Mouse Pineal Gland Research Articles

ETMR-14. THE SINGLE-CELL LANDSCAPE OF PINEOBLASTOMA IDENTIFIES AN ONCOGENIC PHOTORECEPTOR PROGRAM AS A TUMORIGENIC VULNERABILITY UNIFYING DISTINCT EMBRYONAL CNS TUMOR ENTITIES

Abstract BACKGROUND Pineoblastoma (PB) is a rare and aggressive childhood brain tumor with highly variable outcomes. PB is diagnostically characterized by expression of the photoreceptor identity transcription factor CRX, while tumors can be further classified into molecular subgroups (PB-miRNA1, PB-miRNA2, PB-MYC/FOXR2, and PB-RB1), each marked by unique clinico-molecular features. However, developmental origins of PB subgroup heterogeneity and mechanisms governing malignancy remain unknown. METHODS We assembled a single-nucleus RNA-sequencing cohort (n=25) of primary PB tumors, including cases from all subgroups. A single-cell transcriptional atlas of the mouse pineal gland, covering 11 developmental stages (E11-P21), was created as a reference to map PB subgroup origins. Developmental trajectory analysis in the pineal gland showed a progression from pinealocyte progenitors to an early-, mid- and mature stage of pinealocyte differentiation. RESULTS Cross-species bioinformatics identified significant associations between PB subgroups and pinealocyte progenitors, mitotic cells expressing Crx, Otx2 and Neurod1. Single-nuclei gene regulatory networks in PB tumors identified CRX, OTX2, and NEUROD1 as master regulators of an oncogenic photoreceptor program conserved across PB subgroups. Surprisingly, this photoreceptor program was also highly expressed in retinoblastoma (RETB) and Group3 medulloblastoma (G3-MB). The molecular association between PB, RETB and G3-MB was further supported by DNA methylation and bulk RNA-seq, suggesting up-regulation of the oncogenic photoreceptor program is a unifying molecular connection of distinct CNS tumor entities. Querying this photoreceptor program across the developing pineal gland, retina, and cerebellum, identified conserved yet transient expression within each developmental lineage, suggesting the photoreceptor program may be a developmental vulnerability. In-vivo dependency assays for Otx2 and Neurod1 performed in a novel genetically engineered mouse model of the PB-RB subgroup confirmed their selective essentiality in photoreceptor-positive tumors. CONCLUSIONS Collectively, these findings uncover the photoreceptor program as a shared oncogenic dependency underlying anatomically distinct embryonal CNS tumors and provide a path towards rational clinical translation.

Connexin36 localization to pinealocytes in the pineal gland of mouse and rat.

Several cell types in the pineal gland are known to establish intercellular gap junctions, but the connexin constituents of those junctions have not been fully characterized. Specifically, the expression of connexin36 (Cx36) protein and mRNA has been examined in the pineal, but the identity of cells that produce Cx36 and that form Cx36-containing gap junctions has not been determined. We used immunofluorescence and freeze fracture replica immunogold labelling (FRIL) of Cx36 to investigate the cellular and subcellular localization of Cx36 in the pineal gland of adult mouse and rat. Immunofluorescence labelling of Cx36 was visualized exclusively as puncta or short immunopositive strands that were distributed throughout the pineal, and which were absent in pineal sections from Cx36 null mice. By double immunofluorescence labelling, Cx36 was localized to tryptophan hydroxylase-positive and 5-hydroxytryptamine-positive pinealocyte cell bodies and their large initial processes, including at intersections of those processes and at sites displaying a confluence of processes. Labelling for the cell junction marker zonula occludens-1 (ZO-1) either overlapped or was closely associated with labelling for Cx36. Pinealocytes thus form Cx36-containing gap junctions that also incorporate the scaffolding protein ZO-1. FRIL revealed labelling of Cx36 at ultrastructurally defined gap junctions between pinealocytes, most of which was at gap junctions having reticular, ribbon or string configurations. The results suggest that the endocrine functions of pinealocytes and their secretion of melatonin is supported by their intercellular communication via Cx36-containing gap junctions, which may now be tested by the use of Cx36 null mice.

Characterization of the Expression of Basigin Gene Products Within the Pineal Gland of Mice.

