Although a wide variety of genetic tools has been developed to study learning and memory, the molecular basis of memory encoding remains incompletely understood. Here, we undertook an unbiased ...approach to identify novel genes critical for memory encoding. From a large-scale, in vivo mutagenesis screen using contextual fear conditioning, we isolated in mice a mutant, named
Clueless
, with spatial learning deficits. A causative missense mutation (G434V) was found in the voltage-gated potassium channel, subfamily C member 3 (
Kcnc3)
gene in a region that encodes a transmembrane voltage sensor. Generation of a Kcnc3
G434V
CRISPR mutant mouse confirmed this mutation as the cause of the learning defects. While G434V had no effect on transcription, translation, or trafficking of the channel, electrophysiological analysis of the G434V mutant channel revealed a complete loss of voltage-gated conductance, a broadening of the action potential, and decreased neuronal firing. Together, our findings have revealed a role for
Kcnc3
in learning and memory.
Changes in CaMKII-regulated synaptic excitability are a means through which experience may modify neuronal function and shape behavior. While behavior in rodent addiction models is linked with CaMKII ...activity in the nucleus accumbens (NAc) shell, the key cellular adaptations that forge this link are unclear. Using a mouse strain with striatal-specific expression of autonomously active CaMKII (T286D), we demonstrate that while persistent CaMKII activity induces behaviors comparable to those in mice repeatedly exposed to psychostimulants, it is insufficient to increase AMPAR-mediated synaptic strength in NAc shell. However, autonomous CaMKII upregulates A-type K(+) current (IA) and decreases firing in shell neurons. Importantly, inactivating the transgene with doxycycline eliminates both the IA-mediated firing decrease and the elevated behavioral response to cocaine. This study identifies CaMKII regulation of IA in NAc shell neurons as a novel cellular contributor to the sensitization of cocaine reward.
Background Excitatory synaptic transmission in the nucleus accumbens (NAc) regulates the reinstatement of drug seeking, an animal model of relapse in human drug addicts. However, the functional ...adaptations at NAc synapses that mediate reinstatement are not clearly understood. Methods We assessed the behavioral responses of mice to cocaine administration by measuring locomotor stimulation and the acquisition, extinction, and reinstatement of conditioned place preference. Synaptic function was then examined by preparing acute brain slices and performing whole cell voltage-clamp recordings from individual medium spiny neurons in the NAc shell. Results We find that reduced excitatory synaptic strength in the NAc shell is a common functional adaptation induced by multiple experiences known to cause reinstatement, including stress and drug re-exposure. The same synaptic adaptation is observed shortly after reinstatement of conditioned place preference by a cocaine priming injection. Conclusions This common synaptic modification associated with stress, drug re-exposure, and reinstatement defines a potential synaptic gateway to relapse.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Developmental and epileptic encephalopathies (DEE) are rare and serious neurological disorders characterized by severe epilepsy with refractory seizures and a significant developmental delay. ...Recently, DEE73 was linked to genetic alterations of the RNF13 gene, which convert positions 311 or 312 in the RNF13 protein from leucine to serine or proline, respectively (L311S and L312P). Using a fluorescence microscopy approach to investigate the molecular and cellular mechanisms affected by RNF13 protein variants, the current study shows that wild-type RNF13 localizes extensively with endosomes and lysosomes, while L311S and L312P do not extensively colocalize with the lysosomal marker Lamp1. Our results show that RNF13 L311S and L312P proteins affect the size of endosomal vesicles along with the temporal and spatial progression of fluorescently labeled epidermal growth factor, but not transferrin, in the endolysosomal system. Furthermore, GST-pulldown and co-immunoprecipitation show that RNF13 variants disrupt association with AP-3 complex. Knockdown of AP-3 complex subunit AP3D1 alters the lysosomal localization of wild-type RNF13 and similarly affects the size of endosomal vesicles. Importantly, our study provides a first step toward understanding the cellular and molecular mechanism altered by DEE73-associated genetic variations of RNF13.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Exposure to abused drugs and stressful experience, two factors that promote the development of addiction, also modify synaptic function in the mesolimbic dopamine system. Here, we show that exposure ...to a novel environment produces functional synaptic adaptations in the nucleus accumbens (NAc) that mirror the effect of conventional forms of stress. We find an enhancement of excitatory synaptic strength in the NAc shell one day after exposure to a novel environment for 60
min – an effect not observed in NAc core. This effect disappeared following repeated exposure to the same environment, but then reappeared if mice are returned to the same environment 10–14 days later. There were no interactions between the effects of environmental novelty and a single exposure to cocaine (15
mg/kg), with no effect of the latter on synaptic strength in NAc shell. These results have important implications for designing studies of NAc synapses in the context of behavioral analysis, and expand our understanding of how different forms of stress modify NAc synaptic function.
This article is part of a Special Issue entitled ‘Synaptic Plasticity and Addiction’.
