Modulation of neuronal excitability by endogenous adenosine in the absence of synaptic transmission

Rat hippocampal slices were superfused with low calcium, high magnesium medium. Reductions in flow rate were associated with a marked depression of antidromically elicited afterpotentials with little change in the initial antidromic population spike recorded from CA1 pyramidal neurons. The depression of the afterpotential at the lower flow rates was largely reversed by the adenosine antagonist, theophylline (100 μM), by adenosine deaminase (10 μg/ml) and was mimicked by the application of the adenosine reuptake blocker, dipyridamole (100 μM). Since synaptic transmission was blocked, it is concluded that sufficient endogenous adenosine exists in the absence of synaptic function to alter neuronal excitability.     

Adenosine A1 receptor-mediated inhibition of evoked glutamate release is coupled to calcium influx decrease in goldfish brain synaptosomes

Binding of [³H]cyclohexyladenosine (CHA) to the cellular fractions and P₂ subfractions of the goldfish brain was studied. The A₁ receptor density was predominantly in synaptosomal membranes. In goldfish brain synaptosomes (P₂), 30 mM K⁺ stimulated glutamate, taurine and GABA release in a Ca²⁺-dependent fashion, whereas the aspartate release was Ca²⁺-independent. Adenosine, R-phenylisopropyladenosine (R-PIA) and CHA (100 μM) inhibited K⁺-stimulated glutamate release (31%, 34% and 45%, respectively). All of these effects were reversed by the selective adenosine A₁ receptor antagonist, 8-cyclopentyltheophylline (CPT). In the same synaptosomal preparation, K⁺ (30 mM) stimulated Ca²⁺ influx (46.8±6.8%) and this increase was completely abolished by pretreatment with 100 nM ω-conotoxin. Pretreatment with 100 μM R-PIA or 100 μM CHA, reduced the evoked increase of intra-synaptosomal Ca²⁺ concentration, respectively by 37.7±4.3% and 39.7±9.0%. A possible correlation between presynaptic A₁ receptor inhibition of glutamate release and inhibition of calcium influx is discussed.     

Prostacyclin release from the coronary vascular wall by vasoactive substances

Which vasoactive substances that are synthesized in vivo could induce the release of a sufficient amount of prostacyclin (PGI₂) to inhibit platelet aggregation from the vascular wall was investigated in the isolated dog heart perfused by a modified method of Langendorff. Infusion of 5 μM bradykinin or 25 u/ml crude thrombin into the heart for 30 sec resulted in the transient appearance of inhibitory activity of platelet aggregation. The inhibitory activity was stable at alkaline pH but unstable at acidic pH and thermolabile. The appearance of the inhibitory activity was prevented by treatment of the coronary vessel with 30 μM indomethacin or 1 mM tranylcypromine. These results indicated that the inhibitory activity was caused by PGI2. When 25 μM acetylcholine, 25 μM noradrenaline, 25 μM isoproterenol, 10 μM adenosine triphosphate (ATP 5 μM adenosine, 1 μM angiotensin II, 25 μM histamine or 1 μM serotonin was infused for 30 sec, no inhibitory activity of platele aggregation was observed. Bradykinin (5 × 10⁻⁹ 5 × 10⁻⁶ M) and purified thrombin (1 × 10⁻⁹ 1 × 10⁻⁷ M) induced a dose-dependent release of PGI₂ which was assayed using a radioimmunoassay for 6-keto-prostaglandin F₁ (6-keto-PGF₁).     

