Tooth Discoloration from 2 Silver Fluorides Used in Adults with Special Needs: A Randomized Trial.

BACKGROUND: Discoloration of carious lesions after application of silver diamine fluoride lowers patient acceptance and limits its wider use for caries arrest. OBJECTIVE: To assess lesion and tooth color changes from 2 novel silver fluoride (AgF) products and its relationship to caries activity (clinical visuo-tactile scores) and bacterial load (using laser fluorescence with the DIAGNOdent). METHODS: A split-mouth design was followed, with matched smooth surface carious lesions in the same arch in adults with special needs randomized for 1-min treatments with AgF/potassium iodide (KI) (Riva Star Aqua, SDI) and AgF/stannous fluoride (SnF2) (Caries Status Disclosing Solution; Whiteley). Standardized images taken at baseline, immediately postoperatively, and at 3-mo review were subjected to digital image analysis to calculate delta-E and to track changes in luminosity of carious lesions. RESULTS: Twelve participants were recruited in the study. A total of 56 teeth (28 pairs) were included. Significantly greater changes were seen in treated lesions than in the adjacent noncarious natural tooth structure, both immediately after treatment and at the 3-mo review (P < 0.0001). Color change and caries activity were not affected by tooth type, tooth location, plaque status, salivary status, or special needs condition. AgF/SnF2 caused transitory darkening immediately on application, while AgF/KI caused the immediate formation of yellow deposits (silver iodide). Both products caused significant darkening of treated lesions at 3 mo (P = 0.0009; P = 0.0361), with no differences between them (P = 0.506). Responding lesions showed larger and more perceptible color changes immediately after either AgF application (P = 0.002; P = 0.024). CONCLUSIONS: Both AgF products were highly effective for caries arrest in this patient population. Despite minor differences in the appearance of treated lesions at the time of application, both products lead to similar darkening of treated sites at 3 mo. KNOWLEDGE TRANSFER STATEMENT: This study shows the usefulness of silver fluoride used in conjunction with potassium iodide or stannous fluoride for achieving caries arrest in smooth surface lesions in adults with special needs. Patients need to be informed that long-term staining of the lesion occurs with both, similar to silver diamine fluoride.

Skin Color Representation in Teaching Photographs: One Institution's Approach to Addressing Visual Racism in Medical Education.

Problem: Visual racism refers to both the underrepresentation and inappropriate representation of darker skin types in medical education. By not teaching medical students and resident physicians to recognize common conditions in darker skin, it perpetuates biases that contribute to healthcare disparities for racial and ethnic minoritized groups. In this paper we describe our efforts to engage in institutional anti-racism work by addressing imbalances in representation of darker skin types in visual teaching images within our institution's curriculum. Intervention: We initially surveyed preclinical medical students regarding their perceptions of skin color representation in two courses. Researchers recorded the skin types of all teaching photographs in these courses in 2020. We then provided feedback and education to faculty, proposing that they increase brown and black skin color representation in educational content. During 2021, we reviewed the same courses and surveyed students again to ascertain the implementation and impact of our proposal. Context: We applied our intervention to two courses, Host & Defense (H&D) and Skin, Muscle, Bone, and Joint (SMBJ) since both courses utilize a large number of teaching images. Impact: From 2020 to 2021, both H&D and SMBJ significantly increased the proportion of visual teaching images that included darker skin types, with an increase from 28% to 42% in H&D and 20% to 30% in SMBJ. Significantly more students in the courses' 2021 iterations (73% in H&D, 93% in SMBJ) felt that lectures had appropriate representations of darker skin types when compared to students who took the course in 2020 (8% in H&D, 51% in SMBJ). Students in 2021 felt more confident in recognizing dermatological signs and symptoms in patients with darker skin than students in 2020. The majority of students in both 2020 and 2021 reported wanting to see a gradient of skin types for every dermatological condition discussed. Lessons learned: Our work suggests that addressing visual racism can be achieved partly by setting expectations for increased visual representation, collaborating across educational departments, and establishing clear metrics for assessing implementation. Future interventions will require a continual feedback loop of monitoring learning material, assessing faculty and student perception, refining resources, and recommending revisions to improve visual representation across the entire curriculum.