The expression of Basigin gene products and monocarboxylate transporter-1 (MCT1) has been investigated within the mammalian neural retina and suggests a role for these proteins in cellular metabolism within that tissue. The purpose of the present study was to investigate the expression of these same proteins in the pineal gland of the mouse brain. Mouse pineal gland and neural retina RNA and protein were subjected to quantitative reverse transcription-polymerase chain reaction and immunoblotting analyses. In addition, paraffin-embedded sections of each tissue were analyzed for expression of Basigin gene products and MCT1 via immunohistochemistry. The results indicate that MCT1 and Basigin variant-2, but not Basigin variant-1, are expressed within the mouse pineal gland. The expression of Basigin variant-2 and MCT1 was localized to the capsule surrounding the gland. The position and relative amounts of the gene products suggest that they play a much less prominent role within the pineal gland than in the neural retina.

Role of calcium in the gating of isoproterenol‐induced arylalkylamine N‐acetyltransferase gene expression in the mouse pineal gland

Melatonin and its autonomic regulation serve important physiological functions. We recently demonstrated that stimulation of beta-adrenergic receptors only increases nighttime arylalkylamine N-acetyltransferase (Aa-Nat, the rate-limiting enzyme in melatonin synthesis) mRNA levels in mouse pineal gland in vitro, which suggests that pineal clocks may gate Aa-Nat gene expression. In the present study, our data reveal that cAMP analog increased Aa-Nat at any time of day but only in the presence of ionomycin. Using Fura-2AM in ratiometric calcium measurements, we show that isoproterenol stimulation increased intracellular free calcium levels at night, contrary to previous reports. Further, intra- or extracellular calcium depletion suppressed the isoproterenol-induced calcium responses as well as Aa-Nat gene expression. These results suggest calcium may be a critical factor in isoproterenol-induced Aa-Nat gene expression, which may be limited in the daytime. We also found that basal intracellular calcium levels were lower during the night and responses to isoproterenol and KCl depolarization were more robust. In addition, pineals of Cryptochrome mutant mice exhibited no significant difference between day and nighttime basal calcium or isoproterenol response. Together, these results suggest that basal calcium levels in the pineal may be controlled by the endogenous pineal clock, which may influence calcium dynamics, cellular homeostasis and sensitivity to external stimulation. Although the mechanism underlying Aa-Nat gene expression has been well studied, the role of calcium as a link between the pineal clock and Aa-Nat gene expression has been underestimated in rodent pineals.

The Perivascular Phagocyte of the Mouse Pineal Gland: an Antigen‐Presenting Cell

The perivascular space of the rat pineal gland is known to contain phagocytic cells that are immunoreactive for leukocyte antigens, and thus they appear to belong to the macrophage/microglial cell line. These cells also contain MHC class II proteins. We investigated this cell type in the pineal gland of mice. Actively phagocytosing cells with a prominent lysosomal system were found in the pericapillary spaces of the mouse pineal gland following intravenous injection of horseradish peroxidase. The cells also exhibited strong acid phosphatase activity. Perivascular cells were immunopositive for MHC class II protein and for CD68, a marker of monocytes/phagocytes. This study verifies that perivascular phagocytes with antigen‐presenting properties are present in the mouse pineal gland.

The expression of α-internexin and peripherin in the developing mouse pineal gland

The mammalian pineal gland contains pinealocytes, interstitial glial cells, perivascular macrophages, neurons and neuron-like cells. The neuronal identity of neurons and neuron-like cells was an enigma. alpha-Internexin and peripherin are specific neuronal intermediate filament proteins and are expressed differentially in the CNS and PNS. We investigated the development of immunoreactivity and expression patterns of mRNAs for alpha-internexin and peripherin in the mouse pineal gland to determine the neuronal identity of these cells. Both alpha-internexin- and peripherin-immunoreactive cells were readily visualized only after birth. Both proteins were at the highest level on the postnatal day 7 (P7), rapidly declined at P14, and obtained their adult level at P21. Both protein and mRNA of alpha-internexin are expressed in some cells and nerve processes, but not all, of adult mouse pineal gland. Less number of peripherin immunoreactive or RNA-expressing cells and nerve processes were identified. Accumulations of alpha-internexin and peripherin proteins were also found in the cells from the aged pineal gland (P360). We concluded that some cells in the developing mouse pineal gland may differentiated into neurons and neuron-like cells expressing both alpha-internexin and/or peripherin only postnatally, and these cells possess dual properties of CNS and PNS neurons in nature. We suggested that they may act as interneurons between the pinealocyte and the distal neurons innervating the pinealocytes, or form a local circuitry with pinealocytes to play a role of paracrine regulatory function on the pinealocytes.