Rhythm generation in organotypic medullary cultures of newborn rats

Organotypic transverse medullary slices (obex level) from six-day-old rats, cultured for two to four weeks in chemically defined medium contained rhythmically discharging neurones which were activated by CO₂ and H⁺. The mechanisms underlying this rhythmicity and the spread of excitation and synaptic transmission within this organotypic tissue were examined by modifying the composition of the external solution. Our findings showed that (1) Exposure to tetrodotoxin (0.2 μM) or to high magnesium (6 mM) and low calcium (0.2 mM) concentrations abolished periodic activity. (2) Neither the blockade of GABAergic potentials with bicuculline methiodide (200 μM) and/or hydroxysaclofen (200 μM) nor the blockade of glycinergic potentials with strychnine hydrochloride (100 μM) abolished rhythmicity. (3) While atropine sulphate (5 μM) was ineffective in modulating periodic discharges nicotine (100 μM) — like CO₂ — shortened the intervals between the periodic events; hexamethonium (50–100 μM) reduced both periodic and aperiodic activity. (4) Exposure to the NMDA antagonist 2-aminophosphonovaleric acid (50 μM) suppressed periodic events only transiently. In the presence of 2-aminophosphonovaleric acid rhythmicity recovered. However, the AMPA-antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (10–50 μM), abolished periodic activity reversibly within less than 5 min. When 6-cyano-7-nitroquinoxaline-2,3-dione and nicotine were administered simultaneously periodic events persisted for up to 10 min. These findings indicate that synaptic excitatory drive is a prerequisite for the generation of rhythmic discharges of medullary neurones in this preparation. This drive may activate voltage-dependent channels or it may facilitate endogenous cellular mechanisms which initiate oscillations of intracellular calcium concentration. To test the latter possibility (5) calcium antagonists were added to the bath saline. The organic calcium antagonists verapamil and flunarizine (50–100 μM each) and the inorganic calcium antagonists cobalt (2 mM) and magnesium (6 mM) suppressed periodic activity and abolished or weakened the chemosensitivity towards CO₂/acidosis. (6) Dantrolene (10 μM), an inhibitor of intracellular calcium release decreased the periodicity, while thapsigargin (2 μM) which blocks endoplasmic Ca²⁺-ATPase, transiently accelerated the occurrence of periodic events. (7) Oscillations of intracellular free calcium concentrations in Fura-2 AM-loaded cells were weakened or abolished by cobalt (2 mM). The results of (5)–(7) indicate that transmembrane calcium fluxes as well as intracellular Ca²⁺-release and -clearance mechanisms are a prerequisite for intracellular free calcium oscillations which may be important in the generation of rhythmic discharges in medullary neurones.     

Partial antagonism of 8-OH-DPAT'S effects on male rat sexual behavior with a D2, but not a 5-HT1A, antagonist

The serotonin agonist 8-hydroxy-di-propylaminotetralin (8-OH-DPAT), injected systemically or directly into the medial preoptic area (MPOA), reduces the ejaculatory threshold in male rats. While 8-OH-DPAT has been characterized as an agonist at the 5-HT_1A receptor, it also acts at other receptor sites including the dopamine D₂ receptor. The current experiments investigated whether 8-OH-DPAT injected into the MPOA facilitates male sexual behavior through stimulation of the 5-HT_1A receptor or the dopamine D₂ receptor. Experiment 1 co-administered 8-OH-DPAT (6 μg) with either the 5-HT_1A antagonist 4-iodo-N-[2-[4-(methoxyphenyl)-1-piperazinyl]ethyl]-N-2-pyridinyl-benzamide hydrochloride (MPPI) (10 μg) or the D₂ antagonist raclopride (10 μg). Raclopride blocked 8-OH-DPAT's facilitative effects on ejaculation frequency and latency, while the 5-HT_1A antagonist was ineffective. In Experiment 2, 8-OH-DPAT (500 μM), retrodialyzed into the MPOA through a microdialysis probe, enhanced male copulatory behavior similarly to the microinjection, increasing ejaculation frequency and decreasing ejaculation latency, postejaculatory interval and mount frequency. Retrodialyzing 8-OH-DPAT through a microdialysis probe in the MPOA had been previously shown to increase extracellular levels of dopamine and serotonin. The data from the present studies suggest that the effects of 8-OH-DPAT in the MPOA on male rat copulatory behavior may be mediated, at least in part, either directly through 8-OH-DPAT's activity at D₂ receptors or indirectly through 8-OH-DPAT's ability to increase extracellular dopamine.     

