Adolescent Rats Respond Differently to Methylphenidate as Compared to Rats- Concomitant VTA Neuronal and Behavioral Recordings

Abstract

Methylphenidate (MPD) is the most widely prescribed psychostimulant used in adolescents and adults to treat attention-deficit/hyperactivity disorder (ADHD). The recreational use of MPD is becoming more prevalent because of its ability to improve cognitive enhancement. The ventral tegmental area (VTA) of the brain is highly associated with reward, cognition and addiction to drugs including psychostimulants like MPD. The VTA neuronal activity was recorded alongside the horizontal behavioral activity from freely behaving non-anesthetized rats. Four adolescent and four adult groups were treated with either saline, 0.6, 2.5 and 10.0 mg/kg MPD. In both adolescent and adult animals, the animals responded to MPD in a dose-dependent manner, such that as the dose of MPD increased, more animals and more VTA unit responded to the drug. The same doses of MPD elicited in some animals’ behavioral and neuronal sensitization and in other animals’ behavioral and neuronal tolerance. In the 0.6 and 10.0 mg/kg MPD dose groups there were significant differences between the age groups for how many animals expressed behavioral sensitization and behavioral tolerance to chronic MPD exposure. Additionally, the animal’s behavioral response to MPD by excitation or attenuation of activity did not always correlate to the VTA neuronal response, and the age group with significantly higher behavioral response did not always correlate to the age group with significantly higher VTA neuronal responses for a given MPD dose. These findings differ from similar studies recorded from the prefrontal cortex (PFC), which exhibited behavioral response continuously directly correlated to PFC responses for increasing MPD doses. This demonstrates that unlike other areas of the brain, there is not a directly relationship between VTA firing and behavioral activity, suggesting that there is input or modulation of this area from elsewhere in the brain. Further investigation is needed to clearly understand the relationship between VTA firing rates and behavioral responses to different MPD doses, especially given the significant differences in response between young and adult animals and the increasing use of the drug in adolescent populations.

Introduction

Attention deficit hyperactivity disorder (ADHD) is one of the most common psychiatric disorders in childhood (Wilens et al., 2002). From 2003 to 2007, the percentage of children aged 4–17 diagnosed with ADHD increased 21.8% (Center for Disease Control and Prevention 2010). Methylphenidate (MPD) is the most widely prescribed psychostimulant used in children and adolescents to treat ADHD (Lee et al., 2012). Though MPD may effectively treat the disorder in patients who are diagnosed with ADHD, recreational use of the drug is becoming more prevalent because of its ability to improve cognitive enhancement (Arria and Wish, 2006). The mechanism of action of MPD is similar to that of cocaine. MPD impacts the nervous system by binding to the dopamine transporters (DAT) and norepinephrine transporters (NET), preventing the reuptake of dopamine (DA) and norepinephrine (NE) from the synaptic cleft to the presynaptic terminal, thus increasing the amount of DA and NE available to stimulate the post-synaptic receptors (Patrick and Markowitz, 1997; Kuczenski and Segal, 2001; CNS Stimulants, 2012; Amphetamines, 2016; Faraone, 2018).

During adolescence, the human brain undergoes vast synaptic changes in the process of synaptogenesis and pruning, which could potentially be disturbed by the use of these psychostimulants (Brenhouse and Andersen, 2011). The unmonitored uses of MPD can lead to negative behavioral changes in the brain as well as more severe outcomes including depression, death, and the potential for addiction and withdrawal symptoms (Imbert et al., 2014).

The ventral tegmental area (VTA) is a part of the brain that involves reward, motivation and pleasure. The VTA contains many dopaminergic neurons and receptors and is also a major site of DA production. These features make the VTA highly associated with behavioral functions such as the reward system, cognition, and addiction to drugs including psychostimulants such as MPD. Several studies have explored the effects of MPD on the VTA regarding behavioral activities (Ihezie et al., 2019). Chronic administration of 0.6, 2.5 and 10.0 mg/kg MPD in rats resulted in some animals exhibiting behavioral sensitization while other animals exhibited behavioral tolerance. Behavioral tolerance occurs when repetitive administration of the same drug dose leads to decreasing effects when compared to the initial effects, i.e. a higher dose of the drug is needed to elicit the same initial response. Behavioral sensitization is the opposite of behavioral tolerance, where repetitive administrations of the same drug dose leads to increasing effects when compared to the initial dose, meaning a lower dose is required to elicit the same response (Jones and Dafny, 2014). For this experiment with MPD, behavioral tolerance would mean that overtime the same dose of the drug leads to a reduction in behavior, while behavioral sensitization would demonstrate increased behavioral to the same dose over time. Behavioral tolerance and sensitization are important markers that demonstrate a drug can elicit dependence and addiction.

Other experiments have also looked at the dose response effects of MPD on adolescent VTA neuronal activity (Jones et al., 2014; Karim et al. 2017). They found that increasing MPD doses resulted in more VTA neurons responding to the MPD. In general, low doses of the drug led to a decrease in neuronal activity, while higher doses of the drug results in increased neuronal activity. Additionally, the VTA neuronal activity responses recorded from behaviorally tolerant animals was shown to be significantly different than the VTA unit responses recorded from behaviorally sensitized animals (Jones and Dafny, 2014). The present study seeks to evaluate the VTA neuronal recording based on the behavioral responses to chronic MPD, i.e. VTA neuronal recording from behaviorally sensitized animals compared to the VTA neuronal responses to MPD recorded from behaviorally tolerant animals in both adolescent and adult male SD rats.

The rising trend in ADHD prevalence, paired with the increasing recreational use of MPD, makes it imperative to study the effects of the psychostimulants on adolescents, as their brains are still developing, compared to adults. Most experiments have only studied the impact of MPD administration on behavioral activity in either adults or adolescents separately. Thus, the objective of the current study is to compare the VTA electrophysiological and behavioral responses to MPD between adult and adolescent rats. We hypothesize that each acute MPD dose (0.6, 2.5 and 10.0 mg/kg MPD) will elicit about the same effect on adolescent and adult animals, but that repetitive (chronic) MPD doses will elicit different behavioral and neuronal responses between adolescent and adult animals. Additionally, the ratio of how many VTA units respond to chronic MPD between adolescent and adult animals will be significantly different. Finally, we hypothesize that the changes in neuronal activity will elicit either behavioral sensitization or behavioral tolerance that correlate with the neuronal response to the drug.

Methods

Animals

156 adolescent and 151 adult Sprague Dawley (SD) rats were purchased at P-30 and P-50, respectively, from Harlan (Indianapolis, IN). The rats were placed individually in their home cages, which also served as their test cages. The cages were in a vivarium room operating on a 12-hour light/dark schedule (lights on 06:00), and animals were given food and water as needed. After 3 to 4 days of acclimation, recording electrodes were implanted bilaterally within the VTAs. The resident Animal Welfare Committee approved the experiment, which was carried out according to the National Institute of Health Guide for Care and Use of Laboratory Animals.

