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.     

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.     

Endogenous adenosine facilitates neurotransmission via A2A adenosine receptors in the rat superior colliculus in vivo

The concentration of endogenous adenosine in the cerebrospinal fluid increased 2–3-fold of the original level in the area of rat superior colliculus after the intraperitoneal administration of an adenosine deaminase inhibitor, EHNA (erythro-9-(2-hydroxy-3-nonyl)adenosine, 10 mg/kg). Potentials evoked in the superior colliculus by optic tract stimulation were also facilitated by 120–160% of their initial amplitudes. A selective A₁ adenosine receptor antagonist, DPCPX (8-cyclopentyl-1,3-dipropylxanthine), failed to reduce such EHNA-induced facilitation. However, a selective A_2A adenosine receptor antagonist, KF17837 (8(3,4-dimethoxystyryl)-1,3-dipropyl-7-methylxanthine) completely eliminated the facilitatory effects of EHNA. Northern blot analysis demonstrated abundant expression of A₁ adenosine receptor mRNA in the superior colliculus. RT-PCR analysis was able to detect the concomitant expression of A_2A adenosine receptor mRNA, but at levels lower than one-tenth of the striatal expression. In the superior colliculus, A_2A adenosine receptors function predominantly on the facilitatory effects of adenosine, irrespective of the ubiquitous expression of A₁ adenosine receptors.     

Diadenosine polyphosphates facilitate the evoked release of acetylcholine from rat hippocampal nerve terminals

Diadenosine polyphosphates are present in synaptic vesicles, are released upon nerve stimulation and possess membrane receptors, namely in presynaptic terminals. However, the role of diadenosine polyphosphates to control neurotransmitter release in the CNS is not known. We now show that diadenosine pentaphosphate (Ap(5)A, 3-100 microM) facilitated in a concentration dependent manner the evoked release of acetylcholine from hippocampal nerve terminals, with a maximal facilitatory effect of 116% obtained with 30 microM Ap(5)A. The selective diadenosine polyphosphate receptor antagonist, diinosine pentaphosphate (Ip(5)I, 1 microM), inhibited by 75% the facilitatory effect of Ap(5)A (30 microM), whereas the P(2) receptor antagonists, suramin (100 microM) and pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS, 10 microM) only caused a 18-24% inhibition, the adenosine A(1) receptor antagonist, 1,3-dipropyl-8-cyclopentylxanthine (20 nM), caused a 36% inhibition and the adenosine A(2A) 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, 20 nM), was devoid of effect. These results show that diadenosine polyphosphates act as neuromodulators in the CNS, facilitating the evoked release of acetylcholine mainly through activation of diadenosine polyphosphate receptors.     

Potential use of DPCPX as probe for in vivo localization of brain A1 adenosine receptors

The suitability of (³H)DPCPX (8-cyclopentyl-1,3-dipropylxanthine), a xanthine derivative, as an vivo probe for labelling adenosine A₁ receptors was studied in rats. [³H]DPCPX (nM) penetrated largely into the brain (0.8% of the injected dose per gram of brain tissue 5 min after injection). Brain concentrations stayed at a plateau level from 5 to 15 min after the injection. The distribution in the different brain regions was heterogeneous with the highest amount of [³H]DPCPX in cerebellum and hippocampus and the lowest concentrations in hypothalamus and brain stem. Displacement (45–70% of total radioactivity) was obtained by the injection of 250 nM of cold DPCPX or cyclopentylxanthine, an analog of DPCPX. The ex vivo autoradiographic distribution of [³H]DPCPX was similar to the in vitro autoradiographic distribution of tritiated A₁ adenosine receptor ligand as [³H]CHA. These results suggest the potential use of DPCPX for further in vivo investigation of A₁ adenosine receptors with techniques such as positron emission tomography.     

