Myo T. Thwin

Regulation of parkinsonian motor behaviours by optogenetic control of basal ganglia circuitry

Neural circuits of the basal ganglia are critical for motor planning and action selection. Two parallel basal ganglia pathways have been described, and have been proposed to exert opposing influences on motor function. According to this classical model, activation of the ‘direct’ pathway facilitates movement and activation of the ‘indirect’ pathway inhibits movement. However, more recent anatomical and functional evidence has called into question the validity of this hypothesis. Because this model has never been empirically tested, the specific function of these circuits in behaving animals remains unknown. Here we report direct activation of basal ganglia circuitry in vivo, using optogenetic control of direct- and indirect-pathway medium spiny projection neurons (MSNs), achieved through Cre-dependent viral expression of channelrhodopsin-2 in the striatum of bacterial artificial chromosome transgenic mice expressing Cre recombinase under control of regulatory elements for the dopamine D1 or D2 receptor. Bilateral excitation of indirect-pathway MSNs elicited a parkinsonian state, distinguished by increased freezing, bradykinesia and decreased locomotor initiations. In contrast, activation of direct-pathway MSNs reduced freezing and increased locomotion. In a mouse model of Parkinson’s disease, direct-pathway activation completely rescued deficits in freezing, bradykinesia and locomotor initiation. Taken together, our findings establish a critical role for basal ganglia circuitry in the bidirectional regulation of motor behaviour and indicate that modulation of direct-pathway circuitry may represent an effective therapeutic strategy for ameliorating parkinsonian motor deficits.

Aberrant Excitatory Neuronal Activity and Compensatory Remodeling of Inhibitory Hippocampal Circuits in Mouse Models of Alzheimer's Disease

Neural network dysfunction may play an important role in Alzheimers disease (AD). Neuronal circuits vulnerable to AD are also affected in human amyloid precursor protein (hAPP) transgenic mice. hAPP mice with high levels of amyloid-beta peptides in the brain develop AD-like abnormalities, including cognitive deficits and depletions of calcium-related proteins in the dentate gyrus, a region critically involved in learning and memory. Here, we report that hAPP mice have spontaneous nonconvulsive seizure activity in cortical and hippocampal networks, which is associated with GABAergic sprouting, enhanced synaptic inhibition, and synaptic plasticity deficits in the dentate gyrus. Many Abeta-induced neuronal alterations could be simulated in nontransgenic mice by excitotoxin challenge and prevented in hAPP mice by blocking overexcitation. Aberrant increases in network excitability and compensatory inhibitory mechanisms in the hippocampus may contribute to Abeta-induced neurological deficits in hAPP mice and, possibly, also in humans with AD.

Mice lacking Dlx1 show subtype-specific loss of interneurons, reduced inhibition and epilepsy

Dlx homeodomain transcription factors are essential during embryonic development for the production of forebrain GABAergic interneurons. Here we show that Dlx1 is also required for regulating the functional longevity of cortical and hippocampal interneurons in the adult brain. We demonstrate preferential Dlx1 expression in a subset of cortical and hippocampal interneurons which, in postnatal Dlx1 mutants, show a time-dependent reduction in number. This reduction preferentially affects calretinin+ (bipolar cells) and somatostatin+ subtypes (for example, bitufted cells), whereas parvalbumin+ subpopulations (basket cells and chandelier cells) seem to be unaffected. Cell transplantation analysis demonstrates that interneuron loss reflects cell-autonomous functions of Dlx1. The decrease in the number of interneurons was associated with a reduction of GABA-mediated inhibitory postsynaptic current in neocortex and hippocampus in vitro and cortical dysrhythmia in vivo. Dlx1 mutant mice show generalized electrographic seizures and histological evidence of seizure-induced reorganization, linking the Dlx1 mutation to delayed-onset epilepsy associated with interneuron loss.

Distinct Roles of GABAergic Interneurons in the Regulation of Striatal Output Pathways

Striatal GABAergic microcircuits are critical for motor function, yet their properties remain enigmatic due to difficulties in targeting striatal interneurons for electrophysiological analysis. Here, we use Lhx6-GFP transgenic mice to identify GABAergic interneurons and investigate their regulation of striatal direct- and indirect-pathway medium spiny neurons (MSNs). We find that the two major interneuron populations, persistent low-threshold spiking (PLTS) and fast spiking (FS) interneurons, differ substantially in their excitatory inputs and inhibitory outputs. Excitatory synaptic currents recorded from PLTS interneurons are characterized by a small, nonrectifying AMPA receptor-mediated component and a NMDA receptor-mediated component. In contrast, glutamatergic synaptic currents in FS interneurons have a large, strongly rectifying AMPA receptor-mediated component, but no detectable NMDA receptor-mediated responses. Consistent with their axonal morphology, the output of individual PLTS interneurons is relatively weak and sparse, whereas FS interneurons are robustly connected to MSNs and other FS interneurons and appear to mediate the bulk of feedforward inhibition. Synaptic depression of FS outputs is relatively insensitive to firing frequency, and dynamic-clamp experiments reveal that these short-term dynamics enable feedforward inhibition to remain efficacious across a broad frequency range. Surprisingly, we find that FS interneurons preferentially target direct-pathway MSNs over indirect-pathway MSNs, suggesting a potential mechanism for rapid pathway-specific regulation of striatal output pathways.

