Anna Maria Pugliese

Serotonin blocks the long-term potentiation induced by primed burst stimulation in the CA1 region of rat hippocampal slices

The effect of 5-hydroxytryptamine on the induction of long-term potentiation by a train of high frequency pulses (100 Hz; 1 s) or by a stimulation consisting of one burst of five pulses at 100 Hz delivered 170 ms after a single pulse (primed burst) was investigated in the CA1 region of the rat hippocampal slice in vitro with extracellular recordings. Superfusion with 5-hydroxytryptamine (3-30 microM) produced a concentration-dependent decrease in amplitude of the population spikes evoked by test stimuli. The presence of 5-hydroxytryptamine (30 microM) did not affect the magnitude of long-term potentiation produced by the high-frequency stimulation but it prevented the long-term potentiation induced by a primed burst. The action of 5-hydroxytryptamine was mimicked by the 5-hydroxytryptamine1A agonist 5-carboxamidotryptamine (0.3 microM) and blocked by the 5-hydroxytryptamine2/5-hydroxytryptamine1A antagonist spiperone (3 microM) or by the 5-hydroxytryptamine1/5-hydroxytryptamine2 antagonist methiothepin (1-10 microM). The selective 5-hydroxytryptamine2 antagonist ritanserin (1 microM) did not antagonize the block of long-term potentiation produced by 5-hydroxytryptamine. The selective 5-hydroxytryptamine3 antagonists (3-tropanyl)-1H-indole-3-carboxylic acid ester (ICS 205-930; 1 nM) and ondansetron (GR-38032; 30 nM) did not affect the reduction in the population spike produced by application of 5-hydroxytryptamine. In contrast, a primed burst delivered at the fifth minute of 5-hydroxytryptamine application in the presence of a 5-hydroxytryptamine3 antagonist induced a long-term potentiation.(ABSTRACT TRUNCATED AT 250 WORDS)

Investigations into the Adenosine Outflow from Hippocampal Slices Evoked by Ischemia‐Like Conditions

Abstract: The characteristics of adenosine and inosine outflow evoked by 5 min of ischemia‐like conditions in vitro (superfusion with glucose‐free Krebs solution gassed with 95% N2/5% CO2) were investigated on rat hippocampal slices. The viability of the slices after “ischemia” was evaluated by extracellular recording of the evoked synaptic responses in the CA1 region. The evoked dendritic field potentials were abolished after 5 min of superfusion under “ischemia” but a complete recovery occurred after 5 min of reperfusion with normal oxygenated Krebs solution. No recovery took place after 10 min of “ischemia.” The addition of the adenosine A, receptor antagonist 8‐phenylthe‐ ophylline to the superfusate antagonized the depression of the evoked field potentials caused by 5 min of “ischemia.” Five minutes of “ischemia” brought about a six‐ and fivefold increase in adenosine and inosine outflow, respectively, within 10 min. Tetrodotoxin reduced the outflow of adenosine and inosine by 42 and 33%, respectively, whereas the removal of Ca2+ caused a further increase. The NMDA receptor antagonist d(‐)‐2‐amino‐7‐ phoshonoheptanoic acid and the non‐NMDA antagonist 6,7‐dinitroquinoxaline‐2,3‐dione brought about small, not statistically significant decreases of adenosine and inosine outflow. The glutamate uptake inhibitor dihydrokainate did not affect the outflow of adenosine and inosine. Inhibition of ecto‐5′‐nucleotidase by α, β‐methylene ADP and GMP did not affect basal adenosine outflow but potentiated “ischemia”‐evoked adenosine outflow. It is concluded that ischemia‐like conditions in vitro evoke a Ca2+‐independent adenosine and inosine outflow, through a mechanism that partly depends on propagated nervous activity but does not involve excitatory amino acids. The efflux of adenosine is probably responsible for the depression of the evoked synaptic electrical activity during “ischemia” in the hippocampal slices.

