A consistent map in the medial entorhinal cortex supports spatial memory.

The medial entorhinal cortex (MEC) is hypothesized to function as a cognitive map for memory-guided navigation. How this map develops during learning and influences memory remains unclear. By imaging MEC calcium dynamics while mice successfully learned a novel virtual environment over ten days, we discovered that the dynamics gradually became more spatially consistent and then stabilized. Additionally, grid cells in the MEC not only exhibited improved spatial tuning consistency, but also maintained stable phase relationships, suggesting a network mechanism involving synaptic plasticity and rigid recurrent connectivity to shape grid cell activity during learning. Increased c-Fos expression in the MEC in novel environments further supports the induction of synaptic plasticity. Unsuccessful learning lacked these activity features, indicating that a consistent map is specific for effective spatial memory. Finally, optogenetically disrupting spatial consistency of the map impaired memory-guided navigation in a well-learned environment. Thus, we demonstrate that the establishment of a spatially consistent MEC map across learning both correlates with, and is necessary for, successful spatial memory.

A consistent map in the medial entorhinal cortex supports spatial memory.

The medial entorhinal cortex (MEC) is hypothesized to function as a cognitive map for memory-guided navigation. How this map develops during learning and influences memory remains unclear. By imaging MEC calcium dynamics while mice successfully learned a novel virtual environment over ten days, we discovered that the dynamics gradually became more spatially consistent and then stabilized. Additionally, grid cells in the MEC not only exhibited improved spatial tuning consistency, but also maintained stable phase relationships, suggesting a network mechanism involving synaptic plasticity and rigid recurrent connectivity to shape grid cell activity during learning. Increased c-Fos expression in the MEC in novel environments further supports the induction of synaptic plasticity. Unsuccessful learning lacked these activity features, indicating that a consistent map is specific for effective spatial memory. Finally, optogenetically disrupting spatial consistency of the map impaired memory-guided navigation in a well-learned environment. Thus, we demonstrate that the establishment of a spatially consistent MEC map across learning both correlates with, and is necessary for, successful spatial memory.

Further Studies on the Role of BTBD9 in the Cerebellum, Sleep-like Behaviors and the Restless Legs Syndrome.

Genetic analyses have linked BTBD9 to restless legs syndrome (RLS) and sleep regulation. Btbd9 knockout mice show RLS-like motor restlessness. Previously, we found hyperactivity of cerebellar Purkinje cells (PCs) in Btbd9 knockout mice, which may contribute to the motor restlessness observed. However, underlying mechanisms for PC hyperactivity in Btbd9 knockout mice are unknown. Here, we used dissociated PC recording, brain slice recording and western blot to address this question. Our dissociated recording shows that knockout PCs had increased TEA-sensitive, Ca2+-dependent K+ currents. Applying antagonist to large conductance Ca2+-activated K+ (BK) channels further isolated the increased current as BK current. Consistently, we found increased amplitude of afterhyperpolarization and elevated BK protein levels in the knockout mice. Dissociated recording also shows a decrease in TEA-insensitive, Ca2+-dependent K+ currents. The result is consistent with reduced amplitude of tail currents, mainly composed of small conductance Ca2+-activated K+ (SK) currents, in slice recording. Our results suggest that BK and SK channels may be responsible for the hyperactivity of knockout PCs. Recently, BTBD9 protein was shown to associate with SYNGAP1 protein. We found a decreased cerebellar level of SYNGAP1 in Btbd9 knockout mice. However, Syngap1 heterozygous knockout mice showed nocturnal, instead of diurnal, motor restlessness. Our results suggest that SYNGAP1 deficiency may not contribute directly to the RLS-like motor restlessness observed in Btbd9 knockout mice. Finally, we found that PC-specific Btbd9 knockout mice exhibited deficits in motor coordination and balance similar to Btbd9 knockout mice, suggesting that the motor effect of BTBD9 in PCs is cell-autonomous.

Deficiency of Meis1, a transcriptional regulator, in mice and worms: Neurochemical and behavioral characterizations with implications in the restless legs syndrome.

