Entorhinal Cortical Deep Brain Stimulation Rescues Memory Deficits in Both Young and Old Mice Genetically Engineered to Model Alzheimer's Disease.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by progressive cognitive decline. Deep brain stimulation (DBS) has been used to treat a variety of brain disorders and shows promise in alleviating cognitive symptoms in some AD patients (Laxton et al, 2010). We previously showed that DBS of the entorhinal cortex (EC) enhances spatial memory formation in normal (wild-type) mice (Stone et al, 2011). Here we tested the effects of EC-DBS on the progressive cognitive deficits in a genetically-based mouse model of AD. TgCRND8 (Tg) transgenic mice express human amyloid precursor protein harboring the Swedish and Indiana familial AD mutations. These mice exhibit age-related increases in Aß production, plaque deposition, as well as contextual fear and spatial memory impairments. Here, we found EC stimulation in young mice (6 weeks old) rescued the early contextual fear and spatial memory deficits and decreased subsequent plaque load in Tg mice. Moreover, stimulation in older mice (6 months old) was also sufficient to rescue the memory deficits in Tg mice. The memory enhancement induced by DBS emerged gradually (over the course of weeks) and was both persistent and specific to hippocampal-based memories. These results provide further support for the development of novel therapeutics aimed to resolve the cognitive decline and memory impairment in AD using DBS of hippocampal afferents.

Parvalbumin interneurons constrain the size of the lateral amygdala engram.

Memories are thought to be represented by discrete physiological changes in the brain, collectively referred to as an engram, that allow patterns of activity present during learning to be reactivated in the future. During the formation of a conditioned fear memory, a subset of principal (excitatory) neurons in the lateral amygdala (LA) are allocated to a neuronal ensemble that encodes an association between an initially neutral stimulus and a threatening aversive stimulus. Previous experimental and computational work suggests that this subset consists of only a small proportion of all LA neurons, and that this proportion remains constant across different memories. Here we examine the mechanisms that contribute to the stability of the size of the LA component of an engram supporting a fear memory. Visualizing expression of the activity-dependent gene Arc following memory retrieval to identify neurons allocated to an engram, we first show that the overall size of the LA engram remains constant across conditions of different memory strength. That is, the strength of a memory was not correlated with the number of LA neurons allocated to the engram supporting that memory. We then examine potential mechanisms constraining the size of the LA engram by expressing inhibitory DREADDS (designer receptors exclusively activated by designer drugs) in parvalbumin-positive (PV+) interneurons of the amygdala. We find that silencing PV+ neurons during conditioning increases the size of the engram, especially in the dorsal subnucleus of the LA. These results confirm predictions from modeling studies regarding the role of inhibition in shaping the size of neuronal memory ensembles and provide additional support for the idea that neurons in the LA are sparsely allocated to the engram based on relative neuronal excitability.

Neurons are recruited to a memory trace based on relative neuronal excitability immediately before training.

Memories are thought to be sparsely encoded in neuronal networks, but little is known about why a given neuron is recruited or allocated to a particular memory trace. Previous research shows that in the lateral amygdala (LA), neurons with increased CREB are selectively recruited to a fear memory trace. CREB is a ubiquitous transcription factor implicated in many cellular processes. Which process mediates neuronal memory allocation? One hypothesis is that CREB increases neuronal excitability to bias neuronal recruitment, although this has not been shown experimentally. Here we use several methods to increase neuronal excitability and show this both biases recruitment into the memory trace and enhances memory formation. Moreover, artificial activation of these neurons alone is a sufficient retrieval cue for fear memory expression, showing that these neurons are critical components of the memory trace. These results indicate that neuronal memory allocation is based on relative neuronal excitability immediately before training.

Hippocampal neurogenesis regulates forgetting during adulthood and infancy.

Throughout life, new neurons are continuously added to the dentate gyrus. As this continuous addition remodels hippocampal circuits, computational models predict that neurogenesis leads to degradation or forgetting of established memories. Consistent with this, increasing neurogenesis after the formation of a memory was sufficient to induce forgetting in adult mice. By contrast, during infancy, when hippocampal neurogenesis levels are high and freshly generated memories tend to be rapidly forgotten (infantile amnesia), decreasing neurogenesis after memory formation mitigated forgetting. In precocial species, including guinea pigs and degus, most granule cells are generated prenatally. Consistent with reduced levels of postnatal hippocampal neurogenesis, infant guinea pigs and degus did not exhibit forgetting. However, increasing neurogenesis after memory formation induced infantile amnesia in these species.

p63 Regulates adult neural precursor and newly born neuron survival to control hippocampal-dependent Behavior.

