A Study of Somatostatin Receptors in Amygdaloid-Kindled Rat Brain

Abstract. The present study indicates that in kindled rats there are no differences in the total number or affinity of the binding sites in the temporal cortex and a slight increase in the total number of binding sites in the cortex when compared with controls. These results, in view of our other observations, suggest that in the kindled rat brain there may be an increased release of SRIF but no down-regulation of SRIF receptors in the temporal cortex and cortex. There appears to be a significant decrease in the number of SRIF receptors in kindled hippocampus. The mechanism by which this occurs remains unclear.     

Localization and Characterization of Insulin-Like Growth Factor-I Receptors in Rat Brain and Pituitary Gland Using in vitro Autoradiography and Computerized Densitometry* A Distinct Distribution from Insulin Receptors

In order to identify likely sites of action of insulin-like growth factor-I (IGF-I) in rat brain and pituitary gland, we have used the technique of in vitro autoradiography and computerized densitometry to map, characterize and quantify its receptors in coronal and sagittal sections. A discrete and characteristic distribution of IGF-I receptor binding was demonstrated, with specific binding representing 85% of total binding. Displacement and specificity competition curves in the olfactory bulb were typical for authentic IGF-I receptors and computer analysis indicated a single class of binding site with a dissociation constant (K_d) of 13 nM for the choroid plexus and 5.1 nM for the olfactory cortex. IGF-I receptor density was very high in the choroid plexus in ail ventricles, but the binding in other circumventricular organs was variable, with high levels in the median eminence and the sub-fornical organ, and low levels in the organum vasculosum of the lamina terminalis. Highest binding was seen in the glomerular layer of the olfactory bulb and its associated regions the taenia tecta and anteromedial olfactory nucleus. The preoptic and septal regions showed moderate binding, while the hypothalamus, with the exception of the median eminence, showed low IGF-I binding. The pituitary gland showed very high binding density in both anterior and posterior lobes, similar to the median eminence. The thalamus had high IGF-I binding density, while it was low in basal ganglia. In the limbic system the hippocampal CA2, CAS, CA4 layers showed high binding, with little in CA1, while binding was high also in the adjacent amygdala. Binding was low in the mid and hindbrain, with the exception of the geniculate bodies, and the sensory nucleus of the trigeminal nerve. Binding was high in the primary olfactory and endopyriform cortex and in specific superficial layers. Cerebellar binding was also high in the molecular layer. Fibre layers showed no binding. Comparison with insulin receptors revealed common distribution in the choroid plexus, paraventricular nucleus, cerebellum, entorhinal cortex and amygdala, with receptor density three- to five-fold higher for IGF-I than for insulin. In contrast, in the hippocampus, insulin binding was high in the CA1 field, and low in CA2, CA3, CA4 while for IGF-I binding the converse was seen. The arcuate nucleus showed prominent insulin labelling and minimal IGF-I binding, while the median eminence showed low insulin and high IGF-I binding. The hypothalamus was more widely labelled with insulin, while in the thalamus the converse was true. Olfactory bulb laminae were labelled with differing intensity by insulin and IGF-I. In common with insulin receptor distribution was the high density of IGF-I receptors over areas of extensive dendritic arborizations which receive rich synaptic inputs, in the cerebellum, hippocampus and olfactory bulb. We conclude that IGF-I receptors are widespread throughout rat brain and pituitary gland, with concentration in regions concerned with olfaction, autonomie and sensory processing, as well as in regulation of growth hormone release, via feedback at the median eminence and pituitary gland. Many of these regions have in common high rates of metabolic and synthetic activity, which may be mediated by IGF-I and its receptors.     

