Alcohol, a cultural component in most of the western world, is a trillion dollars contributor to the global economy as well as a billion dollars of medical burden worldwide. Whether the percentage of consumers ranges from 1 to 90 percent across countries, alcohol is – unarguably – a global product. Statistically, alcohol use disorders affect more than 100 million people across the globe according to the current Oxford world data.
One of the various forms of alcohol use disorders is the Korsakoff Syndrome, which is characterized by vitamin B1 or thiamine deficiency. Although the majority of Korsakoff cases result from chronic alcohol consumption, there are rare cases where the thiamine deficiency results from processes other than alcohol abuse. Alcohol hinders the GI tract absorption of thiamine as well as thiamine storage in the liver: resulting in the nutritional deficiency of thiamine.
Thiamine is an indispensable co-factor for multiple enzymes of neural cells. Therefore, thiamine deficiency in Korsakoff cases impairs the metabolism of multiple cellular components, including ATP – the energy source of neurons. Neuronal dysfunction gradually causes edema, reactive gliosis, and loss of neurons across various brain regions.
Thiamine deficiency which is the hallmark of Korsakoff syndrome (KS) causes major alterations at the cellular and molecular level which is then expressed in the form of certain key behavioral patterns (discussed below). Thiamine as mentioned earlier is involved in enzymatic activities, one of them being mitochondrial glucose oxidation. Loss of oxidation leads to oxidative stress in the cerebral neurons, eventually causing cytotoxicity.
Moreover, the mitochondrial oxidative stress and disruption in enzymatic activity lead to an imbalance in the amount of neurotransmitters. Increased excitatory neurotransmitters induce excitotoxicity of neurons and the formation of lesions. This form of cytotoxicity affects specific regions of the brain and thereby disrupts specific neurological functions and generates abnormal behavioral patterns as discussed below.
Abnormal walking pattern where the person uses a range of support. The person follows a walking path that strays from a straight line (wider than 2-4 inches); additionally veers in different directions, that resembles stumbling or drunken walk. This is referred to as a wide-based gait. Key behavioral parameter: Ataxia
Many nerves are damaged, especially those connecting the brain and spinal cord with peripheral organs (peripheral neuropathy). Peripheral neuropathy affects the functioning of peripheral organs and causes weakness of the arms and legs, thereby inducing difficulties in walking. Key behavioral parameter: Polyneuropathy
Increased heart rate, drop in blood pressure especially upon sudden standing from a sitting or lying down position, as well as complete loss of consciousness. Key behavioral parameter: Cardiovascular abnormalities
Inability to form and retain new memories, long-term and short-term memory loss. Sometimes, individuals may also lose a few long-term memories. Key behavioral parameter: Memory impairment
Amnesia, disorientation, and confabulations (creating false memories to replace lost ones) Key behavioral parameter: Altered mental status
Underlying Molecular Mechanisms
The mechanism and complexity of neuronal dysfunction in Korsakoff syndrome is still an ongoing research topic. However, thiamine deficiency lies at the core of all these dysfunctions. Interestingly, thiamine deficiency is also a distinguishing trait of Wernicke’s encephalopathy that was first described by Carl Wernicke in the late 1800s. Wernicke’s encephalopathy (WE) is characterized by a triad of symptoms including ataxia, involuntary eye movement or paralysis, and confused or altered consciousness/mental state.
Soon after the reports from Wernicke, Sergei Korsakoff described another condition with overlapping symptoms specifically in chronic alcoholics and additionally characterized by severe memory loss. Currently, 85% of Wernicke’s encephalopathy cases are known to advance into Korsakoff syndrome. Therefore, these two conditions are usually studied together under the name Wernicke Korsakoff Syndrome. Consequently, most disease models used for studying the underlying mechanisms of Korsakoff syndrome also fall into the category of Wernicke Korsakoff Syndrome.
One of the main reasons behind the severe neurological effects of thiamine deficiency is the high oxidative metabolism within the nervous system, which renders neural cells more vulnerable than any other cell type. Thiamine in the form of thiamine pyrophosphate (TPP) acts as a coenzyme in the cerebral glucose metabolism.
Upon thiamine deficiency, the TPP levels are also reduced, which disrupts the production of TPP-dependent enzyme alpha-ketoglutarate dehydrogenase (αKGDH). αKGDH is an essential component of the TCA cycle for the production of ATP in the mitochondria. Accordingly, reduced αKGDH in thiamine deficient neurons of Korsakoff patients (or disease models) manifest oxidative stress-induced cytotoxicity.
