Frank Angenstein

Toxoplasma gondii Actively Inhibits Neuronal Function in Chronically Infected Mice

Upon infection with the obligate intracellular parasite Toxoplasma gondii, fast replicating tachyzoites infect a broad spectrum of host cells including neurons. Under the pressure of the immune response, tachyzoites convert into slow-replicating bradyzoites, which persist as cysts in neurons. Currently, it is unclear whether T. gondii alters the functional activity of neurons, which may contribute to altered behaviour of T. gondii–infected mice and men. In the present study we demonstrate that upon oral infection with T. gondii cysts, chronically infected BALB/c mice lost over time their natural fear against cat urine which was paralleled by the persistence of the parasite in brain regions affecting behaviour and odor perception. Detailed immunohistochemistry showed that in infected neurons not only parasitic cysts but also the host cell cytoplasm and some axons stained positive for Toxoplasma antigen suggesting that parasitic proteins might directly interfere with neuronal function. In fact, in vitro live cell calcium (Ca2+) imaging studies revealed that tachyzoites actively manipulated Ca2+ signalling upon glutamate stimulation leading either to hyper- or hypo-responsive neurons. Experiments with the endoplasmatic reticulum Ca2+ uptake inhibitor thapsigargin indicate that tachyzoites deplete Ca2+ stores in the endoplasmatic reticulum. Furthermore in vivo studies revealed that the activity-dependent uptake of the potassium analogue thallium was reduced in cyst harbouring neurons indicating their functional impairment. The percentage of non-functional neurons increased over time In conclusion, both bradyzoites and tachyzoites functionally silence infected neurons, which may significantly contribute to the altered behaviour of the host.

The BOLD Response in the Rat Hippocampus Depends Rather on Local Processing of Signals than on the Input or Output Activity. A Combined Functional MRI and Electrophysiological Study

Functional magnetic resonance imaging (fMRI) of the human brain is widely used in neuroscience, but the exact relationship between measured fMRI response and the underlying changes in neuronal activity is still elusive. To obtain further information about the specific roles of synaptic (input) and spiking activity (output) for the generation of fMRI-related signals, we used an approach that combines electrophysiological and MRI measurements in the anatomically and physiologically well defined rat hippocampus. Direct electrical stimulation of the perforant pathway enabled us to control synchronized input activity to the dentate gyrus, whereas recorded population spikes from the granular cell layer indicated the dentate output activity. The perforant pathway was first stimulated with 15 identical pulse trains (10 Hz for 8 s), and evoked blood oxygenation level-dependent (BOLD) responses and population spikes were measured for each individual stimulus train. Spatial and magnitude aspects of the elicited BOLD responses differentially changed in the dentate gyrus and hippocampus from early to late stimulus trains together with population spike latencies in the dentate indicating delayed inhibitory network processing. Furthermore, the same number of stimuli presented in different patterns within trains (i.e., bursts of 10 stimuli at 50, 100, or 200 Hz) clearly altered the BOLD responses. Similarly, variations in the BOLD response also occurred when different stimulus patterns were chosen that caused the same number of population spikes. The results indicate that neuronal network activity including inhibitory interneurons rather than exclusively the input or spiking activity of the principal neurons determine a BOLD response to repetitive stimuli.

Important Contribution of α-Neurexins to Ca2+-Triggered Exocytosis of Secretory Granules

α-Neurexins constitute a family of neuronal cell surface molecules that are essential for efficient neurotransmission, because mice lacking two or all three α-neurexin genes show a severe reduction of synaptic release. Although analyses of α-neurexin knock-outs and transgenic rescue animals suggested an involvement of voltage-dependent Ca2+channels, it remained unclear whether α-neurexins have a general role in Ca2+-dependent exocytosis and how they may affect Ca2+ channels. Here we show by membrane capacitance measurements from melanotrophs in acute pituitary gland slices that release from endocrine cells is diminished by >50% in adult α-neurexin double knock-out and newborn triple knock-out mice. There is a reduction of the cell volume in mutant melanotrophs; however, no ultrastructural changes in size or intracellular distribution of the secretory granules were observed. Recordings of Ca2+ currents from melanotrophs, transfected human embryonic kidney cells, and brainstem neurons reveal that α-neurexins do not affect the activation or inactivation properties of Ca2+ channels directly but may be responsible for coupling them to release-ready vesicles and metabotropic receptors. Our data support a general and essential role for α-neurexins in Ca2+-triggered exocytosis that is similarly important for secretion from neurons and endocrine cells.

