Insulin differentially modulates GABA signalling in hippocampal neurons and, in an age-dependent manner, normalizes GABA-activated currents in the tg-APPSwe mouse model of Alzheimer's disease.

AIM: We examined if tonic γ-aminobutyric acid (GABA)-activated currents in primary hippocampal neurons were modulated by insulin in wild-type and tg-APPSwe mice, an Alzheimer's disease (AD) model. METHODS: GABA-activated currents were recorded in dentate gyrus (DG) granule cells and CA3 pyramidal neurons in hippocampal brain slices, from 8 to 10 weeks old (young) wild-type mice and in dorsal DG granule cells in adult, 5-6 and 10-12 (aged) months old wild-type and tg-APPSwe mice, in the absence or presence of insulin, by whole-cell patch-clamp electrophysiology. RESULTS: In young mice, insulin (1 nmol/L) enhanced the total spontaneous inhibitory postsynaptic current (sIPSCT ) density in both dorsal and ventral DG granule cells. The extrasynaptic current density was only increased by insulin in dorsal CA3 pyramidal neurons. In absence of action potentials, insulin enhanced DG granule cells and dorsal CA3 pyramidal neurons miniature IPSC (mIPSC) frequency, consistent with insulin regulation of presynaptic GABA release. sIPSCT densities in DG granule cells were similar in wild-type and tg-APPSwe mice at 5-6 months but significantly decreased in aged tg-APPSwe mice where insulin normalized currents to wild-type levels. The extrasynaptic current density was increased in tg-APPSwe mice relative to wild-type littermates but, only in aged tg-APPSwe mice did insulin decrease and normalize the current. CONCLUSION: Insulin effects on GABA signalling in hippocampal neurons are selective while multifaceted and context-based. Not only is the response to insulin related to cell-type, hippocampal axis-location, age of animals and disease but also to the subtype of neuronal inhibition involved, synaptic or extrasynaptic GABAA receptors-activated currents.

Tonic GABA-activated synaptic and extrasynaptic currents in dentate gyrus granule cells and CA3 pyramidal neurons along the mouse hippocampal dorsoventral axis.

The hippocampus is a medial temporal lobe structure in the brain and is widely studied for its role in memory and learning, in particular, spacial memory and emotional responses. It was thought to be a homogenous structure but emerging evidence shows differentiation along the dorsoventral axis and even microdomains for functional and cellular markers. We have examined in two cell-types of the hippocampal projection neurons, the dentate gyrus (DG) granule cells and CA3 pyramidal neurons, if the GABA-activated tonic current density varied between the dorsal (septal) and the ventral (temporal) poles of the male mouse hippocampus. Tonic synaptic currents, arising from spontaneous and miniature inhibitory postsynaptic currents (sIPSC, mIPSC), and extrasynaptic tonic currents were evaluated. The results revealed different levels of sIPSC but not mIPSC density between the dorsal and ventral hippocampal neurons for both the DG granule cells and the CA3 pyramidal neurons. The extrasynaptic tonic current density was larger in the DG granule cells as compared to the CA3 pyramidal neurons but did not vary between the dorsal and ventral regions. IPSC bursting was observed in both cell-types in the ventral hippocampus but was more common in the CA3 pyramidal neurons. Only in the dorsal DG granule cells was the level of the sIPSC and mIPSC density similar. The results indicate that the tonic GABAergic inhibition is particularly strong in the ventral hippocampal DG granule cells and enhanced in the dorsal as compared to the ventral hippocampal CA3 pyramidal neurons.

Characterization of Dmrt3-Derived Neurons Suggest a Role within Locomotor Circuits.

