Expression of insulin-like growth factor binding protein-4 and -5 mRNAs in adult rat forebrain

Accumulating evidence indicates that the insulin-like growth factors (IGFs) can act as neurotrophic factors. A family of at least six IGF binding proteins (IGFBPs) has been characterized. The IGFBPs prolong the half-life of IGFs in plasma and may modulate IGF action in a cell- or tissue-specific fashion. Two recently characterized IGFBPs, IGFBP-4 and -5, have been shown by northern blot hybridization to be expressed in rat brain, but their cellular sites of synthesis are poorly characterized. Because IGFBP-4 and IGFBP-5 could potentially modulate IGF actions in the brain, we used in situ hybridization histochemistry and ³⁵S-labeled IGFBP-4 and IGFBP-5 riboprobes to localize sites of IGFBP-4 and -5 mRNA expression in adult rat brain. The two IGFBP mRNAs are abundantly expressed within discrete regions of brain. The expression patterns of the two genes are largely nonoverlapping. Notably, IGFBP-4 mRNA is highly expressed within hippocampal and cortical areas, whereas IGFBP-5 mRNA is not detected above background in these areas. Within the hippocampus, abundant IGFBP-4 mRNA expression is detected in pyramidal neurons of the subfields of Ammon's horn and the subiculum and in the granule cell layer of the anterior hippocampal continuation. In the cortex, IGFBP-4 mRNA is widely expressed in most areas and layers. In contrast, IGFBP-5, but not IGFBP-4, mRNA is detected within thalamic nuclei, leptomeninges, and perivascular sheaths. The distinct expression patterns of IGFBP-4 and -5 mRNAs within the brain suggest that these IGFBPs may modulate paracrine/autocrine actions of the IGFs in discrete brain regions or compartmentalization of the IGFs within the brain. © 1994 Wiley-Liss, Inc.     

Expression of ghrelin receptor mRNA in the rat and the mouse brain

Ghrelin is a hormone that stimulates growth hormone secretion and signals energy insufficiency via interaction with its receptor, the growth hormone secretagogue receptor (GHSR). The GHSR is located in both the central nervous system and the periphery. Its distribution in the CNS, as assessed by in situ hybridization histochemistry (ISHH), has been described previously in a few mammalian species, although these studies were limited by either the detail provided or the extent of the regions examined. In the present study, we systematically examined the distribution of GHSR mRNA in the adult rat and mouse brains and cervical spinal cords by using ISHH with novel cRNA probes specific for the mRNA encoding functional GHSR (the type 1a variant). We confirmed GHSR mRNA expression in several hypothalamic nuclei, many of which have long been recognized as playing roles in body weight and food intake. GHSR also was found in several other regions previously unknown to express GHSR mRNA, including many parasympathetic preganglionic neurons. Additionally, we found GHSR mRNA within all three components of the dorsal vagal complex, including the area postrema, the nucleus of the solitary tract, and the dorsal motor nucleus of the vagus. Finally, we examined the coexpression of GHSR with tyrosine hydroxylase and cholecystokinin and demonstrate a high degree of GHSR mRNA expression within dopaminergic, cholecystokinin-containing neurons of the substantia nigra and ventral tegmental area.     

Localization of insulin-like growth factor binding protein-4 expression in the developing and adult rat brain: Analysis by in situ hybridization

We have previously isolated insulin-like growth factor binding protein-4 (IGFBP-4) from media conditioned by a neuronal cell line and have detected IGFBP-4 mRNA in selected regions of the developing and adult rat brain by Northern blot analysis. In this study, the ontogeny and regional distribution of IGFBP-4 expression were determined by in situ hybridization histochemistry. While IGFBP-4 mRNA expression at embryonic day 15 was restricted to choroid plexus primordium and meninges, by embryonic day 20 IGFBP-4 mRNA was also localized in the basal ganglia. In the postnatal rat, at days 1 and 5, IGFBP-4 was also present in the meningeal cell layer surrounding the developing cerebellum and in the hippocampal formation. The distribution of IGFBP-4 mRNA in the adult brain was considerably more widespread. The principal areas where IGFBP-4 mRNA was detected were the cerebral cortex (layers II and IV), olfactory peduncle (anterior olfactory nuclei), limbic system (hippocampus and amygdala), thalamus and basal ganglia, as well as choroid plexus and meninges. The widespread and persistent expression of IGFBP-4 is in marked contrast with IGFBP-2, the other IGFBP in the brain, whose localization by in situ hybridization is reported to be restricted to choroid plexus and meninges. The spatial pattern of IGFBP-4 expression in areas known to either overlap, be adjacent to, or project to regions that express the IGFs or their receptors may reflect a role for IGFBP-4 as a modulator of IGF action in the brain. © 1993 Wiley-Liss, Inc.     

