Holger Jastrow

A differential role of CREB phosphorylation in cAMP-inducible gene expression in the rat pineal

In the rat pineal gland cAMP mediates nocturnal induction of the enzyme arylalkylamine N-acetyltransferase (AA-NAT) as well as of transcription factors such as inducible cAMP early repressor (ICER), Fos-related antigen-2 (Fra-2) and JunB. Cyclic AMP stimulates the phosphorylation of the DNA binding protein cAMP response element binding protein (CREB). While cAMP-induced CREB phosphorylation appears to be a prerequisite for AA-NAT and ICER gene expression, it is not known whether CREB phosphorylation accounts for the full cAMP response of the two genes. Furthermore, the significance of CREB phosphorylation in cAMP-activated Fra-2 and JunB transcription is unknown. In the present in vitro study we used the serine/threonine protein phosphatase inhibitor okadaic acid (OA) to phosphorylate CREB without altering intrapineal cAMP concentration. It was observed that OA (10(-7) M) was less effective than dibutyryl cAMP (dbcAMP; 10(-3) M) in inducing AA-NAT mRNA and ICER mRNA, respectively. On the basis of this finding, it is concluded that CREB phosphorylation alone is apparently not sufficient for the full cAMP response of the two genes. By contrast, OA and dbcAMP equally stimulated the accumulation of the mRNAs of Fra-2 and JunB. Therefore cAMP may induce Fra-2 and JunB transcripts via CREB phosphorylation. Our observations suggest that CREB phosphorylation plays a critical role in diversification of cAMP-dependent gene induction in the rat pineal.

The shape of synaptic ribbons in the rat pineal gland.

Abstract.Under the transmission electron microscope, synaptic ribbons (SRs) of the mammalian pineal gland appear as rod-like organelles. Their three-dimensional structure is not precisely known. In the present study, pineal SRs were investigated using serial sections obtained from rats killed at noon and midnight. The shape of the SRs was reconstructed based on SR profile length and the number of sections in which the profiles were contained. The results obtained show that SRs are basically flat plate-like structures with polymorphic lateral edges. Reconstructions of SRs revealed that they had average dimensions of 300×150×35u2005nm and were 19.3% larger at night than at day; the difference in SR size points to perhaps major differences in synaptic function between day and night.

Adaptation of the disector method to rare small organelles in TEM sections exemplified by counting synaptic bodies in the rat pineal gland

The disector is the only objective method for quantifying particles of variable size in a given volume. With this method, cell organelles are identified on adjacent sections, but only those present in one section are counted. When counting extremely rare structures in transmission electron microscope sections (physical disector), the usual procedure of counting on electron micrographs is limited for economic reasons (e.g. micrographs highly outnumbering the investigated structures). Hence, to apply this unbiased stereological method, a modification of the physical disector concerning 3 aspects has been developed. (1) The prerequisite of screening large corresponding tissue areas (here ∼65000 μm2) was fulfilled by examining tissue areas along the edges of ultrathin sections. (2) The size of the counting frame was determined by measuring the lengths of the section margins (minus a guard area) by means of a Morphomat. This value was multiplied by the width of the investigated tissue zone, corresponding to the diameter of the electron microscope viewing screen. (3) Disector counting was carried out simultaneously on both sections (bidirectional disector) to improve efficiency. In the present study tiny synaptic bodies (SBs) were quantitated by disector in a rat pineal gland, yielding ∼30 SBs/1000 μm3. By contrast, single section profile counts of SBs amounted to 90 SBs/20000 μm2. Since the presently described adaptation of the disector is time‐consuming, it is proposed to determine a proportion factor allowing to estimate number of structures per volume based on single section profile counts. This would decrease the evaluation time by more than 50%.

Unilateral absence of the terminal nerve and distribution of gonadotropin-releasing hormone immunoreactive neurons in the brain of the common mole-rat (Cryptomys, Rodentia)

A paired terminal nerve with gonadotropin-releasing hormone-immunoreactive (GnRHir) neurons was found in five of six specimens of the Zambian common mole-rat (Cryptomys sp.). In these animals the distribution of GnRHir neurons in the CNS was approximately even on both sides. One adult female lacked a right terminal nerve, yet exhibited a comparable total number of GnRHir neurons, most of which were located on the left side of the brain, i. e., on that side where the terminal nerve was present. An additional population of GnRHir cells was detected in the area of the parafascicular and dorsomedial thalamic nuclei of three non-reproductive adult females, but not in young animals (one female, two males). The additional GnRHir cells, referred to as dark spot cells (DSCs) since their perikarya exhibit large or small strongly immunoreactive vacuoles, were present on both sides of the brain in equal numbers even in the specimen with unilateral absence of the terminal nerve. Obviously, the lack of one terminal nerve correlates with a drastic reduction in the number of ipsilateral genuine neurons but leaves the DSCs unaffected.

Changes in the Three Dimensional Structure of Synaptic Ribbons in the Pineal Gland of the Guinea-Pig Caused by Constant Light

Synaptic bodies (SBs) are dynamic synaptic organelles of afferent synapses of the retina, inner ear, lateral line organ and pineal gland in vertebrates. When investigated in a transmission electron microscope, their electron-dense rod-like, round or irregular profiles are surrounded by electron-lucent vesicles. The three-dimensional structure of pineal SBs is not precisely known. Pineal glands of two guinea-pigs (one kept under an LD cycle of 12:12 h; one kept in constant light for 8 weeks) were investigated. SBs were reconstructed in three dimensions to visualise morphological changes in constant light. Transmission electron microscope micrographs from up to 18 serial sections with a known distance (50 nm) of groups of SBs were scanned and processed (controlled super-imposition of corresponding organelles from adjacent sections, involving rotation and shifting of the scanned image) by a self-written programme in the Interactive Data Language (IDL). The resulting 3-D array was further processed by NeurOPS (neurosurgical operation-planning and simulation software developed at the Institut fur Medizinische Statistik und Dokumentation), allowing 3-D visualisation of SBs. The three-dimensional images were printed from different angles to elucidate the appearance of SBs in space. It was found that under constant light, SBs lie opposite one another in adjacent pinealocytes, closely related to the cell membrane. Further, bent plates appearing under the form of V- and U-shaped shields were seen. Smaller lumps or spherical masses of SB material were more often seen and plates orientated in parallel were less frequent.