A New Neural Pathway from the Ventral Striatum to the Nucleus Basalis of Meynert with Functional Implication to Learning and Memory

Abstract

The cholinergic neurons in the nucleus basalis of Meynert (NBM) are among the first group of neurons known to become degenerated in Alzheimer's disease, and thus the NBM is proposed to be involved in learning and memory. The marginal division (MrD) of the striatum is a newly discovered subdivision at the ventromedial border of the mammalian striatum and is considered to be one part of the ventral striatum involved in learning and memory. The present study provided evidence to support the hypothesis that the MrD and the NBM were structurally connected at cellular and subcellular levels with functional implications in learning and memory. First, when wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP) was stereotaxically injected into the NBM, fusiform neurons in the MrD were retrogradely labeled with WGA-HRP gray-blue particles and some of them were double stained in brown color by AchE staining method. Thus, cholinergic neurons of the MrD were shown to project to the neurons in the NBM. Second, in anterograde tract-tracing experiments where WGA-HRP was injected to the MrD, the labeled WGA-HRP was found to be anterogradely transported in axons from the MrD to the synaptic terminals with dendrites, axons, and perikaryons of the cholinergic neurons in the NBM when observed under an electronic microscope, indicating reciprocal structural connections between the MrD and the NBM. Third, when bilateral lesions of the MrD were injured with kainic acid in rats, degenerative terminals were observed in synapses of the NBM by an electronic microscope and severe learning and memory deficiency was found in these rats by the Y-maze behavioral test. Our results suggest reciprocal cholinergic connections between the MrD of the ventral striatum and the NBM, and implicate a role of the MrD-NBM pathway in learning and memory. The efferent fibers of cholinergic neurons in the NBM mainly project to the cortex, and severe reduction of the cholinergic innervation in the cortex is the common feature of Alzheimer's patients. The newly discovered cholinergic neural pathway between the MrD of the ventral striatum and the NBM is supposed involved in the memory circuitries of the brain and probably might play a role in the pathogenesis of the Alzheimer's disease.

Keywords: Alzheimer’s disease; Electronic microscopy; Immunocytochemistry; Learning and memory; The nucleus basalis of Meynert (NBM); The striatum; Tract tracing; Y-maze.

Fig. 1

Location and cytoarchitectural characteristics of the MrD in the ventral portion of the striatum in the rat brain. The diagram on the left side indicates two frontal sections (A and B) of the rat brain. The upper right panel shows that the PHA-L-labeled neuronal bodies in the dorsal part of the striatum (caudoputamen, CPu) are mostly of round or triangular shape. The lower right panel illustrates the MrD located in the ventromedial portion of the striatum (CPu) and dorsolateral to the globus pallidus (GP). The MrD consists of a band of fusiform neurons labeled by PHA-L, which distinguishes the MrD from the rest of the striatum (CPu) and the GP. Cpu, caudoputamen of the striatum corpus; GP, globus pallidus; MrD, marginal division of the striatum

Fig. 2

Determination of the projection from the MrD to the NBM by WGA-HRP retrograde tracing method. a The WGA-HRP injecting site in the NBM is marked (arrow a) where little WGA-HRP tracer shown as blue substance could be detected. Two cholinergic neurons of NBM were observed at the right lower corner (arrow b). b WGA-HRP stained as gray-blue particles retrogradely labeled fusiform somata of neurons in the MrD of the ventral striatum (arrow c) in the rat brain. Some of the WGA-HRP-labeled neurons were stained with brown color background, which were AchE-WGA-HRP double-labeled neurons (arrow d). The AchE-WGA-HRP double-labeled neurons were cholinergic neurons in MrD, which projected to the NBM. MrD, marginal division of the striatum; NBM, basal nucleus of Meynert. Magnification = ×400

