Cellular and subcellular sites for noradrenergic action in the monkey dorsolateral prefrontal cortex as revealed by the immunocytochemical localization of noradrenergic receptors and axons.

A series of electron microscopic immunocytochemical studies was performed to analyze subcellular sites for noradrenergic modulation in monkey prefrontal cortex. One out of 12 noradrenergic varicosities, identified by dopamine beta-hydroxylase immunocytochemistry within single ultrathin sections, forms morphologically identifiable junctions with small dendrites and spines. Accordingly, alpha2-adrenergic receptors, almost all of which are of the A-subtype, that occur in spines are localized discretely over postsynaptic membranes. alpha2-Adrenergic receptors are also found at sites along axons, dendritic shafts and astrocytic processes lacking morphologically identifiable synaptic junctions, suggesting that these receptors are activated by volume transmission. In particular, axonal alpha2-adrenergic receptors occur mostly at pre-terminal regions, suggesting that axo-axonic interactions may mediate reduction of neurotransmitter release at sites other than axo-spinous junctions by closing voltage-dependent calcium channels. These results indicate that noradrenergic modulation of prefrontal cortex involves synaptic interactions at spines of pyramidal neurons and nonsynaptic volume transmission to glia, dendritic shafts and axons.

Intrinsic connections of the rat amygdaloid complex: projections originating in the accessory basal nucleus.

The amygdaloid complex plays an important role in the detection of emotional stimuli, the generation of emotional responses, the formation of emotional memories, and perhaps other complex associational processes. These functions depend upon the flow of information through intricate and poorly understood circuitries within the amygdala. As part of an ongoing project aimed at further elucidating these circuits, we examined the intra-amygdaloid connections of the accessory basal nucleus in the rat. In addition, we examined connections of the anterior cortical nucleus and amygdalahippocampal area to determine whether portions of these nuclei should be included in the accessory basal nucleus (as some earlier studies suggest). Phaseolus vulgaris leucogglutinin was injected into different rostrocaudal levels of the accessory basal nucleus (n = 12) or into the anterior cortical nucleus (n = 3) or amygdalahippocampal area (n = 2). The major intra-amygdaloid projections from the accessory basal nucleus were directed to the medial and capsular divisions of the central nucleus, the medial division of the amygdalohippocampal area, the medial division of the lateral nucleus, the central division of the medial nucleus, and the posterior cortical nucleus. The projections originating in the anterior cortical nucleus and the lateral division of the amygdalohippocampal area differed from those originating in the accessory basal nucleus, which suggests that these areas are not part of the accessory basal nucleus. The present findings and our previous data suggest that each of the deep amygdaloid nuclei have different intra-amygdaloid connections. The pattern of these various connections suggests that information entering the amygdala from different sources can be integrated only in certain amygdaloid regions.

Cellular and subcellular distribution of alpha 2A-adrenergic receptors in the visual cortex of neonatal and adult rats.

Activation of alpha 2-adrenergic receptors (alpha 2AR) in the cerebral cortex has been shown to modulate visually guided delayed response tasks as well as anxiety and depression. We used an antiserum directed specifically against the A subtype of alpha 2AR (alpha 2AAR) to determine the cell types and subcellular sites for noradrenergic reception mediated by this receptor in the adult and the developing rat visual cortices. Light microscopic examination of adult tissue revealed numerous labeled perikarya in layers II-VI, many of which appeared distinctly pyramidal. A few perikarya in layer I also were immunoreactive. In all layers, alpha 2AAR immunoreactivity (alpha 2AAR-ir) was present within proximal dendrites and fine processes. In neonatal tissue, there was an intense, distinct band of immunoreactivity spanning the layer composed of tightly packed immature cell bodies, i.e., the cortical plate. The band dissipated as this tier differentiated postnatally into the supragranular layers. Electron microscopy showed that the supragranular layers, which contain the highest density of noradrenergic fibers, also contain the highest areal density of labeled postsynaptic junctions beyond 2 weeks of age. Throughout the ages, the majority of immunoreactivity occurred at sites which, in single ultrathin sections, appeared to be nonjunctional sites of axons, dendrites, and in glial processes. Our observations indicate that (1) both pyramidal and nonpyramidal neurons are receptive to norepinephrine via alpha 2AAR, (2) alpha 2AAR synthesis is robust prior to synaptogenesis, and (3) alpha 2AAR operates both pre- and postsynaptically.