Localization of mRNA Coding for the Three Subtypes of Atrial Natriuretic Factor (ANF) Receptors in Rat Anterior Pituitary Gland Cells

Atrial Natriuretic Factor (ANF) action is mediated by highly selective and specific receptors. Three subtypes have been characterized and cloned: ANF receptor-A, -B and -C. These subtypes are all expressed in the anterior pituitary of the rat. In the present study, the mRNA for each subtype was detected by in situ hybridization. The amounts of ANFR-A and -B mRNA were found to be similar, and to be twice that of ANFR-C mRNA. At the ultrastructural level, the three types of ANFR mRNA were expressed in three anterior pituitary cell types, namely lactotrophs, corticotrophs, and gonadotrophs, identified by their hormonal content. No signal was revealed in somatotrophs or thyrotrophs. The different forms of mRNA were similar in terms of subcellular localization: in the cytoplasmic matrix and the nuclear euchromatin. These data indicate that the anterior pituitary is an important target tissue for ANF action.     

Immunohistochemical Localization of Five Members of the KV1 Channel Subunits: Contrasting Subcellular Locations and Neuron-specific Co-localizations in Rat Brain

A large variety of potassium channels is involved in regulating integration and transmission of electrical signals in the nervous system. Different types of neurons, therefore, require specific patterns of potassium channel subunit expression and specific regulation of subunit coassembly into heteromultimeric channels, as well as subunit-specific sorting and segregation. This was investigated by studying in detail the expression of six different α-subunits of voltage-gated potassium channels in the rat hippocampus, cerebellum, olfactory bulb and spinal cord, combining in situ hybridization and immunocytochemistry. Specific polyclonal antibodies were prepared for five α-subunits (K_V1.1, K_V1.2, K_V1.3, K_V1.4, K_V1.6) of the Shaker-related subfamily of rat K_v channels, which encode delayed-rectifier type and rapidly inactivating A-type potassium channels. Their distribution was compared to that of an A-type potassium channel (K_V3.4), belonging to the Shaw-related subfamily of rat K_v channels. Our results show that these K_v channel α-subunits are differentially expressed in rat brain neurons. We did not observe in various neurons a stereotypical distribution of K_v channel α-subunits to dendritic and axonal compartments, but a complex differential subcellular subunit distribution. The different K_v channel subunits are targeted either to presynaptic or to postsynaptic domains, depending on neuronal cell type. Thus, distinct combinations of K_v1 α-subunits are co-localized in different neurons. The implications of these findings are that both differential expression and assembly as well as subcellular targeting of K_v channel α-subunits may contribute to K_v channel diversity and thereby to presynaptic and postsynaptic membrane excitability.     

Distribution of Tyrosine Hydroxylase mRNA in the Rat Central Nervous System Visualized by Alkaline Phosphatase in situ Hybridization Histochemistry

The expression of tyrosine hydroxylase mRNA in the rat brain was examined using a novel alkaline phosphatase labelled antisense oligodeoxynucleotide probe. The alkaline phosphatase labelled probe revealed the presence of tyrosine hydroxylase mRNA in all the major cell groups and cell bodies previously described as containing catecholamine fluorescence or known to contain tyrosine hydroxylase immunoreactivity. Using standardized development protocols qualitative comparisons between the amount of mRNA signal in different adrenergic, noradrenergic or dopaminergic cell groups could be made. These studies showed that of the three known catecholaminergic cell types the level of tyrosine hydroxylase mRNA signal was high in the noradrenergic and dopaminergic cells, but much lower in the adrenergic cell groups. The sensitivity of this nonradioactive method of in situ hybridization is excellent and has considerable potential for studies of coexistence or coexpression of two mRNA signals for the localization of mRNA signals at the electron-microscope level.     

Distribution of mRNAs for Chromogranins A and B and Secretogranin II in Rat Brain

The mRNA distribution of chromogranins A and B and secretogranin II was determined in rat brain. In Northern blots the oligonucleotide probes used hybridized with single mRNA species of the expected sizes. With tissue hybridization the mRNA signals for these three proteins were found throughout the brain. However, each of the three messages had a distinct distribution, which was exemplified by the fact that in the various regions either all three proteins, a combination of two or only one of them were apparently synthesized. Significant levels of all three mRNAs were found in several regions of the hippocampus and of the amygdala, in some thalamic nuclei and in the pyriform cortex. On the other hand the subiculum contained only the message for chromogranin A, the granule cell layer of the cerebellum only that for chromogranin B, and in posterior intralaminar thalamic and medial geniculate nuclei and in the nucleus of the solitary tract only secretogranin II mRNA was found. The distinct distributions of mRNAs for the chromogranins in various brain regions support the concept that these proteins are propeptides giving rise to functionally active components.     

