In this webinar, Dr. Tahl Holtzman, Founder of Cambridge NeuroTech, describes a new generation of silicon neural probes offering dozens of recording channels in precisely spaced, high-resolution arrays, built using sophisticated fabrication techniques borrowed from the electronics industry, along with simple-to-follow surgical implantation schemes for both acute and chronic animals. Watch to learn how to take advantage of ultra-small chronic drives to open up scalability to span multiple brain areas in parallel and to achieve excellent chronic stability. In addition, Dr. Holtzman demonstrates integration of novel probes and drives offered by Cambridge NeuroTech with optogenetics that thereby enable your experiments to have the combined capability for measurement AND manipulation of neuronal activity in both acute and freely behaving settings. This webinar will benefit both established electrophysiologists who wish to increase their data yield and experimental reach as well as those investigators whose expertise is centred in and around the animal behavioural, neuropharmacological, and optogenetics arenas. Viewers will learn what silicon neural probes are and how to use them in both acute and chronic experiments, best-practice techniques for surgical implantation in species ranging from mice to monkeys and how to integrate fibre optic cannulas with your probes to enable simultaneous opto-electrophysiology.

1. Employing Electrophysiology and Optogenetics
to Measure and Manipulate Neuronal Activity in
Laboratory Animals
Tahl Holtzman, PhD presents an in-depth tour of the latest
neurotechnology for large-scale, high-resolution extracellular single
unit recording combined with optogenetics in anaesthetized, head-
fixed and freely behaving animals

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3. Employing Electrophysiology and Optogenetics
to Measure and Manipulate Neuronal Activity in
Laboratory Animals
Tahl Holtzman, PhD
Founder and CEO,
Cambridge NeuroTech
Cambridge NeuroTech www.cambridgeneurotech.com

4. 1. Overview of neuronal ensemble recording using silicon probes
alongside optogenetics
2. Main Topics:
• Features, benefits and advantages of silicon probes vs alternatives
• Integrating optogenetic stimulation with silicon probes
• Optimal surgery techniques for acute and chronic experiments; small
and large animals
What are we going to cover today?

5. Why combine electrophysiology with optogenetics?
Electrophysiology
= correlative observation
Optogenetics
= causal intervention (?)
Combining both provides the ability to
monitor whilst manipulating
Silencing in CamKII-eNpHR3.0-YFP expressing cortical neurones in rats
Tahl Holtzman and Ole Paulsen – University of Cambridge, UK

6. Silicon Probes – Live data in a freely behaving animal
• Raw, streaming data from
a silicon probe implanted
in freely behaving rat
cortex (wireless
transmission)
• Note nearly every channel
has spikes on it; often
more than one waveform
shape

7. Silicon Probes – What Are They?
• Planar microelectrode arrays with uniform
properties (site impedance ~50 kOhm)
• Variety of designs applicable to the smallest
neurons in the brain vs. spanning multiple
cortical layers
• Miniaturized devices for minimal tissue
damage
H1
P
E
H2
H3
Hp
20 m
Silicon Probe
(planar electrode array)
80m
150m
200m
300m
790m
1275m

8. Silicon Probes – Microelectrodes for Microanatomy
• Probes are like fishing nets: to catch
small fish you need fine nets…
• Placing multiple electrodes close to
each target neuron produces spatial
over-sampling = better spike-sorting.
• Reach extends from the smallest
units in the CNS (cerebellar granule
cells) to span across multiple cortex
layers without sacrificing single unit
resolution (i.e. no gaps in the fishing net!)

