Specific deletion of NaV1.1 sodium channels in inhibitory interneurons causes seizures and premature death in a mouse model of Dravet syndrome

Heterozygous loss-of-function mutations in the brain sodium channel Na(V)1.1 cause Dravet syndrome (DS), a pharmacoresistant infantile-onset epilepsy syndrome with comorbidities of cognitive impairment and premature death. Previous studies using a mouse model of DS revealed reduced sodium currents and impaired excitability in GABAergic interneurons in the hippocampus, leading to the hypothesis that impaired excitability of GABAergic inhibitory neurons is the cause of epilepsy and premature death in DS. However, other classes of GABAergic interneurons are less impaired, so the direct cause of hyperexcitability, epilepsy, and premature death has remained unresolved. We generated a floxed Scn1a mouse line and used the Cre-Lox method driven by an enhancer from the Dlx1,2 locus for conditional deletion of Scn1a in forebrain GABAergic neurons. Immunocytochemical studies demonstrated selective loss of Na(V)1.1 channels in GABAergic interneurons in cerebral cortex and hippocampus. Mice with this deletion died prematurely following generalized tonic-clonic seizures, and they were equally susceptible to thermal induction of seizures as mice with global deletion of Scn1a. Evidently, loss of Na(V)1.1 channels in forebrain GABAergic neurons is both necessary and sufficient to cause epilepsy and premature death in DS.     

Impaired differentiation and apoptosis of hematopoietic precursors in a mouse model of myelodysplastic syndrome

Expression of a NUP98-HOXD13 (NHD13) fusion gene, initially identified in a patient with myelodysplastic syndrome, leads to a highly penetrant myelodysplastic syndrome in mice that recapitulates all of the key features of the human disease. Expansion of undifferentiated lineage negative (lin(neg)) hematopoietic precursors that express NHD13 was markedly inhibited (30-fold) in vitro. Decreased expansion was accompanied by decreased production of terminally differentiated cells, indicating impaired differentiation of NHD13 precursors. Rather than differentiate, the majority (80%) of NHD13 lin(neg) precursors underwent apoptotic cell death when induced to differentiate. These findings demonstrate that NHD13 lin(neg) cells provide a tractable in vitro system for studies of myelodysplastic syndrome.     

Impaired hippocampal rhythmogenesis in a mouse model of mesial temporal lobe epilepsy

Mesial temporal lobe epilepsy (mTLE) is one of the most common forms of epilepsy, characterized by hippocampal sclerosis and memory deficits. Injection of kainic acid (KA) into the dorsal hippocampus of mice reproduces major electrophysiological and histopathological characteristics of mTLE. In extracellular recordings from the morphologically intact ventral hippocampus of KA-injected epileptic mice, we found that theta-frequency oscillations were abolished, whereas gamma oscillations persisted both in vivo and in vitro. Whole-cell recordings further showed that oriens-lacunosum-moleculare (O-LM) interneurons, key players in the generation of theta rhythm, displayed marked changes in their intrinsic and synaptic properties. Hyperpolarization-activated mixed cation currents (Ih) were significantly reduced, resulting in an increase in the input resistance and a hyperpolarizing shift in the resting membrane potential. Additionally, the frequency of spontaneous excitatory postsynaptic currents (sEPSCs) was increased, indicating a stronger excitatory input to these neurons. As a consequence, O-LM interneurons increased their firing rate from theta to gamma frequencies during induced network activity in acute slices from KA-injected mice. Thus, our physiological data together with network simulations suggest that changes in excitatory input and synaptic integration in O-LM interneurons lead to impaired rhythmogenesis in the hippocampus that in turn may underlie memory deficit.     

