Caudate neuronal recording in freely behaving animals following acute and chronic dose response methylphenidate exposure

Abstract

The misuse and abuse of the psychostimulant, methylphenidate (MPD) the drug of choice in the treatment of attention deficit hyperactivity disorder (ADHD) has seen a sharp uprising in recent years among both youth and adults for its cognitive enhancing effects and for recreational purposes. This uprise in illicit use has lead to many questions concerning the long term consequences of MPD exposure. The objective of this study was to record animal behavior concomitantly with the caudate nucleus (CN) neuronal activity following acute and repetitive (chronic) dose response exposure to methylphenidate (MPD). A saline control and three MPD dose (0.6, 2.5, and 10.0 mg/kg) groups were used.

Behaviorally, the same MPD dose in some animals following chronic MPD exposure elicited behavioral sensitization and other animals elicited behavioral tolerance. Based on this finding, the CN neuronal population recorded from animals expressing behavioral sensitization were also evaluated separately from CN neurons recorded from animals expressing behavioral tolerance to chronic MPD exposure, respectively.

Significant differences in CN neuronal population responses between the behaviorally sensitized and the behaviorally tolerant animals was observed for the 2.5 and 10.0 mg/kg MPD exposed groups. For 2.5 mg/kg MPD, behaviorally sensitized animals responded by decreasing their firing rates while behaviorally tolerant animals showed mainly an increase in their firing rates. The CN neuronal responses recorded from the behaviorally sensitized animals following 10.0 mg/kg MPD responded by increasing their firing rates whereas the CN neuronal recordings from the behaviorally tolerant animals showed that approximately half decreased their firing rates in response to 10.0 mg/kg MPD exposure. The comparison of percentage change in neuronal firing rates showed that the behaviorally tolerant animals trended to exhibit increases in their neuronal firing rates at ED1 following initial MPD exposure and oppositely at ED10 MPD rechallenge. While the behaviorally sensitized animals in general increased in their percentage change of firing rats were observed following acute 10.0 mg/kg MPD and the behaviorally sensitized 10.0 mg/kg MPD animals and a robust increase in neuronal firing rates at ED1 and ED10 rechallenge.

These results suggest the need to first individually analyze animal behavioral activity, and than to evaluate the neuronal responses to the drug based on the animals behavioral response to chronic MPD exposure.

1. Introduction

Methylphenidate (MPD) is an effective and readily prescribed treatment for attention deficit hyperactivity disorder (ADHD). MPD has been used successfully since the 1960s to treat ADHD symptoms. Despite its decade’s old use, the long term effects of this medication have remained largely uninvestigated. Owing to MPD’s pharmacological similarity to cocaine (Bogle and Smith, 2009; Gainetdinov and Caron, 2003; Zhang et al., 2004), it is not surprising that there has been a rise in the illicit use of MPD (Greely et al., 2008). The neurochemical mechanism whereby MPD exerts its effects is still uncertain. It has been previously established that MPD acts predominately on the dopamine (DA) and norepinephrine (NE) system by binding to their transporter and thus blocking the reuptake of DA and NE into the presynaptic terminal, thus increasing the extracellular DA and NE levels in the synaptic cleft (Kuczenski and Segal, 2001; Teo et al., 2003; Nestler, 2001; Volkow et al., 1995). This increase in extracellular DA and NE following repetitive MPD exposure can lead to the phenomenon termed behavioral sensitization or behavioral tolerance.

Behavioral sensitization was defined as a significant further acceleration of locomotor activity following repetitive chronic MPD exposure when compared to the initial acute MPD response. Behavioral tolerance has been defined as an attenuation in locomotor activity following chronic drug exposure when compared to the initial effect of the drug or when no change in locomotion is observed from experimental day 10 (ED10) compared to ED1 (Dafny and Yang, 2006; Kalivas and Stewart, 1991; Robinson, 1984). Generally, it is believed that behavioral sensitization is due to synaptic plasticity in the CNS motive circuit, including the ventral tegmental area (VTA), the nucleus accumbens (NAc), prefrontal cortex (PFC), and CN (Pierce and Kalivas, 2007; Rebec 2006). Some studies report that MPD elicits behavioral sensitization, whereas other studies report that the same dose of MPD results in behavioral tolerance (Barron et al., 2009; Eckerman et al., 1991; Gaytan et al., 1996, 2000; Yang et al., 2003; 2006; 2007). The above observations lead to our hypothesis; some individual animals will display either behavioral sensitization while other individual animals will express behavioral tolerance to the same repetitive dose of MPD (0.6, 2.5, and 10.0 mg/kg).

Neuronal activation is known to be the underlying mechanism regulating behavior, therefore we endeavored to evaluate the CN neuronal responses recorded in animals exposed to acute and/or chronic MPD (0.6, 2.5, and 10.0 mg/kg) based on their behavioral response to repetitive (chronic) MPD exposure. Our second hypothesis is that the CN neuronal population responses following acute and chronic MPD exposure recorded from animals expressing behavioral sensitization to chronic MPD exposure will be different from the CN neuronal population responses to MPD recorded from animals expressing behavioral tolerance.

2. Methods

2.1 Animals

Forty-seven male Sprague dawley rats (Harlan, Indianapolis, IN, USA) were housed individually in clear acrylic cages upon arrival. Animals were allowed access to food and water ad libitum. The home cage was used also as the test cage throughout the experiment to eliminate environment interaction. Animals were allowed 5–7 days acclimation with 12 hour light/dark schedules (lights on at 6:00 am) prior to bilateral electrode implantation into the CN and during all the experimental days. Experiments were approved by our Animal Welfare Committee and carried out in accordance with the National Institute of Health Guide for Care and Use of Laboratory Animals.

