Microdissection of Mouse Brain into Functionally and Anatomically Different Regions

Summary

We present a hands-on, step-by-step, rapid protocol for mouse brain removal and dissection of discrete regions from fresh brain tissue. Obtaining brain regions for molecular analysis has become routine in many neuroscience labs. These brain regions are immediately frozen to obtain high quality transcriptomic data for system level analysis.

Abstract

The brain is the command center for the mammalian nervous system and an organ with enormous structural complexity. Protected within the skull, the brain consists of an outer covering of grey matter over the hemispheres known as the cerebral cortex. Underneath this layer reside many other specialized structures that are essential for multiple phenomenon important for existence. Acquiring samples of specific gross brain regions requires quick and precise dissection steps. It is understood that at the microscopic level, many sub-regions exist and likely cross the arbitrary regional boundaries that we impose for the purpose of this dissection.

Mouse models are routinely used to study human brain functions and diseases. Changes in gene expression patterns may be confined to specific brain areas targeting a particular phenotype depending on the diseased state. Thus, it is of great importance to study regulation of transcription with respect to its well-defined structural organization. A complete understanding of the brain requires studying distinct brain regions, defining connections, and identifying key differences in the activities of each of these brain regions. A more comprehensive understanding of each of these distinct regions may pave the way for new and improved treatments in the field of neuroscience. Herein, we discuss a step-by-step methodology for dissecting the mouse brain into sixteen distinct regions. In this procedure, we have focused on male mouse C57Bl/6J (6-8 week old) brain removal and dissection into multiple regions using neuroanatomical landmarks to identify and sample discrete functionally-relevant and behaviorally-relevant brain regions. This work will help lay a strong foundation in the field of neuroscience, leading to more focused approaches in the deeper understanding of brain function.

Introduction

The brain, along with the spinal cord and retina, comprise the central nervous system that executes complex behaviors, controlled by specialized, precisely positioned, and interacting cell types throughout the entire body¹. The brain is a complex organ with billions of interconnected neurons and glia with precise circuitry performing numerous functions. It is a bilateral structure with two distinct lobes and diverse cellular components². The spinal cord connects the brain to the outside world and is protected by bone, meninges, and cerebrospinal fluid and routes messages to and from the brain^2,3,4. The surface of the brain, the cerebral cortex, is uneven and has distinct folds, called gyri, and grooves, called sulci, that separate the brain into functional centers⁵. The cortex is smooth in mammals with a small brain^6,7. It is important to characterize and study the architecture of the human brain in order to understand the disorders related to the different brain regions, as well as its functional circuits. Neuroscience research has expanded in recent years and a variety of experimental methods are being used to study the structure and function of the brain. Developments in the fields of molecular and systems-level biology have ushered in a new era of exploring the complex relationship between brain structures and the functioning of molecules. Additionally, molecular biology, genetics, and epigenetics are rapidly expanding, enabling us to advance our knowledge of the underlying mechanisms involved in how systems function. These analyses can be carried out on a much more localized basis, to help target the investigation and development of more effective therapies.

The mammalian brain is structurally defined into clearly identifiable discrete regions; however, the functional and molecular complexities of these discrete structures are not yet clearly understood. The multi-dimensional and multi-layered nature of the brain tissue makes this landscape difficult to study at the functional level. In addition, the fact that multiple functions are performed by the same structure and vice versa further complicates the understanding of the brain⁸. It is vital that the experimental approach executed for the structural and functional characterization of brain regions uses precise research methodologies to achieve consistency in sampling for correlating neuroanatomical architecture with function. The complexity of brain has been recently explained using single cell sequencing^9,10 such as the temporal gyrus of the human brain which is composed of 75 distinct cell types¹¹. By comparing this data to those from an analogous region of the mouse brain, the study not only reveals similarities in their architecture and cell types but also presents the differences. To unravel the complex mechanisms, it is therefore important to study diverse regions of the brain with full precision. Conserved structures and function between a human and mouse brain enable the use of a mouse as a preliminary surrogate for elucidating human brain function and behavioral outcomes.

