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Chronic intracerebroventricular infusion of nerve growth factor improves recognition memory in the rat
Neuropharmacology 75 (2013) 255e261
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Chronic intracerebroventricular infusion of nerve growth factor improves recognition memory in the rat Amy M. Birch, Áine M. Kelly* Department of Physiology, School of Medicine, Trinity College Institute of Neuroscience, University of Dublin, Trinity College, Dublin 2, Ireland
a r t i c l e i n f o
a b s t r a c t
Article history: Received 6 June 2013 Received in revised form 25 July 2013 Accepted 29 July 2013
Nerve Growth Factor (NGF) plays pivotal roles in neuronal survival in the adult mammalian brain and may modulate forms of structural and functional plasticity, including neurogenesis. We have shown previously that six weeks of housing in an enriched environment (EE) that did not include access to running wheels resulted in improved recognition memory concomitant with increased NGF expression and neurogenesis in the hippocampus. Here we have attempted to probe a causal link between NGF and the observed enrichment-induced changes in hippocampal function by assessing the effects of six weeks continuous intracerebroventricular (i.c.v.) infusion of NGF on recognition memory and cell proliferation. We report that NGF-infused rats show enhanced recognition memory when compared with vehicletreated controls. Expression of NGF and its receptor, TrkA, was increased in treated rats, as was expression of the synaptic vesicle protein, synapsin. Finally, we observed an increase in cell proliferation in the dentate gyrus of NGF-treated rats. These data indicate that chronic infusion of NGF can stimulate an improvement in learning and memory that is associated with specific cellular changes in the hippocampus, including synaptogenesis and cell proliferation. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: Nerve growth factor Learning and memory Dentate gyrus Synaptogenesis Neurogenesis
1. Introduction Nerve Growth Factor (NGF) is crucial for neuronal survival and growth (Levi-Montalcini and Hamburger, 1951) and also plays a number of roles in mediating plasticity in several regions of the adult brain, including the hippocampus. For example, NGF is involved in maintenance of long-term potentiation (LTP) in dentate gyrus (Kelly et al.,1998) while blockade of hippocampal NGF reduces LTP and impairs spatial memory (Conner et al., 2009). Furthermore, intrahippocampal infusion of NGF has been reported to enhance memory in an inhibitory avoidance task (Walz et al., 2000) while several studies have reported a positive correlation between NGF expression and learning (O’callaghan et al., 2009, Pham et al., 2002). NGF can activate the low-affinity p75 neurotrophin receptor (p75NTR; Lee et al., 2001) but it is believed that its roles in learning and memory are mediated mainly via its activation of the high-affinity receptor TrkA. p75NTR is a member of the tumour necrosis factor (TNF) family of receptors and binding to p75NTR is associated with the activation of pro-apoptotic pathways (Roux and Barker, 2002). It has been reported that conditional TrkA knockout mice display selective impairments in cognitive function (Sanchez-Ortiz
* Corresponding author. Tel.: þ353 18963794. E-mail address: [email protected] (Á.M. Kelly). 0028-3908/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neuropharm.2013.07.023
et al., 2012), although it must be noted that there are some reports of intact learning in TrkA knockout mice (Muller et al., 2012). The mechanisms by which NGF may promote plasticity are manifold. Activation of TrkA induces receptor autophosphorylation, recruitment of adaptor proteins and subsequent activation of the MAP kinase, PI3Kinase/AKt and CAMKII signalling pathways, all of which have been implicated in learning and memory (Patapoutian and Reichardt, 2001). NGF can enhance synaptic communication via increased release of glutamate (Knipper et al., 1994b) and enhancement of synaptogenesis (Garofalo et al., 1992) and may thereby underpin some mechanisms of learning and memory. In the context of adult neurogenesis, increasingly investigated as a key mechanism of plasticity, continuous infusion of exogenous NGF has been shown to enhance the survival of new neurons in the granule cell layer of the dentate gyrus and increase activity in hippocampal cholinergic neurons (Frielingsdorf et al., 2007; Knipper et al., 1994a). These studies indicate that NGF may promote learning and memory via its effects on neuronal signalling, neuronal communication and neuronal morphology. We have shown that a six-week period of housing in an enriched environment (but without access to exercise equipment) improves recognition memory in the rat (Birch et al., 2013). As previous data from our lab has shown exercise alone is a potent memory enhancer and link BDNF to these improvements (Griffin et al., 2009, 2011), we sought to assess the role of additional
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sensory stimulation and novelty in environmental enrichment paradigms in the absence of exercise. The improvement in cognitive function was coincident with increased expression of NGF, but not BDNF, in the hippocampus, increased expression of synaptic vesicle proteins and increased cell proliferation in the dentate gyrus. Given the suggested link between NGF and plasticity, here we sought to investigate whether a six-week period of chronic intracerebroventricular infusion of NGF could mimic the previouslyobserved effects of environmental enrichment on recognition memory, synaptogenesis and cell proliferation and hence examine the potential causal relationship between NGF and enrichmentinduced improvement in cognitive function. 2. Materials and methods 2.1. Animals Male Wistar rats (250e300 g; n ¼ 17) were obtained from BioResources Unit, Trinity College Dublin. Rats were housed 3 per cage with food and water ad libitum with a 12:12 light:dark cycle in a temperature-controlled environment (20e22 C). To assess cell proliferation, all rats were injected with 5-bromo-20 -deoxyuridine (BrdU; 50 mg kg1, SigmaeAldrich) on days 30, 32, 34, 37, 39 and 41 of the 42-day experiment. This dosing regime was used to directly mimic the schedule used in Birch et al. (2013). Experiments were conducted under national law and European Union directives on animal experiments. Every effort was made to minimise animal suffering and to reduce the number of animals used. 2.2. Osmotic minipump implantation Intracerebroventricular infusions were carried out using AlzetÒ Osmotic Pumps and Brain Infusion Kits (2006, Charles River, Cambridge, UK). Pumps were primed with bNGF (n ¼ 6) or cytochrome c (VEH; n ¼ 5) in artificial CSF (150 mM NaCl, 3 mM KCL, 0.19 mM CaCl2, 0.8 mM MgCl2, 0.8 mM Na2HPO4, 0.2 mM NaH2PO4) following manufacturer’s instructions to maintain a continuous infusion of 4 ng h1 for 42 days. Cytochrome c is a commonly used control protein for neurotrophin infusions as it is an inert protein of a similar molecular weight to bNGF (w12,000 Da) with no known extracellular actions (Willson et al., 2008; Kobayashi et al., 1997). The concentration of the recombinant bNGF solution infused was calculated with reference to the increase in bNGF protein observed in rats housed in enriched conditions for 6 weeks in a previous study in our laboratory (Birch et al., 2013). Rats were anaesthetised with 4% isoflurane in pure O2 and injected with preoperatively carprofen (5 mg kg1, s.c.). A single hole was drilled in the skull over the right lateral ventricle (0.9 mm posterior to bregma and 0.14 mm lateral to midline). AlzetÒ Osmotic Pumps were inserted into a pocket under the skin between the scapula, the cannula was slowly lowered to the lateral ventricle to a depth of 3.5 mm and secured using dental cement. The incision was closed using surgical staples. Following surgery, rats were housed three per cage and their food/water intake and weight were monitored daily for the duration of the study. To control for any effects of surgery, an additional group of surgery-naïve rats (CON; n ¼ 6) was included in behaviour experiments. 2.3. Object recognition task The apparatus consisted of a circular black arena (diameter: 1 m, height: 0.5 m) placed in a dimly-lit room. Rats were handled daily for at least one week prior to behavioural testing and habituated to the apparatus as follows; two days prior to the start of behavioural testing, rats were allowed to explore the arena in pairs for 10 min, and on the following day singly for 10 min. Objects used in the task were constructed from large toy bricks and were fixed to the floor of the arena at the start of the task, 15 cm from the wall and equidistant from each other. In the training phase, rats explored three different novel objects during three trials of 5 min with an inter-trial interval of 5 min. In the testing phase, one of the objects was replaced by a novel object (D) in the same position 24 h post-training, the rats were placed back into the arena for 5 min and allowed to explore. Exploration was strictly classed as active; the rats had to be touching the object with at least their noses. To ensure the absence of olfactory cues, objects were cleaned thoroughly between trials. During both the training and testing phases, the time spent exploring each object was recorded in seconds using stopwatches and calculated as a percentage of the total time spent exploring all objects. Rats were trained in the object recognition task on day 41 of infusion and tested on day 42. 2.4. Tissue preparation Immediately after testing, rats were sacrificed by decapitation, the brain quickly dissected free and the right hemisphere was covered in OCTÔ compound (Tissue Tek) and flash frozen in liquid nitrogen for later immunohistochemical analysis. Coronal sections of 10 mm thickness were taken through the dentate gyrus. The
