Estrogen receptor beta and progesterone receptor mRNA in the intergeniculate leaflet of the female rat.

The present study was undertaken to explore the possibility that the integration of hormonal cues in the regulation of neuroendocrine mechanisms may occur outside of the hypothalamus at the level of the lateral geniculate body. In situ hybridization for mRNA encoding estrogen receptor beta and progesterone receptor was carried out on sections containing the lateral geniculate body using [35S]-labeled antisense riboprobes. Labeled cells were present in different limbic and hypothalamic sites as described previously. Populations of cells distributed homogeneously in the ventral lateral geniculate nucleus and intergeniculate leaflet were also found to express mRNA for estrogen receptor beta and progesterone receptor. The dorsal lateral geniculate nucleus lacked specific labeling for either type of gonadal steroid hormone receptor mRNA. The present observation together with the recent demonstration of a direct pathway between the intergeniculate leaflet and hypothalamic neuroendocrine cells indicate that integration of hormonal and photic stimuli in the central regulation of endocrine mechanisms occurs outside of the hypothalamus in the lateral geniculate body.     

Regional distribution of the cells expressing glycine receptor β subunit mRNA in the rat brain

The expression of mRNA of the β subunit of the glycine receptor was investigated in the rat by in situ hybridization histochemistry using an oligonucleotide probe specific to the sequence of the β subunit. Neurons expressing β subunit mRNA were widely and abundantly distributed in the rat brain from the olfactory bulb to the spinal cord. The pattern of distribution of cells containing β subunit mRNA in the lower brainstem was very similar to that of cells containinga₁ subunit mRNA. In addition, β subunit mRNA was strongly expressed by the neurons of the cerebral cortex, hippocampal formation and diencephalon as well as by the Purkinje cells wherea₁ subunit mRNA expression is rare. These findings indicated that the glycine receptor is heterogeneous. The sites where strong labeling was noted were as follows. In the forebrain and diencephalon, strongly labeled neurons were abundant in the olfactory region, hippocampal formation, cerebral cortex, and thalamus. In the hippocampal formation, neurons in the subiculum, pyramidal cells in Ammon's horn, and neurons in the polymorphic layer of the dentate gyrus were strongly labeled. In the thalamus, the anterodorsal, reticular, parafascicular, and the subthalamic nuclei were strongly labeled. In the brainstem, the red nucleus, almost all of the motor neurons in the cranial motor nuclei innervating striated muscles, the trigeminal mesencephalic nucleus, the ventral tegmental nucleus of Gudden, and the pontine nucleus were strongly labeled. In the cerebellum, Purkinje cells in the Purkinje cell layer and all of the cerebellar nuclei were strongly labeled.     

Down-regulation of β-adrenergic receptor following long-term monocular deprivation in cat visual cortex

To examine how adrenergic receptor binding is modified by experimental manipulation of sensory afferent, we carried out binding experiments (membrane fraction and in vitro autoradiography) for both ₂- and β-adrenergic receptors in the brain of cats which had been deprived of vision in one eye. In the cerebral cortex of control animals, β-adrenergic receptor (β-AR) binding was found to be higher in the occipital regions than in other regions, while ₂-AR binding was relatively uniform. Monocular deprivation throughout the postnatal sensitive period (1–7 month of age) significantly decreased β-AR binding in the visual cortex and lateral geniculate nucleus. Scatchard plot analysis in the visual cortex showed ca. 50% reduction in B_max and little change in K_d No significant difference was found in ₂-AR binding following monocular deprivation. Similar extent of down-regulation in β-AR binding was confirmed in all layers of visual cortex using autoradiography.     

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.     

Ischemia-induced changes in α-tubulin and β-actin mRNA in the gerbil brain and effects of bifemelane hydrochloride

Using in situ hybridization histochemistry, we examined changes in the cytoskeletal protein α-tubulin and β-actin mRNAs in the gerbil brain 14 days after transient ischemia. In an attempt to identify the changes induced in the synthesis of cytoskeletal protein by schemia, we also evaluated the effects of post-ischemia administration of bifemelane on these cytoskeletal proteins. α-Tubulin and α-actin mRNAs were decreased in the CA1 region 14 days after transient ischemia. These decreases coincided with the loss of CA1 pyramidal cells, suggesting that they may have been related to delayed neuronal death. The β-actin mRNA level in ischemic controls was significantly increased in the dentate gyrus, habenular nucleus, and medial and lateral thalamic nuclei, where some afferent nerves project into the hippocampal pyramidal cells. The increased β-actin mRNA suggests that there may be a compensatory enhancement of actin synthesis in the afferent neurons that restores loosened synaptic connections with the ischemic cells in the CA1-4 fields. Administration of bifemelane just after recirculation prevented most of the ischemia-induced mRNA reductions in the CA1 field. Bifemelane's effect may be related to inhibition of Ca²⁺ influx and its radical scavenging activity. When bifemelane was administered to the ischemic group, α-tubulin mRNA levels significantly increased in the dentate gyrus and amygdaloid nucleus, and β-actin mRNAs showed a tendency to increase in the CA3 and CA4 fields, dentate gyrus, and medial and lateral thalamic nuclei. These findings suggest that bifemelane may enhance synthesis of cytoskeletal protein, especially in the ischemic brain, inducing axon outgrowth or synapse formation.     

