Specific distribution of Ca / calmodulin-dependent protein kinase II α and β isoforms in some structures of the rat forebrain

The immunohistochemical distribution of Ca²⁺ / calmodulin-dependent protein kinase II (CaM kinase II) α and β isoforms in the rat forebrain was examined by using monoclonal antibodies specific to each isoform. The present study confirmed that α and β immunoreactives are localized only in neuronal elements. At the light microscopic level, specific distribution patterns of these isoforms and staining characteristics were recognized in some regions of the forebrain as follows. Firstly, α-immunoreactive neurons were more homogeneously distributed throughout the cellular layers of the cerebral cortex (i.e., layers II-IV) than β-immunoreactive ones. Secondly, neurons in the globus pallidus were immunostained by the anti-β antibody, but not by the anti-α antibody. Thirdly, neurons in the medial habenular nucleus, the subthalamic nucleus and the reticular thalamic nucleus were more densely stained with the anti-β antibody than with the anti-α antibody. However, marked differences were not observed in the hippocampal formation at the light microscopic level. The electron microscopic analysi of the cerebral cortex demostrated that subcellular localizations of α- and β-immunoreactive products within the cortical neurons were quite dissimilar: (i) the nucleus was stained only with the anti-α antibody, but not with the antiβ antibody, and (ii) β-immunoreactive products were more sporadically localized in the cytoplasms of the perikarya and dendrited than the α-immunoreactive ones. These rigional and subcellular differences between the distribution patterns of α and β immunoreactivities suggest the functional diversity of CaM kinase II α and β isoforms in the central system.     

Regional differences between the immunohistochemical distribution of Ca/calmodulin-dependent protein kinase II α and β isoforms in the brainstem of the rat

The distribution of Ca²⁺/calmodulin-dependent protein kinase II (CaM kinase II) α and β isoforms in the brainstem of adult rats was investigated using an immunohistochemical method with two monoclonal antibodies which specifically recognize the α and β isoform, respectively. We found that these isoforms were differentially expressed by neurons in the substantia nigra, red nucleus, dorsal cochlear nucleus, pontine nuclei and inferior olivary nucleus. Neurons in the inferior olivary nucleus express the α isoform, but not the β isoform. In contrast, neurons in the substantia nigra, red nucleus and pontine nuclei were immunostained with the β antibody, but not with the α antibody. In the dorsal cochlear nucleus, neurons in layers I and II were α-immunopositive, whereas neurons in layers III and IV were β-immunopositive. Therefore, the distribution of the CaM kinase II α-immunopositive neurons is completely different from that of CaM kinase II β-immunopositive neurons. Next we examined the possible coexistence of CaM kinase II α isoform and glutamate or that of CaM kinase II β isoform and glutamic acid decarboxylase (GAD) in the single neuron by double immunofluorescence labelling using a pair of anti-α and anti-glutamate antibodies, or a pair of anti-β and anti-GAD antibodies. The results indicated that neurons expressing anti-α immunoreactivity were also immunopositive against anti-glutamate antibody, and neurons expressing β isoform were also immunopositive against anti-GAD antibody, suggesting that α-immunopositive neurons are classified as excitatory-type neurons, and on the contrary, β-immunopositive neurons are classified as inhibitory-type neurons. In conclusion, the present study confirmed that α- and β-isoforms of CaM kinase II are differentially expressed in the nuclei of the brainstem and have different roles.     

Expression of calcium transporters in the retina of the tiger salamander (Ambystoma tigrinum)

