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. Author manuscript; available in PMC: 2013 Apr 1.
Published in final edited form as: Neurobiol Aging. 2011 Sep 9;33(4):832.e1–832.e14. doi: 10.1016/j.neurobiolaging.2011.07.015

Aging and infection reduce expression of specific BDNF mRNAs in hippocampus

Timothy R Chapman 1, Ruth M Barrientos 1, Jared T Ahrendsen 1, Jennifer M Hoover 1, Steven F Maier 1, Susan L Patterson 1
PMCID: PMC3237944  NIHMSID: NIHMS316871  PMID: 21907460

Abstract

Aging increases the likelihood of cognitive decline after negative life events such as infection or injury. We have modeled this increased vulnerability in aged (24-month-old), but otherwise unimpaired F344xBN rats. In these animals, but not in younger (3-month-old) counterparts, a single intraperitoneal injection of E. coli leads to specific deficits in long-term memory and long-lasting synaptic plasticity in hippocampal area CA1 –processes strongly dependent on BDNF. Here we have investigated the effects of age and infection on basal and fear-conditioning-stimulated expression of Bdnf in hippocampus. We performed in situ hybridization with six probes recognizing: total (pan-)BDNF mRNA, the four predominant 5' exon-specific transcripts (I, II, IV, and VI), and BDNF mRNAs with a long 3'untranslated region (3'UTRs). In CA1, aging reduced basal levels and fear-conditioning-induced expression of total BDNF mRNA, Exon IV-specific transcripts, and transcripts with long 3'UTRs; effects of infection were similar and sometimes compounded the effects of aging. In CA3, aging reduced all of the transcripts to some degree; infection had no effect. Effects in dentate were minimal. Northern blot analysis confirmed an aging-associated loss of total BDNF mRNA in areas CA1 and CA3, and revealed a parallel, preferential loss of BDNF mRNA transcripts with long 3′ UTRs.

Keywords: Aging, Inflammation, Interleukin-1, IL-1β, LTP, Learning, Memory, Hippocampus, Infection, BDNF mRNA, long 3′ UTR, Exons, transcripts, cytokines, Lipopolysaccharide, dendrites

Introduction

Cognitive deficits, particularly those related to learning and memory, are emerging as one of the major disorders of aging (Bishop et al.). Although it is not clear that these deficits are a normal feature of aging - some older individuals retain a very high level of cognitive function - their incidence continues to increase with increasing age. This occurs in part because aging renders the brain more vulnerable to a variety of physiological and psychological stressors (Wofford et al., 1996; Bekker and Weeks, 2003; VonDras et al., 2005). Because relatively little is known about the mechanisms that underlie aging-associated increases in cognitive vulnerability, we have developed a rodent model to study them (Barrientos et al., 2006).

As we have previously reported, the cognitive abilities of aging (24-month-old) F344xBN rats appear comparable to those of their younger (3-month-old) counterparts, however, they are much more vulnerable to the consequences of a peripheral immune challenge (an i.p. injection of live E. coli). Four days after the injection - after recovering from the active infection - the aging, but not the young rats show deficits in contextual fear conditioning, a hippocampus-dependent long-term memory task (Barrientos et al., 2006) and in theta burst-evoked, late-phase long-term potentiation (L-LTP) in the CA1 region of the hippocampus (Chapman et al., 2010), a form of long-lasting synaptic plasticity thought to be associated with consolidation of some spatial/contextual memories. These deficits are intriguingly specific. The infection does not compromise initial learning or formation of short-term memories, nor does it disrupt basal synaptic function or short-term synaptic plasticity in animals of either age. Together, these results suggest that age and a secondary immune challenge might selectively interfere with production of molecular substrates necessary for consolidation of memory-related synaptic plasticity without affecting more basic neuronal and synaptic functions.

In this study we investigated the impact of aging and infection on transcription of brain derived neurotrophic factor (BDNF), a molecule that has been shown to be important for consolidation of hippocampus-dependent memory, and for theta burst evoked L-LTP (Tyler et al., 2002; Bramham and Messaoudi, 2005; Lu et al., 2005). The transcriptional organization of the BDNF gene is complex. At least eight differentially regulated promoters give rise to multiple mRNA transcripts - each of which contains a distinct 5' exon spliced to a common 3' coding exon, and all of which encode an identical BDNF protein (Aid et al., 2007). Some of these promoters are strongly regulated by Ca+2-responsive transcription factors, like CREB, CaRF, and MeCP2 (Tao et al., 1998; Tao et al., 2002; Chen et al., 2003; Hong et al., 2008; West, 2008), and are activated in the hippocampus by behaviorally relevant stimuli like fear conditioning (Rattiner et al., 2004; Lubin et al., 2008). The functional significance of the different BDNF mRNA transcripts is not yet understood, but the different exons within these transcripts may help to orchestrate not only when BDNF can be made, but also for how long, and where in the cell (Tongiorgi et al., 1997; Righi et al., 2000). In addition, regardless of which promoter is activated, the gene uses two alternative polyadenylation sites, leading to mRNAs with either short or long 3′ unstranslated regions (UTR). Recent data suggest that the BDNF mRNAs with the short 3'UTRs are restricted to the cell body, whereas those with the long 3'UTRs are also trafficked to dendrites (An et al., 2008). Thus, the BDNF gene can create at least 16 different mRNA transcripts with distinct 5′ and 3′ exons that might differently contribute to synaptic plasticity and memory consolidation.

