Biomedical subjects

Thomas Knöpfel

Publications and source records attributed to Thomas Knöpfel.

At least 19 recordsLinked to original sources

PubMed2006

In vivo calcium imaging from genetically specified target cells in mouse cerebellum.

Genetically encoded fluorescent calcium indicator proteins provide the potential to monitor activity from genetically specified target cells without a need for single cell resolution. Here we report the use of transgenic mice expressing the fluorescent calcium indicator protein GCaMP2 in cerebellar granule cells to image parallel fiber activity transcranially in vivo. We demonstrated reliable measurements of calcium transients from beams of parallel fibers in response to electrical stimulation in the molecular layer through the intact skull. These parallel fiber calcium transients differed from intrinsic postsynaptic autofluorescence signals in their faster kinetics and resistance to blockers of synaptic transmission. Finally, we used 2P laser-scanning microscopy to demonstrate reliable measurements of calcium transients from beams of parallel fibers at high spatial resolution in living mice. We expect that genetically targeted fluorescent calcium indicator proteins along with optical imaging techniques will be instrumental for the construction of macroscopic and microscopic maps of the function of specific brain circuits.

Animals↗

PubMed2006

Morphological and electrophysiological properties of GABAergic and non-GABAergic cells in the deep cerebellar nuclei.

The deep cerebellar nuclei (DCN) integrate inputs from the brain stem, the inferior olive, and the spinal cord with Purkinje cell output from cerebellar cortex and provide the major output of the cerebellum. Despite their crucial function in motor control and learning, the various populations of neurons in the DCN are poorly defined and characterized. Importantly, differences in electrophysiological properties between glutamatergic and GABAergic cells of the DCN have been largely elusive. Here, we used glutamate decarboxylase (GAD) 67-green fluorescent protein (GFP) knock-in mice to unambiguously identify GABAergic (GAD-positive) and non-GABAergic (GAD-negative, most likely glutamatergic) neurons of the DCN. Morphological analysis of DCN neurons patch-clamped with biocytin-containing electrodes revealed a significant overlap in the distributions of the soma sizes of GAD-positive and GAD-negative cells. Compared with GAD-negative DCN neurons, GAD-positive DCN neurons fire broader action potentials, display stronger frequency accommodation, and do not reach as high firing frequencies during depolarizing current injections. Furthermore, GAD-positive cells display slower spontaneous firing rates and have a more shallow frequency-to-current relationship than the GAD-negative cells but exhibit a longer-lasting rebound depolarization and associated spiking after a transient hyperpolarization. In contrast to the rather homogeneous population of GAD-positive cells, the GAD-negative cells were found to consist of two distinct populations as defined by cell size and electrophysiological features. We conclude that GABAergic DCN neurons are specialized to convey phasic spike rate information, whereas tonic spike rate is more faithfully relayed by the large non-GABAergic cells.

Action Potentials↗

PubMed2006

Involvement of protein synthesis and degradation in long-term potentiation of Schaffer collateral CA1 synapses.

Expression of synaptic plasticity involves the translation of mRNA into protein and, probably, active protein degradation via the proteasome pathway. Here, we report on the rapid activation of synthesis and degradation of a probe protein with the induction of long-term potentiation (LTP) in the hippocampal Schaffer collateral CA1 pathway. The proteasome inhibitor MG132 significantly reduced the field EPSP slope potentiation and LTP maintenance without acutely affecting basal synaptic transmission. To visualize protein dynamics, CA1 pyramidal cells of hippocampal slices were transfected with Semliki Forest virus particles expressing a recombinant RNA. This RNA contained the coding sequence for a degradable green fluorescence protein with a nuclear localization signal (NLS-d1EGFP) followed by a 3'- untranslated region dendritic targeting sequence. NLS-d1EGFP fluorescence remained stable in the low-frequency test stimulation but increased with LTP induction in the cell body and in most dendritic compartments of CA1 neurons. Applying anisomycin, a protein synthesis inhibitor, caused NLS-d1EGFP levels to decline; a proteasome inhibitor MG132 reversed this effect. In the presence of anisomycin, LTP induction accelerated the degradation of NLS-d1EGFP. When both inhibitors were present, NLS-d1EGFP levels remained unaffected by LTP induction. Moreover, LTP-induced acceleration of NLS-d1EGFP synthesis was blocked by rapamycin, which is consistent with the involvement of dendritic mammalian target of rapamycin in LTP-triggered translational activity. Our results clearly demonstrate that LTP induction not only leads to a rapid increase in the rate of protein synthesis but also accelerates protein degradation via the proteasome system.

