Interactive effects of morphine and nicotine on memory function depend on the central amygdala cannabinoid CB1 receptor function in rats.

The present study investigated the possible involvement of the central amygdala (CeA) cannabinoid receptors type-1 (CB1Rs) in the interactive effects of morphine and nicotine on memory formation in a passive avoidance learning task. Our results showed that systemic administration of morphine (3 and 6mg/kg, s.c.) immediately after training phase impaired memory consolidation and induced amnesia. Administration of nicotine (0.3 and 0.6mg/kg, s.c.) before testing phase significantly restored morphine-induced amnesia, suggesting a cross state-dependent learning between morphine and nicotine. The results showed that while the administration of the lower dose of nicotine (0.1mg/kg, s.c.) per se did not induce a significant effect on morphine-induced amnesia, intra-CeA injection of arachidonylcyclopropylamide (ACPA), a cannabinoid CB1 receptor agonist (3 and 4ng/rat), significantly potentiated the nicotine response. Furthermore, the blockade of the CeA cannabinoid CB1 receptors by the injection of AM251 (0.75 and 1ng/rat) reversed the potentiative effect of nicotine (0.6mg/kg, s.c.) on morphine-induced amnesia. It should be considered that bilateral injection of the same doses of ACPA or AM251 (0.5-1ng/rat) into the CeA by itself had no effect on morphine response in a passive avoidance learning task. Confirmed by the cubic interpolation planes, the dose-response data revealed a cross-state-dependent learning between morphine and nicotine which may be mediated by the CeA endocannabinoid system via CB1 receptors.

Contribution of the basolateral amygdala NMDA and muscarinic receptors in rat's memory retrieval.

The present study was designed to investigate the involvement of the muscarinic cholinergic receptors in the basolateral amygdala (BLA) in memory retrieval. Also, the possible relationship between the BLA muscarinic cholinergic and the NMDA receptor systems was evaluated in the inhibitory avoidance learning. Male Wistar rats were bilaterally cannulated into the BLAs and memory retrieval was measured in a step-through type inhibitory avoidance apparatus. Intra-BLA microinjection of different doses of a non-selective muscarinic receptor antagonist, scopolamine (0.5-1µg/rat, intra-BLA), 5min before the testing phase dose-dependently induced amnesia. Pre-test intra-BLA microinjection of different doses of NMDA (0.005-0.05µg/rat) reversed scopolamine-induced amnesia and improved memory retrieval. In addition, different doses of a selective NMDA receptor antagonist, D-AP5 (0.001-0.005µg/rat, intra-BLA) potentiated the response of an ineffective dose of scopolamine (0.5µg/rat) to inhibit memory retrieval. It should be considered that pre-test intra-BLA microinjection of the same doses of NMDA or D-AP5 by themselves had no effect on memory retrieval. Similar to ANOVA analysis, our cubic interpolation analysis also predicted that the activation or inactivation of the NMDA receptors by different doses of drugs can affect the scopolamine response. On the other hand, pre-test intra-BLA microinjection of D-AP5 inhibited the reversal effect of NMDA on scopolamine-induced amnesia. It can be concluded that the BLA cholinergic system, via muscarinic receptors, has a critical role in memory retrieval. Our results also suggest that a cooperative interaction between the BLA NMDA and muscarinic acetylcholine receptors modulates memory formation of inhibitory avoidance task in rats.

Arabidopsis thaliana root elongation growth is sensitive to lunisolar tidal acceleration and may also be weakly correlated with geomagnetic variations.

