A major chondroitin sulfate proteoglycan in the brain, 6B4 proteoglycan/phosphacan, corresponds to the extracellular region of a receptor-like protein-tyrosine phosphatase, PTPzeta/RPTPbeta. Here, we purified and characterized 6B4 proteoglycan-binding proteins from rat brain. From the CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid) extract of brain microsomal fractions, 18-, 28-, and 40-kDa proteins were specifically isolated using 6B4 proteoglycan-Sepharose. N-terminal amino acid sequencing identified the 18-kDa protein as pleiotrophin/heparin-binding growth-associated molecule (HB-GAM). Scatchard analysis of 6B4 proteoglycan-pleiotrophin binding revealed low (Kd = 3 nM) and high (Kd = 0.25 nM) affinity binding sites. Chondroitinase ABC digestion of the proteoglycan decreased the binding affinities to a single value (Kd = 13 nM) without changing the number of binding sites. This suggested the presence of two subpopulations of the proteoglycan with different chondroitin sulfate structures. Heparin potently inhibited binding of 6B4 proteoglycan to pleiotrophin (IC50 = 3.5 ng/ml). Heparan sulfate and chondroitin sulfate C inhibited moderately (IC50 = 150 and 400 ng/ml, respectively), but, in contrast, chondroitin sulfate A and keratan sulfate were poor inhibitors (IC50 > 100 microg/ml). Immunofluorescence and immunoblotting analyses indicated that both 6B4 proteoglycan and PTPzeta are located on cortical neurons. Anti-6B4 proteoglycan antibody added to the culture medium suppressed pleiotrophin-induced neurite outgrowth of cortical neurons. These results suggested that interaction between 6B4 proteoglycan and pleiotrophin is required for the action of pleiotrophin, and chondroitin sulfate chains on 6B4 proteoglycan play regulatory roles in its binding.

Humans and animals can learn that specific sensory cues in the environment predict aversive events through a form of associative learning termed fear conditioning. This learning occurs when the sensory cues are paired with an aversive event occuring in close temporal proximity. Activation of lateral amygdala (LA) pyramidal neurons by aversive stimuli is thought to drive the formation of these associative fear memories; yet, there have been no direct tests of this hypothesis. Here we demonstrate that viral-targeted, tissuespecific expression of the light-activated channelrhodopsin (ChR2) in LA pyramidal cells permitted optical control of LA neuronal activity. Using this approach we then paired an auditory sensory cue with optical stimulation of LA pyramidal neurons instead of an aversive stimulus. Subsequently presentation of the tone alone produced behavioral fear responses. These results demonstrate in vivo optogenetic control of LA neurons and provide compelling support for the idea that fear learning is instructed by aversive stimulus-induced activation of LA pyramidal cells.ear conditioning is a simple form of associative learning that provides a powerful model system to study associative plasticity and memory formation (1-4). During fear conditioning, a neutral stimulus [termed the conditioned stimulus (CS)], often an auditory tone, is paired repeatedly with an aversive stimulus [termed the unconditioned stimulus (US)] and animals learn that the CS predicts the occurrence of the US. When the CS is encountered after learning, animals emit a stereotyped group of adaptive responses, including behavioral freezing and associated physiological adjustments, which together are termed the fear response.The lateral nucleus of the amygdala (LA) is a site of associative plasticity, where US-evoked depolarization of LA pyramidal neurons is thought to instruct plasticity at synapses formed by CS inputs onto the same neurons (5-7). Several lines of indirect evidence support the idea that this plasticity occurs as a result of a Hebbian mechanism through which depolarization of LA pyramidal neurons by the shock US coincident with weaker activation of the same cells by auditory CS inputs results in fear learning (8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18). This hypothesis makes the strong prediction that pairing an auditory CS with direct activation of LA pyramidal neurons as an US should be sufficient, in the absence of a shock US, to support fear learning and memory formation. Here we tested this hypothesis by substituting the aversive US with optical stimulation (19,20) of LA pyramidal neurons during learning, and we report that physiological activation of these cells results in fear conditioning. ResultsThe light activated channelrhodopsin (ChR2) (19,20) has been used in other neural systems to activate specific cell populations and produce learning (21-23). We took advantage of this technology and targeted ChR2 to pyramidal cells by in vivo viralmediated gene transfer. We used an adeno-associated virus (AAV) to express a...

