# High-accuracy electrode implantation in deep brain structures using multi-camera neuronavigation in non-human primates

Ankur Gupta, Adrien Boissenin, Nikolaos Vardalakis, Mathieu Taillade, Hugues Orignac, Nathalie Biendon, Sandra Dovero, Tho Hai Nguyen, Amirouche Sadoun, Fabien Bertrand Paul Wagner

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## To cite this version:

Ankur Gupta, Adrien Boissenin, Nikolaos Vardalakis, Mathieu Taillade, Hugues Orignac, et al. High-accuracy electrode implantation in deep brain structures using multi-camera neuronavigation in non-human primates. Journal of Neural Engineering, 2025, 22 (5), pp.056039. 10.1088/1741-2552/ae135b hal-05356028

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PAPER • OPEN ACCESS

# High-accuracy electrode implantation in deep brain structures using multi-camera neuronavigation in non-human primates

To cite this article: Ankur Gupta et al 2025 *J. Neural Eng.* 22 056039

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PAPER

# High-accuracy electrode implantation in deep brain structures using multi-camera neuronavigation in non-human primates

Ankur Gupta<fnref n="1" />,<fnref n="4" /> ID, Adrien Boissenin<fnref n="1" />,<fnref n="4" /> ID, Nikolaos Vardalakis<fnref n="1" />,<fnref n="5" /> ID, Mathieu Taillade<fnref n="1" />, Hugues Orignac<fnref n="1" />, Nathalie Biendon<fnref n="2" /> ID, Sandra Dovero<fnref n="2" /> ID, Tho Hai Nguyen<fnref n="1" />, Amirouche Sadoun<fnref n="1" /> ID and Fabien B Wagner<fnref n="1" />,\* ID

E-mail: fabien.wagner@u-bordeaux.fr

**Keywords:** neuronavigation, deep brain stimulation, stereo-EEG, stereotactic surgery, hippocampus, non-human primates

Supplementary material for this article is available [online](#)

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10 June 2025

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ACCEPTED FOR PUBLICATION

13 October 2025

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27 October 2025

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Abstract

**Objective.** Precise electrophysiological recordings and stimulation of deep brain structures in large animal models such as non-human primates require highly accurate targeting methods that are currently lacking. **Approach.** To address this limitation, we have integrated advanced multi-camera neuronavigation, high-resolution multimodal neuroimaging (magnetic resonance imaging and computed tomography), and tailored surgical methods to chronically implant electrodes in deep structures of the macaque brain, with an accuracy below or at the millimeter scale. **Main results.** We first illustrate the advantage of multi-camera neuronavigation over traditional two-camera systems in 3D-printed head models, demonstrating that a submillimeter accuracy requires at least five cameras. We then introduce a detailed experimental protocol using skull-based registration for reaching submillimeter accuracies *in vivo*. This protocol was optimized and tested in three macaque monkeys across seven implantations targeting the hippocampus or entorhinal cortex, including five performed with skull-based registration. Next, we quantify our targeting accuracy for these two structures by *in vivo* neuroimaging and histological analyses. We show that our skull-based registration procedure enabled to target the entorhinal cortex with submillimetric accuracy (0.55 mm and 0.89 mm in two animals), while implantation errors for the hippocampus were slightly higher (1.11 mm and 1.68 mm in two animals). Finally, we validate our approach with electrophysiological recordings of these targeted structures during drowsiness and stimulation-induced epileptiform afterdischarges in two chronically implanted animals. **Significance.** This technological and surgical framework provides a means to record and stimulate deep brain structures in large animal models with submillimetric to millimetric accuracy.

1. Introduction

Stereotactic and functional neurosurgery critically relies on the ability to target specific brain regions accurately and precisely with intracranial electrodes. These electrodes can be used to monitor brain activity during stereo-electroencephalography (sEEG) recordings for epilepsy surgery planning and cognitive neurophysiology research [1], or to deliver electrical stimulation during deep brain stimulation

(DBS) for Parkinson's disease and other neurological disorders [2]. To achieve accurate targeting of the desired brain structures, DBS electrode implantations are either guided by microelectrode recordings or by magnetic resonance imaging (MRI) data [3]. These procedures have been traditionally performed using a stereotactic frame rigidly attached to the skull and defining a reference frame based on anatomical landmarks. Alternatively, a surgical neuronavigation system may be used to carry out frameless stereotactic

<footnote n="1">¹ University Bordeaux, CNRS, IMN, UMR 5293, F-33000 Bordeaux, France</footnote>

<footnote n="2">² University Bordeaux, CNRS, HistoCARE Facility, IMN, UMR 5293, F-33000 Bordeaux, France</footnote>

<footnote n="3">³ Motac Neuroscience, F-33270 Floirac, France</footnote>

<footnote n="4">⁴ These authors contributed equally to this work.</footnote>

<footnote n="5">5 Current affiliation: Department of Neurosurgery, University of Pennsylvania, Philadelphia, PA, United States of America.</footnote>

<footnote>* Author to whom any correspondence should be addressed.</footnote>
<table><caption>**Table 1.** Targeting accuracies reported in animal studies using neuronavigation.</caption><thead><tr><th>Study</th><th>System</th><th>Model</th><th>Target</th><th>Procedure</th><th>Reported error</th></tr></thead><tbody><tr><td>Frey et al [11]</td><td>Brainsight</td><td>Phantom/NHP</td><td>Ventrolateral prefrontal cortex</td><td>Injection</td><td>&lt;1.2 mm</td></tr><tr><td>Chen et al [12]</td><td>Brainsight</td><td>Canine</td><td>Caudate, Thalamus, Midbrain</td><td>Biopsy needle</td><td>1.79 ± 0.87 mm</td></tr><tr><td>Long et al [13]</td><td>Brainsight</td><td>Canine</td><td>Thalamus and Hippocampus</td><td>DBS electrode</td><td>4.6 ± 1.5 mm</td></tr><tr><td>Owen et al [14]</td><td>Brainsight</td><td>Canine</td><td>Pituitary fossa</td><td>Transsphenoidal surgery</td><td>3.533 mm</td></tr><tr><td>Wetzel et al [15]</td><td>Brainsight</td><td>NHP</td><td>Cerebellar dentate nucleus</td><td>Injection</td><td>7 out of 8 probes within target area</td></tr><tr><td>Tasserie et al [16]</td><td>Brainsight</td><td>NHP</td><td>Central thalamus</td><td>DBS electrode</td><td>Not reported</td></tr><tr><td>Shinn et al [17]</td><td>Brainsight</td><td>Canine</td><td>Various deep brain areas</td><td>Pointer</td><td>0.64-4.04 mm</td></tr><tr><td>Crayen et al [18]</td><td>Cortexplore</td><td>Phantom/NHP</td><td>Middle temporal cortex</td><td>Electrode in microdrive</td><td>0.4</td></tr><tr><td>Mayer et al [19]</td><td>Cortexplore</td><td>NHP</td><td>Middle temporal cortex</td><td>Microdialysis probe</td><td>Not reported</td></tr></tbody></table>

procedures [4]. Thanks to optical or electromag- netic tracking of the surgical instruments, these neuronavigation systems enable intraoperative visual- ization of the electrode's position on the brain MRI images. The accuracy of clinically approved surgical neuronavigators (e.g. StealthStation S7, Medtronic) revolves around 2 mm [5, 6] and can even exceed 3 mm in some cases [7].

While routinely used in clinical settings, the implantation of depth electrodes for sEEG or DBS is not standardized in preclinical animal models such as non-human primates (NHPs), despite their importance for translational studies and neurotech- nology development [8, 9]. Since the macaque brain is on average 15 times smaller than the human brain in terms of volume [10], high-accuracy targeting in these species is more difficult to achieve than in humans. This consideration becomes even more important when targeting deep regions located near the skull base, such as the entorhinal area and hip- pocampus. Even small offsets or angular deviations at the electrode entry point on the skull surface can translate into major errors in deep structures, pre- venting the ability to target them reliably.

The first neuronavigation system designed for large animal models was introduced in 2004 (Brainsight Research Navigator, Rogue Research Inc.) [11]. It relies on a two-camera optical tracking sys- tem emitting infrared (IR) light and able to track the 3D positions of a set of IR-reflective fiducial markers (Polaris Vicra, NDI), similar to the clinically approved optical navigators (e.g. StealthStation S7, Medtronic). This two-camera system has been used in numerous animal studies with varying precisions depending on the target and procedure [11–17] (table 1). In the original study, the system was shown to have a precision below 1.2 mm using an MRI-compatible phantom, which was sufficient to inject anatomical

tracers into the ventrolateral prefrontal cortex of a macaque monkey [11]. In a follow-up study, the mean distance to target was estimated to be 1.79 mm when implanting a biopsy needle across 17 sites of the caudate nucleus, thalamus, and midbrain of a canine cadaver brain [12]. The same system was later used to implant DBS electrodes into the thalamus and hip- pocampus of three dogs, resulting in a mean accuracy of 4.6 mm, with the largest placement error being 6.49 mm [13]. This two-camera neuronavigation sys- tem has also been employed to target the pituitary fossa in ten canine cadavers [14], which resulted in a mean error of 3.533 mm, or to successfully target the deep cerebellar nuclei in seven out of eight injec- tions in four NHPs [15], or again the thalamus using clinical DBS electrodes in two macaque monkeys [16]. Additionally, a recent study has reported a median error of 3.28 mm for targeting 60 different target points sampling the entire brain in five canine cadavers [17].

Overall, these various studies highlight the impossibility to reach a submillimeter accuracy for deep brain structures using current two-camera neur- onavigation systems. Recently, a novel multi-camera neuronavigation system containing at least four IR cameras (Cortexplorer SCI System, Cortexplore GmbH) has been developed to palliate these limita- tions, leading to a submillimeter targeting accuracy in denatured egg whites and in the middle temporal cor- tical area of two macaque monkeys [18]. This system has been successfully applied to brain microdialysis in NHPs [19].