2-Amino-5-phosphonovalerate↗

PubMed2006

Interaction of Kv3 potassium channels and resurgent sodium current influences the rate of spontaneous firing of Purkinje neurons.

Purkinje neurons spontaneously generate action potentials in the absence of synaptic drive and thereby exert a tonic, yet plastic, input to their target cells in the deep cerebellar nuclei. Purkinje neurons express two ionic currents with biophysical properties that are specialized for high-frequency firing: resurgent sodium currents and potassium currents mediated by Kv3.3. How these ionic currents determine the intrinsic activity of Purkinje neurons has only partially been understood. Purkinje neurons from mutant mice lacking Kv3.3 have a reduced rate of spontaneous firing. Dynamic-clamp recordings demonstrated that normal firing rates are rescued by inserting artificial Kv3 currents into Kv3.3 knock-out Purkinje neurons. Numerical simulations indicated that Kv3.3 increases the spontaneous firing rate via cooperation with resurgent sodium currents. We conclude that the rate of spontaneous action potential firing of Purkinje neurons is controlled by the interaction of Kv3.3 potassium currents and resurgent sodium currents.

Action Potentials↗

PubMed2006

Optical probing of neuronal circuit dynamics: genetically encoded versus classical fluorescent sensors.

During the past few decades, optical methods for imaging activity in networks composed of thousands of neurons have been developed. These techniques rely mainly on organic-chemistry-based dyes as indicators of Ca(2+) and membrane potential. However, recently a new generation of probes, genetically encoded fluorescent protein sensors, has emerged for use by physiologists studying the operation of neuronal circuits. We critically review the development of these new probes, and analyze objectives and experimental conditions in which classical probes are likely to prevail and where the fluorescent protein sensors will open paths to previously unexplored territories of functional neuroimaging.

Animals↗

PubMed2005

Long-term depression at olfactory nerve synapses.

The synapses formed by the olfactory nerve (ON) convey sensory information to olfactory glomeruli, the first stage of central odor processing. Morphological and behavioral studies suggest that glomerular odor processing is plastic in neonate rodents. However, long-term synaptic plasticity, a cellular correlate of functional and structural plasticity, has not yet been demonstrated in this system. Here, we report that ON-->mitral cell (MC) synapses of 5- to 8-d-old mice express long-term depression (LTD) after brief low-frequency ON stimulation. Pharmacological techniques and imaging of presynaptic calcium signals demonstrate that ON-MC LTD is expressed presynaptically and requires the activation of metabotropic glutamate receptors but does not require fast synaptic transmission. LTD at the ON--> MC synapse is potentially relevant for the establishment, maintenance, and experience-dependent refinement of odor maps in the olfactory bulb.

Animals↗

PubMed2005

Activation of cerebellar parallel fibers monitored in transgenic mice expressing a fluorescent Ca2+ indicator protein.

Genetically encoded fluorescent Ca2+ indicator proteins (FCIPs) are promising tools to study Ca2+ signaling in large assemblies of nerve cells. Currently, there are few examples of stable transgenic mouse lines that functionally express such sensors in well-defined neuronal cell populations. Here we report the generation and characterization of transgenic mice expressing an FCIP under the 5' regulatory sequences of the Kv3.1 potassium channel promoter. In the cerebellar cortex, expression was restricted to granule cells. We first demonstrated reliable measurements of Ca2+ transients from beams of parallel fibers and compared the FCIP signals with intrinsic autofluorescence signals. We demonstrate that, in a transgenic line that exhibits a high expression level of the FCIP, autofluorescence signals are negligible and stimulation-induced fluorescence transients represent FCIP signals. Using frontal cerebellar slices we imaged antidromic activation of granule cells following electrical stimulation of parallel fibers and orthodromic activation of beams of parallel fibers following electrical stimulation of granule cells. We found that paired pulse-induced presynaptic Ca2+ transients of parallel fibers are not affected by blockade of N-methyl-D-aspartate receptors.

