A secretin i.v. infusion activates gene expression in the central amygdala of rats

For the last 100 years secretin has been extensively studied for its hormonal effects on digestion. Recent observations that the deficits in social reciprocity skills seen in young (3-4-year-old) autistic children are improved after secretin infusions suggest an additional influence on neuronal activity. We show here that i.v. administration of secretin in rats induces Fos protein expression in the neurons of the central amygdala as well as the area postrema, bed nucleus of the stria terminalis, external lateral parabrachial nucleus and supraoptic nucleus. However, secretin infusion did not induce Fos expression in the solitary tract nucleus or paraventricular nucleus, regions normally activated by related peptides such as cholecystokinin. The peak blood levels of secretin that induce Fos protein expression in rat brain are similar to the peak blood levels observed during i.v. treatment with secretin in humans. The amygdala is known to be critical for developing reciprocal social interaction skills and abnormalities in this brain region have been demonstrated in autistic children.     

Impaired hippocampal synaptic function in secretin deficient mice

Secretin is a gut peptide hormone that is also expressed in the CNS. To explore the potential neuroactive role of secretin in the brain, we have generated secretin deficient mice. Secretin deficient mice demonstrated impairment in synaptic plasticity (significant reduction in long term potentiation (LTP) induction and LTP maintenance) in the CA1 area of the hippocampus. Using a beta-galactosidase (lacZ) reporter in the targeted allele and secretin antibody staining, we have detected secretin gene expression in the hippocampus, cerebellum, and the brain stem in adult mouse brain. In the hippocampus, secretin was expressed in the dentate gyrus, the hilus, and the molecular layer. These findings suggest that secretin is involved in synaptic function in the adult brain.     

Ontogeny of neurotensin-containing neuron system of the rat: immunohistochemical analysis--II. Lower brain stem

The ontogeny of the neurotensin neuron system in the lower brain stem of the rat was investigated by means of indirect immunofluorescent method. Neurotensin-like immunoreactivity-containing cells first appear in the primordium of the n. tractus solitarii, n. tractus spinalis nervi trigemini, reticular formation just medial to the latter nucleus, n. reticularis parvocellularis, n. laterodorsalis tegmenti, and midbrain reticular formation of the fetus at gestational day 17. At gestional day 18, neurotensin-immunoreactive cells newly appear in the n. raphe dorsalis. Between gestational day 19 and postnatal day 7, the animals show a remarkable increase in number of immunoreactive cells and fibers in various lower brain stem areas except for n. tractus spinalis nervi trigemini and n. tractus solitarii. Moreover, during this stage, neurotensin-immunoreactive cells located in the n. prepositus hypoglossi and n. vestibularis lateralis appear for the first time at birth and postnatal day 5, respectively. Since postnatal day 7, although the majority of immunoreactive cells located in the lower brain stem decrease in number as the rats grow, immunoreactive cells in the n. tractus spinalis nervi trigemini, on the contrary, increase in number from after birth until postnatal day 10, and maintain more or less their immunoreactivity even in the adult rat. In addition, neurotensin-immunoreactive cells in the nucleus of the solitary tract increase in number during the fetal period, reach the maximum content at postnatal day 7-10, and maintain their immunoreactivity even in the adult rats. Thus, the present study demonstrated that neurotensin-like immunoreactive structures appear at a very early ontogenetical stage, suggesting that neurotensin plays an important role in the development of the lower brain stem of the rat. In addition, the present study further showed that neurotensin-immuno-reactivity shows various fluctuations during the ontogeny, suggesting multiple functions of neurotensin in the central nervous system.     

Endogenous release and multiple actions of secretin in the rat cerebellum

Previous studies demonstrated that secretin could modulate synaptic transmission in the rat cerebellum. In the present report, we provide evidence for the endogenous release of secretin in the cerebellum and further characterize the actions of secretin in this brain area. First, to show that secretin is released endogenously, blocks of freshly dissected cerebella were challenged with a high concentration of KCl. Incubation with KCl almost doubled the rate of secretin release. This KCl-induced release was sensitive to tetrodotoxin and cadmium suggesting the involvement of voltage-gated sodium and calcium channels. The use of specific channel blockers further revealed that L-type and P/Q-type calcium channels underlie both basal and KCl-evoked secretin release. In support of this, depolarization of Purkinje neurons in the presence of NMDA, group II mGluR and cannabinoid CB1 receptor blockers resulted in increased inhibitory postsynaptic current frequency. Second, we found that the previously reported facilitatory action of secretin on GABAergic inputs to Purkinje neurons is partly dependent on the release of endogenous glutamate. In the presence of CNQX, an AMPA/kainate receptor antagonist, the facilitatory effect of secretin on GABA release was significantly reduced. In support of this idea, application of AMPA, but not kainate receptor agonist, facilitated GABA release from inhibitory terminals, an action that was sensitive to AMPA receptor antagonists. These data indicate that a direct and an indirect pathway mediate the action of secretin in the basket cell-Purkinje neuron synapse. The results provide further and more solid evidence for the role of secretin as a neuropeptide in the mammalian CNS.     

