GAT-1 regulates both tonic and phasic GABA(A) receptor-mediated inhibition in the cerebral cortex

γ-Aminobutyric acid 1 (GAT-1) is the most copiously expressed GABA transporter; we studied its role in phasic and tonic inhibition in the neocortex using GAT-1 knockout (KO) mice. Immunoblotting and immunocytochemical studies showed that GAT-2 and GAT-3 levels in KOs were unchanged and that GAT-3 was not redistributed in KOs. Moreover, the expression of GAD65/67 was increased, whereas that of GABA or VGAT was unchanged. Microdialysis studies showed that in KOs spontaneous extracellular release of GABA and glutamate was comparable in WT and KO mice, whereas KCl-evoked output of GABA, but not of glutamate, was significantly increased in KOs. Recordings from layer II/III pyramids revealed a significant increase in GABA_AR-mediated tonic conductance in KO mice. The frequency, amplitude and kinetics of spontaneous inhibitory post-synaptic currents (IPSCs) were unchanged, whereas the decay time of evoked IPSCs was significantly prolonged in KO mice. In KO mice, high frequency stimulation of GABAergic terminals induced large GABA_AR-mediated inward currents associated with a reduction in amplitude and decay time of IPSCs evoked immediately after the train. The recovery process was slower in KO than in WT mice. These studies show that in the cerebral cortex of GAT-1 KO mice GAT-3 is not redistributed and GADs are adaptively changed and indicate that GAT-1 has a prominent role in both tonic and phasic GABA_AR-mediated inhibition, in particular during sustained neuronal activity.     

Anesthetic and postanesthetic effects of isoflurane on neuronal activity in the rat hippocampus

In this study, we explored anesthetic and postanesthetic effects of isoflurane on GABA-ergic and glutamatergic systems in the rat hippocampus. Our results demonstrate that different neuronal targets affected by isoflurane recover from anesthesia at dissimilar rates. Neirofiziologiya/Neurophysiology, Vol. 39, Nos. 4/5, pp. 371–373, July–October, 2007.     

Alternating sources of perisomatic inhibition during behavior

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Differential subcellular redistribution of protein kinase C isozymes in the rat hippocampus induced by kainic acid

Protein kinase C (PKC) consists of a family of Ca2+/phospholipid-dependent isozymes that has been implicated in the delayed neurotoxic effects of glutamate in vitro. In the present study, we assessed the effect of the glutamate analogue kainic acid (KA) on the subcellular expression of PKC isozymes in the hippocampus (HPC) in the period preceding (0.5, 1.5, 12, and 24 h) and during (120 h) hippocampal necrosis using western blot analysis and PKC isozyme-specific antibodies. Before subcellular fractionation (cytosol + membrane), hippocampi were microdissected into HPC (fields CA1-CA3) and dentate gyrus (DG; granule cells + hilus) regions. Four general patterns of alterations in PKC isozyme expression/distribution were observed following KA treatment. The first pattern was a relative stability in expression following KA treatment and was most apparent for cytosol PKCalpha (HPC + DG) and membrane (HPC) and cytosol (DG) PKCbetaII. The second pattern, observed with PKCgamma and PKCepsilon, was characterized by an initial increase in expression in both membrane and cytosolic fractions before seizure activity (0.5 h) followed by a gradual decrease until significant reductions are observed by 120 h. The third pattern, exhibited by PKCdelta, involved an apparent translocation, increasing in the membrane and decreasing in the cytosol, followed by down-regulation in both fractions and subsequent recovery. The fourth pattern was observed with PKCzeta only and entailed a significant reduction in expression before and during limbic motor seizures followed by a dramatic fivefold increase in the membrane fraction during the period of hippocampal necrosis (120 h). Although these patterns did not segregate according to conventional PKC isozyme classifications, they do indicate dynamic isozyme-specific regulation by KA. The subcellular redistribution of PKC isozymes may contribute to the histopathological sequelae produced by KA in the hippocampus and may model the pathogenesis associated with diseases involving glutamate-induced neurotoxicity.     

