No interaction between methyl jasmonate and ozone in Pima cotton: growth and allocation respond independently to both.

Ozone (O3) is damaging to plants, inducing signalling pathways involving antagonism between jasmonates and ethylene. These pathways mediate O3 responses, particularly to acute exposure, and their manipulation protected several species against acute and chronic O3. We use chronic daily exposure of up to 163 ppb O3, and twice weekly application of up to 320 microg plant(-1) methyl jasmonate (MeJA) to test two hypothesizes: 1) a low rate of MeJA does not affect growth but increases O3 sensitivity; 2) a high rate inhibits growth but reduces O3 sensitivity. Both hypotheses were rejected. Growth declined with increases in both MeJA and O3. MeJA at 40 microg plant(-1) caused no direct effect, and at 160 microg plant(-1) reduced growth similarly at all O3. Neither rate altered O3 sensitivity. These additive responses are not consistent with protection by MeJA in this system. They may reflect inter-specific differences in signalling, since O3 concentrations used here exceeded some reported acute exposures. Alternatively, parallel responses to O3 and MeJA may suggest that O3-induced jasmonates play a developmental role in chronic response but no protective role in the absence of lesions characteristic of acute exposure. MeJA appears useful as a probe of these mechanisms.

Resetting the biological clock: mediation of nocturnal circadian shifts by glutamate and NO.

Circadian rhythms of mammals are timed by an endogenous clock with a period of about 24 hours located in the suprachiasmatic nucleus (SCN) of the hypothalamus. Light synchronizes this clock to the external environment by daily adjustments in the phase of the circadian oscillation. The mechanism has been thought to involve the release of excitatory amino acids from retinal afferents to the SCN. Brief treatment of rat SCN in vitro with glutamate (Glu), N-methyl-D-aspartate (NMDA), or nitric oxide (NO) generators produced lightlike phase shifts of circadian rhythms. The SCN exhibited calcium-dependent nitric oxide synthase (NOS) activity. Antagonists of NMDA or NOS pathways blocked Glu effects in vitro, and intracerebroventricular injection of a NOS inhibitor in vivo blocked the light-induced resetting of behavioral rhythms. Together, these data indicate that Glu release, NMDA receptor activation, NOS stimulation, and NO production link light activation of the retina to cellular changes within the SCN mediating the phase resetting of the biological clock.

A novel adenylyl cyclase-activating serotonin receptor (5-HT7) implicated in the regulation of mammalian circadian rhythms.

We report the cloning and characterization of a novel serotonin receptor, designated as 5-HT7, which is coupled to the stimulation of adenylyl cyclase. 5-HT7 mRNA is expressed discretely throughout the CNS, predominantly in the thalamus and hypothalamus. 5-HT7 has a unique pharmacological profile that redefines agonist and antagonist classification of ligands previously thought to be "selective." The circadian phase of spontaneous neuronal activity of the rat suprachiasmatic nucleus of the hypothalamus advances in response to serotonin ligands with a pharmacological profile consistent exclusively with that of 5-HT7. These findings suggest a physiological role in the regulation of circadian rhythms for one subtype of serotonin receptor, 5-HT7, and provide a pharmacological test to evaluate its role in other neuronal systems.

5-HT1B receptor-mediated presynaptic inhibition of retinal input to the suprachiasmatic nucleus.

