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. Author manuscript; available in PMC: 2016 Jun 1.
Published in final edited form as: Genes Brain Behav. 2015 May 21;14(5):411–418. doi: 10.1111/gbb.12222

Physiological and behavioral effects of amphetamine in BACE1−/− mice

R Madelaine Paredes †, E Piccart †, E Navaira †, D Cruz †, M A Javors †, W Koek †, M J Beckstead †, C Walss-Bass ‡
PMCID: PMC4586110  NIHMSID: NIHMS724054  PMID: 25912880

Abstract

β-Site APP-cleaving Enzyme 1 (BACE1) is a protease that has been linked to schizophrenia, a severe mental illness that is potentially characterized by enhanced dopamine (DA) release in the striatum. Here, we used acute amphetamine administration to stimulate neuronal activity and investigated the neurophysiological and locomotor-activity response in BACE1-deficient (BACE1−/−) mice. We measured locomotor activity at baseline and after treatment with amphetamine (3.2 and 10 mg/kg). While baseline locomotor activity did not vary between groups, BACE1−/− mice exhibited reduced sensitivity to the locomotor-enhancing effects of amphetamine. Using high-performance liquid chromatography (HPLC) to measure DA and DA metabolites in the striatum, we found no significant differences in BACE1−/− compared with wild-type mice. To determine if DA neuron excitability is altered in BACE1−/− mice, we performed patch-clamp electrophysiology in putative DA neurons from brain slices that contained the substantia nigra. Pacemaker firing rate was slightly increased in slices from BACE1−/− mice. We next measured G protein-coupled potassium currents produced by activation of D2 autoreceptors, which strongly inhibit firing of these neurons. The maximal amplitude and decay times of D2 autoreceptor currents were not altered in BACE1−/− mice, indicating no change in D2 autoreceptor-sensitivity and DA transporter-mediated reuptake. However, amphetamine (30 μM)-induced potassium currents produced by efflux of DA were enhanced in BACE1−/− mice, perhaps indicating increased vesicular DA content in the midbrain. This suggests a plausible mechanism to explain the decreased sensitivity to amphetamine-induced locomotion, and provides evidence that decreased availability of BACE1 can produce persistent adaptations in the dopaminergic system.

Keywords: Amphetamine, BACE1, dopamine, schizophrenia, striatum

β-Site APP-cleaving Enzyme 1 (BACE1) is an endopeptidase responsible for proteolytic processing of several proteins including amyloid-precursor protein (APP) and neuregulin 1 (NRG1), a promising schizophrenia candidate gene (Petryshen et al. 2005; Stefansson et al. 2003a,b; Walss-Bass et al. 2006; Williams et al. 2003; Yang et al. 2003; Zhao et al. 2004). BACE1 cleaves NRG1 at the extracellular site, liberating the NRG1 N-terminal domain for interaction with ErbB tyrosine-kinase receptors and activation of downstream signaling events. BACE1 knockout mice (BACE1−/−) show an increase in full-length NRG1, reflecting lack of protein cleavage. These animals exhibit behaviors similar to mice with impaired NRG1 function, including impaired prepulse inhibition and novelty-induced hyperactivity (Luo et al. 2014; Savonenko et al. 2008; Willem et al. 2009), that are considered rodent analogs of schizophrenia behavior in humans. BACE1−/− furthermore exhibit morphological characteristics resembling those found in schizophrenia, including hypomyelination, decreased CA1 spine density and neuronal number, all of which are potentially consequences of altered NRG1-ErbB4 signaling (Hu et al. 2006). Importantly, genetic variants within BACE1 have been shown to correlate significantly with variants within other genes in the NRG1-ErbB4 signaling pathway in schizophrenia families (Hatzimanolis et al. 2013).

