Renato Corradetti

Adenosine decreases aspartate and glutamate release from rat hippocampal slices

The effect of adenosine and related compounds on the release of endogenous aspartate and glutamate from isolated, superfused rat hippocampal slices was studied at rest and during electrical stimulation of the stratum radiatum in the CA3/CA2 region, using a sensitive mass-spectrometric technique. Evoked extracellular potentials were recorded from the CA1 region. Adenosine, at 3 X 10(-4) M concentration, inhibited the stimulation-evoked potentials and prevented the stimulation-induced release of aspartate and glutamate. Similarly, 1-phenylisopropyladenosine (10(-6) M) and cyclohexyladenosine (10(-6) M) depressed both electrical and neurochemical responses to stimulation of the stratum radiatum. 8-Phenyltheophylline (5 X 10(-6) M) increased the release of aspartate and glutamate and antagonized the cyclohexyladenosine-induced inhibition of amino acid release. Our results support the hypothesis that adenosine modulates the electrophysiological responses to stimulation of stratum radiatum through a reduction of the release of the excitatory amino acids aspartate and glutamate.

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)

Extracellular adenosine concentrations during in vitro ischaemia in rat hippocampal slices

The application of an ischaemic insult in hippocampal slices results in the depression of synaptic transmission, mainly attributed to the activation of A1 adenosine receptors by adenosine released in the extracellular space. To estimate the concentration of endogenous adenosine acting at the receptor level during an ischaemic episode, we recorded field e.p.s.ps (fe.p.s.ps) from hippocampal slices, and evaluated the ability of the selective A1 receptor antagonist, 8‐cyclopentyl‐1,3‐dipropylxanthine (DPCPX), to reverse the fe.p.s.p. depression induced by in vitro ischaemia. A relationship between the IC50 of an antagonist and the endogenous concentration of a neurotransmitter has been used for pharmacological analysis. The complete and reversible depression of fe.p.s.p. in the CA1 region induced by 5 min ischaemia was decreased in the presence of DPCPX (50–500 nM). 8‐Phenyltheophylline (10 μM) abolished the depression of fe.p.s.ps during the ischaemic period, while a small (peak effect 12±4%) decrease in fe.p.s.ps was observed during the initial phase of reperfusion. In the time‐interval of maximal depression of fe.p.s.ps., IC50 and adenosine concentration changed as function of time with a good degree of correlation. The maximal value of adenosine concentration was 30 μM. Our data provide an estimation of the adenosine concentration reached at the receptor level during an ischaemic episode, with a higher time discrimination (15 s) than that achieved with any biochemical approach. This estimation may be useful in order to establish appropriate concentrations of purinergic compounds to be tested for their pharmacological effects during an ischaemic episode.

Sporadic Autonomic Dysregulation and Death Associated with Excessive Serotonin Autoinhibition

Sudden infant death syndrome is the leading cause of death in the postneonatal period in developed countries. Postmortem studies show alterations in serotonin neurons in the brainstem of such infants. However, the mechanism by which altered serotonin homeostasis might cause sudden death is unknown. We investigated the consequences of altering the autoinhibitory capacity of serotonin neurons with the reversible overexpression of serotonin 1A autoreceptors in transgenic mice. Overexpressing mice exhibited sporadic bradycardia and hypothermia that occurred during a limited developmental period and frequently progressed to death. Moreover, overexpressing mice failed to activate autonomic target organs in response to environmental challenges. These findings show that excessive serotonin autoinhibition is a risk factor for catastrophic autonomic dysregulation and provide a mechanism for a role of altered serotonin homeostasis in sudden infant death syndrome.

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.

Antagonist properties of (−)‐pindolol and WAY 100635 at somatodendritic and postsynaptic 5‐HT1A receptors in the rat brain