hippocampus was subdissected from the left hemisphere and divided in 3; a small piece of each sample was placed in RNA later (200 ml) and stored at 4 C, snap frozen with liquid nitrogen within seven days, and stored at 80 C for later analysis by RTPCR. The remaining tissue pieces were homogenised either in ice cold Krebs solution (250 ml; 136 Mm NaCl, 16 mM NaHCO3, 10 mM glucose, 2.5 mM KCl, 1.18 mM KH2PO4, 1.18 mM MgSO4, 2 mM CaCl2) for later analysis by ELISA or in lysis buffer (250 ml; 1% v/v NP-40, 20 mM (pH8.0) Tris Base, 136 mM NaCl, 10% v/v glycerol, 2 mM EDTA, 1 mM NaN3, 23 mM aprotinin, 1.54 mM leupeptin) for later analysis by SDS-PAGE and Western immunoblotting. 2.5. Immunohistochemical analysis BrdU immunostaining was performed on 3e4 sections, taken one in six serially, from each rat brain using 3,30 -diaminobenzidine (DAB)-linked staining. Sections were fixed with ice-cold 100% methanol (SigmaeAldrich) and incubated in 2N HCl (Fluka) at 37 C for 30 min to denature DNA. Sections were neutralised with 0.1M Borate buffer (pH 8.5) and endogenous peroxidases were quenched by incubation in 0.3% H2O2 in PBS (137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, 1.5 mM KH2PO4; pH 7.2) for 10 min (SigmaeAldrich). Sections were blocked in 10% normal rabbit serum in PBS with 1% BSA (Vector Laboratories) for 2 h and incubated in anti-BrdU (AbCam; 1:200 in blocking buffer) overnight at 4 C. Sections were washed and incubated in biotinylated rabbit anti-chicken IgG (AbCam; 1:1000 in blocking buffer) for 30 min at room temperature then an avidinebiotin peroxidase complex for 30 min at room temperature (VectastainÒ standard ABC kit, Vector Laboratories). Sections were incubated with DAB to reveal positive staining for BrdU (SigmaeAldrich). All sections were counterstained with haematoxylin (SigmaeAldrich) to enable quantification of nuclei. The number of BrdUþ nuclei in the dentate gyrus was counted and calculated as a percentage of the total number of nuclei in the dentate gyrus in each section. Counting of positive cells was restricted to the blades of the dentate gyrus only, to ensure that all cells that were counted were early progenitor cells (restricted mainly to the subgranular zone of the dentate gyrus). 2.6. ELISA Homogenates prepared in Kreb’s solution were centrifuged (20 min at 11,000 g at 4 C) and supernatant was removed. Protein concentrations of the supernatant were quantified using the Bradford method (Bradford, 1976) and samples were equalised for protein concentration. NGF protein concentration was analysed using Rat bNGF DuoSet ELISA Development system (R&D Systems, UK) according to manufacturer’s instructions. Briefly, 96-well plates were coated with 0.4 mg ml1 concentration of capture antibody (goat anti-rat bNGF in phosphate buffered saline (PBS; 50 ml/well)) and incubated overnight at room temperature. The plate was washed with PBS-T (0.05% TweenÒ20 in PBS) using an automated plate washer (Columbus Plus, Tecan, Austria) and blocked with a reagent diluent (1% BSA in PBS; 150 ml/well) for 1 h at room temperature. After washing, the samples and standards were added (50 ml/well) and incubated for 2 h at room temperature. The plates were washed again and detection antibody (100 ng ml1 biotinylated goat anti-rat bNGF in reagent diluent; 50 ml/well) was added and incubated for 2 h at room temperature. After washing, the plates were reacted with Streptavidin-HRP (50 ml/well) for 20 min at room temperature, washed and substrate solution was added (50 ml/well) and incubated in the dark at room temperature for 20 min. To stop the reaction, 1M H2SO4 was added (50 ml/well). The absorbances of the samples and standards were read at 450 nm in a plate reader (Labsystems Fluoroskan Ascent FL). The regression equation of the standard curve was used to calculate the bNGF concentrations of the samples, and results were expressed as pgNGF/mg protein. 2.7. SDS-PAGE and Western immunoblotting Analysis of TrkA, synapsin I and synaptophysin was performed by SDS-PGE and Western immunoblotting. Briefly, samples that had been homogenised in lysis buffer and equalised for protein concentration were diluted 1:2 in sample buffer (0.5 mM (pH 6.8) TriseHCl, 10% v/v glycerol, 0.05% w/v SDS, 0.5% v/v b-mercaptoethanol, 5% v/v bromophenol blue) and boiled for 5 min at 95 C. Samples (10 ml) were electrophoresed on 10% SDS-PAGE gels and transferred to nitrocellulose membranes at 225 mA for 75 min in a semi-dry transfer block (Apollo Instruments, Alpha Technologies, Dublin, Ireland). All blots were blocked in 5% BSA in TBS with 0.5% TweenÒ 20 for 2 h at room temperature and incubated overnight at 4 C in primary antibodies diluted in 2% BSA in TBS with 0.5% TweenÒ 20 (Trk A, 1:500 [Cell Signalling Technology]; synapsin I, 1:4000 [Cell Signalling Technology]; synaptophysin, 1:4000 [Millipore]). Blots were incubated in HRP-conjugated secondary antibodies (for TrkA, 1:2000 goat anti-rabbit IgG in BSA (2% (w/v) in TBS-T); for synapsin I, 1:6000 goat anti-rabbit IgG in BSA (2% (w/v) in TBS-T); for synaptophysin, 1:6000 goat anti-mouse IgG in BSA (2% (w/v) in TBS-T)) and developed using SupersignalÒ West Dura chemiluminescence reagent (Pierce). Images were captured and analysed using the LAS-3000 Intelligent Dark Box, LAS-3000 Image Reader and Multigauge V2.2 (Fujifilm). Blots were stripped using ReBlot Plus strong antibody stripping solution and reprobed for the housekeeping proteins b-actin (1:2000; SigmaeAldrich) or GAPDH (1:1000; AbCam) to control for protein loading.
A.M. Birch, Á.M. Kelly / Neuropharmacology 75 (2013) 255e261 2.8. Real-time PCR Total RNA was extracted from stored tissue samples using Nucleospin RNA II kits (Macherey-Nagel). The integrity of extracted RNA was assessed by electrophoresis and the concentration was determined using a UVevis spectrophotometer (Beckman Coulter Inc., Ireland). cDNA synthesis was performed on 1e2 mg RNA using a High Capacity cDNA RT Kit (Applied Biosystems, USA). Real-time PCR was performed using Taqman Gene Expression Assay for NGF (assay no. Rn01533872_m1) and TrkA (assay no. Rn00572130_m1; Applied Biosystems, USA). Primer kits contained forward and reverse primers, and a FAM-labelled MGB Taqman probe for TrkA. Rat b-actin was used as an endogenous control and expression was conducted using a gene expression assay containing forward and reverse primers and a VIC-labelled MGB Taqman probe (Applied Biosystems, USA). All RT-PCR measurements were conducted using an ABI Prism 7300 instrument (Applied Biosystems). Forty cycles were run as follows: 10 min at 95 C and for each cycle, 15 s at 95 C and 1 min at 60 C. Fluorescence was read during the annealing and extension phase (60 C) throughout the program and gene expression was calculated relative to the endogenous control. Analysis was performed using the 2-DDCT method. Data are presented as mean relative quotient (RQ) values that represent fold changes relative to the mean value for controls. 2.9. Statistical analysis All data are expressed as mean standard error of the mean (SEM). For behavioural analyses, repeated measures two-way ANOVA were used, where the dependent variable was percentage object exploration, the within subjects factor was ‘object’ and the between subjects factor was ‘treatment’. Other data were analysed using one-way ANOVA. All post-hoc analyses were performed using Bonferroni multiple comparison tests. Data were considered statistically significant when p < 0.05.
