GABA and GABA(A) receptor antagonists alter developing cone photoreceptor development in neonatal rabbit retina.

Gamma aminobutyric acid (GABA) has been established as an important developmental signal in a number of regions of the central nervous system (CNS), including retina. Our previous studies have shown that GABAergic horizontal cells act as the initial synaptic target for developing cone photoreceptors in neonatal rabbit retina. Since intraocular injections of the GABA(A) receptor antagonists, picrotoxin or bicuculline, disrupt cone synaptogenesis in vivo, GABA released from horizontal cells may provide a necessary signal for cone axon growth and/or synapse formation. In the current report, we have used cultured retinal explants to examine the effects of GABA(A) receptor antagonists on other aspects of developing cones. These include the distribution pattern of cone cell bodies across the outer surface of the retina and the expression of GABA(A) receptors within both cone cell bodies and axonal processes. Peanut agglutinin (PNA), a plant lectin that specifically labels cone plasma membrane and extracellular matrix, was used to monitor cone development, and a GABA(A) receptor antibody against the beta2/3 subunits of the protein was used to label GABA(A) receptors. Results showed that cones maintained in the explant culture express GABA(A) receptors in a temporal and spatial pattern similar to that observed in vivo, namely a low expression of receptors on cone cell bodies at postnatal day 1 (P1), peaking around P3 and diminishing by P7. Neonatal retinal explants exposed to the GABA(A) receptor antagonists, bicuculline (10 microM) or SR95531 (5 microM), for 24 h in culture showed disruption of the normal distribution of cone cell bodies. When GABA (100 microM) was added along with either antagonist, cone cell bodies appeared normal. Neither bicuculline nor SR95531 alone had any effect on the general morphology of other retinal layers, suggesting that these GABA(A) receptor antagonists at the concentrations used were not acting as nonspecific disruption agents. The effects of GABA antagonists were confined to the first week after birth with no disruption seen in P9 or adult explant cultures. These data provide a direct demonstration of the necessity for GABAergic input to cones during active synaptogenesis. As we have previously shown, GABA(A) receptor activation causes a substantial increase in intracellular calcium concentrations in cones and thereby could provide a mechanism by which GABA regulates cone maturation.

GABA(A) receptor immunoreactivity is transiently expressed in the developing outer retina.

Extensive evidence has suggested a trophic role of gamma-aminobutyric acid (GABA) on developing cone photoreceptors in postnatal retina. In a previous study, we showed that GABA raises intracellular calcium levels in the developing cones via activation of GABA(A) receptors. Using confocal microscopy in conjunction with immunocytochemistry, we have now demonstrated that (1) GABA(A) receptor subunits are localized on cone cell bodies as well as on cone pedicles, indicating that GABA has a direct, rather than indirect, effect on cones and (2) the temporal expression of GABA(A) receptor subunits coincides with the developmental effects of GABA on cone synaptogenesis. An antibody against the beta 2/3 subunits of the GABA(A) receptor and a specific cone marker peanut-agglutinin lectin (PNA) were used to double-label wholemount neonatal retinal preparations. Results show that GABA(A) receptors are transiently expressed on cone photoreceptors in the early stages of postnatal retinal development. GABA (A)receptor immunoreactivity is clearly present on cone cell bodies and their processes and on other--as yet unidentified--elements (horizontal cells?) in the outer plexiform layer. Immunoreactivity decreases within cone photoreceptor somata after postnatal day 5, but persists in the processes of the outer plexiform layer until day 7. Our results provide support for the hypothesis that GABA acts as an important developmental regulator of cone photoreceptor maturation.

Multivariate analysis of factors associated with postoperative pulmonary complications following general elective surgery.

