Rat Pineal Gland Research Articles

Determination of Melatonin in Rat Pineal Gland and Drug with Flow‐Injection Chemiluminescence

AbstractA flow‐injection chemiluminescence (CL) method for the determination of melatonin based on the CL reaction of melatonin with hydrogen peroxide and sodium hypochlorite (NaOCl) in a basic alkaline solution was developed. The possible CL mechanism has been discussed, and a proposal for the reaction pathway was given that singlet oxygen was clarified to be produced in this reaction system and was responsible for the CL emission. Under the optimized conditions, the linear concentration range of application was 1.0×10−7–2.5×10−4 mol·L−1 with a detection limit of 5.0×10−8 mol·L−1 (S/N=3). The relative standard deviation for eight repeated measurements of 1.0×10−6 mol·L−1 melatonin was 2.8%. The interferences of several important biological substances, some indole compound, cations and anions were studied. No interference was found for the anions, glucose, starch, most of cations and low concentration (less than 3.0×10−6 mol·L−1) of some biological substances and indole compound. The method was applied to the determination of melatonin in rat pineal gland and drug with satisfactory results. The sample throughput was 90 injections per hour.

NeuroD1: developmental expression and regulated genes in the rodent pineal gland

NeuroD1/BETA2, a member of the bHLH transcription factor family, is known to influence the fate of specific neuronal, endocrine and retinal cells. We report here that NeuroD1 mRNA is highly abundant in the developing and adult rat pineal gland. Pineal expression begins in the 17-day embryo at which time it is also detectable in other brain regions. Expression in the pineal gland increases during the embryonic period and is maintained thereafter at levels equivalent to those found in the cerebellum and retina. In contrast, NeuroD1 mRNA decreases markedly in non-cerebellar brain regions during development. Pineal NeuroD1 levels are similar during the day and night, and do not appear to be influenced by sympathetic neural input. Gene expression analysis of the pineal glands from neonatal NeuroD1 knockout mice identifies 127 transcripts that are down-regulated (>twofold, p < 0.05) and 16 that are up-regulated (>twofold, p < 0.05). According to quantitative RT-PCR, the most dramatically down-regulated gene is kinesin family member 5C ( approximately 100-fold) and the most dramatically up-regulated gene is glutamic acid decarboxylase 1 ( approximately fourfold). Other impacted transcripts encode proteins involved in differentiation, development, signal transduction and trafficking. These findings represent the first step toward elucidating the role of NeuroD1 in the rodent pinealocyte.

Mitogen‐activated protein kinase phosphatase‐1 (MKP‐1) preferentially dephosphorylates p42/44MAPK but not p38MAPK in rat pinealocytes

We recently reported a diurnal and norepinephrine (NE) -induced expression of mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1) in the rat pineal gland and postulated that this MKP-1 expression might impact adrenergic-regulated arylalkylamine-N-acetyltransferase (AA-NAT) activity via modulation of MAPKs. In this study, we investigated the effect of depletion of MKP-1 expression by using doxorubicin, a topoisomerase inhibitor that suppresses the expression of MKP-1 in other cell types and small interfering RNA targeted against Mkp1 in NE-stimulated pinealocytes. We found that both treatments were effective in inhibiting NE induction of MKP-1 expression. Moreover, both treatments also resulted in a prolonged activation of p42/44MAPK and an increase in AA-NAT induction by NE. In contrast, treatment of pinealocytes with PD98059, an inhibitor of MAPK kinase, reduced NE-stimulated AA-NAT activity. Interestingly, suppressing MKP-1 expression had no effect on the time profile of NE-stimulated p38MAPK activation. These results indicate that MKP-1 modulates the profile of AA-NAT activity by selectively shaping the activation profile of p42/44MAPK but not that of p38MAPK.