2-Amino-5-phosphonovalerate↗

PubMed2004

Transgenic mice expressing a fluorescent in vivo label in a distinct subpopulation of neocortical layer 5 pyramidal cells.

The neuronal components of cortical circuits have been characterized on the basis of their morphological and functional properties, and further refined by correlation of marker proteins with particular cell types. This latter approach has been very fruitful for GABA-containing neurons, but comparable diagnostic markers for subpopulations of excitatory pyramidal cells have been more elusive. An emerging new approach consists of transgenic mice that express fluorescent proteins under the control of promoters that are active in specific cell types. Here, we analyzed a line of transgenic mice that carries a transgene consisting of regulatory sequences of the potassium channel Kv3.1 and enhanced yellow fluorescent protein (EYFP). In these mice, a set of neurons in neocortical layer 5 expresses high levels of the transgenic marker protein. EYFP-expressing, and nonexpressing layer 5 cells were easily identified in living tissue under conditions suitable for patch-clamp electrophysiology. By using immunolabeling, retrograde Fast Blue labeling and electrophysiological recordings with biocytin injections, we identified the fluorescent neurons as a population of pyramidal cells with distinct morphological and electrophysiological properties when compared with nonfluorescent neighboring layer 5 pyramidal cells. The most prominent morphological difference between these two populations was a much smaller number of apical oblique dendrites in EYFP-positive as compared with the EYFP-negative cells. The most prominent electrophysiological feature was a steady spike frequency adaptation in EYFP-positive cells, whereas EYFP-negative cells responded to a depolarizing current injection with a closely spaced spike doublet followed by constant frequency firing. The in vivo labeled transgenic mice provide an experimental tool for further functional differentiation of these populations of layer 5 pyramidal cells.

Action Potentials↗

PubMed2004

Imaging of gene expression during long-term potentiation.

Long term potentiation (LTP) at hippocampal Schaffer collateral-CA1 synapses involves an early and a late phase, where only the latter is sensitive to protein synthesis inhibitors. Here we characterized the dynamics of protein synthesis associated with the induction of L-LTP using a transgenic mouse model in which a cAMP responsive element (CRE)-regulated promoter drives production of an enhanced yellow fluorescent protein (eYFP). We found that eYFP fluorescence increased after less than 30 min following L-LTP induction. Application of transcription and translation suppressors and the NMDA receptor antagonist D-AP5 inhibited the L-LTP and prevented the rise in eYFP levels. The early-phase of LTP was not affected by inhibiting protein synthesis.

2-Amino-5-phosphonovalerate↗

PubMed2004

Allele-dependent changes of olivocerebellar circuit properties in the absence of the voltage-gated potassium channels Kv3.1 and Kv3.3.