Oxidative stress in Korsakoff syndrome not only has direct cytotoxic effects due to enhanced Reactive Oxygen Species (ROS) but also an indirect impact via impaired buffering of the excitatory neurotransmitter, glutamate. Oxidative stress is known to inhibit glutamate transport via peroxynitrite-induced transporter breakdown thereby generating excitotoxicity.
Additionally, production of the inhibitory neurotransmitter GABA is dependent on the TCA cycle and as thiamine deficiency impairs the TCA cycle, GABA levels are also significantly reduced in Korsakoff syndrome. The reduction in GABA neurotransmitters further aggravates neuronal excitotoxicity.
Disturbances in other neuronal mechanisms: vesicular transport and function of TPP-dependent enzyme pyruvate dehydrogenase lead to an imbalance in the neurotransmitters, dopamine and acetylcholine respectively, in Korsakoff syndrome. Furthermore, acute thiamine deficiency in Korsakoff patients also promotes elevated histamine levels in thalamic brain regions rendering them prone to neurodegeneration.[2,3]
Mouse Models of Korsakoff Syndrome
The underlying mechanisms in most diseases start ahead of the onset of the symptoms and they are primarily responsible for the disease symptoms. However, as the disease mechanisms affect at the cellular and molecular level, which are generally needed to be studied using invasive and controlled experimental settings – animal disease models allow investigation of such disease mechanisms. However, one of the important initial steps to study animal models is a close recapitulation of the disease etiology and symptoms in these animal models.
Korsakoff models can be generated using the following methods: by exposing the animals to chronic alcohol abuse and/or via induction of dietary thiamine deficiency and/or incorporating pyrithiamine, a thiamine inhibitor in their regular diet.
All these approaches and their combinations are known to cause limbic circuit dysfunction in cortical, hippocampal, and thalamic regions — similar to the Korsakoff patients’ brain. However, the degree of neuropathology and behavioral dysfunction varies for each treatment method.
1. Thiamine deficient diet model
To induce neurological symptoms solely via thiamine-deficient (TD) diet requires weeks to months. Moreover, the TD diet protocol alone cannot generate lesions in the thalamus or mammillary bodies, which is an important disease marker in KS patients. Therefore, the TD diet more closely models the WE phase rather than the KS phase of Wernicke-Korsakoff Syndrome (WKS).
2. Thiamine deficient diet with chronic alcohol abuse model
On other hand, a thiamine-deficient diet along with chronic ethanol exposure as well as daily injections of pyrithiamine after 4 weeks of treatment initiation: led to the loss of neurons in frontal cortical regions, suggesting a critical temporal window that allows the synergistic effect of ethanol exposure and TD. Nevertheless, no significant structural differences were observed at the hippocampus or thalamus implying the absence of a synergistic effect of TD and ethanol exposure. These contradicting studies rendered an inconclusive understanding of the effect of TD and ethanol combination.
3. Thiamine deficient diet with pyrithiamine injections model
Interestingly, when a TD diet is incorporated alongside systemic injections of pyrithiamine (PTD), an accelerated progression of the neurological symptoms of KS was observed within 15 to 18 days. Animals exhibit a typical progression of the KS neurological symptoms in both anatomy and neurochemistry. Furthermore, the PTD neurological features in rodents mirror the stereotypic brain pathology as well as behavioral dysfunction found in KS patients.
Lastly, unlike the clinical setting where patients can only be monitored as per individual’s desire of medical assessment, animal models allow regular assessment and monitoring of the progression of the symptoms, in addition to highly controlled monitoring of their diet. Therefore, animal models have been the founding pillars of our increasingly deeper understanding of the various underlying mechanisms of the Korsakoff Syndrome.[6.7]
There are multiple research techniques that are currently being used to further deepen the insight into the disease mechanism and etiology of Korsakoff Syndrome. These techniques can be categorized based on the molecular, cellular, circuit or behavioral level phenotypes.
The phenotypes of brain disorders at the molecular level, neurons and synapses, as well as neural circuits can be termed together as microendophenotypes. Techniques to study microendophenotypes can be invasive and many of these are performed only after behavioral phenotypes of the animal clearly indicate the disease symptoms.
