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Keywords:

  • interleukin-2;
  • hippocampal neuron;
  • dendritic filopodia;
  • dendritic arborization;
  • spines

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. LITERATURE CITED

In this study, we investigated the effects of interleukin-2 (IL-2) on dendritic filopodia, dendritic arborization, and spine maturation during the development of cultured rat hippocampal neurons. The cultured hippocampal neurons were transfected with F-GFP (farnesylated enhanced green fluorescent protein) at DIV5 to display the subtle structure of dendrites, and were then treated with IL-2 at various concentrations for different time before living cell image observation. We found that both the dendritic arborization and the length of dendrites per neuron at DIV7, DIV10, and DIV14 were increased under IL-2 treatment in a dose-dependent manner, and the strongest IL-2 effects on both dendritic number and length were observed at DIV7. Also, there was a significant increase in the mobility of dendritic filopodia in neurons at DIV7 treated with 10 ng/mL IL-2 for 48 hr from DIV5. In addition, IL-2 caused an increase in spine density of neurons at DIV14 either treated with IL-2 from DIV5 to DIV7 or from DIV5 to DIV14, but did not affect neurons treated from DIV12 to DIV14. These results indicate that IL-2 affects the dendritic development and spinogenesis of cultured hippocampal neurons, especially during the early developmental stage. Anat Rec, 2010. © 2010 Wiley-Liss, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. LITERATURE CITED

Interleukin-2 (IL-2), a 15–20 kDa single-chain glycoprotein, is a well-known cytokine that plays important roles in multiple immunoregulatory functions related to T-cells and in the central nervous system (CNS) (Hanisch and Quirion, 1995; Jiang and Lu, 1998; de Araujo et al., 2009). IL-2 produced by peripheral lymphocytes can access the CNS across the blood-brain barrier by a nonsaturable transport mechanism (Waguespack et al., 1994). IL-2 in the brain is potentially produced by neurons and astrocytes and is widely distributed with its receptors throughout the brain (Luber-Narod and Rogers, 1988; Lapchak et al., 1991; Eizenberg et al., 1995; Kowalski et al., 2004). It is also considered that brain-derived IL-2 may be involved in neuroregulatory effects and CNS disorders (Jiang and Lu, 1998). IL-2 has multiple effects on hippocampal neurons where receptors for this cytokine are enriched, for example, affecting cognitive behavioral performances in animals (Nemni et al., 1992; Hanisch et al., 1997; Lacosta et al., 1999), and modifying cellular and molecular substrates of learning and memory such as long-term potentiation (Tancredi et al., 1990). IL-2 can provide trophic support to primary cultured neurons from several regions of the rat brain including the hippocampus (Awatsuji et al., 1993; Sarder et al., 1993; Beck et al., 2005) and positively affect the morphology of neurite branching of rat hippocampal cultures (Sarder et al., 1993; Sarder et al., 1996). IL-2 may play roles in the development and regulation of neurons and have impact on spatial learning and memory (Sarder et al., 1993; Lacosta et al., 1999); exogenous administration of IL-2 induces cognitive impairment (Lacosta et al., 1999). IL-2 knockout (IL-2 KO) mice exhibit cytoarchitectural alterations in the hippocampus (Beck et al., 2005), impaired learning and memory performance, and altered hippocampal neurodevelopment (Petitto et al., 1999) because of the loss of IL-2. Changes of neuronal morphology and connectivity have been proposed as a plausible mechanism involved in behavioral changes (Kolb et al., 1998; Petitto et al., 1999). Thus, morphological studies on developing neurons in vitro by living cell imaging is preferred for further investigating the cellular and molecular mechanisms underlying the effects of IL-2 on brain functions.

In this study, we attempted to determine whether and how treatment with IL-2 at different concentrations during neuronal development from 5 days in vitro (DIV5) to DIV14 interferes with dendritic development and spinogenesis in cultured hippocampal neurons by living cell imaging.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. LITERATURE CITED

Chemicals

Recombinant rat IL-2 was purchased from Peprotech (Rocky Hill, NJ) and all other chemicals were from Sigma Chemical (St. Louis, MO) unless otherwise specified. Solutions were prepared immediately before each experiment.

