Optimizing the Agrobacterium tumefaciens-mediated transformation conditions in Colletotrichum lindemuthianum: a step forward to unravel the functions of pathogenicity arsenals.

Colletotrichum lindemuthianum is a hemibiotrophic fungal pathogen that causes bean anthracnose and it is rated among the top 10 important diseases infecting beans. Currently our knowledge on molecular mechanisms underlying C. lindemuthianum pathogenesis is limited. About five pathogenicity genes have been identified in C. lindemuthianum using Restricted Enzyme Mediated Integration and the transformation using Agroinfection has not been optimized. In this study, a series of experiments were conducted to optimize the key parameters affecting the Agrobacterium tumefaciens-mediated transformation for C. lindemuthianum. The transformation efficiency increased with increase in spore concentration and co-cultivation time. However, the optimum conditions that yielded significant number of transformants were 106 ml-1 spore concentration, co-cultivation time of 72 h, incubation at 25°C and using a cellulose membrane filter for the co-cultivation. The optimized protocol resulted in establishment of large mutant library (2400). A few mutants were melanin deficient and a few were unable to produce conidia. To determine the altered pathogenicity, two new approaches such as detached leaf and twig techniques proved reliable and require fewer resources to screen the large mutant libraries in a short time. Among the 1200 transformants tested for virulence, 90% transformants were pathogenically similar to wild type (race 2047), 96 and 24 were reduced and impaired, respectively. The altered avirulent transformants can prove vital for understanding the missing link between growth and developmental stages of pathogen with virulence. This platform will help to develop strategies to determine the potential pathogenicity genes and to decipher molecular mechanisms of host-pathogen interactions in more detail.

Integrating CRISPR-Cas and Next Generation Sequencing in Plant Virology.

Plant pathology has been revolutionized by the emergence and intervention of next-generation sequencing technologies (NGS) which provide a fast, cost-effective, and reliable diagnostic for any class of pathogens. NGS has made tremendous advancements in the area of research and diagnostics of plant infecting viromes and has bridged plant virology with other advanced research fields like genome editing technologies. NGS in a broader perspective holds the potential for plant health improvement by diagnosing and mitigating the new or unusual symptoms caused by novel/unidentified viruses. CRISPR-based genome editing technologies can enable rapid engineering of efficient viral/viroid resistance by directly targeting specific nucleotide sites of plant viruses and viroids. Critical genes such as eIf (iso) 4E or eIF4E have been targeted via the CRISPR platform to produce plants resistant to single-stranded RNA (ssRNA) viruses. CRISPR/Cas-based multi-target DNA or RNA tests can be used for rapid and accurate diagnostic assays for plant viruses and viroids. Integrating NGS with CRISPR-based genome editing technologies may lead to a paradigm shift in combating deadly disease-causing plant viruses/viroids at the genomic level. Furthermore, the newly discovered CRISPR/Cas13 system has unprecedented potential in plant viroid diagnostics and interference. In this review, we have highlighted the application and importance of sequencing technologies on covering the viral genomes for precise modulations. This review also provides a snapshot vision of emerging developments in NGS technologies for the characterization of plant viruses and their potential utilities, advantages, and limitations in plant viral diagnostics. Furthermore, some of the notable advances like novel virus-inducible CRISPR/Cas9 system that confers virus resistance with no off-target effects have been discussed.

Molecular phylogeny, pathogenic variability and phytohormone production of Fusarium species associated with bakanae disease of rice in temperate agro-ecosystems.

Bakanae is the emerging disease threating the rice cultivation globally. Yield reduction of 4-70% is recorded in different parts of the world. A total of 119 Fusarium isolates were collected from rice plants at different geographical locations and seeds of different rice cultivars. The isolates were evaluated for morphological, biochemical and pathogenic diversity. The amplification of TEF-1α gene was carried out for exploring the species spectrum associated with the cultivated and pre-released rice varieties. The production of gibberellin varied from 0.53 to 2.26 µg/25 ml, while as that of Indole acetic acid varied from 0.60 to 3.15 µg/25 ml among the Fusarium isolates. The phylogenetic analysis identified 5 different species of the genus Fusarium viz. Fusarium fujikuroi, F. proliferatum, F. equiseti, F.oxysporum and F. persicinum after nucleotide blasting in NCBI. Only two Fusarium spp. F. fujikuroi and F. proliferatum were found to be pathogenic under virulence assays of the isolates. The isolates showed a considerable variation in morphological and pathogenic characters. The isolates were divided into different groups based on morphology and pathogenicity tests. The isolates showed a considerable variation in morphology, phytohormone profile and virulence indicative of population diversity. Three species F. equiseti, F.oxysporum and F. persicinum which have not been reported as pathogens of rice in India were found to be associated with bakanae disease of rice, however their pathogenicity could not be established.

Heat induction in two-dimensional graphene-Fe3O4 nanohybrids for magnetic hyperthermia applications with artificial neural network modeling.

