Axonal maintenance, glia, exosomes, and heat shock proteins

© The Author(s), 2016. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in F1000Research 5 (2016): 205, doi:10.12688/f1000research.7247.1.Of all cellular specializations, the axon is especially distinctive because it is a narrow cylinder of specialized cytoplasm called axoplasm with a length that may be orders of magnitude greater than the diameter of the cell body from which it originates. Thus, the volume of axoplasm can be much greater than the cytoplasm in the cell body. This fact raises a logistical problem with regard to axonal maintenance. Many of the components of axoplasm, such as soluble proteins and cytoskeleton, are slowly transported, taking weeks to months to travel the length of axons longer than a few millimeters after being synthesized in the cell body. Furthermore, this slow rate of supply suggests that the axon itself might not have the capacity to respond fast enough to compensate for damage to transported macromolecules. Such damage is likely in view of the mechanical fragility of an axon, especially those innervating the limbs, as rapid limb motion with high impact, like running, subjects the axons in the limbs to considerable mechanical force. Some researchers have suggested that local, intra-axonal protein synthesis is the answer to this problem. However, the translational state of axonal RNAs remains controversial. We suggest that glial cells, which envelop all axons, whether myelinated or not, are the local sources of replacement and repair macromolecules for long axons. The plausibility of this hypothesis is reinforced by reviewing several decades of work on glia-axon macromolecular transfer, together with recent investigations of exosomes and other extracellular vesicles, as vehicles for the transmission of membrane and cytoplasmic components from one cell to another.Harold Gainer’s contribution to this research was supported by the Intramural Research Program of the NINDS, NIH

Involvement of Purinergic P2X4 Receptors in Alcohol Intake of High-Alcohol-Drinking (HAD) Rats

Background: The P2X4 receptor is thought to be involved in regulating alcohol-consuming behaviors and ethanol (EtOH) has been reported to inhibit P2X4 receptors. Ivermectin is an anti-parasitic agent that acts as a positive allosteric modulator of the P2X4 receptor. The current study examined the effects of systemically- and centrally-administered ivermectin on alcohol drinking of replicate lines of high-alcohol-drinking (HAD-1/HAD-2) rats, and the effects of lentiviral-delivered short-hairpin RNAs (shRNAs) targeting P2rx4 on EtOH intake of female HAD2 rats. Method: For the 1st experiment, adult male HAD-1 & HAD-2 rats were given 24-hr free-choice access to 15% EtOH vs. water. Dose-response effects of ivermectin (1.5 to 7.5 mg/kg i.p.) on EtOH intake were determined; the effects of ivermectin were then examined for 2% w/v sucrose intake over 5 consecutive days. In the 2nd experiment, female HAD-2 rats were trained to consume 15% EtOH under 2-hr limited access conditions, and dose-response effects of intracerebroventricular (ICV) administration of ivermectin (0.5 to 2.0 μg) were determined over 5 consecutive days. The 3rd experiment determined the effects of microinfusion of a lentivirus expressing P2rx4 shRNAs into the posterior ventral tegmental area (VTA) on 24-hr EtOH free-choice drinking of female HAD-2 rats. Results: The highest i.p. dose of ivermectin reduced alcohol drinking (30-45%) in both rat lines, but did not alter sucrose intake. HAD-2 rats appeared to be more sensitive than HAD1 rats to the effects of ivermectin. ICV administration of ivermectin reduced 2-hr limited access intake (∼35%) of femal

Reduced Levels of mGlu2 Receptors within the Prelimbic Cortex Are Not Associated with Elevated Glutamate Transmission or High Alcohol Drinking

Background A Grm2 cys407* stop codon mutation, which results in a loss of the metabotropic glutamate 2 (mGlu2) receptor protein, was identified as being associated with high alcohol drinking by alcohol-preferring (P) rats. The objectives of the current study were to characterize the effects of reduced levels of mGlu2 receptors on glutamate transmission and alcohol drinking. Methods Quantitative no-net-flux microdialysis was used to test the hypothesis that basal extracellular glutamate levels in the prelimbic (PL) cortex and nucleus accumbens shell (NACsh) will be higher in P than Wistar rats. A lentiviral-delivered short-hairpin RNA (shRNA)-mediated knockdown was used to test the hypothesis that reduced levels of mGlu2 receptors within the PL cortex will increase voluntary alcohol drinking by Wistar rats. A linear regression analysis was used to test the hypothesis that there will be a significant correlation between the Grm2 cys407* mutation and level of alcohol intake. Results Extracellular glutamate concentrations within the PL cortex (3.6 ± 0.6 vs. 6.4 ± 0.6 μM) and NACsh (3.2 ± 0.4 vs. 6.6 ± 0.6 μM) were significantly lower in female P than female Wistar rats. Western blot detected the presence of mGlu2 receptors in these regions of female Wistar rats, but not female P rats. Micro-infusion of shRNAs into the PL cortex significantly reduced local mGlu2 receptor levels (by 40%), but did not alter voluntary alcohol drinking in male Wistar rats. In addition, there was no significant correlation between the Grm2 mutation and alcohol intake in 36 rodent lines (r = 0.29, p > 0.05). Conclusions Collectively, these results suggest a lack of association between the loss of mGlu2 receptors and glutamate transmission in the NACsh and PL cortex of female P rats, and between the level of mGlu2 receptors in the PL cortex and alcohol drinking of male Wistar rats

A guide to laboratory use of the squid Loligo pealei

From the Preface: Unfortunately, a guide like this one does not happen simply. It is the product of polite requests (at first polite, anyway) from colleagues, of mutual nagging, and of wives with persistant memories. What we are attempting to achieve with this contribution is a consolidation of many years of personal experience with squid into a source of practical information hopefully useful to the increasing number of people working with the squid. One does not undertake the task of preparing such a guide for a limited audience with unreserved enthusiasm. It always seems the same amount of work could produce something for a larger audience and therefore, hopefully, of greater significance. On the other hand when the hours and days spent on developing handling procedures, techniques, and "tricks of the squid trade" are considered it seems untenable not to pass this otherwise unpublishable data on in hope of saving others like pain and time. Therefore, what we have attempted to do is informally put together information that should prove useful to people interested in all aspects of squid biology. In this way we hope to make the laboratory utilization of the squid more efficient not only from the standpoint of the investigator and the collector but also for the future of the species Loligo pealei