Bilayer Charge Reversal and Modification of Lipid Organization by Dendrimers as Observed by Sum-Frequency Vibrational Spectroscopy.

Polyamidoamine (PAMAM) dendrimers are hyperbranched, nanosized polymers with promising biomedical applications as nanocarriers in targeted drug delivery and gene therapy. For the development of safe dendrimer-based biomedical applications it is necessary to gain an understanding of the detailed mechanism of the interactions of both cationic and anionic dendrimers with cell membranes. To characterize dendrimer-membrane interactions we applied solid-supported lipid bilayers as biomembrane models and utilized infrared-visible sum-frequency vibrational spectroscopy to independently probe the interactions of cationic G5-NH2 and anionic G4.5-COONa dendrimers with the two leaflets of the lipid bilayers. Interaction with both dendrimers led to changes in the interfacial water structure and charge density as evidenced by the changes in the OH band intensities in the sum-frequency spectra of the bilayers. Interaction with the G5-NH2 dendrimer also led to a unique inversion of the sign of the OH-stretch amplitudes, in addition to a decrease in their absolute values. We suggest that the positively charged amino groups on the G5-NH2 dendrimer surface bind to the negatively charged bilayer, while uncompensated positive charges not involved in the binding cause a reversal of the electric field and thus an opposite orientation of the interfacial water molecules. More subtle but nonetheless significant changes were seen in the relative magnitudes of the CH amplitudes. The methyl antisymmetric to symmetric stretch amplitude ratios are altered, implying changes in the tilt angles of the phospholipid alkyl chains. The conformational order of the phospholipid alkyl chains of both leaflets is also influenced by the G5-NH2 dendrimer while G4.5-COONa has no effect on the alkyl chain conformation.

Sodium selective ion channel formation in living cell membranes by polyamidoamine dendrimer.

Polyamidoamine (PAMAM) dendrimers are highly charged hyperbranched protein-like polymers that are known to interact with cell membranes. In order to disclose the mechanisms of dendrimer-membrane interaction, we monitored the effect of PAMAM generation five (G5) dendrimer on the membrane permeability of living neuronal cells followed by exploring the underlying structural changes with infrared-visible sum frequency vibrational spectroscopy (SVFS), small angle X-ray scattering (SAXS) and transmission electron microscopy (TEM). G5 dendrimers were demonstrated to irreversibly increase the membrane permeability of neurons that could be blocked in low-[Na(+)], but not in low-[Ca(2+)] media suggesting the formation of specific Na(+) permeable channels. SFVS measurements on silica supported DPPG-DPPC bilayers suggested G5-specific trans-polarization of the membrane. SAXS data and freeze-fracture TEM imaging of self-organized DPPC vesicle systems demonstrated disruption of DPPC vesicle layers by G5 through polar interactions between G5 terminal amino groups and the anionic head groups of DPPC. We propose a nanoscale mechanism by which G5 incorporates into the membrane through multiple polar interactions that disrupt proximate membrane bilayer and shape a unique hydrophilic Na(+) ion permeable channel around the dendrimer. In addition, we tested whether these artificial Na(+) channels can be exploited as antibiotic tools. We showed that G5 quickly arrest the growth of resistant bacterial strains below 10µg/ml concentration, while they show no detrimental effect on red blood cell viability, offering the chance for the development of new generation anti-resistant antibiotics.

Polyamidoamine dendrimer impairs mitochondrial oxidation in brain tissue.

