Location of release sites and calcium-activated chloride channels relative to calcium channels at the photoreceptor ribbon synapse.

Vesicle release from photoreceptor ribbon synapses is regulated by L-type Ca(2+) channels, which are in turn regulated by Cl(-) moving through calcium-activated chloride [Cl(Ca)] channels. We assessed the proximity of Ca(2+) channels to release sites and Cl(Ca) channels in synaptic terminals of salamander photoreceptors by comparing fast (BAPTA) and slow (EGTA) intracellular Ca(2+) buffers. BAPTA did not fully block synaptic release, indicating some release sites are <100 nm from Ca(2+) channels. Comparing Cl(Ca) currents with predicted Ca(2+) diffusion profiles suggested that Cl(Ca) and Ca(2+) channels average a few hundred nanometers apart, but the inability of BAPTA to block Cl(Ca) currents completely suggested some channels are much closer together. Diffuse immunolabeling of terminals with an antibody to the putative Cl(Ca) channel TMEM16A supports the idea that Cl(Ca) channels are dispersed throughout the presynaptic terminal, in contrast with clustering of Ca(2+) channels near ribbons. Cl(Ca) currents evoked by intracellular calcium ion concentration ([Ca(2+)](i)) elevation through flash photolysis of DM-nitrophen exhibited EC(50) values of 556 and 377 nM with Hill slopes of 1.8 and 2.4 in rods and cones, respectively. These relationships were used to estimate average submembrane [Ca(2+)](i) in photoreceptor terminals. Consistent with control of exocytosis by [Ca(2+)] nanodomains near Ca(2+) channels, average submembrane [Ca(2+)](i) remained below the vesicle release threshold (∼ 400 nM) over much of the physiological voltage range for cones. Positioning Ca(2+) channels near release sites may improve fidelity in converting voltage changes to synaptic release. A diffuse distribution of Cl(Ca) channels may allow Ca(2+) influx at one site to influence relatively distant Ca(2+) channels.

Accessory optic projections upon oculomotor nuclei and vestibulocerebellum.

Displaced retinal ganglion cells in birds are the sole source of the retinal projection onto the nucleus of the basal optic root, the main component of the accessory optic system. This nucleus has direct bilateral axonal projections onto the oculomotor nuclear complex, the trochlear nucleus- and folia IXc,d and paraflocculus of the vestibulocerebellum. The cerebellar projection terminates within a superficial band of the granule cell layer adjacent to the Purkinje cell layer as a mossy fiber system. This bisynaptic projection onto oculomotor neurons and the cerebellum may play a functionally distinct and specific role in oculomotor reflexes.

A novel alpha subunit in rat brain GABAA receptors.

Two cDNAs (alpha 1 and alpha 4) from rat brain cDNA libraries encode isoforms of the alpha subunit of the GABA/benzodiazepine receptor, which differ at 30% of their amino acid residues. Northern blot analysis and in situ hybridization histochemistry show that alpha 1 and alpha 4 mRNAs have distinct sizes and distinct regional and cellular distributions in rat brain: both mRNAs are found in the cortex and hippocampus; however, only the alpha 1 mRNA is detected in the cerebellum. We injected RNA transcribed from alpha 1 and alpha 4 cDNAs into Xenopus oocytes, together with an RNA for a rat beta subunit. We obtained GABA-dependent inward currents that were reversibly blocked by picrotoxin. Picrotoxin alone, applied to oocytes producing the alpha and beta polypeptides, elicited an outward current. We suggest that these polypeptides together produce GABA-gated ion channels that can also open spontaneously.

Somatostatin modulates voltage-gated K(+) and Ca(2+) currents in rod and cone photoreceptors of the salamander retina.

