Behavioral and neuronal recording of the nucleus accumbens in adolescent rats following acute and repetitive exposure to methylphenidate

Abstract

The nucleus accumbens (NAc) has been shown to play a key role in the brain's response to methylphenidate (MPD). The present study focuses on neuronal recording from this structure. The study postulates that repetitive exposure to the same dose of MPD will elicit in some rats behavioral sensitization and in others tolerance. Furthermore, the study postulates that NAc neuronal activity recorded from animals expressing behavioral tolerance after repetitive MPD exposure will be significantly different from NAc neuronal activity recorded from animals expressing behavioral sensitization after repetitive MPD exposure at doses of 0.6, 2.5, 5.0, and 10.0 mg/kg. To test this, behavioral and neuronal activity was recorded concomitantly from the NAc of freely behaving adolescent rats (postnatal day 40) before and after acute and repetitive administration of four different MPD doses. Comparing the acute MPD effect to the repetitive MPD effect revealed that the acute response to MPD exhibited dose-response characteristics: an increase in behavioral activity correlated with increasing MPD doses. On the other hand, following repetitive MPD exposure, some animals exhibited attenuated behavior (tolerance), while others exhibited further increases in the recorded behavior (sensitization). Moreover, the neuronal activity following repetitive MPD exposure recorded in animals exhibiting behavioral sensitization was significantly different from neuronal activity recorded in animals exhibiting behavioral tolerance. This implies that when studying the effects of repetitive MPD administration on adolescent rats, it is advisable to simultaneously record both neuronal and behavioral activity and to evaluate all data based on the animals' behavioral response to the repetitive MPD exposure.

the nucleus accumbens (NAc) has often been studied as an important center with regards to the behavioral response elicited by psychostimulants (Kim et al. 2009; Pierce and Kalivas 1997b; Podet et al. 2010). For example, many studies have demonstrated that repeated administration of methylphenidate (MPD) in rats elicits behavioral sensitization, while others have in contrast shown that repeated administration can elicit behavioral tolerance (Askenasy et al. 2007; Gaytan et al. 1997a; Yang et al. 2007). Behavioral sensitization refers to the augmentation of certain behavioral and locomotor responses following repetitive psychostimulant application, and this experimental observation has been suggested to be an experimental marker and model for drug craving (Kalivas and Steward 1991; Robinson 1993; Robinson and Berridge 1993, 2001). Behavioral tolerance, on the other hand, refers to an attenuation of behavioral and locomotor responses following repetitive psychostimulant application (Askenasy et al. 2007; Izenwasser et al. 1999; Kuczenski and Segal 2002). Similarly to sensitization, behavioral tolerance has been suggested as a key mechanism in drug-seeking behavior and increased self-administration (Stewart and Badiani 1993; Yang et al. 2010). In addition, studies in rats have demonstrated that exposure to MPD during the period equivalent to human adolescence leads to behavioral changes that persist into adulthood, signifying that treatment with MPD at a young age can lead to long-term effects (Askenasy et al. 2007; Barron et al. 2009; Marco et al. 2011; Yang et al. 2010).

These behavioral changes, as induced by repetitive psychostimulant administration, are suggested to involve the “motive circuit” or the “brain reward circuit,” a shorthand for a host of interconnected structures and nuclei including the ventral tegmental area, the NAc, the prefrontal cortex, and other brain sites (Pierce and Kalivas 1997b; Rebec 2006). In particular, modulation of dopaminergic transmission in the NAc has been widely investigated as a central process in the expression of behavioral sensitization to psychostimulants (Kim et al. 2009; Pierce and Kalivas 1997a; Podet et al. 2010; Rebec 2006). MPD has also been shown to act as a rewarding and reinforcing stimulus, as evidenced by its ability to induce and maintain self-administrative behavior (Bergman et al. 1989; Kollins et al. 2001), as well as induce conditioned placed preference (CPP) in rats (Martin-Iverson et al. 1985; Mithani et al. 1986; Meririnne et al. 2001). Similarly to sensitization, these effects are at least partially underpinned by dopaminergic transmission in the motive circuit (Askenasy et al. 2007).

The present study focuses on the NAc of adolescent animals because of the NAc's well-established role in psychostimulant response in behavioral studies (Pierce and Kalivas 1997a; Podet et al. 2010). Moreover, adolescent rats are employed to parallel the age in which MPD use is widespread and controversial in humans (Laviola et al. 2003; Marco et al. 2011) and because it has been shown that there is a difference in the response to MPD between adult and adolescent animals, particularly in that adolescent subjects are often more sensitive to psychostimulants than adults (Canese et al. 2009; Marco et al. 2011). Rat adolescence has been suggested to encompass a range as broad as postnatal days 21–60, although most recently the accepted range has been postnatal days 35–50 (Laviola et al. 2003; Marco et al. 2011), and thus this study uses rats that are at postnatal day 40 at the start of the experiment. In addition, it has been reported that MPD administration in anesthetized animals elicits a decrease in neuronal activity, while nonanesthetized animals exhibit the reverse effect, in that they show an increase in neuronal activity (Ruskin et al. 2001). For this reason, nonanaesthetized, freely behaving animals previously implanted with permanent electrodes are used to preclude any influence of anesthetic drugs on electrophysiological measurements (Claussen and Dafny 2012; Chong et al. 2012; Salek et al. 2012). The ultimate objective of the present study is to evaluate the MPD dose-response characteristics of NAc neuronal activity recorded in adolescent animals exhibiting behavioral sensitization after acute and repetitive MPD administration separately from animals that express behavioral tolerance after repetitive MPD administration. Our hypothesis is that the neuronal activity recorded from animals expressing behavioral tolerance to repetitive MPD exposure will be significantly differently from NAc neuronal activity recorded from animals exhibiting behavioral sensitization at the same MPD dose.

