Classical electron microscopic studies of the mammalian brain revealed two major classes of synapses, distinguished by the presence of a large postsynaptic density (PSD) exclusively at type 1, excitatory synapses. Biochemical studies of the PSD have established the paradigm of the synapse as a complex signal-processing machine that controls synaptic plasticity. We report here the results of a proteomic analysis of type 2, inhibitory synaptic complexes isolated by affinity purification from the cerebral cortex. We show that these synaptic complexes contain a variety of neurotransmitter receptors, neural cell-scaffolding and adhesion molecules, but that they are entirely lacking in cell signaling proteins. This fundamental distinction between the functions of type 1 and type 2 synapses in the nervous system has far reaching implications for models of synaptic plasticity, rapid adaptations in neural circuits, and homeostatic mechanisms controlling the balance of excitation and inhibition in the mature brain.

Summary

GABAA receptors (GABAR) mediate most inhibition in the CNS and are also a target for neuroactive steroids such as 3α,5[α]β-THP (3αOH-5[α]β-OH-pregnan-20-one or [allo]pregnanolone). Although these steroids robustly enhance current gated by α1β2δ GABAR, we have shown that 3α,5[α]β-THP effects at recombinant α4β2δ GABAR depend on the direction of Cl- flux, where the steroid increases outward flux, but decreases inward flux through the receptor. This polarity-dependent inhibition of α4β2δ GABAR resulted from an increase in the rate and extent of rapid desensitization of the receptor, recorded from recombinant receptors expressed in HEK-293 cells with whole cell voltage clamp techniques. This inhibitory effect of 3α,5[α]β-THP was not observed at other receptor subtypes, suggesting it was selective for α4β2δ GABAR. Furthermore, it was prevented by a selective mutation of basic residue arginine 353 in the intracellular loop of the receptor, suggesting that this might be a putative chloride modulatory site. Expression of α4βδ GABAR increases markedly at extrasynaptic sites at the onset of puberty in female mice. At this time, 3α,5[α]β–THP decreased the inhibitory tonic current, recorded with perforated patch techniques to maintain the physiological Cl- gradient. By decreasing this shunting inhibition, 3α,5[α]β–THP increased the excitability of CA1 hippocampal pyramidal cells at puberty: These effects of the steroid were opposite to those observed before puberty when 3α,5[α]β–THP reduced neuronal excitability as a pre-synaptic effect. Behaviorally, the excitatory effect of 3α,5[α]β–THP was reflected as an increase in anxiety at the onset of puberty in female mice. Taken together, these findings suggest that the emergence of α4β2δ GABAR at the onset of puberty reverses the effect of a stress steroid. These findings may be relevant for the mood swings and increased response to stressful events reported in adolescence.

Homeostatic synaptic plasticity (HSP) is important for maintaining neurons' excitability within the dynamic range and for protecting neurons from unconstrained LTP that can cause breakdown of synapse specificity (Turrigiano, 2008). Knowledge of the molecular mechanism underlying this phenomenon remains incomplete, especially for the rapid form of HSP. In order to test whether HSP in adulthood depends on an F-actin binding protein – drebrin A, mice deleted of the adult isoform of drebrin (DAKO) but retaining the embryonic isoform (drebrin E) were generated. HSP was assayed by determining whether the NR2A subunit of NMDA receptors (NMDARs) can rise rapidly within spines following the application of an NMDAR antagonist, D-APV, onto the cortical surface. Electron microscopic immunocytochemistry revealed that, as expected, the D-APV treatment of WT mouse cortex increased the proportion of NR2A-immunolabeled spines within 30 min, relative to basal levels in hemispheres treated with an inactive enantiomer, L-APV. This difference was significant at the postsynaptic membrane and postsynaptic density (i.e., synaptic junction) as well as at non-synaptic sites within spines and was not accompanied by spine size changes. In contrast, the D-APV treatment of DAKO brains did not augment NR2A labeling within the spine cytoplasm or at the synaptic junction, even though basal levels of NR2A were not significantly different from those of WT cortices. These findings indicate that drebrin A is required for the rapid (<30 min) form of HSP at excitatory synapses of adult cortices while drebrin E is sufficient for maintaining basal NR2A levels within spines.