Surgeries

Before surgery, two 60 μm Nickel-Chromium Teflon coated wires, except at the tip, were twisted to make the recording electrodes (Medwire Corp, NY). Two twisted electrodes (i.e. 4 recording electrodes in each animal) were made for the VTA of each hemisphere with all four individual wires being secured to a 1.0 cm copper connector pin (A-M systems, INC.) On the day of surgery, pentobarbital was administered to the animals to induce anesthesia. Their heads were shaved and covered with lidocaine hydrochloride topical gel and they were placed in a stereotaxic apparatus. For adolescents, the dose of pentobarbital was 30 mg/kg while for adults the dose was increased to 50 mg/kg. An incision was made on the scalp to expose the skull by removing the skin, muscle and connective tissue. In adolescent rats, bilateral holes were drilled into the skull above the VTA 4.7 mm posterior to bregma and 0.6 mm lateral to the midline using the Sherwood and Timiras (1970) adolescent brain atlas coordinates. In the adult subjects, bilateral holes were drilled into the skull above the VTA 6.0 mm posterior to the bregma and 0.5 mm lateral from the sagittal suture using Paxinos and Watson (1986) brain atlas coordinates. Additional holes were drilled in the frontal sinuses for the reference electrode and a 1.0 mm stainless steel wire was implanted as the reference/ ground electrode and secured in this hole. At vacant spots on the skull, six anchor screws were inserted. The recording electrodes were inserted into the holes at an initial depth of 8.0 mm for adults and 7.0 mm for adolescents, while tracking neuronal activity with a Grass Emitter Hi Z Probe connected to a Grass P511 series amplifier. When a 3:1 signal to noise ratio spike was observed, the electrode was fixed into the skull with a cyanoacrylate surgical adhesive (Loctite, Henkel Co., CT). If the spike activity ratio was less than 3:1, the electrode was lowered 5 to 10 μm up to a max depth of 8.1 mm until a ratio of 3:1 was observed before fixing it to the skull. This process of inserting and adjusting the electrode was done on the other hemisphere as well. The copper pins from each of the 4 wires and the ground electrode were inserted into Amphenol plugs making up the skull cap and were fixed to the skull with dental acrylic cement (Kerr Laboratory Products, MI). Similar procedures were done with adolescents. Five to seven days were allowed for recovery following the procedure. During this recovery time, the rats were transported daily with their home cage into the experimental room and connected to the wireless headstage (Triangle BioSystems Inc, TBSI, Durham, NC, USA) to monitor VTA neuronal activity for two hours a day to acclimate the animals to the recording system. The subjects at the first recording day were P-40 and P-60 for adolescent and adult, respectively.

Drugs

Methylphenidate hydrochloride (MPD) obtained from Mallinckrot (Hazelwood, MO, U.S.A) was dissolved in 0.9% isotonic saline solution to obtain 0.6, 2.5, 10.0 mg/kg doses of MPD. MPD doses were calculated as a free base. The volumes of all MPD injections were set to 0.8 ml with 0.9% isotonic saline solution to keep the injection volumes constant between all animals. All injections were given intraperitoneally. In previous experiments, MPD dosages ranging from 0.1 mg/kg to 40.0 mg/kg were tested for behavioral and neuronal responses (Gaytan et al., 1996). It was shown that doses under 0.6 mg/kg did not elicit any locomotor response while doses greater than 0.6 mg/kg elicited a significant behavioral response. The 0.6 mg/kg MPD only in some animals elicits significant response, while the 2.5 mg/kg and 10.0 mg/kg doses induced acute and chronic effects, such as behavioral sensitization and/ or tolerance, respectively, which is why these doses were used for this study (Algahim et al., 2009; Gaytan et al., 1996, 2000; Kharas et al., 2017; Podet et al., 2010; Venkataraman et al., 2020; Yang et al., 2003, 2006a, 2006b, 2006c, 2006d, 2007).

Experimental Protocol and Data Acquisition

On experimental day one, (ED1), the home cages, with the rats, were placed in a Faraday testing box which helped to reduce outside noise while recording. A wireless head stage was attached to the electrode pins of the skull cap, and the animals were allowed to acclimate for an additional 20–30 minutes before the recording session. During this acclimation period, the recording software monitored the electrical activity, parameters for spike sorting were set (as described in the neuronal spike sorting and counting section), and the saline and MPD injection solutions were prepared. Once the animals had acclimated, they received a saline injection of 0.8 ml, followed immediately by neuronal and behavioral activity recordings simultaneously for one hour to obtain the baseline (ED1 BL) activity. After an hour of recording, the animals then received an additional injection of saline (control group) or MPD (either 0.6 2.5 or 10.0 mg/kg; ED1 MPD), after which recording resumed immediately for another hour (see Table 1). The neuronal activity was collected and stored on a PC using the Cambridge Electronic Design (CED) Spike2.7 software. On ED2 through ED6, the rats received their respective saline or MPD injections daily (Table 1) in their home cages without recordings to initiate the chronic effect of MPD. ED7 through ED9 were washout days in which no injections were given. On ED10, a saline injection was given followed immediately by neuronal and behavioral activity recordings for one hour. These recordings defined the ED10 baseline (ED10 BL), after which a rechallenge administration of the rats’ respective saline or MPD injections (ED10 MPD) was given, and recordings were immediately resumed for an additional hour, similar to ED1 MPD (Table 1).

Table 1.

displays the 4 animal groups and the dose protocol that was followed for each group of the adolescent animals. 4 similar groups were used for the adult animals.

	Experimental Days (ED)
Treatment Groups	ED 1	ED 2–6	ED 7–9	ED 10
1. Saline	Saline/Saline	Saline	Washout	Saline/Saline
2. 0.6 mg/kg MPD	Saline/0.6 mg/kg MPD	0.6 mg/kg MPD	Washout	Saline/0.6 mg/kg MPD
3. 2.5 mg/kg MPD	Saline/2.5 mg/kg MPD	2.5 mg/kg MPD	Washout	Saline/2.5 mg/kg MPD
4. 10.0 mg/kg MPD	Saline/10.0 mg/kg MPD	10.0 mg/kg MPD	Washout	Saline/10.0 mg/kg MPD

Four groups of animals were used: saline, 0.6, 2.5 and 10.0 mg/kg MPD. On experimental day 1 (ED1), animals are given an initial dose of saline and recordings were taken for one hour followed by one of the four designated injections of saline, 0.6, 2.5 or 10.0 mg/kg of MPD and recordings were resumed for an additional hour post injection. On ED 2–6, the animals are given an injection each morning of the designated dose. ED 7–9 are washout days where the animal gets no injection of any kind. On ED10, the animals are given another dose of saline to obtain BL on ED10 for one hour followed by the designated MPD dose for one hour and recordings were taken, identical to that given on ED1.

A wireless neuronal recording system (Triangle BioSystems Inc, TBSI, Durham, NC, USA) and an open field computerized animal activity monitoring system (opto-M3, Columbus Instruments, Columbus, OH), were used to record VTA neuronal activity while locomotive behavioral activity of the animals was recorded simultaneously.