The suprachiasmatic nucleus (SCN) receives glutamatergic afferents from the retina and serotonergic afferents from the midbrain, and serotonin (5-HT) can modify the response of the SCN circadian oscillator to light. 5-HT1B receptor-mediated presynaptic inhibition has been proposed as one mechanism by which 5-HT modifies retinal input to the SCN (Pickard et al., 1996). This hypothesis was tested by examining the subcellular localization of 5-HT1B receptors in the mouse SCN using electron microscopic immunocytochemical analysis with 5-HT1B receptor antibodies and whole-cell patch-clamp recordings from SCN neurons in hamster hypothalamic slices. 5-HT1B receptor immunostaining was observed associated with the plasma membrane of retinal terminals in the SCN. 1-[3-(Trifluoromethyl)phenyl]-piperazine HCl (TFMPP), a 5-HT1B receptor agonist, reduced in a dose-related manner the amplitude of glutamatergic EPSCs evoked by stimulating selectively the optic nerve. Selective 5-HT1A or 5-HT7 receptor antagonists did not block this effect. Moreover, in cells demonstrating an evoked EPSC in response to optic nerve stimulation, TFMPP had no effect on the amplitude of inward currents generated by local application of glutamate. The effect of TFMPP on light-induced phase shifts was also examined using 5-HT1B receptor knock-out mice. TFMPP inhibited behavioral responses to light in wild-type mice but was ineffective in inhibiting light-induced phase shifts in 5-HT1B receptor knock-out mice. The results indicate that 5-HT can reduce retinal input to the circadian system by acting at presynaptic 5-HT1B receptors located on retinal axons in the SCN.

Twelve-hour phase shifts of hamster circadian rhythms elicited by voluntary wheel running.

Running in a novel wheel can serve as a nonphotic zeitgeber to entrain or phase shift circadian rhythms in hamsters. In this study, hamsters were entrained to a light:dark schedule of 14:10 h but had no access to running wheels. At four different phase points of the light cycle, hamsters were transferred to constant darkness and provided with running wheels. All hamsters began running shortly after transfer and were allowed to continue running at their own volition. Approximately 20% of the hamsters transferred at zeitgeber time (ZT) 23 (ZT 12 = lights out) ran more than 4 h after transfer and showed phase advances of the circadian activity rhythm by as much as 15 h, while hamsters that ran less than 4 h on average did not phase shift. A similar result was observed for hamsters transferred at ZT 2. Hamsters transferred at ZT 5 and 8 also did not phase shift if they ran less than 4 h, although the relation between longer runs and phase shifts became less evident. A sustained run in excess of 4 h appeared to be associated with large phase advances. These results show that under certain conditions, a single sustained bout of wheel-running activity is capable of phase shifting the circadian pacemaker by more than 12 h.

Local administration of serotonin agonists blocks light-induced phase advances of the circadian activity rhythm in the hamster.

Circadian rhythms in mammals are synchronized to environmental light-dark cycles through a direct retinal projection to the suprachiasmatic nucleus (SCN), a circadian clock. This process is thought to be modulated by other afferents to the SCN, including a dense serotonergic projection from the midbrain raphe. Previous work from this laboratory demonstrated that a systemically administered 5-hydroxytryptamine1A/7 (5-HT1A/7) agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) dose dependently attenuates light-induced phase shifts of the circadian activity rhythm of the Syrian hamster. In this study, we demonstrate that local injections (1-100 microM) of the 5-HT1A/7 agonists 8-OH-DPAT or 5-carboxamidotryptamine into the region of the SCN inhibit light-induced phase advances of the circadian wheel-running rhythm. In addition, the inhibitory effects of systemically administered 8-OH-DPAT were unaffected by either radiofrequency-induced lesions of the intergeniculate leaflet or 5,7-dihydroxytryptamine-induced lesions of serotonergic projections to the SCN. These findings support a modulatory role of serotonin in photic regulation of circadian phase through an action at the level of the SCN.

Tryptophan loading modulates light-induced responses in the mammalian circadian system.