Primary Hippocampal Neuronal Cultures and Neuronal Transfections

The care and use of animals in these experiments followed the guidelines and protocol approved by the care and use of animals committee of the Chinese academy of sciences. Primary cultures of hippocampal neurons were prepared from postnatal one-day-old Sprague-Dawley rats as previously reported (Luo et al., 2002; Ning et al., 2007). Briefly, after careful dissection from diencephalic structures, the hippocampi were chopped and then digested in 0.25% trypsin for 15 min at 37°C. Dissociated cells were plated at a density of 1 × 105 cells/cm2 on poly-L-lysine coated coverslips in a 35 mm dish in Dulbecco's modified Eagle medium (DMEM; Invitrogen, Carlsbad, CA) containing 10% fetal bovine serum, 10% horse serum, 2 mM glutamine (all from Invitrogen), and 1% antibiotic, and incubated at 37°C in 5% CO2. After culturing in vitro for 24 hr, the medium was replaced with DMEM containing 5% horse serum, 2 mM glutamine, 1% B27 and 1% N2 (Invitrogen). After another 24 hr, half of the medium was replaced with Neurobasal medium containing 2% B27 supplement, 1% antibiotic, and 2 mM glutamine (Invitrogen). At DIV5, cytosine arabinofuranoside was added at a final concentration of 10 μM. Thereafter, half of the medium was replaced twice a week with Neurobasal medium containing 2% B27 supplement, 1% antibiotic, and 2 mM glutamine. Hippocampal neurons were routinely transfected with F-GFP (3 μg/dish) by Lipofectamine 2000 (Invitrogen) at DIV5 (Ning et al., 2007). For dendritic branch imaging, hippocampal neurons were treated with IL-2 at 1, 10, or 100 ng/mL for 48 hr before image acquisition for dose-dependent analysis and were treated with 10 ng/mL IL-2 for 24, 48, or 72 hr before image acquisition for time-dependent analysis. Neurons were treated with 10 ng/mL IL-2 from DIV5 to DIV7 for dendritic filopodia image acquisition and from DIV5 to DIV7, from DIV5 to DIV14 or from DIV12 to DIV14 for spine image acquisition. Control neurons were treated the same using medium without IL-2.

Image Acquisition and Analysis

The image acquisition and analysis were performed as previously reported (Ning et al., 2007). The cultures were perfused in a recording chamber with an extracellular solution containing (in mM):NaCl 145, KCl 3, HEPES 10, CaCl2 3, glucose 8, MgCl2 2 (310 Osm, pH adjusted to 7.30 with NaOH) (Shen et al., 2006; Xu et al., 2006). Nikon bandpass filter cubes (Tokyo, Japan) were used for detecting GFP. Living neurons transfected with F-GFP were imaged on a TE2000 inverted microscope (Nikon, Japan), equipped with a 40 × 1.0 NA objective lens. Digital images were acquired with a CCD camera (CoolSNAP HQ, Roper Scientific, Tucson, AZ) controlled by MetaMorph 7.0 software (Universal Imaging, West Chester, PA). Each recording was acquired from the same level of focus. Stacks were collected every 30 sec. All images were captured within 30 min after leaving the culture medium with or without IL-2 treatment.

For branches, we adopted the terminology used by Havton and Ohara (1993). The dendrite derived directly from the soma is the first-order branch or primary dendrite, and the daughter branches derived from primary dendrites are second-order branches, and so on. Pictures were acquired with a 20× objective. The lengths of all dendritic branches were manually traced and measured with MetaMorph 7.0 (Universal Imaging, West Chester, PASA).

Eleven stacks of images were acquired for each neuron to measure filopodia motility (40× objective). Motility was calculated as the absolute difference between lengths of a same protrusion from frame to frame, divided by the total imaging time. We selected thin protrusions whose length exceeded 3 μm as filopodia, and its extension or retraction was more than 0.8 μm/min as mobile filopodia (Ning et al., 2007). The density of filopodia was calculated based on the number of filopodia on all second- and third-order branches and their total length in a neuron, expressed as filopodia per 100 μm. The length of all filopodia was manually traced and measured with MetaMorph 7.0.

For spines, images were acquired with a 40× objective. Spine density was calculated based on the number of spines on all second- and third-order branches and their total length in a neuron, expressed as spines per 100 μm.

