Anisatin modulation of temperature-dependent inhibition of [H]muscimol binding by chloride ion

We investigated the effects of anisatin, a pure toxic substance isolated from Illicium anisatum, and chloride ion on [³H]muscimol binding to rat brain membranes at various temperatures (0–16°C). Chloride ion (100 mM) itself slightly inhibited specific [³H]muscimol binding at 0–4°C and this inhibition was more marked at 10 or 16°C than at 0–4°C. Anisatin further decreased [³H]muscimol binding inhibited by 100 mM chloride at 16°C without affecting the basal specific binding. These effects of anisatin were also dependent on the temperature. Scatchard analysis revealed that the effects of chloride and chloride plus anisatin on [³H]muscimol binding were mainly due to reduction in the number of binding sites. The temperature- and chloride-dependent effects of anisatin were similar to those of picrotoxinin. Pre-treatment of membranes with 0.05% Triton X-100 markedly decreased the inhibitory effects of chloride and chloride plus anisatin.Our results indicate that anisatin affects the number of GABA receptor sites probably through the picrotoxin sensitive sites and that chloride ion not only modifies the GABA receptors but also plays an important role in the action of anisation and picrotoxinin.     

Biosynthesis of progesterone derived neurosteroids by developing avian CNS : in vitro effects on the gabaa receptor complex

It has been demonstrated in different vertebrate species that the GABA_A receptor complex is modulated by certain steroids. Theses results prompted work on the synthesis of these neurosteroids in the Central Nervous System. However, there are scarcely any studies analyzing their production or their modulatory effects on this receptor during development. In this work, the biosynthesis of [[14]C]progesterone metabolites as well as the characterization of their in vitro effects on the GABA_A receptor complex in developing chick optic lobe were investigated. Studies on progesterone metabolism indicated that this steroid was converted to 5β-pregnanedione, 5β-pregan-3β-ol-20-one, and a 20-hydroxy derivative. Radioactive progesterone was completely metabolized at early embryonic stages, and a great proportion of 5β-pregnanedione was converted to 5β-pregnan-3β-ol-20-one. Thus, it seems that some of the steroidogenic activities present in chick optic lobe are age-dependent, though greater at embryonic stages. Results from in vitro modulation of [[3]H]flunitrazepam binding by 5β-pregnan-3β-ol-20-one indicated that this steroid produces a one-component-concentration dependent enhancement above control binding. 5β-pregnan-3β-ol-20-one EC₅₀ values were 0.195±0.049, 0.101±0.017, 0.147±0.009, and 0.569±0.114 μM, and E_max were 22.37±1.57, 23.67±4.02, 29.01±1.08, and 15.11±2.67% at embryonic days 11, 14, hatching, and postnatal day 21, respectively.In conclusion, the biosynthesis of 5β-pregnan-3β-ol-20-one from progesterone in developing chick optic lobe, together with its ability to modulate the GABA_A receptor present in such tissues, suggests a physiological role of this neurosteroid in developing avian Central Nervous System.     

Enantioselective modulation of GABAergic synaptic transmission by steroids and benz[e]indenes in hippocampal microcultures

The effects of enantiomers of the neurosteroid analogues, 3α-hydroxy-5α-pregnan-20-one (DHP) and 3α-hydroxy-5α-androstane-17β-carbonitrile (ACN), and the benz[e]indene, BI-1, on synaptic currents were examined in microcultures of rat hippocampal neurons. Over the range of 0.1–10 μM, the (+)-enantiomers were more potent and effective than their (−)-enantiomeric counterparts in enhancing γ-aminobutyric acid (GABA)_A receptor-mediated evoked synaptic currents. The (+)-enantiomers had small effects on peak currents, but slowed the decay of inhibitory synaptic currents, resulting in 2–3-fold increases in charge transfer during inhibitory synaptic events at 10 μM. Similar prolongations of spontaneous miniature inhibitory postsynaptic currents (IPSCs) and responses to brief GABA pulses to outside-out patches suggest that the prolongations of evoked synaptic currents result primarily from postsynaptic effects. In contrast, the (−)-enantiomers had little effect on evoked IPSCs at concentrations ≤1 μM, but enhanced inhibitory transmission at 10 μM. At concentrations ≤1 μM, neither the (+)- nor (−)-enantiomers altered glutamate-mediated excitatory synaptic currents. At 10 μM, (+)-DHP and (+)-ACN depressed excitatory responses in a bicuculline-sensitive fashion, suggesting that direct chloride channel gating by the steroids contributed to the depression. These data indicate that certain steroids and benz[e]indenes augment inhibitory synaptic transmission enantioselectively and provide strong support for the hypothesis that steroids act at specific sites on synaptic GABA_A receptors rather than via alteration of membrane lipids. Synapse 29:162–171, 1998. © 1998 Wiley-Liss, Inc.     

