We thank Ms. Charu Reddy and Professor Matteo Carandini (Cortex Lab) for their advice on surgical protocol and sharing of transgenic mouse strain. We thank Dr Norbert Hogrefe (Inscopix) for his guidance and assistance through the development of the surgery. We thank Ms Andreea Aldea (Sun Lab) for her assistance with the surgical setup and data processing. This work was supported by the Moorfields Eye Charity.

All experiments were conducted according to the UK Animals (Scientific Procedures) Act 1986 under personal and project licenses approved and issued by the UK Home Office following appropriate ethics review. Adult transgenic lines CaMKII-TTA; GCaMP6S-TRE21 were bred and their offspring used in the experiment. For the safety of the experimenters and the maintenance of sterile conditions, all the procedures were performed under aseptic conditions and with full personal protective equipment.
<p cl...

Multilayer Imaging with Microprism in Head-Fixed and Freely Moving Animals

<ol>
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Here, we have shown the ability to observe and directly compare neurons in head-fixed and freely moving conditions in the same neural populations. While we demonstrated the application in the visual cortex, this protocol can be adapted to a multitude of other brain areas, both cortical areas and deep nuclei24,25,26,27,28, as well as other data acquisition and ...

The advent of in vivo two-photon fluorescent imaging1,2, combining the new technologies in optical systems and genetically modified fluorescence indicators, has emerged as a powerful technique in neuroscience to investigate the intricate structure, function, and plasticity in the living brain3,4. In particular, this imaging modality offers an unparalleled advantage over traditional electrophysiology by capturing both the morphology and dynamic activities of neurons, thereby facilitating long-term tracking of identified neurons5,6,7,8.
Despite its noteworthy strengths, the application of high-resolution fluorescence imaging often necessitates a static, head-fixed setup that constrains the animal&#39;s mobility9,10,11. Additionally, the use of a transparent glass surface for visualizing neurons restricts observations to one or more horizontal planes, limiting the exploration of the dynamics of vertical processes that extend across different cortical depths12.
Addressing these limitations, the present study outlines an innovative surgical procedure that integrates head plate fixation, microprism, and miniscope to create an imaging modality with multilayer and multimodal capabilities. The microprism allows the observation of the vertical processing along the cortical column13,14,15,16, which is critical in understanding how information is processed and transformed as it moves through different layers of the cortex and how the vertical processing is altered during plastic changes. Moreover, it permits imaging of the same neural populations in a head-fixed paradigm and in a freely moving setting, encompassing the versatile experimental settings17,18,19: for example, head-fixation is often required for well-controlled paradigms like sensory perception assessment and stable recordings under 2-photon paradigm, while freely moving offers a more natural, flexible environment for behavioral studies. Therefore, the ability to conduct a direct comparison in both modes is crucial to furthering our understanding of the microcircuits that enable flexible, functional responses.
In essence, the integration of head plate fixation, microprism, and miniscope in fluorescence imaging offers a promising platform for probing the intricacies of the brain&#39;s structure and functionality. Researchers can sample identified cell populations across various depths spanning all cortical layers, directly compare their responses in both well-controlled and natural paradigms, and monitor their long-term alterations over months20. This approach offers valuable insight into how these neural populations interact and change over time under different experimental conditions, providing a window into the dynamic nature of neural circuits.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.9% Sodium Chloride solution for infusion (Vetivex 11) 250ml</td>
			<td>Dechra</td>
			<td>20091607</td>
			<td>Saline for hydration and drug reconsitution</td>
		</tr>
		<tr>
			<td>18004-1 Trephine 1.8mm diameter bur</td>
			<td>FST</td>
			<td>18004-18</td>
			<td>Drill bit</td>
		</tr>
		<tr>
			<td>1ml syringe</td>
			<td>Terumo</td>
			<td>MDSS01SE</td>
			<td>1ml syringe</td>
		</tr>
		<tr>
			<td>23G x 5/8 inch 6% LUER needle</td>
			<td>Terumo</td>
			<td>NN-2316R</td>
			<td>23G needle</td>
		</tr>
		<tr>
			<td>71000 Automated stereotaxic apparatus w/ built-in software</td>
			<td>RWD</td>
			<td>-</td>
			<td>RWD</td>
		</tr>
		<tr>
			<td>Absorbable Haemostatic Gelatin Sponge (10x10x10mm)</td>
			<td>Surgispon</td>