Statistics

Data were from three independent preparations of cell cultures. Origin (Microcal Software) and Excel (Microsoft, Seattle, WA) were used for data display and analysis. Statistical analyses were performed by SigmaStat (windows version 3.5) using Student's t-test and Two-way ANOVA. Data are expressed as mean ± SEM.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. LITERATURE CITED

IL-2 Increased Both Dendritic Branching and Length of Hippocampal Neurons in a Dose-Dependent Manner

Sample images from control neurons and neurons treated with IL-2 at various concentrations are shown in Fig. 1A. On average, the total numbers of dendritic branches at DIV7, DIV10, and DIV14 were 24.28 ± 0.72, 32.70 ± 1.26, and 39.53 ± 0.95, respectively, whereas the total lengths of branches at DIV7, DIV10, and DIV14 were 439.31 ± 19.21, 902.05 ± 36.37, and 1450.68 ± 41.80 μm, respectively, showing significant increase in branch total number and length during neuronal development (P < 0.05). After treatment for 48 hr, IL-2 increased both the arborization and the total length of dendritic trees in a dose-dependent manner (Figs. 1B,C). IL-2 (10 ng/mL) increased the total number of dendritic branches, causing 48.21 ± 10.74% (P < 0.001), 17.79 ± 5.64% (P < 0.01) and 21.64 ± 4.89% (P < 0.05) increases at DIV7, DIV10, and DIV14, respectively, whereas the total lengths of branches were increased 104.80 ± 9.05% (P < 0.001), 35.04 ± 7.01% (P < 0.01) and 24.48 ± 5.83% (P < 0.05) at DIV7, DIV10, and DIV14, respectively. The enhancing effects of IL-2 on arborization and dendritic growth were time-dependent (data not shown). The effects of IL-2 on both branch number and length were more pronounced at DIV7 than at DIV10 and DIV14 (P < 0.001), suggesting that effects of IL-2 depend on neuronal development stage.

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Figure 1. IL-2 increased both branching and dendritic growth in a dose-dependent manner. (A) Sample images of control and IL-2-treated neurons at DIV14. (B) Dendritic branching of neurons at DIV7, DIV 10, and DIV14 profoundly increased after IL-2 treatment. (C) Dendritic length of neurons at DIV7, DIV10, and DIV14 also significantly increased after IL-2 treatment. (D) The number of dendritic branches of the second, third, and fourth order was notably increased at DIV14 after 10 ng/mL IL-2 treatment for 48 hr. (E) Average segment lengths for most dendritic orders at DIV14 were notably increased after IL-2 treatment. *P < 0.05, **P < 0.01, ***P < 0.001 versus control neurons in each group; +P < 0.05 versus DIV7 neurons (two-way ANOVA). 20 neurons were measured for each independent culture. Scale bar is 50 μm in (A).

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To further investigate the influence of 10 ng/mL IL-2 treatment on the branching and growth of dendritic arbors, we chose neurons at DIV14 for detailed analyses, because neurons at this age have passed the crucial developmental period and are relatively mature (Ning et al., 2007). We analyzed the number and the average length for each order of dendritic arbor. The number of dendritic branches of the second, third, and fourth order was notably increased after 10 ng/mL IL-2 treatment, and for most dendritic orders, the average length in the treated neurons was notably longer than the control neurons (Figs. 1D,E).

IL-2 Notably Increased the Motility of Dendritic Filopodia

Then we analyzed the effects of IL-2 on the density and motility of dendritic filopodia. Dendritic filopodia in control neurons were distributed along dendritic shafts at a density of 6.65 ± 0.26/100 μm and had an average length of 6.06 ± 0.39 μm. The ratio of mobile/total filopodia was 49.96 ± 18% (Figs. 2, 3). Treatment with 10 ng/mL IL-2 for 48 hr from DIV5 to DIV7 notably increased the motility of dendritic filopodia, causing a 30.93 ± 12.83% increase in motile filopodia/100 μm (P < 0.001, Fig. 3B) and a 44.18 ± 7.81% increase in the ratio of mobile/total filopodia (P < 0.001, Fig. 3D). However, the density and average length of filopodia were unaltered by IL-2 treatment (P > 0.05, Figs. 3A,C).

