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. Author manuscript; available in PMC: 2009 Nov 1.
Published in final edited form as: Genes Brain Behav. 2008 Aug 4;7(8):906–914. doi: 10.1111/j.1601-183X.2008.00430.x

Amphetamine-induced locomotion and gene expression are altered in BDNF heterozygous mice

Alicia J Saylor 1, Jacqueline F McGinty 1,*
PMCID: PMC2605203  NIHMSID: NIHMS76377  PMID: 18681898

Abstract

Administration of amphetamine over-stimulates medium spiny neurons by releasing dopamine and glutamate from afferents in the striatum. However, these afferents also release brain-derived neurotrophic factor (BDNF) that protects striatal medium spiny neurons from over-stimulation. Intriguingly, all three neurochemicals increase opioid gene expression in medium spiny neurons. In contrast, striatal opioid expression is less in naïve BDNF heterozygous (BDNF+/-) versus wildtype mice. This study was designed to determine whether partial genetic depletion of BDNF influences the behavioral and molecular response to an acute amphetamine injection. An acute injection of amphetamine (5 mg/kg, i.p.) or saline was administered to wildtype and BDNF+/- mice. Wildtype and BDNF+/- mice exhibited similar locomotor activity during habituation whereas BDNF+/- mice exhibited more prolonged locomotor activation during the third hour after injection of amphetamine. Three hours after amphetamine injection, there was an increase of preprodynorphin mRNA in the caudate putamen and nucleus accumbens and D3R mRNA levels were increased in the nucleus accumbens of BDNF+/- and wildtype mice. Striatal/cortical trkB and BDNF, and mesencephalic TH mRNA levels were only increased in wildtype mice. These results indicate that BDNF modifies the locomotor responses of mice to acute amphetamine and differentially regulates amphetamine-induced gene expression.

Keywords: brain-derived neurotrophic factor, amphetamine, psychostimulant, tyrosine hydroxylase, dynorphin, enkephalin, TrkB, dopamine D3 receptor

INTRODUCTION

Brain-derived neurotrophic factor (BDNF) is important for various forms of neuronal plasticity, including synapse formation and restructuring, axonal sprouting after injury, and long-term potentiation (Genoud et al. 2004; Zhou & Shine 2003; Kovalchuk et al. 2002). In addition to its classic trophic capacities, BDNF can elicit fast-acting, neurotransmitter-like effects because it is anterogradely transported and released from presynaptic vesicles in an activity-dependent manner, a property that is unique to BDNF among neurotrophins (Fawcett et al. 1997; Altar & DiStefano, 1998; Griesbeck et al. 1999). BDNF is found throughout the brain but the spatial profiles of its mRNA and protein expression do not necessarily overlap. For example, the rodent striatum synthesizes very little BDNF mRNA, but is rich in BDNF protein derived from other sources, including cortex, substantia nigra and amygdala, which synthesize and transport BDNF protein anterogradely to be released and act on postsynaptic striatal medium spiny neurons (MSNs) via the protein tyrosine kinase receptor, TrkB (Sauer et al. 1994; Altar et al. 1997; Conner et al. 1997; Altar & DiStefano, 1998). Activation of the TrkB receptor initiates downstream changes in gene transcription via phosphoinositide-3 kinase, ERK/MAP kinase, and phospholipase C-γ signaling (Patapoutian & Reichardt, 2001).

Several studies have demonstrated that BDNF is an important regulator of gene expression in striatal MSNs, the primary neuron type of this structure. In particular, BDNF directly stimulates expression of dopamine D3 receptor (D3R) mRNA, and the opioid peptides, dynorphin and enkephalin. All three genes have a distinct spatial expression profile within the striatum, and the promoter regions of D3R, preprodynorphin (PPD), and preproenkephalin (PPE) contain cAMP response elements (CREs), to which phosphorylated CREB can bind to regulate their gene expression (Konradi et al. 1993; Hyman et al. 1994; Cole et al. 1995; D’Souza et al. 2001). The D3R is expressed most heavily in the nucleus accumbens (Acb), with very low expression extending to the dorsal striatum (Diaz et al. 2000). BDNF is required for normal expression of D3R in the Acb but does not affect expression of either D1 or D2 receptors (Guillin et al. 2001). Dynorphin-containing MSNs also express D1 receptors, and the heaviest dynorphin expression is located along the medial wall of the CPu and throughout the Acb. These MSNs project to the internal segment of the globus pallidus (rodent entopeduncular nucleus) and the substantia nigra. In contrast, enkephalin-containing MSNs are found throughout the CPu and Acb; they express D2 receptors and project to the external segment of the globus pallidus. Exogenous BDNF infused into the striatum (Sauer et al. 1994) or substantia nigra (Arenas et al. 1996) increases the expression of PPE and preprotachykinin in rats. Additionally, our laboratory has previously shown that the precursors to the opioid peptides, PPD and PPE, are substantially reduced in striatal MSNs of BDNF+/- mice (Saylor et al. 2006).

