Diverse microglial motility behaviors during clearance of dead cells in hippocampal slices

We used two-channel three-dimensional time-lapse fluorescence confocal imaging in live rat hippocampal slice cultures (1-7 days in vitro) to determine the motility behaviors of activated microglia as they engage dead and dying cells following traumatic brain tissue injury. Live microglia were labeled with a fluorescently conjugated lectin (IB(4)), and dead neurons were labeled with a membrane-impermeant fluorescent DNA-binding dye (Sytox Orange or To-Pro-3). Tissue injury during the slicing procedure induced neuronal death and microglial activation, but the density of dead cells diminished approximately 10-fold by 7 days in vitro as resident microglia cleared dead cells. In time-lapse movies (4-20 h long), activated microglia exhibited varying levels of motile and locomotory activity. The motility of microglia could change abruptly following contact by other microglia or death of nearby cells. When neighboring cells died, some microglia rapidly moved toward or extended a process to engulf the dead cell, consistent with a chemotactic signaling response. Dead cell nuclei usually were engulfed and carried along by highly motile and locomoting microglia. The mean time to engulfment was approximately 5 times faster for newly deceased cells (33 min) than for extant dead cells (160 min), suggesting that the efficacy of microglial phagocytosis in situ might vary with time after cell death or mode of cell death. These observations demonstrate that activated microglia are heterogeneous with respect to motile activity following traumatic tissue injury and further indicate that cell motility in situ is temporally regulated at the single cell level, possibly by direct cell-cell contact and by diffusible substances emanating from nearby dead cells.     

Purinergic receptors on microglial cells: functional expression in acute brain slices and modulation of microglial activation in vitro

Microglial cells are the pathologic sensors in the brain. ATP released from damaged cells is a candidate for signalling neural injury to microglia. Moreover, ATP is an extracellular messenger for propagating astrocyte activity in the form of Ca2+ waves. To test for the functional expression of purinoreceptors in microglial cells we employed the patch-clamp technique in acute slices of adult mouse brain. ATP triggered a nonselective cationic and a K+ current. Pharmacological screening with purinergic ligands indicated the presence of P2Y1 and P2Y2/4 receptors linked to the activation of a K+ current and P2X receptors, including P2X7, linked to the activation of a nonselective cationic current. These findings suggest that microglial cells in situ express different purinergic receptors with distinct sensitivity and functional coupling. To test for the involvement of purinoreceptors in microglial activation, we stimulated cultured microglial cells with lipopolysaccharide and measured the release of tumour necrosis factor alpha, interleukin-6, interleukin-12 and macrophage inflammatory protein 1alpha, induction of K+ outward currents and nitric oxide release. All these parameters were reduced in the presence of purinergic ligands, indicating that purinergic receptor activation attenuated indicators of microglial activation.     

Blueberry supplementation attenuates microglial activation in hippocampal intraocular grafts to aged hosts

Transplantation of central nervous tissue has been proposed as a therapeutic intervention for age‐related neurodegenerative diseases and stroke. However, survival of embryonic neuronal cells is hampered by detrimental factors in the aged host brain such as circulating inflammatory cytokines and oxidative stress. We have previously found that supplementation with 2% blueberry in the diet increases graft growth and neuronal survival in intraocular hippocampal grafts to aged hosts. In the present study we explored possible biochemical mechanisms for this increased survival, and we here report decreased microglial activation and astrogliosis in intraocular hippocampal grafts to middle‐aged hosts fed a 2% blueberry diet. Markers for astrocytes and for activated microglial cells were both decreased long‐term after grafting to blueberry‐treated hosts compared with age‐matched rats on a control diet. Similar findings were obtained in the host brain, with a reduction in OX‐6 immunoreactive microglial cells in the hippocampus of those recipients treated with blueberry. In addition, immunoreactivity for the pro‐inflammatory cytokine IL‐6 was found to be significantly attenuated in intraocular grafts by the 2% blueberry diet. These studies demonstrate direct effects of blueberry upon microglial activation both during isolated conditions and in the aged host brain and suggest that this nutraceutical can attenuate age‐induced inflammation. © 2009 Wiley‐Liss, Inc.     

