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Moderate release of the major stress hormones, glucocorticoids (GCs), improves hippocampal function and memory. In contrast, excessive or prolonged elevations produce impairments. Enzymatic degradation and reformation of GCs help to maintain optimal levels within target tissues, including the brain. We hypothesized that expressing a GC-degrading enzyme in hippocampal neurons would attenuate the negative impact of an excessive elevation in GC levels on synaptic physiology and spatial memory. We tested this by expressing 11-β-hydroxysteroid dehydrogenase (type II) in dentate gyrus granule cells during a 3-day GC treatment followed by examination of synaptic responses in hippocampal slices or spatial performance in the Morris water maze. In adrenalectomized rats with basal GC replacement, additional GC treatments for three days reduced synaptic strength and promoted the expression of long-term depression at medial perforant path synapses, increased granule cell and CA1 pyramidal cell excitability, and impaired spatial reference memory (without influencing learning). Expression of 11-β-hydroxysteroid dehydrogenase (type II), mostly in mature dentate gyrus granule cells, reversed the effects of high GC levels on granule cell and pyramidal cell excitability, perforant path synaptic plasticity, and spatial memory. These data demonstrate the ability of neuroprotective gene expression limited to a specific cell population to both locally and trans-synaptically offset neurophysiological disruptions produced by prolonged increases in circulating stress hormones. This report supplies the first physiological explanation for previously demonstrated cognitive sparing by anti-stress gene therapy approaches and lends further insight into the hippocampal processes that are important for memory.
Stress activates the hypothalamic-pituitary-adrenal (HPA) axis and increases circulating levels of the stress hormones, glucocorticoids (GCs) (Kolber et al., 2008). An acute stress response provides physical and mental coping resources, but a prolonged response can have an array of adverse effects, including impairing memory functions dependent on the hippocampus (Kim et al., 2006). Thus, reducing the impact of prolonged elevations of GC levels is crucial to maintaining hippocampal integrity and normal memory function.
Learning and memory components of spatial navigation in rodents rely on hippocampal synaptic plasticity (Shapiro and Eichenbaum, 1999; Pittenger and Kandel, 2003; Martin and Morris, 2002). Stress or elevated GC levels have inverted-U effects on hippocampal synaptic function and spatial learning. For instance, a mild stressor or a moderate GC levels enhances long-term potentiation (LTP) (Diamond et al. 1992; Pavlides et al., 1994) and improves spatial memory (Pugh et al. 1997; Roozendaal and McGaugh, 1997; Liu et al. 1999; Akirav et al., 2004). In fact, moderate GC levels seem necessary for memory consolidation (Oitzl and de Kloet, 1992; Sandi et al. 1997; Conrad et al., 1999a). Conversely, chronic stress or increased GC levels reduces LTP (Foy et al., 1987; Diamond et al. 1992), promotes long-term depression (LTD) (Pavlides et al., 1995; Kim et al., 1996; Coussens et al., 1997; Xu et al., 1997; Yang et al., 2004), and impairs spatial memory (Luine et al., 1994; Oitzl et al. 1994; Diamond et al., 1996; Conrad et al. 1997; de Quervain et al., 1998). Thus, as circumstances shift from “stimulating” to “stressful,” GCs shift from being beneficial to impairing (Kim and Yoon, 1998; de Kloet et al., 1999; Sauro et al., 2003). Inverted-U effects can be explained by high affinity mineralocorticoid receptors (MRs) and lower affinity glucocorticoid receptors (GRs) that act as positive and negative modulators, respectively (Reul and de Kloet, 1985; McEwen et al., 1986). However, the physiological disruptions produced in different subregions of the hippocampus during an excessive stress response are not well understood. Likewise, the specific physiological alterations produced by excessive GR activation that relate to impaired spatial cognition need further investigation (Kim and Diamond, 2002).
The involvement of selective subsets of hippocampal neurons in specific aspects of spatial cognition can be tested by expressing factors that regulate glucocorticoid receptor (GR) activation in distinct neuronal subtypes during a period of excessive GC levels. While 11-β-hydroxysteroid dehydrogenase type I is native to the hippocampus, it works bidirectionally to both create and degrade CORT. The renal 11-β-hydroxysteroid dehydrogenase type II (11βHSDII) acts only to degrade CORT (Seckl, 2004). In this study, we used a herpes amplicon viral vector to express 11βHSDII preferentially in granule cells of the dentate gyrus of adult rats, where MRs and GRs are highly expressed and colocalized (Han et al., 2005). We hypothesized that such expression would protect against the effects of excessive GC exposure and expose the impact of alterations in granule cell excitability and perforant path to dentate gyrus (PP-DG) synaptic transmission on spatial memory.
