Reexamination of Dopaminergic Amacrine Cells in the Rabbit Retina: Confocal Analysis with Double- and Triple-labeling Immunohistochemistry

Dopaminergic amacrine cells (DACs) are among the most well-characterized neurons in the mammalian retina, and their connections to AII amacrine cells have been described in detail. However, the stratification of DAC dendrites differs based on their location in the inner plexiform layer (IPL), raising the question of whether all AII lobules are modulated by dopamine release from DACs. The present study aimed to clarify the relationship between DACs and AII amacrine cells, and to further elucidate the role of dopamine at synapses with AII amacrine cell. In the rabbit retina, DAC dendrites were observed in strata 1, 3, and 5 of the IPL. In stratum 1, most DAC dendritic varicosities—the presumed sites of neurotransmitter release—made contact with the somata and lobular appendages of AII amacrine cells. However, most lobular appendages of AII amacrine cells localized within stratum 2 of the IPL exhibited little contact with DAC varicosities. In addition, double- or triple-labeling experiments revealed that DACs did not express the GABAergic neuronal markers anti-GABA, vesicular GABA transporter, or glutamic acid decarboxylase. These findings suggest that the lobular appendages of AII amacrine cells are involved in at least two different circuits. We speculate that the circuit associated with stratum 1 of the IPL is modulated by DACs, while that associated with stratum 2 is modulated by unknown amacrine cells expressing a different neuroactive substance. Our findings further indicate that DACs in the rabbit retina do not use GABA as a neurotransmitter, in contrast to those in other mammals.

The Calretinin Immunoreactive Ganglion Cell Postsynaptic to the ON-Cholinergic Amacrine Cell in the Guinea Pig

PURPOSE: To demonstrate the characterization calretinin-immunoreactive displaced amacrine cells in the ganglion cell layer using immunohistochemistry and electron microscopy. METHODS: For immunohistochemistry, sections from guinea pig retina were incubated with mouse monoclonal antibody directed against calretinin. For double label studies, sections were incuated in mixture of mouse monoclonal anti-calretinin or rabbit polyclonal anti-calretinin with following antibodies: goat polyclonal anti-ChAT, rabbit polyclonal anti-GABA, mouse monoclonal anti-GABAA receptor alpha1, beta2/3. Sections were analyzed using Bio-rad Radiance Plus confocal scanning microscope. Stained sections from three guinea pig were observed with transmission electron microscope. RESULTS: Calretinin immunoreactivity was present in displaced amacrine cells and ganglion cells gaving rise to processes ramified in the inner part of the inner plexiform layer in stratum 4. The same stratum was also occupied by the dendrites of ON-cholinergic amacrine cells. Double-labeling demonstrated that dendrites and cell bodies of displaced amacrine cells colocalized with ON-cholinergic amacrine cells and dendrites of ganglion cells directly overlapped with dendrites of ON-cholinergic amacrine cells. The synaptic connectivity was identified by electron microscopy. Ganglion cell dendrites received synaptic input from ON-cholinergic amacrine cell. GABAA receptor beta2/3 subunit bands cofaciculates the dendrites of displaced amacrine cell and ganglion cell that are juxtapose to the alpha1 subunit of GABAA receptor. CONCLUSIONS: These results indicate that ON-cholinergic amacrine cells modulate calretinin-labeled ganglion cell via GABAA receptor beta2/3 in the guinea pig retina.

Pre-synaptic Neuronal Changes of AII Amacrine Cells in the Streptozotocin-induced Diabetic Rat Retina / 대한해부학회지

It has been previously reported that parvalbumin expression was downregulated in AII amacrine cells, while upregulated in a subset of cone bipolar cells electrically synapse with AII amacrine cell in the streptozotocin-induced diabetic rat retina. In the present study, we aimed to trace biochemical changes of pre-synaptic neurons to AII amacrine cells in rat retina following diabetic injury. Diabetic condition was induced by streptozotocin injection into Sprague-Dawley rats aged of 8 weeks. The experimental term of induced diabetes was set at 1, 4, 12 and 24 weeks. Changes of pre-synaptic neurons were evaluated by immunohistochemistry and Western blot analysis with anti-protein kinase C (PKC)-alpha and anti-tyrosine hydroxylase (TH) antibodies. Rod bipolar cells immunolocalized with PKC-alpha antibody extended their enlarged axon terminals into stratum 5 of the inner plexiform layer. In later diabetes, the axon was shorten and its terminals of rod bipolar cell are slightly enlarged. The protein levels of PKC-alpha were slightly increased along with the duration of diabetes. TH immunoreactive neurons are morphologically classified into two subtypes of amacrine cells in the inner nuclear layer: one (type 1) has large soma with long and primary dendrites, classified with dopaminergic, and the other (type 2) has small soma with dendritic arborization. In the outermost inner plexiform layer, ring-like structures being composed of type 1 cell processes were densely distributed. In diabetic retina, the intensity of TH immunoreactivity in type 1 neurons was reduced. In accordance with morphological changes, the protein levels of TH were reduced during diabetes. These results demonstrate that TH immunoreactive dopaminergic amacrine cells are more susceptible to diabetic injury than the rod bipolar cells in the rat retina and may suggest that downregulation of parvalbumin expression in AII amacrine cells of diabetic retina is mainly due to dysfunction of pre-synaptic dopaminergic amacrine cells.