大鼠小脑和下橄揽核中含Calbindin－D28KmRNA的神经元的生后发育

本实验应用原位杂交组织化学方法观察了大鼠小脑皮质和下橄榄核中含Ｃａｌｂｉｎｄｉｎ－Ｄ２８ＫｍＲＮＡ的神经元的生后发育过程。发现在刚出生时，小脑浦肯野氏细胞已含Ｃａｌｂｉｎｄｉｎ－Ｄ２８ＫｍＲＮＡ，其表达水平在生后第３周时达高峰并持续至成年期。但在下橄榄核中，含Ｃａｌｂｉｎｄｉｎ－Ｄ２８ＫｍＲＮＡ的神经元在生后第７天时才出现，其数量及标记强度在生后第３、４周时迅速增加，并达成年水平。结合以往的资料分析，在小脑中，Ｃａｌｂｉｎｄｉｎ－Ｄ２８Ｋ可能与浦肯野氏细胞的成熟（突起的形成及伸长、突触的形成）过程有关。而在下橄榄核中，Ｃａｌｂｉｎｄｉｎ－Ｄ２８Ｋ主要参与成年期神经元的正常生理功能。     

In situ localization of tau mRNA in developing rat brain

A microtubule-associated protein, tau, promotes microtubule assembly, forms characteristic short cross-bridges (less than 20 nm) between microtubules, and switches isoforms from juvenile to adult at the end of the first postnatal week in the rat brain. The developmental expression of tau was studied in rat central nervous system, mainly the cerebrum and cerebellum, by in situ hybridization. Tau mRNAs were localized in a wide variety of neural cells. The expression of tau mRNAs in the spinal cord appeared to precede that in the brain, and the expression in the brainstem appeared to precede that in the cerebral cortex and cerebellum. On neural cells throughout the cortical plate of the cerebral cortex, tau mRNAs were expressed in large amounts during the first postnatal week, but by the third postnatal week the expression had become reduced. In the cerebellum, tau mRNAs were enriched in granule cells. The expression in the internal granular layer peaked during the second and third postnatal weeks, and the relatively high level of expression persisted to young adulthood. Thin section transmission electron microscopic study revealed that the proportion of neighboring microtubules in parallel fiber axons of cerebellar granule cells with the distance less than 20 nm was as low as 10% at the end of the first postnatal week, but this proportion increased to as high as 35% at the end of the second postnatal week. Northern blot analysis showed that tau mRNAs were congruent to 6 kb as was reported previously, and those detected in the first postnatal week were three- to five-fold more abundant and approximately 0.2 kb smaller than those detected in the second or third postnatal weeks. The data suggest that (a) tau mRNAs are abundantly expressed in a wide variety of neurons in the central nervous system at the stage of neurite formation, and (b) tau mRNAs are expressed in more basal levels at later stages, but may be important in the formation and maintenance of characteristic microtubule bundles typically found in parallel fiber axons and in other axons.     

Stress induces the expression of heterotrimeric G protein beta subunits and the phosphorylation of PKB/Akt and ERK1/2 in rat brain

Various heterotrimeric G protein betagamma subunits (Gbetagamma) are region-specifically expressed in brain where associated with "stress-axis", however, the role of Gbetagamma-mediated signaling in regulating stress is unknown. This study was designed to examine the changes of Gbetagamma expression and Gbetagamma-mediated signaling in rat brain by stress. Experimental stress was induced by immobilization (2h/day for 7 days) and the level of mRNAs and proteins for Gbeta(1-5), and the phosphorylation of PKB/Akt (phosphatidylinositol 3-kinase-linked protein kinase B) and ERK1/2 (extracellular signal-regulated kinase 1/2) were measured in five different regions of rat brain including frontal cortex, striatum, hypothalamus, hippocampus, and cerebellum. As compared in not-handled non-stressed animals, the expression of both mRNAs and proteins for Gbeta(1-5) in brain regions associated with stress was increased in stressed animals. Especially, a significant increase in Gbetas immunoreactivity in the caudate putamen, the paraventricular nucleus of the hypothalamus (PVN), and the dentate gyrus of the hippocampus (DG) of stressed rats was observed. Stress significantly induced the phosphorylation of PKB/Akt and ERK1/2 in striatum, hypothalamus and hippocampus. Therefore, these results suggest that stress may activate, at least in part, the Gbetagamma-mediated PKB/Akt and ERK1/2 signaling pathway by increasing the expression of Gbetas to regulate the physiological responses.     