Neuronal networks within the spinal cord, collectively known as the central pattern generator (CPG), coordinate rhythmic movements underlying locomotion. The transcription factor doublesex and mab-3-related transcription factor 3 (DMRT3) is involved in the differentiation of the dorsal interneuron 6 class of spinal cord interneurons. In horses, a non-sense mutation in the Dmrt3 gene has major effects on gaiting ability, whereas mice lacking the Dmrt3 gene display impaired locomotor activity. Although the Dmrt3 gene is necessary for normal spinal network formation and function in mice, a direct role for Dmrt3-derived neurons in locomotor-related activities has not been demonstrated. Here we present the characteristics of the Dmrt3-derived spinal cord interneurons. Using transgenic mice of both sexes, we characterized interneurons labeled by their expression of Cre driven by the endogenous Dmrt3 promoter. We used molecular, retrograde tracing and electrophysiological techniques to examine the anatomical, morphological, and electrical properties of the Dmrt3-Cre neurons. We demonstrate that inhibitory Dmrt3-Cre neurons receive extensive synaptic inputs, innervate surrounding CPG neurons, intrinsically regulate CPG neuron's electrical activity, and are rhythmically active during fictive locomotion, bursting at frequencies independent to the ventral root output. The present study provides novel insights on the character of spinal Dmrt3-derived neurons, data demonstrating that these neurons participate in locomotor coordination.SIGNIFICANCE STATEMENT In this work, we provide evidence for a role of the Dmrt3 interneurons in spinal cord locomotor circuits as well as molecular and functional insights on the cellular and microcircuit level of the Dmrt3-expressing neurons in the spinal cord. Dmrt3 neurons provide the first example of an interneuron population displaying different oscillation frequencies. This study presents novel findings on an under-reported population of spinal cord neurons, which will aid in deciphering the locomotor network and will facilitate the design and development of therapeutics for spinal cord injury and motor disorders.

Expression of calcium release-activated and voltage-gated calcium channels genes in peripheral blood mononuclear cells is altered in pregnancy and in type 1 diabetes.

Calcium (Ca2+) is an important ion in physiology and is found both outside and inside cells. The intracellular concentration of Ca2+ is tightly regulated as it is an intracellular signal molecule and can affect a variety of cellular processes. In immune cells Ca2+ has been shown to regulate e.g. gene transcription, cytokine secretion, proliferation and migration. Ca2+ can enter the cytoplasm either from intracellular stores or from outside the cells when Ca2+ permeable ion channels in the plasma membrane open. The Ca2+ release-activated (CRAC) channel is the most prominent Ca2+ ion channel in the plasma membrane. It is formed by ORAI1-3 and the channel is opened by the endoplasmic reticulum Ca2+ sensor proteins stromal interaction molecules (STIM) 1 and 2. Another group of Ca2+ channels in the plasma membrane are the voltage-gated Ca2+ (CaV) channels. We examined if a change in immunological tolerance is accompanied by altered ORAI, STIM and CaV gene expression in peripheral blood mononuclear cells (PBMCs) in pregnant women and in type 1 diabetic individuals. Our results show that in pregnancy and type 1 diabetes ORAI1-3 are up-regulated whereas STIM1 and 2 are down-regulated in pregnancy but only STIM2 in type 1 diabetes. Expression of L-, P/Q-, R- and T-type voltage-gated Ca2+ channels was detected in the PBMCs where the CaV2.3 gene was up-regulated in pregnancy and type 1 diabetes whereas the CaV 2.1 and CaV3.2 genes were up-regulated only in pregnancy and the CaV1.3 gene in type 1 diabetes. The results are consistent with that expression of ORAI, STIM and CaV genes correlate with a shift in immunological status of the individual in health, as during pregnancy, and in the autoimmune disease type 1 diabetes. Whether the changes are in general protective or in type 1 diabetes include some pathogenic components remains to be clarified.

Neuron-specific inactivation of Wt1 alters locomotion in mice and changes interneuron composition in the spinal cord.

Locomotion is coordinated by neuronal circuits of the spinal cord. Recently, dI6 neurons were shown to participate in the control of locomotion. A subpopulation of dI6 neurons expresses the Wilms tumor suppressor gene Wt1. However, the function of Wt1 in these cells is not understood. Here, we aimed to identify behavioral changes and cellular alterations in the spinal cord associated with Wt1 deletion. Locomotion analyses of mice with neuron-specific Wt1 deletion revealed a slower walk with a decreased stride frequency and an increased stride length. These mice showed changes in their fore-/hindlimb coordination, which were accompanied by a loss of contralateral projections in the spinal cord. Neonates with Wt1 deletion displayed an increase in uncoordinated hindlimb movements and their motor neuron output was arrhythmic with a decreased frequency. The population size of dI6, V0, and V2a neurons in the developing spinal cord of conditional Wt1 mutants was significantly altered. These results show that the development of particular dI6 neurons depends on Wt1 expression and that loss of Wt1 is associated with alterations in locomotion.

