In mice, the natural acetylcholinesterase inhibitor …

Galantamine Improves
Down Syndrome

And might enhance learning and help
rescue humans with this cognitive impairment
By Will Block

In springtime, the only pretty ring time,
When birds do sing, Hey ding a ding, ding.
Sweet lovers love the spring.

— Shakespeare, As You Like It

S weet lovers or not, it would seem that we are more keenly aware of smells when spring arrives. Supporting this idea is a new study showing that in songbirds (and lovebirds?), odor sensitivity peaks during the nest-building period, which is typically in the spring.1

It has generally been believed that songbirds lack a well-developed sense of smell because their olfactory bulbs—which transmit smell information from the nose to the brain—are small compared to rodents. But the aforementioned study has found otherwise, with songbird sense of smell intensifying once a year, when they court and build their nests. These interrelated needs are supported by their choice of aromatic herbs with which they construct their nests, so that they can locate the herbs, return to their nest, and fumigate parasites and pathogens to protect nestlings. As a class, birds use their sense of smell in navigation, to avoid insects, for nest identification, for detection of chemical signals during courtship, while searching for food, and for avoiding predators. Oddly, odor sensitivity is almost non-existent for songbirds at non-breeding and non-nesting times of the year.


Down syndrome is the most common
form of congenital (birth defect)
intellectual disability.


Unlike songbirds, rodents exhibit a heightened sense of smell the year round and are polyestrous and breed throughout the year. In evolutionary biology, adaptation is the driving force for the development of traits, such as smell as a tool of cognition. For songbirds, the benefit of a heightened sense of smell is a big plus for survival during the breeding season.

We Were Once More Dependent on Smell

Imagine that our olfactory apparatus was still a core means of acquiring knowledge. With our predecessors, there was a far greater need for olfactory empowerment. Before encephalization (where the brain mass increases its ratio relative to the rest of the body), sensory organs provided key, species-specific information that allowed our primitive forerunners to more effectively forage, find mates, circumvent poisons, and avoid hazards, among other advantages.2 But with the advent of settlement, herding, and the agriculture revolution, the advantage of elevated olfactory function is significantly smaller than it once was, although still important such as when we need to detect rancidity and toxicity in food. Nevertheless, the examination of the role of smell in laboratory animals for which it is still important could provide us with essential clues about our own survival, vitally as well as cognitively. And especially if those animals mimicked a memory disabling disease.

Smell Enhancement in Down Syndrome Mice

In a new study, using a strain of mice developed to mimic Down syndrome (DS), researchers found that the natural acetylcholinesterase inhibitor (AChEI) galantamine was able to improve an important learning faculty of the DS model mice that employs smell.3 This has valuable implications for humans with DS, which is the most common form of congenital (birth defect) intellectual disability. And DS is much more common than you would think. There are more than 400,000 people living with DS in the United States, and according to the CDC, its prevalence has been growing dramatically, increasing by nearly 25% between the early 80s and first few years of this century.4 This is due in part to a significant increase in incidence associated with maternal age; the older the mother at birth, the more likely the defect.


While DS is characterized by
a great many disturbances in a wide
variety of tissues, it always involves
considerable cognitive impairment.


DS in humans is also known as trisomy 21, for a genetic defect manifested by triplication of chromosome (Chr) 21 (with noted exceptions, chromosomes come in pairs). Actually, the model mouse used is a segmental trisomy mouse model of DS and (in the study) contains a third copy of the distal region of mouse Chr 16 that contains 94 genes orthologous to the DS critical region of human Chr 21.

Cognitive Impairment, a Core Disability

While DS is characterized by a great many disturbances in a wide variety of tissues, it always involves considerable cognitive impairment. Mental retardation is obvious in DS with lower verbal and mental performance evident along with a distinct delay in language development, including deficits in auditory sequential processing and verbal short-term memory.

The IQ of DS patients is typically less than ½ of normal (50 vs. 100), and if they live until their 40s or 50s, they are much more likely to be diagnosed with the disease of Alzheimer’s (DA). As children with DS grow into adults, they exhibit DA degeneration with progressive neuronal loss and the early signs of DA—including neurofibrillary tangles and brain plaque buildup—which is associated with learning ability decline and memory loss. Yet despite the considerable cognitive impairment in DS, there is currently no treatment for this aspect of the syndrome.