Arc regulates spine morphology and maintains network stability in vivo.

Long-term memory relies on modulation of synaptic connections in response to experience. This plasticity involves trafficking of AMPA receptors (AMPAR) and alteration of spine morphology. Arc, a gene induced by synaptic activity, mediates the endocytosis of AMPA receptors and is required for both long-term and homeostatic plasticity. We found that Arc increases spine density and regulates spine morphology by increasing the proportion of thin spines. Furthermore, Arc specifically reduces surface GluR1 internalization at thin spines, and Arc mutants that fail to facilitate AMPAR endocytosis do not increase the proportion of thin spines, suggesting that Arc-mediated AMPAR endocytosis facilitates alterations in spine morphology. Thus, by linking spine morphology with AMPAR endocytosis, Arc balances synaptic downscaling with increased structural plasticity. Supporting this, loss of Arc in vivo leads to a significant decrease in the proportion of thin spines and an epileptic-like network hyperexcitability.

Reelin Depletion in the Entorhinal Cortex of Human Amyloid Precursor Protein Transgenic Mice and Humans with Alzheimer's Disease

Reelin regulates nervous system development and modulates synaptic plasticity in the adult brain. Several findings suggest that alterations in Reelin signaling may contribute to neuronal dysfunction associated with Alzheimers disease (AD). Cell surface receptors for Reelin, including integrins and very-low-density lipoprotein receptor/apolipoprotein E2 receptor, may be targets of amyloid-β (Aβ) peptides presumed to play key roles in the pathogenesis of AD. Reelin also regulates the extent of tau phosphorylation. Finally, increased amounts of Reelin fragments have been found in CSF from AD patients, suggesting altered processing of Reelin. We therefore hypothesized that Reelin levels might be altered in the brains of human amyloid precursor protein (hAPP) transgenic mice, particularly in brain regions vulnerable to AD such as hippocampus and entorhinal cortex. Compared with nontransgenic controls, hAPP mice had significantly fewer Reelin-expressing pyramidal cells in the entorhinal cortex, the major population of glutamatergic neurons expressing Reelin in the brain. Western blot analysis of the hippocampus, which receives projections from the entorhinal cortex, revealed significant reductions in Reelin levels. In contrast, the number of Reelin-expressing GABAergic interneurons was not altered in either the entorhinal cortex or the hippocampus. Thus, neuronal expression of hAPP/Aβ is sufficient to reduce Reelin expression in a specific population of entorhinal cortical pyramidal neurons in vivo. Underscoring the relevance of these findings, we found qualitatively similar reductions of Reelin-expressing pyramidal neurons in the entorhinal cortex of AD brains. We conclude that alterations in Reelin processing or signaling may be involved in AD-related neuronal dysfunction.

Many Neuronal and Behavioral Impairments in Transgenic Mouse Models of Alzheimer’s Disease Are Independent of Caspase Cleavage of the Amyloid Precursor Protein

Previous studies suggested that cleavage of the amyloid precursor protein (APP) at aspartate residue 664 by caspases may play a key role in the pathogenesis of Alzheimers disease. Mutation of this site (D664A) prevents caspase cleavage and the generation of the C-terminal APP fragments C31 and Jcasp, which have been proposed to mediate amyloid-β (Aβ) neurotoxicity. Here we compared human APP transgenic mice with (B254) and without (J20) the D664A mutation in a battery of tests. Before Aβ deposition, hAPP–B254 and hAPP–J20 mice had comparable hippocampal levels of Aβ1-42. At 2–3 or 5–7 months of age, hAPP–B254 and hAPP–J20 mice had similar abnormalities relative to nontransgenic mice in spatial and nonspatial learning and memory, elevated plus maze performance, electrophysiological measures of synaptic transmission and plasticity, and levels of synaptic activity-related proteins. Thus, caspase cleavage of APP at position D664 and generation of C31 do not play a critical role in the development of these abnormalities.

A mutation in the pericentrin gene causes abnormal interneuron migration to the olfactory bulb in mice

Precise control of neuronal migration is essential for proper function of the brain. Taking a forward genetic screen, we isolated a mutant mouse with defects in interneuron migration. By genetic mapping, we identified a frame shift mutation in the pericentrin (Pcnt) gene. The Pcnt gene encodes a large centrosomal coiled-coil protein that has been implicated in schizophrenia. Recently, frame shift and premature termination mutations in the pericentrin (PCNT) gene were identified in individuals with Seckel syndrome and microcephalic osteodysplastic primordial dwarfism (MOPD II), both of which are characterized by greatly reduced body and brain sizes. The mouse Pcnt mutant shares features with the human syndromes in its overall growth retardation and reduced brain size. We found that dorsal lateral ganglionic eminence (dLGE)-derived olfactory bulb interneurons are severely affected and distributed abnormally in the rostral forebrain in the mutant. Furthermore, mutant interneurons exhibit abnormal migration behavior and RNA interference knockdown of Pcnt impairs cell migration along the rostal migratory stream (RMS) into the olfactory bulb. These findings indicate that pericentrin is required for proper migration of olfactory bulb interneurons and provide a developmental basis for association of pericentrin function with interneuron defects in human schizophrenia.

