Subcellular localization of adenosine A(1) receptors in nerve terminals and synapses of the rat hippocampus

Adenosine is a neuromodulator in the CNS that mainly acts through pre- and postsynaptic A(1) receptors to inhibit the release of excitatory neurotransmitters and NMDA receptor function. This might result from a highly localized distribution of A(1) receptors in the active zone and postsynaptic density of CNS synapses that we now investigated in the rat hippocampus. The binding density of the selective A(1) receptor antagonist, [3H]1,3-dipropyl-8-cyclopentylxanthine ([3H]DPCPX), was enriched in membranes from Percoll-purified nerve terminals (B(max)=1839+/-52 fM/mg protein) compared to total membranes from the hippocampus (B(max)=984+/-31 fM/mg protein), the same occurring with A(1) receptor immunoreactivity. [3H]DPCPX binding occurred mainly to the plasma membrane rather than to intracellular sites, since the binding of the membrane permeable A(1) receptor ligand [3H]DPCPX to intact hippocampal nerve terminals (B(max)=1901+/-192 fM/mg protein) was markedly reduced (B(max)=321+/-30 fM/mg protein) by the membrane impermeable adenosine receptor antagonist, 8-sulfophenyltheophilline (25 microM). Further subcellular fractionation of hippocampal nerve terminals revealed that A(1) receptor immunoreactivity was strategically located in the active zone of presynaptic nerve terminals, as expected to understand the efficiency of A(1) receptors to depress neurotransmitter release. A(1) Receptors were also present in nerve terminals outside the active zone in accordance with the existence of a presynaptic A(1) receptor reserve. Finally, A(1) receptor immunoreactivity was evident in the postsynaptic density together with NMDA receptor subunits 1, 2A and 2B and with N-and P/Q-type calcium channel immunoreactivity, emphasizing the importance of A(1) receptors in the control of dendritic integration.     

Facilitation by P(2) receptor activation of acetylcholine release from rat motor nerve terminals: interaction with presynaptic nicotinic receptors

ATP is released from motor nerve endings together with acetylcholine. Released adenine nucleotides can be extracellularly metabolized into adenosine, which is a presynaptic neuromodulator at neuromuscular junctions, but it is not known if P(2) receptor activation also modulates acetylcholine release from mature motor nerve endings. We now tested the effect of a stable ATP analogue, beta,gamma-imido ATP on the nerve-evoked release of acetylcholine from adult rat hemidiaphragm preparations. beta,gamma-Imido ATP (10-100 microM) facilitated in a concentration-dependent manner evoked acetylcholine release, and 30 microM beta,gamma-imido ATP caused a 125% facilitation of evoked acetylcholine release. This facilitatory effect of beta,gamma-imido ATP (30 microM) was abolished by the P(2) receptor antagonists, suramin (100 microM) and pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS, 10 microM), but not by the A(1) or A(2A) adenosine receptor antagonists, 1,3-dipropyl-8-cyclopentylxanthine (50 nM) and ZM 241385 (50 nM), respectively. The facilitation of acetylcholine release by beta, gamma-imido ATP (30 microM) was also prevented by the nicotinic acetylcholine receptor antagonist, D-tubocurarine (1 microM) and the facilitatory effect (40%) of the nicotinic acetylcholine receptor agonist, 1,1-dimethyl-4-phenylpiperazinium (1 microM) was abolished by PPADS (10 microM). These results demonstrate a presynaptic facilitatory effect of P(2) receptor activation at the rat phrenic nerve endings, which is tightly coupled with the presynaptic nicotinic autofacilitatory system.     

