Article Figures & Data
Figures
- Figure 1.
Two broad classes of principal neurons can be identified in layer II of the anterior PC, based on their morphologies and intrinsic electrical properties. A, Typical dendritic morphologies of layer II principal neurons from which electrical recordings were made. These examples are drawn from a dataset of n = 61 neurons distributed across the thickness of layer II (C). Cells with more superficial somata (in upper layer IIa) resemble typical SL cells, which lack basal dendrites. Cells with deeper somata (in lower layer IIb) resemble typical SP cells, with profuse apical and basal dendrites. Cells with somata near the middle of layer II have intermediate dendritic morphologies. Dashed lines indicate the approximate locations of the layers of the PC. Aff inputs from the olfactory bulb via the lateral olfactory tract are restricted to layer Ia; assn inputs from intracortical principal cells are restricted to all other layers (Ib, II, III). B, Voltage responses (top) of the neurons in A to 500-ms-long current steps (bottom). The hyperpolarizing step was always −80 pA, and the gray traces show the response to a step to just below rheobase. Horizontal dashed lines represent 0 mV. The electrical traces are shown directly below the cell from which the recordings were made. Note that the leftmost three cells have regular-spiking APs, while the rightmost three cells have burst-firing APs. C, Histogram of the somatic locations of the 61 neurons in our dataset, expressed as a fraction of the distance of the soma across the width of layer II (0, superficial border; 1, deep border). The cell somata were distributed across layer II. Locational color code shown at the bottom is used in D. D, Dendrogram showing the result of an unsupervised cluster analysis of the neurons in our dataset, calculated using 7 morphological parameters and 22 electrical parameters measured for each neuron. Colored bars at right indicate the fractional depth (across layer II) of the soma of each neuron, using the color code in C. Note the separation into two broad classes, as follows: SL-like cells that tend to be found more superficially (mostly blue colors), and SP-like cells that tend to be found deeper (red colors).
- Figure 2.
Excitatory synaptic inputs onto both SL and SP cells in the PC can be selectively inhibited with baclofen. A, Plot of mean peak amplitude of EPSCs, normalized to the mean amplitude during the first 5 min of recording, versus time for SL cells (open symbols, n = 5 cells) and SP cells (filled symbols, n = 5) while stimulating the LOT afferent input from the olfactory bulb. Error bars show ±SEM. (±)-Baclofen (100 μm, bath-applied during period shown by the horizontal bar) had little effect on EPSC amplitude in both SL and SP cells. Insets at right show typical EPSCs recorded at the indicated time points (1, baseline, black traces; 2, at end of baclofen application, gray traces). B, Same as A, except for EPSCs recorded while stimulating the assn input in layer Ib (n = 5 for SL cells; n = 6 for SP cells). The Ib input to both SL and SP cells was strongly inhibited by 100 μm baclofen.
- Figure 3.
Dual recordings from an SL and an SP cell show that SL cells receive stronger LOT (aff) inputs, whereas SP cells receive stronger Ib (assn) inputs. A, Image showing the arrangement of electrodes. Simultaneous whole-cell recordings were obtained from an identified SL cell (layer IIa) and SP cell (layer IIb), and an extracellular stimulating (Stim) electrode was placed in either the LOT (illustrated) or in layer Ib. B, Typical EPSCs recorded in this experiment. Each panel shows two EPSCs elicited by two different stimulus strengths in an SL cell (top) and an SP cell (bottom) while stimulating in the LOT (left) or in layer Ib (right). The stimulus artifact has been clipped. When recording the LOT response, the bath solution contained 100 μm baclofen to block contamination by assn inputs. Baclofen was omitted when recording the response to layer Ib stimulation. C, Summary plot showing the mean EPSC amplitude measured in SL cells versus the mean amplitude in SP cells when stimulating either the LOT (filled symbols above the diagonal dashed line) or layer Ib (open symbols below the diagonal). Each point is the decimated average of 10 individual measurements made at a similar stimulus strength (n = 6 cells). Error bars indicate ±SEM.
- Figure 4.