BACKGROUND: The potential nanocarrier polyamidoamine (PAMAM) generation 5 (G5-NH(2)) dendrimer has been shown to evoke lasting neuronal depolarization and cell death in a concentration-dependent manner. In this study we explored the early progression of G5-NH(2) action in brain tissue on neuronal and astroglial cells. RESULTS: In order to describe early mechanisms of G5-NH(2) dendrimer action in brain tissue we assessed G5-NH(2) trafficking, free intracellular Ca(2+) and mitochondrial membrane potential (Ψ(MITO)) changes in the rat hippocampal slice by microfluorimetry. With the help of fluorescent dye conjugated G5-NH(2), we observed predominant appearance of the dendrimer in the plasma membrane of pyramidal neurons and glial cells within 30 min. Under this condition, G5-NH(2) evoked robust intracellular Ca(2+) enhancements and Ψ(MITO) depolarization both in pyramidal neurons and astroglial cells. Intracellular Ca(2+) enhancements clearly preceded Ψ(MITO) depolarization in astroglial cells. Comparing activation dynamics, neurons and glia showed prevalence of lasting and transient Ψ(MITO) depolarization, respectively. Transient as opposed to lasting Ψ(MITO) changes to short-term G5-NH(2) application suggested better survival of astroglia, as observed in the CA3 stratum radiatum area. We also showed that direct effect of G5-NH(2) on astroglial Ψ(MITO) was significantly enhanced by neuron-astroglia interaction, subsequent to G5-NH(2) evoked neuronal activation. CONCLUSION: These findings indicate that the interaction of the PAMAM dendrimer with the plasma membrane leads to robust activation of neurons and astroglial cells, leading to mitochondrial depolarization. Distinguishable dynamics of mitochondrial depolarization in neurons and astroglia suggest that the enhanced mitochondrial depolarization followed by impaired oxidative metabolism of neurons may be the primary basis of neurotoxicity.

Astrocytes convert network excitation to tonic inhibition of neurons.

BACKGROUND: Glutamate and γ-aminobutyric acid (GABA) transporters play important roles in balancing excitatory and inhibitory signals in the brain. Increasing evidence suggest that they may act concertedly to regulate extracellular levels of the neurotransmitters. RESULTS: Here we present evidence that glutamate uptake-induced release of GABA from astrocytes has a direct impact on the excitability of pyramidal neurons in the hippocampus. We demonstrate that GABA, synthesized from the polyamine putrescine, is released from astrocytes by the reverse action of glial GABA transporter (GAT) subtypes GAT-2 or GAT-3. GABA release can be prevented by blocking glutamate uptake with the non-transportable inhibitor DHK, confirming that it is the glutamate transporter activity that triggers the reversal of GABA transporters, conceivably by elevating the intracellular Na+ concentration in astrocytes. The released GABA significantly contributes to the tonic inhibition of neurons in a network activity-dependent manner. Blockade of the Glu/GABA exchange mechanism increases the duration of seizure-like events in the low-[Mg2+] in vitro model of epilepsy. Under in vivo conditions the increased GABA release modulates the power of gamma range oscillation in the CA1 region, suggesting that the Glu/GABA exchange mechanism is also functioning in the intact hippocampus under physiological conditions. CONCLUSIONS: The results suggest the existence of a novel molecular mechanism by which astrocytes transform glutamatergic excitation into GABAergic inhibition providing an adjustable, in situ negative feedback on the excitability of neurons.

Chronic Cerebral Hypoperfusion-Induced Disturbed Proteostasis of Mitochondria and MAM Is Reflected in the CSF of Rats by Proteomic Analysis.

Declining cerebral blood flow leads to chronic cerebral hypoperfusion which can induce neurodegenerative disorders, such as vascular dementia. The reduced energy supply of the brain impairs mitochondrial functions that could trigger further damaging cellular processes. We carried out stepwise bilateral common carotid occlusions on rats and investigated long-term mitochondrial, mitochondria-associated membrane (MAM), and cerebrospinal fluid (CSF) proteome changes. Samples were studied by gel-based and mass spectrometry-based proteomic analyses. We found 19, 35, and 12 significantly altered proteins in the mitochondria, MAM, and CSF, respectively. Most of the changed proteins were involved in protein turnover and import in all three sample types. We confirmed decreased levels of proteins involved in protein folding and amino acid catabolism, such as P4hb and Hibadh in the mitochondria by western blot. We detected reduced levels of several components of protein synthesis and degradation in the CSF as well as in the subcellular fractions, implying that hypoperfusion-induced altered protein turnover of brain tissue can be detected in the CSF by proteomic analysis.

