Selective corticofugal modulation on sound processing in auditory thalamus of awake marmosets.

Cortical feedback has long been considered crucial for the modulation of sensory perception and recognition. However, previous studies have shown varying modulatory effects of the primary auditory cortex (A1) on the auditory response of subcortical neurons, which complicate interpretations regarding the function of A1 in sound perception and recognition. This has been further complicated by studies conducted under different brain states. In the current study, we used cryo-inactivation in A1 to examine the role of corticothalamic feedback on medial geniculate body (MGB) neurons in awake marmosets. The primary effects of A1 inactivation were a frequency-specific decrease in the auditory response of most MGB neurons coupled with an increased spontaneous firing rate, which together resulted in a decrease in the signal-to-noise ratio. In addition, we report for the first time that A1 robustly modulated the long-lasting sustained response of MGB neurons, which changed the frequency tuning after A1 inactivation, e.g. some neurons are sharper with corticofugal feedback and some get broader. Taken together, our results demonstrate that corticothalamic modulation in awake marmosets serves to enhance sensory processing in a manner similar to center-surround models proposed in visual and somatosensory systems, a finding which supports common principles of corticothalamic processing across sensory systems.

Porcine sperm vitrification I: cryoloops method.

The aims of this study were to evaluate porcine sperm vitrification in cryoloops, with and without two different cryoprotectants and assess two warming procedures. Extended (n = 3; r = 4) and raw (n = 5; r = 2) semen was diluted in media without and with cryoprotectants (4% dimethylformamide and 4% glycerol) to a final concentration of 20 × 106 spermatozoa ml-1 and vitrified using the cryoloops method. Two warming procedures were evaluated: rapid method (30 s at 37°C) and an ultra-rapid method (7 s at 75°C, followed by 30 s at 37°C). Total motility (phase contrast), sperm viability (6-carboxifluorescein diacetate and propidium iodide stain), membrane function (hypo-osmotic swelling test), acrosome integrity (phase contrast), chromatin condensation (toluidine blue stain) and chromatin susceptibility to acid denaturation (acridine orange stain) were evaluated before and after vitrification and analysed using Friedman's test. In all media, the only seminal parameters that were maintained after vitrification were chromatin condensation and integrity. Vitrification of porcine spermatozoon using cryoloops, both in the presence or absence of cryoprotectants and independent of the warming procedure used, permits conservation of sperm chromatin condensation and integrity. It would be interesting to further verify this by producing porcine embryos using vitrified spermatozoon with intracytoplasmic sperm injection.

Meiotic maturation and developmental capability of ovine oocytes at germinal vesicle stage following vitrification using different cryodevices.

In order to assess effects of vitrification on ovine oocytes at the germinal vesicle (GV) stage, the conventional plastic straw (CS), the open-pulled straw (OPS), and Cryoloop were used to vitrify ovine oocytes. Oocytes were randomly divided into five groups: (1) Control; (2) Oocytes exposed to vitrification and dilution solutions without any cryopreservation (toxicity); (3) Oocytes vitrified using CS (CS); (4) Oocytes vitrified using OPS (OPS), and (5) Oocytes vitrified using Cryoloop (Cryoloop). The viability, cumulus cell expansion, nuclear maturation after in vitro maturation (IVM), and developmental capability of vitrified oocytes following parthenogenetic activation (PA) or in vitro fertilization (IVF) were assessed. The pretreatment in the vitrification and dilution solutions without any freezing or thawing did not adversely influence oocytes. The viability of vitrified oocytes were significantly declined compared to unfrozen oocytes (P < 0.05). The viability of oocytes vitrified using open-pulled straws or Cryoloop was significantly higher than that in the CS group (P < 0.05). After IVM, the percentage of oocytes reaching to the metaphase II (MII) stage was significantly higher with Cryoloop and OPS following by CS. However, the in vitro maturing percentage of vitrified oocytes was significantly less than that of unfrozen oocytes (P < 0.05). After PA, the developmental capability of vitrified oocytes was significantly decreased compared to unfrozen oocytes. The cleavage rate of oocytes vitrified using conventional plastic straws was significantly less than those of the other freezing groups (P < 0.05). The cleaving capability of oocytes vitrified using Cryoloop was significantly increased compared to the OPS group. However, there was no significant difference existing amongst the freezing groups as concerning the blastocyst rate. Following IVF, the developmental capability of vitrified oocytes was severely damaged compared to that of unfrozen oocytes. The cleavage rate of oocytes vitrified with Cryoloop was similar to that of oocytes vitrified with open-pulled straws. However, the cleavage rate of vitrified oocytes in the CS group was significantly less than that in the OPS or Cryoloop group (P < 0.05). None of oocytes vitrified using conventional plastic straw developed to the blastocyst stage following IVF. There was no significant difference existing between OPS and Cryoloop with respect to the blastocyst rate. After staining with cFDA and PI, cumulus cells surrounding oocytes were partly damaged by vitrification and thawing while the membrane of vitrified oocyte still remained intact. In conclusion, vitrification can seriously damage ovine immature oocytes and cumulus cells surrounding oocytes, which may subsequently affect their developmental capability. Finally, this study further proves that increasing the freezing and thawing velocity benefits survival of vitrified immature oocytes.