Recently, BDNF has emerged as an important mediator of the neuroplastic changes in mesolimbic circuitry that occur following exposure to drugs of abuse. For example, BDNF mRNA is increased in the prefrontal cortex after a single cocaine injection (Le Foll et al. 2005) and in the basolateral amygdala, piriform cortex, and paraventricular hypothalamus after repeated amphetamine injections (Meredith et al. 2002). BDNF protein is increased in the ventral tegmental area (VTA), nucleus accumbens, and amygdala of rats during withdrawal at 30 and 90, but not 1 day, after chronic cocaine self-administration (Grimm et al. 2003). Further, BDNF infusions into the rat VTA enhance cocaine-induced behavioral sensitization (Horger et al. 1999) and increase cue-induced reinstatement to cocaine seeking behavior, a long term effect that lasts for up to 30 days of withdrawal (Lu et al. 2004). Finally, a single intra-prefrontal cortex infusion of BDNF given immediately following the last self-administration session suppresses reinstatement to cocaine seeking (Berglind et al. 2007).

Narita and colleagues (2003) have shown that an intra-accumbal microinjection of BDNF or TrkB antiserum decreases dopamine release, rearing, sniffing and locomotion induced by an acute injection of methamphetamine. The few studies conducted in mice have produced conflicting results about the role that BDNF plays in the response to psychostimulants. BDNF+/- mice show decreased behavioral sensitization to cocaine (Horger et al. 1999), cocaine-induced hyperlocomotion is decreased and cocaine place preference is reduced in BDNF+/- mice compared to wildtypes (Hall et al. 2003). A single amphetamine injection (5 mg/kg) significantly increased only one specific parameter of locomotor activity (number of movements, but not horizontal activity or stereotypy counts) in BDNF+/- mice on a BALB/c 129 background (Dluzen et al. 2001). Since BDNF colocalizes with tyrosine hydroxylase (TH) in mesencephalic dopamine neurons (Seroogy et al. 1994), much focus has been placed on presynaptic dopamine functioning as the locus of BDNF action within the mesolimbic circuitry. However, the effects of BDNF as they relate to amphetamine exposure may also be mediated at the postsynaptic, dopaminoceptive MSNs that express TrkB receptors (Freeman et al. 2003).

Amphetamine is a powerful psychostimulant that induces dopamine release from vesicular and cytosolic pools via reverse transport through plasma membrane and vesicular monoamine transporters (Kogan et al. 1976; Sulzer et al. 1993; Pifl et al. 1995). Previous work has shown that a single injection of amphetamine induces phosphorylation of CREB and a subsequent increase in the gene expression of striatal PPD and PPE in a D1-dopamine receptor-dependent manner in rats (Hurd and Herkenham, 1992; Konradi et al. 1994; Simpson et al. 1995; Wang et al. 1995; Wang and McGinty, 1996). Similarly, acute injection of cocaine or methamphetamine increases D3R mRNA in the Acb (Le Foll et al. 2005). Further, inhibition of the ERK/MAPK pathway with MEK-specific inhibitors attenuates amphetamine-induced hyperlocomotion and prevents the amphetamine-induced increase in expression of opioid peptide mRNA in the rat striatum (Shi and McGinty, 2006). Taken together, these data indicate that the amphetamine-induced increase of extracellular dopamine causes hyperlocomotion and MAPK/CREB-dependent changes in striatal gene expression via activation of dopamine D1 receptors on postsynaptic MSNs. Since D3R, PPD and PPE promoter regions all contain CREs, signaling via D1 and TrkB receptors may interact to induce a full CREB-driven response of these target genes. Therefore, we hypothesized that an amphetamine-induced increase in striatal D3R and opioid peptide gene expression would be attenuated in BDNF-depleted mice.

