Modulation of Specific Sensory Cortical Areas by Segregated Basal Forebrain Cholinergic Neurons Demonstrated by Neuronal Tracing and Optogenetic Stimulation in Mice

Abstract

Neocortical cholinergic activity plays a fundamental role in sensory processing and cognitive functions. Previous results have suggested a refined anatomical and functional topographical organization of basal forebrain (BF) projections that may control cortical sensory processing in a specific manner. We have used retrograde anatomical procedures to demonstrate the existence of specific neuronal groups in the BF involved in the control of specific sensory cortices. Fluoro-Gold (FlGo) and Fast Blue (FB) fluorescent retrograde tracers were deposited into the primary somatosensory (S1) and primary auditory (A1) cortices in mice. Our results revealed that the BF is a heterogeneous area in which neurons projecting to different cortical areas are segregated into different neuronal groups. Most of the neurons located in the horizontal limb of the diagonal band of Broca (HDB) projected to the S1 cortex, indicating that this area is specialized in the sensory processing of tactile stimuli. However, the nucleus basalis magnocellularis (B) nucleus shows a similar number of cells projecting to the S1 as to the A1 cortices. In addition, we analyzed the cholinergic effects on the S1 and A1 cortical sensory responses by optogenetic stimulation of the BF neurons in urethane-anesthetized transgenic mice. We used transgenic mice expressing the light-activated cation channel, channelrhodopsin-2, tagged with a fluorescent protein (ChR2-YFP) under the control of the choline-acetyl transferase promoter (ChAT). Cortical evoked potentials were induced by whisker deflections or by auditory clicks. According to the anatomical results, optogenetic HDB stimulation induced more extensive facilitation of tactile evoked potentials in S1 than auditory evoked potentials in A1, while optogenetic stimulation of the B nucleus facilitated either tactile or auditory evoked potentials equally. Consequently, our results suggest that cholinergic projections to the cortex are organized into segregated pools of neurons that may modulate specific cortical areas.

Keywords: cholinergic facilitation; cholinergic projections; cortical evoked potentials; diagonal band of Broca; nucleus basalis magnocellularis; transgenic mice.

Figure 1

Location of the injection and deposit of fluorescent retrograde tracers. Microphotographs of coronal brain sections and schematic drawings showing the injection sites of the retrograde tracers. (A) Fast Blue (FB), deposit in the A1 cortex; (B) Fluoro-Gold (FlGo), injection in the S1 cortex. In this and in the following figures, abbreviations are: 3V, 3rd ventricle; A1, primary auditory cortex; AuD, secondary auditory cortex, dorsal area; AuV, secondary auditory cortex, ventral area; cc, corpus callosum; CM, central medial thalamic nucleus; f, fornix; ic, internal capsule; LV, lateral ventricle; mt, mamillothalamic tract; Re, reuniens thalamic nucleus; RSD, retrosplenial dysgranular cortex; S1, primary somatosensory cortex; S1BF, primary somatosensory cortex, barrel field; S1Tr, primary somatosensory cortex, trunk region; S1ULp, primary somatosensory cortex, upper lip region; S2, secondary somatosensory cortex; V1, primary visual cortex; V2L, secondary visual cortex, lateral area; V2MM, secondary visual cortex, mediomedial area; V2MM, secondary visual cortex, mediomedial area. Calibration toolbar 600 μm.

Figure 2

Distribution of labeled neurons in the VDB/HDB. (A–C) Confocal microscope images of retrogradely FlGo (S1 injection) and FB (A1 injection) labeled neurons in the VDB/HDB; (D,E) fluorescence microscope images of both Fluoro Gold and Fast Blue labeled neurons in the VDB and HDB. Note that some neurons project to both cortical areas. Dashed line indicates the medial hemispheric line. (F) Image combining fluorescent microscopy with acetylcholine transferase (ChAT) immunocytochemistry techniques in HDB. Asterisks indicates cholinergic neurons. HDB, nucleus of the horizontal limb of the diagonal band; VDB, nucleus of the vertical limb of the diagonal band. Calibration toolbar for (A–F) 250 μm.