Inhibition of hippocampal acetylcholine release after acute and repeated Δ-tetrahydrocannabinol in rats

The effects of acute and repeated administration of Δ⁹-tetrahydrocannabinol (Δ⁹-THC), the psychoactive principle of marijuana, on acetylcholine release in the hippocampus was studied in freely moving rats by microdialysis. The acute intraperitoneal (i.p.) administration of Δ⁹-THC at the doses of 2.5 and 5 mg/kg reduced acetylcholine release by about 25% and 45%, respectively. A dose of 7.5 mg/kg produced no further reduction. Δ⁹-THC effects were antagonized by the cannabinoid CB₁ antagonist SR141716A at the i.p. dose of 1 mg/kg, per se ineffective in modifying acetylcholine concentrations. After a repeated exposure (twice daily for up to seven days) to Δ⁹-THC (7.5 mg/kg, i.p.) or vehicle (0.3 ml/kg, i.p.), the inhibitory effect of Δ⁹-THC (2.5 and 5 mg/kg, i.p) on acetylcholine release was not reduced. The results confirm previous observations that cannabinoids inhibit acetylcholine release through cannabinoid CB₁ receptors, and indicate that no tolerance to this effects develops after a repeated Δ⁹-THC administration.     

Evidence to suggest that extracellular acetate is accumulated by rat hippocampal cholinergic nerve terminals for acetylcholine formation and release

It is well established that extracellular choline is transported into central cholinergic nerve terminals by `high' and `low' affinity processes to form the neurotransmitter acetylcholine (ACh). The intent of the present investigation was to ascertain whether extracellular acetate might also be transported into central cholinergic nerve terminals to form ACh. To test this possibility, rat hippocampal tissue was incubated with varying concentrations of extracellular [1-¹⁴C]acetate (0.1–100 μM) and the uptake of [1-¹⁴C]acetate and the amount of [¹⁴C]ACh formed by the tissue determined. The results indicated that the uptake of extracellular [1-¹⁴C]acetate was temperature-dependent and saturable having an apparent Michaelis constant (K_m) of 22 μM. The formation of [¹⁴C]ACh in the tissue as a function of extracellular [1-¹⁴C]acetate appeared to occur by both `high' and `low' affinity processes with apparent K_m values of 0.5 and 19.6 μM, respectively. In other experiments, three inhibitors (lithium, allicin and sodium) of acetyl CoA synthetase (EC 6.2.1.1 acetate: CoA ligase), the enzyme which converts acetate to acetyl CoA when ATP and CoA are present, inhibited [1-¹⁴C]acetate uptake and the amount of [¹⁴C]ACh formed from that [1-¹⁴C]acetate. Additionally, vesamicol, an inhibitor of ACh transport into synaptic vesicles, blocked the filling of a synaptic vesicle-enriched fraction of hippocampal tissue with newly synthesized [¹⁴C]ACh formed from extracellular [1-¹⁴C]acetate. High K⁺ depolarization of hippocampal tissue loaded with extracellular [1-¹⁴C]acetate not only increased the synthesis but also the release of [¹⁴C]ACh. These results suggest that extracellular acetate is recycled by rat hippocampal cholinergic nerve terminals for the formation and release of ACh. They also suggest that the enzyme acetyl CoA synthetase mediates extracellular acetate uptake into hippocampal cholinergic nerve terminals by metabolizing it to acetyl CoA and thereby creating a diffusion gradient for it to follow. © 1997 Elsevier Science B.V. All rights reserved.     

Presynaptic inhibitory receptors mediate the depression of synaptic transmission upon hypoxia in rat hippocampal slices

