Biomedical subjects
Arthur Konnerth
Publications and source records attributed to Arthur Konnerth.
Optical monitoring of brain function in vivo: from neurons to networks.
The precise understanding of the cellular and molecular basis of brain function requires the direct assessment of the activity of defined cells in vivo. A promising approach for such analyses is two-photon microscopy in combination with appropriate cell labeling techniques. Here, we review the multi-cell bolus loading (MCBL) method that involves the use of membrane-permeant fluorescent indicator dyes. We show that this approach is useful for the functional analysis of clusters of neurons and glial cells in vivo. Work from our and other laboratories shows that the techniques that were previously feasible only in brain slices, like targeted patch clamp recordings from identified cells or pharmacological manipulations in confined brain regions, can now be used also in vivo. We also show that MCBL and two-photon imaging can be easily combined with other labeling techniques, particularly with those involving the use of genetically encoded, green-fluorescent-protein-based indicators. Finally, we examine recent applications of MCBL/two-photon imaging for the analysis of various brain regions, including the somatosensory and the visual cortex.
Dendritic spikes and activity-dependent synaptic plasticity.
Whereas the regenerative nature of action potential conduction in axons has been known since the late 1940s, neuronal dendrites have been considered as passive cables transferring incoming synaptic activity to the soma. The relatively recent discovery that neuronal dendrites contain active conductances has revolutionized our view of information processing in neurons. In many neuronal cell types, sodium action potentials initiated at the axon initial segment can back-propagate actively into the dendrite thereby serving, for the dendrite, as an indicator of the output activity of the neuron. In addition, the dendrites themselves can initiate action-potential-like regenerative responses, so-called dendritic spikes, that are mediated either by the activation of sodium, calcium, and/or N-methyl-D-aspartate receptor channels. Here, we review the recent experimental and theoretical evidence for a role of regenerative dendritic activity in information processing within neurons and, especially, in activity-dependent synaptic plasticity.
Quantitative single-cell RT-PCR and Ca2+ imaging in brain slices.
We have established a quantitative reverse transcriptase-PCR (RT-PCR) approach for the analysis of RNA transcript levels in individual cells of living brain slices. Quantification is achieved by using rapid-cycle, real-time PCR protocols and high-resolution external cDNA standard curves for the gene of interest. The method consists of several procedures, including cell soma harvest, reverse transcription, and an optimized cDNA purification step, which allowed us to quantify transcripts in small types of neurons, like cerebellar granule cells. Thus, we detected in single granule cells an average of 20 transcript copies of the housekeeping gene glyceraldehyde-3-phosphate-dehydrogenase. We combined two-photon calcium imaging and quantitative RT-PCR in single Purkinje and granule cells, respectively, and identified distinct glutamate receptor-dependent Ca2+ responses in these two cell types. The approach was further tested by profiling the expression of the ionotropic glutamate receptor subunits NR2B and NR2C in the cerebellum. Our study revealed a developmental switch from an average of 15 NR2B copies/cell at postnatal day 8 (P8) to about five NR2C copies/cell after P26. Taken together, our results demonstrate that the new method is rapid, highly sensitive, provides reliable results in neurons of various sizes, and can be used in combination with Ca2+ imaging.
Cortical calcium waves in resting newborn mice.
Using a new optical fiber-based approach, we demonstrate the presence of recurrent Ca(2+) transients in cortical neurons of non-anesthetized newborn mice in vivo. These Ca(2+) waves reflect the correlated activity of thousands of cells and were detected only in resting, but not in moving pups. Our results suggest that Ca(2+)-dependent cortical maturation occurs predominantly during the intermittent sleep-like resting periods that are characteristic for the first days of postnatal life.
Determinants of postsynaptic Ca2+ signaling in Purkinje neurons.
Neuronal integration in Purkinje neurons involves many forms of Ca2+ signaling. Two afferent synaptic inputs, the parallel and the climbing fibers, provide a major drive for these signals. These two excitatory synaptic inputs are both glutamatergic. Postsynaptically they activate alpha-amino-3-hydroxy-5-methyl-4-propionic acid (AMPA) receptors (AMPARs) and metabotropic glutamate receptors (mGluRs). Unlike most other types of central neurons, Purkinje neurons do not express NMDA (N-methyl-D-aspartate) receptors (NMDARs). AMPARs in Purkinje neurons are characterized by a low permeability for Ca2+ ions. AMPAR-mediated synaptic depolarization may activate voltage-gated Ca2+ channels, mostly of the P/Q-type. The resulting intracellular Ca2+ signals are shaped by the Ca2+ buffers calbindin and parvalbumin. Ca2+ clearance from the cytosol is brought about by Ca2+-ATPases in the plasma membrane and the endoplasmic reticulum, as well as the Na+-Ca2+-exchanger. Binding of glutamate to mGluRs induces postsynaptic Ca2+-transients through two G protein-dependent pathways: involving (1) the release of Ca2+ ions from intracellular Ca2+ stores and (2) the opening of the cation channel TRPC1. Homer proteins appear to play an important role in postsynaptic Ca2+ signaling by providing a direct link between the plasma membrane-resident elements (mGluRs and TRPC1) and their intracellular partners, including the IP3Rs.
