Activation of subconductance states by γ-aminobutyric acid and its analogs in chick cerebral neurons

The action of -aminobutyric acid (GABA) and the GABA_A-receptor agonists muscimol, isoguvacine and 4,5,6,7-tetrahydroisoxazolo [5,4–c]pyridin-3-ol (THIP) were studied at the single-channel level in outside-out membrane patches from cultured chick cerebral neurons. All agonists activated channels with multiple-conductance states. The main-state conductance activated by all agonists had a value around 26 pS in symmetrical TRIS/Cl solutions. Subconductance states of around 13 pS and 18 pS were seen with application of each agonist. Muscimol and isoguvacine tended preferentially to activate subconductance states. Gating by all agonists was complex. Open-time distributions for main-state activity gated by GABA, isoguvacine and THIP were best described by the sum of two exponential curves with similar time constants. Muscimol-gated activity was best described by the sum of three exponentials indicating the presence of an additional longer open state. These results indicate that certain GABA_A-receptor agonists are capable of preferentially activating subconductance states.     

Identification of N-carboxyethyl γ-aminobutyric acid in bovine brain and human cerebrospinal fluid

The di-carboxylated derivative of spermidine, N-carboxyethyl γ-aminobutyric acid (CEGABA) has been identified in bovine brain and human cerebrospinal fluid by HPLC. This discovery strongly suggests the existence of a metabolic pathway connecting polyamines and GABA via putreanine and CEGABA through progressive oxidative deamination of the amino terminal groups in spermidine.     

Interaction of ivermectin with γ-aminobutyric acid receptors in Trichinella spiralis muscle larvae

The value of the γ-aminobutyric acid (GABA) receptor of nematodes as a target for ivermectin's mode of action remains unclear. Using binding assays, we examined extracts from Trichinella spiralis muscle larvae for the presence of [³H]-ivermectin and [³H]-GABA binding sites. Tissue preparations displayed affinity binding sites for [³H]-ivermectin with a dissociation constant (K _d) of 83 nM and a receptor density (B_max) of 145 fmol/mg protein. We also identified a specific [³H]-GABA binding activity with a K _d of 1.2 μM and a B_max of 4.78 pmol/mg protein. In competition studies, ivermectin was found to be a competitive inhibitor of specific [³H]-GABA binding activity with an inhibition constant (K _i) of 3.39 nM, suggesting that GABA receptors could be implicated in the mechanism of action of ivermectin in nematodes. Received: 7 August 1998 / Accepted: 22 September 1998     

The effect of stereoisomers of cycloserine on the formation and transformation of free γ-aminobutyric acid in brain tissue

Summary A study was made of the effect produced by the stereoisomers of cycloserine on decarboxylization of glutamic acid and transamination of -aminobutyric acid with -ketoglutaric acid in the homogenates of the rat brain at pH 7.5. As revealed. D.L-cycloserine in a concentration of 10^–3 M depressed the first process by 40% and the second one by 45%. In the same conditions D-cycloserine depressed glutamic acid decarboxylation by 9%; it somewhat activated -aminobutyric acid transamination. A discussion is presented on the possible effect of cycloserine isomers on the content of free -aminobutyric acid in the brain (in vivo) following the administration of isoniazid in large doses.(Presented by Active Member AMN SSSR V. V. Zakusov) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 54, No. 10, pp. 67–69, October, 1962     

Effects of transient global ischemia on the neurons in the marginal division of the rat striatum

<正> Transient global ischemia frequently occurs following cardiac arrest and causes ischemic brain injury and vascular dementia.It wassuppised that the injury of neurons in the hippocampus formation sponsors the cognitive defictis.But recently this view has been challenged bya number of experiments.A memory process,objective recognition,found to be largely intact following selective hippocampal formation lesionsboth in monkeys and rats.Some of the extra-hippocampal damages are involved in the process of learning and memory.The marginal division(MrD)is a new subdivision in the striatum firstly discovered by Si Yun Shu in 1988.It is consisted of a band of fusiform neurons at the caudalmargin of the striatum.Depression of learning and memory occurred following injury of MrD.Global cerebral ischemia models were conducted by4-vessel occlusion.Ischemia induced hydrocephalus in hippocapus,cortex and striatum almost at same level.MrD was the injured by ischemia.Y-maze test demonstrated distinct and stable impairmen     

Dopamine and ��-aminobutyric acid are colocalized in restricted groups of neurons in the sea lamprey brain: insights into the early evolution of neurotransmitter colocalization in vertebrates

