Main Research Findings

1) Animal based studies found that HA-966 has the potential to selectively regulate stress-induced metabolic activation of dopamine systems in the meso-prefrontal cortex.

2) Administration of NMDA antagonist HA-966 to rhesus monkeys was shown to impair short-term memory related to visual recognition.

 

Selected Data

1) The research team of Goldstein et al conducted this study utilizing male albino Sprague-Dawley rats, weighing between 300–400 grams. Prior to any experimental procedures, the rats were individually housed and acclimated in the laboratory’s animal colony for at least two weeks. They were kept on a controlled 12-hour light-dark cycle and provided with food and water freely. Human interaction was minimized to reduce external stress influences [1].

The behavioral experiments took place in a sound-attenuated chamber made from modified Plexiglas and aluminum. To avoid the influence of scent cues from previous animals, the chamber was thoroughly cleaned with 70% ethanol and dried with a fan after each use. The chamber setup included an overhead infrared camera system for remote observation, allowing testing to occur in complete darkness without human presence. An ultrasound microphone, sensitive to 21 kHz, the frequency of rat distress calls, was used to capture vocalizations. The captured audio was sent to a monitoring room where it was filtered, digitized, and analyzed for the number, duration, and amplitude of ultrasonic vocalizations. The footshock intensity used in conditioning was set at 0.4 mA, lasting 0.5 seconds, and measured via a calibrated digital oscilloscope [1].

HA-966 was prepared in sterile saline and administered intraperitoneally at a dose of 15 mg/kg. Preliminary tests determined that this dosage did not induce sedation in rats and further analysis of sedation was conducted using an automated locomotor activity system in a dark test chamber. Rats were monitored for two consecutive 30-minute intervals using infrared beam sensors to track movement. This data was used to confirm the lack of sedative effects of the drug.

The core experimental design examined how HA-966 affected conditioned stress responses. Conditioning was carried out during the rats’ active dark phase to replicate naturalistic threat-response conditions. On day one, all rats received saline injections and were allowed to explore the test chamber for 30 minutes. During the last five minutes of this period, three 5-second, 56 dB white noise tones were presented. This was followed by a 30-minute conditioning period where ten additional white noise tones were each paired with a 0.5-second footshock as a conditioned and unconditioned stimulus, respectively. Control animals were exposed to the tones without receiving foot shocks. On day two, some rats were pretreated with HA-966, while others received saline. During this extinction trial, all rats were exposed to the white noise tones without any footshocks to assess memory of the conditioned stress [1].

Another group of rats received HA-966 or saline before the conditioning phase on day one to investigate the drug’s effect on acquisition of stress responses. Regardless of the group, all rats received saline prior to testing on day two. At the end of day two the rats were decapitated without anesthesia to avoid interference with biological assays. Trunk blood was collected for serum corticosterone analysis, and brains were extracted for neurochemical evaluation.

Behavioral responses were assessed remotely and included freezing as a marker of immobility, grooming, grid crossings, and rearing. These behaviors were coded in real time by a trained observer using a manual toggle switch interface, and results were compiled digitally. Ultrasonic vocalizations were assessed using strict criteria to exclude non-distress sounds such as sniffing or scratching. Criteria included the presence of deep, rhythmic breathing observed via video, specific duration and shape of the audio waveform, and concurrent visual confirmation on digital monitoring equipment [1].

Following behavioral testing, the brains were removed and quickly processed. Coronal brain slices were made using a specialized mold and chilled razor blades, and the tissue was kept cold throughout dissection. Brain punches targeting specific regions were collected for monoamine analysis and tissues were sonicated in perchloric acid containing internal standards for catecholamine and serotonin analysis. Protein content was measured, and the samples were centrifuged. Supernatants were prepared for high-performance liquid chromatography using alumina mini-columns and stored for later analysis. Separate columns and mobile phases were optimized for dopamine and its metabolite DOPAC, and for serotonin and its metabolite 5-HIAA. Neurochemical ratios were calculated to assess neurotransmitter turnover and metabolic activity [1].