Changes in intracellular calcium concentration, [Ca2+]i, modulate the flow of visual signals across all stages of processing in the retina, yet the identities of Ca2+ transporters responsible for these changes are still largely unknown. In the current study, the distribution of plasma membrane and intracellular Ca2+ transporters in the retina of tiger salamander, a model system for physiological studies of retinal function, was determined. Plasma membrane calcium ATPases (PMCAs), responsible for high-affinity Ca2+ extrusion, were highly expressed in the salamander retina. PMCA isoforms 1, 2, and 4 were localized to photoreceptors, whereas the inner retina expressed all four isoforms. PMCA3 was expressed in a sparse population of amacrine and ganglion neurons, whereas PMCA2 was expressed in most amacrine and ganglion cells. Na+/Ca2+ exchangers, a high-capacity Ca2+ extrusion system, were expressed in the outer plexiform layer and in a subset of inner nuclear and ganglion layer cells. Intracellular Ca2+ store transporters were also represented prominently. SERCA2a, a splice variant of the sarcoplasmic-endoplasmic Ca2+ ATPase, was found mostly in photoreceptors, whereas SERCA2b was found in the majority of retinal neurons and in glial cells. The predominant endoplasmic reticulum (ER) Ca2+ channels in the salamander retina are represented by the isoform 2 of the IP3 receptor family and the isoform 2 of the ryanodine receptor family. These results indicate that Ca2+ transporters in the salamander retina are expressed in a cell type-specific manner.     

Regeneration of the newt retina: Order of appearance of photoreceptors and ganglion cells

The adult newt regenerates a functional retina following removal or destruction of the original retina. We studied the order of appearance of cell types in the regenerating retina by using immunohistochemical techniques. An antibody that recognizes the alpha subunit (260 kDa) of voltage-dependent Na⁺ channels was found to label a 255-kDa band in Western blots of crude membrane fractions from the normal retina. Cryosections of normal retina revealed intense Na⁺ channel immunoreactivity in somata and axons of ganglion cells, weaker immunoreactivity in somata of amacrine cells, and no immunoreactivity in the inner plexiform layer. In the same sections, immunoreactivity to a monoclonal antibody (RB-1) specific to newt cones was intense in the photoreceptor layer. In regenerating retinas, double staining with the Na⁺ channel antibody as a possible marker of ganglion cells and RB-1 antibody first revealed immunoreactive cells at the intermediate stage (three to five cells thick), which does not exhibit segregated synaptic layers. Na⁺ channel-immunoreactive ganglion cells appeared before the RB-1-immunoreactive photoreceptors. Because ganglion cells also appear before photoreceptor cells in normal development, common mechanisms may control both the generation and the regeneration of the newt retina. J. Comp. Neurol. 396:267–274, 1998. © 1998 Wiley-Liss, Inc.     

Ca-independent activity of Ca/calmodulin-dependent protein kinase II involved in stimulation of neurite outgrowth in neuroblastoma cells

We investigated the involvement of Ca²⁺-independent activity of Ca²⁺/calmodulin-dependent protein kinase II (CaM kinase II) in stimulation of neurite outgrowth. When neuroblastoma Neruo2a (Nb2a) cells expressing the α isoform of CaM kinase II (Nb2a/α cells) were stimulated by plating, they changed shape from round to flattened, and began to form neurites within 15 min. Numbers of cells bearing neurites increased from 15 min to about 2 h. Neurite length increased markedly from 30 min to 2 h after stimulation. Ca²⁺-independent activity of CaM kinase II increased immediately after stimulation, peaked at about 30 min, and then gradually decreased. Autophosphorylation of Thr-286 followed the same time course as the increase in Ca²⁺-independent activity. The autophosphorylation and appearance of Ca²⁺-independent activity preceded the formation of neurites. The effect of mutation of the autophosphorylation site in the kinase whose Thr-286 was replaced with Ala (αT286A kinase) or Asp (αT286D kinase) was examined. αT286A kinase was not converted to a Ca²⁺-independent form, and αT286D kinase had Ca²⁺-independent activity significantly as an autophosphorylated kinase. Cells expressing αT286A kinase did not form neurites, and were indistinguishable from control Nb2a cells. Cells expressing αT286D kinase had much longer neurites than Nb2a/α cells expressing the wild type kinase, although the initiation of neurite outgrowth was very late. These results indicated that Ca²⁺-independent activity of the kinase autophosphorylated at Thr-286 involves for neurite outgrowth.     