Testing for Olfactory Enhancement

In the Simoes de Souza et al, 2011 Down syndrome study, the researchers designed a multifaceted series of tests to examine their hypothesis: that galantamine could increase an important cognitive faculty in DS-model mice, and thus demonstrate that galantamine might be able to improve cognition in DS humans.

The researchers used a total of 28 DS-model mice and 26 control littermates. Altogether, there were 45 male and 9 female mice between the ages of 3 and 6 months.

GO-No Go Characterization and Mouse Olfactory Behavior

There were 10 DS-model mice and 10 control mice used for characterizing the olfactory learning behavior in the go-no go task (a pass/fail type test). For testing the effect of galantamine on mouse olfactory behavior, a group of 8 control mice was treated with daily intraperitoneal (into their abdominal body cavities) injections of galantamine and another group of 8 controls was treated with daily injections of saline vehicle. In the same way, a group of 9 DS-model mice was treated with galantamine and another 9 with saline. The same 34 mice (8 + 8 + 9 + 9) used for testing the effect galantamine and saline were also used for the hidden peanut butter finding test (see below), via the behavioral go-no-go test.

Eliciting Water as a Reward

To elicit motivation to obtain a water reward, the mice were water-deprived to 80–85% of original body weight, and following this, trained during 3 days to poke in an odor sampling port and then respond by licking the water delivery port in the presence of 10% citral in mineral oil to receive a drop of water.

Each trial was initiated by the mouse inserting its head in the odor sampling port and was followed by a 2.5 sec delivery of the citral odor. Reinforcement was delivered if the mouse licked on the water-delivery tube at least once in each of the last four 0.5 sec intervals of the 2.5 sec odor delivery period. Next, the mice learned to respond to the S+ (rewarded) odor and not to the S– (unrewarded) odor, in a pseudo-random order with two restrictions: 1) equal number of rewarded and unrewarded trials in each block of 20 trials and 2) no more than 3 of the same trials in a row. Each session had a maximum of 10 blocks (200 trials). The session was terminated either when the animal completed 10 blocks or when the animal became satiated and stopped initiating trials for over eight minutes. The percentage of correct responses was then calculated.

Olfactory Stimuli for the Go-No Go Task

Each mouse was tested in two different learning tasks: Task A (odor detection): Citral 10% (S1, rewarded), Mineral oil (S–, unrewarded) and Task B (odor discrimination): 2-Heptanone 1% (S+); 3-Heptanone 1% (S–).

Task A was a simple discrimination task using a strong odor as reward and a weak odor as unreward. Task B was a theoretically more challenging problem presenting a new odor pair. Indeed, 7 controls and 6 DS-model mice were unable to reach 70% correct in the last two blocks of the last day of the 2 day training period. For the others mice, task A was performed followed by task B. Odors were generated by mixing different air mixtures diluted by mineral oil; the results were measured by an olfatometer.

The Galantamine Test

The researchers then tested the effects of galantamine on olfactory learning behavior on a separate group of control mice. During days when the mice were undergoing olfactory learning in the go-no go task, they were injected intraperitoneally daily with 3 µg/g (drug/body weight)* of galantamine hydrobromide and then tested 4 hours after the injection. These chronic injections of galantamine began during the 3-day training period in the presence of S+ (rewarded) odor, and continued throughout the go-no-go test in the presence of S+ and S– (unrewarded). The DS-model mice and the control mice were each split into groups, one of which received the galantamine, the other of which received sham injections of sterile saline alone. Of note, because performance in the olfactometer tended to improve with time, it was not possible to compare the performance of the same individual on different treatments. The effect of galantamine was therefore assessed in independent groups. Treatment with galantamine lasted 10 days.


* The equivalent for a 85 kg human (187 lb) is about 21 mg/day.


Hidden Peanut Butter Finding Test

The researchers then used a “digging test” as an independent determination of mouse olfactory ability in a task to find an odor reward hidden in the cage. Mice of all 4 groups were given peanut butter—an appetitive stimulus they were allowed to eat—on the day before to the test day. The next day, the mice were placed in the middle of a clean cage with a Petri dish with peanut butter reward hidden under a thick bedding. The researchers recorded the time needed to dig and find the peanut butter reward.