Figure 3

Distribution of labeled neurons in B nucleus. (A,B) Microphotographs of coronal sections showing retrogradely-labeled neurons located in B nucleus at two different antero-posterior coordinates. Dashed lines delimitate the B nucleus. (C–H) Confocal microscope detail of FlGo (S1 injection) and FB (A1 injection) labeled neurons in B nucleus. Note that some neurons project to both cortical areas. Calibration toolbar for (A–H) 280 μm.

Figure 4

Distribution of labeled neurons in the VDB/HDB and B nuclei. (A) Graphic representation of number of labeled neurons in the VDB/HDB after deposit in the S1 and A1 cortices. Note that this BF area mainly project to the S1 cortex. (B) Graphic representation of the number of labeled neurons in the B nucleus after deposits in the S1 and A1 cortices. Note that this BF area project to the S1 and A1 cortices in a similar proportion. (C–F) Samples of fluorescent microscope images of single- and double-labeled neurons in HDB. (G,H) Samples of fluorescent microscope images of single- and double-labeled neurons in SI/B nucleus. Red asterisks indicate double-labeled neurons. Calibration toolbars (C,D,F–H) 230 μm, (E) 530 μm.

Figure 5

Blue light stimulation of BF neurons induces spike firing in the BF and desynchronization of the somatosensory and auditory field potentials. (A) A short-lasting blue LED stimuli induced spike firing in a representative B neuron (three superimposed traces are shown). (B) A train of stimuli (20 ms pulse duration, 5 Hz) evokes spike firing in an HDB neuron simultaneously to a desynchronization of cortical field potentials (S1 and A1 cortices). The effect lasted less than 10 s. (C) Wavelet analysis of the same trace shown in (B; S1 upper trace; A1 lower trace). Fast activity increases in both cortical field potentials after the blue light stimulation of the HDB area.

Figure 6

Blue light stimulation of BF neurons induces fast activity in the cortical field potentials. Plots of the percentage of change in the frequency band of 30 s of EEG recording respect to 30 s of a control period (100%) in the S1 and A1 cortices (left and right plots, respectively). Blue light stimulation of the HDB or B nuclei reduced the percentage of delta activity (0.5–4 Hz) and increased theta (4–10 Hz) and beta (10–30 Hz) activity during 30 s after light stimulation.

Figure 7

Blue light stimulation of HDB nucleus induces an increase of somatosensory and auditory evoked potentials. (A) Raw data shows an important increase of the somatosensory evoked potentials and a modest increase of the auditory evoked potentials (control and 5 min after blue light stimulation; thin and thick traces, respectively). (B) Plot shows the time course of evoked potential amplitude in 12 cases. The 100% represent the mean amplitude during the control period. Note the greater increase in somatosensory evoked potentials (S1) than in auditory evoked potentials (A1) by HDB stimulation (reference 0 s; vertical blue arrow). (C) Plot of the mean amplitude measured in control and at 5, 10, 20 or 30 min after blue light stimulation at HDB area. The amplitude increase was larger for somatosensory than auditory evoked potentials.

Figure 8

Blue light stimulation of B nucleus induces an increase of somatosensory and auditory evoked potentials. (A) Raw data shows that only somatosensory evoked potentials were affected by blue light stimulation at the B nucleus (control and 5 min after stimulation; thin and thick traces, respectively). (B) Plot shows the time course of evoked potential amplitude in 12 cases (light stimulation occurs at reference 0 s; vertical blue arrow). The 100% represent the mean amplitude during the control period. (C) Plot of the mean amplitude measured in control and 5, 10, 20 or 30 min after blue light stimulation at the B nucleus. The effect was smaller than after HDB stimulation.