Characterization of GABAergic neurons in the mouse lateral septum: a double fluorescence in situ hybridization and immunohistochemical study using tyramide signal amplification.

Gamma-aminobutyric acid (GABA) neurotransmission in the lateral septum (LS) is implicated in modulating various behavioral processes, including emotional reactivity and maternal behavior. However, identifying the phenotype of GABAergic neurons in the CNS has been hampered by the longstanding inability to reliably detect somal immunoreactivity for GABA or glutamic acid decarboxylase (GAD), the enzyme that produces GABA. In this study, we designed unique probes for both GAD65 (GAD2) and GAD67 (GAD1), and used fluorescence in Situ hybridization (FISH) with tyramide signal amplification (TSA) to achieve unequivocal detection of cell bodies of GABAergic neurons by GAD mRNAs. We quantitatively characterized the expression and chemical phenotype of GABAergic neurons across each subdivision of LS and in cingulate cortex (Cg) and medial preoptic area (MPOA) in female mice. Across LS, almost all GAD65 mRNA-expressing neurons were found to contain GAD67 mRNA (approximately 95-98%), while a small proportion of GAD67 mRNA-containing neurons did not express GAD65 mRNA (5-14%). Using the neuronal marker NeuN, almost every neuron in LS (> 90%) was also found to be GABA-positive. Interneuron markers using calcium-binding proteins showed that LS GABAergic neurons displayed immunoreactivity for calbindin (CB) or calretinin (CR), but not parvalbumin (PV); almost all CB- or CR-immunoreactive neurons (98-100%) were GABAergic. The proportion of GABAergic neurons immunoreactive for CB or CR varied depending on the subdivisions examined, with the highest percentage of colocalization in the caudal intermediate LS (LSI) (approximately 58% for CB and 35% for CR). These findings suggest that the vast majority of GABAergic neurons within the LS have the potential for synthesizing GABA via the dual enzyme systems GAD65 and GAD67, and each subtype of GABAergic neurons identified by distinct calcium-binding proteins may exert unique roles in the physiological function and neuronal circuitry of the LS

Addiction and Reward-related Genes Show Altered Expression in the Postpartum Nucleus Accumbens

Motherhood involves a switch in natural rewards, whereby offspring become highly rewarding. Nucleus accumbens (NAC) is a key CNS region for natural rewards and addictions, but to date no study has evaluated on a large scale the events in NAC that underlie the maternal change in natural rewards. In this study we utilized microarray and bioinformatics approaches to evaluate postpartum NAC gene expression changes in mice. Modular Single-set Enrichment Test (MSET) indicated that postpartum (relative to virgin) NAC gene expression profile was significantly enriched for genes related to addiction and reward in 5 of 5 independently curated databases (e.g., Malacards, Phenopedia). Over 100 addiction/reward related genes were identified and these included: Per1, Per2, Arc, Homer2, Creb1, Grm3, Fosb, Gabrb3, Adra2a, Ntrk2, Cry1, Penk, Cartpt, Adcy1, Npy1r, Htr1a, Drd1a, Gria1, and Pdyn. ToppCluster analysis found maternal NAC expression profile to be significantly enriched for genes related to the drug action of nicotine, ketamine, and dronabinol. Pathway analysis indicated postpartum NAC as enriched for RNA processing, CNS development/differentiation, and transcriptional regulation. Weighted Gene Coexpression Network Analysis identified possible networks for transcription factors, including Nr1d1, Per2, Fosb, Egr1, and Nr4a1. The postpartum state involves increased risk for mental health disorders and MSET analysis indicated postpartum NAC to be enriched for genes related to depression, bipolar disorder, and schizophrenia. Mental health related genes included: Fabp7, Grm3, Penk, and Nr1d1. We confirmed via quantitative PCR Nr1d1, Per2, Grm3, Penk, Drd1a, and Pdyn. This study indicates for the first time that postpartum NAC involves large scale gene expression alterations linked to addiction and reward. Because the postpartum state also involves decreased response to drugs, the findings could provide insights into how to mitigate addictions

Double fluorescence in situ hybridization labeling of GAD65 and GAD67 mRNA-expressing neurons in the LSD (A–D), LSI (E–H) and LSV (I–L).

<p>Low magnification images of the white boxed regions (A, E and I) show neurons expressing mRNAs for GAD65 and GAD67 were counted for colocalization analysis. High magnification images show the colocalization of neurons containing both GAD65 and GAD67 mRNAs in the LSD (B–D), LSI (F–H) and LSV (J–L). Two typical examples of neurons coexpressing mRNAs for GAD65 and GAD67 in each subdivision are indicated in arrows. Note that in all three subdivisions of the LS, GAD65 and GAD67 mRNAs are highly colocalized in a single neuron. Scale bars = 150 µm in A, E and I; 50 µm in B–D, F–H, J-L.</p

Double fluorescence in situ hybridization and immunohistochemistry labeling of neurons expressing mRNAs for GAD65/GAD67 (GAD) and calbindin-immunoreactivity in the Cg (A–C) and MPOA (D–F).

