Multifactorial Modulation of TRH Metabolism

1. Thyrotropin releasing hormone (TRH), synthesized in the paraventricular nucleus of the hypothalamus (PVN), is released in response to physiological stimuli through medianeminence nerve terminals to control thyrotropin or prolactin secretion from the pituitary.2. Several events participate in the metabolism of this neuropeptide: regulation of TRH biosynthesis and release as well as modulation of its inactivation by the target cell.3. Upon a physiological stimulus such as cold stress or suckling, TRH is released and levels of TRH mRNA increase in a fast and transient manner in the PVN; a concomitant increase in cfos is observed only with cold exposure.4. Hypothalamic cell cultures incubated with cAMP or phorbol esters show a rise in TRH mRNA levels; dexamethasone produces a further increase at short incubation times.TRH mRNA are thus controlled by transsynaptic and hormonal influences.5. Once TRH is released, it is inactivated by a narrow specificity ectoenzyme, pyroglu-tamyl peptidase II (PPII).6. In adenohypophysis, PPII is subject to stringent control: positive by thyroid hormones and negative by TRH; other hypothalamic factors such as dopamine and somatostatin also influence its activity.7. These combined approaches suggest that TRH action is modulated in a coordinate fashion.     

Universal nuclear domains of somatic and germ cells: some lessons from oocyte interchromatin granule cluster and Cajal body structure and molecular composition

It is now clear that two prominent nuclear domains, interchromatin granule clusters (IGCs) and Cajal bodies (CBs), contribute to the highly ordered organization of the extrachromosomal space of the cell nucleus. These functional domains represent structurally stable but highly dynamic nuclear organelles enriched in factors that are required for different nuclear activities, especially RNA biogenesis. IGCs are considered to be the main sites for storage, assembly, and/or recycling of the essential spliceosome components. CBs are involved in the biogenesis of several classes of small RNPs as well as the modification of newly assembled small nuclear RNA. We have summarized data on the molecular composition, structure, and functional roles of IGCs and CBs in the nuclei of mammalian somatic cells and oocytes of some animals with a special focus on insects. We have focused on similarities and differences between the IGCs and CBs of oocytes and the well‐studied CBs and IGCs of cultured mammalian somatic cells. We have shown the heterogeneous character of oocyte IGCs and CBs, both in structure and molecular content. We have also demonstrated the unique capacity of oocytes to form close structural interactions between IGC and CB components. We proposed to consider these joint structures as integrated entities, sharing the features of both IGCs and CBs.     

Examination of Rapid Dopamine Dynamics with Fast Scan Cyclic Voltammetry During Intra-oral Tastant Administration in Awake Rats

Rapid, phasic dopamine (DA) release in the mammalian brain plays a critical role in reward processing, reinforcement learning, and motivational control. Fast scan cyclic voltammetry (FSCV) is an electrochemical technique with high spatial and temporal (sub-second) resolution that has been utilized to examine phasic DA release in several types of preparations. In vitro experiments in single-cells and brain slices and in vivo experiments in anesthetized rodents have been used to identify mechanisms that mediate dopamine release and uptake under normal conditions and in disease models. Over the last 20 years, in vivo FSCV experiments in awake, freely moving rodents have also provided insight of dopaminergic mechanisms in reward processing and reward learning. One major advantage of the awake, freely moving preparation is the ability to examine rapid DA fluctuations that are time-locked to specific behavioral events or to reward or cue presentation. However, one limitation of combined behavior and voltammetry experiments is the difficulty of dissociating DA effects that are specific to primary rewarding or aversive stimuli from co-occurring DA fluctuations that mediate reward-directed or other motor behaviors. Here, we describe a combined method using in vivo FSCV and intra-oral infusion in an awake rat to directly investigate DA responses to oral tastants. In these experiments, oral tastants are infused directly to the palate of the rat – bypassing reward-directed behavior and voluntary drinking behavior – allowing for direct examination of DA responses to tastant stimuli.     