However, to our knowledge, multi-camera neur- onavigation has never been used for chronic implant- ation of depth electrodes designed for sEEG or DBS applications in NHPs. Here, we introduce a set of surgical tools and techniques combined with multi-camera neuronavigation for high-accuracy
implantation of custom depth electrodes in deep structures of the macaque brain. We illustrate and quantify our approaches by targeting the entorhinal cortex and hippocampus with a millimeter accuracy in three macaque monkeys, including two of them chronically implanted with two depth electrodes. Finally, we provide detailed guidelines for replicating our methods in similar or different brain structures and for avoiding common pitfalls that would affect the targeting accuracy.

## 2. Methods

### 2.1. Animals

Three healthy macaque monkeys (*Macaca fascicularis*), including two males and one female aged between 4 and 14 years old, were used in these experiments (table 2). Experiments were approved by the Institutional Animal Care and Use Committee of Bordeaux (CEEA-50, France) and the French Ministry of Higher Education and Research (Project Nos. APAFIS#26048-2020061216228969-v5 and APAFIS#25587-2020052908471774-v4) in accordance with the European Union directive of 22 September 2010 (2010/63/EU) on the protection of animals used for scientific purposes. Animals were housed in groups whenever possible, in enclosures designed according to the European direct-ive 2010/63/EU in the NHP facility of the Institute of Neurodegenerative Diseases (CNRS UMR5293, University of Bordeaux, animal experimentation registration number D33063268). When it was not possible to maintain an animal in a group, they were housed in adjacent enclosures to other NHPs to per-mit visual and auditory contact and interaction. Environmental enrichment included background music, foraging toys and bedding, as well as cognitive games performed on a touchscreen system intermittently accessible from their housing enclosure.

### 2.2. Neuronavigation and neurophysiology hardware

#### 2.2.1. Multi-camera neuronavigation system

Neuronavigation was performed using the Cortexplorer SCI system provided by cortEXplore GmbH (Austria), with the following hardware configuration: six IRis-13 cameras to track IR-reflective markers, a navigation computer mounted on a standard navigation station for wirelessly connecting to the cameras and displaying real-time position of the tools, a Multi Pointer Standard with a 1 mm diameter tip for subject registration and electrode holder tracking, two cranial references for providing a fixed reference to stereotactic frame and electrode calibration, a Magicum SCI tool for calibrating the cameras, and the Cortex explore software version 2.3.1. Additionally, a second Multi Pointer standard was purchased from the company, disassembled, and mounted on the

custom electrode holder described below for electrode tracking. The six cameras were mounted on the ceiling in a semi-circular arrangement (260 cm diameter, 200 cm between each camera and the stereotactic frame), with their field of view covering the space around the stereotactic frame (figure 1).

#### 2.2.2. Depth electrodes

Custom depth electrodes (figure 2(A)) were purchased from Dixi Fablab SAS (France), with the following specifications: 8 contacts (0.8 mm diameter, 0.5 mm length, 1.5 mm pitch, platinum/iridium) spanning a length of 12 mm, a 50 mm semi-flexible sheath, a 120 mm long metallic stylet used to hold the electrode during insertion, and 8 insulated platinum wires exiting the sheath with a 45° angle and with a length of more than 40 mm. These electrodes were then shipped to Blackrock Neurotech (Blackrock Microsystems Inc., USA) for connectorization. The wires were cut at 15 mm after they exit the sheath and were then laser-welded to a 9-pin male Omnetics connector (A79000-001).

These depth electrodes were then connected to a customized percutaneous, bone-anchored titanium connector (128-channel LGA connector, Blackrock Neurotech) through intermediate implanted Omnetics connectors, to enable interfacing with external neurophysiology equipment. The customized LGA connector was manufactured by Blackrock Neurotech according to our desired pin layout. Briefly, two 9-pin female Omnetics connectors (A79001-001) were attached to one bank of the LGA connector using 3 mil Pt/Ir wires. Clean LGA pads and soldering pins were then potted with M-31CL epoxy adhesive. Lead wires were finally overpotted with silicone. Omnetics connectors exited the LGA connector at one of the pedestal grooves. Additionally, two reference wires (3 mil Pt/Ir) were attached to the LGA connector and exited the pedestal at a different groove.

#### 2.2.3. Electrode holder

The electrode holder is a critical surgical component that required careful attention during its development (figures 2(B)-(D)). After several iterations using 3D-printed plastic prototypes, we opted to collaborate with an external company (Alsymex SAS, France) specialized in high-precision manufacturing. The final version was fabricated from stainless steel (316L), a material chosen to minimize plastic deformation during handling and to withstand autoclaving without corrosion or staining. This design resulted in a system composed of three primary components:

* A 25 cm central rod with a mount for the neuronavigation tracking tool at its upper extremity, and the electrode base at the lower extremity. The rod length was chosen to ensure that the tracking
<table><caption>**Table 2.** Information about the NHPs (*Macaca fascicularis*) used in this study.</caption><thead><tr><td>Animal ID</td><td>Sex</td><td>Age at implantation</td><td>Weight</td></tr></thead><tbody><tr><td>Monkey O</td><td>M</td><td>4.6 years</td><td>5.9 kg</td></tr><tr><td>Monkey E</td><td>M</td><td>5.8 years</td><td>7.3 kg</td></tr><tr><td>Monkey K</td><td>F</td><td>13.9 years</td><td>4.6 kg</td></tr></tbody></table>

<figure><img src="image_6.png" /><figcaption>**Figure 1.** Technological framework for MRI-guided surgical implantation of depth electrodes in NHPs. (A) Overview of the multicamera neuronavigation setup designed for the surgical implantation of depth electrodes into deep brain structures in NHPs. The animal is placed head-fixed in a standard stereotactic frame. A depth electrode is mounted in a custom electrode holder that contains an inner and outer slotted guide tubes (see figure 2 for more details). The neuronavigation system (Cortexplore) consists of a set of six IR cameras mounted on the ceiling, which detect the 3D position of IR-reflective markers attached to key equipment such as the electrode holder, the cranial reference (attached to the stereotactic frame), and a multipointer tool (used for landmarks and subject skull registration). These cameras are then connected to a computer unit that integrates the position of the electrode and the animal's skull in real time. (B) 3D model of a macaque monkey head implanted with two depth electrodes in the hippocampus (red) and entorhinal cortex (blue), both connected to a percutaneous, bone-anchored titanium connector (Blackrock Neurotech) through intermediate implanted connectors (Omnetics). Panel generated using the MRI volumes of Monkey E exported from 3D slicer and rendered in Blender.</figcaption></figure>

tool remains unobstructed during surgical manipulation. Its stainless-steel construction minimizes bending or deformation due to the weight of the components.

* The electrode base supports both the outer and inner guide tubes and the depth electrode. It consists of a 30 × 15 × 10 mm³ stainless-steel block, shrink-fitted onto the central rod for robust attachment. The outer guide tube is positioned within a dedicated notch in the base and secured using a spring plunger. To ensure precise alignment, a dowel pin was used to index the slot of the outer guide tube relative to the notch on the electrode base.

* A stylet holder (32 × 20 × 6 mm³ block) mounted along the central rod and secured with a locking screw. This piece contains a Ø0.5 mm aperture designed for stylet insertion. The electrode stylet is held in place within the stylet holder by

a locking screw. The stylet stabilizes the electrode and prevents displacement during retraction of the guide tubes from the brain. A longitudinal flat (machined along the central rod) ensures precise seating of the locking screw, maintaining precise alignment between the stylet aperture and the inner guide tube, and minimizing stress or torsion on the implant.

Guide tubes were fabricated from stainless steel grade 304 cylinders (inner guide tubes: 18G; outer diameter: 1.24 mm, inner diameter: 0.85 mm; outer guide tubes: 16G thin wall; outer diameter: 1.65 mm, inner diameter: 1.39 mm) sourced from Phymep SARL, France. A 750 µm-wide slot was precisely cut into the electrode holder using wire electrical discharge machining. For the outer guide tube, the top cap was machined separately and subsequently soldered into place by the electrode holder manufacturer. Both
<figure><img src="image_7.png" /><figcaption>**Figure 2.** Schematic of the custom depth electrode and electrode holder. (A) The custom depth electrode contains 8 contacts (0.8 mm diameter, 0.5 mm length, 1.5 mm pitch, platinum/iridium contacts), a metallic stylet used to hold it during insertion, and a 9-pin connector (A79000-001, Omnetics). The electrode was first manufactured by Dixi Fablab and then soldered to a connector by Blackrock Neurotech. (B)-(D) The custom electrode holder (manufactured by Alsymex) allows chronic electrode implantation into the brain thanks to a system of slotted inner and outer guide tubes. It is mounted on a stereotactic arm for precise manipulation. It consists of a 25 cm central rod equipped at its upper extremity with a placeholder for mounting the neuronavigation tracking tool and at its lower end by the electrode base. The electrode base supports an outer guide tube secured by a screw. Inside, an inner guide tube is inserted. Both are slotted to allow disengaging from the electrode and its connector after implantation. The inner guide tube contains a protrusion that prevents it from fully sliding into the outer guide tube, ensuring that both ends remain at the same level. The electrode is inserted into the inner tube, with the tip of the electrode not protruding. The stylet holder is inserted along the central rod and secured in place with a screw. A small hole in the holder allows for the insertion of the stylet, which is also kept in place using a screw. This electrode holder was built from stainless steel, minimizing plastic deformation during handling and allowing it to be autoclaved.</figcaption></figure>

guide tubes were shortened to final lengths of 65 mm (outer guide tube) and 80 mm (inner guide tube), with beveled tips to facilitate smooth penetration into brain tissue. The inner guide tube was made slightly longer so that it extended above the electrode base, allowing for easier gripping during retraction. To prevent the inner guide tube from fully sliding into the outer guide tube, a small protrusion was created on the inner tube, acting as a stop. This ensured that the distal ends of both tubes remained aligned at the same depth during insertion.

