transporter .

Confocal microscopy of the rat hippocampus (Figure 26.1; Colorplate 12) also indicates that in general, Cx I is colocalized with the vesicular GABA transporter, while Cx II colocalizes with the vesicular glutamate transporter.

Figure 26.1. Confocal Microscopy of Presynaptic Proteins in Rat Hippocampus. All images from the granule cell layer with the exception of the lower left panel obtained from the mossy fiber terminal zone. These images largely support the colocalization of complexin I with inhibitory terminals and complexin II with excitatory terminals. However, in other subfields of the hippocampus this relationship was not as consistently observed. Abbreviations: Cx1 and Cx2, complexins 1 and 2; vGLUT, vesicular glutamate transporter 1; vGAT, vesicular GABA transporter. See Colorplate 12.

Figure 26.1. Confocal Microscopy of Presynaptic Proteins in Rat Hippocampus. All images from the granule cell layer with the exception of the lower left panel obtained from the mossy fiber terminal zone. These images largely support the colocalization of complexin I with inhibitory terminals and complexin II with excitatory terminals. However, in other subfields of the hippocampus this relationship was not as consistently observed. Abbreviations: Cx1 and Cx2, complexins 1 and 2; vGLUT, vesicular glutamate transporter 1; vGAT, vesicular GABA transporter. See Colorplate 12.

4.3. Physiological Functions of Complexins

At present, the physiological functions of Cxs are not well understood. While the presumed role of Cxs is to regulate neurotransmission, there is a surprising amount of conflicting evidence as to whether they are facilitatory or inhibitory to neurotransmission. One earlier study reported that injection of a peptide from the central, syntaxin-binding domain of Cxs into squid giant presynaptic terminals inhibited neurotransmitter release at a late stage of synaptic vesicle exocytosis23. Consistent with these findings, injection of anti-complexin II antibody into mature presynaptic neurons of Aplysia buccal ganglia caused a stimulation of neurotransmitter release, suggesting that Cx II is inhibitory to neurotransmitter release. This hypothesis was further supported by evidence that injection of recombinant Cx II caused depression of neurotransmitter release from nerve terminals24. However, these studies would appear to be in contrast to the data from other research groups, who have shown that there is a reduced Ca2+ sensitivity of neurotransmitter release in mutant genetic mice lacking both Cxs I and II, although evoked EPSC amplitudes and spontaneous neurotransmitter release were normal in single knockouts25. Furthermore, Cx II mice display frequency-dependent deficits in long-term potentiation (LTP) of the hippocampus (see below), suggestive of a facilitatory role for this isoform. Insight into these contrasting sets of data may be provided by Morton and colleagues, who have demonstrated that PC12 cells overexpressing mutant huntingtin protein exhibit decreased levels of Cx II and concomitant decreases in neurotransmitter release. Increased expression of Cx II in PC 12 cells that co-expressed the mutant huntingtin protein partially rescued the phenotype and increased neurotransmission, whereas increased Cx II expression in wild-type PC12 cells decreased neurotransmission26. These combined data suggest that Cx II may be required for neurotransmission when at optimal cytosolic concentrations, but along an inverted-U shaped gradient, so that decreases or increases in levels serve to inhibit neurotransmission. Substantially less research has been undertaken to define the physiological role of Cx I in regulation of neurotransmission.

Some degree of insight into the physiological role of Cxs has been provided by the generation of mice that have been genetically engineered for a deletion of the two isoforms. While the combined loss of both Cxs is perinatally lethal25, the individual loss of either Cx results in viable offspring that can survive to puberty. Complexin I knockout mice display a more striking behavioral phenotype than Cx II mutant mice. In the first mention of the Cx I knockout animals, it was reported that these mutants exhibit a strong ataxia, were unable to reproduce, experience sporadic seizures, and die within 2-4 months after birth25. A more recent study, in which the behavioral phenotype of these mice was characterized extensively, revealed that Cx I knockout mice can survive into full adulthood if fed under special conditions. These mice display pronounced motor deficits, ataxia, reduced neuromuscular strength, and a resting tremor. In addition, there are also deficits in complex behaviors and increased reactivity in affective-related tasks27. By contrast, Cx II knockout mice display a relatively subtle behavioral phenotype. At present, there are two separate lines of Cx II mutant mice, both of which display seemingly normal development through to puberty25,28. However, behavioral and physiological deficits become more pronounced with age. The "Kochi" line of mice exhibit normal reproductive behavior, although hippocampal neurons from these mice display plasticity-associated deficits, indicated by a loss of LTP by high-frequency stimulation, recorded in the hippocampal CA1 area, whereas ordinary neural transmission remains normal28. In the most complete behavioral characterization of Cx II knockout mice by Morton and colleagues, a complex pattern of deficits was observed that were best described as relating to "higher function"29. These included age-dependent loss of social interaction, self-care, investigative activity and increased cognitive impairment, especially on spatially mediated tasks and procedures that required reversal learning. This discrete loss of complex behavior, in the absence of overt motor impairments, bears an interesting parallel to human neuropsychiatric disorders, such as schizophrenia and Huntington's disease.

