Puberty is characterized by mood swings and anxiety, often produced by stress. Here, we show that THP (allopregnanolone), a steroid released by stress, increases anxiety in pubertal female mice, a reversal of its well-known anxiety-reducing effect in adults. Anxiety is regulated by GABAergic inhibition in limbic circuits. Although this inhibition is increased by THP before puberty and in adults, THP reduced tonic inhibition of CA1 hippocampal pyramidal cells at puberty, leading to increased excitability. This paradoxical effect of THP was due to inhibition of α4βδ GABA A receptors. These receptors are normally expressed at very low levels, but at puberty, their expression was increased in CA1 hippocampus where they generated outward currents. THP also decreased outward current at recombinant α4β2δ receptors, an effect dependent on arginine 353 in the α4 subunit, a putative Cl − modulatory site. Thus, inhibition of α4β2δ GABA A receptors by THP provides a mechanism for anxiety at puberty.The onset of puberty is associated with increases in emotional reactivity and anxiety 1,2 . Responses to stressful events are amplified 3 , and anxiety and panic disorder first emerge at this time 2 , twice as likely to occur in girls than in boys 2 . Few studies have addressed the biological basis of this important issue, although suicide risk increases in adolescence, despite the use of adult-based medical strategies 2 .The GABA A receptor plays a pivotal role in the generation of anxiety 4 . This receptor is the target for endogenous steroids such as THP (3α-OH-5α [β]-pregnan-20-one or [allo] pregnanolone), which increase GABA-gated currents at physiological concentrations 5 of the steroid. THP is a metabolite of the ovarian/adrenal steroid progesterone, but is also formed in the brain as a compensatory response to stress 6 . In adults, THP potently reduces anxiety in humans 7 , an effect seen in animal models with direct administration into the dorsal CA1 hippocampus 8 , part of the limbic system that regulates emotion. It is generally accepted that * Correspondence and requests for materials should be addressed to S.S.Smith, Dept. of Physiology and Pharmacology, SUNY Downstate Medical Center, 450 Clarkson Ave., Brooklyn, NY 11203 USA; phone: 718-270-2226; FAX: 718-270-3103; email: Sheryl.smith@downstate NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript the GABA-enhancing action of THP underlies its well-known anxiety-reducing effect in adults, which is similar to other GABA-enhancing drugs such as the benzodiazepines.GABA A receptors are pentamers formed predominantly of 2α, 2β and 1γ subunits 9 which gate a Cl − current and produce most fast synaptic inhibition in the brain. Substitution of the δ subunit for γ2 yields a receptor with the highest sensitivity to steroids such as THP 10-12 . These highly sensitive δ-GABA A receptors are extrasynaptic 13 , and mediate tonic rather than synaptic inhibition in areas such as dentate gyrus 14 . Thus, THP and related steroids enhance inhibition h...

Acetylcholine is a ubiquitous cortical neuromodulator implicated in cognition. In order to understand the potential for acetylcholine to play a role in visual attention, we studied nicotinic acetylcholine receptor (nAChR) localization and function in area V1 of the macaque. We found nAChRs presynaptically at thalamic synapses onto excitatory, but not inhibitory, neurons in the primary thalamorecipient layer 4c. Furthermore, consistent with the release enhancement suggested by this localization, we discovered that nicotine increases responsiveness and lowers contrast threshold in layer 4c neurons. We also found that nAChRs are expressed by GABAergic interneurons in V1 but rarely by pyramidal neurons, and that nicotine suppresses visual responses outside layer 4c. All sensory systems incorporate gain control mechanisms, or processes which dynamically alter input/output relationships. We demonstrate that at the site of thalamic input to visual cortex, the effect of this nAChR-mediated gain is an enhancement of the detection of visual stimuli.