Daily Oscillation and Photoresponses of Clock Gene, Clock, and Clock‐Associated Gene, Arylalkylamine N‐acetyltransferase Gene Transcriptions in the Rat Pineal Gland

This study was conducted to investigate the circadian rhythms and light responses of Clock and arylalkylamine N‐acetyltransferase (NAT) gene expressions in the rat pineal gland under the environmental conditions of a 12 h light (05:00–17:00 h): 12 h‐dark (17:00–05:00 h) cycle (LD) and constant darkness (DD). The pineal gland of Sprague‐Dawley rats housed under a LD regime (n=42) for four weeks and of a regime (n=42) for eight weeks were sampled at six different times, every 4 h (n=7 animals per time point), during a 24 h period. Total RNA was extracted from each sample, and the semiquantitative reverse transcription polymerase chain reaction (RT‐PCR) was used to determine temporal changes in mRNA levels of Clock and NAT genes during different circadian or zeitgeber times. The data and parameters were analyzed by the cosine function software, Clock Lab software, and the amplitude F test was used to reveal the circadian rhythm. In the DD or LD condition, both the Clock and NAT mRNA levels in the pineal gland showed robust circadian oscillation (p<0.05) with the peak at the subjective night or at nighttime. In comparison with the DD regime, the amplitudes and mRNA levels at the peaks of Clock and NAT expressions in LD in the pineal gland were significantly reduced (p<0.05). In the DD or LD condition, the circadian expressions of NAT were similar in pattern to those of Clock in the pineal gland (p>0.05). These findings indicate that the transcriptions of Clock and NAT genes in the pineal gland not only show remarkably synchronous endogenous circadian rhythmic changes, but also respond to the ambient light signal in a reduced manner.

Expression analysis of VGLUT during the postnatal maturation of rat pineal gland

Expression analysis of VGLUT during the postnatal maturation of rat pineal gland

Histone H3 Phosphorylation in the Rat Pineal Gland: Adrenergic Regulation and Diurnal Variation

In this study, we investigated phosphorylation of Ser10 in histone H3 by norepinephrine (NE) in the rat pineal gland. In whole-animal studies, we demonstrated a marked increase in histone H3 phosphorylation in the rat pineal gland during the first half of the dark period. Exposure to light during this period caused a rapid decline in histone H3 phosphorylation with an estimated t1/2 of less than 15 min, indicating a high level of dephosphorylation activity. Corresponding studies in cultured pineal cells revealed that treatment with NE produced an increase in histone H3 phosphorylation that peaked between 2 and 3 h and declined rapidly by 4 h. The NE-induced histone H3 phosphorylation was blocked by cotreatment with propranolol or KT5720, a protein kinase A inhibitor, but not by prazosin or other kinase inhibitors. Moreover, only treatment with dibutyryl cAMP but not other kinase activators mimicked the effect of NE on histone H3 phosphorylation. The NE-stimulated H3 phosphorylation was markedly increased by cotreatment with a serine/threonine phosphatase inhibitor, tautomycin or okadaic acid, supporting a high level of ongoing histone H3 dephosphorylation activity. Together, our results indicate that histone H3 phosphorylation is a naturally occurring event at night in the rat pineal gland that is driven almost exclusively by a NE-->beta-adrenergic-->cAMP/protein kinase A signaling mechanism. This transient histone H3 phosphorylation probably reflects the nocturnal activation of multiple adrenergic-regulated genes in the rat pineal gland.

The role of protein turnover in regulating MKP-1 levels in rat pinealocytes

We have previously shown that mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1) is induced at night under the control of a photoneural system in the rat pineal gland. Because of the established roles of MAPKs, glucocorticoids and proteasome activity in regulating MKP-1 expression in other cell types, their relative contributions to MKP-1 regulation were investigated in rat pinealocytes. We found that neither inhibition of MAPKs nor treatment with dexamethasone affected norepinephrine-stimulated MKP-1 expression. In contrast, treatment with proteasome inhibitors increased norepinephrine-stimulated MKP-1 protein levels and abolished the decline in norepinephrine-stimulated MKP-1 protein levels caused by inhibition of transcription or translation, or blockade of α-adrenergic receptors. Taken together, our results indicate that in rat pinealocytes, the continuous and rapid turnover of MKP-1 protein allows for its rapid induction but is not sufficient to generate the sustained increase in MKP-1 expression post-adrenergic stimulation.

