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ObjectiveLithium carbonate (LiCO) is a mainstay therapeutic for the prevention of mood-episode recurrences in bipolar disorder (BD). Unfortunately, its narrow therapeutic index is associated with complications that may lead to treatment non-compliance. Intriguingly, lithium orotate (LiOr) is suggested to possess uptake properties that would allow for reduced dosing and mitigation of toxicity concerns. We hypothesized that due to differences in pharmacokinetics, LiOr is more potent with reduced adverse effects.MethodsDose responses were established for LiOr and LiCO in male and female mice using an amphetamine-induced hyperlocomotion (AIH) model; AIH captures manic elements of BD and is sensitive to a dose-dependent lithium blockade. Next, the relative toxicities of LiOr and LiCO were contrasted after 14 consecutive daily administrations.ResultsLiCO maintained a partial block of AIH at doses of 15 mg/kg or greater in males and 20 mg/kg or greater in females. In contrast, LiOr elicited a near complete blockade at concentrations of just 1.5 mg/kg in both sexes, indicating improved efficacy and potency. Prior application of an organic-anion transporting polypeptide 1A2 (OATP1A2) inhibitor completely blocked the effects of LiOr on AIH while sparing LiCO, suggesting differences in transport between the two compounds. LiCO, but not LiOr, elicited polydipsia in both sexes, elevated serum creatinine levels in males, and increased serum TSH expression in females.ConclusionsLiOr demonstrates superior efficacy, potency, and tolerability to LiCO in both male and female mice as a result of select transport-mediated uptake.

Astrocytes are highly dynamic cells that modulate synaptic transmission within a temporal domain of seconds to minutes in physiological contexts such as Long-Term Potentiation (LTP) and Heterosynaptic Depression (HSD). Recent studies have revealed that astrocytes also modulate a faster form of synaptic activity (milliseconds to seconds) known as Transient Heterosynaptic Depression (tHSD). However, the mechanism underlying astrocytic modulation of tHSD is not fully understood. Are the traditional gliotransmitters ATP or glutamate released via hemichannels/vesicles or are other, yet, unexplored pathways involved? Using various approaches to manipulate astrocytes, including the Krebs cycle inhibitor fluoroacetate, connexin 43/30 double knockout mice (hemichannels), and inositol triphosphate type-2 receptor knockout mice, we confirmed early reports demonstrating that astrocytes are critical for tHSD. We also confirmed the importance of group II metabotropic glutamate receptors (mGluRs) in astrocytic modulation of tHSD using a group II agonist. Using dominant negative SNARE mice, which have disrupted glial vesicle function, we also found that vesicular release of gliotransmitters and activation of adenosine A1 receptors are not required for tHSD. As astrocytes can release lipids upon receptor stimulation, we asked if astrocyte-derived endocannabinoids are involved in tHSD. Interestingly, a cannabinoid receptor 1 (CB1R) antagonist blocked and an inhibitor of the endogenous endocannabinoid 2-arachidonyl glycerol (2-AG) degradation potentiates tHSD in hippocampal slices. Taken together, this study provides the first evidence for group II mGluR-mediated astrocytic endocannabinoids in transiently suppressing presynaptic neurotransmitter release associated with the phenomenon of tHSD.

It was long thought that astrocytes, given their lack of electrical signaling, were not involved in communication with neurons. However, we now know that one astrocyte on average maintains and regulates the extracellular neurotransmitter and potassium levels of more than 140,000 synapses, both excitatory and inhibitory, within their individual domains, and form a syncytium that can propagate calcium waves to affect distant cells via release of "gliotransmitters" such as glutamate, ATP, or adenosine. Neuromodulators can affect signal-to-noise and frequency transmission within cortical circuits by effects on inhibition, allowing for the filtering of relevant vs. irrelevant stimuli. Moreover, synchronized "resting" and desynchronized "activated" brain states are gated by short bursts of high-frequency neuromodulatory activity, highlighting the need for neuromodulation that is robust, rapid, and far-reaching. As many neuromodulators are released in a volume manner where degradation/uptake and the confines of the complex CNS limit diffusion distance, we ask the question-are astrocytes responsible for rapidly extending neuromodulator actions to every synapse? Neuromodulators are known to influence transitions between brain states, leading to control over plasticity, responses to salient stimuli, wakefulness, and sleep. These rapid and wide-spread state transitions demand that neuromodulators can simultaneously influence large and diverse regions in a manner that should be impossible given the limitations of simple diffusion. Intriguingly, astrocytes are ideally situated to amplify/extend neuromodulator effects over large populations of synapses given that each astrocyte can: (1) ensheath a large number of synapses; (2) release gliotransmitters (glutamate/ATP/adenosine) known to affect inhibition; (3) regulate extracellular potassium that can affect excitability and excitation/inhibition balance; and (4) express receptors for all neuromodulators. In this review article, we explore the hypothesis that astrocytes extend and amplify neuromodulatory influences on neuronal networks via alterations in calcium dynamics, the release of gliotransmitters, and potassium homeostasis. Given that neuromodulatory networks are at the core of our sleep-wake cycle and behavioral states, and determine how we interact with our environment, this review article highlights the importance of basic astrocyte function in homeostasis, general cognition, and psychiatric disorders.

Accumulating data point to a key role of Ca2+-dependent gliotransmitter release as a modulator of neuronal networks. Here, we tested the hypothesis that astrocytes in response to agonist exposure also release lipid modulators through activation of Ca2+-independent phospholipase A2(iPLA2) activity. We found that cultured rat astrocytes treated with selective ATP and glutamatergic agonists released arachidonic acid (AA) and/or its derivatives, including the endogenous cannabinoid 2-arachidonoyl-sn-glycerol (2AG) and prostaglandin E2 (PGE2). Surprisingly, buffering of cytosolic Ca2+resulted in a sharp increase in agonist-induced astrocytic lipid release. In addition, astrocytic release of PGE2enhanced miniature excitatory post-synaptic potentials (mEPSPs) by inhibiting the opening of neuronal Kv channels in brain slices. This study provides the first evidence for the existence of a Ca2+-independent pathway regulating the release of PGE2from astrocytes, and furthermore demonstrates a functional role for astrocytic lipid release in the modulation of synaptic activity.Significance StatementUntil now, the majority of studies implicating astrocytes in modulating synaptic activity have focused on Ca2+-dependent release of traditional gliotransmitters such as D-serine, ATP, and glutamate. Mobilization of intracellular stores of Ca2+occurs within a matter of seconds, but this novel Ca2+-independent lipid pathway in astrocytes could potentially occur on a still faster time scale and thus participate in the rapid signaling processes involved in synaptic potentiation, attention, and neurovascular coupling.