MATERIALS AND METHODS

Animals

BDNF+/-and wildtype mice were generated as described previously (Liebl et al. 1997) and subsequently backcrossed for 10-12 generations onto a C57BL/6J genetic background (Lyons et al. 1999). BDNF+/- mice carry a single null allele for BDNF and are viable, fertile, and have a normal life span, whereas mice homozygous for the mutant BDNF allele do not survive into adulthood. The mice used in this study were bred in HET × HET matings and genotyped at the Medical University of South Carolina as previously described (Saylor et al. 2006). Three month-old male littermates were group-housed in a temperature and humidity controlled room and maintained on a 12-h light/dark cycle with food and water available ad libitum. All procedures used on animals in this study were in strict accordance with the NIH Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee.

Locomotor Activity

Total distance traveled was measured during the light cycle with a Digiscan Animal Activity Monitor system (Omnitech Electronics, Columbus, OH) coupled to a computer running VersaMax software (AccuScan Instruments, Columbus, OH). Each activity chamber was partitioned into 20 cm × 20 cm quadrants with acrylic dividers to allow simultaneous testing of two mice, such that four activity chambers allowed testing of eight mice per session. Each activity unit contained 16 photo beams positioned 5 cm apart, with 8 beams on the x-axis and 8 beams on the y-axis and was enclosed in a 90 × 54 × 35 cm sound-attenuated box. The behavioral data were broken down into one-hour segments (habituation; 0-60, 61-120 and 121-180 mins post-injection) and a separate 2×2 repeated measures ANOVA was conducted for each one-hour bin with genotype and drug treatment as independent factors. When a significant interaction was present, the Tukey-Kramer method was used to perform multiple comparisons.

Experimental Design

At three months of age, total distance traveled during a one-hour habituation period was recorded from wildtype (n=12) and BDNF+/- mice (n=12). Mice of each genotype were then injected with either 0.9% saline or d-amphetamine sulfate (5 mg/kg, i.p.; Sigma-Aldrich Co., St. Louis, MO), resulting in four treatment groups of six mice each. The mice were then returned to the activity chambers for a period of 3 hours, after which time the animals were sacrificed and their brains were extracted and snap frozen in isopentane at -35°C in preparation for in situ hybridization histochemistry.

Semi-quantitative in situ hybridization histochemistry

Semi-quantitative in situ hybridization histochemistry was performed as previously described (Gonzalez-Nicolini and McGinty, 2002). Briefly, sections were cut at 12 μm with a cryostat through the striatum of each mouse and thaw-mounted onto Superfrost/Plus slides (Fisher Scientific, Pittsburgh, PA). The sections were pretreated to fix and defat the tissue and block non-specific hybridization. Synthetic cDNA oligodeoxynucleotide probes (48-mers) complementary to PPD (NCBI GenBank Accession number NM 019374, bases 839-886), PPE (NM 017139, bases 715-762), trkB (X17647, bases 2790-2837), BDNF (X55573, bases 660-707), TH (NM 009377, bases 1437-1484) and D3R (NM 007877, bases 753-800) were radiolabeled with 35S-dATP (1250 Ci/mmol; New England Nuclear, Boston, MA) using terminal deoxynucleotide transferase (Roche Diagnostics, Indianapolis, IN). Sections were immersed in 5.0×105 cpm/20 μl hybridization buffer/section overnight (15h) at 37°C in a humid environment and then washed and air dried before being placed into a film cassette with 14C standards (American Radiolabeled Chemicals, St. Louis, MO) and Kodak Biomax film (Rochester, NY) for 4 days (PPE), 6 days (TH), 10 days (PPD), 12 days (trkB), 21 days (BDNF) or 6 weeks (D3R).

