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Research at the CIN covers a very wide range, from molecules, cells and networks all the way up to questions of cognition and behaviour. Therefore, our research groups also make use of a very wide range of methods, which we cover in more detail here. Many important questions they can tackle only by using suitable animal models. This is in keeping with a general tenet that the CIN has upheld since its early days: if we want to understand how the brain generates function, we can only do so in the actual brain of a live organism. It is in the nature of neuroscience to require the living, functioning organ. Neuronal responses to sensory perception, the propagation of stimuli through neural networks, the buildup of potentials that result in movement or cognition: how could we ever hope to study these in a petri dish? What kind of imaging technique would allow us to see electrical impulses from firing neurons in action? And how could we ever feed our computer models without recordings from the living and working brain?
Animal models are a requirement of our work here at the CIN. In their experiments, our research groups are currently making use of mice, rats, zebrafish and a low double-digit number of individuals from two species of non-human primates: rhesus macaques (Macaca mulatta) and common marmosets (Callithrix jacchus). As is true of all animal research in Germany, mice make up the majority of our experimental animals. Depending on the chosen line of investigation, research on animals at the CIN makes use of a large spectrum of methods. Several projects depend heavily on genetically modified mouse lines: e.g. to ‘knock out’ specific genes resulting in certain enzymes no longer being expressed, or to make use of optogenetic methods where light of a certain frequency induces physiological reactions in cells.
Non-human primates employed at the CIN, but also a part of the mice and rats, are trained and used in long-term experiments. Here they react to stimuli in certain ways while their brain activity is monitored, variously by invasive methods or by non-invasive imaging. This is usually necessary whenever higher brain functions are investigated and mere behavioural observations do not suffice. Research into cognition and behaviour such as decision making requires the willing cooperation of the subject animals, in this case, rhesus macaques, whose responses are analysed by invasive and non-invasive methods in conjunction.
When more direct stimulus-response circuits are analysed on a cellular and network level, mice and rats are used. In visuomotor research (which makes up a substantial portion of research at the CIN), both rodents and monkeys are used, depending on the line of questioning, and again utilising a mix of methods.
As usual, and as required by law, higher-developed animals such as primates are only put to use if mice or other lower-developed animals will not suffice because their brains do not have the necessary structure or capability. But in many cases, comparative approaches that pointedly focus on the differences between animals and humans can generate intriguing insights into the specific qualities that give the human brain its unique capabilities. To a number of CIN researchers who analyse very basic oculomotor functions, zebrafish larvae – the lowest developed animal used at the CIN – even offer a substantial advantage: the animals are naturally transparent, making it much easier to apply optogenetics and microscopy. However, to study a given human capabilities in-depth without using human subjects in the first place requires an animal model that shows these capabilities. These come in a wide range, from subtle hand movement to higher cognitive functions.
The German Animal Protection Act (Tierschutzgesetz, TierSchG) offers animals far-reaching protection. It is considered one of the most restrictive such laws world-wide. The Animal Protection Act explicitly allows animal experiments for the following purposes:
- To prevent, understand or treat diseases in humans and animals
- To detect environmental hazards
- To test substances or products with regard to their safety for the health of humans and animals
- For fundamental research
Moreover, the relevant Section V of the Animal Protection Act explicitly demands that animal use in research must always limit the pain, suffering, and damage inflicted, the number of animals used, and the kind of animals used (with regard to the ability to suffer) to the absolute minimum possible. This goes for the experiments themselves (TierSchG § 7 Abs. 1, Nr. 1), of course, but also applies to keeping and breeding animals for research purposes (ibid. Nr. 2).
Institutions at which animal research is performed must name an animal welfare officer (TierSchG §10), and only trained personnel are allowed to perform animal research at all (TierSchG § 8 Abs. 1, Nr. 2). No experiments on animals can take place before the relevant authorities have granted permission, based on a detailed application that includes information about all aspects of the work to be undertaken on animals. In Baden-Württemberg, the authority deciding these applications is the Regierungspräsidium.
