Na,K-ATPase: comparison of the cellular localization of alpha-subunit mRNA and polypeptide in mouse cerebellum, retina, and kidney

A clone encoding mouse brain Na,K-ATPase alpha-subunit was isolated from a mouse brain lambda gt11 cDNA library by using antisera to mouse and bovine brain alpha-subunit. A comparison of the nucleotide sequence of this clone with published sequences of rat brain alpha-subunit isoform clones showed it to be most similar to rat brain alpha 1. An RNA antisense probe prepared from the cDNA insert of the mouse clone detected a single mRNA of approximately 4.5 kb in Northern blots of mouse brain and kidney RNAs. This probe hybridized only to an alpha 1-cDNA insert from rat brain under high stringency conditions on Northern blots. The RNA antisense probe was used for in situ hybridization to sections of mouse kidney, cerebellum, and retina, and the cellular distribution of alpha-subunit mRNA (alpha-mRNA) was compared with that of alpha-subunit polypeptide (alpha-subunit) detected by immunofluorescence in similar sections. In kidney, alpha-mRNA distribution closely paralleled that of the polypeptide with abundant expression in ascending thick limbs and cortical distal tubules and weaker labeling in cortical proximal tubules. The co-distribution of alpha-mRNA and polypeptide in kidney where Na,K-ATPase localization is well established is consistent with the specificity of these probes. In the retina, prominent labeling with both probes was seen in photoreceptor inner segments, inner nuclear layer, and ganglion cell bodies. Plexiform layers and optic fibers expressed abundant alpha-subunit but little mRNA. Light labeling for both was seen in the outer nuclear layer. In cerebellum, alpha-mRNA and alpha-subunit were associated with soma of granule cells, basket cells, and stellate cells. Glomeruli and basket terminals contained abundant alpha-subunit but exhibited little reactivity with the riboprobe. In Purkinje cell bodies, in contrast, the antibody used to identify the cDNA clone did not resolve significant polypeptide in the somal plasmalemma despite abundant somal mRNA expression. Comparison of distribution of the two probes in cerebellum and retina indicates that message accumulation is primarily in cell bodies, while alpha-subunit epitopes may be co-expressed in cell bodies and/or transported to distant sites in cell-specific patterns.     

Intrastriatal Dopamine-rich Implants Reverse the Increase of Dopamine D2 Receptor mRNA Levels Caused by Lesion of the Nigrostriatal Pathway: A Quantitative In Situ Hybridization Study

Changes in striatal dopamine D2 receptor mRNA levels provoked by unilateral 6-hydroxydopamine-induced lesion of the nigrostriatal dopamine pathway were studied by in situ hybridization. The influence of embryonic dopaminergic neurons implanted into the dopamine-depleted striatum on the lesion-induced changes was also examined. Changes in D2 mRNA levels were compared with changes in D2 receptor densities measured in the same animals by receptor autoradiography using [3H]spiperone or [3H]SDZ 205-501 as ligands. The distribution of D2 mRNA in the striatum of control animals closely paralleled that of the D2 receptor itself, as assessed by autoradiography, and the highest density of D2 mRNA occurred in the lateral part of the striatum. One month after lesion, levels of D2 mRNA were 34% higher in the dorsolateral part of the dopamine-depleted striatum than in the corresponding region of the contralateral control striatum. D2 receptor density in this region was increased by 40% relative to the control level. No significant increases could be measured in the medial part of the striatum. The increases in the lateral part were similar at 7 months post-lesion; however, at this time the increase in both D2 mRNA and receptor levels had spread to the medial part of the striatum as well. In the graft-bearing striatum levels of both D2 mRNA and D2 receptors reverted to control levels. This study shows that the post-lesion increase in striatal dopamine receptor and mRNA level is a biphasic phenomenon with a late-occurring component in the medial striatum. It also shows that once the increase in striatal D2 receptor gene expression is accomplished, it is maintained unchanged for long periods, similar to that of D2 receptor levels themselves. Moreover, grafts of embryonic dopaminergic neurons are able to modulate the expression of the dopamine D2 receptor gene.     

Tumor suppressor gene BRCA-1 is expressed by embryonic and adult neural stem cells and involved in cell proliferation

BRCA-1 is a tumor suppressor gene that plays a role in DNA repair and cellular growth control. Here we show that BRCA-1 mRNA is expressed by embryonic rat brain and is localized to the neuroepithelium containing neuronal precursor cells. The expression of BRCA-1 decreases during rat brain development, but BRCA-1 is expressed postnatally by proliferating neuronal precursor cells in the developing cerebellum. Neural stem cells (NSC) prepared from embryonic rat brain and cultured in the presence of epidermal growth factor were positive for BRCA-1. Induction of NSC differentiation resulted in down-regulation of BRCA-1 expression as shown by RNA and protein analyses. In addition to embryonic cells, BRCA-1 is also present in NSC prepared from adult rat brain. In adult rats, BRCA1 was expressed by cells in the walls of brain ventricles and in choroid plexus. The results show that BRCA-1 is present in embryonic and adult rat NSC and that the expression is linked to NSC proliferation.     