We investigated the cellular localization in the salamander retina of one of the somatostatin [or somatotropin release-inhibiting factor (SRIF)] receptors, sst(2A), and studied the modulatory action of SRIF on voltage-gated K(+) and Ca(2+) currents in rod and cone photoreceptors. SRIF immunostaining was observed in widely spaced amacrine cells, whose perikarya are at the border of the inner nuclear layer and inner plexiform layer. sst(2A) immunostaining was seen in the inner segments and terminals of rod and cone photoreceptors. Additional sst(2A) immunoreactivity was expressed by presumed bipolar and amacrine cells. SRIF, at concentrations of 100-500 nM, enhanced a delayed outwardly rectifying K(+) current (I(K)) in both rod and cone photoreceptors. SRIF action was blocked in cells pretreated with pertussis toxin (PTX) and was substantially reduced by intracellular GDP(beta)S. Voltage-gated L-type Ca(2+) currents in rods and cones were differently modulated by SRIF. SRIF reduced Ca(2+) current in rods by 33% but increased it in cones by 40%, on average. Both effects were mediated via G-protein activation and blocked by PTX. Ca(2+)-imaging experiments supported these results by showing that 500 nM SRIF reduced a K(+)-induced increase in intracellular Ca(2+) in rod photoreceptor terminals but increased it in those of cones. Our results suggest that SRIF may play a role in the regulation of glutamate transmitter release from photoreceptors via modulation of voltage-gated K(+) and Ca(2+) currents.

Heterogeneous transgene expression in the retinas of the TH-RFP, TH-Cre, TH-BAC-Cre and DAT-Cre mouse lines.

Transgenic mouse lines are essential tools for understanding the connectivity, physiology and function of neuronal circuits, including those in the retina. This report compares transgene expression in the retina of a tyrosine hydroxylase (TH)-red fluorescent protein (RFP) mouse line with three catecholamine-related Cre recombinase mouse lines [TH-bacterial artificial chromosome (BAC)-, TH-, and dopamine transporter (DAT)-Cre] that were crossed with a ROSA26-tdTomato reporter line. Retinas were evaluated and immunostained with commonly used antibodies including those directed to TH, GABA and glycine to characterize the RFP or tdTomato fluorescent-labeled amacrine cells, and an antibody directed to RNA-binding protein with multiple splicing to identify ganglion cells. In TH-RFP retinas, types 1 and 2 dopamine (DA) amacrine cells were identified by their characteristic cellular morphology and type 1 DA cells by their expression of TH immunoreactivity. In the TH-BAC-, TH-, and DAT-tdTomato retinas, less than 1%, ∼ 6%, and 0%, respectively, of the fluorescent cells were the expected type 1 DA amacrine cells. Instead, in the TH-BAC-tdTomato retinas, fluorescently labeled AII amacrine cells were predominant, with some medium diameter ganglion cells. In TH-tdTomato retinas, fluorescence was in multiple neurochemical amacrine cell types, including four types of polyaxonal amacrine cells. In DAT-tdTomato retinas, fluorescence was in GABA immunoreactive amacrine cells, including two types of bistratified and two types of monostratified amacrine cells. Although each of the Cre lines was generated with the intent to specifically label DA cells, our findings show a cellular diversity in Cre expression in the adult retina and indicate the importance of careful characterization of transgene labeling patterns. These mouse lines with their distinctive cellular labeling patterns will be useful tools for future studies of retinal function and visual processing.

Activation of µ opioid receptors modulates inflammation in acute experimental colitis.

BACKGROUND: µ opioid receptors (µORs) are expressed by neurons and inflammatory cells, and mediate immune response. We tested whether activation of peripheral µORs ameliorates the acute and delayed phase of colitis. METHODS: C57BL/6J mice were treated with 3% dextran sodium sulfate (DSS) in water, 5 days with or without the peripherally acting µOR agonist, [D-Ala2, N-Me-Phe4, Gly5-ol]-Enkephalin (DAMGO) or with DAMGO+µOR antagonist at day 2-5, then euthanized. Other mice received DSS followed by water for 4 weeks, or DSS with DAMGO starting at day 2 of DSS for 2 or 3 weeks followed by water, then euthanized at 4 weeks. Disease activity index (DAI), histological damage, and myeloperoxidase assay (MPO), as index of neutrophil infiltration, were evaluated. Cytokines and µOR mRNAs were measured with RT-PCR, and nuclear factor-kB (NF-kB), the antiapoptotic factor Bcl-xL, and caspase 3 and 7 with Western blot. KEY RESULTS: DSS induced acute colitis with elevated DAI, tissue damage, apoptosis and increased MPO, cytokines, µOR mRNA, and NF-kB. DAMGO significantly reduced DAI, inflammatory indexes, cytokines, caspases, and NF-kB, and upregulated Bcl-xL, effects prevented by µOR antagonist. In DSS mice plus 4 weeks of water, DAI, NF-kB, and µOR were normal, whereas MPO, histological damage, and cytokines were still elevated; DAMGO did not reduce inflammation, and did not upregulate Bcl-xL. CONCLUSIONS & INFERENCES: µOR activation ameliorated the acute but not the delayed phase of DSS colitis by reducing cytokines, likely through activation of the antiapoptotic factor, Bcl-xL, and suppression of NF-kB, a potentiator of inflammation.