Developmental regulation of two isoforms of Ca/calmodulin-dependent protein kinase I β in rat brain

Subtractive hybridization analysis of region-specific gene expression in brain has demonstrated a mRNA species enriched in rat hypothalamus [K.M. Gautvik, L. de Lecea, V.T. Gautvik, P.E. Danielson, P. Tranque, A. Dopazo, F.E. Bloom, J.G. Sutcliffe, Proc. Natl. Acad. Sci. USA 93 (1996) 8733–8738.]. We here show that this mRNA encodes a Ca²⁺/calmodulin-dependent (CaM) kinase belonging in the CaM kinase I β subgroup. cDNA analysis showed that this enzyme was differentially spliced into two isoforms (designated β1 and β2) with distinct C-termini. The C-terminal of the translated CaM kinase I β2 protein (38.5 kDa molecular size), contained 25 amino acid residues not present in the β1 isoform. The two isoforms were differentially developmentally regulated, with the β1 isoform being present in rat embryos from day 18 and the β2 isoform being present from day 5 postnatally. In situ hybridization analysis of adult rat CNS showed CaM kinase I β2 mRNA being enriched in the hypothalamus and the hippocampal formation. Expression was also observed in a number of ventral limbic structures and in the thalamus. Northern blot analysis showed additional expression of multiple β2 isoforms in heart and skeletal muscle. The human mRNA showed a similar distribution. Our data suggest that the two isoforms of CaM kinase I β, created by a splicing process occurring within a week around birth, may have distinct pre- and postnatal functions in a distinct set of CNS neurons and excitable tissues.     

The ultrastructural localization of acetylcholinesterase-like immunoreactivity in the chicken retina

The localization of acetylcholinesterase (AChE) in the chicken retina was studied using histochemical and immunohistochemical techniques. Using histochemistry, reaction end product was found in amacrine cells, ganglion cells, horizontal cells and in 4 bands in the inner plexiform layer. Ultrastructurally, the reaction end product was located between membranes of the endoplasmic reticulum, between the membranes of the nuclear envelope, surrounding neurites in the inner plexiform layer and filling synaptic clefts. Immunohistochemical techniques using a monoclonal antibody against AChE showed a similar staining pattern to that obtained with histochemistry. Ultrastructurally, AChE-like immunoreactivity was located on, not between, the membranes of the endoplasmic reticulum and nuclear envelope of amacrine cells, ganglion cells and horizontal cells. In the inner plexiform layer, immunoreactivity was on both pre- and postsynaptic membranes, and there was no immunoreactivity in non-terminal regions of the dendritic membranes and none within the synaptic clefts.     

Localization of aspartate-like immunoreactivity in the retina of the turtle (Pseudemys scripta).

Aspartate has been reported to be a putative excitatory neurotransmitter in the retina, but little detailed information is available concerning its anatomical distribution. We used an antiserum directed against an aspartate-albumin conjugate to analyze the anatomy, dendritic stratification, and regional distribution of cell types with aspartate-like immunoreactivity in the turtle retina. The results showed dramatic differences in immunoreactivity in the peripheral versus the central retina. Strong aspartate-like immunoreactivity was shown in the peripheral retina, with many well-labeled processes in the inner plexiform layer. Many bipolar, horizontal, amacrine, and ganglion cells, some photoreceptors, and some unidentified cells were strongly immunoreactive in the peripheral retina. In contrast, although the central retina showed well-labeled horizontal cells, there was only light labeling in the inner plexiform layer with weakly immunoreactive amacrine and ganglion cells and no labeled bipolar cells. There were several strongly immunoreactive efferent nerve fibers which left the optic nerve head and arborized extensively in the retina. At the electron microscopic level, electron-dense reaction product was associated with synaptic vesicles at bipolar and amacrine cell synapses in the inner plexiform layer. These results suggest that aspartate may be involved in many diverse synaptic interactions in both the outer plexiform layer and the inner plexiform layer of the turtle retina.     

