1. Measures electrical activity of neurons near electrode tip Single-electrode Recording The primary tool for investigation of brain- behavior relationships for over 60 years A useful tool for studying the details of properties of individual neurons. Ideal for an understanding at the level of individual neurons. Less appropriate for studying networks and systems of neurons. Does not allow measurements of the precise timing of activity between neurons that give insight into how they communicate and interact. The classic single-electrode approach only allows indirect inferences about neural networks. The result: a piecemeal understanding of brain function

2. A More Global View of Brain Function: FMRI. However…. FMRI measures patterns of blood flow to brain areas (the BOLD signal). Result of neurons needing energy (oxygen) when they fire electrical impulses (“action potentials”). The Good: Provides a global view of which brain areas are engaged by a cognitive function. The Bad: It takes five-six seconds for the BOLD signal to build. A lot can happen in the brain in 5-6 seconds.

3. Our approach: Multiple-electrode Recording in Monkeys Performing Cognitive-demanding Tasks Electrode arrays with 500 um spacing to investigate microcircuitry Electrode arrays in different brain areas to investigate large-scale networks. Allows direct measurements of the networks that underlie cognition.

4. Brain waves are rhythmic, coordinated oscillations between neurons (1 – 100 Hz). They reflect how and when networks of neurons communicate. They allow local networks of neurons to synchronize with one another and with distant networks. This allows the brain to orchestrate billions of neurons to produce elaborate behaviors. The idea is that when neurons fire in synchrony with one another, they are better able to communicate than when they fire out of sync. Mounting evidence that brain waves play a critical role in attention, working memory, memory storage, recall, learning, sequencing, planning and more. Abnormal brain waves are associated with neuropsychiatric disorders. Brain Waves Play a Central Role in Brain Function Parkinson’s patients show increased beta band brain waves (which can be decreased by DA therapy) Schizophrenia patients show decreased gamma band brain waves. Guanfacine (ADHD treatment) increases brain wave (EEG) synchrony in rats. Methylphenidate (ADHD) increases theta brain waves in the hippocampus.

5. Our Approach: Complex Behavioral Paradigms for Complex Cognition 1.We use monkeys. Primates have higher-level, and more flexible, cognition than other animals. 2.The Miller Lab are experts at animal training and design the most sophisticated behavioral paradigms in systems neurophysiology. Learning of abstract categories and concepts Rule learning Multi-tasking Decision-making Task switching Reversal learning Sequence memory Planning Attention Working Memory “Cats vs dogs” Small numbers

6. Functional Endpoints: Proposed Project Our multiple electrodes and sophisticated behavioral paradigms can provide precise diagnostic tools for assessing the effects of guanfacine (and other drugs) on the mechanisms of cognition. 1.Our task: Enhancement of higher-order (prefrontal cortex-dependent) learning Guanfacine does not improve simple learning (subcortical or posterior cortical- dependent). It does improve many prefrontal cortex (PFC) dependent tasks, but its effects on PFC-dependent learning are not known. We will use a learning task (conditional visuomotor learning) that is highly PFC dependent. Guanfacine, at the proper dose, should improve learning. 2.Multiple-electrodes offer a powerful diagnostic for directly measuring the effects of guanfacine on cognition. Arnsten and colleagues have provided elegant evidence that guanfacine improves communication in PFC microcircuits that underlie working memory (“delay”) activity. This was indirectly inferred from the activity of single neurons as well as detailed anatomy. Multiple electrodes allow direct examination of the functioning of microcircuits. This gets directly at the network mechanisms underlying cognition and how they are improved by drug therapy.

7. Novel images Familiar images Fixation Cue DelayTarget onset 800 ms 500 ms1000 msResponse 40 % 10 % The Role of Dopamine (D1R) Receptors in the Prefrontal Cortex During Learning Monkeys learned by trial and error to associate two novel visual cues with either an eye movement to the right or left

8. 1 2 3 4 5 6 7 8 9 … Injection schedules Block number Location of the injections and grid configuration Saline 3 µl SCH 23390 (D1 antagonist) 30 µg in 3 µl Infusion rate: 0.3 µl/min (3 µl in 10 minutes) BaselineDrugWashout ------------//----------- 1 2 3 4 5 6 7 8 9 … BaselineDrugWashout ------------//----------- 1 2 3 4 5 6 7 8 9 … BaselineDrugWashout ------------//----------- Session type #1 Session type #2 Session type #3 Recording with Multiple Electrodes while Injecting a D1R Blocker

9. Blocking D1R Receptors Impairs New Learning But Not Long-Term Memory Performance novel associations Performance familiar associations Baseline Saline Washout Percent correct Baseline SCH Washout Percent correct ns Saline SCH23390 BaselineWashout Baseline Criterion Chance