Since its discovery, the possible corelease of classic neurotransmitters from neurons has received much attention. Colocalization of monoamines and amino acidergic neurotransmitters [mainly glutamate and dopamine (DA) or serotonin] in mammalian neurons has been reported. However, few studies have dealt with the colocalization of DA and γ‐aminobutyric acid (GABA) in neurons. With the aim of providing some insight into the colocalization of neurotransmitters during early vertebrate phylogeny, we studied GABA expression in dopaminergic neurons in the sea lamprey brain by using double‐immunofluorescence methods with anti‐DA and anti‐GABA antibodies. Different degrees of colocalization of DA and GABA were observed in different dopaminergic brain nuclei. A high degree of colocalization (GABA in at least 25% of DA‐immunoreactive neurons) was observed in populations of the caudal rhombencephalon, ventral isthmus, postoptic commissure nucleus, preoptic nucleus and in granule‐like cells of the olfactory bulb. A new DA‐immunoreactive striatal population that showed colocalization with GABA in about a quarter of its neurons was observed. In the periventricular hypothalamus, colocalization was observed in only a few cells, despite the abundance of DA‐ and GABA‐immunoreactive neurons, and no double‐labelled cells were observed in the paratubercular nucleus. The frequent colocalization of DA and GABA reveals that the dopaminergic populations of lampreys are more complex than previously reported. Double‐labelled fibres or terminals were observed in different brain regions, suggesting possible corelease of DA and GABA by these lamprey neurons. The present results suggest that colocalization of DA and GABA in neurons appeared early in vertebrate evolution.     

Loss of glia and neurons in the myenteric plexus of the aged Fischer 344 rat

Over the normal lifespan, a subpopulation of myenteric neurons in the small and large intestines dies. This loss is one possible mechanism for the disruptions of gastrointestinal function seen in the elderly. Little, however, is known about how the glia constituting the supportive cells of the myenteric plexus may change with aging and the losses of the enteric neurons. The goal of the present study, therefore, was to determine what, if any, changes occur in the glia associated with myenteric neurons in the aged gut. Two experimental groups, consisting of adult (5–6 months of age, n=8) or aged (26 months of age, n=8) virgin male Fischer 344 rats, fed ad libitum, were examined. The duodenum, jejunum, ileum, colon, and rectum from each rat were prepared as whole mounts, and indirect immunofluorescence was used to visualize the myenteric glia and neurons (antibodies to S-100 and the HuC/D protein, respectively). Separate counts of glia and neurons from the same specimens were determined, and these counts were expressed both as per ganglionic area and as per ganglion to correct for “dilution” effects resulting from age-associated changes in tissue area. Significant reductions in both the numbers of glia as well as neurons occurred in every region of the small and large intestine sampled from aged rats, except for the rectum, where a nonsignificant decrease was observed. Glial loss was proportional to neuronal death, suggesting an interdependency between the two cell types. Thus, an understanding of the nature of the neuron-glia interaction in the enteric nervous system may provide insight into the deterioration of function seen in the aged gut.     

Does cigarette smoke exposure lead to histopathological alterations in the olfactory epithelium? An electron microscopic study on a rat model

Objective: This study was conducted to examine the influence of smoke exposure of variable duration on the ultrastructure of and histopathologic and morphologic alterations in the olfactory epithelium.

Methods: A total of 24 Wistar albino rats were randomly assigned to three groups and fed a standard rat chow and tap water. Experimental rats in groups I and II were exposed to cigarette smoke in a glass cabin over a period of 2 months for 5 or 15 min, respectively, four times daily; control rats (group III) were not exposed to cigarette smoke. After dissection, all tissue specimens were processed using routine procedures for TEM.

Results: Groups I and II exhibited the presence of intraepithelial inflammatory cells and especially deep invaginations in the nuclear membrane of supporting cells. Extended intercellular spaces, cytoplasmic protrusions on the apical surface of supporting cells, atrophy of microvilli and olfactory neuron cilia as well as numerous electron-dense granular structures and lysosome-like structures were observed to an increasing degree from group I to group II. Particularly in group II, both supporting cells and olfactory neurons exhibited a cytoplasmic edema, mitochondrial degeneration, and numerous vacuolar structures, as well as apoptotic and minimal necrotic changes. In this group, hyperplasia of basal cells was also observed.

Conclusion: Our electron microscopic findings show that cigarette smoke leads to toxic degenerative changes in the rat olfactory mucosa.     

Do cardiac neurons play a role in the intrinsic control of heart rate in the rat?

