Modulation of phospholipase A2 activity in primary cultures of rat cortical neurons

Summary. In neurons, phospholipase A₂ (PLA₂) plays a central role in the regulation of membrane phospholipid metabolism. We have addressed the pharmacological modulation of PLA₂ in primary cultures of rat cortical neurons. Inhibition curves were obtained in 4 day-in-culture neurons treated for 30 minutes with either the dual PLA₂ inhibitor methyl arachidonyl fluorophosphonate (MAFP), or the iPLA₂ inhibitor bromoenol lactone (BEL). Full inhibition was achieved with 100 and 250 μM of MAFP, or 10 and 20 μM of BEL. Conversely, a dose-dependent activation of PLA₂ was obtained with 10–20 μg/ml of melitin. PLA₂ inhibition with MAFP or BEL was not acutely toxic for cultured neurons. However, sustained inhibition of the enzyme precluded the development of neurites, and resulted in long-term loss of neuronal viability. We present a model of pharmacological challenge of PLA₂ in vitro, which can be further used to address the involvement of the enzyme in neurodevelopment and neurodegeneration models.     

Myocardial ischemic nociceptive signaling mediated by P2X3 receptor in rat stellate ganglion neurons

Adenosine 5'-triphosphate (ATP) is implicated in peripheral pain signaling through activation of P2X receptors. P2X(3) receptors have a high level of expression in, and selective location on sensory afferents. P2X receptors, particularly the P2X(3) subtype, are identified as targets for novel analgesics. The stellate ganglion (SG) is peripheral sympathetic ganglia involved in heart function. Surgical interventions of sympathetic afferent pathways abolish or relieve angina pectoris, so it is showed that cardiac pain is mediated by the activation of afferents in sympathetic nerves. The cervicothoracic sympathetic ganglia, including the stellate ganglion, are implicated in sensations associated with myocardial ischemia or cardiac pain. In the present study we have examined P2X(3) involvement in cardiac nociceptive transmission. P2X receptor agonists activated currents (I(ATP)) in SG neurons. The I(ATP) amplitude and P2X(3) mRNA expression in myocardial ischemic injury group were much larger than those obtained in control group. Prostaglandin E(2) (PGE(2)) and substance P (SP) increased ATP-activated currents. P2X(3) receptor antagonist A-317491 reduced P2X agonist activated currents and P2X(3) mRNA expression. The results revealed that the myocardial ischemia induced the upregulation of P2X(3) receptor in function and morphous and P2X(3) receptor antagonist A-317491 inhibited P2X agonist activated currents and P2X(3) mRNA expression. The facts indicated that P2X(3) receptor in SG neurons was involved in cardiac nociceptive transmission.     

Depression by morphine-6-glucuronide of nociceptive activity in rat thalamus neurons: comparison with morphine

To assess the contribution of the active metabolite of morphine, morphine-6-glucuronide (M6G), to the analgesic effect of systemically administered morphine, experiments were carried out on rats under urethane anesthesia in which nociceptive activity was evoked by electrical stimulation of afferent C fibers in the sural nerve and recorded from single neurons in the ventrobasal complex of the thalamus. Intravenous (i.v.) injections of morphine completely blocked the activity at doses of 500 and 1000 μg/kg, the ED,, being 44 μg/kg. M6G administered by i.v. injection reduced the evoked nociceptive activity only by about 40% at 80 and 160 μg/kg, the ED₅₀ being 6 μg/kg. After intrathecal (i.t.) injection, morphine produced maximum depression of 55% of the control activity at 20 μg the ED₅₀ is 18 μg. M6G injected i.t. produced maximum depression of 40% at doses ranging from 0.2 to 10 μg. The ED₅₀ of M6G i.t. is below 0.2 μg. The effects of morphine and M6G were reversed by naloxone (200 μg/kg i.v.). The results show that M6G is more potent than morphine, regardless of the route of administration, while morphine is more effective when injected i.v. Due to the low efficacy of M6G, it seems unlikely that this glucuronide contributes substantially to the analgesic effect of morphine when renal function is normal. The results also make evident that the maximum effect of morphine results from an action at spinal and supraspinal sites.     