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Figure 2. Sample images of control and 10 ng/mL IL-2-treated neurons. (A) Control hippocampal neuron (DIV7). (B) Selected images of a time-lapse sequence of dendrites from neuron in (A). Images were acquired every 30 sec. (C) Hippocampal neuron (DIV7) treated with 10 ng/mL IL-2 from DIV5 to DIV7. (D) Time-lapse movie of dendrites from neuron in (C). Arrows in (B) and (D) indicate motility of a typical dendritic filopodium. Scale bar is 20 μm in (A, C) and 5 μm in (B, D).

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Figure 3. 10 ng/mL IL-2 treatment significantly increased the motility of dendritic filopodia. Motile filopodia (B) and the motility of filopodia (D) were increased by 10 ng/mL IL-2 treatment from DIV5 to DIV7. Density of filopodia (A) and average length of filopodia (C) were unaffected. ***P < 0.001 versus controls (Student's t-test). 18 neurons were measured for each independent culture. The total number of filopodia counted was 1,572.

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IL-2 Increased the Density of Dendritic Spines

As the motility of filopodia was increased by IL-2, we then examined whether IL-2 affected the density of dendritic spines in mature neurons at DIV14. The cultured neurons were treated with 10 ng/mL IL-2 from DIV5 to DIV7, from DIV5 to DIV14 and from DIV12 to DIV14, and then were assessed at DIV14. IL-2 caused an increase in spine density at DIV14 when treated from DIV5 to DIV7 or from DIV5 to DIV14; 12.54 ± 3.78% (P < 0.05) and 24.04 ± 7.12% (P < 0.01), respectively. But IL-2 treatment from DIV12 to DIV14 had no effect on spine density (P > 0.05) (Figs. 4A–C).

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Figure 4. Early treatment of IL-2 increased the density of spines in neurons at DIV14. (A) Typical neuron from control culture. (B) Example neuron after treatment with 10 ng/mL IL-2 from DIV5 to DIV7. (C) Quantification of dendritic spine density from control neurons and neurons treated with IL-2 from DIV5 to DIV7, from DIV5 to DIV14, and from DIV12 to DIV14. Scale bar is 20 μm for upper panels and 5 μm for lower panels. *P < 0.05, **P < 0.01, versus controls (Student's t-test). 18 neurons were measured for each independent culture. The total number of spines counted was 3,933.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. LITERATURE CITED

In this study, we have demonstrated that exogenous treatment with IL-2 induced major changes in dendritic branches, filopodia mobility, and spine density in cultured rat hippocampal neurons. First, IL-2 increased the dendritic arborization and growth of neurons at different developmental stages, with the most marked effects at DIV7. Second, IL-2 increased the motility of dendritic filopodia at DIV7. Third, IL-2 treatment at early stages of neuronal development increased spine density at DIV14. All of these results suggest that IL-2 has promotive effects on dendritic development and spinogenesis in cultured hippocampal neurons, especially at early developmental stages.

Accumulating evidence suggests that IL-2 plays a role in the development and survival of neurons from brain regions involved in spatial learning and memory (Petitto et al., 1999). IL-2 provides trophic support for primary cultured hippocampal neurons, promoting their development and survival (Awatsuji et al., 1993; Sarder et al., 1993), and has multiple effects on their morphology, such as promoting the elongation and branching of neurites in both normal and damaged neurons (Sarder et al., 1993; Sarder et al., 1996). The hippocampus is a region with high plasticity (McEwen, 1999; Breedlove and Jordan, 2001), and hippocampal changes such as fluctuations in dendritic complexity, spine density, and soma sizes are associated with learning and memory performance (Schwegler et al., 1996a; Schwegler et al., 1996b; Woolley, 1998). Many nutrients and stresses affect the formation and function of neuronal circuitry by interfering with the developmental progress of dendrites. All of these findings suggest that IL-2 has an impact on dendritic development and spinogenesis in cultured hippocampal neurons. Our study has shown that IL-2 increased both number and total length of dendritic branches at DIV7, DIV10, and DIV14, with the strongest effects on hippocampal neurons at DIV7, in a dose-dependent manner. This result is consistent with previous studies showing that many intracellular and extracellular factors have effects on both dendritic arborization and neuronal growth (Rajan and Cline, 1998; Rosso et al., 2005). The dendrite is the structure for receiving input signals and the location for integrating and exchanging synaptic information, and the length, diameter and arborization of dendrites are all crucial to neuronal function. Increases in dendritic branching and elongation may lead to enhancement of signal transmission among neurons, consequently enhance local circuit function, changes in these processes may lead to developmental and behavioral disorders (Miller and Jacobs, 1984; Ning et al., 2007). For an example, Greenough and colleagues found that both pyramidal and stellate neurons in trained rats have significantly increased dendritic arbors relative to untrained control rats (Chang and Greenough, 1982; Kolb et al., 2003).