Minimal extracellular stimulation shows that unitary LOT inputs onto SL cells tend to be larger than those onto SP cells, whereas unitary layer Ib inputs onto both cell types are similar. A, Data from a typical cell (SL) showing how unitary responses were measured. The amplitude of the stimulator current (Stim; bottom) was increased in small steps close to the threshold for eliciting an EPSC (top). The mean EPSC amplitude during the plateau response was taken to be the unitary EPSC amplitude (here, 152 pA). Inset at right shows traces recorded at the indicated points on the time course plot. Baclofen was not used in these experiments because stimulation strengths were too weak to elicit contaminating assn inputs. B, Histograms showing the distributions of unitary EPSC amplitudes measured in experiments like in A, for SL cells (top row) or SP cells (bottom row) while stimulating either the LOT (left column) or layer Ib (right column). Numbers to the right of each histogram represent the mean of the unitary amplitudes (±SEM) included in the histogram (n = number of cells).
- Figure 5.
The differential strength of LOT and layer Ib synaptic inputs onto SL and SP cells (as shown in Fig. 3, using voltage clamp) is also manifested as differential AP firing thresholds (measured using current clamp). A1, Simultaneous whole-cell current-clamp recordings from an SL cell (top row) and an SP cell (bottom row) while stimulating (Stim) the LOT with the indicated currents (15, 40, 80 μA). Inset at top shows schematically the recording configuration. A2, Analysis of data from the cells in A1, plotting the probability of firing at least one AP versus the current strength applied to the stimulator in the LOT. The SL cell (open symbols) reaches AP threshold at much lower stimulation currents than the SP cell (filled symbols). Similar results were obtained in six cell pairs. The bath solution contained 100 μm baclofen to block contamination by assn inputs. B1, B2, Similar experiment while stimulating in layer Ib. Now, the pattern of excitability is reversed: with assn inputs, the SP cell reaches AP threshold at much lower stimulation currents than the SL cell. Similar results were obtained in five cell pairs. Baclofen was not present during these measurements.
- Figure 6.
When direct aff input to SP cells is prevented by a tissue cut, weak stimulation of the LOT reveals a feedforward excitatory assn input that is largely driven by SL cells. A, Schematic diagram showing the recording configuration, illustrated for a parasagittal slice containing the LOT. The LOT and layer Ia (gray) are transected by a tissue cut. The extracellular stimulating electrode (Stim) is placed at the LOT/Ia border on one side of the cut, while recordings are made from an SP cell on the other side of the cut. Weak stimulation excites aff input to either an SP cell or an SL cell, which in turn provides assn input to the recorded SP cell. B1, Two superimposed episodes recorded in an SL cell (top) and an SP cell (bottom) showing synaptically evoked APs at stimulus strengths just above threshold for eliciting APs. The bath solution did not contain picrotoxin. B2, Histograms of the latency from the stimulus to the peak of the synaptically evoked AP (arrowed in B1), combining measurements on n = 4 SL cells and n = 4 SP cells. C, Top, Superimposed episodes recorded from an SP cell following weak LOT stimulation with the electrode configuration as in A, showing EPSCs that result from the feedforward excitation of assn inputs. The synaptic latency is arrowed. The mean synaptic latency in this cell was 4.40 ± 0.02 ms (n = 230 trials). C, Bottom, Episodes from the same cell after application of 100 μm baclofen. Inhibition of the EPSCs confirms the involvement of assn fibers. D, Histogram of mean synaptic latencies for 20 experiments as in C. Two classes of cells are apparent, with “Early” (mean 4.6 ± 0.1 ms, n = 14) and “Late” (8.0 ± 0.2 ms, n = 6) EPSC latencies.
- Figure 7.
When larger stimulus currents are applied to the LOT in disinhibited slices, strong polysynaptic assn excitation is apparent in SP cells but not in SL cells. This excitation is blocked by baclofen. A1, Simultaneous whole-cell recordings from an SL cell (top) and an SP cell (bottom) while stimulating the LOT in the presence of 100 μm picrotoxin. Long-latency polysynaptic EPSCs (arrowed) are much larger in the SP cell than in the SL cell. Similar observations were made in n = 4 pairs. A2, A similar experiment involving two SP cells. The polysynaptic EPSCs (arrows) are large in both cells. They also exhibit synchronous latency jitter (not illustrated). B1, Analysis of the data from the cell pairs in A1, A2. Slow charge (a measure of the size of the long-latency polysynaptic EPSC) was calculated by integrating over a 200-ms-long window starting 20 ms after the peak of the averaged first EPSC (see Materials and Methods). The slow charge measured in one cell was plotted against that measured in the other. Sweep-to-sweep fluctuations in the amplitudes of this charge gave rise to a cloud of points distributed along a straight line (correlation coefficients: SL + SP: 0.56; SP + SP, 0.93). The slope was larger for the SP + SP pair, reflecting the larger polysynaptic EPSCs in SP cells (SL + SP: 0.10; SP + SP: 1.71). B2, Summary plot showing the mean slow charge measured in individual cells (open symbols; SL, n = 4; SP, n = 12) and the grand mean for each cell type (filled symbols with error bars, ±SEM). These values were significantly different (p = 0.012). C, Simultaneous whole-cell recordings from an SL cell (top) and an SP cell (bottom) before (left) and after (right) perfusing 100 μm baclofen in the bath solution. Each panel shows two superimposed sweeps recorded in each cell. Baclofen blocks the long-latency polysynaptic EPSCs in both cell types and also blocks a putative polysynaptic component in the initial EPSC recorded in the SP cell. The insets (right) show the initial EPSC expanded (black: before baclofen; gray: after baclofen).
