Date of Degree

2-2014

Document Type

Dissertation

Degree Name

Ph.D.

Program

Biology

Advisor

Abdeslem El Idrissi

Advisor

Andrzej Wieraszko

Subject Categories

Developmental Biology | Neuroscience and Neurobiology | Toxicology

Keywords

Brain Excitability, GABAAR, GABA-Shift, Inhibtiory Learning and Memory, Lead Poisoning, VSCC

Abstract

The toxic effects of Pb2+ on the developing rat nervous system has been investigated to assess early developmental GABAergic disruption and its implications with altering inhibitory learning and memory. This goal was achieved using a multi-systems approach: blood lead levels (clinical physiology), qRT-PCR (molecular genetics), brain and primary neuronal culture immunology (immunohistochemical and cellular approaches), physiological cellular components (synaptosomes and protein expression) and finally through learning and memory assessment with GABA mimetic drug manipulations in the intact animal (behavioral pharmacology). The influence of a 956ppm Pb2+ gestational diet (i.e. from birth to sacrifice) resulted in pup mean blood lead levels (BLLs measured in ìg/dL) (Range 28-47) and Dams (Range 33-51) respectively. In contrast, control pups and dams were Pb2+ negative. These ages were selected to determine neurodevelopmental trajectories of the GABA-shift from excitation-to-inhibition postnatally in our model. qRT-PCR studies evinced a delay in mRNA expression regulating GAD 80, 65, CACAN â3, GABAAR and were differentially regulated cortex and hippocampus as a function of age in response to Pb2+. Brain slice immunohistochemistry revealed an early shift of KCC2 expression in both cortex and hippocampus. Notably, these alterations were differentially regulated by age, brain region and subcellular circuitry within structures (i.e. DG vs. CA3). Neuronal cultures revealed that in response to Pb2+ at low micro molar concentrations induced VSCC-â3 nuclear translocation and GABAAR upregulation. KCC2 expression was inhibiting in cultures by Pb2+. Synaptosomal effects of Pb2+ revealed altered glutamate accumulation and handling with increased spontaneous and decreased evoked release in significantly modulated by Pb exposure suggesting altered brain synaptogenesis. Pb2+ exposure resulted in increased binding suggesting post synaptic modification in cortex and hippocampus increasing brain excitability. Behaviorally, Pb2+ exposure resulted in increased anxiety, impulsivity, stress, and disrupted learning and memory regulated by inhibitory circuits that were recovered with taurine, a GABAAR agonist, administration. Specifically, Pb2+ disrupted contextual and auditory associative learning. Taken together, these results suggest that Pb2+ interferes with early VSCCs and GABAAR synergistic action that establishes GABAergic neural networks and in turn produces increased brain excitability and over reactivity as a consequence of reduced inhibition.

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