<p>High magnification images show the colocalization of neurons expressing GAD and CB. Two typical examples of double-labeled (arrows) or single-labeled (arrowheads) neurons are indicated. Note that GAD and CB are highly coexpressed in a single cell, and most CB-immunoreactive cells express GAD mRNA (GABAergic). Scale bar = 50 µm.</p

Double fluorescence in situ hybridization and immunohistochemistry labeling of neurons expressing mRNAs for GAD65/GAD67 (GAD) and parvalbumin-immunoreactivity in the Cg (A–F).

<p>High magnification images show the colocalization of neurons expressing GAD and PV. Typical examples of double-labeled (arrows) or single-labeled (arrowheads) neurons are indicated. Widely spreading dendrites (D, open arrows) and cell bodies (D, arrows) of PV-immunoreactive neurons are clearly stained. Note that GAD and PV are highly coexpressed in a single cell, and most PV-immunoreactive cells express GAD mRNA (GABAergic). Scale bars = 50 µm in A-C; 50 µm in D–F.</p

Schematic diagram depicting probe design for GAD65 and GAD67 in situ hybridization assay.

<p>For each gene, three sets of oligonucleotide probes were generated to target three distinct sequences of each mRNA. Recognition of GAD65 and GAD67 mRNA sequences and length covered by each oligonucleotide probe are indicated. There was no overlap of sequences among all the probes and the probes designed for the two GADS had no homology to one another to ensure the specificity of the probe. The GAD67 probes were labeled with digoxigenin, while GAD65 probes were labeled with either biotin or digoxigenin. Note that probes for GAD65 target the mRNA 3’-non-coding sequences, whereas probes for GAD67 primarily recognize the mRNA 3’-coding sequences (blue box).</p

Expression and colocalization of neurons expressing mRNAs for GAD65/GAD67 (GAD) and immunoreactivity for calcium binding proteins in the LS, Cg and MPOA.

<p>Each bar represents the mean+SEM obtained from four mice. A: number of calbindin-immunoreactive cells: ^a<i>p</i> < 0.001 <i>versus</i> LSr_D and LSr_I, ^b<i>p</i> < 0.05 <i>versus</i> LSc_D and LSc_I, ^c<i>p</i> < 0.001 <i>versus</i> LSc_D, ^d<i>p</i> < 0.05 <i>versus</i> LSr_I and LSr_V, ^e<i>p</i> < 0.001 <i>versus</i> LSr_I and LSc_D, ^f<i>p</i> < 0.01 <i>versus</i> LSr_V, LSc_D and LSc_I; B: ratio of colocalization of GAD and calbindin. ^a<i>p</i> < 0.001 <i>versus</i> LSc_I, ^b<i>p</i> < 0.001 <i>versus</i> LSc_I and LSc_V, ^c<i>p</i> < 0.001 <i>versus</i> GAD/Calbindin; C: number of calretinin-immunoreactive cells, ^a<i>p</i> < 0.001 <i>versus</i> LSr_D and LSr_I, ^b<i>p</i> < 0.05 <i>versus</i> LSc_D and LSc_I, ^c<i>p</i> < 0.001 <i>versus</i> LSc_D, ^d<i>p</i> < 0.001 <i>versus</i> LSr_I and LSc_D, ^e<i>p</i> < 0.001 <i>versus</i> LSr_V and LSc_D; D, ratio of colocalization of GAD and calretinin, ^a<i>p</i> < 0.05 <i>versus</i> LSr_D and LSr_V, ^b<i>p</i> < 0.001 <i>versus</i> LSc_I and LSc_V, ^c<i>p</i> < 0.001 <i>versus</i> LSc_V, ^d<i>p</i> < 0.001 <i>versus</i> LSc_V, ^e<i>p</i> < 0.001 <i>versus</i> GAD/Calretinin.</p

Schematic representation of the brain areas (black boxed regions) in which expression of mRNA and protein immunoreactivity as well as colocalization was examined.

<p>Figures are adapted and modified from The Allen Mouse Brain Atlas (Reference Atlas Version 1, 2008). Nomenclature is obtained from Paxinos and Franklin [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073750#B108" target="_blank">108</a>]. Distance from bregma in the rostrocaudal planes is indicated. Abbreviations: aco, anterior commissure, olfactory limb; act, anterior commissure, temporal limb; Cg, cingulate cortex; LSD, dorsal part of lateral septal nucleus; LSI, intermediate part of lateral septal nucleus; LSV, ventral part of lateral septal nucleus; MPOA, medial preoptic area; VL, lateral ventricle.</p

Double fluorescence in situ hybridization and immunohistochemistry labeling of neurons expressing mRNAs for GAD65/GAD67 (GAD) and NeuN-immunoreactivity in the LSD (A–D), LSI (E–H) and LSV (I–L).

<p>Low magnification images of the white boxed regions (A, E and I) show the GAD- and NeuN-expressing neurons were counted for colocalization analysis. High magnification images show the colocalization of GAD and NeuN in the LSD (B–D), LSI (F–H) and LSV (J–L). Two typical examples of double-labeled neurons, indicating the colocalization of GAD and NeuN in each subdivision are indicated in arrows. Note that in all three subdivisions of the lateral septum, GAD and NeuN are highly coexpressed in a single cell. Scale bars = 150 µm in A, E and I; 50 µm in B–D, F–H, J-L.</p