Microelectrode Guided Implantation of Electrodes into the Subthalamic Nucleus of Rats for Long-term Deep Brain Stimulation

Deep brain stimulation (DBS) is a widely used and effective therapy for several neurologic disorders, such as idiopathic Parkinson’s disease, dystonia or tremor. DBS is based on the delivery of electrical stimuli to specific deep anatomic structures of the central nervous system. However, the mechanisms underlying the effect of DBS remain enigmatic. This has led to an interest in investigating the impact of DBS in animal models, especially in rats. As DBS is a long-term therapy, research should be focused on molecular-genetic changes of neural circuits that occur several weeks after DBS. Long-term DBS in rats is challenging because the rats move around in their cage, which causes problems in keeping in place the wire leading from the head of the animal to the stimulator. Furthermore, target structures for stimulation in the rat brain are small and therefore electrodes cannot easily be placed at the required position. Thus, a set-up for long-lasting stimulation of rats using platinum/iridium electrodes with an impedance of about 1 MΩ was developed for this study. An electrode with these specifications allows for not only adequate stimulation but also recording of deep brain structures to identify the target area for DBS. In our set-up, an electrode with a plug for the wire was embedded in dental cement with four anchoring screws secured onto the skull. The wire from the plug to the stimulator was protected by a stainless-steel spring. A swivel was connected to the circuit to prevent the wire from becoming tangled. Overall, this stimulation set-up offers a high degree of free mobility for the rat and enables the head plug, as well as the wire connection between the plug and the stimulator, to retain long-lasting strength.     

Sex and exercise interact to alter the expression of anabolic androgenic steroid-induced anxiety-like behaviors in the mouse

Anabolic androgenic steroids (AAS) are taken by both sexes to enhance athletic performance and body image, nearly always in conjunction with an exercise regime. Although taken to improve physical attributes, chronic AAS use can promote negative behavior, including anxiety. Few studies have directly compared the impact of AAS use in males versus females or assessed the interaction of exercise and AAS. We show that AAS increase anxiety-like behaviors in female but not male mice and that voluntary exercise accentuates these sex-specific differences. We also show that levels of the anxiogenic peptide corticotrophin releasing factor (CRF) are significantly greater in males, but that AAS selectively increase CRF levels in females, thus abrogating this sex-specific difference. Exercise did not ameliorate AAS-induced anxiety or alter CRF levels in females. Exercise was anxiolytic in males, but this behavioral outcome did not correlate with CRF levels. Brain-derived neurotrophic factor (BDNF) has also been implicated in the expression of anxiety. As with CRF, levels of hippocampal BDNF mRNA were significantly greater in males than females. AAS and exercise were without effect on BDNF mRNA in females. In males, anxiolytic effects of exercise correlated with increased BDNF mRNA, however AAS-induced changes in BDNF mRNA and anxiety did not. In sum, we find that AAS elicit sex-specific differences in anxiety and that voluntary exercise accentuates these differences. In addition, our data suggest that these behavioral outcomes may reflect convergent actions of AAS and exercise on a sexually differentiated CRF signaling system within the extended amygdala.     

Postactivation treatment with nocodazole maintains normal nuclear ploidy of cloned pig embryos by increasing nuclear retention and formation of single pronucleus

The objective of this study was to investigate the effects of postactivation treatment with nocodazole on morphologic changes of donor nuclei and in vitro and in vivo development of somatic cell nucleus transfer (SCNT) embryos in pigs (Sus scrofa). Somatic cell nucleus transfer oocytes were either untreated (control) or treated with nocodazole or demecolcine after electric activation, then cultured in vitro or transferred to surrogate pigs. Treatment with nocodazole (30%) and demecolcine (29%) after electric activation improved embryo development to the blastocyst stage compared with the control (16%). The rate of oocytes that formed single clusters of chromosomes or a pronucleus 4 h after activation was higher after treatment with nocodazole (82%) and demecolcine (86%) than under the control conditions (66%), and this tendency was not altered even 12 h after activation. Pseudo-polar body extrusion was inhibited by nocodazole and demecolcine, and the rate of embryos with diploid chromosomes was higher after treatment with nocodazole (86%) and demecolcine (77%) than under control conditions (58%). Nocodazole treatment resulted in a farrowing rate of 50% with a 1.7% efficiency of piglet production, whereas controls showed a farrowing rate of 60% and a production efficiency of 3.8%. Our results demonstrate that postactivation treatment with nocodazole maintains normal nuclear ploidy of cloned embryos likely by increasing nuclear retention and formation of single pronuclei. In vivo development could be achieved from the transfer of nocodazole-treated embryos but showed some defects compared with control.     

