Frequency-dependent, cell type-divergent signaling in the hippocamposeptal projection

Abstract

Hippocampal oscillations are critical for information processing, and are strongly influenced by inputs from the medial septum. Hippocamposeptal neurons provide direct inhibitory feedback from the hippocampus onto septal cells, and are therefore likely to also play an important role in the circuit; these neurons fire at either low or high frequency, reflecting hippocampal network activity during theta oscillations or ripple events, respectively. Here, we optogenetically target the long-range GABAergic projection from the hippocampus to the medial septum in rats, and thereby simulate hippocampal input onto downstream septal cells in an acute slice preparation. In response to optogenetic activation of hippocamposeptal fibers at theta and ripple frequencies, we elicit postsynaptic GABAergic responses in a subset (24%) of septal cells, most predominantly in fast-spiking cells. In addition, in another subset of septal cells (19%) corresponding primarily to cholinergic cells, we observe a slow hyperpolarization of the resting membrane potential and a decrease in input resistance, particularly in response to prolonged high-frequency (ripple range) stimulation. This slow response is partially sensitive to GIRK channel and D2 dopamine receptor block. Our results suggest that two independent populations of septal cells distinctly encode hippocampal feedback, enabling the septum to monitor ongoing patterns of activity in the hippocampus.

Keywords: hippocamposeptal; medial septum; optogenetics.

Figure 1.

Optogenetic targeting of HS cells. A, AAV vector design for specific targeting of SST-expressing neurons. B, Viral expression (green) in dorsal and ventral hippocampus, with the pyramidal and granule cell layers highlighted by dense DAPI staining (white). Expression was predominantly localized to stratum oriens (superficial to the pyramidal cell layer (DAPI; white) in CA1 and deep to it in CA3) as well as in the DG hilus. Minimal expression was seen in CA2. C, High-magnification view of CA1, demonstrating overlap (white arrowheads) between SST immunostaining (red) and virally expressed eYFP (green). DAPI (white) delineates the pyramidal cell layer. D, High-magnification view of the DG, demonstrating overlap (white arrowheads) between SST immunostaining (red) and virally expressed eYFP (green). DAPI (white) delineates the granule cell layer. E, Sample current-clamp trace from a virally transduced cell, demonstrating high-fidelity light-evoked spiking at both 8 and 50 Hz. F, ChR2-YFP-expressing axons in the medial septum, indicating successful targeting of HS cells, seen at low magnification (left) and high magnification (right).

Figure 2.

Classification of cell types in the adult medial septum. A, Representative firing patterns and expanded single action potential traces for cells classified as fast-firing, cluster-firing, type 1 slow-firing, and type 2 slow-firing cells. For the cluster-firing cell, note the characteristic subthreshold membrane oscillations during firing pauses. For the type 1 slow-firing cell, note the significantly broader action potential. For the type 2 slow-firing cell, note the characteristic afterdepolarization of the action potential waveform. Scale bars: left, 20 mV, 200 ms; right, 20 mV, 10 ms. B, Summary data for maximum spike rate, showing significantly greater maximal firing rate for fast-firing cells (94 ± 5 Hz, n = 51) and cluster-firing cells (59 ± 11 Hz, n = 13) compared with type 1 slow-firing cells (30 ± 2 Hz, n = 55) and type 2 slow-firing cells (38 ± 3 Hz, n = 33). C, Summary data for membrane potential sag reveals significantly larger sag for fast-firing cells (4.5 ± 0.5 mV, n = 38) and cluster-firing cells (4.3 ± 0.8 mV, n = 9) compared with type 1 slow-firing cells (2.2 ± 0.4 mV, n = 46) and type 2 slow-firing cells (1.5 ± 0.3 mV, n = 29). D, Summary data for action potential half-width reveals that type 1 slow-firing cells had the largest half-width (0.99 ± 0.04 ms, n = 31), followed by type 2 slow-firing cells (0.71 ± 0.04 ms, n = 25), both significantly larger than for fast-firing cells (0.48 ± 0.03, n = 21) and cluster-firing cells (0.49 ± 0.04 ms, n = 13). E, Summary data for action potential rise slope (left) and decay slope (right, shown as −mV/ms). Type 1 slow-firing cells had the slowest rise (206 ± 17 mV/ms, n = 31) and decay (73 ± 4 mV/ms, n = 31), followed by type 2 slow-firing cells (288 ± 21 mV/ms rise and 129 ± 14 mV/ms decay, n = 25). Fast-firing cells (309 ± 21 mV/ms rise and 176 ± 14 mV/ms decay, n = 21) and cluster cells (333 ± 25 mV/ms rise and 181 ± 18 mV/ms decay, n = 13) had significantly steeper slopes. Error bars represent SE; significance calculated using one-way ANOVA with Tukey's post hoc test. F, Left, A patched type 1 slow-firing cell filled with biocytin (red) colabels with ChAT immunostaining (blue, left), whereas a patched fast-firing cell does not (right). Error bars represent SE. *, Significance <0.05; **, significance <0.01; ***, significance <0.001.