### 2.2.4. Electrophysiological recordings and stimulation

Neurophysiological data were sampled at 30 kHz using a digital amplifier headstage and neural data acquisition system (Cereplex μ-series headstage and 256-channel Cereplex Direct, Blackrock Microsystems Inc, USA). Electrical stimulation was

applied through a 96-channel macrostimulation system (Cerestim R96, macrostimulation option, Blackrock Microsystems Inc, USA). Each channel was connected both to the data acquisition system and to the stimulator via a 3-way adapter (Y-connector, Blackrock Microsystems Inc, USA), enabling simultaneous recording and stimulation on the same electrode contacts. The neurostimulator was programmed to deliver 10 s continuous trains of biphasic pulses (cathodic-first, 300 μs pulse width, 100 μs inter-phase interval) at 50 Hz and in a bipolar configuration (applied between two neighboring contacts of the hippocampal electrode). We gradually increased the stimulation amplitude starting at 0.1 mA, in steps of 0.1 mA, and continuing until we recorded epileptiform afterdischarges (but no behavioral seizures) or any visible sign of animal discomfort, while not exceeding 2 mA.
## 2.3. MRI data acquisition

### 2.3.1. *Equipment and animal preparation*

MRI acquisitions were performed at the Biomedical Imaging Platform of the University of Bordeaux (CNRS UAR3767, University of Bordeaux, animal experimentation registration number A33-063-930) with a 3T Prisma Siemens MR scanner (Siemens Healthineers AG, Germany), using the manufacturer's body coil for transmission and 64-channel head coil for signal reception. This coil is typically used for human acquisitions. The antenna was positioned over the monkey's head, and foam was inserted to restrain the head during scanning. All implantations reported here used MRI data acquired in the prone position. For each session, the animal was pre-medicated with intramuscular (IM) injections of diazepam (0.5 mg kg⁻¹) and atropine (0.05 mg kg⁻¹). Following induction of anesthesia by ketamine (10 mg kg⁻¹, IM) and xylazine (1 mg kg⁻¹, IM) in the housing facility, the animals were then transported to the imaging platform in a cart with a microwavable heating pad and blanket. During all acquisitions, we continuously monitored heart rate and oxygen saturation using an MRI-compatible pulse oximeter (Model 7500, Nonin Medical) and respiratory rate using the manufacturer's respiratory belt.

### 2.3.2. *MRI protocol*

The T1-weighted sequence employed a 3D magnetization-prepared 180° radio-frequency pulses in combination with a rapid gradient-echo (3D MPRAGE) sequence at an isotropic resolution of 0.5 × 0.5 × 0.5 mm³ [20]. The scan was conducted in the sagittal orientation with a field of view (FOV) of 160 × 160 × 104 mm³, 4% phase oversampling, and an echo time (TE) of 2.32 ms and repetition time (TR) of 2000 ms. We performed six averages with an inversion time of 1 s using a non-selective inversion pulse. To optimize the scan, we applied in-plane acceleration using a GRAPPA (Generalized Autocalibrating Partially Parallel Acquisitions) factor of 2 with 24 center reference lines, and the bandwidth was set to 250 Hz/pixel. Additionally, a train of 208 small flip water-selective excitations was utilized. The sequence lasted 35 min and 50 s.

The T2-weighted sequence was performed using the vendor's 3D sampling perfection with application-optimized contrasts echo sequence at 0.5 × 0.5 × 0.5 mm³ isotropic resolution, with sagittal orientation, 160 × 160 × 104 mm³ FOV, 4% phase oversampling, 384 ms/3200 ms TE/TR, and 4 averages. In-plane acceleration with a GRAPPA factor of 2 and 24 center reference lines were used. A fat saturation preparation module was used. A bandwidth of 473 Hz/pixel was received. A non-selective refocusing pulse train of 218 pulses with variable flip angles was

used. The T2 acquisition duration was 36 min and 19 s.

### 2.3.3. *MRI pre-processing*

MRI data for each scan were imported into the ITK-SNAP software (ITK-SNAP, https://www.itksnap.org). This NiF1T1 file was subsequently loaded into the MATLAB-based MRI denoising toolkit (available at https://sites.google.com/site/pierrickcoupe/softwares/denoising/mri-denoising). The denoising process utilized the AOLMN method with a Rician noise filter [21]. The resulting output file was then prepared for subsequent registration and pre-surgical planning.

## 2.4. Computed tomography (CT)

### 2.4.1. *Equipment and animal preparation*

All CT scans were acquired using a cone-beam computed tomography (CBCT) scanner classically used in dentistry (ProMax® 3D Classic with extended volume of Ø11 × 8 cm, Planmeca SAS, France). The height of the telescopic column was adjusted to capture the entire scalp with the occipital bone resting on the device's chin support and the frontal bone facing upward toward the ceiling. This position was used to ensure that the skull crown was not affected by the cone-beam effect, which creates an artefact at the top of the scanned volume [22, 23].

This procedure was performed under general anesthesia and lasted no more than 30 min. Prior to induction, the animal received diazepam (0.5 mg kg⁻¹, IM) and atropine (0.05 mg kg⁻¹, IM), followed by sedation with ketamine (10 mg kg⁻¹, IM).

### 2.4.2. *CT protocol*

The vendor-supplied software suite was employed using the '3D single-scan' setting with a XL head size and the largest available scan diameter (indicated as '80 + mm × 80 + mm' in the software and corresponding to a volume of 110 mm diameter and 80 mm height). Prior to the full acquisition, a CT localizer (scout) was performed at 90 kV and 6.3 mA, resulting in a 2D image with 512 × 512 pixels, a resolution of 254 µm, and an absorbed radiation dose of 1 mGy cm². After verifying the scout image for proper positioning and orientation, any necessary adjustments were made using the software's left-right and anterior-posterior (A/P) adjustment buttons or by fine-tuning the CBCT's adjustment wheels. Once the head orientation and position were confirmed, a full CT scan was conducted. The resulting scan volume had a diameter of 110 mm and a height of 80 mm, with a total of 734 × 634 × 534 voxels captured at a resolution of 150 µm. It was performed at 90 kV and 6.3 mA for 15.078 s, leading to an absorbed radiation dose of 903 mGy cm². The captured scan was then
imported into the Romexis 6.4.3.33 software, where
a 3D noise filter was applied with 100% strength and
sharpening enabled. A new volume was generated and
exported for further processing.

**2.5. Neuroimaging data processing**

**2.5.1. MRI and CT co-registration**

To ensure both the MRI and CT volumes were aligned
in the same reference frame, the denoised MRI and
CT volumes were imported into 3D Slicer (version
5.4.0) as separate volumes. To align the MRI with
the stereotactic frame and ensure accurate head ori-
entation during surgery, we utilized distinctly visible
structures: the anterior commissure (AC), posterior
commissure (PC), and the midline, and performed an
AC-PC transformation.

The CT volume was manually rotated and
translated to approximately align with the MRI.
Subsequently, a rigid (6 degrees of freedom) general
registration (BRAINS [24]) was performed to refine
the alignment, with the MRI as the fixed volume and
the CT as the moving volume. We used 100% of the
data as samples, and the initial transformation mode
was set to off. The MRI-CT registration was manu-
ally verified, and the transformations were hardened
to prevent any further modifications. A brain mask
was extracted using the generalizable brain extraction
net algorithm [25] (the trained network weights are
available on Zenodo [26]), and the MRI volume was
cropped to isolate the brain.

**2.5.2. Trajectory planning**

All trajectories were initially designed in the 3D Slicer
software (3D Slicer, www.slicer.org/) [27], and then
imported into the Cortexplore neuronavigation soft-
ware. Trajectories targeted either the hippocampus or
the entorhinal cortex using a transoccipital approach
[28–30]. These structures are situated more than
40 mm from the occipital bone in adult macaques.
Two separate entry points were defined on the skull,
one for each target. The hippocampal trajectory was
designed to target the anterior part of the hippocam-
pus and follow its longitudinal (i.e. anteroposterior)
axis, while avoiding the ventricles. For the entorhinal
cortex, the target was marked 1 mm above the basi-
cranium and the trajectory was designed to follow
the parahippocampal gyrus. We made sure that the
two entry points were not too close to each other,
so that the first implantation does not obstruct the
second one (especially after cementing the electrode
in place). These trajectories were marked as lines in
3D Slicer. Finally, the co-registered MRI, CT, and
brain volumes were exported as NIfTI (\*.nii) files.

These files were then imported into the
Cortexplore neuronavigation software. The module
labeled ‘model’ was used to create a 3D represent-
ation of the skull and brain. The threshold on the
CT volume was set between 600 and 3095, while the

threshold on the MRI volume was adjusted to clearly
visualize the brain sulci and gyri. To ensure accur-
acy in subject registration, we avoided using the ‘fill
hole’ option, as it may modify the structures of the
divots. Finally, the coordinates corresponding to the
entry point and target, derived from 3D Slicer, were
directly entered into the planning module.