In parallel, corticosterone levels in blood serum were quantified using a magnetic radioimmunoassay developed in-house. Blood was centrifuged immediately after collection, and serum was stored at −70°C until analysis. The assay used specific antibodies and radiolabeled corticosterone, with magnetic beads enabling the separation of bound and free hormone. A gamma counter was used to detect radioactivity, and results were compared to a standard curve to determine absolute corticosterone concentrations, with the assay sensitive enough to detect as little as 10 ng/ml in 2 µl of serum [1].

2) The study conducted by the research team of Matsuoka and Aigner investigated the effects of drug treatments on rhesus monkeys using a computerized behavioral task. The subjects of the study were four rhesus monkeys, including three males between 11-12 years old and weighing between 7.8-9.0 kg and one 12 year old female weighing 5.2 kg. These animals had previous testing experience, including prior exposure to acute injections of low doses of physostigmine, scopolamine, and MK-801. Their daily food intake was restricted to the amount earned during each testing session, with an additional afternoon feeding of monkey chow. Fresh fruit was provided daily, and water was freely available in the home cage [2].

The behavioral task used in the study was a computer-automated version of the delayed nonmatching-to-sample task. The testing environment consisted of a sound-attenuated cubicle measuring 120 × 60 × 100 cm, with a ventilation fan mounted on the ceiling to reduce external noise. A color monitor with a touch-sensitive screen was positioned at the rear wall of the cubicle, within easy reach of the monkey, who sat in a primate chair directly in front of the screen. The monkeys were rewarded with 190 mg banana-flavored pellets that were dispensed into a receptacle by a dispenser located outside the cubicle.

The delayed nonmatching-to-sample task was designed to assess the animals’ ability to remember and recognize symbols. During the sample phase, the animal was shown 20 unique graphic symbols on the screen, and it received a reward each time it touched a symbol. After all 20 symbols were presented, they were re-shown in the same order, but each symbol was paired with an unfamiliar one in the choice phase. The symbols in the choice phase appeared on the left and right sides of the screen. The position of the old and new symbols varied and the monkeys earned a pellet by touching the unfamiliar symbol [2].

The time between symbol presentations was 30 seconds, and the interval between the sample and choice phase for each symbol was 10 minutes. Each daily session included two 20-trial tests, lasting about 40 minutes in total. Testing was conducted five days a week. Measures such as percent correct choices, response latency, and response bias were calculated for each session. Response bias was determined using Index Y, which calculated the absolute difference between the left and right alternative hit frequencies divided by their sum [2].

The study also involved drug testing to examine the effects of different compounds on the monkeys’ performance. The drugs used in the experiment were HA-966 and MK-801, both of which were administered through intramuscular injections of sterile physiological saline. The dosages for MK-801 ranged from 3.2 to 32 µg/kg, while HA-966 dosages varied from 0.1 to 10 mg/kg. The effects of HA-966 were tested after MK-801, and combinations of the two drugs were also examined. The dosages were administered in an ascending order, and saline control injections were administered before and after each drug test series [2].

Drug injections were given no more than twice per week, with at least two non-injection control sessions intervening between drug or saline testing sessions. Each injection was administered 30 minutes before the start of the testing session while the animals were seated in the primate chair. When both HA-966 and MK-801 were administered in the same session, the drugs were injected sequentially, 30 minutes before testing began [2].

In summary, the study employed a rigorous experimental design to assess the effects of drug treatments on rhesus monkeys using a behavioral task designed to test memory and recognition. The research team utilized a carefully controlled testing environment and precise dosages of drugs to investigate their impact on the monkeys’ ability to perform the delayed nonmatching-to-sample task. The study adhered to ethical guidelines for animal research and employed a combination of behavioral measures to assess the monkeys’ performance [2].

