Main Research Findings

1) HA-966 was shown to antagonize NMDA receptors by selectively interacting with glycine modulatory sites.

2) 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.

 

Selected Data

1) The study conducted by the research team of Foster and Kemp aimed to investigate the pharmacological properties of the compound HA-966, particularly its interactions with excitatory amino acid receptors in the rat brain. A combination of radioligand binding assays, electrophysiological recordings, and cortical slice preparations were employed to assess the compound’s effects.

To study receptor binding, crude postsynaptic density preparations from rat cerebral cortex were used to evaluate the affinity of L-glutamate for NMDA-sensitive sites. Additional binding experiments included measurements for AMPA and kainate using modified protocols based on previous research. Each assay utilized 50 µg of membrane protein, incubated at 32°C for 30 minutes in 50 mM Tris-acetate buffer at pH 7.0 with appropriate radiolabeled ligands. The following radioligands were utilized: 50 nM L-glutamate (40–60 Ci/mmol), 36 nM AMPA (5–15 Ci/mmol), with 100 mM KSCN added, and 16 nM kainate (30–60 Ci/mmol) [1].

Nonspecific binding was determined using 1 mM L-glutamate. Bound and free radioligand separation was done by centrifugation for glutamate and AMPA assays, and by filtration through Whatman GF/C filters for kainate assays. For glycine receptor studies, synaptic plasma membranes were prepared from rat cerebral cortex without Triton X-100 treatment. These membranes were frozen, thawed, and washed before being resuspended in Tris-acetate buffer. For binding assays, 50–100 µg of membrane protein was incubated at 4°C for 30 minutes with 50 nM glycine (40–60 Ci/mmol) in a final volume of 0.5 ml. Nonspecific binding was determined using 1 mM glycine. Bound radioactivity was separated via centrifugation.

The excitatory effects of amino acids on cortical neurons were studied using brain slices from male Sprague-Dawley rats each weighing approximately 100 g. After decapitation, coronal sections sliced 3–4 mm thick were cut from regions between the olfactory tubercle and optic chiasm. These slices were fixed on a glass slide with cyanoacrylate glue and placed in an Oxford vibratome to obtain 500 µm thick sections. These were further dissected into 1 mm-wide wedges containing cortex, white matter, and striatum.The wedges were placed in a two-compartment chamber so that cortical tissue rested in one compartment and white matter in the other [1].

The cortical compartment was perfused with artificial cerebrospinal fluid that lacked Mg²⁺ in order to unblock NMDA receptors, while also containing tetrodotoxin to suppress spontaneous neural activity. The aCSF composition was as follows: NaCl 124 mM, MgSO₄ 2 mM, KCl 2 mM, KH₂PO₄ 1.25 mM, NaHCO₃ 25 mM, CaCl₂ 2 mM, and glucose 11 mM. A direct current potential between the two compartments was monitored using Ag/AgCl electrodes and the cortical side of the chamber was electrically grounded. Excitatory amino acid agonists dissolved in artificial cerebrospinal fluid were applied for 1-minute periods, with at least 10 minutes between applications. HA-966 was perfused for 15–30 minutes before repeating dose-response experiments to evaluate its modulatory effects [1].

To directly examine the effect of HA-966 on individual neurons, whole-cell patch-clamp recordings were performed on primary cultures of rat cortical neurons. These cultures were prepared from 1-day-old neonatal rats using established protocols from previous research. Cells were plated on poly-D-lysine-coated coverslips and maintained in a medium composed of DMEM and Ham’s F12 in a 2:1 ratio, and supplemented with 10% horse serum and 2 mM glutamine. Cytosine arabinofuranoside was added post-confluency to prevent glial cell growth.

Recordings were made 1–4 weeks after plating. Coverslips were transferred to a recording chamber mounted on an inverted microscope and continuously perfused with artificial cerebrospinal fluid at a rate of 1 ml/min. The artificial cerebrospinal fluid used for recordings was gassed with 95% O₂ and 5% CO₂ and composed of NaCl 124 mM, KCl 2 mM, KH₂PO₄ 1.25 mM, MgCl₂ 2 mM, CaCl₂ 2 mM, NaHCO₃ 25 mM, and glucose 11 mM. Patch pipettes were made from borosilicate glass and were filled with an internal solution containing CsF 120 mM, CsCl 10 mM, HEPES 10 mmM, EGTA 10 mM, NaCl 4 mM, CaCl₂ 0.5 mM, at a pH adjusted to 7.25 with CsOH. Patch formation involved applying gentle suction to form a high-resistance seal and further suction was used to rupture the membrane and establish whole-cell access. The membrane potential was clamped at –60 mV [1].

Drug solutions, including HA-966, were applied to cells via a rapid perfusion system with a dual-barreled pipette, allowing swift switching between control and experimental conditions. This setup allowed precise measurement of HA-966’s direct impact on neuronal receptor currents, particularly those mediated by NMDA and glycine receptors [1].

2) 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 [2].

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 [2].

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 [2].

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 [2].

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 [2].

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 [2].