Somatostatin and Thyrotrophin-Releasing Hormone Response and Receptor Status of a Thyrotrophin-Secreting Pituitary Adenoma: Clinical and in vitro Studies

We have described a patient with a thyrotrophin-secreting pituitary adenoma and correlated a detailed physiological and anatomical investigation of the surgically resected tumour with its in vivo regulation. Thyrotrophin secretion was inhibited by circulating thyroid hormones, dopaminergic agonists and the somatostatin analogue SMS 201–995 but could not be stimulated by thyrotrophin-releasing hormone or further inhibited by exogenous triiodothyronine. Prolonged treatment with SMS 201–995 caused tumour shrinkage as shown by successive computed tomography scans but was accompanied by tumour desensitization and the development of diabetes mellitus. This is the first thyrotroph adenoma in which somatostatin receptors have been directly demonstrated and shown to completely block thyrotrophin-releasing hormone-induced inositol phospholipid accumulation when occupied. In addition, preincubation with triiodothyronine significantly inhibited thyrotrophin-releasing hormone-induced inositol phospholipid turnover in dispersed pituitary cells indicating that in this tumour, circulating thyroid hormones were exerting feedback inhibition at the level of the pituitary either by reducing the number of thyrotrophin-releasing hormone receptors and/or their coupling to second messenger pathways. In keeping with this hypothesis, the acute reduction in intrapituitary triiodothyronine by sodium ipodate in vivo had no effect on peripheral thyrotrophin over 6 h suggesting that the onset of the effect of triiodothyronine withdrawal on thyrotrophin secretion was suitably delayed. The importance of the inositol phospholipid second messenger pathway in transducing the secretory response in this tumour was further corroborated by electrophysiological studies which demonstrated thyrotrophin-releasing hormone-induced changes in K⁺ currents which are dependent on intracellular Ca²⁺ ions, presumably mobilized via the inositol phospholipids. In addition to thyrotrophin and α subunit, growth hormone mRNA and growth hormone were found throughout the tumour as were two populations of cells distinguished electron microscopically by the size of their secretory granules. Although acromegalic features are not unusual in thyrotroph adenomas, our patient showed no evidence of inappropriate growth hormone secretion during surgery or in response to pre- or post-operative insulin stress tests.     

Localization of Insulin-Like Growth Factor-II Receptors in Rat Brain by in vitro Autoradiography and Immunohistochemistry