Aging in the rat hippocampus is associated with widespread reductions in the number of glutamate decarboxylase-67 positive interneurons but not interneuron degeneration

Increased excitability of principal excitatory neurons is one of the hallmarks of aging in the hippocampus, signifying a diminution in the number and/or function of inhibitory interneurons with aging. To elucidate this, we performed comprehensive GABA-ergic interneuron cell counts in all layers of the dentate gyrus and the CA1 and CA3 subfields, using serial sections from adult, middle-aged and aged Fischer 344 rats. Sections were immunostained for glutamate decarboxylase-67 (GAD-67, a synthesizing enzyme of GABA) and GAD-67 immunopositive interneurons were counted using an unbiased cell counting method, the optical fractionator. Substantial declines in the absolute number of GAD-67 immunopositive interneurons were found in all hippocampal layers/subfields of middle-aged and aged animals, in comparison with the adult animals. However, the counts were comparable between the middle-aged and aged groups for all regions. Interestingly, determination of the absolute number of interneurons using neuron-specific nuclear antigen (NeuN) expression in the strata oriens and radiatum of CA1 and CA3 subfields revealed an analogous number of interneurons across the three age groups. Furthermore, the ratio of GAD-67 immunopositive and NeuN positive interneurons decreased from adult age to middle age but remained relatively static between middle age and old age. Collectively, the results underscore that aging in the hippocampus is associated with wide-ranging decreases in the number of GAD-67 immunopositive interneurons and most of the age-related changes in GAD-67 immunopositive interneuron numbers transpire by middle age. Additionally, this study provides novel evidence that age-related reductions in hippocampal GAD-67 immunopositive interneuron numbers are due to loss of GAD-67 expression in interneurons rather than interneuron degeneration.     

Analysis of the major domains of the F fertility inhibition protein, FinO

The mechanism of fertility inhibition of conjugation by the F plasmid depends on the presence of both the FinO protein and an antisense RNA, FinP, which together control the expression of the positive regulator of the transfer operon TraJ. FinO both prevents the degradation of FinP, allowing its intracellular concentration to rise, and promotes duplex formation with its target, the traJ mRNA. In this study, deletions in finO were constructed and fused to gst, encoded by the pGEX-2T expression vector, to give GST-FinO fusions of varying lengths. These fusions were then tested for their ability to bind FinP and traJ mRNA, and to promote duplex formation. Our results suggest that the predicted basic N-terminal α-helix is involved in RNA binding, while the central domain is involved in duplex formation. The presence of the acidic C-terminal domain protects FinP from ribonucleolase degradation and might enhance binding of the N-terminal α-helical domain in a manner reminiscent of the Rom protein of ColE1. Received: 4 May 1998 / Accepted: 15 June 1998     

The alteration of γ-aminobutyric acid-transaminase expression in the gerbil hippocampus induced by seizure

It is well established that GABA degradation may play a key role in epileptogenesis. However, whether or not the expression of GABA-transaminase (GABA-T), which catalyzes GABA degradation and participates in the neuronal metabolism via GABA shunt, changes chronologically after on-set of seizure remains to be clarified. To identify the change of GABA-T expression in seizure, GABA-T expression in the gerbil hippocampus, associated with different sequelae of spontaneous seizures, was investigated. The distribution pattern of GABA-T immunoreactive neurons in the hippocampus between the seizure-resistant and pre-seizure group of seizure sensitive gerbils was similar. Interestingly, at 30 min postictal, the enhancement of GABA-T immunoreactivity in the perikarya was apparently observed. This contrasted with the decline in GABA-T immunoreactivity in the granular and pyramidal layer. At 12–24 h postictal, GABA-T immunoreactivity in the hilar neurons had declined significantly. However, the GABA-T immunoreactivity in the granular layer increased. These findings suggest that in the gerbil, the alteration in GABA-T expressions may play an important role in the self-recovery mechanism from seizure attack via both GABA degradation and regulation of neuronal metabolism.     