Micellar electrokinetic capillary chromatography for fast separation and sensitive determination of melatonin and related indoleamines using end‐column amperometric detection

MEKC was used in conjunction with end-column amperometric detection (AD) at a carbon disc electrode (0.3 mm diameter) for the selective and sensitive determination of melatonin and its five related indoleamines including its precursors and metabolites in the pineal gland. The introduction of a sample stacking technique in injection and the buffer additive SDS in the buffer solution system provided the rapid and sensitive analysis. Optimal buffer conditions (10 mmol/L phosphate containing 20 mmol/L SDS, pH 7.2), detection potential (+1.0 V vs. Ag/AgCl), and electrokinetic injection 10 s with the separation voltage of 24 kV were employed to achieve the baseline separation of six pineal hormones within 15 min. The peak currents and the analyte concentrations have a good linear relationship over the range of 6.0 x 10(-8) 6.0 x 10(-5 )mol/L. The detection limits for six pineal hormones by AD are 9.7 to 41.8 nmol/L (equal to 2.0 to 9.7 ng/mL) (S/N = 3), respectively. It is proved to provide about 30- to 250-fold improvement over UV, and be comparable with the sensitive fluorescence detection, which needs pre-column derivatization. The proposed method has been applied for analysis of melatonin and related indoleamines in rat pineal glands. A very simple sample pretreatment procedure, merely involving the homogenization step in perchloric acid, was enough to achieve recoveries in the range of 71 to 127% for all the analytes in the pineal gland.

The influence of insulin and glucose on norepinephrine stimulated pineal melatonin secretion

Interrelationships between carbohydrate metabolism and melatonin have been discussed for a long time. The melatonin(MT1)-receptor and Gi-mediated decrease of insulin secretion by melatonin is a well known fact. Recently, we found reduced melatonin plasma levels in type 2 diabetic patients and diabetic Goto Kakizaki (GK) rats, a model of non insulin dependent diabetes mellitus (NIDDM). Several mechanisms, such as diabetic neuropathy and oxidative stress were discussed by others to be the basis for this observation. We reported the expression of insulin receptor (InsR)-mRNA in the rat pineal gland. Earlier studies showed the InsR as being located at the cell membrane of the pinealocytes. The aim of this study was to investigate glucose, insulin and melatonin plasma levels by radioimmunoassay, regarding age and metabolic state of metabolic healthy Wistar and diabetic GK rats. Diurnal pineal InsR-mRNA expression was monitored by real-time RT-PCR. The influence of insulin and glucose on norepinephrine (NE) stimulated pineal melatonin secretion. Here we show that GK rats are permanently hyperglycemic and hyperinsulinemic at night-time. Hyperglycemia is stronger in adult than in young animals. The pineal expression level of InsR-mRNA is significantly lower in young, but higher in adult pineal glands of GK rats. No diurnal rhythmicity of expression was observed. Insulin-stimulated pineals from batch culture supernatants of both, GK- and Wistar rats, showed decreased melatonin production. First perifusion experiments revealed a lower melatonin output when stimulating pineals with NE and insulin or NE and glucose than with NE alone. The effects were not cAMP-mediated. Our results suggest that there is an insulin/InsR-mediated interrelationship between beta cells and pinealocytes. A contribution of hyperglycemia and hyperinsulinemia to reduced melatonin plasma levels seems to be likely and should be further investigated.

