Different Subgroups of Cholinergic Neurons in the Basal Forebrain Are Distinctly Innervated by the Olfactory Regions and Activated Differentially in Olfactory Memory Retrieval

Abstract

The mammalian basal forebrain (BF), a heterogenous structure providing the primary cholinergic inputs to cortical and limbic structures, plays a crucial role in various physiological processes such as learning/memory and attention. Despite the involvement of the BF cholinergic neurons (BFCNs) in olfaction related memory has been reported, the underlying neural circuits remain poorly understood. Here, we combined viral trans-synaptic tracing systems and ChAT-cre transgenic mice to systematically reveal the relationship between the olfactory system and the different subsets of BFCNs. The retrograde adeno-associated virus and rabies virus (AAV-RV) tracing showed that different subregional BFCNs received diverse inputs from multiple olfactory cortices. The cholinergic neurons in medial and caudal horizontal diagonal band Broca (HDB), magnocellular preoptic area (MCPO) and ventral substantia innominate (SI; hereafter HMS complex, HMSc) received the inputs from the entire olfactory system such as the olfactory bulb (OB), anterior olfactory nucleus (AON), entorhinal cortex (ENT), basolateral amygdala and especially the piriform cortex (PC) and hippocampus (HIP); while medial septum (MS/DB) and a part of rostral HDB (hereafter MS/DB complex, MS/DBc), predominantly from HIP; and nucleus basalis Meynert (NBM) and dorsal SI (hereafter NBM complex, NBMc), mainly from the central amygdala. The anterograde vesicular stomatitis virus (VSV) tracing further validated that the major target of the OB to the BF is HMSc. To correlate these structural relations between the BFCNs and olfactory functions, the neurons activated in the BF during olfaction related task were mapped with c-fos immunostaining. It was found that some of the BFCNs were activated in go/no-go olfactory discrimination task, but with different activated patterns. Interestingly, the BFCNs in HMSc were more significantly activated than the other subregions. Therefore, our data have demonstrated that among the different subgroups of BFCNs, HMSc is more closely related to the olfactory system, both structurally and functionally. This work provides the evidence for distinct roles of different subsets of BFNCs in olfaction associated memory.

Keywords: BFCNs; neuronal circuit; olfactory associated memory; subpopulation; virus tracing tools.

Figure 1

Retrograde AAV-RV viral tracing. (A) Time-course of AAV-RV injections for trans-monosynaptic labeling. (B) Diagram of RV injection site, medial septum/diagonal band Broca complex (MS/DBc; Left), HMS complex (HMSc; Middle) and nucleus basalis Meynert complex (NBMc; Right). (C) Range of starter cells near the injection sites in coronal brain sections, MS/DBc (Upper), HMSc (Middle) and NBMc (Down). The starter cells (yellow) were these co-infected by AAV (green) and RV (red), which were distributed in the given subpopulation range. Scale Bar, 500 μm. (D) The percent of starter cells in the neurons infected by AAV-Dio-TVA-GFP. (E) The percent of cholinergic starter cells. (F) Enlarged views of the starter cells in MS/DBc (Top), HMSc (Middle) and NBMc (Bottom). AAV-Dio-GFP-TVA (Green), RV-ENVA-ΔG-Desred (Red), starter cell (Yellow). Scale bar, 20 μm. *P < 0.05, **P < 0.01 and ***P < 0.001.

Figure 2

Quantitative analysis of the olfactory inputs onto the different basal forebrain cholinergic neuron (BFCN) subpopulations. (A) The percentage of the olfactory inputs onto the subpopulations of BFCNs out of the total inputs across the brain. (B) Proportion of every olfactory area inputs and whole brain inputs to cholinergic neurons in the different subpopulations of BFCNs, MS/DBc (Top), HMSc (Middle) and NBMc (Bottom). (C) Proportion of every olfactory area inputs and whole olfactory regions to cholinergic neurons in the different subpopulations of BFCNs. MS/DBc (Top), HMSc (Middle) and NBMc (Bottom). *P < 0.05, **P < 0.01 and ***P < 0.001.

Figure 3

The outputs of the olfactory bulb (OB) to the BF. (A) Diagram of vesicular stomatitis virus (VSV) injection site. (B) Schematic of VSV anterograde transsynaptic labeling. (C) VSV infected olfactory relative brain regions by 72 h. (D) VSV infected olfactory relative brain regions by 96 h. Neurons (red) were infected by VSV. Scale Bar, 500 μm. (E) Quantitative analysis of the cholinergic neurons infected by VSV in different subsets of the BF. (F–H) Confirmation of cholinergic neurons infected by VSV in HMSc. ChAT+ neurons by anti-Choline Acetyltransferase (green), VSV+ neurons (Red), Merge (Yellow): ChAT+ neurons infected by VSV. Scale bar, 20 μm. n = 4. *P < 0.05, **P < 0.01 and ***P < 0.001.

Figure 4

BF neurons were activated in go/no-go olfactory discrimination task. (A,B) Diagrams of olfactory go/no-go discrimination task. (C) Behavioral performance of two groups during test (day 6) following 5-day training in olfactory go/no-go discrimination task. (D) Total amount of the activated BF neurons among control (white), ONT (gray) and GNG group (red). The quantity of the activated BF neurons in GNG group was higher than those in control group and ONT group. (E,F) The total quantity and mean density of activated BF neurons in different subpopulations. The quantity and density of activated neurons in each subpopulation of BF in GNG group was higher than those in control group and ONT group. Data are showed as mean ± SEM. *P < 0.05, **P < 0.01 and ***P < 0.001; one-way ANOVA was used. n = 5.

Figure 5

BFCNs were activated and showed a characteristic distribution pattern in go/no-go olfactory discrimination task. (A) The square in coronal atlas example indicated the site of HMSc where the cholinergic neurons were activated. (B–D) Representative coronal sections showing co-expressions of c-Fos+ (Red) and ChAT+ neurons (Green) in HMSc for the three paradigms. Scale bar, 20 μm. The arrows point to neurons co-expressing c-Fos and ChAT. (E) BFCNs were activated and showed similar activated patterns among the three groups in go/no-go olfactory discrimination task. In GNG group, the numbers of activated cholinergic neurons in the whole BF and in different subsets were higher than those in control group and ONT group. (F–G) The total quantity and mean density of the activated BFCNs were higher in HMSc than MS/DBc and NBMc among the three groups, and especially go/no-go group. Data are showed as mean ± SEM. *P < 0.05, **P < 0.01 and ***P < 0.001; one-way ANOVA was used. n = 5.

Figure 6

Proportion of the activated cholinergic neurons in total activated neurons and total cholinergic neurons in different subpopulations. (A) Proportion of the activated BFCNs over the total c-fos+ cells in different behavioral paradigms. In GNG group, the proportion of the activated cholinergic neurons in HMSc was much higher than the other two subpopulations. Moreover, higher percentages of the activated BFCNs were found in HMSc and NBMc in ONT group. (B) Proportion of the activated BFCNs over the total ChAT+ cells in different behavioral paradigms. Among the three sub-groups, the highest proportion of the activated cholinergic neurons was in HMSc GNG group. Data are showed as mean ± SEM. *P < 0.05, **P < 0.01 and ***P < 0.001; one-way ANOVA was used. n = 5.