Development of fluorescence imaging probes for nicotinic acetylcholine α4ß2∗ receptors.

Nicotinic acetylcholine α4ß2∗ receptors (nAChRs) are implicated in various neurodegenerative diseases and smoking addiction. Imaging of brain high-affinity α4ß2∗ nAChRs at the cellular and subcellular levels would greatly enhance our understanding of their functional role. Since better resolution could be achieved with fluorescent probes, using our previously developed positron emission tomography (PET) imaging agent [18F]nifrolidine, we report here design, synthesis and evaluation of two fluorescent probes, nifrodansyl and nifrofam for imaging α4ß2∗ nAChRs. The nifrodansyl and nifrofam exhibited nanomolar affinities for the α4ß2∗ nAChRs in [3H]cytisine-radiolabeled rat brain slices. Nifrofam labeling was observed in α4ß2∗ nAChR-expressing HEK cells and was upregulated by nicotine exposure. Nifrofam co-labeled cell-surface α4ß2∗ nAChRs, labeled with antibodies specific for a ß2 subunit extracellular epitope indicating that nifrofam labels α4ß2∗ nAChR high-affinity binding sites. Mouse brain slices exhibited discrete binding of nifrofam in the auditory cortex showing promise for examining cellular distribution of α4ß2∗ nAChRs in brain regions.

Spectral breadth and laminar distribution of thalamocortical inputs to A1.

The GABAergic agonist muscimol is used to inactivate brain regions in order to reveal afferent inputs in isolation. However, muscimol's use in primary auditory cortex (A1) has been questioned on the grounds that it may unintentionally suppress thalamocortical inputs. We tested whether muscimol can preferentially suppress cortical, but not thalamocortical, circuits in urethane-anesthetized mice. We recorded tone-evoked current source density profiles to determine frequency receptive fields (RFs) for three current sinks: the "layer 4" sink (fastest onset, middle-layer sink) and current sinks 100 µm above ("layer 2/3") and 300 µm below ("layer 5/6") the main input. We first determined effects of muscimol dose (0.01-1 mM) on the characteristic frequency (CF) tone-evoked layer 4 sink. An "ideal" dose (100 µM) had no effect on CF-evoked sink onset latency or initial response but reduced peak amplitude by >80%, implying inhibition of intracortical, but not thalamocortical, activity. We extended the analysis to current sinks in layers 2/3 and 5/6 and for all three sinks determined RF breadth (quarter-octave steps, 20 dB above CF threshold). Muscimol reduced RF breadth 42% in layer 2/3 (from 2.4 ± 0.14 to 1.4 ± 0.11 octaves), 14% in layer 4 (2.2 ± 0.12 to 1.9 ± 0.10 octaves), and not at all in layer 5/6 (1.8 ± 0.10 to 1.7 ± 0.12 octaves). The results provide an estimate of the laminar and spectral extent of thalamocortical projections and support the hypothesis that intracortical pathways contribute to spectral integration in A1.

Tone-detection training enhances spectral integration mediated by intracortical pathways in primary auditory cortex.

Auditory-cued behavioral training can alter neural circuits in primary auditory cortex (A1), but the mechanisms and consequences of experience-dependent cortical plasticity are not fully understood. To address this issue, we trained adult rats to detect a 5 kHz target in order to receive a food reward. After 14 days training we identified three locations within A1: (i) the region representing the characteristic frequency (CF) 5 kHz, (ii) a nearby region with CF ∼10 kHz, and (iii) a more distant region with CF ∼20 kHz. In order to compare functional connectivity in A1 near to, vs. far from, the representation of the target frequency, we placed a 16-channel multiprobe in middle- (∼10 kHz) and high- (∼20 kHz) CF regions and obtained current-source density (CSD) profiles evoked by a range of tone stimuli (CF±1-3 octaves in quarter-octave steps). Our aim was to construct "CSD receptive fields" (CSD RFs) in order to determine the laminar and spectral profile of tone-evoked current sinks, and infer changes to thalamocortical and intracortical inputs. Behavioral training altered CSD RFs at the 10 kHz, but not 20 kHz, site relative to CSD RFs in untrained control animals. At the 10 kHz site, current sinks evoked by the target frequency were enhanced in layer 2/3, but the initial current sink in layer 4 was not altered. The results imply training-induced plasticity along intracortical pathways connecting the target representation with nearby cortical regions. Finally, we related behavioral performance (sensitivity index, d') to CSD responses in individual animals, and found a significant correlation between the development of d' over training and the amplitude of the target-evoked current sink in layer 2/3. The results suggest that plasticity along intracortical pathways is important for auditory learning.

Nicotinic neuromodulation in auditory cortex requires MAPK activation in thalamocortical and intracortical circuits.