Patterns of cation-chloride cotransporter expression during embryonic rodent CNS development

Intracellular Cl- plays a key role in cellular volume regulation, cell cycle control and shaping the polarity of inhibitory postsynaptic responses mediated by anion-permeable GABA and glycine receptors. In this study, we have investigated the expression patterns of members of the cation-chloride cotransporters (CCCs), including the K-Cl cotransporters KCC1-4 and the Na-K-2 Cl cotranporter NKCC1 during rodent embryonic brain development. At the time of neurogenesis (embryonic days; E12.5-14.5), KCC1 was only detectable in the developing choroid plexus. KCC2 mRNA was detectable as early as E12.5 in the ventral part of the (cervical) spinal cord, and by E14.5, the expression had spread to TUJ1-positive differentiating regions of the rhombencephalon, diencephalon and olfactory bulb, in parallel with neuronal maturation. KCC3 mRNA was scarce in the cortical plate at E14.5, and slightly up-regulated at birth. In contrast, KCC4 mRNA was abundantly expressed in the ventricular zone and was down-regulated perinatally. At E14.5, NKCC1 was highly expressed in the vimentin-positive radial glia of the proliferative zone of the subcortical region. At later embryonic stages, during gliogenesis (E17-P0), there was a shift in NKCC1 expression to the neuron specific Class III beta-tubulin (betaIII) positive region of the cortical plate. These unique spatiotemporal expression patterns of distinct CCCs during embryonic development suggests that Cl- regulatory mechanisms are critically involved in the control of neuronal development.     

Antenatal hypoxia: Involvement in the development of CNS pathologies during ontogeny

We summarize the data describing the effects of antenatal hypoxia on the development of the fetal CNS. Hypoxia is one of the most widespread and clinically important stress factors, which may affect embryonic development and result in embryotoxic consequences and various developmental pathologies. Acute hypoxia may appear at different stages of pregnancy as an unspecific side effect of toxicosis, cardiovascular and respiratory diseases, smoking, or alcohol consumption, among other causes. Antenatal hypoxia plays a leading role in the delay of the biological development of the CNS and higher functions of the brain. Original Russian Text ? A.V. Graf, E.N. Goncharenko, N.A. Sokolova, I.P. Ashmarin, 2008, published in Neirokhimiya, 2008, Vol. 25, Nos. 1–2, pp. 11–16.     

Fipronil modulation of glutamate-induced chloride currents in cockroach thoracic ganglion neurons

Fipronil is the first phenylpyrazole insecticide introduced for pest control. It is effective against some insects that have become resistant to other insecticides, and exhibits low mammalian toxicity. Although fipronil is known to block GABA receptors, the mechanisms of its selective toxicity and efficacy against insects with dieldrin-resistant GABA receptors are not fully understood. We studied the effects of fipronil on the inhibitory glutamate receptor-chloride channel complex, which is found only in invertebrates. Glutamate-activated chloride currents were recorded from neurons isolated from cockroach thoracic ganglia using the whole-cell patch clamp technique. When glutamate was applied to a neuron, it evoked inward currents with an EC50 of 36.8 +/- 3.0 microM and a Hill coefficient of 1.56 +/- 0.17. The similarity between the reversal potential and the calculated chloride equilibrium potential indicated that glutamate-induced currents were carried by chloride ions. Fipronil suppressed the glutamate-induced peak currents in a dose-dependent manner with an IC50 of 0.73 +/- 0.27 microM and a Hill coefficient of 0.68 +/- 0.15. The current decay phases were greatly prolonged after fipronil application in a concentration-dependent manner. Picrotoxinin (PTX) at 100 microM slightly suppressed glutamate-induced currents to 87.8 +/- 3.7% of the control, and dieldrin at 100 microM had no effect (96.7 +/- 3.1%). AP5 and CNQX, mammalian glutamate receptor antagonists, were without effect on glutamate-induced Cl- currents. It is concluded that the potent blocking action of fipronil against glutamate-gated chloride channels may contribute to the higher toxicity against insects than mammals, as well as the efficacy against insects resistant to other insecticides.     

