Wide-field microscopy

Using wide-field microscopy we investigate how neuronal activity develops and propagrates across the cortex.

In order to examine functional connectivity across the cortex of mice, we use wide-field microscopy (WFM) to image neuronal activity in vivo across the entire dorsal cortex. WFM enables high-speed imaging over large fields of view and is therefore excellent for assessing spatiotemporal dynamics of neuronal activity across a large number of cortical regions. By parcellating the cortex into distinct brain regions we measure how neuronal activity emerges in each cortical region and how it subsequently spreads and propagates throughout the cortex. WFM is particularly useful for assessing how sensory input is processed by the cortex, how cortical dynamics evolve over the course of a variety of behavioral paradigms, and how spontaneous activity patterns develop as a measure of functional connectivity.

Cortical activity patterns under resting-state conditions show rapid, spontaneous patterns of neuronal activity that are often bilaterally symmetrical. Using these spontaneous activity patterns, functional connectivity between brain regions can be inferred from temporally correlated patterns of activity.

By combining WFM with resting-state imaging, sensory stimulation paradigms, or more complex behavioral tasks, our lab examines how the dynamics of neuronal activity across the cortex are shaped by the underlying neuronal circuit and synaptic architecture, and how these activity patterns play a role in driving behavior. By using WFM in combination with humanized mice, we study how human-specific genes impact the functional organization of cortical circuits. We also examine how cortical circuit function is altered in neurodevelopmental disease, such as autism, and how human-specific genes impact the phenotypic expression of these diseases.

Parcellation of the dorsal cortex according to the Allen Brain Atlas. Upon whisker stimulation, neuronal activity in cortical regions of the left and right hemisphere shows widespread and distinct cortical responses to sensory input.

Two-photon microscopy

We use two-photon microscopy to investigate neuronal response properties at a cellular level.

Increased synaptic density, cortico-cortical connectivity, and prolonged maturation, are considered hallmarks of human neurons and are believed to be critical for the remarkable cognitive capacity observed in humans. However, it remains unknown how such features affect neuronal activity and, consequently, the integration and processing of information. While WFM (see section above) provides a powerful approach for studying changes in neuronal responses at a cortex-wide level, it precludes studying neuronal responses and functional connectivity at a cellular level. We therefore employ in vivo two-photon microscopy to investigate at a cellular level how human-specific genes influence the response properties of cortical neurons.

Neuronal activity imaged in vivo in the barrel field of the somatosensory cortex using GCaMP6f as a reporter. Spontaneous neuronal activity of layer 2/3 pyramidal neurons can clearly be observed, while several neurons show time-locked activity to the moment a whisker stimulus is applied.

We use two-photon microscopy to examine how neurons respond to sensory input and how they encode information in a variety of behavioral tasks. Using optogenetic stimulation experiments we test how changes in connectivity, either local or long-range, modify neuronal response properties. This enables us to study how human-specific genes that play a role in brain development and function, impact how neurons process information.

neuronal response properties upon SRGAP2C expression

Our previous work has shown how expression of the human-specific gene SRGAP2C increases sensory coding of layer 2/3 pyramidal neurons in the barrel field of the somatosensory cortex. Expression of SRGAP2C increases the response probability to whisker stimulus (peaks in shaded areas) while reducing overall spontaneous activity (peaks outside shaded areas).

Neuronal circuit mapping

By employing a variety of tracing techniques we map structural connectivity throughout the brain.

The structural organization of neuronal circuits is a critical determinant of brain function. It is this wiring diagram that determines how information flows through the brain. Indeed, alterations in circuit connectivity can profoundly modify how the brain functions.

Synapses are the point of contact where inputs from other neurons (presynaptic) connect with the target neuron (postsynaptic). Changing how synapses develop can therefore have significant impact on how neurons connect with each other. Our previous work has shown that the human-specific gene (HSG) SRGAP2C uses this mechanism to modify cortical connectivity. By modifying synaptic development, SRGAP2C changes cortical circuits by selectively increasing feedforward and feedback cortical connectivity – projections that are considered critical for human cognition.