Three experiments were performed to see whether cardiac neurons contribute to the intrinsic control of heart rate in right atria of adult rats. The intrinsic heart rate response (IRR) was examined by raising right atrial pressure from 2 to 8 mmHg for 3 min. In isolated preparations of the right atrium, the IRR was not significantly altered by the addition of either 1 microM atropine (n =6; control +19+/- 3 min(-1) ; atropine+18+/-3 min(-1); (mean /+/-S.E.M.)) or 1 microM propranolol (n = 5; control +22+/- 4 min(-1); ; propranolol +21+/-3 min(-1); ).Tetrodotoxin (0.5 microm) had no effect on the IRR (n = 6; control +37+/-5 min(-1); tetrodotoxin 38+/-5 min(-1); ). In another experiment, 2-day-old rat pups were injected with capsaicin (50 mg kg(-1); treated) or with vehicle(control). There was no difference in the IRR of right atrial preparations taken from control and treated animals after they reached adulthood (control (n = 7) and treated (n = 8): +30+/- 4 and +32+/- 4 min(-1)). The influence of right atrial pressure on the efficacy of vagal stimulation was examined. The rate response to vagal stimulation was reduced similarly in control and treated preparations when pressure was elevated from 2 to 4 mmHg (control and treated: -34+/- 5% and -33+/- 3%). The effectiveness of the capsaicin treatment was confirmed by the depletion of substance P-immunoreactive nerve fibres in cardiac tissues. Together, these results strongly suggest that cardiac neurons are not involved in intrinsic heart rate control.     

Estrogen receptor-α in the raphe serotonergic and supramammillary area calretinin-containing neurons of the female rat

It is well established that estrogen affects hippocampal long-term potentiation and hippocampus-related memory processes. Furthermore, theta rhythm, in conjunction with long-term potentiation, influences memory and is regulated by subcortical structures, including the median raphe and supramammillary area. To test the validity of the hypothesis that the effects of estrogen on the hippocampus are mediated, at least partly, via these subcortical structures, it must first be determined whether the neurons of the median raphe and supramammillary area contain estrogen receptors. Light and electron microscopic double immunostaining for estrogen receptor-α plus serotonin and estrogen receptor-α plus calretinin on vibratome sections of the median raphe and supramammillary area, respectively, demonstrated that large populations of the median raphe serotonin and supramammillary area calretinin neurons exhibit estrogen receptor-immunoreactive nuclei. These observations indicate that circulating gonadal hormones can affect hippocampal electric activity indirectly, via those subcortical structures that are involved in theta rhythm regulation. Received: 1 February 1999 / Accepted: 17 May 1999     

Analgesic effect of intrathecally γ-aminobutyric acid transporter-1 inhibitor NO-711 administrating on neuropathic pain in rats

To investigate the analgesic effect of intrathecally administered γ-aminobutyric acid (GABA) transporter-1 inhibitor NO-711 on the sciatic nerve chronic constriction injury (CCI) rats. 5 days after intrathecal catheter placement, neuropathic pain model was established by CCI of sciatic nerve on rats. Withdrawal thresholds for mechanical allodynia and latency for thermal hyperalgesia were measured in all animals. All rats operated upon for CCI displayed decreased withdrawal thresholds for mechanical allodynia and latency for thermal hyperalgesia, which has significant difference compared with sham groups. After intrathecal NO-711 administration, withdrawal thresholds and latency were significantly increased on CCI rats compared with control group after 1 day. The results show that GABA transporter-1 inhibitor could effectively develop analgesic effect in sciatic nerve CCI rats’ model.     

Analgesic effect of intrathecally γ-aminobutyric acid transporter-1 inhibitor NO-711 administrating on neuropathic pain in rats

Neuregulin-1 (NRG1) participates in numerous neurodevelopmental processes and plasticity of the brain. Despite this, little is known about its role in Alzheimer's disease (AD). Amyloid β (Aβ) peptide is generally believed to play a critical role in the pathogenesis of AD. The present study examined the effect of synthetic Aβ_1-42 peptides on long-term potentiation (LTP) in the CA1 region of mice hippocampal slices, a cellular model of learning and memory. We found that application of a test dose of Aβ_1-42 (200 nM) significantly inhibited the development of LTP without affecting basal synaptic transmission. Pretreatment with NRG1 effectively prevented Aβ_1-42-induced impairment of LTP, an effect that was dose-dependent. This LTP-restoring action of NRG1 was almost completely abolished by blocking ErbB4, a key NRG1 receptor, suggesting that NRG1 acts through ErbB4 to exert its protective action on LTP. The present study thus provides the first demonstration that NRG1/ErbB4 protects against Aβ-induced hippocampal LTP impairment, suggesting that NRG1 may be a promising candidate for the treatment of early-stage AD.     