[I]4-Aminobenzyl-5′-N-methylcarboxamidoadenosine ([I]AB-MECA) labels multiple adenosine receptor subtypes in rat brain

Adenosine modulates neuronal activity and neurotransmitter release through interaction with cell surface receptors. Four adenosine receptor subtypes, A₁, A_2A, A_2B, and A₃ receptors, have been cloned and characterized. The agonist ligand, [¹²⁵I]AB-MECA ([¹²⁵I]4-aminobenzyl-5′-N-methylcarboxamidoadenosine) has high affinity for recombinant A₁ and A₃ receptors [Olah et al., Mol. Pharmacol., 45 (1994) 978–982]. Rodent A₃ receptors are relatively insensitive to xanthines; inhibition of A₁ receptors with xanthines allows selective detection of A₃ receptors despite the lack of selectivity of the ligand. We studied whether [¹²⁵I]AB-MECA is useful for localization and characterization of A₃ receptors in rat brain. The autoradiographic distribution of total [¹²⁵I]AB-MECA (400 pM) binding closely resembled the pattern of A₁ receptor binding, with highest levels in cerebellum, hippocampus, and thalamus, and moderate levels in cortex and striatum. Drug competition studies confirmed that almost all [¹²⁵I]AB-MECA binding could be attributed to labeling of A₁ receptors. Xanthine amine congener (1 μM) reduced specific [¹²⁵I]AB-MECA binding by >95%, indicating that xanthine-resistant A₃ receptors represent a quantitatively minor subtype. Despite the use of a radioligand with high affinity and high specific activity, the low density of A₃ receptors in rat brain appears insufficient to allow localization, or even consistent detection, of this receptor subtype. In the presence of DPCPX (50 nM, to block A₁ receptors), residual [¹²⁵I]AB-MECA binding to A_2A receptors was observed in the striatum. Thus, [¹²⁵I]AB-MECA labels primarily A₁ and A_2A adenosine receptors in rat brain.     

Adenosine A1 receptors are crucial in keeping an epileptic focus localized

Adenosine is an endogenous neuromodulator with anticonvulsant and neuroprotective properties presumably mediated by activation of adenosine A1 receptors (A1Rs). To study the involvement of A1Rs in neuroprotection during epileptogenesis, we induced status epilepticus by a unilateral intrahippocampal kainic acid (KA) injection (1 nmol) in wild-type C57BL/6 and homozygous adenosine A1R knock out (A1R-KO) mice of the same genetic background. Whereas the KA injection caused non-convulsive status epilepticus in wild-type mice, in A1R-KO mice KA induced status epilepticus with severe convulsions and subsequent death of the animals within 5 days. 24 h after KA injection, brains from wild-type C57BL/6 mice were characterized by slight neuronal cell loss confined to the immediate location of the KA injection. In contrast, KA-injected A1R-KO mice displayed massive neuronal cell loss in the ipsilateral hippocampus, and, importantly, the contralateral hippocampus was also affected with significant cell loss in the hilus and in the CA1 region of the pyramidal cell layer. We conclude that activation of A1 receptors by ambient adenosine is crucial in keeping epileptic foci localized. These results open up a new dimension of the A1 receptor's role in controlling excitotoxic cell death and further demonstrate its importance in preventing the progression of status epilepticus to lethal consequences.     

Functional changes in postsynaptic adenosine A(2A) receptors during early stages of a rat model of Huntington disease

Huntington disease (HD) is a neurodegenerative disorder involving preferential loss of striatal GABAergic medium spiny neurons. Adenosine A_2A receptors (A_2ARs) are present in the striatum at both presynaptic and post-synaptic levels. Blocking pre-synaptic A_2ARs, localized in glutamatergic terminals that contact striatal GABAergic dynorphinergic neurons, reduces glutamate release, which could be beneficial in HD. On the other hand, blockade of post-synaptic A_2ARs, localized in striatal GABAergic enkephalinergic neurons, could exacerbate the motor dysfunction. To evaluate the function of pre- or post-synaptic A_2ARs in HD we used selective antagonists for these receptors in a transgenic rat model of HD. Locomotor activity after systemic administration of the postsynaptic A_2AR antagonist KW-6002 was used to investigate the function of post-synaptic A_2ARs. The role of pre-synaptic A_2ARs was instead evaluated by measuring the reduction of the electromyographic response of mastication muscles during electrical stimulation of the orofacial motor cortex after the systemic administration of the presynaptic A_2AR antagonist SCH-442416. The ability of KW-6002 to produce locomotor activation was lost at 6 and 12 month-old of age in heterozygous and homozygous transgenic rats, but not in wild-type littermates. Nevertheless, no significant changes were observed up to 12 months of age in the potency of SCH-442416 to decrease the electromyographic response after cortical electrical stimulation. These results agree with a selective impairment of the striatal GABAergic enkephalinergic neuronal function during pre-symptomatic stages in HD. Since presynaptic A_2AR function is not impaired, this receptor could probably be used as a target for the symptomatic treatment of the disease.     