Localization of specific binding sites for atrial natriuretic factor in peripheral tissues of the guinea pig, rat, and human.

Specific, high affinity atrial natriuretic factor (ANF) binding sites were identified and localized by autoradiographic techniques in peripheral tissues of the guinea pig, rat, and human. In the guinea pig kidney, high concentrations of ANF binding sites were located in the glomerular apparatus, outer medulla, and small renal arteries. Other peripheral tissues containing ANF binding sites included the zona glomerulosa of the adrenal cortex, the smooth muscle layer of the aorta and gallbladder, the lung parenchyma, the posterior lobe of the pituitary, the ciliary body of the eye, and the leptomeninges and choroid plexus of the brain. The distribution of ANF binding sites in the rat and human kidney was nearly identical to those seen in the guinea pig kidney; high concentrations were present in the glomerular apparatus, outer medulla, and small renal arteries. These results are consistent with earlier physiological and pharmacological studies that suggested that ANF plays a functional role in the regulation of extracellular fluid volume and blood pressure. There appears to be little species variation in the location and concentration of renal ANF binding sites, suggesting that, at least in the kidney, the results in experimental animals are relevant to the actions of ANF in humans. The finding that ANF binding sites were stable and present in high concentrations in human postmortem kidneys further suggests that these tissues may be amenable to testing for the involvement of ANF receptor dysfunction in diseases such as hypertension and congestive heart failure.

Postnatal development of tyrosine hydroxylase immunoreactive amacrine cells in the rabbit retina: I. Morphological characterization.

The present and accompanying (Casini, G., and N.C. Brecha, J. Comp. Neurol. 326:302-313, 1992) papers investigate the postnatal development of tyrosine hydroxylase (TH)-immunoreactive (IR) amacrine cells in the rabbit retina. This study is focused on a detailed analysis of the patterns of cellular growth and differentiation of TH-IR amacrine cells, which serve as a model to gain insights into the mechanisms underlying developmental changes associated with the maturation of amacrine cells. Faintly staining TH-IR neurons are present in the proximal inner nuclear layer of newborn retinas. They are characterized by a large nucleus and usually a single primary process lacking varicosities. At postnatal day (PND) 6, TH-IR processes display more complex morphological characteristics, including a few varicosities, and second- and third-order ramifications. Growth cones are often seen. At PNDs 10 and 12 (eye opening), TH-IR cells have general morphological characteristics similar to adult TH-IR amacrines. They display 2-5 primary processes, which start forming a complex network of fibers in lamina 1 of the inner plexiform layer (IPL). TH-IR processes are also present in lamina 3 and rarely in lamina 5 of the IPL. Many fibers ending in growth cones are observed. In addition, very rare, thin TH-IR fibers are present in the outer plexiform layer. At PND 19, TH-IR fibers form a complex, dense network in lamina 1 of the IPL, and loose networks in laminae 3 and 5. Growth cones are not observed at this age. At PND 26, a few "ring-like" structures formed by TH-IR fibers in lamina 1 of the IPL are observed for the first time. In adult retinas, the "ring-like" structures are more numerous than at PND 26. A second, rare type of TH-IR cell ("type B") is encountered in all retinal regions beginning at PND 10. These cells are characterized by weak immunostaining and a small soma size. The present findings show that a significant differentiation of TH-IR neurons occurs during the first 10-12 PNDs. Eye opening is an important period for the maturation of TH-IR amacrines and, more generally, for the maturation of the IPL.

Vasoactive intestinal polypeptide-containing cells in the rabbit retina: immunohistochemical localization and quantitative analysis.

