Immunoelectron Microscopy of Cryofixed and Freeze-Substituted Plant Tissues.

Cryofixation and freeze-substitution techniques provide excellent preservation of plant ultrastructure. The advantage of cryofixation is not only in structural preservation, as seen in the smooth plasma membrane, but also in the speed in arresting cell activity. Immunoelectron microscopy reveals the subcellular localization of molecules within cells. Immunolabeling in combination with cryofixation and freeze-substitution techniques provides more detailed information on the immunoelectron-microscopic localization of molecules in the plant cell than can be obtained from chemically fixed tissues. Here, we introduce methods for immunoelectron microscopy of cryofixed and freeze-substituted plant tissues.

The ultrastructure of lamellar stack astrocytes.

Astrocytes support neurons and map out nonoverlapping domains in grey matter of the brain. The astrocytes of the glia limitans, however, do overlap. Using ultrastructural tools and immunogold histochemistry a subtype of astrocyte able to assemble large lamellar stacks was investigated at the ventral surface of the brain near the hypothalamus. Lamellar stacks were subsequently discovered also in the internal glia limitans of the epithalamus. Circular lamellar stacks containing AQP4 water channels surround neuronal processes, and might serve as osmosensors. The lamellar stacks are well-organized and can form over 100 membrane layers between neuropil and the basal membrane, but a barrier function is not obvious from the noncontinuous character of the stacks along the glia limitans.

Inflammation alters trafficking of extrasynaptic AMPA receptors in tonically firing lamina II neurons of the rat spinal dorsal horn.

Peripheral inflammation alters AMPA receptor (AMPAR) subunit trafficking and increases AMPAR Ca(2+) permeability at synapses of spinal dorsal horn neurons. However, it is unclear whether AMPAR trafficking at extrasynaptic sites of these neurons also changes under persistent inflammatory pain conditions. Using patch-clamp recording combined with Ca(2+) imaging and cobalt staining, we found that, under normal conditions, an extrasynaptic pool of AMPARs in rat substantia gelatinosa (SG) neurons of spinal dorsal horn predominantly consists of GluR2-containing Ca(2+)-impermeable receptors. Maintenance of complete Freund's adjuvant (CFA)-induced inflammation was associated with a marked enhancement of AMPA-induced currents and [Ca(2+)](i) transients in SG neurons, while, as we previously showed, the amplitude of synaptically evoked AMPAR-mediated currents was not changed 24 h after CFA. These findings indicate that extrasynaptic AMPARs are upregulated and their Ca(2+) permeability increases dramatically. This increase occurred in SG neurons characterized by intrinsic tonic firing properties, but not in those exhibited strong adaptation. This increase was also accompanied by an inward rectification of AMPA-induced currents and enhancement of sensitivity to a highly selective Ca(2+)-permeable AMPAR blocker, IEM-1460. Electron microcopy and biochemical assays additionally showed an increase in the amount of GluR1 at extrasynaptic membranes in dorsal horn neurons 24h post-CFA. Taken together, our findings indicate that CFA-induced inflammation increases functional expression and proportion of extrasynaptic GluR1-containing Ca(2+)-permeable AMPARs in tonically firing excitatory dorsal horn neurons, suggesting that the altered extrasynaptic AMPAR trafficking might participate in the maintenance of persistent inflammatory pain.

Critical period plasticity is disrupted in the barrel cortex of FMR1 knockout mice.

Alterations in sensory processing constitute prominent symptoms of fragile X syndrome; however, little is known about how disrupted synaptic and circuit development in sensory cortex contributes to these deficits. To investigate how the loss of fragile X mental retardation protein (FMRP) impacts the development of cortical synapses, we examined excitatory thalamocortical synapses in somatosensory cortex during the perinatal critical period in Fmr1 knockout mice. FMRP ablation resulted in dysregulation of glutamatergic signaling maturation. The fraction of silent synapses persisting to later developmental times was increased; there was a temporal delay in the window for synaptic plasticity, while other forms of developmental plasticity were not altered in Fmr1 knockout mice. Our results indicate that FMRP is required for the normal developmental progression of synaptic maturation, and loss of this important RNA binding protein impacts the timing of the critical period for layer IV synaptic plasticity.