Activation of nicotinic acetylcholine receptors (nAChRs) by systemic nicotine enhances sensory-cognitive function and sensory-evoked cortical responses. Although nAChRs mediate fast neurotransmission at many synapses in the nervous system, nicotinic regulation of cortical processing is neuromodulatory. To explore potential mechanisms of nicotinic neuromodulation, we examined whether intracellular signal transduction involving mitogen-activated protein kinase (MAPK) contributes to regulation of tone-evoked responses in primary auditory cortex (A1) in the mouse. Systemic nicotine enhanced characteristic frequency (CF) tone-evoked current-source density (CSD) profiles in A1, including the shortest-latency (presumed thalamocortical) current sink in layer 4 and longer-latency (presumed intracortical) sinks in layers 2-4, by increasing response amplitudes and decreasing response latencies. Microinjection of the MAPK kinase (MEK) inhibitor U0126 into the thalamus, targeting the auditory thalamocortical pathway, blocked the effect of nicotine on the initial (thalamocortical) CSD component but did not block enhancement of longer-latency (intracortical) responses. Conversely, microinjection of U0126 into supragranular layers of A1 blocked nicotine's effect on intracortical, but not thalamocortical, CSD components. Simultaneously with enhancement of CF-evoked responses, responses to spectrally distant (nonCF) stimuli were reduced, implying nicotinic "sharpening" of frequency receptive fields, an effect also blocked by MEK inhibition. Consistent with these physiological results, acoustic stimulation with nicotine produced immunolabel for activated MAPK in A1, primarily in layer 2/3 cell bodies. Immunolabel was blocked by intracortical microinjection of the nAChR antagonist dihydro-ß-erythroidine, but not methyllycaconitine, implicating α4ß2*, but not α7, nAChRs. Thus activation of MAPK in functionally distinct forebrain circuits--thalamocortical, local intracortical, and long-range intracortical--underlies nicotinic neuromodulation of A1.

Nicotine Enhances Amplitude and Consistency of Timing of Responses to Acoustic Trains in A1.

Systemic nicotine enhances neural processing in primary auditory cortex (A1) as determined using tone-evoked, current-source density (CSD) measurements. For example, nicotine enhances the characteristic frequency (CF)-evoked current sink in layer 4 of A1, increasing amplitude and decreasing latency. However, since presenting auditory stimuli within a stream of stimuli increases the complexity of response dynamics, we sought to determine the effects of nicotine on CSD responses to trains of CF stimuli (one-second trains at 2-40 Hz; each train repeated 25 times). CSD recordings were obtained using a 16-channel multiprobe inserted in A1 of urethane/xylazine-anesthetized mice, and analysis focused on two current sinks in the middle (layer 4) and deep (layers 5/6) layers. CF trains produced adaptation of the layer 4 response that was weak at 2 Hz, stronger at 5-10 Hz and complete at 20-40 Hz. In contrast, the layer 5/6 current sink exhibited less adaptation at 2-10 Hz, and simultaneously recorded auditory brainstem responses (ABRs) showed no adaptation even at 40 Hz. Systemic nicotine (2.1 mg/kg) enhanced layer 4 responses throughout the one-second stimulus train at rates ≤10 Hz. Nicotine enhanced both response amplitude within each train and the consistency of response timing across 25 trials. Nicotine did not alter the degree of adaptation over one-second trials, but its effect to increase amplitudes revealed a novel, slower form of adaptation that developed over multiple trials. Nicotine did not affect responses that were fully adapted (20-40 Hz trains), nor did nicotine affect any aspect of the layer 5/6 current sink or ABRs. The overall effect of nicotine in layer 4 was to enhance all responses within each train, to emphasize earlier trials across multiple trials, and to improve the consistency of timing across all trials. These effects may improve processing of complex acoustic streams, including speech, that contain information in the 2-10 Hz range.

Responses of mirror neurons in area F5 to hand and tool grasping observation.

Mirror neurons are a distinct class of neurons that discharge both during the execution of a motor act and during observation of the same or similar motor act performed by another individual. However, the extent to which mirror neurons coding a motor act with a specific goal (e.g., grasping) might also respond to the observation of a motor act having the same goal, but achieved with artificial effectors, is not yet established. In the present study, we addressed this issue by recording mirror neurons from the ventral premotor cortex (area F5) of two monkeys trained to grasp objects with pliers. Neuron activity was recorded during the observation and execution of grasping performed with the hand, with pliers and during observation of an experimenter spearing food with a stick. The results showed that virtually all neurons responding to the observation of hand grasping also responded to the observation of grasping with pliers and, many of them to the observation of spearing with a stick. However, the intensity and pattern of the response differed among conditions. Hand grasping observation determined the earliest and the strongest discharge, while pliers grasping and spearing observation triggered weaker responses at longer latencies. We conclude that F5 grasping mirror neurons respond to the observation of a family of stimuli leading to the same goal. However, the response pattern depends upon the similarity between the observed motor act and the one executed by the hand, the natural motor template.