ATP increases extracellular dopamine level through stimulation of P2Y purinoceptors in the rat striatum

The effect of ATP on release of dopamine (DA) from rat striatum was studied using in vivo microdialysis. ATP increased the striatal extracellular levels of DA dose-dependently. These analogs produced an increase in DA according to this order of potency: 2-methylthio ATP > ATP α, β-methylene ATP > ADP > AMP > adenosine. Adenosine 5′-[β, γ imido]-triphosphate had a more prolonged effect on the increase in DA level than ATP. The ATP-induced increase in DA was inhibited by adding suramin, a nonselective P₂ purinoceptor antagonist, and reactive blue 2, a P_2Y purinoceptor antagonist, but not inhibited by xanthine amine congener, an adenosine receptor antagonist. Pertussis toxin reduced the increase in DA produced by ATP, whih suggests that te P₂ purinoceptor may be coupled with a G-protein in the rat striatum. Results suggest that P_2Y purinoceptors may involve an ATP-induced increase in DA. The ATP-induced release of DA was tetrodotoxin-sensitive, Ca²⁺-dependent and was abolished by ω-conotoxin GVIA, indicating that the opening of voltage-sensitive Na⁺ channel and the Ca²⁺ influx through the N-type voltage-dependent calcium channel are both required for the ATP-induced increase in DA. The ATP-induced increase in DA is presumably due to the release of DA via the stimulation of P_2Y purinoceptors in the rat striatum.     

Antinociceptive involvement of substance P in the spinal cord of mice: dose effects of substance P on the behavior elicited by intrathecally administered NMDA

The functional interaction between substance P (SP) and N-methyl-d-aspartate (NMDA) was studied to clarify the diversity of the roles of SP in nociceptive processes at the spinal level in mice. Behavioral responses elicited by intrathecal co-administration of NMDA (0.25 nmol) with various doses of SP (0.3–12 pmol) were observed for 1 min. The high dose of SP (12 pmol) potentiated NMDA-induced responses, which consisted of caudally directed licking and biting, while the low dose of SP (1 pmol) significantly reduced the responses by 40% compared to control mice administered NMDA alone. The antinociceptive effect of the low dose of SP was negated by co-administration of the opioid receptor antagonist naloxone. Furthermore, the antinociception produced by SP was present in mice pre-treated with systemic administration of capsaicin during the neonatal period. These results suggest that one of the roles SP plays at the spinal level is an involvement in antinociception. The activities of excitatory dorsal horn neurons are considered to be inhibited by endogenous opioid peptides released from inhibitory dorsal horn neurons directly stimulated by SP.     

Ultracytochemical localization of Ca2+-ATPase activity in reactive astrocytes

Summary Ca²⁺-ATPase activity on the astrocyte plasma membrane was investigated ultracytochemically, using the lead salt technique. Normal astrocytes showed a weak cytochemical reaction for Ca²⁺-ATPase activity, deposits of the reaction product being small. At 7 and 15 days after cold lesioning, reactive astrocytes apparently in the process of repair of the edematous lesion were observed; these demonstrated an intense cytochemical reaction for Ca²⁺-ATPase activity in their plasma membranes facing the extracellular fluid, with reaction product accumulation. At 2 months, the lesion had progressed to glial scars containing sporadic microcysts. The reactive astrocytes surrounding the microcysts demonstrated a moderate cytochemical reaction for Ca²⁺-ATPase activity in their free plasma membranes, whereas those arranged in a cell-to-cell pattern showed little reaction product in their plasma membranes. In conclusion, a more intense cytochemical reaction was always observed in the free plasma membrane of reactive astrocytes.     

Prevention of experimental allergic neuritis in the lewis rat with bovine P2 protein

Protective doses of bovine P₂ protein (5, 15 or 50 μg) in incomplete Freund's adjuvant (IFA) were administered to Lewis rats and were followed 2,4 or 10 weeks later by challenging doses of either 250 μg bovine P₂ or 2.5 mg bovine PNS myelin in complete Freund's adjuvant (CFA). Protection from experimental allergic neuritis (EAN) could be achieved with a single dose of 5 μg of P₂ in IFA. There was little difference between prophylactic 5 μg and 15 μg doses of P₂. The degree of protection depended upon the interval between protective innoculation and challenge. Protection was partial at 2 weeks and maximal at 4 weeks at which time there was complete protection against P₂-induced EAN and less complete protection from myelin-induced disease. Complete protection at 4 weeks from myelin-induced EAN was achieved with a 50 μg dose. Protection lasted for at least 10 weeks (the longest interval assessed) and was complete with respect to P₂-induced EAN. Partial protection was observed in myelin-challenged animals after 10 weeks with the level of protection greater than that observed after 2 weeks.     