Vasoactive intestinal polypeptide (VIP) possesses neuroactive properties in the nervous system. In this study we characterized VIP immunoreactive neurons in the rabbit retina to provide a basis for a better understanding of the role of this peptide in retinal functions and to further define the morphology of wide-field amacrine cells. VIP immunoreactivity was demonstrated in colchicine-treated retinas. Immunolabeling was observed in amacrine cells located in the proximal inner nuclear layer and, occasionally, in the ganglion cell layer and inner plexiform layer (IPL). Varicose fibers were distributed in laminae 1, 3, and 5 of the IPL. The distribution of VIP immunoreactive cells showed a peak of approximately 50 cells/mm2 in the visual streak and minimum values of approximately 12 cells/mm2 in the peripheral retina. The total number of VIP immunopositive neurons was estimated to be about 11,000. Cell body diameters in the visual streak (8-9 microns) were slightly smaller than those measured in the dorsal or in the ventral retina (9-10 microns). The distribution of nearest neighbor distances (approximately 109 microns in the visual streak and approximately 99 microns in the peripheral retina) showed that VIP immunoreactive neurons were nonrandomly spaced. Labeled neurons emitted one to three thick primary processes, arborizing in secondary processes and collaterals rich in varicosities; these processes often crossed among different IPL laminae. Arborization fields of individual cells overlapped extensively. In the dorsal retina, estimated areas of single arborization fields were larger and processes had lower branching frequency than in the visual streak and in the ventral retina. On the whole, VIP immunoreactive amacrine cells gave rise to a loose meshwork of fibers in the IPL. These characteristics indicate VIP is contained in a class of wide-field amacrine cells and is likely to be involved in widespread regulatory or modulatory functions rather than in the direct transmission of visual information through the retina.

Expression of GAT-1, a high-affinity gamma-aminobutyric acid plasma membrane transporter in the rat retina.

Gamma-aminobutyric acid (GABA) plasma membrane transporters influence synaptic transmission by high-affinity, Na(+)-dependent transport processes. The cDNA clone, GAT-1, encodes a high-affinity Na(+)- and Cl(-)-dependent GABA plasma membrane transporter, which has kinetic and pharmacological properties similar to those of high-affinity GABA uptake systems associated with neurons. The present study evaluates the distribution and cellular localization of this putative neuronal GABA transporter by RNA blot hybridization and in situ hybridization histochemistry in the rat retina. Northern blot hybridization analysis of total retinal and cerebellar RNA extracts demonstrated a single band of hybridization at 4.2 kilobases. GABA transporter mRNA is expressed by numerous cells that are distributed to the proximal inner nuclear layer and the ganglion cell layer and by a few cells located in the inner plexiform layer. Double label studies combining the retrograde transport of the fluorescent dye Fluorogold from the superior colliculus to identify ganglion cells and in situ hybridization histochemistry demonstrated that most GAT-1 mRNA-containing cells in the ganglion cell layer are displaced amacrine cells, although some ganglion cells containing GAT-1 mRNA were visualized. In freshly dissociated retinal cell preparations, the GAT-1 RNA signal is strong in neurons and weak to moderate in specialized glial cells called Müller cells. Müller cells were identified by both their morphology and the presence of the selective Müller cell marker cellular retinaldehyde-binding protein. Only background labeling is seen with the sense GAT-1 RNA probe in both tissue sections and dissociated retinal cell preparations. These findings demonstrate that GAT-1 mRNA is expressed in both the retina and brain. In the retina, this transporter is predominantly localized to amacrine, displaced amacrine and interplexiform cells, and some ganglion cells. This transporter mRNA is also expressed by Müller cells but at a lower level than by neurons. These observations indicate that GABA transport by GAT-1 plasma membrane transporters in the retina is mediated by both neurons and glia cells.

Postnatal development of tyrosine hydroxylase immunoreactive amacrine cells in the rabbit retina: II. Quantitative analysis.