Brief, repeated, oxygen-glucose deprivation episodes protect neurotransmission from a longer ischemic episode in the in vitro hippocampus: role of adenosine receptors

Ischemic preconditioning in the brain consists of reducing the sensitivity of neuronal tissue to further, more severe, ischemic insults. We recorded field epsps (fepsps) extracellularly from hippocampal slices to develop a model of in vitro ischemic preconditioning and to evaluate the role of A1, A2A and A3 adenosine receptors in this phenomenon. The application of an ischemic insult, obtained by glucose and oxygen deprivation for 7 min, produced an irreversible depression of synaptic transmission. Ischemic preconditioning was induced by four ischemic insults (2 min each) separated by 13 min of normoxic conditions. After 30 min, an ischemic insult of 7 min was applied. This protocol substantially protected the tissue from the irreversible depression of synaptic activity. The selective adenosine A1 receptor antagonist, 8‐cyclopentyl‐1,3‐dipropylxanthine (DPCPX, 100 nM), completely prevented the protective effect of preconditioning. The selective adenosine A2A receptor antagonist 4‐(2‐[7‐amino‐2‐(2‐furyl)[1,2,4]triazolo[2,3‐a][1,3,5]triazin‐5‐ylamino]ethyl)phenol (ZM 241385, 100 nM) did not modify the magnitude of fepsp recovery compared to control slices. The selective A3 adenosine receptor antagonists, 3‐propyl‐6‐ethyl‐5[ethyl(thio)carbonyl]‐2‐phenyl‐4‐propyl‐3‐pyridinecarboxylate (MRS 1523, 100 nM) significantly improved the recovery of fepsps after 7 min of ischemia. Our results show that in vitro ischemic preconditioning allows CA1 hippocampal neurons to become resistant to prolonged exposure to ischemia. Adenosine, by stimulating A1 receptors, plays a crucial role in eliciting the cell mechanisms underlying preconditioning; A2A receptors are not involved in this phenomenon, whereas A3 receptor activation is harmful to ischemic preconditioning.

ATP Modulates Cell Proliferation and Elicits Two Different Electrophysiological Responses in Human Mesenchymal Stem Cells

Bone marrow‐derived human mesenchymal stem cells (hMSCs) have the potential to differentiate into several cell lines. Extracellular adenosine 5′‐triphosphate (ATP) acts as a potent signaling molecule mediating cell‐to‐cell communication. Particular interest has been focused in recent years on the role of ATP in stem cell proliferation and differentiation. In the present work, we demonstrate that hMSCs at early stages of culture (P0–P5) spontaneously release ATP, which decreases cell proliferation. Increased hMSC proliferation is induced by the unselective P2 antagonist pyridoxalphosphate‐6‐azophenyl‐2′,4′‐disulfonate (PPADS) and by the selective P2Y1 antagonist 2′‐deoxy‐N6‐methyladenosine3′,5′‐bisphosphate (MRS 2179). A functional role of extracellular ATP in modulating ionic conductances with the whole‐cell and/or perforated patch‐clamp techniques was also investigated. Exogenous ATP increased both the voltage‐sensitive outward and inward currents in 47% of cells, whereas, in 31% of cells, only an increase in inward currents was found. Cells responding in this dual manner to ATP presented different resting membrane potentials. Both ATP‐induced effects had varying sensitivity to the P2 antagonists PPADS and MRS 2179. Outward ATP‐sensitive currents are carried by potassium ions, since they are blocked by cesium replacement and are Ca2+‐dependent because they are eliminated in the presence of 1,2‐bis(2‐aminophenoxy)ethane‐N,N,N′,N′‐tetraacetic acid. On the basis of different electrophysiological and pharmacological characteristics, we conclude that outward ATP‐sensitive currents are due to Ca2+‐dependent K+‐channel activation following stimulation of P2Y receptors, whereas inward ATP‐sensitive currents are mediated by P2X receptor activation. In summary, ATP released in early life stages of hMSCs modulates their proliferation rate and likely acts as one of the early factors determining their cell fate.

A3 adenosine receptor antagonists delay irreversible synaptic failure caused by oxygen and glucose deprivation in the rat CA1 hippocampus in vitro.

