Neuronal and synaptic morphological alterations in the hippocampus of cannabinoid receptor type 1 knockout mice.

Cannabinoid receptor type 1 (CB1R) modulates synaptic activity and is widely distributed in brain areas such as the hippocampus, cerebellum, cerebral cortex, and striatum, among others. CB1R is involved in processes such as memory, learning, motor coordination, and mood. Genetic deletion of CB1R causes behavioral alterations. In this work, we evaluated neuronal morphology and synaptic structure in the hippocampus of adult male CB1R knockout mice (CB1R-/- ). Morphological changes in the CB1R-/- hippocampus evidenced a decrease in the expression of cytoskeletal proteins neurofilaments 160 KDa, neurofilaments 200 KDa, and microtubule-associated protein 2. CA1 neurons showed decreased arborization and changes in synaptic structure such as lower thickness of postsynaptic density and a reduction in synaptophysin levels. Results obtained in the present work provide evidence of the participation of CB1R in the establishment of neuronal structure and networks that could have an important role in neuronal plasticity. In addition, these changes observed in CB1R-/- could be correlated with behavioral alterations reported.

The expression of adenosine receptors changes throughout light induced retinal degeneration in the rat.

The modulation of adenosine receptors, A1 (A1R) and A2A (A2AR), is neuroprotective in different models of retinal injury. In order to understand the processes underlying retinal degeneration, we studied the expression of adenosine receptors in the retinas of control and continuously illuminated (CI) rats by qRT-PCR, Western blot (WB) and immunohistochemistry (IHC). Significant increases of A1R, A2AR, and A2BR mRNAs at 1, 5, and 7 days of CI (P < 0.0001) were observed by qRT-PCR. Also, a significant increase of A3R mRNA was detected after 5 and 7 days of CI. WB studies showed a significant rise of A1R on day 1 of CI and on days 5 and 7 (P < 0.0001), while A2AR increase was seen from 2 days of CI on (P < 0.001). After 1 day of CI, A1R immunoreactivity (A1R-IR) increased in ganglion cell layer, inner nuclear layer, and in both the outer and inner plexiform layers. After 2 days of CI, the A1R-IR went back to control levels. After 5 days of CI, a second rise in A1R, which persisted until 7 days of CI, was measured (P < 0.0001). A significant rise of A2aR immunoreactivity was also observed at day 2 of CI at GCL and INL and subsided at days 5 and 7 (P < 0.0001). The observed up-regulation of A1R after 1 day of CI, corresponds with the peak of oxidative stress; while the rise of A2aR at day 2 of CI, coincides with the massive apoptosis of photoreceptors. We postulate that an early modulation of adenosine receptors could delay or prevent the degeneration of photoreceptors.

Acute hypoxia differentially affects the NMDA receptor NR1, NR2A and NR2B subunit mRNA levels in the developing chick optic tectum: stage-dependent plasticity in the 2B-2A ratio.

It is known that the NMDA-R NR1 subunit is needed for the receptor activity and that under hypoxia the evolution toward apoptosis or neuronal survival depends on the balance NR2A/NR2B subunits. This paper analyzes the effect of acute hypoxia on the above mentioned subunits mRNAs during development. The mean percentage of NR1+ neurons displayed the higher plasticity during development while the NR2A+ neurons the higher stability. Acute hypoxia increased the mean percentage of NR1+ and NR2B+ neurons at ED12 but only that of NR1+ neurons at ED18. Acute hypoxia increased the levels of expression of NR1 and NR2B mRNAs at ED12 without changes in the NR2A mRNA. During early stages there is a higher sensitivity to change the subunits mRNA levels under a hypoxic treatment. At ED12 acute hypoxia increased the probability of co-expression of the NR1-NR2A and NR1-NR2B subunits combinations, the level of NR1 and NR2B and the ratio NR2B/NR2A. These conditions facilitate the evolution towards apoptosis.

Programmed cell death and differential JNK, p38 and ERK response in a prenatal acute hypoxic hypoxia model.

We previously found that prenatal hypoxia induces a significant increase in the levels of active Caspase 3 at 60 min post-hypoxia (p-h) and in the number of TUNEL-positive pyknotic cells, which peaks at 6h p-h. The aim of this work was to study alterations in MAPKs pathways and the effect of specific inhibitors of the JNK (SP600125) and p38 (SB203580) pathways following acute hypoxia in chick optic lobe at embryonic day (ED) 12. To this end, JNK, p38 and ERK1-2 protein kinase expression levels were determined by Western blot in both their active and inactive forms, evaluated at successive p-h times. At 10 and 30 min p-h the P-JNK/JNK ratio was 1.912+/-0.341 and 1.920+/-0.304, respectively. Concomitantly, at 0 min p-h the P-p38/p38 ratio was 1.657+/-0.203. Lastly, the P-ERK/ERK ratio proving non-significant throughout. When inhibitors for JNK and p38 were used, we observed a decrease in the values of active Caspase 3 at 60 min p-h, which correlated with the control values in the parameters of TUNEL-positive cells at 6h p-h. Analysis for P-ATF-2 demonstrated an increase in hypoxic embryos compared to control ones which was reverted in a dose-dependent manner with the use of both inhibitors. All these results indicate that at ED 12, acute hypoxia might be differentially activating JNK and p38 pathways, without affecting the ERK pathway, which in turn would be activating Caspase 3, thus leading to cell death by apoptosis. Furthermore, JNK and p38 activation precede in time the programmed cell death induced by hypoxia.