3. Results 3.1. Effect of chronic intracerebroventricular infusion of NGF on recognition memory Fig. 1 demonstrates that continuous NGF infusion into the lateral ventricle for 42 days improves object recognition memory. During task training (Fig. 1A), there was no significant difference between
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the exploration of the objects (F2,28 ¼ 0.156, p ¼ 0.86), or exploration of the treatment groups (F2,28 ¼ 0.576, p ¼ 0.58), and no interaction between these variables (F4,28 ¼ 1.055, p ¼ 0.40). There was a significant difference in total exploration time between groups (F2,14 ¼ 5.771, p ¼ 0.015), with the CON group exploring the objects significantly less than the NGF-treated group (p ¼ 0.011). There was no significant difference in total exploration time between the VEH- and NGF-treated groups. Mean total exploration time SEM: CON ¼ 109.76 15.17, VEH ¼ 159.01 23.49, bNGF ¼ 201.86 23.38 (data not shown). During testing (Fig. 1B), when object B was replaced with a novel object D, there was a significant difference between the exploration of the objects (F2,14 ¼ 13.16, p ¼ 0.0001) and a significant interaction between the exploration of the objects and treatment group (F4,14 ¼ 3.233, p ¼ 0.027). The NGF-treated group explored the novel object D significantly more than the familiar objects A and C (mean percentage exploration of objects SEM: A ¼ 22.67 2.47, D ¼ 49.73 3.68, C ¼ 27.61 1.66; A vs D: p ¼ 0.019; B vs D: p ¼ 0.023). The CON group did not explore the novel object D significantly more than either familiar object, however analysis revealed that they did explore the familiar object C significantly more than the familiar object A (A vs C: p ¼ 0.019). There was no significant difference between the exploration of the objects in the VEH group. There was no significant difference in total exploration time between the groups (F2,14 ¼ 3.243, p ¼ 0.07). Mean total exploration time SEM: CON ¼ 48.01 10.49, VEH ¼ 54.57 8.63, bNGF ¼ 74.48 1.74 (data not shown). 3.2. Effect of chronic intracerebroventricular infusion of NGF on expression of NGF mRNA and protein in the hippocampus To confirm the efficacy of the infusion into the lateral ventricle, NGF protein concentration was measured in the contralateral hippocampus by method of ELISA. Rats infused with NGF had significantly higher concentrations of NGF in the contralateral hippocampus when compared with CON (p ¼ 0.007) and VEH (p ¼ 0.03) groups (F2,14 ¼ 7.74, p ¼ 0.005; Fig. 2A), suggesting that the exogenous NGF diffused throughout the lateral ventricle and into the contralateral hippocampus. There was no significant difference between groups in the concentration of NGF mRNA in the hippocampus (Fig. 2B; n ¼ 6 in CON and bNGF, n ¼ 5 in VEH; data expressed as mean SEM). 3.3. Effect of chronic intracerebroventricular infusion of NGF on expression of TrkA mRNA and protein in the hippocampus There was a significant difference between groups in the expression of TrkA mRNA in the hippocampus (Fig. 3B; F2,14 ¼ 3.954, p ¼ 0.048). Bonferroni’s multiple comparison test revealed a significant increase in the expression of TrkA mRNA in the NGF-treated group when compared with the CON (p ¼ 0.025) and VEH (p ¼ 0.043) groups. In parallel, we observed a difference in expression of TrkA protein between groups in the contralateral hippocampus; post-hoc analysis revealed a significantly increased expression of TrkA in the hippocampus of NGF-treated rats when compared with VEH (Fig. 3A; p ¼ 0.025), suggesting an upregulation of Trk A in response to the increased availability of NGF in this region. 3.4. Effect of chronic intracerebroventricular infusion of NGF on expression of synaptic vesicle proteins in the hippocampus
Fig. 1. Effect of chronic intracerebroventricular infusion of NGF on recognition memory. There was no significant difference in the exploration of the objects by any group on the training day of the NOR task (A). Only the NGF-treated group explored the novel object significantly more than the familiar objects during testing (B; ***p < 0.001; difference in exploration of object D between groups þp < 0.05). n ¼ 6 in CON and bNGF, n ¼ 5 in VEH; data expressed as mean SEM.
We report a significant difference between groups in the expression of synapsin I in the contralateral hippocampus (F2,14 ¼ 4.885, p ¼ 0.025; Fig. 4A), with an increase in synapsin I in NGF-treated rats when compared with VEH (p ¼ 0.035). There was
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weekly in the final two weeks of infusion. There was a significant difference in the percentage of BrdUþ nuclei between groups (F2,14 ¼ 4.04, p ¼ 0.041; Fig. 5), with an increase in the number of BrdUþ nuclei in NGF-treated rats, although it did not quite reach statistical significance (p ¼ 0.058). 4. Discussion In this study, we show that intracerebroventricular infusion of
bNGF for a period of six weeks can enhance object recognition
Fig. 2. Effect of chronic intracerebroventricular infusion of NGF on expression of NGF mRNA and protein in the hippocampus. There was a significant difference in the concentration of NGF in the hippocampus of the NGF-treated group compared with CON (**p < 0.01; A) and VEH (þp < 0.05) groups. There was no significant difference between groups in the concentration of NGF mRNA in the hippocampus (B; n ¼ 6 in CON and bNGF, n ¼ 5 in VEH; data expressed as mean SEM).
no significant difference in the expression of synaptophysin between groups in the contralateral hippocampus (F2,14 ¼ 0.054, p ¼ 0.95; Fig. 4B). 3.5. Effect of chronic intracerebroventricular infusion of NGF on cell proliferation in the dentate gyrus To measure the effect of NGF on early cell survival in the ipsilateral dentate gyrus, rats were injected with BrdU three times
memory, synaptic vesicle protein expression and cell proliferation in the rat hippocampus. We have previously reported that environmental enrichment in the absence of exercise enhances these measures of plasticity in a time-dependent manner, concomitant with an increase in expression of NGF in the hippocampus (Birch et al., 2013). The present data indicate that these previouslyreported effects of environmental enrichment may be mediated, at least in part, by NGF. Recognition memory can generally be defined as the ability to discriminate the novelty or familiarity of previous experiences. This can be associated with differences in an individual object, a whole environment or the spatial arrangement of objects within an environment. We and others have shown that successful performance of the novel object recognition task used in this study is associated with molecular and cellular changes in the perirhinal cortex and hippocampus (Ennaceur and Delacour, 1988; Clark et al., 2000; Brown and Aggleton, 2001; Callaghan and Kelly, 2013). Several of these reports have suggested a role for the neurotrophin family of proteins, including NGF (Birch et al., 2013) and brainderived neurotrophic factor (BDNF; Bechara and Kelly, 2013; Seoane et al., 2011; Callaghan and Kelly, 2013) in this form of learning and memory. Here we extend this work to demonstrate a direct effect of NGF treatment on performance of a challenging version of this task and suggest that ability to perform the task is associated with cell proliferation in the hippocampus. We chose a challenging version of this task with a high cognitive load, such that control rats could not learn the task and a ceiling effect in performance would be avoided. The results shown here support other reports in the literature of the memory-enhancing effect of NGF (Conner et al., 2009; Walz et al., 2000), although these studies test spatial and inhibitory avoidance memory respectively. To our knowledge, this is the first study to directly assess the role of NGF on recognition memory in rats. Our findings add to the literature by showing that NGF plays a significant role in several different types of memory, although whether this effect would translate into nonhippocampus dependent memory is not known.
Fig. 3. Effect of chronic intracerebroventricular infusion of NGF on expression of TrkA mRNA and protein in the hippocampus. NGF treatment significantly increased expression of TrkA mRNA compared with control groups (*p < 0.05). In parallel, there was a significant difference in the expression of Trk A protein in the hippocampus of the NGF-treated group compared with VEH (*p < 0.05). Blot is representative image from experiment. n ¼ 6 in CON and n ¼ 5 bNGF & VEH, data expressed per b-actin expression as mean SEM.
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Fig. 4. Effect of chronic intracerebroventricular infusion of NGF on expression of synaptic vesicle proteins in the hippocampus There was a significant difference between groups in the expression of synapsin I in the contralateral hippocampus, with an increase in synapsin I in NGF-treated rats when compared with VEH (*p < 0.05). There was no significant difference between groups in the expression of synaptophysin in the contralateral hippocampus. Blots are representative images from experiment. n ¼ 6 in CON and bNGF, n ¼ 5 in VEH, data expressed per GAPDH expression as mean SEM.
