Holger Kittner

The reaction of astrocytes and neurons in the hippocampus of adult rats during chronic ethanol treatment and correlations to behavioral impairments

Chronic ethanol treatment of Wistar rats to 10% (v/v) ethanol over a period of 4, 12, and 36 weeks produced distinct alterations of the glial fibrillary acidic protein immunoreactivity (GFAP-IR) of dorsal hippocampal astrocytes. Ethanol consumption over a period of 4 weeks caused an increase in the total GFAP-IR of the astrocytes. Down-regulation of the total GFAP-IR was measured in all examined brain regions after 36 weeks of ethanol treatment. Prolonged ethanol treatment induced a significant loss of the total number of hippocampal pyramidal and dentate gyrus granule cells. Regional differences in the vulnerability to the neurotoxic effects of chronic ethanol intake over 36 weeks were found: CA3 > CA1 + CA2 > > CA4 > GD. In agreement with the degree of neuronal cell loss, ethanol-induced behavioral impairments were found. The acquisition of maze performance using a complex elevated labyrinth was deteriorated after 36 weeks of ethanol treatment, suggesting a deficit in learning and memory. These findings illustrate the importance of time-response analysis when determining the structural and functional changes produced by chronic ethanol treatment.

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.

Mechanisms of adenosine 5′‐triphosphate‐induced dopamine release in the rat nucleus accumbens in vivo

The endogenous mechanisms modulating ATP‐induced dopamine release in the nucleus accumbens (NAc) were studied by microdialysis in freely moving rats. The ATP analog 2‐Methylthio ATP (2‐MeSATP) facilitated the release of dopamine in a manner sensitive to pertussis toxin and tetrodotoxin. It is suggested that G‐protein‐coupled P2Y receptors and voltage‐gated sodium channels are involved in this process. N‐methyl‐D‐aspartate (NMDA) applied in a concentration of 100 μM decreased the extracellular dopamine level, whereas 1 and 10 mM NMDA enhanced it. The endogenous agonist glutamate (10 μM) inhibited the basal and facilitated release of dopamine. Infusion with a combination of the ionotropic glutamate receptor antagonists (±)‐3‐(2‐carboxypiperazin‐4‐yl)‐propyl‐1‐phosphonic acid (CPP) and 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX), as well as with the metabotropic glutamate receptor antagonist (±)‐α‐methyl‐4‐carboxyphenylglycine (MCPG) increased the basal level of dopamine and potentiated the 2‐MeSATP‐facilitated dopamine release, suggesting an ATP‐mediated glutamate release. The GABAA receptor antagonist bicuculline infused into the NAc also enhanced the basal level of dopamine; however, the application of 2‐MeSATP in the presence of bicuculline caused an early decrease and a subsequent increase of dopamine release. The facilitatory phase of the 2‐MeSATP effect was comparable with that measured in the absence of bicuculline. By contrast, when bicuculline was infused into the ventral tegmental area (VTA) it elevated the accumbal basal dopamine level and in addition facilitated the 2‐MeSATP‐ and the glutamate‐induced dopamine release above that measured in the absence of bicuculline. These results suggest that ATP in the NAc has a physiologically relevant function in modulating dopaminergic transmission depending on the mesolimbic neuronal activity. The first component of the ATP effect involves a direct stimulation of the terminals of VTA neurons, while the second inhibitory component involves a sequential activation of glutamate and, finally, via ionotropic and metabotropic glutamate receptors, of GABA neurons projecting to the VTA. Synapse 39:222–232, 2001.

Neuroprotective effects of the P2 receptor antagonist PPADS on focal cerebral ischaemia-induced injury in rats.

After acute injury of the central nervous system extracellular adenosine 5′‐triphosphate (ATP) can reach high concentrations as a result of cell damage and subsequent increase in membrane permeability. Released ATP may act as a toxic agent, which causes cellular degeneration and death, mediated through P2X and P2Y receptors. Mechanisms underlying the various effects of purinoceptor modulators in models of cerebral damage are still uncertain. In the present study the effect of P2 receptor inhibition after permanent middle cerebral artery occlusion (MCAO) in spontaneously hypertensive rats was investigated. Rats received either the non‐selective P2 receptor antagonist pyridoxalphosphate‐6‐azophenyl‐2′,4′‐disulphonic acid (PPADS) or artificial cerebrospinal fluid (ACSF) as control by the intracerebroventricular route. First, these treatments were administered 10 min before MCAO and subsequently twice daily for 1 or 7 days after MCAO. The functional recovery of motor and cognitive deficits was tested at an elevated T‐labyrinth. The PPADS‐treated group showed a significant reduction of paresis‐induced sideslips compared with ACSF‐treated animals. Infarct volume was reduced in the PPADS group in comparison with the ACSF group. A significant decrease in intermediately and profoundly injured cells in favour of intact cells in the PPADS group was revealed by quantification of celestine blue/acid fuchsin‐stained cells in the peri‐infarct area. The data provide further evidence for the involvement of P2 receptors in the pathophysiology of cerebral ischaemia in vivo. The inhibition of P2 receptors at least partially reduces functional and morphological deficits after an acute cerebral ischaemic event.