Quantitation of the hybridization signals was performed using NIH image 1.62 (W. Rasband, NIMH) on a Macintosh G3 as previously described (Gonzalez-Nicolini and McGinty, 2002). 14C standards were used to generate a calibration curve. Nonuniform illumination was corrected by saving a “blank field”. The upper limit of the density slice option was set to eliminate film background, and this value was used to measure all images. The lower limit was set at the bottom of the LUT scale. An appropriately sized oval field encompassing the caudate putamen (CPu), nucleus accumbens core (AcbC), nucleus accumbens shell (AcbSh), piriform cortex (Pir), or a polygon approximating the anterior cingulate cortex (AC), sensory cortex (S1), substantia nigra pars compacta (SNpc) or ventral tegmental area (VTA) was used to measure hybridization signals (Figure 1). The hybridization signal was expressed as (1) the number of labeled pixels per unit area (area), (2) mean density of tissue in dpm/mg, and (3) integrated density (product of area x mean density). Integrated density more accurately depicts the area over which changes in optical density occur because mean density alone underestimates these changes (Zhou et al. 2004). The mean integrated density ± SEM in each mouse reflects the average values of three adjacent coronal sections; therefore, measurements of the hybridization signals were strongly correlated and could not be treated independently. For each gene, a nested repeated-measures analysis of variance was conducted on the mean integrated density values with genotype and drug treatment as independent factors, followed by planned multiple comparison tests (Least Squares Means) when an interaction was found or to further analyze the source of main effects.

Figure 1. Selection areas.

Figure 1

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Representative schematics outlining the approximate selection areas used in the image analysis of the hybridization signal. The top panel illustrates the regions measured in the forebrain: anterior cingulate cortex (AC), sensory cortex (S1), piriform cortex (Pir), caudate putamen (CPu), nucleus accumbens core (AcbC), and nucleus accumbens shell (AcbSh). The bottom panel illustrates the regions measured in the midbrain: substantia nigra pars compacta (SNpc) and ventral tegmental area (VTA).

RESULTS

Locomotor activity

Figure 2 illustrates the locomotor activity of wildtype and BDNF+/- mice in response to a single injection of amphetamine (5 mg/kg, i.p.). Total distance traveled was not different between genotypes during the one-hour habitation period prior to the injection. During the first and second hours post-injection, both wildtype and BDNF+/- mice treated with amphetamine displayed a significant increase in total distance traveled compared with saline-injected mice (F(1, 284) = 91.68, p < .0001; F(1, 284) = 170.97, p < .0001). During the third hour after amphetamine injection, wildtype and BDNF+/- mice displayed a differential amphetamine-induced locomotor response. Twoway ANOVA performed on locomotor activity values during the third hour post-injection revealed a significant genotype by drug treatment interaction (F(3, 284) = 79.89, p < .0001). Multiple comparison tests revealed that both wildtype and BDNF+/- mice displayed elevated locomotor activity during this entire time compared to saline-treated controls of the same genotype. Although the behavior of amphetamine-treated wildtype mice did not return to statistical baseline, their locomotor activity during the third hour after a single amphetamine injection was significantly less than that of BDNF+/- mice treated with amphetamine and more comparable to that of saline-treated mice. In contrast, amphetamine-treated BDNF+/- mice displayed a prolonged elevation of locomotor activity compared to amphetamine-injected wildtype mice.

Figure 2. Locomotor behavior.

Figure 2

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Total distance traveled in wildtype and BDNF+/- mice during a one-hour habituation period and during one-hour bins after a single injection of 5 mg/kg amphetamine. *p<0.05.

Gene expression

Two-way ANOVA revealed significant main effects of genotype and drug treatment (F(1,20) = 40.66, p < .0001; F(1,20) = 118.23, p < .0001) for PPD expression in the CPu. As previously reported in a different line of BDNF+/- mice (Saylor et al. 2006), BDNF+/- mice expressed significantly less PPD mRNA in the CPu as compared to wildtype mice, and amphetamine increased PPD gene expression in the CPu of both genotypes (Figure 3a). Similarly, significant main effects of genotype and drug treatment, F(1,20) = 19.34, p = .0003; F(1,20) = 8.37, p = .009, were also observed for PPD expression in the AcbC. Planned comparison tests revealed that in the AcbC, BDNF+/- mice expressed less PPD mRNA than wildtype mice. Amphetamine induced an increase in PPD mRNA in BDNF+/- mice, and also tended to have the same effect in wildtype mice (p = .07). Two-way ANOVA revealed a significant main effect of genotype for PPE expression in the CPu, (F(1,26) = 8.36, p = .007). PPE mRNA was expressed significantly less in the CPu of BDNF+/- mice versus wildtypes; however, in contrast to PPD, amphetamine did not induce an increase in PPE mRNA in either genotype in the CPu or AcbC (Figure 3b). PPD and PPE mRNA expression was comparable in all treatment groups in the AcbSh, regardless of genotype or amphetamine treatment (data not shown).

Figure 3. Striatal gene expression.