Many previous such studies have focused on the potential efficacy of NGF to reverse or ameliorate existing deficits in memory due to ageing (Niewiadomska et al., 2006; Frick et al., 1997; Klein et al., 2000), traumatic brain injury (Dixon et al., 1997) or ischaemia (Yang et al., 2011), frequently using significantly higher doses than the 4 ng/h used in the present study. This dose was chosen in order to mirror the final concentration of NGF that we observed as a result of six weeks of environmental enrichment (Birch et al., 2013), thus allowing us to directly compare the results
from each study. Here we show that this physiologically-relevant dose of NGF enhances recognition memory in young healthy rats. Moreover, we did not observe negative side effects often associated with high-dose NGF treatment, including neuropathic pain and weight loss (Eriksdotter Jonhagen et al., 1998; data not shown). Analysis revealed a significant increase in the concentration of NGF in the contralateral hippocampus of the NGF-treated group, confirming the efficacy of the delivery of NGF to the target region. The lateral ventricles lie adjacent to the hippocampus and therefore
Fig. 5. Effect of chronic intracerebroventricular infusion of NGF on cell proliferation in the dentate gyrus. There was a significant difference between groups in the percentage of BrdUþ nuclei however this is a trend toward an increase in the number of BrdUþ nuclei in rats infused with NGF compared with control groups (p ¼ 0.058). (B) Representative images of BrdU staining, with dentate gyrus (DG) labelled on whole hippocampus image to show region of interest (from Kjonigsen et al., 2011). Scale bar represents 100 mm. n ¼ 6 in CON and bNGF, n ¼ 5 in VEH; data expressed as mean SEM.
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an increase in the concentration of NGF in the hippocampus contralateral to the infusion also suggests that the infused NGF is traversing the ventricular system and potentially impacting on adjacent regions relevant to performance of the object recognition task, such as the parahippocampal and perirhinal cortices. We observed no change in expression of NGF mRNA, indicating that the increase in concentration of NGF protein observed in the hippocampus is not as a result of increased gene transcription induced by NGF, but that the infused NGF is reaching its intended target site and being taken up into hippocampal cells. In parallel with the increase in NGF concentration observed in the contralateral hippocampus in the NGF-treated group, we also observed an increase in TrkA receptor protein and mRNA expression. It is likely that this upregulation is a functional response to the increased availability of NGF in the region to enhance the amount of TrkA available to bind to NGF and induce intracellular signalling cascades. Induction of TrkA mRNA and protein by NGF has previously been reported in the literature (Decouto et al., 2003; Silver et al., 2001). Increased expression of both ligand and receptor in the hippocampus is likely to lead to an enhancement of NGF-TrkA signalling and subsequent changes in plasticity. Stimulating Trk expression is associated with induction of immediate early genes such as c-fos, although NGF-TrkA signalling seems to have a greater impact on neuronal maturation rather than functional differentiation into neuronal phenotypes (Takahashi et al., 1999). Given the six-week timeframe of NGF treatment in the current experiment, we considered that the mechanisms underlying the NGF-induced improvement in learning and memory were likely to be protein synthesis-dependent effects on cell morphology and function. In this context, we hypothesised that the possible underlying mechanisms may include synaptogenesis and neurogenesis. We report here that continuous NGF infusion resulted in a significant increase in the expression of synapsin I, but not synaptophysin, in the hippocampus of bNGF-infused rats. Synapsin I is crucial for synaptic transmission, in particular in the clustering and release of synaptic vesicles into the active zone of the synaptic bouton (Cesca et al., 2010) but is also used as a marker of synaptogenesis (Xu et al., 2010) Phosphorylation of synapsin I via ERK is known to be vital for neurotransmitter release and maintenance of the synaptic vesicle pool (Jovanovic et al., 1996; Giachello et al., 2010; Shupliakov et al., 2011). Activation of TrkA by NGF can result in stimulation of ERK phosphorylation (Patapoutian and Reichardt, 2001), suggesting that NGF can stimulate enhanced synaptic plasticity, which could facilitate enhanced memory performance. The effects of NGF on memory seem to be independent of expression of synaptophysin in the hippocampus. Synaptophysin is the most abundantly expressed synaptic vesicle protein, hence its common use as a marker for synaptogenesis. However, it is not vital for neurotransmission (Mcmahon et al., 1996). This indicates that the NGF-induced enhancement of synaptic plasticity is likely to occur via more efficient transmitter release rather than an increase in the number of functional synapses. In addition to the selective NGF-induced increase in synaptic vesicle protein expression, we observed a significant difference between control, vehicle treated and NGF-treated groups in early cell survival in the dentate gyrus subfield of the hippocampus, suggesting that NGF may play a role in enhancing neuronal progenitor cell survival. While post-hoc analysis revealed that the number of BrdU positive cells in the dentate gyrus of the NGFtreated group was not significantly different from the control or vehicle-treated groups, we believe the p value of 0.058 represents a trend that suggests a potential direct effect of NGF on cell proliferation. We considered that the BrdUþ nuclei counted in this study are most likely to be proliferating neurons because they are mostly localised to the subgranular zone of the dentate gyrus, and therefore our results suggest that NGF can enhance early hippocampal
neuronal survival. However, BrdU is a non-specific proliferative marker and thus the proliferation of other cell types, such as microglia or astrocytes, may also be captured in these experiments. Also, the results presented here may only be limited to a small subsection of the dentate gyrus and may not reflect differences in proliferation in the whole structure. The literature suggests that NGF is important in the survival and growth of neurons rather than directly stimulating an increase in proliferation (Olson et al., 2006; Frielingsdorf et al., 2007; Zhu et al., 2011). We show that a continuous NGF infusion can increase neurogenesis in the dentate gyrus and that this may be associated with memory improvements, Previously, Frielingsdorf et al. (2007) found that six days of i.c.v. infusion of NGF did not increase cell proliferation, but that a longer infusion period of twenty days promoted increased survival of neurons. The present study therefore, provides the first evidence to suggest that a chronic NGF infusion may increase neurogenesis in the dentate gyrus and that this may in turn be associated with memory improvements. 5. Conclusion This study demonstrates that continuous intracerebroventricular infusion of a physiologically-relevant dose of NGF results in a significant improvement in recognition memory associated with upregulation of NGF-TrkA interaction and cell proliferation in the hippocampus. This study helps to further elucidate the role that NGF plays in memory and suggests an underlying role for NGF in enhancement of neuronal proliferation. Grant sponsors Health Research Board, Ireland. References Bechara, R.G., Kelly, A.M., 2013. Exercise improves object recognition memory and induces BDNF expression and cell proliferation in cognitively enriched rats. Behav. Brain Res. 245, 96e100. Birch, A.M., Mcgarry, N.B., Kelly, A.M., 2013. Short-term environmental enrichment, in the absence of exercise, improves memory, and increases NGF concentration, early neuronal survival, and synaptogenesis in the dentate gyrus in a timedependent manner. Hippocampus 23, 437e450. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248e254. Brown, M.W., Aggleton, J.P., 2001. Recognition memory: what are the roles of the perirhinal cortex and hippocampus? Nat. Rev. Neurosci. 2, 51e61. Callaghan, C.K., Kelly, A.M., 2013. Neurotrophins play differential roles in short and long-term recognition memory. Neurobiol. Learn. Mem. 10, 39e48. Cesca, F., Baldelli, P., Valtorta, F., Benfenati, F., 2010. The synapsins: key actors of synapse function and plasticity. Prog. Neurobiol. 91, 313e348. Clark, R.E., Zola, S.M., Squire, L.R., 2000. Impaired recognition memory in rats after damage to the hippocampus. J. Neurosci. 20, 8853e8860. Conner, J.M., Franks, K.M., Titterness, A.K., Russell, K., Merrill, D.A., Christie, B.R., Sejnowski, T.J., Tuszynski, M.H., 2009. NGF is essential for hippocampal plasticity and learning. J. Neurosci. 29, 10883e10889. Decouto, S.A., Jones, E.E., Kudwa, A.E., Shoemaker, S.E., Shafer, A.J., Brieschke, M.A., James, P.F., Vaughn, J.C., Isaacson, L.G., 2003. The effects of deafferentation and exogenous NGF on neurotrophins and neurotrophin receptor mRNA expression in the adult superior cervical ganglion. Brain Res. Mol. Brain Res. 119, 73e82. Dixon, C.E., Flinn, P., Bao, J., Venya, R., Hayes, R.L., 1997. Nerve growth factor attenuates cholinergic deficits following traumatic brain injury in rats. Exp. Neurol. 146, 479e490. Ennaceur, A., Delacour, J., 1988. A new one-trial test for neurobiological studies of memory in rats. 1: Behavioral data. Behav. Brain Res. 31, 47e59. Eriksdotter Jonhagen, M., Nordberg, A., Amberla, K., Backman, L., Ebendal, T., Meyerson, B., Olson, L., Seiger, Shigeta, M., Theodorsson, E., Viitanen, M., Winblad, B., Wahlund, L.O., 1998. Intracerebroventricular infusion of nerve growth factor in three patients with Alzheimer’s disease. Dement. Geriatr. Cogn. Disord. 9, 246e257. Frick, K.M., Price, D.L., Koliatsos, V.E., Markowska, A.L., 1997. The effects of nerve growth factor on spatial recent memory in aged rats persist after discontinuation of treatment. J. Neurosci. 17, 2543e2550.
