Main Research Findings

1) When assessing eyeblink classical conditioning as a representation of behavioral paradigm and cognitive functioning in rabbits, administration of nefiracetam was shown to attenuate learning deficits in aged animals.

2) Treatment with nefiracetam was found to reduce associative learning impairments in aged rabbits after an injection of scopolamine in order to disrupt cholinergic system functioning in the brain.

 

Selected Data

1) The assessment of eyeblink classical condition in rabbits is useful to make inferences regarding learning, memory, and aging as these behavioral and neurobiological impairments are hallmarks of Alzheimer’s disease. Alzheimer’s disease is also characterized by dementia related to the neuropathological loss of neurons and accumulation of amyloid-containing plaques and neurofibrillary tangles. Similar pathology has been observed in autopsies of individuals over the age of 35 with Down’s syndrome; this is a condition referred to as Down’s Syndrome/Alzheimer’s disease (DS/AD).

Previous research shows that the development of Alzheimer’s disease is correlated with impaired eyeblink classical conditioning more than the typical impairments seen with aging. It is interesting to note that adults with DS/AD similarly performed poorly during eyeblink classical conditioning, however adults with Down’s syndrome scored significantly higher than individuals with DS/AD or Alzheimer’s disease. Deficits seen in eyeblink classical conditioning related to Alzheimer’s disease indicate that there is a potential disruption in the functioning of cholinergic systems within the hippocampus. That being said, Alzheimer’s disease seemingly affects hippocampal functioning by disturbing cholinergic innervation of both cortical and hippocampal neurons, resulting in a longer acquisition rate of eyeblink classical conditioning. To identify potential treatment for these impairments, nefiracetam was used due to its proposed ability to promote the release of various neurotransmitters that may facilitate normal hippocampal functioning [2] .

Researcher Diana S. Woodruff-Pak hypothesized that nefiracetam affects eyeblink classical conditioning by improving functioning in the hippocampal pyramidal cells. That being said, administration with the nootropic should improve eyeblink classical conditioning in animals with an intact hippocampus, but will elicit no changes in animals where the hippocampus had been removed. 56 specific pathogen free (SPF) female New Zealand retired breeder rabbits were included in experiment 1 and an additional 11 SPF rabbits with verified lesions were included in experiment 2. The mean age of the rabbits included in experiment 1 was 27.9 months old while the average age of the rabbits included in experiment 2 was 26.8 months old. All animals were maintained under standard laboratory conditions and allowed free access to rabbit chow and tap water [2].

A restraining contraption was used to restrain each rabbit during the eyeblink classical conditions. Retractors held the left eye open while a removable platform holding the heastage in place was secured to the animals’ muzzle behind the ears. The headstage was positioned 1 cm away from the cornea and an unconditioned stimulus was presented through the airpuff. Nictitating membrane (NM) movement measurements were also collected through a minitorque potentiometer attached onto the headstage by a level and a lead to a nylon suture in the NM. The potentiometer on the head mount converted the NM movements into electrical signals that underwent analog-to-digital analysis. Collected values were analyzed immediately after each training session and stored for further examination [2].

The rabbits were restrained as previously described, in order to conduct both training sessions and drug injections. For the purpose of the training sessions, the animals were subcutaneously injected with nefiracetam or a vehicle 15 minutes before training began. The rabbits were then placed into the restrainer, fitted with the head mount, and positioned in a ventilated and sound-proofed experimental chamber where a conditioned stimulus was delivered in combination with the unconditioned stimulus, a corneal airpuff.

The conditioned stimulus was an 850 ms, 85 dB, and 1 kHz sound, followed by the delivery of a 100 ms, 3 psi corneal airpuff, 750 ms later. Delivery of both the conditioned stimulus and the corneal airpuff were terminated at the same time with a random inter-trial interval lasting from 10-20 seconds. The full session lasted 35-45 minutes and training was complete within an hour after nefiracetam or the vehicle was administered. During the trial, a conditioned response was automatically scored by the computer system if movement of the nictitating membrane took place between 25-750 ms after the onset of the conditioned stimulus, as well as if the movement was greater than 0.5 mm [2].