Insulin-like growth factor-ll (IGF-II) and its receptor, which is homologous with the mannose-6-phosphate (M6P) receptor, are found in high levels in adult rat and human brain, though their role remains unclear. In order to point to possible regional functions, we have mapped and quantified IGF-II/M6P receptors in sagittal sections of adult rat brain by in vitro autoradiography/computerized densitometry and immunohistochemistry. While in vitro autoradiography allowed mapping and quantitation, immunohistochemistry both confirmed mapping and allowed more detailed determination of cellular distribution of receptors. The two methods were generally in agreement with few areas of mismatching. By in vitro autoradiography, a discrete and characteristic distribution of IGF-II receptor binding was demonstrated, with specific binding representing 85% of total binding. Displacement and specificity competition curves in arcuate nucleus and choroid plexus were typical for authentic IGF-II receptors with half maximal displacement at 1 nM cold IGF-II. IGF-II receptor density, estimated by in vitro autoradiography, was very high in circumventricular organs, especially the median eminence, which had the highest binding in the brain. In the remainder of the brain there was concordance between the distribution of receptors identified by the two techniques, with greatest densities in the olfactory bulb and olfactory pathways, the hippocampus and discrete regions of the cerebral cortex, cerebellum, hypothalamus, thalamus and brainstem. There were however, some notable mismatches. Autoradiographic binding was high to very high in the median eminence, arcuate nucleus, suprachiasmatic nucleus and anterodorsal thalamic nucleus, whereas these areas were only poorly immunostained. Conversely, the septum showed moderate autoradiographic binding, but very prominent immunostaining of neurons in its dorsolat-eral aspect. Using the immunohistochemical technique IGF-II receptors were localized to specific neuronal groups such as the mitral cells of the olfactory bulb, Purkinje cells of the cerebellum and neurons in the red nucleus. Fibre pathways were not labelled by either technique. We conclude that IGF-II/M6P receptors are widespread throughout rat brain, specifically in neurons and blood vessels, with a similar, but distinct distribution to IGF-I and insulin receptors. Many of these regions have in common high rates of metabolic and synthetic activity, which may be mediated by IGF-II/M6P and their receptors.     

Expression of the mRNA of Seven Metabotropic Glutamate Receptors (mGluR1 to 7) in the Rat Retina. An In Situ Hybridization Study on Tissue Sections and Isolated Cells

We have studied the expression of mRNAs for seven metabotropic glutamate receptors (mGluR1–7) in the retina of the adult rat by in situ hybridization with tissue sections and isolated cells using [α³⁵S]dATP-labelled oligonucleotide probes. Hybridization revealed the expression of six of the metabotropic receptor mRNAs, mGluR1, 2 and 4–7, in the retina, while mGluR3 was not detected. Each of the expressed receptor mRNAs showed a distinct pattern of expression. In the outer nuclear layer, corresponding to photoreceptor somata, no labelling was detected. In the outer part of the inner nuclear layer, putative horizontal cells were labelled for mGluR5. More proximal in this layer, corresponding to the position of bipolar cell somata, there was strong labelling for mGluR6. A small number of bipolar cells were also labelled for mGluR5 and mGluR7. In situ hybridization with isolated cells showed that mGluR6 was expressed by rod bipolar cells. Subsets of amacrine cells, with cell bodies along the border between the inner nuclear layer and the inner plexiform layer, were positive for mGluR1, 2, 4 and 7, suggesting considerable heterogeneity of these receptors among amacrine cells. None of the seven metabotropic receptor mRNAs was expressed in isolated Müller glial cells. In the ganglion cell layer, virtually every ganglion cell and displaced amacrine cell was labelled for mGluR1 and mGluR4. Some cells in this layer (˜20% of the total), most likely both ganglion cells and displaced amacrine cells, were also labelled for mGluR2 and mGluR7. These findings suggest that metabotropic glutamate receptors are considerably more widespread among neurons in the retina than indicated by previous physiological and pharmacological investigations.     

Regulation of Prolactin Messenger Ribonucleic Acid Levels by Estradiol and Dihydrotestosterone as Evaluated by in situ Hybridization Performed on Implanted Pituitary Glands and Anterior Pituitary Cells in Culture in the Male Rat

We have recently demonstrated that 17ß-estradiol (E₂) administration increases protactin (PRL) mRNA levels in the male rat anterior pituitary gland and that this stimulatory effect is partially inhibited by concomitant administration of dihydrotestosterone. In order to gain more information about the site(s) of action of E₂ and dihydrotestosterone on PRL gene expression, we have studied the effects of these two hormones in pituitaries implanted under the kidney capsule as well as in anterior pituitary cells in culture. In implanted pituitaries, PRL mRNA levels were increased by 90% as compared to values obtained in the stalk-connected pituitaries from the same animals. Administration of E₂ induced a further increase of PRL mRNA levels in implanted pituitaries, while dihydrotestosterone did not produce any change in animals which had been treated or not with E₂. In anterior pituitary cells in culture, addition of E₂ to the culture medium resulted in a 60% increase of PRL mRNA levels over control values. Supplementation with dihydrotestosterone did not induce any variation in the concentration of PRL mRNA in cells which were treated or not with E₂. These results indicate that E₂ exerts a direct action on PRL cells at the pituitary level and strongly support the key role of the hypothalamus in the inhibitory effect of androgens on estrogen-induced stimulation of PRL mRNA in the male rat pituitary.     

