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Kloppenburg, P.

Publications and source records attributed to Kloppenburg, P..

8 recordsLinked to original sources

GephyrinΔ199-233 - an epileptogenic microdeletion

Gephyrin, as the main organizer of inhibitory synapses, is crucial for inhibitory signal transmission, and implicated in various neurological disorders. Various studies have identified gephyrin microdeletions in conditions of autism, schizophrenia, and epilepsy. Those deletions affected the N-terminal G-domain and/or the central C-domain of gephyrin while the receptor binding C-terminal E-domain was not affected. Here, we investigated the importance of a specific microdeletion ({Delta}199-233) within the C-domain using a full-body knock-in mouse model. Homozygous mice displayed a severe phenotype characterized by reduced fertility, increased mortality, and neurological deficits at early developmental stages. Analyses in dissociated hippocampal neurons demonstrated disrupted synaptic targeting of gephyrin {Delta}199-233 that harbors the functionally important S-palmitoylation site at Cys212. Simultaneously, we found adaptations at the excitatory synapse, with smaller, but more numerous clusters of the excitatory scaffolding protein PSD95. Although, gephyrin {Delta}199-233 showed unexpectedly a facilitated receptor interaction, inhibitory signal transmission was reduced. We hypothesize, that the gephyrin {Delta}199-233-mediated reduction of inhibition triggers compensatory excitation, which possibly fails and/or disrupts the excitation/inhibition ratio in our mouse model. These findings highlight the critical role of the gephyrin C-domain and its post-translational modifications in synaptic function and neuronal health, offering a novel mouse model for the development of potential therapeutic targets addressing gephyrin-associated neurological disorders.

neuroscience↗

Deficiency of Orexin Receptor Type 1 in Dopaminergic Neurons Increases Novelty-Induced Locomotion and Exploration

Orexin signaling in the ventral tegmental area and substantia nigra promotes locomotion and reward processing, but it is not clear whether dopaminergic neurons directly mediate these effects. We show that dopaminergic neurons in these areas mainly express orexin receptor subtype 1 (Ox1R). In contrast, only a minor population in the medial ventral tegmental area express orexin receptor subtype 2 (Ox2R). To analyze the functional role of Ox1R signaling in dopaminergic neurons, we deleted Ox1R specifically in dopamine transporter-expressing neurons of mice and investigated the functional consequences. Deletion of Ox1R increased locomotor activity and exploration during exposure to novel environments or when intracerebroventricularely injected with orexin A. Spontaneous activity in home cages, anxiety, reward processing, and energy metabolism did not change. Positron emission tomography imaging revealed that Ox1R signaling in dopaminergic neurons affected distinct neural circuits depending on the stimulation mode. In line with an increase of neural activity in the lateral paragigantocellular nucleus (LPGi) of Ox1R{Delta}DAT mice, we found that dopaminergic projections innervate the LPGi in regions where the inhibitory dopamine receptor subtype D2 but not the excitatory D1 subtype resides. These data suggest a crucial regulatory role of Ox1R signaling in dopaminergic neurons in novelty-induced locomotion and exploration.

neuroscience↗

Development of a genetically-encoded sensor for probing endogenous nociceptin opioid peptide release

Nociceptin/orphanin-FQ (N/OFQ) is a recently appreciated critical opioid peptide with key regulatory functions in several central behavioral processes including motivation, stress, feeding, and sleep. The functional relevance of N/OFQ action in the mammalian brain remains unclear due to a lack of high-resolution approaches to detect this neuropeptide with appropriate spatial and temporal resolution. Here we develop and characterize NOPLight, a genetically encoded sensor that sensitively reports changes in endogenous N/OFQ release. We characterized the affinity, pharmacological profile, spectral properties, kinetics, ligand selectivity, and potential interaction with intracellular signal transducers of NOPLight in vitro. Its functionality was established in acute brain slices by exogeneous N/OFQ application and chemogenetic induction of endogenous N/OFQ release from PNOC neurons. In vivo studies with fibre photometry enabled direct recording of NOPLight binding to exogenous N/OFQ receptor ligands, as well as detection of endogenous N/OFQ release within the paranigral ventral tegmental area (pnVTA) during natural behaviors and chemogenetic activation of PNOC neurons. In summary, we show here that NOPLight can be used to detect N/OFQ opioid peptide signal dynamics in tissue and freely behaving animals.