Enhanced endogenous serotonergic activity, stimulated by L-tryptophan (TRYPT) loading, was found to have a substantial impact on neurochemical and behavioral aspects of the circadian response to light in the male Syrian hamster. An intraperitoneal (i.p.) injection of 150 mg/kg TRYPT significantly stimulated serotonin (5-HT) release in the suprachiasmatic nuclear (SCN) region, as reflected by a 205 +/- 30% maximal increase in the extracellular concentration of 5-HT assessed using microdialysis. Administration of TRYPT 1 h before exposure to a light pulse (30 min, 40 lux) delivered during late subjective night dose-dependently suppressed the number of SCN cells expressing light-induced Fos-like immunoreactivity (Fos-LI; maximal suppression @200 mg/kg was 77 +/- 4%, p < 0.001). This action of TRYPT was attenuated by pretreatment with the 5-HT1a antagonist, NAN-190, and was abolished by the 5-HT2/5-HT7 antagonist, ritanserin, or the nonselective 5-HT antagonist, metergoline (all 10 mg/kg). These antagonists alone had no effect on light-induced Fos. In a second experiment, pretreatment of free-running hamsters housed under constant darkness with 150 mg/kg TRYPT 45-60 min prior to light exposure (10 min, 20 lux) during late subjective night (CT 19) significantly attenuated the light-induced phase advances of the circadian activity rhythm (66 +/- 7 min vs. 100 +/- 6 min for vehicle controls; p < 0.001). The same dose of TRYPT given 1 h before lights-on for 5 consecutive days in hamsters maintained under 14L:10D altered the phase angle of entrainment such that activity onsets were delayed by 36 +/- 8 min relative to controls (p < 0.05). The same dose of TRYPT administered during late subjective night also suppressed the extracellular concentration of glutamate in the SCN region assessed using microdialysis (55 +/- 8% suppression; p < 0.05 vs. baseline). These results support the hypothesis that the ascending serotonergic projection to the SCN modulates photic entrainment processes within the circadian oscillator.

Is Fos expression necessary and sufficient to mediate light-induced phase advances of the suprachiasmatic circadian oscillator?

Photic stimulation during the subjective night induces the expression of Fos among a discrete population of cells in the suprachiasmatic nuclei (SCN) region of the Syrian hamster. Light appears to stimulate Fos expression only when administered at circadian times (CTs) at which exposure causes a phase shift. Different populations of SCN cells express Fos in response to light pulses that result in phase advances versus phase delays, raising the possibility that different cell populations in the suprachiasmatic hypothalamus participate in light-induced phase advances and delays of the circadian oscillator. Microinjection of excitatory amino acid (EAA) antagonists into the region of the SCN attenuates light-induced phase advances of the free-running activity rhythm and light-induced Fos expression in the hamster SCN. However, injection of N-methyl-D-aspartate (NMDA) at CT 18, which results in a widespread pattern of Fos expression in the hypothalamus that includes the retinorecipient zone of the SCN, does not produce phase advances of the circadian oscillator. The results demonstrate that both light-induced Fos expression and light-induced phase advances are dependent upon EAA neurotransmission within the SCN region. However, expression of Fos in the SCN induced by the EAA agonist NMDA is not sufficient to cause phase advances of the SCN oscillator.

Suprachiasmatic nucleus neurochemistry in the conscious brain: correlation with circadian activity rhythms.

The aim of the research reported here was to provide information on the neurochemical processes that underlie the generation and entrainment of mammalian circadian rhythms. The studies were centered principally around the in vivo brain microdialysis technique for assessing the daily pattern of neurotransmitter activity in the suprachiasmatic hypothalamus of freely behaving Syrian and Siberian hamsters. This approach yielded several findings related to the activities of serotonergic and excitatory amino acid systems in the region of the suprachiasmatic nuclei (SNC). Specifically, we found that (1) there were daily variations in the extracellular concentrations of 5-hydroxyindoleacetic acid (5-HIAA) and glutamate (GLU) in the SCN region (highest levels of 5-HIAA occurred soon after lights-off, whereas GLU peaked later in the dark phase); (2) the daily rhythm of GLU, but not serotonin, in the SCN region appeared to be circadian in nature and was not driven by an external influence; (3) the rhythm in GLU measured in SCN microdialysate involved a tetrodotoxin-insensitive mechanism and did not appear to be directly linked to the expression of locomotor behavior; and (4) direct application of serotonin receptor agonists via the microdialysis probe suppressed the concentration of extracellular GLU in the SCN region, suggesting that serotonin may modulate GLU release in the SCN.

A role for serotonin in the circadian system revealed by the distribution of serotonin transporter and light-induced Fos immunoreactivity in the suprachiasmatic nucleus and intergeniculate leaflet.

