A detailed new procedure for functional imaging of awake, head-fixed rats is described.
Anesthetics, commonly used in preclinical and fundamental scientific research, have a depressive influence on the metabolic, neuronal, and vascular functions of the brain and can adversely influence neurophysiological results. The use of awake animals for research studies is advantageous but poses the major challenge of keeping the animals calm and stationary to minimize motion artifacts throughout data acquisition. Awake imaging in smaller-sized rodents (e.g., mice) is very common but remains scant in rats as rats are bigger, stronger, and have a greater tendency to oppose movement restraints and head fixation over the long durations required for imaging. A new model of neuroimaging of awake, head-fixed rats using customized hand-sewn slings, 3D-printed head implants, head caps, and a headframe is described. The results acquired following a single trial of single-whisker stimulation suggest an increase in the intensity of the evoked functional response. The acquisition of the evoked functional response from awake, head-fixed rats is faster than that from anesthetized rats, reliable, reproducible, and can be used for repeated longitudinal studies.
Most of the basic, preclinical, and translational scientific neuroimaging investigations are acquired from anesthetized animals1,2. Anesthetics ease experimentation but continuously influence the brain's and body's metabolism, blood pressure, and heart rate3. The type of anesthetic and the duration and route of administration add confounding variables to data interpretation that could contribute to reproducibility and translational failures4. A major bottleneck of awake, head-fixed rat neuroimaging studies is the requirement to keep the rat stationary and calm throughout the preparation and data acquisition processes. Small movements produce unwarranted motion artifacts, which can adversely affect data analysis and interpretations.
A new model of neuroimaging from awake, head-fixed rats using customized slings, three-dimensional (3D)-printed head implants, head caps, and a headframe has been devised that offers several advantages for easy experimentation. The 3D head implant is light and covers a small portion of the skull needed for transfixing. The 3D-printed head implants and caps are designed using computer-aided design (CAD) software. The protocols of whisker stimulation, data acquisition, data analysis, and results from anesthetized rats have been described in detail in previous work5,6,7.
All procedures were compliant with National Institute of Health guidelines and approved by the University of California, Irvine Animal Care and Use Committee. Seven males and one female rat (Sprague-Dawley, weight: 185-350 g) were used in this study. After study completion, the rats were sacrificed using carbon dioxide overdose.
1. Design of different components
2. Initial rat training
3. Sling training
4. Presurgical preparation
5. Surgery
6. Awake imaging
The representative optical imaging signals from a single trial of an anesthetized rat and the summed response (of 40 collected trials) of an awake rat are shown (Figure 4). The signal intensity for single-whisker stimulation of an awake rat can be visualized at a higher threshold than for the anesthetized rat, showing a stronger signal from the awake animal. The C2 whiskers of rats are stimulated at 5 Hz for 1 s, and the functional response is displayed as a fractional change compared to the baseline. The darker areas (below the negative threshold) are the main areas of neuronal activity, and the bright white areas (above the positive threshold) show the oxygenated blood response to stimulation9. The images are aligned so that from left to right is from rostral to caudal (C) and from top to bottom is the medial to lateral (L) direction, as shown by the arrows.
Figure 1: Head cap, head implant, and head frame. (A) The head cap (top view): the side of the top view shows the curvature to align along the curvature of the head to protect the head; the two hollowed rectangular parts are for the metal wires to pass through the head cap. (B) Head cap (bottom view) shows the wider rectangular cut to fit in the top bar of the head implant and the two perpendicular cuts for the wires to move through the implant and the head cap to keep them in place. (C) Head implant with the three cut holes for the anchoring screws. The positions of the anchoring screws on the head implant can be adjusted according to the head of the rat. (D) Head cap and head implant (side view); the side view of the head implant shows the rectangular bar hollowed from the inside to allow the wire to pass through to anchor the head cap to the head implant. (E–G) View of the head implant anchored in the head cap through one wire piece; bottom view, side view, and top view to show how the head implant is fitted inside the head cap. (H) Head frame, (I) head implant anchored in the head frame. The distance between two lines on the scale (as shown by the blue rectangle) is 1 mm. Please click here to view a larger version of this figure.
Figure 2: Slings, head implant, and fixation of the head frame for awake, head-fixed imaging. (A,B) Customized sling with netting material for either bottom only or both sides; (C) rat placed on the plastic sheet, fixed with Velcro strips, during sling training; (D–F) top and side views of the head implant on a rat skull above the contralateral hemisphere. Dotted lines show the imaging area. The top and side views clearly show the three holes to fix the head implant to the skull with the anchoring screw. (E) The side view shows the hollow bar through which the wire passes to anchor the head cap to the head implant when the rats are not imaged. One leg of the head frame passed through the hollow part of the head implant for imaging the rat cortex. (G) Head frame through the head implant for awake, head-fixed rats. (H) The head frame through the head implant with its two legs clamped for awake, head-fixed imaging (I) of awake, head-fixed rats during the imaging sessions. Please click here to view a larger version of this figure.