The aim of the present work was to characterize the 5‐hydroxytryptamine1A (5‐HT1A) antagonistic actions of (−)‐pindolol and WAY 100635 (N‐(2‐(4‐(2‐methoxyphenyl)‐1‐piperazinyl)ethyl)‐N‐(2‐pyridinyl) cyclohexane carboxamide). Studies were performed on 5‐HT1A receptors located on 5‐hydroxytryptaminergic neurones in the dorsal raphe nucleus (DRN) and on pyramidal cells in the CA1 and CA3 regions of the hippocampus in rat brain slices. Intracellular electrophysiological recording of CA1 pyramidal cells and 5‐hydroxytryptaminergic DRN neurones showed that the 5‐HT1A receptor agonist 5‐carboxamidotryptamine (5‐CT) evoked in both cell types a concentration‐dependent cell membrane hyperpolarization and a decrease in cell input resistance. On its own, (−)‐pindolol did not modify the cell membrane potential and resistance at concentrations up to 10 μM, but it antagonized the 5‐CT effects in a concentration‐dependent manner. Similar antagonism of 5‐CT effects was observed in the CA3 hippocampal region. (−)‐Pindolol also prevented the 5‐HT1A receptor‐mediated hyperpolarization of CA1 pyramidal cells due to 5‐HT (15 μM). In contrast, the 5‐HT‐induced depolarization mediated by presumed 5‐HT4 receptors persisted in the presence of 3 μM (−)‐pindolol. In the hippocampus, (−)‐pindolol completely prevented the hyperpolarization of CA1 pyramidal cells by 100 nM 5‐CT (IC50=92 nM; apparent KB=20.1 nM), and of CA3 neurones by 300 nM 5‐CT (IC50=522 nM; apparent KB=115.1 nM). The block by (−)‐pindolol was surmounted by increasing the concentration of 5‐CT, indicating a reversible and competitive antagonistic action. Extracellular recording of the firing rate of 5‐hydroxytryptaminergic neurones in the DRN showed that (−)‐pindolol blocked, in a concentration‐dependent manner, the decrease in firing elicited by 100 nM 5‐CT (IC50=598 nM; apparent KB=131.7 nM) or 100 nM ipsapirone (IC50=132.5 nM; apparent KB=124.9 nM). The effect of (−)‐pindolol was surmountable by increasing the concentration of the agonist. Intracellular recording experiments showed that 10 μM (−)‐pindolol were required to antagonize completely the hyperpolarizing effect of 100 nM 5‐CT. In vivo labelling of brain 5‐HT1A receptors by i.v. administration of [3H]‐WAY 100635 ([O‐methyl‐3H]‐N‐(2‐(4‐(2‐methoxyphenyl)‐1‐piperazinyl)ethyl‐N‐(2‐pyridyl)cyclo‐hexane‐carboxamide) was used to assess their occupancy following in vivo treatment with (−)‐pindolol. (−)‐Pindolol (15 mg kg−1) injected i.p. either subchronically (2 day‐treatment before i.v. injection of [3H]‐WAY 100635) or acutely (20 min before i.v. injection of [3H]‐WAY 100635) markedly reduced [3H]‐WAY 100635 accumulation in all 5‐HT1A receptor‐containing brain areas. In particular, no differences were observed in the capacity of (−)‐pindolol to prevent [3H]‐WAY 100635 accumulation in the DRN and the CA1 and CA3 hippocampal areas. Intracellular electrophysiological recording of 5‐hydroxytryptaminergic DRN neurones showed that WAY 100635 prevented the hyperpolarizing effect of 100 nM 5‐CT in a concentration‐dependent manner (IC50=4.9 nM, apparent KB=0.25 nM). In CA1 pyramidal cells, hyperpolarization induced by 50 nM 5‐CT was also antagonized by WAY 100635 (IC50=0.80 nM, apparent KB=0.28 nM).

Electrical Stimulation of the Stratum Radiatum Increases the Release and Neosynthesis of Aspartate, Glutamate, and γ-Aminobutyric Acid in Rat Hippocampal Slices

Abstract: The release of endogenous aspartic, glutamic, and γ‐aminobutyric acids (Asp, Glu, GABA, respectively) was measured in the effluent from superfused hippocampal slices using a new and sensitive mass spectrometnc method. The stimulation of the stratum radiatum of the rat dorsal hippocampus caused a Ca2+‐dependent increase in the release of these amino acids. This release was accompanied by an increase in the incorporation of [13C2 from [13C]glucose into Asp, Glu, and GABA, suggesting an increase in their neosynthesis. The removal of Ca2+ from the superfusion fluid brought about a marked decrease in Asp and Glu release at rest, and prevented their stimulation‐evoked release and the appearance of population spikes. The results support the hypothesis that Asp and Glu are excitatory neurotransmitters in intrinsic hippocampal circuits and are possibly released from the Schaffer collaterals and commissural fibres. The increase in GABA release and neosynthesis during stimulation of the stratum radiatum could be related to recurrent inhibition evoked by transsynaptic stimulation of the pyramidal cells.