Experimental allergic neuritis in the Lewis rat: Characterization of the activity of peripheral myelin and its major basic protein,P2

Experimental allergic neuritis has been produced in the inbred Lewis rat in the absence of experimental allergic encephalomyelitis (EAE) using bovine intradural root myelin. The lack of EAE is probably because P₁ is only weakly encephalitogenic in the ra rat. One of the basic proteins of bovine peripheral myelin, P₂, was isolated and demonstrated to be pure by amino acid analysis and SDS PAGE. It was found to have a molecular weight of 15,400 and contained 4 mol12-cystine/mol. This P₂ was found to be highly neuritogenic and is probably the sole neuritogenic antigen in this system. The successful demonstration of its neuritogenicity must be due in large part to the use of the inbred Lewis rat and bovine P₂, but an explanation could also involve the omission of denaturing organic solvents, the prevention of oxidative denaturation and presumably the fact that any changes which may occur are not sufficient to prevent recognition of the active site by the immune system of the inbred Lewis rat. P₂ was neuritogenic down to 5 μg/animal. Its activity was enhanced by but not dependent on the presence of Mycobacterium in the adjuvant. This suggested that release of P₂ could possibly break tolerance and produce an auto-immune disease such as the Guillain-Barre syndrome.     

Effect of emodin on neuropathic pain transmission mediated by P2X2/3 receptor of primary sensory neurons

Neuropathic pain is the most difficult type of pain to cure. The P2X_2/3 receptors play a crucial role in facilitating the transmission of pain at neuropathic pain states. Emodin is a natural anthraquinone in rhubarb. The present research investigated the effects of emodin on the pain transmission in neuropathic pain states that was mediated by P2X_2/3 receptor in primary sensory neurons. Chronic constriction injury (CCI) model was used as neuropathic pain model. Emodin was dissolved in 0.5% sodium carboxymethyl cellulose (CMC) as vehicle. Sprague-Dawley male rats had been randomly divided into Sham + vehicle group, CCI + emodin group, and CCI + vehicle group. Mechanical withdrawal threshold and thermal withdrawal latency were measured. P2X_2/3 expression in L4/L5 dorsal root ganglion (DRG) was detected by immunohistochemistry, in situ hybridization (ISH) and RT-PCR. The mechanical withdrawal threshold and thermal withdrawal latency in CCI + vehicle group were lower than those in Sham + vehicle group and CCI + emodin group (p < 0.05), while P2X₂ and P2X₃ receptor expression of L4/L5 DRG in CCI + vehicle group was higher than those in the other two groups (p < 0.05). The co-local staining of P2X₂ and P2X₃ in the DRG of CCI group appeared to be more intense than that in the DRG of the other two groups with double-label fluorescence immunohistochemistry. The results showed that the application of emodin alleviated the hyperalgesia of CCI rats and significantly decreased the P2X_2/3 expression of L4/L5 DRG in CCI + emodin group compared with that in CCI + vehicle group (p < 0.05). The data of ISH and RT-PCR in P2X₂ and P2X₃ mRNA expression suggest that the pharmacologic mechanism of emodin is involved in the nucleic acid level. The results showed that emodin can inhibit the transmission of neuropathic pain mediated by P2X_2/3 receptor of primary sensory neurons to alleviate chronic neuropathic pain.     

