Wen-Jun Gao

Cell Type-Specific Development of NMDA Receptors in the Interneurons of Rat Prefrontal Cortex

In the prefrontal cortex, N-methyl-D-aspartic acid (NMDA) receptors (NMDARs) are critical not only for normal prefrontal functions but also for the pathological processes of schizophrenia. Little is known, however, about the developmental properties of NMDARs in the functionally diverse sub-populations of interneurons. We investigated the developmental changes of NMDARs in rat prefrontal interneurons using patch clamp recording in cortical slices. We found that fast-spiking (FS) interneurons exhibited properties of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and NMDA currents distinct from those in regular spiking (RS) and low-threshold spiking (LTS) interneurons, particularly during the adolescent period. In juvenile animals, most (73%) of the FS cells demonstrated both AMPA and NMDA currents. The NMDA currents, however, gradually became undetectable during cortical development, with most (74%) of the FS cells exhibiting no NMDA current in adults. In contrast, AMPA and NMDA currents in RS and LTS interneurons were relatively stable, without significant changes from juveniles to adults. Moreover, even in FS cells with NMDA currents, the NMDA/AMPA ratio dramatically decreased during the adolescent period but returned to juvenile level in adults, compared with the relatively stable ratios in RS and LTS interneurons. These data suggest that FS interneurons in the prefrontal cortex undergo dramatic changes in glutamatergic receptors during the adolescent period. These properties may make FS cells particularly sensitive and vulnerable to epigenetic stimulation, thus contributing to the onset of many psychiatric disorders, including schizophrenia.

Selective modulation of excitatory and inhibitory microcircuits by dopamine

Dopamine plays an important role in the working memory functions of the prefrontal cortex, functions that are impacted in age-related memory decline, drug abuse, and a wide variety of disorders, including schizophrenia and Parkinsons disease. We have previously reported that dopamine depresses excitatory transmission between pyramidal neurons in the prefrontal cortex. Here, using paired recordings, we have investigated dopaminergic modulation of excitatory transmission from pyramidal neurons to fast-spiking (FS) interneurons. In contrast to its effect on recurrent excitation, dopamine was without effect on excitatory transmission to FS interneurons. However, dopamine has directly enhanced the excitability of the FS interneurons to the extent that even a single excitatory postsynaptic potential could initiate spiking with great temporal precision in some of them. These results indicate that dopamines effects on excitatory transmission are target-specific and that the axon terminals of pyramidal neurons can be selectively regulated at the level of individual synapses. Thus, dopamines net inhibitory effect on cortical function is remarkably constrained by the nature of the microcircuit elements on which it acts.

A specialized NMDA receptor function in layer 5 recurrent microcircuitry of the adult rat prefrontal cortex

In the prefrontal cortex, NMDA receptors are important for normal prefrontal functions such as working memory, and their dysfunction plays a key role in the pathological processes of psychiatric disorders such as schizophrenia. Little is known, however, about the synaptic properties of NMDA receptors in the local circuits of recurrent excitation, a leading candidate mechanism underlying working memory. We investigated the NMDA receptor-mediated currents at monosynaptic connections between pairs of layer 5 pyramidal neurons. We found that NMDA receptor-mediated currents at prefrontal synapses in the adult, but not young, rats exhibit a twofold longer decay time-constant and temporally summate a train of stimuli more effectively, compared to those in the primary visual cortex. Experiments with pharmacological, immunocytochemical, and biochemical approaches further suggest that, in the adult animals, neurons express significantly more NR2B subunits in the prefrontal cortex than the visual cortex. The NR2B-rich synapses in the prefrontal circuitry may be critically implicated in online cognitive computations and plasticity in learning, as well as psychiatric disorders.

NMDA hypofunction as a convergence point for progression and symptoms of schizophrenia

Schizophrenia is a disabling mental illness that is now recognized as a neurodevelopmental disorder. It is likely that genetic risk factors interact with environmental perturbations to affect normal brain development and that this altered trajectory results in a combination of positive, negative, and cognitive symptoms. Although the exact pathophysiology of schizophrenia is unknown, the N-methyl-D-aspartate receptor (NMDAR), a major glutamate receptor subtype, has received great attention. Proper expression and regulation of NMDARs in the brain is critical for learning and memory processes as well as cortical plasticity and maturation. Evidence from both animal models and human studies implicates a dysfunction of NMDARs both in disease progression and symptoms of schizophrenia. Furthermore, mutations in many of the known genetic risk factors for schizophrenia suggest that NMDAR hypofunction is a convergence point for schizophrenia. In this review, we discuss how disrupted NMDAR function leads to altered neurodevelopment that may contribute to the progression and development of symptoms for schizophrenia, particularly cognitive deficits. We review the shared signaling pathways among the schizophrenia susceptibility genes DISC1, neuregulin1, and dysbindin, focusing on the AKT/GSK3β pathway, and how their mutations and interactions can lead to NMDAR dysfunction during development. Additionally, we explore what open questions remain and suggest where schizophrenia research needs to move in order to provide mechanistic insight into the cause of NMDAR dysfunction, as well as generate possible new avenues for therapeutic intervention.