The role of adenosine A3 receptor activation during ischaemia‐like conditions produced by oxygen and glucose deprivation (OGD) was evaluated with extracellular recordings from the CA1 region of rat hippocampal slices. In all, 7 min of OGD evoked tissue anoxic depolarisation (AD, peak at ∼7 min from OGD start, n=20) and were invariably followed by irreversible loss of electrically evoked field epsps (fepsps, n=42). The selective adenosine A3 antagonists 3‐propyl‐6‐ethyl‐5[(ethylthio)carbonyl]‐2‐phenyl‐4‐propyl‐3‐pyridinecarboxylate (MRS 1523, 1–100 nM, n=31), N‐[9‐chloro‐2‐(2‐furanyl)[1,2,4]‐triazolo[1,5‐c]quinazolin‐5‐yl]benzeneacetamide (MRS 1220, 100 nM, n=7), N‐(2‐methoxyphenyl)‐N′‐[2‐(3‐pyrindinyl)‐4‐quinazolinyl]‐urea, (VUF 5574, 100 nM, n=3) and 5‐[[(4‐pyridyl)amino]carbonyl]amino‐8‐methyl‐2‐(2‐furyl)‐pyrazolo[4,3‐e]1,2,4‐triazolo[1,5‐c]pyrimidine hydrochloride (1 nM, n=4), prevented the irreversible failure of neurotransmission induced by 7 min OGD (n=45) and the development of AD in 20 out of 22 monitored slices. When tested on OGD episodes of longer duration (8–10 min, n=18), 100 nM MRS 1523 prevented or delayed the appearance of AD and exerted a protective effect on neurotransmission for episodes of up to 9 min duration. In the absence of AD, the fepsp recovery was almost total, regardless of OGD episode duration. These findings support the notion that A3 receptor stimulation is deleterious during ischaemia and suggest that selective A3 receptor block may substantially increase the resistance of the CA1 hippocampal region to ischaemic damage.

Effects of DAU 6215, a novel 5‐hydroxytryptamine3 (5‐HT3) antagonist on electrophysiological properties of the rat hippocampus

1 The aim of the present study was to test the effects of DAU 6215 (endo‐N‐(8‐methyl‐8‐azabicyclo‐[3.2.1]‐octo‐3‐yl)‐2,3‐dihydro‐2‐oxo‐1H‐benzimidazole‐1‐carboxamide hydrochloride), a newly synthesized, selective 5‐hydroxytryptamine3 (5‐HT3) antagonist, on the cell membrane properties and on characterized 5‐HT‐mediated responses of pyramidal neurones in the hippocampal CA1 region. 2 Administration of DAU 6215, even at concentrations several hundred fold its Ki, did not affect the cell membrane properties of pyramidal neurones, nor modify extracellularly recorded synaptic potentials, evoked by stimulating the Schaffers collaterals. 3 Micromolar concentrations (15–30 μm) of 5‐HT elicited several responses in pyramidal neurones that are mediated by distinct 5‐HT receptor subtypes. DAU 6215 did not antagonize the 5‐HT1A‐induced membrane hyperpolarization and conductance increase, a response that was blocked by the selective 5‐HT1A antagonist NAN‐190 (1‐(2‐methoxyphenyl)‐4‐[4‐(2‐phtalamido)butyl‐piperazine). Similarly, DAU 6215 did not affect the membrane depolarization and decrease in amplitude of the afterhyperpolarization, elicited by the activation of putative 5‐HT4 receptors. 4 5‐HT increased the frequency of spontaneous postsynaptic potentials (s.p.s.ps) recorded in pyramidal neurones loaded with chloride. In agreement with previous observations, most of the s.p.s.ps were reversed GABAergic events, produced by the activation of 5‐HT3 receptors on interneurones, because they persisted in the presence of the glutamate NMDA and non NMDA antagonists, d‐aminophosphonovaleric acid (APV; 50 μm) and 6,7‐dinitroquinoxaline‐2,3‐dione (DNQX; 25 μm), and were elicited by the selective 5‐HT3 agonist, 2‐methyl‐5‐HT (2‐Me‐5‐HT, 50 μm). 5 The increase in frequency of s.p.s.ps induced by 5‐HT was significantly antagonized by DAU 6215 in 70% of the cases, whereas the 5‐HT3 antagonist always suppressed the effect of 2‐Me‐5‐HT, at concentrations as low as 60 nm. 6 The antagonistic effect of DAU 6215 was also tested on the 5‐HT3‐mediated block of induction of long‐term potentiation (LTP), elicited by a primed burst (PB) stimulation. Extracellular recordings showed that low concentrations (60 nm) of DAU 6215 suppressed the inhibitory action of 5‐HT on PB‐induced LTP, without affecting the 5‐HT1A‐induced reduction in the amplitude of the population spike. 7 These results provide evidence that DAU 6215 is an effective antagonist of the 5‐HT3‐mediated responses in the central nervous system and may offer a cellular correlate for the pharmacological effects of DAU 6215 as an anxiolytic and cognition enhancer.

