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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
J Neurosci. Author manuscript; available in PMC Oct 25, 2012.
Published in final edited form as:
PMCID: PMC3365570
NIHMSID: NIHMS373621

Abnormalities in Hippocampal Functioning with Persistent Pain

Abstract

Chronic pain patients exhibit increased anxiety, depression, and deficits in learning and memory. Yet how persistent pain affects the key brain area regulating these behaviors, the hippocampus, has remained minimally explored. In this study we investigated the impact of spared nerve injury (SNI) neuropathic pain in mice on hippocampal-dependent behavior and underlying cellular and molecular changes. In parallel, we measured the hippocampal volume of three groups of chronic pain patients. We found that SNI animals were unable to extinguish to contextual fear and showed increased anxiety-like behavior. Additionally, SNI mice in comparison to sham animals exhibited hippocampal 1) reduced extracellular signal-regulated kinase (ERK) expression and phosphorylation, 2) decreased neurogenesis and 3) altered short-term synaptic plasticity. In order to relate the observed hippocampal abnormalities with human chronic pain, we measured the volume of human hippocampus in chronic back pain (CBP), complex regional pain syndrome (CRPS), and osteoarthritis patients (OA). Compared to controls, CBP and CRPS, but not OA, had significantly less bilateral hippocampal volume. These results indicate that hippocampus-mediated behavior, synaptic plasticity and neurogenesis are abnormal in neuropathic rodents. The changes may be related to the reduction in hippocampal volume we see in chronic pain patients, and these abnormalities may underlie learning and emotional deficits commonly observed in such patients.

Introduction

Recent work in both human patients and laboratory animals has demonstrated that the presence of chronic neuropathic pain causes gross reorganization and functioning in both cortical and subcortical structures, including the medial prefrontal cortex (Baliki et al., 2006; Baliki et al., 2008; Metz et al., 2009), thalamus (Apkarian et al., 2004a), amygdala (Han and Neugebauer, 2005; Ji et al., 2010), and anterior cingulate cortex (ACC) (Li et al., 2010). These areas are associated with functions including learning, memory, fear and emotional responses and map on to cognitive and emotional problems suffered by chronic pain patients, such as elevated anxiety and depression (Gore et al., 2011), problems in emotional decision making (Apkarian et al., 2004b), working memory (Dick and Rashiq, 2007), and difficulty in classical conditioning tasks (Flor et al., 2002). However, the hippocampus, which is one of the crucial limbic areas involved in learning and memory with which many of the above brain areas interact, has been studied less systematically, especially in regards to the impact of chronic pain on its function.

Recent studies have independently shown hippocampal abnormalities in animal models of chronic pain including short-term (Ren et al., 2011) and recognition memory deficits (Kodama et al., 2011), abnormal cytokine expression (Al-Amin et al., 2011; del Rey et al., 2011) , deficits in long-term potentiation (LTP) (Kodama et al., 2007), and impaired enriched-environment neurogenesis (Terada et al., 2008). Additionally, a human patient study suggests reduced hippocampal volume in elderly individuals with chronic pain (Zimmerman et al., 2009). Here we offer a comprehensive within-subject approach to define the cognitive and emotional role of the hippocampus in chronic pain by specifically examining hippocampal dependent contextual fear conditioning and extinction, anxiety-like behavior, hippocampal expression of a protein kinase linked to contextual extinction, hippocampal neurogenesis, and short-term hippocampal synaptic plasticity through the use of spared nerve injury (SNI) mice. Additionally we assess human hippocampal volume in three clinical chronic pain conditions.

The hippocampus is critically involved in anxiety, depression, learning and memory (Barkus et al., 2010) and is essential in contextual conditioning and extinction (Phillips and LeDoux, 1992). We hypothesize that chronic pain can be redefined in terms of context conditioning and extinction, and viewed as a state of continual learning through which aversive emotional associations are constantly made with otherwise incidental events due to the persistent presence of a patient’s pain. Moreover, due to the constant presence of the pain, such chronic pain patients may become unable to extinguish to the conditioned event since at each re-exposure pain may still be present (Apkarian, 2008; Apkarian et al., 2009; Apkarian et al., 2011). Within this context, the hippocampus, due to its specific role in contextual learning, may become dysregulated. In fact, our results indicate a disruption of hippocampal-dependent contextual extinction learning accompanied by a cascade of related hippocampal abnormalities. Relatedly, we also observed a decrease in hippocampal volume in human patients with chronic pain, which may be associated with the behavioral and cellular events we observe in mice.

