Topographical projections from the nucleus basalis magnocellularis (Meynert) to the frontal cortex: A voltage-sensitive dye imaging study in rats

Accepted Manuscript Topographical projections from the nucleus basalis magnocellularis (Meynert) to the frontal cortex: A voltage-sensitive dye imaging study in rats Kazuaki Nagasaka, Yumiko Watanabe, Ichiro Takashima PII:

S1935-861X(17)30840-9

DOI:

10.1016/j.brs.2017.06.008

Reference:

BRS 1075

To appear in:

Brain Stimulation

Received Date: 31 January 2017 Revised Date:

31 May 2017

Accepted Date: 30 June 2017

Please cite this article as: Nagasaka K, Watanabe Y, Takashima I, Topographical projections from the nucleus basalis magnocellularis (Meynert) to the frontal cortex: A voltage-sensitive dye imaging study in rats, Brain Stimulation (2017), doi: 10.1016/j.brs.2017.06.008. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Highlights • Topographical mapping between the nucleus basalis magnocellularis/Meynert (NBM) and frontal cortex was examined.

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• Anterior and posterior NBM was stimulated and frontal activity was visualized using optical imaging.

• The anteroposterior axis of the NBM corresponded to the mediolateral axis of the

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dorsal frontal cortex.

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Short Communication

Title: Topographical projections from the nucleus basalis magnocellularis

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(Meynert) to the frontal cortex: A voltage-sensitive dye imaging study in rats

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Abbreviated Title: Topography between the NBM and frontal cortex

Authors: Kazuaki Nagasaka1,2, Yumiko Watanabe1, Ichiro Takashima1,2 Affiliations: 1Human Informatics Research Institute, National Institute of Advanced 2

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Industrial Science and Technology, 1-1-1 Umezono, Tsukuba 305-8568, Japan; Graduate School of Comprehensive Human Sciences, University of Tsukuba, 1-

1-1 Tennodai, Tsukuba 305-9577, Japan.

Correspondence should be addressed to:

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Ichiro Takashima

Human Informatics Research Institute, National Institute of Advanced Industrial Science and Technology (AIST),

1-1-1 Umezono, Tsukuba, Ibaraki 305-8568, Japan

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Tel: +81-29-861-5563, Fax: +81-29-861-5849

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E-mail: [email protected]

Pages: 14, Figures: 1 Abstract: 151 words

Whole manuscript: 1322 words (Introduction: 194, Materials & Methods: 410, Results: 189, Discussion: 529)

Keywords: basal forebrain, acetylcholine, cognitive function, motor function, DBS

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Abstract Background: The nucleus basalis magnocellularis/Meynert (NBM) has been explored as a new target for deep brain stimulation for neurological disorders. Although anatomical

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studies suggest the existence of cholinergic topographical projections of the NBM, it is still unknown whether NBM subregions differentially activate the frontal cortex. Objective: To investigate the topography between the NBM and frontal cortex.

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Methods: Electrical stimulation was applied to the anterior and posterior sites of the NBM in rats, and the evoked frontal activity was investigated using voltage-sensitive

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dye (VSD) imaging.

Results: VSD imaging revealed the functional topography of the NMB and frontal cortex: the anteroposterior axis of the NBM corresponded to the mediolateral axis of the dorsal frontal cortex.

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Conclusion: The present results suggest site-specific control of frontal neuronal activity by the NBM. These findings have practical implications, as the anterior and posterior

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respectively.

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parts of the NBM could be targeted to improve cognitive and motor function,

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Introduction The nucleus basalis of Meynert (magnocellularis in rodents) (NBM) provides the major source of cholinergic fibers to the cerebral cortex. Recently, the NBM was

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explored as a potential target for deep brain stimulation (DBS) in patients with dementia, with some studies reporting improvement in cognitive and behavioral dysfunction in patients with Alzheimer’s and Parkinson’s disease following NBM DBS [1,2]. In

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humans, the NBM extends from the level of the olfactory tubercle to that of the posterior amygdala, spanning a distance of 13–14 mm on the anteroposterior axis. A

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few anatomical studies have suggested that the posterior part of the NBM projects predominantly to the lateral frontal cortex, whereas the anterior part sends more fibers to the medial and dorsomedial frontal regions in rodents [3,4] and in monkeys [5]. Recently, the topographic organization of basal forebrain (BF) cholinergic neurons has

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been studied extensively [6,7]. However, it remains unknown whether the spatiotemporal dynamics of frontal neuronal activity differ according to the NBM activation site. Therefore, we investigated this issue using voltage-sensitive dye (VSD)

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imaging of the frontal neural activity evoked by electrical stimulation of the anterior and

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posterior sites of the NBM in anesthetized rats.

