Carnosine modulates zinc and copper effects on amino acid receptors and synaptic transmission

Carnosine is a dipeptide which is highly concentrated in mammalian olfactory sensory neurons along with zinc and/or copper, and glutamate. Although carnosine has been proposed as a neurotransmitter or neuromodulator, no specific function for carnosine has been identified. We used whole-cell current- and voltage-clamp recording to examine the direct effects and neuromodulatory actions of carnosine on rat olfactory bulb neurons in primary culture. Carnosine did not evoke a membrane current or affect currents evoked by glutamate, GABA or glycine. Copper and zinc inhibited NMDA and GABA receptor-mediated currents and inhibited synaptic transmission. Carnosine prevented the actions of copper and reduced the effects of zinc. These results suggest that carnosine may indirectly influence neuronal excitability by modulating the effects of zinc and copper.     

Distribution of GABA, glycine, and glutamate immunoreactivities in the vestibular nuclear complex of the frog

This study describes the localization of gamma-aminobutyric acid (GABA), glycine, and glutamate immunoreactive neurons, fibers, and terminal-like structures in the vestibular nuclear complex (VNC) of the frog by using a postembedding procedure with consecutive semithin sections at the light microscopic level. For purposes of this study, the VNC was divided into a medial and lateral region. Immunoreactive cells were observed in all parts of the VNC. GABA-positive neurons, generally small in size, were predominantly located in the medial part of the VNC. Glycine-positive cells, more heterogeneous in size than GABA-positive cells, were scattered throughout the VNC. A quantitative analysis of the spatial distribution of GABA glycine immunoreactive cells revealed a complementary relation between the density of GABA and glycine immunoreactive neurons along the rostrocaudal extent of the VNC. In about 10% of the immunolabeled neurons, GABA and glycine were colocalized. Almost all vestibular neurons were, to a variable degree, glutamate immunoreactive, and colocalization of glutamate with GABA and/or glycine was typical. GABA, glycine, or glutamate immunoreactive puncta were found in close contact to somata and main dendrites of vestibular neurons. A quantitative analysis revealed a predominance of glutamate-positive terminal-like structures compared to glycine or GABA containing profiles. A small proportion of terminal-like structures expressed colocalization of GABA and glycine or glycine and glutamate. The results are compared with data from mammals and discussed in relation to vestibuloocular and vestibulo-spinal projection neurons, and vestibular interneurons. GABA and glycine are the major inhibitory transmitters of these neurons in frogs as well as in mammals. The differential distribution of GABA and glycine might reflect a compartmentalization of neurons that is preserved to some extent from the early embryogenetic segmentation of the hindbrain.     

Effects of the Areca nut constituents arecaidine and guvacine on the action of GABA in the cat central nervous system

Arecaidine and guvacine, constituents of the nut of Areca catechu, inhibited the uptake of GABA and beta-alanine, but not that of glycine, by slices of cat spinal cord. In cats anesthetised with pentobarbitone, electrophoretic arecaidine enhanced the inhibitory actions of GABA and beta-alanine, but not those of glycine or taurine, on the firing of spinal neurones. Similarly, electrophoretic guvacine enhanced the inhibition of spinal neurones by GABA but not that by glycine. The uptake of GABA by slices of cat cerebellum was inhibited by arecaidine, and the effect of electrophoretic GABA on the firing of cerebellar Purkinje cells was enhanced by electrophoretic arecaidine. When administered intravenously arecaidine failed to affect synaptic inhibitions considered to be mediated by GABA. Intravenous arecaidine had no effect on either spinal prolonged (presynaptic) inhibition (20mg/kg), dorsal root potentials (20mg/kg) or basket cell inhibition of Purkinje cells (250 mg/kg), although topical arecaidine (6.6-10 x 10(-3) M) blocked this latter inhibition. Large doses of arecaidine (1 g/kg subcutaneous) marginally reduced the lethal effects of bicuculline in mice but appeared to have little or no anticonvulsant activity.     

