Attila Sik

Trans-synaptic shift in anion gradient in spinal lamina I neurons as a mechanism of neuropathic pain

Modern pain-control theory predicts that a loss of inhibition (disinhibition) in the dorsal horn of the spinal cord is a crucial substrate for chronic pain syndromes. However, the nature of the mechanisms that underlie such disinhibition has remained controversial. Here we present evidence for a novel mechanism of disinhibition following peripheral nerve injury. It involves a trans-synaptic reduction in the expression of the potassium–chloride exporter KCC2, and the consequent disruption of anion homeostasis in neurons of lamina I of the superficial dorsal horn, one of the main spinal nociceptive output pathways. In our experiments, the resulting shift in the transmembrane anion gradient caused normally inhibitory anionic synaptic currents to be excitatory, substantially driving up the net excitability of lamina I neurons. Local blockade or knock-down of the spinal KCC2 exporter in intact rats markedly reduced the nociceptive threshold, confirming that the reported disruption of anion homeostasis in lamina I neurons was sufficient to cause neuropathic pain.

Chromogranin-mediated secretion of mutant superoxide dismutase proteins linked to amyotrophic lateral sclerosis.

Here we report that chromogranins, components of neurosecretory vesicles, interact with mutant forms of superoxide dismutase (SOD1) that are linked to amyotrophic lateral sclerosis (ALS), but not with wild-type SOD1. This interaction was confirmed by yeast two-hybrid screen and by co-immunoprecipitation assays using either lysates from Neuro2a cells coexpressing chromogranins and SOD1 mutants or lysates from spinal cord of ALS mice. Confocal and immunoelectron microscopy revealed a partial colocalization of mutant SOD1 with chromogranins in spinal cord of ALS mice. Mutant SOD1 was also found in immuno-isolated trans-Golgi network and in microsome preparations, suggesting that it can be secreted. Indeed we report evidence that chromogranins may act as chaperone-like proteins to promote secretion of SOD1 mutants. From these results, and our finding that extracellular mutant SOD1 can trigger microgliosis and neuronal death, we propose a new ALS pathogenic model based on the toxicity of secreted SOD1 mutants.

A Mechanism for Ca2+/Calmodulin-Dependent Protein Kinase II Clustering at Synaptic and Nonsynaptic Sites Based on Self-Association

The activity of Ca2+/calmodulin-dependent protein kinase II (CaMKII) plays an integral role in regulating synaptic development and plasticity. We designed a live-cell-imaging approach to monitor an activity-dependent clustering of green fluorescent protein (GFP)-CaMKII holoenzymes, termed self-association, a process that we hypothesize contributes to the translocation of CaMKII to synaptic and nonsynaptic sites in activated neurons. We show that GFP-CaMKII self-association in human embryonic kidney 293 (HEK293) cells requires a catalytic domain and multimeric structure, requires Ca2+ stimulation and a functional Ca2+/CaM-binding domain, is regulated by cellular pH and Thr286 autophosphorylation, and has variable rates of dissociation depending on Ca2+ levels. Furthermore, we show that the same rules that govern CaMKII self-association in HEK293 cells apply for extrasynaptic and postsynaptic translocation of GFP-CaMKII in hippocampal neurons. Our data support a novel mechanism for targeting CaMKII to postsynaptic sites after neuronal activation. As such, CaMKII may form a scaffold that, in combination with other synaptic proteins, recruits and localizes additional proteins to the postsynaptic density. We discuss the potential function of CaMKII self-association as a tag of synaptic activity.

Opening of the blood–brain barrier during isoflurane anaesthesia

In order to produce its desired effect, anaesthesia acts upon neuronal elements by modifying membrane conductances and transmitter interactions. The effect of higher doses of isoflurane, widely used in clinical settings, on the permeability of the blood–brain barrier (BBB) is meanwhile ignored. In this study we investigated the integrity of the BBB during various levels of isoflurane anaesthesia (1% and 3%) in cats by monitoring the extravasation of Evans blue. Simultaneously we measured the electroencephalogram (EEG), with particular emphasis on its direct current (DC) component. High doses of anaesthetic (3%) broke down the BBB in the cortex and thalamus, while milder doses (1%) only opened the BBB in the thalamus. The fluorescent signal of Evans blue was visible over an extravascular length of 23 μm in the cortex and 25 μm in the thalamus, similar to the diffusion of the same dye when the BBB was disrupted with mannitol. The opening of the BBB was associated with (i) a positive DC shift in the EEG measured on the scalp and (ii) an evaluated increase in cerebral volume of 2–2.8%. The opening of the BBB by high doses of isoflurane brings into discussion hitherto unexplored effects of anaesthesia on the brain. The electrophysiological correlate provided by the DC component of the EEG constitutes a promising option for the assessment of the BBB integrity during human anaesthesia.

