Glutamatergic neurons of the mouse medial septum and diagonal band of Broca synaptically drive hippocampal pyramidal cells: relevance for hippocampal theta rhythm

Abstract

Neurons of the medial septum and diagonal band of Broca (MS-DBB) provide an important input to the hippocampus and are critically involved in learning and memory. Although cholinergic and GABAergic MS-DBB neurons are known to modulate hippocampal activity, the role of recently described glutamatergic MS-DBB neurons is unknown. Here, we examined the electrophysiological properties of glutamatergic MS-DBB neurons and tested whether they provide a functional synaptic input to the hippocampus. To visualize the glutamatergic neurons, we used MS-DBB slices from transgenic mice in which the green fluorescent protein is expressed specifically by vesicular glutamate transporter 2-positive neurons and characterized their properties using whole-cell patch-clamp technique. For assessing the function of the glutamatergic projection, we used an in vitro septohippocampal preparation, electrically stimulated the fornix or chemically activated the MS-DBB using NMDA microinfusions and recorded postsynaptic responses in CA3 pyramidal cells. We found that glutamatergic MS-DBB neurons as a population display a highly heterogeneous set of firing patterns including fast-, cluster-, burst-, and slow-firing. Remarkably, a significant proportion exhibited fast-firing properties, prominent I(h), and rhythmic spontaneous firing at theta frequencies similar to those found in GABAergic MS-DBB neurons. Activation of the MS-DBB led to fast, AMPA receptor-mediated glutamatergic responses in CA3 pyramidal cells. These results describe for the first time the electrophysiological signatures of glutamatergic MS-DBB neurons, their rhythmic firing properties, and their capacity to drive hippocampal principal neurons. Our findings suggest that the glutamatergic septohippocampal pathway may play an important role in hippocampal theta oscillations and relevant cognitive functions.

Figure 1.

VGLUT2-eGFP-(+) MS-DBB neurons functionally release glutamate. A, An isolated fluorescent MS-DBB neuron cultured from VGLUT2-eGFP transgenic mice. B, Autaptic neurons were held in voltage-clamp and stimulated to fire an action potential, resulting in neurotransmitter release and postsynaptic autaptic currents. An example of an autaptic EPSC displayed by a VGLUT2-eGFP-(+) MS-DBB neuron is shown. C, A typical VGLUT2-eGFP-(+) cell exhibited an EPSC that was completely and reversibly blocked by 20 μm DNQX, indicating that the neuron released glutamate. Scale bars, 500 pA, 5 ms.

Figure 2.

Electrophysiological characteristics of glutamatergic MS-DBB neurons. A, A VGLUT2-eGFP-(+) MS-DBB neuron (soma indicated by an arrow) in an acute slice. B, Approximate locations of recorded VGLUT2-eGFP-(+) neurons in the MS-DBB are indicated, color coded by firing pattern; cluster- and burst-firing glutamatergic neurons were found in lateral regions of the MS-DBB, fast-firing cells were located more medially, slow-firing neurons were found in all MS-DBB subregions. C, Cluster-firing glutamatergic neurons displayed clusters of spikes observable during 5 s depolarizing steps (right), interspersed with subthreshold oscillations (right, inset). D, Upon hyperpolarization, some fast-firing glutamatergic neurons showed a prominent depolarizing sag that was completely blocked by 50 μm ZD7288 (inset). E, Other fast-firing neurons possessed little or no sag. F, Burst-firing glutamatergic neurons presented bursts of several action potentials when depolarized from −80 mV (right) but fired regularly from −60 mV (left). G, Slow-firing glutamatergic neurons fired in a tonic manner at a low rate and typically showed little sag. Numbers in brackets indicate number of cells. Calibration: 20 mV, 250 ms (voltage traces), 100 pA (current steps).

Figure 3.