Methods

Animals

Experiments 1 and 2

C57BL/6 male mice were obtained at 9 weeks of age from Harlan (Experiment 1). DCX-EGFP (doublecortin-enhanced green fluorescence protein) transgenic mice were used for Experiment 2.The mice were housed individually with a 12/12 dark light cycle (7 am–7 pm) with food and water available ad libitum. All studies were approved by the Animal Care and Use Committee of Northwestern University. The mice were operated on at 9 weeks of age and tactile thresholds were measured before surgical manipulation and again on days 3, 7, 9 and 14 post-surgery to ensure surgical success (except in Fear Conditioning group where measurements were taken at day 27 instead of 14 to not interfere with the experiment).

Experiment 3

C57BL/6 male mice were obtained from Harlan and operated on at p21. Mice were housed with littermates under the same conditions as above. Tactile thresholds were measured before surgical manipulation and again on day 7 post-surgery before sacrificing the animal.

Neuropathic pain model: Spared Nerve Injury

The spared nerve injury model (SNI) has been previously described in detail (Decosterd and Woolf, 2000). Mice were anesthetized with isoflurane 1.5-2% and a mixture of 30% N2O and 70% O2. One hind-leg sciatic nerve (varied over different experiments) was exposed at the level of the trifurcation into the sural, tibial, and common peroneal nerves. The tibial and common peroneal nerves were tightly ligated and severed, leaving the sural nerve intact. Animals in the Sham surgery group had their sciatic nerve exposed as in the SNI procedure but received no further manipulation.

Tactile Sensitivity

Paw withdrawal thresholds to Von Frey filament stimulation (VF) were used to assess mechanical sensitivity of the hind paws. Animals were placed in a Plexiglas box with a wire grid floor and allowed to habituate for 15 minutes. At this point, filaments of various forces (Stoelting, USA) were applied to the plantar surface of each hind paw. Filaments were applied in a descending or ascending pattern, determined by the response of the animal. Each filament was applied for a maximum of 2 seconds, and paw withdrawal in response to the filament was considered a positive response. 50% thresholds were calculated according to Chaplan et al., 1994. To be entered into the SNI testing group, animals undergoing SNI surgery were required to decrease their injured paw withdrawal latency significantly when compared to the control paw and Sham animals.

Experiment 1. Hippocampus-mediated behavior and corresponding protein kinase activity

Fear Conditioning and Extinction

Fear conditioning was performed on a subgroup of mice (SNI n=11, Sham n=10, left side surgery) on postoperative day 10 with an automated system (TSE Inc.) and consisted of a single exposure to context (three minutes) followed by a two second tone paired with foot shock (2 seconds, 0.7 mA, constant current) as described previously (Radulovic et al., 1999). The extinction trials were performed at 24-hour intervals and consisted of nonreinforced 3-minute exposures to the context (Fischer et al., 2007). Context-dependent freezing was measured every 5th second over three minute and expressed as percentage of total number of observations. Mice were followed until Sham animals showed significant extinction. At this time mice were placed in a different conditioning box where the previously used tone was presented for three minutes. These tone extinction trials were performed at 24 hour intervals, and tone-dependent freezing was measured every 5th second over three minutes.

Anxiety Behavioral Testing

Anxiety behavioral testing was performed on a subgroup of mice (SNI n=22 (11 left side surgery, 11 right side surgery), Sham n=20 (10 left side surgery, 10 right side surgery). Experiments began on postoperative day 12. These mice did not undergo fear conditioning or extinction.

Black Box Emergence

This test is used as a measure of anxiety-like behavior, in that it measures fear of exposure to an unfamiliar situation. Animals prefer small enclosed chambers on their initial exposure to environments, and if the environment is made more unfamiliar or aversive they spend more time in the small enclosure before exploring. If the animal is more anxious, it will spend a longer time in the chamber before emerging into an open testing space (Takahashi et al., 1989). A 3-sided covered box was placed in the center of an open field illuminated by bright light (to increase open field aversiveness). The behavior of the mice was recorded by a video camera connected to a computer and analyzed by TSE software (VideoMot). Mice were placed in the dark box and the latency for them to leave the box was recorded. The latency to leave the black box was interpreted as anxiety-like behavior (for more details see Kishimoto et al., 2000).

Novelty Suppressed Feeding

In this test there are competing motivations between the drive to eat and fear of entering a brightly lit center area. Food was removed from the home cage 24 hours prior to testing and water was removed two hours prior to testing. One pellet of food was placed in the center of an open field and mice were placed in one corner of the open field. The behavior of the mice was recorded by a video camera connected to a computer and analyzed by TSE software (VideoMot). The behavior of the mice was recorded for 5 minutes and the latency to start eating was recorded. Increased latency was interpreted as an increased anxiety, since classical anxiolytic drugs and SSRI treatment decrease this behavior (David et al., 2009). Due to the latency being capped at 5 minutes, a Mann-Whitney U-test was used in the analysis. This test was adapted from Santarelli et al., 2003.

