Ute Krügel

Purinergic signalling: From normal behaviour to pathological brain function

Purinergic neurotransmission, involving release of ATP as an efferent neurotransmitter was first proposed in 1972. Later, ATP was recognised as a cotransmitter in peripheral nerves and more recently as a cotransmitter with glutamate, noradrenaline, GABA, acetylcholine and dopamine in the CNS. Both ATP, together with some of its enzymatic breakdown products (ADP and adenosine) and uracil nucleotides are now recognised to act via P2X ion channels and P1 and P2Y G protein-coupled receptors, which are widely expressed in the brain. They mediate both fast signalling in neurotransmission and neuromodulation and long-term (trophic) signalling in cell proliferation, differentiation and death. Purinergic signalling is prominent in neurone-glial cell interactions. In this review we discuss first the evidence implicating purinergic signalling in normal behaviour, including learning and memory, sleep and arousal, locomotor activity and exploration, feeding behaviour and mood and motivation. Then we turn to the involvement of P1 and P2 receptors in pathological brain function; firstly in trauma, ischemia and stroke, then in neurodegenerative diseases, including Alzheimers, Parkinsons and Huntingtons, as well as multiple sclerosis and amyotrophic lateral sclerosis. Finally, the role of purinergic signalling in neuropsychiatric diseases (including schizophrenia), epilepsy, migraine, cognitive impairment and neuropathic pain will be considered.

Extracellular Ca2+ is a danger signal activating the NLRP3 inflammasome through G protein-coupled calcium sensing receptors.

Activation of the NLRP3 inflammasome enables monocytes and macrophages to release high levels of interleukin-1β during inflammatory responses. Concentrations of extracellular calcium can increase at sites of infection, inflammation or cell activation. Here we show that increased extracellular calcium activates the NLRP3 inflammasome via stimulation of G protein-coupled calcium sensing receptors. Activation is mediated by signalling through the calcium-sensing receptor and GPRC6A via the phosphatidyl inositol/Ca2+ pathway. The resulting increase in the intracellular calcium concentration triggers inflammasome assembly and Caspase-1 activation. We identified necrotic cells as one source for excess extracellular calcium triggering this activation. In vivo, increased calcium concentrations can amplify the inflammatory response in the mouse model of carrageenan-induced footpad swelling, and this effect was inhibited in GPRC6A−/− mice. Our results demonstrate that G-protein-coupled receptors can activate the inflammasome, and indicate that increased extracellular calcium has a role as a danger signal and amplifier of inflammation.

P2 receptors and neuronal injury

Extracellular adenosine 5′-triphosphate (ATP) was proposed to be an activity-dependent signaling molecule that regulates glia–glia and glia–neuron communications. ATP is a neurotransmitter of its own right and, in addition, a cotransmitter of other classical transmitters such as glutamate or GABA. The effects of ATP are mediated by two receptor families belonging either to the P2X (ligand-gated cationic channels) or P2Y (G protein-coupled receptors) types. P2X receptors are responsible for rapid synaptic responses, whereas P2Y receptors mediate slow synaptic responses and other types of purinergic signaling involved in neuronal damage/regeneration. ATP may act at pre- and postsynaptic sites and therefore, it may participate in the phenomena of long-term potentiation and long-term depression of excitatory synaptic transmission. The release of ATP into the extracellular space, e.g., by exocytosis, membrane transporters, and connexin hemichannels, is a widespread physiological process. However, ATP may also leave cells through their plasma membrane damaged by inflammation, ischemia, and mechanical injury. Functional responses to the activation of multiple P2 receptors were found in neurons and glial cells under normal and pathophysiological conditions. P2 receptor-activation could either be a cause or a consequence of neuronal cell death/glial activation and may be related to detrimental and/or beneficial effects. The present review aims at demonstrating that purinergic mechanisms correlate with the etiopathology of brain insults, especially because of the massive extracellular release of ATP, adenosine, and other neurotransmitters after brain injury. We will focus in this review on the most important P2 receptor-mediated neurodegenerative and neuroprotective processes and their beneficial modulation by possible therapeutic manipulations.