2.2 Surgeries

Prior to surgery, electrodes were made using twisted Nickel-Chromium diamel coated (fully insulated except at tips) 60 micron diameter wire secured to a 1cm copper connector pin (A-M systems, INC.). On the day of surgery the rats were anesthetized with 50 mg/kg pentobarbital intra-peritoneal (i.p.). The animal’s head was shaved and the animal was placed into the stereotaxic apparatus where an incision was made on the scalp to remove the skin, connective tissue and muscle to expose the skull. Holes were drilled in the skull bilaterally above the CN at 0.5 mm anterior to bregma and 3.0 mm lateral from midline for electrode implantation in the CN. Coordinates were obtained from Paxinos and Watson (1986) brain atlas. Six anchor screws were inserted into the skull at various vacant spots to later secure the skull cap from being ripped out. Electrodes were inserted into the brain at an initial depth of 3.5 mm from the skull and neuronal activity was monitored using a Grass emitter Hi Z Probe connected to a Grass P511 series pre-amplifier aiming to obtain 3:1 signal to noise ratio spike activity. If the neuronal activity exhibited less than a 3:1 signal to noise ratio of spike activity, the electrode was lowered in 10 μm increments until a 3:1 ratio activity was observed to maximum depth of 4.5mm (if ratio not obtained, electrode was removed to repeat steps) at which point the electrode was permanently secured to skull using web glue cyanoacrylate surgical adhesive (Claussen and Dafny; 2012; Dafny, 1980; 1982; Dafny and Terkel, 1990; Frolov et al., 2015). Similar procedures were followed for the second twisted electrode which was implanted in the CN of the opposite hemisphere. The four copper pins from the two twisted electrodes (four electrodes total per animal) were inserted into the amphenol plug which were secured to the skull using dental acrylic cement creating the skull cap. Animals were allowed three to five days recovery after electrode implantation, during these days, animals were placed in the experimental apparatus for two to three hours with their home cage and connected to the wireless (telemetric) head stage transmitter (Triangle BioSystems, Inc (TBIS); Durham, NC, USA) for acclimation. On the first (initial) recording day, rats weighed approximately 200g to 220g.

2.3 Drugs

Methylphenidate hydrochloride (MPD) was obtained from Mallinckrot (Hazelwood, MO U.S.A.). MPD was dissolved in a 0.9% isotonic saline solution and the 0.6, 2.5 and 10.0 mg/Kg MPD doses were calculated as a free base. Control injections consisted of 0.8 ml isotonic saline solution (0.9% NaCl), all injections were given i.p. and were equalized to a volume of 0.8 ml with 0.9% saline to keep injection volumes the same for all animals and were administered between 8:00 and 9:00 am.

2.4 Experimental Protocol

During the recovery period from electrode implantation, the animal was placed in the faraday box with their homecage for daily acclimation to the wireless headstage for two to three hours. On experimental day 1 (ED1), rats were placed again with their home cage in a Faraday testing box to reduce “noise” during recording. The wireless Triangle Bio Systems Int’l, (TBSI), Durham, NC, USA; (Fan et al., 2011) head stage was connected to the electrode pins of the skull cap, and animals were allowed to acclimate to the recording system for an additional 30 minutes prior to the beginning of the recording session. During this acclimation time, the neuronal parameters of the neuronal recording were set to capture the electrophysiological activity. After acclimation, the animal received a saline injection of 0.8 ml (standardized for all injections) and the neuronal and behavioral activity was recorded concomitantly for one hour to obtain the baseline activity, followed by either saline or 0.6, 2.5, or 10.0 mg/kg MPD injection (depending on the group) with recordings resumed for an additional hour. At ED2–6 animals received daily injections in their home cage of saline or 0.6 or 2.5, or 10.0 mg/kg MPD without recordings followed by three days of washout (ED7–9) where no injections were given. At ED10, the animal, in his home cage, was again placed inside the faraday testing box to record the CN neuronal and behavioral activity similar to procedure done at ED1 (Table 1).

Table 1.

Table 1 summarizes the experimental protocol. There were four groups of animals (saline, 0.6, 2.5 and 10.0 mg/kg MPD). On experimental day 1 (ED1) the behavioral and the neuronal activity was recorded for one hour following saline injection and for another hour preceding initial saline, 0.6, 2.5 or 10.0 mg/kg methylphenidate (MPD) administration. On ED2 through ED6 a single saline, 0.6, 2.5 or 10.0 mg/kg MPD injections were given without recordings. Animals went through a washout stage on ED7 through ED9 in which no injections were given, recording (behavioral and neuronal) was resumed at ED10 following a saline injection and one hour later following a rechallenge of either saline, 0.6, 2.5 or 10.0 mg/kg MPD administration.

Experimental Days:	1	2–6	7–9	10
Treatment Group:
Saline	saline / saline	saline	Washout	saline / saline
0.6 mg/kg	saline / 0.6 mg/kg	0.6 mg/kg	Washout	saline / 0.6 mg/kg
2.5 mg.kg	saline / 2.5 mg/kg	2.5 mg/kg	Washout	saline / 2.5 mg/kg
10.0 mg/kg	saline / 10.0 mg/kg	10.0 mg/kg	Washout	saline / 10.0 g/kg

The wireless TBSI head stage sent neuronal activity signals to a receiver connected to a Cambridge Electronic Design (CED) analog–to-digital converter (Micro1401-3; Cambridge, England) which digitized the data and transports the digitized data to a PC using Spike 2.7 CED software for offline evaluation. To eliminate any environmental contribution, such as novel environment, all the recordings and the MPD exposures were carried out in the animal’s home cage. This arrangement warranted that any change from baseline activity; behaviorally or electrophysiologically, is due to the effect of MPD exposure.

2.5 Behavior Apparatus

Locomotor activity was recorded using an open field computerized animal activity monitoring system (Opto-M3, Columbus Instruments, Columbus, OH). The animal’s home cage fit into the behavioral recording apparatus thus allowing for simultaneous recording of the animal’s behavior and its neuronal activity in the animal’s home cage. The Columbus open field behavioral recording system contains a 16×8 infrared beams and their sensors (on the opposite side); 40 cm in length (16 beams) by 20 cm in width (8 beams), and are set 5 cm above the floor of the cage. Movement across any of the infrared beams would result in a beam break and was subsequently compiled and downloaded to a PC in 10 minute bin increments. The software organized the data from beam interruptions into several locomotor indices. The horizontal activity (HA) records the overall locomotor activity and the number of stereotypy (NOS) activity which counts repetitive purposeless movements when there is at least one second between episodes of movement. The total activity/60 min following each injection was summed and calculated for the baseline activity at ED1 and ED10 and following MPD exposure both at ED1 and ED10.

2.6 Behavioral Analysis

The HA and NOS activity of each group was analyzed using an ANOVA with repeated measures as follows: (1) ED1 locomotor activity following initial (acute) MPD exposure was compared to ED1 baseline locomotor activity (see Table 1); (2) ED10 baseline activity following six daily MPD exposures and three days washout was compared to ED1 baseline activity (3) activity following MPD rechallenge at ED10 was compared to activity of ED1 following acute MPD exposure (Table1).