Shock Reactivity

Shock reactivity was measured 2 weeks post-extinction on the same subgroup of mice that underwent fear conditioning and extinction. The technique consisted of three sets with each set given 24 hours apart (modified from Turner et al., 1967) in the same conditioning box as used for cue extinction. A set consisted of a series of eight shock intensities (mA, constant current): 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8. Shocks were given in ascending order with a 10 second interval between shocks. Following each shock, the reaction of the mouse was recorded as either no response, flinch, jump, or vocalize. The intensity at which the mouse first responded with a flinch, jump, or vocalization was recorded.

Protein Levels Analysis

Tissue was collected from a subgroup of mice which had not undergone fear conditioning or extinction (SNI n=6, Sham n=5, right side surgery) for western blot analysis. Both right and left dorsal and ventral hippocampus were collected. Hippocampal lysates were subject to 4-12% Bis-Trist Gel (Invitrogen, Carlesbad CA) electrophoresis, and subsequently blotted to nitrocellulose membranes (Bio-Rad, Hercules CA). Membranes saturated with block were incubated with anti-phos-ERK (dpERK, Sigma-Aldrich, St. Louis MO, 1:250), anti-ERK (Santa Cruz Biotechnology, Santa Cruz CA, 1:500), and anti-βTubulin (Sigma-Aldrich, 1:500). Western blots were developed under maximal chemiluminescence emission with Western-Lighting Plus-ECl (PerkinElmer, Waltham MA) on BioRad QuantityOne. pERK and ERK were normalized to βTubulin levels independently to account for ERK expression differences.

Experiment 2. Hippocampal Neurogenesis

Bromodeoxyuridine (BrdU) labeling

BrdU was used to evaluate cell proliferation. DCX-EGFP mice (SNI n=5, Sham n=6, left side surgery) received intraperitoneal injections of BrdU (Sigma-Aldrich) 50mg/kg dissolved in sterile PBS (10mg/ml) and filtered. Injections were given starting day 3 post-surgery (SNI or Sham) and continued for 5 days. Mice were perfused 1 week after the last injection (14 days post-surgery).

Immunohistochemistry

Mice were transcardially perfused with ice-cold 4% paraformaldehyde in phosphate buffer (pH 7.4, 150 mL/mouse). Brains were post-fixed for 48 hours in 4% PFA and sections were cut on a vibratome (Leica VT 1000S; Leica Microsystems) and collected in cold PBS. Free-floating sections were then incubated with primary antibodies for BrdU (1:500; Fitzgerald, Concord MA) and NeuN (1:200; Millipore, Temecula CA), a marker of mature neurons, to allow for triple-illumination of DCX, BrdU and NeuN. Following primary incubation, the corresponding secondary antibody conjugated with Alexa Fluor 633 (Invitrogen) was used for NeuN and biotin conjugated secondary (Jackson Immunoresearch, West Grove PA) along with streptavidin Alexia 546 (Jackson Immunoresearch) were used for BrdU. Slices were mounted in Vectashield (Vector, Burlingame CA) and observed with a fluorescence microscope (Olympus Fluoview FV10i, Confocal Laser Scanning Microscope) for triple-labeling at 60x. Two consecutive hippocampal sections per mouse were live counted for double and triple-labeling in a blinded fashion (bilateral). Images were taken through the microscope.

Experiment 3. Hippocampal dentate gyrus-CA3 fEPSP potentiation

Preparation of hippocampal slices

Mice (SNI n=13, Sham n=12, left side surgery) were anesthetized using isoflurane and sacrificed by decapitation at day 7 post-surgery. The brain was quickly removed and placed in ice cold sucrose-rich ACSF containing (in mM): 87 NaCl, 25 NaHCO3, 25 Glucose, 75 Sucrose, 2.5 KCl, 1.25 NaH2PO4, 0.5 CaCl2, 7 MgCl2, 1 Sodium Pyruvate, 5uM Glutathione. 400 μm horizontal slices of the contralateral (SNI n=6, Sham n=5) or ipsilateral (SNI n=7, Sham n=7) hippocampus (relative to the peripheral nerve injury) were cut in the ventral to dorsal direction using a tissue slicer (Thermo Scientific). Slices were then stored in ACSF (in mM): 125 NaCl, 25 NaHCO3, 25 Glucose, 2.5 KCl, 1.25 NaH2PO4, 2 CaCl2, 1 MgCl2, 1 Sodium Pyruvate, 5uM Glutathione for 20 minutes at ~30°C, followed by 20 minutes at ~36°C.