Effect of A2A adenosine receptor stimulation and antagonism on synaptic depression induced by in vitro ischaemia in rat hippocampal slices

In the present study we investigated the role of A2A adenosine receptors in hippocampal synaptic transmission under in vitro ischaemia‐like conditions. The effects of adenosine, of the selective A2A receptor agonist, CGS 21680 (2‐[p‐(2‐carboxyethyl)‐phenethylamino]‐5′‐N‐ethylcarboxamidoadenosine), and of selective A2A receptor antagonists, ZM 241385 (4‐(2‐[7‐amino‐2‐(2‐furyl)‐{1,2,4}‐triazolo{2,3‐a}{1,3,5}triazin‐5‐ylamino]ethyl)phenol) and SCH 58261 (7‐(2‐phenylethyl)‐5‐amino‐2‐(2‐furyl)‐pyrazolo‐[4,3‐e]‐1,2,4‐triazolo[1,5‐c]pyrimidine), have been evaluated on the depression of field e.p.s.ps induced by an in vitro ischaemic episode. The application of 2 min of in vitro ischaemia brought about a rapid and reversible depression of field e.p.s.ps, which was completely prevented in the presence of the A1 receptor antagonist DPCPX (1,3‐dipropyl‐8‐cyclopentylxanthine) (100 nM). On the other hand both A2A receptor antagonists, ZM 241385 and SCH 58261, by themselves did not modify the field e.p.s.ps depression induced by in vitro ischaemia. A prolonged application of either adenosine (100 μM) or CGS 21680 (30, 100 nM) before the in vitro ischaemic episode, significantly reduced the synaptic depression. These effects were antagonized in the presence of ZM 241385 (100 nM). SCH 58261 (1 and 50 nM) did not antagonize the effect of 30 nM CGS 21680 on the ischaemia‐induced depression. These results indicate that in the CA1 area of the hippocampus the stimulation of A2A adenosine receptors attenuates the A1‐mediated depression of synaptic transmission induced by in vitro ischaemia.

Suppression of Serotonin Neuron Firing Increases Aggression in Mice

Numerous studies link decreased serotonin metabolites with increased impulsive and aggressive traits. However, although pharmacological depletion of serotonin is associated with increased aggression, interventions aimed at directly decreasing serotonin neuron activity have supported the opposite association. Furthermore, it is not clear if altered serotonin activity during development may contribute to some of the observed associations. Here, we used two pharmacogenetic approaches in transgenic mice to selectively and reversibly reduce the firing of serotonin neurons in behaving animals. Conditional overexpression of the serotonin 1A receptor (Htr1a) in serotonin neurons showed that a chronic reduction in serotonin neuron firing was associated with heightened aggression. Overexpression of Htr1a in adulthood, but not during development, was sufficient to increase aggression. Rapid suppression of serotonin neuron firing by agonist treatment of mice expressing Htr1a exclusively in serotonin neurons also led to increased aggression. These data confirm a role of serotonin activity in setting thresholds for aggressive behavior and support a direct association between low levels of serotonin homeostasis and increased aggression.

The release of endogenous GABA and glutamate from the cerebral cortex in the rat

Summary1.The release of endogenous GABA and glutamate from the cerebral cortex was measured using a cortical cup technique in unanaesthetized freely moving rats and anaesthetized rats by means of a sensitive and specific massspectrometric procedure.2.GABA release was not affected by the presence of the dura mater or by anaesthesia. Glutamate output was reduced by urethane but not by pentobarbital anaesthesia and by the presence of the dura.3.An isotonic solution containing 50 mM KCl placed epidurally within the cup elicited a significant short-lasting increase in glutamate output, a decrease in GABA output and a short-lasting electrocorticogram (ECoG) activation.4.When the dura was removed, a high K+ solution placed on the exposed cerebral cortex elicited a 7–8 fold increase in GABA output accompanied by a marked decrease in glutamate output and by ECoG synchronization. The changes in GABA and glutamate output had parallel time-course and were prevented by the application within the cup of tetrodotoxin (3×10−5 M).5.Amphetamine at the doses of 3.7 and 7.4 μmol·kg−1 i.v. inereased glutamate output and at the dose of 37 μmol ·kg−1 i.v. increased GABA output. Both effects were prevented or reduced by haloperidol pretreatment (0.65 μmol ·kg−1 i.v.).6.It is concluded that GABA and glutamate released from the cerebral cortex and diffused into an epidural or cortical cup originate at least in part from the brain. The rate of their release is influenced by changes in neuronal activity. The measurement of their rate of release offers a useful tool for the study of the functional role of cortical GABA and glutamate-releasing neurons.