Heat shock response enhanced by cell culture treatment in mouse embryonic stem cell-derived proliferating neural stem cells.

Cells have a regulatory mechanism known as heat shock (HS) response, which induces the expression of HS genes and proteins in response to heat and other cellular stresses. Exposure to moderate HS results in beneficial effects, such as thermotolerance and promotes survival, whereas excessive HS causes cell death. The effect of HS on cells depends on both exogenous factors, including the temperature and duration of heat application, and endogenous factors, such as the degree of cell differentiation. Neural stem cells (NSCs) can self-renew and differentiate into neurons and glial cells, but the changes in the HS response of symmetrically proliferating NSCs in culture are unclear. We evaluated the HS response of homogeneous proliferating NSCs derived from mouse embryonic stem cells during the proliferative phase and its effect on survival and cell death in vitro. The number of adherent cells and the expression ratios of HS protein (Hsp)40 and Hsp70 genes after exposure to HS for 20 min at temperatures above 43°C significantly increased with the extension of the culture period before exposure to HS. In contrast, caspase activity was significantly decreased by extension of the culture period before exposure to HS and suppressed the decrease in cell viability. These results suggest that the culture period before HS remarkably affects the HS response, influencing the expression of HS genes and cell survival of proliferating NSCs in culture.

Inhibition of epidermal growth factor receptor stimulates prolactin expression in primary culture of the mouse pituitary gland.

The roles of epidermal growth factor (EGF) in the regulation of prolactin (PRL) gene expression in the normal pituitary gland remain poorly understood. In the present study, the effects of EGF and an inhibitor of the EGF receptor, erlotinib, on PRL gene expression were examined both in the pituitary tumour cell line GH3 and in a primary culture of the mouse pituitary gland under similar experimental conditions. The results showed that EGF stimulated PRL expression in GH3 cells, but not in normal cells. Erlotinib was found to counteract EGF in GH3 cells inhibiting the PRL expression enhanced by EGF. By contrast, erlotinib induced an elevation in the PRL mRNA levels in the primary culture of the adult pituitary gland and the initiation of PRL production in the culture of the foetal pituitary gland in which PRL production had not yet occurred. Western blot analyses showed that EGF induced and erlotinib inhibited the activation of extracellular regulated protein kinase equally in GH3 and normal cells. These results suggest that the consequences of EGF receptor activation in normal PRL cells contradict those in adenomatous PRL cells.

Estradiol and corticosterone stimulate the proliferation of a GH cell line, MtT/S: Proliferation of growth hormone cells.

OBJECTIVES: Estrogens are known as a potent growth-stimulator of the anterior pituitary cells such as prolactin cells and somatomammotroph cell lines, while glucocorticoids often inhibit cellular proliferation in the pituitary gland as well as in the extra-pituitary tissues. In this study, the involvement of these steroid hormones in the regulation of proliferation was examined in the MtT/S cells, secreting growth hormone (GH). DESIGN: Effects of estrogens and glucocorticoids were examined in MtT/S cells grown in the medium containing dextran-coated charcoal treated serum. The relative cell density after culture was estimated by the Cell Titer-Glo Luminescent Cell Viability Assay System, and the proliferation rate was determined by the BrdU incorporation method. The mRNA levels were determined by real-time PCR. RESULTS: Estradiol and the specific agonist for both estrogen receptor (ER) α and ERß stimulated MtT/S growth at a dose dependent manner. The membrane impermeable estrogen, 17ß-estradiol-bovine serum albumin conjugate also stimulated the MtT/S proliferation. The effects of all estrogens were inhibited by an estrogen receptor antagonist, ICI182780. Corticosterone stimulated the proliferation of MtT/S cells at doses lower than 10nM without stimulating GH gene transcription, whereas it did not change the proliferation rate at 1µM. The effects of corticosterone were inhibited by glucocorticoid receptor inhibitor, RU486, but not by the mineralocorticoid receptor antagonist, spironolactone. Both estrogens and glucocorticoids were found to stimulate the proliferation of MtT/S, increasing the mRNA expression of cyclins D1, D3, and E. CONCLUSIONS: The results suggest that estrogens and glucocorticoids may be involved in the mechanisms responsible for the proliferation of GH cells in the course of pituitary development, to maintain the population of GH cells in the adult pituitary gland, and also in the promotion of GH cell tumors.