Interneuron firing precedes sequential activation of neuronal ensembles in hippocampal slices

Neuronal firing sequences that occur during behavioral tasks are precisely reactivated in the neocortex and the hippocampus during rest and sleep. These precise firing sequences are likely to reflect latent memory traces, and their reactivation is believed to be essential for memory consolidation and working memory maintenance. However, how the organized repeating patterns emerge through the coordinated interplay of distinct types of neurons remains unclear. In this study, we monitored ongoing spatiotemporal firing patterns using a multi‐neuron calcium imaging technique and examined how the activity of individual neurons is associated with repeated ensembles in hippocampal slice cultures. To determine the cell types of the imaged neurons, we applied an optical synapse mapping method that identifies network connectivity among dozens of neurons. We observed that inhibitory interneurons exhibited an increase in their firing rates prior to the onset of repeating sequences, while the overall activity level of excitatory neurons remained unchanged. A specific repeating sequence emerged preferentially after the firing of a specific interneuron that was located close to the neuron first activated in the sequence. The times of repeating sequences could be more precisely predicted based on the activity patterns of inhibitory cells than excitatory cells. In line with these observations, stimulation of a single interneuron could trigger the emergence of repeating sequences. These findings provide a conceptual framework that interneurons serve as a key regulator of initiating sequential spike activity.     

Involvement of P2X4 receptors in hippocampal microglial activation after status epilepticus

Within the central nervous system, functions of the ATP‐gated receptor‐channel P2X4 (P2X4R) are still poorly understood, yet P2X4R activation in neurons and microglia coincides with high or pathological neuronal activities. In this study, we investigated the potential involvement of P2X4R in microglial functions in a model of kainate (KA)‐induced status epilepticus (SE). We found that SE was associated with an induction of P2X4R expression in the hippocampus, mostly localized in activated microglial cells. In P2X4R‐deficient mice, behavioral responses during KA‐induced SE were unaltered. However, 48h post SE specific features of microglial activation, such as cell recruitment and upregulation of voltage‐dependent potassium channels were impaired in P2X4R‐deficient mice, whereas the expression and function of other microglial purinergic receptors remained unaffected. Consistent with the role of P2X4R in activity‐dependent degenerative processes, the CA1 area was partially protected from SE‐induced neuronal death in P2X4R‐deficient mice compared with wild‐type animals. Our findings demonstrate that P2X4Rs are brought into play during neuronal hyperexcitability and that they control specific aspects of microglial activation. Our results also suggest that P2X4Rs contribute to excitotoxic damages by regulating microglial activation. GLIA 2013;61:1306–1319     

Cellular basis of a rapid effect of mineralocorticosteroid receptors activation on LTP in ventral hippocampal slices

The ventral hippocampus (VH) was recently shown to express lower magnitude LTP compared to the dorsal hippocampus (DH). Exposure to acute stress reversed this difference, and VH slices from stressed rats expressed larger LTP than that produced in the DH, which was reduced by stress. In an attempt to uncover the mechanisms responsible for this differential action, we found that activation of mineralocorticosteroid receptors (MR) by aldosterone mimics the effects of stress in the VH, to facilitate LTP. We also found that aldosterone reduces GABAergic inhibition in both the DH and VH. We now examined if the reduction in inhibition caused by MRs can underlie the altered LTP in the VH. Rat hippocampal slices were recorded before and after exposure to the GABA antagonist bicuculline and to aldosterone. As expected, blockade of GABA with bicuculline enhanced LTP in both DH and VH. However, its effect did not occlude that of aldosterone in the VH, indicating that the latter drug does not operate by blockade of inhibition. Furthermore, the NMDA receptor antagonist APV blocked LTP induced in the presence of bicuculline, but did not block LTP facilitation by aldosterone, indicating that the effect of aldosterone is not mediated by the conventional NMDA-dependent LTP generating mechanism. Furthermore, rapid effects of aldosterone on LTP were blocked by the L-type calcium channel antagonist nifedipine, indicating that aldosterone facilitates calcium influx via nifedipine-sensitive channels, to enhance LTP in the VH. The locus of effect of aldosterone may be the presynaptic terminal, as it caused a marked facilitation of paired pulse potentiation in the VH but not in the DH. These experiments confirm and extend previous suggestions for the effects of MRs on neuronal plasticity in the hippocampus.     

