Adam James Devall

Dr Thelma A Lovick
email: t.a.lovick@bham.ac.uk

Differential activation of the periaqueductal gray by mild anxiogenic stress at different stages of the estrous cycle in female rats Running title: Stress-induced hyperalgesia in female rats

Keywords: Anxiety-induced hyperalgesia, female rats, tail flick reflex, periaqueductal gray matter, estrous cycle, c-fos

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INTRODUCTION

Sex differences in the perception of pain are well documented. However, despite the generally accepted view that females are more sensitive than males (Greenspan et al, 2007), the underlying cause is not clear and indeed, may be multi-factorial. In women, one factor that appears to affect the pain experience is the cyclical variations in sex hormones that occur during the menstrual cycle. Women with existing painful disease states consistently report worsening of their pain during the late luteal phase of the cycle (premenstrual period) [START_REF] Bajaj | Sensory changes during the ovulatory phase of the menstrual cycle in healthy women[END_REF][START_REF] Houghton | The menstrual cycle affects rectal sensitivity in patients with irritable bowel syndrome but not healthy volunteers[END_REF][START_REF] Powell-Boone | Menstrual cycle affects bladder pain sensation in subjects with interstitial cystitis[END_REF].

Cycle-related changes in pain threshold have also been reported in normal healthy women [START_REF] Riley | A meta-analytic review of pain perception across the menstrual cycle[END_REF][START_REF] Fillingim | Sex-related hormonal influences on pain and analgesic responses[END_REF] although a more recent study has challenged this finding [START_REF] Tousignant-Laflamme | Excitatory and inhibitory pain mechanisms during the menstrual cycle in healthy women[END_REF].

The perception of pain depends not only on the level of excitation of peripheral nociceptors but also on the level of activity in the endogenous descending control systems that facilitate or inhibit spinal nociceptive processing [START_REF] Gebhart | Descending modulation of pain[END_REF]. These two components may be influenced differentially during the menstrual cycle. Thus whilst cutaneous nociceptive threshold per se did not change during the cycle, the efficacy of diffuse noxious inhibitory controls (DNIC), which reduce pain sensitivity by engaging endogenous descending inhibitory control, was reduced during the menstrual and luteal phases [START_REF] Tousignant-Laflamme | Excitatory and inhibitory pain mechanisms during the menstrual cycle in healthy women[END_REF]. The menstrual cycle may also influence the level of descending facilitation of spinal nociceptive processing. It has long been recognised in the clinic that pain worsens when anxiety levels are increased moderately [START_REF] Taenzer | Influence of psychological factors on postoperative pain, mood and analgesic requirements[END_REF][START_REF] Lautenbacher | Relationship between clinical pain complaints and pain sensitivity in patients with depression and panic disorder[END_REF][START_REF] Kain | Preoperative anxiety and postoperative pain in women undergoing hysterectomy. A repeated-measures design[END_REF]. Extreme 4 anxiety or fear on the other hand, elicits hypoalgesia [START_REF] Ford | Clinical correlates of stress-induced analgesia: evidence from pharmacological studies[END_REF]. In many women, the pre-menstrual period is associated with the development of adverse psychological symptoms that include increased anxiety and negative mood changes [START_REF] Steiner | Premenstrual syndromes[END_REF]. Thus hormone-linked changes in anxiety levels could be a causative factor for producing menstrual cycle-related increases in pain sensitivity by influencing the brain's endogenous pain control mechanisms.

In animal studies, the data concerning cyclical changes in pain sensitivity in females is equivocal. Whilst there are several reports that cutaneous nociceptive thresholds do not change significantly throughout the estrous cycle in rodents [START_REF] Aloisi | Sex-dependent effects of restraint on nociception and pituitary-adrenal hormones in the rat[END_REF][START_REF] Vinogradova | The effects of stages of the estrous cycle on pain thresholds in female white rats[END_REF][START_REF] Borta | Inhibitory avoidance, pain reactivity, and plus-maze behavior in Wistar rats with high versus low rearing activity[END_REF][START_REF] Terner | Influence of estrous cycle and gonadal hormone depletion on nociception and opioid antinociception in female rats of four strains[END_REF][START_REF] Devall | Hyperalgesia in the setting of anxiety: Sex differences and effects of the oestrous cycle in Wistar rats[END_REF], many earlier studies did detect cycle related differences [START_REF] Frye | Hormonal milieu affects tail flick latency in female rats and may be attenuated by access to sucrose[END_REF][START_REF] Kayser | Estrous and sex variations in vocalization thresholds to hindpaw and tail pressure stimulation in the rat[END_REF]Martinez-Gomez et al, 1994;[START_REF] Mogil | Sex differences in thermal nociception and morphine antinociception in rodents depend on genotype[END_REF]You et al, 2006). The underlying reason for these inconsistent findings is not clear but may relate to strain differences and/or to differences in the extent to which the testing methodology engaged endogenous descending control systems. Interestingly, in animals in which no cyclerelated change in pain threshold was detected, changes in pain sensitivity linked to hormonal status could be detected following exposure of the rats to environmental stimuli that would be expected to engage descending control systems [START_REF] Devall | Hyperalgesia in the setting of anxiety: Sex differences and effects of the oestrous cycle in Wistar rats[END_REF][START_REF] Ryan | The estrous cycle and estrogen modulate stress-induced analgesia[END_REF][START_REF] Walf | Anti-nociception following exposure to trimethylthiazoline, peripheral or intra-amygdala estrogen and/or progesterone[END_REF]. Thus parallels between gonadal hormone-linked changes in anxiety and nociception in women and female rats can be demonstrated when the experimental conditions are carefully controlled.

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The midbrain periaqueductal gray matter (PAG) is a major source of endogenous descending control of spinal nociceptive processing [START_REF] Gebhart | Descending modulation of pain[END_REF]. It is also involved in integrating anxiety-like emotional behavior (see Lovick, 1996 for review). The changing hormonal profile of the female brain during the estrous cycle has been shown to produce differential effects on neuronal excitability in the PAG [START_REF] Brack | Neuronal excitability in the periaqueductal gray matter during the estrous cycle in female Wistar rats[END_REF]. During the late diestrus phase the fall in progesterone level that occurs at this time is associated with upregulation of GABA A receptor subunit expression in the PAG (Griffiths and Lovick, 2005a;[START_REF] Griffiths | Withdrawal from progesterone increases expression of alpha4, beta1, and delta GABA(A) receptor subunits in neurons in the periaqueductal gray matter in female Wistar rats[END_REF][START_REF] Lovick | Changes in GABA(A) receptor subunit expression in the midbrain during the oestrous cycle in Wistar rats[END_REF]. New extrasynaptic receptors with the α4β1δ configuration are expressed on GABAergic interneurones in the PAG and as a consequence, the output neurones become disinhibited, which leads to the circuitry becoming intrinsically more excitable.

If the functional excitability of the PAG circuitry changes at different stages of the estrous cycle, it is possible that the level of descending control, and hence nociceptor responsiveness changes too. To investigate the possibility that differential functional activation of the PAG at different stages of the estrous cycle might underlie changes in pain sensitivity, we examined expression of the immediate early gene c-fos in neurones in the PAG in brains taken from rats that had undergone nociceptive testing in the setting of mild anxiogenic stress at different stages of their estrous cycle. In addition, we investigated whether artificial manipulation of progesterone levels, designed to mimic the changes that occur naturally during late diestrus, could influence nociceptive thresholds and stress-induced activation of the PAG.

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MATERIALS AND METHODS

Animals

Experiments were carried out on female Wistar rats, 200-250 g body weight. Animals were housed in pairs or groups of three in an ambient temperature of 21±1°C under a 12 h light/dark cycle (lights on at 0700 h) with free access to food and water. In line with UK Home Office guidelines for the care of animals used for research purposes, home cages contained an adventure toy (upturned plastic box with an entrance cut out) and a chewing stick to provide an enriched environment. The rats were transported from the animal holding room to a different room to carry out all procedures (injections, vaginal smears etc) and for behavioral testing. All procedures were undertaken in accordance with the UK Animals (Scientific Procedures) Act 1986.

Determination of the estrous cycle phase

A vaginal smear was taken daily at approximately 0915 h and stained using a Quick-Diff FIX staining set (Reagena, Takojantie, Toivala, Finland) to establish that the animals were cycling normally [START_REF] Brack | Cardiovascular and respiratory responses to a panicogenic agent in anaesthetised female Wistar rats at different stages of the oestrous cycle[END_REF]). An additional smear was taken within 2 h of the final behavioral experiment to check the cycle stage.

Behavioral testing

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A detailed description of the behavioral testing protocol has been published recently [START_REF] Devall | Hyperalgesia in the setting of anxiety: Sex differences and effects of the oestrous cycle in Wistar rats[END_REF]. In brief, on 3 days animals were habituated to being confined for 30 min in a Plexiglass restraint tube. On the 4 th day, the rats were placed in the tube and the tail flick latency (TFL) in response to radiant heat from a 600W projector lamp focused on a 3 mm diameter area on the blackened underside of the tail was measured. The voltage supplied to the lamp was adjusted so that a tail flick occurred between 4 and 5 s of switching on the lamp. TFLs were measured at 5 min intervals over a 20 min period to establish baseline values. At the end of the control period, the restraining tube with the rat inside was vibrated at 4 Hz for 5 min to induce mild anxiogenic stress. On cessation of the vibration, TFLs were measured at 2 min intervals over the next 10 min and then at 5 min intervals for the next 15 min. The next day, when the rats had progressed to a different stage of their cycle, they underwent a second behavioral testing experiment identical to the first one. Control animals underwent the same habituation and testing procedures except that vibration stress was omitted. In control animals, TFLs were consistent throughout the 45 min experimental period indicating that repeated heating of the tail did not cause tissue damage.

