Comparison of the VTA and LC response to methylphenidate: a concomitant behavioral and neuronal study of adolescent male rats

The same dose of 0.6, 2.5, and 10 mg/kg methylphenidate (MPD) elicits either behavioral sensitization or tolerance in adolescent rats. There is a direct correlation between the ventral tegmental area (VTA) and locus coeruleus (LC) neuronal response to chronic MPD exposure. Both the VTA and LC are involved in the behavioral and neurophysiological effects of chronic MPD.

Abstract

Methylphenidate (MPD), also known as Ritalin, is a psychostimulant used to treat attention deficit hyperactivity disorder. However, it is increasingly being misused by normal adolescents for recreation and academic advantage. Therefore, it is important to elucidate the behavioral and neurophysiological effects of MPD in normal subjects. MPD inhibits the reuptake of catecholamines, mainly found in the ventral tegmental area (VTA) and locus coeruleus (LC). The VTA and LC normally mediate attention, motivation, and drug reward behaviors. Selective neuronal connections between the VTA and LC have been identified implicating regular interaction between the structures. The objective of this study was to compare the neuronal responses of the VTA and LC to MPD in normal adolescent rats. Animals were implanted with permanent electrodes in the VTA and LC, and neuronal units were recorded following acute and repetitive (chronic) saline or 0.6, 2.5, or 10.0 mg/kg MPD exposure. Animals displayed either behavioral sensitization or tolerance to all three doses of MPD. Acute MPD exposure elicited excitation in the majority of all VTA and LC units. Chronic MPD exposure elicited a further increase in VTA and LC neuronal activity in animals exhibiting behavioral sensitization and an attenuation in VTA and LC neuronal activity in animals exhibiting behavioral tolerance, demonstrating neurophysiological sensitization and tolerance, respectively. The similar pattern in VTA and LC unit activity suggests that the two structures are linked in their response to MPD. These results may help determine the exact mechanism of action of MPD, resulting in optimized treatment of patients.

NEW & NOTEWORTHY The same dose of 0.6, 2.5, and 10 mg/kg methylphenidate (MPD) elicits either behavioral sensitization or tolerance in adolescent rats. There is a direct correlation between the ventral tegmental area (VTA) and locus coeruleus (LC) neuronal response to chronic MPD exposure. Both the VTA and LC are involved in the behavioral and neurophysiological effects of chronic MPD.

the psychostimulant methylphenidate (MPD), also known as Ritalin and Concerta, is one of the most commonly used medications for the treatment of attention-deficit/hyperactivity disorder (ADHD) in children (Volkow 2012; Zuvekas and Vitiello 2012). However, children are increasingly being misdiagnosed with ADHD, and ~500,000 healthy patients are being inappropriately treated with stimulants, including MPD (Bruchmüller et al. 2012; Elder 2010; Ford-Jones 2015). Moreover, as competitiveness in demanding academic settings rises, adolescents turn to improper use of MPD for its potential enhancement of executive functions, including working memory and cognitive control (Smith and Farah 2011; Volkow and Swanson 2013; Zuvekas and Vitiello 2012). Furthermore, a growing number of young adults are selling and abusing prescription MPD for profit and recreation (Busardò et al. 2016; Chen et al. 2015; Greely et al. 2008; Setlik et al. 2009). Thus it is essential to thoroughly study the effects of MPD on normal adolescent subjects.

Clinical literature and imaging review has shown that MPD elicits varied effects in individuals due to the inherent differences between subjects (Volkow and Swanson 2003). Chronic use of psychostimulants such as MPD can result in either behavioral sensitization or behavioral tolerance (Askenasy et al. 2007; May et al. 2015; Robinson and Berridge 1993). Sensitization to a drug is defined by the augmentation of the response after reexposure. Conversely, tolerance is defined by the adaptations that attenuate the effects of repeated equal doses of the same drug (Chao and Nestler 2004; Dafny and Yang 2006; Lee et al. 2009; Yang et al. 2011).

MPD acts on the central nervous system (CNS) by binding to both dopamine (DA) and norepinephrine (NE) transporters and inhibiting neurotransmitter reuptake from the synaptic cleft into the presynaptic terminal (Kuczenski and Segal 2002; Volkow et al. 2002). Several studies have reported increases in locomotor behavioral activity following the administration of MPD (Bonsall et al. 2015; Gaytan et al. 2000; Somkuwar et al. 2016). Behavioral sensitization and tolerance following chronic MPD exposure have already been reported in adolescent rats (Jones and Dafny 2014; Ponchio et al. 2015; Tang and Dafny 2012). Previous studies have also documented dynamic patterns (either sensitization or tolerance) of neuronal activity in specific areas of the CNS in adolescent rats exposed to MPD, including the ventral tegmental area (VTA) and the locus coeruleus (LC) (Brandon et al. 2003; Jones and Dafny 2014; Tang and Dafny 2012 and 2015). However, to the best of our knowledge, the VTA and LC have never been directly compared in adolescent rats after acute and chronic MPD exposure.

The VTA is the main source of DA signaling in the mesocorticolimbic system, a group of CNS sites that mediate goal-directed behaviors, including drug addiction (Juarez and Han 2016; Young et al. 2011). Dopaminergic pathways play a dominant role in the processing of reward-related stimuli and reinforcement, a fundamental concept in the psychomotor stimulant theory of addiction (Volkow and Morales 2015; Wise 2008). The VTA and DA are also associated with pleasure, reward, attention, motivation, and the conditioning of preferences (Volkow and Swanson 2013; Wise 2008).

The LC is the primary source of NE in the CNS, and LC activity correlates with forebrain NE concentration (Berridge and Waterhouse 2003; Brun et al. 1993; Chandler 2015; Florin-Lechner et al. 1996). Noradrenergic pathways consist of cortical and subcortical structures that regulate attention, memory, and sensation, producing specific behavioral activity (Berridge and Waterhouse 2003; Devilbiss and Waterhouse 2004; Weinshenker and Holmes 2016). Additionally, the LC is directly involved in the alteration of gene expression that determines executive function, behavior, and arousal (Berridge and Waterhouse 2003; Chandler 2015; González and Aston-Jones 2006; Sara 2009; Volkow and Swanson 2013).

Many selective, direct neuronal connections between the VTA and LC have been described in different animal models, including rats (Deutch et al. 1986; Geisler and Zahm 2005; Grenhoff et al. 1993; Jones and Moore 1977; Mejías-Aponte et al. 2009; Miller et al. 2011; Ornstein et al.1987; Phillipson 1979; Sara 2009). Several studies have demonstrated VTA and LC molecular and cellular plasticity following drug exposure, implying the structures are involved in various behavioral and neurophysiological processes, including the determination of drug dependence, withdrawal, and reward prediction (Akaoka and Aston-Jones 1991; Mazei-Robison and Nestler 2012). Therefore, comparison of VTA and LC neuronal responses following an acute and chronic MPD dose response protocol was investigated in freely behaving adolescent animals.

METHODS

Animals

One hundred fifty-six adolescent male Sprague-Dawley rats at postnatal days 30–32 (P30–P32) were purchased (Harlan, Indianapolis, IN). Each animal was individually housed in a clear acrylic standard cage that served as both the home and test cage for the experiment. The animals were allowed 5–7 days to acclimate in a vivarium room under normal light-dark cycles with access to food and water before electrode implantation.