Pineal circadian clocks gate arylalkylamine N‐acetyltransferase gene expression in the mouse pineal gland

In mammals it has been thought that the circadian clock localizes only in the suprachiasmatic nucleus of the hypothalamus. Recent studies have revealed that certain brain regions and peripheral tissues may also have intrinsic circadian clocks. However, the roles played by 'peripheral circadian clocks' have not been fully elucidated. In this study, we investigated their function using mouse pineal glands, and found that expression of the arylalkylamine N-acetyltransferase (Aa-Nat, EC 2.3.1.87, the rate-limiting enzyme of melatonin synthesis) gene after adrenergic receptor stimulation depended on the time of day even in vitro (gating). Phase-dependent Aa-Nat responses were observed in both melatonin-proficient and melatonin-deficient mouse pineal glands. Phosphodiesterases are unlikely to suppress Aa-Nat induction because a phosphodiesterase inhibitor itself had no effect on the mRNA levels. Puromycin was ineffective in inducing Aa-Nat mRNA levels in either the presence or absence of isoproterenol, suggesting that newly synthesized proteins may not be necessary to gate the Aa-Nat responses. We also discovered circadian dependence of the expression of Period1-luminescence in Period1-luciferase transgenic mouse pineal glands: circadian clocks may be functional in culture. Aa-Nat mRNA levels showed no significant circadian rhythms in the absence of isoproterenol, thus suggesting that Aa-Nat mRNA levels are induced by adrenergic mechanisms, not by a pineal circadian clock. Our results suggest that the pineal circadian clock may determine timing when Aa-Nat gene expression can respond to inputs from the master circadian clock in the suprachiasmatic nucleus, e.g. adrenergic stimulation.

Daily rhythms in clock gene expression in the mouse pineal gland

Daily rhythms in clock gene expression in the mouse pineal gland

The effect of treatment with melatonin upon the ultrastructure of the mouse pineal gland: a quantitative study.

In order to evaluate melatonin implication in the regulating of its own secretory process by pinealocytes, we used morphometric techniques for transmission electron microscopy. In mice treated with 100 mg of melatonin (N-acetyl-5-methoxy-tryptamine) by daily subcutaneous injection, we observed a decrease in number and volumetric density of lysosomes. Our results showed that melatonin influences the secretory activity of pinealocytes and participates in a complex secretory regulating mechanism.

Pharmacological characterization of adenosine receptors in PGT-beta mouse pineal gland tumour cells.

1. The adenosine receptor in mouse pinealocytes was identified and characterized using pharmacological and physiological approaches. 2. Expression of the two adenosine receptor subtypes A2B and A3 was detected in mouse pineal glands and PGT-beta cells by polymerase chain reaction and nucleotide sequencing. 3. Adenosine and 5'-N-ethylcarboxamidoadenosine (NECA) evoked cyclic AMP generation but the A2)-selective agonist 2-(4-(2-carboxyethyl)phenylethylamino)adenosine-5'-N-ethylcarboxamideadenosine (CGS 21680) and the A1-specific agonists R-N(6)-(2-phenylisopropyl)adenosine (R-PIA) and N(6)-cyclopentyladenosine (CPA) had little effect on intracellular cyclic AMP levels. The A2B receptor selective antagonists alloxazine and enprofylline completely blocked NECA-mediated cyclic AMP accumulation. 4. Treatment of cells with the A3-selective agonist N(6)-(3-iodobenzyl)-5'-(N-methylcarbamoyl)adenosine (IB-MECA) inhibited the elevation of the cyclic AMP level induced by NECA or isoproterenol in a concentration-dependent manner with maximal inhibition of 40 - 50%. These responses were blocked by the specific A3 adenosine receptor antagonist MRS 1191. Pretreatment of the cells with pertussis toxin attenuated the IB-MECA-induced responses, suggesting that this effect occurred via the pertussis toxin-sensitive inhibitory G proteins. 5. IB-MECA also caused a concentration-dependent elevation in [Ca(2+)]i and IP3 content. Both the responses induced by IB-MECA were attenuated by treatment with U73122 or phorbol 12-myristate 13-acetate. 6. These data suggest the presence of both A2B and A3 adenosine receptors in mouse pineal tumour cells and that the A2B receptor is positively coupled to adenylyl cyclase whereas the A3 receptor is negatively coupled to adenylyl cyclase and also coupled to phospholipase C.