Reduced cortical activity due to a shift in the balance between excitation and inhibition in a mouse model of Rett syndrome

Rett Syndrome (RTT) is a devastating neurological disorder that is caused by mutations in the MECP2 gene. Mecp2-mutant mice have been used as a model system to study the disease mechanism. Our previous work has suggested that MeCP2 malfunction in neurons is the primary cause of RTT in the mouse. However, the neurophysiological consequences of MeCP2 malfunction remain obscure. Using whole-cell patch-clamp recordings in cortical slices, we show that spontaneous activity of pyramidal neurons is reduced in Mecp2-mutant mice. This decrease is not caused by a change in the intrinsic properties of the recorded neurons. Instead, the balance between cortical excitation and inhibition is shifted to favor inhibition over excitation. Moreover, analysis of the miniature excitatory postsynaptic currents (mEPSCs)/inhibitory postsynaptic currents (mIPSCs) in the Mecp2-mutant cortex reveals a reduction in mEPSC amplitudes, without significant change in the average mIPSC amplitude or frequency. These findings provide the first detailed electrophysiological analysis of Mecp2-mutant mice and provide a framework for understanding the pathophysiology of the disease and tools for studying the underlying disease mechanisms.     

Relationship between receptor binding and biopotency of somatostatin-14 and somatostatin-28 in mouse pituitary tumor cells

Somatostatin-14 (S-14) acts via specific receptors to inhibit basal as well as hormone- and forskolin-stimulated ACTH secretion in tumor cells (AtT-20/D16-16) of mouse anterior pituitary. In addition S-14 inhibits the stimulated but not basal cAMP accumulation. The potency of somatostatin-28 (S-28) for regulating these processes in these tumor cells has not been reported. In this study we have investigated the relationship between receptor-binding affinities of S-14 and S-28 and their biopotency in these cells. Membrane receptors for S-14 characterized using [125I-Tyr11]S-14 as the radioligand [maximum binding capacity (Bmax) = 1.28 +/- 0.1 pmol/mg; dissociation constant (Kd) = 1.1 +/- 0.04 nM] bound S-28 with 3-fold greater affinity than S-14. Binding sites quantitated using an S-28 analog [Leu8, D-Trp22, 125I-Tyr25]S-28 as radioligand (Bmax = 1.18 +/- 0.15 pmol/mg; Kd = 0.08 +/- 0.06 nM) also exhibited greater affinity for S-28 than S-14. Forskolin-stimulated cAMP accumulation and ACTH secretion in these cells were inhibited to a greater extent (4- and 9-fold, respectively) by S-28 than S-14. Preincubation of the cells with S-14 and S-28 (10(-7) M) resulted in a marked decrease (36% and 71%, respectively) of S-14 receptor concentration. Coincubation of the cells with both S-14 and S-28 led to 56% decrease in S-14 receptor binding. The responsiveness of the cells to forskolin stimulation of ACTH secretion and cAMP accumulation was significantly enhanced by preincubation with S-14 (10(-7) M) whereas the responsiveness to forskolin was completely abolished by preincubation with S-28. Simultaneous exposure of the cells to both S-14 and S-28 resulted in a partial reversal of the inhibiting effect of S-28 on forskolin-stimulated cAMP accumulation in these cells but did not result in a partial reversal of the inhibitory effect of S-28 on forskolin-stimulated ACTH secretion in these cells. These results demonstrate that S-28 is more potent than S-14 in AtT-20/D16-16 cells, its greater potency arising from its greater affinity for binding to S-14 receptors. The differential effects of these peptides after preincubation on the responsiveness of ACTH secretion and cAMP accumulation in these cells to forskolin stimulation suggests the possibility of existence of distinct S-14 and S-28 receptors, but these could not be identified by direct binding experiments using the S-14 and S-28 analogs employed in the study as radioligands.(ABSTRACT TRUNCATED AT 400 WORDS)     