METHODS

Animals

Male Sprague-Dawley rats ∼28 days of age (n = 177) purchased from Harlan (Indianapolis, IN) were given 5–7 days of acclimation to the sound-attenuated animal room and were kept on a 12-h light-dark schedule, with lights on at 6:00 AM. The room was maintained at a temperature of 21 ± 2°C and a humidity of 58–62%. The experiment lasted ∼3 yr, and every few weeks a new shipment of four rats was received. The age of the rats on experimental day 1 was kept constant at postnatal day 40. The rats were allowed food and water ad libitum for the entire study, and their home cage was used as their test cage. The study was approved by the University of Texas Medical School at Houston Animal Welfare Committee and carried out in accordance with the National Institutes of Health Guide for Care and Use of Laboratory Animals.

Drug

Methylphenidate hydrochloride (MPD) was purchased from Mallinckrodt (St. Louis, MO) and for the experiment was dissolved in 0.9% saline (NaCl) to achieve the required concentrations. Previous behavioral and neurophysiological experiments using MPD doses ranging from 0.1 to 40 mg/kg ip have shown that MPD doses of 0.6, 2.5, and 10.0 mg/kg can elicit behavioral sensitization or tolerance (Askenasy et al. 2007; Dafny and Yang 2006; Gaytan et al. 1997b, 2000a, 2000b; Yang et al. 2006a, 2006b, 2007), and thus for this study the doses 0.6, 2.5, 5, and 10 mg/kg MPD were used, with the dose calculated as free base. All injections were equalized to 0.8 ml using 0.9% saline across all MPD doses, allowing injection of equal volumes into all animals.

Surgery

Surgery was performed as detailed in previous studies (Chong et al. 2012; Claussen and Dafny 2012; Clauseen et al. 2014). Rats were anesthetized on the day of surgery using 30 mg/kg ip pentobarbital, and the Sherwood and Timiras (1970) rat brain atlas was used as a guide to drill one hole above the frontal sinus and bilateral 0.6-mm diameter holes above the NAc (1.7 mm anterior to the bregma and 1.2 mm lateral from the midline). Recording electrodes were created by twisting two nickel-chromium, Teflon-coated, 60-μm diameter wires, fully insulated except at the tips and each secured to a 1-cm copper connector pin. One reference electrode was then placed in the frontal sinus. One twisted electrode (consisting of 2 electrodes twisted together) was introduced into each NAc bilaterally by first inserting the twisted electrode to a depth of 6.8 mm, with concurrent monitoring of activity using a Grass emitter Hi Z Probe connected to a Grass P511 amplifier. Electrodes were fixed to the skull only when spike activity exhibited a noise ratio of at least 3:1 in both electrodes, and if this was not achieved, the electrode was inserted further in increments of ∼10 μm until the 3:1 ratio was attained (Chong et al. 2012; Claussen and Dafny 2012). The rats were allowed to recover for 4–7 days, and every day for 2 h the rats (with their home cages) were placed in the experimental behavioral apparatus and connected to the wireless (telemetric) head stage transmitter (∼4 gm in weight; Triangle BioSystems, Durham, NC) to allow for acclimation to the recording systems.

Experiment

Table 1 summarizes the 10-day experimental protocol, with all recording session starting at ∼8:00 AM. At postnatal age of 40 days, rats were randomly assigned to five groups: saline, 0.6, 2.5, 5, or 10 mg/kg MPD. The saline injection group served as a control for handling, injection, and injection volume. On experiment day 1, the animals were again allowed to acclimate to the recording apparatus for 20–30 min. All animals were then injected intraperitoneally with saline (0.8 ml of 0.9% NaCl), and a 60-min baseline of both neuronal and behavioral activity was recorded concurrently (day 1 baseline). The animals were then injected with either saline or 0.6, 2.5, 5.0, or 10 mg/kg MPD depending on their randomly assigned group (day 1 MPD injection), and recordings of neuronal and behavioral data were resumed for another 60 min immediately following injection. On days 2–6, rats continued to be injected daily in their home cage with the same concentration of MPD (or saline) as on day 1 with no data recording, followed by 3 washout days 7–9, during which no injections were given and no recordings were made. The experimental protocol on day 10 (postnatal day 49) was identical to day 1, with a saline injection followed by 60 min of recording baseline neuronal and behavioral activity (day 10 baseline), and an MPD rechallenge at the same dosage as on the other days, followed by another hour of neuronal and behavioral recording (day 10 rechallenge); thus the experiment lasted for 10 days for each animal.

Table 1.

Experimental protocol

Groups	Day 1*	Days 2–6	Days 7–9	Day 10*
Saline control	Saline/saline	Saline	Washout	Saline/saline
0.6 mg/kg MPD	Saline/0.6 MPD	0.6 MPD	Washout	Saline/0.6 MPD
2.5 mg/kg MPD	Saline/2.5 MPD	2.5 MPD	Washout	Saline/2.5 MPD
5.0 mg/kg MPD	Saline/5.0 MPD	5.0 MPD	Washout	Saline/5.0 MPD
10.0 mg/kg MPD	Saline/10.0 MPD	10.0 MPD	Washout	Saline/10.0 MPD

MPD, methylphenidate.

^*Neuronal and behavioral recordings were done on that day.

Behavioral Apparatus

Rat locomotor data were recorded using an open field computerized animal activity system (Opto-M3; Columbus Instruments, Columbus, OH). The animals remained in their home cages during the period of adaptation and during the length of the experiment. The home cage was a clear acrylic cage (40 cm in length and 20 cm in width) that was fitted into the behavioral recording apparatus. The system uses 16 infrared beams (and their sensors on the opposite side) along the length, and 8 beams and their sensors along the width, set 5 cm above the floor of the cage. Movement along any of the beams constitutes a beam break, and the software program converts these into the counts of horizontal activity (HA) and number of stereotypic movements (NOS) (Claussen et al. 2012). HA represents overall locomotor activity as recorded by beam breaks from one beam to the next. NOS, on the other hand, counts the number of small, repetitive movement episodes during which an animal breaks the same beam repeatedly, with at least 1 s between each episode (Claussen et al. 2012; Eckermann et al. 2001; Gaytan et al. 2000a, 2000b; Podet et al. 2010). Thus, in general, as an animal increases (or decreases) its movements after drug administration, it also increases (or decreases) the number of beam breaks it performs. The counts of beam breaks were downloaded to a PC in 10-min bin increments and evaluated for the 60 min postinjection of the saline baseline, and the 60 min postinjection of MPD (or saline for control) on day 1 and day 10, as described in Experiment. These behavioral data were used to stratify animals in each dosage group on the basis of whether they exhibited behavioral sensitization or behavioral tolerance (see Analysis of Behavioral Data) to use that as a basis for analysis of electrophysiological data.

