Effects of the vitamin derivatives of GABA on inhibitory postsynaptic responses of the rat cortical neurons

With the use of an intracellular recording technique, in experiments on immobilized anesthetized rats, we studied intracortical stimulation (ICS)-evoked responses of the motor cortex neurons before and after applications of pantogam (PG) and GABA ascorbinate (A-GABA) on the cortical surface. Application of PG prolonged the IPSP, suppressed the background spike activity (BA), and increased the membrane potential level of the neurons studied. Applications of A-GABA in low and medium concentrations (below 50 μM) increased the amplitude and duration of inhibitory responses, while that in high concentrations (50 μM and more) evoked depolarization of the neuronal membrane with concurrent intensification of the BA. Probable cellular mechanisms of the effects of the above drugs are discussed.     

Effects of abiotic and biotic elicitors on growth and isoflavonoid accumulation in Pueraria candollei var. candollei and P. candollei var. mirifica cell suspension cultures

This study demonstrates the effects of various concentrations of abiotic and biotic elicitors on the cell growth and isoflavonoid accumulation of P. candollei var. mirifica (PM) and P. candollei var. candollei (PC) cell suspension cultures. The two plant varieties exhibited different growth responses and varied isoflavonoid accumulation after the addition of elicitors. Copper sulfate, methyl jasmonate (MeJA), and yeast extract did not significantly affect the growth of either plant variety, whereas oligosaccharide and the biotic elicitors used in this study [i.e., 50 mg l⁻¹ chitosan and all concentrations of laminarin (LAM)] suppressed the growth of PM. The addition of MeJA to the medium principally induced an effect on the isoflavonoid content in both PM and PC, with 2.0 μM MeJA inducing the highest isoflavonoid content, as indicated by the induction index—4.41 in PM and 9.62 in PC cells on the 12th and ninth day of culture, respectively. A maximum total isoflavonoid content of 40.49 mg g⁻¹ dry weight was achieved in PM 21 days after elicitation with 2.0 μM MeJA. LAM elicited the PM cell suspension culture to produce puerarin, which was not found in the unelicited culture. The results of this study provide information that will be useful for enhancing the accumulation of isoflavonoids in P. candollei cell suspension cultures.     

Evidence for an in vivo and in vitro modulation of endogenous cortical GABA release by α-glycerylphosphorylcholine

The effects of α-glycerylphosphorylcholine (α-GPC) on endogenous cortical GABA release were studied both in vivo and in vitro. In freely moving rats, equipped with epidural cups, α-GPC (30–300 mg/kg i.p.) increased GABA release. This effect was potentiated by atropine, both systematically administered (5 mg/kg i.p.) and locally applied (1.4 μM), but not by mecamylamine (4 mg/kg i.p.). The α-GPC-induced increasein GABA release was abolished in rats pretreated with the α₁ receptor antagonist prazosin (14 μg/kg i.p.). In cortical slices α-GPC (0.4 mM) increased the spontaneous GABA efflux. This effectwas abolished by tetrodotoxin (0.5 μM) and prazosin (1 μM), but not by atropine (0.15 μM) ormecamylamine (2.5μM). These results indicate that the facilitatory response by α-GPC on GABArelease does not depend on a direct activation of either muscarinic or nicotinic receptors, but suggest the involvement of the noradrenergic system.     

Norepinephrine Homogeneously Inhibits α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate- (AMPAR-) Mediated Currents in All Layers of the Temporal Cortex of the Rat

The primary auditory cortex is subject to the modulation of numerous neurotransmitters including norepinephrine (NE), which has been shown to decrease cellular excitability by yet unclear mechanisms. We investigated the possibility that NE directly affects excitatory glutamatergic synapses. We found that bath applications of NE (20 μM) decreased glutamatergic excitatory post-synaptic currents (EPSCs) in all cortical layers. Changes in the kinetics of synaptic EPSCs, invariance of pair pulse ratio and of the coefficient-of-variation, together with the decrease of responses to pressure-application of AMPA (500 μM), indicated the postsynaptic nature of the adrenergic effect. Pharmacological experiments suggested that the NE-induced depression of EPSCs is caused by the activation of α1 adrenoceptors, PLC, and a Ca²⁺-independent PKC. We speculate that the decrease in temporal cortex excitability might promote a posterior-to-anterior shift in cortical activation together with a decrease in spontaneous background activity, resulting eventually in more effective sensory processing.     