We report the synthesis and characterization of graphene functionalized with iron (Fe3+) oxide (G-Fe3O4) nanohybrids for radio-frequency magnetic hyperthermia application. We adopted the wet chemical procedure, using various contents of Fe3O4 (magnetite) from 0-100% for making two-dimensional graphene-Fe3O4 nanohybrids. The homogeneous dispersal of Fe3O4 nanoparticles decorated on the graphene surface combined with their biocompatibility and high thermal conductivity make them an excellent material for magnetic hyperthermia. The morphological and magnetic properties of the nanohybrids were studied using scanning electron microscopy (SEM) and a vibrating sample magnetometer (VSM), respectively. The smart magnetic platforms were exposed to an alternating current (AC) magnetic field of 633 kHz and of strength 9.1 mT for studying their hyperthermic performance. The localized antitumor effects were investigated with artificial neural network modeling. A neural net time-series model was developed for the assessment of the best nanohybrid composition to serve the purpose with an accuracy close to 100%. Six Nonlinear Autoregressive with External Input (NARX) models were obtained, one for each of the components. The assessment of the accuracy of the predicted results has been done on the basis of Mean Squared Error (MSE). The highest Mean Squared Error value was obtained for the nanohybrid containing 45% magnetite and 55% graphene (F45G55) in the training phase i.e., 0.44703, which is where the model achieved optimal results after 71 epochs. The F45G55 nanohybrid was found to be the best for hyperthermia applications in low dosage with the highest specific absorption rate (SAR) and mean squared error values.

Population structure of Venturia inaequalis, a hemibiotrophic fungus, under different host resistance specificities in the Kashmir valley.

Venturia inaequalis is a notorious fungal pathogen and show classical gene for gene interaction with its apple host. Neutral markers provide clues about history, evolutionary potential, genetic diversity and population structure of V. inaequalis. The genetic diversity and population structure of fungus indicates that the pathogen is highly diverse with the capacity to breach the scab resistance genes. In the present study, we collected 108 V. inaequalis isolates from three apple cultivars differing in Rvi1 resistance gene. Based on the AMOVA, the variation was mostly distributed among the isolates, providing evidence of non-existence of subpopulation in orchards thus founder population is difficult to arise in Kashmir apple orchards. Pair wise genetic differentiation is less due to regular occurrence of gene flow between the populations residing on different orchard as infected material is transported without stringent quarantine measures. Based on principal coordinate analysis and clustering algorithm as implemented in STRUCTURE, we observed admixture between the two subpopulations, which is quite low, suggesting the existence of pre-zygotic and post-zygotic barriers to gene flow and we cannot rule out the existence of other structures shared by accessions belonging to different varieties. Due to the continuous increase in introduction and monoculture of apple varieties, mixed orchard with different host resistance specificities are more suitable for managing the apple scab in Kashmir valley.

Can extent of high grade dysplasia in Barrett's oesophagus predict the presence of adenocarcinoma at oesophagectomy?

BACKGROUND: Optimal management of Barrett's oesophagus complicated by high grade dysplasia is controversial. Recently, the extent of high grade dysplasia was described as a predictor of subsequent development of cancer in patients undergoing continued surveillance. However, there is no universal agreement on the definition of extent of high grade dysplasia. AIM: To determine if extent of high grade dysplasia in Barrett's oesophagus is a predictor of the presence of adenocarcinoma at the time of oesophagectomy. METHODS: Forty two patients with Barrett's oesophagus and high grade dysplasia who underwent oesophagectomy between 1985 and 1999 were identified from a prospective database. All pathological specimens, including preoperative endoscopic biopsies and post-oesophagectomy sections, were reviewed in a blinded fashion by one expert gastrointestinal pathologist to determine the extent of high grade dysplasia. The extent of high grade dysplasia was defined using two different criteria, one from the Cleveland Clinic and one from the Mayo Clinic. RESULTS: Twenty four of 42 patients (57%) had unsuspected cancer at the time of oesophagectomy. Using the Cleveland Clinic definition, 10 of 21 (48%) patients with focal high grade dysplasia had carcinoma compared with 14 of 21 patients (67%) with diffuse high grade dysplasia (p=0.35). Using the Mayo Clinic definition, adenocarcinoma was found in five of seven (72%) patients with focal high grade dysplasia compared with 19 of 35 (54%) with diffuse high grade dysplasia (p=0.68). CONCLUSIONS: The extent of high grade dysplasia, regardless of how it is defined, does not predict the presence of unsuspected adenocarcinoma at oesophagectomy. There is no evidence as yet that the extent of high grade dysplasia can be used as a basis for decision making in these patients.

Modulation of ethanol-induced motor incoordination by mouse striatal A(1) adenosinergic receptor.