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Chronic intracerebroventricular infusion of nerve growth factor improves recognition memory in the rat Amy M. Birch, Áine M. Kelly* Department of Physiology, School of Medicine, Trinity College Institute of Neuroscience, University of Dublin, Trinity College, Dublin 2, Ireland
a r t i c l e i n f o
a b s t r a c t
Article history: Received 6 June 2013 Received in revised form 25 July 2013 Accepted 29 July 2013
Nerve Growth Factor (NGF) plays pivotal roles in neuronal survival in the adult mammalian brain and may modulate forms of structural and functional plasticity, including neurogenesis. We have shown previously that six weeks of housing in an enriched environment (EE) that did not include access to running wheels resulted in improved recognition memory concomitant with increased NGF expression and neurogenesis in the hippocampus. Here we have attempted to probe a causal link between NGF and the observed enrichment-induced changes in hippocampal function by assessing the effects of six weeks continuous intracerebroventricular (i.c.v.) infusion of NGF on recognition memory and cell proliferation. We report that NGF-infused rats show enhanced recognition memory when compared with vehicletreated controls. Expression of NGF and its receptor, TrkA, was increased in treated rats, as was expression of the synaptic vesicle protein, synapsin. Finally, we observed an increase in cell proliferation in the dentate gyrus of NGF-treated rats. These data indicate that chronic infusion of NGF can stimulate an improvement in learning and memory that is associated with specific cellular changes in the hippocampus, including synaptogenesis and cell proliferation. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: Nerve growth factor Learning and memory Dentate gyrus Synaptogenesis Neurogenesis
1. Introduction Nerve Growth Factor (NGF) is crucial for neuronal survival and growth (Levi-Montalcini and Hamburger, 1951) and also plays a number of roles in mediating plasticity in several regions of the adult brain, including the hippocampus. For example, NGF is involved in maintenance of long-term potentiation (LTP) in dentate gyrus (Kelly et al.,1998) while blockade of hippocampal NGF reduces LTP and impairs spatial memory (Conner et al., 2009). Furthermore, intrahippocampal infusion of NGF has been reported to enhance memory in an inhibitory avoidance task (Walz et al., 2000) while several studies have reported a positive correlation between NGF expression and learning (O’callaghan et al., 2009, Pham et al., 2002). NGF can activate the low-affinity p75 neurotrophin receptor (p75NTR; Lee et al., 2001) but it is believed that its roles in learning and memory are mediated mainly via its activation of the high-affinity receptor TrkA. p75NTR is a member of the tumour necrosis factor (TNF) family of receptors and binding to p75NTR is associated with the activation of pro-apoptotic pathways (Roux and Barker, 2002). It has been reported that conditional TrkA knockout mice display selective impairments in cognitive function (Sanchez-Ortiz
* Corresponding author. Tel.: þ353 18963794. E-mail address: [email protected] (Á.M. Kelly). 0028-3908/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neuropharm.2013.07.023
et al., 2012), although it must be noted that there are some reports of intact learning in TrkA knockout mice (Muller et al., 2012). The mechanisms by which NGF may promote plasticity are manifold. Activation of TrkA induces receptor autophosphorylation, recruitment of adaptor proteins and subsequent activation of the MAP kinase, PI3Kinase/AKt and CAMKII signalling pathways, all of which have been implicated in learning and memory (Patapoutian and Reichardt, 2001). NGF can enhance synaptic communication via increased release of glutamate (Knipper et al., 1994b) and enhancement of synaptogenesis (Garofalo et al., 1992) and may thereby underpin some mechanisms of learning and memory. In the context of adult neurogenesis, increasingly investigated as a key mechanism of plasticity, continuous infusion of exogenous NGF has been shown to enhance the survival of new neurons in the granule cell layer of the dentate gyrus and increase activity in hippocampal cholinergic neurons (Frielingsdorf et al., 2007; Knipper et al., 1994a). These studies indicate that NGF may promote learning and memory via its effects on neuronal signalling, neuronal communication and neuronal morphology. We have shown that a six-week period of housing in an enriched environment (but without access to exercise equipment) improves recognition memory in the rat (Birch et al., 2013). As previous data from our lab has shown exercise alone is a potent memory enhancer and link BDNF to these improvements (Griffin et al., 2009, 2011), we sought to assess the role of additional
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sensory stimulation and novelty in environmental enrichment paradigms in the absence of exercise. The improvement in cognitive function was coincident with increased expression of NGF, but not BDNF, in the hippocampus, increased expression of synaptic vesicle proteins and increased cell proliferation in the dentate gyrus. Given the suggested link between NGF and plasticity, here we sought to investigate whether a six-week period of chronic intracerebroventricular infusion of NGF could mimic the previouslyobserved effects of environmental enrichment on recognition memory, synaptogenesis and cell proliferation and hence examine the potential causal relationship between NGF and enrichmentinduced improvement in cognitive function. 2. Materials and methods 2.1. Animals Male Wistar rats (250e300 g; n ¼ 17) were obtained from BioResources Unit, Trinity College Dublin. Rats were housed 3 per cage with food and water ad libitum with a 12:12 light:dark cycle in a temperature-controlled environment (20e22 C). To assess cell proliferation, all rats were injected with 5-bromo-20 -deoxyuridine (BrdU; 50 mg kg1, SigmaeAldrich) on days 30, 32, 34, 37, 39 and 41 of the 42-day experiment. This dosing regime was used to directly mimic the schedule used in Birch et al. (2013). Experiments were conducted under national law and European Union directives on animal experiments. Every effort was made to minimise animal suffering and to reduce the number of animals used. 2.2. Osmotic minipump implantation Intracerebroventricular infusions were carried out using AlzetÒ Osmotic Pumps and Brain Infusion Kits (2006, Charles River, Cambridge, UK). Pumps were primed with bNGF (n ¼ 6) or cytochrome c (VEH; n ¼ 5) in artificial CSF (150 mM NaCl, 3 mM KCL, 0.19 mM CaCl2, 0.8 mM MgCl2, 0.8 mM Na2HPO4, 0.2 mM NaH2PO4) following manufacturer’s instructions to maintain a continuous infusion of 4 ng h1 for 42 days. Cytochrome c is a commonly used control protein for neurotrophin infusions as it is an inert protein of a similar molecular weight to bNGF (w12,000 Da) with no known extracellular actions (Willson et al., 2008; Kobayashi et al., 1997). The concentration of the recombinant bNGF solution infused was calculated with reference to the increase in bNGF protein observed in rats housed in enriched conditions for 6 weeks in a previous study in our laboratory (Birch et al., 2013). Rats were anaesthetised with 4% isoflurane in pure O2 and injected with preoperatively carprofen (5 mg kg1, s.c.). A single hole was drilled in the skull over the right lateral ventricle (0.9 mm posterior to bregma and 0.14 mm lateral to midline). AlzetÒ Osmotic Pumps were inserted into a pocket under the skin between the scapula, the cannula was slowly lowered to the lateral ventricle to a depth of 3.5 mm and secured using dental cement. The incision was closed using surgical staples. Following surgery, rats were housed three per cage and their food/water intake and weight were monitored daily for the duration of the study. To control for any effects of surgery, an additional group of surgery-naïve rats (CON; n ¼ 6) was included in behaviour experiments. 2.3. Object recognition task The apparatus consisted of a circular black arena (diameter: 1 m, height: 0.5 m) placed in a dimly-lit room. Rats were handled daily for at least one week prior to behavioural testing and habituated to the apparatus as follows; two days prior to the start of behavioural testing, rats were allowed to explore the arena in pairs for 10 min, and on the following day singly for 10 min. Objects used in the task were constructed from large toy bricks and were fixed to the floor of the arena at the start of the task, 15 cm from the wall and equidistant from each other. In the training phase, rats explored three different novel objects during three trials of 5 min with an inter-trial interval of 5 min. In the testing phase, one of the objects was replaced by a novel object (D) in the same position 24 h post-training, the rats were placed back into the arena for 5 min and allowed to explore. Exploration was strictly classed as active; the rats had to be touching the object with at least their noses. To ensure the absence of olfactory cues, objects were cleaned thoroughly between trials. During both the training and testing phases, the time spent exploring each object was recorded in seconds using stopwatches and calculated as a percentage of the total time spent exploring all objects. Rats were trained in the object recognition task on day 41 of infusion and tested on day 42. 2.4. Tissue preparation Immediately after testing, rats were sacrificed by decapitation, the brain quickly dissected free and the right hemisphere was covered in OCTÔ compound (Tissue Tek) and flash frozen in liquid nitrogen for later immunohistochemical analysis. Coronal sections of 10 mm thickness were taken through the dentate gyrus. The