Behavioral Apparatus

Locomotive behavioral activity was recorded concomitantly with the neuronal activity using an open field computerized activity system (Opto-M3, Columbus Instruments, Columbus, Oh). The home cage (40cm × 20cm) fit into the Opto-M3 recording apparatus, and the animal’s neuronal and behavioral activities were able to be recorded in the home cages. The Opto- M3 field system contained 16 and 8 infrared beams, and in their opposite side sensors were set at 5 cm above the cage floor, respectively. The sensors detected interruptions of the infrared beams created by the animal’s movement, which was collected and counted by the analyzer and compiled by OASIS software to total number of horizontal activity (HA). The data were downloaded to a PC every ten minutes (6 bins/hr.) following saline and MPD exposure each for 60 minutes on ED1 and ED10. Using the 6 bin counts after each hour, temporal graphs and histograms were created of HA for saline (baseline activity), as well as activity after MPD administration for ED1 and ED10, respectively.

Electrophysiological Apparatus

The Triangle BioSystems International System (TBSI) consisted of a wireless head stage (weighing 4.5g) that was connected to the electrodes sending neuronal activity signals (sampling rate up to 200 kHz) wirelessly to a remote receiver. The receiver was connected to an analog-to-digital converter (micro 1401–3 Cambridge Electronic Design- CED) that digitized the recording data and send it to a PC using the Spike 2 version 7 software to be stored for offline statistical analysis.

Neuronal Spike Sorting and Counting

The Spike 2 version 7 software (CED) was used for spike sorting. The software recorded and processed the data (sampling rates up to 200 kHz) using low and high pass filters (0.3–3.0 kHz). There were two window discriminator levels, one for positive-going spikes and one for negative-going spikes. Using 1000 waveform data points, the selected spikes with peak amplitudes within the window were used to create templates. The algorithm parameters that were used to capture a spike pattern allowed the extraction of templates. These templates provided high dimensional reference points which were used to perform accurate spike sorting, despite movement artifacts, noise, false threshold crossing, or waveform overlap. All temporally displaced templates were compared to the incoming spike event to determine the template that best fit the selected template amplitude. This yielded the minimum residue variance with spikes being counted and summed to total number of spikes/ 15 seconds sequentially. Waveforms were rejected when the distance between the template and waveform exceeded some threshold (80%) for the 60-minute recording, i.e. 240 numbers/ 60 minutes. Because of this, the spike sorting accuracy in the reconstructed data was about 95%. To count the same spike amplitude and patterns at ED1 and ED10 following saline and MPD administration from the same electrode, all of the parameters of spike sorting for each electrode at ED1 were stored and reused for the activity sorting at ED10. Spikes with peak amplitudes outside these limits, as well as spikes that did not fit the template, were rejected.

Data Analysis

Behavioral and Neuronal Analysis

Since the VTA firing rates were determined to not hold normality assumptions, the differences in both behavioral activity and differences in firing rates for the VTA units were each evaluated using the statistical critical ratio (CR) test, $\text{CR}=\frac{E-C}{\sqrt{E+C}}\pm 1.96=\text{P}<0.05$ (C = control, E = activity after treatment). CR value greater than 1.96 determined that the drug significantly increased locomotor activity or the VTA firing rates as compared to saline (ED1 MPD/ ED1 BL) or (ED10 MPD/ ED1 MPD), i.e. this further increase on ED10 is considered an expression of behavioral sensitization after chronic MPD exposure. On the other hand, a CR value less than −1.96 determined that the drug, on ED10 compared to ED1, significantly attenuated activity, indicating behavioral or electrophysiological tolerance. Any values in between in the range of −1.96 to 1.96 defined the animal as unchanged.

After behavioral and neuronal activity was recorded, sorted, and counted, the post-saline and post-MPD exposure data were exported to a spreadsheet to produce sequential firing rate graphs of spikes/ sec and count the total neuronal activity/ 60 min. Three comparisons were made using the CR equation. First, for each individual animal, behavioral and electrophysiological recordings taken for 60 minutes following MPD exposure on ED1 (ED1 MPD) were compared to the recordings obtained for 60 minutes following baseline (BL) saline injections on ED1 (ED1 BL) to evaluate the acute effect of the drug on both HA and VTA firing rate using the $\text{CR}\text{test}=\frac{(ED1MPD)-(ED1BL)}{\sqrt{(ED1MPD)+(ED1BL)}}\pm 1.96=\text{P}<0.05$. Second, to determine whether the daily saline or MPD injections for 6 days following by 3 washout days changed baseline activity on the 10^th day, baseline behavioral activity and VTA firing rates following saline injection on ED10 (ED10 BL) were compared to baseline behavioral activity and VTA firing rates following saline injection on ED1 (ED1 BL), $\text{CR}=\frac{(ED10BL)-(ED1BL)}{\sqrt{(ED10BL)+(ED1BL)}}\pm 1.96=\text{P}<0.05$. A final comparison was made between activity and VTA firing rates after MPD administration on ED10 and activity and VTA firing rates after MPD administration on ED1 (ED10 MPD / ED1 MPD) to determine whether chronic administration of MPD modulated the behavioral and electrophysiological response to the drug $\text{CR}=\frac{(ED10MPD)-(ED1MPD)}{\sqrt{(ED10MPD)+(ED1MPD)}}\pm 1.96=\text{P}<0.05$.

Based on this equation for locomotor behavior, the rats were individually classified into three subgroups: either expressing behavioral sensitization or tolerance, as well as unchanged. These subgroups were further statistically analyzed using an analysis of variance (ANOVA: treatments days and drug doses) with statistical differences determined by the post hoc Fischer’s LSD method. A final odds ratio analysis was completed by comparing the ratio of sensitized and tolerant rats in the adult age group to the ratio of sensitized and tolerant rats in the adolescent age group.

All the VTA units from adolescent rats were sorted to one group (Table 2A) and then divided into two subgroups: those VTA units that were recorded from animals expressing behavioral sensitization (Table 2B) and those units that were recorded from animals expressing behavioral tolerance (Table 2C). In addition, to determine the likelihood that VTA neuronal activity recorded from behaviorally sensitized animals was different from VTA neuronal activity recorded from behaviorally tolerant animals, a natural log ratio statistical test was utilized. The test was done for the data comparing ED1 MPD to ED1 BL, ED10 BL to ED1 BL, and ED10 MPD to ED1 MPD. 0.5 was added to all groups for computation of the odds ratio, to make up for the smaller values seen on some days for varying doses. A value of 1 or greater in the odds ratio test indicated a significantly higher likelihood that the neuronal recordings in response to MPD exposure in behaviorally sensitized subjects were indeed different from those recorded in behaviorally tolerant subjects. On the other hand, a value less than 1 indicated a lower likelihood that the two VTA neuronal populations were different.

Table 2.

summarizes the number of VTA units recorded from behaviorally sensitized adolescent and adult animals for each MPD dose that responded significantly (P<0.05) to A. Acute (ED1MPD/ ED1BL), B. Baseline changes (ED10BL/ ED1BL) and C. Chronic (ED10MPD/ ED1MPD) MPD, and the direction (increase or decrease) of how they responded to each MPD dose, respectively.