Using sparse in utero electroporation techniques we target individual neurons and visualize their morphology in high detail, up to individual synapses (e.g. dendritic spines as shown on the right). This enables us to determine whether the number and organization of inputs onto these neurons has changed, for example in response to impaired synaptic development or the expression of HSGs.

We made these discoveries by using a combination of in utero electroporation and viral transduction techniques that enables us to visualize individual neurons and subcellular features, such as dendritic morphology and synapses. This allows us to study if and how the organization of neuronal inputs has changed.

Sparse in utero electroporation and viral tracing techniques, such as monosynaptic rabies tracing, enables us to map the connectivity profile of individual neurons. Visualized here is the presynaptic connectivity (neurons in magenta) for a specific cortical neuron (expressing hGFP) in the mouse primary somatosensory cortex.

In addition, using a variety of tracing techniques, including whole-brain monosynaptic rabies tracing, we map connectivity across the entire brain and assess how HSGs alter the structural organization of neuronal circuits. These approaches also enable us to determine how impaired synaptic development, as observed in neurodevelopmental disease such as Autism Spectrum Disorder, alters the structural organization of neuronal circuits throughout the brain.

Using a method for whole-brain reconstruction and registration of every traced neuron onto the Allen Brain Atlas, we identify the anatomical location of each traced presynaptic neuron that connects with a given cortical neuron (starter neuron). This enables us to quantify how impaired synaptic development or the expression of HSGs modifies the organization of neuronal circuits across the brain.

Behavior

Using a variety of behavioral paradigms we study how changes in neuronal circuit structure and function impact behavior

A critical function of the brain is to integrate information from the environment with internal mental and physiological states. This enables the performance of meaningful actions in order to achieve a variety of goals. It is this producing of meaningful actions that we study as behavior.

Behavior depends on many different and highly complex brain functions – from processing of sensory input, to evaluating the value of these inputs, to setting goals and planning motor actions. And behavior itself ultimately changes the environment and internal states, leading to an ongoing and dynamic loop. Changes in the underlying structure and function of neuronal circuits can therefore directly alter behavior. The challenge, and ongoing quest in neuroscience, is to understand this relationship between brain structure, function, and behavior.

schematic of texture discrimination task

Mice are highly capable of discriminating textures with only their whiskers. Such a texture discrimination task can be made progressively harder by reducing the difference between textures, to a point where mice can no longer differentiate between them. Performance in this task requires multiple brain functions, including processing of sensory information. It also relies on mice learning the rule of this task, which can be cognitively demanding. Impaired or enhanced functioning of brain circuits that mediate these behaviors can therefore reduce or improve performance in this task.

Our lab uses a variety of behavioral paradigms to study how human-specific genes (HSGs) that modify brain structure and function impact behavioral outcome. For example, we have previously shown how humanizing mice for SRGAP2C expression modifies circuits involved in processing sensory information and enhances the ability of these mice to learn in a texture-discrimination task. How these changes in brain circuit and function lead to enhanced behavioral performance is a focus of ongoing investigation. Similarly, mouse models for Autism Spectrum Disorder (ASD) are known to display many behavioral deficits, from impaired sensory processing to disturbed social behavior. Unfortunately, we currently lack a full description of the structural and functional circuit changes that lead to these behavioral deficits. Moreover, HSGs that alter neuronal circuit architecture change the context in which genes implicated in ASD act. In order to better understand how ASD phenotypes arise in humans we therefore need to better understand the relationship between HSGs and the phenotypic expression of neurodevelopmental disorders, a topic our lab actively explores.

behavioral performance of humanized SRGAP2C mice

Humanized SRGAP2C mice perform better in a texture-discrimination task that was modified to be highly difficult for mice. This task requires cortical processing of sensory information and is cognitively demanding. While only 60% of wild-type mice learn this task over 50 sessions, almost all SRGAP2C mice are able to perform in this task.

Our work

Wide-field microscopy

Two-photon microscopy

Neuronal circuit mapping

Behavior