Autonomic Neuroscience: Basic and Clinical 156 (2010) 14

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Autonomic Neuroscience: Basic and Clinical

j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / a u t n e u

HIGHLIGHTS IN BASIC AUTONOMIC NEUROSCIENCES: GLIA AND NEUROMODULATION Prepared by: Benedito H. Machado, Davi J.A. Moraes, Kau M. Costa
University of So Paulo, School of Medicine of Ribeiro Preto, Department of Physiology 14049-900 Ribeiro Preto, SP, Brazil

Section Editor: Michael P. Gilbey

Martn ED, Fernndez M, Perea G, Pascual O, Haydon PG, Araque A, Cea V. Adenosine released by astrocytes contributes to hypoxiainduced modulation of synaptic transmission. Glia. 2007 Jan 1; 55(1): 3645. Article summary Adenosine is an important neuromodulator in the CNS. In response to insults, such as hypoxia, adenosine is released and exerts a neuroprotective effect by inhibiting excitatory synaptic transmission. Martin et al. investigated whether the hypoxia-induced excitatory synaptic depression might be mediated by adenosine release from astrocytes by using electrophysiological and calcium imaging techniques in hippocampal slices of transgenic mice with dominant negativesoluble N-ethylmaleimide-sensitive factor attachment protein receptor (dn-SNARE) and in pure astrocytic cultures,. The inhibition of glial cell metabolism with uorocitrate or the blockade of A1 adenosine receptors with 8-cyclopentyltheophylline was effective in preventing the hypoxia-induced reduction in excitatory synaptic transmission. It was also shown that adenosine transporter inhibitors failed to modify hypoxiainduced effects, demonstrating that hypoxia-induced adenosine release was transporter independent. Moreover, adenosine release under hypoxic conditions was shown to be calcium independent. As under normoxic physiological conditions extracellular adenosine is mainly derived from extracellular catabolism of astrocytic ATP released by exocytosis, the presence of adenosine-mediated modulation of excitatory synaptic transmission during hypoxia in transgenic mice with impaired astrocytic ATP exocytosis (dn-SNARE) indicates that the adenosine mediating the hypoxic effects is not produced by extracellular ATP degradation. Thus, the authors conclude that under hypoxic conditions astrocytes regulate the excitatory synaptic transmission by direct release of adenosine, which acts on A1 receptors to reduce presynaptic transmitter release. Commentary The control of excitatory synaptic transmission under hypoxic conditions is critical for the survival of neural tissue. In their paper Martin et al. studied a form of neuronglia signaling in the CNS in order to address a heavily debated question concerning the effects of hypoxic/ischemic insults: what is the source of the adenosine? The understanding of cellular and biochemical mechanisms for adenosine release in this context is an important issue. The authors used a
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combination of pharmacological and electrophysiological approaches combined with a genetically modied mouse. In this model transgene expression is controlled by doxycycline added to the drinking water. In this way cytosolic synaptobrevin II molecule with dominant negative effects on the SNARE-dependent vesicular complex (dn-SNARE) is expressed specically in astrocytes. This approach blocks vesicular docking in astrocytes via SNARE complex and extracellular adenosine levels fall (see Pascual et al., Science, 2005). This study provides the rst experimental evidence demonstrating the roles of specic cell types, enzymatic pathways and membrane transport processes in regulating extracellular concentrations of adenosine under hypoxic conditions. The results, demonstrate that purine release under hypoxic conditions may occur in a different manner to that occurring in basal conditions by non-calcium dependent release of adenosine from astrocytes. However, the mechanism underlying the astrocytic adenosine release pathway requires further studies. Considering that there are different mechanisms and sites of O2 chemoreception in the CNS, these results indicate a novel mechanism for the control of breathing. Previous studies have demonstrated that the rostral ventrolateral medulla is an oxygen sensing region (see D'Agostino et al., Am J Physiol Regul Integr Comp Physiol, 2008) and the results of Martin et al. raise the possibility of the involvement of purines, released from astrocytes under hypoxic conditions, in the modulation of excitatory synaptic activity. The mechanisms proposed by Martin et al. could be one of the pathways through which glial signaling modulates neuronal activity in several brain regions and their results raise questions about the role of gliotransmission in the control of synaptic activity in homeostatic regulatory systems. To further our understanding of synaptic neuromodulation in the CNS in general and in autonomic and respiratory neural pathways, experimental approaches like those used by Martin et al. will be required.