Involvement of the 12-lipoxygenase pathway of arachidonic acid metabolism in homosynaptic long-term depression of the rat hippocampus

Low-frequency stimulation is associated with long-term depression (LTD) of synaptic efficacy in various brain structures. Like long-term potentiation (LTP), homosynaptic LTD in area CA1 of the hippocampus appears to require NMDA receptor activation, changes in postsynaptic calcium concentration and phospholipase A₂ (PLA₂) activation. Arachidonic acid (AA) is released after the activation of calcium-dependent phospholipases and free AA is rapidly metabolized to a family of bioactive products (the eicosanoids) which are thought to be both intracellular and extracellular messengers. In the present study, we investigated the involvement of the cyclooxygenase and lipoxygenase pathways of AA metabolism in the formation of homosynaptic LTD in the rat hippocampus. Stimulation at 1 Hz for 15 min was used to produce homosynaptic depression in area CA1 of hippocampal slices. LTD induction was partially blocked by bromophenacyl bromide (50–100 μM), a selective PLA₂ inhibitor, and by the a nonselective lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA; 100 μM). In contrast, the specific cyclooxygenase blocker indomethacin (100 μM) did not significantly reduce hippocampal LTD. Since NDGA interferes with LTD formation, we examined whether specific inhibitors of 5- and 12-lipoxygenases were capable of blocking LTD expression. The 12-lipoxygenase inhibitor baicalein at a concentration of 50 μM reduced LTP formation when given in the bath, an effect that was less pronounced with the 5-lipoxygenase inhibitor AA-861. These data suggest that the activation of endogenous PLA₂ and the formation of 12-lipoxygenase metabolites of AA may be important factors controlling the expression of hippocampal LTD.     

Presynaptic D2 dopaminergic receptors mediate inhibition of excitatory synaptic transmission in rat neostriatum

The effect of dopamine (DA) on excitatory synaptic transmission was studied in rat neostriatal neurons using intracellular- and whole-cell voltage clamp-recording methods. Depolarizing excitatory postsynaptic potentials (EPSPs) were evoked by cortical stimulation. Superfusion of DA (0.01–10 μM) reversibly decreases EPSP in a concentration-dependent manner and with a estimated IC₅ of 0.3 μM. In addition, the inhibitory effect induced by DA at a low concentratiion (0.1 μM) was antagonized by sulpiride (1–10 nM), a selective D₂ dopaminergic receptor antagonist. However, D₁ dopaminergic receptor antagonist SKF-83566 (1–5 μM) did not affect the blocking effect by DA 0.1 μM. Based on these findings, we conclude that DA at a low concentration ( 0.1 μM) reduced the excitatory response of neostriatal neurons following cortical stimulation via the activation of D₂, but not D₁ dopaminergic receptors, located on the terminals of corticostriatal neurons.     

Adenosine antagonists increase spontaneous and evoked transmitter release from neuronal cells in culture

To examine the role played by endogenous adenosine in the modulation of transmitter release in the CNS, the effect of adenosine antagonists has been studied. Two systems have been used: EPSPs recorded from pyramidal cells in organotypic hippocampal cultures; and release of newly synthesized [3H]glutamate from cerebellar granule cells in dissociated culture. Bath application of 0.1-1 microM 8-phenyltheophylline (8-PT) reversibly increased both the number and size of spontaneous EPSPs and caused bursting activity in some cells. This effect was blocked by the glutamate antagonist gamma-D-glutamylglycine (DGG) (1 mM) but not by atropine (10 microM) or bicuculline (100 microM). Another adenosine antagonist isobutylmethylxanthine (IBMX, 10 microM) had a similar effect to 8-PT. Spontaneous activity in pyramidal cells and that induced by adenosine antagonists was blocked by the adenosine agonist 2-chloroadenosine (2-CA) (0.2-20 microM). 8-PT (10 microM) markedly potentiated K+-stimulated release of newly synthesized glutamate, and also enhanced basal glutamate release. The agonist (-)-phenylisopropyladenosine ((-)-PIA, 2 microM) which is relatively selective for A1 receptors, reduced by 19 +/- 5% the 8-PT-induced enhancement, and reduced K+-stimulated glutamate release in the absence of 8-PT to a similar extent. In contrast 5'-N-ethylcarboxamido adenosine (NECA, 2 microM), which is a relatively selective A2 agonist, slightly enhanced glutamate release. From these results it is likely that 8-PT potentiates glutamate release in both systems by blocking the effect of endogenous adenosine on presynaptic A1 receptors.     