We have demonstrated that ethanol-induced motor incoordination is modulated by cerebellar adenosine A(1) receptor. This study represents an extension into another important brain motor area, the striatum that, unlike cerebellum, has high density of both A(1) and A(2A) receptors. Direct intra-striatal micro-infusion of Ro15-4513 (0.05, 0.5, 1 ng), a partial inverse-agonist of benzodiazepine, significantly and nearly dose-dependently attenuated ethanol-induced motor incoordination indicating mediation of ethanol's motor incoordination by striatum. Intra-striatal A(1)-selective agonist N(6)-cyclohexyladenosine (CHA; 1, 2, 4 ng), A(1) = A(2A) non-selective agonist, 5'-N-ethylcarboxamidoadenosine (NECA; 1.5, 3, 6 ng), and A(1)-selective antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 25, 50, 100 ng) dose-dependently accentuated and attenuated, respectively, ethanol-induced motor incoordination, strongly suggesting modulation by striatal adenosine A(1) receptor. Intra-striatal DPCPX significantly antagonized not only ethanol-induced motor incoordination but also its potentiation by intra-striatal CHA, R-(+)-N(6)-(2-phenylisopropyladenosine) (R-PIA), or NECA. No change in motor coordination occurred after the highest dose of CHA, R-PIA, or NECA followed by saline. Similarly, the highest intra-striatal dose of Ro15-4513 or DPCPX neither altered motor coordination or locomotor activity indicating relative selectivity of interaction with ethanol. Nearly 25-fold higher dose of A(2A)-selective agonist, CGS-21680, compared to CHA was necessary to produce a comparable potentiation of ethanol's motor incoordination perhaps suggesting a lack of or less significant striatal A(2A) involvement. Intra-striatal pertussis toxin (0.5 microg) pre-treatment markedly attenuated ethanol-induced motor incoordination as well as its potentiation by intra-striatal CHA. These results support that striatum is one of the brain motor areas mediating the motor impairing effects of acute ethanol and that the latter's modulation occurs via A(1)-selective receptors coupled to pertussis toxin-sensitive G proteins.

Involvement of the cerebellar adenosine A(1) receptor in cannabinoid-induced motor incoordination in the acute and tolerant state in mice.

Cannabinoids are known to impair motor function in humans and laboratory animals. We have demonstrated an accentuation of cannabinoid (CP55,940)-induced motor incoordination in mice by the adenosine A(1) receptor-selective agonist N(6)-cyclohexyladenosine (CHA) (4 ng) using an intracerebellar (ICB) microinjection method. This effect was mediated by the A(1) receptor because pre-treatment with ICB 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) (100 ng), an adenosine A(1) receptor selective antagonist, completely abolished the accentuation. Furthermore, ICB pre-treatment with DPCPX (100 ng) before ICB CP55,940 (15 microg) attenuated the motor incoordination suggesting a modulation by an endogenous adenosine A(1) system. ICB microinjection of CHA or DPCPX prior to ICB vehicle had no effect on normal motor coordination. ICB microinjection of dipyridamole (25 microg), an adenosine transport inhibitor, significantly accentuated the motor incoordination by ICB CP55,940 (15 microg), providing further support for the involvement of endogenous adenosine in the action of CP55,940. Tolerance to the motor incoordinating effect of ICB CP55,940 was demonstrated following 3 days of i.p. CP55,940 (0.1, 1 or 2 mg/kg every 12 or 24 h; total of six or three injections, respectively). Interestingly, animals which exhibited tolerance to ICB CP55,940 also demonstrated tolerance to the accentuating effect of ICB CHA suggesting cross-tolerance between adenosine agonists and cannabinoids. Cross-tolerance was also demonstrated following 3 days of i.p. CHA (0.25 or 1 mg/kg every 24 h; total of three injections) as further evidence of the modulatory role of the cerebellar adenosine system in the acute manifestation of CP55,940-induced motor incoordination. The involvement of cerebellar adenosine and the A(1) receptor in cannabinoid actions is circumstantially supported by previous evidence that CB(1) receptors and A(1) receptors are both localized on cerebellar granule cell parallel fiber terminals and basket cell neurons where they serve to inhibit the release of neurotransmitters.

Cannabinoid-induced motor incoordination through the cerebellar CB(1) receptor in mice.

Cannabinoids are known to impair motor function in humans and laboratory animals. We have observed dose-dependent motor incoordination in mice evaluated by rotorod following direct intracerebellar (i.c.b.) microinjection of synthetic cannabinoid agonists CP55,940 (5-25 microg) and HU-210 (1.56-6.25 microg), through permanently implanted stainless steel guide cannulas. The motor incoordination was marked at 15, 35 and 55 min post-microinjection. The motor incoordination elicited by HU-210 (6.25 microg) and CP55,940 (20 microg) was significantly blocked by the CB(1) receptor-selective antagonist SR141716A (25 microg i.c.b.), indicating mediation by a cerebellar CB(1) receptor. Further direct evidence of CB(1) mediation was obtained through a CB(1) receptor antisense/mismatch oligodeoxynucleotide approach (3 microg/12 h; total of six doses). Mice treated with intracerebellar antisense had a significantly diminished motor incoordination response to intracerebellar CP55,940 15 microg compared to mice that received intracerebellar mismatch or no prior treatment. Also, the response to intracerebellar CP55,940 in the CB(1) mismatch-treated mice did not differ from the mice that received only CP55,940. A separate study using a cerebellar tissue punching technique, following intracerebellar [3H]-CP55,940 microinjection, confirmed that cannabinoid drug dispersion following microinjections was exclusively confined to the cerebellum. Microinjection of CP55,940 (20 microg) into the hippocampus, an area with a large density of CB(1) receptors, did not impair motor coordination. Taken together, these results indicate that cannabinoid-induced motor impairment occurs by activation of a CB(1) receptor in the cerebellum. The participation of other brain motor areas in cannabinoid-induced motor incoordination will require future study.

