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.
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.
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 the ability to interrogate and manipulate circuit function in a cell-type specific manner with high spatiotemporal resolution. Optogenetic2 and chemogenetic3 approaches, for instance, allow the rapid and reversible manipulation of activity in genetically defined cell types of freely moving animals. Optogenetics involves the use of light-sensitive ion channels, termed channelrhodopsins, to control neuronal activity. Key to this technique is the gene delivery of channelrhodopsin and a source of light to activate the opsin. A common strategy for gene delivery is through a combination of 1) genetically engineered mice expressing Cre-recombinase in discrete neurons, and 2) Cre-dependent viral vectors encoding channelrhodopsin.
While optogenetics provides an elegant, highly precise means to control neuronal activity, the method is contingent upon successful stereotactic microinjection of the viral vector and fiberoptic placement into a defined brain region. Although stereotactic procedures are commonplace within the modern neuroscience lab (and there are several excellent protocols describing this procedure)4,5,6, being able to consistently and reproducibly target discrete brain regions along the midline (i.e., the mediobasal hypothalamus, a brain area critical to the regulation of homeostatic functions7) presents additional challenges. These challenges include avoiding of the superior sagittal sinus, third ventricle, and adjacent hypothalamic nuclei. In addition, there are significant spatial limitations to the bilateral implantation of hardware that is required for inhibition studies. With these challenges in mind, this protocol herein presents a modifiable procedure for targeting discrete brain regions via an angled stereotactic approach.
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
2. Preparation of the stereotax for angled procedure
3. Preparation of materials for injection/implantation
4. Anesthesia
5. Surgical procedure
6. Aligning the central axes of rotation for angled coordinates
7. Microinjection
8. Fiberoptic implantation
NOTE: After viral injection, bilateral fiberoptic cannulas are implanted at the calculated angle per section 1. Note that these coordinates should already be marked on the skull from section 6.
9. Post-surgical care
10. Optogenetics
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 consistent approach. However, given the VMN’s proximity to the midline, space constraints did not permit the non-angled implantation of bilateral fiberoptics, necessitating the development of a surgical strategy for precisely implanting fiberoptics at an angle (Figure 6).
Using this surgical strategy, we microinjected a Cre-dependent AAV expressing a modified channelrhodopsin anion-conducting channel fused with the fluorescent reporter, referred to as a ”SwiChR++” virus10, bilaterally to the VMN of Nos1-cre mice. This was followed by implantation of an optic fiber dorsolateral to each injection site at a 15° angle from the midline. As expected, viral expression was restricted to the VMN and not detected in other brain areas.
Figure 1: Representative example of calculating angled coordinates targeting the hypothalamic ventromedial nucleus. Angles and line segments are not drawn to scale. (A) This length should be calculated using basic trigonometry. In this example, A = 2.03 mm. (B) Estimated length based upon assignment of arbitrary axis of rotation. In this example, B = 7.576 mm. (C) Calculated hypotenuse. It should be noted that the depth of fiberoptic/needle insertion depends upon the desired proximity to the target region, which requires optimization. This figure has been modified from Faber et al. 201911. Please click here to view a larger version of this figure.
Figure 2: Adjustment knobs for the stereotactic head holder apparatus. This figure has been modified from Faber et al. 201911. Please click here to view a larger version of this figure.
Figure 3: Aligning the head holder center of rotation. (A) Positioning the ear bars. (B) Sighting down the scope during 0° level coronal rotation (left), during 15° rotation before adjusting the vertical shift, and the center of rotation is misaligned (middle), and during 15° rotation after adjusting the vertical shift, and the center of rotation is properly aligned (right). This figure has been modified from Faber et al. 201911. Please click here to view a larger version of this figure.
Figure 4: Assigning bregma and aligning the animal head with central axes of rotation. (A) Representative image indicating typical bregma placement. (B) Drawing a reference mark while head is level, before alignment. (C) Properly aligned axis of rotation, after adjusting the vertical shift and readjusting bregma. Please click here to view a larger version of this figure.