Constant illumination induces Alzheimer-like damages with endoplasmic reticulum involvement and the protection of melatonin.

Most patients with Alzheimer's disease (AD) present decreased levels of melatonin, a day-night rhythm-related hormone. To investigate the role of melatonin deficiency in AD, we used constant illumination to interrupt melatonin metabolism and measured some of the AD-like alterations in rats. Concomitant with decreased serum melatonin, the rats developed spatial memory deficits, tau hyperphosphorylation at multiple sites, activation of glycogen synthase kinase-3 and protein kinase A, as well as suppression of protein phosphatase-1. Prominent oxidative damage and organelle lesions, demonstrated by increased expression of endoplasmic reticulum (ER) stress-related proteins including BiP/GRP78 and CHOP/GADD153, decreased number of rough ER and free ribosome, thinner synapses, and increased superoxide dismutase and monoamine oxidase were also observed in the light exposed rats. Simultaneous supplement of melatonin partially arrested the behavioral and molecular impairments. It is suggested that melatonin deficiency may be an upstream effector responsible for the AD-like behavioral and molecular pathologies with ER stress-involved mechanisms.

Angiotensin II-induced hypertension differentially affects estrogen and progestin receptors in central autonomic regulatory areas of female rats.

Estrogen receptor (ER) activation in central autonomic nuclei modulates arterial blood pressure (ABP) and counteracts the deleterious effect of hypertension. We tested the hypothesis that hypertension, in turn, influences the expression and trafficking of gonadal steroid receptors in central cardiovascular circuits. Thus, we examined whether ER- and progestin receptor (PR)-immunoreactivity (ir) are altered in medullary and hypothalamic autonomic areas of cycling rats following chronic infusion of the hypertensive agent, angiotensin II (AngII). After 1 week AngII-infusion, systolic ABP was elevated from 103+/-4 to 172+/-8 mmHg (p<0.05; N=8/group) and all rats were in diestrus (low estrogen). In AngII-infused rats the number of PR-immunoreactive nuclei was reduced (-72%) compared to saline-infused controls also in diestrus (p<0.05). Furthermore, the intensity of ERalpha-ir increased selectively in nuclei (16%) and cytoplasm (21%) of cells in the commissural nucleus of the solitary tract (cNTS; p<0.05) while neither the number nor intensity of ERbeta-labeled cells changed (p>0.05). Following chronic AngII-infusion, electron microscopy showed a higher cytoplasmic-to-nuclear ratio of ERalpha-labeling selectively in tyrosine hydroxylase (TH)-labeled neurons in the cNTS. Furthermore, AngII-infusion increased ERalpha-ir in the cytosol of TH- and non-TH neuronal perikarya and increased the amount of ERalpha-ir associated with endoplasmic reticulum only in TH-containing perikarya. The data suggest that hypertension modulates the expression and subcellular distribution of ERalpha and PR in central autonomic regions involved in blood pressure control. Considering that ERalpha counteracts the central and peripheral effects of AngII, these receptor changes may underlie adaptive responses that protect females from the deleterious effects of hypertension.

Structural correlates of efficient GABAergic transmission in the basal ganglia-thalamus pathway.