Next, to determine whether chronic MPD exposure elicited behavioral sensitization or behavioral tolerance within individual animals for each dose, each individual animal’s behavioral activity at ED10 post-MPD rechallenge was compared to their acute MPD activity at ED1 using a paired t-test with significance set at p<0.05. The main objective of the behavioral analysis of individual animals was to determine which individual animals exhibited behavioral sensitization or tolerance. All the individual animals expressing behavioral sensitization were combined to one subgroup and all the animals expressing behavioral tolerance to chronic MPD exposure were combined to a separate subgroup. These two subgroups were analyzed separately (using an ANOVA with repeated measures) to determine if these two groups exhibit significant differences in their behavioral response to the drug.

Animals expressing behavioral sensitization were considered when they exhibited a significant (p<0.05) further increase in activity at ED10 following MPD rechallenge as compared to the their initial behavioral response to MPD exposure at ED1. Behavioral tolerance was determined when the animal responded oppositely e.g; at ED1 the animals respond by increasing their activity to the drug and at ED10 following MPD rechallenge after six daily MPD injections and three days of washout the animals did not respond or responded with firing rates in the opposite direction from initial MPD exposure.

2.7 Neuronal Data Acquisition

The rat was placed with their home cage inside faraday testing box on the experimental recording days (ED1 and ED10). The wireless Triangle Bio Systems (Durham, NC, USA) head stage was connected to the electrode pins of the skull cap. The Triangle BioSystem head stage sent neuronal activity signals through a transmitter to a receiver that connects to a Cambridge Electronic Design (CED) analog –to- digital converter (Micro1401-3; Cambridge, England) which collected and stored the recorded data onto a PC. Spike 2.7 software (CED) was used off-line to sort for identical spike amplitude and waveforms recorded from the same electrode at ED1 and at ED10 (See section 2.7.1 on spike sorting for more details). This activity was used to calculate the firing rate in spikes per second and to produce a sequential frequency histogram. Approximately one to three spikes (units) were analyzed per electrode.

2.7.1 Spike Sorting

The Cambridge Electronics Design- (CED) Spike 2 version 7 software was used for offline spike sorting (sampling rates up to 200 kHz). The data was captured by the program and processed using low and high pass filters (0.3–3 kHz). Two window discriminators levels were set, one for positive-going spikes and one for negative-going spikes. Spikes with peak amplitude that were triggered by the two window discriminators were used to create the templates. 1000 waveform data points were used to define a spike. The spikes were extracted when the input signal enters the amplitude window (previously determined). Spikes with peak amplitude outside the window discriminators were rejected. The algorithm used to capture a spike allows the extraction of templates that provide high-dimensional reference points that can be used to perform accurate spike sorting, despite the influence of noise, spurious threshold crossing and waveform overlap. All temporally displaced templates are compared with the one selected to the upcoming spike event to find the best fitting template that yields the minimum residue variance. Secondly, a template matching procedure is then performed; when the distance between the template and waveform exceeds the threshold (80%) the waveforms are rejected. That means that the spike sorting accuracy in the reconstructed data is about 95%. All the parameters of spike sorting for each electrode at ED1 were stored and used at ED10. This assured that identical parameters of spike sorting at ED1 were applied to the spike activity from the same electrode at ED10.

2.7.2 Neuronal Data Evaluation

Once fixed templates were sorted, each sorted 60min neuronal activity was counted and statistically calculated and exported to a spread sheet based on neuronal firing rate before and after MPD administration. The initial 60 minutes following saline injection at ED1 contained the baseline activity of ED1. The 60 minute activity following the MPD injection at E1 of either saline or 0.6 or 2.5 or 10.0 mg/kg MPD administration was calculated and statistically compared to the initial recording post saline. The spreadsheet displayed the rat’s identity number, experimental day, dose of MPD, channel (for multiunit recording labeling) and average firing rate based on 15 second interval bins. Firing rates were evaluated for normality assumptions to determine parametric or non-parametric methods to evaluate differences before and after MPD treatments. The firing rates were determined to not hold normality assumptions, so we assessed differences in firing rates using percent change from baseline control. This data was used to analyze the differences in firing rates following MPD exposure compared to the baseline activity to determine if significant increases or decreases in firing rates in response to the drug administration were expressed. Previous experiments have shown that daily saline injections for three weeks do not alter behavioral locomotor activity, sensory evoked neuronal potentials, or electrophysiological neuronal firing rates (Yang et al., 2006a;b;c;d). Therefore, the activity after saline injection was used as control for the drug effect (Claussen and Dafny 2012; Frolov et al., 2015; Jones and Dafny, 2013; Salek et al., 2012; Tang and Dafny 2012; 2013; Yang et al., 2006b, 2007). To determine if MPD induced a significant change/response, multiple methods were employed as follow: the mean activity after treatment needs to be at least two standard errors (S.E.) difference from the control firing rate. Because the firing rates did not hold up to normality assumptions used to determine parametric or non-parametric, significance was based also on the critical ratio test ( $C.R.=\frac{E-C}{\sqrt{E}+C}=\pm 1.96=p<0.05$) (C = control, E = drug activity) (Chong et al., 2012; Claussen and Dafny 2012; Dafny, 1975; 1982; Frolov et al., 2015; Jones and Dafny, 2013; Salek et al., 2012; Tang and Dafny 2012; 2013; Yang et al., 2006a;b;c). The following comparisons were made, from the three (0.6, 2.5, 10.0 mg/kg MPD) groups 1) neuronal activity recorded from all animals 2) neuronal activity recorded from animals expressing behavioral sensitization, and 3) neuronal activity recorded from animals expressing behavioral tolerance, to chronic MPD exposure.

To determine if the CN neuronal responses were significantly different between the behavioral groups (sensitized/tolerant), a log linear model statistical test with chi square distribution using a likelihood ratio statistic value (p<0.05) was used (Waterson et al., 2012) The following comparisons were made: MPD activity following acute (ED1) exposure was significantly compared to ED1 saline baseline to deteremine the acute effect of MPD exposure. The ED10 baseline activity was compared to ED1 baseline activity to determine if six daily MPD exposures and three washout days altered the ED10 baseline neuronal activity. Chronic MPD exposure at ED10 compared to initial MPD exposure at ED1 to determine the chronic effct of MPD. The evaluations were done to the two groups (behaviorally sensitized separate from behaviorally tolerant) to determine if there were significant differences in their responses for each of the three MPD doses (0.6, 2.5, and 10.0 mg/kg MPD) and how they responded (by increase or decrease in neuronal firing rates) for each dose. Next the intensity of the firing rates changes in spikes per second was calculated and an ANOVA was used to determine if there were significant differences in the change in firing rates between the three groups (overall, sensitized, tolerant) following acute and chronic MPD administration to 0.6, 2.5, and 10.0 mg/kg MPD.