High frequency shoot organogenesis and somatic embryogenesis in juvenile and adult tissues of seabuckthorn (<Emphasis Type="Italic">Hippophae rhamnoides</Emphasis> L.)

Seabuckthorn (Hippophae rhamnoides) is a multipurpose small tree with unique berries of high nutritional and pharmaceutical values. A clonally propagated plant originating from a 20-year-old tree of H. r. rhamnoides × mongolica hybrid cultivar Julia and seedling offspring of this cultivar were investigated regarding induction of shoot organogenesis in leaf explants and in roots of intact seedlings, and induction of direct somatic embryogenesis in explants from shoot tissue. The highest percentage of leaf explants showing shoot organogenesis was achieved (juvenile explants, 65%; adult explants, 75%) when incubated in Murashige and Skoog (MS) medium supplemented with either 4.5 μM of the phenylurea cytokinin thidiazuron (TDZ) or 2.25 μM TDZ plus 2.2 μM 6-benzyladenine (BA), for juvenile and adult explants, respectively, both supplemented with 0.53 μM α-naphthaleneacetic acid (NAA). Juvenile explants developed on average 18 shoots per explant in the MS medium supplemented with 4.5 μM TDZ, a four fold increase over those incubated on the medium supplemented with 2.25 μM TDZ and 2.2 μM BA. Adult leaf explants grown on medium containing 2.25 μM TDZ and 2.2 μM BA medium produced 12 shoots per explant, while those grown on medium containing 4.5 μM TDZ produced 5 shoots per explant. Shoot organogenesis was observed in roots of intact seedlings pre-cultured on plain medium lacking nutrients (PM) or woody plant medium (WPM) salts and then grown on WPM salts supplemented with 4.4 μM BA, 0.29 μM gibberrelic acid (GA3), and 57.0 μM indoleacetic acid (IAA). The number of shoots formed on each seedling root system was ten fold higher when the pre-culture was in WPM medium indicating a promoting effect of mineral nutrients in the pre-culture medium. Somatic embryogenesis was induced in both juvenile and adult leaf explants in 65 and 78% of the explants, respectively, in MS-based medium supplemented with 2.0 μM N-(2-Chloro-4-pyridyl)-N ¹-phenylurea (CPPU), 0.53 μM NAA and varying concentrations of BA. There was an interaction effect between MS salt strength and BA concentration. The most effective medium for inducing somatic embryogenesis in juvenile explants contained half strength MS salts and 2.2 μM BA and full strength MS salts and 13.2 μM BA for adult explants.     

Morphogenetic responses from protoplasts and tissue culture of Laminaria digitata (Linnaeus) J. V. Lamouroux (Laminariales, Phaeophyta): callus and thalloid-like structures regeneration

The regeneration of meristematic tissues from sporophytes of Laminaria digitata was studied by protoplast and tissue culture. Sequential treatment of explants in sterile seawater with 1% Betadine for 5 min, 1% commercial bleach for 1–2 min and 2% antibiotic treatment supplemented with 1 μM GeO₂ overnight enabled viable explants as high as 55%. Different morphogenetic responses were observed from tissue culture on media supplemented with plant growth regulators alone or in combination, mainly filamentous calluses up to 50% according to the media. Dark green compact calluses were observed on two combinations: 4 μM Pi + 2 μM N-(2-chloro-4-pyridyl)-N’-phenylurea (CPPU) and 0.04 μM Pi + 0.44 μM 6-benzylaminopurine. Thalloid-like structures comparable to adventitious buds were regenerated on medium supplemented with 4 μM Pi + 0.45 μM zeatin but at low frequency suggesting a strong genotypic effect. Friable calluses were developed from protoplasts in enriched medium with polyamines and containing 0.40 μM CPPU + 0.45 μM 2,4-dichlorophenoxyacetic acid. In order to produce protoplasts, a one-step enzymatic protocol was developed and yields reached 22 × 10⁶ protoplasts per gram of fresh weight.     