Neurotrophin-mediated rapid signaling in the central nervous system: mechanisms and functions.
Neurotrophins regulate growth, survival, and differentiation of central neurons. In addition to the "classical" effects that are relatively slow neurotrophins also elicit rapid signaling that modulates a variety of cellular functions such as membrane excitability, synaptic transmission, and activity-dependent synaptic plasticity. These rapid actions are mediated mainly through the interaction of Trk receptors with ion channels and ionotropic receptors in the plasma membrane.
Single-shock LTD by local dendritic spikes in pyramidal neurons of mouse visual cortex.
Mammalian dendrites are active structures, capable of regenerative electrical activity. Dendritic spikes can mediate synaptic plasticity and could enrich the computational properties of neurons. Besides sodium-based action potentials, which can propagate throughout the dendritic tree, neocortical pyramidal neurons also sustain dendritic spikes that are spatially restricted. The function of these 'local' dendritic spikes is unknown. We show that local spikes, which require activation of N-methyl-d-aspartate receptors (NMDARs), induce long-term synaptic depression (LTD) in layer 5 pyramidal neurons. This depression does not require somatic spiking and is input specific. Moreover, a single synaptic stimulus can evoke a dendritic spike and a brief local dendritic calcium transient, and is sufficient for the full induction of LTD.
From modulator to mediator: rapid effects of BDNF on ion channels.
Neurotrophins (NTs) are [?AUTHOR] a family of structurally related, secreted proteins that regulate the survival, differentiation and maintenance of function of different populations of peripheral and central neurons.1,2 Among these, BDNF (brain-derived neurotrophic factor) has drawn considerable interest because both its synthesis and secretion are increased by physiological levels of activity, indicating a unique role of this neurotrophin in coupling neuronal activity to structural and functional properties of neuronal circuits. In addition to its classical neurotrophic effects, which are evident within hours or days and which usually result from changes in cellular gene expression, BDNF exerts acute effects on synaptic transmission and is involved in the induction of long-term potentiation. Many of these rapid effects of BDNF are mediated by its modulation of ion channel properties following TrkB-mediated activation of intracellular second messenger cascades and protein phosphorylation. However, recent reports have shown that BDNF not only acts as a modulator of ion channels, but can also directly and rapidly gate a Na(+) channel, thereby assigning BDNF the properties of a classical excitatory transmitter. Thus, BDNF, in addition its role as a potent neuromodulator, emerges as an excitatory transmitter-like substance which acutely controls resting membrane potential, neuronal excitability, synaptic transmission and participates in the induction of synaptic plasticity.
Neurotrophin action on a rapid timescale.
Mechanisms underlying the fast action of neurotrophins include intracellular Ca(2+) signaling, neuronal excitation, augmentation of synaptic excitation by modulation of N-methyl-d-aspartate receptor activity and control of synaptic inhibition through the regulation of the K(+)-Cl(-) cotransporter KCC2. The fastest action of brain-derived neurotrophic factor and neurotrophin-4/5 occurs within milliseconds, and involves activation of TrkB and the opening of the Na(+) channel Na(v)1.9. Through these rapid actions, neurotrophins shape neuronal activity, modulate synaptic transmission and produce instructive signals for the induction of long-term changes in the efficacy of synaptic transmission.
Truncated TrkB-T1 mediates neurotrophin-evoked calcium signalling in glia cells.