Rat oligodendrocytes and astrocytes preferentially express fibroblast growth factor receptor-2 and -3 mRNAs

Fibroblast growth factors (FGFs) exert various effects on glial cells as well as on neurons in the brain. The mRNAs for four FGF receptors (FGFR-1-FGFR-4) are expressed in the brain. Although FGFR-1 and -4 mRNAs are preferentially expressed in neurons, FGFR-2 and -3 mRNAs are preferentially expressed in glial cells. However, the glial cells that express these receptors remained to be identified. In this study, we found that oligodendrocytes and astrocytes in the brain preferentially expressed FGFR-2 and FGFR-3 mRNAs, respectively. The isoforms of immunoglobulin-like domain III (IIIb and IIIc) of the receptors have crucial roles in ligand binding. We also determined the isoforms of FGFR-2 and FGFR-3 expressed in glial cells to be of type IIIc. The expression of FGFR-2 IIIc and FGFR-3 IIIc with different ligand specificities might play important roles in the various effects of FGFs on oligodendrocytes and astrocytes. © 1996 Wiley-Liss, Inc.     

Distribution of angiotensin II type-2 receptor (AT2) mRNA expression in the adult rat brain

Radioactively labeled cRNA probes were used for in situ hybridization histochemistry to establish a detailed map of the sites of expression of the recently cloned angiotensin II, type 2 (AT₂) receptor mRNA in the adult rat brain. The distribution of the AT₂ receptor mRNA was consistent with that of the AT₂ binding sites, which were previously established by autoradiographic binding studies. Thus, high AT₂ receptor mRNA expression was observed in the lateral septum, in several thalamic nuclei, in the subthalamic nucleus, in the locus coeruleus, and in the inferior olive. Due to the superior resolution and sensitivity of in situ hybridization, AT₂ receptor expression was localized at the cellular level, and some additional brain nuclei expressing AT₂ receptor mRNA have been identified. These include the red nucleus, the pedunculopontine tegmental nucleus, the bed nucleus of the supraoptic decussation, the paragenual nucleus, and numerous brainstem nuclei. Several brain nuclei, such as the motor hypoglossal nucleus and the cerebellar nuclei, where AT₂ receptor binding had previously been identified in young animals only, showed a high expression of the AT₂ receptor mRNA in the adult rat. No correlation was found between the expression of the AT₂ and the type 1 (AT₁) receptor mRNAs. A combination of the in situ hybridization and glial fibrillary acidic protein (GFAP) immunohistochemistry shows that the AT₂ receptor in the lateral septum showed that the AT₂ receptor was not detected in GFAP immunoreactive astroglial cells, therefore indicating that AT₂ is neuronal rather than glial in this brain region. © 1996 Wiley-Liss, Inc.     

Opposing Roles for Dopamine D2 and D3 Receptors on Neurotensin mRNA Expression in Nucleus Accumbens

Using in situ hybridization histochemistry in rat nucleus accumbens, we show that the dopamine D₃ receptor mRNA is expressed in the ventromedial part of the shell subdivision, where its gross distribution matches that of neurotensin mRNA. In addition, hybridization studies at the cellullar level show that a large fraction of the neurotensin neurons co-express the D₃ receptor mRNA in this restricted area. In contrast, the dopamine D₂ receptor mRNA is expressed mainly in the core and marginally in the shell, at the level of the cone. In rats treated by haloperidol and sulpiride, two D₂-like receptor antagonists, but not by SCH 23390, a D₁-like receptor antagonist, proneurotensin mRNA was increased in the D₂ receptor mRNA-rich areas but decreased in the D₃ receptor mRNA-rich areas. This suggests that the D₂ and D₃ receptors control neurotensin mRNA expression negatively and positively, respectively.     