For the first experiment that took place, 64 rabbits with an intact hippocampus were included in daily acquisition sessions that included 90 trials pairing the conditioned stimulus of the sound and the unconditioned stimulus of the airpuff. Nefiracetam was dissolved in sterile saline and subcutaneously injected in doses of 1 mg/kg, 3 mg/kg, 5 mg/kg, 10 mg/kg, and 15 mg/kg. For the second experiment, 11 rabbits that were histologically confirmed to have bilateral surgical removal of the hippocampus were included in the study, while an additional 20 rabbits underwent a surgical procedure to produce aspiration lesions in aseptic surgical conditions [2].

The rabbits that underwent surgery to produce aspiration lesions were allowed to recover for 3 weeks before all of the test subjects went through further conditioning trials. Nefiracetam was dissolved in sterile saline and delivered to the animals in doses of 0 mg/kg and were given saline only, or 10 mg/kg. The eyeblink classical conditioning acquisition trials were carried out following the same methods as the previous experiments. Performance in the rabbits that underwent hippocampectomy was compared to those that received an injection of sterile saline, and those that received a 10 mg/kg dose of nefiracetam [2].

2) Further research regarding the effects of nefiracetam on eyeblink classical conditioning was conducted by the research team of Pak et al. For the purpose of this study, 56 female retired breeder SPF New Zealand rabbits ranging from 24 to 58 months of age were utilized. The test subjects were housed in individual cages and maintained under standard laboratory conditions with ad libitum access to food and water. In order to conduct eyeblink classical conditioning testing, customized rabbit restraints were used for both injection of scopolamine and nefiracetam, as well as behavioral training. Eyelid retractors were used to hold the left eye of the rabbits open while a removable platform and headstage was fixed behind the animals’ ears and muzzle to position a nozzle 1 cm away from the cornea. An airpuff unconditioned stimulus was delivered through the nozzle and the nictitating membrane/eyeblink response was measured via minitorque potentiometer. The movements that were measured were then converted into electrical signals for further analysis [3].

Two adaptation sessions took place prior to the acquisition trials. During the second adaptation trial the animals were administered a local ophthalmic anesthetic to the left eye in order for the researcher to place a nylon suture loop in the temporal margin of the nictitating membrane. 5 groups each consisting 8 rabbits received a paired presentation of sound and airpuff, while an additional 2 groups of 8 rabbits received unpaired presentation of stimuli. The 5 paired groups included group 1) 1.5 mg/kg scopolamine + 0 mg/kg nefiracetam; group 2) 1.5 mg/kg scopolamine + 5 mg/kg nefiracetam; group 3) 1.5 mg/kg scopolamine + 10 mg/kg nefiracetam; group 4) 1.5 mg/kg + 15 mg/kg nefiracetam; and group 5) vehicle only [3].

Two days after the second adaptation trial and 15 minutes before the first acquisition trial, the rabbits were subcutaneously injected with combined treatments of nefiracetam and scopolamine or a vehicle and scopolamine. Nefiracetam was delivered to the subjects in doses of 5, 10, or 15 mg/kg and scopolamine was delivered prior to nefiracetam or the vehicle in doses of 1.5 mg/kg. Each drug was prepared by being dissolved in sterile saline before being subcutaneously injected between the shoulder blades. The animals were then placed into a sound dampening chamber where a conditioned stimulus tone was delivered, followed by an unconditioned stimulus airpuff. The tones and airpuffs were delivered at the exact magnitude and duration for each trial and a total of 45 sounds and 45 airpuffs were delivered. There was a randomized 10-20 second interval between each trial while the paired and unpaired conditioning sessions took place over 35-45 minutes [3].

 

Discussion

1) The study conducted by Woodruff-Pak examined how administration of nefiracetam affects eyeblink classical conditioning in rabbits with an intact hippocampus and in those with their hippocampus removed bilaterally. Two experiments were conducted, the first of which included 64 intact rabbits administered varying doses of the nootropic including 1, 3, 5, 10, and 15 mg/kg. The second experiment included 11 rabbits that had their hippocampus removed and 20 rabbits that underwent surgery to produce aspiration lesions on the hippocampus. They were then treated with either sterile saline or 10 mg/kg of nefiracetam [2].