Although activity-dependent BDNF transcription and trafficking has been repeatedly linked to physiological events like fear conditioning (Rattiner et al., 2004; Lubin et al., 2008), seizures (Kokaia et al., 1994; Lauterborn et al., 1996), and ischemia (Kokaia et al., 1995), very little is known about how expression of specific transcripts might be affected by aging and or infection. In the study presented here, we have used in situ hybridization to examine the impact of a peripheral immune challenge on expression of specific BDNF transcripts in the hippocampal subfields of young and aged rats in the basal state, and one hour after contextual fear conditioning. In addition, we performed Northern blot analysis to investigate the possibility that age and or infection might differentially affect levels of transcripts with short or long 3′ UTRs.

Material and Methods

Animals

Rats were 3- and 24- month old male Fisher344/Brown Norway F1 hybrids from the NIA Aged Rodent Colony. They were allowed to acclimate to the animal facility for at least two weeks before experiments began. The animals were pair housed, on a 12-hr light dark cycle, with ad libitum access to food and water. All experiments were conducted in accordance with protocols approved by the University of Colorado Animal Care and Use Committee.

E. coli Infection

Stock E. coli cultures (ATCC 15746; American Type Culture Collection, Manassas, VA) were thawed and cultured overnight (15–20 hours) in 40 ml of BHI (DIFCO) at 37°C. Growth of individual cultures was quantified by extrapolating from previously determined growth curves. Cultures were centrifuged for 15 min at 3000 rpm, supernatant discarded, and bacterial pellets were resuspended in sterile PBS to give a final concentration of 1.0 ×1010 CFU/ml. All animals received an intraperitoneal (i.p.) injection of 250 µl of either E. coli or vehicle (PBS).

Fear Conditioning and Collection of Brains for In Situ Hybridization

All brains were collected 4 days after the injections. This time point was chosen because all of the animals have fully recovered from the acute infection (E.g. fever has subsided)(Barrientos et al., 2009), and the aging, but not the young E. coli-injected rats show significant impairments in long-term memory (Barrientos et al., 2006) and theta burst evoked L-LTP (Chapman et al., 2010).

Rats used for examination of the effects of age and infection on basal levels of BDNF transcripts were left undisturbed in their home cages prior to sacrifice. Rats used to assess the impact of age and infection on the capacity to recruit BDNF mRNAs in response to fear learning were taken two at a time from their home cage and placed in a conditioning chamber. They were allowed to explore the chamber for 2 min before the onset of a 2-s footshock (1.4mA), and they were decapitated one hour later. All brains were flash frozen in isopentane maintained at −30° immediately following removal.

Probe Generation and In Situ Hybridization

We choose to analyze only exon I, II, IV, and VI because they comprise most of the BDNF expression in the adult rat brain (Aid et al., 2007). The following bdnf exon-specific sequences according to Aid et al. (Aid et al., 2007) were generated by standard RT-PCR and subcloned into pSC-A plasmid (Stratagene); exon I (Genbank EF125675.1, nt 242–715), exon IIc (EF125678.1, nt 18–534), exon IV (EF15679.1 nt 16–415), exon VI (EF125680.1, nt 27–423), exon 1X, i.e. coding domain (EF125675.1, nt 661–1410), long 3′ UTR (EF125675.1, nt 2956–3466). Radiolabeled riboprobes were generated by an in vivo transcription reaction consisting of 1.0 ug linearized plasmid DNA, 400 µM GTP, CTP, and ATP, 93.75 µCi UTP[35S] 10mM DTT, 17U T3 RNA polymerase (Promega), 10U RNase Inhibitor (Invitrogen); and incubated at 37°C for 1.5 hrs. Plasmid DNA was removed by DNase I (Promega) and nascent radiolabeled RNA was purified over a gravity-flow G-50 Sephadex column.

Coronal brain sections (10 µm) were sliced on a Leica cyrostat (model 1850), mounted onto poly-l-lysine-coated slides, fixed in 4% paraformaldehyde for 1 hr at room temperature, and washed three times in 2X SSC. Slides were then acetylated in 0.1M triethanalomine, 0.25% (vol/:vol) acetic anhydride for 10 min, washed twice in water, dehydrated in graded ethanol solutions and allowed to air-dry. RNA probes were diluted in hybridization buffer (50% deionized formaldehdye, 3X SSC, 50mM sodium phosphate (pH 7.4), 10% dextran sulfate (w/v), 1X Denhardt’s solution, 0.2mg/ml yeast tRNA) to give a final concentration of 1.0–2.0×106 cpm (exon-specific) per 65ul aliquot added to each slide. Sections were cover-slipped and hybridized overnight at 55°C in sealed trays with 50% formamide. Coverslips were removed and slides were washed three times in 2X SSC before incubating in 200µg/ml RNase A at 37°C for 1 hr to remove any unbound probe. Sections were washed with increasingly stringent SSC at room temperature, with a final wash in 0.1X SSC at 65°C for 1 hour. Sections were dehydrated in graded ethanol and exposed to X-ray film (Kodak) for 1–3 weeks.