Region specific increase of dopamine receptor D1/D2 mRNA expression in the brain of μ-opioid receptor knockout mice

Previous pharmacological studies have indicated the possible existence of functional interactions between opioidergic and dopaminergic neurons in the CNS. In this study, the expression of mRNAs encoding dopamine receptor D1/D2 was examined to investigate whether there is a change in the dopamine pathway of mice lacking the μ-opioid receptor by in situ hybridization technique. In the μ-opioid receptor knockout mice, the expression of dopamine receptor D1 mRNA was increased in the olfactory tubercle, nucleus accumbens, caudate putamen, and the layer VI of the neocortex compared with that of wild-type mice. The expression of dopamine receptor D2 mRNA was also increased in the olfactory tubercle, caudate putamen, and the nucleus accumbens of μ-opioid receptor knockout mice. These results indicate that there are compensational changes in the dopaminergic systems of μ-opioid receptor knockout mice.     

疼痛及针刺镇痛时孤啡呔受体mRNA的变化

目的：观察大鼠在福尔马林致痛及针刺镇痛时孤啡肽受体ｍＲＮＡ在一些与镇痛有关核团的变化情况。方法：采用原位杂交组织化学技术。结果：电针后１０ｈ，导水管周围腹侧区、中缝背核及中缝大核内孤啡肽受体ｍＲＮＡ阳性神经元数增多；而在大鼠脚掌注射福尔马林后，上述核团内孤啡肽受体ｍＲＮＡ阳性神经元数却明显减少；电针并注射福尔马林，脑内孤啡肽受体ｍＲＮＡ水平介于单用电针和福尔马林之间。结论：电针能促进孤啡肽受体的合成而伤害性刺激抑制孤啡肽受体的合成。     

Photoperiodic dependent changes in the number of neurons containing mRNA encoding neuropeptide Y in the intergeniculate leaflet of the Syrian hamster

The intergeniculate leaflet (IGL) is a distinct division of the lateral geniculate complex that participates in the regulation of the circadian rhythm through its projections to the circadian pacemaker located in the suprachiasmatic nuclei of the hypothalamus. A high number of neuropeptide Y (NPY) cell bodies has been described in the IGL by immunohistochemistry and in situ hybridization. The present study investigated whether NPY in the IGL is influenced by the length of the daily photoperiod. By using in situ hybridization we show a significant increase of the number of NPY mRNA containing neurons in the mid-part of the IGL of Syrian hamsters maintained in a short photoperiod compared to those kept in a long photoperiod. On the other hand, NPY mRNA expression per cell in the IGL is similar in both photoperiods tested.     

Estrogen and progesterone treatment: effects on muscarinic M(4) receptor subtype in the rat brain

We investigated the effect of ovariectomy (OVX) and hormonal treatment for 10 weeks by estradiol and progesterone on muscarinic M₄ receptor subtype in different brain areas of female rats. Moreover, motor activity of OVX and hormone-treated rats was measured by automated open field exploration boxes. Receptor quantification in the hippocampus, frontal cortex, parietal cortex, amygdala and hypothalamus was done by receptor autoradiography using a selective ligand for muscarinic M₄ receptors. Ovariectomy up-regulated M₄ receptors in the dentate gyrus, CA1, CA3, frontal cortex and hypothalamus whereas the estrogen treatment restored M₄ binding to that of the sham group. Progesterone treatment had no effect on the ovariectomy-induced up-regulation of M₄ receptors. Ovariectomy significantly decreased the exploratory activity of the rats compared to the sham group. Estrogen treatment restored the exploratory behavior of the ovariectomized rats to that of the sham group whereas the progesterone-treated rats were less alert to the surrounding when compared to the sham and estrogen supplemented rats. The effect of estrogen on the hippocampal muscarinic M₄ receptor subtype is a novel finding and may have functional significance for cholinergic receptors especially in relation to postmenopausal memory problems and neurodegenerative disease like Alzheimer’s disease.     

