Reversible inactivation of macaque frontal eye field.

The macaque frontal eye field (FEF) is involved in the generation of saccadic eye movements and fixations. To better understand the role of the FEF, we reversibly inactivated a portion of it while a monkey made saccades and fixations in response to visual stimuli. Lidocaine was infused into a FEF and neural inactivation was monitored with a nearby microelectrode. We used two saccadic tasks. In the delay task, a target was presented and then extinguished, but the monkey was not allowed to make a saccade to its location until a cue to move was given. In the step task, the monkey was allowed to look at a target as soon as it appeared. During FEF inactivation, monkeys were severely impaired at making saccades to locations of extinguished contralateral targets in the delay task. They were similarly impaired at making saccades to locations of contralateral targets in the step task if the target was flashed for < or =100 ms, such that it was gone before the saccade was initiated. Deficits included increases in saccadic latency, increases in saccadic error, and increases in the frequency of trials in which a saccade was not made. We varied the initial fixation location and found that the impairment specifically affected contraversive saccades rather than affecting all saccades made into head-centered contralateral space. Monkeys were impaired only slightly at making saccades to contralateral targets in the step task if the target duration was 1000 ms, such that the target was present during the saccade: latency increased, but increases in saccadic error were mild and increases in the frequency of trials in which a saccade was not made were insignificant. During FEF inactivation there usually was a direct correlation between the latency and the error of saccades made in response to contralateral targets. In the delay task, FEF inactivation increased the frequency of making premature saccades to ipsilateral targets. FEF inactivation had inconsistent and mild effects on saccadic peak velocity. FEF inactivation caused impairments in the ability to fixate lights steadily in contralateral space. FEF inactivation always caused an ipsiversive deviation of the eyes in darkness. In summary, our results suggest that the FEF plays major roles in (1) generating contraversive saccades to locations of extinguished or flashed targets, (2) maintaining contralateral fixations, and (3) suppressing inappropriate ipsiversive saccades.

Insights into Nonpilus Adhesin Functionality and the Molecular Determinants of Nontypeable Haemophilus influenzae Colonization

Bacterial colonization of the upper respiratory tract is the first step in the pathogenesis of nontypeable Haemophilus influenzae (NTHi) disease. Examination of the determinants of NTHi colonization process has been hampered by the lack of an appropriate animal model. To address this, we have developed a model of NTHi colonization in adult rhesus macaques that involves intranasal inoculation of 1x105 CFU and results in persistent colonization of the upper respiratory tract for at least three weeks with no signs of disease, mimicking asymptomatic colonization of humans. Using this model, we assessed the contributions to colonization of the HMW1 and HMW2 adhesive proteins. In competition experiments, the parent strain expressing both HMW1 and HMW2 was able to efficiently out-compete an isogenic mutant strain expressing neither HMW1 nor HMW2. In experiments involving inoculation of single isogenic derivatives of NTHi strain 12, the strains expressing HMW1 or HMW2 or both were able to colonize efficiently, while the strain expressing neither HMW1 nor HMW2 colonized inefficiently. Furthermore, colonization resulted in antibody production against HMW1 and HMW2 in one-third of the animals, demonstrating that colonization can be an immunizing event. In conclusion, we have established that NTHi is capable of colonizing the upper respiratory tract of rhesus macaques, in some cases associated with stimulation of an immune response. The HMW1 and HMW2 adhesive proteins play a major role in the process of colonization.

After establishing that the HMW1 and HMW2 proteins are colonization factors we further investigated the determinants of HMW1 function. HMW1 is encoded in the same genetic locus as two other proteins, HMW1B and HMW1C, with which HMW1 must interact in order to be functional. Interaction with HMW1C in the cytoplasm results in the glycosylation of HMW1. By employing homologues of HMW1C that glycosylate HMW1 in slightly different patterns we show that the pattern of modification is critical to HMW1 function. Structural analysis showed a change in protein structure when the pattern of HMW1 modification differed. We also identified two specific sites which must be glycosylated for HMW1 to function properly. These point mutations did not have a significant effect on protein structure, suggesting that glycosylation at those specific sites is instead necessary for interaction of HMW1 with its receptor. HMW1B is an outer membrane pore through which HMW1 is transported to reach the bacterial cell surface. We observed that HMW1 isolated from the cytoplasm has a different structure than HMW1 isolated from the bacterial cell surface. By forcing HMW1 to be secreted in a non-HMW1B dependent manner, we show that secretion alone is not sufficient for HMW1 to obtain a functional structure. This leads us to hypothesize that there is something specific in the interaction between HMW1 and HMW1B that aids in proper HMW1 folding.

