test schematics

01-intro.Rmd

@@ -5,8 +5,8 @@
 ## Medial Septum
 The medial septum (MS) a structure located in the forebrain (Figure \@ref(fig:MS-Scheme)) can be found across species.
 
-```{r MS-Scheme, fig.cap = 'Schematic of medial septum in saggital (A) and coronal (B) section. Blue shows the location of the MS, light gray represents ventricular space and dark gray fibre bundels. The dashed line in (A) indicates the position of the section of (B). Modified from Allen Brain Atlas.', fig.pos='H', fig.align='center', echo=FALSE, fig.asp = 0.35, fig.height=6}
-sag  <- grImport2::pictureGrob(grImport2::readPicture(rawToChar(rsvg::rsvg_svg("Figures/Schemes/MS_saggital_clean.svg"))))
+```{r MS-Scheme, fig.cap = 'Schematic of medial septum in sagittal (A) and coronal (B) section. Blue shows the location of the MS, light gray represents ventricular space and dark gray fibre bundels. The dashed line in (A) indicates the position of the section of (B). Modified from Allen Brain Atlas.', fig.pos='H', fig.align='center', echo=FALSE, fig.height=3}
+sag  <- grImport2::pictureGrob(grImport2::readPicture(rawToChar(rsvg::rsvg_svg("Figures/Schemes/MS_sagittal_clean.svg"))))
 coronal  <- grImport2::pictureGrob(grImport2::readPicture(rawToChar(rsvg::rsvg_svg("Figures/Schemes/MS_coronal_clean.svg"))))
 wrap_elements(gridExtra::arrangeGrob(sag), clip = T) + wrap_elements(gridExtra::arrangeGrob(coronal), clip=T) + plot_layout(widths = c(3.2,2)) &
   plot_annotation(tag_levels = "A") &
@@ -17,10 +17,10 @@ wrap_elements(gridExtra::arrangeGrob(sag), clip = T) + wrap_elements(gridExtra::
 The parasubiculum (PaS) on the other hand is part of the parahippocampal formation (Figure \@ref(fig:PaS-Scheme)), the posterior part of the mouse brain, and is found in different species [@ding_comparative_2013]. 
 It is adjacent to the medial entorhinal cortex and presubiculum which, as the PaS, have spatially linked functional cell types coding for different aspects used for navigation (Citation needed).
 
-```{r PaS-Scheme, fig.cap = 'Schematic of parasubiculum in a saggital (A) and horizontal (B) section. turqoise shows the location of the PaS, light gray represents ventricular space and dark gray fibre bundels. The dark green in (B) represents the pyramidal cell layer II of the medial entorhinal cortex. The cornu ammonis and the dentate gyrus are marked in light blue.', fig.show='hold', fig.pos='H',echo=FALSE, fig.asp = 0.35, fig.height=6}
+```{r PaS-Scheme, fig.cap = 'Schematic of parasubiculum in a sagittal (A) and horizontal (B) section. turqoise shows the location of the PaS, light gray represents ventricular space and dark gray fibre bundels. The dark green in (B) represents the pyramidal cell layer II of the medial entorhinal cortex. The cornu ammonis and the dentate gyrus are marked in light blue.', fig.show='hold', fig.pos='H',echo=FALSE, fig.height=3}
 hor  <- grImport2::pictureGrob(grImport2::readPicture(rawToChar(rsvg::rsvg_svg("Figures/Schemes/PaS_horizontal_clean.svg"))))
-sag  <- grImport2::pictureGrob(grImport2::readPicture(rawToChar(rsvg::rsvg_svg("Figures/Schemes/PaS_saggital_clean.svg"))))
-wrap_elements(gridExtra::arrangeGrob(sag), clip = T) + wrap_elements(gridExtra::arrangeGrob(hor), clip=T) + plot_layout(widths = c(3.2,2)) &
+sag  <- grImport2::pictureGrob(grImport2::readPicture(rawToChar(rsvg::rsvg_svg("Figures/Schemes/PaS_sagittal_clean.svg"))))
+wrap_elements(gridExtra::arrangeGrob(sag), clip = T) + wrap_elements(gridExtra::arrangeGrob(hor), clip=T) + plot_layout(widths = c(3.2,1.5)) &
   plot_annotation(tag_levels = "A") &
   theme(plot.tag = element_text(size=24))
 ```
@@ -44,9 +44,11 @@ Reference a figure by its code chunk label with the `fig:` prefix, e.g., see Fig
 ```{r nice-tab, tidy=FALSE}
 knitr::kable(
   head(iris, 20), caption = 'Here is a nice table!',
-  booktabs = TRUE
+  booktabs = TRUE,
+  format = "simple"
 )
 ```
+
 look at figure \@ref(fig:nice-fig)
 
 You can write citations, too. For example, we are using the **bookdown** package [@R-bookdown] in this sample book, which was built on top of R Markdown and **knitr** [@xie2015].

