Alex C. Kwan

Activation of Specific Interneurons Improves V1 Feature Selectivity and Visual Perception

Inhibitory interneurons are essential components of the neural circuits underlying various brain functions. In the neocortex, a large diversity of GABA (γ-aminobutyric acid) interneurons has been identified on the basis of their morphology, molecular markers, biophysical properties and innervation pattern. However, how the activity of each subtype of interneurons contributes to sensory processing remains unclear. Here we show that optogenetic activation of parvalbumin-positive (PV+) interneurons in the mouse primary visual cortex (V1) sharpens neuronal feature selectivity and improves perceptual discrimination. Using multichannel recording with silicon probes and channelrhodopsin-2 (ChR2)-mediated optical activation, we found that increased spiking of PV+ interneurons markedly sharpened orientation tuning and enhanced direction selectivity of nearby neurons. These effects were caused by the activation of inhibitory neurons rather than a decreased spiking of excitatory neurons, as archaerhodopsin-3 (Arch)-mediated optical silencing of calcium/calmodulin-dependent protein kinase IIα (CAMKIIα)-positive excitatory neurons caused no significant change in V1 stimulus selectivity. Moreover, the improved selectivity specifically required PV+ neuron activation, as activating somatostatin or vasointestinal peptide interneurons had no significant effect. Notably, PV+ neuron activation in awake mice caused a significant improvement in their orientation discrimination, mirroring the sharpened V1 orientation tuning. Together, these results provide the first demonstration that visual coding and perception can be improved by increased spiking of a specific subtype of cortical inhibitory interneurons.

Fast modulation of visual perception by basal forebrain cholinergic neurons

The basal forebrain provides the primary source of cholinergic input to the cortex, and it has a crucial function in promoting wakefulness and arousal. However, whether rapid changes in basal forebrain neuron spiking in awake animals can dynamically influence sensory perception is unclear. Here we show that basal forebrain cholinergic neurons rapidly regulate cortical activity and visual perception in awake, behaving mice. Optogenetic activation of the cholinergic neurons or their V1 axon terminals improved performance of a visual discrimination task on a trial-by-trial basis. In V1, basal forebrain activation enhanced visual responses and desynchronized neuronal spiking; these changes could partly account for the behavioral improvement. Conversely, optogenetic basal forebrain inactivation decreased behavioral performance, synchronized cortical activity and impaired visual responses, indicating the importance of cholinergic activity in normal visual processing. These results underscore the causal role of basal forebrain cholinergic neurons in fast, bidirectional modulation of cortical processing and sensory perception.

Electrophysiological Characterization of V2a Interneurons and Their Locomotor-Related Activity in the Neonatal Mouse Spinal Cord

The V2a class of Chx10-expressing interneurons has been implicated in frequency-dependent control of left–right phase during locomotion in the mouse. We have used the Chx10::CFP mouse line to further investigate the properties and locomotion-related activity of V2a interneurons in the isolated neonatal spinal cord. V2a interneurons can be divided into three classes, based on their tonic, phasic, or delayed-onset responses to step depolarization. Electrical coupling is found only between neurons of same class and helps to synchronize neuronal activity within the class. Serotonin (5-HT) excites isolated tonic V2a interneurons by depolarizing the neurons and increasing their membrane input resistance, with no significant effects on action potential properties, a mechanism distinct from 5-HT excitation of commissural interneurons. During NMDA-/5-HT-induced locomotor-like activity, patch-clamp recordings and two-photon calcium imaging experiments show that approximately half of V2a interneurons fire rhythmically with ventral root-recorded motor activity; the rhythmic V2a interneurons fired during one half of the cycle, in phase with either the ipsilateral or the contralateral L2 ventral root bursts. The percentage of rhythmically firing V2a interneurons increases during higher-frequency fictive locomotion, and they become significantly more rhythmic in their firing during the locomotor cycle; this may help to explain the frequency-dependent shift in left–right coupling in Chx10::DTA mice, which lack these neurons. Our results together with data from the accompanying paper (Dougherty and Kiehn, 2009) reinforce earlier proposals that the V2a interneurons are components of the hindlimb central pattern generator, helping to organize left–right locomotor coordination in the neonatal mouse spinal cord.