Hypoxia markedly depresses synaptic transmission in hippocampal slices of the rat. This depression is attributed to presynaptic inhibition of glutamate release and is largely mediated by adenosine released during hypoxia acting through presynaptic adenosine A(1) receptors. Paired pulse facilitation studies allowed us to confirm the presynaptic nature of the depression of synaptic transmission during hypoxia. We tested the hypothesis that activation of heterosynaptic inhibitory receptors localized in glutamatergic presynaptic terminals in the hippocampus, namely gamma-aminobutyric acid subtype B (GABA(B)) receptors, alpha(2)-adrenergic receptors, and muscarinic receptors might contribute to the hypoxia-induced depression of synaptic transmission. Field excitatory postsynaptic potentials were recorded in the CA1 area of hippocampal slices from young adult (5-6 weeks) Wistar rats. Neither the selective antagonist for alpha(2)-adrenergic receptors, rauwolscine (10 microM), nor the antagonist for the GABA(B) receptors, CGP 55845 (10 microM), modified the response to hypoxia. The selective adenosine A(1) receptor antagonist, DPCPX (50 nM), reduced the hypoxia-induced depression of synaptic transmission to 59.2+/-9.6%, and the muscarinic receptor antagonist, atropine (10 microM), in the presence of DPCPX (50 nM), further attenuated the depression of synaptic transmission to 49.4+/-8.0%. In the same experimental conditions, in the presence of DPCPX (50 nM), the muscarinic M(2) receptor antagonist AF-DX 116 (10 microM), but not the M(1) receptor antagonist pirenzepine (1 microM), also attenuated the hypoxia-induced depression to 41.6+/-6.6%. Activation of muscarinic M(2) receptors contributes to the depression of synaptic transmission upon hypoxia. This effect should assume particular relevance during prolonged periods of hypoxia when other mechanisms may become less efficient.     

Hyperexcitability in CA1 of the rat hippocampal slice following hypoxia or adenosine

Participation of adenosine receptors in the depression of synaptic transmission during hypoxia, and the production of multiple populations spikes in the pyramidal neurons following hypoxia, has been investigated in the CA₁ area of the rat hippocampal slice. A method is presented for analysing such hyperexcitability, using input/output curves of the second population spike. This method provides evidence that rebound hyperexcitability following hypoxia or prolonged adenosine-mediated inhibition results from an increase in excitability of the CA₁ pyramidal neurons rather than from an increase in excitatory neurotransmitter release. Hypoxia-induced depression of the synaptic components of evoked field potentials was blocked in a concentration dependent manner by the selective A₁ receptor antagonist 8-cyclopenthyltheophylline (8-CPT), demonstrating extracellular accumulation of adenosine during hypoxia. Upon reoxygenation of slices following 30 min hypoxia, multiple population spikes were evoked by a single orthodromic stimulus in slices that exhibited only a single population spike prior to hypoxia. Such post-hypoxic hyperexcitability was not prevented by superfusion of slices with 8-CPT during hypoxia. Depression of synaptic transmission by 30 min superfusion of slices with 50 μM adenosine was also followed, upon washout, by the appearance of multiple population spikes. However, such hyperexcitability could not be produced by superfusion with adenosine analogues selective for A₁ receptors, cyclopentyladenosine, selective for A_2a receptors, 2-p-(2-carboxyethyl)phenetheylamino-5′-ethylcarboxamidoadenosine (CGS 21680), or active at A_2a and A_2b receptors,N⁶-[2-(3,5-dimethyoxyphenyl)-2-(2-methyl-phenyl)ethyl]adenosine, suggesting that adenosine receptors other than the A₁, A_2a or A_2b subtypes are involved in its generation.     

Hydrogen peroxide opposes the hypoxic depression of evoked synaptic transmission in rat hippocampal slices

Hydrogen peroxide (H₂O₂, 3.3 mM) partially reversed the hypoxic depression of the evoked population spike recorded from CA1 region of rat hippocampal slices. It is known that elevated endogenous adenosine contributes to the hypoxic inhibition of the population spike. Exogenous adenosine (100 μM) inhibited the population spike that had been partially resuscitated by H₂O₂ during maintained hypoxia. It is concluded that the ability of H₂O₂ to oppose hypoxic depression does not occur at the level of the adenosine receptor since added adenosine was still effective in inhibiting the evoked potential in the presence of H₂O₂.     