Enhanced food intake after stimulation of hypothalamic P2Y1 receptors in rats: modulation of feeding behaviour by extracellular nucleotides.

The present study was aimed to clarify the role of purinergic signalling in the regulation of ingestion behaviour. The ATP/ADP analogues 2‐methylthioATP (2‐MeSATP) and adenosine 5′‐O‐(2‐thiodiphosphate) (ADPβS) increased the food intake after intracerebroventricular infusion in 18‐h food‐deprived rats. This effect was abolished by pretreatment with the non‐selective P2X/P2Y receptor antagonist pyridoxalphosphate‐6‐azophenyl‐2′,4′‐disulphonic acid (PPADS) or the selective P2Y1 receptor antagonist MRS 2179, respectively. The stimulation of food intake mediated by ADPβS was also blocked by pretreatment with the nitric oxide synthase (NOS) inhibitor Nw‐nitro‐l‐arginine methylester (L‐NAME), as well as with the inhibitor of the soluble guanylyl cyclase 1H‐[1,2,4]oxadiazolo[4,3‐a]quinoxalin‐1‐one (ODQ), suggesting that the orexigenic effect seems to be closely related with the ensuing formation of nitric oxide. The immunohistochemical staining indicating a co‐localization of P2Y1 receptor‐ and nNOS‐immunoreactivities in a population of neurons in the ventromedial hypothalamic nucleus (VMH) agrees with this assumption. Further experiments with the direct local application of these compounds into the VMH and lateral hypothalamic nucleus (LH) show that the stimulation of P2Y1 receptors in these functionally antagonistic brain regions exerts an increased food intake. Hence, different signal transduction mechanisms may operate in the VMH and LH. Our assumption is supported by distinct effects of the NOS inhibitor L‐NAME in these two hypothalamic nuclei. The present data suggest that ATP/ADP, acting as extracellular signal molecules in the rat brain, are involved in the regulation of food intake, possibly depending on P2Y1‐receptor‐mediated nitric oxide production.

Influence of ethanol on the pentylenetetrazol-induced kindling in rats

Summary. The effects of ethanol on the development of pentylenetetrazol (PTZ)-kindling as well as on fully PTZ-kindled convulsions in rats were investigated. Ethanol (0.5, 1.0 and 1.5 g/kg i.p.) administered 15 min prior to each PTZ-injection (35 mg/kg i.p.; 3 times/week) significantly inhibited the progressive seizure development compared to saline-treated controls. For the higher doses of ethanol the kindling process was restricted to seizure stages of 1 or 2. Tolerance to this antiepileptogenic action did not occur even after 20 PTZ-stimulations. In a second series of experiments, 0.5 g/kg ethanol administered 10 h before each PTZ-injection facilitated the rate of kindling development after 7 to 10 PTZ-injections, while the higher doses of ethanol did not modulate or even slightly reduced the seizure development. In a third test, intermittent administration of a high dose of ethanol (2 g/kg p.o.; twice daily for 6 days) before the kindling procedure (0.5 g/kg i.p. ethanol 10 h prior to each PTZ-injection), significantly intensified the kindling development. In addition, studies with fully PTZ-kindled rats demonstrated that ethanol (0.1 to 1.5 g/kg i.p.), given 15 min prior or 2 min after PTZ, reduced the seizure severity in a dose-dependent manner. In conclusion, the present findings provide evidence for pronounced antiepileptogenic and anticonvulsant effects of ethanol after acute application, whereas repeated administration of high doses with longer withdrawal periods leads to proconvulsant actions, possible mediated via neuroadaptive changes in NMDA and/or GABAA receptor-related mechanisms.

Expression of purinergic receptors in the hypothalamus of the rat is modified by reduced food availability.

ATP-sensitive P2 receptors are suggested to play an important role in the cerebral signal transduction. We examined the expression of the P2Y1 receptor and the possibly downstream-related neuronal nitric oxide synthase (nNOS) in the hypothalamus of rats food-restricted for 3 or 10 days and rats refed after a restriction of 10 days. The restriction caused a reduction of the body weight and plasma triacylglyceride, an increase of non-esterified fatty acid levels correlating with a decrease of leptin levels and an enhancement of plasma corticosterone. All changes returned to basal levels after refeeding. The restriction induced an enhanced intake within 30 min after food presentation and a reduction in the latency. Interestingly, the latter was not abolished by refeeding. The daily food intake induced by refeeding was enhanced at the first day only. The expression of hypothalamic P2Y1 receptor/nNOS mRNA and protein and of leptin receptor mRNA were enhanced after restricted feeding. These changes were abolished after 3 days of refeeding. Immunofluorescence studies indicated that P2Y1 receptor and nNOS immunoreactivities are present in the dorsomedial, ventromedial and lateral hypothalamus and in the nucleus arcuatus. P2Y1 receptor-positive cells were partially also nNOS-positive. The P2Y1 receptor labeling was restricted to cell bodies of obviously non-glial cells, whereas nNOS labeling could be detected also at cellular processes of these cells. In the nucleus arcuatus, astrocytes were identified, expressing P2Y1 receptors at cell bodies and cellular processes. The data suggest that restricted feeding may enhance the sensitivity of the hypothalamus to extracellular ADP/ATP by regulation of the expression of P2Y1 receptors and possibly of their signal transduction pathway via nitric oxide production.