Tyrosine hydroxylase (TH)-immunoreactive (IR) amacrine cells of the rabbit retina mature during the first four postnatal weeks, and their cellular development is described in the preceding paper (Casini, G., and N.C. Brecha, J. Comp. Neurol. 326:283-301, 1992). The present investigation is a quantitative analysis of the postnatal development of the TH-IR amacrine cell population. TH-IR amacrine cells gradually increase in size from birth (soma area of 44.7 +/- 12.4 microns2, mean +/- standard deviation) to adulthood (144.2 +/- 28.0 microns2). Cell density slightly increases from postnatal day (PND) 0 (41.9 +/- 9.5 cells/mm2) to PND 6 (47.2 +/- 7.2 cells/mm2), then markedly decreases from PND 6 to adulthood (17.8 +/- 5.3 cells/mm2) as a consequence of retinal growth. TH-IR cell number almost doubles from PND 0 (about 4,100 cells/retina) to adulthood (about 7,850 cells/retina). The increase in the total number of TH-IR amacrine cells can be explained by the generation of new TH-IR cells in the inner nuclear layer, a delay in the expression of the TH phenotype after neurogenesis by cells committed to be dopaminergic, or the acquisition of this dopaminergic phenotype by uncommitted cells. The development of the TH-IR amacrine cell mosaic was assessed by an evaluation of the distribution of nearest neighbor distances of TH-IR cells. There is a poor correlation between this distribution and a theoretical nonrandom distribution before PND 12. After this age, the nearest neighbor distance distribution shifts towards a nonrandom distribution, and is similar to that of the TH-IR amacrine cell population in the adult retina. The establishment of the TH-IR amacrine cell population mosaic is likely to be achieved through different interacting events, including intrinsic (e.g., genetic) factors, environmental influences, and nonuniform retinal growth. Overall, the population parameters analyzed in the present study approach adult values about the time of eye opening (PND 12) and they reach adult values by PND 26.

Immunocytochemical localization of gamma-aminobutyric acid plasma membrane transporters in the tiger salamander retina.

Gamma-aminobutyric acid (GABA) plasma membrane transporters (GATs) play an important role in regulating GABA neurotransmission in the nervous system. The distribution of two GATs, GAT 1 and GAT 3, in salamander retina was investigated by using affinity-purified polyclonal antisera directed to the predicted C-terminals of rat GAT 1 and rat GAT 3. GAT 1-immunoreactivity (-IR) was found in type IB and IIB orthotopic bipolar cells (BCs) located in the distal and middle of the inner nuclear layer (INL), respectively; in type IIA and IA amacrine cells (ACs) located in the middle and proximal INL, respectively; and in interplexiform cells and cells in the ganglion cell layer (GCL). No detectable staining was found in horizontal cells (HCs) or in structures resembling Müller cells. GAT 1-immunoreactive fibers were present in the outer plexiform layer (OPL) and inner plexiform layer (IPL) in three bands corresponding to the three bands previously reported to be GABA-IR. GAT 3 antibodies labeled fewer cells and cell types than GAT 1 antibodies. GAT 3-IR was localized to type IIA and IA ACs and cells in the GCL, but not to BCs, HCs, or Müller cell-like structures. There was weak labeling of the OPL and stronger labeling of the IPL, with three distinct bands at the same depth as observed with GAT 1-IR. Double-labeling showed that the majority of GAT 1-IR BCs (88%), ACs (88%), and cells in the GCL (78%) colocalized with GABA-IR. The present study provides the first direct evidence of the expression of two GAT subtypes in neurons of nonmammalian retinas. These transporters could regulate GABA neurotransmission by reuptake and termination of GABA's action and, perhaps, by GABA release mechanisms. The presence of GAT 1-IR/GABA-IR bipolar cells further supports our earlier observations that a subgroup of orthotopic bipolar cells are likely to be GABAergic.

Transient features of tachykinin peptide innervation of the dorsal lateral geniculate nucleus of the rabbit during postnatal development.

The tachykinin peptide innervation of the developing dorsal lateral geniculate nucleus of the rabbit was studied with immunocytochemical techniques at the light and electron microscopic levels by using an antibody directed to the C-terminal of the mammalian tachykinin peptides. At birth, the rabbit's lateral geniculate nucleus was densely innervated by a large number of labeled fibers. These relatively unbranched axons were dispersed throughout the nucleus but showed a higher density of fiber packing in the external layer of the alpha sector. Over the next three weeks this pattern of distribution changed dramatically. Immunoreactive fibers were gradually eliminated from most of the nucleus, and, by the end of the third postnatal week, they appeared as a narrow plexus deep to the optic tract. At the same time, these axons showed major morphological alterations as they gradually became thicker and developed terminal arborizations characterized by spherical or elongated enlargements. Overall, by the end of the third postnatal week, the pattern of distribution and appearance of tachykinin-labelled fibers in the dorsal lateral geniculate nucleus were similar to those described in the adult rabbit (Brecha et al., 1987). Ultrastructural analysis has shown that during the first week, tachykinin-immunoreactive profiles appeared as round or elongated varicosities making asymmetrical synapses with dendritic shafts throughout the lateral geniculate nucleus. Thereafter, as they were progressively restricted to the external layer of the alpha sector of the nucleus, they began to form multiple synaptic contacts with neuronal processes in complex glomerular neuropil. On the basis of the present and previous findings, we suggest that tachykinin peptides not only play a role as putative neurotransmitters in the retinogeniculate pathway, but they may also play a role in the development of the lateral geniculate nucleus and of the retinogeniculate projection system in the rabbit.