Regulation of KCC2 and NKCC during development: membrane insertion and differences between cell types

The developmental switch of GABA's action from excitation to inhibition is likely due to a change in intracellular chloride concentration from high to low. Here we determined if the GABA switch correlates with the developmental expression patterns of KCC2, the chloride extruder K+-Cl- cotransporter, and NKCC, the chloride accumulator Na+-K+-Cl- cotransporter. Immunoblots of ferret retina showed that KCC2 upregulated in an exponential manner similar to synaptophysin (a synaptic marker). In contrast, NKCC, which was initially expressed at a constant level, upregulated quickly between P14 and P28, and finally downregulated to an adult level that was greater than the initial phase. At the cellular level, immunocytochemistry showed that in the inner plexiform layer KCC2's density increased gradually and its localization within ganglion cells shifted from being primarily in the cytosol (between P1-13) to being in the plasma membrane (after P21). In the outer plexiform layer, KCC2 was detected as soon as this layer started to form and increased gradually. Interestingly, however, KCC2 was initially restricted to photoreceptor terminals, while in the adult it was restricted to bipolar dendrites. Thus, the overall KCC2 expression level in ferret retina increases with age, but the time course differs between cell types. In ganglion cells the upregulation of KCC2 by itself cannot explain the relatively fast switch in GABA's action; additional events, possibly KCC2's integration into the plasma membrane and downregulation of NKCC, might also contribute. In photoreceptors the transient expression of KCC2 suggests a role for this transporter in development.     

Metallothionein 1+2 protect the CNS during neuroglial degeneration induced by 6-aminonicotinamide

6-Aminonicotinamide (6-AN) is a niacin antagonist, which leads to degeneration of gray matter astrocytes. Metallothionein 1+2 (MT-1+2) are neuroprotective factors in the central nervous system (CNS), and to determine the roles for MT after 6-AN, we have examined transgenic mice overexpressing MT-1 (TgMTI* mice) after an i.p. injection with 6-AN. In control mice injected with 6-AN, astrocytes in specific gray matter areas of the brainstem showed degeneration. Reactive astrocytes surrounded the degenerated areas, which were heavily infiltrated by macrophages and T lymphocytes. MT-1+2 expression was significantly decreased in the damaged brainstem areas, but it increased in reactive astrocytes surrounding these areas and also in infiltrating macrophages. The levels of oxidative stress, as determined by immunoreactivity for inducible nitric-oxide synthase (iNOS), malondialdehyde (MDA), and nitrotyrosine (NITT), and the number of terminal deoxynucleotidyl transferase [TdT]-mediated deoxyuridine triphosphate [dUTP]-digoxigenin nick end labeling-positive (TUNEL+), caspase-3+ apoptotic cells were significantly increased in the brainstem of normal mice after 6-AN. In the TgMTI* mice, the 6-AN-induced tissue damage was decreased in comparison to control mice, and they showed significantly reduced numbers of recruited macrophages and T lymphocytes, and a drastic reduction of oxidative stress and apoptotic cell death. In addition, the accompanying reactive astrogliosis was increased in the transgenic mice. To further study the potential protective role of MT, we administered intraperitoneally Zn-MT-2 to 6-AN-injected normal mice and found essentially the same results as those obtained in TgMTI* mice. Thus, we hereby report that endogenous MT-1 overexpression and exogenous MT-2 treatment have significant neuroprotective roles during CNS pathological conditions.     