Micro-Dissection And Northern Blot Analysis Of Hippocampal Subfields

Hippocampi were removed after decapitation and transverse slices (1 mm) were prepared using conventional techniques (Patterson et al., 1996). Slices were dissected under 10X magnification using a scalpel and fine tipped forceps. The mossy fiber projections were severed, and areas CA3 and CA1 were separate from the dentate along the hippocampal fissure. Specific tissue from one hippocampus was aggregated into separate tubes (CA1/CA3 or dentate), rapidly frozen in liquid nitrogen, and RNA was extracted using Trizol (Invitrogen). To validate the accuracy of each dissection, 1.0 ug of RNA was converted to cDNA and PCR was performed for C1ql2 (NM_001105949.1) – a gene with very strong expression in the dentate and low expression in the remainder of the hippocampus. 10 ug of total RNA from CA1/CA3 samples or 4 ug from dentate samples were mixed with loading buffer (50% formaldehyde, 22% formamide, 2X MOPS, 0.2% bromophenol blue, 0.2% xylene cyanol, 0.4 % EDTA), denatured at 80°C for 5 min, and separated on a 1.2 % agarose/formaldehyde gel. RNA was upwardly transferred overnight in 2X SSC to a Zeta-Probe nylon membrane (Bio-Rad) and cross-linked by UV light. A randomly-primed, 32P-labeled DNA probe was created from exon IX (EF125675.1, nt 661–1410) using the Rediprime II Lableing Kit (Amersham) and purified on an Illustra Microspin G-50 column (GE Healthcare). The membrane was prehybridized overnight in ULTRAHyb hybridization buffer (Ambion) and hybridized with 1×106 cpm of probe/ml of hybridization buffer at 55°C for 48 hours. The membrane was then washed twice for 15 min each in 2X SSPE/0.1% SDS, 0.5X SSPE/0.1% SDS, and finally 0.1X SSPE/0.1% SDS at 65°C. To normalize RNA loading and transfer, blots were stripped by rinsing three times with boiling 0.1% SDS and were re-hybridized for GAPDH (NM_017008.3, nt 29–1190) using the same conditions but for only 4 hrs.

Semi-Quantitative Analysis of mRNA

Brain section images developed on X-ray were digitally captured (CCD camera, model XC-77, Sony) and analyzed using Scion Image version 4.0. A macro written by Dr. Serge Campeau (University of Colorado) calculated signal as 3.5 × standard deviations above mean gray value of background, which was set as the cell poor region of the CA1 dendritic field for each image. The average signal and number of pixels was multiplied to give an integrated density value for each region of interest (dentate, CA3, CA1), and the average of 8–12 measurements per animal was taken from the dorsal half of the hippocampus and used to calculate mRNA levels. Northern blot images were exposed to phosphoimager screen (GE Healthcare) and analyzed by scanning laser densitometry using ImageQuant Software.

Statistical Analysis

All data is presented as a percentage of mean integrated density relative to young, vehicle-injected animals in a basal state. Data was analyzed by a 2×2×2 ANOVA (age × infection × fear conditioning) with significance set at p < .05, followed by Fisher’s Least Significance Difference (LSD) post-hoc comparisons.

Results

Total (pan-)BDNF mRNA

We have previously demonstrated that the combination of aging and a recent history of infection gives rise to specific deficits in forms of memory and memory-related synaptic plasticity (Barrientos et al., 2006; Chapman et al., 2010) known to be strongly dependent on BDNF (Schinder and Poo, 2000; Tyler et al., 2002; Chao, 2003; Lu, 2003; Bramham and Messaoudi, 2005). We began the studies described here by asking if these deficits could be correlated with changes in total BDNF gene expression the hippocampus, either in a basal state, or after a learning task. Young and aged rats were injected with either E. coli or saline and sacrificed four days later - either immediately after being taken from their home cages, or one hour after being subjected to mild fear conditioning. To examine levels of total BDNF mRNA in the three major hippocampal sub-regions (dentate, CA3, CA1), we performed in situ hybridization on the brains using a riboprobe designed to bind within exon IX, the protein-coding domain that is shared by all BDNF transcripts.

We found that expression of total BDNF mRNA in the CA1 region was most susceptible to the effects of aging and infection (Fig 1A). CA1 was also the only hippocampal sub-region that showed a response after fear conditioning (Fig 1A). Basal expression of total BDNF mRNA in CA1 was significantly reduced in aged animals (young/vehicle, 100.0 ± 7.5%, aged/vehicle, 55.1 ± 3.0%, p = .024); interestingly, the addition of infection tended to further reduce expression, but markedly increased it’s variability (aged/vehicle, 55.1 ± 3.0%; aged/infected, 29.9 ± 11.0%, p = .198). Infection produced a large reduction in young animals (young/vehicle, 100.0 ± 7.5%, young/infected, 39.2 ± 1.2%, p = .004) (Fig 1A). Interestingly, fear conditioning up-regulated total BDNF mRNA in young, but not in aged animals, regardless of immune status (young/vehicle, 200.6 ± 17.7%, p < .001, young/infected, 99.6 ± 4.5%, p = .004). Fear-conditioned levels were nearly 2-fold lower in young animals with a recent history of infection, but this might have been a residual effect of reduced basal levels rather than an infection-driven impairment in activity-dependent transcription. In contrast, the complete lack of response in the aged animals may reflect aging-dependent impairments in activity-dependent transcription.

Figure 1.

Figure 1

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Aging reduces levels of total BDNF mRNA in CA1 and CA3 and impairs fear conditioning-induced gene expression. (A) Basal expression in CA1 was reduced by both age (F1,43 = 56.6, p < .001) and infection (F1,43 = 29.3, p < .001). Fear conditioning elicited a ~2–3 fold increase in CA1 of young animals, but did not change levels in aged animals, (F1,43 = 17.1, p < .001) (B) Age reduced expression in CA3 (F1,44 = 116.5, p < .001) but infection did not. There was no effect of fear conditioning. (C) Neither age nor infection altered levels in the dentate. There was no effect of fear conditioning. (D–G) Representative in situ hybridization images of basal expression of total (exon IX) BDNF mRNA in hippocampi of (D) young vehicle, (E) young infected, (F) aged vehicle, and (G) aged infected animals. N = 5–8 animals/group. Error bars indicate SEM. *p < .001, age; **p < .001, infection; ***p < .001, fear conditioning.