Double-mutant mice (DKO) lacking the two voltage-gated K(+) channels Kv3.1 and Kv3.3 display a series of phenotypic alterations that include ataxia, myoclonus, tremor and alcohol hypersensitivity. The prominent cerebellar expression of mRNAs encoding Kv3.1 and Kv3.3 subunits raised the question as to whether altered electrical activity resulting from the lack of these K(+) channels might be related to the dramatic motor changes. We used the tremorogenic agent harmaline to probe mutant mice lacking different K(+) channel alleles for altered olivocerebellar circuit properties. Harmaline induced the characteristic 13-Hz tremor in wildtype mice (WT); however, no tremor was observed in DKO suggesting that the ensemble properties of the olivocerebellar circuitry are altered in the absence of Kv3.1 and Kv3.3 subunits. Harmaline induced tremor in Kv3.1-single mutants, but it was of smaller amplitude and at a lower frequency indicating the participation of Kv3.1 subunits in normal olivocerebellar system function. In contrast, harmaline tremor was virtually absent in Kv3.3-single mutants indicating an essential role for Kv3.3 subunits in tremor induction by harmaline. Immunohistochemical staining for Kv3.3 showed clear expression in the somata and proximal dendrites of Purkinje cells and in their axonal projections to the deep cerebellar nuclei (DCN). In DCN, both Kv3.1 and Kv3.3 subunits are expressed. Action potential duration is increased by approximately 100% in Purkinje cells from Kv3.3-single mutants compared to WT or Kv3.1-single mutants. We conclude that Kv3.3 channel subunits are essential for the olivocerebellar system to generate and sustain normal harmaline tremor whereas Kv3.1 subunits influence tremor amplitude and frequency.

Action Potentials↗

PubMed2004

Glutamate-induced elevations in intracellular chloride concentration in hippocampal cell cultures derived from EYFP-expressing mice.

The homeostasis of intracellular Cl(-) concentration ([Cl(-)](i)) is critical for neuronal function, including gamma-aminobutyric acid (GABA)ergic synaptic transmission. Here, we investigated activity-dependent changes in [Cl(-)](i) using a transgenetically expressed Cl(-)-sensitive enhanced yellow-fluorescent protein (EYFP) in cultures of mouse hippocampal neurons. Application of glutamate (100 microm for 3 min) in a bath perfusion to cell cultures of various days in vitro (DIV) revealed a decrease in EYFP fluorescence. The EYFP signal increased in amplitude with increasing DIV, reaching a maximal response after 7 DIV. Glutamate application resulted in a slight neuronal acidification. Although EYFP fluorescence is sensitive to pH, EYFP signals were virtually abolished in Cl(-)-free solution, demonstrating that the EYFP signal represented an increase in [Cl(-)](i). Similar to glutamate, a rise in [Cl(-)](i) was also induced by specific ionotropic glutamate receptor agonists and by increasing extracellular [K(+)], indicating that an increase in driving force for Cl(-) suffices to increase [Cl(-)](i). To elucidate the membrane mechanisms mediating the Cl(-) influx, a series of blockers of ion channels and transporters were tested. The glutamate-induced increase in [Cl(-)](i) was resistant to furosemide, bumetanide and 4,4'-diisothiocyanato-stilbene-2,2'-disulphonic acid (DIDS), was reduced by bicuculline to about 80% of control responses, and was antagonized by niflumic acid (NFA) and 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB). We conclude that membrane depolarization increases [Cl(-)](i) via several pathways involving NFA- and NPPB-sensitive anion channels and GABA(A) receptors, but not through furosemide-, bumetanide- or DIDS-sensitive Cl(-) transporters. The present study highlights the vulnerability of [Cl(-)](i) homeostasis after membrane depolarization in neurons.

Angiogenesis Inhibitors↗

PubMed2003

Motor dysfunction and altered synaptic transmission at the parallel fiber-Purkinje cell synapse in mice lacking potassium channels Kv3.1 and Kv3.3.

Micelacking both Kv3.1 and both Kv3.3 K+ channel alleles display severe motor deficits such as tremor, myoclonus, and ataxic gait. Micelacking one to three alleles at the Kv3.1 and Kv3.3 loci exhibit in an allele dose-dependent manner a modest degree of ataxia. Cerebellar granule cells coexpress Kv3.1 and Kv3.3 K+ channels and are therefore candidate neurons that might be involved in these behavioral deficits. Hence, we investigated the synaptic mechanisms of transmission in the parallel fiber-Purkinje cell system. Action potentials of parallel fibers were broader in mice lacking both Kv3.1 and both Kv3.3 alleles and in mice lacking both Kv3.1 and a single Kv3.3 allele compared with those of wild-type mice. The transmission of high-frequency trains of action potentials was only impaired at 200 Hz but not at 100 Hz in mice lacking both Kv3.1 and Kv3.3 genes. However, paired-pulse facilitation (PPF) at parallel fiber-Purkinje cell synapses was dramatically reduced in a gene dose-dependent manner in mice lacking Kv3.1 or Kv3.3 alleles. Normal PPF could be restored by reducing the extracellular Ca2+ concentration indicating that increased activity-dependent presynaptic Ca2+ influx, at least in part caused the altered PPF in mutant mice. Induction of metabotropic glutamate receptor-mediated EPSCs was facilitated, whereas longterm depression was not impaired but rather facilitated in Kv3.1/Kv3.3 double-knockout mice. These results demonstrate the importance of Kv3 potassium channels in regulating the dynamics of synaptic transmission at the parallel fiber-Purkinje cell synapse and suggest a correlation between short-term plasticity at the parallel fiber-Purkinje cell synapse and motor performance.