Altered Expression of Parvalbumin in Neural Retina of the Insulin-dependent Diabetic Rat / 대한해부학회지

Calcium-binding proteins in the nervous system are functioned in Ca2+ buffering and Ca2+ transport and regulation of various enzyme systems. They potentially have a number of different effects on cells includingaltering the duration of action potentials, promoting neuronal bursting activity and protecting cells against the damaging effects of excessive calcium influx. The present study has been designed to clarify the differential responding patterns of parvalbumin immunoreactive neurons in the rat retina following diabetic injury, for better understandings of role of parvalbumin in the retinal circuitry and in calcium homeostasis. Experimental diabetes was induced by a single intravenous injection of streptozotocin in a dose of 60 mg/kg body weight. Diabetic rats showing high blood glucose levels (above 300 mg/dL) were cared for 1, 4, 8, 12 and 24 weeks, respectively. The retinas at each time point were processed for immunohistochemistry and Western blotting using antiparvalbumin antibody. In the rat retina at normal, parvalbumin immunoreactivity appeared in AII amacrine cells, amacrine cells of a widefield type and displaced amacrine cells. A few bipolar cells are also showed the reactivity. During diabetes, the intensity of parvalbumin immunoreactivity is decreased especially in the AII amacrine cells. The cell number of parvalbumin immunoreactive neurons has showed no large changes throughout the diabetes, except that of bipolar cells. That population of parv immunoreactive of bipolar cells has increased remarkably at later diabetic periods. The protein levels of parvalbumin have showed transiently a slight increase at earlier diabetic periods, and then decreased to lower than that of normal. Parvalbumin immunoreactive bipolar cells at diabetes are co-localized not with PKC-alpha or recoverin, but with glutamate transporter Glt-1b. AII amacrine cell processes were joined with each other and with axon terminals of presumed cone bipolar cells by gap junction. These results suggest that the calcium buffering activity of parvalbumin is shifted from AII amacrine cells to a certain type of cone bipolar cells, in response to diabetes. This event may be occurred through electrically coupled gap junction in between these cell processes.

Expression of Substance P in the Experimental Diabetic Rat Retina / 대한해부학회지

Substance P (Sub P) being composed of 11 amino acids sequence is a kind of tachykinin family peptides. It has been known that this substance plays a role of neurotransmitter and/or neuromodulator and is a very potent vascular growth factor in the nervous system. This study has been investigated expression pattern of Sub P in the rat retina at normal and alteration of Sub P expression following diabetic injury using immunohistochemistry. Diabetic condition was induced by a single injection of streptozotocin in Sprague-Dawley rats aged 8 weeks. The animals showing high blood glucose levels (above 300 mg/dL) were cared for 1, 4, 8 and 12 weeks, respectively. The whole-mounted or sectional preparations of the retinas were used for Sub P immunohistochemistry. Sub P immunoreactivity has been localized in subsets of amacrine cells in the inner nuclear layer (INL) and displaced amacrine cells in the ganglion cell layer (GCL) in the normal retina. The dendrites from amacrine cells in the INL were ramified with strata 1 and 3, and those from displaced amacrine cells in the GCL with strata 5 of the inner plexiform layer. Sub P immunoreactive neurons in both the INL and the GCL were more densely populated in the superior half of the retina. During diabetes, the cell number of Sub P immunoreactive neurons was decreased to one third of the normal value at 4 weeks of diabetes and then slightly increased to half of the normal value at 12 weeks of diabetes. In addition, Sub P mRNA levels were reduced at 4 weeks but reincreased at 12 weeks. These results suggest that Sub P in the rat retina at normal state may function differentially in the superior or the inferior halves and Sub P synthetic pathway in the retinal neurons maybe irradiated in earlier stages of diabetic retinopathy.