These strict demands are in keeping with European Law. EU Directive 2010/63/EU makes the 3R Principle the basis of animal research: scientists are bound to Reduce, Replace, and Refine their use of animals – they must reduce their number as much as possible, they must replace them with alternate methods or species less capable of experiencing suffering – such as replacing monkeys with mice –, and they must refine their procedures to guarantee they inflict a minimum of suffering.
Most of the details in animal research are not regulated in the Animal Protection Act itself, but in Federal Decrees such as the Animal Protection with regard to Experimental Animals Decree (Tierschutz-Versuchstierverordnung, TierSchVersV), the Decree on Reporting Experimental Animals (Versuchstiermeldeverordnung, VersTierMeldV) and the Decree for the Implementation of the EU Directive 2010/63/EU (Verordnung zur Umsetzung der Richtlinie 2010/63/EU TierSchVersVEV).
Every single one of our ongoing research projects has been carefully examined with regard to the stipulations of law and decree by an independent commission following TierSchG §15, and approved by the Regierungspräsidium. Our adherence to these stipulations and requirements is constantly monitored both by our own animal welfare officers and by the authorities, whose Official Veterinarian has the right to perform unannounced controls multiple times each year.
There are countless examples for insights from animal experiments that, conducted diligently and responsibly, promote medical advances and are of great benefit to humans. A prominent example is the Rhesus blood group system (including the Rhesus factor), which takes its name from its discovery in Rhesus macaques in 1940. Since then, knowledge about the Rhesus incompatibility of different blood types has saved millions of newborns from severe damage or even death.
In neuroscience, the discovery of mirror neurons in the brains of Rhesus macaques exemplifies how unexpected results from fundamental research can quickly incur clinical relevance. By now it is well-known that mirror neurons are also present in humans. They are responsible for our ability to put ourselves in the position of others. On the basis of these fundamental insights, medical research now focuses on disorders in the field of personal interactions, such as in autism.
So Rhesus macaques have specific relevance inside and outside neuroscience. Since monkeys are closely related to humans, that does not seem so surprising. As for other animals, there are possibly even more compelling arguments for transferability of research. In fact, for many decades few if any great biomedical advances have come about without the use of animal experiments.
From the discovery of the malaria cycle in 1898, to the first successful organ transplants (1905) and blood transfusions (1915), to the isolation of insulin (1922), seminal breakthroughs which have since saved countless lives have been grounded in animal research almost since the earliest days of modern medicine.
The track record continues with such indispensable insights as the development of antibiotics (proof for efficacy of penicillin in mice; 1940), the development of polio vaccination (extensive testing in mice, rats, monkeys and great apes; 1955) and the first medication for AIDS (research in mice, rats, and dogs; 1986).
Today, greatly promising research such as that into cancer therapies based on monoclonal antibodies, and into deep brain stimulation as a treatment for Parkinson’s disease, epilepsy, and depression is directly dependent on animal research combined with clinical studies. For more information about the history of animal use in biomedical research, take a look at AnimalResearch.info’s excellent timeline here.
A final note: to be fair, sometimes the transferability of animal research to humans is not even desired. In fact, there are many animal species whose physiological capabilities far surpass those of humans in certain fields. Examples include the Axolotl’s astounding ability to regenerate lost limbs, and the Naked Mole-Rat’s resistance to cancer and its longevity. Decoding these species’ secrets promises to open up whole new fields of research that will, at some point, hopefully turn out to be transferable to humans. But for the time being, science is still seeking a deeper understanding of the core mechanisms involved.
A vital and indispensable approach to understanding the functions of the brain is looking at neuronal functions in actual live brains. It is indispensable because only the structured network of a stupendous number of nerve cells can form the needed basis of our ability to think, feel and communicate, of our memory and our consciousness of ourselves and the world around us. Diseases of the brain can only be understood and treated if their basic neurobiological principles are understood. This requires insight into the functions of the healthy brain on the cell level.