Mechanism of delayed intracranial hypertension after cerebroventricular infusions in conscious rats

Prior studies showed that cerebroventricular infusions of artificial cerebrospinal fluid, 8 μl/min for 10 min, followed by a 10 min rest and a 24 h infusion of 0.5 μl/min, raised cerebrospinal fluid pressure (CSFp) of conscious, unrestrained rats after about 2 h. Here, we report that the 10 min infusion alone evoked a delayed, prolonged rise in CSFp. Pressure during the infusion itself rose and recovered quickly, as is usually reported. Pressure/volume tests, used to calculate resistance to outflow (R_o) and compliance (C), revealed that infusions increasedR_o and decreasedC, after a delay (P < 0.05). The rise in CSFp after infusion was blocked by pretreatment with acetazolamide + ouabain (P < 0.05), but the delayed changes in R_o andC were unaffected. We suggest that the 10 min infusion of a sterile, balanced salt solution has a primary effect that increasesR_o; as CSF synthesis continues, C is exhausted and the delayed rise in CSFp ensues. This non-traumatic method of raising CSFp may be a useful method to study intracranial fluid dynamics.     

Differential serotonergic inhibition of in vitro striatal [3H]acetylcholine release in prenatally cocaine-exposed male and female rats

Previous research indicates that prenatal cocaine (pCOC)-exposure results in greater 5-HT₃ agonist-induced inhibition of electrically evoked [³H]acetylcholine (ACh) overflow in rat striatal slices. The present study examines the effects of fluoxetine (FLU)-induced and exogenous serotonin (5-HT) on electrically evoked ACh release from striatal slices prepared from adult male and female (in periods of diestrus or proestrus) rats exposed to saline or cocaine in utero. Additionally, we assessed the impact of monoaminergic receptor stimulation on evoked ACh release by superfusion with selective 5-HT₂, 5-HT₃ and D₂ receptor antagonists in the presence of FLU-induced and exogenous 5-HT and measuring the capacity of these drugs to reverse inhibitory effects of 5-HT. Given our previous findings of accentuated inhibition of ACh release by 5-HT₃ agonism in striata of pCOC-exposed adult rats, we hypothesized that superfusion of endogenous and exogenous 5-HT would lead to greater suppression of evoked ACh release in this group of animals. Our results indicated that ACh release from slices of all prenatal saline (pSAL) rats was inhibited comparably by FLU (10 μM)-elicited increases in endogenous 5-HT or by increases elicited with application of exogenous 5-HT (5 μM). Robust FLU-mediated inhibition of ACh release was evident in slices from pCOC male and pCOC diestrus female rats vs. their respective PSAL control groups. Superfusion of striatal slices with 5-HT (5 μM) produced a pattern of ACh inhibition similar to that produced by FLU; however, the magnitude of ACh inhibition was consistently greater than that observed with FLU. Inhibition of ACh overflow by FLU was blocked by co-superfusion with ketanserin, a 5-HT₂ receptor antagonist, ICS-205,930, a 5-HT₃ receptor antagonist or sulpiride, a D₂ receptor antagonist. Conversely, serotonergic inhibition of ACh overflow was only blocked by a high concentration of ICS-205,930 (5 μM) and was completely reversed by sulpiride (1 μM). Collectively, these findings demonstrate serotonergic modulation of cholinergic neurons varying as a function of prenatal treatment, sex and, for females, phase of estrous. Inhibition of ACh release by 5-HT appears to be mediated by a complex relationship between 5-HT₂, 5-HT₃ and D₂ receptor regulation, as the blockade of any of these receptors reversed the inhibitory effects of FLU on ACh release. Conversely, in the case of exogenous 5-HT-induced inhibition, only blockade of D₂ receptors and high concentrations of the 5-HT₃ receptor antagonists were capable of reversing monoaminergic inhibition. These data support the hypothesis that the enhanced serotonergic modulation of ACh neurons in pCOC-exposed animals is largely mediated by dopamine (DA) and reflect a major biochemical persistence of neurodevelopmental adaptations elicited by early cocaine exposure.     