Cerebellar CB(1) receptor mediation of Delta(9)-THC-induced motor incoordination and its potentiation by ethanol and modulation by the cerebellar adenosinergic A(1) receptor in the mouse.

The effect of intracerebellar microinfusion of antisense oligodeoxynucleotide to Delta(9)-tetrahydrocannabinol (Delta(9)-THC) and other naturally occurring cannabinoid receptor (CB(1)) mRNA on Delta(9)-THC-induced motor impairment was investigated in mice. Delta(9)-THC (15-30 microgram/microliter intracerebellar) resulted in a significant motor impairment in a dose-related manner. The intracerebellar pretreatment with antisense oligodeoxynucleotide (3.0 microgram/100 nl/12 h; six administrations/mouse) virtually abolished Delta(9)-THC (15 and 25 microgram/1 microliter intracerebellar)-induced motor impairment. However, intracerebellar pretreatment with the mismatched oligodeoxynucleotide in exactly the same manner as the antisense was completely ineffective in altering the Delta(9)-THC-induced motor impairment. These results strongly suggest the involvement of CB(1) receptor in the expression of Delta(9)-THC-induced motor impairment. The intracerebellar microinfusion of adenosine A(1)-selective agonist, N(6)-cyclohexyladenosine (CHA) (4 ng/100 nl) significantly enhanced Delta(9)-THC-induced motor impairment, suggesting a cerebellar A(1) adenosinergic modulation of motor impairment. A pretreatment with the antisense and the mismatched oligodeoxynucleotide also markedly attenuated and did not alter, respectively, the cerebellar A(1) adenosinergic modulation (enhancement) of Delta(9)-THC-induced motor impairment. There was no change in the normal motor coordination due to intracerebellar pretreatment with antisense and its mismatch, in the presence as well as absence of intracerebellar CHA indicating the selectivity of interactions with Delta(9)-THC. The Delta(9)-THC-induced motor incoordination was also significantly enhanced dose-dependently by systemic (i.p.) ethanol administration suggesting behavioral synergism between the two psychoactive drugs. Pretreatment (intracerebellar) with pertussis toxin (PTX) markedly attenuated Delta(9)-THC- and Delta(9)-THC+CHA-induced motor incoordination suggesting coupling of CB(1) receptor to PTX-sensitive G-protein (G(i)/G(o)). These data suggested co-modulation by cerebellar cannabinoid and adenosine system of Delta(9)-THC-induced motor impairment. Conversely, the results in the present study also suggested co-modulation by cerebellar adenosine A(1) and CB(1) receptors of ethanol-induced motor impairment, thereby indicating a possible common signal transduction pathway in the expression of motor impairment produced by Delta(9)-THC as well as ethanol.

Adenosinergic modulation of ethanol-induced motor incoordination in the rat motor cortex.

1. On going work in our laboratory has shown that adenosine modulates ethanol-induced motor incoordination (EIMI) when given systemically as well as directly into the cerebral ventricles, cerebellum and corpus striatum of the rat and/or mouse. 2. The objective of this study was to determine what effect adenosine agonists and antagonists would have within the rat motor cortex on EIMI. 3. The participation of the motor cortex in EIMI was suggested when microinfusion of the anti-ethanol compound, Ro15-4513, an inverse agonist of the benzodiazepine binding site, directly into the motor cortex significantly attenuated EIMI. Further, the adenosine agonists N6-cyclohexyladenosine (CHA) and 2-p-(2-carboxyethyl)-phenethylamino-5'-N-carboxaminoadenosine++ + hydrochloride (CGS-21680) significantly accentuated EIMI in a dose-related manner. The adenosine A1 receptor-selective agonist, CHA, appeared most potent in this modulatory effect when compared to the A2-selective agonist, CGS-21680. 4. The extent of diffusion of the adenosine drugs within the cortical tissue after their microinfusion was also checked by measuring the dispersion of microinfused [3H]CHA. The [3H]CHA dispersion study indirectly confirmed that the results of the present investigation were based on the effect of adenosine drugs within the motor cortex only. 5. Accentuation by the A1- and A2-selective adenosine agonists was significantly attenuated by the A1-selective antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) but not by the A2 receptor-selective antagonist 8-(3-chlorostyryl)caffeine (CSC) further suggesting modulation mainly by the A1-subtype. 6. Pretreatment of the motor cortex with pertussis toxin (PT) significantly reduced the capacity of both A1- and A2-selective adenosine agonists to accentuate EIMI suggesting the involvement of a PT-sensitive Gi/Go protein. 7. These data support earlier work which showed that adenosine modulates EIMI within the central nervous system (CNS), most likely via the A1 receptor, and moreover, extend that work by including the motor cortex as a brain area participating in the adenosinergic modulation of ethanol-induced motor impairment.