Presentation of noise during acute restraint stress attenuates expression of immediate early genes and arginine vasopressin in the hypothalamic paraventricular nucleus but not corticosterone secretion in rats.

The present study investigated the effect of acoustic stimulation on the activation of the hypothalamic-pituitary-adrenal (HPA) axis in rats submitted to acute restraint stress, through semi-quantitative histochemical analysis of expression of immediate early gene products (c-Fos, JunB and phosphorylated c-Jun) and arginine vasopressin (AVP) hnRNA in the paraventricular nucleus (PVN). Simultaneous presentation of white or pink noise with restraint resulted in a significant attenuation of stress-induced c-Fos and JunB expression in the dorsal body of dorsal medial parvicellular subdivision (mpdd) of the PVN, as compared with restraint without noise. However, this presentation did not change phosphorylation of c-Jun and the plasma corticosterone level. Moreover, white noise presentation during restraint led to a reduction in the number of c-Fos- or JunB-expressing corticotropin-releasing hormone (CRH) neurons and the number of neurons expressing AVP hnRNA in the mpdd. Dual-histochemical labeling revealed co-expression of c-Fos and JunB, as well as JunB and AVP hnRNA in mpdd neurons. These data suggest that acoustic stimuli have an attenuation effect on the restraint-induced activation of neuroendocrine CRH neurons, resulting in the reduction in AVP production as an adaptation of HPA axis to repeated stress.

Novel Bernard-Soulier syndrome variants caused by compound heterozygous mutations (case I) or a cytoplasmic tail truncation (case II) of GPIbα.

A defective platelet glycoprotein (GP) Ib/IX/V complex [von Willebrand factor (VWF) receptor] results in Bernard-Soulier syndrome (BSS), which is characterized by macrothrombocytopenia and impaired ristocetin- and thrombin-induced platelet aggregation. We found 2 independent BSS-variant families: Case I [compound heterozygous mutations, p.Glu331X and a frame shift by a deletion at c.1444delA of GPIbα (GP1BA) terminating at a premature stop codon (p.Thr452ProfsX58)], and case II [homozygous nonsense mutation at c.1723C>T, p.Gln545X]. Case I platelets expressed no GPIbα, resulting in absence of ristocetin-induced platelet aggregation (RIPA) and 50% reduction in thrombin-induced aggregation with no shape change. The mother's platelets had 50% the expression level of A-type GPIbα (4-repeated VNTR: variable number of tandem repeats, p.[Thr145Met; Ser399_Pro411[4]]); the father's platelets had the same expression level of C-type GPIbα (2-repeated VNTR, p.Ser399_Pro411dup) as the mother's platelets. The mother's RIPA was significantly higher than the father's. Thrombin-induced aggregation was normal in both parents. Case II platelets expressed a GPIbα with an abnormal cytoplasmic tail, p.Gln545X-truncated GPIbα, which complexed with GPIX and GPV on the cell surface; its expression level of the complex was normal. Case II platelets had reversible RIPA, with no ATP release, and weak thrombin-induced aggregation without shape change. These results suggest that a signaling process through the GPIbα cytoplasmic tail required for full platelet activation is defective in BSS variant case II and a length polymorphism of GPIbα is associated with a modified level of RIPA heterozygous BSS case I.

Voluntary resistance running with short distance enhances spatial memory related to hippocampal BDNF signaling.

Although voluntary running has beneficial effects on hippocampal cognitive functions if done abundantly, it is still uncertain whether resistance running would be the same. For this purpose, voluntary resistance wheel running (RWR) with a load is a suitable model, since it allows increased work levels and resultant muscular adaptation in fast-twitch muscle. Here, we examined whether RWR would have potential effects on hippocampal cognitive functions with enhanced hippocampal brain-derived neurotrophic factor (BDNF), as does wheel running without a load (WR). Ten-week-old male Wistar rats were assigned randomly to sedentary (Sed), WR, and RWR (to a maximum load of 30% of body weight) groups for 4 wk. We found that in RWR, work levels increased with load, but running distance decreased by about half, which elicited muscular adaptation for fast-twitch plantaris muscle without causing any negative stress effects. Both RWR and WR led to improved spatial learning and memory as well as gene expressions of hippocampal BDNF signaling-related molecules. RWR increased hippocampal BDNF, tyrosine-related kinase B (TrkB), and cAMP response element-binding (CREB) protein levels, whereas WR increased only BDNF. With both exercise groups, there were correlations between spatial memory and BDNF protein (r = 0.41), p-CREB protein (r = 0.44), and work levels (r = 0.77). These results suggest that RWR plays a beneficial role in hippocampus-related cognitive functions associated with hippocampal BDNF signaling, even with short distances, and that work levels rather than running distance are more determinant of exercise-induced beneficial effects in wheel running with and without a load.