Alpha-2 adrenergic receptor development in rat CNS: an autoradiographic study

During development norepinephrine plays a role in determining the morphologic organization of the CNS and the density and future responsiveness of adrenergic receptors. alpha-2 Adrenergic receptors, one of three adrenergic receptor types, regulate important adult CNS functions and may have a distinct role during development. We examined alpha-2 receptor distribution and density in the rat brain at postnatal days 1, 5, 10, 15, 21, 28 and in adults using the antagonist [(3)H]RX821002 for autoradiography. Binding kinetics and pharmacology for alpha-2 adrenergic receptors were the same in adults and neonates. There was an overall increase in alpha-2 receptor levels during postnatal development with great variability in pattern and timing of receptor density changes among brain regions. Three major patterns were apparent. First, in many regions receptor density increased during postnatal development, generally reaching adult levels around postnatal day 15. Within this group there was variability in timing between regions and there were several regions with receptor densities higher than adult levels during the postnatal period. Second, there were regions with very high levels of receptors at birth and little or no change in density during the postnatal period. Third, some regions demonstrated decreasing or transient expression of alpha-2 adrenergic receptors in the course of postnatal development, including white matter regions, cerebellum and many brainstem nuclei, suggesting specific roles for alpha-2 receptors during development.This study investigates the development of alpha-2 adrenergic receptors in the rat CNS. It demonstrates there is region-specific regulation of alpha-2 receptor development and identifies brain regions where these receptors may play a specific and critical role in the regulation normal development.     

Distribution of the rhythm-related genes rPERIOD1, rPERIOD2, and rCLOCK,in the rat brain

High densities of mRNAs for three rhythm-related genes, rPeriod1 (rPer1), rPer2, and rClock, which share high homology in Drosophila and mice, were found in the hypothalamic suprachiasmatic nucleus (SCN). The SCN, however, is not the only brain region that expresses these genes. To understand the distributions and possible physiological roles of these rhythm-related genes, we examined the gene expressions of rPer1, rPer2, and rClock in different brain regions by serial coronal, sagittal, and horizontal brain sections in Sprague-Dawley male rats. Animals were housed in a light-controlled room (lights on from 0600 to 1800 h) and killed at 1000 or 1200 h, which corresponds to Zeitgeber time 4 or 6. Semi-quantitative in situ hybridization with (35)S-riboprobes was used to evaluate mRNA levels. The mRNAs of rPer1, rPer2, and rClock were widely distributed in the rat CNS, including the olfactory bulb, cortex, piriform cortex, SCN, ventromedial hypothalamus, arcuate nucleus, hippocampus, mammillary nucleus, pontine nucleus, superior and inferior colliculus, cerebellum, median eminence/pars tuberalis, pineal gland, and pituitary. The expression patterns of mRNAs for rPer1 and rPer2 were almost identical. In contrast, different expression patterns were observed between rClock and rPer1 or rPer2 in several brain regions, including the hypothalamic supraoptic and suprachiasmatic nuclei, the paraventricular zone of the caudate putamen, the superior olivary nucleus, and anterior and intermediate lobes of the pituitary. These findings suggest that the different expression patterns observed for rPer1, rPer2, and rClock might be due to their different physiological role(s) in those brain regions.     