Phenotypes and peripheral mechanisms underlying inflammatory pain-related behaviors induced by BmK I, a modulator of sodium channels

The integrated mechanisms of dynamic signaling of sodium channels involved in clinical pain are still not yet clear. In this study, a new rat inflammatory pain model was developed by using the unilateral intraplantar injection of BmK I, a receptor site 3-specific modulator of sodium channels from the venom of scorpion Buthus martensi Karsch (BmK). It was found that BmK I could induce several kinds of inflammatory pain-related behaviors including spontaneous pain companied with unique episodic paroxysms, primary thermal hypersensitivity, and mirror-image mechanical hypersensitivity with different time course of development, which could be suppressed by morphine, indomethacin, or bupivacaine to a different extent. The dramatic attenuation by pretreatment with resiniferatoxin (RTX), an ultrapotent analog of capsaicin, on BmK I-induced pain-related behaviors, paw edema, and spinal L4-L5 c-Fos expression demonstrated that capsaicin-sensitive primary afferent neurons played important roles in pain induced by BmK I. Furthermore, the electrophysiological recordings showed that BmK I persistently increased whole-cell and tetrodotoxin-resistant (TTX-R) peak sodium currents and significantly delayed the inactivation phase of whole-cell sodium currents but could not enhance capsaicin-evoked inward currents, in acute isolated small dorsal root ganglion neurons of rat. The results strongly suggested that the dynamic modulation of BmK I on sodium channels located in peripheral primary afferent neurons, especially in capsaicin-sensitive neurons, mediated pain sensation. Thus, BmK I may be a valuable pharmacological tool to understand the sodium channel-involved pain mechanisms.     

Differential steroid hormone and neural influences on peptide mRNA levels in CRH cells of the paraventricular nucleus: a hybridization histochemical study in the rat

The three major classes of neurons in the paraventricular nucleus (PVH) provide a rich model for studying hormonal and neural influences on multiple neuropeptides expressed in individual cells. A great deal of previous work has examined this problem at the immunohistochemical level, where hormonal and neural influences on peptide levels have been established. In situ hybridization methods were used here to determine whether these effects are accompanied by measurable changes in neuropeptide mRNA levels. In the first series of experiments, the time-course of corticosterone replacement effects on corticotropin-releasing hormone (CRH) mRNA levels in parvicellular neuroendocrine cells of adrenalectomized animals were determined, and a dose-response curve was established. CRH mRNA hybridization remains maximal with plasma levels of steroid up to about 50 ng/ml, then declines sharply between about 60-130 ng/ml, and is just detectable at higher levels. We confirmed that corticosterone decreases vasopressin mRNA levels in this cell group and showed that levels of preproenkephalin mRNA are also decreased, whereas no significant changes in cholecystokinin, beta-preprotachykinin, and angiotensinogen mRNA levels could be detected. Thus, corticosterone decreases some neuropeptide mRNA levels and has no influence on others in this cell group. Tyrosine hydroxylase mRNA hybridization is also unaffected in this part of the nucleus. In a second group of experiments, the cell-type specificity of corticosterone influences was examined. It was found that while the hormone depresses CRH mRNA levels in parvicellular neurons, it increases such levels in PVH neurons with descending projections, in certain magnocellular neurosecretory neurons, and in a part of the central nucleus of the amygdala, whereas no influence was detected in the rostral lateral hypothalamic area. Furthermore, the stimulatory effects of corticosterone have different threshold levels in different cell groups. Thus, in different types of neurons, corticosterone may increase, decrease, or have no influence on CRH mRNA levels. In contrast, while corticosterone depresses vasopressin mRNA levels in parvicellular CRH neurons, it has no obvious effects on vasopressin mRNA levels in magnocellular or descending neurons; as with CRH, the effects of corticosterone on vasopressin mRNA levels are cell-type specific. In a third series of experiments it was shown that glucocorticoid receptor and mineralocorticoid receptor mRNAs are found in all three cell types in the PVH and that corticosterone tends to produce modest increases in mRNA levels for both receptors. Finally, it was shown that unilateral catecholamine-depleting knife cuts do not change mRNA levels for any of the neuropeptides (or steroid hormone receptors) examined here, although dramatic changes in neuropeptide levels themselves have been shown.4+     

