Comment by Anand Gururjan, Rachel Hill, and Maarten van Den Buuse
Reverse-engineering clinical abnormalities in rodents has been a standard approach to creating models of aspects of psychiatric illness, but with the use of genetic knockouts, we have managed to achieve a level of resolution not previously seen using classical drug-induced, lesion-induced, or neurodevelopmental models. Indeed, the use of knockouts in combination with these techniques would, in theory, provide a more accurate model. However, before such combinations are trialed, we would necessarily need to establish the robustness of the knockout model by itself in terms of its construct and face and predictive validity.

As outlined by Carlisle et al. (2011), a model has been created based on clinical findings that genetic variations in the components of the post-synaptic density fraction (PSD) have been linked to several psychiatric disorders. The PSD machinery plays a very important role in regulating signal strength and also selectivity of signals transmitted to the dendrite. In the context of schizophrenia, the components which are thought to be dysfunctional include DISC1, α-CaMKII, SynGAP, and PSD-95. The authors of this study have investigated the effect of deleting exon 3 of the mouse gene LRRC7, which is the start site for the construction of the PSD scaffolding protein known as densin-180. They then proceeded to characterize the behavioral phenotype of the knockouts, changes in expression of other PSD proteins, the effect on neurotransmission, and the morphology of hippocampal neurons in the knockout mice.

Here, we summarize, highlight, and discuss several findings. In terms of the behavioral phenotype, there are several novel findings which are worth discussing. Densin knockout mice were less active in their home cages, but displayed significant novelty-induced hyperactivity. There were also deficits in two forms of short-term memory, unrelated to changes in locomotor activity. In terms of hippocampus-dependent memory, we wonder whether a more specific test like the Morris water maze would have been more appropriate. Perhaps one of the most important behavioral changes observed in the knockout mice were deficits in prepulse inhibition, but it is unclear as to whether there were any effects on the startle response. If the disruption of PPI in the knockouts was accompanied by changes in the startle response, the interpretation would be entirely different (Csomor et al., 2008). It is also unclear why only male mice were tested for PPI. It is stated that fighting “has been shown to alter the PPI response in mice,” but no references are provided to support this. In experiments in our own laboratory, we often test male mice which had to be isolated because of fighting, and have not found consistent differences with socially housed mice of the same genotype. If this model was to be used to assess antipsychotic potential, it would have probably been important to determine whether drugs (e.g., haloperidol, clozapine) would have any effect on the disruption.

The authors reported a reduction in nesting activity in male knockouts, and even though they relate this to social withdrawal—a key negative symptom of schizophrenia—we wonder whether the use of a social cognition test setup would have been more appropriate. It is unclear why females were not tested. Furthermore, given that nest building is a social activity, for those male mice that were eventually housed individually due to aggressive behaviors, the relevance of this measurement should be questioned. And contrary to the authors discussion point, the use of antipsychotics in fact was reported to disrupt nesting behavior (Li et al., 2004).

There were no differences between knockouts and wild-type mice in terms of their balance and coordination, but knockouts did show increased anxiety in the open field test. Further tests, perhaps using the elevated plus maze, would be required to confirm the anxiogenic effect. Interestingly, even though male and female knockout mice were generated for this study, with the exception of the results for the PPI, nesting, and aggression tests, there was no specific mention as to whether there were any sex-specific differences for the other tests.

In terms of the neuromolecular findings, in the PSD the loss of densin produced a decrease in the level of DISC1 and mGluR5 in the forebrain regions of knockouts, which led the authors to propose the existence of a multiprotein complex of densin, DISC1, and mGluR5. Specifically in the context of schizophrenia, reductions in the expression of DISC1 have been previously reported (Brandon et al., 2009), and there have been rodent models based on mGluR5-hypofunction (Gray et al., 2009). Secondly, there were no effects on CaMKII, SynGAP, or PSD-95 in the PSD. PSD-95 in particular is involved in the trafficking of NMDA receptors (NMDARs). The NMDA receptor hypofunction hypothesis of schizophrenia posits that a reduction in glutamatergic neurotransmission is a key feature and cause of the behavioral phenotype. The deletion of densin has no effect on ionotrophic glutamate receptor expression or localization in the PSD, and, hence, the knockouts would be expected to show normal neurotransmission, and indeed this is what the authors found. So it would be appropriate to conclude that this model does not display the construct validity normally associated with the glutamate hypothesis. Furthermore, from a technical point of view, the analysis of entire forebrain regions of the knockouts may miss out on regionally specific effects of densin deletion which may or may not have been important.