OBJECTIVE: To develop a predictive model identifying perioperative conditions associated with postoperative pulmonary complications (PPCs). DESIGN: A prospective survey of patients whose preoperative history and physical examination, spirometric, PaO2 and PaCO2 analysis, and operative results were recorded. These patients underwent postoperative cardiopulmonary examinations until they were discharged from the hospital; their medical records were also reviewed until they were discharged from the hospital. SETTING: The Louisville Veterans Administration Medical Center, Louisville, Ky. PATIENTS: A randomly chosen sample of patients aged 40 years or older who required elective, nonthoracic surgery under general or spinal anesthesia and who were hospitalized at least 24 hours postoperatively. MAIN OUTCOME MEASURE: An analysis of risk factors associated with the development of 1 or more of the following conditions: acute bronchitis, bronchospasm, atelectasis, pneumonia, adult respiratory distress syndrome, pleural effusion, pneumothorax, prolonged mechanical ventilation, or death secondary to acute respiratory failure. RESULTS: Postoperative pulmonary complications developed in 16 (11%) of 148 patients. The risk factors found to be higher among those with PPCs compared with those without PPCs were postoperative nasogastric intubation (81% vs 16%, P<.001), preoperative sputum production (56% vs 21%, P=.005), and longer anesthesia duration (480 vs 309 minutes, P<.001). Upper abdominal surgery was performed in 11 (69%) of the 16 patients with PPCs and in 20 (15%) of the 132 patients without PPCs (P<.001); this difference lost significance in multivariate analysis. The final linear logistic model included postoperative nasogastric intubation (odds ratio [OR], 21.8), preoperative sputum production (OR, 4.6), and longer anesthesia duration (OR exp[0.01x] for an increase in x minutes) (1 minute of additional anesthesia time increases the OR to 1.01), resulting in 92% accuracy in predicting PPCs. CONCLUSIONS: We identified 3 potentially modifiable risk factors for PPCs. If validated, our results may lead to modifications of perioperative care that will further reduce PPCs.

Pyogenic liver abscesses: a comparison of older and younger patients.

OBJECTIVE: To evaluate whether older patients with pyogenic liver abscess have distinctive presenting features or if their management differs from that of younger patients. DESIGN: Retrospective chart review of all cases occurring from 1982 to 1992. SETTING: A regional trauma centre and two large community hospitals. PATIENTS: A total of 38 individuals with a final diagnosis of pyogenic liver abscess. Seventeen patients aged 70 or older comprised the study group and 21 patients under age 70 the comparison group. MEASUREMENTS: Clinical features, laboratory data, therapeutic interventions and outcomes were sought. The presumed aetiology of the abscess was determined. RESULTS: The study group had fewer men (47% vs 81%, P=0.028), less abdominal tenderness on physical examination (41% vs 76%, P=0.028) and fewer positive blood cultures in those sampled (31% vs 67%, P=0.04) than the comparison group. No study group patient had a history of trauma. Times to diagnosis were 3.2 and 5.9 days (P=0.14) and lengths of stay 21.6 and 29.3 days (P=0.08) for study and comparison groups, respectively. There were no differences in mortality or other demographic, clinical, laboratory or pathological variables. CONCLUSIONS: Elderly patients with pyogenic liver abscess have some subtle differences in clinical and laboratory presentation, but these do not appear to delay diagnosis. Active management is tolerated well, with no difference in mortality.

Bodyfat analysis and perception of body image.

Obesity is a common nutritional problem among children. Using the Futrex 5000A method of bodyfat measurement, this prospective study determined the percentage bodyfat in a self-selected, indigent, predominantly black population and the accuracy of perceived body image. Bodyfat exceeded the optimal range in 39% and 67% of female and male children, respectively. Females tended to view themselves as fatter and males perceived themselves as thinner than their actual composition. Parents were more accurate in their perception of obesity in their daughters (88%) than in their sons (52%). Children did not recognize the importance of exercise in preventing obesity. Bodyfat measurement and counseling should be done at an early age to improve this remarkable lack of perception about obesity.

Infant sleep position instruction and parental practice: comparison of a private pediatric office and an inner-city clinic.

