Hypothalamic Supramammillary Nucleus Selectively Excites Hippocampal CA3 Interneurons to Suppress CA3 Pyramidal Neuron Activity

Abstract

A key mode of neuronal communication between distant brain regions is through excitatory synaptic transmission mediated by long-range glutamatergic projections emitted from principal neurons. The long-range glutamatergic projection normally forms numerous en passant excitatory synapses onto both principal neurons and interneurons along its path. Under physiological conditions, the monosynaptic excitatory drive onto postsynaptic principal neurons outweighs disynaptic feedforward inhibition, with the net effect of depolarizing principal neurons. In contrast with this conventional doctrine, here we report that a glutamatergic projection from the hypothalamic supramammillary nucleus (SuM) largely evades postsynaptic pyramidal neurons (PNs), but preferentially target interneurons in the hippocampal CA3 region to predominantly provide feedforward inhibition. Using viral-based retrograde and anterograde tracing and ChannelRhodopsin2 (ChR2)-assisted patch-clamp recording in mice of either sex, we show that SuM projects sparsely to CA3 and provides minimal excitation onto CA3 PNs. Surprisingly, despite its sparse innervation, the SuM input inhibits all CA3 PNs along the transverse axis. Further, we find that SuM provides strong monosynaptic excitation onto CA3 parvalbumin-expressing interneurons evenly along the transverse axis, which likely mediates the SuM-driven feedforward inhibition. Together, our results demonstrate that a novel long-range glutamatergic pathway largely evades principal neurons, but rather preferentially innervates interneurons in a distant brain region to suppress principal neuron activity. Moreover, our findings reveal a new means by which SuM regulates hippocampal activity through SuM-to-CA3 circuit, independent of the previously focused projections from SuM to CA2 or dentate gyrus.SIGNIFICANCE STATEMENT The dominant mode of neuronal communication between brain regions is the excitatory synaptic transmission mediated by long-range glutamatergic projections, which form en passant excitatory synapses onto both pyramidal neurons and interneurons along its path. Under normal conditions, the excitation onto postsynaptic neurons outweighs feedforward inhibition, with the net effect of depolarization. In contrast with this conventional doctrine, here we report that a glutamatergic input from hypothalamic supramammillary nucleus (SuM) largely evades PNs but selectively targets interneurons to almost exclusively provide disynaptic feedforward inhibition onto hippocampal CA3 PNs. Thus, our findings reveal a novel subcortical-hippocampal circuit that enables SuM to regulate hippocampal activity via SuM-CA3 circuit, independent of its projections to CA2 or dentate gyrus.

Keywords: CA3; dentate gyrus; hippocampus; inhibition; parvalbumin interneuron; supramammillary nucleus.

Figure 1.

Retrograde and anterograde tracing reveals the monosynaptic projection from SuM to CA3. A-C, Identification of SuM-CA3 projection using AAVretro-mCherry. Repeated in 3 mice. A, Schematic view of SuM-CA3 projection and viral injection site in dorsal CA3 in WT mice. B, Sample confocal image represents the injection site of AAVretro-mCherry in dorsal CA3. C, Sample confocal image represents mCherry⁺ cells observed in SuM (left). An expanded view shown in right. f, Fornix. D-F, Pseudotyped rabies tracing in Grik4-Cre mice reveals the monosynaptic projection from SuM to CA3. Repeated in 5 mice. D, Schematic view of the experimental procedure and timeline. E, Sample confocal image represents the injection site of rabies virus in dorsal CA3. F, Sample confocal images represent mCherry⁺ cells observed in SuM (left) and an expanded view (right). G-K, Anterograde tracing represents the SuM axonal projection patterns in the hippocampus. Repeated in 2 mice. G, Schematic view of SuM-CA3 projection and injection of AAV-ChR2-EYFP into SuM in WT mice. H, Sample confocal image showing the injection site in SuM and the expanded view (inset). I-K, ChR2-EYFP⁺ fiber distribution in the hippocampus. Note the dense ChR2-EYFP⁺ fibers in DG, CA2, and distal CA3 (CA3a), and the relatively sparse expression of ChR2-EYFP⁺ fibers observed in proximal CA3 (CA3b/c). Purkinje Cell Protein 4 (PCP4, red) is a marker for CA2. s.o., stratum oriens; s.p., stratum pyramidale; s.l., stratum lucidum; s.r., stratum radiatum; s.l.m., stratum lacunosum moleculare.

Figure 2.

Selective targeting of ChR2-EYFP into CA3-projecting SuM neurons. A, Experimental procedure and timeline. B, Sample confocal image represents the expression of Cre-mCherry in dorsal CA3, the first injection site of AAVretro-Cre-mCherry. C, Left, Sample confocal image represents the expression of ChR2-EYFP in SuM, the second injection site of Cre-dependent AAV-DIO-ChR2-EYFP. Right, Expanded view represents colabeling of Cre-mCherry and ChR2-EYFP in SuM cells. D, E, The distribution of ChR2-EYFP⁺ fibers in the hippocampus. Note the dense ChR2-EYFP⁺ fibers in DG, CA2, and CA2/CA3a border, and the relatively sparse expression in CA3b/c. CA3SR, CA3 stratum radiatum. Repeated in 3 mice.