These insights can obviously only be obtained in animal experiments. Without reliable fundamental research, undirected approaches doing more harm than good are the best we could ever hope for. Non-human primates (monkeys) are the only realistic animal model for many questions related to humans.
This is because monkeys are much more closely related to humans than mice, rats or pigs. During the evolution of the mammal brain, certain structures and principles of functioning have evolved which are common to all primates (including humans, great apes, and monkeys). These can only be investigated in members of this order of mammals. As in other biomedical fields, cognitive neuroscience experiments are only performed on monkeys if they focus on these specific properties, and if species that are less closely related to humans canot provide answers to the research questions posed.
Many examples stress the enormous importance of neurobiological experiments in monkeys, among others, to improve diagnostic and therapeutic methods for psychiatric and neurological diseases:
- The discovery of the effects of microstimulation in the brain, which made possible the development of ‘brain pacemakers’ to treat Parkinson’s disease
- The treatment of deafness using cochlea implants developed in animal research, which transform sound into electrical impulses and directly stimulate the acoustic nerve
- The discovery, rewarded with the Nobel prize, of the neurobiological basis of vision, which prepared the ground for understanding vision disorders in children, such as strabismus and myopia
- The research into the functions of the frontal cortex in Rhesus macaques, which provided vital impulses for the treatment of psychiatric diseases
- Vital advances in the development of neuroprosthetics to be used in rehabilitation of patients suffering from paralysis as the result of paraplegia or a stroke have resulted from experiments on Rhesus macaques
- Investigations in monkeys have become more and more important to research into ageing and into neurodegenerative diseases such as Huntington’s disease
- The investigation, rewarded with a Nobel prize, into transmision mechanisms of prion diseases such as Kuru, Scrapie, and Creutzfeldt-Jakob, with its significant implications for our understanding of Bovine spongiform encephalopathy (BSE; also known as ‘mad cow disease’), which is transmissible to humans.
Institutes at Tübingen are very much aware of the great ethical responsibility associated with animal experiments in biological and medical fundamental research. All such experiments conducted here have been carefully reviewed by the animal ethics commission and approved by the authority in charge. Compliance with necessary standards is regularly monitored.
Husbandry of laboratory animals at the Tübingen research institutions takes into account all stipulations of the German Animal Protection Act, as well as international conventions. Holding facilities not only follow these regulations with regard to size and configuration. They are also 'enriched' to provide suitable occupation and opportunity for activities such as playing and working out. Rodent cages, for example, include gymnastic apparatuses such as carousels, as well as places to retreat to, in order to mitigate stress. Primate enclosures provide swings, climbing scaffolds, and ropes for the animals to engage with.
Animals are kept in social groups whenever possible (e.g., not when an individual is sick or recovering from surgery). Animal keepers, a professional veterinarian and behaviour specialists look after them at all times. These staff are, in turn, monitored by an animal welfare officer, as well as subjected to regular controls by the authorities.
Animals are kept at scientific institutions for many years. They are trained for experiments in which they play an active part. The working schedule of Rhesus macaques, for example, follows a similar timeframe as the work week of a human wage earner – including regular periods of free time. The experiments conducted here require concentrated participation by the animals involved in them. Hence, the success of scientific investigations depends in large part on the health and well-being of these animals.
Therefore, training takes a careful step-by-step approach and is based on positive feedback (rewards). Voluntary participation of laboratory animals in the proceedings is the first step of training and is most of the time quickly achieved. Monkeys, for example, learn to sit in the primate chair of their own volition. This is important not only to the animals' welfare, but also to the scientific results achieved: animals under duress behave much differently and show an altered physiology from voluntarily participating subjects.
Whenever surgical procedures are necessary for experiments, these are performed according to the same standards applied in human surgery, including the use of general anaesthetics and diligent follow-up care. Most laboratory animals quickly adjust to e.g. metallic implants and show unrestricted behaviour typical of the species.