Preventing C31 generation does not prevent many Aβ-dependenent neuronal and behavioral impairments in the happ-J20 mouse model of Alzheimer's disease

Background: Many epidemiological studies indicate a clear link between nutrition and Alzheimer’s Disease (AD). Several nutrient deficiencies have been shown to be risk factors for AD. Prospective studies with nutrients, like omega-3 fatty acids or the Mediterranean diet, show a reduced risk of developing AD. Preclinical studies have shown that combinations of specific nutrients synergistically increased membrane and synapse formation, diagnostic features that have been demonstrated to be reduced in AD, resulting in improved learning behavior in animal models. In addition, this specific combination reduces abeta production, plaque burden and neurodegeneration in transgenic mice. Methods: The effect of a medical food (Souvenaid ), designed to improve synapse formation, on memory and cognitive performance was assessed in a proof-of-concept trial with drug naı̈ve mild AD patients. AD patients (MMSE 20-26) were randomly allocated to Souvenaid , a 125 mL (125 kcal) once-a-day milk-based drink or an iso-caloric control drink in a double-blind 12-week study. Primary outcome parameters were a delayed verbal memory task (Wechsler Memory Scalerevised) and the 13-item ADAS-cog, assessed at 12 weeks. In an optional double-blind 12week extension phase patients continued to receive the same study product. The trial is registered with the Dutch Trial Register (#ISRCTN72254645). Results: 212 patients were included in the primary efficacy analysis (mean MMSE 23.9; mean age 73.7; 50% men). Delayed verbal memory, analysed by non-parametric statistics, was significantly improved in the Souvenaid group. In the statistical model excluding covariates no suggestion for an effect was found for 13-item ADAS-cog. There was no significant difference in tolerance (94% compliance) and (serious) adverse events between the study groups throughout the study period. To confirm and further strengthen the results of this first study 2 additional trials with Souvenaid are designed and will start in 2009: EU study, a 24-week study in drug naı̈ve mild AD patients. US study (S-Connect), a 24-week study in mild-to-moderate AD patients using AD medication. Conclusions: Souvenaid has a good safety and tolerability profile, and improves memory in mild AD. These promising results justify further studies in AD. Two clinical trials testing Souvenaid in mild-to-moderate Alzheimer’s patients will start in 2009.

P1-492: Identifying region-specific contributions to entorhinal-hippocampal network vulnerability in Alzheimer's disease

Background: Why distinct neurodegenerative disorders affect specific neuronal populations is largely unknown. Granule cells of the dentate gyrus are particularly vulnerable to A -induced molecular and functional impairments. A may exert these effects within granule cells, in their immediate extracellular milieu after being released from granule cells or synaptic contacts, or by affecting neurons in distant regions that provide afferent input to granule cells. Objective: To identify where A first acts within the entorhinal-hippocampal network to ultimately elicit molecular and behavioral deficits. Methods: We engineered lentiviral vectors to express either green fluorescent protein (lentiGFP) or human amyloid precursor protein (lenti-hAPP) carrying Swedish and London mutations under control of the human ubiquitin C promoter. Wildtype mice received injections of lenti-hAPP into one dentate gyrus (or entorhinal cortex) and of lenti-GFP into the opposite dentate gyrus (or entorhinal cortex) at 2-3 months of age. The distribution and cellular localization of lentivirusmediated expression of hAPP was analyzed 2 and 6 weeks later by immunohistochemistry, and compared with hAPP transgenic mice from line J20. Results: Six weeks after a single viral injection, hAPP was specifically expressed within the dentate gyrus at high levels throughout the rostral-caudal extent of the hippocampus. Granule neurons represented the clear majority of immunolabeled cells, with little to no labeling of pyramidal neurons in CA regions. Within granule neurons, hAPP was expressed in a punctate pattern that was practically indistinguishable between lentivirus-infected cells and uninfected granule cells from hAPP transgenic mice. In the entorhinal cortex, lentivirus-mediated expression of hAPP was also localized primarily to neurons. Deposition of A into plaques was not detected at these early time points. Ongoing experiments are analyzing the effects of this region-specific neuronal expression of hAPP on molecular alterations previously correlated with behavioral deficits in hAPP transgenic mice. Conclusions: Lentivirus-mediated expression of hAPP/A may be an effective tool with which to probe the source of selective vulnerability within the entorhinal-hippocampal network. Understanding where APP/A first triggers network dysfunction may enable the design of new strategies to better protect the most vulnerable brain regions in AD patients. Supported by NIA grant AG022074.