The percent of correct responses per block were obtained for each animal, and then the mean and standard error of the mean were calculated for each group. The mean percent correct for all blocks per session for each odor pair was analyzed demonstrating that the DS-model mice given galantamine had significant improvements in olfactory learning strongly suggesting that galantamine is likely to have therapeutic potential for improving cognitive abilities in DS.

Galantamine Hypothesis

Because of the occurrence of DA in DS, and because DS-model mice show degeneration of basal forebrain cholinergic neurons that correlates with their cognitive decline—along with the fact that deficits in cholinergic modulation in DS-model mice are likely to affect olfactory learning—the researchers hypothesized that the administration of the AChEI galantamine would ameliorate their olfactory learning behavior. They observed that DS-model mice exhibited significant deficits in olfactory-based learning compared to their non-DS littermate controls and that these deficits could be erased by galantamine treatment. When taken as a whole, the study indicates that olfactory learning performance can be a sensitive tool for evaluating deficits in associative learning in mouse models of DS and that galantamine has therapeutic potential for improving cognition in this syndrome.


Yet despite the considerable
cognitive impairment in DS, there is
currently no treatment for
this aspect of the syndrome.


Peanut Butter and Galantamine Go Together

The researchers used a so-called “complementary hidden peanut butter finding test” to assess whether changes in sensory-motor abilities could affect odor responses. Peanut butter is high in the ranks of mouse-preferred food. Before galantamine was given, an olfactometer was used to measure olfactory learning performances of DS-model and control mice. This demonstrated that olfactory-based associative learning is negatively affected in DS-model mice.


The researchers observed that DS-
model mice exhibited significant
deficits in olfactory based learning
compared to their non-DS controls
and that these deficits could be
erased by galantamine treatment.


Furthermore the poor performance in the associative learning task, combined with documented deficits in spatial cognition, strongly support a widespread negative effect of DS on adaptive behaviors. Thus, these arrays of cognitive and sensory deficits validate the DS-model mouse as a valid model of DS. In all likelihood, the deficit in olfactory-based associative learning is due to the combined deficits in learning and odor perception. Also of importance, the deficits are clearly found in humans with DS, namely, mild to moderate mental retardation in verbal and mental performance and olfactory deficits.


Galantamine has
therapeutic potential for
improving cognition in this syndrome.


Complex Disturbance of Multiple Processes

Trisomy 21 (triplicate Ch21 in humans with DS, and a third copy of Chr 16 in DS-model mouse) results in a complex disturbance of multiple processes involved in neurological development and function. While in humans, Chr 21 is the location of many genes expressing olfactory membrane receptors and similarly for Chr 16-17 in DS-model mice, the total neurological effects of the overexpression of these olfactory genes is unknown.

On one hand, olfactory receptor proteins are expressed from a single allele (one of two or more forms of a gene), and an overrepresentation of the olfactory receptor genes do not necessarily lead to an overexpression of olfactory receptor proteins. This overrepresentation of the olfactory receptor genes, however, could down-regulate the expression of certain olfactory receptor proteins that, in turn, could decrease the sensitivity of the olfactory system to certain odorants.


Perinatal dietary supplementation
with choline acts to
significantly improve cognition and
emotion regulation in
the DS-model mouse.


On the other hand, changes in expression of other genes could mediate an olfactory associative learning deficit. Indeed, other studies have provided evidence of olfactory impairment in patients with DS as well as a role for cholinergic neurodegeneration in DS pathology. This is supported by research that olfactory loss is also a common facet of DA. It is hard to exclude olfactory effects that could be caused by the overrepresentation of olfactory receptor genes from those caused by the neural degeneration associated DA, especially in early onset.

Approach Limitation

Unfortunately, the olfactometer cannot discriminate between learning and olfactory impairment. Accordingly, other studies are needed to know how changes in protein expression induced by DS affect olfactory learning. However, it is clear that another neurological consequence associated to the overexpression of genes in DS-model mice is the diminished number of cholinergic neurons on the basal forebrain. This decrease is likely to be one of the main causes of learning deficits in DS mice, and could underlie their olfactory deficits.