Progesterone withdrawal regimen

The progesterone dosing regimen was based on that described previously (Griffiths and Lovick, 2005a). Female animals received twice daily injections (at approximately 0800 h and 1600 h) of progesterone (5 mg Kg -1 IP in corn oil, Sigma, Poole, Dorset, UK) for 6 days. On the afternoon of day 6 the progesterone withdrawal group (PWD) received vehicle (corn oil, 0.1 ml 100g -1 IP) and again at 0800 h on day 7. The high progesterone 8 group (HP) was maintained on progesterone dosing for 7 days. Behavioral testing was carried out on the afternoon of day 7. Control animals in both the HP and PWD groups underwent the habituation and experimental testing procedures but the vibration stress was omitted. The dose of progesterone was chosen to maintain plasma levels of progesterone at the high end of the physiological range [START_REF] Butcher | Altered secretion of gonadotropins and steroids resulting from delayed ovulation in the rat[END_REF]Doornbos et al, 2009). This dosing regimen disrupted the characteristic daily change in the cytological appearance of vaginal smears seen in spontaneously cycling animals (Figure 1A-D).

Progesterone dosing was started on Mondays regardless of the cycle stage of the rat.

Within 2-3 days the smears took on an appearance similar to rats in early diestrus (Figure 1E), when progesterone levels would have been elevated for a 24 h period [START_REF] Butcher | Altered secretion of gonadotropins and steroids resulting from delayed ovulation in the rat[END_REF]. On withdrawal from progesterone the cycle appeared to restart and smears taken 24 h after the final injection showed similarities to rats in proestrus (Figure 1F).

Naïve rats

In order to determine whether the nociceptive testing regimen and/or confinement in the restraint tube induced functional activation of the PAG a group of spontaneously cycling rats was handled daily to obtain vaginal smears but did not undergo any behavioral testing procedures.

Fixation and histochemical processing

Two hours post vibration stress, when acute stimulation-induced activation of c-fos has 9 been shown to be optimal [START_REF] Williams | Changing patterns of c-fos induction in spinal neurons following thermal cutaneous stimulation in the rat[END_REF]), or at an equivalent time in control (nonstressed) animals, the rats were anesthetized with urethane (0.5 ml 100 g -1 , 20% solution i.p.) and perfused retrogradely through the descending aorta with 100 ml heparinized saline (10 units ml -1 ) followed by 200 ml 4% paraformaldehyde in 0.1M phosphate buffer (PB), pH 7.4. The brain was removed, post fixed for 2 h and transferred to 30% sucrose in 0.1M PB at 4°C for cryoprotection. Naïve animals (see above) were anesthetized and perfused at a similar time of day.

Frozen 40 μm thick coronal midbrain sections were washed 3 times in immunobuffer (IB: phosphate buffered saline (PBS) containing 10 mM Trizma base and 0.003% Triton-X 100). Hereafter the sections were gently agitated by placing the vials on an orbital shaking platform. Endogenous peroxidase activity was blocked by incubation in hydrogen peroxide solution (0.5%, in IB containing 1% normal horse serum (NHS), Gibco, UK) for 60 min at room temperature. The sections were then incubated in anti-Fos antibody (SC-52, Santa CruzBiotech, CA, USA; 1:5000 in IB containing 10% NHS) at 4°C for 48 h and washed 3 times in PBS containing 10 mM Trizma base (TPBS) before incubation in secondary antibody (711-065-152, Jackson ImmunoResearch, PA, USA; 1:500 in IB containing 1% NHS) at 4°C for 24 h. The sections were next washed in TPBS, and incubated with Extravidin™-peroxidase (Sigma, 1:1500 in IB) for 4 h. Following further washes in TPBS, sections were incubated with a nickel-diamino-benzidine solution (DAB; 0.04% nickel ammonium sulphate and 0.01% DAB containing 0.004% ammonium chloride and 0.2% glucose in 0.1M PB) for 10 10 min. An equal volume of this solution containing 2 μl ml -1 glucose oxidase was then added to supply hydrogen peroxide to the peroxidase-DAB precipitation reaction. The progress of the ensuing reaction was monitored by periodic inspection of sections under a microscope and stopped when Fos-positive nuclei could clearly be observed in the tissue but before non-specific background stain developed, by flooding with excess TPBS. After further washes in TPBS sections were mounted onto gelatinized slides, dried in air, dehydrated through an ascending series of alcohols, cleared in Histoclear (National Diagnostics, Atlanta, GA, USA) and mounted using Histomount (National Diagnostics).

All reagents were obtained from Sigma, Poole, Dorset, UK unless otherwise stated.

Quantification of Fos-like immunostaining

In an attempt to minimise the effect of batch variations in immunostaining, material from the brains of 3 rats, usually from different estrous cycle stages or treatment groups, were processed in parallel using the same solutions. For each animal, 4 sections representative of antero-posterior levels -6.04, -7.04, -7.8 and -8.72 [START_REF] Paxinos | The Rat Brain in Stereotaxic Coordinates[END_REF], hereafter termed levels I-IV respectively (see Figure 2), were selected for detailed quantitative analysis of the density of Fos-immunoreactive nuclei. The analysis was carried out by an experimenter who was blinded both to the experimental procedure and to the hormonal status of the rats. The sections were first photographed at low power (4x objective) using phase contrast microscopy, which allowed the borders of the PAG to be readily delineated. Bright-field images (6-9 fields of view per section) covering the whole PAG were then captured using the 10x objective and combined electronically to create a 11 photo-montage using image analysis software (LUCIA G, v4.2.8, Laboratory Imaging, Czech Republic). The phase contrast image was scaled to match the magnification of the bright-field image and overlaid at 50% opacity using imaging software (Adobe Photoshop CS, San Jose, CA, USA). The outlines of the PAG and aqueduct were then delineated using a drawing tablet (WACOM, Krefeld, Germany) and a mask was created over the image so that only the PAG remained visible.

Image analysis software (ImageJ, v1.40, National Institutes of Health, USA) was used to identify Fos-positive nuclei. A threshold was set that excluded all pixels except those that were within the darkest 2.5% of the image. The resulting mask was analysed using ImageJ's analyse particles function, filtering out those particles that had a size <15 or >100 pixels 2 (equating to an area of <14.8 and >98.8μm 2 ) and a circular morphology with the ratio of the shortest to longest axis of ≤0.65:1. Based on initial comparisons between computer generated plots and manual plotting of stained nuclei using a drawing tube, these factors described a particle which was clearly distinguishable from the background and with sufficient circularity and size restrictions to equate to our expectation of PAG nuclear morphology. Following this automated step, a map of the section showing the location of all identified Fos-positive nuclei within the boundaries of the PAG was generated. For each section, the PAG was subdivided into its longitudinal columns as described in the rat brain atlas of [START_REF] Paxinos | The Rat Brain in Stereotaxic Coordinates[END_REF]: dorsal (D), dorsolateral (DL), lateral (subdivided into L1 and LII) and ventrolateral (VL) (Figure 2). In order to sample the density of immunolabeled cells within each column of the PAG a triangular counting frame representing an area of 60,000 μm 2 within the tissue was orientated over the image 12 with the apex pointing towards the aqueduct (Figure 2). The number of Fos-positive nuclei present within each frame was then counted manually.

Data analysis

All statistical analysis was performed on the raw data using Prism software (v4.0, GraphPad Software, La Jolla, CA, USA). Tail flick data both within and between groups were analyzed using a post-hoc Dunnett's test in comparison to appropriate control groups after significant (P<0.05) repeated measures one-way ANOVA. For illustrative purposes data is presented as a percentage of mean baseline TFL, although the level of statistical significance shown on the graphs relates to the raw data. Immunostained cell density in the PAG was analysed using post-hoc Dunnett's and Bonferroni comparisons within appropriate groups after significant (P<0.05) one-way ANOVA.

RESULTS

Effect of estrous cycle on vibration stress-evoked changes in nociceptive threshold and expression of Fos-like immunoreactivity in the PAG

The animals used in this study were exposed to nociceptive testing on two consecutive days when the rats were in different stages of their estrous cycle. A detailed report of the effects of short lasting vibration stress on nociceptive thresholds on day 1 has been published recently [START_REF] Devall | Hyperalgesia in the setting of anxiety: Sex differences and effects of the oestrous cycle in Wistar rats[END_REF]. In brief, exposure to vibration stress evoked 13 hyperalgesia only when the rats were in the late diestrus phase of their cycle. The new data reported here describe the results of a second study on the same cohort of rats, but carried out the next day, when each animal had progressed to a different stage of her estrous cycle. The behavioral results reported here represent data from those animals in which we carried out a quantitative analysis of Fos expression in the PAG, i.e. n=5-6 for each group). In these rats sensitivity to pain was linked to estrous cycle stage, indicating that variability in nociceptor responsiveness within a female population is determined principally by the hormonal profile that characterizes the particular stage of the estrous cycle rather than reflecting trait differences between individuals. Moreover, habituation to the testing paradigm on a second exposure, as has been reported for male rats following repeated exposure to a mild anxiogenic stressor [START_REF] Weinberg | Repeated ferret odor exposure induces different temporal patterns of same-stressor habituation and novel-stressor sensitization in both hypothalamic-pituitary-adrenal axis activity and forebrain c-fos expression in the rat[END_REF][START_REF] Girotti | Habituation to repeated restraint stress is associated with lack of stress-induced c-fos expression in primary sensory processing areas of the rat brain[END_REF], did not seem to be present in females.

