Activity-dependent regulation of inhibitory synaptic transmission in hippocampal neurons

Neural activity regulates the number and properties of GABAergic synapses in the brain, but the mechanisms underlying these changes are unclear. We found that blocking spike activity globally in developing hippocampal neurons from rats reduced the density of GABAergic terminals as well as the frequency and amplitude of miniature inhibitory postsynaptic currents (mIPSCs). Chronic inactivity later in development led to a reduction in the mIPSC amplitude, without any change in GABAergic synapse density. By contrast, hyperpolarizing or abolishing spike activity in single neurons did not alter GABAergic synaptic inputs. Suppressing activity in individual presynaptic GABAergic neurons also failed to decrease synaptic output. Our results indicate that GABAergic synapses are regulated by the level of activity in surrounding neurons. Notably, we found that the expression of GABAergic plasticity involves changes in the amount of neurotransmitter in individual vesicles.     

Effects of n-3 polyunsaturated fatty acids on COX-2 and PGE2 protein levels in articular cartilage chondrocytes

Introduction Previous studies within our laboratory have shown that supplementation with n‐3 polyunsaturated fatty acids (PUFAs), but not other fatty acids, has a beneficial effect on reducing the expression and activity of degradative and inflammatory factors known to cause damage and destruction of cartilage in arthritic diseases ( Curtis et al. 2000 , 2002 ). Cyclooxygenase (COX), also known as prostaglandin H synthase, catalyses the rate‐limiting step in the formation of inflammatory prostaglandins (PGs) ( Hla & Neilson 1992 ). PG synthesis requires conversion of arachidonic acid to PGH₂ by either the constitutive COX‐1 or by COX‐2, which is induced by inflammatory and mitogenic stimuli ( Smith et al. 2000 ). Prostaglandin E₂ (PGE₂) is produced from PGH₂ and has been shown to have a number of functions including both anti‐ and pro‐inflammatory actions ( Christman et al. 1991 ). The aim of this project was to investigate the effects of n‐3 PUFAs on cyclooxygenase‐2 and prostaglandin E₂ protein levels in articular cartilage chondrocytes. Materials and methods Articular cartilage was obtained both from 7‐day‐old bovine metacarpo‐metatarsophalangeal joints and from human patients undergoing total knee replacement surgery for osteoarthritis (Llandough Hospital, S.Wales, UK). Both explant and monolayer cultures were set up in DMEM with or without 10–300 µg/ml n‐3[eicosapentaenoic acid (EPA)] or n‐6[arachidonic acid (AA)] PUFAs (minimum 8 h, at 37°C, in 5%CO₂) and in the absence or presence of IL‐1 (10 ng/ml) for a further 4 days. Results Total RNA was extracted and RT‐PCR performed using oligonucleotide primers specific to COX‐2 (Invitrogen, UK). COX‐2 mRNA was found to be absent or only present at very low levels in both bovine and human control cultures. After treatment with IL‐1, this expression greatly increased. However, supplementation of IL‐1‐treated cultures with n‐3 PUFA (EPA) resulted in a loss of COX‐2 mRNA expression. In contrast, supplementation with n‐6 PUFA (AA) had no effect. Western blot analysis, using a polyclonal antibody specific to COX‐2 (Santa Cruz Biotechnology Inc., USA), showed that COX‐2 protein was absent in all control samples and the IL‐1 induction of bovine COX‐2 protein could also be reduced when supplemented with n‐3 PUFAs but not n‐6 PUFAs. Using a commercially available PGE₂ ELISA Immunoassay kit, it was possible to analyse the PGE₂ levels present in explant media from bovine or human samples. In one example, in a 74‐year‐old female patient, PGE₂ protein was very low in the cartilage cultured without IL‐1 treatment or PUFA supplementation (control). When the cartilage was treated with IL‐1, there was a huge induction of PGE₂ levels by as much as 60 fold. This induction is in turn reduced greatly with the supplementation of n‐3 PUFA (EPA) but not with n‐6 PUFA (AA). Discussion It has long been accepted that COX‐2 plays an important role in inflammation. COX‐2 has been found in joints affected by arthritic diseases ( Hla & Neilson 1992 ) and in cultures induced by IL‐1. The current study has shown that both the IL‐1‐induced COX‐2 message and protein levels can be reduced with supplementation by n‐3 PUFAs but not n‐6 PUFAs. This work has also shown that with a decrease in COX‐2 protein levels, there is also a corresponding decrease in prostaglandin E₂ levels caused by n‐3 PUFA supplementation.     