Early Childhood Exposures to Fluorides and Child Behavioral Development and Executive Function: A Population-Based Longitudinal Study.

It is important to both protect the healthy development and maintain the oral health of the child population. The study examined the effect of early childhood exposures to water fluoridation on measures of school-age executive functioning and emotional and behavioral development in a population-based sample. This longitudinal follow-up study used information from Australia's National Child Oral Health Study 2012-14. Children aged 5 to 10 y at baseline were contacted again after 7 to 8 y, before they had turned 18 y of age. Percent lifetime exposed to fluoridated water (%LEFW) from birth to the age 5 y was estimated from residential history and postcode-level fluoride levels in public tap water. Measures of children's emotional and behavioral development were assessed by the Strength and Difficulties Questionnaire (SDQ), and executive functioning was measured by the Behavior Rating Inventory of Executive Function (BRIEF). Multivariable regression models were generated to compare the associations between the exposure and the primary outcomes and controlled for covariates. An equivalence test was also conducted to compare the primary outcomes of those who had 100% LEFW against those with 0% LEFW. Sensitivity analysis was also conducted. A total of 2,682 children completed the SDQ and BRIEF, with mean scores of 7.0 (95% confidence interval, 6.6-7.4) and 45.3 (44.7-45.8), respectively. Those with lower %LEFW tended to have poorer scores of the SDQ and BRIEF. Multivariable regression models reported no association between exposure to fluoridated water and the SDQ and BRIEF scores. Low household income, identifying as Indigenous, and having a neurodevelopmental diagnosis were associated with poorer SDQ/BRIEF scores. An equivalence test confirmed that the SDQ/BRIEF scores among those with 100% LEFW were equivalent to that of those who had 0% LEFW. Exposure to fluoridated water during the first 5 y of life was not associated with altered measures of child emotional and behavioral development and executive functioning.

Population Impact of Sugar-Sweetened Beverages on Dental Caries and Overweight/Obesity in Australian Children.

KNOWLEDGE TRANSFER STATEMENT: The reported findings greatly consolidated evidence of detrimental effects of sugars intake on child oral health and overweight and obesity, some of the most prevalent chronic conditions in children. Evidence on population impact of sugars intake is directly informative to policy makers and the public about the potential impact of population-based programs targeting sugars intake to prevent dental caries and overweight and obesity.

Socioeconomic Status and Toothbrushing in Indigenous and Non-Indigenous Australian Children.

INTRODUCTION: Dental caries in children is a multifactorial and complex condition. Toothbrushing helps maintain good oral hygiene and delivers fluoride. However, determinants of toothbrushing could vary based on Indigenous status. OBJECTIVE: This study aimed to assess the association between socioeconomic status and adequate toothbrushing practice (brushing twice or more a day) in Indigenous and non-Indigenous Australian children. METHODS: Data were acquired from the National Child Oral Health Survey (NCOHS) 2012 to 2014. NCOHS administered questionnaires to parents of a representative sample of 24,215 Australian children aged 5 to 14 y recruited using a complex sampling method. Data on the frequency of toothbrushing and socioeconomic status were collected through the questionnaires. Statistical analysis was conducted progressively from bivariate to multivariable regression modeling, stratified by Indigenous status. RESULTS: Just over half of Indigenous children and over two-thirds of non-Indigenous children reported adequate toothbrushing. The prevalence of adequate brushing (twice or more a day) was 42% (95% confidence interval [CI], 1.10-1.84) higher among children with an overseas-born parent than those with Australian-born parents. Among non-Indigenous children, sex and age, parents' country of birth, number of children in the family, and other family socioeconomic indicators (education, income, private health insurance) were associated with adequate toothbrushing. The prevalence of adequate brushing was 1.09 (95% CI, 1.03-1.15) and 1.15 (95% CI, 1.10-1.21) times higher when their parent possessed vocational training and tertiary education, respectively, compared to those children whose parents had school-level education. CONCLUSIONS: There were differences in patterns of socioeconomic disparities for toothbrushing practices between Indigenous and non-Indigenous Australian children. KNOWLEDGE TRANSFER STATEMENT: To promote positive toothbrushing practices in children, dental clinicians and public health professionals must be aware of the determinants of toothbrushing practices. Socioeconomic disparities in toothbrushing frequency were more apparent in non-Indigenous children. These results will help develop population-specific interventions that tackle the determinants to help improve oral hygiene behavior in Indigenous and non-Indigenous children.

