Is GABA involved in regulating plant growth and development? /

Rapid and large accumulation of GABA (y-aminobutyric acid) in response to a number of plant stresses has been well documented. But the role(s) of GABA in plants is not well defined. In recent years, the possibility of GABA involvement in regulating plant growth and development has been raised. In the present study, this possibility was examined. First, to rapidly and accurately determine GABA levels in plant tissues, a spectrometric method for GABA determination was developed based on a commercially available enzyme Gabase. Seventy mM LaCb almost completely removed water-soluble pigments from plant tissues which greatly interfere with the absorbance reading at 340nm. Inactivation of GAD (glutamate decarboxylase) by immediately adding methanol to a frozen plant tissue powder was suggested to prevent GABA production during extraction. The recovery of GABA with this method was approximately 100%. Second, the relationship between GABA levels and hypocotyl elongation in soybean seedlings was analyzed using different approaches to regulate in vivo GABA levels and the elongation of hypocotyls. The following major observations were made. (1) Mechanical stimulation by stroking elevated GABA levels and concurrently induced a rapid and significant reduction in hypocotyl elongation. (2) External GABA was demonstrated to penetrate into the hypocotyls using '*C-GABA. Application of external GABA elevated in vivo GABA levels, but failed to inhibit hypocotyl elongation. (3) LaCla and blue light irradiation caused an inhibition in the elongation of dark-grown hypocotyls, whereas GABA levels were not significantly affected. (4) Ca^was suggested to be involved in the signal transduction pathway leading from mechanical stimulation to GABA production, as indicated by the ability of La'* to inhibit GABA production in stimulated hypocotyls. (5) Bicuculline, saclofen and baclofen (agonists and antagonists of GABA receptors in animals) had no effect on hypocotyl elongation. It might indicate that GABA-binding components which are structurally similar to animal GABA receptors and functionally capable of regulating plant growth may not exist in plants. Therefore, the conclusion was drawn that GABA alone is not sufficient to inhibit hypocotyl elongation. Third, chloride influx in isolated Asparagus cells was enhanced by lOmM GABA during a 3 hour incubation, but the effect was not specific for GABA. Chloride efflux was not influenced by GABA. Both influx and efflux of chloride were significantly inhibited by NPPB, a chloride channel blocker. These results suggest that GABA does not influence the activity of plant chloride channels.

The role of the GABAergic system in the production of ultrasonic vocalization in rats /

Ultrasonic vocalization plays an important role in intraspecies communication for rats. It has been well demonstrated that rats will emit 22kHz vocalization in stressfiil or threatening situations. Although the neural mechanism underlying vocahzation is not well understood, it is known that chohnergic input to the basal forebrain induces such alarm calls. A number of experiments have found that intracerebral injection of carbachol, a predominantly muscarinic agonist, into die anterior hypothalamic/preoptic area (AH/POA) rehably induces vocalization similar to naturally emitted ultrasonic calls. It has also been shown that carbachol has extensive inhibitory effects on neuronal firing in the same area. This result impUes that the inhibitory effects of carbachol in the AH/POA could trigger vocahzation, and that the GABAergic system could be involved. The purpose of this study is to investigate the effects ofGABA agonists and antagonists on flie production of carbachol induced 22kHz vocalization. The following hypotheses were examined: 1) apphcation ofGABA (a naturally occurring inhibitory neurotransmitter) will have a synergistic effect with carbachol, increasing vocalization; and 2) tiie apphcation ofGABA antagonists (picrotoxin or bicuculline) will reduce caibachol-induced vocalization. A total of sixty rats were implanted with stainless steel guide cannulae in the AH/POA area. After recovery, animals were locally pretreated with 1) GABA (l-40ng), 2) picrotoxin (1 .5^g) or bicuculhne (0.03ng), or 3) sahne; before injection with carbachol (1 .5^g). The resulting vocalization was measured and quantitated. The results indicate that pretreatment with GABA or GABA antagonists had no significant effect on vocalization. Local pretreatment with GABA did not potentiate the vocal response as measured by its duration, latraicy, and total number of calls. Similarly, pretreatment with picrotoxin or bicuculline had no effects on the same measures of vocalization. The results suggest tfiat chohnoceptive neurons involved in the production of alarm calls are not under direct GABAergic control.

Identification of novel retinoid receptors and their roles in vertebrate and invertebrate nervous systems