Components of the circadian system, the suprachiasmatic nucleus and the intergeniculate leaflet receive serotonin input from the raphe nuclei. Manipulations of serotonin neurotransmission disrupt cellular, electrophysiological, and behavioural responses of the circadian system to light, suggesting that serotonin plays a modulatory role in photic regulation of circadian rhythms. To study the relation between serotonin afferents and light-activated cells in the suprachiasmatic nucleus and intergeniculate leaflet, we used immunostaining for the serotonin transporter and for the transcription factor, Fos. Serotonin transporter, a plasma membrane protein located on serotonin neurons, regulates the amount of serotonin available for neurotransmission by re-accumulating released serotonin into presynaptic neurons; expression of Fos in the suprachiasmatic nucleus identifies light-activated cells involved in photic resetting of circadian clock phase. In the suprachiasmatic nucleus, immunostaining for serotonin transporter revealed a dense plexus of fibres concentrated primarily in the ventrolateral region. In the intergeniculate leaflet, serotonin transporter immunostaining identified vertically-oriented columns of fibres. Serotonin transporter immunostaining was abolished by pretreatment with the serotonin neurotoxin, 5,7-dihydroxytryptamine. Exposure to light for 30 min during the dark phase of the light cycle induced Fos expression in the ventrolateral suprachiasmatic nucleus and intergeniculate leaflet regions. In both structures the Fos-expressing cells were encircled by serotonin transporter-immunoreactive fibres often in close apposition to these cells. These results support the idea that serotonin activity plays a modulatory role in processing of photic information within the circadian system.

Testosterone maintains pituitary and serum FSH and spermatogenesis in gonadotrophin-releasing hormone antagonist-suppressed rats.

Groups of adult male rats were treated continuously for 30 days with either vehicle or the potent gonadotrophin-releasing hormone (GnRH) antagonist. (N-Ac-D-Nal(2)1,D-pCl-Phe2,D-Trp3,D-hArg(Et2)6,D-Ala10 )- GnRH (RS 68439; 35 micrograms/day). In addition, groups of vehicle- and antagonist-treated rats received s.c. testosterone implants sufficient to maintain serum testosterone concentrations 3.5- to 5-fold higher than those of vehicle-treated control rats. After 30 days of antagonist treatment serum LH, FSH and testosterone concentrations were at or below the detection limits of their respective assays and pituitary FSH content and GnRH receptor binding were reduced, relative to control animals, by 77 and 98% respectively. Testis weight in antagonist-treated rats was reduced by 75% and spermatogenesis was suppressed to an extent comparable to that observed in hypophysectomized rats. Testosterone, which caused a 40% reduction in serum FSH relative to control animals, prevented the antagonist-induced fall in both serum and pituitary FSH, but not GnRH receptors, below that observed in the vehicle plus testosterone-treated group. Furthermore, spermatogenesis in the antagonist plus testosterone-treated group was indistinguishable from that observed in control animals. It is concluded that testosterone is capable of maintaining serum and pituitary FSH levels in vivo, under conditions which presumably render the pituitary insensitive to hypothalamic GnRH.

In situ hybridization of antisense mRNA oligonucleotides for AMPA, NMDA and metabotropic glutamate receptor subtypes in the rat suprachiasmatic nucleus at different phases of the circadian cycle.

Eleven oligonucleotides directed against mRNA for AMPA, NMDA and metabotropic glutamate receptor subtypes were hybridized to rat coronal brain sections containing the suprachiasmatic nucleus (SCN). These oligonucleotides were hybridized to tissue samples collected at midday and midnight phases of the circadian cycle. Glutamate receptor mRNA for the AMPA subunits GluR1, GluR2 and GluR4, and the NMDA receptor subtype NMDAR1, were heavily expressed in the SCN and surrounding areas. The mRNA for the metabotropic glutamate subunit mGluR1 was only lightly expressed in the SCN. In contrast, mRNA for NMDAR2A, NMDAR2B, NMDAR2C and GluR3 was not detected in the SCN. The mRNA found to be expressed in the rat SCN was similar in samples collected at midday and midnight, suggesting no circadian variation in endogenous SCN glutamate receptors at these two times of the light-dark cycle.

Serotonergic potentiation of photic phase shifts of the circadian activity rhythm.

