Mice devoid of all known thyroid hormone receptors are viable but exhibit disorders of the pituitary–thyroid axis, growth, and bone maturation

Thyroid hormone (T3) has widespread functions in development and homeostasis, although the receptor pathways by which this diversity arises are unclear. Deletion of the T3 receptors TRalpha1 or TRbeta individually reveals only a small proportion of the phenotypes that arise in hypothyroidism, implying that additional pathways must exist. Here, we demonstrate that mice lacking both TRalpha1 and TRbeta (TRalpha1(-/-)beta-/-) display a novel array of phenotypes not found in single receptor-deficient mice, including an extremely hyperactive pituitary-thyroid axis, poor female fertility and retarded growth and bone maturation. These results establish that major T3 actions are mediated by common pathways in which TRalpha1 and TRbeta cooperate with or substitute for each other. Thus, varying the balance of use of TRalpha1 and TRbeta individually or in combination facilitates control of an extended spectrum of T3 actions. There was no evidence for any previously unidentified T3 receptors in TRalpha1(-/-)beta-/- mouse tissues. Compared to the debilitating symptoms of severe hypothyroidism, the milder overall phenotype of TRalpha1(-/-)beta-/- mice, lacking all known T3 receptors, indicates divergent consequences for hormone versus receptor deficiency. These distinctions suggest that T3-independent actions of T3 receptors, demonstrated previously in vitro, may be a significant function in vivo.     

Expression of connexin 57 in the olfactory epithelium and olfactory bulb

In the visual system, deletion of connexin 57 (Cx57) reduces gap junction coupling among horizontal cells and results in smaller receptive fields. To explore potential functions of Cx57 in olfaction, in situ hybridization and immunohistochemistry methods were used to investigate expression of Cx57 in the olfactory epithelium and olfactory bulb. Hybridization signal was stronger in the olfactory epithelial layer compared to the connective tissue underneath. Within the sensory epithelial layer, hybridization signal was visible in sublayers containing cell bodies of basal cells and olfactory neurons but not evident at the apical sublayer comprising cell bodies of sustentacular cells. These Cx57 positive cells were clustered into small groups to form different patterns in the olfactory epithelium. However, individual patterns did not associate with specific regions of olfactory turbinates or specific olfactory receptor zones. Patched distribution of hybridization positive cells was also observed in the olfactory bulb and accessory olfactory bulb in layers where granule cells, mitral cells, and juxtaglomerular cells reside. Immunostaining was observed in the cell types described above but the intensity was weaker than that in the retina. This study has provided anatomical basis for future studies on the function of Cx57 in the olfactory system.     

Differences between natriuretic peptide receptors in the olfactory bulb and hypothalamus from spontaneously hypertensive and normotensive rat brain

Natriuretic peptide receptor-A (NPR-A) functional characteristics in the hypothalamus and olfactory bulb (OB) have been investigated in spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY). Autoradiographic studies demonstrate a decreased number of atrial natriuretic peptide (ANP) binding sites in the olfactory bulb and hypothalamus in SHR compared to WKY rats. We found that NPR-A showed a lower maximal binding capacity (B(max)) and higher affinity in SHR than in WKY rats both in the olfactory bulb and hypothalamus. However, despite the lower B(max) in SHR, both ANP(1-28) and ANP(5-25) stimulated similar or greater cGMP production than in WKY rats. These differences were found even before the development of hypertension. NPR-A in the olfactory bulb and hypothalamus from 3-week-old SHR showed a lower B(max) and K(d) and a higher cGMP production rate than in WKY rats, suggesting that these characteristics are intrinsic of NPR-A in SHR, instead of being a result of hypertension itself. The present study provides evidences for altered NPR-A receptor properties and function in the olfactory bulb and hypothalamus from SHR, which might be involved in the pathogenesis of hypertension.     

Kindling and electrode effects on the benzodiazepine receptors density of olfactory bulb and hippocampus after olfactory bulb kindling.

