19: Nociception in sharks: Neural tracing of the trigeminal nerve

Topic: Basic Science
Subtopic: Non-specific to any diagnosis (including studies in healthy animals), Developing methodology/technology, Animal imaging (includes Ca2+ imaging, fluorescence microscopy), Neuroanatomical circuit analysis

Background and Aims: There is a debate amongst investigators regarding whether fish feel conscious pain or not (Rose, 2016). The physiology of pain in fish, in general, has not yet been established due to the limited studies on the nociceptor pathways (Diggles et al., 2023). The processing of painful stimuli depends on specific structures within the central nervous system, the most important being the cortex in mammals and its counterpart, the dorsal pallium in bony fish and elasmobranchs (Rose et al., 2014). The target tissue for this project was the trigeminal nerve, the fifth cranial nerve (V), an essential component of the motor and sensory innervation of the jaw (Terrier et al., 2022). This project aims to determine whether there is a direct or indirect connection between the trigeminal nerve and the dorsal pallium to identify a relationship between structures that process painful stimuli in sharks.

Methods: Using retrograde neural tracing, the trigeminal nerve intracerebral pathway was mapped in carpet shark (Cephaloscyllium isabella), n=6, and snappers (Pargus auratus), n=4, as a model for elasmobranch and bony fish, respectively. The animals were euthanised with MS-222, then transcardially perfused with species-specific Ringer's solution (Ferreiro-Galve et al., 2012). A craniotomy was performed to extract the brain. Neurobiotin tracer was injected into the trigeminal nerve innervation, and the tissue was incubated in Ringer's solution at 4 °C for 3 days. Afterwards, it was immersion fixed in 4% PFA, followed by 30% sucrose (Forlano et al., 2014; Gutjahr et al., 2024). The brain was then sectioned and placed into histology moulds with OCT (Optimal Cutting Temperature) medium, hardened with liquid nitrogen. Histological sections were cut, and the slides were examined under fluorescence microscope.

Results: The tracer was active from the starting point of the trigeminal nerve, travelling through the four nuclei: descending, motor, sensory, and mesencephalic nucleus. It is evidenced that the mesencephalic nucleus has a connection with the cerebellum. The tracer arrives at the dorsal cumulus of the granular stratum in the cerebellum. Moving towards to anterior, the tracer travelled through the trigeminothalamic tract (first register in sharks), the thalamus, the thalamotelencephalic tract, finishing in the medial pallium, homologous to the hippocampus, within the telencephalon. To discard false positives, specific structures were identified, a well-defined tracing was performed, and intensity analysis was conducted along the intracerebral pathway, showing stable fluorescence.

Conclusions: A complete intracranial pathway of the trigeminal nerve was obtained. Structures previously mentioned in other species were identified for the first time in sharks, such as the trigeminothalamic tract and the connection with the cerebellum. Although no connection was found with the dorsal pallium, the tracer was positive in the medial pallium, homologous to the hippocampus. This structure is related to spatial memory and learning in fish (Rodríguez-Moldes, 2009). The involved structures can process and transmit painful stimuli, such as the trigeminothalamic tract and the descending nucleus of the trigeminal nerve. The main function of the structures, apart from the painful stimuli transmission, is related to long-term memory, spatial learning, proprioception, motor interaction, and muscle memory. These brain connections may help determine whether sharks perceive a painful stimulus, or with a degree of awareness that can be defined as pain, nociception, or an intermediate sensation.

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Presenting Author: Carolina Hernandez Navarro
Submitting Author: Carolina Hernandez Navarro

Senior Author: Craig Radford

IASP 2026 Global Year theme of Neuropathic Pain: No

• Craig Radford: PhD, Professor - University of Auckland
• Farqad Abdulqader: PhD - University of Auckland