Calcium signals in the nucleus accumbens: activation of astrocytes by ATP and succinate.

BACKGROUND: Accumulating evidence suggests that glial signalling is activated by different brain functions. However, knowledge regarding molecular mechanisms of activation or their relation to neuronal activity is limited. The purpose of the present study is to identify the characteristics of ATP-evoked glial signalling in the brain reward area, the nucleus accumbens (NAc), and thereby to explore the action of citric acid cycle intermediate succinate (SUC). RESULTS: We described the burst-like propagation of Ca2+ transients evoked by ATP in acute NAc slices from rat brain. Co-localization of the ATP-evoked Ca2+ signalling with immunoreactivities of the astroglia-specific gap junction forming channel protein connexin43 (Cx43) and the glial fibrillary acidic protein (GFAP) indicated that the responsive cells were a subpopulation of Cx43 and GFAP immunoreactive astrocytes. The ATP-evoked Ca2+ transients were present under the blockade of neuronal activity, but were inhibited by Ca2+ store depletion and antagonism of the G protein coupled purinergic P2Y1 receptor subtype-specific antagonist MRS2179. Similarly, Ca2+ transients evoked by the P2Y1 receptor subtype-specific agonist 2-(Methylthio)adenosine 5'-diphosphate were also blocked by MRS2179. These characteristics implied that intercellular Ca2+ signalling originated from the release of Ca2+ from internal stores, triggered by the activation of P2Y1 receptors. Inhibition by the gap junction blockers carbenoxolone and flufenamic acid and by an antibody raised against the gating-associated segment of Cx43 suggested that intercellular Ca2+ signalling proceeded through gap junctions. We demonstrated for the first time that extracellular SUC also evoked Ca2+ transients (EC50 = 50-60 µM) in about 15% of the ATP-responsive NAc astrocytes. By contrast to glial cells, electrophysiologically identified NAc neurons surrounded by ATP-responsive astrocytes were not activated simultaneously. CONCLUSIONS: We concluded, therefore, that ATP- and SUC-sensitive Ca2+ transients appear to represent a signalling layer independent of NAc neurons. This previously unrecognised glial action of SUC, a major cellular energy metabolite, may play a role in linking metabolism to Ca2+ signalling in astrocytic networks under physiological and pathological conditions such as exercise and metabolic diseases.

Deciphering the scopolamine challenge rat model by preclinical functional MRI.

During preclinical drug testing, the systemic administration of scopolamine (SCO), a cholinergic antagonist, is widely used. However, it suffers important limitations, like non-specific behavioural effects partly due to its peripheral side-effects. Therefore, neuroimaging measures would enhance its translational value. To this end, in Wistar rats, we measured whisker-stimulation induced functional MRI activation after SCO, peripherally acting butylscopolamine (BSCO), or saline administration in a cross-over design. Besides the commonly used gradient-echo echo-planar imaging (GE EPI), we also used an arterial spin labeling method in isoflurane anesthesia. With the GE EPI measurement, SCO decreased the evoked BOLD response in the barrel cortex (BC), while BSCO increased it in the anterior cingulate cortex. In a second experiment, we used GE EPI and spin-echo (SE) EPI sequences in a combined (isoflurane + i.p. dexmedetomidine) anesthesia to account for anesthesia-effects. Here, we also examined the effect of donepezil. In the combined anesthesia, with the GE EPI, SCO decreased the activation in the BC and the inferior colliculus (IC). BSCO reduced the response merely in the IC. Our results revealed that SCO attenuated the evoked BOLD activation in the BC as a probable central effect in both experiments. The likely peripheral vascular actions of SCO with the given fMRI sequences depended on the type of anesthesia or its dose.

Cariprazine modulates sleep architecture in rats.