大鼠肾性骨病模型皮质骨骨量及结构的变化

目的:观察腺嘌呤联合高磷饮食建立的肾性骨病模型大鼠的皮质骨骨量和骨结构的形态学变化,并探讨其骨矿代谢情况及机制。方法:3月龄雄性SD大鼠24只,随机分为腺嘌呤模型组(M组,1~4周予含腺嘌呤的高磷饮食,5~6周高磷饮食饲养)和高磷饮食对照组(C组,高磷饮食饲养6周);皮下注射钙黄绿素进行体内双荧光标记,处死大鼠后取左侧胫骨中段不脱钙包埋制备骨标本,骨组织形态计量学分析骨量和骨结构变化情况。结米:静态参数显示:与C组相比,M组骨组织总面积明显变小、皮质骨面积百分比和皮质骨厚度显著降低,骨髓腔面积百分比明显增大;胫骨皮质上出现侵蚀小孔。动态参数显示:与C组相比,M组骨外膜面荧光标记周长百分比无显著变化,但骨矿化沉积率、骨形成率明显增加;骨内膜面荧光标记周长百分比、骨矿化沉积率明显提高,骨形成率和骨吸收周长百分数明显增加。结论:腺嘌呤作用下高磷饮食大鼠胫骨皮质骨的骨外膜、内膜的骨形成和吸收活跃;皮质骨内外膜骨量丢失,说明骨吸收大于骨形成、骨偶联失衡而导致皮质厚度减少,处于明显的骨高转化代谢状态。     

Reduced cortical inhibition in a mouse model of familial childhood absence epilepsy

Mutations in the GABA(A) receptor gamma2 subunit are associated with childhood absence epilepsy and febrile seizures. To understand better the molecular basis of absence epilepsy in man, we developed a mouse model harboring a gamma2 subunit point mutation (R43Q) found in a large Australian family. Mice heterozygous for the mutation demonstrated behavioral arrest associated with 6-to 7-Hz spike-and-wave discharges, which are blocked by ethosuximide, a first-line treatment for absence epilepsy in man. Seizures in the mouse showed an abrupt onset at around age 20 days corresponding to the childhood nature of this disease. Reduced cell surface expression of gamma2(R43Q) was seen in heterozygous mice in the absence of any change in alpha1 subunit surface expression, ruling out a dominant-negative effect. GABA(A)-mediated synaptic currents recorded from cortical pyramidal neurons revealed a small but significant reduction that was not seen in the reticular or ventrobasal thalamic nuclei. We hypothesize that a subtle reduction in cortical inhibition underlies childhood absence epilepsy seen in humans harboring the R43Q mutation.     

Uncompensated polyuria in a mouse model of Bartter's syndrome

We have used homologous recombination to disrupt the mouse gene coding for the NaK2Cl cotransporter (NKCC2) expressed in kidney epithelial cells of the thick ascending limb and macula densa. This gene is one of several that when mutated causes Bartter's syndrome in humans, a syndrome characterized by severe polyuria and electrolyte imbalance. Homozygous NKCC2-/- pups were born in expected numbers and appeared normal. However, by day 1 they showed signs of extracellular volume depletion (hematocrit 51%; wild type 37%). They subsequently failed to thrive. By day 7, they were small and markedly dehydrated and exhibited renal insufficiency, high plasma potassium, metabolic acidosis, hydronephrosis of varying severity, and high plasma renin concentrations. None survived to weaning. Treatment of -/- pups with indomethacin from day 1 prevented growth retardation and 10% treated for 3 weeks survived, although as adults they exhibited severe polyuria (10 ml/day), extreme hydronephrosis, low plasma potassium, high blood pH, hypercalciuria, and proteinuria. Wild-type mice treated with furosemide, an inhibitor of NaK2Cl cotransporters, have a phenotype similar to the indomethacin-rescued -/- adults except that hydronephrosis was mild. The polyuria, hypercalciuria, and proteinuria of the -/- adults and furosemide-treated wild-type mice were unresponsive to inhibitors of the renin angiotensin system, vasopressin, and further indomethacin. Thus absence of NKCC2 in the mouse causes polyuria that is not compensated elsewhere in the nephron. The NKCC2 mutant animals should be valuable for uncovering new pathophysiologic and therapeutic aspects of genetic disturbances in water and electrolyte recovery by the kidney.     

