Wolfgang Müller

Endogenous Ca2+ Buffer Concentration and Ca2+ Microdomains in Hippocampal Neurons

Ca2+-binding proteins are ubiquitously expressed throughout the CNS and serve as valuable immunohistochemical markers for certain types of neurons. However, the functional role of most Ca2+-binding proteins has to date remained obscure because their concentration in central neurons is not known. In this study, we investigate the intracellular concentration of the widely expressed Ca2+-binding protein calbindin-D28k in adult hippocampal slices using patch-clamp recordings and immunohistochemistry. First, we show that calbindin-D28k freely exchanges between patch pipette and cytoplasm during whole cell patch-clamp recordings with a time constant of ∼10 min. Substituting known concentrations of recombinant calbindin-D28k in patch pipettes enabled us to determine the endogenous calbindin-D28k concentration by postrecording immunohistochemistry. Using this calibration procedure, we find that mature granule cells (doublecortin-) contain ∼40 μm, and newborn granule cells (doublecortin+) contain 0-20 μm calbindin-D28k. CA3 stratum radiatum interneurons and CA1 pyramidal cells enclose ∼47 and ∼45 μm calbindin-D28k, respectively. Numerical simulations showed that 40 μm calbindin-D28k is capable of tuning Ca2+ microdomains associated with action potentials at the mouth of single or clustered Ca2+ channels: calbindin-D28k reduces the increment in free Ca2+ at a distance of 100 and 200 nm by 20 and 35%, respectively, and strongly accelerates the collapse of the Ca2+ gradient after cessation of Ca2+ influx. These data suggest that calbindin-D28k equips hippocampal neurons with ∼160 μm mobile, high-affinity Ca2+-binding sites (κS ∼200) that slow and reduce global Ca2+ signals while they enhance the spatiotemporal fidelity of submicroscopic Ca2+ signals.

Altered Ca2+ Signaling and Mitochondrial Deficiencies in Hippocampal Neurons of Trisomy 16 Mice: A Model of Down’s Syndrome

It has been suggested that augmented nerve cell death in neurodegenerative diseases might result from an impairment of mitochondrial function. To test this hypothesis, we investigated age-dependent changes in neuronal survival and glutamate effects on Ca2+ homeostasis and mitochondrial energy metabolism in cultured hippocampal neurons from diploid and trisomy 16 (Ts16) mice, a model of Down’s syndrome. Microfluorometric techniques were used to measure survival rate, [Ca2+]ilevel, mitochondrial membrane potential, and NAD(P)H autofluorescence. We found that Ts16 neurons die more than twice as fast as diploid neurons under otherwise identical culture conditions. Basal [Ca2+]i levels were elevated in Ts16 neurons. Moreover, in comparison to diploid neurons, Ts16 neurons showed a prolonged recovery of [Ca2+]iand mitochondrial membrane potential after brief glutamate application. Glutamate evoked an initial NAD(P)H decrease that was found to be extended in Ts16 neurons in comparison to diploid neurons. Furthermore, for all age groups tested, glutamate failed to cause a subsequent NAD(P)H overshoot in Ts16 cultures in contrast to diploid cultures. In the presence of cyclosporin A, an inhibitor of the mitochondrial membrane permeability transition, NAD(P)H increase was observed in both diploid and Ts16 neurons. The results support the hypothesis that Ca2+ impairs mitochondrial energy metabolism and may play a role in the pathogenesis of neurodegenerative changes in neurons from Ts16 mice.

Carbachol-induced changes in excitability and [Ca2+]i signalling in projection cells of medial entorhinal cortex layers II and III

The entorhinal cortex (EC) is a major gateway for sensory information into the hippocampus and receives a cholinergic input from the forebrain. Therefore, we studied muscarinic effects on excitability and intracellular Ca2+ signalling in layer II stellate and layer III pyramidal projection neurons of the EC. In both classes of neurons, local pressure‐pulse application of carbachol (1 mm) caused small, atropine‐sensitive membrane depolarizations that were not accompanied by any detectable changes in [Ca2+]i. At a higher concentration (10 mm), carbachol induced a larger membrane depolarization associated with synaptic oscillations and epileptiform activity in both classes of neurons. In contrast to the intrinsic theta rhythm in stellate cells with one dominant peak frequency at ∼ 7 Hz, the synaptically mediated oscillation induced by carbachol showed three characteristic peaks in the theta and gamma frequency range at ∼ 11, 23 and 40 Hz. Although carbachol‐induced epileptiform activity was associated with increases in intracellular free Ca2+ in both layer II and III cells, the observed [Ca2+]i accumulation was significantly larger in layer III than in layer II cells. Responses to intracellular current injections showed differences in Ca2+ accumulation in layer II and III cells at the same membrane potentials, suggesting a dominant expression of low‐ and high‐voltage‐activated Ca2+ channels in these layer II and III cells, respectively. In conclusion, we present evidence for significant differences in the [Ca2+]i regulation between layer II stellate and layer III pyramidal cells of the medial EC.