Protracted postnatal development of inhibitory synaptic transmission in rat hippocampal area CA1 neurons

In the CNS, inhibitory synaptic function undergoes profound transformation during early postnatal development. This is due to variations in the subunit composition of subsynaptic GABA(A) receptors (GABA(A)Rs) at differing developmental stages as well as other factors. These include changes in the driving force for chloride-mediated conductances as well as the quantity and/or cleft lifetime of released neurotransmitter. The present study was undertaken to investigate the nature and time course of developmental maturation of GABAergic synaptic function in hippocampal CA1 pyramidal neurons. In neonatal [postnatal day (P) 1-7] and immature (P8-14) CA1 neurons, miniature inhibitory postsynaptic currents (mIPSCs) were significantly larger, were less frequent, and had slower kinetics compared with mIPSCs recorded in more mature neurons. Adult mIPSC kinetics were achieved by the third postnatal week in CA1 neurons. However, despite this apparent maturation of mIPSC kinetics, significant differences in modulation of mIPSCs by allosteric agonists in adolescent (P15-21) neurons were still evident. Diazepam (1-300 nM) and zolpidem (200 nM) increased the amplitude of mIPSCs in adolescent but not adult neurons. Both drugs increased mIPSC decay times equally at both ages. These differential agonist effects on mIPSC amplitude suggest that in adolescent CA1 neurons, inhibitory synapses operate differently than adult synapses and function as if subsynaptic receptors are not fully occupied by quantal release of GABA. Rapid agonist application experiments on perisomatic patches pulled from adolescent neurons provided additional support for this hypothesis. In GABA(A)R currents recorded in these patches, benzodiazepine amplitude augmentation effects were evident only when nonsaturating GABA concentrations were applied. Furthermore nonstationary noise analysis of mIPSCs in P15-21 neurons revealed that zolpidem-induced mIPSC augmentation was not due to an increase in single-channel conductance of subsynaptic GABA(A)Rs but rather to an increase in the number of open channels responding to a single GABA quantum, further supporting the hypothesis that synaptic receptors may not be saturated during synaptic function in adolescent neurons. These data demonstrate that inhibitory synaptic transmission undergoes a markedly protracted postnatal maturation in rat CA1 pyramidal neurons. In the first two postnatal weeks, mIPSCs are large in amplitude, are slow, and occur infrequently. By the third postnatal week, mIPSCs have matured kinetically but retain distinct responses to modulatory drugs, possibly reflecting continued immaturity in synaptic structure and function persisting through adolescence.     