Baseline TFLs measured during the 20 min control period before onset of the vibration stress were similar in rats at all stages of the estrous cycle and in the animals undergoing different progesterone treatment regimens (Table 1). During exposure to vibration stress, all the animals showed signs of anxiety such as struggling, vocalization, urination, defecation and lashing of the tail. On cessation of the vibration the animals rapidly became quiet and tail flick testing was resumed. During the immediate post stress period, rats in late diestrus displayed a significant hyperalgesia. The mean TFL during the first 10 min post vibration stress decreased by 16.8±1.8% compared to the mean of the TFLs recorded in the 15 min control period immediately prior to the onset of vibration (P<0.01).

In contrast there was no significant change in TFL in rats at other stages of the cycle 14 (Figure 3). TFLs did not change significantly over the course of experiments in any of the control animals that were not subjected to vibration stress (Figure 3).

Fos-like immunoreactivity in the PAG

In all animals we determined the level of functional activation within the PAG resulting from exposure to the nociceptive testing paradigm as reflected by the presence of Fos-like immunoreactive nuclei in brains harvested 2 h after the delivery of the vibration stress, or equivalent time period in non-stressed control animals. For detailed quantitative analysis of Fos-like immunoreactivity in the PAG, we selected animals (5-6 rats per group) in which immunostaining was judged to be optimal. Fos immunoreactivity in the PAG was visible as rounded immunostained profiles that were present throughout the entire rostrocaudal extent of the nucleus in all the groups of animals used in this study. No Fos-positive profiles were present in sections in which the primary or secondary antibody had been omitted from the staining protocol.

Fos expression in the PAG in naïve and control rats

A few stained nuclei, which were evenly distributed throughout all sectors of the PAG and at all four rostro-caudal levels sampled, were present in the naïve animals. However, the density of immunostained profiles did not differ significantly between rats at different cycle stages (Figure 4). In control animals that underwent nociceptive testing but were not 15 subjected to vibration stress, higher numbers of Fos-positive nuclei were present throughout the PAG at all cycle stages (Figure 4). However, compared to naïve rats, the density of labelled cells was significantly higher only in rats in proestrus (Figure 4).

Fos expression in the PAG following exposure to vibration stress

When the rats had been exposed to short-lasting vibration stress, the density of labelled cells present in the PAG was increased in rats in late diestrus and to a lesser extent in proestrus (Figure 4). When the data from rats in late diestrus was analysed in more detail, the elevation in Fos expression was found to be confined to the caudal half of the PAG (levels III and IV, Figure 5) where the density of Fos-immunoreactive nuclei in the dorsolateral, lateral and ventrolateral columns of the PAG increased by more than 500% compared to control animals (Figure 5). In contrast, a similar analysis applied to the increase in Fos expression in rats in proestrus revealed that the number of labelled cells increased by less than 50% compared to the non-stressed control group. Moreover unlike rats in late diestrus, the upregulation of Fos-like protein was restricted to the ventrolateral column of the PAG (Figure 6).

Effect of progesterone withdrawal

The baseline TFLs of rats that had been chronically treated with progesterone and were either undergoing withdrawal (PWD, n=6) or maintained on high progesterone (HP, n=6) were similar and did not differ significantly from the spontaneously cycling animals 16 (Table 1). Following exposure to the vibration, stressed rats undergoing PWD (n=6) displayed a post stress hyperalgesia (16.9±1.9% decrease in TFL), which was similar to that seen in spontaneously cycling rats in late diestrus (Figure 7). In contrast, stress did not induce any change in TFL in the HP group. Control animals from the HP and PWD groups that went through the behavioral testing protocol but were not subjected to the vibration stress did not show any change in TFL (Figure 7). Note that in order to match as far as possible the hormonal profile of progesterone treated rats with those of rats in late diestrus, progesterone treated rats were sacrificed 24-26 h after withdrawal or equivalent time for the group maintained on high progesterone. Thus only one behavioral testing experiment was carried out in this cohort.

Fos-positive nuclei were present throughout the PAG in control i.e. non-stressed rats in the HP and PWD groups. The density of immunostained profiles was similar to that seen in spontaneously cycling control rats at different stages of the estrous cycle. Following exposure to vibration stress rats undergoing PWD showed a more than four-fold increase in the mean number of Fos-positive nuclei in the PAG compared to unstressed controls (Figure 8). No change in Fos expression was apparent in the group maintained on high progesterone. When the location of the labelled cells was examined more closely, the increase in density of Fos-immunoreactive nuclei in the PWD group was found to be localized largely in the lateral, and ventrolateral columns of the caudal PAG although a significant increase in labelled cells was also seen in the dorsal column in the rostral half of the PAG (Figure 9). 17

DISCUSSION

In the present study in female rats, brief exposure to a mild anxiogenic stressor produced a decrease in nociceptive threshold in rats in the late diestrus phase of their estrous cycle, but not at other stages. This finding contrasts with the results of many previous studies more intense stressors, which evoked hypoalgesia, and concurs with the consensus view that whilst fear evoked by intense stress inhibits pain, anxiety evoked by milder stimuli enhances it [START_REF] Rhudy | Fear and anxiety: divergent effects on human pain thresholds[END_REF][START_REF] Ford | Clinical correlates of stress-induced analgesia: evidence from pharmacological studies[END_REF] for discussions on this point). In our study exposure to mild stress also evoked hyperalgesia in rats undergoing withdrawal from long term dosing with exogenous progesterone, suggesting that the fall in levels of progesterone that occurs naturally during late diestrus [START_REF] Butcher | Altered secretion of gonadotropins and steroids resulting from delayed ovulation in the rat[END_REF] may be the underlying trigger. The appearance of stress-induced hyperalgesia correlated with a 4-5 fold increase in the number of Fos-immunoreactive nuclei in the caudal half of the PAG in rats in late diestrus as well as in the PWD group. During late diestrus the PAG circuitry becomes intrinsically more excitable due to a reduction in ongoing GABAergic tone, which is linked to upregulation of α4, β1 and δ GABA A receptor subunits on GABAergic neurons [START_REF] Brack | Neuronal excitability in the periaqueductal gray matter during the estrous cycle in female Wistar rats[END_REF][START_REF] Lovick | Changes in GABA(A) receptor subunit expression in the midbrain during the oestrous cycle in Wistar rats[END_REF]Griffiths and Lovick 2005a). These neuronal changes are linked with the fall in brain levels of progesterone and hence allopregnanolone, the major neuroactive metabolite of progesterone [START_REF] Butcher | Altered secretion of gonadotropins and steroids resulting from delayed ovulation in the rat[END_REF][START_REF] Purdy | Radioimmunoassay of 3 alpha-hydroxy-5 alpha-pregnan-20-one in rat and human plasma[END_REF]. Withdrawal from dosing with exogenous progesterone also evoked upregulation of α4, β1 and δ GABA A receptor subunits [START_REF] Griffiths | Withdrawal from progesterone increases expression of alpha4, beta1, and delta GABA(A) receptor subunits in neurons in the periaqueductal gray matter in female Wistar rats[END_REF] suggesting that the natural fall in progesterone levels that occurs during late diestrus is the trigger for the functional changes within the circuitry in the PAG. It is 18 worth pointing out that in order to match as far as possible, the hormonal profile of progesterone treated rats with those of rats in late diestrus, only one behavioral testing experiment was possible in the former cohort, after 24 h of progesterone withdrawal. We compared the data from this group to data from spontaneously cycling rats after their second behavioral test. However, since we previously found that the behavioral response of individual spontaneously cycling rats was linked to their estrus cycle stage and not whether they were tested for the first or second time [START_REF] Devall | Hyperalgesia in the setting of anxiety: Sex differences and effects of the oestrous cycle in Wistar rats[END_REF], we considered this comparison to be valid.

Interestingly, although Fos expression was increased in late diestrus and during progesterone withdrawal, there were subtle differences in the distribution of labelled cells within the PAG in the two conditions. In rats in late diestrus, the increase in expression of Fos protein was confined to dorsolateral, lateral and ventrolateral columns in the caudal half of the PAG whilst in rats undergoing progesterone withdrawal, there was an increase in labelled cell numbers in the rostral (dorsal, lateral and ventrolateral columns) as well as the caudal PAG (lateral and ventrolateral columns). In contrast, the dorsomedial PAG showed only moderate activation. It has been suggested that the dorsomedial PAG is a key neural structure for the selection of the most appropriate behavioral response in a specific dangerous context [START_REF] Kincheski | The dorsal periaqueductal gray modulates the increased fear-like behavior exhibited by experienced rats in the elevated plus maze[END_REF]. Since the brief, mild vibration stress used in the present study is unlikely to have been perceived by the rat as a severe anxiogenic challenge, the minimal activation of the dorsomedial PAG seen in the present study is not unexpected.