Relationship between nocturnal serotonin surge and melatonin onset in rodent pineal gland

BackgroundWe have recently reported dynamic circadian rhythms of serotonin (5-HT, 5-hydroxytryptamine) output in the pineal gland of rat, which precedes the onset of N-acetylserotonin (NAS) and melatonin secretion at night. The present study was aimed at investigating in detail the relationship between 5-HT onset (5HT-on) and melatonin onset (MT-on) in multiple strains of rats and comparing them with those of hamsters.MethodsAnimals were maintained in chambers equipped with light (250 lux at cage levels) and ventilation in a temperature-controlled room. Following surgical implantation of a microdialysis probe in the pineal gland, animals were individually housed for on-line pineal microdialysis and for automated HPLC analysis of 5-HT and melatonin. Animals were under a light-dark cycle of 12:12 h for the duration of the experiments.ResultsAll animals displayed dynamic 5-HT and melatonin rhythms at night. In all cases, 5HT-on (taken at 80% of the daily maximum levels) preceded MT-on (taken at 20% of the daily maximum levels). Within the same animals, 5HT-on as well as MT-on across multiple circadian cycles exhibited minimum variations under entrained conditions. Large inter-individual variations of both 5HT-on and MT-on were found in outbred rats and hamsters under entrained conditions. In comparison, inbred rats displayed very small individual variations of 5HT-on and MT-on. Importantly, we have uncovered a species-specific relationship of 5HT-on and MT-on. 5HT-on of rats, regardless of the strain, preceded MT-on of the same rats by 50 min. In contrast, 5HT-on of hamsters led MT-on by as much as 240 min. Thus, while a constant relationship of 5HT-on and MT-on exists for animals of the same species, the relative timings of 5HT-on and MT-on differ between animals of different species.ConclusionThese results suggest that both 5-HT and melatonin could serve as reliable markers of the circadian clock because of their day-to-day precision of onset timings within the same animals or within individuals of the same strain or same species. The results also demonstrate that data for MT-on cannot be compared directly between different species, and that 5HT-on may be a more reliable circadian marker when data from animals of different species are compared.

Cyclic AMP-inducible genes respond uniformly to seasonal lighting conditions in the rat pineal gland

The encoding of photoperiodic information ensues in terms of the daily profile in the expression of cyclic AMP (cAMP)-inducible genes such as the arylalkylamine N-acetyltransferase (AA-NAT) gene that encodes the rate-limiting enzyme in melatonin formation. In the present study, we compared the influence of the photoperiodic history on the cAMP-inducible genes AA-NAT, inducible cyclic AMP early repressor (ICER), fos-related antigen-2 (FRA-2), mitogen-activated protein kinase phosphatase-1 (MKP-1), nerve growth factor inducible gene-A (NGFI-A) and nerve growth factor inducible gene-B (NGFI-B) in the pineal gland of rats. For this purpose, we monitored the daily profiles of each gene in the same pineal gland under a long (light/dark 16:8) and a short (light/dark 8:16) photoperiod by measuring the respective mRNA amounts by real-time polymerase chain reaction analysis. We found that, for all genes under investigation, the duration of increased nocturnal expression is lengthened and, in relation to light onset, the nocturnal rise is earlier under the long photoperiod (light/dark 16:8). Furthermore, with the exception of ICER, all other cAMP-inducible genes tend to display higher maximum expression under light/dark 8:16 than under light/dark 16:8. Photoperiod-dependent changes persist for all of the cAMP-inducible genes when the rats are kept for two cycles under constant darkness. Therefore, all cAMP-inducible genes are also influenced by the photoperiod of prior entrained cycles. Our study indicates that, despite differences regarding the expressional control and the temporal phasing of the daily profile, cAMP-inducible genes are uniformly influenced by photoperiodic history in the rat pineal gland.

Photoperiodism as a Modifier of Effect of Extremely Low-Frequency Electromagnetic Field on Morphological Properties of Pineal Gland