Analysis of Behavioral Data

After the behavioral data were recorded as described in Behavioral Apparatus, the HA and NOS were analyzed for each dosage group using an ANOVA test with repeated measures, with significance set at P < 0.05. The comparisons made were as follows: 1) day 1 baseline compared with day 1 post-MPD injection to determine the acute effect of the MPD at each dosage; 2) day 10 baseline compared with day 1 baseline to determine whether the six daily MPD administrations and subsequent washout had an effect on baseline behavioral activity at day 10; and 3) day 10 postrechallenge with MPD compared with day 1 postinitial MPD exposure to determine whether behavioral sensitization or tolerance was expressed in each rat. The “Total” columns of Fig. 1 portray the average HA data at each dosage for all three analyses and show where significance was demonstrated at each dose and time interval.

Fig. 1.

Summary of the behavioral data using horizontal activity (A) and number of stereotypic movement (B) from animals exposed to acute and repetitive methylphenidate (n = 91). Doses at 0.6 (a), 2.5 (b), 5.0 (c), and 10.0 (d) mg/kg are shown. ^aTotal: summary of the behavioral results from all the animals. ^bSensitization: summary of the behavioral results only from the animals expressing behavioral sensitization. ^cTolerance: summary of the behavioral results only from animals expressing behavioral tolerance. ΔSignificant (P < 0.05) difference compared with baseline on day 1. *Significant (P < 0.05) difference compared with methylphenidate (MPD) day 1. MPD; n = number of animals in each group.

Next, the behavioral activity of each rat was analyzed individually using the paired t-test with significance set at P < 0.05. The same three comparisons described in the previous paragraph were used. Based on the third analysis (activity after rechallenge with MPD on day 10 compared with activity after initial MPD exposure on day 1), the rats were labeled as either “behaviorally sensitized” or “no change/tolerant” within their appropriate MPD dosage groups. The “Sensitization” and “Tolerance” columns of Fig. 1 demonstrate the results of this analysis. If the post-MPD behavioral activity of the rat on day 10, after six daily MPD exposures and 3 washout days, was significantly (P < 0.05) increased compared with the post-MPD behavioral activity on day 1, the rat was classified as expressing behavioral sensitization. On the other hand, if the post-MPD behavioral activity of the rat on day 10, after six daily MPD exposures and 3 washout days, was significantly (P < 0.05) decreased compared with the post-MPD behavioral activity at day 1, the rat was classified as expressing behavioral tolerance.

Electrophysiological Recording

Data acquisition.

During the experiment, the rat in its home cage was placed in a Faraday testing box to reduce noise during signal transmission. The electrodes of the skull cap were then connected to the wireless Triangle BioSystems (Durham, NC) head stage, which transmitted neuronal activity signals through a receiver to a Cambridge Electronic Design (CED) analog-to-digital converter (Micro1401-3; Cambridge, England), which also collected the data and stored it to a PC.

Spike sorting.

Spike sorting from the recording was conducted using Spike 2.7 software (CED) at sampling rates of up to 200 kHz and processed using low- and high-pass filters (0.3–3 kHz), with two window discriminatory levels accounting for both positive- and negative-going spikes. Spikes were processed as described in previous publications (Chong et al. 2012; Claussen and Dafny 2012; Claussen et al. 2014; Salek et al. 2012). The parameters for spike sorting used on data recorded on day 1 from each particular electrode and animal were stored and reused identically on day 10, thus assuring that the spike patterns captured on these days were the same for each particular animal and electrode (see Figs. 3, top, and 4, C and D). Single unit spike activity confirmed to be recorded from the NAc and exhibiting similar amplitudes and wave form patterns before and after MPD administration for day 1 and day 10 was analyzed to create a sequential frequency histogram that provides the firing rate in spikes per second. One to two spikes were analyzed per electrode.

Fig. 3.

Representative frequency histograms of NAc unit baseline activity recorded on experimental day 1 (BL1) and day 10 (BL10) after 6 daily 10.0 mg/kg MPD injections and 3 washout days. Increase (A) and decrease (B) in neuronal activity as the result of previous MPD exposure are shown and were recorded from animals expressing behavioral sensitization and behavioral tolerance, respectively. At top are 20 superimposed spikes showing that the same spike shape and amplitude was evaluated on day 1 and day 10.

Electrophysiological Data Analysis

The calculated firing rates for each 60 min segment were exported to a spreadsheet along with the rat's unique number, experimental date, MPD dose, and channel. These firing rates were subsequently found not to hold normality assumptions, and thus differences in mean firing rates were assessed using the critical ratio (CR) test, wherein CR = $(\mathrm{E}-\mathrm{C})\hspace{0.17em}/\hspace{0.17em}\sqrt{[\mathrm{E}+\mathrm{C}]}$ = ± 1.96 = P < 0.05 (C = activity following saline [control] injection; E = activity following MPD injection). CR values greater than +1.96 indicate that the treatment elicited a significant increase in neuronal activity, while CR values less than −1.96 indicate that the treatment elicited a significant decrease in neuronal activity (Chong et al. 2012; Claussen and Dafny 2012; Salek et al. 2012). Three comparisons were made, similar to the behavioral comparisons in Analysis of Behavioral Data: first, the acute effect of MPD was evaluated by comparing day 1 baseline activity with day 1 activity following MPD administration. Second, the day 10 baseline following six daily MPD injections and 3 days of washout was compared with the day 1 baseline to evaluate whether this schedule of injections and washout altered the baseline activity at day 10. Lastly, the effects of MPD rechallenge on day 10 were compared with the effects elicited following the initial MPD injection on day 1 to determine whether the animal exhibited electrophysiological sensitization (increase in neuronal activity) or tolerance (decrease or no change in neuronal activity). Changes of mean activity induced by MPD treatment were considered statistically significant if the firing rate after treatment differed by at least 2 SE from the mean (Claussen and Dafny 2012; Dafny 1982; Salek et al. 2012).