At the cellular level, enhanced striatal dopamine (DA) release from the terminals of midbrain DA neurons may underlie the positive symptoms of schizophrenia, i.e. hallucinations and delusions (Howes et al. 2012; Lyon et al. 2011). Previous studies have observed functional impairment of the prefrontal striatonigral circuit characterized by task-evoked hyperactivity of the substantia nigra in schizophrenia patients (Yoon et al. 2013). Terminal DA release from midbrain DA neurons is inhibited by D2 DA autoreceptors that are located on their cell bodies, dendrites and axon terminals. In the midbrain, somatodendritic DA release from neighboring DA neurons activates D2 autoreceptors and induces a potassium conductance that inhibits firing frequency (Lacey et al. 1987; Pucak & Grace 1994). Synaptic depression of D2 autoreceptor function can attenuate firing inhibition and thus increases excitability (Beckstead & Williams 2007), which could contribute to midbrain hyperdopaminergia in schizophrenia.

Local perfusion with NRG1 stimulates DA release in the hippocampus (Kwon et al. 2008), and intra-striatal injection of NRG1 evokes an almost immediate overflow of striatal DA (Yurek et al. 2004). However, little is known about how disruption of BACE1-mediated cleavage of NRG1 affects midbrain DA signaling and related behaviors. In light of the previous studies showing NRG1 stimulation of DA release, we hypothesized that because of impaired NRG1 cleavage, mice with a BACE1 deletion would exhibit a diminished behavioral response to amphetamine, an indirect DA agonist, with concurrent alterations in DA neuron excitability. In agreement with our hypothesis, we show that BACE1−/− mice exhibit a decrease in sensitivity to the locomotor activation produced by amphetamine. Striatal levels of DA and DA metabolites were unaltered in these mice. Substantia nigra DA neurons from BACE1−/− exhibited enhanced amphetamine-induced D2 autoreceptor-mediated currents and basal firing rate. The increased autoreceptor-mediated inhibition provides a plausible mechanism to explain the decreased sensitivity to amphetamine-induced locomotion in BACE1−/− mice.

Materials and methods

Animals

Wild-type (C57BL/6J) and BACE1−/− (B6.129-Bace1tm1Pcw /J) age-matched male mice were purchased from Jackson Laboratories (Bar Harbor, Maine, USA). For generation of the BACE1 mutant strain, chimeric animals were crossed to C57BL/6 mice, and then backcrossed to the same for seven generations (http://jaxmice.jax.org/strain/004714.html). Mice were 3 months ± 2 weeks old at the time of the locomotor and HPLC experiments, and ranged from 2 to 6 months old for the electrophysiological experiments. Animals were housed in groups at the UTHSCSA Laboratory Animal Resources facility under a 12-h light/12-h dark cycle in a temperature and humidity controlled room with food and water available ad libitum. All experiments were conducted during the light phase of the light/dark cycle. All procedures were approved by the IACUC at UTHSCSA.

Brain slice electrophysiology

Brain slices were prepared from mice (n = 6 per group) not previously used for behavioral experiments, as previously described (Branch et al. 2013). Briefly, mice were anesthetized with isoflurane and immediately decapitated. The brains were then quickly extracted and placed in ice-cold carboxygenated (95% O2 and 5% CO2) artificial cerebrospinal fluid (ACSF), containing (in mM): 126 NaCl, 2.5 KCl, 1.2 MgCl2, 2.4 CaCl2, 1.4 NaH2 PO4, 25 NaHCO3, 11 D-glucose and 1 kynurenic acid. The ventral midbrain was crudely blocked with a razor blade, and horizontal midbrain slices (200 μm) containing the substantia nigra pars compacta were obtained using a vibrating microtome (Leica, Nusslock, Germany). Slices were incubated for at least 25 min at 34°C with carboxygenated ACSF that also contained the NMDA receptor antagonist MK-801 (10 μM).