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The reason for the differences in expression of Fos during late diestrus and during PWD, when the behavioral manifestation of the response to stress in the two groups of rats i.e. increased pain sensitivity was indistinguishable, is not clear. Previous studies have shown that physical stressors that are perceived as threatening and evoke confrontational (active) coping strategies are associated with increased Fos expression in rostral, dorsal half of the PAG whilst passive coping leads to preferential activation in the caudal ventrolateral PAG [START_REF] Keay | Parallel circuits mediating distinct emotional coping reactions to different types of stress[END_REF]. The spontaneously cycling rats in this study had already been subjected to the vibration stress and nociceptive testing protocol on the previous day (see [START_REF] Devall | Hyperalgesia in the setting of anxiety: Sex differences and effects of the oestrous cycle in Wistar rats[END_REF], whereas rats undergoing progesterone withdrawal were experiencing the stress for the first time. When rats were in late diestrus the behavioral outcome of the exposure to stress was the same i.e. hyperalgesia, regardless of whether the rats were experiencing the stress for the first and second time. However, there may have been subtle differences in the way the stress was perceived e.g. potentially escapable (first exposure) or, as a result of experience, inescapable (second exposure). Since rats undergoing progesterone withdrawal were exposed to stress only once, their perception of the stress may have differed from the rats in late diestrus, leading to a different pattern of activation within the PAG. Activation of structures in the rostral PAG in the PWD group may therefore be associated with an active coping strategy on first exposure to the vibration stress.

The PAG is a source of descending facilitatory and inhibitory pathways to the spinal cord that exert parallel but opposing influences on transmission of noxious input through the dorsal horn [START_REF] Gebhart | Descending modulation of pain[END_REF]. Descending projections from the PAG relay on ON and OFF 20 cells in the rostral ventromedial medulla that respectively facilitate and inhibit nociceptive transmission via their projections to the spinal dorsal horn [START_REF] Morgan | Periaqueductal gray neurons project to spinally projecting GABAergic neurons in the rostral ventromedial medulla[END_REF]. Both the inhibitory and facilitatory systems are thought to be tonically active, although it has been suggested that under normal i.e. non-stressed conditions, a small net facilitatory influence predominates, at least in males [START_REF] Bee | Descending facilitation from the brainstem determines behavioural and neuronal hypersensitivity following nerve injury and efficacy of pregabalin[END_REF]. During late diestrus, the level of ongoing GABAergic inhibition within the PAG decreases, rendering the circuitry intrinsically more excitable [START_REF] Brack | Neuronal excitability in the periaqueductal gray matter during the estrous cycle in female Wistar rats[END_REF]. The presence of a post-stress hyperalgesia in rats in late diestrus suggests that exposure to the vibration stress may have further tipped the balance between the levels of activity in these descending pathways in favor of facilitation. Such an effect could be produced by an increase in tonic descending facilitation and/or a decrease in tonic descending inhibition. Identification of the phenotype of the Fos-immunoreactive cell population that was upregulated in response to stress will however, be needed to resolve this issue.

Another possibility is that the increase in functional activation of the PAG in response to brief exposure to stress during late diestrus could reflect a stress-induced increase in afferent input to the PAG. During the initial assessment phase, determination of the emotional significance of a novel and hence inherently stressful stimulus is likely to occur in cortical and/or limbic regions, which then engage the PAG downstream to initiate and integrate the somatomotor and autonomic components of the animal's response. It is well established that mild psychogenic stress activates neurones in various cortical areas which project to the PAG [START_REF] Beitz | The organization of afferent projections to the midbrain periaqueductal gray of the rat[END_REF][START_REF] Ceccatelli | Expression of c-Fos immunoreactivity in transmitter-characterized neurons after stress[END_REF][START_REF] Cullinan | Pattern and time course of immediate early gene expression in rat brain following acute stress[END_REF][START_REF] Da | An electrophysiological characterization of the projection from the central nucleus of the amygdala to the periaqueductal gray of the rat: the role of opioid receptors[END_REF][START_REF] Figueiredo | Stress activation of cortex and hippocampus is modulated by sex and stage of estrus[END_REF][START_REF] Girotti | Habituation to repeated restraint stress is associated with lack of stress-induced c-fos expression in primary sensory processing areas of the rat brain[END_REF]Salchner and 21 Singewald, 2002;[START_REF] Gerrits | Increased stress vulnerability after a prefrontal cortex lesion in female rats[END_REF][START_REF] Rizvi | Connections between the central nucleus of the amygdala and the midbrain periaqueductal gray: topography and reciprocity[END_REF][START_REF] Rocher | Acute stress-induced changes in hippocampal/prefrontal circuits in rats: effects of antidepressants[END_REF][START_REF] Brown | Mild, short-term stress alters dendritic morphology in rat medial prefrontal cortex[END_REF][START_REF] Floyd | Orbitomedial prefrontal cortical projections to distinct longitudinal columns of the periaqueductal gray in the rat[END_REF][START_REF] Keay | Parallel circuits mediating distinct emotional coping reactions to different types of stress[END_REF]. In particular, activation of the orbitofrontal cortex (OFC) has been shown to induce biphasic changes in nociceptive threshold: hypoalgesia followed by a delayed hyperalgesia, and both effects could be blocked by inhibiting transmission through the vlPAG (Zhang et al, 1997). These findings suggest that there may be two functionally distinct pro-and antinociceptive descending pathways from the ventrolateral OFC to the PAG, which may be activated differentially according to the way the stimulus is perceived by the individual. In support of this idea studies in male rats have shown that whilst exposure to a non-noxious stress induced hyperalgesia [START_REF] Vidal | Hyperalgesia induced by non-noxious stress in the rat[END_REF][START_REF] Jørum | Analgesia or hyperalgesia following stress correlates with emotional behavior in rats[END_REF], a more severe stress induced an increase in pain threshold [START_REF] Rizzi | Endogenous nociceptin signaling and stress-induced analgesia[END_REF][START_REF] Vendruscolo | Strain and sex differences in the expression of nociceptive behavior and stress-induced analgesia in rats[END_REF]. The mild stressor used in the present study would therefore be expected to activate the pro-nociceptive pathways from the PAG. Interestingly, in females stress-induced expression of c-fos mRNA in cingulate, frontal and piriform cortices was estrous cycle dependent, being higher in diestrus compared to proestrus (Figureido et al, 2002). Thus enhanced frontal cortical input to the PAG, combined with an increase in intrinsic excitability of its circuitry, may have been responsible for the enhanced level of functional activation of the PAG by mild stress during the diestrus phase seen in the present study.

An unexpected finding in the present study was the increase in the density of Fos-labelled cells in the PAG seen in proestrus, particularly in the control (non-stressed) group.

Baseline nociceptive thresholds were similar at all cycle stages suggesting that in the absence of stress, there was no change in nociceptive sensitivity in different cycle stages.

22

In any case, it seems unlikely that the brief exposure to the noxious heat stimulus during tail flick testing (less than 60 s total duration over the 45 min experiment) would have been be sufficient to induce a detectable increase in expression of Fos protein.

All the rats used in our study, including those in proestrus, had been habituated to being confined in the tube over 3 sessions prior to the experiment and showed no observable behavioral signs of distress. In the present study the density of Fos-positive nuclei in control rats in estrus, early diestrus or late diestrus that had been habituated to being confined in the restraint tube but not vibrated, was not significantly different to naïve animals. In contrast, rats in proestrus showed a significantly raised expression of Fos protein. There were no overt signs that rats in proestrus were reacting differently to rats in other stages of the cycle. However, we cannot exclude the possibility that rats in proestrus perceived being confined in a different way to animals in other cycle stages and that this led to functional activation within the PAG. Plasma and hence brain levels of estrogen are high in proestrus [START_REF] Butcher | Altered secretion of gonadotropins and steroids resulting from delayed ovulation in the rat[END_REF]. The presence of estrogen has been shown to amplify stress-induced responses in the prefrontal cortex [START_REF] Shansky | Estrogen mediates sex differences in stress-induced prefrontal cortex dysfunction[END_REF]. Since the PAG is driven by afferent input from the PFC [START_REF] Floyd | Orbitomedial prefrontal cortical projections to distinct longitudinal columns of the periaqueductal gray in the rat[END_REF], exposure of rats to the restraining tube in proestrus, when estrogen levels are high may, even with prior habituation to the apparatus, lead to enhanced activation of the PAG compared to other cycle stages.

During proestrus there is a very brief surge in progesterone production [START_REF] Butcher | Altered secretion of gonadotropins and steroids resulting from delayed ovulation in the rat[END_REF]. However the kinetics of this effect are too fast to evoke detectable changes in 23 GABA A receptor subunit expression (See [START_REF] Brack | Neuronal excitability in the periaqueductal gray matter during the estrous cycle in female Wistar rats[END_REF] for discussion of this point). Nevertheless, exposure of rats to vibration stress during proestrus induced a significant increase in the level of Fos expression in the PAG, albeit some 10-fold lower and much less widespread than the upregulation of the gene expression seen during late diestrus. The trigger for the relatively weak upregulation of c-fos in proestrus remains unclear. In any case, in terms of functional outcome it does not appear to have been sufficient to produce a detectable behavioral output related to changes in pain sensitivity.

The present study, which was confined to female rats, highlights how neural responsiveness to salient stimuli can be influenced according to the hormonal milieu in which they are received. It would be of interest to compare these findings with data from male rats that have undergone the same stress protocol, as this may shed further light on the functional significance of the patterns of Fos expression that were observed at different stages of the estrus cycle in females.