9. Silicon Probes versus Tetrodes
• Hand-made vs batch process – Uniform electrode
properties
• No complex assembly required – ready to use
out of the box
• Smaller, simpler implants (low skill burden, faster
surgery; more animals per day)
• Smaller overall tissue-volume displacement
• Shorter lead-time to data
• Cost efficiency – consumables vs labor budget
Go to JoVE article

10. Silicon Probes versus Tetrodes
• Small animals push the limits of
technology
• Payload vs capability / capacity
• Network approaches demand
scalability across multiple targets,
and…
• …convenient integration with
other tools, eg. optogenetics
Probes (64 ch.)
Go to Voigts et al. Frontiers in Neuroscience, 7:8, 2013

11. Silicon Probes – Anatomy of a Probe; Acute vs Chronic
• Acute probes –
anaesthetized, head-fixed;
easy to clean and re-use
• Chronic probes – fully
implantable; designed to
mate seamlessly with the
nanoDrive
• Compatible with all
mainstream data-acquisition
headstages
connectors
PCB
flexible
ribbon
cable
interface chip
probe
electrode
array
connectors
CHRONIC
ACUTE

12. Silicon Probes – Our probe versus the competition
• Size matters! Always 15 um thick; 45
– 78 um wide = flexible probes; less
breakages
• Best-in-class signal to noise –
typically ~50 kOhm electrode
impedance
• Minimally responsive to light
induced photoelectric artefacts
• Seamless alignment of chronic
probes with nano-Drives

13. Long-term Chronic Single Unit Stability
Freely behaving rat with wireless transmitter; medial prefrontal cortex
Day 60, probe not moved for 30 days
Tahl Holtzman, Nick Donnelly, Jeff Dalley – University of Cambridge, UK

14. Silicon Probes – The Poly-trode Effect
Freely behaving rat with wireless transmitter; medial prefrontal cortex, implant day 60
Tahl Holtzman, Nick Donnelly, Jeff Dalley – University of Cambridge, UK

15. Long-term Chronic Single Unit Stability
• Data gathered in rats implanted with probes
in medial prefrontal cortex or dorsomedial
striatum
• Data shown in the graph start at implant day
30; the probes were not moved for the next
30 days (minimum)
• Approximately similar numbers of single units
are present at the end of the experiment
• This is the data that launched the Cambridge
NeuroTech!

16. Silicon Probes – Data examples from other labs
• Data gathered in freely behaving rats
implanted with probes in medial
prefrontal cortex
• Data shown on day 14; the probes were
not moved since surgery
• Multiple single units are visible across
each probe electrode array, which
correspond to different cortical layers

17. Silicon Probes – Data examples from other labs

18. nano-Drive – world’s smallest chronic drive
• Easy, quick alignment between probes and
drive-axis
• Movement resolution: 205 microns per turn
• Footprint: 2 x 4 mm with 5 mm travel
• Weight: 0.5 grams
• Multiple nano-Drives per animal
• Sterilizable and reusable between animals

19. nano-Drive – fibre optic cannula integration
• Micro-machined
channels for fibre
cannula alignment
• Choice of fibre cannula
determined by user
• Parallel alignment with
probe; co-implanted
and moveable
• Electrodes oriented
toward light; defined
distance between fibre
tip and electrodes

20. Fibre optic mirror tips for orthogonal illumination
• Micro-machined channels for
fibre cannula alignment
• Choice of fibre cannula
determined by user
• Parallel alignment with probe
• Co-implanted and moveable
• Electrodes oriented toward
light
• Defined separation between
fibre tip and electrodes – 300
or 650 microns

21. Silicon Probes – Photo-identified single units
• Ai32 mouse line crossed to the Adora2A Cre
line; a marker of indirect pathway medium
spiny neurons (MSNs) which in turn express
ChR2
• Posterior striatum in the awake head-fixed
mouse; acute recording P-probe with 125 core
flat fibre
• Single unit spikes are evoked ~10 ms latency,
due in part to resting hyperpolarization of
MSNs
• See also: Kravitz, A. V., Owen, S. F., & Kreitzer, A. C. (2013).
Optogenetic identification of striatal projection neuron
subtypes during in vivo recordings. Brain Research, 1511,
21–32. http://doi.org/10.1016/j.brainres.2012.11.018

22. Silicon Probes – Photo-identified single units
• Transgenic mouse line expressing vGlut2::Cre with
ReachR opsin
• Recording in the brainstem; freely behaving mouse;
laser coupled to a 200 core fibre optic with mirror
tip