Radial migration of developing microglial cells in quail retina: a confocal microscopy study

Microglial cells spread within the nervous system by tangential and radial migration. The cellular mechanism of tangential migration of microglia has been described in the quail retina but the mechanism of their radial migration has not been studied. In this work, we clarify some aspects of this mechanism by analyzing morphological features of microglial cells at different steps of their radial migration in the quail retina. Microglial cells migrate in the vitreal half of the retina by successive jumps from the vitreal border to progressively more scleral levels located at the vitreal border, intermediate regions, and scleral border of the inner plexiform layer (IPL). The cellular mechanism used for each jump consists of the emission of a leading thin radial process that ramifies at a more scleral level before retraction of the rear of the cell. Hence, radial migration and ramification of microglial cells are simultaneous events. Once at the scleral border of the IPL, microglial cells migrate through the inner nuclear layer to the outer plexiform layer by another mechanism: they retract cell processes, become round, and squeeze through neuronal bodies. Microglial cells use radial processes of s-laminin-expressing Müller cells as substratum for radial migration. Levels where microglial cells stop and ramify at each jump are always interfaces between retinal strata with strong tenascin immunostaining and strata showing weak or no tenascin immunoreactivity. When microglial cell radial migration ends, tenascin immunostaining is no longer present in the retina. These findings suggest that tenascin plays a role in the stopping and ramification of radially migrating microglial cells.     

pH transients due to monosynaptic activation of GABAA receptors in rat hippocampal slices.

Extracellular pH transients were evoked in rat hippocampal brain slices by activation of a monosynaptic inhibitory pathway following pharmacological blockade of glutaminergic transmission. Repetitive stimulation in stratum radiatum near the recording site in stratum pyramidale evoked an immediate alkaline shift which was potentiated by pentobarbital and blocked by picrotoxin but not by 2-hydroxy-saclofen. Benzolamide, a poorly permeant inhibitor of carbonic anhydrase (CA), and prontosil-dextran 5000, a macromolecular CA inhibitor, abolished the alkaline transients evoked by stimulation and by exogenous GABA. Thus an extracellular CA is involved in regulating interstitial pH in brain, and the stimulation-induced alkaline transients are caused by net influx of CO2 into CA1 neurons in response to efflux of bicarbonate across postsynaptic GABAA receptor channels.     

Comparison of glucose and lactate as substrates during NMDA-induced activation of hippocampal slices

It has been postulated that lactate released from astrocytes may be the preferred metabolic substrate for neurons, particularly during intense neuronal activity (the astrocyte-neuron lactate shuttle hypothesis). We examined this hypothesis by exposing rat hippocampal slices to artificial cerebrospinal fluid containing either glucose or lactate and either N-methyl-D-aspartate, which activates neurons without stimulating astrocytic glucose uptake, or alpha-cyano-4-hydroxycinnamate, which blocks monocarboxylate transport across plasma and mitochondrial membranes. When exposed to N-methyl-D-aspartate, slices lost synaptic transmission and K+ homeostasis more slowly in glucose-containing artificial cerebrospinal fluid than in lactate-containing artificial cerebrospinal fluid. After N-methyl-D-aspartate exposure, slices recovered synaptic transmission more completely in glucose. These results suggest that hippocampal neurons can use glucose more effectively than lactate when energy demand is high. In experiments with alpha-cyano-4-hydroxycinnamate, 500 microM alpha-cyano-4-hydroxycinnamate caused loss of K+ homeostasis and synaptic transmission in hippocampal slices during normoxia. When 200 microM alpha-cyano-4-hydroxycinnamate was used, synaptic activity and intracellular pH in slices decreased significantly during normoxia. These results suggest that alpha-cyano-4-hydroxycinnamate may have blocked mitochondrial oxidative metabolism along with lactate transport. Thus, studies using alpha-cyano-4-hydroxycinnamate to demonstrate the presence of a lactate shuttle in the brain tissue may need reevaluation. Our findings, together with observations in the literature that (1) glucose is available to neurons during activation, (2) heightened energy demand rapidly activates glycolysis in neurons, and (3) activation of glycolysis suppresses lactate utilization, suggests that glucose is the primary substrate for neurons during neuronal activation and do not support the astrocyte-neuron lactate shuttle hypothesis.     

