Team RITALIN:Research in Testing ADHD's Link to Impulsivity in Neuroscience

13 June 2013Impact of prenatal nicotine exposure on impulsivity and neural activity in medial prefrontal cortex

Prenatal Nicotine Exposure (PNE)PNE is linked to many psychiatric disordersWomen who smoke during pregnancy are three times as likely to have children diagnosed with ADHD1 in 5 women still smoke during pregnancy

Nicotine causes changes in the development that alters dopaminergic & noradrenergic pathways in the brainSeveral studies show behavioral, neuroanatomical, & neurochemical disturbances after PNEBenefits of methylphenidate, a common ADHD drug, point to PNE as a valuable animal model of impulsivityIntroductionIntroduction

Attention Deficit Hyperactivity Disorder (ADHD)Symptoms: impulsivity, hyperactivity, & inattentionAffects 5-10% of all school age childrenTwentyfold increase in prescription of ADHD drugs in the past 30 yearsDiagnoses based on qualitative observationsLimited research on the neurobiology of the disorderADHD drugs are addictive stimulantsFetal nicotine rats and humans with ADHD have similar deficits on behavioral tasks for hyperactivity & impulsivity

Introduction

Modeling ImpulsivityAnimal model validityFace validityConduct validityPredictive validityWhat areas or circuits are involved?How can we uncover more about the neurobiology?

Introduction

Medial Prefrontal Cortex (mPFC)Introduction

Stop Signal Task (SST)Introduction

Research QuestionsIs there a correlation between neural firing in the mPFC cortex and impulsivity in control rats?Is neural firing in the mPFC cortex disrupted and impulsivity increased in fetal nicotine rats?Is there a correlation between this disrupted firing and increased impulsivity?Introduction

Phase 1: Rat Breeding & SelectionMethodologySignificant differences in water consumption & mother weightsNo significant differences in pregnancy duration, pups per litter, pup birth weight, or locomotion*Randomly selected 8 males each from 37 PNE pups (from 3 dams) and 39 control pups (from 3 dams)

Phase 2: SST Training & SurgeryMethodologyCompare movement times and percent correct on stop and go trials

Results

Results

Results

Results

ResultsPost-Surgery Behavior

Phase 3: Neural Recording & AnalysisMethodology12 rats from the control and PNE groups performed 157 sessions, over which we collected neural firing from 346 cells

Methodology

ResultsHistograms for Above/Below Baseline Firing

ResultsHistograms for Preferred/Nonpreferred Stop/Go Trials

ResultsDistributions for Preferred/Nonpreferred for Go Direction

Resultslegend:go ipsi - bluego contra - greenstop ipsi - redstop contra - yellow legend:go ipsi - bluego contra - greenstop ipsi - redstop contra - yellowHistograms for Contra/Ipsi Stop/Go Trials *

ResultsSubtraction Plots for Preferred/Nonpreferred Stop/Go Trials

Preliminary ConclusionsDiscussionBehaviorPNE rats were more impulsive (as measured by SCRT)However, they were better at basic task procedures (eg responding to spatial cue lights)

Neural recordingsSignals from neurons which encode stopping an already initiated movement were attenuated in PNE rats as compared to controlsSignals from neurons which encode direction towards the correct behavioral response were also attenuated as compared to controlsThis points to a neurophysiological pathway disruption in inhibiting impulsivity caused by PNE

Future DirectionsFor my team:Finish collecting data for additional controlsComplete data analysisPresent data at Society for Neuroscience ConferenceWrite & present our thesis at the Senior Thesis ConferenceFor someone else:Try a different task (or SST with different parameters)Give nicotine postnatallyAdminister ADHD drugs to PNE rats and controls

Discussion

My Team

Questions?Questions?

*Gemstone as a four-year honors, like a group PhDFreshman: learn about research, pick teamsSoph: apply for grants, IACUC approval, lit review, refine topicJunior: data collection; various RIP presentationsSenior: data analysis & thesis writing; final presentation in April

***Obesity, conduct disorder, drug abuseHow do we know its nicotine? How do we know PNE causes the symptoms & not a hidden factor? How does it work?Neural pathfinding is guided by nAchR, so named because it can bind nicotineDA & NE pathways are involved in attention & impulsivityMPH = RitalinMPH affects the dopaminergic system, so this makes sense**Which brings us to ADHD. Why is this such an issue?We hypothesize that this increase in diagnosis was largely due to qualitative diagnoses due to limited knowledge of the neurobiology of the disorderBecause of previous research on MPH and its effects on impulsivity, we turned our attention there**DA is involved, but it is involved in many behavioral disorders (eg Parkinsons)This tells us that we can extend our conclusions to humans as wellBut what controls impulsivity? This is the missing piece of the puzzle**Structural abnormalities caused by PNEActive during impulsivity tasksHomologous area in ratsCan we tie impulsivity to PNE to neural activity?Explain neural recordings hereWe know as much about the cortex as we do about the bottom of the oceanmPFC is between eyebrowsThese coronal sections are for PFC, but not exactly for mPFC**SST - what, rats vs humans, ADHDtests the ability to inhibit an ongoing movementDecreased inhibition in humans w/ ADHD

