Jens Eilers

Importance of the Intracellular Domain of NR2 Subunits for NMDA Receptor Function In Vivo

NMDA receptors, a class of glutamate-gated cation channels with high Ca2+ conductance, mediate fast transmission and plasticity of central excitatory synapses. We show here that gene-targeted mice expressing NMDA receptors without the large intracellular C-terminal domain of any one of three NR2 subunits phenotypically resemble mice made deficient in that particular subunit. Mice expressing the NR2B subunit in a C-terminally truncated form (NR2B(deltaC/deltaC) mice) die perinatally. NR2A(deltaC/deltaC) mice are viable but exhibit impaired synaptic plasticity and contextual memory. These and NR2C(deltaC/deltaC) mice display deficits in motor coordination. C-terminal truncation of NR2 subunits does not interfere with the formation of gateable receptor channels that can be synaptically activated. Thus, the phenotypes of our mutants appear to reflect defective intracellular signaling.

A new class of synaptic response involving calcium release in dendritic spines

In the classical view, transmission of signals across synapses in the mammalian brain involves changes in the membrane potential of the postsynaptic cell. The use of high-resolution cellular imaging has revealed excitatory synapses at which postsynaptic, transient alterations in calcium ion concentration are tightly associated with electrical responses (reviewed in ref. 1). Here, by investigating the synapse between parallel glutamatergic fibres and Purkinje cells in the mouse cerebellum, we identify a class of postsynaptic responses that consist of transient increases in dendritic Ca2+ concentration but not changes in somatic membrane potential. Our results indicate that these synaptic Ca2+ transients are mediated by activation of metabotropic glutamate-responsive mGluR1-type receptors and require inositol-1,4,5-trisphosphate-mediated Ca2+ release, from intradendritic stores. The new type of synaptic response is restricted to postsynaptic microdomains, which range, depending on the frequency of stimulation, from individual spines to small spinodendritic compartments. Thus, the synaptic Ca2+-release signal may be one of the critical cues that determine the input specificity of long-term depression, a well-established form of activity-dependent plasticity at these synapses.

Impaired Synaptic Plasticity and Motor Learning in Mice with a Point Mutation Implicated in Human Speech Deficits

Summary The most well-described example of an inherited speech and language disorder is that observed in the multigenerational KE family, caused by a heterozygous missense mutation in the FOXP2 gene [1]. Affected individuals are characterized by deficits in the learning and production of complex orofacial motor sequences underlying fluent speech and display impaired linguistic processing for both spoken and written language [2]. The FOXP2 transcription factor is highly similar in many vertebrate species, with conserved expression in neural circuits related to sensorimotor integration and motor learning [3, 4]. In this study, we generated mice carrying an identical point mutation to that of the KE family, yielding the equivalent arginine-to-histidine substitution in the Foxp2 DNA-binding domain. Homozygous R552H mice show severe reductions in cerebellar growth and postnatal weight gain but are able to produce complex innate ultrasonic vocalizations. Heterozygous R552H mice are overtly normal in brain structure and development. Crucially, although their baseline motor abilities appear to be identical to wild-type littermates, R552H heterozygotes display significant deficits in species-typical motor-skill learning, accompanied by abnormal synaptic plasticity in striatal and cerebellar neural circuits.

STIM2 regulates capacitive Ca2+ entry in neurons and plays a key role in hypoxic neuronal cell death.