Restless legs syndrome is a sleep-related sensorimotor neurological disease affecting up to 10% of the population. Genetic analyses have identified Myeloid Ecotropic viral Integration Site 1 (MEIS1), a transcriptional regulator, to be associated with not only the restless legs syndrome but also self-reported symptoms of insomnia and sleep. This study is to determine if Meis1 deficiency in mice can lead to restless legs syndrome-like phenotypes, and if it is the case, what the underlying mechanisms are. We used two genetic model systems, Caenorhabditis elegans and mice. Egg retention assay and fluorescent reporters were used with C. elegans. For mice, we performed behavioral tests, serum and brain iron detection, qRT-PCR, western blot, immunohistochemistry, and in vitro brain-slice recording. Our results showed that with C. elegans, the function of dop-3, an orthologue of DRD2, was diminished after the knockdown of unc-62, an ortholog of MEIS1. Additionally, unc-62 knockdown led to enhanced transcription of the orthologue of tyrosine hydroxylase, cat-2. Meis1 knockout mice were hyperactive and had a rest-phase-specific increased probability of waking. Moreover, Meis1 knockout mice had increased serum ferritin and altered striatal dopaminergic and cholinergic systems. Specifically, Meis1 knockout mice showed an increased mRNA level but decreased protein level of tyrosine hydroxylase in the striatum. Furthermore, Meis1 knockout mice had increased striatal dopamine turnover and decreased spontaneous firing regularity of striatal cholinergic interneurons. Our data suggest that Meis1 knockout mice have restless legs syndrome-like motor restlessness and changes in serum ferritin levels. The symptoms may be related to dysfunctional dopaminergic and cholinergic systems.

Mu opioid receptor knockout mouse: Phenotypes with implications on restless legs syndrome.

Restless legs syndrome (RLS) is characterized by an irresistible need to move the legs while sitting or lying at night with insomnia as a frequent consequence. Human RLS has been associated with abnormalities in the endogenous opioid system, the dopaminergic system, the iron regulatory system, anemia, and inflammatory and auto-immune disorders. Our previous work indicates that mice lacking all three subtypes of opioid receptors have a phenotype similar to that of human RLS. To study the roles of each opioid receptor subtype in RLS, we first used mu opioid receptor knockout (MOR KO) mice based on our earlier studies using postmortem brain and cell culture. The KO mice showed decreased hemoglobin, hematocrit, and red blood cells (RBCs), with an appearance of microcytic RBCs indicating anemia. Together with decreased serum iron and transferrin, but increased ferritin levels, the anemia is similar to that seen with chronic inflammation in humans. A decreased serum iron level was also observed in the wildtype mice treated with an MOR antagonist. Iron was increased in the liver and spleen of the KO mice. Normal circadian variations in the dopaminergic and serotoninergic systems were absent in the KO mice. The KO mice showed hyperactivity and increased thermal sensitivity in wakefulness primarily during what would normally be the sleep phase similar to that seen in human RLS. Deficits in endogenous opioid system transmission could predispose to anemia of inflammation and loss of circadian variations in dopaminergic or serotonergic systems, thereby contributing to an RLS-like phenotype.

The Role of BTBD9 in the Cerebellum, Sleep-like Behaviors and the Restless Legs Syndrome.

Recent genome-wide association studies (GWAS) have found cerebellum as a top hit for sleep regulation. Restless legs syndrome (RLS) is a sleep-related sensorimotor disorder characterized by uncomfortable sensations in the extremities, generally at night, which are often relieved by movements. Clinical studies have found that RLS patients have structural and functional abnormalities in the cerebellum. However, whether and how cerebellar pathology contributes to sleep regulation and RLS is not known. GWAS identified polymorphisms in BTBD9 conferring a higher risk of sleep disruption and RLS. Knockout of the BTBD9 homolog in mice (Btbd9) and fly results in motor restlessness and sleep disruption. We performed manganese-enhanced magnetic resonance imaging on the Btbd9 knockout mice and found decreased neural activities in the cerebellum, especially in lobules VIII, X, and the deep cerebellar nuclei. Electrophysiological recording of Purkinje cells (PCs) from Btbd9 knockout mice revealed an increased number of non-tonic PCs. Tonic PCs showed increased spontaneous activity and intrinsic excitability. To further investigate the cerebellar contribution to RLS and sleep-like behaviors, we generated PC-specific Btbd9 knockout mice (Btbd9 pKO) and performed behavioral studies. Btbd9 pKO mice showed significant motor restlessness during the rest phase but not in the active phase. Btbd9 pKO mice also had an increased probability of waking at rest. Unlike the Btbd9 knockout mice, there was no increased thermal sensation in the Btbd9 pKO. Our results indicate that the Btbd9 knockout influences the PC activity; dysfunction in the cerebellum may contribute to the motor restlessness found in the Btbd9 knockout mice.

BTBD9 and dopaminergic dysfunction in the pathogenesis of restless legs syndrome.