Figure 3.

CA3-projecting SuM neurons project to the regions outside of the hippocampus. Experimental procedure is the same as in Figure 2. SuM fibers are detected in other SuM targetregions: A, lateral septum (LS), medial septum (MS), and diagonal band of Broca (DB); B, lateral hypothalamus (LH); C, entorhinal cortex (EC); D, reuniens thalamic nucleus (Re); and E, mediodorsal thalamic nucleus (MD).

Figure 4.

Optogenetic simulation of SuM input leads to minimal excitation, but strong feedforward inhibition onto CA3 PNs. A, Sample confocal images represent biocytin-filled CA2 and CA3a PNs from a transverse hippocampal slice that underwent whole-cell recording. Expanded views (right) represent the presence and absence of thorny excrescences (arrowheads) in CA3a and CA2 PNs, respectively. B, Membrane voltage responses of CA2 (left) and CA3a (right) PNs in response to 1 s somatic current injections. C, Voltage-clamp recording represents the light-evoked EPSC responses in PNs in CA2 and CA3a, but not in CA3b or CA3c. D, Percent of CA2 and CA3 PNs that receive light-evoked EPSCs along the transverse axis. Numbers of cells shown above bars. E, Group data of the amplitude of light-evoked EPSC of CA2 (n = 10 cells/7 slices/6 mice) and CA3a PNs (n = 7 cells/5 slices/5 mice). *p < 0.05 (unpaired t test). F, Sample confocal images represent biocytin-filled CA3 PNs from a hippocampal slice that underwent whole-cell recordings. At least three CA3 PNs from a, b, and c subregions were recorded in a same slice. G, Voltage-clamp recording represents the light-evoked IPSC (holding potential at 0 mV) from CA3a, b, and c. Sample IPSC traces of three CA3 cells were from the same slice. H, Summary data of light-evoked ISPCs in CA3a, b, and c. Each line indicates CA3 PNs from the same slice. n = 5 slices/3 mice. *p < 0.05 (one-way repeated-measures ANOVA followed by a Tukey test). I, Schematic illustration of the position of CA3 PNs that displayed light-evoked IPSC responses. Light-evoked IPSCs were detected in all CA3 PNs tested along the transverse axis. J, K, Sample traces (J) and group data (K) of light-evoked ISPCs in the absence or presence of DNQX (20 µm) and APV (50 µm). n = 5 cells/5 slices/3 mice. *p < 0.05 (paired t test). L, M, Sample traces (L) and group data (M) of light-evoked ISPCs in the absence or presence of DAMGO (100 nm). n = 5 cells/5 slices/2 mice. *p < 0.05 (paired t test).

Figure 5.

Pseudotyped rabies virus tracing reveals the monosynaptic projection from SuM to PV⁺ neurons in CA3. A, Experimental procedure and timeline. B, Top left, Sample confocal image represents the viral injection site in dorsal CA3 from a PV-Cre mouse. Top right, Expanded view represents a starter cell (mCherry⁺/GFP⁺) that is also immunopositive for PV staining (arrowhead). Bottom, Expanded views of the starter cell. C, Sample confocal images represent mCherry⁺ cells observed in SuM (top). Two mCherry⁺ cells in SuM shown in the expanded views (bottom). D, Sample confocal image represents the viral injection site in dorsal CA3 from a SOM-Cre mouse. E, No mCherry⁺ cells detected in SuM (repeated in 4 SOM-Cre mice).

Figure 6.