Excess Risk of Dental Caries from Higher Free Sugars Intake Combined with Low Exposure to Water Fluoridation.

The study aimed to quantify the excess risk of interaction between high free sugars (sugars) intake and lack of exposure to water fluoridation on child dental caries. Data from the Australian National Child Oral Health Study, a population-based survey of 24,664 children aged 5 to 14 y, were collected using parental questionnaires and oral epidemiological examinations by trained examiners. Information on socioeconomic status, dental health behaviors, and dental service use was used as covariates. The number of servings of sugars-containing foods and drinks consumed in a usual day was assessed as the main exposure, categorized into 5 groups. Residential history was used to calculate lifetime exposure to fluoridated water (LEFW), categorized as low (<25%), medium (25% to <75%), or high (75%-100%). Caries prevalence (dmfs/DMFS >0) and experience (dmfs/DMFS) in the primary (ages 5-10 y) and permanent (ages 8-14 y) dentitions were the main dependent variables. The association of sugars intake and LEFW with each outcome was estimated in multivariable log-Poisson regression models with robust standard error estimation, adjusted for covariates. The relative excess risk due to interaction (RERI) between sugars intake and LEFW was estimated. Strong positive gradients in all outcomes were observed across sugars intake groups. Relative to the lowest intake group, the 3 highest intake groups had significantly higher adjusted prevalence ratios for having caries and higher adjusted mean ratios of caries experience in both dentitions, after controlling for all covariates. LEFW strongly and consistently attenuated the effects of all levels of sugars intake on the outcomes. RERI estimates indicated that a combination of lack of exposure to fluoridated water and high sugars intake resulted in greater excess risk of primary and permanent caries than if there was no interaction. Evidently, children with high sugars intakes and low exposure to water fluoridation are at disproportionately higher risk of dental caries.

Fluoridated Water Modifies the Effect of Breastfeeding on Dental Caries.

Breastfeeding is important for health and development. Yet, the interaction between breastfeeding duration and usage of fluoridated water on caries experience has not been investigated. This study examined exposure to fluoridation as an effect modifier of the association between breastfeeding duration and caries. The 2012 to 2014 national population-based study of Australian children involved parental questionnaires and oral epidemiological assessment. Children were grouped by parent-reported breastfeeding duration into minimal (none or <1 mo), breastfed for 1 to <6 mo, breastfed for 6 to 24 mo, and sustained (>24 mo). Residential history and main water source used for the first 2 y of life were collected to group children into exposed (WF) and nonexposed (NF) to fluoridation. Socioeconomic status, infant formula feeding, and sugar-sweetened beverage (SSB) consumption data were collected. The prevalence and severity of caries in children aged 5 to 6 y were primary outcomes. Multivariable regression models with robust error estimation were generated to compute prevalence ratios (PRs) and mean ratios (MRs) for 3 breastfeeding groups against the reference (breastfed for 6-24 mo). Of the 5- to 6-y-old children, 2,721 were in the WF and 1,737 were in the NF groups. The groups had comparable distributions of socioeconomic factors, infant formula feeding, and SSB consumption. There were U-shape distributions of caries experience among breastfeeding groups, being more pronounced among NF children. Among NF children, the minimal and sustained breastfeeding groups had significantly higher PR (1.4 [1.1-1.9] and 1.8 [1.4-2.4]) and MR (2.1 [1.4-3.3] and 2.4 [1.4-4.1]) than the reference group. However, among the WF children, this association between breastfeeding duration and caries attenuated after adjustment for other factors. The study contributes evidence of a nonlinear (U-shape) association between breastfeeding duration and dental caries. Early life exposure to fluoridated drinking water attenuated the potential cariogenic effect of both lack of and sustained breastfeeding.