P2 receptor-mediated proliferative effects on astrocytes in vivo

Astrogliosis in response to injury usually represents up‐regulation of glial fibrillary acidic protein (GFAP), hypertrophy, and proliferation. Following pathological events in brain tissue, purine nucleotides and nucleosides are released into the extracellular space. The (patho)physiological importance and molecular mechanisms of the purinoceptor‐mediated effects are nearly unknown. In the present study, the involvement of extracellular ATP in astrogliotic processes via stimulation of P2 receptors was investigated. The structural analogue, 2‐methylthio ATP (2‐MeSATP) and its antagonists reactive blue 2 and pyridoxal‐phosphate‐6‐azophenyl‐2,4‐disulphonic acid (PPADS) were microinfused in situ into the rat nucleus accumbens. The reaction of astrocytes in the nucleus accumbens was investigated by GFAP‐ and 5‐bromo‐2′‐deoxyuridine (BrdU)‐ immunocytochemistry. Tissue injury due to the microinjection procedure caused astrogliosis, which was increased further by 2‐MeSATP. Up‐regulation of GFAP‐immunoreactivity, hypertrophy of astrocytes, and an increase in the number of GFAP‐positive and of GFAP‐/BrdU‐double labeled cells were observed. Reactive blue 2 and PPADS decreased the consequences of tissue injury on astrocytic proliferation when given alone. In addition, both antagonists counteracted the 2‐MeSATP‐induced astrogliosis, supporting the hypothesis that purine nucleotides are involved in these processes via stimulation of P2 receptors in vivo. GLIA 28:190–200, 1999.

P2 receptor‐types involved in astrogliosis in vivo

In the nucleus accumbens (NAc) of rats, the involvement of P2X and P2Y receptors in the generation of astrogliosis in vivo, was investigated by local application of their respective ligands. The agonists used had selectivities for P2X1,3 (α,β‐methylene adenosine 5′‐triphosphate; α,β‐meATP), P2Y1,12 (adenosine 5′‐O‐(2‐thiodiphosphate; ADP‐β‐S) and P2Y2,4,6 receptors (uridine 5′‐O‐(3‐thiotriphosphate; UTP‐γ‐S). Pyridoxalphosphate‐6‐azophenyl‐2,4‐disulphonic acid (PPADS) was used as a non‐selective antagonist. The astroglial reaction was studied by means of immunocytochemical double‐labelling with antibodies to glial fibrillary acidic protein (GFAP) and 5‐bromo‐2′‐deoxyuridine (BrdU). The agonist‐induced changes in comparison to the artificial cerebrospinal fluid (aCSF)‐treated control side reveal a strong mitogenic potency of ADP‐β‐S and α,β‐meATP, whereas UTP‐γ‐S was ineffective. The P2 receptor antagonist PPADS decreased the injury‐induced proliferation when given alone and in addition inhibited all agonist effects. The observed morphogenic changes included hypertrophy of astrocytes, elongation of astrocytic processes and up‐regulation of GFAP. A significant increase of both GFAP‐immunoreactivity (IR) and GFA‐protein content (by using Western blotting) was found after microinfusion of α,β‐meATP or ADP‐β‐S. In contrast, UTP‐γ‐S failed to increase the GFAP‐IR. The morphogenic effects were also inhibited by pre‐treatment with PPADS. A double immunofluorescence approach with confocal laser scanning microscopy showed the localisation of P2X3 and P2Y1 receptors on the GFAP‐labelled astrocytes. In conclusion, the data suggest that P2Y (P2Y1 or P2Y12) receptor subtypes are involved in the generation of astrogliosis in the NAc of rats, with a possible minor contribution of P2X receptor subtypes.

