The major findings from this study in male C57BL/6J mice were: (1) acute treatment with TPA-023 transiently restored episodic memory and executive functioning deficits in scPCP-treated mice; (2) TPA-023 treatment administered prior to each of the 14 injections of PCP significantly prevented scPCP-induced NOR deficit for up to 5 weeks and showed prolonged reversal of scPCP-induced NOR deficit for 1 week; (3) co-administration of SED TPA-023 and SED lurasidone acutely restored NOR in scPCP-treated mice; (4) TPA-023 at two doses and two different time points diminished PCP-induced increase in LMA between 45 and 120 min time intervals; (5) TPA-023 inhibited the increases in cortical ACh, DA, and glutamate as well as the dSTR increases in DA and Glu produced by lurasidone; and (6) TPA-023 inhibited PCP-induced cortical and striatal DA, 5-HT, NE, and Glu efflux.

Acute rescue of NOR by TPA-023

NOR task is dependent upon synaptic plasticity, which includes long-term potentiation (LTP) and successful integrated activity of a variety of GABAergic interneurons and principal neurons in the HIP, PFC, and other brain regions [33, 34]. We observed significant rescue of scPCP-induced LTP impairment by acute TPA-023 treatment (unpublished). Acute treatment with TPA-023 was effective to rescue NOR in scPCP-treated mice. However, this lasted <24 h. The extent of the recovery and the time course matched with that of a single dose of a variety of AAPDs and contrast with the negative effects of haloperidol in this model. The effect of TPA-023 is also identical to the combined effect of 5-HT 2A R inverse agonism, 5-HT 1A R partial agonism, and a weak D 2 R blockade [10, 19, 35, 36]. The rapidity with which TPA-023 and lurasidone restore NOR suggests that all the molecular components needed for the synaptic changes crucial for learning and memory are present in the scPCP-treated mice and that neither new protein synthesis nor neurogenesis is needed. Thus the effects of the PCP regimen are to disrupt the cascades of molecular events that are required for LTP and other cognitive mechanisms and perhaps what TPA-023 and AAPDs do is to inhibit or remove the molecular changes that cause PCP to disrupt NOR when given acutely and are present in some form after washout of PCP following scPCP treatment. This effect of TPA-023 is consistent with a previous report that gaboxadol, a GABA A R agonist, restores NOR in scPCP-treated rats [28]. We have also found that gaboxadol rescues NOR in scPCP-treated male C57BL/6J mice (Rajagopal et al. unpublished data). PregS, an endogenous neurosteroid, which negatively modulates GABA A Rs, has a similar effect suggesting that some specific components of GABA A R signaling are involved in each case.

Combination of SED TPA-023 and Lur rescues NOR

SED of TPA-023 (0.03 mg/kg) in combination with SED lurasidone (0.1 mg/kg) reversed scPCP-induced NOR deficits. We used lurasidone (Fig. 2) as this AAPD, with multi-receptor mechanism, has shown significant efficacy in several domains of cognition, negative symptoms, as well as positive symptoms without any undue side effect profiles in our preclinical studies ([23]. Our initial goal is to investigate the transient effect of interactions between TPA-023 and lurasidone. The long-term effects of the combination treatment of TPA-023 and Lur in the future is indicated.

This suggests that TPA-023 may be a valuable adjunctive treatment for CIAS in combination with Lur or other AAPDs with similar pharmacology, e.g., olanzapine and risperidone. The effect of Lur itself to rescue NOR in the scPCP-treated mice is blocked by pretreatment with the GABA A R antagonist, gabazine, which indicates that stimulation of GABA A R by gaboxadol is required for the transient rescue of Lur (Meltzer et al., unpublished). This supports the conclusion that the efficacy of the combination of PregS and Lur to rescue NOR is based on stimulation of GABA A Rs. Whether TPA-023 would be effective to treat CIAS in patients receiving effective doses of AAPDs for treatment of psychosis, or perhaps only at SEDs, remains to be determined.