The onset of puberty defines a developmental stage when some learning processes are diminished, but the mechanism for this deficit remains unknown. We found that, at puberty, expression of inhibitory α4βδ γ-aminobutyric acid type A (GABAA) receptors (GABAR) increases perisynaptic to excitatory synapses in CA1 hippocampus. Shunting inhibition via these receptors reduced N-methyl-D-aspartate receptor activation, impairing induction of long-term potentiation (LTP). Pubertal mice also failed to learn a hippocampal, LTP-dependent spatial task that was easily acquired by δ−/− mice. However, the stress steroid THP (3αOH-5α[β]-pregnan-20-one), which reduces tonic inhibition at puberty, facilitated learning. Thus, the emergence of α4βδ GABARs at puberty impairs learning, an effect that can be reversed by a stress steroid.

SUMMARY

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.

Presenilins are the major causative genes of familial Alzheimer's disease (AD). Our previous study has demonstrated essential roles of presenilins in memory and neuronal survival. Here, we explore further how loss of presenilins results in age-related, progressive neurodegeneration in the adult cerebral cortex, where the pathogenesis of AD occurs. To circumvent the requirement of presenilins for embryonic development, we used presenilin conditional double knockout (Psen cDKO) mice, in which presenilin inactivation is restricted temporally and spatially to excitatory neurons of the postnatal forebrain beginning at 4 weeks of age. Increases in the number of degenerating (Fluoro-Jade B+, 7.6-fold) and apoptotic (TUNEL+, 7.4-fold) neurons, which represent ∼0.1% of all cortical neurons, were first detected at 2 months of age when there is still no significant loss of cortical neurons and volume in Psen cDKO mice. By 4 months of age, significant loss of cortical neurons (∼9%) and gliosis was found in Psen cDKO mice. The apoptotic cell death is associated with caspase activation, as shown by increased numbers of cells immunoreactive for active caspases 9 and 3 in the Psen cDKO cortex. The vulnerability of cortical neurons to loss of presenilins is region-specific with cortical neurons in the lateral cortex most susceptible. Compared to the neocortex, the increase in apoptotic cell death and the extent of neurodegeneration are less dramatic in the Psen cDKO hippocampus, possibly in part due to increased neurogenesis in the aging dentate gyrus. Neurodegeneration is also accompanied with mitochondrial defects, as indicated by reduced mitochondrial density and altered mitochondrial size distribution in aging Psen cortical neurons. Together, our findings show that loss of presenilins in cortical neurons causes apoptotic cell death occurring in a very small percentage of neurons, which accumulates over time and leads to substantial loss of cortical neurons in the aging brain. The low occurrence and significant delay of apoptosis among cortical neurons lacking presenilins suggest that loss of presenilins may induce apoptotic neuronal death through disruption of cellular homeostasis rather than direct activation of apoptosis pathways.

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 3′-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.

To identify glutamate transporters expressed in forebrain neurons, we prepared a cDNA library from rat forebrain neuronal cultures, previously shown to transport glutamate with high affinity and capacity. Using this library, we cloned two forms, varying in the C terminus, of the glutamate transporter GLT1. This transporter was previously found to be localized exclusively in astrocytes in the normal mature brain. Specific antibodies against the C-terminal peptides were used to show that forebrain neurons in culture express both GLT1a and GLT1b proteins. The pharmacological properties of glutamate transport mediated by GLT1a and GLT1b expressed in COS-7 cells and in neuronal cultures were indistinguishable. Both GLT1a and GLT1b were upregulated in astrocyte cultures by exposure to dibutyryl cAMP. We next investigated the expression of GLT1b in vivo. Northern blot analysis of forebrain RNA revealed two transcripts of ~3 and 11 kb that became more plentiful with developmental age. Immunoblot analysis showed high levels of expression in the cortex, hippocampus, striatum, thalamus, and midbrain. Pre-embedding electron microscopic immunocytochemistry with silver-enhanced immunogold detection was used to localize GLT1b in vivo. In the rat somatosensory cortex, GLT1b was clearly expressed in neurons in presynaptic terminals and dendritic shafts, as well as in astrocytes. The presence of GLT1b in neurons may offer a partial explanation for the observed uptake of glutamate by presynaptic terminals, for the preservation of input specificity at excitatory synapses, and may play a role in the pathophysiology of excitotoxicity.