BACKGROUND: Correlative evidence suggests a relationship between the lunisolar tidal acceleration and the elongation rate of arabidopsis roots grown under free-running conditions of constant low light. METHODS: Seedlings of Arabidopsis thaliana were grown in a controlled-climate chamber maintained at a constant temperature and subjected to continuous low-level illumination from fluorescent tubes, conditions that approximate to a 'free-running' state in which most of the abiotic factors that entrain root growth rates are excluded. Elongation of evenly spaced, vertical primary roots was recorded continuously over periods of up to 14 d using high temporal- and spatial-resolution video imaging and were analysed in conjunction with geophysical variables. KEY RESULTS AND CONCLUSIONS: The results confirm the lunisolar tidal/root elongation relationship. Also presented are relationships between the hourly elongation rates and the contemporaneous variations in geomagnetic activity, as evaluated from the disturbance storm time and ap indices. On the basis of time series of root elongation rates that extend over ≥4 d and recorded at different seasons of the year, a provisional conclusion is that root elongation responds to variation in the lunisolar force and also appears to adjust in accordance with variations in the geomagnetic field. Thus, both lunisolar tidal acceleration and the geomagnetic field should be considered as modulators of root growth rate, alongside other, stronger and more well-known abiotic environmental regulators, and perhaps unexplored factors such as air ions. Major changes in atmospheric pressure are not considered to be a factor contributing to oscillations of root elongation rate.

High-throughput phenotyping of root growth dynamics.

Plant organ phenotyping by noninvasive video imaging techniques provides a powerful tool to assess physiological traits, circadian and diurnal rhythms, and biomass production. In particular, growth of individual plant organs is known to exhibit a high plasticity and occurs as a result of the interaction between various endogenous and environmental processes. Thus, any investigation aiming to unravel mechanisms that determine plant or organ growth has to accurately control and document the environmental growth conditions. Here we describe challenges in establishing a recently developed plant root monitoring platform (PlaRoM) specially suited for noninvasive high-throughput plant growth analysis with highest emphasis on the detailed documentation of capture time, as well as light and temperature conditions. Furthermore, we discuss the experimental procedure for measuring root elongation kinetics and key points that must be considered in such measurements. PlaRoM consists of a robotized imaging platform enclosed in a custom designed phytochamber and a root extension profiling software application. This platform has been developed for multi-parallel recordings of root growth phenotypes of up to 50 individual seedlings over several days, with high spatial and temporal resolution. Two Petri dishes are mounted on a vertical sample stage in a custom designed phytochamber that provides exact temperature control. A computer-controlled positioning unit moves these Petri dishes in small increments and enables continuous screening of the surface under a binocular microscope. Detection of the root tip is achieved by applying thresholds on image pixel data and verifying the neighbourhood for each dark pixel. The growth parameters are visualized as position over time or growth rate over time graphs and averaged over consecutive days, light-dark periods and 24 h day periods. This setup enables the investigation of root extension profiles of different genotypes in various growth conditions (e.g., light protocol, temperature, growth media) and is especially suited for the detection of diurnal or circadian growth rhythms.

Arabidopsis thaliana root growth kinetics and lunisolar tidal acceleration.

• All living organisms on Earth are continually exposed to diurnal variations in the gravitational tidal force due to the Sun and Moon. • Elongation of primary roots of Arabidopsis thaliana seedlings maintained at a constant temperature was monitored for periods of up to 14 d using high temporal- and spatial-resolution video imaging. The time-course of the half-hourly elongation rates exhibited an oscillation which was maintained when the roots were placed in the free-running condition of continuous illumination. • Correlation between the root growth kinetics collected from seedlings initially raised under several light protocols but whose roots were subsequently in the free-running condition and the lunisolar tidal profiles enabled us to identify that the latter is the probable exogenous determinant of the rhythmic variation in root elongation rate. Similar observations and correlations using roots of Arabidopsis starch mutants suggest a central function of starch metabolism in the response to the lunisolar tide. The periodicity of the lunisolar tidal signal and the concomitant adjustments in root growth rate indicate that an exogenous timer exists for the modulation of root growth and development. • We propose that, in addition to the sensitivity to Earthly 1G gravity, which is inherent to all animals and plants, there is another type of responsiveness which is attuned to the natural diurnal variations of the lunisolar tidal force.

Mutations in leaf starch metabolism modulate the diurnal root growth profiles of Arabidopsis thaliana.