Intrinsic connections of the rat amygdaloid complex: projections originating in the basal nucleus.

The amygdaloid complex is involved in associational processes, such as the formation of emotional memories about sensory stimuli. However, the anatomical connections through which the different amygdaloid nuclei process incoming information and communicate with the other amygdaloid nuclei, is poorly understood. As part of an ongoing project aimed at elucidating the intrinsic connections of the rat amygdaloid complex, we injected the anterograde tracer PHA-L (Phaseolus vulgaris-leucoagglutinin) into different rostrocaudal levels of the basal nucleus of the amygdala in 21 rats and analyzed the distribution of labeled fibers and terminals throughout the amygdaloid complex. The connectional analysis, together with cytoarchitectonic observations, suggested that contrary to previous notions the basal nucleus in the rat has three divisions: magnocellular, intermediate, and parvicellular. The magnocellular division has heavy reciprocal connections with the lateral portion of the parvicellular division and the intermediate division projects weakly to the parvicellular division, whereas the projection from the medial portion of the parvicellular division to the intermediate division is heavy and the lateral and medial portions of the parvicellular division are only weakly interconnected, as are the magnocellular and intermediate divisions. The main intraamygdaloid targets of the basal nucleus projections are the nucleus of the lateral olfactory tract, the anterior amygdaloid area, the medial and capsular divisions of the central nucleus, the anterior cortical nucleus, and the amygdalohippocampal area. Our findings provide the most detailed understanding of the intra-amygdala connections of the basal nucleus to date and show that the connections within the basal nucleus and between the basal nucleus and other amygdaloid areas are more widespread and topographically organized than previously recognized.

Ethnic trends in survival curves and mortality.

The nature of secular trends in survival curves has been widely debated. Fries (1984) has argued for increasing rectangularization, while Myers and Manton (1984a; 1984b) have observed increases in mean age at death with little or no change in standard deviation--arguing against rectangularization. We hypothesize that ethnic differences in mortality trends may shed light on this argument. Using California population data for 1970, 1980, and 1990, we examined ethnic differences in rectangularization using both visual and means and standard deviations analyses. The resulting patterns varied by ethnicity, gender, and type of analyses. Nearly all female groups demonstrated modest rectangularization, regardless of mean age of death, while most of the male groups did not.

The endoscope and the radiofrequency unit in DCR surgery.

Endoscopic laser-assisted dacryocystorhinostomy (DCR) offers several advantages over standard external (SE) DCR. The technique eliminates the cutaneous scar and cosmetic blemish of an external dissection and causes less surgical trauma and bleeding than SE-DCR, with shortened postoperative recovery time and lessened postoperative pain. However, the equipment is expensive. We modified this technique using simple instruments such as the curette, Kerrison punch, Freer elevator, Storz endoscope, Ellman Surgitron unit, and the Javate DCR electrodes instead of the laser. Fifty patients with epiphora and nasolacrimal obstruction underwent surgery with our new technique. Fifty age-matched, paired external DCR were performed, and comparisons were made with the endoscopic procedure described. If preoperative epiphora was resolved and nasolacrimal patency was confirmed by lacrimal irrigation 3 months after tube removal, the operation was considered a success. The study attained a 90% success rate for endoscopic radiofrequency-assisted DCR, as compared to a 94% success rate (p > 0.05, not statistically significant) for the SE-DCR.

Cellular and subcellular localization of NMDA-R1 subunit immunoreactivity in the visual cortex of adult and neonatal rats.

