ChR2 expression in AON neurons by virus injection
We began by investigating the properties of associational connections between the AON and the PC. The AON receives direct input from the OB and projects bilaterally to the PC (Haberly, 1998
). We characterized expression of ChR2 a few weeks after injection of adeno-associated virus (AAV2) carrying the gene for ChR2-EYFP into the AON of postnatal 5–7 day old rats. Strong expression of ChR2 in the soma and dendrites of AON neurons was observed, as well as in axons leaving the area (Figure ). Although retrograde axonal transport has been reported for certain types of AAV viruses (Mazarakis et al., 2001
; Zheng et al., 2010
), we detected no labeled mitral cells in the OB with the injection of AAV into the AON. Occasional accidental labeling of migrating newborn neurons in the rostral migratory stream (RMS), which is close to the AON, resulted in the labeling of a few newborn granule and periglomerular cells in the OB. These cells have only local inhibitory connections in the OB and no axonal projections to the olfactory cortex (Haberly, 1998
To confirm functional expression of ChR2, we obtained whole-cell patch-clamp recordings from AON neurons in acute slices. Neurons in the injected area showed characteristic inward currents when stimulated with blue light, with a rapid onset and partial inactivation (Figure ) (Boyden et al., 2005
; Ishizuka et al., 2006
; Wang et al., 2007
; Zhang et al., 2007
). In the recording illustrated in Figure (middle), most of the current recorded under voltage clamp at −70 mV was through ChR2 itself, since addition of TTX (to block evoked synaptic activity) diminished the peak and steady state responses by less than 20% (Figure , middle). In current clamp, light stimulation induced action potentials in ChR2-expressing cells, which could be reliably entrained at 10 Hz (Figure , right).
Anatomy of projections from the AON to the PC
Axons of virally infected AON neurons could be visualized by their EYFP fluorescence and were seen coursing into several cortical areas, including the olfactory tubercle and the PC (Figure ). Consistent with previous studies (Luskin and Price, 1983a
), ChR2-EYFP expressing axons were found in all layers of the PC except the LOT, with notably higher density in layer 1b (Figures and ). Unexpectedly, some EYFP-positive fibers were detected in layer 1a of the PC, which is thought to contain only inputs from the OB (Haberly, 1998
). These layer 1a fibers could be mitral cell axons accidentally labeled during AAV injection into the AON, for example via retrograde labeling of mitral cells. To test this possibility, we injected the same virus into the OB. Despite very strong expression of ChR2-EYFP in the injection area, very few mitral cells could be labeled and only a few fibers were detected in the LOT (Appendix
Figure ). This result indicated that AAV2/9 infects mitral cells with a very low probability that is not enough to account for the labeling found in layer 1a after AON injection. An additional point is that after infection by direct OB injection, mitral cell axons were clearly visible in the LOT and collaterals arose perpendicular to layer 1a (Appendix
Figure ). This is in contrast to the horizontal pattern of axonal innervation in layer 1a after AON injection, with no labeling in the LOT (Figure ). Since we detected no ChR2-EYFP positive mitral cells and no axon fibers projecting in the LOT after AAV injection into the AON, we concluded that no retrograde labeling of mitral cells occurred in our study and that the axons found in layer 1a were in fact from AON neurons. Interestingly, the presence of putative associational fibers in layer 1a has been noted in some early studies using classical Timm staining methods (Friedman and Price, 1984
Properties of light-evoked EPSCs at AON → PC synapses
We next characterized the functional properties of synapses made by axons of AON neurons using whole-cell patch recording from PC neurons (Figure ). At −70 mV, whole-field light stimulation evoked fast inward currents similar to those evoked by electrical stimulation of the LOT or associational fibers (Franks and Isaacson, 2005
) (Figure ). At +40 mV, where the magnesium block of NMDA receptors is relieved, outward currents had a slower time course (Figure ). Light-evoked responses were completely blocked by the sodium channel blocker TTX (1 μM) (Figures ), suggesting that the responses were synaptic. This was confirmed by the application of AMPA and NMDA receptor antagonists (10 μM, CNQX, and 25 μM, DL-AP5), which reduced light-evoked inward currents to 3.60 ± 0.14% (Figures ). These experiments indicate that light-evoked synaptic responses of the AON to PC projections were mediated by both AMPA and NMDA receptors.
