Article Figures & Data
Figures
- Figure 1.
Detailed photographs of the surgical procedure. A, Induce anesthesia and verify the absence of reflexes. After careful fixation to the stereotaxic frame, scrub the surgical site with antiseptic to avoid contamination. B, Make an incision along the sagittal fissure. Expose the skull by scrapping periosteum off the bone. C, Identify the stereotaxic landmarks (Bregma and Lambda) using the sutures on the skull surface. Validate horizontality of the skull. D, Mark the outline of the craniotomy on the skull surface. E, Lift the single bone piece using a double hand technique.Use forceps on one hand to slowly lift the bone flap and a cauterizer with the smallest tip attached on the other hand to scrape the dura of the bone as it is gradually lifted. F, Open each durotomy at the lateral limit of the craniotomy, next to the bone, and extend it carefully along the anterior and posterior boundaries of the craniotomy until the border of the sagittal sinus. G, Slowly lift and fold the bilateral dura flaps over the sinus. H, Elevate the attached sinus and thread a suture beneath it, using a vascular ligation instrument. I, Use one hand to lift the sinus and the other to slide the tip of the instrument under the sinus, carefully avoiding cerebral veins. Once the instrument’s tip is visible on the opposite site, lower the sinus and carefully thread the suture under it without removing the instrument, stabilizing it very carefully. J, Once a 2-3cm loop of suture is threaded, carefully retract the instrument while firmly holding the suture in place at the opposite side of the sinus. K, L, Cut the loop in the middle leaving the suture in place, crossing underneath the sinus, with two strands of thread perpendicular to the sinus. Gently pull one strand towards the anterior limit of the craniotomy and the second towards the posterior limit. M, Double ligate the one at the posterior border, followed shortly by the anterior one. N, Gently lifting the sinus with one hand, cauterize the mid-point between sutures and extend the severed borders of the sinus posteriorly and anteriorly, exposing the longitudinal fissure. O, Cover the whole extension of the craniotomy with a single drop of 1.5% agarose at body temperature (∼37°C) to mechanically stabilize the remaining sinus edges and the suture knots left on them. P, Suture the skin incision, sterilize the wound and apply lidocaine analgesic ointment. Revert anesthesia.
- Figure 2.
Motor behavior is not impaired by SSS transection. Locomotion and exploratory behavior were assessed by open-field test before surgery (Extended Data Fig. 2-1) and at four and eight weeks postsurgery. A, Representative trackplots of the same ST and sham-operated animal after surgery. No significative differences were observed in total distance traveled (B), average speed (C), and total resting time (D) in all time points tested (n = 8, means ± SEM, ‡p > 0.05, ST comparing to sham, repeated measures two-way ANOVA followed by Bonferroni’s multiple comparisons post hoc test). Permanence in the open-field’s subregions was similar between groups in all time points tested (Extended Data Fig. 2-1).
- Figure 3.
Short-term spatial memory is not affected by SSS transection. Spatial memory performance was assessed by the Y-maze test before surgery (Extended Data Fig. 3-1) and at four and eight weeks postsurgery. A, Schematic representation of the Y-maze test. B, top, Quantification of the time spent by sham and ST animals in novel versus other arm at four and eight weeks postsurgery. Both groups showed preference for the novel arm (n = 8, ****p < 0.0001, novel arm compared with other arm, two-way ANOVA followed by Tukey’s multiple comparisons post hoc test). The performance of ST and Sham animals was comparable in both time points (n = 8, ‡p > 0.9999, ST compared with sham, two-way ANOVA followed by Tukey’s multiple comparisons post hoc test). No significant differences were found between the performance of ST animals at different time points (n = 8, #p = 0.9999, time spent in the novel arm at four weeks postsurgery compared with eight weeks postsurgery, two-way ANOVA followed by Tukey’s multiple comparisons post hoc test). All values are mean ± SEM (B, bottom). The number of transitions between arms was not different between groups at each time point (Extended Data Fig. 3-1). Representative trackplots of the same ST and sham-operated animal at four and eight weeks postsurgery. Within subject analysis showed no significant differences in the performance across time points (Extended Data Fig. 3-1).
- Figure 4.
Transection of SSS does not affect brain tissue viability. Histopathological analysis indicates no extensive neural damage or focal lesion 10 weeks after SSS transection. ST animals are comparable to sham-operated animals in all features analyzed. A, Low magnification of brain, skull and meninges stained with hematoxylin and eosin. Scale bar: 5000 μm. B, Brain and skull magnification. Features of normal surgical wound healing were found within the skull and meninges in the craniotomy area of both groups. Note the scar tissue (scored as moderate). No alterations were found in the cerebral parenchyma. Scale bar: 1000 μm. C, Magnification of scar tissue (scored as moderate), composed mainly of fibroblasts, neovascularization, and macrophages (black arrows) with occasional hemosiderin accumulation (blue arrows). Scale bar: 250 μm. D, High magnification of cerebral parenchyma. No alterations were observed in the neurons and glial cells. Scale bar: 100 μm. Histologic damage is not correlated with behavioral performance and did not involve ventricular alterations (Extended Data Fig. 4-1).
- Figure 5.