Isoflurane-induced impairment of synaptic transmission in hippocampal neurons

Summary The effects of anaesthetic applications of isoflurane were studied using intracellular recording techniques in 82 CA₁ neurons of in vitro hippocampal slice preparations (guinea pigs). Various parameters of their excitabilities such as membrane electrical properties, action potentials evoked by intracellular current pulse injections and spike afterhyperpolari-zations, as well as synaptic potentials evoked by electrical stimulation of stratum radiatum, were determined during bath perfusion of clinical concentrations of isoflurane which were measured with ¹⁹fluorine-nuclear magnetic resonance techniques. The vaporizer settings of 1–4% isoflurane corresponded to concentrations of 100 M to 500 M. Isoflurane applications did not produce consistent effects on the resting potentials or passive membrane properties. However, when spike-evoked synaptic activity was blocked by tetrodotoxin, isoflurane application induced a hyperpolarization (3–5 mV) without greatly affecting input conductance and the slopes of current-voltage relations. The threshold, amplitude and duration of single or multiple spikes evoked by current injections also were not greatly altered by isoflurane applications. However, marked reductions were observed in the amplitudes of the long-lasting hyperpolarizations following an evoked train of a constant number (4 or 5) of spikes. The amplitudes of excitatory postsynaptic potentials evoked by electrical stimulation of stratum radiatum were diminished markedly during isoflurane applications; these effects, like those on the afterhyperpolarizations, were closely dependent on the dose and duration of the application. Low doses (<1%) of isoflurane reduced the amplitudes of inhibitory postsynaptic potentials whereas higher doses (1–4%) increased their amplitudes and durations. The effects on afterhyperpolarizations and synaptic potentials could not be attributed to anaesthetic related changes in the resting potentials of the neurons. The investigations provide evidence for presynaptic, as well as postsynaptic, sites of isoflurane actions in hippocampal neurons which presumably may involve direct alterations in intracellular Ca²⁺-disposition or inward Ca²⁺-currents.     

Presynaptic plasma membrane Ca2+ ATPase isoform 2a regulates excitatory synaptic transmission in rat hippocampal CA3

Plasma membrane calcium ATPase isoforms (PMCAs) are expressed in a wide variety of tissues where cell-specific expression provides ample opportunity for functional diversity amongst these transporters. The PMCAs use energy derived from ATP to extrude submicromolar concentrations of intracellular Ca²⁺ ([Ca²⁺]_i) out of the cell. Their high affinity for Ca²⁺ and the speed with which they remove [Ca²⁺]_i depends upon splicing at their carboxy (C)-terminal site. Here we provide biochemical and functional evidence that a brain-specific, C-terminal truncated and therefore fast variant of PMCA2, PMCA2a, has a role at hippocampal CA3 synapses. PMCA2a was enriched in forebrain synaptosomes, and in hippocampal CA3 it colocalized with the presynaptic marker proteins synaptophysin and the vesicular glutamate transporter 1, but not with the postsynaptic density protein PSD-95. PMCA2a also did not colocalize with glutamic acid decarboxylase-65, a marker of GABA-ergic terminals, although it did localize to a small extent with parvalbumin-positive presumed inhibitory terminals. Pharmacological inhibition of PMCA increased the frequency but not the amplitude of mEPSCs with little effect on mIPSCs or paired-pulse depression of evoked IPSCs. However, inhibition of PMCA activity did enhance the amplitude and slowed the recovery of paired-pulse facilitation (PPF) of evoked EPSCs. These results indicated that fast PMCA2a-mediated clearance of [Ca²⁺]_i from presynaptic excitatory terminals regulated excitatory synaptic transmission within hippocampal CA3.     

Transient enhancement of inhibitory synaptic transmission in hippocampal CA1 pyramidal neurons after cerebral ischemia

Pyramidal neurons in hippocampal CA1 regions are highly sensitive to cerebral ischemia. Alterations of excitatory and inhibitory synaptic transmission may contribute to the ischemia-induced neuronal degeneration. However, little is known about the changes of GABAergic synaptic transmission in the hippocampus following reperfusion. We examined the GABA_A receptor-mediated inhibitory postsynaptic currents (IPSCs) in CA1 pyramidal neurons 12 and 24 h after transient forebrain ischemia in rats. The amplitudes of evoked inhibitory postsynaptic currents (eIPSCs) were increased significantly 12 h after ischemia and returned to control levels 24 h following reperfusion. The potentiation of eIPSCs was accompanied by an increase of miniature inhibitory postsynaptic current (mIPSC) amplitude, and an enhanced response to exogenous application of GABA, indicating the involvement of postsynaptic mechanisms. Furthermore, there was no obvious change of the paired-pulse ratio (PPR) of eIPSCs and the frequency of mIPSCs, suggesting that the potentiation of eIPSCs might not be due to the increased presynaptic release. Blockade of adenosine A1 receptors led to a decrease of eIPSCs amplitude in post-ischemic neurons but not in control neurons, without affecting the frequency of mIPSCs and the PPR of eIPSCs. Thus, tonic activation of adenosine A1 receptors might, at least in part, contribute to the enhancement of inhibitory synaptic transmission in CA1 neurons after forebrain ischemia. The transient enhancement of inhibitory neurotransmission might temporarily protect CA1 pyramidal neurons, and delay the process of neuronal death after cerebral ischemia.     