The vesicular GABA transporter, VGAT, localizes to synaptic vesicles in sets of glycinergic as well as GABAergic neurons

A transporter thought to mediate accumulation of GABA into synaptic vesicles has recently been cloned (McIntire et al., 1997). This vesicular GABA transporter (VGAT), the first vesicular amino acid transporter to be molecularly identified, differs in structure from previously cloned vesicular neurotransmitter transporters and defines a novel gene family. Here we use antibodies specific for N- and C-terminal epitopes of VGAT to localize the protein in the rat CNS. VGAT is highly concentrated in the nerve endings of GABAergic neurons in the brain and spinal cord but also in glycinergic nerve endings. In contrast, hippocampal mossy fiber boutons, which although glutamatergic are known to contain GABA, lack VGAT immunoreactivity. Post-embedding immunogold quantification shows that the protein specifically associates with synaptic vesicles. Triple labeling for VGAT, GABA, and glycine in the lateral oliva superior revealed a higher expression of VGAT in nerve endings rich in GABA, with or without glycine, than in others rich in glycine only. Although the great majority of nerve terminals containing GABA or glycine are immunopositive for VGAT, subpopulations of nerve endings rich in GABA or glycine appear to lack the protein. Additional vesicular transporters or alternative modes of release may therefore contribute to the inhibitory neurotransmission mediated by these two amino acids.     

Synaptic inhibition in the isolated respiratory network of neonatal rats

Gramicidin-perforated patch-clamp recording revealed phasic Cl(-)-mediated hyperpolarizations in respiratory neurons of the brainstem-spinal cord preparation from newborn rats. The in vitro respiratory rhythm persisted after block of gamma-aminobutyric acid (GABA), i.e. GABAA, receptor-mediated inhibitory postsynaptic potentials (IPSPs) with bicuculline and/or glycinergic IPSPs with strychnine. In one class of expiratory neurons, bicuculline unmasked inspiration-related excitatory postsynaptic potentials (EPSPs), leading to spike discharge. Bicuculline also blocked hyperpolarizations and respiratory arrest due to bath-applied muscimol, whereas strychnine antagonized similar responses to glycine. The reversal potential of respiration-related IPSPs and responses to GABA, muscimol or glycine was not affected by CO2/HCO3(-)-free solutions, but shifted from about -65 mV to values more positive than -20 mV upon dialysis of the cells with 144 instead of 4 mM Cl-. Impairment of GABA uptake with nipecotic acid or glycine uptake with sarcosine evoked a bicuculline- or strychnine-sensitive decrease of respiratory frequency which could lead to respiratory arrest. Also, the GABAB receptor agonist baclofen led to reversible suppression of respiratory rhythm. This in vitro apnoea was accompanied by a K+ channel-mediated hyperpolarization (reversal potential -88 mV) of tonic cells, whereas membrane potential of neighbouring respiratory neurons remained almost unaffected. Both baclofen-induced hyperpolarization and respiratory depression were antagonised by 2-OH-saclofen, which did not affect respiration-related IPSPs per se. The results show that synaptic inhibition is not essential for rhythmogenesis in the isolated neonatal respiratory network, although (endogenous) GABA and glycine have a strong modulatory action. Hyperpolarizing IPSPs mediated by GABAA and glycine receptors provide a characteristic pattern of membrane potential oscillations in respiratory neurons, whereas GABAB receptors rather appear to be a feature of non-respiratory neurons, possibly providing excitatory drive to the network.     

Amino-acid release from human cerebral cortex during simulated ischaemia in vitro

The aim of the present study was to investigate the release of amino-acids in human cerebral cortex during membrane depolarization and simulated ischaemia (energy deprivation). Superfluous tissue from temporal Iobe resections for epilepsy was cut into 500 microns thick slices and incubated in vitro. Membrane depolarization with 50 mM K+ caused a release of glutamate, aspartate, GABA and glycine, but not glutamine or leucine. The release of glutamate and GABA was Ca(++)-dependent. Slices were exposed to simulated ischaemia (energy deprivation; ED) by combined glucose/oxygen deprivation. This caused a Ca(++)-independent release of glutamate, aspartate, GABA, glycine, and taurine which started after 8 min, peaked at the end or shortly after the 27 min period of ED, and returned to control levels within 11 min following termination of ED. Preloaded D-[3H]aspartate was released both during K(+)-stimulation and ED. Release of D-[3H]aspartate during ED was delayed compared to glutamate supporting an initial phase of synaptic glutamate release. Uptake of L-[3H]glutamate was increased during the period of glutamate release, suggesting passive diffusion across the cell membrane or enhanced transport efficacy in cellular elements with functioning uptake mechanisms.     