hippocampus was subdissected from the left hemisphere and divided in 3; a small piece of each sample was placed in RNA later (200 ml) and stored at 4 C, snap frozen with liquid nitrogen within seven days, and stored at 80 C for later analysis by RTPCR. The remaining tissue pieces were homogenised either in ice cold Krebs solution (250 ml; 136 Mm NaCl, 16 mM NaHCO3, 10 mM glucose, 2.5 mM KCl, 1.18 mM KH2PO4, 1.18 mM MgSO4, 2 mM CaCl2) for later analysis by ELISA or in lysis buffer (250 ml; 1% v/v NP-40, 20 mM (pH8.0) Tris Base, 136 mM NaCl, 10% v/v glycerol, 2 mM EDTA, 1 mM NaN3, 23 mM aprotinin, 1.54 mM leupeptin) for later analysis by SDS-PAGE and Western immunoblotting. 2.5. Immunohistochemical analysis BrdU immunostaining was performed on 3e4 sections, taken one in six serially, from each rat brain using 3,30 -diaminobenzidine (DAB)-linked staining. Sections were fixed with ice-cold 100% methanol (SigmaeAldrich) and incubated in 2N HCl (Fluka) at 37 C for 30 min to denature DNA. Sections were neutralised with 0.1M Borate buffer (pH 8.5) and endogenous peroxidases were quenched by incubation in 0.3% H2O2 in PBS (137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, 1.5 mM KH2PO4; pH 7.2) for 10 min (SigmaeAldrich). Sections were blocked in 10% normal rabbit serum in PBS with 1% BSA (Vector Laboratories) for 2 h and incubated in anti-BrdU (AbCam; 1:200 in blocking buffer) overnight at 4 C. Sections were washed and incubated in biotinylated rabbit anti-chicken IgG (AbCam; 1:1000 in blocking buffer) for 30 min at room temperature then an avidinebiotin peroxidase complex for 30 min at room temperature (VectastainÒ standard ABC kit, Vector Laboratories). Sections were incubated with DAB to reveal positive staining for BrdU (SigmaeAldrich). All sections were counterstained with haematoxylin (SigmaeAldrich) to enable quantification of nuclei. The number of BrdUþ nuclei in the dentate gyrus was counted and calculated as a percentage of the total number of nuclei in the dentate gyrus in each section. Counting of positive cells was restricted to the blades of the dentate gyrus only, to ensure that all cells that were counted were early progenitor cells (restricted mainly to the subgranular zone of the dentate gyrus). 2.6. ELISA Homogenates prepared in Kreb’s solution were centrifuged (20 min at 11,000 g at 4 C) and supernatant was removed. Protein concentrations of the supernatant were quantified using the Bradford method (Bradford, 1976) and samples were equalised for protein concentration. NGF protein concentration was analysed using Rat bNGF DuoSet ELISA Development system (R&D Systems, UK) according to manufacturer’s instructions. Briefly, 96-well plates were coated with 0.4 mg ml1 concentration of capture antibody (goat anti-rat bNGF in phosphate buffered saline (PBS; 50 ml/well)) and incubated overnight at room temperature. The plate was washed with PBS-T (0.05% TweenÒ20 in PBS) using an automated plate washer (Columbus Plus, Tecan, Austria) and blocked with a reagent diluent (1% BSA in PBS; 150 ml/well) for 1 h at room temperature. After washing, the samples and standards were added (50 ml/well) and incubated for 2 h at room temperature. The plates were washed again and detection antibody (100 ng ml1 biotinylated goat anti-rat bNGF in reagent diluent; 50 ml/well) was added and incubated for 2 h at room temperature. After washing, the plates were reacted with Streptavidin-HRP (50 ml/well) for 20 min at room temperature, washed and substrate solution was added (50 ml/well) and incubated in the dark at room temperature for 20 min. To stop the reaction, 1M H2SO4 was added (50 ml/well). The absorbances of the samples and standards were read at 450 nm in a plate reader (Labsystems Fluoroskan Ascent FL). The regression equation of the standard curve was used to calculate the bNGF concentrations of the samples, and results were expressed as pgNGF/mg protein. 2.7. SDS-PAGE and Western immunoblotting Analysis of TrkA, synapsin I and synaptophysin was performed by SDS-PGE and Western immunoblotting. Briefly, samples that had been homogenised in lysis buffer and equalised for protein concentration were diluted 1:2 in sample buffer (0.5 mM (pH 6.8) TriseHCl, 10% v/v glycerol, 0.05% w/v SDS, 0.5% v/v b-mercaptoethanol, 5% v/v bromophenol blue) and boiled for 5 min at 95 C. Samples (10 ml) were electrophoresed on 10% SDS-PAGE gels and transferred to nitrocellulose membranes at 225 mA for 75 min in a semi-dry transfer block (Apollo Instruments, Alpha Technologies, Dublin, Ireland). All blots were blocked in 5% BSA in TBS with 0.5% TweenÒ 20 for 2 h at room temperature and incubated overnight at 4 C in primary antibodies diluted in 2% BSA in TBS with 0.5% TweenÒ 20 (Trk A, 1:500 [Cell Signalling Technology]; synapsin I, 1:4000 [Cell Signalling Technology]; synaptophysin, 1:4000 [Millipore]). Blots were incubated in HRP-conjugated secondary antibodies (for TrkA, 1:2000 goat anti-rabbit IgG in BSA (2% (w/v) in TBS-T); for synapsin I, 1:6000 goat anti-rabbit IgG in BSA (2% (w/v) in TBS-T); for synaptophysin, 1:6000 goat anti-mouse IgG in BSA (2% (w/v) in TBS-T)) and developed using SupersignalÒ West Dura chemiluminescence reagent (Pierce). Images were captured and analysed using the LAS-3000 Intelligent Dark Box, LAS-3000 Image Reader and Multigauge V2.2 (Fujifilm). Blots were stripped using ReBlot Plus strong antibody stripping solution and reprobed for the housekeeping proteins b-actin (1:2000; SigmaeAldrich) or GAPDH (1:1000; AbCam) to control for protein loading.
A.M. Birch, Á.M. Kelly / Neuropharmacology 75 (2013) 255e261 2.8. Real-time PCR Total RNA was extracted from stored tissue samples using Nucleospin RNA II kits (Macherey-Nagel). The integrity of extracted RNA was assessed by electrophoresis and the concentration was determined using a UVevis spectrophotometer (Beckman Coulter Inc., Ireland). cDNA synthesis was performed on 1e2 mg RNA using a High Capacity cDNA RT Kit (Applied Biosystems, USA). Real-time PCR was performed using Taqman Gene Expression Assay for NGF (assay no. Rn01533872_m1) and TrkA (assay no. Rn00572130_m1; Applied Biosystems, USA). Primer kits contained forward and reverse primers, and a FAM-labelled MGB Taqman probe for TrkA. Rat b-actin was used as an endogenous control and expression was conducted using a gene expression assay containing forward and reverse primers and a VIC-labelled MGB Taqman probe (Applied Biosystems, USA). All RT-PCR measurements were conducted using an ABI Prism 7300 instrument (Applied Biosystems). Forty cycles were run as follows: 10 min at 95 C and for each cycle, 15 s at 95 C and 1 min at 60 C. Fluorescence was read during the annealing and extension phase (60 C) throughout the program and gene expression was calculated relative to the endogenous control. Analysis was performed using the 2-DDCT method. Data are presented as mean relative quotient (RQ) values that represent fold changes relative to the mean value for controls. 2.9. Statistical analysis All data are expressed as mean standard error of the mean (SEM). For behavioural analyses, repeated measures two-way ANOVA were used, where the dependent variable was percentage object exploration, the within subjects factor was ‘object’ and the between subjects factor was ‘treatment’. Other data were analysed using one-way ANOVA. All post-hoc analyses were performed using Bonferroni multiple comparison tests. Data were considered statistically significant when p < 0.05.