Hyperactivity in a mouse model of the antiphospholipid syndrome

In the antiphospholipid syndrome (APS), antibodies to a complex of phospholipids and beta2-glycoprotein I (beta2-GPI) are associated with recurrent thromboembolic events, spontaneous abortions, thrombocytopenia and central nervous system (CNS) disturbances. Animals immunized with beta2-GPI develop the systemic manifestations of APS but the involvement of the (CNS) in these animals has not been studied. The objective of the present study was to examine mice with induced experimental APS for behavioral changes. Female Balb/C mice were immunized once with beta2-GPI in complete Freund's adjuvant (CFA) or with CFA alone. Four months after immunization the mice were tested in the staircase apparatus and the following two variables were measured: (1) number of rears: and (2) number of stairs climbed by the mice. Immunization with beta2-GPI resulted in elevated levels of circulating anti-negatively charged phospholipids and anti-beta2-GPI antibodies. The APS mice exhibited hyperactive behavior as reflected by more frequent rears (P < 0.023) and higher number of stairs climbed (P < 0.019) by the mice in 3 min. This simple test demonstrated that experimental APS animals are significantly hyperactive and may serve as a marker for CNS involvement in this model.     

Dynamics of the leading process,nucleus, and Golgi apparatus of migrating cortical interneurons in living mouse embryos

Precisely arranged cytoarchitectures such as layers and nuclei depend on neuronal migration, of which many in vitro studies have revealed the mode and underlying mechanisms. However, how neuronal migration is achieved in vivo remains unknown. Here we established an imaging system that allows direct visualization of cortical interneuron migration in living mouse embryos. We found that during nucleokinesis, translocation of the Golgi apparatus either precedes or occurs in parallel to that of the nucleus, suggesting the existence of both a Golgi/centrosome-dependent and -independent mechanism of nucleokinesis. Changes in migratory direction occur when the nucleus enters one of the leading process branches, which is accompanied by the retraction of other branches. The nucleus occasionally swings between two branches before translocating into one of them, the occurrence of which is most often preceded by Golgi apparatus translocation into that branch. These in vivo observations provide important insight into the mechanisms of neuronal migration and demonstrate the usefulness of our system for studying dynamic events in living animals.     

Impaired motor coordination and Purkinje cell excitability in mice lacking calretinin

In the cerebellum, the parallel fiber-Purkinje cell synapse can undergo long-term synaptic plasticity suggested to underlie motor learning and resulting from variations in intracellular calcium concentration ([Ca2+]i). Ca2+ binding proteins are enriched in the cerebellum, but their role in information processing is not clear. Here, we show that mice deficient in calretinin (Cr-/-) are impaired in tests of motor coordination. An impairment in Ca2+ homeostasis in Cr-/- Purkinje cells was supported by the high Ca2+-saturation of calbindin-D28k in these cells. The firing behavior of Purkinje cells is severely affected in Cr-/- alert mice, with alterations of simple spike firing rate, complex spike duration, and simple spike pause. In contrast, in slices, transmission at parallel fiber- or climbing fiber-Purkinje cell synapses is unaltered, indicating that marked modifications of the firing behavior in vivo can be undetectable in slice. Thus, these results show that calretinin plays a major role at the network level in cerebellar physiology.     

