A Pool of Postnatally Generated Interneurons Persists in an Immature Stage in the Olfactory Bulb

Abstract

Calretinin (CR)-expressing periglomerular (PG) cells are the most abundant interneurons in the glomerular layer of the olfactory bulb. They are predominately generated postnatally from the septal and dorsal subventricular zones that continue producing them well into adulthood. Yet, little is known about their properties and functions. Using transgenic approaches and patch-clamp recording in mice of both sexes we show that CR(+) PG cells of both septal and dorsal origin have homogeneous morphological and electrophysiological properties. However, unlike other PG cells, these axonless neurons express a surprisingly small repertoire of voltage-activated channels and do not fire or fire at most a single and often small action potential. Moreover, they are not innervated by olfactory sensory neurons and receive little synaptic inputs from mitral or tufted cells at excitatory synapses where NMDA receptors predominate. These membrane and synaptic properties, that resemble those of newborn immature neurons not yet integrated in the network, persist over time and limit the recruitment of CR(+) PG cells by afferent inputs that strongly drive local network activity. Together, our results show that postnatally generated CR(+) PG cells continuously supply a large pool of neurons with unconventional properties. These data also question the contribution of CR(+) PG cells in olfactory bulb computation.SIGNIFICANCE STATEMENT Calretinin-expressing PG cells are by far the most abundant interneurons in the glomerular layer of the olfactory bulb. They are continuously produced during postnatal life, including adulthood, from neural stem cells located in the subventricular zones. Surprisingly, unlike other postnatally generated newborn neurons that quickly integrate into preexisting olfactory bulb networks, calretinin-expressing PG cells retain immature properties that limit their recruitment in local network activity for weeks, if not months, as if they would never fully mature. The function of this so far unsuspected pool of latent neurons is still unknown.

Keywords: calretinin; neurogenesis; olfactory bulb; periglomerular cell.

Figure 1.

Spatiotemporal origin and morphology of postnatally generated CR(+) PG interneurons. A, We modified a previously established targeted EPO approach for permanent labeling and fate mapping of neural stem cells that reside in defined microdomains of the postnatal SVZ. A plasmid expressing the Cre recombinase was electroporated in dorsal, lateral, and septal SVZ of Rosa^YFP Cre-reporter mice allowing the recombination and permanent expression of a YFP in neural stem cells and their progeny. The picture on the right shows the accumulation of YFP(+) cells in the olfactory bulb 45 d after a septal EPO. Scale bar, 100 μm. B, Quantification of the fraction of CR(+) PG interneurons among DCX(+)/YFP(+) PG cells at 1.5 and 3 months reveal their stable spatial origin over time. The number of cells expressing CR was negligible following lateral electroporation, and was not quantified. Scale bar, 5 μm. C, Long-term fate mapping was combined with immunodetection of BrdU and DCX to discriminate between cohorts of neurons sequentially produced at distinct postnatal times over a period of 1.5 months. Inset, Representative images of YFP(+) cells expressing BrdU or DCX. Scale bars, 5 μm. D, E, Quantification of YFP(+) PG cells born at early (BrdU−/DCX−), intermediate (BrdU+/DCX−) and late time (BrdU−/ DCX+) during the first 1.5 months of postnatal life reveals the temporal dynamic of neurogenesis from the septal and dorsal SVZ microdomains. Data were normalized in E for better clarity. F, Neurolucida reconstruction of YFP(+)/CR(+) PG interneurons originating from the dorsal and septal SVZ at 21 d post-EPO. G, Quantification of dendritic arborization length and number of nodes in CR(+) PG interneurons originating from the dorsal (red) and septal (green) SVZ. Values for CB(+) PG cells reconstructed at 21 d after dorsal EPO are given for comparison. Scale bars, 10 μm. *p < 0.05, **p < 0.01. H, Sholl analysis representing the number of dendrite intersections with concentric circles of gradually increasing radius. Cre, Cre recombinase; GL, glomerular layer; GCL, granule cell layer; RMS, rostral migratory stream.

Figure 2.

Labeling of a representative population of CR(+) PG interneurons in the CR::EGFP transgenic mice. A, Fate mapping of EGFP(+)/CR(+) PG interneurons from dorsal and septal origin by EPO of a tdTomato-expressing plasmid in CR::EGFP transgenic mice. Captions show marker expression by select cells: Tom(+)/CR(+) appear in orange/yellow, while those also expressing EGFP appear in pink. Scale bars: left, 500 μm; right, 50 μm. B, Proportion of CR(+) PG interneurons expressing EGFP following dorsal and septal EPO. Note that EGFP expression can be observed in both populations of CR(+) PG interneurons. C, Percentage of EGFP expression among the CR(+) PG population at different postnatal ages. D, Neurolucida reconstruction of EGFP(+) and EGFP(−) Tom(+)/CR(+) PG interneurons reveals comparable dendritic arborization length, surface and number of nodes. E, Sholl analysis representing the number and distribution of dendrite intersections with concentric circles of gradually increasing radius. F, Birth dating of CR(+) PG interneuron demonstrates stable EGFP expression in CR(+) PG interneurons up to 60 d after their generation. Scale bar, 10 μm.

Figure 3.