Acetylcholine (ACh) is believed to underlie mechanisms of arousal and attention in mammals. ACh also has a demonstrated functional effect in visual cortex that is both diverse and profound. We have reported previously that cholinergic modulation in V1 of the macaque monkey is strongly targeted toward GABAergic interneurons. Here we examine the localization of m1 and m2 muscarinic receptor subtypes across subpopulations of GABAergic interneurons—identified by their expression of the calcium-binding proteins parvalbumin, calbindin, and calretinin—using dual-immunofluorescence confocal microscopy in V1 of the macaque monkey. In doing so, we find that the vast majority (87%) of parvalbumin-immunoreactive neurons express m1-type muscarinic ACh receptors. m1 receptors are also expressed by 60% of calbindin-immunoreactive neurons and 40% of calretinin-immunoreactive neurons. m2 AChRs, on the other hand, are expressed by only 31% of parvalbumin neurons, 23% of calbindin neurons, and 25% of calretinin neurons. Parvalbumin-immunoreactive cells comprise ≈75% of the inhibitory neuronal population in V1 and included in this large subpopulation are neurons known to veto and regulate the synchrony of principal cell spiking. Through the expression of m1 ACh receptors on nearly all of these PV cells, the cholinergic system avails itself of powerful control of information flow through and processing within the network of principal cells in the cortical circuit.

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 organdies. 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.

Synaptic activities alter synaptic strengths at the axospinous junctions, and such changes are often accompanied by changes in the size of the postsynaptic spines. We have been exploring the idea that drebrin A, a neuron-specific actin-binding protein localized on the postsynaptic side of excitatory synapses, may be a molecule that links synaptic activity to the shape and content of spines. Here, we performed electron microscopic immunocytochemistry with the nondiffusible gold label to explore the relationship among levels of drebrin A, the NR2A subunit of N-methyl-D-aspartate receptors, and the size of spines in the perirhinal cortex of adult mouse brains. In contrast to the membranous localization within neonatal spines, most immunogold particles for drebrin A were localized to the cytoplasmic core region of spines in mature spines. This distribution suggests that drebrin within adult spines may reorganize the F-actin network at the spine core, in addition to its known neonatal role in spine formation. Drebrin A-immunopositive (DIP) spines exhibited larger spine head areas and longer postsynaptic densities (PSDs) than drebrin A-immunonegative (DIN) spines (P < 0.001). Furthermore, spine head area and PSD lengths correlated positively with drebrin A levels (r = 0.47 and 0.40). The number of synaptic NR2A immunolabels was also higher in DIP spines than in DIN spines, whereas their densities per unit lengths of PSD were not significantly different. These differences between the DIP and the DIN spines indicate that spine sizes and synaptic protein composition of mature brains are regulated, at least in part, by drebrin A levels.

Mice with knock-in of two mutations that affect beta amyloid processing and levels (2xKI) exhibit impaired spatial memory by 9–12 months of age, together with synaptic plasticity dysfunction in the hippocampus. The goal of this study was to identify changes in the molecular and structural characteristics of synapses that precede and thus could exert constraints upon cellular mechanisms underlying synaptic plasticity. Drebrin A is one protein reported to modulate spine sizes and trafficking of proteins to and from excitatory synapses. Thus, we examined levels of drebrin A within postsynaptic spines in the hippocampus and entorhinal cortex. Our electron microscopic immunocytochemical analyses reveal that, by 6 months, the proportion of hippocampal spines containing drebrin A is reduced and this change is accompanied by an increase in the mean size of spines and decreased density of spines. In the entorhinal cortex of 2xKI brains, we detected no decrement in the proportion of spines labeled for drebrin A and no significant change in spine density at 6 months, but rather a highly significant reduction in the level of drebrin A immunoreactivity within each spine. These changes are unlike those observed for the somatosensory cortex of 2xKI mice, in which synapse density and drebrin A immunoreactivity levels remain unchanged at 6 months and older. These results indicate that brains of 2xKI mice, like those of humans, exhibit regional differences of vulnerability, with the hippocampus exhibiting the first signatures of structural changes that, in turn, may underlie the emergent inability to update spatial memory in later months.