beta-Alanine and taurine microinjected into the rat caudal ventrolateral medulla increase blood pressure

Unilateral microinjections of GABA, glycine, beta-alanine and taurine into the caudal ventrolateral medulla (CVLM) of the rat, led to an increase in blood pressure and heart rate. The responses to glycine, beta-alanine and taurine but not to GABA could be blocked by strychnine. The responses to taurine and beta-alanine but not to GABA and glycine could be blocked by 6-aminomethyl-3-methyl-4H-1,2,4-benzothiadiazine-1,1-dioxide (TAG), an antagonist of taurine. The taurine antagonist alone injected bilaterally into the CVLM produced a decrease in blood pressure. From CVLM areas microperfused with Krebs solution, spontaneous release of GABA, glycine, beta-alanine and taurine was detected and high K+ stimulation caused a calcium-dependent release of GABA, beta-alanine and taurine. These results suggest that beta-alanine and taurine as well as GABA may be involved in modulation of the cardiovascular control within the CVLM.     

Currents evoked by GABA and glycine in acutely dissociated neurons from the rat medial preoptic nucleus

The responses of acutely dissociated medial preoptic neurons to application of GABA, and glycine were studied using the perforated-patch whole-cell recording technique under voltage-clamp conditions. GABA, at a concentration of 1 mM, evoked outward currents in all cells (n = 33) when studied at potentials positive to -80 mV. The I-V relation was roughly linear. The currents evoked by GABA were partially blocked by 25-75 microM picrotoxin and were also partially or completely blocked by 100-200 microM bicuculline. Glycine, at a concentration of 1 mM, did also evoke outward currents in all cells (n = 12) when studied at potentials positive to -75 mV. The I-V relation was roughly linear. The currents evoked by glycine were largely blocked by 1 microM strychnine. In conclusion, the present work demonstrates that neurons from the medial preoptic nucleus of rat directly respond to the inhibitory transmitters GABA and glycine with currents that can be attributed to GABAA receptors and glycine receptors respectively.     

Qualitative and quantitative analysis of glycine- and GABA-immunoreactive nerve terminals on motoneuron cell bodies in the cat spinal cord: a postembedding electron microscopic study

The distribution of glycine- and gamma-aminobutyric acid (GABA)-like immunoreactivity (LI) in nerve terminals on the cell soma of motoneurons in the aldehyde-fixed cat L7 spinal cord was examined using postembedding immunogold histochemistry in serial ultrathin sections. Quantitative examination of 405 terminals on eight neurons of alpha-motoneuron size in the L7 motor nuclei from one animal was performed. A majority of the terminals (69%) were immunoreactive to glycine and/or GABA. These terminals contained flat or oval synaptic vesicles, thus classifying them as F type or as C type in one case. In no case was a type-F terminal unlabeled for both glycine and GABA. Most of the immunolabeled terminals were immunoreactive to glycine only (62.5%), whereas 35.4% contained both glycine- and GABA-LI. A very small number of immunolabeled terminals (2%) were immunoreactive to GABA only. In those terminals, where glycine- and GABA-LI coexisted, the gold particle density for each amino acid was only half of that seen in boutons containing only one of the two amino acids. The involvement of glycine and GABA in postsynaptic inhibition of spinal alpha-motoneurons is discussed, with particular reference to the possibility that these two inhibitory amino acids may be coreleased from a significant proportion of the nerve terminals impinging on the cell bodies.     

Significance of serum ferritin and lactate dehydrogenase in benign and malignant disease of breast

We previously reported that corymine, an alkaloidal compound extracted from the leaves of Hunteria zeylanica native to Thailand, potentiated convulsions induced by either picrotoxin or strychnine. Therefore, to clarify the mechanism of action of corymine, the effects of corymine on gamma-aminobutyric acid (GABA) and glycine receptors were examined. We used Xenopus oocytes expressing these receptors and the two-electrode voltage-clamp method. The receptors expressed in oocytes injected with rat brain and spinal cord RNA showed the pharmacological properties of GABAA and glycine receptors, respectively. Corymine (1-100 microM) partially (20-30%) reduced the GABA responses in oocytes injected with rat brain RNA, while marked (up to 80%) dose-dependent reductions were observed in the glycine responses in oocytes injected with rat spinal cord RNA. These observations suggest that corymine was more effective against the glycine receptors than the GABA receptors. The ED50 of corymine on the glycine response was 10.8 microM. Corymine, at 30 microM, caused a shift to the right, with a lower maximal response, of the glycine concentration-response curve. This indicated that the action of corymine on glycine receptors is neither competitive nor purely non-competitive. These observations suggest that a binding site other than the glycine recognition site of the glycine receptors is the site of action of corymine.     

