Recent advances in neuroscience techniques and methods ushered in a new era in the research of neuronal function with unprecedented selectivity and temporal resolution. One of the main ...characteristics of these technical advances is the ability to selectively target and/or modulate specific neuronal subpopulations and circuits in both healthy and diseased brains. Although initially designed as tools to help researchers better understand the mechanisms underlying neuronal activity and complex behaviors, these novel approaches may also accelerate the process of drug discovery in many areas of neuroscience, and some may even potentially serve as novel therapeutic strategies. The application of different electrophysiological techniques is still considered essential in studying ion channel function and pharmacology, as well as network-level changes in brain activity. The cutting-edge methods for investigation of brain function include opto-and chemogenetics in freely behaving animals; both approaches enable highly selective control of neuronal activity using either a light stimulation (optogenetics) or a chemical ligand (chemogenetics) in both loss-and gain-of-function experiments. In this review paper, we aim to summarize recent scientific evidence on the state-of-the-art and provide information on these advances, taking into account both academic and pharmaceutical industry points of view.
Over the last years, genetic studies have greatly improved our knowledge on the receptor subtypes mediating various pharmacological effects of positive allosteric modulators at GABA
A receptors. This ...stimulated the development of new benzodiazepine (BZ)-like ligands, especially those inactive/low-active at GABA
A receptors containing the α
1 subunit, with the aim of generating more selective drugs. Hereby, the affinity and efficacy of four recently synthesized BZ site ligands: SH-053-2′N, SH-053-S-CH3-2′F, SH-053-R-CH3-2′F and JY-XHe-053 were assessed. They were also studied in behavioral tests of spontaneous locomotor activity, elevated plus maze, and water maze in rats, which are considered predictive of, respectively, the sedative, anxiolytic, and amnesic influence of BZs. The novel ligands had moderately low to low affinity and mild to partial agonistic efficacy at GABA
A receptors containing the α
1 subunit, with variable, but more pronounced efficacy at other BZ-sensitive binding sites. While presumably α
1 receptor-mediated sedative effects of GABA
A modulation were not fully eliminated with any of the ligands tested, only SH-053-2′N and SH-053-S-CH3-2′F, both dosed at 30
mg/kg, exerted anxiolytic effects. The lack of clear anxiolytic-like activity of JY-XHe-053, despite its efficacy at α
2- and α
3-GABA
A receptors, may have been partly connected with its preferential affinity at α
5-GABA
A receptors coupled with weak agonist activity at α
1-containing subtypes. The memory impairment in water-maze experiments, generally reported with BZ site agonists, was completely circumvented with all four ligands. The results suggest that a substantial amount of activity at α
1 GABA
A receptors is needed for affecting spatial learning and memory impairments, while much weaker activity at α
1- and α
5-GABA
A receptors is sufficient for eliciting sedation.
Highlights • We assessed physical dependence to diazepam 24 h after treatment withdrawal in rats. • Withdrawal of 2 mg/kg diazepam after 28, but not 14 or 21 days increased anxiety. • ...Pentylenetetrazole-induced seizures were enhanced after 21 and 28 days of treatment. • Daily co-treatment with βCCt affected seizures, but not anxiety during withdrawal. • The role of α1 GABAA receptors in development of physical dependence may vary.
► PWZ-029 is an α5 GABAA selective inverse agonist. ► We tested PWZ-029, on its own and with scopolamine, in two memory paradigms. ► It improved object recognition in the novel object recognition ...test. ► PWZ-029 was ineffective in the Morris water maze test. ► Testing of putative procognitive compounds needs use of a variety of models.
Inverse agonism at the benzodiazepine site of α5 subunit-containing GABAA receptors is an attractive approach for the development of putative cognition-enhancing compounds, which are still far from clinical application. Several ligands with binding and/or functional selectivity for α5 GABAA receptors have been synthesized and tested in a few animal models. PWZ-029 is an α5 GABAA selective inverse agonist whose memory enhancing effects were demonstrated in the passive avoidance task in rats and in Pavlovian fear conditioning in mice. In the present study we investigated the effects of PWZ-029 administration in novel object recognition test and Morris water maze, in normal and scopolamine-treated rats. All the three doses of PWZ-029 (2, 5 and 10mg/kg) improved object recognition after the 24-h delay period, as shown by significant differences between the exploration times of the novel and old object, and the respective discrimination indices. PWZ-029 (2mg/kg) also successfully reversed the 0.3mg/kg scopolamine-induced deficit in recognition memory after the 1-h delay. In the Morris water maze test, PWZ-029 (5, 10 and 15mg/kg) did not significantly influence swim patterns, either during five acquisition days or during the treatment-free probe trial. PWZ-029 (2, 5 and 10mg/kg) also proved to be ineffective in the reversal of the 1mg/kg scopolamine-induced memory impairment in the water maze. The present mixed results encourage use of a variety of tests and experimental conditions in order to increase the predictability of preclinical testing of selective α5 GABAA inverse agonists.