A rhythmic change of vesicular glutamate transporter (VGLUT) 2 expression in the rat pineal gland.

The pineal gland secretes melatonin under circadian control via nocturnal noradrenergic stimulation, and expresses vesicular glutamate transporter (VGLUT) 1, VGLUT2 and a VGLUT1 splice variant (VGLUT1v). Although we previously reported that VGLUT2 mRNA level of rat pineal gland at postnatal day 21 is higher in the nighttime than in daytime, questions remained as to the time of postnatal onset of this phenomenon and a 24-h change in the mRNA or protein level at postnatal days. The day-night difference in VGLUT2 mRNA level was evident 14 days after birth. In the adult, VGLUT2 mRNA and protein levels increased in the dark phase, with the protein level showing a 6-h delay. The nocturnal elevation in VGLUT2 mRNA level diminished under the constant light condition but persisted under the constant dark condition. The present data suggest that VGLUT2 in the rat pineal gland is involved in some nocturnal glutamatergic function.

Regional distribution and ontogeny of the first exon variants of the rat growth hormone receptor mRNA in the brain and the pituitary gland.

Expression of the first exon variants of the rat growth hormone receptor mRNA was studied in the brain and the pituitary gland. Four of the five different variant mRNA previously identified in the liver were detected in the cerebral cortex by a conventional reverse-transcription polymerase chain reaction, and, unlike the data reported previously, a quantitative analysis revealed that approximately 90% of the total growth hormone receptor mRNA in the cerebral cortex is V1 form. The present results suggest that the V1 was also a dominant transcript in other brain areas and the pituitary gland, not only in adult but also in fetal and postnatal period. The growth hormone receptor expression in the brain was lower during fetal period than in adults, while in the pituitary gland, the expression is markedly higher in fetuses, suggesting a yet unknown role of growth hormone in the development of this organ.

Hormonal regulation of prolactin cell development in the fetal pituitary gland of the mouse.

The developmental process of prolactin (PRL) cells in the fetal pituitary gland was studied in mice. Although PRL cells were hardly detectable in the pituitary gland of intact fetuses, a treatment with 17beta-estradiol (E(2)) in vitro induced a number of PRL cells that varied drastically in number depending on the stage of gestation with a peak at embryonic d 15. This effect was specific to E(2), with epidermal growth factor, insulin, and forskolin failing to induce PRL cells. Although both estrogen receptor (ER)alpha and ERbeta were expressed in the fetal pituitary gland, the results from ER knockout models showed that only ERalpha mediates E(2) action on PRL cells. A few PRL cells were observed in ERalpha-deficient mice as well as in their control littermates, suggesting that estrogen is not required for the phenotype determination of PRL cells. Unexpectedly, the effect of E(2) on the induction of PRL cells in vitro was diminished after embryonic d 15. Present results suggest that the exposure of fetal PRL cells to glucocorticoids (GCs) results in a reduction of sensitivity to E(2). The mechanism underlying the down-regulation of estrogen sensitivity by GCs was found not to be down-regulation of ER levels, induction of annexin 1, a GC-inducible inhibitor of PRL secretion, or a decrease in the number of PRL precursors by apoptosis. The effect of GCs appeared within 2 h and did not require a de novo protein synthesis. GCs are considered to be involved in the mechanisms of silencing pituitary PRL in gestation possibly through a novel mechanism.

Functional maturation of growth hormone cells in the anterior pituitary gland of the fetus.

Recent studies have disclosed the molecular mechanisms responsible for the phenotype determination of the anterior pituitary cell types. However, as far as growth hormone (GH) cells are concerned, particular extra-cellular cues are required for the initiation of GH and GH-releasing hormone (GHRH)-receptor gene production in addition to the expression of the cell type specific transcription factor, pit-1. The glucocorticoids play a principal role in the functional maturation of nascent GH cells in the fetal pituitary glands in rodents, inducing GH and GHRH-receptor gene expression, and establish the GH secretory system regulated by the brain in late gestation. Research supporting this role for glucocorticoid in the development of GH cells is discussed.

