Michael N. Economo

Optical Dissection of Odor Information Processing In Vivo Using GCaMPs Expressed in Specified Cell Types of the Olfactory Bulb

Understanding central processing requires precise monitoring of neural activity across populations of identified neurons in the intact brain. In the present study, we used recently optimized variants of the genetically encoded calcium sensor GCaMP (GCaMP3 and GCaMPG5G) to image activity among genetically and anatomically defined neuronal populations in the olfactory bulb (OB), including two types of GABAergic interneurons (periglomerular [PG] and short axon [SA] cells) and OB output neurons (mitral/tufted [MT] cells) projecting to the piriform cortex. We first established that changes in neuronal spiking can be related accurately to GCaMP fluorescence changes via a simple quantitative relationship over a large dynamic range. We next used in vivo two-photon imaging from individual neurons and epifluorescence signals reflecting population-level activity to investigate the spatiotemporal representation of odorants across these neuron types in anesthetized and awake mice. Under anesthesia, individual PG and SA cells showed temporally simple responses and little spontaneous activity, whereas MT cells were spontaneously active and showed diverse temporal responses. At the population level, response patterns of PG, SA, and MT cells were surprisingly similar to those imaged from sensory inputs, with shared odorant-specific topography across the dorsal OB and inhalation-coupled temporal dynamics. During wakefulness, PG and SA cell responses increased in magnitude but remained temporally simple, whereas those of MT cells changed to complex spatiotemporal patterns reflecting restricted excitation and widespread inhibition. These results suggest multiple circuit elements with distinct roles in transforming odor representations in the OB and provide a framework for further study of early olfactory processing using optical and genetic tools.

Spike Resonance Properties in Hippocampal O-LM Cells Are Dependent on Refractory Dynamics

During a wide variety of behaviors, hippocampal field potentials show significant power in the theta (4–12 Hz) frequency range and individual neurons commonly phase-lock with the 4–12 Hz field potential. The underlying cellular and network mechanisms that generate the theta rhythm, however, are poorly understood. Oriens-lacunosum moleculare (O-LM) interneurons have been implicated as crucial contributors to generating theta in local hippocampal circuits because of their unique axonal projections, slow synaptic kinetics and the fact that spikes are phase-locked to theta field potentials in vivo. We performed experiments in brain slice preparations from Long–Evans rats to investigate the ability of O-LM cells to generate phase-locked spike output in response to artificial synaptic inputs. We find that O-LM cells do not respond with any preference in spike output at theta frequencies when injected with broadband artificial synaptic inputs. However, when presented with frequency-modulated inputs, O-LM spike output shows the ability to phase-lock well to theta-modulated inputs, despite their strong low-pass profiles of subthreshold membrane impedance. This result was dependent on spike refractory dynamics and could be controlled by real-time manipulation of the postspike afterhyperpolarization. Finally, we show that the ability of O-LM cells to phase-lock well to theta-rich inputs is independent of the h-current, a membrane mechanism often implicated in the generation of theta frequency activity.

Development of Theta Rhythmicity in Entorhinal Stellate Cells of the Juvenile Rat

Mature stellate cells of the rat medial entorhinal cortex (EC), layer II, exhibit subthreshold membrane potential oscillations (MPOs) at theta frequencies (4-12 Hz) in vitro. We find that MPOs appear between postnatal days 14 (P14) and 18 (P18) but show little further change by day 28+ (P28-P32). To identify the factors responsible, we examined the electrical responses of developing stellate cells, paying attention to two currents thought necessary for mature oscillation: the h current I(h), which provides the slow rectification required for resonance; and a persistent sodium current I(NaP), which provides amplification of resonance. Responses to injected current revealed that P14 cells were often nonresonant with a relatively high resistance. Densities of I(h) and I(NaP) both rose by about 50% from P14 to P18. However, I(h) levels fell to intermediate values by P28+. Given the nonrobust trend in I(h) expression and a previously demonstrated potency of even low levels of I(h) to sustain oscillation, we propose that resonance and MPOs are limited at P14 more by low levels of I(NaP) than of I(h). The relative importance of I(NaP) for the development of MPOs is supported by simulations of a conductance-based model, which also suggest that general shunt conductance may influence the precise age when MPOs appear. In addition to our physiological study, we analyzed spine densities at P14, P18, and P28+ and found a vigorous synaptogenesis across the whole period. Our data predict that functions that rely on theta rhythmicity in the hippocampal network are limited until at least P18.