Distribution of cholecystokinin-like immunoreactivity in the rat main olfactory bulb.

The anatomical localization of cholecystokinin-like immunoreactivity (CCK-I) within the rat main olfactory bulb was analyzed by using the peroxidase-antiperoxidase immunocytochemical technique. Neurons or neuronal processes containing CCK-I were localized within all laminae of the olfactory bulb except the olfactory nerve fiber layer. A large population of CCK-I neurons, with morphology, size, and distribution corresponding to that of the middle and external tufted cells, was observed within a zone extending from the deep periglomerular region through the superficial one-half to one-third of the external plexiform layer. A smaller number of immunoreactive perikarya were found in the deep external plexiform layer, the glomerular layer, and rarely within the inner plexiform layer. These CCK-I neurons appeared to correspond to internal tufted cells, periglomerular cells, and deep short-axon cells, respectively. Dense CCK-I staining of fibers and terminals was present within the internal plexiform layer and, less densely, within the neuropil of the granule cell layer. In addition, terminal-like CCK-I was localized within layer 1A of the anterior olfactory nucleus, the olfactory tubercle, and the most rostral piriform cortex. This observation provides corroboration for the identification of the principal CCK-I neuron in the rat olfactory bulb as the centrally projecting middle tufted cell. The present results, demonstrating the localization of CCK-I to both local circuit and projection neurons of the olfactory bulb and to terminal-like puncta in the internal plexiform and granule cell layers, suggest that CCK may be significantly involved in olfactory processing at several levels.

Projections of the nucleus of the basal optic root in the pigeon: an autoradiographic and horseradish peroxidase study.

The efferent projections of the nBOR complex, have been studied with both anterograde autoradiographic and retrograde horseradish peroxidase (HRP) techniques. The nBOR complex includes three distinct subdivisions: the nucleus of the basal optic root (nBOR), the nBOR pars dorsalis (nBORd) and the nBOR pars lateralis (nBOR1). Unilateral injections of 3H-leucine or 3H-proline/3H-leucine mixtures into the nBOR complex have demonstrated prominent bilateral projections upon (1) the vestibulocerebellum, (2) the inferior olivary complex, (3) the oculomotor nuclear complex, (4) the nucleus interstitialis, contralateral projections upon (5) the contralateral nBOR complex and ipsilateral projections upon (6) a pretectal nucleus, the nucleus lentiformis mesencephali, pars magnocellularis. Unilateral injections of HRP confined to folia IXc,d and paraflocculus of the cerebellum, the contralateral nBOR complex or the nucleus lentiformis mesencephali resulted in retrograde labeling of predominantly medium and large size cells within the entire nBOR complex. Unilateral injections of HRP within the inferior olive resulted in retrograde labeling of small, spindle-shaped cells within nBOR and nBORd. Unilateral injections of the oculomotor complex which included the trochlear nucleus resulted in retrograde labeling of small cells within the ipsilateral nBORd and predominantly medium and large cells in the contralateral nBOR. The displaced ganglion cells of the retina give rise to a prominent and distinct projection upon the nBOR complex (Karten et al., '77). The nBOR complex in turn projects upon the oculomotor nuclear complex, the nucleus interstitialis and the vestibulocerebellum, regions which have been implicated in oculomotor function. These findings strongly suggest that the displaced ganglion cells and the accessory optic system have a major influence upon oculomotor reflexes including eye and head movements.

Habenular asymmetry and the central connections of the parietal eye of the lizard.

Histochemical and autoradiographic analyses of the axonal transport of horseradish peroxidase and tritiated amino acids were employed to study the central connectivity of the lizard parietal eye. Somata and processes of centrifugal fibers to the parietal eye were localized in tissue of the dorsal sac and in the leptomeningeal sheath of the pineal gland. Analyses of series of transverse sections of the brain showed the left medial habenular nucleus to be subdivided into pars dorsolateralis and pars ventromedialis, and the right medial habenular nucleus not to be so subdivided. Centripetal fibers of parietal eye ganglion cells project to only the pars dorsolateralis of the left medial habenular nucleus and terminate there in two distinct fields. The asymmetry of the lizard habenula may be a specialization associated with the unilateral projection from the parietal eye.