Differential effects of the intraventricular administration of 6-hydroxydopamine on the induction of type II β-tubulin and tyrosine hydroxylase mRNA in the locus coeruleus of the aging Fischer 344 rat

Noradrenergic neurons of the locus coeruleus have been shown to respond to injury by increasing the synthesis of neurotransmitter (via the activation and induction of tyrosine hydroxylase, the rate-limiting catalyst in the production of catecholamines) and initiating compensatory axonal sprouting. However, this laboratory has recently described a significant deficit in the activation of tyrosine hydroxylase in the aged Fischer 344 rat, in contrast to the young and mature rat, following partial damage to cortical and hippocampal noradrenergic terminals induced by the neurotoxin 6-hydroxydopamine. To extend these observations, we measured changes in the relative levels of neuron-specific type II β-tubulin and tyrosine hydroxylase mRNA in locus coeruleus neurons of 2, 12, and 24-month-old Fischer 344 rats following intraventricular infusions of 6-hydroxydopamine by using in situ hybridization histochemistry. These measures were used as markers of the responsiveness of these neurons to injury. 6-Hydroxydopamine treatment induced a persistent increase (at least 10 days) in the expression of type II β-tubulin mRNA only in 2-month-old animals; this marker decreased in the 12 and 24-month-old animals. Relative levels of tyrosine hydroxylase mRNA increased in 2 and 12-month-old lesioned animals both 3 and 10 days post-treatment. In contrast, the induction of tyrosine hydroxylase mRNA in 24-month-old animals, seen three days post-treatment, was attenuated by 10 days. These data indicate that the capacity of locus coeruleus neurons to compensate for injury by either initiating a potential sprouting response or increasing their capacity to synthesize neurotransmitter is reduced in older animals. © 1996 Wiley-Liss, Inc.     

Disabled-1 mRNA and protein expression in developing human cortex

Disabled-1 (Dab1) forms part of the Reelin-Dab1 signalling pathway that controls neuronal positioning during brain development; Dab1 deficiency gives rise to a reeler-like inversion of cortical layers. To establish a timetable of Dab1 expression in developing human brain, Dab1 mRNA and protein expression were studied in prenatal human cortex. The earliest Dab1 signal was detected at 7 gestational weeks (GW), the stage of transition from preplate to cortical plate, suggesting a role of the Reelin-Dab1 signalling pathway in preplate partition. From 12 to 20 GW, the period of maximum cortical migration, Dab1 expression was prominent in the upper tiers of the cortical plate, to decline after midgestation. Radially orientated apical dendrites of Dab1-expressing neurons indicated a predominant pyramidal phenotype. Pyramidal cells in hippocampus and entorhinal cortex displayed a more protracted time of Dab1 expression compared to neocortex. In addition, at later stages (18-25 GW), Dab1 was also expressed in large neurons scattered throughout intermediate zone and subplate. From 14 to 22 GW, particularly high levels of Dab1 mRNA and protein were observed in cells of the ventricular/subventricular zone displaying the morphology of radial glia. The partial colocalization of vimentin and Dab1 in cells of the ventricular zone supported a radial glia phenotype. The concentration of Dab1 protein in ventricular endfeet and initial portions of radial processes of ventricular-zone cells points to a possible involvement of Dab1 in neurogenesis. Furthermore, a subset of Cajal-Retzius cells in the marginal zone colocalized Dab1 and Reelin, and may thus represent a novel target of the Reelin-Dab1 signalling pathway.     

Postnatal ontogeny of cells expressing prepro-neurotensin/neuromedin N mRNA in the rat forebrain and midbrain: a hybridization histochemical study involving isotope-labeled and enzyme-labeled probes.

The postnatal ontogeny of cells expressing prepro-neurotensin/neuromedin N messenger RNA (prepro-NT/NN mRNA) in the rat forebrain and midbrain was investigated by in situ hybridization histochemistry. According to the pattern of expression during development, the cells which express prepro-NT/NN mRNA can be roughly divided into 2 groups. In type I cells, prepro-NT/NN mRNA expression reaches a maximum in terms of content during the postnatal period. After this early peak, cells of this type express the same or less prepro-NT/NN mRNA, reaching a plateau at an adult level that still contains a high level of expression. In type II cells, prepro-NT/NN mRNA appears during the postnatal period, and the expression decreases dramatically after the first postnatal week, being almost undetectable by a few weeks after birth. Type I cells were observed in the following areas: the piriform cortex, field CA1 of Ammon's horn, subiculum, vertical, and horizontal limbs of the diagonal band of Broca, intermediate part of the lateral septal nucleus, bed nucleus of the stria terminalis, medial preoptic area, lateral hypothalamus, caudal part of the caudate putamen, medial, cortical, and central amygdaloid nuclei, ventral tegmental area, deep mesencephalic nucleus, cuneiform nucleus, dorsal raphe nucleus, laterodorsal tegmental nucleus, parabrachial nucleus, and oral part of the pontine reticular nucleus. Cells of type II were observed in the following areas: the mitral cell layer of the olfactory bulb, rostral part of the caudate putamen, (anterior) cingulate cortex, and retrosplenial cortex (posterior cingulate cortex).     