Action Potentials↗

PubMed2003

Sodium imaging of climbing fiber innervation fields in developing mouse Purkinje cells.

Maturation of specific neuronal connections in the mature nervous system includes elimination of redundant synapses formed earlier during development. In the cerebellum of adult animals, each Purkinje cell (PC) is innervated by a single climbing fiber (CF). In early postnatal development each PC is innervated by multiple CFs and elimination of synapses formed by supernumerary CFs occurs until monoinnervation is established at around postnatal day 20 (P20) in mice. It is not clear whether multiple CFs, or only a single CF, translocate from the cell body of immature PCs to the developing dendrite and, in case several CFs translocate, whether they share or segregate their innervation fields. To localize CF innervation fields, we imaged changes in postsynaptic sodium concentration resulting from CF-mediated postsynaptic currents. We found that more than one CF translocates from an innervation field on the cell body of the PC to the developing dendrite and that these CFs share rather than segregate their innervation fields. We concluded that both the soma and the proximal dendrite of the PC are territories of competition for the developing CFs and that the overlapping of their termination fields may be the prerequisite for a local process of elimination of all but one CF, as previously demonstrated in the developing neuromuscular junction.

Aging↗

PubMed2003

Optical recordings of membrane potential using genetically targeted voltage-sensitive fluorescent proteins.

Optical imaging of electrical activity using voltage-sensitive dyes has been envisaged for many years as a powerful method to investigate multineuronal representation of information processing in brain tissue. This article describes the advent of novel genetically targeted voltage-sensitive fluorescent proteins. This new class of membrane voltage sensors overcomes previous limitations related to the nonselective staining of membranes associated with conventional voltage-sensitive dyes. Here, we discuss the methodology, applications, and potential advantages of this novel technique.

Animals↗

PubMed2002

Transgenic mice expressing a pH and Cl- sensing yellow-fluorescent protein under the control of a potassium channel promoter.

During the last few years a variety of genetically encodable optical probes that monitor physiological parameters such as local pH, Ca2+, Cl-, or transmembrane voltage have been developed. These sensors are based on variants of green-fluorescent protein (GFP) and can be synthesized by mammalian cells after transfection with cDNA. To use these sensor proteins in intact brain tissue, specific promoters are needed that drive protein expression at a sufficiently high expression level in distinct neuronal subpopulations. Here we investigated whether the promoter sequence of a particular potassium channel may be useful for this purpose. We produced transgenic mouse lines carrying the gene for enhanced yellow-fluorescent protein (EYFP), a yellow-green pH- and Cl- sensitive variant of GFP, under control of the Kv3.1 K+ channel promoter (pKv3.1). Transgenic mouse lines displayed high levels of EYFP expression, identified by confocal microscopy, in adult cerebellar granule cells, interneurons of the cerebral cortex, and in neurons of hippocampus and thalamus. Furthermore, using living cerebellar slices we demonstrate that expression levels of EYFP are sufficient to report intracellular pH and Cl- concentration using imaging techniques and conditions analogous to those used with conventional ion-sensitive dyes. We conclude that transgenic mice expressing GFP-derived sensors under the control of cell-type specific promoters, provide a unique opportunity for functional characterization of defined subsets of neurons.