Surgery

Each animal was anesthetized with 30 mg/kg pentobarbital by intraperitoneal injection. The head of each animal was shaved and lidocaine hydrochloride topical gel applied. The animals were then placed in a stereotaxic instrument. One-inch scalp incisions were made, muscle and connective tissue were excised, and the skull was exposed. Bilateral holes were drilled above the VTA (4.7 mm posterior to the bregma and 0.5 mm to midline) and LC (8.0 mm posterior to the bregma and 1.1 mm lateral to midline) using coordinates from a rat brain atlas (Sherwood and Timiras 1970). Six anchor screws were inserted to secure the implanted electrodes and skull cap. Two low-impedance (80 MΩ) nickel-chromium Teflon-coated (except at the tips) wires of 60-µm diameter were twisted to make two recording electrodes. Each wire was secured to a 1.0-cm copper connector pin (A-M Systems). The electrodes were placed individually at an initial depth of 7.0 mm into the VTA and LC in both hemispheres of the brain (i.e., each animal had a total of 8 recording electrodes). The copper connector pins were inserted into a Grass cathode follower, which was attached to a P511 Grass amplifier. During electrode placement, the neuronal activity of each electrode was monitored by a Tektronix oscilloscope. When spike activity reached a signal-to-noise ratio of at least 3:1, the electrode was permanently fixed to the skull with web glue cyanoacrylate surgical adhesive. If the signal-to-noise ratio was less than 3:1, the electrode was lowered by 5- to 10-µm increments (to a maximum depth of 7.6 mm below the skull) until a ratio of 3:1 was reached (Chong et al. 2012; Dafny and Terkel 1990; Salek et al. 2012). The copper pins from the eight electrodes were inserted into Amphenol plugs, which were fixed to the skull with dental acrylic cement. Each animal was given 4–7 days to recover from the procedure in its home cage. During this time, a wireless head stage [Triangle BioSystems (TBSI), Durham, NC] was placed and connected to the Amphenol plug of each animal for at least 2 h/day for acclimation to the recording systems. On the first experimental day, the age of each animal was about P40. All experimental procedures were approved by our Animal Welfare Committee and were in accordance with The National Institute of Health Guide for Care and Use of Laboratory Animals.

Drugs

Methylphenidate hydrochloride (MPD) was purchased (Mallinckrot, Hazelwood, MO) and dissolved in 0.9% isotonic saline solution to create doses of 0.6, 2.5, and 10.0 mg/kg. These doses were selected on the basis of previous MPD dose-response experiments that elicited behavioral sensitization or tolerance (Chong et al. 2012; Frolov et al. 2015; Gaytan et al. 2000; Yang et al. 2003). MPD doses were calculated as a free base. The MPD injections were equalized to a volume of 0.8 ml with 0.9% isotonic saline. Control injections consisted of 0.8 ml of 0.9% isotonic saline. All MPD and control injections were given intraperitoneally in the morning (i.e., during the light phase of the light-dark cycle).

Behavioral Data Acquisition

Behavioral activity was measured using an open-field computerized animal activity system (Opto-M3; Columbus Instruments, Columbus, OH). The use of open field apparatuses to observe locomotor activity has been described in detail (Aragão et al. 2011; Bellinger et al. 2006; Dunne et al. 2007; Gaytan et al. 2000; Prut and Belzung 2003; Yang et al. 2003). Each animal was recorded within its home cage (measuring 40 cm × 20 cm) by the Opto-M3 system equipped with 16 × 8 infrared beams and sensors 5 cm above the floor of the cage. The system checked each sensor at 100 Hz for interruptions of individual infrared beams. The beam interruptions were counted and stored on a personal computer every 10 min (i.e., 6 bins/h). OASIS software was used to organize the collected information into locomotor indexes including horizontal activity (HA) and number of stereotypic activity (NOS).

Neuronal Data Acquisition

Neuronal activity was recorded concomitantly with behavioral activity using the TBSI wireless recording system. After the wireless head stage was placed and connected, the animals were allowed to acclimate for 30 min before the start of recording (Fan et al. 2011). Neuronal signals were sent from either the VTA or LC through a transmitter to the remote receiver connected to a Cambridge Electronic Design (CED) analog-to-digital converter (Micro1401-3; Cambridge, UK). Recorded data were collected and stored on a personal computer, and firing rate graph spikes were sorted and analyzed using CED Spike 2.7 software offline.

Experimental Protocol

The animals were randomly assigned to four groups: saline (control) and 0.6, 2.5, or 10 mg/kg MPD. On experimental day 1 (ED1), each animal and its home cage were placed in a Faraday testing box to reduce noise during recording. After a period of acclimation, each animal received a saline injection followed immediately by a 60-min concomitant neuronal and behavioral recording (i.e., ED1 baseline activity level). An additional injection of either saline or an MPD dose was given, followed by another 60-min recording (i.e., acute MPD effect or ED1 MPD). On ED2 to ED6, daily saline or MPD injections were administered without recordings to elicit the chronic MPD effect and either behavioral sensitization or tolerance (Claussen et al. 2014; Jones and Dafny 2014; Tang and Dafny 2015; Yang et al. 2003). This period was followed by 3 washout days, ED7–ED9, in which no injections were given. On ED10, an identical protocol to that done on ED1 was performed resulting in a 60-min recording postsaline (i.e., ED10 baseline activity level) followed by another recording after saline injection or MPD rechallenge (i.e., chronic MPD effect or ED10 MPD; Table 1).

Table 1.

Experimental protocol of MPD exposure and days on which behavioral and neuronal activity were recorded

	Experiment Days
Treatment	ED1*	ED2–ED6	ED7–9	ED10*
Saline	Saline/saline	Saline	Washout	Saline/saline
MPD (0.6 mg/kg)	Saline/0.6	0.6	Washout	Saline/0.6
MPD (2.5 mg/kg)	Saline/2.5	2.5	Washout	Saline/2.5
MPD (10.0 mg/kg)	Saline/10.0	10.0	Washout	Saline/10.0

On experimental day 1 (ED1), saline was injected, followed immediately by a 60-min recording to be used as an ED1 baseline (ED1 BL). An additional injection of either saline or an MPD dose (ED1 MPD) was given, followed by another 60-min recording. On ED2–ED6, daily saline or MPD injections were administered without recordings to elicit the chronic MPD effect. This period was followed by 3 washout days (ED7–ED9). On ED10, an identical protocol to that done on ED1 was performed, resulting in a 60-min recording postsaline (ED10 BL) and another after saline injection or MPD rechallenge (ED10 MPD).

^*Data for ED1 and ED10 indicate the behavioral and neuronal activity recording days.

Data Analysis

Behavioral activity evaluation.

Locomotor activity (HA and NOS) from each 60-min recording on ED1 and ED10 were summed into 6 bins (i.e., 12 bins total on each ED). These totals were used to make three comparisons for statistical analysis: 1) locomotor activity after acute MPD exposure on ED1 (ED1 MPD) compared with baseline locomotor activity after saline injection on ED1 (ED1 BL) was used to determine the acute MPD effect (ED1 MPD/ED1 BL); 2) baseline locomotor activity after saline injection on ED10 (ED10 BL) compared with baseline activity after saline injection on ED1 (ED1 BL) was used determine whether daily MPD injections for 6 days and 3 washout days altered baseline locomotor activity (ED10 BL/ED1 BL); and 3) locomotor activity after chronic MPD exposure on ED10 (ED10 MPD) compared with activity after acute MPD exposure on ED1 (ED1 MPD) was used to determine the chronic MPD effect (ED10 MPD/ED1 MPD). Student’s t-test and the critical ratio (CR) test,

$$\text{CR}=\frac{\text{E}-\text{C}}{\sqrt{\text{E}+\text{C}}}=\text{±1.96}=P<0.05,$$

where C = control and E = activity after MPD treatment, were used to evaluate for significantly increased or decreased locomotor activity due to drug effect (Chong et al. 2012; Jones and Dafny 2014; Salek et al. 2012; Tang and Dafny 2015). For the first comparison above, E = ED1 MPD and C = ED1 BL. For the second comparison, E = ED10 BL and C = ED1 BL. For the third comparison, E = ED10 MPD and C = ED1 MPD. Each animal was then placed into subgroups exhibiting behavioral sensitization or tolerance. A one-way ANOVA (P < 0.05) was used to evaluate significant differences among the animal subgroups.

Spike sorting.