Materials and Methods All experiments were conducted in accordance with the National Institutes of

Health Guide for the Care and Use of Laboratory Animals and were approved by the Animal Care and Use Committee of the National Institute of Advanced Industrial Science and Technology. We used adult male Wistar rats (220−380 g; n = 12). The rats were anesthetized

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by intraperitoneal injection of urethane (1.3 g/kg) and placed in a stereotaxic frame (Narishige Group, Tokyo, Japan). A craniotomy was performed over the frontal cortex [anteroposterior (AP) −0.5 to 5.5 mm and mediolateral (ML) 0.0 to 4.5 mm from the

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bregma]. A concentric bipolar electrode (IMB-9002; Inter Medical, Nagoya, Japan) was used to stimulate the NBM. The dimensions of the electrode are shown in Fig. 1A (inset in the middle panel). The electrode was inserted obliquely to avoid interference between

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the objective lens and the electrode [8], after making another small craniotomy (diameter 1 mm) in the parietal bone. The insertion angle was 40° to the posterior. The

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NBM was identified during the experiments by monitoring the change in the power spectrum of the frontal local field potential (LFP). The stimulation electrode was advanced in 100-µm steps, and a stimulus train (150 µA, 500 ms, 100 Hz) was applied. When the NBM was stimulated, cortical desynchronization (i.e., from large-amplitude

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slow fluctuations to small-amplitude fast oscillation; Fig. 1A) was induced [9–11]. The power spectrum density of the LFP was analyzed using Thomson’s multitaper method [11]. Based on a cortical desynchronization index, the electrode that targeted the

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anterior NBM was placed at AP −0.9 ± 0.4 mm, ML 2.5 ± 0.2 mm, and depth 6.5 ± 0.5

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mm from the bregma. Similarly, the electrode targeting the posterior NBM was placed at AP −2.2 ± 0.5 mm, ML 3.6 ± 0.4 mm, and depth 7.0 ± 0.8 mm from the bregma. VSD imaging was performed according to a method described previously [12–

14]. After removing the dura, the exposed cortex was stained for 1 h by VSD RH-795 (Life Technologies, Carlsbad, CA, USA). Neural activity was recorded as fractional changes in fluorescence (∆F/F) using a Micam01 system (Brainvision, Tokyo, Japan). The fluorescence signals were recorded with 64 × 60 pixels at a 500-Hz frame rate covering an area of 4.6 × 4.3 mm. In each trial, single-pulse electrical stimulation (300

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µs in duration and 150 µA in intensity) was applied to the NBM, and the average values of 12 consecutive trials with 12-s intervals were obtained.

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Results Single-pulse NBM stimulation-evoked neural activity was assessed in frontal cortical regions (Fig. 1C–1E). As exemplified in Fig. 1C and 1D, single-pulse

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stimulation of the anterior part of the NBM elicited a depolarizing response around the medial part of the dorsal frontal cortex, corresponding to the medial agranular cortex

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(AGm), whereas stimulation of the posterior part of the NBM elicited a depolarizing response around the lateral part of the dorsal frontal cortex, including the lateral agranular cortex (AGl). Figure 1E illustrates the cortical activation bias corresponding to the site of anterior-posterior NBM stimulation. Each activation map was constructed

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by compiling the VSD images of the animals (n = 5 each).

Finally, we evaluated the volume of tissue activated by our electrical stimulation. Figure 1F shows mapping results indicating that cortical desynchronization was induced

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only when a small area (assumed to be the NBM) was stimulated [15]. Desynchronization did not occur after moving the electrode 500 µm, suggesting that the

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stimulus current did not spread beyond 500 µm. As the distance between the anterior and posterior NBM targeted for stimulation was about 1,700 µm, each NBM site was stimulated independently.

Discussion In this study, we succeeded in visualizing neural activity in the dorsal region of the rat frontal cortex, which was evoked by electrical stimulation of the NBM. As

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shown in Fig. 1C, VSD imaging revealed that the anterior NBM activated the AGm region of the dorsal frontal cortex, which is considered to be a part of the prefrontal cortex in rodents [16]. In contrast, the posterior NBM activated the frontal cortex more

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laterally toward the AGl, and the induced activity spread over the secondary and primary motor cortices.