3. Results 3.1. Effect of chronic intracerebroventricular infusion of NGF on recognition memory Fig. 1 demonstrates that continuous NGF infusion into the lateral ventricle for 42 days improves object recognition memory. During task training (Fig. 1A), there was no significant difference between
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the exploration of the objects (F2,28 ¼ 0.156, p ¼ 0.86), or exploration of the treatment groups (F2,28 ¼ 0.576, p ¼ 0.58), and no interaction between these variables (F4,28 ¼ 1.055, p ¼ 0.40). There was a significant difference in total exploration time between groups (F2,14 ¼ 5.771, p ¼ 0.015), with the CON group exploring the objects significantly less than the NGF-treated group (p ¼ 0.011). There was no significant difference in total exploration time between the VEH- and NGF-treated groups. Mean total exploration time SEM: CON ¼ 109.76 15.17, VEH ¼ 159.01 23.49, bNGF ¼ 201.86 23.38 (data not shown). During testing (Fig. 1B), when object B was replaced with a novel object D, there was a significant difference between the exploration of the objects (F2,14 ¼ 13.16, p ¼ 0.0001) and a significant interaction between the exploration of the objects and treatment group (F4,14 ¼ 3.233, p ¼ 0.027). The NGF-treated group explored the novel object D significantly more than the familiar objects A and C (mean percentage exploration of objects SEM: A ¼ 22.67 2.47, D ¼ 49.73 3.68, C ¼ 27.61 1.66; A vs D: p ¼ 0.019; B vs D: p ¼ 0.023). The CON group did not explore the novel object D significantly more than either familiar object, however analysis revealed that they did explore the familiar object C significantly more than the familiar object A (A vs C: p ¼ 0.019). There was no significant difference between the exploration of the objects in the VEH group. There was no significant difference in total exploration time between the groups (F2,14 ¼ 3.243, p ¼ 0.07). Mean total exploration time SEM: CON ¼ 48.01 10.49, VEH ¼ 54.57 8.63, bNGF ¼ 74.48 1.74 (data not shown). 3.2. Effect of chronic intracerebroventricular infusion of NGF on expression of NGF mRNA and protein in the hippocampus To confirm the efficacy of the infusion into the lateral ventricle, NGF protein concentration was measured in the contralateral hippocampus by method of ELISA. Rats infused with NGF had significantly higher concentrations of NGF in the contralateral hippocampus when compared with CON (p ¼ 0.007) and VEH (p ¼ 0.03) groups (F2,14 ¼ 7.74, p ¼ 0.005; Fig. 2A), suggesting that the exogenous NGF diffused throughout the lateral ventricle and into the contralateral hippocampus. There was no significant difference between groups in the concentration of NGF mRNA in the hippocampus (Fig. 2B; n ¼ 6 in CON and bNGF, n ¼ 5 in VEH; data expressed as mean SEM). 3.3. Effect of chronic intracerebroventricular infusion of NGF on expression of TrkA mRNA and protein in the hippocampus There was a significant difference between groups in the expression of TrkA mRNA in the hippocampus (Fig. 3B; F2,14 ¼ 3.954, p ¼ 0.048). Bonferroni’s multiple comparison test revealed a significant increase in the expression of TrkA mRNA in the NGF-treated group when compared with the CON (p ¼ 0.025) and VEH (p ¼ 0.043) groups. In parallel, we observed a difference in expression of TrkA protein between groups in the contralateral hippocampus; post-hoc analysis revealed a significantly increased expression of TrkA in the hippocampus of NGF-treated rats when compared with VEH (Fig. 3A; p ¼ 0.025), suggesting an upregulation of Trk A in response to the increased availability of NGF in this region. 3.4. Effect of chronic intracerebroventricular infusion of NGF on expression of synaptic vesicle proteins in the hippocampus
Fig. 1. Effect of chronic intracerebroventricular infusion of NGF on recognition memory. There was no significant difference in the exploration of the objects by any group on the training day of the NOR task (A). Only the NGF-treated group explored the novel object significantly more than the familiar objects during testing (B; ***p < 0.001; difference in exploration of object D between groups þp < 0.05). n ¼ 6 in CON and bNGF, n ¼ 5 in VEH; data expressed as mean SEM.
We report a significant difference between groups in the expression of synapsin I in the contralateral hippocampus (F2,14 ¼ 4.885, p ¼ 0.025; Fig. 4A), with an increase in synapsin I in NGF-treated rats when compared with VEH (p ¼ 0.035). There was
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weekly in the final two weeks of infusion. There was a significant difference in the percentage of BrdUþ nuclei between groups (F2,14 ¼ 4.04, p ¼ 0.041; Fig. 5), with an increase in the number of BrdUþ nuclei in NGF-treated rats, although it did not quite reach statistical significance (p ¼ 0.058). 4. Discussion In this study, we show that intracerebroventricular infusion of
bNGF for a period of six weeks can enhance object recognition
Fig. 2. Effect of chronic intracerebroventricular infusion of NGF on expression of NGF mRNA and protein in the hippocampus. There was a significant difference in the concentration of NGF in the hippocampus of the NGF-treated group compared with CON (**p < 0.01; A) and VEH (þp < 0.05) groups. There was no significant difference between groups in the concentration of NGF mRNA in the hippocampus (B; n ¼ 6 in CON and bNGF, n ¼ 5 in VEH; data expressed as mean SEM).
no significant difference in the expression of synaptophysin between groups in the contralateral hippocampus (F2,14 ¼ 0.054, p ¼ 0.95; Fig. 4B). 3.5. Effect of chronic intracerebroventricular infusion of NGF on cell proliferation in the dentate gyrus To measure the effect of NGF on early cell survival in the ipsilateral dentate gyrus, rats were injected with BrdU three times
memory, synaptic vesicle protein expression and cell proliferation in the rat hippocampus. We have previously reported that environmental enrichment in the absence of exercise enhances these measures of plasticity in a time-dependent manner, concomitant with an increase in expression of NGF in the hippocampus (Birch et al., 2013). The present data indicate that these previouslyreported effects of environmental enrichment may be mediated, at least in part, by NGF. Recognition memory can generally be defined as the ability to discriminate the novelty or familiarity of previous experiences. This can be associated with differences in an individual object, a whole environment or the spatial arrangement of objects within an environment. We and others have shown that successful performance of the novel object recognition task used in this study is associated with molecular and cellular changes in the perirhinal cortex and hippocampus (Ennaceur and Delacour, 1988; Clark et al., 2000; Brown and Aggleton, 2001; Callaghan and Kelly, 2013). Several of these reports have suggested a role for the neurotrophin family of proteins, including NGF (Birch et al., 2013) and brainderived neurotrophic factor (BDNF; Bechara and Kelly, 2013; Seoane et al., 2011; Callaghan and Kelly, 2013) in this form of learning and memory. Here we extend this work to demonstrate a direct effect of NGF treatment on performance of a challenging version of this task and suggest that ability to perform the task is associated with cell proliferation in the hippocampus. We chose a challenging version of this task with a high cognitive load, such that control rats could not learn the task and a ceiling effect in performance would be avoided. The results shown here support other reports in the literature of the memory-enhancing effect of NGF (Conner et al., 2009; Walz et al., 2000), although these studies test spatial and inhibitory avoidance memory respectively. To our knowledge, this is the first study to directly assess the role of NGF on recognition memory in rats. Our findings add to the literature by showing that NGF plays a significant role in several different types of memory, although whether this effect would translate into nonhippocampus dependent memory is not known.
Fig. 3. Effect of chronic intracerebroventricular infusion of NGF on expression of TrkA mRNA and protein in the hippocampus. NGF treatment significantly increased expression of TrkA mRNA compared with control groups (*p < 0.05). In parallel, there was a significant difference in the expression of Trk A protein in the hippocampus of the NGF-treated group compared with VEH (*p < 0.05). Blot is representative image from experiment. n ¼ 6 in CON and n ¼ 5 bNGF & VEH, data expressed per b-actin expression as mean SEM.
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Fig. 4. Effect of chronic intracerebroventricular infusion of NGF on expression of synaptic vesicle proteins in the hippocampus There was a significant difference between groups in the expression of synapsin I in the contralateral hippocampus, with an increase in synapsin I in NGF-treated rats when compared with VEH (*p < 0.05). There was no significant difference between groups in the expression of synaptophysin in the contralateral hippocampus. Blots are representative images from experiment. n ¼ 6 in CON and bNGF, n ¼ 5 in VEH, data expressed per GAPDH expression as mean SEM.