Diminished dosage of 22q11 genes disrupts neurogenesis and cortical development in a mouse model of 22q11 deletion/DiGeorge syndrome

The 22q11 deletion (or DiGeorge) syndrome (22q11DS), the result of a 1.5- to 3-megabase hemizygous deletion on human chromosome 22, results in dramatically increased susceptibility for “diseases of cortical connectivity” thought to arise during development, including schizophrenia and autism. We show that diminished dosage of the genes deleted in the 1.5-megabase 22q11 minimal critical deleted region in a mouse model of 22q11DS specifically compromises neurogenesis and subsequent differentiation in the cerebral cortex. Proliferation of basal, but not apical, progenitors is disrupted, and subsequently, the frequency of layer 2/3, but not layer 5/6, projection neurons is altered. This change is paralleled by aberrant distribution of parvalbumin-labeled interneurons in upper and lower cortical layers. Deletion of Tbx1 or Prodh (22q11 genes independently associated with 22q11DS phenotypes) does not similarly disrupt basal progenitors. However, expression analysis implicates additional 22q11 genes that are selectively expressed in cortical precursors. Thus, diminished 22q11 gene dosage disrupts cortical neurogenesis and interneuron migration. Such developmental disruption may alter cortical circuitry and establish vulnerability for developmental disorders, including schizophrenia and autism.     

Hyperdynamic circulatory syndrome in a mouse model transgenic for SerpinB3

Introduction and objectivesSerpinB3 is a cysteine protease inhibitor involved in several biological activities. It is progressively expressed in chronic liver disease, but not in normal liver. The role in vascular reactivity of this serpin, belonging to the same family of Angiotensin II, is still unknown. Our aim was to evaluate the in vivo and in vitro effects of SerpinB3 on systemic and splanchnic hemodynamics.Material and methodsDifferent hemodynamic parameters were evaluated by ultrasonography in two colonies of mice (transgenic for human SerpinB3 and C57BL/6J controls) at baseline and after chronic carbon tetrachloride (CCl₄) treatment. In vitro SerpinB3 effect on mesenteric microvessels of 5 Wistar-Kyoto rats was analyzed measuring its direct action on: (a) preconstricted arteries, (b) dose–response curves to phenylephrine, before and after inhibition of angiotensin II type 1 receptors with irbesartan. Hearts of SerpinB3 transgenic mice and of the corresponding controls were also analyzed by morphometric assessment.ResultsIn SerpinB3 transgenic mice, cardiac output (51.6 ± 21.5 vs 30.1 ± 10.8 ml/min, p = 0.003), hepatic artery pulsatility index (0.85 ± 0.13 vs 0.65 ± 0.11, p < 0.001) and portal vein blood flow (5.3 ± 3.2 vs 3.1 ± 1.8 ml/min, p = 0.03) were significantly increased, compared to controls. In vitro, recombinant SerpinB3 had no direct hemodynamic effect on mesenteric arteries, but it increased their sensitivity to phenylephrine-mediated vasoconstriction (p < 0.01). This effect was suppressed by inhibiting angiotensin II type-1 receptors.ConclusionsIn transgenic mice, SerpinB3 is associated with a hyperdynamic circulatory syndrome-like pattern, possibly mediated by angiotensin receptors.     

Inhibitory interneurons in a cortical column form hot zones of inhibition in layers 2 and 5A

Although physiological data on microcircuits involving a few inhibitory neurons in the mammalian cerebral cortex are available, data on the quantitative relation between inhibition and excitation in cortical circuits involving thousands of neurons are largely missing. Because the distribution of neurons is very inhomogeneous in the cerebral cortex, it is critical to map all neurons in a given volume rather than to rely on sparse sampling methods. Here, we report the comprehensive mapping of interneurons (INs) in cortical columns of rat somatosensory cortex, immunolabeled for neuron-specific nuclear protein and glutamate decarboxylase. We found that a column contains ~2,200 INs (11.5% of ~19,000 neurons), almost a factor of 2 less than previously estimated. The density of GABAergic neurons was inhomogeneous between layers, with peaks in the upper third of L2/3 and in L5A. IN density therefore defines a distinct layer 2 in the sensory neocortex. In addition, immunohistochemical markers of IN subtypes were layer-specific. The "hot zones" of inhibition in L2 and L5A match the reported low stimulus-evoked spiking rates of excitatory neurons in these layers, suggesting that these inhibitory hot zones substantially suppress activity in the neocortex.