The molecular mechanisms that regulate adult neural precursor cell (NPC) survival, and thus maintain adult neurogenesis, are not well defined. Here, we investigate the role of p63, a p53 family member, in adult NPC function in mice. Conditional ablation of p63 in adult NPCs or p63 haploinsufficiency led to reduced numbers of NPCs and newborn neurons in the neurogenic zones of the hippocampus and lateral ventricles and in the olfactory bulb. These reductions were attributable to enhanced apoptosis of NPCs and newborn neurons and were rescued by inhibition of caspase activity, p53, or the p53 apoptotic effector PUMA (p53-upregulated modulator of apoptosis). Moreover, these cellular deficits were functionally important because they led to perturbations in hippocampus-dependent memory formation. These results indicate that p63 regulates the numbers of adult NPCs and adult-born neurons as well as neural stem cell-dependent cognitive functions, and that it does so, at least in part, by inhibiting p53-dependent cell death.

Vertebrate intersectin1 is repurposed to facilitate cortical midline connectivity and higher order cognition.

Invertebrate studies have highlighted a role for EH and SH3 domain Intersectin (Itsn) proteins in synaptic vesicle recycling and morphology. Mammals have two Itsn genes (Itsn1 and Itsn2), both of which can undergo alternative splicing to include DBL/PH and C2 domains not present in invertebrate Itsn proteins. To probe for specific and redundant functions of vertebrate Itsn genes, we generated Itsn1, Itsn2, and double mutant mice. While invertebrate mutants showed severe synaptic abnormalities, basal synaptic transmission and plasticity were unaffected at Schaffer CA1 synapses in mutant mice. Surprisingly, intercortical tracts-corpus callosum, ventral hippocampal, and anterior commissures-failed to cross the midline in mice lacking Itsn1, but not Itsn2. In contrast, tracts extending within hemispheres and those that decussate to more caudal brain segments appeared normal. Itsn1 mutant mice showed severe deficits in Morris water maze and contextual fear memory tasks, whereas mice lacking Itsn2 showed normal learning and memory. Thus, coincident with the acquisition of additional signaling domains, vertebrate Itsn1 has been functionally repurposed to also facilitate interhemispheric connectivity essential for high order cognitive functions.

CREB regulates spine density of lateral amygdala neurons: implications for memory allocation.

Neurons may compete against one another for integration into a memory trace. Specifically, neurons in the lateral nucleus of the amygdala with relatively higher levels of cAMP Responsive Element Binding Protein (CREB) seem to be preferentially allocated to a fear memory trace, while neurons with relatively decreased CREB function seem to be excluded from a fear memory trace. CREB is a ubiquitous transcription factor that modulates many diverse cellular processes, raising the question as to which of these CREB-mediated processes underlie memory allocation. CREB is implicated in modulating dendritic spine number and morphology. As dendritic spines are intimately involved in memory formation, we investigated whether manipulations of CREB function alter spine number or morphology of neurons at the time of fear conditioning. We used viral vectors to manipulate CREB function in the lateral amygdala (LA) principal neurons in mice maintained in their homecages. At the time that fear conditioning normally occurs, we observed that neurons with high levels of CREB had more dendritic spines, while neurons with low CREB function had relatively fewer spines compared to control neurons. These results suggest that the modulation of spine density provides a potential mechanism for preferential allocation of a subset of neurons to the memory trace.

MEF2 negatively regulates learning-induced structural plasticity and memory formation.

Memory formation is thought to be mediated by dendritic-spine growth and restructuring. Myocyte enhancer factor 2 (MEF2) restricts spine growth in vitro, suggesting that this transcription factor negatively regulates the spine remodeling necessary for memory formation. Here we show that memory formation in adult mice was associated with changes in endogenous MEF2 levels and function. Locally and acutely increasing MEF2 function in the dentate gyrus blocked both learning-induced increases in spine density and spatial-memory formation. Increasing MEF2 function in amygdala disrupted fear-memory formation. We rescued MEF2-induced memory disruption by interfering with AMPA receptor endocytosis, suggesting that AMPA receptor trafficking is a key mechanism underlying the effects of MEF2. In contrast, decreasing MEF2 function in dentate gyrus and amygdala facilitated the formation of spatial and fear memory, respectively. These bidirectional effects indicate that MEF2 is a key regulator of plasticity and that relieving the suppressive effects of MEF2-mediated transcription permits memory formation.