The neurotrophin receptor TrkB is essential for normal function of the mammalian brain. It is expressed in three splice variants. Full-length receptors (TrkB(FL)) possess an intracellular tyrosine kinase domain and are considered as those TrkB receptors that mediate the crucial effects of brain-derived neurotrophic factor (BDNF) or neurotrophin 4/5 (NT-4/5). By contrast, truncated receptors (TrkB-T1 and TrkB-T2) lack tyrosine kinase activity and have not been reported to elicit rapid intracellular signalling. Here we show that astrocytes predominately express TrkB-T1 and respond to brief application of BDNF by releasing calcium from intracellular stores. The calcium transients are insensitive to the tyrosine kinase blocker K-252a and persist in mutant mice lacking TrkB(FL). By contrast, neurons produce rapid BDNF-evoked signals through TrkB(FL) and the Na(v)1.9 channel. Expression of antisense TrkB messenger RNA strongly reduces BDNF-evoked calcium signals in glia. Thus, our results show that, unexpectedly, TrkB-T1 has a direct signalling role in mediating inositol-1,4,5-trisphosphate-dependent calcium release; in addition, they identify a previously unknown mechanism of neurotrophin action in the brain.
Impairment of LTD and cerebellar learning by Purkinje cell-specific ablation of cGMP-dependent protein kinase I.
The molecular basis for cerebellar plasticity and motor learning remains controversial. Cerebellar Purkinje cells (PCs) contain a high concentration of cGMP-dependent protein kinase type I (cGKI). To investigate the function of cGKI in long-term depression (LTD) and cerebellar learning, we have generated conditional knockout mice lacking cGKI selectively in PCs. These cGKI mutants had a normal cerebellar morphology and intact synaptic calcium signaling, but strongly reduced LTD. Interestingly, no defects in general behavior and motor performance could be detected in the LTD-deficient mice, but the mutants exhibited an impaired adaptation of the vestibulo-ocular reflex (VOR). These results indicate that cGKI in PCs is dispensable for general motor coordination, but that it is required for cerebellar LTD and specific forms of motor learning, namely the adaptation of the VOR.
In vivo two-photon calcium imaging of neuronal networks.
Two-photon calcium imaging is a powerful means for monitoring the activity of distinct neurons in brain tissue in vivo. In the mammalian brain, such imaging studies have been restricted largely to calcium recordings from neurons that were individually dye-loaded through microelectrodes. Previous attempts to use membrane-permeant forms of fluorometric calcium indicators to load populations of neurons have yielded satisfactory results only in cell cultures or in slices of immature brain tissue. Here we introduce a versatile approach for loading membrane-permeant fluorescent indicator dyes in large populations of cells. We established a pressure ejection-based local dye delivery protocol that can be used for a large spectrum of membrane-permeant indicator dyes, including calcium green-1 acetoxymethyl (AM) ester, Fura-2 AM, Fluo-4 AM, and Indo-1 AM. We applied this dye-loading protocol successfully in mouse brain tissue at any developmental stage from newborn to adult in vivo and in vitro. In vivo two-photon Ca2+ recordings, obtained by imaging through the intact skull, indicated that whisker deflection-evoked Ca2+ transients occur in a subset of layer 2/3 neurons of the barrel cortex. Thus, our results demonstrate the suitability of this technique for real-time analyses of intact neuronal circuits with the resolution of individual cells.
Calbindin in cerebellar Purkinje cells is a critical determinant of the precision of motor coordination.
Long-term depression (LTD) of Purkinje cell-parallel fiber synaptic transmission is a critical determinant of normal cerebellar function. Impairment of LTD through, for example, disruption of the metabotropic glutamate receptor-IP3-calcium signaling cascade in mutant mice results in severe deficits of both synaptic transmission and cerebellar motor control. Here, we demonstrate that selective genetic deletion of the calcium-binding protein calbindin D-28k (calbindin) from cerebellar Purkinje cells results in distinctly different cellular and behavioral alterations. These mutants display marked permanent deficits of motor coordination and sensory processing. This occurs in the absence of alterations in a form of LTD implicated in the control of behavior. Analysis of synaptically evoked calcium transients in spines and dendrites of Purkinje cells demonstrated an alteration of time course and amplitude of fast calcium transients after parallel or climbing fiber stimulation. By contrast, the delayed metabotropic glutamate receptor-mediated calcium transients were normal. Our results reveal a unique role of Purkinje cell calbindin in a specific form of motor control and suggest that rapid calcium buffering may directly control behaviorally relevant neuronal signal integration.
Absence epilepsy and sinus dysrhythmia in mice lacking the pacemaker channel HCN2.
