Nicole K. Horst

Visualizing Synaptic Ribbons in the Living Cell

Visual and auditory information is encoded by sensory neurons that tonically release neurotransmitter at high rates. The synaptic ribbon is an essential organelle in nerve terminals of these neurons. Its precise function is unknown, but if the ribbon could be visualized in a living terminal, both its own dynamics and its relation to calcium and vesicle dynamics could be studied. We designed a short fluorescent peptide with affinity for a known binding domain of RIBEYE, a protein unique to the ribbon. When introduced via a whole-cell patch pipette, the peptide labeled structures at the presynaptic plasma membrane of ribbon-type terminals. The fluorescent spots match in size, location, number, and distribution the known features of synaptic ribbons. Furthermore, fluorescent spots mapped by confocal microscopy directly match the ribbons identified by electron microscopy in the same cell. Clearly the peptide binds to the synaptic ribbon, but even at saturating concentrations it affects neither the morphology of the ribbon nor its tethering of synaptic vesicles. It also does not inhibit exocytosis. Using the peptide label, we observed that the ribbon is immobile over minutes and that calcium influx is concentrated at the ribbon. Finally, we find that each ribbon in a retinal bipolar cell contains ∼4000 molecules of RIBEYE, indicating that it is the major component of the synaptic ribbon.

Hypocretin and Nicotine Excite the Same Thalamocortical Synapses in Prefrontal Cortex: Correlation with Improved Attention in Rat

Thalamic projections to prefrontal cortex are important for executive aspects of attention. Using two-photon imaging in prefrontal brain slices, we show that nicotine and the wakefulness neuropeptide hypocretin (orexin) excite the same identified synapses of the thalamocortical arousal pathway within the prefrontal cortex. Although it is known that attention can be improved when nicotine is infused directly into the midlayer of the prefrontal cortex in the rat, the effects of hypocretin on attention are not known. The overlap in thalamocortical synapses excited by hypocretin and nicotine and the lack of direct postsynaptic effects prompted us to compare their effects on a sustained and divided attention task in the rat. Similar to nicotine, infusions of hypocretin-2 peptide into the prefrontal cortex significantly improved accuracy under high attentional demand without effects on other performance measures. We show for the first time that hypocretin can improve attentional processes relevant to executive functions of the prefrontal cortex.

The role of rat dorsomedial prefrontal cortex in spatial working memory

We used an operant delayed spatial alternation task to examine the role of rat dorsomedial prefrontal cortex (dmPFC) in spatial working memory. The task was designed to restrict movements during the delay period to minimize use of motor-mediating strategies. Inactivation of dmPFC (muscimol) resulted in increased errors and increased the temporal variability of responding. Animals did not show perseveration after errors (i.e., responding again at the erroneous location). Under control conditions, the time between spatial responses was greater and more variable before errors as compared to correct responses. These effects were eliminated when muscimol was infused into dmPFC. Trial outcome also affected movement and delay times in the next trial. This effect was diminished with muscimol in dmPFC. By contrast, when muscimol was infused in dorsal agranular insular cortex (AId)-a region that is strongly interconnected with dorsomedial prefrontal regions-there was no effect on delayed spatial alternation performance. These experiments confirm that dmPFC is necessary for successful delayed spatial alternation and establish that there is a relationship between response time variability and trial outcome that depends on dorsomedial prefrontal function.

Working with memory: evidence for a role for the medial prefrontal cortex in performance monitoring during spatial delayed alternation.

Neuronal spike activity was recorded in the medial prefrontal cortex (mPFC) as rats performed an operant spatial delayed alternation task. The sensitivities of neurons to choice, outcome, and temporal information-related aspects of the task were examined. About one-third of neurons were sensitive to the location of delayed responding while animals were at one of two spatially distinct response ports. However, many fewer neurons (<10%) maintained choice information over the delay, each exhibiting persistent differences in firing rates for only a portion of the delay. Another third of cells encoded information about behavioral outcomes, and some of these neurons (>20% of all cells) fired at distinct rates in advance of correct and incorrect responses (i.e., prospective encoding of outcome). Other cells were sensitive to reward-related feedback stimuli (>20%), the outcome of the preceding trial (retrospective encoding, 5-10%), and/or the time since a trial was last performed (10-20%). An anatomical analysis of the recording sites found that cells that were sensitive to choice, temporal, and outcome information were commingled within the middle layers of the mPFC. Together, our results suggest that spatial processing is only part of what drives mPFC neurons to become active during spatial working memory tasks. We propose that the primary role of mPFC in these tasks is to monitor behavioral performance by encoding information about recent trial outcomes to guide expectations and responses on the current trial. By encoding these variables, the mPFC is able to exert control over action and ensure that tasks are performed effectively and efficiently.

