A Transgenic Mouse Model to Selectively Identify α3 Na,K-ATPase Expressing Cells in the Nervous System.

The α3 Na+,K+-ATPase (α3NKA) is one of four known α isoforms of the mammalian transporter. A deficiency in α3NKA is linked to severe movement control disorders. Understanding the pathogenesis of these disorders is limited by an incomplete knowledge of α3NKA expression in the brain as well as the challenges associated with identifying living cells that express the isoform for subsequent electrophysiological studies. To address this problem, transgenic mice were generated on the C57BL/6 genetic background, which utilize the mouse α3 subunit gene (Atp1a3) promoter to drive the expression of ZsGreen1 fluorescent protein. Consistent with published results on α3NKA distribution, a ZsGreen1 signal was detected in the brain, but not in the liver, with Atp1a3-ZsGreen1 transgenic mice. The intensity of ZsGreen1 fluorescence in neuronal cell bodies varied considerably in the brain, being highest in the brainstem, deep cerebellar and select thalamic nuclei, and relatively weak in cortical regions. Fluorescence was not detected in astrocytes or white matter areas. ZsGreen1-positive neurons were readily observed in fresh (unfixed) brain sections, which were amenable to patch-clamp recordings. Thus, the α3NKA-ZsGreen1 mouse model provides a powerful tool for studying the distribution and functional properties of α3NKA-expressing neurons in the brain.

Electrophysiological and Immunohistochemical Evidence for an Increase in GABAergic Inputs and HCN Channels in Purkinje Cells that Survive Developmental Ethanol Exposure.

Ethanol exposures during the early postnatal period of the rat result in significant death of Purkinje cells (PCs). The magnitude, time-course, and lobular specificity of PC death have been well characterized in several studies. Additionally, significant reduction of climbing fiber inputs to the surviving PCs has been characterized. This study investigates whether further alterations to the cerebellar cortical circuits might occur as a result of developmental ethanol exposures. We first examined the firing pattern of PCs in acute slice preparations on postnatal days 13-15. While the basic firing frequency was not significantly altered, PCs from rat pups treated with ethanol on postnatal days 4-6 showed a significantly increased number of inhibitory postsynaptic potentials (IPSCs) and a larger Ih current. We conducted immunofluorescent studies to identify the probable cause of the increased IPSCs. We found a significant 21 % increase in the number of basket cells per PC and a near doubling of the volume of co-localized basket cell axonal membrane with PC. In addition, we identified a significant (~147 %) increase in HCN1 channel volume co-localized to PC volume. Therefore, the cerebellar cortex that survives targeted postnatal ethanol exposure is dramatically altered in development subsequent to PC death. The cerebellar cortical circuit that results is one that operates under a significant degree of increased resting inhibition. The alterations in the development of cerebellar circuitry following ethanol exposure, and the significant loss of PCs, could result in modifications of the structure and function of other brain regions that receive cerebellar inputs.

Sublethal total body irradiation leads to early cerebellar damage and oxidative stress.

The present study aimed at identifying early damage index in the cerebellum following total body irradiation (TBI). Adult male CD2F1 mice (n=18) with or without TBI challenge (8.5 Gy irradiation) were assessed for histology and expression of selected immunohistochemical markers including malondiadehyde (MDA), 8-hydroxy-2'-deoxyguanosine (8-OHdG), protein 53 (p53), vascular endothelial growth factor receptor 2 (VEGF-R2), CD45, calbindin D-28k (CB- 28) and vesicular glutamate transport-2 (VGLUT2) in cerebellar folia II to IV. Compared to sham-controls, TBI significantly increased vacuolization of the molecular layer. At high magnification, deformed fiber-like structures were found along with the empty matrix space. Necrotic Purkinje cells were identified on 3.5 days after TBI, but not on 1 day. Purkinje cell count was reduced significantly 3.5 days after TBI. Compared with sham control, overall intensities of MDA and 8-OHdG immunoreactivities were increased dramatically on 1 and 3.5 days after TBI. Expression of VEGF-R2 was identified to be co-localized with 8-OHdG after TBI. This validates microvessel endothelial damage. The p53 immunoreactivities mainly deposited in the granular layer and microvessels after TBI and co-localization of the p53 with the CD45, both which were found within the microvessels. After TBI, CB28 expression decreased whereas the VGLUT2 expression increased significantly; Purkinje cells exhibited a reduced body size and deformity of dendritic arbor, delineated by CB28 immunoreactivity. Substantial damage to the cerebellum can be detectable as early as 1- 3.5 days in adult animals following sublethal TBI. Oxidative stress, inflammatory response and calcium neurotoxicity-associated mechanisms are involved in radiation-induced neuronal damage.

