Ventral Hippocampal Inputs Preferentially Drive Corticocortical Neurons in the Infralimbic Prefrontal Cortex

Abstract

Inputs from the ventral hippocampus (vHPC) to the prefrontal cortex (PFC) play a key role in working memory and emotional control. However, little is known about how excitatory inputs from the vHPC engage different populations of neurons in the PFC. Here we use optogenetics and whole-cell recordings to study the cell-type specificity of synaptic connections in acute slices from the mouse PFC. We first show that vHPC inputs target pyramidal neurons whose cell bodies are located in layer (L)2/3 and L5 of infralimbic (IL) PFC, but only in L5 of prelimbic (PL) PFC, and not L6 of either IL or PL. We then compare connections onto different classes of projection neurons located in these layers and subregions of PFC. We establish vHPC inputs similarly contact corticocortical (CC) and cortico-amygdala neurons in L2/3 of IL, but preferentially target CC neurons over cortico-pontine neurons in L5 of both IL and PL. Of all these neurons, we determine that vHPC inputs are most effective at driving action potential (AP) firing of CC neurons in L5 of IL. We also show this connection exhibits frequency-dependent facilitation, with repetitive activity enhancing AP firing of IL L5 CC neurons, even in the presence of feedforward inhibition. Our findings reveal how vHPC inputs engage defined populations of projection neurons in the PFC, allowing preferentially activation of the intratelencephalic network.SIGNIFICANCE STATEMENT We examined the impact of connections from the ventral hippocampus (vHPC) onto different projection neurons in the mouse prefrontal cortex (PFC). We found vHPC inputs were strongest at corticocortical neurons in layer 5 of infralimbic PFC, where they robustly evoked action potential firing, including during repetitive activity with intact feedforward inhibition.

Keywords: circuit; hippocampus; interneuron; prefrontal cortex; projection neuron; synapse.

Figure 1.

vHPC projects to the infralimbic and prelimbic PFC. A, Left, RV-mCherry injection into the PFC. Right, Fluorescent images of injection site (PFC), lack of retrograde labeling in dHPC, and prominent retrograde labeling in vHPC. Red, mCherry; gray, DAPI. Scale bars, 800 μm. B, Left, AAV-mCherry injection into the vHPC. Right, Fluorescent images of injection site (vHPC) and prominent anterograde labeling in PFC (bregma +2.0 mm; scale bars, 800 μm) and expanded view of IL (1400–2000 μm from dorsal surface) and PL (500–1400 μm from dorsal surface) regions. Scale bar, 200 μm. Red, mCherry; gray, DAPI. C, Left, vHPC axon distribution in PL (measured at 900–1200 μm from dorsal surface) and IL (measured at 1600 μm–1900 μm from dorsal surface), as a function of the distance from the midline pia along the medial-lateral axis. Right, vHPC axon distribution along dorsal-ventral axis in L2/3 (measured at 250–350 μm from pia) and L5 (measured at 400–500 μm from pia) of IL and PL regions, as a function of the distance from the dorsal surface along the dorsal-ventral axis. Plots show axon fluorescence normalized to the peak fluorescence within the slice (n = 3 animals).

Figure 2.

vHPC inputs contact L2/3 and L5 pyramidal neurons in the PFC. A, Left, AAV-ChR2 injection into the vHPC. Right, Reconstructed dendritic morphologies of pyramidal neurons recorded in L2/3, L5 and L6 of the IL and PL PFC. B, Left, Average vHPC-evoked EPSCs recorded from pairs or triplets of pyramidal neurons in L2/3, L5, and L6 of the IL region. For each group, the amplitudes of EPSCs are normalized to those of the L5 pyramidal neuron. Blue arrow = 3 ms light stimulation. Middle, Summary of EPSC amplitudes. Right, Summary of EPSC amplitude ratios (n = 7 pairs, 6 animals). C, Same as B but for neurons in PL (n = 7 pairs, 6 animals). *p < 0.05.

Figure 3.

vHPC inputs contact L2/3 CC and CA neurons in IL PFC. A, Left, AAV-ChR2 injection into the vHPC, and simultaneous injection of CTB-488 or -647 into the BLA and cPFC. Right, Example fluorescent image of injection sites in the BLA. Scale bar, 800 μm. B, Left, Distribution of CC (white) and CA (green) neurons in the IL PFC. Scale bar, 100 μm. Right, Summary of CC and CA neuron distribution in IL. C, Left, Reconstructed dendritic morphologies of CC and CA neurons. Scale bar, 100 μm. Right, Average vHPC-evoked EPSCs at −70 mV from pairs of L2/3 CC (black) and L2/3 CA (green) neurons in IL. Blue arrow = 3 ms light stimulation. D, Left, Summary of EPSC amplitudes from recordings in C. Right, Summary of EPSC amplitude ratios (n = 7 pairs, 4 animals). Note the logarithmic axis.