10. Blocking D1Rs Decreases Attention and Increases Impulsivity Fixation breaks per blockEarly trials per block Baseline Treatment Washout Saline SCH Effect on attentionEffect on impulsivity ***

11. D1R blockade induces negative deflections on the LFPs Injection

12. Neuronal avalanches are generated by super-synchronous activity Avalanches appeared in 47 of 68 electrodes (~70% of 9 sessions) Duration 18 ± 5min (~10-30 min) Frequency of deflections 0.44  0.03 Hz (0.2-0.6 Hz) Amplitude of deflections is huge: in most cases over 500  V Performance 7 sessions with impairment: drops to 56 ± 15 % Amplitude (  V)

13. Blocking D1R Receptors Causes a Broad-Band Increase in PFC Brain Waves Cue Delay Response Normalized spectrum dB Baseline SCH Task Interval: Brain wave frequency Normalized spectrum dB Abnormal brain waves are a bad thing Puig, M.V. and Miller, E.K. (in preparation)

14. Functional Endpoints: Other Projects 1.Improve functioning of orbital frontal cortex (OFC) networks Reversal learning is highly dependent on the OFC. By adding the requirement to reverse associations to our conditional visuomotor task, we can make it an OFC task. Guanfacine should improve reversal learning and our multiple electrodes can directly measure the network cellular basis for that improvement. 2.Guanfacine as an intelligence enhancer We know that guanfacine improves working memory (WM) for a single to-be- remembered item and helps alleviate ADHD. Can it improve general intelligence? The ability to hold a single item in WM does not correlate well with general intelligence and single item WM is not impaired in many neuropsychiatric disorders. By contrast, WM capacity (how many items you can hold in WM simultaneously) correlates highly with intelligence measures and is reduced in virtually every neuropsychiatric disorder and in aging. In other words, WM capacity may be a great diagnostic of WM function. Multiple-electrode neurophysiology provides a direct and powerful measure of the cellular basis for the network properties that underlie cognitive enhancements by guanfacine.

15. Cognitive capacity: How many things can you hold in mind simultaneously? Individual differences in capacity limits can explain about 25-50% of the individual differences in tests of intelligence It is linked to normal cognition and intelligence: Capacity is highest in younger adults and reduced in many neuropsychiatric disorders Schizophrenia Parkinson’s Disease Vogel et al (2001); Gold et al (2003); Cowan et al (2006); Hackley et al (2009) www.ekmiller.org Cognitive capacity is the bandwidth of cognition. It may be directly related to brain waves.

16. Guanfacine as an intelligence enhancer The Miller Lab used cutting edge multiple-electrode technology to that has yielded the first neurophysiological insight in WM capacity limitations: gamma-band oscillations (brain waves) in the prefrontal cortex. Siegel, Warden, and Miller (2009) showed that PFC gamma-band brain waves provide “memory slots” for holding multiple items in WM. WM capacity is due to a limited number of slots per wave. In theory (soon to be tested), we can increase WM capacity by slowing down the brain wave frequency or increasing its amplitude. This could add 1-2 more memory slots and effectively increase general intelligence. Functional Endpoints: Other Projects

17. Brain waves are central to brain function. They regulate communication between neurons and there is mounting evidence that they play specific and important roles in higher cognition. Abnormal brain waves are apparent in neuropsychiatric disorders. Multiple-electrodes offer a new tool for directly measuring the effects of potential drug therapies on cognition. They allow direct examination of the functioning of microcircuits and large-scale networks of neurons. This gets directly at the network mechanisms underlying cognition. The combination of cutting-edge multiple-electrode technology and sophisticated behavioral paradigms in monkeys can provide a powerful diagnostic of the cellular mechanisms that underlie cognitive enhancements by potential drug therapies. CONCLUSIONS

18. What the Miller Lab can offer Shire 1.Investigation of brain-pharmacology-neurophysiology relationships using cutting-edge multiple-electrode recording techniques. The Miller Lab has invented and pioneered the use of multiple electrodes in behaving monkeys. This has yielded new and direct insight into the communication within networks of neurons during high-level cognition. This can provide a power diagnostic for assessing potential drug therapies. 2.Investigation of the highest levels of cognitive function using the most sophisticated animal training in neuroscience. Most neurophysiological studies of cognition use relatively basic tasks (“pay attention here.” “hold one thing in mind”) The Miller Lab has taken monkey training to a higher level than any other lab. We have taught monkeys to juggle multiple things in memory, anticipate and imagine forthcoming events, make cognitive decisions, to recognize abstract categories and concepts (“cat vs dog”, “same vs different”, numbers 1-5). More complex behavioral methods are needed to understand the pharmacology of truly intelligent behavior.

