GABAergic projections from the medial septum selectively inhibit interneurons in the medial entorhinal cortex

Abstract

The medial septum (MS) is required for theta rhythmic oscillations and grid cell firing in the medial entorhinal cortex (MEC). While GABAergic, glutamatergic, and cholinergic neurons project from the MS to the MEC, their synaptic targets are unknown. To investigate whether MS neurons innervate specific layers and cell types in the MEC, we expressed channelrhodopsin-2 in mouse MS neurons and used patch-clamp recording in brain slices to determine the response to light activation of identified cells in the MEC. Following activation of MS axons, we observed fast monosynaptic GABAergic IPSPs in the majority (>60%) of fast-spiking (FS) and low-threshold-spiking (LTS) interneurons in all layers of the MEC, but in only 1.5% of nonstellate principal cells (NSPCs) and in no stellate cells. We also observed fast glutamatergic responses to MS activation in a minority (<5%) of NSPCs, FS, and LTS interneurons. During stimulation of MS inputs at theta frequency (10 Hz), the amplitude of GABAergic IPSPs was maintained, and spike output from LTS and FS interneurons was entrained at low (25-60 Hz) and high (60-180 Hz) gamma frequencies, respectively. By demonstrating cell type-specific targeting of the GABAergic projection from the MS to the MEC, our results support the idea that the MS controls theta frequency activity in the MEC through coordination of inhibitory circuits.

Keywords: gamma; interneuron; lamina organization; medial entorhinal cortex; medial septum; theta.

Figure 1.

Labeling of axonal projections from MS to MEC. A, Diagram illustrating AAV injection site in the MS and labeled axonal projections (red) to the MEC. B, ChR2-mCherry-expressing cells (red) in a horizontal section containing the MS and also labeled with NeuroTrace (green). C, Horizontal section of the MEC, with cell bodies labeled with NeuroTrace (top left), MS axons expressing ChR2-mCherry (top right), and merged image (bottom left). Normalized mCherry fluorescence quantification reveals differences between layers (bottom right; *p < 0.05, Tukey's test). Scale bars, 200 μm.

Figure 2.

Responses to activation of MS fibers across different MEC cell types. A, Examples of averaged (n = 5) IPSPs and EPSPs evoked by photostimulation (blue bar) of ChR2-positive MS axons recorded at holding potentials on either side of the chloride reversal potential. No stimulation (gray), GABA_AR (red), and iGluR (blue) blockade traces are also shown. B, Representative responses of FS interneurons, LTS interneurons, NSPCs, and SCs to injection of positive and negative current steps. The cells are the same as in A. C, Streptavidin (red) staining of biocytin-filled cells recorded in A and B. ChR2-venus MS axons are in green. Inset displays 40× image of dendritic regions indicated in the main image, showing the absence and presence of spines in inhibitory and excitatory neurons, respectively. Scale bar, 50 μm.

Figure 3.

MS GABAergic fibers target FS and LTS interneurons in MEC. A, Fraction of neurons of each cell type responding to photostimulation in MEC. Numbers indicate responding cells and total number of recorded cells. B, Cumulative probability plots of IPSP amplitude, latency, rise, and decay time constants for responsive inhibitory neurons. C, Fraction of FS and LTS interneurons in each layer receiving GABAergic input from the MS.

Figure 4.

MS GABAergic fibers entrain FS and LTS interneuron firing. A, Voltage-clamp responses in control (black), in the presence of iGluR and AChR antagonists (blue); and in the presence of iGluR, AChR, and GABA_AR antagonists (red). Holding potential, 0 mV. B, Example responses of an FS interneuron to activation of MS GABAergic input at theta frequency (10 Hz). Mean trace, black; individual traces, gray. C, Ten consecutive responses from B on an expanded time scale. D, Normalized IPSP amplitude plotted as a function of stimulus number. E, Example response during the injection of current sufficient for maintained action potential firing without (left) and with (right) theta frequency activation of MS inputs to an FS neuron. F, Ten consecutive responses triggered from the time of each light stimulus illustrate entrainment of rebound spikes. G, Binned counts from 100 stimuli of action potentials as a function of time from the light pulse, illustrating measurement of pause time (red arrow) and frequency (blue arrow).