Guo F, Liu B, Tang F, Lane S, Souslova EA, Chudakov DM, Paton JF, Kasparov S. Astroglia are a possible cellular substrate of angiotensin (17) effects in the rostral ventrolateral medulla. Cardiovasc Res. 2010 Mar 22. Epub ahead of print. Article summary The response of glia to neurotransmitters and their ability to regulate brain microcirculation and neuronal activity are a growing eld of

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investigation. Guo et al., using a combination of adenoviral vectors, organotypic slice cultures of rostral ventrolateral medulla (RVLM) from Wistar rats and spontaneously hypertensive rats (SHR), calcium imaging methods and patch-clamp recordings, showed that astrocytes are a target for Angiotensin 17 (Ang17) in the RVLM. Astrocytes, catecholamine-containing (CA) and non-CA neurons were targeted with the expression of genes under specic promoters. CA neurones expressed EGFP under control of the noradrenaline expressing neuronspecic cis-regulatory element (PRS) 8 promoter and non-CA-neurones expressed EGFP under control of the synapsin-1 promoter. Astrocytic calcium signaling was monitored using a genetically engineered calcium sensor under the control of the GFAP promoter. Calcium signaling in neurons was visualized with Rhod-2. The authors found that the exogenous application of increasing doses of Ang17 produced an elevation in intracellular calcium only in astrocytes. In addition, the intracellular calcium response to Ang17 was signicantly attenuated in SHR. The Ang17 receptor antagonist A779 and the blockade of intracellular calcium release suppressed Ang17-induced intracellular calcium elevations. Considering these results, and previous studies showing the link between astrocytic function and brain microcirculation, the authors conclude that Ang17 modulates astrocyte activity, which may in turn affect local metabolism and microcirculatory ow in RVLM and that these mechanisms may be compromised in SHR. Commentary This study observed that astrocytes in the RVLM are responsive to Ang17 and that in the SHR Ang17 signaling is altered. As the expression of transgenes in different cell types may complicate the interpretation of experimental ndings, the authors used specic promoters as a reliable way to discriminate between neuronal and glial targets and evaluate astrocytic intracellular calcium levels without loading the cells with calcium sensitive dyes. Although the authors have demonstrated that Ang17 initiates calcium intracellular changes only within astrocytes, it is not yet known whether these calcium elevations can induce the release of gliotransmitters and consequently modulate local microcirculation or neuronal activity in the RVLM. To answer this question, further electrophysiological studies are necessary to provide a concise evaluation of the effects of Ang17 on the RVLM synaptic network following the blockade of glial function with specic inhibitors such as uorocitrate. These ndings have important implications regarding our understanding of neuronal and glial signaling in a brainstem region critical for the control of autonomic and respiratory functions. A question that naturally arises from the aforementioned data is whether astrocyte mediated modulation of neuronal activity in the RVLM could be important for the regulation of basal respiratory and sympathetic activity and alterations in these parameters observed in experimental models of hypertension (e.g., SHR, chronic intermittent hypoxia and Angiotensin II dependent: see Zoccal et al., J. Physiol, 2008; Simms et al., J. Physiol, 2009; Toney et al., Exp Physiol, 2010). Considering the recent literature showing neuronglia interactions in other CNS areas, this study raises intriguing possibilities concerning the control of synaptic activity in the RVLM and how such control might impact on the regulation of autonomic and respiratory functions.