Light-evoked responses of AON → aPC synapses had rise times that were similar to EPSCs evoked by electrical stimulation of associational fibers, but distinct from those of EPSCs evoked by LOT stimulation (association LED, 6.69 ± 0.65 ms, n
= 8 vs. association electrode, 5.70 ± 0.47 ms, n
= 8, p
= 0.24. Student's t
-test; LOT electrode, 8.21 ± 0.66 ms, n
= 9, p
< 0.01 for comparisons between association and LOT electrical stimulation by t
-test). The EPSC latency was longer with photostimulation than with electrical stimulation (association LED, 5.28 ± 0.36 ms, n
= 8; association electrode, 3.17 ± 0.18 ms, n
= 8; p
< 0.01, t
-test), presumably because of the time it takes to depolarize axons to threshold by current through ChR2. Paired-pulse stimulation of associational inputs with light revealed similar modulation as that seen with electrical stimulation (association electrode paired-pulse ratio, PPR = 0.85 ± 0.1, n
= 8; association LED PPR = 0.97 ± 0.06, n
= 8; 500 ms inter-stimulus interval). However, this result should be interpreted with caution since paired responses reflect ChR2 inactivation (Lin et al., 2009
) in addition to synaptic plasticity. Association inputs are known to be selectively suppressed by GABAB
receptor agonists (Tang and Hasselmo, 1994
), and accordingly we found that light evoked EPSCs were largely blocked by baclofen (11.6 ± 1.6%, Figure ).
Association inputs to pyramidal cells in the PC have a lower AMPA:NMDA ratio than inputs from the LOT (Franks and Isaacson, 2005
). Neurons were held at −70 mV to measure the contribution of AMPA current, and then at +40 mV to measure the mixture of both AMPA and NMDA currents triggered by electrical or light stimulation. The AMPA:NMDA ratio was operationally calculated from the peak amplitude at −70 mV for AMPA and the amplitude measured 50 ms after the response at +40 mV for NMDA, a time by which AMPA currents have decayed to zero (Poncer and Malinow, 2001
; Lee et al., 2010
). Consistent with previous reports (Franks and Isaacson, 2005
), the AMPA:NMDA ratio was significantly higher for synapses made by LOT axons than for associational synapses when stimulation was through electrodes (LOT electrode, 3.48 ± 0.69, n
= 9 vs. association electrode, 0.988 ± 0.48, n
= 4, p
< 0.01, t
-test). The ratio with photostimulation was similar to that with electrical stimulation of associational inputs (association LED, 0.987 ± 0.13, n
= 6 vs. association electrode, p
= 1.0, t
Our results indicate that selective stimulation of AON inputs by whole-field light stimulation evokes synaptic currents that are very similar to those evoked by electrical stimulation of associational inputs.
Monosynaptic inputs from the AON to the PC
The activation of ChR2-expressing axons projecting from the AON to PC often led to generation of action potentials in monosynaptically connected pyramidal cells. Therefore, EPSCs evoked by light stimulation may include not only monosynaptic inputs from the AON, but also disynaptic recurrent inputs from pyramidal cells in the PC, especially when picrotoxin was used to remove potential contamination from GABAergic synaptic currents (Figure ). Although these multisynaptic inputs could be prevented by the reduction of incident light intensity, a reduced intensity also decreased the amplitude of monosynaptic inputs, reducing the signal to noise ratio in the recordings. In addition, because of the variable shapes of the evoked events, we could not preclude polysynaptic contamination in our recordings. To overcome this uncertainty in isolating monosynaptic inputs, we used a previously described technique (Petreanu et al., 2007
; Cruikshank et al., 2010
) to depolarize ChR2-positive presynaptic terminals directly by blocking action potentials using TTX and potassium channels using 4-aminopyridine (4-AP, 100 μM). TTX prevents recurrent activity, and 4-AP is used to allow more efficient depolarization of axonal and presynaptic membranes.