Prevention of scPCP-induced deficit in NOR by combined pretreatment with scTPA-023

Administration of Lur prior to each injection of PCP has previously been shown to block the effect of PCP to produce a persistent deficit in NOR, but for only 14 days, with re-emergence of the NOR deficit between day 14 and day 21 [23, 36]. TPA-023 was also able to prevent scPCP from impairing NOR that persisted 5 weeks, with the deficit in NOR partially present at week 6, indicating a step-wise decrease in the rescue mechanism. The length of the prevention of the deficit in NOR was 3 weeks longer than that of Lur alone, indicating that TPA-023 may have particular benefit for prevention of adverse effects of PCP. However, higher doses of Lur need to be studied to affirm this conclusion. When used in a regimen comparable to that of Lur, risperidone (0.5 mg/kg, i.p.), another AAPD, did not show a protective effect for the prevention of the deficit in NOR [37]. The prevention of the PCP-induced deficit for a prolonged period suggests that GABA A R agonism may be able to prevent cumulative effects of PCP, perhaps on gene expression, that interfere with learning and memory. These effects persist long after both PCP and TPA-023 are no longer present in the brain. It is conceivable that neurogenesis or restoration of connectivity between different brain areas contributes to this recovery but that without additional treatment with TPA-023 these processes cease to occur or be effective. We tested higher doses in order to have higher concentrations of TPA-023 present throughout the period in which PCP might produce enduring damage to the process required for learning and memory. It is of particular interest that treatment of rats with PCP, 2.5 mg/kg daily for 5 days, reduced cortical PV mRNA levels and expression of PV mRNA and a subunit of the Kv3.1 voltage-gated potassium channel, which is believed to confer fast spiking properties on PV interneurons [38]. These changes were reversed by chronic clozapine treatment (time not specified). The authors suggest that PCP may have caused cell death but that seems unlikely in light of the ability of clozapine in that study or Lur or TPA-023, in our studies, to reverse PCP-induced NOR deficit. Interestingly, we have found the most robust genetic predictor of improvement in psychopathology by Lur in acutely psychotic schizophrenic patients was a polymorphism in the potassium two-pore channel gene, KCNK9 (Li et al., in press).

Persistent rescue of the scPCP-induced deficit in NOR by post-PCP administration of TPA-023

TPA-023 given daily for 7 days to scPCP mice with a persistent deficit in NOR was able to produce a prolonged rescue of NOR but only for 1 week. This contrasts with the brief rescue (24 h) produced by a single dose of TPA-023 (Fig. 1a–c) and the prolonged prevention of the deficit in NOR produced by TPA-023 when given with PCP. During the time when TPA-023 restored NOR, the synaptic events needed for encoding, consolidation, and retrieval of novel object memory were able to function at least equal to that of the normal mice (Huang et al., unpublished). However, the normal mice and mice given TPA-023 may not have performed these activities in the same manner. We have measured neurotransmitter release during NOR in the TPA-023-rescued, Lur-rescued, and normal mice and noted significant differences in the release of DA, ACh, Glu, and GABA in the HIP and PFC regions in these groups (Huang et al, unpublished).

Novel mechanisms that enable NOR may be the consequence of these post-PCP treatments. The return of the NOR deficit in the TPA-023 mice at the end of the second week suggests that PCP-initiated disruptive genomic processes were suppressed but not totally eliminated by scTPA-023 treatment (Li et al., unpublished). Identification of those re-emerging abnormalities in synaptic plasticity could provide information about the development of CIAS and the mechanism of action of TPA-023 and AAPDs and future studies is indicated.