Community trial of silver fluoride treatment for deciduous dentition caries in remote Indigenous communities.

BACKGROUND: Silver fluoride 40% followed by 10% stannous fluoride (AgF) has been used in dental practice in some parts of Australia for many years. This study compared the effectiveness of application of AgF with atraumatic restorative technique (ART) in managing cavitated carious primary molars. METHODS: The study was a community effectiveness trial in two remote Aboriginal communities where AgF has been used for some time. Children between 4 and 8 years of age with caries on primary molars were randomized by birth date to receive AgF or ART. There were 210 children who were included in the study with 384 eligible teeth. Children were followed up for periods between 9 months and 4 years. Negative outcomes such as dental pain, extraction, use of antibiotics and more extensive restorative treatment were considered as the primary outcomes of the study. RESULTS: Those negative outcomes were infrequent in both treatment groups. The prevalence ratio of negative sequelae for children treated with AgF compared with those treated with ART was lower at 0.18. CONCLUSIONS: Silver fluoride was well accepted, easy to use and at least as effective a treatment as ART for dental caries in primary molars in young Aboriginal children in remote areas.

Race- and Income-Related Inequalities in Oral Health in Australian Children by Fluoridation Status.

Inequality in child oral health exists by race and income. Water fluoridation (WF) is effective in caries prevention, but evidence for WF reducing inequality in caries experience is equivocal. This study tested the hypothesis that WF reduces race- and income-related inequality in child caries experience. A cross-sectional national population-based study of child oral health was conducted across 2012 to 2014 for Australian children aged 5 to 14 y, involving a parental questionnaire and an oral epidemiological examination. Children were stratified by fluoridated (F) and nonfluoridated (NF) area of residence, equivalized household income quartiles, and Indigenous and non-Indigenous status. Directly standardized caries experience (measured by the decayed, missing, or filled tooth surfaces [dmfs/DMFS] in both primary [age 5-10] and permanent dentitions [age 9-14]) was estimated for each stratum accounting for the complex sampling design. Differences in caries experience by Indigenous status and equivalized income quartiles were examined between F and NF strata. Socioeconomic inequality in caries experience was examined using the Absolute Concentration Index (ACI), Slope Index of Inequality (SII), Relative Concentration Index (RCI), and Relative Index of Inequality (RII). A total of 21,328 (86.5%) children had complete data. Caries experience was higher in NF than F strata. Race- and income-related gradients in caries experience were observed in both F and NF areas. All indexes of inequality indicated that caries experience was concentrated among lower income groups. Absolute inequalities were consistently lower in F than in NF areas. For the primary dentition, SII values were -4.18 versus -6.20 in the F and NF areas, respectively. The respective values were -0.60 versus -1.66 for the permanent dentition. Income-related inequality in caries was lower in F than in NF areas for both Indigenous and non-Indigenous children. WF was associated with lower caries experience and reduced inequality among children. Knowledge Transfer Statement: The results of this study greatly increase the evidence base that water fluoridation is effective and socially equitable. Dental caries has remained one of the most prevalent chronic conditions in children worldwide. The presented evidence can be used by policy makers and the profession to support the maintenance or expansion of this important public health program to benefit those most at risk of dental caries, the groups at the lowest socioeconomic position in any society.

Trend of income-related inequality of child oral health in Australia.

It is important that we monitor socio-economic inequality in health. Inequality in child oral health has been expected to widen because of widening socio-economic inequality. This study aimed to evaluate trends in income-related inequality in caries experience of Australian children. Cross-sectional studies in 1992/93 and 2002/03 collected data on deciduous caries experience of 5- to 10-year-olds and permanent caries experience of 6- to 12-year-olds. Household composition and income was used to calculate quartiles of equivalized income. Slope Index of Inequality (SII), Concentration Index (CI), and regression-based rate ratios were used to quantify income-related inequality and to evaluate trends. Income-related inequality in caries experience was evident regardless of time and dentition. The three indicators of inequality indicate a significant increase in income-related inequality in child deciduous caries experience during the decade. The income inequality in permanent caries experience did not change significantly. Income inequalities increased in deciduous teeth, but not in permanent teeth, among Australian children.