Functional effects of chronic electroconvulsive shock on serotonergic 5-HT(1A) and 5-HT(1B) receptor activity in rat hippocampus and hypothalamus

The aims of this work were to determine the influence of chronic electroconvulsive shock (ECS) on presynaptic 5-HT(1A) receptor function, postsynaptic 5-HT(1A) receptor function in hippocampus and hypothalamus, and presynaptic 5-HT(1B) receptor function in hippocampus and hypothalamus. This represents part of an on-going study of the effects of ECS on serotonergic receptor activity in selected brain areas which may be relevant to the effects of electroconvulsive therapy (ECT) in humans. Chronic ECS reduced the ability of the 5-HT(1A) receptor agonist 8-hydroxy-2(di-n-propylamino)tetraline (8-OH-DPAT) (0.2 mg/kg s.c.) to decrease 5-HT levels in hypothalamus as shown by in vivo microdialysis, indicative of a reduction in sensitivity of presynaptic 5-HT(1A) autoreceptors. The ability of the 5-HT(1B) receptor antagonist GR 127935 (5 mg/kg s.c.) to increase 5-HT levels in both hippocampus and hypothalamus was unaffected by chronic ECS. 8-OH-DPAT (0.2 mg/kg s.c.) increased cyclic AMP levels in hippocampus measured by in vivo microdialysis approximately 2-fold. The degree of stimulation of cyclic AMP formation was not altered by chronic ECS. However the cyclic AMP response to forskolin (50 micro M) administered via the microdialysis probe, which was approximately 4-fold of basal in sham-treated rats, was almost completely abolished in ECS-treated rats. Since this indicates that either adenylate cyclase catalytic unit activity or Gs protein activity is reduced in the hippocampus after chronic ECS, the lack of change in 8-OH-DPAT-induced cyclic AMP formation may be taken as possible evidence of an increase in sensitivity of postsynaptic 5-HT(1A) receptors in the hippocampus by chronic ECS. Chronic ECS increased basal plasma levels of corticosterone, ACTH and oxytocin. The ACTH response to s.c. injections of 0.2 mg/kg or 0.5 mg/kg 8-OH-DPAT was reduced by chronic ECS. Postsynaptic 5-HT(1A) receptor activity in the hypothalamus, in contrast to the hippocampus, thus appears to be desensitized after chronic ECS. We conclude that chronic ECS has regionally specific effects on both pre- and post-synaptic 5-HT(1A) receptors, but, in contrast to some antidepressant drugs, does not affect presynaptic 5-HT(1B) receptor activity.     

The calcium-dependent [H]acetylcholine release from synaptosomes of brown trout (Salmo trutta) optic tectum is inhibited by adenosine A1 receptors: Effects of enucleation on A1 receptor density and cholinergic markers

