation of the vestibulo-ocular reflex. Although impairment of baseline motor performance was relatively mild, the ability to adapt the phase of the vestibulo-ocular reflex and to consolidate gain adaptations was strongly compromised. Purkinje cells showed abnormal patterns of simple spikes, both during and in the absence of evoked compensatory eye movements. On the basis of modeling our experimental data, we propose that feedforward inhibition, by controlling the fine-scale patterns of Purkinje cell activity, enables the induction of plasticity in neurons of the cerebellar and vestibular nuclei.

Parvalbumin-positive CA1 interneurons are required for spatial working but not for reference memory

Parvalbumin-positive GABAergic interneurons in cortical circuits are hypothesized to control cognitive function. To test this idea directly, we functionally removed parvalbumin-positive interneurons selectively from hippocampal CA1 in mice. We found that parvalbumin-positive interneurons are dispensable for spatial reference, but are essential for spatial working memory.

Impaired Regulation of Renal K+ Elimination in the sgk1-Knockout Mouse

Serum- and glucocorticoid-regulated kinase 1 (Sgk1) contributes to Na+ reabsorption in the aldosterone-sensitive distal nephron. Sgk1-knockout (sgk1-/-) and littermate wild-type mice (sgk1+/+) were used to test the importance of Sgk1 in renal elimination of K+ . Intravenous application of K+ load under anesthesia increased plasma K+ concentration by 1.3 to 1.4 mM in both sgk1-/- (n = 6) and sgkl+/+ (n = 7) mice. However, the increase of absolute and fractional renal K+ excretion observed in sgk1+/+ was significantly blunted in sgk1-/- animals. Both groups of mice decreased or increased renal K+ excretion to a similar extent after a low (<0.03%) or high (5%) K+ diet for 6 d, respectively. In sgk1+/+, plasma K+ concentration was not significantly modified by either high or low K+ diet. In sgk1-/-, however, high K+ diet enhanced plasma K+ concentration by about 1.6 mM, despite an excessive increase of plasma aldosterone concentration reaching values about sixfold higher than in sgk1+/+. Electrophysiological and immunohistochemical studies under high K+ diet indicated that reduced epithelial Na+ channel ENaC and/or Na+/K+-ATPase activity in the aldosterone-sensitive distal nephron accounted for the impaired response in sgk1-/- and that an enhanced apical abundance of renal outer medullary K+ channel ROMK partly compensated for the defect. The acute and chronic regulation of renal K+ elimination involves Sgk1.

From synapse to behavior: Rapid modulation of defined neuronal types with engineered GABAA receptors

In mammals, identifying the contribution of specific neurons or networks to behavior is a key challenge. Here we describe an approach that facilitates this process by enabling the rapid modulation of synaptic inhibition in defined cell populations. Binding of zolpidem, a systemically active allosteric modulator that enhances the function of the GABAA receptor, requires a phenylalanine residue (Phe77) in the γ2 subunit. Mice in which this residue is changed to isoleucine are insensitive to zolpidem. By Cre recombinase–induced swapping of the γ2 subunit (that is, exchanging Ile77 for Phe77), zolpidem sensitivity can be restored to GABAA receptors in chosen cell types. We demonstrate the power of this method in the cerebellum, where zolpidem rapidly induces significant motor deficits when Purkinje cells are made uniquely sensitive to its action. This combined molecular and pharmacological technique has demonstrable advantages over targeted cell ablation and will be invaluable for investigating many neuronal circuits.

Molecular interrogation of hypothalamic organization reveals distinct dopamine neuronal subtypes

The hypothalamus contains the highest diversity of neurons in the brain. Many of these neurons can co-release neurotransmitters and neuropeptides in a use-dependent manner. Investigators have hitherto relied on candidate protein-based tools to correlate behavioral, endocrine and gender traits with hypothalamic neuron identity. Here we map neuronal identities in the hypothalamus by single-cell RNA sequencing. We distinguished 62 neuronal subtypes producing glutamatergic, dopaminergic or GABAergic markers for synaptic neurotransmission and harboring the ability to engage in task-dependent neurotransmitter switching. We identified dopamine neurons that uniquely coexpress the Onecut3 and Nmur2 genes, and placed these in the periventricular nucleus with many synaptic afferents arising from neuromedin S+ neurons of the suprachiasmatic nucleus. These neuroendocrine dopamine cells may contribute to the dopaminergic inhibition of prolactin secretion diurnally, as their neuromedin S+ inputs originate from neurons expressing Per2 and Per3 and their tyrosine hydroxylase phosphorylation is regulated in a circadian fashion. Overall, our catalog of neuronal subclasses provides new understanding of hypothalamic organization and function.

Abolition of zolpidem sensitivity in mice with a point mutation in the GABAA receptor γ2 subunit

Agonists of the allosteric benzodiazepine site of GABAA receptors bind at the interface of the alpha and gamma subunits. Here, we tested the in vivo contribution of the gamma2 subunit to the actions of zolpidem, an alpha1 subunit selective benzodiazepine agonist, by generating mice with a phenylalanine (F) to isoleucine (I) substitution at position 77 in the gamma2 subunit. The gamma2F77I mutation has no major effect on the expression of GABAA receptor subunits in the cerebellum. The potency of zolpidem, but not that of flurazepam, for the inhibition of [3H]flunitrazepam binding to cerebellar membranes is greatly reduced in gamma2I77/I77 mice. Zolpidem (1 microM) increased both the amplitude and decay of miniature inhibitory postsynaptic currents (mIPSCs) in Purkinje cells of control C57BL/6 (34% and 92%, respectively) and gamma2F77/F77 (20% and 84%) mice, but not in those of gamma2F77I mice. Zolpidem tartrate had no effect on exploratory activity (staircase test) or motor performance (rotarod test) in gamma2I77/I77 mice at doses up to 30 mg/kg (i.p.) that strongly sedated or impaired the control mice. Flurazepam was equally effective in enhancing mIPSCs and disrupting performance in the rotarod test in control and gamma2I77/I77 mice. These results show that the effect of zolpidem, but not flurazepam, is selectively eliminated in the brain by the gamma2F77I point mutation.

The hippocampus in aging and disease: From plasticity to vulnerability.

The hippocampus has a pivotal role in learning and in the formation and consolidation of memory and is critically involved in the regulation of emotion, fear, anxiety, and stress. Studies of the hippocampus have been central to the study of memory in humans and in recent years, the regional specialization and organization of hippocampal functions have been elucidated in experimental models and in human neurological and psychiatric diseases. The hippocampus has long been considered a classic model for the study of neuroplasticity as many examples of synaptic plasticity such as long-term potentiation and -depression have been identified and demonstrated in hippocampal circuits. Neuroplasticity is the ability to adapt and reorganize the structure or function to internal or external stimuli and occurs at the cellular, population, network or behavioral level and is reflected in the cytological and network architecture as well as in intrinsic properties of hippocampal neurons and circuits. The high degree of hippocampal neuroplasticity might, however, be also negatively reflected in the pronounced vulnerability of the hippocampus to deleterious conditions such as ischemia, epilepsy, chronic stress, neurodegeneration and aging targeting hippocampal structure and function and leading to cognitive deficits. Considering this framework of plasticity and vulnerability, we here review basic principles of hippocampal anatomy and neuroplasticity on various levels as well as recent findings regarding the functional organization of the hippocampus in light of the regional vulnerability in Alzheimers disease, ischemia, epilepsy, neuroinflammation and aging.