- Figure 8.
SL and SP cells show distinctive forms of short-term synaptic plasticity during trains of stimulation (Stim). A–D, Plots of normalized EPSC amplitude versus time during trains of 10 stimuli at different frequencies, recorded in SL cells (A, B) and SP cells (C, D) while stimulating the LOT (A, C) or layer Ib (B, D). Amplitudes are normalized to the amplitude of the first EPSC in the train. Each panel shows averaged data from n = 6–9 cells; error bars have been omitted for clarity. Insets show example EPSCs, each an average of 10 episodes recorded in a single cell (50 Hz stimulation; stimulus artifacts have been blanked). The scale bars in D refer to the insets in all panels. The bath solution contained 1 μm DNQX and 50 μm d-APV to minimize polysynaptic excitation (as well as 100 μm picrotoxin to block GABAA receptors).
- Figure 9.
Trains of stimuli dynamically modify the differential synaptic input strengths onto SL and SP cells. A, Simultaneous whole-cell current-clamp recordings from an SL cell (top row) and an SP cell (bottom row) while stimulating the LOT with a 50 Hz train of three stimuli at the indicated current strengths (15, 25, 50 μA). Insets show, expanded, the synaptic facilitation present in the SP cell (bottom) but absent in the SL cell (top). The bath solution contained 100 μm baclofen to block contamination by assn inputs. B, Analysis of data from the cells in A. Black lines and symbols plot the probability of firing at least one AP on any EPSP in the train versus stimulus (Stim) current strength. Red lines and symbols plot the probability of firing at least one AP on the first EPSP in the train versus stimulus strength (equivalent to the plots in Fig. 5A2). By permitting APs to occur anywhere in the train, the responsiveness of the SP cell is shifted left (red arrow), closer to that of the SL cell. Similar results were obtained in 10 cell pairs.
Tables
Electrical parameters Morphological parameters SL-like SP-like p SL-like SP-like p Resting potential (mV) −67.2 ± 0.7 −76.3 ± 0.7 <0.001 Soma cross-sectional area (μm2) 268 ± 7 315 ± 9 <0.001 Input resistance (MΩ) 221.2 ± 15.2 55.0 ± 3.5 <0.001 n apical dendrites at soma 2.68 ± 0.12 1.41 ± 0.13 <0.001 Membrane time constant (ms) 26.0 ± 1.7 10.1 ± 0.7 <0.001 n basal dendrites at soma 2.15 ± 0.32 5.96 ± 0.38 <0.001 Sag 0.91 ± 0.01 0.93 ± 0.01 0.22 Total length of apical dendrites (μm) 2594 ± 83 3646 ± 142 <0.001 Action potential threshold (mV) −36.0 ± 0.5 −41.5 ± 0.6 <0.001 Total length of basal dendrites (μm) 448 ± 84 2022 ± 159 <0.001 Action potential halfwidth (ms) 0.74 ± 0.01 0.76 ± 0.01 0.29 n apical dendritic branch points 13.0 ± 0.5 18.5 ± 0.9 <0.001 Action potential height (mV) 76.2 ± 1.1 92.9 ± 0.9 <0.001 n basal dendritic branch points 1.5 ± 0.3 8.6 ± 0.8 <0.001 Values were calculated for cells in the SL-like cluster (n = 34) and the SP-like cluster (n = 27) in Fig. 1D, as described in Materials and Methods. The p values indicate that sag and action potential half widths do not provide a distinction between SL-like and SP-like cells, but all the other parameters listed here do support a clear distinction, on average.