Slices were placed in a recording chamber attached to either an upright microscope (Carl Zeiss, Oberkochen, Germany) or a tube lens (Infinity Photo-Optical Company, Boulder, CO, USA) and maintained at 34-35°C with ACSF perfused at a rate of approximately 1.5 ml/min. Putative DA neurons of the substantia nigra were first visually identified based on their size, appearance and location in relation to the midline, third cranial nerve and the medial terminal nucleus of the accessory optic tract. Dopamine neurons were further identified physiologically by the presence of spontaneous pacemaker firing of wide action potentials (1 – 5 Hz firing rate, >1.1 milliseconds spike width) and a hyperpolarization-activated IH current exceeding 100 pA. Recording pipettes (4 – 7 MΩ for cell-attached recordings, 2.0 – 2.5 MΩ for whole cell recordings) were made from boroscilicate capillaries (World Precision Instruments, Sarasota, FL, USA) with a PC-10 puller (Narishige International USA Inc., East Meadow, NY, USA). A Na HEPES-based pipette filling solution (plus 20 mM NaCl) was used for cell-attached configuration experiments (pH 7.40, 290 mOsm/l). Whole-cell recordings were obtained using an internal solution containing (in mM): 115 K-methylsulfate, 20 NaCl, 1.5 MgCl2, 0.025 EGTA, 10 HEPES, 2 ATP and 0.4 GTP, pH 7.35 – 7.40, 269 – 272 mOsm.

Dopamine (1 M) was applied through iontophoresis to determine the maximum possible D2 receptor-mediated current for each neuron. The iontophoretic pulse length was 10 seconds in naïve slices or 2 seconds in slices that had been exposed to amphetamine. All currents were deemed maximal as the current amplitude reached a plateau while the drug was still being ejected. Amphetamine (30 μM) was applied by bath perfusion, and a large concentration was used to induce rapid vesicle depletion and DA efflux through reverse transport.

Locomotor activity

The locomotor activity of wild-type and BACE1−/− mice was assessed using completely dark customized boxes (Instrumentation Services, University of Texas Health Science Center at San Antonio, Antonio, TX, USA) equipped with infrared light beams (Multi-Varimex, Columbus Instruments, Columbus, OH, USA). On day 1 of behavioral testing, locomotor activity of 42 wild-type and 42 BACE1 knockout mice was recorded immediately following intraperitoneal (i.p.) injection of saline. On day 2 of behavioral testing, locomotor activity was recorded immediately following i.p. injection of 3.2 mg/kg amphetamine, 10 mg/kg amphetamine or saline vehicle (n = 14 per dose, per genotype). Locomotor activity was measured as number of beam breaks recorded in nine consecutive 5-min intervals.

Brain tissue preparation for HPLC measurements

Immediately after locomotor activity testing mice (n = 8 per dose, per genotype) were euthanized by cervical dislocation and brains were harvested, frozen in dry ice and stored at −80°C until assayed. Tissue from the striatum was isolated by punch dissections (2 mm diameter) to a depth of ~1 mm per hemisphere. Approximately, 100 mg of tissue was homogenized in 10× volume of 0.34 M perchloric acid and centrifuged at 4000 g for 10 min. Supernatants were extracted and centrifuged at 15 000 g for 10 min. Filtered samples were injected into the HPLC-EC system at a flow rate of 0.2 ml/min and detected at 220 mV. Dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) were measured in a single run with concentrations in striatum expressed as pg/mg.

Statistical analysis

All data were analyzed using GRAPHPAD Prism version 5.0 or 6.0 (GraphPad.com). Locomotor activity measures were analyzed by two-factor analysis of variance [ANOVA; factors: genotype and time (Fig. 1a); dose and time (Fig. 1b,c); dose and genotype (Fig. 1d)] followed by multiple comparisons of means (Dunnett’s test and Sidak’s test). DA and metabolite levels were analyzed by ANOVA followed by Sidak’s multiple comparisons test (∝= 0.05). Electrophysiology measures were analyzed by Student’s t-test. P value < 0.05 was considered significant. Data are reported as mean ± standard error.

Figure 1. Differences in locomotor response to amphetamine (AMPH) suggest BACE1−/− mice to be less sensitive to amphetamine than wild-type mice.

