Sprouty2 in the Dorsal Hippocampus Regulates Neurogenesis and Stress Responsiveness in Rats

Both the development and relief of stress-related psychiatric conditions such as major depression (MD) and post-traumatic stress disorder (PTSD) have been linked to neuroplastic changes in the brain. One such change involves the birth of new neurons (neurogenesis), which occurs throughout adulthood within discrete areas of the mammalian brain, including the dorsal hippocampus (HIP). Stress can trigger MD and PTSD in humans, and there is considerable evidence that it can decrease HIP neurogenesis in laboratory animals. In contrast, antidepressant treatments increase HIP neurogenesis, and their efficacy is eliminated by ablation of this process. These findings have led to the working hypothesis that HIP neurogenesis serves as a biomarker of neuroplasticity and stress resistance. Here we report that local alterations in the expression of Sprouty2 (SPRY2), an intracellular inhibitor of growth factor function, produces profound effects on both HIP neurogenesis and behaviors that reflect sensitivity to stressors. Viral vector-mediated disruption of endogenous Sprouty2 function (via a dominant negative construct) within the dorsal HIP of adult rats stimulates neurogenesis and produces signs of stress resilience including enhanced extinction of conditioned fear. Conversely, viral vector-mediated elevation of SPRY2 expression intensifies the behavioral consequences of stress. Studies of these manipulations in HIP primary cultures indicate that SPRY2 negatively regulates fibroblast growth factor-2 (FGF2), which has been previously shown to produce antidepressant- and anxiolytic-like effects via actions in the HIP. Our findings strengthen the relationship between HIP plasticity and stress responsiveness, and identify a specific intracellular pathway that could be targeted to study and treat stress-related disorders

Sprouty2 in the Dorsal Hippocampus Regulates Neurogenesis and Stress Responsiveness in Rats

Both the development and relief of stress-related psychiatric conditions such as major depression (MD) and post-traumatic stress disorder (PTSD) have been linked to neuroplastic changes in the brain. One such change involves the birth of new neurons (neurogenesis), which occurs throughout adulthood within discrete areas of the mammalian brain, including the dorsal hippocampus (HIP). Stress can trigger MD and PTSD in humans, and there is considerable evidence that it can decrease HIP neurogenesis in laboratory animals. In contrast, antidepressant treatments increase HIP neurogenesis, and their efficacy is eliminated by ablation of this process. These findings have led to the working hypothesis that HIP neurogenesis serves as a biomarker of neuroplasticity and stress resistance. Here we report that local alterations in the expression of Sprouty2 (SPRY2), an intracellular inhibitor of growth factor function, produces profound effects on both HIP neurogenesis and behaviors that reflect sensitivity to stressors. Viral vector-mediated disruption of endogenous Sprouty2 function (via a dominant negative construct) within the dorsal HIP of adult rats stimulates neurogenesis and produces signs of stress resilience including enhanced extinction of conditioned fear. Conversely, viral vector-mediated elevation of SPRY2 expression intensifies the behavioral consequences of stress. Studies of these manipulations in HIP primary cultures indicate that SPRY2 negatively regulates fibroblast growth factor-2 (FGF2), which has been previously shown to produce antidepressant- and anxiolytic-like effects via actions in the HIP. Our findings strengthen the relationship between HIP plasticity and stress responsiveness, and identify a specific intracellular pathway that could be targeted to study and treat stress-related disorders

Forced swimming and locomotor activity behaviors 28 days after gene transfer.

<p>(<b>a</b>) During the first exposure to the FST, there were no differences in immobility (IM; <i>F</i>_2,41 = 1.20, not significant), swimming (SW; <i>F</i>_2,41 = 1.60, not significant) or climbing (CL; <i>F</i>_2,41 = 0.05, not significant) behaviors (mean ± SEM) among rats given the dnSPRY2, LacZ, or wtSPRY2 vector into the dorsal HIP. (<b>b</b>) Differences in immobility (<i>F</i>_2,41 = 5.77, <i>P</i><0.01) and swimming (<i>F</i>_2,41 = 6.18, <i>P</i><0.01) emerged upon re-exposure to forced swimming 24 hr later. (<b>c</b>) Vectors did not affect activity in an open field (<i>F</i>_22,451 = 0.09, not significant). *<i>P</i><0.05 compared to LacZ, ^^<i>P</i><0.01 compared to dnSPRY2, Fisher’s protected <i>t</i>-tests, 14–15 rats/group.</p

Effect of viral-mediated gene transfer in the dorsal HIP.