Acute amygdaloid response to systemic inflammation

The amygdala, a group of nuclei located in the medial temporal lobe, is a key limbic structure involved in mood regulation, associative learning, and modulation of cognitive functions. Functional neuroanatomical studies suggest that this brain region plays also an important role in the central integration of afferent signals from the peripheral immune system. In the present study, intracerebral electroencephalography and microdialysis were employed to investigate the electrophysiological and neurochemical consequences of systemic immune activation in the amygdala of freely moving rats. Intraperitoneal administration of bacterial lipopolysaccharide (100 μg/kg) induced with a latency of about 2 h a significant increase in amygdaloid neuronal activity and a substantial rise in extracellular noradrenaline levels. Activated neurons in the amygdaloid complex, identified by c-Fos immunohistochemistry, were mainly located in the central nucleus and, to a lesser extent, in the basolateral nucleus of the amygdala. Gene expression analysis in micropunches of the amygdala revealed that endotoxin administration induced a strong time-dependent increase in IL-1β, IL-6, and TNF-α mRNA levels indicating that these cytokines are de novo synthesized in the amygdala in response to peripheral immune activation. The changes in amygdaloid activity were timely related to an increase in anxiety-like behavior and decreased locomotor activity and exploration in the open-field. Taken together, these data give novel insights into different features of the acute amygdaloid response during experimental inflammation and provides further evidence that the amygdala integrates immune-derived information to coordinate behavioral and autonomic responses.

Changes in purinergic signaling after cerebral injury – involvement of glutamatergic mechanisms?

Extracellular purines act as neuromodulators on transmitter release and may exert toxic effects at higher concentrations. In microdialysis studies, endogenous ATP facilitated the extracellular concentration of glutamate in the nucleus accumbens (NAc) of rats. Additionally, P2 receptors are involved in astrogliosis in vivo after a stab wound injury in the same region, suggesting that these receptors, preferentially the metabotropic P2Y1 receptor subtype, mediate also trophic responses. Two sets of experimental findings support the involvement of purinergic and glutamatergic mechanisms in the response of brain to mechanical damage. First, in the present studies, the initial time course of extracellular ATP and glutamate was analyzed after a mechanical injury. The concentration of ATP in microdialysates was elevated only in the first 15‐min sample whereas glutamate returned to a basal concentration not before a 90‐min period had elapsed. We suggest, that the acute injury‐evoked stimulation of P2 receptors contributes to glutamate‐mediated excitotoxicity. Second, the expression of P2Y1 receptors and their possible relation to glutamatergic structures, identified by neuronal vesicular glutamate transporters (VGLUTs), were elucidated in non‐treated and mechanically injured animals after 4 days. The number of P2Y1‐positive cells was significantly increased after injury. Furthermore, P2Y1 receptor‐labeled cells do not exhibit immunoreactivity for VGLUT1 and VGLUT2 without and after injury. However, after injury, a co‐expression of the P2Y1 receptor on VGLUT3‐immunopositive cells in the NAc was observed. No VGLUT1‐, 2‐ and 3‐immunoreactivity was found on P2Y1‐positive glial fibrillary acidic protein‐immunopositive astrocytes at both conditions.

Antidepressant effects of TNF-α blockade in an animal model of depression

Pro-inflammatory cytokines such as tumour necrosis factor-alpha (TNF-α) have repeatedly been shown to play a pivotal role in the pathophysiology of depression. Therefore, we tested the possible antidepressant-like effect of the anti-TNF-α drug etanercept in an animal model of chronic mild stress. Male Wistar rats were assigned to a non-restrained and a restrained protocol for 5 weeks. From beginning of the third week the animals were treated either with Ringer solution daily or with etanercept twice a week (0.3 mg/kg, i.p.) instead of Ringer solution (n = 12 each). As reference, imipramine (10 mg/kg, i.p.) was administered in a third restraint group daily. Naïve non-treated non-restrained rats served as healthy controls (n = 12). In the forced swim test (FST) depression-like behaviour induced by restraint was recorded as enhanced immobile time and reduced climbing activity of the vehicle-treated group in comparison to the naïve and the non-restrained vehicle treated group. The treatment with etanercept significantly reduced the depression-like effects resulting in reduced immobile time in the FST and intensified climbing behaviour (p < 0.01, p < 0.05), both similar to the antidepressive-like effect of imipramine (p < 0.01 both). The repeated restraint induced a loss of body weight gain in the Ringer-treated group which was not reversed, neither by imipramine nor by etanercept. The antidepressant effects of blocking TNF-α using etanercept may be caused by enhancement of serotonergic or noradrenergic neurotransmission or normalization of stress hormone secretion which has to be substantiated in further studies.

