Lane K. Bekar

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.

Forebrain Engraftment by Human Glial Progenitor Cells Enhances Synaptic Plasticity and Learning in Adult Mice

Human astrocytes are larger and more complex than those of infraprimate mammals, suggesting that their role in neural processing has expanded with evolution. To assess the cell-autonomous and species-selective properties of human glia, we engrafted human glial progenitor cells (GPCs) into neonatal immunodeficient mice. Upon maturation, the recipient brains exhibited large numbers and high proportions of both human glial progenitors and astrocytes. The engrafted human glia were gap-junction-coupled to host astroglia, yet retained the size and pleomorphism of hominid astroglia, and propagated Ca2+ signals 3-fold faster than their hosts. Long-term potentiation (LTP) was sharply enhanced in the human glial chimeric mice, as was their learning, as assessed by Barnes maze navigation, object-location memory, and both contextual and tone fear conditioning. Mice allografted with murine GPCs showed no enhancement of either LTP or learning. These findings indicate that human glia differentially enhance both activity-dependent plasticity and learning in mice.

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.

Locus Coeruleus α-Adrenergic–Mediated Activation of Cortical Astrocytes In Vivo

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.

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 Ca2+ 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 Ca2+ spikes and that deletion of Aqp4 reduces these signals. The Ca2+ 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.

Astrocytes Modulate Neural Network Activity by Ca2+-Dependent Uptake of Extracellular K+

Astrocytes modulate synaptic activity by changing the local concentration of extracellular K+. Increasing Synaptic Fidelity Although astrocytes have long been implicated in maintenance of extracellular K+ homeostasis and in the mediation of propagated Ca2+ signals, the relationship between these two properties has been unclear. Here, Wang et al. show that increases in astrocyte cytosolic Ca2+ secondary to activation of G protein–coupled receptors (GPCRs) lead to increased Na+ influx through the Na+/Ca2+ exchanger, which, in turn, stimulates astrocyte Na+,K+-ATPase activity and K+ uptake and consequently decreases the local extracellular K+ concentration. Decreased K+ increased neuronal hyperpolarization, with a consequent suppression of basal—but not evoked—synaptic activity, and enhanced synaptic fidelity. The authors thus conclude that Ca2+-dependent activation of the astrocyte Na+,K+-ATPase through GPCR-activated signaling pathways enables astrocytes to dynamically regulate the extracellular K+ concentration and thereby modulate neuronal function. 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.

Potassium homeostasis in the ischemic brain

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.

The locus coeruleus-norepinephrine network optimizes coupling of cerebral blood volume with oxygen demand.

Given the brains 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.

Vimentin-expressing proximal reactive astrocytes correlate with migration rather than proliferation following focal brain injury

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.