Expression of D1 receptor mRNA in projections from the forebrain to the ventral tegmental area

In situ hybridization was combined with Fluoro-Gold retrograde labeling to determine if cells projecting from the forebrain to the ventral tegmental area (VTA) express D₁ receptor mRNA. Cell counts were made in the prefrontal cortex, shell of the nucleus accumbens, and ventral pallidum to estimate the percentage of neurons projecting to the VTA that express D₁ receptor mRNA. Retrogradely labeled cells were observed in the infralimbic and prelimbic regions of the prefrontal cortex, and up to 37% of the retrogradely labeled cells expressed D₁ receptor mRNA. Double-labeled cells constituted up to 89% of retrogradely labeled neurons in the rostral shell and up to 68% in the caudal shell of the nucleus accumbens. The number of retrogradely labeled cells in the ventral pallidum that were double-labeled ranged from 13% in the rostral to less than 10% in the caudal portions. These data provide anatomical support for a role of D₁ receptors in the reciprocal innervation between the forebrain and VTA. Synapse 25:205–214, 1997. © 1997 Wiley-Liss, Inc.     

Developmental study of the gene expression for α and γ subunits of enolase in the rat brain by in situ hybridization histochemistry

The gene expression for α and γ subunits of enolase, a dimeric enzyme in the glycolytic pathway, was examined in the developing brain of rats by in situ hybridization. The expression for the γ subunit of enolase was first detected in post-mitotic neurons settled in the mantle zone at E13, and it increased progressively until the adult stage. Expression signals for the α subunit were discerned in two discrete regions showing different developmental changes: the signals in the proliferative ventricular zone were intense at E13 and decreased and eventually disappeared around birth, whereas the signals in the mantle zone persisted until the adult stage. In the adult brain, mRNAs for the α and γ subunits were expressed widely in neurons, resulting in almost similar temporal patterns in the brain except for the cerebellum. Expression levels of the α subunit in adult glial cells were below the detection threshold of the in situ hybridization analysis. These findings suggest that both α and γ enolase subunits participate in energy production in neurons of the mature brain and that marked changes in the subunit composition of enolase occur according to both neuron type and maturation. (c) 1993 Wiley-Liss, Inc.     

Different postnatal development profiles of neurons containing distinct GABAA receptor beta subunit mRNAs (beta 1, beta 2, and beta 3) in the rat forebrain.

The expression of three beta subunit (beta 1, beta 2, and beta 3) mRNAs for gamma-aminobutyric acidA receptor in the postnatal rat forebrain was examined by in situ hybridization histochemistry with probes synthesized for the respective subunit mRNAs. The developmental expression of these subunit mRNAs conformed to one of three patterns. Pattern I was high expression of the mRNA at birth and a constant or increasing expression thereafter. In contrast, pattern II was no or very low expression of the mRNA at birth, with expression quickly increasing to reach the adult level in the early postnatal period. Pattern III was the transient expression of the subunit mRNA or else a marked decrease of its expression after a peak in the early postnatal period. On the basis of this classification, the expression of beta 3 subunit mRNA followed pattern I in most regions of the forebrain, such as the isocortex, the olfactory bulb and some of its related areas, the hippocampal formation, the amygdala, the septum, the bed nucleus of the stria terminalis, the caudate-putamen, the nucleus accumbens, the globus pallidus, the ventral pallidum, and the hypothalamus. In some areas, such as the magnocellular preoptic nucleus, the thalamus, and the subthalamic nucleus, pattern III was seen for this subunit. However, none of the regions of the brain showed pattern II expression of beta 3 subunit mRNA. In contrast, the expression of beta 1 and beta 2 subunit mRNAs followed pattern II in most regions of the forebrain. These included the expression of beta 1 subunit mRNA in the isocortex, the olfactory bulb, the hippocampal formation, the amygdala, the septum, the bed nucleus of the stria terminalis, the thalamus, and the hypothalamus, and the expression of beta 2 subunit mRNA in the isocortex, the olfactory bulb and some of its related areas, the amygdala, the nucleus of the diagonal band, the caudate-putamen, the thalamus, and the hypothalamus. Pattern I was not found for beta 1 subunit mRNA, although it was seen in some areas for beta 2 subunit mRNA, such as the ventral pallidum, the globus pallidus, and the magnocellular preoptic nucleus. On the other hand, pattern III was followed by beta 1 subunit mRNA in the anterior olfactory nucleus, the olfactory tubercle, and the piriform cortex, and the same pattern for the beta 2 subunit was also found in the olfactory tubercle, the hippocampal formation, the septum, the bed nucleus of the stria terminalis, and the nucleus accumbens.(ABSTRACT TRUNCATED AT 400 WORDS)     