BACKGROUND: Cariprazine is a dopamine D3-preferring D3/D2 receptor partial agonist compound recently introduced to treat schizophrenia and bipolar disorder. Although cariprazine is clinically classified as a low-somnolence drug, to date no detailed polysomnographic study is available on its effect on sleep. AIMS: This study examined the acute systemic effects of cariprazine on the rat sleep architecture and electroencephalography spectral power. METHODS: Sprague Dawley rats were recorded during their normal sleep period for four hours, and their sleep stages were classified. RESULTS: Cariprazine (0.3 mg/kg i.p.) reduced the time spent in rapid eye movement (REM) sleep and increased REM latency. This dose of cariprazine decreased the gamma (40-80 Hz) band frequency oscillations and increased the theta (4-9 Hz) and alpha (9-15 Hz) frequencies during the wake periods but not during slow-wave sleep. The 0.03 mg/kg dose of cariprazine only increased the alpha power during the wake periods, while the 0.003 mg/kg dose was without any effect. CONCLUSION: Taken together, the present results suggest that the REM-suppressing effect of cariprazine may be related to its effectiveness in improving depressive symptoms, as various drugs with similar REM-reducing properties effectively treat the depressive state, whereas the gamma power-reducing effect of cariprazine may be indicative of its efficacy in schizophrenia or mania, as similar effects have been observed with other D2 and 5-HT2 receptor antagonist drugs. These data contribute to our understanding of the complex mechanism of action that may stand behind the clinical efficacy of cariprazine.

Chronic stepwise cerebral hypoperfusion differentially induces synaptic proteome changes in the frontal cortex, occipital cortex, and hippocampus in rats.

During chronic cerebral hypoperfusion (CCH), the cerebral blood flow gradually decreases, leading to cognitive impairments and neurodegenerative disorders, such as vascular dementia. The reduced oxygenation, energy supply induced metabolic changes, and insufficient neuroplasticity could be reflected in the synaptic proteome. We performed stepwise bilateral common carotid occlusions on rats and studied the synaptic proteome changes of the hippocampus, occipital and frontal cortices. Samples were prepared and separated by 2-D DIGE and significantly altered protein spots were identified by HPLC-MS/MS. We revealed an outstanding amount of protein changes in the occipital cortex compared to the frontal cortex and the hippocampus with 94, 33, and 17 proteins, respectively. The high alterations in the occipital cortex are probably due to the hypoxia-induced retrograde degeneration of the primary visual cortex, which was demonstrated by electrophysiological experiments. Altered proteins have functions related to cytoskeletal organization and energy metabolism. As CCH could also be an important risk factor for Alzheimer's disease (AD), we investigated whether our altered proteins overlap with AD protein databases. We revealed a significant amount of altered proteins associated with AD in the two neocortical areas, suggesting a prominent overlap with the AD pathomechanism.

Synchronization of GABAergic inputs to CA3 pyramidal cells precedes seizure-like event onset in juvenile rat hippocampal slices.

Here we address how dynamics of glutamatergic and GABAergic synaptic input to CA3 pyramidal cells contribute to spontaneous emergence and evolution of recurrent seizure-like events (SLEs) in juvenile (P10-13) rat hippocampal slices bathed in low-[Mg(2+)] artificial cerebrospinal fluid. In field potential recordings from the CA3 pyramidal layer, a short epoch of high-frequency oscillation (HFO; 400-800 Hz) was observed during the first 10 ms of SLE onset. GABAergic synaptic input currents to CA3 pyramidal cells were synchronized and coincided with HFO, whereas the glutamatergic input lagged by approximately 10 ms. If the intracellular [Cl(-)] remained unperturbed (cell-attached recordings) or was set high with whole cell electrode solution, CA3 pyramidal cell firing peaked with HFO and GABAergic input. By contrast, with low intracellular [Cl(-)], spikes of CA3 pyramidal cells lagged behind HFO and GABAergic input. This temporal arrangement of HFO, synaptic input sequence, synchrony of GABAergic currents, and pyramidal cell firing emerged gradually with preictal discharges until the SLE onset. Blockade of GABA(A) receptor-mediated currents by picrotoxin reduced the inter-SLE interval and the number of preictal discharges and did not block recurrent SLEs. Our data suggest that dynamic changes of the functional properties of GABAergic input contribute to ictogenesis and GABAergic and glutamatergic inputs are both excitatory at the instant of SLE onset. At the SLE onset GABAergic input contributes to synchronization and recruitment of pyramidal cells. We conjecture that this network state is reached by an activity-dependent shift in GABA reversal potential during the preictal phase.