Haloperidol treatment at pre-exposure phase reduces the disturbance of latent inhibition in rats with neonatal ventral hippocampus lesions

Animals with neonatal ventral hippocampal lesions develop during or after adolescence abnormal behaviors related to schizophrenia such as anxiety and latent inhibition disruption. The aim of this study was to test whether haloperidol injection prior to pre-exposure session in the latent inhibition test would facilitate latent inhibition.     

(56)Fe-particle radiation reduces neuronal output and attenuates lipopolysaccharide-induced inhibition of long-term potentiation in the mouse hippocampus

Exposure to space radiation consisting of high-energy charged (56)Fe particles represents a significant health risk for astronauts. (56)Fe-particle radiation affects the synaptic plasticity of the hippocampus and alters its response to the experimental immunological stressor lipopolysaccharide (LPS). We previously showed in mice that 1 month after exposure to (56)Fe-particle radiation, the LPS-induced inhibition of hippocampal long-term potentiation (LTP) was significantly attenuated, resulting in seemingly normal LTP. In the current study, we investigated this phenomenon further at longer times postirradiation. We exposed mice to accelerated iron particles ((56)Fe; 600 MeV/nucleon; 1, 2, 4 Gy; brain only), and 1, 3, 6 or 12 months postirradiation we administered LPS. Four hours after the intraperitoneal LPS injection, we prepared hippocampal slices to measure synaptic excitability and plasticity between CA3-CA1 neurons. In unexposed mice, we confirmed that LPS inhibited LTP at all times. However, in mice exposed to 2 Gy, the LPS-induced LTP inhibition was attenuated and reversed to control values. Such reversal was evident at 1 and 3 months but not 6 and 12 months postirradiation. In addition, at 6 and 12 months postirradiation, we observed inhibition of population spike (PS) amplitudes at 4 Gy that correlated with decrements in dendritic potentials, suggesting synaptic damage. Our data show that (56)Fe-particle radiation affects the response of the hippocampus to an immunological stressor and that the alterations progress over time.     

GABA-ergic Inhibition in the CNS: Types of GABA Receptors and Mechanisms of Tonic GABA-Mediated Inhibitory Action

This review considers such aspects of the problem of GABA-ergic inhibition in the CNS as the fundamental molecular mechanisms of GABA-ergic synaptic transmission, current questions on the principles underlying the classification of the GABA receptors, the diversity of the types of GABA-ergic inhibition, as well as possible mechanisms and functional importance of the tonic GABA-mediated inhibitory action.     

8-[2-(2-pentyl-cyclopropylmethyl)-cyclopropyl]-octanoic acid stimulates GABA release from interneurons projecting to CA1 pyramidal neurons in the rat hippocampus via pre-synaptic alpha7 acetylcholine receptors

Nicotinic acetylcholine (ACh) receptors, such as alpha7, alpha3beta4 and alpha4beta2 receptors in the hippocampus, are suggested to modulate neurotransmitter release. 8-[2-(2-Pentyl-cyclopropylmethyl)-cyclopropyl]-octanoic acid (DCP-LA) (100 nM), a linoleic acid derivative, potentiated responses of alpha7, alpha3beta4 and alpha4beta2 ACh receptors expressed in Xenopus oocytes that are blocked by 3-(1-[dimethylaminopropyl] indol-3-yl)-4-[indol-3-yl] maleimide (GF109203X), a selective inhibitor of protein kinase C (PKC), except for alpha3beta4 ACh receptors. DCP-LA enhanced the nicotine-triggered release of GABA from rat hippocampal slices in the presence of tetrodotoxin in a bell-shaped dose-dependent manner at concentrations ranging from 10 nM to 10 microM, although DCP-LA by itself had no effect on GABA release. The DCP-LA action was inhibited by GF109203X or alpha-bungarotoxin, an inhibitor of alpha7 ACh receptors, but not by mecamylamine or dihydro-beta-erithroidine, an inhibitor of alpha3beta4 and alpha4beta2 ACh receptors. A similar effect on GABA release was obtained with 12-O-tetradecanoylphorbol 13-acetate, a PKC activator. DCP-LA (100 nM) also enhanced GABA release triggered by choline, an agonist of alpha7 ACh receptors, but not 3-[2(s)-azetidinylmethoxy] pyridine, an agonist of alpha4beta2 ACh receptors. In addition, DCP-LA (100 nM) increased the rate of nicotine-triggered GABA(A) receptor-mediated miniature inhibitory post-synaptic currents, monitored from CA1 pyramidal neurons of rat hippocampal slices, and the effect was also inhibited by GF109203X or alpha-bungarotoxin but not by mecamylamine. Thus, the results of the present study indicate that DCP-LA stimulates GABA release by enhancing activity of pre-synaptic alpha7 ACh receptors present on the GABAergic terminals of interneurons that transmit to CA1 pyramidal neurons via a PKC pathway.     