To test the major hypothesis of this article, that neuronal activity in animals showing behavioral sensitization will be significantly different from neuronal activity in animals showing behavioral tolerance, a log linear model statistical test was used (Claussen et al. 2014). The log linear test is a type of χ²-square analysis that calculates the values in question by means of weighted natural logarithms. This test is able to determine the degree of interaction between variables in a three-way contingency table, as well as two-way interactions between any two of the variables. At each MPD dose, this test was used to analyze the neuronal firing of all the rats that showed behavioral sensitization, compared with the neuronal firing of all the rats that showed behavioral tolerance, as described in Analysis of Behavioral Data. P < 0.05 obtained from this test was considered to show statistically significant difference between the groups in question, and the results are detailed in Statistical Comparison Between the Behavioral Groups at Each MPD Dose.

Electrode Placement Verification

At the end of the experiment, the rats were deeply anesthetized with sodium pentobarbital, at which point the brain was transcardially perfused with 10% formalin solution containing 3% potassium ferrocyanide. To produce a small lesion and mark placement of the electrode in the brain, a 2-mA DC current was passed through the electrode for 40 s. The brain was then removed and stored in 10% formalin for histological processing. Sixty-micrometer thick coronal sections were then created and stained with cresyl violet. The locations of the lesions (representing electrode tips) were established using the Rat Brain Atlas (Sherwood and Timiras 1970). The data from those rats in which the electrode was found to be in the nucleus accumbens were analyzed and included in this study.

RESULTS

Locomotor Behavior

Ninety nine rats were included in this behavioral and neuronal study after meeting the histological criteria for verification of electrode placement within the NAc and after verification that their neuronal spike amplitudes and durations at day 10 were the same as on day 1 (saline, n = 8; 0.6 mg/kg, n = 24; 2.5 mg/kg, n = 21; 5.0 mg/kg, n = 10; and 10 mg/kg, n = 36). The saline (control) group showed no change in behavioral activity after both acute and repetitive saline injection, demonstrating that handling of the animals, the procedure of injection, and the experimental environment had no effect on the animals' behavior and that the data obtained at day 1 following saline injection can be used as control.

Figure 1, A and B, summarizes the HA and the NOS of all the groups exposed to MPD, with baseline activity on experimental day 1, as well as activity after MPD administration on day 1 and after MPD rechallenge on day 10 following six daily MPD injections and 3 washout days. At each dose, data are shown for all the animals that received that dose (“Total”), for those animals that showed behavioral sensitization (“Sensitization”) to repetitive MPD exposure, and those that showed behavioral tolerance (“Tolerance”) to repetitive MPD exposure. In sum, Fig. 1 demonstrates that acute exposure to MPD at doses of 5.0 and 10.0 mg/kg on day 1 significantly (P < 0.05) increases HA when all animals at each dose are examined together (Fig. 1, Ac, a; and Ad, a). When examined based on behavior, the HA of animals that go on to show behavioral sensitization and behavioral tolerance is increased significantly (P < 0.05) at MPD doses of 2.5, 5.0, and 10.0 mg/kg on day 1 (Fig. 1, Ab, b and c; Ac, b and c; and Ad, b and c). In addition, this figure demonstrates that at MPD doses of 0.6, 2.5, 5.0, and 10.0 mg/kg, MPD exposure on day 10 tends to significantly (P < 0.05) increase HA in some animals compared with day 1 (expressing behavioral sensitization), and tends to significantly (P < 0.05) decrease HA in some animals compared with day 1 (expressing behavioral tolerance).

An overall similar pattern of behavior can be seen when the data for NOS are examined (Fig. 1B). In particular, acute exposure to MPD at doses of 2.5, 5.0, and 10.0 mg/kg on day 1 significantly (P < 0.05) increases NOS when all animals at each dose are examined together (Fig. 1, Bb, a; Bc, a; and Bd, a). In addition, acute exposure to MPD on day 1 significantly (P < 0.05) increases the NOS of animals that go on to show behavioral sensitization and behavioral tolerance at MPD doses of 5.0 and 10.0 mg/kg (Fig. 1, Bc, b and c; and Bd, b and c) but not at 2.5 mg/kg MPD. Finally, as with the HA data, MPD exposure on day 10 tends to significantly (P < 0.05) increase the NOS in some animals compared with day 1 (expressing behavioral sensitization) and tends to significantly (P < 0.05) decrease NOS in some animals compared with day 1 (expressing behavioral tolerance). This effect is especially evident at 5.0 and 10.0 mg/kg for both behavioral metrics.

Electrophysiological Responses

In total, 396 electrodes were implanted in the experimental animals, and 337 were subsequently identified to be in the NAc after histological verification (see Electrode Placement Verification). In most cases, each electrode recorded one individual unit, although occasionally a single electrode recorded two units. Some of the units did not exhibit activity that could be evaluated on day 1 and day 10 (see Spike sorting). In sum, a total of 289 NAc units from 99 rats were evaluated on experimental days 1 and 10: 73 units from the 0.6 mg/kg group, 79 units from the 2.5 mg/kg group, 29 units from the 5.0 mg/kg group, 79 units from the 10.0 mg/kg group, and 29 units from the saline control group.

The baseline activity of the above units was between 0.6 and 3.7 spikes per second. Variations were observed between animals and between batches of rats received. However, the rats acted as their own controls during the experiment (see Electrophysiological Data Analysis), and the response to MPD treatment was not related to the initial baseline.

