Comprehensive analysis of physiological and metabolomic responses to drought reveals specific modulation of acquired tolerance mechanisms in rice.

Mild stresses induce "acquired tolerance traits" (ATTs) that provide tolerance when stress becomes severe. Here, we identified the genetic variability in ATTs among a panel of rice germplasm accessions and demonstrated their relevance in protecting growth and productivity under water-limited conditions. Diverse approaches, including physiological screens, association mapping and metabolomics, were adopted and revealed 43 significant marker-trait associations. Nontargeted metabolomic profiling of contrasting genotypes revealed 26 "tolerance-related-induced" primary and secondary metabolites in the tolerant genotypes (AC-39000 and AC-39020) compared to the susceptible one (BPT-5204) under water-limited condition. Metabolites that help maintain cellular functions, especially Calvin cycle processes, significantly accumulated more in tolerant genotypes, which resulted in superior photosynthetic capacity and hence water use efficiency. Upregulation of the glutathione cycle intermediates explains the ROS homeostasis among the tolerant genotypes, maintaining spikelet fertility, and grain yield under stress. Bioinformatic dissection of a major effect quantitative trait locus on chromosome 8 revealed genes controlling metabolic pathways leading to the production of osmolites and antioxidants, such as GABA and raffinose. The study also led to the identification of specific trait donor genotypes that can be effectively used in translational crop improvement activities.

Stereotaxic assembly and procedures for simultaneous electrophysiological and MRI study of conscious rat.

A stereotaxic restraining assembly was designed and developed for simultaneous electrophysiological recordings and functional MRI (fMRI) data acquisition from a conscious rat. The design of the nonmagnetic stereotaxic apparatus facilitated the restraining of head and body of the unanesthetized conscious animal during MRI experiments. The apparatus was made of Teflon and Perspex materials with an appropriate size and shape for a 4.7 T / 40 cm animal MRI scanner. Electrodes made from nonmagnetic silver wire were implanted on the skull for recording the electroencephalogram (EEG), the electro-oculogram (EOG), and the electromyogram (EMG), while polycarbonate screws were used for anchoring the electrode assembly. There were no major distortions or artifacts observed in the electrophysiological tracings and MR images. Electrophysiological recordings during fMRI acquisitions are useful to study different neurophysiological mechanisms of sleep and pathophysiology of seizure activity. Integration of electrophysiological recordings (with their good temporal resolution) and MRI (with its superior spatial resolution) is helpful in characterizing the functional state of different brain regions.

Changes in sleep-wakefulness after 6-hydroxydopamine lesion of the preoptic area.

This study was undertaken to assess the role of catecholamine fibers, terminating in the preoptic area, in regulating sleep-wakefulness in rats. Sleep-wakefulness was assessed on the basis of 24h electroencephalogram, electromyogram and electro-oculogram recordings before and after destruction of catecholaminergic terminals at the medial preoptic area by bilateral intracerebral injection of 6-hydroxydopamine (8 microg in 0.2 microl). There was a mild reduction in sleep and increase in wakefulness after the lesion. The increase in active wakefulness observed after eight days of lesion persisted even on the 12th day. In spite of the reduction in sleep, the day-night sleep ratio was not affected by 6-hydroxydopamine lesion of the preoptic area. The results indicate that the noradrenergic fibers at the preoptic area have a hypnogenic role.

Magnetic resonance imaging of NMDA-induced lesion of the medial preoptic area and changes in sleep, temperature and sex behaviour.

Destruction of the medial preoptic area (mPOA) neurons of rat brain, induced by intracerebral injection of N-methyl D-aspartic acid (NMDA), has been studied by employing the non-invasive Magnetic Resonance Imaging (MRI) technique. Changes in the MRI images are compared and correlated with the functional changes after the mPOA lesion. The progress of the lesion at the injected site has been monitored (using MRI) from 15 min to 1 month after the stereotaxic microinfusion of NMDA (5 micrograms in 0.2 microliter). This study shows that the localised hyperintense (bright) area starts appearing at the mPOA from 3 h after NMDA injection, and the brightness increases progressively for about 2 days. The size and brightness of hyperintense area decrease thereafter. It has not been possible to locate the lesion site after 3 days, using MRI, except in one rat where a vacuole-like area was seen at the NMDA injected site on postmortem histological examination. The reduction in sleep after the mPOA lesion does not show any correlation with the changes in MRI, as it persists throughout the 3 weeks of recording. On the other hand, the initial drastic reduction in male sex behaviour and the increase in body temperature correlated to some extent with the increased brightness in MRI at the site of lesion. The size and location of the hyperintense area, observed during the first 2 days, match with the lesioned area which was histologically identified after 1 month of NMDA administration. Control administration of normal saline into the mPOA did not produce any alteration in the brightness of the MRI image and practically no loss of neurons at the injected site. Though some functional changes have correlation with the alteration in MRI, this cannot be used to interpret the changes in all the physiological parameters. This study also demonstrates that the disappearance of the brightness in MRI should not be taken to indicate a positive prognosis. Though the lesion could not be seen in MRI within 2 hours, its detection after 3-4 h (but within 3 days) after NMDA lesion would give very valuable information for long term studies.