Estrogen and progesterone affect cocaine pharmacokinetics in female rats

Several studies have reported sex differences in behavioral responses to cocaine whereby females display a greater degree of locomotor activity. Fluctuations in estrogen and progesterone during the estrous cycle have been postulated to underlie these behavioral differences. In this study, we tested the hypothesis that hormonal replacement (estrogen or progesterone) in ovariectomized rats affects cocaine pharmacokinetics. We found that estrogen replacement did not affect cocaine-induced locomotor activity, but progesterone attenuated locomotor counts in comparison with control groups receiving only sesame oil. Estrogen, however, decreased brain levels of cocaine and norcocaine 30 min after cocaine administration in comparison to the group-receiving vehicle at that time point. In addition, in progesterone-treated rats, levels of benzoylecgonine and ecgonine methylester were higher at 30 min post-administration than at 15 min. No changes were found in blood levels of the metabolites. These findings suggest that while progesterone has an impact on locomotor behavior, pharmacokinetic effects may have a limited role in mediating behavioral responses to cocaine.     

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.     

Distribution of substance P and neurokinin-1 receptor immunoreactivity in the suprachiasmatic nuclei and intergeniculate leaflet of hamster, mouse, and rat

The circadian pacemaker in the hypothalamic suprachiasmatic nuclei (SCN) receives photic information directly via the retinohypothalamic tract (RHT) and indirectly from retinally innervated cells in the thalamic intergeniculate leaflet (IGL) that project to the SCN. Using standard immunohistochemical methods, we examined the presence and distribution of substance P (SP) and the neurokinin-1 receptor (NK-1) in the SCN and IGL of rat and determined whether the patterns of immunostaining generalized to the SCN and IGL of Syrian hamster, Siberian hamster, and mouse. Terminals immunoreactive for SP were sparse within the SCN of Siberian and Syrian hamsters and mouse but were intense in the ventral, retinally innervated portion of the rat SCN. Immunostaining for the NK-1 receptor was mainly absent from the SCN of hamster and mouse. In contrast, a plexus of NK-1-ir cells and processes that was in close proximity to SP-ir terminals was found in the ventral SCN of the rat. Substance P-ir terminals were observed in the IGL of all four species, as were NK-1-ir cells and fibres. Double-labelled IGL sections of hamster or rat revealed SP-ir terminals in close apposition to NK-1-immunostained cells and/or fibres. These data indicate that SP could be a neurotransmitter of the RHT in rat, but not in hamster or in mouse, and they highlight potential species differences in the role of SP within the SCN circadian pacemaker. Such species differences do not appear to exist at the level of the IGL, where SP-ir and NK-1-ir were similar in all species studied.     

Localization of cannabinoid receptor mRNA in rat brain

Cannabinoid receptor mRNA was localized in adult rat brain by ³⁵S-tailed oligonucleotide probes and in situ hybridization histochemistry. Labelling is described as uniform or non-uniform depending on the relative intensities of individual cells expressing cannabinoid receptor mRNA within a given region or nucleus. Uniform labelling was found in the hypothalamus, thalamus, basal ganglia, cerebellum and brainstem. Non-uniform labelling that resulted from the presence of cells displaying two easily distinguishable intensities of hybridization signals was observed in several regions and nuclei in the forebrain (cerebral cortex, hippocampus, amygdala, certain olfactory structures). Olfactory-associated structures, basal ganglia, hippocampus, and cerebellar cortex displayed the heaviest amounts of labelling. Many regions that displayed cannabinoid receptor mRNA could reasonably be identified as sources for cannabinoid receptors on the basis of well documented hodologic data. Other sites that were also clearly labelled could not be assigned as logical sources of cannabinoid receptors. The localization of cannabinoid receptor mRNA indicates that sensory, motor, cognitive, limbic, and autonomic systems should all be influenced by the activation of this receptor by either exogenous cannabimimetics, including marijuana, or the yet unknown endogenous “cannabinoid” ligand. © 1993 Wiley-Liss, Inc.     

Different expressions of α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid and N-methyl-D-aspartate receptor subunit mRNAs between visceromotor and somatomotor neurons of the rat lumbosacral spinal cord

The glutamatergic transmission system plays a key role in afferent and efferent pathways involved in micturition. By in situ hybridization combined with retrograde Fast Blue labeling, expression of α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor (GluR-A to -D) and N-methyl-D -aspartate (NMDA) receptor (NR1 and NR2A-D) subunit mRNAs were examined in visceromotor and somatomotor neurons of the rat lumbosacral spinal cord. Parasympathetic preganglionic neurons (PGNs) in the intermediolateral nucleus highly expressed GluR-A and GluR-B subunit mRNAs, with very low levels for GluR-C and GluR-D subunits. As for the NMDA receptor, PGNs were associated with abundant signals for NR1 subunit mRNA, but without any NR2 subunit mRNAs. On the other hand, somatomotor neurons in the ventral horn (dorsolateral nucleus) express all four AMPA receptor subunit mRNAs, showing relatively abundant expressions of GluR-C and GluR-D subunit mRNA compared with PGNs. In addition to high levels of NR1 subunit mRNA, dorsolateral nucleus neurons moderately expressed NR2A and NR2B subunit mRNAs. These results suggest that molecular organization of both AMPA and NMDA receptor channels are distinct between PGNs and dorsolateral nucleus neurons. Considering that native NMDA receptors are heteromeric channels composed of NR1 and NR2 subunits, it seems likely that dorsolateral nucleus neurons, not PGNs, are provided with functional NMDA receptors, which could induce activity-dependent changes in synaptic transmission in the efferent pathway for the lower urinary tract. J. Comp. Neurol. 404:172–182, 1999. © 1999 Wiley-Liss, Inc.     

