Neonatal Exposure to CTM selectively affects SERT immunoreactivity in cortical, but not subcortical structures
Given the possibility that changes in the expression profiles of serotonergic cell bodies (Maciag et al., 2006
) translate into changes within terminal processes of raphe target sites, the SERT-ir fiber density within the HIP (target of midline DR, caudal DR and MR), VB (target of DR lateral wing), and C-P (target of rostral DR) was assessed. Representative photomicrographs in demonstrate that a decrease in SERT-ir fiber density is observed within the dHIP (dCA1: A1, B1) and vHIP (vCA1: A2, B2), but not within the VB (A3, B3), nor C-P (A4, B4) of CTM10 treated rats compared to saline controls. Quantitative analysis revealed that the decrements in CTM10-induced SERT density were significant within each hippocampal subdivision (). The median SERT-ir fiber density within the dCA1 of CTM10 treated rats was 4.37% of the sampled area, while saline treated subjects revealed a median thresholded area of 5.78%. When SERT-ir fiber density was expressed as a function of baseline scores in controls, a decrease of ~24.4% was noted. Similarly, the median SERT-ir fiber density in dCA3 of CTM10 treated animals was 5.41% of the sampled area, while saline controls were found to exhibit a thresholded area of 7.43%. The corresponding decrement between the two treatment groups was calculated to be ~27%. Comparisons in the hilus revealed a median SERT-ir fiber density of 2.48% in the CTM10 treatment group and 3.67% in the saline treatment group. The reduction corresponded to an apparent loss of ~32% of SERT-ir innervation. Within the subdivisions of the vHIP (vCA1 and vCA3), the median SERT-ir density of CTM10 treated rats was 3.47% and 4.87% of the sampled area, respectively. Saline controls from the corresponding experimental subsets exhibited thresholded areas of 5.07% and 6.51%. Normalization of this data indicated a loss of SERT-ir fibers on the order of ~32% and ~25% from baseline values in saline treated rats. Overall, CTM treatment was found to promote a loss of ~28.5% of SERT-ir fibers in the HIP.
Figure 1 Representative photomicrographs of SERT expression, visualized with Cy3, within dCA1, vCA1, VB, and C-P in adult rats treated with saline (A1-A4) and CTM10 (B1-B4) from PN8-21. Note that a decrease in SERT-ir fiber density can be seen within the dHIP (more ...)
Change in SERT-ir fiber density within HIP subdivisions of CTM10 treated rats (compared to saline)
In addition, the relative differences in SERT-ir fiber density that were observed between subdivisions of the dorsal and ventral hippocampus were unaltered by neonatal CTM10 treatment (). In saline-treated rats, the density within the CA3 was the highest, followed by the CA1 subdivision, then the hilus. This trend in relative SERT density is reasonably consistent in CTM10 treated rats even though the SERT density was decreased. This observation suggests that all hippocampal subdivisions are, for the most part, equally affected by neonatal SSRI treatment.
Chart showing relative SERT density within subdivisions of dorsal and ventral hippocampus of one experimental subset (dCA3 = vCA3 > dCA1 = vCA1 > hilus). Note that the relative SERT density does not change with CTM10 treatment.
Since neonatal CTM treatment appears to affect all hippocampal subdivisions similarly, data across different HIP subdivisions was combined. graphically displays the results of a semi-quantitative analysis performed on various forebrain terminal regions. The median SERT-ir fiber density within the dHIP of CTM10 treated rats was 4.08%, while saline treated animals demonstrated a thresholded area of 5.62%; a ~27% difference between the two groups when CTM values were expressed as a function of saline controls. In the vHIP, CTM10-treated rats showed a thresholded area of 4.17%, while saline treated subjects exhibited a SERT-ir fiber density of 5.79%; an apparent drop in SERT-positive profiles that practically mirrored dHIP (~28%). The reductions in SERT density within these regions reached statistical significance (both dHIP and vHIP; p ≤ 0.008). On the other hand, the change in SERT density within the VB of CTM10 treated rats was negligible. Compared to saline controls, which demonstrated a SERT-ir fiber density of 1.61%, the median thresholded area for CTM10-treated rats was 1.62%. Lastly, there was a small decrease in SERT-ir fiber density that was observed in the C-P of CTM10 treated rats. The median thresholded area for CTM10-treated subjects was 2.55%, while saline controls exhibited a sampled area of 2.83%. This reduction, approximately 10% from baseline values, did not reach statistical significance.