Cumulative data point to a key role of Ca 2+ -dependent gliotransmitter release as a modulator of neuronal networks. Here, we tested the hypothesis that astrocytes in response to agonist exposure also release lipid modulators through Ca 2+ -independent phospholipase A 2 (iPLA 2 ) activity. We found that cultured rat astrocytes in response to agonist exposure, released Arachidonic Acid (AA) and/or its derivatives, including the endogenous cannabinoid, 2-arachidonoyl-sn-glycerol (2AG) and the prostaglandin E2 (PGE 2 ). Surprisingly, buffering of cytosolic Ca 2+ was linked to a sharp increase in astrocytic lipid release. In addition, astrocytic release of PGE 2 enhanced mEPSPs by inhibiting the opening of neuronal Kv channels in acute brain slices. This study provides the first evidence for the existence of a Ca 2+ -independent pathway for the release of PGE 2 from astrocytes and furthermore demonstrates a functional role for astrocytic lipid release in the modulation of synaptic activity.

Changes in extracellular potassium ([K+]e) modulate neuronal networks via changes in membrane potential, voltage-gated channel activity and alteration of transmission at the synapse. Given the limited extracellular space in the CNS, potassium clearance is crucial. As activity-induced potassium transients are rapidly managed by astrocytic Kir4.1 and astrocyte-specific Na+/K+-ATPase (NKA), any neurotransmitter/neuromodulator that can regulate their function may have indirect influence on network activity. Neuromodulators differentially affect cortical/thalamic networks to align sensory processing with differing behavioral states. Given serotonin (5HT), norepinephrine (NE), and acetylcholine (ACh) differentially affect spike frequency adaptation and signal fidelity (“signal-to-noise”) in somatosensory cortex, we hypothesize that [K+]e may be differentially regulated by the different neuromodulators to exert their individual effects on network function. This study aimed to compare effects of individually applied 5HT, NE, and ACh on regulating [K+]e in connection to effects on cortical evoked response amplitude and adaptation in male mice. Using extracellular field and K+ ion-selective recordings of somatosensory stimulation, we found that differential effects of 5HT, NE, and ACh on [K+]e regulation mirrored differential effects on amplitude and adaptation. 5HT effects on transient K+ recovery, adaptation and field post-synaptic potential amplitude were disrupted by barium (200 µM), whereas NE and ACh effects were disrupted by ouabain (1 µM) or iodoacetate (100 µM). Considering the impact [K+]e can have on many network functions; it seems highly efficient that neuromodulators regulate [K+]e to exert their many effects. This study provides functional significance for astrocyte-mediated buffering of [K+]e in neuromodulator-mediated shaping of cortical network activity.Significance statementWe demonstrate that the neuromodulators serotonin, norepinephrine, and acetylcholine all have distinct effects on astrocyte-mediated extracellular potassium regulation and that these differential actions are associated with the different effects of the neuromodulators on cortical networks. By affecting astrocytic potassium regulation, long-range neuromodulatory networks can rapidly and efficiently affect broad areas of the brain. Given that neuromodulatory networks are at the core of our behavioral state and determine how we interact with our environment, these studies highlight the importance of basic astrocyte function in general cognition and psychiatric disorders.

In the somatosensory cortex, inhibitory networks are involved in low frequency sensory input adaptation/habituation that can be observed as a paired-pulse depression when using a dual stimulus electrophysiological paradigm. Given that astrocytes have been shown to regulate inhibitory interneuron activity, we hypothesized that astrocytes are involved in cortical sensory adaptation/habituation and constitute effectors of the 5HT-mediated increase in frequency transmission. Using extracellular recordings of evoked excitatory postsynaptic potentials (eEPSPs) in layer II/III of somatosensory cortex, we used various pharmacological approaches to assess the recruitment of astrocyte signaling in paired-pulse depression and serotonin-mediated increase in the paired-pulse ratio (pulse 2/pulse 1). In the absence of neuromodulators or pharmacological agents, the first eEPSP is much larger in amplitude than the second due to the recruitment of long-lasting evoked GABAA-dependent inhibitory activity from the first stimulus. Disruption of glycolysis or mGluR5 signaling resulted in a very similar loss of paired-pulse depression in field recordings. Interestingly, paired-pulse depression was similarly sensitive to disruption by ATP P2Y and adenosine A2A receptor antagonists. In addition, we show that pharmacological disruption of paired-pulse depression by mGluR5, P2Y, and glycolysis inhibition precluded serotonin effects on frequency transmission (typically increased the paired-pulse ratio). These data highlight the possibility for astrocyte involvement in cortical inhibitory activity seen in this simple cortical network and that serotonin may act on astrocytes to exert some aspects of its modulatory influence.

North American incidence of Alzheimer’s disease (AD) is expected to more than double over the coming generation. Although genetic factors surrounding the production and clearance of amyloid-β and phosphorylated tau proteins are known to be responsible for a subset of early-onset AD cases, they do not explain the pathogenesis of the far more prevalent sporadic late-onset variant of the disease. It is thus likely that lifestyle and environmental factors contribute to neurodegenerative processes implicated in the pathogenesis of AD. Herein, we review evidence that (1) excess sucrose consumption induces AD-associated liver pathologies and brain insulin resistance, (2) chronic stress overdrives activity of locus coeruleus neurons, leading to loss of function (a common event in neurodegeneration), (3) high-sugar diets and stress promote the loss of neuroprotective sex hormones in men and women, and (4) Western dietary trends set the stage for a lithium-deficient state. We propose that these factors may intersect as part of a “perfect storm” to contribute to the widespread prevalence of neurodegeneration and AD. In addition, we put forth the argument that exercise and supplementation with trace lithium can counteract many of the deleterious consequences associated with excessive caloric intake and perpetual stress. We conclude that lifestyle and environmental factors likely contribute to AD pathogenesis and that simple lifestyle and dietary changes can help counteract their effects.