The olfactory bulb (OB) kindling is a model of limbic secondary generalized epilepsy. Ten days after the completion of OB kindling, we have studied the long term effects of both electrode insertion and kindling on the binding of [3H]diazepam to crude mitochondrial fractions. On the one hand, we have shown that electrode implantation in sham-operated controls induced an obvious increase in benzodiazepine (BZD) receptor density (Bmax) only at the site of the electrode in comparison to sham-unoperated rats. These results might indicate an additional mechanism extending earlier observations reported by others, who have shown that prolonged electrode implantation induced changes in sham-operated and kindled rats. On the other hand, the long lasting effect of OB kindling on the binding parameters of [3H]diazepam was examined in the focus and in the hippocampus. The results indicate a bilateral increase of BZD receptors in the OB and an ipsilateral increase in the hippocampus. These changes might be a regulation phenomenon in response to a hyperexcitability state and to focal stimulations.     

Ciliary neurotrophic factor in the olfactory bulb of rats and mice

Ciliary neurotrophic factor (CNTF) is primarily regarded as an astrocytic lesion factor, promoting neuronal survival and influencing plasticity processes in deafferented areas of the CNS. Postnatal loss of neurons in CNTF-deficient mice indicates a function of the factor also under physiological conditions. In the olfactory bulb, where neurogenesis, axo- and synaptogenesis continue throughout life, CNTF content is constitutively high. The cellular localization of CNTF in the rat olfactory bulb is not fully resolved, and species differences between mouse and rat are not yet characterized. In the present study, four different CNTF antibodies and double immunolabeling with specific markers for glial and neuronal cells were used to study the cellular localization of CNTF in rat and mouse olfactory bulb. Specificity of the detection was checked with tissue from CNTF-deficient mice, and investigations were complemented by immunolocalization of reporter protein in mice synthesizing beta-galactosidase under control of the CNTF promoter (CNTF lacZ-knock-in mice). In both species, CNTF localized to ensheathing cell nuclei, cell bodies and axon-enveloping processes. Additionally, individual axons of olfactory neurons were CNTF immunoreactive. Both CNTF protein content and immunoreaction intensity were lower in mice than in rats. Scattered lightly CNTF-reactive cells were found in the granular and external plexiform layers in rats. Some CNTF-positive cells were associated with immunoreactivity for the polysialylated form of the neural cell adhesion molecule, which is expressed by maturing interneurons derived from the rostral migratory stream. In CNTF lacZ-knock-in mice, beta-galactosidase reactivity was found in ensheathing cells of the olfactory nerve layer, and in cells of the glomerular, external plexiform and granular layers. The study proves that CNTF is localized in glial and neuronal structures in the rodent olfactory bulb. Results in mice provide a basis for investigations concerning the effects of a lack of the factor in CNTF-deficient mice.     

Expression of adenosine A2a receptors gene in the olfactory bulb and spinal cord of rat and mouse

The expression of adenosine A2a receptors (A2aR) in the mammalian striatum is well known. In contrast the exact distribution of A2aR in other regions of the central nervous system remains unclear. The aim of this study was to investigate the A2aR gene expression in the rat olfactory bulb and spinal cord, two regions which are seldom included in mapping studies. Secondly, we compared the A2aR expression in the rat and in the mouse brain. Hybridization histochemistry was performed with an S35-labelled radioactive oligonucleotide probe. The results show strong expression of A2aR in the mouse and rat striatum in accordance with previous reports. In the olfactory bulb a weak but specific expression of A2aR was found in the granular cell layer in both species. In contrast, no significant expression of the A2aR gene was observed in other parts of the brain or the rat spinal cord. The presence of the A2aR in the mammalian olfactory bulb suggests a functional role for this receptor in olfaction.     