CED Spike 2.7 fixed template matching system software was used to collect the electrophysiological data at rates up to 200 kHz and to process the information using low- and high-pass filters (0.3–3 kHz). There was one window discriminator level for positive-going spikes and one window discriminator level for negative-going spikes. Spikes with peak amplitudes within the window were discriminated and used to create a template of 1,000 waveform data points. The algorithm used to capture a spike pattern allowed for the extraction of templates that provide high-dimensional reference points resulting in consistently accurate spike sorting. All temporally displaced templates were compared with the incoming spike event to find the best-fitting template that yielded the minimum residue variance. If the distance between the template and waveform exceeded the threshold (80%), the waveform was rejected. Consequently, the spike-sorting accuracy in the reconstructed data was ~95%. The sorted spikes were digitized and counted into 15-s bins. The parameters of spike sorting of electrode data from ED1 were stored and reused for spike sorting of the same electrode data from ED10. Thus the spike amplitudes and patterns sorted from ED1 were identical to those sorted from ED10. Neuronal units with spikes that did not fit the template and spikes with peak amplitudes outside the window were rejected (0.49%; 4/812).

Neuronal activity evaluation.

After spike sorting, the data were exported into a spreadsheet. The average total neuronal unit firing rates were calculated and sequential firing rate histograms produced (Fig. 1). These values were used to make three comparisons for statistical analysis: 1) neuronal activity after acute MPD exposure on ED1 (ED1 MPD) was compared with baseline neuronal activity after saline injection on ED1 (ED1 MPD/ED1 BL); 2) baseline neuronal activity after saline injection on ED10 (ED10 BL) compared with baseline activity after saline injection on ED1 (ED1 BL) was used to determine whether daily MPD injections for 6 days and 3 washout days altered baseline locomotor activity (ED10 BL/ED1 BL); and 3) neuronal activity after chronic MPD exposure on ED10 (ED10 MPD) compared with activity after acute MPD exposure on ED1 was used to determine the chronic MPD effect (ED10 MPD/ED1 MPD). The CR test (see Behavioral activity evaluation) was used to evaluate for significantly increased or decreased neuronal activity due to drug effect (Chong et al. 2012; Jones and Dafny 2014; Salek et al. 2012; Tang and Dafny 2015). For the first comparison above, E = ED1 MPD and C = ED1 BL. For the second comparison, E = ED10 BL and C = ED1 BL. For the third comparison, E = ED10 MPD and C = ED1 MPD. Pearson's χ² test (P < 0.05) was used to evaluate significant differences in neuronal activity among units within each animal subgroup in addition to significant differences in neuronal activity recorded among VTA units and LC units.

Fig. 1.

Three representative sequential firing rate histograms of VTA units after 2.5 mg/kg MPD exposure. A: histogram of unit activity at baseline on experimental day 1 (ED1 BL) and unit activity following acute MPD exposure (ED1 MPD). ED1 MPD activity exhibited an attenuation compared with ED1 BL. B: histogram of unit activity at ED1 BL and unit activity following saline injection on ED10 after daily MPD injections for 6 days and 3 washout days (ED10 BL). ED10 BL exhibited similar activity to ED1 BL. C: histogram of unit activity at ED1 MPD and unit activity following chronic MPD exposure (ED10 MPD). ED10 MPD activity exhibited an attenuation compared with ED1 MPD. The spike insets above each histogram represent 20 superimposed spikes obtained randomly during the 60-min recordings and show that the same spike patterns and amplitudes were counted during recording sessions; N = total no. of spikes per 60 min.

Histological verification of electrode placement.

After experiments were completed on ED10, each animal was administered an overdose of pentobarbital sodium and perfused intracardially with 10% formalin solution that contained 3% potassium ferrocyanide. A 2-mA DC current was passed through the tip of each electrode for 30 s to create a small lesion at the tip of the recording electrode. Each animal brain was excised from the skull and placed in 10% formaldehyde for several days. The brains were then sliced into 40- to 50-mm sections and stained with cresyl violet. The electrode positions were confirmed using the rat brain atlas (Sherwood and Timiras 1970). Only recordings obtained from electrodes histologically verified to be in the correct target sites (i.e., the VTA and LC) were analyzed and presented in this study.

RESULTS

Behavioral Responses to Acute and Chronic MPD Exposure

One hundred fifty-six adolescent rats were divided into four experimental groups: 11, 45, 49, and 51 animals treated with saline and 0.6, 2.5, and 10 mg/kg MPD, respectively. No animals were excluded from the study.

Control.

Following saline injection on ED1 and ED10, all animals exhibited similar locomotor activity. Therefore, locomotor activity after the initial saline injection on ED1 (i.e., ED1 BL) was an appropriate baseline control. Any significant difference in behavioral activity after MPD exposure compared with ED1 BL could be attributed to the effects of the drug.

Effect of 0.6 mg/kg MPD.

Forty-five animals were treated with acute and chronic 0.6 mg/kg MPD. There was a significant (P < 0.05) increase in locomotor activity after acute MPD exposure compared with baseline activity on ED1 (i.e., ED1 MPD/ED1 BL; Fig. 2A, All). There was no significant difference in activity after chronic MPD exposure compared with activity after acute MPD exposure (i.e., ED10 MPD/ED1 MPD; Fig. 2A, All).

Fig. 2.

Summary of behavioral responses (i.e., total horizontal activity) to initial saline injections (ED1 BL) and to acute MPD (ED1 MPD) and chronic MPD exposure (ED10 MPD). A–C: summary of the locomotor activity after 0.6 (A), 2.5 (B), and 10 mg/kg MPD exposure (C). Set of 3 columns at left of each panel represent the activity of all animals for each dose. The sets of 3 columns at middle and right of each panel represent the activity of animals that exhibited behavioral sensitization and tolerance to chronic MPD exposure, respectively; N = no. of animals in each group. ED1 BL, locomotor activity recorded following the initial saline injection on ED1; ED1 MPD, locomotor activity recorded following acute MPD exposure on ED1; ED10 MPD, locomotor activity recorded following chronic MPD exposure on ED10. *P < 0.05, significant difference when ED1 MPD was compared with ED1 BL. †P < 0.05, significant difference when ED10 MPD was compared with ED1 MPD.

These 45 animals were separated into two subgroups based on individual responses to chronic MPD exposure. Individual animals that exhibited a further increase or a further decrease in activity after MPD exposure on ED10 compared with the acute response on ED1 were considered to be behaviorally sensitized or tolerant, respectively, and were sorted into either the sensitized or tolerant subgroup.

Twenty-three (23/45) animals exhibiting behavioral sensitization displayed no significant difference in locomotion as a group for the comparison ED1 MPD/ED1 BL (Fig. 2A, Sensitized). However, the behaviorally sensitized animals (23/45) expressed a significant (P < 0.05) increase in activity as a group for the comparison ED10 MPD/ED1 MPD (Fig. 2A, Sensitized).

The remaining animals (22/45) animals exhibiting behavioral tolerance showed a significant (P < 0.05) increase in locomotion as a group for the comparison ED1 MPD/ED1 BL but no significant difference in locomotor activity as a group for ED10 MPD/ED1 MPD (Fig. 2A, Tolerant).

Effect of 2.5 mg/kg MPD.

Forty-nine animals were treated with acute and chronic 2.5 mg/kg MPD. There was a significant (P < 0.05) increase in locomotion for the comparison ED1 MPD/ED1 BL (Fig. 3B, All). There was no significant difference in activity for the comparison ED10 MPD/ED1 MPD (Fig. 2B, All).

Fig. 3.

Representative analogs of neuronal activity from one VTA electrode 15 min after each 2.5 mg/kg MPD injection. ED1 BL represents unit activity recorded following the initial saline injection on ED1. ED1 MPD represents unit activity recorded following acute MPD exposure on ED1. ED10 BL represents unit activity recorded following saline injection on ED10 after daily MPD injections for 6 days and 3 washout days. ED10 MPD represents unit activity recorded following chronic MPD exposure on ED10.

