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Causality is often the end goal of scientific study. Experiments are designed to prove that the relationship we have hypothesised is true, often by ruling out the influence of extraneous variables. Determining causality in biological sciences is often challenged by the presence of probabilistic and so-called ‘hidden variables’ [1]. Neural and cognitive sciences are two fields that have had to innovate to overcome this challenge. An organism is exposed to a stimulus, a ‘hidden’ process occurs in the brain and nervous system, and the organism outputs a behaviour. The goal of many in these fields is to determine the mechanisms that process the environmental inputs, compare with bodily needs and motivations, and decide upon a behavioural or motor output. Estimates suggest that the human brain consists of around 86 billion neural cells, and a similar number of non-neural cells [2]. The large number of connections between neural cells form networks to decode, process and compare stimuli, leading to changes in behaviours, movement and homeostasis. Communication between neurons is very energetically costly, requiring the transport of soluble ions to generate action potentials to carry a signal within a nerve cell, plus the secretion and reabsorption of neurotransmitters to communicate between cells. Consequently, the brain requires a large supply of oxygen [3]. Many studies in neural science infer network activity by measuring the oxygenated blood flow to an area by Magnetic Resonance Imaging (MRI). Combining this method with a task like reading text can be used to suggest that active areas are involved in the process of reading. Whilst this kind of approach is very useful for identifying potential relationships between brain and behaviour, more direct approaches are required to test the causality of a relationship. Both the measurement of targets, and the manipulation of the relationships need to be direct to determine causality. Immunostaining and so-called ‘reversible-lesion’ approaches are essential tools, allowing for experimental discovery and causal testing of neurocognitive relationships.
Seeing what’s there – Immunochemical techniques
Immunostaining takes advantage of the high specificity binding between antibodies and antigens to visualise the distribution and density of targets within cells and tissues. Such techniques can be used to identify the neurotransmitters a nerve cell uses, and the distribution of specific proteins in cells. A recent study used immunohistochemistry (IHC) to show that the effects of the antidepressant fluoxetine appear to be mediated by the serotonin receptor 3 (5-HT3) [4]. Fluoxetine has been shown in several studies to increase the formation of new neurons (neurogenesis) in the dentate gyrus of the adult brain. IHC showed that neural cells in the dentate gyrus of adult rodents expressed 5-HT3 whilst developing, no longer expressing the receptor once mature. Rodents were treated with fluoxetine alone, or fluoxetine and ondansetron, a 5-HT3 antagonist that inhibits the activity of 5-HT3. Rodents treated with fluoxetine showed increased neurogenesis as expected. Those treated with fluoxetine that had 5-HT3 receptors inhibited by ondansetron did not show increased neurogenesis, suggesting that 5-HT3 receptors must be activated normally for the increase in neurogenesis to occur. IHC was used in this study to identify the expression of 5-HT3 in proliferating cells, confirming that this receptor is present during cell development. However, the experimental design does not causally prove that the effects of fluoxetine on neurogenesis require 5-HT3 activation. This study is an excellent example of experimental discovery, and provides a robust building block for causal experiments, such as the ‘reversible-lesion’ approach to build upon.
The ‘Reversible-Lesion’ Method
The best causal evidence in neurocognitive science comes from so-called reversible lesion studies. Neurocognitive sciences have utilised a wide range of experimental methods to investigate the relationship between brain and behaviour. Some of the first methods used to investigate functions of the brain were lesion studies. Medical case studies in humans where a part of the brain had been damaged, or lesioned due to injury, provided some of the earliest evidence of particular functions occurring in specific parts of the brain. The function of a specific region, network, or cell-type in the brain can be impaired through methods such as RNA interference (RNAi), optogenetics, or chemical blockade. RNA interference methods have causally implicated the role of a dopamine transporter protein in the developmental origins of Parkinson’s Disease (PD) [5]. Rodents were transfected with a small-hairpin RNA molecule designed to interfere with the expression of a transporter protein VMAT2, essential for dopamine homeostasis in the substantia nigra. Successful transfection was confirmed using immunohistochemistry, and the absence of VMAT2 was linked with increased oxidative stress, the death of dopaminergic neurons, and development of PD-like motor deficits. A separate group of rodents with VMAT2 RNAi were also transfected with a functional version of human VMAT2 altered to evade the active RNAi. This group of rodents did not suffer from dopaminergic cell death or PD-like symptoms. This shows that the loss of the function that VMAT2 provides, and not the experimental manipulation led to the development of PD-like symptoms and neurodegeneration. The reversible lesion approach is perhaps one of the strongest experimental designs in neurocognitive research, and allows for sound investigation of cause and effect.
Immunochemical techniques when paired with manipulations like RNAi allow for the direct investigation of relationships at the cellular level. Neural and cognitive sciences often rely upon indirect measurements like MRI to determine the contribution of the brain to behaviours. Direct approaches using immunological and interference tools are needed to confirm relationships identified through indirect methods. The increasing availability of tools like RNAi and IHC will allow for causal investigation of a wider range of targets in the future.
The Author: Alex Roberts, Scientific Technical Support Assistant at Abbexa
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References
[1] Elidan, G., Lotner, N., Friedman, N., & Koller, D. (2000). Discovering hidden variables: A structure-based approach. Advances in Neural Information Processing Systems, 13.
[2] Herculano-Houzel S. The human brain in numbers: a linearly scaled-up primate brain. Frontiers in Human Neuroscience. 2009;3. doi:https://doi.org/10.3389/neuro.09.031.2009
[3] Jain, V., Langham, M. C., & Wehrli, F. W. (2010). MRI estimation of global brain oxygen consumption rate. Journal of Cerebral Blood Flow & Metabolism, 30(9), 1598-1607. – “2% of the body’s weight, 20% of the body’s oxygen”
[4] Olivas-Cano I, Rodriguez-Andreu JM, Blasco-Ibañez JM, Crespo C, Nácher J, Varea E. Fluoxetine increased adult neurogenesis is mediated by 5-HT3 receptor. Neuroscience Letters. 2023;795:137027. doi:https://doi.org/10.1016/j.neulet.2022.137027
[5] Bucher, M.L., Barrett, C.W., Moon, C.J. et al. Acquired dysregulation of dopamine homeostasis reproduces features of Parkinson’s disease. npj Parkinsons Dis. 6, 34 (2020). https://doi.org/10.1038/s41531-020-00134-x