High perfusate PO2 impairs thermosensitivity of hypothalamic thermosensitive neurons in slice preparations

The thermosensitivity of neurons in the preoptic area (POA) of guinea pigs was examined in slice preparations during perfusion with artificial cerebrospinal fluid (ACSF) gassed with 95% O₂/5% CO₂ (95% O₂-ACSF) or 21% O₂/5% CO₂74% N₂ (21% O₂-ACSF). Perfusate P_o2, P_co2, and pH of 95% O₂-ACSF measured in the incubation chamber were, respectively, 402.9 mmHg, 39.1 mmHg, and 7.38; those of 21% O₂-ACSF were 154.0 mmHg, 33.1 mmHg, and 7.41. All the thermosensitive neurons (n = 6) originally identified during perfusion with 95% O₂-ACSF also exhibited their thermosensitivity when examined again in 21% O₂-ACSF. However, 63% of the thermosensitive neurons (n = 8) originally identified with 21% O₂-ACSF lost their thermosensitivity in 95% O₂-ACSF. The addition of Superoxide dismutase to 95% O₂-ACSF prevented the loss of thermosensitivity in 88% of the neurons (n = 8). These results suggest that oxygen toxicity due to free radical formation may account for the impairment of neuronal thermosensitivity in 95% O₂-ACSF.     

Oxidative stress increases expression and activity of BACE in NT2 neurons

Recently an aspartyl protease with beta-secretase activity called BACE was identified. In the present paper we showed that BACE is modulated by the oxidative stress product 4-hydroxynonenal (HNE). Exposure of NT(2) neurons to the two classical pro-oxidant stimuli ascorbate/FeSO(4) and H(2)O(2)/FeSO(4) resulted in a significant generation of HNE, which is temporally followed by an increased production of BACE protein levels. HNE mediated BACE induction is accompanied by a proportional elevation of carboxy-terminal fragments of amyloid precursor protein. Moreover, the direct relationship between BACE induction and lipid peroxidation products was strongly confirmed by the protection exerted by a short pretreatment with alpha-tocopherol, the most important antioxidant known to prevent the formation of aldehydic end-products of lipid peroxidation, including HNE. Our results support the hypothesis that oxidative stress and A beta production are strictly interrelated events and suggest that inhibition of BACE may have a therapeutic effect synergic with antioxidant compounds.     

Serotonin potentiation of glycine-activated whole-cell currents in the superficial laminae neurons of the rat spinal dorsal horn is mediated by protein kinase C

The modulatory effects of serotonin (5-HT) on glycine (Gly)-activated whole-cell currents were investigated in neurons acutely dissociated from the superficial laminae (I and II) of the rat spinal dorsal horn using the nystatin-perforated patch recording configuration under voltage-clamp conditions. Our results demonstrate that (1). Gly acted on strychnine (STR)-sensitive Gly receptors and elicited inward Cl(-) currents (I(Gly)) at a holding potential of -40 mV; (2). 5-HT potentiated I(Gly) without affecting the reversal potential of I(Gly); (3). the agonist (alpha-methyl-5-HT) and antagonist (ketanserine) of 5-HT(2) receptor mimicked and blocked the potentiating effect of 5-HT on I(Gly), respectively; (4). bisindolylmaleimide I (BIM), a selective inhibitor of protein kinase C (PKC), reduced the potentiating effect of 5-HT on I(Gly); and (5). 5-HT-induced enhancement of I(Gly) was not affected by pretreatment with 1,2-bis-(2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid tetrakis (acetoxy-methyl) ester (BAPTA AM), a Ca(2+) chelator. These results indicate that (1). the potentiation of 5-HT on I(Gly) is mediated by 5-HT(2) receptor and through Ca(2+)-independent PKC intracellular signal transduction pathway; and (2). the interactions between 5-HT and Gly might modulate the transmission of nociceptive information through the spinal cord.     

Prostaglandin E2 excites neurons of the nucleus tractus solitarius by activating cation channels

Nucleus tractus solitarius (NTS) has a high density of prostaglandin E₂ (PGE₂)-binding sites. Action of PGE₂ (10⁻⁹–10⁻⁶ M) was tested on neurons in a NTS slice with patch-clamp recording under synaptic blockade. PGE₂ raised the firing rate in approximately half of the neurons in cell-attached patch mode. In whole-cell current clamp, PGE₂ depolarized membrane potential accompanied by an increase in firing rate. In whole-cell voltage clamp (−58 mV), PGE₂ induced the inward current with an increase in conductance. The current was linearly related to voltage from −100 mV to −10 mV and suppressed between −10 mV and 20 mV. The current-voltage curve remained similar under low external Cl⁻ or high internal Cl⁻ conditions and after external Na⁺ was replaced by Cs⁺. It is concluded that PGE₂ excites NTS neurons by activating cation conductance.     