Abstract Enormous progress in understanding the role of four populations of benzodiazepine-sensitive GABAA receptors was paralleled by the puzzling findings suggesting that substantial separation of ...behavioral effects may be accomplished by apparently non-selective modulators. We report on SH-I-048A, a newly synthesized chiral positive modulator of GABAA receptors characterized by exceptional subnanomolar affinity, high efficacy and non-selectivity. Its influence on behavior was assessed in Wistar rats and contrasted to that obtained with 2 mg/kg diazepam. SH-I-048A reached micromolar concentrations in brain tissue, while the unbound fraction in brain homogenate was around 1.5%. The approximated electrophysiological responses, which estimated free concentrations of SH-I-048A or diazepam are able to elicit, suggested a similarity between the 10 mg/kg dose of the novel ligand and 2 mg/kg diazepam; however, SH-I-048A was relatively more active at α1 - and α5 -containing GABAA receptors. Behaviorally, SH-I-048A induced sedative, muscle relaxant and ataxic effects, reversed mechanical hyperalgesia 24 h after injury, while it was devoid of clear anxiolytic actions and did not affect water-maze performance. While lack of clear anxiolytic actions may be connected with an enhanced potentiation at α1 -containing GABAA receptors, the observed behavior in the rotarod, water maze and peripheral nerve injury tests was possibly affected by its prominent action at receptors containing the α5 subunit. The current results encourage further innovative approaches aimed at linking in vitro and in vivo data in order to help define fine-tuning mechanisms at four sensitive receptor populations that underlie subtle differences in behavioral profiles of benzodiazepine site ligands.
Abstract Despite significant advances in understanding the role of benzodiazepine (BZ)-sensitive populations of GABAA receptors, containing the α1 , α2 , α3 or α5 subunit, factual substrates of ...BZ-induced learning and memory deficits are not yet fully elucidated. It was shown that α1 -subunit affinity-selective antagonist β -CCt almost completely abolished spatial learning deficits induced by diazepam (DZP) in the Morris water maze. We examined a novel, highly (105 fold) α1 -subunit selective ligand—WYS8 (0.2, 1 and 10 mg/kg), on its own and in combination with the non-selective agonist DZP (2 mg/kg) or β -CCt (5 mg/kg) in the water maze in rats. The in vitro efficacy study revealed that WYS8 acts as α1 -subtype selective weak partial positive modulator (40% potentiation at 100 nM). Measurement of concentrations of WYS8 and DZP in rat serum and brain tissues suggested that they did not substantially cross-influence the respective disposition. In the water maze, DZP impaired spatial learning (acquisition trials) and memory (probe trial). WYS8 caused no effect per se , did not affect the overall influence of DZP on the water-maze performance and was devoid of any activity in this task when combined with β -CCt. Nonetheless, an additional analysis of the latency to reach the platform and the total distance swam suggested that WYS8 addition attenuated the run-down of the spatial impairment induced by DZP at the end of acquisition trials. These results demonstrate a clear difference in the influence of an α1 subtype-selective antagonist and a partial agonist on the effects of DZP on the water-maze acquisition.
The clinical use of benzodiazepines (BZs) is hampered by sedation and cognitive deterioration. Although genetic and pharmacological studies suggest that α1- and α5-containing GABAA receptors mediate ...and/or modulate these effects, their molecular substrate is not fully elucidated. By the use of two selective ligands: the α1-subunit affinity-selective antagonist β-CCt, and the α5-subunit affinity- and efficacy-selective antagonist XLi093, we examined the mechanisms of behavioural effects of diazepam in the tests of spontaneous locomotor activity and water-maze acquisition and recall, the two paradigms indicative of sedative- and cognition-impairing effects of BZs, respectively. The locomotor-activity decreasing propensity of diazepam (significant at 1.5 and 5 mg/kg) was antagonized by β-CCt (5 and 15 mg/kg), while it tended to be potentiated by XLi093 in doses of 10 mg/kg, and especially 20 mg/kg. Diazepam decreased acquisition and recall in the water maze, with a minimum effective dose of 1.5 mg/kg. Both antagonists reversed the thigmotaxis induced by 2 mg/kg diazepam throughout the test, suggesting that both GABAA receptor subtypes participate in BZ effects on the procedural component of the task. Diazepam-induced impairment in the declarative component of the task, as assessed by path efficiency, the latency and distance before finding the platform across acquisition trials, and also by the spatial parameters in the probe trial, was partially prevented by both, 15 mg/kg β-CCt and 10 mg/kg XLi093. Combining a BZ with β-CCt results in the near to control level of performance of a cognitive task, without sedation, and may be worth testing on human subjects.
Display omitted
•Effects of LPS applied in late embryogenesis were tested at postnatal days 40 and 60.•Male rats exhibited spatial learning and memory impairments only in early adulthood.•Female rats ...displayed decreased reactivity to amphetamine only in early adulthood.•Gender differences may mimic cognitive dysfunction and deficit symptoms, respectively.