Basal ganglia pathways to the tectum: the afferent and efferent connections of the lateral spiriform nucleus of pigeon.

Previous studies have demonstrated that the lateral spiriform nucleus (SpL) of the avian pretectum receives a major input from the ipsilateral basal ganglia (Karten and Dubbeldam, '73) and projects to the ipsilateral optic tectum (Brecha et al., '76). The present study has further detailed the anatomical organization of the afferent and efferent connections of SpL, with particular reference to (1) the sources of afferent inputs to SpL, (2) the projection targets of SpL, and (3) the laminar termination pattern of the SpL projection to the tectum. The SpL was found to receive clear-cut major inputs from only three nuclei: (1) the ipsilateral paleostriatum primitivum (PP) of the basal ganglia (the avian homologue of the mammalian globus pallidus), (2) the ipsilateral anterior nucleus of the ansa lenticularis (ALa) of the diencephalon, and (3) the ipsilateral nucleus tegmentipedunculopontinus (TPc) of the mesencephalon. Both TPc and ALa have previously been noted themselves to receive major inputs from the ipsilateral PP (Karten and Dubbeldam, '73). Two other cell groups may give rise to a slight projection to the ipsilateral SpL: (1) the posterior nucleus of the ansa lenticularis (ALp) of the diencephalon and (2) the nucleus semilunaris (SLu) of the isthmic brainstem. The ALp also receives a major input from PP (Karten and Dubbeldam, '73). Two other cell groups may give rise to a slight projection to the ipsilateral SpL: (1) the posterior nucleus of the ansa lenticularis (ALp) of the diencephalon and (2) the nucleus semilunaris (SLu) of the isthmic brainstem. The ALp also receives a major input from PP (Karten and Dubbeldam, '73), while SLu receives a major tectal projection (Hunt and Kunzle, '76a). The ipsilateral tectum was found to be the only projection target of SpL. The present data suggest that the SpL projection to the tectum is restricted to layers 8-13, with layers 11-13 receiving the heaviest projection from SpL. Among layers 8-10, layer 9 receives the lightest projection from SpL. The present results indicate that SpL receives only a limited number of inputs, which in all likelihood relay largely basal ganglia input to SpL. Since SpL projects only to the tectum, the sole function of SpL apparently is the transmission of ipsilateral basal ganglia influences to the avian optic tectum. Tectal layers 8-15 have been previously found to represent the layers of origin of the descending pathways of the avian tectum to hindbrain motor and "premotor" cell groups (Reiner and Karten, '82). In view of the purported involvement of the basal ganglia in motor functions, the basal ganglia pathway to the ipsilateral tectum via SpL may represent a major route by which the avian basal ganglia exert influences over motor functions.

Immunohistochemical localization of avian pancreatic polypeptide-like immunoreactivity in the rat hypothalamus.

The distribution of avian pancreatic polypeptide-like (APP) immunoreactivity within the rat hypothalamus was investigated with the indirect immunoperoxidase method. APP immunoreactive perikarya are found in largest numbers in the retrochiasmatic area, the arcuate nucleus, and the supracommissural portion of the interstitial nucleus of the stria terminalis. Small clusters of immunoreactive neurons are also consistently observed in the ventral aspect of the medial preoptic area and lateral hypothalamic area, immediately dorsolateral to the optic chiasm and tracts. These neurons are apparent in all animals but are more intensely strained and occur in larger numbers following colchicine pretreatment. Other immunoreactive neurons are visible only in colchine-treated rats and are scattered throughout the anterior and lateral hypothalamic areas and the supramammillary nucleus. Immunoreactive axons and terminal fields present an extensive and highly characteristic distribution throughout the hypothalamus, which in many instances exhibits differential distribution within specific subfields of hypothalamic nuclei and areas. The heaviest concentrations of APP immunoreactive axons are present in the periventricular nucleus throughout the rostrocaudal extent of the hypothalamus, the ventrolateral portion of the suprachiasmatic nucleus, the retrochiasmatic area, the parvocellular paraventricular nucleus, the ventral supraoptic nucleus, the perifornical nucleus, the ventral dorsomedial nucleus, and the arcuate nucleus. Moderate plexuses of immunoreactive fibers are also present in the medial preoptic area, the anterior and lateral hypothalamic areas, the nucleus circularis, the median eminence, and the ventral premammillary area. Other areas, such as the ventromedial nucleus, contain virtually no immunoreactive axons but are encapsulated by a dense plexus of immunoreactive terminals. The distribution of a major component of APP immunoreactive fibers exhibits a marked similarity to that of previously described norepinephrine-containing hypothalamic afferents. Other groups of APP immunoreactive perikarya and fibers appear to represent components of intrinsic diencephalic systems.