Presynaptic inhibition is one of the major control mechanisms in the CNS. Previously we reported that A₁ adenosine receptors are highly concentrated in the brain, including optic tectum, of trout and that they inhibited the release of glutamate. The optic tectum is heavily innervated by cholinergic nerve terminals. We have investigated whether A₁ receptors inhibit the presynaptic release of acetylcholine and whether the inhibition is triggered by calcium. The release of [³H]ACh evoked by 30 mM KCl was Ca²⁺ dependent and it was dose-dependently inhibited by the A₁ adenosine receptor agonist 2-chloro-N⁶-cyclopentyladenosine (CCPA) ranging between 10 nM to 100 μM. The maximum of inhibition was reached at 10 μM. The A₁ receptor antagonist 8-cyclopentyltheopylline (CPT, 10 μM), reversed almost completely the inhibition induced by CCPA 10 μM. In Fura-2/AM loaded synaptosomes, K⁺ depolarization raised [Ca²⁺]_i by about 64%. CCPA (10 μM) reduced the K⁺-evoked Ca²⁺ influx increase by about 48% and this effect was completely antagonised by CPT 10 μM. Synaptosome pretreatment with different Ca²⁺ channel blockers differently affected K⁺-evoked Ca²⁺ influx. This was not significantly modified by nifedipine (1 μM, L-type blocker) nor by ω-agatoxin IVA (0.3 μM, P/Q-type blocker), whereas about 50% reduction was shown by 0.5 μM ω-conotoxin GVIA (N-type blocker). Neurochemical parameters associated with cholinergic transmission and the density of A₁ adenosine receptors were measured in the trout optic tectum 12 days after unilateral eye ablation. A significant drop of both acetylcholinesterase (AChE) activity (24%) and choline acetyltransferase (CAT) activity (32%) was observed in deafferentated optic tectum, whereas the high affinity choline uptake did not parallel the decrease in enzyme activity. Eye ablation caused a marked decrease (43%) of A₁ receptor density without changing the affinity. The K⁺-evoked release of [³H]ACh from synaptosomes of deafferentated was not modify as well as the efficacy of 10 μM CCPA in decreasing [³H]ACh release was not apparently modified.     

Involvement of adenosine A2A and dopamine receptors in the locomotor and sensitizing effects of cocaine

Recent data indicate that cocaine locomotor responses may be influenced by dopamine (DA) neurotransmission and adenosine neuromodulation involving the A2A receptor (A2AR). Male Wistar rats were injected with MSX-3 (1-25 mg/kg; an antagonist of A2AR), CGS 21680 (0.05-0.2 mg/kg; an agonist of A2AR), SCH 23390 (0.125-0.25 mg/kg; an antagonist of DA D1/5R), raclopride (0.1-0.8 mg/kg; an antagonist of DA D2/3R), nafadotride (0.2-0.4 mg/kg; an antagonist of DA D3R) or 7-OH-PIPAT (0.01-1 mg/kg; an agonist of DA D3R) to verify the hypothesis that adenosine A2AR and DA receptors and their antagonistic interactions may control locomotor and sensitizing effects of cocaine. In well-habituated animals, MSX-3 (5 mg/kg) increased, while raclopride (0.4-0.8 mg/kg) decreased basal locomotor activation; the other drugs were inactive. The locomotor hyperactivation induced by acute cocaine (10 mg/kg) was enhanced by MSX-3 (5-25 mg/kg) or nafadotride (0.4 mg/kg), while CGS 21680 (0.2 mg/kg), SCH 23390 (0.25 mg/kg), raclopride (0.2-0.8 mg/kg) or 7-OH-PIPAT (0.1 mg/kg) decreased this effect of cocaine. Given during the development of sensitization (in combination with 5-daily cocaine, 10 mg/kg, injections), MSX-3 (5-25 mg/kg) increased, but CGS 21680 (0.2 mg/kg) and raclopride (0.8 mg/kg) reduced the locomotor response to a cocaine challenge dose (10 mg/kg) on day 10. When injected acutely with a cocaine challenge dose (on day 10), CGS 21680 (0.2 mg/kg), raclopride (0.2-0.8 mg/kg) or 7-OH-PIPAT (1 mg/kg) reduced, while MSX-3 (5 mg/kg) or nafadotride (0.4 mg/kg) enhanced the expression of cocaine sensitization. The present results show that adenosine A2ARs and DA D3Rs exert inhibitory actions on acute locomotor responses to cocaine and on the expression of cocaine sensitization, while DA D2Rs had an opposing role in such effects. Pharmacological stimulation of adenosine A2ARs protected against both the development and expression of cocaine sensitization, which may offer a therapeutic potential of A2AR agonists in the treatment of cocaine dependence. The results suggest an antagonistic role of A2ARs in D2R-mediated cocaine actions based at least in part on the existence of A2A/D2 heteromeric receptor complexes.     