The 49 animals were evaluated separately by subgroup (i.e., animals exhibiting behavioral sensitization and behavioral tolerance). Thirty-one (31/49) animals exhibiting behavioral sensitization displayed a significant (P < 0.05) increase in activity as a group for the comparison ED1 MPD/ED1 BL and a further significant (P < 0.05) increase in locomotion as a group for ED10 MPD/ED1 MPD (Fig. 2B, Sensitized).

Eighteen (18/49) animals exhibiting behavioral tolerance expressed a significant (P < 0.05) increase in activity as a group for the comparison ED1 MPD/ED1 BL and a significant (P < 0.05) decrease in activity as a group for ED10 MPD/ED1 MPD (Fig. 2B, Tolerant).

Effect of 10 mg/kg MPD.

Fifty-one animals were treated with acute and chronic 10 mg/kg MPD. There was a significant (P < 0.05) increase in locomotor activity for the comparison ED1 MPD/ED1 BL (Fig. 2C, All). There was no significant difference in activity for ED10 MPD/ED1 MPD (Fig. 2C, All).

The 51 animals were evaluated separately by subgroup (i.e., animals exhibiting behavioral sensitization and behavioral tolerance). Thirty-seven (37/51) animals exhibiting behavioral sensitization displayed a significant (P < 0.05) increase in locomotion as a group for the comparison ED1 MPD/ED1 BL and a further significant (P < 0.05) increase as a group in activity for ED10 MPD/ED1 MPD (Fig. 2C, Sensitized).

Fourteen (14/51) animals exhibiting behavioral tolerance expressed a significant (P < 0.05) increase in locomotion as a group for the comparison ED1 MPD/ED1 BL (Fig. 2C, Tolerant) and a significant (P < 0.05) decrease in locomotor activity as a group for ED10 MPD/ED1 MPD (Fig. 2C, Tolerant).

The daily MPD injections for 6 days (0.6, 2.5, and 10 mg/kg) and 3 washout days had no significant effect on baseline activity when baseline locomotor activity on ED10 and baseline activity on ED1 were compared (i.e., ED10 BL/ED1 BL).

Neuronal Responses to Acute and Chronic MPD Exposure

A total of 812 (403 VTA and 409 LC) neuronal units were recorded and histologically confirmed to be within either the VTA or LC. Only units with similar spike amplitudes and patterns on ED1 and ED10 were included. Thirty-six, 115, 135, and 117 VTA units were evaluated following saline and 0.6, 2.5, and 10 mg/kg MPD exposure, respectively. Fifty-six, 109, 132, and 112 LC units were evaluated following saline and 0.6, 2.5, and 10 mg/kg MPD exposure, respectively.

Effect of saline on VTA and LC units recorded from all animals.

Thirty-six VTA and 56 LC units were recorded following initial and repetitive saline treatment. The baseline activity of only a few VTA and LC units showed a significant (P < 0.05) change following single and multiple saline injections (Tables 2–4). Because firing rates were mostly unaltered after saline injections on ED1 and ED10, recordings of units after the initial saline exposure were an appropriate control. Any significant difference in neuronal activity after MPD exposure compared with neuronal activity after saline treatment on ED1 could be attributed to MPD.

Table 2.

Summary of statistically calculated VTA and LC neuronal responses for ED1 MPD compared with ED1 BL

	ED1 MPD/ED1 BL (Acute)
MPD Dose, mg/kg	VTA				LC
n	↑	↓	≠	n	↑	↓	≠
Units recorded from all animals
Saline	36	0 (0%)	2 (5.6%)	34 (94.4%)	56	1 (1.8%)	3 (5.3%)	52 (92.9%)
0.6	115	26 (22.6%)	20 (17.4%)	69 (60%)	109	21 (19.3%)	37 (33.9%)	51 (46.8%)
2.5	135	43 (31.9%)	30 (22.2%)	62 (45.9%)	132	52 (39.4%)	29 (22%)	51 (38.6%)
10	117	86 (73.5%)	8 (6.8%)	23 (19.7%)	112	89 (79.5%)	13 (11.6%)	10 (8.9%)
Units recorded from sensitized animals
0.6	38	11 (28.9%)	2 (5.3%)	25 (65.8%)	47	9 (19.1%)	5 (10.7%)	33 (70.2%)
2.5	80	36 (45%)	17 (21.3%)	27 (33.7%)	61	28 (45.9%)	16 (26.2%)	17 (27.9%)
10	92	73 (79.4%)	4 (4.3%)	15 (16.3%)	81	63 (77.8%)	11 (13.6%)	7 (8.6%)
Units recorded from tolerant animals
0.6	77	15 (19.5%)	18 (23.4%)	44 (57.1%)	62	12 (19.4%)	32 (51.6%)	18 (29%)
2.5	55	7 (12.7%)	13 (23.7%)	35 (63.6%)	71	24 (33.8%)	13 (18.3%)	34 (47.9%)
10	25	13 (52%)	4 (16%)	8 (32%)	31	26 (83.9%)	2 (6.4%)	3 (9.7%)

Values are statistically calculated VTA and LC neuronal responses on ED1 following acute 0.6, 2.5, and 10 mg/kg MPD exposure compared with the initial neuronal responses to saline on ED1 (ED1 MPD/ED1 BL); n = no. of units in each group. Up-arrow columns include the number and percentage of VTA and LC units that exhibited significant (P < 0.05) increases in neuronal unit activity after acute MPD exposure. Down-arrow columns include the number and percentage of units that exhibited significant (P < 0.05) decreases in neuronal unit activity after acute MPD exposure. Unequal-to columns include the number and percentage of units that exhibited no significant change after acute MPD exposure. Data are shown for VTA and LC units recorded from all animals, for units recorded from animals that exhibited behavioral sensitization after chronic MPD exposure, and for units recorded from animals that exhibited behavioral tolerance after chronic MPD exposure.

Table 4.

Summary of statistically calculated VTA and LC neuronal responses for ED10 MPD compared with ED1 MPD

	ED10 MPD/ED1 MPD (Chronic)
MPD Dose, mg/kg	VTA				LC
n	↑	↓	≠	n	↑	↓	≠
Units recorded from all animals
Saline	36	0 (0%)	0 (0%)	36 (100%)	56	2 (3.6%)	3 (5.4%)	51 (91%)
0.6	115	39 (33.9%)	19 (16.5%)	57 (49.6%)	109	37 (33.9%)	20 (18.4%)	52 (47.7%)
2.5	135	59 (43.7%)	27 (20%)	49 (36.3%)	132	55 (41.7%)	35 (26.5%)	42 (31.8%)
10	117	64 (54.7%)	42 (35.9%)	11 (9.4%)	112	75 (67%)	29 (25.9%)	8 (7.1%)
Units recorded from sensitized animals
0.6	38	19 (50%)	3 (7.9%)	16 (42.1%)	47	23 (48.9%)	3 (6.4%)	21 (44.7%)
2.5	80	47 (58.8%)	15 (18.7%)	18 (22.5%)	61	35 (57.4%)	15 (24.6%)	11 (18%)
10	92	53 (57.6%)	32 (34.8%)	7 (7.6%)	81	63 (77.8%)	12 (14.8%)	6 (7.4%)
Units recorded from tolerant animals
0.6	77	20 (26%)	16 (20.8%)	41 (53.2%)	62	14 (22.6%)	17 (27.4%)	31 (50%)
2.5	55	12 (21.8%)	12 (21.8%)	31 (56.4%)	71	20 (28.2%)	20 (28.2%)	31 (43.6%)
10	25	11 (44%)	10 (40%)	4 (16%)	31	12 (38.7%)	17 (54.9%)	2 (6.4%)

Values are statistically calculated VTA and LC neuronal responses on ED10 following 0.6, 2.5, and 10 mg/kg MPD exposure compared with the neuronal responses following acute MPD exposure on ED1 (ED10 MPD/ED1 MPD); n = no. of units in each group. Up-arrow columns include the VTA and LC units that exhibited significant (P < 0.05) increases in neuronal unit activity after chronic MPD exposure. Down-arrow columns include the units that exhibited significant (P < 0.05) decreases in neuronal unit activity after chronic MPD exposure. Unequal-to columns include the units that exhibited no significant change after chronic MPD exposure. Data are shown for VTA and LC units recorded from all animals, for units recorded from animals that exhibited behavioral sensitization after chronic MPD exposure, and for units recorded from animals that exhibited behavioral tolerance after chronic MPD exposure.