Age-dependent changes in MRI of motor brain stem nuclei in a mouse model of ALS

Mice over-expressing the mutant human G93A-SOD1 are widely used as an animal model of amyotrophic lateral sclerosis (ALS). ALS is characterized by progressive degeneration of motor neurons in the motor cortex, brain stem and spinal cord. The underlying mechanisms for the selective death of motor neurons are still uncertain. To study factors that cause selective neuron degeneration or therapeutic approaches to delay the progression of the disease, a method is required to monitor the state of motor neurons under in-vivo conditions. Here, we demonstrate that in G93A-SOD1 mice the MRI signal intensities of nucleus V, VII, XII, and nucleus ambiguus show a time-dependent increase starting around day 90, parallel to first behavioral signs of a motoneuron disorder.

Hippocampal long-term potentiation in vivo induces translocation of protein kinase Cγ

The possible involvement of the Ca(2+)-dependent protein kinase C (PKC) isoenzymes alpha/beta and gamma in mechanisms of long-term potentiation (LTP) was investigated after tetanic stimulation of the perforant path in vivo. Brief tetanic stimulation of the perforant path resulted in a 150% increase in population spike amplitude recorded from the dentate gyrus synapses in response to test stimuli 5 and 10 min after tetanization. Immunoblot analysis of PKC immunoreactivity in cytosolic and membrane fractions revealed a LTP-induced translocation of gamma PKC but not alpha/beta PKC into the cytosol in dentate gyrus but also in the other ipsilateral hippocampal regions. These data suggest different physiological roles of Ca(2+)-dependent PKC isoenzymes in activity-dependent synaptic plasticity.

Receptor-mediated activation of protein kinase C in hippocampal long-term potentiation: facts, problems and implications.

During the last decade hippocampal long-term potentiation has become one of the most frequently used models to study cellular mechanisms of learning and memory. Receptor-mediated activation of protein kinase C is thought to be involved in LTP stabilisation. In the present review, 1. the molecular structure and activation mechanisms of PKC isoenzymes, 2. the biochemical evidences for PKC activation after induction of LTP using different stimulation paradigms as well as 3. the involvement of metabotropic glutamate receptors in PKC activation after induction of LTP are critically discussed.

Age-dependent differences in glutamate-induced phosphorylation systems in rat hippocampal slices

Glutamate receptor induced changes in the activity of different phosphorylation systems were measured in hippocampal slices from 12‐ and 56‐day‐old rats, by determining the endogenous phosphorylation of 2.5% perchloric acid (PCA) soluble proteins. We identified among these proteins an 85, 80 kDa and the tau protein as specific substrates for protein kinase A (PKA), MARCKS, and neurogranin as specific substrates for protein kinase C (PKC), and prostaglandin‐D‐synthase as substrate for casein kinase II (CKII). In addition, a 35 kDa protein was phosphorylated by calcium/calmodulin dependent kinase II and protein kinase C and a 21 kDa protein was a substrate for all investigated kinases.