The plasmids containing the eGFP (enhanced green fluorescent protein, Genbank accession number U55761) and the 11βHSDII genes (Genbank accession number U14631) have been described previously (Kaufer et al, 2004) and are variants of the standard bicistronic plasmid developed in our laboratory (Ho, 1994). For virus construction, E5 cells were transfected with pα4eGFP (a plasmid carrying the eGFP gene under the control of the HSV a4 promoter), or with pα411βHSDIIpα22eGFP (a bipromoter plasmid carrying eGFP and 11βHSDII genes, Fig. 1A) and then superinfected with d120 helper virus (DeLuca et al., 1985). After maximum infection, virus was released by sonication, partially purified (overlayed on 25 % sucrose, 75 Kg for 19 hr at 4 °C) and resuspended in PBS. For simplicity, the vector with the pα22eGFP plasmid is termed the eGFP vector and the vector with the pα411βHSDIIpα22eGFP plasmid is called the 11βHSDII vector. Animals that express eGFP alone are referred to as controls.
Male Sprague Dawley rats (250-350 g, Simonsen, Gilroy, CA) were anesthetized with isoflurane (3-5 % vapor) and underwent bilateral ADX and subcutaneous implantation of a corticosterone (CORT) pellet (100 mg, 15 % CORT, 85 % cholesterol). Drinking water was replaced with saline (0.9 %) after surgery. For animals in the behavioral experiments, the anesthetic was a 1 mL/Kg injection of a ketamine, acepromazine, and xylazine cocktail (10:2:1 ratio, 100 mg/mL, 10 mg/ mL, and 100 mg/ mL stock solutions, respectively).
Bilateral intracranial surgeries were performed 2-5 days after ADX surgery (11βHSDII and eGFP injections were performed on the same day for each cohort). Each animal was positioned in a stereotaxic device and had burr holes drilled in its skull to deliver eGFP (amplicon:helper: 3.0 × 107 particles/mL:4.8 × 107 particles/mL) or 11βHSDII vector (1.2 × 107 particles/mL:2.6 × 107 particles/mL) into the dentate gyrus (−3.6 A/P and +3.0 M/L mm from bregma, −3.0 D/V mm from dura) (0.5 μL/min for 3 min). Following surgery, animals received the first of three daily subcutaneous injections of either CORT (10 mg/kg) or vehicle (peanut oil) and a single intraperitoneal injection of bromodeoxyuridine, BrDU (in sterile saline, 40 mg/Kg). When added to the basal CORT released by the subcutaneous pellet, these CORT injections produce chronically high levels of serum CORT (approximately 28 mg/dL) for approximately 20 hours per day (Stein-Behrens et al. 1994). Three days of CORT injections were performed so that the animal healed adequately from intracranial surgery and the vector still expressed at high levels at the time of testing (while at the same time assuring the CORT pellets maintained basal serum CORT levels throughout the experiment). All animal experimentation reported in this paper was conducted in accordance with the guidelines specified by the National Institutes of Health (NIH Guide for the Care and Use of Laboratory Animals), the Stanford Department of Veterinary Services and Care, and the Institutional Animal Care and Use Committee of George Mason University.
Twenty-four hours after the final (third) CORT injection, hippocampal slices were prepared and electrically-evoked perforant path to dentate gyrus granule field responses were recorded (Dumas et al., 2000). Briefly, rats were anesthetized with isoflurane, decapitated and the brain was placed into ice-cold oxygenated (95% O2, 5% CO2) artificial cerebrospinal fluid (ACSF in mM: NaCl 124, KCl 2, MgSO4 2, CaCl2 2, KH2PO4 1.25, NaHCO3 26, and dextrose 10, pH 7.4). The hippocampus was dissected free and sliced parallel to the alvear fibers (400-450 μm). A small penetration mark designating the cannula tract was apparent on each hippocampus. Slices close to, but not containing, the injection site were selected and transferred to an interface recording chamber (room temp.) to incubate for at least 2 hr before recording.