Figure 3.

Optogenetically evoked IPSCs in medial septum in response to stimulation of HS fibers. Cells were held in voltage clamp at −40 mV for all IPSC recordings. A, GABA(A)-receptor-mediated IPSCs in a fast-firing cell, evoked by 5 ms blue light pulses delivered at 8 Hz (above) or 50 Hz (below). Blue bars indicate light stimuli. Scale bars: 50 pA, 200 ms. B, Sample trace illustrating inhibition of IPSCs by application of 50 μm picrotoxin. A complete block was seen in four of four cells tested. Scale bars: 20 pA, 200 ms. C, Comparison of optogenetically evoked IPSCs in the fast-firing cell from A and B (black trace) and a type 2 slow-firing cell (gray trace) to illustrate differences in IPSC kinetics. Traces are normalized to peak and aligned to the light stimulus. D, Summary data showing rise time, decay time constant, and half-width of evoked IPSCs in the four cell types: fast firing (n = 18), cluster firing (n = 3), type 1 slow firing (n = 8), and type 2 slow firing (n = 8). Tukey's post hoc tests revealed no significant differences in rise time or half-width, but the decay time constant for type 2 slow-firing cells (10.3 ± 1.1 ms) was significantly larger than that of both fast-firing (5.8 ± 0.6 ms) and cluster-firing (3.5 ± 0.4 ms) cells. Error bars represent SE. **, Significance <0.01.

Figure 4.

Prolonged stimulation leads to hyperpolarization and decreased input resistance in a subset of cells. Cells were recorded in current clamp between −60 and −65 mV. Brief current injections (10–25 pA) were given at a rate of 1–2 Hz to monitor input resistance. A, Current-clamp trace illustrating slow-onset and persistent hyperpolarization and decreased input resistance in response to pulses of blue light delivered at 50 Hz. B, Summary membrane potential data of all responsive cells (n = 24, red), illustrating that on average responding cells have a small light-evoked decrease in membrane potential. Data were binned (n = 3) and linearly detrended to correct for a small (∼1 mV) hyperpolarizing drift over the course of the recording. A matched control group of the same number of nonresponding cells patched on the same days was plotted for comparison (n = 24, black). C, Estimated reversal potentials of the response ranged from −64 to −85 mV (mean, −74.9 ± 1.1 mV, n = 23). D, Hyperpolarization and normalized input resistance decrease were highly correlated (ρ₍₂₁₎ = 0.57, p = 0.0043). Input resistance was averaged across all responsive cells with a statistically significant response (n = 24, red), binned (n = 3), and plotted over time to show either the normalized (E) or absolute (F) decline in input resistance. For plots over time (B, E, F), error bars represent SE.

Figure 5.