In CA3, total BDNF mRNA levels were only reduced by age (young/vehicle, 100.0 ± 4.6%; aged vehicle, 69.5 ± 6.2%, p < .001), and levels were not susceptible to infection in either age group, nor were they affected by fear conditioning (Fig 1B). The dentate region, which robustly expresses BDNF mRNA, was the least vulnerable to age or infection, and did not respond to fear conditioning (Fig 1C).

Exon I mRNA

After determining that age and infection altered levels of total BDNF mRNA in the hippocampus, we decided to more thoroughly investigate their effects on alternative splicing of BDNF mRNA, which could reveal specific transcriptional influences of age and infection. Exon I has a calcium-responsive promoter and shows slow, but sustained transcription under constant neural activity (Hara et al., 2009). A riboprobe specific for exon I-bearing mRNAs, revealed very low expression in CA1, but greater expression in CA3 and dentate (Fig 2A–C). In sharp contrast to the effects on total BDNF mRNA levels in CA1, none of the conditions examined produced any detectable changes in exon I levels (Fig 2A). However, in CA3 (Fig. 2B) age reduced exon I mRNA expression by more than half, (young/vehicle, 100.0 ± 8.9%; aged vehicle, 39.7 ± 5.3%, p < .001), but infection had no effect in either age group. In the dentate, expression was not significantly altered by age (p = .105), but infection was associated with a reduction in exon I transcripts in aged animals following fear conditioning (aged/vehicle, 116.6 ± 5.9%, aged/infected, 73.6 ± 4.9%, p = .033) (Fig 2C).

Figure 2.

Figure 2

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Aging reduces BDNF exon I transcripts in CA3. (A) Expression in CA1 was not changed by age or infection, nor was there a detectable increase following fear conditioning. (B) Age severely reduced expression in CA3 (F1,38 = 293.2, p < .001), but infection did not. There was no effect of fear conditioning. (C) In dentate, age did not significantly alter exon I expression (p = .105); however, the addition of infection did cause a reduction in aged animals (F1,38 = 4.6, p = .038). (D–G) Representative in situ hybridization images of basal expression of exon I-derived mRNA in hippocampi of (D) young vehicle, (E) young infected, (F) aged vehicle, and (G) aged infected animals. N = 4–6 animals/group. Error bars indicate SEM. *p < .001, age; **p < .05, infection.

Exon II mRNA

The probe for Exon II revealed a pattern of expression in the hippocampus very similar to that of exon I (Fig. 3D–G). Although there was slightly more signal intensity in CA1, there was still no significant effect of age or infection (Fig 3A). In CA3, aged animals had less basal expression of exon II than young animals (young/vehicle, 100.0 ± 4.6%; aged/vehicle, 55.9 ± 0.4%, p < .001) (Fig 3B), and infection potentiated the reduction (aged/vehicle, 55.9 ± 0.4%; aged/infected, 28.1 ± 3.3%, p = .004). In aged animals, levels of exon II were slightly elevated in dentate (p < .001), but they were reduced by infection (aged/vehicle, 117.4 ± 6.7%, aged/infected, 77.1 ± 6.3%, p < .001). Fear conditioning had no effect (Fig 3C).

Figure 3.

Figure 3

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Aging reduces BDNF exon II transcripts in CA3. (A) Expression in CA1 was not significantly altered by age, infection, or fear conditioning. (B) Age severely reduced expression in CA3 (F1,37 = 205.8, p < .001). Infection further reduced expression in aged animals,(F1,37 = 6.8, p = .013). (C) Expression in the dentate was decreased by infection in aged animals only (F1,37 = 4.7, p = .036). There was no effect of fear conditioning. (D–G) Representative in situ hybridization images of basal expression of exon II-derived mRNA in hippocampi of (D) young vehicle, (E) young infected, (F) aged vehicle, and (G) aged infected animals. N = 5–6 animals/group. Error bars indicate SEM. *p < .001, age; **p < .05, infection.

Exon IV mRNA

The classification of BDNF as an immediate early gene is largely due to the rapid and robust transcription of exon IV following neural activity. In area CA1, transcripts containing exon IV were expressed at much higher levels than those containing exons I and II. We found that aging reduced basal expression of exon IV mRNA by more than half in both CA1 (young/vehicle, 100.0 ± 2.5%; aged/vehicle, 47.4 ± 5.2%, p < .001)(Fig 4A) and CA3 (young/vehicle, 100.0 ± 5.1%; aged/vehicle, 48.6 ± 4.9%, p < .001) (Fig 4B). Infection also decreased basal expression in CA1 of young animals (young/infected, 67.6 ± 4.4%, p < .01), but did not further reduce levels in aged animals. There was a significant increase in the CA1 region after fear conditioning; but again, this only occurred in young animals (percent increase above basal: young/vehicle, + 50.1 ± 5.7%; young/infected, + 31.1 ± 4.0%, p < .001). The dentate did not change with any of the conditions examined (Fig 4C). Therefore, it appears that aged animals have reduced constitutive and activity-dependent expression of exon IV mRNA in CA1 and CA3.

Figure 4.