To elicit PP-DG synaptic responses a bipolar stimulating electrode (insulated platinum-iridium) was placed in contact with the middle molecular layer in the superior blade. A recording electrode (ACSF-filled glass pipette, 2-8 MW tip resistance) was positioned ˜1 mm from the stimulating electrode in the middle molecular layer to record the fiber potential (FP) and excitatory postsynaptic potential (EPSP). A second recording electrode was placed in the hilus adjacent to the first recording electrode to record the population spike (PS). Responses were evoked at 25, 50, 100, and 200 μA. FP or PS amplitude was calculated as the voltage difference between cursors set at the initiation and negative peak of the response. EPSP slope was extracted from a 0.8 ms epoch at the initial downward deflection of the response (example waveforms are presented in Fig. 2). Response values were averaged at each stimulation level to construct input/output (I/O) curves. After collection of I/O curves, paired-pulse responses were elicited (25, 50, 100, and 200 ms interstimulus intervals) to create interstimulus interval (ISI) curves and test for changes in presynaptic function. For LTD experiments, stimulation intensity was adjusted to elicit a stationary 1 mV baseline EPSP for at least 15 min (0.033 Hz interpulse interval). LTD was induced with 900 pulses at 1 Hz and recording continued for 30 min at baseline frequency starting 15 seconds after LTD induction.
In some slices, Schaffer collateral field responses were recorded in the stratum pyramidale and stratum radiatum of area CA1 with a stimulating electrode positioned approximately 1 mm away in the stratum radiatum near the CA2-CA1 border.
In a second group of subjects, spatial training ensued 24 hr following the final CORT injection. Animals were trained in cohorts of 4-6 animals and all groups were represented within each cohort. The maze consisted of a black circular pool (1.7 m diameter) filled with tap water (24-27 °C) to a level that covered a stationary black escape platform (15 cm diameter) by 1-2 cm. The testing room (2.7 × 6.0 m) had one black wall, one beige wall and one checkered wall (0.6 × 0.6 m squares) surrounding the pool. The fourth side was an open space containing the animal tracking equipment. Training was performed in 6 blocks of 3 trials each with a 20-30 min inter-block interval. The goal quadrant was varied across testing cohorts. Four starting locations, equally spaced from each other were offset from the goal location by 45°. Starting location was chosen pseudorandomly from these positions for each trial. At the end of each trial, a 10 sec latency on the platform was imposed. After the sixth training block (trials 16-18), a probe trial was performed where the platform was removed and the rat was allowed to swim freely for 1 min. With the platform replaced, 3 more trials were performed (trials 19-21). At 24 hr post-training, a second probe trial was performed. For each training trial, latency to escape, path length to escape, and mean swim speed were calculated. For each probe trial, the amount of time searching in each quadrant (dwell time) was recorded.
Immediately after electrophysiological recording, hippocampal slices were fixed in 3% paraformaldehyde (PF, dissolved in PBS) for 10-20 min and refrigerated (4 °C) in PBS overnight. Slices were then embedded in gelatin (300 bloom, 14 %) and postfixed in PF (24-48 hr). Forty μm sections were cut by vibratome and mounted on gelatin-coated slides.
Immediately following the 24 hr probe trial, rats were perfused with roughly 60-80 mL of heparinized saline (2000 units/L, Acros Organics, Geel, Belgium) followed by a similar volume of 3 % PF. Brains were placed in 30 % sucrose in PF for 48-96 hr after which they were frozen to −17 to −19 °C and cut in the coronal plane with a cryostat. Brains from 4 animals were hemisected, embedded in gelatin, and sectioned by vibratome (33 μm, all sections containing dorsal hippocampus were kept). For cryostat-cut sections, beginning with the first section that showed a distinct DG granule cell layer, every third section (33 μm thickness) was saved and mounted on a slide until the dorsal and ventral hippocampus were no longer viewed as distinct structures.
Immunohistochemistry was performed on vibratome-cut sections from animals trained in the MWM. Sections were first washed in Tris-buffered saline (TBS) and those designated for BrDU labeling were incubated in 2N HCl for 10 min. All tissue was made permeable in 1% Triton-X (in TBS, 20 minutes) and blocked with 10 % normal goat serum (in TBS, 20 minutes) followed by overnight incubation in primary antibody (anti-BrDU, Millipore or anti-DCX, Santa Cruz) at a 1:200 dilution in TBS plus Triton-X (0.3 %) and normal goat serum (3 %). Sections were washed 3 times in TBS and incubated in secondary antibody (TRITC-conjugated anti-goat or Cy3-conjugated anti-mouse, Millipore) at a 1:200 dilution in TBS plus Triton-X (0.3 %) and normal goat serum (3 %). Granule cells were visualized with epifluorescence at 475/505 nm (peak excitation/peak emission) for eGFP and 555/565 nm for Cy3 (Olympus BX51WIF). Exposure times and lamp intensity values were identical when imaging sections treated with and without primary antibody.
