Intrinsic membrane properties and cholinergic modulation of mouse basal forebrain glutamatergic neurons in vitro

Abstract

The basal forebrain (BF) controls sleep-wake cycles, attention and reward processing. Compared to cholinergic and GABAergic neurons, BF glutamatergic neurons are less well understood, due to difficulties in identification. Here, we use vesicular glutamate transporter 2 (vGluT2)-tdTomato mice, expressing a red fluorescent protein (tdTomato) in the major group of BF glutamatergic neurons (vGluT2+) to characterize their intrinsic electrical properties and cholinergic modulation. Whole-cell, patch-clamp recordings were made from vGluT2+ neurons in coronal BF slices. Most BF vGluT2+ neurons were small/medium sized (<20µm), exhibited moderately sized H-currents and had a maximal firing frequency of ∼50Hz. However, vGluT2+ neurons in dorsal BF (ventral pallidum) had larger H-currents and a higher maximal firing rate (83Hz). A subset of BF vGluT2+ neurons exhibited burst/cluster firing. Most vGluT2+ neurons had low-threshold calcium spikes/currents. vGluT2+ neurons located in ventromedial regions of BF (in or adjacent to the horizontal limb of the diagonal band) were strongly hyperpolarized by the cholinergic agonist, carbachol, a finding apparently in conflict with their increased discharge during wakefulness/REM sleep and hypothesized role in wake-promotion. In contrast, most vGluT2+ neurons located in lateral BF (magnocellular preoptic area) or dorsal BF did not respond to carbachol. Our results suggest that BF glutamatergic neurons are heterogeneous and have morphological, electrical and pharmacological properties which distinguish them from BF cholinergic and GABAergic neurons. A subset of vGluT2+ neurons, possibly those neurons which project to reward-related areas such as the habenula, are hyperpolarized by cholinergic inputs, which may cause phasic inhibition during reward-related events.

Keywords: Alzheimer’s disease; cortical activation; patch-clamp; sleep; vesicular glutamate transporter; whole-cell.

Figure 1. Subregional location and intrinsic membrane properties of BF vGluT2+ neurons recorded in this study

A and C Schematic diagram showing the BF subregions where electrophysiological recordings were made (Bregma 0.14 mm. Adapted from (Franklin and Paxinos, 2008). HDB: horizontal limb of the diagonal band. LPO: lateral preoptic area. MCPO: magnocellular preoptic nucleus. VP: ventral pallidum. B-D. Top, Black and white infrared differential interference contrast (IR-DIC) and fluorescent (tdTomato) images, scale bar: 10 μm. Bottom, recordings of the voltage response to hyperpolarizing and depolarizing steps. B1 and B2. Examples of two different types of vGluT2+ neurons in the ventromedial BF. Both types of neurons showed a small depolarizing sag during hyperpolarizing steps. Neurons showed either tonic (B1) or burst firing (labeled with asterisks in B2) during depolarizing steps. C and D. Images and intrinsic membrane properties of vGluT2 neurons in lateral and dorsal BF respectively. Note that dorsal vGluT2+ neurons (D) showed a larger sag (black arrow) during hyperpolarizing steps when compared to others types of BF vGluT2+ neurons (B and C).

Figure 2. A selective blocker for hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, ZD7288 (50 μM), blocked hyperpolarization activated inward currents in BF vGluT2+ neurons

A. A representative neuron was stepped to -135 mV for 1s to induce an inward current (Ih) (black trace) which was blocked by ZD7288 (grey trace). The amplitude of the current was measured as the difference between the two points labeled with arrows. B. I-V plot with the voltage step values as the x-axis and Ih amplitudes shown on the y axis. Data were plotted as mean ± SEM (n=7).

Figure 3. The selective T-type calcium channel inhibitor, TTA-P2, blocked low-threshold spikes/currents in BF vGluT2+ neurons

A. In current clamp, a representative vGluT2+ neuron showed rebound spikes following 1 s hyperpolarizing current injections (-180 or -300 pA). The neuron was initially held at ∼-70 mV. B. The same neuron shown in A was incubated with 500 nM TTX to block sodium-dependent action potentials. A hyperpolarizing current injection induced a low-threshold spike (control: black trace) which was blocked by TTA-P2 (red trace). C. In voltage clamp and in the presence of TTX, a representative vGluT2+ neuron showed an inward current at the removal of a 1s voltage step to -125 mV (control: black trace). TTA-P2 also blocked this rebound inward current (red trace). D. I-V plot with the voltage steps shown on the x-axis and the amplitude of rebound inward currents shown on the y-axis. Data were plotted as mean± SEM (n=6).

Figure 4. The cholinergic agonist, carbachol, hyperpolarized vGluT2+ neurons located in the ventromedial basal forebrain

Carbachol (50 μM) was bath applied for 2 min in the presence of TTX (500 nM). A current step of -50 pA was applied every 15 s to test for changes in input resistance. Hyperpolarizations induced by carbachol were associated with a decrease in input resistance, indicating opening of ion channels. Abbreviation: DC – direct current.