The final wiring of the brain occurs after birth and is governed by early experience. A protein called MAP2 seems to take part in the molecular events that underlie the brain’s ability to change

NMDA receptor (NMDAR) activation requires concurrent membrane depolarization, and glutamatergic synapses lacking AMPA receptors (AMPARs) are often considered “silent” in the absence of another source of membrane depolarization. During the second postnatal week, NMDA currents can be enhanced in rat auditory cortex through activation of the α7 nicotinic acetylcholine receptor (α7nAChR). Electrophysiological results support a mainly presynaptic role for α7nAChR at these synapses. However, immunocytochemical evidence that α7nAChR is prevalent at postsynaptic sites of glutamatergic synapses in hippocampus and neocortex, along with emerging electrophysiological evidence for postsynaptic nicotinic currents in neocortex and hippocampus, has prompted speculation that α7nAChR allows for activation of NMDAR postsynaptically at synapses lacking AMPAR. Here we used dual immunolabeling and electron microscopy to examine the distribution of α7nAChR relative to AMPAR (GluR1, GluR2, and GluR3 subunits combined) at excitatory synapses in somatosensory cortex of adult and 1-week-old rats. α7nAChR occurred discretely over most of the thick postsynaptic densities in all cortical layers of both age groups. AMPAR immunoreactivity was also detectable at most synapses; its distribution was independent of that of α7nAChR. In both age groups, approximately one-quarter of asymmetrical synapses were α7nAChR positive and AMPAR negative. The variability of postsynaptic α7nAChR labeling density was greater at postnatal day (PD) 7 than in adulthood, and PD 7 neuropil contained a subset of small AMPA receptor-negative synapses with a high density of α7nAChR immunoreactivity. These observations support the idea that acetylcholine receptors can aid in activating glutamatergic synapses and work together with AMPA receptors to mediate postsynaptic excitation throughout life.

The formation of neuronal circuits during development involves a combination of synapse stabilization and elimination events. Synaptic adhesion molecules are thought to play an important role in synaptogenesis, and several trans-synaptic adhesion systems that promote the formation and maturation of synapses have been identified. The neuroligin–neurexin complex is a heterophilic adhesion system that promotes assembly and maturation of synapses through bidirectional signaling. In this protein complex, postsynaptic neuroligins are thought to interact trans-synaptically with presynaptic neurexins. However, the subcellular localization of neurexins has not been determined. Using immunoelectron microscopy, we found that endogenous neurexins and epitope-tagged neurexin-1β are localized to axons and presynaptic terminals in vivo. Unexpectedly, neurexins are also abundant in the postsynaptic density. cis-expression of neurexin-1β with neuroligin-1 inhibits trans-binding to recombinant neurexins, blocks the synaptogenic activity of neuroligin-1, and reduces the density of presynaptic terminals in cultured hippocampal neurons. Our results demonstrate that the function of neurexin proteins is more diverse than previously anticipated and suggest that postsynaptic cis-interactions might provide a novel mechanism for silencing the activity of a synaptic adhesion complex.