Changes in Retinal Neuronal Population in DBA/2J Mice with Topical Intraocular Pressure Lowering Drugs

PURPOSE: The DBA/2J (D2) mouse is a transgenic mouse with pigmentary glaucoma. In a previous study, we found a reduction of inner retinal thickness in D2 mice. We attempted to discover the effect of eye drops on the retina of D2 mice. METHODS: Ten-month-old D2 mouse eyes were treated with Timoptic XE(R), Cosopt(R), and Xalacom(R) eye drops for a 1-month period. Immunohistochemical staining was performed on the mouse eye sections for analysis. RESULTS: In the control group, GABA and OPN immunoreactivity were markedly decreased and NOS immunoreactivity was increased. In all experimental group, GABA and OPN immunoreactivity were increased, and OPN immunoreactivity was markedly increased especially in the Cosopt(R) group. NOS immunoreactivity was decreased in all experimental groups. There was no difference in glycine immunoreactivity between the control and experimental groups. CONCLUSIONS: Combination anti-glaucoma eye-drops to the D2 mouse changed the retinal neuronal population and these drugs might play an important role in the mechanisms of retinal neuronal death; potential strategies for neuroprotection should therefore be evaluated.

Maturation of the Neurokinin 1 (NK1) Receptor Immunoreactive Amacrine Cells in the Rat Retina during Postnatal Development / 대한해부학회지

We examined the morphological maturation of amacrine cells expressing neurokinin 1 (NK1) receptor, whose ligand is substance P, in the rat retina, focusing on the period from postnatal day 5 (P5) when the outer plexiform layer is formed, to postnatal day 13 (P13) when the eyes open, with immunohistochemistry using a specific antiserum against NK1 receptor, and we compared maturing NK1 receptor-immunoreactive (NK1 receptor-IR) amacrine cells with adult one. In the adult retina, numerous NK1 receptor-IR amacrine cells were located in the inner part of the inner nuclear layer (INL) adjacent to the inner plexiform layer (IPL), and their processes emerging from the somata branched and stratified at 1, 2, and 5 strata of within the IPL. NK1 receptor-IR amacrine cells were already observed at P5. The cell bodies were located in the inner INL away from the IPL and their processes branched and formed two distinct bands in the IPL. Afterwards, somata of NK1 receptor-IR amacrine cells moved toward the inner part of the INL, and thus, were located in the INL adjacent to the IPL. Their processes formed three distinct bands at P10 and then, at P13, three bands occupied the same strata as those of the adult, which were posed at 1, 2, and 5 strata of the IPL. During the postnatal development, most of NK1 receptor-IR amacrine cells directly extended one or a few primary dendrites toward the IPL and formed the strata. However, some of the labeled cells located at the outermost row had horizontal processes emerging from their primary dendrites, and these horizontal processes branched and formed plexuses in the INL. The NK1 receptor-IR amacrine cells with horizontal processes were frequently observed at P7, rarely at P10, and not at P13 and in the adult. These results indicate that the NK1 receptor-IR amacrine cells of the rat retina morphologically mature by way of migration of their somata within the INL and formation of distinct processes during postnatal development, and suggest that they morphologically and functionally complete the maturation process about the time of P13.

Morphology and Distribution of the Vasoactive Intestinal Polypeptide (VIP)-Immunoreactive Amacrine Cells in the Mouse Retina / 대한해부학회지

Vasoactive intestinal polypeptide (VIP) is a neuroactive substance that is widely expressed in both non-mammalian and mammalian retinas. In this study, we immunocytochemically identified and investigated the VIP-containing neurons in the mouse retina, which has become an important model for the study of the structure and function of the mammalian retina, mainly because of the wide availability of transgenic animals. VIP immunoreactivity was observed in the somata of the amacrine cells in the inner nuclear layer (INL) and their varicose processes ramifying in strata 1 and 3 of the inner plexifrom layer (IPL). The distribution of VIP-immunoreactive (IR) amacrine cells showed a peak of 430 cells/mm2 in the central retina and minimum values of 50 cells/mm2 in the peripheral one. Double-label experiments demonstrated that all VIP-IR amacrine cells possessed GABA immunoreactivity. These results demonstrate that VIP-IR amacrine cells of the mouse retina make up a neurochemically and morphologically distinct subpopulation of the GABAergic amacrine cell population.