Curcumin inhibits glutamate release in nerve terminals from rat prefrontal cortex: possible relevance to its antidepressant mechanism

There is abundant evidence suggesting the relevance of glutamate to depression and antidepressant mechanisms. Curcumin, a major active compound of Curcuma longa, has been reported to have the biological function of antidepressant. The aim of the present study was to investigate the effect of curcumin on endogenous glutamate release in nerve terminals of rat prefrontal cortex and the underlying mechanisms. The results showed that curcumin inhibited the release of glutamate that was evoked by exposing synaptosomes to the K⁺ channel blocker 4-aminopyridine (4-AP). This phenomenon was blocked by the chelating the extracellular Ca²⁺ ions, and by the vesicular transporter inhibitor bafilomycin A1, but was insensitive to the glutamate transporter inhibitor DL-threo-β-benzyl-oxyaspartate (DL-TBOA). Further experiments demonstrated that curcumin decreased depolarization-induced increase in [Ca²⁺]_C, whereas it did not alter the resting membrane potential or 4-AP-mediated depolarization. Furthermore, the inhibitory effect of curcumin on evoked glutamate release was prevented by blocking the Ca_v2.2 (N-type) and Ca_v2.1 (P/Q-type) channels, but not by blocking intracellular Ca²⁺ release or Na⁺/Ca²⁺ exchange. These results suggest that curcumin inhibits evoked glutamate release from rat prefrontocortical synaptosomes by the suppression of presynaptic Ca_v2.2 and Ca_v2.1 channels. Additionally, we also found that the inhibitory effect of curcumin on 4-AP-evoked glutamate release was completely abolished by the clinically effective antidepressant fluoxetine. This suggests that curcumin and fluoxetine use a common intracellular mechanism to inhibit glutamate release from rat prefrontal cortex nerve terminals.     

Persistent blockade of potassium-evoked serotonin release from rat frontocortical terminals after fluoxetine administration

We examined 5-HT and 5-HIAA release from frontal cortex evoked by high potassium chloride concentrations in rats pretreated for 3 days with high doses of the 5-HT uptake blocker fluoxetine or of dexfenfluramine, which both releases 5-HT and blocks its reuptake. The standard fluoxetine dose (30 mg/kg i.p.) was about 4 times the drug's ED₅₀ in producing a serotonin-related behavioral effect, anorexia, while the dexfenfluramine dose (7.5 mg/kg i.p.) was about 6 times its ED₅₀. These high doses were chosen in order to elucidate the mechanism by which similar doses of fluoxetine and dexfenfluramine had been found to produce long-term changes in serotonin dynamics. Fluoxetine decreased the basal release of both compounds; dexfenfluramine decreased basal 5-HIAA efflux without affecting the release of 5-HT release. Potassium-evoked 5-HT release was unchanged after dexfenfluramine pretreatment but was suppressed by fluoxetine doses as low as 7.5 mg per kg per day. Basal release of 5-HT and 5-HIAA returned to normal after 7 days of fluoxetine pretreatment, but evoked relase continued to be suppressed. These data suggest that long-term changes in brain serotonin dynamics after high doses of dexfenfluramine or fluoxetine are related to the drug's mechanisms of action, specifically their blockade of 5-HT reuptake.     

Subtype-specificity of the presynaptic α2-adrenoceptors modulating hippocampal norepinephrine release in rat