for respiratory rhythm generation. As within respiratory networks where ATP and P2 receptor signaling contributes to homeostatic ventilatory responses the authors speculated that glial cells were involved in ATP induced neuroexcitations within the pre-Btzinger complex (pre-BtC). In order to investigate this hypothesis, the authors used a variety of electrophysiological and calcium imaging techniques in experimental models such as rhythmic medullary brainslices and glial cell cultures obtained from the pre-BtC. The authors found that glial inhibition with uoroacetate or methionine sulfoximine reduced the basal inspiratory frequency in rhythmic medullary brain-slices. These primary effects were reversed with bath application of glutamine, an indication that they were derived from the blockade of neuronglia metabolic coupling (see Hlsmann et al., Eur J Neurosci, 2000). Both gliotoxins also specically reduced the effect of ATP on ventilatory activity even in the presence of bath-applied glutamine, which indicates an effect on gliotransmitter release. It was also shown that putative glia in these brain-slices respond to bath applications of ATP with an increase in free cytoplasmatic calcium concentration. ATP and the specic P2Y1 receptor agonist MRS2365 also evoked depolarizing currents in electrophysiologically identied glia in medullary brain-slices. Isolated cells obtained from primary cultures of glia from the ventral medulla at the rostrocaudal level of the pre-BtC responded to ATP with a marked increase in intracellular calcium levels. ATP application also evoked a signicant increase in bath glutamate concentration in these primary glial cultures. Even though pre-BtC neurons can respond to ATP independently of glial glutamate release, the accumulated data clearly show that glia contribute to the purinergic excitation of pre-BtC rhythm generating networks and strongly indicate that this mechanism is dependent on glial P2 receptor activation and subsequent glutamate release. Commentary The authors provide a robust set of data showing the effects of purinegic signaling in glial cells embedded within the respiratory rhythm generating network. The effects of glial function in this homeostatic neuronal network were relatively indirect, with a high level of subtlety and complexity, needing a series of well planned experiments to be properly conrmed. The intricacies of how this mechanism inuences respiratory activity, however, still need to be extensively investigated. Although the authors demonstrate that P2Y1 receptors have a distinctive effect in glial ATP sensing, the role of other P2 receptors is unclear. The mechanisms of the observed ATP mediated glutamate release also need further investigation. In spite of these considerable shortcomings the ndings of this study raise many questions for future studies in the eld of autonomic regulation and neuropathologies. Does this form of signaling promote only an increase in the general excitatory drive, or could this mechanism also inuence glial-mediated neuronal coupling (see Fellin et al, Neuron, 2004)? Could this mechanism have a role in the purine mediated neurotransmission of respiratory reexes and sympatheticrespiratory coupling? How do short and long term modulations of glial ATP sensing inuence respiratory control? How may we compile these ndings in current computational models of respiratory rhythm generation? Is this mechanism compromised in certain pathological situations, such as in sudden infant death syndrome or in nonobstructive sleep apnea? Given recent ndings in the literature that many previously overlooked glial dysfunctions may be involved in a wide range of medical maladies, this and future work in the eld may be relevant to our understanding of autonomic disorders.

Huxtable, A.G., Zwicker, J.D., Alvares, T.S., Ruangkittisakul, A., Fang, X., Hahn, L.B., de Chaves, E.P., Baker, G.B., Ballanyi, K., Funk, G.D. 2010. Glia Contribute to the Purinergic Modulation of Inspiratory RhythmGenerating Networks. J Neurosci. 2010; 30(11): 39473958. Article summary Huxtable et al. investigated the role of glial cells in the ATPdependent modulation of a brainstem neuronal network responsible

Park, J.B., Jo, J.Y., Zheng, H., Patel, K.P., Stern, J.E. Regulation of tonic GABA inhibitory function, presympathetic neuronal activity and sympathetic outow from the paraventricular nucleus by astroglial GABA transporters. J Physiol. 2009; 587(19): 46454660.