Electrophysiological recordings

Recordings were conducted in a chamber that was continuously perfused with ACSF containing 2.5mM CaCl2, 1mM MgCl2, 100μM Picrotoxin (Ascent Scientific) and 50μM D-APV (Ascent). The hippocampus was visualized using DIC infrared microscopy. A bipolar stimulating electrode (FHC) was positioned in the hilar region of the dentate gyrus. Glass (SG16, Dagan) recording electrodes containing 3M NaCl and having tip resistances of 1-3MΩ were placed in stratum lucidum of the CA3 region. Electrophysiological recordings were performed at 31°C ± 1 using an Axopatch 200B amplifier and the stimulation was delivered by way of an A360 Stimulus Isolator (WPI). Field potential recordings were sampled at 10kHz and low pass filtered at 5kHz. Data were digitalized using a Digidata 1300 interface and acquired on a PC for offline analysis.

The potentiation induction protocol was as follows: current stimulation levels were set to elicit excitatory field potentials of 40 to 60% of the maximum observed response. Baseline recordings were comprised of one 200μsec pulse every 15sec (0.067Hz) for 10 minutes. Tetanic stimulation consisted of four 1 second 100Hz trains at 30 second intervals. Post tetanic potentiation (PTP) was calculated as the averaged slope of the first two responses following tetanic stimulation relative to control (average response in the 10 minutes preceding the tetanus).

Experiment 4. Hippocampal Volume of Human Chronic Neuropathic Pain Patients

Subjects

50 healthy volunteers (22 males, 28 females, mean age ± SD: 40.1 ± 11.3), 38 chronic back pain (CBP) patients (21 males, 17 females, mean age ± SD: 48.4 ± 9.7), 30 complex regional pain syndrome (CRPS) patients (4 males, 26 females, mean age ± SD: 41.5 ± 11.6), and 20 knee osteoarthritis (OA) patients (16 males, 4 females, mean age ± SD: 53.1 ± 7.5) were recruited for this study. All were right-handed and gave full informed consent to procedures approved by the Northwestern University IRB committee. These subjects are also included in the analysis of Baliki et al., 2011.

Scanning Parameters

All high-resolution T1-anatomical brain images were scanned on a Siemens 3.0 Tesla whole body system with an 8-channel head coil and MPRAGE type image using the following parameters: voxel size = 1× 1× 1 mm, TR = 2500 ms, TE = 3.36 ms, flip angle = 90°, in-plane matrix resolution = 256× 256, slices = 160 and field of view = 256 mm.

Data Analyses

Data were analyzed using the FSL 4.1 (Smith et al., 2004). First, the skull for each T1 image was extracted and the hippocampus was then segmented using FMRIB’s integrated registration and segmentation tool (FIRST, (Patenaude, 2007)). After excluding those subjects who had inaccurate hippocampus segmentation (see details below), the volumes of right and left hippocampi were calculated for each subject. Finally, the group analyses of right and left hippocampus volumes, individually, were achieved after removing the effect of whole brain volume, gender, and age as covariates of no interest.

Segmentation Results Assessment

We developed an objective procedure to assess the reliability of segmentation and consequently excluded those subjects that have unreliable segmentation results. This was a three-step procedure: First, to achieve a gross alignment of T1 images, the T1 image of each subject was initially co-registered to MNI152 standard template by a rigid body transformation. Following this, 15 subjects’ images from each group were randomly selected, averaged, and merged as a new template for each group, from which hippocampi were segmented as a target. Using the transformation matrix from above co-registration, the hippocampus in subject space was transformed into standard space. Subjects whose standard hippocampus volume was outside of its group mean volume ± 1.0 SD were considered unreliable and were not included in the analysis. 5 subjects were excluded with this method.

Results

Hippocampus-mediated behavior and corresponding protein kinase activity

While both cue and context fear conditioning and extinction involve amygdala circuitry, contextual fear conditioning and extinction is unique in its involvement of the hippocampus (Phillips and LeDoux, 1992). Due to our hypothesis being that chronic pain is associated with dysregulation of contextual learning, we first examined whether there is a difference in context and cue acquisition and extinction in a mouse model of neuropathic pain, to determine whether there is a specific hippocampus conditioned learning deficit in contrast to the broader fear conditioning circuitry.