2.8 Histological verification of electrode placement

Upon completion of ED10 recording session, the animals were overdosed with sodium pentobarbital. The rat was then perfused intracardially with 10% formaldehyde solution containing 3% potassium ferrocyanide. A 2mA DC current was passed through the tip of each electrode for 40 seconds to create a small lesion to identify the electrode tip location. The brain was then extracted from the skull and placed in 10% formaldehyde for several days. The brains were sliced in 40–60μ sections and histologically stained with Cresyl violet. The position of the electrode tip was identified by the location of the lesion and the Prussian blue spot using the brain atlas of Paxinos and Watson (1986). All the units reported in this study were confirmed to be from the CN, and exhibited similar spike amplitude and pattern at ED1 and at ED10.

3. Results

3.1 Behavioral Data (Fig 1)

Figure 1.

Figure 1 summarizes the horizontal activity and the number of stereotypic activity (NOS) activity for each of the MPD doses used (0.6, 2.5 and 10.0 mg/kg). Under A, B, and C for HA and a, b, and c for NOS represent the overall, the animals expressing behavioral sensitization, and the animals expressing behavioral tolerance to 0.6 mg/kg MPD, respectively. Under D, E, and F for HA and NOS under d, e, and f represent the overall, the animals expressing behavioral sensitization, and the animals expressing behavioral tolerance to 2.5 mg/kg MPD, respectively. Similary the 10.0 mg/kg MPD behavioral activity is decribed in figure 1G, H and I for HA while NOS is represented in figure 1g, h and i. * indicates significace (p<0.05) at ED1 acute MPD exposure. ▲ indicates significance (p<0.05) at ED10 MPD rechallenge.

A total of forty-seven animals were used as follows: saline (control) group N=11; 0.6 mg/kg N = 12; 2.5 mg/kg N = 12; 10.0 mg/kg MPD N = 12, respectively. The control group following saline injection showed minor fluctuation with no significant changes in behavioral activity following acute and chronic saline administration.

The 12 animals exposed to 0.6 mg/kg MPD group showed no significant changes in HA and NOS activity as a group (Fig 1A,a) when using an ANOVA with repeated measures analysis. However when the animals were separated based on their individual behavioral responses to MPD exposure and analyzed using a paired t-test, four of the animals exhibited a significant attenuation in activity following acute MPD exposure and significant (p<0.05) increases in their activity at ED10 following MPD as compared to ED1 after MPD exposure, thus expressing behavioral sensitization [F_{(1, 12)}= 5.99, p=0.001] (Fig 1B, b), while eight of the animals showed significant (p<0.05) increased acute MPD activity and no significant [F_{(1, 14)}=1.409, p=0.25] change in their HA and NOS activity at ED10 post-MPD exposure compared to ED1 post-initial MPD exposure (Fig 1C, c).

The group exposed to 2.5 mg/kg MPD exhibited significant (p<0.05) changes following acute (initial) MPD administration compared to ED1 baseline activity, however no significant changes in HA and NOS activity at ED10 following MPD rechallenge compared to locomotor activity obtained at ED1 following MPD exposure was observed using an ANOVA with repeated measures (Fig 1D, d). When animals were analyzed individually using a paired t-test, seven animals exhibited behavioral sensitization with further significant (p<0.05) increases in their HA and NOS (Fig 1E, e; F_(1,12)=4.74, p=0.008), while the remaining five animals exhibited significant (p<0.05) decreases or no change in their HA (but not NOS) therefore expressing behavioral tolerance (Fig 1F, f; F_(1,14)=4.6, p=0.12).

The locomotor activity following acute 10.0 mg/kg MPD exhibited significant increases in their HA and NOS when all the animals were analyzed together (n=12; F_{(1, 28)}=5.6, p=0.025), and significant (p<0.05) decrease in HA (not NOS) at ED10 following MPD rechallenge when compared to ED1 after acute MPD exposure using an ANOVA with repeated measures (Fig 1G, g; F_{(1, 28)}=5.04, p=0.02). When each animal was evaluated individually using a paired t-test, five of those animals exhibited further significant (p<0.05) increase in their HA and NOS locomotor activity at ED10 following MPD rechallenge compared to ED1 MPD exposure (Fig 1H, h; F_(1,8)=5.3, p=0.001), while seven animals when individually analyzed exhibited significant (p<0.05) attenuation (tolerance) at ED10 following MPD rechallenge compared to ED1 post MPD exposure (Fig 1I, i; F_{(1, 18)}=5.3931, p=0.03).

3.2 Electrophysiological Results

Recordings from forty-seven animals with a total of one hundred eightyeight (188) electrodes that were histologically verified to be in the CN were evaluated. 451 CN units exhibited similar activity pattern and wave form at ED1 and at ED10 were analyzed. 98, 101, 117, and 135 units were evaluated following: saline, 0.6, 2.5 or 10.0 mg/kg i.p. MPD administration groups, respectively.

3.2.1 The effect of saline on CN units (Table 2a)

Table 2.

Table 2 summarizes the numbers of CN units responding significantly (p<0.05) by changing their firing rate to the three different animal groups following acute and chronic MPD exposure to 0.6, 2.5, and 10.0 mg/kg, respectively. N= number of units and in brackets their percentage; (a) all – summarizes the neuronal responses to saline, 0.6, 2.5, and 10.0 mg/kg recorded for all animals; (b) sensitized – summarizes the neuronal responses to 0.6, 2.5, and 10.0 mg/kg MPD recorded from animals expressing behavioral sensitization; (c) tolerant – summarizes the neuronal responses to 0.6, 2.5, and 10.0 mg/kg MPD recorded from animals expressing behavioral tolerance.