02-methods.Rmd

@@ -2,26 +2,72 @@
 
 ## Animals
 
-## Stereotactic Injections
+## Surgical Procedures
 
+### Stereotactic Injections {#injections}
+For all surgical procedures mice were anaesthetised with isoflurane (1.5% vol/vol in oxygen) and received carprofen (5 $\frac{mg}{kg} \textstyle$) as analgesia additionally to subcutaneous application of lidocaine as local analgesia at the side of incision. Animals were placed into a stereotaxic frame where they were fixed and stabilised with ear bars. After disinfecting the place of incision with iodide (###### find exact one####) an one cm incision was performed on the midline. The skin was pushed and fixed to the side so that the skull was easily accessible.
 
-## In-Vitro
+```{r MS-Injection-Scheme, fig.cap = 'Schematic of medial septum (blue) and the injection canula (black). A 10° angle is used to avoid damage to superior sagittal sinus which is located on the midline on top of the brain surface (not shown).', fig.show='hold', fig.pos='H',echo=FALSE, fig.height=3}
+Inj  <- grImport2::pictureGrob(grImport2::readPicture(rawToChar(rsvg::rsvg_svg("Figures/Schemes/MS_schema.svg"))))
+wrap_elements(gridExtra::arrangeGrob(Inj), clip=T)
+```
+
+The skull was levelled using ########## and a craniotomy drilled at the position of entry (in mm from bregma: 0.7 anteriorposterior, 0.7 mediolateral). A NanoFil syringe with a 34‐gauge needle with adeno-associated virus 1 (AAV1) containing the plasmid for the yellow fluorescent protein (YFP) and channelrhodopsin (*pAAV-EF1*$\alpha$*-double floxed-hChR2(H134R)-EYFP-WPR* at 6.26·10^11^ VG/ml) was placed with a 10° angle through the hole and slowly moved 3.8 mm downwards along the dorsoventral axis to the MS (Figure \@ref(fig:MS-Injection-Scheme)). The position was held for 5 min before 200 nl of the virus was injected at a rate of 40 nl/min. The needle was left for further 5 min until it was slowly withdrawn. During the whole procedure the animal's temperature was maintained at 37°C, eyes were covered with eye-cream to provide moisture, and anaesthesia was checked regularly. The incision was closed and the animal was put back to the home-cage to recover where it woke up after less than 15 min. Animals were checked daily and were allowed to recover for at least 4 weeks.
+
+### Headbar Implant
+Previously injected animals as part of *in-vivo* awake head-fixed recordings were given a headbar implant. Surgical procedures were similar to \@ref(injections), however, after the incision a craniotomy was made at a more anterolateral position (in mm from bregma: 1.2 anteriorposterior, 1.2 mediolateral) to place a screw for grounding. The screw was fixed without damaging the surface of the brain. The skull surface was then sealed using OptiBond™ Solo Plus (KerrHawe SA, Bioggio, Switzerland) and the golden pin attached to the screw was stabilised using CHARISMA® ABC A1 (Kulzer GmbH, Hanau, Germany). A headbar was placed over the skull, aligned to midline and fixed with Paladur® dental cement (Kulzer GmbH, Hanau, Germany). The animal was then released back to the home-cage where it could recover for a at least 2 days before habituation began.
+
+## *In-Vitro* Recordings
 
 ### Slice Preparation
-For slices animals (n = XX PV-Cre mice, n = XX ChAT-Cre mice ) were deeply anaesthetised using isoflurane decapitated, the brain removed and quickly transferred to cold (~4°C) slicing solution (sucrose artificial cerebral spinal fluid - SACF). SACF contained (mM): 87 NaCl, 26 NaHCO~3~, 10 Glucose, 50 Sucrose, 2.5 KCl, 1.25 NaH~2~PO~4~, 0.5 CaCl~2~, 3 MgCl~2~ · 6H~2~O.