All animals demonstrated similar fear conditioned acquisition on testing day 1 (SNI n=11, Sham n=10). A significant difference was seen in context extinction ( F19,190=3.6, p<0.001) with SNI animals showing an inability to extinguish to context. These animals showed no reduction in freezing behavior and continued to freeze 40% of observed time through extinction day 11. In contrast, Sham animals continued to diminish their freezing behavior over 11 extinction days to levels below 20% of observed time (Figure 1a). In cue fear extinction, however, there was no difference between groups and both groups showed successful extinction in 3-4 days (F19,57=2.02, ns) (Figure 1b). An illustration of the animals’ mechanical thresholds can be seen in Figure 1c, which demonstrates that SNI mice (mean=0.02g) exhibit mechanical allodynia on the side of nerve injury while Sham animals do not (mean=0.44g, t(19)=5.935, p<0.001 between groups post surgery). SNI and Sham animals do not significantly differ from each other regarding shock intensity that first elicits flinching (SNI mean=0.13mA, Sham mean=0.14mA, t(16)=0.46), jumping (SNI mean=0.25mA, Sham mean=0.26mA, t(19)=0.18) and vocalizing (SNI mean=0.35mA, Sham mean=0.36mA, t(19)=0.18) (Figure 1d), and no differences were seen for these measures between trial days. This illustrates that the shock stimulation, inducing fear acquisition, was not confounded by the difference in mechanical allodynia between groups. Together, these results indicate that chronic neuropathic pain diminishes hippocampus-mediated fear extinction behavior without affecting a hippocampal-independent form of fear extinction (cue extinction).

Figure 1
Hippocampus-mediated behavior significantly modulated by chronic neuropathic pain

Latency to emerge from the black box was compared between SNI and Sham animals and a significant difference was seen between the two groups (t(39)=3.57, p=0.001) with SNI animals having a longer latency (mean=16.41s) than Sham animals (mean=2.00s) to leave the dark box (SNI n=21, Sham n=20) (Figure 2a). SNI animals also had a prolonged latency until eating (mean=271.09s) in the Novelty Suppressed Feeding (NSF) task compared to Sham animals (mean=224.95), (Mann-Whitney U-test, U=129, Z=2.28, p=0.02) (Figure 2a). This demonstrates that SNI animals show evidence of hippocampal-dependent anxiety-like behaviors. However, this anxiety cannot be considered the cause of the deficit in context extinction since cue extinction is also diminished in studies of anxiety disorders (Green et al., 2011). Mechanical thresholds for these animals, also used for ERK analysis, are shown in Figure 2b. As with all SNI and Sham groups, SNI animals have significantly lower VF thresholds compared to Sham animals on the injured paw at every time point post surgery (F1,144)=89.51, p<0.001. d3: SNI mean=0.03g, Sham mean=0.41g, t(19)=4.82, p<0.001; d7: SNI mean=0.02g, Sham mean=0.43g, t(19)=5.25, p<0.001; d9: SNI mean=0.03, Sham mean=0.50, t(19)=4.04, p<0.001; d14: SNI mean=0.02, Sham mean=0.61, t(19)=4.60, p<0.001 (Figure 2b). The uninjured paw shows no decrease in threshold in either group (Figure 2b).

Figure 2
Increased anxiety-like behavior and mechanical allodynia in SNI animals

Previous research has revealed that ERK1/2 phosphorylation in the hippocampus is critical for contextual fear extinction (Fischer et al., 2007; Tronson et al., 2008), consequently we examined basal levels of pERK and ERK in non-fear conditioned SNI and Sham animals. In the ipsilateral to injury dorsal hippocampus, SNI animals showed a significant reduction in both pERK2:βTubulin, t(9)=4.44, p=0.002) and ERK2:βTubulin (t(9)=3.73, p=0.004) compared to Sham animals (Figure 3a and 3b). Due to the decrease seen in ERK2 expression levels, the pERK2 expression difference can be attributed to a reduction in the total ERK2 gene expression. In contrast, in the ipsilateral ventral hippocampus, SNI animals show a significant reduction in pERK expression, t(9)=3.68, p=0.005, compared to Sham animals (Figure 3c). No significant differences were seen in pERK2 expression or ERK expression (Figure 3d). Due to the lack of changes in ERK expression, the difference in pERK1 expression in the ipsilateral ventral hippocampus is due to differences in ERK activity and phosphorylation, and not due to changes in gene expression.

Figure 3
Ipsilateral hippocampal ERK expression and activity changes in SNI animals

In the contralateral hippocampus to injury (Figure 4), ERK2 expression in ventral hippocampus was significantly altered, with SNI animals again showing a reduction in ERK expression compared to Sham animals, t(6)=2.95, p=0.025, indicating a gene expression difference in this brain region in SNI animals (Figure 4d).

Figure 4
Contralateral hippocampal ERK expression and activity changes in SNI animals

While initially unexpected that changes would be greater in the ipsilateral than contralateral hippocampus, the experiment was replicated numerous times and with surgery alternating on either the left or right hind paw. In each repetition these results were replicated. Therefore, we conclude that there is distinctive decrease in ERK1 and ERK2 phosphorylation and decrease in ERK2 gene expression levels in SNI animals. It is important to note, that while ERK expression changes are not identical bilaterally, both hemispheres show reduced expression. This decrease in basal ERK expression and phosphorylation could be interfering with the normal action of pERK in contextual fear extinction and influencing the behavioral deficit we observe.