Recording and parameter extraction were performed blind to experimental condition. For each animal, EPSP slope values were averaged at each level of the I/O curve and these values were averaged by treatment group. I/O curves and ISI curves were first analyzed by 3-way repeated-measures ANOVA and significant tests were followed by 2-way repeated-measures ANOVAs. Parameters extracted from CA1 recordings were compared by 2-way ANOVA and unpaired t-tests (Bonferroni-corrected).
For LTD analysis, response values from the final 10 minutes of baseline stimulation were averaged and all responses were divided by this mean to produce normalized proportion of baseline values (x 100 for % of baseline). LTD values were compared between groups by 2-way ANOVA (hormone × vector). Within each group , induction of LTD was determined by a paired t-test comparing the mean EPSP value at 25-30 minutes after 1 Hz stimulation to the baseline mean.
For analysis of spatial learning in the MWM, escape latencies, dwell times and mean swim speeds for each animal were averaged by training block. Block means were averaged by treatment group and compared by 3-way repeated-measures ANOVA (hormone × vector × block). To assess memory performance in the immediate and 24 hr probe trials, dwell times for each quadrant were averaged by treatment group. Chi square tests were performed to determine if any quadrant bias was displayed among the subjects in each group. A two-way ANOVA was used to compare dwell time in goal quadrant between groups. One-group t-tests were performed to compare dwell time in the goal quadrant to chance (15 seconds) during the immediate probe trial (same day as training).
Error bars in all figures are standard error of the mean (SEM).
Under fluorescent microscopy, eGFP-positive cells in infected hippocampi were observed in the superior and inferior blades of the DG, with 20-30% of granule cells infected (Fig. 1B). No eGFP-positive neurons were observed in area CA1 and only a handful of non-granule eGFP-positive cells were seen elsewhere across all animals and slices. Higher magnification images of sections from slices used for electrophysiology show distinct fluorescent granule cells and dendrites (Fig. 1C). Fluorescent granule cells were typically observed at both faces of the 400 mm thick slice and at 3 slices away from the injection site, amounting to approximately 3 mm total spread along the longitudinal axis of the hippocampus; this is in agreement with prior studies using this system using a colorimetric (McLaughlin et al., 2000; Dumas et al., 2004) or fluoresecent reporter (Nicholas et al., 2006; Ferguson and Sapolsky, 2008).
Electrically-evoked medial PP-DG synaptic field responses were recorded simultaneously in the molecular and granule cell layers of the superior blade at varying stimulation intensities to produce I/O curves (Fig. 2A). At the 25 μA stimulation intensity, EPSP values in numerous slices were below or near threshold, making calculations unreliable; thus, this stimulation level was omitted from further analyses. A 3-way repeated-measures ANOVA for the effects of CORT level, vector type, and stimulation intensities on the FP amplitudes showed only a repeated-measures effect [F(3,287) = 11.47, p < 0.0001], indicating that the FP responses increased across stimulation intensities but were not affected by hormone or vector conditions. In contrast, the EPSP slope was decreased in slices from High CORT animals compared to slices from Low CORT animals [F(1, 287) = 8.93, p < 0.005], but there was no effect of vector, reflecting a synaptic depression produced by High CORT that was not altered by vector delivery. Likewise, the ratio of the EPSP to FP (EPSP/FP) was reduced by High CORT [F(1, 287) = 19.09, p < 0.0001], but not altered by vector, further supporting a CORT-induced synaptic depression that was not reversed by expression of 11βHSDII or GFP (Fig. 2B).