Figure 3

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Representative digitized photomicrographs and image analysis illustrate the mRNA expression of PPD (a), PPE (b) and D3R (c) in wildtype and BDNF+/- mice 3 hours after a single saline or amphetamine injection (5 mg/kg). *p<.05 vs WT + sal; #p<.05 vs BDNF+/- + sal.

Since the D3R is known to be expressed only in the ventral, but not dorsal striatum (Sokoloff et al. 1990; Diaz et al. 2000, and see Figure 3c), we only measured D3R mRNA in the AcbC and not the CPu. The expression of D3R mRNA was very light even in the ventral striatum after prolonged film exposure, and anatomical landmarks were not distinct enough to measure D3R consistently in the AcbSh. Two-way ANOVA revealed a significant main effect of genotype and drug treatment (F(1,26) = 10.04, p = .0039; F(1,26) = 12.77, p = .0014). BDNF+/- mice expressed only about 65% of wildtype D3R mRNA levels in the AcbC (Figure 3c). After a single amphetamine injection, an increase in D3R mRNA in the AcbC of BDNF+/- mice occurred, with a proportionally smaller increase in wildtype mice that did not reach statistical significance (p = .06; Figure 3c).

Two-way ANOVA revealed a significant genotype by drug treatment interaction for trkB expression in the CPu (F(1,20) = 6.25, p = .02). TrkB mRNA levels were comparable in the CPu of BDNF+/- and wildtype mice treated with saline; however, a single injection of amphetamine induced a significant increase of trkB mRNA in the CPu only in wildtype, but not BDNF+/- mice (Figure 4a). Similarly, a significant genotype by drug treatment interaction was observed in the AC cortex (F(1,20) = 8.77, p = .008) and multiple comparison tests revealed similar trkB expression between mice of either genotype treated with saline, whereas amphetamine induced an increase of trkB mRNA in wildtype mice only (Figure 4a). In addition, measurements from other cortical areas, including piriform and sensory cortex, revealed that trkB expression throughout the cortex is affected in a similar manner as in the AC cortex (data not shown). In the AcbC, trkB expression was similar in all treatment groups, regardless of genotype or drug treatment (data not shown).

Figure 4. Cortical and striatal gene expression.

Figure 4

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Representative digitized photomicrographs and image analysis illustrate the mRNA expression of trkB (a) and BDNF (b) in wildtype and BDNF+/- mice 3 hours after a single saline or amphetamine injection (5 mg/kg). *p<.05 vs WT + sal.

Two-way ANOVA revealed a significant main effect of genotype and drug treatment (F(1,20) = 15.67, p = .0008; F(1,20) = 5.49, p = .03) for BDNF mRNA in the AC cortex. BDNF mRNA in the AC cortex tended to be less in saline-treated BDNF+/- mice than in wildtype mice (p = .08), and an amphetamine-induced increase in BDNF mRNA occurred only in wildtype mice (Figure 4b). Baseline striatal BDNF expression was very low in both genotypes; in the CPu, a main effect for drug treatment approached significance (F(1,18) = 3.34, p = .08), and wildtype mice treated with amphetamine had an increase in BDNF mRNA, whereas BDNF expression in BDNF+/- mice was unaffected by amphetamine (Figure 4b). BDNF expression in the AcbC was similar in all treatment groups, regardless of genotype or drug treatment (data not shown).

A significant genotype by drug treatment interaction was present for TH expression in the SNpc (F(1,19) = 52.64, p < .0001). TH mRNA was equivalent in the SNpc of wildtype and BDNF+/- mice treated with saline. An amphetamine-induced increase of TH mRNA was only observed in the SNpc of wildtype, but not BDNF+/- mice (Figure 5). TH expression was comparable in all treatment groups in the VTA, regardless of genotype or amphetamine treatment (Figure 5).

Figure 5. Mesencephalic gene expression.

Figure 5

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Representative digitized photomicrographs and image analysis illustrate the mRNA expression of TH in wildtype and BDNF+/- mice 3 hours after a single saline or amphetamine injection (5 mg/kg). *p<.05 vs WT + sal.