Nerve growth factor and proprotein convertases furin and PC7 in transected sciatic nerves and in nerve segments cultured in conditioned media: Their presence in Schwann cells,macrophages, and smooth muscle cells

Synthesis of proteins such as nerve growth factor (NGF) is induced after nerve lesion. The NGF precursor (pro-NGF) requires a posttranslational processing by proprotein convertases to become active. In this report, we re-examine the localization of NGF protein and mRNA in injured nerve and show that the candidate pro-NGF convertases furin and PC7 colocalize with NGF in non-neuronal cells in nerve. By Northern blot analysis, 1.5-kb and 1.3-kb NGF mRNAs were shown to be increased in distal and immediately proximal nerve segments on days 1, 4, and 14 after lesion; by Western blot analysis, NGF proteins of high molecular weight were detected after injury. In vivo, two phases of NGF immunopositivity were observed, in macrophages and perivascular cells shortly after lesion and in endoneurial cells on day 1 and 4. To identify the cells containing NGF, nerve segments were incubated in serum-containing medium with or without conditioning by white blood cells isolated from the circulation. Both hybridization and immunoreactivity signals for NGF were elevated after incubation of nerve segments for 4 hours in conditioned media, so that cells with NGF immunoreactivity could be identified by antibodies to specific cell markers. In these nerve fragments, Schwann cells, perivascular smooth muscle cells, and macrophages contained NGF immunoreactivity. The concentration of furin and PC7 mRNA also increased in lesioned nerves. By immunocytochemical investigation of nerve explants, furin and PC7 were detected in endoneurial cells, macrophages and perivascular cells and were colocalized with NGF. These in vitro and in vivo findings suggest that both furin and PC7 are associated with NGF in several cell types of the sciatic nerve and, hence, may be implicated in intracellular processing of pro-NGF. J. Comp. Neurol. 403:471–485, 1999. © 1999 Wiley-Liss, Inc.     

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).     

Differential expression of 5HT-1A, alpha 1b adrenergic, CRF-R1, and CRF-R2 receptor mRNA in serotonergic, gamma-aminobutyric acidergic, and catecholaminergic cells of the rat dorsal raphe nucleus

The dorsal raphe nucleus (DR) has a topographic neuroanatomy consistent with the idea that different parts of this nucleus subserve different functions. Here we use dual in situ hybridization to describe the rostral-caudal neurochemical distribution of three major cell groups, serotonin (5-hydroxytryptamine; 5-HT), gamma-aminobutyric acid (GABA), and catecholamine, and their relative colocalization with each other and mRNA encoding four different receptor subtypes that have been described to influence DR responses, namely, 5HT-1A, alpha(1b) adrenergic (alpha(1b) ADR), and corticotropin-releasing factor type 1 (CRF-R1) and 2 (CRF-R2) receptors. Serotonergic and GABAergic neurons were distributed throughout the rostral-caudal extent of the DR, whereas catecholaminergic neurons were generally restricted to the rostral half of the nucleus. These phenotypes essentially represent distinct cell populations, because the neurochemical markers were rarely colocalized. Both 5HT-1A and alpha(1b) ADR mRNA were highly expressed throughout the DR, and the vast majority of serotonergic neurons expressed both receptors. A smaller percentage of GABAergic neurons also expressed 5HT-1A or alpha(1b) ADR mRNA. Very few catecholaminergic cells expressed either 5HT-1A or alpha(1b) ADR mRNA. CRF-R1 mRNA was detected only at very low levels within the DR, and quantitative colocalization studies were not technically feasible. CRF-R2 mRNA was mainly expressed at the middle and caudal levels of the DR. At midlevels, CRF-R2 mRNA was expressed exclusively in serotonin neurons, whereas, at caudal levels, approximately half the CRF-R2 mRNA was expressed in GABAergic neurons. The differential distribution of distinct neurochemical phenotypes lends support to the idea of functional differentiation of the DR.     