Figure 4

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Aging reduces BDNF exon IV transcripts in CA1 and CA3, and impairs fear-conditioning-induced expression. (A) Basal expression in CA1 is significantly reduced by age (F1,38 = 451.0, p < .001). Although infection caused a decrease in young animals (F1,38 = 26.3, p < .001), it did not further reduce expression in aged animals. Fear conditioning elicited a ~0.3–0.5 fold increase above basal levels in CA1 of young animals (F1,38 = 8.1, p = .007), but did not increase expression in aged animals. (B) In CA3, age reduced expression (F1,48 = 146.1, p < .001), but infection did not. There was no effect of fear conditioning. (C) Expression in DG was not altered by age of infection. (D–G) Representative in situ hybridization images of exon IV-derived mRNA expression in hippocampi of (D) young vehicle, (E) young infected, (F) aged vehicle, and (G) aged infected animals. N = 6–8 animals/group. Error bars indicate SEM. *p < .001, age; **p < .001, infection; ***p < .01, fear conditioning.

Exon VI mRNA

Little is known about the regulation of BDNF gene expression from exon VI. Our riboprobe revealed that exon VI has an expression pattern in the hippocampus (Fig. 5D–G) similar to Exon IV. We found that basal expression of exon VI mRNA in CA1 (Fig. 5A) was significantly reduced by age (young/vehicle, 100.0 ± 8.1%; aged/vehicle, 47.4 ± 5.2%, p < .001), although this change was not as large as with other BDNF exons. Fear conditioning significantly increased expression in only in CA1 of young animals that had not received an infection (percent over basal: young/vehicle, + 55.8 ± 16.1%, p = .02). Age also reduced expression in CA3 (Fig. 5B), but only significantly in the fear conditioned group (basal: young/vehicle, 101.9 ± 9.6%; aged/vehicle, 58.3 ± 3.3%, p < .122; fear conditioned: young/vehicle, 100.0 ± 12.1%; aged/vehicle, 72.1 ± 10.8%, p < .001) (Fig 5F). In the dentate of aged animals (Fig. 5G), there was a significant reduction in basal levels after infection (aged/vehicle, 125.2 ±12.6%; aged/infected, 79.5 ± 15.6, p < .004), but this difference was not significant after fear conditioning (p = .284). In summary, Exon VI expression was reduced in CA1, and CA3 with aging, but not to same extent as the other exons examined here.

Figure 5.

Figure 5

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Aging reduces BDNF exon VI transcripts in CA1 and CA3. (A) Basal expression in CA1 was moderately decreased by age (F1,31 = 22.5, p < .001), but was not significantly altered by infection. Fear conditioning increased expression only in young animals without an infection (p = .02). (B) Age reduced expression in CA3 (F1,31 = 48.43, p < .001). Infection also tended to reduce expression (F1,31 = 3.5, p < .07), but only in aged animals. There was no effect of fear conditioning. (C) Expression in DG was not significantly altered by age or infection. (D–G) Representative in situ hybridization images of basal expression of exon VI-derived mRNA in hippocampi of (D) young vehicle, (E) young infected, (F) aged vehicle, and (G) aged infected animals. N = 5–6 animals/group. Error bars indicate SEM. *p < .001, age; **p = .07, infection; ***p < .05, fear conditioning.

Long 3′ UTR mRNA

BDNF mRNAs are polyadelynated at two alternative sites, leading to mRNAs with either short or long 3′ untranslated regions (UTR). Recent findings suggest that BDNF mRNAs that carry the long 3′UTR may play a particularly important role in activity-dependent synaptic plasticity in hipocampal dendrites (Lau et al., 2010; An et al., 2008). We therefore designed a probe that binds to the untranslated region downstream of the first polyadenylation site and thus specifically to the larger, full-length BDNF transcripts. Our probe revealed that the hippocampal expression pattern of the long 3' BDNF mRNAs (Fig. 6) is quite similar to that of total BDNF mRNAs (Fig. 1). Aging severely reduced basal levels of the long transcripts in CA1 (p < .001) (Fig 6A). Infection produced a similar reduction in young animals (p < .001), but no further reduction in aged animals (p = .664)(young/vehicle, 100 ± 3.1%: young/infected, 31.0 ± 4.0%, aged/vehicle, 26.3 ± 6.0%: aged/infected, 19.2 ± 3.5%). Interestingly, fear-conditioning significantly increased levels of long 3' BDNF mRNAs in all of the young animals (both those with and without a recent history of infection, p< .001), and in the saline-injected aged animals (p = .001), but failed to do so in the E. coli-injected aged animals (p = .101) (percent increase over basal: young/vehicle, +92 ± 18.6%, young/infected +68.1 ± 7.2%, aged/vehicle, +52.3 ± 7.6%, aged/infected, +24.5 ± 4.8%).

Figure 6.

Figure 6

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Aging reduces long 3′ UTR BDNF transcripts in CA1 and CA3; and impairs fear-conditioning-induced expression of long 3′ UTR BDNF transcripts. (A) Basal expression in CA1 was severely reduced by both age (F1,42 = 74.1, p < .001) and infection (F1,42 = 47.1, p < .001); infection altered levels in young animals, and further reduced expression in aged animals. Fear conditioning elicited robust increases in expression in CA1 of young animals and aged/vehicle animals, but a more modest increase in CA1 of aged/infected animals (F1,42 = 63.6, p < .001). (B) In CA3, age reduced expression (F1,45 = 155.5, p < .001), but infection did not. There was no effect of fear conditioning. (C) Neither age or infection significantly altered levels in DG. There was no effect of fear conditioning. (D–G) Representative in situ hybridization images of basal expression of long 3′ UTR BDNF transcripts in hippocampi of (D) young vehicle, (E) young infected, (F) aged vehicle, and (G) aged infected animals. N = 6–8 animals/group. Error bars indicate SEM. *p < .001, age; **p < .001, infection; ***p < .001, fear conditioning.