DISCUSSION

The findings of this study indicate that wildtype and BDNF+/- mice displayed similar baseline locomotor activity and a similar biphasic increase in total distance traveled within the first two hours after an acute injection of amphetamine. However, BDNF+/- mice displayed elevated and prolonged total distance traveled during the third hour post-injection, whereas the locomotor activity of wildtype mice approached baseline levels during this time. In general, striatal PPD and D3R mRNA, striatal/cortical trkB and BDNF mRNA, and nigral TH mRNA tended to be increased after an acute injection of amphetamine in wildtype mice. In contrast, BDNF+/- mice only exhibited an amphetamine-induced increase of PPD and D3R mRNA, but not BDNF, trkB or TH mRNA levels. Although it is well-documented that acute amphetamine induces an increase in PPE mRNA expression in rats (Wang and McGinty, 1996), neither wildtype nor BDNF+/- mice displayed an amphetamine-induced increase in PPE mRNA, highlighting an important difference in the response to psychostimulants between rodent species.

Amphetamine-induced locomotor activity

The locomotor-activating effects of amphetamines are due to increased synaptic dopamine levels and subsequent activation of dopamine D1 receptors (Hurd and Herkenham, 1992; Konradi et al. 1994; Simpson et al. 1995; Wang et al. 1995). The biphasic locomotor response to amph in both genotypes is typical of an immediate increase in ambulation during the first hour, followed by a rapid and marked decrease in ambulation, indicating the emergence of stereotypical behaviors, in the second hour, and finally, re-emergence of ambulation as stereotypical behaviors wane in the third hour (Wolf et al, 1995). No significant alterations in this pattern were observed until the end of the third hour when the ambulation of BDNF+/- mice treated with amph did not decline at the rate of WT mice. It is thought that kappa opioid receptor stimulation, among others, is necessary to terminate amphetamine-induced behavior (Gray et al. 1999; Tzaferis and McGinty, 2001). In the present study, when wildtype mice approached baseline activity during the third hour after amphetamine injection, BDNF+/- mice displayed prolonged, elevated locomotor activity that may be attributable to a weak signal to terminate the locomotor response. Since dynorphin expression and release (You et al. 1994) are upregulated in response to psychostimulants, the subsequent stimulation of presynaptic kappa opioid receptors (KORs), which are located on striatal glutamatergic and dopaminergic terminals (Svingos et al, 1999; Meshul and McGinty, 2000), may initiate a negative-feedback process that decreases glutamate and dopamine release and returns the system to homeostasis (Gray et al. 1999; Rawls and McGinty, 1998). Despite the elevation of PPD mRNA levels in BDNF+/- mice , dynorphin stimulation of KORs may not have been sufficient to terminate the locomotor response as quickly as in wildtype mice. Alternatively, other neurotransmitter systems that were not measured in this study may have contributed to the prolonged locomotor response to amphetamine.

TrkB and D1 receptors independently regulate opioid mRNA

Present and previous results indicate that BDNF is an important regulator of opioid peptide gene expression because PPD and PPE mRNA levels are decreased in BDNF+/- mice as compared to wildtype mice (Saylor et al. 2006). In rats, opioid peptide gene expression is regulated by amphetamine-induced extracellular dopamine activation of D1 receptors on MSNs (Hurd and Herkenham, 1992; Konradi et al. 1994; Simpson et al. 1995; Wang et al. 1995; Wang and McGinty, 1996). Although BDNF+/- mice express less PPD mRNA than wildtype mice at baseline, both genotypes show a large increase in amphetamine-induced striatal PPD mRNA. We predicted that the amphetamine-induced increase in striatal opioid peptide gene expression would be attenuated in BDNF-depleted mice. However, while the gene expression of PPD is regulated by both BDNF and D1 receptors, the present data suggest that acute amphetamine administration (and therefore D1R activation) is sufficient to increase PPD mRNA expression, independent of TrkB and despite decreased baseline PPD mRNA levels in BDNF+/- mice.

Amphetamine induces BDNF gene expression in wildtype mice

As expected, BDNF mRNA in the AC cortex was decreased in BDNF+/- mice. In fact, BDNF mRNA was generally decreased in all measured cortical areas, including sensory and piriform cortex. A single injection of amphetamine induced an increase in BDNF mRNA in the AC cortex of wildtype mice, which is in agreement with a previous study showing that acute injection of cocaine or methamphetamine increases BDNF mRNA in the mouse frontal cortex (Le Foll et al. 2005). In the rodent striatum, BDNF mRNA levels are very low (Conner et al. 1997), and although the detectable expression of BDNF mRNA was equally minimal in the CPu of both genotypes, amphetamine induced a robust increase of BDNF mRNA in the striatum of wildtype, but not BDNF+/- mice. Thus, although acute amphetamine induced BDNF expression in wildtype mice, genetically altered mice that express only one functional allele of BDNF are unable to further upregulate BDNF mRNA in either cortex or striatum. Studies conducted in rats have also reported psychostimulant-induced alterations in BDNF mRNA and/or protein levels. Repeated (but not acute) amphetamine increased BDNF immunoreactivity in the rat striatum and amygdala (Meredith et al. 2002) and withdrawal from cocaine self administration produced an early decrease (24 hours) in BDNF mRNA in the medial prefrontal/AC cortex of the rat (McGinty et al. submitted).