PCP and MK-801, both non-competitive NMDAR antagonists, prevent the induction of LTP in the dentate gyrus and hippocampal CA1 regions [34, 39, 40]. Loss of PV interneurons or markers thereof has been reported in scPCP-treated rodents [37, 38, 41,42,43] and in individuals with schizophrenia [44]. Other possibilities for the disruption of cognition in the NMDAR antagonists-treated rodents other than abnormalities in parvalbumin-positive interneurons should be considered, including, but not restricted to elevated density of cFOS positive interneurons in the hippocampus after repeated NMDAR antagonist treatment [45]. Most recently, we observed that the excitatory synaptic transmission in scPCP-treated animals in CA1 pyramidal neurons needed for LTP induction was more strongly inhibited by excessive GABAergic activity vs control mice, as indicated by significant enhancement in the amplitude of GABA inhibitory postsynaptic currents in hippocampal slices from scPCP-treated mice vs controls [34]. We also observed a strong trend of reversal of scPCP-induced LTP impairment in the scPCP mice given acute TPA-023 (unpublished). These results suggest a novel and previously unreported elevation in GABA signaling in the HIP that could contribute to the cognitive dysfunction in NMDAR antagonist-treated rodents. Because TPA-023 is a subtype-selective partial agonist at α 2,3 and an α 1/5 antagonist, further study is needed to determine which of these effects, or perhaps other unknown but related actions, is responsible for its effects in the scPCP-treated mice.

Effect of TPA-023 on LMA

Acute administration of PCP to rodents increases LMA, stereotypy, and ataxia [46]. The increase in LMA and stereotypy are thought to mimic psychosis in schizophrenia [47]. By itself, at the doses tested, TPA-023, 0.3 mg/kg, enhanced the LMA produced by PCP 10 mg/kg for the first 60 min after administration, while 0.1 mg/kg showed enhanced LMA between 15 and 45 min. However, 1.0 mg/kg significantly reduced LMA during the initial 30 min, after which it was comparable to the PCP group (with no enhanced LMA). Pretreatment with TPA-023, 0.3 and 1 mg/kg, produced partial and complete inhibition of PCP- induced LMA between 90 and 120 min (Fig. 4). The increased LMA produced by PCP has been suggested to be the result of 5-HT or DA release, or both, in the striatum [20]. Acute administration of PCP, 10 mg/kg, to mice significantly increases efflux of cortical and dSTR ACh, DA, 5-HT, NE, and Glu (Fig. 5; [48]. These increases are partially inhibited by the GABA A R agonist gaboxadol and TPA-023 (Meltzer and Huang, unpublished). As will be discussed, TPA-023 alone significantly inhibited PCP-induced neurotransmitter efflux.

Effect of TPA-023 on Lur- and PCP-induced neurotransmitter efflux in mPFC and dSTR

Neurotransmitter release and cognitive function

We, and others, have previously suggested that release of DA, ACh, and Glu in mPFC, HIP, or dSTR by AAPDs could contribute to their ability to improve NOR and RL in scPCP-treated mice [49, 50]. TPA-023, by itself, did not alter the release of neurotransmitters in the mPFC, although it might do so during the course of the NOR task. We did not detect any effect of Lur on basal release of DA, NE, ACh, Glu, GABA, or 5-HT in normal or scPCP-treated mice in this study (Huang et al. unpublished data). However, TPA-023, 0.5 mg/kg, 30 min prior to Lur, 1.0 mg/kg, inhibited the release of DA, ACh, and Glu in the mPFC and DA and Glu in dSTR. It had no effect on the efflux of 5-HT or 5-HIAA in either region. TPA-023 completely eliminated the release of Glu in both regions but only partially blocked the release of DA and ACh. This indicates that Glu release in these two regions is not necessary for the recovery of NOR. Some increase in DA release in the mPFC and dSTR and ACh release in the mPFC may be necessary for the recovery of NOR. The doses of TPA-023 and Lur used in microdialysis are close to the doses used in the ORL but much higher than those used in the NOR tests. We did not test the lower dose of TPA-023 in microdialysis since TPA-023, 0.5 mg/kg had no effect on basal NT efflux. At the lower doses used in NOR, it is possible that TPA-023, could induce other effects, e.g., release of neurotrophins such as BDNF or neuregulin, which could, in part, rescue scPCP-induced NOR deficit.

Neurotransmitter release and LMA