OBJECTIVE: To determine infant sleep instructions that hospital personnel in our community were giving to parents and actual positions practiced after the April 15, 1992 American Academy of Pediatrics recommendation for nonprone positioning. DESIGN: Survey of mothers of infants

Primary care physician access and gatekeeping: a key to reducing emergency department use.

Use of the Emergency Department (ED) for nonurgent conditions results in increased cost and discontinuous health care. This prospective study evaluated a program (KenPAC) that required 24-hour access to a primary care physician (PCP) with ED gatekeeping responsibility. Following established criteria, medical records were reviewed for appropriateness of ED use by an urban indigent pediatric population. Emergency Department visits declined (10% to 7.6% (P = 0.00005) and inappropriate visits dropped (41% to 8%) (P < 0.00001) before KenPAC and after KenPAC, respectively. Parental experience, as judged by age and number of children, played a significant role in ED use. The institution of gatekeeping activity contributed to the reduced overall and inappropriate use of the ED.

N-methyl-D-aspartate-mediated glutamate toxicity in the developing rabbit retina.

Extracellular levels of endogenous glutamate are relatively high in the developing rabbit retina but nonetheless appear to promote cell survival and developmental processes at concentrations considered toxic in the adult. We wished to examine the development of retinal susceptibility to glutamate toxicity as well as the protective effects of two N-methyl-D-aspartate (NMDA) antagonists, 2-amino-5-phosphono-5-valeric acid (APV) and dextromethorphan (Dex), and the nitric oxide synthase (NOS) inhibitor, NG-methyl-L-arginine (metARG). One day in vitro retinal explants of adult and neonatal rabbits were incubated with various agonists and antagonists, and stained with trypan blue to visualize necrotic cells. The density of the necrotic cells was analyzed using the Zeiss Videoplan 2. Immature neurons were approximately 10-fold less sensitive to NMDA toxicity compared to the adult. Although both NMDA antagonists and metARG provided marked protection for adult retinal neurons against glutamate toxicity, the modest susceptibility of the immature neuron was blocked only by Dex and not APV or metARG. At least two factors may contribute to the ability of the neonatal retina to survive in the presence of high levels of endogenous extracellular glutamate. First, the 10-fold developmental increase in NMDA toxicity occurs simultaneously with a 12-15-fold downregulation of extracellular glutamate, probably through the actions of maturing Muller cells. Second, the NMDA/NO excitotoxic pathway may not be active at birth since an NOS inhibitor had little effect at this stage and our previous morphological data demonstrate that NOS-containing cells are not present in their mature configuration until the second postnatal week.

GABA and GABA-A receptors are maximally expressed in association with cone synaptogenesis in neonatal rabbit retina.

Previous studies have shown the cone photoreceptors form reciprocal synapses with horizontal cells during the first week after birth in rabbits. These synapses constitute pioneering elements of the developing outer plexiform layer. We now report that antibodies against the alpha-1 and against the beta-2/3 subunits of the GABA-A receptor label a highly restricted sublamina in the developing outer plexiform layer known to contain nascent cone photoreceptor terminals. Staining is relatively weak at birth, increases to maximal levels between postnatal days 5 and 7, and is significantly reduced in the adult. These results support recent calcium imaging studies which have shown that the activation of GABA-A receptors causes an increase in intracellular free calcium in cones, an effect which is observed only at 3-9 days after birth. The transient expression of GABA-A receptors in this region coincides with the period of peak expression of GABA immunoreactivity in horizontal cells. A direct functional link between GABAergic transmission and cone synaptogenesis is suggested by previous reports that GABA-A receptor antagonists cause disruption of cone synaptogenesis. Together these findings support the notion that GABA functions as a developmental neurotransmitter which is produced by horizontal cells and interacts with developing cone axons in order to facilitate synaptic linkage between these two cells types.

Expression and downregulation of the GABAergic phenotype in explants of cultured rabbit retina.