Changes in preoptic and hypothalamic levels of progesterone receptor mRNA across the oestrous cycle of the ewe

We measured the levels of progesterone receptor (PR) mRNA in the hypothalamus and preoptic area (POA) of the ewe across the oestrous cycle. Perfusion-fixed hypothalamic tissue was collected from sheep killed during the luteal and follicular phases and during behavioural oestrus. Blood samples taken at the time of tissue collection verified that the oestrous ewes were undergoing a preovulatory luteinizing hormone (LH) surge. Matched sections were taken from the POA, periventricular nucleus (PeVN), ventromedial nucleus (VMN) and arcuate nucleus of each animal. In situ hybridization was performed using a sheep specific, (35)S-labelled riboprobe for PR and semiquantitative image analysis was conducted on emulsion-dipped slides. The number of silver grains per cell was greater in the VMN and arcuate nucleus of oestrous ewes than in luteal and follicular phase ewes; there was no cyclic variation in the level of PR mRNA expression in the cells of the POA and PeVN. The number of labelled cells per mm2 in the VMN was higher in the oestrous ewes than in luteal phase and follicular phase ewes. The number of labelled cells in the PeVN was also higher in the oestrous ewes than in follicular phase ewes, but there was no cyclic variation in the POA and arcuate nucleus. In the ewe, the onset of behavioural oestrus corresponds to the onset of the preovulatory LH surge and increased PR mRNA expression at this time is likely to be due to the high concentrations of circulating oestrogen that precede this period.     

Glucocorticoid receptor mRNA in Alzheimer's diseased hippocampus

Hypothalamic-pituitary-adrenal (HPA) axis dysfunction is a common finding in Alzheimer's dementia. Since there is loss of hippocampal corticosteroid receptors in animal models of aging, and since hippocampal cell loss occurs in Alzheimer's disease (AD), it has been suggested that a loss of hippocampal glucocorticoid receptors (GR) may underlie some aspects of HPA axis dysfunction in patients with AD. Levels of corticosteroid receptor protein are not reliably determined in postmortem human brain due to rapid lability. In contrast, levels of mRNA coding for GR are stable in postmortem tissue. We report here initial observations from in situ hybridization experiments which indicate that regional levels of glucocorticoid receptor mRNA in hippocampus, as determined by film autoradiography, are significantly higher in AD hippocampus than in controls. While neuronal levels of GR mRNA in AD, revealed by emulsion autoradiography, were equal in control and AD tissue. Taken together these results suggest that adrenal dysfunction in AD may relate to defects in receptor function rather than corticosteroid receptor loss in the hippocampus.     

Developmental regulation of two isoforms of Ca/calmodulin-dependent protein kinase I β in rat brain

Subtractive hybridization analysis of region-specific gene expression in brain has demonstrated a mRNA species enriched in rat hypothalamus [K.M. Gautvik, L. de Lecea, V.T. Gautvik, P.E. Danielson, P. Tranque, A. Dopazo, F.E. Bloom, J.G. Sutcliffe, Proc. Natl. Acad. Sci. USA 93 (1996) 8733–8738.]. We here show that this mRNA encodes a Ca²⁺/calmodulin-dependent (CaM) kinase belonging in the CaM kinase I β subgroup. cDNA analysis showed that this enzyme was differentially spliced into two isoforms (designated β1 and β2) with distinct C-termini. The C-terminal of the translated CaM kinase I β2 protein (38.5 kDa molecular size), contained 25 amino acid residues not present in the β1 isoform. The two isoforms were differentially developmentally regulated, with the β1 isoform being present in rat embryos from day 18 and the β2 isoform being present from day 5 postnatally. In situ hybridization analysis of adult rat CNS showed CaM kinase I β2 mRNA being enriched in the hypothalamus and the hippocampal formation. Expression was also observed in a number of ventral limbic structures and in the thalamus. Northern blot analysis showed additional expression of multiple β2 isoforms in heart and skeletal muscle. The human mRNA showed a similar distribution. Our data suggest that the two isoforms of CaM kinase I β, created by a splicing process occurring within a week around birth, may have distinct pre- and postnatal functions in a distinct set of CNS neurons and excitable tissues.