The NTHi HMW1C glycosyltransferase mediates unconventional N-linked glycosylation of HMW1. In this system, HMW1 is modified in the cytoplasm by sequential transfer of hexose residues. To determine if this mechanism of N-linked glycosylation is employed by species other than NTHi, we examined Kingella kingae and Aggregatibacter aphrophilus homologues of HMW1C. We found both homologues to be functional glycosyltransferases and identified their substrates as the K. kingae Knh and the A. aphrophilus EmaA trimeric autotransporter proteins. LC-MS/MS analysis revealed multiple sites of N-linked glycosylation on Knh and EmaA. Without glycosylation, Knh and EmaA failed to facilitate wild type levels of bacterial autoaggregation or adherence to human epithelial cells, establishing that glycosylation is essential for proper protein function.

Decision-Making in the Primate Brain

Making decisions is fundamental to everything we do, yet it can be impaired in various disorders and conditions. While research into the neural basis of decision-making has flourished in recent years, many questions remain about how decisions are instantiated in the brain. Here we explored how primates make abstract decisions and decisions in social contexts, as well as one way to non-invasively modulate the brain circuits underlying decision-making. We used rhesus macaques as our model organism. First we probed numerical decision-making, a form of abstract decision-making. We demonstrated that monkeys are able to compare discrete ratios, choosing an array with a greater ratio of positive to negative stimuli, even when this array does not have a greater absolute number of positive stimuli. Monkeys’ performance in this task adhered to Weber’s law, indicating that monkeys—like humans—treat proportions as analog magnitudes. Next we showed that monkeys’ ordinal decisions are influenced by spatial associations; when trained to select the fourth stimulus from the bottom in a vertical array, they subsequently selected the fourth stimulus from the left—and not from the right—in a horizontal array. In other words, they begin enumerating from one side of space and not the other, mirroring the human tendency to associate numbers with space. These and other studies confirmed that monkeys’ numerical decision-making follows similar patterns to that of humans, making them a good model for investigations of the neurobiological basis of numerical decision-making.

We sought to develop a system for exploring the neuronal basis of the cognitive and behavioral effects observed following transcranial magnetic stimulation, a relatively new, non-invasive method of brain stimulation that may be used to treat clinical disorders. We completed a set of pilot studies applying offline low-frequency repetitive transcranial magnetic stimulation to the macaque posterior parietal cortex, which has been implicated in numerical processing, while subjects performed a numerical comparison and control color comparison task, and while electrophysiological activity was recorded from the stimulated region of cortex. We found tentative evidence in one paradigm that stimulation did selectively impair performance in the number task, causally implicating the posterior parietal cortex in numerical decisions. In another paradigm, however, we manipulated the subject’s reaching behavior but not her number or color comparison performance. We also found that stimulation produced variable changes in neuronal firing and local field potentials. Together these findings lay the groundwork for detailed investigations into how different parameters of transcranial magnetic stimulation can interact with cortical architecture to produce various cognitive and behavioral changes.

Finally, we explored how monkeys decide how to behave in competitive social interactions. In a zero-sum computer game in which two monkeys played as a shooter or a goalie during a hockey-like “penalty shot” scenario, we found that shooters developed complex movement trajectories so as to conceal their intentions from the goalies. Additionally, we found that neurons in the dorsolateral and dorsomedial prefrontal cortex played a role in generating this “deceptive” behavior. We conclude that these regions of prefrontal cortex form part of a circuit that guides decisions to make an individual less predictable to an opponent.