Effects of Chronic D-Serine Elevation on Animal Models of Depression and Anxiety-Related Behavior.

NMDA receptors are activated after binding of the agonist glutamate to the NR2 subunit along with a co-agonist, either L-glycine or D-serine, to the NR1 subunit. There is substantial evidence to suggest that D-serine is the most relevant co-agonist in forebrain regions and that alterations in D-serine levels contribute to psychiatric disorders. D-serine is produced through isomerization of L-serine by serine racemase (Srr), either in neurons or in astrocytes. It is released by astrocytes by an activity-dependent mechanism involving secretory vesicles. In the present study we generated transgenic mice (SrrTg) expressing serine racemase under a human GFAP promoter. These mice were biochemically and behaviorally analyzed using paradigms of anxiety, depression and cognition. Furthermore, we investigated the behavioral effects of long-term administration of D-serine added to the drinking water. Elevated brain D-serine levels in SrrTg mice resulted in specific behavioral phenotypes in the forced swim, novelty suppression of feeding and olfactory bulbectomy paradigms that are indicative of a reduced proneness towards depression-related behavior. Chronic dietary D-serine supplement mimics the depression-related behavioral phenotype observed in SrrTg mice. Our results suggest that D-serine supplementation may improve mood disorders

Cannabinoid Type 2 Receptors Mediate a Cell Type-Specific Plasticity in the Hippocampus

Endocannabinoids (eCBs) exert major control over neuronal activity by activating cannabinoid receptors (CBRs). The functionality of the eCB system is primarily ascribed to the well-documented retrograde activation of presynaptic CB1Rs. We find that action potential-driven eCB release leads to a long-lasting membrane potential hyperpolarization in hippocampal principal cells that is independent of CB1Rs. The hyperpolarization, which is specific to CA3 and CA2 pyramidal cells (PCs), depends on the activation of neuronal CB2Rs, as shown by a combined pharmacogenetic and immunohistochemical approach. Upon activation, they modulate the activity of the sodium-bicarbonate co-transporter, leading to a hyperpolarization of the neuron. CB2R activation occurred in a purely self-regulatory manner, robustly altered the input/output function of CA3 PCs, and modulated gamma oscillations in vivo. To conclude, we describe a cell type-specific plasticity mechanism in the hippocampus that provides evidence for the neuronal expression of CB2Rs and emphasizes their importance in basic neuronal transmission

Depression-related behavior in SrrTg mice.

<p>(A) Forced-swim test. Immobility time, defined as a lack of activity aside from small movements needed to keep the body floating, was measured throughout the last 4 min of the session. (Wt: n = 21 (10 males, 11 females); Srr2: n = 17 (8 males, 9 females); Srr12: n = 24 (9 males, 15 females)). (B) Locomotor activity was recorded over a 30 min time period and expressed as distance traveled. (n = 15 per group). (C) Novelty suppressed feeding test. Srr12 mice displayed a significantly reduced latency to approach and eat food compared to Wt mice (Wt: n = 26 (14 males, 12 females); Srr12: n = 28 (15 males, 13 females)). (D) Horizontal (distance traveled) activity 2 weeks after surgery of sham-operated and bulbectomized SrrTg mice and their sham operated littermates in the open-field apparatus (Wt: female 16 (sham 10, bulb 6), male 18 (sham 14, bulb 4); Srr12: female 15 (sham 8, bulb 7), male 13 (sham 5, bulb 7). (E) Ultrasonic vocalization. Measurements of social isolation-induced ultrasonic vocalizations (USV) in SrrTg and Wt pups revealed a significant increase in Srr12 mice in number (left) and duration (right) of calls (Wt: n = 22 (12 males, 10 females); Srr2: n = 23 (9 males, 13 females); Srr12: n = 35 (20 males, 15 females)). All values are expressed as mean ±SEM. *p<0.05 and ***p<0.001.</p

Serine racemase expression and brain D-serine levels in transgenic mice.

<p>(A) Schematic representation of the construct for serine racemase overexpression in glial cells under a GFAP promoter. The GFAP promoter is represented as a black line, the serine racemase ORF as a black bar and the SV40 intron/polyA signal as a grey bar. The cutting sites for the endonucleases <i>Bgl</i>II, <i>Eco</i>RV and <i>Dra</i>III are listed. (B) Quantitative analysis of serine racemase expression in adult mouse brains by TaqMan® Assay. Serine racemase expression was calculated as ΔC_T value, normalized to GAPDH. Shown are the average values of 4 animals, expressed as mean ±SEM. (C) Western blot analysis of serine racemase in whole brain of a Srr12 mouse and a Wt littermate. Images of <i>Western</i> blot analysis of serine racemase and β-actin were performed with specific antibodies using the same extract (n = 4). All values are expressed as mean ±SEM of 5 animals; *p<0.05 and **p<0.01. (D) Quantitative analysis of absolute brain D-serine and L-serine levels per wet weight in 5 Srr12 mice and corresponding Wt littermates by HPLC. D-serine levels of cortex, hippocampus and forebrain were measured. Mice were treated with D-serine (D), L-serine (L), glycine (G) and water (W).</p

Specific serine racemase immunofluorescence in Srr12 mice.