Postnatal differentiation of local networks in the nucleus of the tractus solitarius

Whole-cell voltage-clamp recordings from rat brain slice preparation were used to investigate a possible developmental change in the patterns of synaptic interactions among the nucleus tractus solitarii neurons by analysing spontaneous postsynaptic current activity. Three types of patterns of spontaneous postsynaptic current activity were distinguished in the nucleus tractus solitarii neurons which showed high activities in terms of current frequency and amplitude. The first type was characterized by the presence in an individual cell of high frequencies and large amplitudes of both spontaneous glutamatergic and GABAergic postsynaptic currents, and observed exclusively in postnatal day 0-7 rats. The second and third types of cells showed predominant either inhibitory or excitatory postsynaptic currents, respectively. After postnatal day 5, nucleus tractus solitarii neurons with high background activity were shown to differentiate into either the second or the third type, with the latter of about 70% in the adult caudal/intermediate nucleus tractus solitarii. Axon collaterals of some medium to large neurons seemed to be decreased by pruning during postnatal development.The early postnatal differentiation of background synaptic activity observed in the nucleus tractus solitarii presumably reflects the local network reorganization and may be related to maturational changes in cardiovascular and respiratory functions.     

含GABA_A受体α_6亚单位mRNA神经元在大鼠脑内的生后发育

用原位杂交组织化学方法观察了大鼠小脑皮质和耳蜗核中含ＧＡＢＡＡ受体α６亚单位ｍＲＮＡ神经元的生后发育过程。结果发现小脑在生后发育中，杂交信号在生后第５ｄ最早出现于内颗粒层，在生后第２１ｄ达到高峰并持续至成年期。但在外生发展始终未见到阳性信号。在耳蜗核中，α６亚单位ｍＲＮＡ到生后第７ｄ方出现，其表达水平在此后的阶段内迅速增加，从生后第１４ｄ开始，杂交信号的增强趋于缓慢，至生后第３ｗ达到成年水平。α６亚单位ｍＲＮＡ在小脑及耳蜗核的生后发育早期即有较强表达，提示其与上述两个系统的成熟过程有关。     

Regional and temporal expression of sodium channel messenger RNAs in the rat brain during development

Summary The distribution of mRNA expression for three types of voltage gated neuronal sodium-channels was studied in the rat brain at different developmental stages (embryonal day E18, postnatal day P5 and adult). With the in-situ hybridization technique, using synthetic DNA-oligomer probes, pronounced regional and temporal variations in the expression levels of the different channel subtypes could be detected. In comparison with types I and III, sodium channel II mRNA was the most abundant subtype at all developmental stages. Maximal expression of sodium channel II mRNA was seen at P5 in virtually all parts of the grey matter, except for the cerebellum. In adult rat brain in contrast, sodium channel II mRNA levels were maximal in the granular layer of the cerebellum, whereas in all other regions expression had decreased to roughly 50% of postnatal levels. Na channel I expression was virtually absent at E18 and showed highest levels at P5, with maxima in the caudate nucleus and hippocampus. In the adult brain, expression of Na-channel I was nearly absent in the neocortex, but well detectable in the cerebellum and, at lower levels in the striatum and thalamus. Sodium channel III was mainly expressed at the embryonal stage and showed a decrease to very low levels with little regional preferences in the adult.Supported by Deutsche Forschungsgemeinschaft grant no.: Cr 30/16     

Region-specific expression of messenger RNAs encoding GABAA receptor subunits in the developing rat brain.

The distribution and levels of messenger RNAs encoding the alpha 1, beta 1, beta 2, beta 3, and gamma 2 subunits of the GABAA receptor in the developing and adult rat brain were investigated using quantitative in situ hybridization histochemistry and subunit-specific probes. Regional localization of the subunit messenger RNAs was determined with film autoradiography and expression in identified neuronal cell populations was examined using higher resolution techniques. Each of the GABAA receptor subunit messenger RNAs exhibits a distinct pattern of localization in the developing and adult brain. Of the subunits examined, the alpha 1, beta 2, and gamma 2 are the most abundant and are found in many brain regions, including the olfactory bulb, cortex, hippocampus, thalamic nuclei, and inferior colliculus. In addition, these subunit messenger RNAs are prominent in the cerebellum where virtually all cells of the deep cerebellar nuclei and Purkinje cell layer are labeled. The levels of most of the subunit messenger RNAs, with the exception of that encoding the beta 1 subunit, increase during postnatal development. While the alpha 1, beta 2, and gamma 2 subunit messenger RNAs rise in parallel in many regions and identified cell populations, different subsets of receptor subunit messenger RNAs are co-ordinately expressed at other sites. The greatest increases in subunit messenger RNA levels occur in the cerebellar cortex during the second postnatal week, a period coincident with cerebellar maturation. The co-distribution of different GABAA receptor subunit messenger RNAs in various regions of the developing and adult nervous systems supports the hypothesis that multiple receptor compositions exist. Moreover, that different subunit messenger RNAs exhibit coordinate changes in expression in different regions and cell populations suggests that receptor gene expression is modulated by cell type-specific signals. The temporal changes in subunit messenger RNA levels in the cerebellum raise the possibility that synaptogenesis may play a role in receptor gene regulation in this brain region.     