Ultrastructural demonstration ofl-glutamate decarboxylase and cysteinesulfinic acid decarboxylase in rat retina by immunocytochemistry

The γ-aminobutyric acid (GABA) synthesizing enzyme,l-glutamate decarboxylase (GAD), and the taurine synthesizing enzyme, cysteinesulfinic acid decarboxylase (CSAD) have been localized in rat retina at the ultrastructural level by indirect immunoelectron microscopy. GAD immunoreactivity (GAD-IR) was seen only in some amacrine cells and their terminals. CSAD immunoreactivity (CSAD-IR) was found in most retinal neuronal types and their processes including photoreceptor cells (rod and cone cells), bipolar cells, amacrine cells and ganglion cells. The GAD-IR positive amacrine terminals have been found to make synaptic contact with other GAD-IR negative bipolar and amacrine terminals, and ganglion cell dendrites. Most of the GAD-IR positive terminals are presynaptic. Occasionally, GAD-IR positive amacrine terminals are postsynaptic to another amacrine terminal or ganglion cell body. In the inner plexiform layer, CSAD-IR positive amacrine terminals also make synaptic contacts with other nerve terminals, similar to that of GAD-IR positive amacrine terminals. In addition, CSAD-IR positive bipolar terminals make synaptic contact with some CSAD-IR positive as well as negative amacrine terminals. Both CSAD-IR positive amacrine and bipolar terminals are mostly presynaptic to other CSAD-IR negative terminals. In the outer plexiform layer, CSAD-IR was found to be associated with synaptic vesicles and the synaptic membrane in certain cone pedicles and rod spherules. It is concluded that only a fraction of amacrine cells in rat retina may use GABA as a neurotransmitter. The presence of CSAD-IR in some amacrine, bipolar, photoreceptor and ganglion cells in rat retina is compatible with the notion that taurine may play some important roles, such as those of neurotransmitter or neuromodulator in mammalian retina.     

Immunocytochemical localization of glycine receptors in the mammalian retina

The distribution of glycinergic synapses in the mammalian retina was studied with monoclonal antibodies against glycine receptors and a glycine receptor-related protein (gephyrin). Monoclonal antibody 2b is specific for the α1 subunit of the glycine receptor; monoclonal antibody 4a is specific for all known α subunits and the β subunit, and monoclonal antibody 7a is specific for gephyrin. The three antibodies were applied to the retina of cat, macaque monkey, rat, and rabbit. The general staining pattern is comparable in all these species and it is similar but distinct with all of the three antibodies. Labeling is characterized by a punctate appearance indicating that it occurs at synapses. In the inner plexiform layer, labeling is concentrated in two bands. One band is located close to the inner nuclear layer; the other band is located in the middle of the inner plexiform layer. In the outer plexiform layer, sparse punctate labeling is seen. The distribution of gephyrin was also studied at the ultrastructural level in cat and monkey retina. Gephyrin is present on the postsynaptic membrane of amacrine cells and ganglion cells. The presynaptic profile to gephyrin immunoreactivity is always of an amacrine cell. The AII amacrine cell, the crucial glycinergic interneuron of the rod pathway, is presynaptic to gephyrin immunoreactivity in the OFF-sublamina and is itself gephyrin-positive at an input synapse from another (possibly GABAergic) amacrine cell in the ON-sublamina. © 1993 Wiley-Liss, Inc.     

Identification and characterization of an aquaporin 1 immunoreactive amacrine-type cell of the mouse retina

