Epileptiform bursting in the disinhibited neonatal cerebral cortex

The cerebral cortex, which include the neocortex and hippocampus, is an elaborate neuronal network communicating mainly through glutamate and gamma-aminobutyric acid (GABA). Glutamate, operating via AMPA, kainate, and NMDA receptors excites neurons, and operating via metabotropic glutamate receptors can either increase or decrease the excitation in the neuronal network. GABA, operating through GABAA and GABAB receptors, inhibits the mature neuronal network, and GABAA receptor blockade in the adult cerebral cortex leads to epileptiform bursts. In contrast, in the neonatal cerebral cortex, GABAA has been proposed to function as an excitatory neurotransmitter, and glutamatergic synapses are claimed to be underdeveloped. It is important to understand the mechanisms underlying epileptiform activity in the neonate, because epileptiform activity in the neonate can potentially damage the developing cerebral cortex. In this dissertation I explore the role of GABA in controlling epileptiform activity in the neonatal cerebral cortex. Bath application of GABAA receptor antagonists induced spontaneous generation of large-amplitude population discharges resembling interictal bursts, a form of epileptiform activity; activation of GABAA receptors reduced the amplitude of interictal bursts. Interictal bursts were mediated by glutamatergic neurotransmission, demonstrating that glutamate synapses are functional in the neonate. We conclude that GABA is inhibitory in the neonatal cerebral cortex because it serves to suppress excitatory synchronous activity. Interictal bursts in the neonatal hippocampus were generated in a temporally precise rhythm. The rhythmicity of interictal bursts was not modulated by GABAB receptors, calcium activated potassium conductances, or internally released calcium, butmanipulations that facilitate or suppress the hyperpolarization-activated cation current, Ih, increased or decreased, respectively, the frequency of the bursts. We conclude Ih plays a major role in pacing neonatal interictal bursts. Immunocytochemistry illustrated that Ih channel subunits in neonatal pyramidal neurons were distributed predominately in somata, while in the juvenile and mature hippocampus and neocortex the subunits were mostly found in GABAergic terminals and in the membrane of apical dendrites of pyramidal neurons, with diminished or no expression inside the somata. We conclude that the unique expression of Ih channel subunits in the neonatal hippocampus could contribute to the increased temporal precision of interictal bursts at this developmental stage

Investigating the mechanisms of action of phytocannabinoids and a novel cognitive enhancer to target the comorbidity of temporal lobe epilepsy

Temporal lobe epilepsy (TLE) is the most common type of epilepsy and exists with memory loss as a comorbidity. The conventional therapy available to treat these disorders achieves only modest therapeutic efficacy at best. This study investigates two potential treatments: phytocannabinoids to alleviate seizures, and a novel cognitive enhancer to restore/halt memory deficits. The anti-convulsant properties of cannabidiol (CBD) were first examined with regards to the neuropathology of two major types of hippocampal interneurons expressing parvalbumin (PV) and cholecystokinin (CCK) which are thought to dysfunction during epilepsy. Immunohistochemistry experiments using an in vivo kainic-acid induced epileptic rat model, revealed that PV- and CCK-immunopositive interneurons were significantly affected during epilepsy. This effect was greatly reduced following CBD treatment, suggesting that CBD exerts a neuroprotective function. The effects of CBD on the intrinsic membrane properties of these interneurons, together with hippocampal pyramidal cells, were further investigated in acute brain slices of rat seizure models of TLE (in vivo kainic acid-induced and in vitro Mg2+ free-induced). Whole-cell recordings revealed that bath application of CBD (10 µM) normalised the firing frequency of epileptic adapting pyramidal cells to healthy control levels. A similar effect was seen in hippocampal CCK-immunopositive Schaffer collateral associated (SCA) interneurons. In contrast, CBD resulted in an increased firing of PV-immunopositive interneurons, thus increasing their excitability and restoring the impaired membrane properties of the cells apparent in the epileptic models. The effects of cannabidivarin (CBDV), a similar cannabinoid compound, on the intrinsic membrane properties of these cell types were also evaluated. Additionally, CBDV affected excitatory postsynaptic currents by reducing excitation. In an attempt to address the memory impairment aspect associated with TLE, I investigated the neuronal effects of a5AM21, a novel potential memory enhancer. Electrophysiological experiments revealed that a5AM21 preferentially acts on 5-containing gamma (γ)-aminobutyric acid (GABA) type A (GABAA) receptors, reducing their inhibitory effects. Furthermore, data obtained using behavioural experiment paradigm, the eight-arm radial maze, suggest a significant improvement in short- and long-term memory retrieval in rats treated with a5AM21. In conclusion, the results reveal the potential mechanisms of action of two therapies to alleviate seizures and memory impairment, and the future goals would be to combine CBD/CBDV and a5AM21 as a promising novel targeted therapy for TLE