NMDA receptor activation can alter synaptic strength, cause cell death, and may modulate the release of glutamate and other neurotransmitters. Using a specific and selective antiserum directed against the R1 subunit of the NMDA receptor, we examined (1) whether NMDA receptors in the adult rat visual cortex are exclusively postsynaptic or also presynaptic and (2) whether NMDA-R1 subunits are incorporated into the plasma membrane prior to, contemporaneously, or following the formation of synapses during postnatal development. By light microscopy, NMDA-R1 immunoreactivity in the adult visual cortex is easily detectable within perikarya and proximal dendrites in laminae 2-6. Many of them have the morphological features of pyramidal neurons. In addition, fine punctate labeling is evident throughout the neuropil. Electron microscopy reveals these puncta to reside at postsynaptic densities of axospinous junctions and at fine astrocytic processes and axon terminals. In the deeper laminae, the majority of labeled profiles are astrocytic. Visual cortices of animals in their first postnatal week show concentrated immunoreactivity in a few nonpyramidal neurons within laminae that have just differentiated from the cortical plate. Electron microscopy reveals diffuse labeling along the plasma membrane of dendritic shafts lacking morphologically identifiable synaptic junctions or appositions to axons. Immunoreactivity is detectable in dendritic processes by postnatal day (PND) 2, in axonal processes by PND 4, and in astrocytic profiles by PND 14. Immunoreactivity also is detectable along the postsynaptic membrane of presumably transient axosomatic junctions. At all ages, the prevalence of NMDA-R1-immunoreactive profiles is lamina 1 > 4/5 > 6/6B. These results provide the cellular basis for NMDA receptors' participation in (1) postsynaptic membrane excitability, (2) regulation of transmitter release, (3) and, in the deeper laminae, astrocyte responses. During development, NMDA-R1 subunits are associated with the plasma membrane prior to axons' arrival while clustering of receptors to junctions may be promoted by axonal contact. Finally, spatial segregation of axonal growth cones may be mediated by NMDA-R1 subunits on these axonal processes.

Perikaryal and synaptic localization of alpha 2A-adrenergic receptor-like immunoreactivity.

Through molecular cloning, the existence of three distinct subtypes of alpha 2-adrenergic receptors (alpha 2AR)--A, B and C--has been established and are referred to as alpha 2A AR, alpha 2B AR and alpha 2CAR. Due to limitations in pharmacological tools, it has been difficult to ascribe the role of each subtype to the central functions of alpha 2AR. In situ hybridization studies have provided valuable information regarding their distribution within brain. However, little is known about their subcellular distribution, and in particular, their pre- versus postsynaptic localization or their relation to noradrenergic neurons in the CNS. We used an antiserum that selectively recognizes the A-subtype of alpha 2AR to determine: (1) the regional distribution of the receptor within brains of rat and monkey; (2) the subcellular distribution of the receptor in locus coeruleus (LC) of rats and prefrontal cortex of monkeys; and (3) the ultrastructural relation of the receptor to noradrenergic processes in LC. Light microscopic immunocytochemistry revealed prominent immunoreactivity in LC, the brainstem regions modulating the baroreflex, the granule cell layer of the cerebellar cortex, the paraventricular and supraoptic nuclei of the hypothalamus (PVN, SON), the basal ganglia, all thalamic nuclei, the hippocampal formation and throughout cerebral cortical areas. Comparison of results obtained from rat and monkey brains revealed no apparent interspecies-differences in the regional distribution of immunoreactivity. Immunoreactivity occurred as small puncta, less than 1 micron in diameter, that cluster over neuronal perikarya. Besides these puncta, cell bodies, proximal dendrites and fine varicose processes--most likely to be axonal--of the PVN and SON and the hippocampal granule cells also exhibited homogeneously intense distribution of immunoreactivity. Subcellularly, alpha 2AAR-ir in LC and prefrontal cortex were associated with synaptic and non-synaptic plasma membrane of dendrites and perikarya as well as perikaryal membranous organelles. In addition, cortical tissue, but not LC, exhibited prominent immunoreactivity within spine heads. Rat brainstem tissue immunolabeled dually for alpha 2AAR and dopamine beta-hydroxylase (D beta H, the noradrenaline-synthesizing enzyme) revealed that alpha 2AAR-li occurs in catecholaminergic terminals but is also prevalent within non-catecholaminergic terminals. Terminals exhibiting alpha 2AAR-li formed symmetric and asymmetric types of synapses onto dendrites with and without D beta H-immunoreactivity. These results indicate that: (1) the A-subtype of alpha 2AR is distributed widely within brain; (2) alpha 2AAR-li reflects the presence of newly synthesized alph 2AAR in perikarya as well as those receptors along the plasma membrane of perikarya, dendritic trunks and spines; and (3) alpha 2AAR in LC may operate as heteroreceptors on non-catecholaminergic terminals as well as autoreceptors on noradrenergic terminals.