Effect of 0.6 mg/kg MPD on VTA and LC units recorded from all animals.

One hundred fifteen VTA and 109 LC units were recorded from all animals that received 0.6 mg/kg MPD treatment. Forty percent (46/115) of VTA and 53% (58/109) of LC units demonstrated a significant (P < 0.05) change in firing rate for the comparison ED1 MPD/ED1 BL. The majority of VTA (57%; 26/43) and LC (64%; 37/58) units expressed a significant (P < 0.05) increase and decrease in activity, respectively (Table 2). Fifty percent (58/115) of VTA and 50% (55/108) of LC units displayed a significant (P < 0.05) change in activity for the comparison ED10 BL/ED1 BL. Most of these VTA (52%; 30/58) and LC (62%; 34/55) units exhibited a significant (P < 0.05) decrease in firing rate (Table 3). Fifty percent (58/115) of VTA and 52% (57/109) of LC units showed a significant (P < 0.05) change in firing rate for the comparison ED10 MPD/ED1 MPD. The majority of VTA (67%; 39/58) and LC (65%; 37/57) demonstrated a significant increase in activity (Table 4).

Table 3.

Summary of statistically calculated VTA and LC neuronal responses for ED10 BL compared with ED1 BL

	ED10 BL/ED1 BL
MPD Dose, mg/kg	VTA				LC
n	↑	↓	≠	n	↑	↓	≠
Units recorded from all animals
Saline	36	0 (0%)	1 (2.8%)	35 (97.2%)	56	3 (5.4%)	3 (5.4%)	50 (89.2%)
0.6	115	28 (24.3%)	30 (26%)	57 (49.6%)	109	21 (19.3%)	34 (31.2%)	54 (49.5%)
2.5	135	55 (40.8%)	30 (22.2%)	50 (37%)	132	59 (44.7%)	40 (30.3%)	33 (25%)
10	117	74 (63.3%)	34 (29%)	9 (7.7%)	112	84 (75%)	14 (12.5%)	14 (12.5%)
Units recorded from sensitized animals
0.6	38	10 (26.3%)	4 (10.5%)	24 (63.2%)	47	9 (19.1%)	13 (27.7%)	25 (53.2%)
2.5	80	46 (57.5%)	18 (22.5%)	16 (20%)	61	36 (59%)	12 (19.7%)	13 (21.3%)
10	92	61 (66.3%)	24 (26.1%)	7 (7.6%)	81	63 (77.8%)	9 (11.1%)	9 (11.1%)
Units recorded from tolerant animals
0.6	77	18 (23.4%)	26 (33.8%)	33 (42.8%)	62	12 (19.4)	21 (33.9%)	29 (46.8%)
2.5	55	9 (16.4%)	12 (21.8%)	34 (61.8%)	71	23 (32.4%)	28 (39.4%)	20 (28.2%)
10	25	13 (52%)	10 (40%)	2 (8%)	31	21 (67.8%)	5 (16.1%)	5 (16.1%)

Values are statistically calculated VTA and LC neuronal responses following saline injection on ED10 after daily MPD injections (0.6, 2.5, and 10 mg/kg) for 6 days and 3 washout days compared with the initial neuronal responses to saline on ED1 (ED10 BL MPD/ED1 BL); n = no. of units in each group. Up-arrow columns include the number and percentage of VTA and LC units that exhibited significant (P < 0.05) increases in neuronal unit activity after saline exposure on ED10 compared with ED1. Down-arrow columns include the units that exhibited significant (P < 0.05) decreases in neuronal unit activity after saline exposure on ED10 compared with ED1. Unequal-to columns include the units that exhibited no significant change after saline exposure on ED10 compared with ED1. Data are shown for VTA and LC units recorded from all animals, for units from animals that exhibited behavioral sensitization after chronic MPD exposure, and for units from animals that exhibited behavioral tolerance after chronic MPD exposure.

Effect of 2.5 mg/kg MPD on VTA and LC units recorded from all animals.

One hundred thirty-five VTA and 132 LC units were recorded from all animals that received 2.5 mg/kg MPD treatment. Fifty-four percent (73/135) of VTA and 61% (81/132) of LC units showed a significant (P < 0.05) change in firing rate for the comparison ED1 MPD/ED1 BL. Most of these VTA (59%; 43/73) and LC (64%; 52/81) units displayed a significant (P < 0.05) increase in activity (Table 2 and Fig. 3). Sixty-three percent (55/85) of VTA and 75% (99/132) of LC units expressed a significant (P < 0.05) change in activity for the comparison ED10 BL/ED1 BL. The majority of VTA (65%; 55/85) and LC (60%; 59/99) units exhibited a significant (P < 0.05) increase in firing rate (Table 3 and Fig. 3). Sixty-four percent (86/135) of VTA and 68% (90/132) of LC units demonstrated a significant (P < 0.05) change in for the comparison ED10 MPD/ED1 MPD. Most of these VTA (69%; 59/86) and LC (61%; 55/90) units showed a further significant (P < 0.05) increase in firing rate (Table 4 and Fig. 3).

Effect of 10 mg/kg MPD on VTA and LC units recorded from all animals.

One hundred seventeen VTA and 112 LC units were recorded from all animals that received 10 mg/kg MPD treatment. Eighty percent (94/117) of VTA and 91% (102/112) of LC units demonstrated a significant (P < 0.05) for the comparison ED1 MPD/ED1 BL. The majority of VTA (91%; 86/94) and LC (87%; 89/102) units expressed a significant (P < 0.05) increase in activity (Table 2). Ninety-two percent (108/117) of VTA and 88% (98/112) of LC units displayed a significant (P < 0.05) change in activity for the comparison ED10 BL/ED1 BL. Most of these VTA (69%; 74/108) and LC (86%; 84/98) units showed a significant (P < 0.05) increase in firing rate (Table 3). Ninety-one percent (106/117) of VTA and 93% (104/112) of LC units exhibited a significant (P < 0.05) change in activity for the comparison ED10 MPD/ED1 MPD. The majority of VTA (60%; 64/106) and LC (72%; 75/104) units showed a further significant (P < 0.05) increase in firing rate (Table 4).

Statistical comparison of VTA and LC units recorded from all animals.

After 0.6 mg/kg MPD application, the acute response recorded from VTA units from all animals was significantly (df: 1; χ² = 4.27; P < 0.05) different from the acute response recorded from LC units from all animals (Fig. 4A). After acute 2.5 and 10 mg/kg MPD treatment, the firing rate of VTA units from all animals did not differ significantly (df: 1 and 1; χ² = 0.46 and 0.92; P = 0.50 and 0.34) from the firing rate of LC units from all animals (Fig. 4A). There was no significant (df: 1 and 1; χ² = 1.73 and 0.51; P = 0.19 and 0.48) difference in activity at ED10 BL after 0.6 and 2.5 mg/kg MPD treatment between VTA and LC units from all animals (Fig. 4B). However, activity at ED10 BL after 10 mg/kg MPD treatment of VTA units from all animals was significantly (df: 1; χ² = 8.5; P < 0.01) different from activity at ED10 BL 10 mg/kg MPD treatment of LC units from all animals (Fig. 4B). After chronic 0.6, 2.5, and 10 mg/kg MPD exposure, the response recorded from VTA and LC units from all animals did not differ significantly (df: 1, 1, and 1; χ² = 0.07, 1.08, and 3.23; P = 0.79, 0.30, and 0.07; Fig. 4C).

Fig. 4.