Metabotropic glutamate receptor antagonists, like GABA(B) antagonists, potentiate dorsal root-evoked excitatory synaptic transmission at neonatal rat spinal motoneurons in vitro

Recordings of whole-cell synaptic current responses elicited by electrical stimulation of dorsal roots were made from motoneurons, identified by antidromic invasion, in isolated spinal cord preparations from five- to eight-day-old Wistar rats. Supramaximal electrical stimulation of the dorsal root evoked complex excitatory postsynaptic currents with mean latencies (+/- S.E.M.) of 6.1 +/- 0.26 ms, peak amplitude of -650 +/- 47 pA and duration of 4.30 +/- 0.46 s (n=34). All phases of excitatory postsynaptic currents were potentiated to approximately 20% above control levels in the presence of the metabotropic glutamate receptor antagonists S-2-amino-2-methyl-4-phosphonobutanoate (MAP4; 200 microM; n=15) and 2S, 1'S,2'S-2-methyl-2-(carboxycyclopropyl)glycine (MCCG; 200 microM; n=9). A similar level of potentiation was produced by the GABA(B) receptor antagonist 3-N[1-(S)-(3,4-dichlorophenyl)ethyl]amino-2-(S)-hydroxypropyl-P-benzyl-p hosphinic acid (CGP55845; 200 nM; n=5). MAP4 (200 microM) produced a six-fold rightward shift in the concentration-effect plot for the depressant action of the metabotropic glutamate receptor agonist S-2-amino-4-phosphonobutanoate (L-AP4), whereas CGP55845 produced no significant change in the potency of L-AP4. MAP4 did not antagonize the depressant actions of baclofen (n=8), 1S,3S-1-aminocyclopentane-1,3-dicarboxylate (n=4) or 2-S,1'S,2'S-2-(carboxycyclopropyl)glycine (n=4). The metabotropic glutamate receptor antagonists produced no change in the holding current of any of the neurons, indicating that they had no significant postsynaptic excitatory actions. These results are the first to indicate a possible physiological role for metabotropic glutamate receptors in the spinal cord. Like GABA(B) receptors, they control glutamatergic synaptic transmission in the segmental spinal pathway to motoneurons. This is likely to be a presynaptic control mechanism.     

GABA-Induced intrinsic light-scattering changes associated with voltage-sensitive dye signals in embryonic brain stem slices: coupling of depolarization and cell shrinkage

We have found new evidence for gamma-aminobutyric acid (GABA)-induced intrinsic optical changes associated with a voltage-sensitive dye signal in the early embryonic chick brain stem slice. The slices were prepared from 8-day-old embryos, and they were stained with a voltage-sensitive dye (NK2761). Pressure ejection of GABA to one site within the preparation elicited optical changes. With 580-nm incident light, two components were identified in the GABA-induced optical change. The first component was wavelength dependent, whereas the second, slower change was independent of wavelength. Comparison with the known action spectrum of the dye indicates that the first component reflects a depolarization of the membrane and that the second, slow component is a light-scattering change resulting from cell shrinkage coupled with the depolarization. Similar optical changes also were induced by glycine, although the amplitude of both the first and second signals was much smaller than for GABA. The optical changes induced by GABA persisted in the presence of picrotoxin and 2-hydroxysaclofen, suggesting that these optical responses include a novel GABA response, which has been termed GABAD in our previous reports.     