Figure 5: Fiberoptic implantation procedure. (A) Centering scope view of pilot holes for microinjection (m), fiberoptic (f), and anchor screws (*). (B) Centering scope view of implanted anchor screws, and bone wax covered microinjection drill holes. (C) Positioning the fiberoptic into place during angled implantation. (D) Representative bilateral angled fiberoptic placement. Dotted black arrows indicate areas in which super glue is used to anchor the fiberoptic to the anchor screws and ipsilateral fiberoptic. Please click here to view a larger version of this figure.
Figure 6: Representative results for bilateral targeting of the ventromedial hypothalamus. (A) Schematic representing bilateral microinjection and angled fiberoptic strategy for targeting the VMN. (B) Representative image showing bilateral expression of SwiChR-GFP and tissue damage from angled fiberoptic tracts. 3V = third ventricle, ARC = arcuate nucleus, and VMN = ventromedial nucleus. Please click here to view a larger version of this figure.
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 a defined cell type in freely moving animals. However, effective employment of these technologies relies upon successful stereotactic surgery to target the region of interest. While there are several established protocols describing these methods, which are suitable for targeting many brain regions, targeting deep brain regions along the midline represents significant additional challenges. Demonstrated here is a detailed surgical technique for targeting discrete brain regions via an angled stereotactic approach. Importantly, this technique can be adapted and applied to a diverse range of neuroscience techniques (i.e., optogenetics, chemogenetics, and fiber photometry approaches).
Using this approach, it is shown that acute optogenetic silencing of VMN neurons expressing neuronal nitric oxide synthase (VMNNOS1 neurons) blunts glucagon responses to insulin-induced hypoglycemia in mice9. Using a slightly modified approach, it is further demonstrated that unilateral activation of VMNNOS1 neurons 1) elicits robust hyperglycemia that is driven by counterregulatory responses that are normally reserved for the response to hypoglycemia, and 2) elicits defensive immobility behavior. Furthermore, these behavioral and metabolic responses involve neuronal projections to distinct brain areas. Specifically, the activation of VMNNOS1 neurons projecting to the anterior bed nucleus of the stria terminalis are involved in glycemic responses, whereas VMNNOS1 neurons projecting to the periaqueductal gray are linked to fear-induced behavior responses9.
It should be noted that the protocol is highly specific to the Kopf Model 1900 stereotax and accompanying accessories. While this system enables precise, reproducible implantation as well as microinjection to discrete brain regions (with a common centerline position across multiple tools), the strategy and approach can be adapted to suit other stereotaxic frames. Specifically, instead of rotating the head to perform angled microinjections and implantations, an alternative approach is to utilize the same principles and rotate the dorsal-ventral manipulator instead (see Correia et al.12).
As with any new method, it is critical for individuals to optimize the technique to improve an experiment’s reliability, consistency, and accuracy. In addition, it is important to include the necessary appropriate controls for proper analysis and interpretation of data. These include the use of Cre-negative littermate controls, viral reporter controls (i.e., AAV-GFP), verification of light-dependent neuronal firing modulation using electrophysiology, and (upon study completion) the validation of viral targeting and fiberoptic placement in the region of interest. It is recommended to refer to the publication by Cardozo and Lammel13 for a detailed review of technical considerations and suggested controls.
In summary, the introduction of more advanced and precise neuroscience techniques has supported a significant advancement and understanding of the role of the brain in behavior, cognition, and physiology, and these advancements may lead to potential therapies for CNS-related disorders.
The authors have nothing to disclose.
This work was supported by the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) grants F31-DK-113673 (C.L.F.), T32-GM-095421 (C.L.F.), DK-089056 (G.J.M.), an American Diabetes Association Innovative Basic Science Award (#1-19-IBS-192 to G.J.M.) and the NIDDK-funded Nutrition Obesity Research Center (DK-035816), Diabetes Research Center (DK-017047) and Diabetes, Obesity and Metabolism Training Grant T32 DK0007247 (T.H.M) at the University of Washington.
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 |