Characterization of the norepinephrine uptake system and the role of norepinephrine in the expression of the adrenergic phenotype by quail neural crest cells in clonal culture

This study investigates the role norepinephrine (NE) may play in regulating the differentiation of quail neural crest cells into sympathoadrenal cells. Cues originating from the embryonic microenvironment are thought to play an important role during development. It is conceivable that NE has a positive regulatory function because adrenergic expression by quail neural crest cells in clonal culture can be inhibited by NE uptake inhibitors such as desipramine (DMI). This possibility is further supported by the notion that in the avian embryo presumptive adrenergic neural crest cells are likely to encounter catecholamines shortly after they have acquired the NE uptake mechanism. Our present data indicate that neural crest cells in clonal culture express a high affinity NE uptake system that can be inhibited by desipramine. As in the embryo, it appears before noticeable levels of catecholamines are accumulated by neural crest cells, as judged by formaldehyde-induced catecholamine fluorescence (FIF). A comparison of the time course of appearance of different adrenergic markers suggests that immunoreactivity against the biosynthetic enzyme tyrosine hydroxylase (TH) may appear first, and that it is followed very closely by the appearance of detectable levels of dopamine-β-hydroxylase (DBH) and the NE uptake mechanism. Accumulation of catecholamines (FIF) is observed last. Addition of exogenous NE leads to an increase in adrenergic expression in vitro as judged by an increase in the number of colonies containing FIF-positive cells as well as cells expressing the biosynthetic enzymes TH and DBH. This suggests that exogenous NE can play a positive regulatory role in the differentiation of quail neural crest cells intossympathoadrenal cells.     

GABAA/benzodiazepine receptor α6 subunit mRNA in granule cells of the cerebellar cortex and cochlear nuclei: Expression in developing and mutant mice

The gamma aminobutyric acid_A/benzodiazepine (GABA_A/BZ) receptor is a multisubunit (α, β, γ, δ, and ρ) ligand-gated chloride channel; there are several variants of the α, β, and γ subunits, each of which has been localized throughout the central nervous system. A large number of GABA_A/BZ subunit variants are expressed within the cerebellar cortex. In previous studies from other laboratories, α₆ subunit mRNA has been reported to be present exclusively in cerebellar granule cells. The developmental expression of α₆ mRNA in cerebellar and cochlear granule cells is of interest because it has been suggested that each of these cell types is derived from a common precursor pool. The polymerase chain reaction was used to generate a cDNA fragment encoding a portion of the M3–M4 intracellular loop of the α₆ subunit of the GABA_A/BZ receptor. A [³⁵S] riboprobe, transcribed from this cDNA fragment, was used to examine the expression of the α₆ subunit mRNA by in situ hybridization in developing normal mice and in adult mutant mice with known deficits in synaptic circuitry. A strong hybridization signal was observed over the granule cell layers of both the cerebellum and cochlear nuclei in adult mice. The signal over the cochlear nuclei appeared after birth toward the end of postnatal week 1, coinciding with the appearance of labeling in the cerebellar cortex. The intensity of the hybridization signal in both regions increased rapidly until postnatal day 14, after which it increased more gradually, reaching adult levels during postnatal week 3. In the weaver mutant, α₆ labeling was detected in surviving granule cells, but not in cerebellar regions devoid of granule cells. Significant levels of the α₆ hybridization signal were also present in cerebellar granule cells of Purkinje cell degeneration, lurcher, and staggerer mutants, suggesting that aberrations in synaptic circuitry do not prevent α₆ subunit gene expression. Our results demonstrate that α₆ subunit mRNA is not limited to the cerebellum, but is expressed in other neurons which share a common cellular precursor pool. These data also suggest that these granule cell precursors may be intrinsically programmed to acquire a specific form of the GABA_A/BZ receptor, irrespective of their final location and lack of connections with target neurons. © 1994 Wiley-Liss, Inc.     

The close homologue of the neural adhesion molecule L1 (CHL1): patterns of expression and promotion of neurite outgrowth by heterophilic interactions

The close homologue of L1 (CHL1), a member of the L1 family of neural adhesion molecules, is first expressed at times of neurite outgrowth during brain development, and is detectable in subpopulations of neurons, astrocytes, oligodendrocyte precursors and Schwann cells of the mouse and rat. Aggregation assays with CHL1-transfected cells show that CHL1 does not promote homophilic adhesion or does it mediate heterophilic adhesion with L1. CHL1 promotes neurite outgrowth by hippocampal and small cerebellar neurons in substrate-bound and soluble form. The observation that CHL1 and L1 show overlapping, but also distinct patterns of synthesis in neurons and glia, suggests differential effects of L1-like molecules on neurite outgrowth.     