Restless legs syndrome (RLS) is characterized by an urge to move legs, usually accompanied by uncomfortable sensations. RLS symptoms generally happen at night and can be relieved by movements. Genetic studies have linked polymorphisms in BTBD9 to a higher risk of RLS. Knockout of BTBD9 homolog in mice (Btbd9) and fly results in RLS-like phenotypes. A dysfunctional dopaminergic system is associated with RLS. However, the function of BTBD9 in the dopaminergic system and RLS is not clear. Here, we made use of the simple Caenorhabditis elegans nervous system. Loss of hpo-9, the worm homolog of BTBD9, resulted in hyperactive egg-laying behavior. Analysis of genetic interactions between hpo-9 and genes for dopamine receptors (dop-1, dop-3) indicated that hpo-9 and dop-1 worked similarly. Reporter assays of dop-1 and dop-3 revealed that hpo-9 knockout led to a significant increase of DOP-3 expression. This appears to be evolutionarily conserved in mice with an increased D2 receptor (D2R) mRNA in the striatum of the Btbd9 knockout mice. Furthermore, the striatal D2R protein was significantly decreased and Dynamin I was increased. Overall, activities of DA neurons in the substantia nigra were not altered, but the peripheral D1R pathway was potentiated in the Btbd9 knockout mice. Finally, we generated and characterized the dopamine neuron-specific Btbd9 knockout mice and detected an active-phase sleepiness, suggesting that dopamine neuron-specific loss of Btbd9 is sufficient to disturb the sleep. Our results suggest that increased activities in the D1R pathway, decreased activities in the D2R pathway, or both may contribute to RLS.

Probe the relationship between BTBD9 and MEIS1 in C. elegans and mouse.

Restless legs syndrome (RLS) is a neurological disorder characterized by an urge to move and uncomfortable sensations. Genetic studies have identified polymorphisms in up to 19 risk loci, including MEIS1 and BTBD9. Rodents deficient in either homolog show RLS-like phenotypes. However, whether MEIS1 and BTBD9 interact in vivo is unclear. Here, with C. elegans, we observed that the hyperactive egg-laying behavior caused by loss of BTBD9 homolog was counteracted by knockdown of MEIS1 homolog. This was further investigated in mutant mice with Btbd9, Meis1, or both knocked out. The double knockout mice showed an earlier onset of the motor deficit in the wheel running test but did not have increased sensitivity to the heat stimuli as observed in single KOs. Meis1 protein level was not influenced by Btbd9 deficiency, and Btbd9 transcription was not affected by Meis1 haploinsufficiency. Our results demonstrate that MEIS1 and BTBD9 do not regulate each other.

The role of BTBD9 in the cerebral cortex and the pathogenesis of restless legs syndrome.

Restless legs syndrome (RLS) is a nocturnal neurological disorder affecting up to 10% of the population. It is characterized by an urge to move and uncomfortable sensations in the legs which can be relieved by movements. Mutations in BTBD9 may confer a higher risk of RLS. We developed Btbd9 knockout mice as an animal model. Functional alterations in the cerebral cortex, especially the sensorimotor cortex, have been found in RLS patients in several imaging studies. However, the role of cerebral cortex in the pathogenesis of RLS remains unclear. To explore this, we used in vivo manganese-enhanced MRI and found that the Btbd9 knockout mice had significantly increased neural activities in the primary somatosensory cortex (S1) and the rostral piriform cortex. Morphometry study revealed a decreased thickness in a part of S1 representing the hindlimb (S1HL) and M1. The electrophysiological recording showed Btbd9 knockout mice had enhanced short-term plasticity at the corticostriatal terminals to D1 medium spiny neurons (MSNs). Furthermore, we specifically knocked out Btbd9 in the cerebral cortex of mice (Btbd9 cKO). The Btbd9 cKO mice showed a rest-phase specific motor restlessness, decreased thermal sensation, and a thinner S1HL and M1. Both Btbd9 knockout and Btbd9 cKO exhibited motor deficits. Our results indicate that systematic BTBD9 deficiency leads to both functional and morphometrical changes of the cerebral cortex, and an alteration in the corticostriatal pathway to D1 MSNs. Loss of BTBD9 only in the cerebral cortex is sufficient to cause similar phenotypes as observed in the Btbd9 complete knockout mice.

Hyperactivity, dopaminergic abnormalities, iron deficiency and anemia in an in vivo opioid receptors knockout mouse: Implications for the restless legs syndrome.