PURPOSE: To study the morphologic and neurochemical development of the rabbit retina in explant culture. METHODS: Explants of retina from newborn rabbits were cultured in defined medium in the absence of serum or soluble growth factors. The morphology of the explant and the neurochemical development of the GABAergic system were examined by light microscopy, autoradiography, and immunohistochemistry for 7 days and compared to those of the postnatal rabbit retina in vivo. RESULTS: Cultured explants from newborn rabbit retina develop and maintain well-defined plexiform and cellular layers up to 7 days. Exogenous 3H-gamma-aminobutyric acid (GABA) and antibodies against GABA labeled a population of horizontal, amacrine and displaced amacrine cells in the ganglion cell layer during the first 3 days in culture. After 4 days in culture, the extent of uptake and immunolabeling was diminished among all three cell types, but labeled horizontal cells were markedly rare. At 7 days in culture, uptake and GABA-like immunoreactivity could not be detected in horizontal cells, but antibodies to calbindin-D reacted with horizontal and amacrine cells in the appropriate retinal layers. Peanut agglutinin lectin binding studies revealed a mosaic of cone photoreceptor inner segments indistinguishable from that of neonatal retina in vivo. CONCLUSIONS: The experiments show that the maturation of cellular layers and the developmental expression of the GABAergic phenotype can be observed in retinal explants cultured under chemically defined conditions. Histochemical evidence is presented that indicates cultured explants of newborn rabbit retinas express markers of the GABAergic phenotype in a manner consistent with that observed in vivo. The authors show that horizontal cells continue to survive in culture after the diminution in GABA immunoreactivity.

Calbindin immunoreactivity of horizontal cells in the developing rabbit retina.

Horizontal cells are retinal interneurons that establish inhibitory feedback loops within the outer plexiform layer of the primary visual pathway. Most mammalian retinas contain two types of horizontal cells. A-type horizontal cells have neuritic branches that contact cone photoreceptors exclusively, while the B-type horizontal cells have dendritic branches that contact cones, in addition to axons that form synapses with rod photoreceptors. Immunoreactivity for calbindin, a calcium binding protein involved in calcium transport, was used as a marker for horizontal cells during post-natal development of the rabbit retina. On post-natal days 1, 3 and 5, calbindin immunoreactivity is limited to a single population of A-type horizontal cells. They appear as a monolayer of cells with broad tapering processes, establishing the proximal border of the nascent outer plexiform layer and forming a target for ingrowing cone photoreceptor terminals. The size and density of the cell bodies and the length of neuritic processes are essentially unchanged during this period, which corresponds to the time of peak expression of GABAergic markers in horizontal cells. Coincident with a decrease in GABAergic markers and the completion of cone-to-horizontal cell synaptogenesis by day 7, changes within the horizontal cell mosaic are detected morphometrically. A delayed phase of overall cell growth results in a 70% increase in average somal diameter (representing a 3.7-fold increase in spherical volume), a six-fold increase in mean neurite length and a decrease in cell density to one-third of that found in the newborn. We conclude that the process of terminal differentiation of horizontal cells is not complete until some time after the second post-natal week. Furthermore, the expression of GABAergic markers is associated primarily with early maturational events, whereas expression of calbindin is sustained throughout post-natal development, suggesting a prominent role for calcium dependent mechanisms at all development stages.

Factors associated with improved immunization rates for urban minority preschool children.

Urban, poor, preschool children are noted for having low immunization rates. To determine factors related to completion of immunization, vaccine records of 479 3-year-old children from an inner-city pediatric clinic were reviewed. Complete immunization was defined as four diphtheria-tetanus-pertussis doses, three oral polio vaccine doses, one measles-mumps-rubella dose, and one Haemophilus influenzae type b vaccine dose. Seventy percent of our patients were up-to-date by 2 years of age. The administration of all age-appropriate vaccines at a single visit for patients 15 months and older, the establishment of a continuous primary-care relationship, earlier age at first immunization, and lower birth weight were significantly associated with higher immunization levels in our study.