 

Discussion

1) The study conducted by Goldstein et al investigated the effects of the nootropic compound, HA-966 on the behavior, neurochemistry, and stress response of rats. In the first experiment, the rats were injected with either saline or HA-966 45 minutes prior to being placed into a novel environment. No differences in locomotor activity were observed between the two groups, as both groups exhibited a similar pattern of habituation to the test chamber, indicated by a significant effect of time but no interaction between the treatment group and time [1].

The study also explored the drug’s effects on basal dopamine and serotonin utilization, as well as serum corticosterone levels in rats that had not undergone any behavioral conditioning. Rats were injected with saline or HA-966 and returned to their home cages for 45 minutes before being euthanized. No differences were found in dopamine or serotonin utilization or basal corticosterone concentrations between the saline and HA-966 groups.

The main experimental focus involved examining how HA-966 affected the conditioned stress response. Serum corticosterone levels were significantly elevated in response to conditioned stress in rats that had been shocked compared to those not shocked. However, pretreatment with HA-966 did not attenuate this increase in corticosterone levels. Behavioral observations revealed that the group of rats that had been shocked spent 76.2% of the time freezing during the extinction trial compared to the group of rats that had not been shocked that only spent 0.99% of the time freezing. The group of the rats that were shocked and pretreated with HA-966 exhibited a significant reduction in freezing behavior during the extinction trial, freezing only 37.5% of the time, which represents a 48.3% decrease in freezing compared to the non-shocked group [1].

A repeated measures analysis revealed a significant interaction between treatment and time, showing that freezing behavior in the group of rats shocked and pretreated with the nootropic, was consistently lower throughout the extinction trial, with the difference being most notable in the later stages of the trial. In terms of locomotion, the group of rats that were shocked and treated with saline showed reduced locomotor activity compared to the group of rats not shocked and treated with saline. Pretreatment with HA-966 resulted in a substantial increase in locomotion in the group of rats shocked and treated with HA-966 compared to the group of rats that were shocked and treated with saline, however, HA-966 did not increase spontaneous locomotion in a novel environment [1].

Further neurochemical analysis focused on dopamine utilization in various brain regions. In the medial and lateral prefrontal cortices, dopamine utilization was significantly increased in the shocked group treated with saline compared to the not shocked group treated with saline, as indicated by a higher DOPAC:dopamine ratio. However, this stress-induced increase in dopamine utilization was completely blocked by pretreatment with HA-966. No such effect was observed in the nucleus accumbens, where stress led to a modest increase in dopamine utilization that was not blocked by treatment with HA-966.

Additional analyses in the anterior ventromedial and posterior dorsolateral caudate-putamen did not reveal any significant changes in dopamine utilization in response to stress or HA-966 treatment. The effects of stress on serotonin utilization were also investigated showing that in the medial prefrontal cortex, stress resulted in an increase in the 5-HIAA:serotonin ratio, indicating increased serotonin utilization, but this was not blocked by HA-966 pretreatment. No effects were observed in other regions, such as the nucleus accumbens and anterior ventromedial cortex [1].

The final experiment explored the effects of pretraining administration of HA-966 on the acquisition of conditioned stress responses. Rats pretreated with HA-966 prior to training on day 1 exhibited significantly less freezing during the extinction trial on day 2 compared to saline-pretreated rats. Despite this attenuation of freezing, rats in the HA-966 pretreated group still showed substantial freezing, indicating they had learned the association between the conditioned stimulus and the footshock. No differences in serum corticosterone levels between the saline and HA-966 pretreated groups were observed on day 2. Additionally, a reduction in dopamine utilization in the medial prefrontal cortex was observed on day 2 in the HA-966 pretreated group [1].