Neurochemical identification of stereotypic burst-firing neurons in the rat dorsal raphe nucleus using juxtacellular labelling methods.

Recent electrophysiological studies have discovered evidence of heterogeneity of 5‐hydroxytryptamine (5‐HT) neurons in the mesencephalic raphe nuclei. Of particular interest is a subpopulation of putative 5‐HT neurons that display many of the electrophysiological properties of presumed 5‐HT‐containing neurons (regular and slow firing of single spikes with a broad waveform) but fire spikes in short, stereotyped bursts. In the present study we investigated the chemical identity of these neurons in rats utilizing in vivo juxtacellular labelling methods. Of ten dorsal raphe nucleus (DRN) neurons firing short stereotyped bursts within an otherwise regular firing pattern, all exhibited immunoreactivity for either 5‐HT (n = 6) or the 5‐HT synthesizing enzyme, tryptophan hydroxylase (TRH; n = 2) or both (n = 2). Supporting pharmacological experiments demonstrated that the burst firing DRN neurons demonstrated equal sensitivity to 5‐HT1A agonism and α1‐adrenoceptor antagonism to single spiking DRN neurons that we have previously identified as 5‐HT‐containing. Collectively these data provide direct evidence that DRN neurons that exhibit stereotyped burst firing activity are 5‐HT containing. The presence of multiple types of electrophysiologically distinct midbrain 5‐HT neurons is discussed.

GABAB receptors in the median raphe nucleus: distribution and role in the serotonergic control of hippocampal activity

Previous studies have shown that serotonergic neurons of the median raphe nucleus have a suppressive effect on theta synchronization in the hippocampus. Median raphe lesion, suppression of 5-HT neuronal activity by administration of GABA(A) receptor antagonist or by glutamate blockade or depletion produced long-lasting non-interrupted hippocampal theta in freely behaving rats independent of behavior and in rats anesthetized with urethane. Serotonergic neurons show a characteristic sleep-wake pattern of activity and there is evidence that GABAergic mechanisms play an important role in their regulation. In this study we analyzed the distribution and subcellular localization of GABA(B) receptors in the midbrain raphe complex using combined 5-HT/GABA(B) receptor immunohistochemistry at the light and electron microscopic levels and studied the effects of their pharmacological manipulation on hippocampal electroencephalographic activity in urethane-anesthetized rats. We found that sustained infusion of the GABA(B) receptor agonist baclofen into the median raphe nucleus, using the microdialysis technique, elicited lasting theta activity in the hippocampus. The effect was antagonized by selective GABA(B) receptor antagonists. The predominant localization of GABA(B) receptors in the median, as well as in dorsal raphe was found on serotonergic neurons which strongly indicates that the increase in theta occurrence after baclofen injection resulted from suppression of the serotonergic output originating from the median raphe. On the electron microscopic level, we found GABA(B) receptors located extrasynaptically indicating that these receptors are preferentially activated by strong inputs, i.e. when GABA released from the synaptic terminals is sufficient to spill over from the synaptic cleft. Such conditions might be satisfied during rapid eye movement sleep when GABAergic neurons in the raphe are firing at their highest rate and in rhythmic synchronized bursts. Our data indicate that midbrain raphe GABA(B) mechanisms play an important role in behavioral state control and in hippocampal activity, in particular.

Dendroarchitecture of Relay Cells in Thalamic Barreloids: A Substrate for Cross-Whisker Modulation

A double-labeling protocol was used to determine how the dendroarchitecture of relay cells relates to the three-dimensional structure of barreloids in the ventral posterior medial nucleus of the rat thalamus. Single barreloids were retrogradely labeled by injecting Fluoro-Gold in identified barrel columns, and single relay cells activated by the same whisker, or by an adjacent whisker located on the same arc, were juxtacellularly labeled with biotinylated dextran. Results show that the dendritic field of relay cells is asymmetric, variously oriented with respect to the geometry of the barreloids, and that all cells extend dendrites in surrounding barreloids. Extrabarreloid dendrites are of small size (<1.5 μm) and represent up to 54% (range, 11–54%) of the total dendritic length. In contrast, the thick proximal dendrites remain confined to the home barreloid of the cell, being directed toward its center or along its margin. There is a trend for cells located dorsally in barreloids to form more elaborate trees with a larger proportion of extrabarreloid dendrites. Electron microscopic examination of labeled cells shows that extrabarreloid dendrites are exclusively contacted by synaptic terminals of cortical and reticular thalamic origin, whereas intrabarreloid dendrites also receive contacts from lemniscal terminals. Because corticothalamic and reticular thalamic cells establish point-to-point connections with homotopic barreloids, it is proposed that the spatial arrangement of dendrites determines the combination of whisker deflection that best modulates cell firing. Because relay cell responses are direction sensitive, maximal modulation would occur if dendritic field orientation relates to the direction selectivity of responses.