A Calcium-Receptor Agonist Induces Gustatory Neural Responses in Bullfrogs

The effect of calcium-sensing receptor (CaR) agonists on frog gustatory responses was studied using glossopharyngeal nerve recording and whole-cell patch-clamp recording of isolated taste disc cells. Calcimimetic NPS R-467 dissolved in normal saline solution elicited a large transient response in the nerve. The less active enantiomer of the compound, NPS S-467 induced only a small neural response. The EC₅₀ for NPS R-467 was about 20 μM. Cross-adaptation experiments were performed to examine the effect of 30 μM NPS R-467 and 100 μM quinine on the gustatory neural response. The magnitude of the R-467-induced response after adaptation to quinine was approximately equal to that after adaptation to normal saline solution, indicating that the receptor site for NPS R-467 is different from the site for quinine. NPS R-467 (100 μM) also induced an inward current accompanied with conductance increase and large depolarization in two (13%) of 15 rod cells, and a sustained decrease in outward current and small depolarization in six (40%) other rod cells. NPS S-467 (100 μM) induced a sustained decrease in outward current and depolarization in five (50%) of 10 rod cells. Another calcimimetic cinacalcet (100 μM) induced an inward current accompanied with conductance increase in three (27%) of 11 rod cells. The results suggest that NPS R-467 induces neural responses through cell responses unrelated to a resting K⁺ conductance decrease.     

Differential responses of pea seedlings to indole acetic acid under manganese toxicity

Present study showed the responses of pea seedlings to exogenous indole acetic acid (IAA; 10 and 100 μM) application under manganese (Mn; 50, 100 and 250 μM) toxicity. Manganese and 100 μM IAA alone as well as in combination decreased growth of pea seedlings compared to control. Moreover, some parameters of oxidative stress—hydrogen peroxide (H₂O₂) and malondialdehyde (MDA) were also increased by single and combined treatments of Mn and 100 μM IAA compared to control. In contrast, addition of 10 μM IAA together with Mn, alleviated Mn toxicity symptoms and promoted growth led to the decrease in H₂O₂ and MDA levels compared to Mn treatments alone. Under single and combined treatments of Mn and 100 μM IAA, catalase activity decreased while superoxide dismutase and ascorbate peroxidase activities increased and glutathione reductase and dehydroascorbate reductase exhibited differential responses. However, addition of 10 μM IAA together with Mn, increased activities of studied enzymatic antioxidants. Root and shoot reduced ascorbate (AA) and reduced glutathione (GSH) and, their reduced/oxidized ratios decreased while dehydroascorbate (DHA) and oxidized glutathione (GSSG) contents increased compared to control following single and combined treatments of Mn and 100 μM IAA. However, supply of 10 μM IAA together with Mn, increased AA and GSH, and their reduced/oxidized ratios in root and shoot compared to Mn treatments alone. This study thus suggests that 10 μM of IAA was able to increase Mn tolerance in pea seedlings under Mn toxicity while opposite was noticed for 100 μM IAA.     

