Disruption of centrifugal inhibition to olfactory bulb granule cells impairs olfactory discrimination

Abstract

Granule cells (GCs) are the most abundant inhibitory neuronal type in the olfactory bulb and play a critical role in olfactory processing. GCs regulate the activity of principal neurons, the mitral cells, through dendrodendritic synapses, shaping the olfactory bulb output to other brain regions. GC excitability is regulated precisely by intrinsic and extrinsic inputs, and this regulation is fundamental for odor discrimination. Here, we used channelrhodopsin to stimulate GABAergic axons from the basal forebrain selectively and show that this stimulation generates reliable inhibitory responses in GCs. Furthermore, selective in vivo inhibition of GABAergic neurons in the basal forebrain by targeted expression of designer receptors exclusively activated by designer drugs produced a reversible impairment in the discrimination of structurally similar odors, indicating an important role of these inhibitory afferents in olfactory processing.

Keywords: habituation; uncaging.

Fig. 1.

Selective labeling of GABAergic neurons of the HDB/MCPO reveals a profuse projection into the OB. (A) Confocal imaging of HDB/MCPO sections of a GAD65-Cre mouse immunostained against GAD65 (green) and GAD67 (red). GABAergic neurons express one of the markers exclusively (white arrowhead), but a few cells are positive for GAD65 and GAD67 (yellow, white arrow). (Right) After targeted injections, GABAergic neurons expressing ChR2 (red) are intermingled with cholinergic neurons stained for ChAT (green). (Scale bar: 25 μM.) (B) Confocal imaging of a sagittal section of the OB from a GAD65-Cre mouse showing extensive labeling of GABAergic fibers expressing ChR2 (B2, red) throughout the inner layers of the OB after injection of ChR2 virus into the HDB/MCPO (B1). (Scale bar: 500 μM.) (B3) The afferents mostly innervate the GC layer with only sparse fibers reaching beyond the MC layer to the external plexiform and glomerular layers (GL). (Scale bar: 100 μM.)

Fig. 2.

Stimulation of ChR2 expressed in HDB/MCPO GABAergic afferents inhibits GCs in the OB. (A) (Left) Diagram showing the recording array used; we recorded from GCs surrounded by ChR2⁺ GABAergic fibers and stimulated with blue light. (Center) Stimulation of GABAergic fibers in the vicinity of a recorded GC (50 pulses, 10 Hz) decreases the frequency of firing elicited by a depolarizing current stimulus in the MOB (16 pA, 10 s) (Upper) and in the AOB (24 pA, 15 s) (Lower). The raster plots represent the activity on three cells in each region, highlighting the reversible decrease in action potential frequency induced by LightStim. (Right) LightStim produced a frequency-dependent increase in inhibition of firing in GCs. Bar graph summarizing the reduction in GC firing produced by LightStim at 10 Hz (MOB, n = 7, *P < 0.004; AOB, n = 4, P < 0.02). (Scale bars: 1 s and 20 mV.) (B) (Upper Left) In voltage-clamp conditions, LightStim (10 Hz) elicited robust eIPSCs in a MOB GC. The Inset shows the expanded time axis; most light pulses produced a synchronized eIPSC. (Lower Left) In the same cell, the eIPSCs were not affected by the presence of blockers of glutamatergic transmission (CNQX, 10 µM; APV, 100 µM; LY367385 100 µM) but were completely abolished by the addition of TTX (0.5 µM). (Right) The integral under the area of the LightStim-induced eIPSCs at 10 Hz, or charge transfer (black), was not different in the presence of blockers (orange) but was completely abolished in the presence of TTX (gray bar; *P < 0.03).

Fig. 3.

GABA responses occur throughout the soma and dendritic tree of GCs. (A) Experimental setup used for GABA uncaging experiments. We recorded from the soma of GCs filled with Alexa 594 (red cell) while the slices were perfused with DPNI-GABA (2 mM) and photolysis was elicited by single-photon activation with a 405-nm laser. (B) GC filled with Alexa 594 (20 µM). Colored circles show representative uncaging spots: The blue circle is 10 µm from soma; the red circle indicates a control uncaging event 4 µm from the blue spot; the orange circle indicates a basal dendrite 28 µm from soma; and the green circle indicates a distal dendrite 99 µm from soma. (Scale bars: 10 µm.) (C) Scatter plot of the amplitude (Upper) and rise times (10–90%) (Lower) of laser-evoked GABA IPSC on GC dendrites, as a function of distance from soma, for AOB (n = 4, orange) and MOB (n = 4, black) (107 spots, 1,070 events). Larger IPSC amplitudes were observed within the proximity of the soma (∼30 µm), but the rise time did not vary significantly. (D) (Upper) Representative laser-evoked IPSCs at the specified colored spots shown in B (average of 10 traces per spot). The purple asterisk indicates the time of photolysis. (Scale bars: 20 pA and 100 ms.) (Lower) Bar graph comparing amplitude and kinetics of the representative photolysis-evoked IPSCs shown above. Note that the response evoked at 4 µm from the dendrite (red) has a significantly lower amplitude and larger rise time.

Fig. 4.

Disruption of inhibition from the HDB/MCPO affects odor discrimination. (Upper) A virus encoding for hM₄D_i was injected into the HDB/MCPO of GAD65-Cre mice. Four to five weeks later, animals received an i.p. injection of either PBS (control condition) or CNO (treated condition) and were tested for odor discrimination using the habituation/dishabituation paradigm or were tested for odor-detection threshold (SI Materials and Methods). (Lower) Before the CNO injection, mice habituated to C7 (grey bar) and showed a significant increase in investigation time in the presence of the dishabituated odor, C8 (red bar). Two hours after the CNO injection, mice habituated to C7 failed to show an increase in investigation time for C8. However, the investigation time was increased significantly for a pair of odors differing by two carbons (C6/C8). The inability to discriminate between C7 and C8 was recovered completely 4 h after CNO administration. *P < 0.05; **P < 0.02.