Visualising microglial activation in vivo

In health, microglia reside as quiescent guardian cells ubiquitously, but isolated without any cell-cell contacts amongst themselves, throughout the normal CNS. In disease, however, they act as swift "sensors" for pathological events, including subtle ones without any obvious structural damage. Once activated, microglia show a territorially highly restricted involvement in the disease process. This property, peculiar to microglia, confers to them diagnostic value for the accurate spatial localisation of any active disease process, acute or chronic. In the brain, the isoquinoline PK11195, a ligand for the peripheral benzodiazepine binding site (PBBS), binds with relative cellular selectivity to activated, but not resting, microglia. Labelled with carbon-11, (R)-PK11195 and positron emission tomography (PET) have been used for the study of inflammatory and neurodegenerative brain disease in vivo. These studies demonstrate meaningfully distributed patterns of regional [(11)C](R)-PK11195 signal increases that correlate with clinically observed loss of function. Increased [(11)C](R)-PK11195 binding closely mirrors the histologically well-described activation of microglia in the penumbra of focal lesions, as well as in the distant, anterograde, and retrograde projection areas of the lesioned neural pathway. There is also some indication that in long-standing alterations of a neural network with persistent abnormal input, additional signals of glial activation may also emerge in transsynaptic areas. These data suggest that the injured brain is less static than commonly thought and shows subtle glial responses even in macroanatomically stable appearing regions. This implies that glial activation is not solely a sign of tissue destruction, but possibly of disease-induced adaptation or plasticity as well. Whilst further technological and methodological advances are necessary to achieve routine clinical value and feasibility, a systematic attempt to image glial cells in vivo is likely to furnish valuable information on the cellular pathology of CNS diseases and their progression within the distributed neural architecture of the brain.     

Monitoring extracellular glutamate in hippocampal slices with a microsensor

The direct local assessment of glutamate in brain slices may improve our understanding of glutamatergic neurotransmission significantly. However, an analytical technique that monitors glutamate directly in brain slices is currently not available. Most recording techniques either monitor derivatives of glutamate or detect glutamate that diffuses out of the slice. Microsensors provide a promising solution to fulfill this analytical requirement. In the present study we have implanted a 10 microm diameter hydrogel-coated microsensor in the CA1 area of hippocampal slices to monitor extracellular glutamate levels. The influence of several pharmacological agents, which facilitate glutamate release from neurons or astrocytes, was investigated to explore the applicability of the microsensor. It was observed that KCl, veratradine, alpha-latrotoxine (LTX), DL-threo-beta-benzyloxyaspartate (dl-TBOA) and L-cystine rapidly increased the extracellular glutamate levels. As far as we know this is the first study in which a microsensor is applied to monitor dynamic changes of glutamate in brain slices and in our opinion this type of research may contribute greatly to improve our understanding of the physiology of glutamatergic neurotransmission.     