It is possible that the 11βHSDII vector did not protect against the reduction in baseline synaptic strength produced by High CORT because it was expressed postsynaptically and the source of the synaptic depression was presynaptic in origin. To test for presynaptic involvement, we delivered paired stimulation pulses and examined presynaptically mediated short-term plasticity. While Schaffer collateral, mossy fiber, and lateral perforant path synapses express paired-pulse facilitation (PPF), medial perforant path synapses typically do not, and even display slight paired-pulse depression (Kahle and Cotman, 1993; DiScenna and Teyler, 1994). In agreement, under Low CORT conditions, slices that expressed 11βHSDII or controls showed no PPF across ISI curves (Fig. 2C) and paired-pulse responses were not different between these 2 groups (2-way repeated-measures ANOVA), indicating no effect of 11βHSDII on presynaptic function. The PPF ratio observed in slices from High CORT animals was significantly greater than that observed for slices from Low CORT animals [3-way repeated-measures ANOVA, F(1, 127) = 14.82, p < 0.001]. An elevation in PPF is normally indicative of a decrease in transmitter release (Creager et al., 1980; Foster and McNaughton, 1991). Thus, the findings strongly suggest that the synaptic depression produced by elevated CORT was due at least in part as a reduction in presynaptic transmitter release, which may explain the inability of postsynaptic 11βHSDII expression to reverse the depression of transmission.
Recording in the granule cell layer allowed for observation of changes in the PS, a measure of granule cell excitability. The PS amplitude to EPSP slope ratio (PS/EPSP) was calculated for each data point and averaged at each stimulus level for each experimental group (Fig. 2D). A 3-way ANOVA revealed effects of hormone [F(1, 219) = 12.85, p < 0.001], vector [F(1, 211) = 5.96, p < 0.02], and a hormone × vector interaction [F(1, 287) = 4.23, p < 0.05]. Compared to the Low CORT animals, PS/EPSP values for the High CORT/eGFP [F(1, 191) = 18.97, p < 0.0001] and High CORT/11βHSDII animals were increased [F(1, 191) = 4.31, p < 0.05], reflecting a CORT-induced increase in granule cell excitability. The PS/EPSP values for the High CORT/11βHSDII animals were significantly reduced when compared to the High CORT/eGFP [F(1,143) = 5.50, p < 0.025, denoted by the asterisk], indicating protection against the CORT-induced increase in granule cell excitability by expression of 11βHSDII.
For plasticity experiments, baseline responses were set to 1 mV in amplitude, roughly 1 third to 1 half of the maximum EPSP amplitude for each group. The baseline EPSP slope was not different between groups (in mV/ms - Low CORT/11βHSDII: −0.35 ± 0.02, n = 10; Low CORT/eGFP: −0.29 ± 0.02, n = 10; High CORT/11bHSDII: 0.26 ± 0.03, n = 9; High CORT/eGFP: 0.29 ± 0.02, n = 10). However, a 2-way ANOVA (hormone × vector) comparing mean normalized EPSP slope values at 25-30 minutes after LTD induction was significant [F(3,38) = 6.91, p < 0.005]. When comparing the EPSP slope during the last 10 minutes of baseline and 25-30 minutes after LTD induction, no difference was observed for the Low CORT/11βHSDII (% of baseline, 101 ± 5) or Low CORT/eGFP slices (% of baseline, 95 ± 6). Thus, as previously shown in slices from naïve adult rats, LTD was not induced under basal CORT conditions (Dudek and Bear, 1993, Wagner and Alger, 1995) (Fig. 3A). In agreement with Kim et al (1996), in High CORT control animals, 1 Hz stimulation produced LTD [% of baseline, 77 ± 7; paired t-test, t(18) = 2.1, p < 0.0001]. In contrast, when 11βHSDII was expressed in slices from High CORT animals, no LTD was observed (% of baseline, 100 ± 5), indicating protection against the increased susceptibility to LTD induction produced by High CORT.
In some slices, simultaneous recordings were made in the stratum pyramidale and stratum radiatum in area CA1 upon stimulation of Schaffer collateral axons. Stimulation was set to evoke a 1 mV EPSP in the stratum radiatum. The stimulation intensities, EPSP slopes, and PPF (50 ms ISI) for 1 mV EPSPs were not different between groups (Table 1), indicating that CORT condition or vector expression in DG granule cells did not affect baseline synaptic function at Schaffer collateral synapses.
CA1 pyramidal cell excitability was assessed by measuring the amplitude of the PS recorded in the stratum pyramidale at stimulation intensitites that elicited 1 mV EPSPs in the stratum radiatum. Two-way ANOVAs revealed no differences in stimulation intensity to achieve a 1 mV EPSP or the EPSP slope at a 1 mV amplitude (Table 1). However, for PS analyses, there was a main effect of vector [F(3, 47) = 8.05, p < 0.01] and a hormone × vector interaction [F(3, 47) = 9.09, p < 0.005]. The effect of hormone likely did not reach significance (p < 0.07) due to the reduction in the PS amplitude in the High CORT/11βHSDII below the PS amplitude of the Low CORT groups. The PS amplitudes were larger in the High CORT/eGFP than in the combined Low CORT slices [t(24) = 2.1, P < 0.05], indicating that High CORT increased CA1 pyramidal cell excitability. In contrast, the High CORT/11βHSDII animals displayed PS amplitudes that were of reduced magnitude when compared to either the Low CORT animals [t(26) = 2.1, P < 0.005] or the High CORT/eGFP animals [t(20) = 2.1, P < 0.0001]. Combined, the data show that 11βHSDII expression in DG granule cells, 2 synapses away, prevented the CORT-induced increase in CA1 pyramidal cell excitability.