Cells that Express Brain-Derived Neurotrophic Factor mRNA in the Developing Postnatal Rat Brain

Brain-derived neurotrophic factor (BDNF) is a member of a family of related neurotrophic proteins which includes nerve growth factor (NGF) and hippocampus-derived neurotrophic factor/neurotrophin-3 (NT-3). To obtain information regarding possible roles for BDNF during postnatal brain development, we have examined the temporal and spatial expression of this trophic factor using in situ hybridization. In specific neocortical regions BDNF mRNA-expressing cells were seen at 2 weeks of age and thereafter. One particular neuronal cell type strikingly labelled was the inverted pyramidal cell population in the deep layers of parietotemporal cortex. In pyriform and cingulate cortices, BDNF mRNA was detected at postnatal day 1 and 1 week of age, respectively, with increasing levels during ontogeny. Several forebrain regions, including the thalamic anterior paraventricular nucleus, hypothalamic ventromedial nucleus as well as the preoptic area, contained moderate levels of BDNF mRNA throughout development. BDNF mRNA was detected transiently in several brainstem structures, notably in the substantia nigra and interpeduncular nucleus. Expression of this trophic factor in hippocampus was relatively low in the early neonatal brain, but attained high levels in the CA3 and CA4 regions as well as in the dentate gyrus by 2 weeks of age. At this early age, which is still during the period of neurogenesis in the dentate gyrus, labelling was restricted to the outer layer, which contained cells with a more mature appearance. However, by 3 weeks of age labelling was distributed throughout the granule cell layer. Our results show both transient and persistent expression of BDNF mRNA in various regions of the developing rat brain and suggest that there is a caudal to rostral gradient of BDNF expression during postnatal brain development, which may be correlated to neuronal maturation.     

In situ hybridization detection of trka mRNA in brain: Distribution,colocalization with p75NGFR and up-regulation by nerve growth factor

In situ hybridization techniques were used to examine the distribution and the nerve growth factor (NGF) regulation of trkA mRNA in the adult rat brain in order to identify neurons in discrete regions of the brain that may be NGF responsive. In agreement with previous studies, trkA mRNA was detected within cells located in the medial septum (MS), diagonal band of Broca (DBB), and caudate. trkA mRNA was also detected in many other regions of the brain, including the nucleus basalis of Meynert, substantia innominata, paraventricular nucleus of the thalamus, interpeduncular nucleus, prepositus hypoglossal nucleus, vestibular nudei raphe obscuris, cochlear nucleus, sensory trigeminal nuclei, and gigantocellular as well as perigigantocellular neurons in the medullary reticular formation. By combining in situ hybridization detection of trkA mRNA with immunocytochemical detection ofp75^NGFR, it was determined that the vast majority (> 90%) of the trkA mRNA-containing cells detected in the MS and DBB also express p75^NGFR. Likewise, the vast majority of p75^NGFR-IR cells detected in the MS and DBB expressed trkA mRNA. Intracerebroventricular infusions of NGF into the third ventricle adjacent to the preoptic area resulted in a 58% increase in relative cellular levels of trkA mRNA in the horizontal limb of the DBB. These data provide evidence that both p75^NGFR and trkA are expressed by NGF-responsive neurons in the MS and DBB. In addition, we note that areas that contained trkA mRNA and that also have been reported to contain p75^NGFR are areas where high-affinity NGF binding sites have been observed autoradio-graphically, whereas areas that contain either trkA or p75^NGFR alone are areas where no high-affinity NGF binding has been reported. Together, these findings suggest that both trkA and p75^NGFR play an important role in the formation of high-affinity NGF receptors in brain and, furthermore, suggest that NGF may have physiological effects within many regions of the brain outside of the basal forebrain.