Units recorded from behaviorally sensitized animals
			A. Acute			B. Baseline			C. Chronic
	Dose	N	↑	↓	≠	↑	↓	≠	↑	↓	≠
Adolescent	0.6	38	10	3	25	8	4	26	13	3	22
Adult	0.6	29	2	23	4	8	15	6	13	15	1
Adolescent	2.5	80	36	17	27	46	18	16	47	15	18
Adult	2.5	40	18	14	8	10	28	2	16	22	2
Adolescent	10	92	73	4	15	61	24	7	53	32	7
Adult	10	42	21	18	3	15	27	0	16	26	0

Finally, a log linear model statistical test with chi square distribution and a likelihood ratio statistic value of p < 0.05 was utilized to compare VTA neuronal response to MPD between animals expressing behavioral sensitization and animals expressing behavioral tolerance.

Histological Verification of Electrode Placement

After the final recording on ED10, the animals were overdosed using sodium pentobartbital. Each animal was then perfused intracardially with 10% formaldehyde solution containing 3% potassium ferricyanide. A 2 mA DC current was sent through the electrode tip for 40 seconds to create a small lesion. The lesion at the tip of the electrode indicated where the electrode was placed. The brain was then taken out of the skull and left in 10% formaldehyde for several days. The brains were cut into 40–60 μm slices and stained using Cresyl Violet. The electrode tip position was identified as a lesion with a Prussian blue spot that was created from the ferrous reaction of the electrode current with the staining. The location of the lesion was confirmed using the Paxinos and Watson (1986) and Sherwood and Timiras (1970) brain atlases for the adults and adolescents, respectively. Only those recordings obtained from the VTA that had both similar spike amplitudes on ED1 and ED10 and verified electrodes placements were evaluated.

Results

Eleven, 45, 49, and 51 adolescent animals and 20, 52, 37, and 42 adult SD rats were used. All were treated with single and repetitive saline, 0.6, 2.5 or 10.0 mg/kg MPD (See Table 1).

Behavior- Control

Eleven and 20 animals served as the control group for the adolescent and adult rats respectively, being injected with only saline during experimental day 1 (ED1) to ED6 and ED10. The 2^nd saline injection on ED1 and the saline injection at ED10 were compared to the activity following the first saline injection. They exhibit similar behavioral activity in both adult and adolescent as compared to ED1, indicating that neither handling of the animals, injection procedure, or the volume of the injection alter the rats’ locomotive behavior for adolescent and adult rat groups. This verifies that the saline injection on ED1 (ED1 BL) can be used as the control, i.e. baseline (BL) for the drug effects.

1.0. Behavior- Effect of 0.6 mg/kg MPD on Adolescent and Adult Rats (Figure 1)

Figure 1.

summarizes all the behavioral data of each experimental MPD dose (0.6 mg/kg, 2.5 mg/kg and 10.0 mg/kg); N represents the number in each group. All animal groups for each experimental dose were also divided into 2 subgroups; sensitized animals and tolerant animals. All- summarizes all the animals for a particular MPD dose. Sensitized summarizes only animals that expressed behavioral sensitization following chronic MPD. Tolerant summarizes only animals that expressed behavioral tolerance to chronic MPD. The left column of each group shows the total horizontal activity (HA) following saline injection i.e. baseline activity on experimental day 1 (ED1BL), the middle column shows the activity after acute MPD (ED1 MPD), and the third column shows the activity following chronic MPD of experimental day 10 (ED10MPD). The numbers in the brackets are the total number of animals in each group and subgroup. ED- experimental day; BL- baseline; MPD- methylphenidate.

For each group the HA of ED1 MPD is compared to HA ED1 BL to obtain the acute MPD effect; the HA of ED10 MPD is compared to HA of ED1 MPD to obtain the chronic MPD effect. The HA of adolescent ED1MPD is compared to HA of adult ED1MPD to examine the difference in acute response between adolescent and adult; and HA of adolescent ED10MPD is compared to HA of adult ED10MPD to examine the difference in chronic MPD response between adolescent and adult animals.

The symbols below are used on the figure to represent significant differences (P<0.05) found using the ANOVA

* - Significant acute MPD response (ED1MPD/ ED1BL)

○ – Significant chronic MPD response (ED10MPD/ ED1MPD)

▽- Significant difference between adolescent and adult acute MPD response

● – Significant difference between adolescent and adult chronic MPD response

1.1. Comparing ED1MPD to ED1BL on animals’ behavior

Acute 0.6 mg/kg MPD elicits significant (F (3, 9) = 35.82; P<0.05) increases in locomotion activity using one way ANOVA in the adolescent (N=45) and adult animals (N= 52). The ED10 BL locomotor activity compared to ED1 BL (ED10 BL/ ED1 BL) after six daily 0.6 mg/kg MPD and three washout days was significantly (P<0.05) decreased in the adolescent group, but significantly (F (3,9) = 21.47; P<0.05) increased in the adult group.

Rechallenge 0.6 mg/kg MPD at ED10 (ED10MPD) compared to 0.6 mg/kg ED1MPD on animal’s behavior resulted in significant (F (3,21) = 19.96; P<0.05) increases for only the adolescent group, using the ANOVA (Figure 1).

However, when each individual animal was evaluated using the C.R. test comparing 0.6 mg/kg ED10 MPD to 0.6 mg/kg ED1 MPD, both age groups had some animals expressing behavioral sensitization while others expressed behavioral tolerance as follows: 23/45 (51%) and 22/45 (49%) adolescent and 32/52 (62%) and 20/52 (38%) adult animals responded with significant (F (3,21) = 36.24; P<0.05) increases (i.e. sensitization) or decreases (i.e. tolerance) in behavioral locomotor activites, respectively. When comparing the locomotor activity of ED1MPD to ED1BL, for both the behaviorally sensitized and tolerant groups, the adolescent and adult rats showed significant differences in their acute response to MPD, with the adolescent rats showing significantly (F (3,9) = 38.17; P<0.05) higher locomotor activity in both behavioral groups (2002 and 1884 HA for sensitized adolescent and adult animals; 2451 and 1971 HA for tolerant adolescent and adult animals, respectively; Figure 1).

1.2. Comparing ED10BL to ED1BL on Locomotor Activities after six daily 0.6 mg/kg MPD and three washout days

In both the behaviorally sensitized and behaviorally tolerant groups, when ED10BL was compared to ED1BL locomotor activity of the adolescent and adult rats showed significant (F (3,9) = 23.48; P<0.05), with adult rats showing significantly (F (3,9) = 23.92; P<0.05) higher baseline locomotor activity in both groups as compared to adolescent animals (1472 and 1617 HA for sensitized; 1506 and 2148 for tolerant adolescent and adult animals respectively; Figure 1).