The transient potassium current, the A-current, is involved in spike frequency adaptation in rat amygdala neurons

The possible functional roles of the transient K⁺ current, I_A, in basolateral amygdala (BLA) neurons were studied using a rat brain slice preparation and conventional intracellular recording techniques. Conditioning depolarization, which inactivates I_A slowed the action potential repolarization while conditioning hyperpolarization accelarated the action potential repolarization. 4-Aminopyridine (4-AP, 100 μM), a specific I_A antagonist, also caused a clear delay in spike repolarization similar to the effect of conditioning depolarization suggesting that I_A is involved in the action potential repolarization.

When BLA neurons were excited by injecting long depolarizing current pulses (500 ms), they responded with an initial rapid discharge of action potentials which slowed or accommodated; an afterhyperpolarization (AHP) followed the depolarizing current pulses. Superfusion of 4-AP (100 μM) blocked accommodation resulting in an increase in action potential discharge in 74% (32 out of 43) neurons tested. The remaining 11 cells responded with an increased frequency of discharge of the first few action potentials. Unlike the effect of cadmium (Cd²⁺, 100 μM), a calcium channel blocker, 4-AP did not reduce the AHP. In the presence of norepinephrine (NE, 10 μM), a neurotransmitter which has been shown to block calcium-activated potassuim conductance, 4-AP caused a further increase in the number and frequency of action potential discharge. In addition, in BLA neurons, spontaneous interictal and ictal-like events were observed at low and high concentrations of 4-AP, respectively. We conclude that I_A is involved in the action potential repolarization as well as spike frequency adaptation in BLA neurons and that these actionsmay contribute to the convulsant effect of 4-AP     

Differential effects of the adenosine A(1) receptor allosteric enhancer PD 81,723 on agonist binding to brain and adipocyte membranes.

The benzoylthiophene analog, PD 81,723, has been shown to allosterically enhance agonist binding and functional activation of the mammalian adenosine (ADO) A₁ receptor subtype by putatively maintaining the receptor in a high affinity state. The present studies were conducted to evaluate the ability of PD 81,723 to enhance the binding of [³H]cyclohexyladenosine ([³H]CHA) to A₁ receptors of neural (cerebral cortex) and non-neural (adipocyte) origin in three different species; rat, guinea pig and dog. PD 81,723 (0.3–100 μM) produced a concentration-dependent enhancement of [³H]CHA binding to rat brain A₁ receptors. These effects were also species-dependent with larger enhancements (150–200% of control) observed in guinea pig and dog brain membranes as compared to the rat (120% of control). In contrast, PD 81,723 did not produce any enhancement of [³H]CHA binding to A₁ receptors in adipocyte membranes from any of the species examined. Additional binding studies were conducted using pharmacological manipulations that have previously been shown to enhance the allosteric effects of PD 81,723. In the presence of 1 mM GTP, the allosteric effects of PD 81,723 (15 μM) were increased in rat, guinea pig and dog brain membranes, however, in adipocyte membranes from each species, no significant alteration in agonist binding was observed. Similarly, the A₁ receptor selective antagonist 8-cyclopentyl-1,3-dipropylxanthine (added to effectively reduce the intrinsic antagonist properties of PD 81,723) was found to enhance the allosteric effects of PD 81,723 (15 μM) in brain, but produce no alteration of agonist binding in adipocyte membranes from each species. Examination of the dissociation kinetics of [³H]CHA binding from rat brain and adipocyte membranes revealed that PD 81,723 (15 μM) differentially slowed agonist dissociation from brain, but not adipocyte, membranes. Taken together, the present data support the hypothesis that in tissues that are sensitive to PD 81,723, this benzyolthiophene functions to maintain the A₁ receptor in a high-affinity state and that the relative proportions of high-affinity A₁ receptors present in specific tissues may contribute, at least in part, to the apparent differential effects of PD 81,723 on agonist binding. The tissue specific modulation of A₁ receptor function by PD 81,723 also illustrates the possibility that the locus of allosteric modulation by PD 81,723 may be manifest via a specific, but indirect and tissue-dependent, interaction with the A₁ receptor.     