The initial results of the first experiment found that in the rabbits administered nefiracetam there was a significant improvement in the amount of time it took to learn the association between the stimuli delivered, in comparison to the rabbits that were treated only with a vehicle. The improved learning times are indicative of enhanced cognitive functioning; the dose that was shown to be most effective was 10 mg/kg of nefiracetam. It was also found that the amplitude of the unconditioned response was affected most significantly with 3 mg/kg doses of nefiracetam, in comparison to the animals treated with a vehicle. It is important to mention that in the rabbits administered 10 mg/kg of the nootropic, the average amplitude of the unconditioned response was comparable to the rabbits treated with a vehicle. The amplitude in the 10 mg/kg treatment group was recorded as 4.38 mm while the amplitude in the vehicle treated group was recorded as 5.00 mm [2]. This was despite the previous findings that reported the 10 mg/kg dose of nefiracetam attenuates learning deficits and improves cognitive functioning.

Figure 1: The number of trials it took for the older rabbits included in each experimental treatment group to learn the association between the unconditioned stimulus and conditioned stimulus.

The results of the second experiment conducted including hippocampectomized rabbits found that there was a significantly better rate of leaning in intact older rabbits administered 10 mg/kg of nefiracetam, compared to those treated with a vehicle. That being said, treatment with 10 mg/kg of nefiracetam did not elicit any significant effects on learning in the hippocampectomized rabbits. These findings supported the hypothesis developed by Woodruff-Pak that treatment with the nootropic did not affect cognitive functioning in rabbits that did not have a hippocampus but improved it in rabbits that remained intact. This indicates that the mechanism of action that drives the benefits of nefiracetam is associated with the hippocampal pyramidal cells [2].

Figure 2: The number of trials it took for hippocampectomized and intact rabbits from each experimental treatment group to learn the association between the unconditioned stimulus and conditioned stimulus.

The overall results of both experiment 1 and 2 suggest that 10 mg/kg of nefiracetam had the potential to reduce the number of trials it took for aged rabbits to learn the association between an unconditioned and conditioned stimulus delivered during eyeblink classical conditioning testing. Additionally, this dose was only effective in the rabbits that did not have their hippocampus removed, indicating that the nootropic works to attenuate eyeblink classical conditioning by acting on the cells of the hippocampus. Specifically, the CA1 pyramidal neurons are most affected by aging with adult animals losing 15-20% of these cells [2].

That being said, nefiracetam has been shown to have the potential to protect membrane dysfunction in hippocampal CA1 neurons after episodes of glucose and oxygen deprivation. Additionally, nefiracetam may combat learning deficits in older animals by enhancing the function of the hippocampal CA1 pyramidal neurons that are still remaining. It is important to mention that the results regarding the effects of nefiracetam were supported by research focused on another cognition enhancing supplement, nimodipine. The findings suggest that the improvement of deficits in eyeblink classical conditioning in older rabbits is related to the facilitation of hippocampal pyramidal neuron functioning [2].

2) A group versus training session repeated measures ANOVA was conducted by the research team of Pak et al to determine the interactions between the treatment each rabbit received based on the group they were in and the number of training sessions they underwent. Results of the statistical analysis reported that there was a significant interaction between the group that received nefiracetam and the number of training sessions undergone by the group. Additionally, Tukey HSD test indicated that over 20 training sessions the percentage of conditioned responses in vehicle treated rabbits was significantly higher than rabbits treated with scopolamine and a vehicle. However, in the groups of rabbits treated with 15 mg/kg of nefiracetam and scopolamine, the percentage of conditioned responses was remarkably increased to the same level as the vehicle group, suggesting the nootropic completely reverses the degenerative effects of scopolamine [3].

As for the amplitude of the conditioned response similar results were reported based off of the repeated measures ANOVA. Tukey HSD was also used to analyze the changes in conditioned response amplitude over 20 training sessions The results found that vehicle-treated rabbits exhibited a far greater amplitude of conditioned responses in comparison to all of the training groups. This suggests that while nefiracetam was able to improve scopolamine-related deficits they were not deemed significant by the research team. The findings related to the conditioned response were related to the changes in response latency as the animal’s were measured on how long it took to shift from the unconditioned response period to the conditioned response period.

