Kristina J. Nielsen

Targeting Single Neuronal Networks for Gene Expression and Cell Labeling In Vivo

To understand fine-scale structure and function of single mammalian neuronal networks, we developed and validated a strategy to genetically target and trace monosynaptic inputs to a single neuron in vitro and in vivo. The strategy independently targets a neuron and its presynaptic network for specific gene expression and fine-scale labeling, using single-cell electroporation of DNA to target infection and monosynaptic retrograde spread of a genetically modifiable rabies virus. The technique is highly reliable, with transsynaptic labeling occurring in every electroporated neuron infected by the virus. Targeting single neocortical neuronal networks in vivo, we found clusters of both spiny and aspiny neurons surrounding the electroporated neuron in each case, in addition to intricately labeled distal cortical and subcortical inputs. This technique, broadly applicable for probing and manipulating single neuronal networks with single-cell resolution in vivo, may help shed new light on fundamental mechanisms underlying circuit development and information processing by neuronal networks throughout the brain.

Object features used by humans and monkeys to identify rotated shapes

Humans and rhesus monkeys can identify shapes that have been rotated in the picture plane. Recognition of rotated shapes can be as efficient as recognition of upright shapes. Here we investigate whether subjects showing view-invariant performance use the same object features to identify upright and rotated versions of a shape. We find marked differences between humans and monkeys. While humans tend to use the same features independent of shape orientation, monkeys use unique features for each orientation. Humans are able to generalize to a greater degree across orientation changes than rhesus monkey observers, who tend to relearn separate problems at each orientation rather than flexibly apply previously learned knowledge to novel problems.

Orthogonal micro-organization of orientation and spatial frequency in primate primary visual cortex

Orientation and spatial frequency tuning are highly salient properties of neurons in primary visual cortex (V1). The combined organization of these particular tuning properties in the cortical space will strongly shape the V1 population response to different visual inputs, yet it is poorly understood. In this study, we used two-photon imaging in macaque monkey V1 to demonstrate the three-dimensional cell-by-cell layout of both spatial frequency and orientation tuning. We first found that spatial frequency tuning was organized into highly structured maps that remained consistent across the depth of layer II/III, similarly to orientation tuning. Next, we found that orientation and spatial frequency maps were intimately related at the fine spatial scale observed with two-photon imaging. Not only did the map gradients tend notably toward orthogonality, but they also co-varied negatively from cell to cell at the spatial scale of cortical columns.

Dissociation Between Local Field Potentials and Spiking Activity in Macaque Inferior Temporal Cortex Reveals Diagnosticity-Based Encoding of Complex Objects

Neurons in the inferior temporal (IT) cortex respond selectively to complex objects, and maintain their selectivity despite partial occlusion. However, relatively little is known about how the occlusion of different shape parts influences responses in the IT cortex. Here, we determine experimentally which parts of complex objects monkeys are relying on in a discrimination task. We then study the effect of occlusion of parts with different behavioral relevance on neural responses in the IT cortex at the level of spiking activity and local field potentials (LFPs). For both spiking activity and LFPs, we found that the diagnostic object parts, which were important for behavioral judgments, were preferentially represented in the IT cortex. Our data show that the effects of diagnosticity grew systematically stronger along a posterior–anterior axis for LFPs, but were evenly distributed for single units, suggesting that diagnosticity is first encoded in the posterior IT cortex. Our findings highlight the power of combined analysis of field potentials and spiking activity for mapping structure to computational function in the brain.

Eye movements of monkey observers viewing vocalizing conspecifics

Primates, including humans, communicate using facial expressions, vocalizations and often a combination of the two modalities. For humans, such bimodal integration is best exemplified by speech-reading - humans readily use facial cues to enhance speech comprehension, particularly in noisy environments. Studies of the eye movement patterns of human speech-readers have revealed, unexpectedly, that they predominantly fixate on the eye region of the face as opposed to the mouth. Here, we tested the evolutionary basis for such a behavioral strategy by examining the eye movements of rhesus monkeys observers as they viewed vocalizing conspecifics. Under a variety of listening conditions, we found that rhesus monkeys predominantly focused on the eye region versus the mouth and that fixations on the mouth were tightly correlated with the onset of mouth movements. These eye movement patterns of rhesus monkeys are strikingly similar to those reported for humans observing the visual components of speech. The data therefore suggest that the sensorimotor strategies underlying bimodal speech perception may have a homologous counterpart in a closely related primate ancestor.

Fixations in natural scenes: Interaction of image structure and image content

Explorative eye movements specifically target some parts of a scene while ignoring others. Here, we investigate how local image structure--defined by spatial frequency contrast--and informative image content--defined by higher order image statistics-are weighted for the selection of fixation points. We measured eye movements of macaque monkeys freely viewing a set of natural and manipulated images outside a particular task. To probe the effect of scene content, we locally introduced patches of pink noise into natural images, and to probe the interaction with image structure, we altered the contrast of the noise. We found that fixations specifically targeted the natural image parts and spared the uninformative noise patches. However, both increasing and decreasing the contrast of the noise attracted more fixations, and, in the extreme cases, compensated the effect of missing content. Introducing patches from another natural image led to similar results. In all paradigms tested, the interaction between scene structure and informative scene content was the same in any of the first six fixations on an image, demonstrating that the weighting of these factors is constant during viewing of an image. These results question theories, which suggest that initial fixations are driven by stimulus structure whereas later fixations are determined by informative scene content.