neuroscience↗

Autophagy regulates neuronal excitability by controlling cAMP/Protein Kinase A signaling

Autophagy provides nutrients during starvation and eliminates detrimental cellular components. However, accumulating evidence indicates that autophagy is not merely a housekeeping process. Here, we show that the protein AuTophaGy 5 (ATG5) functions in neurons to regulate the cAMP-dependent protein kinase A (PKA)-mediated phosphorylation of a synapse-confined proteome. This function of ATG5 is independent of bulk turnover of synaptic proteins and requires the targeting of PKA inhibitory R1 subunits to autophagosomes. Neuronal loss of ATG5 causes synaptic accumulation of PKA R1, which sequesters the PKA catalytic subunit and diminishes the cAMP/PKA-dependent phosphorylation of postsynaptic cytoskeletal proteins mediating AMPAR trafficking. Glutamatergic neurons-confined ATG5 deletion augments AMPAR-dependent excitatory neurotransmission and causes the appearance of spontaneous recurrent seizures in mice. Our findings identify a novel role of autophagy in regulating PKA signaling at glutamatergic synapses and suggest the PKA as a target for restoration of synaptic function in neurodegenerative conditions with autophagy dysfunction.

neuroscience↗

CaMK1D signaling in AgRP neurons promotes ghrelin-mediated food intake

Hypothalamic AgRP/NPY neurons are key players in the control of feeding behavior. Ghrelin, a major hormone released under fasting conditions, activates orexigenic AgRP/NPY neurons to stimulate food intake and adiposity. However, cell-autonomous ghrelin-dependent signaling mechanisms in AgRP/NPY neurons remain poorly defined. Here we demonstrate that calcium/calmodulin-dependent protein kinase ID (CaMK1D), a genetic hot spot in type 2 diabetes, is activated in hypothalamus upon ghrelin stimulation and acts in AgRP neurons to promote ghrelin-dependent food intake. Global CaMK1D knockout mice are resistant to the orexigenic action of ghrelin, gain less body weight and are protected against high-fat diet-induced obesity. Deletion of CaMK1D in AgRP but not in POMC neurons is sufficient to recapitulate above phenotypes. Lack of CaMK1D attenuates phosphorylation of CREB and CREB-dependent expression of the orexigenic neuropeptides AgRP/NPY as well as the amount of AgRP fiber projections to the Paraventricular nucleus (PVN), while electrical activity of AgRP neurons and 5 AMP-activated protein kinase (AMPK) signaling are unaffected. Hence, CaMK1D links ghrelin action to transcriptional control of orexigenic neuropeptide availability in AgRP neurons. HighlightsO_LIWhole-body deletion of CaMK1D in mice reduces food intake, ghrelin sensitivity and protects against obesity. C_LIO_LIAgRP/NPY neuron-specific deletion of CaMK1D reduces food intake, ghrelin sensitivity, energy expenditure and protects against obesity. C_LIO_LICaMK1D is dispensable for ghrelin-stimulated electrical activity of AgRP neurons and hypothalamic AMPK signaling. C_LIO_LICaMK1D controls phosphorylation of CREB and CREB-dependent expression of the orexigenic neuropeptides AgRP and NPY. C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=174 HEIGHT=200 SRC="FIGDIR/small/471546v1_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@40d7d7org.highwire.dtl.DTLVardef@11a8c64org.highwire.dtl.DTLVardef@1f6fad3org.highwire.dtl.DTLVardef@1cd5581_HPS_FORMAT_FIGEXP M_FIG C_FIG

physiology↗

Alternative splicing of auxiliary β2-subunits stabilizes Cav2.3 Ca2+ channel activity in continuously active midbrain dopamine neurons

In dopaminergic (DA) substantia nigra (SN) neurons Cav2.3 R-type Ca2+-currents contribute to somatodendritic Ca2+-oscillations. These may contribute to the selective degeneration of these neurons in Parkinsons disease (PD) since Cav2.3-knockout is neuroprotective in a PD mouse model. However, the typical Cav2.3 gating would predict complete channel inactivation during SN DA neuronal firing. Here we show that in tsA-201-cells the membrane-anchored {beta}2-splice variants {beta}2a and {beta}2e stabilize Cav2.3 gating properties allowing sustained Cav2.3 availability during simulated pacemaking and enhanced Ca2+-currents during bursts. We confirmed the expression of {beta}2a and {beta}2e-subunits in the SN and identified SN DA neurons. Patch-clamp recordings of SN DA neurons in mouse brain slices revealed R-type Ca2+-currents similar to {beta}2a- or {beta}2e-stabilized Cav2.3-currents and recordings in cultured murine DA neurons confirmed their activity during pacemaking. Taken together, our data support an important (patho)physiological role of {beta}-subunit alternative splicing for Cav2.3 Ca2+-signaling in highly vulnerable SN DA neurons.

