Seizures regulate the cation-Cl− cotransporter NKCC1 in a hamster model of epilepsy: implications for GABA neurotransmission

BackgroundThe balance between the activity of the Na+/K+/Cl− cotransporter (NKCC1) that introduces Cl− into the cell and the K+/Cl− cotransporter (KCC2) that transports Cl− outside the cell is critical in determining the inhibitory or excitatory outcome of GABA release. Mounting evidence suggests that the impairment of GABAergic inhibitory neurotransmission plays a crucial role in the pathophysiology of epilepsy, both in patients and animal models. Previous studies indicate that decreased KCC2 expression is linked to audiogenic seizures in GASH/Sal hamsters, highlighting that Cl− imbalance can cause neuronal hyperexcitability. In this study, we aimed to investigate whether the Na+/K+/Cl− cotransporter NKCC1 is also affected by audiogenic seizures and could, therefore, play a role in neuronal hyperexcitability within the GASH/Sal epilepsy model.MethodsNKCC1 protein expression in both the GASH/Sal strain and wild type hamsters was analyzed by immunohistochemistry and Western blotting techniques. Brain regions examined included cortex, hippocampus, hypothalamus, inferior colliculus and pons-medulla oblongata, which were evaluated both at rest and after sound-inducing seizures in GASH/Sal hamsters. A complementary analysis of NKCC1 gene slc12a2 expression was conducted by real-time PCR. Finally, protein and mRNA levels of glutamate decarboxylase GAD67 were measured as an indicator of GABA release.ResultsThe induction of seizures caused significant changes in NKCC1 expression in epileptic GASH/Sal hamsters, despite the similar brain expression pattern of NKCC1 in GASH/Sal and wild type hamsters in the absence of seizures. Interestingly, the regulation of brain NKCC1 by seizures demonstrated regional specificity, as protein levels exclusively increased in the hippocampus and hypothalamus. Complementary real-time PCR analysis revealed that NKCC1 regulation was post-transcriptional only in the hypothalamus. In addition, seizures also modulated GAD67 mRNA levels in a brain region-specific manner. The increased GAD67 expression in the hippocampus and hypothalamus of the epileptic hamster brain suggests that NKCC1 upregulation overlaps with GABA release in these regions during seizures.ConclusionsOur results indicate that seizure induction causes dysregulation of NKCC1 expression in GASH/Sal animals, which overlaps with changes in GABA release. These observations provide evidence for the critical role of NKCC1 in how seizures affect neuronal excitability, and support NKCC1 contribution to the development of secondary foci of epileptogenic activity

GSK3β Inhibition by Phosphorylation at Ser³⁸⁹ Controls Neuroinflammation

The inhibition of Glycogen Synthase Kinase 3 β (GSK3β) by Ser9 phosphorylation affects many physiological processes, including the immune response. However, the consequences of GSK3β inhibition by alternative Ser389 phosphorylation remain poorly characterized. Here we have examined neuroinflammation in GSK3β Ser389 knock-in (KI) mice, in which the phosphorylation of Ser389 GSK3β is impaired. The number of activated microglia/infiltrated macrophages, astrocytes, and infiltrated neutrophils was significantly higher in these animals compared to C57BL/6J wild-type (WT) counterparts, which suggests that the failure to inactivate GSK3β by Ser389 phosphorylation results in sustained low-grade neuroinflammation. Moreover, glial cell activation and brain infiltration of immune cells in response to lipopolysaccharide (LPS) failed in GSK3β Ser389 KI mice. Such effects were brain-specific, as peripheral immunity was not similarly affected. Additionally, phosphorylation of the IkB kinase complex (IKK) in response to LPS failed in GSK3β Ser389 KI mice, while STAT3 phosphorylation was fully conserved, suggesting that the NF-κB signaling pathway is specifically affected by this GSK3β regulatory pathway. Overall, our findings indicate that GSK3β inactivation by Ser389 phosphorylation controls the brain inflammatory response, raising the need to evaluate its role in the progression of neuroinflammatory pathologies

Gap junctions in the hypothalamic arcuate neurons of ovariectomized and estradiol-treated rats

Freeze-fracture methodology was used to study the organization of the neuronal plasma membrane in the rat arcuate nucleus, an estrogen sensitive area of the hypothalamus. Freeze-fracture replicas were prepared from 6 adult ovariectomized rats injected with a single dose of 17β-estradiol and from 6 ovariectomized littermates injected with vehicle. Rats were sacrificed 2 days after the injection. Occasional gap junctions were observed in freeze-fractured neuronal membranes from both groups of animals and their incidence was increased (P < 0.01) in estradiol treated rats. This study demonstrates gap junctions in arcuate neurons and suggests that these structures may be affected by gonadal hormones.Peer Reviewe