AII amacrine cell population in the rabbit retina: identification by parvalbumin immunoreactivity.

Parvalbumin (PV) is a calcium-binding protein localized to selected neurons in the nervous system, including the retina. This investigation evaluated the distribution of PV immunoreactivity in the rabbit retina using immunohistochemistry with a monoclonal antibody directed to carp PV. In the inner nuclear layer (INL), PV immunoreactivity was present in horizontal and amacrine cells. In the ganglion cell layer, PV immunostaining was confined to somata that are likely to be both displaced amacrine cells and ganglion cells. PV-immunoreactive (IR) amacrine cells were positioned in the proximal INL adjacent to the inner plexiform layer (IPL). These cells usually gave rise to a single primary process, which arborized into two distinct bands in the IPL. In sublamina a, the processes were thin and had large, irregular endings. In sublamina b, multiple processes branched from the primary process and were characterized by varicosities and spines. PV-IR amacrine cell bodies measured from 8 to 10 microns in diameter. Their density was highest in the visual streak and lowest in the periphery of the superior retina. The average number of PV-IR amacrine cells was 464,045 cells per retina (N = 3), and the average regularity index of the PV-IR cell mosaic was 3.23. PV-IR amacrine cells were further characterized by double-label immunofluorescence experiments using antibodies to PV and tyrosine hydroxylase (TH). Varicose TH-IR processes were in close apposition to many PV-IR amacrine cells and often formed "ring structures" around them. Together, these morphological, quantitative, and histochemical observations indicate that PV immunoreactivity in the INL is localized predominantly to AII amacrine cells, and therefore it is a valuable marker for the identification of this cell type.

Cellular localization and laminar distribution of AMPA glutamate receptor subunits mRNAs and proteins in the rat cerebral cortex.

The cellular and laminar distributions of the alpha-amino-3-hydroxy-5- methyl-4-isoxazole propionate (AMPA) receptor subunits GluR1-4 have been investigated in the cerebral cortex of adult rats by in situ hybridization with 35S-labeled cRNA probes and by immunocytochemistry with subunit-specific antibodies. In sections incubated with the GluR1-4 antisense probes, specific hybridization signal was observed in many but not all cortical cells. Experiments with in situ hybridization and antibodies to glial fibrillary acidic protein (GFAP) showed that percentages of GFAP-immunoreactive cells labeled by the GluR1-4 probes were 20%, 9.4%, 8.2%, and 57.3%, respectively. A semiquantitative evaluation revealed that about 56% of cortical neurons contained the GluR1 subunit, 80% the GluR2, 63% the GluR3, and 44% the GluR4. The number of grains associated with every neuron was determined from sections exposed for 15 days, the background level was subtracted, and labeled neurons were divided into four groups: A (< or = 10 grains), B (11-20 grains), C (21-30 grains), and D (> 30 grains). The number of neurons belonging to each of these groups was then evaluated for their occurrence in each cortical layer. Immunocytochemistry with subunit-specific antibodies showed that 1) GluR1-immunoreactive neurons were mostly layers V and VI nonpyramidal neurons; 2) GluR2/3-immunoreactive neurons were more numerous in layers II-III and V-VI, and most of them were pyramidal; and 3) GluR4-positive cells were the least numerous, and they were either neurons (pyramidal and nonpyramidal) or astrocytes. These observations indicate that cortical neurons exhibit a remarkable degree of heterogeneity with regard to both the composition and the number of AMPA receptors and suggest that this diversity might be correlated with the functional attributes of neurons receiving glutamatergic afferents and with the physiological features of corticifugal neurons.