Association between dental caries and fluorosis among South Australian children.

There is an ongoing debate whether early exposure to fluoride is necessary. The effect of early exposure to fluoride needs to be understood to inform the appropriate population oral health policy. This study aimed to evaluate the effects of systemic fluoride intake in early childhood on caries experience of the permanent dentition observed at the age of 8 or 9 years, controlling for the effects of topical exposures to fluoride at the age of 5 years. The fluorosis experience of children was used as a proxy indicator for early exposure to fluoride, while exposures to fluoride at the age of 5 years, which were collected retrospectively, were used to control for the topical effect on permanent caries experience of the same children. Caries experience recorded at 8 or 9 years of age of 571 children was compared between groups with or without fluorosis. Bivariate analysis and multivariate analysis controlling for exposures to fluoride at the age of 5 years and other potential contributory factors were conducted. A quarter of the sample had very mild to mild fluorosis, while 15% had caries experience in the permanent dentition. Multivariate models showed that children who did not have fluorosis had a higher prevalence and severity of caries compared to those with fluorosis after controlling for other factors. In conclusion, there was a significant negative association between fluorosis status and caries experience in the permanent dentition.

Evidence for target preferences by cholinergic axons originating from different subdivisions of the basal forebrain.

Possible target preferences of basal forebrain cholinergic neurons were studied in organotypic slice cultures. Cholinergic neurons in slices of medial septum or substantia innominata send axons into both hippocampus and neocortex when co-cultured together. However, septal cholinergic axons course through adjacent slices of neocortex to reach and branch densely in slices of hippocampus, but septal axons seldom grow beyond adjacent hippocampal tissue to reach neocortex. In contrast, cholinergic axons from substantia innominata commonly grow through hippocampus to reach neocortex, and also grow through neocortex to reach hippocampus, with similar branching densities in each target. The greater density of septal axonal branches in hippocampus than in neocortex suggests a preference of septal axons for the hippocampal target.

Electrical conduction through poly(dA)-poly(dT) and poly(dG)-poly(dC) DNA molecules.

We report direct measurements of electrical transport through poly(dA)-poly(dT) and poly(dG)-poly(dC) DNA molecules containing identical base pairs. The observed experimental results suggest that electrical transport through DNA molecules occurs by polaron hopping. We have also investigated the effect of gate voltage on the current-voltage curve. It demonstrates the possibility of a DNA field-effect transistor operating at room temperature. Moreover, the gate-voltage dependent transport measurements show that poly(dA)-poly(dT) behaves as an n-type semiconductor, whereas poly(dG)-poly(dC) behaves as a p-type semiconductor.

Cutaneous hamartoma of the hand: MR imaging findings.

We report two cases of magnetic resonance imaging of the cutaneous hamartoma on the hand, which is a rare benign soft tissue tumor.

Lipoblastoma of the parietal pleura in a 7-month-old infant.

A 7-month-old infant boy with pleural lipoblastoma is presented. The diagnosis was confirmed by operation. Radiologic findings consisted of a pleura-based mass with well-defined margin. The main differentiation is from liposarcoma, which is extremely rare in children under 3 years of age.

Enhanced survival and morphological features of basal forebrain cholinergic neurons in vitro: role of neurotrophins and other potential cortically derived cholinergic trophic factors.