共聚焦激光扫描显微镜研究大鼠脑缺血谷氨酸载体GLAST mRNA和EAAT1的表达与其细胞分布

目的通过应用共聚焦激光扫描显微镜技术(confocal laser scanning microscope, CLSM)观察脑缺血后谷氨酸载体GLAST mRNA和EAAT1蛋白表达的细胞定位,探讨CLSM技术中三维重建和三维显示在观察基因和蛋白在神经细胞上空间定位的应用.方法对脑缺血后采用原位杂交和免疫组织化学相结合的荧光双标技术标记的脑片进行双通道断层扫描以及三维数据重组.结果脑缺血后,荧光免疫组化双标显示大脑皮质缺血半暗区内的谷氨酸载体EAAT1蛋白与星形胶质细胞和神经元均有共表达;原位杂交结合免疫组化的荧光双标显示缺血周边区谷氨酸载体GLAST mRNA与星形胶质细胞和神经元有明显的共表达.结论共聚焦激光扫描显微镜观察脑缺血后谷氨酸载体GLAST mRNA和EAAT1蛋白在神经胶质细胞和神经元上的空间定位,为进一步研究脑缺血后谷氨酸载体系统作用机制提供了更为准确的形态依据.     

Induction of heat shock (stress) genes in the mammalian brain by hyperthermia and other traumatic events: a current perspective

Is the heat shock response physiologically relevant? For example, following hyperthermia or ischemia, what neural cell types show induction of heat shock genes and what is the time course of the effect? Initial experiments in this area demonstrated the prominent Induction of a 70 kDa heat shock protein (hsp70) when labeled brain proteins isolated from hyperthermic animals were analyzed. Recently, in situ hybridization and immunocytochemistry have been utilized to map out the pattern of expression of both constitutively expressed and stress-inducible members of the hsp70 multigene family. Different types of neural trauma have been found to induce characteristic cellular responses in the mammalian brain with regard to the type of brain cell that responds by inducing hsp70 and the timing of the induction response. Fever-like temperature causes a dramatic induction of hsp70 mRNA within 1 hr in fiber tracts of the fore-brain and cerebellum, a pattern consistent with a strong glial response to heat shock. Tissue injury, namely, a small surgical cut in the cerebral cortex, induces a rapid and highly localized induction of hsp70 mRNA in cells proximal to the injury site. Using an immunocytochemical approach, a neuronal pattern of induction of hsp70 has been demonstrated following ischemia or kainic acid–induced seizures. It is apparent that the pattern of induction of hsp70 may be a useful early marker of cellular injury and may identify previously unrecognized areas of vulnerability in the nervous system.     

Detection of the 5-HT1A receptor and 5-HT1A receptor mRNA in the rat bowel and pancreas: Comparison with 5-HT1Preceptors

We tested the hypothesis that the rat bowel and pancreas contain 5-HT_1A receptors. ₃H-8-hydroxy-2-(di-n-propylamino) tetralin (₃H-8-OH-DPAT) was used as a radioligand. Binding of ₃H-8-OH-DPAT to membranes derived from the myenteric plexus and the pancreas was investigated by rapid filtration. Alternatively, radioautography was employed to locate ₃H-8-OH-DPAT binding sites in frozen sections of unfixed bowel or pancreas. An excess of 5-HT (10 μM) was used to define nonspecific binding. Saturable, high affinity binding of ₃H-8-OH-DPAT to enteric (K_d= 2.8 ± 1.1 nM; B_max =83.8 ± 4.3 fmol/mgproteim) and pancreatic (K_d = 6.6 ± 1.3 nM; B_max = 44 ± 2.2 fmol/mg protein) membranes was found. The binding of ₃H-8-OH-DPAT to enteric and pancreatic membranes was inhibited by 8-OH-DPAT, NAN-190, and spiperone. In contrast, the binding of ₃H-8-OH-DPAT to enteric and pancreatic membranes was not inhibited by 5-carboxyamidotryptamine, or by avariety of compounds known to bind to other subtypes of 5-HT receptor. Digoxigenin-labeled oligonucleotides were found to detect mRNA encoding the 5-HT_1A receptor in a subset of neurons in myenteric and submucosal ganglia. In contrast, 5-HT_1A mRNA was not found in the pancreas. Radioautography revealed that the highest density of ₃H-8-OH-DPAT binding sites was found in the stomach. These sites were especially numerous in the lamina propria adjacent to gastric glands, and in myenteric ganglia. Pancreatic 5-HT_1Areceptors were located on nerves, lymphoid tissue (especially the capsule of nodes), and on cells scattered in the pancreatic parenchyma. The concentration of ₃H-8-OH-DPAT binding sites in the rat bowel and pancreas was less than that of ₃H-5-HT binding sites; however, the distribution of ₃H-8-OH-DPAT binding sites was similar to that of sites that bind ₃H-5-HT. It is concluded that the rat gut and its extension in the pancreas contains 5-HT_1A receptors. Many, if not all, of the nerve cells and processes that express 5-HT_1A receptors express 5-HT_1p receptors as well. The function of these receptors in the physiology of the entero-pancreatic innervation remains to be determined. © 1993 Wiley-Liss, Inc.