Nonlinear brain dynamics as macroscopic manifestation of underlying many-body field dynamics

Neural activity patterns related to behavior occur at many scales in time and space from the atomic and molecular to the whole brain. Here we explore the feasibility of interpreting neurophysiological data in the context of many-body physics by using tools that physicists have devised to analyze comparable hierarchies in other fields of science. We focus on a mesoscopic level that offers a multi-step pathway between the microscopic functions of neurons and the macroscopic functions of brain systems revealed by hemodynamic imaging. We use electroencephalographic (EEG) records collected from high-density electrode arrays fixed on the epidural surfaces of primary sensory and limbic areas in rabbits and cats trained to discriminate conditioned stimuli (CS) in the various modalities. High temporal resolution of EEG signals with the Hilbert transform gives evidence for diverse intermittent spatial patterns of amplitude (AM) and phase modulations (PM) of carrier waves that repeatedly re-synchronize in the beta and gamma ranges at near zero time lags over long distances. The dominant mechanism for neural interactions by axodendritic synaptic transmission should impose distance-dependent delays on the EEG oscillations owing to finite propagation velocities. It does not. EEGs instead show evidence for anomalous dispersion: the existence in neural populations of a low velocity range of information and energy transfers, and a high velocity range of the spread of phase transitions. This distinction labels the phenomenon but does not explain it. In this report we explore the analysis of these phenomena using concepts of energy dissipation, the maintenance by cortex of multiple ground states corresponding to AM patterns, and the exclusive selection by spontaneous breakdown of symmetry (SBS) of single states in sequences.Comment: 31 page

AAV Vector-Mediated Overexpression of CB1 Cannabinoid Receptor in Pyramidal Neurons of the Hippocampus Protects against Seizure-Induced Excitoxicity

The CB1 cannabinoid receptor is the most abundant G-protein coupled receptor in the brain and a key regulator of neuronal excitability. There is strong evidence that CB1 receptor on glutamatergic hippocampal neurons is beneficial to alleviate epileptiform seizures in mouse and man. Therefore, we hypothesized that experimentally increased CB1 gene dosage in principal neurons would have therapeutic effects in kainic acid (KA)-induced hippocampal pathogenesis. Here, we show that virus-mediated conditional overexpression of CB1 receptor in pyramidal and mossy cells of the hippocampus is neuroprotective and moderates convulsions in the acute KA seizure model in mice. We introduce a recombinant adeno-associated virus (AAV) genome with a short stop element flanked by loxP sites, for highly efficient attenuation of transgene expression on the transcriptional level. The presence of Cre-recombinase is strictly necessary for expression of reporter proteins or CB1 receptor in vitro and in vivo. Transgenic CB1 receptor immunoreactivity is targeted to glutamatergic neurons after stereotaxic delivery of AAV to the dorsal hippocampus of the driver mice NEX-cre. Increased CB1 receptor protein levels in hippocampal lysates of AAV-treated Cre-mice is paralleled by enhanced cannabinoid-induced G-protein activation. KA-induced seizure severity and mortality is reduced in CB1 receptor overexpressors compared with AAV-treated control animals. Neuronal damage in the hippocampal CA3 field is specifically absent from AAV-treated Cre-transgenics, but evident throughout cortical areas of both treatment groups. Our data provide further evidence for a role of increased CB1 signaling in pyramidal hippocampal neurons as a safeguard against the adverse effects of excessive excitatory network activity

Specific In Vivo Staining of Astrocytes in the Whole Brain after Intravenous Injection of Sulforhodamine Dyes