Regulation of Newborn and Adult Monocytes by PGE2: Evidence of Comparable Differentiation Status

PGE₂, which is produced in large amounts by monocytes, is considered to be an important autocrine factor in the regulation of the immune response. Altered sensitivity to PGE₂ is known to reflect the differentiation status of monocytes/macrophages. Since newborn monocytes are thought to be less differentiated than adult peripheral blood monocytes, this study was designed to determine whether the diminished newborn monocyte function, relative to adult peripheral blood monocytes, might be the result of an altered autocrine responsiveness to PGE₂. Phosphodiesterase activity as well as cAMP and TNFα levels in response to PGE₂ were measured in newborn and adult monocytes, as an index of sensitivity to PGE₂. Basal intracellular cAMP levels were reduced in cord monocytes compared with adult monocytes. Addition of increasing concentrations of PGE₂ (10  m ⁻¹⁰–10  m ⁻⁶) caused similar fold increases in intracellular levels of cAMP for both populations. Lipopolysaccharide-induced TNFα production was significantly reduced in a similar fashion for both cord and adult monocytes at high (≥ 10⁻⁷  m ) PGE₂ concentration. Phosphodiesterase activity was comparable in lysates from both populations. This study shows that PGE₂ does not differentially influence newborn and adult monocyte responses and therefore that the diminished functional abilities of newborn monocytes is not due to altered autocrine responsiveness.     

Human alcohol dehydrogenase ADH1, ADH2 and ADH3 loci in a mixed population of Bahia, Brazil

1. The three structural gene loci of human alcohol dehydrogenase have been studied in liver, jejunum and lung from 300 newborns in a triracially mixed population of Bahia, Brazil. 2. The frequency of the ADH23 allele was 0-1392, suggesting that the ADH23 allele is less frequent in Negroes. 3. A new ADH2 variant was identified. The electrophoretic pattern was interpreted as due to a new allele which is provisionally called ADH2Bahia. 4. By electrophoretic classification the 'atypical' variant was found in 2-8% of the sample. A question is raised regarding the ancestral origin of the 'atypical' variant in the population. Because this variant is common in Japanese it may have reached the present day population of Bahia through their American Indian ancestors. 5. Subjective estimation of the proportions of beta chains by giving scores to the liver isozymes alphaalpha, alphabeta and betabeta showed a clear relationship between the fetal weight and the beta chain activity. 6. The proportion of beta chains in the liver is significantly less when there is no enzyme activity in the lung, indicating some synchronous 'turning on' mechanism for alcohol dehydrogenase synthesis in both tissues.     

Enhanced G protein-dependent modulation of excitatory synaptic transmission in the cerebellum of the Ca2+ channel-mutant mouse, tottering