Activity of Hb9 Interneurons during Fictive Locomotion in Mouse Spinal Cord

Hb9 interneurons (Hb9 INs) are putative components of the mouse spinal locomotor central pattern generator (CPG) and candidates for the rhythm-generating kernel. Studies in slices and hemisected spinal cords showed that Hb9 INs display TTX-resistant membrane potential oscillations, suggesting a role in rhythm generation. To further investigate the roles of Hb9 INs in the locomotor CPG, we used two-photon calcium imaging in the in vitro isolated whole neonatal mouse spinal cord preparation to record the activity of Hb9 INs, which were subsequently stained for unambiguous genetic identification. We elicited fictive locomotion by transmitter application or by electrically stimulating the caudal tip of the spinal cord. Although most Hb9 INs were rhythmically active during fictive locomotion, their activity was sparse and they failed to fire with each cycle of the episode. If Hb9 INs are the principal pacemakers of the CPG in the hemisegment in which they are located, they should direct the firing of motor neurons, with their activity preceding that of their ipsilateral segmental ventral roots. Instead, during each locomotor cycle, onset of Hb9 IN activity lagged behind the onset of the ipsilateral ventral root burst by a mean phase of 0.21 during electrical stimulation and 0.28 during transmitter application. Whole-cell recordings in intact and hemisected spinal cords confirmed the imaging results. Our data suggest that Hb9 INs participate in fictive locomotion, but the delayed onset of activity relative to ipsilateral motoneurons suggests that Hb9 INs are unlikely to be the sole intrasegmental rhythm-generating kernel of the CPG.

Polarized microtubule arrays in apical dendrites and axons

The polarization of microtubules within neurons in vivo is crucial in their role of determining the directions and speeds of intracellular transport. More than a decade ago, electron microscopy studies of mature hippocampal cultures indicated that their axons contained microtubules of uniform polarity and that dendrites contained microtubules of mixed polarity. Here, we evaluated polarity distributions in native brain tissues and in cultures by using multiphoton microscopy and second-harmonic generation from microtubules. We confirmed the expected polarized microtubule arrays in axons; however, we also unexpectedly found them ubiquitously in apical dendrites of mature hippocampal CA1 and cortical layer V pyramidal neurons. Some of these organized dendritic microtubule arrays extended for >270 μm with overall polarity of >80%. Our research indicates neurite-specific and age-dependent microtubule organizations that have direct implications for neuronal cargo transport.

Interneuron subtypes and orientation tuning

arising from B. V. Atallah, W. Bruns, M. Carandini & M. Scanziani 73, 159–170 (2012)10.1016/j.neuron.2011.12.013; N. R. Wilson, C. A. Runyan, F. L. Wang & M. Sur 488, 343–348 10.1038/nature11347(2012); S.-H. Lee et al. 488, 379–383 10.1038/nature11312(2012)Parvalbumin-positive (PV+) and somatostatin-positive (SST+) interneurons are two principal subtypes of cortical GABAergic neurons that differ in morphology, physiological properties and postsynaptic targeting. Although GABAergic inhibition is known to be crucial for shaping orientation tuning in the visual cortex, it is unclear whether PV+ and SST+ interneurons have different roles. Recently, Atallah et al., Wilson et al. and Lee et al. addressed this issue by optogenetically manipulating each interneuron subtype, reaching different conclusions: some investigators found that SST+ neuron activation sharpens orientation tuning, whereas PV+ neuron activation has little effect, but others found that activation of PV+ but not SST+ interneurons sharpens orientation tuning. To understand the cause for the discrepancy we examined the impact of several experimental differences among the studies—anaesthesia and the level and duration of optogenetic stimulation—and found that the discrepancies can be largely explained by differences in the level and duration of interneuron activation. There are replies to this Brief Communication Arising by Atallah, B. V., Scanziani, M. & Carandini, M. Nature 508, http://dx.doi.org/10.1038/nature13129 (2014) and El-Boustani, S., Wilson, N. R., Runyan, C. A. & Sur, M. Nature 508, http://dx.doi.org/10.1038/nature13130 (2014).