Pharmacological characterization of ionotropic glutamate receptors in the zebrafish olfactory bulb

The distribution of N-methyl-D-aspartate- (NMDA) and kainic acid- (KA) sensitive ionotropic glutamate receptors (iGluR) in the zebrafish olfactory bulb was assessed using an activity-dependent labeling method. Olfactory bulbs were incubated with an ion channel permeant probe, agmatine (AGB), and iGluR agonists in vitro, and the labeled neurons containing AGB were visualized immunocytochemically. Preparations exposed to 250 microM KA in the presence of a NMDA receptor antagonist (D-2-amino-5-phosphono-valeric acid) and an alpha-amino-3-hydroxyl-5-methylisoxazole-4-propionic acid (AMPA) receptor antagonist (sym 2206), revealed KA receptor-mediated labeling of approximately 60-70% of mitral cells, juxtaglomerular cells, tyrosine hydroxylase-positive cells and granule cells. A higher proportion of ventral olfactory bulb neurons were KA-sensitive. Application of 333 microM NMDA in the presence of an AMPA/KA receptor antagonist (6-cyano-7-nitroquinoxaline-2,3-dione) resulted in NMDA receptor-mediated labeling of almost all neurons. The concentrations eliciting 50% of the maximal response (effective concentration: EC(50)s) for NMDA-stimulated labeling of different cell types were not significantly different and ranged from 148 microM to 162 microM. These results suggest that while NMDA receptors with similar binding affinities are widely distributed in the neurons of the zebrafish olfactory bulb, KA receptors are heterogeneously expressed among these cells and may serve unique roles in different regions of the olfactory bulb.     

Expression of nuclear and mitochondrial thyroid hormone receptors in postnatal rat tongue muscle

In this quantitative study, a competitive RT-PCR analysis was used to measure the level of the thyroid hormone receptors (TRs) in rat tongue muscle during the development of male Wistar rats aged 0, 5, 10, 15 and 21 postnatal days. There were differences between the expression of TR-alpha1 mRNA and the mRNAs for TR-beta1 and TR-beta2 in rat tongue muscle. Using Western blot analysis, a difference in expression between TR-alpha1 protein (c-ErbAalpha1 protein) and 43-kD c-ErbAalpha1 protein (T(3)-binding 43-kD mitochondrial protein) was detected during the development of the rat tongue muscle. Immunohistochemical examination using electron microscopy showed that TR-alpha1 was found in the mitochondria and nuclei in contrast to TR-beta1 detected in rat tongue muscle. In mitochondrial fractions from rat tongue muscle, the expression of 43-kD c-ErbAalpha1 protein was increased dramatically at 15 and 21 days, and a similar tendency was seen in cytochrome c proteins using Western blot analysis. We presume that the 43-kD c-ErbAalpha1 protein plays a role in regulating mitochondrial RNA synthesis during the postnatal development of rat tongue. The mRNA and protein myosin heavy chain isoforms of muscle also had a different expression during development. The slow myosin isoform protein was not found from day 10 in contrast to fast myosin isoforms. It is likely that the expression of TR-alpha1 mRNA from the rat tongue muscle may be related to a specific phase in muscle phenotype during the development.     

General organization of the perinatal and adult accessory olfactory bulb in mice

The vomeronasal system is currently a topical issue since the dual functional specificity, vomeronasal system-pheromones, has recently been questioned. Irrespective of the tools used to put such specificity in doubt, the diversity of the anatomy of the system itself in the animal kingdom is probably of more importance than has previously been considered. It has to be pointed out that a true vomeronasal system is integrated by the vomeronasal organ, the accessory olfactory bulb, and the so-called vomeronasal amygdala. Therefore, it seems reasonable to establish the corresponding differences between a well-developed vomeronasal system and other areas of the nasal cavity in which putative olfactory receptors, perhaps present in other kinds of mammals, may be able to detect pheromones and to process them. In consequence, a solid pattern for one such system in one particular species needs to be chosen. Here we report on an analysis of the general morphological characteristics of the accessory olfactory bulb in mice, a species commonly used in the study of the vomeronasal system, during growth and in adults. Our results indicate that the critical period for the formation of this structure comprises the stages between the first and the fifth day after birth, when the stratification of the bulb, the peculiarities of each type of cell, and the final building of glomeruli are completed. In addition, our data suggest that the conventional plexiform layers of the main olfactory bulb are not present in the accessory bulb.     