Using immunocytochemistry, a type of amacrine cell that is immunoreactive for aquaporin 1 was identified in the mouse retina. AQP1 immunoreactivity was found in photoreceptor cells of the outer nuclear layer (ONL) and in a distinct type of amacrine cells of the inner nuclear layer (INL). AQP1-immunoreactive (IR) amacrine cell somata were located in the INL and their processes extended through strata 3 and 4 of the inner plexiform layer (IPL) with thin varicosities. The density of the AQP1-IR amacrine cells increased from 100/mm(2) in the peripheral retina to 350/mm(2) in the central retina. The AQP1-IR amacrine cells comprise 0.5% of the total amacrine cells. The AQP1-IR amacrine cell bodies formed a regular mosaic, which suggested that they represent a single type of amacrine cell. Double labeling with AQP1 and glycine, gamma-aminobutyric acid (GABA) or GAD(65) antiserum demonstrated that the AQP1-IR amacrine cells expressed GABA or GAD(65) but not glycine. Their synaptic input was primarily from other amacrine cell processes. They also received synaptic inputs from a few cone bipolar cells. The primary synaptic targets were ganglion cells, followed by other amacrine cells and cone bipolar cells. In addition, gap junctions between an AQP1-IR amacrine process and another amacrine process were rarely observed. In summary, a GABAergic amacrine cell type labeled by an antibody against AQP1 was identified in the mouse retina and was found to play a possible role in transferring a certain type of visual information from other amacrine or a few cone bipolar cells primarily to ganglion cells.     

Immunological evidence that the beta isoform of Ca2+/calmodulin-dependent protein kinase IV is a cerebellar granule cell-specific product of the CaM kinase IV gene.

Ca2+/calmodulin-dependent protein kinase IV (CaM kinase IV) exists as two monomeric isoforms, alpha and beta. In this study, we raised an antibody against the beta isoform and provided immunohistochemical evidence for specific expression of the beta isoform in cerebellar granule cells as a single gene-derived translational product distinct from the alpha isoform. Immunohistochemical examination showed that the beta-immunoreactivity was confined to the nuclei of the cerebellar granule cells, in contrast to the more widespread immunoreactivity for the alpha isoform in both nuclei and cytoplasm of the cerebellar granule cells and many other neurons with dominant nuclear localization. In developing cerebella, the beta-immunoreactivity gradually appeared in the internal granule cells during the postnatal 2nd and 3rd weeks, while the alpha-immunoreactivity had already appeared in the internal granule cells in the 1st postnatal week. Unlike the alpha isoform, beta-immunoreactivity was not detected in the Purkinje cells at any developmental stages. The differential expression of the alpha and beta isoforms suggests that each isoform may be involved in different cerebellar functions.     

Corticotropin releasing factor-like immunoreactive neurons in the rat retina

Corticotropin releasing factor (CRF)-like immunoreactive neurons have been identified in the rat retina by immunohistochemical methods using antisera directed against ovine and rat CRF. CRF-like immunoreactivity was observed in both amacrine and ganglion cells which projected fine varicose processes to the inner plexiform layer of the retina. It is suggested that CRF may play a role in retinal function.     

Starburst amacrine cells in cat retina are associated with bistratified, presumed directionally selective, ganglion cells

Starburst amacrine cells of cat retina are similar in form, though more delicate and less profusely branched, when compared to the starburst/cholinergic amacrine cells of rabbit retina, as identified in Golgi preparations. In both species, type a cells branch in the middle of sublamina a of the inner plexiform layer (IPL), but type b (displaced) starburst amacrine cells of cat branch near the a/b sublaminar border (stratum 3) of the IPL, not in the middle of sublamina b (stratum 4), as do those of rabbit. Nevertheless, in each species, this starburst substratum in sublamina b coincides with the sublamina b-level branching of a bistratified ganglion cell, which in rabbit retina shows directionally selective responses. It is proposed that starburst amacrine cells of cat retina are cholinergic and, as in rabbit retina, make selective connections with on-off directionally selective ganglion cells.     

Distribution and synaptic connectivity of neuropeptide Y-immunoreactive amacrine cells in the rat retina