23. Silicon Probes + Optogenetics
• Ai32 mouse line crossed with Pde1c Cre line;
thus ChR2 is expressed in all mossy fiber
terminals projecting to the cerebellum
• Cerebellar cortex in the awake head-fixed
mouse; acute recording with H1-probe and
200 core flat fibre
• Recording from a granule cell (putatively
identified using a published method) which
shows modest increase in firing resulting
from stimulation of pre-synaptic mossy fibre
terminals
• See also: Van Dijck G, Van Hulle MM, Heiney SA, Blazquez PM,
Meng H, Angelaki DE, Arenz A, Margrie TW, Mostofi A, Edgley
S, Bengtsson F, Ekerot CF, Jorntell H, Dalley JW, Holtzman T.
Probabilistic identification of cerebellar cortical neurones
across species. Plos One. 8: e57669. PMID 23469215 DOI:
10.1371/journal.pone.0057669

24. Surgery Protocol

25. Chronic implants – How to….
• In this section you will learn how to use the
nano-Drives and probes to form compact
implants
• A single probe + nano-Drive implant can be
completed in < 2hrs
• The method can be easily adapted to
implant multiple targets

26. Stereotaxy – 3D atlas correction and automated drill / inject
• Experiments are expensive – labor, trained
animals, probes, funding / milestone deadlines
• Targetting errors are common, but easily mitigated
using a computer-guided, atlas-integrated surgery
robot
• Individual animal corrections: roll, pitch, yaw and
scaling with single / double angle approaches
• Automated, impedance-sensing drilling for perfect
craniotomies
• Integrated micro-injections for viral delivery
• More info: cambridgeneurotech/surgery
Closed-loop, ultraprecise,
automated craniotomies
Nikita Pak, Joshua H.
Siegle, Justin P.
Kinney, Daniel J.
Denman, Timothy J.
Blanche, Edward S.
Boyden
Journal of
Neurophysiology 1 June
2015 Vol. 113 no. 10, 3943
-
3953 DOI: 10.1152/jn.0105
5.2014

27. Surgery Protocol – Step 1
a) Place a nano-Drive into the stereotaxic
holder by inserting the drive screw in to the
gripper
b) Ensure correct alignment of shuttle surface
with respect to the cut-out on the
stereotaxic holder
c) Tighten the thumb-wheel to grip the drive
securely

28. Surgery Protocol – Step 2
a) Release the probe from its shipping
container using a mild solvent to dissolve
the adhesive securing the probe
b) Gently prize the connector and interface-
chip free
c) Hold the probe by the connector and flip the
dish upside down to allow the probe to fall
free from the now dissolved adhesive

29. Surgery Protocol – Step 3
a) Apply a small amount of superglue gel to the
nano-Drive shuttle surface
b) Carefully drag the interface chip of the
probe in to position on the shuttle
c) Gently nudge the interface chip in to
alignment with the shuttle

30. Surgery Protocol – Step 4
a) To bond the probe to the nano-Drive
shuttle, apply gentle downward pressure to
the interface chip
b) Take care to support the connector during
this step

31. Surgery Protocol – Step 5
a) Secure the probe connector to the
stereotaxic holder by means of tape or
adhesive putty
b) Apply a thin film of Vaseline to any surface
of the assembly that is required to be
moveable, once implanted
c) Apply a thin-film of vaseline to the to the
stereotaxic holder tool (this will prevent cement
sticking to it – see later)
d) Use a low-temperature cautery to apply and
gently melt the Vaseline in to place

32. Surgery Protocol – Step 6
a) Melt a small amount of solder in to each of
the vias on the probe connector printed
circuit board
b) Solder a short length of suitable wire (e.g.
stainless steel, silver, etc) to the vias (consult
your headstage input map for different configurations)

33. Surgery Protocol – Step 7
a) This probe and drive are now
ready to implant…
b) Immediately prior to
implantation, clean the probe
shanks by rinsing with 70%
ethanol
c) Carefully insert the free end
of the stereotaxic holder in to
your frame