			<td>SSP-101010</td>
			<td>gel-foam</td>
		</tr>
		<tr>
			<td>Alcohol pads 70% isopropyl alcohol</td>
			<td>Braun</td>
			<td>9160612</td>
			<td>Alcohol pads</td>
		</tr>
		<tr>
			<td>Aluminium foil</td>
			<td>Any retailer</td>
			<td>-</td>
			<td>Foil to cover eyes during surgery</td>
		</tr>
		<tr>
			<td>Articifical Cerebrospinal Fluid&#160;</td>
			<td>Tocris Bioscience a Bio-Techne Brand</td>
			<td>3525/25ML</td>
			<td>ACSF</td>
		</tr>
		<tr>
			<td>Automated microinjection pump</td>
			<td>WPI</td>
			<td>8091</td>
			<td></td>
		</tr>
		<tr>
			<td>Betadine solution (10% iodinated Povidone) 500ml</td>
			<td>Videne/Ecolab</td>
			<td>3030440</td>
			<td>Betadine</td>
		</tr>
		<tr>
			<td>Bruker Ultime 2Pplus (customised)</td>
			<td>Bruker</td>
			<td>-</td>
			<td>Two-photon imaging system&#160;</td>
		</tr>
		<tr>
			<td>Cardiff Aldasorber</td>
			<td>Vet-Tech</td>
			<td>AN006</td>
			<td>Anaesthesia absorber</td>
		</tr>
		<tr>
			<td>CFI S Plan Fluor ELWD ADM 20XC</td>
			<td>Nikon</td>
			<td>MRH48230</td>
			<td>20x objective lens</td>
		</tr>
		<tr>
			<td>Compact Anaesthesia system - single gas - isoflurane K/F, with oxygen concentrator model: ZY-5AC and scavenging unit</td>
			<td>Vet-Tech</td>
			<td>AN001</td>
			<td>Compact anaesthesia system&#160;</td>
		</tr>
		<tr>
			<td>Contec Prochlor&#160;</td>
			<td>Aston Pharma</td>
			<td>AP2111L1</td>
			<td>Disinfectant (hypochlorous acid)</td>
		</tr>
		<tr>
			<td>Dexamethasone Sodium Phosphate Injection, USP, 4mg/ml, NDC: 0641-6145-25</td>
			<td>Hikma</td>
			<td>Covetrus:70789</td>
			<td>Dexamethasone</td>
		</tr>
		<tr>
			<td>Dissecting Knife, cutting edge 4mm, thickness 0.5mm, stainless steel</td>
			<td>Fine Science Tools</td>
			<td>10055-12</td>
			<td>Knife for incisino of cortex</td>
		</tr>
		<tr>
			<td>Dual-Sided, Non-Puncture Mouse &#38; Neonatal Rat Ear Bars</td>
			<td>Stoelting</td>
			<td>51649</td>
			<td>Ear bar</td>
		</tr>
		<tr>
			<td>Dummy microscope</td>
			<td>Inscopix</td>
			<td>Dummy microscope</td>
			<td>To help with implantation</td>
		</tr>
		<tr>
			<td>Ethanol (100%)&#160;</td>
			<td>VWR</td>
			<td>40-1712-25</td>
			<td>Used to make 70% ethanol&#160;</td>
		</tr>
		<tr>
			<td>Fisherbrand&#160;Nitrile Indigo Disposable Gloves PPE Cat III</td>
			<td>FischerScientific</td>
			<td>17182182</td>
			<td>Gloves</td>
		</tr>
		<tr>
			<td>Homeothermic Monitor 50-7222-F</td>
			<td>Harvard Apparatus</td>
			<td>50-7222-F</td>
			<td>Homeothermic monitoring system/heating pad</td>
		</tr>
		<tr>
			<td>Image processing software</td>
			<td>ImageJ</td>
			<td>-</td>
			<td>Image processing software</td>
		</tr>
		<tr>
			<td>Inscopix Data Processing Software (IDPS)</td>
			<td>Inscopix</td>
			<td>-</td>
			<td>One-photon calcium imaging processing software</td>
		</tr>
		<tr>
			<td>Insight Duals-232, S/N 2043</td>
			<td>InSight</td>
			<td>Insight Spectra X3</td>
			<td>Two-photon imaging laser</td>
		</tr>
		<tr>
			<td>IsoFlo 250ml 100% w/w inhalation</td>
			<td>Zoetis</td>
			<td>WM 42058/4195</td>
			<td>Isoflurane</td>
		</tr>
		<tr>
			<td>Kwik-Sil Low Toxicity Silicone Adhesive</td>
			<td>World Precision Intruments (WPI)</td>
			<td>KWIK-SIL</td>
			<td>Silicone adhesive</td>
		</tr>
		<tr>
			<td>MICROMOT mains adapter NG 2/S, w/ Drill unit 60/E</td>
			<td>PROXXON</td>
			<td>NO 28 515</td>
			<td>Handheld drill</td>
		</tr>
		<tr>
			<td>nVoke Integrated Imaging and Optogenetics System package</td>
			<td>Inscopix</td>
			<td>-</td>
			<td>One-photon Imaging system and software</td>
		</tr>
		<tr>
			<td>ProView Implant Kit</td>
			<td>Inscopix</td>
			<td>ProView Implant Kit</td>
			<td>Dummy microscope, stereotaxic arm and attachment&#160;</td>
		</tr>
		<tr>
			<td>ProView Prism Probe</td>
			<td>Inscopix</td>
			<td>1050-002203</td>
			<td>Microprism lens</td>
		</tr>
		<tr>
			<td>Rimadyl (50mg/ml)</td>
			<td>Zoetis</td>
			<td>VM 42058/4123</td>
			<td>Carprofen&#160;</td>
		</tr>
		<tr>
			<td>Stereotaxis Microscope on Articulated arm with table clamp</td>
			<td>WPI</td>
			<td>PZMTIII-AAC&#160;</td>
			<td>Microscope</td>
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		<tr>
			<td>Super-Bond Universal kit, SUN Medical</td>
			<td>Prestige-Dental</td>
			<td>K058E</td>
			<td>Adhesive cement</td>
		</tr>
		<tr>
			<td>Two-photon calcium image software</td>
			<td>Suite2P</td>
			<td>-</td>
			<td>Two-photon calcium imaging processing software</td>
		</tr>
		<tr>
			<td>Vapouriser</td>
			<td>Vet-Tech</td>
			<td>-</td>
			<td>Isoflurane vapouriser</td>
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		<tr>
			<td>Xailin Lubricating Eye Ointment 5g</td>
			<td>Xailin-Night</td>
			<td>MLG/28/1551</td>
			<td>Ophthalmic ointment&#160;</td>
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long-term imaging of identified neural populations using microprisms in freely moving and head-fixed animals