Diabetes is a prevalent metabolic disorder that has long been associated with changes in different regions of the brain, including the hippocampus. Changes in hippocampal synaptic plasticity and subsequent impairment in cognitive functions such as learning and memory, are well documented in animal models of type 1 and type 2 diabetes. It is known that RAGE contributes to peripheral micro- and macro-vascular complications of diabetes. However, it is still unknown if RAGE plays a similar role in the development of CNS complications of diabetes. Therefore, we hypothesize that RAGE contributes to cognitive dysfunction, such as learning and memory impairments, in a mouse model of STZ-induced hyperglycemia. Control and STZ-induced hyperglycemic mice from WT and RAGE-KO groups were used for the behavioral experiments. While STZ-induced hyperglycemia decreased locomotor activity in the open field (OF) test, it did not affect the recognition memory in the novel object recognition (NOR) test in either genotype. Spatial memory, however, was impaired in STZ-induced hyperglycemic mice in WT but not in RAGE-KO group in both the Barnes maze (BM) and the Morris water maze (MWM) tests. Consistently, the RAGE antagonist FPS-ZM1 protected WT STZ-induced hyperglycemic mice from spatial memory impairment in the BM test. Our findings indicate that the parameters associated with locomotor activity and recognition memory were independent of RAGE in STZ-induced hyperglycemic mice. In contrast, the parameters associated with hippocampal-dependent spatial memory were dependent on RAGE expression.

Alzheimer’s disease (AD) is the sixth leading cause of death in the United States where it is estimated that one in three seniors dies with AD or another dementia. Are modern lifestyle habits a contributing factor? Increased carbohydrate (sugar) consumption, stress and disruption of sleep patterns are quickly becoming the norm rather than the exception. Interestingly, seven months on a non-invasive high sucrose diet (20% sucrose in drinking water) has been shown to induce behavioral, metabolic and pathological changes consistent with AD in wild-type mice. As chronic stress and depression are associated with loss of locus coeruleus (LC) noradrenergic neurons and projections (source of anti-inflammatory and trophic factor control), we assessed the ability for a selective LC neurotoxin (DSP4) to accelerate and aggravate a high-sucrose mediated AD-related phenotype in wild-type mice. Male C57/Bl6 mice were divided into four groups: 1) saline injected, 2) DSP4 injected, 3) high sucrose drinking water (20%) or 4) DSP4 injected and high sucrose drinking water. We demonstrate that high sucrose consumption and DSP4 treatment promote an early-stage AD-related phenotype after only 3–4 months, as evidenced by elevated fecal corticosterone, increased despair, spatial memory deficits, increased AChE activity, elevated NO production, decreased pGSK3β and increased pTau. Combined treatment appears to accelerate and aggravate pathological processes consistent with Alzheimer disease and dementia. Developing a simple model in wild-type mice will highlight environmental and lifestyle factors that need to be addressed to slow, prevent or even reverse the rising trend in dementia patient numbers and cost.

Background and Purpose Phosphorylation and degradation of myosin light chain 1 (MLC1) during myocardial ischaemia/reperfusion (I/R) injury is a well-established phenomenon. It has been established that MMP-2 is involved in MLC1 degradation and that this degradation is increased when MLC1 is phosphorylated. We hypothesized that simultaneous inhibition of MLC1 phosphorylation and MMP-2 activity will protect hearts from I/R injury. As phosphorylation of MLC1 and MMP-2 activity is important for normal heart function, we used a cocktail consisting combination of low (subthreshold for any protective effect alone) doses of MLC kinase, MMP-2 inhibitors and subthreshold dose of an MLC phosphatase activator. Experimental Approach Isolated rat hearts were subjected to 20 min of global, no-flow ischaemia and 30 min reperfusion in the absence and presence of inhibitors of MLC1 phosphorylation and degradation. Key Results The recovery of cardiac function was improved in a concentration-dependent manner by the MLC kinase inhibitor, ML-7 (1–5 μM), the MLC phosphatase activator, Y-27632 (0.05–1 μM) or the MMP inhibitor, doxycycline (Doxy, 1–30 μM). Co-administration of subthreshold doses of ML-7 (1 μM) and Y-27632 (0.05 μM) showed a potential synergistic effect in protecting cardiac contractility and MLC1 levels in I/R hearts. Further combination with a subthreshold concentration of Doxy (1 μM) showed additional protection that resulted in full recovery to control levels. Conclusions and Implications The results of this study exemplify a novel low-dose multidrug approach to pharmacological prevention of reperfusion injury that will enable a reduction of unwanted side effects and/or cytotoxicity associated with currently available MMP-2 and kinase inhibiting drugs.

Given the brain's uniquely high cell density and tissue oxygen levels bordering on hypoxia, the ability to rapidly and precisely match blood flow to constantly changing patterns in neural activity is an essential feature of cerebrovascular regulation. Locus coeruleus-norepinephrine (LC-NE) projections innervate the cerebral vasculature and can mediate vasoconstriction. However, function of the LC-mediated constriction in blood-flow regulation has never been addressed. Here, using intrinsic optical imaging coupled with an anesthesia regimen that only minimally interferes with LC activity, we show that NE enhances spatial and temporal aspects of functional hyperemia in the mouse somatosensory cortex. Increasing NE levels in the cortex using an α2-adrenergic receptor antagonist paradoxically reduces the extent of functional hyperemia while enhancing the surround blood-flow reduction. However, the NE-mediated vasoconstriction optimizes spatial and temporal focusing of the hyperemic response resulting in a sixfold decrease in the disparity between blood volume and oxygen demand. In addition, NE-mediated vasoconstriction accelerated redistribution to subsequently active regions, enhancing temporal synchronization of blood delivery. These observations show an important role for NE in optimizing neurovascular coupling. As LC neuron loss is prominent in Alzheimer and Parkinson diseases, the diminished ability to couple blood volume to oxygen demand may contribute to their pathogenesis.