In summary, we have shown that brief exposure of female rats to a non-noxious anxiogenic stress in late diestrus induced hyperalgesia that was associated with functional activation of neurones in the caudal half of the PAG. We propose that falling concentrations of progesterone in the brain during late diestrus trigger changes in the functional excitability of the descending pro-nociceptive control pathways in the PAG, which renders them more sensitive to activation by mild anxiogenic stress during late diestrus compared to other stages of the estrous cycle. These effects may be directly

  Figure 1.

Figure 2 .

 2 Figure 2. Schematic representation of method used to quantify Fos-like immunoreactive

Figure 3 .

 3 Figure 3. Graph shows time course of the effect of 5 min of vibration stress (grey bar) on

Figure 4 .

 4 Figure 4. Mean density of Fos-like immunoreactive nuclei in the whole PAG of

Figure 5 .Figure 6 .

 56 Figure 5. Effect of exposure to 5min vibration stress on the density of Fos-

Figure 7 .

 7 Figure 7. Graph shows time course of the effect of 5 min of vibration stress (grey bar) on

Figure 8 .Figure 9 .

 89 Figure 8. Mean density of Fos-like immunoreactive nuclei in the whole PAG of rats in the

ACKNOWLEDGMENTS

The work was supported by MRC project grant G0700379-81940 awarded to TAL. We wish to acknowledge the invaluable technical support provided by Mr Mike Summerfield.

DISCLOSURE/CONFLICT OF INTEREST

The authors declare that, except for income received from my primary employer, no financial support or compensation has been received from any individual or corporate entity over the past three years for research or professional service and there are no personal financial holdings that could be perceived as constituting a potential conflict of interest.


Michele Bellesi

Fiorenzo Conti
email: f.conti@univpm.it

The mGluR2/3 agonist LY379268 blocks the effects of GLT-1 up-regulation on prepulse inhibition of the startle reflex in adult rats

Keywords:

come

The mGluR2/3 agonist LY379268 blocks the effects of GLT-1 up-regulation on prepulse inhibition of the startle reflex in adult rats

INTRODUCTION

GLT-1 (EAAT2; SLC1A2), the main glutamate transporter in mammalian forebrain, is responsible for the largest proportion of total glutamate transport [START_REF] Rothstein | Knockout of glutamate transporters reveals a major role for astroglial transport in excitotoxicity and clearance of glutamate[END_REF][START_REF] Tanaka | Epilepsy and exacerbation of brain injury in mice lacking the glutamate transporter GLT-1[END_REF][START_REF] Conti | Shaping excitation at glutamatergic synapses[END_REF][START_REF] Danbolt | Glutamate uptake[END_REF]. Owing to its perisynaptic localization [START_REF] Rusakov | Perisynaptic asymmetry of glia: new insights into glutamate signalling[END_REF]Melone, et al 2009a) GLT-1 regulates glutamate spillover, thereby modulating the activation of glutamate receptors [START_REF] Scanziani | Use-dependent increases in glutamate concentration activate presynaptic metabotropic glutamate receptors[END_REF][START_REF] Min | Activation of AMPA, kainate, and metabotropic receptors at hippocampal mossy fiber synapses: role of glutamate diffusion[END_REF][START_REF] Mitchell | Glutamate spillover suppresses inhibition by activating presynaptic mGluRs[END_REF][START_REF] Tzingounis | Glutamate transporters: confining runaway excitation by shaping synaptic transmission[END_REF].

For its fundamental role in regulating Glu levels, GLT-1 is involved in the pathophysiology of several neuropsychiatric diseases [START_REF] Beart | Transporters for L-glutamate: an update on their molecular pharmacology and pathological involvement[END_REF][START_REF] Lauriat | EAAT2 regulation and splicing: relevance to psychiatric and neurological disorders[END_REF][START_REF] Sheldon | The role of glutamate transporters in neurodegenerative diseases and potential opportunities for intervention[END_REF]. Some evidence suggests a role for GLT-1 in the pathophysiology of schizophrenia: 1) a susceptibility locus for schizophrenia is probably located within or near the GLT-1 gene [START_REF] Deng | Association study of polymorphisms in the excitatory amino acid transporter 2 gene (SLC1A2) with schizophrenia[END_REF], and this gene is reportedly dysregulated in schizophrenia patients [START_REF] Shao | Shared gene expression alterations in schizophrenia and bipolar disorder[END_REF]; 2) GLT-1 immunoreactivity is increased in the thalamus, striatum, and prefrontal cortex of schizophrenia patients (Matute, et al 2005;[START_REF] Mccullumsmith | Striatal excitatory amino acid transporter transcript expression in schizophrenia, bipolar disorder, and major depressive disorder[END_REF][START_REF] Smith | Expression of excitatory amino acid transporter transcripts in the thalamus of subjects with schizophrenia[END_REF]; 3) the antipsychotic clozapine down-regulates specifically GLT-1 expression and function in in vivo and in vitro [START_REF] Melone | The expression of glutamate transporter GLT-1 in the rat cerebral cortex is down-regulated by the antipsychotic drug clozapine[END_REF][START_REF] Melone | Clozapine-induced reduction of glutamate transport in the frontal cortex is not mediated by GLAST and EAAC1[END_REF]Vallejo-Illarramendi, et al 2005); 4) the psychotomimetics PCP up-regulates specifically GLT-1 expression and function [START_REF] Fattorini | GLT-1 expression and Glu uptake in rat cerebral cortex are increased by phencyclidine[END_REF]; and 5) pharmacologically induced GLT-1 up-regulation is associated to an impairment of the prepulse inhibition (PPI) of the startle reflex, a neurophysiological parameter altered in schizophrenia patients and in animal models of schizophrenia, in a dihydrokainate (DHK)reversible manner and worsens PCP-induced PPI alterations (Bellesi, et al 2009;Melone, et al 2009b). These data support the hypothesis that, in concert with NMDARs and non-NMDARs [START_REF] Lewis | Pathophysiologically based treatment interventions in schizophrenia[END_REF][START_REF] Lewis | Cognitive dysfunction in schizophrenia: convergence of gamma-aminobutyric acid and glutamate alterations[END_REF], GLT-1 plays a role in the dysfunction of glutamatergic transmission that contributes to the disturbance of information processing occurring in schizophrenia [START_REF] Venables | The effect of auditory and visual stimulation on the skin potential response of schizophrenics[END_REF][START_REF] Mcghie | Disorders of attention and perception in early schizophrenia[END_REF][START_REF] Andreasen | Linking mind and brain in the study of mental illnesses: a project for a scientific psychopathology[END_REF][START_REF] Andreasen | Schizophrenia: the fundamental questions[END_REF][START_REF] Tononi | Schizophrenia and the mechanisms of conscious integration[END_REF].

Targeting metabotropic glutamate receptors (mGluRs) is a practical strategy for regulating abnormal glutamatergic transmission [START_REF] Moghaddam | Targeting metabotropic glutamate receptors for treatment of the cognitive symptoms of schizophrenia[END_REF]. The selective mGluR2/3 agonist LY379268 attenuates behavioural abnormalities in animal models of schizophrenia, and its analog 404039 is effective in a schizophrenia phase 2 clinical trial [START_REF] Cartmell | The metabotropic glutamate 2/3 receptor agonists LY354740 and LY379268 selectively attenuate phencyclidine versus d-amphetamine motor behaviors in rats[END_REF][START_REF] Cartmell | Attenuation of specific PCP-evoked behaviors by the potent mGlu2/3 receptor agonist, LY379268 and comparison with the atypical antipsychotic, clozapine[END_REF][START_REF] Clark | Effects of the mGlu2/3 receptor agonist LY379268 on motor activity in phencyclidine-sensitized rats[END_REF][START_REF] Imre | Effects of the mGluR2/3 agonist LY379268 on ketamine-evoked behaviours and neurochemical changes in the dentate gyrus of the rat[END_REF][START_REF] Imre | The preclinical properties of a novel group II metabotropic glutamate receptor agonist LY379268[END_REF][START_REF] Patil | Activation of mGlu2/3 receptors as a new approach to treat schizophrenia: a randomized Phase 2 clinical trial[END_REF][START_REF] Harrison | Metabotropic glutamate receptor agonists for schizophrenia[END_REF]. Recently, we have shown that ceftriaxone-induced GLT-1 up-regulation impairs hippocampal long-term depression (LTD) in a DHK-reversible manner by limiting activation of mGluR2/3, thus indicating that a reduced function of mGluR2/3 mediates the effects of GLT-1 up-regulation [START_REF] Omrani | Up-regulation of GLT-1 severely impairs LTD at mossy fibres-CA3 synapses[END_REF].

Here we tested the hypothesis that administration of the selective mGluR2/3 agonist LY379268 blocks the impairment of PPI of the startle reflex associated to GLT-1 up-regulation.

MATERIAL and METHODS

Animals

Adult Sprague-Dawley male rats (initial weight 280-300 g; Charles River, Milano, Italy) were used. Their care and handling were approved by the Ethical Committee for Animal Research of Università Politecnica delle Marche. Experiments were carried out in accordance with the European Communities Council Directive (86/609/EEC), and all efforts were made to minimize animal suffering and to reduce the number of animals used. Animals were kept under a 12 h dark/light cycle and permitted food and water ad libitum. Rats were caged in pairs and handled within 24 h of arrival and then over at least 1 week before testing; to minimize stress during behavioural testing, rats were briefly handled prior to any procedure [START_REF] Geyer | Measurement of startle response, prepulse inhibition, and habituation[END_REF].