Purkinje cell number-correlated cerebrocerebellar circuit anomaly in the valproate model of autism.

While cerebellar alterations may play a crucial role in the development of core autism spectrum disorder (ASD) symptoms, their pathophysiology on the function of cerebrocerebellar circuit loops is largely unknown. We combined multimodal MRI (9.4 T) brain assessment of the prenatal rat valproate (VPA) model and correlated immunohistological analysis of the cerebellar Purkinje cell number to address this question. We hypothesized that a suitable functional MRI (fMRI) paradigm might show some altered activity related to disrupted cerebrocerebellar information processing. Two doses of maternal VPA (400 and 600 mg/kg, s.c.) were used. The higher VPA dose induced 3% smaller whole brain volume, the lower dose induced 2% smaller whole brain volume and additionally a focal gray matter density decrease in the cerebellum and brainstem. Increased cortical BOLD responses to whisker stimulation were detected in both VPA groups, but it was more pronounced and extended to cerebellar regions in the 400 mg/kg VPA group. Immunohistological analysis revealed a decreased number of Purkinje cells in both VPA groups. In a detailed analysis, we revealed that the Purkinje cell number interacts with the cerebral BOLD response distinctively in the two VPA groups that highlights atypical function of the cerebrocerebellar circuit loops with potential translational value as an ASD biomarker.

gamma-Hydroxybutyrate binds to the synaptic site recognizing succinate monocarboxylate: a new hypothesis on astrocyte-neuron interaction via the protonation of succinate.

Succinate (SUC), a citrate (CIT) cycle intermediate, and carbenoxolone (CBX), a gap junction inhibitor, were shown to displace [3H]gamma-hydroxybutyrate ([3H]GHB), which is specifically bound to sites present in synaptic membrane subcellular fractions of the rat forebrain and the human nucleus accumbens. Elaboration on previous work revealed that acidic pH-induced specific binding of [3H]SUC occurs, and it has been shown to have a biphasic displacement profile distinguishing high-affinity (K(i,SUC) = 9.1 +/- 1.7 microM) and low-affinity (K(i,SUC) = 15 +/- 7 mM) binding. Both high- and low- affinity sites were characterized by the binding of GHB (K(i,GHB) = 3.9 +/- 0.5 microM and K(i,GHB) = 5.0 +/- 2.0 mM) and lactate (LAC; K(i,LAC) = 3.9 +/- 0.5 microM and K(i,LAC) = 7.7 +/- 0.9 mM). Ligands, including the hemiester ethyl-hemi-SUC, and the gap junction inhibitors flufenamate, CBX, and the GHB binding site-selective NCS-382 interacted with the high-affinity site (in microM: K(i,EHS) = 17 +/- 5, K(i,FFA) = 24 +/- 13, K(i,CBX) = 28 +/- 9, K(i,NCS-382) = 0.8 +/- 0.1 microM). Binding of the Na+,K+-ATPase inhibitor ouabain, the proton-coupled monocarboxylate transporter (MCT)-specific alpha-cyano-hydroxycinnamic acid (CHC), and CIT characterized the low-affinity SUC binding site (in mM: K(i,ouabain) = 0.13 +/- 0.05, K(i,CHC) = 0.32 +/- 0.07, K(i,CIT) = 0.79 +/- 0.20). All tested compounds inhibited [3H]SUC binding in the human nucleus accumbens and had K(i) values similar to those observed in the rat forebrain. The binding process can clearly be recognized as different from synaptic and mitochondrial uptake or astrocytic release of SUC, GHB, and/or CIT by its unique GHB selectivity. The transient decrease of extracellular SUC observed during epileptiform activity suggested that the function of the synaptic target recognizing protonated succinate monocarboxylate may vary under different (patho)physiological conditions. Furthermore, we put forward a hypothesis on the synaptic activity-regulated signaling between astrocytes and neurons via SUC protonation.