Neurons lacking the calcium sensor STIM2 are protected from hypoxia-induced cell death. Resisting Ischemia Loss of blood flow to the brain—as can occur during a stroke—leads to the death of neurons, a process that involves a pathological increase in intracellular calcium. Berna-Erro et al. investigated the role of capacitive calcium entry (CCE), a process in which depletion of calcium from intracellular stores triggers its entry across the plasma membrane, in ischemia-induced calcium entry and neuronal death. The calcium-sensing molecule STIM1 is known to play a crucial role in mediating CCE in various cell types; in neurons, however, Berna-Erro et al. found that CCE depended instead on the closely related molecule STIM2. Neurons from mice lacking STIM2 were resistant to the effects of hypoxia in vitro; moreover, mice lacking STIM2 showed less neurological damage than did wild-type mice in a model of ischemic stroke. Thus, the authors conclude that STIM2 is critical to neuronal CCE and that CCE plays a role in neuronal death in ischemia. Excessive cytosolic calcium ion (Ca2+) accumulation during cerebral ischemia triggers neuronal cell death, but the underlying mechanisms are poorly understood. Capacitive Ca2+ entry (CCE) is a process whereby depletion of intracellular Ca2+ stores causes the activation of plasma membrane Ca2+ channels. In nonexcitable cells, CCE is controlled by the endoplasmic reticulum (ER)–resident Ca2+ sensor STIM1, whereas the closely related protein STIM2 has been proposed to regulate basal cytosolic and ER Ca2+ concentrations and make only a minor contribution to CCE. Here, we show that STIM2, but not STIM1, is essential for CCE and ischemia-induced cytosolic Ca2+ accumulation in neurons. Neurons from Stim2−/− mice showed significantly increased survival under hypoxic conditions compared to neurons from wild-type controls both in culture and in acute hippocampal slice preparations. In vivo, Stim2−/− mice were markedly protected from neurological damage in a model of focal cerebral ischemia. These results implicate CCE in ischemic neuronal cell death and establish STIM2 as a critical mediator of this process.

Mutational analysis of dendritic Ca2+ kinetics in rodent Purkinje cells: role of parvalbumin and calbindin D28k

The mechanisms governing the kinetics of climbing fibre‐mediated Ca2+ transients in spiny dendrites of cerebellar Purkinje cells (PCs) were quantified with high‐resolution confocal Ca2+ imaging. Ca2+ dynamics in parvalbumin (PV−/−) and parvalbumin/calbindin D28k null‐mutant (PV/CB−/−) mice were compared with responses in wild‐type (WT) animals. In the WT, Ca2+ transients in dendritic shafts were characterised by double exponential decay kinetics that were not due to buffered Ca2+ diffusion or saturation of the indicator dye. Ca2+ transients in PV−/− PCs reached the same peak amplitude as in the WT but the biphasic nature of the decay was less pronounced, an effect that could be attributed to PVs slow binding kinetics. In contrast, peak amplitudes in PV/CB−/− PCs were about two times higher than in the WT and the decay became nearly monophasic. Numerical simulations indicate that the residual deviation from a single exponential decay in PV/CB−/− is due to saturation of the Ca2+ indicator dye. Furthermore, the simulations imply that the effect of uncharacterised endogenous Ca2+ binding proteins is negligible, that buffered diffusion and dye saturation significantly affects spineous Ca2+ transients but not those in the dendritic shafts, and that neither CB nor PV undergoes saturation in spines or dendrites during climbing fibre‐evoked Ca2+ transients. Calbindins medium‐affinity binding sites are fast enough to reduce the peak amplitude of the Ca2+ signal. However, similar to PV, delayed binding by CB leads to biphasic Ca2+ decay kinetics. Our results suggest that the distinct kinetics of PV and CB underlie the biphasic kinetics of synaptically evoked Ca2+ transients in dendritic shafts of PCs.

Bassoon Speeds Vesicle Reloading at a Central Excitatory Synapse

Summary Sustained rate-coded signals encode many types of sensory modalities. Some sensory synapses possess specialized ribbon structures, which tether vesicles, to enable high-frequency signaling. However, central synapses lack these structures, yet some can maintain signaling over a wide bandwidth. To analyze the underlying molecular mechanisms, we investigated the function of the active zone core component Bassoon in cerebellar mossy fiber to granule cell synapses. We show that short-term synaptic depression is enhanced in Bassoon knockout mice during sustained high-frequency trains but basal synaptic transmission is unaffected. Fluctuation and quantal analysis as well as quantification with constrained short-term plasticity models revealed that the vesicle reloading rate was halved in the absence of Bassoon. Thus, our data show that the cytomatrix protein Bassoon speeds the reloading of vesicles to release sites at a central excitatory synapse.

Rapid Active Zone Remodeling during Synaptic Plasticity

How can synapses change the amount of neurotransmitter released during synaptic plasticity? Although release in general is intensely investigated, its determinants during plasticity are still poorly understood. As a model for plastic strengthening of synaptic release, we here use the well-established presynaptic homeostatic compensation during interference with postsynaptic glutamate receptors at the Drosophila neuromuscular junction. Combining short-term plasticity analysis, cumulative EPSC analysis, fluctuation analysis, and quantal short-term plasticity modeling, we found an increase in the number of release-ready vesicles during presynaptic strengthening. High-resolution light microscopy revealed an increase in the amount of the active zone protein Bruchpilot and an enlargement of the presynaptic cytomatrix structure. Furthermore, these functional and structural alterations of the active zone were not only observed after lifelong but already after minutes of presynaptic strengthening. Our results demonstrate that presynaptic plasticity can induce active zone remodeling, which regulates the number of release-ready vesicles within minutes.