Are oxidative mechanisms primary in ethanol induced Purkinje neuron death of the neonatal rat?

Rat cerebellar Purkinje neurons are vulnerable to ethanol exposure during the brain growth spurt, especially during early postnatal exposure. A prominent hypothesis is that ethanol induces oxidative types of alterations that result in the neurodegeneration. The purpose of this study was to test this hypothesis in two ways. One was to determine if the reactive oxidative species, nitrotyrosine (NT), was produced in the cerebellum following ethanol exposure. Second, was to determine if co-administration of the clinically useful antioxidant N-acetylcysteine (NAC) afforded any protection from Purkinje neuron loss. Rat pups were treated on postnatal day 4 with a single ethanol (6.0 g/kg) or isocaloric intragastric intubation. The cerebelli were analyzed for NT with ELISA assays at 2, 4, 6, or 8 h following the single exposure. No evidence of NT was found at any of these time points. Another group of animals received ethanol exposure on PN4, or ethanol exposure plus NAC. Control groups included isocaloric intubated controls (IC), IC plus NAC, and mother reared controls. Twenty-four hours following the exposures, the pups were perfused and the cerebellum processed for cell counting. Ethanol exposure reduced the number of Purkinje neurons in the cerebellum. Concurrent treatment with antioxidant did not protect the Purkinje neurons from ethanol-related cell loss. These in vivo analyses do not support a robust oxidative mechanism involving the production of reactive nitrogen species as a significant means of Purkinje cell neurodegeneration.

Target-determined expression of alpha3 isoform of the Na+,K+-ATPase in the somatic nervous system of rat.

Factors that determine the differential expression of isoforms of Na(+),K(+)-ATPase in the nervous system of vertebrates are not understood. To address this question we studied the expression of alpha(3) Na(+),K(+)-ATPase in the L5 dorsal root ganglia (DRG) of developing rat, the normal adult rat, and the adult rat after peripheral axotomy. During development, the first alpha(3) Na(+),K(+)-ATPase-positive DRG neurons appear by embryonic day 21. At birth, the L5 DRG have a full complement (14 +/- 2%) of these neurons. By 15 days after sciatic nerve transection in adult rat, the number of alpha(3) Na(+),K(+)-ATPase-positive DRG neurons and small myelinated L5 ventral root axons decreases to about 35% of control counts. These results combined with data from the literature suggest that the expression of alpha(3) Na(+),K(+)-ATPase by rat somatic neurons is determined by target-muscle spindle-derived factors.

Altered expression of Bcl2, Bad and Bax mRNA occurs in the rat cerebellum within hours after ethanol exposure on postnatal day 4 but not on postnatal day 9.

Previous studies have demonstrated that ethanol exposure during the vulnerable postnatal (PN) day 4-6 period results in a dose-dependent loss of Purkinje neurons in rats by apoptosis. Although the mechanism of ethanol action and the reasons for Purkinje cell vulnerability are unknown, we hypothesize that during the PN4-6 vulnerable period Purkinje cells are dependent on active trophic factor suppression of apoptosis. Furthermore, ethanol acts to prevent the reception of this trophic signaling resulting in the execution of the apoptotic pathway that includes specific alterations of proteins in the Bcl2 gene family. Ethanol exposure that occurs after this vulnerable period (i.e. PN9) would not be expected to demonstrate alterations in these apoptotic proteins since the Purkinje cells no longer demonstrate vulnerability to ethanol. The current study was undertaken to identify the alterations in mRNA expression for members of the Bcl2-family within the initial hours following ethanol administration on PN4 or PN9. Semi-quantitative reverse transcriptase with polymerase chain reaction (PCR) techniques were used to determine the expression levels of pro-apoptotic factors Bad and Bax, and anti-apoptotic Bcl(2) mRNA. Ethanol was administered at four different doses (1.5, 3.0, 4.5, and 6.0 g/kg) on PN4 and analyses of whole cerebellar mRNA was conducted at 1, 4, 6, and 8 h after treatment. Doses greater than 1.5 g/kg produced significant decreases in Bcl(2) and significant increases in Bad and Bax mRNA during the 8-h period after treatment. In stark contrast, when ethanol was administered at 3.0 or 6.0 g/kg to PN9 pups, no significant alterations of these apoptotic factors were identified at either 1 or 4 h after treatment. These results are in agreement with and provide further support for our hypothesis that ethanol interrupts the active suppression of apoptosis that is a crucial feature of Purkinje cell vulnerability during this time period.