Relation of exocytotic release of γ-aminobutyric acid to Ca2+ entry through Ca2+ channels or by reversal of the Na+/Ca2+ exchanger in synaptosomes

The specific inhibitor of the -aminobutyric acid (GABA) carrier, NNC-711, {1-[(2-diphenylmethylene) amino]oxyethyl}-1,2,5,6-tetrahydro-3-pyridinecarboxylic acid hydrochloride, blocks the Ca²⁺-independent release of [³H]GABA from rat brain synaptosomes induced by 50 mM K⁺ depolarization. Thus, in the presence of this inhibitor, it was possible to study the Ca²⁺-dependent release of [³H]GABA in the total absence of carrier-mediated release. Reversal of the Na⁺/Ca²⁺ exchanger was used to increase the intracellular free Ca²⁺ concentration ([Ca²⁺]_i) to test whether an increase in [Ca²⁺]_i alone is sufficient to induce exocytosis in the absence of depolarization. We found that the [Ca²⁺]_i may rise to values above 400 nM, as a result of Na⁺/Ca²⁺ exchange, without inducing release of [³H]GABA, but subsequent K⁺ depolarization immediately induced [³H]GABA release. Thus, a rise of only a few nanomolar Ca²⁺ in the cytoplasm induced by 50 mM K⁺ depolarization, after loading the synaptosomes with Ca²⁺ by Na⁺/Ca²⁺ exchange, induced exocytotic [³H]GABA release, whereas the rise in cytoplasmic [Ca²⁺] caused by reversal of the Na⁺/Ca²⁺ exchanger was insufficient to induce exocytosis, although the value for [Ca²⁺]_i attained was higher than that required for exocytosis induced by K⁺ depolarization. The voltage-dependent Ca²⁺ entry due to K⁺ depolarization, after maximal Ca²⁺ loading of the synaptosomes by Na⁺/Ca²⁺ exchange, and the consequent [³H]GABA release could be blocked by 50 M verapamil. Although preloading the synaptosomes with Ca²⁺ by Na⁺/Ca²⁺ exchange did not cause [³H]GABA release under any conditions studied, the rise in cytoplasmic [Ca²⁺] due to Na⁺/Ca²⁺ exchange increased the sensitivity to external Ca²⁺ of the exocytotic release of [³H]GABA induced by subsequent K⁺ depolarization. Thus, our results show that the vesicular release of [³H]GABA is rather insensitive to bulk cytoplasmic [Ca²⁺] and are compatible with the view that GABA exocytosis is triggered very effectively by Ca²⁺ entry through Ca²⁺ channels near the active zones.     

Can the gracilis be used to replace the anterior cruciate ligament in the knee? A cadaver study

PurposeThe purpose of this study was to evaluate whether a four-strand gracilis-only graft can be used in anterior cruciate ligament (ACL) reconstruction.Study designCadaver study.MethodsThis study involved 16 cadaver knees. The length and diameter of the native ACL were measured in each one. The same measurements were performed on a four-strand graft of the gracilis only, the semitendinosus only and both tendons. Student's t-test was used to compare the various conditions.ResultsThe average diameter of the G4 construct was 0.07 mm greater (1%) than the native ACL (p = 0.044). The average cross-sectional area of the G4 construct was 1.2 mm² greater (3.9%) than the native ACL (p = 0.049). The G4 was on average 38.9 mm longer than the intra-articular portion of the ACL (p < 0.001).ConclusionA four-strand gracilis construct meets the anatomical specifications for use as an ACL reconstruction graft. By using the gracilis only, the morbidity associated with harvesting the gracilis and semitendinosus tendons should be reduced. Further studies must be performed to compare the biomechanical properties of this graft with other graft types and also to evaluate how this four-strand gracilis graft behaves in a clinical setting.     

Glutamatergic neurons in the Kölliker-Fuse nucleus project to the rostral ventral respiratory group and phrenic nucleus: a combined retrograde tracing and in situ hybridization study in the rat

K?lliker-Fuse nucleus (KF) neurons are considered to excite motoneurons in the phrenic nucleus (PhN) during inspiration through its projection to the PhN and/or to the rostral ventral respiratory group (rVRG), which in turn projects to the PhN, probably by releasing glutamate from their axon terminals. Using a combined retrograde tracing and in situ hybridization technique, here we demonstrate that most of the KF neurons projecting to the PhN and rVRG contain vesicular glutamate transporter 2 (VGLUT2) mRNA but not glutamic acid decarboxylase 67 (GAD67) mRNA, providing definitive evidence that these neurons are glutamatergic. Together with previous data by Stornetta et al. [Stornetta, R.L., Sevigny, C.P., Guyenet, P.G., 2003b. Inspiratory argumenting bulbospinal neurons express both glutamatergic and enkephalinergic phenotypes. J. Comp. Neurol. 455, 113-124], indicating that PhN-projecting rVRG neurons are VGLUT2 mRNA-positive, the present results suggest that the glutamatergic KF-PhN pathway and/or the glutamatergic KF-rVRG-PhN pathway transmit excitatory outputs of KF neurons to the PhN neurons during inspiration.