Dynamic Clamp: Alteration of Response Properties and Creation of Virtual Realities in Neurophysiology

### Introduction The vast repertoire of electrical activity displayed by neurons, cardiac myocytes, and various endocrine and sensory cells is the result of membrane-bound ion channels each producing a distinct conductance that facilitates current flux through the membrane. These conductances may

Imaging activity in astrocytes and neurons with genetically encoded calcium indicators following in utero electroporation

Complex interactions between networks of astrocytes and neurons are beginning to be appreciated, but remain poorly understood. Transgenic mice expressing fluorescent protein reporters of cellular activity, such as the GCaMP family of genetically encoded calcium indicators (GECIs), have been used to explore network behavior. However, in some cases, it may be desirable to use long-established rat models that closely mimic particular aspects of human conditions such as Parkinsons disease and the development of epilepsy following status epilepticus. Methods for expressing reporter proteins in the rat brain are relatively limited. Transgenic rat technologies exist but are fairly immature. Viral-mediated expression is robust but unstable, requires invasive injections, and only works well for fairly small genes (<5 kb). In utero electroporation (IUE) offers a valuable alternative. IUE is a proven method for transfecting populations of astrocytes and neurons in the rat brain without the strict limitations on transgene size. We built a toolset of IUE plasmids carrying GCaMP variants 3, 6s, or 6f driven by CAG and targeted to the cytosol or the plasma membrane. Because low baseline fluorescence of GCaMP can hinder identification of transfected cells, we included the option of co-expressing a cytosolic tdTomato protein. A binary system consisting of a plasmid carrying a piggyBac inverted terminal repeat (ITR)-flanked CAG-GCaMP-IRES-tdTomato cassette and a separate plasmid encoding for expression of piggyBac transposase was employed to stably express GCaMP and tdTomato. The plasmids were co-electroporated on embryonic days 13.5–14.5 and astrocytic and neuronal activity was subsequently imaged in acute or cultured brain slices prepared from the cortex or hippocampus. Large spontaneous transients were detected in slices obtained from rats of varying ages up to 127 days. In this report, we demonstrate the utility of this toolset for interrogating astrocytic and neuronal activity in the rat brain.

Control of Mitral/Tufted Cell Output by Selective Inhibition among Olfactory Bulb Glomeruli.

Inhibition is fundamental to information processing by neural circuits. In the olfactory bulb (OB), glomeruli are the functional units for odor information coding, but inhibition among glomeruli is poorly characterized. We used two-photon calcium imaging in anesthetized and awake mice to visualize both odorant-evoked excitation and suppression in OB output neurons (mitral and tufted, MT cells). MT cell response polarity mapped uniformly to discrete OB glomeruli, allowing us to analyze how inhibition shapes OB output relative to the glomerular map. Odorants elicited unique patterns of suppression in only a subset of glomeruli in which such suppression could be detected, and excited and suppressed glomeruli were spatially intermingled. Binary mixture experiments revealed that interglomerular inhibition could suppress excitatory mitral cell responses to odorants. These results reveal that inhibitory OB circuits nonlinearly transform odor representations and support a model of selective and nonrandom inhibition among glomerular ensembles.

GenNet: A Platform for Hybrid Network Experiments

We describe General Network (GenNet), a software plugin for the real time experimental interface (RTXI) dynamic clamp system that allows for straightforward and flexible implementation of hybrid network experiments. This extension to RTXI allows for hybrid networks that contain an arbitrary number of simulated and real neurons, significantly improving upon previous solutions that were limited, particularly by the number of cells supported. The benefits of this system include the ability to rapidly and easily set up and perform scalable experiments with hybrid networks and the ability to scan through ranges of parameters. We present instructions for installing, running and using GenNet for hybrid network experiments and provide several example uses of the system.