The present study examined survival- and growth-enhancing effects of cortical cells on basal forebrain cholinergic neurons (BFCNs) in culture and the degree to which endogenous nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) contribute to those trophic effects. When fetal (17 days of gestation) basal forebrain (BF) cells were grown for 5 days in coculture with cortical neurons, staining for acetylcholinesterase (AChE) showed a threefold increase in the number of BFCNs relative to BF cultures without cortex. Most of these labeled cells also displayed enhanced somatic, dendritic, and axonal growth. Coculturing cortical neurons with BF cells taken from postnatal animals produced similar results but with a somewhat greater degree of morphologic enhancement. Function-neutralizing antibodies to NGF, BDNF, and NT-3 were employed to determine whether they would block the trophic effects of cortical neurons on postnatal BFCNs. Although no significant changes in numbers or morphological features of AChE(+) neurons were observed with treatment with individual antibodies, cocultures treated with a combination of all three antibodies displayed fewer morphologically enhanced AChE(+) cells and more nonenhanced cells; the total number of AChE(+) neurons was not significantly changed. Treatment of pure BF cultures with exogenous NGF, BDNF, and NT-3 increased the number of AChE(+) neurons but did not reproduce the morphologic enhancement of cortical cells on BFCNs. These results suggest that neurotrophins by themselves can increase survival of postnatal BFCNs in culture and may work in concert with other unknown cortically derived factors to enhance BFCN morphologic differentiation. The unidentified cortical factors may also have strong survival-enhancing effects on BFCNs that are independent of the known neurotrophins.

Distinctive morphological features of a subset of cortical neurons grown in the presence of basal forebrain neurons in vitro.

Basal forebrain cholinergic neurons (BFCNs) provide the major subcortical source of cholinergic input to cerebral cortex and play an important role in regulating cortical activity. The present study examined the ability of BFCNs to influence neocortical neuronal growth by examining effects of the presence of BFCNs on certain cortical neurons grown under the controlled conditions of dissociated cell culture. Initial experiments demonstrated distinctive morphological features of a population of neurons (labeled with SMI-32, a monoclonal antibody to nonphosphorylated neurofilament proteins that labels pyramidal neurons in vivo) in cocultures containing basal forebrain (BF) and cortical cells. These neurons (large neurons immunoreactive for SMI-32 [SMI-32(+) neurons]) were characterized as having extensive axons, greater soma size, and more dendritic growth than did most SMI-32(+) neurons in the cultures. Staining for SMI-32 in cocultures in which the cortical neurons were labeled with a fluorescent marker before adding the BF cells indicated that virtually all large SMI-32(+) neurons were of cortical origin. Eliminating BFCNs with the selective cholinergic immunotoxin 192 IgG-saporin resulted in a >80% decrease in the number of large SMI-32(+) neurons, although causing little damage to other cells in the treated cultures; this suggests that survival or maintenance of large SMI-32(+) neurons may depend on ongoing trophic support from BFCNs. Thus, present findings suggest that BFCNs may provide powerful growth- and/or survival-enhancing signals to a subset of cortical neurons.

Neonatal treatment with 192 IgG-saporin produces long-term forebrain cholinergic deficits and reduces dendritic branching and spine density of neocortical pyramidal neurons.

The role of basal forebrain-derived cholinergic afferents in the development of neocortex was studied in postnatal rats. Newborn rat pups received intraventricular injections of 192 IgG-saporin. Following survival periods ranging from 2 days to 6 months, the brains were processed to document the cholinergic lesion and to examine morphological consequences. Immunocytochemistry for choline acetyltransferase (ChAT) and in situ hybridization for ChAT mRNA demonstrate a loss of approximately 75% of the cholinergic neurons in the medial septum and nucleus of the diagonal band of Broca in the basal forebrain. In situ hybridization for glutamic acid decarboxylase mRNA reveals no loss of basal forebrain GABAergic neurons. Acetylcholinesterase histochemistry demonstrates a marked reduction of the cholinergic axons in neocortex. Cholinergic axons are reduced throughout the cortical layers; this reduction is more marked in medial than in lateral cortical areas. The thickness of neocortex is reduced by approximately 10%. Retrograde labeling of layer V cortico-collicular pyramidal cells reveals a reduction in cell body size and also a reduction in numbers of branches of apical dendrites. Spine densities on apical dendrites are reduced by approximately 20-25% in 192 IgG-saporin-treated cases; no change was detected in number of spines on basal dendrites. These results indicate a developmental or maintenance role for cholinergic afferents to cerebral cortical neurons.