Aquaporin-4 (AQP4) is a primary influx route for water during brain edema formation. Here, we provide evidence that brain swelling triggers Ca 2+ signaling in astrocytes and that deletion of the Aqp4 gene markedly interferes with these events. Using in vivo two-photon imaging, we show that hypoosmotic stress (20% reduction in osmolarity) initiates astrocytic Ca 2+ spikes and that deletion of Aqp4 reduces these signals. The Ca 2+ signals are partly dependent on activation of P2 purinergic receptors, which was judged from the effects of appropriate antagonists applied to cortical slices. Supporting the involvement of purinergic signaling, osmotic stress was found to induce ATP release from cultured astrocytes in an AQP4-dependent manner. Our results suggest that AQP4 not only serves as an influx route for water but also is critical for initiating downstream signaling events that may affect and potentially exacerbate the pathological outcome in clinical conditions associated with brain edema.

Both serotonin (5-HT) and stress exert changes in cortical inhibitory tone to shape the activity of cortical networks. Because astrocytes are also known to affect inhibition through established purinergic pathways, we assessed the role of GABA and purinergic pathways with respect to the effects of rapid corticosterone (CORT) and 5-HT on cortical inhibition. We used a paired-pulse paradigm (P1 and P2) in acutely isolated mouse brain slices to evaluate changes in cortical evoked inhibition. Normally, 5-HT decreases the amplitude of the first pulse P1, whereas it increases the amplitude of P2 (increasing frequency transmission). Interestingly, it was observed that CORT application decreased P1 and increased P2 similar to that of 5-HT application. Given that CORT and 5-HT are known to modulate inhibition, we applied the GABAA antagonist bicuculline in the presence of both and found that the increase in P2 and the P2/P1 was lost, providing evidence for a common mechanism involving GABAA receptor signalling. Additional occlusion experiments (ie, 5-HT in presence of CORT and CORT in presence of 5-HT) provide further support for a common mechanism. Because both 5-HT and CORT blocked the increase in P2 and P2/P1 with respect to the other, we suggest 5-HT/CORT already utilise the shared mechanism to affect cortical inhibition. Using low concentrations of the GAPDH inhibitor iodoacetate, as commonly used to selectively disrupt astrocyte metabolism, we found that the increase in P2 and P2/P1 was similarly blocked in response to both CORT and 5-HT. Because astrocyte signalling depends in large part on purinergic pathways, the purinergic contribution was assessed using Ab129 (P2Y antagonist) and SCH 58261 (A2A antagonist). Once again, P2Y and A2A receptor blockade similarly disrupted 5-HT- or CORT-mediated increases in P2 and P2/P1. Taken together, these results support the common involvement of GABAergic and purinergic pathways in the effects of CORT and 5-HT that may also involve astrocytes.

The locus coeruleus (LC) provides the sole source of norepinephrine (NE) to the cortex for modulation of cortical synaptic activity in response to salient sensory information. NE has been shown to improve signal-to-noise ratios, sharpen receptive fields and function in learning, memory, and cognitive performance. Although LC-mediated effects on neurons have been addressed, involvement of astrocytes has thus far not been demonstrated in these neuromodulatory functions. Here we show for the 1st time in live mice, that astrocytes exhibit rapid Ca(2+) increases in response to electrical stimulation of the LC. Additionally, robust peripheral stimulation known to result in phasic LC activity leads to Ca(2+) responses in astrocytes throughout sensory cortex that are independent of sensory-driven glutamate-dependent pathways. Furthermore, the astrocytic Ca(2+) transients are competitively modulated by alpha(2)-specific agonist/antagonist combinations known to impact LC output, are sensitive to the LC-specific neurotoxin N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine, and are inhibited locally by an alpha-adrenergic antagonist. Future investigations of LC function must therefore consider the possibility that LC neuromodulatory effects are in part derived from activation of astrocytes.

Chloride concentration has been shown to have a dramatic impact on protein folding and subsequent tertiary conformation [K.D. Collins, Ions from the Hofmeister series and osmolytes: effects on proteins in solution and in the crystallization process, Methods 34 (2004) 300-311; I. Jelesarov, E. Durr, R.M. Thomas, H.R. Bosshard, Salt effects on hydrophobic interaction and charge screening in the folding of a negatively charged peptide to a coiled coil (leucine zipper), Biochemistry 37 (1998) 7539-7550]. As it is known that Kv channel gating is linked to the stability of the cytoplasmic T1 multimerization domain conformation [D.L. Minor, Y.F. Lin, B.C. Mobley, A. Avelar, Y.N. Jan, L.Y. Jan, J.M. Berger, The polar T1 interface is linked to conformational changes that open the voltage-gated potassium channel, Cell 102 (2000) 657-670; B.A. Yi, D.L. Minor Jr., Y.F. Lin, Y.N. Jan, L.Y. Jan, Controlling potassium channel activities: interplay between the membrane and intracellular factors, Proc. Natl. Acad. Sci. U.S.A. 98 (2001) 11016-11023] and that intracellular chloride concentration has been linked to Kv channel kinetics [L.K. Bekar, W. Walz, Intracellular chloride modulates A-type potassium currents in astrocytes, Glia 39 (2002) 207-216; W.B. Thoreson, S.L. Stella, Anion modulation of calcium current voltage dependence and amplitude in salamander rods, Biochim. Biophys. Acta 1464 (2000) 142-150], the objective of the present study was to address how chloride concentration changes affect Kv channel kinetics more closely in an isolated expression system. Initially, no significant chloride concentration-dependent effects on channel steady-state gating kinetics were observed. Only after disruption of the cytoskeleton with cytochalasin-D did we see significant chloride concentration-dependent shifts in gating kinetics. This suggests that the shift in gating kinetics is mediated through effects of intracellular chloride concentration on cytoplasmic domain tertiary conformation as cytoskeletal interaction appears to mask the effect. Furthermore, as cytoskeletal disruption only impacts channel gating kinetics at low physiological intracellular chloride concentrations, these studies highlight the importance of paying close attention to anion concentrations used under experimental conditions.