In vivo brain microdialysis and high-performance liquid chromatography with electrochemical detection were used to study the effect of different selective α₂-antagonists on hippocampal norepinephrine (NE) release in freely moving awake rat. Systemic administration (0.5 mg/kg i.p.) of either the α_2AD-antagonist BRL 44408 or the α-_2BC-antagonist ARC 239 did not significantly change the basal release of NE. At a higher dose (5 mg/kg i.p.) ARC 239 was still ineffective, whereas BRL 44408 caused a significant increase of the extracellular level of NE. Similar results were obtained from in vitro perfusion experiments. Rat hippocampal slices were loaded with [³H]NE and the electrical stimulation-evoked release of [³H]NE was determined. The α₂-antagonists were applied in a concentration range of 10⁻⁸ to 10⁻⁶ M. ARC 239 was ineffective, whereas BRL 44408 significantly increased the electrically induced release of [³H]NE. In agreement with the data of microdialysis and perfusion experiments. BRL 44408 displaced [³H]yohimbine from hippocampal and cortical membranes of rat brain with high affinity whereas ARC 239 was less effective. The pK_i values of eight different α₂-adrenergic compounds showed a very good correlation (r = 0.98, slope = 1.11 P < 0.0001) in hippocampus and frontal cortex where the α₂-adrenoceptors have been characterized as α_2D-subtype. Our data indicate that hippocampal NE release in rat is regulated by α_2D-adrenoceptors, a species variation of the human α_2A-subtype.     

Modulation of presynaptic glucocorticoid receptors on glutamate release from rat hippocampal nerve terminals

In this study, we have examined the role of corticosterone (CORT) in the regulation of neuronal glutamate release using nerve terminals (synaptosomes) isolated from the rat hippocampus. Adult male Sprague‐Dawley rats received either a chronic systemic administration of CORT (daily 25 mg/kg in sesame oil, subcutaneously) or long‐term bilateral adrenalectomy (ADX) (3–4 weeks), and then the release of 4‐aminopyridine (4AP)‐evoked endogenous glutamate and the levels of glucocorticoid receptor (GR) expression from hippocampal nerve terminals were studied. Chronic administration of CORT resulted in a significant increase of 4AP‐evoked glutamate release from hippocampal nerve terminals, whereas ADX reduced 4AP‐evoked glutamate release. In addition, chronic administration of CORT and ADX induced a significant reduction and increase in GR expression in hippocampal synaptosomes, respectively, as detected by Western blots. Furthermore, acute treatment of CORT or dexamethasone facilitated 4AP‐evoked glutamate release from synaptosomes freshly isolated from naïve rat hippocampus and this effect can be significantly prevented by pretreatment of GR antagonist mifepristone, but not by mineralocorticoid receptor (MR) antagonist RU28318. Together, our results strongly support the presence of GRs on presynaptic nerve terminals in the rat hippocampus acting to facilitate the release of neuronal glutamate. Synapse 63:745–751, 2009. © 2009 Wiley‐Liss, Inc.     

Inhibition of glutamate release by bupropion in rat cerebral cortex nerve terminals

Central glutamate neurotransmission has been postulated to play a role in pathophysiology of depression and in the mechanism of antidepressants. The present study was undertaken to elucidate the effect and the possible mechanism of bupropion, an atypical antidepressant, on endogenous glutamate release in nerve terminals of rat cerebral cortex (synaptosomes). Result showed that bupropion exhibited a dose-dependent inhibition of 4-aminopyridine (4-AP)-evoked release of glutamate. The effect of bupropion on the evoked glutamate release was prevented by the chelating the intrasynaptosomal Ca²⁺ ions, and by the vesicular transporter inhibitor, but was insensitive to the glutamate transporter inhibitor. Bupropion decreased depolarization-induced increase in [Ca²⁺]_C, whereas it did not alter the resting synaptosomal membrane potential or 4-AP-mediated depolarization. The effect of bupropion on evoked glutamate release was abolished by the N-, P- and Q-type Ca²⁺ channel blocker, but not by the ryanodine receptor blocker, or the mitochondrial Na⁺/Ca²⁺ exchanger blocker. In addition, the inhibitory effect of bupropion on evoked glutamate release was prevented by the mitogen-activated/extracellular signal-regulated kinase kinase (MEK) inhibitors. Western blot analyses showed that bupropion significantly decreased the 4-AP-induced phosphorylation of extracellular signal-regulated kinase 1 and 2 (ERK1/2), and this effect also was blocked by MEK inhibitor. These results are the first to suggest that, in rat cerebrocortical nerve terminals, bupropion suppresses voltage-dependent Ca²⁺ channel and MEK/ERK activity and in so doing inhibits evoked glutamate release. This finding may provide important information regarding the beneficial effects of bupropion in the brain.     