Interactions between serotonin and substance P in the spinal regulation of nociception

Interactions between 5-hydroxytryptamine (5-HT) and substance P (SP) in the mouse spinal cord were investigated using the tail-flick test and the behavioral response evoked by intrathecal (i.th.) SP or i.th. 5-HT. I.th. injection of 5-HT (20 μg) or the 5-HT₁ receptor agonists(+)-8-hydroxy-2-(di-n-propylamino)tetralin ((+)-8-OH-DPAT) (20 μg) or 5-methoxy-3(1,2,3,6-tetrahydropyridine-4-yl)-1H-indole (RU 24969) (20 μg) markedly inhibited the tail-flick reflex. The effect of these compounds was reduced when SP (5 μg) was given i.th. 55 min, or 55 and 45 min before the agonists. The tail-flick latencies recorded 5 min before injection of a 5-HT agonist were similar in animals treated with SP or vehicle. The changes in the tail-flick test were not due to changes in tail skin temperature since only minimal differences in the skin temperature were recorded between the groups injected with SP or vehicle. I.th. injection of SP (10 ng) or 5-HT (2 μg) produced a similar behavioral response consisting of biting, licking and scratching of the caudal part of the body, indicative of nociceptive stimulation. The responses both to i.th. SP and 5-HT were reduced after i.th. application of SP receptor antagonist [d-Arg¹,d-Trp^7,9,Leu¹¹]-SP (Spantide) (5 μg), as well as 5 min after i.th. injection of the 5-HT receptor antagonist metergoline (4 μg). The data may indicate functional interactions between SP and 5-HT in the mouse spinal cord, which may take place in neurons involved in the processing of nociception.     

Modulation of net outward current in cultured neurons by angiotensin II: involvement of AT1 and AT2 receptors.

In this study we have used whole-cell, voltage-clamp procedures to determine the effects of angiotensin II (AII) on net outward current (I_no) in neurons co-cultured from the hypothalamus and brainstem of 1-day-old rats. I_no is the sum of all inward and outward membrane currents (minus Na⁺, which is blocked by tetrodotoxin) which occur during the repolarization phase of the action potential. We have determined that AII elicits two separate effects on I_no in cultured neurons. AII caused a reversible and concentration (0.1 nM–10 μM)-dependent increase in I_no. This effect is inhibited by the AT₂ receptor-selective antagonists, PD123177 and PD123319 (both 100 nM), but not by the AT₁-selective receptor blocker, DuP753 (Losartan; 100 nM), and so it is mediated by AT₂ receptors. In a smaller number of neurons AII induced a reversible and concentration (0.01 nM–10 μM)-dependent decrease in I_no that was blocked by Losartan (100 nM) but not by PD123177 (100 nM). Thus the decrease in I_no is mediated by AT₁ receptors. Additionally, some neurons displayed both AT₁- and AT₂ receptor-mediated effects on I_no. Our results demonstrate two distinct actions of AII on membrane ionic currents in cultured neurons, effects that are mediated by different AII receptor subtypes.     