In addition, we found that the enhancing effects of IL-2 on both branch number and length were strongest on neurons at DIV7, that is, the early developmental stage in culture. It is well-known that the crucial developmental stage for dendrites in cultured neurons is from DIV7 to DIV14, thus our results indicated that the promoting effects of IL-2 on dendritic development may be different at different times, with the strongest effects in the early development of neurons in culture.

We also showed that 10 ng/mL IL-2 notably increased the motility of dendritic filopodia but not their density or average length at DIV7 after treatment for 48 hr from DIV5. Dendrite filopodia, which are highly dynamic and occur predominantly during early development stage of the mammalian CNS, are thought to be responsible for the formation of dendritic spines and synapses in later development (Dailey and Smith, 1996; Ziv and Smith, 1996; Fischer et al., 1998). As described in previous studies (Portera-Cailliau et al., 2003; Ning et al., 2007), rapid movements of filopodia were observed between DIV7 and DIV8, and then filopodia became shorter, less motile, more stable, and mostly forming spines around DIV14. As filopodia are involved in dendritic arborization and neuronal growth, the ability of IL-2 to increase filopodia motility could be a factor for promoting dendritic development (Vaughn, 1989; Sorra and Harris, 2000; Ning et al., 2007).

Moreover, dendritic filopodia may participate in the formation of spines and synapses. Rapid extensions and movements of filopodia are necessary for neurons to find new contact sites that can then evolve into nascent synapses and mature into functional synaptic connections (Vaughn, 1989; Ziv and Smith, 1996; Jontes and Smith, 2000). As the dendritic surfaces connect to and receives information from more than 95% of the synapses on a neuron, it is, therefore, reasonable to infer changes in synapse number from changes of dendritic extent and spine density that we can measure (Kolb et al., 1998). So the effects of IL-2 on the extent of dendritic arborization and spine density are highly correlated with the number of synapses. Consistently, our study showed that at DIV14 the spine density increased after IL-2 treatment from DIV5 to DIV7 and from DIV5 to DIV14, but treatment from DIV12 to DIV14 had no effect, indicating that IL-2 also promoted spinogenesis and the effect was more potent in early development in cultures. These results were consistent with the effects of IL-2 on dendritic development and filopodia motility. In general, spines increase the surface area of dendrites, the excitatory synaptic density, and the number of connections between neurons (Sorra and Harris, 2000). In rats, spine density in the dentate gyrus and CA1 region of the hippocampus is positively correlated with water-maze learning and memory performance (Pyter et al., 2005). Thus, the density of hippocampal spines may be an indication of the efficiency of the synaptic network involved in spatial learning and memory. In addition, IL-2-induced changes during dendritic development and spinogenisis might influence animal behavior. For example, studies in IL-2 knockout (IL-2 KO) mice report reduction in hippocampal infrapyramidal mossy fiber length (Petitto et al., 1999), a factor which correlates positively with spatial learning ability (Schwegler et al., 1988; Schwegler and Crusio, 1995), and impaired learning and memory performance and sensorimotor gating, indicating that loss of endogenous IL-2 results in abnormal development and behavior.

IL-2 is used as an antitumor agent in clinical, but its anticancer effects are often associated with cognitive disturbances (Vial and Descotes, 1992). Chronic administration of IL-2 has been found to impair spatial learning behavior (Lacosta et al., 1999). However, our study showed IL-2 had trophic effects in early development of neurons in culture. The effects of IL-2 may be differential from different developmental stages.

In conclusion, our study demonstrated that IL-2 promoted dendritic branching and elongation in cultured hippocampal neurons, and increased the motility of dendritic filopodia and spine density in a development-dependent manner. This helps us to further understand the effects of IL-2 on the development of hippocampal neurons. However, the possible molecular and cellular mechanisms of the effects in vivo remain to be determined.

LITERATURE CITED

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. LITERATURE CITED