In the case of Korsakoff Syndrome, the behavioral phenotypes mainly include loss of motor coordination, hippocampus dependent long-term aversive memory, spatial memory as well as social non-aversive memory.
Motor Coordination: Using the Rotarod Test and fTIR Walkway
PTD treatment induces acute physical deficits in mice including body weight loss, loss of peripheral nerves and muscle weakness, ataxia, and seizures. The rotarod test setup is long known to be used in analyzing the motor coordination in rodents. Rotarod setup employs a rotating rod (that is as wide as a drum) that is placed horizontally on top of a touch sensitive platform that also gives mild shocks as the motivation for animals to be on the rod.
The animals are placed on this rod and then allowed to run on the rotating rod by continuously moving in a forward direction. The rotarod can be slowly accelerated about 10X the initial speed. As soon as the animal falls off the rod either due to inability to maintain motor coordination or out of sheer tiredness, the time of fall is recorded by the electronically sensitive floor.
The time that each animal takes to fall off since the accelerating rod starts rotating is called the latency to fall. Shorter latency to fall means weaker motor coordination for the individual animal. PTD mice generally show a shorter latency to fall compared to controls.
As mentioned earlier PTD mice model of Korsakoff syndrome also show ataxia as one of the hallmark symptoms. Ataxia, like in humans, can be detected via observation of the gait of the animals.
Traditional methods like gait test or balance beam test track animal’s paw movements and ability to balance on a thin rod, respectively. However, both the methods employ manual quantification whereas fTIR walkway is an automated device that quantifies step patterns, inter-leg coordination as well as footprint positioning. Moreover, fTIR walkway also removes the unwanted effects such as fear and anxiety of falling off a high raised rod.
The device is based on a simplified and inexpensive frustrated total internal reflection (fTIR) set-up. It has a chamber equipped with a guillotine door, separate testing area and start area on a wide enough (~ 8 inches) walking platform. Chamber is supported by post clamps and is raised at ~20 inches from the surface. An angled mirror at 45° is placed under the chamber along with a cooling LED for setting up the fTIR phenomena. The fTIR Walkway can be used to detect the ataxic gait in the PTD mice.
Hippocampus Dependent Memory Formation: Spatial Memory, Aversive Memory and Social Memory
Wernicke–Korsakoff syndrome (WKS) patients exhibit memory formation impairment associated with thiamine deficiency (TD). TD triggers cytotoxicity and excitotoxicity in neuronal cells in specific brain areas including thalamic regions as well as hippocampal regions like CA1, CA3 and Dentate Gyrus.
Hippocampus function in formation of new memories has been thoroughly established by a large number of studies. Moreover, in the formation of the hippocampal memories, the role of hippocampal connections to distinct brain regions of thalamus and cortex is indispensable; considering the neuronal loss in most of these regions in WKS animal models, investigation of the memory functions in these model animals is a prerequisite for further analysis of their condition.
Morris water maze test
PTD mice show deficits in the hippocampus-dependent memory formation, although they show normal hippocampus-independent memory. The Morris Water Maze task is a classical paradigm that is used to measure the hippocampus-dependent spatial learning and memory in rodents.
Morris Water Maze test has a simple experimental setup: a large pool of water and a small platform. To test the memory function, the animal is first left to swim in this pool of water, as this is a stressful situation for the animal, it tries to escape. The small platform placed in the pool provides the much needed escape to animals; a few visual cues are also placed to mark the location of the platform.
Experiment begins with a pre-training where animals are introduced to the location of the escape platform. To test long-term memory, animals are again left into the pool after a few weeks of pre-training. However, this time the platform is hidden under the water surface, with only visual cues left for them to help locate the platform: and recognition of visual cues require use of hippocampal-dependent spatial reference memory.
Based on the location of the platform the pool of water is divided in four quadrants namely: opposite, adjacent right, target quadrant, and adjacent left. The amount of time that individual mice spend in each quadrant is calculated, along with the time they take to locate the hidden platform, which is termed as escape latency.
PTD mice models of WKS require significantly longer escape latency than control mice; thus, demonstrating the impairment in hippocampus-dependent spatial learning and memory, specifically long-term memory.
Contextual fear conditioning test
Although fear is generated primarily in the amygdala region, recognizing the context of fear takes place in the hippocampus; especially when the recognition involves understanding the spatial environment specifically performed by place cells of the hippocampus. Due to significant loss of neurons in the CA1 and CA3 regions of the hippocampus, PTD mice show significantly impaired response to contextual fear.