Figure 3: Head implant placement. (A) The thin skull preparation for awake, head-fixed imaging. (B) Head implant fixed on the rat skull and the thin-skull imaging area covered with the rubber silicone. (C) Head cap placed on the head implant. (D,E) Head cap anchored to the head implant using coated metal wires. (F) The head cap and the surrounding area covered with rubber-silicone for further support in the fixation and protection of the skull. Please click here to view a larger version of this figure.
Figure 4: Functional responses of C2 whisker stimulations. (A) A representative single trial functional response of a 5 Hz C2 whisker stimulation for 1 s of awake, head-fixed rat imaging, with each trial lasting for 7 s with an inter-trial interval of 3 s ± 2 s. The threshold of grayscale representation of fractional change from baseline (−3.5 × 10−3 to 3.5 × 10−3). (B) A representative single trial functional response of a 5 Hz C2 whisker stimulation for 1 s of an anesthetized (sodium pentobarbital) rat. The threshold of grayscale representation of fractional change from baseline (−2.5 × 10−4 to 2.5 × 10−4). The functional response of the awake, head-fixed rat is 140 times stronger than that of the anesthetized rat. Each frame is a 0.5 s frame. The images are aligned so that from left to right is from rostral to caudal and from top to bottom is from the medial to lateral direction as shown by the arrows. The darker areas (below the negative threshold) are the main areas of neuronal activity, and the bright white areas (above the positive threshold) show the oxygenated blood response to stimulation. Scale bar = 1 mm. Abbreviations: C = caudal; L = lateral. Please click here to view a larger version of this figure.
Supplemental File 1: 3D printing file for the head implant. Please click here to download this File.
Supplemental File 2: 3D printing file for the head cap. Please click here to download this File.
The use of awake, head-fixed rat imaging offers many advantages in terms of ease and customization. The custom-designed slings allow the rats to be wrapped through breathable netting material, eliminating the need to enclose animals in closed, plastic restraining chambers for extended periods of time10,11. Rats are kept calm and stress-free throughout the long durations of successive imaging sessions using a very low dose of acepromazine below the levels of mild sedation in rats (1.0-2.5 mg/kg)12. To keep the rat steady and further eliminate motion artifacts during the imaging sessions, Velcro strips are used. The Velcro strips are placed at 3-6 mm from each other to avoid unnecessary body constriction for long hours. The rats are trained and habituated with slings at a young age to ensure that they remain calm and comfortable resting in their slings during preparation and data acquisition. Based on the preliminary results, young rats weighing about 150-175 g are easier and faster to train than older rats.
The head implant on the rat head weighs only 0.174 g, and the removable head cap weighs 1.483 g. The head implant covers an area of 0.5 cm to 1.5 cm on one hemisphere, allowing complete accessibility of the other hemisphere for neuroimaging. The size of the head cap ensures full coverage of the surgical site. The weights of the head implant and head cap do not seem to hinder the mobility and daily activities, and the rats can be housed together in standard cages. Using this head and body restraining method, the rats can be imaged for 2-3 h each time on different days for longitudinal studies. Multiple imaging sessions can be performed on a single rat for at least up to 3 months using this setup. It takes a total of 25 min to 3D print the head implant and the head cap. The parts are easily customizable depending on the size of the rodent and can also be customized to be used in mice. For studies that require differentiation of the rats, different colors and materials can provide easy identification. In addition, the top part of the cap can be customized to add symbols, numbers, or letters for easy identification.
There are several important steps for successful implantation and imaging, the most important of which is the training and habituation of the rats. The rats are randomly presented with sensory stimuli to minimize the potential for associative learning, which can influence the imaging results. The surgery and all surgical instruments need to be sterile to prevent infection, and the use of local antibiotics is imperative. The use of acepromazine at the start of the imaging is important for keeping the animals calm and quiet to avoid unnecessary movements during the imaging sessions. The skull of the rat needs to be dry for proper fixation, and the layer of deposited dental cement needs to be thin enough for the head cap to fit in the head implant.
For the current study, the imaging area was centered on the somatosensory cortex. The thinned area measures approximately 7.5 mm x 7.5 mm, which is the extent of the area that can be imaged in the current study. However, the imaged area can be increased to 11 mm x 11 mm if needed. Another advantage of this design is that it allows imaging of the entire thinned area despite the curvature of the cortex.