Activation of metabotropic glutamate receptors increases endogenous protein kinase C substrate phosphorylation in adult hippocampal slices

We previously reported (Staak, S., Behnisch, T. and Angenstein, F., Hippocampal long-term potentiation: transient increase but no persistent translocation of protein kinase C (PKC) isoenzymes alpha and beta, Brain Res., 682 (1995) 55-62) that Ca(2+)-dependent PKC isoenzymes alpha/beta and gamma are not translocated between subcellular compartments after stimulation of glutamate receptor subtypes in hippocampal slices. Extending our previous work in this study in situ phosphorylation of endogenous PKC substrates and the translocation of novel PKC isoenzymes delta and epsilon was analysed to detect PKC activation. Two proteins of approximately 94 kDa and 18 kDa were first characterised to be specific PKC substrates. As control of the technique carbachol was shown to increase in situ phosphorylation of the two substrates without any measurable translocation of PKC protein. Activation of metabotropic glutamate receptors by 50 microM DHPG also increased the situ-phosphorylation by 43.9% (94 kDa) and 32.8% (18 kDa) compared to controls but did not induce a measurable subcellular redistribution of conventional and novel PKC isoenzymes. Stimulation by 50 microM trans-ACPD or 0.1 mM quisqualate enhanced the situ phosphorylation in the same range, whereas 0.1 mM NMDA was ineffective. To our knowledge this is the first report showing a direct link between metabotropic glutamate receptor activation and increased endogenous PKC substrate phosphorylation in adult hippocampal slices. This PKC activation was not detectable by a redistribution of enzyme protein between subcellular compartments. We, therefore, conclude, that the failure to detect PKC translocation in physiological experiments is not an indicator for unchanged enzyme activity.

Hippocampal enlargement in Bassoon-mutant mice is associated with enhanced neurogenesis, reduced apoptosis, and abnormal BDNF levels

Mice mutant for the presynaptic protein Bassoon develop epileptic seizures and an altered pattern of neuronal activity that is accompanied by abnormal enlargement of several brain structures, with the strongest size increase in hippocampus and cortex. Using manganese-enhanced magnetic resonance imaging, an abnormal brain enlargement was found, which is first detected in the hippocampus 1xa0month after birth and amounts to an almost 40% size increase of this structure after 3xa0months. Stereological quantification of cell numbers revealed that enlargement of the dentate gyrus and the hippocampus proper is associated with larger numbers of principal neurons and of astrocytes. In search for the underlying mechanisms, an approximately 3-fold higher proportion of proliferation and survival of new-born cells in the dentate gyrus was found to go hand in hand with similarly larger numbers of doublecortin-positive cells and reduced numbers of apoptotic cells in the dentate gyrus and the hippocampus proper. Enlargement of the hippocampus and of other forebrain structures was accompanied by increased levels of brain-derived neurotrophic factor (BDNF). These data show that hippocampal overgrowth in Bassoon-mutant mice arises from a dysregulation of neurogenesis and apoptosis that might be associated with unbalanced BDNF levels.

Hippocampal long-term potentiation: transient increase but no persistent translocation of protein kinase C isoenzymes α and β

Abstract Using a monoclonal antibody the translocation of the Ca 2+ -dependent protein kinase C (PKC) isoenzymes α/β was studied in hippocampal slices after stimulation of glutamate receptors or induction of long-term potentiation. In submerged slices preincubated for 60 min in a medium usually used in electrophysiological studies, cytosolic PKC was not detectable and the amount of membrane-associated enzyme was increased. The treatment of these slices with 10 −6 M phorbol-12,13-dibutyrate induced a time-dependent translocation of α/β PKC from the membrane-associated into the membrane-inserted state. The glutamatergic agonists N -methyl- d -aspartate, quisqualate and trans-ACPD did not cause a membrane insertion of α/β PKC as observed for the phorbol ester when applied alone or in combination. Furthermore, 2 min and 15 min after induction of LTP in the Schaffer collateral-CA1 pathway the distribution of α/β PKC between the two membrane fractions remained unchanged. An increase in the total amount of PKC immunoreactivity was measured immediately after tetanization (142.6% of controls). The data suggest that a membrane insertion of α/β PKC is not a prerequisite for the LTP-induced increased phosphorylation of PKC substrates and that the enzyme might be recruited from a previously inactive pool.