Hyperpolarization-activated cation (HCN) channels are believed to be involved in the generation of cardiac pacemaker depolarizations as well as in the control of neuronal excitability and plasticity. The contributions of the four individual HCN channel isoforms (HCN1-4) to these diverse functions are not known. Here we show that HCN2-deficient mice exhibit spontaneous absence seizures. The thalamocortical relay neurons of these mice displayed a near complete loss of the HCN current, resulting in a pronounced hyperpolarizing shift of the resting membrane potential, an altered response to depolarizing inputs and an increased susceptibility for oscillations. HCN2-null mice also displayed cardiac sinus dysrhythmia, a reduction of the sinoatrial HCN current and a shift of the maximum diastolic potential to hyperpolarized values. Mice with cardiomyocyte- specific deletion of HCN2 displayed the same dysrhythmia as mice lacking HCN2 globally, indicating that the dysrhythmia is indeed caused by sinoatrial dysfunction. Our results define the physiological role of the HCN2 subunit as a major determinant of membrane resting potential that is required for regular cardiac and neuronal rhythmicity.
Neurotrophin-evoked depolarization requires the sodium channel Na(V)1.9.
Brain-derived neurotrophic factor (BDNF) and other neurotrophins are essential for normal brain function. Many types of neurons in the central nervous system are excited by BDNF or neurotrophin-4/5, an action that has recently been implicated in synaptic plasticity. The mechanisms involved in this transmitter-like action of neurotrophins remains unclear. Here, by screening candidate genes with an antisense messenger RNA expression approach and by co-expressing the receptor tyrosine kinase TrkB and various sodium channels, we demonstrate that the tetrodotoxin-insensitive sodium channel Na(V)1.9 underlies the neurotrophin-evoked excitation. These results establish the molecular basis of neurotrophin-evoked depolarization and reveal a mechanism of ligand-mediated sodium channel activation.
Two-photon chloride imaging in neurons of brain slices.
Two-photon laser scanning microscopy has been used successfully for imaging activity-dependent changes of intracellular calcium and sodium levels. Here we introduce a simple technique for two-photon chloride imaging in intact neurons. It involves the use of the membrane-permeable Cl(-) indicator dye MQAE [N-(6-methoxyquinolyl) acetoethyl ester]. Brief incubation with MQAE produced a robust loading of cells in slices from various brain regions including hippocampus, cortex and cerebellum. In contrast to conventional fluorescence measurements using MQAE, two-photon imaging was not affected in a major way by dye bleaching and phototoxic damage. Instead, it allowed prolonged recordings of time-resolved changes in intracellular chloride concentration in somata and dendrites. As an example of an application we imaged GABA-mediated Cl(-) transients in pyramidal cells of cortical and hippocampal slices as well as in cerebellar Purkinje neurons. By combining Cl(-) imaging with the gramicidin-based perforated-patch-clamp technique we showed that changes in MQAE fluorescence are proportional to the magnitudes of GABA-evoked transmembrane Cl(-) fluxes. Thus, MQAE-based two-photon microscopy promises to be a valuable technique for many applications requiring chloride imaging in single cells.
Functional reconstitution of vascular smooth muscle cells with cGMP-dependent protein kinase I isoforms.
The cGMP-dependent protein kinase type I (cGKI) is a major mediator of NO/cGMP-induced vasorelaxation. Smooth muscle expresses two isoforms of cGKI, cGKIalpha and cGKIbeta, but the specific role of each isoform in vascular smooth muscle cells (VSMCs) is poorly understood. We have used a genetic deletion/rescue strategy to analyze the functional significance of cGKI isoforms in the regulation of the cytosolic Ca(2+) concentration by NO/cGMP in VSMCs. Cultured mouse aortic VSMCs endogenously expressed both cGKIalpha and cGKIbeta. The NO donor diethylamine NONOate (DEA-NO) and the membrane-permeable cGMP analogue 8-bromo-cGMP inhibited noradrenaline-induced Ca(2+) transients in wild-type VSMCs but not in VSMCs genetically deficient for both cGKIalpha and cGKIbeta. The defective Ca(2+) regulation in cGKI-knockout cells could be rescued by transfection of a fusion construct consisting of cGKIalpha and enhanced green fluorescent protein (EGFP) but not by a cGKIbeta-EGFP construct. Fluorescence imaging indicated that the cGKIalpha-EGFP fusion protein was concentrated in the perinuclear/endoplasmic reticulum region of live VSMCs, whereas the cGKIbeta-EGFP protein was more homogeneously distributed in the cytoplasm. These results suggest that one component of NO/cGMP-induced smooth muscle relaxation is the activation of the cGKIalpha isoform, which decreases the noradrenaline-stimulated cytosolic Ca(2+) level.