Incidence of urinary tract infection in an inner-city outpatient population.

Premature neonates have been shown to be at increased risk for the development of urinary tract infections. To determine whether this risk persists after the newborn period, we evaluated the incidence of urinary tract infection in children attending an inner-city clinic during a 4-year period. Patients were categorized as premature if born at < 36 weeks' gestational age. Urinalyses were done on 13,683 samples obtained at ages 1, 6, and 12 months and yearly from 2 through 12 years for screening or to evaluate symptoms. The urinalysis had abnormal results in 17%, but only 3.6% of these subsequently had positive urine culture results. Forty children had 85 episodes of bacteriuria, with 37 (44%) of the cases associated with symptoms. The incidence of bacteriuria was 0.9% and 0.1% in premature and term infants, respectively (p < 0.0001). All seven male children had the initial infection in the first year of life. We found a significant association between prematurity and the risk for bacteriuria persisting through the first year of life.

A simple strategy to improve patient adherence to outpatient fecal occult blood testing.

Poor patient adherence reduces the effectiveness of fecal occult blood testing for colon cancer. Patients at the inner-city clinic involved in the study have historically completed only one-third of the tests administered to them. The authors studied three ways of returning test kits (by hand, by mail, and by mail with prepaid postage). Among 146 randomly assigned patients, the completion rates were 37%, 57%, and 71%, respectively. The difference was significant between the first and third groups (p = 0.003), and the cost was less for the third group ($1.71 vs $2.24 per completed test). The authors recommend that clinics serving indigent populations use postage-paid return envelopes with fecal occult blood testing to improve its effectiveness and save money.

Toxic epidermal necrolysis--an unusual cause of colonic perforation. Report of a case.

Toxic epidermal necrolysis (TEN) is a rare and severe reaction to certain drugs that results in full-thickness epidermal necrosis of the skin. Other mucosal surfaces such as the oropharynx, esophagus, conjunctiva, and genitalia may also be affected. Specific involvement of the colon is distinctly unusual. A case of TEN that resulted in complete small bowel necrosis and colonic perforation is reported. Early and aggressive surgical resection dealt successfully with this complication and, along with supportive care, allowed resolution of the systemic condition to occur. Five additional cases of colonic necrosis complicating TEN are reviewed.

Development of the glutamate system in rabbit retina.

We have investigated two characteristics of the glutamate system in the developing rabbit retina. 1) Glutamate immunoreactivity was observed at birth within developing processes of four cell types; two of which, photoreceptors and ganglion cells, are known to be glutamatergic in the adult. Two other cell types, type A horizontal cells and amacrine cells, are immunoreactive to both glutamate and GABA at birth, suggesting that endogenous pools of glutamate in GABAergic neurons serve as precursor for GABA synthesis. Thus it appears that endogenous glutamate pools are present within neurons prior to synaptogenesis as part of the early expression of either the glutamate or GABA transmitter phenotype. 2) Analysis of 3H-glutamate metabolism during retinal development showed that rapid conversion of glutamate to glutamine does not occur until the second postnatal week, coincident with the expression of Muller (glial) cell activity. In the absence of glial metabolism in the neonate, extracellular concentrations of glutamate remain relatively high and are likely to have major effects on neuronal maturation.

Melatonin inhibits ACh release from rabbit retina.

Previous studies have suggested that melatonin, released from photoreceptors, may modulate retinal dark-adaptive responses by inhibition of dopamine release from retinal interneurons. We have broadened these studies to examine the effect of melatonin on release of another retinal neurotransmitter, acetylcholine (ACh). The ACh system in rabbit retina has been localized to starburst amacrine cells, which release ACh in response to a variety of experimental stimuli, including direct potassium depolarization, flashing light, and glutamatergic as well as GABAergic inputs. The effect of melatonin on release of endogenously synthesized [3H]-ACh was measured in perfusates from retinas or retinal synaptosomes preloaded with [3H]-choline chloride. Melatonin significantly inhibited ACh release stimulated by potassium in intact retina but not in synaptosomes. Stimulation of intact retina by flashing light or by the glutamate receptor agonist, kainic acid, was also inhibited by melatonin. In contrast, there was no significant effect of melatonin on picrotoxin-induced release. These findings suggest that melatonin does have an inhibitory effect on ACh release, either by direct interaction with the cholinergic amacrine cell, or indirectly via GABAergic but not glutamatergic neurons.