**2.6. Surgical procedures**

**2.6.1. Surgical preparation and postoperative care**

All procedures were performed under aseptic condi-
tions. A few weeks before surgery, implanted hard-
ware pieces (depth electrodes and percutaneous con-
nector) were shipped to an external service pro-
vider (Steriservices SAS, France) for ethylene oxide
sterilization. On the day of surgery, animals were
pre-medicated with intramuscular (IM) injections of
diazepam (0.5 mg kg⁻¹) and atropine (0.05 mg kg⁻¹)
about 15 min prior to anesthesia induction with ket-
amine (10 mg kg⁻¹), followed by an analgesic injec-
tion of meloxicam (0.2 mg kg⁻¹, IM). The anim-
als were then shaved, intubated with an endotracheal
tube, and placed in a prone position in a stereotactic
frame with a heating pad. Anesthesia was main-
tained using 1%-3% isoflurane or sevoflurane mixed
with pure oxygen. During the whole intervention, we
monitored heart rate, temperature, oxygen satura-
tion, capnography, and respiratory rate. Prior to skin
incision, the animals received a subcutaneous injec-
tion of lidocaine (1 ml, 20 mg ml⁻¹). Post-operative
analgesia was achieved with an injection of bupren-
orphine (0.03 mg kg⁻¹, IM) at the end of surgery,
followed by meloxicam (0.2 mg kg⁻¹ d⁻¹, IM) for
three days. Additionally, the animals were given pro-
phylactic antibiotics (cefalexine 20 mg kg⁻¹, twice per
day for 5 d) and were closely monitored for any sign
of infection, discomfort or sign of traumatic brain
injury for at least two weeks after each surgery.

**2.6.2. Overview of surgical procedures**

Three separate surgical procedures were performed:
drilling of rigid cranial landmarks (surgery 1, ~1.5 h),
insertion of the depth electrodes (surgery 2, ~7 h),
and implantation of the percutaneous connector
(surgery 3, ~1.5 h). Two consecutive surgeries were
separated by a period of at least two weeks to give
enough time for the animal to recover.

During the first surgery, a 2 × 4 cm² area of the
skull was exposed. Then, four landmarks (15 mm
apart) were drilled on either side of the sagittal crest
using a 1.8 mm drill bit. After suturing the skin, we
performed a CT scan prior to the animal's recovery
from anesthesia. The different steps of the second sur-
gery are described in detail in the results section. The
third procedure was integrated into another surgical
intervention not included in this study and which will
not be described in detail here. Importantly, proced-
ures 2 and 3 could technically be merged into a single
surgery, as long as the total time under anesthesia is deemed appropriate for the animal health status.

### 2.6.3. *Setting up the neuronavigation system*

Neuronavigation cameras were calibrated immediately prior to surgery, using the provided calibration wand to sample the regions in space where the neuronavigation markers would be tracked (i.e. around the cranial reference and where the electrode assembly would be placed). Calibration was deemed appropriate when the wand score displayed in the software was below 0.075, the mean wand error below 0.075, and the standard deviation below 0.05. If any of these values exceeded these thresholds, the calibration was repeated.

Following calibration of the cameras, we mounted the cranial reference onto the stereotactic frame, ensuring it remained unoccluded even when the animal would be positioned and draped. This cranial reference served as a rigid frame of reference for all subsequent animal registration and tool tracking steps during the procedure. After securing the cranial reference, the anesthetized animal was positioned in the stereotactic frame.

Meanwhile, the heat-sterilized electrode assembly was assembled on a sterile drape. To minimize the time spent on assembly during surgery, components were preassembled before sterilization whenever possible. Specifically, we placed the stylet holder block onto the central rod and added the multipointer (without markers), allowing us to integrate the outer guide tube, inner guide tube, and electrode into the assembly. After securing the outer guide into the electrode base with a screw, the inner guide tube was inserted. The stylet block was positioned 65 mm away from the top of the outer guide tube to accommodate the 55 mm length of the inner guide. The electrode's stylet was then inserted into the small hole in the stylet holder, and the electrode was carefully advanced into the inner guide tube. Once the electrode exited the outer guide tube, we secured the stylet with a screw. Finally, we attached IR-reflective markers to the multipointer, thereby completing the electrode assembly and preparing it for calibration.

### 2.6.4. *Subject registration*

We began the registration process by placing a multipointer with a 1 mm ball tip on one of the reference points provided and then verifying the multipointer tool in the software. In the 'landmark registration' section of the registration module, we placed four points on the virtual skull, each corresponding to a divot hole. We then positioned the multipointer tip at these points sequentially and sampled each one. This initial step provided an approximate position of the animal in the virtual world, resulting in an RMS registration error of less than 0.3 mm.

Next, we refined registration through skull surface scanning. In the registration module, we selected 'surface registration' using the previously created skull model as a reference. We acquired datasets with the 'apply surface registration' and 'remove outlier' options enabled. During this step, we moved the multipointer tip over the skull around the areas surrounding the divots and the approximate location of the entry points (around the occipital ridge). We sampled approximately 800 data points, including the most posterior, anterior, and lateral accessible areas. If the pointer tip lost contact with the skull at any point, we discarded the dataset. After surface registration, the resulting RMS registration error was under 0.07 mm.

### 2.6.5. *Electrode calibration*

After registering the subject in the virtual space, we covered a movable table with a sterile drape and placed a sterile block with two A/P bars (similar to those on the stereotactic frame) on the table. One bar was used to mount a separate cranial reference, while the other bar held a stereotactic arm. We then placed the electrode assembly on the stereotactic arm. This method of calibrating the electrode on a separate table allowed for faster calibration due to greater maneuverability. In the Cortexplore software, we selected 'tool axis calibration' and chose the appropriate cranial reference from the calibration tool dropdown menu. We then selected the 'referenceAxis1' calibration point. To calibrate the tip, we kept the outer guide tube orthogonal to the plane of the five cranial reference arms. The electrode assembly was lowered to position the electrode tip at the top plane of the cranial reference arms, aligning it with the center of the referenceAxis1 notch. Once aligned, we acquired the tip point. We then lowered the electrode by approximately 3-4 cm and acquired the shaft point. We saved this calibration and repeated the process for the second electrode. After calibration, we moved the table with the electrode assembly out of the camera's field of view. Caution was exercised to ensure that neither the electrode tip nor shaft came into contact with any object, as this would invalidate the calibration.

### 2.6.6. *Entry point drilling and angle adjustment*

The multipointer tool was used to identify the approximate location of the entry points on the skull, which were marked using a sterile pencil. We then drilled the marked entry points using a 1.8 mm diamond burr until we reached the dura mater. The hole was expanded to approximately 4 mm to ensure smooth entry for the outer guide tube (outer diameter 1.7 mm). Next, a titanium screw was anchored in the skull a few millimeters above the two burr holes. The purpose of this screw was to enhance fixation and support cement anchorage during the final steps of the procedure.
Once the hole was drilled, we placed a stereotactic arm on the stereotactic bar and carefully mounted the electrode assembly, ensuring that the outer guide did not touch any surface. In the Cortex explore software, we loaded the previously saved tool calibration by accessing the tool axis calibration menu. At this stage, the tool tip and axis appeared in the software. We made angle adjustments to the stereotactic arm to align one of its axes with the planned trajectory. The electrode tip was then moved closer to the skull, maintaining an approximately 1 cm distance. We made final adjustments to minimize the angles to less than 0.05 degrees on each of the three axes. Once angles were optimized, we locked the stereotactic arm angles to prevent further changes. We then minimized the distance to the entry point by translating the electrode tip, achieved by rotating the left-right and superior-inferior stereotactic arm screws.

### 2.6.7. Electrode insertion into the brain

After piercing the dura with a dura hook, the electrode assembly was carefully lowered along the planned trajectory using the stereotactic arm. To prevent damage to the targeted area, we stopped the guide tube assembly 12 mm before the target, corresponding to the total length of the electrode contacts. Throughout the process, we continuously monitored the three angles in the software and the tool axis to ensure proper alignment with the target. If any deviations exceeded acceptable thresholds (typically due to dura piercing), we withdrew the electrode assembly, repositioned it, and then reinserted it. We performed this reinsertion procedure once across our seven implantations.

We then marked the stylet 12 mm above the stylet holder block, providing a reference point when subsequently lowering the electrode. Next, we unscrewed the stylet screw to release the stylet. Using sterile tweezers, we gently and slowly advanced the stylet into the brain, gradually approaching the target depth. Once the stylet mark reached the top of the stylet block, the electrode had reached the intended location. Finally, we carefully tightened the stylet screw to secure the electrode in place, ensuring it had not shifted during the process.

### 2.6.8. Guide tube removal and electrode fixation

After securely placing the electrode, we carefully removed the inner guide tube from the outer guide tube using sterile tweezers. Next, we clamped the stylet above the electrode base and below the stylet-holding block, so that the stylet and electrode were held in place by a secondary free arm (with six degrees of freedom) fixed on the stereotactic frame. The stylet screw was then unlocked to release the stylet. We aligned the electrode wires with the outer guide slot and slowly withdrew the outer guide tube along the same axis used for electrode insertion, using the same

stereotactic screw that had previously lowered the electrode into the brain. Once the outer guide tube was removed, the electrode remained securely in place in the brain by the stylet. We then carefully removed the entire electrode assembly and stereotactic arm.

Bone cement (Refobacin Plus Bone Cement, Zimmer Biomet) was added to cover the burr hole and nearby support screw, so that the electrode was rigidly attached to the skull. The cement should be viscous enough so that it does not enter in contact with the dura mater. Afterwards, the stylet was clipped from its attachment to the electrode sheath. This implantation procedure was first performed for the entorhinal electrode and then repeated for the hippocampal one.

### 2.6.9. Percutaneous connector fixation

The percutaneous connector (aka pedestal) was first connected to the implanted electrodes through the intermediate Omnetics connectors while holding it manually. Its position on the skull was adjusted to minimize strain on the cables. Next, it was anchored to the skull using 6 titanium screws (length: 6 mm) on the side contralateral to the electrodes. An additional seventh screw (length: 7 mm) served as a reference electrode and was placed a few millimeters anterior to the pedestal on the same side. We ensured that the screw was long enough to make electrical contact with the dura mater and provide a low-impedance reference electrode. The two reference wires from the percutaneous connector were stripped off from their insulation layer and firmly rolled around the reference screw. As a final step, a cement dome was applied, maintaining in place and protecting the electrodes, cables, screws, and the area surrounding the pedestal.

