Lone Helboe

Distribution and pharmacology of alanine–serine–cysteine transporter 1 (asc‐1) in rodent brain

A polyclonal antibody against the Na+‐independent alanine–serine–cysteine transporter 1 (asc‐1) was raised and the specificity of the antibody verified by Western blots performed on membranes prepared from HEK293 cells transiently transfected with the cloned murine asc‐1. The antibody was then used to localize the transporter in the brain of two rodent species by using immunohistochemistry at the light and electron microscopical level. asc‐1‐immunoreactivity (asc‐1‐ir) was widely distributed throughout the mouse and rat brain. Areas with high levels of asc‐1‐ir included hypothalamus, the medial septal area, globus pallidus, entopeduncular nucleus, cingulate and retrosplenial cortices. Moderate asc‐1‐ir was observed in several areas including layers III and V of the neocortex, thalamus, nucleus accumbens, caudate putamen, bed nucleus of stria terminalis, all amygdaloid nuclei, hippocampus (CA1–CA3 and hilus of the dentate gyrus), as well as several brainstem nuclei. asc‐1‐ir was observed as punctuate staining consistent with varicosities matching neuronal cell bodies and dendritic fields. At the ultrastructural level, asc‐1‐ir was mainly confined to presynaptic terminals. Immunostaining in either glial cell bodies or perivascular sites was not observed and white matter was completely devoid of asc‐1‐ir. Furthermore, the pharmacology of the Na+‐independent uptake site for [3H]d‐serine in rat brain synaptosomal P2 fractions was compared with the substrate specificity of the cloned human asc‐1 transporter and a high degree of correlation was demonstrated. We conclude that asc‐1‐ir is widespread in the brain and limited to neuronal structures and that asc‐1 may contribute to synaptic clearance of d‐serine in brain.

In Vivo Internalization of the Somatostatin sst2A Receptor in Rat Brain: Evidence for Translocation of Cell-Surface Receptors into the Endosomal Recycling Pathway

To determine whether cellular compartmentalization of somatostatin receptors can be regulated in vivo, we examined the immunocytochemical distribution of the sst2A receptor (sst2AR) after stereotaxical injections of somatostatin analogs into the rat parietal cortex. Whereas CH-275, a sst1R agonist, failed to induce changes in the diffuse sst2AR immunostaining pattern characteristic of control animals, somatodendritic profiles displaying intracytoplasmic immunoreactive granules became apparent short-term after injection of either somatostatin or the sst2R agonist octreotide. Confocal microscopy revealed that 90% of sst2AR-immunoreactive endosome-like organelles displayed transferrin receptor immunoreactivity. At the electron microscopic level, the percentage of sst2AR immunoparticles dramatically decreased at the plasmalemma of perikarya and dendrites after octreotide injection. Conversely, it significantly increased in endosomes-like organelles. These results demonstrate that sst2ARs undergo, in vivo, rapid and massive internalization into the endocytic recycling compartment in response to acute agonist stimulation and provide important clues toward elucidating somatostatin receptor signaling in the mammalian brain.

Immunohistochemical localization of the somatostatin receptor subtype 2 (sst2) in the central nervous system of the golden hamster (Mesocricetus auratus).

The many actions of somatostatin in the central nervous system are mediated through specific membrane receptors of which five have been cloned. In this study, we have investigated the distribution of one of these receptors, the sst2 subtype, in the brain and spinal cord of the golden hamster (Mesocricetus auratus). Immunohistochemistry was carried out by using polyclonal antibodies raised against the C‐terminal part of the human sst2 receptor. sst2 immunoreactivity was found in the forebrain, brainstem, cerebellum, and spinal cord. In the forebrain, strong immunoreactivity was observed in the deep layers of the neocortex as well as in the endopiriform cortex, claustrum, and basolateral amygdaloid nucleus. Immunoreactivity was also found in the CA1 area of the hippocampus and in the subiculum. In the diencephalon, staining was observed in the periventricular area, the dorsomedial and arcuate nuclei of the hypothalamus, and the medial habenular nucleus. Other areas such as the thalamus, striatum, and globus pallidus were almost devoid of staining. In the brainstem, strong immunoreactivity was observed in the locus coeruleus and the parabrachial nucleus. In addition, immunostaining was observed in the cortex of the cerebellum. In the spinal cord, intense immunoreactivity was seen in lamina I and II of the dorsal horn. Finally, immunoreactive cells were widely distributed in the anterior pituitary. The localization of the sst2 receptor in many brain regions suggests that this receptor subtype is involved in different neuromodulatory actions of somatostatin such as somatosensory, motor, memory, and neuroendocrine functions. J. Comp. Neurol. 405:247–261, 1999.