With the advancement of systems biology approaches, obtaining information from discrete brain regions in rodents has become a key procedure in neuroscience research.While some protocols such as laser capture microdissection¹² can be expensive, mechanical protocols are inexpensive and performed using commonly available tools^13,14. We have used multiple brain regions for transcriptomic assays¹⁵ and have developed a hands-on and rapid procedure to dissect mouse brain regions of interest in a step-by-step manner in a short time. Once dissected, these samples can be stored immediately in cold conditions to preserve the nucleic acids and proteins of these tissues. Our approach can be performed faster leading to high efficiency and permitting less chances for tissue deterioration. This ultimately, increases the chances of generating high quality, reproducible experiments using brain tissues.

Protocol

Animal handling and experimental procedures were conducted in accordance with European, national and institutional guidelines for animal care. All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) at the US Army Center for Environmental Health Research now Walter Reed Army Institute of Research (WRAIR) and performed in a facility accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care International (AAALAC). NOTE: The procedure will be p…

Representative Results

Our understanding of the complex brain structure and function is rapidly evolving and improving. The brain contains multiple distinct regions and building a molecular map can help us better understand how the brain works. In this method paper, we have discussed the dissection of the mouse brain into multiple distinct regions (Table 1). In this protocol, the structures are identified based on the critical landmarks and is achieved by keeping the tissue moist with saline solution by retaining its sturdines…

Discussion

The mammalian brain is a complex organ composed of an array of morphologically distinct and functionally unique cells with diverse molecular signatures and multiple regions that perform specialized and discrete functions. The dissection procedure reported here can have multiple goals depending on the requirements of the lab. In our lab we assessed transcription in multiple brain regions collected from mice exposed to PTSD like stress¹⁶ . We would like to study further the impact of strain genetic …

Materials

Brain Removal
Deaver scissors	Roboz Surgical Store	RS-6762	5.5" straight sharp/sharp
Deaver scissors	Roboz Surgical Store	RS-6763	5.5" curved sharp/sharp
Delicate operating scissors	Roboz Surgical Store	RS-6703	4.75" curved sharp/sharp
Delicate operating scissors	Roboz Surgical Store	RS-6702	4.75" straight sharp/sharp
Light operating scissors	Roboz Surgical Store	RS-6753	5" curved Sharp/Sharp
Micro spatula, radius and tapered flat ends			stainless steel mirror finish
Operating scissors 6.5"	Roboz Surgical Store	RS-6846	curved sharp/sharp
Tissue forceps	Roboz Surgical Store	RS-8160	4.5” 1X2 teeth 2mm tip width
Rongeur (optional)	Roboz Surgical Store	RS-8321 many styles to choose	Lempert Rongeur 6.5" 2X8mm
Pituitary Dissection
Scalpel handle	Roboz Surgical Store	RS-9843	Scalpel Handle #3 Solid 4"
and blades	Roboz Surgical Store	RS-9801-11	Sterile Scalpel Blades:#11 Box 100 40mm
Super fine forceps Inox	Roboz Surgical Store	RS-4955	tip size 0.025 X 0.005 mm
Brain Dissection
A magnification visor	Penn Tool Col	40-178-6	2.2x Outer and 3.3x Inner Lens Magnification, Rectangular Magnifier
Dissection cold plate	Cellpath.com	JRI-0100-00A	Iceberg cold plate & base
Graefe forceps, full curve extra delicate	Roboz Surgical Store	RS-5138	0.5 mm Tip 4” (10 cm) long
Light operating scissors	Roboz Surgical Store	RS-6753	5" curved sharp/sharp
Scalpel handle	Roboz Surgical Store	RS-9843 (repeated above)	Scalpel Handle #3 Solid 4"
and blades (especially #11)	Roboz Surgical Store	RS-9801-11 (repeated above)	Sterile Scalpel Blades:#11 Box 100 40mm
Spatula	Amazon	MS-SQRD9-4	Double Ended Spatula Square AND Round End
Tissue forceps	Roboz Surgical Store	RS-8160 (repeated above)	4.5” 1X2 teeth