## 2.7. Histological analyses

### 2.7.1. Histology/Nissl staining

Histological processing and Nissl staining were conducted in one of the implanted animals (monkey K). Briefly, the brain was extracted postmortem and post-fixed for one week in 4% formaldehyde prepared in 0.1 M phosphate-buffered saline (PBS) at 4 °C. The hemispheres were separated and cryoprotected by sequential immersion in 20% and then 30% PBS-sucrose solutions until the tissue sank. The hemispheres were then rapidly frozen by immersion in isopentane at -50 °C. Serial coronal sections (50 µm thick) were obtained using a cryostat and collected free-floating throughout the entire rostro-caudal extent. Every twelfth section was mounted on gelatin-coated slides, air-dried, and stained with 0.1% cresyl violet. The stained sections were digitized using a 3DHistech Pannoramic Scanner II at 20× magnification. Image analysis and extraction were performed using SlideViewer® 2.5 software (3DHistech Kft, Hungary), in conjunction with
an histological atlas [31]. Electrode tracks were confirmed by comparing the histological data with preoperative targeting information and MRI acquisitions (see next sections for more detailed information).

### 2.7.2. Registration between histology and MRI

Co-registration between histology and MRI was performed using a three-step process: (1) registration between consecutive 2D histological sections to create a 3D histological volume, (2) 3D co-registration between this histological volume and the brain MRI, (3) refined 2D co-registration between individual histological sections and brain MRI based on a set of anatomical landmarks. These three steps are detailed below. Following this process, tears observed in the histological slices were visually compared with electrode locations identified in the post-operative CT scan, thereby confirming the spatial correspondence between implanted electrode trajectories and histology.

First, high-resolution coronal histological sections (imaged at 0.1725 µm resolution) were downsampled to a lower resolution of 4.6 µm using QuPath v0.5.1 [32]. Grayscale conversion was performed on each image in MATLAB 2024b (The MathWorks, Inc., USA). The contrast was adjusted to a normalized intensity range between 0.7843 and 1, corresponding to pixel intensities from 200 to 255. Following this, each image was downsampled by a factor of 0.6 and then stored. The processed grayscale images were automatically registered using intensity-based multimodal registration with default parameters in MATLAB 2024b. Subsequently, a 3D volume was constructed and saved in NiFtI format.

Second, this reconstructed histological volume was imported into 3D Slicer and manually aligned with the pre-operative MRI using rigid transformation. Fine registration was refined using BRAINS general registration module with both 'rigid' (6° of freedom) and 'rigid + scale' (7° of freedom) transformations, leveraging 100% of the available samples. Upon successful registration, individual histological slices were identified within the 3D context and their A/P coordinates were determined relative to MRI.

Third, the previously generated contrast-adjusted individual slices were further imported into 3D Slicer, with lateral resolution corrected to 4.3 µm to match physical geometry. Fiducial-based registration was then applied, using matched anatomical features in both MRI and histology (e.g. as denoted by the yellow cross in figure 8). To mitigate deformation artifacts introduced during tissue processing, cropped histological regions were utilized instead of full slices, as global alignment was rendered unfeasible by *ex-vivo* brain distortion.

## 2.8. Neuronavigation accuracy on plastic heads and in vivo

The implantation accuracy on 3D-printed plastic skulls was determined by aligning a 21G needle to a pre-determined 1 mm hole. The needle was positioned in the center of the hole via a stereotactic manipulator under visual guidance. The error was quantified as the 'tip-to-trajectory error' reported by the neuronavigation software. This procedure was repeated when varying the number of tracking cameras from three to six. Each time, the set of cameras was recalibrated, yielding a quantification of neuronavigation accuracy as a function of the number of cameras.

The implantation accuracy in vivo was determined based on the co-registration between the pre-operative MRI and the post-operative CT scan. The implantation error for each electrode contact was defined as the minimum 3D Euclidean distance between the planned trajectory and the contact's center observed on the CT. This was repeated for all eight contacts of each electrode. The mean and standard deviation of implantation errors across contacts were then computed for each implanted electrode.

## 2.9. Intracranial EEG signal processing

Raw intracranial EEG data were acquired at 30 kHz. The data were then low-pass filtered offline below 500 Hz (fourth-order Butterworth filter) and downsampled at 1 kHz. For analysis of the trials with electrical stimulation, the signals were additionally high-pass filtered above 10 Hz (second-order Butterworth filter) to remove the slow transient induced by the stimulation artefact and be able to visualize afterdischarges properly.

Time-frequency analysis was performed by short-time Fourier transform using the 'spectrogram' function in SciPy library [33] (v1.11.4, Python). Identical parameters were applied for both sleep and afterdischarge recordings: 1 s Hanning windows with 95% overlap, zero-padding to obtain a 0.5 Hz frequency granularity (i.e. nfft was set to twice the sampling frequency), and linear detrending. Spectrograms were plotted using the 'pcolormesh' function from Matplotlib [34], with no interpolation to preserve raw resolution. The spectrogram scales were adjusted to best capture the relevant variations in biological rhythms (0-20 Hz for sleep recordings and 0-80 Hz for afterdischarge recordings). To enhance the visibility of low-intensity spectral components, the color scale of the spectrograms was capped at 50% of the maximal power.

# 3. Results

## 3.1. Multi-camera neuronavigation for surgical implantation of depth electrodes in NHPs

We first developed and optimized neuronavigation and surgical methods for high-accuracy MRI-guided
surgical implantation of depth electrodes in NHPs (figure 1). Standard neuronavigation systems employ only two IR cameras separated by less than 30 cm (e.g. StealthStation S7, Medtronic plc; Brainsight Research Navigator, Rogue Research Inc.; Polaris Vicra, NDI). These cameras are in turn able to detect IR-reflective markers placed on the surgical tools and on a cranial reference rigidly attached to the stereotactic frame. According to the manufacturers' specifications, two-camera systems result in accuracies on the order of 1-2 mm. Based on our own experience and the studies highlighted above, the actual experimental errors for the entry point are usually higher than 2 mm and depend on various factors such as the distance and orientation of the IR-reflective markers with respect to the pair of cameras. These deviations at the entry point can, in turn, translate into unacceptable errors (3-7 mm) in deep brain structures [13, 17]. The macaque hippocampus being about 4 mm in diameter, such deviations can lead to electrode trajectories that completely miss the target.

To circumvent this limitation, we performed a thorough characterization of a commercially available multi-camera neuronavigation system (Cortexplorer SCI System, Cortexplore GmbH) comprising multiple IR cameras mounted on the ceiling in a semi-circular arrangement (260 cm diameter, 200 cm between each camera and the stereotactic frame, three to six cameras in the current setup), with their field of view covering the space around a standard stereotactic frame for NHPs (figure 1(A)). We first aimed to determine the minimum number of cameras required to reach a submillimeter accuracy at the skull level. To this end, we prepared a 3D-printed macaque skull with 1 mm holes used as neuronavigation landmarks, based on the CT scan from one of our animals (figure 3).

Using this setup, we characterized the ability to reach one of these landmarks with a 21G needle attached to a stereotactic holder equipped with IR-reflective markers, which allowed the software to uniquely identify the tool and determine its position and orientation within the 3D virtual environment. After performing the standard registration procedure, we aligned the needle with the hole in physical space and observed the error in the virtual space estimated by the neuronavigation software. We performed this test sequentially when varying the numbers of cameras from three to six (figure 3). We found that the estimation of the landmark location reached a submillimeter accuracy of about 0.4 mm when using five or six cameras, which is substantially improved compared to the 2 mm accuracy achieved with two-camera systems. Moreover, this estimated accuracy was robust to changes in the orientation of the tracked tools. This provided us with a strong rationale for using a six-camera neuronavigation system for depth electrodes implantation into deep brain structures.

Next, we sought to develop custom depth electrodes and associated tools for chronic implantation in macaque monkeys for sEEG and/or DBS applications (figure 2). Commercially available sEEG or DBS electrodes feature an inter-contact spacing too large for applications in macaques. Furthermore, they cannot be easily interfaced with standard percutaneous connectors and associated hardware for neurophysiology experiments in NHPs. We thus established partnerships with three different companies: an established sEEG electrode manufacturer (Dixi Medical/Dixi FabLab), a provider of neurophysiology equipment tailored for NHP experiments (Blackrock Neurotech), and a company with expertise in high-precision manufacturing of medical-grade stainless steel (Alsymex). Through these companies, we manufactured a depth electrode comprising 8 contacts (0.5 mm length, 1.5 mm pitch) with an outer diameter of 800 µm and connected to a 9-pin flat connector (Omnetics) (figure 2(A)). We also customized a widely used bone-anchored percutaneous connector designed for NHP applications (LGA connector, Blackrock Neurotech) to allow interfacing with several depth electrodes through flat connectors fixed on the skull (figure 1(B)). Finally, we designed an electrode holder equipped with slotted guide tubes that enable implantation of these electrodes through neuronavigation (figures 2(B)-(D)).

Once designed and manufactured, we integrated this custom electrode holder with our multi-camera neuronavigation system. Overall, we performed optical tracking of three key instruments: the electrode holder, a cranial reference, and a multipointer device (figure 1(A)). The cranial reference was securely mounted on the standard stereotactic frame, thereby serving as a proxy for the subject's head, and was used as a reference frame for tracking the other tools. A second cranial reference (not shown in figure 1) was also used for calibrating the electrode, without disturbing the subject tracking reference. Finally, the multipointer was used as a multipurpose tool for pointing at skull landmarks, scanning the surface of the skull during subject registration, and identifying the entry point of the electrode trajectories. The multi-camera system, connected to a computer in the operating room, provided in real-time the 3D position and orientation of each tool in the physical space co-registered with preoperative MRI and CT data.

