Modulation schneller neuronaler Netzwerkoszillationen im Hippocampus

The present dissertation comprises three articles published in international peer-reviewed journals. The experiments were planned and conducted at the Institute of Neurophysiology (Charité-Universitätsmedizin Berlin). Freely moving animals display two prominent states of network activity that are related to memory formation. Explorative behaviour and rapid-eye-movement (REM) sleep are characterised by short periods of gamma oscillations nested in theta oscillations, whereas sharp wave-ripple (SPW-R) activity is observed during immobility, consummatory behaviour and slow-wave sleep. I studied different aspects involved in maintenance and stabilisation of SPW-R activity, their emergence and suppression, and the acetylcholine metabolism which is involved in the switch from SPW-R activity to gamma oscillations. I show that application of nicotine led to a dose-dependent shift from SPW-R activity to seizure like events (SLEs). The transition could be linked to a reduced inhibition of pyramidal cells in area CA3. We found that elevation of [K+]0 from 3 to 8.5 mM led to REDs in naïve slices whereas their generation was prevented during episodes of stimulus-induced SPW-R activity. Under this condition the inhibitory conductance was significantly increased. In the next study I demonstrate the role of GABAB receptor mediated inhibition in presynaptic Ca2+ dependent transmitter release. Stimulus-induced SPW-Rs were transiently blocked by application of the GABAB receptor agonist baclofen, an effect that could be prevented by co-application with the antagonist CGP55846. However, CGP55846 itself had no effect on the incidence of SPW-Rs. Interestingly, the induction of SPW-Rs was still possible when baclofen was applied during tetanic stimulation. Lastly, I investigated the acetylcholine metabolism and its implications for the generation of gamma oscillations. Blocking the acetylcholinesterase to inhibit the extracellular degradation of ACh induced gamma oscillations. Additional application of ACh dose dependently increased power and coherence of the oscillations. I further employed experiments to provide evidence for an extracellularly located choline- acetyltransferase that would allow synthesising ACh from ambient residual choline. However, taken together, the results did not support the idea of an extracellular source of ACh.Die vorliegende Dissertation basiert auf drei Artikeln, die vor ihrer Publikation einen peer-review Prozess durchlaufen haben. Die zugrundeliegenden Experimente wurden im Institut für Neurophysiologie der Charité- Universitätsmedizin Berlin geplant und durchgeführt. Experimente an sich frei bewegenden Tieren haben gezeigt, dass zwei Arten von Netzwerkaktivität für die Gedächtnisbildung von Bedeutung sind. Während exploratives Verhalten und paradoxer (REM) Schlaf durch Gamma-Oszillationen gekennzeichnet sind, tritt eine als „sharp wave-ripple“ (SPW-R) bezeichnete Netzwerkaktivität vor allem während Ruhephasen, der Nahrungsaufnahme und orthodoxen (non-REM) Schlaf auf. Ich habe Mechanismen untersucht, die für die Aufrechterhaltung, das Entstehen sowie die Unterdrückung der SPW-R Aktivität verantwortlich sind. Der Metabolismus des Neurotransmitters Acetylcholin wurde ebenfalls untersucht, da er für den Wechsel zwischen den beiden Netzwerkaktivitäten von Bedeutung ist. Ich konnte zeigen, dass induzierte SPW-R durch Gabe von Nikotin dosisabhängig in krampfähnliche Aktivitätsmuster (REDs) transformiert werden können. Die Ursache hierfür ist eine reduzierte Inhibition von Pyramidenzellen in der hippocampalen CA3 Region. Durch unspezifische Erregung (Erhöhung der extrazellulären Kaliumkonzentration von 3 auf 8.5 mM) konnte in unstimulierten Schnitten RED ausgelöst werden, was in Gegenwart von SPW-Rs aufgrund erhöhter Inhibition nicht möglich war. In der nächsten Studie habe ich den Einfluss von GABAB Rezeptoren auf die Calcium-abhängige Transmitterfreisetzung untersucht. Die transiente Unterdrückung der SPW-R durch Aktivierung des GABAB Rezeptors mit Baclofen konnte durch Co-Applikation des Antagonisten CGP55846 verhindert werden. In Gegenwart von Baclofen war die Induktion der SPW-R dennoch möglich. Schließlich habe ich den Acetylcholinmetabolismus untersucht. Durch Hemmung des Acetylcholinabbaus durch die Acetylcholinesterase konnte ich dosisabhängig unterschiedlich starke Formen von Gamma-Oszillationen auslösen, die durch Zugabe von Acetylcholin noch stärker ausgeprägt wurden. Der experimentelle Nachweis, einer Möglichkeit zur extrazellulären Bildung von Acetylcholin, konnte nicht erbracht werden

