Involvement of dopamine receptor in the actions of non-psychoactive phytocannabinoids.

Nematode Caenorhabditis elegans (C. elegans) exhibited a vigorous swimming behavior in liquid medium. Addition of dopamine inhibited the swimming behavior, causing paralysis in 65% of wild-type nematodes. Interestingly, phytocannabinoids cannabidiol (CBD) or cannabidivarin (CBDV), caused paralysis in 40% of the animals. Knockout of DOP-3, the dopamine D2-like receptor critical for locomotor behavior, eliminated the paralysis induced by dopamine, CBD, and CBDV. In contrast, both CBD and CBDV caused paralysis in animals lacking CAT-2, an enzyme necessary for dopamine synthesis. Co-administration of dopamine with either CBD or CBDV caused paralysis similar to that of either phytocannabinoid treatment alone. These data support the notion that CBD and CBDV act as functional partial agonists on dopamine D2-like receptors in vivo. The discovery that dopamine receptor is involved in the actions of phytocannabinoids moves a significant step toward our understanding of the mechanisms for medical uses of cannabis in the treatment of neurological and psychiatric disorders.

Gender Differences in Cardiac Remodeling Induced by a High-Fat Diet and Lifelong, Low-Dose Cadmium Exposure.

Childhood obesity, which is prevalent in developed countries, is a metabolic risk factor for cardiovascular disease. Cadmium (Cd), a ubiquitous environmental toxic metal, also has deleterious effects on the cardiovascular system. However, the combined effects of a high-fat diet (HFD) and lifelong, low-dose Cd exposure on cardiac remodeling remain unclear. This study aims to determine the effects of combined HFD and Cd exposure on cardiac remodeling, as well as gender-specific differences in the response. C57BL/6J mice were exposed to Cd at a low dose (L-Cd, 0.5 ppm) or high dose (H-Cd, 5 ppm) via drinking water from conception to sacrifice. After being weaned, the offspring mice were fed with a HFD (42% kcal from fat) for an additional 10 weeks. H-Cd exposure significantly increased Cd accumulation in the hearts of both parents and their offspring; a HFD showed no added effects on cardiac Cd content. H-Cd exposure increased cardiac metallothionein protein levels only in female mice, regardless of dietary intake. Histological analysis revealed that H-Cd exposure combined with a HFD induced cardiac hypertrophy and fibrosis only in female mice. This was further supported by elevated expression of ANP and COL1A1 protein levels along with COL1A1, COL1A2, and COL3A1 mRNA levels. Profibrotic markers PAI-1, CTGF, and FN were also elevated in HFD/H-Cd-exposed female mice. Levels of the oxidative stress marker 3-NT significantly increased in the hearts of HFD-fed female mice, whereas Cd exposure showed no additional effects. Of all the antioxidant markers examined, levels of CAT significantly increased in mice fed a HFD, regardless of gender and Cd exposure. In summary, a HFD combined with lifelong, low-dose Cd exposure induces cardiac hypertrophy and fibrosis in female but not male mice, a response that is independent of oxidative stress.

A novel conserved RNA-binding domain protein, RBD-1, is essential for ribosome biogenesis.

Synthesis of the ribosomal subunits from pre-rRNA requires a large number of trans-acting proteins and small nucleolar ribonucleoprotein particles to execute base modifications, RNA cleavages, and structural rearrangements. We have characterized a novel protein, RNA-binding domain-1 (RBD-1), that is involved in ribosome biogenesis. This protein contains six consensus RNA-binding domains and is conserved as to sequence, domain organization, and cellular location from yeast to human. RBD-1 is essential in Caenorhabditis elegans. In the dipteran Chironomus tentans, RBD-1 (Ct-RBD-1) binds pre-rRNA in vitro and anti-Ct-RBD-1 antibodies repress pre-rRNA processing in vivo. Ct-RBD-1 is mainly located in the nucleolus in an RNA polymerase I transcription-dependent manner, but it is also present in discrete foci in the interchromatin and in the cytoplasm. In cytoplasmic extracts, 20-30% of Ct-RBD-1 is associated with ribosomes and, preferentially, with the 40S ribosomal subunit. Our data suggest that RBD-1 plays a role in structurally coordinating pre-rRNA during ribosome biogenesis and that this function is conserved in all eukaryotes.

Neuropeptide Y inhibits the hyperexcitability of type A neurons in chronically compressed dorsal root ganglion of the rat.

Our recent data revealed adrenergic sensitivity in chronically compressed dorsal root ganglion (DRG) of rats. As neuropeptide Y (NPY) is a common sympathetic co-transmitter, we investigated the effect of NPY on injured DRG neurons. The expression of NPY Y1 and Y2 receptors and the effect of NPY on chronically compressed DRG neurons were studied using in situ hybridization and extracellular single fiber recording in vitro, respectively. After DRG compression, the expression of Y1 receptor was distinctly increased in large and medium-sized DRG neurons, while Y2 receptor was increased in small DRG neurons. NPY inhibited both the spontaneous activity and the excitatory effect of norepinephrine in injured DRG A-neurons. The results suggest a possibility that NPY may inhibit the hyperexcitability of injured DRG A-neurons via increased Y1 receptor following chronic compression.

Conditions for dye-filling of sensory neurons in Caenorhabditis elegans.

Dye-filling is a common method used to stain Caenorhabditis elegans sensory neurons in vivo. While the amphids and phasmids are easy to stain, a subset of sensory neurons, the IL2 neurons, are difficult to stain reproducibly. Here we examined the conditions under which the IL2 neurons take up the lipophilic fluorescent dye DiI. We find that IL2 dye-filling depends on salt concentration, but not osmolarity. Low salt prior and during incubation is important for efficient dye uptake. Additional parameters that affect dye-filling are the speed of shaking during incubation and the addition of detergents. Our modified dye-filling procedure provides a reliable method to distinguish mutant alleles that stain amphids and phasmids, IL2 neurons, or both. An additional benefit is that it can also stain the excretory duct. The method allows genetic screens to be performed to identify mutants that selectively affect only one of the sensory structures or the excretory duct.

Peripheral axotomy induces only very limited sprouting of coarse myelinated afferents into inner lamina II of rat spinal cord.

Peripheral axotomy-induced sprouting of thick myelinated afferents (A-fibers) from laminae III-IV into laminae I-II of the spinal cord is a well-established hypothesis for the structural basis of neuropathic pain. However, we show here that the cholera toxin B subunit (CTB), a neuronal tracer used to demonstrate the sprouting of A-fibers in several earlier studies, also labels unmyelinated afferents (C-fibers) in lamina II and thin myelinated afferents in lamina I, when applied after peripheral nerve transection. The lamina II afferents also contained vasoactive intestinal polypeptide and galanin, two neuropeptides mainly expressed in small dorsal root ganglion (DRG) neurons and C-fibers. In an attempt to label large DRG neurons and A-fibers selectively, CTB was applied four days before axotomy (pre-injury-labelling), and sprouting was monitored after axotomy. We found that only a small number of A-fibers sprouted into inner lamina II, a region normally innervated by C-fibers, but not into outer lamina II or lamina I. Such sprouts made synaptic contact with dendrites in inner lamina II. Neuropeptide Y (NPY) was found in these sprouts in inner lamina II, an area very rich in Y1 receptor-positive processes. These results suggest that axotomy-induced sprouting from deeper to superficial layers is much less pronounced than previously assumed, in fact it is only marginal. This limited reorganization involves large NPY immunoreactive DRG neurons sprouting into the Y1 receptor-rich inner lamina II. Even if quantitatively small, it cannot be excluded that this represents a functional circuitry involved in neuropathic pain.