Figure 3 Graph comparing SERT-ir fiber density within dHIP, vHIP, VB, and C-P of saline- and CTM10-treated rats. Data represent the median, 25% and 75% percentile (box) and the minimum and maximum (whiskers) values of 8–11 subsets per group. *p = 0.001, (more ...)
We also examined hippocampal tissue sections for qualitative changes in SERT fiber morphology, because neonatal CTM10 treatment has been shown to influence the morphology of SERT labeled axons within the cortex of rats that were sacrificed as adults (Maciag et al., 2006
). Axons have been reported to take on a discontinuous or “beaded” appearance due to the lack of immunolabeling within intervaricose segments. In addition, the current study has revealed a second population of axons. Thick fibers, exhibiting intense fluorescence, were found in conjunction with CTM treatment. Representative photomicrographs in demonstrate a few examples of these thick axons within dCA1 (A), dCA3 (B), and C-P (C) of CTM10 treated rats. These thick axons are readily observed within all subdivisions of the dHIP and vHIP, but are rarely seen in saline treated rats. Furthermore, alterations in axonal morphology are infrequently observed within the VB.
Figure 4 Representative photomicrographs of SERT-ir fibers visualized with Cy3 within subdivisions of dCA1 (A), dCA3 (B), and C-P (C) of CTM10 treated rats. These images show changes in axonal morphology (presence of “thick axons” depicted by arrows: (more ...)
SERT-ir fiber density following exposure of neonates to different doses of CTM
Adult rats (n=3–6 subsets) were examined after they had been treated neonatally with different doses of CTM. In specific, SERT-ir fiber density was evaluated in dHIP, vHIP, VB, and C-P after rats had been exposed to CTM5, CTM10, and CTM20. Representative photomicrographs through the dCA1 (and VB) in demonstrate the dramatic step-wise decrease in SERT-ir fiber density that was observed within all subdivisions of dHIP and vHIP with increasing concentrations of CTM. This pattern of declining SERT-ir is similar from HIP subregion to subregion, and is illustrated in and the bar graph. More specifically, the median thresholded area for CTM5 treated subjects in dCA1, dCA3, the hilus, vCA1, and vCA3 was 5.145, 7.460, 3.355, 4.450, and 5.520, compared to baseline values in saline treated rats of 6.170, 8.940, 3.670, 5.185, and 6.860, respectively. The percentage of lost immunoreactivity corresponded to 16.6%, 16.6%, 8.6%, 14.2%, and 19.5%. Likewise, for CTM10 treated subjects, the sampled SERT-ir fiber density was revealed to be 3.930, 5.720, 2.620, 3.470, and 4.855. Normalized values indicated a decrease of 36.3%, 36.0%, 28.6%, 33.1%, and 29.2%, respectively. Calculations performed on data collected in CTM20-treated rats showed thresholded areas of 2.730, 3.985, 1.480, 2.470, and 2.920. Staining decrements for these subdivisions were determined to be on the order of 55.8%, 55.4%, 59.7%, 52.4%, and 57.4%, respectively. Taken together, CTM5 treatment promotes a loss of 8.6–19.5% of SERT-ir fibers, CTM10 treatment results in a loss of 28.6–36.3% of SERT-ir fibers, and CTM 20 treatment is associated with a reduction in SERT-ir innervation density on the order of 52.4–9.7%.
Figure 5 Representative photomicrographs of SERT/Cy3 labeled fibers within dCA1 and VB of saline-, CTM5-. CTM10-, and CTM20-treated rats. Within the dCA1, the trend is for decreased labeling with increasing doses of CTM. Note: this trend is not evident within (more ...)