Neuropeptide Y (NPY) is a potent bioactive peptide that is widely expressed in the nervous system, including the retina. Here we show that specific NPY immunoreactivity was localized to amacrine and displaced amacrine cells in the rat retina. Immunoreactive cells had a regular distribution across the retina and an overall cell density of 280 cells/mm(2) in the inner nuclear layer (INL) and 90 cells/mm(2) in the ganglion cell layer (GCL). In the INL, most immunoreactive cells were characterized by small cell bodies and fine processes that appeared to ramify primarily in stratum 1 of the inner plexiform layer (IPL). A few cells in the INL also ramified in stratum 3 of the IPL. In the GCL, small to medium immunoreactive cells appeared to ramify primarily in stratum 5 of the IPL. A few immunoreactive processes, originating from somata in the INL and processes in the IPL, ramified in the OPL. NPY-immunoreactive cells contained GABA immunoreactivity, and some amacrine cells also contained tyrosine hydroxylase immunoreactivity. NPY-immunostained processes were most frequently presynaptic to nonimmunostained amacrine and ganglion cell processes and postsynaptic to nonimmunostained amacrine cell processes and cone bipolar cell axonal terminals. These findings indicate that NPY immunoreactivity is present in two populations of amacrine cells, one located in the INL and the other in the GCL, and that these cells mainly form synaptic contacts with other amacrine cells. These observations suggest that NPY-immunoreactive cells participate in multiple circuits mediating visual information processing in the inner retina.     

Localization of putative GABAergic neurons in the larval tiger salamander retina by immunocytochemical and autoradiographic methods

Putative GABAergic neurons in the larval tiger salamander retina were localized by a comparative analysis of glutamate decarboxylase immunoreactivity (GAD-IR), GABA-like immunoreactivity (GABA-IR), and high-affinity 3H-GABA uptake at the light microscopical level. Preliminary data showed that all GAD-IR neurons were double labeled for GABA-IR. However, because the weak somatic labeling with GAD-IR, we could not determine if the converse were true. Neurons commonly labeled with GABA-IR and 3H-GABA uptake include horizontal cells, type I (outer) and type II (inner) bipolar cells, type I (inner) and type II (outer) amacrine cells, and cell bodies in the ganglion cell layer (GCL). In addition, interplexiform cells were identified with GABA-IR. The presence of GABA-IR ganglion cells was indicated by GABA-IR fibers in the optic fiber layer and optic nerve as well as by a GABA-IR cell in the GCL that included a labeled axon. The percentage of labeled somas in the inner nuclear layer (INL) compared to all cells in each layer was similar for the two methods: 30% in INL 1 (outer layer of somas), 15% in INL 2 (middle layer), 43-52% in INL 3 (inner layer), and about 21-26% in the GCL. Labeled processes were found in three bands in the inner plexiform layer, with the densest band located in the most proximal part. Postembedding labeling of 1-micron Durcupan resin sections for GABA-IR showed the same general pattern as obtained with 10-microns cryostat sections, with additional staining, however, of type II (inner) bipolar cell Landolt's clubs. Extensive colocalization of labeling was indicated, and we conclude that GABA-IR can serve as a valid and reliable marker for GABA-containing neurons in this retina and suggest that GABA serves as a transmitter for horizontal cells, several types of amacrine cell, a type of interplexiform cell, and perhaps a small percentage of type I and type II bipolar cells and ganglion cells.     

Differential distributions of the NMDA receptor channel subunit mRNAs in the mouse retina

In the retina, the ε2 and ζ1 subunit mRNAs of the NMDA receptor channel were expressed from embryonic stages and found in ganglion cell layer and whole layer of inner nuclear layer at postnatal day 21 (P21). The ε1 subunit mRNA appeared postnatally and was distributed in ganglion cell layer and an inner third of inner nuclear layer at P21. These findings suggest that molecular organization of the NMDA receptor channel may alter during the retinal development.     

Light and electron microscopic analysis of aquaporin 1-like-immunoreactive amacrine cells in the rat retina