Increasing CREB function in the CA1 region of dorsal hippocampus rescues the spatial memory deficits in a mouse model of Alzheimer's disease.

The principal defining feature of Alzheimer's disease (AD) is memory impairment. As the transcription factor CREB (cAMP/Ca(2+) responsive element-binding protein) is critical for memory formation across species, we investigated the role of CREB in a mouse model of AD. We found that TgCRND8 mice exhibit a profound impairment in the ability to form a spatial memory, a process that critically relies on the dorsal hippocampus. Perhaps contributing to this memory deficit, we observed additional deficits in the dorsal hippocampus of TgCRND8 mice in terms of (1) biochemistry (decreased CREB activation in the CA1 region), (2) neuronal structure (decreased spine density and dendritic complexity of CA1 pyramidal neurons), and (3) neuronal network activity (decreased arc mRNA levels following behavioral training). Locally and acutely increasing CREB function in the CA1 region of dorsal hippocampus of TgCRND8 mice was sufficient to restore function in each of these key domains (biochemistry, neuronal structure, network activity, and most importantly, memory formation). The rescue produced by increasing CREB was specific both anatomically and behaviorally and independent of plaque load or Aß levels. Interestingly, humans with AD show poor spatial memory/navigation and AD brains have disrupted (1) CREB activation, and (2) spine density and dendritic complexity in hippocampal CA1 pyramidal neurons. These parallel findings not only confirm that TgCRND8 mice accurately model key aspects of human AD, but furthermore, suggest the intriguing possibility that targeting CREB may be a useful therapeutic strategy in treating humans with AD.

Maze training in mice induces MRI-detectable brain shape changes specific to the type of learning.

Multiple recent human imaging studies have suggested that the structure of the brain can change with learning. To investigate the mechanism behind such structural plasticity, we sought to determine whether maze learning in mice induces brain shape changes that are detectable by MRI and whether such changes are specific to the type of learning. Here we trained inbred mice for 5 days on one of three different versions of the Morris water maze and, using high-resolution MRI, revealed specific growth in the hippocampus of mice trained on a spatial variant of the maze, whereas mice trained on the cued version were found to have growth in the striatum. The structure-specific growth found furthermore correlated with GAP-43 staining, a marker of neuronal process remodelling, but not with neurogenesis nor neuron or astrocyte numbers or sizes. Our findings provide evidence that brain morphology changes rapidly at a scale detectable by MRI and furthermore demonstrate that specific brain regions grow or shrink in response to the changing environmental demands. The data presented herein have implications for both human imaging as well as rodent structural plasticity research, in that it provides a tool to screen for neuronal plasticity across the whole brain in the mouse while also providing a direct link between human and mouse studies.

Selective erasure of a fear memory.

Memories are thought to be encoded by sparsely distributed groups of neurons. However, identifying the precise neurons supporting a given memory (the memory trace) has been a long-standing challenge. We have shown previously that lateral amygdala (LA) neurons with increased cyclic adenosine monophosphate response element-binding protein (CREB) are preferentially activated by fear memory expression, which suggests that they are selectively recruited into the memory trace. We used an inducible diphtheria-toxin strategy to specifically ablate these neurons. Selectively deleting neurons overexpressing CREB (but not a similar portion of random LA neurons) after learning blocked expression of that fear memory. The resulting memory loss was robust and persistent, which suggests that the memory was permanently erased. These results establish a causal link between a specific neuronal subpopulation and memory expression, thereby identifying critical neurons within the memory trace.

Development and validation of a sensitive entropy-based measure for the water maze.