Computer-assisted image analysis of caveolin-1 involvement in the internalization process of adenosine A2A-dopamine D2 receptor heterodimers

A functional aspect of horizontal molecular networks has been investigated experimentally, namely the heteromerization between adenosine A_2A and dopamine D₂ receptors and the possible role of caveolin-1 in the cotrafficking of these molecular complexes. This study has been carried out by means of computer-assisted image analysis procedure of laser images of membrane immunoreactivity of caveolin-1, A_2A, D₁, and D₂ receptors obtained in two clones of Chinese hamster ovary cells—one transfected with A_2A and dopamine D₁ receptors and the other one with A_2A and D₂ receptors. Cells were treated for 3 h with 10 μM D₁ receptor agonist SKF 38393, 50 μM D₂-D₃ receptor agonist quinpirole, and 200 nM A_2A receptor agonist CGS 21680. In A_2A-D₁-cotransfected cells, caveolin-1 was found to colocalize with both A_2A and D₁ receptors and treatment with SKF 38393 induced internalization of caveolin-1 and D₁ receptors, with a preferential internalization of D₁ receptors colocalized with caveolin-1. In A_2A-D₂-cotransfected cells, caveolin-1 was found to colocalize with both A_2A and D₂ receptors and either CGS 21680 or quinpirole treatment induced internalization of caveolin-1 and A_2A and D₂ receptors, with a preferential internalization of A_2A and D₂ receptors colocalized with caveolin-1. The results suggest that A_2A and D₂ receptors and caveolin-1 likely interact forming a macrocomplex that internalizes upon agonist treatment. These observations are discussed in the frame of receptor oligomerization and of the possible functional role of caveolin-1 in the process of co-internalization and, hence, in controlling the permanence of receptors at the plasma membrane level (prerequisite for receptor mosaic organization and plastic adjustments) and in the control of receptor desensitization. This paper is dedicated to Luciano Martini, Professor of Endocrinology in Milano and to Faustino Savoldi, Professor of Neurolgy in Pavia. Department of Human Anatomy and Physiology, Section of Antomy, University of Padova, 35121 Padova, Italy     

Involvement of the cerebellar adenosine A1 receptor in cannabinoid-induced motor incoordination in the acute and tolerant state in mice

Cannabinoids are known to impair motor function in humans and laboratory animals. We have demonstrated an accentuation of cannabinoid (CP55,940)-induced motor incoordination in mice by the adenosine A₁ receptor-selective agonist N⁶-cyclohexyladenosine (CHA) (4 ng) using an intracerebellar (ICB) microinjection method. This effect was mediated by the A₁ receptor because pre-treatment with ICB 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) (100 ng), an adenosine A₁ receptor selective antagonist, completely abolished the accentuation. Furthermore, ICB pre-treatment with DPCPX (100 ng) before ICB CP55,940 (15 μg) attenuated the motor incoordination suggesting a modulation by an endogenous adenosine A₁ system. ICB microinjection of CHA or DPCPX prior to ICB vehicle had no effect on normal motor coordination. ICB microinjection of dipyridamole (25 μg), an adenosine transport inhibitor, significantly accentuated the motor incoordination by ICB CP55,940 (15 μg), providing further support for the involvement of endogenous adenosine in the action of CP55,940. Tolerance to the motor incoordinating effect of ICB CP55,940 was demonstrated following 3 days of i.p. CP55,940 (0.1, 1 or 2 mg/kg every 12 or 24 h; total of six or three injections, respectively). Interestingly, animals which exhibited tolerance to ICB CP55,940 also demonstrated tolerance to the accentuating effect of ICB CHA suggesting cross-tolerance between adenosine agonists and cannabinoids. Cross-tolerance was also demonstrated following 3 days of i.p. CHA (0.25 or 1 mg/kg every 24 h; total of three injections) as further evidence of the modulatory role of the cerebellar adenosine system in the acute manifestation of CP55,940-induced motor incoordination. The involvement of cerebellar adenosine and the A₁ receptor in cannabinoid actions is circumstantially supported by previous evidence that CB₁ receptors and A₁ receptors are both localized on cerebellar granule cell parallel fiber terminals and basket cell neurons where they serve to inhibit the release of neurotransmitters.     