Giant inhibitory terminals with multiple synapses, the counterparts of excitatory "detonator" or "driver" terminals, have not been described in the forebrain. Using three-dimensional reconstructions of electron microscopic images, we quantitatively characterize a GABAergic pathway that establishes synaptic contacts exclusively via multiple synapses. Axon terminals of the nigrothalamic pathway formed, on average, 8.5 synapses on large-diameter dendrites and somata of relay cells in the ventromedial nucleus of the rat thalamus. All synapses of a given terminal converged on a single postsynaptic element. The vast majority of the synapses established by a single terminal were not separated by astrocytic processes. Nigrothalamic terminals in the macaque monkey showed the same ultrastructural features both in qualitative and quantitative terms (the median number of synapse per target was also 8.5). The individual synapses were closely spaced in both species. The nearest-neighbor synaptic distances were 169 nm in the rat and 178 nm in the monkey. The average number of synapses within 0.75 microm from any given synapse was 3.8 in the rat and 3.5 in the monkey. The arrangement of synapses described in this study creates favorable conditions for intersynaptic spillover of GABA among the multiple synapses of a single bouton, which can result in larger charge transfer. This could explain faithful and efficient GABAergic signal transmission in the nigrothalamic pathway in the healthy condition and during Parkinson's disease. In addition, our structural data suggest that the rodent nigrothalamic pathway can be a valid model of the primate condition, when the mechanism of GABAergic transmission is studied.

Neurokinin-1 receptors in cholinergic neurons of the rat ventral pallidum have a predominantly dendritic distribution that is affected by apomorphine when combined with startle-evoking auditory stimulation.

Cholinergic neurons of the basal forebrain are implicated in startle reflex inhibition by a prior weak stimulus often referred to as prepulse inhibition (PPI) and used as an index of sensorimotor gating deficits in schizophrenia. Gating deficits can be produced in rodent models by acute systemic administration of apomorphine, a non-selective dopamine D1 and D2 receptor agonist that also affects trafficking of neurokinin-1 (NK(1)) receptors induced by startle evoking auditory stimulation (AS) in midbrain neurons. We used electron microscopic immunolabeling of NK(1) receptors and the vesicular acetylcholine transporter (VAchT) to test the hypothesis that the subcellular distributions of these receptors in cholinergic neurons of the rat ventral pallidum are subject to a similar regulation. In vehicle controls, NK(1) immunogold was often seen near cytoplasmic endomembranes in somata and large dendrites, but was more equally distributed in cytoplasmic and plasmalemmal compartments of medium dendrites, and principally located on the plasma membrane of small dendrites. These labeling patterns appeared to be largely independent of whether the NK(1) receptor was co-expressed with VAchT, however only the medium and small VAchT-labeled dendrites showed significant treatment-specific differences in NK(1) immunogold distributions. The NK(1) receptor immunogold particle density on the plasma membrane of medium cholinergic dendrites was significantly enhanced by combined apomorphine and AS, while neither alone affected either the plasmalemmal density or the equality of the plasmalemmal and cytoplasmic distributions of NK(1) receptors in these dendrites. Small cholinergic dendrites showed a significant AS-induced increase in both the plasmalemmal and cytoplasmic density of NK(1) gold particles, and an apomorphine-induced disruption of the preferential plasmalemmal targeting of the NK(1) receptors. These results provide ultrastructural evidence that NK(1) receptors in cholinergic neurons of the ventral pallidum have subcellular locations and plasticity conducive to active involvement in dopamine-dependent sensorimotor processing.

Fewer driver synapses in higher order than in first order thalamic relays.

We used electron microscopy to determine the relative numbers of the three synaptic terminal types, RL (round vesicle, large terminal), RS (round vesicles, small terminal), and F (flattened vesicles), found in several representative thalamic nuclei in cats chosen as representative examples of first and higher order thalamic nuclei, where the first order nuclei relay subcortical information mainly to primary sensory cortex, and the higher order nuclei largely relay information from one cortical area to another. The nuclei sampled were the first order ventral posterior nucleus (somatosensory) and the ventral portion of the medial geniculate nucleus (auditory), and the higher order posterior nucleus (somatosensory) and the medial portion of the medial geniculate nucleus (auditory). We found that the relative percentage of synapses from RL terminals varied significantly among these nuclei, these values being higher for first order nuclei (12.6% for the ventral posterior nucleus and 8.2% for the ventral portion of the medial geniculate nucleus) than for the higher order nuclei (5.4% for the posterior nucleus, and 3.5% for the medial portion of the medial geniculate nucleus). This is consistent with a similar analysis of first and higher order nuclei for the visual system (the lateral geniculate nucleus and pulvinar, respectively). Since synapses from RL terminals represent the main information to be relayed, whereas synapses from F and RS terminals are modulatory in function, we conclude that there is relatively more modulation of the thalamic relay in the cortico-thalamo-cortical higher order pathway than in first order relays.