多巴胺受体在大鼠嗅球的表达及其在帕金森病大鼠嗅觉障碍中的作用

目的 观察多巴胺受体在大鼠嗅球（OB）的表达与分布,探讨左旋多巴（L-DOPA）治疗对帕金森病（PD）大鼠嗅觉的影响。 方法 采用免疫印迹、免疫荧光等方法观察多巴胺受体在大鼠OB中的表达;6-羟多巴胺（6-OHDA）双侧注射建立PD大鼠模型,检测L-DOPA治疗对PD大鼠嗅觉功能及谷氨酸脱羧酶（GAD）和脑源性神经营养因子（BNDF）表达的影响。 结果 嗅球内D1和D2两种多巴胺受体亚型表达含量高。D1和D2在颗粒细胞层（GCL）内GAD阳性的γ-氨基丁酸（GABA）能神经元上大量表达,被酪氨酸羟化酶（TH）阳性神经纤维终末包绕。PD大鼠OB内GCL层TH蛋白表达明显下降（0.05±0.01 vs 0.01±0.00,P<0.001）。L-DOPA治疗后,PD大鼠找寻食物小球时间显著降低［（624.4±113.4）s vs(312.4±79.35)s,P<0.05］,OB内BDNF表达显著升高（0.02±0.01 vs 0.07±0.01,P<0.01）。 结论 D1和D2在GCL层GABA能神经元大量表达。L-DOPA治疗可缓解PD大鼠嗅觉障碍,可能与激活OB内GABA能神经元上的D1和D2复合体,进而改善BDNF表达有关。     

Expression of sex steroid hormone receptors in C cell hyperplasia and medullary thyroid carcinoma

Previous studies have shown that C cells are twice as numerous in male than in female thyroids and that C cell hyperplasia (CCH) is much more frequent in men. These findings suggest regulation involving sex steroid hormones through the expression of sex steroid hormone receptors on C cells. To investigate this hypothesis, we performed an immunohistochemical study of estrogen receptors α (ERα) and β (ERβ), progesterone receptors (PR), and androgen receptors (AR) on specimens from a series of 40 patients operated on for a medullary thyroid carcinoma (MTC; n = 28; female 18, male 10) and/or CCH (n = 19; female 6, male 13). ERβ was the only receptor to be consistently expressed in CCH (100%) and MTC (96.5%), whereas ERα was never expressed. PR and AR were rarely expressed in MTC (7 and 14%, respectively). AR was expressed in half the CCH cases (53%), with a trend to male predominance (61% in men vs 33% in women). Our study is the first to describe ERβ expression in CCH. In addition, our findings suggest that CCH, and possibly MTC, might be influenced by sex steroid hormones, namely, estrogens and androgens, through the expression of ERβ and AR on C cells.     

Physiological relevance of thyroid stimulating hormone and thyroid stimulating hormone receptor in tissues other than the thyroid

Decades of research have provided strong evidence for a reciprocal relationship between the immune system and hormones of the hypothalamus-pituitary-thyroid (HPT) axis. Thyroid stimulating hormone (TSH), in particular, has been shown to have a variety of immune-regulating cytokine-like activities that can influence the outcome of T cell development in the thymus and intestine, and can affect the magnitude of antibody and cell-mediated responses of peripheral lymphocytes. Production of TSH and the expression of the TSH receptor are widely but selectively distributed across many different types of hematopoietic cells in the bone marrow, as well as among subsets of dendritic cells, monocytes and lymphocytes in the spleen and lymph nodes. In addition to their role in immunity, the involvement of TSH-producing hematopoietic cells in the microregulation of thyroid hormone activity represents a novel and potentially important aspect of the TSH-mediated immune-endocrine circuit.     

