Adaptable Angled Stereotactic Approach for Versatile Neuroscience Techniques

Summary

Described here is a stereotactic procedure that can target challenging and difficult-to-reach brain regions (due to spatial limitations) using an angled coronal approach. This protocol is adaptable to both mouse and rat models and can be applied to diverse neuroscientific applications, including cannula implantation and microinjections of viral constructs.

Abstract

Stereotactic surgery is an essential tool in the modern neuroscience lab. However, the ability to precisely and accurately target difficult-to-reach brain regions still presents a challenge, particularly when targeting brain structures along the midline. These challenges include avoiding of the superior sagittal sinus and third ventricle and the ability to consistently target selective and discrete brain nuclei. In addition, more advanced neuroscience techniques (e.g., optogenetics, fiber photometry, and two-photon imaging) rely on targeted implantation of significant hardware to the brain, and spatial limitations are a common hindrance. Presented here is a modifiable protocol for stereotactic targeting of rodent brain structures using an angled coronal approach. It can be adapted to 1) mouse or rat models, 2) various neuroscience techniques, and 3) multiple brain regions. As a representative example, it includes the calculation of stereotactic coordinates for targeting of the mouse hypothalamic ventromedial nucleus (VMN) for an optogenetic inhibition experiment. This procedure begins with the bilateral microinjection of an adeno-associated virus (AAV) encoding a light-sensitive chloride channel (SwiChR++) to a Cre-dependent mouse model, followed by the angled bilateral implantation of fiberoptic cannulae. Using this approach, findings show that activation of a subset of VMN neurons is required for intact glucose counterregulatory responses to insulin-induced hypoglycemia.

Introduction

Neural control of behavior, feeding, and metabolism involves coordination of highly complex, integrative, and redundant neurocircuits. A driving goal of the neuroscience field is to dissect the relationship between neuronal circuit structure and function. Although classical neuroscience tools (i.e., lesioning, local pharmacological injections, and electrical stimulation) have uncovered vital knowledge regarding the role of specific brain regions that control behavior and metabolism, these tools are limited by their lack of specificity and reversibility¹.

Recent advances in the neuroscience field have greatly improved....

Protocol

All procedures were approved in accordance with the National Institutes of Health, the Guide for the Care and Use of Animals and were approved by both the Institutional Animal Care and Use Committee (IACUC) and Environmental Health and Safety at the University of Washington.

1. Calculation of angled coordinates

1. Using a coronal brain atlas, mark a right triangle so that the hypotenuse passes through the target region of interest. In the representative example (Figure 1), the hypothalamic ventromedial nucleus (VMN) is targeted at a 15° angle from the coronal midline.
   NOTE: The placement of the ax....

Results

This protocol describes a surgical procedure for performing optogenetics studies to interrogate the role of hypothalamic VMN neurons in glycemic control⁹. First utilized was a standard (non-angled) stereotactic approach for the bilateral microinjection of an inhibitory channelrhodopsin virus to the VMN. While an angled approach would also be suitable, the standard (non-angled) approach was selected because it is sufficient to target the brain region of interest and is an easy, reliable and consist.......

Discussion

Recent advances in neuroscience have supported advanced insight and understanding into the activity and function of brain neurocircuits. This includes the application of optogenetic and chemogenetic technologies to activate or silence discrete neuronal populations and their projection sites in vivo. More recently, this has included the development of genetically encoded calcium indicators (e.g., GCaMP, RCaMP) and other fluorometric biosensors (e.g., dopamine, norepinephrine) for in vivo recording of neuronal activity in .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Fiberoptic Cannulae	Doric Lenses	MFC_200/230-0.57_###_MF1.25_FLT	Customizable
Kopf Model 1900 Stereotaxic Alignment System	Kopf	Model 1900
Kopf Model 1900-51 Center Height Gauge	Kopf	Model 1900-51
Kopf Model 1905 Alignment Indicator	Kopf	Model 1905
Kopf Model 1911 Stereotaxic Drill	Kopf	Model 1911
Kopf Model 1915 Centering Scope	Kopf	Model 1915
Kopf Model 1922 60-Degree Non-Rupture Ear Bars	Kopf	Model 1922
Kopf Model 1923-B Mouse Gas Anesthesia Head Holder	Kopf	Model 1923-B
Kopf Model 1940 Micro Manipulator	Kopf	Model 1940
Micro4 Microinjection System	World Precision Instruments	--
Mouse bone screws	Plastics One	00-96 X 1/16
Stereotaxic Cannula Holder, 1.25mm ferrule	Thor Labs	XCL
Surgical Drill	Cell Point Scientific	Ideal Micro Drill