Mechanism of the slow response. A–D, Normalized input resistance (R_in) in response to 500 pulses of 50 Hz light delivered at baseline, with drug application, and after wash-out. R_in in each case was averaged across cells that are responsive at baseline, binned (n = 3), and plotted over time (columns 1–3). The population data were fit with splines to determine the relative and absolute magnitude of the input-resistance fall (columns 4 and 5, respectively). A, R_in was stable to repetitive 50 Hz light delivery (n = 7, 7, and 3, respectively). B, Barium, a GIRK channel antagonist, partially blocked the response (n = 7). Normalized R_in fell significantly from 18.5% (95% CI, 16.3–20.8%) at baseline to 8.2% (95% CI, 5.5–10.9%) with barium, and absolute R_in fell significantly from 50.9 MΩ (95% CI, 45.1–56.8 MΩ) at baseline to 28.6 MΩ (95% CI, 20.0–37.3 MΩ) with barium. C, Cyclosomatostatin, a nonspecific SSTR antagonist, partially blocked the response (n = 4). Normalized R_in trended down from 18.0% (5–95% CI, 14.8–21.2%) at baseline to 12.5% (5–95% CI, 8.8–16.9%) with cyclosomatostatin, while the absolute R_in fell significantly from 53.0 MΩ (5–95% CI, 42.8–63.1 MΩ) at baseline to 26.5 MΩ (5–95% CI, 18.3–36.7 MΩ) with cyclosomatostatin. D, Prochlorperazine, a D2 receptor antagonist, significantly blocked the response (n = 5). Normalized R_in fell significantly from 21.4% (5–95% CI, 19.0–23.8%) at baseline to 5.9% (5–95% CI, 3.5–8.2%) with prochlorperazine, and absolute R_in significantly fell from 69.0 MΩ (5–95% CI, 61.2–76.8 MΩ) at baseline to 22.2 MΩ (5–95% CI, 14.2–30.2 MΩ) with prochlorperazine. E, Sample current-clamp trace (top left) illustrates slow-onset hyperpolarization and decreased R_in in response to quinpirole (red line), a D2-receptor agonist (scale bar: 5 mV, 1 min). R_in for that trace was calculated, binned (n = 3), and plotted over time (bottom left). Voltage deflections in response to current injections at baseline (1) and after quinpirole administration (2), at the times indicated on the full trace on the left (scale bar: 1 mV, 1 s). Absolute R_in for all three responsive cells at baseline and in response to quinpirole demonstrates large fall in each case (plot on right). For A–E, error bars on the plots over time (columns 1–3) represent SE, whereas error bars on the summary plots (columns 4–5) represent 5–95% CIs. Significance (*) was defined as nonoverlapping 95% CIs, which are generated by bootstrapping.

Figure 6.

The slow response is evoked more efficiently by 50 Hz stimulation than by pulse number-matched 8 Hz stimulation. Cells were recorded in current clamp between −60 and −65 mV. Brief current injections (10–25 pA) were given at a rate of 1 Hz to monitor input resistance. A, Sample current-clamp trace illustrates a response to 500 pulses of blue light delivered at 50 Hz, but not at 8 Hz. Scale bar: 5 mV, 15 s. B, Summary data showing input resistance of the same cells (n = 8) in response to either 50 Hz stimulation (red) or 8 Hz stimulation (blue). Blue bars indicate light stimulation (note that the duration of 8 Hz stimulation is longer). Data were binned (n = 3) for plotting. C, Percentage input-resistance decrease for 50 Hz (16.1%) versus 8 Hz (3.3%). D, Absolute input-resistance decrease for 50 Hz (48.0 MΩ) versus 8 Hz (14.6 MΩ). E, Time to reach minimum input resistance was faster for 50 Hz (14 s) than for 8 Hz (41 s). Ninety-five percent CIs, calculated by bootstrapping of population fits, were nonoverlapping for all three comparisons (C–E). Error bars on the plots over time (B) represent SE, whereas error bars on the summary plots (C–E) represent 5–95% CIs. Significance (*) was defined as nonoverlapping 95% CIs, which are generated by bootstrapping.

Figure 7.

IPSCs and slow responses are seen in independent populations of septal cells. A, Locations of patched septal cells with IPSCs and/or slow responses. Blue circles indicate fast-firing cells, red diamonds indicate cluster-firing cells, green squares indicate type 1 slow-firing cells, and purple triangles indicate type 2 slow-firing cells. Symbols filled on the left indicate IPSCs only, symbols filled on the right indicate slow-response only, and entirely filled symbols indicate both response types. For display purposes, fast-firing and cluster-firing cells are indicated on the left and type 1 and type 2 slow-firing cells are indicated on the right, although locations of all cell types were in fact symmetric around the midline. B, Pie charts showing distribution of the four characterized cell types: fast (blue), cluster (red), type 1 slow (green), and type 2 slow (purple). Left, Distribution of all cells that could be classified by cell type (n = 248). Center, Distribution of all cells that had light-evoked IPSCs (n = 53). Right, Distribution of all cells that had light-evoked input resistance decrease in response to prolonged 50 Hz stimulation (n = 24). Note that fast cells disproportionately respond with IPSCs, while type 1 (but not type 2) slow cells disproportionately exhibit the slow response. On each pie chart, the numbers indicate the n for each cell type. C, Percentage of cells exhibiting IPSCs and slow responses for each cell type and for all cells, illustrating that fast-firing and type 2 slow-firing cells are more likely to have IPSC responses, whereas type 1 slow-firing cells are more likely to have slow responses. Responses were found to be highly variable by cell type (p = 0.0024 with a logistic regression interaction test). Numbers above each bar indicate the number of positive cells divided by the total number of assayed cells in each case.