Leveraging Fiber Photometry to Decipher Neural Circuits Underlying Anxiety in Mice

Abstract

Anxiety disorders rank among the most prevalent mental health conditions worldwide, significantly affecting patients' lives. They are frequently comorbid with other psychiatric disorders, often exacerbating their severity. Current pharmacological treatments; selective serotonin reuptake inhibitors (SSRIs) and benzodiazepines, remain limited in efficacy and are associated with undesirable side effects, underscoring the urgent need for alternative therapeutic approaches. However, progress in developing new treatments has been hindered by an incomplete understanding of the neural mechanisms underlying these disorders. Bridging this knowledge gap requires advanced research tools capable of providing deeper insight into the neural circuits involved in anxiety. Fiber photometry (FP) has emerged as a powerful and cost-effective technique for measuring neural activity in freely moving animal models. By enabling real-time monitoring of calcium dynamics in specific neural populations within defined brain regions, this method offers invaluable insights into both normal physiological processes and pathological states. In this review, we first present an accessible introduction to FP, detailing its apparatus, procedures, and key advantages and limitations. We then conducted a comprehensive analysis of 39 studies indexed in PubMed that have employed FP to investigate neural circuits implicated in anxiety. Our review reveals the techniques' significant contributions across different research domains, including physiological (33%), pathological (53%), and dual-purpose studies (13%). Beyond summarizing its utility, our goal is to make FP more accessible to researchers. By providing a foundational guide for its integration into future scientific projects, we aim to facilitate advances in anxiety research and contribute to the development of novel therapeutic strategies.

Keywords: anxiety disorders; fiber photometry; mice; neural circuits.

FIGURE 1

Protocol to study and confirm pyramidal neural activity in the ventral hippocampus. Step 1: Local injection (ventral hippocampus) of the virus (AAV9‐CamKII.GCaMP6s.WPRE.SV40) through stereotaxic surgery under isoflurane anesthesia ⟶ recovery 2–5 weeks. Step 2: Fiber photometry recording (see Figure 2). Step 3: Confirmation of virus expression by immunohistochemistry (5× and 10×).

FIGURE 2

Fiber photometry apparatus, recording, and signal processing in the ventral hippocampus. (A) Fiber photometry apparatus and experimental setup. A laser beam is directed through a dichroic mirror into an objective lens and further down an optical patch cord, and to the brain region of interest. Within the brain, the laser excites calcium‐bound GCaMP, inducing a conformational change that triggers fluorescence emission by the green fluorescent protein (GFP). The emitted fluorescence travels back through the optical patch cord, passes through the objective lens again, is separated from the laser light by the dichroic mirror, and then is directed towards a photodetector. The resulting signal is amplified and processed by a data acquisition system. (B) Example of a signal recorded in the vHPC of a Balb/c mouse during basic home cage exploration. We used FP3002 apparatus (Neurophotometrics, MBF Bioscience, USA) for signal recordings as well as Bonsai (open‐source, V 2.9.0) for system control. The raw signal includes a calcium‐dependent fluorescence trace at 470 nm and an isosbestic control trace at 415 nm. Signal processing involves calculating the mean fluorescence (F) of both the calcium‐dependent and isosbestic signals. The data are then normalized as ΔF/F (%), where ΔF/F represents the percentage change in fluorescence relative to the baseline fluorescence (F ₀). This provides a clearer representation of calcium dynamics in the recorded region. Signal processing was done using MATLAB (MathWorks, USA, R2024b). (C) Zoom on calcium transients (red arrow).

FIGURE 3

Illustration summarizing the brain regions recorded across 39 studies. The illustration represents the zones recorded using fiber photometry across the selected studies, categorized into three types: Studies using naïve mice (13 studies, 54%, green) focused on 16 zones, with the BNST and PVN being prominent. Studies using anxiety‐inducing pathophysiological model mice (17 studies, 43.5%, purple) examined 17 zones, notably the NAc, PVN, ACC, and mPFC. Studies using both naïve and anxiety‐inducing pathophysiological model mice (5 studies, 13%, blue) investigated eight zones, with emphasis on the HPC and related areas. The BLA, PVN, IPN, NAc, and dmPFC stand out as the most frequently studied zones overall. *Studies from various hippocampal subregions.