Rat striatal adenosinergic modulation of ethanol-induced motor impairment: possible role of striatal cyclic AMP.

We have previously reported the involvement of the striatum in acute ethanol-induced motor incoordination and the striatal adenosinergic modulation of ethanol-induced motor incoordination through A1 receptor-mediated mechanism(s). The present study, a continuation of our previous work, was carried out to investigate the possible functional correlation between striatal cyclic AMP and ethanol-induced motor incoordination, and its modulation by striatal adenosine in Sprague-Dawley rats. Forskolin (0.1, 0.5 and 1.0 pmol), a known activator of adenylate cyclase, significantly attenuated ethanol-induced motor incoordination in a dose-dependent manner following its direct intrastriatal microinfusion. Forskolin also antagonized the accentuating effect of intrastriatal N6-cyclohexyladenosine on ethanol-induced motor incoordination. These results suggested that ethanol-induced motor incoordination might be functionally correlated to a decrease in the striatal cyclic AMP levels and that the striatal adenosine A1 receptors might modulate ethanol-induced motor incoordination through cyclic AMP signaling mechanism(s). Further support to this hypothesis was obtained by the actual measurement of the striatal cyclic AMP levels in the same experimental conditions as in motor coordination studies using high-performance liquid chromatography with fluoroscence detection. Regardless of the method (focused microwave irradiation, cervical dislocation or decapitation into a dry ice-ethanol mixture) used to kill the animals, a significant decrease in the striatal cyclic AMP levels was observed due to ethanol. Intrastriatal adenosine A1-selective agonist, N6-cyclohexyladenosine (24 ng), caused a further significant decrease in the striatal cyclic AMP levels in the ethanol- but not in the vehicle-treated animals. The further enhancement in the ethanol-induced decrease in the striatal cyclic AMP levels by intrastriatal N6-cyclohexyladenosine, therefore, functionally correlated with the observed potentiating effect of intrastriatal N6-cyclohexyladenosine on ethanol-induced motor incoordination. The effects of intrastriatal N6-cyclohexyladenosine+ethanol and of ethanol alone on the striatal cyclic AMP levels were blocked by intrastriatal pertussis toxin (500 ng) pretreatment, indicating the involvement of pertussis toxin-sensitive G-proteins (Gi, Go) and possibly of the adenosine A1 receptor coupled to the G-proteins in the striatum. Furthermore, ethanol alone significantly decreased the basal as well as the cyclic AMP-stimulated catalytic activities of the striatal cyclic AMP protein kinase, which were further reduced by intrastriatal N6-cyclohexyladenosine. The results of the present study therefore support an involvement of a cyclic AMP signaling pathway in the striatal adenosinergic modulation of ethanol-induced motor incoordination at the post-adenosine A1 receptor level.

Involvement of kappa-opioids in the mouse cerebellar adenosinergic modulation of ethanol-induced motor incoordination.

Using rotorod performance as the test response, possible modulation and co-modulation of ethanol-induced motor incoordination by the cerebellar kappa-opioid and adenosine A1 receptors was studied. A dose-related accentuation of ethanol-induced motor incoordination was observed after direct cerebellar microinfusion of three kappa-opioid receptor agonists: U-50488, U-62066, and bremazocine. On the contrary, significant and dose-related attenuation of ethanol's motor impairment was produced by intracerebellar nor-binaltorphimine, a kappa-opioid receptor antagonist. Furthermore, the accentuation by kappa-agonists was virtually abolished by kappa-antagonist nor-binaltorphimine. Therefore, the accentuation and attenuation by kappa-opioid receptor agonists/antagonist, respectively, was through specific kappa-opioid receptors. Pretreatment with the intracerebellar adenosine A1-selective agonist, N6-cyclohexyladenosine, further enhanced the ethanol-induced motor incoordination and its accentuation by intracerebellar kappa-opioid receptor agonists. Ethanol-induced motor incoordination was markedly attenuated by intracerebellar pertussis toxin (PTX) pretreatment, suggesting an involvement of PTX-sensitive G protein in the expression of motor incoordinating effect of ethanol. Additionally, the intracerebellar PTX also markedly attenuated the accentuation by kappa-opioid agonists of ethanol-induced motor impairment, suggesting participation of PTX-sensitive GTP-binding G protein (Gi, Go) in the kappa-opioid modulation of ethanol's motor impairment. It also confirms that kappa-opioid receptors are linked to PTX-sensitive G protein. The functional similarity between kappa-opioid and adenosine A1 receptors in increasing ethanol's motor incoordination, together with their anatomical co-localization primarily on the axons and axonal terminals of the cerebellar granule cells, suggests a possible common catalytic unit of adenylate cyclase as the basis of modulation of ethanol-induced motor incoordination by both receptor mechanisms.