Day:		A. MPD ED1 vs BL ED1			B. BL ED10 vs BL ED1			C. MPD ED10 vs MPD ED1
Drug Challenge:		increased ↑	Decreased ↓	No change ≠	Increased ↑	decreased ↓	No change ≠	increased ↑	Decreased ↓	No change ≠
(a)all	saline (control)	0 (0%)	0 (0%)	0 (0%)	0(0%)	0(0%)	0 (0%)	0 (0%)	0 (0%)	0 (0%)
	0.6 mg/kg N=101	48 (48%)	32 (31%)	21 (21%)	31 (31%)	70 (69%)	0 (0%)	36 (36%)	65 (64%)	0 (0%)
	2.5 mg/kg N=117	66 (56%)	31 (26%)	20 (18%)	38 (33%)	78 (67%)	1 (1%)	88 (75%)	29 (25%)	0%
	10 mg/kg N=135	90 (67%)	31 (23%)	14 (10%)	34 (25%)	101 (75%)	0 (0%)	98 (73%)	37 (27%)	0 (0%)
(b) sensitized	0.6 mg/kg N=32	19 (59%)	13 (41%)	0%	15 (47%)	17 (53%)	0 (0%)	20 (63%)	12 (37%)	0 (0%)
	2.5 mg/kg N=51	18 (35%)	23 (45%)	10 (20%)	30 (59%)	21 (41%)	0 (0%)	33 (65%)	12 (23%)	6 (12%)
	10 mg/kg N=22	22 (100%)	0 (0%)	0 (0%)	17 (77%)	5 (23%)	0 (0%)	15 (68%)	7 (32%)	0 (0%)
(c) tolerant	0.6 mg/kg N = 69	29 (42%)	19 (28%)	21 (30%)	16 (23%)	53 (77%)	0 (0%)	21 (30%)	48 (70%)	0(0%)
	2.5 mg/kg N= 51	43 (84%)	3 (6%)	5 (10%)	8 (16%)	43 (84%)	0 (0%)	42 (82%)	9 (18%)	0 (0%)
	10 mg/kg N = 113	68 (60%)	31 (28%)	14 (12%)	19 (17%)	89 (79%)	5 (4%)	84 (74%)	29 (26%)	0 (0%)

ED = experimental day; BL = baseline;

^↑indicates significantly increased activity;

^↓significantly decreased activity;

^≠no change.

Ninety-eight CN units were recorded following acute and repetitive saline administration. All these CN units exhibited similar firing rate following the second saline administration at ED1 when compared to the neuronal activity recorded following the initial saline (baseline) administration. The baseline activity following saline administration at ED10 exhibited similar neuronal firing rates when compared to their firing rates at ED1. Moreover, the neuronal activity following the second saline injection at ED10 exhibited similar firing rates to ED10 baseline and also to those obtained at ED1 (Table 2). This lack of change provides evidence that the handling and injection volume did not alter the CN neuronal firing rates and suggests that any changes in neuronal activity observed following MPD exposure compared to saline is due to the drug effect.

3.2.2 Overall CN units without separation for behavioral response (Table 2a)

Table 2a summarizes all the CN units recorded and evaluated, 80%, 82%, and 90% of CN units responded to the initial 0.6, 2.5, and 10.0 mg/kg MPD exposure, by changing significantly (p<0.05)their firing rates. The majority of CN neurons (60%, 68%, and 74%, respectively) responded to 0.6, 2.5, and 10.0 mg/kg MPD by significantly (p<0.05) increasing their neuronal activity (Table 2Aa).

Baseline neuronal activity at ED10 prior to MPD rechallenge was compared to ED1 baseline activity prior to MPD exposure for 0.6, 2.5 and 10.0 mg/kg MPD groups. All doses exhibited predominate decreases in baseline activity; 69%, 67% and 75%, respectively (Table 2Ba)

The neuronal activity obtained at ED10 rechallenge following six daily MPD injections and three days of washout was compared to the ED1 initial MPD exposure. 100%, 91%, and 100% of the CN neuronal population responded significantly (p<0.05) to the rechallenge of 0.6, 2.5, and 10.0 mg/kg MPD exposure, respectively. From the responsive CN units, 36%, 83%, and 73% responded significantly (p<0.05) to the drug by increasing their neuronal activity following rechallenge of 0.6, 2.5, and 10.0 mg/kg MPD exposure as compared to ED1 MPD exposure, respectively (Table 2Ca).

3.3 CN units recorded from animals expressing behavioral sensitization to chronic MPD. (Table 2b)

3.3.1 Acute neuronal responses (Table 2Ab)

The acute effect of MPD on CN neuronal activity recorded from those animals expressing behavioral sensitization (to chronic MPD) showed a significant (p<0.05) change in firing rate by 100%, 77%, and 100% of the recorded neurons to initial 0.6, 2.5, and 10.0 mg/kg MPD, respectively. Of those significantly responding CN units, 59%, 35%, and 100% increased their neuronal activity, while 41%, 45%, and 0% decreased their neuronal activity to 0.6, 2.5, and 10.0 mg/kg MPD, respectively (Table 2Ab, Fig 2A).

Figure 2.

Figure 2 summarizes the number of CN units responding significantly (p<0.05) to MPD (outer column) and the response direction of the firing rate (increase/decrease (inner column)). Histograms above A summarized CN units responded to the acute effect of 0.6, 2.5, and 10.0 mg/kg MPD recorded from animals expressing behavioral sensitization and in the histogram above B are the CN units recorded from animals expressing behavorial tolerance to acute MPD exposure. Histogram C summarize CN unts responded to the chronic effect of 0.6, 2.5, and 10.0 mg/kg M recorded from animals expressing behavioral sensitization and above D are the histograms that summarize the CN units response direction recorded from animals expressing behaviorally tolerance to chronic MPD exposure.

3.3.2 Baseline neuronal responses (Table 2Bb)

The baseline neuronal firing rates at ED10 prior to MPD rechallenge were compared to the ED1 baseline activity prior to acute MPD exposure for the animals expressing behavioral sensitization. The 0.6 mg/kg MPD group showed a 53% attenuation in firing rates whereas the 2.5 and 10.0 mg/kg MPD group showed a 59% and 77% increase in their neuronal firing rates, respectively (Table 2Bb).