-Hemispheres were placed on a vibratome (VT1200S, Leica Biosystems, Wetzlar, Germany) to produce 400 µm horizontal slices (Figure \@ref(fig:PaS-Scheme) B) which were then transferred and stored in an interface chamber for up to 1-6 h. Slices were perfused with ACSF (in mM: 119 NaCl, 26 NaHCO~3~, 10 Glucose, 2.5 KCl, 1 NaH~2~PO~4~, 2.5 CaCl~2~, 1.3 MgCl~2~ · 6H~2~O) and oxygenated during the whole period.
+For slices animals (n = XX PV-Cre mice, n = XX ChAT-Cre mice ) were deeply anaesthetised under isoflurane and then decapitated. The brain was quickly removed and transferred to ice-cold (~4°C) slicing solution (sucrose artificial cerebral spinal fluid -- sACSF). sACSF contained (mM): 87 NaCl, 26 NaHCO~3~, 10 Glucose, 50 Sucrose, 2.5 KCl, 1.25 NaH~2~PO~4~, 0.5 CaCl~2~, 3 MgCl~2~·6H~2~O.
+The forebrain was separated from the rest by making a coronal cut. The posterior part of the brain was separated with an sagittal cut in the two hemispheres. The two posterior Hemispheres and the forebrain were placed on a vibrating blade microtome (VT1200S, Leica Biosystems, Wetzlar, Germany) to produce 400 µm horizontal and coronal slices (Figure \@ref(fig:PaS-Scheme) B) which were then transferred to an interface chamber for up to 1-6 h storage. Slices were superfused constantly with oxygenated ACSF (in mM: 119 NaCl, 26 NaHCO~3~, 10 Glucose, 2.5 KCl, 1 NaH~2~PO~4~, 2.5 CaCl~2~, 1.3 MgCl~2~·6H~2~O). All solutions were saturated in oxygen using carbogen (95% O~2~ and 5% CO~2~).
+To verify channelrhodopsin expression fluorescence was checked in medial septal coronal sections under a fluorescence microscope (DM3000, Leica Biosystems, Wetzlar, Germany) prior to recordings.
 
 ### Slice Recordings
-Slices were transferred from the interface storage to the recording chamber which contained constantly flowing oxygenated ACSF. Cells were identified using XXXX DIC XXXX and recorded in the whole-cell patch-clamp configuration using a glass electrode filled with intracellular solution (in mM: 120 K-Gluconate, 10 Hepes, 10 KCl, 5 EGTA, 2 MgSO~4~ · 7H~2~, 3 MgATP, 1 NaGTP, 5 Phosphocreatine Na, 0.2% Biocytin) to record current or voltage changes and later identify cells via Biocytin staining.
+Slices were transferred from the interface storage to the recording chamber where they were constantly superfused (at 3.5 ml/min) with oxygenated ACSF and maintained at 32-34°C. Cells were visually identified using an upright microscope (BX51W1, Olympus, Tokyo,  Japan) and infrared differential interference contrast microscopy through a digital camera (XM10-IR, Olympus, Tokyo, Japan). To record in the the whole-cell patch-clamp configuration a borosilicate glass electrode (Harvard Apparatus, Holliston, Massachusetts, USA) was pulled on a DMZ-Universal-Electrode-Puller (Zeitz-Instruments, Martinsried, Germany) to 2.5-6 M$\Omega$ and filled with intracellular solution (in mM: 120 K-Gluconate, 10 HEPES, 10 KCl, 5 EGTA, 2 MgSO~4~·7H~2~O, 3 MgATP, 1 NaGTP, 5 Phosphocreatine Na, 0.2% Biocytin). Data was recorded with a Multiclamp 700A/B (Molecular Devices, LLC., San Jose, CA, USA) were signals were filtered at 10 kHz, sampled at 20 kHz and then digitised. Data was either digitised by a Digidata 1550 (Molecular Devices, LLC., San Jose, CA, USA) for use in pClamp. In recordings in IGOR Pro 6.12 (WaveMetrics, Inc., Portland , OR, USA) or WinWCP V5.3.7 [@dempster_new_1997] a BNC-2090 interface board (PCI 6035E A/D Board, National Instruments, Austin, Texas, USA) or a USB-6229 BNC (National Instruments, Austin, Texas, USA) was used for digitisation. After opening, cells were held in voltage-clamp at -60 mV to measure series resistance and assess access. Cells were then switched to current-clamp configuration to perform a characterisation of somatic properties (current steps of 40-100 pA). To assess septal connectivity to the parasubiculum Channelrhodopsin expressing fibres in the area were activated by 10 ms light pulses from an LED (CoolLED