GM-CSF induces noninflammatory proliferation of microglia and disturbs electrical neuronal network rhythms in situ

Background!#!The granulocyte-macrophage colony-stimulating factor (GM-CSF) (or CSF-2) is involved in myeloid cell growth and differentiation, and, possibly, a major mediator of inflammation in body tissues. The role of GM-CSF in the activation of microglia (CNS resident macrophages) and the consequent impacts on neuronal survival, excitability, and synaptic transmission are widely unknown, however. Here, we focused on electrical neuronal network rhythms in the gamma frequency band (30-70 Hz). Gamma oscillations are fundamental to higher brain functions, such as perception, attention, and memory, and they are exquisitely sensitive to metabolic and oxidative stress.!##!Methods!#!We explored the effects of chronic GM-CSF exposure (72 h) on microglia in male rat organotypic hippocampal slice cultures (in situ), i.e., postnatal cortex tissue lacking leukocyte invasion (adaptive immunity). We applied extracellular electrophysiological recordings of local field potential, immunohistochemistry, design-based stereology, biochemical analysis, and pharmacological ablation of microglia.!##!Results!#!GM-CSF triggered substantial proliferation of microglia (microgliosis). By contrast, the release of proinflammatory cytokines (IL-6, TNF-α) and nitric oxide, the hippocampal cytoarchitecture as well as the morphology of parvalbumin-positive inhibitory interneurons were unaffected. Notably, GM-CSF induced concentration-dependent, long-lasting disturbances of gamma oscillations, such as slowing (beta frequency band) and neural burst firing (hyperexcitability), which were not mimicked by the T lymphocyte cytokine IL-17. These disturbances were attenuated by depletion of the microglial cell population with liposome-encapsulated clodronate. In contrast to priming with the cytokine IFN-γ (type II interferon), GM-CSF did not cause inflammatory neurodegeneration when paired with the TLR4 ligand LPS.!##!Conclusions!#!GM-CSF has a unique role in the activation of microglia, including the potential to induce neuronal network dysfunction. These immunomodulatory properties might contribute to cognitive impairment and/or epileptic seizure development in disease featuring elevated GM-CSF levels, blood-brain barrier leakage, and/or T cell infiltration

Status epilepticus induces chronic silencing of burster and dominance of regular firing neurons during sharp wave-ripples in the mouse subiculum

Summary: Sharp wave-ripples (SWRs) are hippocampal oscillations associated with memory consolidation. The subiculum, as the hippocampal output structure, ensures that hippocampal memory representations are transferred correctly to the consolidating neocortical regions. Because patients with temporal lobe epilepsy often develop memory deficits, we hypothesized that epileptic networks may disrupt subicular SWRs. We therefore investigated the impact of experimentally induced status epilepticus (SE) on subicular SWRs and contributing pyramidal neurons using electrophysiological recordings in mouse hippocampal slices. Subicular SWRs expressed hyperexcitable features post-SE, including increased ripple and unit activity. While regular firing neurons normally remain silent during SWRs, selective disinhibition recruited more regular firing neurons for action potential generation during SWRs post-SE. By contrast, burster neurons generated fewer action potential bursts during SWRs post-SE. Furthermore, altered timing of postsynaptic and action potentials suggested distorted neuronal recruitment during SWRs. Distorted subicular SWRs may therefore impair information processing and memory consolidation in epilepsy

Early alterations in hippocampal perisomatic GABAergic synapses and network oscillations in a mouse model of Alzheimer's disease amyloidosis.

