Molecular identity of periglomerular and short axon cells

Abstract

Within glomeruli, the initial sites of synaptic integration in the olfactory pathway, olfactory sensory axons terminate on dendrites of projection and juxtaglomerular (JG) neurons. JG cells form at least two major circuits: the classic intraglomerular circuit consisting of external tufted (ET) and periglomerular (PG) cells and an interglomerular circuit comprised of the long-range connections of short axon (SA) cells. We examined the projections and the synaptic inputs of identified JG cell chemotypes using mice expressing green fluorescent protein (GFP) driven by the promoter for glutamic acid decarboxylase (GAD) 65 kDa, 67 kDa, or tyrosine hydroxylase (TH). Virtually all (97%) TH+ cells are also GAD67+ and are thus DAergic-GABAergic neurons. Using a combination of retrograde tracing, whole-cell patch-clamp recording, and single-cell three-dimensional reconstruction, we show that different JG cell chemotypes contribute to distinct microcircuits within or between glomeruli. GAD65+ GABAergic PG cells ramify principally within one glomerulus and participate in uniglomerular circuits. DAergic-GABAergic cells have extensive interglomerular projections. DAergic-GABAergic SA cells comprise two subgroups. One subpopulation contacts 5-12 glomeruli and is referred to as "oligoglomerular." Approximately one-third of these oligoglomerular DAergic SA cells receive direct olfactory nerve (ON) synaptic input, and the remaining two-thirds receive input via a disynaptic ON-->ET-->SA circuit. The second population of DAergic-GABAergic SA cells also disynaptic ON input and connect tens to hundreds of glomeruli in an extensive "polyglomerular" network. Although DAergic JG cells have traditionally been considered PG cells, their interglomerular connections argue that they are more appropriately classified as SA cells.

Figure 1.

A, Venn diagram showing the heterogeneity in expression of GAD65, GAD67, and TH. Sphere area is proportional of the number of cells of each chemotype (∼300,000 GAD65+ cells, GAD67+ cells, and TH+ cells). B, Schematic showing the relationship between different PG/SA chemotype, morphotype, and synaptotypes. GABAergic (GAD65+) cells are functionally uniglomerular with 30% of cells receiving input via an ON→PG circuit (ONd) and the remaining 70% via an ON→ET→PG circuit (ETd). DAergic/GABAergic (GAD67+) cells exhibit oligoglomerular (contacting up to 15 glomeruli) and polyglomerular (contacting upwards of 30 glomeruli) morphotypes. Oligoglomerular cells are similar to GAD65+ cells with 30% of cells receiving input via an ON→PG circuit and 70% via an ON→ET→PG circuit. All polyglomerular cells receive input via the ON→ET→PG circuit.

Figure 2.

Retrograde labeling of interglomerular neurons. A, Fluorogold-labeled cells (red) at a glomerular injection site in a GAD67-GFP (green) transgenic mouse, immunostained for TH (blue). A small number of mitral cells located below the injection site are labeled. B, FG-labeled (red) and TH-labeled (blue) juxtaglomerular neurons (arrows) 500 μm distant to a glomerular injection. C, FG+ cell within the glomerular layer 400–500 μm distant to the FG injection site expressing GAD67 and TH (C¹ shows DAPI nuclear stain, C² shows FG, C³ shows GAD67-GFP, C⁴ shows TH, and C⁵ shows triple overlay). D, FG+ cell within the glomerular layer 400–500 μm distant to the injection site expressing GAD67 but not TH. E, FG+ cell within the glomerular layer 400–500 μm distant to the injection site negative for GAD67 and TH. F, Percentage of FG-labeled cells plotted as a function of distance from the injection site. The distribution of labeled cells was not significantly different from previous DiI and microbead mouse glomerular injections (data from Aungst et al., 2003). G, Proportion of FG-labeled cells containing FG only, FG and TH/GAD67, or FG and GAD67 as a function of distance from the injection site. ONL, Olfactory nerve layer; MCL, mitral cell layer; GCL, granule cell layer. Scale bars: A (in B), 20 μm; B, 10 μm; C–E (in C), 20 μm.

Figure 3.

TH+ cells differ in their sEPSCs and ON-evoked EPSC inputs. A, TH+GAD67+ cells exhibit single sEPSCs (left traces, green, ∼33% of cells) or bursts of sEPSCs (right traces, red, ∼67% of cells). The lower trace is an expansion of the EPSCs in the boxed region. B, Histogram of the probability of sEPSC bursting for 65 TH+GAD67+ cells (abscissa). Bursting cells are defined as observed sEPSC bursts exceeding chance by ≥2.5 SDs (dashed line; see Materials and Methods; single-sEPSC cells, green bars; burst-sEPSC cells, red bars). C, Superimposed traces of ON-evoked EPSCs in a single-EPSC cell (left trace, green, 15 sweeps) and a burst sEPSC cell (right trace, red, 15 sweeps). D, Histogram showing the number of EPSCs elicited by ON stimulation in 65 TH+GAD67+ cells. Regions of single- and burst-sEPSC cell overlap in the histogram are shown in yellow. E, Histogram showing latency to ON-evoked EPSCs in burst-sEPSC cells. Latency measured from the artifact generated by ON stimulation to the onset of the first evoked current evoked by minimum effective stimulation intensity. F, Histogram showing the jitter in response to ON-evoked EPSCs in burst-sEPSC cells.

Figure 4.

Biocytin filling of TH+GAD67+ cells in the glomerular layer. A, Neurons expressing GFP are easily visualized in the glomerular layer using an epifluorescent microscope. B, The same cell is readily visible in DIC optics allowing whole-cell patch-clamp recording and biocytin filling of identified neurons (patch pipette attached to GFP+ cell, arrow in A and B). C, D, Biocytin-filled cell processes from two different cells stained with NiDAB and glomerular cellular boundaries visualized with neutral red staining. Inset, Biocytin-filled cell dendrites in the absence of neutral red staining. Dotted lines indicate outline of glomerular neuropil.

Figure 5.

Three-dimensional reconstructions of biocytin-filled cells with individual glomeruli shown in different colors. A, GABAergic (GAD65+) uniglomerular ONd and ETd cells. B, DAergic/GABAergic (GAD67+) oligoglomerular ONd and ETd cells. R, rostral. Scale bar, 100 μm.

Figure 6.

Three-dimensional reconstructions of DAergic/GABAergic (GAD67+) polyglomerular cells with and without individual glomeruli shown in different colors. Scale bar, 100 μm.

Figure 7.

DAergic/GABAergic (GAD67+) neurons project greater distances and contact more glomeruli than GABAergic (GAD65+) cells. A, Percentage of biocytin-labeled GABAergic (GAD65+) or DAergic/GABAergic (GAD67+) cells as a function of the projection distance of their most distal process from the soma. B, Scatter plot showing the numbers of glomeruli contacted for each biocytin-filled cell. Uniglomerular, oligoglomerular, and polyglomerular groupings are indicated by the gray ellipses.

Figure 8.

Glomerular projections of GABAergic (GAD65+) and DAergic/GABAergic (GAD67+) cells. The percentage of processes entering each glomerulus was calculated for each cell and sorted in descending order with the primary, or highest percentage innervated, glomerulus demarcated as #1. The graph shows the population mean of process percentage within each glomerulus. A, GABAergic (GAD65+) ONd (left; n = 10) and ETd (right; n = 10) cells. B. DAergic/GABAergic (GAD67+) ONd (left; n = 10) and ETd (right; n = 10) oligoglomerular cells. C, DAergic/GABAergic (GAD67+) ETd polyglomerular cells (n = 5).