We employed the MWM (Morris, 1981) to assess the effects of High CORT and 11βHSDII on hippocampal-dependent spatial learning. Three days following vector delivery and the onset of CORT injections, animals were trained to find a hidden platform in a circular pool. As a whole, all groups displayed learning by reducing their escape latencies across training blocks [F(5, 190) = 20.40, p < 0.001, does not include training data taken after the immediate probe trial, Low CORT/11βHSDII, n = 9; Low CORT/eGFP, n = 10; High CORT/11βHSDII, n = 11; High CORT/eGFP, n = 8]. There was no difference between groups in the average latency (Fig. 4A) or mean path length (not shown) to find the platform across learning curves. Thus, granule cell infection and 3 days of systemically elevated CORT do not produce measurable changes in sensorimotor abilities, working memory, or spatial navigation.
Search strategy probes for the platform location were performed both on the day of training (Fig. 4B) and 24 hr later (Fig. 4C). Chi square tests were performed to detect spatial biases. On the same day of training, all groups showed a quadrant bias (Low CORT/11βHSDII, χ(3) = 13.67, p < 0.01; Low CORT/ eGFP, χ(3) = 11.60, p < 0.01; High CORT/11βHSDII, χ(3) = 9.73, p < 0.02; High CORT/ eGFP, χ(3) = 12.00, p < 0.01). As well, all groups spent more time in the goal quadrant than would be predicted by chance (15 sec, 25%) [Low/11βHSDII: t(6) = 3.40, p < 0.01; Low/eGFP: t(9) = 7.60, p < 0.0001; High/11βHSDII: t(9) = 5.20, p < 0.005; High/eGFP: t(6) = 3.99, p < 0.01], and a 2-way ANOVA (hormone × vector) for dwell time in the goal quadrant showed no main effects or interaction, indicating that all groups utilized a spatial search strategy on the same day of training.
For the 24-hr probe, Chi square tests were significant for the Low-CORT/eGFP group [χ(3) = 11.60, p < 0.01] and the High CORT/11βHSDII group [χ(3) = 9.23, p < 0.05], with a strong trend for significance for the Low CORT/11βHSDII group [χ(3) = 6.56, p < 0.088] suggesting unequal dwell times in the four quadrants for all but the High CORT/eGFP animals. A 2-way ANOVA for dwell time in the goal quadrant produced an effect of vector [F(1, 35) = 4.29, p < 0.05], but not hormone and no interaction. Low CORT animals spent more time in the goal quadrant than would be predicted by chance [Low/11βHSDII: t(8) = 3.30, p < 0.05; Low/eGFP: t(9) = 3.17, p < 0.05], thus displaying memory for the goal quadrant. Animals that received high CORT and expressed eGFP did not show a bias for the goal quadrant, indicating that High CORT impaired spatial memory. However, similar to the Low CORT groups, animals that received high CORT and expressed 11βHSDII spent more time in the goal quadrant [t(10) = 2.50, p = 0.05], supporting the idea that 11βHSDII expression in DG granule cells reduced the negative effects of High CORT on spatial memory.
It is possible that physiological and behavioral sparing was mediated via an effect on neurogenesis or on differential effects of CORT or viral infection on neurons of different maturational states. Vibratome-cut hemisections from four animals trained in the MWM were subjected to immunohistochemistry for the neurogenesis marker, BrDU, and the developmentally-restricted neurofilament protein, doublecortin (DCX). Nearly all sections containing eGFP-positive granule cells also showed positive labeling for BrDU and DCX. There was no obvious difference in the number of BrDU- or DCX-positive cells across animals. There was no evidence of colocalization of eGFP with BrDU (Fig. 5A1, A2), nor in most instances, with DCX (Fig. 5B1, B2). However, there were cases in both Low CORT and High CORT animals in which eGFP was colocalized with DCX (Fig. 5B1a, 5B1b, respectively). These data indicate that, while newly generated cells in the dentate gyrus are not subject to amplicon infection, both mature and immature neurons can be infected, but with no preferential bias toward infecting the latter.