Recent results indicate that astrocytic β-adrenergic receptors (βAR) participate in noradrenergic modulation of synaptic activity. In this study, we sought to examine whether neural activity can, in turn, regulate astrocytic βAR. To address this question, an antiserum that recognizes β-adrenergic receptors (βAR) specifically in astrocytes was used to assess the distribution of the receptors across ocular dominance columns in V1 of two monocular and four visually intact adult monkeys. Cytochrome oxidase histochemistry (CO) was used to identify the position of the cortical laminae and of the ocular dominance columns receiving visual inputs from the intact and enucleated eyes. This stain revealed the expected pattern within V1 of monocular monkeys –i.e. darker and lighter bands of equal widths (ca. 500 μm) spanning laminae 4–6, each associated with larger and smaller blobs, respectively, in lamina 2/3. Alignment of CO sections with adjacent sections stained for astrocytic βAR by the immunoperoxidase method revealed intense βAR-like immunoreactivity (βAR-li) in the superficial laminae, a slightly weaker staining in the infragranular laminae and weakest staining in lamina 4C. Within lamina 4C, a prominent striped pattern was evident. The darker bands of the stripe closely matched widths and positions of the lighter CO columns associated with the enucleated eye. On the other hand, immunocytochemical staining for the astrocytic intermediate filament protein, GFAP, within V1 of monocular monkeys revealed no inter-columnar difference in the density of astrocytic cell bodies or processes. Nissl stain also revealed no overt inter-columnar differences in cell density. V1 of visually intact monkeys exhibited a similar laminar distribution pattern of βAR-li and of CO. Within lamina 4C, βAR-li was uniformly faint and CO staining was uniformly intense. This suggests that the striped pattern of βAR-li seen in lamina 4C of monocular monkeys results from elevation of the βAR-antigen within the inactive columns. The results indicate that astrocytic βAR density is regulated by local neural activity. The mechanisms regulating βAR density are likely to be independent of those regulating glial cell proliferation or GFAP synthesis. In vitro experimental results of others suggest that elevation of astrocytic βAR may be a mechanism compensating for chronic neural inactivity, since the coincident release of noradrenaline with visual stimulation would elevate neuropil excitability via the astrocytic mechanism of (1) decreasing the uptake of neuronally released L-glutamate; (2) increasing GABA uptake; and (3) stimulating glycogenolysis. Alternatively, the changes in βAR-li may reflect an up-regulation of the receptors within inactive columns due to reduced levels of noradrenaline release.

It has long been recognized that noradrenaline, the most abundant catecholamine within the visual cortex, plays important roles in modulating the sensitivity of cortical neurons to visual stimuli. However, whether or not these noradrenaline effects are confined to a discrete synaptic specialization or mediated by diffuse modulation of a group of synapses has remained an issue open for debate. The aim of this study was to examine the cellular basis for noradrenaline action within the visual cortex of adult rats and cats. To this end, I used electron microscopic immunocytochemistry to examine the relationship between (1) catecholamine axon terminals and β-adrenergic receptors (βAR), which, together, may define the effective sphere of noradrenaline modulation; and then (2) these putative sites for catecholamine modulation and axospinous asymmetric junctions where excitatory neurotransmission is likely to dominate. Antibodies against βAR were used at light and electron microscopic levels on the visual cortex of rat and cat. Rat visual cortex was also labeled simultaneously for βAR and the catecholamine-synthesizing enzyme, tyrosine hydroxylase (TH), to determine the ultrastructural relationships between catecholamine terminals and βAR. Immunoperoxidase labeling revealed that βAR404, a polyclonal antibody directed against the C-terminal tail of hamster lung βAR (β2-type), recognized astrocytic processes predominantly. In contrast, βAR248, a polyclonal antibody directed against the third cytoplasmic loop, recognized neuronal perikarya as observed in previous studies. Dual labeling for βAR404 and TH revealed that catecholamine axon terminals that contained numerous vesicles formed direct contacts with astrocytic processes exhibiting βAR404 immunoreactivity. However, some catecholamine axon terminals that lacked dense clusters of vesicles were positioned away from βAR404-immunoreactive astrocytes. Frequently, βAR-immunoreactive astrocytic processes surrounded asymmetric axospinous junctions while also contacting catecholamine axon terminals. These observations support the possibility that, through activation of astrocytic βAR, noradrenaline modulates astrocytic uptake mechanism for excitatory amino acids, such as L-glutamate. Astrocytic βAR might also define the effective sphere of catecholamine modulation through alterations in the morphology of distal astrocytic processes and the permeability of gap junctions formed between astrocytes.