Fluorescent staining of astrocytes without damaging or interfering with normal brain functions is essential for intravital microscopy studies. Current methods involved either transgenic mice or local intracerebral injection of sulforhodamine 101. Transgenic rat models rarely exist, and in mice, a backcross with GFAP transgenic mice may be difficult. Local injections of fluorescent dyes are invasive. Here, we propose a non-invasive, specific and ubiquitous method to stain astrocytes in vivo. This method is based on iv injection of sulforhodamine dyes and is applicable on rats and mice from postnatal age to adulthood. The astrocytes staining obtained after iv injection was maintained for nearly half a day and showed no adverse reaction on astrocytic calcium signals or electroencephalographic recordings in vivo. The high contrast of the staining facilitates the image processing and allows to quantify 3D morphological parameters of the astrocytes and to characterize their network. Our method may become a reference for in vivo staining of the whole astrocytes population in animal models of neurological disorders

Controversies in epilepsy: Debates held during the Fourth International Workshop on Seizure Prediction

Debates on six controversial topics were held during the Fourth International Workshop on Seizure Prediction (IWSP4) convened in Kansas City, KS, USA, July 4–7, 2009. The topics were (1) Ictogenesis: Focus versus Network? (2) Spikes and Seizures: Step-relatives or Siblings? (3) Ictogenesis: A Result of Hyposynchrony? (4) Can Focal Seizures Be Caused by Excessive Inhibition? (5) Do High-Frequency Oscillations Provide Relevant Independent Information? (6) Phase Synchronization: Is It Worthwhile as Measured? This article, written by the IWSP4 organizing committee and the debaters, summarizes the arguments presented during the debates

Generation and Characterization of the Cation-Chloride Cotransporter KCC2 Hypomorphic Mouse

The cation-Cl- cotransporter (CCC) family comprises of Na+-Cl- cotransporter (NCC), Na+-K+-2Cl- cotransporters (NKCC1-2), and four K+-Cl- cotransporters (KCC1-4). These proteins are involved in several physiological activities, such as cell volume regulation. In neuronal tissues, NKCC1 and KCC2 are important in determining the intracellular Cl- levels and hence the neuronal responses to inhibitory neurotransmitters GABA and glycine. One aim of the work was to elucidate the roles for CCC isoforms in the control of nervous system development. KCC2 mRNA was shown to be developmentally up-regulated and follow neuronal maturation, whereas NKCC1 and KCC4 transcripts were highly expressed in the proliferative zones of subcortical regions. KCC1 and KCC3 mRNA displayed low expression throughout the embryogenesis. These expression profiles suggest a role for CCC isoforms in maturation of synaptic responses and in the regulation of neuronal proliferation during embryogenesis. The major aim of this work was to study the biological consequences of KCC2-deficiency in the adult CNS, by generating transgenic mice retaining 15-20% of normal KCC2 levels. In addition, by using these mice as a tool for in vivo pharmacological analysis, we investigated the requirements for KCC2 in tonic versus phasic GABAA receptor-mediated inhibition. KCC2-deficient mice displayed normal reproduction and life span, but showed several behavioral abnormalities, including increased anxiety-like behavior, impaired performance in water maze, alterations in nociceptive processing, and increased seizure susceptibility. In contrast, the mice displayed apparently normal spontaneous locomotor activity and motor coordination. Pharmacological analysis of KCC2-deficient mice revealed reduced sensititivity to diazepam, but normal gaboxadol-induced sedation, neurosteroid hypnosis and alcohol-induced motor impairment. Electrophysiological recordings from CA1-CA3 subregions of the hippocampus showed that KCC2 deficiency affected the reversal potentials of both the phasic and tonic GABA currents, and that the tonic conductance was not affected. The results suggest that requirement for KCC2 in GABAergic neurotransmission may differ among several functional systems in the CNS, which is possibly due to the more critical role of KCC2 activity in phasic compared to tonic GABAergic inhibition.Kationi-kloridi kuljetusproteiini-perhe koostuu natrium-kloridi (NCC), natrium-kalium-kloridi (NKCC1-2), sekä kalium-kloridi (KCC1-4) kuljetusproteiineista. Nämä proteiinit ovat osallisena useissa solujen fysiologissa prosesseissa, kuten solujen tilavuuden säätely. KCC2 ja NKCC1 ovat erityisen tärkeitä hermokudoksessa, jossa ne säätelevät solujen kloridipitoisuutta ja täten hermoston estävien välittäjäaineiden γ-aminovoihapon (GABA) ja glysiinin toimintaa. Yksi työn tavoitteista oli selventää näiden proteiinien rooleja hermoston kehityksessä. KCC2 mRNA:n ilmenemisen havaittiin voimistuvan tuntuvasti kehityksen kuluessa ja seuraavan hermoston kehitystä. NKCC1 ja KCC4 mRNA:t ilmentyivät voimakkaimmin niillä aivojen alueilla, joissa solut jakaantuvat neurogeneesin aikana. KCC1 ja KCC3 mRNA:t ilmentyivät vähäisesti koko sikiönkehityksen ajan. Tulokset puoltavat mahdollista roolia kationi-kloridi kuljetusproteiineille synapsien vasteiden kehityksessä, sekä hermosolujen jakautumisen säätelyssä. Työn päätavoitteena oli tuottaa KCC2-hypomorfinen hiiri (n. 80 % puutos) ja tutkia tämän hiiren ilmiasun muutoksia. Käytimme kyseistä hiirimallia myös selvittääksemme farmakologisesti KCC2:n osuutta GABAA-reseptorikompleksin välittämässä faasisessa ja toonisessa neurotransmissiossa. KCC2:n puutos ilmeni käyttäytymisen tasolla. Hiiri oli ahdistushäiriöinen ja selkeästi normaalia kouristusherkempi, sekä osoitti muistihäiriöitä ja normaalia korkeamman kipukynnyksen. Hiiren motorinen koordinaatio ja spontaani lokomotorinen aktiivisuus olivat sitä vastoin ilmeisen normaalilla tasolla. KCC2-hypomorfisen hiiren farmakologinen analyysi osoitti hiiren olevan epäherkkä bentsodiatsepiini diatsepaamille, mutta normaalisti herkkä gaboksadolin sedaatiovasteelle, neurosteroidin hypnoosivasteelle sekä alkoholin motoriikkaa huonontavalle vasteelle. Sähköfysiologiset mittaukset hippokampuksessa osoittivat KCC2 puutoksen vaikuttavan GABAA-välitteisten faasisten ja toonisten sähkövirtojen käänteispotentiaaleihin, mutta GABAA-välitteinen tooninen konduktanssi oli ilmeisen normaali. Nämä tulokset antavat olettaa että GABA neurotransmissiossa KCC2:n rooli voi vaihdella riippuen siitä, mitä keskushermoston eri funktionaalisista järjestelmistä aktivoidaan. Tämä voi johtua KCC2:n kriittisemmästä roolista GABAA-välitteisessä faasisessa neurotransmissiossa verrattuna tooniseen neurotransmissioon