Temporal change in local forces and total force all over the surface of the sea urchin egg during cytokinesis

We determined the tension over the entire surface of the sea urchin eggs during cytokinesis, on the basis of the intracellular pressure and cell shape. This allowed us to determine the temporal changes in both the distribution of local forces and the total force produced in the whole cell cortex. A spike-like peak at anaphase and a broader peak at the onset of furrowing were observed in the time-course of the total force. Treatment of the eggs with cytochalasin D, blebbistatin, ML-9, or ML-7 significantly lowered the total force when they inhibited cytokinesis, suggesting that the tension results mainly from the interaction between intact actin filaments and activated myosin II. Myosin II would function as a motor, not only in the furrow region, but over a wide area of the cell surface, because the sum of the tensions outside the furrow region was larger than that inside the furrow region throughout cytokinesis. The distribution of the local force revealed that a global increase in the cortical force started well before the onset of furrowing, and that the force inside the furrow region continued to increase despite the decrease in the force outside the furrow region after the onset of furrowing. The spatial and temporal patterns of the force over the entire surface support the hypothesis that there are two separate but coordinated actomyosin activation mechanisms, one of which induces global activation of the cortex and the other of which then maintains the contractility only inside the furrow region.     

Sustained elevation of extracellular adenosine and activation of A1 receptors underlie the post-ischaemic inhibition of neuronal function in rat hippocampus in vitro

Adenosine is released from the compromised brain and exerts a predominately neuroprotective influence. However, the time-course of adenosine release and its relationship to synaptic activity during metabolic stress is not fully understood. Here, we describe experiments using an enzyme-based adenosine sensor to show that adenosine potently (IC50 approximately 1 microm) inhibits excitatory synaptic transmission in area CA1 during oxygen/glucose deprivation ('ischaemia'), and that the prolonged post-ischaemic presence of extracellular adenosine sustains the depression of the field excitatory postsynaptic potential (fEPSP). N-methyl-D-aspartate (NMDA) receptor antagonism promotes post-ischaemic recovery of the fEPSP, in parallel with reduced release of adenosine. Paradoxically, however, after ischaemia the fEPSP recovers in the face of concentrations of adenosine capable of fully eliminating synaptic transmission during ischaemia. This hysteresis is not prevented by NMDA receptor antagonism, is observed during repeated ischaemia when adenosine release is reduced, and does not reflect desensitization of adenosine A1 receptors. We conclude that adenosine exerts powerful inhibitory actions on excitatory synaptic transmission both during, and for some considerable time after, ischaemia. Therapeutic strategies designed to exploit both the continued presence of adenosine and activity of A1 receptors could provide benefits in individuals who have suffered acute injury to the CNS.     

Neural inhibition of egg-laying in the locust, Locusta migratoria

The role of the oviducal nerves during egg-laying in Locusta migratoria has been examined. Section of the oviducal nerves did not inhibit egg-laying in any observable way. Electrical stimulation of the oviducal nerves resulted in a contraction of the common and lower lateral oviducts which propelled ovulated eggs up towards the ovaries. Recordings from oviducal nerves using chronically implanted electrodes showed that electrical activity was low during actual egg-laying, but high at times when egg-laying was not occurring (i.e. during digging behaviour, or following interruption of egg-laying). During these periods of high activity recurrent bursts of action potentials occurred. Similar patterns of electrical activity were recorded in semi-intact preparations using suction electrodes applied to exposed oviducal nerves of locusts which had been interrupted during the process of egg-laying. High frequency bursts of activity were recorded simultaneously from both left and right oviducal nerves.It is concluded that one function of the oviducal nerves is to inhibit egg-laying at inappropriate times, by inducing contractions of the oviducts which propel eggs back towards the ovaries. These nerves therefore provide a physiological basis for part of the adaptive ovipositional activities of locusts.     