To be more specific, the cholinergic neurons in the basal forebrain innervate the cerebral cortex in an extensive manner, and the acetylcholine (ACh) release from these basal forebrain cholinergic neurons modulates the cortical neurons and thus the cerebral cortex. Furthermore, research has shown that the manifestations of dementia in DS have been associated with a frontal lobe dysfunction, which could be associated with the reduced number of cholinergic neurons.

Rescuing Neurological and Behavioral Deficits in DS

Of added interest, decreased number of cholinergic neurons in the horizontal diagonal band of Broca (a region in the brain with functions linked to speech production) in the basal forebrain could result in decreased projection of axons to the olfactory bulb thereby affecting olfactory processing. And if the deficit in neural activity is due to decreased ACh neuron expression, it should be compensated by using nutritional activators of cholinergic neurons, such as galantamine. This natural nutrient boosts ACh responses, both in the neural terminals and nicotinic ACh receptors. In this manner, its activation of cholinergic neurons in the basal forebrain (that project to cortical neurons and olfactory areas) could possibly activate the cholinergic neurons and rescue some of the neurological and behavioral deficits characteristic of DS. This possibility is consistent with previous work that has shown that perinatal dietary supplementation with choline acts to significantly improve cognition and emotion regulation in the DS-model mouse. It’s possible that choline combined with galantamine could have a greater impact (see “Alzheimer’s Breakthrough” in the April issue.)


Overall, galantamine treatment
enabled DS-model mice to
reach a performance level
comparable with that of controls.


Galantamine Elicits Improved Performance, Unlike Donepezil

Overall, galantamine treatment enabled DS-model mice to reach a performance level comparable with that of controls. There was a significant improvement in the number of correct choices in DS-model mice when chronically treated with galantamine, which improved the performance of DS-model mice in both tasks, compared to the performance of control mice where it only had an effect on the most difficult. But the control mice were already achieving high scores on the smell task leaving little scope for improvement. Galantamine elicited improved performance in an olfactory learning task in DS-model mice, whereas in another study using the Morris Water Maze, no benefit was found after treatment with the AChEI donepezil.5


We can reasonably conclude that
galantamine has significant
therapeutic potential for alleviating
learning deficits in humans with DS.


Finally, while olfactory deficits cannot be dissociated from learning deficits at a behavioral level, the positive action of galantamine in the DS-model mice shows that non-olfactory learning deficits in DS-model are linked to a diminished number of cholinergic neurons in the basal forebrain. This reinforces the hypothesis that the lower scores on the olfactory discrimination task is more likely due to an impairment in the acquisition of the learning, resulting from impaired basal forebrain-neocortex circuitry rather than from a olfactory system malfunction.

Only further studies will determine whether these are olfactory or sensory deficits. What is true is that galantamine has been clinically shown to stabilize cognition in patients with DA. And since DS has been strongly linked with the early development of DA—autopsies of patients with DA reveal lesions in the cholinergic neurons of the basal forebrain—we can reasonably conclude that galantamine may help alleviate learning deficits in humans with DS.

Collectively these results indicate that DS-models exhibit impaired learning when compared to controls. As the researchers state in their summary, “Collectively, our study indicates that olfactory learning can be a sensitive tool for evaluating deficits in associative learning in mouse models of DS and that galantamine has therapeutic potential for improving cognitive abilities. [Emphasis added]”

References

  1. De Groof G, Gwinner H, Steiger S, Kempenaers B, Van der Linden A.Neural correlates of behavioural olfactory sensitivity changes seasonally in European starlings. PLoS One 2010 Dec 15;5(12):e14337.
  2. Liman ER. Changing senses: chemosensory signaling and primate evolution. Adv Exp Med Biol 2012;739:206-17.
  3. Simoes de Souza FM, Busquet N, Blatner M, Maclean KN, Restrepo D. Galantamine improves olfactory learning in the Ts65Dn mouse model of Down syndrome. Sci Rep 2011;1:137.
  4. Centers for Disease Control and Prevention. Down Syndrome Cases at Birth Increased. http://www.cdc.gov/Features/dsDownSyndrome/ Updated July 2011. Accessed March 21, 2012.
  5. Rueda N, Flórez J, Martínez-Cué C. Chronic pentylenetetrazole but not donepezil treatment rescues spatial cognition in Ts65Dn mice, a model for Down syndrome. 2008 Neurosci Lett 433, 22–7.


Will Block is the publisher and editorial director of Life Enhancement magazine.

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