Regional changes in phenylethanolamine-N-Methyltransferase of rat brain during development

The changes of phenylethanolamine-N-methyltransferase (PNMT, EC 2.1.1.28), the enzyme that catalyzes the final step in the biosynthesis of adrenaline, were studied during the development of several regions of rat brain. PNMT is present in medulla oblongata-pons, hypothalamus, cerebellum, and midbrain five days before birth, and a progressive increase in the enzyme activity is observed during development. The adult levels are attained between 15 and 20 days, depending on the region. The increases in PNMT activity in the rostral regions are higher than in the caudal regions. PNMT attains adult levels earlier than tyrosine hydroxylase and dopamine-β-hydroxylase. The apparatus for adrenaline synthesis seems to be mature at three weeks after birth in the medulla oblongata-pons, which contains the cell bodies of adrenaline-containing neurons. In the other regions the adult levels of enzyme activity are attained at 15 days after birth.     

Excitotoxic death induced by released glutamate in depolarized primary cultures of mouse cerebellar granule cells is dependent on GABAA receptors and niflumic acid-sensitive chloride channels

Excitotoxic neuronal death has been linked to neurological and neurodegenerative diseases. Several studies have sought to clarify the involvement of Cl(-) channels in neuronal excitotoxicity using either N-methyl-D-aspartic acid (NMDA) or alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate/kainic acid agonists. In this work we induced excitotoxic death in primary cultures of cerebellar granule cells by means of endogenously released glutamate. Excitotoxicity was provoked by exposure to high extracellular K(+) concentrations ([K(+)](o)) for 5 min. Under these conditions, a Ca(2+)-dependent release of glutamate was evoked. When extracellular glutamate concentration rose to between 2 and 4 microM, cell viability was significantly reduced by 30-40%. The NMDA receptor antagonists (MK-801 and D-2-amino-5-phosphonopentanoic acid) prevented cell death. Exposure to high [K(+)](o) produced a (36)Cl(-) influx which was significantly reduced by picrotoxinin. In addition, the GABA(A) receptor antagonists (bicuculline, picrotoxinin and SR 95531) protected cells from high [K(+)](o)-triggered excitotoxicity and reduced extracellular glutamate concentration. The Cl(-) channel blockers niflumic acid and 5-nitro-2-(3-phenylpropylamino)benzoic acid also exerted a neuroprotective effect and reduced extracellular glutamate concentration, even though they did not reduce high [K(+)](o)-induced (36)Cl(-) influx. Primary cultures of cerebellar granule cells also contain a population of GABAergic neurons that released GABA in response to high [K(+)](o). Chronic treatment of primary cultures with kainic acid abolished GABA release and rendered granule cells insensitive to high [K(+)](o) exposure, even though NMDA receptors were functional. Altogether, these results demonstrate that, under conditions of membrane depolarization, low micromolar concentrations of extracellular glutamate might induce an excitotoxic process through both NMDA and GABA(A) receptors and niflumic acid-sensitive Cl(-) channels.     

CNS pattern of metabolic activity during tonic pain: evidence for modulation by beta-endorphin

CNS correlates of acute prolonged pain, and the effects of partial blockade of the central beta-endorphin system, were investigated by the quantitative 2-deoxyglucose technique in unanaesthetized, freely moving rats. Experiments were performed during the second, tonic phase of the behavioural response to a prolonged chemical noxious stimulus (s.c. injection of dilute formalin into a forepaw), or after minor tissue injury (s.c. saline injection). During formalin-induced pain, local glucose utilization rates in the CNS were bilaterally increased in the grey matter of the cervical spinal cord, in spinal white matter tracts and in several supraspinal structures, including portions of the medullary reticular formation, locus coeruleus, lateral parabrachial region, anterior pretectal nucleus, the medial, lateral and posterior thalamic regions, basal ganglia, and the parietal, cingulate, frontal, insular and orbital cortical areas. Pretreatment with anti-beta-endorphin antibodies, injected i.c.v., led to increased metabolism in the tegmental nuclei, locus coeruleus, hypothalamic and thalamic structures, putamen, nucleus accumbens, diagonal band nuclei and dentate gyrus, and in portions of the parietal, cingulate, insular, frontal and orbital cortex. In formalin-injected rats, pretreated with anti-beta-endorphin, behavioural changes indicative of hyperalgesia (increased licking response) were found, which were paralleled by a significant enhancement of functional activity in the anterior pretectal nucleus and in thalamo-cortical systems. A positive correlation was found between the duration of the licking response and metabolic activity of several forebrain regions. These results provide a map of the CNS pattern of metabolic activity during tonic somatic pain, and demonstrate a modulatory role for beta-endorphin in central networks that process somatosensory inputs.     