Drugs and treatments

Ceftriaxone sodium (CEF, Rocefin, Roche, Milano, Italy) was used to up-regulate GLT-1 [START_REF] Rothstein | Betalactam antibiotics offer neuroprotection by increasing glutamate transporter expression[END_REF]Bellesi, et al 2009;[START_REF] Omrani | Up-regulation of GLT-1 severely impairs LTD at mossy fibres-CA3 synapses[END_REF]. CEF was dissolved in saline (0,9% NaCl) and administered intraperitoneally (i.p.) at a dose of 200 mg/kg for 8 consecutive days (Bellesi, et al 2009). LY379268 (2453, Tocris Bioscience, Bristol, UK) was dissolved in saline and administered i.p. in a single dose of 1 mg/kg at day 10, i.e., 2 days after CEF withdrawal. Control rats, matched for age, sex and weight, received saline i.p. All injections were administered between 10:00 and 11:00 AM.

Prepulse inhibition of the startle reflex

Experimental design. Four experimental groups (n = 11 for each group) were studied: the first group received saline for 8 days, followed 2 days after by a single injection of saline (saline group); the second group received saline for 8 days, followed 2 days after by a single injection of LY379268 (saline + LY group); the third group received CEF for 8 days, and an injection of saline 2 days after CEF withdrawal (CEF group); and the fourth group was administered CEF for 8 days, followed by a single injection of LY379268 2 days after CEF withdrawal (CEF + LY group). All acoustic startle experiments were performed in an acoustic startle chamber (SR-LAB; San Diego Instruments, San Diego, CA, USA). Two days before treatment inception, a brief startle session was performed to obtain experimental and control groups matched for startle reactivity (matching session; [START_REF] Geyer | Measurement of startle response, prepulse inhibition, and habituation[END_REF][START_REF] Martinez | Effects of sustained phencyclidine exposure on sensorimotor gating of startle in rats[END_REF]. In all experimental groups, PPI test was performed at day 10 (2 days after CEF withdrawal, to allow complete clearance of the antibiotic [START_REF] Rebuelto | Daily variations in ceftriaxone pharmacokinetics in rats[END_REF]), 30 min after the injection of saline or LY379268.

Testing procedures. For matching startle sessions, rats were placed in a startle chamber and exposed to 5 min of 70 dB background noise followed by 11 pulse only trials (40 ms noise burst at 120 dB); intertrial interval was 15 s [START_REF] Geyer | Measurement of startle response, prepulse inhibition, and habituation[END_REF].

PPI session lasted approximately 14 min, and consisted of 5 min of acclimation (70 dB background) followed by 4 trial types: pulse only trials (40 ms noise burst at 120 dB) and 3 prepulse trials (20 ms noise bursts 3, 6 or 12 dB above background followed 100 ms later by a 40 ms noise burst at 120 dB). Sessions included 3 blocks: block 1 (6 pulse only trials); block 2 (10 pulse only trials and 5 prepulse trials for each prepulse tone presented in pseudorandom order); and block 3 (5 pulse only trials). Intertrial interval was 15 s.

Data collection and analysis. Experimental groups were matched for startle reactivity, which was calculated by averaging pulse alone startle amplitude (2-11 trials) of each animal during matching session [START_REF] Geyer | Measurement of startle response, prepulse inhibition, and habituation[END_REF].

In all animals, startle reflex magnitude was measured by averaging the startle amplitude of pulse only trials of block 2 (10 trials). The degree of PPI (in %) was calculated according to the formula: PPI = 100 x [pulse only units -(prepulse + pulse units)]/(pulse only units) [START_REF] Geyer | Measurement of startle response, prepulse inhibition, and habituation[END_REF], where pulse only units were startle magnitudes recorded from block 2.

Habituation was assessed as the decrement in response magnitude across blocks (block 1, trials 2-6, and block 3, trials 32-36).

Western blotting

After completion of PPI studies (i.e., at day 10), 4 control rats and 4 CEF-treated rats were anaesthetized with chloral hydrate (300 mg/kg i.p.) and decapitated; the brains were dissected and frontal cortex, hippocampus, and striatum were removed. Homogenization and crude synaptic plasma membranes preparation were carried out as described [START_REF] Danbolt | Purification and reconstitution of the sodium-and potassium-coupled glutamate transport glycoprotein from rat brain[END_REF]. Bio-Rad Protein Assay (Bio-Rad Laboratories, GmbH, München, Germany) and a Beckman DU 530 spectrophotometer (Beckman Coulter, Fullerton, CA, USA) were used to determine the total amount of protein in each homogenate (3-4 measurements/homogenate). A standard curve with 2, 4, 6, 8 and 10 µg of bovine serum albumine (BSA; A4503, Sigma Chemicals, St. Louis, MO, USA) was drawn for each dosing run. Since housekeeping proteins (e.g., α-actin and β-tubulins) are sensitive to experimental treatments (particularly pharmacologic ones) and to diverse physiological conditions and have therefore some limitations as internal standard [START_REF] Ferguson | Housekeeping proteins: a preliminary study illustrating some limitations as useful references in protein expression studies[END_REF], 3-6 measurements were made for each brain region of each animal; to minimize procedural variables, homogenates from treated and control animals were loaded onto the same gel [START_REF] Bragina | GLT-1 down-regulation induced by clozapine in rat frontal cortex is associated with synaptophysin up-regulation[END_REF].

For semiquantitative analysis, we drew standard curves of increasing concentration of total protein from controls (n=4) to define a linear range for immunoblot densitometric analysis [START_REF] Bragina | GLT-1 down-regulation induced by clozapine in rat frontal cortex is associated with synaptophysin up-regulation[END_REF]. Nitrocellulose filters were probed with mGluR2/3 antibodies (AB1553 Millipore, Billerica, MA, USA; 1:200; 20 µg of total protein; [START_REF] Testa | Immunohistochemical localization of metabotropic glutamate receptors mGluR1a and mGluR2/3 in the rat basal ganglia[END_REF]. After exposure to secondary antibodies (Vector; Burlingame, CA, USA), bands were visualized by BioRad Chemidoc and Quantity One software using the SuperSignal West Pico (Rockford, IL, USA) chemiluminescent substrate (Bragina, et al 2006 for details).

Immunocytochemistry

After completion of PPI studies (day 10), 4 control rats and 4 CEF-treated rats were anaesthetized with chloral hydrate (300 mg/kg i.p.) and perfused transcardially with a flush (~1 min) of saline followed by 4% paraformaldehyde in phosphate buffer (PB). Brains were removed, post-fixed in the same fixative for 7 days [START_REF] Chen | The glutamate transporter GLT1a is expressed in excitatory axon terminals of mature hippocampal neurons[END_REF] and cut on a Vibratome in 50 µm parasagittal sections, which were collected in PB in serial groups of 5 and then used for immunocytochemistry [START_REF] Minelli | The glial glutamate transporter GLT-1 is localized both in the vicinity of and at distance from axon terminals in the rat cerebral cortex[END_REF][START_REF] Chen | The glutamate transporter GLT1a is expressed in excitatory axon terminals of mature hippocampal neurons[END_REF]Melone, et al 2005). Sections were incubated in a solution containing a mixture of GLT-1 (1:1000; Millipore; AB1783; [START_REF] Bragina | GLT-1 down-regulation induced by clozapine in rat frontal cortex is associated with synaptophysin up-regulation[END_REF] and mGlurR-2/3 (1:50; Millipore; AB1553; Testa, et al 1998) primary antibodies. Sections were then exposed to a solution containing a mixture of affinity-purified fluorescein isothiocyanate (FITC, 1:250; Vector; FI1000/J0114)-or tetramethylrhodamine isothiocyanate (TRITC, 1:250; Molecular Probes, PoortGebouw, The Netherlands, T-2762/6691-1)-conjugated secondary antibodies. Sections were washed, mounted, air-dried and coverslipped using Vectashield mounting medium (H-1000; Vector), and finally examined with a Leica TCS SL confocal laser scanning microscope equipped with an argon (488 nm) and helium/neon (543 nm) laser. Control experiments with single-labelled sections and sections incubated with two primary antibodies and one secondary antibody or with one primary and two secondary antibodies revealed neither bleed-through nor cross-reactivity. For all immunocytochemical studies, sections from controls and CEF-treated animals were processed in parallel to minimize procedural variables.

Data collection and analysis. Fields from layers II-III of the frontal neocortex anterior to the bregma, from stratum lucidum of hippocampal CA1, and from striatum of control (n=4) and CEFtreated (n=4) animals were randomly selected for each fluorescent as 1024 x 1024 pixel images with a planapo x63 objective (numerical aperture 1.4) and pinhole 1.0 Airy unit. To improve signal/noise ratio, 8 frames of each images were averaged; microscopic fields were scanned with a pixel size of 80 nm. Quantitative analysis of puncta staining was performed in randomly selected 8

x 8 µm fields from each 1024 x 1024 pixel image (10 fields for each region for each animal). Image processing was performed on Photoshop CS2 (Adobe Systems, San Jose, CA, USA); optimal resolution of punctate staining was obtained by setting threshold values as described elsewhere (Bellesi, et al 2009). Threshold values did not differ across conditions (i.e., between control and treated groups). Following thresholding, images were transformed in binary images using Image J software (v.1.38, NIH, USA.) [START_REF] Bragina | GLT-1 down-regulation induced by clozapine in rat frontal cortex is associated with synaptophysin up-regulation[END_REF] and mean size and number of mGluR2/3 and GLT-1 puncta was calculated. To verify whether GLT-1 up-regulation occurred at sites where mGluR2/3 was expressed, the mean size of GLT-1 puncta contacting mGluR2/3 puncta (a contact was defined as the presence of fluorescence signals overlap) was estimated.