Stepwise occlusion of the carotid arteries of the rat: MRI assessment of the effect of donepezil and hypoperfusion-induced brain atrophy and white matter microstructural changes.

Bilateral common carotid artery occlusion (BCCAo) in the rat is a widely used animal model of vascular dementia and a valuable tool for preclinical pharmacological drug testing, although the varying degrees of acute focal ischemic lesions it induces could interfere with its translational value. Recently, a modification to the BCCAo model, the stepwise occlusion of the two carotid arteries, has been introduced. To acquire objective translatable measures, we used longitudinal multimodal magnetic resonance imaging (MRI) to assess the effects of semi-chronic (8 days) donepezil treatment in this model, with half of the Wistar rats receiving the treatment one week after the stepwise BCCAo. With an ultrahigh field MRI, we measured high-resolution anatomy, diffusion tensor imaging, cerebral blood flow measurements and functional MRI in response to whisker stimulation, to evaluate both the structural and functional effects of the donepezil treatment and stepwise BCCAo up to 5 weeks post-occlusion. While no large ischemic lesions were detected, atrophy in the striatum and in the neocortex, along with widespread white matter microstructural changes, were found. Donepezil ameliorated the transient drop in the somatosensory BOLD response in distant cortical areas, as detected 2 weeks after the occlusion but the drug had no effect on the long term structural changes. Our results demonstrate a measurable functional MRI effect of the donepezil treatment and the importance of diffusion MRI and voxel based morphometry (VBM) analysis in the translational evaluation of the rat BCCAo model.

Chronic Cerebral Hypoperfusion Induced Synaptic Proteome Changes in the rat Cerebral Cortex.

Chronic cerebral hypoperfusion (CCH) evokes mild cognitive impairment (MCI) and contributes to the progression of vascular dementia and Alzheimer's disease (AD). How CCH induces these neurodegenerative processes that may spread along the synaptic network and whether they are detectable at the synaptic proteome level of the cerebral cortex remains to be established. In the present study, we report the synaptic protein changes in the cerebral cortex after stepwise bilateral common carotid artery occlusion (BCCAO) induced CCH in the rat. The occlusions were confirmed with magnetic resonance angiography 5 weeks after the surgery. Synaptosome fractions were prepared using sucrose gradient centrifugation from cerebral cortex dissected 7 weeks after the occlusion. The synaptic protein differences between the sham operated and CCH groups were analyzed with label-free nanoUHPLC-MS/MS. We identified 46 proteins showing altered abundance due to CCH. In particular, synaptic protein and lipid metabolism, as well as GABA shunt-related proteins showed increased while neurotransmission and synaptic assembly-related proteins showed decreased protein level changes in CCH rats. Protein network analysis of CCH-induced protein alterations suggested the importance of increased synaptic apolipoprotein E (APOE) level as a consequence of CCH. Therefore, the change in APOE level was confirmed with Western blotting. The identified synaptic protein changes would precede the onset of dementia-like symptoms in the CCH model, suggesting their importance in the development of vascular dementia.

Suppression of spike-wave discharge activity and c-fos expression by 2-methyl-4-oxo-3H-quinazoline-3-acetyl piperidine (Q5) in vivo.