We validated this neuronavigation and surgical approach by implanting two macaques (Macaca fascicularis) with two depth electrodes each, targeting respectively the hippocampus and the entorhinal cortex. To this end, we planned a transoccipital surgical approach, positioning the hippocampal electrode along its anteroposterior axis [28–30], and the entorhinal electrode along the parahippocampal gyrus (figure 1(B)). We also provide data from one
<figure><img src="image_8.png" /><figcaption>**Figure 3.** Neuronavigation system accuracy as a function of number of tracking cameras. (A) Tests were performed on a 3D-printed skull derived from a macaque CT scan. A 1 mm hole was drilled in the occipital area of the skull to estimate the error at the entry point of a trajectory. A new CT was then acquired to include this hole and additional landmarks in the neuronavigation virtual space. A standard procedure was used to calibrate an electrode holder attached to a 21G needle used to precisely estimate positioning errors. (B) The error was quantified when varying the number of tracking cameras between three and six. The error range from other two-cameras systems is reported as well, according to the manufacturers' specifications (StealthStation, Medtronic plc; Brainsight, Rogue Research Inc).</figcaption></figure>

additional animal that was used to optimize this neuronavigation procedure, resulting in a total of seven implantations across three animals.

## 3.2. Pre-implantation procedure for trajectory planning

To target these deep brain regions with high accuracy, we optimized the following pre-implantation procedure (figure 4). First, we acquired a whole-brain anatomical (T1- and/or T2-weighted) MRI at an isotropic resolution of 0.5 mm (35 min per acquisition) to identify the target location (figure 4(A)). MRI acquisition was performed in the prone position as during stereotactic surgery, to minimize gravity-induced brain shifts.

Next, we designed a strategy for accurate registration between the physical space defined by the animal during surgery and the previously acquired MRI images. In the Cortexplorer SCI software, registration is performed in two steps: first through the identification of a set of pre-defined landmarks, and then through a skull scanning procedure. The accuracy of initial landmark registration is critical as it influences how the animal in physical space is registered to the MRI data. An incorrect registration may significantly deviate the electrode planned trajectory and result in large inaccuracies at the target. We found that placing these landmarks on the skin, which is a soft and deformable tissue, did not provide satisfactory accuracy. Furthermore, identifying unique natural landmarks on the skull with a submillimeter precision

turns out to be challenging, primarily because most of the skull has a smooth, spherical, and therefore non-unique shape, which makes it unsuitable for registration. To overcome these challenges, we drilled four divots directly on the skull surface (using a 1.8 mm drill bit) to create unique reference points during a stand-alone minimally invasive surgery (figure 4(B)).

After drilling these landmarks, a CT scan was performed at an isotropic resolution of 0.15 mm using a CBCT scanner that is classically used in dentistry and has been previously used for brain surgery in macaques [35] (ProMax® 3D Classic, Planmeca). The scan was performed with the animal's occipital bone resting on the CBCT's chin support and the frontal bone facing upward toward the ceiling (figure 4(C)). Scanning the animal in this position was crucial to ensure that the skull crown was not affected by the cone-beam effect, which creates an artefact at the top of the scanned volume and could affect subsequent MRI/CT co-registration [22, 23].

Following acquisition of the brain MRI and skull CT, both volumes were co-registered in an open-source software for 3D image processing (3D Slicer, www.slicer.org/) [27] (figure 4(D)). To co-register these two modalities, we first identified the AC, PC, and midline to perform an AC-PC transformation. This transformation aligns the volume to the correct orientation. Subsequently, we imported the CT and aligned it manually to approximately match the unique MRI features corresponding to the skull in the MRI volume. Thereafter, we performed a rigid,
<figure><img src="image_9.png" /></figure>

6 degrees of freedom general registration (BRAINS operation in 3D Slicer) and visually inspected the results for consistency.

Finally, we determined the depth electrode implantation trajectories based on anatomical features from MRI (figure 4(E)). For the hippocampal trajectory, the goal was to ensure that all eight electrode contacts reached the bulk of the hippocampus along its longitudinal (anteroposterior) axis. After marking the anterior pole of the hippocampus as the target, the entry point was positioned on the occipital bone so that the implantation followed this axis. The trajectory for the entorhinal cortex was defined as being proximal to the angular bundle and parahippocampal gyrus [36, 37]. Special care was taken to avoid major blood vessels and ventricles.

### 3.3. Depth electrode implantation procedure using neuronavigation

Upon completion of the pre-operative planning procedure, we developed and refined a precise depth electrode implantation protocol to ensure high accuracy and repeatability across animals (figure 5 and movie jneae135bsupp1.mp4). For brevity, we will only explain the main implantation steps and not the standard surgical procedures such as animal placement on the stereotactic frame, sterile disinfection or skin opening (see methods for more details). The following protocol was rigorously tested and thoroughly

practiced on personalized 3D-printed skulls prior to each surgery, which we highly recommend to anyone trying to tailor it to their own applications.

Immediately prior to the implantation surgery, we calibrated the neuronavigation cameras by sampling enough points in the space around the stereotactic frame using the calibration wand (see methods). The first surgical step was to expose the skull and divots drilled during the previous surgery (figure 5(A), left). We then pointed at each of these four divots using the multipointer to register them as discrete landmarks in the Cortexplore software (figure 5(A), middle). This process of landmark registration places the four divots from the animal's skull in the virtual space (figure 5(A), right, red spheres). The initial landmark registration based on only 4 divots is critical but remains relatively coarse (estimated root mean square —RMS— error: 0.3 mm). To refine animal registration, we proceeded to the next step by scanning the skull surface with the multipointer and sampling approximately 800 points in total (figure 5(A), right, cyan dots). We also ensured that the exposed skull around the divots was sampled along with the areas where the entry points would be located. We verified registration accuracy by checking that unique areas on the skull (such as the bregma) had a good correspondence between the physical and virtual space, and that the software reported an estimated root mean square error smaller than 0.1 mm.
<figure><img src="image_10.png" /></figure>
We proceeded with the electrode calibration process, which is done to ensure that the software accurately estimates the electrode's tip position and orientation (figure 5(B)). The electrode was mounted inside the guide tubes of the electrode holder (see also figure 2), with its distal tip not protruding beyond the tube's opening. We then calibrated both the distal tip and the shaft of the electrode, the latter being obtained after lowering the electrode by 3-4 cm. Note that for the implantation of several electrodes, we recommend mounting the electrodes on distinct electrode holders and calibrating them at the same time at the beginning of surgery to maximize efficiency.

Following electrode calibration, we identified the entry point of the planned electrode trajectory by moving the multipointer tip over the skull while the software displayed its estimated position in real time. We drilled a burr hole at the entry point using a 1.8 mm diamond burr and expanded the hole to approximately 4 mm in diameter to ensure that the outer guide tube (diameter 1.7 mm) would not touch the edge of the craniotomy during insertion. Next, we positioned the electrode holder onto the predefined trajectory by adjusting all 3D angles and ensuring that the angular error was smaller than 0.05° in each dimension (figure 5(C)).

In the next step, we pierced the dura using a needle or dura hook and slowly lowered the guide tube assembly, which comprised the inner and outer guide tubes, as well as the electrode (figure 5(D)). We ensured that the stereotactic arm remained aligned with the planned trajectory and that there was no major translation or angular changes that occurred when going through the dura. To prevent damage to the targeted area, we stopped the guide tube assembly 12 mm before the target, corresponding to the total length of electrode contacts. We then marked the stylet (metallic rod used to manually handle the electrode) 12 mm above the stylet holder block, providing a reference point when subsequently lowering the electrode (figure 5(E)). After loosening the stylet screw, we gently pushed the electrode by 12 mm into the brain using a pair of tweezers, without affecting the position of the electrode holder and associated guide tubes (figure 5(F)). Once the electrode reached the desired target location, we tightened the screw to secure the stylet and carefully removed the inner guide tube from the brain with a pair of tweezers (figure 5(G)). Due to the slot along the length of the inner guide tube, we could withdraw the inner guide tube without providing any obstruction to the wires attached to the Omnetics connector.

As the penultimate implantation step, we clamped the stylet above the electrode base and below the stylet-holding block, so that the stylet and electrode were held in place by a secondary free arm fixed on the stereotactic frame. This enabled us to withdraw the holder containing only the outer guide tube

without affecting the position of the electrode in the brain (figure 5(H)). After the outer guide tube was withdrawn, the electrode was disengaged from the outer guide tube via the slit along the outer guide tube length. At this stage, only the electrode remained inserted in the brain on one side and was maintained by the stylet on the other side.

Finally, we added bone cement to cover the burr hole and fix the electrode in place (figure 5(I)). Once cured, we clipped the stylet. We followed the same procedure to implant both depth electrodes and target the hippocampus and entorhinal cortex. Postoperatively, we performed a CT scan with an isotropic resolution of 0.15 mm to verify electrode placement (figure 5(J)).

### 3.4. High-accuracy electrode implantation in the hippocampus and entorhinal cortex

To determine the accuracy of our depth electrodes implantation procedure, we performed a co-registration between the pre-operative MRI and the post-operative CT scan (figures 6, 7, and movie jneae135bsupp2.mp4). We quantified the implantation error for each electrode contact as the minimum 3D Euclidean distance between the planned trajectory and the individual contact's center observed on the CT [4]. This method offers a valuable opportunity to reliably assess electrode location within deep brain structures in vivo, prior to histological validation. We then computed the mean and standard deviation of implantation errors across contacts for each implanted electrode (figures 6 and 7). In total, seven leads were implanted in three NHPs, including five with skull-based registration as described in the previous section, and two with skin-based registration during the initial development of the technique in the first animal (table 3). Note that in the first animal, we explanted the hippocampal electrode initially implanted with skin-based registration and re-implanted another one using skull-based registration.