Nitric oxide synthase in the visual cortex of monocular monkeys as revealed by light and electron microscopic immunocytochemistry.

Recent results indicate that nitric oxide (NO) can play an important role in neuronal excitability by modifying the strength of activated synapses and regulating local cerebral blood flow. We sought to determine whether the level of NO synthase (NOS) could, in turn, also be regulated by neural activity. Results using a polyclonal anti-NOS antibody showed that, in cortical area V1 of monocular monkeys, NOS-immunoreactivity is diminished in lamina 4C neuropil of the deprived ocular dominance columns relative to the immediately adjacent non-deprived columns. Closer examination of lamina 4C indicated that the intercolumnar difference in NOS-immunoreactivity does not reflect differences in the distribution of NOS-labeled perikarya, since relatively few neurons were immunoreactive for NOS in lamina 4C of either monocular or normal binocular monkeys. Electron microscopy revealed that the majority (> 80%) of NOS-immunoreactive profiles in lamina 4C are axon terminals. NOS-immunoreactive spines and dendritic shafts also are present but these are more prevalent in the superficial laminae. In order to determine whether the intercolumnar differences in lamina 4C neuropil correspond to altered densities of NOS cells in the superficial laminae, we performed a series of quantitative analyses. In the superficial laminae, NOS-cells occur as two distinguishable classes: a few that are large and intensely NOS-immunoreactive and many more (ca. 24-fold) that are small and lightly immunoreactive. Analysis of the distribution of 559 small and 105 large NOS-immunoreactive cells within 40-microns-thick tangential sections spanning laminae 2-3 showed that the number of cells (large and small together) associated with each blob is approximately 14 for both deprived (lighter) and non-deprived (darker) blobs. These cells are distributed evenly from the center to periphery of columns. Analysis of the distribution of NOS-cells in the infragranular laminae also did not reveal any columnar differences. These observations suggest that local neural activity may be coupled to NO release via alteration of NOS protein levels specifically within distal axonal processes of neurons. This mechanism could operate in conjunction with the more instantaneous catalytic activation of NOS. Ultrastructural analyses further suggest that NO may act as an anterograde and retrograde messenger arising from terminals in addition to its proposed role as a retrograde messenger arising from dendrites.

Light and electron microscopic localization of alpha subunits of GTP-binding proteins, G(o) and Gi, in the cerebral cortex and hippocampus of rat brain.

Antibodies that recognize alpha subunits of G(o), Gi2 and Gi3 were used to evaluate their association with synaptic junctions. G(o), but not Gi, was concentrated within perikaryal and dendritic cytoplasm of a small population of bipolar neurons. All three G-proteins were associated with the intracellular surface of dendritic, axonal and astrocytic plasma membranes and postsynaptic densities (PSDs). However, association with PSDs was more prevalent for the two Gi's than for G(o) while the association with terminals forming putatively excitatory synapses was more prevalent for G(o) and Gi3 than for Gi2. Thus, neuromodulators may modulate the release of excitatory transmitters via activation of presynaptic Gi3 and G(o) and also regulate the opening of Ca2+ and/or K+ channels via activation of Gi's and G(o) at PSDs.