► Exposure to a novel environment alters synaptic function in nucleus accumbens shell. ► Conventional forms of stressful experience cause similar synaptic changes. ► This effect is no longer observed after repeated exposure to the same context. ► The synaptic impact of the same context is renewed 10–14 days after repeated exposure.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, SAZU, SBCE, UL, UM, UPCLJ, UPUK
Major depressive disorder (MDD) is considered a 'circuitopathy', and brain stimulation therapies hold promise for ameliorating MDD symptoms, including hippocampal dysfunction. It is unknown whether ...stimulation of upstream hippocampal circuitry, such as the entorhinal cortex (Ent), is antidepressive, although Ent stimulation improves learning and memory in mice and humans. Here we show that molecular targeting (Ent-specific knockdown of a psychosocial stress-induced protein) and chemogenetic stimulation of Ent neurons induce antidepressive-like effects in mice. Mechanistically, we show that Ent-stimulation-induced antidepressive-like behavior relies on the generation of new hippocampal neurons. Thus, controlled stimulation of Ent hippocampal afferents is antidepressive via increased hippocampal neurogenesis. These findings emphasize the power and potential of Ent glutamatergic afferent stimulation-previously well-known for its ability to influence learning and memory-for MDD treatment.
► Use embryonic striatal and cortical tissue to culture medium spiny neurons (MSNs). ► Cultures can be made from a single embryo – good for studies with transgenics. ► Electroporated MSNs can retain ...expression for more than 21 days. ► Produces MSNs with complex dendritic morphology and abundant dendritic spines. ► MSNs have
in vivo-like morphological and electrophysiological characteristics.
Dendritic spines of striatal
Medium
Spiny
Neurons (MSNs) receive converging dopaminergic and glutamatergic inputs. These spines undergo experience-dependent structural plasticity following repeated drug administration and during disease states like Huntington's and Parkinson's. Thus, understanding the molecular mechanisms leading to structural plasticity is an important step toward establishing a clear relationship between spine structure and function, and will ultimately contribute to understanding how changes in dendritic spine structure relate to behaviors or diseases. One major difficulty faced when studying MSN development is the lack of a detailed, standardized
in vitro model system that produces MSNs with
in vivo-like morphologies. For example, unlike cultured pyramidal neurons, MSNs grown in mono-cultures display stunted dendritic arborization and fail to develop a full cohort of mature dendritic spines.
Here we report the generation of an embryonic mouse cortical–striatal co-culture that generates high cell yields from a single embryo. Unlike MSNs in striatal mono-culture, MSNs in co-culture develop
in vivo-like morphologies and high densities of dendritic spines. Morphological identification of co-cultured MSNs expressing a soluble fluorescent protein can be confirmed by immunochemical detection of DARPP-32 (
Dopamine and cyclic
AMP
regulated
phospho
protein of 32
kDa). Additionally, co-cultured MSN spines contain PSD-95 puncta and are apposed to SV2 puncta, indicating the spines express synaptic machinery. Finally, whole-cell recordings of co-cultured MSNs exhibit higher mEPSC frequency compared to mono-cultured MSNs, suggesting that the spines are functionally mature. These studies establish that this co-culture system is suitable for studying the morphological and physiological development and function of MSN dendritic spines.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
To study the role of NMDA receptors in dopamine signaling of the striatum, the brain area that receives glutamatergic inputs from various cortical areas and most dopaminergic inputs, we generated ...striatum-specific NMDA receptor-deficient mice. The mutant pups showed reduced food intake and retarded growth starting at the second postnatal week and died on approximately postnatal day 20 (P20). The time course of postnatal lethality is similar to that of compound mutant, double knockout of dopamine D1/D2 receptors, or genetically engineered dopamine-deficient mouse. In vivo electrophysiological recordings in the mutant pups showed that frequencies in the range of gamma oscillation were reduced in the striatal circuits. Moreover, the number of functional dopamine receptors in the striatum as measured by D1- and D2-binding experiments was greatly diminished in the mutants as compared with control animals. A consequence of diminished dopamine binding in the striatum manifested in an increase of locomotor activity. The administration of D1/D2 agonists paradoxically reduced the hyperactivity of the mutant mice as compared with an increase in locomotor activity in control mice. These results demonstrate that the NMDA receptor plays an essential role in the integration of dopamine signaling in the striatum and that is required in behavioral function.
The entorhinal cortex receives a large projection from the piriform cortex, and synaptic plasticity in this pathway may affect olfactory processing. In vitro whole cell recordings have been used here ...to investigate postsynaptic signalling mechanisms that mediate the induction of long-term synaptic depression (LTD) in layer II entorhinal cortex cells. To induce LTD, pairs of pulses, using a 30-millisecond interval, were delivered at 1 Hz for 15 minutes. Induction of LTD was blocked by the NMDA receptor antagonist APV and by the calcium chelator BAPTA, consistent with a requirement for calcium influx via NMDA receptors. Induction of LTD was blocked when the FK506 was included in the intracellular solution to block the phosphatase calcineurin. Okadaic acid, which blocks activation of protein phosphatases 1 and 2a, also prevented LTD. Activation of protein phosphatases following calcium influx therefore contributes to induction of LTD in layer II of the entorhinal cortex.
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