KATP channel blockers selectively interact with A1-adenosine receptor mediated modulation of acetylcholine release in the rat hippocampus

In this study the role of ATP-sensitive K⁺ channels (K_ATP channels) in the A₁ receptor mediated presynaptic inhibitory modulation of acetylcholine release was investigated in the rat hippocampus. N⁶-Cyclohexyladenosine (CHA), the selective A₁-adenosine receptor agonist, reduced concentration-dependently the stimulation-evoked (2 Hz, 1 ms, 240 shocks) [³H]acetylcholine ([³H]ACh) release, from in vitro superfused hippocampal slices preloaded with [³H]choline, an effect prevented by the selective A₁ receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX). By themselves, neither K_ATP channel openers, i.e. diazoxide, pinacidil and cromakalim, nor glibenclamide and glipizide, the inhibitors of K_ATP channels, exerted a significant effect on the resting and evoked release of [³H]ACh. Glibenclamide and glipizide (10–100 μM) completely prevented the inhibitory effect of 0.1 μM CHA and shifted the concentration response curve of CHA to the right. 4-Aminopyridine (10–100 μM), the non-selective potassium channel blocker, increased the evoked release of [³H]ACh, but in the presence of 4-aminopyridine, the inhibitory effect of CHA (0.1 μM) still persisted. Oxotremorine, the M₂ muscarinic receptor agonist, decreased the stimulation-evoked release of [³H]ACh, but its effect was not reversed by glibenclamide. 1,3-Diethyl-8-phenylxanthine (DPX), the selective A₁-antagonist, effectively displaced [³H]DPCPX in binding experiments, while in the case of glibenclamide and glipizide, only slight displacement was observed. In summary, our results suggest that K_ATP channels are functionally coupled to A₁ receptors present on cholinergic terminals of the hippocampus, and glibenclamide and glipizide, by interacting with K_ATP channels, relieve this inhibitory neuromodulation.     

Depolarization-dependent actions of dihydropyridines on synaptic transmission in the in vitro rat hippocampus

Field potential and intracellular recordings were obtained in the in vitro hippocampal slice to study the effects on synaptic transmission of dihydropyridine (DHP) derivatives. Nimodipine or nifedipine by itself had little effect upon the postsynaptic response as determined by field potential analysis. However, facilitation became evident when DHP application was coupled with manipulations which induced a moderate degree of membrane depolarization. In accordance with the hydrophobic nature of these compounds, extensive washing in normal Krebs' solution failed to reverse the facilitation indicating that the DHP effects outlasted the induced depolarization. Nifedipine is photolabile and its actions were reversed when intense light was applied to the slice. Application of the DHP Bay K 8644, resulted in a similar depolarization-dependent increase in neuronal excitability which, upon washout and exposure to light, was at first attenuated and then reversed, resulting in a long-lasting depression of the EPSP that was sensitive to caffeine. This depressant action of Bay K 8644 appeared to be mediated at a site presynaptic to the pyramidal cell because the postsynaptic component of the field potential response to pulsed applications of glutamate was not altered. Intracellular recording from CA1 neurons supports a presynaptic locus for the depressant actions of Bay K 8644; spike threshold for synaptically evoked responses was greatly increased while spike threshold to direct depolarization of the soma was unchanged. These results indicate that DHPs can exert effects on synaptic transmission in hippocampal brain slice under conditions of moderate membrane depolarization.     