With the advancement of multi-photon microscopy and molecular technologies, fluorescence imaging is rapidly growing to become a powerful approach for studying the structure, function, and plasticity of living brain tissues. In comparison to conventional electrophysiology, fluorescence microscopy can capture the neural activity as well as the morphology of the cells, enabling long-term recordings of the identified neuron populations at single-cell or subcellular resolution. However, high-resolution imaging typically requires a stable, head-fixed setup that restricts the movement of the animal, and the preparation of a flat surface of transparent glass allows visualization of neurons at one or more horizontal planes but is limited in studying the vertical processes running across different depths. Here, we describe a procedure to combine a head plate fixation and a microprism that gives multilayer and multimodal imaging. This surgical preparation not only gives access to the entire column of the mouse visual cortex but allows two-photon imaging in a head-fixed position and one-photon imaging in a freely moving paradigm. Using this approach, one can sample identified cell populations across different cortical layers, register their responses under head-fixed and freely moving states, and track the long-term changes over months. Thus, this method provides a comprehensive assay of the microcircuits, enabling direct comparison of neural activities evoked by well-controlled stimuli and under a natural behavioral paradigm.

Author Spotlight: Comparative Imaging of Neural Activity in Awake and Freely Moving States

Long-Term Imaging of Identified Neural Populations using Microprisms in Freely Moving and Head-Fixed Animals

System neuroscience benefits greatly from advancement in imaging technology, especially combined with molecular and genetic approaches. Researchers are increasingly using high resolution, chronic recording, and cell type-specific targeting to dissect the brain's function at finer scale, longer time, and more position. One notable shift in experimental paradigms is moving away from the anesthetized recording towards awake or freely-moving states.This approach allows the characterization of brain activity in natural conditions, but also imposes challenges for precise control on stimulation and measurements. With this protocol, we can image the same neural populations in head-fixed and freely-moving animals. We can directly compare their activities in both well-controlled and natural paradigms.Our protocol enables the study of sensory information processing across cortical layers. It allows us to compare neural responses to controlled sensory stimuli in head-fixed states and its behavioral tasks during free movement, thus facilitating our understanding of sensory processing dynamics.

The method of conducting chronic multilayer in vivo calcium imaging of the same neuronal population over a period of several weeks, using both one- and two-photon imaging modalities, under freely moving and head-fixed conditions has been shown. Here, the ability to identify matching neuronal populations under one-photon imaging while the animal explored an open arena in the dark has been demonstrated (Figure 7A). Calcium traces were extracted from the identified neurons and z-scored...

Watch this Scientific Journal Video about Long-Term Imaging of Identified Neural Populations using Microprisms in Freely Moving and Head-Fixed Animals at JoVE.com

When integrated with a head-plate and an optical design compatible with both single- and two-photon microscopes, the microprism lens presents a significant advantage in measuring neural responses in a vertical column under diverse conditions, including well-controlled experiments in head-fixed states or natural behavioral tasks in freely moving animals.

With the advancement of multi-photon microscopy and molecular technologies, fluorescence imaging is rapidly growing to become a powerful approach for ...

long-term-imaging-identified-neural-populations-using-microprisms