The central amygdala (CeA) plays a role in the relationship among stress, corticotropin-releasing factor (CRF), and alcohol abuse. In whole-cell recordings, both CRF and ethanol enhanced gamma-aminobutyric acid-mediated (GABAergic) neurotransmission in CeA neurons from wild-type and CRF2 receptor knockout mice, but not CRF1 receptor knockout mice. CRF1 (but not CRF2) receptor antagonists blocked both CRF and ethanol effects in wild-type mice. These data indicate that CRF1 receptors mediate ethanol enhancement of GABAergic synaptic transmission in the CeA, and they suggest a cellular mechanism underlying involvement of CRF in ethanol's behavioral and motivational effects.

Emerging evidence suggests that myocyte enhancer factor 2 (MEF2) transcription factors act as effectors of neurogenesis in the brain, with MEF2C the predominant isoform in developing cerebrocortex. Here, we show that conditional knockout of Mef2c in nestin-expressing neural stem/progenitor cells (NSCs) impaired neuronal differentiation in vivo, resulting in aberrant compaction and smaller somal size. NSC proliferation and survival were not affected. Conditional null mice surviving to adulthood manifested more immature electrophysiological network properties and severe behavioral deficits reminiscent of Rett syndrome, an autism-related disorder. Our data support a crucial role for MEF2C in programming early neuronal differentiation and proper distribution within the layers of the neocortex.neurogenesis ͉ synaptogenesis ͉ autism ͉ Rett syndrome K nockdown of the transcription factor MEF2C in mature cerebrocortical neurons results in increased synaptic number and activity (1). To facilitate analysis of MEF2C function in early neuronal development, we engineered a conditional knockout in NSCs by crossing floxed Mef2c mice with Nestin-Cre mice. In contrast to the findings in more mature neurons, we found a striking alteration in the distribution of new neurons in the neocortex and the opposite effect on synaptic activity, i.e., decreased neurotransmission persisting into adulthood.MEF2C belongs to the myocyte enhancer factor 2 (MEF2) subfamily of the MADS (MCM1-agamous-deficiens-serum response factor) gene family (2, 3). We cloned MEF2C from developing mouse brain, and Eric Olson and colleagues then discovered it in the heart (2, 4, 5). In cerebrocortex, MEF2 transcriptional activity is restricted to differentiated cortical neurons in a specific laminar pattern, and its distribution increases along the rostrocaudal axis (2, 4, 6). These features led to speculation on the potential role of MEF2C in the architechtonics of the cerebral cortex (2). Previous studies demonstrated an important role for MEF2C in heart development (7). In the CNS, MEF2C is involved in neuronal apoptosis (8) and synapse formation (1, 9) in vitro or in brain slices. Most recently, our laboratory discovered that a constitutively active form of MEF2C induces embryonic stem cells to commit to a neuronal fate in a virtually exclusive fashion (10). However, studies on the effect of endogenous MEF2C on CNS neurons in vivo were impeded by the embryonic lethality of conventional Mef2c-null mice because of cardiovascular defects at embryonic day (E) 9.5, before brain development (7). Here, we report that conditionally knocking out the Mef2c gene in neural progenitors causes abnormal aggregation and compaction of neurons migrating into the lower layers of the neocortex during development. Knockout mice surviving to adulthood manifest smaller, apparently less mature neurons and smaller whole brain size, with resultant aberrant electrophysiology and behavior.

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Expression of the NR3A subunit with NR1/NR2 in Xenopus oocytes or mammalian cell lines leads to a reduction in N-methyl-d-aspartate (NMDA)-induced currents and decreased Mg(2+) sensitivity and Ca(2+) permeability compared with NR1/NR2 receptors. Consistent with these findings, neurons from NR3A knockout (KO) mice exhibit enhanced NMDA-induced currents. Recombinant NR3A can also form excitatory glycine receptors with NR1 in the absence of NR2. However, the effects of NR3A on channel properties in neurons and synaptic transmission have not been fully elucidated. To study physiological roles of NR3A subunits, we generated NR3A transgenic (Tg) mice. Cultured NR3A Tg neurons exhibited two populations of NMDA receptor (NMDAR) channels, reduced Mg(2+) sensitivity, and decreased Ca(2+) permeability in response to NMDA/glycine, but glycine alone did not elicit excitatory currents. In addition, NMDAR-mediated excitatory postsynaptic currents (EPSCs) in NR3A Tg hippocampal slices showed reduced Mg(2+) sensitivity, consistent with the notion that NR3A subunits incorporated into synaptic NMDARs. To study the function of endogenous NR3A subunits, we compared NMDAR-mediated EPSCs in NR3A KO and WT control mice. In NR3A KO mice, the ratio of the amplitudes of the NMDAR-mediated component to alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor-mediated component of the EPSC was significantly larger than that seen in WT littermates. This result suggests that NR3A subunits contributed to the NMDAR-mediated component of the EPSC in WT mice. Taken together, these results show that NR3A subunits contribute to NMDAR responses from both synaptic and extrasynaptic receptors, likely composed of NR1, NR2, and NR3 subunits.

Both the nucleus accumbens (NAcc) and central amygdala (CeA) are thought to play roles in tolerance to, and dependence on, abused drugs. Although our past studies in rat brain slices suggested a role for NMDA receptors (NMDARs) in NAcc neurons in the effects of acute and chronic opiate treatment, the cellular and molecular mechanisms remained unclear. Therefore, we examined the effects of morphine dependence on electrophysiological properties of NMDARs in freshly isolated NAcc neurons and on expression of mRNA coding for NR2A-C subunits using single-cell RT-PCR. Chronic morphine did not alter the affinity for NMDAR agonists glutamate, homoquinolinate, or NMDA, but decreased the affinity of the coagonist glycine. Chronic morphine altered the NMDAR inhibition by two NMDAR antagonists, 7-Cl-kynurenate and ifenprodil, but not that by d-APV or Mg2+. Chronic morphine accelerated the NMDA current desensitization rate in NAcc neurons. In single-cell RT-PCR, chronic morphine predominantly reduced the number of neurons expressing multiple NR2 subunits. Ethanol also alters NMDARs. We found that low ethanol concentrations (IC50 = 13 mM) inhibited NMDA currents and NMDA-EPSPs in most NAcc neurons in a slice preparation. NAcc neurons from ethanol-dependent rats showed enhanced NMDA sensitivity. In CeA neurons, acute ethanol decreased (by 10-25%) non-NMDA- and NMDA-EPSPs in most neurons. In CeA neurons from ethanol-dependent rats, acute ethanol decreased the non-NMDA-EPSPs to the same extent as in naïve rats, but inhibited (by 30-40%) NMDA-EPSPs significantly more than in controls, suggesting sensitization to ethanol. Preliminary studies with microdialysis and real-time PCR analysis support this idea: local ethanol administration in vivo had no effect on glutamate release, but chronic ethanol nearly tripled the expression of NR2B subunits (the most ethanol sensitive) in CeA. These combined findings suggest that changes in glutamatergic transmission in NAcc and CeA may underlie the neuroadaptions that lead to opiate and ethanol dependence.