 

Discussion

1) The research team of Foster and Kemp evaluated HA-966, a compound known for its NMDA receptor antagonist properties in previous electrophysiological studies, for its ability to interact with various excitatory amino acid receptor sites. Radioligand binding studies revealed that HA-966 exhibited negligible affinity for the NMDA receptor’s glutamate recognition site. At a concentration of 1 mM, HA-966 inhibited NMDA-sensitive glutamate binding to crude postsynaptic densities from rat cerebral cortex by only 5 ± 3%. Similarly, it showed minimal inhibition of AMPA and kainate binding by 4 ± 3% and 24 ± 6%, respectively. This indicates poor interaction with the recognition sites of the quisqualate and kainate receptors as well [1].

To investigate whether HA-966 exerted its effects through interaction with the glycine modulatory site on the NMDA receptor complex, glycine binding to synaptic plasma membranes from rat cerebral cortex was assessed. This binding, which is known to label the glycine site on the NMDA receptor, was shown to be insensitive to strychnine and inhibited by amino acids that act as co-agonists at the glycine site. HA-966 was found to inhibit glycine binding at a concentration of 17.5 µM across four experiments. This suggests a moderate affinity for the glycine modulatory site. However, HA-966 had no significant effect on the classical inhibitory glycine receptor. When strychnine binding was measured in rat brain stem and spinal cord membranes, HA-966 at a concentration of 1 mM resulted in only 11 ± 10% inhibition, suggesting its action is specific to the glycine site of the NMDA receptor and not to the inhibitory glycine receptor [1].

Following these observations, cortical slice experiments were conducted to determine whether the antagonistic effects of HA-966 on NMDA responses was mediated through the glycine site. Using this electrophysiological approach, HA-966 was shown to antagonize NMDA-induced depolarizing responses in cortical tissue. However, HA-966 did not alter depolarizing responses to quisqualate or kainate, even at high concentrations, reinforcing its specificity for the NMDA receptor complex. The minimum concentration at which HA-966 began to antagonize NMDA responses was 100 µM, with a stronger effect seen at 250 µM. Further increasing the concentration to 500 or 1000 µM did not significantly enhance the degree of antagonism, indicating a possible ceiling effect in its antagonistic capability.

It is important to note that the NMDA antagonism by HA-966 was reversible by glycine. When 1 mM glycine was co-applied, it completely reversed the NMDA response inhibition induced by 100 µM HA-966. On average, this reversal significantly reduced the concentration-ratio shift caused by HA-966. A similar effect was observed with D-serine, a known co-agonist at the glycine site. When 100 µM of D-serine was applied, it almost completely abolished the effect of 1 mM HA-966. The average concentration-ratio decreased from 0.47 ± 0.02 to 0.08 ± 0.018, demonstrating that the inhibitory action of HA-966 is mediated by its antagonism at the glycine site of the NMDA receptor [1].

Further investigation was carried out using whole-cell patch-clamp recordings on cultured rat cortical neurons. These experiments allowed precise control over extracellular glycine levels and more direct measurement of the effect of HA-966 on NMDA receptor-mediated currents. Under basal conditions, without added glycine, application of 30 µM of NMDA elicited a moderate response while adding 1 µM of glycine caused a significant potentiation of the NMDA response, increasing the induced current by as much as 50-fold [1].

Interestingly, when HA-966 was applied up to 1 mM, it did not antagonize the basal NMDA response in the absence of glycine, reinforcing that HA-966 does not act at the glutamate recognition site. However, when 1 µM of glycine was co-applied with NMDA, the potentiated responses were reduced in the presence of HA-966 applied at concentrations between 100 and 300 µM, demonstrating its antagonistic action at the glycine site. For example, in one set of recordings from four neurons, 30 µM HA-966 markedly reduced the response potentiated by 300 nM glycine. The response to 10 µM NMDA plus 300 nM glycine was 770 ± 90% relative to baseline, but when HA-966 was added, this was reduced to 160 ± 17%.

Overall, these findings strongly suggest that HA-966 functions as a selective antagonist at the glycine modulatory site on the NMDA receptor complex. It does not significantly interact with the glutamate, AMPA, kainate, or inhibitory glycine receptor binding sites. The specificity of its action is supported by both ligand-binding assays and electrophysiological data from cortical slices and cultured neurons. Its ability to inhibit glycine-facilitated NMDA receptor activation without affecting basal glutamate responses highlights its potential as a pharmacological tool for studying NMDA receptor modulation [1].

2) 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 [2].

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 [2].

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 [2].

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 [2].

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 [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] Singh L, Donald AE, Foster AC, Hutson PH, Iversen LL, Iversen SD, Kemp JA, Leeson PD, Marshall GR, Oles RJ, et al. Enantiomers of HA-966 (3-amino-1-hydroxypyrrolid-2-one) exhibit distinct central nervous system effects: (+)-HA-966 is a selective glycine/N-methyl-D-aspartate receptor antagonist, but (-)-HA-966 is a potent gamma-butyrolactone-like sedative. Proc Natl Acad Sci U S A. 1990 Jan;87(1):347-51. doi: 10.1073/pnas.87.1.347. PMID: 2153294; PMCID: PMC53260.

[2] 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

 

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