Hippocampal CA3 and CA2 have distinct bilateral innervation patterns to CA1 in rodents

Ipsilateral and contralateral hippocampal CA3–CA1 and CA2–CA1 projections were investigated in adult male Long–Evans rats by retrograde tracing. Injection of the retrograde tracer cholera toxin subunit B in the strata oriens and radiatum of dorsal CA1 resulted in labeling of predominantly pyramidal cells in ipsilateral and contralateral CA3 and CA2. The contralateral and ipsilateral anterior–posterior extents of CA3 innervation to CA1 were similar. Fifteen to twenty per cent of the hippocampus proper cells that give rise to CA1 stratum oriens innervation were CA2 pyramidal cells, whereas CA2 cells were a mere 3% for CA1 stratum radiatum innervation. The preferred projection of CA2 pyramidal cells to the CA1 stratum oriens was also manifested in transgenic mice that express GFP under the control of the CACNG5 promoter, in which CA2 cells express high amounts of GFP. The ratios of ipsilateral to contralateral projections were compared. For the CA3–CA1 connection, we found that dorsal CA1 stratum radiatum received more ipsilateral projections whereas CA1 stratum oriens received more contralateral innervation. Interestingly, ipsilateral connections dominated for both CA2–CA1 stratum oriens and CA2–CA1 stratum radiatum. These results demonstrate that the primary intrahippocampal target of CA2 pyramidal cells is the ipsilateral CA1 stratum oriens, in contrast to CA3 cells which project more diversely to bilateral CA1 regions. Such innervation patterns may suggest differential dendritic information processing in apical and basal dendrites of CA1 pyramidal cells.

Expression pattern of voltage-dependent calcium channel subunits in hippocampal inhibitory neurons in mice.

Different subtypes of voltage-dependent calcium channels (VDCCs) generate various types of calcium currents that play important role in neurotransmitter release, membrane excitability, calcium transients and gene expression. Well-established differences in the physiological properties and variable sensitivity of hippocampal GABAergic inhibitory neurons to excitotoxic insults suggest that the calcium homeostasis, thus VDCC subunits expression pattern is likely different in subclasses of inhibitory cells. Using double-immunohistochemistry, here we report that in mice: 1) Cav2.1 and Cav3.1 subunits are expressed in almost all inhibitory neurons; 2) subunits responsible for the L-type calcium current (Cav1.2 and Cav1.3) are infrequently co-localized with calretinin inhibitory cell marker while Cav1.3 subunit, at least in part, tends to compensate for the low expression of Cav1.2 subunit in parvalbumin-, metabotropic glutamate receptor 1alpha- and somatostatin-immunopositive inhibitory neurons; 3) Cav2.2 subunit is expressed in the majority of inhibitory neurons except in calbindin-reactive inhibitory cells; 4) Cav2.3 subunit is expressed in the vast majority of the inhibitory cells except in parvalbumin- and calretinin-immunoreactive neurons where the proportion of expression of this subunit is considerably lower. These data indicate that VDCC subunits are differentially expressed in hippocampal GABAergic interneurons, which could explain the diversity in their electrophysiological properties, the existence of synaptic plasticity in certain inhibitory neurons and their vulnerability to stressful stimuli.

Optimisation of Embryonic and Larval ECG Measurement in Zebrafish for Quantifying the Effect of QT Prolonging Drugs

Effective chemical compound toxicity screening is of paramount importance for safe cardiac drug development. Using mammals in preliminary screening for detection of cardiac dysfunction by electrocardiography (ECG) is costly and requires a large number of animals. Alternatively, zebrafish embryos can be used as the ECG waveform is similar to mammals, a minimal amount of chemical is necessary for drug testing, while embryos are abundant, inexpensive and represent replacement in animal research with reduced bioethical concerns. We demonstrate here the utility of pre-feeding stage zebrafish larvae in detection of cardiac dysfunction by electrocardiography. We have optimised an ECG recording system by addressing key parameters such as the form of immobilization, recording temperature, electrode positioning and developmental age. Furthermore, analysis of 3 days post fertilization (dpf) zebrafish embryos treated with known QT prolonging drugs such as terfenadine, verapamil and haloperidol led to reproducible detection of QT prolongation as previously shown for adult zebrafish. In addition, calculation of Z-factor scores revealed that the assay was sensitive and specific enough to detect large drug-induced changes in QTc intervals. Thus, the ECG recording system is a useful drug-screening tool to detect alteration to cardiac cycle components and secondary effects such as heart block and arrhythmias in zebrafish larvae before free feeding stage, and thus provides a suitable replacement for mammalian experimentation.