Using skull-based registration, we could target the entorhinal cortex/parahippocampal gyrus with a sub-millimeter accuracy (Monkey O: 0.89 ± 0.07 mm, Monkey E: 0.55 ± 0.11 mm; figure 6, rows 1-2). When implanting the hippocampus, we observed slightly higher implantation errors (Monkey O: 1.68 ± 0.09 mm, Monkey K: 1.11 ± 0.18 mm; figure 6, rows 3-4), which we attribute to brain shifts due to several potential factors. First, since the hippocampal electrode was always implanted after the entorhinal electrode, it could have been affected by brain shift or swelling due to the first implantation. Second, the hippocampal trajectory passed very close to the lateral ventricle. If the outer guide tube (which structurally supported the electrode) went through the ventricle, a release of ventricular pressure could have caused brain shift, thereby affecting electrode position [38]. Overall, these results across four different electrode
<figure><img src="image_11.png" /></figure>
<table><caption>**Table 3.** Electrode implantation accuracy using skin-based and skull-based registration methods.</caption><thead><tr><th>Animal ID</th><th>Targeted area</th><th>Registration method</th><th>Notes</th><th>Accuracy (mm)</th><th>Figure panel</th></tr></thead><tbody><tr><td>Monkey K</td><td>Entorhinal cortex (EC)</td><td>Skin-based</td><td>/</td><td>1.73 ± 0.32</td><td>Figure 7, row 1</td></tr><tr><td>Monkey K</td><td>Hippocampus (HC)</td><td>Skin-based</td><td>Electrode later explanted</td><td>2.97 ± 0.44</td><td>Figure 7, row 2</td></tr><tr><td>Monkey K</td><td>Hippocampus (HC)</td><td>Skull-based</td><td>Electrode re-implanted</td><td>1.11 ± 0.18</td><td>Figure 6, row 4</td></tr><tr><td>Monkey E</td><td>Entorhinal cortex (EC)</td><td>Skull-based</td><td>/</td><td>0.55 ± 0.11</td><td>Figure 6, row 2</td></tr><tr><td>Monkey E</td><td>Hippocampus (HC)</td><td>Skull-based</td><td>Mechanical instability of electrode holder</td><td>2.29 ± 0.10</td><td>Figure 7, row 3</td></tr><tr><td>Monkey O</td><td>Entorhinal cortex (EC)</td><td>Skull-based</td><td>/</td><td>0.89 ± 0.07</td><td>Figure 6, row 1</td></tr><tr><td>Monkey O</td><td>Hippocampus (HC)</td><td>Skull-based</td><td>/</td><td>1.68 ± 0.09</td><td>Figure 6, row 3</td></tr></tbody></table>

insertions indicate that our optimized implantation procedure enables to reliably insert multiple contacts within a targeted area in deep structures of the macaque brain with accuracies that range from 0.5 to less than 2 mm.

To highlight the criticality of the steps described above, we also report the implantation errors obtained when deviating from these procedures (figure 7). Prior to optimizing the registration protocol based on four divots directly drilled in the skull, we performed two electrode implantations using skin-based registration. In this case, the first step of the registration procedure (figure 5(A), middle) was based on anatomical landmarks placed on the skin rather than the skull. Since the skin is a deformable tissue prone to registration errors, we attempted to mitigate this issue by 3D-printing a personalized helmet with pre-defined holes, which was used to mark landmarks on the skin prior to incision. Despite this mitigation measure, this procedure resulted in implantation errors around 2-3 mm in deep brain structures (Monkey K, hippocampal electrode: 2.97 ± 0.44 mm, entorhinal electrode: 1.73 ± 0.32 mm; figure 7, rows 1-2), which caused the hippocampal electrode to completely miss its target. Note that in this animal, we were able to explant the hippocampal electrode and reimplant it later using skull landmarks, which resulted in a substantially improved accuracy as reported above (1.11 ± 0.18 mm; figure 6, row 4).

Beyond registration errors, hardware limitations can also impact implantation accuracies. During one implantation, the weight of the electrode assembly produced a slight mechanical instability of the stereotactic arm when used in full extension, which led to a deviation of the guide tube during dura piercing and resulted in an implantation error larger than 2 mm at the target (Monkey E, hippocampal electrode: 2.29 ± 0.10 mm; figure 7, row 3). This example shows that any mechanical instability of the surgical equipment should be minimized, and that all the steps should be practiced on a personalized 3D-printed head model prior to surgery. In subsequent

surgeries, the stereotactic arm was not used in full extension, which eliminated this source of variability.

Finally, we performed histology on one of the implanted animals (Monkey K) and verified the implantation accuracy ex vivo (figure 8). To this end, we employed semi-automatic methods for registering the histological sections to the brain MRI and, by extension, to the post-operative CT scan. We then visually inspected the histological sections corresponding to the electrode trajectories estimated from the CT. We observed a clear track for the hippocampal electrode, which precisely co-localized with the estimated electrode position (figure 8, red circle on histology sections). These results confirmed that the hippocampal electrode in Monkey K was indeed implanted in the CA1 field of the hippocampus as planned pre-operatively (figure 6, bottom row).

### 3.5. Electrophysiological validation: sEEG recordings and stimulation of the hippocampal formation

Additionally, we sought to validate the electrophysiological capabilities of our depth electrodes for intracranial EEG recordings and electrical stimulation, as in standard sEEG experiments. Some of the most prominent features in intracranial EEG signals are the ones characterizing different states of sleep and wakefulness. To assess signal quality from our electrodes, we recorded electrophysiological activity during periods of drowsiness in two chronically implanted animals as they sat passively in a primate chair in the context of other behavioral experiments not described here (figures 9(A) and 10(A)). We observed a combination of sleep spindles (10-15 Hz), theta (4-8 Hz) and delta (0-4 Hz) waves, as previously reported for this state of consciousness in both humans [39, 40] and NHPs [29, 41].

We further aimed to assess the ability to stimulate electrically and record meaningful electrophysiological responses characteristic of the hippocampal formation using these depth electrodes. To this end, we applied electrical stimulation pulse trains consisting of biphasic pulses (300 µs pulse width, 100 µs
<figure><img src="image_12.png" /></figure>

inter-phase interval) at a frequency of 50 Hz for a duration of 10 s between two neighboring contacts of the hippocampal electrode. In response to stimulation above a certain threshold, we observed epileptiform afterdischarges that outlasted the stimulation period and propagated to adjacent contacts and connected areas (figures 9(B) and 10(B)). This induction of afterdischarges is commonly used during intraoperative neuromonitoring for epilepsy or brain tumor surgery. Based on their frequency and length, these afterdischarges correspond to sequential spikes [42]. Though only the hippocampal electrode

was stimulated, they were apparent both in the hippocampus and entorhinal cortex, in an amplitude-dependent manner, and showed different powers and delays across different electrode contacts. This relative heterogeneity across contacts indicates propagation to local and connected circuits.

Overall, these electrophysiological results corroborate the suitability of our depth electrodes design and implantation procedures for performing sEEG recordings and electrical stimulation in deep brain structures of the macaque brain, such as the hippocampal formation.
<figure><img src="image_13.png" /></figure>

## 4. Discussion

Overall, we integrated advanced multi-camera neuro-
onavigation, high-resolution multimodal neuroima-
ging, and tailored surgical methods to chronically
implant depth electrodes in deep structures of the
NHP brain, such as the hippocampus and entorhinal
cortex, with an accuracy below or at the millimeter
scale. Since this level of accuracy was previously

unattainable in NHP models using standard neur-
onavigation techniques, we believe that our proposed
approach will provide a useful resource to the neur-
oscience community, for both basic neurophysiology
research and translational studies involving DBS.
Hereafter, we highlight some key points to con-
sider when using our method, and discuss its
advantages and drawbacks compared to alternative
approaches.
<figure><img src="image_14.png" /><figcaption>**B. Depth electrode recordings during afterdischarges following hippocampal stimulation**</figcaption></figure>

<figure><img src="image_15.png" /></figure>

**Figure 9.** Electrophysiological validation in Monkey E. All data shown in this figure were recorded from Monkey E. (A) Intracranial EEG recordings during drowsiness, represented for contacts 1, 4 and 7 of the two electrodes implanted in the hippocampus (HC) and parahippocampal gyrus (PHG). Left: estimated location of each contact in the hippocampus (red), entorhinal cortex (blue), and parahippocampal gyrus (yellow). Characteristic features of drowsiness in macaques including sleep spindle and theta-delta waves are highlighted in orange and green respectively. Spectrograms of the most distal contact for each electrode are displayed between 0 and 20 Hz. Dotted lines indicate 4, 8, and 12 Hz, delineating standard delta, theta, and alpha frequency bands. (B) Intracranial EEG recordings during epileptiform afterdischarges following electrical stimulation of the hippocampus (10 s bipolar stimulation between contacts 2 and 3, 50 Hz, biphasic pulses, 300 µs pulse width), represented for contacts 1, 4, and 7 of each electrode (4 s zoom-in period highlighted in blue). Spectrograms of the most distal contact for each electrode are displayed between 0 and 80 Hz (white dotted box indicates the zoom-in area). Two stimulation amplitudes are represented (left: 1.5 mA, right: 1.6 mA), highlighting two different regimes of afterdischarges.