Age differences in contingent negative variation activity of healthy young adults and presenile subjects

20 selected right-handed very healthy subjects (10 young adults and 10 presenile subjects mean age 28.3 and 59.6) were tested for CNV activity with a simple warned reaction time (RT) paradigm. EEG and CNV components (post-S1, N1, P2, P3; early CNV; N1200 late CNV; CNV resolution) were recorded from Fz, C3, Cz, C4, P3, Pz, and P4, referenced to linked mastoid electrodes. EOG, RT and stimuli were also recorded. The presenile group differed significanty from the younger group in the auditory post-S1 N1 and P3, and in the early (O-wave) and late (P-wave) CNV complex components. A progressive amplitude reduction only in frontal leads between O-wave and P-wave with the lowest point being reached in the P-wave was characteristic in the presenile group. Further, presenile subjects showed relatively flat CNV waveshapes of low amplitude and, as a whole, performed a little less well than young persons. This finding suggests that the statistically significant changes in post-S1 EPRs and CNV activity recorded in our presenile subjects, without appreciable deficits in behavioral and mental performance, could be alerting signs of early brain involutional processes related to minimal and subclinical decrement of orienting, attentiveness and response preparation capabilities. If such is the case and it could be confirmed in a larger sample of very healthy subjects, these age-related changes in the presenium could be of considerable practical importance for clinical and research applications.

---

Sammario Scopo della presente ricerca è la raccolta di dati normativi ottenuti con una nuova metodica d'analisi dell'onda d'aspettativa, delle sue principali componenti (complesso CNV) e del tempo di reazione motorio (RT) in due gruppi si soggetti normali, adulti ed in età presenile. In 20 soggetti sani (rispettivamente 10 con età media di 28.3 e 10 di 59.6 anni) per evocare il complesso CNV/RT si è adottato un parametro standard di stimolazione molto semplice ed idoneo anche per pazienti con deterioramento mentale: S1 (click)—2 sec d'intervallo —S2 (stimolo visivo)—risposta motoria con RT. Si è derivata l'attività di tipo CNV da regioni frontali, centrali e parietali. In confronto ai soggetti più giovani, modificazioni significative di alcune componenti della CNV si sono rilevate nel gruppo di soggetti in età presenile senza apparente declino delle performances comportamentali e cognitive. Tali modificazioni appaiono indicative delle fasi iniziali dei processi involutivi encefalici fisiologici associati a minimi e sublinici deficit delle risposte d'orientamento, attenzione, concentrazione, memoria a breve termine, capacità di preparazione alle risposte motorie sollecitate da S2. Vengono discusse le implicazioni cliniche ed in particolare l'importanza diagnostica di questo tipo di indagini nel riconoscimento dei primi stadi nel presenium dell'involuzione fisiologica o patologica dell'encefalo.

     

Effects of D2 dopamine receptor agonist and antagonist on brain activity in the rat assessed by functional magnetic resonance imaging

The effects of D₂ dopamine receptor agonist, bromocriptine (BROMO), and antagonist, haloperidol (HPD), on brain activity were investigated in rats by functional magnetic resonance imaging. T2^*-weighted signal intensity was increased in the hypothalamus at 120 min after acute administration of BROMO, and in the ventral posterior and dorsomedial nuclei of the thalamus from 30 to 120 min. In contrast, the signal intensity was decreased in the caudate–putamen at 30 min after acute administration of HPD, in the hypothalamus from 30 to 60 min, and in the perirhinal cortex at 30 min. After chronic (2 weeks) HPD treatment, acute administration of HPD decreased signal intensity in the caudate–putamen at 60 min, in the hypothalamus at 30 min, the perirhinal cortex from 2 to 120 min, the dorsomedial and ventral posterior nuclei of the thalamus from 2 to 120 min, and the medial nucleus of the amygdala from 60 to 120 min. These results suggest that (1) the D₂ receptor agonist increased the activity of the thalamic nuclei and the hypothalamus, while the D₂ receptor antagonist suppressed brain activity in the regions where D₂ receptors were present, (2) the suppression of brain activity in the thalamic nuclei and the perirhinal cortex by acute HPD administration was enhanced by chronic HPD treatment, and (3) the effects of antipsychotic drugs on the thalamus, amygdala, and perirhinal cortex may be related to their therapeutic efficacy, since clinical improvement in schizophrenic patients appears several days after the start of HPD treatment.     