The aim of our study was to determine, using histological and stereological methods, whether photoperiodism has any impact on the effects that chronic (three-month long) exposure to LF-EMF (50Hz) has on morphological characteristics on rat's pineal gland. The experiment was performed on 48 Mill Hill male rats (24 experimental and 24 control). Upon birth, 24 rats were exposed for 7h a day, 5 days a week for 3 months to LF-EMF (50 Hz, 50-500microT, 10V/m). In the winter (short days, long nights), the activity of the pineal gland and neuroendocrine sensitivity is increased. The study was performed both during summer and winter, following the identical protocol. After sacrifice of animals, samples of pineal gland were processed for HE staining and then were analyzed using the methods of stereology. The most significant changes in epiphysis in the first group of animals in wintertime are: altered glandular feature, hyperemia, reduced pinealocytes with pale pink, poor cytoplasm and irregular, stick-form nuclei. In the second group (II) pinealocytes are enlarged, with vacuolated cytoplasm and hyper chromatic, enlarged nucleus. Morphological changes of pineal gland at rats in the summertime were not as intense as in the winter and finding of the gland in the group II is compatible with those from the control group. Stereological results show both in winter and summer in the first group the decrease of volume density of pinealocytes, their cytoplasm and nuclei and in the second group in winter increase the volume density of pinealocytes, cytoplasm and nuclei, while in the second group the results in summertime are equal to those from the control group. Photoperiodism is modifier of effect of LF-EMF on morphological structure of pineal gland, because the gland recovery is incomplete in winter and reversible in summer.

Effect of TNF‐α on the melatonin synthetic pathway in the rat pineal gland: basis for a ‘feedback’ of the immune response on circadian timing

A retino-hypothalamic-sympathetic pathway drives the nocturnal surge of pineal melatonin production that determines the synchronization of pineal function with the environmental light/dark cycle. In many studies, melatonin has been implicated in the modulation of the inflammatory response. However, scant information on the feedback action of molecules present in the blood on the pineal gland during the time course of an inflammatory response is available. Here we analyzed the effect of tumor necrosis factor-alpha (TNF-alpha) and corticosterone on the transcription of the Aa-nat, hiomt and 14-3-3 protein genes in denervated pineal glands of rats stimulated for 5 hr with norepinephrine, using real-time reverse transcription-polymerase chain reaction. The transcription of Aa-nat, a gene encoding the key enzyme in melatonin biosynthesis, together with the synthesis of the melatonin precursor N-acetylserotonin, was inhibited by TNF-alpha. This inhibition was transient, and a preincubation of TNF-alpha for more than 24 hr had no detectable effect. In fact, a protein(s) transcribed, later on, as shown by cycloheximide, was responsible for the reversal of the inhibition of Aa-nat transcription. In addition, corticosterone induced a potentiation of norepinephrine-induced Aa-nat transcription even after 48 hr of incubation. These data support the hypothesis that the nocturnal surge in melatonin is impaired at the beginning of an inflammatory response and restored either during the shutdown of an acute response or in a chronic inflammatory pathology. Here, we introduce a new molecular pathway involved in the feedback of an inflammatory response on pineal activity, and provide a molecular basis for understanding the expression of circadian timing in injured organisms.

Differential Expression of Activator Protein-1 Proteins in the Pineal Gland of Syrian Hamster and Rat May Explain Species Diversity in Arylalkylamine N-Acetyltransferase Gene Expression

Species differences have been reported for the nighttime regulation of arylalkylamine N-acetyltransferase (AA-NAT), the melatonin rhythm-generating enzyme. In particular, de novo synthesis of stimulatory transcription factors is required for Aa-nat transcription in the Syrian hamster but not in the rat pineal gland. The present work investigated the contribution of phosphorylated cAMP-responsive element-binding protein, c-FOS, c-JUN, and JUN-B in the regulation of Aa-nat transcription in Syrian hamsters compared with rats. The nighttime pattern of cAMP-responsive element-binding protein phosphorylation and regulation by norepinephrine observed in the Syrian hamster was similar to those reported in the rat. On the contrary, strong divergences in c-FOS, c-JUN, and JUN-B expression were observed between both species. In Syrian hamster, predominant expression of c-FOS and c-JUN was observed at the beginning of night, whereas a predominant expression of c-JUN and JUN-B was observed in the late night in rat. The early peak of c-FOS and c-JUN, known to form a stimulatory transcription dimer, suggests that they are involved in the nighttime stimulation of Aa-nat transcription. Indeed, early-night administration of a protein synthesis inhibitor (cycloheximide) markedly decreased AA-NAT mRNA levels in Syrian hamster. In the rat, high levels of JUN-B and c-JUN, constituting an inhibitory transcription dimer, are probably involved in the late-night inhibition of Aa-nat transcription. Early-night administration of cycloheximide actually increased AA-NAT mRNA levels toward the late night. Therefore, composition and timing of the pineal activator protein-1 complexes differ between rat and Syrian hamster and may be an activator (Syrian hamster) or an inhibitor (rat) of Aa-nat transcription.