**4.1. Critical aspects to ensure high-accuracy neuronavigation**

In our experience, one of the main factors influencing neuronavigation accuracy is the number of optical tracking cameras. Two-camera systems, although common, produce errors in the 1-2 mm range at the entry point, which can result in inaccuracies of 3-5 mm in deep brain structures [5–7, 13, 14, 17]. We quantified the relationship between the number of cameras and targeting accuracies in the

Cortexplore system, using a 3D-printed skull with 1 mm divot holes used as neuronavigation landmarks. We observed that the tooltip-to-trajectory error dropped significantly as the number of cameras increased. A three-camera setup resulted in an average error of 1.36 mm, while a six-camera configuration reduced this to 0.38 mm. Across five- and six-camera trials, errors consistently remained below 0.45 mm. Accordingly, we adopted a six-camera setup for our optimized protocol.
A. Depth electrode recordings during drowsiness

<figure><img src="image_16.png" /></figure>

B. Depth electrode recordings during afterdischarges following hippocampal stimulation

<figure><img src="image_17.png" /><figcaption>**Figure 10.** Electrophysiological validation in Monkey O. This figure is replicated from figure 9, but with data recorded from Monkey O.</figcaption></figure>

The second aspect to consider for correct targeting is the quality of landmark registration during surgery. When optimizing our procedure, we realized that using anatomical landmarks placed on the skin, which is a soft and deformable tissue, produced variable and often inaccurate results. We initially attempted to mitigate this issue by 3D-printing a personalized helmet with pre-defined landmarks. A similar approach based on a cranial cap surgically screwed into the skull had been previously reported in the literature [43]. In our case, the helmet was used prior to skin incision and was therefore not screwed to the skull, which made it prone to inaccuracies related to skin deformation and resulted in targeting errors of 2-3 mm in the hippocampal formation. Therefore, we strongly encourage other groups

to use skull instead of skin landmarks when performing multi-camera neuronavigation. To this end, we consider our approach of drilling four divot holes during a first surgery prior to a CT scan to be both minimally invasive and highly reliable.

Another aspect to take into account is the potential impact of positional brain shift between MRI acquisition and surgery. Indeed, prior work in humans has demonstrated that gravitational brain shifts of 0.52-1.7 mm can occur between the prone and supine positions [44, 45]. While seemingly minor, such displacements are non-negligible when targeting regions like the hippocampus, which narrows to about 4 mm in diameter in macaque monkeys. To quantify this effect, we acquired high-resolution (0.5 mm isotropic) MRI scans in both the
supine and prone positions. After co-registering the volumes, we detected a positional brain shift of no more than one voxel (0.5 mm) across the anterior and PC. To mitigate this risk entirely, we conducted the pre-operative MRI with the animal placed in the same prone position as during stereotactic surgery, thereby standardizing head orientation and minimizing brain shift-related trajectory errors.

Finally, the quality of the MRI and CT co-registration is critical to achieve accurate targeting. It is important to stress that the co-registration step can be affected by imaging artefacts in each modality. For instance, we performed CT scans using a dental CBCT scanner, which produces a cone-beam artifact at the top of the scanned volume due to beam scattering [22, 23]. When we initially positioned the animals with the lower mandible resting on a chin support (mirroring typical dental CBCT protocols), the scan revealed intense scattering artifacts across the cranial vertex, which obscured the skull surface and impeded accurate surface registration. To address this, we repositioned the animal with the nose facing upward and the occipital bone supported, which shifted the artifact to the frontal bone (a region that was not used for registration). Although artifacts persisted, their location no longer compromised the registration process. We deliberately avoided post-acquisition modifications to the CT scans, limiting adjustments solely to denoising and filtering as provided by the CBCT software to preserve anatomical integrity.

## 4.2. Alternative approaches to multi-camera neuronavigation

It should be mentioned that submillimeter targeting of deep brain structures may also be achieved through two alternative approaches: surgical robots with laser scanning capabilities (e.g. for humans: ROSA ONE Brain robotic system, Zimmer Biomet Holdings, Inc.; for large animal models: Brainsight Vet Robot, Rogue Research Inc.), or an MRI-compatible platform for real-time MRI-guided stereotactic procedures (ClearPoint system, ClearPoint Neuro Inc.).

Laser scanning enables surface registration of the skull or skin with high accuracy. Combined with motorized robotic systems for surgical implantation, this technology can enable submillimeter targeting accuracies [46], similar to what we achieved using multi-camera neuronavigation. Robotic systems have been used previously in macaque monkeys, for example to target the corticospinal tract within the internal capsule using the ROSA ONE Brain robot [47], and selected tracks including deep areas in the ventromedial prefrontal cortex using the Brainsight Vet Robot system [48], as well as in smaller species such as marmoset monkeys [49]. Robotic systems have also been used to implant sEEG electrodes in the macaque hippocampus, which resulted in targeting accuracies of 2.68 ± 1.03 mm (mean ± standard deviation) [50], a number higher than the errors reported in our work.

The other alternative to optical neuronavigation and surgical robots is the use of the ClearPoint platform, a set of MRI-compatible hardware and dedicated software that provides real-time MRI guidance of stereotactic procedures [51]. This system has been used for testing the real-time delivery of viral vectors or cell grafts in large animal models including swine and NHPs [52–58]. Notably, the ClearPoint platform has also been applied to the implantation of DBS electrodes in patients with Parkinson’s disease or other motor disorders, resulting in accurate placement, which was deemed comparable to frame-based methods [59, 60]. However, this method is relatively expensive and cannot be easily applied to preclinical experiments in NHPs except in specialized facilities.

Additionally, it should be noted that custom methods have also been developed by individual labs to optimize targeting accuracy for specific applications. We already mentioned how a personalized craniial cap screwed to the skull and equipped with fiducial markers could improve neuronavigation accuracy in NHPs [43]. We would also like to mention that MRI-compatible stereotactic frames may be used for accurate targeting of deep brain regions, provided that the positioning of the animal’s head in the stereotactic frame is similar between the MRI acquisition and the surgery. For example, such a frame for NHPs was recently released as an open-source material that can be readily 3D-printed [61]. However, ensuring the consistency in head positioning between the MRI and surgery can turn out to be challenging.

## 4.3. Depth electrodes for NHP applications

Lastly, we introduced a set of tools, including depth electrodes, an electrode holder, an inner and outer guide tubes, which enable to perform sEEG and DBS studies in NHP models. Until recently, most groups studying DBS in NHPs relied on custom versions of cardiac catheters with a diameter of 625-750 µm and up to eight platinum-iridium cylindrical contacts, each separated by 500 µm (NuMED for Children, LLC). These surgical leads have been used in numerous studies conducted in large animal models, mainly NHPs, between 2012 and 2024 [13, 62–72]. However, these custom electrodes are no longer available from this company. We therefore reached out to one of the leading manufacturers of sEEG electrodes, Dixi Medical/Dixi Fablab, to develop a similar electrode design with an outer diameter of 800 µm. This is below the diameter of clinical DBS leads (about 1.27-1.5 mm), which have nevertheless been used in some NHP studies [16]. An additional complexity for conducting similar experiments stems from the fact that clinical DBS and sEEG electrode systems typically come with a set of surgical tools for electrode insertion and securing on the skull. In this work, we
developed custom tools tailored to chronic applications in NHPs. Additionally, we decided to share all our technical drawings for easy replication of our methods by other groups.

## 5. Conclusion

We developed novel methods for implanting depth electrodes in deep brain regions of the NHP brain using multi-camera neuronavigation. Our experiments underscore the necessity of rigorous methodological validation at every stage of the procedures that we developed, from optimizing camera configuration and registration strategies, to minimizing brain shift and controlling imaging artifacts. In summary, we provide a technological framework for high-accuracy electrode implantation in large animal models, especially NHPs, which will enable future neuroscience studies that aim at investigating the effects of neuronavigation in deep brain regions such as the hippocampal formation [9, 73]. In future applications, we also anticipate that a combination of anatomical and diffusion MRI data, acquired at a submillimeter resolution (e.g. [20]), will further improve our ability to target specific anatomical structures and white matter tracts.

## Data availability statement

The data that support the findings of this study are openly available at the following URL/DOI: https://doi.org/10.5281/zenodo.17193907.

## Acknowledgment

This work was supported by the European Union's Horizon Europe research and innovation program under European Research Council (ERC) Starting Grant Agreement No. 101040391 (F.B.W., MEMOPROSTHETICS), and the Bordeaux Neurocampus junior chair program co-funded by the Région Nouvelle-Aquitaine and the University of Bordeaux Initiative of Excellence (IdEx) (F.B.W., Neurocampus junior chair, 2020-2023). We would also like to thank the technical support and inputs provided by the following companies: Cortexplore GmbH, Rogue Research Inc., Dixi Medical/Dixi Fablab, Blackrock Neurotech Inc., Alsymex Bordeaux-Mérignac. The authors declare no competing interests.

## Author contributions

F.W. secured funding and coordinated the study. A.G., A.S., and F.W. designed the neuronavigation and surgical procedures. A.G., A.B., and F.W. designed the custom surgical tools. A.G., A.B., N.V. and F.W. evaluated the procedures on plastic heads. A.G., A.B., N.V., and T.H.N. acquired MRI and CT

data. A.S. and T.H.N. performed the surgeries, with help from A.G, A.B, N.V, and F.W. for all neuronavigation steps. M.T, H.O, T.H.N. oversaw animal care. N.B. and S.D. performed histology. A.G., A.B., and M.T. conducted electrophysiological recording sessions. A.G. performed the image processing. A.B. performed the neural data analysis. A.G. and A.B. prepared figures with feedback from F.W. A.G, A.B., and F.W. wrote the first version of the manuscript, which was then edited and approved by all authors.

## ORCID iDs

Ankur Gupta ID 0000-0002-5333-0092

Adrien Boissenin ID 0000-0001-7714-4975

Nikolaos Vardalakis ID 0000-0002-5436-4091

Nathalie Biendon ID 0000-0002-5180-5656

Sandra Dovero ID 0000-0002-5781-1328

Amirouche Sadoun ID 0000-0003-0606-8738

Fabien B Wagner ID 0000-0002-9582-6109

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