Systemic Nicotine Increases Gain and Narrows Receptive Fields in A1 via Integrated Cortical and Subcortical Actions.

Nicotine enhances sensory and cognitive processing via actions at nicotinic acetylcholine receptors (nAChRs), yet the precise circuit- and systems-level mechanisms remain unclear. In sensory cortex, nicotinic modulation of receptive fields (RFs) provides a model to probe mechanisms by which nAChRs regulate cortical circuits. Here, we examine RF modulation in mouse primary auditory cortex (A1) using a novel electrophysiological approach: current-source density (CSD) analysis of responses to tone-in-notched-noise (TINN) acoustic stimuli. TINN stimuli consist of a tone at the characteristic frequency (CF) of the recording site embedded within a white noise stimulus filtered to create a spectral "notch" of variable width centered on CF. Systemic nicotine (2.1 mg/kg) enhanced responses to the CF tone and to narrow-notch stimuli, yet reduced the response to wider-notch stimuli, indicating increased response gain within a narrowed RF. Subsequent manipulations showed that modulation of cortical RFs by systemic nicotine reflected effects at several levels in the auditory pathway: nicotine suppressed responses in the auditory midbrain and thalamus, with suppression increasing with spectral distance from CF so that RFs became narrower, and facilitated responses in the thalamocortical pathway, while nicotinic actions within A1 further contributed to both suppression and facilitation. Thus, multiple effects of systemic nicotine integrate along the ascending auditory pathway. These actions at nAChRs in cortical and subcortical circuits, which mimic effects of auditory attention, likely contribute to nicotinic enhancement of sensory and cognitive processing.

Nicotinic filtering of sensory processing in auditory cortex.

Although it has been known for decades that the drug nicotine can improve cognitive function, the nature of its effects and the underlying mechanisms are not well understood. Nicotine activates nicotinic acetylcholine (ACh) receptors (nAChRs) that normally are activated by endogenous ACh, presumably "hijacking" the cholinergic contribution to multiple cognitive functions, notably attention. Thus, studying nicotine's effects helps to better understand a commonly used drug as well as functions of nAChRs. Moreover, nicotinic agonists are being developed to treat a variety of disorders that involve attention-related or age-related cognitive dysfunction. Studies have shown that nicotine can enhance processing of attended stimuli and/or reduce processing of distracters; that is, nicotine enhances attentional filtering. To examine potential mechanisms within sensory cortex that may contribute to cognitive functions, here we describe nicotinic actions in primary auditory cortex, where well-characterized neural "filters"-frequency receptive fields-can be exploited to examine nicotinic regulation of cortical processing. Using tone-evoked current-source density (CSD) profiles, we show that nicotine produces complex, layer-dependent effects on spectral and temporal processing that, broadly speaking, enhance responses to characteristic frequency (optimal) stimuli while simultaneously suppressing responses to spectrally distant stimuli. That is, nicotine appears to narrow receptive fields and enhances processing within the narrowed receptive field. Since basic cortical circuitry and nAChR distributions are similar across neocortex, these findings may generalize to neural processing in other sensory regions, and to non-sensory regions where afferent inputs are more difficult to manipulate experimentally. Similar effects across sensory and non-sensory cortical circuits could contribute to nicotinic enhancement of cognitive functions.

Null mutations in EphB receptors decrease sharpness of frequency tuning in primary auditory cortex.

Primary auditory cortex (A1) exhibits a tonotopic representation of characteristic frequency (CF). The receptive field properties of A1 neurons emerge from a combination of thalamic inputs and intracortical connections. However, the mechanisms that guide growth of these inputs during development and shape receptive field properties remain largely unknown. We previously showed that Eph family proteins help establish tonotopy in the auditory brainstem. Moreover, other studies have shown that these proteins shape topography in visual and somatosensory cortices. Here, we examined the contribution of Eph proteins to cortical organization of CF, response thresholds and sharpness of frequency tuning. We examined mice with null mutations in EphB2 and EphB3, as these mice show significant changes in auditory brainstem connectivity. We mapped A1 using local field potential recordings in adult EphB2(-/-);EphB3(-/-) and EphB3(-/-) mice, and in a central A1 location inserted a 16-channel probe to measure tone-evoked current-source density (CSD) profiles. Based on the shortest-latency current sink in the middle layers, which reflects putative thalamocortical input, we determined frequency receptive fields and sharpness of tuning (Q(20)) for each recording site. While both mutant mouse lines demonstrated increasing CF values from posterior to anterior A1 similar to wild type mice, we found that the double mutant mice had significantly lower Q(20) values than either EphB3(-/-) mice or wild type mice, indicating broader tuning. In addition, we found that the double mutants had significantly higher CF thresholds and longer onset latency at threshold than mice with wild type EphB2. These results demonstrate that EphB receptors influence auditory cortical responses, and suggest that EphB signaling has multiple functions in auditory system development.