Extracellular calcium alters frequency modulation of [H]acetylcholine release from rat hippocampal slices

The concentration of extracellular Ca²⁺ has been shown to enhance or attenuate [³H]acetylcholine (ACh) release subsequent to a conditioning stimulus in rat brain hippocampal slices. Slices were incubated in vitro in [³H]choline solution. Subsequently the slices were subjected to two consecutive electrical stimulations separated by 15 or 30 min at 0.25, 1, 4 and 16 Hz and [³H]ACh release was assessed. It was found that a conditioning stimulus may reduce [³H]ACh release during a second stimulation. This phenomenon is frequency related and disappears when the two stimulations are 30 min apart. High extracellular Ca²⁺ (4.0 mM) further attenuated [³H]ACh release during the second stimulation whereas low Ca²⁺ (0.32 mM) abolished the decrease in [³H]ACh release following the second stimulation in all frequencies tested.     

Secretion from rat neurohypophysial nerve terminals (neurosecretosomes) rapidly inactivates despite continued elevation of intracellular Ca

Cytoplasmic calcium concentration was measured in neurosecretory nerve terminals (neurosecretosomes) isolated from rat neurohypophyses by fura-2 fluorescence measurements and digital video microscopy. Hormone release and cytoplasmic calcium concentration were measured during depolarizations induced by elevated extracellular potassium concentration. During prolonged depolarizations with 55 mM [K⁺]₀, the cytoplasmic calcium concentration remained elevated as long as depolarization persisted, while secretion inactivated after the initial sharp rise. The amplitude and duration of the increase in [Ca²⁺]_i was dependent on the degree of depolarization such that upon low levels of depolarizations (12.5 mM or 25 mM [K⁺]₀), the calcium responses were smaller and relatively transient, and with higher levels of depolarization (55 mM [K⁺]₀) the responses were sustained and were higher in amplitude. Responses to low levels of depolarization were less sensitive to the dihydropyridine calcium channel blocker, nimodipine, while the increase in [Ca²⁺]_i induced by 55 mM [K⁺]₀ became transient, and was significantly smaller. These observations suggest that these peptidergic nerve terminals possess at least two different types of voltage-gated calcium channels. Removal of extracellular sodium resulted in a significant increase in [Ca²⁺]_i and secretion in the absence of depolarizing stimulus, suggesting that sodium-calcium exchange mechanism is operative in these nerve terminals. Although the [Ca²⁺]_i increase was of similar magnitude to the depolarization-induced changes, the resultant secretion was 10-fold lower, but the rate of inactivation of secretion, however, was comparable.     

Effects of haloperidol and clozapine on glutamate release from nerve terminals isolated from rat prefrontal cortex

The present study was conducted to understand the effect of haloperidol, a typical antipsychotic, and clozapine, an atypical one, on the release of endogenous glutamate in nerve terminals isolated from rat prefrontal cortex using an on-line enzyme-linked fluorometric assay. We found that both haloperidol and clozapine significantly inhibited 4-aminopyridine (4AP)-evoked and veratridine-evoked but not KCl-evoked glutamate release from prefrontocortical synaptosomes. This inhibition produced by these two drugs was concentration-dependent with different potency, and associated with a reduction both in the depolarization-evoked increase in the intrasynaptosomal free Na(+) concentration ([Na(+)](i)) and in 4AP or KCl-evoked depolarization of the synaptosomal plasma membrane potential. Additionally, in the presence of calcium-free medium containing 0.2 mM EGTA, the Ca(2+)-independent component of 4AP-evoked glutamate release was also inhibited by haloperidol or clozapine. Based on these results, we suggest that haloperidol and clozapine inhibit glutamate release from rat prefrontocortical nerve terminals by affecting ion-channel activities determining nerve terminal excitability, probably as a result of Na(+) channel blockage or K(+) channel activation.     