Hippocampal Neurogenesis

Adult hippocampal neurogenesis is indicated to be involved in learning and memory (Deng et al., 2010) and decreases in neurogenesis have been associated with depression (Sahay and Hen, 2007), anxiety (Revest et al., 2009), stress (Dranovsky and Hen, 2006) and impairment in contextual fear conditioning (Saxe et al., 2006). Therefore, we examined the impact of chronic neuropathic pain on hippocampal neurogenesis.

SNI mice showed significantly fewer double-labeled DCX/BrdU cells bilaterally in the subgranular zone of the dentate gyrus of the hippocampus as Sham animals (ipsilateral: t(9)=5.42, p<0.001, contralateral: t(9)=4.27, p=0.002) indicating a decrease in neurogenesis. SNI mice had an average of 14.4 and 14 cells per hippocampal slice (ipsilateral and contralateral, respectively), while Sham animals had an average of 42.5 cells in ipsilateral and 37.7 cells in contralateral dentate gyrus over corresponding slices (Figure 5). There were no significant differences in the percentage of double-labeled EGFP/BrdU cells which were also triple-labeled with NeuN (t(8)=1.66, ns) between SNI (mean=57.66%) and Sham animals (mean=47.50%). At the time of tissue collection, SNI animals (mean=0.01) had significantly lower VF thresholds on the injured side than Sham animals (mean=0.35g) t(8)=3.1, p=0.01.

Figure 5
Reduced hippocampal neurogenesis in SNI animals

These results demonstrate that chronic neuropathic pain animals show a bilateral reduction in adult hippocampal neurogenesis measured by the double-labeling of DCX/BrdU. This reduction in newly born cells suggests a mechanism by which learning, memory, contextual extinction, and other behaviors affected by chronic pain are being modulated. There is no change in the percentage of newly-born cells that are maturing into neurons, determined by triple-labeling with NeuN, suggesting no difference in maturation speed or atrophy post initial growth of the cells.

Hippocampal dentate gyrus-CA3 fEPSP potentiation

The MAP/ERK pathway plays a key role in synaptic plasticity, and intriguingly, MAPKs have been found in synaptic terminals (Boggio et al., 2007). In particular, it has been shown that ERK activity affects short-term synaptic plasticity (post-tetanic potentiation) through phosphorylation of synapsin in cultured systems (Giachello et al., 2010). We therefore investigated whether the large change observed in ERK signaling and decreased neurogenesis in SNI animals may influence short-term synaptic plasticity in the hippocampus of SNI animals at this mossy fiber-CA3 synapse.

Plasticity was induced by 4 tetanic stimulations (1s each, 100 Hz) and we investigated two parameters: the post-tetanic potentiation (within 30s from the end of the tetanus) and the kinetics of the intra-tetanus plasticity. We found that SNI animals differed significantly in both these parameters, and in both ipsilateral and contralateral hippocampi relative to the peripheral nerve injury. Synapses from SNI animals showed a marked use dependent depression between the first and last of the four tetani (Figure 6a,b), while in Sham animals no difference was detected; additionally, PTP, when normalized to baseline slope values, was significantly reduced from 2.13 ± 0.36% (mean ± SEM) in ipsilateral hippocampus in Sham animals (n=7) to 1.30 ± 0.20% in SNI animals (n=7, p< 0.05, Figure 6d). PTP was also significantly reduced from 2.75 ± 0.24% (n=6) in contralateral hippocampus in Sham animals to 1.46 ± 0.58% (n=5, p<0.05, Figure 6d) in SNI animals. Therefore, short-term plasticity in the hippocampus of SNI animals is significantly altered, in agreement with the observed differences found in ERK signaling.

Figure 6
Alterations in short-term plasticity in CA3-mossy fiber pathway in SNI animals

Hippocampal Volume of Human Chronic Neuropathic Pain Patients

To this point all of the work presented has been performed in a mouse model of chronic neuropathic pain. If we desire, however, to understand the results in terms of human chronic pain patients we need to investigate the impact of persistent pain on the hippocampus in such patients. We therefore analyzed the hippocampal volume of several types of chronic pain patients and compared these values to a normal subject group. To determine whether there are differences in hippocampal morphology between various types of chronic pain conditions, we included CBP, CRPS, and OA patients in the analysis. Previous studies by Baliki et al., 2011 and Apkarian et al., 2009 indicate that these pain conditions show distinct cortical functional activity patterns and regional gray matter density decreases relative to one another. Thus, here we test whether hippocampal volume is also differentially influenced by distinct chronic pain conditions.