pE-2, Andover, UK) or an halogen lamp (### check for model ####) coupled to the 60x objective. To elicit inhibitory (IPSPs) or excitatory postsynaptic potentials (EPSPs) and inhibitory (IPSCs) or excitatory postsynaptic currents (EPSCs) a range of light pulse frequencies (10, 20, 40 Hz) was used. Additionally, inputs hidden due to low driving force or simultaneously occurring synaptic inputs were unmasked by changing the holding potential of the cell from -60, to -80 and then to -50 mV after at least 10 trials respectively. In some recordings (##### N number #####) monosynaptic inputs were validated by at first blocking with 1 µM tetrodotoxin (TTX) and then reinstating inputs by depolarising the terminals with ##µM## 4-Aminopyridine (4-AP).
+Liquid junction potential was not corrected for. Slices were fixed in 4% paraformaldehyde (PFA) overnight for immunohistochemistry stainings. 
 
-After opening, cells were hold in voltage-clamp at -60 mV to measure series resistance and assess access.
 
-Then cells were switched to current-clamp configuration to perform a characterisation of cell properties (current steps of 40-100 pA). To assess septal connectivity to the parasubiculum Channelrhodopsin expressing fibres in the area were activated using 10 ms light pulses.
-<!-- check for which light sources --> To elicit inhibitory (IPSPs) or excitatory postsynaptic potentials (EPSPs) and inhibitory (IPSCs) or excitatory postsynaptic currents (EPSCs) a range of pulse frequencies (10, 20, 40 Hz) was used.
+Drugs used:
 
-To detect masked inputs due to low driving force or simultaneously occurring synaptic inputs the holding potential of the cell was changed from -60, to -80 and then to -50 mV after at least 10 trials respectively.
+## *In-Vivo* Recordings
 
+### Anaesthesised Recordings
+Before recordings the animals was anaesthetised as described under \@ref(injections). A craniotomy was performed at 1 anteroposterior and 1 mediolateral (in mm from bregma) and two additional ones at ±2.5 mediolateral and 0 ####### anteroposterior (in mm from lamda). The animal was injected with ### $\frac{mg}{kg} \textstyle$ urethane and subsequently transferred to the recording setup where two ISO-3x-tet-lin optrodes (NeuroNexus, Ann Arbor, MI, USA) with 32 channels each were used to record in the MS and PaS simultaneously and allowed for light stimulation of both regions. The probe targeting the medial septum was placed with an angle of 10° and lowered 4.3 mm from the surface. The optrode targeting the PaS was lowered perpendicular to the brains surface with 2.5-35 mm distance from the surface. 
 
-Drugs used:
+### Head-Fixed Awake Immobile Recordings {#immobile}
+For head-fixed recordings in immobile mice animals were habituated to the setup and trained to stand still in a paper roll whilst being fixed at the headbar. Animals were trained for 5-7 days with increasing time duration until they were able to remain still for up to an hour. After training sessions mice were rewarded with condense milk. After the training period animals were anaesthetised as described in \@ref(injections). A craniotomy was performed at 1 anteroposterior and 1 mediolateral (in mm from bregma) and another craniotomy at -2.5 mediolateral and 0 ####### anteroposterior (in mm from lamda). The probe targeting the medial septum was placed with an angle of 10° and lowered 4.3 mm from the surface. The optrode targeting the PaS was lowered perpendicular to the brains surface with 2.5-35 mm distance from the surface. After the recording session Kwik-Cast (World Precision Instruments Ltd, Hertfordshire, UK) was applied to the craniotomies to protect the brain from drying out.
+
+### Head-Fixed Awake Recordings on Treadmill
+For head-fixed recordings on the treadmill animals were water deprived to encourage them to drink during the training sessions. During the training sessions animals were trained to run on the treadmill whilst being fixed at the headbar. 