Animals↗

PubMed2002

Optical imaging of odor preference memory in the rat olfactory bulb.

Early olfactory preference learning in rat pups occurs when novel odors are paired with reinforcing tactile stimulation that activate the noradrenergic locus coeruleus. Pairing of odor and a noradrenergic agonist in the olfactory bulb is both necessary and sufficient for odor preference learning. This suggests the memory change occurs in the olfactory bulb. Previous electrophysiological experiments demonstrated that odor preference training induces an increase in the field excitatory postsynaptic potential to olfactory nerve input and an alteration, after training, in glomerular [14C]2- deoxyglucose uptake and in single-unit responses of principal cells. We investigate here whether, 24 h after olfactory preference training, there is an alteration in intrinsic optical signals at the glomerular level. Six-day-old rat pups were trained, as previously, for a peppermint odor preference. Trained pups and control littermates were subjected to imaging of odor-induced intrinsic optical signals 1 day after the training session. Trained pups exhibited significantly larger responses to the peppermint compared with untrained littermates previously exposed to the same odor. The response of trained pups to a control odor (amyl acetate) was, however, not significantly different from that of untrained littermates. These observations demonstrate that odor preference memory can be read-out by optical imaging techniques.

Animals↗

PubMed2002

Metabotropic glutamate receptors: electrical and chemical signaling properties.

Over the last two decades, glutamate has been established as the main excitatory neurotransmitter in the mammalian brain. Glutamate released from synapses activates ion channel-forming receptors at postsynaptic cells and consequently mediates fast postsynaptic potentials. These receptors are termed ionotropic glutamate receptors (iGluRs). The subsequent discovery of metabotropic glutamate receptors (mGluRs) revealed that glutamate can also mediate slow synaptic potentials, modulate ion channels, and directly couple to GTP binding proteins. In contrast to the iGluRs, the mGluRs possess seven transmembrane domains and a large intracellular C-terminus that involves interactions with a variety of other intracellular signaling systems. Eight functionally distinct mGluR subtypes are known to be localized to specific neuron types at presynaptic and/or postsynaptic membranes. Their physiological functions involve the generation of slow excitatory and inhibitory synaptic potentials, modulation of synaptic transmission, synaptic integration, and plasticity. The classical role of glutamate as a fast excitatory synaptic transmitter was largely extended by mGluRs acting as a neuromodulator and even as an activator of inhibitory mechanisms at certain synapses.

Animals↗

PubMedDate not supplied

Metabotropic glutamate receptors in the cerebellum with a focus on their function in Purkinje cells.

Metabotropic glutamate receptors (mGluRs) are a family of proteins that have seven transmembrane segments and that couple to G proteins. They differ from ionotropic glutamate receptors in that they do not form ion channels but instead affect intracellular chemical messenger systems. Eight genes coding for different subtypes of mGluRs have been identified to date and numbered accordingly in the order in which the cDNAs were cloned. Based on their principal signal-transduction capabilities in recombinant expression systems and sequence similarities, the family of mGluR subtypes is subdivided into three groups. Group 1 mGluRs (consisting of mGluR1 and 5) functionally couple to phospholipase C and affect the IP3/Ca2+ signaling pathway. The subtypes of group 2 (mGluR2 and 3) and group 3 (mGluR4, 6 7 and 8) inhibit adenylate cyclase and, thereby, mediate a decrease in cAMP concentration. All mGluR subtypes are found in the cerebellar cortex with the exception of mGluR6 which is exclusively expressed in the retina. At the parallel fiber-Purkinje cell synapses mGluR1 is localized in the peri- and extra-synaptic membrane of Purkinje cells. The main focus of this review deals with the functions of this postsynaptically localized mGluR1. These functions include (i) mediation of an inward current and a slow excitatory postsynaptic potential, and (ii) a role in induction of parallel fiber-Purkinje cell long-term depression. We discuss the mechanism underlying the mGluR1-mediated postsynaptic current as well as current theories on the role of mGluR1 in parallel fiber-Purkinje cell long-term depression.

Animals↗