Light-evoked synaptic responses in the presence of TTX and 4-AP qualitatively resembled those obtained in their absence (Figure ), with the peak amplitude of light-evoked EPSCs reduced to 50.6 ± 3.8% with TTX and 4-AP (n
= 3). The pharmacologically isolated monosynaptic input was blocked by AMPA and NMDA receptor antagonists (CNQX and APV) to 5.84 ± 0.54%, confirming that the AON projection to the PC was primarily glutamatergic. Additionally, GABAB
receptor agonists (baclofen) also blocked the EPSC to 20.5 ± 2.4% as expected from previous experiments using electrical stimulation of associational fibers. The synaptic response had longer latencies with TTX and 4-AP than without these blockers (Figure ). This longer latency is likely to be due to the fact that depolarization of the axons under normal conditions is aided by sodium channels, which have a much higher conductance than ChR2; the single channel conductance of ChR2 is ~1 pS (Lin et al., 2009
) while that of a sodium channel is 10–20 pS (Hille, 2001
). When sodium channels are blocked, it would take longer to depolarize the axonal membrane to the threshold for activation of voltage-dependent calcium channels. The AMPA:NMDA ratio of synaptic responses in TTX and 4-AP was not significantly different from control (0.987 ± 0.13, n
= 6 vs. 1.20 ± 0.12, n
= 17, p
> 0.2, t
-test, Figure ).
We next used this method of isolating monosynaptic responses to examine functional connectivity from the AON to PC.
AON connections on principal neurons in the aPC
The PC is generally divided into anterior and posterior parts, with potentially other subdivisions (Haberly, 1998
) (Figure ). We first examined connections from AON to the aPC. The principal neurons in the aPC are heterogeneous in terms of their structural and functional properties. Historically, they have been divided into three classes: superficial pyramidal (SP) cells, deep pyramidal (DP) cells, and semilunar (SL) cells (Haberly, 1998
). Recent studies have determined that the neuronal cell types may lie along a continuum, with the most superficial cells belonging to the SL class, but many properties gradually changing with depth (Suzuki and Bekkers, 2011
; Wiegand et al., 2011
). These studies also noted that SL and SP cells may differ in their synaptic inputs using electrical stimuli that did not separate the different sources (Suzuki and Bekkers, 2006
). Here, we characterize specific synaptic inputs from the AON to SP and SL cells.
We first recorded from putative superficial pyramidal cells, based on their morphology and input resistance (129.5 ± 11.4 MΩ, n
= 18) (Figure ). Pyramidal cells exhibited AMPA and NMDA receptor mediated EPSCs with light stimulation in the presence of TTX and 4-AP, indicating that they receive monosynaptic input from AON neurons (Figure ). We next identified SL cells based on their location, morphology, and higher input resistance (163.7 ± 20.3 MΩ, n
= 7). Morphologically, we chose to characterize cells as SL only if they lacked basal dendrites (Figure ), even though some cells of this class can possess basal dendrites (Wiegand et al., 2011
). The use of TTX to obtain monosynaptic light-evoked responses precluded the characterization of firing patterns. Light-evoked EPSCs recorded in SL cells at −70 mV were very small and often difficult to detect even when neighboring pyramidal cells exhibited large amplitude EPSCs (Figure ). The average amplitude was 64.2 ± 15 pA for SP cells (n
= 18) and only 11.0 ± 3.0 pA for SL cells (n
= 7, p
< 0.01, t
-test). To account for the variability across injections that could lead to different infection rates and expression levels, we compared each of the 7 SL cells with a corresponding SP cell that was recorded in the same slice (Figure ; there are nine points since two SL cells were paired with two SP cells each). This analysis confirmed that SL cells receive significantly smaller AMPA inputs from AON than SP cells. Interestingly, the average amplitude of light-evoked AMPA EPSCs in DP cells was not significantly larger than that of SP cells (DP, 136 ± 86 pA, n
= 3 vs. SP, 64.2 ± 15 pA, n
= 18, p
= 0.50, t
-test), perhaps due to the small sample size for DP cells. Unexpectedly, robust EPSCs could be recorded at +40 mV in SL cells, even though AMPA responses at −70 mV were weak (Figures ). Consequently, the AMPA:NMDA ratio was significantly smaller in SL cells (0.447 ± 0.15, n
= 7) than in SP or DP cells (SL vs. SP, 1.28 ± 0.14, n
= 18, p
< 0.01, t
-test, and SL vs. DP, 1.18 ± 0.23, n
= 3, p
< 0.01, t
-test). Pairwise comparison of SP and SL cells from matched slices ruled out the possibility that this difference was due to variability in expression of ChR2 across experiments (Figure ). The lower AMPA:NMDA ratio may be a general property of all associational synapses made on SL cells, since this was also observed when using electrical stimulation to activate associational fibers more indiscriminately (0.660 ± 0.14).