An analysis of covariance indicates that both CBP and CRPS patient groups have significantly less left and right hippocampal volume as compared to normal subjects. For the left hippocampus, (F3,122=3.79, p=0.01), CBP (t=3.09, p=0.002), and CRPS (t=2.27, p=0.02) patient groups differ significantly from normal subjects through planned comparisons (Figure 7a) (categorical predictors ‘group’ and ‘sex’, continuous predictors of ‘age’ and ‘brain volume’). The same is true in the right hippocampus (F3,122=2.60, p=0.05), where CBP (t=2.09, p=0.04) and CRPS (t=2.44, p=0.02) again differ significantly from normal subjects (Figure 7b). OA subjects did not differ significantly from normal subjects in hippocampal volume in left (t=1.09, p=0.28) or right (t=1.25, p=0.21) hemispheres. Therefore, there is a reduction in hippocampal volume in certain chronic pain conditions but not others.

Figure 7
Decreases in hippocampal volume in human chronic pain patients

Discussion

In this study we reveal multiple hippocampal abnormalities in animals with chronic pain and demonstrate a potential underlying mechanism for hippocampal-mediated behavioral abnormalities in these animals through the reduction of hippocampal neurogenesis and altered short-term synaptic plasticity. Moreover, we are the first to demonstrate decreased hippocampal volume in humans with specific chronic pain conditions, which may be a consequence of some of the mechanisms we observe in the rodent model for chronic pain.

Our results demonstrate that SNI and Sham animals learn fear conditioning for context and cue equally well but only SNI animals are unable to extinguish context fear conditioning. This confirms our primary hypothesis that the presence of ongoing pain may interfere particularly with hippocampus-dependent context extinction, while hippocampus-independent cue extinction remains unaffected. The specificity of this deficit points to a modulation of hippocampal functioning, and not to a more general change in fear conditioning circuitry.

SNI animals displayed increased anxiety-like behavior. However, they did not show diminished cue extinction as seen in studies of anxiety disorders (Green et al., 2011) and the ERK expression changes were different than what is seen in studies of anxiety and depression in response to fear conditioning and extinction (Tronson et al., 2008). The bilateral but dissimilar changes may be consistent with previous persistent pain research demonstrating lateralization of ERK changes in the amygdala (Carrasquillo and Gereau, 2007). This suggests that the observed effects are not due to a general stress on the animal, but rather to the presence of pain. Therefore, while this animal model exhibits anxiety-like behavior similar to what is often seen in clinical pain conditions (Gore et al., 2011), it cannot be considered the cause of the observed hippocampal molecular changes and deficits in contextual fear extinction.

The lack of context extinction in SNI animals demonstrates memory deficit in these animals, which expands and complements previous work that revealed impairments in working and short-term memory in SNI (Ren et al., 2011). While behavioral changes are typically considered linked to long-term synaptic changes, mutant mice lacking synapsin II showed profound learning impairments, which at cellular level are associated with decreased PTP, without any detectable alteration of long-term plasticity (Silva et al., 1996); intriguingly, such impairments were selective for contextual conditioning, without detectable abnormalities in spatial learning. Previous reports revealed that ERK is present in spines and synaptic terminals (Boggio et al., 2007) and that short-term plasticity is dependent on the MAP/ERK pathway (Giachello et al., 2010). This framework ideally fits our data, which show significant changes in hippocampal ERK expression and impaired short-term plasticity (PTP) in neuropathic animals. Additionally, experiments performed in the calyx of Held, another glutamatergic synapse, demonstrated that deletion of synapsins 1 and 2, which are targets of MAP/ERK phosphorylation, increases the use-dependent synaptic depression induced by a high-frequency stimulus train. This effect was selective for high frequency trains and was produced through a reduction of the fraction of the readily releasable vesicle pool late in the train (Sun et al., 2006). It is possible that a similar mechanism is at work at the mossy fiber-CA3 synapse. Furthermore, an increase in dentate gyrus granule cells is seen following high frequency stimulation of the mossy fiber efferents of granule cells (Derrick et al., 2000). This indicates that the changes we observe in synaptic plasticity may tie to neurogenesis and suggests a mechanistic link between the two.

While it is well established that decreases in hippocampal neurogenesis are associated with many comorbid conditions of chronic pain and that this decrease plays an important role in many of the observed cognitive deficits (Revest et al., 2006; Dranovsky and Hen, 2006; Sahay and Hen, 2007; Saxe et al., 2006) only one study has investigated the decrease in neurogenesis in chronic neuropathic pain (Terada et al., 2008). However, these investigators were interested in how chronic neuropathic pain alters enriched-environment increases in neurogenesis. Our study, in contrast, indicates a more direct baseline impact of chronic pain on neurogenesis and not only a modulatory role. Our current work is the first to demonstrate that normal neurogenesis is decreased in the presence of chronic neuropathic pain, which is consistent with the observed abnormal context extinction and altered synaptic plasticity and supports our theory that the hippocampal learning and memory system may be critically involved in chronic pain. The mechanism of this reduction in neurogenesis, which we propose then leads to changes in mossy fiber-CA3 plasticity and observed context extinction deficits, is still being explored. It is known, however, that ERK2 is necessary for cell survival (Aouadi et al., 2006), so the reduction of ERK2 observed in the hippocampus of SNI animals may influence the reduction of neurogenic cells.