Statistical analysis

Startle reactivity was analyzed using one-way ANOVA. Startle reflex magnitudes were analysed with two-way ANOVA with CEF and LY379268 as between-subject factors. PPI scores were analyzed with three-way ANOVA with prepulse intensities as within-subject factor and CEF and LY379268 as between-subject factors. Habituation was analyzed with three-way ANOVA with block as within-subject factor and CEF and LY379268 as between-subject factors. In the presence of interaction, pair-wise comparisons were conducted with two-way ANOVA with CEF or LY379268 as a between-subject factor and prepulse intensity or block as a within-subject factor. For multiple comparisons, α was set at 0.01. Statistical significance for western blotting and immofluorescence studies was evaluated by two-sided t-test (α=0.05). Statistical analysis was performed using SPSS (v.13.0).

RESULTS

Effects of LY379268 on PPI impairment induced by CEF treatment

Mean values of startle reactivity in all experimental groups were 95.3 ± 10.5 for the Saline group; 83.8 ± 10.3 for the Saline + LY group; 94.2 ± 14.9 for the CEF group; and 80.9 ± 14.4 for the CEF + LY group. One-way ANOVA did not reveal any significant difference among the four groups (F[(3,40)=0.44; NS]).

Table 1 about here

Effects on startle magnitude. CEF treatment and a single LY379268 injection had no significant effect on startle magnitude (Table 1). Two-way ANOVA of startle magnitude with CEF and LY379268 as between-subject factors revealed neither significant effects of CEF (F[1,40]=0.02; NS) and LY379268 (F[1,40]=0.12; NS) nor CEF x LY379268 interaction (F[1, 40]=0.15; NS).

Figure 1 about here

Effects on prepulse inhibition. LY379268 blocked PPI impairment induced by CEF administration (Figure 1). Three-way ANOVA of PPI with CEF and LY379268 as between-subject factors and prepulse intensity as a within-subject factor showed a significant effect of CEF (F[1,40]=14.96; P<0.0001) and LY379268 (F[1,40]=8.07; P=0.007), a significant CEF x LY379268 interaction (F[1,40]=17.52; P<0.0001), and a significant effect of prepulse intensity (F[2,80]=95.3; P<0.0001). Prepulse intensity x CEF, prepulse intensity x LY379268, or prepulse intensity x CEF x LY379268 interactions were not significant. For defining CEF x LY379268 interaction, we evaluated pair-wise comparisons among the different treatment conditions. Comparison of group Saline and group CEF with two-way ANOVA, with CEF as a between-subject factor and prepulse intensity as a within subject factor, showed a significant effect of prepulse intensity (F[2,40]=41.83; Effects on habituation. CEF treatment and a single LY379268 injection had no effects on startle habituation to repeated acoustic pulses (Table 1). Three-way ANOVA of startle magnitudes, with CEF and LY379268 as between-subject factors and trial block as within-subject factor, revealed neither significant effect of CEF (F[1,40]=0.25; NS) and LY379268 (F[1,40]=0.01; NS), nor CEF x LY379268 interaction (F[1,40]=0.12; NS), but a significant effect of trial block (F[1,40]=57.06; P<0.0001). CEF x block, LY379268 x block, CEF x LY379268 x trial block interactions were not significant.

These studies showed that PPI alterations induced by CEF treatment were blocked by the selective mGluR2/3 agonist LY379268, indicating that reducing mGluR2/3 activation contributes to PPI impairment associated to CEF treatment.

Effects of CEF treatment on mGluR2/3 expression

To verify whether reduced activation of mGluR2/3 was associated to an alteration of its expression, we studied mGluR2/3 protein levels in forebrain regions regulating PPI (Swerdlow, et al 2001). In rats treated with CEF for 8 days, western blotting studies showed that mGluR2/3 expression was not significantly different from controls in frontal cortex, hippocampus and striatum, thus indicating that CEF treatment does not change the amount of mGluR2/3 expressed (Figure 2). In addition, to rule out the possibility that CEF altered the distribution of mGluR2/3, we performed immunocytochemical studies in the same forebrain regions, and found that in CEFtreated rats size and number of mGluR2/3 immunoreactive puncta were comparable to controls (Figure 3).

Figure 2 and 3 about here

These results showed that PPI impairment induced by CEF treatment was not associated to an altered expression of mGluR2/3.

Spatial relationship between GLT-1 up-regulation and mGluR2/3

CEF treatment up-regulates GLT-1 [START_REF] Rothstein | Betalactam antibiotics offer neuroprotection by increasing glutamate transporter expression[END_REF], and CEF-induced GLT-1 upregulation can be visualized immunocytochemically (Bellesi, et al 2009;[START_REF] Omrani | Up-regulation of GLT-1 severely impairs LTD at mossy fibres-CA3 synapses[END_REF]. Here, we measured mean size of GLT-1 puncta in CEF-treated animals, and showed that it was significantly increased in frontal cortex (by 49.9 ± 12.9%; P=0.03), hippocampus (by 46.2 ± 10.8%; P=0.03) and striatum (by 93 ± 26.1%; P=0.02) compared to controls.

Since both GLT-1 (Melone, et al 2009a) and mGluR2/3 [START_REF] Ferraguti | Metabotropic glutamate receptors[END_REF] are localized perisynaptically, we sought to define the anatomical relationship between GLT-1 upregulation and mGluR2/3 expression.

Figure 4 about here

In both controls and CEF-treated animals, confocal images showed a consistent degree of overlap between the two fluorescent signals, indicating a close spatial relationship between mGluR2/3 and GLT-1. To verify whether the increased expression of GLT-1 occurs also at the very sites expressing mGluR2/3, we estimated the mean size of GLT-1 puncta showing clear overlap with mGluR2/3 puncta (Figure 4), and showed that it was significantly increased in frontal cortex (by 61.8 ± 11.9%; P=0.009), hippocampus (41.4 ± 14.4%; P=0.04) and striatum (103.8 ± 27.2%; P=0.02) (Figure 4). These results indicated that GLT-1 up-regulation occurred in the vicinity of mGluR2/3 receptors, thus suggesting that GLT-1 up-regulation plays a role in determining the reduced activation of mGluR2/3 observed in PPI experiments.

DISCUSSION

The major result of the present study is that PPI alterations associated to GLT-1 upregulation are blocked by the selective mGluR2/3 agonist LY379268. PPI of the startle reflex is a model of sensorimotor gating that is tested in a similar fashion in humans and rats; for this reason it is widely used to study the neurobiology of schizophrenia and the effects of medications [START_REF] Geyer | Measurement of startle response, prepulse inhibition, and habituation[END_REF][START_REF] Dawson | On the clinical and cognitive meaning of impaired sensorimotor gating in schizophrenia[END_REF][START_REF] Swerdlow | Animal models of deficient sensorimotor gating: what we know, what we think we know, and what we hope to know soon[END_REF]Geyer, et al 2001[START_REF] Geyer | Mouse genetic models for prepulse inhibition: an early review[END_REF]. Most importantly, although impaired startle PPI is shared by several neuropsychiatric diseases, it is consistently observed in schizophrenia, where it is believed to be an index of the deficient sensorimotor gating underpinning sensory flooding and cognitive fragmentation [START_REF] Braff | Sensorimotor gating and schizophrenia. Human and animal model studies[END_REF][START_REF] Braff | Gating and habituation of the startle reflex in schizophrenic patients[END_REF]Braff, et al , 2001[START_REF] Braff | Prepulse inhibition and P50 suppression are both deficient but not correlated in schizophrenia patients[END_REF][START_REF] Swerdlow | Assessing the validity of an animal model of deficient sensorimotor gating in schizophrenic patients[END_REF][START_REF] Swerdlow | Startle gating deficits in a large cohort of patients with schizophrenia: relationship to medications, symptoms, neurocognition, and level of function[END_REF][START_REF] Minassian | The relationship between sensorimotor gating and clinical improvement in acutely ill schizophrenia patients[END_REF]. Rothstein and colleagues (2005) showed that CEF robustly stimulates GLT-1 expression, but not that of the other glutamate transporters, through increased GLT-1 gene transcription. This finding has been replicated in different laboratories (e.g., [START_REF] Chu | Pharmacological induction of ischemic tolerance by glutamate transporter-1 (EAAT2) upregulation[END_REF][START_REF] Ouyang | Selective dysfunction of hippocampal CA1 astrocytes contributes to delayed neuronal damage after transient forebrain ischemia[END_REF][START_REF] Lee | Mechanism of ceftriaxone induction of excitatory amino acid tranporter-2 expression and glutamate uptake in primary human astrocytes[END_REF], including ours (Bellesi, et al 2009;[START_REF] Omrani | Up-regulation of GLT-1 severely impairs LTD at mossy fibres-CA3 synapses[END_REF]. We also showed that CEF increases GLT-1a expression in forebrain regions that regulate PPI of the startle (Bellesi, et al 2009). Whether CEF alters expression of synaptically-active molecules at non-glutamatergic synapses is currently unknown, although to date no evidence for an effect of CEF on other neurotransmitter system(s) has been published. As far as the present results are concerned, it must be emphasized that, regardless of whether CEF acts on other transmitter systems or not, its effects on PPI are prevented by the GLT-1 antagonist DHK, indicating that GLT-1 up-regulation is the major mediator of the effects of CEF on PPI.

