Optical dissection of odor information processing in vivo using GCaMPs expressed in specified cell types of the olfactory bulb

Abstract

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.

Figure 1.

Cre-dependent transgene expression in GAD2⁺, TH⁺, and PCdh21⁺ neurons in the OB. A–C, Labeling of OB neurons after crossing Cre driver lines with a reporter mouse line (left, “reporter”) or after injection of Cre-dependent virus into the OB (right, “virus”) for GAD2-Cre (A), TH-Cre (B), and PCdh21-Cre (C) mice. Top panels are overview images (bar,100 μm), middle panels show higher-magnification images in different OB layers (bar, 50 μm), and bottom panels show magnified images of the glomerular layer of reporter-driven (A,B) or virus-driven (C) GCaMP expression. Reporter lines are GCaMP3 (Ai38) for GAD2-Cre and TH-Cre and tdTomato (Ai9) for PCdh21-Cre. GL indicates glomerular layer; MC, mitral cell layer. Note that the expression in GAD2-Cre:reporter crosses (A) appeared brighter in granule cells but was still evident in PG cells (arrowheads). Expression in TH-Cre:reporter crosses included sparsely distributed neurons in the EPL and superficial granule cell layer (B), and expression in PCdh21-Cre:GCaMP3 reporter crosses included ORNs (C); these neurons did not show expression after virus injection. D, Expression of GCaMP after injection of Cre-dependent viral vector into the aPC of a PCdh21-Cre mouse (pseudocoloring in cyan to distinguish retrograde expression from direct expression in C). Expression is widespread throughout the OB (left) and occurs preferentially in mitral and deep tufted cells and their dendrites throughout the EPL (middle). Right: Axon terminals of GCaMP-expressing MT cells in piriform cortex. Note the lack of GCaMP-expressing somata in piriform.

Figure 2.

Immunohistochemical verification of cell-type-selective expression in OB neurons. A–E, Immunohistochemical staining of OB sections of GAD2-Cre:GCaMP3 reporter mice (A,C), TH-Cre:GCaMP3 reporter mice (B,D), and Cdrh1-Cre mice injected with GCaMP3-expressing virus into aPC (E). Images in A–D show anti-GFP enhanced GCaMP expression (green, left), immunostaining for Tbx21 (A,B,E), or TH (C,D) (red, middle) and an overlay of both channels including a magnified section of the glomerular layer (right). Bars indicate 100 and 50 μm for full and magnified views, respectively. Tbx21 antibody labels mitral and tufted cells but not GAD2⁺ or TH⁺ neurons expressing GCaMP (A,B). TH antibody labels only a minority of GCaMP-expressing juxtaglomerular neurons in GAD2-Cre:GCaMP3 reporter crosses (C), but labels most such neurons in TH-Cre:GCaMP3 reporter crosses (D). TH-Cre:GCaMP3 reporter crosses also show GCaMP3 expression in sparsely distributed infraglomerular neurons not labeled by TH antibody.

Figure 3.

Deriving quantitative relationships between action potential firing and GCaMP signals. Ai, In vitro whole-cell recordings were made from putative MT neurons expressing either GCaMP3 or GCaMP5G. Aii, Fluorescence transients corresponding to spike bursts elicited by square current steps in a GCaMP5G expressing MT neuron. Numbers indicate number of spikes evoked by each step. Aiii, Peak fluorescence amplitude in response to increasing numbers of spikes during a 500 ms current step. Dotted line in Aiii indicates the approximate threshold for detectable spike-related fluorescence transients. Inset is a magnification of the region around the threshold. Aiv, Minimum number of spikes occurring during the 500 ms window to elicit a detectable fluorescence response. The threshold for detecting spiking activity was lower when GCaMP5G was virally expressed in MT cells (6.2 ± 0.8; n = 5) than in piriform neurons (12.1 ± 3.8 spikes; n = 8) or in MT cells expressing reporter-driven GCaMP3 (14.2 ± 4.9 spikes; n = 8). Bi, Linearized fluorescence trace (red) and spike rate (black) calculated by optimizing the time constants τ_on and τ_off and the exponent a for the nonlinear fit during spike bursts (see text for details). Inset illustrates supralinear relationship between raw fluorescence and spike rate. Bii, Peak linearized fluorescence (adjusted after exponent derivation) increased approximately linearly in response to successively larger spike bursts. Biii, Linearized fluorescence waveform (red) and time-averaged spike rate (black) during irregular spiking (raw voltage trace is shown at bottom). Biv, Scatterplot of all linearized fluorescence values and corresponding spike rates for this entire trial. Ci, Linearized fluorescence (red) and spike rate (black) during a period of sustained high-frequency spiking resulting in saturation of the indicator (timing indicated by cyan shading). Cii, Scatterplot of fluorescence values as a function spike rate showing that the relationship between these quantities is disrupted for a period of time after saturation (indicated by cyan shaded region and points). D, Box plots comparing time constants τ_on (“Rise”) and τ_off (“Decay”) and the nonlinearity exponent a obtained for all cells (open circles) expressing GCaMP3 or GCaMP5G via virus or GCaMP3 reporter. The exponent was significantly lower for GCaMP3 reporter-expressing MT cells (asterisk). Ei, Simultaneous recordings of GCaMP5G fluorescence at the soma and two dendritic locations (circles) in a putative MT neuron. Spike bursts elicited by current injection at the soma elicit fluorescence transients that decay faster in dendritic compartments, even for locations adjacent to the soma (Eii).

