Changes in apical dendritic structure correlate with sustained ERK1/2 phosphorylation in medial prefrontal cortex of a rat model of dopamine D1 receptor agonist sensitization

Abstract

Rats lesioned with 6-hydroxydopamine (6-OHDA) as neonates exhibit behavioral and neurochemical abnormalities in adulthood that mimic Lesch-Nyhan disease, schizophrenia, and other developmental disorders of frontostriatal circuit dysfunction. In these animals a latent sensitivity to D1 agonists is maximally exposed by repeated administration of dopamine agonists in the postpubertal period (D1 priming). In neonate-lesioned, adult rats primed with SKF-38393, we found selective, persistent alterations in the morphology of pyramidal neuron apical dendrites in the prelimbic area of the medial prefrontal cortex (mPFC). In these animals, dendrite bundling patterns and the typically straight trajectories of primary dendritic shafts were disrupted, whereas the diameter of higher-order oblique branches was increased. Although not present in neonate-lesioned rats treated with saline, these morphological changes persisted at least 21 days after repeated dosing with SKF-38393, and were not accompanied by markers of neurodegenerative change. A sustained increase in phospho-ERK immunoreactivity in wavy dendritic shafts over the same period suggested a relationship between prolonged ERK phosphorylation and dendritic remodeling in D1-primed rats. In support of this hypothesis, pretreatment with the MEK1/2-ERK1/2 pathway inhibitors PD98059 or SL327, prior to each priming dose of SKF-38393, prevented the morphological changes associated with D1 priming. Together, these findings demonstrate that repeated stimulation of D1 receptors in adulthood interacts with the developmental loss of dopamine to profoundly and persistently modify neuronal signaling and dendrite morphology in the mature prefrontal cortex. Furthermore, sustained elevation of ERK activity in mPFC pyramidal neurons may play a role in guiding these morphological changes in vivo.

Fig. 1

Experimental timelines for animal treatments and analyses. For each study, the number of experimental subjects is provided in parentheses, and tissue analyses performed post-sacrifice are indicated on the right. All experiments consisted of 6-OHDA or sham lesioning on PND 4, followed by behavioral priming with weekly treatments of SKF-38393 or saline beginning at PND 42 (arrows over darkened band). Locomotor activity was monitored immediately following each dose. Seven days after the final treatment, animals were perfused transcardially (S), except for a subset of subjects in study A that were sacrificed on PND 84 (21 days after the final dosing, as indicated with an asterisk). In B, cannula implantation (CI) was performed on PND 35 to prepare for preinfusions with MEK inhibitor. Preinfusions were administered 30 min prior to each weekly dose of SKF-38393. To transduce neuronal GFP expression, rats in 2 studies (C and D) received prefrontal cortical injections of AAV-GFP at PND 30 (GFP), followed by agonist dosing beginning on PND 42. In study D, animals expressing GFP were pretreated with systemic injections of SL-327, 30 min before each dose of SKF-38393.

Fig. 2

MAP2 immunohistochemistry is altered in mPFC of D₁-primed rats. Roman numerals indicate approximate location of cortical layers. All animals were euthanized 7 d after the last dose of SKF-38393 or saline except where indicated. (A) Representative mPFC section from a sham-lesioned rat treated with weekly injections of saline (Sham-Sal). (B) Higher power image from the same region in a sham-lesioned rat treated with weekly injections of SKF-38393 (Sham-SKF). (C) Neonate-lesioned rat treated with weekly injections of saline (Les-Sal). (D, E) Lesioned rat treated with weekly injections of SKF-38393 (D₁-primed, Les-SKF). (F) MAP2 in a Les-SKF rat euthanized 21 d after the final dose of SKF-38393. (G, H) Confocal images of MAP2 immunofluorescence in Sham-SKF and Les-SKF rat mPFC, respectively. Note the coarser, more granular appearance of punctate elements in the neuropil of primed rats (arrows in H), and the thickening of dendritic branches at the interface of layers II/III and I (arrowheads in H) compared to those of control rats. (I) Schematic diagram of region of interest, adapted from Paxinos and Watson (1998). Gray box represents area depicted in A and D. (J) MAP2 immunostaining in Les-SKF visual cortex was unaltered. Scale bars for A, D and J, 100 μm. Scale bars for B, C and E-H, 50 μm. Note: a magenta-green version of this figure can be viewed online as Supplementary Figure 1.

Fig. 3

MAP2, αII-spectrin, total cell counts and cortical thickness are all unchanged in mPFC of D₁-primed rats. (A) Representative Western blot of MAP2 and β-actin in mPFC of SKF-38393-treated sham (Sham-SKF) and lesioned rats (Les-SKF) and their saline-treated counterparts (Sham-Sal and Les-Sal, respectively). Faint bands in the center of each lane are consistent with MAP2 degradation products. (B) Spectrin and its breakdown products (SBDPs) in mPFC homogenates from Sham-Sal and Les-SKF rats. Neither the 145-150 kDa calpain-mediated cleavage products, nor the 120 kDa caspase-3 SBDP were altered by D₁ priming. (C) Cresyl violet-stained neurons counted at day 21 after dosing across superficial (I-III) and deep (V-VI) mPFC layers. Data are expressed as percent of total cell counts across all layers: black bars, Sham-Sal, 4810.5 ± 130.7 total cells; dark gray bars, Sham-SKF, 4237.7 ± 289.2 cells; white bars, Les-Sal, 4781.0 ± 441.9 cells; light gray bars, Les-SKF, 4326.5 ± 269.2 cells counted across all layers. (D) Mean cortical layer thickness at day 21 plotted as percent of total mPFC thickness. Filled squares, Sham-Sal; filled circles, Sham-SKF; open squares, Les-Sal; open circles, Les-SKF.