Upregulated expression of neuropeptide Y in hypothalamic-pituitary system of rats by chronic dexamethasone administration.

To study the effect of adrenal steroids on neuropeptide Y (NPY) synthesis in the hypothalamic-pituitary system, we examined NPY expression in rats treated with dexamethasone (a synthetic glucocorticoid) by in situ hybridization and immunohistochemistry. Rats were injected daily with dexamethasone (0.2mg/100g/day for 10 days, sc) or sesame oil (vehicle control), or non-injected (intact control). Relative staining area for corticotropin-releasing hormone or neurophysin II, a vasopressin carrier protein, was increased in the external zone of the median eminence in vehicle control, but was equivalent to that of intact control in the dexamethasone-injected group. Density of NPY-stained fiber varicosities was drastically increased in the external, but not the internal, zone of dexamethasone-injected group, coinciding with the increased NPY hybridization signal level in the arcuate nucleus. Dual-labeling experiments revealed no colocalization of NPY with hypophysiotropic or other peptides examined in single fibers of the median eminence. In the dexamethasone-injected group, expressions of NPY mRNA and peptide were detectable in a few pituitary cells, with some being corticotropes. These results suggest that NPY plays hormonal roles in the hypothalamic-pituitary-adrenal axis.

Cajal-Retzius cells and subplate neurons differentially express vesicular glutamate transporters 1 and 2 during development of mouse cortex.

In the light of the various neurobiological effects of glutamate in brain development, although some embryonic cells are a probable source of glutamate involved in the development of precursor cells and/or immature neurons, little is known about when and where glutamate plays its crucial roles during corticogenesis. To investigate these roles, we focused on the developmental expression of vesicular glutamate transporter (VGLUT)1 and VGLUT2, which are regarded as the best markers for verifying glutamatergic neuron identity, especially the spatiotemporal distributions of their transcripts and proteins in the developing mouse cortex and hippocampus. In situ hybridization studies revealed that VGLUT1 mRNA is expressed in preplate and marginal zone cells at embryonic day (E)10 and in subplate cells by E13, whereas VGLUT2 mRNA is expressed in preplate and marginal zone cells at E10 and in cells of the subventricular zone by E13. Reverse transcriptase-polymerase chain reaction analysis detected full-length VGLUT1 and VGLUT2 gene transcripts in the embryonic brain. By dual labeling combined with immunostaining for microtubule-associated protein 2 (MAP2) or reelin, we showed that MAP2-positive preplate and marginal zone neurons and subplate neurons express VGLUT1, while reelin-positive preplate and marginal zone cells and MAP2-negative subventricular zone cells express VGLUT2. The present study is the first to provide morphologically reliable evidence showing that Cajal-Retzius cells and subplate neurons are glutamatergic, and that the two cells differentially express VGLUT1 and VGLUT2, respectively, as the specific transport system of glutamate in some events orchestrated by these cells during the cortical development of mice.

Regulation of 5'-promoter activity of the rat growth hormone and growth hormone-releasing hormone receptor genes in the MtT/S and MtT/E cells.

The MtT/E and MtT/S cells have been established from a mammotrophic pituitary tumor, and postulated to be progenitor and premature growth hormone (GH) cells, respectively. The difference in the regulation of GH and GH-releasing hormone (GHRH) receptor gene transcription in relation to the developmental stage of GH cells were examined in these two cell lines. In MtT/S cells, triiodothyronine (T3), all-trans retinoic acid (RA) and 9-cis retinoic acid (9cRA) stimulated GH promoter activity but dexamethasone (DEX) did not. On the other hand, DEX stimulated GHRH-receptor promoter alone. T3, RA and 9cRA showed little effect alone but each of them augmented the effect of DEX when used together with DEX. In MtT/E cells, DEX, RA and 9cRA showed similar effect as observed in MtT/S cells on both GH and GHRH-receptor promoter activity. However, T3 neither stimulated GH promoter activity nor augmented the DEX-induced GHRH-receptor gene transcription in MtT/E cells. RT-PCR analyses revealed that both cell types expressed TRalpha1, TRbeta1 and TRalpha2, but expression of TRbeta2, a pituitary specific isoform of TR, was only detected in MtT/S cells. However, the deficiency of TRbeta2 for its own sake does not appear to be a reason why T3 action was not observed in MtT/E cells, because co-transfection of expression plasmids for TRbeta2 and RXRalpha failed in conferring on the cells an ability to respond to T3 by increased GH or GHRH-receptor promoter activity. These results suggest that the acquisition of mechanisms responsible for the regulation of GH or GHRH-receptor transcription by T3 may be involved in the process of functional development of GH cells.

