α7 nAChR agonists decrease VTA DA population activity in MAM rats via the vHipp

In our work, midbrain DA neurotransmission was affected by α7 nAChR agonists and type II PAM by impacting the proportion of spontaneously active DA neurons in the VTA. Our data clearly showed that α7 nAChR agonists PNU282987 and SSR180711 can counteract the hyper-DA state of MAM rats with their likely site of action being the vHipp. This profile is consistent with the potential use of α7 nAChR agonists for schizophrenia treatment not only focusing on cognitive dysfunction, but also as a potential treatment for positive symptoms; and fits with the current search for drugs able to control DA function acting in structures upstream in the brain circuit. Dopamine dysfunction is a core feature in schizophrenia. Neuroimaging studies of schizophrenia patients showed the greatest increase in [18F]-DOPA uptake [28] and amphetamine-induced DA release [29] in the AST that correlates with exacerbation of psychosis. In humans, the dorsal tier of the substantia nigra projects to the AST; this is functionally equivalent to the lateral VTA projection to the core/lateral associative striatum in the rodent [30, 31]. In the MAM model, the most significant increase in DA neuron population activity is in the lateral VTA, which is different from the change in population activity in the limbic medial VTA associated with amphetamine sensitization [23] and depression [32]. Therefore, the ability of the α7 nAChR agonists to normalize activity in the lateral VTA of the MAM model would be consistent with an antipsychotic effect. This selectivity also suggests that α7 nAChR agonists may have less impact on aggravating negative symptoms of schizophrenia in comparison to antipsychotic drugs.

Our data showed that local infusion of the α7 nAChR agonists in the vHipp reduced VTA DA neuron population activity in MAM rats, showing that activation/desensitization of α7 nAChR in this structure is sufficient to normalize VTA DA neuron activity in MAM rats. The rat hippocampus has a complex pattern of α7 nAChR expression; however, the majority of receptors are located in GABAergic interneurons [33, 34]. Activation of these receptors induces a long-lasting GABA release, increasing the frequency of inhibitory post-synaptic currents in pyramidal neurons [34, 35]. In this way, it is possible that potentiation of the activity of vHipp GABAergic interneurons could attenuate the hyperactivity of pyramidal neurons and, consequently, underlie the effects induced by the α7 nAChR agonists in MAM rats. In contrast, the lack of effect in controls is consistent with the fact that attenuation of vHipp activity in normal animals does not impact VTA DA neuron population activity [20, 36]. Due to its widespread localization, α7 nAChR can potentiate both inhibitory and excitatory synapses in the hippocampus and, consequently, the net effect of drug administration can be dependent on the regional excitatory/inhibitory balance. Since there is not a disruption of the hippocampal excitatory/inhibitory balance in control animals, α7 nAChR agonist-induced modulation of hippocampal excitability may not be sufficient to trigger changes in VTA DA function through this pathway. Thus, the hippocampal hyperactivity likely makes MAM rats more sensitive to the effects of α7 nAChR agonists. Moreover, confirmation that α7 nAChR agonists can control hippocampal hyperactivity in MAM rats may further support their potential efficacy on cognitive disturbances.

However, changes in the cholinergic tone may also play a role in this differential sensitivity. Although there is little data regarding alterations in cholinergic transmission in the MAM GD17 model, increases in acetylcholine levels and ChAT activity were detected in the brain of MAM GD16.5 [37] and GD15 exposed rats [38] and a higher number of ChAT immunopositive cells in the medial septum was reported after GD12 exposure [39]. These data are consistent with the increase in acetylcholine and choline levels reported in schizophrenia patients [1]. While data regarding possible changes in the expression of α7 nAChR in the MAM model are absent, these alterations may impact dose dependency of the drugs without significantly impacting potential therapeutic actions.