Recent evidence suggests that serotonin may function to regulate the sensitivity of the circadian clock to the resetting effect of environmental light. Here we report that systemic administration of NAN-190, a drug that acts at both postsynaptic and somatodendritic serotonin receptors, potentiates light-induced phase shifts by as much as 250%. The effects of the drug are dose-related and are significant at light intensities between 0.2 and 200 lux. It is proposed that drugs with pharmacological properties similar to NAN-190 may prove useful as chronobiologics to adjust the sensitivity of the circadian system to natural 'zeitgebers'.

Detection of zinc in isolated nerve terminals using a modified Timm's sulfide-silver method.

An ultrastructural method for detecting the presence of zinc in isolated nerve terminals from the mammalian brain is described. This method is based on the well-known Timm's sulfide-silver technique that has been used by many investigators to detect and localize zinc-containing pathways in sections of intact brain tissue. We report here a modification of this technique that we have used to assess the homogeneity, at the electron microscopic level, of a zinc-enriched synaptosomal fraction from the rat hippocampus. This technique allows biochemical assays to be performed on samples of the same tissue if desired, and also provides the large amounts of tissue needed for synaptosomal isolation. Results indicated that all of the mossy fiber synaptosomes, identified on the basis of their large size and characteristic morphology, stained for zinc using this method, as did about one-third of the smaller synaptic profiles present in the same fraction. The method described here should be useful for determining zinc retention and localization in isolated synaptosomes from other regions of the mammalian central nervous system.

Different populations of cells in the suprachiasmatic nuclei express c-fos in association with light-induced phase delays and advances of the free-running activity rhythm in hamsters.

Circadian rhythmicity is controlled by a light-entrainable pacemaker located in the suprachiasmatic nuclei (SCN) of the mammalian hypothalamus. Brief light exposure during the subjective night causes phase shifts of the free-running activity rhythm and expression of c-fos-related proteins (Fos) among a population of cells in the hamster SCN. Light exposure (30 lux for 15 min) during the early subjective night (CT13) causes phase delays (-60 +/- 12 min), while exposure at mid-subjective night (CT18) causes phase advances (114 +/- 48 min) of the free-running activity rhythm. Light exposure at mid-subjective day (CT6) does not cause phase alterations of the rhythm. Similarly, only light exposure at CT13 or CT18 induces Fos expression in the SCN. The distribution of Fos-immunoreactive cells in the SCN is more widespread in animals stimulated with light at CT18. In addition, a group of cells located dorsal and anterior to the SCN express Fos only after stimulation at CT18. The data are consistent with the hypothesis that Fos expression represents an event in the signal transduction pathway leading to light-induced alterations in circadian pacemaker function. Furthermore, the data raise the possibility that different populations of cells in the suprachiasmatic hypothalamus may participate in light-induced phase advances and delays of the circadian pacemaker.

Adenosine A1 receptors regulate the response of the mouse circadian clock to light.

The purine nucleoside, adenosine, has been implicated in the regulation of circadian phase in the hamster. In the current report, we present pharmacological evidence supporting the involvement of adenosine A1 receptors in the regulation of the response of the circadian clock to light in mice. Systemic injection of the selective adenosine A1 receptor agonist, N(6)-cyclohexyladenosine (CHA; 0.3 mg/kg) resulted in a 49% reduction (P<0.05) in the magnitude of light-induced phase delays. The inhibitory effect of CHA on light-induced phase delays was dose dependent over a range of 0.1 to 5 mg/kg with an apparent EC(50) of 0.3 mg/kg. Prior administration of the selective adenosine A1 receptor antagonist dipropylcyclopentylxanthine (DPCPX; 1 mg/kg) completely blocked the effect of CHA on photic phase delays. Finally, CHA significantly attenuated light-induced phospho-extracellular signal-related kinases (ERK) immunoreactivity in the mouse suprachiasmatic nucleus (SCN), consistent with a mode of action involving events that occur early in the signaling cascade through which photic information is conveyed to the circadian clock. These data indicate that the role of adenosine in the regulation of circadian phase is similar in mice and hamsters.

Glutamate receptor immunoreactivity in the rat suprachiasmatic nucleus.