ATP-induced synaptic potentiation in hippocampal slices

The purpose of this study was to investigate the influence of different adenosine triphosphate (ATP) concentrations (ranging from 400 nM to 250 microM) on hippocampal potentials recorded from pyramidal neurons. ATP applied at a concentration of 400 nM induced a 100% increase in the size of the population spike (potentiation). The potential started to increase 30-60 s after ATP application, reached a maximum after 20 min, and remained potentiated for longer than 1.5 h. Washing the slices with fresh Ringer solution did not reverse the effect. ATP applied at a concentration of 50-150 microM, temporarily depressed the potential. This depression, however, was transient, as the potential gradually recovered by itself and reached a value higher than that observed before ATP application. ATP applied at the concentration of 250 microM caused a long-lasting depression of the potential. The potential was not restored by washing the slices, but recovered after addition of 0.7 microM 3,4-diaminopyridine. These data show a concentration-dependent mode of ATP action on hippocampal neurons and suggest a role for ATP in regulating synaptic efficiency.     

Imaging microglial activation in Huntington's disease

Activated microglia have been proposed to play a major role in the pathogenesis of Huntington's Disease (HD). PK11195 is a ligand which binds selectively to peripheral benzodiazepine binding sites, a type of receptor selectively expressed by activated microglia in the central nervous system. Using (11)C-(R)-PK11195 positron emission tomography (PET), we have recently shown in vivo evidence of increased microglial activation in both symptomatic and presymptomatic HD gene carriers and that the degree of microglial activation in the striatum correlates with the severity of striatal dopamine D2 receptor dysfunction measured with (11)C-raclopride PET. Our findings indicate that microglial activation is an early process in the HD pathology, occurring before the onset of symptoms. The close spatial and temporal relationship between microglial activation and neuronal dysfunction lends further support to the pathogenic link between the two processes in HD. Further longitudinal studies are needed to fully elucidate this link.     

Astrocyte-derived GDNF is a potent inhibitor of microglial activation

Neuroinflammation is recognized as a major factor in Parkinson's disease (PD) pathogenesis and increasing evidence propose that microglia is the main source of inflammation contributing to the dopaminergic degeneration observed in PD. Several studies suggest that astrocytes could act as physiological regulators preventing excessive microglia responses. However, little is known regarding how astrocytes modulate microglial activation. In the present study, using Zymosan A-stimulated midbrain microglia cultures, we showed that astrocytes secrete factors capable of modulating microglial activation, namely its phagocytic activity and the production of reactive oxygen species since both parameters were highly diminished in cells incubated with astrocytes conditioned media (ACM). Glial cell line-derived neurotrophic factor (GDNF), cerebral dopamine neurotrophic factor (CDNF) and brain-derived neurotrophic factor (BDNF), known to have a neuroprotective role in the nigrostriatal system, are among the candidates to be astrocyte-secreted molecules involved in the modulation of microglial activation. The effect of ACM on Zymosan A-induced microglial activation was abolished when the GDNF present in the ACM was abrogated using a specific antibody, but not when ACM was neutralized with anti-CDNF, anti-BDNF or with a heat-inactivated GDNF antibody. In addition, media conditioned by astrocytes silenced for GDNF were not able to prevent microglial activation, whereas supplementation of non-conditioned media with GDNF prevented the activation of microglia evoked by Zymosan A. Taken together, these results indicate that astrocyte-derived GDNF plays a major contribution to the control of midbrain microglial activation, suggesting that GDNF can protect from neurodegeneration through the inhibition of neuroinflammation.     

Glucose utilization of ischemic hippocampal slices

Hippocampal brain slices that were 1000 mu thick were prepared from Sprague-Dawley rats and studied using in vitro glucose utilization under well-oxygenated conditions or after a 15 min anoxic insult produced with a nitrogen atmosphere. Autoradiography reveals that glucose utilization is increased in CA1 and CA3 stratum radiatum of 1000 mu slices, even with full oxygenation, compared to the same regions in 540 mu slices. Following anoxia, there is an initial addition increase in stratum oriens of CA1 and CA3 glucose utilization that is followed by a decline in glucose utilization in all slice regions within an hour of the insult. Because increased glucose utilization is apparent at the slice surfaces as well as at the interior, it is suggested that thick brain slices are a model of brain ischemia, not just hypoxia.     