Suppression of the micturition reflex in urethane-anesthetized rats by intracerebroventricular injection of WAY100635, a 5-HT(1A) receptor antagonist

The influence of supraspinal 5-HT_1A receptors on reflex bladder activity was evaluated in anesthetized rats by studying the effects of intracerebroventricular (i.c.v.) administration of WAY100635 (1–100 μg), a selective 5-HT_1A receptor antagonist. The drug dose-dependently decreased the frequency and/or amplitude of isovolumetric reflex bladder contractions. Low doses (1–10 μg) increased the interval between contractions but only slightly reduced the amplitude of the contractions. However, 100 μg of WAY100635 elicited an initial complete block of bladder reflexes followed by a recovery period lasting 10–15 min during which the frequency of reflex contractions was normal but the amplitude was markedly suppressed by 70–80%. Mesulergine (0.1 mg/kg, i.v.), a 5-HT_2C antagonist, which transiently eliminated bladder activity in some rats (five of 11), blocked the inhibitory effect of WAY100635 (10 or 100 μg, i.c.v.) in only two of six rats. Our data coupled with the results of previous studies suggest that spinal and supraspinal 5-HT_1A receptors are involved in multiple inhibitory mechanisms controlling the spinobulbospinal micturition reflex pathway. The regulation of the frequency of bladder reflexes is presumably mediated by a suppression of afferent input to the micturition switching circuitry in the pons, whereas the regulation of bladder contraction amplitude may be related to an inhibition of the output from the pons to the parasympathetic nucleus in the spinal cord.     

Caffeine inhibits post-tetanic potentiation but does not alter long-term potentiation in the rat hippocampal slice

The effects of caffeine were investigated on the extracellular excitatory postsynaptic potentials (EPSPs) recorded in the stratum radiatum of CA₁ of the rat hippocampal slice in response to stimulation of the Schaffer collaterals. Caffeine in concentrations from 500 μM to 10 mM caused an increase in the amplitude of the EPSP, which reached a peak after 5–10 min perfusion. This increase was antagonized by pretreatment with 20 μM trifluoperazine. Paired-pulse facilitation, augmentation and potentiation were strongly inhibited by caffeine. Augmentation was most sensitive to caffeine, being abolished by 500 μM caffeine. Long-term potentiation (LTP) induced by high-frequency stimulation was not significantly inhibited by caffeine either by 5–10 min or by 60 min perfusion with 10 mM caffeine. Moreover, the caffeine-induced increase in the low-frequency EPSP could be reversed by 30 min washout, demonstrating that it was not an LTP type increase.     

Pharmacological characterization of grooming induced by a selective NK-1 tachykinin receptor agonist

Bilateral intranigral administration of the selective NK-1 tachykinin receptor agonist [AcArg⁶, Sar⁹, Met(O₂)¹¹]SP_6–11 (0–11 nmol total bilateral dose) selectively induced grooming in rats. This response was blocked by concurrent intranigral administration of the NK-1 tachykinin receptor antagonist RP 67580 (2 nmol), but not by NK-2 (L-659, 877) or NK-3 ([Trp⁷, β-Ala⁸]NKA_4–10) antagonists. Pretreatment with systemic opioid (naloxone 1.5 mg/kg) and D₁ dopamine (SCH 23390 100 μg/kg) receptor antagonists also attenuated tachykinin-induced grooming, which was unaffected by D₂ dopamine (sulpiride 30 mg/kg) or 5-HT_2A+C (ritanserin 2 mg/kg) antagonists. Grooming induced by intranigral [AcArg⁶, Sar⁹, Met(O₂)¹¹]SP_6–11 was also attenuated by bilateral 6-hydroxydopamine lesions of te substantia nigra. These findings indicate that grooming induced by intranigral tachykinins reflects activation of NK-1 receptors and is dependent upon endogenous dopamine and consequent selective stimulation of D₁ dopamine receptors.     

GABA, acting at both GABAA and GABAB receptors, inhibits the release of cholecystokinin-like material from the rat spinal cord in vitro.