Summary of the statistically calculated neuronal responses of VTA units compared with LC units recorded from all animals. Each bar represents a percentage of all units recorded within either the VTA or LC after exposure to 0.6, 2.5, or 10 mg/kg MPD. A positive percentage indicates that proportion of units that showed a significant (P < 0.05) increase in activity. A negative percentage indicates that proportion of units that showed a significant (P < 0.05) decrease in activity. A: comparison of VTA and LC unit responses after acute MPD exposure (ED1 MPD/ED1 BL). B: comparison of VTA and LC unit activity at baseline on ED10 (ED10 BL/ED1 BL). C: comparison of VTA and LC unit responses after chronic MPD exposure (ED10 MPD/ED1 MPD). *P < 0.05; **P < 0.01, significant difference when LC units were compared with VTA units after exposure to the same dose.

Effect of 0.6 mg/kg MPD on VTA and LC units recorded from animals that exhibited behavioral sensitization.

Thirty-eight VTA and 47 LC units were recorded from behaviorally sensitized animals that received 0.6 mg/kg MPD treatment. Thirty-four percent (13/38) of VTA and 30% (14/47) of LC units showed a significant (P < 0.05) change in firing rate for the comparison ED1 MPD/ED1 BL. The majority of VTA (85%; 11/13) and LC (64%; 9/14) units expressed a significant (P < 0.05) increase in activity (Table 2). Thirty-seven percent (14/38) of VTA and 47% (22/47) of LC units displayed a significant (P < 0.05) change in activity for the comparison ED10BL/ED1 BL. Most of these VTA (71%; 10/14) and LC (59%; 13/22) units exhibited a significant (P < 0.05) increase and decrease in firing rate, respectively (Table 3). Fifty-eight percent (22/38) of VTA and 55% (26/47) of LC units showed a significant (P < 0.05) change in firing rate for the comparison ED10 MPD/ED1 MPD. The majority of VTA (83%; 19/22) and LC (88%; 23/26) demonstrated a further significant (P < 0.05) increase in activity (Table 4).

Effect of 2.5 mg/kg MPD on VTA and LC units recorded from animals that exhibited behavioral sensitization.

Eighty VTA and 61 LC units were recorded from behaviorally sensitized animals that received 2.5 mg/kg MPD treatment. Sixty-six percent (53/80) of VTA and 72% (44/61) of LC units showed a significant (P < 0.05) change in firing rate for the comparison ED1 MPD/ED1 BL. Most of these VTA (68%; 36/53) and LC (64%; 28/44) units displayed a significant (P < 0.05) increase in activity (Table 2). Eighty percent (64/80) of VTA and 79% (48/61) of LC units expressed a significant (P < 0.05) change in activity for the comparison ED10 BL/ED1 BL. The majority of VTA (72%; 46/64) and LC (75%; 36/48) units exhibited a significant (P < 0.05) increase in firing rate (Table 3). Seventy-eight percent (62/80) of VTA and 82% (50/61) of LC units demonstrated a significant (P < 0.05) change for the comparison ED10 MPD/ED1 MPD. The majority of VTA (76%; 47/62) and LC (70%; 35/50) showed a further significant (P < 0.05) increase in firing rate (Table 4).

Effect of 10 mg/kg MPD on VTA and LC units recorded from animals that exhibited behavioral sensitization.

Ninety-two VTA and 81 LC units were recorded from behaviorally sensitized animals that received 10 mg/kg MPD treatment. Eighty-four percent (77/92) of VTA and 91% (74/81) of LC units exhibited a significant (P < 0.05) change in firing rate for the comparison ED1 MPD/ED1 BL. The majority of VTA (95%; 73/77) and LC (85%; 63/74) units expressed a significant (P < 0.05) increase in activity (Table 2). Ninety-two percent (85/92) of VTA and 89% (72/81) of LC units displayed a significant (P < 0.05) change in activity for the comparison ED10 BL/ED1 BL. Most of these VTA (72%; 61/85) and LC (88%; 63/72) units showed a significant (P < 0.05) increase in firing rate (Table 3). Ninety-two percent (85/92) of VTA and 93% (75/81) of LC units exhibited a significant (P < 0.05) change in activity for the comparison ED10 MPD/ED1 MPD. The majority of VTA (62%; 53/85) and LC (84%; 63/75) units showed a further significant (P < 0.05) increase in firing rate (Table 4).

Statistical comparison of VTA and LC units recorded from animals that exhibited behavioral sensitization.

Following acute 0.6 and 2.5 mg/kg MPD treatment, the activity of VTA units recorded from animals that exhibited behavioral sensitization did not differ significantly (df: 1 and 1; χ² = 1.45 and 0.20; P = 0.23 and 0.65) from the activity of LC units recorded from animals that exhibited behavioral sensitization (Fig. 5A). Yet, after acute 10 mg/kg MPD treatment, the firing rate of VTA units from behaviorally sensitized animals was significantly (df: 1; χ² = 3.94; P < 0.05) different from the firing rate of LC units from behaviorally sensitized animals (Fig. 5A). There was no significant (df: 1 and 1; χ² = 3.2 and 0.14; P = 0.07 and 0.71) difference in activity at ED10 BL after 0.6 and 2.5 mg/kg MPD treatment between VTA and LC units from behaviorally sensitized animals (Fig. 5B). However, the activity at ED10 BL after 10 mg/kg MPD of VTA units from behaviorally sensitized animals was significantly (df: 1; χ² = 5.81; P < 0.05) different from activity at ED10BL of LC units from behaviorally sensitized animals (Fig. 5B). Following chronic 0.6 and 2.5 mg/kg MPD administration, there was no significant (df: 1 and 1; χ² = 0.05 and 0.48; P = 0.83 and 0.49) difference in activity between VTA and LC units from behaviorally sensitized animals (Fig. 5C). After chronic 10 mg/kg MPD administration, the response recorded from VTA units from behaviorally sensitized animals was significantly (df: 1; χ² = 9.36; P < 0.01) different from the response recorded from LC units from behaviorally sensitized animals (Fig. 5C).

Fig. 5.

Summary of the statistically calculated neuronal responses of VTA units compared with LC units recorded from animals that exhibited behavioral sensitization after chronic MPD exposure. Each bar represents a percentage of all units recorded within either the VTA or LC after exposure to 0.6, 2.5, or 10 mg/kg MPD. A positive percentage indicates that proportion of units that showed a significant (P < 0.05) increase in activity. A negative percentage indicates that proportion of units that showed a significant (P < 0.05) decrease in activity. A: comparison of VTA and LC unit responses after acute MPD exposure (ED1 MPD/ED1 BL). B: comparison of VTA and LC unit activity at baseline on ED10 (ED10 BL/ED1 BL). C: comparison of VTA and LC unit responses after chronic MPD exposure (ED10 MPD/ED1 MPD). *P < 0.05; **P < 0.01, significant difference when LC units were compared with VTA units after exposure to the same dose.

Effect of 0.6 mg/kg MPD on VTA and LC units recorded from animals that exhibited behavioral tolerance.

Seventy-seven VTA and 62 LC units were recorded from behaviorally tolerant animals that received 0.6 mg/kg MPD treatment. Forty-three percent (33/77) of VTA and 71% (44/62) of LC units demonstrated a significant (P < 0.05) change for the comparison ED1 MPD/ED1 BL. The majority of VTA (55%; 18/33) and LC (73%; 32/44) units showed a significant (P < 0.05) decrease in firing rate (Table 2). Fifty-seven percent (44/77) of VTA and 53% (33/62) of LC units exhibited a significant (P < 0.05) change in activity for the comparison ED10 BL/ED1 BL. Most of these VTA (59%; 26/46) and LC (64%; 21/33) units displayed a significant (P < 0.05) decrease in firing rate (Table 3). Forty-seven percent (36/77) of VTA and 50% (31/62) of LC units showed a significant (P < 0.05) change in activity for the comparison ED10 MPD/ED1 MPD. The majority of VTA (56%; 20/36) and LC (55%; 17/31) expressed a significant (P < 0.05) increase and decrease in firing rate, respectively (Table 4).