Figure 1

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(a) Locomotor activity on day 1, after an i.p. injection of saline in wild type (WT) and BACE1−/− mice (n = 42 per genotype). (b) Amphetamine-induced locomotion in WT mice (n = 14 per dose). (c) Amphetamine-induced locomotion in BACE1−/− mice (n = 14 per dose). (d) Amphetamine-induced locomotion, averaged over the first 15 min of the session, as a function of genotype and dose (n = 14 per group). Results in each panel were analyzed by two-factor ANOVA [factors: genotype, time (panel a); dose, time (panels b and c); dose, genotype (panel d)] and are shown as mean (±SEM) values; error bars that are not shown are contained within the symbol. For each ANOVA, a statistically significant interaction between both factors was followed by multiple comparisons of means: *P < 0.05 compared with saline (Dunnett’s test; panels b, c and d); #P < 0.05 compared with WT (Sidak’s test; panel d).

Results

Amphetamine-induced locomotor activation is reduced in BACE1−/− mice

We first aimed to determine the sensitivity of BACE1−/− mice to amphetamine, an indirect DA agonist and a psychostimulant considered to be an analog of positive symptoms in schizophrenia, using a locomotor activity test. We performed preliminary studies in wild-type mice (n-4) to assess the minimum amphetamine dose to increase locomotion. Amphetamine significantly increased locomotion at 3.2 mg/kg, but not at a lower (1 mg/kg) or a higher (10 mg/kg) dose in wild-type mice (data not shown). Thus, amphetamine appeared to increase locomotion along an inverted U-shaped dose – response curve. Because we expected BACE1−/− mice to be less sensitive to amphetamine, we chose to use 3.2 and 10 mg/kg doses, to examine a possible rightward shift of the inverted U-shaped dose – response relation. Based on our preliminary studies in wild-type mice, we expected that testing these two doses would be sufficient to discriminate between decreased or enhanced locomotor-activity sensitivity in BACE1 mice compared with wild type. No genotypic differences in locomotor activity were observed with saline injection at baseline, 24 h prior to drug testing [genotype: (F 1,82) = 1.13, P = 0.29; genotype × time interaction: (F 8,656) = 1.45, P = 0.17; Fig. 1a]. During drug testing, 3.2 mg/kg amphetamine increased locomotion in wild-type (Fig. 1b) and in BACE1−/− (Fig. 1c) mice. However, wild-type mice appeared to exhibit greater activity compared with BACE1−/− during the first 15 min of testing. Further, 10 mg/kg amphetamine, which increased locomotion in wild-type mice only at time block 2 (Fig. 1b), markedly increased locomotion in BACE1−/− mice at time blocks 2 – 5 (Fig. 1c). Averaging beam breaks across the first 15 min of the session revealed a significant dose × genotype interaction [(F 2,78) = 7.16, P = 0.0014; Fig. 1d]. A significant dose × genotype interaction was also obtained during the second 15 min of the session [(F 2,78) = 5.78, P = 0.0046] but not during the last 15 min [(F 2,78) = 0.44, P = 0.64] (data not shown). During the first 15 min of the session, amphetamine significantly increased locomotion only at 3.2 mg/kg in wild-type mice, and only at 10 mg/kg in BACE1−/− mice (Fig. 1d). These results suggest a shift to the right of the inverted U-shaped dose – response curve for amphetamine-induced locomotion in BACE1−/− mice.

DA and metabolite levels in BACE1−/− and wild-type mice

Decreased sensitivity to amphetamine-induced locomotion in BACE1−/− mice suggests that alterations may be present in nigrostriatal DA signaling, which is a key mediator of voluntary movement (Hnasko et al. 2006). We therefore assessed levels of DA and its metabolites, DOPAC and HVA in striata extracted from BACE1−/− and wild-type mice after locomotor activity measurements. There were no significant differences at baseline (saline vehicle), 3.2, or 10 mg/kg amphetamine between genotype: DA (F 5,41) = 2.27, P = 0.0652; DOPAC (F 5,40) = 3.275, P = 0.0140; HVA (F 5,41) = 1.551, P = 0.1955. Within genotype, at 10 mg/kg amphetamine there was a significant increase in the amount of DA in BACE1−/− mice compared with the levels detected after saline treatment in these mice (Fig. 2a), and a significant decrease in levels of DOPAC in wild-type mice after saline treatment compared with mice treated with 3.2 mg.kg amphetamine (Fig. 2b). In regard to DA turnover, analyzed as [DOPAC + HVA] divided by DA (F 5,41) = 10.63, P < 0.0001, no differences between genotypes were observed. Within genotype, DA turnover was significantly decreased in both wild-type and BACE1−/− mice after treatment with 3.2 or 10 mg/kg amphetamine (Fig. 2d).