In CA3 (Fig 6B), there was a significant reduction of long transcripts with age (young/vehicle, 100 ± 8.0%; aged/vehicle, 65.4 ± 6.5%, p<0.001); and although young animals displayed no change in CA3 after infection, aged animals did have a further reduction in levels of the long transcripts (aged/vehicle, 65.4 ± 6.5%; aged/infected, 49.7 ± 4.2%, p = .033). Again, expression in the dentate was not affected by age or infection (Fig. 6C). Overall, the effects of age and infection on expression of BDNF mRNAs with long 3′ UTRs resemble those on expression of total (pan-)BDNF mRNA, with the largest changes occurring in the CA1 and CA3 regions in aged animals.

Northern Blot Analysis of BDNF mRNA

The similarity of the expression pattern detected by the probe for total BDNF mRNA (which binds variants with long and short 3’UTRs) with that detected by the probe specific for mRNAs with a long 3’ UTR, suggested two alternative possibilities that could not be resolved using in situ hybridization - either a general reduction of mRNA of both sizes, or a more specific reduction in transcripts with the long 3’ UTR. To address this issue, we employed a Northern blot assay. BDNF mRNA is expressed at very high levels in the dentate, but our in situs revealed that this expression is largely unaffected by age or infection. We therefore dissected the dentate from areas CA1 and CA3 (CA1/3) to avoid diluting effects largely specific to the pyramidal cells (Fig. 7A). Sub-region specific mRNA was separated by size and probed using single-stranded DNA complementary to the coding domain of BDNF, revealing two distinct bands at ~2.0 kb and ~4.0kb (Fig. 4B), in agreement with previous reports (Lapchak et al., 1993b; Oyesiku et al., 1999). Consistent with the results of our in situs, aging was associated with significant reductions in BDNF mRNA expression in CA1 and CA3, but had no effect in the dentate (Fig. 8). Interestingly, the aging-evoked reduction of BDNF mRNA in the pyramidal layers proved to be fairly specific - there was nearly a two-fold greater decrease in BDNF mRNA containing a long 3’ UTR (young/vehicle, 100.0 ± 4.2%, aged/vehicle, 62.1 ± 4.6%, p < .001) than in mRNA with a short 3' UTR (young/vehicle, 100.0 ± 5.4% : aged/vehicle, 78.6 ± 5.8%, p < .001). In contrast to what we observed with our in situs, infection did not further change levels of total or long 3' UTR BNDF mRNA in either young or aged animals. However, this isn’t particularly surprising since the in situ data showed that infection compounded the effect of aging, but only in area CA1, and we used a combination of areas CA1 and CA3 for our Northern blots. Overall, aging leads to preferential depletion of BDNF mRNA containing a long 3’ UTR within the CA1 and CA3 regions.

Figure 7.

Figure 7

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Northern blot analysis of BDNF mRNA in hippocampal subregions. (A) Hippocampal slices (1.0 mm) were micro-dissected to separate the dentate from CA1 and CA3. (B) Equal amounts of total RNA from dentate and a combined sample of CA1 and CA3 were loaded together and probed for BDNF, revealing two bands at ~2 and ~4kb. (C) In young/vehicle animals, expression of total BDNF mRNA was more than 2-fold higher in the dentate than in CA1/CA3. GAPDH served as a loading control. To ensure that dissections were accurate, reverse- transcription PCR was performed for C1ql2 because it has enriched expression in dentate relative to CA1 and CA3 (B). N= 3 animals/group. Error bars indicate SEM.

Figure 8.

Figure 8

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Aging causes a preferential loss of BDNF mRNA containing the long 3’ UTR in CA1 and CA3 regions, but does not affect expression in dentate gyrus. (A,B) Aged animals have reduced levels of both BDNF mRNA transcripts in the CA1 and CA3 regions relative to young animals, F(1,28) = 61.9, p < .001. However, there is a significantly greater reduction of the long 3’ UTR relative to the short 3’ UTR. (C,D) There was no change in BDNF mRNA expression in dentate. Infection did not cause any changes detectable by northern blot. N = 6–8 animals/group. Error bars indicate SEM.

Discussion

We have previously demonstrated that in 24-month-old, but not in 3-month-old F344xBN rats, a single i.p. injection of E. coli leads to specific deficits in forms of long-term memory and long-lasting synaptic plasticity in hippocampal area CA1 (Barrientos et al., 2006; Chapman et al., 2010) that are known to be strongly dependent on BDNF (Tyler et al., 2002; Bramham and Messaoudi, 2005; Lu et al., 2005). The studies described here extend these observations, investigating the possibility that the combination of aging and an infection might reduce basal levels of BDNF mRNAs, or more specifically, might reduce the capacity to recruit particular transcripts for memory-related plasticity in the hippocampus.