Insulin enhances GABAA receptor-mediated inhibitory currents in rat central amygdala neurons.

Insulin, a pancreatic hormone, can access the central nervous system, activate insulin receptors distributed in selective brain regions and affect various cellular functions such as neurotransmission. We have previously shown that physiologically relevant concentration of insulin potentiates the GABAA receptor-mediated tonic inhibition and reduces excitability of rat hippocampal CA1 neurons. The central nucleus of the amygdala (CeA) comprises heterogeneous neuronal populations that can respond to hormonal stimulus. Using quantitative PCR and immunofluorescent labeling, we report that the mRNA and protein of the insulin receptor are abundantly expressed in the rat CeA. The insulin receptor mRNA is also detected in the CeA from post-mortem human brain samples. Furthermore, our whole-cell patch-clamp recordings show that the application of insulin (5 and 50 nM) selectively enhances the frequency and amplitude of spontaneous inhibitory postsynaptic currents (sIPSCs) in rat CeA neurons. Our findings reveal that GABAergic synaptic transmission is a target in the CeA for insulin receptor signaling that may underlie insulin modulation of emotion- and feeding-related behaviors.

Transport of L-glutamine, L-alanine, L-arginine and L-histidine by the neuron-specific Slc38a8 (SNAT8) in CNS.

Glutamine transporters are important for regulating levels of glutamate and GABA in the brain. To date, six members of the SLC38 family (SNATs) have been characterized and functionally subdivided them into System A (SNAT1, SNAT2 and SNAT4) and System N (SNAT3, SNAT5 and SNAT7). Here we present the first functional characterization of SLC38A8, one of the previous orphan transporters from the family, and we suggest that the encoded protein should be named SNAT8 to adhere with the SNAT nomenclature. We show that SLC38A8 has preference for transporting L-glutamine, L-alanine, L-arginine, L-histidine and L-aspartate using a Na+-dependent transport mechanism and that the functional characteristics of SNAT8 have highest similarity to the known System A transporters. We also provide a comprehensive central nervous system expression profile in mouse brain for the Slc38a8 gene and the SNAT8 protein. We show that Slc38a8 (SNAT8) is expressed in all neurons, both excitatory and inhibitory, in mouse brain using in situ hybridization and immunohistochemistry. Furthermore, proximity ligation assay shows highly similar subcellular expression of SNAT7 and SNAT8. In conclusion, the neuronal SLC38A8 has a broad amino acid transport profile and is the first identified neuronal System A transporter. This suggests a key role of SNAT8 in the glutamine/glutamate (GABA) cycle in the brain.

PAT4 is abundantly expressed in excitatory and inhibitory neurons as well as epithelial cells.

PAT4, the fourth member of the SLC36/proton dependent amino acid transporter (PAT) family, is a high-affinity, low capacity electroneutral transporter of neutral amino acids like proline and tryptophan. It has also been associated with the function of mTORC1, a complex in the mammalian target of rapamycin (mTOR) pathway. We performed in situ hybridization and immunohistological analysis to determine the expression profile of PAT4, as well as an RT-PCR study on tissue from mice exposed to leucine. We performed a phylogenetic analysis to determine the evolutionary origin of PAT4. The in situ hybridization and the immunohistochemistry on mouse brain sections and hypothalamic cells showed abundant PAT4 expression in the mouse brain intracellularly in both inhibitory and excitatory neurons, partially co-localizing with lysosomal markers and epithelial cells lining the ventricles. Its location in epithelial cells around the ventricles indicates a transport of substrates across the blood brain barrier. Phylogenetic analysis showed that PAT4 belongs to an evolutionary old family most likely predating animals, and PAT4 is the oldest member of that family.