Analysis of synaptic bodies in the Sprague–Dawley rat pineal gland under extreme photoperiods

Synaptic bodies (SBs) are small, prominent organelles in pinealocytes, most probably involved in signal transduction processes. To check the influence of the photoperiod on their shape plasticity and number we chose two extreme lighting conditions, i.e. 20 h of illumination followed by 4 h of darkness (LD 20:4) versus (LD 4:20). Pineal glands were assessed at 0, 4 and 13 h after dark onset. Under both conditions reconstructed SBs were plates or ribbons but never spheres and there were no obvious differences in morphology. Photoperiodic changes in SB profile size and number were investigated: application of the established method for SB quantification based on single section profile counts (SSPC) of areas showed a significant increase of SB profiles under LD 20:4. However, it has to be noted that SSPC depend on both, number and size of the structures. In contrast to this, modification of the disector counting method, also applied for unbiased quantification of whole SBs, revealed that rat pinealocytes show insignificantly more SBs under LD 20:4 than under 4:20 conditions. The lengths of the SB profiles, which were first measured under different conditions in this study, depend on SB size. They increased significantly under LD 20:4. In conclusion, we detected only an increase in SB size but not in their number. We further prove that, at least for SBs, it is of no value to calculate disector levels from SSPCs.

Expression of luteinizing hormone/chorionic gonadotropin receptor in the rat pineal gland

Luteinizing hormone (LH) influences the secretion of melatonin (N-acetyl-5-methoxytryptamine) from the pineal gland. The present study examined the possible presence of LH/chorionic gonadotropin (CG) receptor in the pineal gland of adult female rats. Reverse transcriptase-polymerase chain reaction analyses demonstrated that LH/CG receptor mRNA is expressed in the pineal gland. Western blotting showed that the pineal gland, like the ovary, contains an 80 kDa receptor protein. Immunohistochemistry revealed that LH/CG receptor, arylalkylamine N-acetyltransferase (a regulatory enzyme in melatonin biosynthesis) and serotonin (a melatonin precursor) are localized primarily to the same cells of the pineal gland. We further found that the levels of pineal LH/CG receptor protein in normal cycling female rats change significantly during the estrous cycle, being lowest at early metestrus. These results demonstrate that LH/CG receptor is expressed in the pineal gland, primarily in melatonin-synthesizing cells, namely pinealocytes. Furthermore, it is suggested that LH influences pineal melatonin secretion through binding to this receptor. In addition, LH/CG receptor levels in the pineal gland are regulated during the estrous cycle under normal physiological conditions.

Cannabinoids attenuate norepinephrine‐induced melatonin biosynthesis in the rat pineal gland by reducing arylalkylamine N‐acetyltransferase activity without involvement of cannabinoid receptors

Cannabinoids modulate neuronal and neuroendocrine circuits by binding to cannabinoid receptors acting upon cAMP/Ca(2+)-mediated intracellular signaling cascades. The rat pineal represents an established model to investigate intracellular signaling processes because a well defined input, the neurotransmitter norepinephrine, is transformed via cAMP/Ca(2+)-dependent mechanisms into an easily detectable output signal, the biosynthesis of melatonin. Here we investigated the impact of cannabinoids on norepinephrine-regulated melatonin biosynthesis in the rat pineal. We demonstrated that treatment of cultured rat pineals with 9-carboxy-11-nor-delta-9-tetrahydrocannabinol (THC), cannabidiol or cannabinol significantly reduced norepinephrine-induced arylalkylamine N-acetyltransferase (AANAT) activity and melatonin biosynthesis. These effects were not mimicked by the cannabinoid receptor agonist WIN55,212-2 and were not blocked by cannabinoid 1 and 2 receptor antagonists. The cannabinoids used did not affect norepinephrine-induced increases in cAMP/Ca(2+) levels. Notably, cannabinoids were found to directly inhibit AANAT activity in lysates of the pineal gland. This effect was specific in so far as cannabinoids did not influence the activity of hydroxyindole-O-methyltransferase (HIOMT), the last enzyme in melatonin biosynthesis. Taken together, our data strongly suggest that cannabinoids inhibit AANAT activity and attenuate melatonin biosynthesis through intracellular actions without involvement of classical cannabinoid receptor-dependent signaling cascades.