Figure 2. Levels of striatal DA, DOPAC and HVA are not significantly different between BACE1−/− and wild-type mice.

Figure 2

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Dopamine (a), DOPAC (b) and HVA (c) were measured in a single run with concentrations expressed as pg/mg tissue in BACE1−/− and wild-type mice treated with saline, 3.2 or 10 mg/kg amphetamine, n = 8 mice per dose per genotype. DA turnover (d) was analyzed as [DOPAC + HVA]/DA. Results in each panel were analyzed by ANOVA followed by Sidak’s multiple comparisons test (∝= 0.05). *P < 0.05 (Sidak’s test).

Amphetamine-induced inhibitory currents are enhanced in BACE1−/− mice

We next used patch-clamp electrophysiology to investigate intrinsic characteristics of putative substantia nigra DA neurons, as well as D2 DA autoreceptor-mediated currents in brain slices from BACE1−/− and wild-type mice. Using the minimally invasive loose cell-attached configuration, we found elevated basal firing rates in BACE1−/− mice compared with wild type (t64 = 2.8, P = 0.007, Fig. 3a,b), while width of the action potential waveform was not different between groups (BACE1−/− 1.482 ± 0.037 milliseconds; wild type 1.487 ± 0.037 milliseconds; t74 = 0.066, P = 0.95, not shown). In whole-cell mode, we did not observe an effect of genotype on other general physiological parameters (not shown) including cell capacitance (BACE1−/− 39.9 ± 1.78 pF; wild type 40.2 ± 1.81 pF; t42 = 0.12, P = 0.90), input resistance (BACE1−/− 111 ± 7.1 MΩ; wild type 108 ± 4.7 MΩ; t42 = 0.38, P = 0.71) and the hyperpolarization-activated current IH [genotype (F 1,17) = 1.44, P = 0.25; voltage (F 4,68) = 1693, P < 0.0001; genotype-voltage interaction (F 4,68) = 0.68, P = 0.61].

Figure 3. Putative DA neurons from BACE1−/− mice exhibit enhanced pacemaker firing.

Figure 3

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(a) Sample traces of pacemaker firing observed using the loose cell-attached configuration in wild type (WT) and BACE1 knockout mice (BACE1−/−). (b) Firing rate (Hz) is significantly larger in DA cells from BACE1−/− mice (white) compared with their WT littermates (black). **P < 0.01.

We then measured DA D2 autoreceptor sensitivity and DA reuptake in slices from BACE1−/− and wild-type mice. With the cell voltage clamped at −55 mV we applied a 10-s pulse of DA (1 M) from an iontophoretic pipette placed adjacent to the cell being recorded. This produced a maximal G-protein coupled inwardly rectifying potassium (GIRK) channel-mediated current via activation of D2 autoreceptors. There was no difference in peak amplitude of the autoreceptor-mediated current (BACE1−/− 219 ± 26.3 pA; wild type 250 ± 31.6 pA; t19 = 0.76, P = 0.46). Further, when normalized to maximum and analyzed over the first 30 seconds, there was no effect of genotype on the current’s decay kinetics (not shown; genotype: F 1,570 = 0.0011, P = 0.97; time: F 29,570 = 74.83, P < 0.0001; genotype – time interaction: F 29,570 = 0.40, P = 0.998). This indicated that there is no significant difference in maximal autoreceptor response to DA or DA uptake in the midbrain of BACE1−/− compared with wild-type mice.