Alterations of cerebellar mRNA specific for BDNF, p75NTR, and TrkB receptor isoforms occur within hours of ethanol administration to 4-day-old rat pups.

Developing cerebellar Purkinje cells of the rat are extremely sensitive to ethanol during postnatal days (PN) 4-6, but not at later times during development. Ethanol exposure during this vulnerable window induces rapid apoptotic Purkinje cell death that is hypothesized to result from ethanol inhibition in brain-derived nerve growth factor (BDNF)-TrkB neurotrophic signaling that results in loss of apoptotic suppression. In this study, the effect that different concentrations of ethanol (1.5, 3.0, 4.5 and 6.0 g/kg) have on steady-state mRNA expression of BDNF and different TrkB receptor isoforms in the cerebellum on PN4 was determined at 1, 4, 6, and 8 h after treatment. Significant decreases in mRNA specific for BDNF and TrkB isoforms were detected within 1 h after ethanol administration. No significant alterations in expression of mRNA specific to the low affinity p75(NTR) receptor were identified. These alterations are concurrent with the PN4 vulnerable period for Purkinje cells since equivalent treatment of PN9 rat pups does not produce significant alterations in mRNA specific to BDNF or TrkB at 4 h after exposure. These results support the hypothesis that ethanol induces a disruption of BDNF-TrkB signaling that results in loss of apoptotic suppression in vulnerable Purkinje cells by growth factor withdrawal.

Detection of Purkinje cell loss following drug exposures to developing rat pups using reverse transcriptase-polymerase chain reaction (RT-PCR) analysis for calbindin-D28k mRNA expression.

A technique is described that allows for the identification and quantification of Purkinje cell loss in cerebellum subsequent to developmental toxic exposures. This technique relies upon the extensively validated findings that the Purkinje cell is the only site of expression in the cerebellum of the calcium binding protein calbindin-D28k. Thus, analysis of mRNA expression specific to this protein by comparison to matched controls provides a reliable means of determining whether cell loss has occurred. Purkinje cell loss was induced in rat pups by ethanol exposure on postnatal day (PN) 4 or valproic acid administration to pregnant dams on gestational day 13. Analysis was conducted on PN5 or PN10 and the results compared to parallel groups of pups where the Purkinje cells were counted by traditional means. When compared to matched control rat pups the decrease in calbindin-D28k mRNA expression indicates Purkinje cell loss regardless of whether the cell loss was induced by prenatal valproic acid or postnatal ethanol exposure. The availability of a biochemical alternative to histological cell counting allows for more detailed analyses of the mechanisms of Purkinje cell death induced by these two toxicants, including analyses of the early alterations in signal transduction proteins.

Ethanol-induced alterations of neurotrophin receptor expression on Purkinje cells in the neonatal rat cerebellum.

Ethanol causes loss of Purkinje cells in the cerebellum during the early stages of differentiation and maturation by a presently unknown mechanism. Neuronal vulnerability in the cerebellum parallels the prominent temporal and anatomical gradients of development (i.e. early to late interlobular and posterior to anterior, respectively). Development of Purkinje cells is known to require binding of the neurotrophins, including brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT3), to the tyrosine-kinase (Trk) receptors TrkB and TrkC, respectively. In addition, Purkinje cells are reported to experience a critical switch between BDNF dependence and NT3 dependence during the period of highest ethanol sensitivity between postnatal days (PN) 4-6. To test the hypothesis that ethanol alters neurotrophin signaling leading to Purkinje neuronal death, the immunohistochemical expression of TrkB and TrkC receptors on Purkinje cells of rat pups following a moderate dose of ethanol was determined at various times surrounding the period of postnatal ethanol vulnerability. Ethanol selectively decreased Purkinje cell expression of TrkB and TrkC receptors following exposures within the vulnerable period (PN4-6). These results suggest that ethanol may induce loss of Purkinje cells by alteration of neurotrophic regulation at this critical stage.