Mouse cerebellar adenosinergic modulation of ethanol-induced motor incoordination: possible involvement of cAMP.

As an extension of our previous work pertaining to brain adenosinergic modulation of ethanol-induced motor incoordination, the effect of direct intracerebellar administration of the A1-selective adenosine agonist, N6-cyclohexyladenosine (CHA) on ethanol-induced motor incoordination was evaluated. Marked accentuation of ethanol-induced motor impairment by CHA was observed. No change in the normal motor coordination was noted when CHA administration was followed by saline instead of ethanol. Intracerebellar cAMP or its analog, 8-(4-chlorophenylthio)-cAMP, significantly inhibited ethanol's motor impairment in a dose-related manner as well as abolished CHA's accentuating effect on ethanol-induced motor incoordination. These observations suggested a possible involvement of cAMP in the adenosinergic modulation and in the expression of ethanol-induced motor incoordination. Further support was provided by the observation of a marked accentuation and attenuation in a dose-related manner of ethanol-induced motor impairment as well as CHA's accentuation of ethanol's motor impairment by intracerebellar miconazole and forskolin, respectively. However, equimolar intracerebellar doses of miconazole and forskolin (inhibitor and stimulator of adenylyl cyclase, respectively) failed to significantly alter ethanol-induced motor incoordination probably due to their mutual functional antagonism. The expression of adenosinergic modulation and that of ethanol-induced motor impairment most likely involved Gi protein-coupled receptor(s) (such as adenosine receptors). The involvement of receptors linked to pertussis toxin-sensitive G-proteins was suggested because intracerebellar pertussis toxin pretreatment markedly inhibited ethanol-induced motor incoordination as well as CHA's accentuation of ethanol's motor impairment. Finally, cAMP, unlike its antagonism to CHA's accentuation, failed to antagonize the accentuation of ethanol-induced motor impairment by intracerebellar GABA(A) agonist (+)-muscimol. This indicated selectivity of cAMP participation in G protein coupled receptor (such as adenosine)-mediated response and not in ionic channel coupled receptor (such as GABA(A))-mediated mechanism. Overall, the data suggested a possible involvement of cerebellar adenylyl cyclase-cAMP signalling pathway in the adenosinergic modulation of ethanol's ataxia.

The striatal adenosinergic modulation of ethanol-induced motor incoordination in rats: possible role of chloride flux.

Previous studies from our laboratory have provided strong evidence that brain adenosine modulates acute ethanol (i.p.)-induced motor incoordination (MI) through receptor mediated mechanism(s). Recently, we have reported the involvement of the striatum in ethanol-induced MI as well as the striatal adenosinergic modulation of the ethanol-induced motor deficit. The present study was thus designed to further characterize the modulatory effect of striatal adenosine on ethanol-induced MI and to look for its functional correlation with chloride flux within the rat striatum. Intrastriatal microinfusion of adenosine A1 receptor agonist N6-cyclohexyladenosine (CHA) and antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), significantly accentuated and attenuated, respectively, the motor incoordinating effect of ethanol while having no effect on the normal motor coordination in saline-treated control animals. These data confirmed the role of striatal adenosine in ethanol-induced MI. The selectivity of interactions between adenosine A1 agonist and antagonist and ethanol was further confirmed by the study in which neither intrastriatal CHA nor DPCPX significantly altered the MI induced by sodium pentobarbital. Previously, we have shown that intrastriatal Ro15-4513 not only significantly attenuated ethanol-induced MI but also blocked its accentuation by intrastriatal CHA. It is well known that Ro15-4513 antagonizes many, but not all, CNS effects of ethanol by blocking the ethanol potentiation of GABA-stimulated uptake of chloride. Therefore, experiments using striatal microsac preparations were carried out to investigate the possible modulation of chloride conductance by CHA and its relationship to ethanol. High concentrations of CHA (10 and 100 nM) increased the total chloride uptake by the striatal microsacs. Corresponding to the ethanol-adenosine interaction observed behaviorally, a much lower concentration (1 nM) of CHA, being ineffective itself, significantly enhanced the stimulatory action of ethanol on chloride uptake. This effect was blocked by either Ro15-4513 (100 nM) or DPCPX (10 nM). The modulatory effect of GABA and/or ethanol on chloride influx was also evaluated, and the results supported the appropriateness to use striatal microsac preparations in the present study. Overall, the data suggested a functional interaction between ethanol and striatal adenosine and further supported the hypothesis that striatal adenosine might, in part, modulate ethanol-induced MI through its effect on chloride conductance through chloride channels coupled to GABA-benzodiazepine receptor complex.

Dispersion characteristics of [3H]-labeled adenosine agonist/antagonist following their intrastriatal microinfusion.