Inhibition of male sexual behavior by serotonin application in the medial preoptic area.

The study investigated the possible involvement of serotonin in the medial preoptic area in the regulation of sexual behavior of male rats. Injection of serotonin in the medial preoptic area resulted in an inhibition, whereas cyproheptadine (a serotonin antagonist) produced a slight facilitation, of male sexual behavior.

Influence of rostral and caudal brain stem reticular formation on thalamic neurons.

Single neuronal activity was recorded from the diffuse thalamic system. Influence of the rostral desynchronizing and caudal synchronizing structures of the brain stem reticular formation on these neurons was studied. Rostral stimulation produced an increase and caudal stimulation a decrease in the thalamic unit firing. A possible mechanism by which the brain stem reticular structures influence the cortical neurons is proposed on the basis of these findings.

Alpha adrenergic system in medial preoptic area involved in sleep-wakefulness in rats.

The study is aimed at investigating the possible involvement of adrenergic mechanisms in the medial preoptic area (mPOA) for modulation of sleep-wakefulness in rats. In this study, saline, norepinephrine (NE), phenoxybenzamine (PBZ) and propranolol (PROP) were injected in the mPOA in different groups of male rats during the day and night. NE and PBZ were injected, during the day and the night respectively, in some control areas adjoining the mPOA in two other groups of animals. Arousal was produced by NE, and sleep by PBZ when they were applied in the mPOA. All other procedures, including application of NE and PBZ in the control areas and beta blocker (PROP) in the mPOA, did not produce alterations in sleep-wakefulness. These findings provide support for a physiological role played by the alpha adrenergic system in the mPOA for arousal, and area specificity of action of this system.

Influence of preoptico-anterior and posterior hypothalamus on midline thalamic neurons.

The present study was aimed at understanding the influence of the posterior hypothalamus (PH) and the preoptico-anterior hypothalamus (PO-AH) on the neurons of the midline thalamus (MTh) in encéphale isolé cats. A majority of the influenced neurons of the MTh showed increased firing on stimulation of the PH. Although the number of neurons showing increased or decreased firing on PO-AH stimulation were nearly equal, stimulus bound increased firing in many neurons was followed by a prolonged decreased firing. It is likely that the hypothalamo-thalamic circuit constitutes a parallel pathway to the reticulo-thalamic circuit for alteration of the cortical EEG.

Tonic activity of medial preoptic norepinephrine mechanism for body temperature maintenance in sleeping and awake rats.

The study was aimed at investigating the possible involvement of tonic activity of the adrenergic receptors in the medial preoptic area (mPOA) for maintenance of body temperature in rats. Differences in drug-induced changes in body temperatures during sleep and wakefulness--during the day and at night--were also investigated. Norepinephrine injected in the mPOA, produced a fall in body temperature, whereas phenoxybenzamine produced an increase. Saline and propranolol produced no alteration. Norepinephrine and phenoxybenzamine produced alterations in sleep-wakefulness also. These changes in sleep-wakefulness did not completely explain the thermal changes. The findings highlight the need for taking into account the basal activity and the time of application of drug in studies on thermo-regulation, using free moving animals.

Mapping of regions in the caudal brain stem that produce stimulus-bound synchronization in the cortical EEG.