Localization of Neuropeptide Y Y1 mRNA in the Human Brain: Abundant Expression in Cerebral Cortex and Striatum

Many neurobiological functions have been ascribed to the NPY Y1 receptor subtype, but autoradiographic analysis has failed to detect Y1 binding sites in most human brain areas, in contrast to the rat. We examined the regional distribution of Y1 mRNA-containing cells in the post-mortem human brain to clarify if there is a major species difference in terms of the existence of Y1 receptors in the human telencephalon, in particular the striatum and cortex. In situ hybridization experiments revealed widespread distribution of Y1 mRNA signals in all layers of most limbic and neocortical regions, predominantly in layer IV (most cortical regions) and layer VI. The striatum showed moderate Y1 receptor mRNA expression levels with intensely expressing cells localized to the nucleus accumbens. The highest Y1 receptor mRNA expression was apparent within the dentate gyrus, and the lowest in the subiculum, parahippocampal gyrus, cerebellum, and thalamus. In vitro autoradiography using [¹²⁵I]Leu³¹Pro³⁴-PYY and [¹²⁵I]PYY with NPY (13–36) or Leu³¹ Pro³⁴ NPY; confirmed the presence of low Y1–like binding in the human brain despite abundant Y1 mRNA expression. However, using a rat model of the human autopsy process, it was apparent that the inability to reveal high Y1– versus Y2–like receptors in the human brain was related in part to marked reductions of Y1–like, but not Y2–like, receptors within a 4 h post-mortem delay. Altogether, the results indicate that the Y1 receptor gene is abundant in the human brain and this receptor may have important roles in cognitive, limbic and motor function.     

Anatomical localization of calmodulin mRNA in the rat brain with cloned cDNA and synthetic oligonucleotide probes

Calmodulin is a small, acidic calcium-binding protein that regulates a number of calcium-dependent enzyme activities and is thought to be involved in neurotransmission. To begin to explore further the regulation of this important protein in the brain, we have cloned a rat calmodulin cDNA and designed an oligonucleotide probe based on this sequence. Both the cDNA and oligonucleotide probes revealed a markedly heterogeneous distribution of hybridization signal for calmodulin mRNA in the rat brain. The greatest apparent abundance of mRNA for calmodulin was seen in the hippocampus and cerebral cortex, whereas many brain regions showed relatively low hybridization signal, including the striatum and portions of the hypothalamus and brainstem.     

Expression of melanocortin 4 receptor mRNA in the central nervous system of the rat

The melanocortin 4 receptor (MC4-R) plays a pivotal role in maintaining energy homeostasis in rodents and humans. For example, MC4-R deletion or mutation results in obesity, hyperphagia, and insulin resistance. Additionally, subsets of leptin-induced autonomic responses can be blocked by melanocortin receptor antagonism, suggesting that MC4-R-expressing neurons are downstream targets of leptin. However, the critical autonomic control sites expressing MC4-Rs are still unclear. In the present study, we systematically examined the distribution of MC4-R mRNA in the adult rat central nervous system, including the spinal cord, by using in situ hybridization histochemistry (ISHH) with a novel cRNA probe. Autonomic control sites expressing MC4-R mRNA in the hypothalamus included the anteroventral periventricular, ventromedial preoptic, median preoptic, paraventricular, dorsomedial, and arcuate nuclei. The subfornical organ, dorsal hypothalamic, perifornical, and posterior hypothalamic areas were also observed to express MC4-R mRNA. Within extrahypothalamic autonomic control sites, MC4-R-specific hybridization was evident in the infralimbic and insular cortices, bed nucleus of the stria terminalis, central nucleus of the amygdala, periaqueductal gray, lateral parabrachial nucleus, nucleus of the solitary tract, dorsal motor nucleus of the vagus (DMV), and intermediolateral nucleus of the spinal cord (IML). By using dual-label ISHH, we confirmed that the cells expressing MC4-R mRNA in the IML and DMV were autonomic preganglionic neurons as cells in both sites coexpressed choline acetyltransferase mRNA. The distribution of MC4-R mRNA is consistent with the proposed roles of central melanocortin systems in feeding and autonomic regulation.