Nonlinearity of two-photon Ca2+ imaging yields distorted measurements of tuning for V1 neuronal populations

We studied the relative accuracy of drifting gratings and noise stimuli for functionally characterizing neural populations using two-photon calcium imaging. Calcium imaging has the potential to distort measurements due to nonlinearity in the conversion from spikes to observed fluorescence. We demonstrate a dramatic impact of fluorescence saturation on functional measurements in ferret V1 by showing that responses to drifting gratings strongly violate contrast invariance of orientation tuning, a fundamental property of the spike rates. The observed relationship is consistent with saturation that clips the high-contrast tuning curve peaks by ∼40%. The nonlinearity was also apparent in mouse V1 responses to drifting gratings, but not as strong as in the ferret. Contrast invariance holds, however, for tuning curves measured with a randomized grating stimulus. This finding is consistent with prior work showing that the linear portion of a linear-nonlinear system can be recovered with reverse correlation. Furthermore, we demonstrate that a noise stimulus is more effective at keeping spike rates in the linear operating regime of a saturating nonlinearity, which both maximizes signal-to-noise ratios and simplifies the recovery of fast spike dynamics from slow calcium transients. Finally, we uncover spatiotemporal receptive fields by removing the nonlinearity and slow calcium transient from a model of fluorescence generation, which allowed us to observe dynamic sharpening of orientation tuning. We conclude that for two-photon recordings it is imperative that one considers the nonlinear distortion when designing stimuli and interpreting results, especially in sensory areas, species, or cell types with high firing rates.

Efficient Receptive Field Tiling in Primate V1.

The primary visual cortex (V1) encodes a diverse set of visual features, including orientation, ocular dominance (OD), and spatial frequency (SF), whose joint organization must be precisely structured to optimize coverage within the retinotopic map. Prior experiments have only identified efficient coverage based on orthogonal maps. Here we used two-photon calcium imaging to reveal an alternative arrangement for OD and SF maps in macaque V1; their gradients run parallel but with unique spatial periods, whereby low-SF regions coincide with monocular regions. Next we mapped receptive fields and found surprisingly precise micro-retinotopy that yields a smaller point-image and requires more efficient inter-map geometry, thus underscoring the significance of map relationships. While smooth retinotopy is constraining, studies suggest that it improves both wiring economy and the V1 population code read downstream. Altogether, these data indicate that connectivity within V1 is finely tuned and precise at the level of individual neurons.

Building maps from maps in primary visual cortex.

Neurons in the visual system respond to more complex and holistic features at each new stage of processing. Often, these features are organized into continuous maps. Could there be a fundamental link between continuous maps and functional hierarchies? Here, we review recent studies regarding V1 maps providing some of the most noteworthy advances in our understanding of how and why maps exist. In particular, we focus on the common theme that some maps are inherited from the input of parallel pathways, which are then intimately linked to the emergence of new functional properties and their corresponding maps. These results on V1 maps may prove to be a unifying framework for hierarchical representations in the visual cortex.

Viral vector-based reversible neuronal inactivation and behavioral manipulation in the macaque monkey

Viral vectors are promising tools for the dissection of neural circuits. In principle, they can manipulate neurons at a level of specificity not otherwise achievable. While many studies have used viral vector-based approaches in the rodent brain, only a few have employed this technique in the non-human primate, despite the importance of this animal model for neuroscience research. Here, we report evidence that a viral vector-based approach can be used to manipulate a monkeys behavior in a task. For this purpose, we used the allatostatin receptor/allatostatin (AlstR/AL) system, which has previously been shown to allow inactivation of neurons in vivo. The AlstR was expressed in neurons in monkey V1 by injection of an adeno-associated virus 1 (AAV1) vector. Two monkeys were trained in a detection task, in which they had to make a saccade to a faint peripheral target. Injection of AL caused a retinotopic deficit in the detection task in one monkey. Specifically, the monkey showed marked impairment for detection targets placed at the visual field location represented at the virus injection site, but not for targets shown elsewhere. We confirmed that these deficits indeed were due to the interaction of AlstR and AL by injecting saline, or AL at a V1 location without AlstR expression. Post-mortem histology confirmed AlstR expression in this monkey. We failed to replicate the behavioral results in a second monkey, as AL injection did not impair the second monkeys performance in the detection task. However, post-mortem histology revealed a very low level of AlstR expression in this monkey. Our results demonstrate that viral vector-based approaches can produce effects strong enough to influence a monkeys performance in a behavioral task, supporting the further development of this approach for studying how neuronal circuits control complex behaviors in non-human primates.