Figure 4.

In vivo two-photon imaging of spontaneous and evoked activity in defined OB neurons. A, GCaMP signals imaged in vivo with two-photon microscopy after retrograde virus infection in PCdh21-Cre mice. Ai, Left: Resting fluorescence and pseudocolor overlay of odorant-evoked fluorescence change in the glomerular layer. Odorant stimulation evoked GCaMP signals restricted to a subset of glomeruli, the borders of which were demarcated by the tufts of MT cell primary dendrites. Middle: Time course of GCaMP fluorescence in two glomeruli from left image. Transients in lower trace are coupled to inhalation (timing not shown). Right: Resting fluorescence and pseudocolor overlay of evoked GCaMP signals in the external plexiform layer, showing odorant-specific activation of lateral dendritic processes. Aii, GCaMP signals imaged from mitral cell somata. Traces show GCaMP fluorescence imaged simultaneously from six mitral cells in one field of view. Neurons 1 and 2 did not respond to the two odorants tested, but showed coherent spontaneous signals; neurons 3–6 each responded to both odorants but with distinct, reproducible dynamics. Aiii, Pseudocolor plot of mitral cell-odorant-evoked GCaMP signals in 83 cell-odorant pairs (one pair per row, imaged from the soma) showing considerable heterogeneity in temporal dynamics relative to the timing of odorant presentation (each row is the average of four trials normalized to peak response and ordered by latency to peak. Aiv, Spontaneous GCaMP signals in neurons 1–3 from Aii, including the signals from the soma (purple) and proximal apical dendrite (pink) of neuron 1. Neurons 1 and 2 showed persistent spontaneous activity fluctuations that were nearly perfectly correlated. GCaMP fluorescence transients decayed more quickly in the dendritic segments compared with the soma of neuron 1. B, GCaMP signals imaged from the glomerular layer in GAD2-Cre:GCaMP3 reporter crosses. Bi, Resting fluorescence and pseudocolor overlay of odorant-evoked fluorescence change imaged at low magnification. Glomerular borders could not be identified from resting fluorescence images, but odorants evoked signals in odorant-specific foci resembling individual glomeruli. Bii, GCaMP fluorescence imaged at higher magnification and traces showing GCaMP signal imaged from six PG cell somata, all of which respond with similar dynamics to odorant presentation and show little or no spontaneous signals. Biii, Pseudocolor plot of odorant-evoked GCaMP signals in 138 PG cell-odorant pairs. In contrast to MT responses, PG cell responses were limited largely to the duration of odorant presentation. C, Resting and odorant-evoked GCaMP fluorescence imaged from the glomerular layer in TH-Cre:GCaMP3 reporter crosses. Ci, Resting and evoked GCaMP fluorescence imaged at low magnification, showing odorant-specific patterns of discrete signal foci resembling glomeruli. Cii, Higher-magnification resting fluorescence image and traces showing GCaMP signal from five SA cell somata in one field of view. The temporal dynamics of many cells within a given field of view tended to be similar, with little evidence of spontaneous activity. Ciii, Pseudocolor plot of odorant-evoked GCaMP signals in 97 SA cell-odorant pairs. Although all cells within a given field of view (demarcated by horizontal white lines) showed similar response dynamics, temporal patterns could vary across the population of imaged neurons. In a subset of these data (bottom), odorant presentation was 2 s instead of 4 s. Long-duration odorant responses were observed even for these short stimuli.

Figure 5.

Odorant-evoked response maps imaged from different ON neuron types share similar topography. A, Odorant-evoked response maps imaged in vivo from (left to right columns): ORNs (from OMP-spH mice), GAD2⁺ neurons, TH⁺ neurons, and PCdh21⁺ MT cells. All maps in the same column were imaged from the same animal, except when indicated by an asterisk. Odorant name and concentration are given at left. All maps are normalized to their own maximum; maximal ΔF values are given below each map. Maps were made using ΔF rather than ΔF/F for consistency with OMP-spH maps, in which evoked fluorescence changes are not correlated with resting fluorescence (Bozza et al., 2004). In addition to similar topography, all response maps shared features including a recruitment of additional foci and an increase in peak amplitude with increasing odorant concentration. SpH, GAD2⁺, and TH⁺ maps consisted of discrete glomerular foci and a relatively small diffuse component. PCdh21⁺ MT maps had a more prominent diffuse component, especially at higher odorant concentrations. B, Consensus topographies for response maps evoked by 2-hexanone (red), isopentylamine (blue), and butyric acid (green) overlaid on a reference bulb image. Lines indicate the 40% contour of response maps for each odorant averaged across multiple bulbs (n = 5–8 bulbs per cell type).

Figure 6.