Fig. 4

Confocal imaging of mPFC neurons transduced with AAV-GFP. (A) Clusters of fluorescent neurons surrounding the injection site in Sham-SKF mPFC. Prominent first order dendrites were long and straight, branching into tufts at the interface of layers II/III and I. (B) Representative high power image of GFP-positive dendrites in mPFC of a Les-SKF rat sacrificed at day 7 after the priming regimen. Arrows indicate the presence of dendritic spines. (C) Overlay of the image in B with the corresponding image of MAP2 immunofluorescence showing colocalization in larger dendritic shafts but not in spines (arrows). (D) Image of a single 1 μm optical section (D.a), and the corresponding reconstructed projection of 10 optical sections (D.b), of Sham-SKF mPFC. Most dendrites are visible along their entire length in both single slice images and projections (indicated by arrowheads). (E) Single 1 μm optical section (E.a), and corresponding stacked reconstruction (E.b), of dendrites in Les-SKF mPFC. Several dendrites disappeared out of the single section images (arrowheads in E.a), and could only be followed for their entire length by stacking multiple images as shown in E.b. Scale bar for A, 50 μm. Scale bar for B-E, 20 μm. Note: a magenta-green version of this figure can be viewed online as Supplementary Figure 2.

Fig. 5

Sustained ERK phosphorylation may contribute to mPFC morphological abnormalities in D₁-primed rats. Antigen retrieval was used to intensify phospho-ERK staining in Sham-SKF (A and A’) and Les-SKF (B and B’) mPFC. Note the wavy phospho-ERK-positive dendritic shafts (arrows) and enhanced immunostaining of dendrite apical tufts (arrowheads) in sections from Les-SKF (D₁-primed) rat mPFC compared to that of Sham-SKF. Scale bars, 200 μm. Specificity of the antibody under antigen retrieval conditions is shown by abolition of staining using a phosphorylated (C), but not a non-phosphorylated (C’) blocking peptide. (D) MEK inhibitors administered prior to each weekly dose of SKF-38393 prevented the sustained increase in phospho-ERK observed at day 7 in D₁-primed rats. Sham-lesioned (filled bars) and neonate-lesioned (open bars) animals were administered icv infusions of vehicle, PD98059 or SL327, 30 min before each weekly systemic injection of SKF-38393. Cell counts were obtained from mPFC sections immunostained for phospho-ERK without antigen retrieval and represent total counts across all cortical layers. Represented are means ± S.E.M. ** p < 0.0001 vs. sham-lesioned rats preinfused with vehicle prior to SKF-38393 (Sham-Veh-SKF); ‡‡‡ p < 0.0001 vs. D₁-primed rats preinfused with vehicle (Les-Veh-SKF). (E and E’) Representative photomicrographs of MAP2 immunohistochemistry in Les-Veh-SKF and Les-SL-SKF mPFC, respectively. Scale bars, 50 μm. (F) Quantitative analysis of MAP2 immunostaining using linear area fraction measurements. The format of the graph is the same as in D. Preinfusions of either PD98059 or SL327 had no effect in sham rats treated with SKF-38393 (filled bars), but prevented the loss of linear dendritic immunostaining in lesioned rats that were primed with the agonist (open bars). * p < 0.001 vs. Sham-Veh-SKF; ‡‡ p < 0.001, and ‡ p < 0.05 vs. Les-Veh-SKF; ANOVA with Fisher’s PLSD.

Fig. 6

Systemic treatment with SL327 prevents the structural abnormalities induced by D₁-priming. Rats were administered 100 mg/kg ip SL327 (sSL) or vehicle (sVeh), 30 min prior to each weekly dose of SKF-38393 and sacrificed 7 days following the final treatment. (A-C), Reconstructed confocal images of GFP-positive dendrites in Sham-sVeh-SKF (A), Les-sVeh-SKF (B), and Les-sSL-SKF (C) rat mPFC. Scale bars, 50 μm. (D) Linearity index of dendritic elements in GFP-expressing mPFC of Sham-sVeh-SKF (black bars), Les-sVeh-SKF (open bars), and Les-sSL-SKF rats (gray bars). Represented are means ± S.E.M. *** p < 0.0001 vs. Sham-sVeh-SKF. (E) Diameter of dendritic elements in mPFC. The format is the same as in D. ** p < 0.005, and * p < 0.05 vs. Sham-sVeh-SKF; ANOVA with Fisher’s PLSD.