1.3. Comparing the Effect of 0.6 mg/kg MPD ED10MPD to 0.6 mg/kg ED1MPD on Locomotor Activities

In both the behaviorally sensitized and behaviorally tolerant rat groups, when comparing the 0.6 mg/kg ED10MPD to ED1MPD, the adolescent and adult rats showed significant (F (3,9) = 36.87; P<0.05) differences in response to chronic MPD, with the adolescent rats showing significantly higher locomotor activity than their adult counterparts (2632 and 2136 HA for sensitized; 2106 and 1730 for tolerant adolescent and adult animals, respectively; Figure 1).

1.4. Comparing the Behaviorally Sensitized Group to the Behaviorally Tolerant Group Treated with 0.6 mg/kg MPD (Figure 2)

Figure 2.

summarizes the behavioral data for each experimental MPD dose (0.6, 2.5 and 10.0 mg/kg). Each group consists of three columns. The left column represents All animals in the group, the middle column represents the animals that exhibit behavioral Sensitization to chronic MPD, and the right column represents the animals that exhibit behavioral Tolerance to chronic MPD. The number above each column represents the number of animals in the group. The figure shows the difference between adolescent and adult animals in terms of the amount of all animals in each age group and each dose that display behavioral sensitization versus behavioral tolerance. There were significant (P<0.05) differences between the ratios of animals that showed behavioral sensitization and behavioral tolerance for adolescent and adult animals only in the 0.6 mg/kg MPD and 10.0 mg/kg MPD doses.

*Indicates a significant (P<0.05) difference found using ANOVA in the ratio of animals who respond to chronic MPD with sensitization or tolerance between adolescent and adult animals. The symbol above the columns for a given dose indicates that for that dose of MPD the proportion of sensitized and tolerant animals are significantly different between adolescent and adult animals.

The ratio of how many animals expressed behavioral sensitization compared to those animals that expressed behavioral tolerance in response to repetitive 0.6 mg/kg MPD was significantly different (F (3,9) = 25.72; P < 0.05) between the adolescent group as compared to the adult group (Figure 2) using the odds ratio test. While both age groups showed a majority of animals expressing behavioral sensitization, the adult animals were significantly more likely (F (3,9) = 21.18; P<0.05) to exhibit behavioral sensitization than adolescent animals (62% and 51%, respectively). This shows additional important differences between adolescent and adult rats’ response to chronic MPD.

2.0. Effect of 2.5 mg/kg MPD on Adolescent and Adult Rats on Animal Behavior (Figure 1)

2.1. Comparing the Effect of ED1MPD to ED1BL

Acute 2.5 mg/kg MPD elicits significant (F (3,9) = 24.06; P<0.05) increases in locomotion in both age groups (Fig. 2). The ED10 BL after six daily 2.5 mg/kg MPD and three washout days exhibits significant (F (3,9) = 17.82); P<0.05) increases in locomotor activities in both the adolescent and adult groups. Rechallenge with 2.5 mg/kg MPD at ED10 compared to 2.5 mg/kg ED1 MPD resulted in further significant (F (3,9) = 19.93; P<0.05) increases in locomotion in both age groups, i.e. both age groups express behavioral sensitization using the ANOVA (Figure 1).

However, when each individual animal was evaluated using the C.R. test comparing ED10 MPD to ED1 MPD, 31/49 (63%) and 18/49 (37%) adolescent and 23/37 (62%) and 14/37 (38%) adult animals responded significantly (F (3,9) = 35.17; P<0.05) with further increases or decreases in their locomotor activities, respectively. Therefore, in each age group some animals expressed behavioral sensitization while other animals expressed behavioral tolerance. When comparing ED1MPD to ED1BL, only the animals expressing behavioral tolerance showed a significant (F (3,9) = 22.36; P<0.05) difference between adolescent and adult MPD response, with the adult rats showing significantly (F (3,9) = 22.93; P<0.05) higher locomotor activity (4008 and 4356 HA for tolerant adolescent and adult animals, respectively; Figure 1).

2.2. Comparing ED10BL to ED1BL Locomotor Activities

When comparing ED10BL to ED1BL locomotor activities, the animals exhibiting behavioral tolerance to chronic 2.5 mg/kg MPD showed a significant (F (3,9) = 18.76; P<0.05) difference in the locomotor activities between adolescent and adult animals, with the adult rat group showing significantly (F (3,9) = 20.82; P<0.05) higher locomotor activity (1611 and 2549 HA for tolerant adolescent and adult animals respectively; Figure 1)

2.3. Comparing 2.5 mg/kg ED10MPD to ED1MPD on Locomotor Activities

When comparing ED10MPD to ED1MPD the adolescent and the adult rats showed significant (F (3,9) = 19.21; P<0.05) differences in response to MPD in both the behaviorally sensitized and tolerant groups, with the adult rats showing significantly higher locomotor responses to chronic MPD (5936 and 6902 HA for sensitized; 3129 and 3604 HA for tolerant adolescent and adult animals respectively; Figure 1).

2.4. Comparing the Behaviorally Sensitized Group to the Behaviorally Tolerant Group Treated with 2.5 mg/kg MPD (Figure 2)

There were no significant differences in the ratio of how many animals expressed behavioral sensitization compared to how many animals expressed behavioral tolerance in response to repetitive 2.5 mg/kg MPD between the adolescent and adult animal groups.

3.0. Effect of 10.0 mg/kg MPD on Adolescent and Adult Rats Locomotor Behavior (Figure 1)

3.1. Comparing the Effect of ED1MPD 10.0 mg/kg to ED1BL

Acute 10.0 mg/kg MPD elicits robust increases in locomotion in both adolescent and adult rat groups (Fig. 1 10.0 mg/kg All). When each individual animal was evaluated using the C.R. test comparing ED10 MPD/ ED1 MPD 37/57 (73%) and 14/57 (37%) adolescents and 14/42 (33%) and 28/42 (67%) adult animals responded with further significant (F (3,9) = 41.37; P<0.05) increases or decreases in locomotion, respectively. In each age group some of the animals expressed behavioral sensitization and some expressed behavioral tolerance to 10.0 mg/kg MPD. When comparing ED1MPD to ED1BL between animals that expressed behavioral sensitization and animals that expressed behavioral tolerance, only the behaviorally tolerant rat group showed significant (F (3,9) = 42.33; P<0.05) differences between adolescent and adult animals, with the adolescent rats showing significantly (F (3,9) = 40.57; P<0.05) higher locomotor activity (10366 and 8934 HA for tolerant adolescent and adult animals respectively; Figure 1).

3.2. Comparing ED10BL to ED1BL of Rats

When comparing ED10BL to ED1BL of these groups, only the animals expressing behavioral sensitization to chronic 10.0 mg/kg MPD showed significant (F (3,9) = 20.59; P<0.05) differences between adolescents and adults, with the adolescent animals showing significantly (F (3,9) = 7.58; P<0.05) higher locomotor activity (2561 and 2042 HA for sensitized adolescent and adult animals respectively; Figure 1).