The reduction in neurogenesis may also involve proinflammatory cytokines, such as TNF-α (Ren et al., 2011), IL-1β (del Rey et al., 2011), or others found to be affected in the hippocampus post-peripheral injury (Al-Amin et al., 2011; del Rey et al., 2011). It is suggested by Ren et al., 2011 that overproduction of TNF-α in the hippocampus may contribute to the memory deficits seen in these animals. TNF-α production is stimulated by ERK1/2 activation and is involved in signal transduction (Lee et al., 2011), so this observation is consistent with the changes of hippocampal ERK expression and decreased neurogenesis we observe. Furthermore, TNF-α seems to have an effect on adult hippocampal neurogenesis (Montgomery and Bowers, 2011). IL-1β is another potential cytokine that may underlie the behavioral and neurogenesis changes in SNI animals. Work by del Rey et al, 2011, found that IL-1β is upregulated in the hippocampus post-SNI and this upregulation is correlated with neuropathic pain behavior. IL-1β has been suggested to lead to decreased neurogenesis in the elderly (Kuzumaki et al., 2010) and could play a similar role in neuropathic pain.

Our results in the SNI model tie together in a consistent and complementary fashion. A hippocampal-specific behavioral deficit in context extinction is seen, which has previously been shown to depend on ERK phosphorylation (Fischer et al., 2007) and short-term hippocampal plasticity (Silva et al., 1996). In addition, ERK expression and phosphorylation changes are observed in SNI animals, as well as alterations of short-term synaptic plasticity at the mossy fiber-CA3 synapse. This type of potentiation is known to rely on ERK phosphorylation (Selcher et al., 2003; Giachello et al., 2010) and dentate gyrus neurogenesis (Derrick et al., 2000), which is decreased in SNI animals. Future investigation can determine any functional significance to the bilateral but dissimilar changes in ERK expression. Finally, dentate gyrus neurogenesis, known to be involved in context extinction (Saxe et al., 2006), is closely tied to hippocampal volume (Erickson et al., 2011), which is decreased in human chronic pain patients. Thus, we can presume that the presence of pain per se interferes with mossy-fiber-CA3 synaptic plasticity and dentate gyrus neurogenesis. The changes in these hippocampal properties may relate to the hippocampal volume decreases in chronic pain patients.

Until this current study there has been very little data available on the hippocampus and pain patients. A single human study of hippocampal volume examined elderly chronic pain patients but observed only a borderline decrease of volume correlated with increased pain (Zimmerman et al., 2009). In our current study we show robust decreases in hippocampus volume in two of the three chronic pain conditions examined. The specificity in these results between patient groups supports the conclusion from previous work that different chronic pain conditions may have unique brain profiles (Apkarian et al., 2009; Baliki et al., 2011). As CBP and CRPS can have a larger neuropathic component than OA, which is regarded as more inflammatory in nature, neuropathic chronic pain conditions seem to have a larger impact on hippocampal volume. Decreased hippocampal volume has been repeatedly observed for depression (Cole et al., 2011), and recently been tied to problems in contextual fear conditioning in humans (Pohlack et al., 2012). Even though human chronic pain is commonly associated with increased depression (Gore et al., 2011), in the current study depression values were not correlated to hippocampal volume and volume decreases in the patients were determined after correcting for depression. Thus, obtained results are due to the presence of chronic pain independent of accompanying depression. This decreased volume may in turn contribute to emotional and cognitive problems seen in chronic pain patients and observed in the present study of an animal chronic pain model.

In summary, we observe robust behavioral, molecular, and synaptic changes in the hippocampus of animals with SNI and, in human chronic pain patients we show decreased hippocampal volume which may be a consequence of processes similar to those we report in the rodent model of chronic pain. We conclude that these unique hippocampal disruptions in chronic pain cannot be explained by comorbidity with other disorders and that these are specific cellular correlates of the observed cognitive and emotional problems seen both in animal models of chronic pain and human pain patients. Therefore, targeting the reversal of these systematic changes in chronic pain could improve both patient quality of life and actual pain behavior.

Acknowledgements

This study was supported by NIH-NINDS Grants NS57704, NS5064091, NIH-NIMH MH73669, and NIH T32 AG020418-09.

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