Reward-related activity in the medial prefrontal cortex is driven by consumption

An emerging literature suggests that the medial prefrontal cortex (mPFC) is crucial for the ability to track behavioral outcomes over time and has a critical role in successful foraging. Here, we examine this issue by analyzing changes in neuronal spike activity and local field potentials in the rat mPFC in relation to the consumption of rewarding stimuli. Using multi-electrode recording methods, we simultaneously recorded from ensembles of neurons and field potentials in the mPFC during the performance of an operant-delayed alternation task and a variable-interval licking procedure. In both tasks, we found that consummatory behavior (licking) activates many mPFC neurons and is associated with theta-band phase locking by mPFC field potentials. Many neurons that were modulated by the delivery of reward were also modulated when rats emitted bouts of licks during the period of consumption. The majority of these licking-modulated neurons were found in the rostral part of the prelimbic cortex, a region that is heavily interconnected with the gustatory insular cortex and projects to subcortical feeding-related centers. Based on the tight coupling between spike activity, theta-band phase locking, and licking behavior, we suggest that reward-related activity in the mPFC is driven by consummatory behavior.

Reversible Inactivation of Rat Premotor Cortex Impairs Temporal Preparation, but not Inhibitory Control, During Simple Reaction-Time Performance.

Previous studies by our lab and others have established a role for medial areas of the prefrontal cortex (mPFC) in the top–down control of action during simple reaction-time (RT) tasks. However, the neural circuits that allow mPFC to influence activity in the motor system have remained unclear. In the present study, we used a combination of tract-tracing and reversible inactivation methods to examine the role of a motor-related area in the rat frontal cortex, called the rostral forelimb area (RFA), in the top–down control of action. Neural tracing studies involved used electrical microstimulation to identify RFA and injections of biotinylated dextran amines (BDA) to map out connections of RFA with other parts of the frontal cortex. Connections were found between RFA and mPFC, the agranular insular cortex, and the primary motor cortex. Reversible inactivations using muscimol infusions into RFA increased response times and eliminated delay-dependent speeding, but did not increase premature responding. These results are markedly different from what is obtained when muscimol is infused into mPFC, which leads to excessive premature responding and a reduction of RTs to stimuli at short delays (Narayanan et al., 2006). We also tested animals during the RT task after inactivating the agranular insular cortex, which contains neurons that projects to and receives from RFA and mPFC, and found no effects on RT performance. Together, these studies suggest that RFA is a premotor region in the rat frontal cortex that competes with mPFC to control action selection. We suggest that RFA controls the threshold that is used to initiate responding and generates prepotent excitation over responding that is crucial for temporal preparation.

Lost in Transition: Aging-Related Changes in Executive Control by the Medial Prefrontal Cortex

Neural correlates of aging in the medial prefrontal cortex (mPFC) were studied using an operant delayed response task. The task used blocks of trials with memory-guided (delayed alternation) and visually-guided (stimulus-response) responding. Older rats (24 months) performed at a slow pace compared with younger rats (6 months). They wasted time engaged in nonessential behaviors (e.g., licking on spouts beyond the period of reward delivery) and were slow to respond at the end of the delay period. Aged mPFC neurons showed normal spatial processing. They differed from neurons in younger rats by having reduced modulations by imperative stimuli indicating reward availability and reduced activity associated with response latencies for reward collection. Older rats showed reduced sensitivity to imperative stimuli at three levels of neural activity: reduced fractions of neurons with changes in firing rate around the stimulus, reduced correlation over neurons at the time of the stimulus as measured with analysis of population activity, and reduced amplitudes of event-related fluctuations in intracortical field potentials at the time of the imperative stimulus. Our findings suggest that aging alters the encoding of time-sensitive information and impairs the ability of prefrontal networks to keep subjects “on task.”