NMDA-Dependent, But Not Group I Metabotropic Glutamate Receptor-Dependent, Long-Term Depression at Schaffer Collateral–CA1 Synapses Is Associated with Long-Term Reduction of Release from the Rapidly Recycling Presynaptic Vesicle Pool

Postsynaptic alterations have been suggested to account for NMDA receptor (NMDAR)-dependent long-term depression (LTD) and long-term potentiation of synaptic strength, although there is substantial evidence supporting changes in presynaptic release. Direct chemical activation of either NMDA or group I metabotropic glutamate receptor (mGluR1) elicits LTD of similar magnitudes, but it is unknown whether they share common expression mechanisms. Using dual-photon laser-scanning microscopy of FM1-43 [N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino)styryl)pyridinium dibromide] to directly visualize presynaptic vesicular release from the rapidly recycling vesicle pool (RRP) at Schaffer collateral terminals in field CA1 of rat hippocampal slices, we found that a persistent reduction in vesicular release from the RRP is induced by NMDA-LTD but not by mGluR1-LTD. Variance-mean analyses of Schaffer collateral release probability (Pr) at varying extracellular calcium concentrations confirmed that NMDA-LTD was associated with reduced Pr, whereas mGluR1-LTD was not. Pharmacological isolation of NMDAR-dependent and mGluR-dependent forms of stimulus-evoked LTD revealed that both are composed of a combination of presynaptic and postsynaptic alterations. However, when group I mGluR-dependent LTD was isolated by combining an NMDAR blocker with a group II mGluR antagonist, this form of LTD was purely postsynaptic. The nitric oxide synthase inhibitor Nω-nitro-l-arginine blocked the induction of NMDA-LTD but did not alter mGluR-LTD, consistent with a selective role for nitric oxide as a retrograde messenger mediating NMDA-LTD. These data demonstrate that single synapses can express multiple forms of LTD with different sites of expression, that NMDA-LTD is a combination of presynaptic and postsynaptic alterations, but that group I mGluR-LTD appears to be expressed entirely postsynaptically.

Muscarinic control of intracortical connections to layer II in rat entorhinal cortex slice.

The cholinergic system is critically involved in oscillatory network activity and synaptic plasticity in the entorhinal cortex (EC) hippocampal formation. Here we demonstrate robust inhibition of field potentials in layer II of the medial EC evoked by stimulation in the deep EC or in the lateral layer II by carbachol (CCh, 0.1-100 microM, K(D) approximately 1 microM). This effect appears not to be mediated by suppression of presynaptic Ca(2+)-signals since paired pulse facilitation was increased by CCh. Blockade of the effect by the muscarinic antagonists atropine and pirenzepine demonstrates mediation by muscarinic receptors, most likely of the M1 subtype. The effect is characterized by absence of desensitization and should be important for laminar shaping of oscillatory activity and synaptic plasticity during acetylcholine-dependent theta-rhythmic activity.

Two-photon imaging of spontaneous vesicular release in acute brain slices and its modulation by presynaptic GABAA receptors.

Action potential-independent spontaneous vesicular release of gamma-aminobutyric acid (GABA) in the CNS mediates miniature inhibitory postsynaptic currents (mIPSCs) and exerts an important control on central excitability. Using dual-photon laser scan microscopy and hyperosmotic loading of the readily releasable vesicle pool with the fluorescent styryl dye FM1-43 in hippocampal slice, we demonstrate action potential-independent release of vesicles (fluorescence destaining) from proximal perisomatic, presumed GABAergic terminals and significant inhibition of this release by the specific GABA(A) receptor agonist muscimol in the presence of tetrodotoxin and glutamate receptor antagonists CNQX and AP5. These data agree with reduction of mIPSCs by muscimol in whole-cell recordings from CA3 pyramidal neurons. In contrast, rate of vesicle release from distal, presumably glutamatergic terminals, was significantly lower and not changed by muscimol. The effect of muscimol on mIPSCs was not blocked but rather enhanced in the absence of external calcium. Our data directly demonstrate a potent disinhibitory reduction of GABA release by GABA(A) receptor activation. Those novel methods should be well suited to study pathophysiological changes in inhibition in resections obtained from neurosurgical treatment of epilepsy patients.