Voltage-gated potassium channels are well established as critical for setting action potential frequency, membrane potential, and neurotransmitter release in neurons. However, their role in the “nonexcitable” glial cell type is yet to be fully understood. We used whole cell current kinetics, pharmacology, immunocytochemistry, and RT-PCR to characterize A-type current in hippocampal astrocyte cultures to better understand its function. Pharmacological analysis suggests that ∼70, 10, and 2+-sensitive, cytoplasmic subunits that appear to be specifically localized to subcellular membrane compartments. Function of these channels remains to be determined in a physiological setting. However, this study suggests that they enable astrocytes to respond rapidly with membrane voltage oscillations to high-frequency incoming signals, possibly synchronizing astrocyte function to neuronal activity.

Vimentin-expressing astrocytes in the adult are commonly associated with the proximal, most reactive gliotic response ultimately leading to the formation of a new glial limitans. It was thought, since vimentin expression and astroglial proliferation are most prominent nearest the lesion site, that vimentin may be a characteristic of immature newly divided astrocytes. We recently established a unique distribution of vimentin-expressing reactive astrocytes at the base of a focal cortical ischemic lesion in rats. The purpose of the present study was to assess the correlation of proliferation and migration with this unique distribution following focal injury. With the use of bromodeoxyuridine (BrdU) and immunohistochemistry for astrocytes and microglia/macrophages, proliferation and migration of cells was shown to be throughout the ipsilateral hemisphere on day one and become progressively more centralized to the lesion by day 3. The vimentin-expressing area at the base of the lesion does not exhibit distinguishable proliferation rates from non-vimentin-expressing regions surrounding the lesion and did not demonstrate obvious double labeling with BrdU+ cells, although on occasion vimentin expression is closely associated with BrdU. However, this region did become a focal point for migration into and around the lesion by day 3. Additionally, asymmetrical distribution of vimentin was shown in four different injury models with vimentin+ cells always situated between the lesion and the corpus callosum. It is concluded that although vimentin-expressing cells did not correlate with proliferating cells in these focal injury models, perhaps this distinct population of reactive astrocytes serve as a source of cytokines or as a physical conduit for migrating cells from distant sites through the corpus callosum.

The effects of CLCA protein expression on the regulation of Cl− conductance by intracellular Ca2+ and cAMP have been studied previously in nonepithelial cell lines chosen for low backgrounds of endogenous Cl− conductance. However, CLCA proteins have been cloned from, and normally function in, differentiated epithelial cells. In this study, we examine the effects of differentiation of the Caco-2 epithelial colon carcinoma cell line on modulation of Cl− conductance by pCLCA1 protein expression. Cl− transport was measured as 36Cl− efflux, as transepithelial short-circuit currents, and as whole cell patch-clamp current-voltage relations. The rate of 36Cl− efflux and amplitude of currents in patch-clamp studies after the addition of the Ca2+ ionophore A-23187 were increased significantly by pCLCA1 expression in freshly passaged Caco-2 cells. However, neither endogenous nor pCLCA1-dependent Ca2+-sensitive Cl− conductance could be detected in 14-day-postpassage cells. In contrast to Ca2+-sensitive Cl− conductance, endogenous cAMP-dependent Cl− conductance does not disappear on Caco-2 differentiation. cAMP-dependent Cl− conductance was modulated by pCLCA1 expression in Caco-2 cells, and this modulation was observed in freshly passaged and in mature 14-day-postpassage Caco-2 cultures. pCLCA1 mRNA expression, antigenic pCLCA1 protein epitope expression, and pCLCA1 function as a modulator of cAMP-dependent Cl− conductance were retained through differentiation in Caco-2 cells, whereas Ca2+-dependent Cl− conductance disappeared. We conclude that pCLCA1 expression may increase the sensitivity of preexisting endogenous Cl− channels to Ca2+ and cAMP agonists but apparently lacks inherent Cl− channel activity under growth conditions where endogenous channels are not expressed.

Members of the CLCA protein family are expressed in airway and intestinal epithelium, where they may participate in secretory activity as mediators of chloride conductance. A calcium-dependent chloride conductance has been observed upon expression of CLCA proteins in non-epithelial cell lines. The pCLCA1 gene, cloned in our laboratory, codes for a product containing a unique A-kinase consensus acceptor site not found in other CLCA proteins. Calcium-dependent, but not cAMP-dependent, chloride conductance increased when pCLCA1 was expressed in NIH/3T3 fibroblasts. We transfected the Caco-2 human colon carcinoma cell line with pCLCA1 to investigate the regulation of CLCA-associated chloride conductance in this differentiated epithelial cell line. Expression of pCLCA1 in the Caco-2 cell line enhanced cAMP-responsive 36 Cl efflux, short circuit current, and whole cell chloride current in these cells. This cAMP-dependent chloride conductance was localized to the apical membrane of polarized Caco-2 cells.

Systemic lipopolysaccharide (LPS) is widely used to induce a neuroinflammatory response that is associated with short-term 'sickness'-behavior that can include fever, loss of activity, loss of appetite, impaired cognition, anxiety and depression. If large enough or left unchecked, this neuroinflammatory response can become self-perpetuating and lead to long-term neurodegenerative processes. In this study, we assess the longer-term effects of a single systemic LPS injection on electrophysiological neuromodulator effects and basic behavioral analysis in mice. Five months after LPS injection, we find a mild reduction in cortical inhibition and altered temporal dynamics of acetylcholine but not norepinephrine or serotonin neuromodulator effects. Consistent with electrophysiological findings, LPS treated mice showed a deficit in memory performance in the novel object recognition test with no effect on measures of anxiety or despair as measured in the open field test and tail suspension test, respectively. Furthermore, LPS-treated mice showed an increase in acetylcholinesterase activity. As increased acetylcholinesterase activity is associated with reduced acetylcholine signaling and impaired cognitive ability, these studies demonstrate the potential for a single inflammatory event to initiate processes that may lead to long-term neurodegeneration.