34. Surgery Protocol – Step 8
a) Make a small craniotomy
b) Perform a durotomy just big enough to
permit insertion of the probe; make a
mechanical durotomy or a chemical
durotomy with collagenase
c) Insert the probe in to the brain to rest a
short distance above your intended target

35. Surgery Protocol – Step 9
a) Repair the durotomy using Dura-Gel; allow
a few minutes to cure
b) Apply Vaseline to the nano-Drive shuttle,
using the low temperature cautery
c) Gently melt the Vaseline to aid in
downward flow to underfill the nano-Drive
shuttle
For dural repair, see: Jackson, N., & Muthuswamy, J. (2008).
Artificial dural sealant that allows multiple penetrations of
implantable brain probes. Journal of Neuroscience
Methods, 171(1), 147–152.
http://doi.org/10.1016/j.jneumeth.2008.02.018

36. Surgery Protocol – Step 9
a) Continue the process of melting Vaseline to
form a column which surrounds the probe
and drive shuttle
b) Carefully remove any excess Vaseline
TIP – create a small cement dam around the edge of the
craniotomy (see Step 8) to catch any stray Vaseline.

37. Surgery Protocol – Step 10
a) Apply a small amount of cement to the back and
sides of the nano-Drive
b) Release the probe connector and position such
that the flex cable forms an S-bend (take care not to
crimp the flex) – tack the connector in place with a
small amount of cement
c) Apply Vaseline to the flex cable surfaces, using the
low-temperature cautery, and fill the ‘eye’ of the
loop formed in the flex cable
d) Attach your Gnd / Ref wires to a nearby skull screw
TIP – pre-apply the vaseline to the flex cable during the probe mounting
step – the surface tension helps to ‘stick’ the cable in to and S-bend
position

38. Surgery Protocol – Step 11
a) Apply cement to cover the flex cable and
secure the connector, taking care to fully
surround the nano-Drive and connector
printed circuit board
b) Apply cement around the shoulders of the
nano-Drive and up the outer surface of the
stereotaxic tool

39. Surgery Protocol – Step 12
a) Release the tension on the stereotaxic
holder and carefully retract to reveal the
nano-Drive screw head (the vaseline you applied to
the holder earlier aids in this by preventing cement
adhesion)
b) Apply additional cement as required
c) Clean the wound and repair soft-tissue
d) Start gathering data!

40. Surgery Protocol – the finished implant

41. Dural repair – large animal and head-fixed acute

42. Multi-target implant in a large animal

43. Features Advantages and Benefits
Small sized, thin probes
15 microns thick, 45 -78 microns wide
• Minimal tissue damage
• Excellent chronic stability
• Flexible, robust probes = less breakages
Densely-spaced electrodes
7.5 – 40 micron site spacing; 64 channels per probe
• Excellent spike-sorting via ‘super-tetrode’ effect
• Record the smallest neurons all the way to multiple cortex layers
Low electrode impedance
Typically 50 kOhm
• Best-in-class signal to noise
• Higher data yield
Small-footprint mating chronic drives
4 x 2 mm footprint, 0.5g weight
• Compact implants - reduced payload on animal
• Integrate fibre optics, etc with probes
Optogenetics-’safe’
Minimal electrode and probe light-sensitivity
• Artefact-free single unit recording during photo-stimulation
Simple, scalable surgery • Lower skill burden; Increased throughput
• Target multiple brain areas in small animals
Silicon Probes – Why you should use them…

44. • How to get best results from your silicon probes
• How to integrate fibre optics with silicon probes
• Best-practice surgical implantation of probes in acute and chronic
experiments
Additional questions? Need help?
info@cambridgeneurotech.com
cambridgeneurotech.com
Summary And Where To Seek Help

45. Tahl Holtzman, PhD
Founder and CEO,
Cambridge NeuroTech
info@cambridgeneurotech.com
www.cambridgeneurotech.com
Thank You
For additional information on the products and applications presented during
this webinar please visit www.cambridgeneurotech.com