We have recently demonstrated that Cxs are associated with cognitive-related synaptic plasticity in rodents. To confirm that Cxs in the hippocampus and frontal cortex were relevant to cognition, we conducted a series of experiments in rats to examine plasticity-associated changes in Cxs during learning and memory tasks. Animals were trained on either working or reference memory tasks and regional levels of Cxs were measured with quantitative immunohistochemistry. In the hippocampus, we observed that learning increased levels of Cx II and Cx II:I ratio throughout most of the hippocampus22. These findings imply that increases in Cx II and a relative increase in excitatory/inhibitory terminals in hippocampus may be important for working and reference memory.

4.4. Preclinical Data on Complexins

Converging data from preclinical animal studies implicate Cxs as molecules of relevance to neuropsychiatric disorders. We and others have shown previously that neurodevelopmental rodent models of schizophrenia, similar to the disorder itself, may be characterized by regional alterations in presynaptic proteins30. In a neurodevelopmental model of schizophrenia based on exposure to variable prenatal stress, it was noted that this manipulation decreased levels of Cx I in the frontal pole31, although cognition was not assessed in these animals. We have recently observed decreased levels of both Cxs in the frontal cortex of rats in a model of prenatal ethanol exposure32. In this paradigm, in utero exposure to high blood alcohol levels of pregnant dams that voluntarily consume ethanol results in a wide range of neurohormonal and cognitive deficits in the offspring. By measuring levels of presynaptic proteins in the frontal cortex and hippocampus, where we had previously observed cognitive-related plastic changes in Cxs, we noted a selective loss of both Cx I and II in the frontal cortex. There was no effect of prenatal ethanol exposure on Cxs in the hippocampus, and decreased levels of Cxs in the frontal cortex were protein specific, as there was no effect of ethanol exposure on the presynaptic protein synaptophysin, which provides a global marker of presynaptic terminal density. The absence of changes in levels of hippocampal Cxs is somewhat surprising, given the well-characterized memory deficits in this paradigm and previous research associating Cxs with memory tasks. However, a possible explanation for the failure to observe decreases in hippocampal complexins is that while basal levels of the proteins remained unaltered, prenatal ethanol exposed rats may display specific plasticity-associated deficits, which would only be apparent after extended training in cognitive paradigms.

Interestingly, Cx II has also been implicated in Huntington's disease. As mentioned above, expression of mutant huntingtin protein - whose polyglutamine expansion within the N-terminus is a contributing factor to the disease - in PC12 cells decreases levels of Cx II and reduces exocytosis. Overexpression of Cx II in these cells reverses the decreases in neurotransmitter release, indicating a specific link between huntingtin and Cx II26. The progressive, neurocognitive deficits that are evident in Cx II knockout mice also exhibit a strong degree of homology with the neuropsychiatric symptoms of the earlier stages of Huntington's disease. In further support of this link, it was noted that there is a highly selective and consistent loss of Cx II in the R6/2 mouse model of Huntington's disease33, which is correlated with progressive neuropathology. In this study, multiple presynaptic proteins were measured throughout the brain, and only the two presynaptic proteins Cx II and a-SNAP were reduced compared to wild-type mice. The loss of Cx II was significantly greater, and occurred significantly earlier, than with a-SNAP. In human postmortem brain, two independent research groups have reported a selective loss of Cx II. In one study, Cx II exhibited a marked reduction in the striatum of Huntington's disease34, while another group reported a selective loss of Cx II in the frontal cortex of early pathological-grade Huntington's disease35. It is unknown why Cx II should be selectively decreased in this disorder, but if Cx II is predominantly localized to excitatory, glutamatergic terminals, there may be a link to neurotoxicity. There is less evidence of a role for Cx I in neurological disorders, although one recent study reported increased levels of Cx I in the substantia nigra of Parkinson's disease36.