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Article summary Park et al. used patch-clamp techniques to investigate the role of glial GABA transporters (GATs) in the modulation of the phasic and tonic modalities of GABAA mediated inhibitory control in hypothalamic paraventricular nucleus (PVN) neurons retrogradely labeled with rhodamine-labelled microspheres microinjected the rostral ventrolateral medulla (RVLM). They also used hemodynamic and renal sympathetic nerve activity (RSNA) recordings in anesthetized rats to evaluate how glial GATs participate in the control of PVN-driven changes in RSNA and blood pressure. The authors show that the non-selective blockade of GATs with nipecotic acid causes a large inward shift of tonic GABAA mediated currents, decreases the frequency of spontaneous GABAA IPSCs and decreases the basal ring activity of PVN-RVLM neurons. The selective blockade of GAT3 with -alanine also resulted in an inward shift of the tonic GABAA currents and decreased the ring activity of PVNRVLM neurons. However, the selective blockade of GAT1 with SKF 89976A caused no signicant alteration in the measured parameters. It was conrmed, using immunohistochemical labeling, that GAT3s were predominantly expressed in glial cells, while GAT1 were mostly located in neurons. The authors then show that bath application of the gliotoxin L--aminoadipic acid had similar effects to the blockade of GATs inducing an inward shift in the tonic GABA current and decreasing the frequency of IPSCs. In anesthetized rats, the microinjection of nipecotic acid or -alanine both elicited a signicant decrease in RSNA. However, there was no change in blood pressure and only nipecotic acid induced a signicant decrease in heart rate. These results all favor the conclusion that the tonic and, to some extent, the phasic components of GABAA mediated inhibition in PVN-RVLM neurons are predominantly under the inuence of glial GAT3 transporters and that this mechanism plays a key role in the control of PVN-driven changes in sympathetic activity. Commentary Although the control of extracellular neurotransmitter concentration by specic transporters is well known, relatively little attention has been given to the participation of the glia in neurotransmitter uptake. Here, Park et al. not only present a very interesting example of such a mechanism, but also show that it has distinct effects on different modes of neuronal excitability control and probably inuences basal levels of sympathetic activity. Although the authors did not use specic gliotoxins, such as uorocitrate, the participation of glia is conrmed by the immunohistochemical analysis. The fact that the tonic modality of GABAA mediated inhibition is more affected by GAT blockade than the phasic modality suggests that GATs are spatially distant from the synaptic cleft. However, their hypothesis that glial control of the phasic modality of GABA inhibition may be due to presynaptic events, in a true tripartite synapse fashion (see Hallassa and Haydon, Neuropharmacology, 2009), must be investigated in future studies. It would be interesting to investigate if the sympathetic over activity observed in experimental models of hypertension can be linked to dysfunctions of this glial mechanism. In a recent study, Prabha et al. (J. Physiol., 2010) have shown that disinhibition of RVLM projecting PVN neurons with a selective GABAA receptor antagonist increases arterial blood pressure, heart rate and ventilatory activity. Furthermore, Prabha et al. demonstrate that the increase of sympathetic and respiratory activity in rats exposed to chronic intermittent hypoxia is related to vasopressin released from RVLM projecting PVN neurons. Therefore, given the ndings of Park et al., it seems plausible that some of these alterations could be related to changes in glial-mediated GABA uptake and, consequently, in the tonic modality of GABAA mediated inhibition of PVN neurons. Whether alterations in this mechanism can account for the autonomic dysfunctions observed in other experimental models or pathophysiological conditions is certainly an issue worth further investigation.

Gordon, G.R.J., Iremonger, K. J., Kantevari, S., Ellis-Davies, G. C.R., MacVicar, B. A., Bains, J.C. Astrocyte-Mediated Distributed Plasticity at Hypothalamic Glutamate Synapses. Neuron. 2009; 64 (3): 391403. Article summary The control of neuronal excitability and synchronization by increasing synaptic strength can affect the output of a given neuron. In this study, Gordon et al. hypothesized that astrocytes regulate synaptic strength in response to afferent activity and scale glutamate synapses on magnocellular neurosecretory cells (MNCs) in the PVN. Glial metabotropic glutamate receptors (mGluR) activation by group I mGluR agonists, ultraviolet (UV) photolysis of caged-glutamate on astrocytes or synaptic stimulation of glutamatergic afferents evoked calcium transients in astrocytes. However, only calcium changes in nearby astrocytes correlated with an increase in synaptic strength in the MNCs. Moreover, one important observation was that the increase in synaptic strength occurred by scaling glutamate synapses in all recorded events in a single given MNC. Therefore, this paper demonstrated that the scaling of glutamatergic synapses in the MNCs is mediated by the activation of astrocytes located close to the soma or dendrite of these neurons. Commentary The authors used a combination of two-photon microscopy, caged molecule photolysis, whole-cell voltage-clamp recordings and homeostatic challenges to investigate whether the feed-forward strengthening of glutamatergic synapses on MNCs in the PVN occurs by scaling and depends on astrocytic activity. Several studies have documented scaling of synaptic strength in several brain regions, but the mechanisms underlying this form of synaptic plasticity are unclear. This is an important issue in the eld of autonomic control of blood volume/osmolarity levels, especially for the understanding of how neuronglia interactions following the tripartite synapse model determine the output of this system, i.e. the release of vasopressin and oxytocin. Moreover, the scaling of synaptic strength observed by Gordon et al. is an example thought to represent the electrophysiological basis of memory and learning in the CNS, a fact that raises questions about synaptic memory processes in the autonomic nervous system. Although elevations in the strength of glutamatergic synapses probably result in an increased release of neurohormones in the bloodstream by amplifying excitatory signals, further investigations are required to determine whether this synaptic plasticity occurs in situations of acute hyperosmolarity or dehydration. A very interesting nding is that activation of glial mGluR does not change the strength of glutamatergic synapses on MNCs after the retraction of astrocytic processes that occur during dehydration. Gordon et al. have signposted a new direction for further investigations on neuronglia signaling. One could speculate whether these mechanisms could also be important in the control of systemic blood pressure, body temperature or other homeostatic parameters.