Fig. 3

The projection from the MrD to the NBM by WGA-HRP retrograde tracing method. The conditions were the same as in Fig. 2. a WGA-HRP stained as gray-blue particles retrogradely labeled fusiform somata of neurons (arrows) in the MrD of the ventral striatum in the rat brain. b High magnification of the boxed area in a. Some fusiform neurons were double-stained in brown color with some blue particles in their cell bodies. This AchE-WGA-HRP double-stained neurons were cholinergic neurons in the MrD which project to NBM. GP, globus pallidus; MrD, marginal division of the striatum; St, striatum; scale bar = 400 μm (a) and 100 μm (b)

Fig. 4

A dot plot showing the ratio as percentage of AchE-WGA-HRP double-labeled neurons to Total WGA-HRP-labeled neurons

Fig. 5

Loss of fusiform neurons in the MrD after KA injection shown by Nissl staining. At the injection site, fusiform neurons in the MrD (upper longer arrow) were lost (pointed by two shorter parallel arrows), while the morphology of neurons in the adjacent striatum (caudoputamen, CPu) and globus pallidus (GP) is still normal. Scale bar = 100 μm

Fig. 6

Identification of AchE-containing neurons and neuronal processes in the MrD, striatum, and the NBM. The cholinergic neurons and nerve fibers were stained in dark brown color by AchE staining method. A row of cholinergic medium-size fusiform neurons stained by AchE method in brown color was observed in the MrD of the striatum (black arrow on the left). The large multi-angular cholinergic neurons in the NBM were heavily stained by AchE method in brown color (black arrow on the right). The rest portion of the striatum (St) is fully filled with AchE-positive substrates. MrD, marginal division of the striatum; NBM, nucleus basalis of Meynert; St, striatum; scale bar = 400 μm

Fig. 7

Demonstration of degenerated terminals in the NBM after KA injection into the MrD—electron microscopic analysis. Anterograde degenerative axon terminals are observed in the NBM following KA injection to the MrD of the rat brain. A terminal (a) with synaptic vesicles and swollen mitochondria is found to form an asymmetric axo-dendritic synapse (arrow) on a dendrite (d). A completely damaged axon (a1, black triangle) with decreased numbers of synaptic vesicles and a disrupted axon membrane is found just above the dendrite (d). A cholinergic neuron with a big nucleus in the NBM is occupied the right panel of the figure. MrD, marginal division of the striatum; NBM, nucleus basalis of Meynert; scale bar = 0.5 μm

Fig. 8

Electron photomicrography showing anterograde WGA-HRP-labeled terminals projected from the MrD to the NBM after WGA-HRP injection in to the MrD. Following WGA-HRP injection to the MrD, anterogradely transported WGA-HRP, which appeared as rod-like electron dense substances, labeled axon terminals (a) from the MrD to form symmetric axo-somatic synapses (arrows) with a cholinergic neuron (cn) of the NBM. MrD, marginal division of the striatum; NBM, nucleus basalis of Meynert; scale bar = 0.5 μm

Fig. 9

Summary of the neuronal connections from the MrD to the NBM using the WGA-HRP tracing method. Schematic summary of the connection from the fusiform neuron (red) in the marginal division (MrD) of the ventral striatum to the nucleus basalis of Meynert (NBM). The axons of round-shaped neurons (blue) in the other parts of the striatum (St) project to the globus pallidus (GP). b Following WGA-HRP injection to the NBM, retrogradely transported WGA-HRP, which are stained as black-blue particles, labeled some fusiform neurons in the MrD of the ventral striatum. MrD, marginal division of the striatum; NBM, nucleus basalis of Meynert. Scale bar = 100 μm. c The efferent projections (white lines) from the MrD to the NBM are observed in a dark field under a light microscope at low magnification. MrD, marginal division of the striatum; NBM, nucleus basalis of Meynert. Scale bar = 800 μm. d Following WGA-HRP injection to the MrD, anterogradely transported WGA-HRP, which appeared as rod-like electron dense substances in axon terminals from the MrD, were found in the axon-somatic synapses(arrows) of cholinergic neurons in the NBM. MrD, marginal division of the striatum; NBM, nucleus basalis of Meynert. Scare bar = 1 μm