Superfusion of slices of the dorsal zone of the lumbar enlargement of the rat spinal cord with an artificial cerebrospinal fluid allowed the collection of cholecystokinin-like material (CCKLM) whose Ca²⁺-dependent release could be evoked by tissue depolarization with 30 mM K⁺. Studies on the possible influence of GABA and related agonists on this process showed that the amino acid, the GABA_A agonist, muscimol, and the GABA_B agonist, baclofen, inhibited the K⁺-evoked release of CCKLM from the rat spinal cord in a concentration-dependent manner. Maximal inhibition did not exceed −40% with either agonist. Furthermore, the effects of GABA_A and GABA_B receptor stimulation were not additive. Whereas the effects of muscimol (10 μM) and baclofen (1 μM) could be completely antagonized by bicuculline (1 μM) and phaclofen (10 μM), respectively, complete blockade of the inhibition by GABA (1 μM) could only be achieved in the presence of both antagonists. These data indicate that both GABA_A and GABA_B receptors are involved in the negative influence of GABA onto CCK-containing neurones within the dorsal horn of the rat spinal cord. Apparently, these receptors are not located on CCK-containing neurones themselves, since the inhibitory effect of GABA on the K⁺-evoked release of CCKLM could be completely prevented by tetrodotoxin (1 μM). As CCK acts centrally as an endogenous opioid antagonist, such a GABA-inhibitory control of spinal CCK-containing neurones might participate in the analgesic action of the amino acid via the intrathecal route.     

Effects of micromolar and nanomolar calcium concentrations on non-synaptic bursting in the hippocampal slice

To determine how [Ca²⁺]₀ affects non-synaptic epileptogenesis in the CA1 area of hippocampal slices, we compared the extracellularly recorded hyperactivity induced by ACSF containing either micromolar (‘low’-Ca²⁺, LC-ACSF) or nanomolar concentrations of Ca²⁺ (‘zero’-Ca²⁺, ZC-ACSF). Both solutions effectively blocked chemical synaptic transmission but spontaneous bursts developed more quickly and consistently in ZC-ACSF and were longer in duration and more frequent than those recorded in LC-ACSF. Antidromically evoked bursts were less epileptiform, i.e., they exhibited fewer population spikes (PSs), in ZC-ACSF. Increasing [Mg²⁺]₀ or decreasing [K⁺]₀ suppressed spontaneous LC-ACSF bursting but only decreased the intensity and frequency of bursting in ZC-ACSF. Either manipulation increased the epileptiform nature of the antidromically evoked field potential, thereby mimicking the effect of increasing [Ca²⁺]₀ from nanomolar to micromolar levels. Bath application of 250–500 μM GABA commonly arrested spontaneous bursting in LC-ACSF. In ZC-ACSF, GABA decreased the burst frequency but paradoxically superimposed high amplitude PSs on each burst. These effects were reversed by the GABA_A receptor antagonists bicuculline methiodide or picrotoxin (50–100 μM). These results indicate that simply lowering [Ca²⁺]₀ from micromolar to nanomolar concentrations increases the burst propensity and intensity of the CA1 population and can dramatically alter responses to pharmacological agents.     

Long-term activation of adenosine A(2a) receptors blocks glutamate excitotoxicity in cultures of avian retinal neurons

Previous work showed the presence of adenosine receptors as well as adenosine uptake and release mechanisms in developing chick retinal neurons in culture. In the present work we show that exogenous glutamate or kainate promotes extensive cell death in these cultures which is blocked when the cultures are previously incubated with adenosine. Addition of glutamate or kainate to purified cultures of retinal neurons and photoreceptors induced massive death of cultured cells which was inhibited in both cases by preincubation with MK801, an NMDA antagonist, or DNQX, an AMPA/kainate antagonist. Cell death was also greatly attenuated by preincubation with adenosine plus EHNA, an adenosine deaminase inhibitor, NBI, an adenosine uptake blocker, the permeable cAMP analogs 8-Br cAMP and Sp cAMP and the A_2a agonists CGS 21680 and DPMA, but not with the A₁ receptor agonist CHA. Kinetic studies performed determining the intracellular LDH activity showed that maximal death was observed after 8 h and in concentrations of glutamate as low as 50 μM. We also observed a time-dependent protective effect of adenosine beginning after 1 h of preincubation and reaching a maximal effect after 24 h, indicating its association with changes in cellular metabolism induced by long-term exposure of cells to the nucleoside. The results show that adenosine inhibits glutamate toxicity in retinal neurons through a long-term activation of A_2a receptors and elevation of intracellular cyclic AMP levels.