This study was aimed at filling the lacunae in our knowledge regarding the localization of the regions in the caudal brain stem that bring about cortical EEG synchronization on electrical stimulation and characteristic features of synchronized waves elicited from those regions. Studies were conducted on 40 encéphale isolé cats. Stimulation of ventromedial regions of the caudal brain stem, with low frequency, elicited stimulus-bound synchronized waves in the cortex which were more prominent ispsilaterally. On the other hand, low-frequency stimulation of dorsal and lateral areas produced synchronized waves which were either equally prominent on both sides, or more prominent on the contralateral side. The loci in the brain stem that produce synchronization were very specific. The induced synchronized waves showed amplitude modulation and did not outlast the train of stimuli. The results are further confirmation of the role of caudal brain stem structures in cortical EEG synchronization. They also provide information regarding the nature of cortical synchronization elicited from these brain stem structures.

Alterations in preoptic unit activity on stimulation of caudal brain stem EEG-synchronizing structures.

Effects of stimulation of EEG-synchronizing structures of the caudal brain stem reticular formation with low (6 Hz) and high (100 Hz) frequencies were studied on 42 neurons of the preoptic area, in encéphale isolé cats. Though low-frequency stimulation produced excitation and inhibition, the majority of the influenced neurons of the preoptic area had effects of the former type. Cortical EEG synchronization was also induced by low-frequency stimulation of the caudal brain stem. High-frequency stimulation, on the other hand, produced inhibition in a majority of the influenced neurons and induced, mostly, desynchronization of the cortical EEG. A majority of the neurons that were inhibited on high-frequency stimulation, remained unaffected during low-frequency stimulation. The influence induced on the preoptic area neurons by low-frequency stimulation could be obtained even in the absence of cortical EEG synchronization. Changes induced on preoptic neurons by high-frequency stimulation may be partially related to induced cortical EEG desynchronization.

Sleep-awake responses elicited from medial preoptic area on application of norepinephrine and phenoxybenzamine in free moving rats.

The study makes an attempt to find out the action of norepinephrine (NE) and phenoxybenzamine (PB), applied at medial preoptic area (mPOA), in altering the sleep-awake responses, in free moving rats, which has not yet been reported. Effects of application of NE during day and PB during night, at mPOA, on sleep-awake responses, were compared with application of saline during identical periods. Application of NE produced arousal, whereas, PB produced the opposite effect. The findings are suggestive of the involvement of NE terminals at mPOA in normal arousal.

Influence of ascending reticular activating system on preoptic neuronal activity.

There is evidence to suggest that the reticular activating system may have an influence on the neurons of the preoptic area (POA). We studied the responses of POA neurons and their relation to the cortical EEG, on stimulation of the midbrain reticular formation (MRF) at various frequencies, in unanesthetized, encéphale isolé cats. Stimulation of the MRF at high frequency produced inhibition of the firing rate in a majority of the responsive neurons of the POA. Stimulation of the MRF with lower frequencies, on the other hand, induced excitation in a majority of the responsive POA neurons. The possibility exists of a summation of stimuli and an involvement of structures bringing about changes in the EEG in causing the shift in the POA neuronal excitability.

Responses of preoptic neurons to stimulation of caudal and rostral brain stem reticular structures.

Effect of stimulation (1 Hz) of rostral and caudal brain stem reticular formation was studied on 41 neurons of preoptic area in encéphale isolé cats. Primary excitation was seen on almost all the 25 neurons influenced by stimulation of either of the areas. Many of these influenced neurons received inputs from both areas and showed poststimulatory oscillations in excitability. The two brain stem reticular structures, which have antagonistic influence on cortical EEG, cortical and subcortical neuronal activity, had identical influence on preoptic area neurons when stimulated at 1 Hz.

Mesencephalic and bulbar reticular influences on somatosensory cortical neurons: short- and long-latency effects.

Somatosensory cortical neurons were intracellularly and extracellularly recorded in cats encéphale isolé, and after acute lesions of midthalamic nuclei or after chronic hemisection of the brain stem at the pretrigeminal level. Intracellular recordings showed postsynaptic facilitatory and inhibitory effects at very low latency by stimulating both the mesencephalic (MRF) and bulbar reticular formation (BRF). Inhibitory effects dominated by stimulating the BRF. Neither midthalamic lesions nor pretrigeminal hemisection changed the quality of latency of postsynaptic responses. Extracellular recordings revealed long-latency inhibition of discharge following MRF stimulation after midthalamic lesion. In these experimental conditions long-latency BRF effects were abolished. No differences were found in responses of pyramidal tract (PT) or non-PT neurons during BRF and MRF stimulation. The results are discussed on the basis of a possible extrathalamic differential reticular control, from caudal and rostral brain stem, of somatosensory cortical neurons.