<p>(<b>a</b>) Detection of dnSPRY2 (tagged with a-flag epitope) 3 days after gene transfer (scale bars: 200 μm or [<i>inset</i>] 100 μm). (<b>b</b>) Net number (mean ± SEM) of BrdU-labeled cells (per 40 μm section) 28 days after gene transfer, expressed as the difference between the hemisphere treated with HSV-dnSPRY2, HSV-LacZ, or HSV-wtSPRY2 and the contralateral hemisphere treated with HSV-LacZ (<i>F</i>_2,28 = 7.27, <i>P</i><0.01). (<b>c</b>) Viral vector treatments did not differentially affect the percentage (mean ± SEM) of cells expressing neuronal (NeuN; <i>F</i>_2,28 = 1.99, not significant), glial (S100ß; <i>F</i>_2,28 = 0.98, not significant) or undefined (other; <i>F</i>_2,28 = 1.57, not significant) cell markers. (<b>d</b>) Brightfield microscopy showing BrdU-labeled cells after HSV-dnSPRY2 or (<b>e</b>) HSV-LacZ. (<b>f</b>) Confocal microscopy showing a BrdU-labeled cell (red), (<b>g</b>) NeuN-labeled neurons (green), and (<b>h</b>) a double-labeled neuron (<i>overlay</i>, yellow; scale bar: 10 μm). (<b>i</b>) BrdU-labeled cells (red) were occasionally double-labeled with S100ß (<b>j</b>, blue) indicating glial cells (<b>k</b>, <i>overlay</i>, violet; scale bar: 10 μm). *<i>P</i><0.05 compared to LacZ, ^^<i>P</i><0.01 compared to dnSPRY2, Fisher’s protected <i>t</i>-tests, 9–11 rats/group.</p

Highly simplified schematic depicting a potential pathway by which disruption of SPRY2 function produces elevated neurogenesis and antidepressant-like behaviors.

<p>Binding of growth factors (GF) such as FGF2 at RTKs activates ERK-dependent signaling cascades (green arrows), resulting in processes that tend to increase neurogenesis and produce antidepressant-like behaviors. However, this process also results in elevated SPRY2 transcription and activation (phosphorylation), which tends to inhibit these processes. Electroconvulsive seizure (ECS) activates GFs and inhibits SPRY2, both of which promote neurogenesis and antidepressant-like behaviors.</p

Effect of ECS on the dorsal HIP.

<p>(<b>a</b>) Repeated ECS increased the number of BrdU-labeled cells and decreased the number (mean ± SEM) of SPRY2-labeled cells within the DG (linear regression: <i>t</i>₈ = 3.41, <i>P</i><0.01); N = 5/group. (<b>b</b>) Expression of SPRY2 in the HIP of sham-treated rats at low or (<i>inset</i>) high magnification. (<b>c</b>) Expression of SPRY2 after repeated ECS. Scale bar: 200 μm or (<i>inset</i>) 50 μm.</p

Expression of growth factor mRNA after treatment of HIP primary cell cultures with viral vectors.

<p>Data are presented as fold induction of the gene of interest (normalized to mean starting quantity of reference genes) relative to LacZ controls, ±SEM. (<b>a</b>) Viral vector treatment altered expression of FGF2 mRNA (<i>F</i>_2,22 = 6.11, <i>P</i><0.01); levels were significantly higher after dnSPRY treatment. (<b>b</b>) The same vectors had no effect on BDNF mRNA (<i>F</i>_2,15 = 0.33, not significant). **<i>P</i><0.01 compared to LacZ, Fisher’s protected <i>t</i>-tests, N = 8–9 per condition.</p

Learning and memory behaviors beginning 28 days after gene transfer.

<p>(<b>a</b>) Over the course of training in the MWM, there we no group differences in latencies to find the platform (main effect of treatment: <i>F</i>_2,27 = 0.99, not significant; interaction: <i>F</i>_14,189 = 1.54, not significant), and latencies decreased over days (<i>F</i>_7,189 = 40.2, <i>P</i><0.01). (<b>b</b>) There were no group differences in the time spent in the quadrant within which the platform was located (Northeast; NE) in probe tests conducted after training sessions 4 and 8 (red arrows) (main effect of treatment: <i>F</i>_2,27 = 1.08, not significant; interaction: <i>F</i>_2,27 = 0.23, not significant), and times increased between probe tests 1 and 2 (main effect of trial: <i>F</i>_1,27 = 40.3, <i>P</i><0.01). Times were higher during the second probe test for rats given the dnSPRY2 (<i>F</i>_1,10 = 13.1, <i>P</i><0.01), LacZ (<i>F</i>_1,10 = 29.1, <i>P</i><0.01), and wtSPRY2 (<i>F</i>_1,10 = 12.3, <i>P</i><0.01) vector. **<i>P</i><0.01 within-subject comparison with probe trial 1, Simple Main Effects tests.</p

Fear and anxiety-like behaviors 28 days after gene transfer.

<p>(<b>a</b>) During the first test session, there were no differences in the time course (<i>F</i>_4,40 = 0.71, not significant) or overall mean (<i>inset</i>: <i>F</i>_2,20 = 0.13, not significant) of FPS (mean ± SEM). (<b>b</b>) Upon re-testing 48 hr later, there were differential effects of the viral vectors in the time course (<i>F</i>_4,40 = 2.63, <i>P</i><0.05) and overall mean (<i>inset</i>: <i>F</i>_2,20 = 7.61, <i>P</i><0.01) of FPS. (<b>c</b>) In separate rats, there were no differences in time spent on the open arms of the EPM (<i>F</i>_2,20 = 0.34, not significant), *<i>P</i><0.05, **<i>P</i><0.01 compared to LacZ at the 10-min time point, ^^<i>P</i><0.01 compared to wtSPRY2 at the 10-min time point, Fisher’s protected <i>t</i>-tests, 7–8 rats/group.</p