+
+
+```{r image-ref-for-in-text, echo = FALSE, message=FALSE, fig.align='center', fig.cap='Some cool caption', out.width='0.75\\linewidth', fig.pos='H'}
+knitr::include_graphics("Figures/Schemes/TestBeaconed3.svg")
+knitr::include_graphics("Figures/Schemes/TestNonBeaconed3.svg")
+```
+
+```{r LinearTrackScheme, fig.cap = 'Schematic of linear track. In A the wall of the reward zone (120-140 cm) is striped so that it can be distingushed by the mouse. and the injection canula (black). A 10° angle is used to avoid damage to superior sagittal sinus which is located on the midline on top of the brain surface (not shown).', fig.show='hold', fig.pos='H',echo=FALSE, fig.height=3}
+
+#![Alt](Figures/Schemes/BeaconedSchemePDF.pdf)
+#BeaconedScheme  <- grImport2::pictureGrob(grImport2::readPicture(rawToChar(rsvg::rsvg_svg("Figures/Schemes/TestBeaconed3.svg"))))
+#NonBeaconedScheme  <- grImport2::pictureGrob(grImport2::readPicture(rawToChar(rsvg::rsvg_svg("Figures/Schemes/NonBeaconedScheme.svg"))))
+#grImport2::grid.picture(grImport2::readPicture(rawToChar(rsvg::rsvg_svg("Figures/Schemes/TestBeaconed2.svg"))), ext = "clipbbox")
+#wrap_elements(gridExtra::arrangeGrob(BeaconedScheme), clip = T)
+#wrap_elements(gridExtra::arrangeGrob(BeaconedScheme), clip=T) / wrap_elements(gridExtra::arrangeGrob(NonBeaconedScheme), clip=T) &
+#  plot_annotation(tag_levels = "A") &
+#  theme(plot.tag = element_text(size=24))
+```
+
+The virtual reality (VR) task entailed a linear track which was 200 cm long and had a reward zone at 120 cm to 140 cm. The animal had to at first reach the reward zone and spent a fixed amount of time (3 s) until a water reward was delivered via a pump. Subsequently, the animal had to reach the end of the track where it was then teleported back to the start. After a waiting period of 5 s a new trial began. During training animals 
+Animals were trained for 3 weeks with increasing time duration until they were able to run and perform a distance estimation task. For the recording session animals were surgically prepared as described in \@ref(immobile) and placed on the treadmill to perform the task. Light stimulation was used for a whole trial after a minimum of 5 trials. At the end of the trial light was switched back off and the animal continued with the trials. The alternating procedure was repeated for the whole recording session.
+
+## Histological Processing
+Animals from *in-vivo* recordings were perfused after the final recording session. Therefore, they were deeply anaesthetised using urethane (2.5 $\frac{g}{kg} \textstyle$ body weight) and transcardially perfused with phosphate-buffered saline (PBS) followed by 4% PFA. The fixed brain was removed and stored in 4% PFA over night. After post-fixation brains were washed in PBS before being they were dissected into two hemispheres and a forebrain part. The three parts were blocked onto a vibrating blade microtome (Leica VT1000S, Leica Biosystems, Wetzlar, Germany) and sliced into 75 µm thick sagittal (hemispheres containing the PaS) and coronal (forebrain containing the MS) sections.
+
+
+### Acquisition and Stimulation
+Signals were acquired and digitised using a RHD2000 system (Intan Technologies, Los Angeles, CA, USA). The light stimulation was performed using Spike2 coupled to an Power1401-3A data acquisition interface (Cambridge Electronic Design Limited, Cambridge, UK). Custom scripts were used to drive pulse and sine wave stimulations at different frequencies (1 Hz, 2 Hz, 4 Hz, 8 Hz, 16 Hz, 32 Hz). 1 Hz (2 ms pulse length) stimulation was used to identify channelrhodopsin expressing cells in the MS whereas higher frequencies were used to drive network activity in the MS and the PaS. For pulse stimulations between 2 and 8 Hz nested pulse bursts (5 times 5 ms single pulse) where used to imitate theta burst activity. Pulse frequencies at 16 and 32 Hz were achieved through single 10 ms long pulses.
 
 ## Statistical Analysis