Tottering , a mouse model for absence epilepsy and cerebellar ataxia, carries a mutation in the gene encoding class A (P/Q-type) Ca²⁺ channels, the dominant exocytotic Ca²⁺ channel at most synapses in the mammalian central nervous system. Comparing tottering to wild-type mice, we have studied glutamatergic transmission between parallel fibres and Purkinje cells in cerebellar slices. Results from biochemical assays and electrical field recordings demonstrate that glutamate release from parallel fibre terminals of the tottering mouse is controlled largely by class B Ca²⁺ channels (N-type), in contrast to the P/Q-channels that dominate release from wild-type terminals. Since N-channels, in a variety of assays, are more effectively inhibited by G proteins than are P/Q-channels, we tested whether synaptic transmission between parallel fibres and Purkinje cells in tottering mice was more susceptible to inhibitory modulation by G protein-coupled receptors than in their wild-type counterparts. GABA_B receptors and α₂-adrenergic receptors (activated by bath application of transmitters) produced a three- to fivefold more potent inhibition of transmission in tottering than in wild-type synapses. This increased modulation is likely to be important for cerebellar transmission in vivo , since heterosynaptic depression, produced by activating GABAergic interneurones, greatly prolonged GABA_B receptor-mediated presynaptic inhibition in tottering as compared to wild-type slices. We propose that this enhanced modulation shifts the balance of synaptic input to Purkinje cells in favour of inhibition, reducing Purkinje cell output from the cerebellum, and may contribute to the aberrant motor phenotype that is characteristic of this mutant animal.     

Slow excitatory synaptic transmission mediated by P2Y1 receptors in the guinea-pig enteric nervous system

Electrophysiological recording was used to study a type of slow excitatory postsynaptic potential (slow EPSP) that was mediated by release of ATP and its action at P2Y₁ receptors on morphologically identified neurones in the submucosal plexus of guinea-pig small intestine. MRS2179, a selective P2Y₁ purinergic receptor antagonist, blocked both the slow EPSP and mimicry of the EPSP by exogenously applied ATP. Increased conductance accounted for the depolarization phase of the EPSP, which occurred exclusively in neurones with S-type electrophysiological behaviour and uniaxonal morphology. The purinergic excitatory input to the submucosal neurones came from neighbouring neurones in the same plexus, from neurones in the myenteric plexus and from sympathetic postganglionic neurones. ATP-mediated EPSPs occurred coincident with fast nicotinic synaptic potentials evoked by the myenteric projections and with noradrenergic IPSPs evoked by sympathetic fibres that innervated the same neurones. The P2Y₁ receptor on the neurones was identified as a metabotropic receptor linked to activation of phospholipase C, synthesis of inositol 1,4,5-trisphosphate and mobilization of Ca²⁺ from intracellular stores.     

Phospholipase A2 activation enhances inhibitory synaptic transmission in rat substantia gelatinosa neurons

Phospholipase A(2) (PLA(2)) activation enhances glutamatergic excitatory synaptic transmission in substantia gelatinosa (SG) neurons, which play a pivotal role in regulating nociceptive transmission in the spinal cord. By using melittin as a tool to activate PLA(2), we examined the effect of PLA(2) activation on spontaneous inhibitory postsynaptic currents (sIPSCs) recorded at 0 mV in SG neurons of adult rat spinal cord slices by use of the whole cell patch-clamp technique. Melittin enhanced the frequency and amplitude of GABAergic and glycinergic sIPSCs. The enhancement of GABAergic but not glycinergic transmission was largely depressed by Na(+) channel blocker tetrodotoxin or glutamate-receptor antagonists (6-cyano-7-nitroquinoxaline-2,3-dione and/or dl-2-amino-5-phosphonovaleric acid) and also in a Ca(2+)-free Krebs solution. The effects of melittin on glycinergic sIPSC frequency and amplitude were dose-dependent with an effective concentration of approximately 0.7 microM for half-maximal effect and were depressed by PLA(2) inhibitor 4-bromophenacyl bromide or aristolochic acid. The melittin-induced enhancement of glycinergic transmission was depressed by lipoxygenase inhibitor nordihydroguaiaretic acid but not cyclooxygenase inhibitor indomethacin. These results indicate that the activation of PLA(2) in the SG enhances GABAergic and glycinergic inhibitory transmission in SG neurons. The former action is mediated by glutamate-receptor activation and neuronal activity increase, possibly the facilitatory effect of PLA(2) activation on excitatory transmission, whereas the latter action is due to PLA(2) and subsequent lipoxygenase activation and is independent of extracellular Ca(2+). It is suggested that PLA(2) activation in the SG could enhance not only excitatory but also inhibitory transmission, resulting in the modulation of nociception.