Multiscale imaging characterization of dopamine transporter knockout mice reveals regional alterations in spine density of medium spiny neurons

Abstract

The dopamine transporter knockout (DAT KO) mouse is a model of chronic hyperdopaminergia used to study a wide range of neuropsychiatric disorders such as schizophrenia, attention deficit hyperactivity disorder (ADHD), drug abuse, depression, and Parkinson's disease (PD). Early studies characterizing this mouse model revealed a subtle, but significant, decrease in the anterior striatal volume of DAT KO mice accompanied by a decrease in neuronal cell body numbers (Cyr et al., 2005). The present studies were conducted to examine medium spiny neuron (MSN) morphology by extending these earlier reports to include multiscale imaging studies using correlated light microscopy (LM) and electron microscopy (EM) techniques. Specifically, we set out to determine if chronic hyperdopaminergia results in quantifiable or qualitative changes in DAT KO mouse MSNs relative to wild-type (WT) littermates. Using Neurolucida Explorer's morphometric analysis, we measured spine density, dendritic length and synapse number at ages that correspond with the previously reported changes in striatal volume and progressive cell loss. Light microscopic analysis using Neurolucida tracings of photoconverted striatal MSNs revealed a highly localized loss of dendritic spines on the proximal portion of the dendrite (30 μm from the soma) in the DAT KO group. Next, thick sections containing MSN dendritic segments located at a distance of 20-60 μm from the cell soma, a region of the dendrite where spine density is reported to be the highest, were analyzed using electron microscope tomography (EMT). Because of the resolution limits of LM, the EM analysis was an extra measure taken to assure that our analysis included nearly all spines. Spine density measurements collected from the EMT data revealed only a modest decrease in the DAT KO group (n=3 mice) compared to age-matched WT controls (n=3 mice), a trend that supports the LM findings. Finally, a synaptic quantification using unbiased stereology did not detect a difference between DAT KO mice (n=6 mice) and WT controls (n=7 mice) at the EM level, supporting the focal nature of the early synaptic loss. These findings suggest that DAT KO mice have MSNs with highly localized spine loss and not an overall morphologically distinct cell shape. The characterization of morphological changes in DAT KO mice may provide information about the neural substrates underlying altered behaviors in these mice, with relevance for human neurological disorders thought to involve altered dopaminergic homeostasis. Results from this study also indicate the difficulty in correlating structural changes across scales, as the results on fine structure revealed thus far are subtle and non-uniform across striatal MSNs. The complexities associated with multiscale studies are driving the development of shared online informatics resources by gaining access to data where it is being analyzed.

Figure 1. 3D renderings of photoconverted medium spiny neurons (MSNs)

Data from cell tracings of photoconverted cells were gathered using Neurolucida Explorer. WT and KO cell tracings were then rendered in 3D using ViQI. [Scale bar = 15 microns; WT = wild-type; KO = dopamine transporter knockout].

Figure 2. Striatal MSN spine density is reduced in DAT KO mice

Spine density measurements collected from a morphometric analysis comparing WT and KO animals. Graph shows a significant reduction in spine density (spine number/10 microns) at 30 microns away from the cell body in KO animals. [MSN = medium spiny neuron; WT = wild-type; KO = dopamine transporter knockout].

Figure 3. Total MSN dendritic length is not significantly different in DAT KO mice

Measurements of total dendritic length collected from a morphometric analysis comparing MSN cell tracings from WT and KO animals. No significant differences were found in total dendritic length between KO and WT mice. [MSN = medium spiny neuron; WT = wild-type; KO = dopamine transporter knockout].

Figure 4. Dendritic intersections are not significantly different in DAT KO mice

Number of dendritic intersections collected from a morphometric analysis comparing MSN cell tracings from WT and DAT KO animals. The number of dendritic intersections between WT and KO animals was not significantly different. [MSN = medium spiny neuron; WT = wild-type; KO = dopamine transporter knockout].

Figure 5. No alterations in DAT KO MSN primary dendrites

Bar graphs comparing the number of primary dendrites between groups. No significant differences were found between DAT KO and WT mice. [MSN = medium spiny neuron; WT = wild-type; KO = dopamine transporter knockout].

Figure 6. 3D rendered dendritic segments from EMT volumes

A representative dendritic segment from each group is shown. 3D-models were reconstructed using IMOD image modeling software [EMT = electron microscopic tomography; WT = wild-type; KO = dopamine transporter knockout; Scale bar = 2 microns].

Figure 7. MSN spine density using EMT volumes is slightly decreased in DAT KO mouse

Bar graphs comparing the spine density (number of spines per unit surface area) between groups. A moderate decrease (did not reach statistical significance) in spine density was recorded between KO and WT mice. [MSN = medium spiny neuron; WT = wild-type; KO = dopamine transporter knockout].

Figure 8. No change in DAT KO striatal synapse to neuron ratio

Bar graphs comparing the synapse to neuron ratio between groups. No significant differences were found between KO and WT mice. [WT = wild-type; KO = dopamine transporter knockout].

Figure 9. Striatal neuron cell density is not altered in DAT KO

Bar graphs comparing striatal neuron density between groups. Neuron density was found to be stable. No significant differences were found between DAT KO and WT mice. [WT = wild-type; KO = dopamine transporter knockout].