We examined basal and fear-conditioning induced expression of total BDNF mRNA, four 5' exon-specific transcripts (I, II, IV, and VI), and BDNF mRNA transcripts with a long 3’untranslated region (3’UTR) in hippocampal areas CA1 and CA3, and in the dentate. We found that area CA1 was the only hippocampal sub-region showing increases in BDNF mRNAs one hour after a relatively mild fear-conditioning paradigm; it was also the area most sensitive to the individual and combined effects of aging and infection. Area CA3 proved more sensitive to the effects of aging alone, while expression in the dentate was only minimally affected by any of the conditions examined. More specifically, in CA1 and CA3, aging produced reductions in total BDNF mRNA, in Exon IV-specific transcripts, and in transcripts with long 3′ UTRs. Aging also impaired fear-conditioning-induced expression of these transcripts. In area CA1, infection produced reductions similar to those seen with aging, and exacerbated the effects of aging on transcripts with long 3′ UTRs. Northern blot analysis confirmed an aging-associated, sub-region specific loss of total BDNF mRNA in areas CA1 and CA3, and revealed a parallel, preferential loss of BDNF mRNA transcripts with long 3′ UTRs.

The idea that aging-associated cognitive deficits may be mediated, at least in part, by reductions in levels of BDNF or its receptors is not new, but it remains attractive given the importance of these molecules for hippocampal function, and many investigators have sought to demonstrate such links. Results have been mixed, with comparisons complicated by differences in species, strains, ages and experimental procedures (reviewed in (Pang and Lu, 2004; Tapia-Arancibia et al., 2008)). However, although levels of BDNF are clearly decreased in some neurodegenerative diseases (e.g. (Phillips et al., 1991)), the bulk of the available evidence suggests that there are not widespread reductions in basal levels of BDNF mRNA and TrkB mRNA in the brain simply as a result of aging (Pang and Lu, 2004; Tapia-Arancibia et al., 2008).

Still, there are indications of more focal, and sometimes more subtle, alterations associated with aging. An early study using in situ hybridization to examine the impact of aging on levels of total BDNF mRNA in F344/N rats found reductions in hippocampal areas CA3 and CA1, but not in dentate (Smith and Cizza, 1996) - our results are very similar. A more recent, and more nuanced study grouped aged (30-month-old) Brown Norway rats based on their performance on a Morris water maze task (Schaaf et al., 2001). Aged rats with unimpaired learning showed a significant increase in BDNF mRNA in the CA1 region following the task, while those with impairments did not. Interestingly, although our aging, but pre-senescent 24-month-old F344xBN hybrid rats did not show overt deficits in a simple fear-learning task prior to infection, they failed to show an increase in total BDNF mRNA in CA1 following the task, and infection tended to slightly exacerbate this effect. This may represent a limited, early stage decrease in the capacity to recruit production of some BDNF mRNA transcripts for memory related processes.

A consistent finding here and in other studies is that aging does not uniformly affect BDNF mRNA levels across the hippocampus, but rather reduces them primarily in areas CA1 and CA3 while leaving them relatively intact in the dentate. There is now some evidence that gene expression in hippocampal pyramidal neurons may be more vulnerable to age than in dentate granule cells (Haberman et al., 2009; Zeier et al., 2010). There are several possibilities as to why this may occur. The hippocampus undergoes a number of morphological, electrophysiological, and metabolic changes with age. Aging can cause a subtle loss of synaptic contacts in the CA3 region and a silencing of synaptic contacts in the CA1 region (Burke and Barnes, 2010). Aged pyramidal neurons in the CA regions also seem particularly sensitive to disruptions in Ca+2 homeostasis, showing a decrease in NMDA function but an increase in voltage gated calcium channels (Kumar et al., 2009). It has been suggested that although the aged dentate also loses synaptic inputs originating from the perforant pathway and experiences a loss of NMDA function, granule cells may maintain excitability and Ca+2 entry through an increase of surface AMPA receptors (Burke and Barnes, 2010). Another recent study demonstrated that aged rats have enhanced glutamate release onto the dentate but diminished released in areas CA1 and CA3 (Stephens et al., 2009). Since BDNF transcription is heavily dependent on neural activity and the appropriate Ca+2 entry, these different compensatory mechanisms could explain why the aged dentate is able to maintain BDNF mRNA levels, while aged CA regions are not.

It is also possible that, age-dependent epigenetic changes, including DNA methylation and histone acetylation (Penner et al., 2010a), could be causing down-regulation of BDNF gene expression in select pyramidal neurons of the hippocampus. Interestingly, it has been reported that aged rats have less basal transcription of Arc, a memory related gene, in CA1, but not in dentate. This was further correlated with increased DNA methylation of the Arc promoter region in CA1, but again, not in dentate (Penner et al., 2010b).

The effects of infection on BDNF mRNA have not been studied as much as those of aging. However, several studies have now examined the effects of immune challenge or proinflamatory cytokines on BDNF mRNA in the hippocampus. Lipopolysaccharide (LPS) is a component of the cell wall of Gram-negative bacteria used to mimic infection. A potent endotoxin, it leads to the release of pro-inflammatory cytokines such as interleukin-1beta (IL-1β) and tumor necrosis factor-alpha (TNF-α)(Drum and Oppemtom, 1989) in the periphery and in the brain (Maier et al., 2001; Konsman et al., 2002). BDNF mRNA in the hippocampus was strongly down-regulated 4 hours after an intraperitoneally injection of LPS or IL-1β (Lapchak et al., 1993a). Intraperitoneally injection of LPS has also been shown to lower BDNF mRNA in hippocampal extracts from both young and old mice (Richwine et al., 2008). Consistent with these observations, we found that a recent peripheral E. coli infection (which triggers production of IL-1β in the hippocampus (Barrientos et al., 2006)) produced a significant reduction in expression of total BDNF mRNA in young rats, and tended to produce some further reduction in aged rats.