Difference in organization of corticostriatal and thalamostriatal synapses between patch and matrix compartments of rat neostriatum.

The neostriatum, which possesses a mosaic organization consisting of patch and matrix compartments, receives glutamatergic excitatory afferents from the cerebral cortex and thalamus. Differences in the synaptic organization of these striatopetal afferents between the patch and matrix compartments were examined in the rat using confocal laser scanning and electron microscopes. Thalamostriatal terminals immunopositive for vesicular glutamate transporter (VGluT) 2 were less dense in the patch than in the matrix compartment, although the density of VGluT1-immunopositive corticostriatal terminals was almost evenly distributed in both the compartments. Quantitative analysis of ultrastructural images revealed that 84% of VGluT2-positive synapses in the patch compartment were formed with dendritic spines, whereas 70% in the matrix compartment were made with dendritic shafts. By contrast, VGluT1-positive terminals display a similar preference for specific synaptic targets in both compartments: about 80% made synapses with dendritic spines. In addition, VGluT2-positive axospinous synapses in the patch compartment were larger than the VGluT1-positive axospinous synapses in both compartments. As axospinous synapses are generally found in neuronal connections showing high synaptic plasticity, the present findings suggest that the thalamostriatal connection requires higher synaptic plasticity in the patch compartment than in the matrix compartment.

Dynamic role of postsynaptic caspase-3 and BIRC4 in zebra finch song-response habituation.

Activation of the protease caspase-3 is commonly thought to cause apoptotic cell death. Here, we show that caspase-3 activity is regulated at postsynaptic sites in brain following stimuli associated with memory (neural activation and subsequent response habituation) instead of cell death. In the zebra finch auditory forebrain, the concentration of caspase-3 active sites increases briefly within minutes after exposure to tape-recorded birdsong. With confocal and immunoelectron microscopy, we localize the activated enzyme to dendritic spines. The activated caspase-3 protein is present even in unstimulated brain but bound to an endogenous inhibitor, BIRC4 (xIAP), suggesting a mechanism for rapid release and sequestering at specific synaptic sites. Caspase-3 activity is necessary to consolidate a persistent physiological trace of the song stimulus, as demonstrated using pharmacological interference and the zenk gene habituation assay. Thus, the brain appears to have adapted a core component of cell death machinery to serve a unique role in learning and memory.

Differential synaptology of vGluT2-containing thalamostriatal afferents between the patch and matrix compartments in rats.

The striatum is divided into two compartments named the patch (or striosome) and the matrix. Although these two compartments can be differentiated by their neurochemical content or afferent and efferent projections, the synaptology of inputs to these striatal regions remains poorly characterized. By using the vesicular glutamate transporters vGluT1 and vGluT2, as markers of corticostriatal and thalamostriatal projections, respectively, we demonstrate a differential pattern of synaptic connections of these two pathways between the patch and the matrix compartments. We also demonstrate that the majority of vGluT2-immunolabeled axon terminals form axospinous synapses, suggesting that thalamic afferents, like corticostriatal inputs, terminate preferentially onto spines in the striatum. Within both compartments, more than 90% of vGluT1-containing terminals formed axospinous synapses, whereas 87% of vGluT2-positive terminals within the patch innervated dendritic spines, but only 55% did so in the matrix. To characterize further the source of thalamic inputs that could account for the increase in axodendritic synapses in the matrix, we undertook an electron microscopic analysis of the synaptology of thalamostriatal afferents to the matrix compartments from specific intralaminar, midline, relay, and associative thalamic nuclei in rats. Approximately 95% of PHA-L-labeled terminals from the central lateral, midline, mediodorsal, lateral dorsal, anteroventral, and ventral anterior/ventral lateral nuclei formed axospinous synapses, a pattern reminiscent of corticostriatal afferents but strikingly different from thalamostriatal projections arising from the parafascicular nucleus (PF), which terminated onto dendritic shafts. These findings provide the first evidence for a differential pattern of synaptic organization of thalamostriatal glutamatergic inputs to the patch and matrix compartments. Furthermore, they demonstrate that the PF is the sole source of significant axodendritic thalamic inputs to striatal projection neurons. These observations pave the way for understanding differential regulatory mechanisms of striatal outflow from the patch and matrix compartments by thalamostriatal afferents.