Lack of coupling between GABA release and GABA synthesis in the rat brain via GABAB autoreceptors

Summary. GABA is synthesized within GABA terminals through a highly compartmentalized process in which glial-derived glutamine is a major precursor and its release is modulated by GABA_B autoreceptors. The aim of this work was to ascertain whether or not GABA synthesis and release are coupled in the rat brain through a GABA_B autoreceptor-mediated modulation. It was found that (−)baclofen (30 μM) reduces the K⁺ stimulated release of [³H]GABA in synaptosomes and prisms (10 μM) from cerebral cortex, while at the same concentrations (−)baclofen failed to modify the synthesis of [³H]GABA from [³H]glutamine in cortical and hypothalamic slices, prisms and in cortical synaptosomes. In this latter preparation, identical results were observed when (−)baclofen was added to Krebs-Tris media, containing 5 or 15 mM K⁺ concentration. In agreement with these latter results, glutamic acid decarboxylase (GAD) activity from cortical and hypothalamic prisms was not affected by 1–100 μM (−)baclofen. Similar results on GABA synthesis were also observed when 1–100 μM 3-aminopropil(methyl)-phosphinic acid or GABA was used instead of (−)baclofen to stimulate GABA_B autoreceptors. [³H]GABA release, [³H]GABA synthesis from [³H]glutamine and GAD activity were also insensitive to the action of the GABA_B antagonist CGP 52432 (10–100 μM). Likewise, muscimol (0.3–100 μM) did not affect GABA synthesis. Our results indicate that unlike GABA release, GABA synthesis is not modulated by GABA_B autoreceptors. Received August 31, 1999 Accepted September 20, 1999     

Effects of bilobalide on amino acid release and electrophysiology of cortical slices

Summary.  This study investigated the effects of bilobalide, a constituent of Ginkgo biloba, on potassium and veratridine-induced release of glutamate and aspartate from mouse cortical slices. We also studied its effects on spontaneous and N-methyl-D-aspartate (NMDA)-induced depolarizations elicited in magnesium-free artificial cerebrospinal fluid (aCSF) as well as its effect on NO-711 (a γ-aminobutyric acid (GABA) uptake inhibitor)-induced depolarizations. Bilobalide, 100 μM significantly reduced both glutamate and aspartate release elicited by potassium or veratridine. Bilobalide (5–100 μM) also significantly reduced the frequency of NO-711 induced depolarizations, however, it had no effect on spontaneous or on NMDA-induced depolarizations at 5–200 μM. These results suggest that the neuroactive properties of bilobalide may be mediated by a reduction in excitatory amino acid neurotransmitter release. Received June 25, 2001 Accepted October 4, 2001     

Glutathione in adaptation of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> to cadmium stress

The role of glutathione (GSH) in the adaptation of wild type Arabidopsis thaliana plants to Cd stress was investigated. The nutrient solution (control or containing 50 or 100 μM Cd) was supplemented with buthionine sulfoximine (BSO; 50, 100, 500 μM, to decrease the GSH content in plants) or GSH (50, 100, 500 μM, to increase its content in plants) in order to find how GSH content could regulate Cd stress responses. BSO application did not influence plant biomass, while exogenous GSH (especially 500 μM) reduced root biomass. BSO (500μM) in combination with Cd (100 μM) increased Cd toxicity on root growth (by over 50 %), most probably due to reduced GSH content and phytochelatin (PC) accumulation (by over 96 %). On the other hand, combination of exogenous GSH (500 μM) with Cd (100 μM) was also more toxic to plants than Cd alone despite a significant increase in GSH and PC accumulation (up to 2.7 fold in the roots). This fact could indicate that the natural content of endogenous GSH in wild type A. thaliana plants is sufficient for Cd-tolerance. A decrease in this GSH content led to decreased Cd-tolerance of the plants but an increase in GSH content did not enhance Cd-tolerance, and it showed even toxic effect on the plants.     