While pharmacological elevations in serum CORT are not equivalent to behavioral stress, the selective effect of CORT on spatial memory corroborates previous research showing that 3 days of rotation stress, 7 days of restraint stress, or 21 days of CORT treatments impairs reference memory, leaving learning and working memory intact. Our finding that High CORT elicits LTD also mimics the facilitating effect of behavioral stress on hippocampal LTD induction (Pavlides et al., 1995; Kim et al., 1996; Coussens et al., 1997; Xu et al., 1997; Yang et al., 2004). The current experiments further demonstrate that a 3-day CORT treatment increases neuronal excitability and produces a lasting depression of baseline PP-DG synaptic transmission that is, in part, presynaptic in origin. The effects of elevated CORT on hippocampal synaptic transmission and spatial reference memory may be indirect, involving a change in sleep patterns or other systemic influence of HPA axis manipulation. Our findings implicate the actions of CORT in DG granule cells as the alterations in granule cell excitability, PP-DG synaptic LTD, and spatial memory were all reversed by 11βHSDII expression specifically within these neurons.
Roughly 20-30 % of the DG granule cell population was infected, with a bias toward mature neurons, which corroborates more detailed previous analyses (McLaughlin et al., 2000). No pyramidal cell infection was observed. Thus, many unprotected neurons in the animals that did not express 11βHSDII could have contributed to the inability of 11βHSDII vector delivery to fully preserve spatial memory. Since 11βHSDII expression reduced the impact of High CORT on granule cell excitability, LTD induction, and spatial memory, it appears that plasticity at DG inputs or DG excitability could regulate spatial memory. While changes in memory consolidation cannot be ruled out, sparing effects on recall are supported by the demonstration that expression of a transdominant GR in DG granule cells can prevent the disruption of long-term spatial memory produced by a CORT injection delivered 30 minutes before the memory test (Ferguson et al., 2007).
Cognitive disruption and dendritic retraction in area CA3 pyramidal cells result from chronic stress or repeated CORT treatment (Woolley et al., 1990; Arbel et al., 1994; Conrad et al., 1996). It is unlikely that dendritic atrophy was created by our 3-day CORT treatment because it is not produced by 2 weeks of stress (Magarinos and McEwen, 1995) and the chronic stress paradigms that produce dendritic atrophy also impair learning (Arbel et al., 1994; Conrad et al., 1996), which was not apparent in this study. More recent work showed that reducing CORT levels during memory assessment prevents chronic stress-induced impairments in spatial memory (Wright et al., 2006), suggesting that the cognitive effect is more closely related to CORT level than to dendritic atrophy.
A persistent alteration in synaptic plasticity 24 hr after the final CORT injection corroborates previous findings in area CA1 and the DG following the termination of a chronic stressor (Pavlides et al., 1993; Alfarez et al., 2003). Both PP-DG and SC-CA1 synapses are more resistant to LTP and more susceptible to LTD induction following chronic stress (Kim et al., 1996; Coussens et al., 1997; Xu et al., 1997). Regional differences in CORT sensitivity are supported by a reduction in baseline synaptic strength at PP-DG synapses not observed at SC-CA1 synapses (current results), and a greater stress-induced shift in metaplasticity at PP-DG synapses (Abraham and Tate, 1997). Whether the effects of CORT on synaptic transmission and plasticity are an indirect result of a more global cellular alteration such as a change in neuronal excitability (Joëls and de Kloet, 1992), calcium channels (Joëls et al., 2003), or cellular metabolism (Takahashi et al., 2002), is an interesting question for follow-up research.
It is unclear whether the increase in neuronal excitability we observed reflects the state of the system during CORT treatment or is a rebound effect. Earlier studies have shown suppressed hippocampal activity with an acute CORT injection (Pfaff, et al., 1971) or an early increase in excitability followed by a suppression (Joëls and de Kloet, 1990). Given the same timecourse, modest increases in CORT can suppress excitability, while larger increases augment excitability, due to differences in MR and GR occupation (Joëls et al., 1994). Thus, when interpreting effects of stress or CORT on neuronal excitability, it is clearly important to consider both level and timecourse of the treatment as well as the time of assay relative to treatment (during or after). Interesting follow-up experiments might include a CORT injection on the day of assay or multiple assessments at differing timepoints after the final CORT delivery.