Finally, we tested sensitivity to amphetamine by perfusing a very large concentration (30 μM) to deplete vesicular DA content, drive DA efflux through reversal of the DA transporter (Covey et al. 2013), and activate somatodendritic autoreceptors. Once the amphetamine-induced outward current reached a plateau, exogenous DA was applied with iontophoresis (2 seconds) to measure the maximum possible current for each individual cell (Fig. 4a). When we took the ratio of these two current amplitudes, we found that amphetamine was more efficacious at inducing an outward current in BACE1−/− mice (t19 = 2.7, P = 0.015, Fig. 4b), consistent with larger vesicular DA content. When we added the maximum possible currents to our previous analysis of current amplitudes, we confirmed that there was no effect of genotype on autoreceptor current amplitude (t40 = 1.48, P = 0.15, Fig. 4c).

Figure 4. Amphetamine-mediated currents are enhanced in the substantia nigra of BACE1−/− mice.

Figure 4

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(a) Sample traces of voltage clamp recordings show response to bath perfusion of amphetamine (30 μM AMPH), followed by a 2 seconds iontophoretic application of DA in a putative substantia nigra DA neuron from a wild type (WT) and BACE1 knockout (BACE1−/−) mouse. (b) The amphetamine-induced D2 autoreceptor-mediated currents were larger in substantia nigra DA neurons from BACE1−/− mice (white) compared with WT (black). (c) The amplitudes of the maximum possible autoreceptor-mediated currents were comparable between cells from WT (black) and BACE1−/− mice (white). *P < 0.05.

Discussion

Although BACE1 has been linked to schizophrenia (Luo et al. 2014; Savonenko et al. 2008; Willem et al. 2009), its role in dopaminergic signaling in the midbrain has not been studied. Here, we show that BACE1−/− mice show an increase in amphetamine-mediated autoreceptor inhibition in the substantia nigra, and a concurrent reduced sensitivity to the locomotor-enhancing effects of amphetamine. DA and DA metabolite levels in the striatum were largely unaltered suggesting that the dopaminergic alterations in BACE1−/− may be in the somatodendritic compartment and not at the axon terminals. This conclusion is based on postmortem measures of DA and DA metabolites. Further studies using in vivo methods such as microdialysis or voltammetry may be needed to replicate these results and validate the current interpretation.

Among its many substrates, BACE1 catalyzes extracellular cleavage of NRG1 transmembrane proteins, thereby releasing the bioactive N-terminal fragment. This fragment contains an epidermal growth factor (EGF) domain that activates different downstream signaling pathways via auto- and trans-phosphorylation of ErbB receptor internal domains. Aberrant NRG1-ErbB4 signaling has been reported in schizophrenia postmortem human brains (Hahn et al. 2006) and in BACE1−/− mice that exhibit schizophreniform behaviors (Savonenko et al. 2008). However, while potentially playing a major role in schizophrenia (Howes et al. 2012), no previous studies had explored whether midbrain dopaminergic signaling is affected by disruption of BACE1-mediated cleavage processes. Here, we studied the effects of BACE1 depletion on dopaminergic neurotransmission in the substantia nigra using patch clamp electrophysiology. In brain slice preparations such as the one we use here, putative DA neurons lack active glutamatergic inputs that in vivo induce burst firing and promote goal-directed behavior. Rather, DA neuron firing in brain slices is autonomously driven by several intrinsic conductances, including voltage sensitive sodium currents (Shi 2009). We found that pacemaker firing is enhanced in BACE1 knockout animals. BACE1 cleaves the B-subunit of type 1 voltage-gated sodium channel, and an over expression of BACE1 reduces sodium current density and surface expression of functional sodium channel proteins in B104 neuroblastoma and hippocampal neurons (Kim et al. 2007). BACE1−/− mice consistently exhibit enhanced surface levels of sodium channel proteins and increased neuronal activity in the hippocampus (Hu et al. 2010). It is conceivable that a similar increase in expression of voltage-gated sodium channels in midbrain DA neurons may be responsible for the enhanced pacemaker firing in BACE1−/− mice.