NAc units exposed to saline (control).

Most (83%) of the NAc units recorded showed no changes in neuronal activity following the second saline injection on day 1 compared with the first saline injection on day 1 and on day 10 (Table 2). These data show that animal handling, volume injection, and the behavioral apparatus do not significantly affect NAc neuronal activity, and any significant change following MPD administration is due to the MPD itself.

Table 2.

Summary of the responses of 289 NAc units following saline and acute and chronic MPD exposure

Groups	Units	Unresponsive	Responsive	Increase	Decrease
Acute effect
Saline	29	24 (82.76%)	5 (17.24%)	1 (3.45%)	4 (13.79%)
0.6 mg/kg	73	52 (71.23%)	21 (28.77%)	16 (21.92%)	5 (6.85%)
2.5 mg/kg	79	40 (50.63%)	39 (49.37%)	27 (34.18%)	12 (15.19%)
5.0 mg/kg	29	8 (27.59%)	21 (72.41%)	18 (62.07%)	3 (10.34%)
10.0 mg/kg	79	12 (15.19%)	64 (81.01%)	56 (70.88%)	8 (10.13%)
Day 10 BL vs. day 1 BL
Saline	29	24 (82.76%)	5 (17.24%)	2 (6.90%)	3 (10.34%)
0.6 mg/kg	73	51 (69.86%)	22 (30.14%)	8 (10.96%)	14 (19.18%)
2.5 mg/kg	79	38 (48.10%)	41 (51.90%)	32 (40.51%)	9 (11.39%)
5.0 mg/kg	29	4 (13.79%)	25 (86.21%)	18 (62.07%)	7 (24.14%)
10.0 mg/kg	79	24 (30.38%)	55 (69.62%)	43 (54.43%)	12 (15.19%)
MPD day 10 vs. MPD day 1
Saline	29	25 (86.21%)	4 (10.79%)	1 (3.45%)	3 (10.34%)
0.6 mg/kg	73	45 (61.64%)	28 (38.36%)	21 (28.77%)	7 (9.59%)
2.5 mg/kg	79	33 (41.77%)	46 (58.23%)	39 (49.37%)	7 (8.86%)
5.0 mg/kg	29	3 (10.34%)	26 (89.66%)	23 (79.31%)	3 (10.35%)
10.0 mg/kg	79	4 (5.06%)	75 (94.94%)	64 (81.01%)	11 (13.93%)

NAc, nucleus accumbens; BL, baseline.

All NAc units exposed to MPD.

Table 2 summarizes the effect of MPD on electrophysiological data of all the MPD doses after acute injection on day 1, the baseline activity on day 10 after six daily MPD injections and 3 washout days compared with baseline activity on day 1, and after rechallenge with MPD on day 10 compared with acute administration of MPD on day 1. For each MPD dose group, the percentage of units that showed increased activity (Fig. 2A), decreased activity (Fig. 2B), or no change following MPD exposure is summarized. Table 3, “Acute effect on day 1,” separates those NAc units recorded from animals expressing behavioral sensitization to repetitive MPD exposure (n = 138) from those NAc units recorded from animals expressing behavioral tolerance to repetitive MPD exposure (n = 122). Table 3 shows the percentage of NAc units that exhibit increased activity, decreased activity or no change following each MPD dosage after acute administration on day 1. Table 3, “Baseline effect on day 10,” shows a similar summary of the activity of the 138 NAc units from animals that demonstrated behavioral sensitization, and the 122 NAc units from animals that demonstrated behavioral tolerance, comparing the day 10 baseline to the day 1 baseline. Finally, Table 3, “Rechallenge effect on day 10,” summarizes the NAc unit responses after MPD rechallenge on day 10 compared with their response to MPD on day 1 in animals that demonstrated behavioral sensitization and tolerance at each MPD dosage.

Fig. 2.

Representative frequency histograms of nucleus accumbens (NAc) units before and following acute exposure to 10.0 mg/kg of MPD recorded from adolescent rats. A: representative NAc unit recorded from an animal expressing behavioral sensitization that responded to MPD by increasing its firing rate. B: NAc unit recorded from an animal expressing behavioral tolerance that responded to 10.0 mg/kg MPD by decreasing its neuronal firing rate. Both effects started about 20 min after the intraperitoneal application and lasted between 30 and 45 min. Arrows indicate when MPD was injected after the baseline recording.

Table 3.

The acute, baseline, and rechallenge effect of 0.6, 2.5, 5.0, and 10. mg/kg MPD on 260 NAc units recorded from animals expressing behavioral sensitization and tolerance