Potassium-induced long-term potentiation in rat hippocampal slices

We observed that a transient increase in extracellular potassium concentration (50 mM for 40 s) was sufficient to induce long-term potentiation (LTP) of synaptic transmission in area CA1 of the hippocampal slice. Potassium-induced potentiation of the Schaffer collateral/commissural synapses demonstrated several features characteristic of tetanus-induced LTP: (1) population excitatory post-synaptic potential (EPSP) amplitudes were enhanced to a similar magnitude (on average 70% above baseline) which (2) lasted for more than 20 min; (3) induction was blocked by bath application of the specific N-methyl-d-aspartate (NMDA) receptor antagonistd-2-amino-5-phosphonovalerate (d-APV), and (4) was attenuated by reduction of the concentration of calcium in the extracellular medium. Induction of either potassium-induced LTP or tetanus-induced LTP occluded the subsequent expression of the other. Finally, exposure to high potassium in the absence of electrical stimulation was sufficient to induce LTP. Taken together, these data indicate that brief depolarizing stimuli other than tetanus can induce LTP. Because potassium-induced LTP is not restricted to the subset of afferents examined electrophysiologically, such a method could facilitate analyses of the biochemical events underlying both the induction and expression of LTP.     

Cyclic AMP-generating systems in rat hippocampal slices

Properties of the norepinephrine- (NE) stimulated, cAMP-generating system were studied in rat hippocampal slices. NE but not other putative neurotransmitters, caused a 3–4-fold rise in cAMP levels in the slices. All 3 main subdivisions of the hippocampus (HPC), the dentate gyrus, areas CA3 and CA1, possessed the capacity to produce cAMP. The latency to the NE stimulation of cAMP formation was about 20 sec but maximal stimulation was reached only after 5–10 min of incubation.Intrahippocampal injection of kainic acid (KA) caused a nearly complete destruction of hippocampal neurons and a marked increase in number of glial cells. NE caused a 12–15-fold rise in cAMP levels in KA-treated HPC. Compared to normal HPC where potency order of noradrenergic agonists indicated activation of a beta-1 receptor type, the pattern for the KA-treated HPC indicated the dominance of beta-2 receptors. The beta-1 antagonist, practolol, and the beta-2 antagonist, H35/25, were about equipotent in blocking the NE-stimulated cAMP formation in normal HPC. In KA-treated HPC, on the other hand, H35/25 was much more potent than practolol in inhibiting NE-stimulated cAMP formation.It is suggested that in the HPC beta-1 adrenergic receptors are primarily neuronal and beta-2 receptors, glial, and that activation of both receptor species results in activation of a cAMP-generating system.     

Nuclear shrinkage in live mouse hippocampal slices

Brain slices are used extensively for biochemical, electrophysiological and molecular investigations. However, only the time frame for electrophysiological and biochemical investigations has as yet been defined. The goal of the present study was to investigate the time course of nuclear structure in live brain slices. Hippocampal slices (300 microm) were prepared from male CD1 mice (25-30 g), stained with Hoechst 33342 (10 microM), calcein-AM (2 microM) and ethidium homodimer (4 microM), and imaged with single- and dual-photon microscopy. The volume of CA1 pyramidal cell nuclei decreased from 759+/-229 microm3 in 40-50 microm depth 25 min after preparation to 453+/-169 microm3 (P<0.001) after 60 min, 315+/-112 microm3 (P<0.001) after 120 min and 128+/-71 microm3 (P<0.001) after 8 h. Similar results were obtained on a prolonged time scale in 70-80 microm depth and with an accelerated time scale in 20-30 microm depth. Live-dead staining showed that cell damage is progressing from the surface to deeper layers of the slices in a time-dependent fashion. We conclude that nuclei of CA1 hippocampal pyramidal cells show a time- and depth-dependent shrinkage converging 8 h after slice preparation to a volume of 90-130 microm; in any depth between 20 and 80 microm. The nucleus in the superficial 80 microm of each side appears dysfunctional even at times suitable for electrophysiological and biochemical experimentation in hippocampal slices. Molecular analysis of cell regulation in brain slices may, therefore, be time-dependently distorted by progressing cell death in at least half of the tissue under investigation.