19. We can also apply this approach to a wide range of cognitive functions

20. Category Learning Freedman, Riesenhuber, Poggio, and Miller (2001) Science Freedman, Riesenhuber, Poggio, and Miller (2002) J. Neurophysiology Freedman, Riesenhuber, Poggio, and Miller (2003) J. Neuroscience Roy, Riesenhuber, Poggio, and Miller (submitted) Cromer, Roy, Riesenhuber, Poggio, and Miller (in preparation) Fundamental to normal human cognition because they imbue the world with meaning. They allow discarding detailed information in favor of general concepts. Disrupted in neuropsychiatric disorders such as autism, schizophrenia, and learning disorders. Patients become mired in details (they lose the forest in the trees).

21. Freedman, Riesenhuber, Poggio, and Miller (2001) Science Freedman, Riesenhuber, Poggio, and Miller (2002) J. Neurophysiology Freedman, Riesenhuber, Poggio, and Miller (2003) J. Neuroscience Roy, Riesenhuber, Poggio, and Miller (submitted) Cromer, Roy, Riesenhuber, Poggio, and Miller (in preparation) For example, my concept of dogs is inextricably linked to every dog I've ever known. It's as if I have a card catalog of dogs I have seen, complete with pictures, which continually grows as I add more examples to my video library. Temple Grandin, Ph.D. Thinking in Pictures Categories and Concepts

22. Category boundary 50% Prototypes 100% Cat 80% Cat Morphs 60% Cat Morphs 60% Dog Morphs 80% Dog Morphs Prototypes 100% Dog “Cats” Versus “Dogs” Learned categories: monkeys had no prior experience with cats and dogs and could learn to categorize the stimuli after their reassignment to arbitrary categories.

23. “cats” “dogs” category boundary C1 C2 C3 D2 D3D1 Activity to individual stimuli along the 9 morph lines that crossed the category boundary Single neuron: 0 0.5 1.0 Normalized firing rate C1 C2 C3 D1 D2 D3 D1 D2 D3 D2 D3 D1 About 1/3 of Prefrontal Neurons Respond to Category Membership not Physical Appearance Freedman, Riesenhuber, Poggio, and Miller (2001) Science Freedman, Riesenhuber, Poggio, and Miller (2002) J. Neurophysiology Freedman, Riesenhuber, Poggio, and Miller (2003) J. Neuroscience

24. Theories of Prefrontal Cortex Function

25. Basal ganglia (striatum): Fast, reward-gated, plasticity Prefrontal cortex: Slower, reward-shaded, plasticity Balance between advantages and disadvantages of slow and fast learning Slower learning of more elaborate, generalized, less error-prone, representations that include the regularties across experiences. Fast learning (“snapshots”) of the specific experiences that predict reward. Error prone.

26. The integrative anatomy of the PFC and BG allows rapid acquisition of the logic of a goal-directed task. This is a “roadmap” that specifies which pattern of “tracks” (neural pathways) are needed to solve a given task. This pattern is activated and maintained in the PFC during task performance, producing “top-down” signals that bias the flow of activity in the cortex along task-relevant pathways. Goal-direction Overcome habits Flexibility Miller, E.K. (2000) Nature Reviews Neuroscience, 1:59-65 Miller, E.K. and Cohen, J.D. (2001) Annual Review of Neuroscience, 24:167-202 A switch operator in a system of railroad tracks: Active Inactive BG loops PFC Sensory cortex Motor cortex “Cue” “Context” Don’t answer Answer DA reward Phone rings At home Guest

27. Goal-direction Overcome habits Flexibility A switch operator in a system of railroad tracks: PFC Sensory cortex Motor cortex “Cue” “Context” Don’t answer Answer Phone rings At home Guest Active Inactive Miller, E.K. (2000) Nature Reviews Neuroscience, 1:59-65 Miller, E.K. and Cohen, J.D. (2001) Annual Review of Neuroscience, 24:167-202 The integrative anatomy of the PFC and BG allows rapid acquisition of the logic of a goal-directed task. This is a “roadmap” that specifies which pattern of “tracks” (neural pathways) are needed to solve a given task. This pattern is activated and maintained in the PFC during task performance, producing feedback signals that bias the flow of activity in the cortex along task-relevant pathways.

28. Goal-direction Overcome habits Flexibility A switch operator in a system of railroad tracks: BG loops PFC Sensory cortex Motor cortex “Cue” “Context” Don’t answer Answer DA reward Phone rings At home Guest Active Inactive Miller, E.K. (2000) Nature Reviews Neuroscience, 1:59-65 Miller, E.K. and Cohen, J.D. (2001) Annual Review of Neuroscience, 24:167-202 The integrative anatomy of the PFC and BG allows rapid acquisition of the logic of a goal-directed task. This is a “roadmap” that specifies which pattern of “tracks” (neural pathways) are needed to solve a given task. This pattern is activated and maintained in the PFC during task performance, producing feedback signals that bias the flow of activity in the cortex along task-relevant pathways.