After the appropriate measurements were collected regarding the shift from an unconditioned to conditioned response period, a Tukey HSD analysis was performed. The data reported that after 20 training sessions response latency of the vehicle treated rabbits was significantly shorter than animals treated with scopolamine and a vehicle and animals treated with scopolamine and 10 mg/kg of nefiracetam. That being said, rabbits treated with scopolamine and 5 or 15 mg/kg of nefiracetam were shown to have similar response latencies to the animals only administered a vehicle compound. These findings suggest that 5 mg/kg and 15 mg/kg of nefiracetam had the potential to completely reverse the degenerative effects of scopolamine. It was noted by the research team that response latencies were found to be faster earlier in training for the rabbits administered 5 mg/kg of nefiracetam and faster later in the training for the rabbits administered 15 mg/kg [3].

Figure 3: Changes in response latency based on the experimental treatment group and the number or training sessions completed by each experimental treatment group

Finally, the amplitude of the unconditioned response was measured to make inferences regarding the strength of the reflexive response to the unconditioned stimulus. A Tukey HSD analysis was performed to evaluate the collected data and revealed that after 20 training sessions the amplitude of the unconditioned response of the vehicle treated rabbits was significantly shorter than animals treated with scopolamine and a vehicle and animals treated with scopolamine and 10 mg/kg of nefiracetam. That being said, rabbits treated with scopolamine and 5 or 15 mg/kg of nefiracetam were shown to have similar response latencies to the animals only administered a vehicle compound. These findings suggest that 5 mg/kg and 15 mg/kg of nefiracetam have the potential to completely reverse the degenerative effects of scopolamine. The researchers noted that the amplitude of the unconditioned response was lower during the initial training sessions but became stable after several trials [3].

There were an additional 2 experimental groups that were presented unpaired stimuli in order for the researchers to determine whether the animals’ performance was affected by sensitization or habituation. One of these groups received a vehicle while the other was treated with 1.5 mg/kg of scopolamine and 15 mg/kg of nefiracetam. A response to the sound of the conditioned stimulus was scored based on whether a blink was observed within the period 750 ms after the sound. There were no significant interactions found between the unpaired group the rabbits were included in or the number of training sessions had been completed. Additionally, there was no change in the rate of reflexive blinking to the sound in the group administered scopolamine and nefiracetam. Findings of this portion of the study allowed the research team to conclude that the nootropic did not sensitize response to the sound of the conditioned stimulus, nor did it cause habituation to occur more regularly [3].

Figure 4: Unconditioned response amplitude in paired and unpaired experimental groups of rabbits.

 

Disclaimer

**LAB USE ONLY**
*This information is for educational purposes only and does not constitute medical advice. THE PRODUCTS DESCRIBED HEREIN ARE FOR RESEARCH USE ONLY. All clinical research must be conducted with oversight from the appropriate Institutional Review Board (IRB). All preclinical research must be conducted with oversight from the appropriate Institutional Animal Care and Use Committee (IACUC) following the guidelines of the Animal Welfare Act (AWA).

 

Citations

[1] DeFord SM, Wilson MS, Gibson CJ, Baranova A, Hamm RJ. Nefiracetam improves Morris water maze performance following traumatic brain injury in rats. Pharmacol Biochem Behav. 2001 Jul-Aug;69(3-4):611-6. doi: 10.1016/s0091-3057(01)00559-7. PMID: 11509223.

[2] Woodruff-Pak DS. Nefiracetam ameliorates learning deficits in older rabbits and may act via the hippocampus. Behav Brain Res. 1997 Feb;83(1-2):179-84. doi: 10.1016/s0166-4328(97)86065-2. PMID: 9062680.

[3] Pak J, Green J, Heifets B, Pak M, Woodruff-Pak D. Nefiracetam ameliorates associative learning impairment in the scopolamine-injected older rabbit. Med Sci Monit. 2002 Apr;8(4):BR105-12. PMID: 11951055.