The ability of 11βHSDII expression in DG granule cells to maintain low excitability levels in area CA1 during high CORT was unexpected and was not the result of extra-DG infection. The most harmonious explanation is that, even in the slice preparation, activity levels in the DG (or entorhinal cortex) affect activity levels in downstream regions. Resulting caveats are that the impact of CORT in CA1 is not entirely due to direct effects in this region and the 11βHSDII-dependent reduction in CA1 excitability is likely secondary to the reduction in DG excitability.
The observation that 3 days of elevated CORT reduced baseline PP-DG synaptic transmission is comparable to the timecourse of synaptic depression that occurs at this relay following ADX without CORT replacement (Stienstra et al., 1998), consistent with the inverted-U effects of CORT in the hippocampus. The High CORT-induced synaptic depression was not reversed by expression of 11βHSDII, possibly due to a difference in the site of the impairment and 11βHSDII expression (pre- and postsynaptically, respectively). Alternatively, it has been shown that CORT mediates a suppression of PP-DG synaptic transmission via effects in the basolateral amygdala (Vouimba et al., 2007), which might not be offset by 11βHSDII expression in DG granule cells. Interestingly, the reduction in baseline synaptic function did not impact spatial learning and was not related to the improvement in spatial memory in animals expressing 11βHSDII.
Occupation of MRs, perhaps with a very low level of GR occupancy, is necessary to maintain synaptic function (Stienstra et al., 1998) and is clearly demonstrated by recent experiments that utilize MR-specific agonists and antagonists (Avital et al., 2006). If 11βHSDII were to completely degrade CORT, one might expect a far left shift on the inverted U curve where MRs are not occupied by CORT, resulting in no benefits for synaptic transmission and memory. This was not observed. It is possible that 11βHSDII does not completely degrade CORT or that cortisone has sufficient efficacy at MRs. Combined with the higher affinity of MRs for CORT, it appears that 11βHSDII expression preferentially reduces occupancy of GRs. This idea is supported by a previous report from our lab showing that 11βHSDII is equally efficacious as a virally-expressed transdominant GR in reducing the endangering effects of CORT on excitotoxicity in vitro and in vivo (Kaufer et al., 2004).
New neurons are continually generated in the DG, many of which mature to become incorporated into the hippocampal circuit (Li et al., 2009). The rate of neurogenesis in the DG is reduced by chronic stress (Gould et al., 1991; Tanapat et al, 1998; Malberg and Duman, 2003), leaving open the possibility that CORT impacted DG physiology and MWM performance in our study via a reduction in neurogenesis. It seems unlikely that our 3-day CORT treatment would reduce proliferation enough to produce observable effects on memory or on excitability and synaptic transmission measured at the population level (Kempermann, 2002), although it should be noted that, at any given time, neurons of all developmental stages are present in the DG. As such, there could be maturation-dependent effects of CORT and/or viral infection. Newborn cells that do not express GR or MR for the first three days are those that are presumed to survive to become fully functional neurons (Garcia et al., 2004). Immature neurons express both MR and GR and, as shown here, can be infected by herpes amplicons. Mature and immature neurons contribute differentially to perforant path synaptic plasticity (Wang et al., 2000; Snyder et al., 2001; Schmidt-Heiber et al., 2004) and to memory function (Snyder et al., 2005; Saxe et al., 2007), so it is important to consider developmental state. Nevertheless, the physiology and memory restoring effects of 11βHSDII in this study appear to be due to its expression in mature DG granule cells as that was the predominant site of eGFP expression.
The current findings demonstrate that various negative impacts on hippocampal function resulting from prolonged elevations in circulating stress hormones are offset by neuroprotective gene expression in a subset of DG granule cells. Effects include normalized PP-DG synaptic plasticity and both local and trans-synaptic neuronal excitability. The current physiological results may explain reports of cognitive sparing produced by similar anti-stress gene therapy approaches. Relationships between the physiological and behavioral changes under High CORT conditions and with 11βHSDII expression allow for a greater understanding of the specific neuronal mechanisms that support learning and memory.
The authors thank Dr. W. Ogle, Dr. D. Kaufer and Z. Pincus for supplying the 11βHSDII vector. Grants from the National Institutes of Health (RMS, RO1NS32848, RO1AGO20633, NIH5T32-NS07280) and the Adler Foundation and internal funds from the College of Science at George Mason University supported this research.