Change in SERT-ir fiber density within HIP subdivisions of CTM 5, 10, and 20 treated rats (compared to saline)
Figure 6 Change in SERT density within HIP subdivisions of CTM5-, CTM10-, and CTM20-treated rats. Data represent the median, 25% and 75% percentile (box) and the minimum and maximum (whiskers) values of 5–6 subsets per group. Kruskal-Wallis statistic = (more ...)
The striking loss of SERT-ir fibers in HIP is even more evident when data across HIP subdivisions is combined into just two sectors, dHIP and vHIP, and analyzed with respect to VB and C-P. These results are graphically depicted in , where the incremental decrease in the HIP SERT-ir fiber population stands in sharp contrast to the unchanging number of labeled axon profiles in VB and C-P across the CTM dosing range. It is worthwhile to mention, that although there was an obvious decrease in the density of SERT-ir fibers in each zone of HIP at lower doses of CTM, statistical significance relative to the saline treatment group was only achieved following exposure to CTM20 (p < 0.05). These findings differ slightly from data demonstrated in , where values for CTM10 were indeed also found to be statistically significant. However, as will be remarked upon in the discussion, this discrepancy can be attributed to a smaller number of cases per treatment group in the dose-response analysis, and also to inherent staining variability, that we, in fact, attempted to compensate for through the use of “subset” tissue processing.
Specific values for each of the treatment groups (that appear in ) are listed according to brain region. Following CTM5 treatment, median thresholded areas of SERT-ir in the dHIP, vHIP, VB, and C-P, were found to be 5.403, 5.015, 1.560, and 2.440, respectively. Corresponding calculations in saline treated groups were determined to be 6.308, 6.030, 1.560, and 2.680. Expression of these data as percentages revealed a 14.3% loss of SERT-ir fibers in dHIP, and a 16.8% reduction in vHIP after CTM5 exposure. Reductions in SERT innervation density within sampled areas after CTM10 treatment were recorded as follows: 4.088, 4.215, 1.605, and 2.400. These numbers translated into a 35.2% loss of SERT-ir fibers in dHIP, and a 30.1% decline in vHIP. The median area of labeling detected after CTM20 exposure was found to be 2.763, 2.695, 1.510, and 2.350, respectively. By normalizing these values, it was determined that dHIP sustained a 56.2% drop in SERT-ir fiber density, while a loss of 55.3% was incurred by the vHIP. In VB, the percentage of lost SERT-ir fibers ranged from 0–3.2% over the CTM5-CTM20 dose spectrum, while the proportion of decreased labeling in C-P spanned a measure of 9.0–12.3%.
Increased frequency of “thick” SERT-ir fibers with elevated levels of neonatal CTM
Careful examination of morphological changes in raphe terminal fields revealed two distinguishing features regarding SERT-ir axons. First, there is a clear association between increasing doses of neonatal CTM and a predisposition for increased numbers of thick SERT-ir fibers. Secondly, these fibers, when studied at high magnification, often exhibit a smooth, non-varicose profile (). As mentioned before, these alterations were most readily observed in dHIP, and vHIP. Such intensely fluorescent, thick axonal profiles were rarely evident in VB and C-P. The dose-dependence of this response was particularly evident after the distribution of SERT-ir fibers in HIP was mapped. Thick, SERT-ir fibers were found to be more prevalent after the administration of higher doses of CTM (CTM10 and CTM20), than with lower doses (CTM5)().
Figure 8 Representative photomicrographs of SERT-ir fibers visualized with Cy3 within dCA1 of a saline (A) and a CTM20 (B) treated rat. The image in (B) illustrates characteristic changes in axonal morphology that occur after CTM treatment. Notice the presence (more ...)
Figure 9 Schematic illustration of the distribution of “thick” SERT-ir axons after increasing doses of CTM. Each pane represents one section through the dorsal HIP of one rat in each treatment group. Note the clear predominance of “thick” (more ...)