Differential thermal sensitivity between the recipient ooplasm and the donor nucleus in Holstein and Taiwan native yellow cattle

The objective of this study was to compare thermal sensitivity of recipient ooplasm and donor nucleus from Holstein and Taiwan native yellow (TY) cows. Oocytes and cumulus cells from each breed were incubated at 43 °C (heat shock) or 38.5 °C (control) for 1 h prior to nucleus transplantation. Reconstructed embryos cloned by transfer of non-heated Holstein donor cells to heat-shocked Holstein ooplasm (Ho⁺-Hd⁻) had a lower (P < 0.05) blastocyst rate than those cloned from non-heated Holstein ooplasm receiving heated (Ho⁻-Hd⁺) or non-heated (Ho⁻-Hd⁻) Holstein donor cells (11.3 vs. 34.3 or 36.8%). Heat-shocked donor cells from either Holstein or TY cows did not significantly affect blastocyst rates of reconstructed embryos produced from Holstein ooplasm (30.6-32.9%). In contrast, blastocyst rates of reconstructed embryos generated with heat-shocked Holstein ooplasm were lower (P < 0.05) than that with heat-shocked TY ooplasm (11.2 vs 45.2%). Without heat shock, embryos reconstructed by transferring donor cells to ooplasm of Holstein or TY cows had similar (P > 0.05) blastocyst rates (28.9-33.3%). Transplantation of reconstructed embryos (n = 30) to recipients (n = 23) resulted in three live calves, derived from embryos cloned with TY ooplasm and donor nuclei from either Holstein (n = 2) or TY cows (n = 1). In conclusion, ooplasm of TY cattle was more resistant to heat stress than that derived from Holsteins; therefore, ooplasm may be a major determinant for thermal sensitivity in bovine oocytes and embryos.     

Background activity of neurons of the supraoptic hypothalamic nucleus in rats under conditions of vibrational stimulation and electromagnetic extrahigh-frequency irradiation

We analyzed background impulse activity of neurons of the supraoptic nucleus of the rat hypothalamus in the course of 15-day-long isolated action of generalized vibrational stimulation and combination of such stimulation with irradiation of the animal’s head with low-intensity extrahigh-frequency (EHF, millimeter-range) electromagmetic waves. The distributions of the neurons by the level of regularity and dynamics of spike trains, separate frequency ranges of impulsation, and pattern of interspike interval (ISI) histograms were estimated. We also calculated the mean frequency of discharges and coefficient of variation of ISIs. A trend toward decreases in the deviations of some parameters of neuronal spike activity generated by supraoptic neurons, which were evident within early time intervals of isolated action of vibration (5 to 10 days), was observed under the influence of EHF electromagnetic irradiation; thus, the latter factor probably exerts a sedative effect. Neirofiziologiya/Neurophysiology, Vol. 39, No. 6, pp. 433–442, November–December, 2007.     

Three-dimensional pore space quantification of apple tissue using X-ray computed microtomography

The microstructure and the connectivity of the pore space are important variables for better understanding of the complex gas transport phenomena that occur in plant tissues. In this study, we present an experimental procedure for image acquisition and image processing to quantitatively characterize in 3D the pore space of apple tissues (Malus domestica Borkh.) for two cultivars (Jonagold and Braeburn) taken from the fleshy part of the cortex using X-ray computer microtomography. Preliminary sensitivity analyses were performed to determine the effect of the resolution and the volume size (REV, representative elementary volume analysis) on the computed porosity of apple samples. For comparison among cultivars, geometrical properties such as porosity, specific surface area, number of disconnected pore volumes and their distribution parameters were extracted and analyzed in triplicate based on the 3D skeletonization of the pore space (medial axis analysis). The results showed that microtomography provides a resolution at the micrometer level to quantitatively analyze and characterize the 3D topology of the pore space in apple tissue. The computed porosity was confirmed to be highly dependent of the resolution used, and the minimum REV of the cortical flesh of apple fruit was estimated to be 1.3 mm³. Comparisons among the two cultivars using a resolution of 8.5 μm with a minimum REV cube showed that in spite of the complexity and variability of the pore space network observed in Jonagold and Braeburn apples, the extracted parameters from the medial axis were significantly different (P-value < 0.05). Medial axis parameters showed potential to differentiate the microstructure between the two evaluated apple cultivars.