Modulation of feeding behaviour by blocking purinergic receptors in the rat nucleus accumbens: a combined microdialysis, electroencephalographic and behavioural study

The nonspecific P2 receptor antagonist pyridoxalphosphate‐6‐azophenyl‐2′,4′‐disulphonic acid (PPADS), the nonspecific P1 receptor antagonist 8‐(p‐sulphophenyl)‐theophylline (8‐SPT) and the combination of both were applied by retrograde microdialysis into the nucleus accumbens (NAc) before and during feeding of 18‐h food‐deprived rats. In addition to the registration of behavioural parameters, such as the amount and duration of food intake, the feeding‐induced changes in dopamine (DA) concentration and the concomitant changes of neuronal activity in the NAc and the ventral tegmental area (VTA) were simultaneously determined. The perfusion with PPADS (20 µm) diminished the amount of food intake and the duration of feeding. Furthermore, the P2 receptor antagonist blocked the feeding‐induced DA release and prevented the feeding‐elicited changes of the electroencephalography (EEG) power distribution which was characterised by an increase in the power of the 8.0–13.0‐Hz frequency band in the NAc and the VTA. The effects of PPADS could be completely prevented by the concomitantly perfused adenosine receptor antagonist 8‐SPT (100 µm). When given alone, 8‐SPT increased the amount of food ingested, the duration of feeding and the EEG power of the higher frequency range, particularly between 19.0 and 30.0 Hz, in both the NAc and the VTA. The feeding‐elicited DA release was supplemented to the enhanced DA level caused by the perfusion with 8‐SPT in an additive manner. The P2 and P1 receptor antagonists interact antagonistically in the modulation of feeding behaviour and the feeding‐induced changes of EEG activity suggesting that both endogenous extracellular ATP and adenosine are involved in the regulation of the feeding‐associated mesolimbic neuronal activity in a functionally antagonistic manner.

P2 receptors are involved in the mediation of motivation-related behavior

The importance of purinergic signaling in the intact mesolimbic–mesocortical circuit of the brain of freely moving rats is reviewed. In the rat, an endogenous ADP/ATPergic tone reinforces the release of dopamine from the axon terminals in the nucleus accumbens as well as from the somatodendritic region of these neurons in the ventral tegmental area, as well as the release of glutamate, probably via P2Y1 receptor stimulation. Similar mechanisms may regulate the release of glutamate in both areas of the brain. Dopamine and glutamate determine in concert the activity of the accumbal GABAergic, medium-size spiny neurons thought to act as an interface between the limbic cortex and the extrapyramidal motor system. These neurons project to the pallidal and mesencephalic areas, thereby mediating the behavioral reaction of the animal in response to a motivation-related stimulus. There is evidence that extracellular ADP/ATP promotes goal-directed behavior, e.g., intention and feeding, via dopamine, probably via P2Y1 receptor stimulation. Accumbal P2 receptor-mediated glutamatergic mechanisms seem to counteract the dopaminergic effects on behavior. Furthermore, adaptive changes of motivation-related behavior, e.g., by chronic succession of starvation and feeding or by repeated amphetamine administration, are accompanied by changes in the expression of the P2Y1 receptor, thought to modulate the sensitivity of the animal to respond to certain stimuli.

Purinergic modulation of extracellular glutamate levels in the nucleus accumbens in vivo

In the present study, the P2 receptor‐mediated modulation of the extracellular glutamate concentration was investigated by microdialysis in the nucleus accumbens (NAc) of freely moving rats. Because of the known interference of dopaminergic and glutamatergic mechanisms in this area the experiments were performed with animals intra‐accumbally treated with 6‐hydroxydopamine (6‐OHDA) to deplete dopamine pools. Perfusion of the NAc with the prototypic P2 receptor agonist 2‐methylthioadenosine 5′‐triphosphate (2‐MeSATP, 0.1, 1 and 10 mM) concentration‐dependently increased the extracellular level of glutamate in this area. Pretreatment with the P2 receptor antagonist pyridoxalphosphate‐6‐azophenyl‐2′,4′‐disulphonic acid (PPADS, 0.1 mM) decreased the basal extracellular glutamate concentration and inhibited the 2‐MeSATP‐induced outflow of glutamate. In rats treated with 6‐OHDA, 2‐MeSATP increased the total extracellular glutamate to an extent about fivefold larger than in sham‐lesioned rats. The perfusion of the dopamine‐depleted NAc with the D2/D3 dopamine receptor agonist quinpirole (0.1 mM) diminished the basal concentration of glutamate and reduced the effect of 2‐MeSATP on the extracellular glutamate.