3.3.3 Chronic neuronal responses (Table 2Cb)

CN units recorded following MPD rechallenge at ED1 from animals expressing behavioral sensitization showed 100%, 88%, and 100% significant response by significantly (p<0.05) changing their firing rate following a rechallenge of 0.6, 2.5, and 10.0 mg/kg MPD when compared to ED1 neuronal activity following acute MPD exposure, respectively. Of those responding CN units 63%, 65%, and 68% showed an increase in their neuronal activity, while 37%, 23%, and 32% showed a decrease in their neuronal activity to 0.6, 2.5, and 10.0 mg/kg MPD compared to ED1 acute MPD activity, respectively (Table 2Cb, Fig 2C).

3.4 CN units recorded from animals expressing behavioral tolerance to chronic MPD. (Table 2c)

3.4.1 Acute neuronal responses (Table 2Ac)

The acute effect of MPD on CN neuronal activity recorded from animals exhibiting behavioral tolerance; exhibited significant (p<0.05) change in firing rates in 70%, 90%, and 88% of the CN neuronal population to acute 0.6, 2.5, and 10.0 mg/kg MPD exposure, respectively. Of those responding CN units; 42%, 84%, and 60% exhibited an increase in their neuronal activity and 28%, 6% and 28% showed a decrease in their neuronal activity to acute 0.6, 2.5 and 10.0 mg/kg MPD, respectively (Table 2Ac, Fig 2B).

3.4.2 Baseline neuronal response (Table 2Bc)

The baseline neuronal activity at ED10 prior to MPD rechallenge was compared to the ED1 baseline activity prior to acute MPD exposure for the animals expressing behavioral tolerance. All MPD dose groups (0.6, 2.5 and 10.0 mg/kg) showed a robust decrease in their baseline firing rates: 77%, 84%, and 79%, respectively (Table 2Bc).

3.4.3 Chronic neuronal responses (Table 2Cc)

The CN neuronal recording from animals exhibiting behavioral tolerance (to chronic MPD exposure) exhibited 100%, 100%, and 100% significant changes (p<0.05) in their firing rate in response to the rechallenge of 0.6, 2.5, and 10.0 mg/kg MPD exposure. Of those responding CN units 30%, 82%, and 74% exhibited increases in their neuronal activity and 70%, 18%, and 26% exhibited a decrease in their neuronal activity to rechallenge administration of 0.6, 2.5, and 10.0 mg/kg MPD compared to ED1 initial MPD exposure, respectively (Table 2Cc, Fig 2D).

3.5 Statistical comparison of CN units responding to MPD recorded from animals expressing behavioral sensitization to those recorded from animals expressing behavioral tolerance

The log linear model statistical test was used to analyze the number of CN units responding (in percentage) to 0.6, 2.5 and 10.0 mg/kg MPD recorded from animals expressing behavioral sensitization to chronic MPD compared to the CN units recorded from animals expressing behavioral tolerance. The results show that the number of responding CN units recorded from animals that exhibited behavioral sensitization to 0.6 mg/kg MPD dose were not significantly different from the CN units firing patterns recorded from animals that exhibited behavioral tolerance (df 2; X² :3.08; p = 0.2). However, the responses of CN units to MPD recorded from animals that exhibited behavioral sensitization following 2.5 and 10.0 mg/kg MPD were significantly different from the CN units which were recorded from animals exhibiting behavioral tolerance (df 2: X² :30.41; p = 0.001; df 2: X² :13.19; p = 0.001). Specifically, recordings obtained from the behaviorally sensitized group following ED1 acute 2.5 mg/kg MPD initially showed a split between neuronal populations increasing and neuronal populations decreasing their firing rates when compared to their baseline firing rates at ED1 whereas the behaviorally tolerant animals following initial acute MPD exposure showed robust increase in their neuronal responses. At ED10 MPD rechallenge of 2.5 mg/kg MPD a more robust (70%) increase in neuronal activity in the CN units recorded from the behaviorally sensitized animals whereas the CN units recorded from the behaviorally tolerant animals showed an attenuation in their neuronal responses following MPD rechallenge at ED10.

3.6 Change in CN neuronal firing rate intensity following MPD exposure recorded from animals expressing behavioral sensitization

3.6.1 Acute responses

The CN neuronal activity recorded from behaviorally sensitized animals following acute 0.6, 2.5, and 10.0 mg/kg MPD exhibited 0%, 13%, and 45% increases in their neuronal firing rate in response to the drug compared to their baseline (baseline arbitrarily set at 0%), and 12%, 24%, and 0% showed a decrease in neuronal firing rate in response to 0.6, 2.5 and 10.0 mg/kg MPD, respectively (Fig 3A).

Figure 3.

Figure 3 summarizes the percentage intensity of firing rate changes from control of CN units responses following acute and chronic 0.6, 2.5, and 10.0 mg/kg MPD, respectively (control set arbitrarily at 0%). The histograms are broken into two groups. Histogram A and B represents the change in percent firing rate changes following acute and chronic 0.6, 2.5, and 10.0 mg/kg MPD exposure from animals expressing behavioral sensitization. Histograms C and D represent the percent change in firing rate following acute and chronic 0.6, 2.5, and 10.0 mg/kg MPD, respectively recorded from animals expressing behavioral tolerance. Acute summarizes the data following the initial MPD exposure; Chronic – summarizes the ED10 firing rate following MPD exposure compared to the initial ED1 firing following MPD exposure. Negative percent (i.e., −20) indicated the percent decrease of firing rates following MPD exposure.

3.6.2 Chronic responses

The MPD rechallenge exposure at ED10 for CN units recorded from animals expressing behavioral sensitization shows a 33%, 13%, and 44% increase (ED1 neuronal activity post MPD set arbitrarily at 0%) in their neuronal firing rate, while 11%, 25% and 0% showed a decrease in their neuronal firing rate to 0.6, 2.5, and 10.0 mg/kg MPD at ED10 compared to ED1, respectively (Fig 3B). An ANOVA was used to statistically compare the overall MPD acute effect at ED1 with the effect of MPD at ED10. A significant difference was observed (Fig 3A compared to Fig 3B; (F = 1.59, p<0.011).

3.7 Change in CN neuronal firing rate intensity following MPD exposure recorded from animals expressing behavioral tolerance

3.7.1 Acute neuronal responses

The neuronal activity recorded from animals expressing behavioral tolerance shows a 16%, 8%, and 13% increased intensity in their neuronal firing rate to acute MPD exposure compared to their baseline neuronal activity, while 21%, 3%, and 39% decreased their neuronal firing rate in response to 0.6, 2.5, and 10.0 mg/kg MPD, respectively (Fig 3C).