Kainate-stimulated Zn2+ uptake labels cortical neurons with Ca2+-permeable AMPA/kainate channels.

The endogenous cation, Zn2+, is synaptically released and may trigger neurodegeneration after permeating through NMDA channels, voltage sensitive Ca2+ channels (VSCC), or Ca2+ permeable AMPA/kainate channels (Ca-A/K). Neurons expressing Ca-A/K can be identified by a histochemical stain based upon kainate-stimulated Co2+ uptake (Co2+(+) neurons). The primary objective of this study was to determine whether a similar approach could be employed to visualize agonist-stimulated intracellular Zn2+ accumulation, and, thus, to test the hypothesis that Ca-A/K permit particularly rapid Zn2+ flux. Substituting Zn2+ for Co2+ during agonist-stimulated uptake, followed by Timm's sulfide-silver staining to visualize intracellular Zn2+, resulted in distinct labeling of a subpopulation of cortical neurons (Zn2+(+) neurons) closely resembling Co2+(+) neurons, suggesting that, like Co2+, Zn2+ may permeate Ca-A/K with particular rapidity. Neither NMDA nor high K+ triggered comparable Zn2+ accumulation, indicating substantially greater permeation through Ca-A/K than through NMDA channels or VSCC. Both fluorescence studies of intracellular Zn2+ accumulation and double staining studies (using SMI-32 and anti-glutamate decarboxylase antibodies, both markers of cortical neuronal subsets), support the contention that Zn2+ and Co2+ labeling identify a common set of neurons characterized by expression of AMPA/kainate channels directly permeable to Zn2+ and Co2+ as well as Ca2+. Furthermore, the preferential destruction of Zn2+(+) neurons (like Co2+(+) neurons) after brief kainate exposures in the presence of lower, more physiologic concentrations of Zn2+ suggests that Zn2+ permeation through Ca-A/K could contribute to selective neurodegeneration in disease. Finally, the study provides a novel and potentially advantageous histochemical approach for kainate-stimulated Co2+ or Zn2+ uptake labeling, using a room temperature technique (Timm's staining) rather than the usual hot AgNO3 development of the Co2+ stain.

Specificity of attachment and neurite outgrowth of dissociated basal forebrain cholinergic neurons seeded on to organotypic slice cultures of forebrain.

Development and differentiation of basal forebrain-derived cholinergic neurons were studied using a new technique that combines dissociated cell cultures with organotypic slice cultures. Slices of cerebral cortex or entire forebrain hemispheres were taken from early postnatal rat pups and maintained as organotypic cultures on membranes. Dissociated cell suspensions of basal forebrain tissue, taken from rat or mouse fetuses at gestational day 15-17, were seeded on to the slice cultures. Combined cultures were maintained for two to 14 days in vitro. Cultures processed for acetylcholinesterase histochemical staining demonstrated that stained neurons display regional variation in attachment to the slice, with most attachment occurring on cortex and with no detectable attachment on the caudate-putamen. Regional differences in attachment occur between cortical areas, with medial (cingulate) cortex showing much denser cell attachment than lateral (parietal) cortex, and across cortical layers, with layer I and deep layers showing more attachment than middle cortical layers. Similar patterns were observed on slices from rat brain irrespective of whether rat or mouse dissociated cells were used. Tyrosine hydroxylase-stained dissociated cells from ventral midbrain displayed a different pattern of attachment, with prominent attachment to the caudate putamen and less apparent specificity of regional and cortical laminar attachment. Little evidence of neurite outgrowth occurred during the first two days in vitro, but by four days, acetylcholinesterase-positive basal forebrain cells displayed several short and thick neurites that appeared to be dendrites, and one long process that appeared to be an axon. By seven days in vitro, dendrites are well developed and the presumed axon has extended branches over wide areas of cortex. These studies revealed several different types of cell-tissue interaction. The degree of cell growth and differentiation ranged from robust growth when dissociated cells were seeded on to slice cultures of normal target tissue, to apparently no attachment or growth when cells were seeded on to non-target tissue. This combined technique appears to be a useful method for studies of specificity of cell attachment and patterns of neurite outgrowth.