The very large G-protein-coupled receptor VLGR1: a component of the ankle link complex required for the normal development of auditory hair bundles.

Sensory hair bundles in the inner ear are composed of stereocilia that can be interconnected by a variety of different link types, including tip links, horizontal top connectors, shaft connectors, and ankle links. The ankle link antigen is an epitope specifically associated with ankle links and the calycal processes of photoreceptors in chicks. Mass spectrometry and immunoblotting were used to identify this antigen as the avian ortholog of the very large G-protein-coupled receptor VLGR1, the product of the Usher syndrome USH2C (Mass1) locus. Like ankle links, Vlgr1 is expressed transiently around the base of developing hair bundles in mice. Ankle links fail to form in the cochleae of mice carrying a targeted mutation in Vlgr1 (Vlgr1/del7TM), and the bundles become disorganized just after birth. FM1-43 [N-(3-triethylammonium)propyl)-4-(4-(dibutylamino)styryl) pyridinium dibromide] dye loading and whole-cell recordings indicate mechanotransduction is impaired in cochlear, but not vestibular, hair cells of early postnatal Vlgr1/del7TM mutant mice. Auditory brainstem recordings and distortion product measurements indicate that these mice are severely deaf by the third week of life. Hair cells from the basal half of the cochlea are lost in 2-month-old Vlgr1/del7TM mice, and retinal function is mildly abnormal in aged mutants. Our results indicate that Vlgr1 is required for formation of the ankle link complex and the normal development of cochlear hair bundles.

Beacon immunoreactivity in the rat hypothalamus.

Beacon (BC) is a peptide of 73 amino acids, whose gene expression was first reported in the hypothalamus of Psammomys obesus (or Israeli sand rat). To appreciate better the functional role of BC in normal rats and sand rats, the distribution of BC immunoreactivity (irBC) and its subcellular localization were studied in the brain of Sprague-Dawley rats. In the hypothalamus, intense staining was present in neurons of the supraoptic (SO), paraventricular (PVH), and accessory neurosecretory nuclei and in cell processes of median eminence. Double labeling of the hypothalamic sections with mouse monoclonal oxytocin (OT) antibody and rabbit polyclonal BC antiserum revealed that nearly all OT-immunoreactive cells from SO, PVH, and accessory neurosecretory nuclei were irBC. Double labeling of the sections with guinea pig vasopressin (VP) antiserum and BC antiserum showed that a population of VP-immunoreactive neurons was irBC. By immunoelectron microscopy, immunoreactive product was associated with mitochondrial membranes or appeared as electron-dense bodies in many PVH and SO neurons. Most of the neurosecretory granules were unstained for BC. Taken together, our results indicate the presence of beacon in the OT-containing neurons and a population of VP-containing neurons, mostly associated with mitochondrial membrane. Insofar as the amino acids sequence of beacon is identical to that of ubiquitin-like 5, it is possible that the distribution of BC immunoreactivity noted in our study is that of ubiquitin-like 5 peptide in the rat hypothalamus.

Traumatic axonal injury in the perisomatic domain triggers ultrarapid secondary axotomy and Wallerian degeneration.