Action of amino acids and convulsants on cerebellar spontaneous action potentials in vitro: effects of deprivation of C1-, K+ of Na+

(1) The inhibition of spontaneous action potentials in guinea pig cerebellar cortex slices by GABA, glycine, taurine and beta-alanine is maintained when C1- in the superfusion medium is almost completely replaced by NO3- or I-('permeant' anion), but the inhibition decreases in magnitude with repeated application of the amino acid. Replacement of C1- by sulfate or isethionate ('impermeant' anion) causes a conversion of inhibition by these amino acids to excitation. The initial excitation which is sometimes seen with these inhibitory amino acids in high C1- media is abolished when C1- is replaced by either permeant or impermeant anions. (2) Reduction of K+ in the medium causes an increase of inhibition by the inhibitory amino acids in the presence of high C1- and reduction of excitation when C1- is replaced by impermeant anion. (3) Excitation by GABA in impermeant anion (low C1-) media is unaffected by reduction of Na+ in the media by 50% but excitations by glycine, taurine, beta-alanine and L-glutamate are greatly reduced. (4). Excitation by GABA in impermeant anion (low C1-) media is abolished by picrotoxin and bicuculline which both suppress inhibition by GABA in a high C1- medium. Strychnine suppresses the effects of glycine, taurine and beta-alanine in either a low or high C1- medium. Bicuculline blocks the inhibitory effect of these three amino acids in a high C1- medium but does not affect their excitatory effects in a low C1- medium. (5) These results are consistent with the hypothesis that the inhibitory amino acids, GABA, glycine, taurine and beta-alanine, cause inhibition via increase of C1- (and perhaps K+) permeability and that glycine, taurine and beta-alanine also interact with strychnine-sensitive receptors mediating (perhaps indirectly) increased permeability to Na+ and, therefore, excitation in low C1- media.     

Properties of GABA(A) receptors in cultured rat oligodendrocyte progenitor cells

We have studied the properties of GABA responses in oligodendrocyte-type 2 astrocyte (O-2A) progenitor cells derived from primary cultures of the neonatal rat brain. In whole cell voltage clamp recordings, rapid application of 1-10 mM GABA elicited current responses in > 85% of the cells examined. The dose-response relationship pooled from nine progenitor cells was best fit by a logistic function of EC50=113 microM and Hill coefficient=0.9. In contrast to the rate of current deactivation, the rate of current activation exhibited marked concentration-dependence. Pharmacologically, GABA, muscimol and ZAPA ((Z)-3[(aminiiminomethyl)thio]prop-2-enoic acid sulphate) produced responses with ligand-specific kinetics, whereas glycine and the GABA(C) receptor agonist CACA were without effect; bicuculline methochloride acted as a competitive antagonist. Neither the amplitude nor the kinetics of currents produced by 100 microM GABA were affected by the benzodiazepine flunitrazepam (1 microM). Similarly the benzodiazepine receptor inverse agonist DMCM (1 microM) was also without effect. GABA-activated currents reversed polarity within 2 mV of the calculated Cl- equilibrium potential. With brief agonist pulses deactivation was monoexponential, however, unlike neurones the rate of deactivation was voltage-independent. Desensitisation of responses to 10 mM GABA was bi-exponential and accelerated at depolarised membrane potentials. Increasing the amount of GABA(A) receptor desensitisation (by increasing the duration of the agonist exposure) consistently produced a slowing of deactivation.     

Posttraumatic stress and maladjustment among adult burn survivors 1 to 2 years postburn. Part II: the interview data

Using native plasma membrane vesicle suspensions from the rat cerebral cortex under conditions designed to alter intravesicular [Ca2+], we found that Ca2+ induced 47 +/- 5% more influx of [3H]GABA, [3H]D-aspartate and [3H]glycine at 37 degrees C with half-times 1.7 +/- 0.5, 1.3 +/- 0.4 and 1.3 +/- 0.4 min, respectively. We labelled GABA transporter sites with the uptake inhibitor, [3H]-(R,S)-N-[4,4-bis(3-methyl-2-thienyl)but-3-en-1-yl]nipecotic acid and found that Ca2+ induced a partial dissociation of the bound inhibitor from GABA transporter sites with a similar half-time. By means of rapid kinetic techniques applied to native plasma membrane vesicle suspensions, containing synaptic vesicles stained with the amphipathic fluorescent styryl membrane probe N-(3-triethylammoniumpropyl)-4-[4-(dibutylamino)styryl]pyrid inium dibromide, we have measured the progress of the release and reuptake of synaptic vesicles in response to Ca2+ and high-[K+] depolarization in the 0.0004-100 s range of time. Synaptic vesicle exocytosis, strongly influenced by external [Ca2+], appeared with the kinetics accelerated by depolarization. These results are consistent with the potential involvement of Ca2+ in taking low-affinity transporters to the plasma membrane surface via exocytosis.     