In vertebrates, signaling by retinoic acid (RA) is known to play an important role in embryonic development, as well as organ homeostasis in the adult. In organisms such as adult axolotls and newts, RA is also important for regeneration of the CNS, limb, tail, and many other organ systems. RA mediates many of its effects in development and regeneration through nuclear receptors, known as retinoic acid receptors (RARs) and retinoid X receptors (RXRs). This study provides evidence for an important role of the RA receptor, RAR~2, in ,( '. regeneration ofthe spinal cord and tail of the adult newt. It has previously been proposed that the ability of the nervous system to regenerate might depend on the presence or absence of this RAR~2 isoform. Here, I show for the very first time, that the regenerating spinal cord of the adult newt expresses this ~2 receptor isoform, and inhibition of retinoid signaling through this specific receptor with a selective antagonist inhibits tail and spinal cord regeneration. This provides the first evidence for a role of this receptor in this process. Another species capable of CNS ~~generation in the adult is the invertebrate, " Lymnaea stagnalis. Although RA has been detected in a small number of invertebrates (including Lymnaea), the existence and functional roles of the retinoid receptors in most invertebrate non-chordates, have not been previously studied. It has been widely believed, however, that invertebrate non-chordates only possess the RXR class of retinoid receptors, but not the RARs. In this study, a full-length RXR cDNA has been cloned, which was the first retinoid receptor to be discovered in Lymnaea. I then went on to clone the very first full-length RAR eDNA from any non-chordate, invertebrate species. The functional role of these receptors was examined, and it was shown that normal molluscan development was altered, to varying degrees, by the presence of various RXR and RAR agonists or antagonists. The resulting disruptions in embryogenesis ranged from eye and shell defects, to complete lysis of the early embryo. These studies strongly suggest an important role for both the RXR and RAR in non-chordate development. The molluscan RXR and RAR were also shown to be expressed in the adult, nonregenerating eNS, as well as in individual motor neurons regenerating in culture. More specifically, their expression displayed a non-nuclear distfibution, suggesting a possible non-genomic role for these 'nuclear' receptors. It was shown that immunoreactivity for the RXR was present in almost all regenerating growth cones, and (together with N. Farrar) it was shown that this RXR played a novel, non-genomic role in mediating growth cone turning toward retinoic acid. Immunoreactivity for the novel invertebrate RAR was also found in the regenerating growth cones, but future work will be required to determine its functional role in nerve cell regeneration. Taken together, these data provide evidence for the importance of these novel '. retinoid receptors in development and regeneration, particularly in the adult nervous system, and the conservation of their effects in mediating RA signaling from invertebrates to vertebrates.

The role of skeletal muscle PLIN proteins at rest and following lipolytic stimulation

This thesis investigated the subcellular location of skeletal muscle PLIN proteins (PLIN2, PLIN3, and PLIN5) as well as protein interactions with ATGL and HSL at rest and following lipolytic stimulation. In addition, the serine phosphorylation state of PLIN2, PLIN3, and PLIN5 was determined at rest and following lipolytic stimulation. An isolated whole muscle technique was used to study the effects of contraction and epinephrine-induced lipolysis. This method allowed for the examination of the effects of contraction and epinephrine alone and in combination. Further, the soleus was chosen for investigating the role of PLIN proteins in skeletal muscle lipolysis due to its suitability for isolated incubation, and the fact that it is primarily oxidative in nature (~80% type I fibres). It has also been previously shown to have the greatest reliance on lipid metabolism and for this reason is ideal for investigating the role of PLIN proteins in lipolysis. Immunofluorescence microscopy revealed that skeletal muscle lipid droplets are partially co-localized to both PLIN2 and PLIN5 and that contraction does not affect the amount of colocalization, indicating that PLIN5 is not recruited to lipid droplets with contraction (PLIN2 ~65%; PLIN5 ~56%). Results from the immunoprecipitation studies revealed that with lipolysis in skeletal muscle the interaction between ATGL and CGI-58 is increased (study 2: 128% with contraction, p<0.05; study 3: 50% with contraction, 25% epinephrine, 80% contraction + epinephrine, p>0.05). Further PLIN2, PLIN3, and PLIN5 all interact with ATGL and HSL, while only PLIN3 and PLIN5 interact with CGI-58. Among these interactions, the association between PLIN2 and ATGL decreases with lipolytic stimulation (study 2: 21% with contraction, p<0.05). Finally our results demonstrate that PLIN3 and PLIN5 are serine phosphorylated at rest and that the level of phosphorylation remains unchanged in the face of either contractile or adrenergic stimulation. In summary, the regulation of skeletal muscle lipolysis is a complex process involving multiple proteins and enzymes. The skeletal muscle PLIN proteins likely play a role in skeletal muscle lipid droplet dynamics, and the data from this thesis indicate that these proteins may work together in regulating lipolysis by interaction with both ATGL and HSL.

Predicting the Pose of β-Casomorphin-5 and 7 in the Opioid Receptors

The opioid receptors consist of three main subtypes; μ, δ, and κ. Previous binding studies have shown that fragments of the milk protein, β-casein, known as β-casomorphins are agonists of these receptors which are selective for the μ receptor subtype. Using the crystal structures of these three receptors, computational molecular docking studies were done using the software GOLD to determine the conformation of β-casomorphin-5 and 7 when they bind to these three opioid receptors. GOLD was able to discriminate among the three receptors when docking the rigid ligands co-crystalized with the receptors. However, GOLD could not discriminate among the three receptors for either of the highly flexible β-casomorphins. A per amino acid scoring method was developed to overcome this problem. This method was used to predict the conformation of both β-casomorphin-5 and 7 in the μ receptor and determine that the two amino acid residues, Lys303 and Trp318 of the μ receptor are responsible for discriminating among the three receptor subtypes for binding of the β-casomorphin-5 and 7.

Pharmacological analysis of 50 kHz vocalizations in the male rat

The production of 50 kHz ultrasonic vocalizations in rats has been associated with both positive social interactions and appetitive behavioural situations. Furthermore, there is significant evidence showing that these vocalizations are controlled by the meso-limbic dopamine system. The purpose of this study was to perform a pharmacological analysis of 50 kHz calls by using dopamine and two dopamine agonists amphetamine and apomorphine, to induce calls. The acoustic parameters of the different call types were compared across each agonist. All three agonists were able to significantly induce more 50 kHz vocalizations compared to the vehicle control. Furthermore, calls elicited by apomorphine had a significantly higher bandwidth compared to those elicited by dopamine and amphetamine. All three agonists also had significantly different pharmacokinetic properties. These observations suggest that the D2 receptor sub-type is involved in the length of call bandwidths.