Calcium permeable AMPA receptors and autoreceptors in external tufted cells of rat olfactory bulb

Glomeruli are functional units of the olfactory bulb responsible for early processing of odor information encoded by single olfactory receptor genes. Glomerular neural circuitry includes numerous external tufted (ET) cells whose rhythmic burst firing may mediate synchronization of bulbar activity with the inhalation cycle. Bursting is entrained by glutamatergic input from olfactory nerve terminals, so specific properties of ionotropic glutamate receptors on ET cells are likely to be important determinants of olfactory processing. Particularly intriguing is recent evidence that AMPA receptors of juxta-glomerular neurons may permeate calcium. This could provide a novel pathway for regulating ET cell signaling. We tested the hypothesis that ET cells express functional calcium-permeable AMPA receptors. In rat olfactory bulb slices, excitatory postsynaptic currents (EPSCs) in ET cells were evoked by olfactory nerve shock, and by uncaging glutamate. We found attenuation of AMPA/kainate EPSCs by 1-naphthyl acetyl-spermine (NAS), an open-channel blocker specific for calcium permeable AMPA receptors. Cyclothiazide strongly potentiated EPSCs, indicating a major contribution from AMPA receptors. The current-voltage (I-V) relation of uncaging EPSCs showed weak inward rectification which was lost after > approximately 10 min of whole-cell dialysis, and was absent in NAS. In kainate-stimulated slices, Co(2+) ions permeated cells of the glomerular layer. Large AMPA EPSCs were accompanied by fluorescence signals in fluo-4 loaded cells, suggesting calcium permeation. Depolarizing pulses evoked slow tail currents with pharmacology consistent with involvement of calcium permeable AMPA autoreceptors. Tail currents were abolished by Cd(2+) and (+/-)-4-(4-aminophenyl)-2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide (NBQX), and were sensitive to NAS block. Glutamate autoreceptors were confirmed by uncaging intracellular calcium to evoke a large inward current. Our results provide evidence that calcium permeable AMPA receptors reside on ET cells, and are divided into at least two functionally distinct pools: postsynaptic receptors at olfactory nerve synaptic terminals, and autoreceptors sensitive to glutamate released from dendrodendritic synapses.     

母源性甲状腺功能亢进对仔鼠心脏甲状腺激素受体mRNA表达的影响

目的 探讨母源性甲状腺功能亢进（简称甲亢）对新生仔鼠心脏中甲状腺激素受体的影响。方法 首先对24只雌性Wistar大鼠建立妊娠合并甲亢模型,取新生5d、10d、15d仔鼠心脏进行解剖检查,取部分心脏组织石蜡切片Masson染色,取部分心脏组织采用实时定量PCR法检测甲
状腺激素受体（TR）α₁、TRα₂、TRβ₁ mRNA水平表达量的变化。结果 在心脏组织中TRα1 mRNA的表达量较TRα₂、TRβ₁的表达量高,且在出生10d的仔鼠心脏（包括甲亢组和对照组）中TRα₁mRNA的表达量呈现峰值;与同期对照仔鼠相比,母源性甲亢仔鼠出生5d 与出生10d心肌中TRα₁
mRNA的表达量显著上调,出生15d的表达量与对照组相比表达量下调;TRα₂在母源性甲亢和对照中差异无统计学意义;TRβ₁在甲亢出生5d仔鼠中表达量显著下调,10d组表达量上调,15d组表达量差异无显著性。结论 母源性甲亢会对仔鼠心肌产生影响,引起甲状腺激素受体的差异表达;
这种变化随出生后时间延长而减弱。甲状腺激素受体中TRα₁的变化最显著,可能在甲亢引起的心肌损伤中起重要作用。     

Influence of isolation and training on fighting in mice with olfactory bulb lesions

Isolation-induced aggressive mice receiving total bilateral bulbectomy failed to fight after chronic training. Animals receiving sub-total olfactory bulbectomy were capable of being trained to attack but the latency to attack was increased. When mice were bulbectomized before being isolated they were incapable of attack regardless of training or completeness of the lesion. These results indicated that isolation influences aggressive behavior in bilaterally bulbectomized mice.     