The role of ATP and adenosine in the brain under normoxic and ischemic conditions.

By taking advantage of some recently synthesized compounds that are able to block ecto-ATPase activity, we demonstrated that adenosine triphosphate (ATP) in the hippocampus exerts an inhibitory action independent of its degradation to adenosine. In addition, tonic activation of P2 receptors contributes to the normally recorded excitatory neurotransmission. The role of P2 receptors becomes critical during ischemia when extracellular ATP concentrations increase. Under such conditions, P2 antagonism is protective. Although ATP exerts a detrimental role under ischemia, it also exerts a trophic role in terms of cell division and differentiation. We recently reported that ATP is spontaneously released from human mesenchymal stem cells (hMSCs) in culture. Moreover, it decreases hMSC proliferation rate at early stages of culture. Increased hMSC differentiation could account for an ATP-induced decrease in cell proliferation. ATP as a homeostatic regulator might exert a different effect on cell trophism according to the rate of its efflux and receptor expression during the cell life cycle. During ischemia, adenosine formed by intracellular ATP escapes from cells through the equilibrative transporter. The protective role of adenosine A1 receptors during ischemia is well accepted. However, the use of selective A1 agonists is hampered by unwanted peripheral effects, thus attention has been focused on A2A and A3 receptors. The protective effects of A2A antagonists in brain ischemia may be largely due to reduced glutamate outflow from neurones and glial cells. Reduced activation of p38 mitogen-activated protein kinases that are involved in neuronal death through transcriptional mechanisms may also contribute to protection by A2A antagonism. Evidence that A3 receptor antagonism may be protective after ischemia is also reported.

Effect of the nootropic drug oxiracetam on field potentials of rat hippocampal slices

1 The effect of the nootropic drug oxiracetam on hippocampal neurotransmission was investigated in the CA1 region of the rat hippocampal slice in vitro by use of extracellular recordings. 2 Superfusion of oxiracetam (0.1–100 μm) produced a concentration‐dependent, wash‐resistant (>90 min), increase in initial slope and amplitude of the dendritic field excitatory postsynaptic potential (e.p.s.p.). This increase was maximal at a concentration of 1 μm (70%). 3 Input‐output curves relating the initial slope to the amplitude of the afferent volley were significantly (P < 0.05) steeper and showed a greater maximal response in the presence of 1 μm oxiracetam than in control conditions. 4 Two trains of high frequency stimulation (100 Hz, 0.4 s, 5 min apart) delivered in the stratum radiatum 30 min after washout of oxiracetam (1 μm) still elicited a long‐term potentiation (LTP) of the field e.p.s.p. However, the absolute magnitude of the LTP produced did not differ from that obtained in untreated slices. 5 After induction and establishment of LTP, oxiracetam (1 μm) had a smaller (27%) and reversible effect on the evoked field e.p.s.p. 6 d‐2‐Amino‐5‐phosphonopentanoic acid (AP‐5), at the same concentration (50 μm) which in our conditions prevented the induction of LTP, blocked the action of 1 μm oxiracetam and strongly depressed the effect of higher concentrations of the nootropic drug. 7 It is concluded that oxiracetam provokes an enduring increase of neurotransmission in the CA1 rat hippocampal region. This action appears to share some features with LTP as indicated by its persistence, sensitivity to AP‐5 and lack of additivity with electrically‐induced LTP.