Note that all experiments performed on animals require permits that are only approved after careful weighing of the burdens borne by the animals involved, as compared to the benefits expected for humans or the expected scientific insight. Possible alternatives are taken into account. Approved experiments are closely supervised. Members of the authorities have full-time access into the experimental and holding facilities. All this applies doubly and triply in cases where experimental animals are required to undergo extended training periods or surgery.
Alternative Methods are valuable and highly welcome. They are employed and refined in biomedical research whenever usefully possible. However, the current state of science does not allow the total substitution of animal research by alternative methods. Non-invasive imaging methods like Functional Magnetic Resonance Imaging (fMRI) allow a glance inside the brain and constitute a substantive expansion of our spectrum of methods.
However, the possibilities of this technology are often overrated. fMRI, for example, measures blood flow in the brain. This is an indirect measure of nerve cell activity, which fluctuates far too rapidly to be resolvable by nuclear imaging. Animal experiments conducted on Rhesus macaques at the MPI for Biological Cybernetics show how simply equating fMRI signals with nerve cell activity can lead to severe misinterpretations. To be exact, a recording of electrical signals of nerve cells requires the insertion of microelectrodes into the brain.
Only if imaging methods are combined with animal experiments will it be possible to produce reliable results where brain functions are concerned. In the future, this will also allow refinement of imaging methods to the point where we can diagnose neurological disorders in human brains without the need for surgery.
Outside of imaging methods, other non-invasive methods are often touted as making experiments on live animals obsolete. For example, cell cultures and similar in vitro-methods seem to hold a lot of potential where substance testing is concerned. But even a fully emulated or even reconstructed organ, which may one day be possible using 3D biochips, simply do not suffice. A complete immune system will still be necessary to test new drugs and understand disease vectors.
And where our field of neuroscience is concerned: modelling a brain inside a vat, or a computer as some seek to do, will exceed our capabilities for decades or even centuries to come, if it turns out to be possible at all. In fact, computer models would have to be fed raw data which can only be gleaned from research in actual live brains - which in turn can only be animal brains, even given the advanced imaging technology that we have on our hands today (or are likely to possess in the future).
As stated above, today’s science cannot completely substitute animal experiments with alternative methods – even though those methods are of course being used and refined in biomedical research whenever possible. In neuroscience, functional imaging methods are often quoted as alternatives to electrophysiological ablation by electrodes, so it will be profitable to give these methods their own section.
Non-invasive imaging such as functional magnetic resonance imaging (fMRI) allow a glance inside the brain. They provide a significant extension of our available methods. However, it is becoming increasingly clear that the opportunities and predictive power of this technology have been much overestimated.
Nerve cells constantly ‘talk to’ each other using tiny, rapid electrical signals called action potentials. fMRI monitors blood flow in the brain’s blood vessels, providing an indirect measurement of nerve cell activity (not their electrical activity). The basic idea is the following: the more blood is pumped through the vessels within a specific brain region, the more active the nerve clusters there must be. This is comparable to monitoring a running motor by means of a thermal imaging camera: working motor parts would look brighter due to heat generation, and energy consumption could be visualised.
But this does not provide direct insight into how the motor works. The functions and interactions of the individual parts would remain hidden. But to treat malfunctions, understanding each part’s function is important. For this reason, functional imaging must remain a complementary method and cannot completely supplant ablations with microelectrodes.
To ‘listen to’ and understand physiological processes in the brain, microelectrodes as thin as a hair are inserted into the monkey brain right next to nerve cells. In a surgical procedure using general anaesthetic, the brain regions that are of interest are accessed. Since the brain is not susceptible to pain, measurements using these microelectrodes do not induce distress: just compare human brain surgery, which is often done without use of an anaesthetic. These pain-free microelectrode measurements allow us to pinpoint the function of nerve cells and brain regions in the live brain. Understanding how nerve cells perform when healthy makes possible the understanding and, ultimately, treatment of psychiatric and neurological brain malfunctions.