Role of dysbindin in dopamine receptor trafficking and cortical GABA function

Dysbindin has been implicated in the pathogenesis of schizophrenia, but little is known about how dysbindin affects neuronal function in the circuitry underlying psychosis and related behaviors. Using a dysbindin knockout line (dys−/−) derived from the natural dysbindin mutant Sandy mice, we have explored the role of dysbindin in dopamine signaling and neuronal function in the prefrontal cortex (PFC). Combined cell imaging and biochemical experiments revealed a robust increase in the dopamine receptor D2, but not D1, on cell surface of neurons from dys−/− cortex. This was due to an enhanced recycling and insertion, rather than reduced endocytosis, of D2. Disruption of dysbindin gene resulted in a marked decrease in the excitability of fast-spiking (FS) GABAergic interneurons in both PFC and striatum. Dys−/− mice also exhibited a decreased inhibitory input to pyramidal neurons in layer V of PFC. The increased D2 signaling in dys−/− FS interneurons was associated with a more pronounced increase in neuronal firing in response to D2 agonist, compared to that in wild-type interneurons. Taken together, these results suggest that dysbindin regulates PFC function by facilitating D2-mediated modulation of GABAergic function.

Heterogeneous organization of the locus coeruleus projections to prefrontal and motor cortices

Significance The locus coeruleus projection system in the brain is thought to exert uniform and synchronous modulatory effects on cells and circuits throughout the CNS by widespread release of its transmitter, norepinephrine. We challenge this notion by demonstrating that neurons in the locus coeruleus maintain segregated connections to brain regions with distinctly different functions. Specifically, cells that communicate with the prefrontal cortex, a region involved in cognition and executive function, are characterized by properties that allow for independent and asynchronous modulation of operations in this area, compared with those that project to the motor cortex and regulate movement generation. These findings have important implications for understanding the role of this system in normal brain physiology and pathologic neuropsychiatric conditions. The brainstem nucleus locus coeruleus (LC) is the primary source of norepinephrine (NE) to the mammalian neocortex. It is believed to operate as a homogeneous syncytium of transmitter-specific cells that regulate brain function and behavior via an extensive network of axonal projections and global transmitter-mediated modulatory influences on a diverse assembly of neural targets within the CNS. The data presented here challenge this longstanding notion and argue instead for segregated operation of the LC–NE system with respect to the functions of the circuits within its efferent domain. Anatomical, molecular, and electrophysiological approaches were used in conjunction with a rat model to show that LC cells innervating discrete cortical regions are biochemically and electrophysiologically distinct from one another so as to elicit greater release of norepinephrine in prefrontal versus motor cortex. These findings challenge the consensus view of LC as a relatively homogeneous modulator of forebrain activity and have important implications for understanding the impact of the system on the generation and maintenance of adaptive and maladaptive behaviors.

Development of calcium‐permeable AMPA receptors and their correlation with NMDA receptors in fast‐spiking interneurons of rat prefrontal cortex

Abnormal influx of Ca2+ is thought to contribute to the neuronal injury associated with a number of brain disorders, and Ca2+‐permeable AMPA receptors (CP‐AMPARs) play a critical role in the pathological process. Despite the apparent vulnerability of fast‐spiking (FS) interneurons in neurological disorders, little is known about the CP‐AMPARs expressed by functionally identified FS interneurons in the developing prefrontal cortex (PFC). We investigated the development of inwardly rectifying AMPA receptor‐mediated currents and their correlation with NMDA receptor‐mediated currents in FS interneurons in the rat PFC. We found that 78% of the FS interneurons expressed a low rectification index, presumably Ca2+‐permeable AMPARs, with only 22% exhibiting AMPARs with a high rectification index, probably Ca2+ impermeable (CI). FS interneurons with CP‐AMPARs exhibited properties distinct from those expressing CI‐AMPARs, although both displayed similar morphologies, passive membrane properties and AMPA currents at resting membrane potentials. The AMPA receptors also exhibited dramatic changes during cortical development with significantly more FS interneurons with CP‐AMPARs and a clearly decreased rectification index during adolescence. In addition, FS interneurons with CP‐AMPARs exhibited few or no NMDA currents, distinct frequency‐dependent synaptic facilitation, and protracted maturation in short‐term plasticity. These data suggest that CP‐AMPARs in FS interneurons may play a critical role in neuronal integration and that their characteristic properties may make these cells particularly vulnerable to disruptive influences in the PFC, thus contributing to the onset of many psychiatric disorders.