Figure 3 Properties of monosynaptic input from the AON to aPC. (A) Monosynaptic inputs to three types of principal neurons in the aPC. Light evoked monosynaptic EPSCs in SP, superficial pyramidal; DP, deep pyramidal; and SL, semilunar cells. Images show representative (more ...)
These results indicate that AON projections make strong functional synaptic contacts on pyramidal cells and weaker connections to SL cells. Interestingly, the NMDA component of AON inputs to SL cells may become important when SL cells are depolarized by direct input from the OB. The AMPA:NMDA ratios are also significantly different in the two classes of synapses.
Connections from the AON to the pPC
There is some uncertainty about the extent of AON projections to the pPC (Haberly and Price, 1978
; Luskin and Price, 1983a
; Brunjes et al., 2005
). We could readily identify ChR2-EYFP labeled axons in the ipsilateral pPC when virus particles were injected into the AON (Figure ). The distribution of axons was similar to that in the aPC, but the overall fluorescence intensity in the pPC was less than 50% of that in the aPC (Figures ). Since AON axons in the pPC are more distal than those in the aPC, it is possible that the amount of ChR2-EYFP is substantially reduced, accounting for the dimmer signals. Allowing more time for transport of protein (3 months post injection) did not lead to greater labeling in pPC. Distance alone is unlikely to account for decreased intensity in pPC since AON projections to the contralateral OB, which is at least as far (Haberly and Price, 1978
; Brunjes et al., 2005
), had an intensity 63.4% of that in ipsilateral aPC. This high fluorescence intensity was observed even within two weeks of injection, indicating that this protein is adequately transported to distal axons. Finally, we obtained higher resolution images of the aPC and the pPC using confocal microscopy under identical conditions. We then quantified the fluorescence intensity of individual axons and varicosities in the two regions. The average fluorescence intensity of EYFP (an indicator of the amount of ChR2-EYFP in the axons) at the single axon/varicosity level was not different in the aPC and pPC (100 ± 2.9% vs. 95.2 ± 3.3%, p
> 0.2, t
= 400 boutons each; Figure ). We also measured the number of fluorescent pixels that crossed an intensity threshold within standardized regions of interest in the two regions, and found that this value is lower in the pPC than in the aPC. These data indicate that the AON does send projections to the pPC, but the density of projections is lower than that in the aPC.
Figure 4 Properties of AON projection to pPC. (A,B) Axons from the AON can be seen projecting to the ipsilateral aPC and pPC. Fluorescence intensity was high in layer 1b where most of associational fibers are found. The intensity was lower in the pPC, but the (more ...)
To characterize functional connections from the AON to the pPC, we obtained whole-cell recordings from neurons in the pyramidal cell layer. Light-evoked synaptic currents were recorded as before. Large synaptic currents were obtained only rarely (Figure , top), and most recordings yielded very small or no currents (Figure , bottom). The probability of recording functional connections from the AON was 0.22 (two out of nine cells) for pPC neurons, which is significantly (p < 0.001 Fisher's exact test) lower than that for aPC (0.84, 54 out of 64 cells) (Figure ).
These data indicate that although AON axons reach the pPC, their projection density as well as their connection probability is much lower than for the aPC.