Sexual differentiation of the neuronal plasma membrane: Neonatal levels of sex steroids modulate the number of exo-endocytotic images in the developing rat arcuate neurons

Exo-endocytotic images and intramembrane protein particles (IMP) were quantitatively assessed in freeze-fracture replicas from the plasma membrane of arcuate neurons of rats aged 0 (newborns), 10, 20 and 100 days postpartum. Membranes contained significantly (P < 0.02) more IMPs in females than in males. Exo-endocytotic images were increased in newborn and 10-day-old males when compared to adult males or to developing females (48 ± 6 vs 6 ± 1 images/100 μm2 in 10-day-old male and female rats, respectively). Androgenization of females with a single injection of testosterone propionate on the day of birth resulted in an increased number of exo-endocytotic images in developing animals (75 ± 9 images/100 μm2, 10-day-old rats) and in the abolishment of the sex differences in the number of IMPs.Peer Reviewe

Sex differences in plasma membrane concanavalin A binding in the rat arcuate neurons

Previous studies have shown that synaptic connections and organization of neuronal membranes are sexually dimorphic in the arcuate nucleus of developing and adult rats. These sex differences can be abolished by the perinatal androgenization of females. In this study the label-fracture method of Pinto da Silva and Kan was used in order to determine whether membrane sex differences are related to the glycoconjugates in neuronal plasma membranes. Six Sprague-Dawley female rats treated with testosterone on the day of birth, six control females injected with vehicle and six intact males were studied when they were 100 days old. The arcuate nucleus was dissected and incubated for 2 hours in a solution of 0.25 mg/ml concanavalin A, washed in buffer and incubated for 3 hours in a suspension of horseradish peroxidase-coated colloidal gold. Then, freeze-fracture replicas of the arcuate nucleus were prepared. Colloidal gold labeling was observed to be codistributed with intramembrane particles in the outer membrane face of the neuronal perikaryal plasma membrane. The numerical density of small (<10 nm) intramembrane particles and colloidal gold particles was significantly greater in control female membranes when compared to males or to androgenized females. The labeling was significantly reduced when the arcuate nucleus was incubated with concanavalin A in presence of 0.5 M methyl-α-manopyranoside. These findings indicate a sex difference in the density and distribution of glycoconjugates and intramembranous particles in the neuronal plasma membrane that is dependent on the perinatal levels of sex steroids and is concordant with, and could be the cause of, sex differences in the pattern of synaptic contacts. © 1989.Peer Reviewe

Synaptic remodeling in the rat arcuate nucleus during the estrous cycle

Adult female rats showing regular vaginal cycles were studied in order to determine the number of axosomatic synapses in thin sections of the arcuate nucleus. The number of synapses per length of perikaryal membrane was significantly decreased in estrus, compared to other days of the estrous cycle (P < 0.05). The reduction in the number of synapses in estrus was accompanied by a decrease in the percentage of the average length of perikaryal membrane covered by presynaptic terminals and by an increase in the percentage of membrane in close apposition of glial processes. Since the average perikaryal perimeter was not significantly changed during the estrous cycle, these results indicate a net decrease in the number of arcuate nucleus axosomatic synapses between proestrus and estrus, with a reinnervation of arcuate neurons between estrus and metestrus. These results suggest that there is a physiological synaptic turnover in the arcuate nucleus of the rat during the estrous cycle.Peer Reviewe

Estrogen-induced synaptic remodelling in adult rat brain is accompanied by the reorganization of neuronal membranes

Adult cycling female rats were injected with estradiol valerate (2 mg/100 g body wt.), a treatment which has previously been shown to result in synaptic remodelling in the arcuate nucleus and constant vaginal estrus. During the 32 weeks following estrogen treatment, arcuate nucleus neuronal plasma membranes were quantatively assessed for intramembrane particle (IMP) number and size using freeze-fracture techniques. Neuronal membranes from untreated cycling females, females injected with oil and untreated males were also studied. Untreated rats had dimorphic sexual phenotypes in membrane organization; female rats had more IMP than males, mainly due to greater numbers of small (<10 nm) particles. These sex differences were observed in perikarya and dendritic shafts, but not in dendritic spines. Following estrogen treatment the density of IMP in membranes from females decreased. The IMP changes were found only in neuronal perikarya and dendritic shafts, not in dendritic spines, and were mainly due to a massive decrease in the number of small (< 10 nm) IMP which was only partially offset by an increase in the number of large (≥10 nm) IMP. Thus, by 32 weeks after estradiol valerate treatment, the number and size of IMP in neuronal membranes from females were not different from those seen in normal males. These results strengthen the idea that estradiol may affect the turnover of certain neuronal membrane components in sex-steroid sensitive areas of the brain.Peer Reviewe