P2 receptor-mediated stimulation of the PI3-K/Akt-pathway in vivo

ATP acts as a growth factor as well as a toxic agent by stimulating P2 receptors. The P2 receptor‐activated signaling cascades mediating cellular growth and cell survival after injury are only incompletely understood. Therefore, the aim of the present study was to identify the role of the phosphoinositide 3 kinase (PI3‐K/Akt) and the mitogen‐activated protein kinase/extracellular signal regulated protein kinase (MAPK/ERK) pathways in P2Y receptor‐mediated astrogliosis after traumatic injury and after microinfusion of ADPβS (P2Y1,12,13 receptor agonist) into the rat nucleus accumbens (NAc). Mechanical damage and even more the concomitant treatment with ADPβS, enhanced P2Y1 receptor‐expression in the NAc, which could be reduced by pretreatment with the P2X/Y receptor antagonist PPADS. Quantitative Western blot analysis indicated a significant increase in phosphorylated (p)Akt and pERK1/2 2 h after ADPβS‐microinjection. Pretreatment with PPADS or wortmannin abolished the up‐regulation of pAkt by injury alone or ADPβS‐treatment. The ADPβS‐enhanced expression of the early apoptosis marker active caspase 3 was reduced by PPADS and PD98059, but not by wortmannin. Multiple immunofluorescence labeling indicated a time‐dependent expression of pAkt and pMAPK on astrocytes and neurons and additionally the colocalization of pAkt, pMAPK, and active caspase 3 with the P2Y1 receptor especially at astrocytes. In conclusion, the data show for the first time the involvement of PI3‐K/Akt‐pathway in processes of injury‐induced astroglial proliferation and anti‐apoptosis via activation of P2Y1 receptors in vivo, suggesting specific roles of P2 receptors in glial cell pathophysiology in neurodegenerative diseases.

Stimulation of P2Y1 receptors causes anxiolytic-like effects in the rat elevated plus-maze: Implications for the involvement of P2Y1 receptor-mediated nitric oxide production

The widespread and abundant distribution of P2Y receptors in the mammalian brain suggests important functions for these receptors in the CNS. To study a possible involvement of the P2Y receptors in the regulation of fear and anxiety, the influences of the P2Y1,11,12 receptor-specific agonist adenosine 5′-O-(2-thiodiphosphate) (ADPβS), the P2X1,3 receptor agonist α,β-methylene ATP (α,βmeATP), the unspecific P2 receptor antagonist pyridoxalphosphate-6-azopheny l-2′,4′-disulfonic acid (PPADS), and the specific P2Y1 receptor antagonist N6-methyl-2′-deoxyadenosine-3′,5′-bisphosphate (MRS 2179) on the elevated plus-maze behavior of the rat were investigated. All tested compounds were given intracerebroventricularly (0.5 μl). ADPβS (50 and 500 fmol) produced an anxiolytic-like behavioral profile reflected by an increase of the open arm exploration. The anxiolytic-like effects were antagonized by pretreatment with PPADS (5 pmol) or MRS 2179 (5 pmol). Both compounds caused anxiogenic-like effects when given alone. Furthermore, the anxiolytic-like effects of ADPβS could be antagonized by pretreatment with the nitric oxide synthase (NOS) inhibitor Nw-nitro-L-arginine methyl ester (L-NAME). In addition, the anxiogenic-like effects of PPADS were reversed by the pretreatment with L-arginine (500 pmol), which is the natural substrate for NOS, but not by D-arginine (500 pmol), which is not. Immunofluorescence staining revealed the presence of P2Y1 receptors on neurons in different brain regions such as hypothalamus, amygdala, hippocampus and the periaqueductal gray. Furthermore, the colocalization of P2Y1 receptors and neuronal NOS (nNOS) on some neurons in these regions could be demonstrated. The highest density of P2Y1- and nNOS-immunoreactivity was detected in the dorsomedial hypothalamic nucleus. Taken together, the present results suggest that P2Y1 receptors are involved in the modulation of anxiety in the rat. The anxiolytic-like effects after stimulation of P2Y1 receptors seem to be in close connection with the related nitric oxide production.