Reactions of olfactory bulb neurons to different stimulus intensities in laboratory mice

Summary The activity of 219 cells in the olfactory bulb of waking, freely breathing mice was analysed, and it was found that their spontaneous discharge rate varied between 0.3 and 33 AP/s. Both butyric acid and geraniol elicited responses of four types: 38% of the 98 responses were of type 1 (excitation), 43% of type 2 (inhibition), 10% of type 3 (complex structure), and 8% were characterized by a change in the temporal pattern of the activity. Response duration varied from less than 500 ms to more than 1 min. 52 secondary neurons were stimulated with four different concentrations of the odor substances. All of the responsive cells showed a clear ability to discriminate concentration. That is, response magnitude varied with intensity, producing non-monotonie curves. Most of the neurons responded only in a region of a more or less limited concentration, and in no case was a saturation curve observed. Approx. 40% of the neurons responded to the lowest concentration tested (10^–8 vol. % of butyric acid or geraniol). The strongest stimuli, 10^–2 vol.%, were relatively ineffective.The work was supported by the Deutsche Forschungsgemeinschaft (Schm 322/7).     

Comparative laminar distribution of various autoradiographic cholinergic markers in adult rat main olfactory bulb

To provide anatomical information on the complex effects of acetylcholine (ACh) in the olfactory bulb (OB), the distribution of different cholinergic muscarinic and nicotinic receptor sub-types was studied by quantitative in vitro autoradiography. The muscarinic M1-like and M2-like sub-types, as well as the nicotinic bungarotoxin-insensitive (α4β2-like) and bungarotoxin-sensitive (α7-like) receptors were visualized using [³H]pirenzepine, [3H]AF-DX 384, [³H]cytisine and [¹²⁵I]α-bungarotoxin (BTX), respectively. In parallel, labelling patterns of [³H]vesamicol (vesicular acetylcholine transport sites) and [³H]hemicholinium-3 (high-affinity choline uptake sites), two putative markers of cholinergic nerve terminals, were investigated. Specific labelling for each cholinergic radioligand is distributed according to a characteristic laminar and regional pattern within the OB revealing the lack of a clear overlap between cholinergic afferents and receptors. The presynaptic markers, [³H]vesamicol and [³H]hemicholinium-3, demonstrated similar laminar pattern of distribution with two strongly labelled bands corresponding to the glomerular layer and the area around the mitral cell layer. Muscarinic M1-like and M2-like receptor sub-types exhibited unique distribution with their highest levels seen in the external plexiform layer (EPL). Intermediate M1-like and M2-like binding densities were found throughout the deeper bulbar layers. In the glomerular layer, the levels of muscarinic receptor subtypes were low, the level of M2-like sites being higher than M1. Both types of nicotinic receptor sub-types displayed distinct distribution pattern. Whereas [¹²⁵I]α-BTX binding sites were mostly concentrated in the superficial bulbar layers, [³H]cytisine binding was found in the glomerular layer, as well as the mitral cell layer and the underlying laminae. An interesting feature of the present study is the visualization of two distinct cholinoceptive glomerular subsets in the posterior OB. The first one exhibited high levels of both [³H]vesamicol and [³H]hemicholinium-3 sites. It corresponds to the previously identified atypical glomeruli and apparently failed to express any of the cholinergic receptors under study. In contrast, the second subset of glomeruli is not enriched with cholinergic nerve terminal markers but displayed high amounts of [³H]cytisine/nicotinic binding sites. Taken together, these results suggest that although muscarinic receptors have been hypothesized to be mostly involved in cholinergic olfactory processing and short-term memory in the OB, nicotinic receptors, especially of the cytisine/α4β2 sub-type, may have important roles in mediating olfactory transmission of efferent neurons as well as in a subset of olfactory glomeruli.