Interhemispheric associational connections
Another set of associational connections whose functional properties have not been selectively studied are the interhemispheric projections. Optogenetic methods offer an unprecedented ability to examine functional connections made by such long-distance projections from identified neurons (Petreanu et al., 2007
). We were able to visualize the projections from AON to the contralateral olfactory cortical areas. Light-evoked synaptic currents could be recorded from contralateral neurons (Figures ), but the probability of finding connections was much lower than the ipsilateral connections from AON to aPC (0.38 vs. 0.84, p
< 0.01 Fisher's exact test, n
= 11 out of 29 cells, and 54 out of 64 cells respectively). The average amplitude of AMPA currents recorded in matched experiments was also significantly smaller in contralateral AON compared to ipsilateral aPC (15.4 ± 4.06 pA, n
= 23 cells vs. 72.9 ± 16.7 pA, n
= 14 cells, p
< 0.001, t
-test). Ipsilateral AON to AON connections could not be studied in detail because of the significant contamination of ChR2 currents.
Figure 5 Contralateral projection from AON. (A,B) ChR2-EYFP signal was readily observed in the contralateral AON. Light-evoked synaptic currents were recorded in the contralateral AON neuron (B), but the probability of finding a connection was only 0.38. (C) Axonal (more ...)
ChR2-EYFP positive fibers were mainly distributed in layer 1b in the contralateral aPC, (Figure ), and the intensity was less than 50% of that in layer 1b in the ipsilateral aPC. This reduction was due to reduced density of labeled axons and not reduced levels of ChR2-EYFP in single axons (contralateral axon intensity was 92 ± 3.3% that of ipsilateral intensity, p > 0.2, t-test). Comparing the projection pattern from the AON to ipsilateral and contralateral aPC revealed an interesting difference (Figure ). While AON axons were found in layer 1a in the ipsilateral aPC, there were few fibers in contralateral aPC. Similarly the relative density of AON axons in layer 2/3 (compared to density in layer 1b) was lower in the contralateral side (Figure ). This resulted in a very clear band of projection in layer 1b on the contralateral side.
Whole-cell recordings revealed low probability of finding functional connections from the AON to contralateral aPC (Figures ), with only 4 out of 11 cells showing responses above noise. This proportion is significantly lower than that for ipsilateral aPC cells in matched recordings (12 out of 14 connections, p < 0.02 Fisher's exact test). Intriguingly, the AMPA:NMDA ratio was larger for contralateral projections (4.1 ± 0.68) than for ipsilateral ones (0.5 ± 0.09, p < 0.01, t-test). The average amplitude of contralateral synaptic currents was also lower than the ipsilateral ones (21.32 ± 8.12 pA vs. 72.9 ± 16.96 pA, p < 0.02, t-test).
These data suggest that ipsilateral and contralateral projections from the AON have different connection properties in the aPC.
Intrinsic connections within the PC
We next examined the properties of recurrent connections within the PC by injecting ChR2-EYFP virus into a single location within the aPC (Figure ; see Methods). The axonal projections from aPC neurons were distributed from layer 1 to layer 3 in both the aPC and pPC, with the highest density in layer 1b (Figures ). Notably, labeled fibers were observed in layer 1a (Figures ), similar to what was found for AON fibers (Figures ). Labeled fibers were also abundant in the pPC, although the fluorescence intensity was lower than that in aPC (Figure ).
Figure 6 Intrinsic connections within the PC. (A) AAV coding for ChR2-EYFP was injected into aPC. The bright signal in the anterior part of the aPC is due to ChR2-EYFP expression in pyramidal and glial cells. (B) EYFP labeled axons projecting to the aPC and pPC (more ...)