The calcium channel antagonist, ω-conotoxin, and electric organ nerve terminals: binding and inhibition of transmitter release and calcium influx

We have previously shown that the calcium channel antagonist ω-conotoxin M-VII-A blocks neurotransmitter release from isolated nerve terminals (synaptosomes) from the electric organ of the electric ray (Yeager et al., J. Neurosci., 7 (1987) 2390–2396). We now demonstrate that a related but more readily available peptide, ω-conotoxin G-VI-A (CgTx), also blocks the release of transmitter from these terminals and, in addition, inhibits depolarization-dependent uptake of Ca²⁺ into these terminals. The half-maximal inhibitory concentration (IC₅₀ for block of depolarization-evoked release and for depolarization-dependent uptake of Ca²⁺ are approximately 3 and 2 μM, respectively. These results suggest the inhibitory effects of CgTx are due to inhibition of Ca²⁺ entry into synaptosomes through voltage-sensitive calcium channels. Assays of radioiodinated CgTx binding to electric organ synaptosomal membranes and synaptosomes appear to show a single binding site with a apparent dissociation constant (K_d of 3–5 μM and toxin receptor densities of 290 and 52 pmol/mg protein, respectively. These CgTx receptor densities are equivalent to 6% of the total synaptosomal membrane protein and 1% of the total synaptosomal protein (assuming a molecular weight of 200 kDa for the toxin receptor). If the observed CgTx receptor densities reflect the actual densities of voltage-sensitive calcium channels in electric organ synaptosomal membranes and synaptosomes, these preparations would be the richest source of these channels yet described.     

Gene expression of cyclooxygenase-1 and Ca(2+)-independent phospholipase A(2) is altered in rat hippocampus during normal aging

Brain aging is associated with inflammatory changes. However, data on how the brain arachidonic acid (AA) metabolism is altered as a function of age are limited and discrepant. AA is released from membrane phospholipids by phospholipase A(2) (PLA(2)) and then further metabolized to bioactive prostaglandins and thromboxanes by cyclooxygenases (COX)-1 and -2. We examined the phospholipase A(2) (PLA(2))/COX-mediated AA metabolic pathway in the hippocampus and cerebral cortex of 4-, 12-, 24- and 30-month-old rats. A two-fold increase in brain thromboxane B(2) level in 24 and 30 months was accompanied by increased hippocampal COX-1 mRNA levels at 12, 24, and 30 months. COX-2 mRNA expression was significantly decreased only at 30 months. Hippocampal Ca(2+)-independent iPLA(2) mRNA levels were decreased at 24 and 30 months without any change in Ca(2+)-dependent PLA(2) expression. In the cerebral cortex, mRNA levels of COX and PLA(2) were not significantly changed. The specific changes in the AA cascade observed in the hippocampus may alter phospholipids homeostasis and possibly increase the susceptibility of the aging brain to neuroinflammation.     

Effects of angiotensin III and angiotensin IV on pentylenetetrazol seizure susceptibility (threshold and kindling): interaction with adenosine A1 receptors

The effects of angiotensin (ANG) III and ANG IV on pentylenetetrazol (PTZ) seizure susceptibility—threshold and kindling in mice—as well as the influence of adenosine A₁ receptor agents (agonist and antagonist) on these effects were studied. It was found that ANG III and ANG IV increased dose-dependently the PTZ seizure threshold and decreased the seizure intensity in PTZ kindled mice. Cyclohexyladenosine (CHA), an adenosine A₁ receptor agonist, potentiated the effects of ANG III and ANG IV on the seizure threshold and kindling, whereas DPCPX (an A₁ receptor antagonist) reversed peptide-induced effects on the PTZ kindling. Taken together, ANG III and ANG IV decrease the PTZ seizure susceptibility. We could suggest that these effects are realized in part through interaction with adenosine A₁ receptors.     

Effects of chronic administration of caffeine on adenosine A1 and A2 receptors in rat brain

Chronic administration of caffeine (75 mg/kg/day) to rats for 12 days increased [3H]R-PIA binding in the cerebral cortex and cerebellum and [3H]NECA binding to high affinity receptor sites in the striatum. The results indicate that both adenosine A1 and A2 receptor subtypes possess mechanisms of adaptation to chronic caffeine treatment. In addition, adenosine A1 receptor binding shows heterogenous neuroanatomical pattern indicating that the A1 response to caffeine treatment presents regional variation in the rat brain.     

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.