Mechanisms of Action of Nefiracetam

Nefiracetam is a nootropic compound of the racetam group. Nefiracetam is derived from another popular nootropic, piracetam. While it is derived from piracetam, nefiracetam is most structurally similar to aniracetam. Both of these compounds are considered fat soluble and are most commonly used for enhancing cognition and improving memory. Like many other racetam compounds, nefiracetam is not usually effective with just one dose. Improved memory formation tends to be observed when nefiracetam is administered for longer than 7 days. Prolonged supplementation of the compound also tends to lead to neurogenesis and assists in facilitating cognitive enhancement.

Currently there are two proposed mechanisms of action for nefiracetam. The first pathway is the prolonged opening of calcium channels. This results in an enhanced receptor signaling that is independent of the synapse. The second pathway is related to the activity of protein kinase C (PKC) and calcium calmodulin-dependent protein kinase II (CaMKII). This pathway leads to the augmentation of cholinergic receptor signaling and results in the release of excitatory neurotransmitters from the presynaptic level. There are benefits to both pathways. For example, the opening of calcium channels is crucial for long term potentiation while the PKC/CaMKII pathway is important for the enhancement of neuronal signaling.

 

Effects of Nefiracetam on Neurotransmission Systems

Nefiracetam has been shown to be capable of regulating the actions of many of the main neurotransmitters in the brain. A multitude of animal-based studies have been conducted examining how the effects of nefiracetam are dependent on cholinergic signaling and acetylcholine (ACh) release. A notable study conducted on rabbits identified improved performance on delayed eye blinking conditions. Researchers Pak et. Al were able to determine that results of this test are dependent on cholinergic signaling in the hippocampus. Since nefiracetam is dependent on the hippocampus in this study, they came to the conclusion that the cognitive enhancing effects of nefiracetam are related to cholinergic signaling.

Additional studies mentioned how the memory improvement effects of nefiracetam seem to depend on ACh signaling in cases of protein synthesis inhibition. Researchers Nabeshima et. Al observed how the effects of nefiracetam can be reversed through administration of scopolamine. This mechanism of action allowed them to hypothesize that the anti-amnesiac effects of nefiracetam could potentially be related to muscarinic acetylcholine receptors. Follow up studies implemented a passive avoidance test for mice where amnesia was induced through the inhibition of protein synthesis. The results of the study found that proper release of ACh proved to be protective against induced amnesia. Nefiracetam can assist in this effort as it has shown potential in preserving ACh levels.

Evidence has noted that nefiracetam seems to activate presynaptic acetylcholine receptors in the hippocampus, resulting in an increase in glutamate release. The enhanced glutaminergic effect leads to more postsynaptic activity as well as the activation of NMDA and AMPA receptors. Because nefiracetam has the ability to increase glutamate release, reports show the nootropic plays a role in improving long term potentiation (LTP). Through the PKC mechanism, low concentrations of the compound enhance LTP that is dependent on NMDA receptors and at high concentrations it enhances LTP dependent on AMPA receptors. Pairing this knowledge with the effects nefiracetam has on the cholinergic system, researchers conclude that the compound effectively recruits both of the major stimulatory neurotransmitters, ACh and glutamate.

Nefiracetam has also shown promise in its ability to regulate functioning of GABA and GABA A receptors. Despite the lack of affinity for the GABA binding sites, reports show that nefiracetam is capable of enhancing the signaling to these receptors when concentrations of GABA are low. When GABA concentrations are high the opposite effect is observed meaning that signaling is suppressed.

Researchers Luthman et. Al discovered that nefiracetam plays a more covert role on the serotonergic system. Their study suggests that doses varying from 10-30 mg/kg of nefiracetam are effective at counteracting the loss of serotonin due to induced ischemia. Additional research has found that in doses ranging from 3-10 mg/kg given 30 minutes after administration of a serotonergic antagonist led to an improvement in performance on various choice recognition tasks.

Similar results were seen when examining the effects of nefiracetam on the dopaminergic system. However, the results varied between studies and benefits were only seen when high doses of the compound were administered. Further research needs to be conducted in order to come to a strong conclusion regarding the relationship between nefiracetam and dopamine levels (https://examine.com/supplements/nefiracetam/research/#RQbokJY-neurology-1_RQbokJY-g-protein-and-calcium-signalling-1).

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