Effect of 2.5 mg/kg MPD on VTA and LC units recorded from animals that exhibited behavioral tolerance.

Fifty-five VTA and 71 LC units were recorded from behaviorally tolerant animals that received 2.5 mg/kg MPD treatment. Thirty-six percent (20/55) of VTA and 52% (37/71) of LC units showed a significant (P < 0.05) change in firing rate for the comparison ED1 MPD/ED1 BL. Most of these VTA (65%; 13/20) and LC (65%; 24/37) of these units exhibited a significant (P < 0.05) decrease and increase in activity, respectively (Table 2). Thirty-eight percent (21/55) of VTA and 72% (51/71) of LC units expressed a significant (P < 0.05) change in activity for the comparison ED10 BL/ED1 BL. The majority of VTA (57%; 12/21) and LC (55%; 28/51) units displayed a significant (P < 0.05) decrease in firing rate (Table 3). Forty-four percent (24/55) of VTA and 56% (40/71) of LC units demonstrated a significant (P < 0.05) change in activity for the comparison ED10 MPD/ED1 MPD. Fifty percent (12/24) of VTA and 50% (20/40) of LC units showed a significant (P < 0.05) increase in firing rate (Table 4).

Effect of 10 mg/kg MPD on VTA and LC units recorded from animals that exhibited behavioral tolerance.

Twenty-five VTA and 31 LC units were recorded behaviorally tolerant animals that received 10 mg/kg MPD treatment. Sixty-eight percent (17/25) of VTA and 90% (28/31) of LC units demonstrated a significant (P < 0.05) change in activity for the comparison ED1 MPD/ED1 BL. The majority of VTA (76%; 13/17) and LC (93%; 26/28) units expressed a significant (P < 0.05) increase in firing rate (Table 2). Ninety-two percent (23/25) of VTA and 84% (26/31) of LC units displayed a significant (P < 0.05) change in activity for the comparison ED10 BL/ED1 BL. Most of these VTA (57%; 13/23) and LC (81%; 21/26) units exhibited a significant (P < 0.05) increase in firing rate (Table 3). Eighty-four percent (21/25) of VTA and 94% (29/31) of LC units showed a significant (P < 0.05) change in firing rate for the comparison ED10 MPD/ED1 MPD. The majority of VTA (52%; 11/21) and LC (59%; 17/29) units exhibited a significant (P < 0.05) increase and decrease in activity, respectively (Table 4).

Statistical comparison of VTA and LC units recorded from animals that exhibited behavioral tolerance.

Following 0.6 and 10 mg/kg MPD application, the acute response of VTA units recorded from behaviorally tolerant animals was not significantly (df: 1 and 1; χ² = 2.74 and 2.46; P = 0.10 and 0.12) different from the acute response of LC units recorded from behaviorally tolerant animals (Fig. 6A). However, after acute 2.5 mg/kg MPD exposure, the activity of VTA units from behaviorally tolerant animals was significantly (df: 1; χ² = 4.67; P < 0.05) different from the activity of LC units from behaviorally tolerant animals (Fig. 6A). The activity at ED10 BL after 0.6, 2.5, and 10 mg/kg MPD treatment of VTA units from behavioral tolerant animals did not differ significantly (df: 1, 1, and 1; χ² = 0.16, 0.03, and 3.38; P = 0.69, 0.86, and 0.07) from the activity at ED10 BL of LC units from behaviorally tolerant animals (Fig. 6B). Following chronic 0.6, 2.5, and 10 mg/kg MPD exposure, the response recorded from VTA units from behaviorally tolerant animals was not significantly (df: 1, 1, and 1; χ² = 0.72, 0, and 0.59; P = 0.40, 1, and 0.44) different from the response recorded from LC units from behaviorally tolerant animals (Fig. 6C).

Fig. 6.

Summary of the statistically calculated neuronal responses of VTA units compared with LC units recorded from animals that exhibited behavioral tolerance after chronic MPD exposure. Each bar represents a percentage of all units recorded within either the VTA or LC after exposure to 0.6, 2.5, or 10 mg/kg MPD. A positive percentage indicates that proportion of units that showed a significant (P < 0.05) increase in activity. A negative percentage indicates that proportion of units that showed a significant (P < 0.05) decrease in activity. A: comparison of VTA and LC unit responses after acute MPD exposure (ED1 MPD/ED1 BL). B: comparison of VTA and LC unit activity at baseline on ED10 (ED10 BL/ED1 BL). C: comparison of VTA and LC unit responses after chronic MPD exposure (ED10 MPD/ED1 MPD). *P < 0.05, significant difference when LC units were compared with VTA units after exposure to the same dose.

DISCUSSION

MPD is believed to have significant effects on dopaminergic and noradrenergic systems within the CNS by increasing the levels of DA and NE (Florin-Lechner et al. 1996; Kuczenski and Segal 1997, 2002; Volkow et al. 2002; Young et al. 2011). The objective of this study was to investigate and compare the acute and chronic effect of MPD on the neuronal activity of the VTA and LC, the main CNS sources of DA and NE, respectively, in freely behaving adolescent animals.

Behavioral activity of adolescent rats was recorded concomitantly with neuronal activity of the VTA and LC after acute and chronic MPD exposure. In previous studies the same chronic MPD dose (0.6, 2.5, or 10 mg/kg) elicited behavioral sensitization in some animals and behavioral tolerance in others. When all animals were statistically evaluated as one group, there was no observed significant chronic drug effect on locomotor activity. To detect a significant MPD effect on behavior, it was necessary to divide the animals into subgroups and evaluate the subjects on the basis of individual responses to chronic MPD exposure, i.e., behavioral sensitization and tolerance. In the present study we present behavioral and neurophysiological data from all animals as one group in addition to data from animal subgroups.

All animals displayed a significant (P < 0.05) increase in locomotor activity after acute 2.5 and 10 mg/kg MPD exposure. Individual animals exhibiting behavioral sensitization showed a significant (P < 0.05) increase in locomotion as a group after chronic 2.5 and 10 mg/kg MPD exposure. Individual animals exhibiting behavioral tolerance showed a significant (P < 0.05) decrease in locomotion as a group after chronic MPD exposure of the same doses.

Acute and chronic MPD administration elicited a significant (P < 0.05) increase in neuronal activity in the majority of VTA and LC units. In general, there was a significant (P < 0.05) difference in neuronal responses between VTA units recorded from animals exhibiting behavioral sensitization and VTA units recorded from animals exhibiting behavioral tolerance. Similarly, there was a significant (P < 0.05) difference in neuronal responses between LC units recorded from behaviorally sensitized animals and behaviorally tolerant animals.

The majority of VTA and LC units recorded from behaviorally sensitized animals expressed a significant (P < 0.05) increase in firing rate (i.e., neurophysiological sensitization) in response to acute and chronic MPD exposure of all doses (i.e., 0.6, 2.5, and 10 mg/kg). This amplified activity may be a consequence of stimulation of local dopaminergic and adrenergic receptors in the VTA and LC caused by MPD induced elevations in DA and NE levels in presynaptic clefts. Previous studies have identified dopaminergic receptors (e.g., D1 and D2) in the VTA that are involved in drug-related behavior such as neurophysiological sensitization and reward prediction (Ranaldi and Wise 2001; Reisi et al. 2014). There is also evidence of neuronal autoregulation through VTA autoreceptors, which, when suppressed, can produce the net effect of excitation (Wang 1981; White and Wang 1984). Similarly, the LC has been found to contain both adrenergic receptors and autoreceptors that are regularly activated by NE during various processes, including attention and memory formation (Cedarbaum and Aghajanian 1977; Gibbs et al. 2010; Igata et al. 2014; Ramirez and Wang 1986). Thus it is possible that the observed excitation of VTA and LC units from behaviorally sensitized animals is due to a local DA- and NE-mediated autoactivation in a positive-feedback system.