SuM provides monosynaptic excitation into CA3 PV⁺ neurons. A, Genetic strategy to target tdTomato into PV⁺ neurons by crossing a floxed tdTomato reporter line (Ai9) with a PV-Cre line. B, Experimental procedure and timeline. C, Representative confocal image shows the hippocampal slice from a PV-tdTomato mouse that was injected with AAV-ChR2-EYFP into SuM. D, Schematic diagram of a likely SuM-to-CA3 circuit. Rec, Recording patch pipette. E, Sample confocal image represents a biocytin-filled tdTomato⁺ neuron in CA3 (arrows) that underwent whole-cell recording. F, Current-clamp recording shows the membrane voltage responses to 1 s somatic injections of indicated currents from a tdTomato⁺ neuron in CA3. G, H, Sample traces (G) and group data (H) of light-evoked EPSCs in the absence and presence of DNQX/APV. n = 4 cells/4 slices/3 mice. *p < 0.05 (paired t test). I, Sample light-evoked EPSP (top) and EPSC (bottom) under current-clamp and voltage-clamp recordings in the presence of TTX (0.5 µm) and 4-AP (1 mm). Repeated in 3 cells/3 slices/1 mice. J, Group data of latencies of EPSCs in CA2/3 PNs (PN-EPSC, n = 12 cells/8 slices/6 mice), EPSCs in CA3 PV⁺ neurons (PV-EPSC, n = 16 cells/12 slices/6 mice), and IPSCs in CA3 PNs (PN-IPSC, n = 17 cells/6 slices/3 mice). ***p < 0.001; n.s., Not significant; one-way ANOVA followed by a Tukey test. K, Schematic illustration of the position of the recorded tdTomato⁺ cells in CA3. The cells that received light-evoked EPSC responses were evenly distributed along the transverse axis. n = 36 cells/18 slices/7 mice. L, Amplitude of light-evoked EPSC of each cell plotted against their normalized position along the transverse axis, with “0” as the most distal and “1” as the most proximal. M, Group data of light-evoked EPSC amplitude from CA3a (n = 6 cells/6 slices/4 mice), CA3b (n = 2 cells/2 slices/2 mice), and CA3c (n = 6 cells/6 slices/4 mice). n.s., Not significant (one-way ANOVA).

Figure 7.

The absence of monosynaptic GABAergic connection between SuM and CA3 PV⁺ neurons. A, A schematic view of SuM-to-CA3 circuit and experimental configuration showing target recording of PV⁺ neurons. PV⁺ neurons provide mutual inhibition. B, Voltage-clamp recording traces from a PV⁺ neuron show light-evoked IPSC (hold potential = 0 mV) and EPSC (holding potential = –75 mV) in the absence (black) and presence (red) of DNQX/APV. C, Expanded view shown in B. IPSC onset is slower than EPSC onset. D, Group data of light-evoked IPSC amplitude in the absence and presence of DNQX/APV. n = 5 cells/5 slices/2 mice. *p < 0.05 (paired t test). E, Group data of light-evoked EPSC latencies (n = 16 cells/12 slices/6 mice) versus IPSC (n = 5 cells/5 slices/2 mice). ***p < 0.001 (unpaired t test).

Figure 8.

Activation of SuM excites both CA3 PV⁺ neurons and DG granule cells. A, Sample confocal image represents a biocytin-filled DG granule cell (GC) that underwent whole-cell recording. B, Current-clamp recording shows membrane voltage responses to 1 s depolarizing or hyperpolarizing current injections in a DG granule cell. C, Voltage-clamp recording shows light-evoked EPSC recorded from a DG granule cell. D, Percent of CA3 PV⁺ neurons and DG granule cells that show light-evoked EPSC responses. Number of cells shown above bars. E, Group data of light-evoked EPSCs from individual CA3 PV⁺ neurons (n = 14 cells/10 slices/6 mice) and DG granule cells (n = 11 cells/7 slices/6 mice). n.s., Not significant (unpaired t test). F, Group data of light-evoked EPSCs from individual animals. Each circle represents the mean EPSC value from 1 animal. n = 4 mice; paired t test. G, Sample traces (top) and group data (bottom) of EPSC responses in CA3 PV⁺ neurons (n = 7 cells/6 slices/4 mice) and DG granule cells (n = 5 cells/4 slices/3 mice) in response to a 20 Hz train of 10 pulse light stimulation. **p < 0.01 (two-way repeated-measures ANOVA followed by a Tukey test). H, Sample traces of four trials represent suprathreshold spikes evoked by a 20 Hz train of 10 pulse light stimulation from a CA3 PV⁺ neuron. I, Sample voltage trace represents subthreshold membrane response evoked by a 20 Hz train of 10 pulse light stimulation in a DG granule cell. J, Probability of spikes evoked by light stimulation in CA3 PV⁺ neurons versus DG granule cells. Numbers of cells shown above bars.

Figure 9.

SuM simultaneously excites DG granule cells and suppresses CA3 PNs. A, Sample traces (left) and group data (right) of membrane voltage responses evoked by single light pulses under current clamp in CA3 PNs (n = 19 cells/11 slices/5 mice) versus DG granule cells (n = 6 cells/4 slices/3 mice) at resting potential. ***p < 0.001 (unpaired t test). B, Sample traces (left) and group data (right) of membrane voltage responses evoked by single light pulses in CA3 PNs (n = 19 cells/11 slices/5 mice) versus DG granule cells (n = 6 cells/4 slices/3 mice) while holding the membrane potential at –50 mV. ***p < 0.001 (unpaired t test). C, D, Sample traces (C) and group data (D) of membrane voltage responses evoked by a 10 Hz train of 10 pulse light stimulation at resting potential (n = 4 cells/3 slices/1 mouse) or at –50 mV (n = 7 cells/5 slices/3 mice) in CA3 PNs.