3.7.2 Chronic neuronal responses

The MPD rechallenge exposure at ED10 for CN units recorded from animals expressing behavioral tolerance shows a 22%, 11%, and 75% increase in their neuronal firing rate intensity responses, while 22%, 15% and 0% showed a decrease in their neuronal firing rate to 0.6, 2.5, and 10.0 mg/kg MPD, respectively (Fig 3D). An ANOVA was used to compare the overall MPD acute effect at ED1 with the effect of MPD at ED10, no statistical significance was observed (Fig 3C compared to Fig 3D; F = 1.14, p<0.196).

3.8 Statistical Comparison of groups (behaviorally sensitized with behaviorally tolerant)

To determine if the percent change in the intensity of CN neuronal firing patterns following MPD administration was significantly different between the two neuronal population groups, an ANOVA with repeated measures statistical test was used. The responses from acute MPD exposure of CN recorded neuronal units recorded from animals exhibiting behavioral sensitization was compared to those exhibiting behavioral tolerance (Fig 3A vs Fig 3C). The animal group exposed to 0.6 mg/kg MPD did not show significant differences in their neuronal firing rates between the two neuronal populations, however, the CN neuronal responses recorded from the 2.5 and 10.0 mg/kg MPD exposure animal group exhibited a significant [P=0.002, F=10.493; P=0.009, F=7.236] difference in the direction of their change in firing rate between those expressing behavioral sensitization when compared to those CN units recorded from animals expressing behavioral tolerance, respectively (Fig 3A vs Fig 3C).

Similar comparisons were performed for the recordings obtained following MPD rechallenge at ED10. The 0.6 and 2.5 mg/kg MPD rechallenge at ED10 recorded from behaviorally sensitized animals did not show a significant difference in response direction compared to the CN neuronal activity recorded from animals exhibiting behavioral tolerance. However, the 10.0 mg/kg MPD grouped showed a significant [P=0.04, F=4.82] difference between the two neuronal populations.

4. Discussion

The objective of this study was to determine if there are individual differences in the animal’s behavioral response to the same dose of repeated MPD exposure, and furthermore if these differences in behavioral responses can be correlated to the CN neuronal firing response. Therefore freely behaving animals previously implanted with permanent semi micro electrodes in the CN were concomitantly recorded neuronal and behavioral activity following acute and repetitive (chronic) MPD exposure. Merchant et al. (1992) showed that while most psychostimulants affect both the limbic and basal ganglia circuitry, MPD selectively appears to act preferentially on the basal ganglia circuit, including the CN. The CN is known to contain an elevated concentration of DA receptors, a specific target of MPD. MPD binds to the dopamine transporter (DAT) and prevents the DA reuptake from the synaptic cleft to the presynaptic termind therefore indirectly increasing the DA levels in the synaptic cleft (Heijtz et al., 2004; Kuczenski and Segal 1997; Nestler 2001; Volkow et al 1995).

The main behavioral findings of this study show that the 0.6 mg/kg MPD group overall behavioral activity exhibited no significant response to acute and chronic MPD. The 2.5 mg/kg MPD group showed a significant increase in their locomotor activity following acute MPD exposure but no significant changes were observed to chronic MPD exposure. The 10.0 mg/kg MPD group showed a significant increase in their locomotor activity following acute MPD exposure, and a significant decrease in their locomotor activity to MPD rechallenge at ED10. When the animals in each dose group were evaluated individually, it was observed that in each MPD group (0.6, 2.5, or 10.0 mg/kg), chronic MPD exposure elicited in some animals behavioral sensitization and some animals elicited behavioral tolerance to chronic MPD exposure. Furthermore all animals expressing behavioral tolerance showed a more robust increase in locomotor activity following the initial MPD exposure compared to the acute responses recorded from individual animals expressing behavioral sensitization. Some differences in the HA and the NOS were observed, this may indicate that the neuronal circuit that regulates the HA or the NOS is not the same.

This finding substantiates our first hypothesis that the same chronic dose of MPD can elicit in some animals behavioral sensitization and behavioral tolerance in others. Current literature posits that each rat can have different metabolism levels and different phenotypes that affect their response to a treatment, resulting in individual differences (Arnsten and Dudley, 2005). Volkow and Swanson (2003) studied the effect of MPD on ADHD and normal patients using an MRI, and reported differences in the responses to MPD among the different subjects. In the present study 47 rats were used, no more than 4 rats came from the same shipment. Thus they may exhibit different phenotypes that can in part explain why we obtained differences in behavioral responses to chronic MPD.

The CN electrophysiological responses also show a significant different responses to MPD exposure recorded from animals expressing behavioral sensitization compared to the CN neuronal activity recorded from animals exhibiting behavioral tolerance following MPD exposure. These significant differences in neuronal responses recorded from behaviorally sensitized and tolerant animals shows the importance of utilizing behavioral outcomes when evaluating neurophysiological events. In addition, behaviorally sensitized animals showed a mixture of moderate increases and decreases in their neuronal activity responses at ED1 following acute MPD exposure whereas the CN units recorded from the animals expressing behavioral tolerance exhibited a robust increase in their neuronal response to the drug. This finding suggests a correlation between the behavioral findings and the electrophysiological observations.

The baseline activity at ED10 after six daily MPD exposures and three washout days compared to ED1 baseline activity was significantly modulated by either an increase or decrease in their firing rates. It is possible to assume that the change in baseline neuronal activity at ED10 determined how these units will respond to the MPD rechallenge at ED10. It can also be said that the changes in the baseline activity at ED10 indicated that the CN units are expressing expectation to receive the drug and/or expressing withdrawal from the drug following the previously given six daily MPD injections (Algahim et al., 2009; 2010; Lee et al., 2011; Bergheim et al., 2012).

Behavioral sensitization or tolerance was defined as a further acceleration or attenuation of locomotor activity following repetitive (chronic) drug exposure when compared to the initial effect of the drug, respectively (Dafny and Yang, 2006; Kalivas and Stewart, 1991; Robinson, 1984). This definition was also used to determine if CN units were expressing neurophysiological sensitization or tolerance.