Whole-cell patch-clamp recordings from infected pyramidal cells in the injected area of the aPC showed the typical adapting ChR2 response in the presence of TTX. To examine synaptic responses, we obtained recordings from non-infected pyramidal cells in the aPC, and pyramidal cells in the pPC. Monosynaptic currents were again isolated using TTX and 4-AP as before. Both AMPA and NMDA currents could be recorded in target cells in both regions (Figures ). The probability of obtaining synaptic currents was almost 100% within the aPC, similar to that reported recently (Franks et al., 2011
), and that in the pPC was 93.3% (14 out of 15 cells). AMPA:NMDA ratio was similar for the two target regions (1.44 ± 0.19 vs. 1.22 ± 0.20, n
= 18 and 14 respectively, p
> 0.4, t
-test). Matched recordings from the same slices revealed that the average monosynaptic response amplitudes were lower for aPC to pPC connection compared to intrinsic connectivity within the aPC (69.3 ± 16.9 pA vs. 129.3 ± 19.7 pA, p
< 0.05, t
Calcium imaging reveals denser local connectivity in the pPC than in the aPC
Experiments with ChR2 did not readily lend themselves to detecting connections originating from single neurons. To examine the functional connectivity of neurons within the aPC, we turned to combined patch-clamp recordings and calcium imaging. Several groups have developed methods to detect the functional targets of single neurons by looking for changes in postsynaptic calcium concentration when a single neuron is made to emit one or more spikes. We labeled a population of cells in the aPC using multicell bolus labeling of OGB-1 (Stosiek et al., 2003
; Apicella et al., 2010
). Labeled cells were visualized with a CCD camera and patch-clamp recording was obtained from a single neuron within the aPC. In early experiments, we stimulated the LOT at increasing intensities and observed that a large number of labeled neurons in the field of view showed robust increase in fluorescence intensity (Figure ). In control experiments, we used high-speed line scanning to confirm that clear fluorescence intensity changes were invariably associated with action potentials (Appendix
Figure ), in line with many previous studies. LOT stimulation also allowed us to assess the health of slices, based on robust responses in a large population of cells. Since intrinsic connections are likely to be weak, we raised the excitability of neurons by increasing the extracellular potassium concentration to 7.5 mM, allowing even weaker synaptic inputs to generate action potentials and the associated calcium signals.
When single neurons within the aPC were stimulated, changes in calcium concentration could be detected in only a small number of cells in the field of view (Figures ). Responding neurons were identified using a threshold of three standard deviations above baseline fluctuations (see Methods; Figure ). On average, we obtained responses in 0.4 ± 0.16 cells (n = 28 slices) out of an average of more than 200 cells in the field of view, with the number of responding cells across experiments following an exponential distribution (Figure ). This suggests that the probability of functional connections within a local area is very low in the aPC.
We next examined the intrinsic connectivity in the pPC using calcium imaging. We found that a burst of five action potentials in a single pPC neuron could activate several neighboring neurons (Figure ). On average, the number of cells within the imaged field that could be activated was 1.2 ± 0.3 for pPC (n = 16 slices), significantly larger (p < 0.01 Fisher's exact test) than the fraction in aPC (0.4 ± 0.16, n = 28 slices) (Figure ). The fraction calculated includes experiments where no cells were activated, and the entire distribution of number of activated cells is shown in Figure .
Figure 8 Connectivity in the pPC. (A) Example of a group of cells in pPC labeled with OGB-1 (green), along with the response image when a single neuron was stimulated (grayscale image). The response image was obtained by taking the difference of 10 frames each (more ...)
Our experiments with ChR2 above indicated that the aPC has strong projections to pPC (Figure ). We wondered whether the activity of individual aPC cells could drive cells in pPC. After labeling cells in the pPC with OGB1, we obtained whole-cell patch-clamp recordings in the aPC. By moving the field of view to the pPC after obtaining a patch recording in aPC, we were able to determine if cells in the pPC could be activated by a burst of spikes in a single aPC cell. To our surprise, more superficial pPC cells (4.7 ± 0.74 cells, n = 9 slices with at least one pPC cell was active) could be activated by a single aPC neuron than even local aPC cells (1.5 ± 0.46 cells, n = 9 slices with at least one aPC cell active; p < 0.01 Fisher's exact test; Figure ). This result was obtained even though our ChR2 experiments indicated that monosynaptic connections were stronger locally within aPC than on pPC neurons. As we discuss below, this was likely due to the denser local connectivity within the pPC which amplifies activation from the aPC. Interestingly, in more than 10 experiments, we never obtained calcium responses in the aPC when stimulating individual pPC cells (Figure ).