Membrane potential‐dependent integration of synaptic inputs in entorhinal stellate neurons

Stellate cells (SCs) of the medial entorhinal cortex exhibit robust spontaneous membrane‐potential oscillations (MPOs) in the theta (4–12 Hz) frequency band as well as theta‐frequency resonance in their membrane impedance spectra. Past experimental and modeling work suggests that these features may contribute to the phase‐locking of SCs to the entorhinal theta rhythm and may be important for forming the hexagonally tiled grid cell place fields exhibited by these neurons in vivo. Among the major biophysical mechanisms contributing to MPOs is a population of persistent (non‐inactivating or slowly inactivating) sodium channels. The resulting persistent sodium conductance (GNaP) gives rise to an apparent increase in input resistance as the cell approaches threshold. In this study, we used dynamic clamp to test the hypothesis that this increased input resistance gives rise to voltage‐dependent, and thus MPO phase‐dependent, changes in the amplitude of excitatory and inhibitory post‐synaptic potential (PSP) amplitudes. We find that PSP amplitude depends on membrane potential, exhibiting a 5–10% increase in amplitude per mV depolarization. The effect is larger than—and sums quasi‐linearly with—the effect of the synaptic driving force, V ‐ Esyn. Given that input‐driven MPOs 10 mV in amplitude are commonly observed in MEC stellate cells in vivo, this voltage‐ and phase‐dependent synaptic gain is large enough to modulate PSP amplitude by over 50% during theta‐frequency MPOs. Phase‐dependent synaptic gain may therefore impact the phase locking and phase precession of grid cells in vivo to ongoing network oscillations.

Targeted Path Scanning: An Emerging Method for Recording Fast Changing Network Dynamics across Large Distances

Attention is being increasingly focused on the dynamical behavior of large networks of neurons and astrocytes and the changes in these dynamics that occur during the progression of diseases like epilepsy. Recording from large numbers of identified cell types has been traditionally difficult, but the advent of fluorescent indicators capable of detecting changes in the internal calcium levels of cells has led to the ability to visually record the activity of large numbers of cells. However, for most imaging techniques the temporal resolution is sharply limited by the time it takes lasers to traverse the typical raster scan. As network dynamics can evolve quite rapidly, this is a serious limitation. The present paper describes the Targeted Path Scan technique, which dramatically increases the scanning frequency by allowing the user selection of trajectories through cells of interest. TPS is discussed in the context of a study of altered network dynamics in a common rat model of epilepsy. In this study, traveling waves of calcium transients that were frequently encountered in astrocytes imaged in brain slices obtained from control rats were dramatically reduced in astrocytes imaged in brain slices obtained from rats that had experienced status epilepticus. The speed of these traveling waves would have made them impossible to identify using traditional scanning techniques.

Using “Hard” Real-Time Dynamic Clamp to Study Cellular and Network Mechanisms of Synchronization in the Hippocampal Formation

We report on development and use of dynamic-clamp technology to understand how synchronous neuronal activity is generated in the hippocampus and entorhinal cortex. We find that “hard” real-time dynamic-clamp systems, characterized by very small maximal errors in timing of feedback, are necessary for cases in which fast voltage-gated channels are being mimicked in experiments. Using a hard real-time system to study cellular oscillations in entorhinal cortex, we demonstrate that the stochastic gating of persistent Na+ channels is necessary for cellular oscillations, and that cellular oscillations lead to dynamic changes in gain for conductance-based synaptic inputs. At the network level, we review experiments demonstrating that oscillating entorhinal stellate cells synchronize best via mutually excitatory interactions. Next, we show that cellular oscillations are volatile in the hypothesized “high-conductance” state, thought to occur in vivo, and suggest alternate means by which coherent activity can be generated in the absence of strong cellular oscillations. We close by discussing future developments that will increase the utility and widespread use of the dynamic-clamp method.