<p>(A) Immunoreactivity for serine racemase is higher in transgenic animals than in Wt controls. Arrows - indicate serine racemase positive cell soma. * - indicates immunoreactivity of the neuropil which is much higher in Srr12 transgenic mice than Wt controls, possibily due to higher expression in astrocytic processes. (B) Serine racemase expression is specifically increased in astrocytes as labeled by the astrocyte marker glial fibrillary acidic protein (GFAP). Arrowheads - indicate GFAP⁺ processes bearing SR⁺ puncta, although both Wt and Srr12 astrocytes express serine racemase, immunoreactivity and colocalization is more pronounced in Srr12 mice. Asterisks indicate GFAP positive cells which seemed to be devoid of serine racemase expression in WT mice. All images were acquired using the same exposure times. Scale bar - 100 µm top panels and 50 µm all other panels. ctx - cortex, cc - corpus callosum.</p

Expression Analysis of CB2-GFP BAC Transgenic Mice

<div><p>The endocannabinoid system (ECS) is a retrograde messenger system, consisting of lipid signaling molecules that bind to at least two G-protein-coupled receptors, Cannabinoid receptor 1 and 2 (CB1 and 2). As CB2 is primarily expressed on immune cells such as B cells, T cells, macrophages, dendritic cells, and microglia, it is of great interest how CB2 contributes to immune cell development and function in health and disease. Here, understanding the mechanisms of CB2 involvement in immune-cell function as well as the trafficking and regulation of CB2 expressing cells are crucial issues. Up to now, CB2 antibodies produce unclear results, especially those targeting the murine protein. Therefore, we have generated BAC transgenic GFP reporter mice (CB2-GFPTg) to trace CB2 expression <i>in vitro</i> and <i>in situ</i>. Those mice express GFP under the CB2 promoter and display GFP expression paralleling CB2 expression on the transcript level in spleen, thymus and brain tissue. Furthermore, by using fluorescence techniques we show that the major sources for GFP-CB2 expression are B cells in spleen and blood and microglia in the brain. This novel CB2-GFP transgenic reporter mouse line represents a powerful resource to study CB2 expression in different cell types. Furthermore, it could be used for analyzing CB2-mediated mobilization and trafficking of immune cells as well as studying the fate of recruited immune cells in models of acute and chronic inflammation.</p></div

Cognition in SrrTg mice.

<p>(A) In the Y-maze task, SrrTg mice displayed no significant differences in the percentage of spontaneous alternations (left) and total number of arm visits (right) compared to Wt mice (Wt: n = 24; Srr2: n = 19; Srr12: n = 27). (B) Morris water maze test. Average water maze escape latencies across 5 training days of Wt and Srr12 mice (n 9–10 male mice per group). (C) Operant conditioning test. The learning performance of SrrTg mice did not differ compared to Wt mice (Wt: n = 16; Srr12: n = 16). All values are expressed as mean ±SEM indicated animals.</p

Brain D-serine levels and behavior of Wt mice after D-serine administration.

<p>(A) Quantitative analysis of brain D-serine and L-serine levels per wet weight in 4–8 dietary-treated Wt mice by HPLC. D-serine levels of cortex, hippocampus and forebrain were measured. Mice were treated with D-serine (D), L-serine (L), glycine (G) and water (W). (B) Forced-swim test. Immobility time, defined as a lack of activity aside from small movements needed to keep the body floating, was measured throughout the last 4 min of the session. D-serine treated mice spent less time immobile compared to controls. (C) Novelty suppressed feeding test. The mice that were treated with D-serine displayed a significantly reduced latency to approach the food and to start eating. (D) Open field test. Distance traveled in the open field arena of Wt mice. Behavior in this paradigm was not changed after amino acid treatment. (E) Operant conditioning test. The Hill equation indicates learning improvement during the training days. Data of all groups resulted in similar learning curves. (F) Water maze test. Escape latencies across 5 training days of D-serine treated and control mice were recorded and did not differ between the groups. *p<0.05 and **p<0.01. Each error bar represents the mean ±SEM of 10 animals.</p