Matrix metalloproteinases (MMPs) significantly contribute to ischemia reperfusion (I/R) injury, namely, by the degradation of contractile proteins. However, due to the experimental models adopted and lack of isoform specificity of MMP inhibitors, the cellular source and identity of the MMP(s) involved in I/R injury remain to be elucidated. Using isolated adult rat cardiomyocytes, subjected to chemically induced I/R-like injury, we show that specific inhibition of MMP-2 expression and activity using MMP-2 siRNA significantly protected cardiomyocyte contractility from I/R-like injury. This was also associated with increased expression of myosin light chains 1 and 2 (MLC1/2) in comparison to scramble siRNA transfection. Moreover, the positive effect of MMP-2 siRNA transfection on cardiomyocyte contractility and MLC1/2 expression levels was also observed under control conditions, suggesting an important additional role for MMP-2 in physiological sarcomeric protein turnover. This study clearly demonstrates that intracellular expression of MMP-2 plays a significant role in sarcomeric protein turnover, such as MLC1 and MLC2, under aerobic (physiological) conditions. In addition, this study identifies intracellular/autocrine, cardiomyocyte-produced MMP-2, rather than paracrine/extracellular, as responsible for the degradation of MLC1/2 and consequent contractile dysfunction in cardiomyocytes subjected to I/R injury.

This study investigates the importance of chemoreceptive and trigeminal information during voluntarily initiated diving in rats. The heart rate responses to simulated diving are unaffected by chemoreceptor drive [McCulloch, P.F., and N. H. West. Am. J. Physiol. 263 (Regulatory Integrative Comp. Physiol. 32): R1049-R1056, 1992] but are reversibly eliminated by infusion of glutamate receptor antagonists into the spinal trigeminal nuclei [McCulloch, P. F., I. A. Paterson, and N. H. West. Am. J. Physiol. 269 (Regulatory Integrative Comp. Physiol. 38): R669-R677, 1995]. To investigate the role of chemoreceptor drive in conscious dives, rats were made hypercapnic, hyperoxic, or hypoxic predive. The role of trigeminal input was explored by infusing the glutamatergic antagonists D-2-amino-7-phosphoheptanoic acid and 6,7-dinitroquinoxaline-2,3-dione into the region of the trigeminal nuclei. The alteration of arterial blood gases predive had no effect on diving bradycardia. Trigeminal blockade reduced the intensity of the bradycardia but did not abolish it. Chemoreceptor input does not play a significant role in determining heart rate during conscious diving in rats. The attenuation, rather than abolition, of bradycardia on trigeminal blockade suggests either that we achieved incomplete blockade or that an additional spectrum of sensory inputs not present in simulated diving is important in determining the underwater heart rate during conscious diving in rats.

Glial cells are historically known for their role in regulating extracellular potassium concentration. It was postulated almost 50 years ago (1) that astrocytes were important for clearing activity-induced rises in extracellular potassium and that astrocytes, via manipulation of extracellular potassium concentration, may regulate neural network activity. Subsequent work in the leech central-nervous-system preparation (2) and isolated retinal Muller cells (3) helped solidify glial ability to buffer extracellular potassium, but it was only recently shown that astrocytes can be recruited to actively regulate extracellular potassium concentration with indirect effects on neural network activity (4). Potassium spatial buffering is the capacity of astrocytes to redistribute locally elevated extracellular potassium following neuronal activity. Evidence from acute slices and astrocytic cocultures suggests redistribution is directed to sites of low extracellular potassium, to prevent subsequent changes in membrane excitability. Due to the tortuosity and lack of extracellular space in the complex mammalian central nervous system, the bulk of this redistribution is achieved through the high selective potassium conductance of astrocytes associated with significant astrocytic gap junction coupling. The general understanding has been that astrocytic inwardly rectifying potassium channels are important for establishing baseline extracellular potassium levels and astrocytic resting membrane potentials, in addition to playing a role in minimizing the postactivity undershoot in extracellular potassium concentration. Active uptake of potassium by the Na+/K+ ATPase has been considered the major driving force behind buffering of activity-induced potassium rises (5,6). Based on early computer modeling, astrocytes were originally suggested to regulate cerebral blood flow by K+ siphoning (7). However, subsequent experimental testing of this model in retinal explants was unable to confirm a role for potassium in neurovascular coupling (8). Despite this lack of a role for K+ in neurovascular coupling in the retina, evidence strongly supporting a role for K+ in cortical neurovascular coupling has been experimentally confirmed (9,10). In this issue, Witthoft et al. (11) incorporate and highlight the importance of astrocytic inwardly rectifying channels in a new computer model showing that these channels accelerate siphoning of potassium into the perivascular space. This helps reconcile the long delay to functional hyperemia predicted by previous models that did not consider astrocytic inward rectifying channels. Witthoft et al. (11) also address and provide a framework to integrate astrocytic IP3 and Ca2+-mediated release of epoxyeicosatrienoic acids (EETs) into the K+ siphoning hypothesis. As such, it would suggest that all Gq-coupled receptors (mGluR1, mGluR5 glutamatergic, α1-adrenergic, M1, M3, M5 muscarinic, 5-HT2 serotonergic, and H1 histaminergic receptors) on astrocytes may elicit similar effects on the vasculature. The model proposed by Witthoft et al. (11) relies on mGluR and IP3 signaling pathways. However, this signaling pathway may not apply to adult animals lacking mGluR-mediated calcium signaling (12). Interestingly, they provide data that suggest the mGluR/IP3 signaling pathway is only important with weaker stimulations. The relatively large potassium rises used in their model were sufficient to result in very similar vasodilation responses when glutamatergic signaling was omitted. Thus, the potassium-siphoning model for neurovascular coupling appears to be a very basic, and possibly ubiquitous, mechanism for coupling dilation to large activity-induced changes in extracellular potassium. Smaller, more refined effects on the vasculature likely result from the integration of nitric oxide, EET, and cyclooxygenase pathways that are closely linked to the local tissue metabolic state (13). In fact, potassium-mediated neurovascular coupling may explain the residual cortical vasodilation seen during vibrissal stimulation under conditions where all the above-mentioned pathways are blocked pharmacologically (14). As Witthoft et al. (11) point out, posttranslational modifications of TRPV4 and BK channels may alter channel kinetics that would alter the relative contribution of this pathway. Thus, potassium contribution to vascular responses probably varies considerably depending on levels of local activity and metabolic demands. It may be reasonable to assume, however, that K+ siphoning always contributes to vasodilation under conditions of high or excessive neural activity. Another very interesting aspect of the model proposed by Witthoft et al. (11) is the inclusion of a bidirectional communication scenario whereby vessels can also affect astrocytic stretch-activated TRPV4 channels on the foot processes via changes in vessel diameter. Stretch or strain activation of surrounding astrocytic foot processes opens TRPV4 channels, allowing calcium to depolarize the foot process. This activates BK channels and permits potassium to flood the perivascular space, leading to smooth muscle inward rectifier-mediated hyperpolarization and vasodilation. This pathway may be important for quelling vasomotion or spasticity as the authors suggested in a previous work (15), but may also have interesting implications for propagating astrocytic endfoot calcium-mediated (BK channel) dilation along the vascular tree to dilate feeding arteries/arterioles (Fig. 1). Thus, astrocyte endfeet alone may be sufficient for rapid propagation of K+-mediated dilatory signals along the vasculature. Figure 1 Back-propagation of vasodilation through astrocyte vascular endfeet. As outlined in Witthoft et al. (11), large-activity-induced potassium increases will result in astrocyte depolarization and siphoning of excess potassium to the gliovascular ... In summary, the model proposed by Witthoft et al. (11) integrates Gq-coupled IP3 and EET production with existing potassium homeostatic mechanisms to provide an elegant means for neurovascular coupling. Extending this model as a framework to include astrocytic nitric oxide, EET, and cyclooxygenase pathways under more modest activity scenarios firmly places astrocytes as primary conduits for neurovascular coupling.