We furthermore investigated the effects of BACE1 depletion on DA D2 autoreceptor-mediated inhibition of midbrain DA neurons. Activation of these receptors via dendrodendritic synapses between DA neurons produces a slow GIRK channel-mediated current that limits firing (Lacey et al. 1987). Amphetamine application activates D2 autoreceptors by increasing cytosolic DA levels through depletion of vesicular content and reverse transport through the DA transporter (Covey et al. 2013; Seiden et al. 1993; Sulzer et al. 1995), and thus provides us with a useful tool to investigate vesicular DA content. BACE1−/− mice exhibited larger amphetamine-induced currents in substantia nigra DA neurons, suggesting enhanced DA content in the somatodendritic compartment. There was no apparent alteration in maximal DA currents in BACE1−/− animals, nor was there evidence for altered DA transporter function.

We found no differences in average baseline locomotor activity between BACE1−/− and wild-type mice. This is in apparent disagreement with earlier reports of novelty-induced hyperactivity and increased locomotor activity in BACE1 knockout mice (Dominguez et al. 2005; Savonenko et al. 2008). However, the mice used in our study were on average younger (3 months ± 2 weeks, compared with 4 – 8 months and 3 – 9 months in the prior studies). In addition, our study used a completely dark enclosed locomotor activity box, compared with transparent boxes used in the previous studies. These procedural differences (e.g. different ages, differently designed locomotor chambers) could be responsible for the apparent discrepancy in results in regard to locomotor activity at baseline. We observed a decrease in sensitivity to amphetamine-induced locomotion in BACE1−/− mice compared with wild type, particularly during the first 15 min of testing (Fig. 1d). Amphetamine significantly increased locomotion only at 3.2 mg/kg in wild-type mice, and only at 10 mg/kg in BACE1−/− mice. These results suggest a shift to the right of the inverted U-shaped dose – response curve for amphetamine-induced locomotion in BACE1−/− mice.

The observed increase in amphetamine-induced D2 autoreceptor-mediated inhibition in BACE1−/− mice could explain the decreased sensitivity to amphetamine-induced locomotion in these animals, because we would expect autoreceptor-mediated inhibition to decrease locomotor activity. Our finding that there is no effect of genotype on autoreceptor current amplitude suggests that the enhanced effect of amphetamine on D2 autoreceptor-mediated outward currents is due to increased efflux of endogenous DA, rather than D2 autoreceptor sensitization or attenuated DA uptake in BACE1−/− mice.

It is important to note that while the dopaminergic system is apparently altered in mice lacking BACE1, enhanced inhibition of dopaminergic signaling is contrary to the midbrain hyperdopaminergia previously reported in schizophrenia (Lyon et al. 2011). Schizophrenia patients exhibit elevated levels of functional NRG1 protein (consistent with increased BACE1 levels), and chronic antipsychotic treatment decreases NRG1 signaling and expression (Pan et al. 2011). Despite this apparent contradiction, BACE1−/− and NRG1−/− both exhibit schizophreniform behaviors including impaired latent inhibition (Rimer et al. 2005), social behavior (Deakin et al. 2009; Kato et al. 2010) and paired pulse inhibition (Deakin et al. 2009; Kato et al. 2010; Savonenko et al. 2008). However, these cognitive and negative symptoms are largely regulated by extradopaminergic areas of the brain, and the areas we studied here have not been extensively investigated in these animal models of schizophrenia. It is also possible that BACE1/NRG1 regulation is an adaptive process that occurs in response to the disease state per se. Our studies suggest that deletion of BACE1 in mice, present from birth, produces persistent adaptations in the dopaminergic system. These alterations could have severe consequences on brain development. In this regard, mice with BACE1 deletion have been shown to have impairments in learning, memory and sensorimotor processes (Kobayashi et al. 2008), alterations that could be a result of dopaminergic signaling dysregulation.

Taken together, our data show that disruption of BACE1 affects dopaminergic signaling in the midbrain, an area thought to play a major role in schizophreniform behaviors. We show that, upon amphetamine treatment, BACE1−/− mice exhibit significant neurophysiological and behavioral differences compared with wild-type mice. Future studies will be needed to determine the role of these adaptations in the schizophreniform behaviors exhibited by mouse models of the disease.

Acknowledgments

This work was supported in by a NARSAD Brain and Behavior Research Young Investigator award to CWB, and NIH grant R01 DA32701 to MJB.

Footnotes

The authors declare no conflicts of interest.

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