Efficient cryopreservation of mouse embryos by modified droplet vitrification (MDV).

The aim of this study was to assess modified droplet vitrification (MDV) for the cryopreservation of early developmental mouse embryos. Mouse embryos were equilibrated in holding solution for 3 min followed by immersion in vitrification solution for 30-45 s, and then three embryos per 3-µL vitrification droplet were directly dropped into liquid nitrogen. Vitrified embryos were warmed to examine their developmental potential both in vitro and in vivo. The results demonstrated that MDV vitrified and warmed embryos had a survival rate of 98.1-99.6% (P>0.05); however, blastocyst development post warming and culture in vitro demonstrated that vitrified 4-celled, 8-celled, 16-celled, morulae, and blastocyst embryos had significant higher developmental potentials (94.7-99.5%) than those from zygotes (9.2%) and 2-celled embryos (85.7%) (P<0.05). Compared to CryoLoop and CryoTech vitrification, MDV showed similar results with regards to rates of survival, blastocyst development, but with the higher hatching rate (76.1% vs. 64.0-67.3%) (P<0.05). Cryopreservation by MDV resulted in a similar blastocyst developmental potential in 4-celled and 16 celled embryos from ICR (94.7-99.5%), C57BL/6J (94.7-96.4%), and their crossbred F1 strain (97.9-98.9%) (P>0.05). After embryo transfer of vitrified ICR embryos from 4-celled, 16-celled, morulae and blastocyst stage, 40.7-43.7% of the embryos developed into live offspring (P>0.05), but MDV vitrification resulted in the highest birth rate (43.8%) compared to CryoLoop (38.3%) and CryoTech (35.4%) (P<0.05), when 4-celled mouse embryos were used for vitrification. Our study clearly demonstrated that MDV is the most efficient vitrification to cryopreserve embryos at least 4-celled and advanced stages, which can be used to preserve important mouse genomes from different strains and different developmental stages.

Effects of various freezing containers for vitrification freezing on mouse oogenesis.

BACKGROUND: In the present study, various freezing containers were tested for mouse embryos of respective developmental stages; embryos were vitrified and then their survival rate and developmental rate were monitored. Mouse two cell, 8 cell, and blastula stage embryos underwent vitrification freezing-thawing and then their recovery rate, survival rate, development rate, and hatching rate were investigated. METHODS: EM-grid, OPS, and cryo-loop were utilized for vitrification freezing-thawing of mouse embryos. RESULTS: It was found that recovery rate and survival rate were higher in the group of cryo-loop compared to those of EM-grid (p < 0.05). Embryonic development rate, two cell embryos to blastocyst, as well as hatching rate were higher in the control group compared to the EM-grid group and OPS group (p < 0.05), yet no difference was noted between the control group and cryo-loop group. Development rate and hatching rate of eight cell morulae and blastocysts were all lower in the treatment groups than the control group whilst hatching rate of blastocysts was higher in the control group compared to the groups of EM-grid and OPS (p < 0.05); although the cryo-loop group was shown to be slightly higher than other groups, it was not statistically significant. CONCLUSIONS: In the study, we investigate effects of freezing containers on vitrified embryos of respective developmental stages; it was demonstrated that higher developmental rate was shown in more progressed (or developed) embryos with more blastomeres. There was however, no difference in embryonic development rate was shown amongst containers. Taken together, further additional studies are warranted with regards to 1) manipulation techniques of embryos for various vitrification freezing containers and 2) preventive measures against contamination via liquid nitrogen.

Cortical inactivation by cooling in small animals.

Reversible inactivation of the cortex by surface cooling is a powerful method for studying the function of a particular area. Implanted cooling cryoloops have been used to study the role of individual cortical areas in auditory processing of awake-behaving cats. Cryoloops have also been used in rodents for reversible inactivation of the cortex, but recently there has been a concern that the cryoloop may also cool non-cortical structures either directly or via the perfusion of blood, cooled as it passed close to the cooling loop. In this study we have confirmed that the loop can inactivate most of the auditory cortex without causing a significant reduction in temperature of the auditory thalamus or other subcortical structures. We placed a cryoloop on the surface of the guinea pig cortex, cooled it to 2°C and measured thermal gradients across the neocortical surface. We found that the temperature dropped to 20-24°C among cells within a radius of about 2.5 mm away from the loop. This temperature drop was sufficient to reduce activity of most cortical cells and led to the inactivation of almost the entire auditory region. When the temperature of thalamus, midbrain, and middle ear were measured directly during cortical cooling, there was a small drop in temperature (about 4°C) but this was not sufficient to directly reduce neural activity. In an effort to visualize the extent of neural inactivation we measured the uptake of thallium ions following an intravenous injection. This confirmed that there was a large reduction of activity across much of the ipsilateral cortex and only a small reduction in subcortical structures.