Hermann, G.E., Van Meter, M.J., Rood, J.C., Rogers, R.C. ProteinaseActivated Receptors in the Nucleus of the Solitary Tract: Evidence for GlialNeural Interactions in Autonomic Control of the Stomach. J Neurosci. 2009; 29(29): 92929300. Article summary This study by Hermann et al. demonstrates for the rst time that changes in autonomic control can be directly signaled by glial

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detection of local chemical stimuli. The initial aim of the study was to conrm the hypothesis that the interaction of thrombin with certain proteinase receptors (PAR1) in the dorsal vagal complex causes disruption of the vagal control of the stomach. This was achieved by monitoring gastric emptying following the injection of thrombin or a specic PAR1 agonist (SFLLRN-NH2) into the fourth ventricle. They also evaluated the distribution of PAR1 in the nucleus of the solitary tract (NST) using double immunohistochemical staining techniques and changes in calcium signaling in NST cells in response to PAR1 activation. The authors conrm the initial hypothesis by showing that the activation of PAR1 with thrombin or the specic agonist SFLLRNNH2 signicantly impairs gastric transit in rats. However, their immunohistochemical assays showed that the distribution of these receptors overlapped with that of glial brillary acidic protein (GFAP) but not neuronal nuclei (NeuN), indicating that this receptor is only expressed in the NST by glial cells. Furthermore, stimulation of PAR1 in NST slices produced a rapid, dramatic and oscillatory increase in glial cytoplasmic calcium, followed by a smaller, delayed monotonic increase in calcium signal in NST neurons. Pre-treatment with a mixture of various glutamate receptor antagonists eliminated the effect of PAR1 activation on neurons while reducing the glial response by only one-third. Together, these data suggest not only that glial cells are the primary targets of PAR1 agonists in the NST, but also that astrocytes probably use glutamate to activate the adjacent NST neurons. Commentary The ndings of this study are not only consistent with the hypothesis that post-traumatic gastric dysfunction is caused by

bleeding-induced systemic increases in thrombin levels and the subsequent activation of PAR1 receptors in the CNS, but also show that glia can act as sensors in this trauma sensing pathway. While this discovery may cause a major impact on how we analyze and treat post-traumatic physiological disorders, perhaps its major contribution is to shed light on the importance of glial cells as sensors of visceral and systemic stimuli. Although there are many recent studies showing the role of glia in the control of synaptic physiology, regeneration of nervous tissue and electric and metabolic neuronal coupling, usually the measured effects of glial activation or inhibition are very subtle and primarily of a modulatory nature. In contrast, this study demonstrates that glial cells play a direct chemosensory function in an important homeostatic response. The authors also speculate that vagal afferents may modulate the chemosensitivity of NST glia. Similar regulatory mechanisms do indeed occur in hippocampus synapses (the tripartite synapse model); however more experimental evidence is needed to conrm this hypothesis. It is important to note the novel importance of thrombin and proteinase receptors in the modulation of neuronal circuits shown in this paper. It can be speculated that perhaps the glial chemoreception of thrombin can account for some of the previously shown effects of this protease in neuronal excitation and some of the neuronal dysfunctions observed in cerebral hemorrhage, a condition in which the levels of thrombin in the brain are highly elevated.