Antiepileptic and network inhibitory actions of Q5 (2-methyl-4-oxo-3H-quinazoline-3-acetyl piperidine) have recently been described in hippocampal slices. Here we present evidence on the in vivo antiabsence effect of Q5. All doses of Q5 tested (0.3 mg/kg, 0.9 mg/kg, 2.8 mg/kg) decreased the number, but not the duration and the frequency of absence spike-wave discharges (SWDs) in freely moving WAG/Rij rats. In vivo network inhibitory action of Q5 was monitored by following c-fos expression in different brain areas of Wistar rats. Significant depletion of c-fos expression was observed after single or repeated injections of Q5 (2.8 mg/kg and 2x2.8 mg/kg) in various brain areas, including hypothalamic paraventricular nucleus, medial amygdaloid nucleus, piriform cortex, somatosensory cortex, periventricular thalamic nucleus and periaqueductal central gray. Thus, our in vivo results demonstrate that in addition to the prevention of absence seizures, Q5 effectively suppresses neuronal activation in various stress- and pain-sensitive brain areas.

Central sensitization-related changes of effective and functional connectivity in the rat inflammatory trigeminal pain model.

Central sensitization is a key mechanism in the pathology of several neuropathic pain disorders. We aimed to investigate the underlying brain connectivity changes in a rat model of chronic pain. Non-noxious whisker stimulation was used to evoke blood-oxygen-level-dependent (BOLD) responses in a block-design functional Magnetic Resonance Imaging (fMRI) experiment on 9.4T. Measurements were repeated two days and one week after injecting complete Freund's adjuvant into the rats' whisker pad. We found that acute pain reduced activation in the barrel cortex, most probably due to a plateau effect. After one week, increased activation of the anterior cingulate cortex was found. Analyses of effective connectivity driven by stimulus-related activation revealed that chronic pain-related central sensitization manifested as a widespread alteration in the activity of the somatosensory network. Changes were mainly mediated by the anterior cingulate cortex and the striatum and affected the somatosensory and motor cortices and the superior colliculus. Functional connectivity analysis of nested BOLD oscillations justified that the anterior cingular-somatosensory interplay is a key element of network changes. Additionally, a decreased cingulo-motor functional connectivity implies that alterations also involve the output tract of the network. Our results extend the knowledge about the role of the cingulate cortex in the chronification of pain and indicate that integration of multiple connectivity analysis could be fruitful in studying the central sensitization in the pain matrix.

Extracellular level of GABA and Glu: in vivo microdialysis-HPLC measurements.

In spite of several studies showing specific physiological functions of changes in the extracellular level of the major excitatory and inhibitory transmitters, Glu and GABA within the brain ([Glu](EXT), [GABA](EXT)) the exact origin (neuronal vs. astroglial, synaptic vs. extrasynaptic) of Glu and GABA present in dialysate samples is still a matter of debate. For better understanding the significance of in vivo microdialysis data, here we discuss methodological details and problems in addition to regulation of [Glu](EXT) and [GABA](EXT). Changes in [Glu](EXT) and [GABA](EXT) under pathological conditions such as ischemia and epilepsy are also reviewed. Based on recent in vivo microdialysis data we argue that ambient [Glu](EXT) and [GABA](EXT)may have a functional role. It is suggested that specific changes in concentrations of Glu and GABA in dialysate samples together with their alterations independent of neuronal activity indicate the involvement of Glu and GABA in the information processing of the brain as essential signaling molecules of nonsynaptic transmission as well. Since various drugs are able to interfere with extrasynaptic signals in vivo, studying the extracellular cell-to-cell communication of brain cells represents a new aspect to improve drugs modulating Gluergic as well as GABAergic neurotransmission.

Preconditioning-specific reduction of c-fos expression in hippocampal granule and pyramidal but not other forebrain neurons of ischemic brain: a quantitative immunohistochemical study.