The GABAA receptor channel mediated chloride ion translocation through the plasma membrane: New insights from 36Cl− ion flux measurements

GABA_A receptors in plasma membranes of neurons are integral oligomers which form chloride channels. The binding of GABA molecules at recognition sites for channel opening triggers a transient increase in transmembrane chloride ion flux. The multiplicity and drug specificity of GABA_A receptor, kinetics of channel opening, and desensitization of GABA_A receptor and its short- and long-term regulation have been investigated by the use of tracer amounts of the radioactive chloride isotope, ³⁶Cl^? ion. Results and new insights from ³⁶Cl^? ion flux measurements have been reviewed. © 1993 Wiley-Liss, Inc.     

Experience influences environmental modulation of function at the benzodiazepine (BZD)/GABA receptor chloride channel complex

Function of the benzodiazepine (BZD)/GABA receptor chloride channel complex was selectively altered by specific aspects of an environmental challenge, i.e. encountering a stranger in a familiar versus an unfamiliar environment. Chloride (Cl-) enhancement of [3H]flunitrazepam [( 3H]flu) binding was facilitated in rats tested in an unfamiliar environment relative to that in rats tested in a familiar environment. Basal [3H]flu binding (binding in the absence of NaCl) also was greater in rats tested in the unfamiliar environment than in rats tested in the familiar environment, and Scatchard analysis of [3H]flu binding indicated that increased [3H]flu binding in the unfamiliar environment reflected an increase in both binding affinity and maximal binding capacity. In addition, both the sensitivity of [3H]flu binding to Cl- and the affinity of BZD recognition sites were decreased in handled control rats relative to non-handled control rats as well as to environmentally-challenged (prehandled) rats, suggesting that the experience of daily handling as well as familiarization with the environment modulates function at the BZD/GABA receptor complex. GABA-mediated 36Cl- uptake was facilitated by testing in either the familiar or unfamiliar environment relative to that measured in non-handled control rats. Thus, changes in GABA-gated chloride channel function may reflect a more fundamental response of this complex to challenging situations. These findings suggest that components of the BZD/GABA receptor complex are differentially influenced by specific aspects of an environmental challenge. Furthermore, function at the BZD recognition site/chloride channel component of this receptor complex is influenced by both repeated and single exposure to specific environments.     

Immunohistochemical and biochemical analysis of N-cadherin expression during CNS development.

The expression of the calcium-dependent adhesion molecule N-cadherin during chick embryo central nervous system (CNS) development was examined by immunohistochemistry and electrophoresis and immunoblotting. During histogenesis, N-cadherin is expressed at high levels in a uniform fashion in many regions of the CNS. However, during later stages of development, expression becomes restricted to the ependymal cells lining the ventricular system and in the choroid plexus. This down-regulation was confirmed by both immunohistochemical and biochemical techniques. The program of expression lags behind in the cerebellum in concert with the delayed development of this region of the brain. A high level of N-cadherin was found to be expressed in the brainstem and spinal cord floorplate, while a low level was detected at the optic nerve head. The results indicate that while, in general, the program of N-cadherin expression is similar in the retina and the brain, certain structures unique to the eye and brain express locally high or low levels of this adhesion protein.     

Expression of sodium channel subtypes during development in rat skeletal muscle.