Regeneration of zoysia grass (Zoysia matrella L. Merr.) cv. Konhee from young inflorescences and stem nodes

We have optimized conditions for efficient regeneration of the vegetatively propagated zoysia grass (Zoysia matrella L. Merr) cultivar “Konhee”. Two explants, young inflorescences, and stem nodes, were used and they displayed different responses to combinations and concentrations of plant growth regulators in callusing, embryogenic callus formation, and regeneration. The highest callus initiation rate from young inflorescences was obtained on medium supplemented with 4.5 to 9.0 μM 2,4-dicholorophenoxy acetic acid (2,4-D) and 0.44 μM 6-benzyl amino purine (BA). When the BA concentration was lowered to 0.044 μM, the highest percent embryogenic callus induction from young inflorescences was achieved. The highest callus initiation rate from stem nodes was obtained, when young inflorescences were cultured on MS medium supplemented with 4.5 to 9.0 μM 2,4-D, 0.44 μM BA, and 0.037 μM abscisic acid (ABA). But embryogenic callus formation from the stem node was highest in the presence of 4.5 to 9.0 μM 2,4-D, 0.044 μM BA, and 0.037 μM ABA. Addition of ABA significantly increased embryogenic callus formation from stem nodes, but not from young inflorescences. Regeneration percentage was variable in response to BA level, and inclusion of α-naphthalene acetic acid (NAA) and gibberellic acid (GA₃) further increased the regeneration percentage. The highest regeneration percentages obtained from the young inflorescences and stem nodes were 82% and 67%, respectively. This is the first report showing that plants can be regenerated from young inflorescences and stem nodes of vegetatively propagated zoysia grass.     

Pharmacological characterization of a nicotinic autoreceptor in rat hippocampal synaptosomes

The modulation of [³H]ACh release by nicotinic compounds was studied in superfused rat hippocampal synaptosomes loaded with [³H]choline. (−)-Nicotine (0.1–10 μM) evoked a dose-dependent increase in [³H]ACh release; higher concentrations were less effective. Nicotine-evoked release was Ca²⁺-dependent, and blocked by the nicotinic antagonists dihydro-β-erythroidine, mecamylamine, and pempidine. The α7-selective antagonist methyllycaconitine did not inhibit nicotine-evoked release when tested at 1 μM, although at 10 μM some attenuation of the response was observed. Six agonists tested were equally efficacious in stimulating [³H]ACh release, as judged by the maximum responses, and gave the following EC₅₀ values: (±)-epibatidine 0.12 μM; (+)-anatoxin-a 0.14 μM; (−)-nicotine 0.99 μM; (−)-cytisine 1.06 μM; ABT-418 2.6 μM; isoarecolone 43 μM. Each agonist generated a “bell-shaped” dose response curve, suggesting desensitisation at higher concentrations. This is supported by analysis of repetitive stimulation with (−)-nicotine and (−)-cytisine: S2/S1 ratios declined sharply with increasing concentration, whereas subsequent KCl-evoked release remained constant. These results are discussed in terms of possible nicotinic receptor subtypes that might be present on hippocampal nerve terminals. Special issue dedicated to Dr. Herman Bachelard.     

MDMA (Ecstasy) Decreases the Number of Neurons and Stem Cells in Embryonic Cortical Cultures

Ecstasy, 3,4-methylenedioxymetamphetamine (MDMA), is a recreational drug used among adolescents, including young pregnant women. MDMA passes the placental barrier and may therefore influence fetal development. The aim was to investigate the direct effect of MDMA on cortical cells using dissociated CNS cortex of rat embryos, E17. The primary culture was exposed to a single dose of MDMA and collected 5 days later. MDMA caused a dramatic, dose-dependent (100 and 400 μM) decrease in nestin-positive stem cell density, as well as a significant reduction (400 μM) in NeuN-positive cells. By qPCR, MDMA (200 μM) caused a significant decrease in mRNA expression of the 5HT3 receptor, dopamine D₁ receptor, and glutamate transporter EAAT2-1, as well as an increase in mRNA levels of the NMDA NR1 receptor subunit and the 5HT_1A receptor. In conclusion, MDMA caused a marked reduction in stem cells and neurons in embryonic cortical primary cell cultures, which was accompanied by changes in mRNA expression of specific receptors and transporters for glutamatergic and monoaminergic neurotransmitters.     