Ca2+-independent muscarinic excitation of rat medial entorhinal cortex layer V neurons

Cholinergic activation of entorhinal cortex (EC) layer V neurons plays a crucial role in the medial temporal lobe memory system and in the pathophysiology of temporal lobe epilepsy. Here, we demonstrate that muscarinic activation by focal application of carbachol depolarizes EC layer V neurons and induces epileptiform activity in rat brain slices. These seizure‐like bursts are associated with a somatic [Ca2+]i increase of 293 ± 82 nm and are blocked by the glutamate receptor antagonists CNQX and APV. Muscarinic activation did not directly evoke a [Ca2+]i increase, but subthreshold and suprathreshold depolarization did. Functional axon mapping revealed local axon branching as well as axon collaterals ascending to layers II and III. During blockade of ionotropic glutamatergic AMPA and NMDA receptors, carbachol depolarized layer V neurons by +7.5 ± 3.4 mV. This direct muscarinic depolarization was associated with a conductance increase of 35 ± 10.3% (+4.3 ± 1.25 nS). Intracellular buffering of [Ca2+]i changes did not block this depolarization, but prolonged action potential duration and reduced adaptation of action potential firing. The muscarinic depolarization was neither blocked by combining intracellular Ca2+‐buffering (EGTA or BAPTA) with non‐specific Ca2+‐channel inhibition by Ni+ (1 mm), nor by Ba2+ (1 mm) nor during inhibition of the h‐current by 2 mm Cs+. In whole‐cell patch‐clamp recording, reversal of the muscarinic current occurred at about −45 mV and −5 mV with complete substitution of intrapipette K+ with Cs+. Thus, muscarinic depolarization of EC layer V neurons appears to be primarily mediated by Ca2+‐independent activation of non‐specific cation channels that conduct K+ about three times as well as Na+.

Impaired Ca-signaling in astrocytes from the Ts16 mouse model of Down syndrome.

The trisomy 16 mouse model of Down syndrome has been used to compare calcium (Ca)-homeostasis and Ca-signaling in astrocytes from trisomic mice and from diploid littermates. Ratio calcium-imaging of Fura-2/AM loaded primary astroglial cultures prepared from the hippocampus shows that resting Ca levels are on average significantly higher in trisomic than in the control astrocytes (280 vs. 120 nM). Serotonin (3 microM) and glutamate (30-300 microM) evoked transient Ca-increases from 400 to 600 nM in euploid but from only 20 to 150 nM in trisomic astrocytes. Imaging of ATP-driven Ca-accumulation in cellular organelles revealed a significantly stronger uptake of Ca in trisomic astrocytes that might buffer cytosolic Ca-increases. Our results demonstrate major disturbances in Ca-signaling in trisomic astrocytes that are likely to be of pathophysiological relevance.

Direct monitoring of vesicular release and uptake in brain slices by multiphoton excitation of the styryl FM ® 1-43

Fluorescence imaging using FM 1-43 and related styryl dyes has provided invaluable insights into presynaptic function of synapses in culture preparations, but has been limited in use for studying central synapses in vivo or in brain slices, because of excessive fluorescence background due to nonspecific membrane binding of dye. We demonstrate here that focal excitation of FM dyes using two-photon laser-scanning microscopy (TPLSM) provides high resolution of FM 1-43-labeled nerve terminals in brain slices by suppressing out-of-focus background and that a readily releasable pool of vesicles can be selectively and stably labeled by hypertonic shock despite slice diffusion barriers. We find direct TPLSM of FM 1-43-labeled nerve terminals to be superior to treatment of slices with either the fluorescent quencher sulforhodamine 101 or dye scavenger ADVASEP-7 in resolving nerve terminal against background fluorescence, enabling continuous monitoring of vesicular uptake, and release of styryl dyes from individual nerve terminals in brain slices.

Subcellular muscarinic enhancement of excitability and Ca2+-signals in CA1-dendrites in rat hippocampal slice.

The cholinergic system is involved in Ca2+-dependent models of learning. To study subcellular modulation, we evoked 50-100 microm long dendritic Ca2+-responses by focal pressure application of glutamate. These Ca2+-responses were augmented by +70% by focally applied carbachol. This atropine-sensitive augmentation started within 1 s concurrent to an augmentation of the glutamate-evoked somatic depolarization and firing. Tetrodotoxin reduced the Ca2+-response to glutamate by 60-80% while, after having restored the Ca2+-signal by increasing the application of glutamate, its muscarinic augmentation was reduced from +73 to +30%. Lithium (2 mM, >2 h) slowed and reduced augmentation of Ca2+-signals and blocked augmentation of the glutamate-evoked depolarization and firing, but not suppression of the slow after-hyperpolarization following repetitive discharge. Thus, several mechanisms contribute to muscarinic augmentation of Ca2+-signals.