Characterising the frequency response of impedance changes during evoked physiological activity in the rat brain

OBJECTIVE: Electrical impedance tomography (EIT) can image impedance changes associated with evoked physiological activity in the cerebral cortex using an array of epicortical electrodes. An impedance change is observed as the externally applied current, normally confined to the extracellular space is admitted into the conducting intracellular space during neuronal depolarisation. The response is largest at DC and decreases at higher frequencies due to capacitative transfer of current across the membrane. Biophysical modelling has shown that this effect becomes significant above 100 Hz. Recordings at DC, however, are contaminated by physiological endogenous evoked potentials. By moving to 1.7 kHz, images of somatosensory evoked responses have been produced down to 2 mm with a resolution of 2 ms and 200 μm. Hardware limitations have so far restricted impedance measurements to frequencies  2 kHz using improved hardware. APPROACH: Impedance changes were recorded during forepaw somatosensory stimulation in both cerebral cortex and the VPL nucleus of the thalamus in anaesthetised rats using applied currents of 1 kHz to 10 kHz. MAIN RESULTS: In the cortex, impedance changed by -0.04 ± 0.02 % at 1 kHz, reached a peak of -0.13 ± 0.05 % at 1475 Hz and decreased to -0.05 ± 0.02 % at 10 kHz. At these frequencies, changes in the thalamus were -0.26 ± 0.1%, -0.4 ± 0.15 % and -0.08 ± 0.03 % respectively. The signal-to-noise ratio was also highest at 1475 Hz with values of -29.5 ± 8 and -31.6 ±10 recorded from the cortex and thalamus respectively. Signficance: This indicates that the optimal frequency for imaging cortical and thalamic evoked activity using fast neural EIT is 1475 Hz