SSS transection allows accurate microinjection of deep hypothalamic nuclei. A, Schematic representation of the target areas used for microinjection: (1) MPOA and (2) SCN. B, Photomicrograph of coronal sections showing extensive AAV8-hSyn-hM4D(Gi)-mCherry expression in the MPOA at five weeks postinfection (1) and strong BDA–Texas Red uptake in the SCN at 11 d postinjection (2). Scale bars: 250 μm. C, Schematic drawing of the ventral surface of the rat brain detailing hypothalamus’ location (left). Whole-brain imaging confirms the presence of fluorescence in deep hypothalamic structures surrounding the optical chiasm, five weeks after injection in MPOA (1) and 11 d postinjection in SCN (2; right). All injections performed using the same virus and tracer showed similar patterns (Extended Data Fig. 5-1).
- Figure 6.
SSS Transection enables multisite recording of deep midline brain areas. A, Representation of the SLIQ hyperdrive used to chronically record neural activity across multiple regions in freely moving animals. B, Schematic representation of the experimental approach (left). An array of 15 octrodes was chronically implanted to simultaneously record neural activity from different hypothalamic nuclei (right). C, Photograph of ventral brain surface, confirming octrode targeting of deep hypothalamic areas surrounding optical chiasm. D, Schematic of tetrode recording of neural activity from SCN, ReCh, and Arc (left). Two seconds of example LFP recorded simultaneously from SCN, ReCh, and Arc (right). Anatomical location of octrode tips was confirmed by histologic analysis (Extended Data Fig. 6-1B).
- Figure 7.
Single units are well isolated and stable over the recording period. A, Amplitude over the duration of an example recording session for three units simultaneously recorded from the same octrode. This recording was performed inside a sleep-box, where the animal was free to move. Units B and C increase their firing rates at a later moment of the recording session when the animal starts to sleep or stays immobile for long periods of time. B, First and last 100 spikes for each unit recorded from the channel where the highest amplitude was detected. Note the significant overlap between early and late waveforms. C, PC scores for the same units as above.
Extended Data
Extended Data Figure 2-1
ST rats exhibit locomotor and exploratory behavior similar to sham-operated animals in all time points tested. Locomotion and exploratory behavior were assessed by the open-field test before surgery and at four and eight weeks postsurgery. A, Representative trackplots of ST and sham-operated animals before surgery. B, No significant differences were found in total distance traveled, average speed and total resting time (n = 8, ‡p > 0.05, ST compared to sham, two-tailed unpaired t test) during presurgery testing. After basal assessment, animals were randomly assigned to experimental (ST) and control (sham) groups. Permanence in the open-field’s subregions was similar between groups in all time points tested (n = 8, ‡p > 0.05, ST compared to sham, two-way ANOVA followed by Tukey’s multiple comparisons post hoc test; C). All values are mean ± SEM. Download Figure 2-1, TIF file.
Extended Data Figure 3-1
Short-term spatial memory performance of ST rats is similar to sham-operated animals in all time points tested. Spatial memory performance was assessed by the Y-maze test before surgery and at four and eight weeks postsurgery. A, Representative trackplots of ST and sham-operated animals before surgery. B, Quantification of the time spent by sham and ST animals in novel versus other arm at baseline assessment. Both groups showed preference for the novel arm (n = 8, means ± SEM, ****p < 0.0001, novel arm compared to other arm, two-way ANOVA followed by Tukey’s multiple comparisons post hoc test). After basal assessment, animals were randomly assigned to experimental (ST) and control (sham) groups. C, Quantification of the number of transitions between arms. No differences were found between groups (n = 8, means ± SEM, ‡p > 0.05, ST compared to sham, repeated measures two-way ANOVA followed by Bonferroni’s multiple comparisons post hoc test). D, Quantification of the time spent by the same animal in the novel arm at different assessments. Download Figure 3-1, TIF file.
Extended Data Figure 4-1
Histological damage is not correlated with performance and did not involve ventricular alterations. A, The extent of damage was quantified in three animals from each group, by measuring the lesioned area in five serial sections per animal (across 1.5 mm) and normalizing to the whole-brain area. No correlation was found between histological damage and Y-maze performance at eight weeks postsurgery (sham: r2 = 0.19, p = 0.71; ST: r2 = 0.87, p = 0.23; Pearson’s correlation). No significant differences were found in histological damage between groups (n = 3, p > 0.05, ST compared to sham, two-tailed unpaired t test). All values are mean ± SEM. B, Normodimensioned lateral ventricles of sham and ST animals. Scale bar: 500 μm. Download Figure 4-1, TIF file.
Extended Data Figure 5-1
SSS transection allows consistent microinjection of deep hypothalamic nuclei. Photomicrograph of coronal sections showing extensive AAV8-hSyn-hM4D(Gi)-mCherry expression in the MPOA at five weeks postinfection (1) and strong BDA–Texas Red uptake in the SCN at 11 d postinjection (2). SSS allows consistent targeting of MPOA and SCN across subjects. Scale bar: 250 μm. Download Figure 5-1, TIF file.
Extended Data Figure 6-1
SSS transection allows multisite recording from deep midline brain structures. A, Postmortem analysis revealed accurate bilateral targeting of SCN in preliminary targeting test, as indicated by the electrolytic lesion and DiI staining. Cell nuclei are stained with Hoechst in blue fluorescence. Octrode tips were dipped in DiI stain (red fluorescence). Scale bar: 250 μm. B, Histological verification of octrode tracks confirms targeting of SCN, ReCh, and Arc. Cell nuclei are stained with Hoechst in blue fluorescence. White arrows indicate octrode tips. Scale bar: 0.50 cm. Download Figure 6-1, TIF file.
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