Astrocytes-The Ultimate Effectors of Long-Range Neuromodulatory Networks?

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.

Astrocytic Endocannabinoids Mediate Hippocampal Transient Heterosynaptic Depression.

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.

Evidence for astrocyte purinergic signaling in cortical sensory adaptation and serotonin-mediated neuromodulation.

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.

Astrocytes modulate neural network activity by Ca²+-dependent uptake of extracellular K+.

Astrocytes are electrically nonexcitable cells that display increases in cytosolic calcium ion (Ca²+) in response to various neurotransmitters and neuromodulators. However, the physiological role of astrocytic Ca²+ signaling remains controversial. We show here that astrocytic Ca²+ signaling ex vivo and in vivo stimulated the Na+,K+-ATPase (Na+- and K+-dependent adenosine triphosphatase), leading to a transient decrease in the extracellular potassium ion (K+) concentration. This in turn led to neuronal hyperpolarization and suppressed baseline excitatory synaptic activity, detected as a reduced frequency of excitatory postsynaptic currents. Synaptic failures decreased in parallel, leading to an increase in synaptic fidelity. The net result was that astrocytes, through active uptake of K+, improved the signal-to-noise ratio of synaptic transmission. Active control of the extracellular K+ concentration thus provides astrocytes with a simple yet powerful mechanism to rapidly modulate network activity.

Critical role of aquaporin-4 (AQP4) in astrocytic Ca2+ signaling events elicited by cerebral edema.

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.

Adenosine A1 receptors mediate local anti-nociceptive effects of acupuncture.

Acupuncture is an invasive procedure commonly used to relieve pain. Acupuncture is practiced worldwide, despite difficulties in reconciling its principles with evidence-based medicine. We found that adenosine, a neuromodulator with anti-nociceptive properties, was released during acupuncture in mice and that its anti-nociceptive actions required adenosine A1 receptor expression. Direct injection of an adenosine A1 receptor agonist replicated the analgesic effect of acupuncture. Inhibition of enzymes involved in adenosine degradation potentiated the acupuncture-elicited increase in adenosine, as well as its anti-nociceptive effect. These observations indicate that adenosine mediates the effects of acupuncture and that interfering with adenosine metabolism may prolong the clinical benefit of acupuncture.

Systemic administration of an antagonist of the ATP-sensitive receptor P2X7 improves recovery after spinal cord injury.

Traumatic spinal cord injury is characterized by an immediate, irreversible loss of tissue at the lesion site, as well as a secondary expansion of tissue damage over time. Although secondary injury should, in principle, be preventable, no effective treatment options currently exist for patients with acute spinal cord injury (SCI). Excessive release of ATP by the traumatized tissue, followed by activation of high-affinity P2X7 receptors, has previously been implicated in secondary injury, but no clinically relevant strategy by which to antagonize P2X7 receptors has yet, to the best of our knowledge, been reported. Here we have tested the neuroprotective effects of a systemically administered P2X7R antagonist, Brilliant blue G (BBG), in a weight-drop model of thoracic SCI in rats. Administration of BBG 15 min after injury reduced spinal cord anatomic damage and improved motor recovery without evident toxicity. Moreover, BBG treatment directly reduced local activation of astrocytes and microglia, as well as neutrophil infiltration. These observations suggest that BBG not only protected spinal cord neurons from purinergic excitotoxicity, but also reduced local inflammatory responses. Importantly, BBG is a derivative of a commonly used blue food color (FD&C blue No. 1), which crosses the blood-brain barrier. Systemic administration of BBG may thus comprise a readily feasible approach by which to treat traumatic SCI in humans.

Adenosine is crucial for deep brain stimulation-mediated attenuation of tremor.

Deep brain stimulation (DBS) is a widely used neurosurgical approach to treating tremor and other movement disorders. In addition, the use of DBS in a number of psychiatric diseases, including obsessive-compulsive disorders and depression, is currently being tested. Despite the rapid increase in the number of individuals with surgically implanted stimulation electrodes, the cellular pathways involved in mediating the effects of DBS remain unknown. Here we show that DBS is associated with a marked increase in the release of ATP, resulting in accumulation of its catabolic product, adenosine. Adenosine A1 receptor activation depresses excitatory transmission in the thalamus and reduces both tremor- and DBS-induced side effects. Intrathalamic infusion of A1 receptor agonists directly reduces tremor, whereas adenosine A1 receptor-null mice show involuntary movements and seizure at stimulation intensities below the therapeutic level. Furthermore, our data indicate that endogenous adenosine mechanisms are active in tremor, thus supporting the clinical notion that caffeine, a nonselective adenosine receptor antagonist, can trigger or exacerbate essential tremor. Our findings suggest that nonsynaptic mechanisms involving the activation of A1 receptors suppress tremor activity and limit stimulation-induced side effects, thereby providing a new pharmacological target to replace or improve the efficacy of DBS.

pCLCA1 lacks inherent chloride channel activity in an epithelial colon carcinoma cell line.

The effects of CLCA protein expression on the regulation of Cl(-) conductance by intracellular Ca(2+) 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 (36)Cl(-) efflux, as transepithelial short-circuit currents, and as whole cell patch-clamp current-voltage relations. The rate of (36)Cl(-) efflux and amplitude of currents in patch-clamp studies after the addition of the Ca(2+) ionophore A-23187 were increased significantly by pCLCA1 expression in freshly passaged Caco-2 cells. However, neither endogenous nor pCLCA1-dependent Ca(2+)-sensitive Cl(-) conductance could be detected in 14-day-postpassage cells. In contrast to Ca(2+)-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 Ca(2+)-dependent Cl(-) conductance disappeared. We conclude that pCLCA1 expression may increase the sensitivity of preexisting endogenous Cl(-) channels to Ca(2+) and cAMP agonists but apparently lacks inherent Cl(-) channel activity under growth conditions where endogenous channels are not expressed.

pCLCA1 becomes a cAMP-dependent chloride conductance mediator in Caco-2 cells.

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 36Cl 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.