Synaptic plasticity is dependent upon the differential sorting, delivery and retention of neurotransmitter receptors, yet the mechanisms underlying these processes are poorly understood. In the present study, we have found that differential sorting of glutamate receptor subtypes begins within the endoplasmic reticulum (ER) of rat hippocampal neurons. While AMPARs are trafficked to the plasma membrane via the conventional somatic Golgi network, NMDARs are diverted from the somatic ER into a specialized ER sub-compartment that bypasses somatic Golgi, merging instead with dendritic Golgi outposts. Intriguingly, this ER sub-compartment is composed of highly mobile vesicles containing the NMDAR subunits NR1 and NR2B, the microtubule-dependent motor protein KIF17, and the postsynaptic adaptor proteins CASK and SAP97. Furthermore, our data demonstrate that the retention and trafficking of NMDARs within this ER sub-compartment requires both CASK and SAP97. These data indicate that NMDARs are sorted away from AMPARs via a non-conventional secretory pathway that utilizes dendritic Golgi outposts.

Greater than 90% of familial Alzheimer’s disease (AD) is linked to mutations of presenilin (PS), and the loss of PS function altogether within mouse brains by conditional double knockout of the PS 1 and 2 genes (PS-cDKO) leads to age-dependent emergence of AD phenotypes, including neurodegeneration and reduced synaptic plasticity in the hippocampal CA1. The goal of our study was to identify the ultrastructural and molecular changes at synapses in the hippocampal CA1 of this PS-cDKO mouse model of AD. We examined the asymmetric (excitatory) synapses formed on apical dendrites of CA1 pyramidal neurons at 2 months postnatal, an age when AD-like symptoms emerge but brain morphology, as assessed by light microscopy, is still normal. Our quantitative electron microscopic analyses confirm that PS-cDKO hippocampi at 2 months postnatal do not yet exhibit synapse losses or spine size alterations. However, immunocytochemistry reveals that the same region exhibits a 28% increase in the proportion of spines labeled for the NR2A subunits of NMDA receptors (NMDAR), with a 31% increase specifically at postsynaptic densities and a concomitant reduction of these subunits at nonsynaptic sites within spine heads. In contrast, no change in levels or the distribution pattern of NR2B subunit levels were detected within spine heads. Presynaptically, NR2A levels are elevated at axo-spinous junctions and these may contribute to the timing-dependent, long-term depression. These observations point to an early-onset trapping of NMDAR at synapses that are subtle but may underlie the reduced synaptic plasticity at 2 months of age and excitotoxicity at later stages.

We have shown previously that auditory experience regulates the maturation of excitatory synapses in the auditory cortex (ACx). In this study, we used electron microscopic immunocytochemistry to determine whether the heightened excitability of the ACx following neonatal sensorineural hearing loss (SNHL) also involves pre- or postsynaptic alterations of GABAergic synapses. SNHL was induced in gerbils just prior to the onset of hearing (postnatal day 10). At P17, the gamma-aminobutyri acid type A (GABAA) receptor's β2/3-subunit (GABAAβ2/3) clusters residing at plasma membranes in layers 2/3 of ACx was reduced significantly in size (P < 0.05) and number (P < 0.005), whereas the overall number of immunoreactive puncta (intracellular + plasmalemmal) remained unchanged. The reduction of GABAAβ2/3 was observed along perikaryal plasma membranes of excitatory neurons but not of GABAergic interneurons. This cell-specific change can contribute to the enhanced excitability of SNHL ACx. Presynaptically, GABAergic axon terminals were significantly larger but less numerous and contained 47% greater density of glutamic acid decarboxylase immunoreactivity (P < 0.05). This suggests that GABA synthesis may be upregulated by a retrograde signal arising from lowered levels of postsynaptic GABAAR. Thus, both, the pre- and postsynaptic sides of inhibitory synapses that form upon pyramidal neurons of the ACx are regulated by neonatal auditory experience.