Following their intrastriatal microinfusion, the dispersion patterns of an adenosine receptor agonist (N6-cyclohexyladenosine) and an antagonist (8-cyclopentyl-1,3-dipropylxanthine) within the striatal tissue were investigated in Sprague-Dawley rats. The [3H]-labeled ligands were microinfused into the striatum of conscious rats through preimplanted guide cannulae in the volume of either 200 (0.1 microCi) or 1000 nl (0.5 microCi) and the animals were killed 15 or 30 min later. The diffusion of the radioactive ligands was evaluated by measuring the radioactivity in the striatal tissue samples using a tissue punching technique. When the volume of microinfusion was 200 nl, the diffusion within the striatum was limited as the radioactivity remained confined to the immediate vicinity of microinfusion site regardless of the postmicroinfusion time (15 or 30 min). The pattern of tissue diffusion was similar at 15 min after the intrastriatal microinfusion of 1000 nl of [3H]-ligands. At 30 min after the intrastriatal microinfusion of 1000 nl volume, a relatively larger area of striatal tissue was covered by the drug solution. In addition, the 1000 nl intrastriatal microinfusion probably resulted in the diffusion of some of the drug solution into the extrastriatal area since small but significant radioactivity was detected at sites outside the striatum. The intrastriatal diffusion of the [3H]-ligand solution was not uniform in all directions from the site of microinfusion. The relationship between the amount of radioactivity remaining at the site of microinfusion and the postmicroinfusion time was inverse. Additionally, at the same postmicroinfusion time (15 or 30 min), a lower percent of the total microinfused radioactivity was found remaining at the microinfusion site with the 1000 nl microinfusion volume than that with the 200 nl volume. Overall, the diffusion patterns of intrastriatal adenosine agonist and antagonist were similar. The results of the present investigation suggest that both the microinfusion volume and the postmicroinfusion time may be important factors in determining the diffusion pattern and tissue content of intrastriatally microinfused adenosine drugs. This information could be important for the correct understanding and interpretation of the data from studies involving drug microinfusions.

Mouse cerebellar GABAB participation in the expression of acute ethanol-induced ataxia and in its modulation by the cerebellar adenosinergic A1 system.

The possible modulation and of co-modulation by the cerebellar GABAB and adenosine A1 receptors of ethanol-induced motor impairment were investigated in the mice using rotorod performance as the test response. Direct cerebellar microinfusion of GABAB agonist, baclofen, and antagonist, phaclofen, into the permanently cannulated mice, produced a dose-dependent accentuation and attenuation, respectively of ethanol-induced motor impairment. The baclofen and phaclofen exhibited accentuation and attenuation, respectively, via GABAB receptors linked to pertussis toxin-sensitive G protein. A comodulation by the cerebellar adenosine A1 receptors was also observed because intracerebellar microinfusion of adenosine agonists NB-cyclohexyladenosine (CHA), 5'-N-ethylcarbox-amidoadenosine (NECA), and 2-p-(2-carboxyethyl)-phenyl-ethylamino-5'-N-ethylacarbox- amidoadenosine (CGS-21680), and antagonist, 8-cyclopentyl-1, 3-dipropylxanthine (DPCPX), also accentuated and attenuated, respectively, ethanol-induced motor impairment. The accentuation of ethanol-induced motor impairment by baclofen was further enhanced after the intracerebellar microinfusion of CHA, suggesting a co-modulation by the co-localized adenosine A1 receptors. A similar response was observed after the intracerebellar microinfusion of adenosine A1 = A2 agonist NECA and the several-fold higher dose of adenosine A2-selective agonist CGS-21680. Ethanol-induced motor impairment was markedly blocked by intracerebellar A1-selective antagonist, DPCPX, as well as by the intracerebellar pertussis toxin pretreatment suggesting again a co-modulation by the adenosine A1 receptors and the involvement of pertussis toxsin-sensitive G protein, respectively. The almost 25-fold higher dose of CGS-21680 to accentuate and DPCPX to attenuate, respectively, ethanol-induced motor impairment together with the reported cerebellar localization of adenosine A1 subtype only, suggested A1 receptor activation by NECA and CGS-21680. The functional similarity between GABAB and adenosine A1, receptors associated with their anatomical co-localization on the cerebellar granule cells, mainly axons and axonal terminals, may suggest a possible common adenylate cyclase catalytic unit as the basis of modulation of ethanol's motor impairment by these two receptor mechanisms.

Antagonism by intracerebellar Ro15-4513 of acute ethanol-induced motor incoordination in mice.