Traumatic axonal injury (TAI) arising from diffuse brain injury (DBI) results in focally impaired axonal transport with progressive swelling and delayed disconnection over several hours within brainstem axons. Neocortical DBI-mediated perisomatic axotomy does not result in neuronal death, suggesting that a comparably delayed axotomy progression was responsible for this unanticipated response. To evaluate delayed perisomatic axotomy, the current study was initiated. Rats received intracerebroventricular 10-kDa dextran followed by moderate midline/central fluid percussion injury (FPI) or FPI alone. At 15, 30, 60, and 180 min post-injury, light and transmission electron microscopy identified impaired axonal transport via antibodies targeting amyloid precursor protein (APP), while double-label fluorescent microscopy explored concomitant focal axolemmal alterations via dextran-APP co-localization. At 15 min post-injury, perisomatic TAI was identified with LM within dorsolateral and ventral posterior thalamic nuclei. Using TEM, many sustaining somata and related proximal/distal axonal segments revealed normal ultrastructural detail that was continuous with focal axonal swellings characterized by cytoskeletal and organelle pathology. In other cases, axotomy was confirmed by loss of axonal continuity distal to the swelling. By 30 min post-injury, perisomatic axotomy predominated. By 60-180 min, somatic, proximal axonal segment, and swelling ultrastructure were comparable to earlier time points although swelling diameter increased. Distal axonal segment ultrastructure now revealed the initial stages of Wallerian degeneration. The site of perisomatic axotomy did not internalize dextran, suggesting that its pathogenesis occurred independent of altered axolemmal permeability. Collectively, this DBI-mediated ultrarapid perisomatic axotomy and its sequelae further illustrate the varied axonal responses to trauma.

Endocytosis of cell surface material mediates cell plate formation during plant cytokinesis.

Dividing plant cells perform a remarkable task of building a new cell wall within the cytoplasm in a few minutes. A long-standing paradigm claims that this primordial cell wall, known as the cell plate, is generated by delivery of newly synthesized material from Golgi apparatus-originated secretory vesicles. Here, we show that, in diverse plant species, cell surface material, including plasma membrane proteins, cell wall components, and exogenously applied endocytic tracers, is rapidly delivered to the forming cell plate. Importantly, this occurs even when de novo protein synthesis is blocked. In addition, cytokinesis-specific syntaxin KNOLLE as well as plasma membrane (PM) resident proteins localize to endosomes that fuse to initiate the cell plate. The rate of endocytosis is strongly enhanced during cell plate formation, and its genetic or pharmacological inhibition leads to cytokinesis defects. Our results reveal that endocytic delivery of cell surface material significantly contributes to cell plate formation during plant cytokinesis.

Genotoxic effects of chromium(VI) and cadmium(II) in human blood lymphocytes using the electron microscopy in situ end-labeling (EM-ISEL) assay.

Evaluation of genotoxic effects of potassium chromate (K2CrO4) and cadmium chloride (CdCl2) was carried out in human blood lymphocytes in vitro as measured by the electron microscopy in situ end-labeling (EM-ISEL). EM-ISEL was used to assess DNA single-strand breaks (SSBs) expressed as number of immunogold particles per microm2 of chromatin at both chromosomal and nuclear DNA levels. Human lymphocytes were cultured in supplemented RPMI medium for 72 h including treatment for 2 h with K2CrO4 (0-150 microM), CdCl2 (0-150 microM) or methyl methanesulfonate (500 microM) as a positive control. Quantification of SSBs by EM-ISEL showed that both compounds are genotoxic agents at non-cytotoxic concentrations. This study brings new information on the utility of EM-ISEL for the evaluation of genotoxicity and confirms the genotoxic effects induced by chromium and cadmium.

Dominant and recessive COL6A1 mutations in Ullrich scleroatonic muscular dystrophy.