Immunocytochemical Study on the Development of the Rod Pathway in the Rat Retina / 대한해부학회지

Rod bipolar cells constitute the second-order neuron in the rod pathway. Previous investigations of the rat retina have evaluated the development of other components of the rod pathway namely the AII amacrine cell and GABAergic amacrine cell populations. To gain further insights into the maturation of this retinal circuitry, we studied the development of rod bipolar cells, immunocytochemistry with antibodies directed to the protein kinase C (PKC), in the rat retina. PKC immunoreactivity first appeared in postnatal day 9 (P9), faint PKC immunoreactivity was observed in the cell bodies located at the distal inner nuclear layer (INL), dendrites in the outer plexiform layer (OPL) and immunoreactive bands in the proximal inner plexiform layer (IPL). PKC immunoreactive cells and terminal bulbs at P10 show stronger immunostaining. At P15, the time of eye opening, PKC immunoreactive cells display stronger immunostaining than those of P10 and more mature characteristics like in the adult retina. Double fluorescence immunocytochemistry using an antiserum against parvalbumin, a marker for the AII amacrine cells, or GABA revealed that PKC immunoreactive rod bipolar cell terminals make contact with AII amacrine cells and GABAergic neurons in the proximal IPL from P9. Given these results, the different components of the rod pathway follow a similar pattern of maturation, presumably allowing the rod pathway to function at the early developmental stage of retina.

Distribution Pattern and Synaptic Circuitry of Cholinergic Neurons in the Rat Retina / 대한해부학회지

The role of acetylcholine as an excitatory neurotransmitter is well established, and cholinergic neurons appear to play an important role in the mammalian retinae. Though it has been reported that certain conventional and displaced amacrine cells are consistently labeled with anti-choline acetyltransferase antiserum in the mammalian retinae, little has been studied on the synaptic circuitry of cholinergic neurons to clarify mechanism of its action in the visual processing of the mammalian retinae. This study was conducted to localize cholinergic neurons and to define their synaptic circuitry in the rat retina by immunocytochemical method using anti-choline acetyltransferase antiserum. The results were as follows: 1. Cholinergic neurons of the rat retina were conventional amacrine cells located in the inner nuclear layer and displaced amacrine cells in the ganglion cell layer. 2. Cholinergic amacrine cells were branched in the middle of the sublamina a of the inner plexiform layer, and cholinergic displaced amacrine cells branched in the sublamina b, forming one prominent band, respectively. 3. Presynaptic processes to cholinergic amacrine cell processes were axon terminals of invaginating and flat cone bipolar cells, and unlabelled amacrine cell processes in the inner plexiform layer. Postsynaptic dyads at the ribbon synapses of axon terminals of cone bipolar cells were cholinergic amacrine cell process and dendrite of ganglion cell, cholinergic amacrine cell process and unlabelled amacrine cell process and cholinergic amacrine cell process and cholinergic amacrine cell process. In addition, cholinergic amacrine cell process formed postsynaptic monoad at the ribbon synapse. 4. Cholinergic amacrine cell processes made output conventional chemical synapses onto the dendrites of ganglion cells, unlabelled amacrine cell processes and cholinergic amacrine cell processes in the inner plexiform layer. These results demonstrate that (1) cholinergic neurons are conventional amacrine cells and displaced amacrine cells of which somata are located in the inner nuclear layer and ganglion cell layer, respectively, (2) cholinergic conventional amacrine cells are involved in OFF pathway, and cholinergic displaced amacrine cells play an important role in ON pathway in visual processing of lightness, and (3) acetylcholine released from cholinergic neurons by light excites directly ON and OFF ganglion cells or indirectly ON and OFF ganglion cells via non-cholinergic amacrine cells.

IMMUNOHISTOCHEMICAL STUDY ON THE COEXISTENCE OF ENKEPHALIN AND SOMATOSTATIN IN AMACRINE CELLS OF THE CHICKEN RETINA / 解剖学报

The immunohistochemical single and double label techniques were used to study the localization and coexistence of immunoreaetive enkephalin (ENK) and somatostatin (SOM) in amacrine cells of the chicken retina. The single label experiments showed that certain SOM-immunoreactive amacrine cells are similar in morphology, location in the inner nuclear layer (INL) and the manner of processes ramified in the inner plexiform layer (IPL) to some ENK-immunoreactive amacrine cells, although the plexus of SOM-immunoreactive processes in sublamina 3 and 4 were not as dense as ENK-immunoreactive plexus and SOM-immunoreactive processes in sublamina 5 were not observed. The double label studies indicated that some amacrine cells showed both SOM and ENK positive immunoreactivity and some other amacrine cells showed only SOM or ENK positive immunoreactivity.The coexistence of two neuropeptides or a neuropeptide and a classical neurotransrnitter in the retinal amacrine cells were also discussed.