The limited success of immunogold labeling for pre-embedding immunocytochemistry of neuronal antigens is largely attributed to poor penetration of large (5-20 nm) colloidal gold particles. We examined the applicability of using silver intensification of 1 nm colloidal gold particles non-covalently bound to goat anti-rabbit immunoglobulin (1) for single labeling of a rabbit antiserum against the catecholamine synthesizing enzyme, tyrosine hydroxylase (TH), and (2) for immunogold localization of rabbit anti-TH simultaneously with immunoperoxidase labeling of a mouse monoclonal antibody against the opiate peptide, leucine-enkephalin (LE). Vibratome sections were collected from acrolein fixed brains of adult rats. These sections were immunolabeled without use of freeze-thawing or other methods that enhance penetration, but damage ultrastructure. By light microscopy, incubations in the silver intensifier (Intense M, Janssen) for less than 10 min at room temperature resulted in a brownish-red reaction product for TH. This product was virtually indistinguishable from that seen using diaminobenzidine reaction for detection of peroxidase immunoreactivity. Longer incubations produced intense black silver deposits that were more clearly distinguishable from the brown immunoperoxidase labeling. However, by light microscopy, the gold particles seen by electron microscopy were most readily distinguished from peroxidase reaction product with shorter silver intensification periods. The smaller size of gold particles with shorter periods of silver intensification also facilitated evaluation of labeling with respect to subcellular organelles. Detection of the silver product did not appear to be appreciably changed by duration of post-fixation in osmium tetroxide. In dual-labeled sections, perikarya and terminals exhibiting immunogold-silver labeling for TH were distinct from those containing immunoperoxidase labeling for LE. These results (1) define the conditions needed for optimal immunogold-silver labeling of antigens while maintaining the ultrastructural morphology in brain, and (2) establish the necessity for controlled silver intensification for light or electron microscopic differentiation of immunogold-silver and peroxidase reaction products and for optimal subcellular resolution.

Postsynaptic density-95 (PSD-95/SAP-90) is a palmitoylated peripheral membrane protein that scaffolds ion channels at excitatory synapses. To elucidate mechanisms for postsynaptic ion channel clustering, we analyzed the cellular trafficking of PSD-95. We find that PSD-95 transiently associates with a perinuclear membranous compartment and traffics with vesiculotubular structures, which migrate in a microtubule-dependent manner. Trafficking of PSD-95 with these vesiculotubular structures requires dual palmitoylation, which is specified by five consecutive hydrophobic residues at the NH2 terminus. Mutations that disrupt dual palmitoylation of PSD-95 block both ion channel clustering by PSD-95 and its synaptic targeting. Replacing the palmitoylated NH2 terminus of PSD-95 with alternative palmitoylation motifs at either the NH2 or COOH termini restores ion channel clustering also induces postsynaptic targeting, respectively. In brain, we find that PSD-95 occurs not only at PSDs but also in association with intracellular smooth tubular structures in dendrites and spines. These data imply that PSD-95 is an itinerant vesicular protein; initial targeting of PSD-95 to an intracellular membrane compartment may participate in postsynaptic ion channel clustering by PSD-95.

Oxytocin is a neuropeptide important for social behaviors such as maternal care and parent-infant bonding. It is believed that oxytocin receptor signaling in the brain is critical for these behaviors, but it is unknown precisely when and where oxytocin receptors are expressed or which neural circuits are directly sensitive to oxytocin. To overcome this challenge, we generated specific antibodies to the mouse oxytocin receptor and examined receptor expression throughout the brain. We identified a distributed network of female mouse brain regions for maternal behaviors that are especially enriched for oxytocin receptors, including the piriform cortex, the left auditory cortex, and CA2 of the hippocampus. Electron microscopic analysis of the cerebral cortex revealed that oxytocin receptors were mainly expressed at synapses, as well as on axons and glial processes. Functionally, oxytocin transiently reduced synaptic inhibition in multiple brain regions and enabled long-term synaptic plasticity in the auditory cortex. Thus modulation of inhibition may be a general mechanism by which oxytocin can act throughout the brain to regulate parental behaviors and social cognition.