3.3. Comparing the Effect of ED10MPD to ED1MPD 10.0 mg/kg

When comparing the effect of ED10MPD to ED1MPD 10.0 mg/kg, both the behaviorally sensitized and behaviorally tolerant rat groups showed significant (F (3,9) = 38.41; P<0.05) differences in their responses between the adolescents and adults, with the adolescent rat group showing significantly (F (3,9) = 38.04; P<0.05) higher locomotor activity for both sensitized and tolerant behavioral groups (12112 and 11650 HA for sensitized; 8803 and 7115 HA for tolerant adolescent and adult animals respectively; Figure 1).

3.4. Comparing the Behaviorally Sensitized Group to the Behaviorally Tolerant Group Treated with 10.0 mg/kg MPD (Figure 2)

The ratio of how many animals expressed behavioral sensitization compared to how many animals expressed behavioral tolerance in response to chronic 10.0 mg/kg MPD was significantly different (F (3,9) = 41.14; P<0.05) between the adolescent group as compared to the adult group (Figure 2) using the odds ratio test. In response to the highest 10.0 mg/kg MPD dose, the adult animals were more likely to exhibit behavioral tolerance (67%), while the adolescent animals were more likely to exhibit behavioral sensitization (73%). These findings are different from those of the 0.6 mg/kg MPD group, where both age groups resulted in a majority of animals exhibiting behavioral sensitization. We see that with increasing dosages, there is a trend for the adolescent animals to exhibit increasing rates of sensitization, while the adult animals begin to exhibit significant rates of behavioral tolerance. This shows additional important differences between adolescent and adult rats’ response to MPD.

Neurophysiologic Results

Eight hundred and eleven (N=811) VTA units were recorded and evaluated; 403 units from adolescent animals and 408 units from adult animals. Specifically, 36, 115, 135, and 117 VTA units recorded from adolescent animals, and 36, 171, 102, and 99 VTA units recorded from adult animals were evaluated following saline, 0.6, 2.5 and 10.0 mg/kg MPD administration, respectively (see Tables 2 and 3). Of the 36 units recorded from adolescent animals treated with saline, 94.4% (34/36) and 100% (36/36) showed no changes in their neuronal firing rates on ED1 and ED10 as compared to the effect of the initial saline injection respectively (see Tables 2 and 3). Of the 36 units recorded from adults treated with saline, 91% (33/36) and 98% (34/36) showed no significant changes in firing rates when treated with saline at ED1 and ED10 as compared to the initial saline injection respectively (see Table 3). This suggests that the observations after saline can be used as the control for handling and injections.

Table 3.

summarizes the number of VTA units recorded from behaviorally tolerant adolescent and adult animals that for each dose of MPD that responded significantly (P<0.05) to A. Acute (ED1MPD/ ED1BL), B. Baseline changes (ED10BL/ ED1BL) and C. Chronic (ED10MPD/ ED1MPD), and the direction (increase or decrease) of how they responded to each dose, respectively.

Units recorded from behaviorally tolerant animals
			A. Acute			B. Baseline			C. Chronic
	Dose	N	↑	↓	≠	↑	↓	≠	↑	↓	≠
Adolescent	0.6	77	15	17	45	20	26	31	20	24	33
Adult	0.6	142	61	42	39	55	77	10	56	72	14
Adolescent	2.5	55	11	12	32	12	11	32	14	17	24
Adult	2.5	62	52	5	5	44	15	3	27	35	0
Adolescent	10	25	11	6	8	10	13	2	7	15	3
Adult	10	57	38	3	16	11	46	0	9	48	0

4.0. Effect of 0.6 mg/kg MPD on VTA Units Recorded from Adolescent and Adult Rats (Table 2, 3)

4.1. Comparing ED1MPD to ED1BL of the VTA Units

40% (46/115) and 65% (112/171) of adolescent and adult VTA units responded significantly (P < 0.05) to acute 0.6 mg/kg MPD, respectively (Tables 2 and 3). Thirty-eight and 77 VTA units were recorded from behaviorally sensitized and behaviorally tolerant adolescent animals, respectively. In adult rats, 29 and 142 VTA units were recorded from behaviorally sensitized and behaviorally tolerant animals, respectively. In the recordings from the behaviorally sensitized animals, there was a significant difference (F (1,23) = 4.42; P<0.05) between the adolescent and adult response to acute 0.6 mg/kg MPD, with a majority of the adolescent rats responding to 0.6 mg/kg MPD by increasing their firing rates, while the majority of the VTA units recorded from adult rats responded by decreased their firing rates. There were no significant differences in the VTA neuronal recordings between behaviorally tolerance adolescent and adult animals (Figure 3).

Figure 3.

A summarizes the Ventral Tegmental Area (VTA) firing rate unit changes for each MPD dose between behaviorally sensitized adolescent and adult animals. Figure 3B summarizes the VTA firing rate changes for each MPD dose between behaviorally tolerance adolescent and adult animals. The top figures represent the acute VTA firing rate changes for each dose between adolescent and adult animals (ED1MPD/ ED1BL), the middle figures represent the baseline VTA firing rate changes (ED10BL/ ED1BL), and the bottom figures represent the chronic VTA firing rate changes (ED10MPD/ ED1MPD).

*Indicates significant (P<0.05) differences found using ANOVA in VTA firing rates between adolescent and adult animals for the given MPD dose

4.2. Comparing ED10BL to ED1BL of the VTA Units

The ED10BL of the VTA units after six daily 0.6 mg/kg MPD injections and three washout days was compared to ED1BL neuronal activities. The neuronal activities were significantly (F (1,15) = 7.11; P<0.05) different in the adolescent rats as compared to the VTA units recorded from behaviorally sensitized adult counterparts (see Table 2, 3), i.e. additional differences between adolescent and adult animals when comparing ED10BL to ED1BL. There were no significant differences in the VTA firing rates between adolescent and adult animals in the behaviorally tolerant groups (Figure 3).

4.3. Comparing the effect of ED10MPD to ED1MPD of the VTA Units

The neuronal activity following repetitive (chronic) MPD (ED10MPD) after six daily 0.6 mg/kg MPD injections from ED1 to ED6 and three washout days from ED7 to ED9 was compared to the neuronal activity obtained from animals after the initial (acute) MPD injection (ED1MPD). Upon MPD 0.6 mg/kg rechallenge on ED10, there was a significant (F (1,23) = 4.51; P<0.05) difference in the responses to MPD between the adolescent and adult VTA firing rates, with a majority of the VTA units recorded from behaviorally sensitized adolescent animals showing significant increases in their firing rates, while a majority of the VTA units recorded from the behaviorally sensitized adult animals showed significant (F (1,23) = 4.09; P<0.05) decreases in their firing rates. For the animals that expressed behavioral tolerance to chronic MPD, there were no significant difference between the VTA units recorded from adolescent animals and the VTA units recorded from adult animals (Figure 3).