Selective inhibition of beta(2)-adrenergic receptor-mediated cAMP generation by activation of the P2Y(2) receptor in mouse pineal gland tumor cells.

Rhythmic noradrenergic signaling from the hypothalamic clock in the suprachiasmatic nucleus to the pineal gland causes an increase in intracellular cAMP which regulates the circadian fluctuation of melatonin synthesis. The activation of phospholipase C (PLC)-coupled P2Y(2) receptors upon treatment with ATP and UTP exclusively inhibited the isoproterenol-stimulated cAMP production in mouse pineal gland tumor cells. However, the activation of other PLC-coupled receptors including P2Y(1) and bombesin receptors had little or no effect on the isoproterenol-stimulated cAMP production. Also, ATP did not inhibit cAMP production caused by forskolin, prostaglandin E(2), or the adenosine analog NECA. These results suggest a selective coupling between signalings of P2Y(2) and beta(2)-adrenergic receptors. The binding of [(3)H]CGP12177 to beta(2)-adrenergic receptors was not effected by the presence of ATP or UTP. Ionomycin decreased the isoproterenol-stimulated cAMP production, whereas phorbol 12-myristate 13-acetate slightly potentiated the isoproterenol response. Chelation of intracellular Ca(2+), however, had little effect on the ATP-induced inhibition of cAMP production, while it completely reversed the ionomycin-induced inhibition. Treatment of cells with pertussis toxin almost completely blocked the inhibitory effect of nucleotides. Pertussis toxin also inhibited the nucleotide-induced increase in intracellular Ca(2+) and inositol 1,4,5-trisphosphate production by 30-40%, suggesting that the ATP-mediated inhibition of the cAMP generation and the partial activation of PLC are mediated by pertussis toxin-sensitive G(i)-protein. We conclude that one of the functions of P2Y(2) receptors on the pineal gland is the selective inhibition of beta-adrenergic receptor-mediated signaling pathways via the inhibitory G-proteins.

Clock gene protein mPER1 is rhythmically synthesized and under cAMP control in the mouse pineal organ.

The mammalian clock gene Per1 is an important element of endogenous oscillators that control daily rhythms in central and peripheral tissues. Although such autonomous clock function is lost in the mammalian pineal gland during evolution, mPer1 mRNA and mPER1 protein were found to be strongly elevated in the mouse pineal organ during the dark period compared to daytime values. In vitro studies showed that mPer1 mRNA and mPER1 protein in mouse pineal gland are induced following the activation of a signalling pathway of fundamental importance for pineal physiology, the norepinephrine/cAMP/phosphoCREB cascade. mPER1 may function in the mouse pineal gland as a time-measuring molecule to participate in regulating rhythmic cellular responses in vivo.

Determination of Pineal Melatonin by Precolumn Derivatization Reversed-Phase High-Performance Liquid Chromatography and Its Application to the Study of Circadian Rhythm in Rats and Mice

Determination of minute amounts of endogenous melatonin in rat and mouse pineal gland was performed using an RP-HPLC system. Melatonin was separated following precolumn derivatization and determined with a fluorescence detector at the emission wavelength of 380 nm with the excitation at 245 nm. The calibration curve of melatonin constructed by adding known amounts of melatonin to the homogenates of mouse pineal gland was linear over the range of 1–500 fmol (injection amount/20 μl). The detection limit of added melatonin was 1 fmol (S / N = 5). Repeatability and day-to-day precision for the melatonin spiked sample of mouse pineal gland was 4.0 and 3.8% (RSD), respectively. Using the present method, circadian changes of melatonin content in rat (Wistar) and mouse (C3H) pineal gland were determined. In addition, a minute amount of melatonin in ddY mouse pineal gland was determined, because pineal melatonin of many inbred mouse strains has been reported to be lower than the detection limit.

Pharmacological characterization of beta2-adrenoceptor in PGT-beta mouse pineal gland tumour cells.