GLT-1 (EAAT2; slc1a2) is the major glutamate transporter in the brain, and is predominantly expressed in astrocytes, but at lower levels also in excitatory terminals. We generated a conditional GLT-1 knock-out mouse to uncover cell-type-specific functional roles of GLT-1. Inactivation of the GLT-1 gene was achieved in either neurons or astrocytes by expression of synapsin-Cre or inducible human GFAPCreERT2. Elimination of GLT-1 from astrocytes resulted in loss of ϳ80% of GLT-1 protein and of glutamate uptake activity that could be solubilized and reconstituted in liposomes. This loss was accompanied by excess mortality, lower body weight, and seizures suggesting that astrocytic GLT-1 is of major importance. However, there was only a small (15%) reduction that did not reach significance of glutamate uptake into crude forebrain synaptosomes. In contrast, when GLT-1 was deleted in neurons, both the GLT-1 protein and glutamate uptake activity that could be solubilized and reconstituted in liposomes were virtually unaffected. These mice showed normal survival, weight gain, and no seizures. However, the synaptosomal glutamate uptake capacity (V max ) was reduced significantly (40%). In conclusion, astrocytic GLT-1 performs critical functions required for normal weight gain, resistance to epilepsy, and survival. However, the contribution of astrocytic GLT-1 to glutamate uptake into synaptosomes is less than expected, and the contribution of neuronal GLT-1 to synaptosomal glutamate uptake is greater than expected based on their relative protein expression. These results have important implications for the interpretation of the many previous studies assessing glutamate uptake capacity by measuring synaptosomal uptake.

Ca2+ release from inositol 1,4,5-trisphosphate (IP3)-sensitive and ryanodine-sensitive intracellular Ca2+ stores is mediated by distinct proteins identified as IP3 receptors (IP3R) and ryanodine receptors (RyR), respectively. We have compared the immunohistochemical localizations of IP3R and RyR in the brain at the light and electron microscopic levels and have also evaluated the distribution of the major brain intracellular Ca(2+)-pumping ATPase. IP3R and RyR occur in overlapping populations of neurons in widespread areas of the brain, but labeling is distinct in a number of areas. For example, IP3R is enriched in cerebellar Purkinje cells and hippocampal CA1 pyramidal cells, while RyR is present at relatively low levels in these cells. RyR is most enriched in the dentate gyrus and CA3/4 areas of the hippocampus, where IP3R levels are low. In the cortex, IP3R is found in pyramidal cell bodies and proximal dendrites, whereas RyR is located predominantly in long, thin apical dendrites of pyramidal cells. In deep cerebellar nuclei, RyR is located in cell bodies that appear devoid of IP3R, whereas IP3R is enriched in terminals surrounding cell bodies. Electron microscopy in the hippocampus reveals RyR in axons, dendritic spines, and dendritic shafts near dendritic spines while IP3R is primarily identified in dendritic shafts and cell bodies. These results suggest that the IP3- and ryanodine-sensitive Ca2+ pools have largely distinct roles in controlling intracellular Ca2+ levels, though in some sites they may interact to varying degrees.

GLT1 is the major glutamate transporter of the brain and has been thought to be expressed exclusively in astrocytes. Although excitatory axon terminals take up glutamate, the transporter responsible has not been identified. GLT1 is expressed in at least two forms varying in the C termini, GLT1a and GLT1b. GLT1 mRNA has been demonstrated in neurons, without associated protein. Recently, evidence has been presented, using specific C terminus-directed antibodies, that GLT1b protein is expressed in neurons in vivo. These data suggested that the GLT1 mRNA detected in neurons encodes GLT1b and also that GLT1b might be the elusive presynaptic transporter. To test these hypotheses, we used variant-specific probes directed to the 3Ј-untranslated regions for GLT1a and GLT1b to perform in situ hybridization in the hippocampus. Contrary to expectation, GLT1a mRNA was the more abundant form. To investigate further the expression of GLT1 in neurons in the hippocampus, antibodies raised against the C terminus of GLT1a and against the N terminus of GLT1, found to be specific by testing in GLT1 knock-out mice, were used for light microscopic and EM-ICC. GLT1a protein was detected in neurons, in 14 -29% of axons in the hippocampus, depending on the region. Many of the labeled axons formed axo-spinous, asymmetric, and, thus, excitatory synapses. Labeling also occurred in some spines and dendrites. The antibody against the N terminus of GLT1 also produced labeling of neuronal processes. Thus, the originally cloned form of GLT1, GLT1a, is expressed as protein in neurons in the mature hippocampus and may contribute significantly to glutamate uptake into excitatory terminals.