Expression of the D3 receptor is responsive to amphetamine

Expression of the D3 receptor is anatomically confined in mesolimbic structures, including the ventral tegmental area, Acb and amygdala, which together mediate emotion, motivation and reward processing (Sokoloff et al. 1990; Diaz et al. 2000). Expression of the D3 receptor is regulated both by BDNF and drugs of abuse. More than 70% of neurons in the Acb co-express mRNA for both TrkB and D3 receptors (Guillin et al. 2001), and these same neurons are innervated by BDNF-containing axon terminals (Conner et al. 1997). BDNF is required for normal expression of D3Rs in the Acb during development, but it does not affect expression of either D1 or D2 receptors (Guillin et al. 2001). Further, a single injection of cocaine or methamphetamine induces an early, transient increase of BDNF mRNA in the prefrontal cortex beginning 2 hours after the stimulant injection. This early, transient increase in BDNF expression is followed by a subsequent, long-lasting elevation of D3R expression in the Acb that reaches its maximum apex at 4 hours after drug administration and is maintained for at least 16 hours afterwards (Le Foll et al. 2005). The results from the present study indicate that D3R mRNA is increased in BDNF+/- mice three hours after acute amphetamine; additionally, in wildtype mice, the amphetamine-induced increase in D3R mRNA falls just short of statistical significance (p = .06). Thus, it is likely that other regulators of D3R gene expression, such as D1-dependent phosphorylation of CREB, are active in BDNF+/- mice. A hyperdopaminergic environment is known to increase D3R expression because levodopa administration increases D3R expression in the accumbens of both the normal and denervated striatum (Guillin et al, 2001) and expression of the D3 receptor is elevated in the brains of cocaine addicts (Staley et al. 1996).

BDNF mediates amphetamine-induced trkB and TH expression

In contrast to our previous findings in a different line of BDNF+/- mice (Saylor et al. 2006), baseline trkB and mesencephalic TH mRNA were similar in both wildtype and BDNF+/- mice. An acute injection of amphetamine stimulated an increase of trkB in the dorsal striatum and the overlying AC cortex only in wildtype, but not in BDNF+/- mice. Interestingly, the amphetamine-induced increase of trkB mRNA in wildtype mice corresponds with a parallel upregulation of BDNF mRNA in the same brain regions. Similarly, TH expression was significantly increased in the SNpc of wildtype mice three hours after a single amphetamine injection, whereas TH mRNA levels did not change in BDNF+/- mice after amphetamine. Previous reports indicated that striatal dopamine concentrations were elevated and K+-stimulated dopamine release was greatly reduced in BDNF+/- versus wildtype mice on a mixed Balb/c genetic background (Dluzen et al. 1999, 2001, 2002). Therefore, the lack of increased presynaptic TH synthesis after amphetamine in BDNF+/- mice may be indicative of abnormal nigrostriatal dopamine neurotransmission and/or dopamine release. TH mRNA levels in the VTA were comparable in all groups and thus not responsive to amphetamine administration or dependent on the BDNF genotype.

Conclusion

BDNF has the ability to alter the typical locomotor response to acute psychostimulant administration because the characteristic period of hyperactivity induced by amphetamine is prolonged in BDNF+/- mice. Furthermore, in mice with a single functional BDNF allele, amphetamine does not increase BDNF, trkB and TH mRNA as in wildtype mice. Of the genes measured in this study, only PPD and D3R were increased after an acute injection of amphetamine, despite a reduction of normal levels of endogenous BDNF. These results indicate that BDNF is an important regulator of psychostimulant-induced locomotor activity and gene expression.

ACKNOWLEDGEMENTS

We thank Joe Vallone for his technical assistance and Scott W. Miller for invaluable statistical consultation. This research was supported by NIH PO1 AG023630, DA03982 (JFM), and F31 DA020238 (AJS).

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