2) The results of the study conducted by Matsuoka and Aigner found that when HA-966 was administered intramuscularly 30 minutes prior to testing, it produced a dose-dependent impairment in the monkeys’ choice accuracy. The doses tested ranged from 0.1 to 10 mg/kg. The data showed that HA-966 at 3.2 mg/kg significantly reduced the percentage of correct choices by 63.4%, compared to the saline control that reduced the percentage of correct choices by 77.3%. A 10 mg/kg dose was the highest that was administered and was shown to cause the most severe impairment, with only 56.9% correct choices [2].

While lower doses of HA-966 did not affect response latency, the 10 mg/kg dose increased response latency during both the sample and choice phases of the task. This increase in latency was significant in the choice phase, but not in the sample phase. The study also measured response bias using Index Y, a metric that quantifies the tendency of the monkeys to favor one side over the other in the choice phase. While HA-966 administration tended to increase response bias, the change was not statistically significant.

The study also investigated effects of the drug MK-801, which was previously reported to impair memory in similar experiments. The results showed a dose-dependent impairment in delayed nonmatching-to-sample task performance. MK-801 was administered at doses ranging from 3.2 to 32 µg/kg, and the highest dose of 32 µg/kg was shown to significantly reduce the monkeys’ choice accuracy, with only 60.6% correct responses compared to the saline control that had the correct responses 76.8% of the time. MK-801 also significantly increased the response bias at the highest dose, however, MK-801 did not significantly affect response latencies during either the sample or choice phases [2].

The study then examined the effects of combining HA-966 and MK-801. First, the researchers tested lower doses of both drugs that did not significantly impair memory when administered individually. When 1 mg/kg of HA-966 was combined with 10 µg/kg of MK-801, there was no noticeable effect on choice accuracy, response latency, or response bias. However, when higher doses of both drugs were administered together, they caused a significant impairment in delayed nonmatching-to-sample task performance.

Specifically, the combination of 3.2 mg/kg of HA-966 and 32 µg/kg of MK-801 resulted in performance that was nearly indistinguishable from chance, with only 55.0% correct choices. This was a significant decrease compared to the results from the highest doses of either drug alone. This combination did not affect response latency, suggesting that the impairment in choice accuracy was not due to slower responses. However, the combined treatment did lead to a significant increase in response bias, comparable to the response bias seen with MK-801 alone at 32 µg/kg. Despite the significant impairments in accuracy and bias, all monkeys still participated in every choice trial during the experiment [2].

In conclusion, the administration of HA-966 and MK-801 individually both impaired memory performance in the delayed nonmatching-to-sample task in a dose-dependent manner, with HA-966 particularly affecting choice accuracy and MK-801 increasing response bias. The combination of these drugs at higher doses produced the most severe impairment in performance, almost reaching chance levels of accuracy, while also influencing response bias. However, the combination of these drugs did not appear to affect response latency. These results suggest that both HA-966 and MK-801 influence memory and decision-making processes, and their combined effects could have synergistic impacts on cognitive performance in this task [2] .

 

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] Goldstein LE, Rasmusson AM, Bunney BS, Roth RH. The NMDA glycine site antagonist (+)-HA-966 selectively regulates conditioned stress-induced metabolic activation of the mesoprefrontal cortical dopamine but not serotonin systems: a behavioral, neuroendocrine, and neurochemical study in the rat. J Neurosci. 1994;14(8):4937-4950. doi:10.1523/JNEUROSCI.14-08-04937.1994

[2] Matsuoka N, Aigner TG. The glycine/NMDA receptor antagonist HA-966 impairs visual recognition memory in rhesus monkeys. Brain Res. 1996;731(1-2):72-78. doi:10.1016/0006-8993(96)00463-5

 

HA-966 HCl is sold for laboratory research use only. Terms of sale apply. Not for human consumption, nor medical, veterinary, or household uses. Please familiarize yourself with our Terms & Conditions before ordering.

 

HA-966 and Its Enantiomers: Divergent Mechanisms and Behavioral Effects in Preclinical Models