Chronic levodopa administration followed by a washout period increased number and induced phenotypic changes in striatal dopaminergic cells in MPTP-monkeys.

In addition to the medium spiny neurons the mammalian striatum contains a small population of GABAergic interneurons that are immunoreactive for tyrosine hydroxylase (TH), which dramatically increases after lesions to the nigrostriatal pathway and striatal delivery of neurotrophic factors. The regulatory effect of levodopa (L-Dopa) on the number and phenotype of these cells is less well understood. Eleven macaques (Macaca fascicularis) were included. Group I (n = 4) received 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine (MPTP) and L-Dopa; Group II (n = 4) was treated with MPTP plus vehicle and Group III (n = 3) consist of intact animals (control group). L-Dopa and vehicle were given for 1 year and animals sacrificed 6 months later. Immunohistochemistry against TH was used to identify striatal and nigral dopaminergic cells. Double and triple labeling immunofluorescence was performed to detect the neurochemical characteristics of the striatal TH-ir cells using antibodies against: TH, anti-glutamate decarboxylase (GAD(67)) anti-calretinin (CR) anti-dopa decarboxylase (DDC) and anti-dopamine and cyclic AMP-regulated phosphoprotein (DARPP-32). The greatest density of TH-ir striatal cells was detected in the striatum of the L-Dopa treated monkeys and particularly in its associative territory. None of the striatal TH-ir cell expressed DARPP-32 indicating they are interneurons. The percentages of TH-ir cells that expressed GAD67 and DDC was approximately 50%. Interestingly, we found that in the L-Dopa group the number of TH/CR expressing cells was significantly reduced. We conclude that chronic L-Dopa administration produced a long-lasting increase in the number of TH-ir cells, even after a washout period of 6 months. L-Dopa also modified the phenotype of these cells with a significant reduction of the TH/CR phenotype in favor of an increased number of TH/GAD cells that do not express CR. We suggest that the increased number of striatal TH-ir cells might be involved in the development of aberrant striatal circuits and the appearance of L-Dopa induced dyskinesias

General characteristics of the animals and TH-ir cell counting in the striatum and substantia nigra.

*<p>: <i>P</i>≤0.05 compared to vehicle or L-Dopa with control group.</p><p>#: <i>P</i>≤0.05 compared to L-Dopa with vehicle group. The data show the mean and its corresponding SEM.</p

Graphic representation of the distribution of striatal TH-ir neurons in each experimental group.

<p>Note that L-Dopa group has the highest number of TH-ir cells, particularly in the more rostral areas. The delineation of territory boundaries based on anatomical assessments is also showed. Sensorimotor (SM), Associative (ASS) and Limbic (Lim) territories of the caudate (Cd), Putamen (Pu) and part of nucleus accumbens (Acc) are included. The distances from the anterior commissure (AC) are indicated.</p

Experimental study design, treatments and group assignments.

<p>Experimental study design, treatments and group assignments.</p

Striatal TH-ir cell distribution.

<p>(A) Mean of density of TH- ir cells in the pre- commissural and post-commissural striatum of control (n = 3), vehicle (n = 4) and L-Dopa (n = 4) groups. Mann and Whitney U: * = p<0.05. (B) Mean density of TH-ir cells in the anterior and posterior parts of pre-commissural striatum of control (n = 3), vehicle (n = 4) and L-Dopa (n = 4) groups. Mann and Whitney U: * = p<0.05.</p

The graphic represents the results of triple immunofluorescence studies in the pre-commissural striatum of every experimental group and the percentage of TH-ir cells that co-localizes with glutamate decarboxylase (GAD67+) and calretinin (CR+).

<p>Note that phenotype TH+/GAD67+/CR- was significantly increased in the L-Dopa group with respect to control group (* = p<0.05 using Tukey test following one way ANOVA repeated measures). Error bars represent SEM.</p

Striatal coronal sections immunostained with TH and counterstained with Nissl showing the morphology of the striatal dopaminergic cells.

<p>Control monkey (A) vehicle-treated monkey (B) and L-Dopa-treated monkey (C). Scale bar, 100 µm (A-C) and 20 µm (A’–C’).</p

Double immunofluorescence images of the striatal TH-ir cells showing one TH-ir cell that also express dopa-decarboxilase (DDC) (A), absence of TH-ir cells immunolabeled with the dopamine and cyclic AMP-regulated phosphoprotein (DARPP-32) (B) TH-ir interneurons expressing calretinin (CR) (C) and glutamate decarboxylase (GAD67) (D).

<p>Scale bars 20 µm.</p

The Guanine Nucleotide Exchange Factor RIC8 Regulates Conidial Germination through Gα Proteins in Neurospora crassa

Heterotrimeric G protein signaling is essential for normal hyphal growth in the filamentous fungus Neurospora crassa. We have previously demonstrated that the non-receptor guanine nucleotide exchange factor RIC8 acts upstream of the Gα proteins GNA-1 and GNA-3 to regulate hyphal extension. Here we demonstrate that regulation of hyphal extension results at least in part, from an important role in control of asexual spore (conidia) germination. Loss of GNA-3 leads to a drastic reduction in conidial germination, which is exacerbated in the absence of GNA-1. Mutation of RIC8 leads to a reduction in germination similar to that in the Δgna-1, Δgna-3 double mutant, suggesting that RIC8 regulates conidial germination through both GNA-1 and GNA-3. Support for a more significant role for GNA-3 is indicated by the observation that expression of a GTPase-deficient, constitutively active gna-3 allele in the Δric8 mutant leads to a significant increase in conidial germination. Localization of the three Gα proteins during conidial germination was probed through analysis of cells expressing fluorescently tagged proteins. Functional TagRFP fusions of each of the three Gα subunits were constructed through insertion of TagRFP in a conserved loop region of the Gα subunits. The results demonstrated that GNA-1 localizes to the plasma membrane and vacuoles, and also to septa throughout conidial germination. GNA-2 and GNA-3 localize to both the plasma membrane and vacuoles during early germination, but are then found in intracellular vacuoles later during hyphal outgrowth