The authors went on to discover that in double knockouts of both densin and NMDARs (GluN1), there was reduced colocalization of CaMKII with PSD-95 in the PSD (~50 percent) compared to NMDAR knockouts alone (~15 percent), even though there was no effect on PSD-95 expression itself. This led the authors to suggest that if either densin or the NMDAR is missing, there is a partial docking of CaMKII together with PSD-95, but if both are missing, there is much less colocalization. The authors then hypothesized that in densin knockouts, CaMKII would bind more to NMDARs as a compensatory response, and, in turn, this would enhance glutamatergic neurotransmission. They found that, while basal CaMKII activity was lower in knockouts, in the presence of the GABAA antagonist bicuculline, phosphorylation of CaMKII was double that observed in wild-types. The authors concluded based on this finding that even though the deletion of densin appears to have no direct impact on the level of expression or localization of CaMKII within the PSD, there are indirect consequences on its localization with respect to other PSD components, and this in turn affects its basal activity and how it responds to neuronal stimuli.

These densin-related changes in PSD architecture and function were thought to result in effects on synaptic plasticity in hippocampal preparations, but this was limited only to LTD. The application of low-frequency stimulation or the application of an mGluR agonist, DHPG, induced no LTD in the knockouts compared to the wild-types. As the authors suggest, the deletion of densin impairs a biochemical step from the point of receptor activation (NMDAR or mGluR5), but to determine whether this was related to the reduced CaMKII microlocalization with PSD-95, the authors could have performed another experiment with the use of the CaMKII inhibitor KN-62, which is known to facilitate DHPG-induced LTD (Schnabel et al., 1999). DHPG is an mGluR1 and mGluR5 receptor agonist, so it could also be suggested that the densin-related decrease in mGluR5 expression in the PSD is not offset by a compensatory increase in mGluR1-receptor activity. It was not reported whether there was any effect of densin deletion on mGluR1 expression in the PSD.

In terms of hippocampal dendritic spine morphology, the loss of densin did not result in any significant changes in overall spine density, but there were significant effects on mushroom spine length (longer) and spine neck diameter (thinner). Bigger, voluminous headed spines are believed to have a greater PSD area, and hence are stronger synaptic contact points. In this study, the average spine head volume as calculated using the diameter seems unaffected by densin loss in all three types (mushroom, stubby, and thin). Strictly speaking, however, the association between spine morphology and psychiatric illness is weak and difficult to interpret given the highly plastic nature of their shape, and that it is often unclear whether changes are due to a primary cause or a downstream effect (Nimchinsky et al., 2002).

Overall, densin-180 knockout mice do show some behavioral changes which resemble some of the positive and negative symptoms of schizophrenia, but future studies would be necessary to confirm the findings using more specific testing paradigms and alternative protocols. To their credit, the authors have done a tremendous amount of work to produce the results, but no attempt was made to make a link between the molecular and behavioral changes. Unfortunately, this "missing link" is what many in the field would describe as a significant limitation of this animal model.

References:

Brandon NJ, Millar JK, Korth C, Sive H, Singh KK, Sawa A (2009) Understanding the role of DISC1 in psychiatric disease and during normal development. J Neurosci 29:12768-12775. Abstract

Carlisle HJ, Luong TN, Medina-Marino A, Schenker L, Khorosheva E, Indersmitten T, Gunapala KM, Steele AD, O'Dell TJ, Patterson PH, Kennedy MB (2011) Deletion of densin-180 results in abnormal behaviors associated with mental illness and reduces mGluR5 and DISC1 in the postsynaptic density fraction. J Neurosci 31:16194-16207. Abstract

Csomor PA, Yee BK, Vollenweider FX, Feldon J, Nicolet T, Quednow BB (2008) On the influence of baseline startle reactivity on the indexation of prepulse inhibition. Behav Neurosci 122:885-900. Abstract

Gray L, van den Buuse M, Scarr E, Dean B, Hannan AJ (2009) Clozapine reverses schizophrenia-related behaviours in the metabotropic glutamate receptor 5 knockout mouse: association with N-methyl-D-aspartic acid receptor up-regulation. Int J Neuropsychopharmacol 12:45-60. Abstract

Li M, Davidson P, Budin R, Kapur S, Fleming AS (2004) Effects of typical and atypical antipsychotic drugs on maternal behavior in postpartum female rats. Schizophr Res 70:69-80. Abstract

Nimchinsky EA, Sabatini BL, Svoboda K (2002) Structure and Function of Dendritic Spines. Annual Review of Physiology 64:313-353. Abstract

Schnabel R, Palmer MJ, Kilpatrick IC, Collingridge GL (1999) A CaMKII inhibitor, KN-62, facilitates DHPG-induced LTD in the CA1 region of the hippocampus. Neuropharmacology 38:605-608. Abstract

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