Immunocytochemical localization of GABA and glycine in amacrine and displaced amacrine cells of macaque monkey retina

Recent studies have varied widely in the percentages of GABA- and glycine-immunoreactive (GABA+, GLY+) amacrines reported for primate retina. We compared the distributions of GABA+ and GLY+ amacrines and displaced amacrines at seven locations along the horizontal meridian of macaque retina using postembedding immunogold labeling with silver intensification. The percentage of GABA+ amacrine profiles was higher in central retina (50-55%) than peripheral retina (30-40%), whereas the percentage of GLY+ amacrine profiles did not vary much with eccentricity (52-57%). GABA and glycine were colocalized in 5-20% of amacrines, depending on the eccentricity, whereas 5-30% of amacrines were not immunoreactive for either neurotransmitter. GABA+ amacrines were slightly larger than GLY+ amacrines or Müller cells. In the ganglion cell layer, 5-20% of neurons were labeled for either GABA or glycine and were identified as displaced amacrines. Of these, 53% were GABA+ only, 11% were GLY+ only, and 37% were double-labeled. A few large, very lightly labeled GABA+ cells were identified as ganglion cells. Other features that varied with eccentricity included the linear density of GABA+ and GLY+ amacrines, and the ratio of amacrines to Müller cells.     

The pharmacology of amino-acid responses in septal neurons

Septal cholinergic neurons are known to play an important role in cognitive processes including learning and memory through afferent innervation of the hippocampal formation and cerebral cortex. The septum contains not only cholinergic neurons but also various types of neurons including GABA (gamma-aminobutyric acid)-ergic neurons. Although synaptic transmission in the septum is mediated primarily by the activation of excitatory and inhibitory amino-acid receptors, it is possible that a distinct phenotype of neuron is endowed with a different type for each of the amino-acid receptors and thus they play different roles from each other, since it has been demonstrated within the septum that there is a regional distribution of various types of amino-acid receptor subunits, their expression as different combinations within a specific cell may produce receptor channels with disparate functional properties. As a first step towards knowing the various functions of septal cholinergic neurons, we characterized the functional properties of glutamate, GABA (type A; GABAA) and glycine receptor channels on cultured rat septal neurons which were histologically identified to be cholinergic. These were similar to those of receptor channels on other types of neurons, except for the actions of some neuromodulators. The septal N-methyl-D-aspartate receptor channel was distinct in being less sensitive to Mg2+ and in a voltage-dependent action of Zn2+. The septal GABAA receptor channel exhibited a lanthanide site whose activation resulted in a positive allosteric interaction with a binding site of pentobarbital. The septal glycine receptor channel was only positively modulated by Zn2+; this action of Zn2+ was not accompanied by an inhibitory effect. Our data suggest that the amino-acid receptors on septal cholinergic neurons may play a distinct role compared to other types of neurons; this difference depends on the actions of neuromodulators and metal cations. It would be interesting to compare these effects recorded in tissue culture to those observed with septal cholinergic neurons in slice preparations.     

Modulation of calcium by inhibitory systems in the developing auditory midbrain

Inhibitory synaptic transmission is of fundamental importance during the maturation of central auditory circuits, and their subsequent ability to process acoustic information. The present study investigated the manner in which inhibitory transmission regulates intracellular free calcium levels in the gerbil inferior colliculus using a brain slice preparation. Inhibitory and excitatory postsynaptic potentials were evoked by electrical stimulation of the ascending afferents at the level of the dorsal nucleus of the lateral lemniscus. Pharmacologically isolated inhibitory synaptic potentials were able to attenuate a calcium rise in collicular neurons that was generated by depolarizing current injection. In addition, GABA(A) and glycine receptor antagonists typically led to an increase of calcium in collicular neurons during electrical stimulation of the ascending afferent pathway at the level of the dorsal nucleus of the lateral lemniscus. Bath application of GABA or muscimol, a GABA(A) receptor agonist, evoked a brief hyperpolarization followed by a long-lasting depolarization in inferior colliculus neurons. This treatment also induced a transient calcium increase that correlated with the membrane depolarization phase. Baclofen, a GABA(B) receptor agonist, had no effect on either membrane potential or calcium levels. Ratiometric measures indicated that the muscimol-evoked rise in calcium was approximately 150 nM above basal levels. The muscimol-evoked responses were completely antagonized by bicuculline and attenuated by picrotoxin. Together, these results suggest that inhibitory synaptic transmission participates in the regulation of postsynaptic calcium during the developmental period. Inhibitory transmission may attenuate a calcium influx that is evoked by excitatory synapses, but it can also produce a modest influx of calcium when activated alone. These mechanisms may help to explain the influence of inhibitory transmission on the development of postsynaptic properties.     