Groups/Behavior	Units	Unresponsive	Responsive	Increase	Decrease
Acute effect on day 1
0.6 mg/kg
Sensitization	24	11 (45.83%)	13 (54.17%)	9 (37.50%)	4 (16.67%)
Tolerance	49	41 (83.67%)	8 (16.33%)	7 (14.29%)	1 (2.04%)
2.5 mg/kg
Sensitization	39	9 (23.08%)	30 (76.92%)	24 (61.54%)	6 (15.38%)
Tolerance	40	31 (77.50%)	9 (22.50%)	3 (7.50%)	6 (15.00%)
5.0 mg/kg
Sensitization	22	5 (22.73%)	17 (77.27%)	15 (68.18%)	2 (9.09%)
Tolerance	7	3 (42.86%)	4 (57.14%)	3 (42.86%)	1 (14.28%)
10.0 mg/kg
Sensitization	53	12 (22.64%)	41 (77.36%)	36 (67.93%)	5 (9.43%)
Tolerance	26	3 (11.54%)	23 (88.46%)	20 (76.92%)	3 (11.54%)
Baseline effect on day 10
0.6 mg/kg
Sensitization	24	13 (54.17%)	11 (45.83%)	3 (12.50%)	8 (33.33%)
Tolerance	49	38 (77.55%)	11 (22.45%)	5 (10.20%)	6 (12.25%)
2.5 mg/kg
Sensitization	39	9 (23.08%)	30 (76.92%)	26 (66.67%)	4 (10.25%)
Tolerance	40	29 (72.50%)	11 (27.50%)	6 (15.00%)	5 (12.50%)
5.0 mg/kg
Sensitization	22	3 (13.64%)	19 (86.36%)	14 (63.63%)	5 (22.73%)
Tolerance	7	1 (14.29%)	6 (85.71%)	4 (57.14%)	2 (28.57%)
10.0 mg/kg
Sensitization	53	11 (20.76%)	42 (79.24%)	36 (67.92%)	6 (11.32%)
Tolerance	26	13 (50.00%)	13 (50.00%)	7 (26.92%)	6 (23.08%)
Rechallenge effect on day 10
0.6 mg/kg
Sensitization	24	5 (20.83%)	19 (79.17%)	14 (58.34%)	5 (20.83%)
Tolerance	49	40 (81.63%)	9 (18.37%)	7 (14.29%)	2 (4.08%)
2.5 mg/kg
Sensitization	39	7 (17.95%)	32 (82.05%)	29 (74.36%)	3 (7.69%)
Tolerance	40	26 (65%)	14 (35.00%)	10 (25.00%)	4 (10.00%)
5.0 mg/kg
Sensitization	22	1 (4.55%)	21 (95.45%)	19 (86.36%)	2 (9.09%)
Tolerance	7	2 (28.57%)	5 (71.43%)	4 (57.14%)	1 (14.29%)
10.0 mg/kg
Sensitization	53	1 (1.89%)	52 (98.11%)	49 (92.45%)	3 (5.66%)
Tolerance	26	3 (11.54%)	23 (88.46%)	15 (57.69%)	8 (30.77%)

Number of NAc units: n = 138 for behavioral sensitization and n = 122 for tolerance.

In sum, Table 2, “Acute effect,” demonstrates that acute administration of MPD at all doses studied causes some animals to change their firing rates, while others remain unresponsive. A dose-response pattern is observed, wherein increasing MPD dosage increases the proportion of NAc units significantly (P < 0.05) altering their firing rates, from 28.77% at 0.6 mg/kg MPD to 81.01% at 10.0 mg/kg MPD (Table 2, “Acute effect,” “Responsive” column). Likewise, of those units that did change their firing rate, a higher proportion did so by increasing their firing rate the higher the MPD dosage administered from 21.92% at 0.6 mg/kg MPD to 70.88% at 10.0 mg/kg MPD (Table 2, “Acute effect,” “Increase” column).

The baseline activity at day 10 after six daily MPD injections and 3 washout days was compared with the day 1 baseline activity. The neuronal activity was modulated in 30.14, 51.90, 86.21, and 69.62% NAc units following 0.6, 2.5, 5.0, and 10.0 mg/kg MPD, respectively, and the majority did so by increasing the baseline firing rate (Table 2, “Day 10 BL vs. day 1 BL,” and Fig. 3A).

Rechallenge with 0.6, 2.5, 5.0, and 10.0 mg/kg MPD on day 10 following six daily MPD injections and 3 days of washout significantly (P < 0.05) altered the firing rates of NAc units in a dose-response pattern, with 38.36% changing the firing rate at 0.6 mg/kg MPD, up to 94.94% changing their firing rate at 10.0 mg/kg (Table 2, “MPD day 10 vs. MPD day 1,” “Responsive” column). Of those units that did change their firing rate, a higher proportion did so by increasing their firing rate in a dose-response pattern from 28.77% at 0.6 mg/kg MPD to 81.01% at 10.0 mg/kg MPD (Table 2, “MPD day 10 vs. MPD day 1,” “Increase” column).

NAc units recorded from animals expressing behavioral sensitization and behavioral tolerance.

To determine if there is a difference in the neuronal firing response to MPD exposure in the two behavioral groups, Table 3 summarizes the electrophysiological data recorded from animals showing behavioral sensitization separately from the electrophysiological data recorded from animals showing behavioral tolerance following repetitive MPD exposure.

In sum, after acute administration of MPD on day 1 at all doses, a majority of NAc units recorded from animals showing behavioral sensitization significantly (P < 0.05) changed their firing rates, and a majority of them did so by increasing their firing rates (Table 3, “Acute effect on day 1,” “Sensitization” rows, and Fig. 2A). On the other hand, in NAc units recorded from animals demonstrating behavioral tolerance, smaller proportions of units significantly changed their firing rates at all doses except 10.0 mg/kg MPD. Furthermore, there was a more varied type of response in the NAc units recorded from the animals expressing behavioral tolerance, with the majority of those units that did respond at 0.6, 5.0, and 10.0 mg/kg MPD increasing their firing rate, and the majority of responding units at 2.5 mg/kg decreasing their firing rate (Table 3, “Acute effect on day 1,” “Tolerance” rows; and Fig. 2B).

In animals demonstrating behavioral sensitization, the baseline activity at day 10 after six daily MPD injections and 3 washout days compared with the day 1 baseline was significantly (P < 0.05) different in 45.8% of NAc units at 0.6 mg/kg MPD, 86.36% of units at 5.0 mg/kg MPD, with the other doses ranging between these percentages (Table 3, “Baseline effect on day 10,” “Sensitization” rows). On the other hand, in animals demonstrating behavioral tolerance, the baseline activity at day 10 compared with the day 1 baseline was significantly (P < 0.05) different in 22.45% of units at 0.6 mg/kg MPD and different in 85.71% of units at 5.0 mg/kg MPD, with other doses ranging between these percentages (Table 3, “Baseline effect on day 10,” “Tolerance” rows).