neuroscience↗

A Simple Method for Getting Standard Error on the Ratiometric Calcium Estimator

1.The ratiometric fluorescent calcium indicator Fura-2 plays a fundamental role in the investigation of cellular calcium dynamics. Despite of its widespread use in the last 30 years, only one publication [2] proposed a way of obtaining confidence intervals on fitted calcium dynamic model parameters from single calcium transients. Shortcomings of this approach are its requirement for a 3 wavelengths protocol (excitation at 340 and 380 nm as usual plus at 360 nm, the isosbectic point) as well as the need for an autofluorence / background fluorescence model at each wavelength. We propose here a simpler method that eliminates both shortcommings: O_LIa precise estimation of the standard errors of the raw data is obtained first, C_LIO_LIthe standard error of the ratiometric calcium estimator (a function of the raw data values) is derived using both the propagation of uncertainty and a Monte-Carlo method. C_LI Once meaningful standard errors for calcium estimates are available, standard errors on fitted model parameters follow directly from the use of nonlinear least-squares optimization algorithms. 2. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=158 SRC="FIGDIR/small/424015v1_fig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@c830a6org.highwire.dtl.DTLVardef@f20cbforg.highwire.dtl.DTLVardef@122fb2aorg.highwire.dtl.DTLVardef@197b331_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 1:C_FLOATNO How to get error bars on the ratiometric calcium estimator? The figure is to be read clockwise from the bottom right corner. The two measurements areas (region of interest, ROI, on the cell body and background measurement region, BMR, outside of the cell) are displayed on the frame corresponding to one actual experiment. Two measurements, one following an excitation at 340 nm and the other following an excitation at 380 nm are performed (at each time point) from each region. The result is a set of four measures: adu340 (from the ROI), adu340B (from the BMR), adu380 and adu380B. These measurements are modeled as realizations of Gaussian random variables. The fact that the measurements as well as the subsequent quantities derived from them are random variable realization is conveyed throughout the figure by the use of Gaussian probability densities. The densities from the MRB are tighter because there are much more pixels in the MRB than in the ROI (the standard deviations of the densities shown on this figure have been enlarged for clarity, but their relative size has been preserved, the horizontal axis in black always starts at 0). The key result of the paper is that the standard deviation of the four Gaussian densities corresponding to the raw data (bottom of the figure) can be reliably estimated from the data alone, [Figure 1], where V is the product of the CCD chip gain squared by the number of pixels in the ROI by the CCD chip readout variance. The algebric operations leading to the estimator (top right) are explicitely displayed. The paper explains how to compute the standard deviation of the derived distributions obtained at each step of the calcium concentration estimation. C_FIG Method nameStandard error for the ratiometric calcium estimator

neuroscience↗

Task-specific roles of local interneurons for inter -and intraglomerular signaling in the insect antennal lobe

Local interneurons (LNs) mediate complex interactions within the antennal lobe, the primary olfactory system of insects, and the functional analog of the vertebrate olfactory bulb. In the cockroach Periplaneta Americana, as in other insects, several types of LNs with distinctive physiological and morphological properties can be defined. Here, we combined whole-cell patch clamp recordings and Ca2+ imaging of individual LNs to analyze the role of spiking and nonspiking LNs in inter- and intraglomerular signaling during olfactory information processing. Spiking GABAergic LNs reacted to odorant stimulation with a uniform rise in [Ca2+]i in the ramifications of all innervated glomeruli. In contrast, in nonspiking LNs, glomerular Ca2+ signals were odorant specific and varied between glomeruli, resulting in distinct, glomerulus-specific tuning curves. The cell type-specific differences in Ca2+ dynamics support the idea that spiking LNs play a primary role in interglomerular signaling, while they assign nonspiking LNs an essential role in intraglomerular signaling.

neuroscience↗