In the water maze, mice are trained to navigate to an escape platform located below the water's surface, and spatial learning is most commonly evaluated in a probe test in which the platform is removed from the pool. While contemporary tracking software provides precise positional information of mice for the duration of the probe test, existing performance measures (e.g., percent quadrant time, platform crossings) fail to exploit fully the richness of this positional data. Using the concept of entropy (H), here we develop a new measure that considers both how focused the search is and the degree to which searching is centered on the former platform location. To evaluate how H performs compared to existing measures of water maze performance we compiled five separate databases, containing more than 1600 mouse probe tests. Random selection of individual trials from respective databases then allowed us to simulate experiments with varying sample and effect sizes. Using this Monte Carlo-based method, we found that H outperformed existing measures in its ability to detect group differences over a range of sample or effect sizes. Additionally, we validated the new measure using three models of experimentally induced hippocampal dysfunction: (1) complete hippocampal lesions, (2) genetic deletion of alphaCaMKII, a gene implicated in hippocampal behavioral and synaptic plasticity, and (3) a mouse model of Alzheimer's disease. Together, these data indicate that H offers greater sensitivity than existing measures, most likely because it exploits the richness of the precise positional information of the mouse throughout the probe test.

Increasing CREB in the auditory thalamus enhances memory and generalization of auditory conditioned fear.

Although the lateral nucleus of the amygdala (LA) is essential for conditioned auditory fear memory, an emerging theme is that plasticity in multiple brain regions contributes to fear memory formation. The LA receives direct projections from the auditory thalamus, specifically the medial division of the medial geniculate nucleus (MGm) and adjacent posterior intralaminar nucleus (PIN). While traditionally viewed as a simple relay structure, mounting evidence implicates the thalamus in diverse cognitive processes. We investigated the role of plasticity in the MGm/PIN in auditory fear memory. First we found that auditory fear conditioning (but not control manipulations) increased the levels of activated CREB in both the MGm and PIN. Next, using viral vectors, we showed that exogenously increasing CREB in this region specifically enhanced formation of an auditory conditioned fear memory without affecting expression of an auditory fear memory, formation of a contextual fear memory, or basic auditory processing. Interestingly, mice with increased CREB levels in the MGm/PIN also showed broad auditory fear generalization (in contrast to control mice, they exhibited fear responses to tones of other frequencies). Together, these results implicate CREB-mediated plasticity in the MGm/PIN in both the formation and generalization of conditioned auditory fear memory. Not only do these findings refine our knowledge of the circuitry underlying fear memory but they also provide novel insights into the neural substrates that govern the degree to which acquired fear of a tone generalizes to other tones.

Neuronal competition and selection during memory formation.

Competition between neurons is necessary for refining neural circuits during development and may be important for selecting the neurons that participate in encoding memories in the adult brain. To examine neuronal competition during memory formation, we conducted experiments with mice in which we manipulated the function of CREB (adenosine 3',5'-monophosphate response element-binding protein) in subsets of neurons. Changes in CREB function influenced the probability that individual lateral amygdala neurons were recruited into a fear memory trace. Our results suggest a competitive model underlying memory formation, in which eligible neurons are selected to participate in amemorytrace as a function of their relative CREB activity at the time of learning.

Hypoglycemic seizures during transient hypoglycemia exacerbate hippocampal dysfunction.

Severe hypoglycemia constitutes a medical emergency, involving seizures, coma and death. We hypothesized that seizures, during limited substrate availability, aggravate hypoglycemia-induced brain damage. Using immature isolated, intact hippocampi and frontal neocortical blocks subjected to low glucose perfusion, we characterized hypoglycemic (neuroglycopenic) seizures in vitro during transient hypoglycemia and their effects on synaptic transmission and glycogen content. Hippocampal hypoglycemic seizures were always followed by an irreversible reduction (>60% loss) in synaptic transmission and were occasionally accompanied by spreading depression-like events. Hypoglycemic seizures occurred more frequently with decreasing "hypoglycemic" extracellular glucose concentrations. In contrast, no hypoglycemic seizures were generated in the neocortex during transient hypoglycemia, and the reduction of synaptic transmission was reversible (<60% loss). Hypoglycemic seizures in the hippocampus were abolished by NMDA and non-NMDA antagonists. The anticonvulsant, midazolam, but neither phenytoin nor valproate, also abolished hypoglycemic seizures. Non-glycolytic, oxidative substrates attenuated, but did not abolish, hypoglycemic seizure activity and were unable to support synaptic transmission, even in the presence of the adenosine (A1) antagonist, DPCPX. Complete prevention of hypoglycemic seizures always led to the maintenance of synaptic transmission. A quantitative glycogen assay demonstrated that hypoglycemic seizures, in vitro, during hypoglycemia deplete hippocampal glycogen. These data suggest that suppressing seizures during hypoglycemia may decrease subsequent neuronal damage and dysfunction.