Several lines of evidence imply changes in inhibitory interneuron connectivity and subsequent alterations in oscillatory network activities in the pathogenesis of Alzheimer's Disease (AD). Recently, we provided evidence for an increased immunoreactivity of both the postsynaptic scaffold protein gephyrin and the GABAA receptor γ2-subunit in the hippocampus of young (1 and 3 months of age), APPPS1 mice. These mice represent a well-established model of cerebral amyloidosis, which is a hallmark of human AD. In this study, we demonstrate a robust increase of parvalbumin immunoreactivity and accentuated projections of parvalbumin positive (PV+) interneurons, which target perisomatic regions of pyramidal cells within the hippocampal subregions CA1 and CA3 of 3-month-old APPPS1 mice. Colocalisation studies confirmed a significant increase in the density of PV+ projections labeled with antibodies against a presynaptic (vesicular GABA transporter) and a postsynaptic marker (gephyrin) of inhibitory synapses within the pyramidal cell layer of CA1 and CA3. As perisomatic inhibition by PV+-interneurons is crucial for the generation of hippocampal network oscillations involved in spatial processing, learning and memory formation we investigated the impact of the putative enhanced perisomatic inhibition on two types of fast neuronal network oscillations in acute hippocampal slices: 1. spontaneously occurring sharp wave-ripple complexes (SPW-R), and 2. cholinergic γ-oscillations. Interestingly, both network patterns were generally preserved in APPPS1 mice similar to WT mice. However, the comparison of simultaneous CA3 and CA1 recordings revealed that the incidence and amplitude of SPW-Rs were significantly lower in CA1 vs CA3 in APPPS1 slices, whereas the power of γ-oscillations was significantly higher in CA3 vs CA1 in WT-slices indicating an impaired communication between the CA3 and CA1 network activities in APPPS1 mice. Taken together, our data demonstrate an increased GABAergic synaptic output of PV+ interneurons impinging on pyramidal cells of CA1 and CA3, which might limit the coordinated cross-talk between these two hippocampal areas in young APPPS1 mice and mediate long-term changes in synaptic inhibition during progression of amyloidosis

Long-term changes in the CA3 associative network of fear-conditioned mice

<div><p></p><p>The CA3 associative network plays a critical role in the generation of network activity patterns related to emotional state and fear memory. We investigated long-term changes in the corticosterone (CORT)-sensitive function of this network following fear conditioning and fear memory reactivation. In acute slice preparations from mice trained in either condition, the ratio of orthodromic population spike (PS) to antidromic PS was reduced compared to unconditioned animals, indicating a decrease in efficacy of neuronal coupling within the associative CA3 network. However, spontaneous sharp wave–ripples (SW-R), which are thought to arise from this network, remained unaltered. Following CORT application, we observed an increase in orthodromic PS and a normalization to control levels of their ratio to antidromic PS, while SW-R increased in slices of fear conditioned and fear reactivated mice, but not in slices of unconditioned controls. Together with our previous observations of altered hippocampal gamma activity under these learning paradigms, these data suggest that fear conditioning and fear reactivation lastingly alters the CORT-sensitive configuration of different network activity patterns generated by the CA3 associational network. Observed changes in the mRNA expression of receptors for glutamate, GABA and cannabinoids in the stratum pyramidale of area CA3 may provide a molecular mechanism for these adaptive changes.</p></div

Long-term changes in the CA3 associative network of fear-conditioned mice

<div><p></p><p>The CA3 associative network plays a critical role in the generation of network activity patterns related to emotional state and fear memory. We investigated long-term changes in the corticosterone (CORT)-sensitive function of this network following fear conditioning and fear memory reactivation. In acute slice preparations from mice trained in either condition, the ratio of orthodromic population spike (PS) to antidromic PS was reduced compared to unconditioned animals, indicating a decrease in efficacy of neuronal coupling within the associative CA3 network. However, spontaneous sharp wave–ripples (SW-R), which are thought to arise from this network, remained unaltered. Following CORT application, we observed an increase in orthodromic PS and a normalization to control levels of their ratio to antidromic PS, while SW-R increased in slices of fear conditioned and fear reactivated mice, but not in slices of unconditioned controls. Together with our previous observations of altered hippocampal gamma activity under these learning paradigms, these data suggest that fear conditioning and fear reactivation lastingly alters the CORT-sensitive configuration of different network activity patterns generated by the CA3 associational network. Observed changes in the mRNA expression of receptors for glutamate, GABA and cannabinoids in the stratum pyramidale of area CA3 may provide a molecular mechanism for these adaptive changes.</p></div