Roots of Arabidopsis thaliana exhibit stable diurnal growth profiles that are controlled by the circadian clock. Here we describe the effects of mutations in leaf starch metabolism on the diurnal root growth characteristics of Arabidopsis thaliana. High temporal and spatial resolution video imaging was performed to quantify the growth kinetics of Arabidopsis wild-type as well as pgm, sex1, mex1, dpe1 and dpe2 starch metabolism mutants grown in three different photoperiods. As a result, root growth patterns of all genotypes displayed characteristic modifications in their diurnal kinetics that were also affected by the photoperiod. To further investigate the role of starch derived substrate deficiency on root growth, the effect of 0.05% extracellular sucrose was studied in 12 h-12 h light-dark cycles.

Circadian control of root elongation and C partitioning in Arabidopsis thaliana.

Plants grow in a light/dark cycle. We have investigated how growth is buffered against the resulting changes in the carbon supply. Growth of primary roots of Arabidopsis seedlings was monitored using time-resolved video imaging. The average daily rate of growth is increased in longer light periods or by addition of sugars. It responds slowly over days when the conditions are changed. The momentary rate of growth exhibits a robust diel oscillation with a minimum 8-9 h after dawn and a maximum towards the end of the night. Analyses with starch metabolism mutants show that starch turnover is required to maintain growth at night. A carbon shortfall leads to an inhibition of growth, which is not immediately reversed when carbon becomes available again. The diel oscillation persists in continuous light and is strongly modified in clock mutants. Central clock functions that depend on CCA1/LHY are required to set an appropriate rate of starch degradation and maintain a supply of carbon to support growth through to dawn, whereas ELF3 acts to decrease growth in the light period and promote growth in the night. Thus, while the overall growth rate depends on the carbon supply, the clock orchestrates diurnal carbon allocation and growth.

Analysis of Arabidopsis thaliana root growth kinetics with high temporal and spatial resolution.

BACKGROUND: Methods exist to quantify the distribution of growth rate over the root axis. However, non-destructive, high-throughput evaluations of total root elongation in controlled environments and the field are lacking in growth studies. A new imaging approach to analyse total root elongation is described. SCOPE: High pixel resolution of the images enables the study of growth in short time intervals and provides high temporal resolution. Using the method described, total root elongation rates are calculated from the displacement of the root tip. Although the absolute root elongation rate changes in response to growth conditions, this set-up enables root growth of Arabidopsis wild-type seedlings to be followed for more than 1 month after germination. The method provides an easy approach to decipher root extension rate and much simpler calculations compared with other methods that use segmental growth to address this question. CONCLUSIONS: The high temporal resolution allows small modifications of total root elongation growth to be revealed. Furthermore, with the options to investigate growth of various mutants in diverse growth conditions the present tool allows modulations in root growth kinetics due to different biotic and abiotic stimuli to be unravelled. Measurements performed on Arabidopsis thaliana wild-type (Col0) plants revealed rhythms superimposed on root elongation. Results obtained from the starchless mutant pgm, however, present a clearly modified pattern. As expected, deviation is strongest during the dark period.

High throughput phenotyping of root growth dynamics, lateral root formation, root architecture and root hair development enabled by PlaRoM.

Plant organ phenotyping by non-invasive video imaging techniques provides a powerful tool to assess physiological traits and biomass production. We describe here a range of applications of a recently developed plant root monitoring platform (PlaRoM). PlaRoM consists of an imaging platform and a root extension profiling software application. This platform has been developed for multi parallel recordings of root growth phenotypes of up to 50 individual seedlings over several days, with high spatial and temporal resolution. PlaRoM can investigate root extension profiles of different genotypes in various growth conditions (e.g. light protocol, temperature, growth media). In particular, we present primary root growth kinetics that was collected over several days. Furthermore, addition of 0.01% sucrose to the growth medium provided sufficient carbohydrates to maintain reduced growth rates in extended nights. Further analysis of records obtained from the imaging platform revealed that lateral root development exhibits similar growth kinetics to the primary root, but that root hairs develop in a faster rate. The compatibility of PlaRoM with currently accessible software packages for studying root architecture will be discussed. We are aiming for a global application of our collected root images to analytical tools provided in remote locations.