Increases and decreases in CN neuronal firing rates following each of the MPD doses were observed. This may be explained by the following mechanisms. The CN is comprised of approximately 90% medium spiny neurons (MSNs) expressing DA receptors. The acute MPD exposure produces DA over flow in the CN that results in neuronal excitation and increased locomotion. Repeated exposure of psychostimulants results in altering gene regulation in striatal output pathways (Yano and Steiner 2005) and neuroplasticity that involves the upregulation of the transcription factors such as CREB or ΔFos B, as well as structural plasticity of CN neuropil, both implemented in the expression of sensitization and tolerance, respectively (Chao and Nestler, 2004; Nestler 2004, 2008; Kim et al., 2009; Russo et al., 2010). The striatal ascending is comprised of two main pathways; a direct excitatory pathway and an indirect inhibitory pathway (Zhang et al., 2004). The CN neurons that contribute to the direct pathway comprise with D1 DA excitatory receptors when activated by psychostimulant that results in increased firing rate to the globus pallidus internal GPi (Hummel and Unterwald, 2002). The CN neurons that contribute to the indirect pathway contains D2 DA inhibitory receptors and when activated by the drug their neuronal activity is attenuated. The CN direct pathway contains an overabundance of D1 DA receptors which control extracellular signal-kinase (ERK). Upregulation of ERK gene expression has been reported to elicit upregulation of the transcription factor ΔFos B, inducing long-term neuroadaptions in the brain (Shi and McGinty, 2011; Zhang et al., 2004). ΔFos B upregulation was reported to correlate with increased reliance to the behavioral effects and increased motivation for the drug (Nestler, 2001; 2004; 2008). Moreover, the upregulation of ΔFosB increases the MSN D1 DA receptors density, when the D1 DA like receptors activates by chronic MPD exposure, the CN units will respond to the drug by increasing their firing rates as seen in this study. The indirect CN pathway is regulated by inhibitory DA D2 receptors, activation of this receptor results in attenuation of its neuronal activity that results in an increase in enkephalin and an increase in cAMP production in the indirect pathway (Henry & White, 1995; Nikolaus et al 2011). Nikolaus et al. (2011) used in vivo experiments to measure the level of D2 DA inhibitory receptors in the striatum of both baboons and rats and reported that there was a decrease in the amount of D2 DA receptors when animals were exposed to increasing doses of MPD. It is therefore postulated that electrodes recorded from units containing DA D1 like receptors will exhibit increased neuronal activity to MPD exposure, while electrodes that are recorded from CN units containing DA D2 like receptors will express attenuation in their firing rates to MPD exposure. In these study 60μ extracellular recordings (approximately 80 kohms) electrodes previously implanted permanently into the CN were used, these recordings are not able to identify whether the recording is from a neuron expressing a majority of D1 or D2 DA receptors. Therefore, it has been postulated that when excitatory responses are observed, across all doses, that this is due to MPD binding and activation of D1 receptor activity; whereas, when attenuation responses was observed, it is probably due to MPD binding to D2 receptors. Using high impendence electrodes with a smaller electrode tip (1 to 5μ) and higher resistance, it is possible to determine if the recordings are from DA neurons or not, based on the neuronal firing rates (Prieto-Gomez et al., 2004; 2005).

The same dose of psychostimulants in some animals elicits an increase in their activity and in others a decrease in their activity can be posited from molecular studies. Several reports state that psychostimulant exposure in some animals elicits CREB upregulation, while the same drug and dose in other animals elicits ΔFosB upregulation (Alburges et al., 2011; Brandon and Steiner, 2003; Nikolaus et al., 2011; Yano and Steiner, 2005). Direct correlations between upregulation of ΔFosB or CREB as a result of repetitive psychostimulant exposure were reported to correlate with the behavioral responses such as behavioral sensitization or tolerance, respectively (Chao and Nestler, 2004; Kim et al., 2009; Nestler, 2004; 2008). It has also been shown that repetitive exposure to psychostimulants results in super sensitivity of D1-like DA receptors in the nucleus accumbens (NAc), which leads to the expression of the sensitized response (Wolf, 2002; Wolf et al., 2003). This increase results in the upregulation of ΔFosB and in D1 like DA receptors that may cause the CN units to express a sensitized state the response to MPD rechallenge. Indeed, in neuronal recordings from behaviorally sensitized animals, when animals were exposed to either 2.5 or 10.0 mg/kg of MPD, the majority of CN neuronal units showed a greater increase in firing rates and locomotor activity compared to the initial MPD dose i.e., exhibit sensitization. In addition, using both in vitro and in vivo methodologies have demonstrated that the increased induction of the transcription factors ΔFosB following psychostimulant exposure further stabilizes the protein by approximately ten-fold (Kelz et al., 1999). This intrinsic protein stability provides a molecular mechanism by which psychostimulant changes in gene expression can persist long after the administration of MPD is ceased (Robison and Nestler 2011).

Another possible mechanism to explain the present results is based on the reports that the same dose of psychostimulants elicits in some animals an increase in its structural plasticity of the neuropil (soma and dendrite spine morphology and density) in the brain reward circuitry, while in other animals the same dose of psychostimulant elicited a reduction in the neuropil components as a result of repetitive psychostimulant exposure (Diatz-Heijtz et al., 2009; Robinson and Kolb, 2004; Russo et al., 2010). It has been demonstrated that following chronic exposure of psychostimulants, an increase in the density of dendrite spines in MSNs that express D1 DA receptors occurs (Kim et al., 2009) and MPD rechallenge exposure to the increase in neuropil density may also result in neurophysiological and behavioral sensitization. In some animals however, the same dose of the drug elicits a decrease in D1 DA receptors and a decrease in neurophil density that would result in neurophysiological and behavioral tolerance to repetitive psychostimulant exposure. Further studies are needed to further dissect the mechanisms of MPD on neuronal and behavioral activities.

In conclusion, experiments in which both neuronal and behavioral responses were recorded from freely behaving animals concomitantly using an acute and chronic MPD dose response protocol, revealed that the same dose of MPD can elicit different locomotor and neuronal responses to acute and chronic MPD exposures. Also, as hypothesized, we observed a correlation between the animals behavioral and CN neuronal responses to MPD exposure. Understanding the differences in animals’ reactions to chronic exposure of MPD, in terms of both the behavioral and neuronal aspects, following chronic MPD exposure, is imperative in future research for analyzing the mechanismof MPD action. Further studies which would include using specific DA antagonists are needed to investigate their specific roles in the behavioral and neuronal sensitization/tolerance responss.

Highlights.

• Acute MPD exhibited dose response increase in behavior

• Chronic MPD elicited both behavioral sensitization and tolerance in same dose

• CN neurons in sensitized animals was significantly different from tolerant animals