The possible antiethanol effect of intracerebellarly microinjected Ro15-4513 was investigated using motor incoordination as the test response. The results of this study further confirmed reports from this and other laboratories that this partially negative ligand of benzodiazepine selectively attenuated and nearly reversed the motor impairment of acute ethanol. The attenuation observed after microinjections of doses of 0.05, 0.1, and 0.5 ng was significant and dose related. There was no effect on normal coordination when the highest dose, 0.5 ng, was administered followed by saline instead of a test dose of ethanol. When 0.5 ng of Ro15-4513 alone was microinjected into the cerebellum, no significant change in the locomotor activity was observed. Even a 10-fold higher intracerebellar dose (5 ng) of Ro15-4513 administered alone produced no significant changes in locomotor activity. This suggests that attenuation of ethanol-induced motor incoordination was most likely due to the selective antiethanol effect of Ro15-4513 at the dose range used in the present investigation. The antiethanol effect of intracerebellar Ro15-4513 also reaffirmed the well-known belief that the cerebellum is an important brain region for ethanol's motor-impairing effect. The results also indirectly suggest the inhibition of GABAA-gated chloride ion channel activity as the most likely basis of Ro15-4513's antiethanol effect.

Possible role of striatal adenosine in the modulation of acute ethanol-induced motor incoordination in rats.

Several reports from our laboratory have suggested the involvement of the brain adenosinergic system in ethanol-induced motor incoordination (EIMI). This study is an extension of the previous work and pertains to the evaluation of the role of the striatal adenosine in EIMI in male Sprague-Dawley rats. Using the motor incoordination induced by 1.5 g/kg of ethanol (ip) as a test response, the possible behavioral interactions between ethanol and adenosine agonists and antagonists in the striatum were investigated. Intrastriatal (IST) administration of adenosine A1-, A1 = A2-, and As-selective agonists, R(-)N6-(2-phenylisopropyl)adenosine (R-PIA), 5'-N-ethylcarboxamido-adenosine (NECA), and 5'-(N-cyclopropyl)-carboxamidoadenosine, respectively, significantly and dose-dependently accentuated EIMI when evaluated by rotorod test, suggesting the striatal adenosinergic modulation of EIMI. No significant change in normal motor coordination was noted, even when the highest IST doses of adenosine agonists were followed by saline instead of ethanol, suggesting that the observed behavioral interactions of these drugs were selective to ethanol. Hippocampus, which is known not to be involved in the normal motor functions, was selected as a control brain area because of the presence of high density of adenosine receptors, as well as the high levels of adenosine. Intrahippocampal NECA failed to alter EIMI, indicating the specific role of striatal and not hippocampal adenosinergic system in the modulation of EIMI. The potentiating effects of adenosine agonists N6-cyclohexyladenosine (CHA) and CGS-21680 on EIMI were blocked by adenosine A1- and A2-selective antagonists, 8-cyclopentyl-1,3-dipropylxanthine and 3,7-dimethyl-1-propargylxanthine, respectively, suggesting the participation of specific adenosine receptors in this functional interaction. A role for the adenosine A1 receptor in the striatal adenosinergic modulation of EIMI was favored based on the rank-order potency of adenosine agonists. IST pretreatment with pertussis toxin (PT), but not with PT beta-oligomer, nearly completely eliminated the accentuation of EIMI by CHA, further supporting the favored role of adenosine A1 receptors in EIMI. Histological and IST [3H]R-PIA distribution data confirmed that the observed behavioral effects were caused by exclusive striatal distribution of intrastriatally microinjected drugs. Data obtained suggested modulation of acute EIMI by striatal adenosine receptor-mediated mechanism(s) and the coupling of these adenosine receptor to the PT-sensitive Gi protein.

Possible central adenosinergic modulation of ethanol-induced alterations in [14C]glucose utilization in mice.

The possible role of brain adenosine in acute ethanol-induced alteration in glucose utilization in the whole brain, as well as in the specific brain areas (cerebellum and brain stem), was investigated. Mice were killed 20-min postethanol, and the fresh tissue slices (300 microns) of brain and/or specific brain areas were incubated for 100 min in a 5.5 mM glucose medium in Warburg flasks using [6-(14)C]glucose as a tracer. Trapped 14CO2 was counted to estimate glucose utilization. Ethanol (2 g/kg, i.p.) markedly increased the glucose utilization in whole brain and in both motor areas of brain. Theophylline (50 mg/kg, i.p.), an adenosine antagonist, significantly reduced ethanol-induced increase in glucose utilization in whole brain, as well as in brain areas. However, adenosine agonist N6-cyclohexyladenosine (CHA; 0.1 mg/kg, i.p.) on the contrary, significantly accentuated ethanol-induced increase in glucose utilization in these tissues that was nearly completely blocked by theophylline pretreatment. Theophylline alone did not produce any significant change in glucose utilization, whereas CHA alone (in vivo and in vitro) significantly increased glucose utilization, as well as ethanol-induced increase in glucose utilization in an additive manner. Relevant supportive data were obtained by experiments in which adenosine deaminase (ADA), p-sulfophenyltheophylline (8-SPT), and CHA were administered in vitro to the slice preparations. Both ADA and 8-SPT were effective in almost completely blocking the ethanol-induced increase in glucose utilization, whereas CHA further enhanced the ethanol-induced increase in glucose utilization in an additive manner.(ABSTRACT TRUNCATED AT 250 WORDS)