Developmentally-regulated expression of tissue-specific splice variant of rat vesicular glutamate transporter 1 in retina and pineal gland.

Three distinct subtypes of vesicular glutamate transporters (VGLUTs) have been identified to date that are expressed basically in a cell type-specific manner. We have found a splice variant of VGLUT1 mRNA that is expressed almost exclusively in photosensitive tissues, i.e. the retina and the pineal gland. The variant mRNA, termed VGLUT1v, contains an additional 75 base pair sequence derived from part of a second intron (designated as exon IIa) between exons 2 and 3. The variant accounted for approximately 70% and 25%of VGLUT1 mRNA in the adult retina and pineal gland, respectively. The expression of VGLUT1v was developmentally regulated in both tissues. Organ culture showed that expression of the variant in the retina increased in association with the development of rod cells, suggesting that VGLUT1v is expressed in rod cells. In situ hybridization with variant-specific probes showed expression of VGLUT1v in the inner segment layer of photoreceptor cells. On the other hand, variant expression did not parallel the development of rhodopsin-positive cells in the pineal gland. As rod cells and pinealocytes are known to release glutamate continuously at ribbon synapses, it is possible that the variant has some functional advantage over the wild-type transporter in such a specialized manner of glutamate release.

Particular subpopulations of midbrain and hypothalamic dopamine neurons express vesicular glutamate transporter 2 in the rat brain.

Vesicular glutamate transporters (VGLUT1, -2, and -3) mediate the accumulation of transmitter glutamate into synaptic vesicles in glutamatergic neurons. VGLUT1 and VGLUT2 are more reliable glutamatergic neuron markers, since VGLUT3 also exists in other neuron types. To study whether the dopaminergic neuron uses glutamate as a cotransmitter, we analyzed VGLUTs expression in dopamine neurons of adult male rats by in situ hybridization and immunohistochemistry. In the ventral midbrain, in situ hybridization analysis revealed no VGLUT1 mRNA expression, a widespread but discrete pattern of VGLUT2 mRNA expression, and a highly limited expression of VGLUT3 mRNA. Reverse-transcriptase polymerase chain reaction analysis detected full-length VGLUT2 gene transcripts in the ventral midbrain. Using in situ hybridization combined with tyrosine hydroxylase (TH) immunostaining, only VGLUT2 signals were detectable in some TH-labeled neurons of A10 dopamine neuron groups, with the highest incidence (20%) in the rostral linear nucleus of the ventral tegmental area. In the forebrain, VGLUT2 signals were demonstrated in half of the A11 TH-labeled neurons in the hypothalamus. Double-label immunostaining for VGLUT2 and vesicular monoamine transporter 2 or TH showed that double-labeled varicosities are rarely observed in any target regions examined of A10 and A11 dopamine neuron groups. These results indicate that VGLUT2 is expressed in subsets of A10 and A11 dopamine neurons, which might release dopamine and glutamate separately from different varicosities in the majority of their single axons.

VGLUT2 expression is up-regulated in neurohypophysial vasopressin neurons of the rat after osmotic stimulation.

A second vesicular glutamate transporter (VGLUT2) is detected in magnocellular neurons in the rat hypothalamus. The present study revealed what phenotype of neurons express VGLUT2 mRNA by the histological method. We found that most vasopressin (VP) neurons and several oxytocin (OT) neurons express VGLUT2 mRNA. VGLUT2 gene expression in VP and OT neurons is enhanced with osmotic challenges. In the neurohypophysis, VGLUT2-staining in OT terminals was reduced with osmotic stimulation. These results indicate that VGLUT2 is principally expressed in VP neurons and also in some OT neurons and that VGLUT2 in VP and OT neurons is involved in osmotic regulation.

The role of pit-1 in the regulation of the rat growth hormone-releasing hormone receptor gene transcription by glucocorticoids.