Rhythmic spontaneous firing of glutamatergic and GABAergic MS-DBB neurons. Ai, A subset of fast-firing glutamatergic MS-DBB neurons displayed highly rhythmic spontaneous firing in theta frequencies that was not driven by rhythmic extrinsic input (top: raw continuous voltage traces for 10 s at Vrest and at −70 mV; bottom: two 1 s segments enlarged to show the extremely regular spontaneous firing and the lack of rhythmic synaptic input). Aii, The autocorrelogram of the spontaneous spikes recorded for 30 s with the theta index value indicated. Notice the clearly defined peaks and the high theta index value, indicating highly rhythmic spontaneous firing. B, The theta index was calculated from the autocorrelogram, defined as the ratio a/b where b is the amplitude of the second peak and a is the amplitude of the rhythmic portion of the second peak. Ci, Cii, Some fast-firing GABAergic MS-DBB neurons also showed highly rhythmic spontaneous firing. D, A scatterplot of theta index values for glutamatergic and GABAergic MS-DBB neurons shows that the degree of rhythmicity in spontaneous firing was not significantly different between the two groups. Ei, Eii, Glutamatergic MS-DBB neurons showed highly rhythmic spontaneous firing in the presence of blockers for synaptic inputs (10 μm DNQX, 5 μm bicuculline, 5 μm tubocurarine, 25 μm AP-5, 10 μm atropine). Calibration: 10 mV, 1 s.

Figure 4.

Electrical stimulation of septohippocampal fibers induced fast-onset glutamatergic responses in CA3 pyramidal cells. Ai, The septohippocampal preparation containing a hemiseptum, ipsilateral hippocampus, and intact fornix-fimbria pathway was cut at an angle to reveal the CA3 pyramidal layer and placed in the recording chamber as shown. A stimulating electrode was positioned on the septal side of the fornix and CA3 pyramidal cells were recorded using a patch pipette. Aii, Aiii, CA3 pyramidal cells were identified by their location (Aiii) and electrophysiological criteria (Aii) (calibration: 20 mV, 250 ms). Bi, At −45 mV, in most cases fornix stimulation led to both an early-onset EPSP (inset) and a late-onset IPSP. Applying bicuculline (5 μm) revealed a potent EPSP that could depolarize some pyramidal cells to spike threshold (right). Bii, Some pyramidal cells responded with only the late-onset IPSP that was blocked by bicuculline. C, In those cells displaying both EPSP and IPSP, bicuculline was applied to isolate the EPSP and this component was subtracted from control to calculate the “IPSP” component. D, Examination of the onset latencies for EPSPs, calculated IPSPs, versus actual IPSPs indicates that the EPSPs occurred with a significantly faster onset compared with the other responses (p < 0.0001). Numbers in brackets indicate number of cells. E, F, The EPSP was mediated by glutamate as it was completely blocked by 20 μm DNQX (E), and repeated stimuli at 5 Hz could evoke rhythmic EPSPs (F). Calibration, unless otherwise noted: 2 mV, 50 ms. a.c., Anterior commissure.

Figure 5.

Local NMDA microinfusions to the MS-DBB triggered glutamatergic responses in CA3 pyramidal cells. Ai, In the septohippocampal preparation, MS-DBB neurons were locally activated by infusing small amounts of NMDA directly into the MS-DBB area using a puffer pipette while recording from CA3 pyramidal cells. Aii, A single NMDA application (200 μm, 100 ms) robustly depolarized a nearby VGLUT2-eGFP-(+) MS-DBB neuron. B, CA3 pyramidal cells responded to NMDA injections in the MS-DBB with EPSPs that were clearly time-locked to the stimuli (left: four consecutive trials at −80 mV; right top: averaged trace at −80 mV; right bottom: a raw trace at −60 mV). C, The EPSPs were abolished by 20 μm DNQX, indicating that the response was mediated by glutamate. D, A new model of the septohippocampal network: a subset of glutamatergic MS-DBB neurons may serve as intrinsic rhythm generators that can contribute a rhythmic excitatory drive to the local septal network as well as to the hippocampus. In the model, solid lines represent physiologically confirmed pathways and dotted lines indicate pathways that remain to be demonstrated. ACh, Acetylcholine; Glut, Glutamate; LS, lateral septum; S.P., stratum pyramidale; S.O., stratum oriens; P, pyramidal cell.