Figure 4.

vHPC inputs target L5 CC neurons in both IL and PL PFC. A, Left, AAV-ChR2 injection into the vHPC, and simultaneous injection of CTB-488 or -647 into the pons and cPFC. Right, Example confocal image of injection sites in the pons. Scale bar, 600 μm. B, Left, Distribution of CC (white) and CP (purple) neurons in the IL and PL PFC. Scale bar, 100 μm. Right, Summary of CC and CP neuron distributions in IL and PL. C, Left, Reconstructed dendritic morphologies of L5 CC and CP neurons in IL. Right, Average vHPC-evoked EPSCs at −70 mV from pairs of L5 CC (black) and L5 CP (purple) neurons in IL (n = 13 pairs, 5 animals). Blue arrow = 3 ms light stimulation. D, Similar to C for L5 CC and CP neurons in PL (n = 10 pairs, 5 animals). E, Left, Summary of EPSC amplitudes from C. Right, Summary of EPSC amplitude ratios from C. Note the logarithmic axis. F, Similar to E for L5 CC and CP neurons in PL, summarized from D. G, Summary of CC/CP EPSC amplitude ratios from recordings in E and F between pairs of cells in PL and IL. Note the logarithmic axis. *p < 0.05.

Figure 5.

vHPC inputs differentially contact the dendrites of L5 neurons. A, Recording schematic. Focused light was delivered along the main apical dendrite of the recorded cell, with 50 μm increments between stimulation spots. Darker spots represent the soma and a location +200 μm along the dendrite, as shown in B. B, Average vHPC-evoked EPSCs recorded at −70 mV when stimulating at either the soma (dark) or apical dendrites (+200 μm from the soma; light) in L5 CC (black) and L5 CP (purple) neurons in either IL or PL. For each cell, EPSCs were normalized to the response evoked from stimulation of the soma. Blue arrow = 3 ms light stimulation (n = 7 cells, 3 animals for each cell type). C, Summary of EPSC charges (arithmetic mean and SEM, normalized to the responses evoked at soma) measured at different distances from the soma in L5 CC (black) or L5 CP (purple) neurons in either IL (left) or PL (right). D, Ratios of EPSC charges evoked in proximal apical dendrites (+200 μm) and soma. Individual values, arithmetic mean, and SEM are plotted for each cell type. *p < 0.05.

Figure 6.

vHPC inputs are weaker onto CT neurons in both L5 and L6. A, AAV-ChR2 injection into the vHPC, and coinjection of red retrobeads (RB) and CTB-647 into the MD and cPFC. B, Reconstructed dendritic morphologies of L5 CC, L5 CT, and L6 CT neurons in IL. C, Average vHPC-evoked EPSCs at −70 mV from triplets of L5 CC (black), L5 CT (red), and L6 CT (dark red) neurons in IL (n = 8 triplets, 5 animals). Blue arrow = 3 ms light stimulation. D, Left, Summary of EPSC amplitudes from C. Right, Summary of EPSC amplitude ratios from C. Note the logarithmic axis. *p < 0.05.

Figure 7.

vHPC inputs preferentially activate L5 CC neurons in IL. A, Left, Average vHPC-evoked EPSCs recorded from triplets of CC neurons in IL L2/3, IL L5 and PL L5. Blue arrow = 3 ms light stimulation. Middle, Summary of EPSC amplitudes. Right, Summary of EPSC amplitude ratios (n = 10 triplets, 5 animals). Note the logarithmic axis. B, vHPC-evoked EPSCs in the six studied cell types, normalized to responses in IL L5 CC neurons, based on the geometric means of their vHPC-evoked EPSC amplitude ratios from experiments in Figures 3, 4, and 6. C, Left, Synaptic conductances used for dynamic-clamp recordings in IL L5 CC neurons, shown across a range of linear scale factors (1–10×), corresponding to conductances of 3.5–35 nS. Right, AP firing of an IL L5 CC neuron in response to these synaptic conductances at resting membrane potential (n = 7 cells, 3 animals). D, Summary of AP firing probability of the six cell types shown in B, as a function of scale factor (n = 7 cells, 3 animals for each cell type). *p < 0.05.

Figure 8.

Activation of IL L5 CC neurons persists in the presence of inhibition. A, Average vHPC-evoked EPSCs at −70 mV and IPSCs at +10 mV, recorded from triplets of IL L2/3 CC neurons, IL L5 CC neurons and PL L5 CC neurons. Blue arrow = 3 ms light stimulation. B, Left, Summary of EPSC amplitudes. Middle, Summary of IPSC amplitudes. Right, Summary of E/I ratio (n = 11 triplets, 6 animals). C, vHPC-evoked EPSPs and APs recorded in current-clamp at resting membrane potentials from an example triplet of CC neurons, with 10 traces superimposed for each cell. D, Summary of AP firing probability (n = 7 triplets, 3 animals). *p < 0.05.

Figure 9.

vHPC activation of IL L5 CC neurons depends on input frequency. A, Average vHPC-evoked 20 Hz trains of EPSCs at −70 mV and IPSCs at +10 mV at IL L5 CC neurons (n = 9 cells, 3 animals). B, Left, Summary of EPSC PPR (EPSC_N/EPSC₁) as a function of pulse number for 20, 8, and 4 Hz trains. Right, Similar for IPSC PPR. Gray dashes line: EPSC_N/EPSC₁ = 1. C, vHPC-evoked 20 Hz trains of EPSPs and APs in an example IL L5 CC neuron, recorded in current-clamp at resting membrane potential, with five staggered traces. Blue arrow: five 3 ms light pulses at 20 Hz. D, Summary of AP firing probability as a function of pulse number for 20, 8, and 4 Hz trains (n = 9 cells, 3 animals). E, Summary schematic of vHPC to PFC projections. vHPC inputs activate CC and CA networks by targeting superficial and deep IL, along with deep PL.