To test the contextual fear conditioning in mice, specially designed conditioning chambers are used. The chamber consists of a shocker grid floor and acrylic patterned walls of 17 x 17 x 25cm (width x depth x height) cm. The acrylic walls can be easily replaced to change the contextual pattern of the spatial ambience, that is recognized by the mice.
Shocker floor grid is connected to a shock generator placed outside of the isolation chamber and is used for multiple shock delivery methods. The shock is always in the range of 01mA to 0.4 mA which is safe for the animals and can be done manually or via the software.
Contextual fear conditioning test is performed in two steps: in the first step the animals are trained by placing them in the chamber, allowing them to explore the spatial ambience (conditioned stimulus) and delivering an electrical footshock (unconditioned stimulus). The animal forms the spatial memory of the ambience and the aversive memory of the footshock associated with it.
In the second step, the associative memories are tested by placing the animal back in the chamber with the same spatial pattern and the shocker grid. However, this time no footshocks are delivered. Nevertheless, animals with functional associative memory recognize the spatial pattern and associate it with the aversive footshock memory and exhibit freezing behavior (an absolute lack of movement, except respiration).
Their associative memory is measured by calculating the amount of time they spent freezing. Animals with functional hippocampus form a hippocampus-dependent aversive memory, however PTD mice show impaired consolidation of contextual fear memory and do not exhibit any significant freezing behavior.
Social recognition test
Another hippocampal function of the formation of non-aversive social memory is also disturbed in the PTD model of Korsakoff mouse models. This particular social function can be assessed using the social recognition task. The social recognition task test an animal’s ability to recognize conspecifics; this ability is critical for the formation of social relationships in the context of mate-finding, territory establishment, and parent-offspring interactions.
The social recognition test setup: sociobox, has a central circular open arena surrounded by an outer ring which consists of five rectangular removable boxes separated by fixed dividers. Initially, in the training protocol, individual subjects (central arena), as well as stimulus animals (rectangular boxes) are placed in the sociobox separately, 2-3 times for a small duration.
After the animals are familiar with the setup and are no more anxious of the novel environment, the experiment protocol begins. Exposure 1, the subject animal is placed in the central arena and the stimulus animals are in the rectangular boxes, with a perforated inner panel which allows a safe social interaction.
The duration of the subject’s social investigation behavior is quantified using a stopwatch. A few hours later (2h for STM and 24h for LTM) in exposure 2, the social memory of the subject is assessed; using an overhead camera to record the length of social investigation time demonstrated by the subject towards one of the pre-exposed stimulus animals.
Finally, a recognition index is calculated which is the ratio of the social investigation time demonstrated by the individual subjects during the test and training sessions.
The PTD mouse models of Korsakoff syndrome show a significantly worse LTM recognition index compared to controls: PTD mice fail to reduce investigation time for the same pre-exposed stimulus animal during test protocol. Therefore, one of the behavioral hallmarks of the Korsakoff syndrome is impaired consolidation of social recognition memory.
Korsakoff syndrome in most cases is a result of chronic alcohol abuse. Alcohol hinders the GI tract absorption of an essential vitamin: thiamine, resulting in the nutritional deficiency of thiamine. Thiamine is indispensable for neuronal metabolism and its energy source.
Therefore, thiamine deficiency causes neuronal dysfunction and loss of neurons across various brain regions; these cellular disruptions are expressed in the form of behavioral patterns like ataxia as well as memory impairment in Korsakoff patients.
Understanding of the underlying mechanisms requires animal models of the disease that allow the use of invasive, controlled experimental settings and regular assessment/monitoring for a thorough investigation. Most of these models are generated using a thiamine deficient diet. As the techniques used to study microendophenotypes can be invasive and are performed post-mortem, analysis of behavioral phenotypes of these models to clearly verify disease symptoms is a crucial prerequisite.
In the case of Korsakoff Syndrome, the behavioral phenotypes mainly include loss of motor coordination, hippocampus-dependent long-term aversive memory, spatial memory as well as social non-aversive memory. All these behavioral phenotypes can be easily assessed using simple but sophisticated experimental setups available on our website.
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- Kim, W.B., Cho, JH. Encoding of contextual fear memory in hippocampal–amygdala circuit. Nat Commun 11, 1382 (2020). https://doi.org/10.1038/s41467-020-15121-2