Darkness stimulates rapid synthesis and release of melatonin in rat retina.

The presence of melatonin in retina has been widely reported for over two decades although studies of its functional importance within the retina have only recently been emphasized. We have analyzed the biochemical characteristics of melatonin synthesis and release, focusing on rapid changes in response to light/dark conditions. Our major findings are consistent with the following conclusions: (1) melatonin synthesis is stimulated within minutes after exposure to darkness, and may reflect an increase in N-acetyl transferase activity; (2) melatonin is not stored, but rather it diffuses freely throughout the retina immediately after it is synthesized; and (3) the dark-induced increase in retinal melatonin release is a synthesis-coupled response and does not involve separate secretion mechanisms. The characteristics of melatonin synthesis and release described herein would be consistent with the proposed role of melatonin as a local paracrine effector of dark-adaptive responses in retina.

AP4 inhibits chloride-dependent binding and uptake of [3H]glutamate in rabbit retina.

Glutamate is one of the major neurotransmitters used by primary and secondary neurons of the visual pathway in retina. AP4(2-amino-4-phosphonobutyric acid) preferentially blocks the activity of one functional subclass of retinal neurons, ON bipolar cells, apparently by acting as an agonist at a hyperpolarizing glutamate receptor. We have used in vitro binding assays to examine different subclasses of presumptive glutamate receptors in retinal membrane fractions. One subclass consists of AP4-sensitive binding sites which require calcium and chloride for maximal binding and which are inhibited by freeze-thaw procedures. In addition, AP4 inhibits chloride-dependent [3H]glutamate uptake into retinal synaptosomes and intact retina. [3H]glutamate which is accumulated via the AP4-sensitive mechanism can be subsequently released by depolarizing levels of potassium. The pharmacological selectivity of AP4-sensitive glutamate receptors on ON bipolar cells measured electrophysiologically is very similar to that of AP4-sensitive, [3H]glutamate binding and uptake, measured biochemically in subcellular fractions. These results raise the possibility that AP4-sensitive glutamate recognition sites in retina may be linked to two separate effectors, one which gates ion channels and leads to hyperpolarization, and another which acts as a glutamate transporter.

Analysis of pre- and postsynaptic factors of the serotonin system in rabbit retina.

[3H]Serotonin is accumulated by a specific set of amacrine cells in the rabbit retina. These cells also accumulate the neurotoxin, 5,7-dihydroxytryptamine, and show signs of necrosis within 4 h of in vivo exposure to the drug. Biochemical analysis of [3H]serotonin uptake reveal a sodium- and temperature-dependent, high affinity uptake system with a Km of 0.94 microM and Vmax of 1.08 pmol/mg protein/min. [3H]Tryptophan is also accumulated in rabbit retinal homogenates by a high affinity process. Accumulated [3H]serotonin is released in response to potassium-induced depolarization of intact, isolated retinas. In vitro binding studies of rabbit retinal homogenate membranes demonstrate specific sets of binding sites with characteristics of the postsynaptic serotonin receptor. These data strongly suggest that rabbit retina contains virtually all of the molecular components required for a functional serotonergic neurotransmitter system. The only significant difference between the serotonin system in rabbit retina and that in the well-established serotonin transmitter systems in nonmammalin retinas and in brains of most species is the relatively low concentration of endogenous serotonin in rabbit retinas, as demonstrated by high-performance liquid chromatography, histofluorescence, or immunocytochemistry.