Parikh et al. showed cue-evoked transient increases in cholinergic activity in the

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medial prefrontal cortex but not in the motor cortex in rats performing a detection task [17]. Conner et al. demonstrated that cholinergic signaling to the motor cortex was

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essential for motor learning and cortical reorganization following motor cortex damage [18]. Hence, it may be speculated that the anterior part of the NBM could play a role in modulating higher cognitive functions, while the posterior part might be involved in modulating motor functions. Considering studies in primates [5, 19], the anterior and

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posterior sites we stimulated are supposed to correspond to the anterior (Ch4a) and intermediate (Ch4i) NBM subdivisions in humans, respectively [2, 20]. The effects of NBM DBS have been reported in patients in the early stages of

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Alzheimer’s dementia, indicating a possible benefit of the treatment [21]. Hence, although further research is required, NBM DBS seems to be promising for enhancing

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memory function. In contrast, NBM DBS has not received much attention as a therapeutic option for patients with motor symptoms. However, patients with Parkinson’s disease and freezing of gait have cholinergic deficits that are selectively driven by NBM-neocortical denervation [22]. Therefore, the present results indicate that NBM DBS may be effective for improving motor-related functions (e.g., improvement in postural instability and gait initiation). The role of noncholinergic signaling to the cortex from the BF has attracted

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attention [23]. For example, gamma aminobutyric acid (GABA)ergic neurons in the BF are involved in activating the cerebral cortex and generating gamma band oscillations [24]. With nonselective electrical stimulation in the present study, the VSD imaging

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results represent the overall cortical response due to activation of both cholinergic and noncholinergic neurons distributed in the BF [6,25]. A recent study demonstrated a fine temporal relationship between frontal event-related potentials and bursting activity in

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BF neurons that were presumed to be noncholinergic [26]. Considering the short latency of the evoked VSD response (about 10 ms), which is consistent with that reported by

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Nguyen and Lin [26], the VSD imaging results may reflect the considerable contribution of noncholinergic actions. Further studies are necessary to elucidate this question.

Notably, the present findings have important practical implications if the NBM

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is to be considered a therapeutic target for neurological diseases: the anterior and posterior parts of the NBM could be targeted for improvement of cognitive and motor function, respectively. We will address this possibility in future work by using animal

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models of neurological deficits. The present study employed only single-pulse stimulation of the NBM; therefore, our next study will examine spatiotemporal changes

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in frontal cortex activity following burst stimulation, in accordance with typical clinical DBS procedures.

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Acknowledgements We thank Drs. Nobuo Kunori and Riichi Kajiwara for their helpful advice on optical

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imaging. This work was supported by a Grant-in-Aid for Scientific Research to I.T. (15K12780, 17H01810) and Y.W. (24730641, 16K04443) from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and by the New Energy

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and Industrial Technology Development Organization (NEDO).

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Figure Legends

Figure 1. (A) Localization of the nucleus basalis of Meynert (NBM). Middle panels,

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schematic view of the experiment, electrode dimensions in the inset, and Nissl-stained coronal sections showing the stimulation site (arrow). Left and right panels, cortical desynchronization following anterior and posterior NBM burst stimulation, respectively.

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Representative local field potentials (LFPs) in the frontal cortex (top traces). Change in LFP power spectral density before (blue) and after (red) NBM stimulation (bottom

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boxes). Data are an average of 5 trials in 1 animal. Shaded area = ± standard error of the mean (SEM). The LFP showed marked changes, characterized by a decrease in power at low frequencies (<15 Hz) and a broad increase at higher frequencies (>20 Hz). (B) Schematic view of voltage-sensitive dye (VSD) imaging. The dotted line indicates the

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approximate border between the medial (AGm) and lateral agranular (AGl) cortical regions. (C) Spatiotemporal dynamics of evoked neural activity after single pulse stimulation of the anterior (upper panels) and posterior (lower panels) NBM. The

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electrical stimulus was applied at 0 ms. The VSD signal was color-coded and superimposed on the background cortical image. (D) Representative time course of the

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VSD signals in the medial (blue line) and lateral regions of the frontal cortex (red line). Each trace shows the signal recorded by the pixels, indicated by a blue or red rectangle in the first frame (t = 0 ms) in (C). Plots of peak signal amplitude as a function of stimulus current (bottom panel; n = 5, mean ± SEM). A stimulus current of 150 µA was used for imaging. (E) Spatial distribution of the evoked activity following anterior (left panel) and posterior (right panel) NBM stimulation. In each panel, the optically detected activation areas were superimposed using the results of 5 animals. The color intensity

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(1–5) indicates the degree of overlap among animals. (F) Cortical desynchronization mapping along dorso-ventral penetration passing through the anterior (left panel) and posterior (right panel) NBM. This experiment was performed independent of VSD

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imaging; hence, the electrode was perpendicularly penetrated into the cortex. BL, basolateral amygdaloid nucleus; BST, bed nucleus of the stria terminalis; Ce, central amygdaloid nucleus; CPu, caudate putamen; HDB, nucleus of the horizontal limb of the

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diagonal band; ic, internal capsule; LGP, lateral globus pallidus; MeA, medial amygdaloid nucleus; MCPO, magnocellular preoptic nucleus; SI, substantia innominate;

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st, stria terminalis; VT, ventral thalamus; A, anterior; M, medial. Scale bars in A = 300

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µm, C = 1.0 mm.

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A

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