Timing of mitogen-activated protein kinase (MAPK) activation in the rat pineal gland

Activation of members of the mitogen-activated protein kinase (MAPK) family of signaling cascades is a tightly controlled event in rat pinealocytes. Cell culture studies indicate that whereas the NE → cGMP activation of p42/44MAPK is rapid and transient, the NE → cAMP activation of p38MAPK is slower and more sustained. The decline in the p42/44MAPK response is in part due to the induction of MAPK phosphatase-1 by NE. In comparison, p38MAPK activation is tightly coupled to the synthesis and degradation of an upstream element in its activation cascade. Whole animal studies confirm activation of p42/44MAPK occurring during the early part of night and precedes p38MAPK activation. Studies with selective MAPK inhibitors reveal a modulating effect of MAPKs on arylalkylamine- N-acetyltransferse (AA-NAT) activity, with involvement of p42/44MAPK in the induction of AA-NAT and p38MAPK participating in the amplitude and duration of the AA-NAT response. These effects of p42/44MAPK and p38MAPK on AA-NAT activity match their timing of activation. Taken together, our studies on the timing of MAPK activation and regulation of AA-NAT by MAPKs add to the importance of MAPKs in regulating the circadian biology of the pineal gland.

Protein/DNA interaction profiling reveals novel regulators of the pineal transcriptome

The rat pineal gland transcriptome exhibits dynamic daily variation that reflects nocturnally restricted hormone production. Here we have used a protein/DNA interaction array to screen for day–night changes in DNA binding activity that are associated with transcriptional rhythms. Overall, 47 of 54 potential consensus binding sequence activities were detected, and of these, 29 (62%) were found to exhibit day:night differences in level. In addition to known, rhythmic pineal DNA binding activities (CRE and AP-1), multiple novel activities were observed including nocturnally elevated AP-2 consensus sequence binding activity. This array result was validated using conventional DNA binding assays, and we have also demonstrated AP-2β and AP-2γ proteins in the pineal gland, in addition to a nocturnally elevated AP-2α isoform. Our results have confirmed the presence of a complex assembly of transcriptional rhythms in the rat pineal gland and have provided details of more factors that contribute to this aspect of circadian neuroendocrine function.

Chronic stress induces upregulation of brain-derived neurotrophic factor (BDNF) mRNA and integrin α5 expression in the rat pineal gland

Chronic stress affects brain areas involved in learning and emotional responses. These alterations have been related with the development of cognitive deficits in major depression. Moreover, stress induces deleterious actions on the epithalamic pineal organ, a gland involved in a wide range of physiological functions. The aim of this study was to investigate whether the stress effects on the pineal gland are related with changes in the expression of neurotrophic factors and cell adhesion molecules. Using reverse transcription-polymerase chain reaction (RT-PCR) and Western blot, we analyzed the effect of chronic immobilization stress on the BDNF mRNA and integrin α5 expression in the rat pineal gland. We found that BDNF is produced in situ in the pineal gland. Chronic immobilization stress induced upregulation of BDNF mRNA and integrin α5 expression in the rat pineal gland but did not produce changes in β-actin mRNA or in GAPDH expression. Stressed animals also evidenced an increase in anxiety-like behavior and acute gastric lesions. These results suggest that BDNF and integrin α5 may have a counteracting effect to the deleterious actions of immobilization stress on functionally stimulated pinealocytes. Furthermore, this study proposes that the pineal gland may be a target of glucocorticoid damage during stress.