Neurochemical characterization of receptor-expressing cell populations by in vivo agonist-induced internalization: Insights from the somatostatin sst2A receptor

Characterization of both neurochemical phenotype of G protein‐coupled receptor (GPCR)‐expressing cells and receptor compartmentalization is a prerequisite for the elucidation of receptor functions in the central nervous system. However, it is often prevented by the diffuse and homogeneous distribution of receptor immunoreactivity. This is particularly true for the somatostatin (SRIF) sst2A receptor, which is largely distributed in the mammalian brain. By using this receptor as a model, we investigated whether receptor internalization, a biochemical property shared by numerous GPCRs, would reveal sst2A‐expressing cell populations in the rat dorsolateral septum (LSD), a region in which SRIF might play an important modulatory role. Thirty minutes to 1 hour after intracerebroventricular injection of the sst2A receptor agonist octreotide, numerous sst2A‐immunoreactive neurons and processes became apparent due to intracytoplasmic accumulation of intensely stained granules. Double‐immunolabeling experiments with synaptophysin and MAP2 provided evidence that internalized sst2A receptors are predominantly localized in the somatodendritic compartment. Revealing sst2A receptor‐expressing cell bodies permitted to analyze their neurotransmitter content. Quantitative analysis demonstrated an extensive overlap (∼85%) between SRIF‐ and sst2A‐expressing neuronal populations. Additionally, numerous SRIF‐immunoreactive axon‐like terminals were found in close apposition with sst2A‐positive cell bodies and dendrites. Taken together, these data suggest that the sst2A receptor is predominantly expressed in LSD neurons as a postsynaptic autoreceptor, thus providing novel neuroanatomic clues to elucidate SRIF neurotransmission in this region. More generally, in vivo agonist‐induced internalization appears as a rapid and powerful tool for the neurochemical characterization of GPCR‐expressing cell populations in the mammalian brain. J. Comp. Neurol. 454:192–199, 2002.

Somatostatin and somatostatin receptors in the pig pineal gland during postnatal development: An immunocytochemical study

An immunohistochemical study of the pineal gland of the domestic pig was carried out using rabbit antisera raised against synthetic peptide fragments corresponding to different amino acid sequences of the prosomatostatin, the somatostatin‐14, and the somatostatin‐28 molecule. The study was supplemented by immunohistochemical staining with rabbit antisera raised against five subtypes of somatostatin receptors. The pineal glands were taken from the newborn, 21‐day‐old and 7‐month‐old pigs. Immunoreactive nerve fibers and cells were observed in the pineal gland with all the antisera against somatostatin and prosomatostatin. The nerve fibers were located throughout the pineal gland—in the capsule, connective septa, and parenchyma—with the highest density in proximo‐ventral part of the gland. The somatostatin positive fibers were also found in the habenular and posterior commissurae areas. Somatostatin‐immunoreactive cell bodies were observed mostly in the central part of the gland. These results point to the existence of two somatostatin sources in the pig pineal gland: 1) nerve fibers, probably of central origin; and 2) cells that may represent intrapineal neurons or specialised pinealocytes. A clear difference in the immunoreactivity between newborn, 21‐day‐old, and 7‐month‐old pigs was found. Generally, the density of nerve fibers was lower in adult than young animals. The number of the cells also decreased with age. By using the antisera against the five somatostatin receptors, only sst3 ‐ receptor immunoreactivity could be detected. The receptor‐immunoreactivity was confined to varicose and smooth fibers and some cells. The sst3‐receptor positive structures were localised in all parts of the gland and their number was higher in younger pigs. Anat Rec 259:141–149, 2000.

Highly specific and selective anti-pS396-tau antibody C10.2 targets seeding-competent tau

The abnormal hyperphosphorylation of the microtubule‐associated protein tau plays a crucial role in neurodegeneration in Alzheimers disease (AD) and other tauopathies.