Activation of glycogen synthase kinase-3 beta is required for hyperdopamine and D2 receptor-mediated inhibition of synaptic NMDA receptor function in the rat prefrontal cortex.

The interactions between dopamine and glutamate systems play an essential role in normal brain functions and neuropsychiatric disorders. The mechanism of NMDA receptor regulation through high concentrations of dopamine, however, remains unclear. Here, we show the signaling pathways involved in hyperdopaminergic regulation of NMDA receptor functions in the prefrontal cortex by incubating cortical slices with high concentration of dopamine or administering dopamine reuptake inhibitor 1-(2-[bis-(4-fluorophenyl)methoxy]ethyl)- 4-(3-phenylpropyl)piperazine (GBR12909) in vivo. We found that, under both conditions, the synaptic NMDA receptor-mediated currents were significantly attenuated by excessive dopamine stimulation through activation of D2 receptors. Furthermore, high dose of dopamine failed to affect NMDA receptor-mediated currents after blockade of NR2B subunits but triggered a dynamin-dependent endocytosis of NMDA receptors. The high-dose dopamine/D2 receptor-mediated suppression of NMDA receptors was involved in the increase of glycogen synthase kinase-3β (GSK-3β) activity, which in turn phosphorylates β-catenin and disrupts β-catenin–NR2B interaction, but was dependent on neither Gq11 nor PLC (phospholipase C). Moreover, the hyperdopamine induced by GBR12909 significantly decreased the expression of both surface and intracellular NR2B proteins, as well as NR2B mRNA levels, suggesting an inhibition of protein synthesis. These effects were, however, completely reversed by administration of either GSK-3β inhibitor or D2 receptor antagonist. These results therefore suggest that GSK-3β is required for the hyperdopamine/D2 receptor-mediated inhibition of NMDA receptors in the prefrontal neurons and these actions may underlie D2 receptor-mediated psychostimulant effects and hyperdopamine-dependent behaviors in the brain.

Development of inhibitory circuitry in visual and auditory cortex of postnatal ferrets: immunocytochemical localization of GABAergic neurons.

The goal of this study was to describe the development of gamma‐aminobutyric acid (GABA)‐containing neurons in visual and auditory cortex of ferrets. The laminar and tangential distribution of neurons containing excitatory, inhibitory, and neuromodulatory substances constrain the potential circuits which can form during development. Ferrets are born at an early stage of brain development, allowing examination of inhibitory circuit formation in cerebral cortex prior to thalamocortical ingrowth and cortical plate differentiation.

Development of inhibitory circuitry in visual and auditory cortex of postnatal ferrets: immunocytochemical localization of calbindin- and parvalbumin-containing neurons.

The inhibitory neurotransmitter γ‐aminobutyric acid (GABA) is thought to play an important role in activity‐dependent stages of brain development. Previous studies have shown that different functional subclasses of cortical GABA‐containing neurons can be distinguished by antibodies to the calcium‐binding proteins parvalbumin and calbindin. Thus insight into the development of distinct subsets of inhibitory cortical circuits can be gained by studying the development of these calcium‐binding protein‐containing neurons. Previous studies in several mammalian species have suggested that calcium‐binding proteins are upregulated in sensory cortex when thalamocortical afferents arrive. In ferrets, the ingrowth of thalamic axons into cortex occurs well into postnatal development, allowing access to early stages of cortical development and calcium‐binding protein expression. We find in ferrets that both parvalbumin‐ and calbindin‐immunoreactivity are present in primary visual and primary auditory cortex long before thalamocortical synapse formation, but that there is a sharp decline in immunoreactivity by postnatal day 20. Day 20 in ferrets corresponds to postnatal day 1 in cats, and thus previous studies in postnatal cats would have missed this early pattern of calcium‐binding protein distribution. Another surprising finding is that the proportion of parvalbumin‐ and calbindin‐immunoreactive neurons peaks secondarily late in development, between P60 and adulthood. This result suggests that the parvalbumin‐ and calbindin‐containing subclasses of nonpyramidal neurons remain immature until late in the critical period for cortical plasticity, and that they are positioned to play an important role in experience‐dependent modification of cortical circuits. J. Comp. Neurol. 422:140–157, 2000.