Previously reported head implants require almost 7-12 anchoring screws to fix the head implant on the rat's head13,14. This precludes the imaging of a bigger area through thinned skull preparation. Another fixation method requires the fixation of a resin material over a large area using head screws, making the skull inaccessible for imaging14. The awake imaging of rats using MRI requires immobilization of animals in cylindrical tubes, making the imaging experiences stressful for the animals11,15. In some other setups, the head implant protrudes out of the head and could become entangled in standard cages16,17. The head implant and head cap eliminate the use of fixation of glass slides and flattening of the thin skull for chronic imaging18,19. The size of the head implant and the use of curvature on the head cap eliminate the need for making changes to the standard cages as in other chronic procedures18,19. The head implants in mice are easier because only a single nut and screw configuration are used, which is not possible in rats, as rats are much stronger and more difficult to be kept steady20.
The limitation of the head implant is that, despite its small size, it requires anchoring of the implant to the skull using screws. The head implant is necessary to keep the animal's head steady but limits imaging of the whole rat brain. However, an advantage of using this head implant is that it can be used to image a wider area for evoked sensory stimulation using various neuroimaging modalities such as intrinsic signal optical imaging, doppler optical coherence tomography, and laser speckle imaging.
The cortical functional representations based on intrinsic signals of awake, head-fixed rats tend to be stronger in intensity than in anesthetized rats using the same whisker stimulation protocol. A similar increase in the strength of evoked intrinsic signal response has been reported in awake monkeys21,22. Current work is ongoing to improve the head implant and head cap design for more challenging environments such as the naturalistic habitat23.
The authors have nothing to disclose.
We acknowledge Clara Jones, James Stirwalt, Linh Hoang, Young Joon Ha, and Amirsoheil Zareh for their help during training of the rats and preparation of the slings. Funding was provided by the National Institutes of Health (NIH, Grant Number: NS119852) and Leducq Foundation (Grant Number:15CVD02).
Rats | Charles River | Sprague Dawley | |
Isoflurane | Pivetal | 21295098 | General anesthetic |
Lidocaine HCl 2% injection | Phoenix | L-2000-04 | Local anesthetic |
Atropine sulfate injection | Vedco | 5098907512 | Help in respiration |
Lactated Ringer's injection solution | Vedco | 50989088317 | |
Flunixin injection | Vedco | 6064408670 | Pain management |
Enrosite injection (Enrofloxacin 2.27%) | VetOne | 501084 | Avoid infection |
PromAce injection (Acepromazine maleate) | Beohringer Ingelheim | 136059 | |
Animax ointment | Dechra Veterinary Products | 122-75 | active ingredients of nystatin 1000units per gram, neomycin sulfate 2.5mg per gram, thiostrepton 2500 units per gram, and triamcinolone acetonide 1mg per gram |
Puralube ophthalmic ointment | Dechra Veterinary Products | 211-38 | |
Povidone-iodine PVP prep pads | Medline | MDS093917 | Betadine generic |
Isopropyl alcohol swabs | BD | 326895 | |
Vetbond tissue adhesive | 3M | 1469SB | |
Bur (drill bit), standard operatory carbide | SS White Burs | 14829 | #3 bur |
Screws, 00-90 x 1/8 flat head stainless steel | J.I. Morris | F0090CE125 | Anchor screws |
Stereotaxic system | Kopf Instruments | 1430 | |
Homeothermic heating blanket | Harvard Apparatus | 50-7220-F | |
Pulse oximeter & heart rate monitor | Kent Scientific | MouseStat Jr. | |
Petrolatum | Fisher Scientific | P66-1LB | Vaseline generic |
Wire, bare copper | Fisher Scientific | 15-545-2C | 20 gauge |
Teets Cold Cure powder | Pearson Dental | C73-0054 | active ingredient: Methyl Methacrylate |
Teets Cold Cure liquid | Pearson Dental | C73-0078 | active ingredient: Methyl Methacrylate |
Silicone mold rubber | Smooth-On | Body Double Fast | silicon polymer |
Metricide 28 (Germicide) | Metrex | Oct-05 | |
India ink, black | Pelikan | 301051 | |
Dental drill | NSK Dental | Ultimate XL-F | |
3D printer | Prusa Research | i3 MK3S+ | |
Sew on fasteners | Velcro | 90030 | |
Pet screening utility fabric | Joann | 10173334 | Netting material |
Bur (drill bit), standard operatory carbide | SS White Burs | 14829 | #1 bur |