This study contrasts the developmental patterns of expression of 2 subtypes of the voltage-dependent sodium channel in rat muscle that are differentiated by their immunoreactivity with monoclonal antibodies raised to the purified muscle sodium channel protein. One subtype is found in the transverse tubular (T) system of slow twitch fibers as well as the plasma membrane of fast and slow twitch fibers in the anterior tibial and soleus muscles. The second is present in the plasma membrane in all fibers of both muscles. The transverse tubular subtype exhibits 2 immunocytochemical staining patterns within muscle fibers, reticular and homogeneous, which may represent labeling of the developing T tubular system and of a cytoplasmic pool of alpha subunits of the sodium channel respectively. The reticular pattern eventually disappears in fast twitch fibers but persists into the adult stage in slow twitch fibers. The homogeneous pattern is also seen with antibodies to the plasma membrane subtype and disappears in early development as immunoreactivity to both subtypes gradually appears in the surface membrane. A reticular pattern is never seen with the plasma membrane subtype. The factors that modulate the expression of these subtypes is unknown.     

DNA fragmentation in normal development of the human central nervous system: a morphological study during corticogenesis

Refinement of the cell number by programmed cell death is a major morphogenetic mechanism of the developing central nervous system (CNS) in vertebrates including mammals, which determines to a significant degree its mature cytoarchitecture. We have examined the topography and the extent of cell death in different regions of the human CNS prenatally (11 fetuses), and in the early post‐natal weeks (three newborns). Attention was focused on the wall of the telencephalon during a relatively short time period (12th–23rd week of gestation), corresponding to the time of major proliferation in the ventricular zone and to the peak of neuronal migration; both these mechanisms are crucial for corticogenesis. The TUNEL method was used, allowing the recognition of cell death because of its ability to label blunt ends of double‐stranded DNA breaks. Morphological features of nuclei at different stages of apoptosis were identified, providing better evidence of the extent of the process than histological stains. Cell labelling was seen in either post‐mitotic elements in the ventricular zone, or along the migratory pathways in the intermediate zone and subplate at all prenatal ages examined. No apoptotic nuclei were seen in the cortical plate. These findings suggest that apoptotic cell death drives the selection of cells which are committed to play a role during the early stages of corticogenesis. Lack of evidence of clonally related apoptotic cells also indicates that cell death occurs randomly. Therefore, molecular signals from the surrounding microenvironment seem to be necessary for the apoptotic pathway to be turned on, thus determining the fate of post‐mitotic cells.     

GABA-stimulated chloride uptake and enhancement by diazepam in synaptoneurosomes from rat brain during prenatal and postnatal development

The present study was undertaken to evaluate the ability of diazepam (DZ) to elicit a response in the brains of developing animals. gamma-Aminobutyric acid (GABA)-stimulated 36Cl- uptake in the presence and absence of DZ was measured in synaptoneurosomal preparations from whole brain of fetal rats at 20 and 21 days gestation and from cerebral cortex of rats at 7, 14, 21, 28 and 60-90 days postnatal age. The ability of GABA to stimulate 36Cl- uptake in a concentration-dependent manner was evident from gestational day 20. The EC50 for GABA stimulation but not the maximum stimulation increased significantly from day 20 to day 21 of gestation. Postnatally, only moderate changes in the EC50 were evident, but the maximum 36Cl- transported increased significantly from 7 to 14 days and remained stable thereafter. DZ enhanced GABA-mediated 36Cl- influx from 20 days gestation, and this enhancement was seen as a decrease in the EC50 for GABA stimulation. However, DZ also significantly increased maximum GABA-stimulated 36Cl- transport in synaptoneurosomal preparations at 21 days gestation and at 7 days postnatal age, a response to DZ not seen at older ages. DZ had a less robust effect on GABA-mediated 36Cl- transport at 28 days postnatal age than at any other age, a result consistent with functional observations of decreased responsivity to DZ in late juvenile, early adolescent rats. The benzodiazepine antagonist Ro 15-1788 prevented the effect of DA on GABA-stimulated 36Cl- uptake in tissue from 21-day fetuses and from 60- to 90-day-old adults.(ABSTRACT TRUNCATED AT 250 WORDS)