Glucocorticoids are involved in the regulation of the rat growth hormone-releasing hormone (GHRH) receptor gene expression, but they act only in the presence of the pituitary specific transcription factor, pit-1. In this study, the role of pit-1 in the glucocorticoid stimulation of the GHRH-receptor gene transcription was examined. The results suggest the presence of a silencer element in the promoter and it is postulated that pit-1 permits glucocorticoid action through suppressing the inhibitory effect of an as yet unknown factor that binds to this element. The present results also suggest that the synergistic activation of the rat GHRH-receptor gene transcription depends on the proper distance between the proximal glucocorticoid response element and the pit-1 binding site.

Up-regulation of VGLUT2 expression in hypothalamic-neurohypophysial neurons of the rat following osmotic challenge.

A second vesicular glutamate transporter (VGLUT2) has been reported to be expressed in neurosecretory neurons of the hypothalamic-neurohypophysial system. To study its role in the neurosecretory neurons, we evaluated the expression of the VGLUT2 gene in the paraventricular (PVN) and supraoptic (SON) nuclei as well as the immunoreactivity in the neurohypophysis under euhydrated and chronic hyperosmotic conditions with in situ hybridization and immunohistochemistry. The intensity of hybridization signals in the PVN, SON and thalamus of rats subjected to water deprivation for 7 days, or drinking 2% NaCl for 4 or 7 days, was compared with that of euhydrated rats (control). The overall intensity in the entire PVN or SON, but not the thalamus, was higher in osmotically stimulated rats than in controls. Within the PVN, a significantly higher intensity of signals than that of controls was found only in the dorsolateral posterior magnocellular region in 4-day salt-loaded rats and in all subregions in water-deprived or 7-day salt-loaded rats. The intensity in the SON was higher in the stimulated rats than in controls, regardless of subregions. In the neurohypophysis, VGLUT2 staining was frequently localized in vasopressin terminals of control rats and was apparently reduced in stimulated rats. These results indicate that VGLUT2 is principally expressed in magnocellular vasopressin neurons, suggesting some local effect of intrinsic glutamate on neurohypophysial hormone secretion.

Expression of inorganic phosphate/vesicular glutamate transporters (BNPI/VGLUT1 and DNPI/VGLUT2) in the cerebellum and precerebellar nuclei of the rat.

Expression of inorganic phosphate/vesicular glutamate transporters (BNPI/VGLUT1 and DNPI/VGLUT2) was studied in the cerebellum and precerebellar nuclei of rats using immunohistochemistry and in situ hybridization. DNPI/VGLUT2-stained mossy fibers were principally seen in the vermis (lobules I and VIII-X) and flocculus, whereas BNPI/VGLUT1-stained mossy fibers were localized throughout the cortex. Some vermal and floccular mossy fibers were stained for both transporters. High levels of DNPI/VGLUT2 mRNA hybridization signals were demonstrated in many neurons throughout the vestibular nuclear complex as well as the lateral reticular, external cuneate, inferior olivary and deep cerebellar nuclei. Significant BNPI/VGLUT1 mRNA signals were demonstrated in the lateral reticular nucleus and vestibular nuclear complex but not in the inferior olivary nucleus, indicating that climbing fibers have DNPI/VGLUT2 only. These results show that DNPI/VGLUT2 is expressed preferentially to vestibulo-, reticulo- and cuneocerebellar neurons, some of which also possess BNPI/VGLUT1, suggesting some differential and co-operative functions between DNPI/VGLUT2 and BNPI/VGLUT1 in the cerebellum.

Differential expression of two distinct vesicular glutamate transporters in the rat retina.

Expression and cellular localization of vesicular glutamate transporters (BNPI and DNPI) were studied in the rat retina. RT-PCR showed expression of both transporter mRNAs. hybridization demonstrated BNPI mRNA signals in the inner segments of photoreceptors and the inner nuclear layer, whereas DNPI mRNA signals were confined to the ganglion cell layer. Punctate BNPI immunoreactivity was localized in the inner and outer plexiform layers, and weak DNPI immunoreactivity was detectable only in some cells and fibers of the ganglion cell layer. The present study suggests that BNPI exists in photoreceptors and bipolar cells, while DNPI is present in ganglion cells, as specific systems in distinct glutamatergic neurons of the retina.