After rechallenge with MPD on day 10 following six daily MPD doses and 3 washout days, the majority of NAc units recorded from animals demonstrating behavioral sensitization significantly (P < 0.05) changed their firing rates at all doses, and the majority of the responding units did so by increasing their firing rates at all doses (Table 3, “Rechallenge effect on day 10,” “Sensitization” rows, and Fig. 4A), and fewer did so by decreasing their firing rates (Fig. 4, B and C). On the other hand, after rechallenge with MPD on day 10, NAc units recorded from animals demonstrating behavioral tolerance did not change their firing rates as robustly, with the majority of units at 0.6 and 2.5 mg/kg not changing their firing rates. However, of those units that did change their firing rates, the majority also did so by increasing their neuronal activity at all doses (Table 3, “Rechallenge effect on day 10,” “Tolerance” rows).

Fig. 4.

Representative frequency histograms of NAc unit firing rates recorded in rats that express behavioral sensitization. In these animals, the effect of 2.5 mg/kg MPD observed on experimental day 1 (MPD D1) was an increase in activity, and the effect of the same dose on day 10 (MPD D10) elicited a further increase in firing rate (A). B: MPD 2.5 mg/kg elicits the opposite effect: on day 1 the drug attenuates the NAc firing rate, and on day 10 further attenuation is observed following administration of the same MPD dose. These 2 NAc responses represent neurophysiological sensitization. C: raw analog recording of an NAc unit on day 1 and day 10 following injection of 2.5 mg/kg MPD, recorded 20 min postinjection. A decrease in firing rate is demonstrated at day 10 compared with day 1. D: 50 superimposed spikes from the same units, sorted by our software on day 1 and day 10, showing that the same spike shape and amplitude was evaluated on both days.

Statistical Comparison Between the Behavioral Groups at Each MPD Dose

NAc units recorded from animals expressing behavioral sensitization and behavioral tolerance above summarizes the electrophysiological results in NAc units exposed to MPD and compares these results between animals that show behavioral sensitization and those that show behavioral tolerance. As explained in Electrophysiological Data Analysis, a log linear model statistical test was used to formally analyze the differences between the neuronal firing response patterns, behavior, and doses, with significance set at P < 0.05. The analysis demonstrates that, as hypothesized, at each dose of MPD (0.6, 2.5, 5.0, and 10.0 mg/kg), neuronal firing rates are significantly different between animals demonstrating behavioral tolerance and animals demonstrating behavioral sensitization (0.6 mg/kg: df = 1, χ² = 4.29, P = 0.0384; 2.5 mg/kg: df = 1, χ² = 16.96, P < 0.0001; 5.0 mg/kg: df = 1, χ² = 15.01, P = 0.0001; 10.0 mg/kg: df = 1, χ² = 51.37, P < 0.0001). The saline (control) group did not show such significance between behavioral groups with regard to firing rates (df = 1, χ² = 0.36, P = 0.5493).

DISCUSSION

The NAc has historically been considered a key center in the behavior response elicited by psychostimulants such as MPD (Kim et al. 2009; Pierce and Kalivas 1997b; Podet et al. 2010). For example, it has been demonstrated that MPD can act as a rewarding and reinforcing stimulus in inducing self-administrative behavior and conditioned place preference (Bergman et al. 1989; Kollins et al. 2001; Martin-Iverson et al. 1985; Meririnne et al. 2001; Mithani et al. 1986). In behavioral experiments using adult animals, repetitive MPD administration has been shown to elicit either dose-dependent behavioral sensitization or behavioral tolerance, leading both to increased and decreased MPD-induced behavioral responses after repetitive MPD exposure, respectively (Askenasy et al. 2007; Eckermann et al. 2001; Gaytan et al. 2002; Podet et al. 2010; Yang et al. 2010). In addition, psychostimulants act upon the neurotransmitter systems that undergo maturation during adolescent development in the form of increases or decreases in dendritic branches, receptor density pruning, and neuronal pathway reorganization, and the immature nervous system tends to react differently from the mature nervous system after exposure to psychostimulants (Huttenlocher 1979; Izenwasser 2005; Kim et al. 2009; Marco et al. 2011; Rakic 1986; Russo et al. 2010). Thus this study uses adolescent rats because most electrophysiological studies on the effects of MPD have been performed in adult animals, while the majority of MPD users are in fact young (Laviola et al. 2003).

In our own previous studies using adult animals and an identical protocol to this study (Claussen et al. 2014; Jones and Dafny 2013, 2014; Tang and Dafny 2013), it was observed that at each dose of MPD, repetitive administration elicits in some animals behavioral sensitization, and in others behavioral tolerance. Moreover, it was found that the HA and NOS exhibit similar responses as metrics of behavior. Thus HA and NOS were used as tools to separate the animals based on their behavioral activity, allowing for the analysis of neuronal activity separately in each behavioral group, as neuronal activity is presumed to regulate this behavioral expression. Specifically, NOS and HA were selected for use in this study because they are regulated by different mechanisms. For example, it is possible that a decrease in recorded HA can be a result of an increase in NOS, and vice versa, and this was in fact demonstrated in previous studies using amphetamine (Gaytan et al. 1998). However, if NOS and HA are found to show similar responses (increase or decrease) as a result of a drug treatment, this can lend further evidence to the fact that real behavioral sensitization or tolerance is being observed. In the present study, the NOS and HA exhibited similar responses following MPD exposure (Fig. 1, A and B; and see Locomotor Behavior), indicating that these metrics are appropriate to use as measures of behavioral response.

The main behavioral findings of the present study show that following repetitive exposure to MPD at all doses examined, some adolescent animals exhibit behavioral sensitization, while others exhibit behavioral tolerance. Thus the electrophysiological recordings from animals expressing behavioral sensitization were evaluated separately from the recordings from animals expressing behavioral tolerance. The acute response to MPD exposure elicited in both groups a similar dose-response effect. However, the baseline activity at day 10 after six daily MPD exposures and 3 washout days was altered in a dose-response pattern: as the MPD dose was increased, more units exhibited a change in their baseline neuronal activity. Moreover, the direction of change (increase or decrease) was significantly different between these two neuronal populations. In addition, those NAc units recorded from animals that show behavior tolerance respond to acute and repetitive MPD exposure statistically differently than those NAc units recorded from animals expressing behavioral sensitization (see Statistical Comparison Between the Behavioral Groups at Each MPD Dose). Specifically, the ratio of NAc units that respond to repetitive MPD exposure by excitation or attenuation is statistically different between the two behavioral groups. This verifies our hypothesis that the same dose of repetitive MPD can elicit either behavioral sensitization or behavioral tolerance and that NAc neuronal response to MPD exposure is different in animals that express behaviorally sensitization and those that express behaviorally tolerance. These observations are of critical importance during the study of psychostimulant response, as they suggest that any behavioral effects studied must be correlated to their electrophysiological underpinnings and vice versa.