Extracellular [K+] can range within 2.5-3.5 mM under normal conditions to 50-80 mM under ischemic and spreading depression events. Sustained exposure to elevated [K+]o has been shown to cause significant neuronal death even under conditions of abundant glucose supply. Astrocytes are well equipped to buffer this initial insult of elevated [K] through extensive gap junctional coupling, Na+/K+ pump activity (with associated glycogen and glycolytic potential), and endfoot siphoning capability. Their abundant energy availability and alkalinizing mechanisms help sustain Na+/K+ ATPase activity under ischemic conditions. Furthermore, passive K+ uptake mechanisms and water flux mediated through aquaporin-4 channels in endfoot processes are important energy-independent mechanisms. Unfortunately, as the length of ischemic episode is prolonged, these mechanisms increase to a point where they begin to have repercussions on other important cellular functions. Alkalinizing mechanisms induce an elevation of [Na+]i, increasing the energy demand of Na+/K+ ATPase and leading to eventual detrimental reversal of the Na+/glutamate- cotransporter and excitotoxic damage. Prolonged ischemia also results in cell swelling and activates volume regulatory processes that release excessive excitatory amino acids, further exacerbating excitotoxic injury. In the days following ischemic injury, reactive astrocytes demonstrate increased cell size and process thickness, leading to improved spatial buffering capacity in regions outside the lesion core where there is better neuronal survival. There is a substantial heterogeneity among reactive astrocytes, with some close to the lesion showing decreased buffering capacity. However, it appears that both Na+/K+ ATPase activity (along with energy production processes) as well as passive K+ uptake mechanisms are upregulated in gliotic tissue outside the lesion to enhance the above-mentioned homeostatic mechanisms.

Reactive astrocytes appear within days of a disturbance or injury to the brain. They form part of a gliotic or scar tissue, which can take on a very complex form, depending on the type and intensity of the disturbance. Reactive astrocytes are usually characterized by morphological changes and increases in intermediate filaments. However, other more functional characteristics are not as well-characterized. In particular, glial interactions with surviving neurones are not very well-defined. Some attention has been paid to glial upregulation of protective factors and changes in expression of extracellular matrix, but changes in membrane properties and homeostatic mechanisms have received very little regard. This is true for injury in general, but is especially true following ischemic injury.

Application of the GABAA receptor agonist muscimol to astrocytes in situ or in vitro results in a receptor-mediated Cl− current with a concomitant block of outward K+ currents. The effect on K+ current is largely selective for the inactivating A-type current. Parallel experiments with various Cl− pipette concentrations show a significant reduction in A-type current under low Cl− conditions with minimal effect on delayed current. In addition, lower Cl− conditions caused a depolarizing shift of steady-state inactivation (V1/2, −68 to −57 mV) and activation (V1/2, −5.8 to 34 mV) kinetics of A-type current only. Cl− had no effect on the time course of inactivation or reactivation kinetics, suggesting the Cl−-mediated effect is largely on activation kinetics, indirectly affecting steady-state inactivation. Muscimol application to astrocytes under perforated patch control (gramicidin) displayed a similar block of A-type current to that of conventional whole cell patch at 40 or 20 mM pipette Cl− concentrations. With barium application under perforated patch conditions, the study of muscimol-mediated Cl− current in isolation of the effect on K+ currents was possible. This allowed estimation of intracellular Cl− concentration from receptor current reversal information. The average intracellular Cl− concentration was found to be 29 ± 3.2 mM. The effect on activation kinetics and lack of effect on time course of inactivation or reactivation suggest that intracellular anion concentrations have an effect on the K+ channel voltage sensor region. Cl− may modulate K+ currents by altering membrane field potentials surrounding K+ channel proteins. GLIA 39:207–216, 2002. © 2002 Wiley-Liss, Inc.

