Physiological Properties of Cholinergic and Non-Cholinergic Magnocellular Neurons in Acute Slices from Adult Mouse Nucleus Basalis

The basal forebrain is a series of nuclei that provides cholinergic input to much of the forebrain. The most posterior of these nuclei, nucleus basalis, provides cholinergic drive to neocortex and is involved in arousal and attention. The physiological properties of neurons in anterior basal forebrain nuclei, including medial septum, the diagonal band of Broca and substantia innominata, have been described previously. In contrast the physiological properties of neurons in nucleus basalis, the most posterior nucleus of the basal forebrain, are unknown.Here we investigate the physiological properties of neurons in adult mouse nucleus basalis. We obtained cell-attached and whole-cell recordings from magnocellular neurons in slices from P42-54 mice and compared cholinergic and non-cholinergic neurons, distinguished retrospectively by anti-choline acetyltransferase immunocytochemistry. The majority (70-80%) of cholinergic and non-cholinergic neurons were silent at rest. Spontaneously active cholinergic and non-cholinergic neurons exhibited irregular spiking at 3 Hz and at 0.3 to 13.4 Hz, respectively. Cholinergic neurons had smaller, broader action potentials than non-cholinergic neurons (amplitudes 64+/-3.4 and 75+/-2 mV; half widths 0.52+/-0.04 and 0.33+/-0.02 ms). Cholinergic neurons displayed a more pronounced slow after-hyperpolarization than non-cholinergic neurons (13.3+/-2.2 and 3.6+/-0.5 mV) and were unable to spike at high frequencies during tonic current injection (maximum frequencies of approximately 20 Hz and >120 Hz).Our results indicate that neurons in nucleus basalis share similar physiological properties with neurons in anterior regions of the basal forebrain. Furthermore, cholinergic and non-cholinergic neurons in nucleus basalis can be distinguished by their responses to injected current. To our knowledge, this is the first description of the physiological properties of cholinergic and non-cholinergic neurons in the posterior aspects of the basal forebrain complex and the first study of basal forebrain neurons from the mouse

Electrophysiology of ChAT⁺ and ChAT⁻ magnocellular neurons in adult mouse nucleus basalis.

<p>Electrophysiology of ChAT⁺ and ChAT⁻ magnocellular neurons in adult mouse nucleus basalis.</p

Identification of cholinergic and non-cholinergic neurons in nucleus basalis.

<p>(<b>A</b>) Examples of retrospective streptavidin (green) and anti-ChAT (red) fluorescence for two recorded neurons. In one recording the streptavidin-positive soma co-labels with anti-ChAT. Anti-ChAT fails to label the other neuron, although nearby cells are ChAT⁺, indicating that the anti-ChAT staining was successful in this slice. (<b>B</b>) Summary schematic showing the locations of nucleus basalis and of all recordings. Red and blue markers indicate the somatic locations of recordings from ChAT⁺ and ChAT⁻ neurons, respectively. Grey shading indicates the probability distribution for nucleus basalis (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011046#s2" target="_blank">Methods</a> section and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011046#pone-0011046-g001" target="_blank">figure 1</a>).</p

Location and dimensions of nucleus basalis.

<p>(<b>A</b>) Widefield image of anti-ChAT immunofluorescence of a coronal section, approximately 1mm posterior to bregma, with prominent anti-ChAT staining in nucleus basalis and caudate putamen. (<b>B</b>) A sub-region of the section in A, showing ChAT-immunoreactive somata in nucleus basalis. (<b>C</b>) Illustration of the method used to describe the location of nucleus basalis. To pool the locations of nucleus basalis from multiple animals, for each section we first overlaid the anti-ChAT image onto a brightfield image of a representative coronal section (panel 1). This aligned all anti-ChAT images to the common brightfield image. For each section we manually drew an outline around all ChAT⁺ somata in nucleus basalis (panel 2) and converted this outline to a binary mask (panel 3). These masks were averaged to give a probability density distribution for nucleus basalis (panel 4). (<b>D</b>) The probability density distribution was then aligned with a schematic of the coronal slice for display purposes. To calculate the dimensions of nucleus basalis we measured the probability density along major and minor axes of the approximately elliptical probability density distribution (dashed lines). (<b>E</b>) A threshold of 10% of the peak intensity (dashed line) in each axis gave dimensions for nucleus basalis of 2.52 mm in the ventromedial-dorsolateral (major) axis and 1.12 mm in the dorsomedial-ventrolateral (minor) axis.</p

Comparison of action potential waveforms.