5.0. Effect of 2.5 mg/kg MPD on VTA Units Recorded from Adolescent and Adult Rats (Table 2, 3)

5.1. Comparing ED1MPD to ED1BL

54% (73/135) and 88% (90/102) of adolescent and adult VTA units responded significantly (F (1,23) = 3.75; P<0.05) by changing their firing rates to acute 2.5 mg/kg MPD, respectively (Tables 2 and 3). Eighty and 55 VTA units were recorded from adolescent behaviorally sensitized and tolerant animals, respectively. 40 and 62 VTA units were recorded from adult behaviorally sensitized and behaviorally tolerant animals, respectively. The majority of the VTA units recorded from both behaviorally sensitized adolescent and behaviorally sensitized adult groups responded to acute 2.5 mg/kg MPD with significant (F (3,23) = 4.16; P<0.05) increases in their firing rates (Table 2,3), with no significant difference between the two age groups. There was a significant difference (F (3,23) = 4.84; P<0.05) in VTA units recorded from behaviorally tolerant adolescent and adult animals, with the majority of the VTA units recorded from adolescent rats responding to 2.5 mg/kg MPD by decreasing their firing rates, while the majority of the VTA units recorded from adult animals responded to the drug by increasing their firing rates (Figure 3).

5.2. Comparing ED10BL to ED1BL of the VTA Units

When comparing the VTA neuronal activities at ED10 BL to ED1 BL, there were significant differences (F (1,23) = 4.87; P<0.05) in VTA firing rates between adolescent and adult animals in both the behaviorally sensitized and the behaviorally tolerant groups. The majority of the VTA units recorded from behaviorally sensitized adolescents showed further significant (F (1,23) = 4.76; P<0.05) increases in baseline firing rates, while those recorded from behaviorally sensitized adults showed more units with significant decreases in their baseline firing rates. In the VTA neuronal recordings obtained from the behaviorally tolerant groups, the majority of VTA units recorded from both the adolescent and adult animals showed significant increases in their neuronal baseline firing rates, with the VTA units recorded from behaviorally tolerant adult animals showing significantly (F (1,23) = 4.08; P<0.05) higher firing rates (Figure 3).

5.3. Comparing ED10MPD to ED1MPD of the VTA Units

Upon rechallenge of MPD on ED10, compared to the initial MPD exposure at ED1MPD, there was a significant difference (F (1,23) = 4.11; P<0.05) in the firing rates of VTA units recorded from the behaviorally sensitized adolescent animals and the units recorded from behaviorally sensitized adult animals, with a majority of the VTA units recorded from adolescent animals showing a significant (F (1,23) = 5.07; P<0.05) increase in firing rates, while the majority of the VTA units recorded from adult animals showed a significant (F (1,23) = 4.88; P<0.05) decrease in firing rates. There was no significant difference in the VTA units recorded from adolescent and adults in the animal groups expressing behavioral tolerance (Figure 3).

6.0. Effect of 10.0 mg/kg MPD on in the VTA Units Recorded from Adolescent and Adult Rats (Table 2, 3).

6.1. Comparing ED1MPD to ED1BL of the VTA Units

80% (94/117) and 81% (80/99) of adolescent and adult VTA units, respectively, responded significantly (F (1,23) = 4.36; P<0.05) to acute 10.0 mg/kg MPD by changing their firing rates. 92 and 25 VTA units were recorded from behaviorally sensitized and behaviorally tolerant adolescent animals, respectively, and 42 and 57 VTA units were recorded from behaviorally sensitized and behaviorally tolerant adult animals, respectively (Table 2 and 3). The majority of the VTA units recorded in both behaviorally sensitized adolescent and behaviorally sensitized adult animals responded to acute MPD (ED1 MPD) significantly (F (1,23) = 4.40; P<0.05) by increasing their firing rates (Table 2, 3). There was a significant difference between the age groups with the VTA units recorded from sensitized adolescent animals showing significantly (F (1,23) = 4.19; P<0.05) higher firing rates than their adult counterparts. Similarly, in the behaviorally tolerant group, both the VTA units recorded from adolescent and the VTA units recorded from adult animals responded with significant (F (2,49) = 3.75; P<0.05) increases in their firing rates, however, the VTA units recorded from the behaviorally tolerant adult animals showed significantly higher firing rates than their adolescent counterparts. These results highlight not only a significant difference between the age groups, but also a difference between the sensitized and tolerant groups, following acute response to 10.0 mg/kg MPD (Figure 3).

6.2. Comparing ED10BL to ED1BL of the VTA Units after six daily 10.0 mg/kg MPD and three washout days

When comparing ED10 BL to ED1 BL VTA neuronal activities of animals exposed to six daily (chronic) 10 mg/kg MPD and three days of no dosing treatment (washout), there were significant differences (F (2,49) = 4.53; P<0.05) in firing rates between the VTA units recorded from adolescent and adult animals in both behaviorally sensitized and the behaviorally tolerant groups, each of which showed the adolescent animals having significantly (F (2,49) = 4.03; P<0.05) higher firing rates than their adult counterparts. In the VTA units recorded from adolescent animals expressing behavioral sensitization, a majority exhibited increases in firing rates on ED10 BL as compared to ED1 BL, while in the VTA units recorded from adult animals a majority of VTA units exhibited decreases in their firings rates. The VTA units recorded from both the adolescent and adult behaviorally tolerant animals showed significant (F (2,49) = 4.33; P<0.05) decreases in their ED 10 BL compared to ED1 BL firing rates, however the adolescent VTA ED10 BL firing rates were still significantly (F (2,49) = 4.45; P<0.05) higher than the adult VTA ED10 BL firing rates (Figure 3).

6.3. Comparing ED10MPD 10.0 mg/kg to ED1MPD 10.0 mg/kg VTA units

Upon rechallenge with MPD on ED10 as compared to ED1MPD, there was a significant difference (F (2,49) = 3.89; P<0.05) in the VTA unit firing rates recorded from behaviorally sensitized adolescent animals as compared to those recorded from adult animals, with the majority of the VTA neuronal recordings from behaviorally sensitized adolescent animals showing significant (F (2,49) = 4.27); P<0.05) increases in their firing rates, while the majority of the VTA neuronal recordings from sensitized adult animals showed significant (F (2,49) = 4.61; P<0.05) decreases in their firing rate. In the VTA units recorded from the behaviorally tolerant animal groups, both the adolescent and adult animals responded to chronic MPD with significant (F (2,49) = 3.96; P<0.05) decreases in their firing rates as compared to the effect observed at ED1 MPD 10.0 mg/kg, however the adolescent animals still had significantly (P<0.05) higher firing rates than their adult counterparts (Figure 3).

Discussion

Methylphenidate is one of the most widely prescribed drugs for ADHD, and its pharmacological profile resembles several drugs that are abused for cognitive enhancement and recreational use, including cocaine and methamphetamine (Gatley et al., 1999; Volkow et al., 2002; Patrick et al., 2005; Yang et al., 2011). MPD impacts the nervous system by binding to DAT and NET and preventing the re-uptake of DA and NE from the synaptic cleft into the presynaptic terminals, thus prolonging the effects of DA and NE at the post-synaptic receptors (Patrick and Markowitz, 1997; Kuczenski and Segal, 2001; CNS Stimulants, 2012; Amphetamines, 2016; Faraone, 2018). Since catecholamine (CA) imbalance is believed to be an underlying cause of ADHD, the significant effects of MPD on CAergic systems in the CNS explain the drug’s role in treating the disorder (Swanson and Volkow, 2008). The VTA is one of the major sources of DA and seems to play a critical role in the effects of addictive drugs, such as eliciting withdrawal and dependence, as well as sensitization and tolerance (Kalivas et al. 1993).