GABAergic deafferentation hypothesis of brain aging and Alzheimer's disease revisited

Considering the mechanisms responsible for age- and Alzheimer's disease (AD)-related neuronal degeneration, little attention was paid to the opposing relationships between the energy-rich phosphates, mainly the availability of the adenosine triphosphate (ATP), and the activity of the glutamic acid decarboxylase (GAD), the rate-limiting enzyme synthesizing the gamma-amino butyric acid (GABA). Here, it is postulated that in all neuronal phenotypes the declining ATP-mediated negative control of GABA synthesis gradually declines and results in age- and AD-related increases of GABA synthesis. The Ca2+-independent carrier-mediated GABA release interferes with Ca2+-dependent exocytotic release of all transmitter-modulators, because the interstitial (ambient) GABA acts on axonal preterminal and terminal varicosities endowed with depolarizing GABA(A)-benzodiazepine receptors; this makes GABA the "executor" of virtually all age- and AD-related neurodegenerative processes. Such a role of GABA is diametrically opposite to that in the perinatal phase, when the carrier-mediated GABA release, acting on GABA(A)/chloride ionophore receptors, positively controls chemotactic migration of neuronal precursor cells, has trophic actions and initiates synaptogenesis, thereby enabling retrograde axonal transport of target produced factors that trigger differentiation of neuronal phenotypes. However, with advancing age, and prematurely in AD, the declining mitochondrial ATP synthesis unleashes GABA synthesis, and its carrier-mediated release blocks Ca2+-dependent exocytotic release of all transmitter-modulators, leading to dystrophy of chronically depolarized axon terminals and block of retrograde transport of target-produced trophins, causing "starvation" and death of neuronal somata. The above scenario is consistent with the following observations: 1) a 10-month daily administration to aging rats of the GABA-chloride ionophore antagonist, pentylenetetrazol, or of the BDZ antagonist, flumazenil (FL), each forestalls the age-related decline in cognitive functions and losses of hippocampal neurons; 2) the brains of aging rats, relative to young animals, and the postmortem brains of AD patients, relative to age-matched controls, show up to two-fold increases in GABA synthesis; 3) the aging humans and those showing symptoms of AD, as well as the aging nonhuman primates and rodents--all show in the forebrain dystrophic axonal varicosities, losses of transmitter vesicles, and swollen mitochondria. These markers, currently regarded as the earliest signs of aging and AD, can be reproduced in vitro cell cultures by 1 microM GABA; the development of these markers can be prevented by substituting Cl- with SO4(2-); 4) the extrasynaptic GABA suppresses the membrane Na+, K+-ATPase and ion pumping, while the resulting depolarization of soma-dendrites relieves the "protective" voltage-dependent Mg2+ control of the N-methyl-D-aspartate (NMDA) channels, thereby enabling Ca2+-dependent persistent toxic actions of the excitatory amino acids (EAA); and 5) in whole-cell patch-clamp recording from neurons of aging rats, relative to young rats, the application of 3 microM GABA, causes twofold increases in the whole-cell membrane Cl- conductances and a loss of the physiologically important neuronal ability to desensitize to repeated GABA applications. These age-related alterations in neuronal membrane functions are amplified by 150% in the presence of agonists of BDZ recognition sites located on GABA receptor. The GABA deafferentation hypothesis also accounts for the age- and AD-related degeneration in the forebrain ascending cholinergic, glutamatergic, and the ascending mesencephalic monoaminergic system, despite that the latter, to foster the distribution-utilization of locally produced trophins, evolved syncytium-like connectivities among neuronal somata, axon collaterals, and dendrites, to bidirectionally transport trophins. (ABSTRACT TRUNCATED)