Antibodies selective for the glutamate receptor subunits GluR1 and GluR2/3 were used to localize glutamate receptor immunoreactivity in the rat suprachiasmatic nuclei (SCN). These antisera identified two distinct cell populations within the SCN. Cells immunoreactive to GluR2/3 antiserum were located predominantly in the ventral SCN while antiserum selective for GluR1 stained a population located along the dorsal and lateral borders of the nucleus. There were no apparent day-night differences in GluR immunoreactivity observed in the SCN.

Regional distribution of cholinergic parameters within the rat striatum.

The topographical distribution of choline acetyltransferase, muscarinic receptor binding and high affinity choline uptake was studied in 21 separate areas of the rat striatum. The areas of the nucleus chosen represented dorsolateral, dorsomedial, ventrolateral and ventromedial regions along the rostrocaudal aspect of the striatum, such that a three-dimensional distribution of the cholinergic parameters could be obtained. Within any given rostrocaudal section, no significant dorsoventral differences were noted for any of the cholinergic parameters. On the other hand, marked differences were found in a comparison of the medial and lateral striatum. Choline acetyltransferase, muscarinic receptor binding and high affinity choline uptake were more concentrated in the lateral striatum than the medial striatum, and the magnitude of this medio-lateral disparity increased from rostral to caudal regions of the nucleus. The lateral striatum exhibited no significant rostrocaudal variations in the cholinergic parameters; however, the medial portion of the striatum did exhibit differences along its rostrocaudal extent, with the rostral-most sections being enriched relative to the more caudal sections. These results suggest that the cholinergic system in the striatum is heterogeneously distributed within this nucleus, with the lateral portion possessing a greater cholinergic innervation than the medial portion. They further suggest that future neurochemical studies of cholinergic alterations in the striatum should include a consideration of the possibility of regional effects within the nucleus rather than treating the striatum as a homogeneous tissue.

Evidence for developmental synaptic regression of cholinergic afferents to the rat main olfactory bulb.

Choline acetyltransferase (ChAT) activity, acetylcholinesterase (AChE) activity and muscarinic cholinergic receptor binding were determined in homogenates of olfactory bulbs from rats killed at intervals from 4 days before through 60 days after birth. In addition, the localization of muscarinic receptors was determined using an in vitro autoradiographic technique in 6-millimicrons thick coronal sections of olfactory bulbs from rats killed at similar intervals after birth. All 3 cholinergic parameters were present in measurable quantities at birth and showed substantial increases between 1 and 20 days after birth. The most rapid increase in cholinergic parameters occurred between days 10 and 20 after birth. ChAT activity and muscarinic receptor binding decreased between days 20 and 35 and increased again between postnatal days 35 and 60. A similar developmental pattern was observed for autoradiographic grain density overlying the granule cell layer of the neonatal bulb. These data suggest that (1) centrifugal cholinergic afferents are present in the rat olfactory bulb at birth, (2) during the early postnatal period (between 10 and 20 days) synaptogenesis occurs resulting in an overproduction of cholinergic synapses and (3) between postnatal days 20 and 35, a period of synaptic reorganization occurs characterized by substantial regression.

A 5-HT(1B) receptor agonist inhibits light-induced suppression of pineal melatonin production.

Serotonin (5-HT) modulates the phase adjusting effects of light on the mammalian circadian clock through the activation of presynaptic 5-HT(1B) receptors located on retinal terminals in the suprachiasmatic nucleus (SCN). The current study was conducted to determine whether activation of 5-HT(1B) receptors also alters photic regulation of nocturnal pineal melatonin production. Systemic administration of the 5-HT(1B) receptor agonist TFMPP attenuated the inhibitory effect of light on pineal melatonin synthesis in a dose-related manner with an apparent ED(50) value of 0.9 mg/kg. The effect of TFMPP on light-induced melatonin suppression was blocked by the 5-HT(1) receptor antagonist, methiothepin, but not by the 5-HT(1A) antagonist, WAY 100,635, consistent with the involvement of 5-HT(1B) receptors. The results are consistent with the interpretation that activation of presynaptic 5-HT(1B) receptors on retinal terminals in the SCN attenuates the effect of light on pineal melatonin production, as well as on circadian phase.