A long-standing hypothesis termed "Hebbian plasticity" suggests that memories are formed through strengthening of synaptic connections between neurons with correlated activity. In contrast, other theories propose that coactivation of Hebbian and neuromodulatory processes produce the synaptic strengthening that underlies memory formation. Using optogenetics we directly tested whether Hebbian plasticity alone is both necessary and sufficient to produce physiological changes mediating actual memory formation in behaving animals. Our previous work with this method suggested that Hebbian mechanisms are sufficient to produce aversive associative learning under artificial conditions involving strong, iterative training. Here we systematically tested whether Hebbian mechanisms are necessary and sufficient to produce associative learning under more moderate training conditions that are similar to those that occur in daily life. We measured neural plasticity in the lateral amygdala, a brain region important for associative memory storage about danger. Our findings provide evidence that Hebbian mechanisms are necessary to produce neural plasticity in the lateral amygdala and behavioral memory formation. However, under these conditions Hebbian mechanisms alone were not sufficient to produce these physiological and behavioral effects unless neuromodulatory systems were coactivated. These results provide insight into how aversive experiences trigger memories and suggest that combined Hebbian and neuromodulatory processes interact to engage associative aversive learning.Hebbian plasticity | amygdala | neuromodulation | instructive signals | associative learning H ebbian plasticity refers to the strengthening of a presynaptic input onto a postsynaptic neuron when both pre-and postsynaptic neurons are coactive (1). This was originally proposed as a mechanism for memory formation. Findings from in vitro and in vivo physiological studies suggest that Hebbian processes control synaptic strengthening (2-10). However, other results and theories suggest that Hebbian mechanisms alone are not normally sufficient for producing synaptic plasticity and that synaptic strengthening mediating memory formation involves interactions between Hebbian and neuromodulatory mechanisms (3,4,7,(11)(12)(13)(14)(15)(16)(17)(18)(19). Although molecules that may mediate Hebbian processes in memory formation have been identified (3,11,16,17,(20)(21)(22), it has been difficult to directly test whether Hebbian plasticity alone or in combination with neuromodulation is necessary and sufficient to produce neural plasticity and memories in behaving animals (especially in mammals). This is because of technical limitations in controlling correlated activity between pre-and postsynaptic neurons involved in memory storage in a temporally/spatially precise manner while measuring behavioral memory formation and neural plasticity.To overcome these problems, we used optogenetic techniques to directly manipulate Hebbian mechanisms in pyramidal neurons in the lateral nucl...

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During sleep and awake rest, the neocortex generates large-scale slow-wave activity. Here we report that the claustrum, a poorly understood subcortical neural structure, coordinates neocortical slow-wave generation. We established a transgenic mouse line allowing genetic and electrophysiological interrogation of a subpopulation of claustral glutamatergic neurons.

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The possibility that some of the brain proteoglycans are receptor-like protein tyrosine phosphatases (PTPases) was investigated. Membrane-bound proteoglycan fractions were prepared from the postnuclear membrane fraction of &day-old rat brain by DEAE ion-exchange chromatography and CsCl density gradient centrifugation. The isolated proteoglycan fractions showed high PTPase specific activities together with the typical PTPase characteristics. Renaturation experiments indicated that chondroitin sulfate proteoglycans with 380-and 170-kDa core proteins carried the PTPase activity. The proteoglycan with 380-kDa core protein was identified as RPTP/S/[ bearing HNK-1 carbohydrate.

Inheritance of the apolipoprotein (apoE) epsilon4 allele is a risk factor for developing Alzheimer's disease (AD). The purpose of the present study was to determine effects of apoE-isoforms on the transbilayer distribution of cholesterol in synaptic plasma membranes (SPM) using mice expressing human apoE3 and apoE4. Total SPM cholesterol levels did not differ among the wild-type and apoE3 and apoE4 knock-in mice. However, a striking difference was observed in the transbilayer distribution of SPM cholesterol. ApoE4 knock-in mice showed an approximately 2-fold increase in exofacial leaflet cholesterol compared with apoE3 knock-in mice and wild-type mice. The results of this study suggest that pathogenic effects of apoE4 on AD development could be closely linked to alteration of cholesterol distribution in SPM.