1. The adrenoceptor in a mouse pineal gland tumour cell line (PGT-beta) was identified and characterized using pharmacological and physiological approaches. 2. Adrenaline and noradrenaline, adrenoceptor agonists, stimulated cyclic AMP generation in a concentration-dependent manner, but had no effect on inositol 1,4,5-trisphosphate production. Adrenaline was a more potent activator of cyclic AMP generation than noradrenaline, with half maximal-effective concentrations (EC50) seen at 175+/-22 nM and 18+/-2 microM for adrenaline and noradrenaline, respectively. 3. The addition of forskolin synergistically stimulated the adrenaline-mediated cyclic AMP generation in a concentration-dependent manner. 4. The pA2 value for the specific beta2-adrenoceptor antagonist ICI-118,551 (8.7+/-0.4) as an antagonist of the adrenaline-stimulated cyclic AMP generation were 3 units higher than the value for the betaI-adrenoceptor antagonist atenolol (5.6+/-0.3). 5. Treatment of the cells with adrenaline and forskolin evoked a 3 fold increase in the activity of serotonin N-acetyltransferase with the peak occurring 6 h after stimulation. 6. These results suggest the presence of beta2-adrenoceptors in mouse pineal cells and a functional relationship between the adenylyl cyclase system and the regulation of N-acetyltransferase expression.

Two distinct P2 purinergic receptors, P2Y and P2U, are coupled to phospholipase C in mouse pineal gland tumor cells.

We found that extracellular ATP can increase the intracellular Ca2+ concentration ([Ca2+]i) in mouse pineal gland tumor (PGT-beta) cells. Studies of the [Ca2+]i rise using nucleotides and ATP analogues established the following potency order: ATP, adenosine 5'-O-(3-thiotriphosphate) > or = UTP > 2-chloro-ATP > 3'-O-(4-benzoyl)benzoyl ATP, GTP > or = 2-methylthio ATP, adenosine 5'-O-(2-thiodiphosphate) (ADP beta S) > CTP. AMP, adenosine, alpha,beta-methyleneadenosine 5'-triphosphate, beta,gamma-methyleneadenosine 5'-triphosphate, and UMP had little or no effect on the [Ca2+]i rise. Raising the extracellular Mg2+ concentration to 10 mM decreases the ATP- and UTP-induced [Ca2+]i rise, because the responses depend on the ATP4- and UTP4- concentrations, respectively. The P2U purinoceptor-selective agonist UTP and the P2Y purinoceptor-selective agonist ADP beta S induce inositol 1,4,5-trisphosphate generation in a concentration-dependent manner with maximal effective concentrations of approximately 100 microM. In sequential stimulation, UTP and ADP beta S do not interfere with each other in raising the [Ca2+]i. Costimulation with UTP and ADP beta S results in additive inositol 1,4,5-trisphosphate generation to a similar extent as is achieved with ATP alone. Pretreatment with pertussis toxin inhibits the action of UTP and ATP by maximally 45-55%, whereas it has no effect on the ADP beta S response. Treatment with 1 microM phorbol 12-myristate 13-acetate inhibits the ADP beta S-induced [Ca2+]i rise more effectively than the ATP- and UTP-induced responses. These results suggest that P2U and P2Y purinoceptors coexist on PGT-beta cells and that both receptors are linked to phospholipase C.

The presence of nerve fibers immunoreactive for vasoactive intestinal peptide (VIP), peptide histidine isoleucine (PHI), and preproVIP(111–122) in the mouse pineal gland

A low to moderate number of vasoactive intestinal peptide (VIP) and peptide histidine isoleucine (PHI)-immunoreactive nerve fibers with prominent boutons-en-passage were demonstrated in the pineal gland of the mouse. The two peptides, which are parts of the same precursor molecule, were distributed identically in the gland. Positive fibers were present in the connective tissue septae in the gland, in the pineal capsule, and in the pineal parenchyma. No VIP-PHI-immunoreactive elements were found in the deep pineal gland, in the pineal stalk, or in habenular and posterior commissures. This morphological distribution of immunoreactive nerve fibers, which is similar to the distribution in other mammals, indicates that the VIP/PHI fibers of the mouse pineal gland originate exclusively from perikarya in a peripheral ganglion, presumably one of the cholinergic ganglia of the head. No evidence for a VIPergic central innervation was found. VIP and PHI are connected via a bridging peptide equivalent to amino acids 111-122 of the precursor (preproVIP(111-122)). In order to demonstrate the possible existence of this peptide in intra-pineal nerve fibers, antisera directed against a synthetic sequence identical to preproVIP(111-122) and immunohistochemistry were applied. PreproVIP(111-122)-immunoreactive nerve fibers were observed in the mouse pineal gland, with the same distribution pattern and morphology as those immunoreactive for VIP and PHI. To quantify the peptide-immunoreactivities, 50 mice pineals were pooled, extracted, and the concentrations were measured radioimmunologically. The concentrations of the VIP and preproVIP(111-122) immunoreactivities were 1.7 and 2.0 pmol/g, respectively, whereas the concentration of PHI was 0.9 pmol/g.(ABSTRACT TRUNCATED AT 250 WORDS)