In this study, we characterized five Ullrich scleroatonic muscular dystrophy patients (two Italians, one Belgian, and two Turks) with a clinical phenotype showing different degrees of severity, all carrying mutations localized in COL6A1. We sequenced the three entire COL6 complementary DNA. Three of five patients have recessive mutations: two patients (P1and P3) have homozygous single-nucleotide deletions, one in exon 9 and one in exon 22; one patient (P2) has a homozygous single-nucleotide substitution leading to a premature termination codon in exon 31. The nonsense mutation of P2 also causes a partial skipping of exon 31 with the formation of a premature termination codon in exon 32 in 15% of the total COL6A1 messenger RNA. The remaining two patients carry a heterozygous glycine substitution in exons 9 and 10 inside the triple-helix region; both are dominant mutations because the missense mutations are absent in the DNA of their respective parents. As for the three homozygous recessive mutations, the apparently healthy consanguineous parents all carry a heterozygous mutated allele. Here, for the first time, we report a genotype-phenotype correlation demonstrating that heterozygous glycine substitutions in the triple-helix domain of COL6A1 are dominant and responsible for a milder Ullrich scleroatonic muscular dystrophy phenotype, and that recessive mutations in COL6A1 correlate with more severe clinical and biochemical Ullrich scleroatonic muscular dystrophy phenotypes.

Mediodorsal thalamic afferents to layer III of the rat prefrontal cortex: synaptic relationships to subclasses of interneurons.

The mediodorsal nucleus of the thalamus (MD) represents the main subcortical structure that projects to the prefrontal cortex (PFC) and it regulates key aspects of the cognitive functions of this region. Within the PFC, GABA local circuit neurons shape the activity patterns and hence the "memory fields" of pyramidal cells. Although the connections between the MD and PFC are well established, the ultrastructural relationships between projecting fibers from the MD and different subclasses of GABA cells in the PFC are not known. In order to address this issue in the rat, we examined MD axons labeled by tract-tracing in combination with immunogold-silver to identify different calcium-binding proteins localized within separate populations of interneurons. Electron micrographic examination of PFC sections from these animals revealed that MD terminals made primarily asymmetric synapses onto dendritic spines and less commonly onto dendritic shafts. Most of the dendrites receiving MD synaptic input were immunoreactive for parvalbumin (ParV), whereas MD synapses onto dendrites labeled for calretinin or calbindin were less frequent. We also observed that some MD terminals were themselves immunoreactive for calcium-binding proteins, again more commonly for ParV. These results suggest that the MD exerts a dual influence on PFC pyramidal cells: direct inputs onto spines and an indirect influence mediated via synapses onto each subclass of interneurons. The apparent preferential input to ParV cells endows MD afferents with a strong indirect inhibitory influence on pyramidal neuron activity by virtue of ParV cell synapses onto soma, proximal dendrites, and axon initial segments.

Aging and subcellular localization of m2 muscarinic autoreceptor in basalocortical neurons in vivo.

By using immunohistochemical approaches at the light and electron microscopic levels, we have shown that aging modifies the subcellular distribution of the m2 muscarinic autoreceptor (m2R) differentially at somato-dendritic postsynaptic sites and at axonal presynaptic sites in cholinergic basalocortical neurons, in vivo. In cholinergic perikarya and dendrites of the nucleus basalis magnocellularis (NBM), aging is associated with a decrease of the density of m2R at the plasma membrane and in the cytoplasm, suggesting a decrease of the total number of m2R in the somato-dendritic field. In contrast, the number of substance P receptors per somato-dendritic surface was not affected. In the frontal cortex (FC), we have shown a decrease of cytoplasmic m2R density also leading to a decrease of the number of m2R per surface of varicosities but with no change of the density of m2R at the membrane. Our results suggest that the decrease of m2R in the somato-dendritic field of the NBM, but not a modification of the number of presynaptic m2 autoreceptors at the plasma membrane in the FC, could contribute to the decrease of the efficacy of cholinergic transmission observed with aging in the rat.