While this study does not test the molecular mechanisms behind the effects described above, we posit that the effects of MPD on dopaminergic transmission can potentially underlie these changes in behavior and firing rates. In particular, it has been shown that activation of D1 dopamine receptors results in excitation, while activation of D2 dopamine receptors results in attenuation of activity, and this change in neuronal activity in turn modulates behavior (Chao and Nestler 2004; Nestler 2004). With repetitive exposure to MPD, the above is repeated, and this elicits alterations in the molecules available, as well as a modulation of receptor density and neuronal morphology. These elicited changes include increases or decreases in the dendritic branch points and spine densities of the neurons in the NAc medium spiny neurons (MSN) (Kim et al. 2009; Russo et al. 2010). Furthermore, it has been demonstrated that repetitive MPD exposure increases the density of MSN-D1 dopamine receptors and not MSN-D2 dopamine receptors in some animals, while in others the same dose of psychostimulant can induce overexpression of D2 dopamine receptors (Kim et al. 2009). Thus increases in NAc neuropil density as a result of repetitive MPD exposure may cause an increase in baseline activity and in the response to MPD rechallenge at day 10, and also vice versa, a decrease in NAc neuropil density may result in baseline attenuation and a decrease in the response to MPD rechallenge. It is possible to postulate that the totality of this plasticity can underlie both behavioral tolerance and sensitization (Chao and Nestler 2004; Kim et al. 2009; Nestler 2004; Russo et al. 2010), although this remains to be further studied.

Following repetitive MPD exposure, some animals express behavioral sensitization, while others express behavioral tolerance at the same MPD dose, and similarly some NAc units express electrophysiological sensitization and others electrophysiological tolerance at the same MPD dose. While the present study does not use molecular assays to address the mechanisms underlying this dichotomy, published studies that target these molecular systems may potentially be used to interpret the present observations (Chao and Nestler 2004; Kim et al. 2009; Nestler 2004; Russo et al. 2010). These reports demonstrate that repetitive exposure to psychostimulants elicits in some animals overexpression of the transcription factor ΔFosB, which results in the expression of sensitization. Conversely, the same dose of psychostimulant elicits in other animals upregulation of the phosphorylation of the transcription factor cAMP receptor binding protein (CREB) in the NAc, which mediates the expression of tolerance (Chao and Nestler 2004; Kim et al. 2009; Nestler 2004, 2005). This neuroplasticity elicited by repetitive psychostimulant exposure can potentially explain how the same dose of MPD elicits both tolerance and sensitization, as observed in this study.

Finally, the differential responses to the same doses of MPD observed in this study can also be explained by reports that such differences may be due to diverse phenotypes among individual animals, especially in terms of rates of drug metabolism (Volkow and Swanson 2003). As no more than four animals came from the same shipment in the present study, each shipment may have contained different breeds. Because of this, it is possible to postulate that the animals within this study exhibit varied phenotypes of drug metabolism and this may have contributed to the differential responses to MPD exposure.

A comparison between the data on adolescent rats presented in the current study and similar data on adults rats (Chong et al. 2012) can also be made. With the use of a similar protocol to the present study, it was demonstrated that at a dose of 2.5 mg/kg MPD, about half of the NAc units recorded changed their firing rates following acute MPD administration on day 1 in both adolescent and adult rats. Moreover, of those NAc units that responded significantly to acute MPD administration, a similarly higher percentage showed an increase in neuronal activity than a decrease in both adults and adolescents. In comparing the baseline on day 10 (after 6 days of MPD administration and 3 days of washout) with the baseline on day 1, only about half of the NAc units from adolescent rats showed a change in baseline neuronal activity, while ∼85% of the NAc units from adult rats showed a change in baseline neuronal activity. Finally, after MPD rechallenge on day 10, ∼58% of NAc units from adolescent rats and 63% of NAc units from adult rats showed a change in their neuronal activity. However, in adolescent rats more of the NAc units that responded to the drug did so by increasing their neuronal firing rate following MPD rechallenge compared with NAc units recorded from adult animals, respectively, 85 vs. 62%. This comparison indicates that the neuronal responses to repetitive psychostimulant administration may differ depending on whether the animal studied is adolescent or adult, and further study is needed to statistically quantify this difference.

In summary, the present study indicates that the behavioral and electrophysiological changes observed after repetitive MPD administration are intricately linked. In particular, not only does the same dose of MPD elicit in some animals behavioral tolerance and in others behavioral sensitization following repetitive MPD exposure, but this behavioral effect is related to neuronal activity in the NAc. This neuronal activity shows statistically significant differences in firing rates in animals demonstrating behavioral tolerance compared with those demonstrating behavioral sensitization. Thus, in studying repetitive psychostimulant exposure in adolescent animals, it is advisable to simultaneously record both behavioral and neuronal activity, as the same dose of MPD can cause an animal to respond with either behavioral sensitization or tolerance, with a potentially different neuronal response underpinning each of these behaviors.

GRANTS

Support for this study was provided by National Institute of Drug Abuse Grant R01-DA-027222.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

Author contributions: A.F. and N.D. interpreted results of experiments; A.F. and N.D. prepared figures; A.F. and N.D. drafted manuscript; A.F. and N.D. edited and revised manuscript; A.F., C.R.-V., and N.D. approved final version of manuscript; C.R.-V. and N.D. performed experiments; C.R.-V. and N.D. analyzed data; N.D. conception and design of research.