Lack of "synaptic" ribbons in the pineal gland of BALB/c mice.

In mammalian pinealocytes "synaptic" ribbons (SR) are regularly occurring organelles that are functionally poorly understood. Since in a number of studies on the mouse pineal gland the presence of SR has not been mentioned, it was the aim of this investigation to quantitate SR in mice. BALB/c mice were chosen, which have recently been shown to have a genetic defect for melatonin synthesis. The pineals of 15 mice killed at night, when SR numbers are normally high, were examined electron microscopically, scanning an area of greater than 20,000 micron 2 per gland. In none of these pineals were SR detected. It is concluded that the lack or extreme rarity of SR in laboratory mice may be related to the inability of the gland to synthesize melatonin, not to the absence of melatonin per se, as melatonin administration in mice does not result in the occurrence of SR.

Glial cells in the pineal gland of mice and rats. A combined immunofluorescence and electron-microscopic study.

Antigenic markers characteristic of astrocytes and their differentiative states (i.e., glial fibrillary acidic protein (GFAP), vimentin, and M1 and C1 antigens) were investigated in the pineal gland of mouse and rat using double immunolabeling techniques. In both species the so-called interstitial cells as characterized by TEM were shown to be astrocytes, since they expressed vimentin, but neither fibronectin (a marker for fibroblasts and endothelial cells) nor the neuron-specific L1 antigen or tetanus toxin receptors. Subpopulations of vimentin-positive pineal astrocytes were also GFAP- and C1- antigen-positive. M1- antigen-positive cells were not detected. It is concluded that a considerable proportion of interstitial cells in the pineal gland of rat and mouse are immature astrocytes which, in contrast to other parts of the central nervous system, persist into adulthood.

Mechanism involved in the response of granulated vesicles in the mouse pinealocyte to acute cold exposure.

Quantitative electron microscopic studies on the mouse pineal gland were done to elucidate the mechanism of the response of the granulated vesicles in pinealocytes to acute cold exposure. An increase in the number of the granulated vesicles in the mouse pinealocytes occurs following administration of isoproterenol and this increase is prevented by pretreatment with the beta-adrenergic blocking agent, propranolol. In addition, a numerical increase of the granulated vesicles in response to acute cold exposure is abolished in mice pretreated with propranolol or in adrenalectomized animals. The results suggest that catecholamines released from the adrenal medulla, but not from sympathetic nerve endings, may cause the response of the granulated vesicles to cold by way of beta-receptors.

Vascular permeability to proteins and peptides in the mouse pineal gland.

The permeability of fenestrated capillaries in the mouse pineal gland to proteins and peptides was demonstrated by means of ultrastructural tracers. Horseradish peroxidase (HRP) and microperoxidase (MP) were injected intravenously and allowed to circulate for approximately 30 s, 1 min, 5 min, 1 or 2 h. The tissue was then fixed by vascular perfusion or by immersion with aldehydes. In all experiments a pronounced extravasation of HRP and MP occurred. Transendothelial vesicular transport seemed to have occurred across the fenestrated capillaries. The most pronounced tracer labeling of vesicles was found after 1 min of MP- or HRP-circulation. The vesicles were uncoated and more than 70% of the HRP- and MP-containing vesicles exhibited diameters between 50 and 110 nm. Furthermore, three other transcapillary pathways taken by the tracers are suggested: 1) via intercellular junctions, 2) through fenestrae and 3) via channels formed by fusion of vesicles with the luminal and abluminal cell membranes. Based on these results, it is assumed that the capillaries in the mouse pineal gland also permeable to peptides synthesized and secreted by the pineal gland.