An alternate approach to understanding a possible link between Cxs and neuropsychiatric disorders is based on determining the effects of pharmaco-therapies on levels of these proteins. For example, research from our laboratory has demonstrated that schizophrenia is associated with a loss of the presynaptic protein SNAP-25 (see below), while treatment of rats with the antipsychotic drugs haloperidol or chlorpromazine produces increases in levels of SNAP-25, opposite the direction of change in the disorder. With regard to Cxs, there are mixed data concerning the effects of antipsychotic drugs. We have previously reported that there is no effect in rats of treatment with the neuroleptic haloperidol for 21 days on levels of Cx proteins, measured by ELISA and quantitative immuno-histochemistry, in the frontal cortex or hippocampus22,37. A study in which rats were administered haloperidol for 28 days according to a "depot" schedule determined that there was no effect of this drug on levels of Cx II mRNA in multiple brain regions. However, haloperidol significantly decreased levels of Cx I mRNA in the medial prefrontal cortex, nucleus accumbens and ventral tegmental area38. A more extensive study which evaluated the effects of both typical and atypical antipsychotics on Cxs reported diverse findings. In this study, levels of Cx II mRNA were increased in the frontoparietal cortex after treatment with the typical antipsychotic chlorpromazine, while levels of Cx I mRNA were elevated by the atypical antipsychotic olanzapine in the dorsolateral striatum and frontoparietal cortex; both olanzapine and haloperidol decreased the Cx II:I mRNA ratio in these latter two brain regions. These combined findings reveal apparently conflicting results, and further studies will be required, using more comparable doses and durations of drug administration between laboratories. Interestingly, a recent study determined the effects of different classes of antidepressant drugs on brain levels of Cxs39. The authors of this study noted that Cx I was induced only in habenular nuclei after treatment with fluoxetine. However, Cx II was significantly increased throughout multiple hippocampal subregions following treatment with the tricyclic antidepressant desipramine and the monoamine oxidase inhibitor tranylcypromine, but not with the selective serotonin reuptake inhibitor fluoxetine. These findings warrant further study of a possible link between Cxs and affective disorders.

4.5. Clinical Data on Complexins

Given the physiological role of Cxs in higher cognitive processes, and the convincing preclinical evidence for Cxs in multiple disease models, a number of studies have investigated a link between Cxs and mental illness. As described above, two independent groups have demonstrated a selective loss of Cx II in the frontal cortex and striatum in the earlier stages of Huntington's disease34,35. Our research group has primarily been interested in a role for the Cxs in psychiatric disorders. We have shown previously that Cxs are lower in both schizophrenia and MDD in frontal cortex. Using enzyme-linked immunoadsorbent assay (ELISA), we confirmed that Cx I immunoreactivity was significantly decreased in schizophrenia and MDD, and the ratio of Cx II:I was significantly increased by 32-34%37. This finding is particularly relevant to a putative loss of functional inhibition in frontal cortex in psychiatric disorders, as a selective loss of Cx I (inhibitory terminals) is consistent with reduced inhibitory synapses formed by GABAergic interneurons in prefrontal cortex, which are hypothesized to contribute to cognitive deficits.

More recently, we have investigated changes in levels of Cxs in the hippocampus in schizophrenia. Initial studies by other groups demonstrated that levels of both Cx I and Cx II mRNA were lower in hippocampus in schizophrenia, with a relatively greater loss of Cx II mRNA; protein studies found a selective loss of Cx II40. We confirmed these findings, and reported that Cx II was lower in hippocampal subregions of individuals with cognitive impairment than those not impaired, resulting in a significantly lower Cx II:I ratio in the hippocampus in cognitively impaired schizophrenics22. These findings are especially important for two reasons. Firstly, the confirmation of findings from a different laboratory, using different post mortem samples, indicates that changes in Cxs in the hippocampus are one of the most consistent molecular changes in schizophrenia. Even changes in excitatory and inhibitory markers, such as GABAergic or glutamatergic terminals, show inconsistent findings in the hippocampus (for example, two recent papers found contradictory changes of vGLUT1 in schizophrenia hippocampus41,42). The second important conclusion from our findings is that the Cxs, and the Cx II:I ratio, are the first synaptic changes to be confirmed as being associated specifically with cognitive deficits in schizophrenia. These links with cognitive impairment - including memory deficits - did not extend to other presynaptic proteins that we have measured in post mortem hippocampus from the same samples, including synaptophysin, suggesting a selective link between Cxs and cognitive dysfunction. These findings indicate that although Cxs are generally decreased in psychiatric disorders, the relative effects on Cx I versus Cx II depend on the specific brain region, as well as the type of disorder. It is therefore of interest that a recent study has confirmed a significant genetic link between a Cx II haplotype and schizophrenia in a Korean sample43. Currently, priorities for the study of Cxs and their relevance to psychiatric disorders include a better understanding of the functional role of Cxs in cognitive plasticity, which will help to provide insights into the nature of disease-related changes, as well as pharmacotherapy-induced changes.

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