Inhalation-linked dynamics of odorant responses are similar across OB neuron types. Response maps (left) and traces (right) showing calcium signals evoked in response to artificial inhalation of the odorant ethyl butyrate imaged from ORNs (A), GAD2⁺ neurons (B), TH⁺ neurons (C), and PCdh21⁺ MT cells (D). Traces show fluorescence from one caudal-lateral region of interest (1, red) and one anteromedial region of interest (2, cyan). Inhalation pulses (data not shown) were 150 ms in duration and were repeated at 2 Hz for ORNs (A) and 1 Hz for all postsynaptic neurons (B–D). E, Plots of difference (Δ) in latency relative to inhalation (left) or rise time (right; time from 10–90% of peak) between caudal-lateral and anteromedial glomeruli imaged from ORNs (n = 2 mice for latency, 3 for rise-time), GAD2⁺ neurons (n = 6), TH⁺ neurons (n = 5), and PCdh21⁺ MT cells (n = 6). See Materials and Methods for details. Error bars indicate SD. Circles in ORN column indicate values from each mouse and only include data not already reported in Spors et al. (2006). In all cases, the caudal-lateral glomerulus reaches peak levels earlier and decays faster than the anterior-medial glomerulus. The p value (paired t test) for comparison between anteromedial and caudal-lateral OB is shown above each bar. The calcium reporter was OGB-1 dextran for ORNs and GCaMP3 for all postsynaptic neurons. Traces are averages of four trials.

Figure 7.

OB neurons show robust odorant-driven and respiration-modulated responses in the awake mouse. Examples of spontaneous and odorant-evoked calcium signals imaged from ORNs using OGB-1 dextran (A), GAD2⁺ neurons (B), TH⁺ neurons (C), and PCdh21⁺ MT cells (D) in the awake, head-fixed mouse. Top trace shows respiration measured via intranasal pressure (A) or thermocouple (B–D), with inhalation up in all cases. Left traces show three successive odorant presentations (black bar) separated by 20–30 s; records are continuous in (B–D). Right traces show a single odorant presentation with expanded time-scale. In B–D, signals from two regions of interest in the same trial are shown. Robust inhalation-linked transients are apparent during odorant presentation in all cell types, although not in all regions of interest (see upper trace in B, for example). In all postsynaptic neuron types, bouts of high-frequency sniffing or a single strong sniff (arrowheads) drive fluorescence increases in the absence of odorant. Responses in ORNs, GAD2⁺, and TH⁺ neurons consist solely of fluorescence increases, whereas responses in PCdh21⁺ MT cells consist of dynamic sequences of brief increases and stronger and longer-lasting decreases, including “off” responses (asterisks). The calcium reporter for all postsynaptic neurons (B–D) is GCaMP5G.

Figure 8.

Complex spatiotemporal sequences of excitation and inhibition among MT cells revealed by GCaMP5 imaging in the awake mouse. Shown are fluorescence traces (bottom) and snapshots of odorant-evoked response maps (top) taken at different times during odorant presentation in an awake, head-fixed PCdh21⁺ mouse expressing GCaMP5G in MT cells. Images show maps of fluorescence changes relative to the preodor “baseline” taken from the indicated times. Each map is scaled so that zero ΔF is in the middle of the pseudocolor range (green). Fluorescence traces are averages of 6–12 pixels taken from the indicated regions and show distinct sequences of fluorescence decrease or increase. Images are taken from the average of four presentations aligned on odorant presentation and so do not reflect inhalation-driven transients.

Figure 9.

Differential effects of anesthesia on PG, SA, and MT cell odorant responses. A–C, Traces showing GCaMP5G fluorescence evoked by odorant presentation imaged from the same region of interest in an awake, head-fixed mouse (red trace) and, <5 min later, after brief application of isoflurane anesthesia (black trace). Isoflurane was removed ∼60 s before imaging. Top traces show respiration measured via intranasal thermocouple during wakefulness (upper) and anesthesia (lower). Traces are shifted slightly to be aligned on the first inhalation after odorant onset. Inhalation-evoked fluorescence increases in GAD2⁺ (A) and TH⁺ neurons (B) are attenuated after anesthesia, whereas strong fluorescence decreases in PCdh21⁺ MT cells are replaced by slow (but small) increases (C). C, Traces from two different regions of interest. Dashed line indicates preodor baseline. D–F, Effect of anesthesia depth on odorant-evoked GCaMP responses imaged in GAD2⁺ (D), TH⁺ (E), and PCdh21⁺ MT (F) cells. Plots show peak inhalation-evoked signals imaged under isoflurane concentrations ranging from 0–2% (0% levels were measured <60 s after adjusting the vaporizer and returned to 0.5% immediately after the imaging trials). Each plot shows mean ± SD responses imaged across one to six regions of interest in each preparation, with values normalized to those at 0.5% isoflurane. In all preparations, isoflurane was delivered via a tracheotomy tube and odorant was sampled (in air) via artificial inhalation (see Materials and Methods). Both GAD2⁺ (n = 4 mice) and TH⁺ responses (n = 5 mice) decrease with increasing isoflurane concentration, whereas PCdh21⁺ responses (n = 4 mice) increase. See text for statistical analysis.