Endogenous adenosine delays the onset of hypoxic depolarization in the rat hippocampus in vitro via an action at A1 receptors

The effect of endogenous adenosine on the delay to hypoxic depolarization (HD) was examined utilizing in vitro slices of gerbil hippocampus. Adenosine receptor antagonists were used to block the actions of adenosine during hypoxia, and the delay to HD was measured in the CA1 region. Both a broad spectrum antagonist (theophylline) and an A₁ receptor-specific antagonist (8-cyclopentyl-1,3-dimethylxanthine; CPT) shortened the delay to HD. These findings indicate that endogenous adenosine working through A₁ receptors prolongs the delay to HD. This effect may contribute to the neuroprotective influence of adenosine and its analogs.     

The effects of manipulation of presynaptic 5-HT nerve terminals on postsynaptic 5-HT1 and 5-HT2 binding sites of the rat brain

Summary The effects of long-term treatment of rats with alaproclate and amiflamine on the number and kinetics of 5-HT₁ and 5-HT₂ binding sites were investigated usingin vitro receptor binding techniques. Some other studies have reported down-regulatory effects of alaproclate and amiflamine on 5-HT₂ binding sites in certain regions of the rat forebrain, but no such effects could be detected in the present study. Induction of a high-affinity binding site for³H-5-HT after long-term antidepressant treatment, as has been reported elsewhere, was not obtained in the present study. The results are compared to the effects obtained by treatment of rats with para-chloroarnphetamine (PCA), which depletes the presynaptic neurons of monoamines. These different types of treatment do not cause any change in the binding properties of the specific 5-HT binding sites. It is thus concluded that such manipulations of the presynaptic 5-HT neurons do not affect the postsynaptic 5-HT₁ and 5-HT₂ binding sites.     

Modulation of acetylcholine and 5-hydroxytryptamine release in hippocampal slices of rats with fimbria-fornix lesions and intrahippocampal grafts containing cholinergic and/or serotonergic neurons

Three-month-old Long-Evans female rats sustained aspirative lesions of the dorsal septohippocampal pathways and, 2 weeks later, received intrahippocampal suspension grafts containing fetal cells from the mesencephalic raphe (rich in serotonergic neurons; RAPHE), the medial septum and the diagonal band of Broca (rich in cholinergic neurons; SEPT), or a mixture of both (COTR). Lesion-only (LES) and sham-operated rats (SHAM) were used as controls. Hippocampal slices of these rats (5-9 month after surgery) were preincubated with [3H]choline or [3H]5-HT, superfused continuously (in the presence of hemicholinium-3 or fluvoxamine) and stimulated electrically (360 pulses, 2 ms, 3 Hz, 26-28 mA) in order to study the presynaptic modulation of acetylcholine (ACh) and serotonin (5-HT) release. The accumulation of [3H]choline and the evoked overflow of [3H]ACh were significantly reduced in slices from LES and RAPHE rats, but reached a close-to-normal level in SEPT and COTR rats. As to accumulation and overflow of [3H]5-HT, the lesion-induced reduction was compensated for only in RAPHE and COTR rats. The relative amount of evoked [3H]5-HT release (in % of tissue-3H) was significantly increased in LES and SEPT rats. Only slight differences (group LES) were found in the sensitivity of muscarinic and serotonergic autoreceptors towards oxotremorine and CP 93,129, respectively. Moreover, CP 93,129 induced a significantly weaker inhibition of ACh release in slices of COTR rats than in all other groups. Using the 5-HT1A receptor agonist 8-OH-DPAT and antagonist Way 100,635, no evidence for a modulatory influence of 5-HT1A receptors was found in RAPHE and COTR rats. It is concluded that despite substantial lesion- and graft-induced changes in the amount of ACh and 5-HT released by hippocampal slices of lesion-only or grafted rats, the presynaptic modulation of these transmitters is only slightly affected by changes in the neuronal environment.     