Acute hypoxia differentially affects the gamma-aminobutyric acid type A receptor alpha(1), alpha(2), beta(2), and gamma(2) subunit mRNA levels in the developing chick optic tectum: stage-dependent sensitivity.

This investigation analyzes the effect of an acute hypoxic treatment on the level of four (alpha(1), alpha(2), beta(2), and gamma(2)) subunit mRNAs of the GABA(A) receptor in layer "i" of the developing chick optic tectum. Our results show that 1 hr of normobaric acute hypoxia significantly changes the subunit mRNA levels. Different subunit mRNAs display different sensitivity to hypoxia: alpha(1), beta(2), and gamma(2) mRNAs are highly sensitive, whereas alpha(2) mRNA is almost not affected. The sensitivity of the mRNA levels to hypoxia is stage dependent. The mean percentages of variation produced by the hypoxia in the level of expression of the four subunits were 20% at ED12, 5% at ED16, and only 2% at ED18. These changes in the mean percentages of expression modify the probability of coexpression. In the case of double mRNA combinations, the hypoxia produced a mean variation in the probability of coexpression of 37% at ED12, 8% at ED16, and only 4% at ED18. With regard to the triple subunit mRNAs combinations, the variations were 206% at ED12, 11% at ED16, and only 7% at ED18. The quadruple combination values were 1,500% at ED12, 21% at ED16, and only 11% at ED18. This study demonstrates that the subunit mRNA levels are highly sensitive during the early stages, suggesting that GABA(A) receptor composition might undergo environment-dependent plastic changes providing a high degree of plasticity to the GABA neurotransmitter system development.

Hypoxia-induced cell death and activation of pro- and anti-apoptotic proteins in developing chick optic lobe.

Exposure of the CNS to hypoxia is associated with cell death. Our aim was to establish a temporal correlation between cellular and molecular alterations induced by an acute hypoxia evaluated at different post-hypoxia (p-h) times and at two stages of chick optic lobe development: embryonic days (ED) 12 and 18. TUNEL assays at ED12 disclosed a significant increase (300%) in pyknotic cells at 6 h p-h, while at ED18 no morphological changes were observed in hypoxic versus controls. At ED12 there was a significant increase (48%) in Bcl-2 levels at the end of the hypoxic treatment, followed by a significant increase of active caspase-9 (49%) and active caspase-3 (58%) at 30 and 60 min p-h, respectively, while at ED18 no significant changes were observed. These findings indicate that prenatal hypoxia produces an equilibrated imbalance in both pro- and anti-apoptotic proteins that culminates in a process of cell death, present at earlier stages of development.

Development and localisation of GABA(A) receptor alpha1, alpha2, beta2 and gamma2 subunit mRNA in the chick optic tectum.

An in situ hybridisation technique was used to analyse the spatial and temporal pattern of expression of the mRNA encoding the four gamma-aminobutyric acid A (GABA(A)) receptor subunits (alpha1, alpha2, beta2, and gamma2) in the developing chick optic tectum. As a rule, layer i, layer h, and transient cell compartment 3 (TCC3) show the highest levels of expression, especially of alpha1, alpha2 and beta2, which undergo striking changes as a function of time. Apart from these common features, the global pattern is highly complex and dynamic. Such complexity derives from the fact that each subunit exhibits a characteristically distinct pattern of expression and the temporal evolution of each differs in the different layers of the tectum. The influence of several developmental cell behaviours such as proliferation, neuronal migration, programmed cell death, and differentiation must be taken into account to understand pattern complexity and dynamics. Our results suggest that differences in the rate of subunit expression, particularly of alpha1, alpha2, and beta2, could have significant consequences on GABA(A) receptor complex subunit composition along development and on the functional properties of the GABA neurotransmitter system.

Vesicular stomatitis virus induces apoptosis at early stages in the viral cycle and does not depend on virus replication.

We detected apoptosis induction in the vesicular stomatitis virus (VSV) infected mammalian cell lines Vero-76, Cos-7, and BHK-21. Cell lines were analyzed by chromosomal DNA fragmentation and nuclear morphology. In order to determine the step in the viral cycle at which apoptosis of infected cells is triggered, chemical and physical agents were used to block viral infection at different times and then the apoptotic response of infected cells was examined. The treatment of Vero-76 infected cells with a lysosomotrophic agent, such as NH4Cl, was shown to abrogate virus apoptosis induction. On the other hand, VSV-induced apoptosis was not blocked by the presence of cycloheximide, suggesting that the de novo viral protein synthesis is not required for this process. UV-inactivated viruses were also capable of inducing apoptosis in Vero-76 cells, indicating that the activation of a programmed cell death process by VSV does not require viral replication. We conclude from these findings that VSV induces apoptosis at early stages of infection.