Numerous basic and epidemiological studies have connected prenatal maternal immune activation with the occurrence of schizophrenia and/or autism. Depending on subtle differences in protocols of the used animal model, a variety of behavioral abnormalities has been reported. This study investigated behavioral differences in Wistar rat offspring of both genders, exposed to the 100μg/kg per day dose of lipopolysaccharide (LPS) in late embryogenesis (embryonic days 15 and 16), while tested at their adolescent and young adult age (postnatal days 40 and 60, respectively). Immune activation was confirmed by detecting high levels of TNF-α and IL-6 in dam blood withdrawn 2h after the first dose of LPS. The animals were assessed in three consecutive trials of locomotor activity (novelty exploration, response to i.p. saline injection and challenge with 0.5mg/kg amphetamine), Morris water maze and social interaction tests. Overt behavioral dysfunction was perceived in adult rats only, and these changes were gender-distinctive. When compared with control rats, LPS females displayed baseline hypolocomotion and a decreased reactivity to amphetamine, while LPS males exhibited spatial learning (acquisition trials) and memory (probe trial) impairments. Prenatal treatment did not affect the time spent in social interaction. As maternal exposure to LPS in late gestation resulted in behavioral changes in offspring in early adulthood, it may model schizophrenia-like, but not autism-like endophenotypes. However, lack of a potentiated response to amphetamine testified that this model could not mimic positive symptoms, but rather certain traits of cognitive dysfunction and deficit symptoms, in males and females, respectively.
Synthesis of ligands inactive or with low activity at α₁ GABA(A) receptors has become the key concept for development of novel, more tolerable benzodiazepine (BZ)-like drugs. WYS8, a remarkably (105 ...times) α₁-subtype selective partial positive modulator, may serve as a pharmacological tool for refining the role of α₁ GABA(A) receptors in mediation of BZs' effects.
Here, the effects of WYS8 on GABA-induced currents and on diazepam-induced potentiation of recombinant BZ-sensitive GABA(A) receptors were studied in more detail. In addition, the behavioral profile of WYS8 (0.2, 1, and 10 mg/kg i.p.), on its own and in combination with diazepam, was tested in the spontaneous locomotor activity, elevated plus maze, grip strength, rotarod, and pentylenetetrazole tests.
WYS8, applied at an in vivo attainable concentration of 100 nM, reduced the stimulation of GABA currents by 1 μM diazepam by 57 % at α₁β₃γ₂, but not at α₂β₃γ₂, α₃β₃γ₂, or α₅β₃γ₂ GABA(A) receptors. The administration of WYS8 alone induced negligible behavioral consequences. When combined with diazepam, WYS8 caused a reduction in sedation, muscle relaxation, and anticonvulsant activity, as compared with this BZ alone, whereas ataxia was preserved, and the anxiolytic effect of 2 mg/kg diazepam was unmasked.
Hence, a partial instead of full activation at α₁ GABA(A) receptors did not necessarily result in the attenuation of the effects assumed to be mediated by activation of these receptors, or in the full preservation of the effects mediated by activation of other GABA(A) receptors. Thus, the role of α₁ GABA(A) receptors appears more complex than that proposed by genetic studies.
► Benzodiazepines cause anterograde rather than retrograde amnesia. ► We studied the effects of midazolam on the water maze behavior in rats. ► It impaired acquisition and retention, but not ...consolidation of spatial learning. ► Midazolam administered before the probe test impaired retrieval of reference memory. ► There is a possibility of retrograde amnesia when midazolam is clinically used.
Amnesia is one of the most discussed properties of the benzodiazepine class of drugs. The effects of benzodiazepines on human memory are usually anterograde, while changes in retrograde memory functions were seldom reported. Such inconsistent findings have prompted numerous animal studies investigating the influences of these positive modulators of inhibitory neurotransmission on different stages of memory. Among the benzodiazepines, memory effects of midazolam are of special interest due to its many and varied clinical applications. The present Morris water maze study in adult male Wistar rats was performed in three experiments in which midazolam was administered at doses of 0.5, 1 and 2mg/kg intraperitoneally, before or immediately after each of five daily learning sessions, with two trials in a session, as well as before the probe test. Midazolam impaired acquisition and subsequent retention of spatial learning of the position of the hidden platform even at a pre-training dose of 0.5mg/kg. This low dose was not associated with impairment of the procedural component of learning, manifested by increased time spent in the periphery of the pool. The lack of midazolam effect on consolidation has not been confounded by the observed below-chance performance of the control group since our additional experiment using diazepam also administered immediately after each of five learning sessions has revealed a similar pattern of results. Finally, midazolam administered before the probe test impaired retrieval of reference memory at all tested doses. Hence, induction of retrograde, besides anterograde amnesia should be kept in mind as a possibility when midazolam is used in clinical settings.
PDF