To specify targets for an ischemic preconditioning paradigm (ischemic tolerance), c-fos expressions in ischemic (induced by 10 min bilateral carotid-occlusions subsequent to coagulation of vertebral arteries) and preconditioned rats (treated for 4 min carotid-occlusions 72 h before ischemia) were compared in 12 forebrain areas/nuclei. Fos immunostaining was applied to serial sections of the forebrain and the density (cell number/area measured) of Fos-immunopositive (Fos+) neurons, as well as their percentile changes were determined in five hippocampal and seven extrahippocampal areas/nuclei of ischemic and preconditioned rats. The ratio of counts found in ischemic over control animals showed several fold increase of Fos+ cells in the three layers (granule cell, molecular and polymorphic) of the dentate gyrus, CA3 and CA1 pyramidal neurons, as well as in thalamic and hypothalamic nuclei and limbic cortical areas. In contrast, preconditioning did not alter c-fos expressions significantly in the extrahippocampal brain areas investigated. These results strengthen the hypothesis that the hippocampal and dentate neurons are more susceptible to ischemic tolerance than cells in other brain regions. In fact stress-response and induction of ischemic tolerance in different forebrain areas can be distinguished.

GABA(B) receptor antagonist CGP-36742 enhances somatostatin release in the rat hippocampus in vivo and in vitro.

Here, we show the modulation of somatostatin functions in the hippocampus by the orally active 'cognition enhancer' GABA(B) receptor antagonist, (3-aminopropyl)n-butylphosphinic acid (CGP-36742), both in vivo and in vitro. Using high-pressure liquid chromatography-coupled electrospray mass spectrometry, we measured a two-fold increase in the extracellular level of somatostatin to CGP-36742 application in the hippocampus of anaesthetised rats. The basal release of [125I]somatostatin in the synaptosomal fraction was increased by CGP-36742 in concentrations lower than 1 muM. Simultaneous measurement of [14C]Glu and [3H]gamma-aminobutyric-acid ([3H]GABA) showed that CGP-36742 increased their basal release. However, prior [125I]somatostatin application suppressed the increase in the basal release of [14C]Glu and induced a net decrease in the basal release of [3H]GABA. Somatostatin application had a similar effect. In slices, CGP-36742 increased the postsynaptic effect of somatostatin on CA1 pyramidal cells. These results suggest a pre- and postsynaptic functional 'cross-talk' between coexisting GABA(B) and somatostatin receptors in the rat hippocampus.

Neuronal and astroglial correlates underlying spatiotemporal intrinsic optical signal in the rat hippocampal slice.

Widely used for mapping afferent activated brain areas in vivo, the label-free intrinsic optical signal (IOS) is mainly ascribed to blood volume changes subsequent to glial glutamate uptake. By contrast, IOS imaged in vitro is generally attributed to neuronal and glial cell swelling, however the relative contribution of different cell types and molecular players remained largely unknown. We characterized IOS to Schaffer collateral stimulation in the rat hippocampal slice using a 464-element photodiode-array device that enables IOS monitoring at 0.6 ms time-resolution in combination with simultaneous field potential recordings. We used brief half-maximal stimuli by applying a medium intensity 50 Volt-stimulus train within 50 ms (20 Hz). IOS was primarily observed in the str. pyramidale and proximal region of the str. radiatum of the hippocampus. It was eliminated by tetrodotoxin blockade of voltage-gated Na(+) channels and was significantly enhanced by suppressing inhibitory signaling with gamma-aminobutyric acid(A) receptor antagonist picrotoxin. We found that IOS was predominantly initiated by postsynaptic Glu receptor activation and progressed by the activation of astroglial Glu transporters and Mg(2+)-independent astroglial N-methyl-D-aspartate receptors. Under control conditions, role for neuronal K(+)/Cl(-) cotransporter KCC2, but not for glial Na(+)/K(+)/Cl(-) cotransporter NKCC1 was observed. Slight enhancement and inhibition of IOS through non-specific Cl(-) and volume-regulated anion channels, respectively, were also depicted. High-frequency IOS imaging, evoked by brief afferent stimulation in brain slices provide a new paradigm for studying mechanisms underlying IOS genesis. Major players disclosed this way imply that spatiotemporal IOS reflects glutamatergic neuronal activation and astroglial response, as observed within the hippocampus. Our model may help to better interpret in vivo IOS and support diagnosis in the future.