Muscarinic acetylcholine receptor subtype mRNA expression and ligand binding in the aged rat forebrain

Previous studies indicate that a 20-30% decline in muscarinic acetylcholine receptor binding occurs in localized areas of rat brain during aging. In this study, reduced [3H]-quinuclidinyl benzilate binding was observed in striata from 24-25-month-old rats relative to 5-6-month-old animals using homogenate binding assays. To determine if the decline in receptor concentration occurs as a result of decreased receptor synthesis, the expression of the m1, m3, and m4 muscarinic receptor mRNAs as well as [3H]-QNB binding were determined in adjacent sections of young and old male rats using in situ hybridization and in vitro receptor autoradiography respectively. A significant decline in collective muscarinic receptor binding as assessed by [3H]-QNB was observed in the caudate putamen, olfactory tubercle, nucleus accumbens, and several frontal and parietal cortical areas. The only difference observed in muscarinic mRNA expression for any of the three subtypes examined was a decline in m1 hybridization in the olfactory tubercle. The results of this study demonstrate that the regional brain areas displaying age-related decreases in receptor binding do not correlate with those areas showing a decrease in muscarinic receptor expression. Apparently, the decline in muscarinic acetylcholine receptor density with age does not result from a decline in receptor gene expression.     

Expression of mKirre, a mammalian homolog of Drosophila kirre, in the developing and adult mouse brain

mKirre, a mammalian homolog of the Drosophila kirre, is expressed in bone marrow stromal cells and the brain. Although mKirre has been shown to support the hematopoietic stem cells, little is known about the function of mKirre in the brain. In the present study, to gain insights into the function of mKirre, we investigated the expression pattern of mKirre gene in the developing and adult mouse brain using in situ hybridization. In the adult brain, mKirre mRNA was highly expressed in the olfactory bulb, the piriform cortex, the cochlear nucleus, and the cerebellum. At embryonic day (E) 11.5, we could observe mKirre mRNA in the differentiating zones of various regions, such as the caudate-putamen, the geniculate body, the thalamus, the amygdala, and the brainstem. Its gene expression in these regions at E11.5 also persisted to the adult, in which its expression levels were much less prominent. After birth, we could first observe high expression of mKirre mRNA in the glomerular and mitral layers of the olfactory bulb, the cortical plate of the neocortex, the cochlear nucleus, and the molecular and granule cell layers of the cerebellum. In the hippocampus, its gene expression was first observed in the dentate gyrus at postnatal day 7. The spatiotemporal expression pattern of mKirre mRNA suggests important roles of mKirre in later developmental processes, especially the synapse formation.     

Differential messenger RNA distribution of lactate dehydrogenase LDH-1 and LDH-5 isoforms in the rat brain

The role of lactate in brain energy metabolism has recently received renewed attention. Although blood-borne monocarboxylates such as lactate poorly cross the blood-brain barrier in the adult brain, lactate produced within the brain parenchyma may be a suitable substrate for brain cells. Lactate dehydrogenase is crucial for both the production and utilization of lactate. In this article, we report the regional distribution of the messenger RNAs for lactate dehydrogenase isoforms 1 and 5 in the adult rat brain using in situ hybridization histochemistry with specific [alpha-(35)S]dATP 3' end-labeled oligoprobes. The autoradiographs revealed that the lactate dehydrogenase-1 messenger RNA is highly expressed in a variety of brain structures, including the main olfactory bulb, the piriform cortex, several thalamic and hypothalamic nuclei, the pontine nuclei, the ventral cochlear nucleus, the trigeminal nerve and the solitary tractus nucleus. In addition, the granular and Purkinje cell layers of the cerebellum showed a strong labeling. The neocortex (e.g., cingular, retrosplenial and frontoparietal cortices) often exhibits a marked laminar pattern of distribution of lactate dehydrogenase-1 messenger RNA (layers II/III, IV and VI being most strongly labeled). In contrast, expression of the lactate dehydrogenase-5 messenger RNA generally seemed more diffusely distributed across the different brain regions. Expression was particularly strong in the hippocampal formation (especially in Ammon's horn and dentate gyrus) and in the cerebral cortex, where no laminar pattern of distribution was observed. Overall, these data are consistent with the emerging idea that lactate is an important energy substrate produced and consumed by brain cells.