Aquaporin 1 (AQP1; also known as CHIP, a channel-forming integral membrane protein of 28 kDa) is the first protein to be shown to function as a water channel and has been recently shown to be present in the rat retina. We previously showed (Kim et al. [1998] Neurosci Lett 244:52-54) that AQP1-like immunoreactivity is present in a certain population of amacrine cells in the rat retina. This study was conducted to characterize these cells in more detail. With immunocytochemistry using specific antisera against AQP1, whole-mount preparations and 50-microm-thick vibratome sections were examined by light and electron microscopy. These cells were a class of amacrine cells, which had symmetric bistratified dendritic trees ramified in stratum 2 and in the border of strata 3 and 4 of the inner plexiform layer (IPL). Their dendritic field diameters ranged from 90 to 230 microm. Double labeling with antisera against AQP1 and gamma-aminobutyric acid or glycine demonstrated that these AQP1-like-immunoreactive amacrine cells were immunoreactive for glycine. Their most frequent synaptic input was from other amacrine cell processes in both sublaminae a and b of the IPL, followed by a few cone bipolar cells. Their primary targets were other amacrine cells and ganglion cells in both sublaminae a and b of the IPL. In addition, synaptic output onto bipolar cells was rarely observed in sublamina b of the IPL. Thus, the AQP1 antibody labels a class of glycinergic amacrine cells with small to medium-sized dendritic fields in the rat retina.     

Immunocytochemical localization of LANT-6-like immunoreactivity within neurons in the inner nuclear and ganglion cell layers in vertebrate retinas

LANT-6 is a hexapeptide (H-Lys-Asn-Pro-Tyr-Ile-Leu-OH) isolated from chicken small intestine, which resembles the COOH-terminal half of neurotensin, except for the amino acid substitutions Lys/Arg and Asn/Arg. The present report concerns the immunocytochemical staining of vertebrate retinas using an antiserum directed against LANT-6. In the retinas from goldfish, bird and turtle, cells in both the inner nuclear and ganglion cell layers were labeled, but in the frog cells were labeled specifically and in the rat only cells in the ganglion cell layer were labeled. Labeled cell bodies in the inner nuclear layer gave rise to processes which were seen primarily within the following laminas of the inner plexiform layer (IPL): in the goldfish, lamina 3; chicken, laminae 1,3 and 4; and turtle, laminae 3,4 and 5. The cell bodies of the labeled neurons in the ganglion cell layer gave rise to dendrites which entered the IPL and axons which descended to the optic fiber layer. The cells with LANT-6-like immunoreactivity were distributed in both the central and peripheral parts of the retina in all the species examined except frog. Measured by radioimmunoassay, the levels of LANT-6-like-immunoreactivity in extracts of turtle, chicken, and goldfish retinas were 5–30 times those for neurotensin-like immunoreactivity, however no LANT-6-like immunoreactivity was detected in frog. Multiple chromatographic analyses indicated that while the LANT-6-like immunoreactivity in chicken retina was indistinguishable from synthetic LANT-6, LANT-6 like immunoreactivity in turle and goldfish retinas was primarily associated with large molecular forms. Treatment of turtle LANT-6-like immunoreactivity with pepsin, an enzyme known to mimic processing for neurotensin precursors, yielded 3 major peptides, one of which co-chromatographed with synthetic LANT-6. The present immunocytochemical localization of LLI within cells in the inner nuclear and ganglion cell layers, coupled with the biochemical characterization of LANT-6 in the vertebrate retinas and brains, suggests that neuropeptides such as LANT-6 may play a role in visual processing both within the retina and within the visual pathways to the brain.     

Developmental changes of protein substrates of Ca / calmodulin-dependent protein kinase II in teh rat forebrain

We previously reported that the level of Ca²⁺ / calmodulin-dependent protein kinase II (CaM kinase II) α and β proteins increases with postnatal age. In the present study, we investigated the development changes in whole protein substrates of CaM kinase II as compared with those of cAMP-dependent protein kinase (A-kinase) in the rat forebrain. Protein substrates were phosphorylated with [γ-³³P]ATP, and analysed by two-dimensional gel electrophoresis. More than 50 substrates for CaM kinase II were found in the soluble and particulate fractions The phosphorylation level of more than 15 substrates increased in the particulate fraction during development. Similarly, that of more than 3 substrates increased in the soluble fraction. Some substrates for A-kinase also increased during development, although some decresed. These findings suggest that the expression of some substrates is regulated during development and that the phosphorylation reaction involves the regulation of neuronal development.