When we compared the VTA and LC units recorded from behaviorally sensitized animals exposed to 0.6 and 2.5 mg/kg MPD, there was no significant difference between the acute MPD effect (i.e., ED1 MPD/ED1 BL), baseline activity (i.e., ED10 BL/ED1 BL), or chronic MPD effect (i.e., ED10 MPD/ED1 MPD). This finding suggests that the VTA and LC in behaviorally sensitized animals exhibit similar dynamics in neuronal activity after exposure to an acute and chronic MPD dose-response protocol. However, when we compared the VTA and LC units recorded from behaviorally sensitized animals exposed to 10 mg/kg MPD, there was a significant (P < 0.05) difference between the acute MPD effect, baseline activity, and chronic MPD effect. This variation in firing rate of VTA and LC units may be a result of altered pharmacokinetics of high-dose MPD (Aoyama et al. 1990). Furthermore, the discrepancy could have been caused by potential MPD dose-dependent effects, including inflammatory stress, toxicity, and ultrastructural changes in the CNS following chronic MPD exposure (Bahcelioglu et al. 2009; Motaghinejad et al. 2016; Thanos et al. 2015). The difference may not be entirely applicable considering the large majority of units from either structure displayed a significant (P < 0.05) increase in firing rate, albeit at different proportions.

The majority of VTA and LC units recorded from behaviorally tolerant animals expressed a significant (P < 0.05) decrease in neuronal activity (i.e., neurophysiological tolerance) in response to acute and chronic MPD treatment of 0.6 and 2.5 mg/kg doses. The observed attenuation in firing rate may have been elicited by local inhibition and/or autoinhibition by the VTA and LC. The D1-like receptor family and the D2-like receptor family typically have antagonistic effects given that the former most often causes neuronal excitation and the latter commonly causes neuronal inhibition (Kebabian and Calne 1979; Trantham-Davidson et al. 2004). Increased D2-like receptor or D2 autoreceptor stimulation leads to the reduction in neuronal activity within the VTA (Ford 2014; Rahman and McBride 2000). Moreover, neuronal circuits triggered by D2 receptor activation have been reported to decrease drug-seeking behavior in rodents (de Jong et al. 2015; Xue et al. 2011).

Neurons within the LC are subject to glutamate induced postactivation inhibition, ultimately producing diminished neuronal firing (Andrade and Aghajanian 1984; Ennis and Aston-Jones 1986; Ramirez and Wang 1986). This autoregulation is likely mediated by AMPA/kainate receptors (Zamalloa et al. 2009). The LC also contains inhibitory α₂-adrenoceptors that modulate neuronal activity (Cedarbaum and Aghajanian 1977; Guiard et al. 2008). Therefore, it is possible that the recorded attenuation of VTA and LC unit activity from behaviorally tolerant animals is due to disproportionate expression of inhibitory D2-like receptor, D2 autoreceptor, AMPA receptor, and/or α₂-adrenoreceptors over excitatory receptors in a negative-feedback system.

When we compared the VTA and LC units recorded from behaviorally tolerant animals exposed to 2.5 mg/kg MPD, there was a significant (P < 0.05) difference between the acute MPD effect (i.e., ED1 MPD/ED1 BL) but no significant difference between baseline activity (i.e., ED10 BL/ED1 BL) or chronic MPD effect (i.e., ED10 MPD/ED1 MPD). Furthermore, when we compared the VTA and LC units recorded from behaviorally tolerant animals exposed to 0.6 and 10 mg/kg MPD, there was no other significant difference between the acute MPD effect, baseline activity, and chronic MPD effect. This observation suggests that the VTA and LC in behaviorally tolerant animals exhibit similar dynamics in neuronal activity after exposure to an acute and chronic MPD dose-response protocol.

The VTA contains noradrenergic receptors that, when exposed to NE, lead to the modification of VTA neuronal activity through either excitation or inhibition (Lee et al. 1998; Paladini and Williams 2004). Previous studies have found that α₁-adrenoceptor (excitatory) stimulation and α₂-adrenoceptor (inhibitory) antagonism cause substantial increases in firing rate of dopaminergic neurons in the VTA (Grenhoff and Svensson 1993). Other studies have observed that D2-like receptors can modulate LC neuronal activity through excitation or inhibition (Cedarbaum and Aghajanian 1977; Guiard et al. 2008; Ornstein et al. 1987).

DA and NE directly activate noradrenergic and dopaminergic receptors in the LC and VTA, respectively, demonstrating cross-reactivity of DA and NE receptors (Guiard et al. 2008; Lin et al. 2008; Newman-Tancredi et al. 1997). This dynamic two-way interaction between the VTA and LC in combination with autoregulation may explain why the two structures share many similarities in neuronal response after acute and chronic MPD exposure. MPD-induced elevations of local DA and NE levels may strengthen neuronal communication between the VTA and LC, resulting in a parallel pattern in firing rates.

ΔFosB is an important transcription factor in the molecular pathways leading to drug-related behaviors, including addiction (Chen et al. 1995; Ruffle 2014). Upregulation of ΔFosB expression in response to acute and chronic psychostimulant administration causes increased locomotion in rodents; i.e., ΔFosB contributes to behavioral sensitization (Chen et al. 1997; McClung and Nestler 2003). Moreover, MPD exposure results in the upregulation of ΔFosB activity, leading to increased spine density and modulation of synaptic strength in medium spiny neurons (MSN) within the nucleus accumbens (NAc) of rodents (Grueter et al. 2013; Robison et al. 2013).

cAMP-response element binding protein (CREB) is another transcriptional activator involved in the regulation of drug responses. When subjects are treated with drugs such as cocaine, there is an elevation in CREB expression, leading to a decrease in behavioral activity possibly through the inhibition of DA release; i.e., CREB elicits behavioral tolerance (Carlezon et al. 1998; Chao and Nestler 2004; Kim et al. 2009; McClung and Nestler 2003; Nestler 2012; Sakai et al. 2002). CREB also stimulates the enlargement of dendritic spines and increases NAc MSN excitability which may limit behavioral sensitization following cocaine administration (Dong et al. 2006; Murphy and Segal 1997).

Differential expression of ΔFosB and CREB throughout the CNS of rodents has been previously demonstrated. Several studies found the degree of induction of ΔFosB isoforms after treatment with cocaine in the prefrontal cortex to be distinct from both the NAc and hippocampus (Perrotti et al. 2008; Vialou et al. 2014). It also has been reported that the NAc expressed higher levels of CREB compared with the VTA in rodents following cocaine treatment (Walters et al. 2003). This evidence in addition to the findings of the current study suggests that the neuronal responses to MPD exposure in the VTA and LC may be controlled by transcription factors such as ΔFosB and CREB in an analogous manner. For example, it is possible that certain animals produce behavioral and neurophysiological sensitization through a specific pattern of ΔFosB and CREB expression within the VTA, LC, and other CNS structures. The expression in these sensitized animals is likely different from transcriptional activity in tolerant animals.

We report, through behavioral and neurophysiological analysis, a clear similarity between the responses of the VTA and LC after acute and chronic MPD exposure. The recorded neuronal activity of the VTA indicates that MPD elicits neurophysiological sensitization in behaviorally sensitized animals and neurophysiological tolerance in behaviorally tolerant animals. The data from LC units reflect the same pattern. Our results reveal a possible interaction or interdependence between the VTA and LC in response to acute and chronic MPD. Future research will focus on quantifying levels of DA and NE in the synaptic clefts within the VTA and LC and observing specific dopaminergic and noradrenergic receptor activity. Increasing the understanding of the exact mechanism of action of MPD will lead to an improvement in the medical management of patients.

GRANTS

This study was supported by National Institute on Drug Abuse Grant R01DA027222.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

N.D. conceived and designed research; C.R.-V. and N.D. performed experiments; T.J.K., C.R.-V., and N.D. analyzed data; T.J.K. and N.D. interpreted results of experiments; T.J.K. and N.D. prepared figures; T.J.K. and N.D. drafted manuscript; T.J.K. and N.D. edited and revised manuscript; T.J.K. and N.D. approved final version of manuscript.