In vitro stem elongation of sweet pepper in media containing 24-epi-brassinolide

In vitro regeneration of sweet pepper (Capsicum annuum L. cvs Jupiter and Pimiento Perfection) has been performed via direct organogenesis. The resulting shoot-buds were placed on media containing 24-epi-brassinolide (EBR) 0.1 μM, a plant steroid lactone, in the presence or absence of zeatin 9.1 μM plus GA3 5.2 μM for further stem elongation. Different responses to these treatments were recorded depending upon the protocols used and the genotypes tested. It appears that EBR does not always act directly on stem elongation but may be an elicitor and/or an enhancer of elongation in concert with endogenous and other exogenously added growth regulators. Elongated shoots were easily rooted with alpha-naphtalenacetic acid 0.5 μM (0.1 mgl-1) and transfered to soil, and following acclimation were taken to maturity in the greenhouse. This revised version was published online in June 2006 with corrections to the Cover Date.     

In vitro regeneration from leaf explants of Neoregelia cruenta (R. Graham) L.B. Smith,an endemic bromeliad from Eastern Brazil

An efficient plant regeneration system was developed for the induction of direct shoot formation from leaves derived from seedlings of Neoregelia cruenta, an endemic Bromeliaceae of Eastern Brazil. Shoot differentiation occurred directly from the leaf bases. In vitro responses were influenced by seedling age and growth regulator combinations. Highest regeneration rates were obtained from explants excised from 7-week-old seedlings cultured in the presence of 22 μM BA and 2.5 μM NAA. Shoot conversion to whole plants was most effective in shoots formed in response to 4.4 or 8.8 μM BA combined with 2.5 μM NAA. This revised version was published online in June 2006 with corrections to the Cover Date.     

Two types of P2x-purinoceptors in neurons of the guinea pig ileum submucous plexus

Effects of exogenous adenosine 5′-triphosphate (ATP) on dissociated guinea pig ileum submucous neurons were studied using a conventional whole-cell patch-clamp technique. With the holding potential of −50 mV, application of 50–1,000 μM ATP evoked an inward current (ATP-induced current) in most (90%) of the tested neurons (n-35). ATP-induced currents were observed regardless of whether or not guanosine 5′-triphosphate (GTP, 0.2 mM) and ATP (2 mM) were present in the intracellular solution, or GTP was replaced with equimolar concentration of guanosine 5′-O-3-thiotriphosphate (n-5). In 26 of 29 neurons studied, which responded to ATP, applications of 50–1,000 μM ATP induced slowly declining currents. ATP receptors did not appear to be completely desensitized during a long pulse (up to 4 min) of 200 μM ATP. Suramin (200 μM) accelerated an increase to peak of the current induced by 200 μM ATP without affecting the maximum response amplitude (n−4_. In about 10% of the neuronsn−3), 50 μM ATP evoked rapidly declining (about 1 sec) currents. Application of 100 μM α,β-Me-ATP to these neurons evoked similar responses. The above results suggest that submucous neurons express two specific subtypes of ionotropic P_2x-purinoceptors, which might be involved in distinct excitatory processes in these neurons.     

An involvement of BDNF and PI3-K/Akt in the anti-apoptotic effect of memantine on staurosporine-evoked cell death in primary cortical neurons