It has also been noted that behaviorally induced alterations in levels of BDNF mRNA can be antagonized by alterations in IL-1β signaling. BDNF mRNA is down regulated by social isolation, but this effect can be blocked by an intra-hippocampal injection of an IL-1β receptor antagonist (Barrientos et al., 2003). Conversely, contextual fear conditioning produces an increase in BDNF mRNA in the hippocampus; this increase is reduced by an intra-hippocampal injection of IL-1β immediately after the learning experience (Barrientos et al., 2004). Similarly, we found that a recent history of infection reduced fear–conditioning-induced increases in total BDNF mRNA in our rats.

Most studies examining the impact of aging on expression of Bdnf have focused exclusively on total BDNF mRNA. Very little is know about the effects of aging on expression of specific BDNF mRNA transcripts, but exercise-induced expression of BDNF mRNA transcripts within the hippocampus is reported to vary across life-span (Adlard et al., 2005). Virtually nothing is known about the effects of inflammation on expression of specific BDNF transcripts in the hippocampus. The experiments presented here have examined the effects of aging and infection on total BDNF mRNA, on the four predominant 5' exon-specific transcripts (I, II, IV, and VI) (Aid et al., 2007), and on BDNF mRNAs with long 3'UTRs (An et al., 2008).

Our probes for the four 5' exon-specific transcripts revealed that under control conditions (young vehicle-injected animals), only exon IV and VI specific transcripts were expressed at substantial levels in CA1, but all four transcripts were more strongly expressed in CA3 and the dentate. In aged animals, expression of exons IV and VI was reduced in CA1; expression of all four transcripts was significantly reduced in CA3, and slightly elevated in the dentate. Infection reduced expression of exon IV in CA1 in young animals, of exons II and VI in CA3 of aged animals, and of exons I and II in the dentates of both young and aged animals. Fear condition had its effects exclusively in CA1 of young animals; it drove increased expression of exons IV (which has a promoter rich in calcium-responsive DNA elements (Greer and Greenberg, 2008)), and VI.

BDNF mRNA transcripts with long 3'UTRs are reported to play important roles in dentritic spine morphology and long-lasting synaptic plasticity (An et al., 2008). We found that the expression of these transcripts in area CA1 was significantly reduced by age and infection, and by the interaction of the two. Fear conditioning increased expression of these transcripts in CA1 in both young and aged animals. However, in the aged animals, basal levels were markedly lower and fear-learning-driven increases were quite small compared to those seen in young animals. Northern blot analysis confirmed an aging-associated, preferential loss of BDNF mRNA transcripts with long 3′ UTRs in CA1/CA3. Intriguingly, a recent study found that mutant mice, unable to produce BDNF mRNA transcripts with long 3′ UTRs, show specific deficits in theta burst evoked L-LTP in hippocampal area CA1 (An et al., 2008) that are strikingly similar to those we see in the aged rats following infection (Chapman et al., 2010).

In sum, these data suggest that aging led to a majority of the alterations in basal BDNF mRNA levels observed here, significantly reducing levels of all major transcripts in CA1 and or CA3, while having minimal effects in the dentate. Furthermore, aging severely impaired fear learning-induced transcription of BDNF. Interestingly, although we have previously demonstrated that in aged animals, addition of an immune challenge produces profound deficits in BDNF-dependent forms of long-term memory and synaptic plasticity (Barrientos et al., 2006; Chapman et al., 2010), here we show that effects of the added immune challenge on expression of BDNF mRNA in aged animals are generally modest. Infection does sometimes exacerbate the effects of aging, but the additional reductions in BDNF mRNAs are small and limited to a few specific transcripts in specific hippocampal sub-regions.

There are several possible explanations for this apparent discrepancy. It may be that the reduction in BDNF mRNA transcripts with long 3′ UTRs, produced by the combination of age and infection, plays a significant role in the observed deficits in memory and plasticity. It is also possible that the reduced levels of BDNF mRNAs in aged animals suffice to support simple learning and memory processes, but may create a vulnerability to a secondary stressor, such as an infection, that manifests itself downstream, or independently, of BDNF mRNA levels. For example, we have recently determined that young and aged rats have comparable basal levels of BDNF protein at hippocampal synapses, but following an infection, levels are significantly reduced in the aged, but not the young animals (Cortese et al., 2011). Deficits in memory and plasticity triggered by high levels of pro-inflammatory cytokines like IL-1β are also likely to involve additional substrates and signaling cascades not directly linked to BDNF and its receptor TrkB. A number of recent studies suggest that pro-inflammatory cytokines may modulate responses to glutamate through actions on NMDA and AMPA receptors (reviewed in Viviani and Boraso, 2011). IL-1β has also been shown to activate p38MAPK, c-jun NH2-terminal kinase (JNK), caspase 1, and NFkB (Vereker et al., 2000b; Vereker et al., 2000a; Curran et al., 2003; Kelly et al., 2003). Thus, it seems plausible that aging-associated reductions in BDNF mRNAs in the hippocampus may contribute to reduced availability of BDNF for memory related plasticity processes following an immune challenge. This may in turn contribute to selective, early stage impairments in long-term memory and synaptic plasticity.

Acknowledgements

This work was supported by an Innovative Seed Grant Award from the University of Colorado (to SLP) and National Institute on Aging Grants 1R21AG031467 (to SLP), and 1R01AG02827 (to SFM).

Footnotes

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Disclosure Statement: None of the authors have actual or potential conflicts of interest with the work reported here.

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