In this study, the VTA neuronal activity was recorded concomitantly with behavioral activity in freely behaving adolescent and adult rats, both before and after the administration of acute and repetitive (chronic) MPD (0.6, 2.5 and 10.0 mg/kg). The main findings of this study are: 1) Behavioral activity recorded from both the adolescent and the adult rats responded following MPD in a dose-dependent manner; 2) In both adolescent and adult animals, the same MPD dose elicited in some rats behavioral sensitization and in others behavioral tolerance; 3) For the 0.6 mg/kg and 10.0 mg/kg MPD doses, there were significant differences in the ratios of how many animals expressed behavioral sensitization as compared to those animals that expressed behavioral tolerance between adolescents and adults; 4) There were also significant differences between the neuronal responses to MPD for each age group with each dosage 5) the animals behavioral response to chronic MPD i.e. sensitization or tolerance did not always correlate to the VTA neuronal response; 6) the age group with the significantly higher behavioral response did not always correlate to the age group with the significantly higher VTA neuronal response to the drug.

Several previous studies had similar findings with increasing doses of drugs such as cocaine and amphetamine (Kim et al., 2009), including excitation and attenuation of VTA units and behavioral sensitization compared to behavioral tolerance. These studies have proposed that the mechanism underlying behavioral sensitization and behavioral tolerance is an upregulation of transcription factor ΔFosB and transcription factor CREB. It was reported that the same dose of MPD causes some animals to express upregulation of ΔFosB, which is suggested to underly behavioral sensitization, and other animals express an upregulation of CREB, which is suggested to underly behavioral tolerance (Hyman and Malenka 2001; McClung et al., 2005; Perrotti et al., 2005; Kim et al., 2009; Chao and Nestler 2004; Nestler, 2012; Ruffle, 2014;). Specific different patterns of ΔFosB and CREB expression within the VTA, LC and other CNS structures, as well as differences in medium spiny neurons may be the mechanism underlying the expression of behavioral and neurophysiological sensitization and tolerance (Jones and Dafny, 2014; Karim et al., 2017).

Important to note also is that the adolescent and adult animals showed significant differences between their rates of behavioral sensitization and behavioral tolerance to the same doses of MPD. For the lower dose, (0.6 mg/kg MPD) the majority of both adolescent and adult animals responded with behavioral sensitization, though the motor activity was significantly higher for the adult group. However, in response to the higher dose (10.0 mg/kg MPD), the adolescent animals continued to display significantly increasing rates of behavioral sensitization, while the majority of adult animals began to express behavioral tolerance. Perhaps this observation is due to differences in the aforementioned transcription factor densities, synaptic pruning and other metabolic differences between adolescent and adult animals which may influence how the animals respond to the drug as they age. These findings have important clinical implications; the fact that the 2.5 mg/kg MPD group showed no significant differences in the rates of sensitization and tolerance between adolescents and adults suggests that this middle dose may be the most effective for use across both age groups. While it is difficult to extrapolate the data in rats directly to humans, the fact that 10.0 mg/kg MPD was more likely to elicit tolerance in adult animals and sensitization in adolescent animals may suggest that adults are less responsive to higher doses of this medication and therefore it may be less effective as a treatment in this age group. This theory is further confirmed by the findings that the adult rats had higher rates of sensitization at the lower 0.6 mg/kg MPD, i.e. they were relatively more responsive to the drug at lower doses.

Our study also shows that there are significantly different neuronal responses in the VTA between adolescent and adult animals at each MPD dose, and the VTA neuronal response does not always correlate to the behavioral response for each animal. It has previously been suggested that the VTA response to the same drug dose with either excitation or attenuation may be due to the animals’ levels and activation of different receptors, D1 and D2 DA, resulting in some animals responding to MPD with excitation and others inhibition, respectively (Jones et al. 2014; Reisi et al. 2014; Kharas and Dafny, 2016). The same study also discussed the possibility that the glutamatergic neurons from the prefrontal cortex (PFC) may influence D1 and D2 DA neurons differently in the VTA and cause differing responses to MPD. Additionally, previous studies have shown that in the PFC, the animal’s behavioral response with either sensitization or tolerance was found to correlate directly with their neuronal response, either increasing or decreasing firing rates, respectively (Venkataraman et. al, 2020). Each of these studies, alongside our findings, suggest that there are inputs outside the VTA that modulate the animal’s behavior and explain why the VTA neuronal response may not directly correlate with the behavioral response.

These behavioral and neuronal differences, especially between the two age groups, could also be due to immature dopamine vesicular release in adolescent animals (Fukui et al., 2003). Plus, while dopamine is the primary neurotransmitter affected by MPD exposure, an increase in secondary neurotransmitters such as norepinephrine (NE) and serotonin (5-HT) may also contribute to the drug’s effects (Tang and Dafny, 2013). It has also been shown in the PFC that there are significant changes in the GABAergic interneurons and D2 DA receptor activation between prepubertal and adult rats, representing developmental shifts in neuromodulation and drug response (McCutcheon and Marinelli, 2009) between adolescent and adult animals. Perhaps similar age-related differences in neuromodulation for the VTA help to explain the findings from our study between young and adult animals.

The significant differences in response to MPD between the two age groups help highlight the importance of assessing adolescent and adult animals separately when examining the effects of drugs such as MPD, especially considering the risks and high usage in our current society. Additionally, the findings that the behavioral response does not always correlate completely with the VTA neuronal response for each age groups highlights that the VTA is not the only brain structure to participate in behavioral regulation, but rather one of many nuclei that are affected by the drug and essential to the modulation of the animal’s behavior. Further investigation is needed to clearly understand the relationship between VTA neuronal and behavioral responses to different MPD doses.

Ultimately, the results of this study illustrate that in general VTA units recorded from adolescent rats are more responsive to MPD exposure than VTA units recorded from adult rats, a concerning finding given the increase in potential MPD abuse in adolescents. Secondly, adolescent and adult animals have significantly different rates of sensitization and tolerance in response to MPD, which needs to be considered carefully as ADHD diagnosis and treatment increases in the US. Additionally, the behavioral response does not always correlate to the VTA neuronal response, meaning that using these drugs as treatments for certain behavioral effects may mask different and potentially opposite neuronal behavior occurring simultaneously in many different structures in the brain. In conclusion, the study has shown that adolescent animals respond significantly different to MPD as compared to adult animals in their behavior as well as their VTA neuronal response in which further investigation is warranted.

Highlights.

• Behavioral activity responded following MPD in a dose-dependent manner

• Some rats exhibit behavioral sensitization and others behavioral tolerance

• There were significant differences in rates of sensitization and tolerance

• Significant differences between VTA neuronal responses for adult and adolescent

• Behavioral response did not always correlate to the VTA neuronal response