Taken together, our results suggest that chronic administration of methylphenidate in mice, at doses that approximate those at the higher therapeutic range in humans, results in a reduced expression of neurotrophic factors, increased neuroinflammation, and a small, but significant loss of SNpc dopamine neurons. These results can only be interpreted in the context on normal brain structure and function, and thus would have direct implications for the illicit/neurocognitive use of MPH. Since the underlying anatomy and biochemistry of ADHD has not been definitively characterized, our findings may or may not be generalizable to the vast majority of humans who are properly diagnosed with ADHD and are prescribed methylphenidate. Nevertheless, this work supports studies that demonstrate that drugs shown to increase the levels of dopamine in the synaptic cleft can contribute to degenerative changes in the basal ganglia. DA neuron and Iba-1-positive microglial cell number in the SNpc were estimated using standard model-based stereological methods. Briefly, for neuronal counts, brains were blocked and serially-sectioned at 10 mm from the rostral hippocampus to the cerebellar-midbrain junction. Serial sections were mounted 5 sections per slide onto polyionic slides. TH-positive neurons and TH-negative, Nissl-positive cells within the SNpc that had the Salvianolic-acid-B characteristics of dopaminergic neurons were counted using a 406 objective. Specifically, neurons from both left and right sides of the SNpc within one section per slide were counted. Both Iba-1 resting and activated microglia were counted. Stringent measures were adopted to classify Iba-1 positive microglia as resting or activated based on morphology based on the detailed description by Graeber and Streit. Microglial cells would be deemed as resting if they contained a small oval Iba-1-positive cell body that averaged 3 microns in diameter with long slender processes. Microglia would be classified as activated when the cell body was slightly increased in size compared to resting microglia and had an irregular shape. Based on cell size of the counting particle in 12 micron sections, we used a high NA lens and a total magnification of 10006 in which we were able to clearly define approximately 18 focal planes within our section. From in vitro studies, expression of 1a-OHase by macrophages at the site of an infection seems to be an important part of innate immunity. Montoya et al have shown that upregulation of the vitamin D pathway occurs in leprosy lesions of patients with self limiting forms of the disease, however, beyond that study there is no evidence that 1a-OHase is expressed 14α-hydroxy-Sprengerinin-C by macrophages in vivo as a result of experimental infection. Intra-mammary infections during lactation offers a model of bacterial infection to determine if 1a-OHase is expressed in response to bacterial infection in vivo. Common pathogens that cause mammary infections include Escherichia coli, Staphylococcus aureus, and Streptococcus uberis. During mammary infection the number of somatic cells secreted in milk will often exceed 106 cells/mL. Approximately 80 to 90 percent of somatic cells in milk from an infected mammary gland are neutrophils and the remainder of the cells are macrophages and lymphocytes. The advantage of using a mammary infection model is that the infiltrating cells during mammary infection can easily isolated from milk using non-invasive procedures; allowing us to study the in vivo immune responses of immune cells to bacterial infection. TLRs are present in the bovine mammary gland and invasion of the mammary gland by bacteria triggers an innate immune response by TLR signaling.