SPEAKER NOTES:First, we have go trials. In a go trial, the rat will initiate the task. Then a directional light will flash and the rat will go to the correct well to receive a reward. *SPEAKER NOTES: There are also stop trials. In a stop trial, the rat again initiates the task. Then a directional light will flash, and the rat begins to respond to that direction. However, the other light flashes so the rat must change direction to receive a reward.***Here, we ask if PNE impacts behavior and neural signals in medial prefrontal cortex (mPFC) as rats perform a stop-signal task**Long-Evans rats selected for behavioral profileGive nicotine via water in proportions equal to 2 packs a day high enough to cause behavioral differences, low enough to prevent serious developmental delaysCross fosteringMales only (brain & behavior homology)Pregnancy duration & pups per litter show that the levels of nicotine we used were not lethal to fetal ratsCompared at selection to ensure that pups were about the same across all metrics**Train for 2 hours every day (~150 trials per session)Mild water deprivation for motivationSurgery is risky; we lost 5 rats out of 16 after our first round (were able to collect from 12)post-op recovery to normal scoresUpcoming data was plotted to see where we were going/get a status update, isnt polishedSurprisingly, the nicotine rats learned the task significantly faster than controls*Stop has a lower percent correct as expected, but nicotine rats are doing better*Not significant*Stop trials are slower as expected, but nicotine rats are significantly slower than controls on stop trials*Error bars represent the standard error of the mean. After surgery, we saw significant differences in movement times as well as continued significant differences in stop & go correctnessAs expected, we found that PNE rats had longer stop change reaction times (SCRTs), which were measured by the difference in movement times on STOP and GO trials. This statistic provides evidence that the PNE rats are more impulsive than control rats. Since PNE rats took longer to alter behavior on STOP trials (SCRT) we predict that activity in mPFC, which we think is necessary for stopping, will be decreased in PNE rats. Since PNE rats were better at responding to spatial signals that instruct behavior, we expect that activity in mPFC of PNE rats was more spatially tuned as compared to controls.***Thousands of action potentials per sessionElectrode implanted in mPFC based on skull landmarks & heights from atlas; progressed each day such that it starts & ends in the mPFCSingle unit extracellular recordings return waveforms must select range of waveforms (AP, after, shape) on channels that have cells to cut out noisehistology - verify mPFC, perfuse, slice, mount, stain, compare to atlas, won't to into detailThese figures and figures on next slide are NOT our data; just for demonstration

**Cut cells with Neuroexplorer: select cluster from 2D feature plot plot based on prediction from live selectionExamine raster plot to ensure that cell is a cellexamine in epochs, combine all cells as one: rats are identical, allow sample = 3 & still significant, cross section of brain too large to pinpoint exactly)DA - group according to characteristics, then repeat**Baseline firing is described as the neural activity before the trial time begins. Firing rate plots are aligned on port exit. X-axis is time from stop-signal onset, measured in seconds. Y-axis is spike rate, defined as spikes or action potentials per second. Blue lines are go trials, and red lines are stop trials. Data was analyzed in Matlab to determine average firing frequency over time.**All four populations are significantly higher for preferred vs nonpreferred direction, suggesting there may be a spatiotopic organization of movement towards rewards in the PFC.Some good questions: where is the reward given? Does that correlate with the peak? The early peak in the nicotine above baseline... we need an explanation for that.above baseline: are driving behaviorbelow baseline: they are allowing behavior to occur.**nice signal for gos, but weaker for stops**Only significant differences is control above baseline for go trials: on go ipsi vs go contra, ipsi is significantly higher than contraIn controls, for neurons that fire above baseline, firing is higher when the trial direction is the same side as the hemisphere being recorded from.**Taking into account the different scales of these graphs, we see that the nicotine below baseline do not have a difference in spikes/s greater than .05. The control below baseline, nicotine above baseline, and control above baseline, on the other hand, have greater different spikes visible around time 0. Most critically the differences in stop spikes for the nicotine groups are significantly lower than the differences in stop for controls (point to the pink line). This indicates that the PFC is more critically involved in the control's stop reaction than the nicotine's stop reaction. it suggests that the controls do a better job of counteracting the competition between the stop and go responses.**Higher go correctness & faster go response timeHigher stop correctness & slow stop response timeThis may be related to a recent study showing that children with ADHD are better than non-diagnosed children at directed, uninterrupted, internally motivated work in a distraction-free environmentFrom neurons which encode this inhibition**ADHD & smoking in adolescence is linked (PFC not completely developed yet)1st month after birth in rats is equivalent to 3rd trimester in humansMention locomotion task (not the results as the methodology paper we read using the same nicotine dosage)***Qs: why can we extrapolate single cells to a whole region? more accurate than BFwhat other parameters could we examine?what's the point of an animal model of impulsivity? limitations (cognitive therapy, diff motivations) can't test a cancer drug on healthy rats (Adderall toxicity, DAT, larger NT implications, 3 circuits)