Oral nicotine consumption does not affect maternal care or early development in mice but results in modest hyperactivity in adolescence

Nicotine exposure during development can alter behavior in adulthood in mice. One route of nicotine administration that can mimic some of the dynamics of human smoking is administration of the drug to pregnant and nursing mice through the drinking water. It is critical to determine if nicotine administration has an impact on maternal behavior as such changes could lead to persistent behavioral alterations in the offspring, independent of the neuropharmacological effects of the drug. While a number of studies have detected nicotine exposure-induced changes, the effects of nicotine administration through the drinking water on maternal behavior in mice have not been examined comprehensively. In the current study we have compared maternal behaviors of C57BL/6J mice exposed to nicotine in the drinking water to behaviors of animals exposed to saccharin (vehicle) in the drinking water for the first 7days after birth of their litters and find no significant between-group differences in any behaviors measured except passive nursing. We have also assessed the effects of nicotine administration through the drinking water on postnatal weight gain of the pups and find no significant differences between groups. Open-field locomotor activity differences between exposed and unexposed offspring in adolescence were also assessed, with transient hyperactivity detected in nicotine-exposed mice. These data suggest that behavioral differences identified between animals exposed to nicotine through maternal drinking water administration are primarily due to the neuropharmacological effects of the drug and not due to effects of exposure on maternal behavior.

Regional inactivations of primate ventral prefrontal cortex reveal two distinct mechanisms underlying negative bias in decision making.

Significance Fear of negative outcomes has a powerful adverse influence on decision making in anxiety disorders. Although neuroimaging studies of patients with anxiety disorders have revealed dysregulation in numerous frontal brain regions including the orbitofrontal and ventrolateral prefrontal cortex, the causal involvement of this dysregulation is unknown. Here we demonstrate that in the marmoset monkey, inactivation of anterior orbitofrontal or ventrolateral prefrontal cortex increases negative bias in decision making via two distinct cognitive mechanisms—elevated uncertainty and attentional disruption, respectively. These findings provide, to our knowledge, the first direct evidence that dysregulation of distinct neurocognitive mechanisms within the prefrontal cortex may underlie the mixed etiology of anxiety disorders. Such insight will allow the development of more precise diagnostics and individually tailored therapeutic approaches. Dysregulation of the orbitofrontal and ventrolateral prefrontal cortices is implicated in anxiety and mood disorders, but the specific contributions of each region are unknown, including how they gate the impact of threat on decision making. To address this, the effects of GABAergic inactivation of these regions were studied in marmoset monkeys performing an instrumental approach–avoidance decision-making task that is sensitive to changes in anxiety. Inactivation of either region induced a negative bias away from punishment that could be ameliorated with anxiolytic treatment. However, whereas the effects of ventrolateral prefrontal cortex inactivation on punishment avoidance were seen immediately, those of orbitofrontal cortex inactivation were delayed and their expression was dependent upon an amygdala–anterior hippocampal circuit. We propose that these negative biases result from deficits in attentional control and punishment prediction, respectively, and that they provide the basis for understanding how distinct regional prefrontal dysregulation contributes to the heterogeneity of anxiety disorders with implications for cognitive-behavioral treatment strategies.

Impaired auditory discrimination learning following perinatal nicotine exposure or β2 nicotinic acetylcholine receptor subunit deletion

Maternal smoking during pregnancy can impair performance of the exposed offspring in tasks that require auditory stimulus processing and perception; however, the tobacco component(s) responsible for these effects and the underlying neurobiological mechanisms remain uncertain. In this study, we show that administration of nicotine during mouse perinatal development can impair performance in an auditory discrimination paradigm when the exposed animals are mature. This suggests that nicotine disrupts auditory pathways via nicotinic acetylcholine receptors (nAChRs) that are expressed at an early stage of development. We have also determined that mice which lack nAChRs containing the β2 subunit (β2* nAChRs) exhibit similarly compromised performance in this task, suggesting that β2* nAChRs are necessary for normal auditory discrimination or that β2* nAChRs play a critical role in development of the circuitry required for task performance. In contrast, no effect of perinatal nicotine exposure or β2 subunit knockout was found on the acquisition and performance of a differential reinforcement of low rate task. This suggests that the auditory discrimination impairments are not a consequence of a general deficit in learning and memory, but may be the result of compromised auditory stimulus processing in the nicotine-exposed and knockout animals.