Dissection of Cortical Microcircuits by Single-Neuron Stimulation In Vivo

BACKGROUND A fundamental process underlying all brain functions is the propagation of spiking activity in networks of excitatory and inhibitory neurons. In the neocortex, although functional connections between pairs of neurons have been studied extensively in brain slices, they remain poorly characterized in vivo, where the high background activity, global brain states, and neuromodulation can powerfully influence synaptic transmission. To understand how spikes are transmitted in cortical circuits in vivo, we used two-photon calcium imaging to monitor ensemble activity and targeted patching to stimulate a single neuron in mouse visual cortex. RESULTS Burst spiking of a single pyramidal neuron can drive spiking activity in both excitatory and inhibitory neurons within a ∼100 μm radius. For inhibitory neurons, ∼30% of the somatostatin interneurons fire reliably in response to a presynaptic burst of ≥5 spikes. In contrast, parvalbumin interneurons showed no detectable responses to single-neuron stimulation, but their spiking is highly correlated with the local network activity. CONCLUSIONS Our results demonstrate the feasibility of mapping functional connectivity at cellular resolution in vivo and reveal distinct operations of two major inhibitory circuits, one detecting single-neuron spike bursts and the other reflecting distributed network activity.

Optical visualization of Alzheimer’s pathology via multiphoton-excited intrinsic fluorescence and second harmonic generation

Intrinsic optical emissions, such as autofluorescence and second harmonic generation (SHG), are potentially useful for functional fluorescence imaging and biomedical disease diagnosis for neurodegenerative diseases such as Alzheimers disease (AD). Here, using multiphoton and SHG microscopy, we identified sources of intrinsic emissions in ex vivo, acute brain slices from AD transgenic mouse models. We observed autofluorescence and SHG at senile plaques as well as characterized their emission spectra. The utility of intrinsic emissions was demonstrated by imaging senile plaque autofluorescence in conjunction with SHG from microtubule arrays to assess the polarity of microtubules near pathological lesions. Our results suggest that tissues from AD transgenic models contain distinct intrinsic emissions, which can provide valuable information about the disease mechanisms.

Fast and slow transitions in frontal ensemble activity during flexible sensorimotor behavior

The ability to shift between repetitive and goal-directed actions is a hallmark of cognitive control. Previous studies have reported that adaptive shifts in behavior are accompanied by changes of neural activity in frontal cortex. However, neural and behavioral adaptations can occur at multiple time scales, and their relationship remains poorly defined. Here we developed an adaptive sensorimotor decision-making task for head-fixed mice, requiring them to shift flexibly between multiple auditory–motor mappings. Two-photon calcium imaging of secondary motor cortex (M2) revealed different ensemble activity states for each mapping. When adapting to a conditional mapping, transitions in ensemble activity were abrupt and occurred before the recovery of behavioral performance. By contrast, gradual and delayed transitions accompanied shifts toward repetitive responding. These results demonstrate distinct ensemble signatures associated with the start versus end of sensory-guided behavior and suggest that M2 leads in engaging goal-directed response strategies that require sensorimotor associations.

Spatiotemporal Dynamics of Rhythmic Spinal Interneurons Measured With Two-Photon Calcium Imaging and Coherence Analysis

In rhythmic neural circuits, a neuron often fires action potentials with a constant phase to the rhythm, a timing relationship that can be functionally significant. To characterize these phase preferences in a large-scale, cell type-specific manner, we adapted multitaper coherence analysis for two-photon calcium imaging. Analysis of simulated data showed that coherence is a simple and robust measure of rhythmicity for calcium imaging data. When applied to the neonatal mouse hindlimb spinal locomotor network, the phase relationships between peak activity of >1,000 ventral spinal interneurons and motor output were characterized. Most interneurons showed rhythmic activity that was coherent and in phase with the ipsilateral motor output during fictive locomotion. The phase distributions of two genetically identified classes of interneurons were distinct from the ensemble population and from each other. There was no obvious spatial clustering of interneurons with similar phase preferences. Together, these results suggest that cell type, not neighboring neuron activity, is a better indicator of an interneurons response during fictive locomotion. The ability to measure the phase preferences of many neurons with cell type and spatial information should be widely applicable for studying other rhythmic neural circuits.