Acetylcholine (ACh) influences attention, short-term memory, and sleep/waking transitions, through its modulatory influence on cortical neurons. It has been proposed that behavioral state changes mediated by ACh result from its selective effects on the intrinsic membrane properties of diverse cortical inhibitory interneuron classes. ACh has been widely shown to reduce the strength of excitatory (glutamatergic) synapses. But past studies using extracellular stimulation have not been able to examine the effects of ACh on local cortical connections important for shaping sensory processing, Here, using dual intracellular recording in slices of rat somatosensory cortex, we show that reduction of local excitatory input to inhibitory neurons by ACh is coupled to differences in the underlying short-term synaptic plasticity (STP). In synapses with short-term depression, where successive evoked excitatory postsynaptic potentials (EPSPs; >5 Hz) usually diminish in strength (short-term depression), cholinergic agonist (5–10 μM carbachol (CCh)) reduced the amplitude of the first EPSP in an evoked train, but CCh's net effect on subsequent EPSPs rapidly diminished. In synapses where successive EPSPs increased in strength (facilitation), the effect of CCh on later EPSPs in an evoked train became progressively greater. The effect of CCh on both depressing and facilitating synapses was blocked by the muscarinic antagonist, 1–5 μM atropine. It is suggested that selective influence on STP contributes fundamentally to cholinergic ″ switching ″ between cortical rhythms that underlie different behavioral states.

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 GABAA 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 GABAA receptors by THP provides a mechanism for anxiety at puberty.

Developmental hearing impairments compromise sound discrimination, speech acquisition, and cognitive function; however, the adjustments of functional properties in the primary auditory cortex (A1) remain unknown. We induced sensorineural hearing loss (SNHL) in developing gerbils and then reared the animals for several days. The intrinsic membrane and synaptic properties of layer 2/3 pyramidal neurons were subsequently examined in a thalamocortical brain slice preparation with whole-cell recordings and electron microscopic immunocytochemistry. SNHL neurons displayed a depolarized resting membrane potential, an increased input resistance, and a higher incidence of sustained firing. They also exhibited significantly larger thalamocortically and intracortically evoked excitatory synaptic responses, including a greater susceptibility to the NMDA receptor antagonist AP-5 and the NR2B subunit antagonist ifenprodil. This correlated with an increase in NR2B labeling of asymmetric synapses, as visualized ultrastructurally. Furthermore, decreased frequency and increased amplitude of miniature EPSCs (mEPSCs) in SNHL neurons suggest that a decline in presynaptic release properties is compensated by an increased excitatory response. To verify that the increased thalamocortical excitation was elicited by putative monosynaptic connections, minimum amplitude ventral medial geniculate nucleus-evoked EPSCs were recorded. These minimum-evoked responses were of larger amplitude, and the NMDAergic currents were also larger and longer in SNHL neurons. These findings were supported by significantly longer AP-5-sensitive durations and larger amplitudes of mEPSCs. Last, the amplitudes of intracortically evoked monosynaptic and polysynaptic GABAergic inhibitory synaptic responses were significantly smaller in SNHL neurons. These alterations in cellular properties after deafness reflect an attempt by A1 to sustain an operative level of cortical excitability that may involve homeostatic mechanisms.

We studied the cholinergic modulation of glutamatergic transmission between neighboring layer 5 regular-spiking pyramidal neurons in somatosensory cortical slices from young rats (P10-P26). Brief bath application of 5-10 μM carbachol, a nonspecific cholinergic agonist, decreased the amplitude of evoked unitary excitatory postsynaptic potentials (EPSPs). This effect was blocked by 1 μM atropine, a muscarinic receptor antagonist. Nicotine (10 μM), in contrast to carbachol, reduced EPSPs in nominally magnesium-free solution but not in the presence of 1 mM Mg+2, indicating the involvement of NMDA receptors. Likewise, when the postsynaptic cell was depolarized under voltage clamp to allow NMDA receptor activation in the presence of 1 mM Mg+2, synaptic currents were reduced by nicotine. Nicotinic EPSP reduction was prevented by the NMDA receptor antagonist D-AP5 (50 μM) and by the nicotinic receptor antagonist mecamylamine (10 μM). Both carbachol and nicotine reduced short-term depression of EPSPs evoked by 10 Hz stimulation, indicating that EPSP reduction happens via reduction of presynaptic glutamate release. In the case of nicotine, several possible mechanisms for NMDAR-dependent EPSP reduction are discussed. As a result of NMDA receptor dependence, nicotinic EPSP reduction may serve to reduce the local spread of cortical excitation during heightened sensory activity.

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.