Cell-type-specific recruitment of GABAergic interneurons in the primary somatosensory cortex by long-range inputs

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Involvement of P2X7 receptors in the regulation of neurotransmitter release in the rat hippocampus

Although originally cloned from rat brain, the P2X7 receptor has only recently been localized in neurones, and functional responses mediated by these neuronal P2X7 receptors (P2X7 R) are largely unknown. Here we studied the effect of P2X7 R activation on the release of neurotransmitters from superfused rat hippocampal slices. ATP (1-30 mm) and other ATP analogues elicited concentration-dependent [3 H]GABA outflow, with the following rank order of potency: benzoylbenzoylATP (BzATP) > ATP > ADP. PPADS, the non-selective P2-receptor antagonist (3-30 microm), Brilliant blue G (1-100 nm) the P2X7 -selective antagonist and Zn2+ (0.1-30 microm) inhibited, whereas lack of Mg2+ potentiated the response by ATP. In situ hybridization revealed that P2X7 R mRNA is expressed in the neurones of the cell body layers in the hippocampus. P2X7 R immunoreactivity was found in excitatory synaptic terminals in CA1 and CA3 region targeting the dendrites of pyramidal cells and parvalbumin labelled structures. ATP (3-30 microm) and BzATP (0.6-6 microm) elicited concentration-dependent [14 C]glutamate efflux, and blockade of the kainate receptor-mediated transmission by CNQX (10-100 microm) and gadolinium (100 microm), decreased ATP evoked [3 H]GABA efflux. The Na+ channel blocker TTX (1 microm), low temperature (12 degrees C), and the GABA uptake blocker nipecotic acid (1 mm) prevented ATP-induced [3 H]GABA efflux. Brilliant blue G and PPADS also reduced electrical field stimulation-induced [3 H]GABA efflux. In conclusion, P2X7 Rs are localized to the excitatory terminals in the hippocampus, and their activation regulates the release of glutamate and GABA from themselves and from their target cells.     

Early events of target deprivation/axotomy-induced neuronal apoptosis in vivo: oxidative stress,DNA damage,p53 phosphorylation and subcellular redistribution of death proteins

The mechanisms of injury- and disease-associated apoptosis of neurons within the CNS are not understood. We used a model of cortical injury in rat and mouse to induce retrograde neuronal apoptosis in thalamus. In this animal model, unilateral ablation of the occipital cortex induces apoptosis of corticopetal projection neurons in the dorsal lateral geniculate nucleus (LGN), by 7 days post-lesion, that is p53 modulated and Bax dependent. We tested the hypothesis that this degenerative process is initiated by oxidative stress and early formation of DNA damage and is accompanied by changes in the levels of pro-apoptotic mediators of cell death. Immunoblotting revealed that the protein profiles of Bax, Bak and Bad were different during the progression of neuronal apoptosis in the LGN. Bax underwent a subcellular redistribution by 1 day post-lesion, while Bak increased later. Bad showed an early sustained increase. Cleaved caspase-3 was elevated maximally at 5 and 6 days. Active caspase-3 underwent a subcellular translocation to the nucleus. A dramatic phosphorylation of p53 was detected at 4 days post-lesion. DNA damage was assessed immunocytochemically as hydroxyl radical adducts (8-hydroxy-2-deoxyguanosine) and single-stranded DNA. Both forms of DNA damage accumulated early in target-deprived LGN neurons. Transgenic overexpression of superoxide dismutase-1 provided significant protection against the apoptosis but antioxidant pharmacotreatments with trolox and ascorbate were ineffective. We conclude that overlapping and sequential signaling pathways are involved in the apoptosis of adult brain neurons and that DNA damage generated by superoxide derivatives is an upstream mechanism for p53-regulated, Bax-dependent apoptosis of target-deprived neurons.