Many previous such studies have focused on the potential efficacy of NGF to reverse or ameliorate existing deficits in memory due to ageing (Niewiadomska et al., 2006; Frick et al., 1997; Klein et al., 2000), traumatic brain injury (Dixon et al., 1997) or ischaemia (Yang et al., 2011), frequently using significantly higher doses than the 4 ng/h used in the present study. This dose was chosen in order to mirror the final concentration of NGF that we observed as a result of six weeks of environmental enrichment (Birch et al., 2013), thus allowing us to directly compare the results
from each study. Here we show that this physiologically-relevant dose of NGF enhances recognition memory in young healthy rats. Moreover, we did not observe negative side effects often associated with high-dose NGF treatment, including neuropathic pain and weight loss (Eriksdotter Jonhagen et al., 1998; data not shown). Analysis revealed a significant increase in the concentration of NGF in the contralateral hippocampus of the NGF-treated group, confirming the efficacy of the delivery of NGF to the target region. The lateral ventricles lie adjacent to the hippocampus and therefore
Fig. 5. Effect of chronic intracerebroventricular infusion of NGF on cell proliferation in the dentate gyrus. There was a significant difference between groups in the percentage of BrdUþ nuclei however this is a trend toward an increase in the number of BrdUþ nuclei in rats infused with NGF compared with control groups (p ¼ 0.058). (B) Representative images of BrdU staining, with dentate gyrus (DG) labelled on whole hippocampus image to show region of interest (from Kjonigsen et al., 2011). Scale bar represents 100 mm. n ¼ 6 in CON and bNGF, n ¼ 5 in VEH; data expressed as mean SEM.
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an increase in the concentration of NGF in the hippocampus contralateral to the infusion also suggests that the infused NGF is traversing the ventricular system and potentially impacting on adjacent regions relevant to performance of the object recognition task, such as the parahippocampal and perirhinal cortices. We observed no change in expression of NGF mRNA, indicating that the increase in concentration of NGF protein observed in the hippocampus is not as a result of increased gene transcription induced by NGF, but that the infused NGF is reaching its intended target site and being taken up into hippocampal cells. In parallel with the increase in NGF concentration observed in the contralateral hippocampus in the NGF-treated group, we also observed an increase in TrkA receptor protein and mRNA expression. It is likely that this upregulation is a functional response to the increased availability of NGF in the region to enhance the amount of TrkA available to bind to NGF and induce intracellular signalling cascades. Induction of TrkA mRNA and protein by NGF has previously been reported in the literature (Decouto et al., 2003; Silver et al., 2001). Increased expression of both ligand and receptor in the hippocampus is likely to lead to an enhancement of NGF-TrkA signalling and subsequent changes in plasticity. Stimulating Trk expression is associated with induction of immediate early genes such as c-fos, although NGF-TrkA signalling seems to have a greater impact on neuronal maturation rather than functional differentiation into neuronal phenotypes (Takahashi et al., 1999). Given the six-week timeframe of NGF treatment in the current experiment, we considered that the mechanisms underlying the NGF-induced improvement in learning and memory were likely to be protein synthesis-dependent effects on cell morphology and function. In this context, we hypothesised that the possible underlying mechanisms may include synaptogenesis and neurogenesis. We report here that continuous NGF infusion resulted in a significant increase in the expression of synapsin I, but not synaptophysin, in the hippocampus of bNGF-infused rats. Synapsin I is crucial for synaptic transmission, in particular in the clustering and release of synaptic vesicles into the active zone of the synaptic bouton (Cesca et al., 2010) but is also used as a marker of synaptogenesis (Xu et al., 2010) Phosphorylation of synapsin I via ERK is known to be vital for neurotransmitter release and maintenance of the synaptic vesicle pool (Jovanovic et al., 1996; Giachello et al., 2010; Shupliakov et al., 2011). Activation of TrkA by NGF can result in stimulation of ERK phosphorylation (Patapoutian and Reichardt, 2001), suggesting that NGF can stimulate enhanced synaptic plasticity, which could facilitate enhanced memory performance. The effects of NGF on memory seem to be independent of expression of synaptophysin in the hippocampus. Synaptophysin is the most abundantly expressed synaptic vesicle protein, hence its common use as a marker for synaptogenesis. However, it is not vital for neurotransmission (Mcmahon et al., 1996). This indicates that the NGF-induced enhancement of synaptic plasticity is likely to occur via more efficient transmitter release rather than an increase in the number of functional synapses. In addition to the selective NGF-induced increase in synaptic vesicle protein expression, we observed a significant difference between control, vehicle treated and NGF-treated groups in early cell survival in the dentate gyrus subfield of the hippocampus, suggesting that NGF may play a role in enhancing neuronal progenitor cell survival. While post-hoc analysis revealed that the number of BrdU positive cells in the dentate gyrus of the NGFtreated group was not significantly different from the control or vehicle-treated groups, we believe the p value of 0.058 represents a trend that suggests a potential direct effect of NGF on cell proliferation. We considered that the BrdUþ nuclei counted in this study are most likely to be proliferating neurons because they are mostly localised to the subgranular zone of the dentate gyrus, and therefore our results suggest that NGF can enhance early hippocampal
neuronal survival. However, BrdU is a non-specific proliferative marker and thus the proliferation of other cell types, such as microglia or astrocytes, may also be captured in these experiments. Also, the results presented here may only be limited to a small subsection of the dentate gyrus and may not reflect differences in proliferation in the whole structure. The literature suggests that NGF is important in the survival and growth of neurons rather than directly stimulating an increase in proliferation (Olson et al., 2006; Frielingsdorf et al., 2007; Zhu et al., 2011). We show that a continuous NGF infusion can increase neurogenesis in the dentate gyrus and that this may be associated with memory improvements, Previously, Frielingsdorf et al. (2007) found that six days of i.c.v. infusion of NGF did not increase cell proliferation, but that a longer infusion period of twenty days promoted increased survival of neurons. The present study therefore, provides the first evidence to suggest that a chronic NGF infusion may increase neurogenesis in the dentate gyrus and that this may in turn be associated with memory improvements. 5. Conclusion This study demonstrates that continuous intracerebroventricular infusion of a physiologically-relevant dose of NGF results in a significant improvement in recognition memory associated with upregulation of NGF-TrkA interaction and cell proliferation in the hippocampus. This study helps to further elucidate the role that NGF plays in memory and suggests an underlying role for NGF in enhancement of neuronal proliferation. Grant sponsors Health Research Board, Ireland. References Bechara, R.G., Kelly, A.M., 2013. Exercise improves object recognition memory and induces BDNF expression and cell proliferation in cognitively enriched rats. Behav. Brain Res. 245, 96e100. Birch, A.M., Mcgarry, N.B., Kelly, A.M., 2013. Short-term environmental enrichment, in the absence of exercise, improves memory, and increases NGF concentration, early neuronal survival, and synaptogenesis in the dentate gyrus in a timedependent manner. Hippocampus 23, 437e450. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248e254. Brown, M.W., Aggleton, J.P., 2001. Recognition memory: what are the roles of the perirhinal cortex and hippocampus? Nat. Rev. Neurosci. 2, 51e61. Callaghan, C.K., Kelly, A.M., 2013. Neurotrophins play differential roles in short and long-term recognition memory. Neurobiol. Learn. Mem. 10, 39e48. Cesca, F., Baldelli, P., Valtorta, F., Benfenati, F., 2010. The synapsins: key actors of synapse function and plasticity. Prog. Neurobiol. 91, 313e348. Clark, R.E., Zola, S.M., Squire, L.R., 2000. Impaired recognition memory in rats after damage to the hippocampus. J. Neurosci. 20, 8853e8860. Conner, J.M., Franks, K.M., Titterness, A.K., Russell, K., Merrill, D.A., Christie, B.R., Sejnowski, T.J., Tuszynski, M.H., 2009. NGF is essential for hippocampal plasticity and learning. J. Neurosci. 29, 10883e10889. Decouto, S.A., Jones, E.E., Kudwa, A.E., Shoemaker, S.E., Shafer, A.J., Brieschke, M.A., James, P.F., Vaughn, J.C., Isaacson, L.G., 2003. The effects of deafferentation and exogenous NGF on neurotrophins and neurotrophin receptor mRNA expression in the adult superior cervical ganglion. Brain Res. Mol. Brain Res. 119, 73e82. Dixon, C.E., Flinn, P., Bao, J., Venya, R., Hayes, R.L., 1997. Nerve growth factor attenuates cholinergic deficits following traumatic brain injury in rats. Exp. Neurol. 146, 479e490. Ennaceur, A., Delacour, J., 1988. A new one-trial test for neurobiological studies of memory in rats. 1: Behavioral data. Behav. Brain Res. 31, 47e59. Eriksdotter Jonhagen, M., Nordberg, A., Amberla, K., Backman, L., Ebendal, T., Meyerson, B., Olson, L., Seiger, Shigeta, M., Theodorsson, E., Viitanen, M., Winblad, B., Wahlund, L.O., 1998. Intracerebroventricular infusion of nerve growth factor in three patients with Alzheimer’s disease. Dement. Geriatr. Cogn. Disord. 9, 246e257. Frick, K.M., Price, D.L., Koliatsos, V.E., Markowska, A.L., 1997. The effects of nerve growth factor on spatial recent memory in aged rats persist after discontinuation of treatment. J. Neurosci. 17, 2543e2550.
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