GABA-mediated Ca2+ signalling in developing rat cerebellar Purkinje neurones

1 Cellular responses to GABAA receptor activation were studied in developing cerebellar Purkinje neurones (PNs) in brain slices obtained from 2‐ to 22‐day‐old rats. Two‐photon fluorescence imaging of fura‐2‐loaded cells and perforated‐patch recordings were used to monitor intracellular Ca2+ transients and to estimate the reversal potential of GABA‐induced currents, respectively. 2 During the 1st postnatal week, focal application of GABA or the GABAA receptor agonist muscimol evoked transient increases in [Ca2+]i in immature PNs. These Ca2+ transients were reversibly abolished by the GABAA receptor antagonist bicuculline and by Ni2+, a blocker of voltage‐activated Ca2+ channels. 3 Perforated‐patch recordings were used to measure the reversal potential of GABA‐evoked currents (EGABA) at different stages of development. It was found that EGABA was about −44 mV at postnatal day 3 (P3), it shifted to gradually more negative values during the 1st week and finally equilibrated at −87 mV at around the end of the 2nd postnatal week. This transition was well described by a sigmoidal function. The largest change in EGABA was −7 mV day−1, which occurred at around P6. 4 The transition in GABA‐mediated signalling occurs during a period in which striking changes in PN morphology and synaptic connectivity are known to take place. Since such changes were shown to be Ca2+ dependent, we propose that GABA‐evoked Ca2+ signalling is one of the critical determinants for the normal development of cerebellar PNs.

Local proliferation of macrophages in adipose tissue during obesity-induced inflammation

Aims/hypothesisObesity is frequently associated with low-grade inflammation of adipose tissue (AT), and the increase in adipose tissue macrophages (ATMs) is linked to an increased risk of type 2 diabetes. Macrophages have been regarded as post-mitotic, but recent observations have challenged this view. In this study, we tested the hypothesis that macrophages proliferate within AT in diet-induced obesity in mice and humans.MethodsWe studied the expression of proliferation markers by immunofluorescence, PCR and flow cytometry in three different models of mouse obesity as well as in humans (n = 239). The cell fate of dividing macrophages was assessed by live imaging of AT explants.ResultsWe show that ATMs undergo mitosis within AT, predominantly within crown-like structures (CLS). We found a time-dependent increase in ATM proliferation when mice were fed a high-fat diet. Upregulation of CD206 and CD301 in proliferating ATMs indicated preferential M2 polarisation. Live imaging within AT explants from mice revealed that macrophages emigrate out of the CLS to become resident in the interstitium. In humans, we confirmed the increased expression of proliferation markers of CD68+ macrophages in CLS and demonstrated a higher mRNA expression of the proliferation marker Ki67 in AT from obese patients.Conclusions/interpretationLocal proliferation contributes to the increase in M2 macrophages in AT. Our data confirm CLS as the primary site of proliferation and a new source of ATMs and support a model of different recruitment mechanisms for classically activated (M1) and alternatively activated (M2) macrophages in obesity.

Axonal calcium entry during fast ‘sodium’ action potentials in rat cerebellar Purkinje neurones.

1. Using laser‐scanning confocal microscopy, fast Ca2+ transients were recorded in individual not yet myelinated axons of Purkinje neurones in cerebellar slices from young rats. Axonal Ca2+ transients could be detected during a single action potential and had progressively larger amplitudes when the number of action potentials was increased. 2. Under voltage‐clamp conditions, axonal Ca2+ transients were as large as those observed in dendrites and in the cell body. Axonal Ca2+ transients were completely blocked by 100 nM of the neurotoxin omega‐agatoxin IVA, indicating that they were caused by Ca2+ entry through P‐type voltage‐gated Ca2+ channels. 3. In conclusion, our results demonstrate action potential‐mediated Ca2+ entry through voltage‐gated Ca2+ channels in axons of cerebellar Purkinje neurones. Experimental evidence indicates that the resulting transient Ca2+ accumulations regulate the frequency of action potentials travelling along the axon.