In the present paper, we showed that LY379268, a compound effective in animal models of schizophrenia [START_REF] Cartmell | The metabotropic glutamate 2/3 receptor agonists LY354740 and LY379268 selectively attenuate phencyclidine versus d-amphetamine motor behaviors in rats[END_REF][START_REF] Cartmell | Attenuation of specific PCP-evoked behaviors by the potent mGlu2/3 receptor agonist, LY379268 and comparison with the atypical antipsychotic, clozapine[END_REF][START_REF] Clark | Effects of the mGlu2/3 receptor agonist LY379268 on motor activity in phencyclidine-sensitized rats[END_REF][START_REF] Imre | Effects of the mGluR2/3 agonist LY379268 on ketamine-evoked behaviours and neurochemical changes in the dentate gyrus of the rat[END_REF][START_REF] Imre | The preclinical properties of a novel group II metabotropic glutamate receptor agonist LY379268[END_REF], blocked PPI impairment associated to GLT-1 up-regulation. Although the present findings do not allow predictions about clinical efficacy of LY379268, it is worth noting that the better assimilated analog LY404039 ameliorates positive and negative symptoms in schizophrenia patients [START_REF] Patil | Activation of mGlu2/3 receptors as a new approach to treat schizophrenia: a randomized Phase 2 clinical trial[END_REF][START_REF] Harrison | Metabotropic glutamate receptor agonists for schizophrenia[END_REF]. Some studies reported that LY379268 failed to block PPI alterations (but not other behavioural abnormalities) induced by administration of NMDA antagonists [START_REF] Cartmell | The metabotropic glutamate 2/3 receptor agonists LY354740 and LY379268 selectively attenuate phencyclidine versus d-amphetamine motor behaviors in rats[END_REF][START_REF] Cartmell | Attenuation of specific PCP-evoked behaviors by the potent mGlu2/3 receptor agonist, LY379268 and comparison with the atypical antipsychotic, clozapine[END_REF][START_REF] Clark | Effects of the mGlu2/3 receptor agonist LY379268 on motor activity in phencyclidine-sensitized rats[END_REF][START_REF] Imre | Effects of the mGluR2/3 agonist LY379268 on ketamine-evoked behaviours and neurochemical changes in the dentate gyrus of the rat[END_REF]. The different results may be explained by the different paradigms used to impair PPI; indeed, it is conceivable that the reduction of glutamatergic transmission induced by NMDA antagonism is more intense than that induced by up-regulation of GLT-1 and that the modulating effect of LY379268 on glutamatergic transmission is not sufficient to restore PPI induced by NMDA antagonists.

The ability of LY379268 to prevent PPI abnormalities associated to GLT-1 up-regulation suggests that the reduced activation of mGluR2/3 is a mechanism by which GLT-1 up-regulation impairs PPI. mGluR2/3 activation depends on the spread of glutamate from release sites to the perisynaptic zone, where these receptors are predominantly located [START_REF] Scanziani | Use-dependent increases in glutamate concentration activate presynaptic metabotropic glutamate receptors[END_REF][START_REF] Min | Activation of AMPA, kainate, and metabotropic receptors at hippocampal mossy fiber synapses: role of glutamate diffusion[END_REF][START_REF] Mitchell | Glutamate spillover suppresses inhibition by activating presynaptic mGluRs[END_REF][START_REF] Ferraguti | Metabotropic glutamate receptors[END_REF]; and GLT-1 controls extracellular levels of glutamate and it is mainly localized perisynaptically (Melone, et al 2009a). Thus, by modulating glutamate spillover, GLT-1 influences activation of mGluR2/3 [START_REF] Rusakov | Extrasynaptic glutamate diffusion in the hippocampus: ultrastructural constraints, uptake, and receptor activation[END_REF][START_REF] Barbour | An evaluation of synapse independence[END_REF][START_REF] Diamond | A broad view of glutamate spillover[END_REF][START_REF] Tzingounis | Glutamate transporters: confining runaway excitation by shaping synaptic transmission[END_REF][START_REF] Zheng | Receptor actions of synaptically released glutamate: the role of transporters on the scale from nanometers to microns[END_REF][START_REF] Omrani | Up-regulation of GLT-1 severely impairs LTD at mossy fibres-CA3 synapses[END_REF]. Our western blotting and immunocytochemical studies are consistent with this view. First, we demonstrated that both expression and distribution of mGluR2/3 in forebrain regions regulating PPI were unchanged in CEF-treated rats compared to controls. Second, we showed that GLT-1 up-regulation occurred also at the very sites where mGluR2/3 was expressed. To do this, we considered the overlap of fluorescence signals in double-labelled sections as a marker of nearness, a procedure validated by recent electron microscopy studies (Melone, et al 2009a). Up-regulation of GLT-1 at sites close to those of mGluR2/3 expression may thus play a crucial role in reducing the amount of glutamate available for mGluR2/3 activation.

GLT-1 up-regulation induced by CEF determines a strong, dihydrokainate sensitive, impairment of mGluR-dependent LTD [START_REF] Omrani | Up-regulation of GLT-1 severely impairs LTD at mossy fibres-CA3 synapses[END_REF]. The possibility that a widespread impairment of LTD can induce cognitive alterations, including PPI abnormalities, has been investigated in animal models of schizophrenia. Mice lacking the dopamine transporter display PPI deficits [START_REF] Ralph | Prepulse inhibition deficits and perseverative motor patterns in dopamine transporter knock-out mice: differential effects of D1 and D2 receptor antagonists[END_REF][START_REF] Barr | The selective serotonin-2A receptor antagonist M100907 reverses behavioral deficits in dopamine transporter knockout mice[END_REF][START_REF] Yamashita | Norepinephrine transporter blockade can normalize the prepulse inhibition deficits found in dopamine transporter knockout mice[END_REF], and exhibit an impairment of hippocampal LTD [START_REF] Morice | Parallel loss of hippocampal LTD and cognitive flexibility in a genetic model of hyperdopaminergia[END_REF]. On these bases, it has been suggested that LTD could play a key role in modulating cognitive flexibility as well as in other complex cognitive functions [START_REF] Morice | Parallel loss of hippocampal LTD and cognitive flexibility in a genetic model of hyperdopaminergia[END_REF][START_REF] Nicholls | Transgenic mice lacking NMDAR-dependent LTD exhibit deficits in behavioral flexibility[END_REF]. It is therefore possible that the alterations of LTD observed in animals with GLT-1 up-regulation [START_REF] Omrani | Up-regulation of GLT-1 severely impairs LTD at mossy fibres-CA3 synapses[END_REF] are responsible for the alterations of the PPI described here, and may conceivably contribute to the cognitive impairment observed in schizophrenia patients.

P<0. 0001 ]

 0001 and of CEF (F[1,20]=25.71; P<0.0001], but not of CEF x prepulse intensity interaction (F[2,40]=0.57; NS]. Comparison of group Saline and group Saline/LY with two-way ANOVA, with LY379268 as a between subject factor and prepulse intensity as a within subject factor, showed a significant effect of prepulse intensity (F[2,40]=80.47; P<0.0001] but not of LY379268 (F[1,20]=2.58; NS] or of LY379268 x prepulse intensity interaction (F[2,40]=0.15; NS]. Comparison of group CEF and group CEF/LY with two-way ANOVA, with LY379268 as between subject factor and prepulse intensity as within subject factor, showed a significant effect of prepulse intensity (F[2,40]=33.11; P<0.0001] and of LY379268 (F[1,20]=14.96; P=0.001] but not of LY379268 x prepulse intensity interaction (F[2,40]=1.03; NS]).

FIGURE LEGEND Figure 1 .

 LEGEND1 FIGURE LEGEND

Figure 2 .

 2 Figure 2. CEF treatment is not associated to alterations of mGluR-2/3 expression in

Figure 3 .

 3 Figure 3. CEF treatment does not affect the distribution of mGlur-2/3 in frontal cortex,

Figure 4 .

 4 Figure 4. GLT-1 up-regulation occurs also in the vicinity of sites of mGluR2/3 expression in

Table 1 .

 1 Startle reflex magnitude and habituation (mean SEM) of experimental groups.

	Group	Startle magnitude	Habituation
		second trial block	first trial block	last trial block
	Saline group (n=11)	102.3 17.7	134.5 21.8	68.2 15.1
	Saline+LY group (n=11)	92.6 25.8	145.2 27.7	73.5 12.9
	CEF group (n=11)	98.9 18.7	139.1 26.9	84.9 22.5
	CEF+LY group (n=11)	95.5 18.4	152.3 38.7	64.5 14.2

Funded by grants from MIUR and Università Politecnica delle Marche to F.C. We gratefully acknowledge A. Frontini's assistance with confocal microscopy.

DISCLOSURE/CONFLICT OF INTEREST

The authors declare that, except for income received from their primary employer, no financial support or compensation has been received from any individual or corporate entity over the past three years for research or professional service and there are no personal financial holdings that could be perceived as constituting a potential conflict of interest.