To elucidate the pathogenic mechanisms in Huntington's disease (HD) elicited by expression of full-length human mutant huntingtin (fl-mhtt), a bacterial artificial chromosome (BAC)-mediated transgenic mouse model (BACHD) was developed expressing fl-mhtt with 97 glutamine repeats under the control of endogenous htt regulatory machinery on the BAC. BACHD mice exhibit progressive motor deficits, neuronal synaptic dysfunction, and late-onset selective neuropathology, which includes significant cortical and striatal atrophy and striatal dark neuron degeneration. Power analyses reveal the robustness of the behavioral and neuropathological phenotypes, suggesting BACHD as a suitable fl-mhtt mouse model for preclinical studies. Additional analyses of BACHD mice provide novel insights into how mhtt may elicit neuropathogenesis. First, unlike previous fl-mhtt mouse models, BACHD mice reveal that the slowly progressive and selective pathogenic process in HD mouse brains can occur without early and diffuse nuclear accumulation of aggregated mhtt (i.e., as detected by immunostaining with the EM48 antibody). Instead, a relatively steady-state level of predominantly full-length mhtt and a small amount of mhtt N-terminal fragments are sufficient to elicit the disease process. Second, the polyglutamine repeat within fl-mhtt in BACHD mice is encoded by a mixed CAA-CAG repeat, which is stable in both the germline and somatic tissues including the cortex and striatum at the onset of neuropathology. Therefore, our results suggest that somatic repeat instability does not play a necessary role in selective neuropathogenesis in BACHD mice. In summary, the BACHD model constitutes a novel and robust in vivo paradigm for the investigation of HD pathogenesis and treatment.

The electrophysiological properties of distinct subpopulations of striatal medium-sized spiny neurons (MSSNs) were compared using enhanced green fluorescent protein as a reporter gene for identification of neurons expressing dopamine D1 and D2 receptor subtypes in mice. Whole-cell patch-clamp recordings in slices revealed that passive membrane properties were similar in D1 and D2 cells. All MSSNs displayed hyperpolarized resting membrane potentials but the threshold for firing action potentials was lower in D2 than in D1 neurons. In voltage clamp, the frequency of spontaneous excitatory postsynaptic currents was higher in D2 than in D1 cells and large-amplitude inward currents (> 100 pA) were observed only in D2 cells. After tetrodotoxin this difference was reduced, suggesting that sodium conductances contribute to the increased frequencies in D2 cells. After pharmacological blockade of GABA(A) receptors, a subset of D2 cells also displayed large spontaneous membrane depolarizations and complex responses to stimulation of the corticostriatal pathway. To further characterize ionotropic glutamate receptor function, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) was applied onto dissociated MSSNs. Application of AMPA alone or in the presence of cyclothiazide (an AMPA receptor desensitization blocker) evoked larger currents in D1 than in D2 cells. Together, these data demonstrate significant differences in electrophysiological properties of subpopulations of MSSNs defined by selective expression of D1 and D2 receptors. D2 cells display increased excitability and reflect ongoing cortical activity more faithfully than D1 cells, an effect that is independent of postsynaptic AMPA receptors and probably results from stronger synaptic coupling. This could help to explain the increased vulnerability of D2 MSSNs in neurodegenerative disorders.

Summary:Cortical dysplasia (CD), a frequent pathological substrate of pediatric epilepsy surgery patients, has a number of similarities with immature cortex, such as reduced Mg 2+ sensitivity of N-methyl-D-aspartate (NMDA) receptors and the persistence of subplate-like neurons and undifferentiated cells. Because γ -aminobutyric acid (GABA) is the main neurotransmitter in early cortical development, we hypothesized increased GABA receptor-mediated synaptic function in CD tissue. Infrared videomicroscopy and whole-cell patch clamp recordings were used to characterize the morphology and electrophysiological properties of immature and normal-appearing neurons in slices from cortical tissue samples resected for the treatment of pharmacoresistant epilepsy in children (0.2-14 years). In addition, we examined spontaneous and evoked synaptic activity, as well as responses to exogenous GABA application. We demonstrate both the presence of immature pyramidal neurons and networks in young CD tissue and the predominance of GABA synaptic activity. In addition, spontaneous GABA depolarizations frequently induced action potentials, supporting a potential excitatory role of GABA in CD. Evoked synaptic responses mediated by GABA were also prominent, and bath application of 4-aminopyridine induced rhythmic depolarizations that were blocked by bicuculline. Finally, responses to exogenous application of GABA had depolarized reversal potentials in severe compared to mild and non-CD cases. The present data support the hypothesis that CD shares features of immature cortex, with predominant and potentially excitatory GABA A receptormediated neurotransmission. These results could partially explain the increased excitability of the cortical network in pediatric CD.

Tuberous sclerosis complex (TSC) and cortical dysplasia Type IIB (CDIIB) share histopathologic features that suggest similar epileptogenic mechanisms. This study compared the morphological and electrophysiological properties of cortical cells in tissue from pediatric TSC (n=20) and CDIIB (n=20) patients using whole-cell patch clamp recordings and biocytin staining. Cell types were normal-appearing and dysmorphiccytomegalic pyramidal neurons, interneurons, and giant/balloon cells, including intermediate neuronal-glial cells. In the cortical mantle, giant/balloon cells occurred more frequently in TSC than in CDIIB cases, whereas cytomegalic pyramidal neurons were found more frequently in CDIIB. Cell morphology and membrane properties were similar in TSC and CDIIB cases. Except for giant/balloon and intermediate cells, all neuronal cell types fired action potentials and displayed spontaneous postsynaptic currents. However, the frequency of spontaneous glutamatergic postsynaptic currents in normal pyramidal neurons and interneurons was significantly lower in CDIIB compared with TSC cases and GABAergic activity was higher in all neuronal cell types in CDIIB. Further, acutely dissociated pyramidal neurons displayed higher sensitivity to exogenous application of GABA in CDIIB compared with TSC cases. These results indicate that, in spite of similar histopathologic features and basic cell membrane properties, TSC and CDIIB display differences in the topography of abnormal cells, excitatory and inhibitory synaptic network properties, and GABAA receptor sensitivity. These differences support the notion that the mechanisms of epileptogenesis could differ in patients with TSC and CDIIB. Consequently, pharmacologic therapies should take these findings into consideration.

SUMMARYTuberous sclerosis complex (TSC) and severe cortical dysplasia (CD), or CD type II according to Palmini classification, share histopathologic similarities, specifically the presence of cytomegalic neurons and balloon cells. In this study we examined the morphologic and electrophysiologic properties of cells in cortical tissue samples from pediatric patients with TSC and CD type II who underwent surgery for pharmacoresistant epilepsy. Normal-appearing pyramidal neurons from TSC and CD type II cases had similar passive membrane properties. However, the frequency of excitatory postsynaptic currents (EPSCs) was higher in neurons from TSC compared to severe CD cases, particularly the frequency of mediumand large-amplitude synaptic events. In addition, EPSCs rise and decay times were slower in normal cells from TSC compared to severe CD cases. Balloon cells were found more frequently in TSC cases, whereas cytomegalic pyramidal neurons occurred more often in CD type II cases. Both cell types were similar morphologically and electrophysiologically in TSC and severe CD. These results suggest that even though the histopathology in TSC and severe CD is similar, there are subtle differences in spontaneous synaptic activity and topographic distribution of abnormal cells. These differences may contribute to variable mechanisms of epileptogenesis in patients with TSC compared with CD type II.