Memantine, a clinically used NMDA receptor antagonist possesses neuroprotective properties, but the exact mechanisms of its beneficial action on neuronal survival are poorly recognized. In the present study, some intracellular mechanisms of memantine effects on staurosporine-evoked cell death were investigated in primary cortical neurons. Memantine (0.1–2 μM) suppressed neuronal apoptosis evoked by staurosporine in 7 DIV cortical neurons, whereas other antagonists of NMDA receptor, MK-801 (1 μM) and AP-5 (100 μM) were ineffective. The anti-apoptotic effects of memantine were not connected with any changes in cytoplasmic calcium concentration or reactive oxygen species level. The immunoblot analysis showed that the staurosporine induced a decrease in p-Akt protein kinase level and that this effect was reversed by memantine treatment. Moreover, the PI3-K inhibitors, wortmannin and LY 294002 attenuated the anti-apoptotic action of memantine on staurosporine-induced cell damage. Furthermore, the ELISA studies showed increased cellular and released BDNF protein level after combined treatment with memantine and staurosporine. There was no effect of memantine on the activation and expression of other protein kinases involved in the mechanism of cellular survival, i.e. ERK1/2, JNK and GSK3-β. The obtained data suggest an NMDAR-independent action of memantine in attenuation of neuronal apoptosis and point to the engagement of BDNF and PI3-K/Akt pathway in these processes.     

Somatic embryogenesis and plant regeneration from root segments of Psoralea corylifolia L., an endangered medicinally important plant

Summary An efficient plant regeneration protocol has been developed from root explants of Psoralea corylifolia L., an endangered medicinally important herbaceous plant species belonging to the family Fabaceae. Nodular embryogenic callus was initiated from young root segments cultured on Murashige and Skoog (MS) medium (1962) supplemented with α-naphthaleneacetic acid (NAA; 2.68–13.42 μM) or 2,4-dichlorophenoxyacetic acid (2.4-D; 2.25–11.25 μM) in combination with 6-benzylaminopurine (BA: 2.2. μM). thiamine HCl (2.9 μM), L-glutamine (342.23 μM) and sucrose (3.0% w/v). The highest frequency (95.2%) of embryogenic calluses was obtained on MS medium supplemented with the growth regulators NAA (10.74 μM) and BA (2.2 μM). Development and maturation of somatic embryos was achieved after transfer of embryogenic calluses to MS medium supplemented with 1.34 μM NAA or 1.12 μM 2,4-D and 4.4–13.2 μM BA. The maximum number (13.8±1.34) of cotyledonary stage somatic embryos was obtained on MS medium containing 1.34 μM NAA and 13.2 μM BA. Germination of somatic embryos occurred on MS medium without any growth regulators and also on MS medium enriched with BA (1.1–8.8 μM), although the maximum germination frequency (76.1%) was obtained on 4.4 μM BA plus 1.45 μM gibberellic acid (GA₃). Plant regeneration without complete somatic embryo maturation was also achieved by transferring clumps of nodular embryogenic calluses onto MSO medium or MS medium supplemented with NAA (1.34 μM) and BA (2.2–8.8 μM). The highest frequency of plant regeneration (93.3%) and mean number of plantlets (15.4±0.88) were obtained on MS medium containing 1.34 μM NAA and 4.4 μM BA. Regenerated plants with well-developed root systems were transferred to pots where they grew vigorously, attained maturity and produced fertile seeds.     

A microdialysis study of the effects of the nicotinic agonist RJR-2403 on cortical release of acetylcholine and biogenic amines

Transcortical dialysis was employed to investigate the effects of subcutaneous (s.c.) injections of RJR-2403 (1.2–7.2 μmol/kg) on extracellular levels of acetylcholine (ACh), norepinephrine (NE), dopamine (DA), and serotonin (5-HT) in rat. Systemic administration of RJR-2403 produced a 90% increase of cortical extracellular ACh levels that persisted for up to 90 minutes after injection. Norepinephrine and DA release were increased 124% and 131% above basal values, respectively. Serotonin (5-HT) levels in the dialysate were also significantly elevated by RJR-2403 (3.6 μmol/kg, s.c.) 70% above baseline at 90 minutes post-injection. Comparison of these responses to those of (−)nicotine from a previous study reveals little difference between the two compounds in their ability to influence cortical neurotransmitter release following systemic administration.