Effect of the selective 5-HT1A receptor antagonist WAY 100635 on the inhibition of e.p.s.ps produced by 5-HT in the CA1 region of rat hippocampal slices

The actions of N‐(2‐(‐4(2‐methoxyphenyl)‐1‐piperazinyl)ethyl)‐N‐(2‐pyridinyl) cyclohexane carboxamide (WAY 100635), a novel and selective 5‐hydroxytryptamine1A (5‐HT1A) antagonist, on excitatory postsynaptic potentials (e.p.s.ps) were investigated by use of intracellular recordings in pyramidal cells of the CA1 region of rat hippocampal slices. WAY 100635 (10 nM) did not affect any of the investigated parameters of cell excitability such as membrane potential, total input resistance (Rin), firing threshold, action potential amplitude, action potential frequency adaptation, and slow afterhyperpolarization (sAHP) which follows repetitive firing of action potentials. WAY 100635 did not have any effect on either the slope or the amplitude of e.p.s.ps evoked by stimulation of the CA1 stratum radiatum. Bath application of either 5‐hydroxytryptamine (5‐HT, 10–30 μM) or 5‐carboxamidotryptamine (5‐CT, 300 nM) hyperpolarized the membrane potential (ΔVm=−4.1±0.9 and −6.0±0.9 mV, respectively), and reduced Rin (−25±8% and −18±1%, respectively). 5‐HT blocked the action potential frequency adaptation and significantly reduced the amplitude of the sAHP that follows repetitive firing of action potentials. 5‐HT significantly decreased the amplitude of evoked e.p.s.ps (−14±6%). This effect was greater in the presence of the GABAA receptor antagonist bicuculline (10 μM, −45±12%) and was mimicked by 5‐CT (−49±5%). Both AMPA and NMDA components of e.p.s.ps were significantly reduced in amplitude by 5–HT (−38±8%, n=6, and −29±12%, n=3, respectively; P<0.05). WAY 100635 fully antagonized the hyperpolarization, the reduction of Rin, and the decrease in amplitude of e.p.s.ps elicited by 5‐HT, while it did not affect the action of 5‐HT on the action potential frequency adaptation. In the presence of WAY 100635, 5‐HT elicited a depolarization which was blocked by 10–30 μM RS 23597‐190, a selective 5‐HT4 receptor antagonist. Our data demonstrate that WAY 100635 is devoid of direct effects on CA1 pyramidal cell excitability and on evoked e.p.s.ps, while it fully antagonizes the effects of 5‐HT on excitatory synaptic transmission and on hyperpolarization, without affecting the 5‐HT4 receptor‐mediated response. Since WAY 100635 selectively antagonizes 5‐HT1A receptor‐mediated actions of 5‐HT, our data also demonstrate that the inhibitory action of 5‐HT on excitatory synaptic transmission in CA1 is mediated by 5‐HT1A receptors.

Neurological basis of AMP-dependent thermoregulation and its relevance to central and peripheral hyperthermia

Therapeutic hypothermia is of relevance to treatment of increased body temperature and brain injury, but drugs inducing selective, rapid, and safe cooling in humans are not available. Here, we show that injections of adenosine 5′-monophosphate (AMP), an endogenous nucleotide, promptly triggers hypothermia in mice by directly activating adenosine A1 receptors (A1R) within the preoptic area (POA) of the hypothalamus. Inhibition of constitutive degradation of brain extracellular AMP by targeting ecto 5′-nucleotidase, also suffices to prompt hypothermia in rodents. Accordingly, sensitivity of mice and rats to the hypothermic effect of AMP is inversely related to their hypothalamic 5′-nucleotidase activity. Single-cell electrophysiological recording indicates that AMP reduces spontaneous firing activity of temperature-insensitive neurons of the mouse POA, thereby retuning the hypothalamic thermoregulatory set point towards lower temperatures. Adenosine 5′-monophosphate also suppresses prostaglandin E2-induced fever in mice, having no effects on peripheral hyperthermia triggered by dioxymetamphetamine (ecstasy) overdose. Together, data disclose the role of AMP, 5′-nucleotidase, and A1R in hypothalamic thermoregulation, as well and their therapeutic relevance to treatment of febrile illness.