Inward and, depending on activation state, outward potassium currents are the dominant ion channels in microglial cells in culture. During transition between resting and activated phases, there is also an upregulated expression of stretch/swelling-activated chloride currents. Pharmacological blockade of the specific potassium channels does not prevent the transition, whereas blockade of chloride channels does, suggesting that this current may be involved in phase changes. Interestingly, this chloride current is far less studied than the potassium currents with regard to the different microglial phases. One puzzling finding when studying microglial state is that despite changes in current densities and membrane oscillations during transition, there is no evidence of an accompanying change in membrane potential. In other cells of the immune system, membrane oscillations and alterations in membrane potential are correlated with transitions in cellular phases. This discrepancy in microglia may be a result of the fact that almost all ion channel and membrane potential studies in culture are undertaken with concomitant dialysis of cytoplasm with pipette solution. Further complicating matters is that the few studies that use microglia in situ, find fundamental differences in ion channel current patterns of “resting” microglia as well as different temporal changes to pathological events or stimuli. Microsc. Res. Tech. 54:26–33, 2001. © 2001 Wiley-Liss, Inc.

Cultured rat hippocampal astrocytes were used to investigate the mechanism underlying the suppression of Ba2+-sensitive K+currents by GABAAreceptor activation. Muscimol application had two effects on whole cell currents: opening of the well-known Cl−channel of the GABAAreceptor and a secondary longer-lasting blockade of outward K+currents displaying both peak and plateau phases. This blockade was independent of both Na+(inside and outside) and ATP in the pipette. It also seemed to be independent of muscimol binding to the receptor because picrotoxin application showed no effect on the K+conductance. The effect is blocked when anion efflux is prevented by replacing Cl−with gluconate (both inside and out) and is enhanced with more permeant anions such as Br−and I−. Moreover, the effect is reproduced in the absence of muscimol by promoting Cl−efflux via lowering of extracellular Cl−levels. These results, along with the requirement for Cl−efflux in muscimol experiments, show a strong dependency of the secondary blockade on Cl−efflux through the Cl−channel of the GABAAreceptor. We therefore conclude that changes in the intracellular Cl−concentration alter the outward K+conductances of astrocytes. Such a Cl−-mediated modulation of an astrocytic K+conductance will have important consequences for the progression of spreading depression through brain tissue and for astrocytic swelling in pathological situations.

Glial cells are known for their role in development and expression of GABA receptors. However, there seems to be a lack of in situ studies characterizing GABA receptor expression and function in glial cells from early development to adulthood. Consequently, we examined GABA receptor expression on rat hippocampal glial cells in both neonatal and adult slices using the whole-cell patch-clamp technique. Glial cells in adult and neonatal slices exhibit responses to muscimol (1 mM; GABAA), but not baclofen (1 mM; GABAB), demonstrating that receptor electrophysiology remains qualitatively similar in glial cells throughout development. Adult muscimol current densities however, do show a decrease in size to approximately 36% of the neonatal response. Muscimol responses were found to be sensitive to bicuculline, suggesting that they are mediated by GABAA receptors. In addition to receptor currents, muscimol causes a concomitant long-term blockade of outward K+ currents in glial cells of both neonatal and adult slices. Comparisons of percentage peak blockade in adult and neonatal glial cells show no significant difference. However, when comparing average absolute conductance blockade, we see that adult glial cells display a significantly smaller response than neonatal and cultured astrocytes. Therefore, although the percentage blockade of outward currents remains consistent throughout development, neonatal glial cells display a larger physiological effect. Thus, it can be concluded that, although the complex GABA response in glial cells is affected by development, the receptor current and secondary blockade are a basic mechanism for neuronal–glial interaction throughout life. GLIA 26:129–138, 1999. © 1999 Wiley-Liss, Inc.

Background Lipopolysaccharide (LPS)-mediated sickness behaviour is known to be a result of increased inflammatory cytokines in the brain. Inflammatory cytokines have been shown to mediate increases in brain excitation by loss of GABAA-mediated inhibition through receptor internalization or inactivation. Inflammatory pathways, reactive oxygen species and stress are also known to increase monoamine oxidase-A (MAO-A) and acetylcholinesterase (ACh-E) activity. Given that neuromodulator actions on neural circuits largely depend on inhibitory pathways and are sensitive to alteration in corresponding catalytic enzyme activities, we assessed the impact of systemic LPS on neuromodulator-mediated shaping of a simple cortical network. Methods Extracellular field recordings of evoked postsynaptic potentials in adult mouse somatosensory cortical slices were used to evaluate effects of a single systemic LPS challenge on neuromodulator function 1 week later. Neuromodulators were administered transiently as a bolus (100 μl) to the bath perfusate immediately upstream of the recording site to mimic phasic release of neuromodulators and enable assessment of response temporal dynamics. Results Systemic LPS administration resulted in loss of both spontaneous and evoked inhibition as well as alterations in the temporal dynamics of neuromodulator effects on a paired-pulse paradigm. The effects on neuromodulator temporal dynamics were sensitive to the Monoamine oxidase-A (MAO-A) antagonist clorgyline (for norepinephrine and serotonin) and the ACh-E inhibitor donepezil (for acetylcholine). This is consistent with significant increases in total MAO and ACh-E activity found in hemi-brain samples from the LPS-treated group, supporting the notion that systemic LPS administration may lead to longer-lasting changes in inhibitory network function and enzyme (MAO/ACh-E) activity responsible for reduced neuromodulator actions. Conclusions Given the significant role of neuromodulators in behavioural state and cognitive processes, it is possible that an inflammatory-mediated change in neuromodulator action plays a role in LPS-induced cognitive effects and could help define the link between infection and neuropsychiatric/degenerative conditions.