The effects of prostaglandin E2 on the firing rate activity of thermosensitive and temperature insensitive neurons in the ventromedial preoptic area of the rat hypothalamus

In response to an immune system challenge with lipopolysaccharide (LPS), recent work has shown that Fos immunoreactivity is displayed by neurons in the ventromedial preoptic area of the hypothalamus (VMPO). In addition, neurons in this region show distinct axonal projections to the anterior perifornical area (APFx) and the paraventricular nucleus (PVN). It has been hypothesized that neurons within the VMPO integrate their local responses to temperature with changes in firing activity that result from LPS induced production of prostaglandin E(2) (PGE(2)). This may be an important mechanism by which the set-point regulation of thermoeffector neurons in the APFx and PVN is altered, resulting in hyperthermia. To characterize the firing rate activity of VMPO neurons, single-unit recordings were made of neuronal extracellular activity in rat hypothalamic tissue slices. Based on the slope of firing rate as a function of tissue temperature, neurons were classified as either warm sensitive or temperature insensitive. Neurons were then treated with PGE(2) (200 nM) while tissue temperature was held at a constant level ( approximately 36 degrees C). The majority of temperature insensitive neurons responded to PGE(2) with an increase in firing rate activity, while warm sensitive neurons showed a reduction in firing rate. This suggests that both warm sensitive and temperature insensitive neurons in the VMPO may play critical and contrasting roles in the production of a fever during an acute phase response to infection.     

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.     

Involvement of GABA(A) receptor in modulation of jaw muscle activity evoked by mustard oil application to the rat temporomandibular joint

The effect of intrathecal administration of the GABA(A) receptor antagonist bicuculline methylbromide on jaw muscle electromyographic (EMG) activity evoked by mustard oil injection into the rat temporomandibular joint was studied. Bicuculline given prior to mustard oil augmented the EMG activity evoked by mustard oil, and "rekindling" of EMG activity was induced by bicuculline given 30 min after mustard oil. These results suggest that central GABA(A) receptors modulate reflex responses to noxious craniofacial stimuli.     

Locus coeruleus neurons show reduced alpha2-receptor responsiveness and decreased basal activity in spontaneously hypertensive rats

Summary Previous studies have indicated that brain noradrenaline (NA) neurons in spontaneously (genetically) hypertensive rats (SHR) are implicated in the development of hypertension. Thus, a number of biochemical aberrations in the metabolism of NA in the SHR brain have been detected although the data are not in total agreement. We report here experiments utilizing single cell recording techniques which show directly a reduction in neuronal activity of brain NA neurons in the locus coeruleus (LC) of SHR. This reduction develops gradually with age and in parellel with the increased blood pressure (BP), but is not altered by acute alterations in BP. The SHR were found to display an increased intraneuronal monoamine oxidase (MAO) activity as well as a specifically reduced sensitivity of inhibitory alpha₂-receptors within the LC. It is suggested that in SHR the LC system, in spite of a reduced basal activity, displays increased responsiveness to sensory stimuli, a phenomenon that may contribute to the development of hypertension.     

CRH and the noradrenergic system mediate the antinociceptive effect of central interleukin-1α in the rat

After intracerebroventricular administration, both interleukin-1_α and corticotropin-releasing hormone increase nociceptive thresholds evaluated by the hot-plate test in the rat. Pretreatment with 6-hydroxydopamine or prazosin fully prevents the action of both substances. Moreover, the effect of interleukin-1_α is completely blocked by the intracerebroventricular administration of the corticotropin-releasing hormone antagonist α-helical CRH 9–41. Our results suggest an involvement of CRH and the noredrenergic system in the antinociceptive effect of central interleukin-1_α.