Intrinsic membrane properties of CR(+)/EGFP(+) PG cells. A, Voltage responses of three representative CR(+) PG cells to 500 ms long current steps. Cell 1 did not fire, Cell 2 fired a small non-overshooting action potential and Cell 3 fired a single overshooting action potential followed by a spikelet. Inset, Red traces are a zoom on the first suprathreshold response of Cells 2 and 3. B, Distribution histogram of action potential amplitudes across all the EGFP(+) PG cells tested. C, The amplitude of the action potential did not depend on the age of the mouse. D, Depolarizing voltage steps (5 mV/500 ms) induced fast inward Na⁺ currents and outward K⁺ currents (inset, higher-magnification). The hyperpolarizing prepulse (−40 mV from V_h = −75 mV) evoked an I_h current (arrow). E, Na⁺ currents were isolated in the presence of K⁺ and Ca²⁺ channel blockers (Cs, TEA, 4-AP, and Cd). F, Maximal Na⁺ current amplitudes recorded in CR(+)/EGFP(+) PG cells compared with those recorded in CR(−) PG cells in the same slices. Recordings were done as in E.

Figure 4.

Synaptic inputs of CR(+)/EGFP(+) PG cells. A, Paired recording between an EGFP(+)/CR(+) PG cell and a nonlabeled PG cell projecting into the same glomerulus. Fifteen traces are superimposed for each cell. The dashed line indicates OSN stimulation. B, Spontaneous IPSCs (sIPSCs recorded at V_h = 0 mV; top trace) and EPSCs (sEPSCs recorded at V_h = −75 mV; bottom trace) in a representative EGFP(+)/CR(+) PG cell. Right, Distribution histograms (bin of 1 Hz) of synaptic input frequencies showing the predominance of cells receiving few inputs. Bottom, Summary graphs of sEPSCs and sIPSCs frequencies in cells from mice at different developmental ages. Each dot represents a cell. C, OSN-evoked synaptic responses of an EGFP(+)/CR(+) PG cell recorded in the voltage-clamp mode at a positive holding potential (top traces), at the equilibrium potential for excitation (middle traces) or at a negative potential (middle traces), and in the current-clamp mode (bottom traces). Several traces are superimposed each time. Stimulation, 150 μA. D, Distant stimulations in the glomerular layer or in the EPL failed to evoke any EPSC in EGFP(+)/CR(+) PG cells clamped at V_h = −75 mV. In contrast, an outward IPSC was sometime evoked (traces marked with an asterisk). Traces are representative responses selected over >80 trials (stimulation intensity 500 μA). Inset, Several consecutive IPSCs evoked by a distant stimulation and recorded at V_h = 0 mV.

Figure 5.

Excitatory synapses are enriched with NMDA receptors in CR(+)/EGFP(+) PG cells. Comparison of AMPA/NMDA ratios at excitatory synapses in CR-EGFP(+) PG cells (top traces) and in Kv3.1-EYFP(+) PG cells (bottom traces). EPSCs were elicited by an electrical stimulation in the EPL and recorded at a positive holding potential in control conditions and in the presence of D-AP5 (50 μm). The NMDA component (gray trace) was obtained by subtracting the D-AP5 trace from the control trace. Summary bars show the average AMPA/NMDA ratio in CR(+)/EGFP(+) PG cells and in Kv3.1-EYFP(+) PG cells. Each dot represents a cell.

Figure 6.

Synaptic integration of CR(+)/EGFP(+) PG cells does not change over time. A, Current–voltage relationship of an EGFP(+) PG cell (top) colabeled with Tom (bottom), 63 d after EPO of a Tom-expressing plasmid in a CR::EGFP transgenic mouse. B, Distribution of action potential amplitudes in all the EGFP(+)/Tom(+) cells recorded 19–63 d post-EPO. C, Excitatory (left) and inhibitory (right) input frequencies in EGFP(+)/Tom(+) PG cells recorded at different intervals after EPO. Each dot represents a cell. Horizontal bars show average frequencies for cells recorded at 15–23 d or 54–63 d post-EPO.

Figure 7.

CR(+) PG cells are poorly recruited in vitro and in vivo. A, OSN-driven spiking of EGFP(+)/CR(+) PG cells in olfactory bulb slices. Two PG cells projecting into the same glomerulus, one EGFP(+) and one EGFP(−), were recorded in the loose-cell attached configuration. Their firing was elicited by an electrical stimulation of the OSNs. Raster plots from four pairs are shown with 20 consecutive sweeps for each pair. The EGFP(−) cell is on the left, the EGFP(+) on the right. A typical response is shown for Pair 1. OSNs were stimulated at t = 0, at an intensity of 30–200 μA (stimulation artifact has been blanked). The average evoked capacitive action current is shown on the right for each pair [green trace: EGFP(+) cell; black trace: EGFP(−) cell]. B, C, Summary plots of the results (n = 18 pairs). B, Average number of spikes evoked within the 200 ms following the stimulation. C, Amplitudes of the average capacitive currents evoked by the stimulation. D, c-Fos expression in PG interneuron subtypes (CB+, CR+, TH+) in activated glomeruli in vivo. Insert, Higher-magnifications of select region (boxes on overviews). The graph shows the proportion of each PG interneuron subtypes expressing c-Fos. Scale bars, 25 μm.

Figure 8.

stimulation of CR(+)/EGFP(+) PG cells does not generate self-inhibition. Ten millisecond depolarizing steps from V_h = −70 mV to V_h = 0 mV evoked an inward tail current that was blocked by the GABA_A receptor antagonist gabazine (GBZ; 5 μm) in Kv3.1-EYFP(+) PG cells. Traces are averages of 15 (control) and 20 trials (GBZ). In contrast, self-inhibition was not evoked in CR-EGFP(+) PG cells (average trace from 23 trials). Summary bars show the average GABA-mediated self-inhibition current (measured 10 ms after the end of the stimulation step) in CR(+)/EGFP(+) PG cells and in Kv3.1-EYFP(+) PG cells. Each dot represents a cell.