<p>(<b>A</b>) Example action potentials from a ChAT⁺ (black) and a ChAT⁻ (grey) neuron, illustrating the difference in spike waveform. Spikes are aligned to threshold (filled arrowhead). The ChAT⁺ neuron has a broader action potential with a shoulder on the decaying phase (open arrowhead). (<b>B</b>) The shoulder (open arrowhead) is clearly visible in the phase plot for ChAT⁺ action potential: the shoulder reduces the maximum rate of decay of the action potential. (<b>C</b>) Threshold, amplitude, half width and decay time for action potentials in ChAT⁺ (black) and ChAT⁻ (grey) neurons. n = 9 ChAT⁺ and 30 ChAT⁻ neurons.</p

Comparison of firing rates and after-spike potentials.

<p>(<b>A</b>) Spike frequency as a function of injected current for ChAT⁺ (black symbols) and ChAT⁻ neurons (grey symbols). The shaded areas denote the full range of each population (n = 9 ChAT⁺ and 28 ChAT⁻ neurons). Inset: Spike amplitudes during trials with only three action potentials. ChAT⁺ (black symbols) and ChAT⁻ neurons (grey symbols) displayed weak spike amplitude accommodation. (<b>B</b>) Example action potentials from a ChAT⁺ (black) and a ChAT⁻ (grey) neuron, illustrating the differences in after-spike potentials. Traces were aligned to the threshold (arrowhead). Inset: Same action potentials on an expanded time scale, clearly showing the fAHP and, in the ChAT⁻ neuron, the ADP. (<b>C</b>) Plots showing the amplitudes of fAHP, ADP and sAHP for ChAT⁺ (black) and ChAT⁻ (grey) neurons. n = 9 ChAT⁺ and 30 ChAT⁻ neurons.</p

Examples of whole-cell recordings and subthreshold characteristics.

<p>(<b>A and B</b>) Maximum intensity projections of 2-photon image stacks acquired during recording from two neurons, one ChAT⁺ (A) and one ChAT⁻ (B). In each, the recording pipette is visible to the right of the soma. (<b>C and D</b>) Voltage responses to −200, −150, −100, −50, 0, 50 and +200 pA current steps for 300ms. Dashed lines indicate 0 mV. C is from the ChAT⁺ neuron in A; D from the ChAT⁻ neuron in B. (<b>E</b>) Voltage-current curves for the ChAT⁺ (black, filled symbols) and ChAT⁻ (open, grey symbols) neurons in A and B, both showing anomalous rectification at potentials hyperpolarized to rest. (<b>F</b>) Sag ratio (steady state / peak voltage) during 300 ms, −200 pA current steps in ChAT⁺ and ChAT⁻ neurons.</p

Cell-attached recordings and spontaneous firing activity.

<p>(<b>A</b>) Schematic illustration of a coronal slice approximately 1mm posterior to bregma, based on the atlas of Franklin and Paxinos (2008) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011046#pone.0011046-Sim1" target="_blank">[25]</a>. (<b>B</b>) Brightfield image of a slice showing the region around nucleus basalis during recording from a neuron in nucleus basalis. Arrowheads mark the recording pipette. (<b>C</b>) Example of a cell-attached recording from a neuron which did not exhibit spontaneous activity. Current injection (500 pA, 300 ms) evoked action potentials. Inset: action potential waveform. (<b>D</b>) Raster plots showing 10 seconds of irregular firing in 12 spontaneously firing neurons recorded in the cell-attached configuration. Each vertical line denotes the timing of an action potential and neurons are arranged in two columns, each containing the plots for 6 neurons. (<b>E</b>) Raster plots from two spontaneously firing neurons in the whole-cell configuration. The neurons were identified as ChAT+ (red) and ChAT− (blue) neurons by retrospective immunocytochemistry.</p