Previous studies have uncovered a potential role of the opioid system in iron hemostasis and dopamine metabolism. Abnormalities in both of these systems have been noted in human RLS. Autopsy studies of human RLS have shown an endogenous opioid deficiency in the thalamus. Opioids, particularly prolonged-release oxycodone/naloxone, have been approved in Europe to be a second-line therapy for severe restless legs syndrome (RLS). To study the role of opioid receptors in the pathogenesis of RLS, we used a triple knockout (KO) mouse strain that lack mu, delta, and kappa opioid receptors and explored the behavioral and biochemical parameters relevant to RLS. The triple KO mice showed hyperactivity and a trend of increased probability of waking during the rest period (day) akin to that in human RLS (night). Surprisingly, triple KO mice also exhibit decreased serum iron concentration, evidence of anemia, a significant dysfunction in dopamine metabolism akin to that noted in human RLS, as well as an increased latency in response to thermal stimuli. To our knowledge, this is the first study to demonstrate that the endogenous opioid system may play a role in iron metabolism and subsequently in the pathogenesis of anemia. It is also the first study showing that opioid receptors are involved in the production of motor restlessness with a circadian predominance. Our findings support the role of endogenous opioids in the pathogenesis of RLS, and the triple KO mice can be used to understand the relationship between iron deficiency, anemia, dopaminergic dysfunction, and RLS.

The Role of BTBD9 in Striatum and Restless Legs Syndrome.

Restless legs syndrome (RLS) is a sensory-motor neurological disorder characterized by uncomfortable sensations in the extremities, generally at night, which is often relieved by movements. Genome-wide association studies (GWAS) have identified mutations in BTBD9 conferring a higher risk of RLS. Knockout of the BTBD9 homolog in mice (Btbd9) and fly results in motor restlessness and sleep disruption. Clinical studies have found RLS patients have structural and functional abnormalities in the striatum; however, whether and how striatal pathology contributes to the pathogenesis of RLS is not known. Here, we used fMRI to map regions of altered synaptic activity in basal ganglia of systematic Btbd9 knock-out (KO) mice. We further dissected striatal circuits using patch-clamp electrophysiological recordings in brain slices. Two different mouse models were generated to test the effect of specific knockout of Btbd9 in either striatal medium spiny neurons (MSNs) or cholinergic interneurons (ChIs) using the electrophysiological recording, motor and sensory behavioral tests. We found that Btbd9 KO mice showed enhanced neural activity in the striatum, increased postsynaptic currents in the MSNs, and decreased excitability of the striatal ChIs. Knocking out Btbd9 specifically in the striatal MSNs, but not the ChIs, led to rest-phase specific motor restlessness, sleep disturbance, and increased thermal sensation in mice, which are consistent with results obtained from the Btbd9 KO mice. Our data establish the role of Btbd9 in regulating the activity of striatal neurons. Increased activity of the striatal MSNs, possibly through modulation by the striatal ChIs, contributes to the pathogenesis of RLS.

Differential Dopamine D1 and D3 Receptor Modulation and Expression in the Spinal Cord of Two Mouse Models of Restless Legs Syndrome.

Restless Legs Syndrome (RLS) is often and successfully treated with dopamine receptor agonists that target the inhibitory D3 receptor subtype, however there is no clinical evidence of a D3 receptor dysfunction in RLS patients. In contrast, genome-wide association studies in RLS patients have established that a mutation of the MEIS1 gene is associated with an increased risk in developing RLS, but the effect of MEIS1 dysfunction on sensorimotor function remain unknown. Mouse models for a dysfunctional D3 receptor (D3KO) and Meis1 (Meis1KO) were developed independently, and each animal expresses some features associated with RLS in the clinic, but they have not been compared in their responsiveness to treatment options used in the clinic. We here confirm that D3KO and Meis1KO animals show increased locomotor activities, but that only D3KO show an increased sensory excitability to thermal stimuli. Next we compared the effects of dopaminergics and opioids in both animal models, and we assessed D1 and D3 dopamine receptor expression in the spinal cord, the gateway for sensorimotor processing. We found that Meis1KO share most of the tested behavioral properties with their wild type (WT) controls, including the modulation of the thermal pain withdrawal reflex by morphine, L-DOPA and D3 receptor (D3R) agonists and antagonists. However, Meis1KO and D3KO were behaviorally more similar to each other than to WT when tested with D1 receptor (D1R) agonists and antagonists. Subsequent Western blot analyses of D1R and D3R protein expression in the spinal cord revealed a significant increase in D1R but not D3R expression in Meis1KO and D3KO over WT controls. As the D3R is mostly present in the dorsal spinal cord where it has been shown to modulate sensory pathways, while activation of the D1Rs can activate motoneurons in the ventral spinal cord, we speculate that D3KO and Meis1KO represent two complementary animal models for RLS, in which the mechanisms of sensory (D3R-mediated) and motor (D1R-mediated) dysfunctions can be differentially explored.