α7 nAChR modulators increase VTA DA population activity in control rats: involvement of the BLA

In normal rats, we showed that only the α7 nAChR PAM II PNU120596 increased the number of spontaneously active DA neurons after systemic administration. The absence of effect of PNU282987 and NS1738 on DA population activity is consistent with previous DA microdialysis data, in which systemic administration of these drugs did not impact dopamine overflow in rats NAcc [40]. In contrast, infusion of α7 nAChR agonists in the BLA increased the number of active DA neurons in the VTA. The predominant effect of BLA nAChR on more medial/central regions of the VTA is in agreement with previous studies describing the increased cFos expression in the NAcc shell, but not in the NAcc core or in the dorsal striatum after systemic administration of both SSR180711 and PNU282987 [41, 42]. This profile is also consistent with previous data reporting BLA-mediated stress effects on VTA DA population activity [43], which would be consistent with a greater impact on affect or reward DA systems rather than psychosis [44].

It was recently proposed that the primary role of BLA α7 nAChR is to enhance inhibitory neurotransmission through activation of somatodendritic receptors on GABAergic interneurons [45]. Pharmacological activation of the BLA decreases the number of VTA DA neurons spontaneously firing; however, local infusion of TTX did not change the number of active VTA DA neurons [26]. Thus, a general inhibition of BLA activity induced by α7 nAChR agonists cannot explain the observed results. It is possible that α7 nAChR agonists may be acting on specific subsets of BLA neurons to activate VTA DA neurons. Such a selective effect may also account for the lack of change in VTA DA neuron activity observed in MAM rats. Since the BLA projects to the vHipp [46], it is possible that this BLA-vHipp projection participates in the observed effect, and that the already-present vHipp hyperactivation in the MAM rats obscures any additional activation by BLA afferents. Whether the α7 nAChR can exert this projection-specific activation require further investigation.

Final considerations

In several cases, the α7 nAChR ligands impacted DA neuron firing rate and burst firing. While most of the changes were small in magnitude and may not represent a biologically significant effect, they support a differential α7 nAChR modulation of VTA DA neurons between MAM and control rats: increases in firing frequency and burst activity were mostly shown in control rats, whereas decreases in the same parameters were generally seen in MAM rats. Indeed, our data show that drug effects can vary according to the basal level of activity of specific brain circuits and highlights the importance of using appropriated animal models to make inferences about potential therapeutic use of new neuropsychiatric drug candidates.

We did not find any distinction with respect to functional impact across the drugs tested that could be specifically attributed to differences in their molecular mechanisms of action. Similar results have been reported by others after single drug administration [40, 47, 48]. Thus, it is likely that differences in the pharmacological profile among distinct α7 nAChR ligands will only be disclosed after repeated treatment. Although little tolerance has been seen regarding the procognitive effects of α7 nAChR ligands [42], the maintenance of the effect over DA transmission after repeated exposure has yet to be demonstrated and can elucidate further differences in their pharmacological profile.

Currently, few studies investigated the effects of α7 nAChR modulators in validated neurodevelopmental models of schizophrenia, and all of them focused on cognitive outcomes such as sensory processing, executive function, attention and memory [10, 18]. Beside ours, only two other studies employed the MAM model [49, 50] presenting conflicting results and with neither focusing on the DA system. While opposite effects of drug infusion into the vHipp versus the BLA were observed, our findings with systemic administration showed that, when the brain circuit is disrupted, the action of the α7 nAChR agonists in the vHipp appear to predominate. Finally, the apparent lack of effect of the type I PAM NS1738 in our experimental conditions should be interpreted with caution. Among the tested drugs, NS1738 has the shortest elimination half-life (42 min) in rats [16] and its fast elimination may contribute to data variability.

In summary, α7 nAChR agonists and PAMs impact the DA system of rats in a state-dependent manner. The PAM II PNU120596 increased DA activity in normal rats whereas α7 nAChR agonists reversed the increase in DA neuron activity in the MAM model, and this effect appears to be mediated by their action in the vHipp. Moreover, local BLA infusion of the same agonists increases VTA DA activity in normal rats. Taking together, these data point to α7 nAChR agonism as a promising strategy for the development of new pharmacological therapies for schizophrenia.