Consequences of perinatal hypoxia in developing brain: Changes in GABA transporter functioning in cortical,hippocampal and thalamic rat nerve terminals

Perinatal hypoxia leads to behavioral abnormalities, cognitive disabilities, and epilepsy resulting from alterations in neurodevelopment, maturation and construction of the network. Considering a particular role of γ-aminobutyric acid (GABA) for an immature brain, we analysed transporter-mediated [³H]GABA uptake in the cortical, hippocampal and thalamic nerve terminals isolated from rats of different age in the control and after perinatal hypoxia. The state of hypoxia was induced by exposure of rats at the age of 10 postnatal days (pd) (that corresponds approximately to the time of birth in humans) to a respiratory medium with low O₂ content (4% O₂ and 96%N₂) for 12 min (up to the initiation of clonico-tonic seizures). Here, we found that the initial rate of [³Н]GABA uptake was higher in the young rats (pd 17–19) as compared to the older ones (pd 24–26, 38–40 and 66–73) in both control and hypoxia groups. It decreased abruptly by 50% in the thalamus and by 25% in the cortex for the period from pd 17–19 to pd 66–73. In the hippocampus, a decrease in the rate during the same time interval was 25%. Exposure to hypoxia had no effect on the intensity of [³Н]GABA uptake by the cortical and thalamic nerve terminals, but caused a significant age-dependent attenuation (by 35%) of the uptake intensity in the hippocampal ones. Significant age-dependent hypoxia-independent decrease in [³Н]GABA uptake with step-like dynamics of changes was shown in the thalamus and cortex. Gradual age-dependent hypoxia-dependent decrease in [³Н]GABA uptake was revealed in the hippocampus, and so a particular vulnerability of the latest structure to hypoxia as compared to the cortex and thalamus was revealed.     

Facilitation by arachidonic acid of acetylcholine release from the rat hippocampus

We investigated the effect of arachidonic acid (AA) on the release of [3H]acetylcholine ([3H]ACh) from the rat hippocampus. AA (3-30 microM) increased the basal tritium outflow and the field-electrically evoked release of [3H]ACh from hippocampal slices in a concentration-dependent manner. AA (30 microM) produced a 69+/-7% facilitation of the evoked and a 36+/-3% facilitation of basal tritium outflow. The effect of AA (30 microM) on the evoked tritium release was prevented by bovine serum albumin (BSA, 1%), which quenches AA, and was unaffected by the cyclooxygenase inhibitor, indomethacin (100 microM), and the lipooxygenase inhibitor, nordihydroguaiaretic acid (50 microM). Phospholipase A2 (PLA2, 2 U/ml), an enzyme that releases AA from the sn-2 position of phospholipids, mimicked the facilitatory effect of AA on the evoked tritium release (86+/-14% facilitation), an effect prevented by BSA (1%). The PLA2 activator, melittin (1 microM), enhanced the evoked tritium release by 98+/-11%, an effect prevented by the PLA2 inhibitor, arachidonyl trifluromethylketone (AACOCF3, 20 microM), and by BSA (1%). AA (30 microM), but not arachidic acid (30 microM), also facilitated (72+/-9%) the veratridine (10 microM)-evoked [3H]ACh release from superfused hippocampal synaptosomes, whereas PLA2 (2 U/ml) and melittin (1 microM) caused a lower facilitation (46+/-1% and 38+/-5%, respectively). The present results show that both exogenously added and endogenously produced AA increase the evoked release of [3H]ACh from rat hippocampal nerve terminals. Since muscarinic activation triggers AA production and we now observed that AA enhances ACh release, it is proposed that AA may act as a facilitatory retrograde messenger in hippocampal cholinergic muscarinic transmission as it has been proposed to act in glutamatergic transmission.